GENE EXPRESSION CHARACTERIZATION OF A GLAT1RAMER ACETATE RELATED DRUG PRODUCT IN MAMMALIAN AND HUMAN CELLS

This application claims, benefit of U.S. i½ovisional Application No. 62/394,398. filed September 14, 2016, and U.S. Provisional Application No, 62/402,189, filed September 30, 2016, the entire contents of each, of which are hereby incorporated by reference herein.

Throughout this application various publications are referenced by numerical identifiers in parentheses. Full citations of these references can be found following the Examples. The disclosures of these publications in then entireties are hereby incorporated by refereiice into this application in order to more fully describe the state of the art to which this invention pertains.

Multiple sclerosis (MS) is a chronic, debilitating autoimmune disease of the central nervous system (CNS) with either relapsmg-reniitting (RR) or progressive course leading to neurologic deterioration and disability. At time of initial diagnosis. RRMS is the most common form of the disease (Noseworthy et al. 2000) which is characteiized by unpredictable acute episodes of neurological dysfunction (relapses), followed by variable recovery and periods of clinical stability. The vast majority of RRMS patients eventually develop secondary progressive (SP) disease with or without superimposed relapses. Around 15% of patients develop a sustained deterioration of their neurological function from, the beginning; this form is called primary progressive (PP) MS. Patients who have experienced a single clinical event (Clinically Isolated Syndrome or "CIS") and who show lesion dissemination on subsequent magnetic resonance imaging (MR∑) scans according to McDonald's criteria, are also considered as having relapsing MS (Guideline on clinical investigation of medicinal products for the treatment of multiple sclerosis EMEA, London 16 September 2006).

With a prevalence that varies considerabl around the world, MS is the most common cause of chronic neurological disability in young adults (Bjartmar et al. 2002; Fleming 2002). Anderson et al. (1992) estimated that there were about 350,000 physician-diagnosed patients with MS in the United States in 195)0 (approx. 140 per 100,000 population). It is estimated that about 2.5 million individuals are affected worldwide (Compston et al. 2006). I general, there has been a trend toward an increasing prevalence and incidence of MS worldwide, but the reasons for this trend are not folly understood ( Anderson et al. 1992).

Current therapeutic approaches consist of i) symptomatic treatment ii) treatment of acute relapses with corticosteroids and hi) treatment aimed to modify the comse of the disease. Currently approved therapies target the inflammatory processes of the disease. Most of them are considered to act as immunomodulatory but then mechanisms of action have not been completely elucidated. Immunosuppressants or cytotoxic agents are also used in some patients after failure of conventional therapies. Several medications have been approved and clinicall ascertained as efficacious for the treatment of RR-MS; including BETASERON®, AVONEX® and REBIF®, which are derivatives of the cytokine interferon beta (TFNB), wirose mechanism of action in MS is -generally attributed to its itnnKinoniodalatoiy effects, antagonizing pro-nillainnmioiy reactions-. and hidseing suppressor cells (Revel et al. 2003). Oilier approved drugs- fo the treatment of MS. include Mitoxantrone and atalizumab.

Copaxone* (leva Pharmaceutical dusfnes Ltd.) is a glatiramer acetate drag product approved for treatment of patients w t relapsmg-reiinttmg multiple sclerosis (RRMS) and clinically isolated -syndrome (CIS) (Copaxone, Food and Dra Administratio Approved Labeling (Reference ID: 3443331) [online], TEVA Pharmaceutical Industries Ltd., 2014 [retrieved on December 24, 2014], Retrieved from the Internet: <L!RL: mv .a.ccessdat,a.fda.gov?¾ Glatiramer acetate drag substance (GA), the active substance of Copaxone ^® , is a complex mixture of polypeptides and is the first member of the gjatiraniokl class: i.e., a complex mixture of synthetic polypeptides of varying sizes assembled from four naturally occurring amino acids: L-glutamic acid, L-alanine, L-lysine, and L- tyrosine, in a defined molar ratio (Varkoiily et al. 2009). GA elicits anti-inflammatory as well as neuiOprotective effects in various animal models of chronic inilarnmatoiy and neurodegenerative diseases (Filippi M, et al. (2001); ipnis J. Schwartz M (2002); Teitelbaimi D et al. (1988); Putheti et al. (2003)) and has been shown to be safe and effective in reducing relapses and delaying neurologic disability in MS patients following long-term treatment (Ford et al. 2010).

The mechanisms underlying GA therapeutic activity are not fully elucidated, but GA activity on immune cells has been well demonstrated. GA appears to act as an altered peptide ligand (APL) of encephalitogenic epitopes within myelin basic protein (MBP) (Aharoni et al. 1999) and demonstrates cross-reactivity with MBP at the humoral and cellular levels (Anion R. Aharoni R (2004); Teitelbaimi et al. (1991): Webb et al. (1973): Duda et al. (2000); Brenner T et al. (2001); Aharoni et al. (1997)). The unique antigenic sequences of the GA polypeptide mixture compete with myelin antigens for binding to MHC class II molecules on antigen presenting cells (APCs) and presentation to the T cell receptor (TCR). resulting in the induction of anergy or deletion of autoreactive MBP -reactive T cells and proliferation of GA-reaciive T cells. At initiation of Copaxone* treatment GA-reaetrve CD4+ T- cell lines from MS patients secrete both pro-inflammatory T helper type 1 (Till) and anti-inflammatory Th2 cytokines (Duda et, al. (2000); Wiesemann E, et al. (2003)), but continued exposure to Copaxone ^® induces a shift in GA-reactive T cells toward the Th2 phenotype (Duda et al (2000); Aharoni et al. (1997); Aharoni et al. (1998); Aharoni et al. (2003); Miller et al. (1998); Neuhasu et al. (2000)).

Copaxone ^® also increases the number and suppressive capacity of CD4+CD25+FOXP3+ regulatory T cells, which are functionally impaired in MS patients (Venken et al. (2008); Haas et al. (2009); Hong et al. (2005)). Furthermore, treatment leads to antigen-nonspecific modulation of APC function. Copaxone ^® treatment promotes development of anti-inflammatory type Π monocytes characterized by an increase in mterleiikin (IL)-IO and tiansfonnmg growth factor-beta (TGF-β) and decreased production of IL-12 md tansor /necrosis factor (I (Weber et ai, 2007),

5 SUMMARY Of THE H E XIQ

The .present, invention provi des a process for efen'aeterizing a. gl atimiier acetate related drug subs tance or drug product comprising, the steps of

(a) obtaining _; a hatch of th glatirarner acetate related, drag substance or drag product ;

(b) contacting mamm li ceils with an .amount of the gia&ainer acetate related drug substance or drug product of step (a): and

(c) (i) detennining the level of expression of at least one gene ofMmpl2, Tgfbi, Ciec4n, CIec4a3, Fcerlg, Fcgr2b. Fcgr3, Csf3r, Hlr2 or Tbx2i ; (ii) detennining the level of expression of at least one gene of Ahcyl2, Chi5, Cxcr6, Dhrs3, Egr2, FabpS, Ptp4al, Gprl 83, Lampl. Ncoa4, Ptger4, Qk, Rragd, Sec24a, Sell 11, Sestdl, Slc30a4, Soati or Strip2: (iff) deienniiiing the level of expression of at least one gene of Fam 12a, Gznia, Itprl or Zbtb32; (iv) detennining the level of expression of at least one gene ofGzmb, Faml a3, Nfil3, Gzina. Eaf2, Carl or St7; (v) detennining the level of expression of a least one gene of Crisp3, Apoe, Dapll, Pde5a, Scg5, St8sia6, 4930426D05Rik or Pgapl; (vi) determining the level of expression of at least one gene involved in one or more pathway of collagen metabolic process (GO:0032963), defense response to bacterium (GO:0042742), defense response to Gram- negative bacterium (00:0050829), endocytosis (00:0006897), leukocyte mediated immunity (GO-.0002443), membrane invagination (00:0010324), membrane organization (GO:0016044), mu!ticellalar organisma! lnacrornolecule metabolic process (00:0044259), multicellular organismal metabolic process (00:0044236), myeloid leukocyte activation (GO:0002274), neutrophil chemotaxis (GO:OQ30593), phagocytosis, engulfinent (GO:0006911), positive regulation of cellular component organization (GO:00S1130), positive regulation of endocytosis (00:0045807), positive regulation of foam cell differentiation (GO;0010744),. positive regulation of phagocytosis (00:0050766), regulation of endocytosis (00:0030100), regulation of foam cell differentiation (GO: 0010743), regulation of phagocytosis (GO:0050764), regulation of type I hypersensitivity (GO:0001810), regulation of vesicle- mediated transport (00:0060627) or response to yeast (00:0001878): (vii) deteiininiiig the level of expression of at least one gene involved in one or more pathway of nucleotide binding (00:0000166), double-stranded RNA binding (00:0003725), NAEH- ADP-ribosyitransferase activity (00:0003950), transferase activity, transferring pentosyl groups (GO:0016763), transmission of nerve impulse (00:0019226), regulation of membraue potential (GO:O04239i), cytokine activity (00:0005125), ion homeostasis (GO:0050801) or adenyiylttansferase activity (00 0070566); (viii) detennining the level of expression of at least one gene involved in one or more pathway of Cytokine-cytokine receptor interaction (mmu04060), Jak-STAT signaling pathway (mm«04630). Sterol metabolic process (GO-.0016125), Sterol biosyndietic process (GO:0016126), Hematopoietic cell lineage (mmu04640). Myeloid cell apoptosis (GO: 0033028), Regulation of survival gene product expression (00:0045884), Regulation of leukocyte proliferation (GO:0070663), Positive regulation of survival gene product expression (GO;0045885), Hematopoietic cell lineage (mmu04640) or Metabolism, of xenobioties by cytochrome P450 (airauO09SQ); (ix) determining the level of expression of at least one gene iavo!yed in one or more pathway of Intrinsic to membrane receptor activity. Immune response, Leukocyte migration, Cseffiokine receptor binding, Cytokine-eyto ne receptor interaction. Cytokine activity, lak- ST.AT signaling pathway, Sterol bipsynthetie process. Steroid biosyuthetic process. Ceil surface. Cytokine binding. Cholesterol metabolic process, Cheniokine 'activity. Negative regulation of hormone secretion, Xsoprenoid biosyntlietic process or Positive regulation of peptidyl-serine phosphorylation :(x) determining the level of expression of at least one gene involved in one or more pathway of Glucose metabolic process, Glycolysis, Gluconeogenesis, Hexose catabolic process, Glucose catabolic process, Monosaccharide catabolic process, Hexose metabolic process. Monosaccharide metabolic process. Cellular carbohydrate catabolic process. Alcohol catabolic process. Carbohydrate catabolic process. Carbohydrate binding. Generation of precursor metabolites and energy. Phagocytosis. Leukocyte migration. Leukocyte chemotaxis. Positive regulation of phagocytosis. Pentose phosphate pathway. Galactose metabolism. Carbohydrate kinase activity. Myeloid leukocyte activation, Chemokine receptor binding. Regulation of type I hypersensitivity, Defense response to Gram-negative bacterium or Positive regulation of foam cell differentiation: (xi) determining the level of expression of at least one gene involved in one or more pathway of leukocyte migration (GO:0050900), cell chemotaxis (GO:0060326), leukocyte chemotaxis (00:0030595), phagocytosis (00:0006909), positive regulation of phagocytosis (GO:0050766), cell motion (GO:0006928), regulation of phagocytosis (00:0050764). behavior (GO:0007610), membrane invagination (GO:0010324), endocytosis (GO: 0006897), positive regulation of endocytosis (GO:0045807), phagocytosis, engulfment (GO:0006911), defense response to bacterium (GO:0042742), membrane organization (00:0016044), neutrophil chemotaxis (00:0030593), defense response to Gram-negative bacterium (GO:0050S29), regulation of endocytosis (GO.-0030100), leukocyte mediated immimity (00:0002443), cell migration (GO:0016477), regulation of vesicle-mediated transport (GO:0060627), localization of cell (GO:0051674), cell motility (GO:0048870), collagen metabolic process (00:0032963), multicellular organismal macromolecule metabolic process (GO: 0044259), positive regulation of cellular component organization (GO:0051130), myeloid leukocyte activation (00:0002274), positive regulation of foam cell differentiation (00:0010744), multicellular organismal metabolic process (GO:0044236), response to yeast (00:0001878), regulation of type I hypersensitivity (GO:0001S10), regulation of foam cell differentiation (00:0010743), sugar binding (00:0005529), endopeptidase activity (00:0004175), serine hydrolase activity (GO:0017171), serine-type peptidase activity (GO:0008236), serine-type endopeptidase activity (00:0004252), cytokine activity ((30:000 125), calcium ion binding (00:0005509), immunoglobulin binding (00:0019865), chemokine activity (00:0008009), chemokine receptor binding (GO:0042379), peptidase activity, acting on L-amino acid peptides (00:0070011), peptidase activity (00:0008233), IgG binding (00:0019864). protein complex binding (GO:0032403) or enzyme inhibitor activity (GO:0004857); (xii) detenni ing the level of expression of at least one gene mvofved in one or more pathway of glucose metabolic process (GO:0006006), glycolysis _: (GQ:0OO6096), exose iseiaLwlic process (GO:001931S) _; , monosaccharide iiietaboiic process (GQ:O005996), Jiexose caiabolk process (GO;0019320), glucose catabolie process (GO: 0006007), monosaccharide esiabohc process (GQ:0046365), cellular carbohydrate- eataboiic process (GQ:004427S), alcohol eataboiie process: (GQ:0O46104), carbohydrate, catabolic process (GO:0O16052), generation of precursor metabolites and energy (GO:O0O6O 1), oxidorediictase ^' activity, acting: on single donors with incorporation of molecular oxygen, iricorpomtion of ^' two atoms of oxygen (GO:0016702), oxidorediictase activity, acting on single donors with incorporation of molecular oxygen (GO.-0016701), cytokine activity (00:0005125), carbohydrate kinase activity (00:0019200), Glycolysis / Ghiconeogenesis (inmuOOOlO), Pentose phosphate pathway (inmu00030). Fructose and niannose metabolism (rnmuOOOSl) or Galactose metabolism (mmuO0O52); (xiii) determining the level of expression of at least one gene involved in one or more pathway of RIG-I-like receptor signaling pathway or Adenylyltransferase activity; or (xiv) determining the level of expression of at least one gene of Cxcl3 _t Clec4a3, UgtlalO, Ugtla6b, UgtlaSa, Ugtla7c. Ugtla9, UgtlaS, Ugtlal, Ugiia2, Clec4n, Tgfti, Nov. Ltf Cci6, Mni i2, Ms4a6d, LpL Mrnp8, Cdl77, Tfec, Clec7a, Mpo, Ccl9, Ccrl, Gpnmb, Msrl, Ms4a6d, Lyz2, Ctsl, Thbsl, Dab2, Arg2, Ccl6, Thbsl, Paidl, ClecSa, Cfp, Ifitm6, Lyzl, Oh¾i4. RnfI28, Ms4a3, Clec4d, S100a4, Ctsg. Prto3, Ngp, F10, Hal. Ccl2, Lyzl , Thbsl, Anxal, Lgals3, SiOOa.6, Nftml, CbiI3, Cbii4, Sppl, Stfa211 _t Ptgrl, 111©, Cd68, Olfml, FcgrS, Hlr2, Cregi, Dmxl2, Sirpa, 116, Elane, Csfir, Lcn2, Cd300ib, AloxSap, Illm, Slcl lal, Tyrobp, CdSOOa, Pira2, Piral, Pira7, Pira6, Pka5, Pira4, Gml0693, Lilra6, Pirb, Piral I, Ginl4548, Ms4a7, Mgstl , Camp, Bstl , Slc40al. Cregi , Csfir. Cd36, Rnfl28, Mrcl , MsrL Itgain, Serpinb2, Lilrb4. Gp49a ₅ Wide 17, AnxaS, RgslS, Sk40al, Bstl, C3ari , Mrap9, Histlh4c, Histlh4b, Histlh4a, Hist4h4, Histlh4f, Histlh4d, Histlh4k, Histlh4j, Histlh4i, Histlh4h, Histih4n, HistiMm, Hist2h4, Dstn, Igsf6, Mitf, Csf3r, Pf4, Pygl, Pdpn, S100a9, Fcgr2b, Idl, Emrl, Cd9, CebpdL Pla2g7, Arg2, F10. Gchn, SlOOaS, Ptplad2, Clec4a2. Clec4bl, Marcks, CsEra, Fcerlg, Clec4a2, Ppic, Cd302. Gstml, McptS, Cd3001f, Csf2rb, Bnip3, Ak4. Tpil, Plcxdi, Aldoc, Rgsi l, Ankrd37, PM, Pdkl, Cxel9, Tbx2 i, Pgki , Egln3, Ddit4, Pfkp, Pgiii2, P4hai, Slcl6a3, Ubd, Slc2al , 5330426P16Rik, Grhpr, Cxcli l. Rasa4, Ndrgl, Faml62a, Pkm. EghiL Aldoa, Gm 10480, Bnip31, Galkl, Pgaml , Pgkl , Mi£ Enol , Enol, Pdxp, PpplrSb, Stomli , Gzma, Sed2. Ccng2, Higdla, Eno2. Gfil . Pmi, Ldlia, Eroll, 30000020 lORik, Hk2, Slc2a3, Chchd6, P4hai, Eroll, Gpii, Gimap7, Oasli, Gml l l lO, Syce2, Ndrgl, Pt.gs2, Trappc6a, Prelidl, If 2712a, Zbtb32, Trini30a, Preiidl, Gapdh, Sdc3, BC062258, LOG 102643247, 23i0022B05Rik, Ms4a4c, Iil2rbl . Mthfdll, Smagp, Nampt, Fasl. PafahlbS, Prelidl, Gngl2, S, Kdm3a, Atf5, Tnfsf9, thereb characterizing the glatiramer acetate related drag substance or drag product of step (a).

The present invention also provides a process for characterizing a glatiramer acetate related drug substance or drug product comprising the steps of:

(a) obtaining a batch of the glatiramer acetate related drug substance or drug product: (b) contacting mammalian cells with an amount of the glatiramer . acetate related drug substance .or drug product of step (a): and

(c) detenniaia th level of expression of at least one gene hwolved. in one or more pathways set forth in Table 5, Table 7, Table 8, Table 10, Table 14 or Table 15,

thereby characterizing the glatirainer acetate related .drug substance or drag product of step (a).

The present invention also provides a process for discriminating ^' between gl atlraraer acetate related drug substances or drug products comprising the steps of:

(a) characterizing two or more glatiramer acetate related drag substances or drug products according to the process of the present invention to obtain characteristics of each of the glatiramer acetate related drag substances or drag products; and

(b) comparing tlie characteristics of the giatiranier acetate related drug substances or drag products obtained in step (a),

thereby discriminating between glatiramer acetate related drug substances or drug products.

The present invention also provides a process for producing or releasing a drug product comprisin a glatiramer acetate related drag substance, the improvement comprising the steps of:

(a) characterizing the glatiramer acetate related drug substance according to the process of any one of the present invent ion: and

(b) (i) discarding the batch of the glatiramer acetate related drug substance as unacceptable for inclusion in the drug product if the level of expression of one or more genes of Mnipl2. Tgfbi, Clec4n, C!ec4a3 _t Fcerlg, Fegr2b, Fcgr3. Csilr, I r2 or Tbx21 is not substantially identical to the level of expression by the same type of cells in the presence of the gla tiramer acetate drag substance under the same conditions; (ii) discarding the batch of the glatiramer acetate related drug substance as unacceptable for inclusion in the drug product if the level of expression of one or more genes of Aheyl2, Cln5, Cxcr6, Dhrs3, Egr2 _t Fabp5, Ptp4al _? Gprl83, Lanipl, Ncoa4, Ptger4, Qk, Rragd, Sec24a _f Se ll Sestdl , Slc30a4, Soatl or Strip2 is not substantially identical to the level of expression by the same type of cells in the presence of the glatiramer acetate drug substance under the same conditions; (iii) discarding the batch of the glatiramer acetate related drug substance as unacceptable for inclusion in tlie drug product if the level of expression of one or more genes of Fam212a, Gzma, Itprl or Zbtb32 is no substantially identical to the level of expression by the same type of cells in the presence of the glatiramer acetate drug substance vmder the same conditions: (iv) discarding the batch of the glatiramer acetate related drug substance as unacceptable for inclusion hi the drug product if the level of expression of one or more genes of Gzmb, Fami a3, Nfil3, Gzma, Eaf2. Carl or St? is downregulated or substantially identical to the level of expression by the same type of ceils in the absence of the glatiramer acetate related drug substance under the same conditions; (v) discarding the batch of the glatiramer ace ate-related-dnig-sabstaoee a,s unacceptable for inclusion to;t¾eito¾ r9di«; if ib leveiof $q»e8&taii of one or more genes of Crisp3, Apoe, Dapii, Pde5a, Scg5, StSsiaS, 4 30426DQ5Rik or Pgapl is, upregulated. or substantially identical to th level of expressioii by the sam type of ceils in the absence oftiie gfetiranier acetate related drug substance under die same coiKiifions; (vi) discarding the batch of die giatiramer acetate related drag substanc as unacceptable for iiidiision in the drag product if the set of differentially expressed genes indicates enrichment of one or more pathway of collagen metabolic process (00:0032963), defense response to bacterium (00:0042742) _* defease response to Grani- negative bacterium (00:0050829), endoeytosis (GO:0006897), ieukocyte mediated immunity (GO:0Q02443), membrane invagination (00:0010324), membrane organization (GO:0016044), multicellular organismal niaciOniolecule metabolic process (00:0044259), multicellular organisnial metabolic process (00:0044236), myeloid leukocyte activation (00:0002274), neutrophil chemotaxis (00:0030593), phagocytosis, engulfment (GO:0006911), positive regulation of cellular component organization (GO:0051130), positive regulation of endoeytosis (00:0045807), positive regulation of foam cell differentiation (00:0010744), positive regulation of phagocytosis (00:0050766), regulation of endoeytosis (00:0030100), regulation of foam cell differentiation (00:0010743), regulation of phagocytosis (GO:0050764), regulation of type I hypersensitivity (GO:0001810), regulation of vesicle- mediated transport (00:0060627) or response to yeast (GO.O001878): (vii) discarding the batch of the giatiramer acetate related drug substance as unacceptable for inclusion i the drug product if the set of differentially expressed genes indicates enrichment of one or more pathway of nucleotide binding (00:0000166), double-stranded R A binding (00:0003725), NAD+ ADP-ribosyltransferase activity (00:0003950), transferase activity, transferring pentosyl groups (00:0016763), transmission of nerve impulse (00:0019226), regulation of membrane potential (00 0042391), cytokine activity (00:0005125), ion homeostasis (00:0050801) or aelenyiyitiansfeiase activity (00:0070506); (viii) discarding the batch of the giatiramer acetate related dmg substance as unacceptable for inclusion in the drug product if the set of differentially expressed genes indicate no substantial enrichment of one or more pathway of Cytokine-cytokine recepto interaction (mmu04060), Jak-STAT signaling pathway (mniu04630). Sterol metabolic process (00:0016125), Sterol biosynthetic process (GO:0016126), Hematopoietic cell lineage (mmiiG4540), Myeloid cell apoptosis (GO: 0033028), Regulation of survival gene product expression (GO:0045884), Regulation of leukocyte proliferation (00:0070663), Positive regulation of survival gene product expression (00 0045885), Hematopoietic cell lineage (mmu04640) or Metabolism of xenobiotics by cytochrome P450 (mmu00980); (ix) discardin the batch of the giatiramer acetate related dmg substance as unacceptable for inclusion in the drug product if the set of differentially expressed genes indicate no substantial enrichment of one or more pathway of mtrinsie to membrane receptor activity. Immune response. Leukocyte migration, Cheniokine receptor binding. Cytokine-cytokine receptor interaction, Cytokine activity, Jak-STAT signaling pathway. Sterol biosynthetic process. Steroid biosynthetic process. Cell surface. Cytokine binding, Cholesterol metabolic process, Chemokine activity, Negative regulation of hormone secretion, Isoprenoid brosyrnhetic process or Positive regulation of peptidyi-se iBepiiosp!iorylatipii; fx) discarding the batch of the glatiranier acetate related; drug substance as unacceptable for inclusion in fee drug pr oduc t if th set of differentially expressed genes indicates emich eut of one or more pathway of Gluco se metabolic process. Glycolysis, GlueoneogeHesis, Hexose esiaooiie process, Glucose eatabolic process, Monosacdiaride eatabolic process, Hexose metabolic process. Monosaccharide metabolic process. Cellular carbohydrate eatabolic process, Alcohol eatabolic process. Carbohydrate eatabolic process. Carbohydrate bmding, Generation of precursor metabolites and energy. Phagocytosis, Leukocyte migr ation. Leukocyte chemotaxis. Positive regulation of phagocytosis. Pentose phosphate pathway, Galactose metabolism. Carbohydrate kinase activity. Myeloid leukocyte activation, Chemokine receptor binding. Regulation of type I hypersensitivity. Defense response to Gram-negative bacteriuin or Positive regulation of foam cell differentiation; (xi) discarding the batch of the glatiramer acetate related drug substance as unacceptable for inclusion in the drug product if the set of differentially expressed genes indicates enrichment of one or more pathway of leukocyte migration (00:0050900), cell chemotaxis (00:0060326), leukocyte chemotaxis (00:0030595), phagocytosis (GO:0006909), positive regulation of phagocytosis (GO: 0050766), cell motion (GO:0006928), regulation of phagocytosis (00:0050764), behavior (00:0007610), membrane invagination (00:0010324), endocytosis (00:0006897), positive regulation of endocytosis (GO:0045807), phagocytosis, engulfment (GO:00069i I), defense response to bacterium (00:0042742), membrane organization (00:0016044), neutrophil chemotaxis (00:0030593), defense response to Gram-negative bacterium (GO:0050829), regulation of endocytosis (GO:Q030100), leukocyte mediated immunity (00:0002443), cell migration (GO:0016477), regulation of vesicle-mediated transport (00:0060627), localization of cell (00:0051674), cell motility (GO:0048870), collagen metabolic process (GO:0032963), multicellular organismal macroinolecule metabolic process (00:0044259), positive regulation of cellular component organization (GO:00 1130), myeloid leukocyte activation (00:0002274), positive regulation of foam cell differentiation (00:0010744), multicellular organismal metabolic process (00:0044236), response to yeast (GO:0001878), regulation of type I hypersensitivity (GO:000i 810), regulation of foam cell differentiation (00:0010743), sugar binding (GO:0005529), endopeptidase activity (GO:0004175), serine hydrolase activity (00:0017171), serme-type peptidase activity (GO:0008236), serine-type endopeptidase activity (00:0004252), cytokine activity (00:0005125), calcium ion binding (GO:0005509), immunoglobulin binding (00:0019865), chemokine activity (GO:0008009), chemokine receptor binding (00:0042379), peptidase activity, acting on L-amino acid peptides (00:0070011), peptidase activity (GO:0008233), IgG binding (00:0019864), protein complex binding (00:0032403) or enzyme inhibitor activity (GO:0004857); (xii) discarding the batch of the glatiramer aceta te related drag substance as unacceptable for inclusion in the drag product if the set of differentially expressed genes indicates enrichment of one or more pathway of glucose metabolic process (00:0006006), glycolysis (00:0006096), hexose metabolic process (00:0019318), monosaccharide metabolic process (GO:0005996), hexose eatabolic process (GO:0019320), glucose c, eatabolie process GO:QO 6O07), monosaccharide; eatabolie process. (GQ:0046365), cellular carbohydrate caia olic ^process {GO:GG442753, alcohol caiabolic process. (00:0046164), carboliydraf caiabolic process (GO :0 16052), generation of precursor metabolites and energy (GO: 00060 1), oxidoreduetase activity, acting on single .donors with incorporation of molecular oxyge , incorporation of wo atoms of oxygen (00 0016702), oxidoreduetase activity, actin on single donors: with. incorporation of molecular oxygen (00:0016701), cytokine activity (GO;0OO5125), carbohydrate kinase activity (GO; 0019200), Glycolysis Glticoiieogenesis (inmuOOOlO), Pentose phosphate pathway (mmu0OO3O). Fructose and mannose metabolism (mmu00G51) or Galactose metabolism (mmu0O052); (xiii) discarding the batc of the glatirainer acetate related drag substance as unacceptable for inchision in the drug product if the set of differentially expressed genes indicates enrichment of one or more pathway of RIG-I-like receptor signaling pathway or Adenyiyltransferase activity; or (xiv) determining the level of expression of at least one gene of Cxel3, Clec4a3, UgtlalO, Ugtla6b, Ugtla6a, Ugtla7c, Ugtla9, UgtlaS. Ugtlal, Ugtla2. Ciec4n, Tgfbi, Nov, Ltf Ccl6, Mmpl2, Ms4a.6d, Lpl, Mmp8, Cdl77, Tfec, Ciec7a, Mpo Ccl9, Ccrl, Gpninb, Marl, Ms4a6d. Lyz2, CtsL Thbsl, Dab2, Arg2, Ccl6, Thbsl, Paldl, Clec5a, Cfp, Ifitm6, Lyzl, 01fin4, Rnfl28, Ms4a3, Clec4d, S100a4, Ctsg, Prtn3, gp, F10, Hal, Cci2, Lyzl. Thbsl, Anxal, LgalsS S100a6, Nfaml, Chil3, Chil4, Sppl. Stfa211, Ptgrl, Ilif9. Cd68, Olfrnl, Fcgr3, Hlr2, Cregl, Dmxl2, Sirpa, 116. Elane. Csflr, Lcn2, CdSOGlb, AioxSap. 321m, Slcl lal, Tyrobp, CdSOOa, Pira2, Piral, Pira.7, Pira6, PiraS, Pira4, Ginl0693, Lilra6, Pirb, Pirall, Gml4548, Ms4a7, Mgstl, Camp, Bstl, Sic40a.i, Cregl, Csflr, Cd36, Rafl2S, Mrcl, Msrl, Itgam, Seipinb2, Lslrb4, Gp49a, Wfdcl7, Anxa3, Rgs S, Slc4Qal. Bstl, C3aii, Mmp9, Histlh4c. Histlirfb, Hist.lh4a, Hist4h4, Histlh4i Histlh4d, Histlh4k, Histlh4j, Histlli4i, Hist h4h, Histlli4n, Histlh4m, Hist2h4, Dstn, IgsfS, Miff. Csf3r, Pf4, Pygl, Pclpn. S100a9, Fcgr2b, Ml, Emrl, Cd9, Cebpd, Pla2g7, Arg2, F10, Gclm, S100a8. Ptpiad2, Ciec4a2, Clec4bl, Marcks, Csf2ra, Fcerlg, Clec4a2, Ppic, Cd302, Gstml, McptS. CdSOOlf Csf2rb, Binp3, Ak4. Tpi , Plcxdl. Aldoc, Rgsl 1. Ankrd37. Pfkl, Pdkl, Cxcl9. Tbx21, Pgkl,Egln3, Ddit4,Pfkp, Pgm2, P4hal, Slcl6a3, Ubd, Slc2al, 5330426P16Rik, Griipr, Cxcll l. Rasa4, Ndrgl, Faml62a, Pkm, Eglnl, Aidoa, Gml0480, BnipSL Gaikl, Pgaml, Pgkl, Mil Enolb, Enol, Pdxp, Ppplr3b. Stomll, Gzrna, Scd2, Ccng2, Higdla, Eno2, GfiL Pml, Lclha, ErolL 3000002C10Rik, Hk2, Slc2a3, Chchd6, P4hal, Eroll, Gpsl, Gimap7, Oasll. Gini 1110, Syce2, Ndrgl, Ptgs2, Trappc6a, Prelidl, Ifs2712a, Zbfl 32, TrimSOa. Prelidl, Gapdh, Sdc3, BCQ6225S, LOCI 02643247, 2310022B05Rik _t Ms4a4c, ill2rbl, Mthfdll, Smagp. Nanipt, Fasl, Pafahlfo3 Prelid2, Gngl2, MS, dm3a, Atf3, Tnfsf9.

The present Invention also provides a process fo identifying suboptima! activity of a glatiranier acetate related drug substance or ding product comprising the steps of:

(a) administering a glatiranier acetate related drag substance or ding product to a mammal:

(b) characterizing the glatiranier acetate related cling substance or drag product according to the process of the present invention; and!) (i) identifying the glatirainer acetate related drug substance or drug prqdugt as having a suboptimal activity if the levei of expression of ^* one or more genes of Mmp 12, TgfbL C ^* lec4n, Clec4a3, Fceiig, Fcgr2b. FegrS C ^' siSr, Hlr2 or Tbx21 is not substantially identical to flie level of expression b the sarae type of cells in the presence of the .glatiramer acetate related drug subs.tsn.ee or drug product under the same conditions; (ii) identifying the glatirainer acetate related drug subs.tsn.ee. or drugprodnet as havin a suboptimal activity if the level of expression of one or more genes of Ahcyl2, C!nS, Cxcr6, I ⁾ hrs3, Egr2, FabpS, Ptp4al, Gprl S3, Larn l, Ncoa4, P†ger4, Qk, Rragd, Sec2I Sehll, Sestdl, Sic30a4, Soafi or Sirip2 i not substantially identical to the level of expression by the same type of cells in the presence of the glatirainer acetate related drug substance or drug product under the same conditions; (hi) identifying the glatirainer acetate related drug substance or drug product as having a suboptinial activity if the level of expression of one or more genes of Fain212a, Gzma, Itprl or Zbtb32 is not substantially identic al to the level of expression by the same type of cells in the presence of the glatirainer acetate related drug substance or drug product under the same conditions; (iv) identifying the glatirainer acetate related drug substance or drug product as having a suboptimal activity if the level of expression of one or more genes of Gzmb, Faml9a3, Nfil3, Gznia, Eaf2, Carl or Si 7 is downregulated or substantially identical to the level of expression by the same type of cells in the absence of the glatirainer acetate related drug substance under the same conditions; or (v) identifying the glatiramer acetate related drug substance or drug product as having a suboptimal activity if the level of expression of one or more genes of CrispS, Apoe, Dapll, PdeSa, Scg5, St8sia6, 4930426D05Rik or Pgapl is upregulated or substantially identical to the level of expression by the same type of cells in the absence of the glatiramer acetate related drug substance under the same conditions; (vi) identifying the glatiramer acetate related drug substance or drug product as having a suboptimal activity if the set of differentially expressed genes indicates enrichment of one or more pathway of collagen metabolic process (00:0032963), defense response to bacterium (00:0042742), defense response to Gram- negative bacterium (00:0050829), endocytosis (00:0006897), leukocyte mediated immunity (GO:0Q02443), membrane invagination (00:0010324), membrane organization (GO:001 044), multicellular organismal niacroniolecule metabolic process (00:0044259), multicellular orgaaismal metabolic process (00:0044236), myeloid leukocyte activation (GO:0002274), neutrophil chemotaxis (GO:0030593), phagocytosis, engulfmeiit (GO:0006911), positive regulation of cellular component organization (GO:005113Q), positive regulation of endocytosis (00:0045807), positive regulation of foam cell differentiation (GO:00i0744), positive regulation of phagocytosis (00:0050766), regulation of endocytosis (00:0030100), regulation of foam cell differentiation (GO: 0010743), regulation of phagocytosis (GO:0050764), regulation of type I hypersensitivity (GO:0001810), regulation of vesicle- mediated transport (GO:0060627) or response to yeast (GO:0001878); (vii) identifying the glatiramer acetate related drag substance or drug product as having a suboptimal activity if the set of differentially expressed genes indicates enrichment of one or more pathway of nucleotide binding (GO:0000166), double-stranded SNA binding (00:0003725), NAD+ ADP-ribosyltransferase activity (00:0003950). transferase activity, transferring pentosyl groups (00:0016763), transmission of nerve impulse

II (00:0019226 , regulation of membrane potential Gd:G04239i} _s cytokine ^■ activity (GO: 0905125), ion homeostasis (GO:0G5O801) or adenylyteansierase activity (GO:007e56S); (viii) identifying the. glatiramer acetate related drug substance or drug product _, as. liavin a .subopiinial activity if the set of differentially expressed genes indicate no substantial, enrichment of one or more pathways of Cytokine- cytokine receptor interaction {fimiuO406G} ₅ Jak-STAT signaling: pathway (nimu04630), Sterol -metabolic process (00:0016125), Sterol biosynthetic process (00:0016126), Hematopoietic cell lineage (mmu0464.0).. Myeloid cell apoptosis GO: : 0033028), Regulation of survival gene product expression (00:0045884), Regulation of leukocyte proliferation (GO:0070663), Positive regulation of survival gene product expression (00:0045885), Hematopoietic cell lineage (mmuQ4640) or Metabolism of xenobiotics by cytochrome P450 (mmu00980); (ix) identifying the glatiramer acetate related drug substance or drug product as having a suboptimai activity if the set of differentially expressed genes indica te no substantial enrichment of one or more path wa of Intrinsic to membrane receptor activity. Immune response, Leukocyte migration, Chemokine receptor binding, Cytokine- cytokine receptor interaction, Cytokine activity, Jak-STAT signaling pathway. Sterol biosynthetic process. Steroid biosynthetic process. Cell surface. Cytokine binding. Cholesterol metabolic process, Chemokine activity. Negative regulation of hormone secretion, Isoprenoid biosynthetic process or Positive regulation of peptidyl-serine phosphorylation; (x) identifying the glatiramer acetate related drug substance or drug product as having a suboptimai activity if the set of differentially expressed genes indicates one or more pathway of enrichment of Glucose metabolic process, Glycolysis, Gruconeogenesis, Hexose catabolic process. Glucose catabolic process, Monosaccharide catabolic process, Hexose metabolic process. Monosaccharide metabolic process. Cellular carbohydrate catabolic process. Alcohol catabolic process. Carbohydrate catabolic process. Carbohydrate binding. Generation of precursor metabolites and energy. Pha ocytosis, Leukocyte migration, Leukocyte chemotaxis. Positive regulation of phagocytosis. Pentose phosphate pathway. Galactose metabolism. Carbohydrate kinase activity, Myeloid leukocyte activation, Chemokine receptor binding. Regulation of type I hypersensitivity, Defense response to Gram-negative bacterium or Positive regulation of foam cell differentiation; (xi) identifying the glatiramer acetate related drug substance or drug product as having a suboptimai activity if the set of differentially expressed genes indicates enrichment of one or more pathway of leukocyte migration (00:0050900), cell chemotaxis (GO:0060326), leukocyte chemotaxis (GO:0030595), phagocytosis (GO:0006909), positive regulation of phagocytosis (00:0050766), cell motion (00:0006928), regulation of phagocytosis (00:0050764), behavior (GO:0007610), membrane invagination (GO:0010324), endocytosis (00:0006897). positive regulation of endocytosis (00:0045807), phagocytosis, eiigulfmeiit (GO:0006911), defense response to bacterium (00:0042742), membrane organization (00:0016044), neutrophil chemotaxis (00:0030593), defense response to Gram-negative bacterium (00:0050829), regulation of endocytosis (GO:003Gi00), leukocyte mediated immunity (00:0002443), cell migration (GO:0016477), regulation of vesicle- mediated transport (GO:0060627), localization of cell (00:0051674), cell motility (00:0048870), collages . metabolic process (00:0032963), midtkellular orgain^mal iiiacromoleciileme&boiic process (GQ:0044259), positive regulation of cellular component organization (GQ;0051130), myeloid leukocyte activation (GO:0CK)2274), positive regulation of ioani cell difierentiaiioa (00:0010744}, mrmieeilular orgamsmal metabolic process (GO;0044235) response to yeast (GO:00018 8), regiilatio of ^' type I hypersensitivity (G );00i)18i0) regiilatiQii of foam cell differentiation (00:0010743), sugar binding (GQ;O0 5529), eiidopeptidase a^

seriue-type peptidase activity (G0:OOO8236), serihe-type endopeptidase activity (60:0004252), cytokine activity (00:0005125), calcium ion binding (GO:0005509), immtmoglobulin binding (GO.-0019865), chemokine activity (00:0008009), chemokine receptor binding (GO:0042379), peptidase activity, acting on L-animo acid peptides (GO:0070011), peptidase activity (GO:G008233), IgG binding (00:0019864), protein complex binding (00:0032403) or enzyme inhibitor activity (GO:0004857); (xii) identifying the glatiramer acetate related drag substance or drug product as having a suboptimal activity if the set of differentially expressed genes indicates enrichment of one or more pathway of glucose metabolic process (00:0006006), glycolysis (00:0006096), hexose metabolic process (GO:00i9318), monosaccharide metabolic process (GO:0005996), hexose catabolic process (00:0019320), glucose catabolic process (GO:OOO6O07), monosaccharide catabolic process (00:0046365), cellular carbohydrate catabolic process (GO:0044275), alcohol catabolic process (00:0046164), carbohydrate catabolic process (00:0016052), generation of precursor metabolites and energy (00:0006091), oxidoreductase activity, acting on single donors with incorporation of molecular oxygen, incorporation of two atoms of oxygen (00:0016702), oxidoreductase activity, acting on single donors with incorporation of molecular oxygen (00:0016701), cytokine activity (00:0005125). carbohydrate kinase activity (GO:0019200), Glycolysis / Gluconeogenesis (mmuOOOlO). Pentose phosphate pathway (mmu00030). Fructose and mannose metabolism (mmuO0O51) or Galactose metabolism (nimu00052); (xiii) identifying the glatiramer acetate related drug substance or drug product as having a suboptimal activity if the set of differentially expressed genes indicates enrichment of one or more pathway of RIG-I-like receptor signaling pathway or Adenylyltransferase activity; or (xiv) determining the level of expression of at least one gene of Cxcl3, Clec4a3, UgtlalO, Ugtla6b, Ugtla6a. Ugtla7e, Ugtla9, Ugtla5, Ugtlal , Ugtla2. Clec4n, Tgfbi, Nov, Ltf, Ccl6, Mmpl2, Ms4a6d, Lpl, Mmp8, Cdl77, Tfec, Clec7a. Mpo, Ccl9, Ccrl, Gpnmb, Msrl, Ms4a6d, Lyz2, Ctsl, Thbsl, Dab2, Arg2, Ccl6, Thbsl, Pakll, ClecSa, Cfp, Ifitm6, Lyzl, 01fm4. Rnfl28, Ms4a3, Clec4d, S100a4, Ctsg, PrtnS, Ngp, F10, Hal, Ccl2„ Lyzl, Thbsl, Anxal, Lgals3, S100a6, Nfaml, C 13, Chil4, Sppl, Stfa211, Ptgrl, Iilf , Cd68, Olfinl, FcgrS, lilrl, CregL Dmx52, Sirpa, 116, Elane, Csflr, Lerr2, CdSOOlb, AloxSap, Iilm, Slcl lal, Tyrobp, Cd300a, Pira2, Piral, Pira7, Pira6, Pira5, Pira4, Gml0693, Liira6, Pirb, Piral 1, Gml4548, Ms4a7„ Mgstl, Camp, Bstl, Slc40al, Cregl, Csflr, Cd36, Rnfl28, Mrcl, Msrl, Itgam, Serpinb2, Lilrb4„ Gp49a, Wfdcl7, Anxa3, RgslS, Sk40al, Bstl, C3arl. Mmp9, Histlh4e, Histlh4b, Histlh4a, Hisi4h4, Histlh4f, Histih4d, Histlh4k, Histlh4j, Histlh4i, Hisfih4h, Histl ln, Histlh4m, Hist2h4, Dstn, Igsf6, Mitf, CsSr, Pf4, PygL Pdpn, S100a9, Fcgr2b, Idl, Emrl, Cd9, Cebpd, Pla2g7, Arg2, F10, Gdm. SlOOaS, Ptplad2 _v CIec4a2 _; Clec4bl. Marcfe CsfEra, Fcerlg, Giec4a2, Ppie, Cd302, GsunL McptS, CdSOOlf. Csf2rb, Bnip3, Ak4 _f Tprl, PkxdL Akloe, Kgsl L Aiifad37, PfidL Pdkl, 0∑cl ₅ Tfex21, P kl, Egln¾ Ddit4, Pfkp, Pgro2, P4imE SIc16a3, TJbd, Slc2aL 5330426P16R¾ Grhpr, Cxeli Rasa4, drgl, Faml62a, Pkm EgiaL Aldoa, Gml SO, BnipSi GalkL PgamL PgfcL Mil Eaoib, EnoF Pdxp, PpplrSb, Stomll, Gzma, Scd2, Ceng2, Higdla, Eao2, GfiL Prill, Ldha, Emit 30OO002CtORik, Hk2, Slc2a3, ChchdS, P4hai, ErolL Gpii _f Girnap?, Oas l. Gml 1110,.Syce2. Ndrgl.. Ptgs2, TmppcSa, PrehdL Ifi2712a, ZM02 _? . TritoS a, PreiidL Gapdli, Sdc3, BC06225S, LOC 102643247, 2310022B05Rik. Ms4a4c. I112rbl, Mthfdll ₅ Smagp, Nampt, FasL Pafahlb3, Prelid2, Gngl2, Iri KdiiiSa, Atf3, TnfsS.

The present invention also provides a process for producing or releasing a drag product comprising a glatirarner acetate related drag substance, the improvement comprising the steps of:

(a) obtaining a batch of the glatiramer acetate related drug substance or drag product;

(¥} contacting a first gr oup of niarrmiahan cells with an amount of the glatiramer acetate related drag substance or drag product of step (a);

(c) contacting a second group of mammalian cells of the same type with an amount of a reference standard;

(d) determining a set of genes differentially expressed by the first group after the contacting of step

(b) relative to genes expressed by the second group after the contacting of step (c); and

(e) (i) discarding the batch of the glatiramer acetate related drug substance as unacceptable for inclusion in die drug product if the set of differentially expressed genes includes one or more genes of Mmpl2, Tgfbi, Ciec4n Clec4a3, Fceri g _f Fcgr2b, Fcgr3, Csf3r, Iilr2 or Tbx21 ; (ii) discarding the batch of the glatrramer acetate related dr ug substance as unacceptable for inclusion in the drug product if the set of differentially expressed genes includes one or more genes of Ahcyl2, Cln5, Cxcr6, Dhrs3, Egr2, FabpS, Ptp4al, Gprl83, Lam l, Ncoa4, Ptger4, Qfc. Rragd, Sec24a, Sehll, SestdL Slc30a4, Soatl or Strip2; (iii) discarding the batch of the glatiramer acetate related drag substance as unacceptable for inclusion in the drag product if the set of differentially expressed genes includes one or more genes of Fam212a, Gzma, Itpri or Zbtb32; (iv) discarding the batch of the glatiramer acetate related drag substance as imacceptable for inclusion hi the drug product if the set of differentially expressed genes includes one or more genes of Gznrfa, Faml9a3, Nfil3, Gzma, Eaf2, Carl or St7; (v) discarding tire batch of the glatiramer acetate related drag substance as unacceptable for inclusion in the drug product if the set of differentially expressed genes includes one or more genes of Crisp3, Apoe, Dapil, Pde5a, Scg5, St8sia6, 4 30426D05Rik or Pgapl ; (vi) discarding the batch of the glatiramer acetate related drug substance as unacceptable fo inclusion in the drug product if tire set of differentially expressed genes indicates enrichment of one or more pathway of collagen metabolic process (GO:0032963), defense response to bacterium (GO:0042742), defense response to Gram-negative bacterium (00:0050829), endocytosis {60:0006897), lejikocyte; mediated iminunity ((30:0002443),. ineinbrane it aginatioH (GO:O0iO324), membrane organization (GO:O016044), multicellular organismal maaOnioIeeul metabolic process. (00:0044259), multicellular organisnral metabolic process (00:0044236), myeloid leukocyte activation (00:0002274), neutrophil chemotaxi (GO:0030593), phagocytosis, engulfinent. (00:0006911)., positive regulation, of cellular component organization. (00:0051130), positive regulation of endocytosis (00:0045807), positive regulation of foam cell differentiation (00:0010744), positive regulation of phagocytosis (00:0050766), regulation of endocytosis (00:0030100), regulation of foam cell differentiation (00:0010743), regulation of phagocytosis (00:0050764), regulation of type I hypersensitivity (00:0001810). regulation of vesicle- mediated transport (00:0060627) or response to yeast (GQ:0001878); (vii) discarding the batch of the giatirainer acetate related drug substance as unacceptable for inclusion in the drag product if the set of differentially expressed genes indicates enrichment of one or more pathwa of nucleotide binding (GO-.0000166), double-stranded R A binding (GO:0003725), NAD+ ADP-ribosyltransferase activity (00:0003950), transferase activity, transferring pentosyl groups (GO:00i6763), transmission of nerve impulse (00:0019226), regulation of membrane potential (00:0042391), cytokine activity (GO.-0005125), ion homeostasis (GO:0050801) or adenylyitransferase activity (GO:0070566); (viii) discarding the batch of the giatirainer acetate related drug substance as unacceptable for inclusion in the drug product if the set of differentially expr essed genes indicate no subs tantial enrichment of one or more pathways of Cytokine-cytokine receptor interaction (mmu04060). Jak-STAT signaling pathway (mmu04630), Sterol metaboli process (00:0016125), Sterol hiosynthetic process (00:0016126), Hematopoietic cell lineage (mmuG4540), Myeloid cell apoptosis (GO: 003 028), Regulation of survival gene product expression (GO:0045884), Regulation of leukocyte proliferation (GO:0070663), Positive regulation of survival gene product expression (GO:00458S5), Hematopoietic cell lineage (mmu04640) or Metabolism of xenobiotics by cytochrome P450 (mmu00980); (ix) discarding the batch of the giatirainer acetate related drug substance as unacceptable for inclusion in the drug product if the set of differentially expressed genes indicate no substantial enrichment of one or more pathway of Intrinsic to membrane receptor activity. Immune response. Leukocyte migration, Chemokine receptor binding, Cytokine-cytokine receptor interaction. Cytokine activity, Jak-STAT signaling pathway. Sterol biosynthetic process, Steroid biosynthetic process. Cell surface. Cytokine binding. Cholesterol metabolic process, Chemokine activity. Negative regulation of hormone secretion, Isoprenoid biosynthetic process or Positive regulation of peptidyl-serrne phosphorylation; (x) discarding the batch of the giatirainer acetate related h ug substance as unacceptable for inclusion in the dr ug product if the set of differentially expressed genes indicates enrichment of one or more pathway of Glucose metabolic process. Glycolysis, Gluconeogenesis, Hexose catabolic process. Glucose catabolic process. Monosaccharide catabolic process, Hexose metabolic process. Monosaccharide metabolic process. Cellular carbohydrate catabolic process. Alcohol catabolic process. Carbohydrate catabolic process. Carbohydrate binding. Generation of precursor metabolites and energy, Phagocytosis, Leukocyte migration, Leiikixyte chemotaxis. Pos tive regulation, of ^* phagocytosis.. Pentose .phosphate pathway. Galactose metabolism. Carbohydrate kinase activity, Myeloid leukocyte activation, Chempkihe receptor binding. Regulation oftype I hypersensitivity. Defense response t Gram-negative bacterium or Positive regulation of foafii ceil differentiation: (xi) discarding th batch, of the glatirainer acetate related drag substance as unacceptable for inclusion in the drug product if the set of differentiall expressed, genes indicates enrichment of one or more pathway of leukocyte migration (00:0050900), _. cell ehetnotaxis (00:0060526), leukocyte chemotaxis (60:·ΟΟ3Ο593), phagocytosis (00:0006909·),. positive regulation of phagocytosis (GO:0Q50766), cell motion (GO:0006928), regulation of phagocytosis (00:0050764), behavior (00:0007610), membrane invagination (GO:0010324), endocytosis (GO:0006897), positive regulation of endocytosis (GO:0045807), phagocytosis, engulfinent (00:0006911), defense response to bacterium (GO:0042742), membrane organization (00:0016044), neutrophil chemotaxis (00:0030593), defense response to Gram-negative bacterium (GO-.0050829), regulation of endocytosis (GO:0030100), leukocyte mediated inimunity (00:0002443), cell migration (GO:0016477), regulation of vesicle-mediated transport (GO:0060627), locaiization of cell (00:0051674), cell motility (GO:0048870), collagen metabolic process (GO:0032963), multicellular organismal macromolecule metabolic process (GO:0044259), positive regulation of cellular component organization (GO:00 1130), myeloid leukocyte activatio (00:0002274), positive regulation of foam cell differentiation (00:0010744), multicellular organismal metabolic process (00:0044236), response to yeast (GO:0001878), regulation of type I hypersensitivity (GO:0001810), regulation of foam cell differentiation (00:0010743), sugar binding (00:0005529), eiidopepiidase activity (GO:000 175), serine hydrolase activity (00:0017171), serrne-type peptidase activity (00:0008236), serme-type eiidopepiidase activity (00:0004252), cytokine activity (00:0005125), calcium ion binding (00:0005509), immunoglobulin binding (00 0019865), chemokine activity (00:0008009), chemokine receptor binding (00:0042379), peptidase activity, acting on L-amino acid peptides (00:0070011), peptidase activity (GO:0008233), IgG binding (00:0019864), protein complex binding (GO:0032403) or enzyme inhibitor activity (00:0004857): (xii) discarding the batch of the glatirainer aceta te related drag substance as unacceptable for inclusion in the drug product if the set of differentially expressed genes indicates enrichment of one or more pathway of glucose metabolic process (GO:0006006), glycolysis (00:0006096), hexose metabolic process (00:0019318), monosaccharide metabolic process (00:0005996), hexose catabolic process (00:0019320), glucose catabolic process (00:0006007), monosaccharide catabolic process (00:0046365), cellular carbohydrate catabolic process (00:0044275), alcohol catabolic process (00:0046164), carbohydrate catabolic process (GO:0016052), generation of precursor metabolites and energy (00:0006091), oxidorediictase activity, acting on single donors with mcoiporation of molecular oxygen, incorporation of two atoms of oxygen (00:0016702), oxidorediictase activity, acting on single donors with mcoiporation of molecular oxygen (00:0016701), cytokine activity (00 0005125), carbohydrate kinase activity (00:0019200), Glycolysis / Gluconeogenesas (mniuOOOlO), Pentose phosphate pathway (mimiOGOSO). Fractose and niaimose; metabolism (BimuOOOSl) or Galactose metabolism (mm«0Q052); (xai) discarding the batch of the glaiiramer acetate related drug substance as unacceptable for inclusion iii the dra product, if ^' the set of diiiermtially expressed genes indicates emiehraeni of one or more pathway of RIG-I-like .receptor signaling pathway or Adenylyitraasferase activity: or (xiv) detennining the level of expression of at least one gene of Cxel3, Clec4a3, Ugt ^' lai.O, Ugtla6b, Ugtla6a, Ugtla.7e, Dgtla9, U tla Ug iaL Ugtla¾ Clec4¾ TgfbjL. Nov, Ltil Ccl6, Μήφ12, Ms4a6d, LpL MffipS, Cdi77, Tfec, Clec7a, Mpo, Cc , Ccrl, Gpnrab, Marl, Ms4a6d, L}¾ Ctsl Thbsl, Dab2, Arg2, Ccl6, Thhsi, Paldl, ClecSa, Cfp, Ifito.6, Lyzl, 01fin4, Rnfl28, Ms4a3, Ciec4d, S100a4, Ctsg, Prto3, Ngp, F10, Hal, CcI2, Lyzl. Thbsl, Anxal, LgalsS, SI00a6, NfamL CMS, ChiR Sppl, Stfa21L Ptgrl, III©, Cd68, Olfinl, Fegr3, Hlr2, Cregl. Drnxl2, Sirpa, 316, Eiane, Csflr, Lcn2, CdSOOIb, AloxSap, Him. Slcl lal, Tyrobp, CdSOOa, Pira2, Piral, Pita?, Pira6, PiraS, Pira4, Gmi0693, Lilra6, Pii , Piral 1, Gm 14548, Ms4a7, Mgstl, Camp, Bstl, Slc40al, Cregl, Csflr, Cd36, Rnfl28, Mrcl, Msrl, Itgam, Serpmbl, Lilrb4, Gp49a, Wfdcl7, Anxa3, RgslS, Slc40al. Bstl, C3arL Mmp9, Histlh4c. Histlirfb, Hist.lh4a, H_st4h4, Histlh4f, Histlh4d, Histlh4k, HistlMj, Histih4i. Histlb4h, Hisiih4n, Histlh4m, Hist2h4, Dsto, IgsfS, Mirf, Csf3r, Pf4, Pygl, Pdpn, S100a9, Fcgr2b, Ml, Emrl, Cd9, Cebpd, Pla2g7, Arg2, F10, Gclin, SlOOaS, Ptpiad2, Clec4a2, Clec4bl, Marcks, Csf2ra, Fcerlg, Clec4a2, Ppic, Cd3G2, Gstml, McptS. Cd3001f, Csf2rh Bnip3, Ak4, Tpi , Plcxdl. Aldoc, Rgsl l, Ankrd37, Pikl, Pdkl, Cxcl9. Tbx21, PgkF EginS, Ddit4, Pffcp, Pgm2, P4hal, Sicl6a3, Ubd. Slc2al, 5330426P16Rik. Grtapr, Cxcll 1, Rasa4, Ndrgl, Faml62a, Pkm, Eglnl, Aidoa, Gml0480, Bnip3L Galkl, Pgaml, Pgkl, Mil Enolb, Enoi, Pdxp, PpplrSb, Stomll, Gzma, Sed2. Ccng2, Higdla, Eno2, Gfil. Pml, Ldha, Eroll, 3000002C10Rik, Hk2, Slc2a3, Chchd6, P4hal, Eroll, Gpil, Gimap7, Oasll, Gini 1110, Syce2, Ndrgl, Ptgs2, Trappc6a, Prelidl, Iii2712a, Zbtb32, Trim3Ga, Prelidl, Gapdh, Sdc3, BC06225S, LOC 102643247, 23 iO022BO5Rik, Ms4a4c, IH2rbl, MthfdlL Smagp. Nampi, Fash Pafahlb3, Prelid2, Gngl2, Irf8, dni3a. Atf3, Tnfsf9.

The present invention also provides a process for producing or releasing a drug product comprising a glatiranier acetate related drug substance, the improvement comprising the steps of:

(a) obtaining a hatch of the glatiramer acetate related drug substance or drug product:

(b) contacting a first group of mammalian cells with an amount of the glatiramer acetate related drug substance or drug product of step (a);

(c) contacting a second group of mammalian ceils of the same type with an amount of a reference standard;

(d) detemiining a set of genes differentially expressed by the first group after the contacting of step (b) relative to genes expressed by the second group after the contacting of step (c); and

(e) discarding the batch of the glatiramer acetate related drug substance as unacceptable for inclusion in the drug product if the set of differentially expressed genes indicate no substantial emichment of one or more pathways set fort in. Table 3 or Table 10; or discarding the batch of the gi atiraiBer acetate related drug substance as unacceptable for inclusion m die -drug product if the set of differentii ily expressed geaes indicates emichraent of one or more pathways set forth in Table 5, Table 7, Table 8, Table 1 or Table 15.

Tlie present invention also pro ides a process for identifying xuboptimal activity of a glatiramer acetate related drug substance or drug product comprising the steps of :

(a) obtaining a batch of the glatiramer acetate related ch ug substance or drag product;

(b) contacting a first group of mammalian cells with an amount of the glatiramer acetate related drug substance or drug product of step (a) ;

(c) contacting a second group of mammalian cells of the same type with an amount of a reference standard;

(d) determining a set of genes differentially expressed by the first group after the contacting of step (b) rela tive to genes expressed by the second group after the contacting of step (c); and

(e) (i) identifying the glatiramer acetate related drag substance or drag product as having a suboptimal activity if the set of differentially expressed genes includes one or more genes of Mmpl2, Tgfbi, Clec4n, Clec4a3, Feerlg, Fcgr2b, Fcgr3, Csf3r, II I r 2 or Tbx21 : (ii) identifying the glatiramer acetate related drag substance or drug product as having a suboptimal activity if the set of differentially expressed genes includes one or more genes of Ahcyl2, Cln5, Cxcr6, Dhrs3, Egr2, Fabp5, Ptp4al , Gpri 83, LampL Ncoa4, Ptger4, Qk, Rragd, Sec24a, Sehll, Sestdl, Slc30a4, Soatl or Strip2: (iii) identifying the glatiramer acetate related drug substance or drug product as having a. suboptimal activity if the set of differentially expressed genes mchides one or more genes of Fam212a,. Gzma, Itprl or Zbtb32; (iv) identifying the glatiramer acetate related drug substance or drag product as having a suboptimal activity if the set of differentially expressed genes includes one or more genes of Gzmb, Faml9a3, Nfii3, Gzma,. Eaf2, Carl or St7; (v) identifying the glatiramer acetate related drug substance or drug product as having a suboptimal ac tivity if the set of differentially expressed genes mchides one or more genes ofCrispS, Apoe, Dapll , Pde5a, Scg5, 518813 , 4930426D05Rik or Pga l ; (vi) identifying the glatiramer acetate related drug substance or drug product as having a suboptimal activity if the set of differentially expressed genes indicates enrichment of one or more pathway of collagen metabolic process (00:0032963), defense response to bacterium (GO:0Q42742), defense response to Gram- negative bacterium (GO:0050829), endocytosis (GO:0006897), leukocyte mediated immtmity (GO:0Q02443), membrane invagination (GO:0010324), membrane organization (GO:0016G44), multicellular organismal macromolecule metabolic process (GO:0044259), multicellular organismal metabolic process (00:0044236), myeloid leukocyte activation (GO:0002274), neutrophil chemotaxis (GO:003Q593), phagocytosis, engulfment (GO:0006911), positive regulation of cellular component organization (GO:0051 13Q), positive regulation of endocytosis (GO:0045807), positive regulation of foam cell differentiation (GO:0010744), positive regi ation of phagocytosis (00:0050766.), regulation of endocytesis (00:0030100). regulation of fbani cell differentiation (00:0010743), regulation of phagocytosis (QO:0050764) _> regulation .oftype. I bypei¾ensitiviiy ^' (00:00018 i .0). regi aiion of veskle- inediated transport (00:0060627) or response to yeast (00:000.1.878); (vii) identifying the glatiramer acetate ..related dmg substance o drug product as having a suboptiinal activity if the set of differentially expressed, genes indicates emicimieni of one or more pathway of nucleotide binding (00:0000166), double-stranded' SNA binding (00:0003725), NAD+ ABP-ribosyltransferase activity (00:0003950), transferase activity, tiansfeiiing pentosyl groups (GO:0016763), transmission of nerve impulse (GO:0019226), regulation of membrane potential (GO:0042391) . cytokine activity (00:0005125), ion homeostasis (GO:0050801) or adenyly!transferase activity (GO:0070566); (viii) identifying the giatiramer acetate related drag substance or drag product as having a suboptimai activity if the set of differentially expressed genes indicate no substantial enrichment of one or more pathways of Cytokine- cytokine receptor interaction (mmu04060), Jak-STAT signaling pathway (mmu04630), Sterol metabolic process (00:0016125), Sterol biosynthetic process (00:0016126), Hematopoietic cell lineage (mniuG4640), Myeloid ceil apoptosis (GO: 0033028), Regulation of survival gene product expression (GO:0045884), Regulation of leukocyte proliferation (GO:0070663), Positive regulation of survival gene product expression (GO:0045885), Hematopoietic cell lineage (mmu04640) or Metabolism of xenobiotics by cytochrome P450 (mmu00980); (ix) identifying the glatiramer acetate related drug substance or drag product as having a suboptimai activity if the set of differentially expressed genes indicate no substantial enrichment of one or more pathway of Intrinsic to membrane receptor activity. Immune response, Leukocyte migration, Chemokine receptor binding, Cytokine- cytokine receptor interaction. Cytokine activity, Jak-STAT signaling pathway. Sterol biosynthetic process. Steroid biosynthetic process. Cell surface. Cytokine binding. Cholesterol metabolic process, Chemokine activity. Negative regulation of hormone secretion, Isoprenoid biosynthetic process or Positive regulation of peptidyl-serine phosphorylation; (x) identifying the glatiramer acetate related drug substance or drug product as having a suboptimai activity if the set of differentially expressed genes indicates enrichment of one or more pathway of Glucose metabolic process. Glycolysis, Gluconeogenesis, Hexose catabolic process. Glucose catabolic process. Monosaccharide catabolic process, Hexose metabolic process. Monosaccharide metaboli process. Cellular carbohydrate cataboli process. Alcohol catabolic process. Carbohydrate catabolic process, Carbohydrate binding. Generation of precursor metabolites and energy. Pha ocytosis, Leukocyte migration, Leukocyte chemotaxis. Positive regulation of phagocytosis. Pentose phosphate pathway. Galactose metabolism. Carbohydrate kinase activity. Myeloid leukocyte activation, Chemokine receptor binding. Regulation of type I hypersensitivity. Defense response to Gram-negative bacterium or Positive regulation of foam cell differentiation; (xi) identifying the glatiramer acetate related drug substance or drag product as having a suboptimai activity' if the set of differentially expressed genes indicates enrichment of one or more pathway of leukocyte migration (GO.O050900), cell chemotaxis (GO:0060326), leukocyte chemotaxis (00:0030595), phagocytosis (00:00069091 positive regulation of phagocytosis (00:0050766), eel! motion (00:0006928), regulation of phagocytosis ((¾ :CK 507 } _> behavior (00:0007610), Membra e u agmation (G :0G 10324), eadoeytosis (00:0006:897 ), positive regulation pf eadoeytosis, (00:0045807), phagocytosis, engulfinent (00:00069] 1), defense response to bacterium (00:0042742), membrane organization (00:0016044), neutrophil chemotaxis (00:0030593), defense response to Grain-negative bacteman (00:0050829), regulation of eadoeytosis (00:0030100), leukocyte mediated immunity (00:0002443), cell migration (GO; 0016477), regulation of vesicle- mediated transport (00:0060627), localization of cell (00:0051674), cell motility (00:0048870), collagen metabolic process (00:0032963), multicellular organismal macroniolecule metabolic process (00:0044259), positive regulation of cellular component organization (00:0051130), myeloid leukocyte activation (00:0002274), positive regulation of foam cell differentiation (00:0010744), multicellular organismal metabolic process (00:0044236), response to yeast (00:0001878), regula tion of type I hypersensitivity (00:0001810), regu!atioa of foam cell differentiation (00:0010743), sugar binding (00:0005529), endopeptidase activity (GO:0004175), serine hydrolase activity (00:0017171), serine-type peptidase activity (00:0008236), seriae-type endopeptidase activity (00:0004252), cytokine activity (00:0005125). calcium ion binding (00:0005509), immunoglobulin binding (00:0019865), chemokine activity (00:0008009), chemokine receptor binding (00:0042379), peptidase activity, acting on L-amino acid peptides (00:0070011), peptidase activity (00:0008233), IgG binding (00:0019864), protein complex binding (00:0032403) or enzyme inhibitor activity (00:0004857); (xii) identifying the glatiramer acetate related ding substance or drug product as having a suboptimal activity if the set of differentially expressed genes indicates enrichment of one or more pathway of glucose metabolic process (00:0006006), glycolysis (00:0006096), hexose metabolic process (00:0019318), monosaccharide metabolic process (00:0005996), hexose catabolic process (00:0019320), glucose catabolic process (00:0006007), monosaccharide catabolic process (00:0046365), cellular carbohydrate catabolic process (00:0044275), alcohol catabolic process (00:0046164), carbohydrate catabolic process (00:0016052), generation of precursor metabolites and energy (00:0006091), oxidoreductase activity, acting on single donors with incorporation of molecular oxygen, incorporation of two a toms of oxygen (00:0016702), oxidoreductase activity , ac ting on single donors with incorporation of molecular oxygen (00:0016701), cytokine activity (00:0005125). carbohydrate kinase activity (00:0019200), Glycolysis / Gluconeogenesis (mmuOOOlO), Pentose phosphate pathway (mnmO0O3O), Fructose and mannose metabolism (mmu00051) or Galactose metabolism (mmu00052); (xiii) identifying the glatiramer acetate related drug substance or drug product as having a suboptimal activity if the set of differentially expressed genes indicates enrichment of one or more pathway of RIG-I-like receptor signaling pathway or Adenylyltransferase activity; or (xiv) detennining the level of expression of at least one gene of Cxcl3, Clec4a3, Ugtla iO, Ugtla6b, Ugtla6a, Ugtia7c, Ugtla9, UgtiaS, Ugtlal, Ugtla2. Clec4n, Tgfbi, Nov, Ltf, Ccl6, Mmpl2, Ms4a6d, Lpl, Mmp8, Cdl 77, Tfec, CiecJa. Mpo, Ccl9, Ccrl, Gpnmb, Msrl, Ms4a6d, Lyz2, Ctsl, Thbsl, Dab2, Arg2, Cc ; ThbsL PaMl, CieeSa, Cfp, Ifiii»6 _f LyzL QW≠, RnfI28 _v Ms4a3, Clec4d, S100a4, Ctsg,Prtn3:, Ngp, F10 Hal Cel2, Lyzl, ThbsL Anxal. LgalsS SlOOafi NfaniL ChilS C ^' iii Sppt, Stfa21X, PtgrL flif9, CdSS, Olfml, Fcgr3:, Ilir2 _; Cregl, DmxI2, Sirpa 116, Kane, Csflr, Lena, Cc&dQlb, AloxSap, Iilm, SkJJ al, Twobp, CdSOOa, Pira2, PiraL Pira7, Pira PftaS, Pira4, GmI0S93, Liira6, Pirb, Pira! 1, Gra!4548, Ms4a7, Mgst Camp, Bstl, Ste40al, Cregi, Csflr, Cd36 _? Rnfli.28, ^' SftcJ. Msrl, Itgam, SapK, LUrb4,Gp 9a, _. Wfdcl7, Anxa.3, Rgsl8, Slc40aL BstL C3arl, Mmp9 _? HistlMc, Histl i HistlMa, His 4h4, Histi f Histlh4d, Histlirik. Histl j, His ih4i Fhstlh4h, Histih4n, Histlh4m, His†.2h4, Dstn, IgsfiS, Miff, Csf3r, Pf4, Pygl, Pdpn, SliM3a9, Fegr2b, Idl. Emrl, Cd9, Cebpd, Pla2g7, Arg2, F10, Gckii. S100a8, Ptplad2, C2ec4a2, Clec4bl , Maids, Csf2ra, Fcerlg. Clec4a2, Ppic, Cd302, Gstml , MeptS, Cd3001f, Csfirb, Bnip3, Ak4, Tpi 1 , Plcxdi , Aldoc, Rgsl I. Ankrd37, Pfkl, Pdkl, Cxcl9, Tbx21, Pgkl, Egin3, Ddit4, Pfkp, Pgin2, P4hal, Sicl6a3, Ubd, Slc2al, 5330426P16Rik, Grhpr, Cxcll l, Rasa4, Ndrgl, Faml62a, Pkm, Eglnl . Aldoa, Gml0480, Bnip31, GalkL Pgaml, Pgkl , Mif, Enolb, Enol, Pdxp, PpplrSb, StomlL Gzma, Scd2, Ccng2. Higdla, Eno2. Gfil, Pail, Ldha, Eroll, 3000002C10Rik _t Hk2, Slc2a3, Chchd6, P4hal , Eroll, Gpil , Gimap7, Oas l, Gml 1 i 10, Syce2, Ndrgl, Ptgs2, Trappc6a, Freiidi, Ifi2712a, Zbtb32, TriinSOa, Prelidl, Gapdh, Sdc3, BC062258, LOC 02643247, 2310022B05Rik. Ms4a4c. I112rbl, Mlhfdll ₅ Smagp, Nampt, Fast PafahlbS, Prelid2, Gngl2, MS, Kdm3a, Atf3, Tnfsf .

The present invention also provides a process for identifying suboptimal activity of a glatiramer acetate related drag substance or drag product comprising the steps of:

(a) obtaining a batch of the glatiramer acetate related ding substance or drag product;

(b) contacting a first group of mammalian cells with an amount of the glatiramer acetate related drug substance or drag product of step (a);

(c) contacting a second group of mammalian cells of the same type with an amount of a reference standard;

(d) determining a set of genes differentially expressed by the first group after the contacting of step (b) relative to genes expressed by the second group after the contacting of step (c); and

(e) identifying the glatiramer acetate related drag substance or drug product as ha ving a suboptimal activity if the set of differentially expressed genes indicate no substantial enrichment of one or more pathways set forth in Table 3 or Table 10; or identifying the glatiramer acetate related drag substance or drug product as having a suboptimal activity if the set of differentiall expressed genes indicates enrichment of one or more pathways set forth in Table 4, Table 7, Table 8, Table 14 or Table 15.

The present mvention also provides a process for producing a drug product comprising a glatiramer acetate related drug substance which involves an array of testing, the improvement comprising including in the array of testing the steps of: (a characterizing the glatiramer acetate; related drag substance according to the process of the; present invention; and

(b) (i) meludin the batch of the glatmuner acetate related drag substance in the production of the drug product if the level of espressioii ofoiie or niore genes of mpI2, Tgfbi, Clec4n, Clec4a3, Feerlg, Fcgrib, -Fegi3 _; Csi¾v Iilr2 or Tbx21 is substantially identical, to the le vel of 'expression, by the same 't pe of cells in the presence of the glafiramer acetate drug substance under the same conditions; (ii) including the batch oi the glatiramer acetate related drug substance in the production of the drag product if the level of expression of one or more genes of Aheyl2, Cin5, Cxer6, Dhrs3, Egr2, Fabp5, Ptp4al, GprlS3, LarapL Ncoa4 ₅ Ptger4, Qk, Rragd Sec24a, Sehll, SestdL Slc30a4, Soatl or Strip2 is substantially identical to the level of expression by the same type of cells in the presence of the glatiranier acetate drag substance under the same conditions; (iii) including the batch of the glatiranier acetate related drug substance in the production of the drag product if the level of expression of one or more genes of Fam212a, Gzma, Itprl or Zbt.b32 is substantially identical to the level of expression by the same type of cells in the presence of the glatiramer acetate drug substance under the same conditions; (iv) including the batch of the glatiramer acetate related drug substance in the production of the drag product if the level of expression of one or more genes ofGzmb, Faml9a3, Nfi!3, Gzma, Eaf2, Carl or St7 is upregulated relative to the level of expression by the same type of cells in the absence of the glatiramer acetate related drug substance under the same conditions; or (v) including the batch of the glatiramer acetate related drug substance in the produc tion of the drug product if the level of expression of one or more genes of CrispS, Apoe, Dapll, PdeSa, Scg5„ St8sia6, 4930426D05Rik or Pgapl is dowtiregulated relative to the level of expression by the same type of cells in the absence of the glatiramer acetate related drag substance under the same conditions.

The present invention also provides a process for releasing a drug product comprising a glatiramer acetate related drug substance, which process involves an array of testing, the improvement comprising including in the array of testing the steps of:

(a) characterizing the glatiramer acetate related drag product according to the process of the present invention; and

(b) (i) releasing the batch of the glatiramer acetate related drag substance in the production of the drug product if the level of expression of one or more genes of Mmpl2, Tgfbi Clec4n, Ciec4a3. Feerlg, Fcgr2b, Fcgr3, Csf3r, Blr2 or Tbx21 is substantially identical to the level of expression by the same type of cells in the presence of the glatiramer acetate drug substance under the same conditions; (ii) including the batch of the glatiramer acetate related drug substance in the production of the drug product if the level of expression of one or more genes of Aheyl2, C iS, Cxci S. Dhrs3 Egr2, FabpS, Pt.p4al. Gpi S3, LampL Ncoa4, Ptger4, Qk, Rragd, Sec24a, Sehll, Sestdl, Slc30a4, Soatl or Steip2 is substantially identical to me level of expression by the same type of cells in the presence of the glatiranier acetate drag substance under the same conditions; (lii) including the batch of the glatiiame acetate related drug substance in the prodiieiioa of the drag produc t if the level of expression of one or more genes of Fani212a, Gzma, Itprl or Z b32 is substantially identical to the level of expression b the sa me t ype of cells .in .The presence of the glatiramer acetate thug substance underthe same conditions; (iv) including the batch of the glatiramer acetate related drug substance in. the production of the ding product if fire level of expression of one or more genes of G¾nb, Fami 9a3, fil3 _T Gzrna, Eaf2, Carl or St? is upregulated relati¥e to the level of expression by the same type of cells in the absence of the glatiranier acetate related drag substance under the same conditions; or (v) including the batch of the glatiranier acetate rela ted drag substance in the production of the drag product if the level of expression of one or more genes of C ispS, Apoe, Dapll, PdeSa, Scg5„ St8sia6, 4930426D05Rik or Pgapl is dowmegulated relative to the level of expression by tlie same type of cells in the absence of the glatiramer acetate related drag substance under the same conditions.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure I . Expression Levels of 116 i (left) Microarray Data and (right) Confirmation by KT-PCR.

Figure 2, Volcano plot showing probesets (mcludmg for 116) driving enrichment, of the eytokine- ey&skiue interaction pathway.

Fig, 3. Gene expression levels for control (maanitol). treatment, Copaxone ^® freairaeni, and GLATOPA treatment for key genes relevant to inflarnaiatoiy processes and Copaxone ^® mechanism of action. Levels of anti-inflammatory 1110(A), 114(B) and Foxp3(C) are increased relative to control by both Copaxone ^8' and GLATOPA. Levels of pro-iiiflammatoiy H12a(D) are reduced relative to control by both Copaxone* and GLATOPA. Treatment of splenoeytes from Copaxone ^® -immunized mice; similar results obtained with GLATOPA-iinniimized mice. Fold Change (FC) and adjusted p values are relative to mannitol control.

Figure 4. Pathways enriched among top probesets differentially expressed in response to Copaxone ^® treatment in Copaxone ^s' -iimimnized mice. The dashed line represents a significance level ofBenjamini- eorreeied value 0.05.

Figure 5. GLATOPA and Copaxone ^® differentially modulate a significant subset of probesets (including approximately 7% of probesets modulated by Copaxone ^® ), a) Copaxone®-mimunized samples; b) GLATOPA-mimiinized samples.

Figure 6. Expression levels of Tgfbi were higher with GLATOPA reiative to Copaxone ^® treatment of splenoeytes from Copaxone ^® -inimunized mice, as reflected in multiple probesets for this gene.

Figure 7. Increased expression of Clec4n with GLATOPA versus Copaxone ^® treatment, under bom immunization conditions, for multiple probesets.

Figure 8. Increased expression of Fcei g with GLATOPA versus Copaxone ^® treatment, under both immunization conditions.

Figure 9. Expression of cytokine gene Cxcl3 is increased with GLATOPA treatment relative to Copaxone ^® treatment, under both immunization conditions.

Figure 10. Pathways enriched among top probesets differing between GLATOPA and Copaxone ^® (mannitol-coiiected comparison) under both immunization conditions. The dashed line represents a significance level of Benjammi-corrected p value 0.05.

Figure 11. Volcano plot showing probesets driving enrichment of the cytokine -cytokine receptor interaction pathway. Light dots correspond to Copaxone ^® immunization, and dark dots correspond to GLATOPA immunization. Multiple dots of the same color for a given gene represent separate probesets, adding robustness to the observed results. .Figure 12. Volcano plot of probeseis driving enrichment of the pathway: regulation of adaptive; immune response based on somatic recombination of tmiiuiie receptors built Sam himiimoglobulin supeiianiily domains. Dark and light dots represent data from GLATOPA and Copaxone ^® iminiiriizatioti conditions, respectively.

Fig; 13, Multiple immune genes were differentially expressed between GLATOPA d Copaxone ^® in the mouse splenocyte model system, in both nifcroarray and qRT-PCR studies. Shown here are Cel2 (A), Ciec4n (B), Fcgr3 (C), and C ^' sBr (D).

Figure 14. MMP9 expression is further increased with GLATOPA relative to Copaxone ^® in THP-1 studies in two independent laboratories.

Figure 15. Volcano plot of probeseis (hiving enrichment of the pathway: regulation of adaptive immune response based on somatic recombination of immune receptors built from immunoglobulin superfaniily domains. Dark and light dots represent data from the Hungary and Israel studies, respectively. Multiple dote of the same color for a given gene represent separate probeseis, adding robustness to the observed results.

Figure 16. Volcano plot of probesete in the GSEA leading edge for the membrane-dependent cytokine- cytokine receptor interaction pathway and significantly differentially expressed (FDR < 0.05) in the comparison between GLATOPA and Copaxone* in THP-i cells. Dark and light dots represent data from the Hungary and Israel studies, respectively. Multiple dots of the same color for a given gene represent separate probeseis, adding robustness to the observed results.

Figure 17, Design of reciprocal internal control mouse splenocyte study using Copaxone ^® and

GLATOPA.

Figure IS, The physicochemical and biological observations are linked by immunological effects of altered charge properties of particles taken up by APCs. UT = untreated; P = nanoparticle.

Figure 19. Design of reciprocal internal control mouse splenocyte study using Copaxone ^® and POLIMUNOL.

Figure 20. Effects of Copaxone® versus mannitol control tr eatment on splenocytes from Copaxone£>- immunized mice.

Figure 21. Data shown for CopaxoneS-immunized samples. Similar results for POLIMUNOL immunization.

Figure 22. Copaxone®-immunized samples. Relative coloring scheme according to the row minimum and row maximum. Figure shows ihe 223 present probeseis with adj p<0.05 for POLIMUNOL vs Copaxone's* (Mamiiiol-correeted; CopaxoneC-hnmimized). .Figure 23. Significant -pathway emieiiment among pro esets more highly express ed wi th POLJMU OL relative to Copaxone® (FG> 1.4), Pathway enrichnient conducted using the DAVID plai&rni (Hwang et ai 2009) as described; in [H&sson et ai J Nem'omiinunoL 20161, with &e addition of a fold change cutoff of FC > 1.4. Adj p = Benjamins adjtisied p value from DAVID.

Figure 24. .Probesets ^' in die "response to vims" pa hway differing in expression with F£>! .4 between: POLIMXJ OL vs. Co axoae®.

Figure 25. CYPlBi was significantly more highly expressed with POLIMU QL vs Copaxone®, as detected by all four present probesets for this gene.

Figure 26. The pliyskocheniical and biological observations are linked by literature describing immunological effects of altered charge properties of particles taken up by APCs.

BETAILEP DESCRIPTION ^' OF THE INVENTIO

Effibodimeats of the lave nikm

The present invention provides a process lo characterizing a glatff&rner acetate related drag substance or drag prodoet comprising the steps of:

(a) obtaining a batch of fee gi atiramer acetate related drug substance or drag prodoet:

(b) contacting inaiimiahan cells with an amount of the glatiramer acetate related drug substance or drag product of step (a); and

(c) (i) detenniiiing the level of expression of at. least one gene of Mmpl2, Tgfbi Clec4n. Clec4a3, Fcerlg, Fcgr2b, Fcgr3, Csf3r, I!Ir2 or Tbx2i; (ii) determining the level of expression of at least one gene of Alicyi2, Cln5, CxenS, Dnrs3, Egr2, FabpS, Ptp4al, Gprl83, Lampl, Ncoa4, Ptger4, Qkjagd, Sec24a, SehlL Sestdl . Slc30a4, Soatl or Strip2: (iii) deteiiniiiing the level of expression of at least one gene of Fam212a, Gzma, Itprl orZbtb32; (iv) determining the level of expression of at least one gene of Gzmb, Fanii9a3 Nfil3, Gzma, Eaf2, Carl or St7; (v) detemiining t e level of expression of at least one gene of Crisp3, Apoe, Dapll, PdeSa, Scg5, St8sia6, 4930426D05Rik or Pgapi: (vi) deterniining the level of expression of at least one gene involved in one or more pathway of collagen metabolic process (00:0032963), defense response to bacterium (GO:0042742) ₅ defense response to Gram- negative bacterium (GO:00S0829), endocytosis (GO:0006897). leukocyte mediated immunity (00:0002443), membrane invagination (GO:0010324), membrane organization (GO: 0016044), multicellular organismal macromoleciile metabolic process (GO:Q044259), multicellular organismal metabolic process (GO:Q044236), myeloid leukocyte activation (00:0002274), neutrophil chemotaxis (00:0030593), phagocytosis,, engulfhient (00:0006911),. positive regulation of cellular component organization (GO;005 i 130)„ positive regulation of endocytosis (GO:0045807), positive regulation of foam cell differentiation (GO:0010744), positive regulation of phagocytosis (00:0050766), regulation of endocytosis (GO:0030100), regulation of foam cell differentiation (GO: 0010743), regulation of phagocytosis (00:0050764), regulation of ty e I hypersensitivity (GO:0001810), regulation of vesicle- mediated transport (00:0060627) or response to yeast (GO:000i S78); (vii) detennining the level of expression of at least one gene involved in one or more pathwa of nucleotide binding (00:0000166), double-stranded RNA binding (00:0003725), NAD+ ADP-ribosyltransferase activity (00:0003950), transferase activity, transferring pentosyi groups (00:0016763), transmission of nerve impulse (GO:0019226), regulation of membrane potential (00:0042391), cytokine activity (GO:000512S), ion homeostasis (GO:005Q801) or adenylyltransferase activity (GO:0070566): (viii) detemiining the level of expression of at least one gene involved in one or more pathwa of Cytokine-cytokine receptor interaction (mrnu04060). Jak-STAT signaling pathway (mmu04630). Sterol metabolic process (00:0016125), Sterol biosynthetic process (00:0016126), Hematopoietic cell lineage (mmu04640). Myeloid cell apoptosis (GO: 0033028), Regulation of survival gene product, expressio (00:0045884), Regnkfion of leukocyte ^■ proliferation .(00:0970663), Positive regulation of. survival gene prodiief expression (00:0045885), Hematopoietic cell lineage (inimi94640) or Metabolism of xenobioiies by cytochrome P450 (nnnii009S0): (ix) deiennhiing the level of expression of at least one gene involved in one or more pathway of Intrinsic to membrane receptor activity, itniimne response. Leukocyte migration, C einokine receptor bmdmg, Cytoknie-cytokine receptor iniei¾ction. Cytokine activity. Jafc- STAT signaling pathway. Sterol biosynmetic process. Steroid biosynt etic process. Ceil surface. Cytokine binding. Cholesterol metabolic process, Ciiemokine activity. Negative regulation of hormone secretion, Isoprenoid biosynthetic process or Positive regulation of peptidyl-serine phosphorylation; (x) deiei niniiig the level of expression of at least one gene involved in one or more pathway of Glucose metabolic process. Glycolysis, Glueoneogenesis, Hexose catabolic process. Glucose catabolic process. Monosaccharide catabolic process, Hexose metabolic process. Monosaccharide metabolic process. Cellular carbohydrate catabolic process. Alcohol catabolic process. Carbohydrate catabolic process. Carbohydrate binding. Generation of precursor metabolites and energy. Phagocytosis, Leukocyte migration. Leukocyte chemotaxis. Positive regulation of phagocytosis. Pentose phosphate pathway. Galactose metabolism. Carbohydrate kinase activity. Myeloid leukocyte activation, Ciiemokine receptor binding. Regulation of type I hypersensitivity. Defense response to Gram-negative bacterium or Positive regulation of foam cell differentiation; (xi) determining the level of expression of at least one gene involved in one or more pathway of leukocyte migration (GO:0050900), cell chemotaxis (GO-.006G326), leukocyte chemotaxis (GO:0030595), phagocytosis (GO:0006909), positive regulation of phagocytosis (GO;0050766), cell motion (GO:0006928), regulation of phagocytosis (GO:0G50764), behavior (GO:0007610), membrane invagination (GO;0010324), endocytosis (GO:0006897), positive regulation of endocytosis (GO:0045807), phagocytosis, engulfine t (GO;000691i), defense response to bacterium (00:0042742), membrane organization (00:0016044), neutrophil chemotaxis (GO.0030593), defense response to Gram-negative bacterium (00:0050829), regulation of endocytosis (00:0030100), leukocyte mediated immunity (00:0002443), cell migration (00:0016477), regulation of vesicle-mediated transport (GO:0060627), localization of cell (00:0051674), cell motility (GO:0048870), collagen metabolic process (GO:0032963), iimlticellular organismal inacromolecule metabolic process (GO:0Q44259), positive regulation of cellular component organization (00:0051130), myeloid leukocyte activation (GO:0002274), positive regulation of foam cell differentiation (GO: 0010744), multicellular organismal metabolic process (GO:0044236), response to yeast (00:0001878), regulation of type I hypersensitivity (00:0001810), regulation of foam cell differentiation (00:0010743), sugar binding (GO:0005529), endopeptidase activity (00:0004175), serine hydrolase activity (00:0017171), serme-type peptidase activity (00:0008236), serine-type endopeptidase activity (00:0004252), cytokine activity (GO:0005i25), calcium ion binding (00:0005509), immunoglobulin binding (00:0019865), ciiemokine activity (00:0008009), ciiemokine receptor binding (00:0042379), peptidase activity, acting on L-amino acid peptides (όΟ:ί)070«ϊ 1), pef>Bdase activity (00:0008233), IgG binding (GO:QO 19864;}, protein complex feinditig (00:0032403) or enzyme inhib tor activity (00:0004857); (xii) detenniaingthe level of expression of at. least one gene involved in one or more 'pathway of glucose metabolic process (GO; 0006006), glycolysis {GO:OOO6096}, hexose metabolic process GOrOO 19318), monosacdiaiide metabolic process: (00:0005996), .hexose catabolic process (GQ:0019320), glucose catabolic process (00:0006007), monosaccharide catabolic process (000046365). cellular carbohydrate catabolic process (00:0044275), alcohol catabolic process (G0; O46I64), carbohydrate catabolic process (GO:0016052), generation of precursor metabolites and energy (GO:0006091), oxidorediictase activity, acting on single donors with incorporation of molecular oxygen, incorporation of two atoms of oxygen (GO :0016702), oxidoreductase activity, acting on single donors with incorporation of molecular oxygen (00:0016701), cytokine activity (00:0005125), carbohydrate kinase activity (GO:0019200), Glycolysis / Ghiconeogenesis (mmuOOOlO), Pentose phosphate pathway (mma00030). Fructose and mamiose metabolism (tmimOOGSl) or Galactose metabolism (mmuOO052); (xiii) determining the level of expression of at least one gene involved in one or more pathway of RIG-I-like receptor signaling pathway or Adenylyitransferase activity; (xiv) determining the level of expression of at least one gene of Cxcl3, Clee4a3, UgtlalO, Ugtla6b, Ugtla6a, Ugtla7c, Ugtla9, UgtlaS, Ugtlal, Ugtla2, Clec4n, TgfbL Nov, Ltf Ccl6, mpl2, Ms4a6d. Lpl. Mmp8, Cdl77, Tfec, Clec7a, Mpo, Ccl9, Ccrl, Gpnmb, Msrl, Ms4a6d, Lyz2, Ctsi, Thbsl, Dab2, Arg2. Ccl6, Thbsl, Paldi, CiecSa, Cfp, Iiitm6, Lyzl, 01fhi4, Rnfl28, Ms4a3, Clec4d, S100a4, Ctsg, Prtn3, Ngp, F10, Hal, Cci2, Lyzl, Thbsl, Anxai, Lgals3, SlQ0a6, NfaniL Chil3, C 14, Sppl, Stfa2!l, Ptgrl, 111©, Cd68, Olfml, Fcgr3, Hlr2, Cregl. Dmxl2, Siipa, 116. Elane, Csf r, Lcn2, CdSQOlb, AloxSap, Illrn, Sicllal, Tyrobp, CdSOOa, Pira2, Piral, Pira7, Pira6, Pira5, Pira4, Gmi0693, Lilra6, Pirb, Piral 1, Gin 14548, Ms4a7, Mgstl, Camp, Bstl, Slc40al, Cregl, Csflr, Cd36, Rnfl28, Mrei, Msrl, Itgam, Serpinb2. Lilrb4, Gp49a, Wfdcl7, Anxa3, RgsiS, Slc40al, Bstl , C3arl, Mn_p9, Hist.lh4c, Histlh4b, Histlli4a, Hist4h4, Histlh41 Histlh4d, Histlli4k, Histlh4j, Histlh4i, Histlli4h, Hist.lh4n, Hist.lh4m, Hist2h4, Dstn, IgsiS, Mitt Csf3r, Pf4, PygL Pdpn, S100a9, Fcgr2b,∑dl, Emrl, Cd9, Cebpd, Pia2g7, Arg2, FiO. Gclm, SlOOaS, Piplad2, Clec4a2, Clec4bi, Marcks, Csf2ra, Fcerlg, Clec4a2, Ppic, Cd302, Gstnii, McptS, Cd300if, Csf2rb, Bnip3, Ak4, Tpil, Pfcxdl, Aldoc, Rgsl l, Ankrd37, Pfkl, Pdkl, Cxcl9, Tbx2L Pgkl, Egki3, Ddit4, Pfkp, Pgm2, P4hal, Sfcl6a3, Ubd. Slc2al, 5330426P16Rik, Grlipr, Cxclll, Rasa4, Ndrgl , Fain 162a, Plan, Eglnl. Aldoa, Grn 10480, BnipSl, Ga kl, Pgaml, Pgkl, Mif, Enolb, Enol. Pdxp, Ppplr3b, Stomll, Gzina, Scd2, Ccng2, Higdla, Eno2, Gfil, Pinl, Ldha, Era II, 3000002C10Rik, Hk2, SIc2a3, Chchd6, P4hal, Eroli, Gpil, Gimap7, Oasll, Gml l llO, Syce2, Ndrgl, Ptgs2, Trappc6a, Prelidl, Ifi2712a, Zbtb32, TrimSOa, Prelidl, Gapdh, Sdc3, BC062258, LOC 102643247, 2310022B05Rik, Ms4a4c, I112rbi, Mtbldll, Sniagp, Nampt, Fasl, Pafahlb3, Preiid2, Gngl2, IrfS, Kdni3a, Aif3, TnfsrS,

thereby characterizing the glatiramer acetate related ding substance or drag product of step (a).

23 The present, invention also provides a process for characterizin a glatiranier acetate related dru substance or drug product comprising the steps of:

(a) obtaining a batch of the glathanier acetate related drug substance or drug product ;.

(b) contacting mammalian cells with an amount of the glatiranier acetate related drug substance or drug product of step (a): and

(c) determining the level of expression of at least one geae nwolved. in one or more pathways set forth in Table 3, Table 5, Table 7, Table 8. Table 10, Table 14 or Table 15,

thereby characterizing the glatiranier acetate related drug substance or drug product of step (a).

In some embodiments, step (c)(i) further comprises determining the level of expression of at least one gene of Mmp9, H7r. 116, Ilib, ILK), IL4, Foxp3, 1112a, MmpS, Cxcl3 (GRO-1), Csflr, Ccl2. Cxcl9, Cd40, Cel5, IliOra or TnfsflSb.

In some embodiments, step (c)(ii) ftuther comprises determining the level of expression of at least one gene of Ifi271i, Lonrfl or Pdlim4.

In some embodiments, s tep (e)(iii) ftuther comprises determining the level of expression of at least one gene of AI607873, Daxx, Epstil , Ftsjd2(RniS), Hcfc, Ifi44, Ifitl, IiippSb, Mxl, Nmrall , Oasll, Pyhinl, Sgcb, Tdrd7, Tor3a. Zcchc2 or Zfyve26.

hi some embodiments, step (c)(iv) ftuther comprises determining the level of expression of at least one gene of 114, Ccl3, H6, Tnfisf9, Cish, CD9, IL7R, RAB27B, SLAMF8 or FGL2.

hi some embodiments, ste (c)(v) further comprises determining the level of expression of at least one gene of I17r, Gpnmb, BMP8B, SERPI B10, DEPTOR {DEPDC6} or KCNQ4.

hi some embodiments, step (c) fiirther comprises deiemiining the level of expression of at least one gene of Mxl , Ifhg, Rsad2, Eif2ak2, IfihL Irf7, Oaslaor Gm9706 (IsglS).

In some embodiments, steps (c)(vi) - step (c)(xiv) or step (c) of comprises determining the level of expression of all genes involved in one or more pathways, two or more pathways, three or more pathways, torn' or more pathways, five or more pathways or six or more pathways.

In some embodiments, steps (c)(vi) - step (c)(xiv) or step (c) comprises determhiing the level of expression of at least two genes, at least three genes, at least four genes, at least five genes or at least six genes involved in the same pathway.

hi some embodiments, step (c)(viii) or step (c)(ix) or step (c) ftuther comprises determining the level of expression for all genes involved in one or more additional pathway, two or more additional pathways, three or more additional pathways, four or more additional pathways, five or more additional pathways or six or more additional pathways set forth in Table 3 or Table 10. In some embodiments, step (c)(yrii) or step (c)(ix) or. step (c) ftirtlier, comprises detemiimng the lew! of expression for at least one gene involved, in one or more additional padiway set forth in Table 3 or Table 10,

In some embodiments, step (c}fvm) or step (e¾is) or step (c) fiirtlier comprises detenniaing the level of expression for at least one gene, at least t o genes, at least three genes, at least four genes, at least five genes or at least six genes involved- in one additional pathway set. forth, in Table 3 or Table 10.

In some embodiments, step (c)(vi), step (c)(vii). step (c)(x), step (c)(xi), step (c)(xii) or step (c)(xiii) or step (c) ftirtlier comprises deteriiihiing the level of expression for all genes involved in one or more additional pathway, two or more additional pathways, three or more additional pathways, four or more additional pathways, five or more additional pathways or six or more additional pathways set forth in Table 5, Table 7, Table 8, Table 14 or Table 15.

In some embodiments, step (c)(vi), step (c)(vii), step (c)(x). step (c)(xi), step (c)(xii) or step (c)(xiii) or step (c) further comprises deterniining the level of expression for at least one gene involved in one or more additional pathway set form in Table 5, Table 7, Table 8, Table 14 or Table 15.

In some embodiments, step (c}(vi), step (c){vii} _t step (c){x), step (c)(xi), step (c)(xii) or step (e)(xiii) or step (c) further comprises detemiinhig the level of expression for a leas one gene, at least two genes, at least diree genes, at least four genes, at least five genes or at least six genes involved in one additional pathway set form in Table 5, Table 7, Table 8, Table 14 or Table 15,

In some embodiments, contacting the mammalian cells in ste (b) comprises i) administering to a mammal a predetermined amount of glatiramer acetate related drag substance or drug product of step (a), or ii) incubating the cells with an amoimt of the glatiramer acetate related drug substance or drug product of ste (a), or a combination thereof.

In some embodiments, contacting the mammalian cells hi ste (b) comprises i) administering to a mammal a predetermined amount of glatiramer acetat related drug substance or drug product of ste (a), and ii) obtaining cells from the mamma! at one or more predetermined time points.

In some embodiments, contacting the mammalian cells in ste (b) comprises incubating monocytic cell line cells wit an amount of the glatiramer aceta te rela ted drug substance or drag product of step (a).

In some embodiments, the mammalian cells are THP-1 cells.

In some embodiments, contacting the mammalian cells in step (b) comprises (i) immunizing a mammal with a predetermined amount of glatiramer acetate related drug substance or drug product, (ii) preparing a culture of cells from the mammal of step (i) at one or more predetermined time points after immunization, and (iii) incubating cells from the culture of cells obtained from the mammal with an amoimt of the glatiramer acetate related drug substance or drug product of step (a). In some embodiments , the glaiiramer acetate rela ted dra substance or drag product of step (iii) is the same gktirsaier acetate related, dru substance or drug product of step (i). In some embodiaieBts, the. giatiramer acetate related drug, substance or drag product of step ^' (iii) is a different glatiramer acetate related drug substance or drag product of step ( ^' i).

In some embodiments, fee mciibation is for about 24 hours, for about 1 hoars _* or for about 6 hours.. In some embodimfiiits. the predetenniiied rime point, after immunization is 3 days.

In some embodiments, the contacting of step (b) is in a cell culture. In some embodiments, the culture is a primary culture. In some embodiments, the contacting of step (b) is in a mammal. In some embodiments, the mammal is a rodent or human.

In some embodiments, the giatiramer acetate related drag substance or drug product is othe than giatiramer acetate drug substance or drag product.

hi some embodiments, the cell is of a type of FoxP3+ T cells, regulatory T cells, natural killer T cells, T helper 2 cells. CD8+ T cells, CD4+ T cells, B cells, macrophage cells, monocyte cells, eosinophils, dendritic ceils, granulocytes, megakaryocytes, or myeloid progenitors.

The present invention also provides a process for discriminating between giatiramer acetate related drug substances or drug products comprising the steps of:

(a) characterizing two or more giatiramer acetate related drug substances or drug products according to the process of the present invention to obtain characteristics of each of the giatiramer acetate related drug substances or drug products; and

(b) comparing the characteristics of the giatiramer acetate related drug substances or drag products obtained in step (a),

thereby discriminating between giatiramer acetate related drug substances or drug products.

The present invention also provides a process for producing a drag product comprising a giatiramer acetate related drag substance, the improvement comprising the steps of:

(a) characterizing the giatiramer acetat related drag substance according to the process of any one of the present invention: and

(b) (i) discarding the batch of the giatiramer acetate related drug substance as unacceptable for inclusion in the drug product if the level of expression of one or more genes ofMmpl2, Tgfbi, Clec4n, Clec4a3, Fcerlg, Fcgr2b. Fcgr3, Csf3r, Hlr2 or Tbx21 is not substantially identical to the level of expression by the same type of cells in the presence of the giatiramer acetate drug substance under the same conditions; (ii) discarding the batch of the giatiramer acetate related drag substance as unacceptable for inclusion in the drag product if the level of expression of one or more genes of Ahcyl2, Chi5, Cxcr6, Dhrs3, Egr2, Fabp5, Ptp4al, Gprl83, Lampl, Ncoa4, Ptger4, Qk, Rragd, Sec24a, Sehll, $estdl, Slc30a4, Soatl or Strip? is not substantially ideiitieal to the level of expression by the same ty e of ceils, in the presence of the. giaiiramer acetate drug substaace wider the same conditions; (iii) discarding the hatch of the glatirainer acetate related drug substance as unacceptable tor inclusion in die drag product if the level of ^' expression, of one or .more genes of Fam212a, Gznia, JIJJFI or Zbtb32 is not substantially identical to. die level of expression by the same type of cells in the presence of the glatiramer acetate drug substance under die same conditions; (iv) discarding the batch of the glatiramer acetate related drag substance as tmacceptable ^' for inclusion in the dra product if the level of expression of one or more genes of Gzmb, Faml9a3, NfilS, Gzma, Eaf2, Carl or St7 is do nregulated or substantially identical to the level of expression by the same type of cells in the absence of the glatiramer acetate related drug substance under the same conditions; (v) discarding the batch of the glatiramer acetate related dra substance as unacceptable for inclusion in the drug product if the level of expression of one or more genes of Crisp3, Apoe, Dapll, PdeSa, Scg5, St.8sia,6, 4930426D05Rik or Pgapl is upregulated or substantially identical to the level of expression by the same type of cells in the absence of the glatiramer acetate related drag substance under the same conditions; (vi) discarding the batch of the glatiramer acetate related drag substance as unacceptable for inclusion in the drag product if the set of differentially expressed genes indicates enrichment of one or more pathway of collagen metabolic process (GO:0032963), defense response to bacterium (GO:0042742) ₅ defense response to Gram- negative bacterium (GO:0050S29), endocytosis (GO:0006897). leukocyte mediated immunity (00:0002443),. membrane invagination (GO:0010324), membrane organization (GO:0016044), multicellular organismal macromolecule metabolic, process (00:0044259). multicellular organismal metabolic process (00:0044236). myeloid leukocyte activation (00:0002274), neutrophil chemotaxis (GO:0030593), phagocytosis, engulfinent (00:000691 1), positive regulation of cellular component organization (00:005 i 13Q)„ positive regulation of endocytosis (00:0045807), positive regulation of foam cell differentiation (00:0010744), positive regulation of phagocytosis (00:0050766), regula tion of endocytosis (00:0030100), regulation of foam cell differentiation (00:0010743), regulation of phagocytosis (00:0050764), regulation of ty e I hypersensitivity (00:000 IS 10), regulation of vesicle- mediated transport (GO:0060627) or response to yeast (GO:000187S); (vii) discarding the batch of the glatiramer acetate related drag substance as unacceptable for inclusion in the drag product if the set of differentially expressed genes indicates enrichment of one or more pathwa of nucleotide binding (00:0000166), double-stranded R A binding (00:0003725), NAD+ ADP-ribosylttansferase activity (00:0003950), transferase activity, transferring pentosyl groups (00:0016763), transmission of nerve impulse (00:0019226), regulation of membrane potential (00:0042391), cytokine activity (00:0005125), ion homeostasis (00:0050801) or adenylykransferase activity (00:0070566); (viii) discarding the batch of the glatiramer acetate related drag substance as unacceptable for inclusion in the drug product if the set of differentially expressed genes indicate no subs tantial enrichment of one or more pathway of Cytokine-cytokine recepto interaction (mmu04060), Jak-STAT signaling pathway (mmu04630). Sterol metabolic process (00:0016125), Sterol biosynthetic process (00:0016126). Hematopoietic cell lineage (inmu0464Q ^: ), Myeloid cell apoptosis (GO: 0033028):, Regulation of survival gene roduct expression (GO;004SS } Regulation of leukocyte proliferation ;{GQ;OQ7066|), Positive, regulation of survival gene product expression (GO;0O45§85), Hematopoietic cell lineage (aimii04640) or Metabolism of xenobioties by cytochrome P 50 {mmii00980); (i ) discarding the batch, of the giatiramer acetate related drug substance as unacceptable for inclusion in the drag product if the set of differentially expressed genes indicate no substantial eiffiehr&ent of one or more pathway of I t insic to membrane reeeptea: .activity, Irnmmie response, Leukocyte migration, Chemokme recepto bmding. Cytokine-cytokine receptor interaction. Cytokine activity, iak-STAT signaling pathway. Sterol biosynthetic process. Steroid biosynthetic process, Cell surface. Cytokine binding. Cholesterol metabolic process, Chemokine activity,. Negative regulation of hormone secretion, Isoprenoid biosynthetic process or Positive reg lation of peptidyl-serine phosphorylation; (x) discarding the batch of the g!atiramer acetate related drug substance as unacceptable for inclusion hi the drug prodiict if the set of differentially expressed genes indicates enrichment of one or more pathway of Glucose metabolic process. Glycolysis, Gluconeogenesis, Hexose catabolic process. Glucose catabolic process. Monosaccharide catabolic process, Hexose metabolic process. Monosaccharide metabolic process. Cellular carbohydrate catabolic process. Alcohol cataboli process. Carbohydrate catabolic process. Carbohydrate binding. Generation of precursor metabolites and energy. Phagocytosis, Leukocyte migration. Leukocyte chemotaxis. Positive regulation of phagocytosis. Pentose phosphate pathway. Galactose metabolism. Carbohydrate kinase activity, Myeloid leukocyte activation, Chemokine receptor binding. Regulation of type I hypersensitivity. Defense response to Gram-negative bacteiium or Positive regulation of foam cell differentiation; (xi) discarding the batch of the giatiramer acetate related drag substance as unacceptable for inclusion in the drag product if the set of differentially expressed genes indicates enrichment of one or more pathway of leukocyte migration (GO:Q050900), cell chemotaxis (00:0060326). leukocyte chemotaxis (GO;0030595), phagocytosis (GO;0006909), positive regulation of phagocytosis (GQ:0050766), cell motion (00:0006928), regulation of phagocytosis (GO:0050764), behavior (GG: 0007610), membrane invagination (GO:0010324), endocytosis (GO:0G06897}, positive regulation of endocytosis (GO:0045807), phagocytosis, eiigiilfment (GO:0006911), defense response to bacterium (GO:0042742), membrane organization (00:0016044), neutrophil chemotaxis (00:0030593), defense response to Gram-negative bacteiium (GO:0050829), regulation of endocytosis (GO:0030100), leukocyte mediated immunity (GO:0002443), cell migration (GO;0016477), regulation of vesicie-mediated transport (00:0060627), locaiization of cell (GO:0051674), cell motility (GO:0048870), collagen metabolic process (00:0032963), multicellular organisms! macromolecnle metabolic process (00:0044259), positive regulation of cellular component organization (GO:005i 130), myeloid leukocyte activation (GO:G002274), positive regulation of foam cell differentiation (GO:0010744), multicellular organismal metabolic process (GO:0Q44236), response to yeast (GO;0001878>, regulation of type I hypersensitivity (GO:000i 810), regulation of foam cell differentiation (00:0010743), sugar' binding (00:0005529), eiidopepiidase activity (00:0004175), serine hydrolase activity (00:0017! 71), serine-rype peptidase acti ity (00:0008236), seria - ype eadopeptidase activity (00:0004252), cytokine acti ity (GO:00Q51 5), calcium ion binding (00:0005509), iramiiBogiohiilin binding (GO.00 9865), eaeraokine activity (00:0008009), cheiiiokine receptor binding (00:0042379), peptidase activity, acting on L-amino acid peptides (00:0070011), peptidase activity (00:0008233), IgG binding (00:0019:864), protei complex bindin (00:0032403) or eazyrne inhibitor activity (00:0004857); (xii) discarding the batch of the glaiiramer acetate related drug substance as unacceptable for inciii&ion hi the drug product; if the set of differentially expressed genes indicates enrichment of one or more path way of glucose metabolic process (00:0006006), glycolysis (00:0006096), hexose metabolic process (GO:0019318), monosaccharide metabolic process (00:0005996), hexose catabolic process (00:0019320), glucose catabolic process (00:0006007), monosaccharide catabolic process (00:0046365), cellular carbohydrate catabolic. process (00:0044275), alcohol catabolic process ((30:0046164), carbohydrate catabolic process (00:0016052), generation of precursor metabolites and energy (00:0006091). oxidoreductase activity,, acting on single donors with incorporation of molecular oxygen, incorporation of two atoms of oxygen (00:0016702), oxidoreduciase activity, acting on single donors with incorporation of molecular oxygen (00:0016701), cytokine activity (00:0005125), carbohydrate kinase activity (00:0019200), Glycolysis / Gluconeogenesis (mmuOOOlO), Penrose phosphate pathway (mniiiOOOSO), Fructose and mannose metabolism (nmiu00051) or Galactose metabolism (mniu00052); (xiii) discarding me batch of the glatirarner acetate related dmg substance as unacceptable tor inclusion in the drug product if the set of differentially expressed genes indicates enrichment of one or more pathway of RIG-I-like receptor signaling pathway or Adenylyltransferase activity; or (xiv) determining the level of expression of at least one gene of Cxc , Ciec4a3, UgtlalG, Ugtia6b, Ugtla6a, Ugtla7c, Ugtla9, Ugtla5, UgtlaL Ugtla2, Clec4n, Tgfbi, Nov, Ltf, Cc!6, Mrapl2, Ms4a6d, Lpl, Mmp8. Cdl77, Tfec, Clec7a, Mpo, Ccl9, Ccrl , Gpnmb, Msrl, Ms4a6d. Lyz2, Ctsl. Thbsl , Dab2, Arg2, Ccl6, Thbsl, Paldl, Clec5a, Cfp, Ifitm6, Lyzl, 01fi¾4, Rnfl28, Ms4a3, Ciec4d, SlOOa.4, Ctsg, Prto3, Ngp, F10, Hal, Ccl2, Lyzl, Thbsl, Anxal, Lgals3, S100a6, Nfanii, C 3, C 4, Sppi, Stfa211, Ptgrl, Illf9, Cd68, Olfinl, Fegr3, Illi2, Cregl. Dmxl2, Sirpa, 116, Eiaiie, Csflr, Lcn2, CdSOOlb, AloxSap, Ulro, Slcl iai, Tyrobp, CdSOOa, Pira2, Piral. Pira7, Pira6, PiraS, Pira4, Gml0693, Lilra6, Pirb, Pirall, Gml4548, Ms4a7, Mgstl, Camp, Bstl, Slc40al, Cregl, Csflr, Cd36, Rnfl28, Mixl, Msrl, Itgam, Serpmb2, Lilrb4, Gp49¾ Widcl?, Anxa3, Rgsl8, SIc40ai, Bstl, C3arl, Mmp9. Histlh4c, Histlh4b, Hisilh4a, Hist4h4, Histlh4f, Histlh4d, Histlh4k, Histlhlj, Histih4i. Histlh4h, Histlh4n, Histlh4m, Hisf2h4, Dstii, Igsf6, Mitf, Csf3r, Pf4. Pygl, Pdpn. S100a9, Fcgr2b, Ml, Emrl, Cd9, Cebpd, Pla2g7, Arg2, FIO, Gclm, SlOOaB, Ptplad2, Clec4a2, Clec4bl, Marcks, Csf2ra, Fcerlg, Clec4a2, Ppic, Cd302, Gstml, McptS, Cd300i£ Csf2rb, Bnip3, Ak4, Tpil, Plcxdl, A oe, Rgsl 1, Ankrd37, Pfkl Pdki, Cxd9, Tbx2i, Pgki, Eghi3, Ddit4, Pfkp, Pgm2, P4hal, Skl6a3, Ub& Sk2al, 5330426P16Rik. Grhpr, Cxcll 1, Rasa4, Ndrgl, Faml62a, Pkm, Eglnl, Aidoa, Gml0480, Bnip3i. Gaikl, Pgaml, Pgki, Mif, Enolb, Enoi, Pdxp, PpplrSb, Stomll, Gzma, Sed2. Ccng2, Higdla, Eno2, Gfil. Piiii. Ldha, Eroll, 3000002C10Rik, ¾2, Sk2a3, ChehdS, P4hal ₅ Emit Gpil , Gknap?, Oasli, Gml 1110, ce2, Ndrgl, Ptgs2, Trappe6a, PrelidX, IS2712a, Zbtb32, TruaSOa, Prelidl, Gapdk Sdc3, BC06225S, LQCil 02643-247, 23ί0β22Β05¾& . s4a4c _f . ^' fil2rbX, Mthfdll, Sniagp, Nampt, Fast Pafafclb3, Prelid2 Gngl2. MB, KiiiiiSa, Atf¾ Tnfsf9.

The present, invention also provides a process for releasing a drag product comprising a glatiramer acetate related drug substance, the improvement comprising the steps of;

(a) characterizing the glatiramer acetate related drug substance according to the process of the present invention: and

(b) (i) discarding the batch of the gjatirainer acetate related drag substance as unacceptable for inclusion in the drag product if the level of expression of one or more genes ofMmpl2, Tgfbi, Clec4n. Clec4a3, Fcerlg, Fcgr2b, Fcgr3, Csf3r, Illr2 or Tbx2I is not substantially identical to the level of expression by the same type of cells in the presence of the glatiramer acetate drug substance under the same conditions; (ii) discarding the batch of the glatiramer acetate related drag substance as unacceptable for inclusion in the drag product if the level of expression of one or more genes of Ahcyl2, Cm5, Cxcr6, Dhrs3, Egr2, Fabp5, Ptp4al, Gprl83, Lanipl, Ncoa4, Ptger4, Qk, Rragd, Sec24a, Sehll. Sestdl . Slc30a4. Soatl or Sfiip2 is not substantially identical to the level of expression b the same type of cells in the presence of the glatiramer acetate drug substance under the same conditions; (iii) discarding the batch of the glatiramer acetate related drag substance as unacceptable for inclusion hi the drag product if the level of expression of one or more genes ofFam212a, Gzma, Itpi or Zbtb32 is not substantially identical to the level of expression by the same type of cells in the presence of the glatiramer acetate drug substance under the same conditions; (iv) discaidmg the batch of the glatiranier acetate related drag substance as unacceptable for inclusion in the drag product if the level of expression of one or more genes of Gzmb, Faml9a3, fil3 ₅ Gzma, Eaf2, Carl or St7 is downregiilated or substantially identical to the level of expression by the same type of cells in the absence of the glatiramer acetate related drug substance under the same conditions; (v) discaidmg the batch of the glatiramer acetate related drag substance as una cceptable for inclusion in the drug pr oduct if the le vel of expres sion of one or more genes of Crisp3, Apoe, DaplL PdeSa, Scg5, St8sia6, 4930426D05 ik or Pgapl is upreguiated or substantially identical to the level of expression by the same type of cells in the absence of the glatiramer acetate related drag substance under the same conditions; (vi) discardin the batch of the glatiramer ace tate related drug substance a s unacceptable for inclusion in the drug product if the set of differentially expressed genes indicates enrichment of one or more pathway of collagen metabolic process (GO:0032963). defense response to bacterium (GO:0042742), defense response to Gram- negative bacterium (GO:0050829), endocytosis (GO;0006897), leukocyte mediated immunity (GO:0002443), membrane invagination (GO:0010324), membrane organization (GO:0016044). multicellular organismal macromolecule metabolic process (GO:0044259), multicellular organismal metabolic process (00:0044236), myeloid leukocyte activation (00:0002274), neutrophil chemotaxis (00:0030593), phagocytosis, engulfmeiit (GO;OO069!J), positive; regulation of ^' cellular, component organization (00:0051130), positive regulation of endocytosis (GO;004S807), positive regulation of foam: cell differentiation (GO;00i6 ^' 744 , positive regulation of phagocytosis (00:0050766), regulation of endocytosis (00:0030100), regulation of foam cell differentiatioii (GO:0010743), regulation of phagocytosis (GO:0€50764j, regulation of type I hypersensitivity ^' (Op: 0001810), regulation of vesicle- mediated transport (00:0060627) or response to yeast ^• (GO-tOOOl 878): (vii) discarding the batch of the giatiramer acetate related drag . ^' substance as unacceptable for inclusion in the drag product if the iset of differentially expressed genes indicates enrichment of one or more pathway of nucleotide binding (GO.-0000166), double-stranded RNA binding (GO.O0O3725), NAD+ ADP-ribosyltransferase activity (00:0003950), transferase activity, transfening pentosyi groups (GO:0016763), transmission of nerve impulse (GO:0019226), regulation of membrane potential (GO:0042391), cytokine activity (GO-.0005125), ion homeostasis (GO:0050801) or adenyiyltransferase activity (00:0070566); (viii) discarding the batch of the giatiramer acetate related drug substance as unacceptable for inclusion in the drag product if the set of differentially expressed genes indicate no substantia! enrichment of one or more pathway of Cytokine-cytokine receptor interaction (mniu04060), Jak-STAT signaling pathway (minu04630). Sterol metabolic process (00:0016125), Sterol biosynthetic process (00:0016126), Hematopoietic cell lineage (nimu04640). Myeloid cell apoptosis (GO: 0033028), Regulation of survival gene product expression (00:0045884),. Regulation of leukocyte proliferation (GO: 0070663), Positive regulation of survival gene product expression (GO:0045885), Hematopoietic cell lineage (mmu0464O) or Metabolism of xenobiotics by cytochrome P450 (mmu00980); (ix) discarding the batch of the giatiramer acetate related drug substance as unacceptable for inclusion in the drug product if the set of differentially expressed genes indicate no substantial enrichment of one or more pathway of Intrinsic to membrane receptor activity. Immune response. Leukocyte migration, Chemokine receptor binding. Cytokine-cytokine receptor interaction. Cytokine activity, Jak-STAT signaling pathway. Sterol biosynthetic process. Steroid biosynthetic process, Cell surface. Cytokine binding. Cholesterol metabolic process, Chemokine activity. Negative regulation of hormone secretion, Isoprenoid biosynthetic process or Positive regulation of peptidyl-serine phosphorylation; (x) discarding the batch of the giatiramer acetate related drug substance as unacceptable for inclusion in the drug product if the set of differentially expressed genes indicates enrichment of one or more pathway of Glucose metabolic process. Glycolysis, Gluconeogenesis, Hexose catabolic process. Glucose catabolic process. Monosaccharide catabolic process, Hexose metabolic process. Monosaccharide metabolic process. Cellular carbohydrate catabolic process. Alcohol catabolic process. Carbohydrate catabolic process. Carbohydrate binding, Generation of precursor metabolites and energy. Phagocytosis, Leukocyte migration, Leukocyte chemotaxis. Positive regulation of phagocytosis. Pentose phosphate pathway, Galactose metabolism. Carbohydrate kinase activity, Myeloid leukocyte activation, Chemokine receptor binding. Regulation of type I hypersensitivity, Defense response to Gram-negative bacterium or Positive regulation of foam cell differentiation; (xi) discarding the batch of the giatiramer acetate related drag substance as unacceptabl for inclusion in the dru product if the set of differentially expressed genes indicates eniichnient of one or more■pathway of leukocyte migration (GO; 0050900), cell eheraotaxis (GO;OO60326 , leukocyte cae notaxis (GO;003OS95), phagocytosis (00:0006909), positive regulation of phagocytosis (00:0050766), cell motion (00:0006928), regulation of phagocytosis (00:0050764), behavio (00:0007610), membrane imagin tion (00:0010324), endoeytosis (00:0006897), positive regiiiation of endoeytosis (00:0045807),. phagocytosis, engulftnent {ΟΌ;0ββ69Π).,, defense response to bacterium (GQ;00 2742). _? . membrane organization (GO.O016044), neutrophil chemotaxis (GO:0030593), defense response to Gram-negative bacterium (GO:0050829), regulation of endoeytosis (GO:0030100), leukocyte mediated mimunify (GO:0002443), cell migration (00:0016477),. regulation of vesicle-mediated transport (GO: 0060627), localization of cell (GO:0051674), ceil motility (GO:0048870), collagen metabolic process (GO:0032963) _t multicellular organismal macromolecule metabolic process (GO:Q044259), positive regulation of cellular component organization (00:0051130), myeloid leukocyte activation (GO:0002274), positive regulation of foam eel! differentiation (GO: 0010744), multicellular organismal metabolic process (00:0044236), response to yeast (00:0001878),. regiiiation of type I hypersensitivity (00:0001810), regiiiation of foam cell differentiation (GO:0010743), sugar binding (00:0005529), endopeptidase activit (GO:0004175), serine hydrolase activity (00:0017171). serine-rype peptidase activity (00:0008236), serine-rype endopeptidase activity (00:0004252), cytokine activity (00:0005125), calcium ion binding (00:0005509), immunoglobulin binding (GO:00i9865), cheinokine activity (GO:0008009) ₅ chemokine receptor binding (GO:0042379), peptidase activity, acting on L-amino acid peptides (00:0070011), peptidase activity (GO:0008233), IgG binding (00:0019864), protein complex binding (00:0032403) or enzyme inhibitor activity (00:0004857); (xii) discarding the batch of the glatiramer acetate related drag substance as unacceptable for inclusion in the drug product if the set of differentially expressed genes indicates enrichment of one or more path wa of glucose metabolic process (GO:0006006), glycolysis (GO: 0006096), hexose metabolic process (GO:0019318), monosaccharide metabohc process (00:0005996), hexose catabolic process (00:0019320), glucose catabolic process (GO:0006007), monosaccharide catabolic process (00:0046365), cellular carbohydrate catabolic process (00:0044275), alcohol catabolic process (00:0046164), carbohydrate catabolic process (GO:0016052), generation of precursor metabolites and energy (00:0006091), oxidoreductase activity, acting on single donors with incorporation of molecular oxygen, incorporation of two atoms of oxygen (00:0016702), oxidoreductase activity, acting on single donors with incorporation of molecular oxygen (GO:0016701), cytokine activity (00:0005125), carbohydrate kinase activity (00:0019200), Glycolysis Glucoiieogenesis (mnmOOOlO), Pentose phosphate pathway (mmuOOOSO), Fructose and rnarmose metabolism (mmu00051) or Galactose metabolism (mmu00052); (xiii) discarding me batch of the glatiramer acetate related drag substance as unacceptable for inclusion in the drug product if the set of differentially expressed genes indicates enrichment of one or more pathway of RIG-I-like receptor signaling pathway or Adenylyltraiisferase activity; or (xiv) determining the level of expression of at least one gene of Cxei3, Clec4a3, IJgtlalO, Dgtia6b, Ugtia6a _f UgtlaTe, Ugtia% Ugtla5, U tlaL K rlai, ¾ c4», Tgfbj, Nov, Ltl C<¾ pi2, M$4a6d, LpL MinpS, Cdi 77, Tfec, CtecJa, Mpo, Ccl9, Ceri, Gpanib. MsrL Ms4a6d, Lyz2, CtsL Thbsl, Da¾2, Arg2, Cci6, Thbsl, Paldl, Cle Sa, Cfp, IfifrnS, Lyzl, 01im4, Rnfl2S, Ms4a3, Clec4cl S100a4 ₅ Ctsg, ¾tiLLN , FIO, Hat Cc_2, Lyz , TlibsL AnxaL Lgais3, SIGi S, Nfem ^' l , Chil3, CliiM, Sppl, Stfa21I ₅ Prgrl , HIS, Cd6S, OlfmL Fegr¾ Cregi/Dtnxi2 Sirpa, B6,.Elaae, Csftr. Lcn¾, Cd$0G¾ AioxSap, 1¾ SicUal, Tyrobp, CdSOQa, Pha2, Pkai Pira7, Ph¾6, PiraS, Pira4, Ginl0iS93, Lika.6 Pirb, Pirall, Gml4548, Ms4a7, Mgstl, Camp, Bstl, Slc40al, Cregl. Csfir, Cd36, Rnfl28, Mrcl, Msrl. Itgam, Seipinb2, Lilrb4, Gp49a, Wfdcl7, Anxa3. RgslS, Slc40al, Bstl C3arl , Mmp9, Histlh4c, Histlh4b, Histlh4a, Hist4h4. Η½ί1ίι41 Histlh4d. Histlh4k, Histlh4j, HistlMi, HisiiMii. Histlh4n, HIsilMm Hist2h4, Dsiii IgsiS, Mitf, Csf3r, Pf4, Pygl, Pdpn, S100a9, Fcgr2b, Idl, Enarl, Cd9, Cebpd, Pla2g7, Arg2 FIO, Gclm, S100a8, Plplad2, Clec4a2 Clec4bl, Marcks, Csf2ra, Fcerlg. Ciec4a2, Ppic, Cd302 Gstml McptS, CdSOOlf, Csf2rb ₅ Bnip3. Ak4, Tpil . Pfcxd 1 , Aldoc, Rgsll, Ankrd37, Pfkl, Pdkl , Cxcl9, Tbx21 , Pgfcl, Egm3, Ddit4, Pfkp, Pgm2, P4hal, Sicl6a3, Ubd, Steal, 5330426P16Rik, Grhpr, Cxcil 1, Rasa4, Ndigi, Faini62a, Pkm, Eglnl, AMoa, Gml04S0, Bnip3L Galkl, Pgaml, Pgkl, Mil Enolb, Enol, Pdxp, Fpplr3b, Stonill, Gzrna, Scd2, Ccng2, Higdla Eno2 Gfil, Prol, Ldha, ErolL 3000002C10Rik, Hk2, Sic2a3, Chc 6. P4hal , ErolL Gpil, Gimap7, Gasll, Grai l 110, Syce2, Ndrgl, Ptgs2, Trappc6a, Prelidi, Ifi27i2a, Zbtb32, TrimSOa, Prelidl, Gapdh, Sdc3, BC062258, LOC102643247, 2310022B05Rik, Ms4a4c, U12rbi, Mttrfdll, Smagp, Narnpt, Fasl Pafahib3, Prelid2, Gngl2, IrfS, Kdm3a, Atf3 Tnfsf9.

The present invention also provides a process for identifying suboptimal activity of a glarirainer acetate related drug substance or drug product comprising the steps of:

(a) administering a glatiramer acetate related drug substance or drag product to a mammal;

(b) characterizing the glatiramer acetate related drug substance or drug product according to the process of the present invention; and

(c) (i) identifying the glatirainer acetate related drag substance or drug product as having a. suboptmial activity if the level of expression of one or more genes ofMinpi2, Tgibi, Clec4n, Clec4a3, Fcerlg. Fcgr2b. Fcgr3, Csf3r, Hlr2 or Tbx21 is not substantially identical to the level of expression by the same type of cells in the presence of the glatiramer acetate related drag substance or drug product under the same conditions; (ii) identifying the glatiramer acetate related drag substance or drug product as having a suboptimai activity if the level of expression of one or more genes of Ahcyl2, Cm5, Cxer6, Dhrs3, Egr2 FabpS, Ptp4al, GprlS3, Lampl, Ncoa4, Ptger4, QL Rragd. Sec24a, Sehll, Sestdl. Slc30a4, Soatl or Stiip2 is not substantially identical to the level of expression by the same type of cells in the presence of the glatiramer acetate related drug substance or drug product under the same conditions: (iii) identifying the glatirainer acetate related drag substance or drag product as having a.

33 subopthua! activity if the level of expression of one or more genes of Fam212a.. Gzma. Itprl or Zbtb32 is not substantially identical to the level of expression by tire same type of cells i the presence of ^' the giatiramsr acetate related drag substance or Aug product under the same conditions; (rv) identifying, die giaiiramer acetate related .drag substance or drug product as ha ving a suboptimal activity if the level o expression of one or more genes Of Gzmk Faiui9a3, fiB, Gzma, Eaf2, Carl, or St7 is downregulated or substantially identical to the level of expression by the same type of cell in the absence of the glatiramer acetate related drug substance Under the same conditions: or (v) identify/trig the glaliramer acetate related drag substance or drug product as having a suboptimal activity if the level of expression of one or more genes of Crisp3, Apoe, Dapll, Pde5a, Scg5, St8sia6, 4930426D05Rik or Pgapl is upregulated or substantially identical to the level of expression by the same type of cells in the absence of the glatiramer acetate related drug substance under the same conditions: (vi) identifying the glatiramer acetate related drag substance or drug product as having a suboptimal activity if the set of differentially expressed genes indicates enrichment of one or more pathway of collagen metabolic process (00:0032963), defense response to bacterium (00:0042742), defense response to Grani- iiegaiive bacterium (00:0050829), endocytosis (GO:0006897), leukocyte mediated immunity (GO:0Q02443), membrane invagination (00:0010324), membrane organization (00:0016044), multicellular organismal macromolecule metabolic process (GO:0044259), multicellular organismal metabolic process (00:0044236), myeloid leukocyte activation (00:0002274), neutrophil chemotaxis (00:0030593), phagocytosis, engulfment (GO:0006911), positive regulation of cellular component organization (GO:005113Q), positive regulation of endocytosis (GO:0045807), positive regulation of foam cell differentiation (GO:0010744), positive regulation of phagocytosis (GO:0050766), regulation of endocytosis (GO:0030100), regulation of foam cell differentiation (GO:0010743), regulation of phagocytosis (00:0050764), regulation of type I hypersensitivity (00:0001810), regulation of vesicle- mediated transport, (GO:0060627) or response to yeast (GO:0001878); (vii) identifying the glatiramer' acetate related drug substance or drag product as having a suboptimal activity if the set of differentially expressed genes indicates enrichment of one or more pathway of nucleotide binding (GO:0000166), double-stranded SNA binding (00:0003725), NAD+ ADP-iibosyltransferase activity (00:0003950). transferase activity, transferring pentosyl groups (00:0016763), transmission of nerve impulse (GO-.0019226), regulation of membrane potential (GG.0042391), cytokine activity (00:0005125), ion homeostasis (00:0050801) or adenylyitransfera.se activity ⁷ (00:0070566); (viii) identifying the glatii'amer acetate related drug substance or drug product as having a suboptimal activity if the set of differentially expressed genes indicate no substantial enrichment of one or more pathways of Cytokine- cytokine receptor interaction (mmu04060), Jak-STAT signaling pathway (mmu04630). Sterol metabolic process (GO:0016125), Steroi biosynthetic process (GO:0016126), Hematopoietic cell lineage (mmu04640). Myeloid ceil apoptosis (GO; 0033028), Regulation of survival gene product expression (00:0045884), Regulation of leukocyte proliferation (00:0070663), Positive regulation of survival gene product expression (00:0045885), Hematopoietic cell lineage (mmu04640) or Metabolism of .xenobiotics fay cyt ch o e P450 (nimu0u980); ix) identifying the glatiramer acetate related drug substance or drug .product -as having- a sutopiiiiiai activity if the set of differentially expressed genes indicate no substantial enrichinent of one or more pathway of ^' tiinsic to membrane receptor activity,. Iinmtme response, ^' Leukocyte migration, Chemokine receptor binding, Cytokine- cytokine receptor ^' interaction. Cytokine activity, Jak-STAT signaling pathway. Sterol biosynmeti.c process. Steroid biosyntheiic process. Ceil surface, /Cytokine binding. Cholesterol metabolic process, Chemokine activity, Negative regulation of hormone secretion, Isopreaoid biosyntheiic process o Positive regulation of peptidyl-serine phosphorylation; (x) identifying the glatiramer acetate related drag substance or drug product as having a suboptimal activity if the set of differentially expressed genes indicates one or more patliway of enrichment of Glucose metabolic process, Glycolysis, Glueoneogenesis, Hexose catabolic process. Glucose catabolic process. Monosaccharide catabolic process, Hexose metabolic process. Monosaccharide metabolic process. Cellular carbohydrate catabolic process. Alcohol catabolic process, Carbohydrate catabolic process. Carbohydrate binding. Generation of precursor metabolites and energy. Pha ocytosis, Leukocyte migration, Leukocyte chemotaxis. Positive regulation of phagocytosis. Pentose phosphate pathway. Galactose metabolism. Carbohydrate kinase activity, Myeloid leukocyte activation, Chemokine receptor binding. Regulation of type I hypersensitivity. Defense response to Grain-negative bacterium or Positive regulation of foam cell differentiation; (xi) identifying the giatiranier acetate related drug substance or drug product as having a suboptimal activity if the set of differentially expressed genes indicates enrichment of one or more pathway of leukocyte migration (GO:005090G), cell chemotaxis (GO:0060326), leukocyte chemotaxis (GO-.0030595), phagocytosis (GO:0006909), positive regulation of phagocytosis (00:0050766), cell motion (GG:G006928), regulation of phagocytosis (00:0050764), behavior (GO:0007610), membrane invagination (GO:0010324), endocytosis (00:0006897), positive regulation of endocytosis (GO:0Q45807), phagocytosis, engulfment (GO:0006911), defense response to bacterium (GO.-0042742), membrane organization (GO:0016044), neutrophil chemotaxis (00:0030593), defense response to Gram-negative bacterium (00:0050829), regulation of endocytosis (GO:0030i00), leukocyte mediated immunity (00:0002443), cell migration (GO:0016477), regulation of vesicle- mediated transport (00:0060627), localization of cell (00:0051674), cell motility (00:0048870), collagen metabolic process (00:0032963), multicellular organismal macromolecule metabolic process (00:0044259), positive regulation of cellular component organization (GO:0051130), myeloid leukocyte activation (GO:0002274), positive regulation of foam cell differentiation (00:0010744), multicellular organisms! metabolic process (00:0044236), response to yeast (GO:0001878), regulation of type I hy ersensitivity (00:0001810), regulation of foam cell differentiation (GO:0010743), sugar binding (00:0005529), endopeptidase activity (00:0004175), serine hydrolase activity (GO:0017171), serine-type peptidase activity (00:0008236), serine-type endopeptidase activity (GO:0004252), cytokine activity (00:0005125), calcium ion binding (GO:0005509), immunoglobulin binding (GO:0019865), chemokine activity (GO:0008009), chemokine receptor binding (00:0042379), peptidase activity, acting on L-amino acid peptides (GQ;§07OO1 ), peptidase; activity (GO:QCK)8233) _v XgG bindin (GO:0 19864), protein complex bindin (GO;0O32403) or enzyme hiinbitor activity (GO:O 04§57); (xii) identifying the gktiramer acetate related drag substance or drag proditct as having a svibopomal activity if the set of : differentially expressed genes indicates enrichment of one or more pathway of glucose metabolic process (GO:0006006), glycolysis (GO:0O06i)96), hexose metabolic process <GO.:0O19318), monosaccharide metabolic · process (GO;00 5996), hexose catabolic process (GO:0O1932O), ghtcose catabolic process (GO:OOO6O07), monosaccharide catabolic process (GO:0046365), cellular carbohydrate catabolic process (GO:0044275), alcohol catabolic process (GO.O046164), carbohydrate catabolic process (GO:0016052), generation of precursor metabolites and energy (GO:0006G91), oxidoreduc tase activity, acting on single donors with incorporation of molecular oxygen, incorporation of two atoms of oxygen (GO:G016702), oxidoreductase activity, acting on single donors with incoiporation of molecular oxygen (GO: 0016701 ), cytokine activity (GO:0005125). carbohydrate kinase activity (GO:Q019200), Glycolysis / Gluconeogenesis (minuGOOiO), Pentose phosphate pathway (mmu00030) _t Fructose and niannose metabolism (mmu00051) or Galactose metabolism (mm«0O052); (xiii) identifying the g!atiramer acetate related drug substance or drag product as having a suboptimal acti vity if the set of differentially expressed genes indicates enrichment of one or more pathwa of RIG-I- ike receptor signaling pathway or Adenyryltransferase activity; or (xiv) determining the level of expression of at least one gene of Cxcl3, Clec4a3, Ugtla iO, Ugtla6b, Ugtla6a, Ugtla7c, Ugtla9, UgtiaS, Ugtlal , Ugtl a2. Clec4n, Tgibi, Nov, Ltf, Cci6, Mmpl2, Ms4a6d, Lpl, Mmp8, Cdl 77, Tfec, CiecJa. Mpo, Cc!9, Ccrl, Gpnmb, Msrl, Ms4a6d, Lyz2, C , Thbsl , Dab2, Arg2, Ccl6, Thbsl, Paldl , ClecSa, Cfp. Ifitmfi, LyzL 01frn4, Rnfl28, Ms4a3, Clec4d. S100a4, Ctsg, Prtn3, Ngp, F10, Hal. Ccl2, Lyzi , Thbsl , Anxal , Lgals3, S100a6, faml , C 13, Chil4, Sppl , Stfa211 , Ptgrl , Illf9, Cd68, Olfml, FcgrS, IHr2, Cregi, Dmxi2, Sirpa, 116, Elane, Csfir. Lcn2, CdSQOIb, AloxSap, II 1m, Slcl lal , Tyrobp, Cd300a, Pira2, Pira l , Pira7, Pira6, Pira5, Pira4. Gml0693, Lilra6, Pirb, Pira i l , Gml 4548. Ms4a7, Mgstl, Camp. Bstl , Slc40a L Cregl . Csfir, Cd36, Rnfl28, Mrcl, Msrl , Itgam, Serpinb2, Lihb4, Gp49a, Wfdcl 7, Anxa3, gsl S, Sk40a l, Bstl , C3arl. Mmp9, Histlh4e, Histlb4b, Histlh4a, Hisi4h4, Histlb4f, Histih4d, Histlh4k, Histlb4j, Histlb4i, Hist ib4b, Hisilhln, Histlh4m, Hist2h4, Dstn, Igsf6, Mitf, Csfir, Pf4, PygL Pdpn, S100a9, Fcgr2b, Idl, Emrl . Cd9, Cebpd, Pla2g7, Arg2, FIG. Gclm, S iOOaS, Ptplad2. Clec4a2, Clec4bl, Marcks, Csfira, Fcerlg, C2ec4a2, Ppic, Cd302, Gstml , McptS, CdSOOl Csf2rb, Bnip3, Ak4, Tpil , Pkxdi, Aldoc, Rgsl 1 , Ankrd37, Pikl, Pdkl , Cxcl9, Tbx21, Pgkl, EglnS, Ddit4, Pfkp, Pgm2, P4hal, Sk l6a3, Ubd, Slc2ai , 5330426P16Rik, Grhpr, Cxcll l, Rasa4. Ndrgl . Faml62a, Pkm, Eglnl, Aldoa, Gml0480. Bnip31. Galkl , Pgaml, Pgkl, Mif, Enolb. Enol, Pdxp. Ppplr3b, Stomll , Gziiia, Sed2. Ccng2, Higdl a, Eno2, Gfi i, PinL Ldlia, Eroll, 3000002C 10Rik, Hk2, Sk2a3, Chchd6, P4bai , Eroll, Gpil , Gimap7, Oasll, Gml 1110, Syce2, Ndrgl, Ptgs2, Trappc6a, Prelidl, Ifi2712a, Zbtb32, Tiim30a, Prelidl , Gapdh, Sdc3, BC062258, LOO 02643247, 231OQ22B05Rik, Ms4a4c, U12rb i , Mtfafdll, Sniagp, Nampt, Fasl, PafahlbS, Pre 2, Gngl 2, hfS, Kdm3a, Atf3, Tnfsf9. In some embodiments, the mammai is a rodent or human.

In some embodiments, the level of expression is determined in hematological cells. In. some embodiments, the level of expression is determined in splenoeytes. In some embodiments, the level of expression is. determine in monocytes. In some embodiments, the monocytes ar THP- 1.

In som embodiments, the human has previously been treated, with a glatiiamer acetate related drag subs.Ssn.ee. or drag .product, In some embodiments, the human is a naive human. In some embodiments, the human is a g!atiramoid naive human. In some embodiments, the human is afflicted with RRMS.

In some embodiments, the rodent is a mouse. In some embodiments, the mouse is a female (SJL X BALB/C) Fl mouse. In some embodiments, the mouse is about 8 to 12 weeks old. In some embodiments, the primary culture is a cuitee of spleen cells. In some embodiments, the primary culture is a culture of lymph node cells. In some embodiments, the primary culture of spleen cells is prepared about 3 days after immunization.

In some embodiments, the glatiiamer acetate related drag substance is a glatiramoid or wherein the glatiramer acetate related drug product comprises a glatiranioid. hi some embodiments, the glatiramer acetate related drag substance is a glatiramoid other than glatiramer acetate drag substance, or wherein the glatiramer acetate related drag product comprises a glatiramoid other than glatiramer acetate drug substance.

The present invention also provides a process for producing a drug product comprising a glatiramer acetate related drag substance, the improvement comprising the steps of:

(a) obtaining a batch of the glatiramer acetate related drag substance or drug product;

(b) contacting a first group of mammalian cells with an amount of the glatiramer acetate related drug substance or drag product of step (a);

(c) contacting a second group of mammalian cells of the same type with an amount of a reference standard:

(d) determining a set of genes differen tiall expressed by the first group after the contacting of step (b) rela tive to genes expressed by the second group after the contacting of step (c); and

(e) (i) discarding the batch of the glatiramer acetate related drug substance as unacceptable for inclusion in the drag product if the set of differentially expressed genes includes one or more genes of Mmpl2, Tgffai, Clec4n, Ciec4a3, Fcerlg, Fcgr2b, Fegr3, Csilr, Hlr2 or Tbxll; (ii) discarding the batch of the glatiramer acetate related drug substance as imacceptable for inclusion in the drug product if the set of differentially expressed genes includes one or more genes of Ahcyl2. Cln5. Cxcr6, Dhrs3. Egr2, Fabp5, Ptp4ai, Gpi 83, Lampl, coa4, Ptger4, Qk, Rragd, Sec24a, Sehil, Sestdl, Sk30a4, Soati or Strip! ; (iii) discarding the batch of the glatiramer acetate related drug substance as unacceptable for inclusion in the drag product if the set of differentially expressed genes includes one or more genes. of Fam212a, Gznia, Itpil or Zbi>32: (iy) discaaxfiiig- title batch of the glatiramer acetate related dru substance as unacceptable for inclusion in the drag product if the set of ^■ differentially expressed genes, includes one or more genes of Gzfab, Faml.9a3, fil3, Gzma, Eaf2, Carl or Sf7; (v) discarding the batch of the glatiramer acetate related drag substance as unacce table for inclusion in the drag product if the set of differentially expressed genes includes one or more genes of Crisp3, Apoe, Dapll, PdeSa, Scg5, SiSsiaS, 4930426D 5Rifc or Pgapl ; (vi) discarding the batch of the glathanier acetate related drag substance as unacceptable for inclusion in the dmg product if the set of differentially expressed genes indicates enrichment of one or more pathway of collagen metabolic process (GO:0032953), defense response to bacterium (00:0042742), defense response to Gram-negative bacterium (00:0050829), endocytosis (GO:0006897), leukocyte mediated immunity (00:0002443), membrane invagination (GO: 0010324), membrane organization (GO:0016044), multicellular organismal macrornolecule metabolic process (GO:0044259), multicellular organismal metabolic process (00:0044236), myeloid leukocyte activation (GO:0002274), neutrophil chemotaxis (00:0030593), phagocytosis, engulfnieni (00:0006911), positive regulation of cellular component organization (00:0051130), positive regulation of endocytosis (00:0045807), positive regulation of foam cell differentiation (00:0010744), positive regulation of phagocytosis (00:0050766), regulation of endocytosis (00:0030100), regulation of foam cell differentiation (00:0010743), regulation of phagocytosis (00:0050764), regulation of ty e I hypersensitivity (GO:0001810), regulation of vesicle- mediated transport (GO:0060627) or response to yeast (GO:0001878); (vii) discarding the batc of the giarirarner acetate rela ted drag substance as unacceptable for inclusion in the drug product if the set of differentially expressed genes indicates enrichment of one or more pathway of nucleotide binding (GO:0000166), double-stranded RNA binding (00:0003725), NAD+ ADP-ribosylttansferase activity (00:0003950), transferase activity, transferring pentosyl .groups (GO:0016763), transmission of nerve impulse (00:0019226), regulation of membrane potential (00:0042391), cytokine activity (00:0005125), ion homeostasis (00:0050801) or adenylyltransferase activity (00:0070566); (viii) discarding the batch of the glatiramer acetate related drug substance as unacceptable for inclusion in the drug product if the set of differentially expressed genes indica te no substantial enrichment of one or more pathways of Cytokine-cytokine receptor interaction (mmu0406G), Jak-STAT signaling pathway (mmu04630). Sterol metabolic process (00:0016125), Sterol biosynthetic process (00:0016126), Hematopoietic cell lineage (mmu04640). Myeloid cell apoptosis (GO: 0033028), Regulation of survival gene product expression (GO:0045884), Regulation of leukocyte proliferation (GO:0070663), Positive regulation of survival gene product expression (00:0045885), Hematopoietic cell lineage (mmu04640) or Metabolism of xenobioti.cs by cytochrome P450 (mmu00980); (ix) discarding the batch of the glatiramer acetate related drug substance as unacceptable for inclusion in the drag product if the set of differentially expressed genes indicate no substantial enrichment of one or more pathway ofmtrinsic to membrane receptor activity. Immune response. Leukocyte migration, Chemokine receptor binding. Cytokine-eyt.okine receptor interaction, Cytokine; activity, lak-STAT signaling pathway. Sterol biosy¾theiie process. Steroid biosytithetic process. Ceil surface, Cytokine binding, Cholesterol metabolic process, Clieniokine activity, Negative regulation of hormon secretion, isoprenoi biosynmetie process or Positive regulation of pepttdyl-serine phosphorylation:: ( ) discarding the hatc ofthe glatiramer acetate related dmg substance as unacceptable for inclusion in the dmg product if the set of differentially expressed genes indicates enrichment of one or more pathway of Glucose metabolic process. Glycolysis, Ghiconeogenesis:, Hexose cafabolic process. Glucose catabolic process. Monosaccharide catabolic process, Hexose metabolic process. Monosaccharide metabolic process. Cellular carbohydrate catabolic process. Alcohol cataboli process. Carbohydrate catabolic process. Carbohydrate binding, Generation of precursor metabolites and energy. Phagocytosis, Leukocyte migi¾iion. Leukocyte clieniotaxis. Positive regulation of phagocytosis. Pentose phosphate pathway, Galactose metabolism. Carbohydrate kinase activity. Myeloid leukocyte activation, Chemokine receptor binding. Regulation of type I hypersensitivity. Defense response to Gram-negative bacteiium or Positive regulation of foam cell differentiation; (xi) discarding the batch of the glatiramer acetate related drag substance as unacceptable for inclusion in me drug product if the set of differentially expressed genes indicates enrichment of one or more pathway of leukocyte migration (GO: 0050900), cell chemotaxis (00:0060326). leukocyte chemotaxis (00:0030595), phagocytosis (00:0006909), positive regulation of phagocytosis (00 0050766), cell motion (00:0006928), regulation of phagocytosis (00:0050764), behavior (GO: 0007610), membrane invagination (GO:0010324), endocytosis (GO:0006897), positive regulation of endocytosis (GO.O045807), phagocytosis, eiigivlfmeiit (GO:0006911), defense response to bacterium (00:0042742). membrane organization (00:0016044), neutrophil chemotaxis (GO:0030593), defense response to Gram-negative bacterium (00:0050829), regulation of endocytosis (00:0030100), leukocyte mediated immunity (00:0002443), cell migration (GOrOO 16477), regulation of vesicle-mediated transpoit (GO:0060627), localization of cell (00:0051674), cell motility (GO:0048870), collagen metabolic process (GO:0032963), multicellular orgamsinal macromolecule metabolic process (00:0044259), positive regulation of cellular component organization (00:0051130), myeloid leukocyte activation (GO:0002274), positive regulation of foam cell differentiation (GO:0010744), multicellular organismal metaboli process (GO:0044236) ₅ response to yeast (00:0001878), regulation of type I hypersensitivity (00:0001810), regulation of foam cell differentiation (00:0010743), sugar binding (00:0005529), endopeptidase activity (GO:0004175). serine hydrolase activity (GO:0017i7I), serine-type peptidase activity (GO.-0008236), serine-type endopeptidase activity (00:0004252), cytokine activity (00:0005125), calcium son binding (00:0005509), immunoglobulin binding (00:0019865), clieniokine activity (00:0008009), chemokine receptor binding (00:0042379), peptidase activity, acting on L-amino acid peptides (GO:0070011), peptidase activity (GO:0008233). IgG binding (GO:0019S64), protein complex binding (00:0032403) or enzyme inhibitor activity (00:0004857): (xii) discarding the batch of the glatiramer acetate related dmg substance as unacceptable for inclusion in the dmg product if the set of differentially expressed genes indicates enrichment of one or more path wa of glucose metabolic process (GO:0006OO6), glycolysis (GO: 9006096), hexose metabolic process: (GQ:G0193I8), monosaccharide metabolic process (00:0005996), hexose cafabobc process (00:0019320), ghjeose eatabojic process (00:0006007); monosaccharide catabolic process (00:0046365), cellular carbohydrate catabolic process (G©:0O44275), alcohol catabolic process (00:0046164), carbohydrate catabolic process (00:0016052) generation of precrasor metabolites and energy (00:0006091 oxidoreducta.se activity, acting on single donors with ^corporation of molecular oxygen, incorporation of two atoms of oxygen (00:0016702), oxidoreductase activity, acting on single donors with incorporation of molecular oxygen (00:0016701), cytokine activity (00:0005125), carbohydrate kinase activity (00:0019200), Glycolysis / Gliiconeo enesis (mmuOOOlO), Pentose phosphate pathway (iimiuOOGSO), Fructose and mannose metabolism (inrnuOOOS i) or Galactose metabolism (nirnuOQ052); (xiii) discarding the batch of die glatiramer acetate related drag substance as unacceptable for inclusion in the drug product if die set of differentially expressed genes indicates enrichment of one or more pathway of RIG-I-like receptor signaling pathway or Adenylyltransf erase activity; or (xiv) determining the level of expression of at least one gene of Cxc , Ciec4a3, UgtlalO, Ugtiaoh, Ugt la6a, UgtlaTc, Ugtla9. UgtlaS, Ugtlal . Ugtla2, Clec4n, TgfbL Nov, Ltf Ccl6, Mmpl2, Ms4a6d, LpL Mmp8, Cdl 77, Tfec, Clec7a, Mpo, Ccl9, Ccrl , Gpnmb, Msrl, Ms4a6d. Lyz2, Ctsl. Thbsl, Dab2, Arg2, Ccl6, Thbsl, Paldl, ClecSa, Cfp. Ifitm6. Lyzl, 01fm4, Rnfl28, Ms4a3, Clec4d, Si00a4, Ctsg, Prtn3, Ngp, F10, Hal, Ccl2, Lyzl, Thbsl, Anxal, Lgals3, S100a6, Nfami, C 3, C 4, Sppi, Stfa211 _t Ptgrl, Ill 9, Cd68, Olfinl, Fegr3. Iiii2, Cregl. Dmxl2, Sirpa, 116, Elane, Csflr, Lcn2. Cd3Q01b AloxSap, lilm, Slcl lal, Tyrobp, CdSOOa, Pira2, Piral. Pira7, Pira6, PiraS, Pira4, Gml0693, Liira6, Pirb, Pirall, Gml4548, Ms4a7, Mgsfi, Camp, Bstl, Slc40al, Cregl. Csflr, Cd36, Rnfl28, Mrcl, Msrl, Itgam, Serpinb2 Lilrb4, Gp49a, Wfdcl7, Anxa3 _t Rgsl8, Sk40ai, Bstl, C3arl _t Mmp9. Histlh4c, Histili4b, Histlh4a, Hist4h4, Histlh4£ Histlh4d, Histlh4k, Histih4j, Histlh4i. Histlh4h, Histih4ii. Histlh4m. Hist:2h4, Dst , Igsf6, Miff, Csi3r, Pf4. Pygl, Pdpn. S100a9, Fcgr2b, ML Emrl, Cd9, Cebpd, Pla2g7, Arg2, FIO, Gclm, S100a8, Ptplad2, Clec4a2, Clec4bl, Marcks, Csf2ra, Fcerlg, Clec4a2, Ppic, Cd302, Gstml, McptS, Cd3001£ Csf2rb, Bnip3, Ak4, Tpil, Plcxdl, Akloc, Rgsl 1, Anfcrd37, Pffcl, Pdki, Cxd9, Tbx21, Pgki, Eghi3, Ddit4, Pfkp, Pgm2, P4hal, Slcl 6a3, Ubd Slc2al , 5330426P16Rik. Grhpr, Cxcil 1, Rasa4, Ndrgl , Faml62a, Pkm, Eghil, Aldoa, Giiil0480, BnipSL Galfcl, Pgaml , Pgkl, Mi£ Enolb, Enol, Pdxp, Ppplr3b, Stonill, Gzma, Scd2, Ccng2, Higdla, Eno2, Gfil, Pail, Ldlia, ErolL 3OO0OO2CiORik, Hk2, Sk2a3, Chchd6. P4hai, Erol , Gpil, Giniap7, Oasil, Gml 1110, Syce2, Ndrgl, Ptgs2, Trappc6a, Prelidl, Ifi2712a. Zbtb32, TrimSOa, Prelidl, Gapdh, Sdc3, BC062258, LOC 102643247, 2310022B05Rik, Ms4a4c. I112rbl, Mdifdll, Smagp, Nampt, Fasl, Paiahlb3, Prelid2 Gngl2, IrfS, Kdni3a, Atf3, Tnfsf9.

The present invention also provides a process for producing a drug product comprising a glatiramer acetate related drug substance, the improvement comprising the steps of: (a obtaining a batch of the glatirame acetate related drug substance or drag, product:

(b) contacting a. first group of mammalian cells with an amount of the glatiramer acetate related drug substance or drug product of step (a) :

(c) contacting a second group of mammalian cells of the same type with an amount of a. reference standard:

(d) detemiiniiig a set of genes differed ialJ expressed by the first group after the contactiBg of step (b) relative to genes expressed by the second group after the contacting of step (c); and

(e) discarding the batch of the glatiiamer acetate related drug substance as unacceptable for inclusion in the drag product if the set of differentially expressed genes indicate no substantial enrichment of one or more pathways set forth in Table 3 or Table 10; or discarding the batch of the glatii amer acetate related drag substance as unacceptable for inclusion in the drag product if the set of differentially expressed genes indicates enrichment of one or more pathways set forth in Table 5, Table 7. Table 8, Table 14 or Table 15.

The present invention also provides a process for releasing a ding product comprising a glatiiamer acetate related drug substance, the improvement comprising the steps of:

(a) obtaining a batch of the glatiramer acetate related drug substance or drug product;

(b) contacting a first gr oup of mammalian cells with an amount of the glatiramer acetate related drug substance or drug product of step (a);

(c) contacting a second group of mammalian cells of the same type with an amount of a reference standard;

(d) detennining a set of genes differentially expressed by the first group after the contacting of step (b) relative to genes expressed by the second group after the contacting of step (c); and

(e) (i) discarding the batch of the glatiramer acetate related drag substance as unacceptable for inclusion in the drug product if the set of differentially expressed genes includes one or more genes of Mmpl2, Tgfbi, Clec4n. Clec4a3, Fceri g _; Fcgr2b, Fcgr3, Csf3r, Iilr2 or Tbx21 ; (ii) discarding the batch of the glatiramer acetate related drug substance as unacceptable for inclusion in the drag product if the set of differentially expressed genes includes one or more genes of Ahcyl2, Cln5, Cxcr6, Dhrs3, Egr2, FabpS, Ptp4al, Gprl83, Lam l, coa4, Ptger4, Qk, Riagd, Sec24a, SehlL SestdL Slc30a4, Soatl or Strip2; ( i) discarding the batch of the glatiramer acetate related drag substance as unacceptable for inclusion in the drug product if the set of differentially expressed genes includes one or more genes of Fam212a _» Gzma, Itprl or Zbtb32; (iv) discarding the batch of the glatiramer acetate related drag substance as unacceptable for inclusion in the drug product if the set of differentially expressed genes includes one or more genes of Gzmb, Faml9a3 _t Nfil3, Gzma, Eaf2, Carl or St ^' 7; (v) discarding the batch ^' of the g iramer acetate related drug subs tance as unacceptable- for inclusion in the .drug produc if the set of differentially expressed genes includes one or more genes of Crisps, Ap e, Dapll, PdeSa, Scg5, St8sia6, 4930426D05Rik or Pgapl ; (vi) discarding the batch of the glatlrainer acetate related drug subsiaiice as unacceptable for fficlnsion in the drug -product if the set of differentially expressed genes indicates enrichment of one or more pathway of collage metabolic process (000032963), defense response to bacterium (00:0042742-), defease response to Grain-negative bacterium (00:0050829). endocytosis {(30:0006897), leukocyte mediated imiifflity GQ;fl002443)., membrane invagination (00:0010324), membrane organization (GO:0O16044), multicellular organismal macroinolecule metabolic process (GO:0044259), multicellular organismal metabolic process (00:0044236), myeloid leukocyte activation (00:0002274), neutrophil cheniotaxis (00:0030593), phagocytosis, engulftnent (GO:0006911), positive regulation of cellular component organization (GO:0051130), positive regulation of endocytosis (00:0045807), positive regulation of foam cell differentiation (GO:0010744), positive regulation of phagocytosis (00:0050766), regulation of endocytosis (GO:0030i00), regulation of foam cell differentiation (GO: 0010743), regulation of phagocytosis (00:0050764), regiilaiion of type I hypersensitivity (00:0001810), regiilaiion of vesicle- mediated transport (00:0060627) or response to yeast (00:0001878); (vii) discarding the batch of the glatiramer acetate related drug substance as unacceptable for inclusion in the drag product if the set of differentially expressed genes indicates enrichinent of one or more pathway of nucleotide binding (00:0000166), double-stranded R A binding (00:0003725), NAD+ ADP-ribosyiiransfera.se activity (GO:0003950) ₅ transferase activity, transferring pentosyl groups (GO :0016763), transmission of nerve impulse ((30:001 226), regulation of membrane potential (00:0042391) , cytokine activity (00:0005125), ion homeostasis (GO:0050801) or adenylyltransferase activity (GO:0070566); (viii) discarding the batch of the glatiramer aceiate related drug substance as unacceptable for inclusion in the drug product if the set of differentially expressed genes indicate no substantial enrichment of one or more pathways of Cytokine-cytokine receptor interaction (mniuG4060), Jak-STAT signaling pathway (mmu04630). Sterol metabolic process (00:0016125), Sterol biosynthetie process (00:0016126), Hematopoietic cell lineage (mniu04640). Myeloid cell apoptosis (GO: 0033028), Regulation of survival gene product expression (GO:0045884), Regulation of leukocyte proliferation (00:0070663), Positive regulation of survival gene product expression (00:0045885), Hematopoietic cell lineage (mmu04640) or Metabolism of xenobiotics by cytochrome P450 (mmu00980); (ix) discardin the batch of the glath amer acetate related drag substance as unacceptable for inclusion in the drag product if the set of differentially expressed genes indicate no substantial enrichment of one or more pathway of mtrinsic to membrane receptor activity. Immune response. Leukocyte migration, Cheinokine receptor binding. Cytokine-cytokine receptor interaction, Cytokine activity, Jak-STAT signaling pathway. Sterol biosynthetie process. Steroid biosynthetie process. Ceil surface, Cytokine binding. Cholesterol metabolic process, Cheinokine activity'. Negative regulation of hormone secretion, Isoprenoid biosynthetie process or Positive regulation of peptidyl-serine phosphorylation: (x) discarding die batch of the giatiramer acetate related drug substance as unacceptable for melusion in the drag product if the set of difiereatially expressed genes · indicates enrichment of one or more pathway of Glucose metabolic process. Glycolysis, Gliiconeogenesis, Hexose eatabolic process. Glucose caiabolie process. Monosaccharide eatabolic process, Hexose metabolic process. Monosaccharide metabolic process. Cellular carbohydrate cataboMe process. Alcohol eatabolic process, Carbohydrate eatabolic process. Carbohydrate binding, Generation of precursor metabolites and energy. Phagocytosis, Leukocyte ^' migration. Leukocyte chemotaxis. Positive regulation of phagocytosis. Pentose phosphate pathway. Galactose metabolism. Carbohydrate kinase activity. Myeloid leukocyte activation, Cheniokine receptor binding. Regulation of type I hypersensitivity. Defense response to Gram-negative bacterium or Positive regulation of loam ceil differentiation; (xi) discarding the batch of the giatiramer acetate related drag substance as unacceptable for inclusion in the drag product if the set of differentially expressed genes indicates enrichment of one or more pathway of leukocyte migration (GO:Q050900), cell chemotaxis (00:0060326), leukocyte chemotaxis (GO:0030595), phagocytosis (00:0006909), positive regulation of phagocytosis (GO: 0050766), cell motion (00:0006928), regulation of phagocytosis (00:0050764), behavior (00:0007610), membrane invagination (00:0010324), endocytosis (GO:0006897), positive regulation of endocytosis (00:0045807), phagocytosis, engulfment (00:0006911), defense response to bacterium (00:0042742), membrane organization (GO :0016044), neutrophil chemotaxis (GO:0030593), defense response to Gram-negative bacterium (00:0050829), regulation of endocytosis (GO:0030100), leukocyte mediated immunity (00:0002443), cell migration (GO:0016477), regulation of vesicle-mediated transport (00:0060627), localization of cell (00:0051674), cell motility (00:0048870), collagen metabolic process (00:0032963), multicellular organismal macromoieciile metabolic process (00:0044259), positive regulation of cellular component organization (GO:0051130), myeloid leukocyte activation (00:0002274), positive regulation of foam cell differentiation (00:0010744), multicellular organismal metabolic process (GO.-0044236), response to yeast (GO:0001878) ₅ regulation of type I h^ersensitivity (00:0001810), regulation of foam ceil differentiation (00:0010743), sugar binding (00:0005529), endopeptidase activity (00:0004175), serine hydrolase activity (00:0017171), serme-fype peptidase activity (GO:0008236), serine-type endopeptidase activity (00:0004252), cytokine activity (00:0005125), calcium ion binding (00:0005509), immunoglobulin binding (GO:0019865), chemokme activity (00:0008009), chemokme receptor binding (00:0042379), peptidase activity, acting on L-amino acid peptides (00:0070011), peptidase activity (00:0008233), IgO binding (00:0019864), protein complex binding (00:0032403) or enzyme inhibitor activity (00:0004857): (xii) discarding the batch of the giatiramer acetate related drug substance as unacceptable for inclusion in the drug product if the set of differentially expressed genes indicates enrichment of one or more pathway of glucose metabolic process (00:0006006), glycolysis (GO: 0006096), hexose meiabolic process (00:0019318), monosaccharide metabolic process (00:0005996), hexose eatabolic process (00:0019320), glucose eatabolic process (00:0006007), monosaccharide eatabolic process (00:0046365), cellular carbohydrate eatabohe process (GO;0044275), alcohol catabolic process (GO:00461.64), carbohydrat catabolic process (GO:0016O52), generation of precursor metabolites and energy (GO:00Q¾091), oxi dorediictase activity, acting on single donors with iBcoiporaflon of molecular oxygen, incorporation of twi? atoms of oxygen (GO;0016702), oxidoredoctase activity, actin on single donors: with. iiicor oralioH of molecular oxygen (GO .0016701), cytokine activity (GO ^• .0005125), carbohydrate kina.se activity (GO; 0019200), Glycolysis Glucoheogenesis (mnajOOOlQ), Pentose phosphate pathway (miiruOOO30), Fructose and niamiose metabolism (nraiuOOOS i) or Galactose metabolism (mniii00052); (xiii) discarding the batch of the glatirarner acetate related drag substance as unacceptable for inclusion in the drug product if the set of differentially expressed genes indicates enrichment of one or more pathway of RIG-I-like receptor signaling pathway or Adeny yltransferase activity; or (xiv) determining the level of expression of at least one gene of CxcI3, Clec4a3, UgtlaiG, Ugtla6b, Ugtla6a, Ugt iaTc, Ugtla9, UgtlaS. Ugtlal, Ugtla2. Ciee4n, Tgfbi, Nov, Lrf, Ccl6, Mmpl2, Ms4a6d, Lpl, Mmp8, Cdi77 Tfec. Clec7a, Mpo, Ccl9, CcrL Gpnmb. Msrl, Ms4a.6d, Lyz2 ₅ Ctsl, Thbsl. Dab2, Arg2. Ccl6, Thbsl, Paldl, Clec5a, Cfp, Ifitai6, Lyzl, 01fm4, Rafi28, Ms4a3, Clee4d, S100a4, Ctsg, Prt 3, gp, F10, Hal, Ccl2, Lyzl, Thbsl, Airxal, Lgals3, Si00a6, Nfaml, Chil3, Chii4, Sppl, Stfa2il, Ptgrl, 111©, Cd68. Olfml, Fcgr3, Illr2, Cregl, Dmxl2, Sirpa, 116. Elane. Csflr, Lcn2, CdSOGlb, AioxSap. 321m, Slcl lal, Tyrobp, Cd300a ₅ Pira2, Piral, Prra7, Pira6, PiraS, Pira4, Gml0693, Lilra6, Pirb, Piral l, Gml4S4§, Ms4a7, Mgstl, Camp, Bstl, Slc40aL Cregl, Csflr, Cd36, Rnfl2S, Miel, Msrl, Itgam, Seipinb2, Lihb4, Gp49a, Wfdcl7, AnxaS, Rgsi S, Slc40al, Bstl, CSarl, Mrnp9, Histlh4c, Histlh4b, Histlh4a, Hist4h4, Histlh4f, Histlh4d, Histlh4k, Histlh4j, Histl!i4i, Histlh4h, His†lh4n, Histlh4m, Hist2h4, Dsfn, IgsfS, Mitf, Csf3r, Pf4, F gL Pdpn, S100a9, Fcgr2b, Idl, Emrl, Cd9, Cebpd. Pla2g7, Arg2, F10, Gclm, S100a8, Ptpiad2, Clec4a2, Clec4bl, Marcks, Csf2ra, Fcerlg, Clec4a2, Ppic, Cd302, Gstml, McptS. Cd3001f, Csf2rb, BnipS, Ak4, Tpii, Pkxdl. Aldoc, Rgsl l, Ankrd37, Pfld, Pdkl, Cxc , Tbx21, Pgkl,Egla3, Ddit Pfkp, Pgm2, P4hal, Slcl6a3, Ubd, Slc2aL 5330426P16Rik, Grhpr, Cxcll l. Rasa4, Ndrgl, Fami62a, Pkm, Eglnl, Aldoa, Gml0480, Bnip31, Galkl , Pgaml, Pgkl, Mif, Enolb, E ol, Pdxp, Ppplr3b, Stomll, Gzma, Scd2, Ccng2, Higdla, Eno2, Gill, Pml, Lclha, Eroll, 3000002 10Rik, Hk2, Slc2a3, Chchd6, P4hal. Eroll, Gpil, Giinap7, Oasll, GmllllO, Syce2, Ndrgl, Pigs2, Tmppc6a, Prelidl, Ifs2712a, Zbtb32, TrimSOa. Prelidl, Gapdh, Sdc3, BC06225S, LOCI 02643247, 2310022B05Rik, Ms4a4c, H12rbl, MthfdlL Smagp, Nampt. Fasl, PafahlbS, Prelid2, Gngl2, M8, Kdni3a, Atf3, Tnfsf9.

The present invention also provides a process for releasing a drug product comprising a glatiramer acetate related drag substance, the improvement comprising the steps of:

(a) obtaining a batch of the glatirarner aceta te related drug substance or drag product;

(b) contacting a first group of mammalian ceils with an amount of the glatirarne acetate related drug substance or drug product of step (a); (c ) contacting a second group of mammalian cells of the same type with an amount of a reference standard;

(d) determining a set of genes differentially expressed by the first group after th contacting of s tep (b) relative to genes expressed by the second group after ^' the contacting of step (e); and

(e) discarding the bateh of the glatiranier acetate related drag substance as imaeeeptable for inclusion m the drag, product if the set of differentially expressed genes mdieate no substantial enrichment of one or more pathways set forth in Table 3 or Table 10; or discarding the batch of the glatiramer acetate related drag substance as unacceptable for inclusion in the drag prockct if the set of differentially expressed genes indicates enrichment of one or more pathways set forth in Table 5„ Table 7. Table 8, Table 14 or Table 15.

The present invention also provides a process for identifying suboptimal activity of a glatiramer acetate related ding substance or ding product comprising the steps of :

(a) obtaining a batch of the glatiramer acetate related drug substance or drag product;

(b) contacting a first group of mammalian cells with an amount of the glatiramer acetate related drug substance or drug product of step (a);

(c) contacting a second group of mammalian ceils of the same type with an amount of a reference standard;

(d) determining a set of genes differentially expressed by the first group after the contacting of step (b) relative to genes expressed by the second group after the contac ting of step (c); and

(e) (i) identifying the glatiramer acetate related drug substance or drug product as having a suboptimal activity if the set of differentially expressed genes includes one or more genes of Mmpl2, Tgfbi, Cleoto, Ciec4a3, Fcerlg, Fcgr2b, FcgrS, Csf3r, Illr2 or Tbx21; (ii) identifying the glatiramer acetate related drug substance or drug product as having a suboptimal activity if the set of differentially expressed genes includes one or more genes of Ahcyl2, ClnS, Cxcr6, Dhrs3, Egr2. FabpS, Ptp4aL Gprl83, Lampl, Ncoa4, Ptger4, Qk, Rragd, Sec24a. Seal I, Sestdl, Slc30a4, Soat.1 or Strip2: (iii) identifying the glatiramer acetate related drag substance or drug product as having a suboptimal activity if the set of differentially expressed genes includes one or more genes of Fam212a, Gznia, Itprl or Zbtb32; (iv) identifying the glatiramer acetate related drug substance or drug product as having a suboptimal activity if the set of differentially expressed genes includes one or more genes of Gzmb, Faml9a3 Nfil3, Gznia, EafS, Carl or St7; (v) identifyin the glatiramer acetate related drug substance or drug product as having a suboptimal activity if the set of differentially expressed genes includes one or more genes of Cris S, Apoe, Dapll, Pde5a, Scg5, St8sia6, 4930426D05Rik or Pgap 1 ; (vi) identifying the glatiramer acetate related drug substance or dmg product as having a suboptimal activity if the set of differentially expressed genes mdicates enrichment of one or more pathway of collagen metabolic process (G :0032963), defense response io bacterium (00:0042742), defense response to Gram- aegative bacterium (G0:00S0829), endocytosis (GO:Q0Q6897), leukocyte mediated, immunity (00:0002443), membrane invagination (OO:0010324), membrane organization (GO 0 16044), multicellular organisma] niacromoleciile metabolic proces (00:0044259), imdticeluilar orgaaisiiiai metabolic process (00:0044236), myeloid leukocyte activation (00:0002274), neutrophil chemotaxis (00:0030593), phagocytosis, eugulfmeiat (G ;00O69i 1), positive regulation of cellular component organization (GO;0051130), positive' regulation of endocytosis (00:0045807), positive regulation of foam cell differentiation (GO:0010744), positive regulation of phagocytosis (00:0050766), regulation of endocytosis (GO:00301Q0), regulation of foam cell differentiation (00:0010743), regulation of phagocytosis (00:0050764), regulation of type I hypersensitivity (00:0001810), regulation of vesicle- mediated transport (GO:0060627) or response to yeast (GO:OO0187S); (vii) identifying the glatiramer acetate related drug substance or drag product as having a suboptimal activity if the set of differentially expressed genes indicates enrichment of one or more pathway of nucleotide binding (00:0000166). double-stranded RNA binding (00:0003725), NAEH- ADP-ribosyitransferase activity (00:0003950), transferase activity, transferring pentosyl groups (GO:0016763), transmission of nerve impulse (00:0019226), regulatio of membrane potential (GO:0042391) , cytokine activity (00:0005125), ion homeostasis (GO:0050801) or adenylyl transferase activity (00:0070566); (viii) identifying the glatiramer acetate related drag substance or drag product as having a suboptimal activity if the set of differentially expressed genes indicate no substantial enrichment of one or more pathways of Cytokine- cytokine receptor interaction (mmu04060), Jak-STAT signaling pathway (mmu04630), Sterol metabolic process (00:001 125), Sterol biosynthetic process (00:0016126), Hematopoietic cell lineage (mmu04640). Myeloid cell apoptosis (GO: 0033028), Regulation of survival gene product expression (GO: 0045884), Regulation of leukocyte proliferation (GO:0070663), Positive regulation of survival gene product expression (GO:0045885), Hematopoietic cell lineage (mmu04640) or Metabolism of xenobiotics by cytochrome P450 (mmu00980); (ix) ideffiifying the glatiramer acetate related drug substance or drag product as having a suboptimai activity- if the set of differentially expressed genes indicate no substantial enrichment of one or more pathway of Intrinsic to membrane receptor activity. Immune response, Leukocyte migration, Cheinokine receptor binding. Cytokine- cytokine receptor interaction. Cytokine activity, Jak-STAT signaling pathway. Sterol biosyntheti process. Steroid biosynthetic process. Ceil surface. Cytokine binding. Cholesterol metabolic process, Chemokine activity. Negative regulation of hormone secretion, Isoprenoid biosynthetic process or Positive regulation of peptidyl-serine phosphorylation; (x) identifying the glatiramer acetate related drag substance or drug product as having a suboptimal activity if the set of differentially expressed genes indicates enrichment of one or more pathway of Glucose metabolic process. Glycolysis, Gluconeogenesis, Hexose catabolic process. Glucose catabolic process. Monosaccharide catabolic process, Hexose metabolic process. Monosaccharide metabolic process. Cellular carbohydrate catabolic process. Alcohol catabolic process. Carbohydrate catabolic process. Carbohydrate binding. Generation of precursor ^' metabolites ami energy. Phagocytosis, . Leukocyte, migration, Leukocyte ehemotaxis. Positive regulation of phagocytosis. Pentose phosphate pathway, Galactose metabolism. Carbohydrat kinase activity. Myeloid, leukocyte activation, Chemokine receptor binding, Regulation of type I ¾yfei¾eQsitivity ₅ Defense response to Gram-negative haeteiiiist or Positiv ^■ regulation of foam cell, differentiation:: (xi) identifying the g!atiramer acetate related drug substance or dmg product as having: a suhoptimal ^' activity if the set of differentially expressed genes indicates enrichment of one or more pathway ^' of leukocyte .migration (GO:O0509OO), ceil ehemotaxis (GO;0O6O326), leukocyte cheniotaxis (GO.-0030595), phagocytosis (00:0006909), positive reguiation of phagocytosis (GO:0Q50766), cell motion (GO:0006928), regulation of phagocytosis (00:0050764), behavior (00:0007610), membrane invagination (GO:0010324), endocytosis (00:0006897), positive reguiation of endocytosis (00:0045807), phagocytosis, engulfinent (00:0006911), defense response to bacterium (GO-.0042742), membrane organization (GO:0016044), neutrophil chernotaxis (GO:0030593), defense response to Gram-negative bacterium (GO:0050829), regulation of endocytosis (00:0030100). leukocyte mediated immunity (00:0002443), cell migration (GO:0016477), regulation of vesicle- mediated transport (GO:0060627). localization of ceil (GO:0051674), cell motility (00:0048870), collagen metabolic process (00:0032963). muiticellular organismal macromolecule metabolic process (GO:0Q44259), positive regulation of cellular component organization (00:0051130), myeloid leukocyte activation (00:0002274), positive regulation of foam cell differentiation (GO:0010744), multicellular organismal metabolic process (GO:0044236), response to yeast (GO:0001878), reguiation of type I hypersensitivity (00:0001810). regulation of foam cell differentiation (00:0010743). sugar binding (00:0005529), endopeptidase activity (GO:0004175), serine hydrolase activity (00:0017171), serine-type peptidase activity (GO.0008236), serine-type endopeptidase activity (00:0004252), cytokine activity (GO:0005125), calcium ion binding (GO:0005509), immunoglobulin binding (GO.0019865), chemokine activity (GO:00080 9), chemokine receptor binding (GO:0042379), peptidase activity, acting on L-amino acid peptides (GO:0070011), peptidase activity (GO:0008233), IgG binding (GO:0019S64), protein complex binding (00:0032403) or enzyme inhibitor activity' (00:0004857); (xii) identifying the glatiramer acetate related drag substance or drug product as having a suboptimal activity if the set of differentially expressed genes indicates enrichment of one or more pathway of glucose metabolic process (00:0006006), glycolysis (GO:0006096), hexose metabolic process (GO:0019318), monosaccharide metabolic process (GO:0005996), hexose catabolic process (GO:0019320), glucose catabolic process (GO:0006007), monosaccharide catabolic process (GO.O04636S), cellular carbohydrate catabolic process (GO:0044275), alcohol catabolic process (GO:0Q461 4), carbohydrate catabolic process (GO:0Q16052), genera tion of precm sor metabolites and energy (GO:0006091), oxidoreductase activity, acting on single donors with incorporation of molecular oxygen, incorporation of two atoms of oxygen (GO:0016702) , oxidoreductase activity, acting on single donors with incorporation of molecular oxygen (GO: 0016701), cytokine activity' (GO:0005125), carbohydrate kinase activity (00:0019200), Glycolysis / Gluconeogenesis (mmuOOOlO), Pentose phos hate pathway (nmmOOOSQ), Fructose and mannose ^■ /metabolism (mm«.QD05I) or Galactose metabolism (iaHiu0OO52) (xiii identifying the gktiraaier acetate related drug substance or drug product as having a sufaoptirnal activity- if the set of differentially expressed genes mdicaies enrichraenf of one or more pathway of ¾iG -l¾e receptor signaling pathway or Adenylyiiransferas activity; ^' or (xiv) determining the level of expression of at least, one gene ofCxelS, Oec4a3, UgtlalO, Ugt.la6b, Ugtla6a Ugtla7c, Ugtla9 _? UgtlaS, Ugt ^' lal, Ugtl ^' a2, Clec4 Tgfbt OY, Lri Cc¾ Mmp ^' 12, Ms4aSd XpL Μ ι &, Cdi 77, Tfee, Clec7a _? Mpo, Ccl¾ Ccr L Opamb, Marl , Ms4a6d _f Lyz2, Cisl, Thbsl _f Dab2 Arg2, Ccl6, Thbsi, Paldl, ClecSa, Cft , Ifitm6. Lyzl, 01fei4. Rnfl28, Ms4a3, Clec4d, S100a4, Ctsg, Prtn3, Ngp, FIG. Hal Cell, Lyzl , Thbsi, Anxal, Lgals3, S100a6, Ni¾ml, C!iilS, ChiR Sppl , Stfa21L P grl, &, Cd68, Gffinl, Fcgr3, Ilii'2, Cregl, DmxI2, Sirpa, 116, Elane, Csflr, Lena, Cd3001b, AloxSap, Illm, Slcl lal, Tyrobp, Cd300¾ Pira2 Piral, Pira7, Pira6, PiraS. Pira4, Gnil0693, Lilra6, Pirb, Piral 1, Gml4548, Ms4a7, Mgstl , Camp, Bstl, Slc40al, Cregl, Csfir. Cd36, Rnfi28. Mrel , MsrL Itgam, Serpmb2, Lilrb4, Gp49a, Wfdcl?, Anxa3, Rgsl8, Slc40a l, Bstl, C3arl, Mmp9, Histlh4e, Histlh4L\ Histlh4a, Hist4h4, Hisiiii4f. Histih4d, Histlh4k, HistlMj, Histih4i, Histlh4h, Histlh4n, Histlh4i¾ Hist2h4, Dstn, IgsiB, Mitf, CsCr, Pf4, Pygl, Pdp S100a9. Fcgr2b, Idl. Emrl, Cd9, Cebpd, Pla2g7, Arg2, FIO, Gchii. SlGOaS, Ptplad2, C2ec4a2, Clec4bl , Marcks, CsfZra, Fcerlg. Clec4a2, Ppic, Cd302, Gstml , Mcpt.8, CdJOOif, Csf2rb, Bnip3, Ak4. Tpii , PlcxdL Aldoc, Rgsl L Ankrd37, Pfkl, Pdkl, Cxel9, Tbx21, Pgkl, Egln3, Ddit4, Pflcp, Pgin2 P4hal, Sicl6a3, Ubd, Steal, 5330426P16Rik, Grhpr, Cxcll l, Rasa4 Ndrgl, Fanil62a, Pkrn, EgtoL Aidoa, Ginl048G, Bnip31, Galfcl, Pgaml. Pgkl, Mif, Enolb, Enol , Pdxp, Ppplr3b, Stomil , Gzma, Scd2, Ccng2. Higdla, £no2. Gfil, Pnil, Ldha, Em 11, 3000002C10Rik, Hk2, Slc2a3, Chchd6, P4hal , Eroll, GpiL Gimap7, Oasll , Gml l 1 10. Syce2, Ndrgl, Ptgs2, Trappc6a, Preiidi, Ifi2712a, Zbtb32, TriinSOa, Pre!idl, Gapdli. Sdc3, BC062258, LOC102643247 23i0022B0SRrk. Ms4a4c _t I112rbl, Mtlifdll, Smagp, Nampt, Fasl, Pafahlb3, Prelid2, Gngl2, MS, Kdiii3a, Atf3, TnfsS.

The present inventio also provides a process for identifying suboptimal activity of a glatiramer acetate related hug substance or drug product comprising the steps of:

(a) obtaining a batch of the glatiramer acetate related drug substance or drug product;

(b) contacting a first group of mammalian cells with an amount of the glatiramer acetate related drug substance or drug product of step (a);

(c) contacting a second group of mammalian ceils of the same type with an amount of a reference standard;

(d) determining a set of genes differentially expressed by the first group after the contac ting of s te (b) relative to genes expressed by the second group after the contac ting of step (c); and

(e) identifying the glatiramer acetate related drug substance or drug product as ha ving a suboptimal activity if the se of differentially expressed genes indicate no substantial enrichment of one or more path ays set forth in Table 3 or Table 10; or identilying the; glatiramei' acetate related drug substance or drug product as having a suhoptinial activity if the set of different rally expressed enes indicates, emichment of one or more pathways set forth in Table 5. Table 7, Table 8, Table 1 er Table 15, la some embodhnents, wherein the cells are THP-1 cells.

In some enibodiinents, the referenee standard is gktiranier acetate related, drug substance or drag product in some embodiments, the referenee standard is mamiitQ.!. hi some embodiments, the reference standard is medium.

In some embodiments, the determining step (d) comprises comparing the expression of genes expressed by the first group to the expression of genes expressed by the second group. In some embodiments, the determining step (d) comprises comparing the expression of genes by the first group of cells and by the second group of cells to expression of the genes by the same type of ceils exposed to mannitol or medium.

The present invention also provides a process for producing a drug product comprising a glatiramer acetate related drug substance which involves an array of testing, the improvement comprising including in the array of testing the steps of:

(a) characterizing the glatiramer acetate related drug substance according to the process of the present invention; and

(b) (i) including the batch of the glatiramer acetate related drug substance in the production of the drug product if the level of expression of one or more genes of Mmpl2, Tgihi,€160411, Clec4a3, FceiTg. Fcgr2b _t Fcgr3, Csf3r, Ilii'2 or Tbx21 is substantially identical to the level of expression by the same type of cells in the presence of the glatiramer acetate drag substance under the same conditions; (ii) including the batch of the glatiramer acetate related drug substance in the production of the drag product if the level of expression of one or more genes of Ahcyl2, Cln5, Cxcr6, Dhrs3, Egr2. Fabp5, Ptp4al, Gprl83, Lampl, Ncoa4, Ptger4, Qk, Rragd, Sec24a, SehlL Sestdl, Slc30a4, Soati or Strip2 is substantially identical to the level of expression by the same type of cells in the presence of the glatiramer acetate drag substance under the same conditions; (in) including the batch of the glatiramer acetate related drug substance in the production of the ding product if the level of expression of one or more genes of Fam212a, Gzrna, Itprl or Zbtb32 is substantially identical to the level of expression by the same type of cells in the presence of the glatiramer acetate drag substance under the same conditions; (iv) including the batch of the glatiramer acetate related drug substance in the production of the drug product if the level of expression of one or more genes of Gzmb, Fanil9a3. NJ313, Gzma, Eaf2, Carl or St7 is upregulated relative to the level of expression by the same type of cells in the absence of the glatiramer acetate related drag substance under the same conditions; or (v) including the batch of the glatiramer acetate related drug substance in the production of the drag product if the level of expression of one or more genes of Crisp3, Apoe, Dapll, PdeSa, Scg5, StSsiafS. 4930426D05Rik or Pgap is downi'egulated relative to the level of expression by the same type of cells in the absence of the giatirainer acetate related drag substance under the same conditions.

The present invention also provides a process for releasing a dru product comprising a giaiaarner acetate related drug substa ce:, which process involves an array of testing, the .improvement comprising: including in die array of testing the steps of

(a) characterizing the glariramer acetate related drag product according to the process: of the preseiit invention; and

(b) (i) releasing the batch of the glatiramer acetate related drug substance in the production of the ding product if the level of expression of one or more genes ofMmpl2, Tgfbi, Ciec4n, CIec4a3, Fcerl g, Fcgr2b, Fcgr3, CsSr, Ilii'2 or Tbx21 is substantially identical to the level of expression by the same type of cells in the presence of the giatirainer acetate drug substance under the same conditions; (ii) including the batch of the giatirainer acetate related drag substance in the production of the drag product if the level of expression of one or more genes of AhcyI2, ClnS, Cxer6, Dhrs3, Egr2, FabpS, Ptp4al, Gprl83, Larnpl, Ncoa.4, Ptger4, Qk, Rragd, Sec24a, SehIL Sestdi, Slc30a4, Soati or Strip2 is substantially identical to the level of expression by the same type of cells in the presence of the giatirainer acetate drag substance under the same conditions; (iii) including the batch of the glath anier acetate related drag substance in the production of the drag product if the level of expression of one or more genes of Fam212a, Gzina, Itprl or Zbtb32 is substantially identical to the level of expression by the same type of cells hi the presence of the giatirainer acetate drug substance under the same conditions; (iv) including the batch of the giatirainer acetate related drug substance in the production of the drug product if the level of expression of one or more genes of Gzinb, Fami9a3, NrlB, Gznia, Eaf2 _t Carl or St7 is upregulated relative to the level of expression by the same type of cells in the absence of a glariramer acetate related drug substance under the same conditions; or (v) including the batch of the glariramer acetate related drug substance in the production of the drug product if the level of expression of one or more genes of Cris s, Apoe, Dapll, PdeSa, ScgS, St8sia6, 4930426D05Rik or Pgapl is do nregulated relative to the level of expression by the same type of cells in the absence of a giatirainer acetate related drug substance under the same conditions.

Each embodiment, disclosed herein is contemplated as being applicable to each of the other disclosed embodiments. Thus, all combinations of the various elements described herein are within the scope of the invention.

Definitions

As used herein, a "naive human" is a human that has not been treated with any multiple sclerosis drug.

As used herein, a "glatiramoid naive human" is a hitman that has not been treated with any glatiramoid drug. A glatiiamoid naive human could have been treated with another multiple sclerosis drug. As used, herein, '¾ the blood of is represented by peripheral blood mononuclear cells (PBMCs), lymphocytes, monocytes, macrophages, basophils, dendritic cells or oilier cells derived from a mammal' s blood.

As used herein, "under the same conditions" means that when two or more substances are tested and eornpared, variables or conditions that are not. tested are controlled to allow eoiicltisions to be made regarding the tested variables. For ^■ example, when contacting cells with two substances being tested, according to the methods herein disclosed, (e g , CARDS mid GARS), to determin and compare gene exression levels resulting from contacting the ceils with each of the tested substances, oilier variables should be adjusted, if necessary, so as to be comparable, at the discretion of the person skilled in the art - including, for example, the amount of the cell culture, the amount of each tested substance, and/or the type, components and additions to medium. Depending on the specific test done, the variables that should be controlled may be established according to the knowledge and experience of the person skilled in the art.

As used herein "in the absence of the glatiramer acetate related drug substance," "in the absence of the glatiramer acetate related drug product," or "in the absence of the glatiramer acetate related drug substance or drag product," means in the presense of a negative control, e.g. mannitoi or medium only. The presence of a different glatiramer acetate related drug substance or drug product, such a glatiramer acetate drug substance or drug product, does not fall under the definition of "in the absence of the glatiramer acetate related drug substance" or "in the absence of the glatiramer acetate related drug product."

As used herein, a "reference standard" is a sample or value which serves as a point of comparison for another sample or value which differs from the reference standard with respect to one or more variables. With specific regard to a human, a "reference standard" is a value or range of values that characterizes a defined population in a defined state of health. For example, a reference standard can characterize a healthy human or a human afflicted with multiple sclerosis, and when the hmnan is afflicted with multiple sclerosis the human can be naive or having received glatiramer acetate drag substance.

Glatiramer acetate (GA), the active ingredient of Copaxone®, consists of the acetate salts of synthetic polypeptides, containing four naturally occurring amino acids: L-ghitamic acid, L-alanine, L-fyroshie, and L-lysine with an average molar fraction of 0.141 , 0.427, 0.05)5, and 0.338, respectively. The peak molecular weight of glatiramer acetate is between 5,000 and 9,000 Daltons (Copaxone, Food and Drug Admimstration Approved Labeling (Reference ID; 3443331) [online], TEVA Pharmaceutical Industries Ltd.. 2014 [retrieved on December 24, 2014], Retrieved from the Internet: <URL: ¾ vw.accessdata:.fda.gov i¾igsatfda_docs label 2014/020622s0891bl .pdf>).

Chemically, glatiramer acetate is designated L-glutamk acid polymer with L-alanine, L-lysine and L- tyrosine, acetate (salt). Its structural formula is: (Gl«Ala,Lys J x)x. CH3COOH

(€5H9NQ4.C3H7N02.C6H14N202 C9H1 lN03)x:xG2H402

CAS-1 724S-92-9

CopaxonetHs a clear, colorless to slighdy yellow, sterile, itorrpyrogeme solution for suixitfaneou iBjeetios, Each I niL- o Copaxorie® solution contains 20mg or 40aig of GA, the active ingredient, and 40mg of HiaranioL The pH of the solutions is appi¾xunatel 5.5 to 7,0. Co axone*!; 20ffigmrL in a prefiiled syringe (PFS) is an approved product indicated for the treatment of patients with relapsing forms of multiple sclerosis, the safety and efficacy of winch are supported by over two decades of clinical research and over a decade of post-marketing experience. Copaxone's; 20mg/iiiL is administered daily.

Copaxone's* 40mg/iiiL in a PFS was developed as a new formulation of the active ingredient GA. Copaxone® 40mg/mL is a prescription medicine indicated for the treatment of patients with relapsing forms of multiple sclerosis (Copaxone, Food and Drug Administration Approved Labeling (Reference ID: 3443331) [online], TEVA Pharmaceutical Industries Ltd., 2014 [retrieved on December 24, 2014], Retrieved from the Internet <URL: mvw.accessdata.fda.gov ihiigsatfda^ Copaxone® 4Qing/mL is administered three times per week.

As used herein, the term "glatiramer acetate related drug substance" (GARDS) is intended to include polypeptides with a predetermined sequence as well as mixtures of polypeptides assembled from the four amino acids glutamic acid (E), alanine (A), lysine (K), and tyrosine (Y); fi'om any three of the amino acids Y, E, A and K, i.e. YAK, YEK, YEA or EAK; or from three of the amino acids Y, E, A and and a fourth amino acid. Examples of glariramer acetate related drug substances are iiisclosed in U.S. Patents 6,514,938 Al, 7,299,172 B2, 7,560,100 and 7,655,221 B2 and U.S. Patent Application Publication No. US 2009-0191173 Al, the disclosures of which are hereby incorporated by reference in their entireties. Glatiramer acetate related substances include glatiramoids.

As used herein, a "glatiramer acetate related drug product" (GARDP) contains a glatiramer acetate related drug substance.

As used herein, a "glatiramer acetate related drug substance or drug product" is a glatiramer acetate related drug substance or a glatiramer acetate related drug product.

As used herein a "glatiramoid" is a c omplex mixture of synthetic proteins and polypeptide s of varying sizes assembled from four naturally occurring amino acids: L-glutamic acid, L-aianme, L-lysine, and L-tyrosme. Examples of glatiramoids include glatiramer acetate dr ug substance (the active ingredient in Copaxone'*) as well the active ingredients in other products, e.g. GA-Natco, POLIMUNOL and GLATOPA. As vised .herein, a glahramer acetate drug substanc " (GADS) is glatiramer acetate produced by leva Pharmaceutical Industries, Ltd, and is the active .ingredient in a giatiranier acetate drug product. _.

As useti herein, a "giatiranier acetate drag product" (GADP) contains a glatiramer. acetate drug substance produced by Teva Pharmaceutical Industries, Ltd.

As used herehi, a ^giatiranier: acetate drug substance o drag product" is a giatiranier acetate drug subslan.ee. or a, glatiramer acetate drug product

As used herein a "glatiramer acetate reference standard" is or contains the drag substance found in a glatiramer acetate drug product.

As used herein "suboptimal activity" refers to a negative response or to a response which is less than the response to giatiranier acetate drag substance or glatirame acetate drag product produced by leva Pharmaceutical Industries, Ltd.

As used herein, "release" of a drag product refers to making the product available to consumers.

As used herein, "about" with regard to a stated number encompasses a range of +10 percent to -10 percen of the stated value. By way of example, about 100 mg therefore includes the range 90- 110 rag and therefore also includes 90, 91, 2, 93, 4, 95 96, 97, 98, 99, 100, 101, 102. 103, 104, 105, 106, 107, 108, 109 and 1 10 mg. Accordingly, about 100 nig includes, in an embodiment 100 mg.

As used herein, "hematological cell" comprises neutrophils, erythrocytes, basophils, monocytes, eosinophils, platelets, lyrnphoc-ytes, and spienocytes.

As used herein, an "array of testing" for a. glatiramer acetate related drag substance or drag product includes, but is not limited to, any analytical method test such as in vitro tests or molecular weight tests, biological assays such as the ex vivo tests and clinical efficacy tests which characterize the GARDS or GA DP, or clinical trials. Examples of testing for a giatiranier acetate related drug substance or drag product are disclosed in U.S. Patent Application Publication Nos. US 2012-0309671 and US 2011- 0230413, and hi PCT International Application Publication Nos. WO 2000/018794, WO 2012/051106, WO 2013/055683, WO 2014/058976, the disclosures of which are hereby incorporated by reference in their entireties.

It is understood that where a parameter range is provided, all integers within that range, tenths thereof and hundredths thereof, are also provided by the invention. For example, "0.2-5 nig" is a disclosure of 0.2 mg, 0.21 mg. 0.22 mg, 0.23 mg etc. up to 0.3 mg. 0.31 mg, 0.32 mg, 0.33 mg etc. up to 0.4 mg, 0.5 mg, 0.6 mg etc. up to 5.0 mg.

All combina tions of the various elements described herein are within the scope of the invention.

It is understood that where "at least one" or "one or more" is recited along with a list, then 1. 2, 3, 4... or all members of that list are disclosed in every combination. For example, in a group of 43 genes, "at least one" and "one or more" is a disclosure of one gene, two genes, three genes, etc., in any combination, up to the forty three genes.

As used herein, "characierization" or "characterizmg" is understood, to include obtaining information which was produced, in the. same location .or country, or a different location or country from where any remaining steps of the raerfiod are performed.

As used herein, processes of producing a glatirafiier acetate related drag substance or drag product, are known in the art. Examples of such maniifactaring processes are disclosed in US ^' . Patent 5.800.808. and in PCX International Application Publication Nos. WO 2005/032553, WO 2005/032395, WO 1999/22402, the disclosures of which are hereby incorporated by reference in their entireties.

Biological characterization of a giatiramer acetate related drug product using mammalian and human cells are known in the art e.g. as disclosed in PCX International Application Publication No. WO 2016/004250, the disclosures of which are hereby incorporated by reference in their entireties.

This mvenrion will be better understood by reference to the Experimental Details which follow, but those skilled in the ait will readily appreciate that the specific experiments detailed are only illustrative of the invention as described more fully in the claims which follow thereafter.

Experimental Details

Methods

Alouse Splenocvtes: Gene Expression Analysis— POLIMUNOL (Purported Generic GA) versus Copaxone ^® Gene Expression Studies

Mice

All experimental procedures conformed to accepted ethical standards for use of animals in resear c h and were in accordance with Committee for the Care and Use of Experimental Animals guidelines and approved by the Teva Institutional Animal Care and Use Committee. For these experiments, 8- to 12- week-old female (Balb/c X SJL) Fl mice (Janvier, Fiance) were purchased. Mice were kept at 2 i±3 ^: 'C; the relative humidity was 30-70%, the light/dark cycle was 12/12 h. Animals were maintained on a standard rodent pellet diet and sterile filtered tap water available ad libitum.

Immunization of mice and preparation ofex-vivo mouse spleen cell cultures

To stimulate induction of GA and POLIMUNOL (purported generic) -reactive X cells, mice in each treatment group were injected with 100 uL of a 2.5 mg/mL solution of either Copaxone® (GA drug product, Teva Pharmaceutical Industries, Petach Tikva, Israel) or POLIMUNOL (Synthon, Nijmegen, Netherlands) in phosphate-buffered saline (250 pg GARS per mouse). Mice were housed tor 3 days after immunization; mice were then sacrificed and their spleens were asepticaiiy removed and placed on ice in RPMI 1640. A single cell suspension was prepared. After red blood cells lysis, splenocytes from the same immunization grou were pulled and resuspended to a final eoiieenhation of 10 x 10° cells niL in defined, ceil culture media (BCCMl) (Biological Industries, Beit Haexaek, Israel} (96.7% v/v) enriched with L-ghiiaBiine 2 inM (1 v/v), MEM Non-E-ssential Amino Adds 2 niM (¾ -v/v), sodium pyruvate 1. mM (1% v/v), anfi iotic/srmrnycotie solution (0.2% v/v) and 2-mercaptoethanoi 50mM. (0. i% v/v) . Figures .19 and 22.

In iitra cel activation

Splenocytes were treated with activator samples diluted in medium (125 L per well of 80 iig/ L solutions, final concentration in the well after addition of cells: 40 pg/mL) of: i) 3 different batches of GA drug product manufactured by Tev ii) one batch of POLIMUNOL (purported generic GA). POLIMUNOL {purported generic GA) is a product marketed as generic GA and manufactured by a company other than Teva (i.e. Synthon). The activator samples or marmitol (the nonactive excipient in Copaxone ^® ) were added to 96-weU tissue culture plates (three wells pe sample). Splenocytes (125 uL 10 x If SPL cell mL suspension) were added to the activator solutions. Each activator sample was loaded in two different plates. One tor the cells from mice immunized with GA and one for cells from mice immunized with proposed generic. Plates were incubated for 24 h at 37°C. Cells were collected from the wells and were centrifuged at 300g for 5 minutes. Supernatants were aspirated and cell pellets were resuspended in RLT buffer (from RNEASY MINI KIT OF QIAGEN, Cat # 74106) for cell lysis. The cell lysates were centrifuged and supematants were collected and frozen at -70°C. Samples were sent for further processing. Figures 19 and 22,

Analysis Methods

For additional detail on experimental methods used in mouse studies, please refer to publications [Bakshi et al; Towfic et aljj. Note that in this study, mice were immunized with either Copaxone* or _. POLIMUNOL (purported generic GA), and subsequently splenocytes were isolated and treated ex vivo with Copaxone ^® or POLIMUNOL (purported generic GA), RNA was extracted and expression profiled across the entire genome using the AFFYMETRT MOUSE GENOME 430 2 CHIP. Three lots of Copaxone ^® , and one lot of POLIMUNOL {purported- generic GA)were comparatively tested ia si replicates eacl is dictated by power-analysis calculations performed using the R statistical package ssize.fdr to determine the number of samples needed to detect differentially-expressed genes with a fold-change between treatments of as low as 1.3 with 80% power. Two technical replicates were used, resulting in 12 replicates per condition.

Outlier identification and normalization

Outlier samples were identified using the R package ArrayQCMetrics and excluded from further data processing steps. A sample was considered an outlier if it failed more than half of the included tests either before or after RMA normalization. Data were RMA normalized using the Affy R package. .Batch correction.

Correction for batch, variation was performed using CoinBat (Johnson et a .,. 2007), as implemented in the. SYA R package (Leek JT et aL 2013), Briefly, ConiBat is an empirical Bay siaa approach using location and scale metrics across several genes to adjust for batch effects in datasets, even datasets containing small sample sizes... Date of nheroarray experiment wa used as batch, and t e combination o treatment and immunization w¾s used as covariaie. Principal Component Analysis (a multivariate approach) showed thai the main effect in die first principal component remained due to treatment effects after batcii correction.

Differentia! expression analysis

Differentially expressed probesets were identified across conditions using linear models for microarray data (LIMMA; Smyth, G. K. (2004)), a standard R Bioconductor package. Linear models and empirical Bayes methods for assessing differential expression in microarray experiments. Statistical Applications in Genetics and Molecular Biology 3, No. 1, Article 3). To compare GA aad purported generic, comparisons were collected to compare each treatment relative to mannitol control (e.g., [GA vs mannitol] was compared via LIMMA to [POLIMUNOL (purported generic GA) vs maimitol]). Probesets were filtered by MAS5 calls of presence on the chi (to be considered present, a probeset was required to have on average a call of present or marginal across samples), Probesets were mapped to genes using the annotation available for the Mouse 4302 chip from Afrymetrix. Unless otherwise noted, FDR adjusted p values reported for genes represent the lowest FDR adjusted p value for present probesets for that gene. For the comparison of immunization and activation with GA versus immunization and activation with POLIMUNOL (purported generic GA), an additional correction for run order, performed using LIMMA (Smyth, 2004), was evaluated (as described in Hasson et al, 2016).

Pathway enrichment analysis

Upregulaied and dowmegulaied probesets were analyzed separately for pathway enrichment, using DAVID (Huang et al. Nucleic Acids Res 2009). Pathway enrichment results were visualized using volcano plots, plotting either -log adjusted p values or untraiisformed adjusted p values versus enrichment scores for the pathways. For comparisons between branded GA and purported generic GA, upregulaied or downregulated probesets with FDR-adjusted p values < 0.05 and ibid changes (FC) with absolute value greater than 1.2, or for further analyses greater than 1.4. were used for pathway enrichment. For comparisons between branded GA and mannitol. upregulaied or downregulated probesets with FDR-adjusted p values < 0.05 and FC with absolute value greater than 2 were used for pathway enrichment. DAVID runs were conducted in December 2014, January 2015, and March-April 2016.

Human Monocyte Cell Line: Gene Expression Analysis: - POLI&fUNOL (Purported Generic GA) versus Copaxone Gene Expression Studies JSgpefUfientel design

Cells, from a human monocyte eel! line (THP-1) were Slsniiilated ^' with .either branded glatiiamer acetate - Copaxone ^® , of purported generic PQLIMt!NOL, or vehicle control (mannitol) f r 6 hours based OB prior observations of GA effects in this model system ( olitz et al _. , _. 2015), KNA was extracted and expression profiled i a blinded fashion across the entire genome _f using the iymetrix Human Genome TJ133 Plus: 2.0 e p, mteifogamig a total of over 47,000 transcripts. Three 1.ots _: of Copaxone ^® and one lot of PGLIMU QL (AXBN) were comparatively tested in six replicates each. This entire experiment was performed independently twice, using an identical study design, reagents and compounds by the same technicians on two separate days; resulting in twelve replicates total per condition.

A priori power analysis

Using the R statistical package ssize.fdr, power calculations were performed to determine the number of samples needed to detect differentially expressed genes with a fold-change between treatments of as low as 1.3 with 80% power. Based on the results of these power calculations, the experiment was designed to include six replicates of each condition. The order in which the samples were processed was randomized with respect to treatment and stimulation time in order to avoid creating confounding batch effects.

Outlier identification and normalization

Outlier samples were identified using the R package ArrayQCMetrics and excluded from further data processing steps. A sample was considered an outlier if it tailed more than half of the included tests either before or after RMA normalization. Data were RMA normalized using the Affy R package.

Batch correction

Correction for batch variation was periomied using ComBat (Johnson et aL Biostat, 2007), as implemented in the SVA R package (Leek JT, Johnson WE, Parker HS, Jaffe AE, Storey JD (2013) sva; Surrogate Variable Analysis. Available: wmv.bioeondiietor.org/paekages/release¾ Briefly, ComBat is an empirical

Bayesian approach utilizing location and scale metrics across several genes to adjust for batch effects in datasets, even datasets containing small sample sizes. Date of experiment was utilized as batch. Treatment labels were added as covariates to the batch correction in order to preserve relevant treatment effects. Principal Component Analysis (a multivariate approach) showed that the main effect in the first principal component remained due to treatment effects after batch correction.

Differentia ! expr ssio n a nafys is

Differentially expressed probesets were identified across conditions using linear models tor microaiTay data (LIMMA; Smyth, G. K. (2004)), a standard R Bioconductor package. Linear models and empirical Bayes methods fo assessing differential expression in microarray experiments. Statistical Applications in Genetics and Molecular Biology 3, No. 1.Article 3). To compare Copaxone ^® and a pmported generic, comparisons were corrected to compare each treatmen relative to maenitGl control (e.g., [GA vs maimitol] was compared vi UMMA to {purported generic vs niannitol]}. For us in pathway anal ses, probeseis were filtered. by MASS calls of presence on the chip for the relevant samples in the comparison (e.g., to be considered present, a probeset was required to have on average a call of presen or marginal across samples). An di tk l QC step wax performed to remove probesets determined to be highly variable between mi iple THP-i datasefs, as follows; a probeset was deemed highly variable if across three THP-i studies to date, that probeset was observed to be upregulaied, downregulated, and not modulated by Copaxone ^® across the three studies. This criterion resulted in filtering out 216 probesets. Probesets were mapped to genes using the annotation available for die U133 Phis 2.0 chip from Affymetiix. FDR adjusted p values reported for genes represent the lowest FDR adjusted p value for present probesets for that gene.

Pathway enrichment analysis

Upregulated and dowmeguiated probesets were analyzed separately for pathway enrichment,, using DAVID (Huang et al. Nucleic Acids Res 2009). Pathway enrichment results were visualized using volcano plots, plotting -log p values versus enrichment scores. For comparisons between Copaxone ^® and pmported generic, upregulated or downregulated probesets with FDR-adjusted p values < 0.05 and fold changes (FC) with absolute value greater than 1.1 were used for pathway enrichment. For comparisons between Copaxone ^® and niamiitoi upregulated or downregulated probesets with FDR- adjusted p values < 0.05 ami FC with absolute value greater than 1.3 were used for pathway enrichment.

Gene Expression Analysis— GLATOPA versus Copaxone ⁹ Gene Expression Studies

Experimental methods for mouse spl-enocyte studies

Mice:

For these experiments, 8- to 12-week-old female (Balb c X SJL) Fl mice (Janvier, France) were used. Mice were kept at 21±3°C; the relative humidity was 30-70%. the light/dark cycle was 12/12 li. Animals were maintained on a standard rodent pellet diet and sterile filtered tap water available ad libitum.

Immunization of mice and preparation of ex vivo spleen cell cultures:

To stimulate induction of GA and GLATOPA-reactive T cells, 26 mice in each treatment group were injected with 100 uL of a 2.5 mg/niL solution of either Copaxone® (GA drug product) or GLATOPA'D in phosphate-buffered saline (250 pg GAper mouse). Mice were housed for 3 days after iinmtmizatiom mice were then sacrificed and their spleens were aseptically removed and placed on ice in RPMI 1640. A single cell suspension was prepared. After red blood ceils lysis, splenocytes from the same immmiizaiion grou were pulled and resuspended to a final concentration of 10 x 106 cells/mL in defined cell culture medium (DCCM1) (Biological Industries, Beit Haemek, Israel) (96.7% v v) enriched with L-glntanhiie 2 mM. (1% v v), MEM Nors-Essential Amino Acids 2 niM (1% v v) sodium pyruvate 1 niM (1% v/v), aatibiotie/antiinyeotk solution (0:2% v/v) and 2-mercapioethanol 50mM (0.1% v/v).

In vfiw ceil ac uation:

Splenoeytes. were treated wife activator samples diluted hvrnedkim (125 pL per well of 80 ig/iiiL solutions, final, concentration in me well after addi tion of ike cells: 40 jig/niL. Six different batches of Copaxone® and six different batches of GLATOPA were used for treatment. The activator samples or mannitoi (the nonactive excipient in Copaxone® and GLATOPA, used as contra! in fee experiment) in medium were added to 96-weU tissue culture plates. Ail batches were tested in 6 replicates, each replicate in 3 wells. The study was performed twice resulting in 12 replicates for each batch. Splenocyt.es (125 }iL 10 x 106 SPL ce!l/mL suspension) were added to the activator solutions. Each activator sample was loaded in two different plates, one for the cells from mice immunized with GA and one for cells from mice immunized with GLATOPA. Plates were incubated for 24 h at 37 ^a 'C in 5% C02 atmosphere. Cells were collected from the wells and were centrifuged at 300g for 5 minutes. Supernatants were aspirated and cell pellets were resuspended in RLT buffer (from R easy mini kit of Qiagen, Cat # 74106) for cell lysis. The cell lysates were centiifuged and supernatants were collected and frozen at -70°C. Samples were sent fo further processing.

Experimental methods for THP-1 studies

Cells from a human monocyte cell line (THP-1) were stimulated wife either branded glatiramer acetate - Copaxone®, or purported generic GLATOPA, or vehicle control (mannitoi) for 6 hours based on prior observations ofGA effects in this model system (Kolitz et al.2015). R A was extracted and expression profiled hi a blinded fashion across the entire genome, using the Affymetrix Human Genome Ul 33 Plus 2.0 chip, interrogating a total of over 47,000 transcripts. 5 lots each of Copaxone® and GLATOPA were tested as described below. Two studies were conducted, in two separate laboratories.

Cell culture and treatment:

THP-1 cells (human monocytic cell line) was maintained in RPMI 1640 medium supplemented with 10% Fetal bovine serum, ImM sodium-pyruvate, 0.025mg ml D-glucose. lOmM Hepes, 0.05mM 2- mercaptoeihano!. For the study IniL of THPi cell suspension containing 2x106 cells/mL was added to each well of 6 well plates. ImL of lOOpg/ml Copaxone® or GLATOPA batches were added to the wells. Five different batches of Copaxone® and five different batches of GLATOPA were used tor treatment. Mannitoi (the nonactive excipient in Copaxone® and GLATOPA) used as control in the experiment. Ail batches were tested in 10 replicates. Plates were incubated for 6 h at 37*C in 5% C02 atmosphere. Cells were collected from the wells and were centrifuged at 300g for 5 minutes. Supernatants were aspirated and cell pellets were resuspended in RLT butler (from RNeasy mini kit of Qiagen, Cat # 74106) for cell lysis. The cell lysates were centiifuged and supernatants were collected and frozen at.-?0^C, Samples were sent for furthe processing.

Data atia -sis methods for expression dat

Outlier identification and normalization

Outlier samples were identified using the R package ArrayQCMetri.cs and excluded from further data processing steps. A sample was considered an outlier if it failed more than half of ^' the included ^' tests either befo e or after -RMA normalization. Daia were RMA normalized using the Aff R package.

Batch correction

Correction for batch variation was performed using ComBai (Johnson et at Biostat, 2007), as implemented in the SVA R package (Leek JT, Johnson WE, Parker HS, Jaffe AE, Storey JD (2013) sva: Surrogate Variable Analysis. Available: wmv.bioconducior.org packages/release¾io^^ Briefly, ComBai is an empirical

Bayesian approach utilizing location and scale metrics across several genes to adjust tor batch effects in datasets, eve datasets containing small sample sizes. For the ΊΉΡ-1 dataset ftora the Israel lab. day of lab experiment and date of microarray run were combined for use as batch with treatment as covariate. For the THP-1 dataset from the Hungary lab, no batch correction was needed tor date of experiment or array run. For the mouse splenocyte dataset, date of experiment combined with date of microarray ran was used as batch, and the combination of tieatment and immunization was used as covariate. Principal Component Analysis showed that the main effect in the first principal component remained due to treatment effects after batch correction. Differential expression analysis

Differentially expressed probesets were identified across conditions using linear models for microarray data (LIMMA: Smyth, G. K. (2004)), a standard R Bioconductor package. To compare Copaxone®' and purported generic, comparisons were corrected to compare each treatment rela tive to mannitol control (e.g., [Copaxone¾> vs mannitol] was compared via LIMMA to [GLATOPA vs mannitol]). Probesets were filtered by MAS5 calls of presence on the chip (to be considered present, a probeset was required to ha ve on a verage a call of present or marginal across samples). Probesets were mapped to genes using the annotation available for the Mouse 430 2 chip from Affymetiix in the case of the splenocyte study, and tire HG U133 Plus 2 for the THP-1 study. For the THP-1 studies, run order from the microarray run was used as covariate in the analysis. Unless otherwise noted, FDR adjusted p values reported for genes represent the lowest FDR adjusted p value for present probesets for that gene. FDR in this context refers to the Benjamini Hochberg method, as implemented in the LIMMA R package.

Pathway enrichment, analysis

Top up- and down-regulated probesets wer e analyzed separatel for pathway enrichment, using DAVID (Huang et aL Nucleic Acids Res 2009). Significance of pathway enrichment results was determined using Bersjamffl} p. value < 0.05. Pathway enrichment results were; visualize sing volcano plots, plotting -log adjusted, p ^■ values versus enrichment scores for the pathways. Fold change filters wer used to obtain _. probeset list s. of ^" appropriate length for pathway analysis, with DAVID . Probesets called present on the drip were used as background. For comparisons: bet ee Copaxone® and GLATQPA, up- or down-regulated probesets with. FDR-adjusted p values: < 0.05 and fold changes (FC) with absolute value greater than 1 ,1 under both an uitiz tion conditions (splenocyte data) or FDR-adjtisted p values 0.05 hi both laboratories (THP-1) were used, for pathway eniichnient. For comparisons between Copaxone® and mannitoL up- or down-regulated probesets with FDR-adjusted p values < 0.05 and FC with absolute value greater tha 2 were used for pathway enrichment. DAVID runs were conducted during March-May 2016.

Gene Set Enrichment Analysis (GSEA) (Subramanian et al, 2005) was used to identify pathways enriched in the THP-1 differential expression results between GLATOPA and Copaxone® (mannitoi- corrected). Probesets were filtered to use present probesets only and ranked by fold change for GSEA pre-ranked analysis. The analysis was run using the Ja va interface available from the Broad, in February 2016. Probesets were collapsed to genes usin the HG_UL 3_Phis_2.chip annotation available hi the GSEA tool with the tool default. The cytoknie-eytokine receptor interaction gene set was obtained from Kegg iwwTv.genome.jp/dbget-hm get._^^ for use with the GSEA tool. qRT-PCR measurements

19 genes were chosen for measurement of expression levels by qRT-PCR in the Copaxone®- siiHiiunization samples, based on the findings from the splenocyte microarra results. Experimental assistance was provided by EAjQuintiles. Four reference transcripts (Actb, Ppib, Tbp, and RpllSa) were averaged for use as overall reference, and vehicle control (mannitoi) samples were used as calibrator. RQ was calculated as 2 ^-ΔΔα (Schmittgen and Livak, 2008). Single-sided t tests with unequal variance were used to compare RQ values between treatments.

Example 1

Products that mimic MBP, similar to Copaxone®, cause MS-like symptoms, demonstrating a delicate immune balance dependent on composition. Copaxone®'s broad mechanism of action, affecting multiple immune cell types and pathways, has not yet been fully elucidated:

1. Upon injection, the intact material is hydrolyzed locally through complex mechanisms, at unknown dynamics and at unknown preferential cleavage sites.

2. The fragments are the taken up by antigen presenting cells.

3. Antigen presenting cells present specific, yet undefined, epitopes to T-cells of die immune system, modifying the delicate balance between proinflammatory, anti-inilammatory and regulatory immune cells (Thl, Th2 and Treg, respectively) in the brain. Because ^' o a one® is hydro yzed locally, there are no known pharmacokinetics. In addition, there are aq validated phaniiaeodynarnics markers.

Measures of biological activity, suck as potency. Mocking EAE induction, and cytotoxicity, as well as. iiiHtHH-e-iieeogaitioii specificity to Copaxone® monoclonal and. polyclonal antibodies, were noa- diserinnnatory i the comparison between GLATOPA to Copaxone®. These biological methods are of low resolution and therefore lack sensitivity to detect, medianistie BJQlec»te-diff€^c ^' es ^' that ^:' iaay ^' -iiav significant biological and clinical impact Such molecular differences are ^ of particular relevance in the contest of long-term immunotherapy for a chronic disease, progr essing over the course of multiple decades. For these reasons, higher resolution biological analyses were pursued using genome wide gene expression data.

Copaxone® is an antigen that participates in the immunological triad, along with antigen presenting cells and T cells. In order to model each of these cell types, a mouse spienocyte system and human monocyte (THP-1) cell line were utilized. Unbiased genome-wide gene expression studies were conducted to examine effects of treatment with Copaxone® or GLATOPA in each model system.

Para from reciprocal mouse spienocyte experiment

Reciprocal mouse spienocyte experiment with Copaxone® and GLATOPA was conducted as described above in the methods section and as depicted in Figure 17.

Effects of Copaxone® treatment relative to control

Copaxone® treatment modulated thousands of probesets versus mannitoi control in splenocytes from CopaxoneC-immunized mice (Table 1), Genes consistent with prior literature about Copaxone® mechanism of action were modulated, including prominent immunomodulatory (Fig, 3A-D): 1110, the gene for anti-inflammatoiy cytokine IL-10, was upregulated with Copaxone® treatmen (FC 1.54, ad] p < 1.8e-16). IL-10 is expressed by monocytes and lymphocytes, and has multiple immunological functions, including downregulation of Thl cytokine expression, and enhancement of B cell survival and antibody production. 114, the gene for anti-inflammatory IL-4, was upregulated with Copaxone' ¹ !' treatment (FC 7.4, adj p < 9.6e~46). Foxp3, a well-known marker gene for anti-inflammatory regulatory T cells that is crucial to Treg development and function, was upregulated with Copaxone® treatment (FC 1.3S, adj p < 3.6e-10), 1112a, the gene for pro-inflammatory IL-12, was downregulated witli Copaxone® treatment (FC -2.14, adj p < i.2e-30). These observations are consistent with extensive prior literature from across the globe (Duda 2000, Anion 2004, Vieira et al 2003, Kim et al 2004, Ruggieri 2007, Weber et al 2007. Carpintero 2010, Hasson et al 2016). This consistency with prior studies validates the method and approach.

Table 1. Differentially Expressed Piobesets in Mouse Splenocytes Across Reciprocal Models of Immunization and Ex-Vivo Treatments. Immunization # of probeseis # of profaesefs tip, FDR j O.05 flown, FDR p<0.05

Co axone® Copaxone® vs insanlto! 5,854 6,817

GLATOPA vs inanniiol 5,95 6,71

GLATOPA vs Copaxone® 602 456

(mannitol-correcied)

GLATOPA Copaxone® vs i¾amiito] 7,173 7,344

GLATOPA vs mariiiiioi 7,157 7,204

GLATOPA vs Copaxone® 7S2 1,036 (maiinitol-correeied)

Numbers of present probesets passing FDR<0.05.

Shown in Table 2 are top genes modulated by Copaxone® treatment relative to niamiitol spienocytes fiom CopaxQiie®-iminiBiized mice.

Table 2. Top genes laoilntatefi by Copaxone® ^' treatment relative to maiiiiitol coairol 1M

Among the most pronouncedly modulated probesers (|FC|>2) comparing Copaxone® treatment versus maimitol, 75 pathways (61 up, 14 down) were significantly enriched (adjusted p < 0.05), using the Gene Ontology (Ashbumer et al, 2000) and Kegg pathway databases (Kanehisa et al, 2000). In the up direction, these included 51 pathways in GO Biological Process (BP), 1 in GO Molecular Function (MF), 5 in GO Cellular Component (CC), and 4 in Kegg. In the down direction, 2 pathways in GO BP, 1 in GO MF, 9 in GO CC, and 2 in Kegg were significantly enriched by adjusted 0.05. These pathways are illustrated in Figure 4 and listed in Table 3. Table 3. Pathways enriched by Copaxone® treatmen ^' relative to mannitol on splenocyfes from CepaxaneS-immnaized mite.

Pathway

Cy tbkine-eyto iie receptor interaction (nnn«04060)

Extracellular region (€30:0005576) _.

Extracellular space (GO;000S6i5)

Cell stuface (G0:»O09986)

External side of plasma membrane (GO: 0009897}

Extracellular region part (00:0044421)

Immune response (GO:0006955)

Jak-STAT signaling pathway immu0463G)

Sterol metabolic process (GO;0016125)

Sterol biosynthetic process (00:0016126)

Positive regulation of cell proliferation (00:0008284}

Hematopoietic cell lineage (mm«0 640)

Positive regulation ofleukocyte proliferation (00:0070665)

Myeloid ceil apoptosis (GO: 0033028)

Regulation of survival gene product expression (00:0045884)

Regulation of eukocyte proliferation (00:0070663)

Positive regulation of survival gene product expression (GO:004588S)

Intrinsic to membrane (GO: 0031224)

Cell surface (GO: 0009986)

External side of plasma membrane (00:0009897)

Carbohydrate binding (GO:0030246)

Hematopoietic cell lineage (mniu04640)

Metabolism of xenobiotics by cytochrome P450 (mmu00980)

The top egg pathway enriched among probesets upregulated by Copaxone® treatment was the cytokine-cytokine receptor interaction pathway (adj p < 5e-5). This observation is consisten with prior observations for Copaxone® treatment in this system, as well as other model systems (e.g. THP-1) ( oiitz et a , 2015: Hasson et al, 2016).

Effects of GLATOPA treatment on Copaxone ¹ ® immunized mice

Introduction of AB generic compounds is often associated with switching of treated patients from an originator version to the newly marketed generic. In the context of antigens, therapeutically presciibed as immunomodulators, it is important to assess the similarity in biological and clinical response between the two treatment phases. Therefore gene expression profiles of splenocytes from CopaxoneS- immunized mice under ex-vivo treatment by either GLATOPA or Copaxone® were examined. GLATOPA modulated thousands of probesets similarly to Copaxone®' treatment in the same iimnunrzafion paradigm, including genes importan for OA's mechanism (Fig. 3A-D). For example, GLATOPA treatment increased die expression of 1110 (FC=L46, adj p<9.9e-12), 114 (FC 7.9, adj p < 9.7e-42), and Foxp3 (FC 1.41, adj p < 6.5e-i3), and decreased expression of 1112a (FC -2,15, adj p < 5.6e-35). .In addition to the observed similarities, a sigmfk¾it ^■■ subset of probesets (incl«<fcig approximately 7% of probesets affected by Copaxone® treatment} was modula d to differing degrees by GLATOPA and. Copaxone®, as illustrated in Figure 5. 1.Q58 probesets (6.02 up, 456 down) were significantly differentially expressed (FDR. ρ<0.β5 betwee GLATOPA and Copaxone® (Jnaamtol-corfected) under Copaxone!? kmmHrization, The range of observed fold change difference among these probesets: as -1.56 to 1.46..

GLATOPA ΪΪΠΚ Cepaxoue: Differentially expressed genes common to both . Immunization conditions

A subset of probesets that were differentially expressed between Copaxone® and GLATOPA treatment regardless of whether mice were immunized with Copaxone® or with GLATOPA were focused. This subset represents the most robust signature of dissimilarities, regardless of sequence of switching, thus reflecting a conservative assessment of inherent biological dissimilarities. This subset of probesets, showing differences between GLATOPA and Copaxone® treatment irrespective of immunization condition, comprised 747 present probesets (387 up and 360 down with GLATOPA versus Copaxone® treatment, respectively, in mannitol-corrected comparison, representing approximately 5% of probesets modulated by Copaxone® treatment).

Beyond numerical differentiation, functionally relevant genes and pathways are specifically encoded within this subset of probesets: for instance, expression levels of several matiix metal!oproteinases were higher with GLATOPA versus Copaxone® treatment, including Mmp9, MmpS, and Mmpl.2. The MMP protein is key to blood brain barrier (BBB) disruption, in particular increasing access of immune cells (including autoimmune T cells) to the CNS. High MMP9 levels have been consistently associated with MS (Rosenberg 2005; Romme Christensen et al 2013; Kouwenhoven et al 2002), as well as in MRI active patients with gadohnitim-eiAaiiciiig lesions versus patients without (Waubant 2006). In fact, MMP9 has been proposed as a biomarker for MS diagnosis and progression (Milward et al 2008). GA has been reported to inhibit expression ofMMP9 in healthy human peripheral blood mononuclear cells (Kiiop 2013). Consistent with these data, Copaxone® treatment in the splenocyte model lowered MMP expression relative to maimitol control to a greater degree tha did GLATOPA. This was the case under both immunization conditions, and for both probesets for Mmp9 present on the chip. This gene has been previously observed to differ in level between Copaxone® and other purported generics, including POLIMUNQL (Hasson et al, 2016) and PROBIOGLAT (Kolitz et ai, 2015).

The Tgfbi gene (coding for transforming growth factor beta induced protein) was increased in level between GLATOPA and Copaxone® (adj p < 5.3e-10, FC 1.34: Figure 6). That is. relative to maimitol control, GLATOPA treatment decreased levels of this gene less than Copaxone® treatment. As shown in Figure 6, this result was observed for both immunization conditions. Four probesets were present on the chip for this gene, and all four showed the same pattern, lending additional support to the differential expression of this gene. Tgfbr lias roles in adhesion a d. iiifiaiimiafion, and is induced by transfoniiing growth factor beta, a iactor with cpatext-depeadeat effects on T cell polarization that can induce Thl 7 differentiation (Khnura and KislHiaoto, 2010; Giiicber efc al, 2011).

C!ec4n was also increased in expression with GLATOPA relative to GopaxoneC>ti"eatnient (Figia 7); that is, Copaxone® treatment decreased the expression level of this gene relative to control to a greate extent than GL ATOPA treatment This was true under both immunization conditions., and for both probesets for Clec4s present o the array, A related, mouse gene, Ciec4a3, also shewed similar differences. The Ckc4n gene, representing the mouse homolog of human CLEC6A, codes for the antigen-presenting cell lectin-like receptor complex APLEC, also known as dendritic cell inhibitory receptor 3, which allows transduction of signaling through Fc receptor gamma chain FCER1G (SwissProf). Interestingly and consistent with this functional activity,. Fcei is itself modulated differentially by GLATOPA and Copaxone® (Figure 8), along with several related genes, Fcgr2b and Fcgr3 (each increased with GL ATOPA relative to Copaxone® treatment). AM four probesets tor Fcgr2b tha were called present on the array showed the same result. Fcgr2b encodes a receptor for the Fc region of eomplexed or aggregated immunoglobulins gamma, involved in a range of effector and regulatory functions including modulation of antibody production by B-cells. Fcgr2b receptor binding is reported to lead to downregulatioii of existing cell activation that had been caused by antigen receptors on B-cells (BCR), T-cells (TCR) or by another Fc receptor (UniProf).

Additional genes of interest that differed between GLATOPA and Copaxone® under both immunization conditions included Cxcl3, also known as GRQ-1. a cliemokme that participates in recruitment of neutrophils (Figure 9; FC 1.46, adj p < 3.9e-6 and FC 1.48, adj p < 3.2e-5 for Copaxone®and GLATOPA immunization, respectively): Csf r (FC 1.13. adj p < 1.0e-5 and FC 1.2, adj p < 9.8e-l l for Copaxoiie®and GLATOPA immunization, respectively) and Csf3r (FC 1.12, adj p

< 3.1e-9 and FC 1.1 , adj p < 2.7e-9 fo Copaxone® and GLATOPA immunization, respectively), receptors for colony stimulating iactor cytokines important for differentiation, survival and function of immune cells such as monocytes and neutrophils; 116 (FC 1.15. adj p < 0.008 and FC LIS, adj p < 9.1e- 5 for Copaxone® and GLATOPA immunization, respectively), a cytokine required for generating Thl 7 cells (UniProt); along with another Thl 7 -relevant gene, II7r (FC 1.07, adj p < 0.05 and FC 1.08, adj p

< 0.006 for Copaxone® and GLATOPA immunization, respectively).

GLATOPA versus Copaxone! ¹ : Differentially expressed pathways imder both Immuiiizatioi} conditions

The probesets differentially expressed between Copaxone® and GLATOPA treatment under both immunization conditions were also examined tor imdeiiying pathway enrichment. In order to obtain appropriate number's of probesets for conducting the enrichment using DAVID, this subset of probesets was filtered using a FC cutoff of LI, to yield 1 6 probesets (representing 112 genes) in the up and 1 17 probesets (representing 7 g aes) in. the ^' .down direction.

Table 4. Probese s differentially expressed witii adj ρ Φ_05 between Glatopa. and opaxon regardless of ImmaaizatiOE cottdiiiea, |FGf > 1.1

Higher expression with Gtutopa freetment vs Copaxone treatment:

Copaxone iiamaaizalioH Giatopa i m iiiiizatioa

Probeset Geae FC P. Value adj.P.V FC P.Valae adj.P.

al Val

1438148_at Cxcl3 1.46348 6.61E- 3.92E- 1.48023 2.8663E 3.2237

882 09 06 445 -07 E-05

1429954_af Clec4a3 1.39185 2.40E- 2.41E- 1.50305 7.055E- 2.6516

754 10 07 534 17 E-13

1426260_a_at UgtlalO /// 1.36367 2.711 - 9.73E- 1.3S362 1.4336E 2.1552

Ugtia6fo /// 95 13 10 399 -14 E-l l TJgtla6a

TJgtla7e / /

Ugtla9 ///

UgtlaS ///

Ugtlal ///

Ugtla2

142595 l_a_at CIec4n 1.35291 1.23E- 2.41E- 1.47736 5.7419E 1.7264

244 1 1 08 124 -16 E-12 i448123_s_ai Tgfbi 1.33965 1.10E- 6.26E- 1.39521 8.091 E 1.4037

43 1.3 10 249 -15 E-l l i426852_x_at Nov 1.33004 6.19E- 6.9SE- 1.38238 1.3047E 7.3557

123 16 12 861 -17 E-14

1456250_x_at Tgfbi 1.32231 3.69E- 8.76E- 1 ,41115 3.0563E 4.0542

143 12 09 -14 E-l l

1419627_s_at CIec4n 1.31551 1.03E- 2.89E- 1.42127 3.1858E 7.5622 906· 07 659 425 -1 1 E-08

I421366_ai CleeSa 1.18436 1. E- 0.00030 1.12353 0:00075 0.0209

155 06 074 802 182 8519

1452279_a:t Cfp 1.18410 7..04E- 1.44E- 1.22465 2.:3 ^' 393E 1.055E

004 12 08 116 -17 -13

1440865 a† Ifitm6 1.18228 1.07E- 5.96E- 1.15517 8.8329E 8.2331

\ 7^ 08 06 632 -07 E-05

1436996_x_at Lyzl 1.17918 1.51E- 2.61E- 1.21889 7.4706E i .3765

145 l i 08 752 -15 E-l l

1437060_ai Olfio4 1.17913 6.64E- 2.38E- 1.19020 6.3249E 6.2284

08 05 04 -07 E-05

1418318 a† Riifl28 1.17795 7.86E- 0.00104 1.24705 L0167E 3.0167

468 06 538 639 -09 E-07

1420572_at Ms4a3 1.17599 5.Ϊ0Ε- 4.89E- 1.16888 L 5839E 6.1057

539 10 07 9 -10 E-08 i420S04_s_ai Clec4d 1.17572 4.47E- 1.01E- 1.22977 1.0Ϊ 09Ε 2.6819

359 12 08 599 -15 E-12

1424542_at S 100a4 1.17546 3.94E- 9.34E- 1.24406 3.323 IE 1.7559

617 07 05 824 -1 1 E-08

1 19594 a† Ctsg 1.17110 2.56E- 4.28E- 1.20025 3..4963E 1 :7717

362 11 08 4 3 -11 E-OS

1419669_at PitnS 1.17104 1.43E- L54E- 1.20188 4.4253E 2.1009

745 10 07 285 -11 E-08

141 S722_at Ngp 1.16784 5.82E- 0.00012 1.16200 1.4628E 0.0001

157 07 678 901 -06 2355

1418992_ai F10 1.16782 S.20E- 0.00016 1.16174 6.8935E 6.7467

814 07 816 479 -07 E-05 Ugtiai / /

Ug†la2

1415947_ai Cregl 1.13217 5.00E- 6.84E^ 1.15572 2.19E- 2.1161

648 .11 08 92 13: E-10

1419873_s_at IJ3-20S 2J9E- 1.01E- 1.19451 9.1357E 9.8102

031 08 05 048 -14 E-l l

1450883_a_at Cd36 1.13071 0.00025 0.01522 1.14976 6.6662E 0.0031

921 312 155 502 -05 2205

1449036_at Riifi28 1.12807 7.96E- 0.00636 1.14200 2.1041E 0.0001

862 05 36 476 -06 6678

1450430_ai Mrc l 1.12804 0.00031 0.01787 1.20214 8.866 IE 1.2457

534 316 843 032 -OS E-05

1422062_at Msrl 1.12681 2.13E- 1.55E- 1.14794 8.7503E 3.7586

601 09 06 209 -11 E-08

1422046 a† Itgam 1.12679 3.96E- 2.4SE- 1.17499 1.6325E 93197

782 09 06 394 -11 E-09

1419082_at Seipinb2 1.12670 8.44E- 0.00666 1.14518 0.00047 0.0147

494 05 889 939 243 9139 i420394_s_ai L >4 /// Gp49a 1.12612 3.26E- 5.25E- 1.16192 9.9405E 1.0426

05 11 08 601 -14 E-10

1436530_at Wfdci? 1.12597 2.63E- 1.82E- 1.15603 3.2477E 2.8002

417 09 06 275 -13 E-10

1460330 a† Anxa3 1.12548 4.56E- 1.77E- 1.14836 3.4387E 2.3498

438 OS 05 21 -12 E-09

1420398_at RgslS 1.12373 1.39E- 0.00160 1.15483 7.035 IE 6.8678

737 05 901 795 -07 E-05

1417061_at Slc40al 1.12294 2.41E- 6.39E- 1.14420 1.4346E 2.8885

31 mi 07 05 83 -09 E-07

?.427994_ai CdlOOlf 1.10382 8.75E- 2.83E- 1.12141 6.2495E 3.9147

009 08 05 878. -12 1 -09

1421326_at 1.10174 6.86E- Θ.00Θ93: 1.13253 7.236E- 1.0427

128 409 137 08 E-05

Lower expression with Glatopa treatment vs Copaxone treatment:

Copaxone immunization Glatop immunization

Probeset Gene FC P. Value aclj.P.V FC P. Value adj. P.

al Val i422470_ai Baip3 -1.5561457 USE- 9.05E- -1.5688967 7.3932E 1.6831

OS 06 -09 E-06

14503S7_s_ai Ak4 -1.551748 8.76E- 2.S E- -1.5166541 1.1138E 1.5085

08 05 -07 E-05

1435659_a_at Tpil -1.5127684 6.35E- 2.29E- -1.6118948 7.8498E 1.7614

08 05 -09 E-06

1437842_at Plcxdl -1.4920022 7.98E- 0.00016 -1.4434761 1.7078E 3.32E- 07 5 1 -08 06

1415 18_a_at. Tpil -1.4619861 1.30E- 3.94E- -1.5714854 8.9012E 1.9208

07 05 -09 E-06

1452927_x_a Tpil -1.4465645 9.74E- 3.07E- -1.545345 1.164E- 2.4081 t 08 05 08 E-0

1451461_a_af Aidoc -1.4224336 3.9SE- 1.59E- -1.4620829 6.4S79E 1.5106

08 05 -09 E-06

1421830_at Ak4 -1.4046381 3.51E- 8.85E- -1.327857 2.4308E 0.0001

07 05 -06 8869

1425245_a_at Rgsl i -1.3484295 4.13E- 9.69E- -1.303955 8.7087E 0.0005

35 07 05 -06 6191 i436538_af. Ankrd37 -1.3301616 4.43Έ- 0.00067 - 1.5300535 3.2Ί7Ε- 8.3385

06 08 09 E-07

1439I48_a_at Pfkl -1.3249924 5.901·- 2.14E- -1,3941757 6.2765E 1.993

08 05 -10 E-07

1423747_a_at Pdkl - 1.3150775 3.96E- 9.36E- -1.3513424 3.1518E 5.344E

07 05 -08 -06

141 S652_at Cxcl9 - 1.2966299 0.00109 0.04392 -1.3790931 2.5476E 4.5415

177 502 -08 E-06

1450269_a_at Pfkl -1.2944646 1.64E- 7.96E- - 1.3308814 7.1659E 1.6406

08 06 -09 E-06

1449361 a† T x21 -1.2763681 1.69E- 0.00189 -1.3458518 3.4233E 1.7717

05 966 -1 1 E-08

1417S64_at Pgki - i .2762344 1.1 SE- 3.60E- -1.2679882 8.3021E 7.S995

07 05 -07 E-05

1435S36_at Pdkl -1.2696324 8.79E- 2.83E- - 1.348825 1.794E- 6.687E

08 05 10 -08

1418649_ai Egln3 -1.267839 0.00013 0.00937 -1.2709358 2.9539E 0.0015

302 427 -05 9359

1428306 a† Ddit4 - 1.2598336 1.43E- 7.23E- -1.266315 i 4.5686E 1.5377

08 06 -10 E-07

1416069_at Pfkp - i .2410744 5.S0E- 2. BE-1.2871092 1.2567E 5.1527

08 OS -10 E-08

1451 149_at Pgm2 -1.2393865 1.3 IE- 6.79E- - 1.21904 1.0604E 1.4529

OS 06 -07 E-05

1452094_at P4hal -1.2348958 5.01E- 0.00011 -1.2449254 1 3 ? 191·. 1.7871

07 241 -07 E-05

05 401 07 -05

I418S29_a_at. EHO2 -1.1€63 31 0.0001 1 0.00844 - 1.1341357 7,:457SE 0.0034

567 127 -05

1 1 7679 i GS1 -1.1658749 9.47E- 3.01E- -1.2278918: 5.13 ΠΈ 4.06E-

08 05 -13 10

1456I03_at Pml - 1.1657435 3.75E- 0.00058 -1.1792807 3.6743E 1.2554

06 088 -10 E-07

1419737_a_af Ld a - 1.1642904 2.3SE- 6.39E- -1.1817157 1.363 IE 1.7806

07 05 -07 E-05

1449324_af Eroll -1.163418 5.06E- 0.00074 - 1.1544889 3.0959E 0.0002

06 109 -06 3194

1431997 a† 3000002C - 1.1629206 1.66E- 0.00029 -1.182533 5E-07 5.1485 lORik 06 603 E-05

1422612_at - 1.1621 179 3.66E- 0.00056 -1.151743 1.1408E 0.0007

06 952 -05 0871

145589S_x_a Slc2a3 -1.1562098 6.71E- 0.00092 - 1.1023425 0.00015 0.0060 ί 06 235 Oi l 5016

1434974_at Pdkl - 1.155837 2.0 lE- 0.00215 -1.1354522 0.00012 0.0050

05 902 121 7591.

1437052_s_at Sic2a3 - 1.15534 2.19E- 0.00036 -1.1068932 5.1794E 0.0025

06 816 -05 5575

1440047_at — - 1.1549135 1.95E- 0.00033 -1.1487473 3.3527E 2.S002

06 673 -13 E- 10

1438659_x_a ChcM6 -1.1531792 5.55E- 0.00477 - 1.1825929 2.7788E 0.0002 ί 05 616 -06 1063

1426519_at P4hal -1.1528389 0.00104 0.04268 -1.219446 1.8537E 0.0010

863 439 -05 5961. 1419022_a_ai Eaolb // - 1.1519498 6.19E- 0.00013 -1.1714238 5.1969E 5.3029 Eaol 07 -07 E-05

145008 lji_a Gpi ! -.1.1505073 8.30E- 4.8GE- - 1.1485586 2.412E- 4.3514

09 06 08 E-06 i4I9G29_a:i Eroll -1. 29369 0.00032 0.0184 -1.1648231 1.1546E 0.000 ^"

645 401 -05 1531

1420997_a_at Gpil - 1.1419641 1.56E- 7.65E- -1.1462237 1.8 1E- 3.5834

08 06 08 E-06

1450976_at Ndigl - 1.140721 3.43E- 0.00325 -1.1426626 1.2701E 0.0007

05 S09 -05 7517

I444283_ai Gimap? -1.139852 3.69E- 9.09E- - 1.1302292 1.0258E 9.2526

07 05 -06 E-05

1424339 a† Oasl 1 - 1.1374824 0.00102 0.04227 -1.2255553 1.7618E 4.9975

451 484 -09 E-07

1434S14_x_a Gpi i - 1.1355869 4.16E- 1.65E- -1.1318441 4.685E- 7.31 14 ί 08 05 08 E-06

1441 105_at Gml l l lO -1.1354878 9.88E- 0.00123 - 1.1403744 1 ^" 1961- 4.9472

06 447 -07 E-05 i429270_a_at Syce2 -1.1346884 3.41E- 0.00053 -1.1201579 1.1833E 0.0007

06 364 -05 3004

1423413_at Ndrgl - 1.1336228 0.00037 0.02066 -1.1583939 1.2384E 0.0007

397 -05 5783

1417262_at Ptgs2 -1.1332284 0.00065 0.03118 -1.1083538 0.00196 0.0444

454 709 I f 7118

1417034_ai Trappc6a -1.1314281 3.59E- 8.94E- - 1.1448239 1.4848E 1.8823

07 05 -07 E-05

14 i9030_ai Eroll -1.1297069 0.00030 0.01760 -1.1199521 0.00081 0.0222

These probesets were subjected to pathway enrichment using the NTH DAVID platform tor pathways in GO (Gene Ontology: Ashbumer et al, 2000) and Kegg ( anehisa et al, 2000) (Figure 10 and Table 5). 72 pathways were significantly enriched (adj p < 0.05) among probesets increased in expression, and 19 pathways among probesers decreased in expression with GLATOPA relative to Copaxone® treatment. These pathway enrichments lend further robustness and biological relevance to the observed differences.

Table 5. Selected Pathways enriched among top probesets differing between GLATOPA and Copaxone® under both immunization conditions.

Pathway

Glycolysis Glucose metabolic process

Glycolysis / Gluconeogenesis

Hexose catabolic process

Glucose catabolic process

Monosaccharide catabolic process

Hexose metabolic process

Monosaccharide metabolic process

Cellular carbohydrate catabolic process

Alcohol catabolic process

Carbohydrate catabolic process

Extracellular region

Carbohydrate binding

Immune response

Extracellular space

Extracellular region part

C!iemotaxis

Defense response

Generation of precursor metabolites and energy

Pattern binding

Heparin binding

Locomotory behavior

Phagocytosis

Leukocyte migration

Giycosaminoglycan binding

Leukocyte chemotaxis

Positive regulation of phagocytosis

Inflammatory response

Cytokine-cytokine receptor interaction

Pentose phosphate pathway [CELLREF]

Galactose metabolism [CELLREF] Carboliydraie kitiase acti vity [CELLREF]

Myeloid leukocyte activation

Cheiriokine recepto binding

Regulation ©f ^' cype I hyperseusiUtivity

Defense response to Gram-negative bacterium

Positive regulation of foam cell ciifierentiaiion

Pathways enriched among probesets increased in expression with GLATOPA relative to Copaxone® included immunological pathways such as cytokine-cytokine receptor interaction (adj p 2.066-4), inflammatory response (adj p < 7.78e-5), hematopoietic cell lineage (adj p < 3.75e-4), regulation of adaptive immune response based on somatic recombination of immune receptors built from iminimogiobiilm sitpeifamily domains (adj p<0.048), and leukocyte migration (adj p < 2.82e-7), as well as immunoglobuli binding (adj p < S.9e-4) and regulation of type I hypersensitivity (adj p < 0.025). Figure i 1 shows the probesets imdeiiyiiig the enrichment of the cytokine-cytokine receptor interaction pathway among probesets differing between GLATOPA and Copaxone ¹ !', of particular interest since this pathway is modulated by Copaxone® and relevant for its mechanism of action. Figure 12 shows probesets underlying enrichment of the pathway: regulation of adaptive immune response based on somatic recombination of immune receptors built from immunoglobulin superfaniily domains. Pathways enriched among probesets having lower expression with GLATOPA versus Copaxone® included glycolysis (adj p < 1.7e-37) and multiple additional metabolism-related pathways.

Validation via qRT-PCR from spkiioeytes

Based on the findings from the splenocyte microarray data, 19 genes (listed in Table 6) were chosen for testing by qRT-PCR in the Copaxone®-immunization samples based on potential biological interest Four reference transcripts (Aetb, Ppib„ Top, and Rpll 3a) were averaged for use as overall reference, and mannitol control samples were used as calibrator.

The qRT-PCR results confirmed the significant differences in expression between GLATOPA and Copaxone® treatments for all 19 genes (as described in Table 6): 17 with higher expression with GLATOPA relative to Copaxone®, ami 2 with lower expression, in consistent direction with the array results. Results for several of these genes are illustrated in Fig. 13A-D.

Table 6. qRT-PCR Results for all 19 Tested Genes p.valo adj.p iold Change ( Q

space)

CxclS L43E-08. 2.26E-0S 1.58

Clee4a3 8J55B-11 2.03E-10 ί .44

Ccl 4/23E-14 2.68E-13 1.41

Tgfbi L 4B-14- 1.37E-13 1.38

Ciec4n 2.84E-13 1.08E-.12 ί 3

MnipS 1.22E-09 2.32E-09 1.28

Mm l2 1.32E-07 L94E-07 1 25

CsSr 7.50E-15 1.37E-13 1 25

Csfir 1.69E-11 4.59E-11 1 21

IUi-2 2.09E-13 9.94E-13 1.21

Fcgi'3 5.10E-13 1.62E-12 1.19

Mmp9 1.65E-G4 1.S5E-04 1.17

Fcerl 2.54E-05 3.22E-05 1.14

116 9.35E-07 1.27E-06 1.13

Illb 3.86E-05 4.59E-05 1.10

Fcgr2b 4.82E-04 5.08E-04 1.07

I17r 7.98E-03 7.98E-03 1.06

Tbx21 4.19E-10 8.85E-10 0.79

Cxcl9 1.90E-09 3.29E-09 0.76

Higher expression of Thl7 related genes 116, Illb, and il7r was confirmed as significant in the qRT- PCR data. In addition, many of the genes tested participated in pathways of interest that were enriched among differences between GLATOPA and Copaxone® in the microarray studies. For example, Cxcl3 and Ccl2 encode chemokmes relevant for leukocyte accumulation during inflammation. These genes participate in the chemokine receptor binding pathway, enriched among probesets increased in expression with GLATOPA relative to Copaxone® (adj p < 0.002).

In particular, GLATOPA increases Ccl2 expression, while Copaxone® does not. Fig. 22B. Cc!2 is increased by GLATOPA (FC 1.20, adj p<0.012) but not Copaxone ^® (adj p>0;74) vs control and is up with GLATOPA relative to Copaxone ^® (FC 1.17, adj p<0.0003) in Copaxone ^® immunization array data; similar for GLATOPA immunization. It has been shown that positively charged nanopartides significantly stimulate chemokine (C-C Motif) Ligand Ccl2 expression (a chernoattractant involved in recruitment of immune cells), while negatively charged ones do not (Fromen CA, et al. 2016)). Fig. 22A. Positive surface charge distribution is mechanistically linked to imiiHiomodiilation and cytotoxicity according to extensive literature, potentially linking fee observed immunomodulation signature of GLATOPA to its higher: positivity in surface charge.

Csf 1 r ₅ ;Csf3r, and B lr2 each encode cytokine receptors, and participate in the cytokme-cytokme receptor interaction pathway discussed above, as well as the cytokine, binding pathway, also enriched among such differences between GLATOPA and Copaxone® (adj p 0.009). Fcgr3, Fcgr2b, and Fcerlg. ifiummogiobulm receptor genes, participate in die similarly enriched imnaHioglohii!iii binding pathwa (adj p < S.9e-4), Ciec4n (dendritic cell inhibitory receptor 3), as discussed above, is known to allow signaling through Fcerlg. The three irnmunoglobu!in receptor genes also participate in the regulation of type I hypersensitivity pathway, also enriched among GLATOPA-Copaxone®differences (adj p < 0.025). These immunoglobulin receptor genes, as well as 116, also participate in the regulation of adaptive immune response based on somatic recombination of immune receptors built from immunoglobulin superfamily domains pathway described above.

Several matrix metalloproieinase genes were confirmed as differentially expressed, including Mmp9, Mm l2, and Mmp8. As noted above, Mmp9 also diifered m expression level between GLATOPA and Copaxone® treatment in the THP-1 experiment, as well as between Copaxone® and additional purported generics POLIMUNOL and PROBIOGLAT in prior studies ( olitz et al, 2015; Hasson et al, 2016). As described above, MMP9 is reported to increase immune cell access to the CNS, and high levels have been associated with MS {Kouweiihoven et al. 2012). MmpS plays roles in the regulation of innate immunity (Tester et al, 2007), as well as in macrophage differentiation and polarization (Wen et al, 2015), and Mrn l2 may have a role in antiviral immunity (Marchant et al, 2014).

Tbx21 was less highly expressed with GLATOPA relative to Copaxone® treatment. This gene encodes transcription factor T-bet, which can control mteiferon-gamma expression and development of Thl- polarized T cells (ITniProt). Cxcl9 is an inflammatory cytokine that was also lower in expression with GLATOPA relative to Copaxone®. For both genes, the effect of Copaxone® was to increase the level of expression relative to mannitol control, whereas GLATOPA increased the expression level less than did Copaxone®. Thus a number of genes important for inflammatory processes are represented among the differences between Copaxone® and GLATOPA. What the sum total of these effects would be in terms of downstream safety and efficacy is not clear and would need to be tested.

hi summary, all 19 of the tested genes, chosen for examination due to their immunological functions and participa tion in biological pathways, were found to be significantly differentially expressed by qRT- PCR, confirming the results observed on the microarray.

Gene expression profiles in THP-1 cells

The THP-1 human monocyte cell line, primarily comprised of Antigen Presenting Cells capable of interacting with T and B cells loaded with (auto)anti ens (Chanput et al, 2014), was used for in vitro experiments to characterize unbiased genome-wide expression profiles of treatment, with GL ATOPA and .Copaxone®. The robustness of this model, system to diaracTerizafiou of Copaxone® mode of action and u orted generic differentiation has been studied ^' extensively, including validation in _. primary human monocytes (Kolitz ei al, 2015). In order: to ensure robustness and. repeatability of the herein reported results, two independent experiments were earned out in. two independent labora tories, one in Israel and one in Hungary.

Iii eac o the studies, genes known to be modtilated as part of Copaxone® mechanism of action were affected by Copaxone® treatment versus maiinitoi control.

These included anti-inflammatory IL1EN, for which in each study three probesets were unregulated by Copaxone® (FC 1.3, FDR p < 4e-18 in Hungary study; FC 1.3, FDR < 1.2e-16 in Israel study) and ILIORA, the recepto tor anti-inflammatory cytokine IL-IO, also upregulated in both studies (FC 1.8, FDR p < 2.2 e-27 in Hungary study: FC 1.6, FDR p < 3.9e-17 in Israel study). These observations are consistent with prior reports in this model system (Hasson et al, 2016).

In each of the experiments, similar to the observations in the splenoeyte studies discussed above, both Copaxone® and GLATOPA treatment resulted in modulation of thousands of genes, many of which were commonly modulated between Copaxone® and GLATOPA treatment. However, in addition, similar to the splenoeyte studies, in both of the THP-1 studies a subset of probesets (65 total, 64 up, and 1 down with GLATOPA relative to Copaxone® after filtering for presence/absence calls) differed between GLATOPA and Copaxone® (FC range -1.06 to 1.33).

Several of these probesets were for genes differentially modula ted by GLATOPA also in the splenoeyte study. For example, IL7R, which was also seen to differ between Copaxone® and GLATOPA in the splenoeyte study, was also expressed more highly with GLATOPA treatment relative to Copaxone® treatment in both of the THP-1 studies, for multiple IL7R probesets. As shown in Figure 14, the same was true for MMP9, for the single probeset present on the chip.

The 65 probesets overlapping that differed significantly (FDR p < 0.05) in both independent studies in the two laboratories (representing 57 DAVID IDs) were examined for underlying pathway enrichment Performing a hypothesis-free pathway analysis across all of GO and Kegg, the regulation of adaptive immune response pathway, and specifically the regulation of adaptive immune response based on somatic recombination of immune receptors built from iimiiunogiobulin superfamily domains pathway, were significantly enriched among these probesets (adjusted p value < 7. ie-4 and 1.2e-3, respectively; Figure 15). A targeted Gene Set Enrichment Analysis was also performed to assess emichment of the cytokine-cytokine receptor interaction pathway observed to be enriched in the splenoeyte studies in the comparison between GLATOPA and Copaxone® in each study separately. This pathway was significantly enriched (p<0.001) inborn studies. The significantiy modulated probesets participating in the leading edge of this pathway from each study are illustrated in Figure 16. Both the adaptive immune response athwa s and the cytokme-cyto iie receptor interaction pathway as well as genes. CD40, CCL5, TL 1 OA, TNFSF 13B and MMP9 were enriched in comparing; Copaxone's. ¹ with other purported generics, nicluding ^: POLI U OL and PR.OBIOGLAT (Kolitz et _. al 2915; Hassoa ef a 2016) .

The physieocheniieal and biological observations are linked by immunological effects of altered charge properties of particles taken -up by .Antigen. Presenting Cells (APCs). Positive surface charge dis.firi.bari.on. is mechanistically linked to innmrnomodulation and cytotoxicity (Fromen ei al. 2016), For- -example, positively charged nanoparticles significantly stimulate chemokine Cel2 expression (a chemoattractant involved in recruitment of immune cells), while negatively charged ones do not (Figure 18, left). Notably, GLATOPA increases Cell expression, while Copaxone ^® does not (Figure 18, right). These observations suggest a potential link between the observed innnunomodulation signatures of GLATOPA to its higher positivity in surface charge.

Robustness o f results

The robustness and biological relevance of these results was clearly demonstrated in the following ways:

* Strict statistic al significance: Hundreds of genes differed between Copaxone® and GLATOPA with adjusted p < 0.05, a universally accepted threshold for statistical significance applied after a gold standard adjustment, for multiple hypothesis testing (false discovery rate. FDR) that, filters out noise by reducing the number of differences to only the strongest signals.

* Multiple probesets: Multiple probesets differed between Copaxone® and GLATOPA for many of the differentially expressed genes. This means mat in cases where they mieroarray chip has two different "detectors" for a given gene, both detectors (i.e. probesets) frequently identified the same genes as differing between Copaxone® and GLATOPA - an observation that further reduces the likelihood of any spurious signals.

* Pathway enrichment: The probesets that differed between Copaxone® and GLATOPA were significantly enriched for biologically relevant pathways. In the unlikely event that the hundreds of genes passing the strict p-value threshold above were simply a random set of genes (i.e. noise), they would be scattered across the genome. Instead, the differences fell non-randonily into specific pathways with a strict threshold of adj. p < 0.05 statistical significance applied also to the pathway enrichments. The results consistently indicated differences in expression of immunological genes and pathways between Copaxone® and GLATOPA treatment. The consistent pathways and directionality both support the robustness of the observed differences.

* Multiple experiments: The differences between Copaxone® and GLATOPA were observed consistently across multiple experiments. In the mouse splenocyte data, separate experiments were performe immvmizmg the mice first with, either Copaxone® or GLATGPA, and then comparing the two drags. The THP-1 studies were repeated in two separate late in difierent countries. Ia each ease, flie magnitude and biological relevance of the observation was _: similar across - both studies, i½dmg lipregiilaoon of iinmnne response with GLATOPA, which wo ud not happen if the differences were driven by noise,

* Multiple model systems: Differences between Copaxone® and GLATOPA were observed in both mouse- splenoeytes and human monocytes, in both cases supporting ^re ulation of anime response-associated pathways with GLATOPA relative to Copaxone®, Such consistent differences across model systems are extremely unlikely to occur by chance.

* PCR confirmation: Key differences between Copaxone® and GLATOPA in the splenocyte study were confirmed using a separate assay technology, PCR, which eliminates any possibility that the specific inicroarray technology utilized for the initial assay could have been responsible for the observed differences between Copaxone-D and GLATOPA.

Six layers of robustness support the observation of a biologically relevant differential modulation of immune response genes and pathways by GLATOPA relative to Copaxone®.

Discussion

A mouse splenocyte model and a human monocytic cell line (THP-1) were used to model each of these interdependent cell types in validated model systems. An unbiased genome-wide approach was used to assess effects of Copaxone® and GLATOPA. fn splenocytes, both gk iramoids modulated thousands of genes relative to controls, including genes known to be associated with Copaxone® 's mechanism of action, and demonstrated in prior publications, providing validation of the model systems. Despite shniiarity in modulation of many genes. Copaxone® and GLATOPA differed significantly in then effect on hundreds of genes. 7% of genes modulated by Copaxone® were differentially modulated by GLATOPA re!ative to Copaxone®, and specifically multiple immunologically-relevant gene and pathway differences were observed (Table 7 and Table 8, Figures 1 and 2).

Table 7. Pathways in Kegg, Gene Ontology, Biological Processes, Cellular Component and Molecular Function Enriched Among Top Probesets Higher in Expression with GLATOPA Relative to Copaxone® Treatment Under Both Immunization Conditions.

Term Count PVsdne Fold Beiijam i

Enrichment

Category: GO BP

GO : 0006955 - - illumine response 25 L34E-13 6.51 I .16E-10 Term Coiiat P Valu Fold BefljamiQi

EHrichiBMit

GO::0042330-"ta:?ds 13 3.17E-12 17.87 1.3 E-09

GO: 0006935~ hexB«texis 13 3J7E-I2 17.8? 1.37E-09

GO:OO06952--dei¾nse respojiSe- 20 l,4SE-l i 7,2! 4.Ϊ8Ε-09

G-O:O950900- etiiioc fe aiigi'aiioa 9 1.30E-09 24.75 2..S2E-07

GO::00o0326~eeli chemotaxis 8 2.59E-09 ^■ n-M 4.S0E-Q7

GO:0030595~ leukocyte chemotaxis 8 2.59E-09 31.26 4.50E-07

GOi0007626-4oeoinoiory behavior 13 5.26E-09 9,75 7.61E-07

GO:O0O69O9~-phagocyiosis 8 1.89E-07 18.00 2.34E-05

GO:0050766~positive regulation of phagocytosis 6 2.76E-07 37.12 2.99E-05

GO:0006928~-ceil motion 13 5.26E-07 6.4S 5.07E-05

GO 10050764-regii] at ion of phagocytosis 6 6.82E-07 31.82 5.92E-05

GO:O0O7610~-behavior 13 1.06E-06 6.07 7.67E-05

GO 10006954-4nflamma:tory es use 11 9.86E-07 7.85 7.78E-05

GO:0009611 -response to wounding 13 1.21E-06 5.99 8.09E-05

GO 10010324~nienibiane inva gina tion 11 2.50E-06 7.10 1.55E-04

GO:O0O689?~-eridocytosis 11 2.50E-06 7.10 1.55E-04

GO:00 5807~positive regulation of endoc tosis 6 3.76E-06 23.45 2.17E-04

GO: 0006911 -phagocytosis, engiilfment 5 5.81E-0S 37.12 3.15E-04

GO:0002252~ii-iaiim€ effector process 9 6.49E-06 8.79 3.31E-04

GO:0042742~-defense response to bacterium 7 7.86E-06 14.05 3.79E-04

GOi00226 Ϊ O -biologicai adhesion 12 9.72E-06 5.43 4.44E-04

GO : 0007 i 55-ceil adhesion 12 9.72E-06 5.43 4.44E-04

GO 10010044-"inenibraiie organiza don 12 1.37E-05 5.24 5.65E-04

GO:0030593-iientrophii chemotaxis 5 L34E-05 30.94 5.S2E-04

GOi0050829~deieiise response to Gram-negative 4 2.24E-05 59.40 8.84E-04 bacteiiuin

Gi3:0030i00~-regiilatioii of endocytosis 6 4.10E-05 14.85 0.0015

GO: 0002443 -leukocyte mediated immunity 7.28E-05 9.62 0.0026

GO:001647?-ceil migration 9 1.26E-04 5.86 0.0043

GO:0060627~i¾gulatioa of vesicle-mediated transport 6 1.92E-04 10.87 0.0062

GO: 0051674- localization of ceil 9 1.90E-04 5.52 0.0063

GO:0048870~cell motility 9 1.90E-04 5.52 0.0063

GO:O032963-coliagen metabolic process 4 2.56E-04 29.70 0.0079

GOi0044259-muiticelloiar orgariismal macromoiecule 4 3.49E-04 27.00 0.0104 metabolic process

GO:000 617~respoiise to bactexH&ii 7 4.88E-04 6.84 0.0140 Term Coast P Value Fold Be-ajaiainl

EHriCiUBMit

GO;0031130~pssitive reg atioii of cellular ^■ 5.24E-04 .6:75 0.0141 c inpciieBt orgaHizatioo

G0:¾£K)2274 _^ my«ioid teukoey aetivatien 5 S.42E-04 12,80 0.0141

G :0010744---positive regiilation of f&am eeil 3 5.22E-04 74.24 0.0145 differ entiafioii

4 5.53E-04 .22.84 0.0Ϊ50 process

GO: 0001878- -response to yeast 3 0.0010 55.68 0.0253

GO:::G001810~-reguIation of type I hypersensitivity 3 0.0010 55.68 0.0253

GO:0010743- -regiilation of foam cell differentiation 3 0.0010 55.68 0.0253

GO::0002S22-reguIarion of adapiive immune response 5 0.0020 9.05 0.0482 based on somatic recombination of immune receptors

built from ininranoglobulin supertamily domains

GO::0002S19-reguIarion of adapiive immune response 5 0.0020 9.05 0.0482

Category: GO < <

GO:0005576 -extracellular region 33 3.66E-I6 5.48 4.30E-14

GO:0005615--exiracellulsr space 19 8.15E-12 8.04 3.50E-10

GO:0044421 -extracellular region part 21 7.9 IE- 12 6.90 5.10E-10

GO:00099S0~ceIl surface 10 6.96E-04 4.04 0.0222

GO::00 1224-infrmsic to membrane 48 0.0017 1.45 0.0421

Category: GO M

GO:0030246~c¾:bohydrate binding 18 4.15E-I4 1.2.07 8.99E-12

GO:0008201 - -heparin binding 9 2.59E-10 29.35 1.87E-08

GO:::0030247-pcilysaccliaride binding 10 2.58E-I0 2242 2.79E-08

GO:0001871 - -patters binding 10 2.5SE-10 22.42 2.79E-08

GO:::0005539-glycosaminog1ycsi5 binding 9 2.35E-09 23.06 1.27E-07

GO:0GG5529~sugar binding 9 2.23E-06 10.09 9.70E-05

GO:0004175-endopeptidase activity 12 3.81E-06 5.98 1. 8E-04

GO:00I7171-serine hydrolase activity 8 7.80E-06 10.63 2.121 -01

GO:0008236~serkie-type peptidase activity 8 6.86E-06 10.83 2.13E-04

GO:0004252~sexing-type endopeptidase activity 9.54E-06 13.57 2.30E-04

GO:0005125 -cytokine activity 8 1.59E-05 9.57 3.45E-04

GO:0005509-Ci(fc:iuin ion binding 15 2.17E-05 3.89 4.27E-04

GO : 001 865-inminnoglobiilin bindiag 4 4.91E-05 47.83 8.88E-04

GO:0008009~cljeniokine activity 5 8.90E-05 19.93 0.0015

GO:::0042379-cheiriokine receptor binding 5 1.12E-04 18.88 0.0017

GO:00700I 1- -peptidase activity, acting on L-anniio 13 L5IE-04 3.72 0.0022 acid peptides.

10' Term Const P Value Fold Beajaioinl

Enrichis At

GO::0Q08233--peptidase activit 13 2.26E-04 ^■ 3,56 0.0031

QQ-M 1 955-cytokine binding: 6 6,93E-0 8,28 0.0088

G0:OQ 19864-TgG binding 3 0.00Π ^' 53,81 Q-0I33

GO:0032403~proteia complex binding 5 o.ooie 9.70 Ο.ΟΪ 78

GO;0(KM857 ^; --enzyme iubibitor -activity 0.0040 5.59 0,0428

Category: Kegg

mmu04060 :Cy fokine-c ytokine receptor interaction H 3.56E-06 6.40 2.06E-04 ni ii04640:Heniaiopoieik cell lineage 8 1.29E-05 9.40 3.75E-04

Count: Number of probesets in pathway

* Correction for multiple hypothesis testing

TaMe 8. Pathways Enriched Among Top Probesets Lower in Expression with GLATOPA versus Copaxone® Under Both Immunization Conditions

Term Coiisr PValvte Fold Benjairrkii

Enriehirierri

Category: GO BP

GOi0006006-gracose metabolic process 19 4.70E-21 24.97 2.50E-18

GO:00Q6096~-giyeoiysis 14 1.42E-20 55.88 3.76E-18

GO:00193 iS-hexose metabolic process 20 4.26E-20 19.60 7.53E-18

Gi3:0005996~-iBoriosaccliaiide metabolic process 20 3.34E-1 17.67 4.44E-17

GOi0019320- iexose cataboiie process 14 6.23E-19 44.38 6.62E-17

GO:000600?~-gi«eose catabolie process 14 6.23E-1 44.38 6.62E-17

GOi0046365-"monosaeeharkle cataboli process 14 9.84E-19 43.11 8.71E-17

GO:0Q44275~-ceihiIar carbohydrate cataboiic process 14 2.34E-18 40.78 1.78E-16

GOi004 164~aicoaoi catabolic process 14 3.53E-18 39.71 2.34E-16

GO:0016052~-cfflbohydrate catabolie process 14 1.17E-16 31.43 6.55E-15

GO 100060 1 -generation of precursor metabolites and 16 6.60E-11 9,22 3.50E-09 energy

Category: GO MF

GO:0016702~oxidoreductase activity, acting OH single 5 3. ΠΕ-04 14.78 0.0164 donors with incorporation of molecular oxygen,

incorporation of two atoms of oxygen

GOi00i6701 ~oxidoied«etase activity, acting on single 5 3. ΠΕ-04 14.78 0.0164 donor's with incorporation of molecular oxygen

GO:0005125~cyfolcine activity 6 1.43E-04 11.53 0,0225

GO:00 I9200-'-carbohydiaie kinase activity 4 3.03E-04 28.83 0.0239 Category: Kegg

mmuOOOi 0 Glycol sis / Giucoiieogeaesis 16 2..59E-19 27,84 1,37E-17 nimuOOOS&Pentose phosphate pathway 6 7.26E-0S 19.83 1..92E^Q4 iiimiiG0Q51 :Fmctose sad. maaaose jnetabolism 5,04E-05 33,68: ^' S E-04

$mHuO0052 : Galactose metabolism ^' 5 L68E-04 16.53 0.002

Example 2

As described above, a mouse splenocyte system and human monocyte (THP-1) cell liae were utilized. Unbiased genome-wide gene expression studies were conducted to examine effects of treatment with Copaxone-® or POLIMLI OL in each model system.

Data From Reciprocal Mouse Splenocyte Experiment

Reciprocal mouse spleiiocyie experiment with Copaxone'® and POLIMU OL was conducted as described above in the methods section and as depicted in Figure 19.

Analyzing spJenocytes from mice immunized as well as activated with Copaxone®, 16,647 probesets were significantly modulated relative to mannitol: 8342 (representing 5101 genes) increased in expression level (throughout, termed upregukted) and 8305 (5208 genes) decreased in expression level (throughout, termed downregulated). Table 9.

Table 9. Numbers of probesets modulated by each treatment sequence

Comparison for immunization labeled "corresponding to treatment" refers to treatment and immunization with POLIMUNOL versus treatment and immunization with Copaxone®.

Adj p: adjusted p value, collected for multiple hypothesis testing using Benjamin! Hochberg.

After imposing a conservative fold change filter of IFCj > 2, 411 probesets were upreguiated by Copaxone® relative to niarsnitol (in Copaxone® immunized mice). These probesets enriched for 76 pathways, many of which are immunologically relevant, and include relevant aspects of Co a one's MoA such' as .the eyiokine-eytofcme receptor interaction pathway identified also. in the ΤΉΡ-l data described, below.

Similarly, after filteiiag by jFQ 2, 485 dowiregulated probeseis were detected, enxlc iig for 56 pathways, a number of : which are associated with modulation of the immune system. Both the iipregulated and dowitregula ted pa thways are depicted in Figure 20 and listed i Table 10,

Table 10. Selected pa hways enriched among . ^' top probeseis moflniafed b Copaxone® relativ to mannitol control treatment on splenocytes from Copaxone! -immHiiized mice.

Pathway

Membrane part

Intrinsic to membrane receptor activity

Immune response

Transmembrane receptor activity

Irramme system process

Extracellular region

Carbohydrate binding

External side of plasma membrane

Hematopoietic cell lineage

Response to external stimulus

Leukocyte migration

Chemokine receptor binding

C} okine-cytokine receptor interaction

Cytokine activity

Extracellular space

Immune response

Jak-STAT signaling pathway

Immune system process

Sterol biosynthetie process

Steroid biosyntlietic process

Response to stimulus

Cell surface

Cytokine binding

Cholesterol metabolic process

Chemokine activity

Negative regulation of hormone secretion

Isoprenoid biosyntlietic process

Positive regulation of peptidyl-serine phosphorylation

As expected, Copaxone® treatment iipregulated key anti-inflammatory cytokine genes 114, as well as markers of regulatory T cells, Foxp3. Complementarity, Copaxone® downregiilated proinflammatory cytokine genes such as 1112a (Figure 21).

Analysis of splenocytes from mice immunized as well as activated with POLIMiJNOL revealed that thousands of probesets were modulated similarly by POLIMUNOL and Copaxone®, including several mechanism of action genes such as 114, Foxp3 and 1112a (Figure 21). When spienocytes from Copsxone -imimmized mice were activated ex -vivo with either POLIMUNOL or Copaxone® and. compai ed to each other while eoatiOUiag for maa to!, .hundreds of piofeeseis differ si ffcantly. as showu in Figure 22. Further, hundreds of probesets differ significantly between POLIMUNOL aud Copaxone® treatment in m<¾vidual lot to lot comparisons (Table 1 1).

TaM 11. Comparisons between Copaxone® k*is ₅ verses each oilier and versus POLIMUNOL.

Treatment Comparison Mannitol-corrected Dirt

Copaxone* Immunization

POLIMUNOL vs. Lotl 21S 226

POLIMUNOL vs. Lot.2 97 105

POLIMUNOL vs. Lot3 95 107

Lotl vs Lot2 0 0

Lotl vs Lot 3 0 0

I ol2 vs Lot 3 0 0

POLIMUNOL Immunization

POLIMUNOL vs. Lotl 146 318

POLIMUNOL vs. Lot2 48 153

POLIMUNOL vs. Lot3 134 260

Lotl vs Lot2 0 0

Lotl vs Loi3 0 0

Lot2 vs Loi3 0 0

THP-i data

POLIMUNOL vs. Lotl 398 564

POLIMUNOL vs. Lot2 177 307

POLIMUNOL vs. Lot3 48 90

Lotl vs Lot2 0 0

Lotl vs Lot3 0 0

Lot! vs Lot3 1 0 Shown are numbers of probesets significant by FDR in each limma. comparison.

Gefte expression profiles from splenocytes of Copa one#-immoiiized mice treated ex vivo with Co a one® were compared with gene expression profiles ironi splenocytes of POLIMUNOL- HBffiUuized mic treated ex vivo Willi POLJMUNOL. 235 probesets were difiereHtiaiiy expressed after imposing a fold change filter of jFCj > 1.2 and correction lor multiple .hypothesis ^' testing (FDR p value b 0.05); 206 of those were upregmated and 30 were dowiiregnlared in fee POLIM.U GL- iiiamiiiized mice ^■ treated e vivo with POIJ O OL relative to Copaxone#-in¾nioiized mice treated ex vivo with Copaxone®.

Table 12 below shows top probesets significantly downregukied by POLIMUNOL-inammized mice treated with POLIMUNOL relative to Copaxone^-immumzed mice treated with CopaxonetXTab!e 12. Top probesets significantly dowmegalated by POLIMLTNOL-imnmiiized mice treated with POLIMUNOL relative to Copaxoiie®-raim«iiized mice treated with Copaxone®.

Table 13 below shows top probesets significantly upregulated by POLIMIjNOL-ii«maiiized mice treated with POLIM0 OL relative to CopaxoasiMnimunieed miee treated with Copaxone®,

Table 13. Top probesets sigHifiea ly «preg¾iaied by POLIMUNOL-imittHaizeti mice treated will POLIMU OL relative to Co axoHe^-iHunHi-ized mice treated wits Co axone!;.

As shown in Figure 23 and Table 14, Kegg and GO Biological Process pathways significantly enriched among probesets more highly expressed (adj p<0.05, FC>1.4) by POLIMUNOL relative to Copaxone® included immunological pathways such as immune response (adj p < 6.8e-9), response to virus (adj p < 2.3e-8)„ defense response (adj p < 0.017), cytokine activity (adj p < 0.03), RIG-I-like receptor signaling pathway (adj p < 9.2e-5), and cytokine-cytokine receptor interaction (adj p 0.044). In particular, as shown in Figure 24, probesets in the "response to virus" pathway differing in expression (FC>1.4) between POLIMUNQL and Copaxone ¹ !' include MxL Img _t Rsad2, Eif2ak2, Ifihl, 7, Oasla, Gni9706 flsglS). Table 14. Significant pathway earkiii«i*iif among probesets more highly expressed with POLI IINQL relative to Co axone®,

Pathway

ifiirmme response

Response to vims

RIG-Irlike receptor signaling pathway

C tosoEc DNA-sensing pathway

Defense response

Adenyiyitransferase -activity

'-S'-oligoadenylate synthetase activity

Table 15 below shows a list of pathways enriched among probesets significantly modulated by POLIMU OL relative to Copaxone®.

Table 15. List of pathways enriched among probesets stgBiflcautly niodtiiated by P LEVftJ OL relative to Copaxone®.

103

Gene Expmssi-on Profiles In THP-1 Cells

The THP-1 Irania monocyte cell line was used for in vitro experiments to characterize unbiased genome-wide expression profiles of treatment with POLIMUNOL and Copaxone® as described above.

As shown in Figure 25. POLIMUNOL consistently upregulated CYPIBI relative to Copaxone®, which was detected by all four present probesets for this gene. Several of the same immunological pathways differing in splenocytes. including cytokine-cytokine receptor interaction (adj p<1.9xl0-5) and immune response (adj p<i .5x10-5), were also enriched among POLIMONQL and Copaxone® differences in THP-1 cells at a threshold of FC > 1.1.

Lilt et al. 2014 describes that surface charge of micro-particles affects secretion of T F-alpha and alters immune response (Figure 26, left). Notably, secreted TNF-a!pha level is higher with POLIMUNOL relative to Copaxone® in the THP-1 model system (Figure 26, right). ■References.:

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