STABILIZED COMPOSITIONS OF PROTEINS HAVING A FREE TiMOI,

MOIETY

CROSS-REFERENCE TO RELATEB APPLICATIONS

This app lication claims priority to U.S. Application Serial No. s0/771,555, Hied on February 7, 200s. The disclosure of the prior application is considered pan of (and is incorporated by reference in) the disclosure of this application.

FIELD OF THE INVENTION

The invention relates io compositions of proteins having iree thiols, and to methods of making and methods of using such compositions. The compositions have optimized stability.

BACKGROUND OF THE INVENTION

A drug product (eg., that contains a protein) c;m be stored in liquid or iyophiiizeά, i.e., freeze-dried, fbnn. A iyophdized drug product is. often reconstituted by adding a suitable adm inistration diluent just prior to patient use.

Active protein may bt; lust as a result ϋ£ physical instabilities, including denaturatioπ and aggregation, as well as chemical instabilities, including, tor example, hydrolysis, deaπudadon, and oxidation. The stability of a protein drug in a particular form, e.g.. in a liquid or in a iyopαife ed form, can be an important consideration m selection of & product form.

SUMMARY OF TME INVENTION

In genera!, the invention features a composition which includes a protein having a free thiol (-S-H) (e.g., on a cysteine residue) and/or other moiety subject to oxidation (e.g., Tyr, Trp, or Met moiety) and a carbohydrate, wherein the carbohydrate is present in an amount sufficient to maintain the stability of the protein, and thereby of the composition. In a particularly preferred embodiment, die moiety to be protected is a free thiol.

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Compositions and methods described herein provide for increased stability and storage life by increasing the stability of a protein contained therein.

Compositions described herein, e.g., liquid compositions containing a protein, have prolonged stability. E.g.. under pre-seiecied conditions, e.g., upon storage in a gas tight container, at a temperature of 2-8 Xl for 3 period of op to 3, s, 9, 12, or 24 rπomhs (or in some embodiments longer), a protein in the composition will retain at least 50, 55, s0, s5, 70, 75, 80, 85, 90, 95, 99, or 100 % of the stability it had prior to storage. Stability, as used herein, includes parameters such as protein structure (e.g., minim izing or preventing changes in protein structure, e.g., protein aggregation or protein degradation (e.g., fragmentation)) and/or a biological activity of the protein, e.g., the ability to convert substrate into product.

Protein stability can be measured, e.g., by measuring protein aggregation, protein degradation, or levels of a biological activity of the protein. Protein aggregation can he determined, e.g., by size exclusion chromatography, non -denaturing PAGIl or other methods lor determining size, etc. For example, the composition can have less than a I , 5, H), 15, 20, 25, 30, 35. 40, 45, or 50 % increase m the amount of protein aggregation (e.g., as measured by size exclusion chromatography} as compared to the amount of protein aggregation ihat was m the composition prior to storage (e.g., storage at a temperature of 2-8 ^v € for a period of up to 3, s , ^Q , i 2, or 24 mouths (or longer)}. Protein degradation can be determined, e.g., by reverse phase HPLC, noπ-dεnatαπng PAGE, ion- exchange chromatography, peptide mapping, or similar methods, As an example, the composition can have less than a 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 % increase in the amount of protein degradation (e.g., as measured by reverse phase HPLC) as compared to the amount of protein degradation thai was in the eonφosiήon prior to storage (e.g., storage at a temperature of 2-S ⁰ C for a period of up to 3, 0, 9, S 2, or 24 mouths ( ^' or longer)). The biological activity of a protein can be measured, e.g., by in vitro or in vivo assays, e.g., ELISA (e.g., to measure binding or enzymatic activity) and other enzymatic assays (e.g., spectrophoϊometrie, llαoπsnetπc, caiorimetric. ehomi luminescent radiometric, or chromatographic assays), kinase assays, and so forth. Aa an example, die composition can have less than, a 1 , 5, 10. 15. 20, 25, 30, 35, 40, 45, or 50 % decrease hi a biological activity of the protein (e.g., enzymatic activity, e g.. as

measured by an m vitro assay) as compared to the am ount of the biological activity that was in the composition prior to storage (e.g., storage at a temperature of 2-S°C for a period of up io 3. s, 9, 12, or 24 months (or longer)},

IB one aspect, the protein does not modify, e.g., cleave, any other components of the composition. For example, in one preferred embodiment, in a composition containing gjυeoeerebrosid&se (GCB), the composition doss not contain pαiysorbaie as a surfactant because GCB can recognize poiysorbate us a substrate and can cleave polysorbate to release tree tatty acids.

Embodiments of the invention have stability comparable iυ thai of a iyophiiizeϋ composition of the same protein. A liquid composition described herein can have at least 50, 55, s0, s5. 70, 75, 80, 85, 90, 05, 99, or KK) % of the level of protein stability (e.g., retained activity) of a iyopliilizαi composition after 3, s, 12, 18, or 24 months of storage (e.g., if a lyoplrillzsd composition has retained 90% of Its activity at I S months, the composition of the invention has retained at least 50. 55, s0, s5, 70. 75, SlJ, 85, 90, 95, 99, or 1 (Hl % of that level).

In one aspect, the disclosure features a composition thai, includes a protein having a free thiol and a carbohydrate, wherein the carbohydrate is present in an amount sufficient Io maintain the stability of the protein and wherein the pH of the composition is less than 7.0. In some embodiments, the composition also includes an antioxidant, wherein the antioxidant and carbohydrate are present in amounts sufficient to maintain the stability of the protein, and thereby of the composition, a.od wherein the pH of the composition is less than 7.0, For example, the antioxidant is cysteine, cysteine hydrochloride ^cysteine- HOl), or methionine (e.g., present at between about 0.001 and about 10 % (wt/vol)) and the carbohydrate is sucrose or trehalose (e.g., present at between about ] and about 40 % (wt/vo]}}. hi certain embodiments, the pή' is in the range of about 4.5 to about s.5, e.g., preferably between about 5.0 and s.0, e.g., more preferably between about 5.5 HJUI 5.8 (e.g.. about 5.7). Irs a preferred embodiment, the composition includes a surfactant (e.g., poloxamer 188}.

In a preferred embodiment, the pB of She composition is, e.g., between about 4.5 and about s.5, e.g., between about 5.0 and about s .0, e.g., between about 5.5 and about 5.S (e.w., about 5.7).

hi certain embodiments, the stability is at least 5-80 % greater (e.g., at least about 3%, ai least about 10%, at leas! a bo in 15%, at least about.20%, at least about 25'Ki, at lciiSt about 30%, ai lets! about 35%, HI least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about ϋθ%, ai least about s5%, at least about 70%, m iea^t about 75%, or at least about 80% greater), under pre-seiected conditions, than tbe stability of a composition which differs by lacking the carbohydrate (and the antiox ktant, if used).

In certain embodiments, the carbohydrate (and optionally, an antioxidant) is present in an amount sufficient to stabilize the free thiol of the protein (e.g., the protein shows less aggregate formatiorh e.g., the protein shows about 5%, aboui 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45% ^' , about 50%, about 55%. about s0%, about s5%, about 70%, about 75%, about 80%, about 85%, about 90% ^' about ^<:) 5%> or about 99% less aggregate formation under pre-scteeted conditions than an otherwise identical proiei.n composition that does not contain the carbohydrate (and antioxidant, if used}}.

In certain embodiments, the carbohydrate (and optionally, an antioxidant) Ls present hi an amount sufficient to increase the stability of the protein (e.g., the protein shows iess aggregate formation, e.g., the protein shows about 5%. about 10%, about i 5%, about 20%, about 25%-, about 30%, about 35%, about 40%, about 45% _: about 50%, about 55%, about s0'Mi, about s5%. about 70%, about 75%, about $0%, about S5%, about 90% about 95% or about 99%; less aggregate formation under pre-seiected conditions than an otherwise identical protein composition that does not contain the carbohydrate tarsd antioxidant, if used)}.

In certain embodiments, the carbohydrate iixnύ optionally, an antioxidant) is present in an amount sufficient to inhibit the reaction oi ^' a free thiol on a iirsi rrsoleeuSc of the protein with a free thiol on a second molecule of the protein to form an aggregate,

In certain embodiments, the carbohydrate (and optionally, an antioxidant) is present in ai) amount sufficient to inhibit the formation of an aggregate formed by the reaction of a free thiol on a first molecule of the protein with a free thiol on a second molecule of the protein by at least 5-80 % (e.g., ai least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least abon;

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35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about s(5%, at least about s5%, at least about 70%, at least about 75%, or at least about 80% greater;, under pre-seiecied conditions, as compared to the same composition lacking the carbohydrate (and the antioxidant, if present). in certain embodiments, the carbohydrate {and optionally, an antioxidant) is present in an amount sufficient that upon storage, in a gas tieht container, at a temperature of 2-8 ⁰ C, for a period of U months, the composition, will retain at least 50, 55, s0, s5, 70, 75, 80, 85, ' ^' K), 95, 99, or 100 % of the stability the composition bad prior to storage. In a preferred embodiment., tbe storage occurs in darkness, in certain embodiments, the carbohydrate (and optionally, an antioxidant) is present in an amount sufficient to have stability comparable to that, of a lyophilized composition comprising about 0.01 % poJγsorbate-20, pH s.0, 50 mM Citrate. In certain embodiment the composition further includes about ) -40 % (e.g... about 5 to about 30%, e.g., about 8 to about 24 %, e.g., about 1 s %, e.g., about 3-5 % weight per volume i ^' w/v}} of a carbohydrate, e.g , sucrose or trehalose. In some embodiments, the carbohydrate is preferably sucrose. hi a preferred embodiment, the composition is a liquid.

In certain embodiments, the composition contains less than about 10 % C); (e.g., less than about 5 % (>>, e.g., less thai) about 2% Ch). hi a preferred embodiment, the amount of dissolved O- is less than tbe amount of dissolved inert gases in the composition.

In certain embodiments, the composition is made by a method comprising physical removal of O; from tbe composition (e.g.. degassing tae composition, purging a solution with a gas other than O ₂ , e.g., with an inert gas (e.g., with N? or ArK e.g., bubbling the gas other than O? (e.g., N _?. or Ar) through the composition}.

In certain embodiments, the protein in the composition that contains a fixe thiol has zero, two, four, six, or more thiol groups which form sulflwdryl bridges. In certain embodiments, the protein containing a tree thiol has. lwo _: three, or more tree thiol groups a.od has zero, ϊwo, tour, or more thiol groups which form sulfhycryi bridges, per acuve unit of protein.

hi certain embodiments, the protein containing a free thiol is selected from the group consisting of giucocerebrosidase (GCB). basic fibroblast growih taoto? (bFGF), acidic fibroblast growth factor (aFGFh hemoglobin, thioredoxiπ, eaiciuni- and iπiegπn- binding protein 1 (ClBi ), beta-lacioglobulin B. beϊα-lacLoglobuiiπ AB, seπsm albumin, antibodies (e.g., human antibodies, e.g.. IgA (e.g., tumeric IgA), IgG (e.g., IgG2). and IgM; recombinant human antibodies), antibody fragments ( e.g., Fab' fragments, F(ab'}j fragments, single-chain Pv fragments (scFv)}, antibodies and aniibody fragments (e.g.. Fab ^' , e.g., monoclonal antibody fragment C4s.3; and scPv) engineered (e.g., so that the antibody or antibody fragment can be labeled, e.g., with 99mTc, to clinical imaging) to introduce cysteine residues (e.g., in the third heavy chain constant domain., e.g.. ai position 442 in EϊJ/OU numbering; monoclonal antibody MN- 14 Cs high-affinity anii- carcinoembryonk antigen (CEA) mab)k core 2 beta ! .,s-Ni- aeetyigiucosamiπyitπrnsferase-M (02GnT-M), core 2 beta LCi-N- acety IgI ucosanimyltransferase-l! (("2GnI-I), platelet-derived growth factor receptor-beta (PDGF-beta), adenine niicleαtide transiocase (AN ¹ T), p53 tumor suppressor protein, gluten proteins, acid sphiπgonweUnαse (recomhinoπt acid sphyrsgomyeiinase), desfuroyicetiiofiir (DFC), spolipopvotem BIOO {apoB _. f and other low density lipoprcuein domains, apolipoprotein A-I variants (e.g., αpohpoprotem A-I (Mϊiaπoj and apoHpoproterπ A-I {Pans}), hypoxia-inducible factor- 1 alpha (HlF- I aiphs K von Willebrand factor (VWF), proteins and peptide mimetics that contain the ("AAX motif (e.g., Ras), mucolytics, carboxypeptidase Y, eathepsin B, catbepsiα C, skeletal muscle Ca" ^v release ehaiinel/ryaπodiπe recepior (RyRl ), nuclear factor kappa B (NF-KB), AP-L proieiu-ilisuliϊde isomerase (FDS), glycoprotein ib aiphs (GPI b alpha), easemeurin (CaN), ilbrilUn- i, CD4, S100A3 (also known as SHH)E), ionotropie ghsSanutte receptors, burn aii intcr-alpha-inhibhor heavy chain L aipha2 -antiplasmin falpha2AF). thrombospondin (also known as glycoprotein G), gelsoHn, mucins, creatine kinase (e.g., S-thioπiεihyhmodifiεd creatine kinase). Factor ViIi, phospholipa.se D (PLD), insulin receptor beta siώunlt, acetylcbolisiesierase, proehyinosin, modified alpha 2- niaerogbbuhn (alpha 2M) (e.g.. proteinase- or methysamine-reaeted alpha 2M), glutathione reductase (GR), complement component €2 {e.g., 2&h eompieπient component C3 (e.g , C3b), complement eomponetn 4 (e.g., 4d). com piemen! Factor B

(e.g., Bb), alpiia-laetaibumin, beuvD-galaciosidase, endoplasmic reticulum €a ^';'* -ATPase, RNase inhibitor, Jipocoriin I (also known as annexin 1 ), proliferating cell nuclear antigen (PCNA), &cύn (e.g., globular actin), coenzyme A (CoA), acyl-CoA synthetase (e.g., f:nλtyτy! -coenzyme A synthetase), 3~2traπs^noyWJoA--isorneτase precursor, atrial natriuretic factor (AN FV-seositive gυanylate cyclase, .Px-peptidase, aldehyde dehydrogenase (e.g., acyiated aldehyde dehydrogenase;, P -450 and NADFH- P-450 reductase, giyccraldfihydes-3-pbosphate dehydrogenase (GAPDH), s-pyruvoyl fetrahydropterin synthetase, iutropiπ receptor, low molecolat weight acid phosphatase, serum ehoUnesierase (BChE), adreπodoxύi, hyaluronidase, carnitine acyitransferases, mterleukin~2 (1L-2), phosphoglycenύe kinase, instils n-degradisig enzyme (IDE), cyiochrome cl heme subunit, S -protein, vaJyl-iRNA synthetase (VRS), aipha-amylase L ϊTsuscle AMP deannna.se, lactate dehydrogenase, and sonialostatm-nmdmg protein, in a pi-sierred embodiment, the protein containing a iVec thiol is GCB.

In another preferred embodiment, the protein containing a free thiol is HFGF.

In one aspect, the disclosure features <s liquid composition of GCB that includes GCB, and a carbohydrate at a pH less than 7.0, was produced by exposing the composition to ar? mat gas (e.g., !%}, and the inert gas is present in a concentration higher thars in the ambient atmosphere, e.g., the composition contains at least about 85 %, 90 %, 95 %, or 99 %, or preferably 100 % inert gas. hi certain embodiments, the composition also includes an antioxidant. For example, the antioxidant is cysteine, cy&teine-MCI, or methionine (e.g., present at between about 0,001 and about 10 % (wt/voi)} and the carbohydrate is sucrose or trehalose (e.g., present at between about i and about 40 % (wt/vol)). In. certain embodiments, the pH is in the range of about 4.5 to about s .5, e.g., preferably between about 5.0 and about s .0, e.g., more preferably between about 5,5 and about 5.8 (e.g., about 5.7). In certain embodiments, the composition also contains a surfactant (e.g... poloxyrner ISS).

In one aspect, tbe disclosure features a composition i.hat includes a protein having a tree thiol and a carbohydrate, and is at a pli below the pKa of A iree thiol on the protein, wherein the carbohydrate is present in an amount suiiicicnL to increase the stability of the protein at Che pH.

In certain embodiments, ihe stability is al least 5-80 % greater (e.g., at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least ahoiu 25%.. at least about 30%, at least about 35%, at least about 40%, at least about 45%, ai least about 50%, at least about 55%,.. at least about s0%, at bast about s5%, at least about ?(;%, at least about 75%, or at least about 80% greater), under pre-seSecied conditions, than the stability of a composition which lacks the carbohydrate and which has a pH above the pKa of a free thiol on the protein. in certain embodiments, tbe carbohydrate is present m an amount sufficient to stabilize the free thiol of the protein. in certain embodiments, the carbohydrate is present in an amount sufficient to inhibit the reaction of a free thiol on a first molecule of the protein with a free thiol on a second molecule of the protein to Conn an aggregate.

In certain embodiments, the carbohydrate is present In an amount sufficient to inhibit the formation of an aggregate formed by the reaction of a free thiol on a first molecule of the protein with a free thiol on a second molecule of the protein by at least 50, 55, s0, s5, 70, 75, SO, Sl vϋ, 95, 99, or 100 %, under pre-seleeted conditions, as compared to the same composition lacking the carbohydrate. in a preferred embodiment, the carbohydrate is present in an amount suffi cient that upon storage in darkness, in a gas tight container, at a teniperaαrre of 2-8X for a period of up to 3, s, 9, 1 ~. or 24 months (or longer), the composition will retain at least 50, 55, s0, s5, 70, 75, 80, 85, 90, 95, 99, or 100 % of the stability it had prior to storage.

In certain embodiments, the carbohydrate is present in an. amount sufficient for the composition to have stability comparable to that of a lyophihzed composition,

In a preferred embodiment, the composition is a liquid.

In certain embodiments, the composition contains less than 10 \'4> O; (e.g.. less than 5 % O _? , e.g., less than 2% Q _: ). In certain embodiments, the amount of dissolved O> is Jess than the amount of dissolved inert gases in tbe composition.

In certain embodiments, the composition is made by a method comprising physical removal of O^ from the composition (e.g., degassing the composition, purging n solution with a gas other than O^ e.g., with an inert gas (e.g., with N; or Ar). e.g., bubbling the gas other than (> (e.g., N^ or Ar) through the composition}.

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Ln certain embodiments, the protein m the composition that contains a free thiol has two, three, four, five, or more free thiol groups per active umi of protein.

In certain embodiments, the protein in the composition thai contains a free thiol has two, tour, six, or more thiol groups which form suifnydryl bridges per active unit (e.g.. diπv≥r) of protein. hs certain embodiments, the protein that euntams a tree thiol has two, three, or more free thiol groups and has two, four, or more thiol groups which form siϊlfhydryl bridges, per active unit of protein. in certain embodiments, the protein containing a free thiol is selected from the group consisting of glucoeerebrosidase (GCB), basic fibroblast growth factor (h FGF), acidic fibroblast growth factor (aFGP), hemoglobin, ihioredoxin, calcium- and integrin- biudi πg protein 1. ((!1B i K beta-iacioglobuiiπ B, beia-lαeroglohuhrs AB, serum albumin, antibodies (e.g., human antibodies, e,g. _v !gA (e.g.. dimeric IgA.), IgG ie.g... ϊgG2), and IgM; recombinant human antibodies), antibody fragments { e.g., Fab' iVagmenb, F(ab ^' h fragments, single-chain Fv fragments (scFv)), antibodies and antibody fragments (e.g. _; Fab ^" , e.g., monoclonal antibody fragment C4ό,3; mid scFv) engineered (e.g., so that the antibody or antibody fragment can be labeled, e.g., with 99rπTc, to clinical imaging) to introduce cysteine residues (e.g., m the third heavy chain constant domain, e.g., at position 442 in EU/OiJ numbering; monoclonal antibody MN- 14 (a nigh-aiϊm ^ή y anU- carcinoembryonic antigen (CEAs mab _. )), core 2 beta 1,s-N- acetylgiueosasninyltraiisferase-M {C2GΏT-M}, core 2 beta 1 ,s -N- acetyigiucosaαiiriyitransierase-I (C2GnT--I), platelet-deπved growth factor receptor-beta (POGF-bεta), adenine nucleotide transiocase (ANT), p53 tumor suppressor protein, gknen proteins, acid sphingomyelinase (recombinant acid sphingomyelinase), deslliroyicefliofur (DFC! ¹ ), apolipoproteiii BI OO (apoB) and other low density lipoprotein domains, apoHpoprotein A-I variants (e.g., apolipoprotein A- I (Milano) and apcfiipoproiem A-f (Paris)}, hypoxia-indneible taetor-1 aipba (HlF-i alpba _. i, von WiHεbrarrd factor (VWF), proteins and peptide rnimetics that coniain me CAAX motif (e.g., Ras), mncϋlylics, carboxypεpfidase Y, cathepsln B _s euthepsin C. skeletal nu∑scle Ctr ^'* release cbannel/ryariodine recepior (RyRl }, nuclear factor kappa B (NF-KB), AP-I . proteϊ n-di.su slide isomerase (PDl), glycoprotein Ib alpha lGPib alpha), calcineurin (CaN), fibrillin- 1, CD4, S100A3 (also known as S lOOE), ionotropk glntamaie receptors,

human mter-alpha-inhibitor heavy chain 1, alpha2-aantip lasmin (alp ha2AP), thrombosp ondin (also blown as glycoprotein G), gelsohn, mucins, creatine kinase (e.g., S-thiomethyl-modified creatine kinase). Factor VIII phospholipase D (PLD), ,nsulin receptor beta subumit, acetylcholinesterase, prochymosin, modified alpha 2- macroglobulin (alpha 2M ) (e.g., proteinase- or methylannne-reaeted alpha 2M), glutathione reductase (GR), complement component C2 (e.g., 2a), complement comp onent C3 (e.g., C3b), complement component 4 (e.g., 4d), complement Factor B (e.g., Bb), alpha-lactalbumin, beta-D-galactosidase, endoplasmic reticulum Ca2+-A TPase, RNase inhibitor, lipocoriin 1 (also known as annexin S.), proliferating cell nuclear antigen { PCNA) ,ctin (e.g., globular actin), coenzyme A (CoA), acyl-CoA synthetase (e.g., butyryl-coenzyme A synthetase), 3~2trans-enoyl-CoA-isomerase precursor, atrial natriuretic factor (ANP}-sensiiive guanylate cyclase. Pz-peptidase, aldehyde dehydrogenase (e.g., acylated aldehyde dehydrogenase). P-450 and NADPH-F-450 reductase, gIyceraldehydes-3-phosp hate dehydrogenase (GAFDH), s-ρyruvoyl tetrahydrop terin synthetase, lutropin receptor, low moleculat weight. acid phosphatase, serum cholinesterase (BChE), adrenodoxin, by^luroradase, earmϋne acyltransiexasus, interleukin-2 (11..-2), phosphoglycerate kinase, insulin-degrading enzyme (IDB), cytochrome cl herne subunit, S-protein, valyl-tRNA synthetase (VRS), alpha-amylase 1, muscle AMP deaminase, lactate dehydrogenase, and somatostatin-binding protein, in a preferred embodiment, the protein containing a free thiol is GCB. in another preferred embodiment, the protein containing a A'ee thiol is bFGF, In one aspect, the disclosure features a liquid composition of GCB that includes GCB and a carbohydrate and is at a pH between about 0 and about 7, and the carbohydrate is present in an amount sufficient to maintain the biophysicai/biochemieat integrity (e.g., molecular weight, charge distribution) and bioaciivity characteristics/properties of the GCB at the pH. For example, the composition retains at least 50, 55, s0, s5, 70, 75, 80, 85, 90, 95, 99, or 100 % of the biological activity it had poor to storage (e.g., storage at a temperature of 2 -S ⁰ C for a period oi up to 3, 6, 9, 12, or 24 months (or longer)}, As another examp le, at least 50, 55, s0. s5, 70.. 75. SO, 85, 90, 95, 99, or 100 % of the proteins m the composition retain the average molecular weight

or average charge distribution thai the proteins had prior to storage (e.g., storage at a temperature of 2-Z ⁰ C for a period of up to 3, s , 9, 12, or 24 months (or longer)),

In certain embodiments, {he pll is in the range of about 4,5 to about s .5, e.g., about 5.0 to about s,0 (e g., the pH is about 5.5 to aboui 5.S. e.g.. about 5.?}.

Ln a preferred embodiment, the carbohydrate is sucrose or trehalose (e.g., present in an amount between about i and about 40 %, e.g., between about 3 % and about 5% s'wt/voi}}. hi one aspect, the disclosure features a liquid composition of GCB that includes GCB, an antioxidant, a carbohydrate, at a pH between 4.5-s .5. and the composition was produced by exposing the composition to an inert gas (e g., Ni or Ar). Irs certain embodiments, the pB. is in the range of about 4.5 to about s .5, e.g., about 5.0 to about s.0 (e.g., the pf l is about 5.5 to about 5.8, e.g., about 5.7).

In certain embodiments, the liquid composition includes about 0.1 -40 rng/nsJ QC-B (e.g., more preferably ahotu U.5 to about S O rog/ml e.g., about 2 to about S nig/ml or about 5 mg/ml) (e.g., about 2 nig/nu), about 0.001 -10 % cysteine (e.g., about 0.075%), about 1-40 % sucrose (e.g., about 1s%), at a pH of about 5.5-s .0 {e.g., about 5.7), and the level of dissolved <>> is iess than about 10 % less than about 5 %, e.g., less than about 2 %>. hi a preferred embodiment, the composition also includes a snrtacϊant ( e.g., poloxamer 188).

In one aspect, the disclosure features a gas tight container thai contains a protein component and a heaάspacc wherein the protein component is a protein having a tree thiol and the heatbpace is at least 90%, 05% or 99% (vol/voi) an inert gas.

In certain embodiments, the gas tight container is a prefixed syringe, ;. _* . vial, ur an ampoule. In a more preferred embodiment, the preil lled syringe is a needleless syrirsgt.

In certain embodiments, the protein containing a tree thiol is selected from the group consisting o fglucocerebrosidase (GCB), basic fibroblast growth factor ^bFGF), acidic fibroblast growth iactor (aPGP), hemoglobin, thioredoxin, calcium- and inte grin- binding protein I (CIB i), beta-iacωgiohulm B, beta-laetoglobaiin AB, serum albumin, antibodies (t.g,, human andbodt€s, e.g., IgA (e.g., dimeric IgA), IgG (e.g., Igϋ2), and IgM; recombinant human antibodies), antibody fragments ( e.g., PsV fragments, F(ab ^' b

fragments, singie-chiun Fv fragments {scFv}}, antibodies and antibody fragments (e.g.. Fab ^' , e.g., monoclonal antibody fragment ("4s.3; and so Fv) engineered {e.g.. so that the antibody or antibody fragment can be labeled., e.g., wiih 99m Te. to clinical imaging) to introduce cysteine residues fe.t»., in the third heavy chain constant domain, e.a;., af position 442 in EU/OU numbering; monoclonal antibody MN-14 (a higli-afiinity aπti- caremosmbryonio antigen (CEA) mab}}, core 2 beta 1 ,s-N- acetyiglυcosaminyltraπsicrase-M {C2GnT-M j, core 2 beta 1,s-N- aceiylglucosarninyUransierase-i (C2GnTd), platelet-derived growth factor receptor-beta (FDCJ F-- beta), adenine .nucleotide transiocase (ANT). p53 tumor suppressor protein, gluten proteins, aeid sphingomyelinase (recombinant acid sphingomyelinase), desfuroykefhϋfur (DFC), apolipoprotein Bl OO (apoB) and other IOλV density lipoprotein domains, apoϋpoprotem A-T variants (e.g., apolipoprotein A-T (Miiaπo) and apolipoproiein A-I (Park}}, hypoxia-inducible factor- 1 alpha (.i-llF-i alpha }, von WilJebrand i ^' actor (VWF), proteins and peptide mirnetics that contain the CAAX nioύf (e.g., Ras), mucolytics, carboxypeptidase Y, cathepsiπ B ₅ ctUhepsin C, skeletal muscle CV ⁺ release chαnnel/ryαπodine recepior (RyRI ), nuclear fector kappa B (NF-KB), AP- L protem-distdOάe isomerase (PDl), glycoprotein Ib alpha (GPI b alpha), caJcmeurin (CaN), iibrlHln-1 , CD4, SI00A3 (also known as SIOUE), ionotropic glutamate receptors, human inter-alpha- inhibitor heavy chain L alpha2-autipla&nim (alpha2AP), tlirombospondin (also known as glycoprotein G), gclsolrn, nroeins, creatine kinase (e.g., $-tbiomethyi-modifsed creatine kinase). Factor VlH, phosphoHpasc D (PLD), insulin receptor beta sabunii, acetylcholinesterase, proehymosin, modilssd alpha 2- rn-icrogiobulvn (alpha. 2M) (e.g., proteinase- or racthylamine-reacted alpha 2M), glutathione reductase (GR), complement component C2 S e.g., 2a), complement componetit 03 (e.g., O3b), complement component 4 (e.g., 4d), complement l-actor B (e.g., Bb), aipha-iactaiburmn, beta-D-galactosidase, endoplasmic reticulum RNase inhibitor, iφocortin I (also known as annex in 1 }, proliferating ceil nuclear antigen (PCNA), actin (e.g., globular actin), coenzyme λ (CoA), acyl-C ^' oA synthetase (e.g., butyryl-coenzymo A synthetase), 3-2trans-enoyl-CoA-isomera.se precursor, atrial natriuretic factor (ANF)-sensitivc guanylate cyclase, Pz-peptuiasc, aldehyde dehydrogenase (e.g., acyiated aldehyde dehydrogenase}, P-450 and NA[)Pl--I~P-45()

reductase gly ceraidehydes-3--phosphate dehydrogenase (GAFDH), s-pyruvoyi Ie trah ydropteπn synthetase, luiropm receptor, low moleeuiai weight aesd phosphatase, serum eholkiesterase (BChE), adrersoάoxin, hyaiurouidase. carnitine acyltraissieτases. intCϊieukirs-2 {11,-2}, phosphoglycerate kinase, insulin-degrading enzym e UDE), cytochrome ci heme suburm, S-proLein, vaiyl-tRNA synthetase (VRS), aipha-arnylase I, muscle AMP deaminase, lactate dehydrogenase, and somaiostaiiπ-biπdlπg protein.

In a preferred embodiment, the protein containing a free thiol is GCB.

In another preferred erahodirneni, die protein containing a free thiol is bFGF,

In one aspect, the disclosure features a method of packaging a composition that includes contacting a irεe thiol containing protein with so inert gas (e.g., N? or Ar) to reduce the amount of a reactive species (e.g., Ch), and introducing the protein and the inert gas into a gas tight container. The term "reactive species ^' " includes molecules or ions formed by the incomplete one-electron reduction of oxygen. These reactive species include O ₂ : superoxides; peroxides; hydroxy! radical; and hypoehiorαus acid.

In a preferred embodiment, the inert gas is N; or Ar and the reactive species is O;.

In certain embodiments, the free thiol containing proteirs is seiecied from Lhe group consisting of gkicocerεbrosidase (GCB), basic fibroblast growth (actor (bFGF-X acidic fibroblast growth i actor (aFGP.S, hemoglobin, thioreαoxin. calcium- and integrin- binding protein 1 (ClBl ), beϊa-lactosjlobukn B, beta-lactoglobtuiα AB, seπsm albumin. antibodies (s.g,, human antibodies, e.g., SgA (e.g , dimerie IgAj, lg(j (e.g., IgG2), and IgM; recombinant human anUbodies), antibody fragments { e.g.. Fab' fragments. F{ab"h li-agrøenta, single-chain Fv fragments (scFv)κ antibodies mid antibody fragments (e.g., Fab', e.g., monoclonal antibody fragment C4s 3; and scFv) engineered (e.g., so that the antibody or antibody fragment can be labeled, e.g., with. 99rnTc, u> clinical Imaging} Io introduce cysteine residues {e.g., m the third heavy chain constant domain, e.g.. at position 442 in EU/OU numbering; monoclonal antibody MN- 14 (a high-affinity anti- carcinoembryønie antigen (CEA) aiab}}, core 2 beta Ls-N- acetylgJucosaminyi transferase-M (02GnT-M), core 2 beta 1 ,s-N- acetylgkicosaminyhransferase-I (C2GnT-i), platelet-derived gτx)wth factor receptor-beta (PDGF~bct-i), adenine nucleotide traπsiocase (ANT), pS3 tumor suppressor protein, gluten proteins, acid sphingomyelinase (recombinant acid spbyπgosπyelinase}.

desmroylcεfUoftrr (DfC), apolipaprofem BIOO {apoB} and other low density lipoprotein domains, aρo lipoprotein A-i variants (e.g., apolipoprotein A-I (Msiano) and apo lipoprotein A-I (Pads)), hypoxia-inducible factor- 1 alpha (HIF-I alpha), von WiJlebrand factor (VWF), proteins and peptide rnirøeties that contain the CAAX motif (e.g., Rash mucolytics, carboxypeptidase Y, ciuhepsiπ B, eaihepsin O, skeletal masde Ca ^{" "'} release ehannei/ryanodine receptor I RyRl }, nuclear factor kappa B (NF-KBh AF-I , pfotein-dlsuH ^' ick bomerase (PDI), glycoprotein ib alpha (GPI b alpha;, ealcmeusi n (CaN), iϊbriJlin-1 , C 04, S100A3 (also known as Sl OOE), ioπotropic gUuarøate receptors, human inier-aipha-inhihitor heavy chain 1 , alpha2-ai$tiplasnτhi (alpha2AF ), thronibospoiidsn (also known as glycoprotein G), gel soli π, mucins, creatine kinase (e.g., S-tliioiϊJOthyl-πiodifted creatine kinase). Factor VIfL phospholipase D (PLD), insulin receptor beta snbnnit acetylcholinesterase, proehymosirκ moditled alpha 2« rnacroglobtnin (alpha 2M) {e.g.. proteinase- or methylamine- reacied alpha 2^ K giniatSnone reductase (GR), complement component C2 (e.g., 2a), complement component C3 (e.g.. €3b), conψiεnient component 4 (e.g., 4d), complemerr; factor B {e.g., Bb), alpha-bctaibnniin, beuvD-gaiaotosiciase, endoplasmic reticulum Cs" ^! -ATPasε, RNase inhibitor, iipoconin 1 talso known as annexin 1 ). proliferating cell naclear aiϊtigen (PCNA), acnn (e.g., globular αciin), coenzyme A (CoA), acyl-CoA synthetase (e.g., butyryl -coenzyme A synthetase), 3-2trans-enoyl-C?oA-i3on ^" ierase precursor, atrial uatriureri c føcior { ANPVsensitive guaoylaie cyclase. Pz-pepti<Jase, aldehyde dehydrogenase (eg,, aeylαtsd aldehyde dehydrogenase K P-450 and NADPH-P-45i; reductase, glyceraldehyde$-3-ρhosphatε dehydrogenase (GAPDH), ό-pyruvuyl tctrahydroptcrin synthetase, lutropiπ receptor, low molecuUu weight acid phosphatase, serum cbolin.esterase (BChE), adrenodoxin, hyalurofiida.se, carnitine acyitransierases, interleuktn-2 UL.--2), phosphoglycerate kinase, insulin-degrading enzyme (IDE}, cytochrome el heme subuπit, S-protetn, valyl-tRNA synthetase (VRS), alpha -antylase L muscle AMP deaminase, lactate dehydrogenase, and somatostatm-binding protein.

In a preferred embodiment, the protein containing a iree thiol is GCB. in another preferred embodiment, the protein containing a free thiol is bFGF. hi one aspect, the disclosure features a method of treating a patient (e.g., a pariern in need of treatment with a free-ύπol containing protein, e.g., a patient with a deficiency

of the free-thiol protεm) thai includes administering a composition described herein, e.g.. a composition containing a free-thio! protein (e.g., GCBj ₅ Lo a patient. For example, a pharmaceutics; composition that is administered to a patient can mekidε n. composition described herein, e.g., in a therapeutieaily-efieetive amount.

In a preferred embodiment, lhe administration is by IV infusion or subcutaneous.

In one embodiment, a composition described herein thai contains a free-thiol protein (e.g., GCS) is used irs therapy,

In one embodiment a composition described herein thai contains a free-thiui protein (e.g., GCB) is used for the manufacture of a medicament for the treatment of a condition in which there is a need for the free-thioi containing protein (e.g., the use of a GCB composition described herein for the treatment of a giueocerebrosidase deficiency, e.g., Gaucher disease). For example, a medicament for administration to a patient can include a composition described herein, e.g., In a therapenticaily-eiiective amount

In one aspect, the disclosure features a method of treating a patient having a giucocerebrosidase deficiency that includes administering a GCB composition described herein.

In certain embodiments, the giucocerebrosidase deficiency is Gaucher disease.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the an to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practicing or testing of the present invention, suitable materials and methods are described below. All cited publications, patent app lications, patents, and other references mentioned herein axe incorp orated by reference in their entirely, hi ease of conflict, the present specification, including defhiiUons, wdl control, in addition, the materials, methods, and examples are illustrative only and not intended to he limiting.

Other features, objects, and advantages of the invention will be apparent from the description and from the claims.

LJ

DEl AS LED DESCRIPTION

Overview

Compositions of free thiof-eontalning proteins (e.g., GCB) arc relatively unstable as liquid compositions. The three exposed tree thiol groups in GC B can undergo reactions which lead to reduction in stability, e.g., by aggregation of GCB molecules. For example, In buffer at a pH of s, typ ically 1-2 % of the protein has aggregated upon one month of storage and about I S % has aggregated alter 0 months of storage. While not wishing to be bound sm ctly by theory or mechanism, it is believed that a number of ^[ actors contribute to protein instability, e.g., the aggregation reaction: For example, free Oz in solution ears accelerate cross-linking of the free thiol groups- leading u> aggregation. If the reaction of the free thiol groups is reduced arsd/or if (he protein can be made more compact for example, by burying cysteine residues in hydrophobic domains, protein aggregation can be reduced. Sn addition, protein degradation (e.g., fragmentation) ears be reduced.

Embodiments described herein include one or more measures to address one or more of these issues. Aa examples, various factors have been addressed to increase the stability of compositions (e.g.. liquid compositions} of free fnioi-coiitambig proteins, for example: the presence of reactive spades (e.g., free O?) in solution, the availability of free sυ ^j fhydryi groups (e.g., free thiols) on the protein, the protein conformation, and pH. One, two, three, four, or all of these factors can be altered Oi controlled to increase the stability of a protein of interest.

Free Thiol-Contanimg Proteins

Free ihiol-hearmg proteins are proteins which in active form have one or more -S-.H moieties. In preferred embodiments, the S-H moiety is accessible to a reactant and can react with that reaetam, e.g., a reducing agent such a.s cysteine, under conditions which are optimal for stability. Alternatively, the -S-H moiety am react with a reaelant under physiological conditions with one or more biological fluids that it comes into contact with when administered to a patient, e.g.. the moiety is accessible for reaction in blood.

A particularly preferred lree thioi-coπiamiiig protein is gkicoeerebrosidase (GCB). The structure of GCB in solution provides relatively accessible (as opposed io buried or hindered) free -S-H moieties, which promotes reactions with the - S-H moiety.

Another particularly preferred free thioi-eorstaming protein is basic fibroblast growth iktor (bFGF).

Other examples of iree ϋiioi-containing proteins include: acidic fibroblast growth factor CaFGF), hemoglobin, thioredoxin, calcium- arid integrivi-bmdmg protein 1 (ClBl }. heta-laetogiøbulm B. beta-laetoglobulin AB, serum albumin, antibodies (e.g., human antibodies, e.g., IgA (e.g., dimeric IgA), IgG (e.g., IgG2), and IgM; recombinant human antibodies), antibody .fragm ents ( e.g.. Fab ^' fragments, F(ab"h fragments, smgie-chaήi Fv fragments (scfvjj, antibodies and antibody fragment (e.g., Fab\ e.g., monoclonal antibody fragment C4s .3; and scFv) that have been engineered (e.g., so that the antibody or antibody fragment ears be labeled, e.g.. with 99τnTe, for clinical imaging) to introduce cysteine residues (e.g.. in the third heavy chain constant domain, e.g., at position 442 in EU /OU numbering; monoclonal antibody MN- 14 Ss high-affinity aπti-earclnoembryomc antigen (CEA) mahλk core 2 hela 1 ,0~N-aeetyIgtucosaminyitransferase-M ξC2GnT-M), core 2 beta Kό-N-aeetylglucosarainyltransferase-I (C2GΏT-I}, platelet-derived growtfi factor receptor-beta (PDGF-beJa), adenine naclootide transloease ^ANT), ^* ρ53 tumor suppressor protein, gluten proteins, acid sphingomyelinase (recombinant acid sphingomyelinase), desfnroylceftiofur (DFC), apolipoprotein Bl OO (apoB) and other low density lipoprotein domains, apolipoprotein A--1 variants (e.g., apolipoproteirs A-I (Miianoi and apohpoprotein A-I (Paris)^, hypoxia-inducible factor- 1 alpha (HIF-I alpha), von Willebrand iactor (VWf K proteins and peptide rnϊmeTics that contain the ( ^X AAX motif (e.g., Ras _. h mucolytics, carboxypeptidase Y, catbepd n 8. eathepsin C\ skeletal muscle C<τ ^" release eliannel/ryanodine receptor (RyR i ), nuclear factor kappa B (NF- KB) ₁ AP- \, proidn-disui fide isoraerase (PDl), glycoprotein I b alpha (GPIb alpha), cajcineuriri (CaN), ilbrillin-l , CD4, Sl 00 A3 (also known as SlOOE), ionotropic glutarnate receptors, human inlcr-aiρba~inhibitor heavy el ^{^} sain L alpba2-antip!ai»rnin la1ρba2AP}, thrυmbospondin (also known as glycoprotein G), gelsohn, mucins, creatine kinase (e.g., S-thiomethyl-rnodified creatine kinase). Factor ViIL phospholipast D (PFD), insiitπi receptor beta subttnit, acety Ic ho \w esterase, prochymosin, niodined alpha

2-maerogIobdm (alpha 2M) (e.g., proteinase- or rnethy laniine-reacted alpha 2M), glutathione reductase (GR), complement component C2 (e.g., 2a), complement component. C3 (e.g., C3b), complement coraponent 4 {e.g., 4d), complement Factor B (e.g., Bb), aipha-laci alburairs, beia-D-gaiaciosidase.. endoplasmic reticulum Ca ^" ' ^{" :"} -ATPaae, RNase inhibitor, iipoεorfin \ (also known as annex in ! L proliferating ceti nuclear antigen (PCNA), aciin (e.g., globular actin), coenzyme A (CoA), acyl-CoA synthetase (e.g., buiyryl-coerszyme A synthetase), 3-2trans-ti!oyi-CoA-isorHera$e precursor, atrial natriuretic factor (ANf S-sensiiive guaπylaie cyclase, Pz-peptidase, aldehyde dehydrogenase (e.g., aeyhued aldehyde dehydrogenase), P-450 and NADPH-F-450 reductase, glycεraldehydes~3-phαsphate dehydrogenase (GAPDH), s -pyτuvoy! teimhydropteriu synthetase, luiropiπ receptor, low rnolecuku weighs, acid phosphatase, serum eholinestetase ( BChE), adrenodoxin, hyaHtrottidase, carnitine acvKransferases, uHerleαkii)-2 (31.-2), phoaphoglyeeratc kinase, insulin-degrading enzyme (IDE), cytochrome cl heme subunii, S-protem, va!y!-tRNA synthetase (V RS) , aipha-aπiy!ase L muscle AMP deaminase, lactate dehydrogenase, and somatostatin-biriding prolein. Also included are fragments of sucii proteins (e.g., active domains, structural domains, dormnam negaiive fragments, and so forth). The proteins containing a free thiol group can he naturufly occurring proteins, recombinant proteins, proteins modified (e.g., by recombinant DNA technology} to contain a cysteine residue, or proteins chemically or enzyniatieaily treated so that a suIlhydryS moiety on a cysteine residue is in a reduced state, i.e., to have a tree ~S~H moiety fπ same embodiments, a composition described herein includes a protein having a naturally occurring sequence. In other embodiments, the sequence of the protein will differ at 1 , 2, 3, 4. 5, or up to 10 amino acid residues from a natural Iy occurring sequc ace, In other embodiments, the amino acid sequence of the protein will differ by 1 , 2,. 3. 4, 5.. or up tϋ 10 %, from a naturally occurring sequence.

Reactive Snccics Removal

Reactive species, e.g., (> or peroxides, dissolved in solution eaα decrease the stability of a protein in the composition, e.g., by promoting protein aggregation. However, even in the ahseirce of (X. free -S-H moieties can cross-link,

. 13 ...

To increase protein stability, reactive species, e.g., O ₂ , in a solution can be removed, e.g., chemically, by the use of O ₂ scavengers, e.g., sulfi tes. Chemical scavengers are often less desirable as they can cause protein degradation. O ₂ can also be removed physically from a solution, e.g., by degassing the solution, e.g.. by applying a vacuum to the solution to remove the O ₂ from solution and replacing it with an inert gas, e.g., nitrogen or argon. Reduction of O ₂ levels can also be accomplished physically by purging a solution with a gas other then O ₂ , e.g., an inert gas, e.g.,, .nitrogen or argon. Purging can be accomplished by bubbling the gas through Um solution to be purged of O ₂ .

Wnh protein ⁽ e.g.. GCB) compositions, bubbling or other manipulations which result in interfaces between a gas and a protein-containing solution are often avoided in the treatment of proteins because they can denature proteins, however, these manipulations have been discovered to be well-toierated in the GCB methods disclosed herein,

O ₂ removal can be combined with minimization of contact of a solution with O ₂ . e.g., by manipulation and storage under conditions which minimise (he presence of O ₂ , e,g., filling of containers under a gas other than O ₂ , e.g., an inert gas, e.g., nitrogen or argon, or the sealing of containers with such a gas, In general, it is desirable to minimize the contact of the solution withh O; prior to administration to the patient. O ₂ leveis in head space should he reduced to less than about 10 %, preferably less than about 5%, and more preferably less than about 2%.

Removal of reactive species may also result in increased protein stability, e.g., by minimizing oxidation of other moieties as well, e.g., 1 ^' yr, Trp and/or Met residues, in particular, it is desirable to minimize oxidation of these moieties in GCB,

One can test a candidate method for removal of O ₂ by providing a composition containing 2 mg/ml GCB, 0.075 % cysteine (as an antioxidant), 1 s % sucrose (to decrease -S-H availability}, adjusting the pH to 5.7, and applying the candidate method. The stability of the GCB composition produced by the candidate method, measured, e.g., as a percent aggregation or degradationn, at a predetermined time is compared with one or more standards. For example, a suitable standard would be a composition similar to the test conditions except thai an O: removal method is not applied. The stabilities of the

treated ⁽ wherein the candidate O> removal method is applied) and untreated (wherein on C>2 remova) method is not applied) compositions are compared. Suitability can be shown by the test treatment increasing stability as compared with this standard. Another standard can be a composition similar to the test composition except that in place of the candidate method of removal, 0> is removed by α method described herein, (or example, by purging or degassing with an i.nort gas. Suitability can be shown by the candidate method having comparable or better effects on stability than the method described herein,.

Protein stability can be measured, e.g., by measuring protein aggregation or protein degradation. Protein aggregation can be determined, e.g.. by size exclusion chromatography, non-denaturing PAGE, or other methods for determining size, etc. Protein degradation can be determined, e.g., by reverse phase HPLC. nun-denaturing PAGE, iosvexchange chromatography, peptide mapping, or similar methods.

Antioxidants

The stability of a protein in a composition can he increased (e.g., cross-linking mediated by tree ~S~H moieties can be reduced) by the addition of an antioxidant, and in particular, an anti-oxidant which includes a moiety which reacts with Hie irce -S-H (e.g., an -S-H). e g., cysteine, i-ystesne-HCJ, or methionine. For a protein (e.g., GCBj thai contains both free thiol groups and also interna! disulfide linkages within the protein molecule, the level of antioxidant (e.g., cysteine) used should be high enough to rninbrrizε cross-linking of the free thiol bonds (e.g., aggregation) but tow enough so as not to cause fragmentation, ami/or proteolysis, and/or degradation (e.g., detectable with reverse-phase HPLC). For example, with cysteine, particularly lor GCB, inclusion of about 0.001% to about 10%, e.g., about 0.01 to about 0. ! 5 %, e.g.., about 0.05 % to about 0.1%, is suitable. Levels over 10 % may not be optimal.

For example, one can test a candidate antioxidant (which can be any agent that can remove or reduce dissolved Oi in solution) by providing a composition containing 2 mg/m i GCB, 1s % sucrose (to decrease -S-H availability), adjusting the pH to 5.7, adding the candidate antioxidant (e.g., in an amount described herein, e.g., 0,075%), and purging the composition of CK The stability of the GCB composition containing the candidate antioxidant, measured, e g., as a percenϊ aggregation or degradation, at a

predetermined time is compared with one or more standards. For example, a suitable standard would be a composition similar to the ttrsf conditions except that an antioxidant is not added to the composition. The stabilities of the treated (containing the antioxidant} and untreated (lacking an antioxidant) compositions are compared. Suitability can be shown by the test treatment increasing stability as compared with this standard. Another standard cars be a composition similar to the test composition except that in place of the candidate antioxidant, an ami oxidant described herein, for example, cysteine {e.g.. m aα amount described herein, e.g., 0.075%), is added to the composition. Suitability can be shown by the candidate antioxidant having comparable or better effects on stability than an antioxidant described herein. If the candidate antioxidant is determined to be suitable (e.g., it increases stability of the composition as compared to one of the standards), the concentration of the candidate antioxidant can be refmed. For example, the concentration can be increased or decreased over a range of values and compared ?o the standard and to the other concentrations being iested to determine which concentration ccusses the greatest increase in stability.

Protein stability can be measured, e.g., by measuring protein aggregation or protein degradation. Protein aggregation can be determined, e.g., by size exclusion chromatography, non-denaturing PAGE, or other methods lor determining size, etc. Protein degradation can be determined, e.g., by reverse phase HPLC, non-denaturing PAGB-, ion-exchange chromatography, peptide mapping, or similar methods.

A preferred antioxidant is cysteine. Other antioxidants suitable tor use include. eyststne-BCL reduced glutathione, thioedianoiamine, thtodiglyeoi, thioaeette acid, monotliioglycerol, N -acetylcysteine, dithiothreitol, DL-ihicctic acid, mereaptoethauoL dimereaptopropanol, bisulfite, dihydroacorbate, metahisulftte, sulfite, formaldehyde sulfaxylate, thiosulfats, and acetone bisulfite. In some embodiments, a combination of two or more of these antioxidants is used in the compositions described herein. The suitability of the combination can be tested as described above for a candidate antioxidant.

Addition of anti-ox tdams can result in increased protein stability, e.g., by minimizing oxidation of other moieties as well, e.g., Tyr, Tηp, and/or Met residues. In particular, it is desirable to minimize oxidation of these moieties in GCB.

CarbohyctπueS

In some embodiments, a carbohydrate is included in the: composition. E.g.. a carbohydrate can cause the protein to be more compact, and for example, bury or otherwise hinder access to a moiety, e.g.. a cysteine residue (e.g., a i ^' rez -S-B moiety on a cysteine residue), e.g.., a cysteine residue in a hydrophobic domain. This can (e.g., with GCBs mcresse protein stability, e.g., by reducing protein aggregati on.

Carbohydrates include non-reducing sugars, e.g., non-reducing disaecharides, e.g., sucrose or trehalose, which are suitable for this purpose. ^' The level of sugar in the composition can be critical. A sugar content of about 1 to about 40 %, e.g., about 5 to about 30%, e.g., about S to about 24 %, e.g., abom 1 s %, weight per volume (w/vs is suitable, e.g.. for use with GCB. λ sugar content of about 3 to about 5 % is also suitable.

O«£ can test a candidate substance, e.g., a candidate carbohydrate, for decreasing -S-H availability by providing a composition containing 2 rng/m i GCB, 0,075 % cysteine (as an antioxidant), adjusting the pH to 5.7, adding the candidate substance (e.g., in an amount described herein, e.g., 1 s%), arrd purging the composition of (K The .stability of the GCB composition containing the candidate substance, measured, e.g., as a percent aggregation or degradation, at a predetermined time is compared with one or more standards. For example, a suitable standard would be a composition shrmar to the Lest conditions except thai a substance is not added to the composition. The stabilities of the treated (containing the substance) and untreated (lacking a substance) compositions are compared. Suitability can be shown by the test treatment increasing stability as compared with this standard. Another standard can be a composition similar to the test composition except that in place of the candidate substance, a substance described herein, lor example, sucrose (eg,, in an amount described hereby e.g., 1 s'JxO. is added to the composition. Suitability can be shown by the candidate substance having comparable or better effects OΏ stability than a substance described herein, if the candidate substance is determined to b& suitable (e.g.. it increases stability of the composition as compared to one of the standards), the concentration of the candidate substance can be refined. For example, the concentration can be increased or decreased over u range of values and

compared Io the Standard and to the other concentrations being tested lo determine which concentration causes fee greatest increase in stability.

Protein stability can be measured, e.g.- by measuring protein aggregation or protein degradation. Protein aggregation can be determined, e.g.. by siíse exclusion chromatography. non-dsmaUm ng PAGE, or other methods for determining size. etc. Protein degradation can be determined, e.g., by reverse phase HPLC, non-denaturing PAGE, ion-exchange chromatography, peptide mapping, or similar methods.

Preferred carbohydrates are trehalose or sucrose. Other preferred substances suitable for use include: maltose, raffmose, glucose, sorbitol Other suitable substances that can be used to stabilize the protein include: carbohydrates such as lactose and arabiiiose; poiyois such as mannitol, glycerol , and xylitoi; amino acids such as glycine, arginine, lysine, hisπ ^' dme, alanine, methionine, and leucine; and polymers such as PECi poioxuraers, dextran, polypropylene glycol, polysaccharides, niemykdiidose, sodium carboxyrncihy] cellulose, polyvinyl pyrroiidone (PVP), hydrohzed geiatm, and human albumin. In some embodiments, a combination of two or more of these carbohydrates (e.g., sucrose and trehalose) is used in the compositions described herein. The suitability of the combination can be tested as described above for a candidate carbohydrate.

PH _. pB can be critic;) 1 in achieving an optimized protein composition, e.g., a liquid protein composition with increased stability. pH can work by affecting the conformation and/or aggregation and/or degradation and/or the reactivity of the protein. For example, at a higher pH, O^ can be more reactive. The pPI is preferably less than 7.0, more preferably in die range of about 4,5 to about s .5, more preferably about 5.0 to about s .0, and more preferably about 5.5 to about 5.8, more preferably about 5.7, With some proteins, e.g., GCEJ, aggregation can reach undesirable kveis at u pH above 7.0 and degradation (e.g., fragmentation) can reach undesirable levels at a pϊ l under 4,5 or 5.0, or at a pi-! above s.5 or 7.0.

One can test a candidate pB by providing a composition containing 2 rng/ml GCB, 0.07S % cysteine (as an antioxidant). 1s % sucrose (to decrease -S-H availability), adjusting the composition to a candidate pH, and purging the composition of On, The

stability of the GCB composition at ihe candidate pH, measured, e.g., as s percent aggregation or degradat ion, at a predetermined time ss compared with one or more standards. For example, a suitable standard would be a composition similar to the test conditions except that ihe pϊi of the composition is not adjusted. The stabiliti es of the treated (the composition adjusted io the candidate pH) and untreated (the pH is τκ>ι adjusted} compositions are compared. Suitability can be shown by the test treatment increasing stability as compared with this standard. Another standard can be a composition similar to the test composition except that in place of the candidate pH, the composition bas a pH described herein, tor example, pH SJ. Suitability ear, he shown by the composition at the candidate pfj having comparable or better effects on stability than the composition at pH 5.7.

Protein stability can be measured, e.g., by measuring protein aggregation or protein degradation. Protein aggregation can be determined, e.g., by size exclusion chromatography, non-denaturing PAGE, or other methods for determining size, etc. Protein degradation can be determined, e.g., by reverse phase HPLC, non-denaturing PAGE, ion-exchange chromatography, pspii.de mapping, or similar meifiods.

Buffers that can be used lo adjust the pH of a protein composition include: histidinε, citrate, phosphate, glycine, succinate, acetate, giuiamate, Tris, tartrate, aspartate, male-ate, and lactate, A preferred buffer is citrate.

Pro tein Coricentraύon

A preferred protein (e.g., GCB) concentration can be between about 0,1 to about 40 mg/ml, more preferably about 0.5 to about 10 mg/ml, e.g., aboot 2 to about 8 mg/ml or about 5 sng/rnl

One can test for a suitable protein concentration by providing a composition containing 0.075 % cysteine (as an antioxidant), 1s % sucrose (to decrease -S-H availability ϊ, adjusting the piH to 5,7, adjusting the protein (e.g., GtUB) to a candidate concentration, and purging ihe composition of (>>. The stability of the protein (e.g., GCB) composition, at the candidate concentration, measured, e.g., as a percent aggregation or degradation, at a predetermined time is compared with out' or more standards. For exaranie, a suitable standard would be a composition similar to the test

conditions except thai the protein (e.g., GCB) concentration is a concentration described herein, e.g., 2 mg/mi. The stabilities of the protein (e.g., GCB) at each concentration are compared. Suitability can be shown by the candidate concentration having comparable or better eifects on stability than a concentration described herein.

Protein stability ear* be measured, e.iu by measuring protein aa^re-^ation or protein degradation. Protein aggregation can be determined, e.g., by size exclusion chromatography, non-denaturing PAGE, or other methods for determ ining ύy.c, etc. Protein degradation can be determined, e.g., by reverse phase HPLC-, non-denaturing PACJE, iun-ex change chromatography, peptide mapping, or similar methods.

Surfacjλπts

A surfactant can be added to the liquid protein (e.g., GCB) eomposiiion. hi a preferred embodiment this eun increase protein stability, e.g., reduce protein degradation, e.g., due to arr/iiquid interface upon shaking/shipment. A surfactant that increases protein stability., e.g., does not cause protein degradation, in the liquid composition is selected. A surfactant suitable lor use is e.g., poloxamer 1 SS, e.g., PLURONIC® Fs8. The surfactant can be present in an amount between about 0.005% and about 5%, e.g., between about 0.01 % and about 1%, e.g., about 0.025% and about 0.5%. e.g., about 0.03% and about 0.25%, e.g., about 0.04 to about 0.1%, e.g., about 0 05% to ahom U.075%, e.g., 0.05%.

Ideally, a surfactant selected for use in the protein compositions described herein is one that is not modified, e.g., cleaved, by the protein.

For example, one can test a candidate surfactant by providing a composition containing 2 mg/ml GCB, 0.075 % cysteine (as an antioxidant), 1s % sac-rosy (to decrease - S-Ii availability), adjusting the pH to 5,7, adding the candidate surfactant (e.g., in an amount described herein, e.g., 0.05%), and purging the composition of Ov The stability of the GCB composition containing the candidate surfactant, measured, e.g., as a percent aggregation or degradation, at a predetermined time is eompared with one or nusre standards. For example, a suitable standard would be a composition similar to the test conditions except thai a surfactant is not added to the composition. The stabilities of the treated (containing the surfactant) and untreated (lacking a surfactant) compositions

are compared in conditions simulating "real world" scenarios, e.g.. shipping. Suitability can be shown by the tesi treatment increasing stabiiuy as compared with this standard. Another standard can be a composition similar to the test composition except that in place of the candidate surfactant, a surfactant described herein, for example, poloxaraεr 188 (e.g., m ars amount described herein, e.g., 0,05%), is added to the composition. Suitability can be shown by the candidate surfactant having comparable or better effects uii stability than a surfactant described herein. If the candidate surfactant is determ ined to be suitable ⁽ e.g., it increases stability of the composition as compared to one of the standards), the concentration of the candidate surfactant can be reϋneά. For example, the concentration can be increased or decreased over tx range of values and compared to the standard ana to the other concentrations being tested to determine which concentration causes the greatest increase in stability.

In some embodiments, a combination of two or more surfactants is used in the compositions described herein. The suitability of the combination can be tested as described above for a candidate surfactant.

Protein stability can be measured, e.g.. by measuring protein aggregation, or protein degradation. Protein aggregation cars be determined, e.g., by sixe exclusion chromatography, non-denaturing PACjE, or other methods for determining si?;e, etc. Protein degradation can be determined, e.g., by reverse phase HPLCl non-denaturing PAGE, ion-exchange chromatography, peptide mapping, or similar methods.

GCB

Gaudier disease is an autosomal recessive lysosomal storage disorder characterized by a deficiency in the lysosomal enzyme, glucocerebrosidase (GCS). GCS bydroiyzes the gtyeoϋpid glucocerebroside that is formed after degradation of glycosphmgulipids in the membranes of white blood cells and red blood cells. The deficiency in this enryine causes glycoeerebrøside to accumulate in large quantities in the lysαsomes of phagocytic cells located in the hver, spleen, and bone marrow of Gaucher patients. Accumulation of these molecules causes a range of clinical manifestations including splenomegaly, hepatomegaly, skeletal disorder, thrombocytopenia and anemia.

(Beutier et at Gaucher disease; In: The Metabolic and Molecular Bases of Inherited Disease (McGraw-Hill inc. New York, 1995} pp.2s25~2s39).

Treaments tor patients suffering from this disease include administration of analgesics for relief of bone pain, blood and platelet transfusions and, in some cases, splenectomy, John replacement is sometimes .necessary for patients who experience bone erosion. Enzyme replacement therapy with OCB has been used as a treatment for Gaucher disease.

The structure of GCB in solution provides relatively accessible (as opposed to buried or hindered) free -S-H moieties, which promotes reactions with the -S-H moiety.

GCB can be obtained by methods that, are known i.n the art. For example, WO02/.5927, WO2005/089047, W003/056897, WOO 1 /77307, WO01/0707S, and WO90/07573: European Published App. No. EPI 39282s; U.S. Published Application Nos. 2005-0026249, 2005-00198s 1 , 2002-Oi s8750, 2005-0265988, 2004-0043457, 2003-0215435, and 20034)133924; and U.S. Patent Application No. 10/9s8,870; U.S. Patent Nos. 7,138,262, 6,451,s00, 6,074,864, 5,879,s 80, 5,549,892, 5,23s,838, and 3,910,822 describe methods or preparing (GCB protein. Any of the GCB protein preparations described in these patents and applications can be formulated into a composition described herein.

GCB enzymatic activity can be measured as described in the examples provided herein, or us described in the art , e.g., In U.S. Pal. No. 7 ,138,262.

Packaging and Delivery protein compositions, e.g., GCB compositions, e.g., the compositions described herein and in WO02/15927, US. Published Application Nos, 2005-002s249, 2005- 001986 1 and 2002-01s8750, and U.S. Patent Application Nos. 09/041 ,471 and 10/9s 8.S70. can be packaged in a two chamber syringe. For example, the composition in lyophilized f orm can be placed into a ilrsi syringe chamber and a liquid can be present in a second syringe chamber (see e.g., U.S. Published Application No, 2004-0249339).

Protein compositions, e.g.. GCB compositions, e.g., the compositions described herein and in WO02/ 15927, U.S. Published Application Nos. 2005-002s 249. 2005- 001986 1 , and 2002-016887S0, and U.S. Patent Application Nos. 09/s41 ,471 and

I 0/%070, can be packaged in a needleless syringe (see e g., U.S. Patent Nos. s,40s,455 and s,939,324). Briefly, as one example, the injection device includes; a gas chamber containing a gas or a source of gas; a port which can allow for release of gas irom the gas chamber; u plunger, which: upon the release of gas from, the gas chamber, can cause movement of at least a first piston; a first piston.; a second piston; a first chamber, e.g. a chamber useful for drug storage and mixing; a piston housing, in which are disposed the first piston, the second piston and the first chamber; a displacement member which can. independent of the motive power of gas front the gas chamber, cause movement of one or both of the first and second pistons (the displacement member can be the plunger or a separate member); an orifice suitable for needleless injection in communication with the first chamber; wherein the first and second piston, are slidεab-ly disposed within the piston housing, and the displacement member, the source of gas, and the plunger are disposed such thai; in a first position of the pistons, a second chamber, e.g., a fluid reservoir, is defined within the piston housing by the first piston, die piston housing and the second piston, the displacement member can move one or bolii of the pistons into a second position wherein the first piston rs in a position such fliat the second chamber, which can be a fluid reservoir, is in communication with the first chamber, which can be IJ drug storage and mixing chamber, and the second piston is moved in the direction of the first piston, thereby decreasing the volume of the second chamber and allowing the transfer of fluid from the second chamber to the first cham ber, the plunger, upon release of gas from the gas chamber, causes the first piston to move so as to decrease the volume of the first chamber allowing a substance to he expelled through the orifice and from the chamber and, e.g., to a subject.

The needleless syringe can include separate modules for a first component, e.g., a dry or liquid component, and a second component, e.g., a liquid component, ^' The modules can be provided as two separate components and assembled, e.g., by the subject who will administer the component to himself or herself, or by another person, e.g., by an individual who provides or delivers health care. Together, the modules can lbrm all or part of the piston housing of devices described herein. The devices can be used Lo provide any first and second component where it is desirable to store or provide the components separately and combine them pπor to administration to a subject.

- 2K -

Protein (e.g., GCB) compositions described herein can be incorporated into pharmaceutical compositions suitable for administration to a subject, e.g., a human. A GCB composition can include a sufficient dosage of GCB to treai a subject having Gaucher disease. The pharmaceutical compositions can include one or more pharmaceutical iy acceptable carriers. As used herein the language "p harmaceutically acceptable earner" is intended to include any and all solvents, excipieots,. dispersion media, coatings, antibacterial and antifungal agents, isotonic and adsorption delaying agents, and the like, compatible with pharmaceutical administration. Pharmaceutical formulation is a well-established an, and is farther described, e.g., in Gennaro (ed.), Remington: The Science and Practice of Pharmacy, 2OtSi ed.., Lipnincoti, Williams & Wilkkis (2000) (ISBN: 0s8330s472 }: Ansel et al, Pharmaceutical Dosage Forms arui Drug Delivery Systems, 7th Ed., Lippincott Williams <& Wilkins Publishers (iy99j (ISBN: 0s83305727); and ICibbe (ed.}. Handbook of Pharmaceutical Bxcipients American Pharmaceutical Association, 3rd ed. (2000) (ISBN; 0 ^s )l ?330%X). Except insofar as any conventional media or agent is incompatible with the active compound, such media can be used in the compositions of the invention. Supplementary active compounds can also be incorporated into the compositions,

A pharmaceutical composition may include a "therapeutically effective amount" of a composition described herein. Such effective amounts can he determined based on the effect of the administered composition. A therapeutically effective amount of a composition may also vary according to (actors such as the disease suite,, age, sex, and weight of the individual and the ability of the composition to elicit a desired response in the individual, e.g., amelioration, of at least one symptom of a condition or disorder e.g., a giucocerehrosida.se deficiency, e.g., Gaucher disease. A therapeutically effective amount is also one in which any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects.

A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. For example, the composition can be administered by a parenteral mode (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular injection ). The phrases "parenteral administration" and "administered parenieraiiy as used herein mean modes of administration other than enteral and topical

administration, usually by injectioii, and include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrastermd injection aad infusion. Preferably, the route of administration is intravenous. Solutions or suspensions used for parenteral application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols. glycerine, propylene glycol or other synthetic solvents; antibacterial agents such us benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethyienediamtnetetraacetic acid; butlers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such fits sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can by enclosed hi ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable tor injectable use include sterile aqueous solutions ⁽ where water soluble) or dispersions and sterile powders, e.g., lyopfriiized preparations, for the extemporaneous preparation of steπle injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, CREMOPHOR EL™ (BASF, Parsippany. Ni ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringabiiuy exists. It must be stable under the conditions of manufacture and stomge and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier con be a solvent or dispersion medium comaining, for example, water, etharsol, polyol (for example, glycerol, propylene glycol and liquid poiyetheyiesie glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the acuøn of microorganisms can be achieved by various antibacterial auα antifungal agents, for example, parabens, cblorobutanol, phenol ascorbic acid, thimcrosal, and the like. In many cases, it will be preferable to include isotonic agents, for examp le, sugars, pdy&lcohofs such as maniioL sorbitol sodium

chloride in the composition. Prolonged stability of the injectable corπposmoπs can be brought about by including in the composition an agem which delays adsorption, for example, aluminum monostearate, human stxurn albuπun and ^ehvdn.

Slerile injectable sokuioπs can be prepared by incorporating GCB compositions described herein m the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by Olter sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. Ui the case of sterile powders for the composition of sterile injectable solutions, the preferred methods of composition are vacuum dryiπμ arid freeze- drying, e.g., lyophi li ^g ation, whkh yields a powder of the active ingredient plus any additional desired ingredient from a previously sterite-ftitered solution thereof

The active compounds (e.g., GCB compositions described herein) cars be prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymej-s can be used, such as ethylene vinyl acetate, poSyanhydrides, polyglycoiic acid, collagen, polyorthocsters, and poiylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known Io those skilled in Um ait, for example, as described m U.S. Patent No. 4,522.,SsI 1 .

Protein compositions, e.g., GCB compositions, described herein can be administered with medical devices known in the art. For example, a protein (e.g.. GCB) composition described herein can be administered with a needle-less hypodermic injection device, such as the devices disclosed In U.S. Patent No. 5399,1 s3. 5,383,851, 5,312335, 5,0s4,413, 4,941 ,SSO, 4,790,824, or 4,59s.55s. Examples of well-known implants and modules useful in the invention include; U.S. Patent Mo. 4,487.s03, which discloses an implantable micro-inrusion pump lor dispensing medication at ft controlled rate; U.S. Patent No. 4,4^s, 194, which discloses a therapeutic device for administering

medicares ihroisgh the skin; U.S. Patent No. 4,447,233, which discloses a medication mfusion pump for delivering medication at a precise infusion rate; ^" U.S. Patent No. 4,447,224, which discloses a variable flow Implantable infusion apparatus for continuous drug delivery; U.S. Patau No. 4,439,19s, which discloses at- osmotic drug delivery system having multi-chamber eovnpartments; and IIS, Patent No. 4,475 J %, which discloses an osmotic drug delivery system. Of course, many other such implants, delivery systems, and modules also are known.

EXAMPLES

Example J : Materials aBdJKqo.pmeiit

The following reagents were used in generating ihe results presented in

Examples 2-s ;

GCB: GIucocerebrosidase was prepared using, but not limited to, the methods described in International App. No. PCT/USO i /25882 Other methods known to one of ordinary skill in the an using recombinant DNA technology may also be used, for example, the methods described in Internationa! App. NOB.

PCT/US88/043R PCT/1JS8WO58O1, and PCT/US92/0043I , and US Patent Nos.

5,23ύJ38Bϊ , 5,s4I,s 70B1 , 5,549.892Bl , and s,270,9&9BL

Sucrose; P/N S-124-Oi or S-124-02, Ptaiistiehl (Wαukegcπ. IL)

Cysteine HCI: P/N ^" 2071-05, JTBaker (Phillipsburg, NJ)

Pokoxanier 188: P/N Pl 1s9, Spectrum (New Brunswick, NJ)

Sodium Chrate: P/N 3s49-01 , JTBaker (Phillipsburg, NJ)

20 ml. vials: P/N s800-032 L West Pharmaceuticals Services (Lkmvϋie. PA)

2 nil- vials: P/N s800-0314, West Pharmaceuticals Services (LionviOe, PA)

20 mm stoppers: P/N 1950-0414, West Pharmaceuticals Services (Lionviile, PA)

13 rnnϊ stoppers: P/N 1950-0412, West Pharmaceuticals Services (Lionvilϊe, PAj

N ₂ gas; P/N UN 100s, Airgas (Salem, NH )

Lyopmiixeπ Genesis 35EL, SP industries (Waπninstcr, FA)

Example 2: GCB Stability

GCB was formulated at 2.5 mg/ml in 1s % sucrose 0.03% Cysteine HCL 0.05% pobxamer ! 88, 5O m M sodium citrate, pH s.0. TwerUy-rrsL glass vials were ti lled at 4.5 niL each wish the formulated solutions. The fiUed vials were loaded oruo a shelf of a iyophilizer and vacuum degassed at 500 mϊ with a shelf temperature of 2O ⁰ C for 3 minutes, folknved by backfill with N^ to 950 rπBar and Immediately stoppered with 20 mm gray stoppers. The samples were placed into a 2-8 ⁰ C stability chamber. At 0, s , 12, 18, and 24 months after storage, the samples were pulled and tested for enzyme activity, aggregation by SE-HPLC, and degradation changes by RP-HPLC. Enzyme activity was assayed by a colorimetric assay using p-mtrophenyl β~D-giueopyraoosiue as the substrate (the activity can also be assayed, e.g, using the assay described in US PaL No. 7,138,2s2).

The results are summarized in Table 1. GCB from this composition had less than 5% changes compared to the baseline after 24 months at 2-S ⁰ C.

Table 1 : Stability Summary for Example 2 after 24 Møsitt$ Storage at Z-B ⁰ C

Example 3: Effect _. of O ₂ Levels

GCB was formulated at 2.5 πig/m L GCB in 1s% sucrose, 0,03% Cysteine HCi. 0.05% pobxamer 188, 50 mM sodium citrate, pH s.0. ϊwo-mL glass vέals were filled Lo ! m L each with the formulated sokύions. The headspace of the vsals was treated using a iyophiiker to have O> level of 3%, s%. or 14%. The samples were placed into a 2-S ⁰ C stability chamber. At the s months time point, the samples were pulled and tested for aggregation change by SE-HI ⁵ LC ^' and degradation change by RP-HPLC. The results are summarized m Table 2. GCB from these compositions is sensitive to the oxygen level .in

the headspace of the v ia With O; L-ss than 3%, essentially no changes, were observ ed after (> months a.2- S "C

Table 2; _>tal>ilit Summan' for Exam le 3 after (> .Moaths Stora e af 2-8 ^J C

t'reentaLV setane rom the hasolnc.

GCB was soimiiUtiod at 2,5 ms mi ⁽ ϊCB m ^f \05 ⁿ c Olivine HCL ''.-5 ⁵ O foloxaracr ; ^S, 50 n\M ^odnuii citrate, prl o 0, ctmtauu-ig sϋcr<^c iovcJs vf v>* _> „ > ⁱ \>, ^ ¹ '-., or 1 O^'.i. 1 «o-iiu. glass! \ ials uere tilsed to 1 ml vacii \Mth tiit iorratualed sol»ϋt<ns ] \w \ sals λ\ crc \ aciumi degassed by a iyophih/er and overfed with \; to V>i) mBar, iolk^A'cc by ciosπig xv Uh i 3 mm stoppers. 1 he samples wereplaced into d.2-i> ^c C mihύrtx chamber. λt the o uionth time poiυi. the samples were pulled for tt-snn^ of ayi?,re«uuun change b> SH-HPL V diiύ degradiitiou change by RP-IIPf wCw The results are sαmniuu/cd ύi Table 3.

ϊ ^' iϊhle 3: Stability Summary for Example 4 after 6 MontliN Stura«t>M ¹ 2-8'X"

^■ r c ai'c i anc roπi e* .-^ n.

Exa»ll>k 3; Effect af Cysteine Lεs els

CK B ^as ibrmulated <u 2.5 nuyt^l GCB m !Cλ< suorosSv.-, 0.1/5 ⁰ O poioxomer I ¹ ^S, 50 niNI sodnim cHratc. pll s.0, eoiuαining cysteine HCL of U"'.. or (lO5 ^c ι\ 1wo-mL gkt?.^ vials Y*. ere ftlL-d iu ) n\L each v, ah the fonnu!ait.'d sυluttons. The \ iais were \ aciumi ik'gasso*! h\ a ϊyopiisiizw and overlaid wuh N ^! ; to ⁽⁾ *»C lυBur in the iit\uispαee. ib1!>n>w\l

by closing with 1 3 mm stoppers. These samples were placed into a 2-8 ^o C stability chamber. At the s month time point, the samples were pulled for testin g of aggregation change by SB -HPLC and degradation change by RP-HPLC . The results are summarized in Table 4. Addition of cysteine HCL reduced the aggregation level but increased the degradation level as detected by RP-HPLC.

Table 4: Stability Summary for Example 5 after 6 Months Storage at 2 -8 ^o C

Example 6:Effects of pH. Levels

GCB was formulated at 2.5 nιg/mL GCB iϋ \ b% sucrose. 0.05% cysteine HCi, U.05% potoxamer I ZH, 5(> ru.M sodium citrate with pK of (».0, 5.$ ur 5.5 1 glass vials were tilled to I nd. each svnh the fonnuhned <ιtsiuϊions. The vials were vacuum degassed and overlaid with Nh to by eloaing with 13 ram stoppers. These samples were placed into a 15-1 ? ^C C stability chamber. At the 3 month iirrse point the samples were pulled for testing of aggregation change by SE- HPLC and degradation change by R.P-HPLC. The results are summarized in Table 5. Decreasing pH can reduce hoih the aggregation level and the degradation level.

Table 5: StabslHy Summary for Example s after 3 MOHϊϊIS Storage ui I3-.! 7°C

OTH ER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof the foregoing description is intended to illustrate and not limit the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.