LIQUID ANTIBODY FORMULATION

TECHNICAL FIELD

The present invention relates to a stable pharmaceutical formulation comprising an antibody; preferably the pharmaceutical formulation is liquid.

BACKGROUND

In the context of pharmaceutical product development, a pharmaceutical formulation refers to a mixture of an active pharmaceutical ingredient(s) and inactive ingredients, also known as excipients that are combined to produce a final drug product.

While the active pharmaceutical ingredient (API) has a pharmacological activity that contributes to the diagnosis, treatment, or prevention of a certain disease(s), the inactive ingredients have the role of contributing to product stability, biopharmaceutical profile, appearance and to patient acceptability (P. Furrer, European Pharmaceutical Review, Issue 2, 2013, April 18 2013). In particular the excipients, according to the International Pharmaceutical Excipients Council (IPEC, 1995) "are all substances contained in a dosage form other than the active substance or finished dosage form, which have been appropriately evaluated for safety and are included in a drug delivery system to either aid the processing of the drug delivery system during its manufacture, protect, support, enhance stability, bioavailability, or patient acceptability, assist in product identification, or enhance any other attributes of the overall safety and effectiveness of the drug delivery system during storage or use". Excipients therefore include solvents, diluents, buffering agents, pH-adjusting agents, surfactants, preservatives, tonicity agents, stabilizing agents etc.

When developing a drug product, the choice of the pharmaceutical formulation (API(s) concentration, excipients, excipients concentration) depends on the properties of the API(s), on the drug product presentation, on the route of administration of the drug product and on the administration dosage. Among the factors driving such choice, there is the stability of the final drug product, for which integrity of the API and safety of the formulation for the patients must be assured. At this aim, analytical tests are carried out to assess the stability of the pharmaceutical formulation when subjected to different stresses (i.e. freeze-thaw cycles, shaking) and in different conditions (i.e. short term and long term storage, storage at different temperatures). A first indication of product degradation is the visual appearance, which could disclose large aggregate formation as well as microbial contamination. A part from visible alterations, the presence/absence of sub-visible particle in parenteral products is regulated by Pharmacopeia, in fact, significant presence of sub-visible particles, whether they are product-related or foreign, may lead to potential immunogenicity and may compromise the efficacy of the drug product as well as its safety (A. McAuley et al. BioPharm International Volume 22, Issue 6, Jun 01, 2009).

Different drug product types have different characteristics that affect their stability (M. Rios, BioProcess Int, May 12, 2015), therefore additional appropriate stability tests needs to be adapted to the product. For instance, when considering biological therapeutic products, such as therapeutic antibodies, the stability of the antibody can be determined by assays that show the consistency in protein concentration, the presence/absence of aggregates, fragments, or charge variants and by assays that confirm the capability of the antibody to efficiently bind its target. For the production of therapeutic antibodies, the development of stable pharmaceutical formulations is necessary to obtain an effective and stable drug products, therefore optimizing antibody pharmaceutical formulations, and in parallel performing exhaustive stability tests, is a primary need of the pharmaceutical industry.

SUMMARY The present invention relates to a stable liquid pharmaceutical formulation comprising an anti-OX40 antagonist antibody or fragment thereof, a buffer and one or more stabilizing or tonicity agents.

In one aspect of the present invention, the disclosed pharmaceutical formulation comprises an anti- 0X40 antagonist antibody or fragment thereof that is present within said pharmaceutical formulation at a concentration between about 100 mg/mL and about 200 mg/mL. In another aspect the pharmaceutical formulation has a pH between about 5.0 and about 7.0. In a more particular aspect, the pharmaceutical formulation has a pH of about 6.0 ± 0.5.

In one embodiment the buffer of the pharmaceutical formulation is selected from the group comprising acetate, histidine-HCI, citrate and phosphate, wherein said buffer is present within said pharmaceutical formulation at concentration between about 1 mM and about 50 mM. In a more particular embodiment the buffer is histidine-HCI or citrate, present within said pharmaceutical formulation at a concentration of about 25 mM.

In another embodiment, the pharmaceutical formulation comprises a stabilizing or tonicity agent selected from the group comprising sodium chloride, calcium chloride, mannitol, sucrose, polyethylene glycol 400, sorbitol, trehalose, EDTA, glycine, arginine, arginine-FICI, glutamine, glutathione, methionine, cysteine, proline, lysine, lysine-HCI, Polysorbate 20, Polysorbate 40 and Polysorbate 80, Poloxamer, Poloxamer 188, Poloxamer 407.

In a particular embodiment, the stabilizing or tonicity agent is sodium chloride present within said formulation at a concentration between about 100 mM and about 200 mM; and/or arginine-HCI present with said formulation at a concentration between about 100 mM and about 200 mM; and/or proline present within said formulation at a concentration between about 100 mM and about 200 mM; and/or lysine-HCI present within said formulation at a concentration between about 100 mM and about 200 mM; and/or mannitol present within said formulation at a concentration between about 0.5% and about 5%; and/or sucrose present within said formulation at a concentration between about 5% and about 10%; and/or methionine present within said formulation at a concentration between about 1 mM and about 20 mM, and/or Polysorbate 80 present within said formulation at a concentration between about 0.01% and about 0.15%.

In a particular embodiment the pharmaceutical formulation comprises an anti-OX40 antagonist antibody or fragment thereof present within said pharmaceutical formulation at a concentration of about 150 mg/mL, an histidine-HCI buffer present within said pharmaceutical formulation at a concentration of about 25 mM, arginine-HCI or sodium chloride present within said pharmaceutical formulation at a concentration of about 150 mM, optionally methionine present within said pharmaceutical formulation at a concentration of about 10 mM, and Polysorbate 80 present within said pharmaceutical formulation at a concentration between about 0.03% and about 0.06%, and wherein said pharmaceutical formulation has pH between about 5 and about 7.

In another particular embodiment the pharmaceutical formulation comprises an anti-OX40 antagonist antibody or fragment thereof present within said pharmaceutical formulation at a concentration of about 150 mg/mL, histidine HCI buffer present within said pharmaceutical formulation at a concentration of about 25 mM, mannitol present within said pharmaceutical formulation at a concentration between about 1% and about 5% or sucrose present within said pharmaceutical formulation at a concentration of about 8.5%, optionally proline or lysine-HCI present within said pharmaceutical formulation at a concentration of about 150 mM, or arginine-HCI present within said pharmaceutical formulation at a concentration of about 50 mM, and Polysorbate 80 present within said pharmaceutical formulation at a concentration of about 0.036%, and wherein said pharmaceutical formulation has pH of about 6.

In another particular embodiment the pharmaceutical formulation comprises an anti-OX40 antagonist antibody or fragment thereof present within said pharmaceutical formulation at a concentration of about 150 mg/mL, citrate buffer present within said pharmaceutical formulation at a concentration of about 25 mM, sodium chloride or arginine-HCI present within said pharmaceutical formulation at a concentration of about 150 mM, and Polysorbate 80 present within said pharmaceutical formulation at a concentration of about 0.036%, and wherein said pharmaceutical formulation has pH of about 6.

In a more particular embodiment the pharmaceutical formulation comprises an anti-OX40 antagonist antibody or fragment thereof present within said pharmaceutical formulation at a concentration of about 150 mg/mL, an histidine-HCI buffer present within said pharmaceutical formulation at a concentration of about 25 mM, proline or sodium chloride present within said pharmaceutical formulation at a concentration of about 150 mM, or arginine-HCI present within said pharmaceutical formulation at a concentration between about 50 mM and about 150 mM, optionally methionine present within said pharmaceutical formulation at a concentration of about 10 mM or mannitol present within said pharmaceutical formulation at a concentration between about 2% and about 3.5%, and Polysorbate 80 present within said pharmaceutical formulation at a concentration between about 0.03% and about 0.06%, wherin said pharmaceutical formulation has pH between about 5 and about 7, and wherein said pharmaceutical formulation is stable for at least about 6 months at a temperature of about 25°C.

In another more particular embodiment the pharmaceutical formulation comprises an anti-OX40 antagonist antibody or fragment thereof present within said pharmaceutical formulation at a concentration of about 150 mg/mL, histidine HCI buffer present within said pharmaceutical formulation at a concentration of about 25 mM, arginine-HCI or sodium chloride present within said pharmaceutical formulation at a concentration of about 150 mM and Polysorbate 80 present within said pharmaceutical formulation at a concentration of about 0.036%, wherein said pharmaceutical formulation has pH of about 6 and wherein said pharmaceutical formulation is stable for at least about 18 months at a temperature of about 5°C.

The present invention also relates to a pharmaceutical formulation comprised within a prefilled syringe which comprises a glass barrel characterized by a staked needle having gauge between about 25G and about 30G and a siliconization level between about 0.2mg and about 1 mg per prefilled syringe, and a rubber stopper. In a particular embodiment, the pharmaceutical formulation is comprised within a prefilled syringe which comprises a glass barrel characterized by a staked needle having gauge of about 27G and a siliconization level between about 0.3 mg and about 0.7 mg per prefilled syringe, and a halobutyl stopper. In one aspect of the present invention, the anti-OX40 antagonist antibody or fragment thereof is a humanized monoclonal anti-OX40 antagonist antibody or fragment thereof.

The current invention also relates to a pharmaceutical formulation for use in the treatment of an OX40-mediated disorders. In a particular embodiment, a therapeutically effective amount of the pharmaceutical formulation according to the present invention is administrated subcutaneously using a prefilled syringe.

The current invention also discloses a method of manufacturing the pharmaceutical formulation according to the present invention, and/or a method of manufacturing a vial or a prefilled syringe containing said pharmaceutical formulation according to the present invention. Also disclosed herein is a method to test said pharmaceutical formulation to any assay for stability determination.

The present invention also relates to an article of manufacture comprising: (a) a container which holds the prefilled syringe and (b) instructions for using said prefilled syringe.

Also disclosed in the current invention is a method for obtaining a high concentrated antibody solution comprising the steps of subjecting a clarified cell harvest to an affinity chromatography step, and subjecting the obtained eluate to at least two ion exchange chromatography steps and to at least three UF/DF steps.

The present invention related to a liquid pharmaceutical formulation comprising an anti-OX40 antagonist antibody or fragment thereof, a buffer and one or more stability or tonicity agents. As used herein, the following terms have the following meanings: "a", "an", and "the" as used herein refers to both singular and plural unless the context clearly dictates otherwise.

Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures utilized in connection with, and techniques of cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry, laboratory procedures and techniques of analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Temperatures above 0°C are expressed in the present invention either by preceding the temperature value by "+" or not, e.g. 25°C and +25°C can be used interchangeably according to the present invention.

A "liquid” formulation is one that has been prepared in a liquid format. Such a formulation may be suitable for direct administration to a subject or, alternatively, can be packaged for storage either in a liquid form, in a frozen state or in a dried form (e.g. lyophilized) for later reconstitution into a liquid form or other forms suitable for administration to a subject.

The term "buffer" as used herein refers to a buffered solution that resists changes in pH by the action of its acid-base conjugate components. A buffer of this invention has a pH in the range from about 5.0 to about 7.0; and preferably is 6.0±0.5. Examples of buffers that can control the pH in this range include acetate (e.g. sodium acetate), succinate (such as sodium succinate), gluconate, amino acids, such as histidine (e.g. histidine-HCI), citrate, phosphate, other organic acid buffer, their salts and combinations of buffers. In one embodiment of the present invention the buffer is present within the pharmaceutical formulation at concentration between about 1 mM and about 100 mM; in a more specific embodiment the concentration of the buffer is between about 5 mM and about 50 mM; preferably the concentration of the buffer is about 25 mM. In another aspect of the present invention, the concentration of the buffer is at least about 1 mM, at least about 5 mM, at least about 10 mM, at least about 20 mM, at least about 25 mM, at least about 30 mM, at least about 40 mM, at least about 50 mM, at least about 60 mM, at least about 70 mM, at least about 80 mM, at least about 90 mM, at least about 100 mM. The present invention also includes a buffer with a concentration at any intermediate value of the above said values. In a particular embodiment of the present invention, the buffer is Histidine, e.g. histidine-HCI, present within the pharmaceutical formulation at a concentration of about 25 mM; in another particular embodiment the buffer is citrate, present within the pharmaceutical formulation at a concentration of about 25 mM.

A stabilizing or tonicity agent may be added to the formulation to stabilize the protein in the lyophilized form. Said stabilizing or tonicity agent is selected from the group comprising sodium acetate, sodium bicarbonate, sodium carbonate, sodium chloride (NaCI), potassium acetate, potassium bicarbonate, potassium carbonate, potassium chloride, calcium chloride (CaCI2) sugars such as sucrose, glucose and trehalose, polyols such as mannitol, maltitol, sorbitol, xylitol, erythritol, and isomalt, polyethylene glycol, such as PEG400, Ethylenediaminetetraacetic acid (EDTA), amino acids such as histidine (e.g. histidine-HCI), arginine (e.g. arginine hydrochloride) and glycine, methionine, proline, lysine (e.g. lysine-HCI), glutamic acid, glutamine, cysteine, amines, glutathione, cyclodextrin, such as such as Hydroxypropyl b-cyclodextrin (HPBCD), Hydroxypropyl-sulfobutyl b- cyclodextrin (HPSBCD), Sulfobutylether b-cyclodextrin (SBECD), b-cyclodextrin (BetaCD), cyclodextrin (Alpha CD) and g-cyclodextrin (Gamm CD) and surfactants. Non limiting examples of a typical surfactant include: non- ionic surfactants (HLB 6 to 18) such as sorbitan fatty acid esters (e.g. sorbitan monocaprylate, sorbitan monolaurate, sorbitan monopalmitate), glycerine fatty acid esters (e.g. glycerine monocaprylate, glycerine monomyristate, glycerine monostearate), poly glycerine fatty acid esters (e.g. decaglyceryl monostearate, decaglyceryl distearate, decaglyceryl monolinoleate), polyoxyethylene sorbitan fatty acid esters (e.g. polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan trioleate, polyoxyethylene sorbitan tristearate), polyoxyethylene sorbitol fatty acid esters (e.g. polyoxyethylene sorbitol tetrastearate, polyoxyethylene sorbitol tetraoleate), polyoxyethylene glycerine fatty acid esters (e.g. polyoxyethylene glyceryl monostearate), polyethylene glycol fatty acid esters (e.g. polyethylene glycol distearate), polyoxyethylene alkyl ethers (e.g. polyoxyethylene lauryl ether), polyoxy ethylene polyoxypropylene alkyl ethers (e.g. polyoxyethylene polyoxypropylene glycol ether, polyoxyethylene polyoxypropylene propyl ether, polyoxyethylene polyoxypropylene cetyl ether) , polyoxyethylene alkylphenyl ethers (e.g. polyoxyethylene nonylphenyl ether), polyoxyethylene hydrogenated castor oils (e.g. polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil), polyoxyethylene beeswax derivatives (e.g. polyoxyethylene sorbitol beeswax), polyoxyethylene lanolin derivatives (e.g. polyoxyethylene lanolin), and polyoxyethylene fatty acid amides (e.g. polyoxyethylene stearyl amide); anionic surfactants such as Cio-Cis alkyl sulfates salts (e.g. sodium cetyl sulfate, sodium lauryl sulfate, sodium oleyl sulfate), polyoxyethylene Cio-Cis alkyl ether sulfates salts with an average of 2 - 4 moles of ethylene oxide (e.g. sodium polyoxyethylene lauryl sulfate), and Cs-Cis alkyl sulfosuccmate ester salts (e.g. sodium lauryl sulfosuccmate ester); natural surfactants such as lecithin, glycerophospho lipid, sphingophospho lipids (e.g. sphingomyelin) and sucrose esters of C 12-C 18f atty acids; Poloxamers such as Poloxamer 188, Poloxamer 407, Poloxamer 124, Poloxamer 237, Poloxamer 338; salts and combinations of the above cited components .

In a certain aspect of the present invention, the stabilizing or tonicity agent is present within the pharmaceutical formulation at a concentration between about 0.001 mg/mL and about 300 mg/mL, i.e. between about 50 mg/mL and 100 mg/mL, between about 70 mg/mL and 200 mg/mL, between about 5 mg/mL and 50 mg/mL, between about 1 mg/mL and 20 mg/mL, between about 0.1 mg/mL and 10 mg/mL, between about 0.001 mg/mL and 0.1 mg/mL. In another aspect of the present invention, the stabilizing or tonicity agent is present within the pharmaceutical formulation at a concentration between about 1 mM and about 300 mM, i.e. between about 5 mM and 200 mM, specifically about 10 mM, or about 50 mM, or about 100 mM, or about 150 mM, or about 200 mM. In another aspect of the present invention, the stabilizing or tonicity agent is present within the pharmaceutical formulation at a concentration between about 0.001% and about 15%, i.e. between about 0.01% and about 5% or between about 0.03% and about 3%. In a particular aspect, the pharmaceutical formulation of the present invention comprises one or more stabilizing or tonicity agent(s) selected form the group comprising sodium chloride, arginine-HCI, proline, mannitol, lysine- HCI, sucrose, methionine and a surfactant. In a more particular aspect the pharmaceutical formulation of the present invention comprises NaCI present within the pharmaceutical formulation at a concentration between about 100 mM and about 200 mM, specifically between about 130 mM and about 170 mM, even more specifically NaCI is present within the pharmaceutical formulation at a concentration of about 150 mM. In another particular aspect the pharmaceutical formulation of the present invention comprises arginine-HCI present within the pharmaceutical formulation at a concentration between about 100 mM and about 200 mM, specifically between about 130 mM and about 170 mM, even more specifically arginine-HCI is present within the pharmaceutical formulation at a concentration of about 150 mM. In another particular aspect the pharmaceutical formulation of the present invention comprises proline present within the pharmaceutical formulation at a concentration between about 100 mM and about 200 mM, specifically between about 130 mM and about 170 mM, even more specifically proline is present within the pharmaceutical formulation at a concentration of about 150 mM. In another particular aspect the pharmaceutical formulation of the present invention comprises mannitol present within the pharmaceutical formulation at a concentration between about 0.5% and about 10%, specifically between about 1% and about 5%, even more specifically mannitol is present within the pharmaceutical formulation at a concentration of selected from the group comprising about 1%, about 2%, about 2.5%, about 3%, about 4%, about 4.5%. In another particular aspect the pharmaceutical formulation of the present invention comprises lysine-HCI present within the pharmaceutical formulation at a concentration between about 100 mM and about 200 mM, specifically between about 130 mM and about 170 mM, even more specifically lysine-HCI is present within the pharmaceutical formulation at a concentration of about 150 mM. In another particular aspect the pharmaceutical formulation of the present invention comprises sucrose present within the pharmaceutical formulation at a concentration between about 1% and about 15%, specifically between about 5% and about 10%, even more specifically sucrose is present within the pharmaceutical formulation at a concentration of about 8.5%. In another particular aspect the pharmaceutical formulation of the present invention comprises methionine present within the pharmaceutical formulation at a concentration between about 1 mM and about 50 mM, specifically between about 5 mM and about 25 mM, even more specifically methionine is present within the pharmaceutical formulation at a concentration of about 10 mM. In another aspect the pharmaceutical formulation of the present invention comprises a surfactant present within the pharmaceutical formulation at a concentration between about 0.001% and about 1%, specifically between about 0.005% and 0.5%, more specifically between about 0.01% and 0.1%, even more specifically the surfactant is present within the pharmaceutical formulation at a concentration selected from the group comprising about 0.03%, about 0.04%, about 0.05%, and about 0.1%. Preferably, the surfactant is selected from polyoxyethylene sorbitan fatty acid esters. Particularly preferably the surfactant is Polysorbate 20, 21 , 40, 60, 65, 80, 81 and 85, most preferably Polysorbate 80. Polysorbate 80 is also known by the brand name Tween 80™ (ICI Americas, Inc.). In a specific embodiment of the present invention, the surfactant is Polysorbate 80, present within said pharmaceutical formulation at a concentration of about 0.036%, or about 0.054%, or about 0.1%. The present invention also includes a stabilizing or tonicity agent at any intermediate value of the above said values.

The term "antibody" as referred to herein includes whole antibodies and any antigen binding fragments or single chains thereof. An "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding fragment thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHI, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR) with are hypervariable in sequence and/or involved in antigen recognition and/or usually form structurally defined loops, interspersed with regions that are more conserved, termed framework regions (FR or FW). Each VH and VL is composed of three CDRs and four FWs, arranged from amino- terminus to carboxy-terminus in the following order: FW1, CDR1, FW2, CDR2, FW3, CDR3, FW4. The amino acid sequences of FW1, FW2, FW3, and FW4 all together constitute the "non-CDR region" or "non-extended CDR region" of VH or VL as referred to herein.

The term "heavy chain variable framework region" as referred herein may comprise one or more (e.g., one, two, three and/or four) heavy chain framework region sequences (e.g., framework 1 (FW1), framework 2 (FW2), framework 3 (FW3) and/or framework 4 (FW4)). Preferably the heavy chain variable region framework comprises FW1, FW2 and/or FW3, more preferably FW1, FW2 and FW3. The term "light chain variable framework region" as referred herein may comprise one or more (e.g., one, two, three and/or four) light chain framework region sequences (e.g., framework 1 (FW1), framework 2 (FW2), framework 3 (FW3) and/or framework 4 (FW4)). Preferably the light chain variable region framework comprises FW1, FW2 and/or FW3, more preferably FW1, FW2 and FW3. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the First component (Clq) of the classical complement system.

Antibodies are grouped into classes, also referred to as isotypes, as determined genetically by the constant region. Fluman constant light chains are classified as kappa (CK) and lambda (CX) light chains. Fleavy chains are classified as mu (m), delta (d), gamma (y), alpha (a), or epsilon (e), and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Thus, "isotype" as used herein is meant any of the classes and/or subclasses of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions. The known human immunoglobulin isotypes are IgGl (IGHG1), lgG2 (IGHG2), lgG3 (IGHG3), lgG4 (IGHG4), IgAl (IGHA1), lgA2 (IGHA2), IgM (IGHM), IgD (IGHD), and IgE (IGHE). The so-called human immunoglobulin pseudo-gamma IGHGP gene represents an additional human immunoglobulin heavy constant region gene which has been sequenced but does not encode a protein due to an altered switch region (Bensmana M et al., (1988) Nucleic Acids Res. 16(7): 3108). In spite of having an altered switch region, the human immunoglobulin pseudo-gamma IGHGP gene has open reading frames for all heavy constant domains (CHI -CH3) and hinge. All open reading frames for its heavy constant domains encode protein domains which align well with all human immunoglobulin constant domains with the predicted structural features. This additional pseudo-gamma isotype is referred herein as IgGP or IGHGP. Other pseudo immunoglobulin genes have been reported such as the human immunoglobulin heavy constant domain epsilon PI and P2 pseudo genes (IGHEP1 and IGHEP2). The IgG class is the most commonly used for therapeutic purposes. In humans this class comprises subclasses IgGl, lgG2, lgG3 and lgG4. In mice this class comprises subclasses IgGl, lgG2a, lgG2b, lgG2c and lgG3.

The terms "antagonistic antibody" or "antagonist antibody" are used herein equivalently and include an antibody that is capable of inhibiting and/or neutralising the biological signaling activity of 0X40, for example by blocking binding or substantially reducing binding of 0X40 to 0X40 ligand and thus inhibiting or reducing the signalisation pathway triggered by 0X40 and/or inhibiting or reducing an OX40-mediated cell response like lymphocyte proliferation, cytokine expression, or lymphocyte survival. The term "anti-OX40 antagonist antibody or fragment thereof" is used herein to indicate antibodies or antibody fragments thereof that bind to 0X40 e.g. human 0X40, and are capable of inhibiting and/or neutralising the biological signalling activity of 0X40, for example by blocking binding or substantially reducing binding of 0X40 to 0X40 ligand and thus inhibiting or reducing the signalisation pathway triggered by 0X40 and/or inhibiting or reducing an OX40-mediated cell response like lymphocyte proliferation, cytokine expression, or lymphocyte survival.

Antibody fragments include, but are not limited to, (i) the Fab fragment consisting of VL, VH, CL and CHI domains, including Fab' and Fab'-SH, (ii) the Fd fragment consisting of the VH and CHI domains, (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment (Ward ES et al., (1989) Nature, 341 : 544-546) which consists of a single variable, (v) F(ab')2 fragments, a bivalent fragment comprising two linked Fab fragments (vi) single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird RE et al, (1988) Science 242: 423-426; Huston JS et al, (1988) Proc. Natl. Acad. Sci. USA, 85: 5879-83), (vii) bispecific single chain Fv dimers (PCT/US92/09965), (viii) "diabodies” or "triabodies", multivalent or multispecific fragments constructed by gene fusion (Tomlinson I & Hollinger P (2000) Methods Enzymol. 326: 461-79; WO94/13804; Holliger P et al, (1993) Proc. Natl. Acad. Sci. USA, 90: 6444-48) and (ix) scFv genetically fused to the same or a different antibody (Coloma MJ & Morrison SL (1997) Nature Biotechnology, 15(2): 159-163).

In another embodiment of the present invention, the anti-OX40 antibody is present in the pharmaceutical formulation at a concentration between about 1 mg/mL and about 200 mg/mL. In a specific embodiment the anti-OX40 antibody is present in the pharmaceutical formulation at a concentration between about 1 mg/mL and 100 mg/mL, more specifically at a concentration between about 5 mg/mL and about 50 mg/mL, more specifically at a concentration of about 10 mg/mL. The present invention also includes the anti-OX40 antibody with a concentration at any value between the above cited concentrations. According to another aspect of the present invention, the anti-OX40 antibody or fragment thereof is present within the pharmaceutical formulation at a concentration between about 100 mg/ml and about 200 mg/mL, specifically at a concentration between about 130 mg/ml and 180 mg/ml, more specifically at a concentration of about 150 mg/mL. In one aspect of the present invention, the concentration of anti-OX40 antibody or fragment thereof is selected from the group of at least about 1 mg/mL, at least about 10 mg/mL, at least about 100 mg/mL, at least about 130 mg/mL, at least about 150 mg/mL, at least about 170 mg/mL, at least about 200 mg/mL. The present invention also includes concentrations of an anti-OX40 antagonist antibody or fragment thereof at any intermediate value of the above said values. In a preferred embodiment the concentration of the anti-OX40 antibody or fragment thereof is about 150 rng/rnL

In one particular embodiment of the present invention, the pharmaceutical formulation comprises an anti-OX40 antagonist antibody or fragment thereof present within said pharmaceutical formulation at a concentration of about 10 mg/mL, histidine buffer present within said pharmaceutical formulation at a concentration of about 15 mM, sodium chloride present within said pharmaceutical formulation at a concentration of about 150 mM and Polysorbate 80 present within said pharmaceutical formulation at a concentration of about 0.01%, and said pharmaceutical formulation has pH of about 6.25.

In another particular embodiment of the present invention, the pharmaceutical formulation comprises an anti-OX40 antagonist antibody or fragment thereof present within said pharmaceutical formulation at a concentration between about 150 mg/mL and about 200 mg/mL, more specifically between about 160 mg/mL and about 195 mg/mL, or between about 160 mg/mL and about 175 mg/mL, histidine buffer present within said pharmaceutical formulation at a concentration of about 25 mM, sodium chloride or arginine-HCI present within said pharmaceutical formulation at a concentration of about 150 mM, and said pharmaceutical formulation has pH or about 6. Oil.

In another particular embodiment of the present invention, the pharmaceutical formulation comprises an anti-OX40 antagonist antibody or fragment thereof present within said pharmaceutical formulation at a concentration between about 150 mg/mL and about 200 mg/mL, more specifically between about 165 mg/mL and about 181 mg/mL, histidine-HCI buffer present within said pharmaceutical formulation at a concentration of about 25 mM, arginine-HCI present within said pharmaceutical formulation at a concentration of about 150 mM, and Polysorbate 80 present within said pharmaceutical formulation at a concentration of about 0.1%, and said pharmaceutical formulation has pH or about 6.0±1.

In another embodiment the pharmaceutical formulation comprises an anti-OX40 antagonist antibody or fragment thereof present within said pharmaceutical formulation at a concentration of about 150 mg/mL, an histidine-HCI buffer present within said pharmaceutical formulation at a concentration of about 25 mM, arginine-HCI or sodium chloride present within said pharmaceutical formulation at a concentration of about 150 mM, optionally methionine present within said pharmaceutical formulation at a concentration of about 10 mM, and Polysorbate 80 present within said pharmaceutical formulation at a concentration between about 0.03% and about 0.06%, and wherein said pharmaceutical formulation has pH between about 5 and about 7. In another embodiment the pharmaceutical formulation comprises an anti-OX40 antagonist antibody or fragment thereof present within said pharmaceutical formulation at a concentration of about 150 mg/mL, histidine HCI buffer present within said pharmaceutical formulation at a concentration of about 25 mM, mannitol present within said pharmaceutical formulation at a concentration between about 1% and about 5% or sucrose present within said pharmaceutical formulation at a concentration of about 8.5%, optionally proline or lysine-HCI present within said pharmaceutical formulation at a concentration of about 150 mM or arginine-HCI present within said pharmaceutical formulation at a concentration of about 50 mM, and Polysorbate 80 present within said pharmaceutical formulation at a concentration of about 0.036%, and wherein said pharmaceutical formulation has pH of about 6.

In another embodiment the pharmaceutical formulation comprises an anti-OX40 antagonist antibody or fragment thereof present within said pharmaceutical formulation at a concentration of about 150 mg/mL, citrate buffer present within said pharmaceutical formulation at a concentration of about 25 mM, sodium chloride or arginine-HCI present within said pharmaceutical formulation at a concentration of about 150 mM, and Polysorbate 80 present within said pharmaceutical formulation at a concentration of about 0.036%, and wherein said pharmaceutical formulation has pH of about 6.

In a more particular embodiment of the present invention, the pharmaceutical formulation comprises an anti-OX40 antagonist antibody or fragment thereof present within said pharmaceutical formulation at a concentration of about 150 mg/mL, histidine-HCI buffer present within said pharmaceutical formulation at a concentration of about 25 mM, sodium chloride present within said pharmaceutical formulation at a concentration of about 150 mM and Polysorbate 80 present within said pharmaceutical formulation at a concentration of about 0.036%, and said pharmaceutical formulation has pH of about 6.

In another more particular embodiment of the present invention, the pharmaceutical formulation comprises an anti-OX40 antagonist antibody or fragment thereof present within said pharmaceutical formulation at a concentration of about 150 mg/mL, histidine-HCI buffer present within said pharmaceutical formulation at a concentration of about 25 mM, arginine-HCI present within said pharmaceutical formulation at a concentration of about 150 mM and Polysorbate 80 present within said pharmaceutical formulation at a concentration of about 0.036%, and said pharmaceutical formulation has pH of about 6.

In another more particular embodiment of the present invention, the pharmaceutical formulation comprises an anti-OX40 antagonist antibody or fragment thereof present within said pharmaceutical formulation at a concentration of about 150 mg/mL, histidine-HCI buffer present within said pharmaceutical formulation at a concentration of about 25 mM, arginine-HCI present within said pharmaceutical formulation at a concentration of about 150 mM and Polysorbate 80 present within said pharmaceutical formulation at a concentration of about 0.036%, and said pharmaceutical formulation has pH of about 5.5.

In another more particular embodiment of the present invention, the pharmaceutical formulation comprises an anti-OX40 antagonist antibody or fragment thereof present within said pharmaceutical formulation at a concentration of about 150 mg/mL, histidine-HCI buffer present within said pharmaceutical formulation at a concentration of about 25 mM, arginine-HCI present within said pharmaceutical formulation at a concentration of about 150 mM and Polysorbate 80 present within said pharmaceutical formulation at a concentration of about 0.036%, and said pharmaceutical formulation has pH of about 6.5.

In another more particular embodiment of the present invention, the pharmaceutical formulation comprises an anti-OX40 antagonist antibody or fragment thereof present within said pharmaceutical formulation at a concentration of about 150 mg/mL, citrate buffer present within said pharmaceutical formulation at a concentration of about 25 mM, sodium chloride present within said pharmaceutical formulation at a concentration of about 150 mM and Polysorbate 80 present within said pharmaceutical formulation at a concentration of about 0.036%, and said pharmaceutical formulation has pH of about 6.

In another more particular embodiment of the present invention, the pharmaceutical formulation comprises an anti-OX40 antagonist antibody or fragment thereof present within said pharmaceutical formulation at a concentration of about 150 mg/mL, citrate buffer present within said pharmaceutical formulation at a concentration of about 25 mM, arginine-HCI present within said pharmaceutical formulation at a concentration of about 150 mM and Polysorbate 80 present within said pharmaceutical formulation at a concentration of about 0.036%, and said pharmaceutical formulation has pH of about 6.

In another more particular embodiment of the present invention, the pharmaceutical formulation comprises an anti-OX40 antagonist antibody or fragment thereof present within said pharmaceutical formulation at a concentration of about 150 mg/mL, histidine-HCI buffer present within said pharmaceutical formulation at a concentration of about 25 mM, proline present within said pharmaceutical formulation at a concentration of about 150 mM, mannitol present within said pharmaceutical formulation at a concentration of about 2.5% and Polysorbate 80 present within said pharmaceutical formulation at a concentration of about 0.036%, and said pharmaceutical formulation has pH of about 6.

In another more particular embodiment of the present invention, the pharmaceutical formulation comprises an anti-OX40 antagonist antibody or fragment thereof present within said pharmaceutical formulation at a concentration of about 150 mg/mL, histidine-HCI buffer present within said pharmaceutical formulation at a concentration of about 25 mM, lysine-HCI present within said pharmaceutical formulation at a concentration of about 150 mM, mannitol present within said pharmaceutical formulation at a concentration of about 1% and Polysorbate 80 present within said pharmaceutical formulation at a concentration of about 0.036%, and said pharmaceutical formulation has pH of about 6.

In another more particular embodiment of the present invention, the pharmaceutical formulation comprises an anti-OX40 antagonist antibody or fragment thereof present within said pharmaceutical formulation at a concentration of about 150 mg/mL, histidine-HCI buffer present within said pharmaceutical formulation at a concentration of about 25 mM, arginine-HCI present within said pharmaceutical formulation at a concentration of about 50 mM, mannitol present within said pharmaceutical formulation at a concentration of about 3% and Polysorbate 80 present within said pharmaceutical formulation at a concentration of about 0.036%, and said pharmaceutical formulation has pH of about 6.

In another more particular embodiment of the present invention, the pharmaceutical formulation comprises an anti-OX40 antagonist antibody or fragment thereof present within said pharmaceutical formulation at a concentration of about 150 mg/mL, histidine-HCI buffer present within said pharmaceutical formulation at a concentration of about 25 mM, arginine-HCI present within said pharmaceutical formulation at a concentration of about 150 mM, methionine present within said pharmaceutical formulation at a concentration of about 10 mM and Polysorbate 80 present within said pharmaceutical formulation at a concentration of about 0.036%, and said pharmaceutical formulation has pH of about 6.

In another more particular embodiment of the present invention, the pharmaceutical formulation comprises an anti-OX40 antagonist antibody or fragment thereof present within said pharmaceutical formulation at a concentration of about 150 mg/mL, histidine-HCI buffer present within said pharmaceutical formulation at a concentration of about 25 mM, mannitol present within said pharmaceutical formulation at a concentration of about 4.2% and Polysorbate 80 present within said pharmaceutical formulation at a concentration of about 0.036%, and said pharmaceutical formulation has pH of about 6.

In another more particular embodiment of the present invention, the pharmaceutical formulation comprises an anti-OX40 antagonist antibody or fragment thereof present within said pharmaceutical formulation at a concentration of about 150 mg/mL, histidine-HCI buffer present within said pharmaceutical formulation at a concentration of about 25 mM, sucrose present within said pharmaceutical formulation at a concentration of about 8.5% and Polysorbate 80 present within said pharmaceutical formulation at a concentration of about 0.036%, and said pharmaceutical formulation has pH of about 6.

In another more particular embodiment of the present invention, the pharmaceutical formulation comprises an anti-OX40 antagonist antibody or fragment thereof present within said pharmaceutical formulation at a concentration of about 150 mg/mL, histidine-HCI buffer present within said pharmaceutical formulation at a concentration of about 25 mM, arginine-HCI present within said pharmaceutical formulation at a concentration of about 150 mM and Polysorbate 80 present within said pharmaceutical formulation at a concentration of about 0.054%, and said pharmaceutical formulation has pH of about 6.

The pharmaceutical formulation according to the present invention is stable. A "stable" formulation is one in which the protein therein essentially retains its physical stability and/or chemical stability and/or biological activity upon storage. Various analytical techniques for measuring protein stability are available in the art and are reviewed for example in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, New York, Pubs. (1991) and Jones A (1993) Adv Drug Delivery Rev, 10: 29-90. Stability can be measured at a selected temperature for a selected time period.

Analytical tests useful to determine said stability include but are not limited to: monitoring of the visual appearance as a significant change in the appearance of sample may indicate product degradation and/or microbial contamination leading to safety risk for the patients; sub-visible particles analysis, as the presence of higher sub-visible particles in parental solutions may lead to immunogenic responses; protein content measurement (e.g. by measuring absorbance at 280 nm wavelength (A280) by UV-VIS Spectroscopy or by SoloVPE) as any significant variation from its target concentration would not provide effective dose to patients; pH measurement as changes in pH may be indicative of degradation of buffering agents and lead to protein instability; size variants monitoring (e.g. by SE-HPLC and/or by cGE reduced and non-reduced) as changes in monomeric content toward aggregates (higher size than monomer) or fragments (smaller size than monomer) is an indication of its degradation; charge variants monitoring (e.g. by clEF) as changes in content of charged variants is an indication of its degradation; antibody potency measurement (e.g. by ELISA) as any significant change of binding property of the antibody toward its target would indicate antibody degradation. Additionally, the amino acid sequence as well as post-translational modifications (i.e. deamidation, oxidation, glycation, N-terminal variants, C-terminal variants and glycosylation site occupancy) can be verified, for instance by peptide mapping. Other characteristics of the formulation can be monitored, such as osmolarity and viscosity, as well as the protein thermal stability for instance by nano-format of Differential Scanning Fluorimetry (DSF), syringeability.

In one aspect of the present invention, the pharmaceutical formulation is stable under stresses comprising: rolling stress, storage at temperature higher than the room temperature, such as storage at +40 °C for at least 1 week; shaking such as shaking at 900 rpm for at least 24 hours, at least 48 hours, at least 72 hours at room temperature (25°C); freezing-thawing circles such as freezing at -20°C or at -80°C and thawing at about 5°C, or at about 20°C, or at about 25°C, more in particular such as 3 to 5 freeze-thaw cycles by freezing at -80 °C and thawing at +5 °C, such as 3 to 5 freeze-thaw cycles by freezing at -80 °C and thawing at +25 °C, and such as freeze-thaw cycles by freezing at -20 °C and thawing at 20 °C.

According to one aspect of the present invention, the pharmaceutical formulation is stable at temperature equal to or less than about 40°C, i.e. at a temperature between about -80°C and 40°C, for at least about 1 month, or at least about 3 months, or at least about 6 months, or at least about 9 months, or at least about 12 months, or at least about 18 months, or at least about 24 months, or at least about 30 months, or at least about 36 months. The present invention also includes the disclosed pharmaceutical formulations stable at any value between the above cited temperatures, and at any time intervals between the above cited times. In one embodiment of the present invention, the pharmaceutical formulation of the present invention is stable when stored for at least about 1 month, or at least about 2 months, or at least about 3 months, or at least about 6 months, or at least about 9 months, or at least about 12 months, or at least about 18 months, or at least about 24 months, or at least about 30 months, or at least about 36 months, at a temperature of about of at least about 5°C, more specifically at a temperature of about 5°C or at a temperature of about 25°C; in a specific embodiment the pharmaceutical formulation of the present invention is stable when stored for at least about 9 month at a temperature of about 5°C or at a temperature of about 25°C, for properties comprising glycation, sequence terminal modifications, and potency. In another specific aspect, the pharmaceutical formulation according to the present invention is stable when stored for at least about 36 months at a temperature of about 5°C. In a more specific embodiment, the disclosed pharmaceutical formulation is stable at about +25±2°C for at least 1 month; in another specific embodiment, the disclosed pharmaceutical formulation is stable at about +5±3°C for at least about 1 month, preferably for at least about 3 months; in another specific embodiment, the disclosed pharmaceutical formulation is stable at about -80±20°C or about -20±5°C for at least about 1 month, or at least about 3 months, preferably for at least about 6 months. In another more specific embodiment, the disclosed pharmaceutical formulation is stable at about 40±2°C for at least about 1 month; in another specific embodiment, the disclosed pharmaceutical formulation is stable at about 25±2°C for at least about 1 month, more specifically for least about 3 months, preferably for at least about 6 months; in another specific embodiment, the disclosed pharmaceutical formulation is stable at about 5±3°C for at least about 1 month, specifically for at least about 3 months, more specifically for at least about 6 months, even more specifically for at least about 12 months, preferably for at least about 18 months, more preferably for at least about 24 months.

In a more particular embodiment the pharmaceutical formulation comprising an anti-OX40 antagonist antibody or fragment thereof present within said pharmaceutical formulation at a concentration of about 150 mg/mL, an histidine-HCI buffer present within said pharmaceutical formulation at a concentration of about 25 mM, proline or sodium chloride present within said pharmaceutical formulation at a concentration of about 150 mM, or arginine-HCI present within said pharmaceutical formulation at a concentration between about 50 mM and about 150 mM, optionally methionine present within said pharmaceutical formulation at a concentration of about 10 mM of mannitol present within said pharmaceutical formulation at a concentration between about 2% and about 3.5%, and Polysorbate 80 present within said pharmaceutical formulation at a concentration between about 0.03% and about 0.06%, and having pH between about 5 and about 7, is stable for at least about 6 months at a temperature of about 25°C or less.

In another more particular embodiment the pharmaceutical formulation comprising an anti-OX40 antagonist antibody or fragment thereof present within said pharmaceutical formulation at a concentration of about 150 mg/mL, histidine HCI buffer present within said pharmaceutical formulation at a concentration of about 25 mM, arginine-HCI or sodium chloride present within said pharmaceutical formulation at a concentration of about 150 mM and Polysorbate 80 present within said pharmaceutical formulation at a concentration of about 0.036%, and having pH of about 6 is stable for at least about 18 months at a temperature of about 5°C or less. The liquid formulation of the present invention can be comprised within a container. For instance a container made of glass, plastic, other polymers. Non limiting examples of said container are syringes, such as pre-filled syringe, glass cartridge syringe, autoinjectors, bottles, vials, and test tubes, pens, bags such as storage bags. The liquid formulation according to the present invention can be contained in a prefilled syringe for subcutaneous injection using an autoinjectors.

In one embodiment of the present invention, the pharmaceutical formulation is comprised within a glass vial, such as a 2 mL glass vials stoppered with 13 mm injection rubber stopper or such as a 10 mL glass vial stoppered with a 20 mm injection flurotec-coated stopper.

In another embodiment of the present invention the pharmaceutical formulation is comprised within a prefilled syringe (PFS)). A PFS may be filled with a volume such as about 0.1 mL, or about 0.2 mL, or about 0.5 mL, or about 1 mL, or about 1.5 mL, or about 2 mL, or about 2.5 mL, or about 3 mL of the formulation according to the present invention. In particular, the prefilled syringe comprises a glass barrel characterized by a needle having gauge between 25 and 30G and a siliconization level between 0.2mg and 1 mg per prefilled syringe, and a halobutyl stopper; wherein the needle may be a staked needle or a needle externally mounted for instance on a Luer lock of a syringe tip. Suitable material of which the PFS is made include and are not limited to glass and plastic. In a specific embodiment about 2 mL, such as 2.1 mL of said pharmaceutical formulation is filled in 2.25 mL glass pre-fillable syringe which comprises a glass barrel characterized by a staked needle having gauge of about 27G ½" and a siliconization level between 0.3 mg and 0.7 mg per prefilled syringe, and a halobutyl stopper. Examples of commercially available pre-filled syringe barrels/stoppers are Gerresheimer, Schott, OMPI, BD NovaPure, West Pharmaceutical Services.

To determine the stability of the prefilled syringe filled with the formulation according to the present invention, the analytical tests mentioned above are applicable. Additionally, the performance of syringe (i.e. Syringeability) may be judged based on two forces namely, the force required to initiate plunger movement (break-loose force) and the force required to maintain the movement of plunger (gliding force). Any significant change in these two forces may indicate poor syringe performance which may lead to inaccuracy in dose volume and/or pain to patients during injection.

In one aspect of the present invention, the pharmaceutical formulation is expelled from the PFS in a time between 5s and 30 s, preferably in a time between 10s and 20 s, more preferably in 15 s. In another aspect of the present invention, when the pharmaceutical formulation is expelled from the PFS in a time between 10s and 20 s, the break lose force is between about 2N and about 4N and the gliding force is between about 4N and about 30N, for instance between about 4N and 13N. According to one aspect of the present invention, the pharmaceutical formulation comprised within a 2.25 mL glass prefilled syringe is stable at temperature equal to or less than about 40°C, i.e. at a temperature between about -80°C and 40°C, for at least about 1 month, or at least about 3 months, or at least about 6 months, or at least about 9 months, or at least about 12 months, or at least about 18 months, or at least about 24 months. The present invention also includes the disclosed pharmaceutical formulation stable at any value between the above cited temperatures, and at any time intervals between the above cited times. In a more specific embodiment, the disclosed pharmaceutical formulation is stable at about 40±2°C for at least about 1 month; in another specific embodiment, the disclosed pharmaceutical formulation is stable at about 25±2°C for at least about 1 month, more specifically for least about 3 months; in another specific embodiment, the disclosed pharmaceutical formulation is stable at about 5±3°C for at least about 1 month, specifically for at least about 3 months, more specifically for at least about 6 months, even more specifically for at least about 12 months, preferably for at least about 18 months, more preferably for at least about 24 months.

In a more particular embodiment the pharmaceutical formulation comprising an anti-OX40 antagonist antibody or fragment thereof present within said pharmaceutical formulation at a concentration of about 150 mg/mL, an histidine-HCI buffer present within said pharmaceutical formulation at a concentration of about 25 mM, proline or sodium chloride present within said pharmaceutical formulation at a concentration of about 150 mM, or arginine-HCI present within said pharmaceutical formulation at a concentration between about 50 mM and about 150 mM, optionally methionine present within said pharmaceutical formulation at a concentration of about 10 mM or mannitol present within said pharmaceutical formulation at a concentration between about 2% and about 3.5%, and Polysorbate 80 present within said pharmaceutical formulation at a concentration between about 0.03% and about 0.06%, and having pH between about 5 and about 7, is comprised within a prefilled syringe and it is stable for at least about 6 months at a temperature of about 25°C or less.

In another more particular embodiment the pharmaceutical formulation comprising an anti-OX40 antagonist antibody or fragment thereof present within said pharmaceutical formulation at a concentration of about 150 mg/mL, histidine HCI buffer present within said pharmaceutical formulation at a concentration of about 25 mM, arginine-HCI or sodium chloride present within said pharmaceutical formulation at a concentration of about 150 mM and Polysorbate 80 present within said pharmaceutical formulation at a concentration of about 0.036%, and having pH of about 6, is comprised within a prefilled syringe and it is stable for at least about 18 months at a temperature of about 5°C or less. In a particular embodiment, the anti-OX40 antibody or fragment thereof is a humanized monoclonal anti-OX40 antagonist antibody or fragment thereof.

The term "humanized antibody" or "humanized anti-OX40 antibody" as used herein includes antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences as well as within the CDR sequences derived from the germline of another mammalian species.

More specifically, the anti-OX40 antagonist antibody or fragment is GBR830 (CAS Registry Number 2126777-87-3).

In a certain embodiment, the pharmaceutical formulation of any one of the preceding claims for use in the treatment of patients suffering of an OX40-mediated disorders.

As used herein, the term "OX40-mediated disorder" includes conditions such as allergy, asthma, COPD, rheumatoid arthritis, psoriasis and diseases associated with autoimmunity and inflammation. In particular, according to the present invention, exemplary 0X40 mediated disorders include infections (viral, bacterial, fungal and parasitic), endotoxic shock associated with infection, arthritis, rheumatoid arthritis, asthma, chronic obstructive pulmonary disease (COPD), pelvic inflammatory disease, Alzheimer's Disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, Peyronie's Disease, coeliac disease, gallbladder disease, Pilonidal disease, peritonitis, psoriasis, vasculitis, surgical adhesions, stroke, Type I Diabetes, lyme disease, arthritis, meningoencephalitis, autoimmune uveitis, immune mediated inflammatory disorders of the central and peripheral nervous system such as multiple sclerosis, lupus (such as systemic lupus erythematosus) and Guillain-Barr syndrome, Atopic dermatitis, autoimmune hepatitis, fibrosing alveolitis, Grave's disease, IgA nephropathy, idiopathic thrombocytopenic purpura, Meniere's disease, pemphigus, primary biliary cirrhosis, sarcoidosis, scleroderma, Wegener's granulomatosis, pancreatitis, trauma (surgery), graft-versus-host disease (GVHD), transplant rejection, cardiovascular disease including ischaemic diseases such as myocardial infarction as well as atherosclerosis, intravascular coagulation, bone resorption, osteoporosis, osteoarthritis, periodontitis, hypochlorhydia, hidradenitis and neuromyelitis optica.

Other exemplary 0X40 mediated disorder include infections (viral, bacterial, fungal and parasitic), endotoxic shock associated with infection, arthritis, rheumatoid arthritis, asthma, bronchitis, influenza, respiratory syncytial virus, pneumonia, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), cryptogenic fibrosing alveolitis (CFA), idiopathic fibrosing interstitial pneumonia, emphysema, pelvic inflammatory disease, Alzheimer's Disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, Peyronie's Disease, coeliac disease, gallbladder disease, Pilonidal disease, peritonitis, psoriasis, vasculitis, surgical adhesions, stroke, Type I Diabetes, lyme disease, arthritis, meningoencephalitis, autoimmune uveitis, immune mediated inflammatory disorders of the central and peripheral nervous system such as multiple sclerosis, lupus (such as systemic lupus erythematosus) and Guillain-Barr syndrome, Atopic dermatitis, autoimmune hepatitis, fibrosing alveolitis, Grave's disease, IgA nephropathy, idiopathic thrombocytopenic purpura, Meniere's disease, pemphigus, primary biliary cirrhosis, sarcoidosis, scleroderma, Wegener's granulomatosis, pancreatitis, trauma (surgery), graft-versus-host disease (GVHD), transplant rejection, cardiovascular disease including ischaemic diseases such as myocardial infarction as well as atherosclerosis, intravascular coagulation, bone resorption, osteoporosis, osteoarthritis, periodontitis, hypochlorhydia, hidradenitis and neuromyelitis optica.

In accordance to a preferred aspect of the present invention, the anti-OX40 antagonist antibody is used for the treatment or prevention of an OX40-mediated disorder selected from the group comprising atopic dermatitis, rheumatoid arthritis, autoimmune uveitis, multiple sclerosis, lupus (such as systemic lupus erythematosus), ulcerative colitis, scleroderma and graft-versus-host disease (GVHD), scleroderma, hidradenitis, and ulcerative colitis.

The present invention also provides a method for treating an OX40-mediated disorder, wherein the OX40-mediated disorder is selected from the group comprising atopic dermatitis, rheumatoid arthritis, autoimmune uveitis, multiple sclerosis, lupus (such as systemic lupus erythematosus) and graft-versus-host disease (GVHD) , scleroderma, hidradenitis, and ulcerative colitis.

As used herein, the term "subject" includes any human or nonhuman animal. The term "nonhuman animal" includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. Preferably the subject is human.

A "patient" for the purposes of the present invention includes both humans and other animals, preferably mammals and most preferably humans. Thus the antibodies of the present invention have both human therapy and veterinary applications. The term "treatment" or "treating" in the present invention is meant to include therapeutic treatment, as well as prophylactic, or suppressive measures for a disease or disorder. Thus, for example, successful administration of an antibody prior to onset of the disease results in treatment of the disease. As another example, successful administration of an antibody after clinical manifestation of the disease to combat the symptoms of the disease comprises treatment of the disease.

"Treatment" and "treating" also encompasses administration of an antibody after the appearance of the disease in order to eradicate the disease. Successful administration of an antibody after onset and after clinical symptoms have developed, with possible abatement of clinical symptoms and perhaps amelioration of the disease, comprises treatment of the disease. Those "in need of treatment" include mammals already having the disease or disorder, as well as those prone to having the disease or disorder, including those in which the disease or disorder is to be prevented.

The antibody or of the present invention can be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. Preferred routes of administration include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. More preferred routes of administration are intravenous or subcutaneous. The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. Alternatively, an antibody of the invention can be administered via a non- parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.

The antibody of the present invention can be administered at a single or multiple doses. The term "dose" or "dosage" as used in the present invention are interchangeable and indicates an amount of drug substance administered per body weight of a subject or a total dose administered to a subject irrespective to their body weight.

In one embodiment, a therapeutically effective amount of the pharmaceutical formulation according to the present invention is administrated subcutaneously using a prefilled syringe. In accordance with one aspect of the present invention, the pharmaceutical formulations provided may be administered to individuals. In particular, a therapeutically effective amount of said pharmaceutical formulation is administrated subcutaneously as single-dose drug product in a pre-fillable syringe system. Administration is preferably in a "therapeutically effective amount", this being sufficient to show benefit to a subject. Such benefit may be at least amelioration of at least one symptom. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of medical doctors. Appropriate doses of antibody are well known in the art (Ledermann JA et al., (1999) Int J Cancer 47: 659-664; Bagshawe KD et a/., (1991) Antibody, Immunoconjugates and Radiopharmaceuticals, 4: 915-922). The precise dose will depend upon a number of factors, including the size and location of the area to be treated, body weight of the subject, the precise nature of the antibody (e.g. whole antibody or fragment) and any additional therapeutic agents administered before, at the time of or after administration of the antibody.

The current invention also discloses a method of manufacturing the pharmaceutical formulation according to the present invention, and/or a method of manufacturing a vial or a prefilled syringe containing said pharmaceutical formulation according to the present invention.

Also disclosed herein is a method to test said pharmaceutical formulation to any assay for stability determination. Non limiting examples of stability assay include analytical test such as monitoring of the visual appearance, sub-visible particles analysis, protein content measurement (e.g. by measuring absorbance at 280 nm wavelength), pH measurement, osmolarity measurement, viscosity measurement, protein thermal stability measurement for instance by nano-format of Differential Scanning Fluorimetry (DSF), size variants monitoring (e.g. by SE-HPLC and/or by cGE reduced and non- reduced), charge variants monitoring (e.g. by clEF), antibody potency measurement (e.g. by ELISA) as any significant change of binding property of the antibody toward its target would indicate antibody degradation, peptide mapping, post-translational modifications mapping.

The present invention also relates to an article of manufacture comprising: (a) a container which holds the prefilled syringe and (b) instructions for using said prefilled syringe.

Also disclosed in the current invention is a method for obtaining a high concentrated protein solution, such as a concentrated antibody solution, comprising the steps of subjecting a clarified cell harvest to an affinity chromatography step, and subjecting the obtained eluate to at least two ion exchange chromatography steps and to at least three UF/DF steps.

An antibody can be produced by introducing genetic material encoding said biomolecule of interest in host cells and culture said host cells. The term "host cells" refers to all the cells in which the biomolecule of interest, such as an antibody or antibody fragment thereof, codified by the artificially introduced genetic material is expressed, including those cells in which the foreign nucleic acid is directly introduced and their progeny. In the host cells it can be introduced an expression vectors (constructs), such as plasmids and the like, encoding the biomolecule of interest e.g., via transformation, transfection, infection, or injection. Such expression vectors normally contain the necessary elements for the transcription and translation of the sequence encoding the biomolecule of interest. Methods which are well known to and practiced by those skilled in the art can be used to construct expression vectors containing sequences encoding the protein of interest, as well as the appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Cell lines suitable as host cells include and are not limited to bacteria, mammalian, insect, plant and yeast cells. Cell lines often used for the expression and production of therapeutic antibodies include mammalian cells lines such as Chinese hamster ovary (CHO) cells, NSO mouse myeloma cells, human cervical carcinoma (HeLa) cells and human embryonic kidney (HEK) cells.

The terms "cell culture" and "culture" as used herein are interchangeable and refer to the growth and/or propagation and/or maintenance of cells in controlled artificial conditions, and they indicate a cell culture which comprises a cell culture medium and cell culture material comprising cells, cell debris, for instance generated upon cell death, colloidal particles, such as DNA, RNA and host cell proteins (HCP), and (bio)molecules secreted by the cultured cells, such as the biomolecule of interest, e.g. an antibody or antibody fragment thereof. The cells of a cell culture can be cultured in suspension or attached to a solid substrate, in containers comprising a cell culture medium. For example a cell culture can be grown in tubes, spin tubes, flasks, bags, roller bottles, bioreactors.

When the production of the biomolecule of interest has a commercial purpose, often the host cells are cultured in bioreactors, under conditions that aid their growth and the expression of said biomolecule of interest. The term "bioreactor," as used herein, refers to any manufactured or engineered device or system that supports a biologically active environment. Optimal culturing conditions are obtained by the control and adjustment of several parameters including: the formulation of the cell culture medium, the bioreactor operating parameters, the nutrient supply modality and the culturing time period. The formulation of the culturing medium has to be optimized to favorite cell vitality and reproduction; examples of constituents of the cell culture medium include but are not limited to essential amino acids, salts, glucose, growth factors and antibiotics. Important bioreactor operating parameters include: initial cell seeding density, temperature, pH, agitation speed, oxygenation and carbon dioxide levels. Nutrients can be supplied in different ways: in the batch mode culture all the necessary nutrients are present in the initial base medium and are used till exhausted while wastes accumulate; in the fed-batch culture additional feed medium is supplied to prevent nutrient depletion and prolong the culture; differently, in the perfusion modality, cells in culture are continuously supplemented with fresh medium containing nutrients that flows in the bioreactor removing cell wastes. The culturing period is important as it needs to be long enough to let the cells produce a consistent amount of product but it cannot be too long to impair their viability. Commonly used bioreactors are typically cylindrical, ranging in size from liters to cubic meters, and are often made of stainless steel. In the embodiments described herein, a bioreactor is made of plastic or of stainless steel. It is contemplated that the total volume of a bioreactor may be any volume ranging from 100 ml to up to 20.000 Liters or more, depending on a particular process.

The terms "cell culture medium," and "culture medium" and "medium" are used interchangeably herein and they refer to a nutrient solution used for growing cells, such as animal cells, e.g., mammalian cells. Such a nutrient solution generally includes various factors necessary for cell attachment, growth, and maintenance of the cellular environment. For example, a typical nutrient solution may include a basal media formulation, various supplements depending on the cell type and, occasionally, antibiotics. Besides nutrients and other constituents supporting the growth and production of cells, the cell culture medium may also contain waste products, host cell proteins (HCP) and material from lysed cells. The composition of the culture medium may vary in time during the course of the culturing of cells and at the stage of clarification may be depleted of one or more of the original constituents.

The terms "clarify", "clarification", "clarification step", "clarification process" as used herein are interchangeable and generally refer to one or more steps that aid the removal of a part of the cell culture material from the cell culture, such as removal of cells and cell debris, to obtained clarified cell culture.

The term "clarified cell harvest" refers to a material produced by first harvesting the host cell culture and then subjecting the harvest to a process of clarification.

The clarified cell harvest may be loaded onto a chromatography column.

The term "chromatography" refers to the operation of separating compounds of a mixture based on their capability to interact with a stationary phase of a chromatography column, from which they can be retained or eluted. Non limiting examples of chromatographic techniques, including ion exchange, hydrophobic interaction, affinity, sizing or gel filtration, and reversed-phase chromatography, carried out at atmospheric pressure or at high pressure using systems such as FPLC and HPLC The present invention also relates to a method for obtaining a high concentrated protein solution, such as a high concentrated antibody solution, comprising the steps of subjecting a clarified cell harvest to an affinity chromatography step, and subjecting the obtained eluate to at least two ion exchange chromatography steps and to at least three UF/DF steps.

More in particular, the method for obtaining a high concentrated antibody solution comprising the steps of (i) subjecting a clarified cell harvest to a protein A chromatography; (ii) subjecting the obtained protein A eluate first to a low pH hold step, wherein pH is about 3.5, and then (iii) to a cation exchange chromatography (CEX) step; (iv) concentrate the obtained CEX eluate by a first UFDF step (UFDF1); (v) subjecting the UFDF1 concentrated solution to a anion exchange chromatography followed by (vi) nano-filtration; (vii) concentrate the obtained nano-filtered solution by a second UFDF step (UFDF2); (viii) subjecting the UFDF2 concentrated solution to a third UFDF step (UFDF3).

Even more in particular, the method for obtaining a high concentrated antibody solution according to the present invention comprises a UFDF3 characterized by the use of a tangential flow filtration cassette and by the following steps: (a) equilibrated of the cassette by an equilibration buffer; (b) loading of the cassette with an antibody solution (such as a UFDF2 concentrated solution) wherein the antibody concentration is between about 50 mg/mL and about 100 mg/mL, preferably between about 65 mg/mL and about 75 mg/mL; (c) ultrafiltration 1 (UF1) to concentrate the protein to a concentration between about 100 mg/mL and about 110 mg/mL, preferably to a concentration of about 100 mg/mL; (d) diafiltration using a diafiltration buffer; (e) ultrafiltration 2 (UF2) to concentrate the protein to a concentration of about 260 mg/mL; (f) flush with a flushing buffer to concentrate the protein to a concentration between about 160 mg/mL and about 180 mg/mL, preferably to a concentration between about 150 mg/mL and about 170 mg/mL, preferably to a concentration of about 170 mg/mL.

In a specific embodiment of the present invention, to perform UFDF3 it is used a cassette wherein the pressure is low, an equilibration buffer comprising histidine at a concentration of about 5mM at pH about 6, a diafiltration buffer comprising histidine at a concentration of about 25 mM and arginine- HCI at a concentration of about 150 mM at a pH of about 6, and a flushing buffer comprising histidine at a concentration of about 25 mM and arginine-HCI at a concentration of about 150 mM at a pH of about 6. The present invention also includes buffers components with a concentration at any value between the above cited concentrations

In an even more specific embodiment, UFDF 3 loading is performed at room temperature using a volumetric loading factor less than about 30 L/m ² , preferably about 25 L/m ² ; UF1 and diafiltration are performed at a cross flow rate between about 240 LMFI and 325 LMFI, preferably about 290 LMFI, a feed flow rate between about 260 LMFI and about 350 LMH, preferably about 315 LMH, with a feed pressure equal to or less than about 3 bars and a TMP between 0.6 bars and 1 bar, preferably about 0.8 bars; UF2 is performed at a cross flow rate between about 7 LMH and between about 325 LMH, preferably about 290 LMH, a feed flow rate between about 7 LMH and about 350 LMH, preferably about 315 LMH, with a feed pressure equal to or less than about 3 bars and a TMP equal to or less than about 1.5 bars, preferably about 0.8 bars. The present invention also includes volumetric loading factor, cross flow rate, feed flow rate, feed pressure at any value between the above cited concentrations

In a particular aspect, the method for obtaining a high concentrated antibody solution also comprises the step of (ix) generating a bulk drug substance (BDS) and/or a drug product (DP) wherein the protein is present at a high concentration, i.e. concentration of about 150 mg/mL.

In particular, according to one aspect of the present invention, the UFDF3 concentrated solution is diluted to obtain a protein concentration between about 160 mg/mL and about 170 mg/mL, more preferably the protein concentration is about 150 mg/mL. In a more specific embodiment, the UFDF3 concentrated solution is diluted by the addition of one or more excipient(s), for instance by the addition of one or more stabilizing or tonicity agent(s), such as Polysorbate 80.

The high concentrated antibody solution obtained by the method according to the present invention can be filled into a container, such as a syringe e.g. a prefilled syringe, or it can be stored at low temperature, i.e. stored at a temperature of about -70±15°C, for instance it can be stored in a storage bag at a temperature of about -70±15°C. In a specific aspect of the present invention, the UFDF3 concentrated solution and/or the BSD undergo other steps, such as filtration steps e.g. 0.2 pm bioburden reduction filtration, before being stored or filled into a container.

In one embodiment of the present invention, the method for obtaining a high concentrated antibody solution is used to obtain a high concentrated solution of an anti-OX40 antagonist antibody or fragment thereof. In a more specific embodiment the anti-OX40 antagonistic antibody or fragment is GBR830 (CAS Registry Number 2126777-87-3).

According to the present invention, the disclosed pharmaceutical formulation is suitable to make the a stable bulk drug substance and/or a stable drug product.

Figure 1: Electrophoregrams of samples obtained by cGE analysis (A) REF1; (B) TO; (C) T9M at 25°C; (D) T9M at 5°C. Figure 2: Profiles of samples obtained by clEF analysis (A) REF1; (B) TO; (C) T9M at 25°C; (D) T9M at 5°C.

Figure 3: Profiles of samples obtained by iCE analysis (A) REF1; (B) T9M at 25°C; (C) T9M at 5°C.

Figure 4: Overlay of profiles obtained by iCE analysis. Figure 5: Profiles of samples obtained by CEX-FIPLC analysis (A) REF1; (B) TO; (C) T9M at 25°C; (D) T9M at 5°C.

Figure 6: Trial 1 and trial 2 UFDF process flowcharts Figure 7: Gel theory plot example

Figure 8: Comparison of conductivity trend during diafiltration step between both trials. (A) Trial 1, Buffer 1; (B) Trial 2, Buffer 1; (C) Trial 1, Buffer 2; (D) Trial 2, Buffer 2.

Figure 9: Cgel estimation chart: Normalized filtrate flux = f[log(C _r )], (A) Bufferl; (B) Buffer2.

Figure 10: Size variants repartition of BDS post UFDF by SE-HPLC analysis, %Aggregates (first column), % monomer (second column), % fragments (third column)

Figure 11: Charge variants repartition of BDS post UFDF by IEF with iCE3, %Acid (first column), % Main (second column), %Basic (third column)

Figure 12: pi range of BDS post UFDF by IEF with iCE3, pi start (first column), pi end (second column), pi of main peak (third column)

Figure 13: UFDF 3 process performance (permeate flux / TMP vs. concentration)

Figure 14: SE-HPLC analysis data, %Aggregates (first column), % monomer (second column), % fragments (third column); (A) Abl-Samplel; (B) Abl-Sample2.

Figure 15: clEF by iCE3 analysis data, %Acid (first column), % Main (second column), %Basic (third column; (A) Abl-Samplel; (B) Abl-Sample2.

Figure 16: cGE (non-reduced) analysis data of Abl-Samplel, Assigned peak (first column), IgG peak (second column), others peak (third column). Figure 17: cGE (reduced) analysis data of Abl-Samplel, %LC (first column), %HC (second column), Total other (third column).

Figure 18: cGE (non-reduced) analysis data of Abl-Sample2, Assigned peak (first column), IgG peak (second column), others peak (third column). Figure 19: cGE (reduced) analysis data of Abl-Sample2, %LC (first column), %HC (second column), Total other (third column).

Figure 20: Results of the syringeability measurements before (A and B) and after 2 weeks of storage at 40 °C (C and D); measurements were performed with ejection speeds of 10 (A and C) and 20 s/2 ml (B and D); results represent mean force profiles, n = 3. Figure 21: Results of the cumulative particle concentrations >2 pm obtained by MFI analysis comparing TO (first column) to T-FT (second column) and T2w_40C (third column) of the 50-mg/ml stock dilutions in different PFS; values were not corrected for the dilution factor. Ejection speeds of 10 (A) and 20 s/2 ml (B).

Figure 22: : Results of the cumulative particle concentrations > 5 pm obtained by MFI analysis before and after stressing; cumulative particle number was divided into non-silicone oil droplet-like particle (bottom part of the column) and silicone oil droplet-like particle (upper part of the column) fractions; values were not corrected for the dilution factor. (A) TO, ejection speeds 10 sec; (B) TO, ejection speeds 20 sec; (C) TFT; (D) T2w at 40°C, ejection speeds 10 sec; (E) T2w at 40°C, ejection speeds 20 sec.

Figure 23: Results of the cumulative particle concentrations > 10 pm obtained by MFI analysis before and after stressing; cumulative particle number was divided into non-silicone oil droplet-like particle (bottom part of the column) and silicone oil droplet-like particle (upper part of the column) fractions; values were not corrected for the dilution factor. (A) TO, ejection speeds 10 sec; (B) TO, ejection speeds 20 sec; (C) TFT; (D) T2w at 40°C, ejection speeds 10 sec; (E) T2w at 40°C, ejection speeds 20 sec.

Figure 24: Results of the cumulative particle concentrations >25 pm obtained by MFI analysis before and after stressing; cumulative particle number was divided into non-silicone oil droplet-like particle (bottom part of the column) and silicone oil droplet-like particle (upper part of the column) fractions; values were not corrected for the dilution factor. (A) TO, ejection speeds 10 sec; (B) TO, ejection speeds 20 sec; (C) TFT; (D) T2w at 40°C, ejection speeds 10 sec; (E) T2w at 40°C, ejection speeds 20 sec. Figure 25: Thermal stability evaluation F2, F6, F9 and F10 in Barrell and Barrel 3(ii), (A) Tagg, onset; (B) Tm, onset; (C) Tml.

Figure 26: Thermal stability evaluation FI to F13 in Barrell, (A) Tagg, onset; (B) Tm, onset; (C) Tml

Figure 27: Melting curves F2 vs F7. (A) Ratio fluorescence signal 350nm/330nm; (B) 1 ^st derivative fluorescence signal 350nm/330nm; (C) fluorescence signal 350nm; (D) Back reflection intensity.

Figure 28: Turbidity (OD350) measured for FI to F13 in Barrell (left panel), and for F2, F6, F9, F10 in Barrel3(ii) (right panel) at TO (first column), after TFT (second column) and after T-roll stresses (third column).

Figure 29: Turbidity (OD550) measured for FI to F13 in Barrell (left panel), and for F2, F6, F9, F10 in Barrel3(ii) (right panel) at TO (first column), after TFT (second column) and after T-roll stresses (third column).

Figure 30: Micro-Flow Imaging (MFI), particle level measurement in protein-containing samples compared to formulation buffer.

Figure 31: MFI particle levels > 2 pm results before and after stressing . TO (first column), TFT (second column) and T-roll stresses (third column).

Figure 32: MFI particle levels > 5 pm results before and after stressing and shape analysis. (A) TO; (B) TFT; (C) T-roll.

Figure 33: MFI particle levels > 10 pm results before and after stressing and shape analysis. (A) TO; (B) TFT; (C) T-roll.

Figure 34: SEC monomer content analysis for FI to F13 in Barrell (A), and for F2, F6, F9, F10 in Barrell and Barrel3(ii) (B) at TO (first column), after TFT (second column) and after T-roll stresses (third column).

Figure 35: SEC aggregates analysis (FIMWS) for FI to F13 in Barrell (A), and for F2, F6, F9, F10 in Barrell and Barrel3(ii) (B) at TO (first column), after TFT (second column) and after T-roll stresses (third column). Figure 36: SEC aggregates analysis (LMWS) for FI to F13 in Barrell (A), and for F2, F6, F9, F10 in Barrell and Barrel3(ii) (B) at TO (first column), after TFT (second column) and after T-roll stresses (third column).

Figure 37: Purity analysis (Bioanalyzer)

Figure 38: Purity analysis (Bioanalyzer), results before and after stressing in non-reducing conditions for FI to F13 in Barrell (A), and for F2, F6, F9, F10 in Barrell and Barrel3(ii) (B) at TO (first column), after TFT (second column) and after T-roll stresses (third column).

Figure 39: Purity analysis (Bioanalyzer), results before and after stressing in reducing conditions for FI to F13 in Barrell (A), and for F2, F6, F9, F10 in Barrell and Barrel3(ii) (B) at TO (first column), after TFT (second column) and after T-roll stresses (third column).

Figure 40: iCE main species content for FI to F13 in Barrell (A), and for F2, F6, F9, F10 in Barrell and Barrel3(ii) (B) at TO (first column), after TFT (second column) and after T-roll stresses (third column).

Figure 41: iCE profile of F7

Figure 42: iCE acidic species, results before and after stressing for FI to F13 in Barrell (A), and for F2, F6, F9, F10 in Barrell and Barrel3(ii) (B) at TO (first column), after TFT (second column) and after T- roll stresses (third column).

Figure 43: iCE basic species, results before and after stressing for FI to F13 in Barrell (A), and for F2, F6, F9, F10 in Barrell and Barrel3(ii) (B) at TO (first column), after TFT (second column) and after T- roll stresses (third column).

Figure 44: Thermal stability results, before and after 1 month storage (F350): TO (first column), TIM at 5°C (second column), TIM at 25°C (third column) and TIM at 40°C (forth column). (A) Tm onset, (B) Tml.

Figure 45: Thermal stability results, before and after 1 month storage (back reflection): TO (first column), TIM at 5°C (second column), TIM at 25°C (third column) and TIM at 40°C (forth column). Tagg onset.

Figure 46: Thermal stability results, before and after 1 month storage (Barrell VS Barrel3(ii) PFS) : TO (first column), TIM at 5°C (second column), TIM at 25°C (third column) and TIM at 40°C (forth column). (A) Tagg, onset; (B) Tm, onset; (C) Tml. Figure 47: Turbidity (OD350) results, before and after 1 month storage for FI to F13 in Barrell (A), and for F2, F6, F9, F10 in Barrell and Barrel3(ii) (B): TO (first column), TIM at 5°C (second column), TIM at 25°C (third column) and TIM at 40°C (forth column).

Figure 48: Turbidity (OD550) results, before and after 1 month storage for FI to F13 in Barrell (A), and for F2, F6, F9, F10 in Barrell and Barrel3(ii) (B): TO (first column), TIM at 5°C (second column), TIM at 25°C (third column) and TIM at 40°C (forth column).

Figure 49: MFI results at 5°C, before and after 1 month storage

Figure 50: MFI particle levels > 2 pm results, before and after 1 month storage. TO (first column), TIM at 5°C (second column), TIM at 25°C (third column) and TIM at 40°C (forth column).

Figure 51: MFI particle levels > 5 pm results and shape analysis, before and after 1 month storage. Silicon oil droplet-like particles (upper part of the column), non-silicon oil droplet-like particles (bottom part of the column). TO (A), TIM at 5°C (B), TIM at 25°C (C) and TIM at 40°C (D).

Figure 52: MFI particle levels > 10 pm results and shape analysis, before and after 1 month storage. Silicon oil droplet-like particles (upper part of the column), non-silicon oil droplet-like particles (bottom part of the column). TO (A), TIM at 5°C (B), TIM at 25°C (C) and TIM at 40°C (D).

Figure 53: MFI particle levels > 25 pm results and shape analysis, before and after 1 month storage. Silicon oil droplet-like particles (upper part of the column), non-silicon oil droplet-like particles (bottom part of the column). TO (A), TIM at 5°C (B), TIM at 25°C (C) and TIM at 40°C (D).

Figure 54: SEC monomer content analysis, before and after 1 month storage for FI to F13 in Barrell (A), and for F2, F6, F9, F10 in Barrell and Barrel3(ii) (B): TO (first column), TIM at 5°C (second column), TIM at 25°C (third column) and TIM at 40°C (forth column).

Figure 55: SEC aggregates (FIMWS) analysis, before and after 1 month storage for FI to F13 in Barrell (A), and for F2, F6, F9, F10 in Barrell and Barrel3(ii) (B): TO (first column), TIM at 5°C (second column), TIM at 25°C (third column) and TIM at 40°C (forth column).

Figure 56: SEC aggregates (LMWS) analysis, before and after 1 month storage for FI to F13 in Barrell (A), and for F2, F6, F9, F10 in Barrell and Barrel3(ii) (B): TO (first column), TIM at 5°C (second column), TIM at 25°C (third column) and TIM at 40°C (forth column). Figure 57: Purity analysis (Bioanalyzer) in non-reducing conditions, before and after 1 month storage for FI to F13 in Barrell (A), and for F2, F6, F9, F10 in Barrell and Barrel3(ii) (B): TO (first column), TIM at 5°C (second column), TIM at 25°C (third column) and TIM at 40°C (forth column).

Figure 58: Purity analysis (Bioanalyzer) in reducing conditions, before and after 1 month storage for FI to F13 in Barrell (A), and for F2, F6, F9, F10 in Barrell and Barrel3(ii) (B): TO (first column), TIM at 5°C (second column), TIM at 25°C (third column) and TIM at 40°C (forth column).

Figure 59: iCE3 analysis (main species), before and after 1 month storage for FI to F13 in Barrell (A), and for F2, F6, F9, F10 in Barrell and Barrel3(ii) (B): TO (first column), TIM at 5°C (second column), TIM at 25°C (third column) and TIM at 40°C (forth column).

Figure 60: iCE3 exemplary profiles, before and after 1 month storage

Figure 61: iCE3 analysis (acidic species), before and after 1 month storage for FI to F13 in Barrell (A), and for F2, F6, F9, F10 in Barrell and Barrel3(ii) (B): TO (first column), TIM at 5°C (second column), TIM at 25°C (third column) and TIM at 40°C (forth column).

Figure 62: iCE3 analysis (basic species), before and after 1 month storage for FI to F13 in Barrell (A), and for F2, F6, F9, F10 in Barrell and Barrel3(ii) (B): TO (first column), TIM at 5°C (second column), TIM at 25°C (third column) and TIM at 40°C (forth column).

Figure 63: Dynamic viscosity results before and after 3 months storage for FI to F13 in Barrell (A), and for F2, F6, F9, F10 in Barrell and Barrel3(ii) (B): T3M at 5°C (first column), T3M at 25°C (second column), and T3M at 40°C (third column).

Figure 64: Syringeability results, 3 months storage for FI to F13 in Barrell (A), and for F2, F6, F9, F10 in Barrell and Barrel3(ii) (B): T3M at 5°C (first column), T3M at 25°C (second column), and T3M at 40°C (third column).

Figure 65: Syringeability results, 3 months storage (A) Barrell, T3M, F2; (B) Barrel3(ii), T3M, F2; (C) Barrell, T3M, F6; (D) Barrel3(ii), T3M, F6.

Figure 66: Syringeability results, 3 months storage (A) Barrell, T3M, F9; (B) Barrel3(ii), T3M, F9; (C) Barrell, T3M, F10; (D) Barrel3(ii), T3M, F10. Figure 67: Thermal stability results, before and after 3 months storage (F350). (A) Tm, onset; (B) Tml.

TO (first column), TIM at 5°C (second column), T3M at 5°C (third column), TIM at 25°C (forth column),

T3M at 25°C (fifth column), TIM at 40°C (sixth column), and T3M at 40°C (seventh column).

Figure 68: Thermal stability results, before and after 3 months storage (T agg onset, back reflection).

TO (first column), TIM at 5°C (second column), T3M at 5°C (third column), TIM at 25°C (forth column),

T3M at 25°C (fifth column), TIM at 40°C (sixth column), and T3M at 40°C (seventh column).

Figure 69: Thermal stability results, before and after 3 months storage (Barrell VS Barrel3(ii) PFS). (A) Tagg onset, (B) Tm, onset; (B) Tml. TO (first column), TIM at 5°C (second column), T3M at 5°C (third column), TIM at 25°C (forth column), T3M at 25°C (fifth column), TIM at 40°C (sixth column), and T3M at 40°C (seventh column).

Figure 70: Turbidity (OD350) results, before and after 3 months storage. (A) Barrell, (B) Barrel 1 and Barrel3(ii). TO (first column), TIM at 5°C (second column), T3M at 5°C (third column), TIM at 25°C (forth column), T3M at 25°C (fifth column), TIM at 40°C (sixth column), and T3M at 40°C (seventh column).

Figure 71: Turbidity (OD550) results, before and after 3 months storage. (A) Barrell, (B) Barrel 1 and Barrel3(ii). TO (first column), TIM at 5°C (second column), T3M at 5°C (third column), TIM at 25°C (forth column), T3M at 25°C (fifth column), TIM at 40°C (sixth column), and T3M at 40°C (seventh column).

Figure 72: MFI particle levels > 2 pm results, before and after 3 months storage. TO (first column), TIM at 5°C (second column), T3M at 5°C (third column), TIM at 25°C (forth column), T3M at 25°C (fifth column), TIM at 40°C (sixth column), and T3M at 40°C (seventh column).

Figure 73: MFI particle levels > 5 pm results and shape analysis, before and after 3 months storage.

Silicon oil droplet-like particles (upper part of the column), non-silicon oil droplet-like particles (bottom part of the column). TO (A), T3M at 5°C (B), T3M at 25°C (C) and T3M at 40°C (D).

Figure 74: MFI particle levels > 10 pm results and shape analysis, before and after 3 months storage.

Silicon oil droplet-like particles (upper part of the column), non-silicon oil droplet-like particles (bottom part of the column). TO (A), T3M at 5°C (B), T3M at 25°C (C) and T3M at 40°C (D). Figure 75: MFI particle levels > 25 pm results and shape analysis, before and after 3 months storage. Silicon oil droplet-like particles (upper part of the column), non-silicon oil droplet-like particles (bottom part of the column). TO (A), T3M at 5°C (B), T3M at 25°C (C) and T3M at 40°C (D).

Figure 76: SEC monomer content analysis, before and after 3 months storage for FI to F13 in Barrell (A), and for F2, F6, F9, F10 in Barrell and Barrel3(ii) (B). TO (first column), TIM at 5°C (second column), T3M at 5°C (third column), TIM at 25°C (forth column), T3M at 25°C (fifth column), TIM at 40°C (sixth column), and T3M at 40°C (seventh column).

Figure 77: SEC aggregates (FIMWS) analysis, before and after 3 months storage for FI to F13 in Barrell (A), and for F2, F6, F9, F10 in Barrell and Barrel3(ii) (B). TO (first column), TIM at 5°C (second column), T3M at 5°C (third column), TIM at 25°C (forth column), T3M at 25°C (fifth column), TIM at 40°C (sixth column), and T3M at 40°C (seventh column).

Figure 78: SEC aggregates (LMWS) analysis, before and after 3 months storage for FI to F13 in Barrell (A), and for F2, F6, F9, F10 in Barrell and Barrel3(ii) (B). TO (first column), TIM at 5°C (second column), T3M at 5°C (third column), TIM at 25°C (forth column), T3M at 25°C (fifth column), TIM at 40°C (sixth column), and T3M at 40°C (seventh column).

Figure 79: Purity analysis (Bioanalyzer) in non-reducing conditions, before and after 3 months storage for FI to F13 in Barrell (A), and for F2, F6, F9, F10 in Barrell and Barrel3(ii) (B). TO (first column), TIM at 5°C (second column), T3M at 5°C (third column), TIM at 25°C (forth column), T3M at 25°C (fifth column), TIM at 40°C (sixth column), and T3M at 40°C (seventh column).

Figure 80: Purity analysis (Bioanalyzer) in reducing conditions, before and after 3 months storage for FI to F13 in Barrell (A), and for F2, F6, F9, F10 in Barrell and Barrel3(ii) (B). TO (first column), TIM at 5°C (second column), T3M at 5°C (third column), TIM at 25°C (forth column), T3M at 25°C (fifth column), TIM at 40°C (sixth column), and T3M at 40°C (seventh column).

Figure 81: iCE3 analysis (main species), before and after 3 months storage at 5°C (A), at 25°C (B) and at 40°C (C). TO (first column), TIM (second column), T3M (third column).

Figure 82: iCE3 analysis (acidic species), before and after 3 months storage at 5°C (A), at 25°C (B) and at 40°C (C). TO (first column), TIM (second column), T3M (third column).

Figure 83: iCE3 analysis (basic species), before and after 3 months storage at 5°C (A), at 25°C (B) and at 40°C (C). TO (first column), TIM (second column), T3M (third column). Figure 84: Thermal stability results, before and after 6 months storage (F350) for F2, F7, F9, F10 and F13 in Barrell. (A) Tm onset, (B) Tml. TO (first column), TIM at 5°C (second column), T3M at 5°C (third column), T6M at 5°C (forth column), TIM at 25°C (fifth column), T3M at 25°C (sixth column), and T6M at 25°C (seventh column).

Figure 85: Thermal stability results, before and after 6 months storage (Tagg onset, back reflection) for F2, F7, F9, F10 and F13 in Barrell. TO (first column), TIM at 5°C (second column), T3M at 5°C (third column), T6M at 5°C (forth column), TIM at 25°C (fifth column), T3M at 25°C (sixth column), and T6M at 25°C (seventh column).

Figure 86: Turbidity (OD350) results, before and after 6 months storage for F2, F7, F9, F10 and F13 in Barrell. TO (first column), TIM at 5°C (second column), T3M at 5°C (third column), T6M at 5°C (forth column), TIM at 25°C (fifth column), T3M at 25°C (sixth column), and T6M at 25°C (seventh column).

Figure 87: Turbidity (OD550) results, before and after 6 months storage for F2, F7, F9, F10 and F13 in Barrell. TO (first column), TIM at 5°C (second column), T3M at 5°C (third column), T6M at 5°C (forth column), TIM at 25°C (fifth column), T3M at 25°C (sixth column), and T6M at 25°C (seventh column).

Figure 88: MFI particle levels > 2 pm results, before and after 6 months storage (A, top panel) TO; (A, bottom panel T6M at 25°C; (B) T6M at 5°C.

Figure 89: MFI particle levels > 5 pm results and shape analysis, before and after 6 months storage. Silicon oil droplet-like particles (upper part of the column), non-silicon oil droplet-like particles (bottom part of the column). TO (A), T6M at 25°C (B), T6M at 5°C (C).

Figure 90: MFI particle levels > 10 pm results and shape analysis, before and after 6 months storage. Silicon oil droplet-like particles (upper part of the column), non-silicon oil droplet-like particles (bottom part of the column). TO (A), T6M at 25°C (B), T6M at 5°C (C).

Figure 91: MFI particle levels > 25 pm results and shape analysis, before and after 6 months storage. Silicon oil droplet-like particles (upper part of the column), non-silicon oil droplet-like particles (bottom part of the column). TO (A), T6M at 25°C (B), T6M at 5°C (C).

Figure 92: SEC monomer content analysis, before and after 6 months storage for F2, F7, F9, F10 and F13 in Barrell. TO (first column), TIM at 5°C (second column), T3M at 5°C (third column), T6M at 5°C (forth column), TIM at 25°C (fifth column), T3M at 25°C (sixth column), and T6M at 25°C (seventh column). Figure 93: SEC aggregates (HMWS) analysis, before and after 6 months storage for F2, F7, F9, F10 and F13 in Barrell. TO (first column), TIM at 5°C (second column), T3M at 5°C (third column), T6M at 5°C (forth column), TIM at 25°C (fifth column), T3M at 25°C (sixth column), and T6M at 25°C (seventh column).

Figure 94: SEC aggregates (LMWS) analysis, before and after 6 months storage for F2, F7, F9, F10 and F13 in Barrell. TO (first column), TIM at 5°C (second column), T3M at 5°C (third column), T6M at 5°C (forth column), TIM at 25°C (fifth column), T3M at 25°C (sixth column), and T6M at 25°C (seventh column).

Figure 95: Purity analysis (Bioanalyzer) in non-reducing conditions, before and after 6 months storage for F2, F7, F9, F10 and F13 in Barrell. TO (first column), TIM at 5°C (second column), T3M at 5°C (third column), T6M at 5°C (forth column), TIM at 25°C (fifth column), T3M at 25°C (sixth column), and T6M at 25°C (seventh column).

Figure 96: Purity analysis (Bioanalyzer) in reducing conditions, before and after 6 months storage for F2, F7, F9, F10 and F13 in Barrell. TO (first column), TIM at 5°C (second column), T3M at 5°C (third column), T6M at 5°C (forth column), TIM at 25°C (fifth column), T3M at 25°C (sixth column), and T6M at 25°C (seventh column).

Figure 97: iCE3 analysis (main species), before and after 6 months storage for F2, F7, F9, F10 and F13 in Barrell. TO (first column), TIM at 5°C (second column), T3M at 5°C (third column), T6M at 5°C (forth column), TIM at 25°C (fifth column), T3M at 25°C (sixth column), and T6M at 25°C (seventh column).

Figure 98: iCE3 analysis (acidic species), before and after 6 months storage for F2, F7, F9, F10 and F13 in Barrell. TO (first column), TIM at 5°C (second column), T3M at 5°C (third column), T6M at 5°C (forth column), TIM at 25°C (fifth column), T3M at 25°C (sixth column), and T6M at 25°C (seventh column).

Figure 99: iCE3 analysis (basic species), before and after 6 months storage for F2, F7, F9, F10 and F13 in Barrell. TO (first column), TIM at 5°C (second column), T3M at 5°C (third column), T6M at 5°C (forth column), TIM at 25°C (fifth column), T3M at 25°C (sixth column), and T6M at 25°C (seventh column). EXAMPLES

Example 1. Investigation of the effect of buffer, pH and excipient on an antibody pre- formulation

Pre-formulation studies have been performed to assess the effect of buffer type, pH and excipient on an anti-OX40 antibody, Abl.

Material and methods

To determine the effect of buffer type, pH and excipient on Abl, its stability was determined by SE- HPLC (briefly, by employing a linear flow of aqueous buffer at a flow rate of 1 mL/min and an injection volume of 50 pi; separation was achieved by using a TSK gel G3000 column) and by subjecting the sample to different types of stress, including (i) storage at 40°C for 1 month (M); (ii) Freeze-Thaw (-40 to +25 C° 3 times); (iii) shear stress, applied by placing vials filled with Abl on a horizontal shaker, speed of 900 rpm was applied for 3 days; and (iv) light exposure of lKlux at 25°C.

Results

The effect of pH and solution molarity is shown in Table 1, the effect of buffer type and pH is shown in Table 2 and the effect of the excipients is shown in Table 3. These studies were carried out using a low concentration of Abl equal to 10 mg/ml.

Table 1. Effect of pH and solution molarity. Stress: 1 Month (M) at 40°C. N represents the vial number.

Table 2: Effect of buffer type and pH. Stress: Freeze-thaw and 1 M at 40°C. N represents the vial number.

Tab e 3: Effect of excipient. Stress: Freeze-thaw, Shear stress, Lig it Exposure, 1M at 40°C. N represents the vial number.

Example 2: Study of the stability on low concentration formulation

Part 1

To characterize the stability of a liquid formulation containing a low concentration of Abl, the stability of Abl Drug Product (DP) stored during approximately nine months at 25°C was analyzed. The data were compared to two standards, to the TO of stability study and to the same time point at 5°C. Materials and Methods

Reference materials: REF1 and REF2.

Abl DP formulation: 10 mg/mL Abl, 15 mM Histidine, 150 mM NaCI, 0.01% polysorbate, such as Tween 80, pH 6.25. The samples were tested as follow: pH measurement, appearance, A280 measurement, SDS-PAGE, SE-FIPLC, cGE, clEF (iCE)/CEX-FIPLC, Binding ELISA/Cell-based ELISA. The amino acid sequence and post- translational modifications (deamidation, oxidation, glycation, N-terminal variants, C-terminal variants and glycosylation site occupancy) was verified by peptide mapping RP-HPLC coupled to LC- ESI-MS (MS/MS). Results and Discussion

Table 4 summarizes results of analyses.

Table 4: Results of stability tests. NT: Not Tested

The results of cGE are presented in Figure 1 and in Table 5.

Table 5: Values of Retention Time (RT) and %Area of peaks for cGE analysis

On cGE, TO and T9M at 5°C are similar to reference standard profile except for the shoulder before the main peak which is not visible in REF1. It seems to be specific to the material. The shoulder clearly appears as an independent peak on sample T9M at 25°C (Figure 1). Values of peak area confirm this observation (Table 5). HC and lOOkD fragments increase as well. Regarding values and profiles, cGE clearly highlight the fact that non-glycosylated (NG) species are created during the degradation of Abl as well as HC and lOOkD fragments. The results of clEF are presented in Figure 2 and in Table 6.

Table 6: Values of percentages area of peaks for clEF analysis Due to the complexity of clEF profile, visual observation is difficult. Nonetheless, sample T9M at +25°C does not has a double main peak and the first peaks of the profile seem lower that in the other profiles. So this sample seems to contain less main and basic species (Figure 2). Values of Table 6 confirm the visual observation. Acidic peak represents 41% of total species instead of 22-28% in other samples. Main specie represents 37% of total species instead of 44-45% in other samples. The variation can be explained by a change in the global charge of the molecule like deamidation or oxidation.

The results of iCE are presented in Figure 3, Figure 4 and in Table 7.

Table 7: Values of Retention Time (RT) and percentages area of peaks for iCE analysis

TO sample was not analyzed by iCE as the method was not available for routine at this period. Profiles of iCE are visually comparable (Figure 3). Overlay allows to notice that acidic species are higher and main and basic species are lower in T9M at +25°C compared to the two other profiles. Data of Table 7 confirm this observation. Retention time is similar for all samples. The hypothesis can be made that charge of the molecule changes due to oxidation and/or deamidation.

The results of CEX-FIPLC are presented in Figure 5 and in Table 8.

Table 8: Values of Retention Time (RT) and percentages area for CEX-FIPLC analysis. NA: Not Applicable. Data shows that two peaks increase (peak 3 and 4) and one peak almost disappear (peak 7 at 16.6 minutes) in sample T9M at +25°C. This evolution could be linked to the oxidation and/or deamidation.

Additionally, peptide mapping was performed with RP-HPLC LC-ESI-MS (MS) to confirm amino acid sequence. Table 9 summarizes the results, results confirms 100% identity with the expected sequence. As expected, sequence of all sample are 100% identical.

Table 9: Peptide mapping RP-HPLC LC-ESI-MS (MS) for Abl - confirmation of sequence identity

Data related to deamidation and isomerization analysis are presented in Table 10. Peptide mapping was performed with RP-HPLC LC-ESI-MS (MS) to confirm level of deamidation.

Table 10: Peptide mapping RP-HPLC LC-ESI-MS (MS) - deamidation

A high deamidation level is found on the 326 ^th amino acid of the heavy chain (H326) for the stability sample T9M at +25°C compared to the other samples. Deamidation seems to occur during degradation of the molecule.

Data related to oxidation analysis are reported in Table 11. Peptide mapping was performed with RP- HPLC LC-ESI-MS (MS) to confirm oxidation sites along with the level of oxidations.

Table 11: Peptide mapping RP-HPLC LC-ESI-MS (MS)- Oxidation levels

A higher oxidation level is found on three sites of the heavy chain for the stability sample T9M at +25°C compared to the other samples. Oxidation seems to occur in specific sites during degradation of the molecule. Data related to glycation analysis are summarized in Table 12. Peptide mapping was performed with RP-HPLC LC-ESI-MS (MS) to confirm glycation sites along with the level of glycation.

Table 12: Peptide mapping RP-HPLC LC-ESI-MS (MS)- Glycation levels

Glycation does not go over 4% for all amino acids of all samples. Degradation does not lead to glycation. Data on N-terminal and C-terminal modifications are reported in Table 13. Peptide mapping was performed with RP-HPLC LC-ESI-MS (MS) to understand N and C terminal modifications.

Table 13: Peptide mapping RP-HPLC LC-ESI-MS (MS) - N-terminal and C-termina modifications Results are similar for all samples. PyoGlutamate is slightly higher for the stability example T9M at +25°C but the difference is not significant and can be due to the method variation. No degradation on C-terminal and N-terminal sequences are detected.

Conclusions

Data provide supportive evidence for the conclusion that Abl is stable regarding the glycation, the sequence terminal modifications, pH, appearance and concentration. Testing highlights significant differences in charge variant, oxidation and deamidation levels for stability sample T9M at +25°C compared to reference standard. Hypothesis can be made that the peak at 14min on CEX-HPLC profile could be related to oxidation or deamidation. These modifications are also translated by a decrease in acidic species and an increase in basic species on iCE profile. Nonetheless, potency of Abl is not impacted. A potential explanation could be that modifications identified do not occurs in target binding regions (CDR) or do not cause any change on this particular regions. During nine months, Abl goes through several modifications due to the stability of the molecule (including methionine/tryptophan oxidation and asparagine deamidation). All the modifications leads to a change on the global charge of the molecule and are detected thanks to charge variant tests (iCE and CEX-HPLC). The potency of the molecule is not affected by the charge variation. Part 2

Additionally, the drug Product formulated in 15 mM L-Histidine, 150 mM Sodium Chloride, 0.01% Polysorbate 80, pH 6.25, at a concentration of 10.7 mg/mL was stored at +25 ± 2°C at different time (T) intervals, namely 1 month (M), 2, 3, 6 and 12 months; and also at + 5 ± 3°C (at 1, 2, 3, 6, 12, 18, 24, 30, 36 M) to study the stability. The results are summarized in Table 14 to Table 16.

Table 14. Stability at Long Term Stability Condition of +5 ± 3°C

Table 15. Stabi ity at Long Term Stability Condition of +5 ± 3°C

Table 16. Stability at Accelerated Stability Condition of +25 ± 2°C Abl drug product is stable for 36 months when stored at + 5 ± 3°C in terms of purity (based on SE- HPLC and cGE), charge profile (based on iCE3 and CEX-HPLC), protein content (A280), binding to target (ELISA) and subvisible particle content. Physical appearance and pH values are also within specifications. Example 3: Concentration of the Ab formulation

Part 1: Concentration of Abl liquid formulation

In order to develop high concentration Abl liquid formulation, UFDF (Ultrafiltration Diafiltration), UFDF2, was tested. In particular the target concentration was between about 160 and 195 mg/mL.

Material and Methods

Bulk drug substance (BDS)

The characteristics of the starting material BDS Abl-lotl (30C (thawed at 30°C)) are in Table 17:

Table 17: Starting BDS material data Buffers

The two buffers used for this study are shown in Table 18:

Table 18: UFDF2 buffers composition Equipment

30 kDa cut-off Omega centramate T series cassette with 0.019 m ² area from Pall was used for this UFDF2 development. A new single multiple-use cassette was used for each trial. Cogent pscale Tangential Flow Filtration system (DI-DSP-078) from Millipore was used to perform the UFDF2 development. A 120U/DV peristatic pump from Watson Marlow was used to provide a continuous feed during diafiltration step. Nanodrop 2000 from Thermo Scientific was used for protein concentration determination. SevenExcellence pH-meter was used for pH and conductivity measurements. HIAC 9703 particle counter (HACH, Skan AG) was used for subvisible particles estimation. Lovis 2000 ME microviscosimeter from Anton Paar was used for dynamic viscosity measurements.

Analytics method

Samples protein content was determined by A ₂₈ o using Thermo Scientific Nanodrop 2000. SE-HPLC and Sub-visible particles analysis were performed on pre- and post- concentration steps to determine if any aggregation was induced due to UFDF process. IEF by iCE3 was also run to see any change in the charge variants for the same reason. Conductivity were measured on input, permeate and retentate samples. pH and osmolality were also measured on initial, diafiltrated and concentrated final material.

UFDF process

Two different trials were performed to concentrate Abl BDS in desired buffers. Same starting BDS was used, but parameters were optimized in between. UFDF process flowcharts and parameters are detailed in Figure 6 and Table 19. Two concentration steps had to be performed in trial 1 as the parameters were not optimized. As a consequence, feed overpressures happened with both buffers in trial 1 before concentration target was reached. Thus concentration have been performed in two steps during this trial.

C _gei determination

During concentration step, many data points were taken, by regularly recording feed and retentate pressures, permeate volume and flux, and temperature. By determining theoretical concentration on the retentate (C _r ) at each point (Equation 1), it is possible to use the gel theory to determine the theoretical gel concentration, or C _gei , maximum possible retentate concentration before membrane clogs.

Equation 1: determination of theoretical retentate concentration:

Vretentate ~ V initial ^— Vpermeate and Cr— (Cinitial ' Vinitial ) /v retentate)

Using the linear trend line of Permeate flux = f [ln(C _r )] plot in semi-log scale Figure 7, it is possible to define K which is the slope of the trend line and b its intercept, and finally the gel theory can predict the C _gei value (Equation 2). Permeate flux is expressed in L.m ² .h ¹ (abbreviated LMH in Figure 7).

Equation 2: gel theory to predict gel concentration

Cgei = e ^{(b/ K)}

Results

Diafiltration was performed with 4 diavolumes on each run. 5 diavolumes is a safer margin to be sure the diafiltration step is complete, but the permeate conductivities obtained are within the target conductivity ± 3% (see table 5 for buffer conductivity target). The conductivity trend during diafiltration can be found in Figure 8.

Using calculations explained above and after data collection, theoretical C _gei has been estimated on trial 2 with both buffer conditions (see Table 20). For both buffers, theoretical C _gei reaches about 200 mg/mL.

Table 20: Theoretical Cgei estimation on Trial 2

Yields (or material recovery) of the UFDF processes were estimated. These were estimated on the concentrated product before flush. In production condition, the system must be flushed with fresh buffer and this flushing product must be mixed with the concentrated product. In this study, system was flushed to "clean" the system and membrane, but flushing product was not mixed with the concentrated product because of concern on the final BDS concentration. The yields obtain are compared in Table 21. As better process parameters was used for Trial 2, its yield values are more relevant in the concern of a feasibility study.

Table 21: UFDF process yields (without flush)

Once the diafiltration has ended, the product's pH, conductivity, osmolality and protein concentration was measured to be compared with starting material characteristics. Analysis were also performed after concentration step. These data can be found inTable 22.

Consistency of the process can be interpreted from these results. Flowever some unexpected results were observed; an increasing of pH was observed on each run during the concentration step. This phenomenon is linked to the Gibbs-Donnan's effect, which occurs in this range of protein concentration. This induces a gradient of proton concentration between the two sides of the membrane which explains the pH change during the process.

Differences of conductivity values was also observed between the retentate and permeate after diafiltration, and conductivity of retentate decreased further during concentration step of each runs. This could be linked to the protein concentration but has to be investigated.

Maximum protein concentration achieved in buffer 1 is about 164 mg/mL and could be assimilated to an experimental C _gei , as concentration had to be stopped to avoid feed pump overpressure. Buffer 2 allowed to reach higher protein concentration of 192 mg/mL in trial 1. In trial 2, concentration in buffer 2 was stopped until target concentration was reached (173 mg/mL).

Table 22: IPC physico-chemical data collected during the different UFDF trials

Final BDS post UFDF products were analysed with starting material in SE-H PLC runs. The profile obtained proved consistency in monomer, aggregates and fragments repartition between starting and ending BDS with both buffers, and there is reproducibility between both trials' size variants (see Figure 10). Final BDS post UFDF products were analysed with starting material in IEF runs on iCE 3 system.

The profile obtained proved consistency in charge variants repartition and pi ranges between starting and ending BDS with both buffers, and was comparable with the reference standard used (Figure 11 and Figure 12).

Subvisible particles analysis was also performed to have an insight on the UFDF products content. The values obtained are compiled in Table 23.

In addition to above analysis, the viscosity of these two formulations were also measured using a Rolling-ball viscometer. The Lovis dynamic viscosities were observed to be 12.25 mPa-s and 11.38 mPa-s at 25°C for formulation containing NaCI and L-Arginine HCI respectively. However, during this analysis, the standard (water) was expired and also it did not passed specifications.

Conclusions

The main aim of this study was achieved as it showed that it is possible to concentrate Abl in both the selected buffers at a protein concentration >150 mg/mL. However different optimization have to be performed to assess the feasibility of this process for a scale-up, e.g. including a flush step before retrieving the post-UFDF end product.

This study give an insight on the consistency of the BDS stability against the UFDF process in these conditions, and it can give hints to justify an UFDF buffer selection, as buffer 2 (25 mM L-Hisitidine, 150 mM L-Arginine-HCI, pH 6.0) allows to reach higher concentration compared to buffer 1 (25 mM L- Hisitidine, 150 mM NaCI, pH 6.0).

Part 2: UFDF3

A UFDF3 step has been developed to concentrate the product and reach the target concentration of 170 mg/mL after flushing. The concentration was performed by Tangential Flow Filtration (TFF) which consists in a filtration cassette inside which the flows between two membranes. The flow inside the cassette also generates pressure which applied to the flow perpendicularly, i.e. against the membranes walls. This pressure pushes solvent through the membrane toward the permeate line, and molecules smaller than the membrane cut-off with it, while bigger molecules are kept in recirculation back to the feed / retentate or through the drain.

UFDF load and product storage conditions

Freeze / thaw and hold time studies were performed on UFDF 3 load and product as described in Table 24:

Table 24: Hold time study conditions for Abl UFDF 3 product

Thus UFDF 3 load and product were firstly frozen at - 20 °C, then thaw at room temperature and to finish re-frozen. UFDF 3 loads and products were analyzed by SE-HPLC, CGE (reduced and non- reduced), iCE and CEX-HPLC to assess the product quality. pH, conductivity, concentration and UFDF 3 loads and products osmolality were also measured. Results and Discussion

The selected cassette for the UFDF 3 step is a tangential flow filtration cassette, named Cassette 1. The diafiltration was done using 25 mM Histidine, 150 mM Arginine pH 6.0 (DF buffer) during 7 DVs. The first concentration was made up to 100 g/L prior to be diafiltered into the aforementioned pre- formulation buffer. Then, the diafiltered product was concentrated again until the feed pressure reaches 3 bars (~ 220 g/L). Even if higher pressures (until 5 bars) did not show an impact on product quality, the 3 bars maximum pressure corresponds to the limit of the TFF system (tubing limitation) according to recommendation.

This choice leads to certain constraints which were taken into account during development. They are described in the Table 25.

3 development Cross Flow Rate (CFR)

CFR is calculated using feed and permeate flow rates (CFR = Feed flow rate - Permeate flow rate). For this reason, a pump calibration was performed prior to this run (not shown) in order to ensure an accurate measurement of CFR. CFRs (from 100 LMH to 360 LMH), Figure 13 shows the UFDF 3 process performances (permeate flux and TMP evolution throughout the UFDF 3 step) obtained with different CFRs (from 100 LMH to 360 LMH). As demonstrated, increasing the CFR tends to improve the permeate flux. Indeed the starting permeate flux obtained with 100 LMH CFR is approximately 13 LMH compared to that obtained with 360 LMH CFR which is approximately 25 LMH. Nevertheless, no significant permeate flux improvement from 240 LMH to 360 LMH CFRs is observed (~ 20 LMH and ~ 25 LMH for respectively 240 LMH and 360 LMH CFRs). Regarding the pressure, a pressure drop is observed at the end of concentration. For runs performed with a CFR comprised between 240 and 360 LMH CFRs, this phenomenon occurs around 210 g/L whereas for lower CFR (~ 100 LMH), it appears later at about 250 g/L. However, even if there is an earlier pressure drop for CFR > 100 LMH, working with higher CFR (> 240 LMH) allows twice higher permeate flux, at the beginning and during diafiltration, thus allowing much lower process duration (gain of more than 2 hours of process time). Moreover, the reached pre-flushed concentration of 210 g/L is sufficient for a flush of 3 HUV. Consequently, a CFR range of > 240 LMH - < 360 LMH is the best compromise to reach highest flux without having too much pressure.

Maximum feed pump

The flow rate selection must take into account the maximum feed pump speed capacity of the selected TFF skid. Indeed, as the CFR is expressed in L/h per square meter, it is dependent on the cassette surface. Consequently, depending of the cassette surface used for a run, there is a risk to be above the maximum limit of the feed pump speed (i.e. 1000 L/h). The maximum feed pump (L/h) is the multiplication of the maximum feed flow rate (LMH) and the maximum surface area (m2):

Considering a maximum holder capacity of 2.85 m2, the maximum feed flow rate calculation is :

Maximum feed pump (L/h) 1000 L/h

Maximum feed flow rate (LMH)

Maximum surface (m ² ) 2.85 m ²

= 351 LMH

Knowing that, at this feed flow rate (FFR), the permeate flow rate (PFR) is about 25 LMH, it means that the CFR would be:

CFR (LMH) = FFR (LMH) - PFR (LMH) = 351 LMH - 25 LMH = 326 LMH Consequently, a maximum CFR limit of 326 LMH should be established.

Moreover, to avoid working at the maximum of the pump which is not desirable, a set point at 290 LMH was defined. Based on small scale runs data, the permeate flow rate obtained at the beginning of the concentration with a CFR of 290 LMH was 25 LMH. Considering a maximum total surface area of 2.85 m2, the associated feed flow rate for the defined CFR set point would be:

FFR (LMH) = CFR (LMH) + PFR (LMH) = 290 LMH + 25 LMH = 315 LMH

Which corresponds to a feed pump speed of: Feed pump speed (L/h) FFR (LMH) x max cassettes surface (m ² )

= 315 LMH x 2.85 m ² 898 L/h

As the cassettes surface of 2.85 m2 is the maximum stood for the manufacturing TFF system, the feed pump would speed is still suitable whatever the surface area used. Furthermore the maximum pump speed will never be reached. Thus the recommendation for the CFR is > 240 LMFI - < 325 LMFI with a set point at 290 LMFI.

Volumetric Loading Factor (VLF)

The tested VLFs are described in the Table 26:

Table 26: Abl UFDF 3 VLF evaluation

Table 26 shows that higher the VLF is, longer is the process duration: 5 hours at 15 L/m2, 9 hours at 20 L/m2, and 11.8 hours at 30 L/m2. According to SBL mass balance for a 5000 L batch, the highest expected UFDF 3 load volume would be 75 L. If the maximum VLF is set at 25 L/m2, a cassette surface of 3 m2 would be required which is not suitable for the maximum capacity of the holder (< 2 x 1.14 m2 + 1 x 0.57 m2). Therefore, the VLF of 30 L/m2 was chosen as the upper limit to fit with potential highest volumes. Moreover, no product quality impact was noticed whatever the VLF. Thus the recommendation for the VLF is < 30 L/m2 with a set point at 25 L/m2.

Diafiltration

The scope of this experiment was to define the most appropriate product concentration to start the diafiltration. The choice was made on a compromise between sufficient volume reduction (to fit with the retentate tank volume capacity) while keeping the lowest DF duration (i.e. high permeate flux). Previous experiments allowed to select a start of the DF for a product concentration of 100 g/L. Targeting this concentration allowed to put the product into the diafiltration buffer (25 mM Histidine, 150 mM L-Arginine pH 6.0) before reaching too high pressure caused by the increasing viscosity. Indeed, the diafiltration buffer allows a reduction of viscosity which is high when the product is in the equilibration buffer (5 mM Histidine pH 6.0).

According to SBL mass balance, for a 5000 L batch, the UFDF 3 load volume and concentration upper limits are 75 L at 75 g/L. Targeting 100 g/L for the diafiltration would lead to a DF product volume of 56.3 L which is higher than the maximum volume recommended by the supplier (55 L for the selected retentate tank of 50 L). Consequently, target DF concentration was optimized to mitigate this volume constraint. The results are presented in Table 27.

Table 27: Abl UFDF 3 DF concentration evaluation.

The only difference observed is the process duration which is slightly longer for a DF performed at 110 g/L compared to one performed at 100 g/L (9.8 hours than 9.3 hours respectively). However, this difference can be considered as negligible and no impact on product quality was observed. Thus, for high starting volumes, the recommendation is to start the diafiltration at a product concentration of 110 g/L. If this is not the case, targeting 100 g/L is reducing the whole process duration. The recommendation for the DF concentration is < 110 g/L with a set point at 100 g/L. The actual value to target should be calculated while taking into account the initial volume and the retentate tank capacity. Worst case scenario 1: initial volume upper limit

Following the recommendation mentioned in the previous section, calculations were made to indicate which concentration to choose for different initial volumes (Table 28).

Table 28: DF concentration recommendation for different UFDF 3 load volumes

According to the scale up calculation provided above and in order to fit with the maximum retentate tank capacity of 55 L, it appears that the diafiltration step must be performed at 100 g/L up to 70 L initial volume. Above 70 L, the diafiltration has to be performed at 110 g/L. Worst case scenario 2: Initial volume lower limit

In the case of lowest initial volume, the manufacturing constraint is the minimum working volume of the system. Indeed, the risk is to have a product pre-flushed volume below the minimum working volume which may lead to increase in foam and shear stress.

Therefore, an initial volume lower limit must be established to avoid having a pre-flushed volume below the minimum recommended recirculation volume. Table 29 shows estimated pre-flushed volumes for different potential initial volumes and assuming that the pre-flushed concentration is 220 g/L.

Table 29: UFDF 3 volumes estimation related to low initial volumes and concentration

According to the scale up calculation provided above, it appears that processing low initial volumes can be risky in term of the minimum retentate tank capacity and pre-flushed volumes achieved. This point need to be considered and hence determine before running step at scale. The ecommendation here would be not to processed less than 40 L. Initial volumes able to be processed during this UFDF3 step as well as the DF concentration are:

> 40 L - < 70 L with a diafiltration performed at 100 g/L (maximum tank capacity) > 70 L - < 80 L with a diafiltration performed at 110 g/L (minimum tank capacity).

Product quality

No impact on product quality has been observed during UFDF 3 development.

Table 30 shows two examples of product quality results obtained at the selected optimized conditions:

Table 30: UFDF 3 analytical results

High viscosity observed for Sample C UFDF 3 load is unexpected. Flowever, high deviation level (~ 23 %) occurred during the analysis. The yield obtained for Sample D (93 %) is slightly lower than expected but it is easily explained by the lack of flush (2.8 FIUV), lower than the specification set (> 3 FIUV). Indeed the flush is an important point to consider, especially for such high concentrations, in order to recover as much product as possible. No significant differences were observed with respect to SE- H PLC (loads and products > 98 %) and CGE (loads and products > 92 % with less than 1 % differences in all conditions). Regarding charged variant profiles by clEF, differences of about 1.5 % were observed between small and pilot runs. No significant differences between respective load and product were detected, indicating that UFDF 3 does not charged variant profiles.

Stability studies

During the first assessment hold time and freeze and thaw studies have been performed using UFDF 3 load and product from Sample G. For each sample (load and product), all tested conditions were analyzed within the same sequence in order to minimize the variability. Table 31 and Table 32 represent hold time study results of respectively UFDF 3 load and product at different time points, namely 1 week (w) and 2 weeks, and storage temperatures.

Table 31: UFDF 3 load hold time study

Table 32: UFDF 3 product hold time study

Whatever the sample (load or product), no significant differences were detected regarding charge variant profiles neither by clEF nor by CEX-FIPLC. SE-HPLC are all within method variability (< 0.3 %) as well, observed variations are thus not significant. Slight variations observed with respect to CGE (reduced and non-reduced) are inherent to the method and thus results are considered as comparable. To conclude, UFDF 3 load and product are both stable up to 2 weeks at room temperature (+ 22.5 ± 2.5°C) as well as at + 5 ± 3°C.

Freeze and thaw study

As said before, all tested conditions were analysed within the same sequence in order to minimize the variability. Table 33 represents freeze and thaw study results of the UFDF 3 product.

Table 33: UFDF 3 product freeze and thaw study

There are no significant differences regarding charge variant profiles neither by clEF nor by CEX-FIPLC. The product purity by SE-HPLC is highly similar whatever the F/T number. Fragments content with respect to CGE (reduced and non-reduced) is also comparable. To conclude, the freeze and thaw at - 20°C (up to 3 times) of the UFDF 3 product sample has no significant impact.

Case study

Case study 1

Inputs: Volume to process = 80 L and Concentration = 75 g/L In this particular case, main concerns are the retentate tank capacity, the cassettes surface and hence the related pump speed. The first step is to define the surface required for this UFDF 3 step by targeting 25 L/m2 (VLF set point):

Volume to process 80 L „

- - - = - = 3. 2 m ²

Target VLF 25 L/m ²

The next calculation will be to assess if by using this surface, the VLF will still fit with the specifications:

Volume to process 80

- - ⁱ —z - =

Cassettes surface 2.85

This VLF remains within specifications (< 30 L/m2) even it is higher than the set point of 25 L/m2. The second step is to ensure if the system pump speed is suitable with this cassettes surface taking into account the maximum feed flow rate of 315 LMH (i.e. 290 LMH CFR). Even if the feed flow rate to be applied (898 L/h) is closed to the maximum pump speed limit (< 1000 L/h), it remains suitable for this UFDF 3 step. The third step is to estimate the volume at the start of the diafiltration. Indeed, this volume should be lower than 50 L to fit with the maximum retentate tank capacity (< 55 L). Knowing that the diafiltration concentration set point is 100 g/L, the DF volume is:

C, initiale X V; initial 75 g/L X 80 L

= 60 L

DF 100 g/L

As explained earlier, if this volume does not fit with the tank capacity (higher than 55 L), the target DF concentration should be 110 g/L instead of 100 g/L set point:

75 g/L X 80 L

54. 5 L

110 g/L

Moreover, this calculated volume corresponds to the total retentate volume without including the system hold-up volume (HUV) which includes the flow path void volume (FVV)and the cassette / device void volume (DVV)

The DF volume actually contained into the retentate tank is thus:

V _DF - System HUV = V _DF - ( FVV + (2 X DVV _{1 14 m 2} + 1 X DVV _{Q S7 m} z)) = 54.5 L - (0.650 L + (2 X 0.227 L + l x 0.118 L))

= 54.5 L - 1.222 L

= 53. 3 L This volume fit with the retentate tank maximum capacity and can be processed (< 55 L).

The final step is to estimate the preflushed volume into the tank assuming a related concentration of 220 g/L to ensure that it is higher than the minimum retentate tank capacity:

Cpref lushed 220 g/L There is no issue regarding the minimum retentate tank capacity. It is not a concern with such volumes.

Case study 2

Inputs: Volume to process = 45 L, Concentration = 65 g/L

In this case study, there is only one main concern which is the retentate tank capacity and more precisely its minimum recirculation volume. The same exercise as the case study 1 above is performed to assess the suitability of these inputs by considering same parameters.

The first step is to define the surface required for this UFDF 3 step by targeting 25 L/m2 (VLF set point):

Based on two cassettes sizes suitable with the manufacturing scale (0.57 m2 and 1.14 m2) and in order to achieve this required surface, 2 x 1.14 m2 is used (total surface of 2.28 m2). The next calculation will be to assess if by using this surface, the VLF will still fit with the specifications:

Cassettes surface 2.28 m ²

This VLF remains within specifications (< 30 L/m2) even it is lower than the set point of 25 L/m2. In this case, the feed flow rate to applied targeting a set point of 315 LMH (i.e. 290 LMH CFR) is not on the critical path. Indeed the maximum pump capacity (< 1000 L/h) is not reached using this surface (2.28 m2). It is nevertheless calculated for information only:

Feed flow rate x Cassettes surf ace = 315 LMH x 2.28 m ² = 718 L/h

Within the same way, the diafiltration step can be performed at 100 g/L without any issue. Anyway, the volume content into the retentate tank at this stage does not reach the minimum tank capacity. However, the critical point to consider here is the pre-flushed volume remaining into the retentate tank at the end of concentration. The final step is to estimate this volume assuming a pre-flushed concentration of 220 g/L The goal is to ensure that it is higher than the minimum retentate tank capacity:

Cpref lushed 220 g/L

This calculated volume corresponds to the total retentate volume without including the FIUV thus the pre-flushed volume actually contained into the retentate tank is:

= 13.3 L - (0.650 L + (2 X 0.227 L))

= 13.3 L - 1.104 L

= 12. 2 L Although it is really closed to the minimum, this pre-flushed volume fit with the retentate tank minimum capacity and can be processed.

To conclude, the Table 34 is a summary of process parameters to be applied for performing this UFDF 3 case study:

Table 34: summary of UFDF 3 case studies Conclusions

For the implemented UFDF3 step a TFF cassette with a surface < 2 x 1.14 m ² + 1 x 0.57 m ² is required. Equilibration of the cassette is performed using the following parameter:

• Rinsing: FIPW - < 0.1 mS/cm

• Equilibration buffer: 5 mM L-Histidine pH 6.0 - target pFI/conductivity

• Feed pressure: < 3 bars

UFDF 3 loading is performed at room temperature using:

• Volumetric loading factor: target 25 L/m ² (< 30 L/m ² )

• Concentration: > 65 g/L - < 75 g/L

For UF1 the following parameter are recommended:

• Cross flow rate: target 290 LMH (> 240 LMH - < 325 LMH)

• Feed flow rate: target 315 LMH (> 260 LMH - < 350 LMH)

• Feed pressure: < 3 bars

• TMP: target 0.8 bars (> 0.6 bars - < 1.0 bars)

• Concentration: : target 100 g/L (> 100 g/L - < 110 g/L

For diafiltration the following parameter are recommended:

• Cross flow rate: target 290 LMH (> 240 LMH - < 325 LMH)

• Feed flow rate: target 315 LMH (> 260 LMH - < 350 LMH)

• Feed pressure: < 3 bars

• TMP: target 0.8 bars (> 0.6 bars - < 1.0 bars)

• Diafiltration buffer: 25 mM L-Histidine, 150 mM L-Arginine-HCI pH 6.0 (SR-2167) - > 6 DVs For UF2 the following parameter are recommended:

• Cross flow rate: target 290 LMH (> 7 LMH < 325 LMH)

• Feed flow rate: target 315 LMH (> 7 LMH - < 350 LMH)

• Concentration: < 260 g/L

• Feed pressure: < 3 bars

• TMP: < 1.5 bars

Flushing is performed with:

• Flushing buffer: 25 mM L-Histidine, 150 mM L-Arginine-HCI pH 6.0 - > 3 HUV

• Concentration reached: > 162 g/L - < 179 g/L Example 4: PFS development

Part 1: Force Degradation Studies and PFS characteristics analysis

Two formulation compositions were evaluated for forced degradation studies and for pre-fillable syringe (PFS) development:

(FI) 25 mM L-Histidine, 150 mM Sodium chloride, pH 6.0 and

(F2) 25 mM L-Histidine, 150 mM L-Arginine Hydrochloride, pH 6.0.

Material and Methods

Abl-lotl(30C) was diafiltered into two different formulation buffers separately and were concentrated further to achieve a protein concentration of > 164 mg/mL. To each of the UF/DF 2 retentate solution, a calculated amount of Polysorbate 80 was added while adjusting protein concentration to 150 mg/mL to generate Formulated bulk drug substance. The formulated bulk solution was filtered through 0.22 m PES membrane based filter and was filled into 2.25 mL glass PFS system manually without N ₂ gas flushing. This would provide a worst case scenario. The formulation composition and role of each ingredients of both Abl drug product in PFS (300 mg/2 mL) is summarised in below Table 35.

Table 35: Formulation composition and role of each ingredient

A small scale forced degradation study using Formulated bulk drug substance (150 mg/mL) was performed in order to understand the molecular stability of Abl under various stress conditions. About 1 mL of filtered Formulated bulk drug substance was filled into 2 mL glass vials and was stoppered with 20 mm injection rubber stopper. The vials were subjected to multiple stresses as listed below for defined period:

Storage at + 40 °C for 1 week

Shaking at 900 rpm for 72 hours at room temperature 3 Freeze-thaw cycles by freezing at -80 °C ± -20 °C and thawing at +5 °C ± 3 °C

3 Freeze-thaw cycles by freezing at -80 °C ± -20 °C and thawing at +25 °C ± 2 °C

The control (unstressed) sample was stored at +5 °C ± 3 °C

Abl drug product quality was monitored using several analytical techniques as listed in Table 36.

Table 36: Details of analytical test performed and their justification Results forced degradation study

The data of appearance, pH and protein content (by A280) are populated in below table.

Table 37: Physico-chemical analysis data

The data of SE-HPLC analysis are shown in Figure 14. The clEF by iCE3 analysis data are summarized in Figure 15. The cGE (non-reduced) and cGE (reduced) analysis data of Abl-Samplel are shown in Figure 16 and Figure 17 respectively. The cGE (non-reduced) and cGE (reduced) analysis data of Abl-Sample2 are shown in Figure 18 and Figure 19 respectively.

Based on the data of forced degradation study it can be concluded that no significant change in physico-chemical attributes of Abl-Samplel and Abl-Sample2 were observed across all stressed samples. The data from protein stability (SE-HPLC, cGE (reduced and non-reduced) and clEF by iCE3) indicated that there was no major impact on size and charge variants between the control and stressed samples.

Based on all analytical data, it was observed that heat stress (storage at 40°C for 1 week) was relatively most destabilizing for the Abl in both Abl-Samplel and Abl-Sample2. However both these products seem to be stable concerning multiple F/T cycles and shaking stress.

PFS development

An Abl drug product in PFS (300 mg/2 mL) has been developed for subcutaneous injection using an autoinjector. The final product shall be designed to deliver 2 mL of solution containing Abl concentration at 150 mg/mL with high accuracy of dose and minimum pain to the patients. Hence it's important to select an appropriate combination of PFS barrel and plunger stopper.

The key parameters that would impact the delivery of 2 mL of Abl solution are Syringe plunger diameter, Needle length, Needle diameter, Viscosity of solution. The Hagen-Poiseuille Equation is mentioned below that can be used for calculation of plunger force required for delivery of s solution through a PFS.

F = (128 QpLA) / p * D ⁴

F = Plunger force (in N)

Q = Volumetric flow ate (in cm ³ /min)

m = Dynamic viscosity of solution (in N*min/cm ² )

L = Needle length (in cm)

D = Needle bore diameter (in cm)

A = Syringe plunger area (in cm ² )

p (pi) = 3.14159

Assuming 2 mL of Abl (150 mg/mL) solution with a dynamic viscosity of 0.02 Pa.s (20 centipoise) is to be delivered using an autoinjector, the following two scenarios could be derived suing above equation.

Based on above table formulation composition shall be such selected that not only prevents the Abl (at 150 mg/mL) from degradation but also assist in its complete delivery with maximum patient compliance (lower injection force). Part 2: Testing of different PSF configurations

Based on the analysis above, four different PFS configurations were tested with one exemplary Abl formulation (composition given in Table 39).

Table 39: Composition of exemplary Abl formulation tested.

Table 40 summarizes the different PFS materials and configurations tested. The Abl test formulation (Table 39) covered two main aspects: (i) a high Abl protein content of 165 mg/ml (higher range of target concentration of 150 ± 15 mg/ml), and (ii) a high PS80 content of 0.1% (w/v) (about 3x the target concentration of 0.036 % (w/v)), maximizing potential interactions with the packaging components of PFS. The high protein concentration was achieved by using an ultrafiltration process. The test formulation was fully-formulated in 25 mM L-Histidine, 150 mM L-Arginine-HCI, 0.1% (w/v) Polysorbate 80 at a pH of 6.0, via spiking of excipients. The test formulation was filled manually into four different types of PFS barrels and was closed with a Stopperl PFS stopper (Table 40). The samples were tested directly after preparation (TO) and after stressing under freeze-thaw (T-FT) and heat, namely storage for 2 weeks at 40°C (T2w_40C), conditions. Methods used for characterization included visual inspection, Micro-Flow Imaging (MFI) and syringeability.

Table 40: Overview of PFS configurations tested

Materials

Abl BDS material at a concentration of 71 mg/ml in 5 mM histidine at pH 6 was used. Highly purified water (as defined by the European Pharmacopoeia) and in the following named "water" was prepared in-house by using a Milli-Q system (Merck Millipore). The water is characterized by a resistivity value of 18.2 MW cm (at 25 °C).

Table 41: Overview of PFS materials tested. Sample preparation

Abl BDS material was concentrated up to 185 mg/ml by a TFF process. Sample aliquots of 2.0 ml Abl test formulation were filled manually into 2.25-ml siliconized syringes (Barrell, Barrel4, Barrel2, Barrel3(i)) under LAF conditions. A Multipette Xstream (Eppendorf) equipped with a sterile 10-ml Combitip (suitable for high viscosities) was used for transferring the formulation into the syringes. The syringes were closed with plungers (Stopperl) by using a Groninger SVH 100V inserting and closing machine. The machine was set to create a headspace of 2 - 3 mm above the liquid inside the syringes (complete detachment of stopper and liquid were ensured).

Stress / storage conditions

Freeze-thaw stress: the formulations in PFS (each containing 2.0 ml of formulation) were exposed in horizontal positioning to five (manual) cycles of freeze-thaw stress between room temperature (20 °C to 25 °C) and -20 °C (-15 °C to - 25 °C). Samples equilibrated to room temperature were placed manually into a -20 °C-freezer and kept at -20 °C for a minimum of two hours. Subsequently, the frozen samples were thawed again at room temperature for a minimum of two hours (complete thawing was ensured by visual inspection). This procedure was performed 5 times.

Heat stress: the formulations were stored in PFS (each containing 2.0 ml formulation) in a horizontal positioning at 40 °C/75% RH. in a climate chamber for two weeks. Analytical methods

Table 42 provides an overview of the sampling time points and analytical methods of this study.

Table 42: Overview of stress conditions and analytical methods for characterization of samples; n.a. = not applicable.

Visual inspection

The vials were inspected for the presence or absence of visible particles under gentle, manual, radial agitation for 5 seconds in front of a white background and for 5 seconds in front of a black background according to the European Pharmacopoeia (9 ^th edition; monograph 2.9.20) at 2,000 - 3,750 lux. The inspection was performed independently by two trained examiners.

To classify the observed visible particles, a number score on the basis of the "Deutscher Arzneimittel- Codex" (DAC 2006) was used, as listed in Table 43. Fiber-like structures and particles that are likely non-inherent to the product are not accounted.

Table 43: Number score for visible particles, excluding fibers and particles that are likely non-inherent.

Syringeability

Syringeability testing (n = 6) was performed on PFS samples by using a Zwickline syringeability system (Zwick GmbH, Ulm, Germany). The force profile was recorded for expelling the entire content of each PFS at a pre-defined speed by using TestXpert II software Version V3.7. For three PFS units, the test speed was set to expel the PFS content in 20 seconds and for another three PFS in 10 seconds. The approximate travel distance of the PFS stopper for expelling the entire content of each PFS was estimated by using a ruler and defined individually for each type of syringe (barrel geometries were considered). The test speeds applied are summarized in Table 44. Measurements were terminated after the maximum force of 80 N was exceeded during analysis (STOP criterion for measurements). The three PFS samples expelled at the same speed were collected in a sterile, particle-free container (e.g., a 15-ml Falcon tube) and used directly for MFI analysis.

The (i) break loose force and the (ii) gliding force were determined (i) as the temporary, initial maximum at the beginning of the force profile and (ii) as the plateau level of the force profile, respectively, and reported.

Micro-Flow Imaging (MFI)

Micro-Flow Imaging measurements were conducted with a MFI-5200 particle analyzer system equipped with a silane-coated high-resolution lOO-pm SP3 flow cell.

MFI View System Software (MVSS) version was used to perform the measurements and Lumetics LINK software was used to analyze the samples. The concentrations of particles > 1 pm, 2 pm, 5 pm, 10 pm, and 25 pm as well as exemplary particle images of particles > 5 pm were reported. Particle populations' > 5 pm were divided into silicone oil droplet-like particles and non-silicone oil droplet- like particles, if applicable.

Results

Visual inspection

The results of the visual inspection are shown in Table 45. At TO, all samples were free of visible particles and exhibited a yellow coloration. After freeze-thaw as well as heat-stressing the samples showed no or few visible particles and a yellow coloration irrespective of the PFS barrel type.

Table 45: Results of visual inspection of Abl formulation

Syringeability

Syringeability measurements were performed at TO and T2w_40C. The content (2 ml) of each syringe was expelled within 10 and 20 s (n = 3 each). The staked needle was 27G ½" for all PFS configurations tested. The content expelled in each triplicate measurement was collected in a 15-ml Falcon tube and used for MFI measurements as described above. Results of the break loose and gliding forces are shown in Table 46. The mean force profiles are given in Figure 20.

Table 46: Results of break loose and gliding forces of Abl formulation in WP 3A for expelling e entire PFS content within 10 and 20 s, respectively. Gliding forces were comparable for Barrell, Barrel2 and Barrel3(i). In detail, values for gliding forces remained below 13 N for expelling the PFS content within 10 s and below 7 N for expelling the PFS content within 20 s. Gliding forces required to expel the content of Barrel4 PFS were higher compared to the other PFS, i.e., 30 N for expelling the PFS content within 10 s and 16 N for expelling the PFS content within 20 s. After heat stressing, the break loose forces increased slightly, whereas the gliding forces decreased slightly, irrespective of the PFS barrel type.

Micro-Flow Imaging (MFI)

MFI analysis was performed on the 50-mg/ml stock dilutions of the Abl test formulation in order to assess the number of sub-visible particles. The results of the MFI measurements are shown in Figure 21 to Figure 24, as well as Table 47 to Table 51. For particle levels > 5 pm a shape analysis was performed to distinguish between non-silicone oil droplet-like particles and silicone oil droplet-like particles.

At TO, all samples showed less than 10,000 particles >2 pm per ml, except sample in Barrel2 PFS, which showed a slightly higher number of sub-visible particles (15,000 particles >2 pm per ml). After stressing, the particle levels > 2 pm increased slightly compared to TO, but remained overall low for both stress conditions.

The impact of FT stress was least for Barrell PFS and highest for Barrel4 PFS. The impact of heat stress was highest for Barrel3(i), especially when expelling the PFS content in 10 s. The impact of ejection speed was overall less pronounced for Barrell and Barrel2 PFS compared to Barrel3(i) and Barrel4 (higher particle levels observed when expelled at higher speed of 10 s/2 ml).

Performing shape analysis to distinguish between non-silicone oil droplet-like particle and silicone oil droplet-like particle fractions, Barrel2 PFS exhibited the largest numbers of silicone oil droplet-like particles in the size regions > 5 pm, > 10 pm and > 25 pm, most pronounced at TO.

Overall, all samples were characterized by very low numbers of sub-visible particles with only small differences between the four PFS barrel types.

Table 47: Cumulative concentration of particles > 2 pm determined by MFI; values are reported as the mean of n = 3 measurements after expelling the PFS content; values are reported not corrected for the dilution factor (ca. 3-fold); n.a. = not analyzed.

Table 41: Cumulative concentration of non-silicone oi droplet-like particles > 5 pm determined by MFI

(and LINK software); values are reported as the mean of n = 3 measurements after expelling the PFS content; values are reported not corrected for the dilution factor (ca. 3-fold); n.a. = not analyzed.

Table 42: Cumulative concentration of silicone oil droplet-like particles >5 pm determined by MFI (anc LINK software); values are reported as the mean of n = 3 measurements after expelling the PFS content; values are reported not corrected for the dilution factor (ca. 3-fold); n.a. = not analyzed.

Table 48: Cumulative concentration of non-silicone oil droplet- ike particles >10 pm determined by

MFI (and LINK software); values are reported as the mean of n = 3 measurements after expelling the

PFS content; values are reported not corrected for the dilution factor (ca. 3-fold); n.a. = not analyzed.

Table 49: Cumulative concentration of silicone oil roplet-like particles >10 pm determined by MFI

(and LINK software); values are reported as the mean of n = 3 measurements after expelling the PFS content; values are reported not corrected for the dilution factor (ca. 3-fold); n.a. = not analyzed.

Table 50: Cumulative concentration of non-silicone oil droplet-like particles >25 pm determined by MFI (and LINK software); values are reported as the mean of n = 3 measurements after expelling the PFS content; values are reported not corrected for the dilution factor (ca. 3-fold); n.a. = not analyzed.

Table 51: Cumulative concentration of silicone oil droplet-like particles >25 pm determined by MFI (and LINK software); values are reported as the mean of n = 3 measurements after expelling the PFS content; values are reported not corrected for the dilution factor (ca. 3-fold); n.a. = not analyzed.

Summary and conclusions

Four different PFS configurations were tested with one exemplary Abl formulation. The main finding was that by testing different siliconization levels, no particular sensitivity of Abl towards silicone oil could be identified. Therefore, it can be concluded that all standard PFS solutions would be feasible as primary packaging material configurations for Abl drug product formulated at 150 ± 15 mg/ml protein. The results obtained with respect to differences observed between the different siliconization levels can be summarized as follows:

Differences between PFS configurations:

Syringeability

• Higher forces required for Barrel4 compared to other PFS barrel types

· Results for gliding forces very similar for Barrell, Barrel2 and Barrel3(i)

• No impact of 2 weeks of storage at 40 °C on any force profile

Higher siliconization levels of Barrel2 and Barrel3 (i) (0.7 mg/PFS) may explain lower gliding forces compared to 0.55 mg per PFS (Barrel4). The variability of siliconization between different PFS lots as well as within one PFS lot must be considered high, such as for example 0.5 mg ± 0.2 mg per PFS for Barrell PFS. The syringeability of Barrell PFS, however, was in the same range as the syringeability of PFS barrels with 0.7 mg/PFS.

Subvisible particle levels

· Barrel2 PFS showed highest levels of silicone oil droplet-like particles compared to other PFS barrel types

• No major differences observed in non-silicone oil droplet-like particle fractions

Impact of different PFS configurations on Abl stability upon stressing:

• All samples showed no visible and low subvisible particle levels before stressing

· No impact on subvisible particle levels upon FT stressing observed

• Small impact of heat stress observed: few visible particles in Barrel3 (i) and Barrel4 and slightly increased non-silicone oil droplet-like particle levels in all PFS barrel types (most pronounced for Barrel3 (i))

• No impact of the ejection speed (10 versus 20 s/2 ml) on subvisible particle levels determined This study suggests PFS barrels from Barrell (nominal silicone level 0.5 ± 0.2 mg per PFS) as well as from BD and Barrel2 (nominal silicone level 0.7 ± 0.2 or ± 0.3 mg per PFS) to give gliding force profiles in the target range, i.e., 5-6 N.

Barrell and Barrel3(i) were selected and further tests.

Part 3 Molecular stability analysis

Material and methods

The PFS post exposure to freeze-thaw and heat stresses, were analysed for various Abl quality parameters such as protein content (by A280), purity by (SE-HPLC, cGE-reduced, and cGE-non- reduced, iCE3) and potency (Binding ELISA).

Results and conclusions

The results of protein content analysis, shown in Table 52, indicate that there was no significant change in terms of protein content post stresses which shall be attributed to the analytical method variations.

Table 52. Protein content results Binding ELISA, Table 53, shows that both stresses, did not alter the target binding potency of the molecule irrespective of type of PFS barrels used.

Table 53. Binding ELISA results

In Table 54 the results of iCE3 analysis are shown indicating no significant change in % of charged variants of Abl were noticed post Freeze-Thaw (FT) stress but there was a non-specific increase in the acidic variants across all type of barrels post Heat stress (HS) with Barrel3(i) and Barrel2 barrels showing slightly higher %Acidic variants than Barrell/Barrel4.

Table 54: iCE3 results

Non-reduced cGE analysis (Table 55) shows that Abl is stable (no change in content of different species) post Freeze-Thaw (FT) stress but there was a decrease in %lgG peak across all type of barrels post Heat stress (HS) suggesting a non-barrel-specific degradation of molecule under heat condition.

Other peaks = LC + HC + IgG'.

Reduced cGE analysis (Table 56) shows that there was no observable degradation trend post stress conditions irrespective of type of barrels used.

Table 56: Reduced cGE results In Table 57 the results of SE-HPLC analysis are reported, indicating that Abl remained stable post Freeze Thaw stress whereas heat stress cased loss of %monomer and %aggregates while increased %fragments irrespective of barrel types used.

Table 57: SE-HPLC results

In conclusion all different types of barrels used (with variable silicon oil lubrication content) showed no specific Abl degradations. The molecule was stable under freeze-thaw stress whereas under heat stress, it showed increased fragmentations and acidic variants levels which was largely similar across all barrel types. This confirmed that Abl have no particular sensitivity with silicon oil with the range of 0.3 to 1.0 mg/barrel tested. Example 5: Stability studies

Materials and methods

Stress and stability tests were performed for 13 formulations (Table 58) containing 150 mg/ml of Abl, in different PFS (Table 59).

Table 58. Formulations. (PS: Polysorbate)

Table 59: Tested PFS Sample preparation

Abl BDS material was provided at a nominal protein concentration of about 69.47 mg/ml in 5 mM histidine, pH 6.45. The sample was concentrated to about 190 mg/ml by using TFF. Full formulation was achieved either by spiking (two-step addition of concentrated excipient stock) or by buffer exchange (discontinuous diafiltration via Amicon and dialysis). A third spiking step was performed by spiking of respective volumes of 10% (w/v) PS80 stock solutions to both protein samples and formulation buffers/placebo, followed by the filtration of the samples through 0.22-pm PES filters under laminar air-flow conditions and by the filling of product/placebo formulation buffers into 2.25- ml PFS and stoppering under vacuum. Stress / storage conditions

Stress conditions:

• Freeze-thaw stress (TFT) was performed manually and in horizontal position, 5 cycles from +20°C to -20 °C in freezer, holding times >2 h or until completely frozen or thawed

• Rolling stress (T-roll) was performed in a vertical test tube rotatorat 40 rpm for 2 days at room temperature (> 100,000 bubble rolls in total per barrel)

Storage conditions: Samples were stored in horizontal position for 1 month, 3 and 6 months of storage at 5 °C, and 25 °C/60% r.h. and for up to 3 months at 40 °C/75% r.h.

Analytical methods

Samples were analyzed before (TO) and after stress and storage, by using the analytical methods specified in Table 60.

TO characterization

pH, osmolality and viscosity

First an initial characterization (TO) has been done, measuring pH, osmolality and viscosity of the above described samples. The results of the characterization are shown in Table 61.

Table 61: TO characterization Thermal stability

Next thermal stability of samples was evaluated using NanoDSF that utilizes measurement of intrinsic fluorescence of protein upon heating from 20 to 95 °C to determine T _m values (aggregation onset and unfolding events). Apparent protein melting temperatures (Tm) obtained from the ratio of the fluorescence emission at 350 nm and 330 nm (350/330 nm ratio) as a function of temperature. The Tm values and the onset temperature of unfolding (Tm, onset) are calculated by software. In addition, the intensity of the back reflection was monitored to get information about temperature onset of aggregation (Tagg, onset) by the software. There we two melting events were observed (Tml & Tm2) and their results are summarized in Table 62 and in Figure 25.

Table 62: nanoDSF resu ts

All thermal events were comparable and irrespective of the type of PFS (Barrell or Barrel3(ii)). The results reported above indicate that the thermal stability is not impacted by methionine (F10) or higher PS80 content.Thermal/conformational stability is slightly reduced at reduced pH 5.5 (F3) compared to pH 6.5 (F4). A potentially positive impact of compositions with proline (F7) and His/Man or Sue combinations Fll and F12 is detected. The determination of T _m onset ^was difficult for formulations F7, Fll and F12 due to a different melting profile to the other formulations (see Figure 27 for exemplarily melting curves). Thermal events were determined manually under evaluation of

350 nm/330 nm (+ 1 ^st derivative) as well as 350 nm (+ 1 ^st derivative). Results - Stress analyses

pH

At TO, the pH of all samples was within the acceptance limits of target pH ± 0.1 pH units. The pH was unchanged after freeze-thaw and rolling stress, as shown in Table 63.

Table 63: pH ana ysis results after freeze-thaw anc rolling stress

Protein content

At TO, the protein content of all samples was within the acceptance limits of 150 ± 15 mg/ml. The protein content was unchanged after freeze-thaw and rolling stress (still within target range) as shown in Table 64.

Table 64: Protein content analysis results after freeze-thaw and rol ing stress

Turbidity

No difference in the OD350 and OD550 before and after freeze-thaw and rolling stress were detected as shown in Figure 28 and in Figure 29, respectively.

Visual inspection

At TO, all samples were practically free of visible particles and exhibited a yellowish coloration. All formulations remained unchanged after freeze-thaw and rolling stress as shown in Table 65.

Table 65: Visual inspection results after freeze-thaw rolling stress

MFI

All formulations were measured diluted in their respective formulation buffer (at ca. 50 mg/ml). The particle content of the dilution media (Buffer) was very low and comparable for all formulation buffers. The particle levels of protein-containing samples was higher compared to the formulation buffers, overall highest for F5 (* exceeds y-axis, Figure 30), see Table 66 and Table 67.

Table 66: MFI results after stressing

Table 67: MFI results after stressing

Particle levels > 2 miti were overall low in this study, except for F5 (Figure 31). Silicone oil was found in all PFS samples at TO, mainly in the small size region (> 5 pm and potentially below, but differentiation not possible), likely causing increased particle levels. FT and rolling stress did not affect the non- silicone oil-like particle fractions, but some differences were observed for the silicone oil fractions. Silicone oil fractions increased after FT: F5 (large droplets). Silicone oil fractions increased after rolling stress: largest impact on F10 (large droplets), F6. Silicone oil levels > 5 pm were overall lower in Barrel3(ii) PFS compared to Barrell PFS (Figure 32 and Figure 33). SEC analysis

At TO the SEC profiles of most formulations were comparable, with a monomer content of ca. 97%; a slight reduction of monomer content was observed for F5 (96.4%) > F6, Fll (96.8%). After stressing (FT and rolling stress), the monomer content remained unchanged for all formulations in both types of PFS. There was no difference between samples stressed in Barrell or Barrel3(ii) PFS (Figure 34). Initial FIMWS levels were comparable for F2, F4, F7, F8, F9, F10, F12, and F13 at ca. 1.2%; slightly increased FIMWS levels were found for F5 (1.8%) > F6, Fll (1.5%). All FIMWS levels remained unchanged after FT and rolling stress. There was no difference between samples stressed in Barrell or Barrel3(ii) PFS (Figure 35). Initial LMWS levels were at ca. 1.7% for all formulations and remained unchanged after FT and rolling stress. There was no difference between samples stressed in Barrell or Barrel3(ii) PFS (Figure 36).

Bioanalyzer purity

At TO, the sample was 95-96% under non-reducing conditions, and 98% under reducing conditions, and comparable for all formulations. There was no difference between Barrell and Barrel3(ii) PFS (Figure 37). Similarly, there was no difference in the determined purity (Bioanalyzer) under non reducing conditions before and after freeze-thaw and rolling stress (Figure 38), and no difference in the purity (Bioanalyzer) under reducing conditions before and after freeze-thaw and rolling stress (Figure 39). iCE

At TO the iCE profiles were comparable (main species ca. 49-50%) (slightly different pattern for F7), pi range was between 7.6 to 8.9; the pi range of F8 was slightly more acidic (7.2 to 8.1). After stressing (FT and rolling stress), the main species content remained unchanged for all formulations. There was no difference in the iCE profiles between Barrell and Barrel3(ii) PFS samples (Figure 40). Initial acidic species levels were comparable for all formulations at ca. 28-29%. All acidic species levels remained unchanged after FT and rolling stress. There was no difference between samples stressed in Barrell or Barrel3(ii) PFS (Figure 42). Initial basic species levels were at ca. 21-22% for all formulations and remained unchanged after FT and rolling stress. There was no difference between samples stressed in Barrell or Barrel3(ii) PFS (Figure 43).

Conclusions

• All formulations were successfully prepared meeting the target specifications of 150 mg/ml ± 15 mg/ml protein content and a target pH ± 0.1 pH units • All formulations were free of visible particles and exhibited low levels of subvisible particles (MFI), turbidity (OD350, OD550) and soluble aggregates (SEC); the monomer content (SEC) was 96-97%; and the main isoform (iCE3) content was 49-50% for all formulations (results in consensus with expected values considering the CoA, received on 9^ October 2018 and the expected impact of the sample preparation procedures)

• Viscosity was successfully reduced by addition of 150 mM of viscosity reducing agents, namely.

Viscosities were lowest for 150 mM arginine in combination with histidine-bufferat ca. 6-7 mPa*s and highest for Fll and F12 at 16 and 18 mPa*s (no arginine)

Other formulation-related observations

• Slightly reduced thermal/conformational stability for F3 at pH 5.5

• F7 (proline) different melting curve and slightly different iCE pattern

• the pi range of F8 was slightly shifted towards acidic pi region (pi region 7.2 to 8.1), the pattern as such was comparable to other formulations (pi region 7.7 to 8.7)

• No major impact of methionine on protein stability in F10 so far

• Impact of different stressors on Abl

• Freeze-thaw and rolling stress had no measurable impact on any formulation (protein stability)

• Impact of different stressors on PFS barrels

• Rolling stress increased the silicone oil droplet content in Barrell > Barrel3(ii) PFS

Results - Stability 1 month

The results of the stability test after one month of storage are shown below. pH

At TO, the pH of all samples was within the acceptance limits of target pH ± 0.1 pH units. The pH remained unchanged after 1 month of storage, irrespective of the storage condition, as shown in Table 68.

Table 68. pH before and after one month of storage

Osmolarity

Initial osmolalities were largely in the expected/acceptable range except for F6 which showed lower osmolality than expected possibly due to the complex formulation of citrate with arginine and F7 and F8 which showed higher osmolality up to 460 mOsmol/kg. After 1 month of storage at 40 °C, the osmolalities remained unchanged. At TO, the pH of all samples was within the acceptance limits of target pH ± 0.1 pH units. The pH remained unchanged after 1 month of storage, irrespective of the storage condition, as shown in Table 69.

Thermal stability

nanoDSF results before and after Tim of storage are shown in Figure 44 to Figure 46. At TO, no impact of methionine (F10) or higher PS80 content, slightly reduced thermal/conformational stability at pH 5.5 (F3) compared to pH 6.5 (F4). Values remained unchanged for all formulations after 1 month of storage, irrespective of the storage condition. Similar trends as were observed with the fluorescence signal (F350) and back reflection. Onset of aggregation remained unchanged after storage at 5 °C and 25°C. All thermal events remained comparable to TO after Tim, irrespective of the type of PFS (Barrell or Barrel3(ii)). Protein content

At TO, the protein content of all samples was within the acceptance limits of 150 ± 15 mg/ml. The protein content was unchanged after storage of 1 month at 5 °C, 25 °C and 40°C (Table 70).

Table 70: Protein concentration before and after one month of storage Turbidity

Initial OD350 values were comparable for most formulations, but slightly higher for FI, F5 and F8; there was no difference between samples in Barrell and Barrel3(ii) PFS. No difference in the OD350 before and after storage for 1 month at 5 °C. OD350 values increased after storage at 25 °C < 40 °C. Increase in OD350 at 40 °C was less pronounced for the citrate-based formulations F5, F6 compared to the histidine-based formulations (Figure 47). Initial OD550 values were comparable for most formulations, but slightly higher for FI, and slightly lower for F7; there was no difference between samples in Barrell and Barrel3(ii) PFS. No change upon storage for 1 month at 5 °C and 25 °C. OD550 values increased after storage at 40 °C independent of formulation and syringe type (Figure 48). Visual inspection

At TO, all samples were practically free of visible particles and exhibited a yellowish coloration. After storage for 1 month at 5 °C and 25 °C all formulations remained unchanged. After storage for 1 month at 40 °C, most formulation remained particle-free, but F3 (pH 5.5) showed a slightly increased number of particles (score of 2), F1/F5/F6 appeared turbid after Tim at 40 °C compared to other formulations (Table 71).

Table 71: Visual inspection before and after one month of storage

MFI

All formulations were measured diluted in their respective formulation buffer (at ca. 50 mg/ml). The particle content of the dilution media (Buffer) was very low and comparable for all formulation buffers. The particle levels of protein-containing samples was higher compared to the formulation buffers (dilution medium), overall highest for F5 (* exceeding y-axis), confirming results reported for TO (see Figure 49).

Particle levels > 2 pm were overall low in this study (Figure 50), except for F5 at TO and after storage for 1 month at 5 °C. Silicone oil was found in all PFS samples, mainly in the small size region (> 5 pm and potentially below, but differentiation not possible), likely causing increased particle levels (Figure 51 ). After storage for 1 month at 5 °C, non-silicone oil-like particle fractions increased more than at 25 °C and 40 °C (especially F8, F9, F13, FI, Fll); solely for F6 in Barrel3(ii) PFS particles of all size classes appeared to have increased at 40 °C. Silicone oil fractions (mainly in the size range > 5 and 10 pm) slightly increased after storage for 1 month at 40 °C (Figure 52, Figure 53). Silicone oil levels > 5 pm were overall lower in Barrel3(ii) PFS compared to Barrell PFS. For a summary of the results see Table 72 and Table 73.

Table 73: MFI results before and after 1 month of storage: silicone oil drop et-like particles SEC results

The initial SEC profiles of most formulations were comparable, with a monomer content of ca. 97.6%; a slightly lower monomer content was observed for F5 (97.0%) > F6, Fll (97.3%). The monomer content decreased after storage for 1 month at 25 °C < 40 °C for all formulations in both types of PFS, but remained unchanged at 5 °C; lowest monomer content after storage at 25 °C/ 40 °C was observed for FI, F3, F5, Fll, F12 (ca. 95% at 40 °C). There was no difference between samples stored in Barrell or Barrel3(ii) PFS (Figure 54). Initial FIMWS levels were comparable for F1-F4, F7-F10, F12 and F13 at ca. 1.0%; slightly increased FIMWS levels were found for F5 (1.4%) > F6, Fll (1.3%). FIMWS levels increased after storage for 1 month at 25 °C < 40 °C (especially F5, FI, Fll, F3, F12). There was no difference between samples stored in Barrell or Barrel3(ii) PFS (Figure 55). Initial LMWS levels were at ca. 1.5% for all formulations. LMWS levels increased after storage for 1 month at 25 °C < 40 °C relatively independent of the type of formulation. There was no difference between samples stored in Barrell or Barrel3(ii) PFS (Figure 56).

Bioanalyzer purity

The purity (Bioanalyzer) under non-reducing conditions remained largely unchanged after storage for 1 month. There was no difference between samples stored in Barrell or Barrel3(ii) PFS (Figure 57). The purity (Bioanalyzer) under reducing conditions was not affected after storagefor 1 month irrespective of storage temperature. There was no difference between samples stored in Barrell or Barrel3(ii) PFS (Figure 58). iCE

At TO the iCE profiles were comparable (main species ca. 50%) (slightly different pattern for F7), pi range was between 7.6 to 8.9; only the pi range of F8 was slightly more acidic (7.2 to 8.1). Main species content decreased similarly in all formulations after 1 month of storage at 25 °C < 40 °C, but remained unchanged at 5 °C. There was no difference in the iCE profiles between Barrell and Barrel3(ii) PFS samples (Figure 59 and Figure 60). Initial acidic species levels were comparable for all formulations at ca. 28%. Acidic species content increased in all formulations after 1 month of storage at 25 °C <40 °C with little difference between the formulations; no change after Tim of storage at 5 °C. There was no difference between samples stored in Barrell or Barrel3(ii) PFS (Figure 61). Initial basic species levels were at ca. 21% for all formulations, and remained relatively unaffected after storage at 5 °c and 25 °C. Basic species content decreased after storage for 1 month at 40 °C with little difference between the formulations; no change after Tim of storage at 5 °C. There was no difference between samples stored in Barrell or Barrel3(ii) PFS (Figure 62). Conclusions

• All formulations are within the target specifications of 150 mg/ml ± 15 mg/ml protein content and a target pH ± 0.1 pH units;

• At TO, all formulations were free of visible particles and exhibited low levels of subvisible particles (MFI), turbidity (OD350, OD550) and soluble aggregates (SEC); the monomer content

(SEC) was ca.98%; and the main isoform (iCE3) content was ca. 50% for all formulations

• Viscosity was successfully reduced by addition of 150 mM of viscosity reducing agents. Viscosities were lowest for 150 mM arginine in combination with histidine-buffer at ca. 6-7 mPa*s and highest for Fll and F12 at 16 and 18 mPa*s (no arginine). After storage for 1 month at 5°C

• All samples remained stable with no/little changes after storage at 5 °C; only subvisible particle concentration increased slightly for some formulations

After storage for 1 month at 25°C < 40°C

• pH, osmolality (only measured for 40 °C samples), protein content, thermal stability (nanoDSF), as well as sample purity (Bioanalyzer) remained unchanged after storage at 25 °C and 40 °C for all formulations

• All formulations were similarly affected: increase in OD550 values (turbidity), LMWS species (SEC), as well as acidic and basic species (iCE)

Impact of PFS barrel Barrell versus Barrel3(ii): · No differences observed between Barrell and Barrel3(ii) PFS at 5 °C and 25 °C, solely storage at 40 °C increased content of silicone oil droplet-like particles in Barrell > Barrel3(ii) PFS

Results - Stability 3 months

The results of the stability test after one month of storage are shown below. pH

At TO, the pH of all samples was within the acceptance limits of target pH ± 0.1 pH units. The pH remained unchanged after 3 months of storage, irrespective of the storage condition (Table 74).

Table 74: pH before and after 3 months of storage Osmolarity

Initial osmolalities were largely in the expected/acceptable range except for (i) F6 which showed lower osmolality than expected possibly due to the complex formulation of citrate with arginine and (ii) F7 and F8 which showed higher osmolality of up to 460 mOsmol/kg. No strict acceptance criteria had been defined; selected lead candidates may require adjustments of (i) sample preparation procedure, and/or (ii) mannitol content. After 3 months of storage, the osmolalities remained unchanged, irrespective of the storage condition (Table 75).

Table 75. Osmola ity before and a ^: ter 3 months of storage Dynamic viscosity

Initial values for dynamic viscosity were < 20 mPa*s for all formulations. The most effective viscosity reducing agent was arginine (F2, F3, F4, F6, F9, F10, F13), followed by lysine (F8) and proline (F7); formulations containing no amino acids exhibited higher initial values for dynamic viscosity (FI, F5: NaCI; Fll, F12: polyols). After 3 months of storage, the dynamic viscosity remained largely unchanged, independent of storage condition (Table 76).

Table 76: Dynamic viscosity results before and after 3 months of storage

No difference observed between samples stored in Barrell or Barrel3(ii) PFS (Figure 63).

Syringeability

Gliding forces after storage for 3 months at 5 °C were as expected and compliant with results for dynamic viscosity: highest (17-19 N) for Fll and F12; no difference between samples stored in Barrell or Barrel3(ii) PFS. Comparing 25 °C to 5 °C storage of 3 months, gliding forces increased slightly for samples in Barrel3(ii) PFS, whilst they were comparable for Barrell PFS (no formulation effect observed). Comparing 40 °C to 5 °C storage of 3 months, gliding forces increased clearly for all formulations with similar forces required for Barrell and Barrel3(ii) PFS (Figure 64 - Figure 66).

Thermal stability

At TO, no impact of methionine (F10 vs. F2) or higher PS80 content. At TO, slightly reduced thermal/conformational stability at pH 5.5 (F3) compared to pH 6.5 (F4). Potentially slightly positive impact of composition of histidine with proline (F7) and the His/Man or His/Suc combinations Fll and F12. Values remained unchanged for all formulations after 3 months of storage at 5 °C and 25 °C, but were slightly reduced after storage at 40 °C (similar for all formulations) (Figure 67). Similar trends as were observed with the fluorescence signal (F350) were observed with the back reflection. Onset of aggregation remained unchanged after storage at 5 °C and 25 °C. Reduction in Tagg, onset for all formulations after 3 months of storage at 40 °C, most pronounced for F3, and least pronounced for F7, F9 and F10 (Figure 68). No impact of the type of PFS (Barrell or Barrel3(ii)) on nanoDSF results (Figure 69).

Protein content

At TO, the protein content of all samples was within the acceptance limits of 150 ± 15 mg/ml. The protein content did not change during storage of 3 months at 5 °C, 25 °C and 40 °C (Table 77).

Table 77: Protein concentration before and after 3 months of storage Turbidity

Initial OD350 values were comparable for most formulations, but slightly higher for FI, F5 and F8; there was no difference between samples in Barrell and Barrel3(ii) PFS. No difference in the OD350 before and after storage for up to 3 months at 5 °C. OD350 values increased after storage at 25 °C < 40 °C. Increase in OD350 at 40 °C was highest for FI, F8 and F12, and least pronounced for the citrate- based formulations F5, F6 compared to the histidine-based formulations (Figure 70). Initial OD550 values were comparable for most formulations, but slightly higher for FI and slightly lower for F7; there was no difference between samples in Barrell and Barrel3(ii) PFS. No change upon storage for up to 3 months at 5 °C and 25 °C. OD550 values increased after T3m storage at 40 °C independent of formulation and PFS type (Figure 71). Visual inspection

At TO, all samples were practically free of visible particles and exhibited a yellowish coloration. At 5 °C storage (T3m), all formulations remained free of visible particles, but samples F5, F6 appeared slightly turbid. At 25 °C storage (T3m), most formulations remained unchanged but FI, F5 and F6 exhibited clearly visible particles (score 10). At 40 °C storage (T3m), all formulations exhibited visible particles (turbid appearance), solely F7, Fll, F12 in Barrell, and F9 in Barrel3(ii) PFS remained unchanged (Table 78).

Table 78: Visual inspection before and after 3 months of storage MFI

Particle levels > 2 pm were overall low in this study, except for F5 at TO and after storage for up to 3 months at 5 °C (Figure 72). After storage for 3 months the non-silicone oil-like particle fractions were largely unaffected irrespective of storage condition or formulation (observed for particle levels > 5 pm and larBarrell), solely slightly increased levels for FI and F5 (Figure 73). Silicone oil-like particle fractions increased after storage for 3 months at 25 °C < 40 °C (mainly in the size range > 5 and 10 pm) with no/little difference between samples stored in Barrell and PFS (Figure 74, Figure 75).

SEC results

The initial SEC profiles of most formulations were comparable, with a monomer content of ca. 97.6%; a slightly lower monomer content was observed for F5 (97.0%) > F6, Fll (97.3%). The monomer content remained largely unchanged after storage for up to 3 months at 5 °C. The monomer content of all formulations decreased after storage for 3 months at 25 °C < 40 °C; lowest monomer content after storage was observed for F3 (ca. 88% at 40 °C). There was no difference between samples stored in Barrell or Barrel3(ii) PFS (Figure 76). Initial FIMWS levels were comparable for F1-F4, F7-F10, F12 and F13 at ca. 1.0%; slightly increased FIMWS levels were found for F5 (1.4%) > F6, Fll (1.3%). FIMWS levels increased slightly after storage of 3 months at 5 °C. FIMWS levels increased after storage of 3 months at 25 °C < 40 °C (especially FI, F3, F5, Fll, F12: 4-5% after 3 months of storage at 40 °C). There was no difference between samples stored in Barrell or Barrel3(ii) PFS (Figure 77). Initial LMWS levels were at ca. 1.5% for all formulations. LMWS levels remained unchanged after storage for 3 months at 5 °C. LMWS levels increased after storage for 3 months at 25 °C < 40 °C for all formulations; only formulation effect for F3 with highest LMWS content after 3 months of storage at 40 °C (6.8%). There was no difference between samples stored in Barrell or Barrel3(ii) PFS (Figure 78).

Bioanalyzer purity

The purity (Bioanalyzer) under non-reducing conditions was not affected by storage

of 3 months at 5 and 25 °C. After storage of 3 months at 40 °C, the purity decreased in all formulations (lowest sample purity observed for Fll with ca. 80% and F12 with ca. 84%). There was no difference between samples stored in Barrell or Barrel3(ii) PFS (Figure 79). The purity (Bioanalyzer) under reducing conditions was not affected by storage of 3 months at 5 °C and 25 °C. After storage of 3 months at 40 °C, the purity decreased in all formulations. There was no difference between samples stored in Barrell or Barrel3(ii) PFS (Figure 80). iCE

After storage for 3 months at 5 °C, main species content remained largely unchanged. After storage for 3 months at 25 °C < 40 °C, main species content decreased for all formulations. There was no difference in the iCE profiles between samples stored in Barrell or Barrel3(ii) PFS (Figure 81). After storage for 3 months at 5 °C, acidic species content remained largely unchanged. After storage for 3 months at 25 °C < 40 °C, acidic species content increased for all formulations. There was no difference in the iCE profiles between samples stored in Barrell or Barrel3(ii) PFS (Figure 82). After storage for 3 months at 5 °C, basic species content remained largely unchanged. After storage for 3 months at 25 °C < 40 °C, basic species content decreased for all formulations. There was no difference in the iCE profiles between samples stored in Barrell or Barrel3(ii) PFS (Figure 83).

Conclusions

• All formulations are within the target specifications of 150 mg/ml ± 15 mg/ml protein content and a target pH ± 0.1 pH units • At TO, all formulations were free of visible particles and exhibited low levels of subvisible particles (MFI, except F5), turbidity (OD350, OD550) and soluble aggregates (SEC); the monomer content (SEC) was ca. 98%; and the main isoform (iCE3) content was ca. 50% for all formulations

• Viscosity was successfully reduced by addition of 150 mM of viscosity reducing agents.

Viscosities were lowest for 150 mM arginine in combination with histidine- buffer at ca. 6-7 mPa*s (F2, F3, F4, F10, F13) and highest for Fll and F12 at 16 and 18 mPa*s (no arginine)

After storage for 3 months at 5 °C:

^• All samples remained stable with no/little changes after storage at 5 °C; only

in visual inspection F5 and F6 appeared slightly turbid

After storage for 3 months at 25 °C and 40 °C:

^• pH, osmolality, protein content, non-silicone oil-like particle fractions (MFI) as

well as dynamic viscosity remained largely unchanged after storage at 25 °C and 40 °C for all formulations

• All formulations were affected by 3 months of storage as follows: increase in OD350 and OD550 values (turbidity), gliding forces, silicone oil-like particle fractions (MFI), FIMWS and LMWS content (SEC), and acidic species (iCE). Decrease in thermal/conformational stability (nanoDSF) and sample purity under reducing and non-reducing conditions (Bioanalyzer) at 40 °C storage, and loss of monomer content (SEC) at 25 °C < 40 °C

Impact of PFS barrel Barrell versus Barrel3(ii):

^• Gliding forces increased slightly for samples in Barrel3(ii) PFS after storage at 25 °C compared to 5 °C (no increase of gliding forces observed for samples stored in Barrell PFS)

^• For both PFS types the gliding forces (silicone oil droplet-like particle content) increased similarly after T3m of storage at 40 °C. NOTE: silicone oil droplet-like particle content was slightly increased in Barrell compared to Barrel3(ii) afterTlm of storage at 40 °C and after rolling stress

Results - Stability 6 months

The results of the stability test after 6 months of storage for formulation F2, F7, F9, F10 and F13 are shown below. pH

At TO, the pH of all samples was within the acceptance limits of target pH ± 0.1 pH units. The pH remained unchanged after 6 months of storage, irrespective of the storage condition (Table 79)

Table 79: pH before and after 6 months of storage Dynamic viscosity

Initial values for dynamic viscosity were < 15 mPa*s for all formulations. The most effective viscosity reducing agent was arginine (F2, F9, F10, F13), followed by proline (F7). Viscosity remained unchanged for up to T3m, but appeared reduced by 1.5-3 mPa*s after T6m (Table 80).

Table 80: dynamic viscosity before and after 6 months of storage

Thermal stability

At TO, no impact of methionine or higher PS80 content was observed. Values remained unchanged for all formulations after 6 months of storage at 5 °C and 25 °C (Figure 84). Onset of aggregation remained unchanged after storage at 5 °C and 25 °C (Figure 85).

Protein content

At TO, the protein content of all samples was within the acceptance limits of 150 ± 15 mg/ml. The protein content was unchanged after storage of 6 month at 5 °C and 25 °C (Table 81).

Table 81: Protein content before after 6 months storage

Turbidity

Initial OD350 values were comparable for all five formulations. No difference in the OD350 observed before and after storage for up to 6 months at 5 °C. OD350 values increased after storage at 25 °C, with no clear difference between the formulations (Figure 86). Initial OD550 values were comparable for most formulations (slightly lower for F7). No change upon storage for up to 6 months at 5 °C and 25 °C, all formulations revealed in OD550 values (Figure 87).

Visual inspection

At TO, all samples were practically free of visible particles and exhibited a yellowish coloration. After 6 months of storage at 5 °C, all formulations except F7 (DAC score 2) remained free of visible particles; F2, F9 and F13 appeared slightly turbid. After 6 months of storage at 25 °C, all formulations showed no or few visible particles; F2, F10 and F13 appeared slightly turbid (Table 82).

Table 82: Visual inspection before and after 6 months of storage ME!

After 6 months of storage at 5 °C and 25 °C the particle levels > 2 pm increased slightly, most pronounced for F7, F9 (5 °C) and F2 (25 °C). Particle levels of F10 and F13 remained similar to initial levels and thus lower compared to the other formulations (Figure 88). After 6 months of storage at 5 °C and 25 °C the levels of proteinaceous particles > 5 pm increased compared to TO and T3m. Trend more pronounced for F2, F7, and F9 compared to F10 and F13 (5 °C > 25 °C). Silicone oil droplet-like particle levels higher compared to T0/T3m, but no clear formulation differences (Figure 89). After 6 months of storage at 5 °C and 25 °C the levels of proteinaceous particles > 5 pm increased compared to TO and T3m. Trend more pronounced for F2, F7, and F9 compared to F10 and F13 (5 °C > 25 °C). Silicone oil droplet-like particle levels higher compared to T0/T3m, but no clear formulation differences (Figure 90). Similar trend for particles > 25 pm than for > 5 and 10 pm was observed. Slightly increased particle levels for F2 and F7 compared to F9, F10 and F13. Silicone oil droplet-like particle levels remained low and comparable to T0/T3m (Figure 91).

SEC results

The initial SEC profiles of all five formulations were comparable, with a monomer content of ca. 97.6%. The monomer content decreased marginally after storage for up to 6 months at 5 °C. Upon storage of 6 months at 25 °C, the monomer content of all formulations decreased to 95.7-95.3% with no clear difference between the formulations (Figure 92). Initial FIMWS levels were comparable at ca. 1.0% for all five formulations. FIMWS levels increased slightly after storage of 6 months at 5 °C (to ca. 1.2%). FIMWS levels increased after storage of 6 months at 25 °C (most pronounced for F7 and F9: 1.7%; least pronounced for F10: 1.3%) (Figure 93). Initial LMWS levels were at ca. 1.5% for all formulations. LMWS levels increased slightly to ca. 1.7% upon storage for 6 months at 5 °C for all formulations. Upon storage for 6 months at 25 °C, LMWS levels increased for all formulations to ca. 2.9% (Figure 94).

Bioanalyzer purity

The purity (Bioanalyzer) under non-reducing conditions was overall not affected by storage at 5 and 25 °C for up to 6 months (Figure 95). The purity (Bioanalyzer) under reducing conditions was not affected by storage at 5 °C for up to 6 months. After 6 months of storage at 25 °C, the purity seemed slightly reduced for F2, F7, F9 and F10, not F13 (Figure 96). iCE

After storage for 6 months at 5 °C, main species content remained largely unchanged. After storage for 6 months at 25 °C, main species content decreased for all formulations (no differences between formulations) (Figure 97). After storage for 6 months at 5 °C, acidic species content remained largely unchanged. After storage for 6 months at 25 °C, acidic species content increased for all formulations (Figure 98). After storage for 6 months at 5 °C, basic species content remained largely unchanged. After storage for 6 months at 25 °C, basic species content decreased for all formulations (Figure 99). ELISA

Table 83. Binding ELISA

Binding ELISA Table 83 showed that many formulations were out of specifications after 3M of incubation at 40°C. Conclusions

All formulations are within the target specifications of 150 mg/ml ± 15 mg/ml protein content and a target pH ± 0.1 pH units

^• At TO, all formulations were free of visible particles and exhibited low levels of subvisible particles (MFI, except F5), turbidity (OD350, OD550) and soluble aggregates (SEC); the monomer content (SEC) was ca. 98%; and the main isoform (iCE3) content was ca. 50% for all formulations ^• Viscosity was successfully reduced by addition of 150 mM of viscosity reducing agents. Viscosities were lowest for 150 mM arginine in combination with histidine- buffer at ca. 6-7 mPa*s (F2, F10, F13)

After storage for 6 months at 5 °C:

All formulations remained relatively stable with only little changes observed:

^• in visual inspection F2, F9, and F13 appeared slightly turbid, and F7 exhibited clearly visible particles (no changes for F10)

^• Proteinaceous particles > 5 pm formed particularly for F2 > F7 > F9,

particle levels in F10 and F13 affected least

^• Soluble aggregates increased from 1.0% to 1.3% for F7 and F9, and to 1.1% for F10, and F13

^• LMWS slightly increased by 0.3% for all formulations

^• Viscosity reduced between 2.1 - 3.5 mPa*s, affect lowest for F13, highest for F7

After storage for 6 months at 25 °C:

All formulations were clearly affected after storage at 25 °C; the following changes were observed:

^• in visual inspection F2, F10, and F13 appeared slightly turbid, no change in content of visible particles for any of the formulations

^• Proteinaceous particles > 5 pm formed particularly for F7, particle levels in F10 and F13 affected least

^• OD350 (not OD550) increased for all formulations similarly

^• Soluble aggregates increased from 1.0% to 1.7% for F7 and F9, and to 1.3% for F10

^• LMWS increased by 1.4% for all formulations

^• Viscosity reduced between 1.5 - 2.6 mPa*s, affect lowest for F2/F13, highest for F9

^• Reduction of main iCE species from 50% to 40% (no formulation differences), whilst acidic species increased (from 29% to 42%) and basic species decreased (from 21.5% to 18.5%)

Example 6: Storage stability studies

The molecular stability profile of Abl formulated bulk drug substance (150 mg/mL) was further investigated:

(a) when filled in 10 mL glass vials (target fill volume: 2.1 mL); and (b) when filled in 2.25 mL glass pre-fillable syringe (PFS) system (target fill volume: 2.1 mL) and stored under different temperature storage conditions.

Material and methods

This study was performed using:

(a) Development batches of Abl formulated BDS (150 mg/mL) with lot Abl-Samplel and Abl-

Sample2, manufactured at in-house.

- Lot Abl-Samplel: formulated in 25 mM Histidine, 150 mM Sodium chloride, 0.36 mg/mL Polysorbate 80, pH 6.0

- Lot Abl-Sample2: formulated in 25 mM Histidine, 150 mM L-Arginine hydrochloride, 0.36 mg/mL Polysorbate 80, pH 6.0.

The formulated bulk drug substance at target concentration of 150 mg/mL of Abl was filled into 10 mL glass vials (2.1 mL fill per vial). The required number of Abl formulated BDS vials were stored under multiple temperature conditions of -80 ± 20°C, -20 ± 5°C, +5 ± 3°C and +25 ± 2°C in order to assess its stability. (b) Development batches of Abl drug product in PFS (PFS1) (300 mg/2 mL) with lot Abl-Sample3 and

Abl-Sample4, manufactured in-house.

Lot Abl-Sample3: formulated in 25 mM Histidine, 150 mM Sodium chloride, 0.36 mg/mL Polysorbate 80, pH 6.0

Lot Abl-Sample4: formulated in 25 mM Histidine, 150 mM L-Arginine hydrochloride, 0.36 mg/mL Polysorbate 80, pH 6.0.

The formulated bulk drug substance at target cone of 150 mg/mL of Abl was filled into PFS1. The required number of Abl PFS were stored under multiple temperature conditions of +5 ± 3°C, +25 ± 2°C and +40 ± 2°C in order to access its stability.

The container closure systems for formulated bulk drug substance and PFS are detailed in the Table 84 and Table 85, respectively.

Table 84: Container closure system details for formulated bulk drug substance

Table 85: Container closure system details for PFS

The key quality attributes to be monitored during this stability are listed down along with their justification in Table 86.

Table 86: Details of test to be performed and their justification

Results stability of Abl formulated bulk drug substance

The results of the stability studies on Abl formulated bulk drug substance are reported in Table 87 to Table 94.

Table 87: Abl formulated BDS (150 mg/mL) lot Abl-Samplel under storage of -80 ± 20°C

Table 88: Abl formulated BDS (150 mg/mL) lot Abl-Samplel under storage of -20 ± 5°C

Table 90: Abl formulated BDS (150 mg/mL) lot Abl-Samplel under storage of +25 ± 2°C

Table 91: Abl formulated BDS (150 mg/mL) lot Abl-Sample2 under storage of -80 ± 20°C

Table 92: Abl formulated BDS (150 mg/mL) lot Abl-Sample2 under storage of -20 ± 5°C

Table 94: Abl formulated BDS (150 mg/mL) lot Abl-Sample2 under storage of +25 ± 2°C

According the results above, Abl formulated BDS (150 mg/mL) in vial with lot Abl-Samplel and Abl- Sample2 are within specification for all the stability indicating parameters. Both the lots met specifications in terms of purity (based on SE-HPLC and cGE), charge profile (based on iCE), protein content (A280) and binding to target (Binding ELISA) when stored frozen up to 6 months at -80 ± 20° & -20 ± 5°C. The pH values were also within predefined specifications however the color of solution varied from slightly brownish to brownish with presence of random (dust like) particulates without any observable trend.

Under liquid state and at elevated storage conditions of +5 ± 3°C as well as +25 ± 2°C, both lots (Abl- Samplel and Abl-Sample2) were within specifications for all the stability parameters for up to 3 months and 1 month of storage respectively.

Additionally, solution osmolality were measured at all time-points for both the lots; it was observed to be consistent across all storage conditions. The average value of osmolality for lot Abl-Samplel for all time-points and storage conditions was (329 ± 5) mOsm/kg whereas for lot Abl-Sample2 it was (310 ± 3) mOsm/kg. Considering the method variation of ± 10 mOsm/kg, osmolality values for both these lots are almost the same for each given storage conditions.

Based on available data, it can be concluded that Abl formulated BDS (150 mg/mL) in vial having lot Abl-Samplel and Abl-Sample2, are found to be:

Stable for 6 months when stored frozen at -80 ± 20°C & -20 ± 5°C

- Stable for 3 months when stored refriBarrellated at +5 ± 3°C

Stable for 1 month when stored at +25 ± 2°C

Results stability study ore-filled syringe drug product

The results of the stability studies on Abl formulated bulk drug substance are reported from Table 95 to Table 108.

Table 95: Abl DP in PFS1 (300 mg/2mL) lot Abl-Sample3 under storage of +5 ± 3°C

Table 96: Abl DP in PFS1 (300 mg/2mL) lot Abl-Sample3 under storage of +5 ± 3°C

Table 97: Abl DP in PFS1 (300 mg/2mL) lot Abl-Sample3 under storage of +5 ± 3°C

Table 99: Abl DP in PFS1 (300 mg/2mL) lot Abl-Sample3 under storage of +25 ± 2°C

Table 100: Abl DP in PFS1 (300 mg/2mL) lot Abl-Samp e3 under storage of +25 ± 2°C

Table 101: Abl DP in PFS1 (300 mg/2mL) lot Abl-Sample3 under storage of +40 ± 2°C

Table 103: Abl DP in PFS1 (300 mg/2mL) lot Abl-Sample4 under storage of +5 ± 3°C

Table 104: Abl DP in PFS1 (300 mg/2mL) lot Abl-Sample4 under storage of +5 ± 3°C

Table 105: Abl DP in PFS (300 mg/2mL) lot Abl-Sample4 under storage of +5 ± 3°C

Table 107: Abl DP in PFS1 (300 mg/2mL) lot A 3l-Sample4 under storage of +25 ± 2°C

Table 108: Abl DP in PFS1 (300 mg/2mL) lot Abl-Sample4 under storage of +40 ± 2°C

According to the results reported above, Abl drug product in PFS (300 mg/2 mL) with lot Abl- Sample3 and Abl-Sample 4 are within specification for all the stability indicating parameters when stored up to 18 months at +5 ± 3 °C. Both the lots met specifications in terms of purity (based on SE- HPLC and cGE), charge profile (based on iCE), protein content (A280) and binding to target (Binding ELISA). The pH values were also within predefined specifications however the color of solution varied from slightly yellowish to slightly brownish with presence of random (dust like) particulates at some time points without any observable trend.

Under accelerated storage conditions of +25 ± 2 °C, both the samples were found to be stable for up to 3 months. Due to incorrect PFS storage at +40 ± 2 °C between 3 and 6 months instead of intended storage condition of +25 ± 2 °C, both lots were OOS for SE-HPLC, cGE (non-reduced and reduced) and clEF (by iCE3) at 6 months. Under regular stress condition of +40 ± 2 °C for 1 month, both lots did not met predefined stability specifications for cGE (non-reduced) and clEF (by iCE3) tests.

Additionally, solution osmolality were measured at all time-points for both lots; it was observed to be consistent across all storage conditions. The average value of osmolality for Abl-Sample3 for all time-points and storage conditions was 329 ± 6 mOsm/kg whereas for Abl-Sample4 it was 313 ± 8 mOsm/kg. Considering the method variation of ± 10 mOsm/kg, osmolality values for both these lots are comparable for each storage conditions.

As a part of syringeability testing, the break-loose force and gliding force were measured for 6M samples stored at +5 ± 3 °C and +25 ± 2 °C (3M actual storage). All PFS showed low break loose forces (5.9 N to 7.5 N), except for Abl-Sample3 - 5 °C sample which did not show a break-loose point. The average gliding force for all PFS sample was also low (4.1 N to 5.9 N), except for Abl-Sample3 - 25 °C sample which had a higher gliding force of 12.9 N. These values of gliding force are in close agreement with theoretical value of 5.71 N force needed using Flagen-Poiseuille Equation for delivering Abl (150 mg/mL) solution with a dynamic viscosity of 0.02 Pa.s (20 centipoise) through a standard 27G needle.

The sub-visible particle analysis by MFI revealed that of total particles having size > 5 pm about 97.5 to 98.7% of that were of protein-like for all 6M samples stored at +5 ± 3°C and +25 ± 2°C except for lot# Abl-Sample3 +25 °C sample for which the fraction of protein-like was 92.7% which could have been generated due to excessive heat stress during storage. Abl-Sample4 was also submitted to excessive heat stress and doesn't appear to show the same result. Flowever, further testing beyond n=l would be necessary in order to obtain conclusive results. Also, more orthogonal particle analysis methods may contribute to a more detailed and reliable characterization of particle size distribution beyond the particle size range covered by MFI.

Based on available data, it can be concluded that Abl DP in PFS (300 mg/2 mL), are found to be:

1) Stable for 18 months when stored refrigerated at +5 ± 3°C

2) Stable for 3 months when stored +25 ± 2°C

3) Not stable for 1 month when stored at +40 ± 2°C