Method for selectively controlling the generation of degradants from a biological product exposed to a stress condition

Abstract

The present invention relates to a method for targeted generation of degradants from a biological product exposed to a low pH stress, wherein said method comprises a) selecting a set of different low pH stress conditions, wherein each stress condition is determined by a combination of a low pH and at least two additional factors, b) exposing said biological product to each low pH stress condition of said set, c) analyzing the degradants obtained in each low pH stress condition, d) processing the data obtained from said analysis, to obtain a predictive model of the degradants generated in function of the low pH and of the at least two additional factors and e), optionally, determining from said predictive model, a combination of values or ranges of values of low pH and of the at least two additional factors to be used to generate desired degradants from said biological product exposed to a low pH stress.

Description

[0001] METHOD FOR SELECTIVELY CONTROLLING THE GENERATION OF DEGRADANTS

[0002] FROM A BIOLOGICAL PRODUCT EXPOSED TO A STRESS CONDITION

[0003] Filed of the invention

[0004] The present invention concerns a method of controlling the generation of degradants from a biological product exposed to a stress condition.

[0005] Technical background

[0006] Forced degradation studies are integral to the development of recombinant monoclonal antibody (mAb) therapeutics. These studies are critical for the development of stability indicating methods enabling the monitoring of product degradation and identification of product critical quality attributes (CQAs). Throughout its lifecycle, a mAb may undergo pH and temperature excursions and other stresses, which can lead to the generation of a variety of degradant species. Monitoring low pH stress degradation behavior is critical, as biologic therapeutics are exposed for example to acidic conditions during the viral inactivation step in the platform manufacturing process.

[0007] It is widely known that physical degradation (aggregation, fragmentation) is the predominant degradation pathway experienced upon exposure to low pH. Chemical degradation (deamidation, succinimide and isoAsp formation) may also be observed at low pH; however, this type of degradation typically predominates upon exposure to high pH. The use of extreme low pH conditions can lead to concomitant generation of multiple types of degradants, which can confound the interpretation of functional impact of a particular degradation route. Strategic selection of low pH stress conditions can help to prevent multiple degradation pathways occurring simultaneously, potentially allowing for unambiguous CQA assignment.

[0008] Currently, there exists a general understanding of the molecular mechanisms of IgG aggregation under low pH conditions, where modulation of aggregation behavior can be achieved through the fine-tuning of buffer ionic strength, temperature, pH and protein concentration. The most commonly used approaches to study low pH stress on monoclonal antibodies are: a) pH of 3-5, b) a temperature of 25°C and 40°C (also 2-8°C and higher than 40°C), c) strong acid / base to achieve desired pH (also buffer exchange into low pH buffer, such as citrate, acetate, etc.), d) duration of exposure of one to two weeks, e) monitoring technique: SEC (size exclusion chromatography), f) expected degradation: aggregation / particle formation, fragmentation, g) chemical modifications may also be observed (deamidation, oxidation, succinimide formation, isomerization, etc.) (see J. Halley et al., Journal of Pharm. Sciences 109 (2020) 6-21). However, there is a lack of clear guidance on how forced degradation studies should be conducted.

[0009] There is therefore a need fora method for determining the conditions allowing targeted generation of degradants from a biological product exposed to a low pH stress.

[0010] Brief summary of the invention

[0011] The Inventors succeeded in mapping out the effects of various factors that lead to low pH-induced monoclonal antibody (mAb) degradation, to assist in the selection of suitable stress conditions for targeted generation of mAb degradants. In a preliminary step, the Inventors analyzed the effect of low pH, citrate buffer concentration and temperature on the distinct degradation behaviors observed for an lgG4 antibody. Using key stability-indicating assays, an extended look into the physicochemical and biological activity profiles of the degraded lgG4 antibody revealed insightful differences in the degradation pathways induced by the modulation of low pH citrate buffer conditions. The Inventors then used a Design of Experiment (DoE) approach to define a set of 15 low pH stress conditions to which the antibody was exposed. Degradants generated in each low pH stress condition were analyzed. The experimental data were then processed using a statistic tool, to obtain a predictive model for targeted generation of degradants as a function of pH, temperature and citrate buffer concentration.

[0012] By “stability-indicating assay”, it is herein meant an assay able to separate, identify, and quantify the biological product from any degradants that may arise during storage or under stress conditions.

[0013] As it will be clear from the description, the above method can be applied to other biological products and other parameters.

[0014] In a first aspect is provided a method for analyzing degradation of a biological product, wherein said method comprises: a) selecting a set of different low pH stress conditions, wherein each stress condition is determined by a combination of a low pH and at least two additional factors, the value of the low pH factor being chosen among NO values, the value of the first additional factor being chosen among N1 values, the value of the second additional factor being chosen among N2 values, wherein NO, N1 and N2 are integers greater than or equal to 2, b) exposing said biological product to each low pH stress condition of the set selected in step a), to obtain a mixture comprising the biological product and degradants for each low pH stress condition, c) analyzing the degradants obtained in step b) in each low pH stress condition, to obtain data, d) processing the data obtained in step c), to obtain a predictive model of the degradants generated in function of the low pH and of the at least two additional factors, and e) optionally, determining from said predictive model, (i) a combination of values or ranges of values of low pH and of the at least two additional factors to be used to generate desired degradants from said biological product exposed to a low pH stress or (ii) the nature and / or amount of degradants that will be generated when exposing the biological product to a combination of values or ranges of values of low pH and of the at least two additional factors.

[0015] In some embodiments, the method as defined above is for targeted generation of degradants from a biological product exposed to a low pH stress or is for monitoring degradation of a biological product exposed to a low pH stress.

[0016] In some embodiments, the method as defined above comprises step f) after step e) and step f) comprises: exposing the biological product to a low pH stress condition characterized by a combination of values determined in step e) to favor generation of at least one desired degradant and / or minimize generation of at least one unwanted degradant, or exposing the biological product to a low pH stress condition characterized by a combination of values determined in step e) to conduct a forced degradation study of a biological product.

[0017] In some embodiments, the additional factor as defined above is selected from the group consisting of temperature, duration of exposure, concentration of a chemical compound, initial concentration of the biological product and ionic strength.

[0018] In some embodiments, in step d), processing the data obtained in step c) comprises performing statistical analysis of the data to obtain said predictive model.

[0019] In some embodiments, the method as defined above comprises step ao) before step a), wherein step ao) comprises (i) exposing said biological product to at least one low pH stress condition, wherein said stress condition is characterized by a low pH and at least two additional factors and (ii) analyzing the degradants obtained in said low pH stress condition.

[0020] In some embodiments, step ao) as defined above comprises exposing said biological product to at least two low pH stress conditions, wherein each stress condition is characterized by a low pH and at least two additional factors and wherein the low pH stress conditions differ from each other by opposed values for at least one of the additional factors and (ii) analyzing the degradants obtained in each low pH stress condition.

[0021] In some embodiments, the values of at least one of the additional factors in step a) and / or of the low pH are selected in function of the results obtained in step ao).

[0022] In some embodiments, analyzing the degradants comprises assessing the amount and / or the nature of at least one degradant.

[0023] In some embodiments, step c) as defined above further comprises assessing the biological activity of the biological product in each low pH stress condition.

[0024] In some embodiments, the biological product as defined above is a protein. In some embodiments, the biological product as defined above is an antibody or an antigen-binding fragment thereof. In some embodiments, the biological product as defined above is an IgG antibody, such as an lgG1 , lgG2, lgG3 or lgG4 antibody.

[0025] In some embodiments, analyzing the degradants as defined above comprises assessing the presence and / or amount of at least one degradant selected from the group consisting of low molecular weight species (LMWS), high molecular weight species (HMWS) and charge variants.

[0026] In some embodiments, the additional factors as defined above in step ao) and / or in step a) comprise temperature and concentration of citrate.

[0027] In some embodiments, in step a) as defined above, low pH is selected in the range from 3 to 4, temperature is selected in the range from 22°C to 35°C and the concentration of citrate is selected in the range of 10 mM to 100 mM, for example in the range of 50 mM to 100 mM. In some embodiments, in step a) as defined above, the values for low pH comprise 3.1 , 3.4 and 3.6, the values for the concentration of citrate comprise 50 mM, 75 mM and 100 mM and the values for the temperature comprise 22°C, 25°C and 35°C.

[0028] In some embodiments, the low pH stress conditions of the set selected at step a) have the same duration of exposure and the same initial concentration of the biological product.

[0029] In another aspect is provided the use of the predictive model obtained in step d) of the method as defined above, to select a combination of values or ranges of values of low pH and of at least two additional factors to be used to generate desired degradants from a biological product exposed to a low pH stress.

[0030] In another aspect is provided the use of the predictive model obtained in step d) of the method as defined above, to determine a low pH stress condition able to favor generation of at least one desired degradant and / or minimize generation of at least one unwanted degradant from a biological product, or to conduct a forced degradation study of a biological product. Detailed description

[0031] Before the present disclosure is described in detail, it is to be understood that this disclosure is not limited to particular methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0032] Definitions

[0033] The term “antigen-binding protein”, as used herein, refers to a protein capable of binding specifically to at least one antigen. Examples for antigen-binding protein include, but are not limited to, antibodies or fragments thereof. Antigen-binding proteins may be of non-human (e.g., murine) or human origin. If such antigen-binding proteins are of nonhuman (e.g., murine) origin, these may be “humanized” to decrease immunogenicity or to increase stability. In some embodiments, the antigen-binding protein is a multispecific antigen binding-protein, such as a bispecific or trispecific antigen-binding protein. A multispecific antigen-binding protein typically contains antigen binding domains specific for at least two antigens.

[0034] The term "heavy chain" as used herein encompasses a full-length heavy chain and fragments thereof. A full-length heavy chain includes a variable region domain, VH, and three constant region domains, CH1 , CH2, and CH3. The VH domain is at the aminoterminus of the polypeptide, and the CH3 domain is at the carboxyl-terminus. The term "heavy chain" includes any immunoglobulin polypeptide having sufficient variable region sequence to confer specificity for an antigen.

[0035] The term "light chain" as used herein encompasses a full-length light chain and fragments thereof. A full-length light chain includes a variable region domain, VL, and a constant region domain, CL. Like the heavy chain, the variable region domain of the light chain is at the amino-terminus of the polypeptide. The term "light chain" as used herein includes any immunoglobulin polypeptide having sufficient variable region sequence to confer specificity for an antigen.

[0036] Naturally-occurring antibodies typically comprise a tetramer. Each such tetramer typically is composed of two identical pairs of polypeptide chains, each pair having one full- length light chain (typically having a molecular weight of about 25 kDa) and one full-length heavy chain (typically having a molecular weight of about 50-70 kDa). The amino-terminal portion of each light and heavy chain typically includes a variable region of about 100 to 1 10 or more amino acids that typically is responsible for antigen recognition. The carboxyterminal portion of each chain typically defines a constant region responsible for effector function.

[0037] Human light chains are typically classified as kappa and lambda light chains.

[0038] Heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.

[0039] IgG has several subclasses, including, but not limited to, IgG 1 , lgG2, lgG3, and lgG4.

[0040] IgM has subclasses including, but not limited to, IgM 1 and lgM2.

[0041] IgA is similarly subdivided into subclasses including, but not limited to, lgA1 and lgA2.

[0042] Within full-length light and heavy chains, typically, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 more amino acids.

[0043] The variable regions of each light / heavy chain pair typically form the antigen-binding site. The variable regions typically exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs from the two chains of each pair typically are aligned by the framework regions, which may enable binding to a specific epitope. From N- terminal to C-terminal, both light and heavy chain variable regions typically comprise the domains FR1 , CDR1 , FR2, CDR2, FR3, CDR3, and FR4. The assignment of amino acids to each domain is typically in accordance with the definitions of Kabat et al., 1991 , Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242.

[0044] A bispecific or bifunctional antibody typically is an artificial hybrid antibody having two different heavy chain / light chain pairs and two different binding sites.

[0045] The term “antigen-binding fragment” of an antibody, as used herein, includes any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex.

[0046] Binding fragments include, but are not limited to, F(ab), F(ab'), F(ab')2, Fv, singledomain antibodies such as VHH antibodies (nanobodies) and single-chain antibodies.

[0047] A F(ab) fragment is comprised of one light chain and the CH1 and variable regions of one heavy chain. The heavy chain of a F(ab) molecule cannot form a disulfide bond with another heavy chain molecule.

[0048] A F(ab') fragment contains one light chain and one heavy chain that contains more of the constant region, between the CH1 and CH2 domains, such that an interchain disulfide bond can be formed between two heavy chains to form an F(ab')2 molecule. The Fv region comprises the variable regions from both the heavy and light chains, but lacks the constant regions.

[0049] Single-chain antibodies are Fv molecules in which the heavy and light chain variable regions have been connected by a flexible linker to form a single polypeptide chain, which forms an antigen-binding region.

[0050] The term “monoclonal antibody” or “mAb” as used herein, refers to an antibody molecule of a single amino acid composition and sequence that is directed against at least one specific antigen, and is not to be construed as requiring production of the antibody by any particular method. A monoclonal antibody may be produced by a single clone of B cells or hybridoma, but may also be recombinant, i.e. produced by protein engineering. The monoclonal antibody may for instance correspond to a murine, a chimeric, a humanized or a fully human antibody.

[0051] The term "chimeric antibody" refers to an engineered antibody which in its broadest sense contains one or more regions from one antibody and one or more regions from one or more other antibody(ies). In particular, a chimeric antibody comprises a VH domain and a VL domain of an antibody derived from a non-human animal, in association with a CH domain and a CL domain of another antibody, in particular a human antibody. As the non- human animal, any animal such as mouse, rat, hamster, rabbit or the like can be used. In an embodiment, a chimeric antibody has variable domains of mouse origin and constant domains of human origin.

[0052] The term "humanized antibody" refers to an antibody which is initially wholly or partially of non-human origin and which has been modified to replace certain amino acids, in particular in the framework regions of the heavy and light chains, in order to avoid or minimize an immune response in humans. The constant domains of a humanized antibody are most of the time human CH and CL domains. In an embodiment, a humanized antibody has constant domains of human origin.

[0053] The term “recombinant”, as used herein, refers to antibodies or antigen-binding fragments thereof of the disclosure created, expressed, isolated or otherwise obtained by technologies or methods known in the art as recombinant DNA technology which include, e.g., DNA splicing and transgenic expression. The term refers to antibodies or antigenbinding fragments thereof expressed in a non-human mammal (including transgenic non- human mammals, e.g., transgenic mice), or a cell (e.g., CHO cells) expression system or isolated from a recombinant combinatorial human antibody library.

[0054] Biological product

[0055] In some embodiments, the biological product is a drug or a biological reagent. In some embodiments, the biological product as defined above is a protein.

[0056] In some embodiments, the protein as defined above is a recombinant protein.

[0057] In some embodiments, the protein as defined above is produced by naturally- occurring cells or by genetically-engineered cells.

[0058] In some embodiments, the protein is an antigen-binding protein, an enzyme, a hormone or a cytokine.

[0059] In some embodiments, the protein is an antigen-binding protein, such as an antibody or an antigen-binding fragment thereof.

[0060] The antigen-binding protein, antibody and antigen-binding fragment thereof are as defined above in the section “Definitions”.

[0061] In some embodiments, the antibody as defined above is a monoclonal antibody (mAb). In some embodiments, the monoclonal antibody as defined above is a murine, a chimeric, a humanized or a fully human antibody.

[0062] In some embodiments, the antibody as defined above is a multispecific antibody, such as a bispecific antibody or a trispecific antibody.

[0063] In some embodiments, the antigen-binding fragment as defined above is a F(ab), F(ab'), F(ab')2, Fv, a single-domain antibody, such as a VHH antibody or single-chain antibody.

[0064] In some embodiment, the protein as defined above is an IgM, IgD, IgG, IgA or IgE antigen-binding protein. In some embodiments, the protein as defined above is an IgG antigen-binding protein. In some embodiments, the protein as defined above is an lgG1 , lgG2, lgG3 or lgG4 antigen-binding protein. In some embodiments, the protein as defined above is an lgG4 antigen-binding protein.

[0065] By the expression “IgX antigen-binding protein", it is herein meant that the antigenbinding protein comprises a heavy chain corresponding to a heavy chain of an antibody of isotype IgX.

[0066] In some embodiments, the protein as defined above is an IgM, IgD, IgG, IgA or IgE antibody. In some embodiments, the protein as defined above is an IgG antibody. In some embodiments, the protein as defined above is an lgG1 , lgG2, lgG3 or lgG4 antibody. In some embodiments, the protein as defined above is an lgG4 antibody.

[0067] The expression “IgX antibody” or “antibody of isotype IgX” are herein synonymous.

[0068] Non-limiting examples of antibodies that may be used in the method of the invention comprise: panitumumab, omalizumab, abagovomab, abciximab, actoxumab, adalimumab, adecatumumab, afelimomab, afutuzumab, alacizumab, alemtuzumab, alirocumab, altumomab, amatuximab, anatumomab, apolizumab, atinumab, tocilizumab, basilizimab, bectumomab, belimumab, bevacizumab, biciromab, canakinumab, cetuximab, daclizumab, densumab, eculizumab, edrecolomab, efalizumab, efungumab, ertumaxomab, etaracizumab, etanercept, golimumab, infliximab, natalizumab, palivizumab, panitumumab, pertuzumab, ranibizumab, rituximab, tocilizumab, trastuzumab, dupilumab, sarilumab or fresolimumab.

[0069] In some embodiment, the antigen-binding protein as defined above, such as the antibody or antigen-binding fragment as defined above, is conjugated to a moiety, such as a ligand or a therapeutic moiety.

[0070] In some embodiments, the protein as defined above is an enzyme.

[0071] Non-limiting examples of enzymes that may be used in the method of the invention comprise acid a-glucosidase, a-L-iduronidase, iduronate sulfatase, heparan N-sulfatase, galactose-6-sulfatase, acid P-galactosidase, P-glucoronidase, N-acetylglucosamine-1- phosphotransferase, a-N-acetylgalactosaminidase (a-galactosidase B), acid lipase, lysosomal acid ceramidase, acid sphingomyelinase, P-glucosidase, galactosylceramidase, a-galactosidase A, acid P-galactosidase, P- galactosidase, neuraminidase, hexosaminidase A or hexosaminidase B.

[0072] In some embodiments, the protein as defined above is a cytokine or a hormone.

[0073] Non-limiting examples of hormone or cytokine that may be used in the method of the invention comprise human erythropoietin, tumor necrosis factor (e.g. TNF-a, TNF-P or TNF- K), interferon alpha or interferon beta.

[0074] Degradants from a biological product

[0075] In some embodiments, degradants from a biological product are generated upon exposure of said biological product to a low pH stress condition. The low pH stress condition is for example as defined below in the section "Low pH stress condition”.

[0076] In some embodiments, the degradants result from a physical degradation or a chemical degradation.

[0077] Physical degradation includes aggregation and fragmentation.

[0078] Chemical degradation includes deamidation, succinimide and isoAsp (isoaspartate) formation.

[0079] In some embodiments, the degradants result from physical degradation.

[0080] In some embodiments, the degradant is selected from the group consisting of size variants, such as low molecular weight species (LMWS), high molecular weight species (HMWS), and charge variants.

[0081] LMWS result from fragmentation of the biological product, such as fragmentation of an antigen-binding protein. The presence and amount of LMWS may be assessed by any method well known by the skilled person, such as size exclusion chromatography (SEC), capillary gel electrophoresis (CGE), capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) or nonreduced CE-SDS (NR CE-SDS).

[0082] HMWS result from aggregation of the biological product, such as aggregation of an antigen-binding protein.

[0083] The presence and amount of HMWS may be assessed by any method well known by the skilled person, such a size exclusion chromatography (SEC).

[0084] Charge variants result from an alteration of the biological protein, such as an antigen-binding protein, which modifies its isoelectric pH (pl) values, leading to charge heterogeneity.

[0085] The presence and amount of charge variants may be assessed by any method well known by the skilled person, such capillary isoelectric focusing (clEF), Imaged capillary isoelectric focusing (iCIEF) or cation exchange chromatography (CEX). pH of the low pH stress condition

[0086] In some embodiments, the stress condition is a low pH stress condition.

[0087] In some embodiments, the low pH condition corresponds to a condition to which the biological product is exposed during its lifecycle, for example during its manufacture or purification, such as during a low pH inactivation step.

[0088] In some embodiments, the low pH condition mimics a condition to which the biological product is exposed during its lifecycle, for example during its manufacture or purification, such as during a low pH inactivation step.

[0089] In some embodiments, a low pH stress condition is a condition wherein the biological product is exposed to a pH equal to or lower than 5.

[0090] In some embodiments, the pH of the low pH stress condition as defined above is equal to or lower than 5, equal to or lower than 4.9, equal to or lower than 4.8, equal to or lower than 4.7, equal to or lower than 4.6, equal to or lower than 4.5, equal to or lower than 4.4, equal to or lower than 4.3, equal to or lower than 4.2, equal to or lower than 4.1 . or equal to or lower than 4.

[0091] In some embodiments, the pH of the low pH stress condition as defined above is equal to or greater than 3.

[0092] In some embodiments, the pH of the low pH stress condition as defined above is comprised from 3 to 5, such as from 3 to 4.

[0093] In some embodiments, the pH of the low pH stress condition as defined above is 3, 3.1 , 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9 or 4. Additional factors of the low pH stress condition

[0094] In some embodiments, the low pH stress condition as defined above is characterized by a pH as defined above and at least two additional factors.

[0095] In some embodiments, the additional factor as defined above is selected from the group consisting of temperature, duration of exposure, concentration of a chemical compound, initial concentration of the biological product and ionic strength.

[0096] In some embodiments, the temperature of the low pH stress condition as defined above is equal to or greater than 18°C, 19°C, 20°C, 21 °C or 22°C.

[0097] In some embodiments, the temperature of the low pH stress condition as defined above is equal to or lower than 40°C, 39°C, 38°C, 37°C, 36°C or 35°C.

[0098] In some embodiments, the temperature of the low pH stress condition as defined above is selected in the range from 18°C to 40°C, 20°C to 38°C or 22°C to 35°C.

[0099] In some embodiments, the temperature of the low pH stress condition as defined above is 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31 °C, 32°C, 33°C, 34°C or 35°C.

[0100] The duration of exposure is the duration of exposure of the biological product to the low pH stress condition.

[0101] In some embodiments, the duration of exposure is at least 5 hours, at least 6 hours or at least 7 hours.

[0102] In some embodiments, the duration of exposure is at most 16 days, at most 15 days, at most 14 days or at most 13 days.

[0103] In some embodiments, the duration of exposure is comprised from 5 hours to 16 days, 6 hours to 15 days or 7 hours to 14 days.

[0104] In some embodiments, the duration of exposure is comprised from 5 hours to 15 hours, 6 hours to 14 hours, 7 hours to 12 hours, 7 hours to 10 hours or 7 hours to 9 hours.

[0105] In some embodiments, the duration of exposure is comprised from 6 hours to 8 hours, for example around 7 hours.

[0106] In some embodiments, the concentration of a chemical compound is the concentration of a buffer, such as the buffer used to obtain the pH of the low pH stress condition.

[0107] In some embodiments, the buffer is a citrate buffer, phosphate buffer or acetate buffer.

[0108] In some embodiments, the citrate buffer is a solution of citric acid and sodium citrate. In some embodiments, the acetate buffer is a solution of acetic acid and sodium acetate.

[0109] In some embodiments, the phosphate buffer is a solution of the weak acid monosodium phosphate and disodium phosphate.

[0110] In some embodiments, the concentration of buffer, such as of the citrate, phosphate or acetate buffer, is at least 1 mM, at least 5 mM, at least 10 mM.

[0111] In some embodiments, the concentration of buffer, such as the citrate, phosphate or acetate buffer, is at most 500 mM, at most 400 mM, at most 300 mM, at most 200 mM or at most 100 mM.

[0112] In some embodiments, the concentration of buffer, such as the citrate, phosphate or acetate buffer, is comprised from 1 mM to 500 mM, from 5 mM to 400 mM, from 10 mM to 300 mM, from 10 mM to 200 mM or from 10 mM to 100 mM. In some embodiments, the concentration of buffer, such as the citrate, phosphate or acetate buffer, is comprised from or from 50 mM to 100 mM.

[0113] Ionic strength is a measure of the concentration of charges in a solution.

[0114] In some embodiments, ionic strength is calculated as follows: ^(Ci*z / 2) / 2, where Ci is the molar concentration of ion i in mol / Land Zi is the net charge of ion i.

[0115] Buffering species contribute to ionic strength of solution, along with any other salts, if present.

[0116] In some embodiments, ionic strength is modulated by modulating the concentration of buffer, such as the concentration of citrate, phosphate or acetate buffer.

[0117] The initial concentration of the biological product is the concentration of the biological product before being exposed to a low pH stress condition.

[0118] In some embodiments, the initial concentration of the biological product is at least 1 mg / mL, at least 2 mg / mL, at least 3 mg / mL, at least 4 mg / mL or at least 5 mg / mL.

[0119] In some embodiments, the initial concentration of the biological product is at most 50 mg / mL, at most 40 mg / mL, at most 30 mg / mL, at most 20 mg / mL or at most 10mg / mL.

[0120] In some embodiments, the initial concentration of the biological product is comprised from 1 mg / mL to 50 mg / mL, from 2 mg / mL to 40 mg / mL, from 3 mg / mL to 30 mg / mL, from 4 mg / mL to 20 mg / mL or from 5 mg / mL to 10 mg / mL.

[0121] In some embodiments, the initial concentration of the biological product is around 5 mg / mL. Low pH stress condition

[0122] In some embodiments, the low pH stress condition as defined above is characterized by a pH as defined above in the section “pH of the low pH stress condition" and at least two additional factors as defined above in the section “Additional factors of the low pH stress condition”.

[0123] In some embodiments, the low pH stress condition as defined above is characterized by a pH as defined above and two, three, four, five or at least five additional factors as defined above.

[0124] In some embodiments, the low pH stress condition as defined above is characterized by:

[0125] - a low pH selected in the range from 3 to 4,

[0126] - a temperature selected in the range from 22°C to 35°C,

[0127] - a concentration of citrate selected in the range of 10 mM to 100 mM, for example in the range of 50 mM to 100 mM, optionally, a duration of exposure selected in the range of 6 hours to 14 days, for example in the range of 6 hours to 8 hours and optionally, an initial concentration of the biological product from 5 mg / mL to 10 mg / mL.

[0128] In some embodiments, the low pH stress condition as defined above is characterized by: a low pH of 3.1 , 3.4 or 3.6, a concentration of citrate buffer of 10 mM, 50 mM, 75 mM or 100 mM a temperature comprise 22°C, 25°C or 35°C, optionally a duration of 6 hours, 7 hours or 8 hours, and optionally, an initial concentration of the biological product of 4 mg / mL, 5 mg / mL or 6 mg / mL.

[0129] Method for analyzing degradation of a biological product exposed to a low pH stress

[0130] In some aspects of the present disclosure, a method is provided for analyzing degradation of a biological product, such as degradation of a biological product exposed to a low pH stress. In some embodiments, the method is for analyzing degradation of a biological product exposed to a low pH stress during its manufacture or purification, such as during a low pH inactivation step. In some embodiments, the method as defined above is for targeted generation of degradants from a biological product exposed to a low pH stress or is for monitoring degradation of a biological product exposed to a low pH stress.

[0131] In some embodiments, the method as defined above comprises: ao) optionally, (i) exposing said biological product to at least one low pH stress condition, wherein said stress condition is characterized by a low pH and at least two additional factors and (ii) analyzing the degradants obtained in said low pH stress condition, a) selecting a set of different low pH stress conditions, wherein each stress condition is determined by a combination of a low pH and at least two additional factors, the value of the low pH factor being chosen among NO values, the value of the first additional factor being chosen among N1 values, the value of the second additional factor being chosen among N2 values, wherein NO, N1 and N2 are integers greater than or equal to 2, b) exposing said biological product to each low pH stress condition of the set selected in step a), to obtain a mixture comprising the biological product and degradants for each low pH stress condition, c) analyzing the degradants obtained in step b) in each low pH stress condition, to obtain experimental data, d) processing the experimental data obtained in step c), to obtain a predictive model of the degradants generated in function of the low pH and of the at least two additional factors, and e) optionally, determining from said predictive model, (i) a combination of values or ranges of values of low pH and of the at least two additional factors to be used to generate desired degradants from said biological product exposed to a low pH stress or (ii) the nature and / or amount of degradants that will be generated when exposing the biological product to a combination of values or ranges of values of low pH and of the at least two additional factors.

[0132] By “targeted generation of degradants”, it is herein particularly meant the generation of the desired degradant(s) and / or a minimized generation of unwanted degradant(s).

[0133] In some embodiments, the method as defined above is a method of selectively controlling the generation of degradants from a biological product exposed to a stress condition. By “selectively controlling the generation of degradants”, it is herein particularly meant favoring the generation of the desired degradant(s) and / or minimizing the generation of unwanted degradant(s).

[0134] In some embodiments, selectively controlling the generation of degradants allows mimicking a low pH condition, such as a low pH condition occurring during the lifecycle of the biological product, such as during its purification, in particular in a low pH viral inactivation step. In such case, selectively controlling the generation of degradants for example means obtaining the profile of degradants (in particular the amount and kinds of degradants) generally obtained in the mimicked low pH condition. In some embodiments, this may be useful to optimize the production of a biological product, such as its purification, in particular the low pH viral inactivation step, to study product degradation, or to identify critical quality attributes (CQAs) of the biological product.

[0135] A CQA is a physical, chemical, biological or microbiological property or characteristic that should be within an appropriate limit, range, or distribution to ensure the desired product quality and safety.

[0136] In some embodiments, the method as defined above allows selecting suitable low pH stress conditions for a biological product, such as to favor generation of at least one desired degradant and / or minimize generation of at least one unwanted degradant, or to conduct a forced degradation study of said biological product. In some embodiments, the method as defined above allows selecting suitable low pH stress condition(s) for targeted generation of low pH-induced antibody degradants.

[0137] A forced degradation study for example allows assessing the impact of process changes on degradation of a biological product. A forced degradation study comprises exposure of the biological product to stress conditions, such as a low pH stress condition, which can exceed those used for conventional stability studies.

[0138] In the above methods, the biological product is as defined above in the section “Biological product’.

[0139] In some embodiments, the biological product is a protein as defined above, such as an antigen-binding protein, an enzyme, a hormone or a cytokine.

[0140] In some embodiments, the biological product is an antigen-binding protein, such as an antibody as defined above or an antigen-binding fragment thereof as defined above. In some embodiments, the biological product is an IgG antigen-binding protein as defined above, such as an lgG1 , lgG2, lgG3 or lgG4 antigen-binding protein as defined above. In some embodiments, the biological product is lgG4 antigen-binding protein as defined above. In some embodiments, the biological product is an IgG antibody as defined above, such as an lgG1 , lgG2, lgG3 or lgG4 antibody as defined above. In some embodiments, the biological product is lgG4 antibody as defined above.

[0141] In some embodiments, steps a) to e) are carried out using DoE (Design of Experiment).

[0142] In a DoE, multiple experimental factors are varied in parallel to strategically evaluate their effects on a desired outcome / response. By manipulating multiple inputs at the same time, DoE can help identify important interactions that may be missed when varying only one factor at a time (OFAT). Ultimately, DoE can help optimize and / or better understand the most important factors that affect an observed response. In a DoE, statistical analysis methods utilize multiple linear / nonlinear regressions to fit a model to the experimental response data. In the frame of the invention, DoE is a structured experimental design approach that helps increase product or process understanding and identify CQAs (Critical Quality Attributes).

[0143] Step a0)

[0144] Step a0) is optional in the method as defined above.

[0145] Step a0) is a preliminary step whose aim is to reduce the time needed to perform the method as defined above.

[0146] In some embodiments, step a0) comprises (i) exposing said biological product to at least one low pH stress condition, wherein said stress condition is characterized by a low pH and at least two additional factors and (ii) analyzing the degradants obtained in said low pH stress condition.

[0147] The low pH stress condition is as defined above in the section “Low pH stress condition".

[0148] In some embodiments, the additional factors comprise temperature and buffer concentration, such as citrate or acetate concentration.

[0149] In some embodiments, the low pH stress condition used in step a0) is characterized by:

[0150] - a low pH of 3.1 , 3.4 or 3.6,

[0151] - a concentration of citrate buffer of 10 mM, 50 mM, 75 mM or 100 mM,

[0152] - a temperature of 22°C, 25°C or 35°C,

[0153] - optionally a duration of 6 hours, 7 hours, 8 hours, 10 days or 13 days, and optionally, an initial concentration of the biological product of 4 mg / mL, 5 mg / mL or 6 mg / mL. In some embodiments, the low pH stress condition used in step ao) is characterized by: a low pH of 3.6, a concentration of citrate buffer of 10 mM citrate, a temperature of 35°C, and

[0154] - a duration of exposure of 13 days.

[0155] In some embodiments, the low pH stress condition used in step a0) is characterized by:

[0156] - a low pH of 3.5,

[0157] - a concentration of citrate buffer of 100 mM citrate,

[0158] - a temperature of 22°C, and

[0159] - a duration of exposure of 7 hours.

[0160] In some embodiments, step ao) comprises exposing said biological product to at least two low pH stress conditions, wherein each stress condition is characterized by a low pH and at least two additional factors and wherein the low pH stress conditions differ from each other by opposed values for at least pH or one of the additional factors and (ii) analyzing the degradants obtained in each low pH stress condition.

[0161] In some embodiments, step a0) comprises exposing said biological product to two low pH stress conditions, for example two, three or four low pH stress conditions.

[0162] In some embodiments, the additional factors comprise temperature and buffer concentration, such as citrate or acetate buffer concentration.

[0163] In some embodiments, the low pH stress conditions differ from each other by opposed values for pH and / or one, two, three or more additional factors.

[0164] In some embodiments, the low pH stress conditions differ from each other by opposed values for temperature, buffer concentration (such as citrate or acetate buffer concentration) and / or duration of exposure. In some embodiments, opposed values for temperature differ from at least 3°C, such as 32°C in the first low pH stress condition and 35°C in the second. In some embodiments, opposed values for temperature differ from at least 5°C or at least 10°C. In some embodiments, opposed values for buffer concentration, such as citrate or acetate buffer concentration, differ from at least a factor 2, for example 50 mM in the first low pH stress condition and 100 mM in the second, or 10 mM in the first low pH stress condition and 100 mM in the second. In some embodiments, opposed values for duration of exposure differ from at least four hours, at least one day or at least seven days. In some embodiment, the duration of exposure 13 days in the first low pH stress condition and 7 hours in a second low pH stress condition. In some embodiments, the low pH stress conditions further differ from each other by the pH. In some embodiments, the pH differs from at least 0.1 between each low pH stress condition. In some embodiments, the low pH is 3.5 in a first low pH stress condition and 3.6 in the second low pH stress condition.

[0165] In some embodiments, the first low pH stress condition used in step ao) is characterized by a low pH of 3.6, a concentration of citrate buffer of 10 mM citrate, a temperature of 35°C, and a duration of exposure of 13 days and the second low pH stress condition used in step a0) is characterized by a low pH of 3.5, a concentration of citrate buffer of 100 mM citrate, a temperature of 22°C and a duration of exposure of 7 hours.

[0166] Step ao) then comprises (ii) analyzing the degradants obtained in the low pH stress condition or each low pH stress condition.

[0167] In some embodiments, analyzing the degradants comprises assessing the amount and / or nature of the degradants.

[0168] The degradants are as defined above in the section “Degradants from a biological product’. In some embodiments, the degradant is selected from the group consisting of low molecular weight species (LMWS), high molecular weight species (HMWS) and charge variants.

[0169] The amount and / or nature of the degradants may be assessed as defined above in the section “Degradants from a biological product’.

[0170] The nature of the degradants is the kind of degradant, such as for example LMWS, HMWS or charge variants.

[0171] In some embodiments, analyzing the degradants comprises assessing the amount and / or presence of LMWS, HMWS and charge variants.

[0172] In some embodiments, the amount of a given degradant is expressed as a percentage of total species present (i.e. all the degradants and the biological product).

[0173] In some embodiments, the amount of degradant is expressed as the difference between the amount of degradant after exposure to the low pH stress condition and the amount of degradant in the starting material (i.e. before exposure to the low pH stress condition). For example, if the starting material (unstressed) possesses 1 % aggregate and the degraded material possesses 10% aggregate, a change of +9% aggregate is reported.

[0174] In some embodiments, analyzing the degradants may be performed by (i) size exclusion chromatography (SEC), in particular for assessing the presence and / or amount of HMWS, (ii) capillary gel electrophoresis (CGE), capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) or non-reduced CE-SDS (NR CE-SDS), in particular for assessing the presence and / or amount of LMWS, and / or (iii) capillary isoelectric focusing (clEF), Imaged capillary isoelectric focusing (iCIEF) or cation exchange chromatography (CEX), in particular for assessing the presence and / or amount of charge variants.

[0175] In some embodiments, step ao) further comprises assessing the biological activity of the biological product in the low pH stress condition or each low pH stress condition.

[0176] The biological activity depends on the nature of the biological product.

[0177] For example, the biological activity assessed for an antigen-binding protein includes its antigen binding ability and / orthe biological response induced upon binding to the antigen and / or the effector response induced by the antigen-binding protein.

[0178] For example, the biological activity assessed for a receptor includes its ability to bind its ligand and / or the biological response induced upon binding to its ligand.

[0179] For example, the biological activity assessed for a ligand includes its ability to bind its receptor and / or the biological response induced upon binding to its receptor.

[0180] For example, the biological activity assessed for an hormone or cytokine includes the biological response induced by said hormone or cytokine.

[0181] For example, the biological activity assessed for an enzyme includes its ability to bind its substrate and / or its enzymatic activity.

[0182] The biological activity may be assessed by any test well known by the skilled person, such as ELISA, Surface Plasmon Resonance (SPR), enzyme activity or potency bioassay.

[0183] A potency bioassay for example allows determining the ability of a product to induce a response in a relevant test system, such as in a cell assay.

[0184] In some embodiments, the results obtained in step ao) are used to select the values of pH and / or those of at least one of the additional factors used in step a).

[0185] For example, in some embodiments, step a0) further comprises determining if some values or combinations of values of the low pH and / or of the additional factors induce generation of a profile of degradants close to the target profile of degradants and, optionally, are not associated to degradation of the biological activity of the biological product.

[0186] In some embodiments, the target profile of degradants is characterized by the presence and / or amount of specific desired degradants and / or the absence and / or amount of specific unwanted degradants.

[0187] A non-limitative example of target profile is (i) the presence of HMWS and (ii) the absence or the presence in low amount of both LM WS and charge variants. A non-limitative example of target profile is (i) the presence of 20% to 40% of HMWS and (ii) the absence or minimized presence of both LMWS and charge variants. In some embodiments, the target profile as defined above is further characterized by the absence or low amounts of degradants resulting from chemical degradation.

[0188] In some embodiments, the lower duration of exposure leading to a profile of degradants close to the target profile of degradants is used as a value for the duration of exposure in step a).

[0189] In some embodiments, if one additional factor does not significantly change degradants in a specific range of values, this range of values may not be used as in step a). For example, if there is no significant change of degradants between 10 mM and 50 mM of citrate buffer within an evaluation range of 10 to 100 mM, the range of values in step a) can be chosen starting from 50 mM, such as from 50 mM to and 100 mM.

[0190] Step a)

[0191] In some embodiments, step a) comprises selecting a set of different low pH stress conditions, wherein each low pH stress condition is determined by a combination of a low pH and at least two additional factors, the value of the low pH factor being chosen among NO values, the value of the first additional factor being chosen among N1 values, the value of the second additional factor being chosen among N2 values, wherein NO, N1 and N2 are integers greater than or equal to 2.

[0192] In some embodiments, NO, N1 and N2 are independently integers greater than or equal to 2, 3, 4, 5 or 6.

[0193] In other words, the variables in the set of low pH stress conditions are pH and said at least two additional factors. Some other additional factors may be constant, i.e. identical in each low pH stress condition of the set.

[0194] In some embodiments, two, three, four or at least five values may be used for each variable. The number of values may be identical or different between the variables. In some embodiments, three values are used for each variable.

[0195] The low pH stress condition, the low pH and the additional factors are as defined above in the section “Low pH stress condition”.

[0196] In some embodiments, the additional factor is selected from the group consisting of temperature, duration of exposure, concentration of a chemical compound (such as concentration of buffer), initial concentration of the biological protein and ionic strength.

[0197] In some embodiments, the additional factor is not duration of exposure.

[0198] In some embodiments, the additional factors comprise temperature and buffer concentration, such as citrate or acetate buffer concentration. In some embodiments, the variables between each low pH stress condition are the pH, temperature and the buffer concentration.

[0199] In some embodiments, the initial concentration of the biological product and the duration of exposure are identical in each low pH stress condition.

[0200] In some embodiments, the variables between each low pH stress condition are the pH, temperature and the buffer concentration, whereas the initial concentration of the biological product and the duration of exposure are identical in each low pH stress condition.

[0201] In some embodiments wherein the method as defined above comprises step a0), the values of at least one of the additional factors in step a) and / or of the low pH are selected in function of the results obtained in step a0), in particular as disclosed above.

[0202] In some embodiments, low pH is selected in the range from 3 to 4, temperature is selected in the range from 22°C to 35°C and the concentration of citrate buffer is selected in the range of 10 mM to 100 mM, for example in the range of 50 mM to 100 mM.

[0203] In some embodiments, the values for low pH comprise 3.1 , 3.4 and 3.6, the values for the concentration of citrate buffer comprise 50 mM, 75 mM and 100 mM and the values for the temperature comprise 22°C, 25°C and 35°C.

[0204] In some embodiments, the low pH stress conditions of the set selected at step a) have the same duration of exposure and the same initial concentration of the biological product.

[0205] The set of different low pH stress conditions may be a full set or a fractional set.

[0206] In some embodiments, the set of different low pH stress conditions is a full set, meaning that the set comprises every possible combination of values of the low pH and of the additional factors.

[0207] In some embodiments wherein a full set of different low pH stress conditions is selected, step a) comprises No x Ni x . .. x Nj conditions, wherein No is the number of values for the low pH factor, Ni is the number of values for the first additional factor Nj is the number of values for the jthadditional factor, wherein No, Ni, ... and Nj are independently integers greater than or equal to 2 and wherein j is an integer equal to or greater than 2.

[0208] In some embodiments, the set of different low pH stress conditions is a fractional set, meaning that the set does not comprise every possible combination of values of the low pH and of the additional factors. A fractional set advantageously reduces the number l ' l of conditions and thus the number of analysis of the degradants, thereby reducing both time and costs for carrying out the method.

[0209] In some embodiments wherein a fractional set is used, the number of low pH stress conditions of the set is chosen, so as to achieve enough resolution. Suitable softwares such as JMP® may be used as a tool for DoE to determine the minimal number of conditions required to achieve enough resolution,. The minimal number of conditions required to achieve enough resolution may also be determined manually.

[0210] In some embodiments, the fractional set comprises 15 or at least 15 low pH stress conditions, in particular when three variables are used (the variables being pH and two additional factors).

[0211] In some embodiments, a fractional set comprises every combinations of the lower and upper values of the pH and of the lower and upper values of each additional factor.

[0212] In some embodiments, the fractional set comprises: (i) every combinations of the lower and upper values of the pH and of the lower and upper values of each additional factor, (ii) at least combinations of the lower and upper values of pH with a middle value of each additional factor, (iii) at least the combination of a middle value of the pH and of a middle value of each additional factor, (iv) at least the combinations of a middle value of pH, the lower and upper values of a first additional factor and a middle value of a second additional factor and (v) at least the combinations of a middle value of pH, a middle value of said first additional factor and the lower and upper value of said second additional factor. In some embodiments wherein three variables and three values for each variable are used, this gives a fractional set of 15 conditions, as for example disclosed in Table 1 of the Example.

[0213] By “middle value”, it is herein meant any value between the lower and upper value for a given additional factor or for the pH.

[0214] For example, the lower value of pH is 3.1 , a middle value of pH is 3.4 and the upper value of pH is 3.6; the lower value of buffer concentration is 50 mM, a middle value of buffer concentration is 75 mM and the upper value of buffer concentration is 100 mM; the lower value of temperature is 22°C, a middle value of temperature is 25°C and the upper value of temperature is 35°C.

[0215] Step b)

[0216] In some embodiments, step b) comprises exposing said biological product to each low pH stress condition of the set selected in step a), to obtain a mixture comprising the biological product and degradants for each low pH stress condition.

[0217] In step b), each low stress condition results in the generation of degradants. The degradants are as defined above in the section “Degradants from a biological product". In some embodiments, the degradant is selected from the group consisting of low molecular weight species (LMWS), high molecular weight species (HMWS) and charge variants. In some embodiments, the degradants comprises low molecular weight species (LMWS), high molecular weight species (HMWS) and charge variants.

[0218] In some embodiments, the degradants are the same as in step a0).

[0219] In some embodiments, the low pH stress condition characterized by combination of a middle value of the pH and of a middle value of each additional factor is assessed in duplicate in step b).

[0220] Replicates at the center point of a Design of Experiments (DOE) indeed help detecting if the relationship between inputs and outputs is linear or exhibits curvature and provide a cost-effective way to increase the number of data points, enhancing the experiment’s power to detect significant inputs. Center points allow for the estimation of inherent variability in the system by observing the variation in outcomes under the same settings. The presence of curvature often indicates optimal settings, making center points valuable for insightful results.

[0221] Step c)

[0222] In some embodiments, step c) comprises analyzing the degradants obtained in step b) in each low pH stress condition, to obtain experimental data.

[0223] In some embodiments, analyzing the degradants comprises assessing the amount and / or nature of the degradants.

[0224] The degradants are as defined above in the section “Degradants from a biological product’.

[0225] The amount and nature of the degradants are as defined above in optional step ao).

[0226] In some embodiments, analyzing the degradants comprises assessing the presence and / or amount of at least one degradant selected from the group consisting of low molecular weight species (LMWS), high molecular weight species (HMWS) and charge variants.

[0227] In some embodiments, analyzing the degradants comprises assessing the amount and / or presence of LMWS, HMWS and charge variants.

[0228] The nature the amount and / or nature of the degradants may be assessed as defined above in the section “Degradants from a biological product.

[0229] In some embodiments, analyzing the degradants may be performed by (i) size exclusion chromatography (SEC), in particular for assessing the presence and / or amount of HMWS, (ii) capillary gel electrophoresis (CGE), capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) or non-reduced CE-SDS (NR CE-SDS), in particular for assessing the presence and / or amount of LMWS, and / or (iii) capillary isoelectric focusing (clEF), Imaged capillary isoelectric focusing (iCIEF) or cation exchange chromatography (CEX), in particular for assessing the presence and / or amount of charge variants.

[0230] In some embodiments, analyzing the degradants is as performed in step ao).

[0231] In some embodiments, step c) further comprises assessing the biological activity of the biological product in each low pH stress condition.

[0232] Assessing the biological activity of the biological product is as defined above in step a0).

[0233] For example, the biological activity assessed for an antigen-binding protein includes its antigen binding ability and / or the biological response induced upon binding to the antigen.

[0234] For example, the biological activity assessed for a receptor includes its ability to bind its ligand and / or the biological response induced upon binding to its ligand.

[0235] For example, the biological activity assessed for a ligand includes its ability to bind its receptor and / or the biological response induced upon binding to its receptor.

[0236] For example, the biological activity assessed for an hormone or cytokine includes the biological response induced by said hormone or cytokine.

[0237] For example, the biological activity assessed for an enzyme includes its ability to bind its substrate and / or its enzymatic activity.

[0238] The biological activity may be assessed by any test well known by the skilled person, such as ELISA, Surface Plasmon Resonance (SPR) or potency bioassay.

[0239] A potency bioassay for example allows determining the ability of a product to induce a response in a relevant test system, such as in a cell assay.

[0240] In some embodiments, the experimental data includes the nature and / amount of each degradant and, optionally the biological activity of the biological product, in function of the value of the low pH and of the values of each additional factor, for each low pH stress condition.

[0241] Step d)

[0242] In some embodiments, step d) comprises processing the experimental data obtained in step c), to obtain a predictive model of the degradants generated in function of the low pH and of the at least two additional factors.

[0243] In some embodiments, processing the experimental data obtained in step c) comprises performing statistical analysis of the experimental data, to obtain said predictive model of the degradants generated in function of the low pH and of the at least two additional factors.

[0244] The statistical analysis allows assessing effects of the low pH and of the additional factors, within the assessed ranges, on the generation of degradants (in particular nature of the degradant(s) and, optionally, amount of degradant(s)) and, optionally, on the biological activity of the biological product, to fit a model to the experimental data.

[0245] This model is a predictive model, since it allows predicting future outcomes.

[0246] The statistical analysis used in step d) as defined above may be performed using any relevant means, as a tool for DoE, such as, but not limited to, computer software, such as Microsoft Excel (Microsoft), JMP® (SAS), GraphPad Prism (GraphPad Software), MATLAB (MathWorks®), Custom Sensors PX2 (Custom Sensors), and graphing paper (manual drafting).

[0247] The statistical analysis used in step d) as defined above may be performed using multiple linear and nonlinear regressions, to fit a model to the experimental data.

[0248] In some embodiments, processing the experimental data obtained in step c) as defined above comprises performing statistical analysis of the experimental data, to obtain a predictive model of the degradants and, optionally of the biological activity of the product, generated in function of the low pH, the concentration of citrate and the temperature.

[0249] Step e)

[0250] Step e) is optional. In some embodiments, the method as defined above comprises a further step e).

[0251] In some embodiments, step e) comprises determining from said predictive model, (i) a combination of values or ranges of values of low pH and of the at least two additional factors to be used to generate desired degradants from said biological product exposed to a low pH stress or (ii) the nature and / or amount of degradants that will be generated when exposing the biological product to a combination of values or ranges of values of low pH and of the at least two additional factors.

[0252] In some embodiments, "generate desired degradants” means to favor the generation of the desired degradants and / or minimizing the generation of unwanted degradants.

[0253] In some embodiments, “generate desired degradants” means mimicking degradants obtained in lifecycle of a product, for example to set up a forced degradation study.

[0254] In some embodiments, step e) comprises determining from said predictive model, a combination of values or ranges of values of low pH and of the at least two additional factors to be used to induce aggregation with little or no fragmentation and little or no charge variant formation, in particular when the biological product is an antigen-binding protein, such as an antibody or antigen-binding fragment thereof as defined above.

[0255] In some embodiments, step e) comprises determining from said predictive model, a combination of values or ranges of values of low pH and of the at least two additional factors to be used to generate targeted amounts of HMWS and minimized amounts of LMWS and charge variants, in particular when the biological product is an antigen-binding protein, such as an antibody or antigen-binding fragment thereof as defined above.

[0256] In some embodiments, step e) comprises determining from said predictive model, a combination of values or ranges of values of low pH and of the at least two additional factors to be used to generate 20% to 40% of HMWS and minimized amounts of LMWS and charge variants, in particular when the biological product is an antigen-binding protein, such as an antibody or antigen-binding fragment thereof as defined above.

[0257] In some embodiments, step e) is for analysis studies, such as forced degradation study, or for improving the process of production of the biological product, in particular the viral inactivation step of the purification of a biological product.

[0258] In some embodiments, step e) allows determining the optimal parameters of the viral inactivation step for the purification of a biological product.

[0259] Step f

[0260] Step f) is optional. In some embodiments, the method as defined above comprises step f) after step e).

[0261] In some embodiments, step f) comprises: exposing the biological product to a low pH stress condition characterized by a combination of values determined in step e) to favor generation of at least one desired degradant and / or minimize generation of at least one unwanted degradant, or exposing the biological product to a low pH stress condition characterized by a combination of values determined in step e) to conduct a forced degradation study of the biological product.

[0262] In some aspects of the present disclosure is provided a method of antibody degradation, wherein said method comprises the steps of the method for analyzing degradation of a biological product as defined above, wherein the biological product is an antibody, and, optionally, a step of exposing the antibody to a low pH stress condition characterized by a combination of values determined in step e) to favor generation of at least one desired degradant and / or minimize generation of at least one unwanted degradant,

[0263] In some aspects of the present disclosure is provided a method for studying forced degradation, wherein said method comprises the steps of the method for analyzing degradation of a biological product as defined above and, optionally, a step of exposing the biological product to a low pH stress condition characterized by a combination of values determined in step e).

[0264] In some aspects of the present disclosure is provided a method for monitoring degradation of a biological product exposed to a low pH stress, wherein said method comprises the steps of the method for analyzing degradation of a biological product as defined above and, optionally, a step of exposing the antibody to a low pH stress condition characterized by a combination of values determined in step e) to favor generation of at least one desired degradant and / or minimize generation of at least one unwanted degradant.

[0265] In some aspects of the present disclosure is provided a method for identifying product critical quality attributes (CQAs) of an antibody, wherein said method comprises the steps of the method for analyzing degradation of a biological product as defined above and, optionally, identifying CQAs of the antibody, such as as referred to in ICH Guidelines of Pharmaceutical Development Q8(R2), in particular so that those product characteristics having an impact on product quality can be studied and controlled.

[0266] Use of the predictive model obtained by the method for targeted generation of degradants from a biological product exposed to a low pH stress

[0267] In some aspects is provided the use of the predictive model obtained in step d) of the method for target generation of degradants from a biological product exposed to a low pH stress, said method being a defined above, such as to select a combination of values or ranges of values of low pH and of at least two additional factors to be used to generate desired degradants from a biological product exposed to a low pH stress.

[0268] In some aspects is provided the use of the predictive model obtained in step d) of the method for target generation of degradants from a biological product exposed to a low pH stress, said method being a defined above, such as to determine a low pH stress condition able to favor generation of at least one desired degradant and / or minimize generation of at least one unwanted degradant from a biological product, or to conduct a forced degradation study of a biological product.

[0269] Figures Figure 1 : Preliminary step: conditions A and B (up: factors; middle: results as regards to HMWS, LMWS and charge variants; bottom: relative potency of the monoclonal antibody).

[0270] Figure 2: Scatterplot matrix of the study design

[0271] Figure 3: Aggregation. Surface response plots for HMWS determined by SEC in function of the temperature and pH at a low or high concentration of citrate (up) ad in function the concentration of citrate and pH at low or high temperature.

[0272] Figure 4: Fragmentation. Surface response plots for LMWS determined by NR CE- SDS in function of the temperature and pH at a low or high concentration of citrate (up) ad in function the concentration of citrate and pH at low or high temperature.

[0273] Figure 5: Charge variant formation. Surface response plots for main charge variants determined by iCIEF in function of the temperature and pH.

[0274] Figure 6: Predictive model. A. Percentage of HMWS; B: Percentage of LMWS; C: Percentage of main charge variant; D: desirability.

[0275] EXAMPLES

[0276] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to use the method featured in the invention and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

[0277] Aim of the low pH stress study

[0278] The biological product was an lgG4 antibody. The aim was to determine low pH stress conditions allowing generating HMWS as the main degradant, preferably in the range 20% to 40% (the percentage being expressed with respect to the total content of antibody) with no or little LMWS and charge variants.

[0279] As a preliminary step, degradation behaviors observed using two low distinct pH stress conditions were analyzed. The first low pH stress condition A was as follows: 10 mM citrate, pH 3.6, 35°C, up to 13 days (see Figure 1, top). The second low pH stress condition B was as follows: 100 mM citrate, pH 3.5, 22°C, up to 7 hours.

[0280] In the first condition, 13 days were required to generate at least 30% HMWS. Concomitant generation of chemical and physical degradants (both HMWS and LMWS) was observed. Generation of multiple types of degradants can complicate the interpretation of effects of physical and / or chemical degradation on potency.

[0281] In the second condition, only physical degradation (HMWS formation, no LMWS) was observed as the major degradation pathway with little / no chemical modifications.

[0282] The second condition was a quick degradation route, as more than 30% HMWS were generated after only 7 hours of stress. Besides, no loss in potency was observed in this condition.

[0283] The second low pH stress condition was a good starting point for the DoE approach.

[0284] Design of Experiment

[0285] The effects of citrate buffer concentration, pH and incubation temperature on the level and nature of degradants generated was studied via design of experiment (DoE) approach.

[0286] Size-based purity (using SEC for HMWS, i.e. aggregates, and NR CE-SDS for LMWS, i.e. fragmentation) and charge-based purity (using iCIEF for charge variants) were analyzed after exposure to various low pH stress conditions. Degradant levels were compared to levels present in the starting material (unstressed antibody).

[0287] Based on the preliminary step, the ranges of values for the different factors were as follows:

[0288] - pH (3.1 - 3.6),

[0289] - [Citrate] (50 - 100 mM),

[0290] Temperature (22°C - 35°C),

[0291] Incubation Time: 7 hours (constant) and

[0292] - Protein Concentration: 5 mg / mL (constant).

[0293] JMP® was used to design an experiment to assess effects of these factors, within the specified ranges, on degradant generation. The fractional set of 15 unique low pH stress conditions is shown in Table 1 below.

[0294] Table 1 : DoE design

[0295] Results

[0296] Based on these experimental data, a predictive model was obtained mapping out response surface(s) (using 3-dimensional surface plots) of degradant generation via modulation of the concentration of citrate, pH and incubation temperature.

[0297] As shown in Figure 3, increases in temperature and decreases in pH are associated with a rise in the percentage of HMWS. These two variables interactively affect the percentage of HMWS, such that changes in one variable change the effect of the other. Increases in the concentration of citrate buffer lead to only marginal increases in the percentage of HMWS.

[0298] As shown in Figure 4, increases in temperature and decreases in pH correspond to an increase in the percentage of LMWS. These two variables exhibit an interactive influence on the percentage of LMWS. Increases in the concentration of citrate buffer lead to only marginal increases in the percentage of LMWs.

[0299] As shown in figure 5, decreases in pH and increases in temperature lead to decreased percentage of main charge variants, indicating formation of charge variant species. Both variables do not show interaction with each other.

[0300] A predictive model fitting the experimental data was obtained (see Figure 6). The predictive model can help identify recommended conditions for targeted generation of low pH-induced mAb degradants. For example, recommended low pH stress conditions to generate 20% to 40% of HMWS (in 7 hours) for the antibody are a concentration of citrate buffer of around 100 mM, a pH of 3.55 and an incubation temperature of 22°C.

[0301] Conclusion

[0302] Distinct low pH stress conditions resulted in different degradation behaviors for the mAb. Selective generation of HMWS (aggregates) is possible via guided modulation of low pH stress conditions, as shown by the obtained predictive model.

[0303] This low pH DoE study has shown that: increased concentrations of citrate buffer result in modest increases in HMWS and LMWS, with no effect on main charge variant, within the range studied, and within the ranges studied, pH and temperature have more profound effects on the levels of HMWS, LMWS and main charge variant: o higher temperatures result in increased HMWS and LMWS with significant losses in main charge variants, o lower pH results in substantial increases in HMWS and LMWS with significant losses in main charge variants, and o pH and temperature displayed interaction with each other for HMWS and LMWS: the modulation of one variable impacts the effect on the response of the other.

[0304] Finally, selective generation of mAb degradants is possible via guided modulation of low pH stress conditions: the predictive model allows selectively and quickly inducing aggregation for an lgG4 with little fragmentation and charge variant formation. This can aid in giving a better understanding of the effect of aggregates on function of the IgG therapeutics.

Claims

CLAIMS1. A method for analyzing degradation of a biological product , wherein said method comprises : a) selecting a set of different low pH stress conditions, wherein each stress condition is determined by a combination of a low pH and at least two additional factors, the value of the low pH factor being chosen among NO values, the value of the first additional factor being chosen among N1 values, the value of the second additional factor being chosen among N2 values, wherein NO, N1 and N2 are integers greater than or equal to 2, b) exposing said biological product to each low pH stress condition of the set selected in step a), to obtain a mixture comprising the biological product and degradants for each low pH stress condition, c) analyzing the degradants obtained in step b) in each low pH stress condition, to obtain data, d) processing the data obtained in step c), to obtain a predictive model of the degradants generated in function of the low pH and of the at least two additional factors, and e) optionally, determining from said predictive model, (i) a combination of values or ranges of values of low pH and of the at least two additional factors to be used to generate desired degradants from said biological product exposed to a low pH stress or (ii) the nature and / or amount of degradants that will be generated when exposing the biological product to a combination of values or ranges of values of low pH and of the at least two additional factors.

2. The method according to claim 1 , wherein said method is for targeted generation of degradants from a biological product exposed to a low pH stress or is for monitoring degradation of a biological product exposed to a low pH stress.

3. The method according to claim 1 or 2, comprising step f) after step e), wherein step f) comprises: exposing the biological product to a low pH stress condition characterized by a combination of values determined in step e) to favor generation of at least one desired degradant and / or minimize generation of at least one unwanted degradant, orexposing the biological product to a low pH stress condition characterized by a combination of values determined in step e) to conduct a forced degradation study of a biological product.

4. The method according to any one of claim 1 to 3, wherein the additional factor is selected from the group consisting of temperature, duration of exposure, concentration of a chemical compound, initial concentration of the biological product and ionic strength.

5. The method according to any one of claims 1 to 4, wherein, in step d), processing the data obtained in step c) comprises performing statistical analysis of the data to obtain said predictive model.

6. The method according to any one of claims 1 to 5, wherein said method comprises step a0) before step a), wherein step a0) comprises (i) exposing said biological product to at least one low pH stress condition, wherein said stress condition is characterized by a low pH and at least two additional factors and (ii) analyzing the degradants obtained in said low pH stress condition.

7. The method according to claim 6, wherein step a0) comprises exposing said biological product to at least two low pH stress conditions, wherein each stress condition is characterized by a low pH and at least two additional factors and wherein the low pH stress conditions differ from each other by opposed values for at least one of the additional factors and (ii) analyzing the degradants obtained in each low pH stress condition.

8. The method according to claim 6 or 7, wherein the values of at least one of the additional factors in step a) and / or of the low pH are selected in function of the results obtained in step ao).

9. The method according to any one of claims 1 to 8, wherein analyzing the degradants comprises assessing the amount and / or the nature of at least one degradant.

10. The method according to anyone of claims 1 to 9, wherein step c) further comprises assessing the biological activity of the biological product in each low pH stress condition.

11. The method according to anyone of claims 1 to 10, wherein the biological product is a protein.

12. The method according to claim 11 , wherein the biological product is an antibody or an antigen-binding fragment thereof.

13. The method according to claim 12, wherein the biological product is an IgG antibody, such as an lgG1 , lgG2, lgG3 or lgG4 antibody.

14. The method according to claim 12 or 13, wherein analyzing the degradants comprises assessing the presence and / or amount of at least one degradant selected from the group consisting of low molecular weight species (LMWS), high molecular weight species (HMWS) and charge variants.

15. The method according to any one of claims 12 to 14, wherein the additional factors in step a0) and / or in step a) comprise temperature and concentration of citrate.

16. The method according to any one of claims 1 to 15, wherein in step a), low pH is selected in the range from 3 to 4, temperature is selected in the range from 22°C to 35°C and the concentration of citrate is selected in the range of 10 mM to 100 mM, for example in the range of 50 mM to 100 mM.

17. The method according to claim 16, wherein the values for low pH comprise 3.1 , 3.4 and 3.6, the values for the concentration of citrate comprise 50 mM, 75 mM and 100 mM and the values for the temperature comprise 22°C, 25°C and 35°C.

18. The method according to any one of claims 1 to 17, wherein the low pH stress conditions of the set selected at step a) have the same duration of exposure and the same initial concentration of the biological product.

19. Use of the predictive model obtained in step d) of the method according to any one of claims 1 to 18, to select a combination of values or ranges of values of low pH and of at least two additional factors to be used to generate desired degradants from a biological product exposed to a low pH stress.

20. Use of the predictive model obtained in step d) of the method according to any one of claims 1 to 18, to determine a low pH stress condition able to favor generation of at least one desired degradant and / or minimize generation of at least one unwanted degradant from a biological product, or to conduct a forced degradation study of a biological product.

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