Disulfide isoform modulation

Abstract

This invention relates to a method for modulation of disulfide structural isoform distribution in a recombinant protein. The invention also relates to a method of stabilizing disulfide structural isoform distribution by preventing disulfide bond rearrangement in a recombinant protein, methods for the production of a recombinant protein with modulated disulfide isoforms, and a method for altering disulphide structural isoform distribution by enhancing disulfide bond rearrangement in a recombinant protein. The invention also relates to a use of a reducing agent for modulation of disulfide structural isoform distribution by enhancing disulfide bond rearrangement in a recombinant protein, and to a use of an oxidising agent for stabilizing disulfide structural isoform distribution by preventing disulfide bond rearrangement in a recombinant protein.

Description

Docket No. P38734-WO DISULFIDE ISOFORM MODULATION

[0001] This invention relates to a method for modulation of disulfide structural isoform distribution in a recombinant protein. BACKGROUND

[0002] Disulfide bonds are post-translational modifications formed by the oxidation of two thiol (i.e., sulfhydryl) groups within cysteine residues of proteins. In IgG antibodies, disulfide bond linkages are vital for proper folding, stability and biological activity. All IgG antibodies are composed of two light chain polypeptides and two heavy chain polypeptides connected in a “Y” configuration by inter-chain disulfide bonds.

[0003] IgG antibodies in human serum exist as four different subclasses—IgG1, IgG2, IgG3, and IgG4. Of the four IgG subclasses, the IgG2 subclass bears the least affinity for Fc receptors and is therefore a framework option for therapeutic antibodies that do not need effector function (Isaacs et al., J Immunol., 1998, 161, 8, 3862–3869).

[0004] It is an aim of the present invention to provide a biotherapeutic with improved and / or more consistent activity by controlling IgG2 isoform distribution. BRIEF SUMMARY OF THE DISCLOSURE

[0005] The invention provides methodology for modulating the levels of the different disulfide isoforms in recombinant proteins.

[0006] In accordance with a first aspect of the present invention, there is provided a method for modulation of disulfide structural isoforms in a recombinant protein, comprising: contacting the recombinant protein with a solution comprising a reducing agent.

[0007] Without wishing to be bound by theory, it is thought that treating the recombinant protein with a reducing agent promotes isoform rearrangement from a first isoform of the recombinant protein to a second isoform of the recombinant protein. The resulting recombinant protein is therefore enriched with the second type of isoform over the first type of isoform, thereby optimising the biological activity of the recombinant protein in line with the biological activity of the second isoform.Docket No. P38734-WO

[0008] In accordance with a second aspect of the present invention, there is provided a method for preventing disulfide bond rearrangement in a recombinant protein, comprising: contacting the recombinant protein with a solution comprising an oxidising agent.

[0009] Without wishing to be bound by theory, it is thought that treating the recombinant protein with an oxidising agent decreases disulfide isoform rearrangement, meaning that the amounts of each isoform are maintained in the resulting recombinant protein.

[0010] In accordance with a third aspect of the present invention, there is provided a method for the production of a recombinant protein with modulated disulfide structural isoforms, comprising: (a) culturing a eukaryotic cell line that comprises a polynucleotide encoding the recombinant polypeptide under conditions suitable for production of the polypeptide; and (b) contacting the recombinant protein with a solution comprising a reducing agent.

[0011] In accordance with a fourth aspect of the present invention, there is provided a method for the production of a recombinant protein with modulated disulfide structural isoforms, comprising: (a) culturing a eukaryotic cell line that comprises a polynucleotide encoding the recombinant polypeptide under conditions suitable for production of the polypeptide; and (b) contacting the recombinant protein with a solution comprising an oxidising agent.

[0012] In accordance with a fifth aspect of the present invention, there is provided a method for the production of a recombinant protein that is an IgG2 having an increased amount of IgG2-B structural isoform, the method comprising: (a) culturing a eukaryotic cell line that comprises a polynucleotide encoding the recombinant polypeptide under conditions suitable for production of the polypeptide; and (b) contacting the recombinant protein with a solution comprising a reducing agent.

[0013] In accordance with a sixth aspect of the present invention, there is provided a method for the production of a recombinant protein that is an IgG2 having an increased amount of IgG2-A structural isoform, the method comprising: (a) culturing a eukaryotic cell line that comprises a polynucleotide encoding the recombinant polypeptide under conditions suitable for production of the polypeptide; and (b) contacting the recombinant protein with a solution comprising an oxidising agent.Docket No. P38734-WO

[0014] In accordance with a seventh aspect of the present invention, there is provided a recombinant protein with modulated disulfide structural isoforms obtainable or obtained by the methods of the invention.

[0015] In accordance with an eighth aspect of the invention, there is provided a method for modulating disulfide structural isoform distribution by enhancing disulfide bond rearrangement in a recombinant protein, comprising: contacting the recombinant protein with a solution comprising a reducing agent.

[0016] In accordance with a ninth aspect of the invention, there is provided a use of a reducing agent for modulation of disulfide structural isoform distribution by enhancing disulfide bond rearrangement in a recombinant protein.

[0017] In accordance with a tenth aspect of the invention, there is provided a use of an oxidising agent for stabilizing disulfide structural isoform distribution by preventing disulfide bond rearrangement in a recombinant protein.

[0018] In accordance with an eleventh aspect of the invention, there is provided a recombinant protein obtained or obtainable by any of the method of the eighth aspect, the use of ninth, or the use of the tenth aspect. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which: Figure 1 shows a schematic of three structural IgG2 isoforms—IgG2-A, IgG2- A / B, and IgG2-B—and their interconversions. Figure 2 shows the effect of media additives on mAb X disulfide isoform distribution in cell-free supernatant. Figure 3 shows the effect of L-cysteine and L-glutathione concentration on mAb X disulfide isoform distribution in bioreactor production cultures. Figure 4 shows the effect of L-cysteine, temperature and presence of cells on mAb Y disulfide isoform distribution over incubation time. Figure 5 shows the effect of L-cysteine concentration on mAb Y disulfide isoform distribution.Docket No. P38734-WO Figure 6 shows effect of L-glutathione concentration on extent of mAb Y disulfide isoform distribution. Figure 7 shows the interaction of metal supplements with L- cysteine or L- glutathione on mAb X disulfide isoform distribution. Figure 8 shows the effect of copper and L-cysteine supplementation on mAb Y disulfide isoform distribution. Figure 9 shows the effect of different L-cysteine supplemental strategies during production culture on mAb X disulfide isoform distribution and production cell culture performance. DETAILED DESCRIPTION

[0020] The abbreviations used herein have their conventional meaning within the chemical and biological arts.

[0021] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0022] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract andDocket No. P38734-WO drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0023] The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

[0024] For the avoidance of doubt, it is hereby stated that the information disclosed earlier in this specification under the heading “Background” is relevant to the invention and is to be read as part of the disclosure of the invention.

[0025] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. DEFINITIONS

[0026] The terms used in this specification generally have their ordinary meanings in the art, within the context of this disclosure and in the specific context where each term is used. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the compositions and methods of the present disclosure and how to make and use them.

[0027] As used herein, the use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification can mean “one,” but it is also consistent with the meaning of “one or more,” “at least one” and “one or more than one.”

[0028] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s)” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms or words that do not preclude the possibility of additional acts or structures. The present disclosure also contemplates other embodiments “comprising,” “consisting of” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0029] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurementDocket No. P38734-WO system. For example, “about” can mean within 3 or more than 3 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value.

[0030] The terms “cell culture medium” and “culture medium” refer to a nutrient solution used for growing mammalian cells that typically provides at least one component from one or more of the following categories: 1) an energy source, usually in the form of a carbohydrate such as glucose; 2) all essential amino acids, and usually the basic set of twenty amino acids plus cysteine; 3) vitamins and / or other organic compounds required at low concentrations; 4) free fatty acids; and 5) trace elements, where trace elements are defined as inorganic compounds or naturally occurring elements that are typically required at very low concentrations, usually in the micromolar range.

[0031] The nutrient solution can optionally be supplemented with one or more components from any of the following categories: 1) hormones and other growth factors as, for example, insulin, transferrin, and epidermal growth factor; 2) salts and buffers as, for example, calcium, magnesium, and phosphate; 3) nucleosides and bases such as, for example, adenosine, thymidine, and hypoxanthine; and 4) protein and tissue hydrolysates.

[0032] “Culturing” a cell refers to contacting a cell with a cell culture medium under conditions suitable to the survival and / or growth and / or proliferation of the cell.

[0033] “Batch culture” refers to a culture in which all components for cell culturing (including the cells and all culture nutrients) are supplied to the culturing bioreactor at the start of the culturing process.Docket No. P38734-WO

[0034] “Fed-batch cell culture,” as used herein refers to a batch culture wherein the cells and culture medium are supplied to the culturing bioreactor initially, and additional culture nutrients are fed, continuously or in discrete increments, to the culture during the culturing process, with or without periodic cell and / or product harvest before termination of culture.

[0035] “Perfusion culture,” sometimes referred to as continuous culture, is a culture by which the cells are restrained in the culture by, e.g., filtration, encapsulation, anchoring to microcarriers, etc., and the culture medium is continuously, step-wise or intermittently introduced (or any combination of these) and removed from the culturing bioreactor.

[0036] As used herein, the term “cell,” refers to animal cells (e.g. mammalian cells), fungal cells (e.g. yeast cells), cultured cells, host cells, recombinant cells and recombinant host cells. Such cells are generally cell lines obtained or derived from mammalian tissues or fungi which are able to grow and survive when placed in media containing appropriate nutrients and / or growth factors.

[0037] The term “cell line” as used herein includes reference to a culture of eukaryotic cells that can be propagated repeatedly. The eukaryotic cells of the cell line may be selected from any cell as defined herein.

[0038] The terms “host cell,” “host cell line” and “host cell culture” are used interchangeably and refer to cells and their progeny into which exogenous nucleic acid can be subsequently introduced to create recombinant cells. These host cells may also have been modified (i.e., engineered) to alter or delete the expression of certain endogenous host cell products (e.g., endogenous virus-like particles or endogenous host cell proteins). Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny does not need to be completely identical in nucleic acid content to a parent cell, but can contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein. The introduction of exogenous nucleic acid (e.g., by transfection) to these host cells would create recombinant cells that are derived from the original “host cell,” “host cell line” or “host cell line”. The terms “host cell,” “host cell line” and “host cell culture” may also refer to such recombinant cells and their progeny. The terms “recombinant cell”, “recombinant cell line” and “recombinant cell culture” are used interchangeably and referDocket No. P38734-WO to cells and their progeny into which exogenous nucleic acid has been introduced to enable the expression of recombinant product of interest. The recombinant product expressed by such cells may be a recombinant protein, a recombinant viral particle, or a recombinant viral vector. The term “mammalian host cell” or “mammalian cell” refers to cell lines derived from mammals that are capable of growth and survival when placed in either monolayer culture or in suspension culture in a medium containing the appropriate nutrients and growth factors. The necessary growth factors for a particular cell line are readily determined empirically without undue experimentation, as described for example in Mammalian Cell Culture (Mather, J. P. ed., Plenum Press, N.Y.1984), and Barnes and Sato, (1980) Cell, 22:649. Typically, the cells are capable of expressing and secreting large quantities of a particular protein, e.g., glycoprotein, of interest into the culture medium. Examples of suitable mammalian host cells within the context of the present disclosure can include Chinese hamster ovary cells / -DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:42161980); dp12.CHO cells (EP 307,247 published 15 Mar.1989); CHO-K1 (ATCC, CCL-61); baby hamster kidney cells (BHK, ATCC CCL 10); mouse sertoli cells (TM4, Mather, Biol. Reprod., 23:243-2511980); canine kidney cells (MDCK, ATCC CCL 34); HEK 293 cells; buffalo rat liver cells (BRL 3A, ATCC CRL 1442); mouse mammary tumor (MMT 060562, ATCC CCL51). In certain embodiments, the mammalian cells include Chinese hamster ovary cells (CHO). In certain embodiments, the cells comprise a polynucleotide that encodes a polypeptide. In another embodiment, the cell expresses the polypeptide transiently or expresses the polypeptide stably. In a further embodiment, the cells expressing the polypeptide stably comprises a polynucleotide that is integrated in the cellular genome of the cell at a targeted location. In another further embodiment, the cells expressing the polypeptide stably comprises a polynucleotide that is integrated in the cellular genome of the cell at a random location.

[0039] The term “activity” as used herein with respect to activity of a protein refers to any activity of a protein including, but not limited to, enzymatic activity, ligand binding, drug transport, ion transport, protein localization, receptor binding, and / or structural activity. Such activity can be modulated, e.g., reduced or eliminated, by reducing or eliminating the expression of the protein, thereby reducing or eliminating the presence of the protein. Such activity can also be modulated, e.g., reduced or eliminated, by alteringDocket No. P38734-WO the nucleic acid sequence encoding the protein such that the resulting modified protein exhibits reduced or eliminated activity relative to a wild type protein.

[0040] The term “expression” or “expresses” are used herein to refer to transcription and translation occurring within a host cell. The level of expression of a product gene in a host cell can be determined on the basis of either the amount of corresponding mRNA that is present in the cell or the amount of the protein encoded by the product gene that is produced by the cell. For example, mRNA transcribed from a product gene is desirably quantitated by northern hybridization. Sambrook et al., Molecular Cloning: A Laboratory Manual, pp.7.3-7.57 (Cold Spring Harbor Laboratory Press, 1989). Protein encoded by a product gene can be quantitated either by assaying for the biological activity of the protein or by employing assays that are independent of such activity, such as western blotting or radioimmunoassay using antibodies that are capable of reacting with the protein. Sambrook et al., Molecular Cloning: A Laboratory Manual, pp.18.1-18.88 (Cold Spring Harbor Laboratory Press, 1989). When reference is made to reduction and / or elimination of the expression of one or more endogenous products relative to the expression of the endogenous product(s) in an unmodified cell, such reductions and / or eliminations of expression encompass reductions and / or eliminations of the active endogenous product, notwithstanding the presence of mRNA encoding all or a portion of the endogenous product or the presence of endogenous product translated from such mRNA.

[0041] As used herein, “polypeptide” refers generally to peptides and proteins having more than about ten amino acids. The polypeptides can be homologous to the host cell, or preferably, can be exogenous, meaning that they are heterologous, i.e., foreign, to the host cell being utilized, such as a human protein produced by a Chinese hamster ovary cell, or a yeast polypeptide produced by a mammalian cell. In certain embodiments, mammalian polypeptides (polypeptides that were originally derived from a mammalian organism) are used, more preferably those which are directly secreted into the medium.

[0042] The term “protein” is meant to refer to a sequence of amino acids for which the chain length is sufficient to produce the higher levels of tertiary and / or quaternary structure. This is to distinguish from “peptides” or other small molecular weight drugs that do not have such structure. Typically, the protein herein will have a molecular weight of at least about 15-20 kD, preferably at least about 20 kD. Examples of proteins encompassedDocket No. P38734-WO within the definition herein include host cell proteins as well as all mammalian proteins, in particular, therapeutic and diagnostic proteins, such as therapeutic and diagnostic antibodies, and, in general proteins that contain one or more disulfide bonds, including multi-chain polypeptides comprising one or more inter- and / or intrachain disulfide bonds.

[0043] The term “glycoprotein” refers to a protein which contains an oligosaccharide chain covalently attached to amino acid side-chains. The oligosaccharide(s) may be attached to the protein in a co-translational or post-translational modification, during a process known as glycosylation. Exemplary glycoproteins include antibodies, which typically have an N-linked oligosaccharide on each heavy chain.

[0044] The term “antibody” is used herein in the broadest sense and encompasses various antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies (e.g., antibodies consisting of a single heavy chain sequence and a single light chain sequence, including multimers of such pairings), multispecific antibodies (e.g., bispecific antibodies) and antibody fragments so long as they exhibit the desired antigen-binding activity. A therapeutic antibody is an antibody that may be used in the treatment of a disease.

[0045] An “antibody fragment,” “antigen-binding portion” of an antibody (or simply “antibody portion”) or “antigen-binding fragment” of an antibody, as used herein, refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab’, Fab’-SH, F(ab’)2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv, and scFab); single domain antibodies (dAbs); and multispecific antibodies formed from antibody fragments. For a review of certain antibody fragments, see Holliger and Hudson, Nature Biotechnology 23:1126-1136 (2005).

[0046] The term “chimeric” antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0047] The “class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG and IgM, and several of these can be further divided into subclasses (isotypes),Docket No. P38734-WO e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In certain embodiments, the antibody is of the IgG2 isotype. In certain embodiments, the antibody is of the IgG4 isotype. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called , , , and , respectively. The light chain of an antibody can be assigned to oneof two types, called kappa ( ) and lambda ( ), based on the amino acid sequence of itsconstant domain.

[0048] The “disulfide isoform” or “disulfide structural isoform” of an antibody refer to specific structural isomers of an antibody based on the configuration of the disulfide bonds. For example, as illustrated in Figure 1, an IgG2 may be in one of three disulfide structural isoforms. In the IgG2-A isoform—which represents the IgG2 canonical structure—the classical IgG2 disulfide bond linkage is present between the cysteine residue on the Fab arm of the heavy chain and the c-terminal cysteine of the corresponding light chain, in combination with four symmetrical disulfide bond linkages between the cysteines in the hinge region of the heavy chains. In the IgG2-B isoform, the Fab arm of each heavy chain is linked to the hinge region of the opposite heavy chain, while the c- terminal cysteine of each light chain is linked to the hinge region of its corresponding heavy chain such that only two disulfide bond linkages exist between the heavy chains via cysteines in the hinge region. In the IgG2-A / B isoform, only one Fab arm of a heavy chain and its corresponding light chain are linked by this classical disulfide bond, whereas the other Fab arm (of the other heavy chain) is linked to the hinge region of its opposite heavy chain, while the c-terminal cysteine of the other light chain is linked to the hinge region of its corresponding heavy chain, in combination with three disulfide bond linkages in the hinge region between the heavy chains. Therefore the IgG2-A / B isoform represents an intermediate between the IgG2-A and IgG2-B isoforms. The “disulfide isoform distribution” or “disulfide structural isoform distribution” refer to the distribution of the different relative amounts (usually represented as a percentage) of the three disulfide structural isoforms (IgG2-A, IgG2-A / B and IgG2-B) within an IgG2 product.

[0049] The term “titer” as used herein refers to the total amount of recombinantly expressed antibody produced by a cell culture divided by a given amount of medium volume. Titer is typically expressed in units of milligrams of antibody per milliliter or liter of medium (mg / ml or mg / L). In certain embodiments, titer is expressed in grams of antibody per liter of medium (g / L). Titer can be expressed or assessed in terms of aDocket No. P38734-WO relative measurement, such as a percentage increase in titer as compared obtaining the protein product under different culture conditions.

[0050] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies in accordance with the presently disclosed subject matter can be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.

[0051] A “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody- encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.

[0052] A “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human complementarity determining regions (CDRs) and amino acid residues from human framework regions (FRs). In certain aspects, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDRs correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally can comprise at least a portion of an antibody constantDocket No. P38734-WO region derived from a human antibody. A “humanized form” of an antibody, e.g., a non- human antibody, refers to an antibody that has undergone humanization.

[0053] As used herein, the term “recombinant protein” refers generally to peptides and proteins, including antibodies, that are encoded by a nucleic acid that is “heterologous,” i.e., foreign to the host cell being utilized, such as a nucleic acid encoding a human antibody that is introduced into a non-human host cell.

[0054] The following abbreviations are used herein: CHO Chinese hamster ovary Ig Immunoglobulin IgG Immunoglobulin G RP-HPLC Reversed-phase high performance liquid chromatograph LC / MS Liquid chromatography / mass spectrometry Methods of Modulating Disulfide Structural Isoform Distribution

[0055] In an aspect, the invention provides a method for modulation of disulfide structural isoform distribution in a recombinant protein, comprising: contacting the recombinant protein with a solution comprising a reducing agent.

[0056] In embodiments, the recombinant protein is an antibody or fragment thereof comprising a hinge region. The antibody or fragment thereof may be an immunoglobulin G (IgG) or fragment thereof. For example, the IgG may be an IgG2 or an IgG4. It may be that the IgG is an IgG2.

[0057] In embodiments, the recombinant protein is an IgG2 and the method comprises contacting with a sufficient level of reducing agent to increase the relative amount of IgG2-B disulfide isoform.

[0058] In embodiments, the relative amount of IgG2-B disulfide isoform may be increased by at least about 0.5%. In embodiments, the relative amount of IgG2-B disulfide isoform may be increased by at least about 1%. In embodiments, the relative amount of IgG2-B disulfide isoform may be increased by at least about 2%. In embodiments, the relative amount of IgG2-B disulfide isoform may be increased by at least about 4%. InDocket No. P38734-WO embodiments, the relative amount of IgG2-B disulfide isoform may be increased by at least about 6%. In embodiments, the relative amount of IgG2-B disulfide isoform may be increased by at least about 8%. In embodiments, the relative amount of IgG2-B disulfide isoform may be increased by at least about 10%. In embodiments, the relative amount of IgG2-B disulfide isoform may be increased by at least about 12%. In embodiments, the relative amount of IgG2-B disulfide isoform may be increased by at least about 15%.

[0059] In embodiments, the relative amount of IgG2-B disulfide isoform may be increased by an amount in the range of from about 0.5% to about 15%. In embodiments, the relative amount of IgG2-B disulfide isoform may be increased by an amount in the range of from about 1% to about 10%.

[0060] The relative amount of IgG2-B disulfide isoform and any increase in this may be determined by any suitable method, for example by a method described in the assay “Measurement of relative amount of disulfide isoforms” provided hereinbelow.

[0061] In embodiments, the reducing agent comprises reduced sulfur. The reducing agent may comprises an -SH moiety.

[0062] In embodiments, the reducing agent is at least one antioxidant, optionally selected from 2-mercaptoethanol, alpha-ketoglutarate, alpha-tocopherol, ascorbic acid, citrate, cystamine, cysteamine, cysteine, cysteine hydrochloride, fumaric acid, glutathione, lactic acid, lipoic acid, hypotaurine, N-acetyl-cysteine, N-acetyl-cysteine amine, phenolic compounds, pyruvate, S-carboxylmethylcysteine, selenium, selenocysteine, sorbic acid, taurine, or a combination thereof. In embodiments, the reducing agent comprises cysteine and / or glutathione. It may be that the reducing agent is cysteine. It may be that the reducing agent is glutathione.

[0063] In embodiments, the reducing agent is present at a concentration of at least about 10 times the concentration of the recombinant protein. In embodiments, the reducing agent is present at a concentration of at least about 25 times the concentration of the recombinant protein. In embodiments, the reducing agent is present at a concentration of at least about 50 times the concentration of the recombinant protein.

[0064] In embodiments, the reducing agent is present at a concentration of not more than about 500 times the concentration of the recombinant protein. In embodiments, the reducing agent is present at a concentration of not more than about 400 times theDocket No. P38734-WO concentration of the recombinant protein. In embodiments, the reducing agent is present at a concentration of not more than about 300 times the concentration of the recombinant protein. In embodiments, the reducing agent is present at a concentration of not more than about 200 times the concentration of the recombinant protein.

[0065] In embodiments, the reducing agent is present at a concentration of at least about 10 times and not more than about 500 times the concentration of the recombinant protein. In embodiments, the reducing agent is present at a concentration of at least about 25 times and not more than about 300 times the concentration of the recombinant protein. In embodiments, the reducing agent is present at a concentration of at least about 50 times and not more than about 200 times the concentration of the recombinant protein.

[0066] In embodiments, the solution further comprises at least one metal ion. The metal ion may be a transition metal ion. For example, the metal ion may be selected from copper, iron, manganese or zinc. The metal ion may be an ion of copper (e.g. Cu2+) or iron (e.g. Fe3+or Fe2+).

[0067] Without wishing to be bound by theory, it is thought that the presence of at least one metal ion decreases the ability of the reducing agent to promote disulfide isoform conversion (e.g. to IgG2-B).

[0068] In embodiments, the recombinant protein is an IgG2 and the solution comprises a sufficient level of metal ion to increase the relative amount of IgG2-A isoform.

[0069] In embodiments, the metal ion may be at a concentration of from about 0.1 μM to about 5000 μM. In embodiments, the metal ion may be at a concentration of from about 1 μM to about 1000 μM. In embodiments, the metal ion may be at a concentration of from about 10 μM to about 500 μM. In embodiments, the metal ion may be at a concentration of from about 25 μM to about 250 μM. In embodiments, the metal ion may be at a concentration of from about 50 μM to about 150 μM.

[0070] In embodiments, the contacting comprises at least about 10 minutes. In embodiments, the contacting comprises at least about 15 minutes. In embodiments, the contacting comprises at least about 30 minutes. In embodiments, the contacting comprises at least about 1 hour.Docket No. P38734-WO

[0071] In embodiments, the contacting comprises not more than about 96 hours. In embodiments, the contacting comprises not more than about 48 hours.

[0072] In embodiments, the method further comprises isolating the recombinant protein with modulated distribution of disulfide structural isoforms. The isolating may comprise the use of any suitable method, such as those known in the art. For example, if the recombinant protein with modulated distribution of disulfide isoforms is an IgG (such as an IgG2 or an IgG4), isolation may comprise use of Protein A, Protein G, or Protein L based separation methods (e.g. it may comprise the use of Protein A, or Protein G based separation methods).. Methods for Preventing Disulfide Bond Rearrangement in a Recombinant Protein

[0073] In another aspect, the invention provides a method for preventing disulfide bond rearrangement in a recombinant protein, comprising: contacting the recombinant protein with a solution comprising an oxidising agent.

[0074] In embodiments, the recombinant protein is an antibody or fragment thereof comprising a hinge region. The antibody or fragment thereof may be an immunoglobulin G (IgG) or fragment thereof. For example, the IgG may be an IgG2 or an IgG4. It may be that the IgG is an IgG2.

[0075] In embodiments, the oxidising agent comprises a metal ion. The metal ion may be a transition metal ion. For example, the metal ion may be selected from copper, iron, manganese or zinc. The metal ion may be an ion of copper (e.g. Cu2+) or iron (e.g. Fe3+or Fe2+).

[0076] Without wishing to be bound by theory, it is thought that the presence of at least one metal ion in the oxidising agent decreases disulfide isoform conversion (e.g. to IgG2- B), therefore maintaining amounts of each isoform present.

[0077] In embodiments, the recombinant protein is an IgG2 and the solution comprises a sufficient level of metal ion to increase the relative amount of IgG2-A isoform.

[0078] In embodiments, the metal ion may be at a concentration of from about 0.1 μM to about 5000 μM. In embodiments, the metal ion may be at a concentration of from about 1 μM to about 1000 μM. In embodiments, the metal ion may be at a concentration of from about 10 μM to about 500 μM. In embodiments, the metal ion may be at a concentrationDocket No. P38734-WO of from about 25 μM to about 250 μM. In embodiments, the metal ion may be at a concentration of from about 50 μM to about 150 μM.

[0079] In embodiments, the oxidising agent comprises an oxidised sulfur. The oxidised sulfur may comprise an -S-S- moiety. The oxidised sulfur may be selected from cystine, glutathione disulfide, S-sulfocysteine, or a derivative or combination thereof.

[0080] In embodiments, the oxidising agent is present at a concentration of at least about 2 times the concentration of the recombinant protein. In embodiments, the oxidising agent is present at a concentration of at least about 5 times the concentration of the recombinant protein. In embodiments, the oxidising agent is present at a concentration of at least about 10 times the concentration of the recombinant protein.

[0081] In embodiments, the oxidising agent is present at a concentration of at least about 2 times and not more than about 500 times the concentration of the recombinant protein. In embodiments, the oxidising agent is present at a concentration of at least about 2 times and not more than about 200 times the concentration of the recombinant protein. In embodiments, the oxidising agent is present at a concentration of at least about 5 times and not more than about 150 times the concentration of the recombinant protein. In embodiments, the oxidising agent is present at a concentration of at least about 10 times and not more than about 100 times the concentration of the recombinant protein.

[0082] In embodiments, the contacting comprises at least about 10 minutes. In embodiments, the contacting comprises at least about 15 minutes. In embodiments, the contacting comprises at least about 30 minutes. In embodiments, the contacting comprises at least about 1 hour.

[0083] In embodiments, the contacting comprises not more than about 96 hours. In embodiments, the contacting comprises not more than about 48 hours. Methods of Culturing

[0084] In embodiments, the invention provides a method of culturing mammalian cells comprising a recombinant protein with modulated disulfide structural isoform distribution, further comprising: (a) culturing a eukaryotic cell line that comprises a polynucleotide encoding the recombinant polypeptide under conditions suitable for production of the polypeptide; and (b) contacting the recombinant protein with a solution comprising aDocket No. P38734-WO reducing agent. Any of the below recited embodiments may also apply to this aspect of the invention. Methods for Producing Recombinant Protein with Modulated Disulfide Structural Isoform Distribution

[0085] Another aspect of the invention provides a method for the production of a recombinant protein with modulated disulfide structural isoform distribution, comprising: (a) culturing a eukaryotic cell line that comprises a polynucleotide encoding the recombinant polypeptide under conditions suitable for production of the polypeptide; and (b) contacting the recombinant protein with a solution comprising a reducing agent.

[0086] In embodiments, the cell line is an animal cell line (e.g. a mammalian cell line) or a fungal cell line (e.g. a yeast cell line). In embodiments, the cell line is an animal cell line.

[0087] In embodiments, the cell line is a mammalian cell line. For example, the cell line may be a CHO cell line.

[0088] In embodiments, the cell line is cultured in a cell culture medium. In embodiments, the cell line is cultured under batch or fed-batch culture conditions, or perfusion culture conditions (with continuous or semi-continuous perfusion).

[0089] In embodiments, the cell line is cultured under fed-batch culture conditions.

[0090] In embodiments, (b) the contacting is performed not more than about 4 days before the end of (a) the culturing. In embodiments, (b) the contacting is performed not more than about 3 days before the end of (a) the culturing. In embodiments, (b) the contacting is performed not more than about 2 days before the end of (a) the culturing. In embodiments, (b) the contacting is performed not more than about 1 day before the end of (a) the culturing. In embodiments, (b) the contacting is performed not more than about 0.5 days before the end of (a) the culturing. In embodiments, (b) the contacting is performed after the end of (a) the culturing.

[0091] In embodiments, the cell line is cultured under perfusion culture conditions. The perfusion culture conditions may be semi-continuous perfusion or continuous perfusion.

[0092] In embodiments, (b) the contacting is performed intermittently during (a) the culturing. In embodiments, (b) the contacting is performed throughout (a) the culturing.Docket No. P38734-WO

[0093] In embodiments, the recombinant protein is an antibody or fragment thereof comprising a hinge region. The antibody or fragment thereof may be an immunoglobulin G (IgG) or fragment thereof. For example, the IgG may be an IgG2 or an IgG4. It may be that the IgG is an IgG2.

[0094] In embodiments, the recombinant protein is an IgG2 and the method comprises contacting with a sufficient level of reducing agent to increase the relative amount of IgG2-B disulfide isoform.

[0095] In embodiments, the relative amount of IgG2-B disulfide isoform may be increased by at least about 0.5%. In embodiments, the relative amount of IgG2-B disulfide isoform may be increased by at least about 1%. In embodiments, the relative amount of IgG2-B disulfide isoform may be increased by at least about 2%. In embodiments, the relative amount of IgG2-B disulfide isoform may be increased by at least about 4%. In embodiments, the relative amount of IgG2-B disulfide isoform may be increased by at least about 6%. In embodiments, the relative amount of IgG2-B disulfide isoform may be increased by at least about 8%. In embodiments, the relative amount of IgG2-B disulfide isoform may be increased by at least about 10%. In embodiments, the relative amount of IgG2-B disulfide isoform may be increased by at least about 12%. In embodiments, the relative amount of IgG2-B disulfide isoform may be increased by at least about 15%.

[0096] In embodiments, the relative amount of IgG2-B disulfide isoform may be increased by an amount in the range of from about 0.5% to about 15%. In embodiments, the relative amount of IgG2-B disulfide isoform may be increased by an amount in the range of from about 1% to about 10%.

[0097] The relative amount of IgG2-B disulfide isoform and any increase in this may be determined by any suitable method, for example by a method described in the assay “Measurement of relative amount of disulfide isoforms” provided hereinbelow

[0098] In embodiments, the concentration of the reducing agent in the solution obtained by the (b) contacting step is at least about 1 mM. In embodiments, the concentration of the reducing agent in the solution obtained by the (b) contacting step is at least about 2 mM. In embodiments, the concentration of the reducing agent in the solution obtained by the (b) contacting step is at least about 4 mM. In embodiments, the concentration of the reducing agent in the solution obtained by the (b) contacting step is at least about 6 mM. InDocket No. P38734-WO embodiments, the concentration of the reducing agent in the solution obtained by the (b) contacting step is at least about 8 mM. In embodiments, the concentration of the reducing agent in the solution obtained by the (b) contacting step is at least about 10 mM.

[0099] In embodiments, the concentration of the reducing agent in the solution obtained by the (b) contacting step is in the range of from about 1 mM to about 12 mM. In embodiments, the concentration of the reducing agent in the solution obtained by the (b) contacting step is in the range of from about 1 mM to about 10 mM. In embodiments, the concentration of the reducing agent in the solution obtained by the (b) contacting step is in the range of from about 1 mM to about 8 mM. In embodiments, the concentration of the reducing agent in the solution obtained by the (b) contacting step is in the range of from about 2 mM to about 6 mM.

[0100] In embodiments, the reducing agent comprises reduced sulfur. The reducing agent may comprises an -SH moiety.

[0101] In embodiments, the reducing agent is at least one antioxidant, optionally selected from 2-mercaptoethanol, alpha-ketoglutarate, alpha-tocopherol, ascorbic acid, citrate, cystamine, cysteamine, cysteine, cysteine hydrochloride, fumaric acid, glutathione, lactic acid, lipoic acid, hypotaurine, N-acetyl-cysteine, N-acetyl-cysteine amine, phenolic compounds, pyruvate, S-carboxylmethylcysteine, selenium, selenocysteine, sorbic acid, taurine, or a combination thereof. In embodiments, the reducing agent comprises cysteine and / or glutathione. It may be that the reducing agent is cysteine. It may be that the reducing agent is glutathione.

[0102] In embodiments, the solution further comprises an oxidising agent.

[0103] In embodiments, the oxidising agent comprises a metal ion. The metal ion may be a transition metal ion. For example, the metal ion may be selected from copper, iron, manganese or zinc. The metal ion may be an ion of copper (e.g. Cu2+) or iron (e.g. Fe3+or Fe2+).

[0104] In embodiments, the metal ion may be at a concentration of from about 0.1 μM to about 5000 μM. In embodiments, the metal ion may be at a concentration of from about 1 μM to about 1000 μM. In embodiments, the metal ion may be at a concentration of from about 10 μM to about 500 μM. In embodiments, the metal ion may be at a concentrationDocket No. P38734-WO of from about 25 μM to about 250 μM. In embodiments, the metal ion may be at a concentration of from about 50 μM to about 150 μM.

[0105] In embodiments, the oxidising agent comprises an oxidised sulfur. The oxidised sulfur may comprise an -S-S- moiety. The oxidised sulfur may be selected from cystine, glutathione disulfide, S-sulfocysteine, or a derivative or combination thereof.

[0106] The concentration of oxidising agent in the solution may be in an amount of at least about 1 mM. The concentration of oxidising agent in the solution may be in an amount of at least about 2 mM. The concentration of oxidising agent in the solution may be in an amount of at least about 4 mM. The concentration of the oxidising agent in the solution may be in an amount of not more than about 15 mM. The concentration of the oxidising agent in the solution may be in an amount of not more than about 12 mM. The concentration of the oxidising agent in the solution may be in an amount of not more than about 10 mM. In embodiments, the concentration of oxidising agent in the solution may be in the range of from about 1 mM to about 15 mM, e.g. in the range of from about 1mM to about 10 mM.

[0107] In embodiments, the recombinant protein is an IgG2 and the solution comprises a sufficient level of oxidising agent to increase the relative amount of IgG2-A isoform.

[0108] The increase in the relative amount of IgG2-A may be at least about 1%, or may be at least about 2%. For example, the increase in the relative amount of IgG2-A may be in the range of from about 1% to about 12% (e.g. from about 1% to about 10%). In an example, the increase in the relative amount of IgG2-A may be in the range of from about 1.5% to about 8% (e.g. from about 2% to about 6%).

[0109] In another aspect, there is provided a method for the production of a recombinant protein with modulated distribution of disulfide structural isoforms, comprising: (a) culturing a eukaryotic cell line that comprises a polynucleotide encoding the recombinant polypeptide under conditions suitable for production of the polypeptide; and (b) contacting the recombinant protein with a solution comprising an oxidising agent. The oxidising agent and / or recombinant protein may be as defined in any of the embodiments above.

[0110] In another aspect, there is provided a method for the production of a recombinant protein that is an IgG2 having an increased amount of IgG2-B disulfide structural isoform, the method comprising: (a) culturing a eukaryotic cell line that comprises a polynucleotideDocket No. P38734-WO encoding the recombinant polypeptide under conditions suitable for production of the polypeptide; and (b) contacting the recombinant protein with a solution comprising a reducing agent. The recombinant protein and / or reducing agent may be as defined in any of the embodiments above.

[0111] In embodiments, the solution further comprises an oxidising agent. The oxidising agent may be as defined in any of the embodiments above.

[0112] In another aspect, there is provided a method for the production of a recombinant protein that is an IgG2 having an increased amount of IgG2-A disulfide structural isoform, the method comprising: (a) culturing a eukaryotic cell line that comprises a polynucleotide encoding the recombinant polypeptide under conditions suitable for production of the polypeptide; and (b) contacting the recombinant protein with a solution comprising an oxidising agent. Any of the above recited embodiments may also apply to this aspect of the invention. Recombinant Protein with Modulated Disulfide Structural Isoforms

[0113] In another aspect, there is provided recombinant protein with modulated disulfide structural isoforms obtainable or obtained by a method of the disclosure.

[0114] The methods of the present disclosure involve the production of recombinant proteins having disulfide bonds, such as antibodies or fragments thereof comprising a hinge region and fusion proteins; or conjugates thereof. Where the recombinant protein is an antibody (such as an IgG2, IgG4 or fragment thereof), the antibody may be, but is not limited to, a monospecific antibody, a multispecific antibody and antigen-binding fragments thereof comprising a hinge region.

[0115] In embodiments, the polynucleotide that encodes the polypeptide is integrated in the cellular genome of the cells of the cell line at a targeted location. In alternative embodiments, the polynucleotide that encodes the polypeptide is randomly integrated in the cellular genome of the cells of the cell line.

[0116] In embodiments, the polynucleotide that encodes the polypeptide is an extrachromosomal polynucleotide. The extrachromosomal polynucleotide may comprise extrachromosomal DNA (ecDNA) and / or extrachromosomal circular DNA (eccDNA).Docket No. P38734-WO

[0117] In embodiments, the polynucleotide that encodes the polypeptide is integrated into a chromosome of the cells of the cell line.

[0118] In embodiments, the recombinant polypeptide is an antibody, an antigen, an enzyme, a gene vector or a vaccine.

[0119] In embodiments, the recombinant polypeptide is a therapeutic antibody.

[0120] In embodiments, when the recombinant polypeptide is an antibody, the antibody is a multispecific antibody or antigen-binding fragment thereof.

[0121] In embodiments, the therapeutic antibody is selected from anti-HER receptor family antibodies (such as anti-HER1 (EGFR), anti-HER2, anti-HER3 and anti- HER4); anti-CD protein antibodies (such as anti-CD3, anti-CD4, anti-CD8, anti- CD19, anti-CD20, anti-CD21, anti-CD22, anti-CD25, anti-CD33, anti-CD34, anti- CD38, anti-CD52); anti-IL-8 antibodies; anti-VEGF antibodies; anti-CD40 antibodies, anti-CD11a antibodies; anti-CD18 antibodies; anti-IgE antibodies; anti- Apo-2 receptor antibodies; anti-Tissue Factor (TF) antibodies; anti- cell adhesion molecules such as LFA-1, Mol, p150,95, VLA-4, ICAM-1, VCAM, anti-human 47 integrin antibodies, anti-human v 8 integrin antibodies, anti- v 3antibodies including either or or subunits thereof (e.g. anti-CD11a, anti-CD18 or anti-CD11b antibodies); anti-EGFR antibodies; anti-Fc receptor antibodies; anti- carcinoembryonic antigen (CEA) antibodies; anti-human renal cell carcinoma antibodies; anti-human colorectal tumor antibodies; anti-human melanoma antibody R24 directed against GD3 ganglioside; anti-human squamous-cell carcinoma; antibodies directed against breast epithelial cells; antibodies that bind to colon carcinoma cells; anti-EpCAM antibodies; anti-GpIIb / IIIa antibodies; anti- RSV antibodies; anti-CMV antibodies; anti-HIV antibodies; anti-hepatitis antibodies; anti-CA 125 antibodies; anti-human 17-1A antibodies; and anti-human leukocyte antigen (HLA) antibodies, and anti-HLA DR antibodies, anti-growth factors such as vascular endothelial growth factor (anti-VEGF) or fragments; anti- IgE; anti-blood group antigens; anti-flk2 / flt3 receptor; and anti-obesity (OB) receptor; anti-amyloid antibodies, anti-alpha-synuclein (e.g.: prasinezumab), anti- amyloid-beta, anti-growth hormone (GH), including human growth hormone (hGH) and bovine growth hormone (bGH); anti-growth hormone releasing factor;Docket No. P38734-WO anti-parathyroid hormone; anti-thyroid stimulating hormone; anti-lipoproteins; anti- -1 -antitrypsin; anti-insulin A-chain; anti-insulin B-chain; anti-proinsulin; anti-follicle stimulating hormone; anti-calcitonin; anti-luteinizing hormone; anti- glucagon; anti-clotting factors such as factor VIIIC, tissue factor or von Willebrands factor; anti-clotting factors such as Protein C; anti-atrial natriuretic factor; anti-lung surfactant; anti- a plasminogen activator, such as urokinase or tissue-type plasminogen activator (t-PA); bombazine; thrombin; anti-tumor necrosis factor- and - ; anti-enkephalinase; RANTES (regulated on activationnormally T-cell expressed and secreted); anti-human macrophage inflammatory protein (MIP-1- ); anti- a serum albumin such as human serum albumin (HSA);anti- mullerian-inhibiting substance; anti- relaxin A-chain; anti- relaxin B-chain; anti-prorelaxin; anti- mouse gonadotropin-associated peptide; anti-DNase; anti- inhibin; anti-activin; anti- receptors for hormones or growth factors; anti- protein A or D; anti- rheumatoid factors; anti- a neurotrophic factor such as bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), or a nerve growth factor such as NGF- ; platelet-derived growth factor(PDGF); fibroblast growth factor such as aFGF and bFGF; epidermal growth factor (EGF); anti- transforming growth factor (TGF) such as TGF- and TGF- ,including TGF- 1, TGF- 2, TGF- 3, TGF- 4, or TGF- 5; anti- insulin-likegrowth factor-I and -II (IGF-I and IGF-II); anti- des(1-3)-IGF-I (brain IGF-I); insulin-like growth factor binding proteins (IGFBPs); anti- erythropoietin (EPO); anti- thrombopoietin (TPO); anti- osteoinductive factors; anti- immunotoxins; anti- abone morphogenetic protein (BMP); anti- an interferon such as interferon- , - ,and - ; anti- colony stimulating factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF; anti- interleukins (ILs), e.g., IL-1 to IL-10; superoxide dismutase; anti- T-cell receptors; anti- surface membrane proteins; anti- decay accelerating factor (DAF); anti- a viral antigen such as, for example, a portion of the AIDS envelope; anti- transport proteins; anti- homing receptors; anti- addressins; anti- regulatory proteins; anti- immunoadhesins; and a therapeutic antibody directed against biologically active fragments or variants of any of the above-listed polypeptides.

[0122] In embodiment, the therapeutic antibody is selected from AVASTIN® (bevacizumab), HERCEPTIN® (trastuzumab), LUCENTIS® (ranibizumab),Docket No. P38734-WO RAPTIVA® (efalizumab), RITUXAN® (rituximab), ACTEMRA® (tocilizumab - anti-IL-6 receptor), XOLAIR® (omalizumab), OCREVUS® (ocrelizumab - anti- CD20 antibody), PERJETA® (pertuzumab - HER dimerization inhibitors (HDIs)), TECENTRIQ® (anti-PD-L1 antibody), LUNSUMIO® or COLUMVI TM (anti- CD20 X anti-CD3 bispecific antibody), VABYSMO® (anti- VEGF-A X anti- angiopoietin-2 bispecific antibody), anti-CD79b antibody, anti-OX40 ligand, anti- oxidized LDL (oxLDL), anti-amyloid beta (e.g., trontinemab), anti-CD4 (MTRX1011A), anti- EGFL7 (EGF-like-domain 7), anti-IL13, Apomab (anti- DR5-targeted pro-apoptotic receptor agonist (PARA), anti-BR3 (CD268, anti- BLyS receptor 3, anti-BAFF-R, (BAFF Receptor), anti-TIGIT (anti-T-cell immunoreceptor with immunoglobulin (Ig) and immunoreceptor tyrosine-based inhibitory motif domains) antibodies, astegolimab (anti-ST2, an IL-33 receptor), anti-beta 7 integrin subunit, anti- v 8 integrin antibodies, dacetuzumab (Anti-CD40), GA101 (obinutuzumab - anti-CD20 monoclonal antibody), MetMAb (anti- MET receptor tyrosine kinase), cevostamab (anti-Fc receptor-homolog 5 (FcRH5) X anti-CD3 bispecific antibody), anti-neuropilin-1 (NRP1), and rhuMAb IFN alpha. Method for Enhancing Disulfide Bond Rearrangement in a Recombinant Protein

[0123] In another aspect, there is provided a method for enhancing disulfide bond rearrangement in a recombinant protein, comprising: contacting the recombinant protein with a solution comprising a reducing agent.

[0124] Any of the embodiments relating to the first aspect of the invention may also apply to this aspect.

[0125] In another aspect, there is provided a recombinant protein obtained or obtained by this method for enhancing disulfide bond rearrangement in a recombinant protein described above. ASSAYS Measurement of distribution (relative amount) of disulfide isoforms

[0126] The relative and absolute amounts of disulfide structural isoforms (to determine the distribution of the isoforms) of a protein may be measured using any suitable method.Docket No. P38734-WO Such a method may comprise an affinity separation (e.g. Protein A or Protein G for IgG) to isolate the protein, followed by reversed phase high performance liquid chromatography (RP-HPLC) to separate and quantify the disulfide structural isoforms of the protein. Liquid chromatography / mass spectrometry (LC / MS) methods may also be used, e.g. an affinity separation followed by RP-HPLC, with MS detection of the disulfide structural isoforms in the RP-HPLC eluent.

[0127] An exemplary assay for quantifying IgG2 disulfide structural isoforms is provided hereinbelow in Example 4. This method may be readily adapted to other IgGs; and as the skilled person would appreciate, disulfide structural isoforms of recombinant proteins more generally may be determined with appropriate RP-HPLC and / or LC / MS based methods. EXAMPLES

[0128] The disclosure will be more fully understood by reference to the following examples. They should not, however, be construed as limiting the scope of the disclosure. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. Materials and Methods

[0129] Recombinant IgG2 monoclonal antibodies- mAb X and mAb Y, were expressed in Chinese Hamster Ovary (CHO) cell lines, and then purified by protein A affinity chromatography. L-cysteine stock solution was prepared with L-cysteine hydrochloride monohydrate purchased from Ajinomoto Health and Nutrition. L-glutathione stock solution was prepared with reduced L-glutathione purchased from Millipore Sigma. S- sulfocysteine stock solution was prepared with L-cysteine S-sulfate sodium salt sesquihydrate, purchased from EMD Millipore. L-cystine stock solution was prepared with L-cystine purchased from Millipore Sigma. Copper (II) sulfate stock solution was prepared with cupric sulfate pentahydrate, purchased from Sigma-Aldrich. Iron (II) sulfate stock solution was prepared with ferrous sulfate heptahydrate, purchased from Sigma- Aldrich. Example 1: Cell culturing processDocket No. P38734-WO

[0130] Two different CHO cell lines (cell line X and cell line Y) were used to generate IgG2 monoclonal antibodies mAb X and mAb B, respectively. Both cell lines were cultivated in suspension using proprietary chemically-defined medium and underwent multiple passage expansions in shake flasks prior to inoculating 2-L Applikon bioreactors for the production of IgG2 antibodies. Established control systems for pH, temperature and oxygen specific to each cell line were employed during the production cultures. Concentrated nutrient feeds were added to the production cultures to maintain cell growth, viability and productivity. In the bioreactor study employing different L-cysteine feeding strategies, cell culture supernatants were analyzed post-production to determine cystine concentrations using the free amino acid analysis method with pre-column derivation and reversed-phase high performance liquid chromatograph (RP-HPLC) as described in detail previously (Cohen SA, Michaud DP.1993. Synthesis of a fluorescent derivatizing reagent, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, and its application for the analysis of hydrolysate amino acids via high-performance liquid chromatography. Anal Biochem 211(2):279–287). Example 2: Incubation of cell cultures with supplemental media additives

[0131] The incubation studies used either harvested cell culture fluid (i.e., cell-free supernatant) or CHO cell cultures. After growing CHO cells producing either mAb X or mAb Y in a fed-batch production cultures for ~14 days, the cells were removed by centrifugation and the resulting supernatant was used for the cell-free incubation studies. The studies using CHO cell cultures involved pulling cells sourced from 2-L Applikon bioreactors (during fed-batch production process) and further incubating them in theconditions specified for each experiment. Samples taken were stored in -80 C prior topurification and analytical testing. 2.1. Effect of media additives on IgG2 disulfide isoform distribution for mAb X and mAb Y

[0132] Without wishing to be bound by theory, it was thought that the disulfide isoform distribution of recombinant IgG2 antibodies could be modulated by changing the reduction potential of the extracellular bioprocess environment to impact the extent of interchain disulfide bond rearrangement. Specifically, it is thought that during the different bioprocessing stages—spanning upstream processing through harvest and downstream processing—IgG2 can undergo disulfide bond shuffling and hence isoform conversionDocket No. P38734-WO upon exposure to a different redox environment. During the cell culture production stage of upstream processing, certain media supplements that support cell growth and productivity are also redox active, e.g. L-cysteine and L-glutathione (the free thiol can participate in redox reactions in the extracellular environment and therefore contributes an adequate reduction potential to trigger rearrangement of the IgG2 inter-chain disulfide bonds).

[0133] Cell-free supernatant containing recombinant IgG2 (mAb X) was incubated with the four media supplements with different reduction potentials: L-cysteine, L-cystine, L- glutathione and S-sulfocysteine (Figure 2). The cell-free supernatant was generated by harvesting a 2-L bioreactor mAb X production culture on day 14 and removing the cells by centrifugation; the cell-free supernatant is therefore representative of feedstock that would be supplied to purification for downstream processing of mAb X. After 24 hours of incubation, an increase in IgG2-B isoform for mAb X was observed for supernatants supplemented with 6 mM L-cysteine or 6 mM L-glutathione, with a corresponding decrease in IgG2-A / B and IgG2-A isoforms. By contrast, no clear change in IgG2 isoform distribution was observed after 24 hours of incubation with 3 mM L-cystine or 6 mM S- sulfocysteine.

[0134] To understand the impact of the presence of cells and the concentration of media additives (as reducing agents) on IgG2 disulfide isoform rearrangement, L-cysteine or L- glutathione were supplemented on day 13 to 2-L bioreactor production cultures of recombinant CHO cells expressing mAb X (~6 g / L). The cultures were incubated with the media supplements (at 1 or 6 mM, representing 24 or 141 μM of supplement per μM mAb X) for 24 hours prior to day 14 harvest. The cell culture studies showed that after 24 hours of incubation, an increase in IgG2-B isoform for mAb X was observed for bioreactor cultures supplemented with L-cysteine or L-glutathione in a dose-dependent manner, with a corresponding decrease in IgG2-A / B and IgG2-A isoforms (Figure 3).

[0135] After 24 hours of incubation, the increases in IgG2-B isoform at the highest supplement levels tested (6 mM of L-cysteine or L-glutathione) were similar in this cell culture study and the earlier cell-free supernatant study (Figure 2). It is thought that this is due to a lack of interference by or interaction with the cells during this incubation period. The higher impact of L-cysteine over L-glutathione was also observed in both studiesDocket No. P38734-WO (Figures 2 and 3), thereby pointing to higher potential of L-cysteine over L-glutathione (at the same molar concentration) for modulating IgG2 disulfide isoform distribution.

[0136] The next three studies were conducted with mAb Y. The cell-free incubation study with mAb X indicated that the bulk of the disulfide rearrangement occurred within 24 hours of supplementation with L-cysteine because the increase in IgG2-B isoform between 24 and 48 hours was relatively minor relative to the increase between 0 and 24 hours (Figure 2). Therefore, the first study with mAb Y was conducted with additional sampling between time of L-cysteine addition up to 24 hours of incubation (Figure 4). This study also assessed the additional impact of cells by testing samples taking from the same 2-L bioreactor during the end of production culture (day 14) and incubating them in the presence of supplemental L-cysteine with and without cells. A time-dependent increase in IgG2-B isoform for mAb Y was observed in both the cell-containing and cell- free incubations with L-cysteine (Figure 4). A considerable fraction of the disulfide isoform conversion was observed within the first 15 minutes post L-cysteine addition, and the bulk of the conversion was observed by 8 hours, with some additional conversion between 8 and 24 hours. After 24 hours of incubation, the final IgG2 disulfide isoform distribution for mAb Y was consistent across the different incubation temperatures, L- cysteine levels, and sample types tested. The presence of cells did not have a considerable impact on the extent of disulfide isoform rearrangement. It is thought that this lack of cellular interference is due to the extracellular nature of IgG2 disulfide bond shuffling.

[0137] To further assess the dose dependence of IgG2 disulfide rearrangement on supplemental L-cysteine, a titration curve was generated using mAb Y harvested cell culture fluid (i.e., cell-free supernatant from day 14 of a 2-L bioreactor production culture). The IgG2-B disulfide isoform increased with the amount of supplemental L- cysteine, with corresponding decreases in the IgG2-A and IgG2-A / B isoforms (Figure 5). The amount of L-cysteine supplemented was calculated on a mole per mole ratio relative to the amount of mAb Y, and showed a relatively linear increase up to 150 μM of L- cysteine per μM mAb Y (representing 3 mM L-cysteine in ~3 g / L of mAb Y). Increasing the L-cysteine supplementation further from 3 mM to 6 mM (representing 300 μM of L- cysteine per μM mAb Y) yielded small changes in disulfide isoform rearrangement.Docket No. P38734-WO

[0138] Based on the titration study for mAb Y with L-cysteine, another study with mAb Y was conducted to understand the impact of L-glutathione on IgG2 disulfide isoform rearrangement. The IgG2-B disulfide isoform increased with the amount of supplemental L-glutathione in a time-dependent manner, with corresponding decreases in the IgG2-A and IgG2-A / B isoforms (Figure 6). The amount of L-glutathione supplemented (1, 3 and 6 mM) was presented on a mole per mole ratio relative to the amount of mAb Y used in these incubation studies. Similar to the observations with L-cysteine titration, increasing the L-glutathione supplementation further from 3 mM to 6 mM (representing 375 μM of L-glutathione per μM mAb Y) yielded relatively small changes in disulfide isoform rearrangement at the end of incubation.

[0139] Thus, the media supplements L-cysteine and L-glutathione have an impact in driving disulfide rearrangement from the IgG2-A isoform to the intermediate IgG2-A / B isoform towards the IgG2-B isoform in a dose-dependent and time-dependent manner within the conditions tested. The presence of cells did not have a noticeable impact, thereby indicating that the IgG2 disulfide shuffling is an extracellular phenomenon. These trends are consistent for both mAb X and mAb Y produced by different CHO hosts in different upstream processes, indicating that these findings should be relevant to other IgG2s. 2.2. Interaction with Metal Ions

[0140] Many metal ions (e.g., iron, copper, manganese, zinc, selenium ions) are essential supplements in cell culture because of their critical roles in ensuring proper cellular functioning (e.g., as enzyme co-factors). With the addition (a day before harvest) of supplemental ferrous sulfate or cupric sulfate to CHO cell cultures expressing mAb X, no clear trend on IgG2 disulfide isoform rearrangement was observed in the absence of supplemental L-cysteine or L-glutathione (Figure 7). However, the supplemental ferrous sulfate or cupric sulfate interfered with IgG2 disulfide isoform in the presence of supplemental L-cysteine or L-glutathione during the 24-hour incubation. Specifically, the supplementation of both metals decreased the ability of L-cysteine and L-glutathione to promote disulfide isoform conversion to IgG2-B, with both metals showing a greater interference with L-cysteine, and with the cupric sulfate showing a stronger interference than ferrous sulfate.Docket No. P38734-WO

[0141] The interaction of these transition metals with L-cysteine and L-glutathione is surprising in terms of both the directionality and extent of their impact. For example, ferrous sulfate can oxidize readily to the ferric form in aqueous solution and could therefore show a synergistic effect with the reducing agents L-cysteine and L-glutathione by increasing the overall reduction potential of the extracellular environment to promote IgG2 disulfide rearrangement.

[0142] The impact of cupric sulfate on IgG2 disulfide isoform rearrangement was further investigated with mAb Y. The copper supplementation hindered the shift in disulfide isoform rearrangement towards IgG2-B that would otherwise occur in the presence of L- cysteine during the 24-hour incubation with CHO cell cultures expressing mAb Y (Figure 8). It is thought that this observed minimization of isoform rearrangement is due to an interaction between copper and L-cysteine such that the reduction potential of the latter is diminished, which is consistent with the oxidizing nature of cupric sulfate in cell culture and the ability of copper to catalyze the oxidation of L-cysteine monomer.

[0143] Thus, the presence metals (such as iron and copper) counters the impact of media supplements (such as L-cysteine and L-glutathione) in promoting IgG2 disulfide isoform rearrangement. Additional iron or copper may be supplemented to minimize IgG2 disulfide shuffling and thereby maximize the IgG2-A content in the mAb product. 2.3. Comparison of different L-cysteine addition strategies during upstream processing to modulate IgG2 disulfide isoform distribution

[0144] The practical application and translation of the L-cysteine findings above to a typical bioreactor production culture were investigated. Recombinant CHO cells expressing mAb X were cultured for 14 days under six different conditions in duplicate 2- L bioreactors. The study was designed to assess the impact of the L-cysteine supplementation strategy by varying the following factors across six cases: (1) total L- cysteine concentration supplemented in each condition; (2) total number of L-cysteine feeds; (3) concentration of L-cysteine in each feed; and (4) initial L-cysteine concentration (Figure 9a).

[0145] For the cases with the same total concentration of L-cysteine supplemented (i.e., 6 mM), there was a range in the IgG2-B content, with a notably higher IgG2-B for the case with 5 supplemental L-cysteine feeds instead of 2 or 0 (Figure 9a). For the casesDocket No. P38734-WO with the same total number of L-cysteine feeds (i.e., 2) and also the same initial L-cysteine concentration (i.e., 3 mM), the IgG2-B content increased with the concentration of L- cysteine in the feed and with the total L-cysteine concentration added. For the two cases with the same L-cysteine concentration in the feed (i.e., 1.5 mM), the differences in total L-cysteine supplemented (i.e., 4 mM) and in IgG2-B content (~15%) were also the greatest. For the two cases with the same lowest L-cysteine initial concentration (i.e., 1 mM), the IgG2-B content were the highest in spite of their considerable differences in total L-cysteine supplemented (i.e., 4 mM). In particular, the case with the second highest IgG2-B content (~50%) had the lowest total L-cysteine concentration (i.e., 6 mM), L- cysteine feed concentration (i.e., 1 mM), and initial L-cysteine concentration (i.e., 1 mM), but it had the most number of L-cysteine feeds (i.e., 5), second only to the case with the highest IgG2-B content (~55%). In particular, the addition of L-cysteine during the later stage of the production culture (e.g., days 10-12), was observed to increase the IgG2-B level to a greater extent than when added at earlier stages of culture (e.g., days 3-7).

[0146] In cell culture media, L-cysteine oxidizes within days to form L-cystine. Since L-cysteine cannot be reliably measured by HPLC method, L-cystine was measured instead in this study. The differences in the L-cysteine supplementation strategies can be inferred through their impact on L-cystine profiles (Figure 9b). The three cultures with the highest L-cystine levels at the end of the production culture also showed the three highest IgG2-B levels. By contrast, the two cultures with the lowest L-cystine levels (with depletion days before harvest) showed the two lowest IgG2-B levels.

[0147] These differences in L-cysteine supplementation strategy did not translate into notable differences in cell culture performance (Figure 9c) or final mAb X titers (~ 6 / gL; data not shown). Therefore, the supplementation strategy for L-cysteine during the cell culture production process is can modulate IgG2 disulfide isoform distribution without impacting cell culture performance. Example 3: Affinity purification of mAb using protein

[0148] The antibody was purified using conventional chromatographic procedures (Fahrner, R. L.; et al., Industrial Purification of Pharmaceutical Antibodies: Development, Operation, and Validation of Chromatography Processes. Biotechnol. Genetic Eng. Rev.Docket No. P38734-WO 2001, 18, 301-327). The monoclonal antibodies were captured from the harvested cell culture fluid by protein A via PhyTip (PhyNexus, San Jose, CA) on a LYNX robot platform. Depending on the concentration of the protein, samples may have undergone dilution with deionized water prior to two sets of captures on the PhyTip containing 160 uL of protein A resin. The antibodies were bound onto the resin during a 4-cycle binding step, which include 1mL harvest cell culture fluid per cycle, then eluted with 100 mM acetic acid, before a final neutralization of eluted pool to pH 5.5. After purification, theisolated isoforms were stored in -80 C prior to further evaluation.Example 4: IgG2 Disulfide Isoform Quantitation by RP-HPLC of mAb in-vivo samples

[0149] Protein-A purified disulfide isoforms were separated by reversed-phase high performance liquid chromatography (RP-HPLC) as described by Dillon et al., (Dillon, T. M., Bondarenko, P. V., Rehder, D. S., Pipes, G. D., Kleemann, G. R., and Ricci, M. S. Optimization of a reversed-phase high-performance liquid chromatography / mass spectrometry method for characterizing recombinant antibody heterogeneity and stability. (2006) J. Chromatogr.1120, 112–120), via HALO protein C4 column (Advanced Material Technology) on a UHPLC system (Agilent 1290) and quantified based on UV detection at 280 nm. Mobile phase A consisted of 0.1% Trifluoroacetic acid (TFA) in 98% Water and 2% Isopropyl Alcohol (IPA). Mobile phase B consisted of 0.1% TFA in 70% IPA, 20% Acetonitrile (ACN) and 10% water. The flow rate was 1 mL / min and column temperature was maintained at 85°C. Samples were first diluted to 1 mg / mL with Mobile phase A prior to injection of 2.5uL. The 13 min LC gradient consisted of 25%B for a 0.5 min wash / equilibration, 25-33% B in 7.5 min, 33-65% B in 1 min, 65-90% B in 0.1 min, a column wash (90% B for 1.4 min), 90-0% B in 0.01 minute, and column re-equilibration (25% B for 2.5 min). Peaks were integrated according to the product specific instructions.

Claims

Docket No. P38734-WO WHAT IS CLAIMED IS:

1. A method for modulation of disulfide structural isoforms in a recombinant protein, comprising: contacting the recombinant protein with a solution comprising a reducing agent.

2. The method of claim 1, wherein the wherein the recombinant protein is an antibody or fragment thereof comprising a hinge region.

3. The method of claim 2, wherein the antibody or fragment thereof is an immunoglobulin G (IgG) or fragment thereof.

4. The method of claim 3, wherein the IgG is an IgG2 or an IgG4, optionally wherein the IgG is an IgG2.

5. The method of any preceding claim, wherein the recombinant protein is an IgG2 and the method comprises contacting with a sufficient level of reducing agent to increase the relative amount of IgG2-B disulfide isoform.

6. The method of any preceding claim, wherein the reducing agent comprises reduced sulfur, optionally wherein the reducing agent comprises an -SH moiety.

7. The method of any of claims 1 to 5, wherein the reducing agent is at least one antioxidant, optionally selected from 2-mercaptoethanol, alpha-ketoglutarate, alpha- tocopherol, ascorbic acid, citrate, cystamine, cysteamine, cysteine, cysteine hydrochloride, fumaric acid, glutathione, lactic acid, lipoic acid, hypotaurine, N- acetyl-cysteine, N-acetyl-cysteine amine, phenolic compounds, pyruvate, S- carboxylmethylcysteine, selenium, selenocysteine, sorbic acid, taurine, or a combination thereof.

8. The method of any preceding claim, wherein the reducing agent comprises cysteine and / or glutathione.

9. The method of any preceding claim, wherein the reducing agent is present at a concentration of at least about 10 times the concentration of the recombinant protein.

10. The method of any preceding claim, wherein the reducing agent is present at a concentration of at least about 25 times the concentration of the recombinant protein.Docket No. P38734-WO 11. The method of any preceding claim, wherein the reducing agent is present at a concentration of at least about 50 times the concentration of the recombinant protein.

12. The method of any preceding claim, wherein the reducing agent is present at a concentration of not more than about 500 times the concentration of the recombinant protein.

13. The method of any preceding claim, wherein the reducing agent is present at a concentration of not more than about 300 times the concentration of the recombinant protein; optionally wherein the reducing agent is present at a concentration of not more than about 200 times the concentration of the recombinant protein.

14. The method of any preceding claim, wherein the reducing agent is present at a concentration of at least about 20 times and not more than about 300 times the concentration of the recombinant protein optionally wherein the reducing agent is present at a concentration of at least about 50 times and not more than about 200 times the concentration of the recombinant protein.

15. The method of any preceding claim, wherein the solution further comprises at least one metal ion.

16. The method of claim 15, wherein the metal ion is a transition metal ion, optionally selected from copper, iron, manganese, or zinc.

17. The method of claim 15 or claim 16, wherein the metal ion is an ion of copper (such as Cu2+) or iron (such as Fe3+or Fe2+).

18. The method of any of claims 15 to 17, wherein the recombinant protein is an IgG2 and the solution comprises a sufficient level of metal ion to increase or prevent decrease in the relative amount of IgG2-A isoform.

19. The method of any of claims 15 to 17, wherein the metal ion is at a concentration of from about 0.1 μM to about 5000 μM; optionally wherein the metal ion is at a concentration of from about 10 μM to about 500 μMDocket No. P38734-WO further optionally wherein the metal ion is at a concentration of from about 50 μM to about 150 μM.

20. The method of any preceding claim, wherein the contacting comprises at least about 10 minutes, optionally at least about 15 minutes.

21. The method of any preceding claim, wherein the contacting comprises at least about 1 hour.

22. The method of any preceding claim, wherein the contacting comprises not more than about 96 hours, optionally not more than about 48 hours.

23. The method of any preceding claim, further comprising isolating the recombinant protein with modulated disulfide isoforms.

24. A method for stabilizing the disulfide structural isoform distribution by preventing disulfide bond rearrangement in a recombinant protein, comprising: contacting the recombinant protein with a solution comprising an oxidising agent.

25. The method of claim 24, wherein the wherein the recombinant protein is an antibody or fragment thereof comprising a hinge region.

26. The method of claim 25, wherein the antibody or fragment thereof is an immunoglobulin G (IgG) or fragment thereof.

27. The method of claim 26, wherein the IgG is an IgG2 or an IgG4, optionally wherein the IgG is an IgG2.

28. The method of any of claims 24 to 27, wherein the oxidising agent comprises a metal ion.

29. The method of claim 28, wherein the metal ion is a transition metal ion, optionally selected from copper, iron, manganese, or zinc.

30. The method of claim 28 or claim 29, wherein the metal ion is an ion of copper (such as Cu2+) or iron (such as Fe3+or Fe2+).

31. The method of any of claims 28 to 30, wherein the recombinant protein is an IgG2 and the solution comprises a sufficient level of metal ion to maintain the relative amount of IgG2-A isoform.Docket No. P38734-WO 32. The method of any of claims 28 to 31, wherein the metal ion is at a concentration of from about 0.1 μM to about 5000 μM; optionally wherein the metal ion is at a concentration of from about 10 μM to about 500 μM further optionally wherein the metal ion is at a concentration of from about 50 μM to about 150 μM.

33. The method of any of claims 24 to 32, wherein the oxidising agent comprises an oxidised sulfur, optionally wherein the oxidised sulfur comprises an -S-S- moiety.

34. The method of claim 33, wherein the oxidised sulfur is selected from cystine, glutathione disulfide, S-sulfocysteine, or a derivative or combination thereof.

35. The method of any of claims 28 to 34, wherein the oxidising agent is present at a concentration of at least about 10 times the concentration of the recombinant protein.

36. The method of any of claims 28 to 34, wherein the oxidising agent is present at a concentration of at least about 2 times and not more than about 200 times the concentration of the recombinant protein optionally wherein the oxidising agent is present at a concentration of at least about 10 times and not more than about 100 times the concentration of the recombinant protein.

37. The method of any of claims 28 to 36, wherein the contacting comprises at least about 10 minutes, optionally at least about 15 minutes.

38. The method of any of claims 28 to 37, wherein the contacting comprises at least about 1 hour.

39. The method of any of claims 28 to 38, wherein the contacting comprises not more than about 96 hours, optionally not more than about 48 hours.

40. A method for the production of a recombinant protein with modulated disulfide structural isoforms, comprising: (a) culturing a eukaryotic cell line that comprises a polynucleotide encoding the recombinant polypeptide under conditions suitable for production of the polypeptide; and (b) contacting the recombinant protein with a solution comprising a reducing agent.Docket No. P38734-WO 41. The method of any of claim 40, wherein the cell line is animal cell line (such as a mammalian cell line), or a fungal cell line (such as a yeast cell line).

42. The method of any of claim 40 or claim 41, wherein the cell line is a mammalian cell line, optionally a CHO cell line.

43. The method of any of claims 40 to 42, wherein the cell line is cultured in a cell culture medium.

44. The method of any of claims 40 to 43, wherein the cell line is cultured under batch or fed-batch culture conditions, or perfusion culture conditions (with continuous or semi- continuous perfusion).

45. The method of claim 44, wherein the cell line is cultured under fed-batch conditions or intensified fed-batch culture conditions.

46. The method of any of claims 40 to 45, wherein (b) the contacting is performed not more than about 4 days before the end of (a) the culturing; optionally not more than 3 days before the end of (a) the culturing; further optionally not more than 2 days before the end of (a) the culturing.

47. The method of any of claims 40 to 46, wherein (b) the contacting is performed not more than about 1 day before the end of (a) the culturing; optionally wherein (b) the contacting is performed not more than about 0.5 days before the end of (a) the culturing.

48. The method of any of claims 40 to 47, wherein (b) the contacting is performed after end of (a) the culturing.

49. The method of any of claims 40 to 44, wherein the cell line is cultured under perfusion culture conditions, optionally wherein the perfusion culture conditions are semi- continuous perfusion or continuous perfusion.

50. The method of any of claims 40 to 44 or of claim 49, wherein (b) the contacting is performed intermittently during (a) the culturing.

51. The method of any of claims 40 to 44 or of claim 49, wherein (b) the contacting is performed throughout (a) the culturing.Docket No. P38734-WO 52. The method of any of claims 40 to 51, wherein the wherein the recombinant protein is an antibody or fragment thereof comprising a hinge region.

53. The method of claim 52, wherein the antibody or fragment thereof is an immunoglobulin G (IgG) or fragment thereof.

54. The method of claim 53, wherein the IgG is an IgG2 or an IgG4, optionally wherein the IgG is an IgG2.

55. The method of any of claims 40 to 54, wherein the recombinant protein is an IgG2 and the method comprises contacting with a sufficient level of reducing agent to increase the relative amount of IgG2-B disulfide structural isoform.

56. The method of any of claims 40 to 55, wherein the concentration of the reducing agent in the solution obtained by the (b) contacting step is at least about 1 mM, optionally at least about 2 mM, further optionally at least about 4 mM.

57. The method of any of claims 40 to 56, wherein the reducing agent comprises reduced sulfur, optionally wherein the reducing agent comprises an -SH moiety.

58. The method of any of claims 40 to 57, wherein the reducing agent is at least one antioxidant, optionally selected from 2-mercaptoethanol, alpha-ketoglutarate, alpha- tocopherol, ascorbic acid, citrate, cystamine, cysteamine, cysteine, cysteine hydrochloride, fumaric acid, glutathione, lactic acid, lipoic acid, hypotaurine, N- acetyl-cysteine, N-acetyl-cysteine amine, phenolic compounds, pyruvate, taurine, or a combination thereof.

59. The method of any of claims 40 to 58, wherein the reducing agent comprises cysteine and / or glutathione.

60. The method of claims 40 to 59, wherein the solution further comprises an oxidising agent.

61. The method of claim 60, wherein the oxidising agent comprises at least one metal ion.

62. The method of claim 61, wherein the metal ion is a transition metal ion, optionally selected from copper, iron, manganese, or zinc.

63. The method of claim 61 or claim 62, wherein the metal ion is an ion of copper (such as Cu2+) or iron (such as Fe3+or Fe2+).Docket No. P38734-WO 64. The method of any of claims 61 to 63, wherein the metal ion is at a concentration of from about 0.1 μM to about 5000 μM; optionally wherein the metal ion is at a concentration of from about 10 μM to about 500 μM further optionally wherein the metal ion is at a concentration of from about 50 μM to about 150 μM.

65. The method of any of claims 60 to 64, wherein the oxidising agent comprises an oxidised sulfur, optionally wherein the oxidised sulfur comprises an -S-S- moiety.

66. The method of claim 65, wherein the oxidised sulfur is selected from cystine, glutathione disulfide, S-sulfocysteine, or a derivative or combination thereof.

67. The method of any of claims 60 to 66, wherein the recombinant protein is an IgG2 and the solution comprises a sufficient level of oxidising agent to increase the relative amount of IgG2-A isoform.

68. A method for the production of a recombinant protein with modulated disulfide structural isoform distribution, comprising: (a) culturing a eukaryotic cell line that comprises a polynucleotide encoding the recombinant polypeptide under conditions suitable for production of the polypeptide; and (b) contacting the recombinant protein with a solution comprising an oxidising agent.

69. The method of claim 68, wherein the oxidising agent is as further defined in any of claims 61 to 67.

70. The method of claim 68 or claim 69, wherein the recombinant protein is as further defined in any of claims 52 to 54.

71. A method for the production of a recombinant protein that is an IgG2 having an increased amount of IgG2-B structural isoform, the method comprising: (a) culturing a eukaryotic cell line that comprises a polynucleotide encoding the recombinant polypeptide under conditions suitable for production of the polypeptide; and (b) contacting the recombinant protein with a solution comprising a reducing agent.Docket No. P38734-WO 72. The method of claim 71, wherein the reducing agent is as further defined in any of claims 56 to 59.

73. The method of claim 71 or 72, wherein the solution further comprises an oxidising agent; optionally wherein the oxidising agent is as further defined in any of claims 61 to 67.

74. A method for the production of a recombinant protein that is an IgG2 having an increased amount of IgG2-A structural isoform, the method comprising: (a) culturing a eukaryotic cell line that comprises a polynucleotide encoding the recombinant polypeptide under conditions suitable for production of the polypeptide; and (b) contacting the recombinant protein with a solution comprising an oxidising agent.

75. The method of claim 74, wherein the oxidising agent is as further defined in any of claims 61 to 67.

76. The method of any of claims 68 to 75, further comprising the features of any of claims 41 to 51.

77. A recombinant protein with modulated distribution of disulfide structural isoforms obtainable or obtained by the method of any preceding claim.

78. A method for enhancing disulfide bond rearrangement in a recombinant protein, comprising: contacting the recombinant protein with a solution comprising a reducing agent.

79. The method of claim 78, further comprising a feature as defined in any of claims 2 to 23.

80. Use of a reducing agent for modulation of disulfide structural isoform distribution by enhancing disulfide bond rearrangement in a recombinant protein.

81. Use of an oxidising agent for stabilizing disulfide structural isoform distribution by preventing disulfide bond rearrangement in a recombinant protein.

82. A recombinant protein, obtained or obtainable by the method of claim 78 or claim 79, or by the use of claim 80 or claim 81.

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