Reducing alpha-gal

Abstract

The disclosure relates to methods of producing a glycoprotein with reduced a-Gal content by reducing pH from 7.20 to 6.90, by increasing GlcNAc or zinc concentration and / or by reducing uridine or manganese concentration. Also provided are glycoproteins obtained or obtainable by said methods.

Description

REDUCING ALPHA-GAL

[0001] This disclosure relates to methods of producing a glycoprotein with reduced a-Gal content. Also provided are glycoproteins obtained or obtainable by said methods.BACKGROUND

[0002] Galactose-a-1 ,3-galactose (alpha-Gal or a-Gal) is a common carbohydrate structure expressed in mammalian tissues and is synthesised by the glycosylation enzyme a-1 ,3-galactosyltransferase (FIG. 1 ). Alpha-gal modifies extracellular proteins and lipids, and is expressed across all organs, tissues, and cell types to varying degrees in mammals, except in humans and Old-World primates who lack a functional gene encoding a-1 ,3-galactosyltransferase.

[0003] The absence of this gene in humans leads to the production of anti-Gal antibodies specific to the alpha-Gal antigen, to the extent that said anti-Gal antibodies make up approximately 1 % of all circulating immunoglobulins in humans. Thus, exposure to an alpha-Gal-containing glycan provokes an immunogenic response. Further, individuals with IgE isotype anti-Gal antibodies elicit anaphylaxis in response to exposure to alpha-Gal.

[0004] Due to the presence of some alpha-Gal glycans in some medications and vaccines, such biotherapeutics need to be carefully screened to avoid undesirable immunogenicity.

[0005] It is an aim of the present invention to provide a new approach for reducing alpha-Gal content in glycoprotein production.BRIEF SUMMARY OF THE DISCLOSURE

[0006] The invention provides methodology for producing a glycoprotein with reduced the a-Gal content.

[0007] In accordance with a first aspect of the present invention, there is provided a method of producing a glycoprotein with a reduced a-Gal content, comprising: culturing a eukaryotic cell line comprising a polynucleotide encodingthe polypeptide portion of the glycoprotein under conditions suitable for production of the glycoprotein, wherein the conditions suitable for production comprise one or more of: (a) a pH of below about 7.1 ; (b) a GIcNAc concentration in the culture of at least about 10 mM; (c) a zinc concentration in the culture of at least about 400 pM; (d) a uridine concentration in the culture of less than about 15 mM; and (e) a manganese concentration in the culture of less than about 400 nM.

[0008] Without wishing to be bound by theory, it is thought that controlling one or more of conditions (a) to (e) in the methods of the disclosure results in the production of a glycoprotein having reduced alpha-Gal content compared to a glycoprotein produced without control of said conditions.

[0009] In accordance with a second aspect of the present invention, there is provided a glycoprotein obtained or obtainable by the method of the first aspect.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:FIG. 1 is a schematic equation showing the synthesis of an alpha-gal- containing glycan (e.g. Gala1-3Gal[31-4GlcNAc-R) by reacting the required precursor (e.g. Gal[31-4GlcNAc-R), with a Gal containing moiety (e.g. UDP-Gal), in the presence of an enzyme (e.g. a1-3GT) (Macher et al. (2008)).FIG. 2A (upper panel), FIG. 2B (middle panel) and FIG. 2C (lower panel) show process indicators identified from screening DOE of the ligand fusion protein in the first cell line. Results from control (i.e. conditions that produced elevated alpha-gal), centerpoint (i.e. conditions that are the midpoint of control and test conditions for each parameter tested), and test cases are displayed. A range of growth profiles was observed. The cases containing a pH shift (shown in star makers) had reduced growth.FIG. 3 shows the total galactosylation(G1 F+G2F+G1 S1 F+G2S1 F+G2S2F) of control, centerpoint, and DOE test cases for the ligand fusion protein in the first cell line. Centerpoint conditions reduced total galactosylation by approximately 20% compared to the controls. DOE testconditions showed a range of total galactosylation, where cases with a pH shift (star makers) showed decreases in total galactosylation up to approximately 40%.FIG. 4A shows the total galactosylation(G1 F+G2F+G1 S1 F+G2S1 F+G2S2F), FIG. 4B the final viability, and FIG. 4C the volumetric integral of viable cell concentration (IVCC) for three cell lines, each adapted to express a different product. The first cell line expresses a ligand fusion protein, the second a complex antibody, and the third a complex bispecific antibody. In each case, a control process and process lever with and without a pH shift are compared. Process levers decreased total galactosylation by >10% in 2 of the 3 cell lines. pH shift with process levers further reduced galactosylation in all cell lines tested, ranging from 10 to 30% reduction across all of the cell lines.FIG. 5 shows how the relative abundance of alpha-gal containing glycan correlates with total galactosylation in the three cell lines. The samples were sialidase-treated and analyzed on a HILIC-MS method. Process levers that reduced total galactosylation also reduced abundance of alpha-gal.DETAILED DESCRIPTION

[0011] The abbreviations used herein have their conventional meaning within the chemical and biological arts.

[0012] 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.

[0013] 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 ofthe 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 and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0014] 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.

[0015] 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.

[0016] 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

[0017] 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.

[0018] 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.”

[0019] 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.

[0020] 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 measurement 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.

[0021] 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; and5) 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.

[0022] 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; and4) protein and tissue hydrolysates.

[0023] “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.

[0024] “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.

[0025] “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.

[0026] “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.

[0027] As used herein, the term “cell,” refers to animal cells, mammalian cells, cultured cells, host cells, recombinant cells and recombinant host cells. Such cells are generally cell lines obtained or derived from mammalian tissues which are able to grow and survive when placed in media containing appropriate nutrients and / or growth factors.

[0028] 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.

[0029] 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 refer 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.

[0030] 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 aparticular 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, llrlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 1980); 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., 23:243-251 1980); canine kidney cells (MDCK, ATCC CCL 34); ); 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.

[0031] “Growth phase” of the cell culture refers to the period of exponential cell growth (the log phase) where cells are generally rapidly dividing. The duration of time for which the cells are maintained at growth phase can vary based on the cell-type, the rate of growth of cells and / or the culture conditions, for example. In certain embodiments, during this phase, cells are cultured for a period of time, usually between 1-4 days, and under such conditions that cell growth is maximized. The determination of the growth cycle for the host cell can be determined for the particular host cell envisioned without undue experimentation. “Period of time and under such conditions that cell growth is maximized” and the like, refer to those culture conditions that, for a particular cell line, are determined to be optimal for cell growth and division. In certain embodiments, during the growth phase, cells are cultured in nutrient medium containing the necessary additives generally at about 30°-40°C in a humidified, controlled atmosphere, such that optimal growth is achieved for the particular cell line. In certain embodiments,cells are maintained in the growth phase for a period of about between one and four days, usually between two to three days.

[0032] “Production phase” of the cell culture refers to the period of time during which cell growth is / has plateaued. The logarithmic cell growth typically decreases before or during this phase and protein production takes over. During the production phase, logarithmic cell growth has ended, and protein production is primary. During this period of time the medium is generally supplemented to support continued protein production and to achieve the desired glycoprotein product. Fed-batch and / or perfusion cell culture processes supplement the cell culture medium or provide fresh medium during this phase to achieve and / or maintain desired cell density, viability and / or recombinant protein product titer. A production phase can be conducted at large scale.

[0033] 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 altering the nucleic acid sequence encoding the protein such that the resulting modified protein exhibits reduced or eliminated activity relative to a wild type protein.

[0034] 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 westernblotting 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.

[0035] 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.

[0036] 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 encompassed 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.

[0037] 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.

[0038] 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 antigenbinding activity. A therapeutic antibody is an antibody that may be used in the treatment of a disease.

[0039] 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).

[0040] 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.

[0041] 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), e.g., lgG1 , lgG2, lgG3, lgG4, lgA1 , and lgA2. In certain embodiments, the antibody is of the lgG1 isotype. In certain embodiments, the antibody is of the lgG2 isotype. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called a, 5, s, y and p, respectively. The light chain of an antibody can be assigned to one of two types, called kappa (K) and lambda (A), based on the amino acid sequence of its constant domain.

[0042] 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 a relative measurement, such as a percentage increase in titer as compared obtaining the protein product under different culture conditions.

[0043] 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.

[0044] 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 orderived 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 antigenbinding residues.

[0045] 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 constant 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.

[0046] 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.

[0047] As used herein (unless required otherwise by context), the term “specified level” refers to pH level of below about 7.1 recited in condition (a).

[0048] The following abbreviations are used herein:Alpha-Gal or a-Gal Galactose-a-1 ,3-galactoseDOE Design of experimentIVCC Integral of viable cell concentrationHILIC-MS Hydrophilic interaction liquid chromatography-mass spectrometryCHO Chinese hamster ovaryBHK Baby hamster kidneyBRL Buffalo rat liverMMT Mouse mammary tumorCDR Complementary determining regionFR Framework regionHVR Hypervariable regionVH Heavy chain variable domainVL Light chain variable domainUDP Uridine diphosphateUTP Uridine triphosphateGIcNAc N-acetyl glucosamineGALE UDP-glucose 4-epimerase / UDP-galactose 4-epimeraseUDP-Gal Uridine diphosphate galactoseMethods of Producing a Glycoprotein

[0049] In an aspect, the invention provides a method of producing a glycoprotein with a reduced a-Gal content, comprising: culturing a eukaryotic cell line comprising a polynucleotide encoding the polypeptide portion of the glycoprotein under conditions suitable for production of the glycoprotein, wherein the conditions suitable for production comprise one or more of: (a) a pH of below about 7.1 ; (b) a N-acetyl glucosamine (GIcNAc) concentration in the culture of at least about 10 mM; (c) a zinc concentration in the culture of at least about 400 pM; (d) a uridine concentration in the culture of less than about 15 mM; and (e) a manganese concentration in the culture of less than about 400 nM.

[0050] In embodiments, (a) the pH is from about 7.1 to about 6.7. In embodiments, (a) the pH is from about 7.1 to about 6.8. In embodiments, (a) the pH is from about 7.1 to about 6.9. The pH may be about 6.9.

[0051] In embodiments, the eukaryotic cell line is cultured under fed-batch culture conditions, wherein the condition (a) comprises shifting the pH from a higher pH to the pH of below about 7.1 . The higher pH may be a pH of from about 7.2 to about 7.4. The higher pH may be a pH of about 7.2.

[0052] In embodiments, the shifting the pH may be performed at least 2 days after culturing begins. In embodiments, the shifting the pH may be performed at least 4 days after culturing begins. In embodiments, the shifting the pH may be performed at least 6 days after culturing begins.

[0053] In embodiments, (a) comprises having the pH at the specified level for at least the last 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 days of the conditions suitable for production. In embodiments, (a) comprises having the pH at the specified level for at least the last 5, 6, 7, or 8 days of the conditions suitable for production. In embodiments, (a) comprises having the pH at the specified level for at least the last 6 days of the conditions suitable for production.

[0054] In embodiments, (a) comprises having the pH at the specified level for not more than the last 4, 5, 6, 7, 8, 9, 10, 11 or 12 days of the conditions suitable for production. In embodiments, (a) comprises having the pH at the specified level for not more than the last 5, 6, 7, or 8 days of the conditions suitable for production. In embodiments, (a) comprises having the pH at the specified level for not more than the last 6, 7, or 8 days of the conditions suitable for production.

[0055] Enzymes typically have optimal activities at a particular pH range for a given enzyme. Without wishing to be bound theory, it is thought that having the pH below about 7.1 increases the activity of galactosidases (e.g. CHO galactosidases have optimal activity at lower pH). Therefore having the pH at a lower level or reducing the pH as described in the present disclosure should reduce galactosylation, and thereby a-Gal levels.

[0056] In embodiments, (b) the GIcNAc concentration in the culture is from about 10 mM to about 50 mM. In embodiments, the GIcNAc concentration in the culture is from about 10 mM to about 25 mM. In embodiments, the GIcNAc concentration in the culture is from about 10 mM to about 20 mM. In embodiments, the GIcNAcconcentration in the culture is from about 10 mM to about 18 mM. Without wishing to be bound by theory, it is thought that supplementing the cell culture medium with GIcNAc reduces galactosyltransferase activity. In particular, the enzyme UDP-glucose 4-epimerase / UDP-galactose 4-epimerase (GALE) converts UDP-glucose to UDP-Gal and UDP-GIcNAc to UDP-GalNAc. It is thought that supplementing GIcNAc increases the amount of UDP-GIcNAc, which acts as a completive substrate for GALE and thereby decreases the production of UDP-Gal. Having a lower level of UDP-Gal is expected to reduce a-Gal levels.

[0057] In embodiments, (c) the zinc concentration in the culture is from about 400 pM to about 2,500 pM. In embodiments, (c) the zinc concentration in the culture is from about 800 pM to about 2,000 pM. In embodiments, (c) the zinc concentration in the culture is from about 1 ,200 pM to about 1 ,800 pM. In embodiments, (c) the zinc concentration in the culture is from about 1 ,200 pM to about 1 ,600 pM. Without wishing to be bound by theory, it is thought that zinc (e.g. Zn2+) acts as an inhibitor of galactosyltransferase, such that when the concentration of zinc in the culture is increased above a given level, decreased galactosyltransferase activity is achieved.

[0058] In embodiments, (d) the undine concentration in the culture is from about 0 mM to about 0.2 mM. In embodiments, (d) the undine concentration in the culture is from about 0 mM to about 0.15 mM. In embodiments, (d) the undine concentration in the culture is from about 0 mM to about 0.1 mM. In embodiments, (d) the uridine concentration in the culture is about 0 mM. Without wishing to be bound by theory, it is thought that decreasing the amount of undine may result in a decreased amount of UDP-Gal being produced. In particular, undine is a precursor to uridyltriphosphate (UTP), which is required to convert Gal-1 P to UDP- Gal, which provides the source of the a-Gal epitope in the production of a-Gal- containing glycans. By decreasing the amount of UDP-Gal, the amount of galactosylated glycan will also be reduced. Thus, it is thought that decreasing the amount of uridine results in decreased galactosyltransferase activity.

[0059] In embodiments, (e) the manganese concentration in the culture is from about 0 nm to about 400 nm. In embodiments, (e) the manganese concentrationin the culture is from about 0 nm to about 200 nm. In embodiments, (e) the manganese concentration in the culture is about 0 nm. Without wishing to be bound by theory, it is thought that manganese (e.g. Mn2+) is a cofactor of galactosyltransferase activity such that as the concentration of manganese (e.g. Mn2+) in the culture is decreased, decreased galactosyltransferase activity is achieved.

[0060] In embodiments, the conditions suitable for production comprise (a) and one or more of (b), (c), (d), (e) and (f). In embodiments, the conditions suitable for production comprise (a) and one or more of (b), (c), (d), and (e).

[0061] In embodiments, the conditions suitable for production comprise (d) and (e). In embodiments, the conditions suitable for production further comprise one or more of (a), (b), and (c).

[0062] In embodiments, the conditions suitable for production comprise (a) (b) and (c), and optionally (d) and / or (e). In embodiments, the conditions suitable for production comprise (a) (d) and (e).

[0063] In embodiments, the conditions suitable for production comprise (a) (b), (c), (d) and (e).

[0064] In embodiments, the glycoprotein is a therapeutic glycoprotein.

[0065] In embodiments, the polypeptide portion of the glycoprotein is a recombinant polypeptide.

[0066] In embodiments, the glycoprotein is a recombinant glycoprotein selected from a fusion protein (e.g. a ligand fusion protein), an antibody, an antigen, an enzyme, or a vaccine.

[0067] In embodiments, the antibody is a multispecific antibody or antigenbinding fragment thereof.

[0068] In embodiments, the antibody consists of a single heavy chain sequence and a single light chain sequence or antigen-binding fragments thereof.

[0069] In embodiments, the antibody comprises a chimeric antibody, a human antibody or a humanized antibody.

[0070] In embodiments, the antibody comprises a monoclonal antibody.

[0071] In embodiments, the method further comprises isolating the glycoprotein. The isolating the glycoprotein may be by any suitable method. For example, the isolating may comprise contacting the glycoprotein with a reagent having affinity for the glycoprotein, and optionally washing and / or eluting the glycoprotein from the reagent having affinity for the glycoprotein. The affinity reagent may be provided as part of a stationary phase. Examples of reagents that may have affinity for the glycoprotein include a HILIC stationary phase, or a lectin(s). Where the glycoprotein is a specific class of glycoprotein, the reagent having affinity for the glycoprotein may be selected to have affinity for the specific class. For example, where the glycoprotein is an antibody, the reagent having affinity may be selected from Protein G, Protein A, Protein A / G, Protein L, or a combination of one of more thereof.

[0072] In embodiments, the eukaryotic cell line is an animal cell line. In embodiments, the eukaryotic cell line is a mammalian cell line. For example, the mammalian cell line may be selected from a Chinese hamster ovary (CHO) cell line, a baby hamster kidney (BHK) cell line, a mouse sertoli cell line, a Madin- Darby canine kidney (MDCK) cell line, a buffalo rat liver (BRL) cell line, or a mouse mammary tumor cell line, or their derivatives.

[0073] In embodiments, the mammalian cell line is a modified mammalian cell line. For example, the modified mammalian cell line may be selected from CHO / DHFR (Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 1980), dp12.CHO (EP 307,247 published 15 Mar. 1989), CHO-K1 (ATCC, CCL-61 ), BHK ATCC CCL 10, mouse sertoli (TM4, Mather, Biol. Reprod., 23:243-251 1980), MDCK ATCC CCL 34, HEK 293, BRL 3A ATCC CRL 1442), MMT 060562 ATCC CCL51 , or their derivatives.

[0074] In embodiments, the cell line is a CHO cell line, or its derivative. For example, the cell line may be a CHO K1 cell line, a CHO K1 SV cell line, a DG44 cell line, a DUKXB-11 cell line, a CHOK1 S cell line, or a CHO K1 M cell line, or their derivatives.

[0075] In embodiments, the polynucleotide that encodes the polypeptide portion of the glycoprotein is either an extrachromosomal polynucleotide or an integrated polynucleotide that is integrated into a chromosome of the cells of the cell line. The polynucleotide that encodes the polypeptide portion of the glycoprotein may be an extrachromosomal polynucleotide, the polynucleotide that encodes the polypeptide portion of the glycoprotein is may be integrated into a chromosome of the cells of the cell line. In embodiments where the polynucleotide is an integrated polynucleotide, the integrated polynucleotide may be either a random integration or a targeted integration.

[0076] In embodiments, the eukaryotic cell line is cultured in a cell culture medium.

[0077] In embodiments, the eukaryotic cell line is cultured under batch or fed- batch culture conditions, or perfusion culture conditions (with continuous or semi- continuous perfusion). The fed-batch culture conditions may be intensified fed- batch culture conditions.

[0078] In embodiments, the eukaryotic cell line is cultured under perfusion culture conditions. The perfusion culture conditions may be semi-continuous perfusion or continuous perfusion. As the skilled person would appreciate, where the pH is at a specified level in perfusion culture, the pH may be at the specified level for the duration of the perfusion culture, rather than shifting the pH from a higher pH to a lower pH at during the perfusion culture.

[0079] In embodiments, the reduced a-Gal content comprises a reduction in a- Gal content of at least about 20% when compared to a corresponding control method of producing the glycoprotein, said control method not comprising any of (a), (b), (c), (d) or (e).

[0080] In embodiments, the reduction in a-Gal content is at least about 50%. In embodiments, the reduction in a-Gal content is at least about 75%.

[0081] In embodiments, the reduction in a-Gal content is calculated after determining the level of a-Gal containing glycans using a hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) protocol disclosed herein.

[0082] In embodiments, the recombinant polypeptide is an antibody, an antigen, an enzyme, a gene vector or a vaccine.

[0083] In embodiments, the recombinant polypeptide is a therapeutic antibody.

[0084] 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-lgE 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 a4[37 integrin antibodies, anti-human av[38 integrin antibodies, anti-av[33 antibodies including either a or [3 or subunits thereof (e.g. anti-CD11 a, anti-CD18 or anti- CD11 b 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-Gpl Ib / llla 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- lgE; 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; antiparathyroid hormone; anti-thyroid stimulating hormone; anti-lipoproteins; anti- a-1 -antitrypsin; anti-insulin A-chain; anti-insulin B-chain; anti-proinsulin; anti-follicle stimulating hormone; anti-calcitonin; anti-luteinizing hormone; anti-glucagon; anticlotting 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-a and -[3; anti- enkephalinase; RANTES (regulated on activation normally T-cell expressed and secreted); anti-human macrophage inflammatory protein (Ml P-1 -a); 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; antireceptors 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-[3; platelet-derived growth factor (PDGF); fibroblast growth factor such as aFGF and bFGF; epidermal growth factor (EGF); antitransforming growth factor (TGF) such as TGF-a and TGF-[3, including TGF-[31 , TGF-[32, TGF-[33, TGF-[34, or TGF-[35; anti- insulin-like growth factor-l and -II (IGF-I and IGF-II); anti- des(1-3)-IGF-l (brain IGF-I); insulin-like growth factor binding proteins (IGFBPs); anti- erythropoietin (EPO); anti- thrombopoietin (TPO); anti- osteoinductive factors; anti- immunotoxins; anti- a bone morphogenetic protein (BMP); anti- an interferon such as interferon-a, -|3, and -y; 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.

[0085] In embodiment, the therapeutic antibody is selected from AVASTIN® (bevacizumab), HERCEPTIN® (trastuzumab), LUCENTIS® (ranibizumab), 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- av[38 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.Glycoprotein

[0086] In another aspect, there is provided a glycoprotein obtained or obtainable by a method of the disclosure.

[0087] The methods of the present disclosure involve the production of glycoproteins, such as antibodies and fusion proteins; or conjugates thereof. Where the glycoprotein is an antibody, the antibody may be, but is not limited to, a monospecific antibody (e.g., an antibody consisting of a single heavy chain sequence and a single light chain sequence, including multimers of such pairings), a multispecific antibody and antigen-binding fragments thereof.Multispecific Antibodies

[0088] An antibody may be a multispecific antibody, e.g., a bispecific antibody. “Multispecific antibodies” are monoclonal antibodies that have binding specificities for at least two different sites, i.e. , different epitopes on different antigens (i.e. , bispecific) or different epitopes on the same antigen (i.e., biepitopic). The multispecific antibody may have three or more binding specificities. Multispecific antibodies can be prepared as full length antibodies or antibody fragments as described herein.

[0089] Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537 (1983)) and “knob-in-hole” engineering (see, e.g., U.S. Patent No. 5,731 ,168, and Atwell et al., J. Mol. Biol. 270:26 (1997)). Multispecific antibodies can also be made by engineering electrostatic steering effects for making antibody Fc- heterodimeric molecules (see, e.g., WO 2009 / 089004); cross-linking two or more antibodies or fragments (see, e.g., US Patent No. 4,676,980, and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to produce bi-specific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5): 1547-1553 (1992) and WO 2011 / 034605); using the common light chain technology for circumventing the light chain mis-pairing problem (see, e.g., WO 98 / 50431 ); using “diabody” technology for making bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)); and preparing trispecific antibodies as described, e.g., in Tutt et al. J. Immunol. 147: 60 (1991 ).

[0090] Engineered antibodies with three or more antigen binding sites, including for example, “Octopus antibodies”, or DVD-lg are also included herein (see, e.g., WO 2001 / 77342 and WO 2008 / 024715). Other non-limiting examples of multispecific antibodies with three or more antigen binding sites can be found in WO 2010 / 115589, WO 2010 / 112193, WO 2010 / 136172, WO 2010 / 145792 and WO 2013 / 026831 . The bispecific antibody or antigen binding fragment thereof also includes a “Dual Acting FAb” or “DAF” (see, e.g., US 2008 / 0069820 and WO 2015 / 095539.

[0091] Multispecific antibodies may also be provided in an asymmetric form with a domain crossover in one or more binding arms of the same antigen specificity, i.e., by exchanging the VH / VL domains (see, e.g., WO 2009 / 080252 and WO 2015 / 150447), the CH1 / CL domains (see, e.g., WO 2009 / 080253) or the complete Fab arms (see, e.g., WO 2009 / 080251 , WO 2016 / 016299, also see Schaefer et al, PNAS, 108 (2011 ) 1187-1191 , and Klein at al. , MAbs 8 (2016) 1010-20). Amultispecific antibody may comprise a cross-Fab fragment. The term “cross-Fab fragment” or “xFab fragment” or “crossover Fab fragment” refers to a Fab fragment, wherein either the variable regions or the constant regions of the heavy and light chain are exchanged. A cross-Fab fragment comprises a polypeptide chain composed of the light chain variable region (VL) and the heavy chain constant region 1 (CH1 ), and a polypeptide chain composed of the heavy chain variable region (VH) and the light chain constant region (CL). Asymmetrical Fab arms can also be engineered by introducing charged or non-charged amino acid mutations into domain interfaces to direct correct Fab pairing. See, e.g., WO 2016 / 172485.

[0092] Various further molecular formats for multispecific antibodies are known in the art and are included herein (see, e.g., Spiess et al., Mol. Immunol. 67 (2015) 95-106).

[0093] A particular type of multispecific antibodies, also included herein, are bispecific antibodies designed to simultaneously bind to a surface antigen on a target cell, e.g., a tumor cell, and to an activating, invariant component of the T cell receptor (TCR) complex, such as CD3, for retargeting of T cells to kill target cells.

[0094] Additional non-limiting examples of bispecific antibody formats that can be useful for this purpose include, but are not limited to, the so-called “BiTE” (bispecific T cell engager) molecules wherein two scFv molecules are fused by a flexible linker (see, e.g., WO 2004 / 106381 , WO 2005 / 061547, WO 2007 / 042261 , and WO 2008 / 119567, Nagorsen and Bauerle, Exp Cell Res 317, 1255-1260 (2011 )); diabodies (Holl iger et al., Prot. Eng. 9, 299-305 (1996)) and derivatives thereof, such as tandem diabodies (“TandAb”; Kipriyanov et al., J Mol Biol 293, 41-56 (1999)); “DART” (dual affinity retargeting) molecules which are based on the diabody format but feature a C-terminal disulfide bridge for additional stabilization (Johnson et al., J Mol Biol 399, 436-449 (2010)), and so-called triomabs, which are whole hybrid mouse / rat IgG molecules (reviewed in Seimetz et al., Cancer Treat. Rev. 36, 458-467 (2010)). Particular T cell bispecific antibodyformats included herein are described in WO 2013 / 026833, WO 2013 / 026839, WO 2016 / 020309; Bacac et al., Oncoimmunology 5(8) (2016) e1203498.Antibody Fragments

[0095] The glycoprotein may be an antibody fragment. For example, but not by way of limitation, the antibody fragment may be a Fab, Fab’, Fab’-SH or F(ab’)2 fragment, in particular a Fab fragment. Papain digestion of intact antibodies produces two identical antigen-binding fragments, called “Fab” fragments containing each the heavy- and light-chain variable domains (VH and VL, respectively) and also the constant domain of the light chain (CL) and the first constant domain of the heavy chain (CH1 ). The term “Fab fragment” thus refers to an antibody fragment comprising a light chain comprising a VL domain and a CL domain, and a heavy chain fragment comprising a VH domain and a CH1 domain. “Fab’ fragments” differ from Fab fragments by the addition of residues at the carboxy terminus of the CH1 domain including one or more cysteines from the antibody hinge region. Fab’-SH are Fab’ fragments in which the cysteine residue(s) of the constant domains bear a free thiol group. Pepsin treatment yields an F(ab’)2 fragment that has two antigen-binding sites (two Fab fragments) and a part of the Fc region. For discussion of Fab and F(ab’)2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Patent No. 5,869,046.

[0096] The antibody fragment may be a diabody, a triabody or a tetrabody. “Diabodies” are antibody fragments with two antigen-binding sites that can be bivalent or bispecific. See, for example, EP 404,097; WO 1993 / 01161 ; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0097] The antibody fragment may be a single chain Fab fragment. A “single chain Fab fragment” or “scFab” is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody heavy chain constant domain 1 (CH1 ), an antibody light chain variable domain (VL), an antibody light chain constantdomain (CL) and a linker, wherein said antibody domains and said linker have one of the following orders in N-terminal to C-terminal direction: a) VH-CH1-linker-VL- CL, b) VL-CL-linker-VH-CH1 , c) VH-CL-linker-VL-CH1 or d) VL-CH1-linker-VH-CL. In particular, said linker may be a polypeptide of at least 30 amino acids, preferably between 32 and 50 amino acids. Said single chain Fab fragments are stabilized via the natural disulfide bond between the CL domain and the CH1 domain. In addition, these single chain Fab fragments might be further stabilized by generation of interchain disulfide bonds via insertion of cysteine residues (e.g., position 44 in the variable heavy chain and position 100 in the variable light chain according to Kabat numbering).

[0098] The antibody fragment may be a single-chain variable fragment (scFv). A “single-chain variable fragment” or “scFv” is a fusion protein of the variable domains of the heavy (VH) and light chains (VL) of an antibody, connected by a linker. In particular, the linker may be a short polypeptide of 10 to 25 amino acids and is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa. This protein retains the specificity of the original antibody, despite removal of the constant regions and the introduction of the linker. For a review of scFv fragments, see, e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93 / 16185; and U.S. Patent Nos. 5,571 ,894 and 5,587,458.

[0099] The antibody fragment may be a single-domain antibody. “Single-domain antibodies” are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. A single-domain antibody may be a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B1 ).

[0100] Antibody fragments may be made by various techniques, including but not limited to proteolytic digestion of an intact antibody.Chimeric and humanized antibodies

[0101] The antibody may be a chimeric antibody. Certain chimeric antibodies are described, e.g., in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81 :6851 -6855 (1984)). The chimeric antibody may comprise a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, or rabbit) and a human constant region. It may be that the chimeric antibody is a “class switched” antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigenbinding fragments thereof.

[0102] A chimeric antibody may be a humanized antibody. Typically, a non- human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody.Generally, a humanized antibody comprises one or more variable domains in which the CDRs (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally will also comprise at least a portion of a human constant region. In certain embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve antibody specificity or affinity.Humanized antibodies and methods of making them are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and are further described, e.g., in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat’l Acad. Sci. USA 86:10029-10033 (1989); US Patent Nos. 5, 821 ,337, 7,527,791 , 6,982,321 , and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991 ) (describing “resurfacing”); Dall’Acqua et al., Methods 36:43-60 (2005) (describing “FR shuffling”); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing the “guided selection” approach to FR shuffling).Commercial Manufacturing

[0103] The methods of the present disclosure may be employed in the production of a molecule of interest at manufacturing scale. “Manufacturing scale" production of therapeutic proteins, or other proteins, utilize cell cultures ranging from about 400 L to about 80,000 L, depending on the protein being produced and the need. Typically, such manufacturing scale production utilizes cell culture sizes from about 400 L to about 25,000 L. Within this range, specific cell culture sizes, such as about 4,000 L, about 6,000 L, about 8,000, about 10,000, about 12,000 L, about 14,000 L, about 16,000 L or about 25,000 L may be utilized.

[0104] The methods of the present disclosure may be employed to support the high-density growth of mammalian cells in suspension cell culture, and I or to support the expression of recombinant protein from mammalian cells, and I or in the production of large quantities of a molecule of interest in a shorter timeframe as compared to conventional media or media used previously in cell culture. In certain embodiments, the methods of the present disclosure can be employed for improved quality of the molecule of interest as compared to conventional methods used previously in cell culture. In certain embodiments, the methods of the present disclosure can be used for the optimal expression of cell culture products such as polypeptides, proteins, antibodies (monoclonal, bispecific, trispecific, multispecific, etc.) antibody fragments, etc.Manufacture of Antibodies using the Media of the present Disclosure

[0105] Therapeutic antibodies that can be produced in the media described in this disclosure include, without limitation, 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-lgE 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 a4[37 integrin antibodies, anti-human av[38 integrin antibodies, anti-av[33 antibodies including either a or [3 or subunits thereof (e.g. anti-CD11 a, anti-CD18 or anti-CD11 bantibodies); 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-Gpl Ib / llla 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. Other exemplary proteins to which therapeutic antibodies are designed include 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); growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoproteins; a-1 -antitrypsin; insulin A- chain; insulin B-chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; clotting factors such as factor VIIIC, tissue factor or von Willebrands factor; anti-clotting factors such as Protein C; atrial natriuretic factor; lung surfactant; a plasminogen activator, such as urokinase or tissue-type plasminogen activator (t-PA); bombazine; thrombin; tumor necrosis factor-a and - [3; enkephalinase; RANTES (regulated on activation normally T-cell expressed and secreted); human macrophage inflammatory protein (MIP-1-a); serum albumin such as human serum albumin (HSA); mullerian-inhibiting substance; relaxin A-chain; relaxin B-chain; prorelaxin; mouse gonadotropin-associated peptide; DNase; inhibin; activin; receptors for hormones or growth factors; protein A or D; rheumatoid factors; 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-P; platelet-derived growth factor (PDGF); fibroblast growth factor such as aFGF and bFGF; epidermal growth factor (EGF); transforming growth factor (TGF) such as TGF-a and TGF-p, including TGF-|31 , TGF-[32, TGF-[33, TGF-[34, or TGF-[35; insulin-like growthfactor-1 and -II (IGF-I and IGF-II); des(1-3)-IGF-l (brain IGF-I); insulin-like growth factor binding proteins (IGFBPs); erythropoietin (EPO); thrombopoietin (TPO); osteoinductive factors; immunotoxins; a bone morphogenetic protein (BMP); an interferon such as interferon-a, -|3, and -y; colony stimulating factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1 to IL-10; superoxide dismutase; T-cell receptors; surface membrane proteins; decay accelerating factor (DAF); a viral antigen such as, for example, a portion of the AIDS envelope; transport proteins; homing receptors; addressins; regulatory proteins; immunoadhesins; and biologically active fragments or variants of any of the above-listed polypeptides. Many other antibodies and / or other proteins may be used in accordance with the instant disclosure, and the above lists are not meant to be limiting.

[0106] Therapeutic antibodies of particular interest include those that are commercially available or are in clinical development such as: AVASTIN® (bevacizumab), HERCEPTIN® (trastuzumab), LUCENTIS® (ranibizumab), 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- av[38 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 ), rhuMAb IFN alpha, etc. Many other antibodies and / or otherproteins may be used in accordance with the instant disclosure, and the above lists are not meant to be limiting.EXAMPLES

[0107] 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

[0108] Recombinant CHO cell lines expressing three different therapeutic proteins were used. For small-scale studies, cells were thawed and expanded to inoculate production cultures in miniaturized systems, either Sartorius Ambr®15 or Ambr®250 systems (Sartorius, Edgewood, NY). The temperature, pH, and dissolved oxygen (DO) in the bioreactors were controlled using their, respective, Sartorious ambr15 and Sartorius ambr250 softwares. Temperature, pH, and DO for all fed-batch production cultures were maintained at 35°C, 7.20, and 30% (of air saturation); and at 48 hours, temperature setpoint was shifted to 33°C and was maintained thereafter. Concentrated nutrient feeds were added to the production cultures at 24, 96 and 192 hours post-inoculation. Cultures were maintained for a total duration of 12 days.

[0109] In various test cases, pH was shifted to 6.90 at 48 or 144 hours in culture. Depending on the test condition, uridine, N-acetyl glucosamine (GIcNAc), manganese, and zinc were excluded from the basal media or added to the culture at the time of inoculation.

[0110] All cultures were sampled multiple times over the course of each production run and analyzed for VCD, viability pH, dissolved oxygen, glucose, lactate, and ammonium levels using the BioProfile FLEX2 (Nova Biomedical, Waltham, MA). Culture supernatants were stored frozen at -80°C until they wereanalyzed for product titer, and glycan distributions. A high throughput HILIC glycan method was used to screen samples, enabling selection for submission to the alpha-gal method for further characterization.Example 1 : Alpha-Gal Method for the determination of glycoform levels

[0111] Alpha-Gal represents a non-human glycosylation pattern. The levels of alpha-Gal containing glycans were determined by hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) analysis of sialidase treated glycans. In this analysis, glycans were first treated with sialidase to remove sialic acids and then enzymatically released from the protein by treatment with PNGase F. The released glycans were subsequently labeled with a procaine-based IPC fluorophore (InstantPC, Agilent Technologies) prior to their separation by hydrophilic interaction liquid chromatography. Relative quantification of the labeled glycans was accomplished by integration of the glycan fluorescence signals and identification of the separated glycans was determined by mass spectrometry.

[0112] While the above method was used in the present disclosure, there are also methods for determination of glycoforms that are known to the person of skill in the art, for example methods as set out in L. Zhang et al., “Glycan analysis of therapeutic glycoproteins”, MABS, 2016, Vol. 8, No. 2, 205-215.Example 2: Effective process parameters on alpha-Gal level in ligand fusion protein, complex antibody and complex bispecific antibody

[0113] Alpha-Gal-containing glycans have a terminal Gala1-3Gal sequence, and expression is catalyzed by the a1-3-galactosyltransferase enzyme using UDP-Gal and a glycoprotein as substrates (FIG. 1 ). Because galactosylated glycan is a required substrate for alpha-gal epitope addition, reducing abundance of total galactosylation should also reduce abundance of alpha-gal-containing glycan.

[0114] A screening study was used to identify process levers that influence galactosylation and therefore alpha-gal-containing glycans. Experimental design methodology and statistical methods used for analysis are detailed in "Design ofExperiments" (Douglas Montgomery, 8thEdition, 2012, John Wiley & Sons). Statistical software JMP 16 was used for both experimental design and analysis. A 16-case quarter-fractional factorial screening experimental design was used, with additional center point cases included, using a cell line that produces a ligand fusion protein. This design was powered to estimate the main effects and some interactions.

[0115] pH conditions tested included: no shift where pH was controlled at 7.20 for the duration of the production culture or pH controlled at 7.20 from production inoculation until 48 hours where the pH control shifted to 6.90 for the remainder of the culture. Media additives were also screened with GIcNAc ranging from 0 to 18 mM, zinc ranging from 111 to 800 pM, undine from 0 to 0.32 mM, manganese from 0 to 850 nM, and copper from 0.001 to 0.003 mM. Center point cases reduced total galactosylation by approximately 20% compared to the experimental control cases, and DOE test cases that included a pH shift showed the greatest changes in galactosylation, up to roughly 40%. As shown by the parameter effect estimates in Table 1 , implementing a low pH shift to 6.9048 hrs after inoculation showed the greatest impact, reducing total galactosylation by 18% compared to a constant pH condition of 7.20. Increasing GIcNAc and zinc concentrations also reduced total galactosylation. The interaction of undine and manganese together also influenced galactosylation.

[0116] Process levers were combined in a subsequent study using three cell lines that each produced ligand fusion protein (first cell line), complex antibody (second cell line) and complex bispecific antibody (third cell line), respectively. Three cases were tested for each cell line: (1 ) control conditions with constant pH setpoint of 7.20 (no pH shift), 0 mM GIcNAc, 111 pM zinc, 1 pM copper, and 0.32 mM uridine, (2) a combination of process levers at 18 mM GIcNAc, 1600 pM zinc, 3 mM copper, and 0 mM undine without pH shift and (3) a combination of process levers at 18mM GIcNAc, 1600 uM zinc, 3mM copper, OmM undine and pH shift from 7.20 to 6.90 on hour 144 of culture. Process levers with and without low pH shift reduced galactosylation in the three cell lines tested. pH shift reducedgalactosylation further in all cases, showing these levers are impactful across multiple cell lines and molecule formats (FIG. 3).Table 1 - parameter estimates from DOE analysis for total glycosylation.Implementing a pH shift, adding GIcNAc, zinc or manganese, and the removal of uridine were shown to decrease total glycosylation.

[0117] Sample characterization by the described HILIC-MS assay indicated that the total galactosylation levels correlate to relative abundance of alpha-gal- containing glycans (FIG. 4). Test cases employing process lever reduced alphagal compared to their respective control cases.

Claims

WHAT IS CLAIMED IS:1 . A method of producing a glycoprotein with a reduced a-Gal content, comprising: culturing a eukaryotic cell line comprising a polynucleotide encoding the polypeptide portion of the glycoprotein under conditions suitable for production of the glycoprotein; wherein the conditions suitable for production comprise one or more of:(a) a pH of below about 7.1 ;(b) a GIcNAc concentration in the culture of at least about 10 mM;(c) a zinc concentration in the culture of at least about 400 pM;(d) a undine concentration in the culture of less than about 15 mM; and(e) a manganese concentration in the culture of less than about 400 nM.

2. The method of claim 1 , wherein (a) the pH is from about 7.1 to about 6.7: optionally the pH is about 6.9.

3. The method of claim 1 or claim 2, wherein the eukaryotic cell line is cultured under fed-batch culture conditions, wherein the condition (a) comprises shifting the pH from a higher pH to the pH of below about 7.1 .

4. The method of any preceding claim, wherein (a) comprises having the pH at the specified level for at least the last 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 days of the conditions suitable for production; optionally wherein (a) comprises having the pH at the specified level for at least the last 4, 5, 6, 7, or 8 days of the conditions suitable for production.

5. The method of any preceding claim, wherein (a) comprises having the pH at the specified level for not more than the last 4, 5, 6, 7, 8, 9, 10, 11 or 12 days of the conditions suitable for production; optionally wherein (a) comprises having the pH at the specified level for not more than the last 5, 6, 7, or 8 days of the conditions suitable for production.

6. The method of any preceding claim, wherein (b) the GIcNAc concentration in the culture is from about 10 mM to about 50 mM;optionally wherein the GIcNAc concentration in the culture is from about 10 mM to about 20 mM.

7. The method of any preceding claim, wherein (c) the zinc concentration in the culture is from about 400 pM to about 2,500 pM; optionally the zinc concentration in the culture is from about 800 pM to about 2,000 pM; further optionally the zinc concentration in the culture is from about 1 ,200 pM to about 1 ,800 pM.

8. The method of any preceding claim, wherein (d) the undine concentration in the culture is from about 0 mM to about 0.2 mM; optionally wherein the uridine concentration in the culture is from about 0 mM to about 0.1 mM; further optionally wherein the uridine concentration in the culture is about 0 mM.

9. The method of any preceding claim .wherein (e) the manganese concentration in the culture is from about 0 nm to about 400 nm; optionally wherein the manganese concentration in the culture is from about 0 nm to about 200 nm; further optionally wherein the manganese concentration in the culture is about 0 nm.

10. The method of any preceding claim, wherein the conditions suitable for production comprise (a) and one or more of (b), (c), (d), and (e).11 . The method of any preceding claim, wherein the conditions suitable for production comprise (d) and (e); optionally further comprising one or more of (a), (b), and (c).

12. The method of any preceding claim, wherein the glycoprotein is a therapeutic glycoprotein.

13. The method of any preceding claim, wherein the polypeptide portion of the glycoprotein is a recombinant polypeptide.

14. The method of any preceding claim, wherein the glycoprotein is a recombinant glycoprotein selected from a fusion protein, an antibody, an antigen, an enzyme, or a vaccine.

15. The method of claim 14, wherein the antibody is a multispecific antibody or antigen-binding fragment thereof.

16. The method of claim 14 or claim 15, wherein the antibody consists of a single heavy chain sequence and a single light chain sequence or antigen-binding fragments thereof.

17. The method of any of claims 14-16, wherein the antibody comprises a chimeric antibody, a human antibody or a humanized antibody.

18. The method of any of claims 14-17, wherein the antibody comprises a monoclonal antibody.

19. The method of any preceding claim, further comprising isolating the glycoprotein.

20. The method of any preceding claim, wherein the eukaryotic cell line is cultured in a cell culture medium.21 . The method of any preceding claim, wherein the eukaryotic cell line is an animal cell line.

22. The method of any preceding claim, wherein the eukaryotic cell line is a mammalian cell line; optionally wherein the mammalian cell line is a modified mammalian cell line.

23. The method of any preceding claim, wherein the cell line is a CHO cell line, or its derivative.

24. The method of any preceding claim .wherein the polynucleotide that encodes the polypeptide portion of the glycoprotein is either an extrachromosomal polynucleotide or an integrated polynucleotide that is integrated into a chromosome of the cells of the cell line.

25. The method of claim 24, wherein the integrated polynucleotide is either a random integration or a targeted integration.

26. The method of any preceding claim, wherein the eukaryotic cell line is cultured under batch or fed-batch culture conditions, or perfusion culture conditions (with continuous or semi-continuous perfusion).

27. The method of claim 24, wherein the eukaryotic cell line is cultured under fed- batch culture conditions; optionally wherein the fed-batch culture conditions are intensified fed-batch culture conditions.

28. The method of any of claims 1 , 2 or 6-24, wherein the eukaryotic cell line is cultured under perfusion culture conditions; optionally wherein the perfusion culture conditions are semi-continuous perfusion or continuous perfusion.

29. The method of any preceding claim, wherein the reduced a-Gal content comprises a reduction in a-Gal content of at least about 20% when compared to a corresponding control method of producing the glycoprotein, said control method not comprising any of (a), (b), (c), (d) or (e).

30. The method of claim 27, wherein the reduction in a-Gal content is at least about 50%; optionally wherein the reduction in a-Gal content is at least about 75%.31 . The method of claim 27 or 28, wherein the reduction in a-Gal content is calculated after determining the level of a-Gal containing glycans using a hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) protocol disclosed herein.

32. A glycoprotein obtained or obtainable by the method of any of claims 1 to 29.

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