Optimization of polypeptide sialic acid content

Abstract

The present inventors provide a method for optimizing the sialic acid content of a recombinant polypeptide. The method includes culturing a cell line engineered to express the recombinant polypeptide under conditions that promote production of the recombinant polypeptide; and monitoring the cell viability of the cultured cell line on at least one of days 7, 8, 9, 10, 11, or 12 of culturing. If the cell viability is below a threshold level, the method also includes stopping the culturing and isolating the recombinant polypeptide after a first predetermined additional time. If the cell viability is above the threshold level, the method also includes stopping the culturing and isolating the recombinant polypeptide after a second predetermined additional time, the second predetermined additional time being at least about 12 hours longer than the first predetermined time.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 431,945, filed December 12, 2022, the entire contents of which are incorporated by reference.

[0002] The present disclosure relates to methods for optimizing the sialic acid content of recombinant polypeptides. [Background technology]

[0003] Commercially important proteins, such as therapeutic proteins, enzymes, and antibodies, can be produced by cell culture. In such cell cultures, a host cell line contains the genetic material encoding the protein of interest. Under ideal conditions, the host cell line expresses the protein of interest. However, variations in post-translational modifications of the expressed protein can affect the quality of the produced protein. This is particularly important for biopharmaceuticals, where product specifications typically specify quality attributes related to post-translational modifications.

[0004] Many important biopharmaceutical protein products are typically produced by culturing cells in bioreactors and purifying the desired product from the extracellular medium. The cell culture process utilizes live cells, typically requiring highly skilled personnel, and because animal cells grow slower than many common contaminants (e.g., bacteria, viruses, and fungi), the technique must be performed using strict aseptic techniques. Furthermore, even relatively modest changes to cell or culture conditions can result in a reduction in the quality of the protein product. One example of a post-translational modification related to critical product quality is the level of sialic acid present in the resulting final protein / polypeptide. Direct analysis of sialic acid levels in expressed proteins is challenging and time-consuming.

[0005] It would therefore be desirable to provide improved and / or more timely methods for optimizing the levels of sialic acid in recombinant polypeptide / protein products. Summary of the Invention

[0006] There remains a need to provide a method for optimizing the sialic acid content of a recombinant polypeptide. "Optimization" in this context means obtaining a recombinant polypeptide product, such as a therapeutic protein (such as an antibody or antibody-related polypeptide), having a desired level of sialic acid per molecule. For example, many recombinant polypeptides (such as therapeutic proteins) have specifications that require the sialic acid level to be at a particular level, in which case optimization involves having the sialic acid content of the recombinant polypeptide within the specifications.

[0007] In the methods used to culture recombinant proteins, the level of sialic acid content often decreases over the culture period. Such a decrease can result in a recombinant polypeptide product with suboptimal sialic acid levels. For example, a therapeutic drug can be unusable because the sialic acid level is below specification. However, even when the same cell culture method is used, due to inherent process variability, the time point can vary from culture to culture. This means that it can be difficult to obtain a recombinant polypeptide product with optimal levels of sialic acid, especially when considering obtaining a high-titer product.

[0008] The present inventors have noted that cell viability predicts sialic acid levels. In particular, the present inventors have determined that cell viability determined at a relatively early stage of cell culture (e.g., one of days 6 to 11) predicts optimal sialic acid levels at a relatively later stage of cell culture (e.g., one or more of days 9 to 14). With this in mind, the present inventors have developed exemplary methods of the present invention.

[0009] The present invention provides a method for optimizing the sialic acid content of a recombinant polypeptide. The method includes culturing a cell line engineered to express the recombinant polypeptide under conditions promoting production of the recombinant polypeptide; and monitoring cell viability of the cultured cell line on at least one of days 6, 7, 8, 9, 10, 11, or 12 of culturing. If cell viability is below a threshold level, the method also includes stopping the culturing and isolating the recombinant polypeptide after a first predetermined additional time. If cell viability is above the threshold level, the method also includes stopping the culturing and isolating the recombinant polypeptide after a second predetermined additional time, the second predetermined additional time being at least about 10 hours longer than the first predetermined time. [Brief explanation of the drawings]

[0010] Hereinafter, embodiments of the present invention will be further described with reference to the accompanying drawings. [Figure 1] This figure provides an example of sialic acid levels of recombinant Fc fusion proteins obtained from cell cultures from about 9 to about 14 days. The Fc fusion proteins had eight N-linked glycosylation sites. The cell cultures comprised a CHO cell line engineered to express the Fc fusion proteins. As shown, the sialic acid levels (moles sialic acid / moles recombinant protein) decreased over time. [Figure 2] Figure 1 shows a typical progression of exemplary abundant glycan morphologies for recombinant Fc-fusion proteins obtained from cell cultures from about 9 to about 14 days. Circles represent galactose (Gal), pentagons represent mannose (Man), squares represent N-acetylglucosamine (GlcNAc), triangles represent fucose (Fuc), and diamonds represent sialic acid (NAc). [Figure 3] A general correlation was identified between cell viability and sialic acid levels in culture. [Figure 4] 1 shows the correlation between cell viability in cell cultures on days 6, 7, 8, 9, 10, and 11 and the level of sialic acid on day 12. [Figure 5] The correlation between viability measured on days 8, 9, and 10 of culture and the level of sialylated glycan species (G2-1SA) at the end of culture (day 12) is shown. G2-1SA is normalized to the highest G2-1SA reported in the optimally sialylated antibody sample set (i.e., a normalized G2-1SA of 0.50 is 50% of the highest G2-1SA value reported in the data set). [Figure 6] A diagram of the correlation between normalized G2-1SA and culture duration is provided, showing that viability measurements earlier in the culture can be used to control the level of sialylation at the end of the culture by varying the culture duration based on earlier viability measurements. DETAILED DESCRIPTION OF THE INVENTION

[0011] Throughout this specification and the claims, the terms "comprise" and "contain" and variations thereof mean "including but not limited to," and they are not intended to (and do not) exclude other moieties, additives, components, integers, or steps. Throughout this specification and the claims, the singular encompasses the plural unless the context requires otherwise. In particular, when the indefinite article is used, the specification should be understood to contemplate plural as well as singular, unless the context requires otherwise.

[0012] It should be understood that any feature, integer, property, compound, chemical moiety, or group described in connection with a particular aspect, embodiment, or example of the invention is applicable to any other aspect, embodiment, or example described herein, except where incompatible. All features disclosed in this specification (including any accompanying claims, abstract, and drawings), and / or all steps of any method or process so disclosed, may be combined in any combination, except combinations in which at least some of such features and / or steps are mutually exclusive. The invention is not limited to the details of any of the foregoing embodiments. The invention extends to any novel one or any novel combination of features disclosed in this specification (including any accompanying claims, abstract, and drawings), or any novel one or any novel combination of steps of any method or process so disclosed.

[0013] The reader's attention is directed to all articles and documents related to this application that have been filed contemporaneously with or prior to this application and that are open to public inspection herewith, and the contents of all such articles and documents are incorporated herein by reference.

[0014] For the avoidance of doubt, it is hereby stated that the information disclosed hereinabove under the heading "Background" is relevant to the present invention and should be read as part of the disclosure of the present invention.

[0015] 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.

[0016] definition The following explanations of terms and methods are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the same.

[0017] The term "about" or "approximately," unless otherwise specified, means within an acceptable error range of a particular value as determined by one skilled in the art, which depends 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 standard deviations or more than 3 standard deviations depending on the technical field. Alternatively, "about" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within one order of magnitude of a value, preferably within 5-fold, and more preferably within 2-fold.

[0018] The terms "polypeptide" and "protein" can be used interchangeably and generally refer to peptides and proteins having more than about 10 covalently linked amino acids linked by peptidyl bonds. The term protein encompasses purified natural products or products that can be produced partially or wholly using recombinant or synthetic techniques. The terms peptide and protein can also refer to protein aggregates, such as dimers or other multimers, fusion proteins, protein variants, or derivatives thereof. The terms also include proteins modified by protein modifications, such as glycosylation, acetylation, phosphorylation, PEGylation, ubiquitination, etc. Proteins can contain amino acids not encoded by nucleic acid codons. Proteins can have an amino acid sequence of sufficient length to produce higher levels of tertiary and / or quaternary structure. Typical proteins herein can have a molecular weight of at least about 15-20 kDa, preferably at least about 20 kDa. Examples of proteins encompassed by the definition herein generally include all mammalian proteins, particularly therapeutic and diagnostic proteins, such as therapeutic and diagnostic antibodies, and proteins containing one or more disulfide bonds, including multi-chain polypeptides containing one or more inter- and / or intra-chain disulfide bonds.

[0019] The term "post-translational modification" (PTM), unless otherwise specified, refers to the covalent modification of a protein after protein biosynthesis. Exemplary PTMs that can be determined by the methods (or using the systems) of the invention include glycosylation, deamidation, succinimidation, isomerization, C-terminal clipping, N-terminal cyclization, oxidation, and proteolysis.

[0020] The term "antibody," unless otherwise specified, 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, as described, for example, in Labrijn, et al., "Bispecific antibodies: a mechanistic review of the pipeline," Nat. Rev. Drug Discov., 2019 Aug. 18(8), 585-608), and antibody fragments, so long as they exhibit the desired antigen-binding activity. Therapeutic antibodies are antibodies that can be used to treat disease.

[0021] The terms "antibody fragment," "antigen-binding portion" (or simply "antibody portion"), or "antigen-binding fragment" of an antibody, unless otherwise specified, refer to a molecule other than an intact antibody that contains a portion of the intact antibody that binds to 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 specific antibody fragments, see Holliger and Hudson, Nature Biotechnology 23:1126-1136 (2005). Therapeutic antibody fragments are antibody fragments that can be used to treat disease.

[0022] 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.

[0023] The term "human antibody," unless otherwise specified, refers to an antibody having an amino acid sequence corresponding to that of an antibody produced by a human or human cell, or derived from a non-human source that utilizes sequences encoding the human antibody repertoire or other human antibodies. This definition of a human antibody specifically excludes humanized antibodies, which contain non-human antigen-binding residues.

[0024] The term "humanized antibody," unless otherwise specified, refers to a chimeric antibody comprising amino acid residues of non-human CDRs and amino acid residues of human FRs. In embodiments, 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 may optionally 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.

[0025] The term "monoclonal antibody," unless otherwise specified, 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 variant antibodies that contain, for example, naturally occurring mutations or that may arise during production of the monoclonal antibody preparation, in which such variants are generally 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 should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies according to the presently disclosed subject matter can be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci; such methods, as well as other exemplary methods for producing monoclonal antibodies, are described herein.

[0026] The term "isolated" refers to a biological component (e.g., a nucleic acid molecule, polypeptide, or protein) that has been substantially separated or purified from other biological components, i.e., other chromosomal and extrachromosomal DNA and RNA, and other biological components of the cells of the organism in which the protein naturally occurs. "Isolated" nucleic acids, polypeptides, and proteins include nucleic acids, polypeptides, and proteins purified by standard purification methods. The term also encompasses nucleic acids, polypeptides, and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acid molecules, polypeptides, and proteins. An isolated component may be purified.

[0027] Purified: This term does not require absolute purity, but rather is intended as a relative term. Thus, for example, a purified product is one that is more enriched than the product (e.g., polypeptide or protein) in its environment within a cell, such that the product is substantially separated from cellular components (nucleic acids, lipids, carbohydrates, and [other] polypeptides) that may accompany it.

[0028] Purified: This term does not require absolute purity, but rather is intended as a relative term. Thus, for example, a purified product is one that is more enriched than the product (e.g., polypeptide or protein) in its environment within a cell, such that the product is substantially separated from cellular components (nucleic acids, lipids, carbohydrates, and [other] polypeptides) that may accompany it.

[0029] In one example, a product of interest (e.g., a recombinant polypeptide) of the present disclosure is purified when at least 50% by weight of the sample is composed of the product, for example, when at least 60%, 70%, 80%, 85%, 90%, 92%, 95%, 98%, or 99% or more of the sample is composed of the polypeptide. Examples of methods that can be used to purify polypeptides include, but are not limited to, those disclosed in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY, 1989, Ch. 17). Protein purity can be determined, for example, by high-pressure liquid chromatography or other conventional methods.

[0030] The term "titer" refers to the total amount of product of interest (e.g., recombinant polypeptide such as an 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 polypeptide per liter of medium (g / L). Titer can be expressed or evaluated in terms of a relative measure, such as the percentage increase in titer compared to obtaining protein product under different medium conditions.

[0031] As used herein, the term "cell" includes reference to a eukaryotic cell. Unless the context requires otherwise, a reference to a cell may include a reference to a plurality of cells. A eukaryotic cell may be an animal cell (e.g., a mammalian cell). A eukaryotic cell may be a mammalian cell, such as a hybridoma, CHO cell, COS cell, VERO cell, HeLa cell, HEK 293 cell, PER-C6 cell, K562 cell, MOLT-4 cell, M1 cell, NSO cell, NS-1 cell, COS-7 cell, MDBK cell, MDCK cell, MRC-5 cell, WI-38 cell, WEHI cell, SP2 / 0 cell, BHK cell (including BHK-21 cell), and derivatives thereof. A cell may be a CHO cell, an NSO cell, or a derivative thereof. CHO cells can be, for example, CHO K1 cells, CHO K1SV cells, DG44 cells, DUKXB-11 cells, CHOK1S cells, CHO K1M cells, CHO cell lines generated by targeted gene integration (TI) and derivatives thereof.

[0032] As used herein, the term "cell line" includes reference to a culture of eukaryotic cells that can be propagated repeatedly. The eukaryotic cells of the cell line can be selected from any cell as defined herein.

[0033] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably herein and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and its progeny, regardless of the number of passages. The progeny need not be completely identical in nucleic acid content to the parent cell, but may 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.

[0034] As used herein, the term "mammalian host cell" or "mammalian cell" refers to cells and cell lines derived from mammals that are capable of growth and survival when placed in either monolayer or suspension culture in a medium containing appropriate nutrients and growth factors. The growth factors required for a particular cell line can be readily determined empirically without undue experimentation, as described, for example, in Mammalian Cell Culture (Mather, JP ed., Plenum Press, NY 1984) and Barnes and Sato, (1980) Cell, 22:649. Typically, the cells are capable of expressing and secreting large amounts of a particular protein of interest, e.g., a glycoprotein, into the culture medium. Examples of suitable mammalian host cells include Chinese hamster ovary cells / -DHFR (Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 1980); dp12. CHO cells (EP 307,247 published March 15, 1989); CHO-K1 (ATCC, CCL-61); SV40-transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651); human embryonic kidney line (293 cells, or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol., 36:59 1977); baby hamster kidney cells (BHK, ATCC CCL 10); mouse Sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251 1980); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); dog kidney cells (MDCK, ATCC CCL34); buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci., 383:44-68 1982); MRC5 cells; FS4 cells; and a human hepatoma line (Hep G2).Mammalian cells include Chinese hamster ovary cells / -DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 1980); dp12.CHO cells (European Patent No. 307,247 published March 15, 1989).

[0035] As used herein, the term "cell culture medium" refers to a nutrient solution for culturing cells. "Cell culture feed" and "cell culture additive" refer to nutritional supplements that can be added to cell culture medium to improve medium performance. For example, cell culture feed and / or cell culture additives can be added to cell culture medium during batch culturing of cells. Cell culture media may be chemically defined or may contain undefined components. For example, cell culture media for mammalian cells typically contain at least one component from one or more of the following categories: 1) A source of energy, usually in the form of carbohydrates such as glucose; 2) A basic group of all essential amino acids, and usually 20 amino acids plus cysteine; 3) Vitamins and / or other organic compounds required in low concentrations; 4) free fatty acids; and 5) Trace elements (trace elements are defined as inorganic compounds or naturally occurring elements that are typically required in very low concentrations, usually in the micromolar range). Cell culture media and similar nutrient solutions may optionally be supplemented with one or more components from any of the following categories: 1) hormones and other growth factors, such as insulin, transferrin, and epidermal growth factor; 2) Salts and buffers, such as calcium, magnesium, and phosphate; 3) nucleosides and bases, such as adenosine, thymidine, and hypoxanthine; and 4) Protein and tissue hydrolysates.

[0036] The term "culturing" refers to contacting cells with cell culture medium under conditions suitable for cell survival and / or growth and / or proliferation. A "cell culture" refers to one or more cells in contact with cell culture medium.

[0037] The term "batch culture" refers to a culture in which all components for cell culture (including cells and all culture nutrients) are supplied to the culture bioreactor at the beginning of the culture process.

[0038] As used herein, the term "fed-batch cell culture" refers to a batch culture in which cells and culture medium are initially fed to a culture bioreactor, and additional culture nutrients are fed to the culture continuously or in discrete increments during the culture process, with or without periodic cell and / or product harvesting before the end of the culture.

[0039] The term "perfusion culture," sometimes referred to as continuous culture, is a culture in which cells are maintained in culture, for example, by filtration, encapsulation, anchoring to microcarriers, etc., and culture medium is introduced and removed from the culture bioreactor continuously, stepwise, or intermittently (or any combination thereof).

[0040] The terms "expression" or "expressing" are used herein to refer to transcription and translation occurring within a host cell. The level of expression of a product gene within a host cell can be measured based on either the amount of corresponding mRNA present in the cell or the amount of protein encoded by the product gene produced by the cell. For example, mRNA transcribed from a product gene can be quantified by Northern hybridization. Sambrook et al., Molecular Cloning: A Laboratory Manual, pp. 7.3-7.57 (Cold Spring Harbor Laboratory Press, 1989). The protein encoded by a product gene can be quantified either by assaying the biological activity of the protein or by using an assay unrelated to such activity, such as Western blot or radioimmunoassay, using an antibody 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).

[0041] cell culture A cell culture comprises a cell culture medium and at least one (typically a plurality) of cells. For example, a cell culture medium can comprise a cell culture medium and a plurality of eukaryotic cells, e.g., cells engineered to produce a protein product.

[0042] Cell culture media contain many components. They provide the nutrients necessary to maintain and grow cells in a controlled, artificial, in vitro environment. The properties and composition of cell culture media vary depending on the specific cellular requirements. Important parameters include osmolality, pH, and nutrient formulation.

[0043] Culture media contain a mixture of amino acids, glucose, salts, vitamins, and other nutrients and are available from suppliers in either powder or liquid form. The requirements for these components vary depending on the cell line. pH regulation is important for optimal culture conditions and is generally achieved using an appropriate buffer system. Chemically defined media (CDMs) are preferred for therapeutic and related applications because they provide reproducible, contaminant-free media when prepared and used under sterile conditions; however, some cell types may require the use of media containing serum, proteins, or other biological extracts (such as yeast extracts or enzymatic digests of plant or animal material).

[0044] Extremely simple defined media consisting essentially of vitamins, amino acids, organic salts, inorganic salts, and buffers have also been used in cell culture. However, such media (often referred to as "basal media") are usually significantly deficient in the nutritional content required by most animal cells. Therefore, these media often need to be supplemented, for example, with feeds or other additives to form a complete medium. Furthermore, in batch culture systems, the medium is often periodically replenished with concentrated feeds or additives to maintain the viability of the cultured cells and / or the production of biological products, such as polypeptides (e.g., antibodies or biologically functional fragments of antibodies), proteins, peptides, hormones, viruses or virus-like particles, nucleic acids, or fragments thereof.

[0045] Ingredients that may be present in a basal medium include amino acids (nitrogen source), vitamins, inorganic salts, sugars (carbon source), buffer salts, and lipids. Basal media for use with certain mammalian cell culture systems may contain ethanolamine, D-glucose, N-[2-hydroxyethyl]-piperazine-N'-[2-ethanesulfonic acid] (HEPES), linoleic acid, lipoic acid, phenol red, PLURON1C F68, putrescine, and sodium pyruvate.

[0046] Amino acid sources that can be included in the medium include L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, and derivatives thereof. These amino acids can be obtained commercially, for example, from Sigma (Saint Louis, Missouri).

[0047] Vitamin sources that can be included in the medium include biotin, choline chloride, and D-Ca. 2+ Pantothenate, folic acid, i-inositol, niacinamide, pyridoxine, riboflavin, thiamine, and vitamin B 12. These vitamins are commercially available, for example, from Sigma (Saint Louis, Missouri).

[0048] Inorganic salt sources that can be used in the medium include one or more calcium salts (e.g., CaCl), Fe(NO), KCl, one or more magnesium salts (e.g., MgCl and / or MgSO), one or more manganese salts (e.g., MnCl), NaCl, NaHCO, NHPO, and ions of the trace elements selenium, vanadium, zinc, and copper. These trace elements can be provided in various forms, preferably in the form of salts such as NaSeO, NHVO, ZnSO, and CuSO. These inorganic salts can be obtained commercially, for example, from Sigma (Saint Louis, Missouri).

[0049] Any of these media may also contain, as needed, hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleosides (such as adenosine and thymidine), antibiotics (such as gentamicin), and the like.(商標)The expression vector may be supplemented with nutrients (such as drugs), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations known to those skilled in the art. Culture conditions, such as temperature, pH, etc., will typically be those already used with the host cell selected for expression and will be apparent to those skilled in the art. Exemplary culture conditions include M. Takagi and K. Ueda, ``Comparison of the optimal culture conditions for cell growth and tissue plasminogen activator production by human embryo lung cells on microcarriers'', Biotechnology, (1994), 41,565-570; H. J. Morton, ``A survey of commercially available tissue culture media'', In Vitro(1970),6(2),89-108;J.Van der Valk,et al.,(2010),''Optimization of chemically defined cell culture media-replacing fetal bovine serum in mammalian in vitro methods,''Toxicology in vitro,24(4),1053-1063;RJGraham et al.,''Consequences of trace metal variability and supplementation on Chinese hamster ovary(CHO)cell culture performance:A review of key mechanisms and considerations'',BIotechnol.Bioeng.(2019),116(12),3446-3456;S.Janoschek et al., "A protocol to transfer a fed-batch platform process into semi-perfusion mode: The benefit of automated small-scale bioreactors compared to shake flasks as a scale-down model," Biotechnol. Prog., (2019), 35(2), e2757; and M. Kuiper et al., "Repurposing fed-batch media and feeds for highly productive CHO perfusion processes," Biotechnology Progress, 15 April 2019, https: / / doi.org / 10.1002 / btpr.2821, all of which are incorporated herein by reference in their entireties.

[0050] Cell cultures intended for the production of recombinant proteins can include eukaryotic cells encoding the recombinant protein. Recombinant proteins can be produced by growing cells expressing the product of interest under a variety of cell culture conditions. For example, cell culture procedures for large-scale or small-scale production of proteins are potentially useful in the context of the present disclosure. Procedures can be used, including, but not limited to, fluidized-bed bioreactors, hollow fiber bioreactors, roller bottle cultures, shake flask cultures, or stirred-tank bioreactor systems (with or without microcarriers in the latter two systems), and can alternatively be operated in batch, fed-batch, or continuous modes.

[0051] In fed-batch culture, host cells (which may be eukaryotic cells, e.g., mammalian host cells) and culture medium are first supplied to a culture vessel, and additional culture nutrients are continuously or individually supplied to the culture during the culture, with or without periodic cell and / or product harvest before the end of the culture. Fed-batch culture may include, for example, semi-continuous fed-batch culture, in which the entire culture (including cells and medium) is periodically removed and replaced with fresh medium. Fed-batch culture is distinct from simple batch culture, in which all components for cell culture (including cells and all culture nutrients) are supplied to the culture vessel at the beginning of the culture process. For example, see F. Li, N. Vijayasankaran, A. Shen, R. Kiss, A. Amanullah; MAbs 2(5), 466-479. Fed-batch culture can further be distinguished from perfusion culture insofar as the supernatant is not removed from the culture vessel during the process (in perfusion culture, cells are maintained in culture, e.g., by filtration, encapsulation, or anchoring to microcarriers, and culture medium is continuously or intermittently introduced and removed from the culture vessel).

[0052] Typically, fed-batch or continuous cell culture conditions are devised to enhance the growth of eukaryotic cells (e.g., mammalian cells) during the growth phase of the cell culture. During the growth phase, cells are grown under conditions and for a period of time optimized for growth. Culture conditions, such as temperature, pH, dissolved oxygen (dO2), etc., used for a particular host will be apparent to those skilled in the art.

[0053] product In the method of optimizing sialic acid in a recombinant polypeptide of the present disclosure, the recombinant polypeptide can be any recombinant polypeptide product of interest that contains sialic acid. The method of the present disclosure can be used to produce polypeptides, such as mammalian polypeptides. Non-limiting examples of such polypeptides include hormones, receptors, fusion proteins, regulatory factors, growth factors, complement system factors, enzymes, coagulation factors, anticoagulants, kinases, cytokines, CD proteins, interleukins, therapeutic proteins, diagnostic proteins, and antibodies. The cells and / or cell lines and / or methods of the present disclosure are typically not specific for the molecule, e.g., the antibody, that is produced.

[0054] In the methods of the present disclosure, the recombinant polypeptide whose sialic acid content is optimized can be an antibody, including a therapeutic or diagnostic antibody or an antigen-binding fragment thereof. The antibody can 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 a multimer of such a pair), a multispecific antibody, and an antigen-binding fragment thereof. For example, but not limited to, the multispecific antibody can be a bispecific antibody, a dual-epitope antibody, a T-cell-dependent bispecific antibody (TDB), a dual-acting FAb (DAF), or an antigen-binding fragment thereof.

[0055] multispecific antibodies The antibody may be a multispecific antibody, e.g., a bispecific antibody. A "multispecific antibody" is a monoclonal antibody that has at least two different moieties, i.e., binding specificities for different epitopes on different antigens (i.e., bispecific), or different epitopes on the same antigen (i.e., dual epitopes). A multispecific antibody may have three or more binding specificities. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments as described herein.

[0056] Techniques for producing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983)) and "knobs-in-holes" engineering (see, e.g., U.S. Pat. No. 5,731,168 and Atwell et al., J. Mol. Biol. 270:26 (1997)). Multispecific antibodies can also be produced by manipulating electrostatic steering effects to create antibody Fc heterodimeric molecules (see, e.g., WO 2009 / 089004); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, 229:81 (1985)); producing bispecific antibodies using leucine zippers (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992) and WO 2011 / 034605); using conventional light chain technology to circumvent light chain mispairing issues (see, e.g., WO 98 / 50431); using "diabody" technology to create bispecific antibody fragments (see, e.g., Hollinger et al., J. Immunol., 148(5):1547-1553 (1992) and WO 2011 / 034605). al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and by the use of single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)); and by the preparation of trispecific antibodies, for example, as described in Tutt et al. J. Immunol. 147:60 (1991).

[0057] Engineered antibodies (including, for example, "octopus antibodies") or DVD-Igs having three or more antigen-binding sites are also included herein (see, e.g., WO 2001 / 77342 and WO 2008 / 024715). Further non-limiting examples of multispecific antibodies having 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. Bispecific antibodies or antigen-binding fragments thereof also include "dual-acting Fabs" or "DAFs" (see, e.g., U.S. Patent Application Publication Nos. 2008 / 0069820 and WO 2015 / 095539).

[0058] Multispecific antibodies can also be provided in asymmetric forms with domain crossovers in one or more binding arms with the same antigen specificity, i.e., by exchanging VH / VL domains (see, e.g., WO 2009 / 080252 and WO 2015 / 150447), CH1 / CL domains (see, e.g., WO 2009 / 080253), or complete Fab arms (see, e.g., WO 2009 / 080251, WO 2016 / 016299; see also Schaefer et al., PNAS, 108 (2011) 1187-1191, and Klein at al., MAbs 8 (2016) 1010-20). Multispecific antibodies can include cross-Fab fragments. The term "cross-Fab fragment" or "xFab fragment" or "crossover Fab fragment" refers to a Fab fragment in which either the variable or constant regions of the heavy and light chains have been exchanged. A cross-Fab fragment contains a polypeptide chain composed of a light chain variable region (VL) and a heavy chain constant region 1 (CH1), and a polypeptide chain composed of a heavy chain variable region (VH) and a light chain constant region (CL). Asymmetric Fab arms can also be engineered by introducing charged or uncharged amino acid mutations at the domain interface to direct correct Fab pairing. See, for example, WO 2016 / 172485.

[0059] A variety of additional molecular formats of multispecific antibodies are known in the art and are included herein (see, e.g., Spiess et al., Mol. Immunol. 67 (2015) 95-106).

[0060] A particular type of multispecific antibody also included herein is a bispecific antibody designed to simultaneously bind to a surface antigen on a target cell, e.g., a tumor cell, and to an activation-invariant component of the T cell receptor (TCR) complex, e.g., CD3, to retarget T cells to kill the target cell.

[0061] Further non-limiting examples of bispecific antibody formats that may be useful for this purpose include so-called "BiTEs" (bispecific T cell engagers), in which 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 (Holliger et al., Prot. Eng. 9, 299-305 (1996)) and derivatives thereof, such as tandem bispecific antibodies ("TandAbs"; 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 further stabilization (Johnson et al., J Mol Biol 399, 436-449 (2010)); and the so-called triomab, a fully hybrid mouse / rat IgG molecule (reviewed in Seimetz et al., Cancer Treat. Rev. 36, 458-467 (2010)). Specific T cell bispecific antibody formats included herein are described in WO 2013 / 026833, WO 2013 / 026839, WO 2016 / 020309; Bacac et al., Oncoimmunology 5(8) (2016) e1203498.

[0062] antibody fragment Antibodies whose sialic acid content can be optimized according to the methods provided herein may be or may include antibody fragments. For example, but not limited to, antibody fragments can be Fab, Fab', Fab'-SH, or F(ab')2 fragments, particularly Fab fragments. Papain digestion of an intact antibody produces two identical antigen-binding fragments (so-called "Fab" fragments), each containing the heavy and light chain variable domains (VH and VL, respectively) as well as the light chain constant domain (CL) and the first heavy chain constant domain (CH1). Thus, the term "Fab fragment" refers to an antibody fragment containing a light chain comprising the VL and CL domains and a heavy chain fragment comprising the VH and CH1 domains. "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 is a Fab' fragment in which the cysteine ​​residue(s) in the constant domains retain a free thiol group. Pepsin treatment yields two antigen-binding sites (two Fab fragments) and an F(ab')2 fragment containing part of the Fc region. See U.S. Patent No. 5,869,046 for a discussion of Fab and F(ab')2 fragments that contain salvage receptor-binding epitope residues and have increased in vivo half-lives.

[0063] The antibody fragment may be a diabody, triabody, or tetrabody. A "diabody" is an antibody fragment having two antigen-binding sites, which may be bivalent or bispecific. See, e.g., 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).

[0064] 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 constant domain (CL), and a linker, wherein the antibody domains and linker have one of the following orders from N- to C-terminus: 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, the linker may be a polypeptide of at least 30 amino acids, preferably between 32 and 50 amino acids. The single-chain Fab fragment is stabilized by a native disulfide bond between the CL and CH1 domains. In addition, these single-chain Fab fragments can be further stabilized by the creation of interchain disulfide bonds through the insertion of cysteine ​​residues (e.g., at position 44 in the variable heavy chain and position 100 in the variable light chain according to the Kabat numbering).

[0065] 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 an antibody's heavy chain (VH) and light chain (VL) linked by a linker. In particular, the linker is a short polypeptide of 10 to 25 amino acids, typically rich in glycine for flexibility and serine or threonine for solubility, and can connect the N-terminus of VH to the C-terminus of VL, or vice versa. This protein retains the specificity of the original antibody despite the removal of the constant region and the introduction of the linker. For a review of scFv fragments, see, for example, Pluckthun, 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.

[0066] The antibody fragment may be a single-domain antibody. A "single-domain antibody" is an antibody fragment that contains all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. The single-domain antibody may be a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516).

[0067] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies.

[0068] Chimeric and humanized antibodies The antibody whose sialic acid content can be optimized according to the method provided herein can be a chimeric antibody. Specific chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a non-human primate such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In another example, a chimeric antibody is a "class-switched" antibody whose class or subclass has been changed from that of the parent antibody. The chimeric antibody includes its antigen-binding fragment.

[0069] 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 parent non-human antibody. Usually, a humanized antibody comprises one or more variable domains in which the CDRs (or portions thereof) are derived from a non-human antibody, and the FRs (or portions thereof) are derived from a human antibody sequence. Optionally, a humanized antibody also comprises 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.

[0070] Humanized antibodies and methods for making them are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and 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); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (description of specificity-determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (description of "surface reconstruction"); Dall'Acqua et al., Methods 36:43-60 (2005) (description of "FR reconstruction"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (description of the "guided selection" approach for FR construction).

[0071] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from consensus sequences of human antibodies of particular subgroups of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening of fr libraries (see, e.g., Baca et al. al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0072] Human antibodies The antibody whose sialic acid content can be optimized according to the methods provided herein can be a human antibody. Human antibodies can be produced using various techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5:368-74 (2001) and Lonberg, Curr. Opin. Immunol., 20:450-459 (2008).

[0073] Human antibodies may be prepared by administering immunogens to transgenic animals that have been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or part of the human immunoglobulin loci that replace the endogenous immunoglobulin loci, or that are extrachromosomally present or randomly integrated into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci are generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). Also see, for example, XENOMOUSE. (商標) See also U.S. Patent Nos. 6,075,181 and 6,150,584, which describe HUMAB® technology; U.S. Patent No. 5,770,429, which describes HUMAB® technology; U.S. Patent No. 7,041,870, which describes KM MOUSE® technology; and U.S. Patent Application Publication No. 2007 / 0061900, which describes VELOCIMOUSE® technology. The human variable regions from intact antibodies produced by such animals can be further modified, for example, by combining with different human constant regions.

[0074] Human antibodies can also be produced by hybridoma-based methods. Human myeloma cell lines and mouse-human heteromyeloma cell lines for producing human monoclonal antibodies have been described. (See, for example, Kozbor J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147:86 (1991)). Human antibodies produced via human B cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Further methods include those described, for example, in U.S. Patent No. 7,189,826 (which describes the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (which describes human-human hybridomas). Human hybridoma technology (trioma technology) is also described in Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0075] target molecule Non-limiting examples of molecules that can be targeted by antibodies with optimized sialic acid content produced by the methods disclosed herein include soluble serum proteins and their receptors, as well as other membrane-bound proteins (e.g., adhesins). In certain examples, the antibodies can bind to one, two, or more cytokines, cytokine-related proteins, and cytokine receptors. Cytokine receptors include 8MPI, 8MP2, 8MP38 (GDF10), 8MP4, 8MP6, 8MP8, CSFI (M-CSF), CSF2 (GM-CSF), CSF3 (G-CSF), EPO, FGF1 (αFGF), FGF2 (βFGF), FGF3 (int-2), FGF4 (HST), FGF5, FGF6 (HST-2), FGF7 (KGF), FGF9, FGF1 O 1A, IL 1B, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL1 0, IL 11, IL 12A, IL 12B, IL 13, IL 14, IL 15, IL 16, IL 17, IL 17B, IL 18, IL 19, IL20, IL22, IL23, IL24, IL25, IL26, IL27, IL28A, IL28B, IL29, IL30, PDGFA, PDGFB, TGFA, TGFB1, TGFB2, TGFBb3, LTA (TNF-β) , LTB, TNF (TNF-α), TNFSF4 (OX40 ligand), TNFSF5 (CD40 ligand), TNFSF6 (FasL), TNFSF7 (CD27 ligand), TNFSF8 (CD30 ligand), TNFSF9 (4-1BB ligand), TNFSF10 (TRAIL), TNFSF11 (TRANCE), TNFSF12 (APO3L), TNFSF13 (April), TNFSF13B, TNFSF14 (HVEM-L), TNFSF15 (VEGI), TNFSF18, HGF (VEGF D), VEGF, VEGFB, VEGFC, IL1R1, IL1R2, IL1RL1, IL1RL2, IL2RA, IL2RB, IL2RG, IL3RA, IL4R, IL5RA, IL6R, IL7R, IL8RA, IL8RB, IL9R, IL10RA, IL10RB, IL IL11RA, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL15RA, IL17R, IL18R1, IL20RA, IL21R, IL22R, IL1HY1, IL1RAP, IL1RAPL1, IL1RAPL2, IL1RN, IL6ST, IL18BP, IL18RAP, IL22RA2, AIF1, HGF, LEP (leptin), PTN, and THPO.

[0076] Antibodies with optimized sialic acid content produced according to the methods disclosed herein include CCLI(1-309), CCL2 (MCP-1 / MCAF), CCL3 (MIP-Iα), CCL4 (MIP-Iβ), CCL5 (RANTES), CCL7 (MCP-3), CCL8 (mcp-2), CCL11 (eotaxin), CCL13 (MCP-4), CCL15 (MIP-Iδ), CCL16 (HCC-4), CCL17 (TARC), CCL18 (PARC), CCL19 (MDP-3b), CCL20 (MIP-3α), CCL21 (SLC / Exodus-2), CCL22 (MDC / STC-1), CCL23 (MPIF-1), CCL24 (MPIF-2 / Eotaxin-2), CCL25(TECK), CCL26(eotaxin-3), CCL27(CTACK / ILC), CCL28, CXCLI(GROI), CXCL2(GR02), CXC L3 (GR03), CXCL5 (ENA-78), CXCL6 (GCP-2), CXCL9 (MIG), CXCL10 (IP10), CXCL11 (1-TAC), CXCL 12(SDFI), CXCL13, CXCL14, CXCL16, PF4(CXCL4), PPBP(CXCL7), CX3CL1(SCYDI), SCYEI, XCLI( lymphotactin), XCL2 (SCM-Iβ), BLRI (MDR15), CCBP2 (D6 / JAB61), CCRI (CKRI / HM145), CCR2 (mcp-IRBIRA), CCR3(CKR3 / CMKBR3), CCR4, CCR5(CMKBR5 / ChemR13), CCR6(CMKBR6 / CKR-L3 / STRL22 / DRY6), CCR7(CKR7 / EBII), CCR8(CMKBR8 / TER1 / CKR-L1), CCR9(GPR-9-6), CCRL1(VS HK1), CCRL2 (L-CCR), XCR1 (GPR5 / CCXCR1), CMKLR1, CMKOR1 (RDC1), CX3CR1 (V28), CXCR 4, GPR2 (CCR10), GPR31, GPR81 (FKSG80), CXCR3 (GPR9 / CKR-L2), CXCR6 (TYMSTR / STRL33 / Bonzo), HM74, IL8RA (IL8Rα), IL8RB (IL8Rβ), LTB4R (GPR16), TCP10, CKLFSF2, CKLFSF3, CKLFSF4, CKLFSF5, CKLFSF6, CKLFSF7, CKLFSF8, BDNF, C5, C5R1, CSF3, GRCC10 (C10), EPO, FY (DARC), GDF5, HDF1, HDF1α, DL8, PRL, RGS3, RGS13, SDF2, SLIT2, TLR2, TLR4, TREM1, TREM2, and VHL.

[0077] In a particular example, the recombinant polypeptide with optimized sialic acid content obtained according to the methods of the present invention is an antibody (e.g., a multispecific antibody, such as a bispecific antibody) capable of binding to one or more target molecules selected from the following: 0772P (CA125, MUC16) (i.e., ovarian cancer antigen), ABCF1; ACVR1; ACVR1B; ACVR2; ACVR2B; ACVRL1; ADORA2A; aggrecan; AGR2; AICDA; AIF1; AIG1; AKAP1; AKAP2; AMH; AMHR2; amyloid beta; ANGPTL; ANGPT2; ANGPTL3; ANGPTL4; ANPEP; APC; APOC1; AR; ASLG659; ASPHD1 (aspartate beta-hydroxylase domain) phospho-containing 1; LOC253982; AZGP1 (zinc-α-glycoprotein); B7.1; B7.2; BAD; BAFF-R (B cell-activating factor receptor, BLyS receptor 3, BR3); BAG1; BAI1; BCL2; BCL6; BDNF; BLNK; BLRI (MDR15); BMP1; BMP2; BMP3B (GDF10); BMP4; BMP6; BMP8; BMPR1A; BMPR1B (bone morphogenetic protein receptor-type IB); BMPR2; BPAG1 (plectin); BRCA1; Brevica ;C19orf10(IL27w);C3;C4A;C5;C5R1;CANT1;CASP1;CASP4;CAV1;CCBP2(D6 / JAB61);CCL1(1-309);CCL11(eotaxin);CCL13(MCP-4); CCL15(MIP1δ);CCL16(HCC-4);CCL17(TARC);CCL18(PARC);CCL19(MIP-3β);CCL2(MCP-1);MCAF;CCL20(MIP-3α);CCL21(MTP-2);SLC; Exodus-2;CCL22(MDC / STC-1);CCL23(MPIF-1);CCL24(MPIF-2 / Eotaxin-2);CCL25(TECK);CCL26(Eotaxin-3);CCL27(CTACK / ILC);CCL2 8;CCL3(MTP-Iα);CCL4(MDP-Iβ);CCL5(RANTES);CCL7(MCP-3);CCL8(mcp-2);CCNA1;CCNA2;CCND1;CCNE1;CCNE2;CCR1(CKRI / HM145);CCR2(mcp-IRβ / RA);CCR3(CKR / CMKBR3);CCR4;CCR5(CMKBR5 / ChemR13);CCR6(CMKBR6 / CKR-L3 / STRL22 / DRY6);CC R7(CKBR7 / EBI1);CCR8(CMKBR8 / TER1 / CKR-L1);CCR9(GPR-9-6);CCRL1(VSHK1);CCRL2(L-CCR);CD164;CD19;CD1 C; CD20; CD200; CD22 (B cell receptor CD22-B isoform); CD24; CD28; CD3; CD37; CD38; CD3E; CD3G; CD3Z; CD4; CD40; CD40L; CD44; CD45RB; CD52; CD69; CD72; CD74; CD79A (CD79α, immunoglobulin-related α, B cell-specific protein); CD79B; CDS; CD80; CD81; CD83; CD 86;CDH1(E-cadherin);CDH10;CDH12;CDH13;CDH18;CDH19;CDH20;CDH5;CDH7;CDH8;CDH9;CDK2;CDK3;CDK4;CDK5;C DK6;CDK7;CDK9;CDKN1A(p21 / WAF1 / Cip1);CDKN1B(p27 / Kip1);CDKN1C;CDKN2A(P16INK4a);CDKN2B;CDKN2C;CDK N3;CEBPB;CER1;CHGA;CHGB;chitinase;CHST10;CKLFSF2;CKLFSF3;CKLFSF4;CKLFSF5;CKLFSF6;CKLFSF7;CKLFSF8;CLDN3;CLDN7 (claudin-7);CLL-1 (CLEC12A, MICL, and DCAL2);CLN3;CLU (clusterin);CMKLR1;CMKOR1 (RDC1);CNR1;COL 18A1;COL1A1;COL4A3;COL6A1;Complement factor D;CR2;CRP;CRIPTO(CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor);CSFI(M-CSF);CSF2(GM-CSF);CSF3(GCSF);CTL A4;CTNNB1(b-catenin);CTSB(cathepsin B);CX3CL1(SCYDI);CX3CR1(V28);CXCL1(GRO1);CXCL10(IP-10);CXCL11(I-TAC / IP-9);CXCL12(SDF1);CXCL13;CXCL14;CXCL16;CXCL2(GRO2);CXCL3(GRO3);CXCL5(ENA-78 / LIX);CXCL6(GCP-2);CXCL9(MIG);CXCR3(GPR9 / CKR-L2);CXCR4;CXCR5(Burkitt's lymphoma receptor 1, G protein-coupled receptor);CXCR6(TYMSTR / STRL33 / Bonzo);CYB5;CYC1;CYSLTR1;DAB2IP;DES;DKFZp451J0118;DNCLI;DPP4;E16(LAT1,SLC7A5);E2F1;ECGF1;ED G1;EFNA1;EFNA3;EFNB2;EGF;EGFR;ELAC2;ENG;ENO1;ENO2;ENO3;EPHB4;EphB2R;EPO;ERBB2 (Her-2);EREG;ERK8;ESR1;ESR2;ETBR (endothelin type B receptor);F3 (TF);FADD;FasL;FASN;FCER1A;FCER2;FCGR3A;FcRH1 (Fc receptor-like protein 1);FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain-containing phosphatase anchor protein 1a), SPAP1B, SPAP 1C);FGF;FGF1(αFGF);FGF10;FGF11;FGF12;FGF12B;FGF13;FGF14;FGF16;FGF17;FGF18;FGF19;FGF2(bFGF);FGF20;FGF21;FGF22;FGF23;FGF3(int-2 );FGF4(HST);FGF5;FGF6(HST-2);FGF7(KGF);FGF8;FGF9;FGFR;FGFR3;FIGF(VEGFD);FELl(EPSILON);FILl(ZETA);FLJ12584;FLJ25530;FLRTI(Fibro nectin); FLT1; FOS; FOSL1 (FRA-1); FY (DARC); GABRP (GABAa); GAGEB1; GAGEC1; GALNAC4S-6ST; GATA3; GDF5; GDNF-Ra1 (GDNF family receptor alpha 1; GFRA1; GDNFR; GDNFRA; RETL1; TRNR1; RET1L; GDNFR-alpha 1; GFR-alpha-1); GEDA; GFI1; GGT1; GM-CSF; GNASI; GNRHI; GPR2 (CCR10); GPR19 (G protein-coupled receptor 19; Mm.4787);GPR31;GPR44;GPR54 (KISS1 receptor; KISS1R; GPR54; HOT7T175; AXOR12);GPR81 (FKSG80);GPR172A (G protein-coupled receptor 172A; GPCR41; FLJ11856; D15Ertd747e);GRCCIO(C10);GRP;GSN (Gelsolin);GSTP1;HAVCR2;HDAC4;HDAC5 ;HDAC7A;HDAC9;HGF;HIF1A;HOP1;histamine and histamine receptor;HLA-A;HLA-DOB (beta subunit of MHC class II molecule (Ia antigen));HLA-DRA;HM74;HMOXI;HUMCYT2A;ICEBERG;ICOSL;1D2;IFN-a;IFNA1;IFNA2;IFNA4;IFNA5;IFNA6;IFNA7;IFNB 1;IFNgamma;DFNW1;IGBP1;IGF1;IGF1R;IGF2;IGFBP2;IGFBP3;IGFBP6;IL-l;IL10;IL10RA;IL10RB;IL11;IL 11RA;IL-12;IL12A;IL12B;IL12RB1;IL12RB2;IL13;IL13RA1;IL13RA2;IL14;IL15;IL15RA;IL16;IL17;IL 17B;IL17C;IL17R;IL18;IL18BP;IL18R1;IL18RAP;IL19;IL1A;IL1B;ILIF10;IL1F5;IL1F6;IL1F7;IL1F8; IL1F9;IL1HY1;IL1R1;IL1R2;IL1RAP;IL1RAPL1;IL1RAPL2;IL1RL1;IL1RL2,ILIRN;IL2;IL20;IL20Rα;IL21 R;IL22;IL-22c;IL22R;IL22RA2;IL23;IL24;IL25;IL26;IL27;IL28A;IL28B;IL29;IL2RA;IL2RB;IL2RG;IL3;IL30;IL3RA;IL4;IL4R;IL5;IL5RA;IL6;IL6R;IL6ST (glycoprotein 130);influenza A;influenza B;EL7;EL7R;EL8;IL8RA;DL8RB;IL8RB;DL9;DL9R;DLK;INHA;INHBA;INSL3;INSL4;IRAK1;IRTA2 (immunoglobulin superfamily receptor translocation associated 2);ERAK2;ITGA1;ITGA2; ITGA3; ITGA6 (a6 integrin); ITGAV; ITGB3; ITGB4 (b4 integrin); α4β7 and αEβ7 integrin heterodimers; JAG1; JAK1; JAK3; JUN; K6HF; KAI1; KDR; KITLG; KLF5 (GC Box BP); KLF6; KLKIO; KLK12; KLK13; KLK14; KLK15; KLK3; KLK4; KLK5; KLK6; KLK9; KRT1; KRT19 (keratin 19); KRT2A; KHTHB6 (hair-specific H-type keratin); LAMAS; LEP (leptin); LGR5 (leucine-rich repeat-containing G protein-coupled receptor 5; GPR49, GPR67); Lingo-p75; Lingo-Tro y; LPS; LTA (TNF-b); LTB; LTB4R (GPR16); LTB4R2; LTBR; LY64 (lymphocyte antigen 64 (RP105), a type I membrane protein of the leucine-rich repeat (LRR) family; Ly6E (lymphocyte antigen 6 complex, locus E; Ly67, RIG-E, SCA-2, TSA-1); Ly6G6D (lymphocyte antigen 6 complex, locus G6D; Ly6-D, MEGT1); LY6K( Lymphocyte antigen 6 complex, locus K; LY6K; HSJ001348; FLJ35226; MACMARCKS; MAG or OMgp; MAP2K7 (c-Jun); MDK; MDP; MIB1; midkine; MEF; MIP-2; MKI67; (Ki-67); MMP2; MMP9; MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin); MS4A1; MSG783 (RNF124, hypothetical protein FLJ20315); MSMB; MT3 (metallothionectin-111); MTSS1; MUC1 (mucin); MYC; MY088; Napi3b (also known as NaPi2b) (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3b); NCA; NCK2; neurocan; NFKB1; NFKB2;NGFB(NGF);NGFR;NgR-Lingo;NgR-Nogo66(Nogo);NgR-p75;NgR-Troy;NME1(NM23A);NOX5;NPPB;NR0B1;NR0B2;NR1D1;NR1D2;NR1H2;NR1H3;NR1H4;NR1 12;NR113;NR2C1;NR2C2;NR2E1;NR2E3;NR2F1;NR2F2;NR2F6;NR3C1;NR3C2;NR4A1;NR4A2;NR4A3;NR5A1;NR5A2;NR6A1;NRP1;NRP2;NT5E;NTN4;ODZI;OPRD1;OX4 0;P2RX7;P2X5 (purinergic receptor P2X ligand-gated ion channel 5);PAP;PART1;PATE;PAWR;PCA3;PCNA;PD-L1;PD-L2;PD-1;POGFA;POGFB;PECAM1;PF4 (CXCL4);PGF;PGR;Phosphacan;PIAS2;PIK3CG;PLAU (uPA);PLG;PLXDC1;PMEL17 (silver homolog;SILV;D12S53E;PMEL17;SI;SIL);PPBP (CXCL7);PPID;PRI;PRKCQ;PRKDI;PRL;PROC;PROK2;PSAP;PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 gene); PTAFR; PTEN; PTGS2 (COX-2); PTN; RAC2 (p21 Rac2); RARB; RET (ret proto-oncogene; MEN2A; HSCR1; MEN2B; MTC1; PTC; CDHF12; Hs.168114; RET51; RET-ELE1); RGSI; RGS13; RGS3; RNF110 (ZNF144); ROBO2; S100A2; SCGB1D2 (lipophilin B); SCGB2A1 (mammaglobin 2); SCGB2A2 (mammaglobin 1); SCYEI (endothelial-monocyte-activating cytokine); SDF2; Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type 1 and type 1-like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5B);SERPINA1;SERPINA3;SERP1NB5 (maspin);SERPINE1 (PAI-1);SERPDMF1;SHBG;SLA2;SLC2A2;SLC33A1;SLC43A1;SLIT2;SPPI;SPRR1B (Sprl);ST6GAL1;STABI;STAT6;STEAP (prostate six-transmembrane epithelial antigen);STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer-related genes 1, prostate cancer-related protein 1, prostate six-transmembrane epithelial antigen 2, prostate six-transmembrane protein); TB4R2; TBX21; TCPIO; TOGFI; TEK; TENB2 (putative transmembrane proteoglycan); TGFA; TGFBI; TGFB1II; TGFB2; TGFB3; TGFBI; TGFBRI; TGFBR2; TGFBR3; THIL; THBSI (thrombospondin-1); THBS2; THBS4; THPO; TIE (Tie-1); TMP3; tissue factor; TLR1; TL R2; TLR3; TLR4; TLR5; TLR6; TLR7; TLR8; TLR9; TLR10; TMEFF1 (transmembrane protein 1 with an EGF-like domain and two follistatin-like domains; tomoregulin-1); TMEM46 (Sesar homolog 2); TNF; TNF-a; TNFAEP2 (B94); TNFAIP3; TNFRSFIIA; TNFRSF1A; TNFRSF1B; TNFRSF21; TNFRSF5; TNFRSF6 (Fas); TNFRSF7 ;TNFRSF8;TNFRSF9;TNFSF10 (TRAIL);TNFSF11 (TRANCE);TNFSF12 (AP03L);TNFSF13 (April);TNFSF13B;TNFSF14 (HVEM-L);TNFSF15 (VEGI);TNFSF18;TNFSF4 (OX40 ligand);TNFSF5 (CD40 ligand);TNFSF6 (FasL);TNFSF7 (CD27 ligand);TNFSFS (CD30 ligand);TNFSF9 (4-1 BB ligand);TOLLIP;Toll-like receptor;TOP2A (topoisomerase Ea);TP53;TPM1;TPM2;TRADD;TMEM118 (Ring finger protein, transmembrane 2;RNFT2;FLJ14627);TRAF1;TRAF2;TRAF3;TRAF4;TRAF5;TRAF6; TREM1; TREM2; TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4); TRPC6; TSLP; TWEAK; tyrosinase (TYR; OCAIA; OCA1A; tyrosinase; SHEP3); VEGF; VEGFB; VEGFC; versican; VHL C5; VLA-4; XCL1 (lymphotactin); XCL2 (SCM-1b); XCRI (GPR5 / CCXCRI); YY1; and ZFPM2. Optimization of sialic acid content It is desirable to optimize the level of sialic acid in many recombinant polypeptides. See, for example, S. Weikert, et al., "Engineering Chinese hamster ovary cells to maximize sialic acid content of recombinant glycoproteins," Nature Biotechnology (1999), vol. 17(11), pp. 1116-1121. Antibodies, for example, contain sialic acid. In many applications using antibodies, it is important that the antibody have an appropriate level of sialic acid, which can be reflected by antibody product specifications that specify that the antibody should be within a given range. See, e.g., Engineering Chinese hamster ovary cells to maximize the sialic acid content of recombinant glycoproteins, S. Weikert, D. Papac, J. Briggs, D. Cowfer, S. Tom, M. Gawlitzek, J. Lofgren, S. Mehta, V. Chisholm, N. Modi, S. Eppler, K. Carroll, S. Chamow, D. Peers, P. Berman & L. Krummen; Nature Biotechnology volume 17, pages 1116-1121 (1999).

[0078] Recombinant polypeptides containing sialic acid are typically produced in cell culture using mammalian cell lines engineered to express the recombinant polypeptide. The present inventors have observed that the level of sialic acid content often decreases in the later stages of culture. Such a decrease can result in a recombinant polypeptide product having a suboptimal sialic acid level. For example, this can result in a therapeutic antibody that cannot be used because the sialic acid level is below specification. Therefore, it is desirable to have a method that maximizes the chances that a recombinant polypeptide is isolated from a cell culture with an optimal level of sialic acid (e.g., a level of sialic acid (mol) / polypeptide (mol) that falls within specification).

[0079] The present inventors have determined that, for example, for antibodies, cell viability predicts the level of sialic acid. In particular, the present inventors have determined that cell viability determined relatively early in cell culture (e.g., one of days 6-11 or 8-11) predicts optimal levels of sialic acid at relatively later stages of cell culture (e.g., one of days 9-14 or more). With this in mind, the present inventors have developed methods for optimizing the sialic acid content of recombinant polypeptides. These methods may, in some cases, allow cell culture to continue for longer, which may also result in relatively higher titers.

[0080] One aspect of the present invention provides a method for optimizing the sialic acid content of a recombinant polypeptide. The method includes culturing a cell line engineered to express the recombinant polypeptide under conditions that promote production of the recombinant polypeptide; and monitoring cell viability of the cultured cell line on at least one of days 6, 7, 8, 9, 10, 11, or 12 of culturing. If cell viability is below a threshold level, the method also includes stopping the culturing and isolating the recombinant polypeptide after a first predetermined additional time. If cell viability is above the threshold level, the method also includes stopping the culturing and isolating the recombinant polypeptide after a second predetermined additional time. The second predetermined additional time is at least about 10 hours (e.g., 12 hours) longer than the first predetermined time.

[0081] In embodiments, monitoring of cell viability is performed on at least one of days 6, 7, 8, 9, 10, or 11. For example, monitoring of cell viability may be performed on at least one of days 7, 8, 9, or 10. Monitoring of cell viability may be performed on at least one of days 8, 9, or 10. Monitoring of cell viability may be performed on day 8. Monitoring of cell viability may be performed on day 9. Monitoring of cell viability may be performed on day 10.

[0082] In embodiments, the threshold level is at least about 65% cell viability. For example, the threshold level may be at least about 70% cell viability. The threshold level may be at least about 75% cell viability. The threshold level may be at least about 80% cell viability. The threshold level may be at least about 85% cell viability. The threshold level may be at least about 90% cell viability. The threshold level may be at least about 95% cell viability.

[0083] In embodiments, the threshold level is selected from about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% cell viability. For example, the threshold level may be selected from about 75%, about 80%, about 85%, or about 90% cell viability.

[0084] The duration of each of the first and second predetermined additional times may be determined from the time that cell viability monitoring is performed. If the monitoring involves sampling followed by online or offline analysis, the additional times are determined from the time of sampling. The duration of the second predetermined additional time may also (or instead) be specified by how much longer it is than the first predetermined additional time.

[0085] In embodiments, the first predetermined additional time is selected such that the culturing is carried out for a total time of at least about 8 days. The first predetermined additional time may be selected such that the culturing is carried out for a total time of at least about 9 days. For example, the first predetermined additional time may be selected such that the culturing is carried out for a total time of at least about 10 days. The first predetermined additional time may be selected such that the culturing is carried out for a total time of at least about 11 days. The first predetermined additional time may be selected such that the culturing is carried out for a total time of at least about 12 days.

[0086] In an embodiment, the first predetermined additional time period may include a time period selected from the range of about 0.5 days to about 4 days. For example, the first predetermined additional time period may include a time period selected from the range of about 0.5 days to about 3 days. For example, the first predetermined additional time period may include a time period selected from the range of about 0.5 days to about 2 days. The first predetermined additional time period may include a time period selected from the range of about 1 day to about 3 days. For example, the first predetermined additional time period may include a time period selected from the range of about 1 day to about 2 days.

[0087] In embodiments, the first predetermined additional time period comprises at least about 10 hours. The first predetermined additional time period may comprise at least about 12 hours. The first predetermined additional time period may comprise at least about 18 hours. The first predetermined additional time period may comprise at least about 24 hours. The first predetermined additional time period may comprise at least about 36 hours. The first predetermined additional time period may comprise at least about 48 hours.

[0088] In embodiments, the first predetermined additional time period includes about 72 hours or less. The first predetermined additional time period may include about 60 hours or less. The first predetermined additional time period may include about 48 hours or less. The first predetermined additional time period may include about 36 hours or less. The first predetermined additional time period may include about 24 hours or less.

[0089] In embodiments, the second predetermined additional time is at least about 1 day longer than the first predetermined additional time. For example, the second predetermined additional time may be at least about 1.5 days longer than the first predetermined additional time. The second predetermined additional time may be at least about 2 days longer than the first predetermined additional time. The second predetermined additional time may be at least about 2.5 days longer than the first predetermined additional time. The second predetermined additional time may be at least about 3 days longer than the first predetermined additional time. The second predetermined additional time may be at least about 3.5 days longer than the first predetermined additional time. The second predetermined additional time may be at least about 4 days longer than the first predetermined additional time. The second predetermined additional time may be at least about 4.5 days longer than the first predetermined additional time.

[0090] In embodiments, the second predetermined additional time is about 1 day longer than the first predetermined additional time. The second predetermined additional time may be about 1.5 days longer than the first predetermined additional time. The second predetermined additional time may be about 2 days longer than the first predetermined additional time. The second predetermined additional time may be about 2.5 days longer than the first predetermined additional time. The second predetermined additional time may be about 3 days longer than the first predetermined additional time. The second predetermined additional time may be about 3.5 days longer than the first predetermined additional time. The second predetermined additional time may be about 4 days longer than the first predetermined additional time. The second predetermined additional time may be about 4.5 days longer than the first predetermined additional time.

[0091] In an embodiment, the second predetermined additional time may include a time selected from the range of about 1 day to about 6.5 days. For example, the second predetermined additional time may include a time selected from the range of about 1.5 days to about 6.5 days. The second predetermined additional time may include a time selected from the range of about 1.5 days to about 5.5 days. The second predetermined additional time may include a time selected from the range of about 1.5 days to about 4.5 days. The second predetermined additional time may include a time selected from the range of about 1.5 days to about 3.5 days. The second predetermined additional time may include a time selected from the range of about 2 days to about 6 days. The second predetermined additional time may include a time selected from the range of about 2 days to about 5 days. The second predetermined additional time may include a time selected from the range of about 2 days to about 4 days. The second predetermined additional time may include a time selected from the range of about 2 days to about 3 days.

[0092] In embodiments, the second predetermined additional time period comprises at least about 2 days. The second predetermined additional time period may comprise at least about 3 days. The second predetermined additional time period may comprise at least about 4 days. The second predetermined additional time period may comprise at least about 5 days.

[0093] In embodiments, the second predetermined additional time period may include about 6.5 days or less. The second predetermined additional time period may include about 5.5 days or less. The second predetermined additional time period may include about 4.5 days or less. The second predetermined additional time period may include about 3.5 days or less. The second predetermined additional time period may include about 2.5 days or less.

[0094] In embodiments, the recombinant polypeptide comprises at least two N-linked glycan sites. For example, the recombinant polypeptide may comprise at least three N-linked glycan sites. The recombinant polypeptide may comprise at least four N-linked glycan sites. The recombinant polypeptide may comprise at least five N-linked glycan sites. The recombinant polypeptide may comprise at least six N-linked glycan sites.

[0095] In embodiments, the recombinant polypeptide comprises 2 to 12 N-linked glycan sites. The recombinant polypeptide may comprise 2 to 10 N-linked glycan sites. The recombinant polypeptide may comprise 4 to 12 N-linked glycan sites. The recombinant polypeptide may comprise 4 to 10 N-linked glycan sites. The recombinant polypeptide may comprise 6 to 12 N-linked glycan sites. The recombinant polypeptide may comprise 6 to 10 N-linked glycan sites.

[0096] In embodiments, the recombinant polypeptide may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 N-linked glycan sites. The recombinant polypeptide may comprise 2, 3, 4, 5, 6, 7, 8, 9, or 10 N-linked glycan sites. The recombinant polypeptide may comprise 4, 5, 6, 7, 8, 9, or 10 N-linked glycan sites. The recombinant polypeptide may comprise 6, 7, 8, 9, or 10 N-linked glycan sites.

[0097] In embodiments, the recombinant polypeptide is an antibody, antigen, enzyme, or vaccine. The antibody may be a multispecific antibody (e.g., a bispecific antibody) or an antigen-binding fragment thereof. The antibody may be a multispecific antibody, such as a bispecific antibody. The antibody may be an antibody or an antigen-binding fragment of a multispecific (e.g., bispecific) antibody. The antibody may comprise or consist of a single heavy chain sequence and a single light chain sequence, or an antigen-binding fragment thereof. The antibody may comprise or consist of a single heavy chain sequence and a single light chain sequence. The antibody may comprise or consist of an antigen-binding fragment of a single heavy chain sequence and a single light chain sequence. The antibody may comprise a chimeric antibody, a human antibody, or a humanized antibody. The antibody may comprise a chimeric antibody. The antibody may comprise a human antibody. The antibody may comprise a humanized antibody. The antibody may comprise a monoclonal antibody.

[0098] In embodiments, the recombinant polypeptide is, for example, a fusion protein comprising an antibody (e.g., a multispecific antibody, e.g., a bispecific antibody) or an antigen-binding fragment thereof. The recombinant polypeptide can be a fusion protein comprising an antibody. The recombinant polypeptide can be a fusion protein comprising an antigen-binding fragment of an antibody. The antibody can comprise or consist of a single heavy chain sequence and a single light chain sequence, or an antigen-binding fragment thereof. The antibody can comprise or consist of a single heavy chain sequence and a single light chain sequence. The antibody can comprise or consist of an antigen-binding fragment of a single heavy chain sequence and a single light chain sequence. The antibody can comprise a chimeric antibody, a human antibody, or a humanized antibody. The antibody can comprise a chimeric antibody. The antibody can comprise a human antibody. The antibody can comprise a humanized antibody. The antibody can comprise a monoclonal antibody.

[0099] In embodiments, the recombinant polypeptide is an Fc fusion protein. The Fc fusion protein may be or comprise a multispecific (e.g., bispecific) antibody. The Fc fusion protein may be or comprise a cytokine Fc fusion protein.

[0100] In an embodiment, the cell line is an animal cell line. For example, the cell line may be a mammalian cell line. The cell line may be a CHO cell line or an NS0 cell line. The cell line may be a CHO cell line. For example, the cell line may be a CHO cell line selected from the group consisting of CHO K1 cell line, CHO K1SV cell line, DG44 cell line, DUKXB-11 cell line, CHOK1S cell line, or CHO K1M cell line, or a derivative thereof. In an embodiment, the CHO cell line may be a CHO cell line generated by targeted gene integration (TI). The cell line may be an NS0 cell line. For example, the cell line may be an NS0 cell line.

[0101] In embodiments, the cell line is cultured in a cell culture medium. The cell line can be cultured under fed-batch or perfusion culture conditions. For example, the cell line can be cultured under fed-batch culture conditions. The fed-batch culture conditions can include enhanced fed-batch culture conditions. For example, the cell line can be cultured under perfusion culture conditions. The perfusion culture conditions can include semi-continuous perfusion or continuous perfusion.

[0102] In embodiments, the isolated recombinant polypeptide comprises a level of sialylation from about 5 moles per mole of recombinant polypeptide to about 20 moles per mole of recombinant polypeptide. The isolated recombinant polypeptide may comprise a level of sialylation from about 7 moles per mole of recombinant polypeptide to about 15 moles per mole of recombinant polypeptide. The isolated recombinant polypeptide may comprise a level of sialylation from about 8 moles per mole of recombinant polypeptide to about 12 moles per mole of recombinant polypeptide. [Example]

[0103] Example 1 - Recombinant Cytokine Fc Fusion Proteins material and method The data presented in this paper are from experiments performed using a recombinant Chinese hamster ovary (CHO) cell line genetically engineered to secrete a recombinant Fc fusion protein with eight N-glycosylation sites.

[0104] Frozen ampoules of this cell line were thawed into a proprietary, chemically defined medium developed in-house and cultured in shake flasks at 37°C in a humidified incubator with 5% CO2 for at least two weeks before use in any bioreactor experiments. After the cells demonstrated good growth characteristics and high viability, they were scaled up for production culture experiments in a 2-liter stirred suspension bioreactor (Applikon, Foster City, CA). Production cultures were typically inoculated at approximately 1.0 million viable cells / mL in a chemically defined basal medium, also developed in-house. Cultures were exposed to the production culture medium through two or three inoculation stage cultures, each lasting three days. Production cultures were operated in fed-batch mode, and a concentrated, proprietary, chemically defined nutrient medium was fed to the cultures on day three at 20% of the culture volume. Glucose concentrations were analyzed daily, and if the glucose concentration fell below a certain threshold, it was replenished from a glucose stock solution to prevent glucose depletion.

[0105] The reactor was equipped with calibrated dissolved oxygen, pH, and temperature probes. Dissolved oxygen was controlled online by sparging with air and / or oxygen. pH was controlled by adding CO2 or Na2CO3. Antifoam was added to the culture as needed. Cultures were maintained at pH 7.0 and temperature 37°C from day 0 to day 3, then 33°C after day 3, with stirring at 275 rpm. Dissolved oxygen levels were controlled to maintain 30% air saturation. Viable cell density (VCC), viability, offline pH, osmolality, and metabolite concentrations were determined daily using a NovaFlex (Nova Biomedical, Waltham, MA). Packed cell volume (PCV) was measured after centrifugation of the cell suspension at 830 g for 10 min using graduated centrifuge tubes (Kimble Science Products, Fullerton, CA). PCV was expressed as a percentage of the total culture volume. Culture supernatant was also collected daily by centrifugation of 1 mL of cell culture medium and used for product titration using high performance liquid chromatography.

[0106] Measurement of sialic acid content For all cultures performed in this study, cell culture fluid was harvested by centrifugation at the end of the culture period (typically 12 days). Monoclonal antibodies in the harvested cell culture fluid were purified using Protein A affinity chromatography. N-acetylneuraminic acid (NANA) content was determined using a sialic acid reverse-phase ultra-high performance liquid chromatography (RP-UHPLC) method. Samples were acid hydrolyzed and subsequently derivatized with o-phenylenediamine (OPD). Analysis was by C-18 RP-UHPLC with fluorescence detection. Concentrations were calculated from an external calibration curve and reported as moles of NANA per mole of protein. To characterize the effect of culture duration on sialic acid content, cell culture fluid samples from earlier culture periods were also analyzed in this manner to measure sialic acid content.

[0107] Results and Discussion The results are shown in Figures 1, 3, and 4. Figure 1 shows that the sialic acid content measured over the course of cell culture decreases with increasing culture duration. Figure 3 shows that the sialic acid content measured at the end of the culture (day 12) correlates with the viability of the culture measured on that day, i.e., the higher the viability of the culture, the higher the sialic acid content. Figure 4 shows that the viability of the culture measured earlier in the period (day 10) correlates with the sialic acid content measured on the final day of culture (day 12), i.e., the sialic acid content at the end of the culture can be predicted by viability measurements from earlier in the culture. Therefore, adjusting the overall duration of the culture based on measurements of cell viability earlier in the culture can be used to control the level of sialic acid content at the end of the culture.

[0108] Example 2 - Recombinant Monoclonal Antibodies A recombinant Chinese hamster ovary (CHO) cell line genetically engineered to secrete monoclonal antibodies was prepared and cultured in a manner similar to that described for Example 1.

[0109] The viability of the cell cultures was measured on days 8, 9, and 10, as well as on the final day of cell culture (day 12). The final level of sialylated species (G2-1SA) present in optimally sialylated antibodies was found to correlate with viability on days 8, 9, and 10. Figure 5 shows the correlation between viability measured on days 8, 9, and 10 of culture and the level of sialylated glycan species (G2-1SA) at the end of culture (day 12). G2-1SA is normalized to the highest G2-1SA reported in the optimally sialylated antibody sample set (i.e., a normalized G2-1SA of 0.50 is 50% of the highest G2-1SA value reported in the data set). Figure 6 also shows the correlation between normalized G2-1SA and culture duration. Thus, this example also demonstrates that viability measurements earlier in the culture can be used to control the final level of sialylation at the end of culture by varying the culture duration based on earlier viability measurements.

Claims

1. 1. A method for optimizing the sialic acid content of a recombinant polypeptide, comprising: Culturing a cell line engineered to express said recombinant polypeptide under conditions that promote production of said recombinant polypeptide; monitoring cell viability of the cultured cell line on at least one of days 6, 7, 8, 9, 10, or 11 of the culture; if the cell viability is below a threshold level, stopping the culturing and isolating the recombinant polypeptide after a first predetermined additional time period; or if the cell viability is above the threshold level, stopping the culturing and isolating the recombinant polypeptide after a second predetermined additional time period; Including, the second predetermined additional time period is at least about 12 hours longer than the first predetermined time period; method.

2. 10. The method of claim 1, wherein said monitoring cell viability is performed on at least one of the seventh, eighth, ninth, tenth, or eleventh day.

3. 10. The method of claim 1, wherein said monitoring cell viability is performed on at least one of the 8th, 9th, 10th, or 11th day.

4. 3. The method of claim 1 or 2, wherein said monitoring of cell viability is performed on at least one of the 8th, 9th, or 10th day.

5. The method of any one of claims 1 to 4, wherein the threshold level is at least about 65% cell viability.

6. The method of any one of claims 1 to 5, wherein the threshold level is at least about 70% cell viability.

7. The method of any one of claims 1 to 6, wherein the threshold level is at least about 75% cell viability.

8. The method of any one of claims 1 to 7, wherein the threshold level is at least about 80% cell viability.

9. The method of any one of claims 1 to 8, wherein the threshold level is at least about 85% cell viability.

10. The method of any one of claims 1 to 9, wherein the threshold level is at least about 90% cell viability.

11. The method of any one of claims 1 to 10, wherein the threshold level is at least about 95% cell viability.

12. 4. The method of claim 1, wherein the threshold level is selected from about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% cell viability.

13. 13. The method of any one of claims 1 to 12, wherein the first predetermined additional time period is selected such that the culturing is carried out for a total time period of at least about 8 days, optionally at least about 10 days, and further optionally at least about 11 days.

14. 14. The method of any one of claims 1 to 13, wherein the first predetermined additional time period comprises a time period selected from the range of about 0.5 days to about 4 days.

15. 15. The method of any one of claims 1-14, wherein the second predetermined additional time period is at least about 1.5 days, at least about 2 days, at least about 2.5 days, at least about 3 days, at least about 3.5 days, or at least about 4 days longer than the first predetermined additional time period.

16. 16. The method of any one of claims 1 to 15, wherein the second predetermined additional time period comprises a time period selected from the range of about 1.5 days to about 6 days.

17. The method of any one of claims 1 to 16, wherein the recombinant polypeptide comprises at least two N-linked glycan sites.

18. The method of any one of claims 1 to 17, wherein the recombinant polypeptide is an antibody, an antigen, an enzyme, or a vaccine.

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

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

21. The method of any one of claims 14 to 16, wherein the antibody comprises a chimeric antibody, a human antibody, or a humanized antibody.

22. The method of any one of claims 14 to 17, wherein the antibody comprises a monoclonal antibody.

23. The method of any one of claims 1 to 22, wherein the recombinant polypeptide is a fusion protein optionally comprising an antibody or an antigen-binding fragment thereof.

24. 20. The method of claim 19, wherein the recombinant polypeptide is an Fc fusion protein.

25. The method of any one of claims 1 to 24, wherein the cell line is a mammalian cell line.

26. 26. The method of any one of claims 1 to 25, wherein said cell line is a CHO cell line or an NSO cell line; optionally a CHO K1, CHO K1SV, DG44, DUKXB-11, CHOK1S or CHO K1M cell line, or a CHO cell line generated by targeted gene integration (TI), or a derivative thereof.

27. The method of any one of claims 1 to 26, wherein the cell line is cultured in a cell culture medium.

28. 28. The method of any one of claims 1 to 27, wherein the cell line is cultured under fed-batch or perfusion culture conditions.

29. 25. The method of claim 24, wherein the cell line is cultured under fed-batch culture conditions, optionally wherein the fed-batch culture conditions are enriched fed-batch culture conditions.

30. 25. The method of claim 24, wherein the cell line is cultured under perfusion culture conditions, optionally wherein the perfusion culture conditions are semi-continuous perfusion or continuous perfusion.

31. 31. The method of any one of claims 1-30, wherein the isolated recombinant polypeptide comprises sialylation at a level of from about 5 mol per mol of recombinant polypeptide to about 20 mol per mol of recombinant polypeptide.

32. 32. The method of any one of claims 1-31, wherein the isolated recombinant polypeptide comprises sialylation at a level of from about 8 mol per mol of recombinant polypeptide to about 12 mol per mol of recombinant polypeptide.

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