B vitamin modulation

Abstract

This disclosure relates to methods of optimizing glycosylation of a recombinant polypeptide. Also provided are methods of optimizing charge pattern of a recombinant polypeptide. Also provided are methods of optimizing size distribution of a recombinant polypeptide. The methods comprise modulating the level of B vitamins in a cell culture comprising a cell line engineered to express the recombinant polypeptide.

Description

B Vitamin Modulation

[0001] This invention relates to methods of optimizing glycosylation of a recombinant polypeptide. Also provided are methods of optimizing charge pattern of a recombinant polypeptide. The methods may comprise modulating the level of B vitamins in a cell culture comprising a cell line engineered to express the recombinant polypeptide.BACKGROUND

[0002] Recombinant therapeutic proteins are predominantly produced using mammalian cells that have been transfected to express the therapeutic product. Recombinant monoclonal antibodies (mAbs) are typically produced using Chinese Hamster Ovary (CHO) cells and exhibit extensive heterogeneity at the time of cell culture harvest. Two of the major forms of heterogeneity observed in CHO-derived mAbs arise from post-translational modifications occurring during cell culture that lead to differences in glycosylation and charge distributions. Another form of heterogeneity in mAbs are size distribution, with the monomeric form (i.e., monomer) being the main desired form of the mAb, and the high molecular weight (HMW) form and the low molecular weight (LMW) forms being undesired forms.

[0003] The most common form of glycosylation heterogeneity occurring in mAbs occurs at the Asn297 on the two IgG heavy chains (Majewska et al., 2020, Annual Review of Chemical and Biomolecular Engineering, 11 , 311-338). Following a glycan transfer to the N-linked glycosylation consensus site of the mAb in the endoplasmic reticulum, the glycan is then trimmed and extended in the Golgi via a series of enzyme-catalyzed reactions. The basic glycan can have additional linkages to fucose at its first GIcNAc (to generate fucosylated mAbs) or to additional mannose (to generate high mannose forms, i.e., mannosylated mAbs) or to GIcNAc and then to galactose (to generate galactosylated mAbs) and finally to sialic acid (to generate sialylated mAbs). For illustration purposes and to aid subsequent discussions on glycosylation heterogeneity, some of the major glycosylation patterns observed in mAbs are illustrated in Figure 1.

[0004] Charge heterogeneity observed in mAbs can stem from modifications that are common across mAbs and processes or are unique to a given mAb and process (Liu et al., 2014, Biotechnology Progress, 32, 1103-1112). A number of variants are typically observed when the mAbs are analyzed by charge-based separation techniques such as isoelectric focusing (IEF) electrophoresis or ion exchange (I EX)chromatography. When analyzed by lEF-based methods, the acidic forms (i.e. , acidic variants or acidic species) have lower apparent isoelectric point (pl) while the basic forms (i.e., basic variants or basic species) have higher apparent pl relative to the main isoform (i.e., main species). When analyzed by IEX chromatographybased methods, the acidic and basic species are defined based on their retention times relative to the main peak.

[0005] Size heterogeneity is observed in mAbs due to the presence of high molecular weight (HMW) forms and I or low molecular weight (LMW) forms in addition to the main desired monomeric form (i.e. the mAb monomer). HMW forms are typically multimers that can arise from non-covalent interactions or the formation of intermolecular disulfide bonds. LMW forms are often fragments of mAbs that may be caused by spontaneous hydrolysis of peptide bonds or proteolytic enzymes present as host cell proteins. LMW forms can also arise from incomplete disulfide bond formation. These mAb size variants (i.e., LMW forms and HMW forms) may be measured by size-exclusion chromatography (SEC) or capillary electrophoresis sodium dodecyl sulfate (CE-SDS). Generally, SEC is more suited for measuring HMW forms and CE-SDS for resolving LMW forms.

[0006] Glycosylation, charge, and size variants in mAbs are usually considered to be critical quality attributes (CQAs) because of their potential impact on pharmacokinetics, safety and efficacy of the product. In particular, the extent of mAb glycosylation in terms of afucosylation, galactosylation, mannosylation, and sialylation can impact antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), PK, and anti-inflammatory activity respectively (Majewska et al., 2020, Annual Review of Chemical and Biomolecular Engineering, 11 , 311-338; Nimmerjahn et al., 2023, Nature Immunology, 24, 1244- 1255). The charge distribution in terms of acidic or basic forms (also referred to as variants or species) can impact biological activity, stability, and PK, depending on the type and location of the charge modification (Hintersteiner et al., 2016, mAbs, 8, 1548-1560; Singh et al., 2016, Electrophoresis, 37, 17-18; Yan et al., 2009, Journal of Pharmaceutical Sciences, 98, 3509-3521). Size variants in the form of HMW or LMW forms (also referred to as species) can impact the immunogenicity and potency of a mAb ((Hermeling et al., 2004, Pharmaceutical Research, 21 , 897- 903; Moussa et al., 2016, Journal of Pharmaceutical Sciences, 105, 417-430; Wang et al., 2018, Journal of Pharmaceutical and Biomedical Analysis, 154, 468- 475; Vlasak and lonescu, 2011, mAbs, 3, 253-263. Therefore, these CQAs need tobe carefully controlled during the cell culture process to ensure consistent product quality.

[0007] It is an aim of the present invention to provide methods of controlling glycosylation, charge, and / or size variants of recombinant polypeptides.BRIEF SUMMARY OF THE DISCLOSURE

[0008] In a first aspect of the invention, there is provided a method of optimizing glycosylation of a recombinant polypeptide, comprising: culturing a cell line engineered to express the recombinant polypeptide in a culture medium under conditions promoting production of the recombinant polypeptide; and modulating the level of B vitamins in the cell culture; optionally wherein the modulating the level of B vitamins comprises providing a lower level of B vitamins in the cell culture, or a higher level of B vitamins in the cell culture.

[0009] Without wishing to be bound by theory, it is thought that the glycosylation profiles of recombinant proteins can be impacted by modulating the levels of several B vitamins during the cell culture process. In particular, higher levels of B vitamins are thought to result in specific glycosylation profile differences in the extent of afucosylation, galactosylation, mannosylation, and sialylation.

[0010] In a second aspect of the invention, there is provided a method of optimizing charge pattern of a recombinant polypeptide, comprising: culturing a cell line engineered to express the recombinant polypeptide under conditions promoting production of the recombinant polypeptide; and modulating the level of B vitamins in the cell culture; optionally wherein the modulating the level of B vitamins comprises providing a lower level of B vitamins in the cell culture, or a higher level of B vitamins in the cell culture.

[0011] Without wishing to be bound by theory, it is thought that the charge pattern can be impacted by modulating the levels of several B vitamins supplemented during the cell culture process. In particular, higher levels of B vitamins are thought to result in overall charge profiles differences in the distribution of acidic, basic, and main species.

[0012] In a third aspect of the invention, there is provided a method of optimizing size pattern of a recombinant polypeptide, comprising: culturing a cell line engineered to express the recombinant polypeptide underconditions promoting production of the recombinant polypeptide; and modulating the level of B vitamins in the cell culture; optionally wherein the modulating the level of B vitamins comprises providing a lower level of B vitamins in the cell culture, or a higher level of B vitamins in the cell culture.

[0013] Without wishing to be bound by theory, it is thought that the size pattern can be impacted by modulating the levels of several B vitamins supplemented during the cell culture process. In particular, higher levels of B vitamins are thought to result in overall size profiles differences in the distribution of HMW forms and LMW forms relative to the desired main monomeric form of the recombinant product.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:Figure 1 shows a schematic indicating the nomenclature used for different mAb N-glycosylation patterns provided in the present disclosure. Total afucosylation is represented by the sum of GO-N, GO, G1, G2, and M5 species. Total galactosylation is represented by the sum of G1 , G2, G1F, G2F, G1S1F, G2S1F, and G2S2F species. Mannosylation is represented by the M5 species. Total sialylation is represented by the sum of the G1S1F, G2S1F, and G2S2F species.Figure 2 shows the integral viable cell count (IVCC) (1e8 cells day / L) at the time of production culture harvest across bioreactors using different mAbs, cell lines, media, cell culture processes, and culture durations with (+) and without (-) supplemental B-vitamins.Figure 3 shows the final viability (%) at the time of production culture harvest in bioreactors across mAbs, cell lines, media, cell culture processes, and culture durations with (+) and without (-) supplemental B-vitamins.Figure 4 shows the harvest titer (g / L) in bioreactor production cultures across mAbs, cell lines, media, cell culture processes, and culture durations with and without supplemental B-vitamins.Figure 5 shows total afucosylation (%) levels across mAbs, cell lines, media, cell culture processes, and culture durations with (+) and without (-) supplemental B-vitamins. Total afucosylation is represented by the sum of GO-N, GO, G1 , G2, and M5 glycan species.Figure 6 shows levels of GO-N afucosylated species across mAbs, cell lines, media, cell culture processes, and culture durations with (+) and without (-) supplemental B-vitamins.Figure 7 shows levels of GO afucosylated species across mAbs, cell lines, media, cell culture processes, and culture durations with (+) and without (-) supplemental B- vitamins.Figure 8 shows levels of G1 afucosylated species across mAbs, cell lines, media, cell culture processes, and culture durations with (+) and without (-) supplemental B- vitamins.Figure 9 shows levels of G2 afucosylated species across mAbs, cell lines, media, cell culture processes, and culture durations with (+) and without (-) supplemental B- vitamins.Figure 10 shows total galactosylation (%) levels across mAbs, cell lines, media, cell culture processes, and culture durations with (+) and without (-) supplemental B- vitamins. Total galactosylation is represented by the sum of G1 , G2, G1 F, G2F, G1S1 F, G2S1 F, and G2S2F species.Figure 11 shows levels of G1 F galactosylated species (%) across mAbs, cell lines, media, cell culture processes, and culture durations with (+) and without (-) supplemental B-vitamins.Figure 12 shows levels of G2F galactosylated species (%) across mAbs, cell lines, media, cell culture processes, and culture durations with (+) and without (-) supplemental B-vitamins.Figure 13 shows levels of M5 mannosylated species (%) across mAbs, cell lines, media, cell culture processes, and culture durations with (+) and without (-) supplemental B-vitamins.Figure 14 shows total sialylation (%) levels across mAbs, cell lines, media, cell culture processes, and culture durations with (+) and without (-) supplemental B-vitamins. Total sialylation is represented by the sum of G1S1 F, G2S1 F, and G2S2F species.Figure 15 shows levels of G1S1 F sialylated species (%) across mAbs, cell lines, media, cell culture processes, and culture durations with (+) and without (-) supplemental B-vitamins.Figure 16 shows levels of G2S1 F sialylated species (%) across mAbs, cell lines, media, cell culture processes, and culture durations with (+) and without (-) supplemental B-vitamins.Figure 17 shows levels of G2S2F sialylated species (%) across mAbs, cell lines, media, cell culture processes, and culture durations with (+) and without (-) supplemental B-vitamins.Figure 18 show levels of main species (%) in terms of charge distribution across mAbs, cell lines, media, and cell culture processes with (+) and without (-) supplemental B- vitamins.Figure 19 shows levels of acidic species (%) in terms of charge distribution across mAbs, cell lines, media, and cell culture processes with (+) and without (-) supplemental B- vitamins.Figure 20 shows the levels of basic species (%) in terms of charge distribution across mAbs, cell lines, media, and cell culture processes with (+) and without (-) supplemental B-vitamins.Figure 21 shows the integral viable cell count (IVCC) (1e8 cells day / L) in bioreactor production cultures on Days 10 and 12 across bioreactors with varying levels of supplemental B-vitamins.Figure 22 shows the final viability (%) in bioreactor production cultures on Days 10 and 12 across bioreactors with varying levels of supplemental B-vitamins.Figure 23 shows the harvest titer (g / L) in bioreactor production cultures on Days 10 and 12 across bioreactors with varying levels of supplemental B-vitamins.Figure 24 shows total afucosylation (%) levels on Days 10 and 12 across bioreactors with varying levels of supplemental B-vitamins. Total afucosylation is represented by the sum of G0-N, GO, G1, G2, and M5 glycan species.Figure 25 shows levels of G0-N afucosylated species on Days 10 and 12 across bioreactors with varying levels of supplemental B-vitamins.Figure 26 shows levels of GO afucosylated species on Days 10 and 12 across bioreactors with varying levels of supplemental B-vitamins.Figure 27 shows levels of G1 afucosylated species on Days 10 and 12 across bioreactors with varying levels of supplemental B-vitamins.Figure 28 shows levels of G2 afucosylated species on Days 10 and 12 across bioreactors with varying levels of supplemental B-vitamins.Figure 29 shows total galactosylation (%) levels on Days 10 and 12 across bioreactors with varying levels of supplemental B-vitamins. Total galactosylation is represented by the sum of G1 , G2, G1 F, G2F, G1S1 F, G2S1 F, and G2S2F species.Figure 30 shows levels of G1 F galactosylated species (%) on Days 10 and 12 across bioreactors with varying levels of supplemental B-vitamins.Figure 31 shows levels of G2F galactosylated species (%) on Days 10 and 12 across bioreactors with varying levels of supplemental B-vitamins.Figure 32 shows levels of M5 mannosylated species (%) on Days 10 and 12 across bioreactors with varying levels of supplemental B-vitamins.Figure 33 shows total sialylation (%) levels on Days 10 and 12 across bioreactors with varying levels of supplemental B-vitamins. Total sialylation is represented by the sum of G1S1 F, G2S1 F, and G2S2F species.Figure 34 shows levels of G1S1 F sialylated species (%) on Days 10 and 12 across bioreactors with varying levels of supplemental B-vitamins.Figure 35 shows levels of G2S1 F sialylated species (%) on Days 10 and 12 across bioreactors with varying levels of supplemental B-vitamins.Figure 36 shows levels of G2S2F sialylated species (%) on Days 10 and 12 across bioreactors with varying levels of supplemental B-vitamins.Figure 37 show levels of main species (%) in terms of charge distribution on Days 10 and 12 across bioreactors with varying levels of supplemental B-vitamins.Figure 38 shows levels of acidic species (%) in terms of charge distribution on Days 10 and 12 across bioreactors with varying levels of supplemental B-vitamins.Figure 39 shows the levels of basic species (%) in terms of charge distribution on Days 10 and 12 across bioreactors with varying levels of supplemental B-vitamins.Figure 40 shows DOE 1 parameter effect estimates and prediction profiler for integral of viable cell concentration (IVCC) (1e8 cells day / L) response.Figure 41 shows DOE 1 parameter effect estimates and prediction profiler for final viability (%) response at the time of production culture harvest.Figure 42 shows DOE 1 parameter effect estimates and prediction profiler for mAb A harvest titer (g / L) response.Figure 43 shows DOE 1 parameter effect estimates and prediction profiler for total afucosylation (%) response. Total afucosylation is represented by the sum of GO-N, GO, G1 , G2, and M5 glycan species.Figure 44 shows DOE 1 parameter effect estimates and prediction profiler for G0- N (%) afucosylated species response.Figure 45 shows DOE 1 parameter effect estimates and prediction profiler for GO (%) afucosylated species response.Figure 46 shows DOE 1 parameter effect estimates and prediction profiler for G1 (%) afucosylated species response.Figure 47 shows DOE 1 parameter effect estimates and prediction profiler for G2 (%) afucosylated species response.Figure 48 shows DOE 1 parameter effect estimates and prediction profiler for total galactosylation (%) response. Total galactosylation is represented by the sum of G1, G2, G1 F, G2F, G1S1 F, G2S1 F, and G2S2F species.Figure 49 shows DOE 1 parameter effect estimates and prediction profiler for G1 F (%) galactosylated species response.Figure 50 shows DOE 1 parameter effect estimates and prediction profiler for G2F (%) galactosylated species response.Figure 51 shows DOE 1 parameter effect estimates and prediction profiler for M5 (%) mannosylated species response.Figure 52 shows DOE 1 parameter effect estimates and prediction profiler for total sialyation (%) response. Total sialylation is represented by the sum of G1S1 F, G2S1F, and G2S2F species.Figure 53 shows DOE 1 parameter effect estimates and prediction profiler for G1S1 F (%) sialylated species response.Figure 54 shows DOE 1 parameter effect estimates and prediction profiler for G2S1 F (%) sialylated species response.Figure 55 shows DOE 1 parameter effect estimates and prediction profiler for G2S2F (%) sialylated species response.Figure 56 shows DOE 1 parameter effect estimates and prediction profiler for main species (%) in terms of charge distribution response.Figure 57 shows DOE 1 parameter effect estimates and prediction profiler for acidic species (%) in terms of charge distribution response.Figure 58 shows DOE 1 parameter effect estimates and prediction profiler for basic species (%) in terms of charge distribution response.Figure 59 shows DOE 1 parameter effect estimates and prediction profiler for sum of LMW forms (%) in terms of size distribution response.Figure 60 shows DOE 1 parameter effect estimates and prediction profiler for sum of HMW forms (%) in terms of size distribution response.Figure 61 shows DOE 2 parameter effect estimates and prediction profiler for final viability (%) response at the time of production culture harvest with cell lines D and E producing mAb C.Figure 62 shows DOE 2 parameter effect estimates and prediction profiler for integral of viable cell concentration (IVCC) (1e8 cells day / L) response with cell lines D and E producing mAb C.Figure 63 shows DOE 2 parameter effect estimates and prediction profiler for harvest mAb C titer (g / L) response with cell lines D and E.Figure 64 shows DOE 2 parameter effect estimates and prediction profiler for Acidic Species (%) in terms of charge distribution response with cell lines D and E producing mAb C.Figure 65 shows DOE 2 parameter effect estimates and prediction profiler for Basic Species (%) in terms of charge distribution response with cell lines D and E producing mAb C.Figure 66 shows DOE 2 parameter effect estimates and prediction profiler for Main Species (%) in terms of charge distribution response with cell lines D and E producing mAb C.Figure 67 shows DOE 2 parameter effect estimates and prediction profiler for sum of LMW forms (%) in terms of size distribution response with cell lines D and E producing mAb C.Figure 68 shows DOE 2 parameter effect estimates and prediction profiler for sum of HMW forms (%) in terms of size distribution response with cell lines D and E producing mAb C.DETAILED DESCRIPTION

[0015] The abbreviations used herein have their conventional meaning within the chemical and biological arts.

[0016] 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.

[0017] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0018] 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.

[0019] 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.

[0020] 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

[0021] 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.

[0022] 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.”

[0023] 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.

[0024] 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.

[0025] 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.

[0026] In the context of the present invention and disclosure, the nutrient solution (or nutrient feed) may comprise one or more B vitamins. The one or more B vitamins may be selected from cyanocobalamin (B12), folic acid (B9), pyridoxine (B6) and riboflavin (B2). The one or more B vitamins may consist of one or more B vitamins selected from cyanocobalamin (B12), folic acid (B9), pyridoxine (B6) and riboflavin (B2). The one or more B vitamins in the nutrient solution (e.g. included in the basal medium) may also comprise one or more of thiamine (B1), niacin (B3), pantothenic acid (B5), biotin (B7).

[0027] 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.

[0028] “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.

[0029] “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.

[0030] “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.

[0031] In a fed-batch culture or a perfusion culture, the composition of the nutrient solution may change over time. For example, as provided in exemplary methods of the invention, the level of one or more B vitamins in the cell culture may be modulated. The one or more B vitamins that have their level modulated may comprise (orconsist) of one or a combination of riboflavin (B2), pyridoxine (B6), folic acid (B9), or cyanocobalamin (B12).

[0032] As used herein, the term “cell,” refers to animal cells (e.g. mammalian cells), fungal cells (e.g. yeast cells), cultured cells, host cells, recombinant cells and recombinant host cells. Such cells are generally cell lines obtained or derived from mammalian tissues or fungi which are able to grow and survive when placed in media containing appropriate nutrients and / or growth factors.

[0033] 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.

[0034] 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.

[0035] 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. 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 forexample in Mammalian Cell Culture (Mather, J. P. ed., Plenum Press, N.Y. 1984), and Barnes and Sato, (1980) Cell, 22:649). Typically, the cells are capable of expressing and secreting large quantities of a particular protein, e.g., glycoprotein, of interest into the culture medium. Examples of suitable mammalian host cells within the context of the present disclosure can include Chinese hamster ovary cells / -DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 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); HEK 293 cells; buffalo rat liver cells (BRL 3A, ATCC CRL 1442); mouse mammary tumor (MMT 060562, ATCC CCL51). In certain embodiments, the mammalian cells include Chinese hamster ovary cells (CHO). In certain embodiments, the cells comprise a polynucleotide that encodes a polypeptide. In another embodiment, the cell expresses the polypeptide transiently or expresses the polypeptide stably. In a further embodiment, the cells expressing the polypeptide stably comprises a polynucleotide that is integrated in the cellular genome of the cell at a targeted location. In another further embodiment, the cells expressing the polypeptide stably comprises a polynucleotide that is integrated in the cellular genome of the cell at a random location.

[0036] 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.

[0037] 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 quantitatedeither by assaying for the biological activity of the protein or by employing assays that are independent of such activity, such as western blotting or radioimmunoassay using antibodies that are capable of reacting with the protein. Sambrook et al., Molecular Cloning: A Laboratory Manual, pp. 18.1-18.88 (Cold Spring Harbor Laboratory Press, 1989). When reference is made to reduction and / or elimination of the expression of one or more endogenous products relative to the expression of the endogenous product(s) in an unmodified cell, such reductions and / or eliminations of expression encompass reductions and / or eliminations of the active endogenous product, notwithstanding the presence of mRNA encoding all or a portion of the endogenous product or the presence of endogenous product translated from such mRNA.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] The term “antibody” is used herein in the broadest sense and encompasses various antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies (e.g., antibodies consisting of a single heavy chain sequence and a single light chain sequence, including multimers of such pairings), multispecific antibodies (e.g., bispecific antibodies) and antibody fragments so long as they exhibit the desired antigen-binding activity. A therapeutic antibody is an antibody that may be used in the treatment of a disease.

[0042] 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).

[0043] 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.

[0044] 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. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, 5, E, 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.

[0045] 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.

[0046] 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.

[0047] A “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.

[0048] 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.

[0049] 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.

[0050] The following abbreviations are used herein:2-AB 2-aminobenzamideADCC Antibody-dependent cellular cytotoxicityB12 CyanocobalaminB2 RiboflavinB6 PyridoxineB9 Folic acidCDC Complement-dependent cytotoxicityCE-SDS Capillary electrophoresis sodium dodecyl sulfateCHO Chinese hamster ovaryCpB Carboxypeptidase BCQA Critical quality attributeDO Dissolved oxygenDOE Design of ExperimentsHI LIC LIPLC Hydrophilic interaction chromatography ultrahigh performance liquid chromatographyHMW High molecular weightIEF Isoelectric focusingI EX Ion exchangeIg ImmunoglobulinIgG Immunoglobulin GIVCC Integral of viable cell concentrationLMW Low molecular weight mAbs Monoclonal antibodies pl Isoelectric pointSEC Size exclusion chromatographyVCD Viable cell densityMethods of Optimizing Glycosylation of a Recombinant Polypeptide

[0051] In an aspect, the invention provides a method of optimizing glycosylation of a recombinant polypeptide, comprising: culturing a cell line engineered to express the recombinant polypeptide in a culture medium under conditions promoting production of the recombinant polypeptide; and modulating the level of B vitamins in the cell culture.

[0052] The step of modulating the level of B vitamins may comprise providing a lower level of B vitamins in the cell culture, or a higher level of B vitamins in the cell culture.

[0053] The step of modulating the level of B vitamins may comprise providing a lower level of B vitamins in the cell culture. In these embodiments, the lower level may comprise maintaining or decreasing the level of B vitamins in the cell culture. Decreasing the level of B vitamins is typically achieved via a dilution. For example, decreasing the level of B vitamins may be achieved by supplementing the cell culture with media and / or nutrient feeds without (-) B vitamins, or by replacing at least part of the cell culture medium with media comprising a low level or without (-) B vitamins.

[0054] The step of optimizing the glycosylation may comprise optimizing the level of afucosylation, and / or galactosylation, and / or mannosylation, and / or sialylation. The step of optimizing the glycosylation may comprise optimizing the level of afucosylation. The step of optimizing the glycosylation may comprise optimizing the level of galactosylation. The step of optimizing the glycosylation may comprise optimizing the level of mannosylation. The step of optimizing the glycosylation may comprise optimizing the level of sialylation.

[0055] It may be that optimizing afucosylation comprises using modulated levels of B vitamins to achieve the desired level of afucosylation.

[0056] It may be that optimizing afucosylation comprises using a higher level of B vitamins to maintain a decreased level of afucosylation. Alternatively, it may be that optimizing afucosylation comprises using a lower level of B vitamins to maintain an increased level of afucosylation.

[0057] It may be that optimizing galactosylation comprises using modulated levels of B vitamins to achieve the desired level of galactosylation.

[0058] It may be that optimizing galactosylation comprises using the higher level of B vitamins to maintain a decreased level of galactosylation. Alternatively, it may be that optimizing galactosylation comprises using the lower level of B vitamins to maintain an increased level of galactosylation.

[0059] It may be that optimizing galactosylation comprises using the higher level of B vitamins to maintain an increased level of galactosylation. Alternatively, it may be that optimizing galactosylation comprises using the lower level of B vitamins to maintain a decreased level of galactosylation.

[0060] It may be that optimizing mannosylation comprises using modulated levels of B vitamins to achieve the desired level of mannosylation.

[0061] It may be that optimizing mannosylation comprises using the higher level of B vitamins to maintain a decreased level of mannosylation. Alternatively, it may be that optimizing mannosylation comprises using the lower level of B vitamins to maintain an increased level of mannosylation.

[0062] It may be that optimizing sialylation comprises using modulated levels of B vitamins to achieve the desired level of sialylation.

[0063] It may be that optimizing sialylation comprises using the higher level of B vitamins to maintain a decreased level of sialylation. Alternatively, it may be that optimizing sialylation comprises using lower level of B vitamins to maintain an increased level of sialylation.

[0064] It may be that optimizing sialylation comprises using the higher level of B vitamins to maintain an increased level of sialylation. Alternatively, it may be that optimizing sialylation comprises using lower level of B vitamins to maintain a decreased level of sialylation.

[0065] The method may comprise optimizing both glycosylation and optimizing the charge pattern of the recombinant polypeptide. In these embodiments, the method may further comprise any of the features of the second aspect, or embodiments thereof, as disclosed herein.

[0066] The higher levels of B vitamins may be achieved by supplementing the cell culture with B vitamins.

[0067] The supplementing with B vitamins may comprise at least partially replacing the cell culture medium with a production culture medium having a high level of B vitamins. Alternatively (or additionally), the supplementing with B vitamins may comprise providing a feed with a high level of B vitamins.

[0068] The higher levels of B vitamins may be achieved by increasing the levels of B vitamins in the production culture medium. Alternatively (or additionally), the higher levels of B vitamins may be achieved by supplementing the production cell culture with nutrient feed(s) containing high levels of B vitamins.

[0069] Further alternatively (or additionally), the higher levels of B vitamins may be achieved by supplementing one or more nutrient feeds with higher levels of B vitamins to the cell culture during the production phase.

[0070] In an embodiment, the nutrient feeds may be added through continuous feeding or daily feeds during a production period from 1-20 days (optionally a period of from 6- 13 days). Alternatively, the nutrient feeds may be added to the cell culture medium 1-4 times during a production period of from 2-15 days (optionally a period of from 6-13 days). Alternatively, the nutrient feeds may be added to the cell culture medium 2-3 times during a period of from 3-14 days (optionally a period of from 6- 13 days).

[0071] The B vitamins may comprise one or a combination of riboflavin (B2), pyridoxine (B6), folic acid (B9), or cyanocobalamin (B12). The B vitamins may consist of one or a combination of riboflavin (B2), pyridoxine (B6), folic acid (B9), or cyanocobalamin (B12).

[0072] The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 200 pM B2. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 150 pM B2. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 100 pM B2. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 75 pM B2. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 50 pM B2. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 40 pM B2. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 30 pM B2. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 20 pM B2. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 10 pM B2. The higher level of B vitamins may comprise providing a level of increase inB vitamins in the culture medium of about 0.1 pM to about 7.5 pM B2. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 5 pM B2.

[0073] The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.5 pM to about 200 pM B2. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 200 pM B2. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1.25 pM to about 200 pM B2. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1.5 pM to about 200 pM B2. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 2 pM to about 200 pM B2.

[0074] The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 200 pM B2 (e.g. of about 0.1 pM to about 20 pM B2). The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 100 pM B2 (e.g. of about 1 pM to about 10 pM B2, or, of about 1 pM to about 50 pM B2). The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 2 pM to about 50 pM B2 (e.g. of about 2 pM to about 5 pM B2).

[0075] The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 2000 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 1500 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 1000 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 800 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 600 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 500 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 400 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 300 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 200pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 150 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 125 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 100 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 90 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 80 pM B6.

[0076] The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 5 pM to about 2000 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 10 pM to about 2000 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 15 pM to about 2000 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 20 pM to about 2000 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 25 pM to about 2000 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 30 pM to about 2000 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 35 pM to about 2000 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 40 pM to about 2000 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 45 pM to about 2000 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 50 pM to about 2000 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 55 pM to about 2000 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 60 pM to about 2000 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 5 pM to about 1000 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 20 pM to about 1000 pM B6. The higher level of Bvitamins may comprise providing a level of increase in B vitamins in the culture medium of about 50 pM to about 1000 pM B6.

[0077] The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 2000 pM B6 (e.g. of about 1 pM to about 200 pM B6). The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 50 pM to about 1000 pM B6 (e.g. of about 50 pM to about 100 pM B6). The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 60 pM to about 800 pM B6 (e.g. of about 60 pM to about 80 pM B6).

[0078] The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 2000 pM B9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 1500 pM B9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 1000 pM B9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 800 pM B9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 600 pM B9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 500 pM B9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 400 pM B9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 300 pM B9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 200 pM B9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 150 pM B9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 100 pM B9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 80 pM B9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 60 pM B9.

[0079] The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 2 pM to about 2000 pM B9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in theculture medium of about 5 pM to about 2000 pM B9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 10 pM to about 2000 pM B9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 15 pM to about 2000 pM B9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 20 pM to about 2000 pM B9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 25 pM to about 2000 pM B9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 30 pM to about 2000 pM B9.

[0080] The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 2000 pM B9 (e.g. of about 1 pM to about 200 pM B9). The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 20 pM to about 800 pM B9 (e.g. of about 20 pM to about 80 pM B9, or 20 pM to about 600 pM B9). The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 30 pM to about 600 pM B9 (e.g of about 30 pM to about 60 pM B9).

[0081] The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 200 pM B12. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 150 pM B12. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 100 pM B12. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 80 pM B12. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 60 pM B12. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 40 pM B12. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 20 pM B12. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 15 pM B12. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 10 pM B12. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 8pM B12. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 6 pM B12.

[0082] The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.5 pM to about 200 pM B12. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.75 pM to about 200 pM B12. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 200 pM B12. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1.2 pM to about 200 pM B12. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1.4 pM to about 200 pM B12. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1.6 pM to about 200 pM B12. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1.8 pM to about 200 pM B12. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 2 pM to about 200 pM B12.

[0083] The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 200 pM B12 (e.g. of about 0.1 pM to about 20 pM B12). The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 100 pM B12 (e.g. of about 1 pM to about 10 pM B12). The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 2 pM to about 60 pM B12 (e.g. of about 2 pM to about 6 pM B12).

[0084] The higher level of B vitamins may comprise a total level of about 1 pM to about 200 pM B2. The higher level of B vitamins may comprise a total level of about 1 pM to about 150 pM B2. The higher level of B vitamins may comprise a total level of about 1 pM to about 100 pM B2. The higher level of B vitamins may comprise a total level of about 1 pM to about 80 pM B2. The higher level of B vitamins may comprise a total level of about 1 pM to about 60 pM B2. The higher level of B vitamins may comprise a total level of about 1 pM to about 40 pM B2. The higher level of B vitamins may comprise a total level of about 1 pM to about 20 pM B2. The higher level of B vitamins may comprise a total level of about 1 pM to about 15 pM B2. The higher level of B vitamins may comprise a total level of about 1 pM to about 10 pM B2. The higher level of B vitamins may comprise a total level of about1 pM to about 8 pM B2. The higher level of B vitamins may comprise a total level of about 1 pM to about 6 pM B2.

[0085] The higher level of B vitamins may comprise a total level of about 1.1 M to about 200 pM B2. The higher level of B vitamins may comprise a total level of about 1.2 pM to about 200 pM B2. The higher level of B vitamins may comprise a total level of about 1.3 pM to about 200 pM B2. The higher level of B vitamins may comprise a total level of about 1.4 pM to about 200 pM B2. The higher level of B vitamins may comprise a total level of about 1.5 pM to about 200 pM B2. The higher level of B vitamins may comprise a total level of about 1.6 pM to about 200 pM B2. The higher level of B vitamins may comprise a total level of about 1.7 pM to about 200 pM B2. The higher level of B vitamins may comprise a total level of about 1.8 pM to about 200 pM B2. The higher level of B vitamins may comprise a total level of about 1.9 pM to about 200 pM B2. The higher level of B vitamins may comprise a total level of about 2 pM to about 200 pM B2.

[0086] The higher level of B vitamins may comprise a total level of about 1 pM to about 200 pM B2 (e.g. of about 1 pM to about 20 pM B2). The higher level of B vitamins may comprise a total level of about 2 pM to about 60 pM B2 (e.g. of about 2 pM to about 6 pM B2).

[0087] The higher level of B vitamins may comprise a total level of about 10 pM to about 2000 pM B6. The higher level of B vitamins may comprise a total level of about 10 pM to about 1500 pM B6. The higher level of B vitamins may comprise a total level of about 10 pM to about 1000 pM B6. The higher level of B vitamins may comprise a total level of about 10 pM to about 800 pM B6. The higher level of B vitamins may comprise a total level of about 10 pM to about 600 pM B6. The higher level of B vitamins may comprise a total level of about 10 pM to about 400 pM B6. The higher level of B vitamins may comprise a total level of about 10 pM to about 200 pM B6. The higher level of B vitamins may comprise a total level of about 10 pM to about 150 pM B6. The higher level of B vitamins may comprise a total level of about 10 pM to about 100 pM B6.

[0088] The higher level of B vitamins may comprise a total level of about 15 pM to about 2000 pM B6. The higher level of B vitamins may comprise a total level of about 20 pM to about 2000 pM B6. The higher level of B vitamins may comprise a total level of about 25 pM to about 2000 pM B6. The higher level of B vitamins may comprise a total level of about 30 pM to about 2000 pM B6. The higher level of B vitamins may comprise a total level of about 35 pM to about 2000 pM B6. The higher level ofB vitamins may comprise a total level of about 40 pM to about 2000 pM B6. The higher level of B vitamins may comprise a total level of about 45 pM to about 2000 pM B6. The higher level of B vitamins may comprise a total level of about 50 pM to about 2000 pM B6. The higher level of B vitamins may comprise a total level of about 55 pM to about 2000 pM B6. The higher level of B vitamins may comprise a total level of about 60 pM to about 2000 pM B6.

[0089] The higher level of B vitamins may comprise a total level of about 10 pM to about 2000 pM B6 (e.g. of about 10 pM to about 200 pM B6). The higher level of B vitamins may comprise a total level of about 50 pM to about 1500 pM B6, such as of about 50 pM to about 200 pM B6 (e.g. of about 50 pM to about 150 pM B6). The higher level of B vitamins may comprise a total level of about 60 pM to about 1000 pM B6 (e.g. of about 60 pM to about 100 pM B6).

[0090] The higher level of B vitamins may comprise a total level of about 10 pM to about 2000 pM B9. The higher level of B vitamins may comprise a total level of about 10 pM to about 1500 pM B9. The higher level of B vitamins may comprise a total level of about 10 pM to about 1000 pM B9. The higher level of B vitamins may comprise a total level of about 10 pM to about 800 pM B9. The higher level of B vitamins may comprise a total level of about 10 pM to about 600 pM B9. The higher level of B vitamins may comprise a total level of about 10 pM to about 400 pM B9. The higher level of B vitamins may comprise a total level of about 10 pM to about 200 pM B9. The higher level of B vitamins may comprise a total level of about 10 pM to about 150 pM B9. The higher level of B vitamins may comprise a total level of about 10 pM to about 125 pM B9. The higher level of B vitamins may comprise a total level of about 10 pM to about 100 pM B9. The higher level of B vitamins may comprise a total level of about 10 pM to about 80 pM B9.

[0091] The higher level of B vitamins may comprise a total level of about 15 pM to about 2000 pM B9. The higher level of B vitamins may comprise a total level of about 20 pM to about 2000 pM B9. The higher level of B vitamins may comprise a total level of about 25 pM to about 2000 pM B9. The higher level of B vitamins may comprise a total level of about 30 pM to about 2000 pM B9.

[0092] The higher level of B vitamins may comprise a total level of about 10 pM to about 2000 pM B9 (e.g. of about 10 pM to about 200 pM B9). The higher level of B vitamins may comprise a total level of about 20 pM to about 1000 pM B9 (e.g. of about 20 pM to about 100 pM B9, or, of about 20 pM to about 600 pM B9). Thehigher level of B vitamins may comprise a total level of about 30 pM to about 800 pM B9 (e.g. of about 30 pM to about 80 pM B9).

[0093] The higher level of B vitamins may comprise a total level of about 1 pM to about 200 pM B12. The higher level of B vitamins may comprise a total level of about 1 pM to about 150 pM B12. The higher level of B vitamins may comprise a total level of about 1 pM to about 100 pM B12. The higher level of B vitamins may comprise a total level of about 1 pM to about 80 pM B12. The higher level of B vitamins may comprise a total level of about 1 pM to about 60 pM B12. The higher level of B vitamins may comprise a total level of about 1 pM to about 50 pM B12. The higher level of B vitamins may comprise a total level of about 1 pM to about 40 pM B12. The higher level of B vitamins may comprise a total level of about 1 pM to about 20 pM B12. The higher level of B vitamins may comprise a total level of about 1 pM to about 15 pM B12. The higher level of B vitamins may comprise a total level of about 1 pM to about 10 pM B12. The higher level of B vitamins may comprise a total level of about 1 pM to about 8 pM B12. The higher level of B vitamins may comprise a total level of about 1 pM to about 6 pM B12.

[0094] The higher level of B vitamins may comprise a total level of about 1 pM to about 200 pM B12 (e.g. of about 1 pM to about 20 pM B12). The higher level of B vitamins may comprise a total level of about 1 pM to about 100 pM B12 (e.g. of about 1 pM to about 10 pM B12). The higher level of B vitamins may comprise a total level of about 1 pM to about 50 pM B12 (e.g. of about 1 pM to about 6 pM B12).

[0095] The lower levels of B vitamins may be achieved by supplementing the cell culture with media and / or nutrient feeds without (-) B vitamins.

[0096] The lower level of B vitamins may comprise a total level of about 0 pM to about 1 pM B2. The lower level of B vitamins may comprise a total level of about 0 pM to about 0.9 pM B2. The lower level of B vitamins may comprise a total level of about 1 pM B2.

[0097] The lower level of B vitamins may comprise a total level of about 0.05 pM to about 1 pM B2. The lower level of B vitamins may comprise a total level of about 0.1 pM to about 1 pM B2. The lower level of B vitamins may comprise a total level of about 0.15 pM to about 1 pM B2. The lower level of B vitamins may comprise a total level of about 0.2 pM to about 1 pM B2.

[0098] The lower level of B vitamins may comprise a total level of about 0.2 pM to about 0.9 pM B2.

[0099] The lower level of B vitamins may comprise a total level of about 0 pM to about 12 pM B6. The lower level of B vitamins may comprise a total level of about 0 pM to about 11 pM B6. The lower level of B vitamins may comprise a total level of about 0 pM to about 10 pM B6. The lower level of B vitamins may comprise a total level of about 0 pM to about 9 pM B6.

[0100] The lower level of B vitamins may comprise a total level of about 0.5 pM to about 12 pM B6. The lower level of B vitamins may comprise a total level of about 1 pM to about 12 pM B6. The lower level of B vitamins may comprise a total level of about 1.5 pM to about 12 pM B6. The lower level of B vitamins may comprise a total level of about 2 pM to about 12 pM B6. The lower level of B vitamins may comprise a total level of about 2.5 pM to about 12 pM B6. The lower level of B vitamins may comprise a total level of about 3 pM to about 12 pM B6.

[0101] The lower level of B vitamins may comprise a total level of about 1 pM to about 50 pM B6.The lower level of B vitamins may comprise a total level of about 1 pM to about 10 pM B6. The lower level of B vitamins may comprise a total level of about 3 pM to about 9 pM B6. The lower level of B vitamins may comprise a total level of about 50 pM B6.

[0102] The lower level of B vitamins may comprise a total level of about 0 pM to about 12 pM B9. The lower level of B vitamins may comprise a total level of about 0 pM to about 11 pM B9. The lower level of B vitamins may comprise a total level of about 0 pM to about 10 pM B9. The lower level of B vitamins may comprise a total level of about 0 pM to about 9 pM B9.

[0103] The lower level of B vitamins may comprise a total level of about 0.5 pM to about 12 pM B9. The lower level of B vitamins may comprise a total level of about 1 pM to about 12 pM B9. The lower level of B vitamins may comprise a total level of about 1.5 pM to about 12 pM B9. The lower level of B vitamins may comprise a total level of about 2 pM to about 12 pM B9. The lower level of B vitamins may comprise a total level of about 2.5 pM to about 12 pM B9. The lower level of B vitamins may comprise a total level of about 3 pM to about 12 pM B9.

[0104] The lower level of B vitamins may comprise a total level of about 1 pM to about 10 pM B9. The lower level of B vitamins may comprise a total level of about 3 pM to about 9 pM B9. The lower level of B vitamins may comprise a total level of about 0 pM to about 20 pM B9. The lower level of B vitamins may comprise a total level of about 0 pM to about 30 pM B9. The lower level of B vitamins may comprise a totallevel of about 20 pM B9. The lower level of B vitamins may comprise a total level of about 30 pM B9.

[0105] The lower level of B vitamins may comprise a total level of about 0 pM to about 2 pM B12. The lower level of B vitamins may comprise a total level of about 0 pM to about 1.8 pM B12. The lower level of B vitamins may comprise a total level of about 0 pM to about 1.6 pM B12. The lower level of B vitamins may comprise a total level of about 0 pM to about 1.4 pM B12. The lower level of B vitamins may comprise a total level of about 0 pM to about 1.3 pM B12. The lower level of B vitamins may comprise a total level of about 0 pM to about 1.2 pM B12. The lower level of B vitamins may comprise a total level of about 0 pM to about 1.1 pM B12. The lower level of B vitamins may comprise a total level of about 0 pM to about 1 pM B12. The lower level of B vitamins may comprise a total level of about 0 pM to about 9 pM B12.

[0106] The lower level of B vitamins may comprise a total level of about 0.05 pM to about 2 pM B12. The lower level of B vitamins may comprise a total level of about 0.1 pM to about 2 pM B12. The lower level of B vitamins may comprise a total level of about 0.15 pM to about 2 pM B12. The lower level of B vitamins may comprise a total level of about 0.2 pM to about 2 pM B12. The lower level of B vitamins may comprise a total level of about 0.1 pM to about 1 pM B12. The lower level of B vitamins may comprise a total level of about 1 pM B12.

[0107] The lower level of B vitamins may comprise a total level of about 0.1 pM to about 1 pM B12. The lower level of B vitamins may comprise a total level of about 0.2 pM to about 0.9 pM B12.

[0108] The B vitamins may comprise riboflavin (B2), pyridoxine (B6), folic acid (B9), and cyanocobalamin (B12). The B vitamins may consist of riboflavin (B2), pyridoxine (B6), folic acid (B9), and cyanocobalamin (B12).

[0109] The recombinant polypeptide may comprise at least one N-linked glycan site. For example, the recombinant polypeptide may comprise at least two N-linked glycan sites.

[0110] The recombinant polypeptide may be an antibody, an antigen, an enzyme, or a vaccine.

[0111] It may be that the recombinant polypeptide is an antibody. The antibody may be a multispecific antibody or antigen-binding fragment thereof. The antibody mayconsist of a single heavy chain sequence and a single light chain sequence or antigen-binding fragments thereof.

[0112] The antibody may comprise a chimeric antibody, a human antibody or a humanized antibody. The antibody may comprise a monoclonal antibody.

[0113] The recombinant polypeptide may be a fusion protein. The fusion protein may comprise an antibody or an antigen-binding fragment thereof. For example, the recombinant polypeptide may be an Fc fusion protein.

[0114] The cell line may be a mammalian cell line.

[0115] The cell line may be a CHO cell line or an NS0 cell line. For example, the cell line may be a CHO K1 cell line, a CHO K1SV cell line, a DG44 cell line, a DUKXB-11 cell line, or a CHOK1S cell line, or a targeted gene integration (Tl) - generated CHO cell line, or their derivatives.

[0116] The cell line may be cultured under fed-batch culture conditions, or perfusion culture conditions.

[0117] It may be that the cell line is cultured under fed-batch culture conditions. The fed- batch culture conditions may be intensified fed-batch culture conditions.

[0118] It may be that the cell line is cultured under perfusion culture conditions. The perfusion culture conditions may be semi-continuous perfusion or continuous perfusion.

[0119] It may be that the method further comprises optimizing the size pattern of the recombinant polypeptide. This may be achieved according to any of the embodiments described in relation to the third aspect.

[0120] It may be that the method further comprises optimizing the charge pattern of the recombinant polypeptide. This may be achieved according to any of the embodiments described in relation to the second aspect.

[0121] It may be that the method further comprises both optimizing the charge pattern of the recombinant polypeptide and optimizing the size pattern of the recombinant polypeptide. This may be achieved according to any of the embodiments described in relation to the second and third aspects.Method of Optimizing Charge Pattern of a Recombinant Peptide

[0122] In another aspect, there is provided a method of optimizing charge pattern of a recombinant polypeptide, comprising: culturing a cell line engineered to express the recombinant polypeptide under conditions promoting production of the recombinant polypeptide; and modulating the level of B vitamins in the cell culture.

[0123] The step of modulating the level of B vitamins may comprise providing a lower level of B vitamins in the cell culture, or a higher level of B vitamins in the cell culture.

[0124] The step of modulating the level of B vitamins may comprise providing a lower level of B vitamins in the cell culture. In these embodiments, the lower level may comprise maintaining or decreasing the level of B vitamins in the cell culture. Decreasing the level of B vitamins is typically achieved via a dilution. For example, decreasing the level of B vitamins may be achieved by supplementing the cell culture with media and / or nutrient feeds without (-) B vitamins, or by replacing at least part of the cell culture medium with media comprising a low level or without (-) B vitamins.

[0125] The step of optimizing charge pattern may comprise optimizing the relative amount of acidic species, and / or main species, and / or basic species of the recombinant polypeptide. The step of optimizing charge pattern may comprise optimizing the relative amount of acidic species. The step of optimizing charge pattern may comprise optimizing the relative amount of main species. The step of optimizing charge pattern may comprise optimizing the relative amount of basic species.

[0126] It may be that optimizing the relative amount of acidic species comprises using the higher level of B vitamins to maintain an increased level of acidic species. Alternatively, it may be that optimizing the relative amount of acidic species comprises using the lower level of B vitamins to maintain a decreased level of acidic species.

[0127] It may be that optimizing the relative amount of main species comprises using the higher level of B vitamins to maintain a decreased level of main species. Alternatively, it may be that optimizing the relative amount of main species comprises using the lower level of B vitamins to maintain an increased level of main species.

[0128] It may be that optimizing the relative amount of basic species comprises using the higher level of B vitamins to maintain a decreased level of basic species. Alternatively, it may be that optimizing the relative amount of basic speciescomprises using the lower level of B vitamins to maintain an increased level of basic species.

[0129] The higher levels of B vitamins may be achieved by supplementing the cell culture with B vitamins.

[0130] The supplementing with B vitamins may comprise at least partially replacing the cell culture medium with a production culture medium having a high level of B vitamins, or may comprise providing a feed with a high level of B vitamins.

[0131] The higher levels of B vitamins may be achieved by increasing the levels of B vitamins in the production culture medium. Alternatively, the higher levels of B vitamins are achieved by supplementing the production cell culture with nutrient feed(s) containing high levels of B vitamins.

[0132] Further alternatively, the higher levels of B vitamins may be achieved by supplementing one or more nutrient feeds with higher levels of B vitamins to the cell culture during the production phase.

[0133] In an embodiment, the nutrient feeds may be added through continuous feeding or daily feeds during a production period from 1-20 days (optionally a period of from 6-13 days). Alternatively, the nutrient feeds may be added to the cell culture medium 1-4 times during a production period of from 2-15 days (optionally a period of from 6-13 days). The nutrient feeds may be added to the cell culture medium 2-3 times during a period of from 3-14 days (optionally a period of from 6-13 days).

[0134] The B vitamins may comprise one or a combination of riboflavin (B2), pyridoxine (B6), folic acid (B9), or cyanocobalamin (B12). The B vitamins may consist of one or a combination of riboflavin (B2), pyridoxine (B6), folic acid (B9), or cyanocobalamin (B12).

[0135] The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 200 pM B2. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 150 pM B2. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 100 pM B2. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 75 pM B2. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 50 pM B2. The higher level of B vitamins may comprise providing a level of increase in Bvitamins in the culture medium of about 0.1 pM to about 40 pM B2. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 30 pM B2. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 20 pM B2. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 10 pM B2. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 7.5 pM B2. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 5 pM B2. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 50 pM B2.

[0136] The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.5 pM to about 200 pM B2. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 200 pM B2. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1.25 pM to about 200 pM B2. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1.5 pM to about 200 pM B2. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 2 pM to about 200 pM B2.

[0137] The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 200 pM B2 (e.g. of about 0.1 pM to about 20 pM B2). The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 100 pM B2 (e.g. of about 1 pM to about 10 pM B2). The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 2 pM to about 50 pM B2 (e.g. of about 2 pM to about 5 pM B2). The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 50 pM B2.

[0138] The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 2000 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 1500 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 1000 pM B6. The higher level of B vitamins may compriseproviding a level of increase in B vitamins in the culture medium of about 1 pM to about 800 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 600 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 500 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 400 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 300 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 200 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 150 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 125 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 100 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 90 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 80 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1000 pM B6.

[0139] The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 5 pM to about 2000 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 10 pM to about 2000 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 15 pM to about 2000 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 20 pM to about 2000 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 25 pM to about 2000 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 30 pM to about 2000 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 35 pM to about 2000 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 40 pM to about 2000 pM B6. The higher level of Bvitamins may comprise providing a level of increase in B vitamins in the culture medium of about 45 pM to about 2000 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 50 pM to about 2000 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 55 pM to about 2000 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 60 pM to about 2000 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1000 pM B6.

[0140] The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 2000 pM B6 (e.g. of about 1 pM to about 200 pM B6). The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 50 pM to about 1000 pM B6 (e.g. of about 50 pM to about 100 pM B6). The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 60 pM to about 800 pM B6 (e.g. of about 60 pM to about 80 pM B6).

[0141] The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 2000 pM B9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 1500 pM B9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 1000 pM B9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 800 pM B9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 600 pM B9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 600 pM B9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 500 pM B9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 400 pM B9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 300 pM B9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 200 pM B9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 150 pM B9. The higher level of B vitamins maycomprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 100 pM B9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 80 pM B9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 60 pM B9.

[0142] The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 2 pM to about 2000 pM B9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 5 pM to about 2000 pM B9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 10 pM to about 2000 pM B9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 15 pM to about 2000 pM B9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 20 pM to about 2000 pM B9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 25 pM to about 2000 pM B9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 30 pM to about 2000 pM B9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 20 pM to about 600 pM B9.

[0143] The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 2000 pM B9 (e.g. of about 1 pM to about 200 pM B9). The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 20 pM to about 800 pM B9 (e.g. of about 20 pM to about 80 pM B9). The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 30 pM to about 600 pM B9 (e.g. of about 30 pM to about 60 pM B9). The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 600 pM B9.

[0144] The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 200 pM B12. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 150 pM B12. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 100 pM B12. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about0.1 pM to about 80 pM B12. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 60 pM B12. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 40 pM B12. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 20 pM B12. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 15 pM B12. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 10 pM B12. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 8 pM B12. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 6 pM B12.

[0145] The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.5 pM to about 200 pM B12. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.75 pM to about 200 pM B12. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 200 pM B12. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1.2 pM to about 200 pM B12. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1.4 pM to about 200 pM B12. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1.6 pM to about 200 pM B12. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1.8 pM to about 200 pM B12. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 2 pM to about 200 pM B12. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 50 pM B12.

[0146] The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 200 pM B12 (e.g. of about 0.1 pM to about 20 pM B12). The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 100 pM B12 (e.g. of about 1 pM to about 10 pM B12). The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culturemedium of about 2 pM to about 60 pM B12 (e.g. of about 2 pM to about 6 pM B12). The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 50 pM B12.

[0147] The higher level of B vitamins may comprise a total level of about 1 pM to about 200 pM B2. The higher level of B vitamins may comprise a total level of about 1 pM to about 150 pM B2. The higher level of B vitamins may comprise a total level of about 1 pM to about 100 pM B2. The higher level of B vitamins may comprise a total level of about 1 pM to about 80 pM B2. The higher level of B vitamins may comprise a total level of about 1 pM to about 60 pM B2. The higher level of B vitamins may comprise a total level of about 1 pM to about 40 pM B2. The higher level of B vitamins may comprise a total level of about 1 pM to about 20 pM B2. The higher level of B vitamins may comprise a total level of about 1 pM to about 15 pM B2. The higher level of B vitamins may comprise a total level of about 1 pM to about 10 pM B2. The higher level of B vitamins may comprise a total level of about 1 pM to about 8 pM B2. The higher level of B vitamins may comprise a total level of about 1 pM to about 6 pM B2.

[0148] The higher level of B vitamins may comprise a total level of about 1.1 pM to about 200 pM B2. The higher level of B vitamins may comprise a total level of about 1.2 pM to about 200 pM B2. The higher level of B vitamins may comprise a total level of about 1.3 pM to about 200 pM B2. The higher level of B vitamins may comprise a total level of about 1.4 pM to about 200 pM B2. The higher level of B vitamins may comprise a total level of about 1.5 pM to about 200 pM B2. The higher level of B vitamins may comprise a total level of about 1.6 pM to about 200 pM B2. The higher level of B vitamins may comprise a total level of about 1.7 pM to about 200 pM B2. The higher level of B vitamins may comprise a total level of about 1.8 pM to about 200 pM B2. The higher level of B vitamins may comprise a total level of about 1.9 pM to about 200 pM B2. The higher level of B vitamins may comprise a total level of about 2 pM to about 200 pM B2.

[0149] The higher level of B vitamins may comprise a total level of about 1 pM to about 200 pM B2 (e.g. of about 1 pM to about 20 pM B2). The higher level of B vitamins may comprise a total level of about 2 pM to about 60 pM B2 (e.g. of about 2 pM to about 6 pM B2).

[0150] The higher level of B vitamins may comprise a total level of about 10 pM to about 2000 pM B6. The higher level of B vitamins may comprise a total level of about 10 pM to about 1500 pM B6. The higher level of B vitamins may comprise a total levelof about 10 pM to about 1000 pM B6. The higher level of B vitamins may comprise a total level of about 10 pM to about 800 pM B6. The higher level of B vitamins may comprise a total level of about 10 pM to about 600 pM B6. The higher level of B vitamins may comprise a total level of about 10 pM to about 400 pM B6. The higher level of B vitamins may comprise a total level of about 10 pM to about 200 pM B6. The higher level of B vitamins may comprise a total level of about 10 pM to about 150 pM B6. The higher level of B vitamins may comprise a total level of about 10 pM to about 100 pM B6.

[0151] The higher level of B vitamins may comprise a total level of about 15 pM to about 2000 pM B6. The higher level of B vitamins may comprise a total level of about 20 pM to about 2000 pM B6. The higher level of B vitamins may comprise a total level of about 25 pM to about 2000 pM B6. The higher level of B vitamins may comprise a total level of about 30 pM to about 2000 pM B6. The higher level of B vitamins may comprise a total level of about 35 pM to about 2000 pM B6. The higher level of B vitamins may comprise a total level of about 40 pM to about 2000 pM B6. The higher level of B vitamins may comprise a total level of about 45 pM to about 2000 pM B6. The higher level of B vitamins may comprise a total level of about 50 pM to about 2000 pM B6. The higher level of B vitamins may comprise a total level of about 55 pM to about 2000 pM B6. The higher level of B vitamins may comprise a total level of about 60 pM to about 2000 pM B6.

[0152] The higher level of B vitamins may comprise a total level of about 10 pM to about 2000 pM B6 (e.g. of about 10 pM to about 200 pM B6). The higher level of B vitamins may comprise a total level of about 50 pM to about 1500 pM B6 (e.g. of about 50 pM to about 150 pM B6). The higher level of B vitamins may comprise a total level of about 60 pM to about 1000 pM B6 (e.g. of about 60 pM to about 100 pM B6).

[0153] The higher level of B vitamins may comprise a total level of about 10 pM to about 2000 pM B9. The higher level of B vitamins may comprise a total level of about 10 pM to about 1500 pM B9. The higher level of B vitamins may comprise a total level of about 10 pM to about 1000 pM B9. The higher level of B vitamins may comprise a total level of about 10 pM to about 800 pM B9. The higher level of B vitamins may comprise a total level of about 10 pM to about 600 pM B9. The higher level of B vitamins may comprise a total level of about 10 pM to about 400 pM B9. The higher level of B vitamins may comprise a total level of about 10 pM to about 200 pM B9. The higher level of B vitamins may comprise a total level of about 10 pM to about 150 pM B9. The higher level of B vitamins may comprise a total level of about 10pM to about 125 pM B9. The higher level of B vitamins may comprise a total level of about 10 pM to about 100 pM B9. The higher level of B vitamins may comprise a total level of about 10 pM to about 80 pM B9.

[0154] The higher level of B vitamins may comprise a total level of about 15 pM to about 2000 pM B9. The higher level of B vitamins may comprise a total level of about 20 pM to about 2000 pM B9. The higher level of B vitamins may comprise a total level of about 25 pM to about 2000 pM B9. The higher level of B vitamins may comprise a total level of about 30 pM to about 2000 pM B9.

[0155] The higher level of B vitamins may comprise a total level of about 10 pM to about 2000 pM B9 (e.g. of about 10 pM to about 200 pM B9). The higher level of B vitamins may comprise a total level of about 20 pM to about 1000 pM B9 (e.g. of about 20 pM to about 100 pM B9). The higher level of B vitamins may comprise a total level of about 30 pM to about 800 pM B9 (e.g. of about 30 pM to about 80 pM B9).

[0156] The higher level of B vitamins may comprise a total level of about 2 pM to about 200 pM B12. The higher level of B vitamins may comprise a total level of about 2 pM to about 150 pM B12. The higher level of B vitamins may comprise a total level of about 2 pM to about 100 pM B12. The higher level of B vitamins may comprise a total level of about 2 pM to about 80 pM B12. The higher level of B vitamins may comprise a total level of about 2 pM to about 60 pM B12. The higher level of B vitamins may comprise a total level of about 2 pM to about 40 pM B12. The higher level of B vitamins may comprise a total level of about 2 pM to about 20 pM B12. The higher level of B vitamins may comprise a total level of about 2 pM to about 15 pM B12. The higher level of B vitamins may comprise a total level of about 2 pM to about 10 pM B12. The higher level of B vitamins may comprise a total level of about 2 pM to about 8 pM B12. The higher level of B vitamins may comprise a total level of about 2 pM to about 6 pM B12.

[0157] The higher level of B vitamins may comprise a total level of about 2 pM to about 200 pM B12 (e.g. of about 2 pM to about 20 pM B12). The higher level of B vitamins may comprise a total level of about 2 pM to about 100 pM B12 (e.g. of about 2 pM to about 10 pM B12). The higher level of B vitamins may comprise a total level of about 2 pM to about 60 pM B12 (e.g. of about 2 pM to about 6 pM B12).

[0158] The lower level of B vitamins may comprise a total level of about 0 pM to about 1 pM B2. The lower level of B vitamins may comprise a total level of about 0 pM to about 0.9 pM B2.

[0159] The lower level of B vitamins may comprise a total level of about 0.05 pM to about 1 pM B2. The lower level of B vitamins may comprise a total level of about 0.1 pM to about 1 pM B2. The lower level of B vitamins may comprise a total level of about 0.15 pM to about 1 pM B2. The lower level of B vitamins may comprise a total level of about 0.2 pM to about 1 pM B2.

[0160] The lower level of B vitamins may comprise a total level of about 0.2 pM to about 0.9 pM B2.

[0161] The lower level of B vitamins may comprise a total level of about 0 pM to about 12 pM B6. The lower level of B vitamins may comprise a total level of about 0 pM to about 11 pM B6. The lower level of B vitamins may comprise a total level of about 0 pM to about 10 pM B6. The lower level of B vitamins may comprise a total level of about 0 pM to about 9 pM B6.

[0162] The lower level of B vitamins may comprise a total level of about 0.5 pM to about 12 pM B6. The lower level of B vitamins may comprise a total level of about 1 pM to about 12 pM B6. The lower level of B vitamins may comprise a total level of about 1.5 pM to about 12 pM B6. The lower level of B vitamins may comprise a total level of about 2 pM to about 12 pM B6. The lower level of B vitamins may comprise a total level of about 2.5 pM to about 12 pM B6. The lower level of B vitamins may comprise a total level of about 3 pM to about 12 pM B6.

[0163] The lower level of B vitamins may comprise a total level of about 1 pM to about 10 pM B6. The lower level of B vitamins may comprise a total level of about 3 pM to about 9 pM B6.

[0164] The lower level of B vitamins may comprise a total level of about 0 pM to about 12 pM B9. The lower level of B vitamins may comprise a total level of about 0 pM to about 11 pM B9. The lower level of B vitamins may comprise a total level of about 0 pM to about 10 pM B9. The lower level of B vitamins may comprise a total level of about 0 pM to about 9 pM B9.

[0165] The lower level of B vitamins may comprise a total level of about 0.5 pM to about 12 pM B9. The lower level of B vitamins may comprise a total level of about 1 pM to about 12 pM B9. The lower level of B vitamins may comprise a total level of about 1.5 pM to about 12 pM B9. The lower level of B vitamins may comprise atotal level of about 2 pM to about 12 pM B9. The lower level of B vitamins may comprise a total level of about 2.5 pM to about 12 pM B9. The lower level of B vitamins may comprise a total level of about 3 pM to about 12 pM B9.

[0166] The lower level of B vitamins may comprise a total level of about 1 pM to about 10 pM B9. The lower level of B vitamins may comprise a total level of about 3 pM to about 9 pM B9.

[0167] The lower level of B vitamins may comprise a total level of about 0 pM to about 2 pM B12. The lower level of B vitamins may comprise a total level of about 0 pM to about 1.8 pM B12. The lower level of B vitamins may comprise a total level of about 0 pM to about 1.6 pM B12. The lower level of B vitamins may comprise a total level of about 0 pM to about 1.4 pM B12. The lower level of B vitamins may comprise a total level of about 0 pM to about 1.3 pM B12. The lower level of B vitamins may comprise a total level of about 0 pM to about 1.2 pM B12. The lower level of B vitamins may comprise a total level of about 0 pM to about 1.1 pM B12. The lower level of B vitamins may comprise a total level of about 0 pM to about 1 pM B12. The lower level of B vitamins may comprise a total level of about 0 pM to about 9 pM B12.

[0168] The lower level of B vitamins may comprise a total level of about 0.05 pM to about 2 pM B12. The lower level of B vitamins may comprise a total level of about 0.1 pM to about 2 pM B12. The lower level of B vitamins may comprise a total level of about 0.15 pM to about 2 pM B12. The lower level of B vitamins may comprise a total level of about 0.2 pM to about 2 pM B12.

[0169] The lower level of B vitamins may comprise a total level of about 0.1 pM to about 1 pM B12. The lower level of B vitamins may comprise a total level of about 0.2 pM to about 0.9 pM B12.

[0170] The B vitamins may comprise riboflavin (B2), pyridoxine (B6), folic acid (B9), and cyanocobalamin (B12). The B vitamins may consist of riboflavin (B2), pyridoxine (B6), folic acid (B9), and cyanocobalamin (B12).

[0171] The recombinant polypeptide may comprise at least one N-linked glycan site. For example, the recombinant polypeptide may comprise at least two N-linked glycan sites.

[0172] The recombinant polypeptide may be an antibody, an antigen, an enzyme, or a vaccine.

[0173] It may be that the recombinant polypeptide is an antibody. The antibody may be a multispecific antibody or antigen-binding fragment thereof. The antibody may consist of a single heavy chain sequence and a single light chain sequence or antigen-binding fragments thereof.

[0174] The antibody may comprise a chimeric antibody, a human antibody or a humanized antibody. The antibody may comprise a monoclonal antibody.

[0175] The recombinant polypeptide may be a fusion protein. The fusion protein may comprise an antibody or an antigen-binding fragment thereof. For example, the recombinant polypeptide may be an Fc fusion protein.

[0176] The cell line may be a mammalian cell line.

[0177] The cell line may be a CHO cell line or an NS0 cell line. For example, the cell line may be a CHO K1 cell line, a CHO K1SV cell line, a DG44 cell line, a DUKXB-11 cell line, or a CHOK1S cell line, or a targeted gene integration (Tl) - generated CHO cell line, or their derivatives.

[0178] The cell line may be cultured under fed-batch culture conditions, or perfusion culture conditions.

[0179] It may be that the cell line is cultured under fed-batch culture conditions. The fed- batch culture conditions may be intensified fed-batch culture conditions.

[0180] It may be that the cell line is cultured under perfusion culture conditions. The perfusion culture conditions may be semi-continuous perfusion or continuous perfusion.Method of Optimizing Size Pattern of a Recombinant Peptide

[0181] In another aspect, there is provided a method of optimizing size pattern of a recombinant polypeptide, comprising: culturing a cell line engineered to express the recombinant polypeptide under conditions promoting production of the recombinant polypeptide; and modulating the level of B vitamins in the cell culture.

[0182] The step of modulating the level of B vitamins may comprise providing a lower level of B vitamins in the cell culture, or a higher level of B vitamins in the cell culture.

[0183] The step of modulating the level of B vitamins may comprise providing a lower level of B vitamins in the cell culture. In these embodiments, the lower level may comprise maintaining or decreasing the level of B vitamins in the cell culture. Decreasing the level of B vitamins is typically achieved via a dilution. For example,decreasing the level of B vitamins may be achieved by supplementing the cell culture with media and / or nutrient feeds without (-) B vitamins, or by replacing at least part of the cell culture medium with media comprising a low level or without (-) B vitamins.

[0184] For a recombinant polypeptide, the size pattern may comprise monomeric form(s) of the recombinant polypeptide, and / or low molecular weight (LMW) form(s) of the recombinant polypeptide, and / or high molecular weight form(s) of the recombinant polypeptide. Exemplary LMW forms include fragments of the recombinant polypeptides, whether caused by hydrolysis of monomeric form(s), the action proteolytic enzymes monomeric form(s), incomplete disulfide bone formation, or any other cause. Exemplary HMW forms include multimers of the recombinant polypeptide, which could arise from, e.g., non-covalent interactions or intermolecular disulfide bonds.

[0185] The step of optimizing size pattern may comprise optimizing the relative amount of LMW forms, monomeric forms, and / or HMW forms of the recombinant polypeptide. The percentage of LMW, HMW, and monomeric forms add up to 100% of the product. In general, it is desirable to maximize the monomeric form and minimize the LMW and HMW forms. The step of optimizing size pattern may comprise optimizing the relative amount of LMW forms. The step of optimizing size pattern may comprise optimizing the relative amount of HMW forms. In preferred examples, the step of optimising size pattern may comprise maximising the relative amount of monomeric forms.

[0186] It may be that optimizing the relative amount of LMW forms comprises using the higher level of B vitamins to maintain an increased level of LMW forms. Alternatively, it may be that optimizing the relative amount of LMW forms comprises using the lower level of B vitamins to maintain a decreased level of LMW forms.

[0187] It may be that optimizing the relative amount of monomeric form(s) comprises using the higher level of B vitamins to maintain a decreased level of monomeric form. Alternatively, it may be that optimizing the relative amount of monomeric form comprises using the lower level of B vitamins to maintain an increased level of monomeric form.

[0188] It may be that optimizing the relative amount of HMW forms may be impacted by the levels of B vitamins. Alternatively, it may be that optimizing the relative amount of HMW forms may not be impacted by the levels of B vitamins.

[0189] The higher levels of B vitamins may be achieved by supplementing the cell culture with B vitamins.

[0190] The supplementing with B vitamins may comprise at least partially replacing the cell culture medium with a production culture medium having a high level of B vitamins, or may comprise providing a feed with a high level of B vitamins.

[0191] The higher levels of B vitamins may be achieved by increasing the levels of B vitamins in the production culture medium. Alternatively, the higher levels of B vitamins are achieved by supplementing the production cell culture with nutrient feed(s) containing high levels of B vitamins.

[0192] Further alternatively, the higher levels of B vitamins may be achieved by supplementing one or more nutrient feeds with higher levels of B vitamins to the cell culture during the production phase.

[0193] In an embodiment, the nutrient feeds may be added through continuous feeding or daily feeds during a production period from 1-20 days (optionally a period of from 6-13 days). Alternatively, the nutrient feeds may be added to the cell culture medium 1-4 times during a production period of from 2-15 days (optionally a period of from 6-13 days). The nutrient feeds may be added to the cell culture medium 2-3 times during a period of from 3-14 days (optionally a period of from 6-13 days).

[0194] The B vitamins may comprise one or a combination of riboflavin (B2), pyridoxine (B6), folic acid (B9), or cyanocobalamin (B12). The B vitamins may consist of one or a combination of riboflavin (B2), pyridoxine (B6), folic acid (B9), or cyanocobalamin (B12).

[0195] The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 200 pM B2. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 150 pM B2. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 100 pM B2. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 75 pM B2. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 50 pM B2. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 40 pM B2. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 30 pM B2. The higher level of B vitamins maycomprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 20 pM B2. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 10 pM B2. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 7.5 pM B2. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 5 pM B2. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 50 pM B2.

[0196] The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.5 pM to about 200 pM B2. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 200 pM B2. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1.25 pM to about 200 pM B2. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1.5 pM to about 200 pM B2. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 2 pM to about 200 pM B2.

[0197] The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 200 pM B2 (e.g. of about 0.1 pM to about 20 pM B2). The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 100 pM B2 (e.g. of about 1 pM to about 10 pM B2). The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 2 pM to about 50 pM B2 (e.g. of about 2 pM to about 5 pM B2). The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 50 pM B2.

[0198] The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 2000 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 1500 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 1000 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 800 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 600 pM B6.The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 500 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 400 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 300 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 200 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 150 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 125 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 100 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 90 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 80 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1000 pM B6.

[0199] The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 5 pM to about 2000 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 10 pM to about 2000 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 15 pM to about 2000 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 20 pM to about 2000 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 25 pM to about 2000 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 30 pM to about 2000 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 35 pM to about 2000 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 40 pM to about 2000 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 45 pM to about 2000 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about50 pM to about 2000 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 55 pM to about 2000 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 60 pM to about 2000 pM B6. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1000 pM B6.

[0200] The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 2000 pM B6 (e.g. of about 1 pM to about 200 pM B6). The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 50 pM to about 1000 pM B6 (e.g. of about 50 pM to about 100 pM B6). The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 60 pM to about 800 pM B6 (e.g. of about 60 pM to about 80 pM B6).

[0201] The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 2000 pM B9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 1500 pM B9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 1000 pM B9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 800 pM B9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 600 pM B9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 600 pM B9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 500 pM B9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 400 pM B9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 300 pM B9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 200 pM B9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 150 pM B9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 100 pM B9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 80 pMB9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 60 pM B9.

[0202] The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 2 pM to about 2000 pM B9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 5 pM to about 2000 pM B9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 10 pM to about 2000 pM B9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 15 pM to about 2000 pM B9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 20 pM to about 2000 pM B9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 25 pM to about 2000 pM B9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 30 pM to about 2000 pM B9. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 20 pM to about 600 pM B9.

[0203] The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 2000 pM B9 (e.g. of about 1 pM to about 200 pM B9). The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 20 pM to about 800 pM B9 (e.g. of about 20 pM to about 80 pM B9). The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 30 pM to about 600 pM B9 (e.g. of about 30 pM to about 60 pM B9). The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 600 pM B9.

[0204] The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 200 pM B12. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 150 pM B12. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 100 pM B12. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 80 pM B12. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 60 pM B12. The higher level of B vitamins may comprise providing a level of increasein B vitamins in the culture medium of about 0.1 pM to about 40 pM B12. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 20 pM B12. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 15 pM B12. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 10 pM B12. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 8 pM B12. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 6 pM B12.

[0205] The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.5 pM to about 200 pM B12. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.75 pM to about 200 pM B12. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 200 pM B12. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1.2 pM to about 200 pM B12. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1.4 pM to about 200 pM B12. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1.6 pM to about 200 pM B12. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1.8 pM to about 200 pM B12. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 2 pM to about 200 pM B12. The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 50 pM B12.

[0206] The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 200 pM B12 (e.g. of about 0.1 pM to about 20 pM B12). The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 1 pM to about 100 pM B12 (e.g. of about 1 pM to about 10 pM B12). The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 2 pM to about 60 pM B12 (e.g. of about 2 pM to about 6 pM B12). The higher level of B vitamins may comprise providing a level of increase in B vitamins in the culture medium of about 50 pM B12.

[0207] The higher level of B vitamins may comprise a total level of about 1 pM to about 200 pM B2. The higher level of B vitamins may comprise a total level of about 1 pM to about 150 pM B2. The higher level of B vitamins may comprise a total level of about 1 pM to about 100 pM B2. The higher level of B vitamins may comprise a total level of about 1 pM to about 80 pM B2. The higher level of B vitamins may comprise a total level of about 1 pM to about 60 pM B2. The higher level of B vitamins may comprise a total level of about 1 pM to about 40 pM B2. The higher level of B vitamins may comprise a total level of about 1 pM to about 20 pM B2. The higher level of B vitamins may comprise a total level of about 1 pM to about 15 pM B2. The higher level of B vitamins may comprise a total level of about 1 pM to about 10 pM B2. The higher level of B vitamins may comprise a total level of about 1 pM to about 8 pM B2. The higher level of B vitamins may comprise a total level of about 1 pM to about 6 pM B2.

[0208] The higher level of B vitamins may comprise a total level of about 1.1 pM to about 200 pM B2. The higher level of B vitamins may comprise a total level of about 1.2 pM to about 200 pM B2. The higher level of B vitamins may comprise a total level of about 1.3 pM to about 200 pM B2. The higher level of B vitamins may comprise a total level of about 1.4 pM to about 200 pM B2. The higher level of B vitamins may comprise a total level of about 1.5 pM to about 200 pM B2. The higher level of B vitamins may comprise a total level of about 1.6 pM to about 200 pM B2. The higher level of B vitamins may comprise a total level of about 1.7 pM to about 200 pM B2. The higher level of B vitamins may comprise a total level of about 1.8 pM to about 200 pM B2. The higher level of B vitamins may comprise a total level of about 1.9 pM to about 200 pM B2. The higher level of B vitamins may comprise a total level of about 2 pM to about 200 pM B2.

[0209] The higher level of B vitamins may comprise a total level of about 1 pM to about 200 pM B2 (e.g. of about 1 pM to about 20 pM B2). The higher level of B vitamins may comprise a total level of about 2 pM to about 60 pM B2 (e.g. of about 2 pM to about 6 pM B2).

[0210] The higher level of B vitamins may comprise a total level of about 10 pM to about 2000 pM B6. The higher level of B vitamins may comprise a total level of about 10 pM to about 1500 pM B6. The higher level of B vitamins may comprise a total level of about 10 pM to about 1000 pM B6. The higher level of B vitamins may comprise a total level of about 10 pM to about 800 pM B6. The higher level of B vitamins may comprise a total level of about 10 pM to about 600 pM B6. The higher level of B vitamins may comprise a total level of about 10 pM to about 400 pM B6. The higherlevel of B vitamins may comprise a total level of about 10 pM to about 200 pM B6. The higher level of B vitamins may comprise a total level of about 10 pM to about 150 pM B6. The higher level of B vitamins may comprise a total level of about 10 pM to about 100 pM B6.

[0211] The higher level of B vitamins may comprise a total level of about 15 pM to about 2000 pM B6. The higher level of B vitamins may comprise a total level of about 20 pM to about 2000 pM B6. The higher level of B vitamins may comprise a total level of about 25 pM to about 2000 pM B6. The higher level of B vitamins may comprise a total level of about 30 pM to about 2000 pM B6. The higher level of B vitamins may comprise a total level of about 35 pM to about 2000 pM B6. The higher level of B vitamins may comprise a total level of about 40 pM to about 2000 pM B6. The higher level of B vitamins may comprise a total level of about 45 pM to about 2000 pM B6. The higher level of B vitamins may comprise a total level of about 50 pM to about 2000 pM B6. The higher level of B vitamins may comprise a total level of about 55 pM to about 2000 pM B6. The higher level of B vitamins may comprise a total level of about 60 pM to about 2000 pM B6.

[0212] The higher level of B vitamins may comprise a total level of about 10 pM to about 2000 pM B6 (e.g. of about 10 pM to about 200 pM B6). The higher level of B vitamins may comprise a total level of about 50 pM to about 1500 pM B6 (e.g. of about 50 pM to about 150 pM B6). The higher level of B vitamins may comprise a total level of about 60 pM to about 1000 pM B6 (e.g. of about 60 pM to about 100 pM B6).

[0213] The higher level of B vitamins may comprise a total level of about 10 pM to about 2000 pM B9. The higher level of B vitamins may comprise a total level of about 10 pM to about 1500 pM B9. The higher level of B vitamins may comprise a total level of about 10 pM to about 1000 pM B9. The higher level of B vitamins may comprise a total level of about 10 pM to about 800 pM B9. The higher level of B vitamins may comprise a total level of about 10 pM to about 600 pM B9. The higher level of B vitamins may comprise a total level of about 10 pM to about 400 pM B9. The higher level of B vitamins may comprise a total level of about 10 pM to about 200 pM B9. The higher level of B vitamins may comprise a total level of about 10 pM to about 150 pM B9. The higher level of B vitamins may comprise a total level of about 10 pM to about 125 pM B9. The higher level of B vitamins may comprise a total level of about 10 pM to about 100 pM B9. The higher level of B vitamins may comprise a total level of about 10 pM to about 80 pM B9.

[0214] The higher level of B vitamins may comprise a total level of about 15 pM to about 2000 pM B9. The higher level of B vitamins may comprise a total level of about 20 pM to about 2000 pM B9. The higher level of B vitamins may comprise a total level of about 25 pM to about 2000 pM B9. The higher level of B vitamins may comprise a total level of about 30 pM to about 2000 pM B9.

[0215] The higher level of B vitamins may comprise a total level of about 10 pM to about 2000 pM B9 (e.g. of about 10 pM to about 200 pM B9). The higher level of B vitamins may comprise a total level of about 20 pM to about 1000 pM B9 (e.g. of about 20 pM to about 100 pM B9). The higher level of B vitamins may comprise a total level of about 30 pM to about 800 pM B9 (e.g. of about 30 pM to about 80 pM B9).

[0216] The higher level of B vitamins may comprise a total level of about 2 pM to about 200 pM B12. The higher level of B vitamins may comprise a total level of about 2 pM to about 150 pM B12. The higher level of B vitamins may comprise a total level of about 2 pM to about 100 pM B12. The higher level of B vitamins may comprise a total level of about 2 pM to about 80 pM B12. The higher level of B vitamins may comprise a total level of about 2 pM to about 60 pM B12. The higher level of B vitamins may comprise a total level of about 2 pM to about 40 pM B12. The higher level of B vitamins may comprise a total level of about 2 pM to about 20 pM B12. The higher level of B vitamins may comprise a total level of about 2 pM to about 15 pM B12. The higher level of B vitamins may comprise a total level of about 2 pM to about 10 pM B12. The higher level of B vitamins may comprise a total level of about 2 pM to about 8 pM B12. The higher level of B vitamins may comprise a total level of about 2 pM to about 6 pM B12.

[0217] The higher level of B vitamins may comprise a total level of about 2 pM to about 200 pM B12 (e.g. of about 2 pM to about 20 pM B12). The higher level of B vitamins may comprise a total level of about 2 pM to about 100 pM B12 (e.g. of about 2 pM to about 10 pM B12). The higher level of B vitamins may comprise a total level of about 2 pM to about 60 pM B12 (e.g. of about 2 pM to about 6 pM B12).

[0218] The lower level of B vitamins may comprise a total level of about 0 pM to about 1 pM B2. The lower level of B vitamins may comprise a total level of about 0 pM to about 0.9 pM B2.

[0219] The lower level of B vitamins may comprise a total level of about 0.05 pM to about1 pM B2. The lower level of B vitamins may comprise a total level of about 0.1 pMto about 1 pM B2. The lower level of B vitamins may comprise a total level of about 0.15 pM to about 1 pM B2. The lower level of B vitamins may comprise a total level of about 0.2 pM to about 1 pM B2.

[0220] The lower level of B vitamins may comprise a total level of about 0.2 pM to about 0.9 pM B2.

[0221] The lower level of B vitamins may comprise a total level of about 0 pM to about 12 pM B6. The lower level of B vitamins may comprise a total level of about 0 pM to about 11 pM B6. The lower level of B vitamins may comprise a total level of about 0 pM to about 10 pM B6. The lower level of B vitamins may comprise a total level of about 0 pM to about 9 pM B6.

[0222] The lower level of B vitamins may comprise a total level of about 0.5 pM to about 12 pM B6. The lower level of B vitamins may comprise a total level of about 1 pM to about 12 pM B6. The lower level of B vitamins may comprise a total level of about 1.5 pM to about 12 pM B6. The lower level of B vitamins may comprise a total level of about 2 pM to about 12 pM B6. The lower level of B vitamins may comprise a total level of about 2.5 pM to about 12 pM B6. The lower level of B vitamins may comprise a total level of about 3 pM to about 12 pM B6.

[0223] The lower level of B vitamins may comprise a total level of about 1 pM to about 10 pM B6. The lower level of B vitamins may comprise a total level of about 3 pM to about 9 pM B6.

[0224] The lower level of B vitamins may comprise a total level of about 0 pM to about 12 pM B9. The lower level of B vitamins may comprise a total level of about 0 pM to about 11 pM B9. The lower level of B vitamins may comprise a total level of about 0 pM to about 10 pM B9. The lower level of B vitamins may comprise a total level of about 0 pM to about 9 pM B9.

[0225] The lower level of B vitamins may comprise a total level of about 0.5 pM to about 12 pM B9. The lower level of B vitamins may comprise a total level of about 1 pM to about 12 pM B9. The lower level of B vitamins may comprise a total level of about 1.5 pM to about 12 pM B9. The lower level of B vitamins may comprise a total level of about 2 pM to about 12 pM B9. The lower level of B vitamins may comprise a total level of about 2.5 pM to about 12 pM B9. The lower level of B vitamins may comprise a total level of about 3 pM to about 12 pM B9.

[0226] The lower level of B vitamins may comprise a total level of about 1 pM to about 10 pM B9. The lower level of B vitamins may comprise a total level of about 3 pM to about 9 pM B9.

[0227] The lower level of B vitamins may comprise a total level of about 0 pM to about 2 pM B12. The lower level of B vitamins may comprise a total level of about 0 pM to about 1.8 pM B12. The lower level of B vitamins may comprise a total level of about 0 pM to about 1.6 pM B12. The lower level of B vitamins may comprise a total level of about 0 pM to about 1.4 pM B12. The lower level of B vitamins may comprise a total level of about 0 pM to about 1.3 pM B12. The lower level of B vitamins may comprise a total level of about 0 pM to about 1.2 pM B12. The lower level of B vitamins may comprise a total level of about 0 pM to about 1.1 pM B12. The lower level of B vitamins may comprise a total level of about 0 pM to about 1 pM B12. The lower level of B vitamins may comprise a total level of about 0 pM to about 9 pM B12.

[0228] The lower level of B vitamins may comprise a total level of about 0.05 pM to about 2 pM B12. The lower level of B vitamins may comprise a total level of about 0.1 pM to about 2 pM B12. The lower level of B vitamins may comprise a total level of about 0.15 pM to about 2 pM B12. The lower level of B vitamins may comprise a total level of about 0.2 pM to about 2 pM B12.

[0229] The lower level of B vitamins may comprise a total level of about 0.1 pM to about 1 pM B12. The lower level of B vitamins may comprise a total level of about 0.2 pM to about 0.9 pM B12.

[0230] The B vitamins may comprise riboflavin (B2), pyridoxine (B6), folic acid (B9), and cyanocobalamin (B12). The B vitamins may consist of riboflavin (B2), pyridoxine (B6), folic acid (B9), and cyanocobalamin (B12).

[0231] The recombinant polypeptide may comprise at least one N-linked glycan site. For example, the recombinant polypeptide may comprise at least two N-linked glycan sites.

[0232] The recombinant polypeptide may be an antibody, an antigen, an enzyme, or a vaccine.

[0233] It may be that the recombinant polypeptide is an antibody. The antibody may be a multispecific antibody or antigen-binding fragment thereof. The antibody may consist of a single heavy chain sequence and a single light chain sequence or antigen-binding fragments thereof.

[0234] The antibody may comprise a chimeric antibody, a human antibody or a humanized antibody. The antibody may comprise a monoclonal antibody.

[0235] The recombinant polypeptide may be a fusion protein. The fusion protein may comprise an antibody or an antigen-binding fragment thereof. For example, the recombinant polypeptide may be an Fc fusion protein.

[0236] The cell line may be a mammalian cell line.

[0237] The cell line may be a CHO cell line or an NS0 cell line. For example, the cell line may be a CHO K1 cell line, a CHO K1SV cell line, a DG44 cell line, a DUKXB-11 cell line, or a CHOK1S cell line, or a targeted gene integration (Tl) - generated CHO cell line, or their derivatives.

[0238] The cell line may be cultured under fed-batch culture conditions, or perfusion culture conditions.

[0239] It may be that the cell line is cultured under fed-batch culture conditions. The fed- batch culture conditions may be intensified fed-batch culture conditions.

[0240] It may be that the cell line is cultured under perfusion culture conditions. The perfusion culture conditions may be semi-continuous perfusion or continuous perfusion.Multispecific Antibodies

[0241] 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.

[0242] 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 orfragments (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 mispairing 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).

[0243] 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.

[0244] 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., \NO 2009 / 080251 , WO 2016 / 016299, also see Schaefer et al, PNAS, 108 (2011) 1187-1191 , and Klein at al., MAbs 8 (2016) 1010-20). A multispecific 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.

[0245] 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).

[0246] 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.

[0247] 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 (Holliger 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 antibody formats included herein are described in WO 2013 / 026833, WO 2013 / 026839, WO 2016 / 020309; Bacac et al., Oncoimmunology 5(8) (2016) e1203498.Chimeric and humanized antibodies

[0248] 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 nonhuman 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 antigen-binding fragments thereof.

[0249] 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 withcorresponding 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.

[0250] 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).ASSAYSDetermination of Glycosylation Distribution

[0251] An assay based on hydrophilic interaction chromatography (HILIC) ultrahigh performance liquid chromatography (UHPLC) was used to determine the relative glycosylation distribution of the mAbs from cell culture samples. Samples were prepared by purification using protein A affinity resin, followed by rapid deglycosylation and aqueous 2-aminobenzamide (2-AB) labelling, and subsequent analysis of the 2-AB labelled glycans by HILIC UHPLC with fluorescence detection.

[0252] In addition to reporting individual glycosylation structures (Figure 1), afucosylation, galactosylation and sialylation were calculated. Total afucosylation was calculated as the sum of G0-N, GO, G1 , G2, and M5 species. Total galactosylation was calculated as the sum of G1 , G2, G1 F, G2F, G1S1 F, G2S1 F, and G2S2F species. Total sialylation was calculated as the sum of G1S1 F, G2S1 F, and G2S2F species. Mannosylation was determined by the amount of the M5 species.Determination of Charge Distribution

[0253] The relative charge distribution for each mAb sample was determined by imaged capillary isoelectric focusing (icIEF) on protein A purified material as previously described (Zhang et al., 2011 , Analytical Chemistry, 83, 8501-8508). In brief, an iCE280 analyzer (Convergent Bioscience) with a fluorocarbon-coated capillarycartridge and ampholyte solution were used. Ultraviolet light (280 nm) was passed through the capillary into the lens of a digital camera with charge-coupled device to obtain an image of the charge variants. Prior to this icIEF analysis, the samples were treated with carboxypeptidase B (CpB) to remove c-terminal lysines on the mAb heavy chains. This CpB treatment removes the contribution of the c-terminal lysines towards charge heterogeneity in the basic region of the electropherogram and thereby improves the resolution of other basic species.Determination of Size Distribution

[0254] The relative molecular size distribution was analyzed with Capillary Electrophoresis (CE-SDS) with laser-induced fluorescence to quantify the size distribution under denaturing conditions and Size Exclusion Chromatography (SEC) as previously described in Gomez et al., 2011. CE-SDS was used to quantify the LMW forms of the mAb. SEC was used to quantify the HMW forms of the mAb.EXAMPLES

[0255] 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 MethodsCell culture experimental design

[0256] Recombinant CHO cell lines stably transfected to express different mAbs were used in the cell culture experiments. The experiments were conducted with two cell lines (A and B) expressing mAb A, and one cell line (C) expressing mAb B.

[0257] Three different cell culture media (1 , 2, and 3) were used, differing in their composition of the basal media and the nutrient feeds. These media and feeds contain the necessary components for CHO cell cultures. These media and feeds are chemically defined, providing a consistent lot-to-lot defined composition. Examples of chemically-defined media have been described in patent literature, such as U.S. Patent Nos. 4,767,704; 5,691,202; 6,048,728; 6,900,056; and 7,601 ,535; as well as in European Patent No. 1482031. Such media are consideredderivatives of DMEM-F12 Media. DMEM-F12 (available from Thermo Fisher Scientific) comprises a composition as set out in Table 1.Table 1: DMEM-F12

[0258] Ten different fed-batch production processes were tested in these experiments, differing in cultivation strategies for temperature, pH, dissolved oxygen (DO), and nutrient feeding. Table 2 provides a summary of relevant conditions. For each specific condition of mAb, cell line, media, and process, duplicate bioreactors were tested with (+) and without (-) supplemental B-vitamins in the nutrient feeds. An additional eleventh experiment (listed as 11 in Table 2) titrated the level of B- vitamins supplemented in the nutrient feeds from 0- to 10-fold in total concentration (Table 6), to further illustrate the effects of supplementing different levels of B- vitamins. The B-vitamins tested were cyanocobalamin (B12), folic acid (B9), pyridoxine (B6), and riboflavin (B2).Table 2: Fed-batch process conditions

[0259] To initiate each small-scale study, cells were thawed and expanded to inoculate fed-batch production cultures in either 2-L glass bioreactors (Applikon) or an Ambr 25O (Sartorius). The temperature, pH, and DO in the bioreactors were controlled using a DeltaV controller for the 2-L glass vessels and Sartorius Ambr®250 Runtime software for the Ambr®250. Temperature, pH, and DO for all fed-batch production cultures were controlled as described in Table 2. Nutrient feeds were added to the production cultures as described in Table 2. The total amount of supplemental B-vitamins added to the production cultures through the nutrient feeds depended on the process tested, as shown in Tables 3, 4,5, 6, 7, and 8. The amount of B-vitamins present in the basal media is shown in Table 9. The total amount of B-vitamins throughout each process is shown in Table 10.Table 3. Increase in total amount of B vitamins provided to production bioreactor cultures from supplementation of nutrient feeds with (+) or without (-) B-vitamins for Process 1, 2, 3, 4, and 5.Table 4. Increase in total amount of B vitamins provided to production bioreactor cultures from supplementation of nutrient feeds with (+) or without (-) B-vitamins for Process 6.Table 5. Increase in total amounts of B vitamins provided to production bioreactor cultures from supplementation of nutrient feeds with (+) or without (-) B-vitamins for Process 7, 8, 9, and 10.Table 6. Titration of total amounts of B vitamins provided to production bioreactor cultures from supplementation of nutrient feeds for Process 11.Table 7. Increase in total amounts of B vitamins provided to production bioreactor cultures from supplementation of nutrient feeds with (+) or without (-) B-vitamins for DOE 1.Table 8. Increase in total amounts of B vitamins provided to production bioreactor cultures from supplementation of nutrient feeds with (+) or without (-) B-vitamins for DOE 2.Table 9. Concentration of B vitamins in basal media.Table 10. Range of total B vitamins tested for each process.

[0260] All cultures were sampled daily and analyzed for viable cell density (VCD), viability, pH, dissolved oxygen (DO), glucose, lactate, and ammonium levels using the BioProfile Flex or Flex2 (Nova Biomedical, Waltham, MA). To provide samples to analyze for product quality (e.g., glycosylation and charge distributions), all production cultures were sampled at time of harvest and some production cultures were additionally sampled 1-3 days prior to time of harvest. Culture supernatants were stored frozen at -80°C until they were analyzed for product titer, glycosylation and charge distribution.

[0261] Across the range of mAbs, cell lines, media, processes and culture duration tested, each unique combination was tested in duplicate production bioreactors with (+) or without (-) supplemental B-vitamins in the nutrient feeds or titrated with different levels of supplemental B-vitamins in the nutrient feeds. The production cultures supplemented with B-vitamins in the nutrient feeds would therefore be provided with a higher total amount of B-vitamins, with the exact increase in B- vitamin levels dependent on the process used (as shown in Tables 3, 4, 5, and 6). While a total of 18 unique conditions were tested for impact of B-vitamins, if the impact of culture duration were removed, 11 unique conditions are represented in this experimental design. The 5 conditions (processes 2, 3, 4, 5, and 11) that spanned a range of culture durations are shown to determine if the impact of B- vitamins is consistent over different culture durations.

[0262] To further investigate and quantify the impact of B vitamins, a structured statistical methodology was taken to design and analyze the cell culture experiments to determine the statistically significant effects of B vitamins on cell culture performance and mAb product quality. Specifically, two design of experiments (DOE) were tested. DOE 1 was conducted with cell line A expressing mAb A andDOE 2 was conducted with cell lines D and E expressing mAb C. DOE 1 was a fractional-factorial DOE evaluating supplemental B-vitamins in the nutrient feed, basal media osmolality, hydrocortisone, galactose, and manganese on cell line A expressing mAb A. DOE 2 was a fractional-factorial DOE evaluating supplemental B-vitamins in the nutrient feed, pH shift, hypotaurine and manganese on cell lines D and E expressing mAb C.Example 1 : Impact on Cell Culture Performance

[0263] The cumulative cell growth as represented by integrated viable cell count (Figure 2 and Figure 21) was similar or higher in the cases with supplemental B-vitamins relative to the identical conditions without supplemental B-vitamins. However, there was no consistent trend on the presence (+) or absence (-) of supplemental B- vitamins on the key cell culture performance indicators of final viabilities at time of harvest (Figure 3 and Figure 22). There was no consistent trend on the presence (+) or absence (-) of supplemental B-vitamins on the key cell culture performance indicators of mAb harvest titers at low levels of B-vitamins (Figure 4). At higher levels of B-vitamins as shown in the B vitamin titration experiment (2-fold and higher), a slight decrease in mAb harvest titers was observed (Figure 23). This means that varying levels of B-vitamins generally provides acceptable cell culture performance.

[0264] The statistical modelling of DOE 1 shows minimal response to integral of viable cell concentration (Figure 40), final viability (Figure 41) and mAb A harvest titer (Figure 42) from supplemental B-vitamins. The statistical modelling of DOE 2 shows an increase in integral of viable cell concentration (Figure 62) and mAb C titer (Figure 63) with cell line E, but minimal response with cell line D from supplemental B-vitamins. This data indicates that varying levels of B-vitamins in combination with other cell culture process parameters generally provides acceptable cell culture performance.Example 2: Impact on Glycosylation Distribution Profiles

[0265] The presence (+) or absence (-) of supplemental B-vitamins in the nutrient feeds provided during the production cultures impacted the glycosylation profiles of mAbs A and B (Figures 5-17, Figures 24-36, and Figures 43-55)).Afucosylation

[0266] In all the 10 unique conditions tested that varied across mAbs, cell lines, media , and processes, the production cultures with (+) supplemental B vitamins consistently showed lower levels of total afucosylation relative to the corresponding cultures without (-) supplemental B vitamins (Figure 5). In the B vitamin titration experiment, higher concentrations of B vitamins in the nutrient feed media continued to decrease the levels of total afucosylation (Figure 24). These trends remain consistent for the 5 conditions tested at different culture durations (processes 2, 3, 4, 5, and 11), representing a total of 18 unique conditions when culture duration is included. These results demonstrate the consistent impact of lowering B vitamin levels in production cultures on enhancing total afucosylation levels in mAbs. The impact of B vitamins on individual afucosylated species of G0- N, GO, G1 , G2, and M5 are shown in Figures 6 and 25, 7 and 26, 8 and 27, 9 and 28, and 13 and 32, respectively. The individual afucosylated species also tended to trend higher on average with the decrease or in the absence of supplemental B vitamins.

[0267] The statistical modelling of DOE 1 shows the addition of supplemental B-vitamins in combination with other cell culture variables resulted in lower levels of total afucosylation (Figure 43). Individual afucosylated species of G0-N, GO, G1 , G2, and M5 also showed the same trend in Figures 44, 45, 46, 47 and 51 respectively, further supporting the above findings.Galactosylation

[0268] The presence (+) or absence (-) of B vitamins shifted the extent of total galactosylation, thereby demonstrating its ability to impact galactosylation (Figure 10). For cell line A expressing mAb A, a general trend of decreased total galactosylation was observed from supplemental B vitamins at moderate levels. In the B vitamin titration experiment, the highest levels of B vitamins (6x and 10x) showed an increase in total galactosylation.. In mAb B, supplemental B vitamins consistently increased total galactosylation across the different production process conditions tested.

[0269] A consistent increase in the G1 galactosylated species was observed across all the 16 conditions without (-) supplemental B-vitamins relative to the corresponding cultures with (+) supplemental B-vitamins (Figure 8). A decrease in other galactosylated species G2, G1 F, G2F, G1S1F, G2S1F, and G2S2F was also observed for certain processes / cell lines / media, as shown in Figures 9, 11 , 12, 15, 16, and 17, respectively. In the B vitamin titration experiment, G1 increased without the presence of B vitamins in the feed media (Figure 27). Similar levels ofG2 was observed across all cases (Figure 28). G1 F and G2F increased with the highest levels of B vitamins (6x and 10x), while G1S1 F, G2S1F, and G2S2F had similar levels across all test conditions, as shown in Figures 30, 31, 34, 35, and 36 respectively.Mannosylation

[0270] Mannose 5 (M5) species was used to represent overall mannosylation trends since other mannosylated structures were of too low a level to be reliably quantified. In the conditions tested - varying across mAbs, cell lines, media, processes, and culture durations - the production cultures with (+) supplemental B vitamins showed generally lower levels of M5 relative to the corresponding cultures without (-) supplemental B vitamins (Figure 13). Additionally, the B vitamin titration experiment showed a similar impact with lower M5 levels across all cases that were supplemented with B vitamins irrespective of the concentration (Figure 32). These results demonstrate the impact of increasing B vitamin levels in production cultures on decreasing mannosylation levels in mAbs.

[0271] The statistical modelling of DOE 1 shows the addition of supplemental B-vitamins in combination with other cell culture variables resulted in lower levels of mannose 5 (M5) species (Figure 51), further supporting the above findings.Sialylation

[0272] The presence (+) or absence (-) of B-vitamins shifted the total amount of sialylated species in some of the conditions to varying extents, thereby demonstrating its potential impact on total sialylation (Figure 14). In particular, a decrease or similar level of different individual sialylated species G1S1F, G2S1 F, and G2S2F (and shown in Figures 15, 16, and 17, respectively) was observed in the presence (+) of B-vitamins compared to the absence (-) of B-vitamins. The B vitamin titration experiment resulted in both total sialylation and the individual sialylated species to be lower at the 1x B vitamin supplementation level and slightly lower at higher concentrations (2x - 10x) as compared to the cases without supplemental B vitamins in the nutrient feed media (Figure 33). For mAb A expressed by cell line A in media 3 and process 5, the highest levels of total sialylation and individual sialylated species (G1S1 F, G2S1F, and G2S2F) were observed both with (+) and without (-) supplemental B-vitamins. This condition also showed the largest decrease in total sialylation with B-vitamin supplementation. These observations indicate that the effect of B-vitamins on total sialylation may be more evident when the levels of total mAb sialylation are higher.Example 3: Impact on Charge Distribution Profiles

[0273] Six of the 16 conditions with (+) and without (-) supplemental B-vitamins in the nutrient feeds and the B vitamin titration experiment were tested for charge distribution (Figures 18, 19, 20, 37, 38, and 39). Only one media and two process conditions were chosen from cell line A expressing mAb A for further analytical evaluation and hence the other 10 conditions for mAb A using cell line A were not analyzed. The conditions tested for glycosylation profiles in mAb A cell line B and mAb B cell line C were also analyzed for charge distribution. DOE 1 and DOE 2 were both analyzed for charge distribution in mAb A and mAb C, respectively.Main Species

[0274] In all the conditions analyzed — varying across mAbs, cell lines, media, and processes — the production cultures with (+) supplemental B vitamins showed consistently lower levels of main species relative to the corresponding cultures without (-) supplemental B vitamins (Figure 18). Additionally, the B vitamin titration experiment showed that at 10-fold increase of supplemental B vitamin levels, main species further decreased (Figure 37). The statistical modelling of DOE 1 (cell line A expressing mAb A) and DOE 2 (both cell lines D and E expressing mAb C) showed a response of decreasing main species with the supplementation of B vitamins in combination with other cell culture variables (Figure 56 and Figure 66), further supporting the above findings.

[0275] These results demonstrate the consistent impact of lowering B vitamin levels in production cultures on enhancing overall main species (in terms of charge distribution) in mAbs. These results are surprising because there has not been any previous reports on the impact of these B vitamins on charge distributions in mAbs, and hence there are also no established underlying mechanism(s) for how these B vitamins can affect charge profiles for mAbs.Acidic Species

[0276] Production cultures with (+) supplemental B vitamins showed consistently higher levels of acidic species relative to the corresponding cultures without (-) supplemental B vitamins (Figure 19). Additionally, the B vitamin titration experiment showed levels of acidic species continued to increase with increasing levels of supplemental B vitamins (Figure 38). The statistical modelling of DOE 1 (cell line A expressing mAb A) and DOE 2 (both cell lines D and E expressing mAb C) showed a response of increasing acidic species with the supplementation of B vitamins incombination with other cell culture variables (Figure 57 and Figure 64) , further supporting the above findings.

[0277] These results demonstrate the consistent impact of lowering B vitamin levels in production cultures on lowering acidic species in mAbs. These results are surprising because a previous report on biotin — which is a different B-vitamin from the B-vitamins tested in this work — showed the opposite trend, i.e., increasing levels of biotin decreased acidic species on an IgG (Gangwar et al., 2021 , Biotechnology Journal, 16, 2000464).Basic Species

[0278] Five of the six conditions showed production cultures with (+) supplemental B vitamins yielded lower levels of basic species relative to the corresponding cultures without (-) supplemental B vitamins (Figure 20). Additionally, the B vitamin titration experiment showed levels of basic species continued to decrease with increasing levels of supplemental B vitamins (Figure 39). The statistical modelling of DOE 1 (cell line A expressing mAb A) and DOE 2 (both cell lines D and E expressing mAb C) showed a response of decreasing basic species with the supplementation of B vitamins in combination with other cell culture variables (Figure 58 and Figure 65), further supporting the above findings.

[0279] These results demonstrate how increasing B vitamin levels in production cultures are likely to decrease basic species in mAbs. These results are surprising because a previous report on biotin — which is a different B-vitamin from the B-vitamins tested in this work — showed the opposite trend, i.e., increasing levels of biotin increased basic species on an IgG (Gangwar et al., 2021 , Biotechnology Journal, 16, 2000464).Example 4: Impact on Size Distribution Profiles

[0280] DOE 1 (with cell line A expressing mAb A) and DOE 2 (with both cell lines D and E expressing mAb C) were analyzed for size distribution with CE-SDS for the sum of LMW forms and SEC for the sum of HMW forms.

[0281] The statistical modelling of DOE 1 (cell line A expressing mAb A) and DOE 2 (both cell lines D and E expressing mAb C) showed a response of increasing sum of LMW forms with the supplementation of B vitamins in combination with other cell culture variables (Figure 59 and Figure 67). These results demonstrate that decreasing B vitamin levels in production cultures are likely to decrease LMW forms.

[0282] In DOE 1 and DOE 2 there was minimal impact to mAb aggregates as shown with sum of HMW forms (Figure 60 and Figure 68). This data demonstrates potential robustness of HMW forms with varying levels of B vitamins.

Claims

CLAIMS1. A method of optimizing glycosylation of a recombinant polypeptide, comprising: culturing a cell line engineered to express the recombinant polypeptide in a culture medium under conditions promoting production of the recombinant polypeptide; and modulating the level of B vitamins in the cell culture; optionally wherein the modulating the level of B vitamins comprises providing a lower level of B vitamins in the cell culture, or a higher level of B vitamins in the cell culture.

2. The method of claim 1 , wherein the lower level comprises maintaining or decreasing the level of B vitamins in the cell culture.

3. The method of claim 1 or claim 2, wherein optimizing the glycosylation comprises optimizing the level of afucosylation, and / or galactosylation, and / or mannosylation, and / or sialylation.

4. The method of claim 3, wherein optimizing afucosylation comprises using higher level of B vitamins to maintain a decreased level of afucosylation.

5. The method of claim 3, wherein optimizing afucosylation comprises using lower level of B vitamins to maintain an increased level of afucosylation6. The method of any of claims 3 to 5, wherein optimizing galactosylation comprises using modulated levels of B vitamins to achieve the desired level of galactosylation.

7. The method of any claims 3 to 6, wherein optimizing mannosylation comprises using modulated levels of B vitamins to achieve the desired level of mannosylation.

8. The method of any of claims 3, 4 or 6, wherein optimizing mannosylation comprises using the higher level of B vitamins to maintain a decreased level of mannosylation.

9. The method of any of claims 3, 5 or 6, wherein optimizing mannosylation comprises using the lower level of B vitamins to maintain an increased level of mannosylation.

10. The method of any claims 3 to 9, wherein optimizing sialylation comprises using modulated levels of B vitamins to achieve the desired level of sialylation.

11. The method of any of claims 3, 4, 6 or 8, wherein optimizing sialylation comprises using the higher level of B vitamins to maintain a decreased level of sialylation.

12. The method of any of claims 3, 5, 6 or 9, wherein optimizing sialylation comprises using lower level of B vitamins to maintain an increased level of sialylation.

13. A method of optimizing charge pattern of a recombinant polypeptide, comprising: culturing a cell line engineered to express the recombinant polypeptide under conditions promoting production of the recombinant polypeptide; andmodulating the level of B vitamins in the cell culture; optionally wherein the modulating the level of B vitamins comprises providing a lower level of B vitamins in the cell culture, or a higher level of B vitamins in the cell culture.

14. The method of claim 13, wherein the lower level comprises maintaining or decreasing the level of B vitamins in the cell culture15. The method of claim 13 or claim 14, wherein the optimizing charge pattern comprises optimizing the relative amount of acidic species, and / or main species, and / or basic species of the recombinant polypeptide.

16. The method of claim 15, wherein optimizing the relative amount of acidic species comprises using the higher level of B vitamins to maintain an increased level of acidic species.

17. The method of claim 15, wherein optimizing the relative amount of acidic species comprises using the lower level of B vitamins to maintain a decreased level of acidic species.

18. The method of claim 15 or claim 16, wherein optimizing the relative amount of main species comprises using the higher level of B vitamins to maintain a decreased level of main species.

19. The method of claim 15 or claim 17, wherein optimizing the relative amount of main species comprises using the lower level of B vitamins to maintain an increased level of main species.

20. The method of any of claims 15, 16 or 18, wherein optimizing the relative amount of basic species comprises using the higher level of B vitamins to maintain a decreased level of basic species.

21. The method of any of claims 15, 17 or 19, wherein optimizing the relative amount of basic species comprises using the lower level of B vitamins to maintain an increased level of basic species.

22. The method of any of claims 1 to 12, wherein the method comprises optimizing both glycosylation and optimizing the charge pattern of the recombinant polypeptide.

23. The method of claim 22, further comprising the features of any of claims 13 to 21.

24. A method of optimizing size pattern of a recombinant polypeptide, comprising: culturing a cell line engineered to express the recombinant polypeptide under conditions promoting production of the recombinant polypeptide; and modulating the level of B vitamins in the cell culture;optionally wherein the modulating the level of B vitamins comprises providing a lower level of B vitamins in the cell culture, or a higher level of B vitamins in the cell culture.

25. The method of claim 24, wherein the lower level comprises maintaining or decreasing the level of B vitamins in the cell culture26. The method of claim 24 or claim 25, wherein the optimizing size pattern comprises optimizing the relative amount of LMW forms, monomeric form, and / or HMW forms of the recombinant polypeptide.

27. The method of claim 26, wherein optimizing the relative amount of LMW forms comprises using the higher level of B vitamins to maintain an increased level of LMW forms.

28. The method of claim 26, wherein optimizing the relative amount of LMW forms comprises using the lower level of B vitamins to maintain a decreased level of LMW forms.

29. The method of claim 26 or claim 27, wherein optimizing the relative amount of monomeric form comprises using the higher level of B vitamins to maintain a decreased level of monomeric form.

30. The method of claim 26 or claim 28, wherein optimizing the relative amount of monomeric form comprises using the lower level of B vitamins to maintain an increased level of monomeric form.31 . The method of any of claims 26, 27 or 29, wherein optimizing the relative amount of HMW forms may or may not be impacted by the levels of B vitamins.

32. The method of any of claims 26, 28 or 30, wherein optimizing the relative amount of HMW forms may or may not be impacted by the levels of B vitamins.

33. The method of any of claims 1 to 12, wherein the method further comprises optimizing the size pattern of the recombinant polypeptide.

34. The method of claim 33, further comprising the features of any of claims 13 to 21 .

35. The method of claim 34, further comprising the features of any claims 24 to 32.

36. The method of any preceding claim, wherein the higher levels of B vitamins are achieved by supplementing the cell culture with B vitamins.

37. The method of claim 24, wherein the supplementing with B vitamins comprises at least partially replacing the cell culture medium with a production culture medium having higher levels of B vitamins, or wherein the supplementing with B vitamins comprises providing one or more nutrient feeds with higher levels of B vitamins.

38. The method of claim 24, wherein the higher levels of B vitamins are achieved by either supplementing or perfusing the production culture with medium containing higher levels of B vitamins.

39. The method of claim 24, wherein the higher levels of B vitamins are achieved by increasing the levels of B vitamins in the production culture medium, or, supplementing the production cell culture with nutrient feed(s) containing higher levels of B vitamins.

40. The method of claim 24, wherein the higher levels of B vitamins are achieved by supplementing one or more nutrient feeds with higher levels of B vitamins to the cell culture during the production phase; optionally adding the nutrient feeds through continuous feeding or daily feeds during a production period from 1-20 days; or optionally adding the nutrient feeds to the cell culture medium 1 -4 times during a production period of from 2-15 days; or optionally adding the nutrient feeds to the cell culture medium 2-3 times during a period of from 3-14 days.41 . The method of any preceding claim, wherein the B vitamins comprise (or consist) of one or a combination of riboflavin (B2), pyridoxine (B6), folic acid (B9), or cyanocobalamin (B12).

42. The method of claim 29, wherein the higher level of B vitamins comprises providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 200 pM B2; optionally about 1 pM to about 50 pM B2; further optionally about 2 pM to about 5 pM B2.

43. The method of claim 29 or claim 30, wherein the higher level of B vitamins comprises providing a level of increase in B vitamins in the culture medium of about 1 pM to about 2000 pM B6; optionally about 50 pM to about 1000 pM B6; further optionally about 60 pM to about 80 pM B6.-SO-44. The method of any of claims 29 to 31, wherein the higher level of B vitamins comprises providing a level of increase in B vitamins in the culture medium of about 1 pM to about 2000 pM B9; optionally about 20 pM to about 600 pM B9; further optionally about 30 pM to about 60 pM B9.

45. The method of any of claims 29 to 32, wherein the higher level of B vitamins comprises providing a level of increase in B vitamins in the culture medium of about 0.1 pM to about 200 pM B12; optionally about 1 pM to about 50 pM B12; further optionally about 2 pM to about 6 pM B12.

46. The method of any of claims 29 to 33, wherein the higher level of B vitamins comprises a total level of about 1 pM to about 200 pM B2; optionally a total level of about 1 pM to about 50 pM B2; further optionally a total level of about 2 pM to about 6 pM B2.

47. The method of any of claims 29 to 34, wherein the higher level of B vitamins comprises a total level of about 10 pM to about 2000 pM B6; optionally a total level of about 50 pM to about 1000 pM B6; further optionally a total level of about 60 pM to about 100 pM B6.

48. The method of any of claims 29 to 35, wherein the higher level of B vitamins comprises a total level of about 10 pM to about 2000 pM B9; optionally a total level of about 20 pM to about 600 pM B9; further optionally a total level of about 30 pM to about 80 pM B9.

49. The method of any of claims 29 to 36, wherein the higher level of B vitamins comprises a total level of about 2 pM to about 200 pM B12; optionally a total level of about 2 pM to about 50 pM B12; further optionally a total level of about 2 pM to about 6 pM B12.

50. The method of any preceding claim, wherein the lower level of B vitamins comprises a total level of about 0 pM to about 1 pM B2; optionally a total level of about 0.2 pM to about 0.9 pM B2.

51. The method of any preceding claim, wherein the lower level of B vitamins comprises a total level of about 0 pM to about 10 pM B6; optionally about 1 pM to about 10 pM B6; further optionally about 3 pM to about 9 pM B6.

52. The method of any preceding claim, wherein the lower level of B vitamins comprises a total level of about 0 pM to about 10 pM B9;optionally about 1 pM to about 10 pM B9; further optionally about 3 pM to about 9 pM B9.

53. The method of any preceding claim, wherein the lower level of B vitamins comprises a total level of about 0 pM to about 2 pM B12; optionally a total level of about 0.1 pM to about 1 pM B12; further optionally a total level of about 0.2 pM to about 0.9 pM B1254. The method of any preceding claim, wherein the B vitamins comprise (or consist) of riboflavin (B2), pyridoxine (B6), folic acid (B9), and cyanocobalamin (B12).

55. The method of any preceding claim, wherein the recombinant polypeptide comprises at least one N-linked glycan sites; optionally wherein the recombinant polypeptide comprises at least two N-linked glycan sites.

56. The method of any preceding claim, wherein the recombinant polypeptide is an antibody, an antigen, an enzyme, or a vaccine.

57. The method of claim 44, wherein the recombinant polypeptide is an antibody; optionally wherein the antibody is a multispecific antibody or antigen-binding fragment thereof.

58. The method of claim 44 or claim 45, wherein the antibody consists of a single heavy chain sequence and a single light chain sequence or antigen-binding fragments thereof.

59. The method of any of claims 44 to 46, wherein the antibody comprises a chimeric antibody, a human antibody or a humanized antibody.

60. The method of any of claims 44 to 47, wherein the antibody comprises a monoclonal antibody.

61. The method of any preceding claim, wherein the recombinant polypeptide is a fusion protein, optionally comprising an antibody or an antigen-binding fragment thereof.

62. The method of claim 49, wherein the recombinant polypeptide is an Fc fusion protein.

63. The method of any preceding claim, wherein the cell line is a mammalian cell line.

64. The method of any preceding claim, wherein the cell line is a CHO cell line or an NS0 cell line; optionally a CHO K1 cell line, a CHO K1SV cell line, a DG44 cell line, aDUKXB-11 cell line, or a CHOK1S cell line, or a targeted gene integration (Tl) - generated CHO cell line, or their derivatives.

65. The method of any preceding claim, wherein the cell line is cultured under fed-batch culture conditions, or perfusion culture conditions.

66. The method of claim 53, wherein the cell line is cultured under fed-batch culture conditions, optionally wherein the fed-batch culture conditions are intensified fed-batch culture conditions.

67. The method of claim 53, wherein the cell line is cultured under perfusion culture conditions, optionally wherein the perfusion culture conditions are semi-continuous perfusion or continuous perfusion.

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