Abstract:
Protein glycosylation greatly influences the structure, function, and pharmacokinetics of recombinant proteins. Here, growth media supplemented with metal ions is shown to modulate the protein glycosylation profile of recombinant proteins expressed in a variety of eukaryotic cell lines. In particular, millimolar amounts of ferric salts (e.g. ferric nitrate, ferric citrate, etc.) significantly increased the percentage of galactosylated N-glycan species. The effect on protein glycosylation was concentration-dependent manner up to 1.0 mM across multiple cell lines without adverse effects on cell viability. The use of iron supplements in cell culture media provides an efficient and effective approach towards the specific re-targeting of N-glycan glycoform profiles of recombinantly expressed proteins.

Description:
RELATED APPLICATIONS 
       [0001]    The present application is a continuation of U.S. patent application Ser. No. 14/506,372, filed on Oct. 3, 2014, which in turn claims priority from U.S. Provisional Patent Application No. 61/886,855, filed on Oct. 4, 2013. The entire contents of each of the foregoing applications are incorporated herein by reference. 
     
    
     FIELD 
       [0002]    The present invention is directed to methods for adjusting the glycosylation profile of recombinantly-expressed proteins by supplementing the culture media metal ions. 
       BACKGROUND 
       [0003]    Protein glycosylation is a well-established post translation modification that influences the folding (Walsh et al. (1990) Biochemistry 29(26):6250-7), solubility (Leavitt et al. (1977) J. Biol. Chem. 252(24):9018-23), immunogenicity (Shields et al., (2002) J. Biol. Chem. 277(30):26733-40; Rudd et al., (2001) Science 291(5512):2370-6; Lowe and Marth, (2003) Annu. Rev. Biochem. 72:643-91, binding (Wallick et al., (1988) J. Exp. Med. 168(3):1099-109), stability (Wyss and Wagner, (1996) Curr. Opin. Biotechnol. 7(4):409-16), and pharmacokinetics (PK) (Millward, T. A., et al. (2008) Biologicals 36(1):41-7; Gross et al., (1988) Eur. J. Biochem. 173(3):653-9; Clincke et al., (2011) BMC Proceedings 5 (Suppl 8): p114-115) of recombinant protein therapeutics (reviewed in Hossler et al. Glycobiology (2009) vol. 19 pp. 936-949). 
         [0004]    During the N-glycosylation biosynthetic pathway, oligosaccharides are added onto asparagine residues of protein therapeutics in a step-wise manner followed by the step-wise removal and addition of individual monosaccharides in the endoplasmic reticulum (ER), and Golgi apparatus. This progression of potential glycoforms is summarized in  FIG. 1 . Briefly, mannose is removed to allow the addition of N-acetylglucosamine (GlcNAc) followed by the addition of galactose. The glycosylation profile for a given therapeutic glycoprotein often includes a mixture of these different glycoforms. 
         [0005]    The impact of protein glycosylation on the physiochemical properties of recombinant proteins is well-documented. The protein glycosylation profile is therefore a critical process parameter in the manufacturing of recombinant protein therapeutics. 
         [0006]    For the foregoing reasons, there is an unmet need in the art for the consistent and reliable glycosylation of recombinant biologics during manufacture. 
       SUMMARY 
       [0007]    The present invention is directed to methods for modulating the glycosylation profile of proteins recombinantly expressed in cultured eukaryotic cells by supplementing a chemically defined cell culture media with millimolar amounts of a metal ion. 
         [0008]    In one embodiment, the metal ion is iron. 
         [0009]    In other embodiments, the N-glycan oligosaccharide profile of recombinant proteins are increased by supplementing the chemically defined cell culture media with ferrous sulfate, ferric citrate, or ferric nitrate. The observed effect of these metal ions on glycosylation is scale or vessel independent and occurs even if the metal irons are introduced to the culture via the feed medium. 
         [0010]    In another embodiment, the present invention provides for a method for modulating the glycosylation profile of a recombinant protein expressed in a eukaryotic cell comprising the culturing of a eukaryotic cell in a protein-free, chemically defined culture medium supplemented with an effective amount of a metal salt and expressing a recombinant protein in the eukaryotic cell, wherein the amount of the metal salt is effective at modulating the glycosylation profile of the recombinant protein. 
         [0011]    The metal salt can be an iron salt such as ferric nitrate, ferric citrate or ferrous sulfate. In certain embodiments, the metal ion further includes zinc, copper, chromium and/or manganese. 
         [0012]    In certain embodiments, the effective amount of a metal salt in the culture medium can be less than about 1 μM. 
         [0013]    For example, in one embodiment, the effective amount of zinc, chromium or copper ions in the culture medium is less than about 1 μM. 
         [0014]    In other embodiments, the effective amount of a metal salt in the culture medium can be from about 1 μM to about 100 μM. 
         [0015]    For example, in one embodiment, the effective amount of ferrous sulfate in the culture media can be about 20 μM. 
         [0016]    In other embodiments, the effective amount of a metal salt in the culture medium can be from about 0.1 mM to about 10 mM of metal ions. 
         [0017]    For example, in one embodiment, the effective amount of magnesium salt in the culture medium can be about 1 to about 10 mM. 
         [0018]    The eukaryotic cell can be a mammalian cell including Chinese hamster ovary (CHO) cells, Chinese hamster ovary (CHO) cells, CHO DUX-B 11 cells, mouse myeloma SP2/0, human HT-1080, NSO myeloma cell line, mouse L cells, mouse A9 cells, baby hamster kidney cells, C127 cells, COS cells and PC8 cells. 
         [0019]    In another embodiment, the recombinantly expressed protein can be any of a number of protein binders, such as an antibody, a fragment or variant thereof, or a fusion protein comprising an antibody, a fragment or variant thereof. 
         [0020]    In another embodiment, the recombinantly expressed protein binder can be humanized. 
         [0021]    In one embodiment, the addition of the metal ion to the chemically defined cell culture medium reduces NGA2F and NGA2F-GlcNAc glycosylation of the expressed recombinant protein. For example, the addition of the metal ion to the chemically defined cell culture medium can reduce NGA2F and NGA2F-GlcNAc glycosylation of the expressed recombinant protein by at least 15%. 
         [0022]    In another embodiment, the addition of the metal ion to the chemically defined cell culture medium increases galactosylaton of the expressed recombinant glycoprotein. 
         [0023]    In yet another embodiment, the addition of the metal ion to the chemically defined cell culture medium increases NA1F and NA2F glycosylation of the expressed recombinant protein. For example, the addition of the metal ion to the chemically defined cell culture medium can increase NA1F glycosylation of the expressed recombinant protein by at least about 4 fold. In another example, the addition of the metal ion to the chemically defined cell culture medium can increase NA2F glycosylation of the expressed recombinant protein by at least about 7 fold. 
         [0024]    In another embodiment, the present invention is directed to a cell culture medium for modulating the glycosylation profile of a recombinantly-expressed protein, comprising a protein-free chemically defined culture medium supplemented with an effective amount of a metal salt effective at modulating the glycosylation profile of a recombinantly-expressed protein, wherein the metal salt is not ferric citrate. 
         [0025]    The metal salt can be an iron salt such as ferric nitrate. In one embodiment, the effective amount of the metal salt is about 0.2 mM to about 1 mM. 
         [0026]    In another embodiment, the recombinantly-expressed protein is a protein binder, such as an antibody, a fragment or variant thereof, or a fusion protein comprising an antibody, a fragment or variant thereof. 
         [0027]    The previously described embodiments have many advantages, including the ability to modulate the glycosylation of recombinantly expressed proteins by supplementing the culture media with millimolar amounts of a metal ion. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0028]    The skilled artisan will understand that the drawings, described below, are for illustration purposes only. The figures are not intended to limit the scope of the teachings in any way. 
           [0029]      FIG. 1  depicts a simplified linear reaction view of the N-glycan biosynthetic pathway in mammalian cells. 
           [0030]      FIG. 2  shows an exemplary depiction of the cell culture performance of Antibody 1 in ferric nitrate supplemented media. 
           [0031]      FIG. 3  shows an exemplary depiction of the N-glycan oligosaccharide results of Antibody 1 in ferric nitrate supplemented media. 
           [0032]      FIG. 4  shows an exemplary depiction of the cell culture performance of Antibody 1 in various iron supplemented media. 
           [0033]      FIG. 5  shows an exemplary depiction of N-glycan oligosaccharide results of Antibody 1 in various iron supplemented media. 
           [0034]      FIG. 6  shows an exemplary depiction of the cell culture performance of Antibody 2 in various iron supplemented media. 
           [0035]      FIG. 7  shows an exemplary depiction of the titer and N-glycan oligosaccharide results of Antibody 2 in various iron supplemented media. 
           [0036]      FIG. 8  shows an exemplary depiction of the cell culture performance of DVD-Ig in various iron supplemented media. 
           [0037]      FIG. 9  shows an exemplary depiction of the titer and N-glycan oligosaccharide results of DVD-Ig in various iron supplemented media. 
           [0038]      FIG. 10  shows an exemplary depiction of the N-glycan oligosaccharide results of Antibody 1 for various iron supplemented feed media. 
           [0039]      FIG. 11  shows an exemplary depiction of the N-glycan oligosaccharide results in bioreactors for Antibody 1 in various iron supplemented media. 
           [0040]      FIG. 12  shows an exemplary depiction of the N-glycan oligosaccharide results in bioreactors for DVD-Ig 2 in various iron supplemented media. 
           [0041]      FIG. 13  shows an exemplary depiction of the N-glycan oligosaccharide results of Antibody 1 in various metal ion supplemented media. 
           [0042]      FIG. 14  shows an exemplary depiction of the N-glycan oligosaccharide results of Antibody 1 in various iron, transferrin, and galactose supplemented media. 
           [0043]      FIG. 15  shows an exemplary depiction of the N-glycan oligosaccharide results of Antibody 1 in various iron, manganese, and galactose supplemented media. 
       
    
    
     DETAILED DESCRIPTION 
       [0044]    Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. The following definitions are provided to help interpret the disclosure and the claims of this application. 
         [0045]    As used herein, the term “chemically defined medium” or “chemically defined media” refers to a synthetic growth medium in which the identity and concentration of all the components are known. Chemically defined mediums do not contain bacterial, yeast, animal, or plant extracts, animal serum or plasma although they may or may not include individual plant or animal-derived components (e.g., proteins, polypeptides, etc). Chemically defined media may contain inorganic salts such as phosphates, sulfates, and the like needed to support growth. The carbon source is defined, and is usually a sugar such as glucose, lactose, galactose, and the like, or other compounds such as glycerol, lactate, acetate, and the like. While certain chemically defined culture media also use phosphate salts as a buffer, other buffers may be employed such as citrate, triethanolamine, and the like. As used herein, a chemically defined cell culture medium contains no more than micromolar amounts of any metal ion. Examples of commercially available chemically defined mediums include, but are not limited to, various Dulbecco&#39;s Modified Eagle&#39;s (DME) mediums (Sigma-Aldrich Co; SAFC Biosciences, Inc), Ham&#39;s Nutrient Mixture (Sigma-Aldrich Co; SAFC Biosciences, Inc), combinations thereof, and the like. Methods of preparing chemically defined culture mediums are known in the art, for example in U.S. Pat. Nos. 6,171,825 and 6,936,441, WO 2007/077217, and U.S. Patent Application Publication Nos. 2008/0009040 and 2007/0212770. In certain embodiments, the chemically defined medium does not contain non-ionic detergents such as Pluronic® F-68. 
         [0046]    As used herein, the term “serum-free culture medium” or “serum-free culture media” refers to a culture medium that is not supplemented with an animal serum. Although oftentimes serum-free mediums are chemically defined mediums, serum-free mediums may be supplemented with discrete animal or plant proteins or protein fractions. Methods of preparing serum-free culture medium are known in the art, for example in U.S. Pat. Nos. 6,171,825; 6,936,441 and EP 1849862. 
         [0047]    As used herein, the term “an effective amount” means a sufficient amount of a metal ion to modulate the glycosylation profile of a recombinantly-expressed protein. 
         [0048]    A metal ion may be any positively charged metal ion selected from, but not limited to, the following Al +3 , Cr +2 , Cr +3 , Cd +1 , Cd +2 , Co +2 , Co +3 , Ca +2 , Mg +2 , Ni +2 , Ti +2 , Ti +3 , Ti +4 , V +2 , V +3 , V +5 , Sr +2 , Fe +2 , Fe +3 , Au +2 , Au +3 , Au +1 , Ag +2 , Ag +1 , Pd +2 , Pd +4 , Pt +2 , Pt +4 , Cu +1 , Cu +2 , Mn +2 , Mn +3 , Mn +4 , Zn +2 , Se +4+2  and mixtures thereof. The ions are generally added to the medium in the form of salts such as chlorides and sulphates. In certain embodiments, the metal ion may not include ferric citrate. 
         [0049]    A mammalian cell can be a mammalian cell line including, but not limited to, CHO cells, human embryonic kidney cells (HEK), baby hamster kidney (BHK), mouse myeloma (NSO), and human retinal cells (PERC.6). 
         [0050]    The term “antibody” as referred to herein includes whole antibodies and any antigen binding fragment (i.e., “antigen-binding portion”) or single chains thereof. An “antibody” refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding portion thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. 
         [0051]    The term “antigen-binding portion” of an antibody (or simply “antibody portion”), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., TNFα, IL-12). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term “antigen-binding portion” of an antibody include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment (Ward et al, (1989) Nature 341:544-546), which consists of a VH or VL domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. In one embodiment of the present disclosure, the antibody fragment is selected from the group consisting of an Fab, an Fd, an Fd&#39;, a single chain Fv (scFv), an scFva, and a domain antibody (dAb). 
         [0052]    Still further, an antibody or antigen-binding portion thereof may be part of a larger immunoadhesion molecule, formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides. These other proteins or peptides can have functionalities that allow for the purification of antibodies or antigen-binding portions thereof or allow for their association with each other or other molecules. Thus, examples of such immunoadhesion molecules include use of the streptavidin core region to make a tetrameric single chain variable fragment (scFv) molecules (Kipriyanov et al. (1995) Human Antibodies and Hybridomas 6:93-101) and the use of a cysteine residue, a marker peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv molecules (Kipriyanov et al. (1994) Mol. Immunol. 31:1047-1058). Antibody portions, such as Fab and F(ab′)2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies. Moreover, antibodies, antibody portions and immunoadhesion molecules can be obtained using standard recombinant DNA techniques. 
         [0053]    Two antibody domains are “complementary” where they belong to families of structures which form cognate pairs or groups or are derived from such families and retain this feature. For example, a VH domain and a VL domain of an antibody are complementary; two VH domains are not complementary, and two VL domains are not complementary. Complementary domains may be found in other members of the immunoglobulin superfamily, such as the Va and VP (or gamma and delta) domains of the T-cell receptor. 
         [0054]    The term “domain” refers to a folded protein structure which retains its tertiary structure independently of the rest of the protein. Generally, domains are responsible for discrete functional properties of proteins, and in many cases may be added, removed or transferred to other proteins without loss of function of the remainder of the protein and/or of the domain. By single antibody variable domain is meant a folded polypeptide domain comprising sequences characteristic of antibody variable domains. It therefore includes complete antibody variable domains and modified variable domains, for example, in which one or more loops have been replaced by sequences which are not characteristic of antibody variable domains, or antibody variable domains which have been truncated or comprise N- or C-terminal extensions, as well as folded fragments of variable domains which retain at least in part the binding activity and specificity of the full-length domain. 
         [0055]    Variable domains of the present disclosure may be combined to form a group of domains; for example, complementary domains may be combined, such as VL domains being combined with VH domains. Non-complementary domains may also be combined. Domains may be combined in a number of ways, involving linkage of the domains by covalent or non-covalent means. 
         [0056]    A “dAb” or “domain antibody” refers to a single antibody variable domain (VH or VL) polypeptide that specifically binds antigen. 
         [0057]    As used herein, the term “antigen binding region” or “antigen binding site” refers to the portion(s) of an antibody molecule, or antigen binding portion thereof, which contains the amino acid residues that interact with an antigen and confers on the antibody its specificity and affinity for the antigen. 
         [0058]    The term “epitope” is meant to refer to that portion of any molecule capable of being recognized by and bound by an antibody at one or more of the antibody&#39;s antigen binding regions. In the context of the present disclosure, first and second “epitopes” are understood to be epitopes which are not the same and are not bound by a single monospecific antibody, or antigen-binding portion thereof. 
         [0059]    The term “DVD-Ig” or “dual-variable-domain-immunoglobulin” refers to a protein containing two or more antigen binding sites and is a tetravalent or multivalent binding protein. The DVD-Ig comprises two heavy chain DVD polypeptides and two light chain DVD polypeptides; each half of a DVD-Ig comprises a heavy chain DVD polypeptide, and a light chain DVD polypeptide, and two antigen binding sites. Each binding site comprises a heavy chain variable domain and a light chain variable domain with a total of six CDRs involved in antigen binding per antigen binding site. The description and generation of DVD-Ig molecules are detailed in the U.S. Pat. No. 7,612,181 B2. 
         [0060]    The phrase “recombinant antibody” refers to antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial antibody library, antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295) or antibodies prepared, expressed, created or isolated by any other means that involves splicing of particular immunoglobulin gene sequences (such as human immunoglobulin gene sequences) to other DNA sequences. Examples of recombinant antibodies include chimeric, CDR-grafted and humanized antibodies. 
         [0061]    The term “human antibody” refers to antibodies having variable and constant regions corresponding to, or derived from, human germline immunoglobulin sequences as described by, for example, Kabat et al. (See Kabat, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). The human antibodies of the present disclosure, however, may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. 
         [0062]    Recombinant human antibodies of the present disclosure have variable regions, and may also include constant regions, derived from human germline immunoglobulin sequences (See Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo. In certain embodiments, however, such recombinant antibodies are the result of selective mutagenesis or backmutation or both. 
         [0063]    The term “backmutation” refers to a process in which some or all of the somatically mutated amino acids of a human antibody are replaced with the corresponding germline residues from a homologous germline antibody sequence. The heavy and light chain sequences of a human antibody of the present disclosure are aligned separately with the germline sequences in the VBASE database to identify the sequences with the highest homology. Differences in the human antibody of the present disclosure are returned to the germline sequence by mutating defined nucleotide positions encoding such different amino acid. The role of each amino acid thus identified as candidate for backmutation should be investigated for a direct or indirect role in antigen binding and any amino acid found after mutation to affect any desirable characteristic of the human antibody should not be included in the final human antibody. To minimize the number of amino acids subject to backmutation those amino acid positions found to be different from the closest germline sequence but identical to the corresponding amino acid in a second germline sequence can remain, provided that the second germline sequence is identical and colinear to the sequence of the human antibody of the present disclosure for at least 10, up to about 12 amino acids, on both sides of the amino acid in question. Backmuation may occur at any stage of antibody optimization. 
         [0064]    The term “chimeric antibody” refers to antibodies which comprise heavy and light chain variable region sequences from one species and constant region sequences from another species, such as antibodies having murine heavy and light chain variable regions linked to human constant regions. 
         [0065]    The term “CDR-grafted antibody” refers to antibodies which comprise heavy and light chain variable region sequences from one species but in which the sequences of one or more of the CDR regions of VH and/or VL are replaced with CDR sequences of another species, such as antibodies having murine heavy and light chain variable regions in which one or more of the murine CDRs (e.g., CDR3) has been replaced with human CDR sequences. 
         [0066]    The term “humanized antibody” refers to antibodies which comprise heavy and light chain variable region sequences from a non-human species (e.g., a mouse) but in which at least a portion of the VH and/or VL sequence has been altered to be more “human-like”, i.e., more similar to human germline variable sequences. One type of humanized antibody is a CDR-grafted antibody, in which human CDR sequences are introduced into non-human VH and VL sequences to replace the corresponding nonhuman CDR sequences. The oligosaccharide structure attached to the peptide chain is known as a “glycan” molecule. The glycan structures found in naturally occurring glycopeptides are typically divided into two classes, “N-linked glycans” or N-linked oligosaccharides” and “0-linked glycans” or O-linked oligosaccharides”. 
         [0067]    Peptides comprising “0-linked glycans” have a saccharide attached to the hydroxy oxygen of serine, threonine, tyrosine, hydroxylysine, and or hydroxyproline residue in the primary protein. 
         [0068]    Peptides expressed in eukaryotic cells typically comprise N-glycans. “N-glycans” are N-glycosylated at an amide nitrogen of an asparagine or an arginine residue in a protein via an N-acetylglucosamine residue. These “N-linked glycosylation sites” occur in the peptide primary structure containing, for example, the amino acid sequence asparagine-X-serine/threonine, where X is any amino acid residue except proline and aspartic acid. 
         [0069]    All N-linked oligosaccharides have a common “pentasaccharide core” of Man 3 GlcNAc 2 . (“Man” refers to mannose; “Glc” refers to glucose; “NAc” refers to N-acetyl; and “GlcNAc” refers to N-acetylglucosamine). The pentasaccharide core is also referred to as the “trimannose core” or the “paucimannose core.” (see  FIG. 1 ) 
         [0070]    N-glycans differ with respect to the presence of, and/or in the number of branches (also called “antennae”) comprising peripheral sugars such as N-acetylglucosamine, galactose, N-acetylgalactosamine, N-acetylneuraminic acid, fucose and sialic acid that are added to the Man 3 GlcNAc 2  core structure. Optionally, this structure may also contain a core fucose molecule and/or a xylose molecule. For a review of standard glycobiology nomenclature see, Essentials of Glycobiology Varki et al. eds., 1999, CSHL Press, the contents of which are incorporated herein by reference. N-glycans are classified according to their branched constituents (e.g., oligomannose-type, complex, or hybrid). An “oligomannose-type” or “high mannose-type” N-glycan has five or more mannose residues. 
         [0071]    A “complex-type” N-glycan typically has at least one GlcNAc attached to the 1,3 mannose arm and at least one GlcNAc attached to the 1,6 mannose arm of a pentasaccharide core. Complex-type N-glycans may also have galactose (“Gal”) or N-acetylgalactosamine residues that are optionally modified with sialic acid or derivatives, e.g., N-acetyl neuraminic acid. Complex-type N-glycans may also have intrachain substitutions comprising “bisecting” GlcNAc, and core fucose (“Fuc”). Complex N-glycans may also have multiple antennae on the pentasaccharide core and are, therefore, also referred to as “multiple antennary-type glycans.” 
         [0072]    A “hybrid-type” N-glycan comprises at least one GlcNAc on the terminal of the 1,3 mannose arm of the pentasaccharide core and zero or more mannoses on the 1,6 mannose arm of the trimannose core. 
         [0073]    NGA2F +NGA2F-GlcNAc refers to the agalactosyl fucosylated biantennary oligosaccharides. 
         [0074]    NGA2F has the structure: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0075]    NA1F and NA2F refer to the fraction of galactose-containing fucosylated biantennary oligosaccharides. 
         [0076]    NGA2F glycan is found on many mammalian glycoproteins including human IgG and is a substructure of bi-antennary N-linked oligosaccharides such as A2F, A1F, and NA2F. 
         [0077]    The term “glycoform” denotes a type of polypeptide with a specific type and distribution of polysaccharides attached to, i.e. two polypeptides would be of the same glycoform if they comprise glycans with the same number, kind, and sequence of monosaccharides, i.e. have the same “glycosylation profile.” Glycoproteins often consist of a number of different glycoforms. 
         [0078]    The “glycosylation profile” refers to the properties of the glycans of a glycosylated polypeptide. These properties include, but are not limited to, the glycosylation sites, or the glycosylation site occupancy, or the identity, structure, composition or quantity of the glycan attached to a recombinantly expressed polypeptide. 
       EXAMPLES 
       [0079]    The following examples describe experiments relating to methods disclosed herein. 
       Example 1 
     Shake Flask Culture of CHO cell Lines Expressing Humanized Monoclonal Antibodies in the Presence of Different Amounts of Ferric Nitrate 
       [0080]    To test the effect of metal ions of protein glycosylation, recombinant Chinese Hamster Ovary (CHO) cell lines (CHO DUX-B 11 origin based on a DHFR (dihydrofolate reductase) expression system) expressing two different humanized monoclonal antibodies (Antibody 1, Antibody 2) or a dual variable domain immunoglobulin (DVD-Ig) were shake flask cultured in chemically defined basal medium (CDBM) and chemically defined feed medium (CDFM) supplemented with selected iron compounds. The cell lines were serially expanded through separate seed train inoculums to generate enough cells for inoculation. All supplemented cultures were performed in triplicate unless otherwise indicated. Supplements and concentrations utilized during the cultures were slightly different between the 2 different cell lines as shown in Table 1. 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Summary of cell culture process conditions &amp; iron supplementation details 
               
             
          
           
               
                   
                 Antibody 1 
                 Antibody 2 
                 DVD-Ig 
               
               
                   
               
             
          
           
               
                 Culture Vessel 
                 500 mL 
                 500 mL 
                 500 mL 
                 500 mL 
                 500 mL 
                 500 mL 
                 500 mL 
                 500 mL 
               
               
                   
                 shake flask 
                 shake flask 
                 shake flask 
                 shake flask 
                 shake flask 
                 shake flask 
                 shake flask 
                 shake flask 
               
               
                 Culture Mode 
                 Fedbatch 
                 Fedbatch 
                 Fedbatch 
                 Fedbatch 
                 Fedbatch 
                 Fedbatch 
                 Fedbatch 
                 Fedbatch 
               
               
                 Initial Culture 
                 36 
                 36 
                 36 
                 36 
                 36 
                 36 
                 36 
                 36 
               
               
                 Temperature (° C.) 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Iron Supplements 
                 Ferric 
                 Ferric 
                 Ferrous 
                 Ferric 
                 Ferric 
                 Ferric 
                 Ferric 
                 Sodium 
               
               
                 Evaluated 
                 Nitrate 
                 Citrate 
                 Sulfate 
                 Nitrate 
                 Citrate 
                 Nitrate 
                 Citrate 
                 Citrate 
               
               
                 Supplement 
                 0.1, 0.5, 1, 
                 0.1, 0.5, 1 
                 1 
                 0.1 
                 0.1, 1 
                 0.1, 1 
                 0.1, 1 
                 0.5 
               
               
                 Concentrations 
                 2, 5 
                   
                   
                   
                   
                   
                   
                   
               
               
                 (mM) a   
               
               
                   
               
               
                   a Higher concentrations were evaluated with Antibody 1, but resulted in significantly detrimental cell culture performance: Ferric Nitrate (10 mM), Ferrous Sulfate (3, 5, 10 mM). 
               
             
          
         
       
     
         [0081]    The cell line producing Antibody 1 (also called ABBV-696) was cultured in shake flasks in fedbatch mode after an abbreviated seed train in two independent experiments. Ferric nitrate was supplemented into chemically-defined basal and feed media at concentrations of 0.1, 1.0, and 5.0 mM (Experiment 1) and 0.1, 0.5, 1.0, and 2.0 mM (Experiment 2), and compared to an unsupplemented control condition. 
         [0082]    Viable cell density (VCD) and cell viability values were measured through trypan blue exclusion via ViCell automated cell counters (Beckman Coulter, Fullerton, Calif.), glucose and lactate values were measured with a ABL-805 (Radiometer Medical, Denmark) blood gas analyzer. 
         [0083]    Antibody titers were measured from crude cell culture harvests on a Poros A™ (Life Technologies, Carlsbad, Calif.) Protein A affinity column using an Agilent (Santa Clara, Calif.) 1200 Series HPLC, or equivalent, operating with a low pH, step elution gradient with detection at 280 nm. Absolute concentrations were assigned with respect to reference standard calibration curves. Purified antibodies subjected to additional analytical characterization were purified using MabSelect™ Protein A (GE Healthcare, Piscataway, N.J.) using a low pH, step elution gradient, followed by buffer exchange using Corning Life Sciences (Tewksbury, Mass.) Spin Concentrator X UF columns according to the manufacturers&#39; recommended procedures. 
         [0084]    The viable cell density, viability, and harvest titer results are shown in  FIG. 2 . At these particular concentrations, ferric nitrate generally facilitated an increase in VCD, with the exception of the highest concentration (5.0 mM) supporting the lowest peak VCD. Viability results were comparable to the control, i.e. unsupplemented conditions, with the exception of the 5.0 mM condition, which facilitated a drop in cell viability that was larger, and earlier in the culture, compared to control conditions. 
         [0085]    Harvest titer results demonstrated that below 2.0 mM ferric nitrate did not adversely impact titer, but a concentration of ferric nitrate of 2.0 and 5.0 mM lowered the harvest titer significantly. Collectively, these results suggest that there are concentrations of ferric nitrate which minimize the drop in viable cell density, while simultaneously ensuring a comparable cell viability and harvest titer profile. 
         [0086]    The glycosylation profile of the recombinantly expressed proteins was then determined using standard procedures. Approximately 200 μg of Protein A purified samples from Cell Lines 1 and 2 were then treated with N-glycanase at 37° C. overnight to remove the N-glycans from the protein. The protein was precipitated and the supernatant was taken for subsequent chemical derivatization of the reducing end of the released glycans with 2-aminobenzamide (2-AB) dye. Following the derivatization step, the excess labeled was removed using clean up cartridges and the samples were analyzed using normal phase HPLC with fluorescent detection. Mobile phase A was 100% acetonitrile and mobile phase B was 50 mM ammonium formate pH 4.4. The glycan were eluted from a polyamide column (Prozyme, Hayward, Calif.) using a shallow gradient. The labeled glycans were detected using a fluorescence detector with an excitation wavelength of 330 nm and an emission wavelength of 420 nm. 
         [0087]    The N-glycan oligosaccharide results were quite different between the variously supplemented cultures and the unsupplemented control as shown in  FIG. 3 . Data in each experiment for each oligosaccharide was normalized to the level observed in the 1.0 mM condition for that particular experiment. The addition of iron shifted the distribution toward increased galactosylation, reducing the total NGA2F (NGA2F and NGA2F-GlcNAc) species thereby increasing the total NA1F (NA1F plus NA1F-GlcNAc) and NA2F species. A concentration-dependent response was observed below 1.0 mM ferric nitrate. The impact of iron on galactosylation was still effective but seemed to reach a plateau at 2.0 mM ferric nitrate. The total NGA2F species was reduced by as much as 25% with 7-fold increases in total NA1F and 50-fold increases in NA2F species. The supplementation of ferric nitrate into the cell culture media is capable of significantly influencing the overall protein galactosylation pathway, and for some commercial protein therapeutics this is the desired product quality distribution. 
       Example 2 
     Shake Flask Cell Culture in the Presence of Additional Iron Supplements 
       [0088]    Additional shake flask experiments were performed with Cell Line 1 to evaluate the impact of other iron compounds, namely ferric citrate and ferrous sulfate. Concentrations of the respective iron compounds were chosen so as to minimize any potential adverse impacts on cell growth and productivity, but still facilitate a measurable impact on the glycoform profile. Both an unsupplemented control and cultures supplemented with ferric nitrate were included in this study. Cell culture process performance indicators were monitored and measured throughout the respective cultures. Viable cell density, cell viability, harvest titer, and harvest N-glycan oligosaccharide data was measured and reported. 
         [0089]      FIG. 4  highlights the cell culture results observed through the use of iron supplementation into the basal medium. All iron supplemented cultures showed a higher viable cell density compared to the unsupplemented control. The results indicate a significant increase in peak VCD from 9×10 6  cells/mL for the control versus 12-14×10 6  cells/mL for the iron supplemented cultures. At the end of the culture period, the final titer ratio was similar to the control suggesting a slight drop in overall antibody productivity upon inclusion of these particular concentrations of iron, consistent with the results observed from the previous shake flask cultures. 
         [0090]    After Protein A purification, the N-glycan glycoform profile was measured with the results highlighted in  FIG. 5 . Similar to the initial ferric nitrate shake flask results, there was a significant increase in overall galactosylation. The total NGA2F species was reduced 15-30% with the least impact from ferrous sulfate and the most significant impact from the ferric nitrate or ferric citrate at higher concentrations. Corresponding increases in the total NA1F and NA2F species were observed as well. Ferrous sulfate supplementation had modest 3-fold increases in both total NA1F and NA2F species. Ferric nitrate supplementation provided 5-fold increases in total NA1F with 7- to 9-fold increases in NA2F. Ferric citrate supplementation provided very similar results to ferric nitrate with a concentration dependence observed below 1.0 mM as previously observed with ferric nitrate. The absolute value of the fold increase in NA2F varied between experiments and relies heavily on quantification of the very small amount of this species present in the control culture. Nonetheless, significant increases were observed in NA2F with the addition of iron to the basal medium. 
       Example 3 
     Evaluation of Alternative Cell Lines for the Impact of Iron Supplementation on the Resulting Protein Glycosylation Profiles 
       [0091]    The cell line expressing Antibody 2 (also called ABT-806) was evaluated in shake flask culture in fedbatch mode. Ferric citrate or ferric nitrate was supplemented into the basal medium at various concentrations to evaluate the resulting impact on cell culture performance, as well as the protein glycosylation profile. The unsupplemented control was performed in duplicate. The viable cell density, viability, and harvest titer results are shown in  FIGS. 6 and 7 . Ferric citrate supplementation at either 0.1 or 1.0 mM improved overall cell growth with higher peak viable cell density (increased by 4×10 6  cells/mL) while ferric nitrate at 0.1 mM had very little impact when compared to the unsupplemented control. Contrary to Antibody 1, all iron-supplemented conditions had a positive impact to the titer for Antibody 2. 
         [0092]    Similar to Antibody 1, there was a very significant impact on the protein glycosylation profiles of harvest samples across all iron concentrations tested ( FIG. 7 ) and the modulation of the protein glycosylation was concentration-dependent. The total NGA2F species declined by approximately 20%, similar to Antibody 1. The total NA1F species increased 4- to 6-fold compared to the unsupplemented control while the NA2F species increased 7- to 10-fold. Hence, as with Antibody 1, there was a very significant impact on the N-glycan glycoform profile, and the individual N-glycans which increased or decreased upon iron supplementation was consistent between both cell lines. 
         [0093]    To confirm that the observed effect on protein glycosylation was not unique to recombinantly expressed monoclonal antibodies, the impact of iron supplementation was evaluated with a cell line expressing a DVD-Ig molecule (denoted DVD 1, also called ABBV-257). The DVD-Ig cell line was cultured in shake flasks with ferric nitrate, ferric citrate, or sodium citrate supplementation at various concentrations. Sodium citrate was included to provide additional evidence that the impact to the oligosaccharide profile with the prior experiments was a result of the iron addition. All conditions were performed in triplicate.  FIG. 8  shows the cell culture viable cell density and viability performance for one representative from each experimental condition. The average harvest titer for the triplicates and the relative oligosaccharide profile results are in  FIG. 9 . 
         [0094]    Iron supplementation had minimal impact to the cell culture growth performance and harvest titer. However, the impact to the galactosylated species is similar to that observed for both Antibody 1 and 2 with iron supplementation. Increased concentration of both ferric nitrate and ferric citrate supplemented to the culture provided increased galactosylation as observed previously with both antibody-expressing cell lines. The total NGA2F species declined by approximately 20%, while the total NA1F species increased 3- to 9-fold, and the NA2F species increased 50- to 100-fold compared to the unsupplemented control. Sodium citrate had no effect on the oligosaccharide profile compared to the unsupplemented control. 
       Example 4 
     Evaluation of Impact of Iron Supplementation in Feed Medium 
       [0095]    Prior examples included iron supplementation via the culture basal medium, thus introducing iron to the cell culture throughout the duration of the shake flask experiment. By introducing iron to the culture via the feed medium, exposure to iron would be more gradual over the length of the culture. The impact to Antibody 1 was evaluated in triplicate shake flasks as described previously. Similar to prior results with Antibody 1, cell growth was enhanced with no improvement to titer.  FIG. 10  shows the impact to the relative oligosaccharide profile and that regardless of where the iron is introduced to the cell culture (basal versus feed medium), the galactosylation of the antibody increases as observed previously. 
       Example 5 
     Evaluation of Impact of Iron Supplementation in Bioreactors 
       [0096]    Prior experiments included iron supplementation in shake flask cultures. Experiments with the cell line expressing Antibody 1 and with a cell line expressing a different DVD-Ig (denoted DVD 2, also called ABT-122) than discussed previously were carried out to confirm the impact from iron supplements would be observed in bioreactors. Operating conditions were similar to shake flask experiments, but now included pH control at pH 6.90 and dissolved oxygen control at a setpoint of 40% air saturation. Antibody 1 bioreactors were run in triplicate with the exception of the 0.08 mM FC condition. DVD 2 bioreactors were run in duplicate. In  FIG. 11 , two independent experiments with the cell line expressing Antibody 1 show the dose-dependence effect of ferric citrate on the increase in total NA1F and NA2F species in bioreactors.  FIG. 12  shows the similar effect with the cell line expressing a DVD-Ig molecule, however the impact to this DVD-Ig molecule is limited to the total NA1F species as the NA2F species is not observed when this cell line is cultured in chemically defined medium. The same cell line expressing this DVD-Ig molecule grown in a complex cell culture medium (containing plant-derived hydrolysates) contains a significantly different oligosaccharide profile and iron supplementation has little impact on altering this profile. 
       Example 6 
     Evaluation of Impact of Alternative Metal Ions on Oligosaccharide Profile 
       [0097]    Given the observations presented here with both ferric and ferrous forms of iron, and studies performed previously with manganese, additional metal ions listed in the table below were explored for their impact on the glycosylation pathway. All evaluations were performed in triplicate in shake flasks with the cell line expressing Antibody 1. 
         [0000]    
       
         
               
               
               
             
           
               
                   
                   
               
               
                   
                 Metal Salt 
                 Concentration Evaluated 
               
               
                   
                   
               
             
             
               
                   
                 Cupric Chloride 
                 0.01, 0.2 mM 
               
               
                   
                 Cupric Sulfate 
                 0.01, 0.2 mM 
               
               
                   
                 Zinc Chloride 
                 0.02, 0.5 mM 
               
               
                   
                 Zinc Sulfate 
                 0.05, 0.5 mM 
               
               
                   
                 Chromium Chloride 
                 0.001, 0.01 mM  
               
               
                   
                   
               
             
          
         
       
     
         [0098]    As seen in  FIG. 13 , these additional metal ions were unsuccessful at increasing the galactosylation for the oligosaccharide profile for Antibody 1. In fact, copper appeared to have a slightly inhibitory effect on the galactosylation. This figure only depicts the lower concentrations evaluated, but similar results were observed at the higher concentrations listed in the table above. 
       Example 7 
     Evaluation of Iron Transport Limitation 
       [0099]    In all experiments with all cell lines evaluated, the galactosylation reaches a plateau between 0.5 and 1.0 mM iron supplemented. Despite the increase in iron with minimal impact to culture performance, the total NA1F and NA2F levels do not increase. One potential explanation is the inability for iron to effectively enter the cell. Another possible explanation is the lack of galactose to incorporate into the oligosaccharide chain. The next set of experiments explored if iron transport into the cell or the galactose availability were the limiting factors. Recombinant transferrin (rTf) was added to triplicate shake flask cultures with the cell line expressing Antibody 1 with varying concentrations of ferric citrate. An independent experiment was performed with the same cell line to also determine if galactose supplementation in addition to iron and transferrin would enhance the galactosylation with triplicate shake flasks for each condition. As seen in  FIG. 14 , no impact was observed to the galactosylation with the addition of transferrin and without galactose, the typical profile for the oligosaccharide species is similar to results shown previously without transferrin. However, when galactose is supplemented with iron, increased galactosylation is observed overall, even though the plateau between 0.5 and 1.0 mM ferric citrate continues to persist. 
       Example 8 
     Evaluation of Iron Synergy with Manganese and Galactose on the Oligosaccharide Profile 
       [0100]    Manganese has been reported in several studies to increase the galactosylation of recombinant proteins with synergistic effects observed when combined with galactose supplementation. Shake flask experiments were conducted in triplicate with the cell line expressing Antibody 1 to determine if the plateau observed between 0.5 and 1.0 mM ferric citrate could be overcome by including these additional factors known to improve galactosylation. Ferric citrate was supplemented to the cultures via the CDBM as described previously. Manganese and galactose were supplemented into both the CDBM and CDFM at the concentrations listed in  FIG. 15 . There is no synergistic effect with ferric citrate and manganese observed. Manganese in combination with galactose is effective at altering the galactosylation for Antibody 1, however ferric citrate with galactose is more effective, and a dose-dependence is observed with galactose supplementation. 
         [0101]    Any patent, patent application, publication, or other disclosure material identified in the specification is hereby incorporated by reference herein in its entirety. Any material, or portion thereof, that is said to be incorporated by reference herein, but which conflicts with existing definitions, statements, or other disclosure material set forth herein is only incorporated to the extent that no conflict arises between that incorporated material and the present disclosure material. 
       Abbreviations 
       [0000]    
       
         DVD-Ig Dual variable domain immunoglobulin 
         EDTA Ethylenediaminetetraacetic acid 
         FC Ferric citrate 
         FN Ferric nitrate 
         FS Ferrous sulfate 
         Fuc Fucose 
         Gal Galactose 
         GlcNAc N-acetylglucosamine 
         Man Mannose 
         PK Pharmacokinetics 
         SC Sodium citrate 
         VCD Viable cell density