Patent Publication Number: US-2017349655-A1

Title: Methods to modulate lysine variant distribution

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     The present application is a continuation application of U.S. patent application Ser. No. 14/923,958, filed on Oct. 27, 2015, allowed, which is a divisional application of U.S. patent application Ser. No. 13/830,976, filed on Mar. 14, 2013, issued as U.S. Pat. No. 9,181,572, which claims priority to U.S. Provisional Application Nos. 61/636,469, filed on Apr. 20, 2012 and 61/696,207, filed on Sep. 2, 2012. The entire disclosures of each of the foregoing applications are incorporated herein by reference. 
    
    
     1. INTRODUCTION 
     The instant invention relates to the field of recombinant protein production and purification of recombinant proteins, and in particular to compositions and processes for controlling the relative distribution of C-terminal lysine variants of proteins (e.g., antibodies) expressed by recombinant host cells. In certain aspects of the invention, controlling the relative distribution of the different C-terminal lysine variants of protein expressed by recombinant host cells is achieved by modifying conditions employed in culturing the cells. 
     2. BACKGROUND OF THE INVENTION 
     Large-scale production of proteins for biopharmaceutical applications involves the use of cell cultures that are known to produce proteins exhibiting varying levels of heterogeneity. One potential source of heterogeneity involves C-terminal lysine residues, such as those typically found on the heavy chains of antibody molecules. C terminal lysines can be lost, so that individual antibodies in a production batch can vary at their C terminus as to whether a lysine residue is present. C-terminal lysines can be potentially present on both the heavy chains of an antibody (Lys 2), on either one of the heavy chains (Lys 1), or neither of them (Lys 0). Since lysine can carry a positive charge, antibodies lacking the basic C-terminal lysine(s) differ in the charge state from the ones that contain the lysine, so that the distribution of lysine variants (% Lys 0, % Lys 1, % Lys 2 of the total Lysine Sum) can be detected by ion-exchange chromatographic methods, such as analysis employing a ProPac WCX-10 Weak Cation-Exchange column for the high-resolution separation of protein isoforms (Dionex, CA), and subsequently quantified. 
     The C-terminal lysine heterogeneity is commonly observed in biopharmaceutical antibodies and proteins. For instance, in the process of manufacture of Remicade (Infliximab), the heterogeneity during the fermentation was approximately 20% (Lys 0 and Lys 1) and 80% (Lys 2) (U.S. Patent Application publication U.S. 2010/0297697A1). Other examples are detailed in a review article on lysine variants (Harris R, “Processing of C-terminal lysine and arginine residues of proteins isolated from mammalian cell culture” Journal of Chromatography A, 705 129-134 (1995)). 
     The present study is directed to cell culture methods to modulate a product quality attribute of recombinant proteins. Specifically, the invention provides methods for influencing the relative distribution of the different C-terminal lysine variants of the product antibody obtained from the cell culture harvest. C-terminal lysine can be potentially present on both the heavy chains of the antibody (Lys 2), on either one of them (Lys 1) or neither of them (Lys 0). Since, antibodies lacking the basic C-terminal lysine(s) differ in the charge state from the ones that contain the lysine, the distribution of lysine variants (% Lys 0, % Lys 1, % Lys 2 of the total Lysine Sum) can be detected by ion-exchange chromatographic methods such as WCX-10 and subsequently quantified ( FIG. 1 ). 3. 
     SUMMARY OF THE INVENTION 
     The present invention is directed to compositions and methods that control (modulate or limit) the extent of lysine variant heterogeneity in a population of proteins produced by cell culture. In certain embodiments, control is exerted over the distribution or amount of lysine variants of protein produced by cell culture. In certain embodiments, the protein is an antibody. In certain embodiments such control is exerted in order to facilitate consistency and reproducibly in obtaining a particular lysine variant distribution, e.g., in the production of a therapeutic antibody. The variant distribution in such contexts may change due to a number of reasons, e.g., processes changes such altering media usage, and the methods described herein allow for the production of compositions of the desired lysine variant specification. 
     Heterogeneity of C-terminal lysine variants, as that term is used herein, can refer to (i) the presence of subspecies of protein molecules which differ from one another based on whether a full or partial complement of C-terminal lysines are present (or whether C-terminal lysine(s) is (are) absent); and/or (ii) to the relative proportion or amount of said subspecies in the population. In certain embodiments, heterogeneity can arise from both a difference in the amount of C-terminal lysine variants in the population of proteins and the subspecies of C-terminal lysine variants present in the population (species) of protein. 
     In certain embodiments, the protein is an antibody, and control is exerted over the distribution or amount of C-terminal lysine variants to decrease the relative amount of a Lys 0 lysine variant in a population of proteins produced by cell culture, wherein the Lys 0 lysine variant comprises an antibody with heavy chains that do not comprise a C-terminal lysine. In certain embodiments, control is exerted over the distribution or amount of C-terminal lysine variants to decrease the amount of a Lys 1 lysine variant in a population of proteins produced by cell culture, wherein the Lys 1 lysine variant comprises an antibody with one heavy chain that comprises a C-terminal lysine. In certain embodiments, control is exerted over the distribution or amount of C-terminal lysine variants to decrease the amount of a Lys 2 lysine variant in a population of proteins produced by cell culture, wherein the Lys 2 lysine variant comprises an antibody wherein both heavy chains comprise a C-terminal lysine. In certain embodiments, control is exerted over the distribution or amount of C-terminal lysine variants to increase the amount of a Lys 0 lysine variant in a population of proteins produced by cell culture. In certain embodiments, control is exerted over the distribution or amount of C-terminal lysine variants to increase the amount of a Lys 1 lysine variant in a population of proteins produced by cell culture. In certain embodiments, control is exerted over the distribution or amount of C-terminal lysine variants to increase the amount of a Lys 2 lysine variant in a population of proteins produced by cell culture. 
     In certain embodiments, control over the distribution or amount of C-terminal lysine variants produced by cell culture is exerted by employing certain media components during production of a protein, for example, an antibody, of interest. In certain embodiments, control over the distribution or amount of C-terminal lysine variants produced by cell culture is exerted by supplementing the media of cells expressing the protein of interest with one or more amino acids. In certain embodiments, one or more of the amino acids belong to a group of basic amino acids. In certain embodiments, the one or more amino acids is arginine, lysine, histidine, or combinations thereof, including combinations of arginine and/or lysine with ornithine. In certain embodiments, supplementing the media of cells expressing the protein of interest with one or more amino acids reduces the relative amount of a Lys 0 lysine variant, and increases the relative amount of a Lys 1 and/or Lys 2 lysine variant in the cell culture or a composition comprising the protein or antibody purified from the cell culture. In certain embodiments, these amino acids may be supplemented as dipeptides or tri-peptides of different combinations for lysine variant modulation 
     In certain embodiments, control over the distribution or amount of C-terminal lysine variants produced by cell culture is exerted by controlling the amount of Zinc present in the media employed in culturing the cells expressing the protein of interest. In certain embodiments, supplementing the media of cells expressing the protein of interest with Zinc to an overall concentration of less than about 10 μM reduces the relative amount of a Lys 0 lysine variant, and increases the relative amount of a Lys 1 and/or Lys 2 lysine variant in the cell culture or a composition comprising the protein or antibody purified from the cell culture. In certain embodiments, modulation of concentration of zinc in combination with that of the basic amino acids is used to modulate the lysine variant distribution. 
     In certain embodiments, control over the distribution or amount of C-terminal lysine variants produced by cell culture is exerted by adjusting the pH, and/or temperature of a cell culture expressing a protein or antibody of interest. In certain embodiments, increasing the pH of the cell culture expressing the protein or antibody of interest reduces the amount of a Lys 0 lysine variant, and increases the amount of a Lys 1 and/or Lys 2 lysine variant in the cell culture, or a composition comprising the protein or antibody purified from the cell culture, compared to a control cell culture at a lower pH. In certain embodiments, the final pH of the cell culture is adjusted to a pH of about 6.7 to about 7.1. In certain embodiments, the temperature of the cell culture is adjusted to a temperature of about 31 to about 37° C. In certain embodiments, decreasing the temperature of the cell culture expressing the protein or antibody of interest reduces the amount of a Lys 0 lysine variant, and increases the amount of a Lys 1 and/or Lys 2 lysine variant in the cell culture, or a composition comprising the protein or antibody purified from the cell culture, compared to a control cell culture at a higher temperature. 
     In certain embodiments, the methods of culturing cells expressing a protein of interest, such as an antibody or antigen-binding portion thereof, or purifying such protein from a sample, as described herein, modulates the distribution, reduces the amount, or increases the amount of lysine variants present in the resulting composition. In certain embodiments, the resulting composition has a reduced amount of Lys 0 lysine variant, and an increased amount of Lys 1 and Lys 2 lysine variants compare to a composition that was not prepared according to the methods of the present application. In one aspect, the sample comprises a cell harvest wherein the cell line is employed to produce specific proteins of the present invention. In a particular aspect, the sample is prepared from a cell line used to produce anti-TNF-α antibodies. 
     In certain embodiments, control over the subspecies and/or amount of C-terminal lysine variants in the protein compositions described herein is exerted by employing one or more of the foregoing methods during the production and purification of the desired proteins, such as antibodies or antigen-binding portions thereof, described herein. 
     The purity/heterogeneity of the proteins of interest in the resultant sample product can be analyzed using methods well known to those skilled in the art, e.g., weak cation exchange chromatography (WCX), capillary isoelectric focusing (cIEF), size-exclusion chromatography, Poros™ A HPLC Assay, Host Cell Protein ELISA, Protein A ELISA, and western blot analysis. 
     In yet another embodiment, the invention is directed to one or more pharmaceutical compositions comprising an isolated protein, such as an antibody or antigen-binding portion thereof, and an acceptable carrier. In another aspect, the compositions further comprise one or more pharmaceutical agents. 
    
    
     
       4. BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a representation of the lysine variants in a sample WCX-10 chromatogram and quantification scheme of each of the variants. 
         FIG. 2  depicts the effect of zinc modulation in adalimumab producing cell line 1, media 1 on viable cell density (n=2). 
         FIG. 3  depicts the effect of zinc modulation in adalimumab producing cell line 1, media 1 on viability (n=2). 
         FIG. 4  depicts the effect of zinc modulation in adalimumab producing cell line 1, media 1 on harvest titer (n=2) 
         FIG. 5  depicts the effect of zinc modulation in adalimumab producing cell line 1, media 1 on day 10 WCX 10 profile relative lysine distribution (n=2). 
         FIG. 6  depicts the effect of zinc modulation in adalimumab producing cell line 1, media 1 on viable cell density (n=2). 
         FIG. 7  depicts the effect of zinc modulation in adalimumab producing cell line 1, media 1 on viability (n=2). 
         FIG. 8  depicts the effect of zinc modulation in adalimumab producing cell line 1, media 1 on harvest titer (n=2). 
         FIG. 9  depicts the effect of zinc modulation in adalimumab producing cell line 1, media 1 on day 10 WCX 10 profile relative lysine distribution (n=2). 
         FIG. 10  depicts the effect of total arginine concentration in adalimumab producing cell line 2, media 1 on viable cell density (n=2). 
         FIG. 11  depicts the effect of total arginine concentration in adalimumab producing cell line 2, media 1 on viability (n=2). 
         FIG. 12  depicts the effect of total arginine concentration in adalimumab producing cell line 2, media 1 on harvest titer (n=2). 
         FIG. 13  depicts the effect of total arginine concentration in adalimumab producing cell line 2, media 1 on day 10 on WCX 10 profile relative lysine distribution (n=2). 
         FIG. 14  depicts the effect of total arginine concentration in adalimumab producing cell line 2, media 1 on day 12 on WCX 10 profile relative lysine distribution (n=2). 
         FIG. 15  depicts the effect of total arginine concentration in adalimumab producing cell line 3, media 1 on viable cell density (n=2). 
         FIG. 16  depicts the effect of total arginine concentration in adalimumab producing cell line 3, media 1 on viability (n=2). 
         FIG. 17  depicts the effect of total arginine concentration in adalimumab producing cell line 3, media 1 on harvest titer (n=2). 
         FIG. 18  depicts the effect of total arginine concentration in adalimumab producing cell line 3, media 1 on WCX 10 profile relative lysine distribution (n=2). 
         FIG. 19  depicts the effect of total arginine concentration in adalimumab producing cell line 1, media 1 on WCX 10 profile relative lysine distribution (n=2). 
         FIG. 20  depicts the effect of arginine addition to adalimumab producing cell line 1, media 2 on day 11 on WCX-10 profile relative lysine distribution (n=2). 
         FIG. 21  depicts the effect of arginine addition to adalimumab producing cell line 2, media 3 on WCX-10 profile relative lysine distribution (n=2). 
         FIG. 22  depicts the effect of total arginine concentration in mAB1 producing cell line on WCX-10 profile relative lysine distribution (n=1). 
         FIG. 23  depicts the effect of total arginine concentration in mAB2 producing cell line on WCX-10 profile relative lysine distribution (n=2). 
         FIG. 24  depicts the effect of total lysine concentration in adalimumab producing cell line 2, media 1 on viable cell density (n=2). 
         FIG. 25  depicts the effect of total lysine concentration in adalimumab producing cell line 2, media 1 on viability (n=2). 
         FIG. 26  depicts the effect of total lysine concentration in adalimumab producing cell line 2, media 1 on harvest titer (n=2). 
         FIG. 27  depicts the effect of total lysine concentration in adalimumab producing cell line 2, media 1 on WCX 10 profile relative lysine distribution (n=2). 
         FIG. 28  depicts the effect of total lysine concentration in adalimumab producing cell line 3, media 1 on viable cell density (n=2). 
         FIG. 29  depicts the effect of total lysine concentration in adalimumab producing cell line 3, media 1 on viability (n=2). 
         FIG. 30  depicts the effect of total lysine concentration in adalimumab producing cell line 3, media 1 on harvest titer (n=2). 
         FIG. 31  depicts the effect of total lysine concentration in adalimumab producing cell line 3, media 1 on WCX 10 profile relative lysine distribution (n=2). 
         FIG. 32  depicts the effect of total lysine concentration in adalimumab producing cell line 1, media 1 on WCX 10 profile relative lysine distribution (n=2). 
         FIG. 33  depicts the effect of lysine addition to adalimumab producing cell line 1, media 2 on WCX-10 profile relative lysine distribution (n=2). 
         FIG. 34  depicts the effect of lysine addition to adalimumab producing cell line 2, media 3 on WCX-10 profile relative lysine distribution (n=2). 
         FIG. 35  depicts the effect of total lysine concentration in mAB1 producing cell line on WCX-10 profile relative lysine distribution (n=1). 
         FIG. 36  depicts the effect of total lysine concentration in mAB2 producing cell line on WCX-10 profile relative lysine distribution (n=2). 
         FIG. 37  depicts the effect of total histidine concentration in adalimumab producing cell line 2, media 1 on viable cell density (n=2). 
         FIG. 38  depicts the effect of total histidine concentration in adalimumab producing cell line 2, media 1 on viability (n=2). 
         FIG. 39  depicts the effect of total histidine concentration in adalimumab producing cell line 2, media 1 on harvest titer (n=2). 
         FIG. 40  depicts the effect of total histidine concentration in adalimumab producing cell line 2, media 1 on WCX 10 profile relative lysine distribution (n=2). 
         FIG. 41  depicts the effect of total histidine concentration in adalimumab producing cell line 3, media 1 on viable cell density (n=2). 
         FIG. 42  depicts the effect of total histidine concentration in adalimumab producing cell line 3, media 1 on viability (n=2). 
         FIG. 43  depicts the effect of total histidine concentration in adalimumab producing cell line 3, media 1 on harvest titer (n=2). 
         FIG. 44  depicts the effect of total histidine concentration in adalimumab producing cell line 3, media 1 on WCX 10 profile relative lysine distribution (n=2). 
         FIG. 45  depicts the effect of total histidine concentration in adalimumab producing cell line 1, media 1 on WCX 10 profile relative lysine distribution (n=2). 
         FIG. 46  depicts the effect of histidine addition to adalimumab producing cell line 1, media 2 on WCX-10 profile relative lysine distribution (n=2). 
         FIG. 47  depicts the effect of histidine addition to adalimumab producing cell line 2, media 3 on WCX-10 profile relative lysine distribution (n=2). 
         FIG. 48  depicts the effect of total histidine concentration in mAB1 producing cell line on WCX-10 profile relative lysine distribution (n=1). 
         FIG. 49  depicts the effect of total histidine concentration in mAB2 producing cell line on WCX-10 profile relative lysine distribution (n=2). 
         FIG. 50  depicts the effect of concentration modulation of multiple amino acids to adalimumab producing cell line 1, media 1 on viable cell density (n=2). 
         FIG. 51  depicts the effect of concentration modulation of multiple amino acids to adalimumab producing cell line 1, media 1 on viability. 
         FIG. 52  depicts the effect of concentration modulation of multiple amino acids to adalimumab producing cell line 1, media 1 on harvest titer. 
         FIG. 53  depicts the effect of concentration modulation of multiple amino acids to adalimumab producing cell line 1, media 1 on WCX 10 profile relative lysine distribution. 
         FIG. 54  depicts the effect of concentration modulation of multiple amino acids to adalimumab producing cell line 3, media 1 on viable cell density (n=2). 
         FIG. 55  depicts the effect of concentration modulation of multiple amino acids to adalimumab producing cell line 3, media 1 on viability. 
         FIG. 56  depicts the effect of concentration modulation of multiple amino acids to adalimumab producing cell line 3, media 1 on harvest titer. 
         FIG. 57  depicts the effect of concentration modulation of multiple amino acids to adalimumab producing cell line 3, media 1 on WCX 10 profile relative lysine distribution. 
         FIG. 58  depicts the effect of concentration modulation of multiple amino acids to adalimumab producing cell line 2, media 1 on WCX 10 profile relative lysine distribution (n=2). 
         FIG. 59  depicts the effect of concentration modulation of zinc and multiple amino acids to adalimumab producing cell line 1, media 1 on WCX 10 profile relative lysine distribution a) overall prediction plot, b) prediction plots for each additive. 
         FIG. 60  depicts the effect of peptides of varying length addition to adalimumab producing cell line 2, media 1 on WCX 10 profile relative lysine distribution (n=2). 
         FIG. 61  depicts the effect of pH modulation to adalimumab producing cell line 1, media 1 on viable cell density (n=2). 
         FIG. 62  depicts the effect of pH modulation to adalimumab producing cell line 1, media 1 on viability (n=2). 
         FIG. 63  depicts the effect of pH modulation to adalimumab producing cell line 1, media 1 on harvest titer (n=2). 
         FIG. 64  depicts the effect of pH modulation to adalimumab producing cell line 1, media 1 on WCX 10 profile relative lysine distribution (n=2). 
         FIG. 65  depicts the effect of pH modulation to adalimumab producing cell line 1, media 2 on viable cell density (n=2). 
         FIG. 66  depicts the effect of pH modulation to adalimumab producing cell line 1, media 2 on viability (n=2). 
         FIG. 67  depicts the effect of pH modulation to adalimumab producing cell line 1, media 2 on harvest titer (n=2). 
         FIG. 68  depicts the effect of pH modulation to adalimumab producing cell line 1, media 2 on WCX 10 profile relative lysine distribution (n=2). 
         FIG. 69  depicts the effect of pH modulation to adalimumab producing cell line 3, media 1 on viable cell density (n=2). 
         FIG. 70  depicts the effect of pH modulation to adalimumab producing cell line 3, media 1 on viability (n=2). 
         FIG. 71  depicts the effect of pH modulation to adalimumab producing cell line 3, media 1 on harvest titer (n=2). 
         FIG. 72  depicts the effect of pH modulation to adalimumab producing cell line 3, media 1 on WCX 10 profile relative lysine distribution (n=2). 
         FIG. 73  depicts the effect of temperature modulation to adalimumab producing cell line 1, media 1 on viable cell density (n=2). 
         FIG. 74  depicts the effect of temperature modulation to adalimumab producing cell line 1, media 1 on viability (n=2). 
         FIG. 75  depicts the effect of temperature modulation to adalimumab producing cell line 1, media 1 on harvest titer (n=2). 
         FIG. 76  depicts the effect of temperature modulation to adalimumab producing cell line 1, media 1 on WCX 10 profile relative lysine distribution (n=2). 
         FIG. 77  depicts the effect of temperature modulation to adalimumab producing cell line 1, media 1 on viable cell density (n=2). 
         FIG. 78  depicts the effect of temperature modulation to adalimumab producing cell line 1, media 1 on viability (n=2). 
         FIG. 79  depicts the effect of temperature modulation to adalimumab producing cell line 1, media 1 on harvest titer (n=2). 
         FIG. 80  depicts the effect of temperature modulation to adalimumab producing cell line 1, media 1 on WCX 10 profile relative lysine distribution (n=2). 
     
    
    
     5. DETAILED DESCRIPTION OF THE INVENTION 
     The instant invention relates to the field of protein production and purification. In particular, the instant invention relates to compositions and processes for controlling the distribution or amount of C-terminal lysine variants expressed by host cells when used to produce a protein of interest, as well as compositions and processes for controlling the distribution or amount of C-terminal lysine variants present in purified preparations of a protein of interest. Certain embodiments of the invention relate to culturing said cells and/or purifying said proteins under conditions that modulate the distribution or amount of C-terminal lysine variants that are expressed by the cells or are present in purified protein preparations. In certain embodiments, the methods described herein employ culturing said cells in the presence of one or more amino acids. In certain embodiments, the methods described herein employ culturing said cells in the presence of Zinc, including, but not limited to, when Zinc is present in combination with one or more amino acids. In certain embodiments, the methods described herein employ culturing said cells under conditions of increased or decreased temperatures compared to a control temperature. In certain embodiments, the methods described herein employ culturing said cells under conditions of increased or decreased pH compared to a control pH. In certain embodiments, the present invention is directed toward pharmaceutical compositions comprising one or more proteins, such as, but not limited to an antibody or antigen-binding portion thereof, purified by a method described herein. In certain embodiments such control is exerted in order to facilitate consistency and reproducibly in obtaining a particular lysine variant distribution, e.g., in the production of a therapeutic antibody. The variant distribution in such contexts may change due to a number of reasons, e.g., processes changes such altering media usage, and the methods described herein allow for the production of compositions of the desired lysine variant specification. 
     For clarity and not by way of limitation, this detailed description is divided into the following sub-portions:
     (i) Definitions;   (ii) Antibody Generation;   (iii) Protein Production;   (iv) Protein Purification; and   (v) Pharmaceutical Compositions. 5.1 Definitions   

     In order that the present invention may be more readily understood, certain terms are first defined. 
     As used herein, the term “lysine variant heterogeneity” refers to a characteristic of a population of proteins wherein the population consists of proteins of substantially identical amino acid sequence, but where the population exhibits variation in the presence or absence of C-terminal lysine residues. Although such lysine variant heterogeneity can be observed under general cell culture conditions, the use of particular cell culture conditions, as detailed below, can increase or decrease the distribution or amount of lysine variant heterogeneity. 
     In certain embodiments, the protein is an antibody, and the distribution of lysine variant heterogeneity comprises a distribution of the lysine variants Lys 0, Lys 1 and Lys 2, wherein the Lys 0 lysine variant comprises an antibody with heavy chains that do not comprise a C-terminal lysine, wherein the Lys 1 lysine variant comprises an antibody with one heavy chain that comprises a C-terminal lysine, and wherein the Lys 2 lysine variant comprises an antibody wherein both heavy chains comprise a C-terminal lysine. 
     In certain embodiments, C-terminal lysine variants are associated with charge heterogeneities present in protein preparations, for example, monoclonal antibody (mAb) preparations, produced through a cell culture process. These heterogeneities can be detected by various methods, such as, for example, WCX-10 HPLC (a weak cation exchange chromatography), or IEF (isoelectric focusing). 
     In certain embodiments, the heterogeneity arises from subspecies of protein differing by the presence or absence of C-terminal lysines. For example, the population of proteins may comprise more than one subspecies of lysine variant. In one non-limiting example, the lysine variants may comprise at least two of Lys 0, Lys 1 and Lys 2 lysine variants which can be detected by weak cation exchange chromatography of the expression product of a host cell expressing adalimumab. 
     In certain embodiments, the heterogeneity arises from the size of subpopulations having different C-terminal lysine profiles. For example, the population of proteins may comprise more than one subspecies of C-terminal lysine variant, and each of the variants may be present in different amounts. In one non-limiting example, the C-terminal lysine variants may be at least two of the Lys 0, Lys 1 and Lys 2 lysine variants detected by weak cation exchange chromatography of the expression product of a host cell expressing adalimumab. In certain embodiments, Lys 0, Lys 1 or Lys 2 subspecies are present in different amounts. 
     In certain embodiments, the heterogeneity arises from both a difference in the amount of lysine variants in the population of proteins and the type of lysine variants present in the population of proteins. 
     The term “antibody” includes an immunoglobulin molecule comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region (CH). 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 LCVR or 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 (CDRs), 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 term “antigen-binding portion” of an antibody (or “antibody portion”) includes fragments of an antibody that retain the ability to specifically bind to an antigen. 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 comprising 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 comprising the VH and CH1 domains; (iv) a Fv fragment comprising the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546, the entire teaching of which is incorporated herein by reference), which comprises a VH 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, the entire teachings of which are incorporated herein by reference). Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody. Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see, e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123, the entire teachings of which are incorporated herein by reference). Still further, an antibody or antigen-binding portion thereof may be part of a larger immunoadhesion molecule, formed by covalent or non-covalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include use of the streptavidin core region to make a tetrameric scFv molecule (Kipriyanov, S. M., et al. (1995) Human Antibodies and Hybridomas 6:93-101, the entire teaching of which is incorporated herein by reference) and use of a cysteine residue, a marker peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv molecules (Kipriyanov, S. M., et al. (1994) Mol. Immunol. 31:1047-1058, the entire teaching of which is incorporated herein by reference). 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, as described herein. In one aspect, the antigen binding portions are complete domains or pairs of complete domains. 
     The phrase “clarified harvest” refers to a liquid material containing a protein of interest, for example, an antibody of interest such as a monoclonal or polyclonal antibody of interest, that has been extracted from cell culture, for example, a fermentation bioreactor, after undergoing centrifugation to remove large solid particles and subsequent filtration to remove finer solid particles and impurities from the material. 
     The term “human antibody” includes antibodies having variable and constant regions corresponding to human germline immunoglobulin sequences as described by 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 invention 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), e.g., in the CDRs and in particular CDR3. The mutations can be introduced using the “selective mutagenesis approach.” The human antibody can have at least one position replaced with an amino acid residue, e.g., an activity enhancing amino acid residue which is not encoded by the human germline immunoglobulin sequence. The human antibody can have up to twenty positions replaced with amino acid residues which are not part of the human germline immunoglobulin sequence. In other embodiments, up to ten, up to five, up to three or up to two positions are replaced. In one embodiment, these replacements are within the CDR regions. However, the term “human antibody”, as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. 
     As used herein a “recombinant expression vector” can be any suitable recombinant expression vector, and can be used to transform or transfect any suitable host. For example, one of ordinary skill in the art would appreciate that transformation or transfection is a process by which exogenous nucleic acid such as DNA is introduced into a cell wherein the transformation or transfection process involves contacting the cell with the exogenous nucleic acid such as the recombinant expression vector as described herein. Non-limiting examples of such expression vectors are the pUC series of vectors (Fermentas Life Sciences), the pBluescript series of vectors (Stratagene, La Jolla, Calif.), the pET series of vectors (Novagen, Madison, Wis.), the pGEX series of vectors (Pharmacia Biotech, Uppsala, Sweden), and the pEX series vectors (Clontech, Palo Alto, Calif.). 
     The phrase “recombinant human antibody” includes human 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 human antibody library, antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see, e.g., Taylor, L. D., et al. (1992) Nucl. Acids Res. 20:6287-6295, the entire teaching of which is incorporated herein by reference) or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences (see, Kabat, E. A., 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 approach or back-mutation or both. 
     An “isolated antibody” includes an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds a particular target protein is substantially free of antibodies that specifically bind antigens other than the target protein). An isolated antibody that specifically binds a human target protein may bind target molecules from other species. Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals. 
     As used herein, the term “adalimumab”, also known by its trade name Humira® (AbbVie) refers to a human IgG antibody that binds the human form of tumor necrosis factor alpha. In general, the heavy chain constant domain 2 (CH2) of the adalimumab IgG-Fc region is glycosylated through covalent attachment of oligosaccharide at asparagine 297 (Asn-297). Weak cation-exchange chromatography (WCX) analysis of the antibody has shown that it has three main charged-variants (i.e. Lys 0, Lys 1, and Lys 2). These variants, or charged isomers, are the result of incomplete posttranslational cleavage of the C-terminal lysine residues. 
     The term “activity” includes activities such as the binding specificity/affinity of an antibody for an antigen and/or the neutralizing potency of an antibody. 
     The phrase “nucleic acid molecule” includes DNA molecules and RNA molecules. A nucleic acid molecule may be single-stranded or double-stranded, but in one aspect is double-stranded DNA. 
     The phrase “isolated nucleic acid molecule,” as used herein in reference to nucleic acids encoding antibodies or antibody portions (e.g., VH, VL, CDR3) and includes a nucleic acid molecule in which the nucleotide sequences encoding the antibody or antibody portion are free of other nucleotide sequences encoding antibodies or antibody portions that bind antigens other than the target antigen, which other sequences may naturally flank the nucleic acid in human genomic DNA. Thus, e.g., an isolated nucleic acid of the invention encoding a VH region of a particular antibody contains no other sequences encoding other VH regions that bind antigens other than the target antigen. The phrase “isolated nucleic acid molecule” is also intended to include sequences encoding bivalent, bispecific antibodies, such as diabodies in which VH and VL regions contain no other sequences other than the sequences of the diabody. 
     The phrase “recombinant host cell” (or simply “host cell”) includes a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein. 
     As used herein, the term “recombinant protein” refers to a protein produced as the result of the transcription and translation of a gene carried on a recombinant expression vector that has been introduced into a host cell. In certain embodiments the recombinant protein is an antibody, preferably a chimeric, humanized, or fully human antibody. In certain embodiments the recombinant protein is an antibody of an isotype selected from group consisting of: IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA1, IgA2, IgD, or IgE. In certain embodiments the antibody molecule is a full-length antibody (e.g., an IgG1 or IgG4 immunoglobulin) or alternatively the antibody can be a fragment (e.g., an Fc fragment or a Fab fragment). 
     As used herein, the term “cell culture” refers to methods and techniques employed to generate and maintain a population of host cells capable of producing a recombinant protein of interest, as well as the methods and techniques for optimizing the production and collection of the protein of interest. For example, once an expression vector has been incorporated into an appropriate host, the host can be maintained under conditions suitable for high level expression of the relevant nucleotide coding sequences, and the collection and purification of the desired recombinant protein. Mammalian cells are preferred for expression and production of the recombinant of the present invention, however other eukaryotic cell types can also be employed in the context of the instant invention. See, e.g., Winnacker, From Genes to Clones, VCH Publishers, N.Y., N.Y. (1987). Suitable mammalian host cells for expressing recombinant proteins according to the invention include Chinese Hamster Ovary (CHO cells) (including dhfr-CHO cells, described in Urlaub and Chasin, (1980) PNAS USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in Kaufman and Sharp (1982) Mol. Biol. 159:601-621, the entire teachings of which are incorporated herein by reference), NS0 myeloma cells, COS cells and SP2 cells. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2), the entire teachings of which are incorporated herein by reference. 
     When using the cell culture techniques of the instant invention, the protein of interest can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. In embodiments where the protein of interest is produced intracellularly, the particulate debris, either host cells or lysed cells (e.g., resulting from homogenization), can be removed by a variety of means, including but not limited to, by centrifugation or ultrafiltration. Where the protein of interest is secreted into the medium, supernatants from such expression systems can be first concentrated using a commercially available protein concentration filter, e.g., an Amicon™ or Millipore Pellicon™ ultrafiltration unit, which can then be subjected to one or more additional purification techniques, including but not limited to affinity chromatography, including protein A affinity chromatography, ion exchange chromatography, such as anion or cation exchange chromatography, and hydrophobic interaction chromatography. 
     As used herein the term “on-line” refers to processes that are accomplished in the context of an on-going cell culture run. For example, the administration of a particular nutrient or change in temperature, or pH occur on-line when such administrations or changes are implemented in an existing cell culture run. Similarly, measurement of certain data is considered on-line if that data is being collected in the context of a particular cell culture run. For example, on-line gas analysis refers to the measurement of gases introduced into or released from a particular cell culture run. In contrast, the term “off-line”, as used herein, refers to actions taken outside the context of a particular cell culture run. For example, the production of cell culture media comprising specific concentrations of particular components is an example of an off-line activity. 
     The term “modifying”, as used herein, is intended to refer to changing one or more amino acids in the antibodies or antigen-binding portions thereof. The change can be produced by adding, substituting or deleting an amino acid at one or more positions. The change can be produced using known techniques, such as PCR mutagenesis. 
     The term “about”, as used herein, is intended to refer to ranges of approximately 10-20% greater than or less than the referenced value. In certain circumstances, one of skill in the art will recognize that, due to the nature of the referenced value, the term “about” can mean more or less than a 10-20% deviation from that value. 
     The term “control”, as used herein, is intended to refer to both limitation as well as to modulation. For example, in certain embodiments, the instant invention provides methods for controlling diversity that decrease the diversity of certain characteristics of protein populations, including, but not limited to, the presence, distribution and/or amounts of lysine variants. Such decreases in diversity can occur by: (1) promotion of a desired characteristic; (2) inhibition of an unwanted characteristic; or (3) a combination of the foregoing. As used herein, the term “control” also embraces contexts where heterogeneity is modulated, i.e., shifted, from one diverse population to a second population of equal, or even greater diversity, where the second population exhibits a distinct profile of the characteristic of interest. 
     5.2 Antibody Generation 
     The term “antibody” as used in this section refers to an intact antibody or an antigen binding fragment thereof. 
     The antibodies of the present disclosure can be generated by a variety of techniques, including immunization of an animal with the antigen of interest followed by conventional monoclonal antibody methodologies e.g., the standard somatic cell hybridization technique of Kohler and Milstein (1975) Nature 256: 495. Although somatic cell hybridization procedures are preferred, in principle, other techniques for producing monoclonal antibody can be employed e.g., viral or oncogenic transformation of B lymphocytes. 
     One preferred animal system for preparing hybridomas is the murine system. Hybridoma production is a very well-established procedure. Immunization protocols and techniques for isolation of immunized splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known. 
     An antibody preferably can be a human, a chimeric, or a humanized antibody. Chimeric or humanized antibodies of the present disclosure can be prepared based on the sequence of a non-human monoclonal antibody prepared as described above. DNA encoding the heavy and light chain immunoglobulins can be obtained from the non-human hybridoma of interest and engineered to contain non-murine (e.g., human) immunoglobulin sequences using standard molecular biology techniques. For example, to create a chimeric antibody, murine variable regions can be linked to human constant regions using methods known in the art (see e.g., U.S. Pat. No. 4,816,567 to Cabilly et al.). To create a humanized antibody, murine CDR regions can be inserted into a human framework using methods known in the art (see e.g., U.S. Pat. No. 5,225,539 to Winter, and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.). 
     In one non-limiting embodiment, the antibodies of this disclosure are human monoclonal antibodies. Such human monoclonal antibodies can be generated using transgenic or transchromosomic mice carrying parts of the human immune system rather than the mouse system. These transgenic and transchromosomic mice include mice referred to herein as the HuMAb Mouse® (Medarex, Inc.), KM Mouse® (Medarex, Inc.), and XenoMouse® (Amgen). 
     Moreover, alternative transchromosomic animal systems expressing human immunoglobulin genes are available in the art and can be used to raise antibodies of the disclosure. For example, mice carrying both a human heavy chain transchromosome and a human light chain transchromosome, referred to as “TC mice” can be used; such mice are described in Tomizuka et al. (2000) Proc. Natl. Acad. Sci. USA 97:722-727. Furthermore, cows carrying human heavy and light chain transchromosomes have been described in the art (e.g., Kuroiwa et al. (2002) Nature Biotechnology 20:889-894 and PCT application No. WO 2002/092812) and can be used to raise antibodies of this disclosure. 
     Recombinant human antibodies of the invention can be isolated by screening of a recombinant combinatorial antibody library, e.g., a scFv phage display library, prepared using human VL and VH cDNAs prepared from mRNA derived from human lymphocytes. Methodologies for preparing and screening such libraries are known in the art. In addition to commercially available kits for generating phage display libraries (e.g., the Pharmacia Recombinant Phage Antibody System, catalog no. 27-9400-01; and the Stratagene SurfZAP™ phage display kit, catalog no. 240612, the entire teachings of which are incorporated herein), examples of methods and reagents particularly amenable for use in generating and screening antibody display libraries can be found in, e.g., Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. PCT Publication No. WO 92/18619; Dower et al. PCT Publication No. WO 91/17271; Winter et al. PCT Publication No. WO 92/20791; Markland et al. PCT Publication No. WO 92/15679; Breitling et al. PCT Publication No. WO 93/01288; McCafferty et al. PCT Publication No. WO 92/01047; Garrard et al. PCT Publication No. WO 92/09690; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; McCafferty et al., Nature (1990) 348:552-554; Griffiths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J Mol Biol 226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrard et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982; the entire teachings of which are incorporated herein. 
     Human monoclonal antibodies of this disclosure can also be prepared using SCID mice into which human immune cells have been reconstituted such that a human antibody response can be generated upon immunization. Such mice are described in, for example, U.S. Pat. Nos. 5,476,996 and 5,698,767 to Wilson et al. 
     In certain embodiments, the methods of the invention include anti-TNFα antibodies and antibody portions, anti-TNFα-related antibodies and antibody portions, and human antibodies and antibody portions with equivalent properties to anti-TNFα, such as high affinity binding to hTNFα with low dissociation kinetics and high neutralizing capacity. In one aspect, the invention provides treatment with an isolated human antibody, or an antigen-binding portion thereof, that dissociates from hTNFα with a Kd of about 1×10 −8  M or less and a Koff rate constant of 1×10 −3  s −1  or less, both determined by surface plasmon resonance. In specific non-limiting embodiments, an anti-TNFα antibody purified according to the invention competitively inhibits binding of Adalimumab to TNFα under physiological conditions. 
     In yet another embodiment of the invention, antibodies or fragments thereof, can be altered wherein the constant region of the antibody is modified to reduce at least one constant region-mediated biological effector function relative to an unmodified antibody. To modify an antibody of the invention such that it exhibits reduced binding to the Fc receptor, the immunoglobulin constant region segment of the antibody can be mutated at particular regions necessary for Fc receptor (FcR) interactions (see, e.g., Canfield and Morrison (1991) J. Exp. Med. 173:1483-1491; and Lund et al. (1991) J. of Immunol. 147:2657-2662, the entire teachings of which are incorporated herein). Reduction in FcR binding ability of the antibody may also reduce other effector functions which rely on FcR interactions, such as opsonization and phagocytosis and antigen-dependent cellular cytotoxicity. 
     5.3 Protein Production 
     To express a protein of the invention, such as an antibody or antigen-binding fragment thereof, DNAs encoding the protein, such as DNAs encoding partial or full-length light and heavy chains in the case of antibodies, are inserted into one or more expression vector such that the genes are operatively linked to transcriptional and translational control sequences. (See, e.g., U.S. Pat. No. 6,914,128, the entire teaching of which is incorporated herein by reference.) In this context, the term “operatively linked” is intended to mean that a gene encoding the protein of interest is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the gene. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. In certain embodiments, the protein of interest will comprising multiple polypeptides, such as the heavy and light chains of an antibody. Thus, in certain embodiments, genes encoding multiple polypeptides, such as antibody light chain genes and antibody heavy chain genes, can be inserted into a separate vector or, more typically, the genes are inserted into the same expression vector. Genes are inserted into expression vectors by standard methods (e.g., ligation of complementary restriction sites on the gene fragment and vector, or blunt end ligation if no restriction sites are present). Prior to insertion of the gene or genes, the expression vector may already carry additional polypeptide sequences, such as, but no limited to, antibody constant region sequences. For example, one approach to converting the antibody or antibody-related VH and VL sequences to full-length antibody genes is to insert them into expression vectors already encoding heavy chain constant and light chain constant regions, respectively, such that the VH segment is operatively linked to the CH segment(s) within the vector and the VL segment is operatively linked to the CL segment within the vector. Additionally or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the protein from a host cell. The gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein). 
     In addition to protein coding genes, a recombinant expression vector of the invention can carry one or more regulatory sequence that controls the expression of the protein coding genes in a host cell. The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the protein coding genes. Such regulatory sequences are described, e.g., in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990), the entire teaching of which is incorporated herein by reference. It will be appreciated by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences may depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. Suitable regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus 40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g., the adenovirus major late promoter (AdMLP)) and polyoma. For further description of viral regulatory elements, and sequences thereof, see, e.g., U.S. Pat. No. 5,168,062 by Stinski, U.S. Pat. No. 4,510,245 by Bell et al. and U.S. Pat. No. 4,968,615 by Schaffner et al., the entire teachings of which are incorporated herein by reference. 
     In addition to the protein coding genes and regulatory sequences, a recombinant expression vector of the invention may carry one or more additional sequences, such as a sequence that regulates replication of the vector in host cells (e.g., origins of replication) and/or a selectable marker gene. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017, all by Axel et al., the entire teachings of which are incorporated herein by reference). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. Suitable selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells with methotrexate selection/amplification) and the neo gene (for G418 selection). 
     An antibody, or antibody portion, of the invention can be prepared by recombinant expression of immunoglobulin light and heavy chain genes in a host cell. To express an antibody recombinantly, a host cell is transfected with one or more recombinant expression vectors carrying DNA fragments encoding the immunoglobulin light and heavy chains of the antibody such that the light and heavy chains are expressed in the host cell and secreted into the medium in which the host cells are cultured, from which medium the antibodies can be recovered. Standard recombinant DNA methodologies are used to obtain antibody heavy and light chain genes, incorporate these genes into recombinant expression vectors and introduce the vectors into host cells, such as those described in Sambrook, Fritsch and Maniatis (eds), Molecular Cloning; A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), Ausubel et al. (eds.) Current Protocols in Molecular Biology, Greene Publishing Associates, (1989) and in U.S. Pat. Nos. 4,816,397 &amp; 6,914,128, the entire teachings of which are incorporated herein. 
     For expression of protein, for example, the light and heavy chains of an antibody, the expression vector(s) encoding the protein is (are) transfected into a host cell by standard techniques. The various forms of the term “transfection” are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like. Although it is theoretically possible to express the proteins of the invention in either prokaryotic or eukaryotic host cells, expression of antibodies in eukaryotic cells, such as mammalian host cells, is suitable because such eukaryotic cells, and in particular mammalian cells, are more likely than prokaryotic cells to assemble and secrete a properly folded and immunologically active protein. Prokaryotic expression of protein genes has been reported to be ineffective for production of high yields of active protein (Boss and Wood (1985) Immunology Today 6:12-13, the entire teaching of which is incorporated herein by reference). 
     Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryote, yeast, or higher eukaryote cells described above. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, e.g., Enterobacteriaceae such as  Escherichia , e.g.,  E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella , e.g.,  Salmonella typhimurium, Serratia , e.g.,  Serratia marcescans , and  Shigella , as well as  Bacilli  such as  B. subtilis  and  B. licheniformis  (e.g.,  B. licheniformis  41P disclosed in DD 266,710 published Apr. 12, 1989),  Pseudomonas  such as  P. aeruginosa , and  Streptomyces . One suitable  E. coli  cloning host is  E. coli  294 (ATCC 31,446), although other strains such as  E. coli  B,  E. coli  X1776 (ATCC 31,537), and  E. coli  W3110 (ATCC 27,325) are suitable. These examples are illustrative rather than limiting. 
     In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for polypeptide encoding vectors.  Saccharomyces cerevisiae , or common baker&#39;s yeast, is the most commonly used among lower eukaryotic host microorganisms. However, a number of other genera, species, and strains are commonly available and useful herein, such as  Schizosaccharomyces pombe; Kluyveromyces  hosts such as, e.g.,  K. lactis, K. fragilis  (ATCC 12,424),  K. bulgaricus  (ATCC 16,045),  K. wickeramii  (ATCC 24,178),  K. waltii  (ATCC 56,500),  K. drosophilarum  (ATCC 36,906),  K. thermotolerans , and  K. marxianus; yarrowia  (EP 402,226);  Pichia pastoris  (EP 183,070);  Candida; Trichoderma reesia  (EP 244,234);  Neurospora crassa; Schwanniomyces  such as  Schwanniomyces occidentalis ; and filamentous fungi such as, e.g.,  Neurospora, Penicillium, Tolypocladium , and  Aspergillus  hosts such as  A. nidulans  and  A. niger.    
     Suitable host cells for the expression of glycosylated proteins, for example, glycosylated antibodies, are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as  Spodoptera frugiperda  (caterpillar),  Aedes aegypti  (mosquito),  Aedes albopictus  (mosquito),  Drosophila melanogaster  (fruitfly), and  Bombyx mori  have been identified. A variety of viral strains for transfection are publicly available, e.g., the L-1 variant of  Autographa californica  NPV and the Bm-5 strain of  Bombyx mori  NPV, and such viruses may be used as the virus herein according to the present invention, particularly for transfection of  Spodoptera frugiperda  cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be utilized as hosts. 
     Suitable mammalian host cells for expressing the recombinant proteins of the invention include Chinese Hamster Ovary (CHO cells) (including dhfr-CHO cells, described in Urlaub and Chasin, (1980) PNAS USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in Kaufman and Sharp (1982) Mol. Biol. 159:601-621, the entire teachings of which are incorporated herein by reference), NSO myeloma cells, COS cells and SP2 cells. When recombinant expression vectors encoding protein genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or secretion of the antibody into the culture medium in which the host cells are grown. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2), the entire teachings of which are incorporated herein by reference. 
     Host cells are transformed with the above-described expression or cloning vectors for protein production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. 
     The host cells used to produce a protein may be cultured in a variety of media. Commercially available media such as Ham&#39;s F10™ (Sigma), Minimal Essential Medium™ (MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco&#39;s Modified Eagle&#39;s Medium™ (DMEM), (Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. No. Re. 30,985 may be used as culture media for the host cells, the entire teachings of which are incorporated herein by reference. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as gentamycin drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan. 
     Host cells can also be used to produce portions of intact proteins, for example, antibodies, including Fab fragments or scFv molecules. It is understood that variations on the above procedure are within the scope of the present invention. For example, in certain embodiments it may be desirable to transfect a host cell with DNA encoding either the light chain or the heavy chain (but not both) of an antibody. Recombinant DNA technology may also be used to remove some or all of the DNA encoding either or both of the light and heavy chains that is not necessary for binding to an antigen. The molecules expressed from such truncated DNA molecules are also encompassed by the antibodies of the invention. In addition, bifunctional antibodies may be produced in which one heavy and one light chain are an antibody of the invention and the other heavy and light chain are specific for an antigen other than the target antigen, depending on the specificity of the antibody of the invention, by crosslinking an antibody of the invention to a second antibody by standard chemical crosslinking methods. 
     In a suitable system for recombinant expression of a protein, for example, an antibody, or antigen-binding portion thereof, a recombinant expression vector encoding the protein, for example, both an antibody heavy chain and an antibody light chain, is introduced into dhfr-CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, the protein gene(s) are each operatively linked to CMV enhancer/AdMLP promoter regulatory elements to drive high levels of transcription of the gene(s). The recombinant expression vector also carries a DHFR gene, which allows for selection of CHO cells that have been transfected with the vector using methotrexate selection/amplification. The selected transformant host cells are cultured to allow for expression of the protein, for example, the antibody heavy and light chains, and intact protein, for example, an antibody, is recovered from the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells and recover the protein from the culture medium. 
     When using recombinant techniques, the protein, for example, antibodies or antigen binding fragments thereof, can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. In one aspect, if the protein is produced intracellularly, as a first step, the particulate debris, either host cells or lysed cells (e.g., resulting from homogenization), can be removed, e.g., by centrifugation or ultrafiltration. Where the protein is secreted into the medium, supernatants from such expression systems can be first concentrated using a commercially available protein concentration filter, e.g., an Amicon™ or Millipore Pellicon™ ultrafiltration unit. 
     Prior to the process of the invention, procedures for purification of protein, for example, antibodies or antigen binding fragments thereof, from cell debris initially depend on the site of expression of the protein. Some proteins can be secreted directly from the cell into the surrounding growth media; others are made intracellularly. For the latter proteins, the first step of a purification process typically involves: lysis of the cell, which can be done by a variety of methods, including mechanical shear, osmotic shock, or enzymatic treatments. Such disruption releases the entire contents of the cell into the homogenate, and in addition produces subcellular fragments that are difficult to remove due to their small size. These are generally removed by differential centrifugation or by filtration. Where the protein is secreted, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, e.g., an Amicon™ or Millipore Pellicon™ ultrafiltration unit. Where the protein is secreted into the medium, the recombinant host cells can also be separated from the cell culture medium, e.g., by tangential flow filtration. protein can be further recovered from the culture medium using the protein purification methods of the invention. 
     Numerous populations of proteins expressed by host cells, including, but not limited to, host cells expressing antibodies, such as adalimumab, may comprise a number of lysine variants, for example, combinations of two or more of Lys 0, Lys 1 and Lys 2, and are therefore amenable to the instant invention&#39;s methods for control of C-terminal lysine variant heterogeneity. For example, weak cation-exchange chromatography (WCX) analysis of adalimumab has shown the presence of the three lysine variants corresponding to Lys 0, Lys 1 and Lys 2. The presence of these lysine variants provides an exemplary system to identify those cell culture conditions that allow for control over lysine variant heterogeneity. 
     The production of C-terminal lysine variants can be dependent upon changes in process parameters. (Lawrence, D. (2008), C-terminal Lysine Variants in Fully Human Monoclonal Antibodies: Investigation of Test Methods and Possible Causes. Biotechnology and Bioengineering. 100: 1132-1143). Native glycoproteins and/or recombinant glycoproteins (e.g., natural antibodies and/or therapeutic antibodies) that are translated at the endoplasmic reticulum (ER) must fold properly and often assemble into multimeric complexes. There are several proteins that help these proteins to fold properly. Some of these proteins only need the cleavage of the ER N-terminal sequence of the protein to become a mature protein. However, other glycoproteins can require further processing (“posttranslational modifications”) to become a mature and fully-functional. Some of these posttranslational modifications include glycosylation, formation of disulfide bonds, N-terminal pyroglutamate, methionine oxidation, asparagine deamination, phosphorylation, acetylation, and enzymatic removal of C-terminal lysine or arginine residues. (Ahrer et al., (2006), Chromatographic and Electrophoretic Characterization of Protein Variants. Journal of Chromatography. 841:110-122; Li et al. (2005), Current Therapeutic Antibody Production and Process Optimization. Bioprocessing Journal; Harris, J. (1995), Processing of C-terminal Lysine and Arginine Residues of Proteins Isolated from Mammalian Cell Culture. Journal of Chromatography. 705: 129-134; and Parkins, M., Theiler, R., et al. (2000), Determination of the Origin of Charge Heterogeneity in a Murine Monoclonal Antibody. Pharmaceutical Research. 17: 1110-1117). 
     Without being bound by theory, studies have demonstrated that the enzymatic removal of the C-terminal Lys residues is the primary contributor to the heterogeneity of recombinant monoclonal antibodies, including, but not limited to, the adalimumab glycoprotein. (Harris et al. (2004), Commercial Manufacturing Scale Formulation and Analytical Characterization of Therapeutic Recombinant Antibodies. Drug Development Research. 61: 137-154). Additional studies have determined that the charge heterogeneity can be generated by: 1) removal of the lysine or arginine C-terminal residue in the IgG heavy chains; 2) conversion of N-terminal glutamate to pyroglutamate; 3) dehydration of aspartate residues; and 4) alternate cleavage of a signal peptide that results in the presence of basic residues. The adalimumab heavy chain terminal sequence is proline-glycine-lysine. However, as noted above, the lysine residues are partially removed during the manufacturing process. Thus, adalimumab can comprise a mixture of antibodies bearing zero, one, or two C-terminal lysine residues. The specific sequence can be detected by cation exchange chromatography. It has been observed that this charge heterogeneity, caused by the incomplete posttranslational cleavage, may not affect the potency of the protein to bind the TNF-α. (Santora et al., (2001), Characterization of Noncovalent Complexes of Recombinant Human Monoclonal Antibody and Antigen Using Cation Exchange, Size Exclusion Chromatography, and BIAcore. Analytical Biochemistry 299: 119-129). Even though the removal of C-terminal lysine residues may not couple with the biological function of the antibody, it is a factor to consider in maintaining batch-to-batch consistency. (Parkins et al. (2000), Determination of the Origin of Charge Heterogeneity in a Murine Monoclonal Antibody. Pharmaceutical Research. 17: 1110-1117). 
     Experiments have shown that the charge heterogeneity of the antibody, due to the incomplete removal of C-terminal lysine residues, can be traced to carboxypeptidase activity/expression differences due to process parameter variability or changes. Since the penultimate residue, glycine, is not removed, this suggests that the carboxypeptidase is specific for basic residues, such as lysine and arginine. (Lawrence, D. (2008), C-terminal Lysine Variants in Fully Human Monoclonal Antibodies: Investigation of Test Methods and Possible Causes. Biotechnology and Bioengineering. 100: 1132-1143; and Harris, J. (1995), Processing of C-terminal Lysine and Arginine Residues of Proteins Isolated from Mammalian Cell Culture. Journal of Chromatography. 705: 129-134). In order to develop strategies to control such charged isomer heterogeneity, as well as heterogeneity in glycosylation, the experiments outlined in the Examples below were conducted. 
     The experiments disclosed herein demonstrate that, in certain embodiments, variation in raw materials used in cell culture, and particularly in the context of media preparation, can vary product quality significantly. For example, as outlined herein, control over the amount of Zinc present in cell culture media can allow for the modulation of C-terminal lysine variant heterogeneity. Zinc has been previously reported in literature to be a cofactor of the enzyme carboxypeptidase (Valee B et.al (1960), The role of zinc in carboxypeptidase, Journal of Biological Chemistry, 235, 1, 64-69). However, it was not appreciated until the filing of the instant application that adjusting the levels of zinc in culture media could allows for modulation of the lysine variants. 
     5.3.1 Adjusting Zinc Concentration to Control Lysine Variation 
     In certain embodiments of the instant invention, control of C-terminal lysine variant heterogeneity can be attained by adjustment of the zinc concentration of the media employed in the cell culture run. In certain non-limiting embodiments, such adjustment will be to decrease the amount of Zinc in the media, while in other non-limiting embodiments the necessary adjustment to achieve the desired control over lysine variant heterogeneity will involve an increase in the amount of Zinc in the media. Such increases or decreases in the amount of Zinc can be of a magnitude of 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, and ranges within one or more of the preceding, of the original amount. 
     In certain embodiments, cell culture media containing no Zinc is supplemented with Zinc to achieve a final Zinc concentration in the cell culture media of less than 60 μM. In certain embodiments, the cell culture will contain a total concentration of Zinc of between about 0.025 and about 10 μM, between about 0.05 and 10 μM, between about 0.1 and 10 μM, between about 0.2 and 10 μM, between about 0.25 and 10 μM, between about 0.5 and 10 μM, between about 1 and 10 μM, between about 1.5 and 9.5 μM, between about 2 and 9 μM, between about 2.5 and 8.5 μM, between about 3 and 8 μM, between about 3.5 and 7.5 μM, between about 4 and 7 μM, between about 4.5 and 6.5 μM, between about 5 and 6 μM. In certain embodiments, the cell culture media containing no Zinc is supplemented with Zinc to achieve a final Zinc concentration in the cell culture media of about 3.4 μM or about 6.7 μM. 
     In certain embodiments, the cell culture media contains Zinc in an amount effective to reduce the amount of one or more C-terminal lysine variants in a protein or antibody sample by about 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, and ranges within one or more of the preceding. 
     In certain embodiments, the cell culture media contains Zinc in an amount effective to increase the amount of one or more C-terminal lysine variants in a protein or antibody sample by about 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, and ranges within one or more of the preceding. 
     In certain embodiments, the cell culture media contains Zinc in an amount effective to reduce the amount of a Lys 0 lysine variant, and to increase the amount of a Lys 1 and/or Lys 2 lysine variant expressed by the cell culture. 
     For example, and not by way of limitation, as detailed in Example 6.1, below, certain embodiments include, but are not limited to when the Zinc concentration of the cell culture medium employed is reduced from a control concentration of about 10 μM to about 3.4 μM, and the % Lys 0 of an adalimumab sample purified from the supplemented culture is reduced to 67.9% from a control amount of 92.7%. Additionally, the amounts of Lys 1 and Lys 2 in the adalimumab sample are increased to 19.9% (Lys 1) and 12.2% (Lys 2) from an adalimumab sample purified from the control culture (10 μM Zinc) having 6.3% Lys 1 and 1.0% Lys 2. Furthermore, as detailed in Example 6.1, below, certain embodiments include, but are not limited to when the Zinc concentration of the cell culture medium employed is reduced from a control concentration of about 10 μM to about 3.4 μM or about 6.7 μM, and the % Lys 0 of an adalimumab sample purified from the supplemented culture is reduced to 69.0% (3.4 μM) or 89.9% (6.7 μM) from a control amount of 92.8%. Additionally, the amounts of Lys 1 and Lys 2 in the adalimumab sample are increased to 21.8% (Lys 1—3.4 μM), 8.7% (Lys 1—6.7 μM), 9.1% (Lys 2—3.4 μM), and 1.4% (Lys 2—6.7 μM) from an adalimumab sample purified from the control culture (10 μM Zinc) having 6.2% Lys 1 and 1.1% Lys 2. 
     In certain embodiments, the cell culture contains Zinc as well as arginine, lysine, and histidine, each at a concentration sufficient to reduce the amount of a Lys 0 lysine variant, and to increase the amount of a Lys 1 and/or Lys 2 lysine variant expressed by the cell culture. For example, but not by way of limitation, the concentration range for arginine and lysine in this can be between about 1 to about 3 g/l, while the concentration range for histidine is between about 0 to about 1 g/l and the concentration range for zinc is about 30 μM to about 60 μM. 
     5.3.2 Adjusting Amino Acid Concentration to Control C-terminal Lysine Variation 
     In certain embodiments of the instant invention, control of C-terminal lysine variant heterogeneity can be attained by adjustment of the amino acid composition of the cell culture media. In certain embodiments, such adjustment will be to increase the amount of one or more amino acids in the media, while in other embodiments the necessary adjustment to achieve the desired control over lysine variant heterogeneity will involve a decrease in the amount of one or more amino acids in the media. Such increases or decreases in the amount of the one or more amino acids can be of a magnitude of 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, and ranges within one or more of the preceding, of the original amount. 
     In certain embodiments, the cell culture media is supplemented with one or more amino acids wherein each of the one or more amino acids is supplemented in an amount of between about 0.025 and 20 g/L, or between about 0.05 and 15 g/L, or between about 0.1 and 14 g/L, or between about 0.2 and 13 g/L, or between about 0.25 and 12 g/L, or between about 0.5 and 11 g/L, or between about 1 and 10 g/L, or between about 1.5 and 9.5 g/L, or between about 2 and 9 g/L, or between about 2.5 and 8.5 g/L, or between about 3 and 8 g/L, or between about 3.5 and 7.5 g/L, or between about 4 and 7 g/L, or between about 4.5 and 6.5 g/L, or between about 5 and 6 g/L. In certain embodiments, the cell culture media is supplemented with one or more amino acids wherein each of the one or more amino acids is supplemented in an amount of about 0.5 g/L, or about 1 g/L, or about 2 g/L, or about 4 g/L, or about 8 g/L. 
     In certain embodiments, the cell culture media is supplemented with one or more amino acids wherein each of the one or more amino acids is supplemented in an amount effective to reduce the amount of one or more lysine variants in a protein or antibody sample by about 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, and ranges within one or more of the preceding. 
     In certain embodiments, the cell culture media is supplemented with one or more amino acids wherein each of the one or more amino acids is supplemented in an amount effective to increase the amount of one or more C-terminal lysine variants in a protein or antibody sample by about 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, and ranges within one or more of the preceding. 
     In certain embodiments, the cell culture media is supplemented with one or more amino acids wherein each of the one or more amino acids is supplemented in an amount effective to reduce the amount of a Lys 0 lysine variant, and to increase the amount of a Lys 1 and/or Lys 2 lysine variant expressed by the cell culture. 
     In certain embodiments, the one or more amino acids used to supplement the cell culture media is arginine, lysine, histidine, or certain combinations of arginine or lysine with ornithine. In certain embodiments, the amino acids are provided as single peptides, as dipeptides, as tripeptides or as longer oligopeptides. In certain embodiments, the di-, tri-, and/or oligopeptides are individually composed of a single amino acid, while in alternative embodiments, the di-, tri-, and/or oligopeptides are individually composed of two or more particular amino acids. 
     For example, and not by way of limitation, as detailed in Example 6.2, below, when the production medium employed in the example was supplemented with 3 g/L arginine and 3 g/L lysine, the % Lys 0 of an adalimumab sample purified from the supplemented culture was reduced to 72.1% from a control amount of 86.7%. Additionally, the amounts of Lys 1 and Lys 2 in the adalimumab sample was increased to 20.8% (Lys 1) and 7.1% (Lys 2) from an adalimumab sample purified from a control culture having 11.1% Lys 1 and 2.2% Lys 2. 
     Similarly, although the percentage of Lys0 relative to Lysine sum in the control sample was 82.9% on day 10, in the sample with the highest concentration of arginine in this experiment (9 g/l), the percentage of relative Lys0 was reduced to 73.4%. This relative modulation of Lys 0, Lys 1 and Lys 2 was directly related to the concentration of arginine in the media. Thus, in certain embodiments, the relative level of Lys0 to lysine sum can be modulated, i.e., increased or decreased, by adjusting the concentration of arginine in the culture media. 
     In certain embodiments, a lysine supplementation of cell culture is employed to modulate the ratio of Lys0 to lysine sum. As detailed Example 6.2, below, in the sample with the highest concentration of lysine (11 g/l), the percentage of relative levels of Lys0 was reduced to 67.7%, which contrasts with the percentage of Lys0 relative to lysine sum (sum of the peak areas corresponding to Lys 0, Lys 1 and Lys 2) in the control sample of 92.5%. A dose dependent decrease in relative Lys0, and a dose dependent increase in relative Lysl and Lys2 regions was observed in test conditions with increased lysine concentration. Thus, in certain embodiments, the relative level of Lys0 to lysine sum can be modulated, i.e., increased or decreased, by adjusting the concentration of lysine in the culture media. 
     In certain embodiments, a histidine supplementation of cell culture is employed to modulate the ratio of Lys0 to lysine sum. As detailed Example 6.2, below, in the sample with the highest concentration of histidine (10 g/l), the percentage of relative Lys0 was reduced to 80.6% from a control sample percentage of 92.5%. The decrease in Lys 0 corresponded with the increase in relative levels of both Lys1 and Lys2. In an alternative example, the percentage of Lys0 relative to lysine sum in the control sample was 94.2%. In the sample with the highest concentration of histidine in this experiment (8 g/l), the percentage of relative Lys0 was reduced to 81.5%. The decrease in Lys0 corresponded with the increase in relative levels of both Lys1 and Lys2. A dose dependent decrease in relative Lys0, and a dose dependent increase in relative Lys1 and Lys2 regions was observed in test conditions with increased histidine concentration. Thus, in certain embodiments, the relative level of Lys0 to lysine sum can be modulated, i.e., increased or decreased, by adjusting the concentration of histidine in the culture media. 
     In certain embodiments, an ornithine/arginine or ornithine/lysine supplementation of cell culture is employed to modulate the ratio of Lys0 to lysine sum. As detailed Example 6.2, below, the combination of ornithine with arginine or lysine reduced the relative level of Lys0 to 81.9% in comparison with the condition with just arginine and lysine increase with a relative level Lys0 of 84.7%. Thus, the increase of ornithine may exhibit synergistic effects in modulating lysine variant distribution when added in combination with arginine and lysine. Thus, in certain embodiments, the relative level of Lys0 to lysine sum can be modulated, i.e., increased or decreased, by adjusting the concentration of a combination of ornithine and either arginine and/or lysine in the culture media. 
     In certain embodiments, an arginine/lysine/histidine/ornithine combination supplementation of cell culture is employed to modulate the ratio of Lys0 to lysine sum. As detailed in Example 6.2, below, in comparison to the lower concentrations, or conditions where amino acids were supplemented individually, a further reduction in Lys0 relative to lysine sum was observed in conditions where combinations of amino acids were increased in the media. A progressive decrease was observed in relative Lys0 when more amino acids were increased in combination. The percentage of relative Lys0 was reduced from 94.9% in the control sample to 73.9% in the sample with all four amino acid concentrations increased Thus, in certain embodiments, the relative level of Lys0 to lysine sum can be modulated, i.e., increased or decreased, by adjusting the concentration of a combination of all four amino acids (arginine, lysine, histidine, and ornithine) in the culture media. 
     In certain embodiments, the three amino acid monomers arginine, lysine, and histidine, the dipeptides lys-lys and arg-lys, or the tripeptides lys-lys-lys, his-arg-lys, and arg-his-lys are used as supplements to cell culture in order to modulate the ratio of Lys0 to lysine sum. As detailed in Example 6.2, below, in comparison to the control, a reduction in Lys0 relative to lysine sum was also observed in conditions where dipeptides and tripeptides were supplemented to the media. Specifically, a decrease was observed in relative Lys0 when polypeptides were supplemented. The percentage of relative Lys0 was reduced from 88.0% in the control sample to 71.9% in a sample supplemented with tripeptide arg-his-lys (4g/1), and to 74.0% in a sample supplemented with his-arg-lys (2 g/l). 
     In certain embodiments, the medium supplements described herein are such that they can be included in the medium at the start of culture, or can be added in a fed-batch or in a continuous manner. The medium supplements could be supplemented to chemically defined or hydrolysate based basal media. The methods described in this invention may be used in combination with different cultivation methods including, but not limited to, batch, fed-batch, chemostat and perfusion, and with various cell culture equipment including, but not limited to, shake flasks with or without suitable agitation, spinner flasks, stirred bioreactors, airlift bioreactors, membrane bioreactors, reactors with cells retained on a solid support or immobilized/entrapped as in microporous beads, and any other configuration appropriate for optimal growth and productivity of the desired cell line. The methods described here may also be combined with the appropriate choice of process parameters as described in section 5.3.3. 
     5.3.3 Adjusting Process Parameters to Control Lysine Variation 
     The variation in the process parameters, such as the temperature and/or pH, at which cells are cultured, can vary product quality significantly. In certain embodiments of the instant invention, control of C-terminal lysine variant heterogeneity can be attained by adjustment of the temperature and/or pH of the cell culture run. In certain embodiments, such adjustment will be to increase the temperature and/or pH at which a cell culture is cultured, while in other embodiments the necessary adjustment to achieve the desired control over lysine variant heterogeneity will involve a modulation of the temperature and/or pH at which a cell culture is cultured. Such increases or decreases in cell culture temperature, and/or pH can be of a magnitude of 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, and ranges within one or more of the preceding, of the original temperature. 
     In certain embodiments, the cell culture is cultured at a temperature of between about 25 and 50° C., or between about 30 and 40° C., or between about 31 and 39° C., or between about 31.5 and 38.5° C., or between about 32 and 38° C., or between about 32.5 and 37.5° C., or between about 33 and 37° C., or between about 33.5 and 36.5° C., or between about 34 and 36° C., or between about 34.5 and 35.5° C. In certain embodiments, the cell culture is cultured at a temperature of about 30, 31, 32, 33, 34, 35, 36, or 37° C. 
     For example, and not by way of limitation, as detailed in Example 6.3, below, when the temperature of a cell culture run was decreased from 37° C. to 31° C., the % Lys 0 of an adalimumab sample purified from the culture was reduced from 84.9% to 72.8%. Additionally, the amounts of Lys 1 and Lys 2 in the adalimumab sample was increased from 13.4% (Lys 1) and 1.7% (Lys 2), to 22.2% (Lys 1) and 5.0% (Lys 2). Thus, in certain embodiments, the relative level of Lys0 to lysine sum can be modulated, i.e., increased or decreased, by adjusting the temperature of the cell culture run. 
     In certain embodiments, pH is either increased or decreased in order to increase or decrease the amount of Lys0 relative to the lysine sum. For example, but not by way of limitation, a reduction in pH to 6.7 from a control pH of 7.1 can be employed to increase the amount of Lys0 relative to the lysine sum. For example, and not by way of limitation, as detailed in Example 6.3, below, when the pH of a cell culture run was decreased from 7.1 to 6.7, the % Lys 0 of an adalimumab sample purified from the culture was increased from 82.0% to 88.7%. In certain embodiments the pH is increased from 6.7 to 6.8, 6.9, 7.0, or 7.1 in order to achieve a decrease in the amount of Lys0 relative to the lysine sum. 
     In certain embodiments, the temperature and/or pH of the cell culture is decreased or increased in an amount effective to reduce the amount of one or more lysine variants in a protein or antibody sample by about 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, and ranges within one or more of the preceding. 
     In certain embodiments, the temperature and/or pH of the cell culture is decreased or increased in an amount effective to increase the amount of one or more lysine variants in a protein or antibody sample by about 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, and ranges within one or more of the preceding. 
     In certain embodiments, the temperature and/or pH of the cell culture is decreased or increased in an amount effective to reduce the amount of a Lys 0 lysine variant, and to increase the amount of a Lys 1 and/or Lys 2 lysine variant expressed by the cell culture. 
     5.3.4 Additional Exemplary Strategies 
     In addition to the above-described embodiments, the present invention is also directed to embodiments wherein the medium supplements described herein are added in a batch-wise fashion, a continuous feeding fashion, or a combination of both during cell culture. In addition, certain embodiments will involve the adding such media supplements one at a time and/or addition at multiple time points during the cell culture process. In certain embodiments, the cell culture process will involve preloading the culture media with excess of one or more medium supplements. In certain embodiments, the addition of one or more supplements will be based on measurements taken on-line, in-line, and/or at line. In certain embodiments, the addition of one or more supplements will occur with other substrates, metal scavengers, and/or combination with other culture conditions such as temperature pH, etc. In certain embodiments, one or more media supplements will be added as multimers, e.g., arg-arg, his-his, arg-his-orn, etc., and/or as chemical variants of amino acids or analogs of amino acids, salt forms of amino acids, controlled release of amino acids by immobilizing in gels, etc, and/or in fully or partially dissolved form. 
     In certain embodiments, the culture process will occur in bags, flasks, disposables, hollow fiber, perfusion, and/or air lift process equipment. In certain embodiments, one or more media supplements will be added to seed bioreactor before transfer to achieve a final concentration in the fermentor. In certain embodiments, achieving a known concentration of one or more of the media supplements can occur either through an in-situ combination resulting the generation of the supplement or a degradation/reaction resulting the generation of the supplement, i.e., adding a substrate and enzyme/catalyst to produce the components necessary. In certain embodiments the addition of one or more media supplement will based on measured amount of lysine distribution. 
     5.4 Protein Purification 
     5.4.1 Protein Purification Generally 
     In certain embodiments, the methods of the present invention can be used in combination with techniques for protein purification to provide for the production of a purified protein preparation, for example, a preparation comprising an antibody or an antigen binding fragment thereof, from a mixture comprising a protein and at least one process-related impurity or product-related substance. 
     For example, but not by way of limitation, once a clarified solution or mixture comprising the protein of interest, for example, an antibody or antigen binding fragment thereof, has been obtained, separation of the protein of interest from the process-related impurities and/or product-related substances can be performed using a combination of different purification techniques, including, but not limited to, affinity separation steps, ion exchange separation steps, mixed mode separation steps, and hydrophobic interaction separation steps. The separation steps separate mixtures of proteins on the basis of their charge, degree of hydrophobicity, or size. In one aspect of the invention, separation is performed using chromatography, including cationic, anionic, and hydrophobic interaction. Several different chromatography resins are available for each of these techniques, allowing accurate tailoring of the purification scheme to the particular protein involved. The essence of each of the separation methods is that proteins can be caused either to traverse at different rates down a column, achieving a physical separation that increases as they pass further down the column, or to adhere selectively to the separation medium, being then differentially eluted by different solvents. In some cases, the antibody is separated from impurities when the impurities specifically adhere to the column and the antibody does not, i.e., the antibody is present in the flow through. 
     As noted above, accurate tailoring of a purification scheme relies on consideration of the protein to be purified. In certain embodiments, the separation steps of employed in connection with the cell culture methods of the instant invention facilitate the separation of an antibody from one or more process-related impurity and/or product-related substance. Antibodies that can be successfully purified using the methods described herein include, but are not limited to, human IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, and IgM antibodies. In certain embodiments, Protein A affinity chromatography can be useful, however, in certain embodiments, the use of Protein A affinity chromatography would prove useful, for example in the context of the purification of IgG3 antibodies, as IgG3 antibodies bind to Protein A inefficiently. Other factors that allow for specific tailoring of a purification scheme include, but are not limited to: the presence or absence of an Fc region (e.g., in the context of full length antibody as compared to an Fab fragment thereof) because Protein A binds to the Fc region; the particular germline sequences employed in generating to antibody of interest; and the amino acid composition of the antibody (e.g., the primary sequence of the antibody as well as the overall charge/hydrophobicity of the molecule). Antibodies sharing one or more characteristic can be purified using purification strategies tailored to take advantage of that characteristic. 
     5.4.2 Primary Recovery and Virus Inactivation 
     In certain embodiments, it will be advantageous to subject a sample produced by the techniques of the instant invention to at least a first phase of clarification and primary recovery. In addition, the primary recovery process can also be a point at which to reduce or inactivate viruses that can be present in the sample mixture. For example, any one or more of a variety of methods of viral reduction/inactivation can be used during the primary recovery phase of purification including heat inactivation (pasteurization), pH inactivation, solvent/detergent treatment, UV and γ-ray irradiation and the addition of certain chemical inactivating agents such as (β-propiolactone or e.g., copper phenanthroline as in U.S. Pat. No. 4,534,972, the entire teaching of which is incorporated herein by reference. 
     The primary recovery may also include one or more centrifugation steps to further clarify the sample mixture and thereby aid in purifying the protein of interest. Centrifugation of the sample can be run at, for example, but not by way of limitation, 7,000×g to approximately 12,750×g. In the context of large scale purification, such centrifugation can occur on-line with a flow rate set to achieve, for example, but not by way of limitation, a turbidity level of 150 NTU in the resulting supernatant. Such supernatant can then be collected for further purification. 
     In certain embodiments, the primary recovery may also include the use of one or more depth filtration steps to further clarify the sample matrix and thereby aid in purifying the antibodies produced using the cell culture techniques of the present invention. Depth filters contain filtration media having a graded density. Such graded density allows larger particles to be trapped near the surface of the filter while smaller particles penetrate the larger open areas at the surface of the filter, only to be trapped in the smaller openings nearer to the center of the filter. In certain embodiments, the depth filtration step can be a delipid depth filtration step. Although certain embodiments employ depth filtration steps only during the primary recovery phase, other embodiments employ depth filters, including delipid depth filters, during one or more additional phases of purification. Non-limiting examples of depth filters that can be used in the context of the instant invention include the Cuno™ model 30/60ZA depth filters (3M Corp.), and 0.45/0.2 μm Sartopore™ bi-layer filter cartridges. 
     5.4.3 Affinity Chromatography 
     In certain embodiments, it will be advantageous to subject a sample produced by the techniques of the instant invention to affinity chromatography to further purify the protein of interest away from process-related impurities and/or product-related substances. In certain embodiments the chromatographic material is capable of selectively or specifically binding to the protein of interest. Non-limiting examples of such chromatographic material include: Protein A, Protein G, chromatographic material comprising, for example, an antigen bound by an antibody of interest, and chromatographic material comprising an Fc binding protein. In specific embodiments, the affinity chromatography step involves subjecting the primary recovery sample to a column comprising a suitable Protein A resin. In certain embodiments, Protein A resin is useful for affinity purification and isolation of a variety of antibody isotypes, particularly IgG1, IgG2, and IgG4. Protein A is a bacterial cell wall protein that binds to mammalian IgGs primarily through their Fc regions. In its native state, Protein A has five IgG binding domains as well as other domains of unknown function. 
     There are several commercial sources for Protein A resin. One suitable resin is MabSelect™ from GE Healthcare. A non-limiting example of a suitable column packed with MabSelect™ is an about 1.0 cm diameter×about 21.6 cm long column (˜17 mL bed volume). This size column can be used for small scale purifications and can be compared with other columns used for scale ups. For example, a 20 cm×21 cm column whose bed volume is about 6.6 L can be used for larger purifications. Regardless of the column, the column can be packed using a suitable resin such as MabSelect™. 
     5.4.4 Ion Exchange Chromatography 
     In certain embodiments, it will be advantageous to subject a sample produced by the techniques of the instant invention to ion exchange chromatography in order to purify the protein of interest away from process-related impurities and/or product-related substances. Ion exchange separation includes any method by which two substances are separated based on the difference in their respective ionic charges, and can employ either cationic exchange material or anionic exchange material. For example, the use of a cationic exchange material versus an anionic exchange material is based on the localized charges of the protein. Therefore, it is within the scope of this invention to employ an anionic exchange step prior to the use of a cationic exchange step, or a cationic exchange step prior to the use of an anionic exchange step. Furthermore, it is within the scope of this invention to employ only a cationic exchange step, only an anionic exchange step, or any serial combination of the two. 
     In performing the separation, the initial protein mixture can be contacted with the ion exchange material by using any of a variety of techniques, e.g., using a batch purification technique or a chromatographic technique. 
     Anionic or cationic substituents may be attached to matrices in order to form anionic or cationic supports for chromatography. Non-limiting examples of anionic exchange substituents include diethylaminoethyl (DEAE), quaternary aminoethyl (QAE) and quaternary amine(Q) groups. Cationic substituents include carboxymethyl (CM), sulfoethyl (SE), sulfopropyl (SP), phosphate (P) and sulfonate (S). Cellulose ion exchange resins such as DE23™, DE32™, DE52™, CM-23™, CM-32™, and CM-52™ are available from Whatman Ltd. Maidstone, Kent, U.K. SEPHADEX®-based and -locross-linked ion exchangers are also known. For example, DEAE-, QAE-, CM-, and SP-SEPHADEX® and DEAE-, Q-, CM-and S-SEPHAROSE® and SEPHAROSE® Fast Flow are all available from Pharmacia AB. Further, both DEAE and CM derivitized ethylene glycol-methacrylate copolymer such as TOYOPEARL™ DEAE-6505 or M and TOYOPEARL™ CM-650S or M are available from Toso Haas Co., Philadelphia, Pa. 
     5.4.5 Ultrafiltration/Diafiltration 
     In certain embodiments, it will be advantageous to subject a sample produced by the techniques of the instant invention to ultrafiltration and/or diafiltration in order to purify the protein of interest away from process-related impurities and/or product-related substances. Ultrafiltration is described in detail in: Microfiltration and Ultrafiltration: Principles and Applications, L. Zeman and A. Zydney (Marcel Dekker, Inc., New York, N.Y., 1996); and in: Ultrafiltration Handbook, Munir Cheryan (Technomic Publishing, 1986; ISBN No. 87762-456-9). A preferred filtration process is Tangential Flow Filtration as described in the Millipore catalogue entitled “Pharmaceutical Process Filtration Catalogue” pp. 177-202 (Bedford, Mass., 1995/96). Ultrafiltration is generally considered to mean filtration using filters with a pore size of smaller than 0.1 μm. By employing filters having such small pore size, the volume of the sample can be reduced through permeation of the sample buffer through the filter while antibodies are retained behind the filter. 
     Diafiltration is a method of using ultrafilters to remove and exchange salts, sugars, and non-aqueous solvents, to separate free from bound species, to remove low molecular-weight material, and/or to cause the rapid change of ionic and/or pH environments. Microsolutes are removed most efficiently by adding solvent to the solution being ultrafiltered at a rate approximately equal to the ultratfiltration rate. This washes microspecies from the solution at a constant volume, effectively purifying the retained protein. In certain embodiments of the present invention, a diafiltration step is employed to exchange the various buffers used in connection with the instant invention, optionally prior to further chromatography or other purification steps, as well as to remove impurities from the protein preparations. 
     5.4.6 Hydrophobic Interaction Chromatography 
     In certain embodiments, it will be advantageous to subject a sample produced by the techniques of the instant invention to hydrophobic interaction chromatography in order to purify the protein of interest away from process-related impurities and/or product-related substances. For example, a first eluate obtained from an ion exchange column can be subjected to a hydrophobic interaction material such that a second eluate having a reduced level of impurity is obtained. Hydrophobic interaction chromatography (HIC) steps, such as those disclosed herein, are generally performed to remove protein aggregates, such as antibody aggregates, and process-related impurities. 
     In performing an HIC-based separation, the sample mixture is contacted with the HIC material, e.g., using a batch purification technique or using a column. Prior to HIC purification it may be desirable to remove any chaotropic agents or very hydrophobic substances, e.g., by passing the mixture through a pre-column. 
     Whereas ion exchange chromatography relies on the charges of the protein to isolate them, hydrophobic interaction chromatography uses the hydrophobic properties of the protein. Hydrophobic groups on the protein interact with hydrophobic groups on the column. The more hydrophobic a protein is the stronger it will interact with the column. Thus the HIC step removes host cell derived impurities (e.g., DNA and other high and low molecular weight product-related species). 
     Hydrophobic interactions are strongest at high ionic strength, therefore, this form of separation is conveniently performed following salt precipitations or ion exchange procedures. Adsorption of the protein of interest to a HIC column is favored by high salt concentrations, but the actual concentrations can vary over a wide range depending on the nature of the protein and the particular HIC ligand chosen. Various ions can be arranged in a so-called soluphobic series depending on whether they promote hydrophobic interactions (salting-out effects) or disrupt the structure of water (chaotropic effect) and lead to the weakening of the hydrophobic interaction. Cations are ranked in terms of increasing salting out effect as Ba ++ ; Ca ++ ; Mg ++ ; Li + ; Cs + ; Na + ; K + ; Rb + ; NH4 + , while anions may be ranked in terms of increasing chaotropic effect as PO −−− ; SO 4   −− ; CH 3 CO 3   − ; Cl − ; Br − ; NO 3   − ; ClO 4   − ; I − ; SCN − . 
     In general, Na, K or NH 4  sulfates effectively promote ligand-protein interaction in HIC. Salts may be formulated that influence the strength of the interaction as given by the following relationship: (NH 4 ) 2 SO 4 &gt;Na 2 SO 4 &gt;NaCl&gt;NH 4 Cl&gt;NaBr&gt;NaSCN. In general, salt concentrations of between about 0.75 and about 2 M ammonium sulfate or between about 1 and 4 M NaCl are useful. 
     HIC columns normally comprise a base matrix (e.g., cross-linked agarose or synthetic copolymer material) to which hydrophobic ligands (e.g., alkyl or aryl groups) are coupled. A suitable HIC column comprises an agarose resin substituted with phenyl groups (e.g., a Phenyl Sepharose™ column). Many HIC columns are available commercially. Examples include, but are not limited to, Phenyl Sepharose™ 6 Fast Flow column with low or high substitution (Pharmacia LKB Biotechnology, AB, Sweden); Phenyl Sepharose™ High Performance column (Pharmacia LKB Biotechnology, AB, Sweden); Octyl Sepharose™ High Performance column (Pharmacia LKB Biotechnology, AB, Sweden); Fractogel™ EMD Propyl or Fractogel™ EMD Phenyl columns (E. Merck, Germany); Macro-Prep™ Mehyl or Macro-Prep™ t-Butyl Supports (Bio-Rad, California); WP HI-Propyl (C3)™ column (J. T. Baker, New Jersey); and Toyopearl™ ether, phenyl or butyl columns (TosoHaas, PA). 
     5.4.7 Multimodal Chromatography 
     In certain embodiments, it will be advantageous to subject a sample produced by the techniques of the instant invention to multimodal chromatography in order to purify the protein of interest away from process-related impurities and/or product-related substances. Multimodal chromatography is chromatography that utilizes a multimodal media resin. Such a resin comprises a multimodal chromatography ligand. In certain embodiments, such a ligand refers to a ligand that is capable of providing at least two different, but co-operative, sites which interact with the substance to be bound. One of these sites gives an attractive type of charge-charge interaction between the ligand and the substance of interest. The other site typically gives electron acceptor-donor interaction and/or hydrophobic and/or hydrophilic interactions. Electron donor-acceptor interactions include interactions such as hydrogen-bonding, π-π, cation-π, charge transfer, dipole-dipole, induced dipole etc. Multimodal chromatography ligands are also known as “mixed mode” chromatography ligands. 
     In certain embodiments, the multimodal chromatography resin is comprised of multimodal ligands coupled to an organic or inorganic support, sometimes denoted a base matrix, directly or via a spacer. The support may be in the form of particles, such as essentially spherical particles, a monolith, filter, membrane, surface, capillaries, etc. In certain embodiments, the support is prepared from a native polymer, such as cross-linked carbohydrate material, such as agarose, agar, cellulose, dextran, chitosan, konjac, carrageenan, gellan, alginate etc. To obtain high adsorption capacities, the support can be porous, and ligands are then coupled to the external surfaces as well as to the pore surfaces. Such native polymer supports can be prepared according to standard methods, such as inverse suspension gelation (S Hjerten: Biochim Biophys Acta 79(2), 393-398 (1964). Alternatively, the support can be prepared from a synthetic polymer, such as cross-linked synthetic polymers, e.g. styrene or styrene derivatives, divinylbenzene, acrylamides, acrylate esters, methacrylate esters, vinyl esters, vinyl amides etc. Such synthetic polymers can be produced according to standard methods, see e.g. “Styrene based polymer supports developed by suspension polymerization” (R Arshady: Chimica e L&#39;Industria 70(9), 70-75 (1988)). Porous native or synthetic polymer supports are also available from commercial sources, such as Amersham Biosciences, Uppsala, Sweden. 
     5.5 Pharmaceutical Compositions 
     The proteins, for example, antibodies and antibody-portions, produced using the cell culture techniques of the instant invention can be incorporated into pharmaceutical compositions suitable for administration to a subject. Typically, the pharmaceutical composition comprises a protein of the invention and a pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In many cases, it is desirable to include isotonic agents, e.g., sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody or antibody portion. 
     The protein compositions of the invention can be incorporated into a pharmaceutical composition suitable for parenteral administration. The protein can be prepared as an injectable solution containing, e.g., 0.1-250 mg/mL antibody. The injectable solution can be composed of either a liquid or lyophilized dosage form in a flint or amber vial, ampule or pre-filled syringe. The buffer can be L-histidine approximately 1-50 mM, (optimally 5-10 mM), at pH 5.0 to 7.0 (optimally pH 6.0). Other suitable buffers include but are not limited to sodium succinate, sodium citrate, sodium phosphate or potassium phosphate. Sodium chloride can be used to modify the toxicity of the solution at a concentration of 0-300 mM (optimally 150 mM for a liquid dosage form). Cryoprotectants can be included for a lyophilized dosage form, principally 0-10% sucrose (optimally 0.5-1.0%). Other suitable cryoprotectants include trehalose and lactose. Bulking agents can be included for a lyophilized dosage form, principally 1-10% mannitol (optimally 24%). Stabilizers can be used in both liquid and lyophilized dosage forms, principally 1-50 mM L-methionine (optimally 5-10 mM). Other suitable bulking agents include glycine, arginine, can be included as 0-0.05% polysorbate-80 (optimally 0.005-0.01%). Additional surfactants include but are not limited to polysorbate 20 and BRIJ surfactants. 
     In one aspect, the pharmaceutical composition includes the protein at a dosage of about 0.01 mg/kg-10 mg/kg. In another aspect, the dosages of the protein include approximately 1 mg/kg administered every other week, or approximately 0.3 mg/kg administered weekly. A skilled practitioner can ascertain the proper dosage and regime for administering to a subject. 
     The compositions of this invention may be in a variety of forms. These include, e.g., liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The form depends on, e.g., the intended mode of administration and therapeutic application. Typical compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for passive immunization of humans with other antibodies. One mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In one aspect, the protein is administered by intravenous infusion or injection. In another aspect, the protein is administered by intramuscular or subcutaneous injection. 
     Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration. Sterile injectable solutions can be prepared by incorporating the active compound (i.e., protein, antibody or antibody portion) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile, lyophilized powders for the preparation of sterile injectable solutions, the methods of preparation are vacuum drying and spray-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The proper fluidity of a solution can be maintained, e.g., by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, e.g., monostearate salts and gelatin. 
     The protein of the present invention can be administered by a variety of methods known in the art, one route/mode of administration is subcutaneous injection, intravenous injection or infusion. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. In certain embodiments, the active compound may be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978, the entire teaching of which is incorporated herein by reference. 
     In certain aspects, a protein of the invention may be orally administered, e.g., with an inert diluent or an assimilable edible carrier. The compound (and other ingredients, if desired) may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the subject&#39;s diet. For oral therapeutic administration, the compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. To administer a compound of the invention by other than parenteral administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation. 
     Supplementary active compounds can also be incorporated into the compositions. In certain aspects, a protein of the invention is co-formulated with and/or co-administered with one or more additional therapeutic agents that are useful for treating disorders. For example, an antibody or antibody portion of the invention may be co-formulated and/or co-administered with one or more additional antibodies that bind other targets (e.g., antibodies that bind other cytokines or that bind cell surface molecules). Furthermore, one or more antibodies of the invention may be used in combination with two or more of the foregoing therapeutic agents. Such combination therapies may advantageously utilize lower dosages of the administered therapeutic agents, thus avoiding possible toxicities or complications associated with the various monotherapies. It will be appreciated by the skilled practitioner that when the protein of the invention are used as part of a combination therapy, a lower dosage of protein may be desirable than when the protein alone is administered to a subject (e.g., a synergistic therapeutic effect may be achieved through the use of combination therapy which, in turn, permits use of a lower dose of the protein to achieve the desired therapeutic effect). 
     It should be understood that the protein of the invention can be used alone or in combination with an additional agent, e.g., a therapeutic agent, said additional agent being selected by the skilled artisan for its intended purpose. For example, the additional agent can be a therapeutic agent art-recognized as being useful to treat the disease or condition being treated by the protein of the present invention. The additional agent also can be an agent which imparts a beneficial attribute to the therapeutic composition, e.g., an agent which effects the viscosity of the composition. 
     Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. In certain embodiments it is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit comprising a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals. 
     An exemplary, non-limiting range for a therapeutically or prophylactically effective amount of a protein of the invention is 0.01-20 mg/kg, or 1-10 mg/kg, or 0.3-1 mg/kg. It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition. 
     6. EXAMPLES 
     6.1 Methods for modulating the Lysine Variant Distribution in Cell Culture by the Modulation of Zinc Concentration in Culture Medium 
     This example provides methods to modulate the lysine variant distribution of monoclonal antibodies by modulating the levels of zinc in medium. The examples included here cover experiments with two different adalimumab producing cell lines in chemically defined media. 
     6.1.1 Materials and Methods 
     Cell Lines and Adaptation Cultures 
     Two adalimumab producing cell lines were employed in the studies discussed herein. Upon thaw, cells were typically cultured in a combination of 250 mL and 500 mL Corning vented non-baffled shake flasks on a shaker platform at 110 RPM for cell line 1 and 180 rpm for cell line 2 in a 35° C., 5% CO 2  incubator. Subsequent to the initial cell growth in the standard IVGN CD basal growth media, cells were adapted for two passages in separate flasks in basal media containing different concentrations of zinc. Only the cultures that demonstrated good cell growth in the adaptation phase were carried forward to the production stage. 
     Cell Culture Media 
     The initial growth media was prepared from proprietary basal CD media GIA1 (Invitrogen, media 1). For the adaptation and production culture stages (in different concentrations of zinc), media was prepared starting from either proprietary basal CD media GIA1 (media 1) or CD media without zinc (Basal 2). The control cultures were carried through adaptation and production stage in Basal 1 media. The test conditions were carried through both adaptation and production stages in Basal 2 media supplemented with different concentrations of zinc. The trace element compounds supplemented to media are listed in Table 1. The detailed descriptions of culture media for the different conditions for both cell lines are listed in Table 2. All media was filtered through Corning 1L filter systems (0.22 μm PES) and stored at 4° C. until usage. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 List of trace element compounds supplemented to culture media 
               
            
           
           
               
               
               
            
               
                   
                 Compound 
                 Catalog No./Source 
               
               
                   
                   
               
               
                   
                 Zinc Chloride 
                 Fluka, 96468 
               
               
                   
                 Zinc Sulfate Heptahydrate 
                 Sigma, Z0251 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Detailed description of culture media for different  
               
               
                 experimental conditions 
               
            
           
           
               
               
               
            
               
                   
                   
                 Estimated final 
               
               
                 Cell 
                   
                 concentration 
               
               
                 line  
                 Condition 
                 of Zinc (μM) 
               
               
                   
               
            
           
           
               
               
               
            
               
                 1 
                 1 
                 10 
               
               
                   
                 2 
                 3.4 
               
               
                 2 
                 1 
                 10 
               
               
                   
                 2 
                 6.7 
               
               
                   
                 3 
                 3.4 
               
               
                   
               
            
           
         
       
     
     Production Cultures 
     Production cultures were initiated in duplicates in 500 mL Corning vented non-baffled shake flasks (200 mL working volume). The shake flasks were kept in incubators maintained at 35° C. and 5% CO 2  on shaker platforms that were either set at 110 rpm for cell line 1 or 180 rpm for cell line 2. In all experiments, the cells were transferred from the adaptation stage to the production stage at a split ratio of 1:5. 
     The harvest procedure of the shake flasks and reactors involved centrifugation of the culture sample at 3,000 RPM for 30 min and storage of supernatant in PETG bottles at −80° C. before submission for protein A purification and WCX-10 analysis. 
     WCX-10 Assay 
     For quantification of charge variants of antibodies, cation exchange chromatography was performed on a Dionex ProPac WCX-10, Analytical column 4 mm×250 mm (Dionex, CA). A Shimadzu LC10A HPLC system was used as the HPLC. The mobile phases used were 10 mM Sodium Phosphate dibasic pH 7.5 (Mobile phase A) and 10 mM Sodium Phosphate dibasic, 500 mM Sodium Chloride pH 5.5 (Mobile phase B). A binary gradient (94% A, 6% B: 0-20 min; 84% A, 16% B: 20-22 min; 0% A, 100% B: 22-28 min; 94% A, 6% B: 28-34 min) was used with detection at 280 nm. Quantitation is based on the relative area percent of detected peaks ( FIG. 1 ). 
     6.1.2 Results and Discussion 
     Effect of Varying Zinc Concentration in Chemically Defined Media with Cell Line 1 
     In this example, the effect of varying total zinc concentration (control (10 μM), 6.7 μM, 3.4 μM) in cell culture media on culture performance and product quality was evaluated using cell line 1. The ratios of the concentration of the two zinc salts (zinc chloride, zinc sulfate) were kept constant between the test conditions. As described in the materials and methods, each of the production stage cultures were initiated from respective adaptation cultures with corresponding levels of total zinc. 
     A difference in cell growth and viability profiles was observed between the test conditions and the control ( FIG. 2 ,  FIG. 3 ). While the peak viable cell density (VCD) in the control condition was about 11×10 6  cells/ml, the peak VCD for the 3.4 μM zinc condition was about 8×10 6  cells/ml. Corresponding to difference in peak VCD, the harvest titer was also slightly reduced in the 3.4 μM zinc condition (1.0 g/l) compared to the control (1.3 g/l) ( FIG. 4 ). The cultures were harvested on day 10 at viability of 50% or lower for each condition and the harvest was taken through protein A purification before WCX-10 analysis. From the WCX-10 analysis, the lysine variant distribution was characterized and the relative proportion of Lys0, Lys 1, and Lys2 variants were quantified as a fraction of the total Lysine sum. A zinc dose dependent decrease in the relative fraction of relative Lys0 was observed from 92.8% in the control condition to 69.0% in the 3.4 μM zinc condition. A corresponding relative increase in both Lys1/Lys2 variants was also observed ( FIG. 5 ). 
     Thus, lowering the zinc concentration provides an effective method to increase the relative proportion of the product antibody with C-terminal lysine on one or both the heavy chains (Lys1/Lys2). 
     Effect of Varying Zinc Concentration in Chemically Defined Media with Cell Line 2 
     In this example, the effect of varying total zinc concentration (control (10 μM), 3.4 μM) in basal cell culture media on cell culture performance and product quality was evaluated using cell line 2. The ratios of the concentration of the two zinc salts (zinc chloride, zinc sulfate) were kept constant between the test conditions. As described in the materials and methods, each of the production stage cultures were initiated from respective adaptation cultures with corresponding levels of total zinc. 
     A significant difference in cell growth and viability profile was observed between the two test conditions ( FIG. 6 ,  FIG. 7 ). While the peak viable cell density (VCD) in the control condition was about 22×10 6  cells/ml, the peak VCD for the 3.4 μM zinc condition was only about 11×10 6  cells/ml. Corresponding to difference in peak VCD, the harvest titer was also significantly reduced in the 3.4 μM zinc condition compared to the control ( FIG. 8 ). The cultures were harvested on day 10 at the target viability of 50% for each condition and the harvest was taken through protein A purification before WCX-10 analysis. From the WCX-10 analysis, the lysine variant distribution was characterized and the relative proportion of Lys0, Lys 1, and Lys2 variants were quantified as a fraction of the total lysine sum. There was a decrease in the relative fraction of Lys0 in the control condition (92.7%) versus the relative Lys0 in the 3.4 μM zinc condition (67.9%). The relative fractions of Lys 1/Lys2 variants were also correspondingly higher ( FIG. 9 ). 
     Thus, the increase in relative proportion of the product antibody with C-terminal lysine on one or both the heavy chains (Lys 1/Lys2) corresponding to reduction in levels of zinc in basal media, was also observed in this example. 
     6.2 Methods for Modulating the Lysine Variant Distribution in Cell Culture by the Addition of Amino Acids 
     This example provides methods to modulate the lysine variant distribution of monoclonal antibodies by supplementing specific components to the cell culture medium. The supplemented medium components included here are several amino acids (arginine, lysine, histidine added individually and ornithine in combination with arginine, lysine and histidine). 
     6.2.1 Materials and Methods 
     Cell Source and Adaptation Cultures 
     Three adalimumab producing cell lines, one mAB1 producing cell line and one mAB2 producing cell line were employed in the studies covered here. 
     For adalimumab producing cell lines, cells were cultured in their respective growth media (chemically defined media (media 1) or a hydrolysate based media (media 2 or media 3)) in a combination of vented non-baffled shake flasks (Corning) on a shaker platform at 110 RPM (cell line 1),180 RPM (cell line 2), 140 RPM (cell line 3) and 10 L or 20 L wave bags (GE). For experiments with cells in the hydrolysate based media (media 3), cells were thawed in media 1 and then adapted to media 3 over a few passages. Cultures were propagated in a 35° C., 5% CO 2  incubator for cell line 1 and 2 and in a 36° C., 5% CO 2  incubator for cell line 3 in order to obtain the required number of cells to be able to initiate production stage cultures. 
     For the mAB1 producing cell line, cells were cultured in chemically defined growth media (media 1) in a combination of vented non-baffled shake flasks (Corning) on a shaker platform at 130 RPM and 20L wave bags (GE). Cultures were propagated in a 36° C., 5% CO 2  incubator to obtain the required number of cells to be able to initiate production stage cultures. 
     For the mAB2 producing cell line, cells were cultured in chemically defined growth media (media 1) in a combination of vented non-baffled shake flasks (Corning) on a shaker platform at 140 RPM and 20 L wave bags (GE). Cultures were propagated in a 35° C., 5% CO 2  incubator to obtain the required number of cells to be able to initiate production stage cultures. 
     Cell Culture Media 
     Growth and production media were prepared from either a chemically defined media formulation (media 1) or hydrolysate-based medium formulations (media 2 and media 3). For preparation of the media 1, the media (IVGN GIA-1, proprietary formulation) was supplemented with L-glutamine, sodium bicarbonate, sodium chloride, and methotrexate solution. Production media consisted of all the components in the growth medium, excluding methotrexate. For cell line 1, mAB1, and mAB2 both growth and production medium were also supplemented with insulin. 
     For the hydrolysate-based formulation (media 2), the growth media was composed of PFCHO (proprietary CD formulation from SAFC), Dextrose, L-Glutamine, L-Asparagine, HEPES, Poloxamer 188, Ferric Citrate, Recombinant Human Insulin, Yeastolate (BD), Phytone Peptone (BD), Mono- and Di-basic Sodium Phosphate, Sodium Bicarbonate, Sodium Chloride and methotrexate. Production media consisted of all the components listed in the growth medium, excluding methotrexate. 
     For the hydrolysate-based formulation (media 3), the growth media was composed of OptiCHO (Invitrogen), L-Glutamine, Yeastolate (BD), Phytone Peptone (BD) and methotrexate. Production media consisted of all the components listed in the growth medium, excluding methotrexate. 
     Amino acids used for the experiments were reconstituted in Milli-Q water to make a 100 g/L stock solution, which was subsequently supplemented to both growth and production basal media. After addition of amino acids, media was brought to a pH similar to non-supplemented (control) media using 5N hydrochloric acid/5N NaOH, and it was brought to an osmolality similar to unsupplemented (control) media by adjusting the concentration of sodium chloride. All media was filtered through Corning 1L filter systems (0.22 μm PES) and stored at 4° C. until used. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 List of Amino Acids Supplemented to Culture Media and the 
               
               
                 Relevant Concentration Ranges Tested 
               
            
           
           
               
               
               
            
               
                   
                 Amino Acid 
                 Catalog No./Source 
               
               
                   
                   
               
               
                   
                 Arginine 
                 Sigma, A8094 
               
               
                   
                 Lysine 
                 Calbiochem, 4400 
               
               
                   
                 Histidine 
                 Sigma, H5659 
               
               
                   
                 Ornithine 
                 Sigma, 06503 
               
               
                   
                   
               
            
           
         
       
     
     Production cultures 
     Production cultures were initiated either in 500 ml shake flasks (Corning) or in 3 L Bioreactors (Applikon). For shake flask experiments, duplicate 500 mL Corning vented non-baffled shake flasks (200 mL working volume) were used for each condition. The shake flasks were kept in incubators either maintained at 35° C. or 36° C. and 5% CO 2  on shaker platforms that were either set at 110 rpm for adalimumab producing cell line 1, 180 rpm for adalimumab producing cell line 2, 140 rpm for adalimumab producing cell line 3, 130 rpm for mAB1 producing cell line, or 140 rpm for mAB2 producing cell line. For the bioreactor experiments, 3 L bioreactors (1.5 L working volume) were run at 37-33° C. (temperature shift), 30% DO (dissolved oxygen), 200 rpm, pH profile from 7.1 to 6.9 in three days and pH 6.9 thereafter. In all experiments, the cells were transferred from the seed train to the production stage at a split ratio of 1:5. 
     Cultures were run in either batch or fed-batch mode. In the batch mode, cells were cultured in the respective production medium. 1.25% (v/v) of 40% glucose stock solution was fed when the media glucose concentration reduced to less than 3 g/L. In the fed-batch mode, cultures were run with either the IVGN feed as per the following feed schedule—(4% (v/v)—day 3, 6%—day 5, 8%—day 7, 10%—day 9, 10%—day 11) or 10X Ex-Cell PFCHO feed (SAFC, 67411)—3% (v/v) on day 3. In fed-batch cultures with IVGN feed, cultures were also fed with 1.25% (v/v) of 40% glucose stock solution when the glucose concentration was below 1.5 g/l on IVGN feed days and when the concentration fell below 2.5 g/l on other days. In fed-batch cultures with 10X PFCHO feed, 1.25% (v/v) of 40% glucose stock solution was fed when the media glucose concentration was below 3 g/L. 
     Retention samples for titer analysis, of 2×1.5 mL, were collected daily for the bioreactor experiments (section 2.2.4) beginning on Day 8, and frozen at −80° C. The samples taken from each were later submitted for titer analysis. 
     The harvest procedure of the shake flasks and reactors involved centrifugation of the culture sample at 3,000 RPM for 30 min and storage of supernatant in PETG bottles at −80° C. before submission for protein A purification and WCX-10 analysis. 
     WCX-10 Assay 
     The acidic species and other charge variants present in cell culture harvest samples were quantified. Cation exchange chromatography was performed on a Dionex ProPac WCX-10, Analytical column (Dionex, CA). A Shimadzu LC10A HPLC system was used as the HPLC. 
     For the adalimumab and mAB1 samples, The mobile phases used were 10 mM Sodium Phosphate dibasic pH 7.5 (Mobile phase A) and 10 mM Sodium Phosphate dibasic, 500 mM Sodium Chloride pH 5.5 (Mobile phase B). A binary gradient (94% A, 6% B: 0-20 min; 84% A, 16% B: 20-22 min; 0% A, 100%B: 22-28 min; 94% A, 6% B: 28-34 min) was used with detection at 280 nm. 
     For mAB2 samples, the mobile phases used were 20 mM (4-Morpholino)ethanesulfonic Acid Monohydrate (MES) pH 6.5 (Mobile phase A) and 20 mM MES, 500 mM Sodium Chloride pH 6.5 (Mobile phase B). An optimized gradient (minute/% B): 0/3, 1/3, 46/21, 47/100, 52/100, 53/3, 58/3 was used with detection at 280 nm. 
     Quantitation is based on the relative area percent of detected peaks. The peaks that elute at relative residence time earlier than the main peak (Lys 0) corresponding to the drug product are together represented as the acidic peaks. The peaks that eluate at a relative residence time later than main peak in the basic region correspond to Lys 1 and Lys 2 respectively. 
     6.2.2 Results and Discussion 
     Effect of Supplementation of Arginine to Culture Media 
     The addition of arginine was tested in several experimental systems covering multiple cell lines, media and monoclonal antibodies. Following is a detailed description of two representative experiments where two different adalimumab producing cell lines were cultured in a chemically defined media (media 1). 
     Cell line 2 was cultured in media 1 with different total concentrations of arginine (1 (control), 1.25, 1.5, 2, 3, 5, 9 g/l). The cultures were performed in shake flasks in batch format with only glucose feed as described in the materials and methods. The cells grew to maximum viable cell densities (VCD) in the range of 18-22×10 6  cells/ml for the different conditions tested. The growth and viability profiles were comparable between the different conditions, although a slight decrease in viable cell density profile was observed in samples with the 9 g/l arginine condition ( FIG. 10 ,  FIG. 11 ). The harvest titers were comparable between the conditions ( FIG. 12 ). On Day 10 and Day 12 of culture, duplicate shake flasks for each of the conditions were harvested and then subsequently analyzed using WCX-10 post protein A purification and the percentages of total peak(s) area corresponding to Lys 0, Lys 1 and Lys 2 were quantified ( FIG. 13 ,  FIG. 14 ). The percentage of Lys0 relative to lysine sum (sum of areas corresponding to peaks Lys 0, Lys 1 and Lys 2) in the control sample was as 91.9% on day 10. In the sample with the highest tested concentration of arginine in this experiment (9 g/l), the relative percentage of Lys0 was reduced to 77.2%. A dose dependent decrease in relative Lys0 was observed in conditions with arginine concentrations beyond 2 g/l ( FIG. 13 ). The decrease in relative Lys 0 corresponded with the increase in relative levels of both Lys 1 and Lys 2. A dose dependent decrease in relative levels of Lys 0, and a corresponding dose dependent increase in Lys1 and Lys2 were observed in conditions with increased arginine. A similar trend in reduction of relative levels of Lys0 with arginine increase was also observed in the day 12 harvest samples ( FIG. 14 ). 
     Cell line 3 was cultured in media 1 with different concentrations of arginine (1 (control), 3, 5, 7, 9 g/l). The cultures were performed in shake flasks in batch format with only glucose feed as described in the materials and methods. The cells grew to maximum viable cell densities (VCD) in the range of 7-10×10 6  cells/ml for the different conditions tested. The growth and viability profiles were comparable between the different test conditions, although a slight decrease in viable cell density and viability profiles was observed in samples with the 9 g/l arginine condition ( FIG. 15 ,  FIG. 16 ). The product titer was also comparable between the conditions ( FIG. 17 ). On Day 10 of culture, duplicate shake flasks for each of the conditions were harvested and then subsequently analyzed using WCX-10 post protein A purification and the percentages of total peak(s) area corresponding to the Lys 0, Lys 1 and Lys2 species were quantified ( FIG. 18 ). The percentage of Lys0 relative to Lysine sum in the control sample was 82.9% on day 10. In the sample with the highest concentration of arginine in this experiment (9 g/l), the percentage of relative Lys0 was reduced to 73.4%. The decrease in Lys0 corresponded with the increase in relative levels of both Lys1 and Lys2. This relative modulation of Lys 0, Lys 1 and Lys 2 was directly related to the concentration of arginine in the media. 
     Thus, although the lysine variant distributions were substantially different between the control conditions in the studies presented above, significant modulation in the relative levels of the lysine variants (decrease in Lys 0 and increase in Lys 1/Lys 2) with increased arginine concentration was observed in both cases. 
     Additional experiments were performed with multiple cell lines in chemically defined or hydrolysate based media to demonstrate the wide range of applicability of this method. The experimental setup for each of these experiments was similar to that described above. The summaries of results of the different experiments performed for adalimumab are summarized in  FIG. 19 ,  FIG. 20  and  FIG. 21 . A reduction in relative Lys0, and increase in relative Lys 1 and Lys2 species with increased arginine concentration was also observed in each case. 
     In addition to adalimumab, the utility of this method for acidic species reduction was also demonstrated for processes involving two other mABs. The experimental setup for each of these experiments was similar to that described in section above and in the materials and methods. The reduction of acidic species with arginine increase for experiments corresponding to each mAB is summarized in  FIG. 22  and  FIG. 23 . A reduction in relative Lys0, and increase in relative Lysl and Lys2 species with increase in arginine concentration was also observed in both cases. 
     Effect of Supplementation of Lysine to Culture Media 
     The addition of lysine was tested in several experimental systems covering multiple cell lines, media and monoclonal antibodies. Following is a detailed description of two representative experiments where two different adalimumab producing cell lines were cultured in a chemically defined media (media 1). 
     Cell line 2 was cultured in media 1 with different concentrations of lysine (1 (control), 5, 7, 9, 11 g/l). The cultures were performed in shake flasks in batch format with only glucose feed as described in the materials and methods. The cells grew to maximum viable cell densities (VCD) in the range of 17-23×10 6  cells/ml for the different conditions tested. A slight dose dependent decrease in viable cell density profile was observed in all test conditions, with no significant effect on viability profiles ( FIG. 24  and  FIG. 25 ). On Days 10 and 11 of culture samples were collected for titer analysis. The harvest titers for all conditions were comparable ( FIG. 26 ). On Day 11 of culture, duplicate shake flasks for each of the conditions were harvested and then subsequently analyzed using WCX-10 post protein A purification and the percentages of total peak(s) area corresponding to the Lys 0, Lys 1 and Lys 2 peaks were quantified ( FIG. 27 ). The percentage of Lys0 relative to lysine sum (sum of the peak areas corresponding to Lys 0, Lys 1 and Lys 2) in the control sample was 92.5%. In the sample with the highest concentration of lysine in this experiment (11 g/l), the percentage of relative levels of Lys0 was reduced to 67.7%. The decrease in Lys0 corresponded with the increase in relative levels of both Lys1 and Lys2. A dose dependent decrease in relative Lys0, and a dose dependent increase in relative Lys1 and Lys2 regions was observed in test conditions with increased lysine concentration. 
     Cell line 3 was cultured in media 1 with different concentrations of lysine (1 (control), 3, 5, 7, 9, 11 g/l). The cultures were performed in shake flasks in batch format with only glucose feed as described in the materials and methods. The cells grew to maximum viable cell densities (VCD) in the range of 9.5-11.5×10 6  cells/ml for the different conditions tested. The growth and viability profiles were comparable between the different conditions, although a slight decrease in viable cell density and viability profiles was observed in samples with lysine concentration greater than 1 g/l, ( FIG. 28 ,  FIG. 29 ). On Days 10, 11 and 12 of culture samples were collected for titer analysis. The harvest titers for all conditions were comparable ( FIG. 30 ). On Day 12 of culture, duplicate shake flasks for each of the conditions were harvested and then subsequently analyzed using WCX-10 post protein A purification and the percentages of total peak(s) area corresponding to the Lys 0, Lys 1 and Lys2 peaks were quantified ( FIG. 31 ). The percentage of Lys0 relative to lysine sum in the control sample was 94.2%. In the sample with the highest concentration of lysine in this experiment (11 g/l), the percentage of relative level of Lys0 was reduced to 76.0%. The decrease in Lys0 corresponded with the increase in relative levels of both Lys1 and Lys2. A dose dependent decrease in relative levels of Lys0, and a corresponding increase in relative levels of Lys1 and Lys2 was observed in test conditions with increased lysine concentration. 
     Additional experiments were performed with multiple cell lines in chemically defined or hydrolysate based media to demonstrate the wide range of applicability of this method. The experimental setup for each of these experiments was similar to that described above. The summaries of results of the different experiments performed for adalimumab are summarized in  FIG. 32 ,  FIG. 33  and  FIG. 34 . A reduction in relative levels of Lys0, and a corresponding increase in relative levels of Lys 1 and Lys2 with increased lysine was also observed in each case. 
     In addition to adalimumab, the utility of this method for acidic species reduction was also demonstrated for processes involving two other mABs. The experimental setup for each of these experiments was similar to that described in section above and in the materials and methods. The reduction of acidic species with arginine addition for experiments corresponding to each mAB is summarized in  FIG. 35  and  FIG. 36 . A reduction in relative levels of Lys0, and increase in relative levels of Lys 1 and Lys2 species with increased lysine was also observed in each case. 
     Effect of Supplementation of Histidine to Culture Media 
     The modulation of histidine concentration was tested in several experimental systems covering multiple cell lines, media and monoclonal antibodies. Following is a detailed description of two representative experiments where two different adalimumab producing cell lines were cultured in a chemically defined media (media 1). 
     Cell line 2 was cultured in media 1 with different concentrations of histidine (0 (control), 4, 6, 8, 10 g/l). The cultures were performed in shake flasks in batch format with only glucose feed as described in the materials and methods. The cells grew to maximum viable cell densities (VCD) in the range of 12-22×10 6  cells/ml for the different conditions tested. A dose dependent decrease in viable cell density profile was observed in all conditions, with the 10 g/l histidine condition having significant reduction in growth ( FIG. 37 ). A corresponding significant impact on the viability profile was also observed ( FIG. 38 ). There was a small dose dependent decrease in titers for all conditions with histidine supplementation ( FIG. 39 ). On Day 11 for control sample and Day 12 for the remaining conditions, duplicate shake flasks were harvested and then subsequently analyzed using WCX-10 post protein A purification and the percentages of total peak(s) area corresponding to the lysine species were quantified ( FIG. 40 ). The percentage of Lys0 relative to lysine sum in the control sample was 92.5%. In the sample with the highest concentration of histidine in this experiment (10 g/l), the percentage of relative Lys0 was reduced to 80.6%. The decrease in Lys 0 corresponded with the increase in relative levels of both Lys1 and Lys2. 
     Cell line 3 was cultured in media 1 with different concentrations of histidine (0 (control), 2, 4, 6, 8 g/l). The cultures were performed in shake flasks in batch format with only glucose feed as described in the materials and methods. The cells grew to maximum viable cell densities (VCD) in the range of 6-10×10 6  cells/ml for the different conditions tested. A dose dependent decrease in viable cell density profile was observed in all samples supplemented with histidine ( FIG. 41 ). In comparison to the impact on VCD profile, the viability profiles were more comparable between the conditions ( FIG. 42 ). The harvest titers for all conditions were comparable ( FIG. 43 ). On Day 12 of culture, duplicate shake flasks for each of the conditions were harvested and then subsequently analyzed using WCX-10 post protein A purification and the percentages of total peak(s) area corresponding to Lys 0, Lys 1 and Lys 2 species were quantified ( FIG. 44 ). The percentage of Lys0 relative to lysine sum in the control sample was 94.2%. In the sample with the highest concentration of histidine in this experiment (8 g/l), the percentage of relative Lys0 was reduced to 81.5%. The decrease in Lys0 corresponded with the increase in relative levels of both Lys1 and Lys2. A dose dependent decrease in relative Lys0, and a dose dependent increase in relative Lys1 and Lys2 regions was observed in test conditions with increased histidine concentration. 
     Additional experiments were performed with multiple cell lines in chemically defined or hydrolysate based media to demonstrate the wide range of applicability of this method. The experimental setup for each of these experiments was similar to that described above. The results of the different experiments performed for adalimumab are summarized in  FIG. 45 ,  FIG. 46  and  FIG. 47 . A reduction in relative Lys0, and increase in relative Lys1 and Lys2 species with increased histidine was also observed in each case. 
     In addition to adalimumab, the utility of this method for acidic species reduction was evaluated for processes involving two other mABs. The experimental setup for each of these experiments was similar to that described in section above and in the materials and methods. The results for experiments corresponding to each mAB are summarized in  FIG. 48  and  FIG. 49 . For mAB1, a dose dependent reduction in relative levels of Lys0 was evident with increased histidine. However, for mAB2, the relative change was minimal within the histidine concentration range tested. 
     Effect of Amino Acid Modulation on Culture Media in 3 L Bioreactors 
     In this study, 3 L bioreactors were set up to confirm the effect of amino acid addition on lysine variant distribution in more controlled conditions (pH and DO) at a larger scale. 8 Bioreactors (1.5 L working volume) were set up with cell line 2 in IVGN production media. The process included a temperature shift from 37° C. to 33° C. when the cell density criterion of 6×10 6  cells/ml was met. The pH was controlled via CO 2  gas flow/0.5 N Sodium Hydroxide base at a starting pH of 7.1 that was subsequently allowed to ramp down to 6.9 over the initial three days of the process. The dissolved oxygen was controlled at 30% and the agitation rate was maintained at 200 rpm. The cultures were fed with Ex-Cell PFCHO (SAFC, 67411) (3% (v/v)) on Day 3 of culture and with 18.8 g of 40% (w/v) glucose solution on days when glucose in the reactor was measured to be below 3 g/l. The test conditions included the amino acid concentration in media to be as follows: Control (1 g/l arginine and 1 g/l lysine), 3 g/l Arginine, 3 g/l Arginine/2 g/l Lysine and 3 g/l Arginine/3 g/l Lysine. Reactors were run in duplicates for each condition. 
     The culture performance was comparable between the different conditions with similar growth and viability profiles ( FIG. 50, 51 ). The cultures were harvested on Day 11 with the harvest viability between 40-50% in all the different conditions. Culture harvests were processed through protein A purification and WCX-10 analysis for quantification of the lysine variants. The lysine variant distribution in the control samples were 86.7% (Lys 0), 11.1% (Lys 1) and 2.2% (Lys 2). The Lys 0 was reduced 72.1% in the condition with the highest concentration of amino acids (3 g/l Arginine/3 g/l Lysine sample) ( FIG. 53 ). The decrease in Lys 0 corresponded with the increase in relative levels of both Lys 1 and Lys 2. Thus, increase of amino acids arginine and lysine can modulate lysine distribution even in 3 L bioreactors under controlled conditions of temperature and pH. 
     Effect of Ornithine Modulation on Culture Media 
     In this example, the effect of increased ornithine concentration was tested both individually as well as in combination with other amino acids arginine and lysine. The study was performed with adalimumab producing cell line 2 in media 1. The experiment was carried out in 500 ml shake flasks (200 ml working volume) and was run on shaker platforms set at 180 rpm in incubators set to be controlled at 35.0° C. and 5% CO 2 . The conditions tested included a control (only 1 g/l arginine and 1 g/l lysine), and test conditions including condition 2 (1 g/l ornithine, 1 g/l arginine, 1 g/l lysine), test condition 3 (4 g/l ornithine, 1 g/l arginine and 1 g/l lysine), test conditions 4 (1 g/l ornithine, 5 g/l arginine, 1 g/l lysine), test condition 5 (0 g/l ornithine, 5 g/l arginine, 2 g/l lysine), and test condition 6 (1 g/l ornithine, 5 g/l arginine, 2 g/l lysine). The cell culture performed comparably between the control and the test conditions with similar growth and viability profiles ( FIG. 54 ,  FIG. 55 ). Samples were collected for all conditions on day 10 for titer, which were comparable ( FIG. 56 ). The cultures were harvested at day 10, processed through protein A purification and WCX-10 analysis, and the relative fractions of lysine variants were estimated. There was no significant change in the lysine variant distribution in the conditions where only the ornithine concentration was increased (at 1 g/l or 4 g/l) compared to the control. However, the combination of ornithine increase with arginine or lysine increase reduced the relative level of Lys0 to 81.9% in comparison with the condition with just arginine and lysine increase with a relative level Lys0 of 84.7% ( FIG. 57 ). Thus, the increase of ornithine may exhibit synergistic effects in modulating lysine variant distribution when added in combination with arginine and lysine. 
     Effect of Increase in Concentration of a Combination of Arginine, Lysine, Histidine, and Ornithine to Culture Media 
     In this experiment, the combined use of the four amino acids arginine, lysine, histidine and ornithine for modulation of the lysine variants is demonstrated. The experiment described here was performed using adalimumab producing cell line 2 in chemically defined media (media 1). The concentration range for arginine and lysine in this experiment was 1-3 g/l while the concentration range for histidine and ornithine in this experiment was between 0-2 g/l In comparison to the lower concentrations, or conditions where amino acids were supplemented individually, a further reduction in Lys0 relative to lysine sum was observed in conditions where combinations of amino acids were increased in the media ( FIG. 58 ). A progressive decrease was observed in relative Lys0 when more amino acids were increased in combination. The percentage of relative Lys0 was reduced from 94.9% in the control sample to 73.9% in the sample with all four amino acid concentrations increased. 
     Effect of Increase in Concentration of a Combination of Arginine, Lysine, Histidine, and Zinc to Culture Media 
     In this experiment, the combined use of zinc and the three amino acids arginine, lysine, and histidine for lysine species modulation is demonstrated. The experiment described here was performed using adalimumab producing cell line 1 in chemically defined media (media 1). The concentration range for arginine and lysine in this experiment was between 1-3 g/l. The concentration range for histidine was between 0-1 g/l. The concentration range for zinc in this experiment was 30 μM-60 μM. Using the data from the experiment, a model predicting the effects of addition of these supplements to media for relative Lys0 reduction (R 2 : 0.98, P=0.09) is described in  FIG. 59 . The model predicted a contribution from each of the amino acids towards relative Lys0 reduction. The model also predicted an increase in relative Lys0 with an increase in zinc, which further supports the claim that reduction of zinc in culture causes a reduction in relative Lys0. It may be also possible to utilize this model to predict the choice of concentrations of these different components to the media, in order to achieve a target reduction in relative Lys0. 
     Effect of Supplementation of Single Peptides, Dipeptides and Tripeptides to Culture Media 
     In this experiment, the use of the three amino acid monomers arginine, lysine, and histidine, use of the dipeptides lys-lys and arg-lys, and use of the tripeptides lys-lys-lys, his-arg-lys, and arg-his-lys for lysine species modulation is demonstrated. The experiment described here was performed using adalimumab producing cell line 1 in chemically defined media (media 1). The concentration range for each peptide set tested in this experiment was between 0-4 g/l. In comparison to the control, a reduction in Lys0 relative to lysine sum was also observed in conditions where dipeptides and tripeptides were supplemented to the media ( FIG. 60 ). A decrease was observed in relative Lys0 when polypeptides were supplemented. The percentage of relative Lys0 was reduced from 88.0% in the control sample to 71.9% in a sample supplemented with tripeptide arg-his-lys (4 g/l), and to 74.0% in a sample supplemented with his-arg-lys (2 g/l). 
     6.2.3 Conclusion 
     The experiments outlined above demonstrate the different methods that can be used either by themselves or in suitable combinations to modulate the lysine variant distribution profile of a protein of interest. Specifically, increasing the concentration in culture media of the amino acids lysine, arginine, histidine, or combinations thereof along with ornithine and limiting the concentration of zinc in media, resulted in the relative modulation of the lysine variants with a decrease in the relative levels of Lys 0 and a corresponding increase in both Lys 1 and Lys 2. 
     6.3 Methods for Modulating the Lysine Variant Distribution in Cell Culture by Adjusting Process Parameters 
     6.3.1 Materials and Methods 
     Cell Source and Adaptation Cultures 
     Three adalimumab producing CHO cell lines were employed in the studies covered here. Upon thaw, adalimumab producing cell line 1 was cultured in a chemically defined basal media (media 1) or hydrolysate based growth media (media 2) in a combination of vented shake flasks on a shaker platform at 110 rpm and 20 L wavebags in a 35° C., 5% CO 2  incubator. In some cases, the culture might be transferred into a seed reactor with pH 7.1, 35° C. and 30% DO. In some cases, the culture was adapted to either media 1 or media 2 by propagated in a 10 L or 20 L wavebag for 7-13 days with one or two passages before initiating production stage cultures. 
     Upon thaw, adalimumab producing cell line 3 was cultured in chemically defined growth media (media 1) in a combination of vented shake flasks on a shaker platform at 140 rpm and 20 L wave bags. Cultures were propagated in a 36° C., and 5% CO 2  incubator to obtain the required number of cells to be able to initiate production stage cultures. 
     Cell Culture Media 
     Media 1, the chemical defined growth or production media, was prepared from basal IVGN CD media GIA1. For preparation of the IVGN CD media formulation, the proprietary media was supplemented with L-glutamine, sodium bicarbonate, sodium chloride, and methotrexate solution. Production media consisted of all the components in the growth medium, excluding methotrexate. For cultures with adalimumab producing cell line 1 and mAb2 producing cell line, the medium was also supplemented with insulin. In some cases, 10 mM or 5 mM of Galactose (Sigma, G5388) and 0.2 μM or 10 μM of Manganese (Sigma, M1787) were supplemented into production medium for cultures with adalimumab producing cell line 3 and adalimumab producing cell line 1, respectively. Osmolality was adjusted by the addition of sodium chloride. All media was filtered through filter systems (0.22 μm PES) and stored at 4° C. until usage. 
     For the hydrolysate-based formulation (media 2), the growth media was composed of PFCHO (proprietary CD formulation from SAFC), Dextrose, L-Glutamine, L-Asparagine, HEPES, Poloxamer 188, Ferric Citrate, Recombinant Human Insulin, Yeastolate (BD), Phytone Peptone (BD), Mono- and Di-basic Sodium Phosphate, Sodium Bicarbonate, Sodium Chloride and methotrexate. Production media consisted of all the components listed in the growth medium, excluding methotrexate. 
     Production Cultures 
     Production cultures were initiated in 3 L Bioreactors (Applikon). The bioreactors (1.5-2.0 L working volume) were run at the following conditions (except for the different experimental conditions): 35° C., 30% DO (dissolved oxygen), 200 rpm, pH profile from 7.1 to 6.9 in three days and pH 6.9 thereafter. In all experiments, the cells were transferred from the wavebag to the production stage at a split ratio of 1:5.6 (except experiments with mAb2 producing cell line where the split ratio was 1:5). When the media glucose concentration reduced to less than 3 g/L, approximately 1.25% (v/v) of 40% glucose stock solution was fed 
     The harvest procedure of reactors involved centrifugation of the culture sample at 3,000 RPM for 30 min and storage of supernatant in PETG bottles at −80° C. before submission for protein A purification and WCX-10 analysis. 
     WCX-10 Assay 
     The acidic species and other charge variants present in cell culture harvest samples were quantified. Cation exchange chromatography was performed on a Dionex ProPac WCX-10, Analytical column (Dionex, CA). 
     For adalimumab samples, the mobile phases used were 10 mM Sodium Phosphate dibasic pH 7.5 (Mobile phase A) and 10 mM Sodium Phosphate dibasic, 500 mM Sodium Chloride pH 5.5 (Mobile phase B). A binary gradient (94% A, 6% B: 0-20 min; 84% A, 16% B: 20-22 min; 0% A, 100% B: 22-28 min; 94% A, 6% B: 28-34 min) was used with detection at 280 nm. The WCX-10 method used for mAb2 samples used different buffers. The mobile phases used were 20 mM (4-Morpholino) ethanesulfonic Acid Monohydrate (MES) pH 6.5 (Mobile phase A) and 20 mM MES, 500 mM Sodium Chloride pH 6.5 (Mobile phase B). An optimized gradient (minute/% B): 0/3, 1/3, 46/21, 47/100, 52/100, 53/3, 58/3 was used with detection at 280 nm. Quantitation is based on the relative area percent of detected peaks, as described above. 
     6.3.2 Results and Discussion 
     Effect of Process pH in Media 1 with cell line 1 
     Five different pH conditions were assessed in this study: 7.1, 7.0, 6.9, 6.8 and 6.7. The cultures were started at pH set point of 7.1; then were ramped down to the target pH set points within 4 days. All cultures reached similar maximum viable cell densities on day 8, except for the culture at pH 6.7 condition, for which the maximum cell density was much lower than the other cultures ( FIG. 61 ). In addition, the viability of the culture at pH 7.1 and pH 7.0 dropped much earlier than the other cultures ( FIG. 62 ). The viability of cultures at pH 7.1 and pH 7.0 were 38% and 54% on day 10, respectively; while the viability of the cultures at lower pH (including pH 6.9, 6.8 and 6.7) was above 70% on the same day. Samples were taken in the last three days of the cultures and measured for titer. The titer of each tested condition increased corresponding to the decrease in pH, from 1.2 g/l in the pH 7.1 condition to 1.8 g/l in the pH 6.8 condition; however, product titer was not continued to increase at pH 6.7 (1.6 g/l) ( FIG. 63 ). The cultures were harvested at ≦50% viability. The harvest was protein A purified, then analyzed using WCX-10. From the WCX-10 analysis, the lysine variant distribution was characterized and the relative proportion of Lys0, Lys 1 and Lys2 variants were quantified as a fraction of the total Lysine sum. The relative fraction of Lys0 increased with decrease in pH from 82.0% in the pH 7.1 condition to 88.7% in the pH 6.7 condition, with corresponding 5.6% decrease in relative levels of Lysl and 1.1% decrease in Lys2 ( FIG. 64 ). 
     Effect of Process pH in Media 2 with cell line 1 
     Three different pH conditions were assessed in this study: 7.0, 6.9, and 6.8. The cultures were started at pH of 7.1; then were ramped down to the target pH set points within 3 days of culture. The viable cell density and viability were comparable across the different pH set points until day 8. After day 8, the viable cell density and viability were slightly higher corresponding to lower pH set points ( FIG. 65 ,  FIG. 66 ). The cultures were harvested at ˜50% viability. The product titer was slightly higher at pH 6.8 comparing to pH 6.9 and 7.0 ( FIG. 67 ). The resulting peak areas from WCX-10 analysis were quantified ( FIG. 68 ). The relative fraction of Lys0 increased with decrease in pH from 76.8% in the pH 7.0 condition to 80.5 in the pH6.8 condition, with corresponding 2.8% decrease in relative levels of Lys 1 and 0.9% decrease in Lys2. 
     Effect of Process pH in Media 1 with Cell Line 3 
     Five different pH conditions were assessed in this study: 7.1 7.0, 6.9, 6.8, and 6.7. The cultures were started at pH set point of 7.1; then were ramped down to the target pH set points within 4 days of culture. The pH set points showed significant effect on the cell growth and viability with this cell line and media. Cell density was lower at higher pH and viability also dropped earlier at higher pH ( FIG. 69 ,  FIG. 70 ). The cells were harvested at an approximate viability of 50%. The titer was slightly increased as the pH was reduced, reached the highest titer at pH 6.8 condition ( FIG. 71 ). The resulting peak areas from WCX-10 analysis were quantified ( FIG. 72 ). The relative fraction of Lys0 increased with decrease in pH from 88.1% in the pH 7.1 condition to 93.9% in the pH 6.7 condition, with corresponding 4.6% decrease in relative levels of Lys1 and 1.7% decrease in Lys2. 
     Effect of Process Temperature in Media 1 with Cell Line 1 
     Three different temperature conditions were assessed: 33° C., 35° C. and 37° C. The cultures were harvested at the target viability of 50% for each condition. At a lower temperature, the culture duration was longer with higher viability through the culture ( FIG. 73 ,  FIG. 74 ). Samples were collected for titer analysis on harvest days. The titer for all conditions was comparable ( FIG. 75 ). The harvest was taken through protein A purification before WCX-10 analysis. From the WCX-10 analysis, the lysine variant distribution was characterized and the relative proportion of Lys0, Lys1 and Lys2 variants were quantified as a fraction of the total Lysine sum ( FIG. 76 ). The relative fraction of Lys0 increased with increase in temperature from 80.3% in the 33° C. condition to 86.6% in the 37° C. condition, with corresponding 5.3% decrease in relative levels of Lys1 and 1.0% decrease in Lys2. Thus, lowering the process temperature seems to provide an effective method to increase the relative proportion of the product antibody with C-terminal lysine on one or both the heavy chains (Lys 1/Lys 2). 
     Effect of Process Temperature in Media 1 with Cell Line 2 
     Three different temperature conditions were assessed: 33° C., 35° C., and 37° C. The cultures were harvested at the target viability of 50% for each condition. The VCD and viability profiles were similar for the 37° C. and 35° C. conditions, but the 33° C. condition took longer to drop to 50% ( FIG. 77 ,  FIG. 78 ). The product titers were comparable at different temperature conditions ( FIG. 79 ). The harvest was taken through protein A purification before WCX-10 analysis. From the WCX-10 analysis, the lysine variant distribution was characterized and the relative proportion of Lys0, Lys1 and Lys2 variants were quantified as a fraction of the total Lysine sum ( FIG. 80 ) The relative fraction of Lys0 increased with increase in temperature from 88.6% in the 33° C. condition to 93.1% in the 37° C. condition, with corresponding 3.8% decrease in relative levels of Lys1 and 0.7% decrease in Lys2. Thus, the results here are consistent with that observed for cell line 1. 
     6.3.3 Conclusion 
     The experiments outlined above demonstrate the different methods that can be used either by themselves or in suitable combinations to control the lysine variant distribution profile of a protein of interest, e.g., the antibody adalimumab. These experiments also indicate that altering cell culture process parameters on-line can be used to modulate the lysine variant distribution. Increasing pH set points or reducing temperature set points can lead to a relative shift in lysine variant distribution from Lys0 to Lys1 Lys2. 
     Patents, patent applications, publications, product descriptions, GenBank Accession Numbers, and protocols that may be cited throughout this application, the disclosures of which are incorporated herein by reference in their entireties for all purposes. For example, but not by way of limitation, patent applications designated by the following U.S. application Ser. Nos. are incorporated herein by reference in their entireties for all purposes: 13/803,808; 13/830,583; 13/829,989; 13/831,181; and 13/804,220.