Methods for controlling the galactosylation profile of recombinantly-expressed proteins

Information

  • Patent Grant
  • 9365645
  • Patent Number
    9,365,645
  • Date Filed
    Wednesday, February 3, 2016
    8 years ago
  • Date Issued
    Tuesday, June 14, 2016
    8 years ago
Abstract
The present invention relates to methods for modulating the glycosylation profile of recombinantly-expressed proteins. In particular, the present invention relates to methods of controlling the galactosylation profile of recombinantly-expressed proteins by supplementing production medium, e.g., a hydrolysate-based or a chemically defined medium, with manganese and/or D-galactose.
Description
1. INTRODUCTION

The present invention relates to methods for modulating the glycosylation profile of recombinantly-expressed proteins. In particular, the present invention relates to methods of controlling the galactosylation profile of recombinantly-expressed proteins by supplementing production media with manganese and/or galactose.


2. BACKGROUND OF THE INVENTION

Utilization of a particular type of production media, e.g., hydrolysate-based media or chemically defined media (“CD” or “CDM”), for CHO cell cultures producing recombinant proteins can enhance cell growth and target protein production. However, recombinant proteins produced in different CD or hydrolysate-based media can exhibit large differences in their product quality profile. In certain instances, this variability can lead to increases in the fraction of the agalactosyl fucosylated biantennary oligosaccharides NGA2F+NGA2F-G1cNAc and decreases in the fraction of galactose-containing fucosylated biantennary oligosaccharides NA1F+NA2F. Shifts in the glycosylation profile of recombinant proteins of this magnitude are significant as these shifts may render the resulting production lots of the target protein out of compliance with approved process specifications.


3. SUMMARY OF THE INVENTION

The present invention relates to methods for modulating the glycosylation profile of recombinantly-expressed proteins. In particular, the present invention relates to methods of controlling the galactosylation profile of recombinantly-expressed proteins by supplementing production media with manganese and/or galactose. In certain embodiments the production media is a hydrolysate-based media or a CD media.


In certain embodiments, the present invention is directed to methods of controlling the galactosylation profile of recombinantly-expressed antibody. In certain embodiments, the recombinantly-expressed antibody is an anti-TNFα antibody. In certain embodiments, the recombinantly-expressed anti-TNFα antibody is adalimumab.


In certain embodiments, the present invention is directed to methods of controlling the galactosylation profile of recombinantly-expressed proteins by supplementing a production medium, e.g., a hydrolysate-based or a CD medium, used in the production of recombinantly-expressed proteins with manganese and/or galactose. In certain embodiments, the manganese supplement can take the form of any biologically-acceptable manganese salt, for example, but not limited to, manganese (II) chloride. In certain embodiments, the galactose supplement can take the form of any biologically-acceptable galactose-containing compound, for example, but not limited to, D-(+)-galactose.


In certain embodiments, the present invention is directed to methods of controlling the galactosylation profile of recombinantly-expressed proteins by supplementing a production medium, e.g., a hydrolysate-based or a CD medium, used in the production of recombinantly-expressed proteins with a sufficient amount of manganese and/or a manganese-containing supplement to achieve the following manganese concentrations in the production media: at least about 0.1, at least about 0.2, at least about 0.5, at least about 1.0, at least about 10, at least about 20, at least about 25, at least about 40, at least about 50, at least about 60, at least about 75, at least about 80, or at least about 100 μM, wherein that production media is used to dilute a supplement-free cell culture growth media containing no supplement by a ratio of about 1:4 or about 1:5 (supplement-free growth media: supplemented production media). In certain embodiments, the present invention is directed to methods of controlling the galactosylation profile of recombinantly-expressed proteins by supplementing a production medium, e.g., a hydrolysate-based or a CD medium, used in the production of the recombinantly-expressed proteins with sufficient galactose and/or galactose-containing supplement to achieve the following galactose concentrations in the production media: at least about 1, at least about 4, at least about 5, at least about 10, at least about 15, at least about 20, at least about 30, at least about 40, at least about 60, or at least about 100 mM, wherein that production media is used to dilute a supplement-free cell culture growth media containing no supplement by a ratio of about 1:4 or about 1:5 (supplement-free growth media: supplemented production media).


In certain embodiments, the present invention is directed to methods of controlling the galactosylation profile of recombinantly-expressed proteins by supplementing a production medium, e.g., a hydrolysate-based or a CD medium, used in the production of recombinantly-expressed proteins with sufficient manganese and/or a manganese-containing supplement and sufficient galactose and/or galactose-containing supplement to achieve at least about the following manganese (Mn) and galactose (Gal) concentrations in the production media presented as Mn (μM)/Gal (mM): 0/1, 0/4, 0/5, 0/10, 0/15, 0/20, 0/30, 0/40, 0/60, 0/100, 0.1/0, 0.2/0, 0.5/0, 1.0/0, 10/0, 20/0, 25/0, 40/0, 50/0, 75/0, 80/0, 100/0, 0.2/1, 0.2/4, 0.2/30, 0.5/1, 0.5/4, 0.5/30, 10/10, 10/20, 10/40, 20/10, 20/20, 20/40, 25/15, 40/10, 40/20, 40/40, 40/100, 50/30, 60/20, 60/40, 60/100, 80/20, 80/40, 80/100, 100/20, 100/40, 100/100, wherein that production media is used to dilute a supplement-free cell culture growth media containing no supplement by a ratio of about 1:4 or about 1:5 (supplement-free growth media: supplemented production media).


In certain embodiments, the present invention is directed to methods of controlling the galactosylation profile of recombinantly-expressed proteins by supplementing a production medium, e.g., a hydrolysate-based or a CD medium, used in the production of recombinantly-expressed proteins with sufficient manganese and/or a manganese-containing supplement and sufficient galactose and/or galactose-containing supplement to achieve at least about the following manganese (Mn) and galactose (Gal) concentrations in the production media presented as Mn (μM)/Gal (mM): 0.2/1, 0.2/4, 0.2/30, 0.5/1, 0.5/4, 0.5/30, 10/10, 10/20, 10/40, 20/10, 20/20, 20/40, 25/15, 40/10, 40/20, 40/40, 40/100, 50/30, 60/20, 60/40, 60/100, 80/20, 80/40, 80/100, 100/20, 100/40, 100/100, wherein that production media is used to dilute a supplement-free cell culture growth media containing no supplement by a ratio of about 1:4 or about 1:5 (supplement-free growth media: supplemented production media).





4. BRIEF DESCRIPTION OF THE FIGURES


FIG. 1A depicts the culture growth of adalimumab-producing CHO cell line in CDM GIA-1 in batch shake flasks. FIG. 1B depicts the viability of adalimumab-producing CHO cell line in CDM GIA-1 in batch shake flasks. FIG. 1C depicts the normalized titer of adalimumab-producing CHO cell line in CDM GIA-1 in batch shake flasks.



FIG. 2A depicts the culture growth of adalimumab-producing CHO cell line in CDM GIA-1 in fed-batch 3 L bioreactors. FIG. 2B depicts the viability of adalimumab-producing CHO cell line in CDM GIA-1 in fed-batch 3 L bioreactors. FIG. 2C depicts the normalized titer of adalimumab-producing CHO cell line in CDM GIA-1 in fed-batch 3 L bioreactors.



FIG. 3A depicts the galactosylation profile (NGA2F+NGA2F-GlcNac) of adalimumab in CHO cell line in CDM GIA-1 in batch shake flasks. FIG. 3B depicts the galactosylation profile (NA1F+NA2F) of adalimumab in CHO cell line in CDM GIA-1 in batch shake flasks.



FIG. 4 depicts the percentage galactosylation change of adalimumab in CDM GIA-1 in batch shake flasks relative to control.



FIG. 5 summarizes the effect of manganese and/or galactose addition to CDM GIA-1 on galactosylation of adalimumab relative to control in CHO cell line.



FIG. 6A depicts the galactosylation profile (NGA2F+NGA2F-GlcNac) of adalimumab in CHO cell line in CDM GIA-1 in fed-batch 3 L bioreactors. FIG. 6B depicts the galactosylation profile (NA1F+NA2F) of adalimumab in CHO cell line in CDM GIA-1 in fed-batch 3 L bioreactors.



FIG. 7A depicts the culture growth of CHO cell line in CDM HyClone CDM4CHO in batch shake flasks. FIG. 7B depicts the viability of CHO cell line in CDM HyClone CDM4CHO in batch shake flasks.



FIG. 8A depicts the galactosylation profile (NGA2F+NGA2F-GlcNac) of adalimumab in CHO cell line in CDM HyClone CDM4CHO in batch shake flasks. FIG. 8B depicts the galactosylation profile (NA1F+NA2F) of adalimumab in CHO cell line in CDM HyClone CDM4CHO in batch shake flasks.



FIG. 9 summarizes the effect of manganese and/or galactose addition to CDM HyClone CDM4CHO on galactosylation of adalimumab relative to control in CHO cell line.



FIG. 10A depicts the culture growth of CHO cell line in hydrolysate media in batch shake flasks. FIG. 10B depicts the viability of CHO cell line in hydrolysate media in batch shake flasks.



FIG. 11A depicts the galactosylation profile (NGA2F+NGA2F-GlcNac) of adalimumab in CHO cell line in hydrolysate media in batch shake flasks. FIG. 11B depicts the galactosylation profile (NA1F+NA2F) of adalimumab in CHO cell line in hydrolysate media in batch shake flasks.



FIG. 12 summarizes the effect of manganese and/or galactose addition to hydrolysate media on galactosylation of adalimumab relative to control in CHO cell line.



FIG. 13A depicts the culture growth of adalimumab-producing CHO cell line #2 in CDM GIA-1 in batch shake flasks. FIG. 13B depicts the viability of adalimumab-producing CHO cell line #2 in CDM GIA-1 in batch shake flasks.



FIG. 14A depicts the galactosylation profile (NGA2F+NGA2F-GlcNac) of adalimumab in CHO cell line #2 in CDM GIA-1 in batch shake flasks. FIG. 14B depicts the galactosylation profile (NA1F+NA2F) of adalimumab in CHO cell line #2 in CDM GIA-1 in batch shake flasks.



FIG. 15 summarizes the effect of manganese and/or galactose addition to CDM GIA-1 on galactosylation of adalimumab relative to control in CHO cell line #2.



FIG. 16A depicts the culture growth of adalimumab-producing CHO cell line #3 in CDM GIA-1 in fed-batch 3 L bioreactors. FIG. 16B depicts the viability of adalimumab-producing CHO cell line #3 in CDM GIA-1 in fed-batch 3 L bioreactors. FIG. 16C depicts the normalized titer of adalimumab-producing CHO cell line #3 in CDM GIA-1 in fed-batch 3 L bioreactors.



FIG. 17A depicts the galactosylation profile (NGA2F+NGA2F-GlcNac) of adalimumab in CHO cell line #3 in CDM GIA-1 in fed-batch 3 L bioreactors. FIG. 17B depicts the galactosylation profile (NA1F+NA2F) of adalimumab in CHO cell line #3 in CDM GIA-1 in fed-batch 3 L bioreactors.



FIG. 18 summarizes the effect of manganese and/or galactose addition to CDM GIA-1 on galactosylation of adalimumab relative to control in CHO cell line #3.



FIG. 19A depicts the culture growth of adalimumab-producing NSO cell line in CDM PFBM-3/PFFM-4 fed-batch shake flasks. FIG. 19B depicts the viability of adalimumab-producing NSO cell line in CDM PFBM-3/PFFM-4 fed-batch shake flasks. FIG. 19C depicts the normalized titer of adalimumab-producing NSO cell line in CDM PFBM-3/PFFM-4 fed-batch shake flasks.



FIG. 20A depicts the galactosylation profile (NGA2F+NGA2F-GlcNac) of adalimumab in NSO cell line in CDM PFBM-3/PFFM-4 fed-batch shake flasks. FIG. 20B depicts the galactosylation profile (NA1F+NA2F) of adalimumab in NSO cell line in CDM PFBM-3/PFFM-4 fed-batch shake flasks.



FIG. 21 summarizes the effect of manganese and/or galactose addition to CDM PFBM-3/PFFM-4 on galactosylation of adalimumab relative to control in NSO cell line.



FIG. 22A depicts the culture growth of CHO cell line producing mAb #1 in CDM GIA-1 in batch shake flasks. FIG. 22B depicts the viability of CHO cell line producing mAb #1 in CDM GIA-1 in batch shake flasks.



FIG. 23A depicts the galactosylation profile (NGA2F+NGA2F-GlcNAc) of mAb #1 in CDM GIA-1 in batch shake flasks. FIG. 23B depicts the galactosylation profile (NA1F+NA2F) of mAb #1 in CDM GIA-1 in batch shake flasks.



FIG. 24 summarizes the effect of manganese and/or galactose addition to CDM GIA-1 on galactosylation of mAb #1 relative to control.



FIG. 25A depicts the culture growth of CHO cell line producing mAb #2 in CDM GIA-1 in fed-batch 3 L bioreactors. FIG. 25B depicts the viability of CHO cell line producing mAb #2 in CDM GIA-1 in fed-batch 3 L bioreactors. FIG. 25C depicts the normalized titer of CHO cell line producing mAb #2 in CDM GIA-1 in fed-batch 3 L bioreactors.



FIG. 26A depicts the glycosylation profile (NGA2F+NGA2F-GlcNAc) of mAb #2 in CDM GIA-1 in fed-batch 3 L bioreactors. FIG. 26B depicts the glycosylation profile (NA1F+NA2F) of mAb #2 in CDM GIA-1 in fed-batch 3 L bioreactors.



FIG. 27 summarizes the effect of manganese and/or galactose addition to CDM GIA-1 on galactosylation of mAb #2 relative to control.





5. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods modulating the glycosylation profile of recombinantly-expressed proteins. In particular, the present invention relates to methods of controlling (e.g., modulating) the galactosylation profile of recombinantly-expressed proteins by supplementing production medium, e.g., a hydrolysate-based or a CD medium, with manganese and/or galactose. For example, but not by way of limitation, the present invention demonstrates that supplementation of particular ranges of manganese and/or galactose concentrations to chemically defined media can be used to fine-tune the galactosylation profile of monoclonal antibodies produced in CHO and NSO cell lines. Similarly, supplementation of galactose alone to hydrolysate-based media is effective to modulate the galactosylation profile of the monoclonal antibody adalimumab produced in a CHO cell line in a concentration dependent manner. In view of such findings, the methods disclosed herein can be used to modulate the galactose content of recombinant proteins by controlling the amounts of manganese and/or galactose present in cell culture media. The studies described herein have also established that the changes in the galactosylation profiles obtained via implementation of the methods of the present invention are not only scale (1.5 L vs. 200 mL) and process independent (fed-batch in controlled bioreactor environment vs. batch in shake flasks), but also that no significant impact on culture growth and productivity is observed for most conditions studied.


A terminal galactose is added to NGA2F by β-galactosyltransferase enzyme in the presence of manganese chloride, to produce NA1F (in the case of an addition of a single terminal galactose) or NA2F (in the case of an addition of two terminal galactose molecules). This galactosyltransferase-mediated reaction employs UDP-galactose as the sugar substrate and Mn2+ as a cofactor for galactosyltransferase. Thus, without being bound by theory, it is believed that a change in protein homogeneity taking the form of an increase in the fraction of N-linked oligosaccharide NGA2F and a decrease in the fraction of NA1F+NA2F N-linked oligosaccharides could be caused by either an insufficient amount of the substrate (UDP-galactose), the cofactor for galactosyltransferase (Mn2+), or both.


In certain embodiments, the present invention is directed to methods of controlling the galactosylation profile of recombinantly-expressed antibody. In certain embodiments, the recombinantly-expressed antibody is an anti-TNFα antibody. In certain embodiments, the recombinantly-expressed anti-TNFα antibody is adalimumab.


In certain embodiments, the present invention is directed to methods of controlling the galactosylation profile of recombinantly-expressed proteins by supplementing a production medium, e.g., a hydrolysate-based or a CD medium, used in the production of recombinantly-expressed proteins with manganese and/or galactose. In certain embodiments, the manganese supplement can take the form of any biologically-acceptable manganese salt, for example, but not limited to, manganese (II) chloride. In certain embodiments, the galactose supplement can take the form of any biologically-acceptable galactose-containing compound, for example, but not limited to, D-(+)-galactose.


In certain embodiments, the present invention is directed to methods of controlling the galactosylation profile of recombinantly-expressed proteins by supplementing a production medium, e.g., a hydrolysate-based or a CD medium, used in the production of recombinantly-expressed proteins with a sufficient amount of manganese and/or a manganese-containing supplement to achieve at least about the following manganese concentrations in the production media: at least about 0.1, at least about 0.2, at least about 0.5, at least about 1.0, at least about 10, at least about 20, at least about 25, at least about 40, at least about 50, at least about 60, at least about 75, at least about 80, or at least about 100 μM, wherein that production media is used to dilute a supplement-free cell culture growth media containing no supplement by a ratio of about 1:4 or about 1:5 (supplement-free growth media: supplemented production media). In certain embodiments, the present invention is directed to methods of controlling the galactosylation profile of recombinantly-expressed proteins by supplementing a production medium, e.g., a hydrolysate-based or a CD medium, used in the production of recombinantly-expressed proteins with sufficient galactose and/or galactose-containing supplement to achieve at least about the following galactose concentrations in the production media: at least about 1, at least about 4, at least about 5, at least about 10, at least about 15, at least about 20, at least about 30, at least about 40, at least about 60, or at least about 100 mM, wherein that production media is used to dilute a supplement-free cell culture growth media containing no supplement by a ratio of about 1:4 or about 1:5 (supplement-free growth media: supplemented production media).


In certain embodiments, the present invention is directed to methods of controlling the galactosylation profile of recombinantly-expressed proteins by supplementing a production medium, e.g., a hydrolysate-based or a CD medium, used in the production ofrecombinantly-expressed proteins with sufficient manganese and/or a manganese-containing supplement and sufficient galactose and/or galactose-containing supplement to achieve at least about the following manganese (Mn) and galactose (Gal) concentrations in the production media presented as Mn (μM)/Gal (mM): 0/1, 0/4, 0/5, 0/10, 0/15, 0/20, 0/30, 0/40, 0/60, 0/100, 0.1/0, 0.2/0, 0.5/0, 1.0/0, 10/0, 20/0, 25/0, 40/0, 50/0, 75/0, 80/0, 100/0, 0.2/1, 0.2/4, 0.2/30, 0.5/1, 0.5/4, 0.5/30, 10/10, 10/20, 10/40, 20/10, 20/20, 20/40, 25/15, 40/10, 40/20, 40/40, 40/100, 50/30, 60/20, 60/40, 60/100, 80/20, 80/40, 80/100, 100/20, 100/40, 100/100, wherein that production media is used to dilute a supplement-free cell culture growth media containing no supplement by a ratio of about 1:4 or about 1:5 (supplement-free growth media: supplemented production media).


In certain embodiments, the present invention is directed to methods of controlling the galactosylation profile of recombinantly-expressed proteins by supplementing a production medium, e.g., a hydrolysate-based or a CD medium, used in the production of recombinantly-expressed proteins with sufficient manganese and/or a manganese-containing supplement and sufficient galactose and/or galactose-containing supplement to achieve at least about the following manganese (Mn) and galactose (Gal) concentrations in the production media presented as Mn (μM)/Gal (mM): 0.2/1, 0.2/4, 0.2/30, 0.5/1, 0.5/4, 0.5/30, 10/10, 10/20, 10/40, 20/10, 20/20, 20/40, 25/15, 40/10, 40/20, 40/40, 40/100, 50/30, 60/20, 60/40, 60/100, 80/20, 80/40, 80/100, 100/20, 100/40, 100/100, wherein that production media is used to dilute a supplement-free cell culture growth media containing no supplement by a ratio of about 1:4 or about 1:5 (supplement-free growth media: supplemented production media).


In certain embodiments, the production medium, e.g., a hydrolysate-based or a CD medium, used in the production of a recombinantly-expressed protein is supplemented with manganese and not galactose. For the shake flasks studies in Example 1, but not by way of limitation, addition of manganese and not galactose to production IVGN CDM GIA-1 lowered the NGA2F+NGA2F-GlcNac sum by 6% to 9% and increased the NA1F+NA2F sum by 8% to 9% (FIGS. 3, 4, and 5). No further increase in manganese concentration was explored in the experimental design due to the growth inhibition observed at about 100 μM.


In certain embodiments, the production medium, e.g., a hydrolysate-based or a CD medium, used in the production of a recombinantly-expressed protein is supplemented with galactose and not manganese. For the shake flasks studies in Example 1, but not by way of limitation, addition of galactose only to production IVGN CDM GIA-1 lowered the NGA2F+NGA2F-GlcNac sum by 3% to 7% and increased NA1F+NA2F by 3% to 7% (FIGS. 3, 4, and 5). These findings indicate that a manganese concentration of about 100 μM and a galactose concentration of about 100 mM represent the maximum range of interest for this Example 1.


In certain embodiments, the production medium, e.g., a hydrolysate-based media or a CD media, used in the production of a recombinantly-expressed protein is supplemented with both manganese and galactose. For example, but not by way of limitation, the studies outlined in Example 1 indicate that the addition of combinations of manganese and galactose to production IVGN CDM GIA-1 resulted in a significant decrease in the NGA2F+NGA2F-GlcNac sum of 11% to 26% and a corresponding significant increase in the NA1F+NA2F sum of 13% to 23% as compared to the control condition where no manganese or galactose were added to the production media (FIGS. 3, 4, and 5). The effect on modulation of galactosylation of adalimumab in production IVGN CDM GIA-1 with the combined addition of manganese chloride and galactose was synergistic. In particular, the combined addition of manganese chloride and galactose decreased the NGA2F+NGA2F-GlcNac sum and increased the NA1F+NA2F sum by a larger percentage than by adding manganese or galactose alone and summing up their individual effects. For example, but not by way of limitation, addition of 40 μM manganese chloride alone reduced the NGA2F sum by 6%, and addition of 40 mM galactose alone decreased the NGA2F+NGA2F-GlcNac sum by 6%. However, the combined addition of manganese chloride and galactose at these same concentrations (i.e. 40 μM manganese+40 mM galactose) led to an 18% reduction in the NGA2F+NGA2F-GlcNac sum, 6% higher than their combined individual contributions to the reduction of the NGA2F+NGA2F-GlcNac sum. We define this effect as being synergistic and maintain this definition throughout the invention. The largest percent decrease in the NGA2F+NGA2F-GlcNac sum of approximately 26% was observed with the combined addition of 100 μM manganese chloride and 100 mM galactose. The largest percent increase in the NA1F+NA2F sum of approximately 23% was recorded with the combined addition of 60 μM manganese chloride and 100 mM galactose.


For the fed-batch bioreactor study described in Example 1, two manganese chloride and galactose combinations were studied and the results indicate that the decrease in the NGA2F+NGA2F-GlcNac sum and the corresponding increase in the NA1F+NA2F sum was scale (1.5 L vs. 200 mL) and process independent (fed-batch in controlled bioreactor environment vs. batch in shake flasks). For example, but not by way of limitation, the combined addition of 40 μM manganese chloride and 20 mM galactose to both production basal CDM GIA-1 and feed CDM JCL-5 decreased the NGA2F+NGA2F-GlcNac sum by 26% and increased the NA2F+NA2F sum by 27% compared to the control cultures (FIG. 6). A further increase in the galactose concentration to 40 mM in addition to manganese supplementation at 40 μM concentration resulted in an additional 3% decrease in the NGA2F+NGA2F-GlcNac sum, and a corresponding 3% increase in the NA1F+NA2F sum


In certain embodiments, the present invention is directed to the supplementation of CD media used in the production of a recombinantly-expressed protein with galactose and/or manganese. That such supplementation is effective across distinct CD media is evidenced by the results outlined in Example 2. Specifically, Example 2 results indicate that the addition of manganese chloride alone within the range of 0 to 40 μM to production CDM HyClone CDM4CHO decreased the NGA2F+NGA2F-GlcNac sum by a maximum of 5% in a concentration dependent manner (FIG. 8). A comparable maximum increase of 4% in the NA1F+NA2F sum was also achieved. Addition of galactose alone up to a maximum concentration of 40 mM yielded a 6% maximum decrease in the NGA2F+NGA2F-GlcNac sum and a corresponding 6% increase in the NA1F+NA2F sum. Modulation of galactosylation was also observed in production CDM HyClone CDM4CHO cultures supplemented with both manganese chloride and galactose. An additive effect was observed in cultures supplemented with both manganese chloride and galactose. The combined addition of manganese chloride and galactose decreased the NGA2F+NGA2F-GlcNac sum and increased the NA1F+NA2F sum by a comparable percentage as when manganese or galactose were added alone and their individual effects were summed up (FIG. 9). For example, but not by way of limitation, addition of 40 μM manganese chloride alone reduced the NGA2F+NGA2F-GlcNac sum by 5%, and addition of 40 mM galactose alone decreased the NGA2F sum by 6%. The combined addition of manganese chloride and galactose at these same concentrations (i.e. 40 μM manganese+40 mM galactose) led to a 12% reduction in the NGA2F+NGA2F-GlcNac sum. We define this effect as being additive and maintain this definition throughout the invention. The highest percentage decrease in the NGA2F sum of 12% and the corresponding 11% increase in the NA1F+NA2F sum was observed for the culture supplemented with 40 μM manganese chloride and 40 mM galactose.


In certain embodiments, the present invention is directed to the supplementation of a hydrolysate-based media used in the production of a recombinantly-expressed protein with galactose and/or manganese. For example, as outlined in Example 3, the addition of manganese chloride alone within the range of 0 to 40 μM to hydrolysate-based production media decreased the NGA2F+NGA2F-GlcNac sum by approximately 1%, although that change is within the oligosaccharide assay variability (FIG. 11). The addition of galactose alone up to a maximum concentration of 40 mM yielded a maximum decrease of 4% in the NGA2F+NGA2F-GlcNac sum and a corresponding 4% maximum increase in the NA1F+NA2F sum. Such oligosaccharide profile changes achieved with the addition of galactose alone are comparable to the changes recorded when combinations of galactose and manganese chloride were added to the hydrolysate-based media. For example, the combined addition of manganese chloride ranging from 0 to 40 μM and galactose ranging from 0 to 40 mM to hydrolysate-based media led to an approximate 5% maximum decrease in the NGA2F+NGA2F-GlcNac sum and a corresponding 3% increase in the NA1F+NA2F sum (FIG. 12). The highest percentage decrease of 5% in the NGA2F+NGA2F-GlcNac sum and the corresponding 4% increase in the NA1F+NA2F sum was observed for the culture supplemented with 40 mM galactose and either 20 μM or 40 μM manganese chloride.


The compositions and methods of the present invention also find use across distinct cell lines. For example, but not by way of limitation, the study described in Example 4 illustrates that the supplementation of a CD media, GIA-1, with galactose and/or manganese is effective to modulate galactosylation of adalimumab produced using a CHO cell line distinct from that employed in Examples 1-3. For example, but not by way of limitation, when using this alternative cell line, the addition of manganese chloride alone within the range of 0 to 20 μM to production CDM GIA-1 decreased the NGA2F+NGA2F-GlcNac sum in a concentration dependent manner and increased the NA1F+NA2F sum by approximately the same percentage. A maximum decrease of 22% in the NGA2F+NGA2F-GlcNac sum and a maximum corresponding increase of 23% in the NA1F+NA2F sum was observed with the addition of 20 μM manganese chloride (FIG. 14). Similarly, a concentration dependent decrease in the NGA2F+NGA2F-GlcNac sum and a corresponding increase in the NA1F+NA2F sum was observed with the addition of galactose alone in the range of 0 to 20 mM. A maximum decrease of 9% in the NGA2F+NGA2F-GlcNac sum and a corresponding maximum increase of 10% in the NA1F+NA2F sum was observed with the addition of 20 mM galactose. Similarly, an additive effect was observed for the oligosaccharide profiles of adalimumab produced in cultures supplemented with the combined addition of manganese chloride and galactose to GIA-1 media (FIG. 15). For example, but not by way of limitation, addition of 10 μM manganese chloride alone reduced the NGA2F+NGA2F-GlcNAc sum by 18%, and addition of 10 mM galactose alone decreased the NGA2F+NGA2F-GlcNAc sum by 6%. The combined addition of manganese chloride and galactose at these same concentrations led to a 24% reduction in the NGA2F+NGA2F-GlcNac sum. The highest percentage decrease of 35% in the NGA2F+NGA2F-GlcNAc sum and the corresponding increase of 37% in the NA1F+NA2F sum were observed for the culture supplemented with 40 μM manganese chloride and 20 mM galactose.


That the compositions and methods of the present invention also find use across distinct cell lines is further reinforced by the results of Example 5, which employs a third adalimumab-producing cell line that is distinct from either of the adalimumab-producing cell lines of Examples 1-4. For example, but not by way of limitation, when using this third cell line, the addition of manganese chloride alone within the range of 0 to 1 μM to production CDM GIA-1 decreased the NGA2F+NGA2F-GlcNAc sum in a concentration dependent manner and increased the NA1F+NA2F sum by approximately the same percentage. A maximum decrease of 26% in the NGA2F+NGA2F-GlcNAc and a corresponding increase of 28% in the NA1F+NA2F oligosaccharides were observed with the addition of 1 μM manganese chloride (FIG. 17). The addition of galactose alone at 30 mM concentration to production CDM GIA-1 decreased the NGA2F+NGA2F-GlcNAc sum by 4% and increased the NA1F+NA2F sum by 3%. Furthermore, when manganese chloride and galactose were supplemented together into the production basal and feed media, the results demonstrated a synergistic benefit towards the decrease in the NGA2F+NGA2F-GlcNAc and the increase in the NA1F+NA2F oligosaccharides which is consistent with the results demonstrated in Example 1 (FIG. 18). For example, but not by way of limitation, at 0.2 μM manganese chloride plus 30 mM galactose the observed 25% decrease in the NGA2F+NGA2F-GlcNAc sum was 6% more than the sum of the decrease observed with the addition of 0.2 μM manganese chloride alone (15%) and that of 30 mM galactose alone (4%). Similarly, the resulting 24% increase in the NA1F+NA2F sum was more than the sum of the increase observed with the addition of 0.2 μM manganese chloride alone (16%) and that of 30 mM galactose alone (3%). The combined supplementation of 0.5 μM manganese chloride+30 mM galactose also demonstrated a synergistic effect on the galactosylation profile of adalimumab produced in this third cell line. A maximum decrease compared to the control condition of 34% in the NGA2F+NGA2F-GlcNac and a corresponding 34% maximum increase in the NA1F+NA2F oligosaccharides was observed with the combined addition of 0.5 μM manganese chloride and 30 mM galactose to chemically defined GIA-1 media.


That the compositions and methods of the present invention also find use across distinct types of cell lines is further reinforced by the results of Example 6, which employs a fourth adalimumab-producing cell line that is distinct from the adalimumab-producing cell lines of Examples 1-5, in that it is an NSO cell line. For example, but not by way of limitation, when using this NSO cell line, the addition of manganese chloride alone within the range of 0 to 0.5 μM to production CDM PFBM-3/PFFM-4 decreased the NGA2F+NGA2F-GlcNac sum in a concentration dependent manner and increased the NA1F+NA2F sum by approximately the same percentage. A maximum decrease of 18% in the NGA2F+NGA2F-GlcNac sum and a corresponding increase of 20% in the NA1F+NA2F sum were observed with the addition of 0.5 μM manganese chloride (FIG. 20). However, manganese doses greater than 0.5 μM were not explored further due to cytotoxicity effects. Similarly, a concentration dependent decrease in the NGA2F+NGA2F-GlcNac sum and a corresponding increase in the NA1F+NA2F sum were observed with the addition of galactose alone in the range of 0 to 10 mM to production CDM PFBM-3/PFFM-4. A maximum decrease of 14% in the NGA2F+NGA2F-GlcNac sum and a corresponding increase of 15% in the NA1F+NA2F sum was observed with the addition of 10 mM galactose. In addition, the effect on modulation of galactosylation of adalimumab produced in a NSO cell line in production CDM PFBM-3/PFFM-4 supplemented with manganese chloride and galactose was synergistic (FIG. 21). For example, but not by way of limitation, addition of 0.2 μM manganese chloride alone reduced the NGA2F+NGA2F-GlcNac sum by 12%, and addition of 4 mM galactose alone decreased the NGA2F+NGA2F-GlcNac sum by 2%. However, the combined addition of manganese chloride and galactose at these same concentrations (i.e. 0.2 μM manganese+4 mM galactose) led to a 19% reduction in the NGA2F+NGA2F-GlcNac sum, 5% higher than their combined individual contributions. A maximum decrease of ˜26% in the NGA2F+NGA2F-GlcNac sum and a corresponding 28% increase in the NA1F+NA2F sum were observed with the combined addition of 0.5 μM manganese chloride and 4 mM galactose.


The compositions and methods of the present invention also find use in the production of diverse antibodies, as evidenced by the results of Example 7, which employs a CHO cell line that produced an antibody distinct from adalimumab. For example, but not by way of limitation, when producing this antibody distinct from adalimumab, the addition of manganese chloride alone within the range of 0 to 40 μM to production CDM GIA-1 decreased the NGA2F+NGA2F-GlcNac sum by a maximum of 26% (FIG. 23). A comparable maximum increase of 27% in the NA1F+NA2F sum was also achieved. Addition of galactose alone up to a maximum concentration of 40 mM yielded a maximum decrease of 12% in the NGA2F+NGA2F-GlcNac sum and a corresponding 13% maximum increase in the NA1F+NA2F sum in a concentration dependent manner. In addition, the combined addition of galactose and manganese chloride to production CDM GIA-1 resulted in a greater percent reduction in the NGA2F+NGA2F-GlcNac sum and, correspondingly, a greater percent increase in the NA1F+NA2F sum as compared to the addition of either component alone (FIG. 24). For example, but not by way of limitation, the addition of 40 μM manganese chloride alone reduced the NGA2F+NGA2F-GlcNac sum by 20%, and the addition of 40 mM galactose alone decreased the NGA2F+NGA2F-GlcNac sum by 12%. However, the combined addition of manganese chloride and galactose at these same concentrations (i.e. 40 μM manganese+40 mM galactose) led to a 27% decrease in the NGA2F+NGA2F-GlcNac sum. The highest percentage decrease of 32% in the NGA2F+NGA2F-GlcNac sum and the corresponding increase of 30% in the NA1F+NA2F sum were observed for the culture supplemented with 20 μM manganese chloride and 20 mM galactose.


That the compositions and methods of the present invention also find use when producing diverse antibodies is further reinforced by the results of Example 8, which employs a CHO cell line producing an antibody distinct from both adalimumab and the antibody of Example 7. For example, but not by way of limitation, when producing this third antibody, the addition of manganese chloride alone in the range of 0 to 75 μM to production CDM GIA-1 decreased the NGA2F+NGA2F-GlcNac sum by a maximum of 18% (FIG. 26). A comparable maximum increase of 16% in the NA1F+NA2F sum was also achieved. Addition of galactose alone up to a maximum concentration of 60 mM yielded a maximum decrease of 12% in the NGA2F+NGA2F-GlcNac sum and a corresponding 11% maximum increase in the NA1F+NA2F sum. In addition, when manganese chloride and galactose were supplemented together into the basal and feed media, the results demonstrated at least an additive effect and sometimes a synergistic effect towards the decrease in the NGA2F+NGA2F-GlcNAc sum and the increase in the NA1F+NA2F sum (FIG. 27). The synergistic effect was observed for the condition supplemented with 25 μM manganese chloride and 15 mM galactose. The observed 22% decrease in the NGA2F+NGA2F-GlcNAc sum was 5% more than the sum of the decrease observed with the addition of 25 μM manganese chloride alone (10%) and 15 mM galactose alone (7%). The additive effect was observed for the condition supplemented with 50 μM manganese chloride and 30 mM galactose. The observed 28% decrease in the NGA2F+NGA2F-GlcNAc sum was comparable to the sum of the decrease observed with the addition of 50 μM manganese chloride alone (18%) and 30 mM galactose alone (12%). A maximum decrease of 28% in the NGA2F+NGA2F-GlcNAc and a corresponding 25% maximum increase in the NA1F+NA2F sum compared to the control condition was observed with the combined addition of 50 μM manganese chloride and 30 mM galactose to chemically defined GIA-1 media.


Although specifically directed to the production of antibodies, the following description outlines general techniques that can be adapted for the production of other recombinantly-expressed proteins. For example, to express a recombinant antibody, nucleic acids encoding partial or full-length light and heavy chains 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 an antibody gene 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 antibody gene. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into a separate vector or, more typically, both genes are inserted into the same expression vector. The antibody genes are inserted into an expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are present). Prior to insertion of the antibody or antibody-related light or heavy chain sequences, the expression vector may already carry antibody constant region sequences. For example, one approach to converting particular 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 antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain 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 the antibody chain genes, a recombinant expression vector of the invention can carry one or more regulatory sequence that controls the expression of the antibody chain 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 antibody chain 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 antibody chain 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 & 6,914,128, the entire teachings of which are incorporated herein.


For expression of the light and heavy chains, the expression vector(s) encoding the heavy and light chains 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 antibodies 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 antibody. Prokaryotic expression of antibody genes has been reported to be ineffective for production of high yields of active antibody (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'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 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 antibodies 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), NS0 myeloma cells, COS cells and SP2 cells. When recombinant expression vectors encoding antibody 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 antibody 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 an antibody may be cultured in a variety of media. Commercially available media such as Ham's F10™ (Sigma), Minimal Essential Medium™ ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle'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. 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 antibodies, such as 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 of this invention. 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 antigen binding. 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 original antigen by crosslinking an antibody of the invention to a second antibody by standard chemical crosslinking methods.


In a suitable system for recombinant expression of an antibody, or antigen-binding portion thereof, of the invention, a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is introduced into dhfr-CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, the antibody heavy and light chain genes are each operatively linked to CMV enhancer/AdMLP promoter regulatory elements to drive high levels of transcription of the genes. 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 antibody heavy and light chains and intact 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 antibody from the culture medium.


When using recombinant techniques, the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. In one aspect, if the antibody 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 antibody 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 antibodies from cell debris initially depend on the site of expression of the antibody. Some antibodies can be secreted directly from the cell into the surrounding growth media; others are made intracellularly. For the latter antibodies, 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 antibody 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 antibody is secreted into the medium, the recombinant host cells can also be separated from the cell culture medium, e.g., by tangential flow filtration. Antibodies can be further recovered from the culture medium using the antibody purification methods of the invention.


6. EXAMPLES
6.1. Example 1
6.1.1. Materials & Methods

In the studies summarized in this example, we investigated the effects on product quality attributes resulting from the addition of manganese chloride and/or galactose to chemically defined Life Technologies Gibco, GIA-1, media (proprietary formulation) in the adalimumab-producing CHO cell line utilized in Example 3, but adapted to GIA-1 media. The studies were performed in either a batch process in shake flasks or a fed-batch process in 3 L bioreactors.


Growth and production media for the adalimumab-producing CHO cell line were prepared using a proprietary Life Technologies Gibco chemically defined media, GIA-1. Basal production and feed media were supplemented with Manganese (II) Chloride (Sigma M1787-100 mL; 1.0 M±0.1 M) and D(+)Galactose (Sigma G5388-1 kg) according to the experimental design described in Table 1. All media were filtered through Corning 0.5 L or 1 L filter systems 0.22 μm Poly(Ether Sulfone) (PES) and stored at 4° C. until use.


The cell line utilized for both studies was generated from the adalimumab-producing CHO cell utilized in Example 3 by adapting it to chemically defined GIA-1 media for 7 (2 to 3 day each) passages in a combination of 250 mL and 500 mL Corning vented non-baffled shake flasks before freezing.


Upon thaw, for the batch shake flask study, cells were expanded for 3 to 5 (2 to 3 day each) passages in a combination of 250 mL and 500 mL Corning vented non-baffled shake flasks. Production cultures were initiated in duplicate 500 mL Corning vented non-baffled shake flasks (200 mL working volume) at an initial viable cell density (VCD) of approximately 0.5×106 cells/mL. Cultures were maintained on orbital shakers at 110 revolutions per minute (RPM) in a dry incubator at 35° C. and 5% CO2. The shake flask study was run in an extended batch mode by feeding a glucose solution (1.25% (v/v) of 40% solution) when the media glucose concentration fell below 3 g/L.


For the fed-batch bioreactor study, cells were expanded for 8 (2 to 3 day each) passages in Corning vented non-baffled shake flasks maintained on orbital shakers at 110 RPM and in 20 L cell bags (3 L to 10 L working volume) maintained at 20-25 RPM, 7.5° angle, and 0.25 SLPM airflow in a dry incubator at 35° C. and 5% CO2. Production cultures were initiated in duplicate 3 L bioreactors (1.5 L working volume) at 35° C., 30% dissolved oxygen, 200 RPM, pH ramp from 7.1 to 6.9 over 3 days, and pH setpoint of 6.9 thereafter. A fixed split ratio of cells to media of 1:5 was utilized to initiate the production stage cultures. In the fed-batch mode, a chemically-defined feed from Life Technologies Gibco, JCL-5 (proprietary formulation), was added as follows: 3% (v/v)—day 3, 5%—day 4, 7%—day 5, 10%—day 6, and 10% —day 7. Additional glucose (1.25% (v/v) of 40% solution) was fed when the media glucose concentration fell below 3 g/L.


For all studies with CHO cell lines described throughout this invention, samples were collected daily and measured for cell density and viability using a Cedex cell counter. Retention samples for titer analysis via Poros A method were collected by centrifugation at 12,000 RPM for 5 min when the culture viability began declining. The cultures were harvested by collecting 125 mL aliquots and centrifuging at 3,000 RPM for 30 min when culture viability was near or below 50%. All supernatants were stored at −80° C. until analysis.


For all studies, the harvest samples were Protein A purified and prepared for the oligosaccharide assay using the following procedures. As a first step in the process of establishing the identity and quantifying the oligosaccharides, they are released from the protein by enzymatic digestion with N-glycanase. Once the glycans are released, the free reducing end of each glycan is labeled by reductive amination with a fluorescent tag, 2-aminobenzamide (2-AB). The resulting labeled glycans are separated by normal-phase HPLC (NP-HPLC) in acetonitrile: 50 mM ammonium formate, pH 4.4, and detected by a fluorescence detector. Quantitation is based on the relative area percent of detected sugars. Throughout this invention, the relative area percentages of the agalactosyl fucosylated biantennary oligosaccharides, denoted as NGA2F+NGA2F-GlcNAc sum, and the galactose-containing fucosylated biantennary oligosaccharides NA1F+NA2F sum are reported and discussed.


6.1.2. Experimental Design

As detailed in Table 1, for the batch shake flask study, manganese chloride was supplemented at the following concentrations in production media: 0, 40, 60, 80, and 100 μM. Galactose was supplemented at the following levels in production media: 0, 10, 20, 40, and 100 mM. Individual and combined additions of manganese chloride and galactose were studied using a comprehensive design divided into 3 sets of experiments. Each experiment had a control culture for direct comparison of culture growth, productivity, and product quality. Production media used for the control cultures was not supplemented with manganese chloride or galactose. Culture growth, productivity, and product quality data for control cultures is the average of the 3 experiments.


For the fed-batch bioreactor study, manganese chloride and galactose were supplemented to both production and feed media in the following combinations: 40 μM manganese chloride and 20 mM galactose; 40 μM manganese chloride and 40 mM galactose (Table 2). Basal and feed media for the control cultures were not supplemented with manganese chloride or galactose.









TABLE 1







Experimental design for the batch shake flasks study (Example 1)











Manganese
Galactose




(μM)
(mM)
ID















0
0
Mn(0) Gal(0)



0
10
Mn(0) Gal(10)



0
20
Mn(0) Gal(20)



0
40
Mn(0) Gal(40)



0
100
Mn(0) Gal(100)



40
0
Mn(40) Gal(0)



80
0
Mn(80) Gal(0)



100
0
Mn(100) Gal(0)



40
10
Mn(40) Gal(10)



40
20
Mn(40) Gal(20)



40
40
Mn(40) Gal(40)



40
100
Mn(40) Gal(100)



60
20
Mn(60) Gal(20)



60
40
Mn(60) Gal(40)



60
100
Mn(60) Gal(100)



80
20
Mn(80) Gal(20)



80
40
Mn(80) Gal(40)



80
100
Mn(80) Gal(100)



100
20
Mn(100) Gal(20)



100
40
Mn(100) Gal(40)



100
100
Mn(100) Gal(100)

















TABLE 1







Experimental design for the fed-


batch 3 L bioreactors study (Example 1)









Manganese
Galactose



(μM)
(mM)
ID












0
0
Mn(0) Gal(0)


40
20
Mn(40) Gal(20)


40
40
Mn(40) Gal(40)









6.1.3. Culture Growth & Productivity

For the shake flasks experiments, cell growth and viability profiles of cultures in production media supplemented with galactose in the 0 to 100 mM concentration range and/or manganese chloride up to 80 μM concentration were comparable to control cultures without manganese and/or galactose added (FIGS. 1A, 1B). Cultures grown in media supplemented with manganese chloride at 100 μM concentration and galactose concentrations in the 0 to 100 mM range experienced growth lag and decreased viability for the first 4 production days, likely due to toxic effects of manganese at this concentration. However, toxicity effects were overcome after production day 4. Harvest titer for most experimental conditions was 3% to 14% higher than the average harvest titer for the control cultures (FIG. 1C). All three control cultures had comparable growth profiles and productivity. For the bioreactors fed-batch experiment, culture growth, viability profiles, and harvest titer were comparable for all conditions (FIGS. 2A, 2B, 2C).


6.1.4. Oligosaccharide Analysis

In this example, the modulation of galactosylation with the addition of manganese chloride and/or galactose to chemically defined media GIA-1 was explored using the adalimumab-producing CHO cell line utilized in Example 3, but adapted to GIA-1 media.


6.1.4.1. Batch Shake Flask Study

For the shake flask study, the addition of manganese chloride alone within the range of 0 to 100 μM to production CDM GIA-1 decreased the NGA2F+NGA2F-GlcNac sum in a concentration dependent manner and increased the NA1F+NA2F sum by approximately the same percentage. A maximum change of 9% in the NGA2F+NGA2F-GlcNac and 8% in the NA1F+NA2F oligosaccharides was observed with the addition of 100 μM manganese chloride (FIG. 3). Manganese doses greater than 100 μM were not explored further due to cytotoxicity effects. Similarly, a concentration dependent decrease in the NGA2F+NGA2F-GlcNac sum and a corresponding increase in the NA1F+NA2F sum were observed with the addition of galactose alone in the range of 0 to 100 mM to production CDM GIA-1. A maximum change of 7% in the NGA2F+NGA2F-GlcNac and the NA1F+NA2F oligosaccharides was observed with the addition of 100 mM galactose.


The effect on modulation of galactosylation of adalimumab in CDM GIA-1 with the combined addition of manganese chloride and galactose was synergistic. Combined addition of manganese chloride and galactose decreased the NGA2F+NGA2F-GlcNac sum and increased the NA1F+NA2F sum by a larger percentage than by adding manganese or galactose alone and summing up their individual effects (FIGS. 4 and 5). For example, addition of 40 μM manganese chloride alone reduced the NGA2F+NGA2F-GlcNac sum by 6%, and addition of 40 mM galactose alone decreased the NGA2F+NGA2F-GlcNac sum by 6%. However, the combined addition of manganese chloride and galactose at these same concentrations (i.e. 40 μM manganese+40 mM galactose) led to an 18% reduction in the NGA2F+NGA2F-GlcNac sum, 6% higher than their combined individual contributions to the reduction of the NGA2F+NGA2F-GlcNac sum. We define this effect as being synergistic and maintain this definition throughout the invention. A maximum decrease compared to the control condition of 26% in the NGA2F+NGA2F-GlcNac sum was observed with the combined addition of 100 μM manganese chloride and 100 mM galactose.


6.1.4.2. Fed-Batch Bioreactor Study

For the fed-batch 3 L bioreactors study, two manganese chloride and galactose combinations were studied and we show that the decrease in the NGA2F+NGA2F-GlcNac and the corresponding increase in the NA1F+NA2F oligosaccharides was scale (1.5 L vs. 200 mL) and process independent (fed-batch in controlled bioreactor environment vs. batch in shake flasks). Combined addition of 40 μM manganese chloride and 20 mM galactose to both production basal CDM GIA-1 and feed CDM JCL-5 decreased the NGA2F+NGA2F-GlcNac sum by 26% and increased the NA2F+NA2F sum by 27% compared to the control cultures (FIG. 6). A further increase in the galactose concentration to 40 mM in addition to manganese supplementation at 40 μM concentration resulted in an additional 3% decrease in the NGA2F+NGA2F-GlcNac sum, and a corresponding 3% increase in the NA1F+NA2F sum.


6.2. Example 2
6.2.1. Materials & Methods

In the study summarized in this example, we investigated the effects on product quality attributes resulting from the addition of manganese chloride and/or galactose to the chemically defined media Thermo Scientific HyClone CDM4CHO using the adalimumab-producing CHO cell line used in Example 1 further adapted to HyClone media.


Growth and production media for the adalimumab-producing CHO cell line were prepared using Thermo Scientific HyClone chemically defined media CDM4CHO without L-glutamine (Catalogue # SH30558.02). Production media was supplemented with Manganese (II) Chloride (Sigma M1787-100 mL; 1.0 M±0.1 M) and D(+)Galactose (Sigma G5388-1 kg) according to the experimental design described in Table 3. All media were filtered through Corning 0.5 L or 1 L (0.22 μm PES) filter systems and stored at 4° C. until use.


Upon thaw, cells were adapted to and expanded in HyClone CDM4CHO media for 5 (2 to 3 day each) passages in a combination of 250 mL, 500 mL, and 1000 mL Corning vented non-baffled shake flasks. Production cultures were initiated in duplicate 500 mL Corning vented non-baffled shake flasks (200 mL working volume) at an initial VCD of approximately 0.5×106 cells/mL. Cultures were maintained on orbital shakers at 110 RPM in a dry incubator at 35° C. and 5% CO2. A glucose solution (1.25% (v/v) of 40% solution) was fed when the media glucose concentration fell below 3 g/L.


6.2.2. Experimental Design

As detailed in Table 3, manganese chloride was supplemented at the following concentrations in production media: 0, 10, 20, and 40 μM. Galactose was supplemented at the following levels in production media: 0, 10, 20, and 100 mM. Production media for the control cultures was not supplemented with manganese chloride or galactose.









TABLE 3







Experimental design for Example 2









Manganese
Galactose



(μM)
(mM)
ID












0
0
Mn(0) Gal(0)


0
10
Mn(0) Gal(10)


0
20
Mn(0) Gal(20)


0
40
Mn(0) Gal(40)


10
0
Mn(10) Gal(0)


20
0
Mn(20) Gal(0)


40
0
Mn(40) Gal(0)


10
10
Mn(10) Gal(10)


20
20
Mn(20) Gal(20)


40
40
Mn(40) Gal(40)









6.2.3. Culture Growth & Productivity

Cell growth and viability profiles of cultures in production HyClone CDM4CHO supplemented with galactose in the 0 to 40 mM concentration range and/or manganese chloride up to 10 μM concentration were comparable to the control cultures without manganese and/or galactose added (FIGS. 7A, 7B). Increasing the concentration of manganese chloride in HyClone CDM4CHO production media to 20 μM or 40 μM slowed down culture growth. Manganese doses greater than 40 μM were not explored further due to the observed growth inhibition effects. Harvest titer for all conditions was comparable to the control (data not shown).


6.2.4. Oligosaccharide Analysis

In this example, the modulation of galactosylation with the addition of manganese chloride and/or galactose to the commercially available HyClone CDM4CHO was explored using the adalimumab-producing CHO cell line used Example 1 further adapted to HyClone media.


The addition of manganese chloride alone within the range of 0 to 40 μM to production CDM HyClone CDM4CHO decreased the NGA2F+NGA2F-GlcNac sum by a maximum of 5% in a concentration dependent manner (FIG. 8). A comparable maximum increase of 4% in the NA1F+NA2F sum was achieved. Addition of galactose alone up to a maximum concentration of 40 mM yielded a 6% maximum decrease in the NGA2F+NGA2F-GlcNac sum and a corresponding 6% increase in the NA1F+NA2F sum.


An additive effect was observed in cultures supplemented with both manganese chloride and galactose. The combined addition of manganese chloride and galactose decreased the NGA2F+NGA2F-GlcNac sum and increased the NA1F+NA2F sum by a comparable percentage as when manganese or galactose were added alone and their individual effects were summed up (FIG. 9). For example, addition of 40 μM manganese chloride alone reduced the NGA2F+NGA2F-GlcNac sum by 5%, and addition of 40 mM galactose alone decreased the NGA2F+NGA2F-GlcNac sum by 6%. The combined addition of manganese chloride and galactose at these same concentrations (i.e. 40 μM manganese+40 mM galactose) led to a 12% reduction in the NGA2F+NGA2F-GlcNac sum. We define this effect as being additive and maintain this definition throughout the invention.


The highest percentage decrease in the NGA2F+NGA2F-GlcNac sum of 12% and the corresponding 11% increase in the NA1F+NA2F sum was observed for the culture supplemented with 40 μM manganese chloride and 40 mM galactose.


6.3. Example 3
6.3.1. Materials & Methods

In the study summarized in this example, we investigated the effects on product quality attributes resulting from the addition of manganese chloride and/or galactose to a hydrolysate-based media (proprietary formulation) in an adalimumab-producing CHO cell line.


Growth and production media for the adalimumab-producing CHO cell line were prepared using yeast and soy hydrolysates according to a proprietary formulation. Production media was supplemented with Manganese (II) Chloride (Sigma M1787-100 mL; 1.0 M±0.1 M) and D(+)Galactose (Sigma G5388-1 kg) according to the experimental design described in Table 4. All media were filtered through Corning 0.5 L or 1 L filter systems (0.22 μm PES) and stored at 4° C. until use.


Upon thaw, cells were expanded for 9 (2 to 3 day each) passages in a combination of 250 mL, 500 mL, and 1000 mL Corning vented non-baffled shake flasks. Production cultures were initiated in duplicate 500 mL Corning vented non-baffled shake flasks (200 mL working volume) at an initial VCD of approximately 0.5×106 cells/mL. Cultures were maintained on orbital shakers at 110 RPM in a dry incubator at 35° C. and 5% CO2. A glucose solution (1.25% (v/v) of 40% solution) was fed when the media glucose concentration fell below 3 g/L.


6.3.2. Experimental Design

As detailed in Table 4, manganese chloride was supplemented at the following concentrations in production media: 0, 10, 20, and 40 μM. Galactose was supplemented at the following levels in production media: 0, 10, 20, and 40 mM. Production media for the control cultures was not supplemented with manganese chloride or galactose.









TABLE 4







Experimental design for Example 3









Manganese
Galactose



(μM)
(mM)
ID












0
0
Mn(0) Gal(0)


0
10
Mn(0) Gal(10)


0
20
Mn(0) Gal(20)


0
40
Mn(0) Gal(40)


10
0
Mn(10) Gal(0)


20
0
Mn(20) Gal(0)


40
0
Mn(40) Gal(0)


10
10
Mn(10) Gal(10)


20
20
Mn(20) Gal(20)


40
40
Mn(40) Gal(40)









6.3.3. Culture Growth & Productivity

Cell growth of most cultures in the hydrolysate-based media supplemented with galactose in the 0 to 40 mM concentration range and/or manganese chloride in the 0 to 40 μM concentration was slower compared to the control cultures without manganese or galactose added (FIG. 10A). However, all cultures reached a comparable peak viable cell density. Some cultures supplemented with 20 μM or 40 μM manganese chloride showed decreased viability by day 3 of production culture, but recovered as the cultures progressed (FIG. 10B). The culture supplemented with 10 μM manganese chloride was studied with a second control condition (B) in a separate experiment. Both these cultures grew to slightly higher maximum VCD compared to all other cultures, however results were within historical variation. Harvest titer for all conditions was comparable to the control (data not shown).


6.3.4. Oligosaccharide Analysis

In this example, the modulation of galactosylation by the addition of manganese chloride and/or galactose to a hydrolysate-based media was explored using an adalimumab-producing CHO cell line in shake flasks.


The addition of manganese chloride alone within the range of 0 to 40 μM to hydrolysate-based production media decreased the NGA2F+NGA2F-GlcNac sum by approximately 1%, a change that is within the oligosaccharide assay variability (FIG. 11). The addition of galactose alone up to a maximum concentration of 40 mM yielded a maximum decrease of 4% in the NGA2F+NGA2F-GlcNac sum and a corresponding 4% maximum increase in the NA1F+NA2F sum.


The oligosaccharide profile changes achieved with the addition of galactose alone are comparable to the changes recorded when combinations of galactose and manganese chloride were added to the hydrolysate-based media. The combined addition of manganese chloride ranging from 0 to 40 μM and galactose ranging from 0 to 40 mM to hydrolysate-based media led to an approximate 5% maximum decrease in the NGA2F+NGA2F-GlcNac sum and a corresponding 3% increase in the NA1F+NA2F sum (FIG. 12). The highest percentage decrease of 5% in the NGA2F+NGA2F-GlcNac sum and the corresponding 4% increase in the NA1F+NA2F sum was observed for the culture supplemented with 40 mM galactose and either 20 μM or 40 μM manganese chloride.


6.4. Example 4
6.4.1. Materials & Methods

In the study summarized in this example, we investigated the effects on product quality attributes resulting from the addition of manganese chloride and/or galactose to chemically defined Life Technologies Gibco GIA-1 media using a different adalimumab producing CHO cell line than in Examples 1, 2, and 3, named CHO cell line #2.


Growth and production media for the adalimumab-producing CHO cell line #2 were prepared using a proprietary Life Technologies Gibco chemically defined media, GIA-1. Production media only was supplemented with with Manganese (II) Chloride (Sigma M1787-100 mL; 1.0 M±0.1 M) and D(+)Galactose (Sigma G5388-1 kg) according to the experimental design described in Table 5. All media were filtered through Corning 0.5 L or 1 L filter systems (0.22 μm PES) and stored at 4° C. until use.


Upon thaw, cells were expanded for 5 to 8 (2 to 3 day each) passages in a combination of 250 mL, 500 mL, and 1000 mL Corning vented non-baffled shake flasks. Production cultures were initiated in duplicate 500 mL Corning vented non-baffled shake flasks (200 mL working volume) at an initial VCD of approximately 0.5×106 cells/mL. Cultures were maintained on orbital shakers at 180 RPM in a dry incubator at 35° C. and 5% CO2. A glucose solution (1.25% (v/v) of 40% solution) was fed when the media glucose concentration fell below 3 g/L.


6.4.2. Experimental Design

As detailed in Table 5, manganese chloride was supplemented at the following concentrations in production media: 0, 10, 20, and 40 μM. Galactose was supplemented at the following levels in production media: 0, 10, and 20 mM. Production media for the control cultures was not supplemented with manganese chloride or galactose. This study was run in 2 blocks.









TABLE 5







Experimental design for Example 4













Manganese
Galactose





(μM)
(mM)
ID
















Block I
0
0
Mn(0) Gal(0)




20
0
Mn(20) Gal(0)




10
10
Mn(10) Gal(10)




20
20
Mn(20) Gal(20)




40
20
Mn(40) Gal(20)



Block II
0
0
Mn(0) Gal(0)




0
10
Mn(0) Gal(10)




0
20
Mn(0) Gal(20)




10
0
Mn(10) Gal(0)










6.4.3. Culture Growth & Productivity

Culture growth, viability profiles, and harvest titer of cultures in production CDM GIA-1 supplemented with galactose in the 0 to 20 mM concentration range and/or manganese chloride in the 0 to 40 μM concentration range were comparable to the control cultures without manganese or galactose added (FIGS. 13A, 13B; harvest titer data not shown).


6.4.4. Oligosaccharide Analysis

In this example, the modulation of galactosylation by the addition of manganese chloride and/or galactose to chemically defined GIA-1 media was explored using the adalimumab-producing CHO cell line #2.


The addition of manganese chloride alone within the range of 0 to 20 μM to production CDM GIA-1 decreased the NGA2F+NGA2F-GlcNac sum in a concentration dependent manner and increased the NA1F+NA2F sum by approximately the same percentage. A maximum decrease of 22% in the NGA2F+NGA2F-GlcNac sum and a maximum corresponding increase of 23% in the NA1F+NA2F sum was observed with the addition of 20 μM manganese chloride (FIG. 14). Similarly, a concentration dependent decrease in the NGA2F+NGA2F-GlcNac sum and a corresponding increase in the NA1F+NA2F sum was observed with the addition of galactose alone in the range of 0 to 20 mM. A maximum decrease of 9% in the NGA2F+NGA2F-GlcNac sum and a corresponding maximum increase of 10% in the NA1F+NA2F sum was observed with the addition of 20 mM galactose.


An additive effect was observed for the oligosaccharide profiles of adalimumab produced in cultures supplemented with the combined addition of manganese chloride and galactose to GIA-1 media (FIG. 15). For example, addition of 10 μM manganese chloride alone reduced the NGA2F+NGA2F-GlcNac sum by 18%, and addition of 10 mM galactose alone decreased the NGA2F+NGA2F-GlcNac sum by 6%. The combined addition of manganese chloride and galactose at these same concentrations led to a 24% reduction in the NGA2F+NGA2F-GlcNac sum. The highest percentage decrease of 35% in the NGA2F+NGA2F-GlcNac sum and the corresponding increase of 37% in the NA1F+NA2F sum were observed for the culture supplemented with 40 μM manganese chloride and 20 mM galactose.


6.5. Example 5
6.5.1. Materials & Methods

In the study summarized in this example, we investigated the effects on product quality attributes resulting from the addition of manganese chloride and/or galactose to chemically defined Life Technologies Gibco GIA-1 media using a different adalimumab-producing CHO cell line than in Examples 1, 2, 3, and 4, named CHO cell line #3.


Growth and production media for the adalimumab-producing CHO cell line #3 were prepared using the proprietary Life Technologies Gibco chemically defined media, GIA-1. Basal production and feed media were supplemented with Manganese (II) Chloride (Sigma M3634-100 g) and D(+)Galactose (Sigma G5388-1 kg) according to the experimental design described in Table 6.


Upon thaw, cells were expanded in Corning vented non-baffled shake flasks maintained on orbital shakers at 140 RPM, and in 10 L cell bags (2 L working volume) maintained at 25 RPM, 7° angle, and 0.25 SLPM airflow in a dry incubator at 36° C. and 5% CO2. Production cultures were initiated in 3 L bioreactors (1.4 L initial working volume) at 36° C., 30% dissolved oxygen, 200 RPM, and pH 6.9±0.2. A fixed split ratio of cells to media of 1:6.7 was utilized to initiate the production stage cultures. A temperature shift was performed when the culture VCD reached a value higher than 5×106 cells/mL. The chemically-defined feed from Life Technologies Gibco JCL-5 was added as follows: 4% (v/v)—day 2, 6%—day 4, 8%—day 6, 10%—day 8, and 10%—day 10. Additional 400 g/L glucose was added to the reactor cultures as needed to ensure glucose levels did not deplete. Bioreactors were harvested at a viability of approximately 50% or on production day 17, whichever condition occurred first.


6.5.2. Experimental Design

As detailed in Table 6, manganese chloride was supplemented at the following concentrations in both production and feed media: 0, 0.1, 0.2, 0.5, and 1.0 μM. Galactose was supplemented at 0 and 30 mM concentrations in both production and feed media. In addition, a combined manganese chloride and galactose supplementation strategy was utilized for the production basal and feed media at either 0.2 or 0.5 μM manganese chloride plus 30 mM galactose. Basal and feed media for the control cultures were not supplemented with manganese chloride or galactose.









TABLE 6







Experimental design for Example 5









Manganese
Galactose



(μM)
(mM)
ID












0
0
Mn(0) Gal(0)


0.1
0
Mn(0.1) Gal(0)


0.2
0
Mn(0.2) Gal(0)


0.5
0
Mn(0.5) Gal(0)


1.0
0
Mn(1.0) Gal(0)


0
30
Mn(0) Gal(30)


0.2
30
Mn(0.2) Gal(30)


0.5
30
Mn(0.5) Gal(30)









6.5.3. Culture Growth & Productivity

Growth profiles of most cultures supplemented with manganese chloride and/or galactose were comparable to the control culture except for the cultures supplemented with 30 mM galactose alone or in combination with 0.2 μM manganese chloride which grew slower and reached a lower peak VCD (FIG. 16A). However, the culture supplemented with 0.5 μM manganese chloride and 30 mM galactose had a growth profile comparable to the control culture indicating that neither manganese chloride nor galactose at the concentrations studied are detrimental to culture growth. Viability profiles and harvest titer were comparable to the control condition (FIGS. 16B, 16C).


6.5.4. Oligosaccharide Analysis

In this example, the modulation of galactosylation with the addition of manganese chloride and/or galactose to chemically defined media GIA-1 was explored using the adalimumab-producing CHO cell line #3.


The addition of manganese chloride alone within the range of 0 to 1 μM to production CDM GIA-1 decreased the NGA2F+NGA2F-GlcNac sum in a concentration dependent manner and increased the NA1F+NA2F sum by approximately the same percentage. A maximum decrease of 26% in the NGA2F+NGA2F-GlcNAc and a corresponding increase of 28% in the NA1F+NA2F oligosaccharides were observed with the addition of 1 μM manganese chloride (FIG. 17). The addition of galactose alone at 30 mM concentration to production CDM GIA-1 decreased the NGA2F+NGA2F-GlcNAc sum by 4% and increased the NA1F+NA2F sum by 3%.


When manganese chloride and galactose were supplemented together into the production basal and feed media, the results demonstrated a synergistic benefit towards the decrease in the NGA2F+NGA2F-GlcNAc and the increase in the NA1F+NA2F oligosaccharides which is consistent with the results demonstrated in Example 1 (FIG. 18). For example, at 0.2 μM manganese chloride plus 30 mM galactose the observed 25% decrease in the NGA2F+NGA2F-GlcNAc sum was 6% more than the sum of the decrease observed with the addition of 0.2 μM manganese chloride alone (15%) and that of 30 mM galactose alone (4%). Similarly the resulting 24% increase in the NA1F+NA2F sum was more than the sum of the increase observed with the addition of 0.2 μM manganese chloride alone (16%) and that of 30 mM galactose alone (3%). The combined supplementation of 0.5 μM manganese chloride+30 mM galactose also demonstrated a synergistic effect on the galactosylation profile of adalimumab produced in the CHO cell line #3. A maximum decrease compared to the control condition of 34% in the NGA2F+NGA2F-GlcNac and a corresponding 34% maximum increase in the NA1F+NA2F oligosaccharides was observed with the combined addition of 0.5 μM manganese chloride and 30 mM galactose to chemically defined GIA-1 media.


6.6. Example 6
6.6.1. Materials & Methods

In the study summarized in this example, we investigated the effects on product quality attributes resulting from the addition of manganese chloride and/or galactose to chemically defined Life Technologies Gibco PFBM-3 basal medium and PFFM-4 feed medium (proprietary formulation) using an adalimumab-producing NSO cell line in a fed-batch process in shake flasks.


Growth and production media for the adalimumab-producing NSO cell line were prepared using a proprietary Life Technologies Gibco chemically defined media, PFBM-3 basal medium plus PFFM-4 feed medium. Production and feed media were supplemented with Manganese (II) Chloride (Sigma M1787-100 mL; 1.0 M±0.1 M) and D(+)Galactose (Sigma G5388-1 kg) according to the experimental design described in Table 7.


Upon thaw, cells were expanded for 3 to 5 (2 days each) passages in a combination of 250 mL, 500 mL, 1 L, 2 L and 3 L Corning vented non-baffled shake flasks. Production cultures were initiated in single 1 L Corning vented non-baffled shake flasks (240 mL initial working volume) at an initial VCD of approximately 2.5×105 cells/mL. Cultures were maintained on orbital shakers at 100 RPM in a dry incubator at 37° C. and 5% CO2. The shake flask study was run in a fed-batch mode and the culture was fed PFFM-4 as follows: 24 mL—day 2, 28.8 mL—day 4, 28.8 mL—day 6, and 28.8 mL—day 8.


Samples were collected every 2 days and measured for cell density and viability using a Cedex cell counter. Retention samples for titer analysis via Poros A method were collected daily beginning on Day 8 by centrifugation at 2,000 g for 10 min and then filtered through 0.2 um PVDF syringe filter. The cultures were harvested on production day 10. The entire culture was collected, chilled on ice to approximately 0° C. for 1.5 hours, the cells and debris flocculated at pH 5.0 by the addition of 1M citric acid and held for 15 minutes, and centrifuged at 4000×g for 15 min at 5° C. The supernatant was passed through 0.20 um Millipore Stericup PES filters, and, immediately post filtration, the acidified clarified cell-free harvest was neutralized with 2M Tris to pH 7.1±0.2. The cell free harvest was transferred to PETG bottles and stored at −80° C. until analysis.


6.6.2. Experimental Design

As detailed in Table 7, manganese chloride was supplemented at the following concentrations in both production and feed media: 0, 0.2, and 0.5 μM. Galactose was supplemented at the following levels in both production and feed media: 0, 1, 4, 5, and 10 mM. Manganese chloride and galactose were added in a full factorial, two level DOE design for the 0, 1, and 4 mM galactose conditions and all concentrations of manganese chloride. Individual and combined additions of manganese chloride and galactose were studied using a comprehensive design divided into 2 sets of experiments. Each experiment had a control culture for direct comparison of culture growth, productivity, and product quality. Production medium for control cultures was not supplemented with manganese chloride or galactose.









TABLE 7







Experimental design for Example 6













Manganese
Galactose





(μM)
(mM)
ID
















Block I
0
0
Mn(0) Gal(0)




0
5
Mn(0) Gal(5)




0
10
Mn(0) Gal(10)



Block II
0
0
Mn(0) Gal(0)




0.2
0
Mn(0.2) Gal(0)




0.5
0
Mn(0.5) Gal(0)




0
1
Mn(0) Gal(1)




0.2
1
Mn(0.2) Gal(1)




0.5
1
Mn(0.5) Gal(1)




0
4
Mn(0) Gal(4)




0.2
4
Mn(0.2) Gal(4)




0.5
4
Mn(0.5) Gal(4)










6.6.3. Culture Growth & Productivity

Culture growth and viability profiles in production media supplemented with galactose in the 0 to 5 mM concentration range and/or manganese chloride up to 0.5 μM concentration were comparable to the control condition without manganese or galactose added (FIGS. 19A, 19B). The addition of galactose at 10 mM concentration had a detrimental effect on culture growth and productivity. The cultures in the Block I experiment had a lower maximum VCD and overall lower viability than the cultures in the Block II experiment. All cultures in the Block II experiment showed similar VCD and viability profiles. Harvest titer for most experimental conditions was comparable to the harvest titer for the control condition except for the titer of the culture supplemented with 10 mM galactose, which was 60% lower (FIG. 19C).


6.6.4. Oligosaccharide Analysis

In this example, the modulation of galactosylation by the addition of manganese chloride and/or galactose to chemically defined PFBM-3/PFFM-4 media was explored using an adalimumab-producing NSO cell line in a fed-batch process in shake flasks.


The addition of manganese chloride alone within the range of 0 to 0.5 μM to CDM PFBM-3/PFFM-4 decreased the NGA2F+NGA2F-GlcNac sum in a concentration dependent manner and increased the NA1F+NA2F sum by approximately the same percentage. A maximum decrease of 18% in the NGA2F+NGA2F-GlcNac sum and a corresponding increase of 20% in the NA1F+NA2F sum were observed with the addition of 0.5 μM manganese chloride (FIG. 20). Manganese doses greater than 0.5 μM were not explored further due to cytotoxicity effects. Similarly, a concentration dependent decrease in the NGA2F+NGA2F-GlcNac sum and a corresponding increase in the NA1F+NA2F sum were observed with the addition of galactose alone in the range of 0 to 10 mM to CDM PFBM-3/PFFM-4. A maximum decrease of 14% in the NGA2F+NGA2F-GlcNac sum and a corresponding increase of 15% in the NA1F+NA2F sum was observed with the addition of 10 mM galactose.


The effect on modulation of galactosylation of adalimumab produced in a NSO cell line in CDM PFBM-3/PFFM-4 supplemented with manganese chloride and galactose was synergistic (FIG. 21). For example, addition of 0.2 μM manganese chloride alone reduced the NGA2F+NGA2F-GlcNac sum by 12%, and addition of 4 mM galactose alone decreased the NGA2F+NGA2F-GlcNac sum by 2%. However, the combined addition of manganese chloride and galactose at these same concentrations (i.e. 0.2 μM manganese+4 mM galactose) led to a 19% reduction in the NGA2F+NGA2F-GlcNac sum, 5% higher than their combined individual contributions. A maximum decrease of ˜26% in the NGA2F+NGA2F-GlcNac sum and a corresponding 28% increase in the NA1F+NA2F sum were observed with the combined addition of 0.5 μM manganese chloride and 4 mM galactose.


6.7. Example 7
6.7.1. Materials & Methods

In the study summarized in this example, we investigated the effects on product quality attributes resulting from the addition of manganese chloride and/or galactose to chemically defined Life Technologies Gibco GIA-1 media in a CHO cell line producing a monoclonal antibody generically named mAb #1.


Growth and production media for the CHO cell line producing mAb #1 were prepared using a proprietary Life Technologies Gibco chemically defined media, GIA-1. Production media only was supplemented with Manganese (II) Chloride (Sigma M1787-100 mL; 1.0 M±0.1 M) and D(+)Galactose (Sigma G5388-1 kg) according to the experimental design described in Table 8. All media were filtered through Corning 0.5 L or 1 L filter systems (0.22 μm PES) and stored at 4° C. until use.


Upon thaw, cells were expanded for 6 (3 day each) passages in a combination of 250 mL, 500 mL, and 1000 mL Corning vented non-baffled shake flasks. Production cultures were initiated in duplicate 500 mL Corning vented non-baffled shake flasks (200 mL working volume) at an initial VCD of approximately 0.5×106 cells/mL. Cultures were maintained on orbital shakers at 125 RPM in a dry incubator at 35° C. and 5% CO2. A glucose solution (1.25% (v/v) of 40% solution) was fed when the media glucose concentration fell below 3 g/L.


6.7.2. Experimental Design

As detailed in Table 8, manganese chloride was supplemented at the following levels in production media: 0, 10, 20, and 40 μM. Galactose was supplemented at the following levels in production media: 0, 10, 20, and 100 mM. Production media for the control cultures was not supplemented with manganese chloride or galactose.









TABLE 8







Experimental design for Example 7









Manganese
Galactose



(μM)
(mM)
ID












0
0
Mn(0) Gal(0)


0
10
Mn(0) Gal(10)


0
20
Mn(0) Gal(20)


0
40
Mn(0) Gal(40)


10
0
Mn(10) Gal(0)


20
0
Mn(20) Gal(0)


40
0
Mn(40) Gal(0)


10
10
Mn(10) Gal(10)


20
20
Mn(20) Gal(20)


40
40
Mn(40) Gal(40)









6.7.3. Culture Growth & Productivity

Cultures supplemented with manganese chloride alone in the concentration range of 0 to 20 μM grew comparable to the control cultures (FIG. 22A). Cultures supplemented with galactose alone or with the combination of 20 μM manganese and 20 mM galactose grew to a lower maximum VCD compared to the control, but had the same growth rate until the peak VCD was achieved on production day 6. These cultures ended a day earlier, on production day 9 (FIG. 22B). Cultures supplemented with 40 μM manganese chloride and galactose at all levels studied along with the culture supplemented with 10 μM manganese chloride and 10 mM galactose experienced slower growth and decreased peak VCD compared to the control. Harvest titer was 3-24% lower than the control condition (data not shown).


6.7.4. Oligosaccharide Analysis

In this example, the modulation of galactosylation by the addition of manganese chloride and/or galactose to chemically defined Life Technologies Gibco GIA-1 media was explored using a CHO cell line producing the monoclonal antibody mAb #1.


The addition of manganese chloride alone within the range of 0 to 40 μM to production CDM GIA-1 decreased the NGA2F+NGA2F-GlcNac sum by a maximum of 26% (FIG. 23). A comparable maximum increase of 27% in the NA1F+NA2F sum was achieved. Addition of galactose alone up to a maximum concentration of 40 mM yielded a maximum decrease of 12% in the NGA2F+NGA2F-GlcNac sum and a corresponding 13% maximum increase in the NA1F+NA2F sum in a concentration dependent manner.


The combined addition of galactose and manganese chloride to production CDM GIA-1 resulted in a greater percent reduction in the NGA2F+NGA2F-GlcNac sum and, correspondingly, a greater percent increase in the NA1F+NA2F sum as compared to the addition of either component alone (FIG. 24). For example, the addition of 40 μM manganese chloride alone reduced the NGA2F+NGA2F-GlcNac sum by 20%, and the addition of 40 mM galactose alone decreased the NGA2F+NGA2F-GlcNac sum by 12%. However, the combined addition of manganese chloride and galactose at these same concentrations (i.e. 40 μM manganese+40 mM galactose) led to a 27% decrease in the NGA2F+NGA2F-GlcNac sum. The highest percentage decrease of 32% in the NGA2F+NGA2F-GlcNac sum and the corresponding increase of 30% in the NA1F+NA2F sum were observed for the culture supplemented with 20 μM manganese chloride and 20 mM galactose.


6.8. Example 8
6.8.1. Materials & Methods

In the study summarized in this example, we investigated the effects on product quality attributes resulting from the addition of manganese chloride and/or galactose to chemically defined Life Technologies Gibco GIA-1 media in a CHO cell line producing the monoclonal antibody generically named mAb #2 in a fed-batch process in 3 L bioreactors.


Growth and production media for the mAb #2 producing CHO cell line were prepared using the proprietary Life Technologies Gibco chemically defined media, GIA-1. Basal production and feed media were supplemented with Manganese (II) Chloride (Sigma M3634) and D(+)Galactose (Sigma G5388-1 kg) according to the experimental design described in Table 9.


Upon thaw, cells were expanded in Corning vented non-baffled shake flasks maintained on orbital shakers at 140 RPM, and in 10 L cell bags (2 L working volume) maintained at 25 RPM, 7° angle, 0.25 SLPM airflow in a dry incubator at 36° C. and 5% CO2. Production cultures were initiated in 3 L bioreactors (1.5 L initial working volume) at 36° C., 25% dissolved oxygen, 200 RPM, and pH 7.0. The chemically-defined feed from Life Technologies Gibco JCL-5 was added as follows: 3% (v/v)—day 3, 5%—day 5, 7%—day 7, 10%—day 9, and 10%—day 11. Additional 400 g/L glucose was added to the bioreactor cultures as needed to ensure the glucose levels did not deplete. Bioreactors were harvested at viability of approximately 70% or below or on production day 15, whichever condition occurred first.


6.8.2. Experimental Design

As detailed in Table 9, manganese chloride was supplemented at the following concentrations in both production and feed media: 0, 25, 50, and 75 μM. Galactose was supplemented at 0, 15, 30, and 60 mM concentrations in both production and feed media. In addition, a combined manganese chloride and galactose supplementation strategy was utilized for the production basal and feed media at 25 μM manganese chloride+15 mM galactose, and 50 μM manganese chloride+30 mM galactose. Basal and feed media for the control cultures were not supplemented with manganese chloride or galactose.









TABLE 9







Experimental design for Example 8









Manganese
Galactose



(μM)
(mM)
ID












0
0
Mn(0) Gal(0)


25
0
Mn(25) Gal(0)


50
0
Mn(50) Gal(0)


75
0
Mn(75) Gal(0)


0
15
Mn(0) Gal(15)


0
30
Mn(0) Gal(30)


0
60
Mn(0) Gal(60)


25
15
Mn(25) Gal(15)


50
30
Mn(50) Gal(30)









6.8.3. Culture Growth & Productivity

Growth profiles of most cultures supplemented with galactose in the 0 to 60 mM concentration range and/or manganese chloride in the 0 to 75 μM range were comparable to the control culture except for the culture supplemented with 25 μM manganese chloride alone which grew slower after production day 7 (FIG. 25A). However, increasing the amount of manganese chloride supplemented to production CDM GIA-1 to 50 μM or 75 μM resulted in cultures with growth profiles comparable to the control culture. Viability profiles and harvest titer were comparable to the control condition (FIGS. 25B, 25C).


6.8.4. Oligosaccharide Analysis

In this example, the modulation of galactosylation with the addition of manganese chloride and/or galactose to chemically defined media GIA-1 was explored using a CHO cell line producing the monoclonal antibody mAb #2.


The addition of manganese chloride alone in the range of 0 to 75 μM to production CDM GIA-1 decreased the NGA2F+NGA2F-GlcNAc sum by a maximum of 18% (FIG. 26). A comparable maximum increase of 16% in the NA1F+NA2F sum was achieved. Addition of galactose alone up to a maximum concentration of 60 mM yielded a maximum decrease of 12% in the NGA2F+NGA2F-GlcNAc sum and a corresponding 11% maximum increase in the NA1F+NA2F sum.


When manganese chloride and galactose were supplemented together into the basal and feed media, the results demonstrated at least an additive effect and sometimes a synergistic effect towards the decrease in the NGA2F+NGA2F-GlcNAc and the increase in the NA1F+NA2F oligosaccharides (FIG. 27). The synergistic effect was observed for the condition supplemented with 25 μM manganese chloride and 15 mM galactose. The observed 22% decrease in the NGA2F+NGA2F-GlcNAc sum was 5% more than the sum of the decrease observed with the addition of 25 μM manganese chloride alone (10%) and 15 mM galactose alone (7%). The additive effect was observed for the condition supplemented with 50 μM manganese chloride and 30 mM galactose. The observed 28% decrease in the NGA2F+NGA2F-GlcNAc sum was comparable to the sum of the decrease observed with the addition of 50 μM manganese chloride alone (18%) and 30 mM galactose alone (12%). A maximum decrease of 28% in the NGA2F+NGA2F-GlcNAc and a corresponding 25% maximum increase in the NA1F+NA2F sum compared to the control condition was observed with the combined addition of 50 μM manganese chloride and 30 mM galactose to chemically defined GIA-1 media.


All patents, patent applications, publications, product descriptions and protocols, cited in this specification are hereby incorporated by reference in their entirety. In case of a conflict in terminology, the present disclosure controls.


While it will be apparent that the invention herein described is well calculated to achieve the benefits and advantages set forth above, the present invention is not to be limited in scope by the specific embodiments described herein. It will be appreciated that the invention is susceptible to modification, variation and change without departing from the spirit thereof.

Claims
  • 1. A composition comprising an antibody comprising the heavy and light chain variable domains of adalimumab, wherein less than 70% of the total N-linked oligosaccharides present on said antibody are of an agalactosyl fucosylated biantennary oligosaccharide form (sum NGA2F+NGA2F-GlcNAc).
  • 2. The composition of claim 1, wherein the antibody is adalimumab.
  • 3. The composition of claim 1, wherein 49-69% of the total N-linked oligosaccharides present on said antibody are of an agalactosyl fucosylated biantennary oligosaccharide form (sum NGA2F+NGA2F-GlcNAc).
  • 4. The composition of claim 1, wherein 55-65% of the total N-linked oligosaccharides present on said antibody are of an agalactosyl fucosylated biantennary oligosaccharide form (sum NGA2F+NGA2F-GlcNAc).
  • 5. The composition of claim 1, wherein 26-40% of the total N-linked oligosaccharides present on said antibody are of a galactose-containing fucosylated biantennary oligosaccharide form (sum of NA1F+NA2F).
  • 6. The composition of claim 1, wherein 35-44% of the total N-linked oligosaccharides present on said antibody are of a galactose-containing fucosylated biantennary oligosaccharide form (sum of NA1F+NA2F).
  • 7. The composition of claim 1, wherein 30-40% of the total N-linked oligosaccharides present on said antibody are of a galactose-containing fucosylated biantennary oligosaccharide form (sum of NA1F+NA2F).
  • 8. The composition of claim 1, wherein said antibody has been produced by culturing a mammalian cell in a cell culture media which has been supplemented with a manganese supplement and a galactose supplement.
  • 9. The composition of claim 8, wherein the cell culture media has been supplemented with 0.2-100 μM of a manganese supplement and 1-100 mM of a galactose supplement.
  • 10. The composition of claim 8, wherein the cell culture media comprises a hydrolysate based media or a chemically defined media.
  • 11. The composition of claim 8, wherein the mammalian cell is a CHO cell or an NSO cell.
  • 12. A pharmaceutical composition comprising the composition of claim 1 and a pharmaceutically acceptable carrier.
  • 13. The pharmaceutical composition of claim 12, wherein the pharmaceutically acceptable carrier comprises a polyalcohol.
  • 14. The pharmaceutical composition of claim 12, wherein the pharmaceutically acceptable carrier comprises mannitol, sodium chloride, sodium phosphate or polysorbate 80.
  • 15. The pharmaceutical composition of claim 12, wherein the pharmaceutical composition is suitable for parenteral administration.
  • 16. A composition comprising adalimumab, wherein less than 70% of the total N-linked oligosaccharides present on said adalimumab are of an agalactosyl fucosylated biantennary oligosaccharide form (sum NGA2F+NGA2F-GlcNAc).
  • 17. The composition of claim 16, wherein 49-69% of the total N-linked oligosaccharides present on said adalimumab are of an agalactosyl fucosylated biantennary oligosaccharide form (sum NGA2F+NGA2F-GlcNAc).
  • 18. The composition of claim 16, wherein 55-65% of the total N-linked oligosaccharides present on said adalimumab are of an agalactosyl fucosylated biantennary oligosaccharide form (sum NGA2F+NGA2F-GlcNAc).
  • 19. The composition of claim 16, wherein 26-40% of the total N-linked oligosaccharides present on said adalimumab are of a galactose-containing fucosylated biantennary oligosaccharide form (sum of NA1F+NA2F).
  • 20. The composition of claim 16, wherein 35-44% of the total N-linked oligosaccharides present on said adalimumab are of a galactose-containing fucosylated biantennary oligosaccharide form (sum of NA1F+NA2F).
  • 21. The composition of claim 16, wherein 30-40% of the total N-linked oligosaccharides present on said adalimumab are of a galactose-containing fucosylated biantennary oligosaccharide form (sum of NA1F+NA2F).
  • 22. The composition of claim 16, wherein said adalimumab has been produced by culturing a mammalian cell in a cell culture media which has been supplemented with a manganese supplement and a galactose supplement.
  • 23. The composition of claim 22, wherein the cell culture media has been supplemented with 0.2-100 μM of a manganese supplement and 1-100 mM of a galactose supplement.
  • 24. The composition of claim 22, wherein the cell culture media comprises a hydrolysate based media or a chemically defined media.
  • 25. The composition of claim 22, wherein the mammalian cell is a CHO cell or an NSO cell.
  • 26. A pharmaceutical composition comprising the composition of claim 16 and a pharmaceutically acceptable carrier.
  • 27. The pharmaceutical composition of claim 26, wherein the pharmaceutically acceptable carrier comprises a polyalcohol.
  • 28. The pharmaceutical composition of claim 26, wherein the pharmaceutically acceptable carrier comprises mannitol, sodium chloride, sodium phosphate or polysorbate 80.
  • 29. The pharmaceutical composition of claim 26, wherein the pharmaceutical composition further comprises polysorbate 80.
  • 30. The pharmaceutical composition of claim 26, wherein the pharmaceutical composition is suitable for parenteral administration.
RELATED APPLICATIONS

This application is a divisional application of U.S. patent application Ser. No. 14/619,799; which is a continuation application of U.S. patent application Ser. No. 14/493,068, filed on Sep. 22, 2014, now U.S. Pat. No. 9,090,688, issued on Jul. 28, 2015; which is a divisional application of U.S. patent application Ser. No. 13/457,020, filed on Apr. 26, 2012, now U.S. Pat. No. 9,062,106, issued on Jun. 23, 2015; which claims the benefit of U.S. Provisional Application Ser. No. 61/479,727, filed on Apr. 27, 2011. The entire contents of each of the foregoing applications are incorporated herein by reference.

US Referenced Citations (500)
Number Name Date Kind
RE30985 Cartaya Jun 1982 E
4399216 Axel et al. Aug 1983 A
4510245 Cousens et al. Apr 1985 A
4560655 Baker Dec 1985 A
4634665 Axel et al. Jan 1987 A
4657866 Kumar Apr 1987 A
4767704 Cleveland et al. Aug 1988 A
4801687 Ngo Jan 1989 A
4816397 Boss et al. Mar 1989 A
4877608 Lee et al. Oct 1989 A
4927762 Darfler May 1990 A
4933435 Ngo Jun 1990 A
4968615 Koszinowski et al. Nov 1990 A
5045468 Darfler Sep 1991 A
5047335 Paulson et al. Sep 1991 A
5096816 Maiorella Mar 1992 A
5110913 Coan et al. May 1992 A
5118796 Prior et al. Jun 1992 A
5122469 Mather et al. Jun 1992 A
5126250 McDonough et al. Jun 1992 A
5168062 Stinski Dec 1992 A
5179017 Axel et al. Jan 1993 A
5231024 Moeller et al. Jul 1993 A
5328985 Sano et al. Jul 1994 A
5378612 Nakashima et al. Jan 1995 A
5429746 Shadle et al. Jul 1995 A
5530101 Queen et al. Jun 1996 A
5545403 Page Aug 1996 A
5545806 Lonberg et al. Aug 1996 A
5545807 Surani et al. Aug 1996 A
5561053 Crowley Oct 1996 A
5569825 Lonberg et al. Oct 1996 A
5605923 Christensen, IV et al. Feb 1997 A
5625126 Lonberg et al. Apr 1997 A
5633162 Keen et al. May 1997 A
5633425 Lonberg et al. May 1997 A
5644036 Ramage et al. Jul 1997 A
5654407 Boyle et al. Aug 1997 A
5656272 Le et al. Aug 1997 A
5661016 Lonberg et al. Aug 1997 A
5672347 Aggarwal et al. Sep 1997 A
5672502 Birch et al. Sep 1997 A
5698195 Le et al. Dec 1997 A
5705364 Etcheverry et al. Jan 1998 A
5721121 Etcheverry et al. Feb 1998 A
5730975 Hotamisligil et al. Mar 1998 A
5770429 Lonberg et al. Jun 1998 A
5789650 Lonberg et al. Aug 1998 A
5795967 Aggarwal et al. Aug 1998 A
5811299 Renner et al. Sep 1998 A
5859205 Adair et al. Jan 1999 A
5876961 Crowe et al. Mar 1999 A
5877293 Adair et al. Mar 1999 A
5929212 Jolliffe et al. Jul 1999 A
5945098 Sarno et al. Aug 1999 A
5976833 Furukawa et al. Nov 1999 A
5989830 Davis et al. Nov 1999 A
5994510 Adair et al. Nov 1999 A
6015558 Hotamisligil et al. Jan 2000 A
6024938 Corbo et al. Feb 2000 A
6036978 Gombotz et al. Mar 2000 A
6048728 Inlow et al. Apr 2000 A
6066719 Zapata May 2000 A
6075181 Kucherlapati et al. Jun 2000 A
6090382 Salfeld Jul 2000 A
6093324 Bertolini et al. Jul 2000 A
6113898 Anderson et al. Sep 2000 A
6150584 Kucherlapati et al. Nov 2000 A
6171586 Lam et al. Jan 2001 B1
6171825 Chan et al. Jan 2001 B1
6235281 Stenzel et al. May 2001 B1
6255458 Lonberg et al. Jul 2001 B1
6258562 Salfeld et al. Jul 2001 B1
6270766 Feldman et al. Aug 2001 B1
6300129 Lonberg et al. Oct 2001 B1
6339142 Basey et al. Jan 2002 B1
6399381 Blum et al. Jun 2002 B1
6406909 Shibuya et al. Jun 2002 B1
6410270 Strittmatter et al. Jun 2002 B1
6413746 Field Jul 2002 B1
6436397 Baker et al. Aug 2002 B1
6448380 Rathjen et al. Sep 2002 B2
6451983 Rathjen et al. Sep 2002 B2
6489447 Basey et al. Dec 2002 B1
6498237 Rathjen et al. Dec 2002 B2
6509015 Salfeld et al. Jan 2003 B1
6528286 Ryll Mar 2003 B1
6593458 Rathjen et al. Jul 2003 B1
6656466 Etcheverry et al. Dec 2003 B1
6673575 Franze et al. Jan 2004 B1
6673986 Kucherlapati et al. Jan 2004 B1
6680181 Castan Jan 2004 B2
6870034 Breece et al. Mar 2005 B2
6872549 Van Ness et al. Mar 2005 B2
6890736 Reddy et al. May 2005 B1
6900056 Lee et al. May 2005 B2
6914128 Salfeld et al. Jul 2005 B1
6924124 Singh Aug 2005 B1
6936441 Reiter et al. Aug 2005 B2
6974681 McGrew Dec 2005 B1
7029982 Zhuang et al. Apr 2006 B1
7070775 Le et al. Jul 2006 B2
7084260 Lonberg et al. Aug 2006 B1
7122641 Vedantham et al. Oct 2006 B2
7189820 Ruben Mar 2007 B2
7192584 Le et al. Mar 2007 B2
7223394 Salfeld et al. May 2007 B2
7250165 Heavner et al. Jul 2007 B2
7276239 Le et al. Oct 2007 B2
7323553 Fahrner et al. Jan 2008 B2
7326681 Gerngross Feb 2008 B2
7332303 Schilling Feb 2008 B2
7390660 Behrendt et al. Jun 2008 B2
7429491 Luan et al. Sep 2008 B2
7449308 Gerngross et al. Nov 2008 B2
7473680 DeFrees et al. Jan 2009 B2
7504485 Salfeld et al. Mar 2009 B2
7517670 Umana et al. Apr 2009 B2
7521206 Heavner et al. Apr 2009 B2
7521210 Knudsen Apr 2009 B2
7541031 Salfeld et al. Jun 2009 B2
7588761 Salfeld et al. Sep 2009 B2
7612181 Wu et al. Nov 2009 B2
7645609 Follstad Jan 2010 B2
7714112 Engstrand et al. May 2010 B2
7750129 Johansson et al. Jul 2010 B2
7767207 Ghayer et al. Aug 2010 B2
7863426 Wan et al. Jan 2011 B2
7883704 Salfeld et al. Feb 2011 B2
7906329 Umana et al. Mar 2011 B2
7919264 Maksymowych et al. Apr 2011 B2
7947471 Knudsen May 2011 B2
7972810 Crowell et al. Jul 2011 B2
8034906 Borhani et al. Oct 2011 B2
8043863 Bosques et al. Oct 2011 B2
8067182 Kelley et al. Nov 2011 B2
8093045 Pla et al. Jan 2012 B2
8192951 Wang et al. Jun 2012 B2
8197813 Salfeld et al. Jun 2012 B2
8206714 Salfeld et al. Jun 2012 B2
8209132 Bosques et al. Jun 2012 B2
8216583 Kruase et al. Jul 2012 B2
8216851 Parsons et al. Jul 2012 B2
8231876 Wan et al. Jul 2012 B2
8304250 Parsons et al. Nov 2012 B2
8313925 Gregory et al. Nov 2012 B2
8338088 Collins et al. Dec 2012 B2
8361705 Parsons et al. Jan 2013 B2
8361797 Osborne et al. Jan 2013 B2
8372400 Salfeld et al. Feb 2013 B2
8372401 Salfeld et al. Feb 2013 B2
8388965 Rao et al. Mar 2013 B2
8414894 Salfeld et al. Apr 2013 B2
8420081 Fraunhofer et al. Apr 2013 B2
8436149 Borhani et al. May 2013 B2
8470318 Ravetch et al. Jun 2013 B2
8470552 Croughan et al. Jun 2013 B2
8512983 Gawlitzek et al. Aug 2013 B2
8586356 Bosques et al. Nov 2013 B2
8623644 Umana et al. Jan 2014 B2
8629248 Umana et al. Jan 2014 B2
8632773 Kasermann et al. Jan 2014 B2
8652487 Maldonado Feb 2014 B2
8663945 Pla et al. Mar 2014 B2
8663999 Parsons et al. Mar 2014 B2
8703498 Parsons et al. Apr 2014 B2
8729241 Liu et al. May 2014 B2
8753633 Salfeld et al. Jun 2014 B2
8821865 Neu et al. Sep 2014 B2
8852889 Prentice Oct 2014 B2
8883146 Fraunhofer et al. Nov 2014 B2
8883156 Wan et al. Nov 2014 B2
8895009 Wan et al. Nov 2014 B2
8895709 Hickman et al. Nov 2014 B2
8906372 Wan et al. Dec 2014 B2
8906646 Pla et al. Dec 2014 B2
8911964 Pla et al. Dec 2014 B2
8916153 Wan et al. Dec 2014 B2
8921526 Chumsae et al. Dec 2014 B2
8946395 Herigstad et al. Feb 2015 B1
9017687 Wang et al. Apr 2015 B1
9035027 Ghayur et al. May 2015 B2
9062106 Bengea Jun 2015 B2
9067990 Wang et al. Jun 2015 B2
9085618 Ramasubramanyan et al. Jul 2015 B2
9085619 Fraunhofer et al. Jul 2015 B2
9090688 Bengea Jul 2015 B2
9096879 Khetan et al. Aug 2015 B2
9103821 Bosques et al. Aug 2015 B2
9150645 Subramanian et al. Oct 2015 B2
9181337 Subramanian et al. Nov 2015 B2
9181572 Subramanian et al. Nov 2015 B2
9182467 Parsons et al. Nov 2015 B2
9200069 Ramasubramanyan et al. Dec 2015 B2
9200070 Ramasubramanyan et al. Dec 2015 B2
9206390 Rives Dec 2015 B2
9234033 Rives Jan 2016 B2
9249182 Herigstad et al. Feb 2016 B2
9255143 Bengea Feb 2016 B2
9265815 Fraser et al. Feb 2016 B2
9266949 Ramasubramanyan et al. Feb 2016 B2
9290568 Rives Mar 2016 B2
20020045207 Krummen et al. Apr 2002 A1
20020132299 Field Sep 2002 A1
20020187526 Ruben et al. Dec 2002 A1
20030012786 Teoh et al. Jan 2003 A1
20030049725 Heavner et al. Mar 2003 A1
20030096414 Ciccarone et al. May 2003 A1
20030125247 Rosen et al. Jul 2003 A1
20030153735 Breece et al. Aug 2003 A1
20030157108 Presta Aug 2003 A1
20030161828 Abdelghany et al. Aug 2003 A1
20030166869 Vedantham et al. Sep 2003 A1
20030175884 Umana et al. Sep 2003 A1
20030178368 van Reis Sep 2003 A1
20030206898 Fischkoff et al. Nov 2003 A1
20030219438 Salfeld et al. Nov 2003 A1
20030229212 Fahrner et al. Dec 2003 A1
20030235585 Fischkoff et al. Dec 2003 A1
20040009172 Fischkoff et al. Jan 2004 A1
20040029229 Reeves et al. Feb 2004 A1
20040033228 Krause et al. Feb 2004 A1
20040033535 Boyle et al. Feb 2004 A1
20040038878 Tanikawa et al. Feb 2004 A1
20040101939 Santora et al. May 2004 A1
20040120952 Knight et al. Jun 2004 A1
20040126372 Banerjee et al. Jul 2004 A1
20040126373 Banerjee et al. Jul 2004 A1
20040131614 Banerjee et al. Jul 2004 A1
20040132140 Satoh et al. Jul 2004 A1
20040136986 Raju Jul 2004 A1
20040136989 Banerjee et al. Jul 2004 A1
20040136990 Banerjee et al. Jul 2004 A1
20040136991 Banerjee et al. Jul 2004 A1
20040151722 Banerjee et al. Aug 2004 A1
20040162414 Santora et al. Aug 2004 A1
20040166111 Kaymakcalan et al. Aug 2004 A1
20040171152 Price et al. Sep 2004 A1
20040191243 Chen et al. Sep 2004 A1
20040191256 Raju Sep 2004 A1
20040214289 deVries et al. Oct 2004 A1
20040219142 Banerjee et al. Nov 2004 A1
20050004354 Salfeld et al. Jan 2005 A1
20050100965 Ghayur et al. May 2005 A1
20050123541 Heavner et al. Jun 2005 A1
20050175611 Mahler et al. Aug 2005 A1
20050249735 Le et al. Nov 2005 A1
20050271654 Rinderknecht et al. Dec 2005 A1
20050272124 Chen et al. Dec 2005 A1
20060009385 Hoffman et al. Jan 2006 A1
20060018907 Le et al. Jan 2006 A1
20060024293 Salfeld et al. Feb 2006 A1
20060057638 Bosques et al. Mar 2006 A1
20060083741 Hoffman et al. Apr 2006 A1
20060127950 Bosques et al. Jun 2006 A1
20060149042 Konstantinov et al. Jul 2006 A1
20060246073 Knight et al. Nov 2006 A1
20060252672 Betenbaugh et al. Nov 2006 A1
20060269479 Colton et al. Nov 2006 A1
20060287432 Christensen et al. Dec 2006 A1
20070003548 Heavner et al. Jan 2007 A1
20070004009 Dixit et al. Jan 2007 A1
20070015239 Bihoreau et al. Jan 2007 A1
20070020260 Presta Jan 2007 A1
20070041905 Hoffman et al. Feb 2007 A1
20070060741 Kelley et al. Mar 2007 A1
20070071747 Hoffman et al. Mar 2007 A1
20070081996 Hoffman et al. Apr 2007 A1
20070110743 Drapeau et al. May 2007 A1
20070111284 Ryll May 2007 A1
20070134256 Lai et al. Jun 2007 A1
20070161084 Crowell Jul 2007 A1
20070172475 Matheus et al. Jul 2007 A1
20070172897 Maksymowych et al. Jul 2007 A1
20070184045 Doctor et al. Aug 2007 A1
20070184529 Etcheverry et al. Aug 2007 A1
20070190057 Wu et al. Aug 2007 A1
20070196373 Le et al. Aug 2007 A1
20070202051 Schuschnig Aug 2007 A1
20070202104 Banerjee et al. Aug 2007 A1
20070212770 Grillberger et al. Sep 2007 A1
20070269463 Donovan Nov 2007 A1
20070292442 Wan et al. Dec 2007 A1
20070298040 Le et al. Dec 2007 A1
20080009040 Grillberger et al. Jan 2008 A1
20080025976 Le et al. Jan 2008 A1
20080095762 Presta Apr 2008 A1
20080112953 McAuley et al. May 2008 A1
20080118496 Medich et al. May 2008 A1
20080131374 Medich et al. Jun 2008 A1
20080160577 Dell'Orco et al. Jul 2008 A1
20080166348 Kupper et al. Jul 2008 A1
20080193466 Banerjee et al. Aug 2008 A1
20080206246 Ravetch et al. Aug 2008 A1
20080219952 Fischer et al. Sep 2008 A1
20080227136 Pla et al. Sep 2008 A1
20080269132 Gomes et al. Oct 2008 A1
20080269468 Vogel et al. Oct 2008 A1
20080274507 Gomes et al. Nov 2008 A1
20080292642 Borhani et al. Nov 2008 A1
20080305114 Salfeld et al. Dec 2008 A1
20080311043 Hoffman et al. Dec 2008 A1
20090017472 Stuhlmuller et al. Jan 2009 A1
20090028794 Medich et al. Jan 2009 A1
20090053786 Kao et al. Feb 2009 A1
20090060910 Johnson et al. Mar 2009 A1
20090068172 Kaymakcalan et al. Mar 2009 A1
20090068705 Drapeau et al. Mar 2009 A1
20090110679 Li et al. Apr 2009 A1
20090123378 Wong et al. May 2009 A1
20090142828 Bucciarelli et al. Jun 2009 A1
20090148513 Fraunhofer et al. Jun 2009 A1
20090151023 Kuvshinov et al. Jun 2009 A1
20090155205 Salfeld et al. Jun 2009 A1
20090175857 Salfeld et al. Jul 2009 A1
20090202546 Harris et al. Aug 2009 A1
20090202557 Argiriadi et al. Aug 2009 A1
20090203055 Ngantung et al. Aug 2009 A1
20090208500 Joly et al. Aug 2009 A1
20090226530 Lassner et al. Sep 2009 A1
20090239259 Hsieh Sep 2009 A1
20090253174 Serber et al. Oct 2009 A1
20090258018 Medich et al. Oct 2009 A1
20090269302 Salfeld et al. Oct 2009 A1
20090271164 Peng et al. Oct 2009 A1
20090280065 Willian et al. Nov 2009 A1
20090291062 Fraunhofer et al. Nov 2009 A1
20090304682 Hoffman et al. Dec 2009 A1
20090317399 Pollack et al. Dec 2009 A1
20100003243 Okun et al. Jan 2010 A1
20100016557 Salfeld et al. Jan 2010 A1
20100021451 Wong Jan 2010 A1
20100034823 Borhani et al. Feb 2010 A1
20100040604 Salfeld et al. Feb 2010 A1
20100040630 Elden et al. Feb 2010 A1
20100113294 Venkataraman et al. May 2010 A1
20100135987 Hickman et al. Jun 2010 A1
20100136025 Hickman et al. Jun 2010 A1
20100151499 Collins et al. Jun 2010 A1
20100160894 Julian et al. Jun 2010 A1
20100167313 Essig et al. Jul 2010 A1
20100172911 Naso Jul 2010 A1
20100189717 Kim et al. Jul 2010 A1
20100221823 McCoy et al. Sep 2010 A1
20100255013 Presta Oct 2010 A1
20100256336 Yuk et al. Oct 2010 A1
20100278808 Ravetch et al. Nov 2010 A1
20100278822 Fraunhofer et al. Nov 2010 A1
20100279306 Bosques et al. Nov 2010 A1
20100297697 Ambrosius et al. Nov 2010 A1
20110002935 Wan et al. Jan 2011 A1
20110003338 Bayer et al. Jan 2011 A1
20110053223 Bayer et al. Mar 2011 A1
20110053265 Follstad et al. Mar 2011 A1
20110054414 Shang et al. Mar 2011 A1
20110081679 Jing et al. Apr 2011 A1
20110081700 Hasslacher et al. Apr 2011 A1
20110086050 Presta Apr 2011 A1
20110086798 Sethuraman et al. Apr 2011 A1
20110097336 Wu et al. Apr 2011 A1
20110123544 Salfeld et al. May 2011 A1
20110124024 Raju et al. May 2011 A1
20110130544 Ram et al. Jun 2011 A1
20110136682 Bosques et al. Jun 2011 A1
20110171227 Okun et al. Jul 2011 A1
20110207676 Callewaert et al. Aug 2011 A1
20110213137 Bosques et al. Sep 2011 A1
20110263828 Wong et al. Oct 2011 A1
20110300151 Okun et al. Dec 2011 A1
20110318340 Collin et al. Dec 2011 A1
20120014956 Kupper et al. Jan 2012 A1
20120015438 Schilling et al. Jan 2012 A1
20120039900 Stuhlmuller et al. Feb 2012 A1
20120039908 Combs et al. Feb 2012 A1
20120077213 Pla et al. Mar 2012 A1
20120093810 Takada et al. Apr 2012 A1
20120107783 Julian et al. May 2012 A1
20120107874 Liu et al. May 2012 A1
20120123688 Ramasubramanyan et al. May 2012 A1
20120129185 Maksymowych et al. May 2012 A1
20120134988 Ravetch et al. May 2012 A1
20120171123 Medich et al. Jul 2012 A1
20120177596 Fischkoff et al. Jul 2012 A1
20120178107 Salfeld et al. Jul 2012 A1
20120183997 Alley et al. Jul 2012 A1
20120190005 Schaub et al. Jul 2012 A1
20120201831 Salfeld et al. Aug 2012 A1
20120213792 Salfeld et al. Aug 2012 A1
20120219564 Salfeld et al. Aug 2012 A1
20120238730 Dong et al. Sep 2012 A1
20120244168 Salfeld et al. Sep 2012 A1
20120251541 Baurin et al. Oct 2012 A1
20120251550 Borhani et al. Oct 2012 A1
20120258114 Salfeld et al. Oct 2012 A1
20120263731 Fraunhofer et al. Oct 2012 A1
20120264920 Wang et al. Oct 2012 A1
20120264927 Parsons et al. Oct 2012 A1
20120276109 Fraser et al. Nov 2012 A1
20120276134 Fraser et al. Nov 2012 A1
20120276155 Kishimoto et al. Nov 2012 A1
20120276157 Fraser et al. Nov 2012 A1
20120276158 Fraser et al. Nov 2012 A1
20120276160 Maldonado Nov 2012 A1
20120276631 Bengea et al. Nov 2012 A1
20120277165 Collins et al. Nov 2012 A1
20120282262 Okun et al. Nov 2012 A1
20120282270 Krause et al. Nov 2012 A1
20120288494 Borhani et al. Nov 2012 A1
20120294888 Kishimoto et al. Nov 2012 A1
20120301498 Altreuter et al. Nov 2012 A1
20120301510 Kishimoto et al. Nov 2012 A1
20120308514 Salfeld et al. Dec 2012 A1
20120329709 Collins et al. Dec 2012 A1
20130004507 Fischkoff et al. Jan 2013 A1
20130028903 Wan et al. Jan 2013 A1
20130065219 Tsang et al. Mar 2013 A1
20130084605 Zhou et al. Apr 2013 A1
20130096283 Khetan et al. Apr 2013 A1
20130115224 Salfeld et al. May 2013 A1
20130122011 Hoffman et al. May 2013 A1
20130122018 Salfeld et al. May 2013 A1
20130149300 Hiatt et al. Jun 2013 A1
20130156760 Fraunhofer et al. Jun 2013 A1
20130195888 Wang et al. Aug 2013 A1
20130205604 Esenwein et al. Aug 2013 A1
20130231255 Collins et al. Sep 2013 A1
20130243786 Banerjee et al. Sep 2013 A1
20130244280 Parikh et al. Sep 2013 A1
20130273059 Wan et al. Oct 2013 A1
20130280267 Wan et al. Oct 2013 A1
20130280274 Subramanian et al. Oct 2013 A1
20130309242 Wan et al. Nov 2013 A1
20130323261 Wan et al. Dec 2013 A1
20130330356 Salfeld et al. Dec 2013 A1
20130330357 Salfeld et al. Dec 2013 A1
20130336957 Wang et al. Dec 2013 A1
20130338344 Ramasubramanyan et al. Dec 2013 A1
20130344084 Subramanian et al. Dec 2013 A1
20140010820 Wang et al. Jan 2014 A1
20140045212 Bosques et al. Feb 2014 A1
20140046032 Blanche et al. Feb 2014 A1
20140065710 Rives et al. Mar 2014 A1
20140072585 Herigstad et al. Mar 2014 A1
20140120583 Prentice May 2014 A1
20140134674 Pla et al. May 2014 A1
20140134675 Pla et al. May 2014 A1
20140141007 Fraunhofer et al. May 2014 A1
20140141008 Fraunhofer et al. May 2014 A1
20140142286 Prentice May 2014 A1
20140154270 Wang et al. Jun 2014 A1
20140178984 Jerums et al. Jun 2014 A1
20140199340 Maldonado Jul 2014 A1
20140206038 Pla et al. Jul 2014 A1
20140234905 Pla et al. Aug 2014 A1
20140255423 Hickman et al. Sep 2014 A1
20140271622 Prentice Sep 2014 A1
20140271623 Parren et al. Sep 2014 A1
20140271626 Chumsae et al. Sep 2014 A1
20140271632 Hossler et al. Sep 2014 A1
20140271633 Hossler Sep 2014 A1
20140273057 Prentice et al. Sep 2014 A1
20140274911 Collins et al. Sep 2014 A1
20140274912 Prentice Sep 2014 A1
20140275494 Wang et al. Sep 2014 A1
20140288278 Nti-Gyabaah et al. Sep 2014 A1
20140296490 Faid et al. Oct 2014 A1
20140301977 Nadarajah et al. Oct 2014 A1
20140314745 Rives et al. Oct 2014 A1
20140363845 Sinacore et al. Dec 2014 A1
20140377275 Neu et al. Dec 2014 A1
20150023977 Fraunhofer et al. Jan 2015 A1
20150110775 Subramanian et al. Apr 2015 A1
20150110799 Ramasubramanyan et al. Apr 2015 A1
20150132320 Chumsae et al. May 2015 A1
20150132801 Ramasubramanyan et al. May 2015 A1
20150133639 Wentz et al. May 2015 A1
20150140006 Ramasubramanyan et al. May 2015 A1
20150141632 Markosyan May 2015 A1
20150158944 Bengea et al. Jun 2015 A1
20150166650 Ramasubramanyan et al. Jun 2015 A1
20150166653 Wang et al. Jun 2015 A1
20150183865 Rives et al. Jul 2015 A1
20150183866 Rives et al. Jul 2015 A1
20150259410 Ramasubramanyan et al. Sep 2015 A1
20150299249 Herigstad et al. Oct 2015 A1
20150320728 Fraser et al. Nov 2015 A1
20150320856 Altreuter et al. Nov 2015 A1
20150320870 Maldonado Nov 2015 A1
20150320884 Fraser et al. Nov 2015 A1
20150328333 Fraser et al. Nov 2015 A1
20150335762 Fraser et al. Nov 2015 A1
20150361170 Fraunhofer et al. Dec 2015 A1
20160017281 Sunstrom Jan 2016 A1
20160022650 Fraser et al. Jan 2016 A1
20160030554 Kishimoto et al. Feb 2016 A1
20160030555 Kishimoto et al. Feb 2016 A1
20160039924 Zeng Feb 2016 A1
20160039925 Subramanian et al. Feb 2016 A1
20160046708 Subramanian et al. Feb 2016 A1
20160068881 Prentice Mar 2016 A1
Foreign Referenced Citations (211)
Number Date Country
1563090 Jan 2005 CN
3631229 Mar 1988 DE
0101681 Mar 1984 EP
0173177 Mar 1986 EP
0186833 Jul 1986 EP
0212489 Mar 1987 EP
230584 Aug 1987 EP
0351789 Jan 1990 EP
0366043 May 1990 EP
374510 Jun 1990 EP
453898 Oct 1991 EP
0460426 Dec 1991 EP
0481791 Apr 1992 EP
0492448 Jul 1992 EP
0523949 Jan 1993 EP
585705 Mar 1994 EP
0612251 Aug 1994 EP
0614984 Sep 1994 EP
0659766 Jun 1995 EP
0746398 Dec 1996 EP
0764719 Mar 1997 EP
0956873 Nov 1999 EP
0956875 Nov 1999 EP
1075488 Feb 2001 EP
1174148 Jan 2002 EP
1176195 Jan 2002 EP
1221476 Jul 2002 EP
1254666 Nov 2002 EP
1308455 May 2003 EP
1308456 May 2003 EP
1418967 May 2004 EP
1568388 Aug 2005 EP
1745141 Jan 2007 EP
1849862 Oct 2007 EP
1851305 Nov 2007 EP
2080809 Jul 2009 EP
2144929 Jan 2010 EP
2152856 Feb 2010 EP
2213726 Aug 2010 EP
2305712 Apr 2011 EP
2357250 Aug 2011 EP
2495307 Sep 2012 EP
2500414 Sep 2012 EP
2528002 Nov 2012 EP
2574677 Apr 2013 EP
2160530 Dec 1985 GB
2279077 Dec 1994 GB
2285MUM2013 Jan 2015 IN
6-292592 Oct 1994 JP
7289288 Nov 1995 JP
WO-8700195 Jan 1987 WO
WO-9003430 Apr 1990 WO
WO-9005144 May 1990 WO
WO-9102078 Feb 1991 WO
WO-9109967 Jul 1991 WO
WO-9201047 Jan 1992 WO
WO-9211383 Jul 1992 WO
WO-9216221 Oct 1992 WO
WO-9216553 Oct 1992 WO
WO-9217583 Oct 1992 WO
WO-9306213 Apr 1993 WO
WO-9311793 Jun 1993 WO
WO-9402602 Feb 1994 WO
WO-9408619 Apr 1994 WO
WO-9420139 Sep 1994 WO
WO-9425585 Nov 1994 WO
WO-9426910 Nov 1994 WO
WO-9429347 Dec 1994 WO
WO-9511317 Apr 1995 WO
WO-9523813 Sep 1995 WO
WO-9633208 Oct 1996 WO
WO-9633735 Oct 1996 WO
WO-9634096 Oct 1996 WO
WO-9704801 Feb 1997 WO
WO-9713852 Apr 1997 WO
WO-9729131 Aug 1997 WO
WO-98-08934 Mar 1998 WO
WO-9823645 Jun 1998 WO
WO-9824883 Jun 1998 WO
WO-9824884 Jun 1998 WO
WO-9824893 Jun 1998 WO
WO-9850433 Nov 1998 WO
WO-9856418 Dec 1998 WO
WO-9858964 Dec 1998 WO
WO-9922764 May 1999 WO
WO-9932605 Jul 1999 WO
WO-9954342 Oct 1999 WO
WO-9957134 Nov 1999 WO
WO-9957246 Nov 1999 WO
WO-0003000 Jan 2000 WO
WO-01-44442 Jun 2001 WO
WO-0147554 Jul 2001 WO
WO-01-59069 Aug 2001 WO
WO-0177362 Oct 2001 WO
WO-0212502 Feb 2002 WO
WO-0212501 Feb 2002 WO
WO-03046162 Jun 2003 WO
WO-03045995 Jun 2003 WO
WO-03059935 Jul 2003 WO
WO-03066662 Aug 2003 WO
WO-2004008100 Jan 2004 WO
WO-2004058944 Jul 2004 WO
WO-2004058800 Jul 2004 WO
WO-2004097006 Nov 2004 WO
WO-2005042569 May 2005 WO
WO-2005-062967 Jul 2005 WO
WO-2005082483 Sep 2005 WO
WO-2005100584 Oct 2005 WO
WO-2006014683 Feb 2006 WO
WO-2006043895 Apr 2006 WO
WO-2006045438 May 2006 WO
WO-2006099308 Sep 2006 WO
WO-2006110277 Oct 2006 WO
WO-2007-005786 Jan 2007 WO
WO-2007024743 Mar 2007 WO
WO-2007055916 May 2007 WO
WO-2007070315 Jun 2007 WO
WO-2007-077217 Jul 2007 WO
WO-2007087384 Aug 2007 WO
WO-2007117490 Oct 2007 WO
WO-2007117505 Oct 2007 WO
WO-2008028686 Mar 2008 WO
WO-2008033517 Mar 2008 WO
WO-2008-057240 May 2008 WO
WO-2008057634 May 2008 WO
WO-2008068879 Jun 2008 WO
WO-2008087184 Jul 2008 WO
WO-2008121616 Oct 2008 WO
WO-2008135498 Nov 2008 WO
WO-2009027041 Jan 2009 WO
WO-2009017491 Feb 2009 WO
WO-2009023562 Feb 2009 WO
WO-2009058769 May 2009 WO
WO-2009073569 Jun 2009 WO
WO-2009079382 Jun 2009 WO
WO-2009135656 Nov 2009 WO
WO-2010036443 Apr 2010 WO
WO-2010-048183 Apr 2010 WO
WO-2010043703 Apr 2010 WO
WO-2010111633 Sep 2010 WO
WO-2010122460 Oct 2010 WO
WO-2010129469 Nov 2010 WO
WO-2010127069 Nov 2010 WO
WO-2010136209 Dec 2010 WO
WO-2010138502 Dec 2010 WO
WO-2011005773 Jan 2011 WO
WO-2011009623 Jan 2011 WO
WO-2011-019619 Feb 2011 WO
WO-2011015926 Feb 2011 WO
WO-2011024025 Mar 2011 WO
WO-2011044180 Apr 2011 WO
WO-2011073235 Jun 2011 WO
WO-2011069056 Jun 2011 WO
WO-2011098526 Aug 2011 WO
WO-2011110598 Sep 2011 WO
WO-2011133886 Oct 2011 WO
WO-2011127322 Oct 2011 WO
WO-2011133902 Oct 2011 WO
WO-2011134919 Nov 2011 WO
WO-2011134920 Nov 2011 WO
WO-2012019160 Feb 2012 WO
WO-2012030512 Mar 2012 WO
WO-2012046255 Apr 2012 WO
WO-2012050175 Apr 2012 WO
WO-2012051147 Apr 2012 WO
WO-2012065072 May 2012 WO
WO-2012062810 May 2012 WO
WO-2012120500 Sep 2012 WO
WO-2012140138 Oct 2012 WO
WO-2012145682 Oct 2012 WO
WO-2012149197 Nov 2012 WO
WO-2012147048 Nov 2012 WO
WO-2012147053 Nov 2012 WO
WO-2012158551 Nov 2012 WO
WO-2013-009648 Jan 2013 WO
WO-2013-011076 Jan 2013 WO
WO-2013006461 Jan 2013 WO
WO-2013006479 Jan 2013 WO
WO-2013013013 Jan 2013 WO
WO-2013021279 Feb 2013 WO
WO-2013-066707 May 2013 WO
WO-2013095966 Jun 2013 WO
WO-2013-158273 Oct 2013 WO
WO-2013-158279 Oct 2013 WO
WO-2013158275 Oct 2013 WO
WO-2013-164837 Nov 2013 WO
WO-2013-176754 Nov 2013 WO
WO-2013-177115 Nov 2013 WO
WO-2013-177118 Nov 2013 WO
WO-2013181585 Dec 2013 WO
WO-2013-186230 Dec 2013 WO
WO-2014018747 Jan 2014 WO
WO-2014039903 Mar 2014 WO
WO-2014052360 Apr 2014 WO
WO-2014096672 Jun 2014 WO
WO-2014099636 Jun 2014 WO
WO-2014125374 Aug 2014 WO
WO-2014-149935 Sep 2014 WO
WO-2014150655 Sep 2014 WO
WO-2014151878 Sep 2014 WO
WO-2014158231 Oct 2014 WO
WO-2014159488 Oct 2014 WO
WO-2014159494 Oct 2014 WO
WO-2014159499 Oct 2014 WO
WO-2014179601 Nov 2014 WO
WO-2014207763 Dec 2014 WO
WO-2015004679 Jan 2015 WO
WO-2015007912 Jan 2015 WO
WO-2015051293 Apr 2015 WO
WO-2015073884 May 2015 WO
WO-2016007764 Jan 2016 WO
Non-Patent Literature Citations (512)
Entry
Raju (BioProcess Intl. Apr. 2003:44-53).
Tebbey et al. (MAbs. Sep. 3, 2015. 7(5):805-11).
“Genentech unveils production capacity hikes,” in-Pharma Technologist.com Jun. 28, 2005, pp. 1-2.
“Memorandum in Support of Centocor's Motion for Summary Judgment No. 1 that All Asserted Claims Are Invalid for Lack of Written Description”, dated Aug. 1, 2013 and submitted by defendant in Civil Action No. 09-40089-FDS, 28 pages.
“Memorandum in Support of Centocor's Motion for Summary Judgment No. 2 that All Asserted Claims Are Invalid for Lack of Enablement”, dated Aug. 1, 2013 and submitted by defendant in Civil Action No. 09-40089-FDS, 22 pages.
“Memorandum in Support of Centocor's Motion for Summary Judgment No. 4 that Claims Encompassing Non-recombinant Human Antibodies Are Invalid for Failing to Meet the Requirements of 35 U.S.C. §112”, dated Aug. 1, 2013 and submitted by defendant in Civil Action No. 09-40089-FDS, 21 pages.
“Memorandum in Support of Centocor's Motion No. 3 for Summary Judgment that the 394 and 031 Patents Are Invalid for Under 35 U.S.C. §102(f) for Failing to Name the Proper Inventors”, dated Aug. 1, 2013 and submitted by defendant in Civil Action No. 09-40089-FDS, 13 pages.
“Memorandum in Support of Centocor's Motion No. 6 for Summary Judgment that References Dated Before Feb. 10, 1997 Qualify as Prior Art to the 394 and 031 Patents”, dated Aug. 1, 2013 and submitted by defendant in Civil Action No. 09-40089-FDS, 16 pages.
“Plaintiffs' Memorandum in Support of Their Motion for Partial Summary Judgment”, dated Aug. 1, 2013 and submitted by plaintiff in Civil Action No. 09-40089-FDS, 49 pages.
“Plaintiffs' Rule 56.1 Statement of Undisputed Material Facts in Support of Their Motion for Partial Summary Judgment”, dated Aug. 1, 2013 and submitted by plaintiff in Civil Action No. 09-40089-FDS, 13 pages.
Abbott Laboratories Press Release, “Abbott Laboratories Receives FDA Approval Earlier Than Expected for HUMIRA (adalimumab) for the Treatment of Rheumatoid Arthritis,” Dec. 31, 2002, pp. 1-4.
Abraham, E., et al., “Efficacy and Safety of Monoclonal Antibody to Human Tumor Necrosis Factor α in Patients with Sepsis Syndrome,” JAMA, vol. 273(12):934-941 (1995).
Adams. et al., “Aggressive cutaneous T-cell lymphomas after TNFα blockade,” J. Am. Acad. Dermatol 2004;51 :660-2.
Ahmed, M. U.et al.; N-(Carboxyethyl)lysine, a product of the chemical modification of proteins by methylglyoxal, increases with age in human lens proteins; Biochem. J. 1997, 324, 565-570.
Ahmed, N. & Thornalley, P. J.; Peptide Mapping of Human Serum Albumin Modified Minimally by Methylglyoxal in Vitro and in Vivo; Ann. N.Y. Acad. Sci. 2005, 1043,260-266.
Ahmed, N. et al.; Peptide Mapping Identifies Hotspot Site of Modification in Human Serum Albumin by Methylglyoxal Involved in Ligand Binding and Esterase Activity; J. Biol. Chem. 2005, 280, 5724-5732.
Ahmed, N.; Thornalley, P. J.; Advanced glycation endproducts: what is their relevance to diabetic complications?; Diabetes, Obes. Metab. 2007, 9, 233-245.
Alessandri, L. et al., “Increased serum clearance of oligomannose species present on a human IgG1 molecule.” mAbs, (2012), 4(4); 509-520.
Alfaro, J. F.; Chemo-Enzymatic Detection of Protein Isoaspartate Using Protein Isoaspartate Methyltransferase and Hydrazine Trapping; Anal. Chem. 2008, 80, 3882-3889.
Alfaro, J. F.; Synthesis of LuxS Inhibitors Targeting Bacterial Cell-Cell Communication; Org. Lett. 2004, 6, 3043-3046.
Altamirano, C., et al., “Strategies for fed batch cultivation of t-PA producing CHO cells: substitution of glucose and glutamine and rational design of culture medium”, J. Biotechn. 110:171-179, 2004.
Andersen DC, The effect of cell-culture conditions on the oligosaccharide structures of secreted glycoproteins. Curr Opin Biotechnol. Oct. 1994;5(5):546-9.
Anonymous, “SACHEM Displacement Chromatography,” Aug. 29, 2012, Retrieved from the internet: www.displacementchromatoaraphy.com, retrieved on Jul. 30, 2014.
Antes et al. “Analysis of lysine clipping of a humanized Lewis-Y specific IgG antibody and its relation to Fc-mediated effector function” Journal of Chromatography B:Biomedical Sciences and Applications, Elsevier, Amsterdam, NL, vol. 852, No. 1-2, May 31, 2007, 250-256.
Anumula et al., “Quantitative glycan profiling of normal human plasma derived immunoglobulin and its fragments Fab and FcO” (2012) J. Immunol. Methods, 382:167-176.
Arend et al., “Inhibition of the production and effects of interleukins-1 and tumor necrosis factor α in rheumatoid arthritis” (1995) Arth. Rheum., 38(2):151-160.
Ashkenazi et al., “Immunoadhesins: An alternative to human monoclonal antibodies” (1995) Methods, 8(2): 104-115.
Averginos, Gab '04 Abstracts—GE Healthcare Life Sciences, “HUMIRA manufacturing: challenges and the path taken”, France, Oct. 3-5, 2004, published 2005, pp. 14-16.
Avgerinos et al. (GAb '04 Abstracts—GE Healthcare Life Sciences, France Oct. 3-5, 2004, pp. 15-16 published 2005).
Awdeh, Z.L., A.R. Williamson, and B.A. Askonas, One cell-one immunoglobulin. Origin of limited heterogeneity of myeloma proteins. Biochem J, 1970. 116(2): p. 241-8.
Azevedo et al., “Integrated Process for the Purification of Antibodies Combining Aqueous Two-Phase Extraction, Hydrophobic Interaction Chromatography and Size-Exclusion Chromatography”, Journal of Chromatography (2008) 1213(2): 154-161.
Babcock, James et al., “Partial Replacement of Chemically Defined Media with Plant-Derived Protein Hydrolysates,” BioPharm International, vol. 23: Jun. 6, 2010, 6 pages.
Ballez, J.S. et al., “Plant protein hydrolysates support CHO-320 cells proliferation and recombinant IFN—[gamma] production in suspension and inside microcarriers in protein-free media”, Cytotechnology 44:3, 103-114, 2004.
Bandyopadhyay S., et al. Physicochemical and functional characterization of a biosimilar adalimumab ZRC-3197, Biosimilars, 2015;5, pages 1-18.
Barb et al., “Branch-specific sialylation of IgG-Fc glycans by ST6GaI-I” Biochemistry, (2009) 48:9705-9707.
Barbuto, J. et al. “Production of Neutralizing Antibodies to Tumor Necrosis Factor by Human Tumor-Infiltrating B Lymphocytes” Proc. Am. Assoc. Cancer Res,. 34:487, Abstr. 2904 (1993).
Barnes et al., “Stability of Protein Production from Recombinant Mammalian Cells,” Biotechnology and Bioengineering, 81:6, Mar. 20, 2003, pp. 631-639.
Bartelds et al., “Development of antidrug antibodies against adalimumab and association with disease activity and treatment failure during long-term follow-up” (2011) JAMA, 305(14):1460-1468.
BD Bioscience Product Description for BBL Phytone Peptone (Advanced Processing, Third Edition) (Sep. 23, 2010) (www.bdbiosciences.com/external—files/Doc—Recon—2.0/ab/others/Phytone—Soytone.pdf www.bdbiosciences.com/external—files/Doc—Recon—2.0/ab/others/Phytone—Soytone.pdf>), (last accessed Jan. 8, 2015), 4 pages.
Bendtzen, K. et al. “Auto-antibodies to IL-1α and TNFα in Normal Individuals and in Infectious and Immunoinflammatory Disorders” The Physiological and Pathological Effects of Cytokines, 447-52 (1990).
Bertolini et al., Stimulation of bone resorption and inhibition of bone formation in vitro by human tumour necrosis factors, (1986) Nature 319:516-518.
Biblia, T.A. et al., “In Pursuit of the Optimal Fed-Batch Process for Monoclonal Antibody Production”, Biotechnol. Prog 11(1):1-13, Jan.-Feb. 1995.
Birch, Jr. et al., “Antibody production”, Adv. Drug Delivery Reviews 58:671-685, 2006.
Bird et al. “Single-chain antigen-binding proteins.” Science. (1988) 242:423-426.
Blaker, GJ, et al., “The Glucose, Insulin and Glutamine Requirements of Suspension Cultures of HeLa Cells in a Defined Culture Medium”, J. Cell Sci. 9:529-537, 1971.
Biastoff, S.; et al.; Colorimetric Activity Measurement of a Recombinant Putrescine N-Methyltransferase from Datura stramonium; Planta Med. 2006, 72, 1136.
Boekstegers, P., et al., “Repeated administration of a F(ab′)2 fragment of an anti-tumor necrosis factor alpha monoclonal antibody in patients with severe sepsis: effects on the cardiovascular system and cytokine levels,” Shock, vol. 1(4):237-245 (1994).
Bollati-Fogolin M., et al., “Temperature Reduction in Cultures of hGM-CSF-expressing CHO Cells: Effects on Productivity and Product Quantity”, Biotechnol. Prog. 21:17-21, 2005.
Bonafede et al. “Cost per treated patient for etanercept, adalimumab, and infliximab across adult indications: a claims analysis” Advances in Therapy, Springer Healthcare Communications, Heidelberg, vol. 29, No. 3, Mar. 9, 2012, 234-249.
Boswell et al. “Effects of Charge on Antibody Tissue Distribution and Pharmacokinetics” Bioconjugate Chem.(21) 2153-2163 (2010).
Boyle, P. et al. “A Novel Monoclonal Human IgM Autoantibody which Binds Recombinant Human and Mouse Tumor Necrosis Factor-α” Cell. Immunol., 152:556-68 (1993).
Boyle, P. et al. “The B5 Monoclonal Human Autoantibody Binds to Cell Surface TNFα on Human Lymphoid Cells and Cell Lines and Appears to Recognize a Novel Epitope” Cell. Immunol., 152:569-81 (1993).
Brekke, O. et al., “Therapeutic Antibodies for Human Diseases at the Dawn of the Twenty-first Century,” Nature, vol. 2:52-62 (2002).
Brock, Jonathan et al., “Detection and identification of arginine modifications on methylglyoxal-modified ribonuclease by mass spectrometric analysis,” Journal of Mass Spectrometry, 2007; 42: 89-100.
Brorson et al., “Bracketed Generic Inactivation of Rodent Retroviruses by Low pH Treatment; for Monoclonal Antibodies and Recombinant Proteins,” Biotechnology and Bioengineering,; vol. 82(3): 321-329 (2003).
Bruggemann et al., “Production of human antibody repertoires in transgenic mice” Cur. Op. Biotechnol. *;455-458 (1997).
Bruggemann, M., Neuberger, M.S., “Strategies for expressing human antibody repertoires in transgenic mice,” Immunol. Today 17:391-397 (1996).
Burteau et al. (in Vitro Cell Dev Biol—Animal, Jul. / Aug. 2003. 39-291-296).
Byun, et al. Archives of Biochemistry and Biophysics, “Transport of anti-IL-6 binding fragments into cartilage and the effects of injury,” 532 (2013), pp. 15-22.
Cai B, et al. “C-Terminal Lysine Processing of Human Immunoglobulin G2 Heavy Chain In Vivo” Biotechnol. Bioeng. 2011;108: 404-412.
Cambridge Antibody Technology, advertisement of phage display services, Science vol. 253, No. 5018 (1991).
Carter et al., “Humanization of an anti-p185HER2 antibody for human cancer therapy,” Proc. Nat. Acad. Sci89:4285-4289 (1992).
Chang KH, et al., “N-Acetylcysteine Increases the Biosynthesis of Recombinant EPO in Apoptotic Chinese Hamster Ovary Cells”, Free Radic Res. 30(2):85-91, 1999.
Chang, T. & Wu, L., Methylglyoxal, oxidative street, and hypertension, Can. J. Physiol.Pharmacol. 84: 1229-1238 (2006).
Chaplen, F.W.R., et al., Effect of endogenous methylgiyoxal on Chinese hamster ovary celis grown in culture Cytotechnology 1996, vol. 22, Issue 1-3, Abstract and references, 6 pages.
Chaplen, F.W.R., Incidence and potential implications of the toxic metabolite methylglyoxal in cell culture: A review, Cytotechnology 26: 173-183, 1998.
Chaplen, FWR; A dissertation entitled Analysis of Methylglyoxal Metabolism in Mammalian Cell Culture; Univ. of Wisconsin-Madison 1996, 218 pages.
Charter, Edward A., “A New Process for the Separation and Purification of Egg Yolk; Antibodies,” BASc., The University of British Columbia; A Thesis; Apr. 1993, 163 pages.
Chelius, D. et al.; Identification and Characterization of Deamidation Sites in the Conserved Regions of Human Immunoglobulin Gamma Antibodies, Anal. Chem. 2005, 77,6004-6011.
Choo et al. “High-level production of a monoclonal antibody in murine myeloma cells by perfusion culture using a gravity settler” Biotechnology Progress, vol. 23, No. 1, Jan. 1, 2007, 225-231.
Chow, A. et al. “Effect of monoclonal antibody on human tumor necrosis factor (TNF MAb) on TNFα, IL-1β, and IL-6 levels in patients with sepsis syndrome” Clinical Research, 42:2 299A (1994).
Chua, FKF et al., “Hyper-stimulation of monoclonal antibody production by high osmolarity stress in eRDF medium”, J. Biotechnology 37(3):265-275, Nov. 15, 1994.
Chumsae, C., et al., Comparison of methionine oxidation in thermal stability and chemically stressed samples of a fully human monoclonal antibody. Journal of Chromatography B, 2007. 850(1-2): p. 285-294.
Chumsae, C., Gaza-Bulseco, G., & Liu, H., Identification and localization of unpaired cysteine residues in monoclonal antibodies by fluorescence labeling and mass spectrometry. Anal Chem, 2009.81(15): p. 6449-57.
Chumsae, Chris et al.: “Arginine modifications by methylglyoxal: discovery in a recombinant monoclonal antibody and contribution to acidic species.”, Analytical Chemistry Dec. 3, 2013, vol. 85, No. 23, Dec. 3, 2013, pp. 11401-11409.
Chung et al., “Utilization of Lysozyme Charge Ladders to Examine the Effects of Protein Surface; Charge Distribution on Bindinq Affinity in Ion Exchange Systems,” Lanqmuir 26(2): 759-768 (2010).
Chung et al. “Cetuximab-induced anaphylaxis and IgE specific for galactose-a-1,3-galactose” NEJM 358:11, 1109-1117 (2008).
Cleland, J. et al., “A Specific Molar Ratio of Stabilizer to Protein is Required for Storage Stability of a Lyophilized Monoclonal Antibody,” Journal of Pharmaceutical Sciences, vol. 90(3):310-321 (2001).
Clincke et al. “Effect of iron sources on the glycosylation macroheterogeneity of human recombinant IFN-y produced by CHO cells during batch processes,” BMC Proceedings (Nov. 22, 2011) 5(Suppl 8):PI14.
Clincke et al. “Characterization of metalloprotease and serine protease activities in batch CHO cell cultures: control of human recombinant IFN-γ proteolysis by addition of iron citrate,” BMC Proceedings (Nov. 22, 2011) 5(Suppl 8):P115.
Clincke, M. et al., “Effect of surfactant pluronic F-68 on CHO cell growth, metabolism, production, and glycosylation of human recombinant IFN-γ in mild operating conditions,” Biotechnol. Prog. 27(1): 181-190, 2011.
Cohen, J., et al., “Intersept: An international, multicenter, placebo-controlled trial of monoclonal anitbody to human tumor necrosis factor-α in patients with sepsis,” Crit Care Med, vol. 24(9):1431-1440 (1996).
Cordoba, A.J., et al., Non-enzymatic hinge region fragmentation of antibodies in solution. Journal of Chromatography B, 2005. 818(2): p. 115-121.
Cox, J. et al. “A directory of human germ-line Vκ segments reveals a strong bias in their usage” Eur. J. Immunol., 24(2):827-36 (1994).
Cromwell (GAB'04 Abstracts—GE Healthcare Life Sciences, Franc Oct. 3-5, 2004, pp. 17-18 published 2005).
Crowell, C.K., et al., Amino acid and manganese supplementation modulates the glycosylation state of erythropoietin in a CHO culture system. Biotechnology and bioengineering, Feb. 15, 2007; 96(3):538-549.
cygnustechnologies.com/product—detail/host-cell-protein-antibodies/anti-cho-h...CYGNUS Technologies, Anti-CHO HCP (Apr. 18, 2012), 1 page.
Dai, S.; An Integrated Proteomic Analysis of Major Isoaspartyl-Containing Proteins in the Urine of Wild Type and Protein Llsoaspartate O-Methyltransferase-Deficient Mice; Anal. Chem. 2013, 85, 2423-2430.
Das et al., “Delivery of rapamycin-loaded nanoparticle down regulates ICAM-1 expression and maintains an immunosuppressive profile in human CD34+ progenitor-derived dendritic cells” (2008) J Biomed Mater Res A., 85(4):983-92.
Daugherty, et al. Formulation and Delivery Issues for Monoclonal Antibody Therapeutics. Advanced Drug Delivery Reviews, 2006. vol. 58, pp. 686-706.
Davies et al., “Antibody VH domains as small recognition units.” Biotechnology, 13:475-479 (1995).
Department of Surgery, University of Toronto, Annual Report (1998-1999)(348 pages).
DePhillips et al., “Determinants of protein retention characteristics on cation-exchange adsorbents,”; Journal of Chromatograph A, 933:57-72 (2001).
deZongotita et al., “Phosphate feeding improves high-cell-concentration NS0 myeloma cell culture performance for monoclonal antibody production” Biotechnology and Bioengineering. 2000, 69: 566-576.
Dick et al: “C-terminal lysine variants in fully human monoclonal antibodies: Investigation of test methods; and possible causes”, Biotechnology and Bioengineering, vol. 100, No. 6, Aug. 15, 2008, pp. 1132-1143.
Dionex Application Note 125 (Monitoring Protein Deamidation by Cation-Exchange Chromatography. 2009; pp. 1-7).
Dobo, A. & Kaltashov, I. A.; Detection of Multiple Protein Conformational Ensembles in Solution via Deconvolution of Charge-State Distributions in ESI MS; Anal. Chem. 2001,73, 4763-4773.
Dolezal, et al., “Escherichia coli Expression of a Bifunctional Fab-peptide Epitope Reagent for the Rapid Diagnosis of HIV-1 and HIV-2”, Immunotechnology, 1:197-209 (1995).
Doring, E., “Identification and Characterization of a TNFa Antagonist Derived from a Monoclonal Antibody” (1994) Mol. Immunol .31(14): 1059-1067.
Drew, Berry et al., “The Effects of Media Formulations on the Biochemical Profile of IgG Expressed in Sp2/0 Cells as Measured by Cation Exchange HPLC,” European Society of Animal Cell Technology Meeting Jan. 2007, Poster #1115, 1 page.
Du et al., “Chromatographic analysis of the acidic and basic species of recombinant monoclonal antibodies” MAbs, Sep.-Oct. 2012; 4(5):578-85.
Eason et al., “Inhibition of the effects of Tnf in renal allograft recipients using recombinant human dimeric tumor necrosis factor receptors” (1995) Transplantation, 59(2):300-305.
Ebersbach et al., “Affilin-novel binding molecules based on human gamma-B-crystallin, an all beta-sheet protein” (2007) J. Mol. Biol., 372 (1): 172-85.
Elliot et al., “Randomised double-blind comparison of chimeric monoclonal antibody to tumour necrosis factor alpha (cA2) versus placebo in rheumatoid arthritis” (1994) Lancet, 344(8930):1105-1110.
Elliot et al., “Repeated therapy with monoclonal antibody to tumour necrosis factor α (cA2) in patients with rheumatoid arthritis” (1994) Lancet, 344:1125-1127.
Elliot, “Treatment of rheumatoid arthritis with chimeric monoclonal antibodies to tumor necrosis factor α” (1993) Arthritis & Rheumatism, 36(12):1681-1690.
Ellison, Jay W. et al., “The Nucleotide Sequence of a Human Immunoglobulin Cγ1 Gene,” Nucleic Acids Research, vol. 10, No. 13 (1982), 9 pages.
Emery, P. “Adalimumab therapy: Clinical findings and implications for integration into clinical guidelines for rheumatoid arthritis.” Drugs of Today, 41(3): p. 155-163. (2005).
ERBITUX (cetuximab) label, Revised Aug. 2013, 8 pages.
European Medicines Agency (EMA Europe), “2004 Report on Scientific Discussion for the Approval of Humira™ (adalimumab),” Last accessed Nov. 12, 2014 at www.ema.europa.eu/docs/en—GB/document—library/EPAR—Scientific—Discussion/human/000481/WC500050867.pdf; 25 pages.
Ewert et al., “Biophysical Properties of Human Antibody Variable Domains,” J. Mol. Biol. 324: 531-; 553 (2003).
Exhibit dated Aug. 1, 2013 and cited by defendant in Civil Action No. 09-40089-FDS providing the Jun. 23, 2009 trial transcript of the PM session in the matter of Centocor, et al. v. Abbott Laboratories, 50 pages.
Exhibit dated Aug. 1, 2013 and cited by defendant in Civil Action No. 09-40089-FDS providing the trial transcript in the matter of Abbott Laboratories, et al. v. The Mathilda and Terrance Kennedy Institute, S.D.N.Y., 90 pages.
Exhibit dated Aug. 1, 2013 and cited by plaintiff in Civil Action No. 09-40089-FDS providing excerpts from the File History of U.S. Appl. No. 12/578,487, 5 pages.
Exhibit dated Aug. 1, 2013 and cited by defendant in Civil Action No. 09-40089-FDS providing the Jun. 24, 2009 trial transcript of the AM session in the matter of Centocor, et al. v. Abbott Laboratories, E.D. TX., 42 pages.
Exhibit dated Aug. 1, 2013 and cited by defendant in Civil Action No. 09-40089-FDS providing the Sep. 20, 2012 Day 8 trial transcript in the matter of Abbott v. Centocor Ortho Biotech Inc., D. MA., 71 pages.
Exhibit dated Aug. 1, 2013 and cited by plaintiff in Civil Action No. 09-40089-FDS providing Declaration by Jochen Salfeld, dated Jan. 17, 2013, 40 pages.
Extended European Search Report for Application No. 13877986.3. Dated Aug. 4, 2014, 11 pages.
Fahrner et al., “Industrial purification of pharmaceutical antibodies: development, operation, and validation of chromatography processes” Biotechnology and Genetic Engineering Reviews, 18, 2001, pp. 301-327.
Fava et al., “Critical role of peripheral blood phagocytes and the involvement of complement in tumour necrosis factor enhancement of passive collagen-arthritis” (1993) Clin. Exp. Immunol., 94(2):261-266.
FDA Package insert for Adalimumab, Sep. 26, 2003, pp. 1-16.
Feldmann, “Anti-TNF-alpha Therapy of Rheumatoid Arthritis: What Have We Learned?” (2001) Annu. Rev. Immunol., 19:163-196.
Felver et al., “Plasma tumor necrosis factor α predicts decreased long-term survival in severe alcoholic hepatitis” (1990) Alcohol. Clin. Exp. Res. 14(2):255-259.
Fernandes, “Demonstrating Comparability of Antibody Glycosylation during Biomanufacturing,” European Biopharmaceutical Review. (2005) pp. 106-110.
Fietze et al., “Cytomegalovirus infection in transplant recipients the role of tumor necrosis factor” (1994) Transplantation, 58(6):675-680.
Figini, “In Vitro assembly of Repertoires of Antibody Chains on the Surface of Phage by Renaturation” (1994) J. Mol. Biol., 239:68-78.
Fishwild et al., “High-avidity human IgG kappa monoclonal antibodies from a novel strain of minilocus transgenic mice” (1996) Nature Biotechnology, 14:845-851.
Fleisher B., Mechanism of glycosylation in the Golgi apparatus. J Histochem Cytochem, Aug. 1983; 31 (8):1033-1040.
Folk et al., “Carboxypeptidase B, Purification and Characterization of the Porcine Enzyme,” J. Biological Chem, 1960, 235:2272-2277.
Fomsgaard, “Auto-antibodies to Tumor Necrosis Factor α in Healthy Humans and Patients with Inflammatory Diseases and Gram-Negative Bacterial Infections” (1989) Scand. J. Immunol. 30:219-23.
Foote, J., “Antibody framework residues affecting the conformation of the hypervariable loops” (1992) J. Mol .Biol., 224(2):487-499.
Freitag et al., “Displacement chromatography in biotechnological downstream processing,” J. Chromatography, (1995) 691(1):101-112.
Gagnon et al., “A Systematic Approach to the Purification of Monoclonal Antibodies,” LC-GC 11 (1):26-34 (1993).
Gagnon, P., “Polishing methods for monoclonal IgG purification” Chapter 17, Taylor & Francis Group, LLC, pp. 491-505, 2007.
Gao et al. “Site-selective modifications of arginine residues in human hemoglobin induced by methylglyoxal.” Biochemistry, 2006; pp. 15654-15660.
Gatto, B. “Biologics targeted at TNF: design, production and challenges”, Reumatismo 58(2):94-103, 2006.
Gauthier, M. A.& Klok, H.-A. Arginine-Specific Modification of Proteins with Polyethylene Glycol Biomacromolecules; 2011, 12, 482-493.
Gaza-Bulseco, G., et al., Characterization of the glycosylation state of a recombinant monoclonal antibody using weak cation exchange chromatography and mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci, 2008. 862(1-2): p. 155-60. Epub Dec. 8, 2007.
Genbank Entry for CHO Cathepsin L., EGW13555, Aug. 25, 2011, pp. 1-2.
Ghaderi, et al., “Implications of the Presence of N-glycolylneuraminic acid in Recombinant Therapeutic Glycoproteins”, Nature Biotechnology, 28(8):863-868 (2010).
Ghaderi, et al., “Production platforms for biotherapeutic glycoproteins. Occurrence, impact, and challenges of non-human sialylation”, Biotechnology and Genetic Engineering Reviews, 28:147-176 (2012).
Gilar et al., “Characterization of glycoprotein digests with hydrophilic interaction chromatography and mass spectrometry” (2011) Analytical Biochem., 417:80-88.
Giroir et al., “Inhibition of tumor necrosis factor prevents myocardial dysfunction during burn shock” (1994) Am. J. Physiol., 267(1 Pt 2):H118-24.
Goetze, A. et al., “High-mannose glycans on the Fc region of therapeutic IgG antibodies increase serum clearance in humans.” Glycobiology (2011), 21(7); 949-959.
Gonzalez et al. “Purification of Lactic Acid from Fermentation Broths by Ion-Exchange Resins” Ind. Eng. Chem. Res. 45:3243 (2006).
Goochee CF the Oligosaccharides of Glycoproteins: Bioprocess Factors Affecting Oligosaccharide Structure and their Effect on Glycoprotein Properties. Nature Biotechnology Dec. 1991 1346-1355.
Goochee, C.F. “Bioprocess Factors Affecting Glycoprotein Oligosaccharide Structure.” Develop. Biol. Standard, vol. 76 (1992). 95-104.
Goswami et al., “Developments and Challenges for mAb-Based Therapeutics,” Antibodies, 2:452-500, 2013.
Grabulovski et al., “A Novel, Non-immunogenic Fyn SH3-derived Binding Protein with Tumor Vascular Targeting Properties” (2007) J Biol Chem 282, (5): 3196-3204.
Graf et al., “Ion exchange resins for the purification of monoclonal antibodies from animal cell culture” Bioseparation 4 (1) :7-20 (Feb. 1994). ;4 (1) :7-20 (Feb. 1994).
Gram et al., “In vitro selection and affinity maturation of antibodies from a naive combinatorial immunoglobulin library” (1992) PNAS, 89:3576-3580.
Gramer et al. “Modulation of antibody galactosylation through feeding of uridine, manganese chloride, and galactose,” Biotechnology and Bioengineering. (Jul. 1, 2011) 108(7):1591-1602.
Gramer et al., “Glycosidase Activities of the 293 and NS0 Cell Lines, and of an Antibody-Producing Hybridoma Cell Line”, Biotechnology and Bioengineering, 43:423-428 (1994).
Gramer M Jet Al: “Modulation of Antibody Galactosylation Through Feeding of Uridine, Manganese Chloride, and Galactose”,Biotechnology and Bioengineering, Wiley & Sons, Hoboken, NJ, US,vol. 108, No. 7, Jul. 1, 2011, pp. 1591-1682.
Gramer, M.J., et al., “Manipulation of Antibody Glycoforms in a High-Yield GS-CHO Process to Meet Comparability Requirements”, Biotechnology and Bioengineering, vol. 108, No. 7, Jul. 2011, pp. 1591-1602.
Green et al., “Antigen-specific human monoclonal antibodies from mice engineered with human Ig heavy and light chain YACs” (1994) Nature Genetics, 7:13-21.
Griffiths et al., “Isolation of high affinity human antibodies directly from large synthetic repertoires” (1994) EMBO J., 13:3245-3260.
Griffiths, “Human anti-self antibodies with high specificity from phage display libraries” (1993) The EMBO J. 12(2):725-34.
Gross et al. “Involvement of various organs in the initial plasma clearance of differently glycosylated rat liver secretory proteins,” Eur. J. Biochem. (1988) 173(3):653-659.
Grosvenor, Sally, “A New Era in Cell Culture Media Development,” BioPharm International, Jul. 2012 vol. 25: 7, 7 pages.
Grunberg, J. et al., “High-Yield Production of Recombinant Antibody Fragments in HEK-293 Cells Using Sodium Butyrate”, BioTechniques 34(5):968-972, May 2003.
Gu, X. et al: “Improvement of interferon-gamma sialylation in Chinese hamster ovary cell culture by feeding of N-acetylmannosamine”,Biotechnology and Bioengineering, Wiley & Sons, Hoboken, NJ, US, vol. 58, No. 6, Jun. 20, 1998, pp. 642-648.
Guile et al., “A rapid high-resolution high-performance liquid chromatographic method for separating glycan mixtures and analyzing oligosaccharide profiles” (1996) Anal Biochem., 240(2):210-26.
Haddadi et al., “Delivery of rapamycin by PLGA nanoparticles enhances its suppressive activity on dendritic cells” (2008) J Biomed Mater Res A., 84A(4):885-98.
Hansen et al., “The role of tumor necrosis factor-alpha in acute endotoxin-induced hepatotoxicity in ethanol-fed rats” (1994) Hepatology, 20(2):461-474.
Harding et al., “Class switching in human immunoglobulin transgenic mice” (1995) Ann. NY Acad. Sci., 764:536-547.
Harlow and Lane, Antibodies A Laboratory Manual, Purification of Antibodies by using a; Deae-matrix (Batch), Storing and Purifying Antibodies; Chapter 8: 302-303 (1988).
Harlow et al., Eds (“Antibodies: A Laboratory Manual” 1988. Cold Spring Harbor Laboratory Press, Chapter 7, pp. 245, 247,and 253).
Harris, “Processing of C-terminal lysine and arginine residues of proteins isolated from mammalian cell culture” Journal of Chromatography, (1995) 705; 129-134.
Harris, R.J., et al., Identification of multiple sources of charge heterogeneity in a recombinant antibody. Journal of Chromatography B: Biomedical Sciences and Applications, 2001. 752(2): p. 233-245.
Harris, Reed J. et al., “Structural Characterization of a Recombinant CD4-IgG Hybrid Molecule,” Eur. J. Biochem. 194:611-620 (1990).
Harrison et al., “Protein N-Glycosylation in the Baculovirus-Insect Cell Expression System and; Engineering of Insect Cells to Produce “Mammalianized” Recombinant Glycoproteins,” Advances in; Virus Research, 68:159-191 (2006).
Hawkins, “Selection of Phage Antibodies by Binding Affinity Mimicking Affinity Maturation” (1992) J. Mol. Biol., 226:889-896.
Heidemann, R. et al., “The use of peptones as medium additives for the production of a recombinant therapeutic protein in high density perfusion cultures of mammalian cells”, Cytotechnology 32:157-167, 2000.
Helms et al., “Destabilizing loop swaps in the CDRs of an immunoglobulin VL domain,” Protein; Science 4:2073-2081 (1995).
Hiatt et al., “Characterization and Applications of Antibodies Produced in Plants”, Intern. Rev. Immunol., 10:139-152 (1993).
Hiatt et al., “Production of Antibodies in Transgenic Plants”, Nature, 342:76-78 (1989).
Hillgren, A. et al., “Protection mechanism of Tween 80 during freeze-thawing of a model protein LDH,” International Journal of Pharmaceutics, vol. 237:57-69 (2002).
Hills, A.E. et al., Metabolic control of recombinant monoclonal antibody N-glycosylation in GS-NS0 cells, Biotechnology and Bioengineering, Oct. 20, 2001; 75(2):239-251.
Hipkiss, A.; Can the beneficial effects of methionine restriction in rats be explained in part by decreased methylglyoxal generation resulting from suppressed carbohydrate metabolism?; Biogerontology 2012, 13, 633-636.
Hober, et al. “Protein a chromatography for antibody purification”, J. Chromatography B, vol. 848 (2007) pp. 40-47.
Hokke et al., “Sialylated Carbohydrate Chains of Recombinant Human Glycoproteins Expressed in Chinese Hamster Ovary Cells Contain Traces of N-glycolylneuraminic acid”, FEBS, 275:9-14 (1990).
Holler, “Modulation of Acute Graft-Versus-Host Disease After Allogeneic Bone Marrow Transplantation by Tumor Necrosis Factor-alpha (TNF-alpha) Release in the Course of Pretransplant Conditioning: Role of Conditioning Regimens and Prophylactic Application of a Monoclonal Antibody Neutralizing Human TNF-alpha (MAK 195F)” (1995) Blood, 86(3):890-899.
Holt, L. et al., “Domain antibodies: proteins for therapy,” Trends in Biotechnology, vol. 21(11):484-490 (2003).
Hoogenboom et al., “By-passing immunisation : Human antibodies from synthetic repertoires of germline VH gene segments rearranged in vitro” (1992) J. Mol. Biol., 227:381-388.
Hoogenboom, “Converting rodent into human antibodies by guided selection” (1996) Antibody Engineering, Oxford University Press, pp. 169-185.
Horvath et al: “Characterization of a Monoclonal Antibody Cell Culture Production Process Using a Quality by; Design Approach”, Molecular Biotechnology, vol. 45, No. 3, Jul. 1, 2010, pp. 203-206.
Hossler et al., “Optimal and consistent protein glycosylation in mammalian cell culture”, Glycobiology; (2009), 19(9):936-949.
Hossler et al.; “Improvement of mammalian cell culture performance through surfactant enabled concentrated feed media”; Biotechnology Progress; 29(4):1023-1033 (2013).
Hossler, Patrick et al., “Targeted Shifting of Protein Glycosylation Profiles in Mammalian Cell Culture through Media Supplementation of Cobalt.” J. Glycobiol vol. 3; 1.(2014). 9 pages.
Huang et al. “Effects of anti-TNF monoclonal antibody infusion in patients with hairy cell leukaemia” (1992) Br. J. Haematol., 81(2):231-234.
Huang, L., et al., In Vivo Deamidation Characterization of Monoclonal Antibody by LC/MS/MS. Analytical Chemistry, 2005. 77(5): p. 1432-1439.
Humira (adalimumab) label, Revised Sep. 2013, 87 pages.
Humira (adalimumab) prescribing information, Dec. 20, 2002, pp. 1-16.
Huse, “Generation of a large combinatorial library of the immunoglobulin repertoire in phage lambda” (1989) Science, 246:1275-81.
Hussain et al., “Hepatic expression of tumour necrosis factor-alpha in chronic hepatitis B virus infection” (1994) J. Clin. Pathol., 47:1112-1115.
Huston et al. “Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli,” Proc. Natl. Acad. Sci. USA(1988) 85:5879-5883.
HyClone™ CDM4CHO Catalog listing (last accessed Nov. 17, 2014).
ICH Topic Q6B “Specifications:Test Procedures and Acceptance Criteria for Biotechnological/Biological Products,” Sep. 1999, pp. 1-17.
Indian Patent Office—IPAIRS application status for 2285/MUM/2013—Application not yet published. Document found on internet at ipindiaonline.gov/in/patentsearch/search/index.aspx. Last accessed Apr. 13, 2015.
International Preliminary Report on Patentability for Application No. PCT/US07/08359, dated Dec. 12, 2011, 5 pages.
International Preliminary Report on Patentability for Application No. PCT/US2011/060388, dated May 30, 2012, 9 pages.
International Preliminary Report on Patentability for Application No. PCT/US2013/031352 dated Nov. 25, 2014, pp. 1-10.
International Preliminary Report on Patentability for Application No. PCT/US2013/031365, dated Mar. 3, 2015, 9 pages.
International Preliminary Report on Patentability for Application No. PCT/US2013/031380, dated Sep. 15, 2015, 13 pages.
International Preliminary Report on Patentability for Application No. PCT/US2013/031389, dated Oct. 21, 2014, pp. 1-10.
International Preliminary Report on Patentability for Application No. PCT/US2013/031485, dated Oct. 21, 2014, pp. 1-8.
International Preliminary Report on Patentability for Application No. PCT/US2013/031681, dated Oct. 21, 2014, pp. 1-8.
International Preliminary Report on Patentability for Application No. PCT/US2013/041954, dated Nov. 25, 2014, pp. 1-14.
International Preliminary Report on Patentability for Application No. PCT/US2013/041958, dated Dec. 4, 2014, pp. 1-2.
International Preliminary Report on Patentability for Application No. PCT/US2013/065720, dated Sep. 24, 2015, 8 pages.
International Preliminary Report on Patentability for Application No. PCT/US2013/065749 dated Sep. 15, 2015, 10 pages.
International Preliminary Report on Patentability for Application No. PCT/US2013/065797, dated Sep. 24, 2015, 8 pages.
International Preliminary Report on Patentability for Application No. PCT/US2013/069702, dated Sep. 15, 2015, 8 pages.
International Preliminary Report on Patentability for Application No. PCT/US2014/024151, dated Sep. 15, 2015, 9 pages.
International Preliminary Report on Patentability for Application No. PCT/US2014/024256, dated Sep. 15, 2015, pp. 1-9.
International Preliminary Report on Patentability for Application No. PCT/US2014/026606, dated Sep. 15, 2015, 13 pages.
International Preliminary Report on Patentability for Application No. PCT/US2014/026636, dated Sep. 15, 2015, 9 pages.
International Search Report and Written Opinion for Application No. PCT/US2008/085066, dated May 12, 2009, 5 pages.
International Search Report and Written Opinion for Application No. PCT/US2010/033387, dated Aug. 7, 2012, 10 pages.
International Search Report and Written Opinion for Application No. PCT/US2013/031380, dated Feb. 5, 2014, 162 pages.
International Search Report and Written Opinion for Application No. PCT/US2013/041954, dated Dec. 17, 2013, 21 pages.
International Search Report and Written Opinion for Application No. PCT/US2013/041958, dated Dec. 17, 2013, 21 pages.
International Search Report and Written Opinion for Application No. PCT/US2013/065720, dated Dec. 16, 2013, 14 pages.
International Search Report and Written Opinion for Application No. PCT/US2013/065797, dated Nov. 26, 2013, 14 pages.
International Search Report and Written Opinion for PCT/US2012/035266, dated Feb. 7, 2013 (corresponds to U.S. Appl. No. 13/547,020), 4 pages.
International Search Report and Written Opinion from PCT/US2013/065749 dated Mar. 18, 2014, 18 pages.
International Search Report and Written Opinion from PCT/US2014/024151 dated Aug. 7, 2014, pp. 1-16.
International Search Report and Written Opinion from PCT/US2015/039773 dated Sep. 25, 2015, pp. 1-14.
International Search Report and Written Opinion from PCT/US2015/042846 dated Feb. 2, 2016, pp. 1-22.
International Search Report for Application No. PCT/IB03/04502, dated May 26, 2004, 6 pages.
International Search Report for Application No. PCT/US2011/060388 dated May 30, 2012, 6 pages.
International Search Report for Application No. PCT/US2013/031352, Dated Apr. 25, 2013, 6 pages.
International Search Report for Application No. PCT/US2013/031389, Dated Jun. 3, 2013, 4 pages.
International Search Report for Application No. PCT/US2013/031485, Dated Jun. 25, 2013, 4 pages.
International Search Report for Application No. PCT/US2013/031681, Dated Jun. 14, 2013, 6 pages.
International Search Report for Application No. PCT/US2014/026606, Dated Dec. 8, 2014, 8 pages.
International Search Report for Application No. PCT/US2014/026636, Dated Jul. 29, 2014, 5 pages.
International Search Report for Application No. PCT/US2015/038819 Dated Sep. 2, 2015, 12 pages.
International Search Report from PCT/US2014/024256 dated Jul. 30, 2014, pp. 1-15.
International Search Report with Written Opinion corresponding to International Patent Application No. PCT/US2014/059127, mailed May 7, 2015, 21 pages.
International Search Report with Written Opinion corresponding to International Patent Application No. PCT/US2014/065793, mailed Jul. 27, 2015, 20 pages.
Invitation to Pay Additional Fees for International Application No. PCT/US2013/031380, Dated Nov. 28, 2013, 5 pages.
Invitation to Pay Additional Fees for International Application No. PCT/US2013/065749, Dated May 27, 2014, pp. 1-8.
Invitation to Pay Additional Fees for International Application No. PCT/US2014/026606, Dated Jul. 8, 2014, pp. 1-8.
Jack, M.; Wright, D.; The Role of Advanced Glycation Endproducts and Glyoxalase I in Diabetic Peripheral Sensory Neuropathy; Transl. Res. 2012, 159, 355-365.
Jakobovits, A., “Production of fully human antibodies by transgenic mice” (1995) Curr. Op. Biotechnol., 6:561-566.
Jakubowski, H., Protein N-homocysteinylation: implications for atherosclerosis. Biomedicine; Pharmacotherapy, 2001. 55(8): p. 443-447.
Jayapal, Karthik P., et al., “Recombinant Protein Therapeutics from CHO Cells—20 Years and Counting,” CHO Consortium, SBE Special Section, 40-47 (2007).
Jayme et al.; “Media formulation options and manufacturing process controls to safeguard against introduction of animal origin contaminants in animal cell culture”; Cytotechnology; 33:27-36 (2000).
Jefferis, R., Glycosylation of Recombinant Antibody Therapeutics. Biotechnology Progress, 2005.21(1): p. 11-16.
Jespers, “Guiding the Selection of Human Antibodies from Phage Display Repertoires to a Single Epitope of an Antigen” (1994) Bio/Technology, 12:899-903.
Johnson et al., “Characterization of cathepsin L secreted by Sf21 insect cells”, Archives of Biochemistry and Biophysics (2005), 444:7-14.
Johnson, K.A., et al., Cation exchange HPLC and mass spectrometry reveal C-terminal amidation of an IqG1 heavy chain. Analytical Biochemistry, 2007. 360(1): p. 75-83.
Kalyanpur, M., “Downstream Processing in the Biotechnology Industry” Molecular Biotechnology, vol. 22:87-98 (2002).
Kanda, et al.: “Comparison of biological activity among nonfucosylated therapeutic IgG1 antibodies with three different N-linked Fc oligosaccharides: the high-mannose, hybrid, and complex types”, Glycobiology, Oxford University Press, US, vol. 17, No. 1, Sep. 2006, pp. 104-118.
Karampetsou et al., “TNF-α antagonists beyond approved indications: stories of success and prospects for the future”, Q J Med (2010) 103:917-928.
Kaschak et al: “Characterization of the basic charge variants of a human IgGI: Effect of copper concentration in cell culture media”, MABS, vol. 3, No. 6, Nov. 1, 2011, pp. 577-583.
Kaufman et al., “Amplification and expression of sequences cotransfected with a modular dihydrofolate reductase complementary dna gene” (1982) Mol. Biol., 159(4):601-621.
Kazuaki F et al “Enhancment of productivity of recombinant a-amidating enzyme by low temperature culture” Cytotechnology 31:85-94, 1999.
Kempeni, “Update on D2E7: a fully human anti-tumour necrosis factor-alpha monoclonal antibody” (2000) Ann. Rheum. Dis., 59(Suppl. I):144-145.
Kempeni, J, “Preliminary results of early clinical trials with the fully human anti-TNFα monoclonal antibody D2E7”, Ann. Rheum. Dis., 1999, pp.1 170-172, vol. 58, (Suppl. I).
Kempf, C, et al. “Virus inactivation during production of intravenous immunoglobulin.” Transfusion 1991; vol. 31: p. 423-427.
Khawli et al, “Charge variants in IgGI: Isolation, characterization, in vitro binding properties and pharmacokinetics in rats”, MABS, vol. 2, No. 6, Nov. 1, 2010, pp. 613-624.
Kim et al.: “Development of serum-free medium supplemented with hydrolysates for the production of therapeutic antibodies in CHO cell cultures using design of experiments”, Applied Microbiology and Biotechnology, Springer, Berlin, DE, vol. 83, No. 4, Mar. 6, 2009, pp. 639-648.
Kim, NS. et al., “Inhibition of sodium butyrate-induced apoptosis in recombinant Chinese hamster ovary cells by constitutively expressing antisense RNA of caspase-3”, Biotechn. & Bioengin. 78(2):217-228, 2002.
Kingkeohoi, S., Analysis of methylglyoxal metabolism in CHO celis grown in culture, Cytotechnology (2005) 48:1-13.
Kipriyanov et al. “Recombinant single-chain Fv fragments carrying C-terminal cysteine residues: production of bivalent and biotinylated miniantibodies,” Molecular Immunology, (1994) 31(14):1047-1058 F.
Kipriyanov et al., “Single-chain antibody streptavidin fusions: tetrameric bifunctional scFv-complexes with biotin binding activity and enhanced affinity to antigen,” Human Antibodies and Hybridomas. (1995) 6(3):93-101.
Knight et al., “Construction and initial characterization of a mouse-human chimeric anti-TNF antibody” (1993) Mol. Immunol., 30(16):1443-1453.
Koide et al., “Monobodies: antibody mimics based on the scaffold of the fibronectin type III domain” (2007), Methods Mol. Biol., 352: 95-109.
Konig et al., “Tumor necrosis factor α and interleukin-1 stimulate bone resorption in vivo as measured by urinary [3H] tetracycline excretion from prelabeled mice” (1988) J. Bone Miner. Res., 3(6):621-627.
Kopaciewicz et al., “Retention Model for High-Performance Ion-Exchange Chromatography,”; Journal of Chromatoqraphy, 266:3-21 (1983).
Krehenbrink et al., “Artificial Binding Proteins (Affitins) as Probes for Conformational Changes in Secretin PuID” (2008) J. Mol. Biol., 383 (5): 1058-68.
Kunkel et al., “Comparisons of the Glycosylation of a Monoclonal Antibody Produced under Nominally Identical Cell Culture Conditions in Two Different Bioreactors” (2000) Biotechnol. Prog., 16(3): 462-470.
Kunkel, Jeremy P., et al., “Dissolved oxygen concentration in serum-free continuous culture affects N-linked glycosylation of a monoclonal antibody,” Journal of Biotechnology, 62 (1998), 55-71.
Kwon et al., “Production of lactic acid by Lactobacillus rhamnosus with vitamin-suppremented soybean hydrolysate”, Enzyme Microb Technol. (2000), 26:209-215.
Leavitt et al. “Impaired Intracellular Migration and Altered Solubility of Nonglycosylated Glycoproteins of Vesicular Stomatitis Virus and Sindbis Virus,” J. Biol. Chem. (1977) 252(24):9018-9023.
Lerner et al., “Tumor necrosis factors α and β can stimulate bone resorption in cultured mouse calvariae by a Prostaglandin-independent mechanism” (1993) J. Bone Miner. Res., 8(2):147-155.
Lerner, “Antibodies without immunization” (1992) Science, 258:1313-1314.
Leusch, “Failure to demonstrate TNFα-specific autoantibodies in human sera by ELISA and Western blot” (1991) J. Immunol. Methods, 139:145-47.
Lewis, “Use of alanine scanning mutagenesis to improve the affinity of an anti gp120 (HIV) antibody” (1994) J. Cell. Biochem., 18D:215.
Li, F. et al., “Current Therapeutic Antibody Production and Process Optimization” BioProcessing Journal, vol. 4(5):23-30 (2005).
Li, Feng, et al., “Cell Culture Processes for Monoclonal Antibody Production,” mAbs 2:5, 466-479 (Sep.-Oct. 2010).
Lifely et al., “Glycosylation and Biological Activity of CAMPATH-1H Expressed in Different Cell Lines and Grown Under Different Culture Conditions”, Glycobiology, 5(8):813-822 (1995).
Liu et al. “Recovery and purificaiton process development for monoclonal antibody production” MABS, 2(5), pp. 480-499 (2010).
Liu et al., “The significance of changes in serum tumour necrosis factor (TNF) activity in severely burned patients” (1994) Burns, 20(1):40-44.
Liu, H., Assessment of antibody fragmentation by reversed-phase liquid chromatography and mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci, 2008. 876(1): p. 13-23. Epub Oct. 15, 2008.
Liu, H., et al., Heterogeneity of monoclonal antibodies. Journal of Pharmaceutical Sciences, 2008. 97(7): p. 2426-2447.
Liu, M, et al.; Discovery of Undefined Protein Cross-Linking Chemistry: A Comprehensive Methodology Utilizing 18O—Labeling and Mass Spectrometry; Anal. Chem. 2013, 5900-5908.
Liu, M.et al.; Protein Isoaspartate Methyltransferase-Mediated 18O—Labeling of Isoaspartic Acid for Mass Spectrometry Analysis; Anal. Chem. 2011, 84, 1056-1062.
Lo, T.W. et al., Binding and modification of proteins by methylglyoxal under physiological conditions. A kinetic and mechanistic study with N alpha-acetylarginine, N alpha-acetyilysine, and N alpha-acetyllysine, and bovine serum albumin, Dec. 23, 1994, The Journal of Biological Chemistry, 269, 32299-32305.
Logan, John S. “Transgenic Animals: Beyond ‘Funny Milk’”, Current Opinion in Biotechnology, 4:591-595 (1993).
Lonberg et al., “Antigen-specific human antibodies from mice comprising four distinct genetic modifications” (1994) Nature, 368:856-859.
Lonberg et al., “Human Antibodies from Transgenic Mice” (1995) Int. Rev. Immunol., 13:65-93.
Low et al., “Mimicking Somatic Hypermutation: Affinity Maturation of Antibodies Displayed on Bacteriophage Using a Bacterial Mutator Strain” (1996) J. Mol. Biol., 260:359-368.
Low, Nigel: thesis extract (1996) Cambridge University.
Lowe et al. “A Genetic Approach to Mammalian Glycan Function,” Annu. Rev. Biochem. (2003) 72:643-691.
Lu et al.: “A T-flask based screening platform for evaluating and identifying plant hydrolysates for a fed-batch cell culture process”, Cytotechnology, Kluwer Academic Publishers, DO, vol. 55, No. 1, Aug. 18, 2007, pp. 15-29.
Luo et al., “Understanding of C-terminal lysine variants in antibody production using mammalian cells” Abstract of papers, ACS, Anaheim, CA, US, Mar. 2011, 1 page.
Luo et al: “Probing of C-terminal lysine variation in a recombinant monoclonal antibody production using Chinese hamster ovary cells with chemically defined media”, Biotechnology and Bioengineering, vol. 109, No. 9, Apr. 11, 2012, pp. 2306-2315.
Luo, Ying et al.: “Development toward rapid and efficient screening for high performance hydrolysate lots in a recombinant monoclonal antibody manufacturing process.”, Biotechnology Progress Jul. 2012, vol. 28, No. 4, Jul. 2012, pp. 1061-1068.
Ma, et al., “Generation and Assembly of Secretory Antibodies in Plants”, Science, 268:716-719 (1995).
MacDonald et al., “Tumour necrosis factor-alpha and interferon-gamma production measured at the single cell level in normal and inflamed human intestine” (1990) Clin. Exp. Immunol, 81(2):301-305.
Maeda, et al., “Analysis of Nonhuman N-Glycans as the Minor Constituents in Recombinant Monoclonal Antibody Pharmaceuticals”, Anal. Chem., 84:2373-2379 (2012).
Mahler, et al. Induction and analysis of aggregates in a liquid IgG1-antibody formulation. Eur J Pharm Biopharm. 2005, 59(3):407-17; p. 408; col. 1-2; p. 409; col. 2, “2.2.2 Stirring stress”.
Manning, M., et al., Stability of Protein Pharmaceuticals: An Update. Pharmaceutical Research, 2010.27(4): p. 544-575.
Marks et al., “Human antibody fragments specific for human blood group antigens from a phage display library” (1993) Bio/Technology, 11:1145-1150.
Marks et al., “Molecular evolution of proteins on filamentous phage. Mimicking the strategy of the immune system” (1992) J. Biol. Chem. 267:16007-16010.
Marks, “By-passing immunization: Human antibodies from V-gene libraries displayed on phage” (1991) J. Mol. Biol., 222:581-597.
Marks, “Human Monoclonal Antibodies from V-gene Repertoires Expressed on Bacteriophage.” In Antibody Engineering, Second Edition, edited by Carl A.K. Borrebaeck (1995), pp. 53-88. New York: Oxford Univ. Press.
Marks, JD., “By-Passing Immunization: Building High Affinity Human Antibodies by Chain Shuffling” (1992) Biotechnology, 10:779-783.
Martin, A.C.R. “Accessing the Kabat antibody sequence database by computer” (1996)Proteins: Structure, Function and Genetics25:130-133.
Martinelle, K. et al., “Effect of different cell culture medium surfactants on cell growth and viability”, Cells and Culture, Proceedings of the 20th ESACT Meeting v4 819-822, Jun. 17-20, 2007.
Matthews, R. G.; et al.; Cobalamin-Dependent and Cobalamin-Independent Methionine Synthases: Are There Two Solutions to the Same Chemical Problem?; Helv. Chim. Acta 2003, 86, 3939-3954.
McAtee et al., “Isolation of monoclonal antibody charge variants by displacement chromatography,” Current Protocols in Protein Science, 8.10-8.10.13, 2012.
McCauley et al., “Altered cytokine production in black patients with keloids” (1992) J. Clin. Immunol., 12(4):300-308.
McClain et al., “Increased tumor necrosis factor production by monocytes in alcoholic hepatitis” (1989) Hepatology, 9(3):349-351.
Medynski, “Phage Display: All Dressed Up and Ready to Role” (1994) Bio/Technology, 12:1134-1136.
Mehta, et al. “Purifying therapeutic monoclonal antibodies,” Chemical Engineering Progress; May 2008, 104, 5; pp. S14-S20.
Mendez et al., “Functional transplant of megabase human immunoglobulin loci recapitulates human antibody response in mice” (1997) Nature Genetics, 15:146-156.
Meuwly, F. et al., “Conversion of a CHO cell culture process from perfusion to fed-batch technology without altering product quality”, J.Biotechn. 123:106-116, 2006.
Miller et al., “Characterization of site-specific glycation during process development of a human therapeutic monoclonal antibody” Journal of Pharmaceutical Sciences, vol. 100, No. 7, Jul. 2011, 2543-2550.
Millipore, “Pellicon 2 Filters and Holders,” 2003, pp. 1-8.
Millward et al. “Effect of constant and variable domain glycosylation on pharmacokinetics of therapeutic antibodies in mice,” Biologicals.(2008) 36(1):41-47.
Mizuochi, T., et al., Structural and numerical variations of the carbohydrate moiety of immunoglobulin G. J Immunol, 1982. 129(5): p. 2016-20.
Moller et al., “Monoclonal antibodies to human tumor necrosis factor α: In vitro and in vivo application” (1990) Cytokine 2(3):162-169.
Moore, A., et al., “Effects of temperature shift on cell cycle, apoptosis and nucleotide pools in CHO cell batch cultures”, Cytotechnology, 23:47-54, 1997.
Moorhouse, K.G., et al., Validation of an HPLC method for the analysis of the charge heterogeneity of the recombinant monoclonal antibody IDEC-C2B8 after papain digestion. Journal of Pharmaceutical and Biomedical Analysis, 1997. 16(4): p. 593-603.
Morgan et al. “Designing Biobetter Monoclonal Antibody Therapeutics by Glycoengineering,” International Pharmaceutical Industry. (2011) pp. 38-44.
Mostafa, A et al.; Plasma protein advanced glycation end products, carboxymethyl cysteine, and carboxyethyl cysteine, are elevated and related to nephropathy in patients with diabetes Mol. Cell. Biochem. 2007, 302, 35-42.
Muller-Spath, et al., “Chromatographic Separation of Three Monoclonal Antibody Variants Using Multicolumn Countercurrent Solvent Gradient Purification (MCSGP)” Biotechnology and Bioengineering, vol. 100. No. 6 (2008), pp. 1166-1177.
Möller, Monoclonal antibodies to human tumor necrosis factor α: in vitro and vivo application (1990) Cytokine, 2(3):162-69.
Neuberger M. et al., “Mice perform a human repertoire” (1997) Nature, 386:25-26.
Ngo et al., “Kosmotropes enhance the yield of antibody purified by affinity chromatography using immobilized bacterial immunoglobulin binding proteins,” Journal of Immunoassay & Immunochemistry, (2008) 29(1):105-115.
Ni, W.; Analysis of Isoaspartic Acid by Selective Proteolysis with Asp-N And Electron Transfer Dissociation Mass Spectrometry; Anal. Chem. 2010, 82,7485-7491.
Nilsson, “Antibody engineering” (1995) Current Opinion in Structural Biology, 5:450-456.
Nixon et al., “Engineered protein inhibitors of proteases” (2006) Curr Opin Drug Discov Devel, 9(2): 261-8.
Nogal, B., Chhiba, K. and Emery, J. C. (2012), Select host cell proteins coelute with monoclonal antibodies in protein a chromatography. Biotechnol Progress, 28: 454-458.
Noguchi et al., “Failure of Human Immunoresponse to N-Glycolylneuraminic Acid Epitope Contained in Recombinant Human Erythropoietin”, Nephron, 72:599-603 (1996).
Noguchi et al., “Immunogenicity of N-Glycolylneuraminic Acid-Containing Carbohydrate Chains of Recombinant Human Erythropoietin Expressed in Chinese Hamster Ovary Cells”, J. Biochem., 117:59-62 (1995).
Nygren et al., “Alternative binding proteins: affibody binding proteins developed from a small three-helix bundle scaffold” (2008) FEBS J., 275 (11): 2668-76.
Oh, D-K. et al., “Increased erythritol production in fed-batch cultures of Torula sp. by controlling glucose concentration”, J. Industrial Microb. & Biotechn. 26(4): 248-252, 2001.
Oh, et al., “Effect of N-Acetylcystein on Butyrate-Treated Chinese Hamster Ovary Cells to Improve the Production of Recombinant Human Interferon-β-1a”, Biotechnol. Prog. 21(4):1154-1164, 2005.
Oh, SKW, et al., “Substantial Overproduction of Antibodies by Applying Osmotic Pressure and Sodium Butyrate”, Biotechn. Bioengin. 42(5):601-610, 1993.
Osbourn, “From rodent reagents to human therapeutics using antibody guided selection” (2005) Methods, 36(1):61-68.
Ouellette, D.; Studies in serum support rapid formation of disulfide bond between unpaired cysteine residues in the VH domain of an immunoglobulin G1 molecule; Anal. Biochem. 2010, 397, 37.
Oya, T. et al. Methylglyoxal Modification of Protein: Chemical and Immunochemical Characterization of Methylglyoxal-Arginine Adducts. J. Bioi Chem. Jun. 25, 1999; vol. 274, No. 26, pp. 18492-19502.
Pacis, et al.: “Effects of cell culture conditions on antibody N-linked glycosylation-what affect high mannose 5 glycoform”, Biotechnology and Bioengineering vol. 108, No. Oct. 10, 2011, pp. 2348-2358.
Packer et al., “A general approach to desalting oligosaccharides released from glycoproteins” (1998) Glycoconj J., 15(8):737-47.
Paoli, T. et al., A Study of D-Lactate and Extracellular Methylglyoxal Production in Lactate ReUtilizing CHO Cultures, Biotechnology and Bioengineering, vol. 107, No. 1, Sep. 1, 2010, pp. 182-189.
Parekh RB N-glycosylation and the production of recombinant glycoproteins vol. 7, Issue 5, p. 117-122, May 1989 Trends in Biotechnology.
Parekh, R.B., et al., Association of rheumatoid arthritis and primary osteoarthritis with changes in the glycosylation pattern of total serum IgG. Nature, 1985. 316(6027): p. 452-7.
Patel, T. P. et al.: “Different culture methods lead to differences in glycosylation of a murine IgG monoclonal antibody”, Biochemical journal, The Biochemical Society, London, GB, vol. 285, No. 3, Jan. 1, 1992, pp. 839-845.
PCT/US2013/069702 International Search Report & Written Opinion mailed Jan. 31, 2014, 13 pages.
Perchiacca, J.M., et al., “Engineering Aggregation-Resistant Antibodies,” Annual Review of Chemical and Biomolecular Engineering 3:263-286 (2012).
Perkins, M.; et. al. Determination of the Origin of Charge Heterogeneity in a Murine Monoclonal Antibody; M. Pharm. Res. 2000, 17, 1110-1117.
Pietersz et al., “In vitro and in vivo Antitumor Activity of a Chimeric anti-CD19 Antibody”, Cancer Immunol. Immunother., 41:53-60 (1995).
Pink, T. et al.: “Regulation of Slayer protein synthesis of bacillus stearothermophilus PV72 through variation of continuous cultivation conditions”, Journal of Biotechnology, Elsevier Science Publishers, Amsterdam, NL, vol. 50, No. 2, Oct. 1, 1996, pp. 189-200.
Potter et al., “Antibody Production in the Baculovirus Expression System”, Intern. Rev. Immunol., 10:103-112 (1993).
Poul et al., “Design of Cassette Baculovirus Vectors for the Production of Therapeutic Antibodies in Insect Cells”, Immunotechnology, 1:189-196 (1995).
Proteus, “Protein A Antibody Purification Handbook,” Pro-Chem Inc., 2005, pp. 1-52.
Quan, C., et al., A study in glycation of a therapeutic recombinant humanized monoclonal antibody: Where it is, how it got there, and how it affects charge-based behavior. Analytical Biochemistry, 2008.373(2): p. 179-191.
Queen, C., “A humanized antibody that binds to the interleukin 2 receptor” (1989) Proc. Natl. Acad. Sci. USA, 86(24):10029-10033.
Rabbani, N.; Thornalley, P. J.; Glyoxalase in diabetes, obesity and related disorders; Semin. Cell Dev. Biol. 2011, 22, 309-317.
Rader et al. “A phage display approach to rapid antibody humanization: Designed combinatorial V gene libraries” (1998) Proc Natl Acad Sci USA, 95:8910-8915.
Raju et al. “Galactosylation variations in marketed therapeutic antibodies,” MABS. (May 1, 2012) 4(3):385-391.
Raju et al., “Glycoengineering of Therapeutic Glycoproteins:? In Vitro Galactosylation and Sialylation of Glycoproteins with Terminal N-Acetylglucosamine and Galactose Residues” (2001) Biochemistry, 40(30):8868-8876.
Raju, TS. “Glycosylation Variations with Expression Systems and Their Impact on Biological Activity of Therapeutic Immunoglobulins”, BioProcess International., 44-53 (2003).
Rankin et al., “The therapeutic effects of an engineered human anti-tumour necrosis factor alpha antibody(CDP571) in rheumatoid arthritis” (1995) Br. J. Rheumatol., 34:334-342.
Rau “Adalimumab (a fully human anti-tumour necrosis factor alpha monoclonal antibody) in the treatment of active rheumatoid arthritis: the initial results of five trials” Ann Rheum Dis 2002,61 (Suppl II): ii70-ii73.
Rea, J. C. et al.: “Validation of a pH gradient-based ion-exchange chromatography method for high-resolution monoclonal antibody charge variant separations”, Journal of Pharmaceutical and Biomedical Analysis, New York, NY, US, vol. 54, No. 2, Jan. 25, 2011, pp. 317-323.
Reichert JM., et al., “Monoclonal antibody successes in the clinic”, Nature Biotech. 23(9):1073-1078, 2005.
Reinhart, “Assessment of the safety and efficacy of the monoclonal anti-tumor necrosis factor antibody-fragment, MAK 195F, in patients with sepsis and septic shock: a multicenter, randomized, placebo-controlled, dose-ranging study” (1996) Crit. Care Med., 24(5):733-742.
Remy et al., “Zinc-finger nucleases: A powerful tool for genetic engineering of animals” (2010) Transgenic Res., 19(3): 363-71.
Ren, D., et al., Reversed-phase liquid chromatography-mass spectrometry of site-specific chemical modifications in intact immunoglobulin molecules and their fragments. Journal of Chromatography A, 2008. 1179(2): p. 198-204.
Restelli, Veronica, et al., “The Effect of Dissolved Oxygen on the Production and the Glycosylation Profile of Recombinant Human Erythropoietin Produced From CHO Cells,” Biotechnology and Bioengineering, vol. 94, No. 3, (2006) 481-494.
Rheinwald JG, et al., “Growth of Cultured Mammalian Cells on Secondary Glucose Sources”, Cell, 287-293, 1974.
Ridder et al., “Generation of Rabbit Monoclonal Antibody Fragments from a Combinatorial Phage Display Library and Their Production in Yeast Pichia pastoris”, Biotechnology, 13:255-260 (1995).
Riechmann, “Phage display and selection of a site-directed randomized single-chain antibody Fv fragment for its affinity improvement” (1993) Biochemistry, 32(34):8848-8855.
Roe, S. “Separation Based on Structure” Chapter 4, § 5.2, In, Protein Purification Methods; A Practical Approach, Harries, et al. Sep. 1989, p. 203.
Rouiller et al. “Effiect of hydrocortisone on the production and glycosylation of an Fc-Fusion protein in CHO cell cultures,” Biotechnology Progress.(May 2012) 28(3):803-813.
Routier, F. H. et al.: “The glycosylation pattern of a humanized IgGI antibody(D1.3) expressed in CHO cells”, Glycoconjugate Journal, Chapman & Hall, GB, vol. 14, No. 2, Jan. 1, 1997, pp. 201-207.
Roy, B.M., et al., Toxic concentrations of exogenously supplied methy!glyoxal in hybridoma cell culture, Cytotechnology (2004) 46:97-107.
Roy, Samar N. et al., “Secretion of Biologically Active Recombinant Fibrinogen by Yeast.” The Journal of Biological Chemistry, vol. 270; 40 (1995). 23761-23767.
Rube et al., “Ewing's sarcoma and peripheral primitive neuroectodermal tumor cells produce large quantities of bioactive tumor necrosis factor-α (TNF-α) after radiation exposure”, Int. J. Radiation Oncology Biol. Phys., (2003), vol. 56, No. 5, pp. 1414-1425.
Rudd et al. “Glycosylation and the Immune System,” Science. (2001) 291(5512):2370-2376.
Rudikoff et al., “Single amino acid substitution altering antigen-binding specificity” (1982) Proc. Natl. Acad. Sci. USA, 70:1979-1983.
Russell et al., “Targets for sepsis therapies: Tumor necrosis factor versus interleukin-1” (1993) Curr. Opin. Biotech., 4:714-721.
Sakai et al.; “Use of nonionic surfactants for effective supply of phosphatidic acid in serum-free culture of Chinese hamster ovary cells”; Journal of Bioscience and Bioengineering; 92(3):256-261 (2001).
Salfeld, “Development of a Fully Human Antibody to TNF by Phage Display Technology,” IBC Conference, Antibody Engineering, San Diego (Dec. 1996), pp. 1-36.
Sandadi, S. et al., “Heuristic Optimization of Antibody Production by Chinese Hamster Ovary Cells”, Biotech. Progress, American Institute of Chem. Engineers: 21(5): 1537-1542, 2005.
Sandhu, J. “Protein engineering of antibodies” (1992) Critical Reviews in Biotechnology, 12:437-462.
Santiago et al., “Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases” (2008) Proc. Natl. Acad. Sci. USA., 105(15):5809-14.
Santora et al., “Characterization of recombinant human monoclonal tissue necrosis factor-alpha antibody using cation exchange HPLC and capillary isoelectric focusing,” Analytical Biochemistry, (1999) 275:98-108.
Santora, “Characterization of Noncovalent Complexes of Recombinant Human Monoclonal Antibody and Antigen Using Cation Exchange, Size Exclusion Chromatography, and BIAcore” (2001) Analytical Biochemistry, 299:119-129.
Sargent (pp. 1-3, Internet Archive captured Aug. 28, 2013, cellculturedish.com/2012/01/cho-cells-the-top-expressionsystem-of-best-selling-biologic-drugs/).
Sato et al, “Stimulation of monoclonal antibody production by human-human hybridoma cells with an elevated concentration of potassium or sodium phosphate in serum-free medium,” Cytotechnology 2:63-67, 1989.
Satoh, Mitsuo et al.: “Non-Fucosylated therapeutic antibodies as next-generation therapeutic antibodies”, Expert opinion on biological therapy, Ashley, London, GB, vol. 6, No. 11, Nov. 1, 2006, pp. 1161-1173.
Saxena, R. K. et al.; Microbial production and applications of 1 ,2-propanediol; Indian J. Microbiol. 2010,50,2-11.
Scales et al., “Hepatic ischemia/reperfusion injury: importance of oxidant/tumor necrosis factor interactions” (1994) Am. J. Physiol., 267 (6 Pt 1):G1122-1127.
Schiestl et al. “Acceptable changes in quality attributes of glycosylated biopharmaceuticals” Nature Biotechnology, 29(4), 310-312 (2011).
Schwieterman, “Immunosuppression in Combination with Monoclonal Antibodies” in Biologic Agents in Autoimmune Disease (Mar. 2-4, 1995), 9 pages.
Scientific Discussion. Retrieved from the Internet: ema.europa.eu/dics/en—GB/document—library/EPAR—Sceintific—Discussion/human/00481/WC500050867.pdf [retrieved on Jun. 29, 2015], EMEA, 2004, 25 pages.
Senczuk et al. “Hydrophobic interaction chromatography in dual salt system increases protein binding capacity” Biotechnology and Bioengineering, 103(5), 930-935 (2009).
Seo, Jin Seok, et al., “Effect of culture pH on recombinant antibody production by a new human cell line, F2N78, grown in suspension at 33.0° C. and 37.0° C.,” Appl. Microbiol Biotechnol., vol. 97 (2013). 5283-5291.
Seresht et al., “The impact of phosphate scarcity on pharmaceutical protein production in S. cerevisiae: linking transcriptomic insights to phenotypic responses” Microbial Cell Factories. 2011, 10: 104.
Serrick et al., “The early release of interleukin-2, tumor necrosis factor-alpha and interferon-gamma after ischemia reperfusion injury in the lung allograft” (1994) Transplantation, 58(11):1158-1162.
Shankar et al., “Evaluation of the role of second messenger systems in tumor necrosis factor-stimulated resorption of fetal rat limb bones” (1993) Bone, 14(6):871-876.
Sheeley et al., “Characterization of Monoclonal Antibody Glycosylation: Comparison of Expression Systems and Identification of Terminal α-Linked Galactose”, Anal. Biochem., 247(1):102-110 (1997).
Sheikh et al., “Studies of the digestion of bradykinin, lysyl bradykinin, and kinin-degradation products by carboxypeptidases A, B, and N;”. Biochemical Pharmacology. 1986, 35: 1957-1963.
Shen, Amy Y. et al., “Recombinant DNA Technology and Cell Line Development,” from “Cell Culture Technology for Pharmaceutical and Cell-Based Therapies,” CRC Press, 1995, 15-40.
Sheron et al., “Increased production of tumour necrosis factor alpha in chronic hepatitis B virus infection” (1991) J. Hepatol., 12(2):241-245.
Shibuya et al., “The elderberry (Sambucus nigra L.) bark lectin recognizes the Neu5Ac(alpha 2-6)Gal/GaINAc sequence”(1987) J. Biol. Chem., 262(4): 1596-1601.
Shields et al. “Lack of Fucose on Human IgGI N-Linked Oligosaccharide Improves Binding to Human FcyRIII and Antibody-dependent Cellular Toxicity,” J. Biol. Chem. (2002) 277(30):26733-26740.
Shih, “Effects of Anions on the Deamidation of Soy Protein”. Journal of Food Science. 1991, 56: 452-454.
Shirato, Ken et al., “Hypoxic regulation of glycosylation via the N-acetylglucosamine cycle.” J. Clin. Biochem. Nutr. vol. 48; 1 (2011). 20-25.
Shubert et al. “Comparison of ceramic hydroxy- and fluoroapatite versus Protein A/G—based resins in the isiolation of a recombinant human antibody from cell culture supernatant” J. Chromatography A, 114 (2007) 106-113.
Shukla et al., “Host cell protein clearance during protein A chromatography: development of an improved column wash step,” Biotechnology Progress, (2008) 24(5):1115-1121.
Shukla et al., “Recent advances in large-scale production of monoclonal antibodies and related proteins,” Trends in Biotechnology, (2010) 28(5):253-261.
Sigma Catalog “RPMI1640” (last accessed Jan. 22, 2015), 3 pages.
Sigma MSDS for RMPI1640 (last accessed Jan. 22, 2015), 6 pages.
Silverman et al., “Multivalent avimer proteins evolved by exon shuffling of a family of human receptor domains” (2005) Nat. Biotechnol., 23 (12): 1556-61.
Sioud et al., “Characterization of naturally occurring autoantibodies against tumour necrosis factor-alpha (TNF-α): in vitro function and precise epitope mapping by phage epitope library” (1994) Clin. Exp. Immunol., 98:520-525.
Skerra et al., “Alternative binding proteins: Anticalins—harnessing the structural plasticity of the lipocalin ligand pocket to engineer novel binding activities kerra” (2008) FEBS J., 275 (11): 2677-83.
Stumpp et al., “DARPins: A new generation of protein therapeutics” (2008) Drug Discov. Today, 13 (15-16): 695-701.
Sun et al., “Bowel necrosis induced by tumor necrosis factor in rats is mediated by platelet-activating factor” (1988) J. Clin. Invest., 81(5):1328-1331.
Sundaram et al., “An innovative approach for the characterization of the isoforms of a monoclonal antibody product,” Mabs, 3(6):505-512, 2011.
Sung, Y.H. et al., “Yeast hydrolysate as a low-cost additive to serum-free medium for the production of human thrombpoietin in suspension cultures of Chinese hamster ovary cells”, Applied Microbilolgy and Biotechnology 63:5, 527-536, 2004.
Suthanthiran et al., “Renal transplantation” (1994) New Engl. J. Med., 331(6):365-375.
Takagi, M. et al., “The effect of osmolarity on metabolism and morphology in adhesion and suspension chinese hamster ovary cells producing tissue plasminogen activator”, Cytochnology 32:171-179, 2000.
Takashima et al., “Characterization of Mouse Sialyltransferase Genes: Their Evolution and Diversity” (2008) Biosci. Biotechnol. Biochem., 72(5):1155-1167.
Tamura et al., “Structural correlates of an anticarcinoma antibody: identification of specificity-determining residues (SDRs) and development of a minimally immunogenic antibody variant by retention of SDFs only,” J. Immun. (2000) 164:1432-1441.
Tan et al., “Expression and purification of a secreted functional mouse/human chimaeric antibody against bacterial endotoxin in baculovirus-infected insect cells”, Biotechnol. Appl. Biochem. (1999), 30:59-64.
Taylor et al. “A transgenic mouse that expresses a diversity of human sequence heavy and light chain immunoglobulins,” Nucleic Acids Research,(1992) 20(23):6287-6295.
Taylor et al.,“Human immunoglobulin transgenes undergo rearrangement, somatic mutation and class switching in mice that lack endogenous IgM” (1994) Int. Immunol., 6:579-591.
Tebbey, Paul W., et al., “Consistency of quality for the glycosylated monoclonal antibody Humira (adalimumab),” MAbs, Sep. 3, 2015;7(5); 805-11.
Teichmann, S. Declaration dated Dec. 17, 2010 from opposition proceedings in EP 0929578, 6 pages.
TESS database “HYCLONE” Trademark #76244963. Filing date Apr. 23, 2001. Live mark. Last accessed Jan. 21, 2015.
TESS database “HYCLONE” Trademark #85769283. Filing date Sep. 30, 2012. Live mark. Last accessed Jan. 21, 2015.
Tharmalingam et al.; “Pluronic Enhances the Robustness and Reduces the Cell Attachment of Mammalian Cells”; Molecular Biotechnology; 39(2):167-177 (2008).
The Difference-Between, “Poly vs. Polyalcohol—What's the difference?” pp. 1-2, downloaded from http://the-difference-between.com/polyalcohol/polyol on Apr. 16, 2016.
The Kennedy Institute of Rheumatology, 1995 Annual Scientific Report, “Anti-TNF trials and studies of mechanisms of action”.
The MW Calculator available at the Sequence Manipulation Suite (see www.bioinformatics.org/sms2/index.html), downloaded Feb. 25, 2014, 2 pages.
The pl Calculator available at the Sequence Manipulation Suite (see www.bioinformatics.org/sms2/index.html, downloaded Feb. 25, 2014, p. 1.
The Statement on a Nonproprietary Name Adopted by the USAN Council for Adalimumab, p. 1, downloaded on May 19, 2011 from www.ama-assn.org/resources/doc/usan/adalimumab.doc. 1 page.
Thompson, “Affinity maturation of a high-affinity human monoclonal antibody against the third hypervariable loop of human immunodeficiency virus: use of phage display to improve affinity and broaden strain reactivity” (1996) J. Mol. Biol., 256(1):77-88.
Thorp, “Tumour Necrosis Factor Induction of ELAM-1 and ICAM-1 on Human Umbilical Vein Endothelial Cells—Analysis of Tumour Necrosis Factor Receptor Interaction” (1992) Cytokine, 4(4): 313-319.
Tomiya et al., “Comparing N-glycan processing in mammalian cell lines to native and engineered; lepidopteran insect cell lines,” Glycoconjuqate Journal 21 :343-360 (2004).
Tomlinson, “The repertoire of human germline VH sequences reveals about fifty groups of VH segments with different hypervariable loops” (1992) J. Mol. Biol., 227:776-98.
Tomlinson, “The structural repertoire of the human Vk domain” (1995) The EMBO J., 14(18):4628-38.
Tracey et al., “Shock and tissue injury induced by recombinant human cachectin” (1986) Science, 234(4775):470-474.
Tracey, “Tumor necrosis factor: A pleiotropic cytokine and therapeutic target” (1994) Annu. Rev. Med., 45:491-503.
Tsuchiyama et al., “Comparison of anti-TNF alpha autoantibodies in plasma and from EBV transformed lymphocytes of autoimmune and normal individuals” (1995) Hum. Antibod. Hybridomas, 6(2):73-76.
United States Food and Drug Administration (FDA) Biological Licensing Application File No. 125057 (Adalimumab) (Dec. 31, 2002) (Last Accessed Mar. 4, 2015 at www.fda.gov/Drugs/DevelopmentApprovalProcess/HowDrugsareDevelopedandApproved/ApprovalApplications/TherapeuticBiologicApplications/ucm080610.htm, 1 page.
Urlaub et al., “Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity” (1980) Proc. Natl. Acad. Sci. USA, 77:4216-4220.
Vallee B et al. “The role of zinc in carboxypeptidase” The Journal of Biological Chemistry, (1960) 235, 1; 64-69.
Valliere-Douglass et al., “Glutamine-linked and Non-consensus Asparagine-linked Oligosaccharides Present in Human Recombinant Antibodies Define Novel Protein Glycosylation Motifs”, J. Biol. Chem., 285:16012-16022 (2010).
Van Der Poll et al., “Activation of coagulation after administration of tumor necrosis factor to normal subjects” (1990) N. Engl. J. Med., 322(23):1622-1627.
Van Der Poll et al., “Comparison of the early dynamics of coagulation activation after injection of endotoxin and tumor necrosis factor in healthy humans” (1991) Prog. Clin. Biol. Res., 367:55-60.
Van Der Poll, “Effect of postponed treatment with an anti-tumour necrosis factor (TNF) F(ab')2 fragment on endotoxin-induced cytokine and neutrophil responses in chimpanzees” (1995) Clin. Exp. Immunol., 100:21-25.
Van Dulleman et al., “Treatment of Crohn's disease with anti-tumor necrosis factor chimeric monoclonal antibody (cA2)” (1995) Gastroenterology, 109(1):129-135.
Van Herreweghe, et al.; Tumor necrosis factor-induced modulation of glyoxalase I activities through phosphorylation by PKA results in cell death and is accompanied by the formation of a specific methylglyoxal-derived AGE; Proc. Natl. Acad. Sci. 2002, 99, 949-954.
Van Lent PL, et al. “The impact of protein size and charge on its retention in articular cartilage” J Rheumatol. Aug. 1987;14(4):798-805.
Varasteh et al. Optimization of Anti-Rh D Immunoglobulin Stability in the Lyphiliization Process. Iranian Journal of Basic Medical Sciences, Spring 2008, vol. 11, No. 1. pp. 55-61.
Varki et al. Essentials of Glycobiology, 2nd edition, (1999) CSHL, available online: ncbi.nlm.nih.gov/books/NBK1908/.
Vasilli, P. et al., The Pathophysiology of Tumor Necrosis Factors, Annu. Rev. Immunol. 10:411-452 (1992).
Vaughan, “Human antibodies by design” (1998) Nature Biotechnology, 16:535-539.
Vlasak, J. & Ionescu, R., Heterogeneity of Monoclonal Antibodies Revealed by Charge-Sensitive Methods. Current Pharmaceutical Biotechnology, 2008. 9(6): p. 468-481.
Wagner et al., “Antibodies generated from human immunoglobulin miniloci in transgenic mice” (1994) Nucl. Acids Res. 22:1389-1393.
Wagner et al., “The diversity of antigen-specific monoclonal antibodies from transgenic mice bearing human immunoglobulin gene miniloci” (1994) Eur. J. Immunol., 24:2672-2681.
Wallick et al. “Glycosylation of a VH residue of a monoclonal antibody against alpha (1-6) dextran increases its affinity for antigen,” J. Exp. Med.(1988) 168(3):1099-1109.
Walsh et al. “Effect of the carbohydrate moiety on the secondary structure of ?2-glycoprotein. I. Implications for the biosynthesis and folding of glycoproteins,” Biochemistry. (1990) 29(26):6250-6257.
Walsh, et al.: “Post-translational modifications in the context of therapeutic proteins”, Nature Biotechnology, vol. 24, No. 10, Oct. 2006, pp. 1241-1252.
Wang et al., “The immobilized leukoagglutinin from the seeds of Maackia amurensis binds with high affinity to complex-type Asn-linked oligosaccharides containing terminal sialic acid-linked alpha-2,3 to penultimate galactose residues” (1988) J Biol. Chem., 263(10): 4576-4585.
Wang, Tina et al., “Exploring Post-translational Arginine Modification Using Chemically Synthesized Methylglyoxal Hydroimidazolones,” J. Am. Chem. Soc., 2012, 134, pp. 8958-8967.
Wang, Z.; et al. Desulfurization of Cysteine-Containing Peptides Resulting from Sample Preparation for Protein Characterization by MS; Rapid Commun. Mass Spectrom. 2010, 24, 267-275.
Ward, “Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli” (1989) Nature, 341:544-546.
Warnock et al., “In vitro galactosylation of human IgG at 1 kg scale using recombinant galactosyltransferase” (2005) Biotechnol. Bioeng., 92(7):831-842.
Watt, S.; et al.; Effect of Protein Stabilization on Charge State Distribution in Positive- and Negative-Ion Electrospray Ionization Mass Spectra; J. Am. Soc. Mass. Spectrom. 2007, 18, 1605-1611.
Wedemayer et al., “Structural insights into the evolution of an antibody combining site” (1997) Science, 276:1665-1669.
Weikert et al., “Engineering Chinese hamster ovary cells to maximize sialic acid content of recombinant glycoproteins” (1999) Nature Biotechnology, 17(11): 1116-1121 6.(Skip) Drickamer K, Taylor M E (2006). Introduction to Glycobiology, 2nd ed.
Weinstein et al., “Primary structure of beta-galactoside alpha 2,6-sialyltransferase. Conversion of membrane-bound enzyme to soluble forms by cleavage of the NH2-terminal signal anchor” (1987) J. Biol. Chem. 262(36):17735-17743.
Wiendl et al., “Therapeutic Approaches in Multiple Sclerosis. Lessons from failed and interrupted treatment trials”, BioDrugs. (2002), 16(3):183-200.
Williams et al., “Kinetic analysis by stopped-flow radiationless energy transfer studies: effect of anions on the activity of carboxypeptidase A”. Biochemistry. 1986, 25, 94-100.
Williams, A. et al., Ion-Exchange Chromatography, Oct. 1998, Supplement 44, pp. 10-10-1-10-10-30.
Winter, “Humanized antibodies” (1993) Immunol. Today, 14(6):243-246.
Winter, “Making antibodies by phage display technology” (1994) Annu. Rev. Immunol., 12:433-455.
Wolff et al., “The Kinetics of Carboxypeptidase B Activity,” J. Biological Chem, 1962, 237:3094-3099.
Wong N.S.C. et al: “An investigation of intracellular glycosylation activities in CHO cells: Effects of nucleotide sugar precursor feeding” Biotechnology and Bioengineering, vol. 187, No. 2,Oct. 1, 2010, pp. 321-336.
Worthington Biochemical Corporation, porcine pancreas carboxypeptidase B, one page, Feb. 25, 2012.
Wurm, FM, “Production of recombinant protein therapeutics in cultivated mammalian cells”, Nature Biotechnology 22(11):1393-1398, 2004.
Wyss, et al. “The structural role of sugars in glycoproteins,” Curr. Opin. Biotechnol. (1996), 7(4); 409-416.
Xiang, T., Chumsae, C. & Liu, H., Localization and Quantitation of Free Sulfhydryl in Recombinant Monoclonal Antibodies by Differential Labeling with 12C and 13C lodoacetic Acid and LC-MS Analysis. Analytical Chemistry, 2009. 81(19): p. 8101-8108.
Yao et al., “The potential etiologic role of tumor necrosis factor in mediating multiple organ dysfunction in rats following intestinal ischemia-reperfusion injury” (1995) Resuscitation, 29(2):157-168.
Yigzaw et al., “Exploitation of the adsorptive properties of depth filters for host cell protein removal during monoclonal antibody purification,” Biotechnology Progress, (2006) 22(1):288-296.
Yuk, I.H. et al., Controlling Glycation of Recombinant Antibody in Fed Batch Cell Cultures, Nov. 2011 , Biotechnology and Bioenqineering, vol. 108, No. 11 pp. 2600-2610.
Yumioka et al., “Screening of effective column rinse solvent for Protein-A chromatography,” Protein Expression and Purification, (2010) 70(2): 218-223.
Zang, T.; et al.; Chemical Methods for the Detection of Protein N-Homocysteinylation via Selective Reactions with Aldehydes; Anal. Chem. 2009, 81, 9065-9071.
Zatarain-Rios E and Mannik M, “Charge-charge interactions between articular cartilage and cationic antibodies, antigens, and immune complexes,” Arthritis Rheum. Nov. 1987;30(11):1265-73.
Zhang et al. “A novel function for selenium in biological system: Selenite as a highly effective iron carrier for Chinese hamster overary cell growth and monoclonal antibody production,” Biotechnology and Bioengineering. (2006) 95(6):1188-1197.
Zhang et al., “CHO glycosylation mutants as potential host cells to produce therapeutic proteins with enhanced efficacy” (2013) Advances in Biochemical Engineering/Biotechnology, 131:63-87.
Zhang et al., “Isolation and characterization of charge variants using cation exchange displacement chromatography,” 1218(31): 5079-5086, 2011.
Zhang, B., et al., Unveiling a Glycation Hot Spot in a Recombinant Humanized Monoclonal Antibody. Analytical Chemistry, 2008. 80(7): p. 2379-2390.
Zhang, F. et al., “The Effect of Dissolved Oxygen (DO) Concentration on the Glycosylation of Recombinant Protein Produced by the Insect Cell-Baculovirus Expression System.” Biotechnology and Bioengineering, (2002), 77(2); 219-224.
Zhang, T.; Identification and Characterization of Buried Unpaired Cysteines in a Recombinant Monoclonal IgG1 Antibody; Anal. Chem. 2012, 84, 7112-7123.
Zhang, W. and Czupryn, M.J., Free Sulfhydryl in Recombinant Monoclonal Antibodies. Biotechnology Progress, 2002. 18(3): p. 509-513.
Zhang, Y. et al., “Specificity and Mechanism of Metal Ion Activation in UDP-galactose: β-Galactoside-α-1,3-galactosyltransferase.” J. Biological Chemistry vol. 276; 15 (2001). 11567-11574.
Zhao, G.; Chemical Synthesis of S-Ribosyl-L-homocysteine and Activity Assay as a LuxS Substrate; Bioorg. Med. Chem. Lett. 2003,13,3897-3900.
Zhou, Z. et al.; An Antibody-Catalyzed Allylic Sulfoxide-Sulfenate Rearrangement; J. Org. Chem. 1999,64,8334-8341.
Zhou, Z. S. et al. An Antibody-Catalyzed Selenoxide Elimination; J. Am. Chem. Soc. 1997, 119, 3623-3624.
Zou et al., “Dominant expression of a 1.3 Mb human Ig kappa locus replacing mouse light chain production” (1996) FASEB J., 10:1227-1232.
Provisional Applications (1)
Number Date Country
61479727 Apr 2011 US
Divisions (2)
Number Date Country
Parent 14619799 Feb 2015 US
Child 15014694 US
Parent 13457020 Apr 2012 US
Child 14493068 US
Continuations (1)
Number Date Country
Parent 14493068 Sep 2014 US
Child 14619799 US