US20160152702A1 - Methods for controlling the galactosylation profile of recombinantly-expressed proteins - Google Patents
Methods for controlling the galactosylation profile of recombinantly-expressed proteins Download PDFInfo
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- US20160152702A1 US20160152702A1 US15/014,694 US201615014694A US2016152702A1 US 20160152702 A1 US20160152702 A1 US 20160152702A1 US 201615014694 A US201615014694 A US 201615014694A US 2016152702 A1 US2016152702 A1 US 2016152702A1
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- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/34—Sugars
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2511/00—Cells for large scale production
Definitions
- the present invention relates to methods for modulating the glycosylation profile of recombinantly-expressed proteins.
- the present invention relates to methods of controlling the galactosylation profile of recombinantly-expressed proteins by supplementing production media with manganese and/or galactose.
- a particular type of production media e.g., hydrolysate-based media or chemically defined media (“CD” or “CDM”)
- CD chemically defined media
- 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.
- the present invention relates to methods for modulating the glycosylation profile of recombinantly-expressed proteins.
- the present invention relates to methods of controlling the galactosylation profile of recombinantly-expressed proteins by supplementing production media with manganese and/or galactose.
- the production media is a hydrolysate-based media or a CD media.
- the present invention is directed to methods of controlling the galactosylation profile of recombinantly-expressed antibody.
- the recombinantly-expressed antibody is an anti-TNF ⁇ antibody.
- the recombinantly-expressed anti-TNF ⁇ antibody is adalimumab.
- 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.
- a production medium e.g., a hydrolysate-based or a CD medium
- the manganese supplement can take the form of any biologically-acceptable manganese salt, for example, but not limited to, manganese (II) chloride.
- the galactose supplement can take the form of any biologically-acceptable galactose-containing compound, for example, but not limited to, D-(+)-galactose.
- 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 ⁇ M, 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).
- a production medium e.g., a hydrolysate-based or a CD medium
- 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).
- a production medium e.g., a hydrolysate-based or a CD medium
- 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 ( ⁇ M)/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
- 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 ( ⁇ M)/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
- 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.
- the present invention relates to methods modulating the glycosylation profile of recombinantly-expressed proteins.
- 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.
- production medium e.g., a hydrolysate-based or a CD medium
- 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.
- a terminal galactose is added to NGA2F by ⁇ -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 Mn 2+ as a cofactor for galactosyltransferase.
- the present invention is directed to methods of controlling the galactosylation profile of recombinantly-expressed antibody.
- the recombinantly-expressed antibody is an anti-TNF ⁇ antibody.
- the recombinantly-expressed anti-TNF ⁇ antibody is adalimumab.
- 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.
- a production medium e.g., a hydrolysate-based or a CD medium
- the manganese supplement can take the form of any biologically-acceptable manganese salt, for example, but not limited to, manganese (II) chloride.
- the galactose supplement can take the form of any biologically-acceptable galactose-containing compound, for example, but not limited to, D-(+)-galactose.
- 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 ⁇ M, 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).
- a production medium e.g., a hydrolysate-based or a CD medium
- 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).
- a production medium e.g., a hydrolysate-based or a CD medium
- 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 ( ⁇ M)/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,
- Mn
- 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 ( ⁇ M)/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
- the production medium e.g., a hydrolysate-based or a CD medium
- the production medium used in the production of a recombinantly-expressed protein
- 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 ⁇ M.
- the production medium e.g., a hydrolysate-based or a CD medium
- the production medium used in the production of a recombinantly-expressed protein
- 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 ).
- a manganese concentration of about 100 ⁇ M and a galactose concentration of about 100 mM represent the maximum range of interest for this Example 1.
- the production medium e.g., a hydrolysate-based media or a CD media
- the production medium used in the production of a recombinantly-expressed protein
- both manganese and galactose are supplemented with both manganese and galactose.
- 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.
- the combined addition of 40 ⁇ M 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 ⁇ M concentration resulted in an additional 3% decrease in the NGA2F+NGA2F-GlcNac sum, and a corresponding 3% increase in the NA1F+NA2F sum
- 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 ⁇ M 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.
- 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 ).
- addition of 40 ⁇ M manganese chloride alone reduced the NGA2F+NGA2F-GlcNac sum by 5%
- 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.
- 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.
- a hydrolysate-based media used in the production of a recombinantly-expressed protein with galactose and/or manganese.
- the addition of manganese chloride alone within the range of 0 to 40 ⁇ M 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 combined addition of manganese chloride ranging from 0 to 40 ⁇ M 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 ⁇ M or 40 ⁇ M manganese chloride.
- compositions and methods of the present invention also find use across distinct cell lines.
- 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.
- the addition of manganese chloride alone within the range of 0 to 20 ⁇ M 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.
- Example 5 employs a third adalimumab-producing cell line that is distinct from either of the adalimumab-producing cell lines of Examples 1-4.
- the addition of manganese chloride alone within the range of 0 to 1 ⁇ M 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.
- 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.
- 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.
- the addition of manganese chloride alone within the range of 0 to 0.5 ⁇ M 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.
- addition of 0.2 ⁇ M manganese chloride alone reduced the NGA2F+NGA2F-GlcNac sum by 12%
- addition of 4 mM galactose alone decreased the NGA2F+NGA2F-GlcNac sum by 2%
- the combined addition of manganese chloride and galactose at these same concentrations led to a 19% reduction in the NGA2F+NGA2F-GlcNac sum, 5% higher than their combined individual contributions.
- 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.
- Example 7 which employs a CHO cell line that produced an antibody distinct from adalimumab.
- the addition of manganese chloride alone within the range of 0 to 40 ⁇ M 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.
- the addition of 40 ⁇ M manganese chloride alone reduced the NGA2F+NGA2F-GlcNac sum by 20%
- the addition of 40 mM galactose alone decreased the NGA2F+NGA2F-GlcNac sum by 12%.
- the combined addition of manganese chloride and galactose at these same concentrations led to a 27% decrease in the NGA2F+NGA2F-GlcNac sum.
- 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.
- Example 8 which employs a CHO cell line producing an antibody distinct from both adalimumab and the antibody of Example 7.
- the addition of manganese chloride alone in the range of 0 to 75 ⁇ M 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.
- 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 ⁇ M manganese chloride alone (10%) and 15 mM galactose alone (7%).
- the additive effect was observed for the condition supplemented with 50 ⁇ M 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 ⁇ M manganese chloride alone (18%) and 30 mM galactose alone (12%).
- 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.
- 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).
- the expression vector Prior to insertion of the antibody or antibody-related light or heavy chain sequences, the expression vector may already carry antibody constant region sequences.
- 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.
- 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).
- 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.
- 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.
- 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.
- CMV cytomegalovirus
- SV40 Simian Virus 40
- AdMLP adenovirus major late promoter
- 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).
- 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).
- DHFR dihydrofolate reductase
- 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.
- 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.
- 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.
- eukaryotic cells such as mammalian host cells
- expression of antibodies in eukaryotic 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.
- Enterobacteriaceae such as Escherichia , e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus
- Salmonella e.g., Salmonella typhimurium
- 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.
- 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.
- 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 K. marxianus
- yarrowia EP 402,226
- Pichia pastoris EP 183,070
- Candida Trichoderma reesia
- Neurospora crassa Schwanniomyces such as Schwanniomyces occidentalis
- 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.
- 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.
- Chinese Hamster Ovary CHO cells
- 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.
- 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.
- 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.
- mice 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 F10TM (Sigma), Minimal Essential MediumTM ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's MediumTM ((DMEM), Sigma) are suitable for culturing the host cells.
- 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.
- 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.
- 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.
- 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.
- the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium.
- the particulate debris either host cells or lysed cells (e.g., resulting from homogenization)
- supernatants from such expression systems can be first concentrated using a commercially available protein concentration filter, e.g., an AmiconTM or Millipore PelliconTM ultrafiltration unit.
- 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.
- supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, e.g., an AmiconTM or Millipore PelliconTM ultrafiltration unit.
- a commercially available protein concentration filter e.g., an AmiconTM or Millipore PelliconTM ultrafiltration unit.
- 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.
- 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.
- 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.
- the harvest samples were Protein A purified and prepared for the oligosaccharide assay using the following procedures.
- 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.
- 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.
- manganese chloride was supplemented at the following concentrations in production media: 0, 40, 60, 80, and 100 ⁇ M.
- 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.
- manganese chloride and galactose were supplemented to both production and feed media in the following combinations: 40 ⁇ M manganese chloride and 20 mM galactose; 40 ⁇ M manganese chloride and 40 mM galactose (Table 2). Basal and feed media for the control cultures were not supplemented with manganese chloride or galactose.
- manganese chloride was supplemented at the following concentrations in production media: 0, 10, 20, and 40 ⁇ M.
- 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.
- 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 ⁇ m PES) and stored at 4° C. until use.
- Manganese (II) Chloride Sigma M1787-100 mL; 1.0 M ⁇ 0.1 M
- D(+)Galactose Sigma G5388-1 kg
- manganese chloride was supplemented at the following concentrations in production media: 0, 10, 20, and 40 ⁇ M.
- 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.
- 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 ⁇ M 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 ).
- 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 ⁇ m PES) and stored at 4° C. until use.
- Manganese (II) Chloride Sigma M1787-100 mL; 1.0 M ⁇ 0.1 M
- D(+)Galactose Sigma G5388-1 kg
- manganese chloride was supplemented at the following concentrations in production media: 0, 10, 20, and 40 ⁇ M.
- 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.
- 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.
- Manganese (II) Chloride Sigma M3634-100 g
- D(+)Galactose Sigma G5388-1 kg
- 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.
- manganese chloride was supplemented at the following concentrations in both production and feed media: 0, 0.1, 0.2, 0.5, and 1.0 ⁇ M.
- Galactose was supplemented at 0 and 30 mM concentrations in both production and feed media.
- a combined manganese chloride and galactose supplementation strategy was utilized for the production basal and feed media at either 0.2 or 0.5 ⁇ M manganese chloride plus 30 mM galactose. Basal and feed media for the control cultures were not supplemented with manganese chloride or galactose.
- 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 ⁇ M manganese chloride ( FIG. 17 ).
- 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.
- manganese chloride was supplemented at the following concentrations in both production and feed media: 0, 0.2, and 0.5 ⁇ M.
- 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.
- FIGS. 19A, 19B 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 ⁇ M concentration were comparable to the control condition without manganese or galactose added.
- 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 ).
- 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 ⁇ m PES) and stored at 4° C. until use.
- Manganese (II) Chloride Sigma M1787-100 mL; 1.0 M ⁇ 0.1 M
- D(+)Galactose Sigma G5388-1 kg
- manganese chloride was supplemented at the following levels in production media: 0, 10, 20, and 40 ⁇ M.
- 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.
- 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 ).
- the addition of 40 ⁇ M manganese chloride alone reduced the NGA2F+NGA2F-GlcNac sum by 20%
- the addition of 40 mM galactose alone decreased the NGA2F+NGA2F-GlcNac sum by 12%.
- the combined addition of manganese chloride and galactose at these same concentrations i.e.
- manganese chloride was supplemented at the following concentrations in both production and feed media: 0, 25, 50, and 75 ⁇ M.
- Galactose was supplemented at 0, 15, 30, and 60 mM concentrations in both production and feed media.
- a combined manganese chloride and galactose supplementation strategy was utilized for the production basal and feed media at 25 ⁇ M manganese chloride+15 mM galactose, and 50 ⁇ M manganese chloride+30 mM galactose. Basal and feed media for the control cultures were not supplemented with manganese chloride or galactose.
- the additive effect was observed for the condition supplemented with 50 ⁇ M 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 ⁇ M 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 ⁇ M manganese chloride and 30 mM galactose to chemically defined GIA-1 media.
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Abstract
Description
- 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.
- 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.
- 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.
- 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).
-
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 CHOcell line # 2 in CDM GIA-1 in batch shake flasks.FIG. 13B depicts the viability of adalimumab-producing CHOcell line # 2 in CDM GIA-1 in batch shake flasks. -
FIG. 14A depicts the galactosylation profile (NGA2F+NGA2F-GlcNac) of adalimumab in CHOcell line # 2 in CDM GIA-1 in batch shake flasks.FIG. 14B depicts the galactosylation profile (NA1F+NA2F) of adalimumab in CHOcell 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 CHOcell line # 2. -
FIG. 16A depicts the culture growth of adalimumab-producing CHOcell line # 3 in CDM GIA-1 in fed-batch 3 L bioreactors.FIG. 16B depicts the viability of adalimumab-producing CHOcell line # 3 in CDM GIA-1 in fed-batch 3 L bioreactors.FIG. 16C depicts the normalized titer of adalimumab-producing CHOcell line # 3 in CDM GIA-1 in fed-batch 3 L bioreactors. -
FIG. 17A depicts the galactosylation profile (NGA2F+NGA2F-GlcNac) of adalimumab in CHOcell line # 3 in CDM GIA-1 in fed-batch 3 L bioreactors.FIG. 17B depicts the galactosylation profile (NA1F+NA2F) of adalimumab in CHOcell 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 CHOcell 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 producingmAb # 1 in CDM GIA-1 in batch shake flasks.FIG. 22B depicts the viability of CHO cell line producingmAb # 1 in CDM GIA-1 in batch shake flasks. -
FIG. 23A depicts the galactosylation profile (NGA2F+NGA2F-GlcNAc) ofmAb # 1 in CDM GIA-1 in batch shake flasks.FIG. 23B depicts the galactosylation profile (NA1F+NA2F) ofmAb # 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 ofmAb # 1 relative to control. -
FIG. 25A depicts the culture growth of CHO cell line producingmAb # 2 in CDM GIA-1 in fed-batch 3 L bioreactors.FIG. 25B depicts the viability of CHO cell line producingmAb # 2 in CDM GIA-1 in fed-batch 3 L bioreactors.FIG. 25C depicts the normalized titer of CHO cell line producingmAb # 2 in CDM GIA-1 in fed-batch 3 L bioreactors. -
FIG. 26A depicts the glycosylation profile (NGA2F+NGA2F-GlcNAc) ofmAb # 2 in CDM GIA-1 in fed-batch 3 L bioreactors.FIG. 26B depicts the glycosylation profile (NA1F+NA2F) ofmAb # 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 ofmAb # 2 relative to control. - 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.
- 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 day day day 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.
- 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) - 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 afterproduction 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 ). - 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.
- 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. - 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. - 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.
- 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) - 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). - 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.
- 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.
- 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) - 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 byday 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). - 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. - 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.
- 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.
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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) - 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). - 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. - 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 day day day 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 onproduction day 17, whichever condition occurred first. - 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.
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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) - 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 ). - 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 CHOcell 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. - 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 onproduction 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. - 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.
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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) - 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 ). - 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. - 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.
- 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.
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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) - 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 onproduction 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). - 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. - 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 day day day 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 onproduction day 15, whichever condition occurred first. - 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.
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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) - 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 ). - 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.
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10513724B2 (en) | 2014-07-21 | 2019-12-24 | Glykos Finland Oy | Production of glycoproteins with mammalian-like N-glycans in filamentous fungi |
Families Citing this family (49)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2012149197A2 (en) | 2011-04-27 | 2012-11-01 | Abbott Laboratories | Methods for controlling the galactosylation profile of recombinantly-expressed proteins |
WO2013114245A1 (en) | 2012-01-30 | 2013-08-08 | Dr. Reddy's Laboratories Limited | Process of modulating man5 and/or afucosylation content of glycoprotein composition |
US9150645B2 (en) | 2012-04-20 | 2015-10-06 | Abbvie, Inc. | Cell culture methods to reduce acidic species |
US9181572B2 (en) | 2012-04-20 | 2015-11-10 | Abbvie, Inc. | Methods to modulate lysine variant distribution |
US9067990B2 (en) | 2013-03-14 | 2015-06-30 | Abbvie, Inc. | Protein purification using displacement chromatography |
WO2013176754A1 (en) | 2012-05-24 | 2013-11-28 | Abbvie Inc. | Novel purification of antibodies using hydrophobic interaction chromatography |
US9206390B2 (en) | 2012-09-02 | 2015-12-08 | Abbvie, Inc. | Methods to control protein heterogeneity |
US9512214B2 (en) | 2012-09-02 | 2016-12-06 | Abbvie, Inc. | Methods to control protein heterogeneity |
US20160185847A1 (en) * | 2012-12-17 | 2016-06-30 | Laboratoire Francais Du Fractionnement Et Des Biotechnologies | Use of monoclonal antibodies for the treatment of inflammation and bacterial infections |
WO2014143205A1 (en) | 2013-03-12 | 2014-09-18 | Abbvie Inc. | Human antibodies that bind human tnf-alpha and methods of preparing the same |
WO2014151878A2 (en) * | 2013-03-14 | 2014-09-25 | Abbvie Inc. | Methods for modulating protein glycosylation profiles of recombinant protein therapeutics using monosaccharides and oligosacharides |
TWI625390B (en) * | 2013-03-14 | 2018-06-01 | 安美基公司 | Methods for increasing mannose content of recombinant proteins |
US9017687B1 (en) | 2013-10-18 | 2015-04-28 | Abbvie, Inc. | Low acidic species compositions and methods for producing and using the same using displacement chromatography |
WO2014159579A1 (en) | 2013-03-14 | 2014-10-02 | Abbvie Inc. | MUTATED ANTI-TNFα ANTIBODIES AND METHODS OF THEIR USE |
EP3391942A1 (en) | 2013-03-26 | 2018-10-24 | Coherus Biosciences, Inc. | Protein production method |
PL3036320T3 (en) * | 2013-08-19 | 2021-11-02 | Biogen Ma Inc. | Control of protein glycosylation by culture medium supplementation and cell culture process parameters |
US20160237399A1 (en) | 2015-02-18 | 2016-08-18 | Biogen Ma Inc. | Control of Protein Glycosylation by Culture Medium Supplementation and Cell Culture Process Parameters |
EP3052640A2 (en) | 2013-10-04 | 2016-08-10 | AbbVie Inc. | Use of metal ions for modulation of protein glycosylation profiles of recombinant proteins |
US10376582B2 (en) | 2013-10-16 | 2019-08-13 | Outlook Therapeutics, Inc. | Buffer formulations for enhanced antibody stability |
US9085618B2 (en) | 2013-10-18 | 2015-07-21 | Abbvie, Inc. | Low acidic species compositions and methods for producing and using the same |
US8946395B1 (en) | 2013-10-18 | 2015-02-03 | Abbvie Inc. | Purification of proteins using hydrophobic interaction chromatography |
US9181337B2 (en) | 2013-10-18 | 2015-11-10 | Abbvie, Inc. | Modulated lysine variant species compositions and methods for producing and using the same |
WO2015073884A2 (en) | 2013-11-15 | 2015-05-21 | Abbvie, Inc. | Glycoengineered binding protein compositions |
JP6797458B2 (en) | 2014-01-29 | 2020-12-09 | エルジー・ケム・リミテッド | How to regulate galactosylation of recombinant proteins by optimizing the culture medium |
EP3110941A4 (en) | 2014-02-25 | 2017-10-18 | Dr. Reddy's Laboratories Ltd. | Process for modifying galactosylation and g0f content of a glycoprotein composition by glutamine supplementation |
WO2015128314A1 (en) | 2014-02-27 | 2015-09-03 | F. Hoffmann-La Roche Ag | Modulation of cell growth and glycosylation in recombinant glycoprotein production |
KR101660580B1 (en) | 2014-04-02 | 2016-09-28 | 프레스티지 바이오파마 피티이. 엘티디. | A method for preparing an antibody by controlling a sugar content of the antibody |
US20160185848A1 (en) * | 2014-07-09 | 2016-06-30 | Abbvie Inc. | Methods for modulating the glycosylation profile of recombinant proteins using sugars |
MA41685A (en) | 2014-10-17 | 2017-08-22 | Biogen Ma Inc | COPPER SUPPLEMENT FOR THE REGULATION OF GLYCOSYLATION IN A MAMMAL CELL CULTURE PROCESS |
US20160115225A1 (en) * | 2014-10-24 | 2016-04-28 | Abbvie Inc. | Methods of Reducing Methylglyoxal (MGO) Modification of Recombinant Proteins in Cell Culture |
WO2016089919A1 (en) * | 2014-12-01 | 2016-06-09 | Amgen Inc. | Process for manipulating the level of glycan content of a glycoprotein |
KR102007930B1 (en) | 2014-12-31 | 2019-08-06 | 주식회사 엘지화학 | A method for controlling glycosylation of recombinant glycoprotein |
EP3247718B1 (en) | 2015-01-21 | 2021-09-01 | Outlook Therapeutics, Inc. | Modulation of charge variants in a monoclonal antibody composition |
US20160280767A1 (en) * | 2015-03-23 | 2016-09-29 | Lonza Ltd. | Methods for controlling protein glycosylation |
HU231463B1 (en) * | 2015-08-04 | 2024-01-28 | Richter Gedeon Nyrt. | Method for increasing the galactose content of recombinant proteins |
WO2017083224A1 (en) | 2015-11-09 | 2017-05-18 | Bristol-Myers Squibb Company | Methods to manipulate quality attributes of polypeptides produced in cho cells |
CA3013336A1 (en) | 2016-02-03 | 2017-08-10 | Oncobiologics, Inc. | Buffer formulations for enhanced antibody stability |
US10519479B1 (en) * | 2016-03-15 | 2019-12-31 | Ares Trading S.A. | Methods for modifying glycosylation using manganese |
US20200347126A1 (en) | 2016-08-03 | 2020-11-05 | Fyb 202 Project Gmbh | Production of Biosimilar Ustekinumab In CHO Cells |
AU2019319970A1 (en) * | 2018-08-10 | 2021-03-11 | Genentech, Inc. | Cell culture strategies for modulating protein glycosylation |
HUP1800376A2 (en) | 2018-11-07 | 2020-05-28 | Richter Gedeon Nyrt | Method for modifying the glycosylation profile of a recombinant glycoprotein produced in cell culture |
SG11202104092UA (en) * | 2018-11-13 | 2021-05-28 | Janssen Biotech Inc | Control of trace metals during production of anti-cd38 antibodies |
CN111321188A (en) * | 2018-12-17 | 2020-06-23 | 嘉和生物药业有限公司 | Formula for modifying antibody glycoform, cell culture method and application in industrial production |
WO2020190031A1 (en) | 2019-03-18 | 2020-09-24 | (주)지플러스 생명과학 | Transgenic plant with suppressed expression of cgl1 and cgl2 and method for producing target protein using same |
KR102348638B1 (en) | 2019-11-21 | 2022-01-11 | (주)지플러스 생명과학 | Antibody produced by using afucosylated n.benthamiana and uses thereof |
CN112779307B (en) * | 2021-01-11 | 2022-04-19 | 苏州药明生物技术有限公司 | Method for two-stage regulation of CHO expression exogenous protein glycoform |
WO2022154762A1 (en) * | 2021-01-18 | 2022-07-21 | Turgut İlaçlari A.Ş. | Method of producing adalimumab |
WO2023021532A1 (en) * | 2021-08-20 | 2023-02-23 | Dr. Reddy’S Laboratories Limited | A process to produce a pharmaceutical composition |
WO2023244746A1 (en) | 2022-06-15 | 2023-12-21 | Abbvie Inc. | Risankizumab compositions |
Family Cites Families (523)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
USRE30985E (en) | 1978-01-01 | 1982-06-29 | Serum-free cell culture media | |
US5179017A (en) | 1980-02-25 | 1993-01-12 | The Trustees Of Columbia University In The City Of New York | Processes for inserting DNA into eucaryotic cells and for producing proteinaceous materials |
US4399216A (en) | 1980-02-25 | 1983-08-16 | The Trustees Of Columbia University | Processes for inserting DNA into eucaryotic cells and for producing proteinaceous materials |
US4634665A (en) | 1980-02-25 | 1987-01-06 | The Trustees Of Columbia University In The City Of New York | Processes for inserting DNA into eucaryotic cells and for producing proteinaceous materials |
DE3280462T2 (en) | 1981-09-08 | 1995-04-20 | Univ Rockefeller | Antibodies to a composition with mediator activity and its use in a pharmaceutical composition. |
US4510245A (en) | 1982-11-18 | 1985-04-09 | Chiron Corporation | Adenovirus promoter system |
US4560655A (en) | 1982-12-16 | 1985-12-24 | Immunex Corporation | Serum-free cell culture medium and process for making same |
US4657866A (en) | 1982-12-21 | 1987-04-14 | Sudhir Kumar | Serum-free, synthetic, completely chemically defined tissue culture media |
GB8308235D0 (en) | 1983-03-25 | 1983-05-05 | Celltech Ltd | Polypeptides |
DD266710A3 (en) | 1983-06-06 | 1989-04-12 | Ve Forschungszentrum Biotechnologie | Process for the biotechnical production of alkaline phosphatase |
US4767704A (en) | 1983-10-07 | 1988-08-30 | Columbia University In The City Of New York | Protein-free culture medium |
US4704366A (en) | 1984-06-22 | 1987-11-03 | Bio-Rad Laboratories, Inc. | Process for binding IgG to protein A |
US5672347A (en) | 1984-07-05 | 1997-09-30 | Genentech, Inc. | Tumor necrosis factor antagonists and their use |
DE3431140A1 (en) | 1984-08-24 | 1986-03-06 | Behringwerke Ag, 3550 Marburg | ENHANCER FOR EUKARYOTIC EXPRESSION SYSTEMS |
US4879231A (en) | 1984-10-30 | 1989-11-07 | Phillips Petroleum Company | Transformation of yeasts of the genus pichia |
IL73883A (en) | 1984-12-20 | 1990-12-23 | Yeda Res & Dev | Monoclonal antibodies against tnf-alpha,hybridomas producing them and method for the purification of tnf-alpha |
US5168062A (en) | 1985-01-30 | 1992-12-01 | University Of Iowa Research Foundation | Transfer vectors and microorganisms containing human cytomegalovirus immediate-early promoter-regulatory DNA sequence |
US5672502A (en) | 1985-06-28 | 1997-09-30 | Celltech Therapeutics Limited | Animal cell culture |
GB8516415D0 (en) | 1985-06-28 | 1985-07-31 | Celltech Ltd | Culture of animal cells |
DE3650150T2 (en) | 1985-08-16 | 1995-04-27 | Univ Rockefeller | Anabolic activity modulator and its uses. |
IT206190Z2 (en) | 1985-12-17 | 1987-07-13 | Iveco Fiat | IMPROVING THE BODYWORK OF A HEAVY VEHICLE |
US4968615A (en) | 1985-12-18 | 1990-11-06 | Ciba-Geigy Corporation | Deoxyribonucleic acid segment from a virus |
US4925796A (en) | 1986-03-07 | 1990-05-15 | Massachusetts Institute Of Technology | Method for enhancing glycoprotein stability |
US5681718A (en) | 1986-03-14 | 1997-10-28 | Celltech Limited | Methods for enhanced production of tissue plasminogen activator in cell culture using alkanoic acids or salts thereof |
US4927762A (en) | 1986-04-01 | 1990-05-22 | Cell Enterprises, Inc. | Cell culture medium with antioxidant |
GB8610600D0 (en) | 1986-04-30 | 1986-06-04 | Novo Industri As | Transformation of trichoderma |
DE3631229A1 (en) | 1986-09-13 | 1988-03-24 | Basf Ag | MONOCLONAL ANTIBODIES AGAINST HUMAN TUMORNESCROSE FACTOR (TNF) AND THEIR USE |
US4801687A (en) | 1986-10-27 | 1989-01-31 | Bioprobe International, Inc. | Monoclonal antibody purification process using protein A |
US5045468A (en) | 1986-12-12 | 1991-09-03 | Cell Enterprises, Inc. | Protein-free culture medium which promotes hybridoma growth |
US4877608A (en) | 1987-11-09 | 1989-10-31 | Rorer Pharmaceutical Corporation | Pharmaceutical plasma protein formulations in low ionic strength media |
US6238891B1 (en) | 1987-11-18 | 2001-05-29 | Cetus Oncology Corporation | Method of increasing product expression through solute stress |
US5118796A (en) | 1987-12-09 | 1992-06-02 | Centocor, Incorporated | Efficient large-scale purification of immunoglobulins and derivatives |
AU626572B2 (en) | 1988-07-18 | 1992-08-06 | Chiron Corporation | Monoclonal antibodies reactive with cachectin |
ATE135397T1 (en) | 1988-09-23 | 1996-03-15 | Cetus Oncology Corp | CELL CULTIVATION MEDIUM FOR INCREASED CELL GROWTH, TO INCREASE THE LONGEVITY AND EXPRESSION OF THE PRODUCTS |
US6048728A (en) | 1988-09-23 | 2000-04-11 | Chiron Corporation | Cell culture medium for enhanced cell growth, culture longevity, and product expression |
US5126250A (en) | 1988-09-28 | 1992-06-30 | Eli Lilly And Company | Method for the reduction of heterogeneity of monoclonal antibodies |
GB8823869D0 (en) | 1988-10-12 | 1988-11-16 | Medical Res Council | Production of antibodies |
EP0366043B1 (en) | 1988-10-24 | 1994-03-30 | Otsuka Pharmaceutical Co., Ltd. | Monoclonal antibody |
AU634186B2 (en) | 1988-11-11 | 1993-02-18 | Medical Research Council | Single domain ligands, receptors comprising said ligands, methods for their production, and use of said ligands and receptors |
ATE147634T1 (en) | 1988-12-19 | 1997-02-15 | American Cyanamid Co | PRODUCTS FOR TREATING ENDOTOXIN SHOCK IN A MAMMAL |
US5047335A (en) | 1988-12-21 | 1991-09-10 | The Regents Of The University Of Calif. | Process for controlling intracellular glycosylation of proteins |
US5530101A (en) | 1988-12-28 | 1996-06-25 | Protein Design Labs, Inc. | Humanized immunoglobulins |
DE4009603A1 (en) | 1989-03-30 | 1990-10-04 | Leybold Ag | Lock chamber for substrate |
US4933435A (en) | 1989-04-05 | 1990-06-12 | Bioprobe International | Antibody purification process |
US5169936A (en) | 1989-04-14 | 1992-12-08 | Biogen, Inc. | Protein purification on immobilized metal affinity resins effected by elution using a weak ligand |
EP0402226A1 (en) | 1989-06-06 | 1990-12-12 | Institut National De La Recherche Agronomique | Transformation vectors for yeast yarrowia |
US6451983B2 (en) | 1989-08-07 | 2002-09-17 | Peptech Limited | Tumor necrosis factor antibodies |
DK0486526T3 (en) | 1989-08-07 | 1996-06-24 | Peptide Technology Ltd | Binding ligands for tumor necrosis factor |
US5959087A (en) | 1989-08-07 | 1999-09-28 | Peptide Technology, Ltd. | Tumour necrosis factor binding ligands |
GB8921123D0 (en) | 1989-09-19 | 1989-11-08 | Millar Ann B | Treatment of ards |
GB8928874D0 (en) | 1989-12-21 | 1990-02-28 | Celltech Ltd | Humanised antibodies |
US5859205A (en) | 1989-12-21 | 1999-01-12 | Celltech Limited | Humanised antibodies |
US6673986B1 (en) | 1990-01-12 | 2004-01-06 | Abgenix, Inc. | Generation of xenogeneic antibodies |
US6075181A (en) | 1990-01-12 | 2000-06-13 | Abgenix, Inc. | Human antibodies derived from immunized xenomice |
US6150584A (en) | 1990-01-12 | 2000-11-21 | Abgenix, Inc. | Human antibodies derived from immunized xenomice |
US5945098A (en) | 1990-02-01 | 1999-08-31 | Baxter International Inc. | Stable intravenously-administrable immune globulin preparation |
AU646695B2 (en) | 1990-04-09 | 1994-03-03 | Immunex Corporation | Isolated viral protein cytokine antagonists |
DE4037604A1 (en) | 1990-04-25 | 1991-10-31 | Bayer Ag | Use of anti-TNF antibodies to treat ischaemia and its sequelae - esp. to increase survival rate after myocardial infarct and transplants |
US5378612A (en) | 1990-05-11 | 1995-01-03 | Juridical Foundation The Chemo-Sero-Therapeutic Research Institute | Culture medium for production of recombinant protein |
US5110913A (en) | 1990-05-25 | 1992-05-05 | Miles Inc. | Antibody purification method |
US5096816A (en) | 1990-06-05 | 1992-03-17 | Cetus Corporation | In vitro management of ammonia's effect on glycosylation of cell products through pH control |
GB9014932D0 (en) | 1990-07-05 | 1990-08-22 | Celltech Ltd | Recombinant dna product and method |
GB9015198D0 (en) | 1990-07-10 | 1990-08-29 | Brien Caroline J O | Binding substance |
US5770429A (en) | 1990-08-29 | 1998-06-23 | Genpharm International, Inc. | Transgenic non-human animals capable of producing heterologous antibodies |
US6300129B1 (en) | 1990-08-29 | 2001-10-09 | Genpharm International | Transgenic non-human animals for producing heterologous antibodies |
US5625126A (en) | 1990-08-29 | 1997-04-29 | Genpharm International, Inc. | Transgenic non-human animals for producing heterologous antibodies |
EP0546073B1 (en) | 1990-08-29 | 1997-09-10 | GenPharm International, Inc. | production and use of transgenic non-human animals capable of producing heterologous antibodies |
US5661016A (en) | 1990-08-29 | 1997-08-26 | Genpharm International Inc. | Transgenic non-human animals capable of producing heterologous antibodies of various isotypes |
US5633425A (en) | 1990-08-29 | 1997-05-27 | Genpharm International, Inc. | Transgenic non-human animals capable of producing heterologous antibodies |
US7084260B1 (en) | 1996-10-10 | 2006-08-01 | Genpharm International, Inc. | High affinity human antibodies and human antibodies against human antigens |
US5545806A (en) | 1990-08-29 | 1996-08-13 | Genpharm International, Inc. | Ransgenic non-human animals for producing heterologous antibodies |
US5789650A (en) | 1990-08-29 | 1998-08-04 | Genpharm International, Inc. | Transgenic non-human animals for producing heterologous antibodies |
US6255458B1 (en) | 1990-08-29 | 2001-07-03 | Genpharm International | High affinity human antibodies and human antibodies against digoxin |
US5122469A (en) | 1990-10-03 | 1992-06-16 | Genentech, Inc. | Method for culturing Chinese hamster ovary cells to improve production of recombinant proteins |
GB9022547D0 (en) | 1990-10-17 | 1990-11-28 | Wellcome Found | Purified immunoglobulin |
GB9022545D0 (en) | 1990-10-17 | 1990-11-28 | Wellcome Found | Culture medium |
GB9022543D0 (en) | 1990-10-17 | 1990-11-28 | Wellcome Found | Antibody production |
GB9109645D0 (en) | 1991-05-03 | 1991-06-26 | Celltech Ltd | Recombinant antibodies |
GB2279077B (en) | 1990-12-21 | 1995-06-14 | Celltech Ltd | Therapeutic compositions comprising recombinant antibodies specific for the TNFalpha |
US5994510A (en) | 1990-12-21 | 1999-11-30 | Celltech Therapeutics Limited | Recombinant antibodies specific for TNFα |
GB9028123D0 (en) | 1990-12-28 | 1991-02-13 | Erba Carlo Spa | Monoclonal antibodies against human tumor necrosis factor alpha |
EP0575545B1 (en) | 1991-03-15 | 2003-05-21 | Amgen Inc. | Pegylation of polypeptides |
US5698195A (en) | 1991-03-18 | 1997-12-16 | New York University Medical Center | Methods of treating rheumatoid arthritis using chimeric anti-TNF antibodies |
US5656272A (en) | 1991-03-18 | 1997-08-12 | New York University Medical Center | Methods of treating TNF-α-mediated Crohn's disease using chimeric anti-TNF antibodies |
US7192584B2 (en) | 1991-03-18 | 2007-03-20 | Centocor, Inc. | Methods of treating psoriasis with anti-TNF antibodies |
US20040120952A1 (en) | 2000-08-07 | 2004-06-24 | Centocor, Inc | Anti-TNF antibodies and peptides of human tumor necrosis factor |
US20070298040A1 (en) | 1991-03-18 | 2007-12-27 | Centocor, Inc. | Methods of treating seronegative arthropathy with anti-TNF antibodies |
US6277969B1 (en) | 1991-03-18 | 2001-08-21 | New York University | Anti-TNF antibodies and peptides of human tumor necrosis factor |
US20060246073A1 (en) | 1991-03-18 | 2006-11-02 | Knight David M | Anti-TNF antibodies and peptides of human tumor necrosis factor |
DE07012626T1 (en) | 1991-03-18 | 2010-01-21 | New York University | Monoclonal and chimeric antibodies to human tumor necrosis factor |
US5328985A (en) | 1991-07-12 | 1994-07-12 | The Regents Of The University Of California | Recombinant streptavidin-protein chimeras useful for conjugation of molecules in the immune system |
IE922287A1 (en) | 1991-07-15 | 1993-01-27 | Wellcome Found | Production of antibodies |
EP0605522B1 (en) | 1991-09-23 | 1999-06-23 | Medical Research Council | Methods for the production of humanized antibodies |
GB9122820D0 (en) | 1991-10-28 | 1991-12-11 | Wellcome Found | Stabilised antibodies |
WO1993011793A1 (en) | 1991-12-17 | 1993-06-24 | Schering Corporation | Use of the combination of anti-tumor necrosis factor plus interleukin-6 to treat septic shock |
EP0636026B1 (en) | 1992-04-02 | 2001-12-05 | Smithkline Beecham Corporation | Compounds useful for treating inflammatory diseases and inhibiting production of tumor necrosis factor |
AU675661B2 (en) | 1992-07-24 | 1997-02-13 | Abgenix, Inc. | Generation of xenogeneic antibodies |
DE69321909T2 (en) | 1992-08-28 | 1999-04-01 | Bayer Ag | Use of anti-TNF monoclonal antibodies for the treatment of bacterial meningitis |
US6270766B1 (en) | 1992-10-08 | 2001-08-07 | The Kennedy Institute Of Rheumatology | Anti-TNF antibodies and methotrexate in the treatment of arthritis and crohn's disease |
DE69312077T2 (en) | 1992-10-08 | 1997-10-30 | Kennedy Inst Of Rheumatology | TREATMENT OF AUTOIMMUNE AND INFLAMMATION DISEASES |
EP0671936A1 (en) | 1992-10-15 | 1995-09-20 | Dana-Farber Cancer Institute, Inc. | TREATMENT OF INSULIN RESISTANCE IN OBESITY LINKED TYPE II DIABETES USING ANTAGONISTS TO TNF-$g(a) FUNCTION |
JPH06292592A (en) | 1993-02-09 | 1994-10-21 | Snow Brand Milk Prod Co Ltd | Production of glycoprotein |
DE69427928T3 (en) | 1993-03-05 | 2012-05-10 | Bayer Healthcare Llc | Human monoclonal anti-TNF alpha antibody |
DE4307508A1 (en) | 1993-03-10 | 1994-09-15 | Knoll Ag | Use of anti-TNF antibodies as a medicine in the treatment of heart failure (heart muscle weakness) |
JPH08509612A (en) | 1993-04-26 | 1996-10-15 | ジェンファーム インターナショナル インコーポレイテッド | Transgenic non-human animal capable of producing heterologous antibody |
JPH08510642A (en) | 1993-05-12 | 1996-11-12 | ゾマ コーポレイション | Immunotoxin consisting of gelonin and antibody |
RU2139092C1 (en) | 1993-06-03 | 1999-10-10 | Терапьютик Антибодиз Инк. | Use of antibody fragment in therapy |
EP0724651B1 (en) | 1993-10-19 | 2008-08-20 | The Scripps Research Institute | Synthetic human neutralizing monoclonal antibodies to human immunodeficiency virus |
EP0659766A1 (en) | 1993-11-23 | 1995-06-28 | Schering-Plough | Human monoclonal antibodies against human cytokines and methods of making and using such antibodies |
NZ278607A (en) | 1994-02-07 | 1999-05-28 | Knoll Ag | Use of tnf antagonists for treating disorders involving elevated serum levels of il-6 wherein the serum levels are 500pg/ml or above |
EP0666312A1 (en) | 1994-02-08 | 1995-08-09 | Wolfgang A. Renner | Process for the improvement of mammalian cell growth |
US5429746A (en) | 1994-02-22 | 1995-07-04 | Smith Kline Beecham Corporation | Antibody purification |
EP0748338A4 (en) | 1994-03-04 | 2001-03-28 | Merck & Co Inc | In vitro antibody affinity maturation using alanine scanning mutagenesis |
US5856179A (en) | 1994-03-10 | 1999-01-05 | Genentech, Inc. | Polypeptide production in animal cell culture |
US5945311A (en) | 1994-06-03 | 1999-08-31 | GSF--Forschungszentrumfur Umweltund Gesundheit | Method for producing heterologous bi-specific antibodies |
JPH10503179A (en) | 1994-06-24 | 1998-03-24 | イミュネックス・コーポレーション | Controlled release polypeptide composition and method of treating inflammatory bowel disease |
ZA955642B (en) | 1994-07-07 | 1997-05-06 | Ortho Pharma Corp | Lyophilized imaging agent formulation |
US5561053A (en) | 1994-08-05 | 1996-10-01 | Genentech, Inc. | Method for selecting high-expressing host cells |
SE503424C2 (en) | 1994-11-14 | 1996-06-10 | Pharmacia Ab | Process for purification of recombinant coagulation factor VIII |
US5641870A (en) | 1995-04-20 | 1997-06-24 | Genentech, Inc. | Low pH hydrophobic interaction chromatography for antibody purification |
EP1978033A3 (en) | 1995-04-27 | 2008-12-24 | Amgen Fremont Inc. | Human antibodies derived from immunized xenomice |
CA2219486A1 (en) | 1995-04-28 | 1996-10-31 | Abgenix, Inc. | Human antibodies derived from immunized xenomice |
US5705364A (en) | 1995-06-06 | 1998-01-06 | Genentech, Inc. | Mammalian cell culture process |
US6656466B1 (en) | 1995-06-06 | 2003-12-02 | Genetech, Inc. | Human tumor necrosis factor—immunoglobulin(TNFR1-IgG1) chimera composition |
US5721121A (en) | 1995-06-06 | 1998-02-24 | Genentech, Inc. | Mammalian cell culture process for producing a tumor necrosis factor receptor immunoglobulin chimeric protein |
US6113898A (en) | 1995-06-07 | 2000-09-05 | Idec Pharmaceuticals Corporation | Human B7.1-specific primatized antibodies and transfectomas expressing said antibodies |
CN1151842C (en) | 1995-07-27 | 2004-06-02 | 基因技术股份有限公司 | Stable isotonic lyophilized protein formulation |
JP4306813B2 (en) | 1995-09-19 | 2009-08-05 | アスビオファーマ株式会社 | New method for culturing animal cells |
US5989830A (en) | 1995-10-16 | 1999-11-23 | Unilever Patent Holdings Bv | Bifunctional or bivalent antibody fragment analogue |
US6090382A (en) | 1996-02-09 | 2000-07-18 | Basf Aktiengesellschaft | Human antibodies that bind human TNFα |
AR005035A1 (en) | 1995-12-11 | 1999-04-07 | Merck Patent Ges Mit Beschränkter Haftung | PROCEDURE TO PREPARE RECOMBINANT PROTEINS IN E. COLI, BY FERMENTATION WITH GREAT CONCENTRATION OF CELLS. |
HU230048B1 (en) | 1996-02-09 | 2015-06-29 | Abbvie Biotechnology Ltd | Use of human tnf-alpha binding antibodies |
ES2180689T3 (en) | 1996-04-19 | 2003-02-16 | Nestle Sa | IMMORTALIZED LINE OF HUMAN COLON EPITHELIAL CELLS. |
TW491855B (en) | 1996-08-07 | 2002-06-21 | Csl Ltd | Purification of immunoglobulins |
EP0953041A4 (en) | 1996-08-30 | 2003-01-29 | Life Technologies Inc | Serum-free mammalian cell culture medium, and uses thereof |
US20040171152A1 (en) | 1996-10-10 | 2004-09-02 | Invitrogen Corporation | Animal cell culture media comprising non-animal or plant-derived nutrients |
PT1864999E (en) | 1996-11-27 | 2009-06-25 | Genentech Inc | Affinity purification of polypeptide on protein a matrix |
ES2301183T3 (en) | 1996-12-03 | 2008-06-16 | Amgen Fremont Inc. | COMPLETELY HUMAN ANTIBODY THAT JOINS THE EGFR RECEIVER. |
GB9625175D0 (en) | 1996-12-04 | 1997-01-22 | Medi Cult As | Serum-free cell culture media |
US5804420A (en) | 1997-04-18 | 1998-09-08 | Bayer Corporation | Preparation of recombinant Factor VIII in a protein free medium |
US6159468A (en) | 1997-04-28 | 2000-12-12 | Eli Lilly And Company | Activated protein C formulations |
US6235883B1 (en) | 1997-05-05 | 2001-05-22 | Abgenix, Inc. | Human monoclonal antibodies to epidermal growth factor receptor |
JP3919235B2 (en) | 1997-06-13 | 2007-05-23 | ジェネンテク,インコーポレイテッド | Antibody preparation |
US6171586B1 (en) | 1997-06-13 | 2001-01-09 | Genentech, Inc. | Antibody formulation |
US6475725B1 (en) | 1997-06-20 | 2002-11-05 | Baxter Aktiengesellschaft | Recombinant cell clones having increased stability and methods of making and using the same |
US20040191256A1 (en) | 1997-06-24 | 2004-09-30 | Genentech, Inc. | Methods and compositions for galactosylated glycoproteins |
PT994903E (en) | 1997-06-24 | 2005-10-31 | Genentech Inc | METHODS AND COMPOSITIONS FOR GALACTOSILED GLICOPROTEINS |
US20020045207A1 (en) | 1997-10-31 | 2002-04-18 | Lynne A. Krummen | Glycoprotein production process |
WO1999022764A1 (en) | 1997-10-31 | 1999-05-14 | Genentech, Inc. | Methods and compositions comprising glycoprotein glycoforms |
US20040136986A1 (en) | 1997-10-31 | 2004-07-15 | Genentech, Inc. | Methods and compositions comprising glycoprotein glycoforms |
DE59813187D1 (en) | 1997-12-03 | 2005-12-15 | Roche Diagnostics Gmbh | PROCESS FOR THE PREPARATION OF POLYPEPTIDES WITH APPROPRIATE GLYCOSILATION |
WO1999032605A1 (en) | 1997-12-19 | 1999-07-01 | Novo Nordisk A/S | Method for producing heterologous proteins in eukaryotic cells on an industrial scale using nucleotide-manipulating agents |
WO1999054342A1 (en) | 1998-04-20 | 1999-10-28 | Pablo Umana | Glycosylation engineering of antibodies for improving antibody-dependent cellular cytotoxicity |
WO1999057246A1 (en) | 1998-05-01 | 1999-11-11 | Life Technologies, Inc. | Animal cell culture media comprising non-animal or plant-derived nutrients |
DE69936946T2 (en) | 1998-05-06 | 2008-05-15 | Genentech, Inc., South San Francisco | Purification of antibodies by ion exchange chromatography |
SI1308456T1 (en) | 1998-05-06 | 2008-02-29 | Genentech Inc | Antibody purification by ion exchange chromatography |
DE19821933C1 (en) | 1998-05-15 | 1999-11-11 | Disetronic Licensing Ag | Device for administering an injectable product |
DE19822031C2 (en) | 1998-05-15 | 2000-03-23 | Disetronic Licensing Ag | Auto injection device |
US6528286B1 (en) | 1998-05-29 | 2003-03-04 | Genentech, Inc. | Mammalian cell culture process for producing glycoproteins |
US6406909B1 (en) | 1998-07-10 | 2002-06-18 | Chugai Seiyaku Kabushiki Kaisha | Serum-free medium for culturing animal cells |
US6914128B1 (en) | 1999-03-25 | 2005-07-05 | Abbott Gmbh & Co. Kg | Human antibodies that bind human IL-12 and methods for producing |
US7883704B2 (en) | 1999-03-25 | 2011-02-08 | Abbott Gmbh & Co. Kg | Methods for inhibiting the activity of the P40 subunit of human IL-12 |
WO2000058362A1 (en) | 1999-03-26 | 2000-10-05 | Human Genome Sciences, Inc. | Neutrokine-alpha binding proteins and methods based thereon |
ES2568899T3 (en) | 1999-04-09 | 2016-05-05 | Kyowa Hakko Kirin Co., Ltd. | Procedure to control the activity of an immunofunctional molecule |
US7297680B2 (en) | 1999-04-15 | 2007-11-20 | Crucell Holland B.V. | Compositions of erythropoietin isoforms comprising Lewis-X structures and high sialic acid content |
WO2000065070A2 (en) | 1999-04-26 | 2000-11-02 | Genentech, Inc. | Cell culture process for glycoproteins |
AU4314900A (en) | 1999-04-28 | 2000-11-17 | Yamanouchi Pharmaceutical Co., Ltd. | Parenteral medicinal composition containing humanized monoclonal antibody fragment and method for stabilizing the same |
AT409379B (en) | 1999-06-02 | 2002-07-25 | Baxter Ag | MEDIUM FOR PROTEIN- AND SERUM-FREE CELL CULTURE |
ATE327033T1 (en) | 1999-07-30 | 2006-06-15 | Genentech Inc | CHARGED FILTRATION MEMBRANES AND USES THEREOF |
US6586206B1 (en) | 1999-09-27 | 2003-07-01 | Genentech, Inc. | Methods for making recombinant proteins using apoptosis inhibitors |
AR026743A1 (en) | 1999-12-09 | 2003-02-26 | Pharmacia Ab | PRODUCTION OF PEPTIDES |
KR20010056451A (en) | 1999-12-15 | 2001-07-04 | 윤재승 | Arginine-enriched medium used for mass-producing recombinant protein in animal cell culture |
US20030124119A1 (en) | 1999-12-28 | 2003-07-03 | Tadao Yamazaki | Stable antibody compositions and injection preparations |
WO2001059089A2 (en) | 2000-02-08 | 2001-08-16 | Genentech, Inc. | Improved galactosylation of recombinant glycoproteins |
TWI373343B (en) | 2000-02-10 | 2012-10-01 | Abbott Gmbh & Co Kg | Antibodies that bind human interleukin-18 and methods of making and using |
GB0003231D0 (en) | 2000-02-11 | 2000-04-05 | Medi Cult As | Cell culture media |
JP2001218840A (en) | 2000-02-14 | 2001-08-14 | Kanegafuchi Chem Ind Co Ltd | Adsorbent for transforming growth factor. beta, adsorbing/removing method, and adsorber |
AU2001246846A1 (en) | 2000-04-06 | 2001-10-23 | Chugai Seiyaku Kabushiki Kaisha | Immunoassay of anti-hm1.24 antibody |
AU2001266557A1 (en) | 2000-04-12 | 2001-10-23 | Human Genome Sciences, Inc. | Albumin fusion proteins |
DE60139720D1 (en) | 2000-06-28 | 2009-10-08 | Glycofi Inc | Process for the preparation of modified glycoproteins |
US7598055B2 (en) | 2000-06-28 | 2009-10-06 | Glycofi, Inc. | N-acetylglucosaminyltransferase III expression in lower eukaryotes |
US7449308B2 (en) | 2000-06-28 | 2008-11-11 | Glycofi, Inc. | Combinatorial DNA library for producing modified N-glycans in lower eukaryotes |
WO2002002793A1 (en) | 2000-07-05 | 2002-01-10 | Japan As Represented By Secretary Of Osaka University | Process for producing glycoprotein |
EP1336410A4 (en) | 2000-08-04 | 2005-10-12 | Chugai Pharmaceutical Co Ltd | Protein injection preparations |
UA81743C2 (en) | 2000-08-07 | 2008-02-11 | Центокор, Инк. | HUMAN MONOCLONAL ANTIBODY WHICH SPECIFICALLY BINDS TUMOR NECROSIS FACTOR ALFA (TNFα), PHARMACEUTICAL MIXTURE CONTAINING THEREOF, AND METHOD FOR TREATING ARTHRITIS |
US7288390B2 (en) | 2000-08-07 | 2007-10-30 | Centocor, Inc. | Anti-dual integrin antibodies, compositions, methods and uses |
US20050249735A1 (en) | 2000-08-07 | 2005-11-10 | Centocor, Inc. | Methods of treating ankylosing spondylitis using anti-TNF antibodies and peptides of human tumor necrosis factor |
US20060018907A1 (en) | 2000-08-07 | 2006-01-26 | Centocor, Inc. | Anti-TNF antibodies and peptides of human tumor necrosis factor |
WO2002016590A2 (en) | 2000-08-21 | 2002-02-28 | Clonex Development, Inc. | Methods and compositions for increasing protein yield from a cell culture |
DE60141908D1 (en) | 2000-10-02 | 2010-06-02 | Novo Nordisk Healthcare Ag | GLYCO FORMS OF FACTOR VII |
US20090151023A1 (en) | 2000-11-13 | 2009-06-11 | Viktor Kuvshinov | Transformation system for Camelina sativa |
EP1360314B1 (en) | 2001-02-15 | 2009-01-14 | Centocor, Inc. | Chemically defined medium for cultured mammalian cells |
US20030096414A1 (en) | 2001-03-27 | 2003-05-22 | Invitrogen Corporation | Culture medium for cell growth and transfection |
WO2002076578A1 (en) | 2001-03-27 | 2002-10-03 | Smithkline Beecham Corporation | Control of glycoforms in igg |
JP2004537290A (en) | 2001-05-24 | 2004-12-16 | ヒューマン ジノーム サイエンシーズ, インコーポレイテッド | Antibodies to tumor necrosis factor δ (APRIL) |
US20030012786A1 (en) | 2001-05-25 | 2003-01-16 | Teoh Leah S. | Use of anti-TNF antibodies as drugs in treating septic disorders of anemic patients |
CA2817619A1 (en) | 2001-06-08 | 2002-12-08 | Abbott Laboratories (Bermuda) Ltd. | Methods of administering anti-tnf.alpha. antibodies |
EP1404813A4 (en) | 2001-06-13 | 2004-11-24 | Genentech Inc | Methods of culturing animal cells and polypeptide production in animal cells |
PT2087908T (en) | 2001-06-26 | 2018-07-16 | Amgen Inc | Antibodies to opgl |
HUP0700103A3 (en) | 2001-08-03 | 2012-09-28 | Glycart Biotechnology Ag | Antibody glycosylation variants having increased antibody-dependent cellular cytotoxicity |
GB0119520D0 (en) | 2001-08-10 | 2001-10-03 | Owen Mumford Ltd | Improvements relating to injection devices |
CN1309825C (en) | 2001-10-02 | 2007-04-11 | 诺和诺德医疗保健公司 | Method for production of recombinant proteins in eukaryote cells |
US7125843B2 (en) | 2001-10-19 | 2006-10-24 | Neose Technologies, Inc. | Glycoconjugates including more than one peptide |
CN100423777C (en) | 2001-10-25 | 2008-10-08 | 杰南技术公司 | Glycoprotein compositions |
DE60231055D1 (en) | 2001-11-12 | 2009-03-19 | Novo Nordisk As | CLEANING OF PEPTIDES USING METAL-ION-AFFINITY CHROMATOGRAPHY |
EP1453948A2 (en) | 2001-11-28 | 2004-09-08 | Sandoz Gmbh | Cell culture process |
MXPA04005191A (en) | 2001-11-28 | 2005-04-29 | Sandoz Ag | Method for producing a recombinant polypeptide. |
US7473680B2 (en) | 2001-11-28 | 2009-01-06 | Neose Technologies, Inc. | Remodeling and glycoconjugation of peptides |
WO2003046162A2 (en) | 2001-11-28 | 2003-06-05 | Polymun Scientific Immunbiologische Forschung Gmbh | Process for the production of polypeptides in mammalian cell cultures |
AU2002359816B2 (en) | 2001-12-21 | 2006-07-13 | Immunex Corporation | Methods for purifying protein |
JP4460302B2 (en) | 2002-02-05 | 2010-05-12 | ジェネンテック インコーポレイテッド | Protein purification method |
US20030161828A1 (en) | 2002-02-19 | 2003-08-28 | Abbott Gmbh & Co. Kg | Use of TNF antagonists as drugs for the treatment of patients with an inflammatory reaction and without suffering from total organ failure |
CA2417689C (en) | 2002-03-05 | 2006-05-09 | F. Hoffmann-La Roche Ag | Improved methods for growing mammalian cells in vitro |
JP2005521401A (en) | 2002-03-27 | 2005-07-21 | イミュネックス・コーポレーション | Methods for increasing polypeptide production |
US20030190710A1 (en) | 2002-03-28 | 2003-10-09 | Devries Ruth L. | Control of glycoforms in IgG |
WO2003085118A1 (en) | 2002-04-09 | 2003-10-16 | Kyowa Hakko Kogyo Co., Ltd. | Process for producing antibody composition |
US20060234226A1 (en) | 2002-04-26 | 2006-10-19 | Fahner Robert L | Non-affinity purification of proteins |
US20040009172A1 (en) | 2002-04-26 | 2004-01-15 | Steven Fischkoff | Use of anti-TNFalpha antibodies and another drug |
US20030206898A1 (en) | 2002-04-26 | 2003-11-06 | Steven Fischkoff | Use of anti-TNFalpha antibodies and another drug |
US20040029229A1 (en) | 2002-05-20 | 2004-02-12 | Reeves Philip J. | High level protein expression system |
CA2492267C (en) | 2002-07-15 | 2013-09-10 | Immunex Corporation | Methods and media for controlling sialylation of proteins produced by mammalian cells |
US20090280065A1 (en) | 2006-04-10 | 2009-11-12 | Willian Mary K | Uses and Compositions for Treatment of Psoriasis |
US20040126372A1 (en) | 2002-07-19 | 2004-07-01 | Abbott Biotechnology Ltd. | Treatment of TNFalpha related disorders |
US20040033228A1 (en) | 2002-08-16 | 2004-02-19 | Hans-Juergen Krause | Formulation of human antibodies for treating TNF-alpha associated disorders |
US7067279B1 (en) | 2002-08-23 | 2006-06-27 | Immunex Corporation | Cell culture performance with betaine |
US6924124B1 (en) | 2002-08-23 | 2005-08-02 | Immunex Corporation | Feeding strategies for cell culture |
US6974681B1 (en) | 2002-08-23 | 2005-12-13 | Immunex Corporation | Cell culture performance with vanadate |
DK1578774T3 (en) | 2002-09-18 | 2014-02-03 | Danisco Us Inc | PURIFICATION OF IMMUNOGLOBULINES |
US6890736B1 (en) | 2002-09-20 | 2005-05-10 | Immunex Corporation | Methods for producing proteins in cultured cells |
MY150740A (en) | 2002-10-24 | 2014-02-28 | Abbvie Biotechnology Ltd | Low dose methods for treating disorders in which tnf? activity is detrimental |
US7674885B2 (en) | 2002-11-01 | 2010-03-09 | Bayer Healthcare Llc | Process for concentration of macromolecules |
US20040086532A1 (en) | 2002-11-05 | 2004-05-06 | Allergan, Inc., | Botulinum toxin formulations for oral administration |
US20040101939A1 (en) | 2002-11-22 | 2004-05-27 | Santora Ling C. | Method for reducing or preventing modification of a polypeptide in solution |
US20040162414A1 (en) | 2002-11-22 | 2004-08-19 | Santora Ling C. | Method for reducing or preventing modification of a polypeptide in solution |
WO2004055164A2 (en) | 2002-12-13 | 2004-07-01 | Abgenix, Inc. | System and method for stabilizing antibodies with histidine |
AU2003303394B2 (en) | 2002-12-23 | 2009-02-19 | Bristol-Myers Squibb Company | Product quality enhancement in mammalian cell culture processes for protein production |
CN101044239B (en) | 2002-12-23 | 2010-12-08 | 布里斯托尔-迈尔斯斯奎布公司 | Mammalian cell culture processes for protein production |
PT1601697E (en) | 2003-02-28 | 2007-09-04 | Lonza Biologics Plc | Antibody purification by protein a and ion exchange chromatography |
JP2006525015A (en) | 2003-05-01 | 2006-11-09 | ディーエスエム アイピー アセッツ ビー.ブイ. | Method for producing biological material by perfusion culture of suspended animal cells |
ATE472597T2 (en) | 2003-05-15 | 2010-07-15 | Wyeth Llc | CONTROLLED GLUCOSE SUPPLY FOR ANIMAL CELL CULTURE |
GB2404665B (en) | 2003-08-08 | 2005-07-06 | Cambridge Antibody Tech | Cell culture |
WO2005035748A1 (en) | 2003-10-10 | 2005-04-21 | Novo Nordisk Health Care Ag | Method for large-scale production of a polypeptide in eukaryote cells and a culture vessel suitable therefor |
FR2861080B1 (en) | 2003-10-20 | 2006-02-17 | Lab Francais Du Fractionnement | ANTIBODIES HAVING AN OPTIMIZED FUCOSE AND GALACTOSE RATE |
EP1687328B1 (en) | 2003-10-24 | 2010-03-31 | Amgen, Inc. | Process for purifying proteins in a hydrophobic interaction chromatography flow-through fraction |
US20050100965A1 (en) | 2003-11-12 | 2005-05-12 | Tariq Ghayur | IL-18 binding proteins |
DE10355251A1 (en) | 2003-11-26 | 2005-06-23 | Merck Patent Gmbh | Water-based pharmaceutical preparation for treatment of tumors has active ingredient effective against receptor of endothelial growth factor receptor |
CA2894300A1 (en) | 2003-12-08 | 2005-06-23 | The Government Of The United States Of America, As Represented By The Secreatary, Department Of Health And Human Services | Monoclonal antibodies that bind or neutralize dengue virus |
AU2004308494B2 (en) | 2003-12-23 | 2010-03-18 | Genentech, Inc. | Novel anti-IL 13 antibodies and uses thereof |
WO2005063813A2 (en) | 2003-12-23 | 2005-07-14 | Applied Research Systems Ars Holding N.V. | Process for the production of tumor necrosis factor-binding proteins |
WO2005077130A2 (en) | 2004-02-11 | 2005-08-25 | Tanox, Inc. | A method for the removal of aggregate proteins from recombinant samples using ion exchange chromatography |
RU2390353C2 (en) | 2004-02-12 | 2010-05-27 | Мерк Патент Гмбх | High-concentration liquid anti-egfr antibody compositions |
WO2005082483A1 (en) | 2004-02-27 | 2005-09-09 | Ge Healthcare Bio-Sciences Ab | A process for the purification of antibodies |
CN1234725C (en) | 2004-04-07 | 2006-01-04 | 陈志南 | High performance quick purifying method for preparing piecewise antibody |
TWI556829B (en) | 2004-04-09 | 2016-11-11 | 艾伯維生物技術有限責任公司 | Multiple-variable dose regimen for treating tnfα-related disorders |
WO2005100584A2 (en) | 2004-04-15 | 2005-10-27 | Glycofi, Inc. | Production of galactosylated glycoproteins in lower eukaryotes |
WO2005111627A2 (en) | 2004-04-15 | 2005-11-24 | Massachusetts Institute Of Technology | Methods and products related to the improved analysis of carbohydrates |
US20060127950A1 (en) | 2004-04-15 | 2006-06-15 | Massachusetts Institute Of Technology | Methods and products related to the improved analysis of carbohydrates |
BRPI0510295A (en) | 2004-05-04 | 2007-11-06 | Novo Nordisk Healthcare Ag | preparation of a glycoprotein, and method for producing the same |
JP2008515772A (en) | 2004-07-21 | 2008-05-15 | グライコフィ, インコーポレイテッド | Immunoglobulin predominantly containing Gal2GlcNAc2Man3GlcNAc2 glycoform |
US20060223147A1 (en) | 2004-08-05 | 2006-10-05 | Kyowa Hakko Kogyo Co., Ltd., | Process for producing glycoprotein composition |
TWI384069B (en) | 2004-08-27 | 2013-02-01 | Pfizer Ireland Pharmaceuticals | Production of polypeptides |
PT2550971T (en) | 2004-09-30 | 2017-10-02 | Bayer Healthcare Llc | Devices and methods for integrated continuous manufacturing of biological molecules |
US20060083741A1 (en) | 2004-10-08 | 2006-04-20 | Hoffman Rebecca S | Treatment of respiratory syncytial virus (RSV) infection |
US20060194301A1 (en) | 2004-10-09 | 2006-08-31 | Doctor Bhupendra P | Large-scale production of human serum butyrylcholinesterase as a bioscavenger |
US7029982B1 (en) | 2004-10-21 | 2006-04-18 | Sharp Laboratories Of America, Inc. | Method of affecting RRAM characteristics by doping PCMO thin films |
WO2006043895A1 (en) | 2004-10-21 | 2006-04-27 | Ge Healthcare Bio-Sciences Ab | A method of antibody purification |
US20060094104A1 (en) | 2004-10-29 | 2006-05-04 | Leopold Grillberger | Animal protein-free media for cultivation of cells |
US20080160577A1 (en) | 2005-02-04 | 2008-07-03 | Glaxo Group Limited | Optimization of Heterologous Polypeptide Expression |
JP5523674B2 (en) | 2005-02-11 | 2014-06-18 | ノボ ノルディスク ヘルス ケア アクチェンゲゼルシャフト | Production of polypeptides in serum-free cell culture media containing plant protein hydrolysates |
MX2007011129A (en) | 2005-03-11 | 2007-11-06 | Wyeth Corp | A method of weak partitioning chromatography. |
WO2006107990A2 (en) | 2005-04-05 | 2006-10-12 | The Johns Hopkins University | Improving protein n-glycosylation of eukaryotic cells using dolichol-linked oligosaccharide synthesis pathway, other n-gylosylation-increasing methods, and engineered hosts expressing products with increased n-glycosylation |
NZ562949A (en) | 2005-04-11 | 2009-05-31 | Medarex Inc | Protein purification using HCIC and ion exchange chromatography |
US9963510B2 (en) | 2005-04-15 | 2018-05-08 | Macrogenics, Inc. | Covalent diabodies and uses thereof |
US20090203055A1 (en) | 2005-04-18 | 2009-08-13 | Massachusetts Institute Of Technology | Compositions and methods for RNA interference with sialidase expression and uses thereof |
WO2006113666A2 (en) | 2005-04-19 | 2006-10-26 | Massachusetts Institute Of Technology | Amphiphilic polymers and methods of use thereof |
NZ591701A (en) | 2005-05-16 | 2012-11-30 | Abbott Biotech Ltd | Use of tnf inhibitor for treatment of erosive polyarthritis |
EP1891207A2 (en) | 2005-06-03 | 2008-02-27 | Biovitrum AB | Process for cultivating animal cells comprising the feeding of plant-derived peptones |
CA2608818A1 (en) | 2005-06-03 | 2006-12-14 | Genentech, Inc. | Method of producing antibodies with modified fucosylation level |
CA2614046C (en) | 2005-06-30 | 2018-05-15 | Centocor, Inc. | Methods of controlling properties of therapeutic proteins, fc-containing therapeutic proteins of the g2s2 alpha-(2,3)-sialylated glycoform, and uses thereof |
EP1913149A4 (en) | 2005-07-26 | 2009-08-05 | Sangamo Biosciences Inc | Targeted integration and expression of exogenous nucleic acid sequences |
US20070041905A1 (en) | 2005-08-19 | 2007-02-22 | Hoffman Rebecca S | Method of treating depression using a TNF-alpha antibody |
US7612181B2 (en) | 2005-08-19 | 2009-11-03 | Abbott Laboratories | Dual variable domain immunoglobulin and uses thereof |
AU2006283560B2 (en) | 2005-08-19 | 2011-12-08 | Centocor, Inc. | Proteolysis resistant antibody preparations |
EA015901B1 (en) | 2005-08-26 | 2011-12-30 | Арес Трейдинг С.А. | Process for the preparation of glycosylated interferon beta |
EP1942935A4 (en) | 2005-09-02 | 2009-12-23 | Glycofi Inc | Immunoglobulins comprising predominantly a glcnacman3glcnac2 glycoform |
AR058140A1 (en) | 2005-10-24 | 2008-01-23 | Wyeth Corp | PROTEIN PRODUCTION METHOD USING ANTI-SENESCENCE COMPOUNDS |
RU2438704C2 (en) | 2005-11-01 | 2012-01-10 | Эбботт Байотекнолоджи Лтд. | Methods and compositions for diagnostics of ankylosing spondylitis with application of bio-markers |
WO2008057634A2 (en) | 2006-10-26 | 2008-05-15 | The Rockefeller University | Polypeptides with enhanced anti-inflammatory and decreased cytotoxic properties and relating methods |
US20080206246A1 (en) | 2006-04-05 | 2008-08-28 | Ravetch Jeffrey V | Polypeptides with enhanced anti-inflammatory and decreased cytotoxic properties and relating methods |
US8470318B2 (en) | 2005-11-07 | 2013-06-25 | The Rockefeller University | Polypeptides with enhanced anti-inflammatory and decreased cytotoxic properties and relating methods |
EP2455100A3 (en) | 2005-11-07 | 2012-11-07 | The Rockefeller University | Reagents, methods and systems for selecting a cytotoxic antibody or variant thereof |
EP3026109B1 (en) * | 2005-12-08 | 2022-03-09 | Amgen Inc. | Improved production of glycoproteins using manganese |
PL2522717T3 (en) | 2006-01-04 | 2014-08-29 | Baxalta Inc | Oligopeptide-free cell culture media |
US20070190057A1 (en) | 2006-01-23 | 2007-08-16 | Jian Wu | Methods for modulating mannose content of recombinant proteins |
US20070202051A1 (en) | 2006-02-10 | 2007-08-30 | Pari Gmbh | Aerosols for sinunasal drug delivery |
JP6084761B2 (en) | 2006-04-05 | 2017-02-22 | ザ ロックフェラー ユニバーシティー | Polypeptides with enhanced anti-inflammatory properties and reduced cytotoxic properties and related methods |
TWI392684B (en) | 2006-04-05 | 2013-04-11 | Abbott Biotech Ltd | Antibody purification |
EP2012586A4 (en) | 2006-04-10 | 2010-08-18 | Abbott Biotech Ltd | Uses and compositions for treatment of ankylosing spondylitis |
US20080118496A1 (en) | 2006-04-10 | 2008-05-22 | Medich John R | Uses and compositions for treatment of juvenile rheumatoid arthritis |
WO2008063213A2 (en) | 2006-04-10 | 2008-05-29 | Abbott Biotechnology Ltd. | Uses and compositions for treatment of psoriatic arthritis |
US9605064B2 (en) | 2006-04-10 | 2017-03-28 | Abbvie Biotechnology Ltd | Methods and compositions for treatment of skin disorders |
US9399061B2 (en) | 2006-04-10 | 2016-07-26 | Abbvie Biotechnology Ltd | Methods for determining efficacy of TNF-α inhibitors for treatment of rheumatoid arthritis |
US20090317399A1 (en) | 2006-04-10 | 2009-12-24 | Pollack Paul F | Uses and compositions for treatment of CROHN'S disease |
US7846724B2 (en) | 2006-04-11 | 2010-12-07 | Hoffmann-La Roche Inc. | Method for selecting CHO cell for production of glycosylated antibodies |
US20080131374A1 (en) | 2006-04-19 | 2008-06-05 | Medich John R | Uses and compositions for treatment of rheumatoid arthritis |
MX2008014744A (en) | 2006-05-19 | 2009-02-10 | Glycofi Inc | Erythropoietin compositions. |
US20080311043A1 (en) | 2006-06-08 | 2008-12-18 | Hoffman Rebecca S | Uses and compositions for treatment of psoriatic arthritis |
US20100021451A1 (en) | 2006-06-08 | 2010-01-28 | Wong Robert L | Uses and compositions for treatment of ankylosing spondylitis |
US20100234577A1 (en) | 2006-06-14 | 2010-09-16 | Smithkline Beecham Corporation | Methods for purifying antibodies using ceramic hydroxyapatite |
TWI527603B (en) | 2006-06-30 | 2016-04-01 | 艾伯維生物技術有限責任公司 | Automatic injection device |
EP3255141B1 (en) | 2006-07-13 | 2021-12-01 | Wyeth LLC | Production of antibodies with improved glycosylation pattern |
WO2008028974A1 (en) | 2006-09-08 | 2008-03-13 | Novo Nordisk A/S | Methods of optimizing chromatographic separation of polypeptides |
BRPI0716997B8 (en) | 2006-09-10 | 2021-05-25 | Glycotope Gmbh | protein or composition of protein molecules, methods for producing and using it |
US8911964B2 (en) | 2006-09-13 | 2014-12-16 | Abbvie Inc. | Fed-batch method of making human anti-TNF-alpha antibody |
EP2500416A1 (en) | 2006-09-13 | 2012-09-19 | Abbott Laboratories | Cell culture improvements |
MY161866A (en) | 2006-09-13 | 2017-05-15 | Abbvie Inc | Cell culture improvements |
US10982250B2 (en) | 2006-09-18 | 2021-04-20 | Genentech, Inc. | Methods of protein production |
JP5631591B2 (en) | 2006-10-06 | 2014-11-26 | アムジエン・インコーポレーテツド | Stable antibody formulation |
MX2009004351A (en) | 2006-10-27 | 2009-05-12 | Abbott Biotech Ltd | Crystalline anti-htnfalpha antibodies. |
US8192951B2 (en) | 2006-11-03 | 2012-06-05 | Wyeth Llc | Glycolysis-inhibiting substances in cell culture |
JP5065391B2 (en) | 2006-12-06 | 2012-10-31 | 日本ケミカルリサーチ株式会社 | Method for producing human erythropoietin |
EP2099914A1 (en) | 2006-12-22 | 2009-09-16 | F. Hoffmann-Roche AG | Selection method |
US20080226635A1 (en) | 2006-12-22 | 2008-09-18 | Hans Koll | Antibodies against insulin-like growth factor I receptor and uses thereof |
RU2466189C2 (en) * | 2006-12-28 | 2012-11-10 | Сентокор Орто Байотек Инк. | POLYPEPTIDE EXPRESSION VECTOR WITH SYALIDASE ACTIVITY, METHOD FOR PROVIDING SYALIDASE ACTIVITY IN CELL CULTURE AND METHOD FOR CONTROLLING PROPERTIES OF Fc-CONTAINING MOLECULES EXPRESSED IN CELL LINE |
US7691980B2 (en) | 2007-01-09 | 2010-04-06 | Bio-Rad Laboratories, Inc. | Enhanced capacity and purification of antibodies by mixed mode chromatography in the presence of aqueous-soluble nonionic organic polymers |
US8168185B2 (en) | 2007-01-17 | 2012-05-01 | Merck Serono Sa | Process for the purification of anti CD-25 antibodies |
DK2115126T3 (en) | 2007-03-02 | 2015-05-04 | Wyeth Llc | Use of copper and glutamate in cell culture for the preparation of polypeptides |
MX2009010361A (en) | 2007-03-29 | 2009-10-16 | Abbott Lab | Crystalline anti-human il-12 antibodies. |
AU2008232902B2 (en) | 2007-03-30 | 2013-10-03 | Medlmmune, Llc | Antibody formulation |
CN104480140B (en) | 2007-04-03 | 2019-12-31 | 奥克西雷恩英国有限公司 | Glycosylation of molecules |
DK2135091T3 (en) | 2007-04-16 | 2011-09-19 | Momenta Pharmaceuticals Inc | MS methods for evaluating glycans |
WO2008128216A1 (en) | 2007-04-16 | 2008-10-23 | Momenta Pharmaceuticals, Inc. | Methods for labeling glycans |
EP2135081B1 (en) | 2007-04-16 | 2012-12-05 | Momenta Pharmaceuticals, Inc. | Methods related to cell surface glycosylation |
CN101681396A (en) | 2007-04-16 | 2010-03-24 | 动量制药公司 | Glucoprotein product that limits and associated method |
WO2008128222A1 (en) | 2007-04-16 | 2008-10-23 | Momenta Pharmaceuticals, Inc. | Analysis of phosphorylated glycans, glycopeptides or glycoproteins by imac |
EP2135089B1 (en) | 2007-04-16 | 2015-09-02 | Momenta Pharmaceuticals, Inc. | Comparative analysis of protein conformations by using 2d noesy nmr spectra |
WO2008128225A1 (en) | 2007-04-16 | 2008-10-23 | Momenta Pharmaceuticals, Inc. | Multi-dimensional chromatographic methods for separating n-glycans |
EP2135088A1 (en) | 2007-04-16 | 2009-12-23 | Momenta Pharmaceuticals, Inc. | Characterization of n-glycan mixtures by nuclear magnetic resonance |
WO2008128230A1 (en) | 2007-04-16 | 2008-10-23 | Momenta Pharmaceuticals, Inc. | Reference glycoprotein products and related methods |
US20100151499A1 (en) | 2007-04-16 | 2010-06-17 | Momenta Pharmaceuticals, Inc. | Proteolytic release of glycans |
JP5552421B2 (en) | 2007-04-16 | 2014-07-16 | モメンタ ファーマシューティカルズ インコーポレイテッド | Characterization of N-glycans using exoglycosidases |
US20110213137A1 (en) | 2007-04-16 | 2011-09-01 | Momenta Pharmaceuticals, Inc. | Isotopically-labeled glycans |
TW200902708A (en) | 2007-04-23 | 2009-01-16 | Wyeth Corp | Methods of protein production using anti-senescence compounds |
WO2008135498A2 (en) | 2007-05-04 | 2008-11-13 | Novo Nordisk A/S | Prevention of protein degradation in mammalian cell cultures |
EP1988101A1 (en) | 2007-05-04 | 2008-11-05 | Novo Nordisk A/S | Improvement of factor VIII polypeptide titers in cell cultures |
CA2687082C (en) | 2007-05-11 | 2014-01-14 | Amgen Inc. | Improved feed media |
EP2165194A4 (en) | 2007-05-31 | 2010-09-08 | Abbott Lab | BIOMARKERS PREDICTIVE OF THE RESPONSIVENESS TO TNF-alpha INHIBITORS IN AUTOIMMUNE DISORDERS |
EP2152318A4 (en) | 2007-06-01 | 2011-12-07 | Abbott Biotech Ltd | Uses and compositions for treatment of psoriasis and crohn's disease |
US20100221823A1 (en) | 2007-06-11 | 2010-09-02 | Amgen Inc. | Method for culturing mammalian cells to improve recombinant protein production |
EP2171451A4 (en) | 2007-06-11 | 2011-12-07 | Abbott Biotech Ltd | Methods for treating juvenile idiopathic arthritis |
KR101643514B1 (en) | 2007-07-09 | 2016-07-27 | 제넨테크, 인크. | Prevention of disulfide bond reduction during recombinant production of polypeptides |
WO2009011782A2 (en) | 2007-07-13 | 2009-01-22 | Abbott Biotechnology Ltd. | METHODS AND COMPOSITIONS FOR PULMONARY ADMINISTRATION OF A TNFa INHIBITOR |
KR100897159B1 (en) | 2007-07-26 | 2009-05-14 | 보령제약 주식회사 | A plant recombinant human CTLA4Ig and a method for producing the same |
US8753839B2 (en) | 2007-08-08 | 2014-06-17 | Abbvie Inc. | Compositions and methods for crystallizing antibodies |
EP3327132A3 (en) | 2007-08-09 | 2018-07-18 | Wyeth LLC | Use of perfusion to enhance production of fed-batch cell culture in bioreactors |
CN101802005B (en) | 2007-08-28 | 2015-09-16 | 艾伯维生物技术有限公司 | Comprise the composition about the conjugated protein of adalimumab and method |
EP2031064A1 (en) | 2007-08-29 | 2009-03-04 | Boehringer Ingelheim Pharma GmbH & Co. KG | Method for increasing protein titres |
WO2009027041A1 (en) | 2007-08-31 | 2009-03-05 | F. Hoffmann-La Roche Ag | Glycosylation profile analysis |
JP2011500032A (en) | 2007-10-12 | 2011-01-06 | シグマ−アルドリッチ・カンパニー | Compositions and methods for improving sialylation of glycoproteins |
EP2050764A1 (en) | 2007-10-15 | 2009-04-22 | sanofi-aventis | Novel polyvalent bispecific antibody format and uses thereof |
EP2205972B1 (en) | 2007-10-29 | 2012-01-04 | University Of Georgia Research Foundation, Inc. | In vivo isotopic labeling method for quantitative glycomics |
WO2009058769A1 (en) | 2007-10-30 | 2009-05-07 | Schering Corporation | Purification of antibodies containing hydrophobic variants |
US8420081B2 (en) | 2007-11-30 | 2013-04-16 | Abbvie, Inc. | Antibody formulations and methods of making same |
US8883146B2 (en) | 2007-11-30 | 2014-11-11 | Abbvie Inc. | Protein formulations and methods of making same |
US20130195888A1 (en) | 2007-11-30 | 2013-08-01 | Abbvie | Ultrafiltration and diafiltration formulation methods for protein processing |
JP5727790B2 (en) | 2007-12-27 | 2015-06-03 | バクスター・インターナショナル・インコーポレイテッドBaxter International Incorp0Rated | Cell culture process |
US8399627B2 (en) | 2007-12-31 | 2013-03-19 | Bayer Pharma AG | Antibodies to TNFα |
WO2009086550A1 (en) | 2008-01-03 | 2009-07-09 | Abbott Laboratories | Predicting long-term efficacy of a compound in the treatment of psoriasis |
CN107119095B (en) | 2008-01-03 | 2022-07-05 | 康乃尔研究基金会有限公司 | Glycosylated protein expression in prokaryotes |
WO2009091912A2 (en) | 2008-01-15 | 2009-07-23 | Abbott Laboratories | Improved mammalian expression vectors and uses thereof |
JP6078217B2 (en) | 2008-01-15 | 2017-02-08 | アッヴィ・ドイチュラント・ゲー・エム・ベー・ハー・ウント・コー・カー・ゲー | Powdered protein composition and method for producing the same |
TWI472339B (en) | 2008-01-30 | 2015-02-11 | Genentech Inc | Composition comprising antibody that binds to domain ii of her2 and acidic variants thereof |
MX2010008364A (en) | 2008-01-30 | 2010-08-23 | Abbott Lab | Compositions and methods for crystallizing antibody fragments. |
US20110129468A1 (en) | 2008-02-29 | 2011-06-02 | Biogen Idec Ma Inc. | Purified immunoglobulin fusion proteins and methods of their purification |
CA2717614A1 (en) | 2008-03-11 | 2009-09-17 | Genentech, Inc. | Antibodies with enhanced adcc function |
MX2010010503A (en) | 2008-03-24 | 2010-11-09 | Abbott Biotech Ltd | Methods and compositions for treating bone loss. |
AU2009233906A1 (en) | 2008-04-08 | 2009-10-15 | Amyris, Inc. | Expression of heterologous sequences |
US8163551B2 (en) | 2008-05-02 | 2012-04-24 | Seattle Genetics, Inc. | Methods and compositions for making antibodies and antibody derivatives with reduced core fucosylation |
WO2009135656A1 (en) | 2008-05-06 | 2009-11-12 | Lonza Biologics Plc. | A method for the purification of antibodies using displacement chromatography |
EP3002299A1 (en) | 2008-06-03 | 2016-04-06 | AbbVie Inc. | Dual variable domain immunoglobulins and uses thereof |
WO2010016943A2 (en) | 2008-08-08 | 2010-02-11 | Biogen Idec Ma Inc. | Nutrient monitoring and feedback control for increased bioproduct production |
US10927144B2 (en) | 2008-08-14 | 2021-02-23 | Genentech, Inc. | Methods for removing a contaminant using indigenous protein displacement ion exchange membrane chromatography |
US20100069617A1 (en) | 2008-09-12 | 2010-03-18 | Ge Healthcare Bio-Sciences Ab | Enhanced protein aggregate removal by mixed mode chromatography on hydrophobic interaction media in the presence of protein-excluded zwitterions |
JP2012503656A (en) | 2008-09-26 | 2012-02-09 | エウレカ セラピューティクス,インコーポレイテッド | Cell lines and proteins with mutant glycosylation patterns |
WO2010043703A1 (en) | 2008-10-17 | 2010-04-22 | Dsm Ip Assets B.V. | Removal of host cell proteins |
NZ592094A (en) | 2008-10-20 | 2013-01-25 | Abbott Lab | Antibodies that bind to il-18 and methods of purifying the same |
SG10201702922VA (en) | 2008-10-20 | 2017-06-29 | Abbvie Inc | Isolation and purification of antibodies using protein a affinity chromatography |
CN104974251A (en) | 2008-10-20 | 2015-10-14 | Abbvie公司 | Viral inactivation during purification of antibodies |
GB0821100D0 (en) | 2008-11-18 | 2008-12-24 | Hansa Medical Ab | Antibodies |
EP2358760B1 (en) | 2008-12-19 | 2015-02-18 | Momenta Pharmaceuticals, Inc. | Characterization of o-linked glycans |
JP2012513030A (en) | 2008-12-19 | 2012-06-07 | モメンタ ファーマシューティカルズ インコーポレイテッド | Methods for modified glycans |
US8614297B2 (en) | 2008-12-22 | 2013-12-24 | Hoffmann-La Roche Inc. | Anti-idiotype antibody against an antibody against the amyloid β peptide |
AU2010203836B2 (en) | 2009-01-08 | 2015-05-28 | Cytiva Bioprocess R&D Ab | Separation method using single polymer phase systems |
NZ593816A (en) | 2009-01-22 | 2012-11-30 | Momenta Pharmaceuticals Inc | Galactose-alpha-1, 3-galactose-containing n-glycans in glycoprotein products derived from cho cells |
US20100292443A1 (en) | 2009-02-26 | 2010-11-18 | Sabbadini Roger A | Humanized platelet activating factor antibody design using anti-lipid antibody templates |
US9127043B2 (en) | 2009-03-05 | 2015-09-08 | Biogen Ma Inc. | Purification of immunoglobulins |
EP2233499A1 (en) | 2009-03-26 | 2010-09-29 | CSL Behring AG | Antibody composition with altered Fab sialylation |
SI2420572T1 (en) | 2009-04-01 | 2015-10-30 | Evec Incorporated | Monoclonal antibody capable of binding to specific discontinuous epitope occurring in ad1 region of human cytomegalovirus gb glycoprotein, and antigen-binding fragment thereof |
CA2757079C (en) | 2009-04-20 | 2015-05-19 | Pfizer Inc. | Control of protein glycosylation and compositions and methods relating thereto |
US20120282654A1 (en) | 2009-04-29 | 2012-11-08 | Schering Corporation | Antibody purification |
MX2011011541A (en) | 2009-04-29 | 2012-02-28 | Abbott Biotech Ltd | Automatic injection device. |
SG175188A1 (en) | 2009-05-04 | 2011-11-28 | Abbott Biotech Ltd | Stable high protein concentration formulations of human anti-tnf-alpha-antibodies |
US20120329709A1 (en) | 2009-05-26 | 2012-12-27 | Brian Edward Collins | Production of glycoproteins |
KR20120042744A (en) | 2009-05-28 | 2012-05-03 | 베링거 인겔하임 인터내셔날 게엠베하 | Method for a rational cell culturing process |
AU2010252230B2 (en) | 2009-05-28 | 2013-07-04 | Eth Zurich | N-glycan core beta-galactosyltransferase and uses thereof |
CA2763164A1 (en) | 2009-06-05 | 2010-12-09 | Momenta Pharmaceuticals, Inc. | Methods of modulating fucosylation of glycoproteins |
ES2752874T3 (en) | 2009-07-06 | 2020-04-06 | Hoffmann La Roche | Eukaryotic cell culture procedure |
KR101498772B1 (en) | 2009-07-24 | 2015-03-04 | 에프. 호프만-라 로슈 아게 | Optimizing the production of antibodies |
US8945895B2 (en) | 2009-07-31 | 2015-02-03 | Baxter International Inc. | Methods of purifying recombinant ADAMTS13 and other proteins and compositions thereof |
WO2011015926A1 (en) | 2009-08-03 | 2011-02-10 | Avesthagen Limited | A process of fermentation, purification and production of recombinant soluble tumour necrosis factor alfa receptor (tnfr) - human igg fc fusion protein |
WO2011019620A1 (en) | 2009-08-10 | 2011-02-17 | Genentech, Inc. | Antibodies with enhanced adcc function |
KR20180010324A (en) | 2009-08-11 | 2018-01-30 | 제넨테크, 인크. | Production of proteins in glutamine-free cell culture media |
WO2011019622A1 (en) | 2009-08-14 | 2011-02-17 | Genentech, Inc. | Cell culture methods to make antibodies with enhanced adcc function |
WO2011024025A1 (en) | 2009-08-28 | 2011-03-03 | Avesthagen Limited | An erythropoietin analogue and a method thereof |
US9540426B2 (en) | 2009-10-06 | 2017-01-10 | Bristol-Myers Squibb Company | Mammalian cell culture processes for protein production |
US8470552B2 (en) | 2009-10-12 | 2013-06-25 | Keck Graduate Institute | Strategy to reduce lactic acid production and control PH in animal cell culture |
EP2325296A1 (en) | 2009-11-20 | 2011-05-25 | LEK Pharmaceuticals d.d. | Production of glycoproteins with low N-glycolylneuraminic acid (Neu5Gc) content |
US9096879B2 (en) | 2009-11-24 | 2015-08-04 | Biogen Ma Inc. | Method of supplementing culture media to prevent undesirable amino acid substitutions |
US10087236B2 (en) | 2009-12-02 | 2018-10-02 | Academia Sinica | Methods for modifying human antibodies by glycan engineering |
EP2507627A2 (en) | 2009-12-04 | 2012-10-10 | Momenta Pharmaceuticals, Inc. | Antennary fucosylation in glycoproteins from cho cells |
WO2011073235A1 (en) | 2009-12-18 | 2011-06-23 | Csl Ltd. | Method of purifying polypeptides |
EP2531613A2 (en) | 2010-02-02 | 2012-12-12 | Abbott Biotechnology Ltd. | Methods and compositions for predicting responsiveness to treatment with tnf-alpha inhibitor |
WO2011095596A1 (en) | 2010-02-04 | 2011-08-11 | Vivalis | Fed-batch process using concentrated cell culture medium for the efficient production of biologics in eb66 cells |
BR112012020254A2 (en) | 2010-02-12 | 2016-05-03 | Dsm Ip Assets Bv | Method for the purification of antibodies from a protein mixture and single operating unit. |
CA2789564C (en) | 2010-03-10 | 2019-08-13 | F. Hoffmann-La Roche Ag | Method for purifying immunoglobulin solutions |
US8981081B2 (en) | 2010-03-12 | 2015-03-17 | Purecircle Usa Inc. | High-purity steviol glycosides |
ES2602108T3 (en) | 2010-04-07 | 2017-02-17 | Momenta Pharmaceuticals, Inc. | Method for quantifying glycoforms containing high mannose |
BR112012027001A2 (en) | 2010-04-23 | 2016-07-19 | Genentech Inc | heteromultimeric protein production |
US20110262965A1 (en) | 2010-04-23 | 2011-10-27 | Life Technologies Corporation | Cell culture medium comprising small peptides |
PT2563906T (en) | 2010-04-26 | 2018-02-16 | Novartis Ag | Process for cultivation of cho cells |
RU2644651C2 (en) | 2010-04-26 | 2018-02-13 | Новартис Аг | Medium for cells cultivation |
JP6050226B2 (en) | 2010-05-28 | 2016-12-21 | ジェネンテック, インコーポレイテッド | Lowering lactic acid levels and increasing polypeptide production by downregulating LDH and PDHK expression |
CN105056232A (en) | 2010-06-03 | 2015-11-18 | 阿布维生物技术有限公司 | Uses and compositions for treatment of hidradenitis suppurativa |
US9428546B2 (en) | 2010-07-30 | 2016-08-30 | Pfizer Inc. | Tandem purification of proteins |
CN103080300B (en) | 2010-08-05 | 2015-11-25 | 安姆根有限公司 | Increase the productive rate of cell culture and the dipeptides of vigor |
DE102010038990A1 (en) | 2010-08-06 | 2012-02-09 | Robert Bosch Gmbh | Shaft bearing device for a hand tool |
WO2012030512A1 (en) | 2010-09-03 | 2012-03-08 | Percivia Llc. | Flow-through protein purification process |
MX2013003038A (en) | 2010-09-17 | 2013-05-01 | Abbvie Inc | Raman spectroscopy for bioprocess operations. |
WO2012040041A1 (en) | 2010-09-20 | 2012-03-29 | Abbott Laboratories | Purification of antibodies using simulated moving bed chromatography |
US20120258496A1 (en) | 2010-09-27 | 2012-10-11 | Boehringer Ingelheim International Gmbh | Production of low fucose antibodies in h4-ii-e rat cells |
JP5888616B2 (en) | 2010-10-08 | 2016-03-22 | カディラ・ヘルスケア・リミテッド | Expression vectors for high level expression of recombinant proteins |
WO2012051147A1 (en) | 2010-10-11 | 2012-04-19 | Abbott Laboratories | Processes for purification of proteins |
US20130224797A1 (en) | 2010-10-15 | 2013-08-29 | Jcr Pharmaceuticals Co., Ltd. | Method for producing glycoprotein having mannose residue as non-reducing end of sugar chain |
EP2450375A1 (en) | 2010-11-09 | 2012-05-09 | Sandoz Gmbh | Cell culture medium and process for protein expression, said medium and process comprising a PAM inhibitor |
ME02506B (en) | 2010-11-11 | 2017-02-20 | Abbvie Biotechnology Ltd | HIGH CONCENTRATION ANTI-TNFalpha ANTIBODY LIQUID FORMULATIONS |
US20130331554A1 (en) | 2010-11-15 | 2013-12-12 | Biogen Idec Inc. | Enrichment and concentration of select product isoforms by overloaded bind and elute chromatography |
EP2649087A1 (en) | 2010-12-08 | 2013-10-16 | Amgen Inc. | Ion exchange chromatography in the presence of an amino acid |
JP2014506132A (en) | 2011-01-07 | 2014-03-13 | アッヴィ・インコーポレイテッド | Anti-IL-12 / IL-23 antibody and use thereof |
TW201309330A (en) | 2011-01-28 | 2013-03-01 | Abbott Lab | Compositions containing glycosylated antibodies and uses thereof |
AR085302A1 (en) | 2011-02-24 | 2013-09-18 | Sanofi Sa | METHOD OF PRODUCTION OF STIRATED ANTIBODIES |
BR112013022832A2 (en) | 2011-03-06 | 2016-11-22 | Merck Serono Sa | low fucose cell lines and uses thereof |
CA2829629A1 (en) | 2011-03-10 | 2012-09-13 | Board Of Regents, The University Of Texas System | Protein nanoparticle dispersions |
US20120238730A1 (en) | 2011-03-15 | 2012-09-20 | Abbott Laboratories | Integrated approach to the isolation and purification of antibodies |
TWI803876B (en) | 2011-03-28 | 2023-06-01 | 法商賽諾菲公司 | Dual variable region antibody-like binding proteins having cross-over binding region orientation |
EP2511293A1 (en) | 2011-04-13 | 2012-10-17 | LEK Pharmaceuticals d.d. | A method for controlling the main complex N-glycan structures and the acidic variants and variability in bioprocesses producing recombinant proteins |
US9133493B2 (en) | 2011-04-21 | 2015-09-15 | Amgen Inc. | Method for culturing mammalian cells to improve recombinant protein production |
WO2012149197A2 (en) | 2011-04-27 | 2012-11-01 | Abbott Laboratories | Methods for controlling the galactosylation profile of recombinantly-expressed proteins |
CA3182519A1 (en) | 2011-04-29 | 2012-11-01 | Selecta Biosciences, Inc. | Tolerogenic synthetic nanocarriers for antigen-specific deletion of t effector cells |
WO2012147048A2 (en) | 2011-04-29 | 2012-11-01 | Biocon Research Limited | Methods for reducing accumulation of lactate during culturing and method for producing polypeptide |
ES2560470T3 (en) | 2011-04-29 | 2016-02-19 | Biocon Research Limited | A method to reduce the heterogeneity of antibodies and a production process of said antibodies |
US20140108084A1 (en) | 2012-10-12 | 2014-04-17 | Crestron Electronics, Inc. | Initiating Schedule Management Via Radio Frequency Beacons |
US9562252B2 (en) | 2011-05-13 | 2017-02-07 | Biogen Ma Inc. | Methods of preventing and removing trisulfide bonds |
PL2726600T3 (en) | 2011-07-01 | 2017-08-31 | Amgen Inc. | Mammalian cell culture |
WO2013006461A1 (en) | 2011-07-01 | 2013-01-10 | Biogen Idec Ma Inc. | Cholesterol-based media supplementals for cell culture |
EP2729496B8 (en) | 2011-07-06 | 2017-10-18 | Genmab A/S | Modulation of complement-dependent cytotoxicity through modifications of the c-terminus of antibody heavy chains |
WO2013009648A2 (en) | 2011-07-08 | 2013-01-17 | Momenta Pharmaceuticals, Inc. | Cell culture process |
GB201112429D0 (en) | 2011-07-19 | 2011-08-31 | Glaxo Group Ltd | Antigen-binding proteins with increased FcRn binding |
WO2013013013A2 (en) | 2011-07-21 | 2013-01-24 | Alnylam Pharmaceuticals, Inc. | Compositions and methods for producing modified glycoproteins |
KR20140101331A (en) | 2011-08-10 | 2014-08-19 | 라보라토이레 프란카이즈 듀 프락티온네먼트 에트 데스 바이오테크놀로지스 | Highly galactosylated antibodies |
US20130149300A1 (en) | 2011-09-27 | 2013-06-13 | Icon Genetics Gmbh | MONOCLONAL ANTIBODIES WITH ALTERED AFFINITIES FOR HUMAN FCyRI, FCyRIIIa, AND C1q PROTEINS |
WO2013066707A1 (en) | 2011-10-31 | 2013-05-10 | Merck Sharp & Dohme Corp. | Chromatography process for resolving heterogeneous antibody aggregates |
CN104023804B (en) | 2011-11-02 | 2017-05-24 | 弗·哈夫曼-拉罗切有限公司 | Overload And Elute Chromatography |
SG10202001596VA (en) | 2011-12-19 | 2020-04-29 | Univ Rockefeller | Non-sialylated anti-inflammatory polypeptides |
AU2013255413C1 (en) | 2012-03-07 | 2016-03-24 | Cadila Healthcare Limited | Pharmaceutical formulations of TNF-alpha antibodies |
SG10201701224UA (en) | 2012-03-12 | 2017-04-27 | Merck Patent Gmbh | Removal of protein aggregates from biopharmaceutical preparations in a flowthrough mode |
US9150645B2 (en) | 2012-04-20 | 2015-10-06 | Abbvie, Inc. | Cell culture methods to reduce acidic species |
US9181572B2 (en) | 2012-04-20 | 2015-11-10 | Abbvie, Inc. | Methods to modulate lysine variant distribution |
WO2013158275A1 (en) | 2012-04-20 | 2013-10-24 | Abbvie Inc. | Cell culture methods to reduce acidic species |
US9067990B2 (en) | 2013-03-14 | 2015-06-30 | Abbvie, Inc. | Protein purification using displacement chromatography |
TW201348247A (en) | 2012-05-21 | 2013-12-01 | Abbvie Inc | Novel purification of non-human antibodies using protein a affinity chromatography |
US20140154270A1 (en) | 2012-05-21 | 2014-06-05 | Chen Wang | Purification of non-human antibodies using kosmotropic salt enhanced protein a affinity chromatography |
WO2013176754A1 (en) | 2012-05-24 | 2013-11-28 | Abbvie Inc. | Novel purification of antibodies using hydrophobic interaction chromatography |
WO2013181585A2 (en) | 2012-06-01 | 2013-12-05 | Momenta Pharmaceuticals, Inc. | Methods related to adalimumab |
WO2013186230A1 (en) | 2012-06-12 | 2013-12-19 | Boehringer Ingelheim International Gmbh | Pharmaceutical formulation for a therapeutic antibody |
WO2014018747A2 (en) | 2012-07-26 | 2014-01-30 | Momenta Pharmaceuticals, Inc. | Glycoproteins with anti-inflammatory properties |
AU2013304237B2 (en) | 2012-08-17 | 2017-12-21 | Christian Frisch | Complex-specific antibodies and antibody fragments and its use |
US9512214B2 (en) | 2012-09-02 | 2016-12-06 | Abbvie, Inc. | Methods to control protein heterogeneity |
US9206390B2 (en) | 2012-09-02 | 2015-12-08 | Abbvie, Inc. | Methods to control protein heterogeneity |
TWI698253B (en) | 2012-09-07 | 2020-07-11 | 美商柯赫勒斯生物科學有限公司 | Stable aqueous formulations of adalimumab |
US20150252108A1 (en) | 2012-09-26 | 2015-09-10 | Momenta Pharmaceuticals, Inc. | Glycoprotein preparations |
US20160185847A1 (en) | 2012-12-17 | 2016-06-30 | Laboratoire Francais Du Fractionnement Et Des Biotechnologies | Use of monoclonal antibodies for the treatment of inflammation and bacterial infections |
US9844594B2 (en) | 2012-12-18 | 2017-12-19 | Merck Sharp & Dohme Corp. | Liquid formulations for an anti-TNF α antibody |
MX2015010427A (en) | 2013-02-13 | 2016-03-17 | Lab Francais Du Fractionnement | Highly galactosylated anti-tnf-alpha antibodies and uses thereof. |
AU2014225272B2 (en) | 2013-03-08 | 2015-10-22 | Neuclone Biologics Pty Ltd | A cell expression system |
US20140271633A1 (en) | 2013-03-14 | 2014-09-18 | Abbvie Inc. | Mammalian cell culture performance through surfactant supplementation of feed media |
US20140271622A1 (en) | 2013-03-14 | 2014-09-18 | Momenta Pharmaceuticals, Inc. | Methods of cell culture |
US9677105B2 (en) | 2013-03-14 | 2017-06-13 | Momenta Pharmaceuticals, Inc. | Methods of cell culture |
US9017687B1 (en) | 2013-10-18 | 2015-04-28 | Abbvie, Inc. | Low acidic species compositions and methods for producing and using the same using displacement chromatography |
EP2836515A1 (en) | 2013-03-14 | 2015-02-18 | AbbVie Inc. | Low acidic species compositions and methods for producing and using the same |
US9217168B2 (en) | 2013-03-14 | 2015-12-22 | Momenta Pharmaceuticals, Inc. | Methods of cell culture |
WO2014151878A2 (en) | 2013-03-14 | 2014-09-25 | Abbvie Inc. | Methods for modulating protein glycosylation profiles of recombinant protein therapeutics using monosaccharides and oligosacharides |
US8956830B2 (en) | 2013-03-14 | 2015-02-17 | Momenta Pharmaceuticals, Inc. | Methods of cell culture |
WO2014159579A1 (en) | 2013-03-14 | 2014-10-02 | Abbvie Inc. | MUTATED ANTI-TNFα ANTIBODIES AND METHODS OF THEIR USE |
WO2014149935A1 (en) | 2013-03-15 | 2014-09-25 | Janssen Biotech, Inc. | Manufacturing methods to control c-terminal lysine, galactose and sialic acid content in recombinant proteins |
EP3754012A1 (en) | 2013-03-15 | 2020-12-23 | Alder Biopharmaceuticals, Inc. | Antibody purification and purity monitoring |
US20160108450A1 (en) | 2013-05-02 | 2016-04-21 | Momenta Pharmaceutcals, Inc. | Sialylated glycoproteins |
KR101569783B1 (en) | 2013-06-05 | 2015-11-19 | 한화케미칼 주식회사 | A Method of Antibody Purification |
AR096713A1 (en) | 2013-06-25 | 2016-01-27 | Cadila Healthcare Ltd | PURIFICATION PROCESS FOR MONOCLONAL ANTIBODIES |
TW201514305A (en) | 2013-07-06 | 2015-04-16 | Cadila Healthcare Ltd | Improved process for production of monoclonal antibodies |
US20160151484A1 (en) | 2013-07-19 | 2016-06-02 | Hexal Ag | Methods and formulations which allow the modulation of immune responses related to the administration of a biopharmaceutical drug |
PL3036320T3 (en) | 2013-08-19 | 2021-11-02 | Biogen Ma Inc. | Control of protein glycosylation by culture medium supplementation and cell culture process parameters |
JP2016527911A (en) | 2013-08-20 | 2016-09-15 | レック・ファーマシューティカルズ・ディー・ディーLek Pharmaceuticals D.D. | Cell culture media and process for controlling α-amidation and / or C-terminal amino acid cleavage of polypeptides |
EP3052640A2 (en) | 2013-10-04 | 2016-08-10 | AbbVie Inc. | Use of metal ions for modulation of protein glycosylation profiles of recombinant proteins |
US9181337B2 (en) | 2013-10-18 | 2015-11-10 | Abbvie, Inc. | Modulated lysine variant species compositions and methods for producing and using the same |
US9085618B2 (en) | 2013-10-18 | 2015-07-21 | Abbvie, Inc. | Low acidic species compositions and methods for producing and using the same |
US8946395B1 (en) | 2013-10-18 | 2015-02-03 | Abbvie Inc. | Purification of proteins using hydrophobic interaction chromatography |
WO2015073884A2 (en) | 2013-11-15 | 2015-05-21 | Abbvie, Inc. | Glycoengineered binding protein compositions |
US20160185848A1 (en) | 2014-07-09 | 2016-06-30 | Abbvie Inc. | Methods for modulating the glycosylation profile of recombinant proteins using sugars |
WO2016022377A2 (en) | 2014-08-05 | 2016-02-11 | Abbvie Inc. | Methods for modulating the glycosylation profile of recombinant proteins using dissolved oxygen |
SI3237432T1 (en) | 2014-12-22 | 2023-12-29 | UCB Biopharma SRL | Protein manufacture |
CN105777896B (en) | 2015-03-19 | 2019-08-16 | 广东东阳光药业有限公司 | A kind of purification process at antibody acidity peak |
CN105777895A (en) | 2015-03-19 | 2016-07-20 | 广东东阳光药业有限公司 | Application of sodium phenylbutyrate in purification of antibody acidic peak |
CN105777904B (en) | 2015-03-23 | 2019-12-10 | 广东东阳光药业有限公司 | cation exchange chromatography purification method of anti-TNF alpha monoclonal antibody |
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10513724B2 (en) | 2014-07-21 | 2019-12-24 | Glykos Finland Oy | Production of glycoproteins with mammalian-like N-glycans in filamentous fungi |
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