Among the up-regulated genes of the 1X M1 medium treatment (highlighted in orange in Table II), Srebf1, Insig1, and Scap are genes for sterol biosynthesis (8). These genes were non-differentially expressed in cells fed with 2.50X + 0.5% P188. P188, a surfactant consisting of hydrophobic and hydrophilic blocks, has been reported to reduce cell death (9, 10). It may play a role similar to lipids, which have hydrophobic tails and hydrophilic heads that confer cell protection from stress. In addition to three sterol synthesis genes, Htra3, a serine peptidase gene, was upregulated more than four-fold from day 9 to day 11 in the 1X M1 medium, while only about two-fold in the concentrated medium. Given that serine peptidase contributes to protein degradation, a higher level of this protease may be one of the reasons for the lower antibody levels observed in the 1X M1 medium compared to that in the 2.50X + 0.5% P188 medium.
Discussion
In this study, different strategies of preparing concentrated feed media were compared. Although higher concentrations of feed media could be obtained by raising pH, there was an optimal pH of feed media in which cell growth and antibody production were not adversely affected. The addition of a surfactant makes it possible to prepare more concentrated feed media while maintaining the pH. In this study, 2.50X concentrated medium supplemented with 0.5% P188 led to a 10-50% increase in titers while feeding as low as 40% of the original feed volume. The surfactant addition to the feed did not have any impact on measured protein quality attributes.
Medium storage stability is a general concern for manufacturing and development using concentrated feed media. In this study, the 2.50X + 0.5% P188 M1 and 1.33X M2 media have a shelf life of 60 days at 2-4 oC and 26 days at ambient temperature respectively (data not shown). There was no precipitation observed and antibody titers were not affected. These data show that concentrating feed medium either by raising pH or adding surfactant is an effective and applicable approach in simplifying the manufacturing process and improving productivity.
Acknowledgements
The authors would like to thank Gautam Nayar, Joel Goldstein, and Frank Ritacco for their review and comments on this manuscript. The authors would also like to thank Amanda Bell, Wenkui Lan, and Ya Fu for their support in protein purification and protein quality analysis.
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About the Authors
Ping Xu is a scientist at Bristol-Myers Squibb, Process Development, 519 Route 173 West, Bloomsbury, NJ 08804, [email protected]; Xiao-Ping Dai is director of Process Development, Biologics at Celgene Corporation, 200 Connell Drive, Berkeley Heights, NJ 07922, and previously manager of Cell Culture Sciences at Bristol-Myers Squibb when this work was done; Albert Kao is an undergraduate student at the University of Michigan, Ann Arbor, MI 48109; Rosario Scott is a senior scientist, and Reb Russell is site head and head of Biologics Process Development, both at Bristol-Myers Squibb, Process Development, 519 Route 173 West, Bloomsbury, NJ 08804.