Biopharmaceuticals continue to be a growing, important class of pharmaceutical products on the market, and many of these products are produced using mammalian cell culture. Monoclonal antibodies, currently the dominant class of biopharmaceuticals, generally require mammalian cell culture production to enable addition of critical post-translational modifications such as glycosylation. Since these products are often given to patients at high doses, total bulk drug requirements for a single product can be as high as 7,000 kg per year. When monoclonal antibodies first entered the market, there was significant concern over the ability of the industry to produce enough product to treat eligible patients worldwide. To be able to meet this high demand, the industry has developed cell line development and cell culture technologies that improve the overall performance and viability of production cell lines in the bioreactor. This article reviews emerging approaches to improving biopharmaceutical production cell lines and cell culture performance through the application of genomics and related technologies. Together these technologies are positioned to cause significant changes that will enable each and every mammalian cell culture run to provide high yields of biopharmaceutical products with desired quality attributes.
Introduction
Demand for biopharmaceutical products, especially monoclonal antibody-related products, continued to grow at a very healthy pace in 2012 with total biopharmaceutical sales reaching nearly $130 billion, or approximately 13% of the total pharmaceutical market1 2. In 2012, there were 34 biopharmaceutical “blockbuster” products with annual sales over $1 billion, of which over 60% were produced in mammalian cell culture. Five of the top ten selling pharmaceutical products are now biopharmaceuticals, four of which are monoclonal antibodies3. These products, Humira, Enbrel, Remicade and Rituxan, combined with Avastin, Herceptin and the long list of monoclonal antibody products in development make monoclonal antibodies the fastest growing class of biopharmaceutical products. In addition, as the patents governing the exclusive rights to many of these blockbuster biopharmaceutical products begin to expire, the pharmaceutical industry worldwide has shown a growing interest in developing follow-on biologics or biosimilars to provide lower cost alternatives to the current innovator products on the market.
Figure 1. Total sales in the US and Europe of traditional pharmaceuticals
(blue) and biopharmaceuticals (green) are shown by year for the past decade.
Sales information was obtained from company annual reports and other publically available sources.
Despite the growing market demand for innovator and biosimilar biopharmaceutical products and the associated requirement for increasing amounts of mammalian cell culture capacity to meet this demand, ongoing advances in cell culture process yields coupled with significant expansion of manufacturing capacity in the last decade, have enabled the industry to meet the market demand for these products to date. However, as these advanced medicinal products reach expanded markets worldwide and as newer, higher dose innovator products progress through clinical trials and onto the market, additional advances in cell line development and cell culture technology will be required to continue to produce enough product using the available mammalian cell culture capacity.
Cell line development and cell culture advances in the past 2-5 years include many orthogonal approaches to improve expression levels and productivity in the bioreactor. These include improvements in the parental cell lines, advances in genetic methods to improve gene expression through a variety of means, improved media and feed composition, alternative supplements or analysis to enable enhanced productivity in the bioreactor, and single use bioreactors that enable diversified manufacturing strategies and local production. Each of these advances utilized alone or in conjunction will contribute to improvements in supply of bulk biopharmaceutical products over the next decade and will enable these life-saving medicines to reach greater patient populations worldwide.
Impact on CHO Cell Line Engineering
In the past decade, numerous companies and academic groups have focused significant efforts on using genomic, proteomic, and transcriptomic studies to generate a complete understanding of CHO cell biology under different conditions. The hope and promise of these studies was that they would provide actionable results that could facilitate CHO host cell and production cell line engineering for greater viability and productivity, and could contribute to better control of overall performance in the bioreactor. Goals of these studies included identification of unique biomarkers that indicate better performance or identification of targets for knock-in or knock-out cell line engineering. Since a rapid rate of cell growth to achieve maximum biomass quickly and sustained viability throughout the culture should lead to greater production of the desired protein from a bioreactor, many of the studies focused on achieving one or both of these goals. The publication of the CHO genome in 20104 provided the extensive information that is necessary to implement such studies and reach actionable conclusions.
Colin Clark and his colleagues at the National Institute for Cellular Biotechnology in Dublin, Ireland, have published numerous studies on differential expression analysis in a vast number of production CHO cell lines. These studies have included proteomics, transcriptomics, and microRNA (miRNA) analysis to filter out false positive and false negative results and obtain a clear understanding of those genes that are actively involved in enabling high viability and productivity of CHO production cells in culture. In one such study, Doolan et al.5 performed microarray and proteomics analysis of CHO cells to identify specific genes that contributed to faster cell growth. The analysis of mRNA levels and protein levels was performed on two CHO production cell lines that expressed the same transgene but grew at different rates. They report that they identified 118 transcripts and 58 proteins that were expressed at different levels in the two cell lines, and of those 21 gene candidates showed up in both sets. By using silencing RNA (siRNA) to inhibit expression of selected gene candidates, the group found that a protein known as valosin-containing protein (VCP) is a major contributor to CHO cell growth and viability. VCP is therefore one example of a protein that could be manipulated by engineering parental or production CHO cell lines to improve growth and viability. In another publication by the same group, Sanchez et al.6 showed that the tumor suppressor MiR-7 also appears to inhibit proliferation of CHO cells in culture. Therefore, inhibition of MiR-7 is another approach to improving cell growth and viability in the bioreactor.
Carlage et al.7 studied the impact of a known inhibitor of apoptosis, Bcl-X(L), on expression patterns of host cell genes in a CHO cell line that was expressing a transgene. Production CHO cells transfected with the gene encoding this apoptosis inhibitor had higher levels of transgene expression than a parallel non-transfected culture. Using proteomic analysis, the two cell lines were compared at various times during cell culture to identify any differential protein expression. Of the 392 proteins that were identified in the proteomic analysis, 32 were differentially expressed. In the high-producing cell culture, several proteins related to protein metabolism were up-regulated, including eukaryotic translation initiation factor 3 and ribosome 40S. In addition, several intermediate filament proteins such as vimentin and annexin, as well as histone H1.2 and H2A, were down-regulated in the high producer. These studies provide additional insight into methods to engineer host CHO cell lines for better growth, viability, and transgene expression.
Nicolas Mermod’s group at the University of Lausanne have focused their efforts on understanding the mechanism of transgene recombination into the genome during the initial phases of cell line development and the secretory pathway functions that are essential for cell health and productivity of the secreted product encoded by the transgene. 8 This work could lead to advances at the cell line development stage that could create production cell lines with greater genetic stability, cell health, and ability to produce the desired protein.
Many mechanisms for integration of transfected plasmids into mammalian cell genomes such as CHO cells have been suggested in publications, but none of the proposed mechanisms explains the results that are obtained when analyzing the genomes and transgene structures of CHO production cell lines. In collaboration with the Swiss Institute of Bioinformatics and Selexis SA, a cell line development and engineering company co-founded by Dr. Mermod, his group has evaluated the data from CHO cell production cell lines and proposed a novel molecular integration model that may explain transgene integration. Dr. Mermod’s laboratory is currently evaluating this model and has shown that when they block competing recombination pathways and use plasmids containing matrix-attachment regions, transgene expression levels are significantly increased compared to control cultures. Therefore, the integration model they have developed has some scientific merits, and it also may have a practical value to allow improved methods for cell line construction.9
Overall, the studies of the CHO genome and differential expression have successfully identified target pathways for molecular engineering of the parental cell lines and for identification of high producers using biomarkers. Exploitation of these advances in cell line engineering has only recently been initiated within the industry but the productivity impact on newer CHO production cell lines can already be seen. For example, Selexis has recently launched a series of engineered versions of their host CHO-M cell line designed to address many of the secretory bottlenecks associated with difficult to express proteins.10
Leveraging their knowledge of the CHO-M cell line genome and transcriptome, Selexis identified potential issues with the CHO-M secretory pathways such as stalled translocation, improper folding, incomplete post-translational modifications or insufficient cellular respiration to handle the increased protein load. They then used transposon-based vectors to express over 100 different auxiliary proteins which could overcome secretion bottlenecks associated with the CHO-M cell line and built combinatorial CHO libraries containing different combinations of these proteins. Using the resulting SURE CHO-M Library, Selexis was able to boost productivity of a non-natural mini-body by over 10 fold without any changes in gene copy number or transcription levels. Such improvements are emerging throughout industry as it exploits the wealth of data that has recently become available through genomics and associated technologies.
Conclusion
As more products enter the market and as biopharm sales expand into new geographic areas, mammalian cell culture must become a more efficient production method to meet this continually increasing market demand. Many have recognized this need for improvement in cell culture and have made significant efforts in understanding and engineering the production cell line allowing for better understanding of culture conditions and paving the way for maximum productivity of the cell line. With these industry-wide advances in understanding CHO cell biology and their application to the production of biopharmaceuticals, the industry continues to evolve to more efficient, more productive, and more robust cell culture approaches that should be able to meet the growing demand for these products.
References
1 Data derived from the BioProcess Technology Consultants Biopharmaceutical Database.
2 IMS Health Inc. Total Unaudited and Audited Global Pharmaceutical Market, 2003 – 2012 [Internet]. Danbury (CT): IMS Health Inc; 2013 Aug [cited 2013 Aug 13]. 1 p. Available from: http://www.imshealth.com/deployedfiles/imshealth/Global/Content/Corporate/Press%20Room/Total_World_Pharma_Market_Topline_metrics_2012.pdf
3 IMS Health Inc. Top 20 Global Products 2012 [Internet]. Danbury (CT): IMS Health Inc; 2013 Apr [cited 2013 Aug 9]. 1 p. Available from: http://www.imshealth.com/deployedfiles/ims/Global/Content/Corporate/Press%20Room/Top-Line%20Market%20Data%20&%20Trends/Top_20_Global_Products_2012_2.pdf
4 Xu X et al. “The genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line,” Nat Biotechnol. 2011;29(8):735–741
5 Doolan P. et al. “Microarray and Proteomics Expression Profiling Identifieds Several Candidates, Including the Valosin-Containing Protein (VCP), Involved in Regulating High Cellular Growth Rate in Production CHO Cell Lines,” Biotechnol Bioeng. 2010 May 1;106(1):42-56
6 Sanchez, N. et al. “MiR-7 triggers cell cycle arrest at the G1/S transition by targeting multiple genes including Skp2 and Psme3,” PLoS One. 2013 Jun 6;8(6):e65671. doi: 10.1371/journal.pone.0065671. Print 2013
7 Carlage T. et al. “Proteomic profiling of a high-producing Chinese hamster ovary cell culture,” Anal Chem. 2009 Sep 1;81(17):7357-62
8 Mermod, N. “CHO genome sequencing and engineering to improve clone selection and expression” Presented at IBCUSA Cell Line Development and Engineering Conference, 20-22 May, 2013, La Jolla, CA
9 Dr. Nicolas Mermod, Personal communication
10 Dr. Pierre-Alain Girod, Personal communication
