BIOMANUFACTURING: THE KEY TO SUCCESS IN BIOTECHNOLOGY

By Mike Butler, PhD

Historical Perspective
Canada led the world of biotechnology in 1921 when Banting and Best extracted a biologically active hormone from the pancreas of dogs. This Nobel Prize-winning work led to the isolation of insulin, which has been used for the treatment of Type 1 diabetes mellitus ever since. Fortunately for these research workers at the University of Toronto (Toronto, ON), insulin has cross-species activity such that the active ingredients from dogs, cows or pigs aid in the metabolism of glucose in humans.
Pig (or porcine) insulin differs from the human protein by only one amino acid residue and so, is an excellent source of material that can be used for substitution therapy in the treatment of patients. In the past, the ready availability of porcine pancreas enabled easy scale up for bioprocess development of this biopharmaceutical product. Only a small proportion of diabetics demonstrated an adverse immunogenic response from prolonged treatment with the xenobiotic therapeutic. This could be solved by chemical conversion of the single variant amino acid, which happens to be at the end of one of insulin’s two peptide chains.
Alternatively, in the 1980s, the authentic human protein could be produced from genetically engineered E. coli into which the human insulin gene was inserted. Humulin® (human recombinant insulin) was the first recombinant protein to be licensed for therapeutic use (1982) and heralded the beginning of a major industry for therapeutic proteins — biopharmaceuticals.
Could this success story be translated to other therapies? Well, yes . . . but not so easily! The choice of insulin as the first protein in the world to be used in substitution therapy was fortunate. It was opportune that the active ingredient from dogs (and other animals) works in humans. It was also fortunate that the two recombinant proteins (insulin and human growth hormone) produced in the early years of recombinant DNA technology from E. coli were biologically active. Later choices of target proteins did not offer such advantages.
Both insulin and growth hormone are fairly small and simple proteins. The structures have no complexities, such as the post-translational modifications necessary for some glycoproteins. E. coli are fast-growing bacteria that are eminently suitable for producing simple proteins but lack the capacity for the production of high molecular weight glycoproteins that characterize the newer products.

Animals Cells and Therapeutic Proteins
Over 150 biopharmaceuticals have now been approved for human therapeutic use. These include hormones, blood factors, thrombolytics, vaccines, interferons, monoclonal antibodies and enzymes. A further 500 or so are presently undergoing clinical trials. This drives the annual global market for biopharmaceuticals to over $30 billion US 1,2. However, this success rate would not have been possible without the incorporation of animal cell cultures into the armoury of technologies for biomanufacture.
Erythropoietin (EPO) is a good example. This natural glycoprotein, synthesized in the kidney, is required for the maturation of red blood cells (erythropoiesis). Kidney damage or dysfunction may lead to anemia that is treatable by the administration of recombinant EPO. This is an extremely efficacious treatment that, in many cases, replaces the need for kidney dialysis and has led EPO to become a blockbuster drug with annual sales exceeding $2 billion US. The method of production is from a genetically engineered animal cell that is transfected with the human EPO gene. Unlike E. coli, animal cells contain the metabolic infrastructure to be able to process and secrete the fully glycosylated EPO, which has a carbohydrate complement of around 40 per cent.
A high proportion of approved biopharmaceuticals now require production by animal cells. Monoclonal antibodies form the major group of newly approved and potential cell culture therapeutic products, with 19 presently on the market and more than 370 in the pipeline, by the latest estimates. Earlier problems such as the immunogenicity associated with these proteins have been solved by “humanizing” the structures and by fusing with other proteins to create novel activities. These have led to a variety of very efficacious biotherapeutic antibodies like Rituxan®, Enbrel®, Remicade®, Synagis® and Herceptin® for the treatment of various medical conditions including cancer, rheumatoid arthritis, genetic disorders and cardiovascular disease3,4.

The Production Process
Chinese hamster ovary (CHO) cells have become the standard mammalian host cells used in such production, largely because they have been well characterized and there is a history of regulatory approval for recombinant proteins produced from these cells. Other cell lines offering alternative platform technologies for production are being pursued and may offer scientific advantages. The major hurdle, however, can be to convince the regulatory authorities to accept such alternatives.
The ability to produce and select a high-producing animal cell line is key to the initial stages of the development of a bioprocess. Transfection of cells with the target gene along with an amplifiable gene, such as dihydrofolate reductase (DHFR) or glutamine synthetase (GS), has offered effective platforms for expression of the required proteins. In these systems, selective pressure is applied to the cell culture with an inhibitor of the DHFR or GS enzymes that causes an increase in the number of copies of the transfected genes including the target gene. More recently developed vectors include matrix, or scaffold, attachment regions (MAR or SAR) that ensure stable and efficient gene expression. This allows the selection of stable clones with high specific productivity over long periods of cell growth.
A producer cell may be grown in batch cultures to above 106 cells per millilitre over three to four days to allow synthesis and product secretion. Specifically designed stirred-tank bioreactors are suitable — with capacities up to 100,000 litres (the highest capacity bioreactor recently built by Genentech Inc., South San Francisco, Calif.). The strategic use of fed-batch or perfusion cultures has enabled considerable enhancement of yields from this process. For example, by directly supplying cells with a continuous, balanced nutrient feed, a fed-batch culture can now be expected to yield upward of 2 g/l of recombinant protein.
This area of bioprocess development becomes of even greater importance as some of the first generation blockbuster drugs start being produced as generics. Eleven biopharmaceuticals, with combined annual sales of $13.5 million US, are set to lose patent protection in 2006. The challenge then will be to produce bioequivalents in efficient low-cost bioprocesses.
There are several key areas of bioprocess development that need to be addressed to ensure the future success of animal cell culture processes — glycomics, serum-free media and the capacity crunch.

Why Glycomics?
Animal cells are used for biomanufacture because of their capabilities in adding carbohydrates (glycans) to synthesized proteins5. These are produced as pools of different glycoforms with varying glycan structures attached to a single peptide backbone with a known amino acid sequence. Consistent protein structures can be produced from expression of genetic vectors, a process that is well understood through recent developments in genomics and proteomics. However, directing the pool of glycan structures (glycomics) is still a bit of a mystery! Small changes in the culture conditions — such as nutrient content, pH, temperature or oxygen levels — may have a significant impact on the distribution of glycan structures found on the resulting recombinant protein. This, of course, is of major concern in trying to produce consistent biopharmaceuticals.
Functional glycomics is an expanding area of science that attempts to understand the function of specific glycoforms and may allow the production of even more efficacious drugs6. The ability to direct such structural changes by controlling the conditions of cell culture or by metabolically engineering the host cell lines is therefore of great value in bioprocess development.
For EPO, it is clear that the number of terminal sialic acid groups on the glycans determines its half-life in the blood stream and therefore, the efficacy of the drug. This has led to a new generation EPO called darbepoetin, which has the potential to incorporate more sialic acid groups and has a three times higher drug half-life. A second example of the value of functional glycomics involves Herceptin, a novel humanized antibody approved for the treatment of breast cancer. Recent work has shown that a novel glycoform of the antibody with no fucose has a 53 times higher binding capacity to a receptor that triggers its therapeutic activity.

Serum-free Media
Bovine serum was used as a supplement of cell culture media for several decades. It is a rich source of hormones, growth factors and trace elements that promote rapid cell growth, and its high albumin content ensures that the cells are well-protected from potentially adverse conditions such as pH fluctuations or shear forces. However, the mad cow crisis in the beef industry raised a concern about the use of animal serum in the production of biotherapeutics, to the extent that there is now a strong demand for cell culture formulations that are free of all animal components. The challenge that this demand poses is the ability to identify effective substitutes for all the growth-promoting factors that are present in serum.
It turns out that producer cell lines are quite fastidious in their growth requirements and that such requirements vary considerably from one cell line to another. In fact, even different clones of CHO cells may require different formulations for optimal growth. This has given rise to a strong drive to develop serum-free and animal component-free formulations that has intensified even further as the number of recombinant products increases.

The Capacity Crunch
Enbrel is a novel biotherapeutic based upon an antibody structure fused to a growth factor receptor. Developed by Immunex Corp. (now a subsidiary of Amgen Inc.,Thousand Oaks, CA), this fusion protein has proven effective in the treatment of rheumatoid arthritis. However, when this product was licensed in 2001 there was insufficient large-scale culture capacity to meet clinical demands. Not only was this capacity unavailable at Immunex, the firm was unable to find any contract manufacturers with suitable capacity for production.
This highlights a general problem that still exists today: that the pipeline of biotherapeutic products is expanding more rapidly than the world capacity for cell culture production7. It is estimated that the present world capacity for cell culture production stands at 475,000 litres, 75 per cent of which is controlled by biopharmaceutical companies and the remainder by contract manufacturers. There are plans to increase this capacity threefold in the next few years, but some estimates suggest that this will still cause a shortfall, with manufacturing demand continuing to exceed the production capacity3.
A rider to the capacity crunch problem is the personnel crunch, with a shortage of highly qualified personnel available to manage the impending demand for production of the new series of cell culture products. To become competitive in this area, Canada needs to mobilize its resources. It is hoped that initiatives like the Cell-Factory Bioprocessing Research Network (Cell-Net), a consortium organized and funded by the Natural Sciences and Engineering Research Council of Canada (NSERC) (Ottawa, ON), will be encouraged to expand to create the new generation of cell biologists, biochemical engineers and biotechnologists needed for the bright new future of biopharmaceuticals. This will ensure that Canada will maintain the world leadership in biotechnology that it established in 1921.

References:
(1)     Walsh, G. 2003. Biopharmaceuticals benchmarks. 2003. Nature/Biotechnology 21: 865-870.
(2)     Pavlou, A.K. 2003. Marketspace: Trends in biotherapeutics. Journal of Commercial Biotechnology 9: 358-363.
(3)     Molowa, D.T. and R. Mazanet. 2003. The state of biopharmaceutical manufacturing. Biotechnology Annual Review 9: 285-302.
(4)     Pavlou, A.K. 2004. The immunotherapies markets, 2003-2008. Journal of Commercial Biotechnology 10: 273-278.
(5)     Butler, M. 2004. Animal Cell Culture and Technology 2nd edition, 256 pages; publ. Bios Scientific Publishers Ltd., Oxford.
(6)     Shriver, Z., Raguram, S. and R. Sasisekharan. 2004. Glycomics: A pathway to a class of new and improved therapeutics. Nature Reviews Drug Discovery 3: 863-873.
(7)     Mallik, A., Pinkus, G.S. and S. Sheffer. 2002. Biopharma’s capacity crunch. The McKinsey Quarterly 9.

Mike Butler is a professor of Animal Cell Technology in the department of microbiology, University of Manitoba (Winnipeg, MB). His research interests lie in the industrial application of animal cells to the large-scale commercial production of biologicals such as monoclonal antibodies, recombinant proteins and viral vaccines. His laboratory has research programs to understand the control of glycosylation during the production of recombinant proteins, the metabolic management and engineering of cells in culture and the production of viruses from microcarrier culture systems. A spinoff company (Biogro Technologies Inc., Winnipeg, Man.) has been created from this work to commercialize and develop serum-free formulations for cell culture bioprocesses. Butler was a 2004 NSERC Synergy Award winner for innovative university-industry collaboration.
A rider to the capacity crunch problem is the personnel crunch, with a shortage of highly qualified personnel available to manage the impending demand for production of the new series of cell culture products. To become competitive in this area, Canada needs to mobilize its resources. It is hoped that initiatives like the Cell-Factory Bioprocessing Research Network (Cell-Net), a consortium organized and funded by the Natural Sciences and Engineering Research Council of Canada (NSERC) (Ottawa, ON), will be encouraged to expand to create the new generation of cell biologists, biochemical engineers and biotechnologists needed for the bright new future of biopharmaceuticals. This will ensure that Canada will maintain the world leadership in biotechnology that it established in 1921.

References:
(1)     Walsh, G. 2003. Biopharmaceuticals benchmarks. Nature/Biotechnology 21: 865-870.
(2)     Pavlou, A.K. 2003. Marketspace: Trends in biotherapeutics. Journal of Commercial Biotechnology 9: 358-363.
(3)     Molowa, D.T. and R. Mazanet. 2003. The state of biopharmaceutical manufacturing. Biotechnology Annual Review 9: 285-302.
(4)     Pavlou, A.K. 2004. The immunotherapies markets, 2003-2008. Journal of Commercial Biotechnology 10: 273-278.
(5)     Butler, M. 2004. Animal Cell Culture and Technology 2nd edition, 256 pages; Bios Scientific Publishers Ltd., Oxford.
(6)     Shriver, Z., S. Raguram, and R. Sasisekharan. 2004. Glycomics: A pathway to a class of new and improved therapeutics. Nature Reviews Drug Discovery 3: 863-873.
(7)     Mallik, A., G.S. Pinkus, and S. Sheffer. 2002. Biopharma’s capacity crunch. The McKinsey Quarterly 9.

Mike Butler, PhD is a professor of Animal Cell Technology in the department of microbiology, University of Manitoba (Winnipeg, MB). His research interests lie in the industrial application of animal cells to the large-scale commercial production of biologicals such as monoclonal antibodies, recombinant proteins and viral vaccines. His laboratory has research programs to understand the control of glycosylation during the production of recombinant proteins, the metabolic management and engineering of cells in culture and the production of viruses from microcarrier culture systems. A spinoff company (Biogro Technologies Inc., Winnipeg, Man.) has been created from this work to commercialize and develop serum-free formulations for cell culture bioprocesses. Butler was a 2004 NSERC Synergy Award winner for innovative university-industry collaboration.