June 1, 2007 (Vol. 27, No. 11)
Susan Aldridge, Ph.D.
Researchers Discuss Improvements for Processing and Manufacturing Biologics
The recent “BioProcess International™ Conference and Exhibition,” held in Paris, provided a forum for updates in manufacturing and process development. The world of biotech, monoclonals in particular, experienced bad press in March 2006, when six Phase I volunteers suffered extreme adverse reactions to TeGenero’s TGN 1412 at London’s Northwick Park Hospital. “This was a unique event in the history of clinical trials,” said Gordon Duff, Ph.D., M.D., of the University of Sheffield in his analysis of the incident and its implications for biologics development.
TGN 1412 is a monoclonal that activates T cells in a superagonist manner and was intended as a treatment for autoimmune disease, including rheumatoid arthritis and leukemia. According to an investigation by Medicines and Healthcare Products Regulatory Agency, no manufacturing issues were actually involved in the TGN 1412 catastrophe—it was a mode of action problem, with the antibody inducing a cytokine storm in the volunteers.
Dr. Duff chaired the group set up by the U.K. Secretary of State for Health to look into the matter and to recommend what more should be done in the transition from preclinical to Phase I for certain biologics. The group looked at molecules acting with novel mechanisms, those with highly species-specific activity (where it is hard to extrapolate from animal data), and those directed toward the immune system. The group, reporting in December 2006, made 22 recommendations, which could find their way into new EMEA regulations regarding first time in humans trials. The recomendations included more sharing of information, a broader approach to dose calculation, sequential administration of a higher risk drug to participants, and inspection and accreditation of centers where such trials are carried out.
Lest the industry regards this as yet another barrier, Dr. Duff said, “we believe our recommendations will increase the safety of the first human volunteers in trials of biologics with higher potency, without stifling innovation in areas of unmet medical need.”
With thousands of new targets now under investigation, the number of high-risk new biologics is sure to increase. There have always been issues around the safety of biologic drugs—first with contamination of material that led to cases of HIV and hepatitis C from blood products, Creutzfeldt-Jakob disease from growth hormone, then immunogenicity, and now, TGN 1412’s overpotency.
Process Changes in mAB Glycosylation
Meanwhile, Keith Wilson, Ph.D., manager of biopharmaceutical analytical development for GlaxoSmithKline R&D (www.gsk.com), U.K., described the impact of process change on mAb glycosylation. Changes often occur when one process is used to get material to GLP quickly but another optimized process is used to get first into human (FIH) material. The GLP material must be at least representative of FIH material.
Investigation of glycosylation patterns can be an important production quality parameter, Dr. Wilson noted. GSK uses an orthogonal approach to glycoprotein analysis: oligosaccharide profiling with HPLC/CE, an analysis of the nonglycosylation heavy chain with CE/SDS, and then total carbohydrate content with HPLC. This allows thorough investigation of the common oligosaccharide patterns found in mAbs.
In one example, a cell line was changed after GLP but before FIH. This change showed differences in a nonglycosylated heavy chain that corresponded with the total carbohydrate content. “This caused some discussion within the company,” reported Dr. Wilson. “We carried out an impact assessment and found no significant differences in pharmacokinetics in guinea pigs and no differences in tertiary structure of the antibody.” On submission to a European regulatory agency, there were no queries to answer and on beginning clinical trials with this antibody, there have been no issues, although monitoring for immunogenicity is ongoing.
Glycosylation differences are of course of interest to regulatory authorities in the context of biosimilar manufacturing. Chris Holloway, group director regulatory affairs at ERA Consulting, discussed differences in the regulatory situation for biosimilars between the EU and the U.S. The EMEA has created a marketing authorization route with guidelines for specific products. It is believed that several products are now up for review, following the approval of Omnitrop and Valtropin and the rejection of Alpheon.
In the U.S., there is much to gain and lose from biosimilars. There is currently massive lobbying on the issue as the amendment to the Public Health Service Act is debated.
Holloway has analyzed scientific aspects of the European versus U.S. approach to regulation and notes that in the latter, “the term similar is a broad umbrella term.” There is also more of an emphasis on postmarketing trials in Europe but in the U.S., this appears only to be proposed in particular cases. It is not a condition of approval. “There appears to be a looser interpretation of the word comparability and more limitations upon the regulatory agency,” Holloway concluded.
Improving Protein Purification
“Should we just make things bigger or go for alternatives?” asked Duncan Low, Ph.D., scientific director, process development, at Amgen (www.amgen.com). The question is whether existing technology can actually deal with more than 100 kg lots, noting that there has been a shift from concern over manufacturing capacity to concern over costs. Purification is the most significant factor in cost of goods, with protein A and viral inactivation being the most expensive steps, according to data from Brian Kelley, Ph.D., director of purification process development at Genetics Institute-Wyeth.
Dr. Low described how he sees the future of mAb purification. Cell culture yields are likely to be more than 10 g per liter in the near future, thanks to greater cell density, which also leads to more host cell protein and more debris. He thinks that protein A may be approaching its performance ceiling of 50 g per liter and a flow rate of 750 cm per hour flow rate.
Alternatives to protein A include bacterial IgG binding protein, lectins, immunoaffinity columns, single domain antibodies (highly selective and the closest to protein A), and synthetic ligands. Tests, however, show that selectivity decreases as complexity of the ligand decreases, but in economic terms, the less complex ligands are cheaper. Engineering solutions to the cost problem might include simulated moving beds instead of columns, which leads to significant savings in resin costs.
Alternatives to chromatography include precipitation, crystallization, magnetic separation, and aqueous two-phase systems. Amgen carried out a comparison of Protein A against precipitation that suggested that the latter gave equivalent or better performance with a clean product. Membranes too can play a role in viral inactivation and in many other processes.
“In the next five years, there will be incremental improvements such as high-throughput resins and increases in selectivity, and we will be asking questions about chromatography and whether there is room for nonchromatography modalities,” Dr. Low concluded
The need for higher capacity and greater selectivity during protein purification could be addressed by rational affinity ligand design, which is being developed by Chris Lowe, Ph.D., director of the Institute of Biotechnology at the University of Cambridge. The institute uses molecular modeling to find ligands that may bind to at least part of the target protein binding site; these are then synthesized and immobilized on a column. Those that do bind target protein can then be optimized, with those combining high-binding capacity with high-elution capacity being the most promising.
mAbs are clearly major targets for this work, and experiments on a synthetic ligand version of both protein A and protein L have been carried out.
Meanwhile, Dr. Lowe has also used the synthetic ligand approach, in application to NovoNordisk’s (www.novonordisk.com) Factor VIIa, to replace the antibody in an immunochromatography purification step with a small molecule alternative. “This antibody saturates its capacity for target protein rapidly,” Dr Lowe said. “The synthetic ligands proved to have a higher capacity, with the final choice molecule having 35 times more binding capacity than the antibody.”
More recently, Dr. Lowe’s team has been targeting glycoforms, looking for synthetic ligands that will bind to proteins with specific glycosylation patterns. This would enable a company to pull out a particular glycoform from a product, something which is likely to gain the approval of the regulatory authorities. Finally, they are also developing ligands for prion proteins that could help in removal of this form of contamination and are interested in 3-D ligands, which can bind in an even more specific way to their target.
Biologics in China
The meeting reflected the global nature of today’s bioprocessing with Yuling Li, senior director of biopharmaceutical development for Human Genome Sciences (HGI; www.hgsi.com) and president of the Chinese Biopharmaceutical Association-USA, describing current opportunities and challenges for biopharmaceutical development in China. There are over 400 companies involved in biotech in China, with a growth rate of 19.2% (compared with 4–5% in the U.S.). State funding for total R&D will increase to $112 billion by 2020. “This is a significant increase,” Li pointed out. “Biotech is a priority.”
At present, China has over 100 science and technology parks and there will be 30 more by 2010, underlining a strong commitment to translational medicine and development of innovative biopharmaceutical products. The government is also offering incentives for business like tax breaks, import/export exemptions, simplified customs regulations.
China is now adopting and enforcing patent law and tightening its regulatory framework, which may end up closer to the European rather than U.S. model, according to Li. In manufacturing, China has some good microbial capacity, but mammalian is still missing. This area could provide opportunities for contract manufacturing. GMP was introduced in 1998, but there are still some gaps in achieving it everywhere.
Sharpening Manufacturing’s Image
Manufacturing is still perceived as dull compared to drug discovery, leading to it being undervalued and underinvested. Kevin Cox, Ph.D., director and president of Avecia Biologics (www.avecia.com), U.K., warned that companies involved in biomanufacturing need to do more to sharpen up their image. “People do not give due care and attention to manufacturing, but a poorly thought-out manufacturing strategy will destroy value,” he said.
Dr. Cox would like to see more integration of technologies in manufacturing, process, and analysis. The introduction of membrane separation, continuous processing, in-line dilution, and in-line testing could make a real difference to efficiency. The introduction of new products beyond mAbs, such as cell-based vaccines, mAb fragments, RNAi, and stem cells, will lead to a need for smaller, multiproduct facilities.
“As an industry, we rarely share problems and issues, although in reality we are all facing the same ones,” Dr. Cox reported. “We do not yet present a strong coordinated voice to the regulators.” However, bioprocessUK has begun to address some of these networking issues and is already seeing positive results, and he urged delegates to learn from their example.