Maximizing Pipeline Value and Operational Efficiency through High Throughput Protein Expression

By Bertrand C. Liang, M.D., Ph.D., M.B.A. and Patrick K. Lucy

At the core of every drug development program is the challenge to successfully express active, high quality product, and this is especially true of biologics. Indeed, very frequently drug developers are faced with protein expression issues, struggling to express a specific protein for months (and sometimes years) with varying degrees of success; low or no expression, insoluble expression, proteolytic clipping of the expressed protein or a combination thereof are examples of the hurdles encountered in product development efforts. Such challenges in protein expression are a major source of biopharmaceutical development delays, particularly as production strain development is typically performed in a linear and iterative fashion. As these delays often occur in the discovery and preclinical development stages of a product’s life cycle, the impact often goes unnoticed or is accepted as the status quo in the industry.

It has been previously documented that delays in drug development effect value loss of at least one million dollars per day1. Moreover, as the biopharmaceutical industry develops more advanced protein engineering technologies, lead molecules are becoming more complex and diverse; hence, the expression of these molecules is and will be much more challenging for legacy protein expression hosts. Additionally, the recent renaissance of subunit vaccines in response to environmental and man-made threats has presented the industry with many antigens typically very difficult to express. This suggests that drug developers face an ever expanding set of challenges, not only in establishing proof-of-concept and mechanism of action for the respective molecules within their portfolios, but also the ability to express such proteins in sufficient quantity, quality and efficiency for robust evaluation. This article will focus on delays caused by protein expression, specifically in early biopharmaceutical development, and will elaborate upon a very powerful technology-based solution, resulting in much more efficient and fruitful product pipeline and product development processes.

At the outset of a non-glycosylated protein expression effort, scientists typically seek out the lowest nominal cost and most accessible expression host to produce the protein of interest. Most often, the host selected is a default option, Escherichia coli (E. coli). E.coli is a Gram-negative, biosafety level one organism typically used in most research laboratories for the recombinant production of heterologous proteins. While most scientists are very familiar with the technical attributes of the E. coli organism from earlier experiences in the academic and/or industry environments, most initially are unfamiliar with the limited success rate of the organism in producing viable quantities of active, high quality recombinant protein. In fact, in more than 75% of cases, the protein expression result achieved with E.coli is insufficient to move a program forward.2-5 Moreover, the toolbox of protein expression elements available for and modifiable to E.coli is limited, and those elements that are available can be incompatible with other elements. These issues limit the degrees of freedom available to the drug developer; hence, a scientist beginning protein expression efforts with E.coli would expect to require other alternatives in the great majority of instances. In some cases, the lack of active, soluble protein may result in expression as an insoluble inclusion body. While suboptimal, since discovery scientists often require only small amounts of protein to complete their studies, refolding of the insoluble protein is often performed. While refolding insoluble protein may address a near term need (with delays), it will usually present challenges when product demand increases as development efforts progress. Unfortunately, this approach defers opportunity (and generates higher real) costs to later and more expensive stages of drug development, manifest by production challenges, inconsistent product performance due to slight variances in the refold process, or having to produce the protein in a different host that more effectively produces the protein of interest. These efforts create delays and cost issues, and may result in ambiguity of performance of a particular protein due to heterogeneity or other quality issues associated with post-translational processing.

Often, as a result of these issues, a development scientist abandons efforts to successfully express a protein in E.coli. Subsequent approaches include evaluating lower eukaryotic systems, such as a variety of yeast host systems and/or baculovirus hosts, which again increase the time required to progress programs expeditiously through development and, thereby delaying time to commercialization and revenue recognition. These host organisms present yet another challenge with regard to non-glycosylated target proteins; expression in eukaryotic organisms results in unrequired glycosylation of the protein being expressed, which not only could impact the performance of the expressed protein by masking potential binding sites, but again create heterogeneity, manifest as the batch to batch variation of the glycosylation pattern and ultimately affecting product performance. This has been seen in recent cases with eukaryotic hosts, and is the source of significant delays and costs, particularly when later in the development value chain.6 Such challenges may be avoided through the selection of a protein expression platform that maintains a high success rate of producing soluble and active protein throughout the product development life cycle, from discovery through commercialization.

The Pfēnex Expression Technology™ platform offers a robust solution to the real and opportunity costs incurred through the maintenance of several recombinant expression platforms (as noted) and the continued screening of each of the platforms to arrive at a viable protein expression solution. For a multitude of reasons, including cost of goods, quality and speed, non-glycosylated proteins are best produced by a prokaryotic host. This ensures the protein of interest can be produced avoiding unnecessary and often heterogeneous post-translational modifications. After screening in excess of eighty proteins that failed to express successfully in E. coli, and in most cases both E.coli and yeast, the Pfēnex Expression Technology platform has maintained a success rate approaching 90%. The platform expresses proteins at high titers, and high quality; a production strain capable of producing soluble active protein can be identified within five weeks. The platform combines a powerful Pseudomonas fluorescens production organism, a vast array of expression components and unique engineered host strains, with an advanced, unique, robotic strain screening platform to achieve this unprecedented success rate. This robotically-enabled combinatorial approach allows Pfenex scientists to screen thousands of unique expression strains in parallel within five weeks while generating titer, functionality and quality data for the expressed protein. The platform also allows for the expression of a single or several genes of interest in parallel thus supporting and integrating the entire product development value chain, including discovery, development and manufacturing needs.

A recent case study presented at the Protein Engineering Summit (PEGS) in Boston by Georg Klima, Boehringer Ingelheim’s Head of Process Science – Microbial, illustrates the power of the parallel screening approach employed by Pfenex Inc.6 The case study details the expression of a Fab fragment through four different approaches including E. coli (periplasmic), E.coli (inclusion body), Pichia pastoris and Pseudomonas fluorescens (Pfēnex Expression Technology). The E.coli expression experiments were performed by Boehringer Ingelheim, the Pichia pastoris experiments were performed by an unnamed technology provider, and Pseudomonas fluorescens expression was performed by Pfenex Inc. The results of the study revealed Pfenex was successful not only in delivering 500 mg of soluble, active, high quality, highly purified protein for pre-clinical evaluation, but was able to do so within ten weeks of receipt of the coding gene; all three other expression approaches failed to express the protein. The value to the drug developer is clear; there are significant opportunity and real costs associated with using the traditional linear, and iterative approach to product protein production. Indeed, data derived from Pfenex partners suggest development program delays in the range of five to 12 months on programs which have utilized such a serial approach. The real costs of such delays, including materials, FTE’s and facility costs are in the millions of dollars and certainly millions of dollars more due to the delays of a product reaching commercialization stage. This value loss is in addition to that due to halting potentially promising programs because of an inability to produce an adequate amount of high quality product to evaluate and/or progress to the ensuing stage of development. As a result, utilizing a platform such as Pfēnex Expression Technology earlier and throughout the development program provides tangible benefits not only in expediting programs by efficient protein production, but also by allowing programs to avoid attrition due to inability to produce material.

Hence, organizations developing protein based therapeutics and vaccines incur significant real and opportunity cost in the pursuit of a viable short and long term expression host capable of producing high titers of soluble active protein. While early discovery efforts are fueled by the rapid acquisition of active protein, further development efforts are sustained by repeated and consistent production of active protein throughout the development lifecycle. Status quo legacy expression hosts simply do not have the technical depth to meet the expression needs of today’s complex protein based biopharmaceuticals. Employing the Pfēnex Expression Technology platform early in the development lifecycle of a biopharmaceutical will result in the potential progression of more programs, and more rapidity through development, resulting in a significant reduction of real and opportunity costs.

References:
1) Maloff, B. (1999) Partnering for success – Performance measurements for sponsors, contract research organizations and the site management organizations. Drug Information Journal 33, 655-661.
2) Graslund, S. et al. Protein production and purification, Nature Methods 5:135-146 (2008)
3) Berrow, N. S. et al. Recombinant protein expression and solubility screening in Escherichia coli: a comparative study. Biological Crystallography. 62: 1218-1226 (2006)
4) Gillette, W. K. et al. Pooled ORF Expression Technology (POET), Molecular and Cellular Proteomics, 4: 1657-1652 (2005)
5) Service, R.F. Tapping DNA for structures produces a trickle, Science 298:948-950 (2002)
7) Taylor, N. Myozyme Becomes Lumizyme After Biologics Scale Up, in-Pharma Technologist.com, February 16, 2009
6) http://www.pfenex.com/admin/uploads/BII_Pfenex_PEGS_2011.pdf

About The Authors

Bertrand C. Liang, M.D., Ph.D., M.B.A.
Chief Executive Officer
Bert Liang is a serial entrepreneur, scientist and clinician who has led efforts in the academic, industry and private equity/venture settings. He is CEO of Pfēnex Inc., a protein company providing solutions for lead therapeutic, reagent, biodefense and biosimilar product needs via strain engineering and production expertise. He is also a General Partner at Forward Medical Sciences/Forward Ventures, a venture capital firm specializing in health care investments particularly partnered with industry. He was previously Vice Chairman at Paramount Biosciences, where he led the private equity activities globally (from Shanghai to Frankfurt), President and CEO of Tracon Pharma (an oncology-focused portfolio company), and Vice President and Site Head, Development (Pre-clinical and Clinical), as well as Vice President, New Ventures (partnered with MPM Capital), at Biogen Idec. Dr. Liang joined Idec from Amgen, where he led the development to approval of various cytokines, including Neulasta® whose sales have eclipsed $8B since introduction into the market in 2002. Dr. Liang serves on the Board of Directors of several organizations, including Tracon Pharma and La Jolla Pharmaceuticals, and he previously held academic positions at the National Cancer Institute, University of Colorado and University of Vermont, where he headed Human Medical Genetics.

Dr. Liang received his medical degree from Northwestern Medical School, his doctorate in molecular biology from the University of Bolton, and attended business school at Regis University and the MIT Sloan School of Management. Dr. Liang trained in internal medicine at Brown University, Neurology and Oncology at the University of Michigan, with post-doctoral studies in molecular biology and genetics at the National Center for Human Genome Sciences and National Cancer Institute of the National Institutes of Health.

Patrick K. Lucy
Vice President of Business Development
Mr. Lucy played a leadership role on the original team that developed Pfēnex Expression Technology™ and subsequently led the successful commercial launch of the platform in 2004. He has led the negotiation and completion of numerous technology partnering, licensing, manufacturing and collaboration agreements with the federal government and pharmaceutical companies worldwide. Mr. Lucy is experienced in the areas of alliance management, licensing, intellectual property, biopharmaceutical operations, and biopharmaceutical facility design, construction and validation.

Prior to his role in Pfēnex, Mr. Lucy was employed by The Dow Chemical Company for nine years in business development roles for the Dowpharma business unit. Mr. Lucy has been in the biotechnology industry for over eighteen years and was previously employed by Collaborative BioAlliance, Inc., Lonza Biologics, Celltech Biologics and Repligen Corporation.

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