Recent Advances and Trends in the Biotechnology Industry - Development and Manufacturing of Recombinant Proteins and Antibodies

Wednesday, October 30, 2013

Manufacturing Operations
Late Stage Cell Culture

Since the early 1980s, biotechnology products have shaped the pharmaceutical industry. A large number of monoclonal antibodies and therapeutic proteins have been approved, delivering meaningful contributions to patients’ lives, and are anticipated to be the major growth driver for the industry in the upcoming years [1-5]. In 2012, the list of the top 20 best-selling drugs included 8 biologics [6] (see Table 1).

Table 1. Top 20 best-selling drugs in 2012 (modified from [6])

Mammalian cells are the expression systems of choice [7-10] due to their ability to properly fold and modify these complex proteins and antibodies. Tremendous process development efforts throughout the last decade have resulted in significantly increased manufacturing scales, product titers (up to 8g/L in fed batch processes), and recovery yields, thereby satisfying heightened market demand for existing and new products. Cell culture manufacturing scales up to 25 m3 operated in a batch, repeated batch, or fed batch mode, followed by a sequence of chromatography, filtration, and concentration steps delivering API batch sizes of up to 50 to 100 kg of protein/antibody, represent state of the art technology – a typical antibody production process is shown in Shukla and Thömmes 2010 [11-13]. Perfusion processes have been reported as an alternative manufacturing strategy to traditional large-scale fed-batch processes. Perfusion processes typically utilize smaller bioreactors in scales up to 1000 L, with high perfusion rates and process durations up to 200 days. [12, 14-23]

Since the generation of murine monoclonal antibodies via hybridoma technology by Kohler and Milstein [24], numerous milestones in the industry have been achieved, and advances in research, development, and manufacturing have been described. These include fully humanized recombinant monoclonal antibodies and the exploitation of antibody fragments for favorable characteristics, as well as the introduction of novel scaffolds based on antibody technology. Recently, the glyco-engineering of antibodies and fusion proteins are exploiting favorable modifications of antibody binding sites, as well as the antibody Fc sequence for enhanced effector function. The first non-radioactive antibody drug conjugates (e.g. Adcetris®, Kadcyla®, Mylotarg®) have received approval and will enable more targeted therapies and – in conjunction with companion diagnostics – open the gate to personalized healthcare. The version “2.0” of future proteins and antibodies based on today’s products will be a “facelift” by manipulating molecular frameworks to further improve binding specificity and efficacy, reduce needed therapeutic dose, conjugation to immunoactive substances, bi-specific binding antibodies, cytokine coupled entities, etc. A major breakthrough comparable to the first antibody technology is still pending. It should be stated that recent progress has been made in developing antibodies which interact more directly with the immune system – up-regulating its behavior towards antigens (e.g. cancer cells), in what is termed immunotherapy.

For today and tomorrow’s molecular formats, the major challenges will be funding and increased success rates in R&D. Although there is clear growth and trend towards the use of biologics, the pharmaceutical industry still faces significant challenges. Patent expiry is one of these major threats for biologics developers and manufacturers, particularly given the high R&D costs associated with bringing these biologics to market. This “patent cliff ” is well known in the small molecules world, and its impact has been felt with several drugs. In upcoming years, several biologics will run out of patent protection, and several “biosimilar” or “biobetter” manufacturers will enter the arena. In addition, increasing safety requirements, shift of significant growth to emerging markets, restricted market access, increased development costs and declining R&D productivity will put more pressure on the value and supply chain of innovation-driven companies.

However, this pressure has been vital to the industry in terms of innovating both, process and product development. Maturation of technologies and processes, higher degrees of automation, and process robustness driven by science and process management has been seen over the last decade.

In process development, high throughput robotics for both upstream and downstream process development have been vital to screen and improve cell lines, media compositions, chromatography media, etc. Increased titer results are due to improved expression vectors and media optimization supported by the higher automation degree and decreased scale (e.g. micro titer plates and tube bioreactors), allowing broader screens.

The “omics” approaches, including transcriptomics, proteomics, and metabolomics, along with the sequencing of the CHO genome have provided tools and data to support and enhance these efforts. Most of the significant improvements to date, however, have been based on the more conventional cell line/media approaches, with “omics” approaches still in search of significant success stories.

Another advancement which has increased efficiencies and enabled rapid startup of new manufacturing operations was the development and implementation of disposable / single-use technologies early on in the development processes. Now, the first concepts are wellestablished in manufacturing processes (e.g., filters, containers, culture flasks, bioreactors, etc.). With increasing titers and smart integrations of mature technologies, even fully disposable facilities have been reported. However, this is not only due to the availability of disposable technologies. The significant improvements in product titers and downstream purification yields driven by resin capacity and process throughput have enabled a highly efficient and commercially viable manufacturing based on disposables. One commercial provider of custom contract manufacturing, development services, and optimized technologies reported titers in their XD technology (integrated perfusion cell culture and product concentration by UF) utilizing a continuous cell line of up to 25g/L in a fully disposable system. Titers in more common fed batch cultures are up to 8 g/L in disposable bioreactors up to 2m3. This means that one batch derived from fully disposable systems that can be installed and started up quickly and at reduced capital cost can deliver approximately 16 kg of product into purification in an extremely small footprint – who would have thought this possible 20 years ago?

Next to bacterial fermentation for “relatively simple” recombinant proteins or antibody fragments, the work horse of the industry is still Chinese Hamster Ovary (CHO) cells. Yeast strains producing high yields of recombinant proteins such as EPO, or specific cell lines like the aforementioned continuous human cell line producing recombinant antibodies have been reported. However, these new cell culturing systems appear unlikely to replace the current work horses of the industry, E. coli bacteria and CHO cells, based on their significant successes and regulatory experience and acceptance. Non-cell culturing systems, such as transgenic animals or plants, have yet to make a significant dent in the established preference for E. coli or CHO cells.

In the late 1990s, the elimination of animal-derived raw materials both upstream (serum, peptones removed to yield chemically-defined medium) and downstream (Veggie Protein A affinity resin) delivered meaningful reductions in the virus contamination risks to cell culture processing. Additional risk mitigation was delivered through the implementation of viral clearance barriers in manufacturing processes (High Temperature Short Time heat treatment, novel virus filters, UV inactivation, etc.).

To increase efficiencies and mitigate risks, most companies have developed and implemented platform bioprocess technologies, beginning with a host cell line and media platform translated into a manufacturing platform based on standardized bioreactor platforms (including their design) and standardized purification processes (2 or 3 chromatography steps followed by formulation and filtration). The partnership with major suppliers – as in other industries – has enabled significant gains in throughput and robustness of processes based on these platforms. The processing options gained through these collaborations help the industry accelerate development work, gain knowledge, use common validation principles, and mitigate risks for scale up and technology transfers.

Today’s biopharmaceutical value and supply chains have matured. Supply chain risk mitigation through applications of mature technologies has become more and more important, as can be seen via examples of recent process contaminations having significant impact on a company’s well-being. In this case, “old” technologies such as HTST (High Temperature Short Time) heat treatment and other technologies analogous to pasteurization go through revitalization. In general, pilot and large scale manufacturing facilities are equipped with high tech sensors, sophisticated data analysis systems, and are highly automated to support process monitoring and continuous improvement. Pursuit of the PAT and QbD initiatives require enhanced analytics and data management capabilities.

While the discussion to this point has been focused on the bioprocess, it is important to note that advances and trends in pharmaceutical R&D have driven improvements in the ability to formulate, manufacture, and deliver biological drug products. Increasing desire for subcutaneous delivery of protein products has driven development of stable high concentration (100-200 mg/mL) formulations. Such applications can trigger the need to address significantly increased product viscosities, which present challenges in delivering the product. These challenges have been met both by improved formulation components as well as through improved delivery devices. Significant growth has occurred in the use of pre-filled syringe devices that can better support targeted self-administered applications (such as for arthritis indications). Development of sophisticated auto-injectors have further improved dosing simplicity for self-administration. Combination products (more than one API) have also advanced, one interesting example being the use of hyaluronidase co-formulated to facilitate subcutaneous delivery for products previously administered only intravenously. Such innovation provided for improvement in patient convenience and reduction of infusion reactions while enabling self-administration. On the protein drug product manufacturing front, manufacturers have adopted greater use of vapor phase hydrogen peroxide as a means of providing greater sterility assurance for these products which cannot be terminally sterilized.

Finally, the role of manufacturing has grown increasingly important. Manufacturing in the 21st century has to be agile, efficient and resilient. Planned and predictable performances along the supply chain are of crucial importance to guarantee uninterrupted supply to patients with high quality and acceptable financial performance. The goal for this part of the organization is to adapt to a changing portfolio of existing and new products, meeting quality and supply expectations and freeing up resources to be invested back into R&D – manufacturing has to become “externally supportive” and a strategic enabler for the industry [26-32].

What to Expect in the Next Decade

New molecular formats will be developed in pursuit of better answers to unmet medical needs. Combination products will be pursued as one means of improving patient convenience while reducing healthcare costs. Further increases in automation capability and efficiency of paperless systems for manufacturing will also be key to reducing costs and improving efficiencies. Two types of biopharmaceutical manufacturing facilities will likely be common – platform-based large scale manufacturing facilities in large-market regions, and small regional disposable plants in emerging markets. Given that large-scale (> 10 m3) plants have the ability to produce great quantities of proteins for blockbuster markets, and that more targeted (and smaller) patient populations can be addressed through moderate-scale (up to 2 m3) disposable systems, cell culture titers will not necessarily need to increase from current productivities. Resources will be spent more on improving control of protein product quality attributes to address Quality by Design and Process Analytical Technologies objectives and increasing downstream processing efficiency to drive costs down further. With increased product titers to a large extent based on increased cell culture growth, the physical characterization of bioreactors regarding mass transfer (especially CO2), mixing, and shear forces becomes more important [33-36]. A renewed focus on traditional engineering principles will be necessary. This will also be important to minimize risks for technology transfers and troubleshoot during technology transfers and process scale-up projects [37]. Expertise in modeling unit operations by Computational Fluid Dynamics and M3C (modeling, monitoring, measurement, and control) of bioprocesses via advanced approaches such as Artificial Neural Networks and Statistical Online Control will be relevant to supporting the next stage of process robustness and reliability [38-40].

Continuous processing trends should be noted. However, to date, only molecules requiring small residence time due to impact on product quality (e.g., Factor VIII, Epoitin, Factor VII, etc.) and specific expression systems have been established in commercial manufacturing processes. Recently, perfusion application in seed train for intermediate storage of large volume starter cultures and increase inoculation cell densities in production scale bioreactors to shorten operation time (subsequently leading to higher facility outputs) have been reported [41-43].

Much progress has been made in establishing reliable supply chains capable of delivering biopharmaceuticals for increasing applications that include numerous oncology and rheumatologic indications. The industry has matured significantly. While much has been achieved, unanswered questions remain. Pressures on the industry, such as the need for new molecular formats in the pipeline, evolving regulatory requirements driven by a desire to benefit from Quality by Design approaches and –most importantly – the need for global access to high quality bio-therapeutics addressing unmet medical needs at competitive costs, will drive the next decade’s achievements.

Author Biographies

Dr. Michael Pohlscheidt is Director of Manufacturing Operations at Genentech, Inc., Oceanside, CA. He received his degree in bioengineering in 2001 from the University of Applied Sciences in Aachen, Germany. His Ph.D. thesis was performed at Bayer HealthCare AG and Bayer Technology Services and guided by the University of Magdeburg, Germany (2005). From 2005 to 2010, he worked in different positions at Pharma Biotech Production & Development, Roche Diagnostics GmbH, Penzberg, Germany.

Dr. Robert Kiss is a Distinguished Engineer and the Director of Late Stage Cell Culture at Genentech, South San Francisco. He has worked in the process development and technical support of cell culture and fermentation processes for more than twenty years in the biopharmaceutical industry. He is a Fellow of the American Institute of Medical & Biological Engineers, and is a licensed professional engineer.

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