Research review paperRational design and optimization of downstream processes of virus particles for biopharmaceutical applications: Current advances
Introduction
Over the last 25 years, the pharmaceutical industry has been shifting a great deal of interest and resources into the development of novel pharmaceutical molecules based on biologicals: biopharmaceuticals (Crommelin et al., 2003). Beyond the exponential market growth on monoclonal antibodies (mAbs), there is today great promise for novel biopharmaceuticals based on virus particles, either for vaccination (e.g., virus-like particles (VLPs) (Buckland, 2005)) or for gene or cell therapies (e.g., recombinant viral vectors (Ferguson et al., 2010)). These products are not only far larger than mAbs – e.g., over 107 Da for an HPV-VLP (Hanslip et al., 2006) when compared to the average 1.5 × 105 Da molecular weight of an immunoglobulin G (IgG) (Shukla et al., 2007) – but are also required to contain a well assembled three-dimensional geometry, properly characterized with the necessary subunits in the proper place and ratio and with the proper post-translational modifications of the exposed proteins. The latter being critical for instance to elicit the desired immune responses (for vaccination purposes) or to allow efficient cell target internalization and ultimately transgene expression (for gene therapy purposes).
Moreover, viral vectors should remain infective, which in some systems, as retroviral vectors, means the presence of specific viral enzymes biologically active for completion of the gene transfer process (Carmo et al., 2009). All this complexity raises both biological and technological challenges (Rodrigues et al., 2007a).
As far as the manufacturing process is concerned, there have been great achievements in the implementation of scalable systems using animal cells for improvement of product titer and quality. However, much less effort has been put to the essential downstream purification processes for the more complex biopharmaceutical particles thus constituting currently a major bottleneck.
Due to the intricate nature of these biological particles, there are critical implications on the downstream processing concerning purity, potency and quality of the final product. According to the desired final target: i) the process-derived impurities such as host cell protein (HCP) and host cell (HC) DNA contents must be below a certain limit – purity –; ii) the concentration (or titer) must be as high as achievable so that the volume of the required dose is the smallest feasible – potency –; iii) the quantity of product-derived impurities, damaged, non-functional virus particles should be as low as attainable compared to the functional virus particles — quality. The regulatory authorities – US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) – require the industry to define strict process and product guidelines that may differ depending upon the application. An infective virus, inactivated virus, VLP or viral vector to be used as a vaccine follows a set of guidelines established for vaccine products (FDA, 2010a). A viral vector as a gene therapy product needs to meet the guidelines set for cell and gene therapy medicines (FDA, 2010b). For example, for adenovirus as a gene therapy viral vector the authorities require a ratio of physical to infective virus titer below 30 (quality); however, the admissible levels of HCP and HC DNA (purity), although monitored consistently, are not requirements per se for lot release (EDQM, 2011a). On the other hand, for a human papillomavirus VLP as a vaccine, besides the mandatory immunological potency tests, the HC DNA levels need to be below 10 ng per human dose for batch release (EDQM, 2011b). The downstream processes should thus be designed to accommodate these requirements according to the final application. Often, a sensible compromise must be made between cost, throughput, and purity to meet both the quality and potency aimed at in a given pre-clinical or clinical trial. It is the goal of the integrated manufacturing process to deliver the product in large quantities (scalability), with high quality (purity) and in high titer (potency), and doing so in a cost-effective manner.
This review discusses the state of the art of the everlasting quest for the design of “the ideal” DSP for these complex biopharmaceuticals. The rising interest in the use of knowledgeable tools for process design and optimization over heuristics-based process development is discussed, addressing relevant case studies where process knowledge and/or product characterization had an impact on improving current recovery yields and productivities.
Section snippets
Current choices in DSP of complex biopharmaceuticals
The state of the art in the purification of animal cell culture-based complex biopharmaceuticals relies on membrane and chromatographic processes (Konz et al., 2008, Peixoto et al., 2007, Przybycien et al., 2004, Rodrigues et al., 2007a, Vicente et al., 2009a, Vicente et al., 2009b). These unit operations are part of some of the commonly named platform technologies for purification; prominent examples include monoclonal antibodies (mAbs) (Kelley et al., 2009, Li et al., 2009) and adenoviral
In pursuit of an ideal DSP platform
The fundamental desideratum in large scale manufacturing of any product is that the technologies and resources involved must be cost-effective. Such “tenet” is also applicable for complex biopharmaceuticals. However, as these products are designed for clinical applications, the safety concerns require complex, thus not inexpensive, DSPs.
The ideal DSP platform for virus particles must decrease the impurity levels to acceptable values while maintaining and concentrating the product in its
Outlook
Rational optimization of downstream processes for complex biopharmaceuticals, as viral vectors or VLPs, is still in its infancy. It is clear that a more knowledgeable approach shall highly reduce the “meandering” of the optimization path. With the strict demands in product purity, safety, potency and quality, QbD shall be key in the manufacturing of these biologicals. Rational tools, overviewed here into more detail for IEX process, will be crucial for a more sustained process design and
Acknowledgments
We thank Dr. Pedro Cruz (ECBio, Portugal) for enlightening discussions. We acknowledge funding from the European Commission (Baculogenes, LSHB-2006-037541 and Clinigene — Network of Excellence, LSHB-2006-018933) and the Portuguese Fundação para a Ciência e a Tecnologia (PTDC/EQU-EQU/71645/2006 and SFRH/BD/31257/2006).
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