Elsevier

Biotechnology Advances

Volume 29, Issue 6, November–December 2011, Pages 869-878
Biotechnology Advances

Research review paper
Rational design and optimization of downstream processes of virus particles for biopharmaceutical applications: Current advances

https://doi.org/10.1016/j.biotechadv.2011.07.004Get rights and content

Abstract

The advent of advanced therapies in the pharmaceutical industry has moved the spotlight into virus-like particles and viral vectors produced in cell culture holding great promise in a myriad of clinical targets, including cancer prophylaxis and treatment. Even though a couple of cases have reached the clinic, these products have yet to overcome a number of biological and technological challenges before broad utilization. Concerning the manufacturing processes, there is significant research focusing on the optimization of current cell culture systems and, more recently, on developing scalable downstream processes to generate material for pre-clinical and clinical trials. We review the current options for downstream processing of these complex biopharmaceuticals and underline current advances on knowledge-based toolboxes proposed for rational optimization of their processing. Rational tools developed to increase the yet scarce knowledge on the purification processes of complex biologicals are discussed as alternative to empirical, “black-boxed” based strategies classically used for process development. Innovative methodologies based on surface plasmon resonance, dynamic light scattering, scale-down high-throughput screening and mathematical modeling for supporting ion-exchange chromatography show great potential for a more efficient and cost-effective process design, optimization and equipment prototyping.

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).

References (117)

  • F. Dismer et al.

    Effects of ionic strength and mobile phase ph on the binding orientation of lysozyme on different ion-exchange adsorbents

    J Chromatogr A

    (2008)
  • E. Dormond et al.

    An efficient process for the purification of helper-dependent adenoviral vector and removal of helper virus by iodixanol ultracentrifugation

    J Virol Methods

    (2010)
  • M.R. Etzel et al.

    Viral clearance using monoliths

    J Chromatogr A

    (2009)
  • T. Gotoh et al.

    Proteolytic activity and recombinant protein production in virus-infected sf-9 insect cell cultures supplemented with carboxyl and cysteine protease inhibitors

    J Biosci Bioeng

    (2001)
  • D.L. Grzenia et al.

    Tangential flow filtration for virus purification

    J Membr Sci

    (2008)
  • I. Gutiérrez-Aguirre et al.

    Concentrating rotaviruses from water samples using monolithic chromatographic supports

    J Chromatogr A

    (2009)
  • R. Hjorth

    Expanded-bed adsorption in industrial bioprocessing: recent developments

    Trends Biotechnol

    (1997)
  • A. Jungbauer et al.

    Polymethacrylate monoliths for preparative and industrial separation of biomolecular assemblies

    J Chromatogr A

    (2008)
  • B. Kalbfuss et al.

    Direct capture of influenza a virus from cell culture supernatant with sartobind anion-exchange membrane adsorbers

    J Membr Sci

    (2007)
  • P. Kramberger et al.

    Concentration of plant viruses using monolithic chromatographic supports

    J Virol Methods

    (2004)
  • A. Lyddiatt

    Process chromatography: current constraints and future options for the adsorptive recovery of bioproducts

    Curr Opin Biotechnol

    (2002)
  • H. Mach et al.

    Disassembly and reassembly of yeast-derived recombinant human papillomavirus virus-like particles (hpv vlps)

    J Pharm Sci

    (2006)
  • M.C. Mellado et al.

    Purification of recombinant rotavirus vp7 glycoprotein for the study of in vitro rotavirus-like particles assembly

    J Chromatogr B Analyt Technol Biomed Life Sci

    (2008)
  • J.M. Mollerup et al.

    Quality by design — thermodynamic modelling of chromatographic separation of proteins

    J Chromatogr A

    (2008)
  • D.P. Nayak et al.

    Downstream processing of mdck cell-derived equine influenza virus

    J Chromatogr B Analyt Technol Biomed Life Sci

    (2005)
  • B.K. Nfor et al.

    Rational and systematic protein purification process development: the next generation

    Trends Biotechnol

    (2009)
  • L. Opitz et al.

    Lectin-affinity chromatography for downstream processing of mdck cell culture derived human influenza a viruses

    Vaccine

    (2007)
  • L. Opitz et al.

    Capture of cell culture-derived influenza virus by lectins: strain independent, but host cell dependent

    J Virol Methods

    (2008)
  • C. Peixoto et al.

    Purification of adenoviral vectors using expanded bed chromatography

    J Virol Methods

    (2006)
  • C. Peixoto et al.

    Downstream processing of triple layered rotavirus like particles

    J Biotechnol

    (2007)
  • M. Prashad et al.

    Depth filtration: cell clarification of bioreactor offloads

    Filtr Sep

    (2006)
  • T.M. Przybycien et al.

    Alternative bioseparation operations: life beyond packed-bed chromatography

    Curr Opin Biotechnol

    (2004)
  • N.S. Pujar et al.

    Electrostatic effects on protein partitioning in size-exclusion chromatography and membrane ultrafiltration

    J Chromatogr A

    (1998)
  • T. Rodrigues et al.

    Screening anion-exchange chromatographic matrices for isolation of onco-retroviral vectors

    J Chromatogr B

    (2006)
  • T. Rodrigues et al.

    Purification of retroviral vectors for clinical application: biological implications and technological challenges

    J Biotechnol

    (2007)
  • D.K. Roper

    Determining surface plasmon resonance response factors for deposition onto three-dimensional surfaces

    Chem Eng Sci

    (2007)
  • D.K. Roper et al.

    Adenovirus type 5 intrinsic adsorption rates measured by surface plasmon resonance

    Anal Biochem

    (2006)
  • K. Saha et al.

    A simple method for obtaining highly viable virus from culture supernatant

    J Virol Methods

    (1994)
  • C.M. Schaldach et al.

    The influence of ionic strength on the interaction of viruses with charged surfaces under environmental conditions

    J Colloid Interface Sci

    (2006)
  • S. Schubert et al.

    Investigation of the interaction mechanism of the recombinant human antibody mdj8 and its fragments with chromatographic apatite phases

    J Chromatogr A

    (2009)
  • A.A. Shukla et al.

    Downstream processing of monoclonal antibodies — application of platform approaches

    J Chromatogr B Analyt Technol Biomed Life Sci

    (2007)
  • E.I. Trilisky et al.

    Sorption processes in ion-exchange chromatography of viruses

    J Chromatogr A

    (2007)
  • E.I. Trilisky et al.

    Relation of structure to performance characteristics of monolithic and perfusive stationary phases

    J Chromatogr A

    (2009)
  • R. van Reis et al.

    Bioprocess membrane technology

    J Membr Sci

    (2007)
  • T. Vicente et al.

    Anion-exchange membrane chromatography for purification of rotavirus-like particles

    J Membr Sci

    (2008)
  • M. Aboud et al.

    Rapid purification of extracellular and intracellular moloney murine leukemia virus

    Arch Virol

    (1982)
  • E. Ayuso et al.

    High aav vector purity results in serotype- and tissue-independent enhancement of transduction efficiency

    Gene Ther

    (2010)
  • J. Barsoum

    Concentration of recombinant baculovirus by cation-exchange chromatography

    Biotechniques

    (1999)
  • C.A. Brooks et al.

    Steric mass-action ion exchange: displacement profiles and induced salt gradients

    AIChE J

    (1992)
  • B.C. Buckland

    The process development challenge for a new vaccine

    Nat Med

    (2005)
  • Cited by (0)

    View full text