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January 15, 2011 (Vol. 31, No. 2)

Optimizing Downstream Processing of Enveloped Viruses

Case Study Demonstrates Utility of Inventive Strategies

  • Click Image To Enlarge +
    Figure 1. (A) Downstream process flow diagram; (B) Selection of optimal TMP and retentate flow rates at the tangential flow filtration step; (C) Sartobind D run of a bind-elute anion-exchange chromatography: baculoviruses are collected in the first elution peak. [B and C reproduced from Gene Therapy, 16:766-775 with permission of Nature Publishing Group.]

    Enveloped viruses have tremendous potential in a number of areas of great interest to biotech and pharma firms including gene therapy and vaccination. These viruses, which are typically produced in animal cell systems, are expensive to manufacture. As a result, identification of biological and engineering strategies that are scalable and cost-effective is paramount.

    The complexity of these biological products, however, has hampered the development of robust downstream processes. Obtaining the quantities required for preclinical and clinical trials is especially problematic.The labile lipid membrane layer that harbors glycoproteins (often critical for infection) over the viral capsid further increases the challenges inherent in the processing of enveloped viruses.

    Scientists at the Animal Cell Technology Unit of iBET  are using recombinant baculoviruses as an enveloped virus model in order to optimize this task. Recombinant baculoviruses are widely used as vectors for the production of recombinant proteins in insect cells.

    More recently, these viruses have been gaining attention due to their emerging potential as gene-therapy vehicles. While their production in stirred bioreactors using insect cells is an established technology, downstream processing of baculoviruses intended for clinical applications is only now catching up.

    This article will discuss the evaluation of a scalable, cost-effective downstream processing strategy based on membrane processes. The evaluated process comprised three steps—depth filtration, ultra/diafiltration, and membrane sorption (Figure 1). Global recovery yields of clinical-grade material reached 40% using easy-to-scale-up technologies under cGMP guidelines. This constituted a major advance over the much lower yield and nonscalable purification process based on ultracentrifugation density gradients (Figure 1).

  • Methods

    Sf9 insect cells were grown in Sf900-II serum-free medium (Life Technologies) to produce recombinant baculoviruses. The cells were infected at a multiplicity of infection of 0.1 infective viruses per viable cell. The bioreaction was carried out using a disposable Wavebag bioreactor with a 25 L working volume. When the cell viability was lowered to about 50%, the bulk was harvested and submitted to clarification.

    Microfiltration was integrated downstream of the bioreactor via a series of two depth filters—3 µm and 0.65 µm (Sartorius Stedim Biotech). This strategy prevented baculoviruses from becoming entrapped within the pores (these rod-shaped viruses are up to 400 nm long). Tangential flow ultrafiltration and diafiltration were performed using 100 kDa Hydrosart cassettes (Sartorius Stedim Biotech). An ÄKTAcrossflow (GE Healthcare) system was used to operate each filtration step autonomously. The bulk was concentrated sixfold and diafiltered with two diafiltration volumes continuously.

    Anion-exchange membrane chromatography was used as a capture step utilizing Sartobind D membrane adsorber units (Sartorius Stedim Biotech). Elution profiles were generated with increasing NaCl concentrations up to 1.5 M NaCl on PBS equilibration buffer supplemented with Ca2+ and Mg2+. An ÄKTAexplorer 100 system (GE Healthcare) was used to perform these runs.

    Virus genomes were quantitated using real-time PCR (Roche Diagnostics) tracing a specific baculovirus gene and by TCID50 end-point dilution assay using Sf9 cells (infective titer).

    Zeta-potential was measured by dynamic light scattering (DLS) using Zetasizer NanoZS (Malvern Instruments) in low conductivity phosphate buffers; hydrodynamic size assessment was carried out using the same equipment. Surface plasmon resonance (SPR) experiments were performed with the Biacore 2000 system (GE Healthcare) at 25ºC, using 20 mM phosphate buffers with the specified NaCl supplementation and at a constant pH 6.8. Sensors chips were derivatized with diethylaminoethyl weak anion-exchanger ligand.

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