Purification of recombinant baculoviruses for gene therapy using membrane processes

Abstract

Recombinant baculoviruses (rBVs) are widely used as vectors for the production of recombinant proteins in insect cells. More recently, these viral vectors have been gaining increasing attention due to their emerging potential as gene therapy vehicles to mammalian cells. Their production in stirred bioreactors using insect cells is an established technology; however, the downstream processing (DSP) of baculoviruses envisaged for clinical applications is still poorly developed. In the present work, the recovery and purification of rBVs aiming at injectable-grade virus batches for gene therapy trials was studied. A complete downstream process comprising three steps-depth filtration, ultra/ diafiltration and membrane sorption-was successfully developed. Optimal operational conditions for each individual step were achieved yielding a scalable DSP for rBVs as vectors for gene therapy. The processing route designed hereby presents global recovery yields reaching 40% (at purities over 98%) and, most importantly, relies on technologies easy to transfer to process scales under cGMP guidelines.

Purification of a recombinant baculovirus of Autographa californica M nucleopolyhedrovirus by ion exchange membrane chromatography

“…Insect viruses are central components of current manufacturing processes producing recombinant proteins such as enzymes (Vicente et al, 2010;Vicente et al, 2009) and human or animal vaccines (Justice et al, 2011) at industrial scale (virus cultures in the order of several m 3 /day, Boehringer Ingelheim Vetmedica). For decades large scale applications of biopesticides (in the order of 10 4 km 2 /year (Moscardi, 1999)) have made use of insect viruses targeting specifically their hosts (Mishra, 1998;Moscardi, 1999;Oliveira et al, 2006).…”

“…This includes the use of ultrafiltration membranes, ion exchange membranes and resin-based chromatography (Barsoum, 1999;Michalsky et al, 2009;Transfiguracion et al, 2011;Wu et al, 2007). However, the purification of AcMPNV is developed poorly (Vicente et al, 2010;Vicente et al, 2009) and materials and processes for the efficient downstream processing of baculoviruses for clinical applications is sought (Vicente et al, 2010;Vicente et al, 2009). …”

Control and optimization of solute transport in a thin porous tube

“…Crossflow filtration achieves this by pumping fluid containing suspended particles or dissolved solute down a tube with porous walls (also referred to as a membrane), and is used in a broad set of applications including water purification, protein separation for gene therapies, soluble antibiotic extraction, blood fractionation, and tissue-engineering bioreactors. [1][2][3][4][5] By controlling the membrane permeability and pressure drop down the tube relative to that across the membrane, the proportion of fluid that permeates across the membrane can be prescribed. This enables separation of particles that are too large to pass through the membrane pores (for example, in water purification processes), as well as controlled delivery of solutes to the extra-membrane space (such as in tissue-engineering applications).…”

Development and validation of a HPLC method for the quantification of baculovirus particles

“…For example, the amount of BV added to the process can drastically affect the production capacity of the system as well as the overall complexity of the process. For this reason, a great deal of effort has been put into investigating the effect of the multiplicity of infection on the production [6][7][8][9][10] as well as into methods to purify [11][12][13] concentrate [14,15] and quantify [16] these viral vectors.…”

“…Given the increased interest in baculoviruses [19] as well as our experience in purifying [12,20,21] and quantifying viruses [22][23][24][25] using chromatography, we have investigated the quantification of BV using high performance liquid chromatography (HPLC). BV has already been shown to be captured from solution using both anion and cation exchange materials [11,13,26], however, the use of HPLC for the quantification of BV has not been explored. This may be due to the low absorbance signals associated with baculovirus when monitoring the eluent at 260 and 280 nm.…”