Towards purification of adenoviral vectors based on membrane technology

Abstract

Given increasing applications of recombinant adenoviruses for gene therapy and vaccination, there is a need for highly robust and fast purification platforms for their large scale manufacture. Traditional chromatographic methods using resins as matrices have several limitations such as high-pressure drops and slow processing rates due to pore diffusion and channelling of the feed through the bed. In contrast, membrane adsorbers offer the advantage of fast, gentle, and effective isolation. Furthermore, membranes are easy to use, no column packing is needed and, when used as disposables, no cleaning validation is necessary, representing a substantial advantage to meet cGMP requirements. In this work, a strategy for purification of adenovirus vectors from cell-culture bulks fully based on membrane devices is presented. Ultrafiltration membranes with molecular weight cutoffs of 300, 500, and 750 kDa were tested for the concentration of cell-culture supernatant after an initial clarification step. The results show that the use of ultrafiltration/diafiltration membranes not only concentrates the virus but also leads to the removal of 90% of host cell DNA and proteins in the retentate. Two membrane adsorbers (Sartobind Q and Sartobind anion direct) were evaluated for adenovirus vectors capture and purification. To define the best operating conditions, the effect of pH, conductivity, and recirculation of load bulk on the recovery yield of infectious adenoviruses were evaluated. Sartobind anion direct allows for higher recovery yields (up to 62%) of infectious adenoviruses than Sartobind Q; identical ratios between total and infectious adenoviruses (TP/IP) were achieved for both membrane adsorbers. The overall recovery yield of the process is $52%; this work credits membrane technology as an alternative for the concentration and purification of adenoviruses and as a promising solution for downstream processing of other viral vectors.

Capturing of cell culture‐derived modified Vaccinia Ankara virus by ion exchange and pseudo‐affinity membrane adsorbers

“…One possibility to economize or even to eliminate the nuclease treatment in a new production process might be the application of adsorption chromatography methods, ideally using chromatography media with predominant convective material transport like membrane adsorbers (MA). There are several examples for the application of ion exchange and affinity MA for the purification of virus particles like adenoviral vectors (Peixoto et al, 2008;Sellick, 2006), Aedes aegypti densonucleosis virus (Enden et al, 2005), baculovirus (Wu et al, 2007), and influenza virus (Kalbfuss et al, 2007a;Opitz et al, 2007aOpitz et al, , 2009. With regard to the low isoelectric point (pI) of $4.0, as estimated for the Lister strain of Vaccinia virus (Douglas et al, 1969), anion exchange MA might be a promising option.…”

Sulfated membrane adsorbers for economic pseudo‐affinity capture of influenza virus particles

“…Membrane adsorbers, which may overcome such drawbacks, have been already used for affinity separation of different target molecules, for example, recombinant proteins (Cattoli et al, 2006;Mellado et al, 2007), antibodies (Boi et al, 2006b;Castilho et al, 2002;Platonova et al, 1999), lectins (Boi et al, 2006a;Sorci et al, 2006) or influenza virus particles (Opitz et al, 2007a). In addition, membrane-based ion-exchange chromatography was successfully applied for purification of several other virus species, including for example insect baculoviruses (Wu et al, 2007), mosquito-specific parvoviruses (Czermak et al, 2008;Specht et al, 2004), alphaherpesviruses (Karger et al, 1998), adenovirus vectors (Peixoto et al, 2008) or influenza viruses (Kalbfuß et al, 2007b). Furthermore, immobilized metal affinity membrane adsorption using zinc ions was applied for separation of adenoviral vectors (Lee et al, 2009).…”

Designing clinical-grade integrated strategies for the downstream processing of human mesenchymal stem cells

“…This requirement is in line with what is advised by regulatory agencies, due to the main drawbacks associated to the use of FBS, such as the variability between different lots and suppliers and the risk of contamination with animal pathogens, which may trigger an immune response upon MSC therapy [5]. Within this context, large efforts have been made towards the development of serum- and xeno- free culture medium formulations for the expansion of hMSC.…”

“…Therefore, the main aim of this work was the design of integrated methodologies (filtration and membrane technology approaches) [5] for the robust and clinical-grade downstream processing of hMSC.…”