Development of a novel adenovirus purification process utilizing selective precipitation of cellular DNA

Abstract

The use of recombinant adenoviral vectors for vaccination and gene therapy requires the development of purification processes that are cost-effective, scalable, and capable of robust host cell DNA clearance. An adenovirus purification process was developed which incorporates selective precipitation of host cell DNA, enabling a reduction in the use of costly nucleases and chromatographic resins while substantially improving DNA and protein clearance capabilities. In this work, three cationic detergents were evaluated for their capacity to selectively precipitate DNA from adenovirus-containing cell lysate. Parameters including pH, sodium chloride concentration, nonionic surfactant concentration, and cell density were investigated during development of the precipitation step. In a novel application, the cationic detergent domiphen bromide was found to have superior selectivity for host cell DNA. In addition, domiphen bromide-induced precipitation of adenovirus was shown to be reversible, which reduces the importance of mixing. Precipitation of DNA in the cell lysate coupled with primary clarification resulted in 3 logs of DNA clearance and improved impurity clearance in the subsequent ultrafiltration step. As a result, nuclease treatment and/or anion exchange chromatography can be eliminated, or included exclusively to improve process robustness.

Development of a platform process for adenovirus purification that removes human set and nucleolin and provides high purity vector for gene delivery

“…These include detergent cell lysis (Peixoto et al, 2006), DNA digestion (Shabram et al, 1997), depth filter clarification (Goerke et al, 2005;Peixoto et al, 2008), Ad capture by anion exchange (Duffy et al, 2005;Peixoto et al, 2008) and Ad Characterization of a representative lot of Ad using the assays described in Materials and Methods Section. These include detergent cell lysis (Peixoto et al, 2006), DNA digestion (Shabram et al, 1997), depth filter clarification (Goerke et al, 2005;Peixoto et al, 2008), Ad capture by anion exchange (Duffy et al, 2005;Peixoto et al, 2008) and Ad Characterization of a representative lot of Ad using the assays described in Materials and Methods Section.…”

A reversed phase HPLC assay for the simultaneous quantitation of non-ionic and ionic surfactants in bioprocess intermediates

“…It was first used in determining the clearance of these surfactants by monitoring of in-process samples in the purification of an adenovirus vaccine candidate under development [13]. In this process, cell lysis is facilitated by the addition of 0.1% Triton X-100 (w/v).…”

Purification of recombinant baculoviruses for gene therapy using membrane processes

“…However, this value can be significantly decreased if an additional operation specifically aimed at reducing DNA is incorporated, for Purification of baculoviruses for gene therapy T Vicente et al example, by adding a nuclease incubation step or DNA precipitation. 22 To further characterize the rBV samples obtained after purification with the membrane adsorber, dynamic light scattering (DLS) was used to estimate the hydrodynamic size of the particles; bovine serum albumin (BSA) and trimers of gp64 (obtained as described elsewhere 23 ) were used to serve as comparison entities (internal controls) against rBV particles coming from sucrose gradient purification and from the proposed purification strategy (Figure 7a). These results show that rBVs purified using the strategy described are comparable with the size obtained from the classical sucrose gradient purified rBV sample.…”