DNA Depletion by Precipitation in the Purification of Cell Culture‐Derived Influenza Vaccines

Abstract

A pilot study for the purification of cell culture-derived human influenza virus is presented, which focuses on the early removal of DNA by precipitation. Strains of influenza virus were propagated using Madin Darby canine kidney cells as a host. A harvesting time of about 72 h postinfection was chosen to minimize the level of impurities (host cell DNA and protein). Cell culture supernatant was clarified by centrifugation and the performance of this operation was characterized on the basis of Sigma theory. An average clarification efficiency of 93 % (based on turbidity) and a product yield of 85 % (based on hemagglutination activity) were obtained at a load of 1.6 · 10 -8 m s -1. Furthermore, the applicability of Sigma theory for scale-up studies using two different laboratory centrifuges was verified. Selective precipitation of DNA was achieved by the addition of polyethyleneimine (PEI). Full factorial design was applied to optimize selectivity considering pH, ionic strength, and the concentration and molecular weight of PEI. Under optimized conditions, treatment with PEI resulted in a reduction of DNA to 15 % of the initial amount, while 86 % of virions (based on neuraminidase activity) were recovered. The subsequent concentration of virions was realized by tangentialflow ultrafiltration. Recovery based on hemagglutination activity was determined to 63 % on average. Including the previous precipitation step, overall reduction in DNA after tangential-flow ultrafiltration was 500-fold. These results indicate that the suggested unit operations are suited for the early depletion of DNA in cell culture-derived influenza vaccine production.

Improved virus purification processes for vaccines and gene therapy

“…For example, it was demonstrated that fine adjustment of the precipitant concentration enables up to 90% DNA removal with more than 90% adenovirus recovery (Goerke et al, ). Kröber et al () implemented a DNA precipitation method for influenza vaccine: polyethyleneimine (PEI) was used, and the optimal conditions were obtained through a DoE approach achieving 85% DNA removal. Selective DNA precipitation has the advantage of being inexpensive, easily scalable—precipitated DNA can be removed by depth filtration—and suitable for high cell density processes.…”

Manufacturing Bacteriophages (Part 1 of 2): Cell Line Development, Upstream, and Downstream Considerations

“…Purification studies have been conducted which demonstrate the utility of precipitation compared to centrifugation for viral vectors such as bovine rotavirus and AAV [78]. In addition, rather than precipitating the virus itself, some researchers have instead precipitated host cell proteins and nucleic acids [75,79]. Like the commonly-used CsCl 2 centrifugation method, however, precipitation may also reduce phage activity (measured in PFU/mL) [74].…”

“…Like the commonly-used CsCl 2 centrifugation method, however, precipitation may also reduce phage activity (measured in PFU/mL) [74]. Another potential drawback of precipitation was elucidated in the Kröber et al study with influenza viruses, which found that the isoelectric point of the particles contributed to product losses by co-precipitation with cationic polymers [75,79]. Precipitation may be improved for utility with phages by enhancing process robustness and identifying nontoxic compounds to be applied in pharmaceutical production [75].…”