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

Abstract

AbstractA 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 tangential‐flow 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.