Use of protamine sulphate for elimination of substrate DNA in poliovaccines produced on continuous cell lines

Amosenko, F. A.; Svitkin, Yuri V.; Popova, V.D.; Terletskaya, E.N.; Тимофеев, А В; Elbert, L. B.; Lashkevich, V A; Drozdov, S. G.

doi:10.1016/0264-410x(91)90156-z

Cited by 5 publications

(3 citation statements)

References 2 publications

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“…One common approach seems to be the digestion of hcDNA by nuclease-treatment Transfiguracion et al, 2003). In other studies hcDNA was precipitated selectively from preparations of poliovirus using protamine sulphate or from adenovirus using cationic detergents (Amosenko et al, 1991;Goerke et al, 2005). Kumar et al (2002) reported the clearance of hcDNA from cell culture-derived rabies vaccine using a second density gradient centrifugation step.…”

Section: Introductionmentioning

confidence: 99%

Purification of cell culture‐derived human influenza A virus by size‐exclusion and anion‐exchange chromatography

Kalbfuss

Wolff

Morenweiser³

et al. 2006

Biotech & Bioengineering

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A process comprising of size-exclusion chromatography (SEC) and anion-exchange chromatography (AEC) was investigated for downstream processing of cell culture-derived influenza A virus. Human influenza virus A/PR/8/34 (H1N1) was propagated in serum-free medium using MDCK cells as a host. Concentrates of the virus were prepared from clarified and inactivated cell culture supernatants by cross-flow ultrafiltration as described before. SEC on Sepharose 4 FF resulted in average product yields of 85% based on hemagglutination (HA) activity. Productivity was maximized to 0.15 column volumes (cv) of concentrate per hour yielding a reduction in total protein and host cell DNA (hcDNA) to 35 and 34%, respectively. AEC on Sepharose Q XL was used to separate hcDNA from virus at a salt concentration of 0.65 M sodium chloride. Product yields >80% were achieved for loads >160 kHAU/mL of resin. The reduction in hcDNA was 67-fold. Split peak elution and bimodal particle volume distributions suggested aggregation of virions. Co-elution with hcDNA and constant amounts of hcDNA per dose indiciated association of virions to hcDNA. An overall product yield of 52% was achieved. Total protein was reduced more than 19-fold; hcDNA more than 500-fold by the process. Estimation of the dose volume from HA activity predicted a protein content at the limit for human vaccines. Reduction of hcDNA was found insufficient (about 500 ng per dose) requiring further optimization of AEC or additional purification steps. All operations were selected to be scalable and independent of the virus strain rendering the process suitable for vaccine production. Biotechnol. Bioeng. 2007;96: 932-944.

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Section: Introductionmentioning

confidence: 99%

Purification of cell culture‐derived human influenza A virus by size‐exclusion and anion‐exchange chromatography

Kalbfuss

Wolff

Morenweiser³

et al. 2006

Biotech & Bioengineering

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“…Co-precipitation by the polycations is, therefore, not surprising. By contrast, DNA precipitation with PS investigated by Amosenko et al [19] did not cause any loss in the yield of the poliovirus. As this virus has an alkaline isoelectric point [52], this could be explained by an overall positive charge (although the authors do not report pH during precipitation).…”

Section: Dna Precipitationmentioning

confidence: 82%

“…Moreover, PEI has been used for the selective separation of nucleic acids from microbial cell extracts by precipitation [12][13][14]. Various other cationic agents had been used for the precipitation of nucleic acids: spermine, spermidine, cobalthexamine [15][16][17], divalent metal ions [18], and protamine sulfate (PS) [19,20].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2010

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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.

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Viral Vaccines Purification

Kalbfuss

Reichl

2014

Vaccine Development and Manufacturing

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Use of protamine sulphate for elimination of substrate DNA in poliovaccines produced on continuous cell lines

Cited by 5 publications

References 2 publications

Purification of cell culture‐derived human influenza A virus by size‐exclusion and anion‐exchange chromatography

Purification of cell culture‐derived human influenza A virus by size‐exclusion and anion‐exchange chromatography

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

Viral Vaccines Purification

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