B. Kalbfuss

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

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.

Size-exclusion chromatography as a linear transfer system: Purification of human influenza virus as an example

Preparative size-exclusion chromatography suffers from low selectivity and productivity. Empirical optimization of operating conditions constitutes a laborious task due to many parameters. Here, a modeling framework based on linear systems theory is presented for predicting the influence of volume overloading. Impulse-responses characterizing system behavior are derived from experimental data by maximum entropy deconvolution. Theoretical derivations are validated experimentally by study of a model system and chromatography of human influenza virus. By application of the theory it is demonstrated how group separation operations can be optimized with respect to yield, purity, productivity and dilution of the product.