Harvesting and concentration of human influenza A virus produced in serum‐free mammalian cell culture for the production of vaccines

Abstract

A process scheme for the harvesting and concentration of cell culture-derived human influenza A virus is presented. The scheme comprises two static filtration steps, chemical inactivation by beta-propiolactone and cross-flow ultrafiltration. Human influenza A virus A/PR/8/34 (H1N1) was produced in roller bottles with serum-free medium using MDCK cells as a host. Cultivations resulted in specific hemagglutination (HA) activities of 393 HAU (100 microL)(-1) and turbidities of 0.479 OD measured as the extinction of light at 700 nm (mean values are presented). The concentrations of soluble protein and DNA in the harvests were 72 microg/mL and 5.73 microg/mL, respectively. An average product yield of 79% based on HA activity was achieved after clarification by depth (85%) and microfiltration (93%). The turbidities of cell culture supernatants were reduced to 2% of their initial value. Concentration with 750 kDa hollow-fiber modules by a factor of 20 resulted in 97% recovery of the product when operated at a constant flux of 28 L/(m(2) h) and a wall shear rate of 9,500 s(-1). The amount of protein and DNA could be reduced to 16% and 33% of their initial amount, respectively. An overall product yield of 77% was achieved. Clarified supernatants and concentrates were further analyzed by non-reducing SDS-PAGE and agarose gel electrophoresis. Particle volume distributions of concentrates were obtained by dynamic light scattering analysis. From the results it can be concluded that the suggested process scheme is well suited for the harvesting and concentration of cell culture-derived influenza A virus.

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

“…Agglutination of erythrocytes by cell debris can therefore be expected to lead to an overestimation in the HA titer of unclarified supernatant. Furthermore, a similar product yield of 79 % was reported by Kalbfuß et al [9] for the clarification of influenza virus-containing supernatant by normal-flow filtration. In their study, two filter media were combined in order to enhance filter capacity: a fibrous pre-filter (0.65 lm) and a membrane microfilter (0.45 lm).…”

“…Tangential . A standard filtration setup with flux control was used for the concentration and diafiltration of influenza virus as already described [9]. Briefly, the retentate was circulated by a gear pump (drive 5130 with ZP1 pump head).…”

“…Average concentrations of total protein showed somewhat higher scatter with 46.7 ± 13.4 lg mL -1 but, in principle, the same assumption should be valid. Although, the influenza virus consists of protein to a large extend, a simple back-of-the-envelope calculation is sufficient to demonstrate that its contribution to total protein concentration can be neglected in most cases [9]. Includes activity found in membrane wash.…”

“…Recently, a process scheme for the purification of influenza A virus was suggested by Kalbfuß et al [9,10]. Clarification of cell culture supernatant was carried out by a combination of mesh and membrane filters (0.65 lm and 0.45 lm, respectively).…”

Broad Spectrum Microbicidal Activity of Photocatalysis by TiO2

“…FCV was propagated by using confluent layers of Crandell-Reese feline kidney (CRFK) cell cultures. The harvested samples were clarified and concentrated by a combination of depth and membrane filters (microfiltration with 0.45-μm polypropylene mesh and cross-flow polysulfone hollow-fiber ultrafiltration with a 100-kDa cut-off, GE Healthcare, Japan) [48]. Subsequently, the virus was purified by sucrose density gradient centrifugation using a linear sucrose gradient of 30%-70% [49].…”