Performance characteristics of qualified cell lines for isolation and propagation of influenza viruses for vaccine manufacturing

Abstract

Cell culture is now available as a method for the production of influenza vaccines in addition to eggs. In accordance with currently accepted practice, viruses recommended as candidates for vaccine manufacture are isolated and propagated exclusively in hens' eggs prior to distribution to manufacturers. Candidate vaccine viruses isolated in cell culture are not available to support vaccine manufacturing in mammalian cell bioreactors so egg-derived viruses have to be used. Recently influenza A (H3N2) viruses have been difficult to isolate directly in eggs. As mitigation against this difficulty, and the possibility of no suitable egg-isolated candidate viruses being available, it is proposed to consider using mammalian cell lines for primary isolation of influenza viruses as candidates for vaccine production in egg and cell platforms. To investigate this possibility, we tested the antigenic stability of viruses isolated and propagated in cell lines qualified for influenza vaccine manufacture and subsequently investigated antigen yields of such viruses in these cell lines at pilot-scale. Twenty influenza A and B-positive, original clinical specimens were inoculated in three MDCK cell lines. The antigenicity of recovered viruses was tested by hemagglutination inhibition using ferret sera against contemporary vaccine viruses and the amino acid sequences of the hemagglutinin and neuraminidase were determined. MDCK cell lines proved to be highly sensitive for virus isolation. Compared to the virus sequenced from the original specimen, viruses passaged three times in the MDCK lines showed up to 2 amino acid changes in the hemagglutinin. Antigenic stability was also established by hemagglutination inhibition titers comparable to those of the corresponding reference virus. Viruses isolated in any of the three MDCK lines grew reasonably well but variably in three MDCK cells and in VERO cells at pilot-scale. These results indicate that influenza viruses isolated in vaccine certified cell lines may well qualify for use in vaccine production.

Keywords: Antigenic stability; Genetic stability; Influenza vaccines; MDCK; VERO; Vaccine-certified cell lines; Virus isolation; Virus purification.

Fig. 1

Goals of the study. The sensitivity of vaccine-certified cell lines for the primary isolation of influenza viruses from clinical specimens; the antigenic and genetic stability of influenza viruses propagated in vaccine-certified cell lines; virus yield in pilot scale production platforms; antigen yield in pilot scale production platforms.

Fig. 2

Virus protein yield in pilot scale production platforms. One representative A(H1N1) and A(H3N2) virus and one virus representing each of the two type B lineages isolated in each of the three cell lines (MDCK-1: gray stipple fill bar; MDCK-2: hatched fill bar; and MDCK-3: black fill bar) were propagated in four vaccine-certified cell lines (indicated in the horizontal axis). Virus was purified from 22.5 mL culture supernatant and the content of virus protein was determined. Values are means of two independent purifications, with the standard error shown as T bar extensions. The large differences among production cell lines are most likely the result of approximately 10-fold lower cell densities relative to optimal bioreactor conditions and do not necessarily diminish their acceptability for large-scale production of influenza vaccines.