Process intensification for Peste des Petites Ruminants Virus vaccine production.

Process intensification for Peste des Petites Ruminants Virus (PPRV) vaccine production in anchorage dependent Vero cells is challenging, involving substantial amount of bioprocess development. In this study, we describe the implementation of a new, scalable bioprocess for PPRV vaccine production in Vero cells using serum-free medium (SFM), microcarrier technology in stirred-tank bioreactors (STB), in-situ cell detachment from microcarriers and perfusion. Vero cells were successfully adapted to ProVero™-1 SFM, reaching growth rates similar to serum-containing cultures (0.030 1/h vs 0.026 1/h, respectively). An in-situ cell detachment method was successfully implemented, with efficiencies above 85%. Up to 2.5-fold increase in maximum cell concentration was obtained using perfusion when compared to batch culture. Combining perfusion with the in-situ cell detachment method enabled the scale-up to 20 L STB directly from a 2 L STB, surpassing the need for a mid-scale platform (i.e. 5 L STB) and thus reducing seed train duration. Head-to-head comparison of cell growth and PPRV production in the 2 L and 20 L STB was performed, and no significant differences could be observed. Estimated infectious PPRV titers in Tissue Culture Infection Dose (TCID50) (TCID50/mL = 5 × 106 and TCID50/cell = 5) are within the log-range reported in literature for PPRV production in STB and SFM by Silva et al. (2008), thus confirming the feasibility and scalability of the seed train designed [1]. The novel and scalable vaccine production process herein proposed has the potential to assist the upcoming Peste des Petites Ruminants (PPR) Global Eradication Program (targeted by FAAO for 2030) by providing African local and/or regional manufacturers with a platform capable of generating over 25,000 doses of Nigeria 75/1 strain in just 19 days using a 20 L STB.

Verification of a new biocompatible single-use film formulation with optimized additive content for multiple bioprocess applications.

Single-use bioprocessing bags and bioreactors gained significant importance in the industry as they offer a number of advantages over traditional stainless steel solutions. However, there is continued concern that the plastic materials might release potentially toxic substances negatively impacting cell growth and product titers, or even compromise drug safety when using single-use bags for intermediate or drug substance storage. In this study, we have focused on the in vitro detection of potentially cytotoxic leachables originating from the recently developed new polyethylene (PE) multilayer film called S80. This new film was developed to guarantee biocompatibility for multiple bioprocess applications, for example, storage of process fluids, mixing, and cell culture bioreactors. For this purpose, we examined a protein-free cell culture medium that had been used to extract leachables from freshly gamma-irradiated sample bags in a standardized cell culture assay. We investigated sample bags from films generated to establish the operating ranges of the film extrusion process. Further, we studied sample bags of different age after gamma-irradiation and finally, we performed extended media extraction trials at cold room conditions using sample bags. In contrast to a nonoptimized film formulation, our data demonstrate no cytotoxic effect of the S80 polymer film formulation under any of the investigated conditions. The S80 film formulation is based on an optimized PE polymer composition and additive package. Full traceability alongside specifications and controls of all critical raw materials, and process controls of the manufacturing process, that is, film extrusion and gamma-irradiation, have been established to ensure lot-to-lot consistency.

Automation of cell line development.

An automated platform for development of high producing cell lines for biopharmaceutical production has been established in order to increase throughput and reduce development costs. The concept is based on the Cello robotic system (The Automation Partnership) and covers screening for colonies and expansion of static cultures. In this study, the glutamine synthetase expression system (Lonza Biologics) for production of therapeutic monoclonal antibodies in Chinese hamster ovary cells was used for evaluation of the automation approach. It is shown that the automated procedure is capable of producing cell lines of equal quality to the traditionally generated cell lines in terms of colony detection following transfection and distribution of IgG titer in the screening steps. In a generic fed-batch evaluation in stirred tank bioreactors, IgG titers of 4.7 and 5.0 g/L were obtained for best expressing cell lines. We have estimated that the number of completed cell line development projects can be increased up to three times using the automated process without increasing manual workload, compared to the manual process. Correlation between IgG titers obtained in early screens and titers achieved in fed-batch cultures in shake flasks was found to be poor. This further implies the benefits of utilizing a high throughput system capable of screening and expanding a high number of transfectants. Two concentrations, 56 and 75 muM, of selection agent, methionine sulphoximine (MSX), were applied to evaluate the impact on the number of colonies obtained post transfection. When applying selection medium containing 75 muM MSX, fewer low producing transfectants were obtained, compared to cell lines selected with 56 muM MSX, but an equal number of high producing cell lines were found. By using the higher MSX concentration, the number of cell line development projects run in parallel could be increased and thereby increasing the overall capacity of the automated platform process.

Process development for functional membrane receptor production in mammalian cells.

Two model G-protein coupled membrane receptors (GPCRs), aserotonin (5HT) and a metabotropic glutamate (mGlu) receptor, stablyexpressed in CHO cells were used to characterize cultureconditions for maximum receptor expression and functionalactivity in membrane preparations. Expression levels of the5HT receptor were affected by the growth phase of the cellculture. Maximum receptor density, as measured by ligandbinding per mg membrane protein, was observed when cells wereharvested in late exponential growth phase. Expression couldbe increased further by addition of 10 mM sodium butyrate andincubation at 31 degrees C for 24 hours prior to cellharvest. In contrast, functional activity as determined byagonist-stimulated GTPgammaS binding was independent of the growthrate. For both receptors, butyrate treatment at decreasedtemperature negatively affected functional activity. The mGlureceptor membranes lost functional activity considerably whenthe cells were cultured in an agitated system either onmicrocarriers or as aggregates in suspension. Functionalactivity could be restored and further improved compared to acontrol grown in T-flasks when the cell culture was incubatedat 31 degrees C for 48 hours following a complete mediumexchange and omission of sodium butyrate.