RESUMO
The generation of stable clones for biomolecule production is a common but lengthy and labor-intensive process. For complex molecules, such as viruses or virus-like particles (VLPs), the timeline becomes even more cumbersome. Thus, in the early stages of development, transient production methods serve as a reasonable alternative to stable clone construction. In this work, an investigation of a polyethylenimine- (PEI-) based transfection method for the transient production of Chikungunya (Chik) VLPs, a vaccine candidate molecule, was undertaken. This effort focuses on tracking cell population responses during transfection, understanding how process changes affect these responses, and monitoring patterns in cell performance over the culture duration. Plasmid labeling and VLP staining were employed to comprehensively track cells via flow cytometry and to draw correlations between plasmid DNA (pDNA) uptake and the resulting VLP expression. The method detected high transfection efficiency (≥97%) in all samples tested and demonstrated the capability to track kinetics of plasmid-cell binding. With varied transfection cell concentrations, the pDNA binding kinetics are altered and saturation binding is observed in the lowest cell concentration sample tested in less than 3 hours of incubation. Interestingly, in all samples, the flow cytometry analysis of relative pDNA amount versus VLP expression staining showed that cells which contained fewer pDNA complexes resulted in the highest levels of VLP stain. Finally, to determine the potential breadth of our observations, we compared daily expression patterns of ChikVLP with a reporter, monomeric GFP molecule. The similarities detected suggest the interpretations presented here to likely be more broadly informative and applicable to PEI-based transient production of additional biological products as well.
RESUMO
An alternating tangential flow (ATF) perfusion-based transient gene expression (TGE) bioprocess has been developed using human embryonic kidney (HEK) 293 cells to produce H1-ss-np, a promising candidate for a universal influenza vaccine. Two major adjustments were taken to improve the process: (1) eliminate the interference of microbubbles during gene transfection; and (2) utilize an ATF perfusion system for a prolonged culture period. As a result, a closed-operation 9-days ATF perfusion-based TGE bioprocess was developed. The TGE bioprocess showed continuous cell growth with high cell viability and prolonged cellular productivity that achieved recombinant product level of ~270 mg/L which was more than two times that of 4-days base-line TGE bioprocess. In addition, the consumables cost per milligram for ATF perfusion-based TGE bioprocess was ~70% lower than that of the base-line TGE bioprocess suggesting high cost savings potential in vaccine manufacturing. Based on the lower contamination risk, higher productivity, and cost efficiency, the ATF perfusion-based TGE bioprocess can likely provide potential benefits to many future applications in vaccine and drug manufacturing.
