As the number of cell and gene therapy clinical trials have rapidly increased, the production of viral vectors has received more attention to support the development and manufacturing of the therapies. Adeno-associated virus (AAV) is the most common vector platform. AAV is non-pathogenic compared to other vectors and has several serotypes which can target different bodily tissues. Traditionally, AAV vectors have been cultured in adherent HEK293 and HEK293T cell lines. Unfortunately, AAV use has exceeded supply, and there is a growing pipeline of potential approved therapies lining up in the next decade. As a result, there is a pressing need for both an increase in vector-manufacturing capacity, and an increase in AAV process productivities beyond what the standard HEK293 adherent culturing practices can support.
A new study from groups at Lausanne University Hospital, Ecole Polytechnique Fédérale de Lausanne (EPFL), and ExcellGene SA demonstrated a platform to generate AAV vectors from suspension processes in orbitally shaken bioreactors (OSRs). The team desired to validate a GMP-compliant process that could scale economically to address the vector requirements from early research through to clinical trials. Both adherent and suspension cell lines were cultivated with an initial cell number at a transfection of approximately 500 x 106 cells. For the two adherent cultures, the cells were grown in five cell factories (T-flasks) each and fed DMEM media containing 10% FBS. At harvest, each batch of cultures had roughly the same number of cells. The batches were transfected with calcium phosphate and had transfection efficiencies of 83% – 86%. In contrast, the suspension cultures were cultivated in two sets of two TubeSpin 600 OSRs at the same cell numbers as the two T-flask batches. The OSRs were fed F17 media supplemented with 4 mM of Glutamax (L-alanyl-L-glutamine). At harvest, each of the OSR cultures increased their cell numbers to approximately 1 x 109 cells. Transfection was carried out with PEI using only half the plasmid DNA amount compared to the calcium phosphate (T-flask) approach. The transfection efficiency (60% – 61%) was slightly lower, but the overall total number of cells expressing the transfected gene was higher in the cells cultivated in OSR suspensions.
All transfected cultures then underwent vector purification. Two different methods were compared, iodixanol gradient centrifugation (IGC) or immunoaffinity chromatography (IAC), to evaluate the more scalable approach. There was double the number of purified viral genomes from the IGC method than IAC. The researchers believed this difference was caused due to the fundamentals of how each approach separates the vectors. IAC relies on the affinity of the resin ligands to the viral capsid and does not discriminate between empty and full capsids. Since IGC relies on purification based on particle density, the IGC approach can separate empty and full vectors.
In vitro potency assay results from SDS-PAGE, western blot analysis, and capsid ELISA analysis of all IGC and IAC preparations demonstrated the presence of the three main viral capsid proteins in the absence of other protein impurities. The assays did show a more intense response from suspension-produced AAV capsids purified by IAC which contained a proportionally higher number of empty capsids. Despite this finding, vectors produced in suspension and purified by IAC resulted in almost double the number of assembled capsids in total. The ratio of full-to-empty viral capsids was determined with transmission electron microscopy, which showed that preparations purified by IGC resulted in higher values compared to IAC. In vivo analysis of the different preparations should high expression of the transfected fluorescent gene (i.e., GFP) over portions of the striatum in mice. IGC purified vectors showed higher median transduction efficiency than IAC purified vectors. Overall, the vectors from the suspension processes elicited a higher transduction efficiency regardless of the purification process. Therefore overall, the vectors produced by the new OSR suspension process had potencies that were like, if not better than, those produced from adherent T-flask cultures.
Challenges for viral vector manufacturing will remain as large amounts of vectors are required for the necessary preclinical and clinical trial studies. As described here, alternative approaches will attempt to solve compliance, production, and scalability issues of adherent cultures grown in serum-supplemented media. This study provided a thorough comparison between culture process (adherent vs. suspension), media strategy (serum-supplemented vs. serum-free), transfection approaches (calcium phosphate vs. PEI), and purification techniques (IGC vs. IAC) for other groups to reproduce or modify to fit their own projects.
By, Glenn A. Harris