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In recent years, AAV manufacturing processes have evolved significantly to not only address the need for later-stage clinical and commercial supplies, but also to address regulatory concerns around process consistency and control. Typically, current production processes are based either on transient expression in HEK293 cells or infection by baculovirus.

While both these approaches have been used for large-scale vector production, product developers and CDMOs are increasingly focusing on the development of platform processes using triple transfection of HEK293 cells.

Initially, production processes employed adherent approaches in cell factories and large-scale systems. Now, most vector producers have migrated to suspension platforms due to the inherent scalability of these systems and the availability of a wider range of development and production platforms.

Upstream Development

The upstream process development impacts vector productivity as well as the quality of the vector in terms of the levels of full and empty capsids generated in the production process.

While standardizing some elements of the upstream process such as base media and cell build processes is possible, the transfection process is clearly critical, in terms of the amount and ratios of plasmid used and the transfection process itself. This process needs to be characterized and controlled to achieve optimal process outcomes and also to allow for predictable scalability to the intended production scale.

According to Pirjo Merilahti, PhD, Technology Manager at Biovian, additional critical aspects in the upstream process include the development of a seed train process that reaches target cell density while keeping the cells in good condition; the development of a cultivation strategy that is suitable for the chosen growth method (whether it is batch, fed-batch, or perfusion); and the optimization of the lysis and filtrations steps to maximize recovery.

Continual Refinement

Biovian continually refines their AAV upstream platform to improve its critical processes. “We are not looking for the easiest way to execute the process,” Merilahti said. “Instead we look for the best way. We think outside the box and search for alternative ways. The target is to maximize the yield with the best possible ratio.”

Some of the refinements Merilahti detailed include optimization of the seed train process to allow for the possibility to use perfusion cultivation to increase the cell density while keeping the concentration of undesirable metabolites as low as possible. Another aspect Biovian employs to increase the efficiency of the lysis operation is testing different detergents and endonuclease possibilities and then implementing the best alternatives.

“Optimization is critical to increase the yield,” Merilahti emphasized. “We also evaluate potential scenarios to shorten the entire process.”

For example, Biovian screens different cell lines to identify the options that provide the best yield and ful-to-empty capsid ratios. It also utilizes a design of experiment (DoE) approach to test different transfection reagents and transfection parameters to determine how to enhance the transfection process in both shake flasks and bioreactors.

An Inside Look

For over two decades, Biovian has been dedicated to providing CDMO excellence in biopharmaceutical manufacturing. The CDMO especially concentrates on viral vector production (adenovirus and AAV), microbial production of recombinant proteins, and plasmid DNA.

With expertise in process development, scale-up, and clinical manufacturing, as well as in technology transfer, Biovian undertakes both drug substance and drug product manufacturing.

If you are attending the Bioprocessing Summit in Boston, August 14–17, attend the presentation on AAV manufacturing given by Eero Mustalahti, Head of Business Development at Biovian, on August 17 at 11:45 am, to get the latest insight into Biovian’s advancements. You can also visit Booth 324 to discuss particulars with company experts.




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