Enhancing rAAV production by HEK293 cells via metabolic profiling

Conference Dates

February 6 – 10, 2022


Viral vector manufacturing is expensive and time-consuming. Demand for rAAV-based vectors has risen

massively in the past decade and continues to rise thanks to urgent healthcare supply demand. The industry is,

however, currently missing a cost-effective and robust manufacturing strategy. One of the major downsides of

rAAV production is the high percentage of “empty” vector particles being produced and harvested. In addition to

complicating downstream purification processes, this characteristic limits the efficiency of rAAV manufacture

and presents uncertainties for scale-up.

Efficiency of the manufacturing process is largely dependent on the productivity of the production cell line. Much

emphasis has been put into understanding the effects of recombinant protein production on mammalian cell

lines (e.g., CHO, HeLa, HEK293) but relatively little is known about the effects of viral vector production on cell

biology and behaviour. Over the years, many clones have been derived, isolated and engineered from HEK293

to induce improvements in productivity and efficiency. However, the high cost of production and licensing, the

expression of potentially undesired elements (e.g., T-antigen) and regulatory approval processes for next

generation cell lines, hinders their use in clinical manufacturing. Increased understanding of HEK293 in relation

to existing processes and process control offers realistic opportunity to enhance the efficiency of rAAV


Our aim is to identify and understand the critical parameters that contribute to setting the productivity in HEK293

cells (in terms of final yield and abundance of full capsids), ranging from the metabolic requirements prior to and

during viral vector production, to cell culture parameter optimisation to maintain the cells in an optimal state of


We tested several commercially available media for rAAV9 production and selected the candidate that provided

the best yield and quality of viral vector. With this medium as our baseline, we investigated the metabolism

during a period of culture via extracellular metabolic profiling of control and rAAV producing cells. The analysis

revealed the rapid use of several amino acids over the first 24 hr post-inoculation and the subsequent

generation of metabolites indicative of metabolic profiles associated with cell growth. rAAV9 producing cells

show lower rates of amino acid and glucose consumption than control cells but the profile of metabolism was

not significantly changed as a result of transfection/production of rAAV9. These data were used to design

medium supplements and the effect of supplement addition on cell proliferation, viability and rAAV

production/quality was assessed. Specific combinations of amino acids generated an increased cell density (up

to 9.3x106 cells/mL at 5 days post-inoculation compared to 4.4x106 cells/mL for cells in non-supplemented

medium). This was associated with retention of improved viability in the presence of the supplement. In addition,

the metabolic profiling we undertook indicated the build-up of potentially toxic/growth inhibitory metabolites

during the period of stock cell preparations prior to setting up transfections. In various dilution experiments we

were able to optimise the pre-treatment, cell density and dilution protocol to generate predictable and

reproducible efficiencies of transfection, cell growth and rAAV production.

Overall, our data contributes metabolic insights to process conditions that generate HEK293 cells of appropriate

health and defined parameters to robust and enhanced production of rAAV, providing work schemes that are

also appropriate to the manufacture of further types of viral vectors.

Poster 8.pdf (83 kB)

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