The cQrex® GQ effect: Boosting viral vector yield and quality in HEK-based AAV-production

Adeno-associated virus (AAV) vectors are a platform technology for gene therapies. Prominent examples authorized for marketing in Europe and the U.S. include Luxturna® and Zolgensma® which are based on AAV2 and AAV9 respectively. For AAV alone, over 260 clinical trials are ongoing. As biopharmaceutical development progresses from clinical to commercial phases, there is an increasing need to improve the productivity of viral vector production processes.

Two key challenges to improving the productivity include obtaining sufficient virus titers during cell culture, and reducing the percentage of empty capsids that do not contain genetic information. The number of full vs. empty capsids is of particular interest because it is a critical quality attribute for purity. Empty capsids may cause dose-limiting side effects based on immunogenicity, and removal of these capsids during downstream processing is difficult.

Ideally, both parameters can be improved by choosing the right supplement for the cell culture process. The following case study shows how this was achieved leveraging the cQrex® effect.

cQrex® GQ enables high titer AAV production

Over the past few years, experts at Evonik have developed a portfolio of highly pure, chemically defined peptides. These products, sold under the cQrex® brand, became a mainstay in the bioprocess media developers’ toolboxes as they can help to improve media performance and simplify production processes.

The dipeptide cQrex® GQ (Glycyl-L-Glutamine hydrate, Gly-Gln) is a superior form of L-glutamine that enables stable media formulations and optimized supply of this key amino acid.

Key features:

  • Animal-origin free with strictly controlled, high purity
  • Manufactured at well-established ISO-certified, cGMP manufacturing facilities in Germany and France

This is why it is used in commercial protein drug manufacturing. Interestingly, Evonik research recently showed that replacement of L-glutamine by cQrex® GQ was also found to be a powerful tool to increase viral vector yield and quality.

Case study

In a lab study in shake flasks, HEK Expi293F cells were pre-cultured in FreeStyleTM F17 Expression Medium, a commercial medium recommended for viral vector production. They were cultivated for five passages, split into three replicates each, and transfected using polyethylenimine (PEI MAX®, Polysciences) with a 2-plasmid system for AAV8 (Plasmid Factory) featuring green fluorescent protein as the gene of interest. During the pre-culture and production phase of AAV8 production, different variants of the medium were tested. As recommended by the manufacturer, the medium was supplemented to a final concentration of 8 mM with L-glutamine (Gln) or L-alanyl-L-glutamine, the latter in the form of a commercial ready-to-use supplement (GlutaMAXTM, Thermo Fisher, supplied as liquid stock at 200 mM in 0.85% NaCl). Alternatively, a 200 mM stock solution of cQrex® GQ was prepared. From this stock solution the peptide was added to the medium at a final concentration of 8 mM.

cQrex® GQ supplementation led to a higher transfection efficiency determined via fluorescence measurement of green-fluorescent protein-positive cells compared to supplementation of L-glutamine alone, but also over GlutaMAXTM, at all measurements points (Figure 1).

Figure 1: Influence of glutamine supplements on viral vector production (AAV8) using HEK Expi293F cells: transfection efficiency determined via relative fluorescence of expressed GFP by flow cytometry 48, 72, and 96 h post-transfection.

In addition to more efficient transfection, higher AAV8 production yield was achieved. Using cQrex® GQ as the only glutamine source led to genomic titers 72 % (96 h post transfection) or 87 % (120 h post transfection) above the results for the other glutamine sources (Figure 2).

Figure 2: Influence of glutamine supplements on viral vector production (AAV8) using HEK Expi293F cells: viral genome titer (mean of n=3 ± SD) determined by qPCR 96 and 120 h post transfection.

By providing an optimized glutamine source via cQrex® GQ, even product quality could be improved. cQrex® GQ supplementation resulted in a lower percentage of empty capsids and a more favorable ratio between full and empty capsids indicating a packaging rate of over 20 % (Figure 3).

Figure 3: Influence of glutamine supplements on viral vector production (AAV8) using HEK Expi293F cells: full/ empty ratio calculated based on viral genome titer determined by qPCR and capsid titer determined by ELISA

Hence, choosing the right, chemically defined, non-animal-derived and highly pure dipeptide to optimize L-glutamine supply was shown to boost viral vector production yield and product quality. Optimizing your cell culture system with cQrex® peptides could help you to drive efficiency in the development and production of gene therapies.

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About the Author

Johanna PetersDr. Johanna Peters, Project Manager Innovation Domain Cell and Tissue Systems, Evonik

Dr. Johanna Peters joined Evonik in 2017 and currently works for Creavis, the company’s strategic innovation unit and business incubator. Johanna has over eight years’ experience in the healthcare industry. She holds a PhD in pharmaceutics and biopharmaceutics from the Henrich-Heine University of Düsseldorf in Germany.

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