Regeneron is integrating single-use chromatographic clarification into its commercial-scale process for manufacturing adeno-associated vectors (AAVs), which enables it to eliminate endonuclease in downstream purification and reduce manufacturing costs.
This decision provides a cost-effective way to scale AAV production to meet the growing need for delivery vehicles for gene therapies. according to the company. With the cost of goods for rAAV products ranging from roughly $500,000 to $1 million per dose, reducing manufacturing costs is one way to start bringing therapeutic costs in line with payers’ budgets.
Several scientists have developed ways to improve downstream purification for AAVs. Typically, however, they incorporate endonuclease to degrade the high levels of host cell protein and host cell DNA.
Scientists led by Andrew D. Tustian, EngD, senior director, preclinical manufacturing, and process development at Regeneron, showed endonuclease isn’t always needed and that eliminating it can save approximately $100,000 per 500-L batch without sacrificing yield.
Similar yield & lower costs
During the study, the team performed 22 filtration runs using single-use chromatography filters and compared filtration results among cell culture lysate that contained zero endonuclease, 10 U/mL of endonuclease for partial host cell DNA digestion, or 100 U/mL of endonuclease for full digestion.
When producing rAAV8 and rAAV9 in HEK293 cell cultures, they report achieving yields “matching or exceeding the filtrate quality of traditional depth filtration.”
Host cell DNA was reduced by up to 3 logs (from 105 ng/mL to less than 10 ng/mL) for rAAV9, and 2.5 logs for rAAV8. That’s comparable to or better than that achieved using traditional depth filtration with endonuclease treatment. Additional benefits include greater ease in sourcing raw material and eliminating the need to perform a residual endonuclease assay. And, they point out, the cost of goods required to harvest the AAVs dropped by more than 90%.
To implement single-use chromatographic filtration in commercial-scale rAAV manufacturing, Tustian and colleagues report that adding 100 mM of salt to the harvest buffer improves filtration throughput, prevents clogging, and limits rAAV binding to the filter itself.
Binding studies showed that a conductivity greater than 25 millisiemens per centimeter prevented rAAV8 capsids from binding to the filtration media when pH ranges between 7.0 to 8.5. “This offers an additional level of process safety,” they note, even though negatively charged impurities inside the bioreactor minimize capsid binding.
When implementing single-use chromatographic clarification, the only caution Tustian highlighted for GEN was this: “Mainly, just consider the impact of endonuclease removal on the capacity of the [single-use chromatography] filters.”
