Reducing the levels of empty AAV capsids, which do not carry the transgene of interest, is a significant challenge in gene therapy process development. One approach to improving the full to empty capsid ratio is to increase the percentage of full capsids generated during cell culture. One challenge with this approach is that the assays typically used to determine the full to empty ratio are not compatible with crude bioreactor feedstreams. Typically, cell harvests are purified one at time using lab scale columns in series.  It is time consuming and requires larger quantities of cell lysate which are limited in gene therapy.  To overcome this challenge, a high-throughput methodology that includes high-throughput cell culture, purification, and analysis of AAV vectors was developed.<br/><br/>This plate-based purification method enables the rapid evaluation of empty/full ratios from up to 96 AMBR® bioreactor cell lysates.  AAV crude lysates are purified using Tecan liquid handler and eluted fractions are analyzed using SEC-HPLC.  Up to 48 samples can be purified and analyzed within 1.5 work days. Our upstream process development team has increased understanding of relationship between culture conditions and empty/full ratio leading to process improvements.  This new tool for gene therapy process development enables us to explore a broader range of bioreactor conditions in less time and with less material. <br/>

Development of a high-throughput purification method to assess the impact of bioreactor conditions on rAAV empty/full capsid ratio

2019 Spring National Meeting

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