Resubmission - Development and scale-up of Continuous Countercurrent Tangential Chromatography (CCTC) for next generation AAV vector manufacturing (Direct to Phase II)

Research Strategy Grant# 1R44GM154572-01A1 In this administrative supplement we will be making a strong case to the NIH to enable funding for key equipment needed for the completion of our development and scale-up efforts to commercialize BioRMB™, formerly known as Continuous Countercurrent Tangential

Chromatography (CCTC), for next generation AAV vector manufacturing. Summary In our initial submission to the NIH in March 2024, we outlined four aims for the provided funds, the first of which was to commercialize BioRMB™ for the purification of rAAV to significantly improve recovery, product quality, and process productivity when compared with column

chromatography. Since receiving these funds, we have already begun the development of a comprehensive analytical and process development toolkit for benchtop experimental design to execute capture and polishing operations for rAAV purification on our BioRMB™ platform, and we were able to execute purification of rAAV8 with excellent yield and product quality. For the capture

step, we started with 2.5 Liters of harvested material, and performed an elution buffer DoE study on BioRMB™, screening different elution pHs. We achieved an overall 80% recovery during steady-state operation, and considering only the lower pH condition, our AAV maximum recovery was at 85%. Further analysis of impurities removal will be performed.

In addition, we have used a commercial rAAV harvest material provided by Spark Therapeutics, concentrated 10 times, in an effort of process intensification to reduce the capture process time. An initial bioreactor volume of 40 Liters was concentrated to 3.9 Liters and was captured using AAVX affinity resin (Thermo Scientific) on Kascade™ BioRMB™, over a 5-hour capture process.

Titer analyses by qPCR showed an average of 89% rAAV recovery during steady-state. Preliminary HEK293 HCP ELISA analyses revealed that host-cell protein was reduced below detectable levels in eluted samples. This purified material has been used for initial AAV polishing characterization bench studies, where a specific resin, target loading, and buffer conditions were

selected to design the polishing process to be used on the BioRMB™ platform. We are still working on optimized elution conditions for this rAAV molecule, and will use the bench-scale data to design the flow path and operational parameters for AAV polishing step on the Kascade™ BioRMB™. Additionally, we have begun developing a suite of analytical assays and QC protocols to better

characterize crude and purified rAAV molecules. Furthermore, we have successfully demonstrated that BioRMB™ can achieve up to 4 log reduction of viral contaminants (LRV) from a mAb harvest spiked with a viral-like particle (RVLP). We have every reason to believe BioRMB™ can achieve similar results with AAV crude harvest.

However, the ability to demonstrate the integration of AAV capture and polishing, resulting in a high purity product, is a key milestone that must also be achieved, and we have identified critical gaps in our capabilities that prevent us from doing so. To that end, we are requesting additional funds for equipment to allow us to address the following:

1. To evaluate the capacity of BioRMB™ to effectively remove impurities, such as HCPs, we require an Automated Immunoassay system. This technology will enable us to conduct, in a high throughput manner, in-depth impurity removal studies for rAAV, to demonstrate equivalent log reductions of such impurities. In addition, we will have an orthogonal

technique for total AAV capsids titer. The following table provides a summary of the equipment manufacturer, project priority, costs, and the overall budget ($202,320 + 6% tax = $214,459.00). Equipment Manufacturer Function Priority Quote xPlore Gyrolab Automated Immunoassays (HCP and total AAV capsids) 1 $214,459.00