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

> **NIH NIH R44** · CHROMATAN CORPORATION · 2024 · $214,459

## Abstract

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 achie...

## Key facts

- **NIH application ID:** 11174147
- **Project number:** 3R44GM154572-01A1S1
- **Recipient organization:** CHROMATAN CORPORATION
- **Principal Investigator:** Oleg Shinkazh
- **Activity code:** R44 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $214,459
- **Award type:** 3
- **Project period:** 2024-05-15 → 2026-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/11174147

## Citation

> US National Institutes of Health, RePORTER application 11174147, Resubmission - Development and scale-up of Continuous Countercurrent Tangential Chromatography (CCTC) for next generation AAV vector manufacturing (Direct to Phase II) (3R44GM154572-01A1S1). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/11174147. Licensed CC0.

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