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Developing a Fluorescence-based Continuous (MCSGP) AEX-HPLC Method for Quantification of Full, Partial and Empty Capsid in AAV products

Develop and validate an analytical technology for full/partial/empty capsid quantification of rAAV product using multi-step HPLC columns with a fluorescence detector.
Categories
Cell and Gene therapies
Project status
100% Completed

Solution

Performance Period: 9/1/2021 to 5/31/2023

The University of Massachusetts Lowell (UML), Mass Biologics, Artemis Biosystems, Merck, and Massachusetts Life Sciences Center, propose to develop and validate an analytical technology for full/partial/empty capsid quantification of rAAV product. A complete differentiation of full, empty, and partial capsids in gene therapy products is an absolute must in terms of product quality and purity. Due to low packaging efficiency, not all capsids successfully encapsidated the desired genome. There is some inevitable population of empty or partial capsids, which have failed to package intact vector DNA, but none or only truncated vector DNA. It is essential to determine a complete ratio of full (drug substance) and partial/empty (impurity) AAVs.

There are several compendial techniques used for capsid contents assessment in the industry that include enzyme-linked immunosorbent assay (ELISA), transmission electron microscopy (TEM), analytical ultracentrifugation (AUC), and high-performance liquid chromatography (HPLC). The industry and regulatory agencies still need a robust, sensitive, reproducible, and sample sparing method that allows complete quantification and characterization of capsid contents.

The primary objective of the proposal is to develop a versatile analytical tool with sufficient sensitivity for differentiation and quantification of all full, partial, and empty AAV capsids in viral products. The project aims to implement multiple strategies to enhance sensitivity and purity without compromising yields. The primary aspect is to use the multi-step HPLC columns along with fluorescence detector. A multi-step IEX-HPLC coupled with a fluorescent detector provides the capability of differentiating full, partial, and empty AAV capsids.

Accurate quantification of all different capsid ratios will lead to significant enhancement in product quality, quality control, and eventually to the more rapid release of gene therapy-related products that are compliant with regulatory guidance.

Outcomes and Impacts

Development of an analytical approach for improved separation of full, partial, and empty AAV capsid contents.

Development of an automated approach for robustness and reproducibility while sparing buffer and sample.

Rapid and at-line identification and accurate quantification each capsid variation in AAV products.

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Project Lead

University of Massachusetts Lowell

University of Massachusetts Lowell

Participating Organizations

Artemis Biosystems Inc.

Artemis Biosystems Inc.

MassBiologics of the University of Massachusetts Medical School

MassBiologics of the University of Massachusetts Medical School

Massachusetts Life Sciences Center

Massachusetts Life Sciences Center

Merck Sharp & Dohme LLC

Merck Sharp & Dohme LLC

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