Logo yposkesi
waves image

Platform optimization for efficient AAV purification – Ask the experts Q&A

In a previous publication, experts Vincent Ravault (Yposkesi) and Nicolas Laroudie (Thermo Fisher Scientific), explained how the French CDMO Yposkesi and Thermo Fisher Scientific optimized AAV purification using the POROS™ CaptureSelect™ AAVX resin.

If you haven’t read the article yet, you can find it here

In this publication, Vincent and Nicolas are answering some questions about this purification tool that can be used as a pan-affinity instrument for the universal capture of AAV vectors, and how Yposkesi optimized the operational parameters to make the resin an efficient, robust, and productive purification platform that fits within the constraints faced by CDMOs.

Cell & Gene Therapy Insights 2022; 8(1), 1–14
DOI: 10.18609/cgti.2022.001

Can the POROS CaptureSelect resin be cleaned and re-used?

Nicolas: Yes, the resin can be cleaned and reused. Many customers use the resin once, particularly CDMOs that deal with multiple serotypes and multiple transgenes and want to avoid spending a lot of time validating cleaning. But the resin can absolutely be cleaned and reused, and many customers are doing that. Notably, the resin is not very caustic stable, and so for cleaning, we do recommend using acidic solutions such as phosphoric or citric acid. In case of very dirty resin, we advise additional cleaning with chaotropic agents, such as guanidine hydrochloride or urea. I would encourage people who want to clean and re-use the resin to reach out to their local application specialist, who can help them develop a process for this.

Apart from AAV2 and AAV8, do you have experience with other AAV serotypes and POROS CaptureSelect AAVX?

Vincent: The goal for Yposkesi now is to expand this platform to a broad range of AAV serotypes. With our experience of AAV2 and AAV8 serotypes, we know that the AAVX resin is a good solution to use as a purification platform. Currently, we are working with AAV5, 6, and 9, and the results so far are promising. We also know that we can work with modified capsids.

Is the resin available in a pre-packed format?

Nicolas: Yes, we do have pre-packed formats. We have 1 and 5 mL pre-packed formats available that are compliant with standard benchtop chromatographic systems. We also have robocolumns available, at 200 μL and 600 μL, for high-throughput screening. Of course, the resin can be purchased as bulk material and our local Field Application Specialists are happy to support customers in packing the resins in their own columns, whatever the scale.

Which additional washing conditions would be suitable for host cell protein and host cell DNA reduction?

Vincent: Several washing conditions are interesting to assess. For example, you can add an extra washing step using high salt concentration. You can also wash your column with a low pH buffer in order to remove impurities from the column before recovery of AAV in the eluate. If you decide to implement the second washing step you have to be careful that your washing condition won’t affect the integrity of your capsid. Moreover, if the washing step pH is too close to the pH of the elution buffer, a significant quantity of capsids could be eluted during your washing step, and as a result, lower your AAV yield during the elution.

Is the resin GMP compliant?

Nicolas: The resin is used in GMP manufacturing by many of our customers. While not manufactured in a cGMP process, the resin is produced under an ISO 13485 environment. When you purchase the resin, you can request the regulatory support package, including documents regarding quality, stability, production, control method, and so on. Those documents are useful when you make a product and submit a dossier to a regulatory agency. For each of our commercially available CaptureSelect resins, we developed an ELISA assay to monitor the level of ligand leakage over the purification process.

Vincent, why did you use two different analytical methods during this study – ELISA for dynamic binding capacity and viral genome titers at termination during your screening?

Vincent: During our DBC study, a lot of fractions were collected in the flowthrough at the outlet of the column in order to calculate 10% breakthrough for AAV vectors, so we needed to use a high throughput assay for the analysis of the first full fractions. The ELISA assay allowed us to test several samples in parallel and to get the results quickly, in around half a day. In the screening study, the number of samples was much lower – only two samples for each set of conditions screened were produced – so here we used an internal assay for the quantification of the viral genome titer in the product. The viral genome titer was determined by qPCR for each serotype.

Which resin can be implemented for a polishing step?

Vincent: Several different resins can be implemented for this step. Commonly, an anion exchanger is implemented in order to reduce host cell protein and host cell DNA. Anion exchange also has the capability to separate empty and full AAV capsids, and some suppliers have developed resins specifically for the polishing step. For more information, you can contact chromatography resin suppliers.

View PDF

Copyright: Published by Cell and Gene Therapy Insights under Creative Commons License Deed CC BY NC ND 4.0 which allows anyone to copy, distribute, and transmit the article provided it is properly attributed in the manner specified below. No commercial use without permission.
Attribution: Copyright © 2022 Thermo Fisher Published by Cell and Gene Therapy Insights under Creative Commons License Deed CC BY NC ND 4.0.
Article source: This article is a transcript of a webinar, which can be found here.
Webinar recorded: Oct 26 2021; Revised manuscript received: Jan 4 2021; Publication date: Jan 24 2022.