Evaluating Improvements and Challenges in Affinity Chromatography for AAV Purification
| dc.contributor.advisor | Betenbaugh, Michael | |
| dc.contributor.advisor | Hadidi, Mahsa | |
| dc.creator | Dachenko, Alexandra | |
| dc.date.accessioned | 2023-11-02T15:36:00Z | |
| dc.date.available | 2023-11-02T15:36:00Z | |
| dc.date.created | 2023-08 | |
| dc.date.issued | 2023-07-31 | |
| dc.date.submitted | August 2023 | |
| dc.date.updated | 2023-11-02T15:36:01Z | |
| dc.description.abstract | Purification process development is crucial to the Research and Development (R&D) sector in the biotechnology industry by designing and optimizing downstream unit operations capable of manufacturing safe treatments for clinical trials. Purification is crucial in manufacturing therapeutics such as monoclonal antibodies (mAbs), mRNA, viral vectors, and non-viral vectors. Recently, viral vector gene therapy modalities using adeno-associated viruses (AAVs) have been shown to be an effective therapeutic agent for both rare and common diseases in humans. One of the key polishing steps in the downstream manufacturing process of AAVs is affinity chromatography, where a ligand with specific binding affinity to an AAV serotype is coupled to a base matrix that separates AAVs from host cell contaminants produced in the cell culture process. Column resin is the most common affinity chromatography matrix, but it possesses mass transfer limitations. This report evaluated a prototype affinity membrane device coupled with AVB ligand designed to overcome these mass transfer limitations and decrease process times, while yielding comparable AAV recovery and purity to column resin. The evaluations included dynamic binding capacity (DBC) studies, ligand lifetime studies, and buffer optimizations. The performance of the device was compared to control experiments using two common column resins with the same ligand as the device. The novel membrane device can bind at least 2e14 vg/mL_membrane and can undergo at least 3 cycles without a major binding capacity drop. The recovery of AAV is comparable to column resins; additional process development studies were required to increase recovery values because process parameters used for resin with the same ligand were not effective. A comparison of AAV product purity cannot be made because impurity profiles were not tested. Some recommendations for future work with the availability of more devices include more DBC studies, buffer optimization studies, and testing of the device performance with different AAV serotypes and load material. The prototype membrane device offers unique benefits over column resin for affinity chromatography in its fast flow rates, reusability, and potentially lower costs due to its “prepacked” design; however, the evaluations demonstrated the device does not provide significant enough process improvements that would favor its implementation in place of column resin for the tested material. | |
| dc.format.mimetype | application/pdf | |
| dc.identifier.uri | https://jscholarship.library.jhu.edu/handle/1774.2/69011 | |
| dc.language.iso | en_US | |
| dc.publisher | Johns Hopkins University | |
| dc.publisher.country | USA | |
| dc.subject | Adeno-associated viruses (AAVs) | |
| dc.subject | purification | |
| dc.title | Evaluating Improvements and Challenges in Affinity Chromatography for AAV Purification | |
| dc.type | Thesis | |
| dc.type.material | text | |
| thesis.degree.department | Chemical and Biomolecular Engineering | |
| thesis.degree.discipline | Chemical & Biomolecular Engineering | |
| thesis.degree.grantor | Johns Hopkins University | |
| thesis.degree.grantor | Whiting School of Engineering | |
| thesis.degree.level | Masters | |
| thesis.degree.name | M.S.E. |
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