Adeno-associated viral capsid stability on anion exchange chromatography column and its impact on empty and full capsid separation
Recombinant adeno-associated viral vector (rAAV) mediated gene therapy is gaining traction in treating genetic disorders. Current rAAV production systems yield a mixture of capsids largely devoid of the transgene (empty capsid) compared with the desired therapeutic product (full capsid). Anion excha...
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| Format: | Article |
| Language: | English |
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Elsevier
2023-12-01
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| Series: | Molecular Therapy: Methods & Clinical Development |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2329050123001511 |
| _version_ | 1797660424607367168 |
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| author | Ohnmar Khanal Vijesh Kumar Mi Jin |
| author_facet | Ohnmar Khanal Vijesh Kumar Mi Jin |
| author_sort | Ohnmar Khanal |
| collection | DOAJ |
| description | Recombinant adeno-associated viral vector (rAAV) mediated gene therapy is gaining traction in treating genetic disorders. Current rAAV production systems yield a mixture of capsids largely devoid of the transgene (empty capsid) compared with the desired therapeutic product (full capsid). Anion exchange chromatography (AEX) is an attractive method for separating empty and full AAV capsids because of its scalability. Resin types and buffer composition are key considerations for AEX and must support capsid stability to be suitable for downstream processing. We examined the impact of binding durations (0–8 h) using various binding ionic strengths (15–75 mM), pH (7.5–9.0), resin chemistry (POROS XQ, POROS HQ, POROS I, and BIA QA monolith), and proprietary Q resins with different ligand densities for effects on capsid stability. Empty capsids were altered upon extended binding, leading to retention time shifts and loss of resolution between empty and full capsids. Viral capsid protein analysis reveals that full capsids have more viral capsid protein 3 (VP3) proteins than empty capsids. Analytical hydrophilic liquid chromatography showed that empty capsid retention time shift is accompanied by changes to the empty capsid’s native VP3 protein. Among the potential stabilizing additives considered, magnesium chloride was the most effective at reducing negative impacts caused by extended binding. |
| first_indexed | 2024-03-11T18:30:37Z |
| format | Article |
| id | doaj.art-19361272f35442399cfc9c903b0f84c6 |
| institution | Directory Open Access Journal |
| issn | 2329-0501 |
| language | English |
| last_indexed | 2024-03-11T18:30:37Z |
| publishDate | 2023-12-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Molecular Therapy: Methods & Clinical Development |
| spelling | doaj.art-19361272f35442399cfc9c903b0f84c62023-10-13T11:04:38ZengElsevierMolecular Therapy: Methods & Clinical Development2329-05012023-12-0131101112Adeno-associated viral capsid stability on anion exchange chromatography column and its impact on empty and full capsid separationOhnmar Khanal0Vijesh Kumar1Mi Jin2Technology Development, Spark Therapeutics, Inc., Philadelphia, PA, USA; Corresponding author: Ohnmar Khanal, Spark Therapeutics, Inc., 3737 Market Street, Philadelphia, PA 19104, USA.Technology Development, Spark Therapeutics, Inc., Philadelphia, PA, USA; Corresponding author: Vijesh Kumar, Spark Therapeutics, Inc., 3737 Market Street, Philadelphia, PA 19104, USA.Technology Development, Spark Therapeutics, Inc., Philadelphia, PA, USARecombinant adeno-associated viral vector (rAAV) mediated gene therapy is gaining traction in treating genetic disorders. Current rAAV production systems yield a mixture of capsids largely devoid of the transgene (empty capsid) compared with the desired therapeutic product (full capsid). Anion exchange chromatography (AEX) is an attractive method for separating empty and full AAV capsids because of its scalability. Resin types and buffer composition are key considerations for AEX and must support capsid stability to be suitable for downstream processing. We examined the impact of binding durations (0–8 h) using various binding ionic strengths (15–75 mM), pH (7.5–9.0), resin chemistry (POROS XQ, POROS HQ, POROS I, and BIA QA monolith), and proprietary Q resins with different ligand densities for effects on capsid stability. Empty capsids were altered upon extended binding, leading to retention time shifts and loss of resolution between empty and full capsids. Viral capsid protein analysis reveals that full capsids have more viral capsid protein 3 (VP3) proteins than empty capsids. Analytical hydrophilic liquid chromatography showed that empty capsid retention time shift is accompanied by changes to the empty capsid’s native VP3 protein. Among the potential stabilizing additives considered, magnesium chloride was the most effective at reducing negative impacts caused by extended binding.http://www.sciencedirect.com/science/article/pii/S2329050123001511AAV stabilityanion exchange chromatographyAEXempty capsidsfull capsidsstability in adsorbed state |
| spellingShingle | Ohnmar Khanal Vijesh Kumar Mi Jin Adeno-associated viral capsid stability on anion exchange chromatography column and its impact on empty and full capsid separation Molecular Therapy: Methods & Clinical Development AAV stability anion exchange chromatography AEX empty capsids full capsids stability in adsorbed state |
| title | Adeno-associated viral capsid stability on anion exchange chromatography column and its impact on empty and full capsid separation |
| title_full | Adeno-associated viral capsid stability on anion exchange chromatography column and its impact on empty and full capsid separation |
| title_fullStr | Adeno-associated viral capsid stability on anion exchange chromatography column and its impact on empty and full capsid separation |
| title_full_unstemmed | Adeno-associated viral capsid stability on anion exchange chromatography column and its impact on empty and full capsid separation |
| title_short | Adeno-associated viral capsid stability on anion exchange chromatography column and its impact on empty and full capsid separation |
| title_sort | adeno associated viral capsid stability on anion exchange chromatography column and its impact on empty and full capsid separation |
| topic | AAV stability anion exchange chromatography AEX empty capsids full capsids stability in adsorbed state |
| url | http://www.sciencedirect.com/science/article/pii/S2329050123001511 |
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