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Article

Multiple-Monitor HPLC Assays for Rapid Process Development, In-Process Monitoring, and Validation of AAV Production and Purification

BIA Separations, Sartorius Company, Mirce 21, 5270 Ajdovscina, Slovenia
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Pharmaceutics 2021, 13(1), 113; https://doi.org/10.3390/pharmaceutics13010113
Received: 17 December 2020 / Revised: 8 January 2021 / Accepted: 13 January 2021 / Published: 17 January 2021
HPLC is established as a fast convenient analytical technology for characterizing the content of empty and full capsids in purified samples containing adeno-associated virus (AAV). UV-based monitoring unfortunately over-estimates the proportion of full capsids and offers little value for characterizing unpurified samples. The present study combines dual-wavelength UV monitoring with intrinsic fluorescence, extrinsic fluorescence, and light-scattering to extend the utility of HPLC for supporting development of therapeutic AAV-based drugs. Applications with anion exchange (AEC), cation exchange (CEC), and size exclusion chromatography (SEC) are presented. Intrinsic fluorescence increases sensitivity of AAV detection over UV and enables more objective estimation of empty and full capsid ratios by comparison of their respective peak areas. Light scattering enables identification of AAV capsids in complex samples, plus semiquantitative estimation of empty and full capsid ratios from relative peak areas of empty and full capsids. Extrinsic Picogreen fluorescence enables semiquantitative tracking of DNA with all HPLC methods at all stages of purification. It does not detect encapsidated DNA but reveals DNA associated principally with the exteriors of empty capsids. It also enables monitoring of host DNA contamination across chromatograms. These enhancements support many opportunities to improve characterization of raw materials and process intermediates, to accelerate process development, provide rapid in-process monitoring, and support process validation. View Full-Text
Keywords: adeno-associated virus; AAV; HPLC; intrinsic fluorescence; extrinsic fluorescence; light scattering; empty capsids; full capsids; process development; in-process analysis; validation adeno-associated virus; AAV; HPLC; intrinsic fluorescence; extrinsic fluorescence; light scattering; empty capsids; full capsids; process development; in-process analysis; validation
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MDPI and ACS Style

Gagnon, P.; Goricar, B.; Mencin, N.; Zvanut, T.; Peljhan, S.; Leskovec, M.; Strancar, A. Multiple-Monitor HPLC Assays for Rapid Process Development, In-Process Monitoring, and Validation of AAV Production and Purification. Pharmaceutics 2021, 13, 113. https://doi.org/10.3390/pharmaceutics13010113

AMA Style

Gagnon P, Goricar B, Mencin N, Zvanut T, Peljhan S, Leskovec M, Strancar A. Multiple-Monitor HPLC Assays for Rapid Process Development, In-Process Monitoring, and Validation of AAV Production and Purification. Pharmaceutics. 2021; 13(1):113. https://doi.org/10.3390/pharmaceutics13010113

Chicago/Turabian Style

Gagnon, Pete, Blaz Goricar, Nina Mencin, Timotej Zvanut, Sebastijan Peljhan, Maja Leskovec, and Ales Strancar. 2021. "Multiple-Monitor HPLC Assays for Rapid Process Development, In-Process Monitoring, and Validation of AAV Production and Purification" Pharmaceutics 13, no. 1: 113. https://doi.org/10.3390/pharmaceutics13010113

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