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. 2021 Jan 17;13(1):113.
doi: 10.3390/pharmaceutics13010113.

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

Pete Gagnon  1 , Blaz Goricar  1 , Nina Mencin  1 , Timotej Zvanut  1 , Sebastijan Peljhan  1 , Maja Leskovec  1 , Ales Strancar  1
Affiliations

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

Pete Gagnon et al. Pharmaceutics. .

Abstract

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.

Keywords: AAV; HPLC; adeno-associated virus; empty capsids; extrinsic fluorescence; full capsids; in-process analysis; intrinsic fluorescence; light scattering; process development; validation.

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Conflict of interest statement

The authors declare no conflict of interest. Sartorius had no role in the design of the study, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Separation of empty and full capsids by anion exchange (AEC) with different monitoring methods. (a) UV absorption at 260 nm and 280 nm. (b) Light scattering. (c) Intrinsic fluorescence.
Figure 2
Figure 2
Picogreen staining requirements for adeno-associated virus (AAV) capsids. (a) Dye concentration. (b) Incubation time.
Figure 3
Figure 3
Picogreen detection of capsid exterior-associated DNA. (a) AEC eluted with a salt gradient. (b) AEC eluted with a pH gradient.
Figure 4
Figure 4
SEC of cation exchange (CEC)-purified AAV capsids stained with Picogreen. (a) Before treatment with proteinase K. (b) After 1 h incubation. (c) After 24 h.
Figure 5
Figure 5
CEC with Picogreen staining for characterization of DNA contamination. (a) Clarified lysate. (b) After CEC capture from clarified lysate. (c) After CEC capture from lysate treated with nuclease and TFF.
Figure 6
Figure 6
SEC with Picogreen staining. (a) Filtered sf9/BEV lysate. (b) Filtered HEK293 lysate. (c) Filtered HEK293 lysate after treatment with CIMasphere H-Bond particles.
Figure 7
Figure 7
SEC with intrinsic fluorescence. (a) Filtered lysate. (b) After treatment with 4 M NaCl at indicated pH values. (c) After treatment with CIMasphere H-Bond particles at 4 M NaCl, pH 3.5.
Figure 8
Figure 8
Light scattering identification of AAV in chromatograms of filtered lysate. (a) SEC. (b) CEC with salt gradient elution. (c) AEC with salt gradient elution.
See this image and copyright information in PMC

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