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.
Highlights
Rapid analytical feedback is a critical element for timely development of processes that yield clinical quality biological drugs
The ratio of UV absorbances at 260 nm and 280 nm enabled identification of an early peak populated dominantly by empty capsids (260/280 = 0.64), and a later peak populated dominantly by full capsid (260/280 = 1.31)
Light scattering estimated that the full capsid peak represented 78%, but that should be understood to be an overestimate due to the influence of higher mass in full capsids [1,54,55]
Summary
Rapid analytical feedback is a critical element for timely development of processes that yield clinical quality biological drugs. It plays an important role in documenting that established processes remain within specified control limits during validation and manufacturing. HPLC monitored by UV absorbance at 280 nm has contributed substantially to fulfillment of these roles with recombinant proteins like monoclonal antibodies. This was reflected the dominance the 280 nm profile. Removal by NaOH [6] This was reflected in the dominance of the 280 nm profile. DNA contamination remains substantial, as shown in panel (a) of Figure 5
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