In-Capillary Immunoassay And Total Protein Detection For Adeno-Associated Virus (AAV) Proteins During Purification From Whole-Cell Lysate

During adeno-associated virus (AAV) manufacture, critical quality attributes must be monitored including the presence, identity, and purity of viral vector proteins. Traditionally, the identity and purity of these proteins is monitored by Western blot using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE). However, SDS-PAGE is notoriously challenging and labor-intensive. Additionally, AAVs are limited in sample size as they are difficult to manufacture, and demand outstrips supply.

Here, we have developed a method to monitor the purification of AAV2 using automated capillary electrophoresis followed by immunoassay and total protein detection directly in the capillary, eliminating the need for SDS-PAGE. AAV2 was purified from HEK293 cell lysate using affinity chromatography, and the steps of the purification process (load, flow-through, wash and elution) were monitored by the capillary-based immunoassay. VP1, VP2 and VP3 capsid proteins were resolved and identified either individually or simultaneously, depending on the AAV antibody used for detection, and the total protein assay monitored the presence of impurities. The sensitivity of this assay reduced sample size down to 3 μL of  sample, corresponding to approximately 400 pg or 1x10⁸ genomic copies loaded per well. We anticipate that this in-capillary immunoassay and total protein detection can replace traditional SDS-PAGE methods in AAV manufacturing workflows.