Determination of AAV Capsid Protein Ratios by Sodium Dodecyl Sulfate Capillary Electrophoresis (CE-SDS)-UV Absorption...

Recombinant adeno-associated viruses (AAVs) have become a leading platform for in vivo gene therapy. The AAV capsid is assembled from 60 individual protein subunits derived from three overlapping viral proteins (VPs): VP1, VP2, and VP3. The VP's ratio distribution affects the capsid trafficking and transgene expression. Quantitative analysis of the ratio is important in monitoring the potency of the AAVs. Sodium dodecyl sulfate capillary electrophoresis (CE-SDS) offers automated and quantitative determination of the VP ratio distribution, and the method has been widely used in the application. The CE-SDS method described was designed to ensure the consistent determination of AAV VP ratios across various sample concentrations. UV-absorption detection was chosen for this purpose as it negated the need for any labeling in the process. To compensate for the lower sensitivity associated with UV-absorption detection, methanol precipitation was employed during sample preparation, and a stacking injection technique was optimized for the samples in the study under CE conditions. These two techniques were found to be highly compatible. The method demonstrated robust linearity for AAV serotype 9 concentrations and exhibited good multiday precision. The VP ratios were successfully determined in AAV9 samples across a concentration range of 6.15 × 10¹² to 1.85 × 10¹³ GC/mL. The relative standard deviation (RSD%) for VP ratio determination within this concentration range was maintained below 7% for VP3, VP2, and VP1. Additionally, this method was further applied to other AAV serotypes, including AAV2 and AAV8, illustrating its versatility and broad applicability.