Characterization and Mechanism of VP3 Variant Peak in AAV Capsid Subunit Purity Analysis
This study aimed to investigate the cause of an unidentified peak that appeared ahead of the VP3 subunit peak during the purity analysis of adeno-associated virus(AAV)capsid subunits by capillary electrophoresis sodium dodecyl sulfate(CE-SDS)technique.Initially,a point mutation was introduced to the second start codon within the base sequence of the recombinant adeno-associated virus(rAAV)capsid protein's VP3 subunit via PCR to generate a mutant rAAV.Subsequently,the relationship between this mutation and the unidentified signal peak was elucidated using CE-SDS technique.Additionally,the relative molecular weight and peptide analysis of the unknown peak was detected by LC-MS.The results indicated that if the VP3 subunit's base sequence contain two initiation codons(ATG),the translation might start from the second initiation codon.And the expression prod-uct was a variant of the VP3 subunit(VP3'),which aligned with the unidentified signal peak observed in CE-SDS analysis.Consequently,this unidentified peak should be regarded as a AAV capsid subunit peak during the purity assessment of AAV capsid proteins.