Systematic comparison of wide pore size exclusion chromatography columns for the characterization of gene therapy products

A new generation of wide pore SEC columns was systematically evaluated for the determination of size variants for mRNAs ranging from 1000 to 5000 nucleotides and for multiple recombinant adeno-associated viruses (rAAVs) serotypes. Among the six SEC columns (450 to 700 Å) employed for rAAV analysis, the DNACore AAV-SEC column shows the highest efficiency (11,000 plates), probably due to its monodisperse 3 µm silica particles, while the SRT SEC-500 column showed the lowest efficiency (< 1000 plates) attributed to its 5 µm particle packing. Optimal selectivity for rAAVs was observed with columns having larger pore sizes (550 - 700 Å). However, the final resolution for different rAAV serotypes was highly sample-dependent, and no single column consistently provided the best separation of size variants. Additionally, the six columns were suitable to study the degradation trends of rAAVs subjected to accelerated stress conditions. Among the five SEC columns (700 - 1000 Å) evaluated for mRNA analysis, the Biozen dSEC-7 LC column (700 Å) column systematically achieved the highest efficiency, and was particularly well suited for analyzing small mRNA (∼ 1000 nts). However, columns with larger pore sizes such as AdvanceBio SEC 1000A were more appropriate for larger mRNA (> 1000 nt). Despite these findings, the separation of low molecular weight species (LMWS) and high molecular weight species (HMWS) of mRNA remains limited across all tested columns, making integration challenging and the quantification of LMWS and HMWS less accurate. However, comparable (but not strictly identical) quantitative performance was obtained among the columns, with only a few exceptions.