Program

Poster Presentations

Day 3, June 12（Fri.）　　Room P (5F 501+502)

3P-46

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Identification of Recombinant Adeno-Associated Virus Serotypes by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

(¹UOsaka, ²Shimadzu, ³UOsaka, ⁴U-Medico)
^oRyoji Nakatsuka^1,2,3, Kenjiro Matsumoto⁴, Yannan Liu¹, Kimitoshi Takeda⁴, Yasuo Tsunaka¹, Tetsuo Torisu¹, Yuki Yamaguchi¹, Susumu Uchiyama¹

Recombinant adeno-associated virus (rAAV) is widely used for gene delivery, but the diversity of serotypes complicates identification of serotypes during manufacturing. Conventional antibody-based assays often lack the specificity required to distinguish closely related serotypes or variants. Here, we present a matrix-assisted laser desorption/ionization mass spectrometry (MALDI–MS) workflow combined with a weighted scoring algorithm for reliable rAAV serotype identification. Wild-type and variant serotypes were produced in-house, enzymatically digested, and analyzed using a benchtop MALDI–TOF instrument. Spectra were processed with Python software that scores peptide mass fingerprints based on both peptide count and specificity, defined by m/z differences from non-target serotypes. This approach enabled unambiguous identification of all tested serotypes, with reproducible scoring across replicate measurements. Variant-specific peptides derived from mutation sites allowed clear discrimination of engineered capsids from their parental serotype despite >98% sequence identity of the viral protein. Overall, this study demonstrates that MALDI–MS with a weighted scoring strategy provides a rapid, antibody-free, and cost-effective method for rAAV serotype and variant identification, supporting its application as an orthogonal identity test in research, development, and manufacturing workflows.

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