Intact and peptide level characterization of nanobodies by high resolution HPLC-MS/MS

Nanobodies are single-domain antibodies derived from heavy-chain-only antibodies (HcAbs) commonly found in camelids, consisting solely of the variable, antigen-binding domain. Their small size affords some unique properties such as higher stability, better tissue penetration with potential access to cryptic tumors and easier, higher yield prokaryotic production, which has attracted growing interest in therapeutic and research use.

 

In this work, we characterized the stochastic labeling of two research-grade nanobodies using the benchtop X500B QTOF LC-MS/MS System. The former nanobody contains an Alexa Fluor dye conjugated via the maleimide linker, while the latter nanobody is biotin-labeled via N-hydroxysuccinimide (NHS) conjugation. A quick and reproducible intact mass measurement was able to confirm the ID, purity, modifications and disulfide bonds. The differences in heterogeneity in labeling between maleimide and NHS chemistry is also readily seen. A rapid trypsin digestion followed by peptide mapping was carried out to pinpoint the specific sites and degrees of labeling. Finally, we were able to adjust the biotin labeling efficiency by varying reagent to nanobody ratio . The expression, purification and characterization of these research-grade nanobodies lay the groundwork for future therapeutic programs.

 

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