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Multi-angle light scattering coupled with size-exclusion chromatography (SEC-MALS) is a standard approach for protein characterization. Recently MALS detection has been coupled with ion-exchange chromatography (IEX) which demonstrated the feasibility and high value of MALS in combination with non-sized-based fractionation methods. In this study we coupled reverse-phase ultra-high pressure liquid chromatography (RP-UPLC) with a low-dispersion MALS detector for the characterization of intact monoclonal antibody (mAbs) and their fragments. We confirmed a constant refractive index increment value for mAbs in RP gradients, in good agreement with the values in literature for other classes of proteins. We showed that the impurities eluting from a RP column can often be related to aggregated species and we confirmed that in most cases those oligomers are present also in SEC-MALS. Yet, in few cases small aggregates fractions in RP-UPLC are an artifact. In fact, proteins presenting thermal and physical stability not suitable for the harsh condition applied during the RP separation of mAbs (i.e. organic solvents at high temperature) can aggregate. Further, we applied RP-UPLC-MALS during a long term stability studies. The different principle of separation used in RP-UPLC- MALS provides an additional critical level of protein characterization compared to SEC-MALS and IEX-MALS.

scientific reports

Coupling Multi-Angle Light Scattering to Reverse-Phase Ultra-High-Pressure Chromatography (RP-UPLC-MALS) for the characterization monoclonal antibodies