Abstract
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.
Highlights
The use of columns with large pores (300 A ) at high temperatures (60–75 °C) in combination with non-traditional solvent system containing ion pairing agents has been consolidated as standard procedure for the analysis of mAbs, overcoming previous difficulties[14,15]
We investigated RP-UPLP-MALS for mAb characterization, focusing on two common applications: (i) analysis and characterization of mAb fragments, which are typically studied by mass spectrometry, (ii) analysis of mAbs after long term storage
The principle of RP-HPLC-MALS is the combination of RP chromatography with an online MALS detector
Summary
The use of columns with large pores (300 A ) at high temperatures (60–75 °C) in combination with non-traditional solvent system containing ion pairing agents has been consolidated as standard procedure for the analysis of mAbs, overcoming previous difficulties[14,15]. Small chemical differences cannot be separated by standard RP-HPLC16, as they are often insufficient to yield significant changes in polarity[17]. We took advantage of ultra-high pressure LC (UPLC) instrumentation to further refine the separation of mAb species and their derivatives. We investigated RP-UPLP-MALS for mAb characterization, focusing on two common applications: (i) analysis and characterization of mAb fragments, which are typically studied by mass spectrometry, (ii) analysis of mAbs after long term storage. The former is a real-time stability testing which permits the establishment of recommended storage condition and shelf life of the bio-therapeutic products. The addition of MALS allows the Mw assignment for each individual peak in the chromatogram enabling differentiation between chemical variants of the monomeric form and other impurities or degradation products as aggregates and fragments
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