But we have also shown that in absence of such changes, the profile remains largely stable for at least 2 months! Thus largely exceeding the IgG1 half-life. This is a lot less than the potential and expected millions or even billions.Īt its current state the approach has shown that each donor has a unique repertoire, and that it may change over time in case of physiological changes, i.e. We found that the human IgG1 repertoire is dominated by only ‘a few’ (tens up to hundreds) of clones. The specificity for IgG1 allowed for use in combination with less specific other parts of the protocol. Right? NO! We have developed a methodology using FabALACTICA to generate profiles of IgG1 Fabs from human plasma samples. Immunoglobulin repertoires in blood are extremely complex and it is impossible to monitor individual clones. Microdroplet Ultrafast Reactions Speed Antibody Characterization. Rapid Characterization of Antibodies via Automated Flow Injection Coupled with Online Microdroplet Reactions and Native-pH Mass Spectrometry. These workflows and enzymes will hugely benefit pharmaceutical research and development where ultrafast digestions will play a pivotal role to speed up antibody characterization!įabRICATOR Product Page FabALACTICA Product Page Referencesġ. Microdroplets are generated to accelerate enzymatic reactions a 7.5 million-fold compared to the same reaction performed in bulk aqueous solvents (Zhao et el., 2021). We are pleased to showcase our most recent work that completely automates antibody characterization workflows using Genovis SmartEnzymes! In our recent paper, Gunawardena et al., 2023, we demonstrate the use of FabRICATOR (IdeS) and FabALACTICA (IgdE) enzymes that digest antibodies in real-time. Online Collision-Induced Unfolding of Therapeutic Monoclonal Antibody Glyco-Variants through Direct Hyphenation of Cation Exchange Chromatography with Native Ion Mobility–Mass Spectrometry. Altogether, our CEX-CIU method opens new possibilities to link conformational changes and resistance to gas-phase unfolding charge variants. Using this approach, we showed that the deglycosylated Fc fragments were more prone to unfolding events, while remodeled glycoforms showed increased resistance to gas-phase unfolding (both compared to the initial product). After transglycosylation and before CEX-CIU analysis, we performed digestion with the FabRICATOR enzyme since the main interest was the modified Fc fragment. During this procedure, the glycans present on the initial mAb were first partially removed leading to the deglycosylated product, whereafter a particular glycoform was attached (in this case, G2S2 with and without fucose). To generate defined glycoforms, we used TransGLYCIT remodeling. The novel CEX-CIU method was applied to derive glycoform-specific information on the gas-phase unfolding patterns of Fc fragments of trastuzumab. How did SmartEnzymes enhance your project?
In this way, we could monitor the unfolding patterns of species with differences in charge and thereby, reveal subtle conformational differences between mAb proteoforms. We have combined the charge-based separation of cation exchange chromatography (CEX) with the ability to investigate the gas-phase behavior of collision-induced unfolding (CIU). In our study, a fast and robust method was developed to characterize the conformational landscape of proteins and proteoforms in an online manner. Therefore, it is key to develop innovative analytical strategies to gain an in-depth understanding of the influence of PTMs both on a structural and functional level. Besides increased structural complexity, altered PTM profiles may impact protein conformation or even biological function (i.e., efficacy and safety of therapeutic mAbs). Therapeutic monoclonal antibodies (mAbs) are heterogeneous proteins that can have a variety of post-translational modifications (PTMs), including glycosylation or deamidation.
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