For Better Biologics

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Therapeutic monoclonal antibodies (mAbs) are heterogeneous proteins that can have a variety of post-translational modifications (PTMs), including glycosylation or deamidation. Besides increased structural complexity, altered PTM profiles may impact protein conformation or even biological function (i.e., efficacy and safety of therapeutic mAbs). 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. In our study, a fast and robust method was developed to characterize the conformational landscape of proteins and proteoforms in an online manner. 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 this way, we could monitor the unfolding patterns of species with differences in charge and thereby, reveal subtle conformational differences between mAb proteoforms.

How did SmartEnzymes enhance your project?

The novel CEX-CIU method was applied to derive glycoform-specific information on the gas-phase unfolding patterns of Fc fragments of trastuzumab. To generate defined glycoforms, we used TransGLYCIT remodeling. 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). After transglycosylation and before CEX-CIU analysis, we performed digestion with the FabRICATOR enzyme since the main interest was the modified Fc fragment. 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). Altogether, our CEX-CIU method opens new possibilities to link conformational changes and resistance to gas-phase unfolding charge variants.

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TransGLYCIT Product Page

References

van Schaick et al., 2023. Online Collision-Induced Unfolding of Therapeutic Monoclonal Antibody Glyco-Variants through Direct Hyphenation of Cation Exchange Chromatography with Native Ion Mobility–Mass Spectrometry. Anal. Chem.

Tell us about your project!

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. 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).

How did SmartEnzymes enhance your project?

These workflows and enzymes will hugely benefit pharmaceutical research and development where ultrafast digestions will play a pivotal role to speed up antibody characterization!

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FabRICATOR Product Page

FabALACTICA Product Page

References

1. Gunawardena et al., 2023. Rapid Characterization of Antibodies via Automated Flow Injection Coupled with Online Microdroplet Reactions and Native-pH Mass Spectrometry. Anal. Chem.
2. Zhao et al., 2021. Microdroplet Ultrafast Reactions Speed Antibody Characterization. Anal. Chem.

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We are working on the glycan engineering of mucin by SmartEnzymes, specifically the SialEXO enzyme. The abundant glycans on mucin are the bioactive ligands binding to biomolecules and cell receptors, modulating cell responses and human physiology.

How did SmartEnzymes enhance your project?

SialEXO is one of the most efficient sialidases we tested that can remove sialic acid from mucin in mucus gel directly. This high activity enhances our aim to modulate the cell responses towards modified mucin in mucus, with potential application as new therapy for mucin-related diseases.

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SialEXO Product Page

Complete desialylation of biopharmaceuticals using SialEXO

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Immunoglobulin repertoires in blood are extremely complex and it is impossible to monitor individual clones. Right? NO! We have developed a methodology using FabALACTICA to generate profiles of IgG1 Fabs from human plasma samples. The specificity for IgG1 allowed for use in combination with less specific other parts of the protocol. We found that the human IgG1 repertoire is dominated by only ‘a few’ (tens up to hundreds) of clones. This is a lot less than the potential and expected millions or even billions. At 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. a sepsis episode. 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.

How did SmartEnzymes enhance your project?

In addition to the knowledge we can gain on antibody biology in vivo using FabALACTICA, we also succeeded in deriving the protein sequence of one of the detected clones. This is an additional huge advantage of the ability to ‘see’ individual clones. Many more biological and technological angles will be worked on with this technology. Stay tuned!

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FabALACTICA Product Page

Generating Fabs using FabALACTICA

Publication

Blog post