Applications of Genovis' SmartEnzymes

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At Genovis, we believe that enzymes with unique properties can be used as biological tools to support the research and development of complex biopharmaceuticals to help bring safe and effective medicines to patients in need. Genovis SmartEnzymes can be used in applications including Antibody Characterization, Antibody Fragmentation, Antibody Conjugation, Antibody Deglycosylation, O-glycan Analysis, EXOglycosidases and Proteomics.

Antibody Characterization

Antibody Conjugation

Antibody Deglycosylation

Antibody Fragmentation

EXOglycosidases

O-glycan Analysis

Proteomics

All Applications

All Applications

Learn about the applications of Genovis' SmartEnzymes.

Genovis SmartEnzyme Reference Tool

SmartEnzyme Reference Tool

Find the right reference for the antibody attribute you are studying

References

References

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SmartStories

SmartStories

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Latest SmartStories

Min Kyung So
KBIO Osong Medical Innovation Foundation

FabRICATOR for Intact Analysis of Biologics

Constantin Blöchl
University of Salzburg

FabULOUS Middle-level Analysis of Murine Polyclonal Antibodies

Hanieh Khalili
University of East London

Antibody mimetics generation using GingisKHAN

Bastiaan Duivelshof
University of Geneva

Generating site-specific ADCs using the GlyCLICK technology

Min Kyung So
KBIO Osong Medical Innovation Foundation

FabRICATOR for Intact Analysis of Biologics

Tell us about your project

The major mission for our team is analytical method development and characterization of protein pharmaceuticals using LC, CE, and MS. When analyzing antibody drug candidates, the intact mass measurement with LC-MS is the first step to ensure the sample quality. There are many tools for intact mass analysis of antibodies, and the FabRICATOR is the one main choice for our workflow because it is very efficient and fast. Although the other products from Genovis have been used in our team for characterization of proteins, the FabRICATOR enzyme remains a workhorse for our mission.

Research Group


FabRICATOR

Constantin Blöchl
University of Salzburg

FabULOUS Middle-level Analysis of Murine Polyclonal Antibodies

Tell us about your project

The development of top-down and middle-level analytical HPLC-MS strategies in recent years mainly focused on the characterization of therapeutic monoclonal antibodies. In our research, we aim at developing similar strategies to analyze polyclonal IgG with regard to subclass abundance and glycosylation patterns, which may provide new insights into immune regulatory processes. However, when dealing with polyclonal IgGs, we faced the challenge of pronounced molecular heterogeneity arising from sequence variability. In this context, we took advantage of the glycosylated Fc domain, which is conserved in polyclonal antibodies and determines the IgG subclass. In our recent work, we investigated the feasibility of middle-level analysis by FabULOUS (SpeB) digestion and HPLC-MS of polyclonal mouse IgG.

How did SmartEnzymes enhance your work?

The FabULOUS enzyme was exploited for the generation of Fc/2 subunits from all murine IgG subclasses, which all were proven amenable for proteolytic cleavage. The obtained subunits enabled the dissociation of Fc and Fab domains required to tackle the immense sequence heterogeneity within the variable regions. Middle-down analysis by HPLC-MS of the Fc/2 subunits allowed the assignment of the specific isotypes, while middle-up analysis provided quantitative information on the subclasses as well as their respective glycosylation variants. The workflow thereby enables global analysis of polyclonal murine IgGs with respect to subclass abundances including closely related isotypes as well as glycosylation profiles and other PTMs. Finally, we demonstrated the capabilities of our workflow in a pilot study dealing with polyclonal IgG from mouse serum after immunization with pollen allergen.

In summary, the described middle-level workflow provides comprehensive information obtained in a single analysis involving swift sample preparation, standard HPLC-MS analysis, and straightforward data evaluation as an attractive extension to the toolbox of analytical strategies for antibodies.

Publication

Blog Post


FabULOUS

Hanieh Khalili
University of East London

Antibody mimetics generation using GingisKHAN

Tell us about your project

To extend the utility of both IgG and Fc-fusion proteins and overcome their stability issues which is a need to develop longer acting medicines, we have developed IgG and Fc-fusion mimetics called Fab-PEG-Fab (FpF) and receptor-PEG-receptor (RpR). Antibody fragments (Fabs) and receptor-binding fragment (VEGFR1-VEGFR2) are obtained by proteolytical digestion of IgG and aflibercept respectively. FpF and RpRs are prepared using disulfide bridging reagents, PEG-di(mono-sulfone) reagent. Each terminus in the PEG reagent undergoes site-specific conjugation with the two cysteine thiols from an accessible disulfide in a Fab or VEGFR1-VEGFR2 by a sequence of addition-elimination reactions resulting in conjugation by the insertion of a 3-carbon methylene bridge to re-anneal the original disulfide into a more stable re-bridged disulfide.

How did SmartEnzymes enhance your project?

To prepare the RpR, we needed to generate receptor-binding fragment (VEGFR1-VEGFR2). We first used immobilised pepsin but the yield and efficiency of obtained fragment was very poor. Using of IdeS enzyme (FabRICATOR®, Genovis), we were able to obtain pure fragment with a yield of above 90%. In case of GingisKHAN, we used it to generate Fab fragment from IgG to prepare Fab-PEG-Fab mimetics. While we initially used immobilized papain to obtain Fab, we later found out that the stability profile of FpF prepared from Papain-Fab was very different from FpF prepared from GingisKHAN-Fab. The Fab generated by GingisKHAN was pure and homogenous and resulted in increased stability of FpF.

Blog Post

Research Group


GingisKHAN

FabRICATOR

Bastiaan Duivelshof
University of Geneva

Generating site-specific ADCs using the GlyCLICK technology

Tell us about your project and how SmartEnzymes enhanced it:

In our project we focused on glycan-mediated conjugation technology to create homogeneous and site-specific ADC products. The aim was to develop an analytical platform that can quickly and accurately monitor this conjugation process. First, we used the innovative GlyCLICK-technology to couple the commonly used maytansine payload DM1 to trastuzumab in a site-specific manner. The glycan-mediated technology allowed to not only control the DAR, but also the drug load distribution (DLD) of the ADC product. Then, to monitor these important CQAs and the conjugation process, we used a middle-up LC/HRMS approach to compare the GlyCLICK product with unconjugated trastuzumab. The subunits were generated by using FabRICATOR and FabALACTICA enzymes and were analyzed in both RPLC- and HILIC-mode. A significant shift in retention of the crystallizable fragment (Fc/2) was observed as result of the lipophilic drug payload conjugation, confirming the site-specific conjugation process. At last, MS detection confirmed the accurate DAR ratio of 2.0 and the absence of randomly conjugated payloads. Therefore, we believe that the GlyCLICK procedure to create novel ADCs combined with the middle-up analysis to monitor important CQAs related to the conjugation process holds a great potential in the field of ADC development.  

Any ideas for new SmartEnzymes and what they could be called?

Tell us more: For middle-up analysis of complex protein therapeutics, with both N- and O-glycans it would be interesting to have an enzyme that enables the removal of the O-glycans without prior removal of the sialic acids. This would be useful when you aim to keep the N-glycans intact during the analysis. Another option would be a sialidase that only removes the sialic acids from O-glycans while keeping the N-glycans intact. This would be useful when analyzing, e.g., highly glycosylated fusion proteins like etanercept, where a judicious combination of enzymatic digestions can provide highly interesting information on the glycoform heterogeneity.

Link to GlyCLICK ADC Development

Link to Paper


GlyCLICK Product Page

GlyCLICK Applications Page


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IgG Protease Digestion Chart

With the IgG protease digestion chart, you see the digestion sites of the Genovis' enzymes and what species and subclass of antibody the enzymes digest.

Species & Subclasses FabRICATOR®
Native IgG
FabULOUS®
Digestion under reduced condition.
* Primary digestion site.
GingisKHAN®
Native IgG
Human IgG1 ..CPAPELLG / GPSVF.. ..KTHT / CPPCPAPEL..* ..KSCDK / THTCPPCP..
Human IgG2 ..CPAPPVA / GPSVF.. Yes (Site not determined) No digestion confirmed
Human IgG3 ..CPAPELLG / GPSVF.. Yes (Site not determined) Multiple sites in Fc
Human IgG4 ..CPAPEFLG / GPSVF.. Yes, with longer incubation time No digestion confirmed
Mouse IgG1 No digestion confirmed Yes, several in the hinge region No digestion confirmed
Mouse IgG2a ..CPAPNLLG / GPSVF.. Yes, several in the hinge region No digestion confirmed
Mouse IgG2b No digestion confirmed Yes, several in the hinge region No digestion confirmed
Mouse IgG3 ..CPPGNILG / GPSVF.. Yes, several in the hinge region No digestion confirmed
Rat IgG2b ..CPVPELLG / GPSVF.. Yes, several in the hinge region Not determined
Rhesus Monkey ..CPAPELLG / GPSVF.. Not determined Not determined
Rabbit ..CPPPELLG / GPSVF.. ..KPT / CPPPE..* Not determined

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