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

All references on Genovis' products

SmartStories

SmartStories

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Latest SmartStories

Bastiaan Duivelshof
University of Geneva

Generating site-specific ADCs using the GlyCLICK technology

Dan Bach Kristensen
Symphogen A/S

FabRICATOR MagIC produce F(ab)2 subunits in less than 30 minutes

Valegh Faid
LFB Biotechnologies

FabALACTICA in non-reducing study of antibody disulphides

Weiming Yang
Johns Hopkins University

OpeRATOR changes the game in the field of O-linked glycoproteomics

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

Dan Bach Kristensen
Symphogen A/S

FabRICATOR MagIC produce F(ab)2 subunits in less than 30 minutes

Tell us about your project and how SmartEnzymes enhanced it

F(ab)2 workflows have for years been an essential part of Symphogen's characterization toolbox. With the introduction of FabRICATOR MagIC we have made a swift transition to automated, fast and robust F(ab)2 production. Combining the FabRICATOR MagIC with the KingFisher Prime Duo robot we can now produce F(ab)2 subunits in less than 30 minutes, and with minimal manual sample handling. Indeed, the only time we handle the antibody sample is when we pipet the sample into the 96 well plate, where the digestion is performed subsequently. In other words, the FabRICATOR MagIC workflow is truly a one-step pipetting workflow. The rest is handled by the FabRICATOR MagIC and the KingFisher Prime Duo robot. After the digestion, the 96 well plate with the digested mAbs is transferred directly to autosampler of our LC MS platform and analyzed. Simple, robust, hands-off and superfast production of F(ab)2 subunits for analytical characterization of antibody biopharmaceuticals.

Have ideas for new SmartEnzymes and what they could be called? Tell us more:

MagIC versions of Fab generating enzymes and enzymes for deglycosylation of mAbs.

Link to FabRICATOR MagIC Evaluation


FabRICATOR MagiC Product page

FabRICATOR MagIC Application page

Valegh Faid
LFB Biotechnologies

FabALACTICA in non-reducing study of antibody disulphides

How did you come up with the idea to combine FabRICATOR (IdeS) and FabALACTICA (IgdE)? 

The middle-up analysis is fast and very informative regarding the protein sequence integrity and post-translational modifications. However, investigating the oxidative state of disulfide bridges is tricky and often involved a time-consuming peptide mapping in non-reducing conditions. The combination of FabRICATOR and FabALACTICA in non-reducing conditions presents the advantage to generate specifically three fragments i.e. hinge, Fc/2 and Fab that are both easily separated by RP-HPLC and analysed by MS.

How does the new enzymatic assay compare to previous methods to study antibody disulphide bonds? 

Peptide mapping in non-reducing condition is time consuming even if dedicated software to improve the treatment of data has largely improved. The combined FabRICATOR and FabALACTICA middle-up approach increases the throughput for the investigation of free thiols and disulphide scrambling.

Would the assay be used in a QC setting relying solely on liquid chromatography separation?

The analytical workflow is robust and requires mere handlings of the antibody samples. Once the identification of each peak of the chromatogram is confirmed by MS, quantitation based on the UV detection is a current practice. Such analytical configuration involving an HPLC and a UV detection is actually common in most of QC labs and thus easily and robustly implementable.

 Why are antibody disulphide bonds important?

Disulphide bonds are highly important because of their critical role in the stabilization of protein conformations. Breaking and/or scrambling of disulphide bond occur during manufacturing and storage of biotherapeutics which is a concern in terms of safety and efficacy. The monitoring of these product-derived impurities is mandatory during development operations in order to minimize these forms.

Link to Paper


FabALACTICA

FabRICATOR

Blog post

Weiming Yang
Johns Hopkins University

OpeRATOR changes the game in the field of O-linked glycoproteomics

Tell us about yourself?

I work as a Research Associate in Mass Spectrometry Core Facility at the Johns Hopkins University. My interest is in O-linked glycosylation and I have developed a series of glycoproteomic methods to study protein N- and O-linked glycosylation.

What is new with the ExoO method you have developed?

Using EXoO, scientists can start to gain new insight into their biological systems regarding O-linked glycoproteins. The EXoO method identifies a large number of O-linked glycosylation sites in the sample that may be easily identified by using other methods such as various enrichments coupled with ETD-MS/MS.

What are the benefits of applying Operator in the workflow?

The OpeRATOR enzyme is a key component in the workflow. The high specificity of OpeRATOR enabled release of site-specific O-linked glycopeptides from solid phase support. Therefore, the resulting glycopeptides are relatively pure for improved identification.

How did you perform the analysis prior to this method?

We tried to released O-glycopeptides using beta-elimination to study site of protein O-linked glycosylation and ETD-MS/MS for O-linked glycopeptide analysis but the number of identification is lower than the use of the current method using EXoO to release the O-glycopeptides.

How would you describe the impact of OpeRATOR on the O-glycan field?

The discovery of OpeRATOR changes of the game in the field of O-linked glycoproteomics. It makes the analysis of large-scale and site-specific O-linked glycoproteome in clinical samples feasible.

What are your thoughts on the future of O-glycan analysis?

EXoO and OpeRATOR provide unique research tools to identify the site of O-linked glycosylation. So far, the evidence supports that core 1 Gal-GalNAc structure can be studied by the use of OpeRATOR. In the future, the structures of O-linked glycans on the specific sites on the proteins can be revealed in a single workflow.

Link to Publication


OpeRATOR

Application

OpeRATOR Poster

Blog post


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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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