Applications of Genovis' SmartEnzymes

Antibody Digestion

Antibody Deglycosylation

Antibody Conjugation

Glycan Profiling

Affinity Purification

Proteomics

Application Notes

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References

References

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SmartStories

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Latest SmartStories

Albert Bondt
Utrecht University

FabALACTICA in a Quantitative IgG1 Clone Profiling Study

Yury Tsybin
Spectroswiss

FabRICATOR Supports Analysis of a Complex Antibody-Oligonucleotide Conjugate

Oliver Krigslund
Finsen Laboratory/BRIC, University of Copenhagen

GlyCLICK for site-specific ADC development

C. Gstöttner & E. Dominguez-Vega
Leiden University Medical Center

Smart strategies to beat Covid-19 using SmartEnzymes

Albert Bondt
Utrecht University

FabALACTICA in a Quantitative IgG1 Clone Profiling Study

Tell us about your project!

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!

FabALACTICA Product Page

Generating Fabs using FabALACTICA

Publication

Blog post

Yury Tsybin
Spectroswiss

FabRICATOR Supports Analysis of a Complex Antibody-Oligonucleotide Conjugate

Tell us about your project!

In the current work, we utilized FabRICATOR (IdeS) digestion to streamline the characterization of a very complex antibody (trastuzumab)-oligonucleotide conjugate (AOC). Both denaturing and native Orbitrap mass spectrometry approaches were employed to analyze the corresponding intermediate and final AOC samples at intact and subunit levels.

How did SmartEnzymes enhance your project?

FabRICATOR has proven its ease of use, efficiency, and excellent specificity! Actually, FabRICATOR-based workflows have been an essential part of the Spectroswiss’ mAb analysis toolbox for almost a decade. We were the first to use FabRICATOR for the electron transfer dissociation (ETD) middle-down mass spectrometry of mAbs in 2014 (https://pubs.acs.org/doi/10.1021/ac4036857). Today, we regularly use FabRICATOR in our research and CRO activities for the mAb and ADC/AOC analysis that we perform on several structural levels.

Publication

Blog post


Discover the SmartEnzymes used in this project!

FabRICATOR

Antibody Digestion using FabRICATOR

Oliver Krigslund
Finsen Laboratory/BRIC, University of Copenhagen

GlyCLICK for site-specific ADC development

Tell us about your project!

Our main project is to develop future cancer drugs, and we have developed a platform around antibody drug conjugates (ADCs), where we test various actionable receptors as novel ADC targets. This project uses the GlyCLICK kit to conjugate antibodies to highly potent linker-toxins (payloads). By using GlyCLICK, we can develop highly homogeneous ADCs with a well-defined drug-to-antibody ratio (DAR). Usually, ADC development relies on conjugation to the cysteine- or lysine-residues of an antibody, which result in highly heterogeneous products with varying DARs. 

How did SmartEnzymes enhance your project?

When comparing the conjugation methods using MALDI-TOF mass spectrometry, the GlyCLICK prepared ADCs result in a narrow peak. In contrast, the cysteine-conjugated ADCs have several and broader peaks, suggesting that the GlyCLICK ADCs are highly homogeneous compared to the cysteine-conjugated ADCs. Even though techniques such as hydrophobic interaction chromatography (HIC) or reverse-phase HPLC can ensure more homogenous ADC batches, these methods are time-consuming, require expensive equipment, and generate lower ADC yield. In our project, drug homogeneity is of the highest importance, making the GlyCLICK preferred to other conjugation methods.

As GlyCLICK prepares the antibody for click-chemistry conjugation, the antibody can be conjugated to other components such as fluorescent dyes. This allows us to investigate how the conjugation method affects antibody functions such as binding affinity, stability, and internalization in target positive cell lines. Compared to interchain cysteine conjugation, we obtain more stable ADCs with the GlyCLICK technology since the cysteine conjugation relies on breaking some of the stabilizing cysteine bonds in the antibody.

Overall, the GlyCLICK kit has dramatically improved our project by making the ADC development more efficient and making the ADCs safer to use without losing potency.

Project Page


Discover the SmartEnzymes used in this project! GlyCLICK
C. Gstöttner & E. Dominguez-Vega
Leiden University Medical Center

Smart strategies to beat Covid-19 using SmartEnzymes

Tell us about your project!

Well characterized recombinant SARS-CoV-2 proteins are essential instruments in the fight against COVID-19. They are currently employed for diagnostic purposes, vaccine development and many other research activities. Our project aimed to perform an in-depth structural and functional characterization of commercial receptor-binding domains (RBDs) expressed in different mammalian systems (CHO and HEK293 cells). To achieve our goal, we applied several SmartEnzymes from the Genovis portfolio including the FucosEXO, GalactEXO, SialEXO, OglyZOR and OpeRATOR enzymes.

How did SmartEnzymes enhance your project?

We analyzed the RBDs by MS after complete removal of the N and O-glycans, permitting us to detect various modifications in the protein backbone (e.g. cysteinylation, pyroglutamic acid). By sequentially trimming of the N- and O-glycans using a combination of different exoglycosidases, we could assign the N- and O-glycans at the intact level, overcoming potential glycoform bias as observed by glycopeptide analysis. Overall, we observed tremendous differences in the glycosylation between CHO- and HEK293 produced RBDs with the latter exhibiting a combination of core 1 and core 2 type O-glycans. RBDs present two potential O-glycosylation sites in close proximity, difficulting the localization of the O-glycosylation site. With the help of the OpeRATOR enzyme, we demonstrated the presence of a single, fully occupied O-glycosylation site to the threonine 323 in both mammalian production systems. Furthermore, we investigated the functional differences between the glycosylated and deglycosylated RBDs with the help of SmartEnzymes. Our data suggested that RBD glycosylation plays a role in conformational stabilization of the spike protein rather than a direct involvement in ACE2 binding.

Link to the publication


Discover the SmartEnzymes used in this project! Exoglycosidases
Enzymes for O-glycans

View all SmartStories »

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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