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

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

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

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
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


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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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