Share your experience, be a SmartScientist

At Genovis, we firmly believe that sharing scientific achievements and experiences will lead to new ideas and insights that will contribute to better biologics for patients.

 

 

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

Aaron Bailey
BGI Americas

Analytical services lab exploit FragIT, OglyZOR and SialEXO

Bo Holmqvist
ImaGene-iT

Dynamic fluorescence imaging using the GlyCLICK technology

Simone Nicolardi
Leiden University Medical Center

GingisKHAN & FabRICATOR assist in-source decay FT-ICR Mass Spectrometry analysis

Your name
Your Affiliation

Present your work here!

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

Aaron Bailey
BGI Americas

Analytical services lab exploit FragIT, OglyZOR and SialEXO

How have you setup your industrial platform? 

Our mass spec center is a service and R&D lab focused on solutions for proteomics research and biologiccharacterization. So we provide LC-MS services for multi-omic researchers as well as pharmaceutical research and development customers. 

How important is sample preparation? 

We have had success using several deglycosidases including SmartEnzymes like FragIT, OglyZOR and SialEXO. In a sort of combinatorial sample prep matrix these enzymes help determine how highly complex glycoform patterns are derived from possibly several types of glycosylation events (i.e., N-linked vs. O-linked vs. sialylated). This sample prep strategy is a powerful way to navigate this challenge. 

What are the advantages of using SmartEnzymes? 

We found the activity and purity of the SmartEnzymes was ideal for native LC-MS because we can efficiently deglycosylate using very small amounts of enzyme. Since the levels are near or below the actual level of detectionwe are not seeing them in our datasetsThis is a critical point because it means we can keep our analyses streamlined and avoid having to address any artifact data that is not specifically related to the therapeutic protein in question. 

What are the next steps for you? 

The San Jose lab uniquely allows BGI to offer state-of-the-art genomic and proteomic analytical services globally. In the coming year we plan to grow our team and add new LC and MS instrumentation to further increase our ability to support this market. We plan to provide even deeper support for our main focus areas of proteomics and biologics characterization. 

Link to Aaron´s ASMS Poster


FabRICATOR

FragIT

OglyZOR

SialEXO

Bo Holmqvist
ImaGene-iT

Dynamic fluorescence imaging using the GlyCLICK technology

Tell us about yourself and ImaGene-iT

I am CSO at ImaGene-iT, an independent contract research company supporting the life science industry and academia. Our projects comprise a variety of questions and involve various types of biological samples on both the histological and cellular level. As a principal investigator, I perform most of the quality assessments using high-resolution confocal microscopy along with imaging for further digital analysis.

What are the key aspects of experimental design? 

From the question at hand and to obtain the best end result, we want to participate in the whole chain of tailor-made procedures. This includes the initial handling of samples, the choice of labeling technology and the best suited detection and imaging equipment.

What are the advantages of using GlyCLICK? 

In our tests, antibodies with GlyCLICK-conjugated fluorophores work very well for imaging in both in vitro cell and tissue analyses. The conjugates provide an excellent signal-to-noise ratio for detection and imaging with fluorescence microscopy. For confocal microscope analysis, GlyCLICK-conjugated antibodies give an optimal range of intensity levels. The optimal range of detected intensity levels improves the quality of assessment as well as the digital imaging.

What does the future of imaging look like? 

In our field, one of the major recent advances in imaging is the ability to extract a large amount of data from images. The use of markers with a known number of conjugation sites per target molecule, such as the GlyCLICK technology offers, can significantly improve quantitation possibilities for all types of imaging-based analysis. The combined use of GlyCLICK-conjugated fluorescence with small animal in vivo imaging could further expand the quality of quantitative data that can be collected for our clients, from the cellular level to the whole animal.

More about ImaGene-iT


GlyCLICK

Applications

Blog post

Simone Nicolardi
Leiden University Medical Center

GingisKHAN & FabRICATOR assist in-source decay FT-ICR Mass Spectrometry analysis

Tell us about yourself? 

My work is focused on the development of ultrahigh resolution mass spectrometry-based methods for the analysis of biomolecules such as glycans, peptides, and proteins. This includes the structural characterization of monoclonal antibodies for determination of primary amino acid sequence and post-translational modifications. 

What is new in your recent publication? 

Our newly developed method combines the advantage of ultrahigh resolution mass measurements with the efficient fragmentation provided by MALDI in-source decay (ISD) using 1,5-diaminonaphthalene as a reducing MALDI matrix. The main advantages are complementary sequence information compared to other mass spectrometric fragmentation techniques and the use of minimal sample preparation procedure that does not require separation techniques such as liquid chromatography. 

What are the advantages of using SmartEnzymes? 

The use of FabRICATOR or GingisKHAN allows for increasing sequence information of heavy chains to more internal protein regions. In addition, after digestion, the mAb Fc portion can be analyzed directly by MALDI-MS allowing for the detection of the most abundant Fc glycoforms. 

How do you view sample preparation prior to MS analysis? 

Liquid chromatography is typically used to separate mAb subunits and remove ESI-non-compatible compounds like salts. Our method is based on MALDI which is known to be more tolerant to salts. Thus, SmartEnzymes combined with MALDI-ISD MS avoids laborious sample preparation steps that can lead to artificial the modification of mAbs. Also, it comes with short analysis times in contrast to other chromatographic techniques.

What are you working on next? 

Our attention is now on bispecific mAbs. We apply our MALDI-based method for the simultaneous analysis of all six different polypeptide chains generated after treatment with FabRICATOR and chemical reduction of the disulfide bonds. Our aim is to develop a fast method for the monitoring of chemically induced mAb modifications. 

Link to publication


GingisKHAN

FabRICATOR

Applications

Your name
Your Affiliation

Present your work here!

At Genovis, we firmly believe that sharing scientific achievements and experiences will lead to new ideas and insights that will contribute to better biologics for patients. Take the opportunity to share your work on our SmartStories stage and inspire others to follow your lead. We invite you to sign up below to receive a SmartScientist kit. Every SmartScientist needs not just smart enzymatic tools but also a few smart necessities for the lab.    

Apply here!

Take the opportunity to share your work on our SmartStories stage and inspire others to follow your lead. We invite you to sign up below to receive a SmartScientist kit. Every SmartScientist needs not just smart enzymatic tools but also a few smart necessities for the lab.

 

Order