Tell us about your project!

Therapeutic monoclonal antibodies (mAbs) are heterogeneous proteins that can have a variety of post-translational modifications (PTMs), including glycosylation or deamidation. Besides increased structural complexity, altered PTM profiles may impact protein conformation or even biological function (i.e., efficacy and safety of therapeutic mAbs). Therefore, it is key to develop innovative analytical strategies to gain an in-depth understanding of the influence of PTMs both on a structural and functional level. In our study, a fast and robust method was developed to characterize the conformational landscape of proteins and proteoforms in an online manner. We have combined the charge-based separation of cation exchange chromatography (CEX) with the ability to investigate the gas-phase behavior of collision-induced unfolding (CIU). In this way, we could monitor the unfolding patterns of species with differences in charge and thereby, reveal subtle conformational differences between mAb proteoforms.

How did SmartEnzymes enhance your project?

The novel CEX-CIU method was applied to derive glycoform-specific information on the gas-phase unfolding patterns of Fc fragments of trastuzumab. To generate defined glycoforms, we used TransGLYCIT remodeling. During this procedure, the glycans present on the initial mAb were first partially removed leading to the deglycosylated product, whereafter a particular glycoform was attached (in this case, G2S2 with and without fucose). After transglycosylation and before CEX-CIU analysis, we performed digestion with the FabRICATOR enzyme since the main interest was the modified Fc fragment. Using this approach, we showed that the deglycosylated Fc fragments were more prone to unfolding events, while remodeled glycoforms showed increased resistance to gas-phase unfolding (both compared to the initial product). Altogether, our CEX-CIU method opens new possibilities to link conformational changes and resistance to gas-phase unfolding charge variants.

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TransGLYCIT Product Page

References

van Schaick et al., 2023. Online Collision-Induced Unfolding of Therapeutic Monoclonal Antibody Glyco-Variants through Direct Hyphenation of Cation Exchange Chromatography with Native Ion Mobility–Mass Spectrometry. Anal. Chem.

Tell us about your project!

We are pleased to showcase our most recent work that completely automates antibody characterization workflows using Genovis SmartEnzymes! In our recent paper, Gunawardena et al., 2023, we demonstrate the use of FabRICATOR (IdeS) and FabALACTICA (IgdE) enzymes that digest antibodies in real-time. Microdroplets are generated to accelerate enzymatic reactions a 7.5 million-fold compared to the same reaction performed in bulk aqueous solvents (Zhao et el., 2021).

How did SmartEnzymes enhance your project?

These workflows and enzymes will hugely benefit pharmaceutical research and development where ultrafast digestions will play a pivotal role to speed up antibody characterization!

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FabRICATOR Product Page

FabALACTICA Product Page

References

1. Gunawardena et al., 2023. Rapid Characterization of Antibodies via Automated Flow Injection Coupled with Online Microdroplet Reactions and Native-pH Mass Spectrometry. Anal. Chem.
2. Zhao et al., 2021. Microdroplet Ultrafast Reactions Speed Antibody Characterization. Anal. Chem.

Tell us about your project!

We are working on the glycan engineering of mucin by SmartEnzymes, specifically the SialEXO enzyme. The abundant glycans on mucin are the bioactive ligands binding to biomolecules and cell receptors, modulating cell responses and human physiology.

How did SmartEnzymes enhance your project?

SialEXO is one of the most efficient sialidases we tested that can remove sialic acid from mucin in mucus gel directly. This high activity enhances our aim to modulate the cell responses towards modified mucin in mucus, with potential application as new therapy for mucin-related diseases.

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SialEXO Product Page

Complete desialylation of biopharmaceuticals using SialEXO

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!

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FabALACTICA Product Page

Generating Fabs using FabALACTICA

Publication

Blog post

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

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Discover the SmartEnzymes used in this project!

FabRICATOR

Antibody Digestion using FabRICATOR

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

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Discover the SmartEnzymes used in this project! GlyCLICK

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

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Discover the SmartEnzymes used in this project! Glycan profiling

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

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FabRICATOR

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

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FabULOUS

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

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GingisKHAN

FabRICATOR

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

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GlyCLICK Product Page

GlyCLICK Applications Page

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

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FabRICATOR MagiC Product page

FabRICATOR MagIC Application page

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

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FabALACTICA

FabRICATOR

Blog post

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

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OpeRATOR

Application

OpeRATOR Poster

Blog post

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 biologics characterization. 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 detection, we are not seeing them in our datasets. This 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

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FabRICATOR

FabRICATOR Immobilized

OglyZOR

SialEXO

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

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GlyCLICK

Applications

Blog post

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 a 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

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GingisKHAN

FabRICATOR

Applications

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