Inter-laboratory Study of mAb Characterisation using Top-Down and Middle-Down Mass Spectrometry
Srzentic et al. present an interlaboratory study investigating the current state of top-down and middle-down mass spectrometry methods for the characterization of therapeutic monoclonal antibodies and explore the potential added value of these methods to the more commonly used bottom-up mass spectrometry.
In the growing market of therapeutic monoclonal antibodies (mAbs), there is an increasing demand to precisely and reproducibly characterize the structural heterogeneity of mAbs. At present, bottom-up mass spectrometry (MS) approaches, using peptide mapping of tryptic peptides, is widely employed for this. In this study, initiated by the Consortium of Top-Down Proteomics, the use of top-down and middle-down MS methods were explored as potential complements to bottom-up MS approaches in regard to primary structure confirmation, variable domain sequencing and post-translational modification (PTM) identification on mAbs.
The three commercial mAbs: SiLuLite, NIST mAb standard and Herceptin were centrally prepared in three different forms before being distributed to 20 participating laboratories worldwide; intact for top-down analysis, and digested using either of the IgG proteases GingisKHAN (above the hinge) or FabRICATOR (below the hinge) for middle-level analysis. The participating laboratories were asked to analyze these using their top-down/middle-down MS workflow of choice.
The study revealed the wide variety of techniques and expertise available among the participants. Nevertheless, top-down/middle-down approaches proved to be a fast and valuable compliment to bottom-up approaches in achieving a high amino acid sequence coverage, avoiding artifacts and confirming bottom-up derived PTM profiling and stoichiometry. They could also deduce if protein modifications observed in bottom-up assays were naturally occurring or sample preparation mediated. Middle-level analysis, using FabRICATOR or GingisKHAN generated subunits, proved especially beneficial for assigning correct monoisotopic masses and identifying glycosylation and glycation sites. The use of IgGZERO to remove the Fc N-glycans and eliminate glycosylation related heterogeneity prior to top-down/middle-down MS analysis was also suggested. Overall, the study shows the added value of top-down/middle-down approaches for mAb analysis and the great potential for further advances in top-down and middle-down MS techniques.
QED Bioscience and Genovis Form a New Team!
We are excited to announce that QED Bioscience and Genovis have joined together and combined their strengths to provide top-quality research tools to scientists across the globe.
QED Bioscience is a US company based in San Diego that has been designing and manufacturing antibodies for 25 years, providing superior antibody products and services to support our customers with the expertise and assistance they should expect from a full-spectrum antibody company.
Whether you are conducting antibody research, optimizing an existing antibody assay, or developing an antibody therapeutic, you need the best antibodies, and characterization tools and methods from QED Bioscience and Genovis. Use the best products, with the right protocols. Our new team will focus on delivering the best antibodies and antibodies conjugated with site-specific technologies to improve antibody-based research. The new conjugation technology GlyCLICK has striking impact on reproducibility and quality of labeling and is available today.
As a Genovis customer, we encourage you to explore the new offerings together with QED Bioscience and thank you for your continued business with us. Read more about QED Bioscience on their website
Best regards,
Eileen Skaletsky,
Managing Director, QED Bioscience
John Lindsay,
President, Genovis Inc.
Antibody Development with QED
Monoclonal Antibody Development
The art of creating hybridomas requires years of experience to be perfected. In the more than 20 years that QED has been providing this service to the scientific community, we have made monoclonal antibodies to numerous different types of antigens including large and small peptides, proteins, haptens, carbohydrates, bacterial and viral antigens, and plasmid DNA-encoded antigens.
Learn more on Monoclonal Antibody Development
Polyclonal Antibody Development
QED offers custom antibody production in rabbits utilizing your protein, peptide, small molecule, plasmid, or other antigens. All programs can be customized to fit your needs, so if you need additional rabbits or want to use your own immunization protocol, just send us your requirements.
Learn more on Polyclonal Rabbit Antibody Production
Mapping the O-glycoproteome Using Site-Specific Extraction of O-linked Glycopeptides
The Tn-antigen is characterized by the presence of a single N-Acetylgalactosamine (GalNAc) residue linked to serine or threonine residues on proteins. It is an immature glycoform without extension to form any of the common O-glycan core structures. The Tn antigen is a hallmark of many forms of cancer and rarely found in healthy tissue. Mapping and comparing Tn-antigens on proteins is therefore of great interest but current methods are laborious and inefficient.
Scientists at Johns Hopkins School of Medicine have developed an ingenious new approach for mapping Tn-antigens from a number of complex samples (Yang et al 2020a). It is based on a technique called ExoO (Yang et al 2018) where tryptic peptides are covalently immobilized on a solid support and digested with OpeRATOR®. As this enzyme can only digest when an O-glycan is present, this results in the specific release of O-glycopeptides which can be analyzed in-depth using LC-MS.
However, OpeRATOR does not digest at sites modified with Tn antigens. To circumvent this limitation, Yang et al. first treated their samples with recombinant C1GalT1, an enzyme able to elongate the Tn antigen into a core 1 structure (Gal-β1,3- GalNAc). Sites modified with core 1 O-glycans are efficiently digested by OpeRATOR and can therefore be mapped in depth using EXoO and LC-MS. To distinguish the Tn-antigen sites from naturally occurring core 1 glycosylation sites, the C1GalT1 reaction was performed in the presence of a heavy isotope labelled donor substrate (13C-UDP-Gal). This introduces a unique mass signature that specifically marks the Tn-antigen sites and is easily detectable by mass spectrometry. Using this new approach, termed EXoO-Tn, Yang et al. were able to identify an approximately 10-fold higher number of Tn-glycosylation sites compared to previous studies.
Link to paper, Yang, W. et al., 2018. Mapping the O-glycoproteome using site-specific extraction of O-linked glycopeptides (EXoO). Molecular systems biology, 14(11), p.e8486.
For a detailed description on how to use OpeRATOR for in-depth mapping of O-glycosylation sites using the EXoO workflow, check also this recent publication in Nature Protocols (Yang et al 2020b)
FabRICATOR® in complete characterization of seven therapeutic monoclonal antibodies
Monoclonal antibodies (mAb) are used in various treatments and new potential targets continue to arise. To ensure a high specificity of the antibody to the intended antigen each mAb needs to be well characterized since they are often heterogeneous. In this paper, Giorgetti et al. combined bottom-up, middle-level and intact level analysis to get the complete structure of seven mAbs with worldwide approval. For the middle-level analysis, FabRICATOR digestion followed by a reduction step was the preferred approach.
On-line capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) compatible with all three levels of analysis was used for the characterization. The overall heterogeneity and high mass post-translational modifications (PTMs) were determined at the intact level. Confirming the results, the middle-level approach expanded on this with more advanced PTM data and information about the backbone structure. Finally, bottom-up analysis provided the precise location of the PTMs and the relative quantity of micro-heterogeneities in the proteoforms. However, while bottom-up data provided the most detailed information it required the most sample preparation which might lead to artifacts. The intact and middle-level analysis avoided this problem. Therefore, the research group suggested that a combination of the three levels efficiently analyzed with CE-ESI-MS was ideal for a representative, complete characterization of the structure of the mAbs.
For middle-level analysis the mAbs were digested with FabRICATOR yielding F(ab)’2 and Fc/2 fragments of about 100 kDa and 25 kDa. This allowed PTMs such as methionine oxidation and lysine clipping to be observed. The N-glycan profile was also determined. Reduction of the F(ab)’2 fragment to light chain (LC) and Fd fragments further reduced complexity to make small PTMs such as asparagine deamidation detectable. In addition, the speed and efficiency along with the high accuracy was appreciated by the researchers.
IgGZERO® used to determine the core fucosylation of antibodies in bioprocess
Scientists at NIBRT in Dublin together with scientists at the University of Manitoba, Winnipeg, have investigated the effect of two different methods to control the level of fucosylation of a model antibody during expression in CHO cells.
Production of biopharmaceuticals in mammalian cells requires that critical quality attributes are controlled for safety and therapeutic efficacy. The efficacy of an IgG antibody for cancer immunotherapy is dependent on its ability to elicit effector functions such as antibody-dependent cell cytotoxcicity (ADCC). The absence of fucose on the core GlcNAc of the Fc glycan in the antibody increases the ADCC and hence there is a desire to control the level of fucosylation during the manufacturing process.
The model antibody investigated in this work was a camelid heavy-chain human Fc fusion of about 80 kDa in size. During expression of the antibody in CHO cells, two factors were were evaluated: the effect of adding a fucosyltransferase inhibitor and the impact of overexpression of a gene that deflects the fucose de novo pathway to a dead-end. The antibodies were harvested from the cell culture supernatant by a protein A column. The N-glycans of the antibodies were then released using PNGaseF, labelled with 2-AB and analyzed by HILIC-HPLC. The fucosylation pattern of the antibodies was identified by electrospray ionization mass spectroscopy (ESI-MS) of the intact control antibody after treatment with IgGZERO (EndoS). Hydrolysis with IgGZERO results in three possible antibody variants with two, one or no fucose per antibody. Since IgGZERO specifically removes the Fc glycans leaving the core GlcNAc (+/- fucose), the observed shift in mass of -146 Da and -292 Da revealed antibody species where one or two fucoses were missing. Using this approach, it was shown that the inhibitor for fucosyltransferase decreased the addition of fucose on the inner GlcNAc during the expression of the antibody in a concentration dependent manner.
By combining data from the released glycan analysis with the mass data of intact antibody after IgGZERO treatment, crucial information about the glycan profile and fucosylation pattern was revealed and evaluated to support the bioprocess design.
IgGZERO® Turns a Toxic Antibody into a Novel Treatment for Sepsis
Genovis SmartEnzyme IgGZERO was used in this recent study by researches from the University of Pennsylvania and the Philadelphia Children’s Hospital.
Sepsis is a dysregulated immune response to an infection that leads to very high levels of inflammation resulting in tissue damage and potential for multiorgan failure. It therefore leads to a high rate of mortality and morbidity. Neutrophil extracellular traps are part of the innate immune systems defence against infections where neutrophils rupture and release DNA, histones and many antimicrobial proteins. This however comes at a cost as NETs are degraded by circulating DNase and toxic degradation products are formed.
In a recent study published in Blood, researchers from the University of Pennsylvania and the Philadelphia Childrens Hosptial used a monoclonal antibody that binds to NETs to stabilize the traps, therefore significantly reducing the collateral tissue damage induced by NET degradation products. However, the mAb also activated the complement system and platelets, therefore negating the positive effect of NET stabilization. Using IgGZERO, the authors were able to remove the Fc glycosylation from their mAb, thereby impairing its ability to elicit an immune response. The deglycosylated mAb was still able to stabilize NETs and its administration lead to a significantly improved outcome in a murine sepsis model.
Fc glycosylation is a major determinant for which effector functions a monoclonal antibody is able to elicit and therefore its mode of action. Genovis IgG-specific endoglycosidases (GlycINATOR and IgGZERO) provide an easy way to remove the Fc glycans from mAbs.
Genovis Business Continuity
Genovis continues operation and remain committed to serve our customers during the ongoing pandemic crisis. Manufacturing and distribution of the Genovis SmartEnzymes™ are running according to our business continuity plan to meet our customers’ needs and to secure supply.
We are monitoring the situation carefully and will notify you immediately if we experience disruptions in fulfilling customer orders. Genovis is fully committed to providing uninterrupted supply of our products to safely keep your research on track.
With these uncertain times “scientists enabling scientists” takes on new meaning. Toward this goal, we continue to provide convenient online access to our customer service, technical support and sales teams. Should you have any questions or concerns, do not hesitate to contact us at info@genovis.com.
Most importantly, we hope your staff as well as ours stay healthy and safe during this time.
SmartEnzymes Poster Highlights 2019
Which are the top five poster from Genovis in 2019? We have selected the posters that are both popular with our website visitors and that describe the most exciting new applications using SmartEnzymes. Click on the images to download the full posters.
1) ASMS Poster, 2019
1 – Analysis of O-glycosylated-Biopharmaceuticals using an O-glycan Dependent Endoprotease and LC-MS (ASMS, 2019)
In this collaborative work, we set out to combine our novel, specific enzymes with the latest LC-MS technologies from Thermo Fisher Scientific in order to improve and simplify analytical workflows for biotherapeutics. We demonstrate in-depth characterization of O-glycosylated biopharmaceuticals and quantitative comparison of O-glycosylation patterns. We also present a workflow for total deglycosylation of heavily glycosylated biopharmaceuticals, allowing for intact mass spectrometry analysis without interference from glycan heterogeneity.
2) World ADC Poster, 2019
2 – Robust Generation of Site-specific and Homogeneous Antibody Conjugates using GlyCLICK® (World ADC, 2019)
In this work, we present details on the enzymatic processing of the Fc-glycans that result in the homogenous conjugates, and confirmed it by mass spectrometry. The immunoreactivity of the conjugated antibodies was studied using surface plasmon resonance and toxicity by a dose-dependent response of a DM1 GlyCLICK conjugated trastuzumab (T-DM1).
CASSS AT Poster, 2019
3 – An Automated Workflow for Analysis of Monoclonal Antibody Subunits (CASSS AT 2019)
Here we present a rapid, automated solution for antibody subunit generation and analysis using a standard HPLC-MS setup with only minor modifications. FabRICATOR® (IdeS) enzyme was immobilized in an HPLC column format to allow for easy on-column digestion of IgG-based biologics followed by RP-HPLC-MS analysis. This facilitates a fully automated, completely hands-off workflow for analysis of several critical quality attributes.
4 – FabALACTICA® Generates Pure and Homogenous mAb Subunits that Facilitate 2D-NMR Spectroscopy Analysis (Festival of Biologics, 2019)
4) Festival of Biologics Poster, 2019
Two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) allows for the precise atomic-level comparison of higher order structure (HOS) for IgG-based biopharmaceuticals. Since most of the approved therapeutic mAbs today have a human IgG1 backbone, the cysteine protease FabALACTICA (IgdE) can simplify the analysis. The enzyme digests human IgG1 at a specific site above the hinge, generating intact Fab and Fc fragments. In this work, we present a workflow for obtaining homogeneous Fab and Fc fragments that are ideal for evaluating HOS of the chimeric mAb infliximab using 2D-NMR.
5 – Complete and Rapid Desialylation of Therapeutic Glycoproteins using Immobilized SialEXO® (AET 2019)
The enzymatic performance of the Immobilized SialEXO column was tested on therapeutic glycoprotein substrates: human C1 inhibitor, etanercept, cetuximab, and human tissue-type plasminogen activator (tPA). Treated and native glycoproteins were then analyzed using released glycan analysis, antibody subunit LC-MS, and capillary iso-electric focusing. The sialidase column delivered complete de-sialylation of 0.5 mg glycoprotein after 30 min at room temperature. In summary, the new Immobilized SialEXO column provides robust and rapid desialylation ideal for routine analysis of biopharmaceuticals with a range of commonly used analytical techniques
AET Poster, 2019
First Paper on the FabRICATOR®-HPLC Column from Genentech
Scientists at Genentech and the University of Geneva have used FabRICATOR-HPLC to set up an automated workflow for the analysis of monoclonal antibodies by LC-MS.
Developing and manufacturing therapeutic antibodies requires the analysis of many product quality attributes. Robust and fast analytical methods are needed to support process development and quality control. In this new paper by Camperi et al., a novel workflow for automated subunit analysis of mAbs is described. Samples were digested on-column using FabRICATOR-HPLC and then transferred to a reverse phase HPLC column where the subunits could be separated and analyzed by mass spectrometry. This allowed for a completely hands-off analysis of critical quality attributes such as Fc glycosylation, glycation and C-terminal lysine clipping. More than 150 injections were performed on the same FabRICATOR-HPLC column with reliable results. Furthermore, analysis of a bispecific antibody revealed product related impurities due to mispairing of the chains.
FabRICATOR-HPLC enables on-line sample preparation for middle-level analysis and therefore reduces operator time and errors due to sample handling.
Link to FabRICATOR-HPLC Product page and Poster below
SmartEnzymes™ in Antibody Subunit Analysis by Janssen and Celgene
Scientists at Janssen and Celgene use SmartEnzymes to analyze symmetric and asymmetric structure–function relationships of modified bispecific antibodies.
Forced degradation studies are useful for quickly evaluating critical quality attributes. Such modifications may have different biological impacts depending on if they occur symmetrically, in both chains of the antibody, or asymmetrically, in only one chain.
In the recently published article, Evans et al. first generated symmetrically and asymmetrically oxidized or deamidated samples. They then used two SmartEnzymes, IgGZERO® and FabRICATOR®, to do middle level mass spectrometry analysis, also referred to as antibody subunit analysis. They first employed IgGZERO to deglycosylated the N-glycans from the Fc region, FabRICATOR was used to digest the antibodies below the hinge and a reduction step resulted in subunits in the range of 25kDa – ideal for MS analysis.
Using this approach, the authors could probe the impact of symmetrical and asymmetrical oxidation or deamidation on IgG1 binding to the FcRn receptor or on protein A, respectively. The authors propose their technique, with subunit analysis, is ideal as a platform method for monitoring these or other modifications where symmetry might be crucial.
Read more about IgGZERO or FabRICATOR.
Full article available here:
Evans, A. R. et al., 2019. Using bispecific antibodies in forced degradation studies to analyze the structure–function relationships of symmetrically and asymmetrically modified antibodies, mAbs, 11:6, 1101-1112, DOI: 10.1080/19420862.2019.1618675