Articles in the Category ”References”
Monitoring Glycation Levels on Bispecific Biologics using FabRICATOR®
Bispecific monoclonal antibodies (BsAbs) are multi-functioning and complex biologics with the ability to recognize two different epitopes for improved therapeutic properties. Characterizing protein modifications such as glycation on biologics is vital to ensure consistency in stability and function. The structural complexity of BsAbs requires robust analytical methods, where conventional top-down and bottom-up strategies may lack in sensitivity or even introduce further modifications. Middle-level analysis using site-specific proteases such as GingisKHAN®(Kgp) and FabRICATOR®(IdeS) is an intermediate strategy that enables complementary analysis of intact or reduced Fab and Fc fragments.
In a recent article by Gstöttner et al. (2019) from Leiden University together with Roche Pharma Technical Development, the authors analyzed a BsAb for protein modification levels, N- and C-terminal sequencing and modification localization using top-down, middle-level and bottom-up strategies. The BsAb was analyzed for changes in glycation levels over time using middle-up FT-ICR MS on Fc/2, LC and Fd’ fragments obtained by FabRICATOR digestion. The scientists also localized glycation hot spots on the heavy chain backbone of the FabRICATOR-digested BsAb using sequential in source decay (ISD) MALDI fragmentation.
Using FabRICATOR in a novel middle-up MS strategy, the scientists were able to analyze all antibody subunits in a single high-resolution mass spectrum. By implementing the method in a forced-glycation experiment, changes in glycation levels were successfully monitored over time. The authors were also able to localize several glycation hot spots by intact top-down and FabRICATOR-assisted middle-down analyses. The use of middle-level strategies in combination with conventional MS-based methods successfully provided complementary data for monitoring the level of glycation.
Learn more about FabRICATOR and our other proteases.
SmartEnzymes™ in a new approach to characterize ADCs
Antibody drug conjugates (ADCs) consist of monoclonal antibodies chemically linked to a cytotoxic agent. The target specificity of the monoclonal antibody in combination with the potency of the cytotoxic drug make ADCs promising therapeutic agents. However, the molecules are often complex, making evaluation of the quality attributes for the ADC challenging.
In order to characterize the ADCs, the predominant analysis of choice is peptide mapping with reversed-phase liquid chromatography (RPLC) coupled to mass spectrometry. However, the sample preparation steps in a bottom-up approach are often time-consuming and a comprehensive view of ADCs with different sequence variants and post-translational modifications is lacking.
In this recently published article by Chen et al., a middle-down RPLC-MS strategy with electron transfer disscociation (ETD) was developed to analyze lysine and cysteine conjugated ADCs at the subunit level. FabRICATOR® (IdeS) and GingisKHAN® (KGB) were used to generate the subunits. FabRICATOR digests below the hinge, generating F(ab’)2 and Fc/2 fragments, and GingisKHAN digests above the hinge, generating intact Fab and Fc fragments. For the deglycosylation, the IgG-specific endoglycosidase GlycINATOR® (EndoS2) was used.
This middle-down approach enabled high-resolution evaluation of several ADC quality attributes at the subunit level, including drug to antibody ratio (DAR), conjugation sites and micro-variants. The approach shows great potential for investigating quality attributes during the development and characterization of novel ADCs.
Read more about FabRICATOR, GingisKHAN and GlycINATOR.
FragIT™ kit in Study Evaluating IgG Charge
Protein charge is a fundamental property that influences both the ability to interact with other molecules and the structure, solubility and stability of the protein.
In this collaborative work by Boehringer Ingelheim Pharmaceuticals, Janssen BioTherapeutics and University of New Hampshire, the charge measurements of twelve monoclonal IgG and their F(ab’)2 and Fc fragments are presented. Besides other interesting findings, it is suggested that mAb charge measurements is valuable when it comes to selecting candidate molecules for development.
FragIT kit was used to achieve the IgG subunits before charge evaluation. This kit consists of spin columns of an immobilized version of the FabRICATOR (IdeS) enzyme for antibody digestion and spin columns for affinity binding of the Fc fragments. Using this kit, the Fc and F(ab’)2 fragments were easily separated from each other, with no enzyme in the final preparation.
For more information about FragIT kit, please visit the following page:
The full-text paper is available online:
Yang et al., 2019. IgG Charge: Practical and Biological Implications. Antibodies, 8(1), 24
Improved antibody-PET tracers for in vivo imaging with GlyCLICK®
Radioactively labelled antibodies are excellent immuno-PET tracers for evaluating in vivo distribution and performance of therapuetic agents. Site-specific conjugation at the antibody Fc glycan site by enzymatic remodeling allows for a uniform label distribution of such PET-tracers, compared to conjugates generated with conventional random labelling strategies.
In an article by Kristensen et al. (2019), the authors evaluated the stability, immunoreactivity and in vivo biodistribution of the radioactively labelled mAb Trastuzumab (Herceptin). Using GlyCLICK, the antibody was enzymatically modified with GlycINATOR (EndoS2) and conjugated with a DIBO-DFO chelator prior to 89Zr radioactive labelling. Comparing the GlyCLICK technology with ß-galactosidase remodelled conjugates and two random labelling techniques, the authors obtained valuable data on the overall performance of the various PET-tracers.
Antibodies subjected to site-specific labelling showed significantly increased in vitro stability and immunoreactivity compared to randomly labeled Trastzumab. Furthermore, using in vivo immuno-PET imaging, these conjugates also displayed superior tumor-targeting properties based on the successful detection of HER2-positive tumors in mouse models. These results highlight the advantages of site-specific antibody conjugation.
For more information on GlyCLICK please visit
FabRICATOR® in capillary zone electrophoresis-native mass spectrometry
The use of capillary electrophoresis for the analysis of therapeutic antibodies and other biopharmaceuticals is growing in popularity. A new article by researchers from CNRS in France showed how capillary zone electrophoresis-native mass spectrometry could be used for the quality control of intact therapeutic monoclonal antibodies. The authors show for the first time the use of a triple layer coated (PB-DS-PB) capillary with mAbs, which helps prevent mAbs adsorption. The intact therapeutic mAb, Infliximab, was analyzed under non-denaturing conditions to retain conformational heterogeneity and avoid denaturation. A middle up approach using FabRICATOR digestion was used to characterize dimer association detected in the stressed mAbs preparation. Digestion below the hinge region of the mAb produced F(ab’)2 and Fc/2 fragments which was subsequently analysed by capillary zone electrophoresis-native mass spectrometry. Using this approach the authors were able to see nature of the dimer association while maintaining the non-covalent interactions of the Fc/2 fragments.
For more information on FabRICATOR please visit the following pages:
- FabRICATOR Product Page
- Generation of Antibody Fragments
Free Thiols using FabRICATOR® and FabALACTICA®
In biopharmaceutical product development and manufacturing, free thiol content is one of the product quality attributes of interest as its presence could impact structure, stability and function of the product.
At Biogen, Yi Pu et al have optimized a label-free LC (UV) / MS method for free thiol quantification at a subunit level of IgG1 and IgG4. The new method, which is based on a method developed by Faid et al*, was compared to two conventional approaches, Ellman’s assay and peptide mapping.
It is very challenging to identify free thiol forms by mass spectrometry at the intact antibody level. By combining the highly specific proteolytic enzymes FabALACTICA (IgdE) and FabRICATOR (IdeS) the authors generated the subunits Fab, hinge and Fc/2, suited for confident mass determination. The subunits were subsequently separated on a polyphenyl reversed phase column in order to separate free thiol forms from their corresponding disulphide bond-linked form. A baseline or near baseline separation was obtained making it possible to calculate the free thiol content on each subunit.
The result of the quantification of free thiols from all three methods were comparable and showed similar trends even though the peptide mapping approach generally gave a higher free thiol content.
The authors conclude that compared to Ellman’s assay, the subunit approach is more sensitive, requires less sample and provides domain-specific information of the free thiol content. Compared to peptide mapping, the subunit method is faster, less labour intensive and lacks dependence on labelling efficiency. Finally, it demonstrated promise in the quantification of free thiols in a high throughput manner with domain specific information available.
The developed method has successfully been applied to several in-house IgG1 mAbs with different hydrophobicity and isoelectric points.
*V. Faid Y. Leblanc N. Bihoreau G. Chevreux Middle-up analysis of monoclonal antibodies after combined IgdE and IdeS hinge proteolysis: Investigation of free sulfhydryls, J. Pharm. Biomed. Anal. 149 (2018) 541-546, https://doi.org/10.1016/j.jpba.2017.11.046
For more information on FabRICATOR and FabALACTICA please visit the following pages:
- FabRICATOR Product Page
- FabALACTICA Product Page
- Generation of Antibody Fragments
FabRICATOR® driven middle-down glycan analysis using NMR
Analysis of the glycosylation of therapeutic antibodies and other biopharmaceuticals is typically done by LC or LC/MS-based methods. However, each analytical technique has its strengths and weaknesses which makes the development of orthogonal methodologies crucial for in-depth characterization. In this study, researchers from the FDA present a middle-down NMR approach for studying glycosylation of therapeutic antibodies. Analogous to middle-down MS methods, the antibodies were digested using FabRICATOR to yield Fc/2 fragments. After chemical denaturation, these exhibited high enough solubility and sufficiently fast molecular dynamics to allow for glycan analysis by 2D-NMR without the need for isotope labeling or glycan release. Using this method, the authors were able to determine the chemical structure, glycosidic linkage position and anomeric configuration of each monosaccharide unit of the major Fc N-glycan structures and were able to quantify important quality attributes such as galactosylation and fucosylation.
For more information on FabRICATOR please visit the following pages:
- FabRICATOR Product Page
- Generation of Antibody Fragments
SmartEnzymes™ assist MALDI in-source decay FT-ICR Mass Spectrometry analysis
The Consortium for Top-down Proteomics is currently conducting a large inter lab study. They are developing methods for the analysis of intact mAbs and antibody subunits generated by digestion with either FabRICATOR or GingisKHAN. In this paper, van der Burgt et al. present a novel method for the analysis of mAbs based on MALDI in-source decay fragmentation coupled with high resolution FT-ICR mass spectrometry. The standard method for antibody sequence confirmation by MS is based on fragmentation using electron transfer dissociation (ETD). The MALDI-ISD based method yielded complementary fragments to those observed in ETD experiments, translating to increased sequence coverage. Using digestion with either FabRICATOR or GingisKHAN, a higher total sequence coverage could be achieved than for the intact mAbs. FabRICATOR digestion also allowed for direct analysis of Fc glycosylation by MALDI-FT-ICR without the need for LC separation.
Meet the Scientist
We got the opportunity to interview the last author of the paper, Dr Simone Nicolardi at Leiden University Medical Center.
Tell us about yourself?
I am a senior researcher at the Center for Proteomics and Metabolomics at Leiden University Medical Center (https://www.lumc.nl/org/proteomics-metabolomics/). 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 with the MALDI in-source decay fragmentation you have published?
Our newly developed method combines the advantage of ultrahigh resolution mass measurements, obtained using high-end instrumentation and novel spectra processing software, with the efficient fragmentation provided by MALDI-in-source decay 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 FabRICATOR or GingisKHAN?
In MALDI-ISD experiments singly charged ions are generated from the protein backbone. Thus, a wide m/z-range is needed for the detection of all fragments generated from large compounds, such as monoclonal antibodies. Even in high-end MS instrumentation, this m/z-range is limited and sequence information is obtained from N- and C-terminal protein portions only. The use of FabRICATOR or GingisKHAN allows extending sequence information of heavy chains to more internal protein regions. In addition, after digestion mAb Fc portion can be analyzed directly by MALDI-MS allowing the detection of most abundant Fc glycoforms.
How do you view sample preparation prior to MS analysis of mAbs?
The structure characterization of mAbs is generally performed using a multi-level approach based on different analytical methods. Many of these methods are based on liquid chromatography (LC) electrospray ionization (ESI) MS. LC is used to separate mAb subunits and to remove ESI-non-compatible compounds such as salts. Our method is based on MALDI which is known to be more tolerant to salts in the sample. Thus, MALDI-ISD MS avoids laborious sample preparation steps that can lead to artificial modification of mAbs. Also, it comes with short analysis times in contrast to other chromatographic techniques.
What are you working on now?
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 disulfide bonds. Our aim is to develop a fast method for the monitoring of chemically induced mAb modifications.
For more information on FabRICATOR or GingisKHAN please visit the following pages:
- FabRICATOR and GingisKHAN Product Page
- Generation of Antibody Fragments
FabRICATOR, SialEXO and OglyZOR in Middle-up HILIC/HRMS Approach
In an article by Valentina D’Atri et al. recently published in Analytical Chemistry (2019), the scientists developed a middle-up HILIC/HRMS workflow for detailed characterisation of the Fc fusion protein etanercept. The etanercept molecule consists of an IgG1 Fc domain fused to a tumour necrosis factor receptor (TNFR) and is used in the treatment of autoimmune diseases such as rheumatoid arthritis. The protein is highly glycosylated and contains numerous O- and N-glycosylation sites that require extensive characterization.
To develop a strategy that would work with a mass spec instrument of limited resolution, the authors used FabRICATOR enzyme to specifically digest the etanercept molecule and generate TNFR and Fc/2 subunits. Combinations of the O- and N- glycosidases SialEXO, OglyZOR and PNGaseF were applied to allow evaluation of the O- and N-glycosylation patterns of TNFR and Fc/2 respectively. In addition, complete deglycosylation allowed for primary structure analysis. By using a wide-pore HILIC stationary phase, appropriate separation of the subunits with different degrees of remaining glycans was achieved, and this significantly facilitated spectra deconvolution.
Applying this workflow, D’Atri and colleagues were able to assess the main PTMs, the subunit distribution of glycans, the overall N/O glycan composition and the sialylation profiles of each subunit.
Read more about the SmartEnzymes in this publication
- FabRICATOR – Enabling middle level analytics
- OglyZOR – Efficient removal of O-glycans
- SialEXO – Getting rid of sialic acids
Reference:
OpeRATOR Publication from Johns Hopkins University
Scientists from the prestigious Johns Hopkins University School of Medicine have used OpeRATOR to develop a workflow to map O-glycosylated sites on proteins in very complex samples. O-glycoproteins are notoriously difficult to study due to the low abundance, high structural heterogeneity and low stability. Previous approaches using affinity enrichment or engineered cell culture systems either lack efficiency or are ill-suited forO-glycoproteomic studies of complex samples.
In the workflow developed by Weiming Yang and colleagues, protein samples such as serum or kidney tissue were digested with trypsin, immobilized onto beads through the N-terminus and treated with OpeRATOR and SialEXO. OpeRATOR is an endoprotease and derived from the gut commensal bacteria Akkermansia muciniphila that specifically cleaves peptides and proteins N-terminally of O-glycosylated serine or threonine residues. Therefore, only O-glycopeptides are released from the solid support and were identified using ETD mass spectrometry.
Using this workflow, Yang et al. were able to map over 3000 O-glycosylation sites from human serum, T cells and kidney tissue, almost doubling the number of known O-glycosylation sites. They were also able to detect and quantify the aberrant O-glycosylation patterns in kidney tumors, showcasing the potential use of such methodologies for both basic research and diagnostic purposes.
Meet the Scientist
We got the opportunity to interview the first author of the paper, Weiming Yang at Johns Hopkins University.
For more information on OpeRATOR go the the following pages:
- OpeRATOR Product Page
- Analysis of O-glycosylated proteins
- Download poster on OpeRATOR from ASMS 2018
