Articles in the Category ”References”

FragIT™ kit in Study Evaluating IgG Charge

September 2, 2019 | References |

 

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:

FragIT kit

 

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

Reference:
Kristensen, L. et al., 2019. Site-specifically labeled 89Zr-DFO-trastuzumab improves immuno-reactivity and tumor uptake for immuno-PET in a subcutaneous HER2-positive xenograft mouse model. Theranostics, 9(15). pp.4409-4420.

FabRICATOR® in capillary zone electrophoresis-native mass spectrometry

June 19, 2019 | References |

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.

 

 

product-box-fabricator

For more information on FabRICATOR please visit the following pages:

The full text paper is available online:

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:

The full text paper is available online:

FabRICATOR® driven middle-down glycan analysis using NMR

March 15, 2019 | References |

Blog 2

 

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.

 

product-box-fabricator

For more information on FabRICATOR please visit the following pages:

The full text paper is available online:

SmartEnzymes™ assist MALDI in-source decay FT-ICR Mass Spectrometry analysis

March 14, 2019 | References |

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?

DSC09608 (003)

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.

 

Sequence Analysis

For more information on FabRICATOR or GingisKHAN please visit the following pages:

The full text paper is available online:

FabRICATOR, SialEXO and OglyZOR in Middle-up HILIC/HRMS Approach

Unknown-4

 

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

 

Reference:

D’Atri, V. et al., 2018. Orthogonal Middle-up Approaches for Characterization of the Glycan Heterogeneity of Etanercept by Hydrophilic Interaction Chromatography Coupled to High-Resolution Mass Spectrometry. Analytical Chemistry, 91(1), pp.873–880.

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.

 

Weiming Yang

Tell us about yourself?
I am a Research Associate in Mass Spectrometry Core Facility in the Center for Biomarker Discovery and Translation (www.biomarkercenter.org) of the Johns Hopkins University. The Mass Spectrometry Core Facility carries large-scale proteomics with particular emphasis on protein glycosylation on proteome scale to elucidate functions of glycoproteins on biology and disease. Before this position, I was a postdoc fellow in the same lab and worked on innovative glycoproteomics methods and HIV research. My interest in protein O-linked glycosylation started from every beginning at Hopkins that I was able to identify an O-linked glycosylation site in HIV gp120 from the infectious virion. Later on, I developed a series of glycoproteomic methods to study protein N- and O-linked glycosylation. The development of novel glycoproteomic methodologies led to new areas toward the discovery of the biomarker for HIV reservoir and new insight into cancer biology.

 

What is new with the ExoO method you have developed?
The major advantage of EXoO is its applicability to analyze clinical samples that is a breakthrough and central to reveal the significance of protein O-linked glycosylation in diseases. Using EXoO, now, scientists can start to gain new insight into their biological systems regarding O-linked glycoproteins. O-linked glycoproteins are ubiquitous on the cell surface and extracellular environment that is highly relevant to new treatment for diseases and diagnostics. Also, the EXoO is advantageous to analyze mucin-type O-linked glycoproteins that cannot be easily analyzed by conventional methods. 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.

 

How did you perform the analysis prior to this method?
We tried to use the same solid phase method to immobilize the peptides but released O-glycopeptides using beta-elimination to study site of protein O-linked glycosylation. Beta-elimination is a chemical reaction that can tag the site of protein O-linked glycosylation but give some background release of peptides from the solid support. We tried 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.

 

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.

 

What can you tell us about what you currently are working on?
Currently, we are applying the method to study different diseases including cancers and HIV reservoir.

 

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. For O-glycans, the specificity of OpeRATOR is not completely clear that will need further investigation. O-glycans have many different structures. The glycomic methods may still be the best way to go.

 

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. Glycomic method focus on the identification of all different O-glycan structures with linkage and quantitative information. In the future, the structures of O-linked glycans on the specific sites on the proteins can be revealed in a single workflow.

 

OpeRATOR-1200px

For more information on OpeRATOR go the the following pages:

 

The full text paper is available online:
OpeRATOR_vit_Kant_500px

SmartEnzymes™ in Multiplexed Middle-Down MS for targeted structure analysis

 

 

 In a recent article by Srzentic et al. (2018) the authors present a multiplexed middle-down MS workflow with improved performance for targeted protein structure analysis. Using GingisKHAN for antibody digestion, the authors analysed the F(ab) subunits of a therapeutic mAb. By implementing spectral and transient averaging of mass spectra across several LC-MS experiments, the authors revealed valuable information on chain pairing in the mAb.

 

To make the analysis, the therapeutic mAb trastuzumab was digested above the hinge using the GingisKHAN enzyme to generate intact F(ab) subunits. Intact myoglobin was subjected to analysis in a top-down MS approach to benchmark the workflow. The GingisKHAN-generated F(ab) subunits were then analysed using the middle-down MS workflow to compare the performance of data averaging approaches.

 

The results show the performances of spectral and transient averaging for tandem mass spectra as separate software tools for structural protein analysis. The transient averaging provided the most extensive sequence coverage for the F(ab) subunits, followed by spectral averaging. Furthermore, utilizing the multiplexed middle-down MS workflow for subunit analysis, the authors detected low-abundance branched product ions revealing valuable information about the light and heavy chain connectivity.

 

GingisKHAN® (Kgp enzyme) is a cysteine protease that digests human IgG1 at a specific site above the hinge region. The enzyme generates intact Fc and Fab subunits in 60 minutes.

 
Learn more about GingisKHAN

 
Srzentic et al., 2018. Multiplexed Middle-Down Mass Spectrometry Reveals Light and Heavy Chain Connectivity in a Monoclonal Antibody

Antibody Sequence Analysis using GingisKHAN® and FabRICATOR®

September 28, 2018 | Applications, References |

  In an article by Luca Fornelli & Kristina Srzentic et al. recently published in Analytical Chemistry the authors present a workflow for antibody sequence determination by combining top-down and middle-down LC/MS. The authors analyzed the therapeutic antibody rituximab in its intact and fragmented form, using FabRICATOR and GingisKHAN to generate antibody subunits. By combining the performance of multiple ion activation techniques and a new software tool with top-level and middle-level strategies, the authors achieved extensive sequence coverage and obtained valuable information on key quality attributes.

  Rituximab was fragmented using members of the SmartEnzymes™ family for the generation of various antibody subunits. GingisKHAN was used for generating intact Fc and Fab subunits by site-specific cleavage of IgG1 above the hinge region. In order to obtain antibody subunits Fc/2, Fd and LC the authors used FabRICATOR-digestion followed by reduction. The intact antibody and the antibody subunits were analyzed using reversed phase LC/MS coupled with three separate ion activation techniques, and analyzed using a new software tool for fragment ion deconvolution.

  The complementing features of the ion activation techniques provided high quality information for a low number of LC/MS experiments. The authors achieved sequence coverage equivalent to what is obtainable with bottom-up strategies. In addition, the authors were able to analyze quality attributes such as PTMs, chain pairing and intact antibody mass determination – properties otherwise lost after extended proteolysis. These results highlight the benefits of combining top-level and middle-level strategies for applications currently performed by bottom-level strategies.

GingisKHAN® (Kgp enzyme) is a cysteine protease that digests human IgG1 at a specific site above the hinge region. The enzyme generates intact Fc and Fab subunits in 60 minutes.

Learn more about GingisKHAN

Fornelli et. al., 2018. Accurate Sequence Analysis of a Monoclonal Antibody by Top-Down and Middle-Down Orbitrap Mass Spectrometry Applying Multiple Ion Activation Techniques.