FabRICATOR driven middle-down glycan analysis using NMR

March 15, 2019 | References |

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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.

 

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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 |

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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.

 

Sequence Analysis

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

The full text paper is available online:

OpeRATOR Presentation at GlycoBioTec

February 13, 2019 | Uncategorized |

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Genovis was selected to present at the GlycoBioTec 2019 Conference in Berlin in January. Andreas Nägeli, Senior Scientist at Genovis, presented novel workflows based on the O-glycan specific OpeRATOR enzyme and GlycOCATCH coupled to LC-MS.

 

 

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

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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.

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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.

 

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For more information on OpeRATOR go the the following pages:

 

The full text paper is available online:
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Poster Presentations at PEGS Europe 2018

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This week, scientists from Genovis are presenting two poster at the Protein Engineering Summit in Lisbon, Portugal. The posters cover our O-glycan specific endoprotease Operator and the recently launched FabRICATOR-HPLC column for automated antibidy subunit generation. Check out the poster abstracts below:

An O-glycan Specific Endoprotease with Applications in Glycoprotein Analysis using LC-MS

Helen Nyhlen, Maria Nordgren, Stephan Björk, Rolf Lood, Fredrik Leo, Fredrik Olsson
Genovis AB, Sweden

Changes in protein glycosylation may have an impact on the structure and function of a glycoprotein and O-glycosylation has drawn more and more attention for its roles in a wide range of biological processes. Characterization of glycosylation is of growing importance for the development and quality control of recombinant glycoprotein drugs and biosimilars. The study of O-linked glycosylation within the field of glycoproteomics is however challenging due to complicated sample preparation, difficult analytical procedures and the lack of O-glycan specific enzymes.

An O-glycan specific protease originating from the mucin degrading bacteria Akkermansia muciniphila has been described previously. The enzyme is dependent on the presence of O-glycans for digestion and hydrolyzes the peptide bond N-terminally to O-glycosylated serine and threonine residues. This feature can be used for the generation of intact O-glycopeptides to study site occupancy and composition of O-glycans in various biologic samples. We present here workflows that enabled determination of O-glycan sites and composition for O-glycosylated biopharmaceuticals and for proteins in human serum.

The O-linked glycosylation sites of biopharmaceuticals were assessed by treatment with PNGaseF, sialidases, O-protease and/or trypsin overnight prior LC/MS. The unique MS/MS peptides obtained revealed and defined the O-glycosylated threonine and serine residues. Enrichment of O-glycoproteins from human serum was achieved in native conditions using an affinity binding resin for O-glycan protein based on agarose beads with immobilized inactive O-protease. The complex protein sample was desialylated during the incubation step for binding. Bound proteins were then eluted by urea and treated with PNGaseF, active O-protease and/or trypsin followed by RP-C18 or HILIC separation and ESI-QTOF/MS analysis. The resin displayed high affinity for core 1 mucin-type glycans. With this workflow peptides and O-glycopeptides, with site-specific information, from several serum proteins were identified.

To summarize, using the characteristics of the O-protease and the O-glycoprotein affinity binding resin, strategies for the characterization of O-glycosylated proteins from pure and complex protein samples have been developed. The O-protease and the O-glycoprotein binding resin are potentially useful tools for deep characterization of O-glycoproteins.

 

Rapid On-column Digestion for Automated Monoclonal Antibody Analysis

Stephan Björk, Andreas Nägeli, Maria Nordgren, Linda Andersson, Helen Nyhlen, Jonathan Sjögren, Fredrik Olsson
Genovis AB, Lund, Sweden

Monoclonal antibodies (mAbs) and other IgG-based biopharmaceuticals are a fast-growing market. The inherent heterogeneity of such biologics necessitates detailed characterization by liquid chromatography and mass spectrometry (LC-MS) during development and production. While bottom-up peptide mapping is still the gold standard for analysis of critical quality attributes, such approaches are resource and time intensive in terms of both data acquisition and analysis. Top-down and middle-down approaches are therefore gaining in popularity. Antibody subunit analysis has become a widely accepted analytical strategy for rapid characterization of therapeutic antibodies and related products. The IdeS enzyme specifically digests IgG just below the hinge, generating F(ab’)2 and Fc/2 fragments. Reduction of disulfide bonds yields fragments of 23-25kDa in size which are amenable to high-resolution mass spectrometry. The IdeS based middle-level LC-MS workflow therefore enables the analysis of multiple antibody quality attributes such as glycosylation, oxidation, and C-terminal lysine clipping.

Here we present a rapid and automatable solution for antibody subunit generation in an HPLC column format. FabRICATOR (IdeS) enzyme was immobilized on the column to allow for automated middle-level analysis in a 2D-HPLC setup. The mAbs are digested on-column in the first dimension and the resulting subunits are separated and analyzed in the second dimension by RP-HPLC. This could be achieved with minor modifications to an HPLC-MS setup and potentially be connected directly to a bioreactor for automated monitoring of an on-going mAb production. The column tolerates continuous operation at 37°C for >10 days without a significant decrease in digestion performance and delivers consistent results for Fc glycan analysis during the entire period of operation. Additionally, other critical quality attributes such as Fab glycosylation and lysine clipping could be monitored. FabRICATOR-HPLC provides a fast solution for antibody subunit generation while reducing sample handling errors and increasing throughput.

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

October 18, 2018 | Applications, References |

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 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 |

Sequence Analysis

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.

FabRICATOR® in service for in-depth 2D-LC MS profiling of therapeutic mAbs

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In an article by Stroll et al. (2018), the authors demonstrate a striking in-depth characterization of three therapeutic mAbs, achieved by combining FabRICATOR® (IdeS) digestion with an online two-dimensional LC-MS approach. The authors generate a highly resolved separation and detection of FabRICATOR-digested N-glycosylated mAb subunits by implementing Active Solvent Modulation (ASM), a method for valve-based effluent dilution between the first and second dimension separations.

Multidimensional Liquid Chromatography constitutes a powerful technology for in-depth profiling of therapeutic proteins, capable of generating rapid and highly resolved separations. The authors demonstrate the advantages of implementing ASM in an online 2D-LC system for deep profiling of antibody glycosylations, subjecting mAbs to FabRICATOR digestion followed by HILIC x RP separation and ESI Mass Spectrometry (ESI-MS) detection.

Three therapeutic antibodies displaying diverse N-glycosylation patterns were submitted to digestion using FabRICATOR for a single site-specific proteolytic cleavage below the hinge, generating Fc/2 and F(ab’)2 fragments. Further reduction of the interchain disulphide bonds of the F(ab’)2 subunit was carried out on the FabRICATOR-digested samples for the additional generation of LC and Fd fragments.

Implementing the ASM method on antibody subunits, the authors achieved a significant increase in detection sensitivity for Fc/2 and Fd fragments, without detectable breakthrough, otherwise associated with larger loading volumes in the second-dimension separation. Furthermore, the authors demonstrated the resolving power of HILIC x RP for analyzing the extent of glycosylations present in heavily glycosylated mAbs, the method showing increased separation and detection for both high and low abundant glycan species, compared to 2D-LC combining CEX and RP separations.

FabRICATOR is a protease with a single digestion site below the hinge of IgG. The enzyme is widely used in middle-level analytical workflows for characterization of antibody based biopharmaceuticals.

 
Learn more about FabRICATOR

 
Stoll, D.R. et al., 2018. Development of Comprehensive Online Two-Dimensional Liquid Chromatography-Mass Spectrometry using Hydrophilic Interaction and Reversed-Phase Separations for Rapid and Deep Profiling of Therapeutic Antibodies. Analytical Chemistry, pp.acs.analchem.8b00776–9.

Introducing Rob Horsefield

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Genovis is expanding its sales and marketing organization at its Lund headquarters and has hired Rob Horsefield as Sales & Business Development Manager. Rob has multiple years of experience in the industry from sales in analytical chemistry, pharmaceutical development and protein chemistry.

We are happy and proud to have you on board, Rob. Welcome to Genovis!

 

Tell us a bit about yourself.
I moved to Sweden in 2004 from the UK and live outside Lund with my Swedish wife and two children. I am now a Swedish citizen and fluent in Swedish. In my spare time I am a passionate road cyclist and enjoy long rides with my friends.

 

If you were to describe yourself using only one word – what would that word be?

Trustworthy.

 

Tell us a bit about your previous working life.

I have a background in protein biochemistry and did my PhD in membrane protein crystallography. After that I worked at Gothenburg University for four years and then AstraZeneca for three and a half years. But most recently I was with Agilent Technologies for six years selling their analytical instruments in Southern Sweden and more recently liquid chromatography as their product specialist for the Nordics.

 

What will your main focus be here at Genovis?

My focus is to be the trusted associate at Genovis for our customers. I believe in always putting our customers first and securing their satisfaction. I like to work in a structured manner, pay attention to details and be on-time.

 

What do you believe will be the biggest opportunity in your new position as a Senior Application and Market Area Manager?

I hope we can leverage my experiences in protein biochemistry research, the biopharma market and LC/LC-MS instrument sales to grow Genovis’s business in Europe and Asia, and further expand our customer base.

 

Four Quick Questions:

Coffee or tea?

Tea, very strong with milk. But not fruit tea. And I never drink coffee, no, really.

 

Aerosmith or Depeche Mode?

Depeche Mode of course.

 

Ice cream or candy?

Both please, lots of.

 

Cricket or Rugby?

Rugby for sure, but there is still a small space left in my heart for cricket too.