Introducing Rob Horsefield

Rob portrait

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.

OpeRATOR™ Decodes O-glycans; Publication by FDA and Genovis

Screen Shot 2018-07-03 at 23.59.42

 

Scientist at the Center for Biologics Evaluation and Research, Food and Drug Administration, have, in collaboration with Genovis, developed a method for analyzing O-glycosylated proteins based on a solid phase chemical modification and followed by OpeRATOR digestion. Using this method, up to 8-fold more O-glycosites were discovered as compared to previously reported data.

 

The method uses an on-bead system to capture tryptic peptides deglycosylated using PNGaseF from a glycoprotein mixture. First, the tryptic peptides are bound via the N-terminus to the beads, and subsequent modifications to the sugars can be carried out. Secondly, the OpeRATOR enzyme is applied to digest the peptide bond, N-terminal of the O-glycosylated serine or threonine. In this way, only O-glycosylated peptides will be cleaved off and enriched. The OpeRATOR digested peptides were then analyzed using LC-MS/MS.

 

OpeRATOR was launched at the American Society for Mass Spectrometry 2017 and the FDA team quickly became interested in this novel tool. The enzyme originates from Akkermansia muciniphila and has been engineered by Genovis for biotech applications and analytical workflows and denoted OpeRATOR. The enzyme binds to musin type O-glycans and cuts the protein backbone, N-terminally of the O-glycosylated site. OpeRATOR can be used to study site occupancy and composition of O-glycans on biopharmaceuticals and for O-glycomic workflows.

 

We establish the method on standard glycoproteins, confirming known O- glycosites with high accuracy and confidence, and reveal up to 8-fold more glycosites than previously reported with concomitant increased heterogeneity” (Shuang et al 2018)

 

The paper has been selected Editor’s choice in Analytical Chemistry and is available using the link below:

 

Shuang Yang et al., “Deciphering Protein O‑Glycosylation: Solid-Phase Chemoenzymatic Cleavage and Enrichment,” Analytical Chemistry, June 3, 2018, 1–9, doi:10.1021/acs.analchem.8b01834.

 

More information on OpeRATOR and its applications:

 

https://www.genovis.com/products/enzymes-for-o-glycans/operator/

 

OpeRATOR_vit_Kant_500px
OpeRATOR-1200px
Digested-guide-to-asms-2018

The Digested Guide to ASMS 2018

 

Several researchers have submitted abstracts for ASMS 2018 in San Diego, in which Genovis’ SmartEnzymes have been used. Below is a selection of these abstracts.

To read the poster abstracts, visit the Online Planner for ASMS, and paste the abstract titel in the search field.

Monday June 4 

10:30AM-2:30PM: Poster Session

MP 045 – GlyCLICK

Development of NISTmAb-derived homogeneous antibody-drug conjugate (ADC) standards

Shanhua Lin1; Terry Zhang2; Brian Agnew3; Trina Mouchahoir4; John Schiel4
1Thermo Fisher Scientific, Sunnyvale, CA; 2Thermo Fisher Scientific, San Jose, CA; 3Thermo Fisher Scientific, Eugene, Oregon; 4NIST, Gaithersburg, MD

MP 046 – FabRICATOR

Discovery and confirmation of glucuronylation as a new acidic post-translational modification on therapeutic monoclonal antibodies

Yuetian Yan1; Anita Liu1; Shunhai Wang1; Thomas Daly1; Ning Li1
1Regeneron Pharmaceuticals, Tarrytown, NY

MP 049 – FabRICATOR

Ultrasensitive Characterization of Size and Charge Heterogeneity of Therapeutic Monoclonal Antibodies by Native Mass Spectrometry

Shunhai Wang1; Yuetian Yan1; Anita Liu1; Thomas Daly1; Ning Li1
1Regeneron Pharmaceuticals, Tarrytown, NY

MP 464 – FabALACTICA

Quantitative UPLC-MSE analysis of disulfide bonds and free sulfhydryls in monoclonal antibodies using IgdE protease assisted digestion

Jeroen de Keijzer1; Peter van Maurik1; Anja Boumeester1; Emile van Corven1; Gideon Oudgenoeg1
1Bioceros, Utrecht, Netherlands

 

Tuesday June 5 

10:30AM-2:30PM: Poster Session

TP 624 – FabRICATOR

Avoiding method induced heterogeneity in the analysis of heterogeneity of monoclonal antibodies using Mass Spectrometry after Single Site Proteolysis

Gideon Oudgenoeg1; Anja Boumeester2; Peter van Maurik2; Jeroen de Keijzer2; Emile van Corven2
1Bioceros, Utrecht, Netherlands; 2Bioceros, Utrecht, Netherlands

 

Wednesday June 6 

10:30AM-2:30PM: Poster Session

WP056 – FabRICATOR

Characterizing and Quantitating Therapeutic Antibody Multimer Degradation Using Affinity Capture Mass

Neha Srikumar1; Wenjing Li1; Robert Tchelepi1; Chen Gu1; Diego Ellerman1; Greg A Lazar1; Yichin Liu1John C. Tran1
1Genentech Inc., South San Francisco, CA

WP 327  – GingisKHAN

Comprehensive Domain-Specific [Fc vs. Fab] N-glycosylation Analysis of Therapeutic Proteins

Charles Nwosu1; Shuangqi Sally Liu2; Lei Wang2; May Zhu2; Anne Kowal2
1Takeda Pharmaceuticals International Co, Cambridge, MA; 2Takeda Pharmaceuticals International Co., Cambridge, MA

WP 340 – FabULOUS

Analysis of Fragmented Porcine Immunoglobulin G (IgG) by MALDI-MS and UPLC-ESI-MS

HELENE PERREAULT1; Claudia Nelson2
1University of manitoba, Winnipeg; 2University of Manitoba, Winnipeg, MB

WP 342 – OpeRATOR and GlycOCATCH

A Novel O-glycoprotease with applications in O-glycan Analysis using mass spectrometry

Rolf Lood1, 2; Maria Nordgren1; Fredrik Leo1; Stephan Björk1; Malin Mejáre1Fredrik Olsson1
1Genovis AB, Lund, Sweden; 2Department of Infectious Diseases, Lund University, Lund, Sweden

WP 350 – OpeRATOR

Deciphering complex o-glycosylation: solid-phase chemoenzymatic cleavage and enrichment

Shuang Yang1; Philip Onigman2; Jonathan Sjogren2; Wells W. Wu3; Rong-fong Shen3; John Cipollo1
1LBP, CBER, FDA, Silver Spring, MD; 2Genovis AB Inc., Boston, MA; 3FBR, CBER, FDA, Silver Spring, MD

WP 676 –FabRICATOR, GlycINATOR, IgGZERO

Monitoring Critical Quality Attributes: Core Fucosylation of N-glycans using an Integrated Subunit LC/MS Workflow Method

Nilini S Ranbaduge1; Henry Y Shion1; Ying Qing Yu1; Weibin Chen1
1Waters Corporation, Milford, MA

WP 678 – FabRICATOR

In-depth Characterization of the Heterogeneous Dimerization Interfaces of A Monoclonal Antibody: from Subdomain Level to Residue Level

Yuetian Yan1; Shunhai Wang1Thomas Daly1; Ning Li1
1Regeneron Pharmaceuticals, Tarrytown, NY

W 691 – OpeRATOR, OglyZOR and SialEXO

LC-MS Characterization of Complex Glycoproteins

Amber Peariso1; Jason X. Tang1
1Eli Lilly & Company, Indianapolis, IN

WP 692 – FabRICATOR

Rapid Identity Assays for mAb Development, Production Control and Release

Anja Resemann1; Waltraud Evers1Yue Ju2; Guillaume Tremintin2; Detlev Suckau1
1Bruker Daltonics, Bremen, Germany; 2Bruker Daltonics, Billerica, MA

 

Thursday June 7 

10:10AM-10:30PM: Oral Presentation

Oral Presentation – FabRICATOR

Classification of Plasma Cell Disorders by 21 Tesla FT-ICR Top-Down and Middle-Down MS/MS Analysis of Monoclonal Immunoglobulins in Human Serum

Lidong He1, 2; Lissa C Anderson2; David R Barnidge3; David L Murray4; Surendra Dasari4; Angela Dispenzieri4; Christopher L Hendrickson1, 2; Alan G Marshall1, 2
1Florida State University, Tallahassee, FL; 2National High Magnetic Field Laboratory, Tallahassee, FL; 3The Binding Site, Rochester, MN; 4Mayo Clinic, Rochester, MN

 

10:30AM-2:30PM: Poster Session

ThP 003 – FabRICATOR

LC-MS in Combination with Multiple Enzymatic Digestion for Sequence Variant Identification in Support of Cell Line Development

Renpeng Liu1; Lintao Wang1; Alexandru C. Lazar1
1ImmunoGen, Waltham, MA

ThP 017 – GingisKHAN

Consortium for Top-Down Proteomics Inter-laboratory Study for Characterizing Monoclonal Antibodies (mAbs) by Top-Down Mass Spectrometry

Kristina Srzentic1; Luca Fornelli1; Yury Tsybin2; Joseph Loo3; Jeffrey Agar4; Julia Chamot-Rooke5Paul Danis6; Ying Ge7; David Goodlett8; Neil Kelleher1; Ljiljana Pasa Tolic9; Lloyd Smith7; Timothy Toby1; Konstantin Nagornov2; JENNIFER BRODBELT10; Sylvester Greer10; Mathieu Dupré5; David Clarke11; Ziqing Lin7; Kim Haselmann12; Christopher Hendrickson13; Lidong He13; Donald Hunt14; Jared Shaw9; Wendy Sandoval15; Richa Sarin16; Detlev Suckau17; Yuri E.M. van der Burgt18; Norelle Wildburger19; Nicolas L. Young20; Alain Beck21; John Yates22; Jolene Diedric22; Sneha Chatterjee23; Frank Sobott24; Anton Kozhinov2; ALAN G. MARSHALL13; LISSA C. ANDERSON13; Natalia Gasilova25; Laure Menin25; Neil Quebbenamm3; Sung Hwan Yoon26; Josh Hinkle14; Simone Nicolardi18; Matthew V. Holt20; Yunqiu Chen16; Nicholas Schmitt4
1Northwestern University, Evanston, IL; 2Spectroswiss Sàrl, Lausanne, Switzerland; 3UCLA, Los Angeles, CA; 4Northeastern University, Boston, MA; 5Institute Pasteur, Paris, France; 6Eastwoods Consulting, Boylston, MA; 7University of Wisconsin, Madison, WI; 8University of Maryland, Baltimore, Baltimore; 9PNNL, Richland, WA; 10University of Texas at Austin, Austin, TX; 11Edinburgh University, Edinburgh, United Kingdom; 12Novo Nordisk, Malov, Denmark; 13National High Magnetic Field Laboratory, Tallahassee, FL; 14University of Virginia, Charlottesville, VA; 15Genentech, Inc., South San Francisco, CA; 16Biogen Inc, Cambridge, MA; 17Bruker Daltonik GmbH, Bremen, Germany; 18Leiden University Medical Centre, Leiden, Netherlands; 19Washington University, St. Louis, St. Louis, MO; 20Baylor College of Medicine, Houston, TX; 21Centre d’immunologie Pierre Fabre, Saint-Julien-en-Genevois, France; 22The Scripps Research Institute, La Jolla, CA; 23University of Antwerp, Antwerp, Belgium; 24University of Leeds, Leeds, United Kingdom; 25Ecole Polytechnique Fédérale de Lausanne, Ch-1015 Lausanne, Switzerland; 26University of Maryland, Baltimore, MD

ThP 028 – FabRICATOR

Top- and Middle-Down CE-ESI-MS Analysis of Intact mAbs Using the ZipChip Coupled to a Fusion Lumos ETD Mass Spectrometer

Tricia C. Ho1; Erik J. Soderblom1; Erin Redman2; Greg M. Waitt1; M. Arthur Moseley1
1Duke University School of Medicine, Proteomics and Metabolomics Shared Resource, Durham, NC; 2908 Devices, Inc., Carrboro, NC

ThP 419 – FabRICATOR

Quantitation of Misincorporations: Strategies and System Suitability

Kathleen Cornelius1; Olga Friese1; Mary Denton2; Jason Rouse2
1Pfizer, Inc, Chesterfield, MO; 2Pfizer Inc., Andover, MA

 

2:30 -2:50 PM: Oral Presentation

Ballroom 20D – FabRICATOR, GingisKHAN, GlycINATOR and IgGZERO

A Suite of Liquid Chromatography Strategies Coupled Online to Top-down High-resolution Mass Spectrometry for Comprehensive Analysis of Antibody Drug Conjugates

Bifan Chen1; Ziqing Lin1; Qingge Xu1; Cexiong Fu2; Qunying Zhang2; Ying Ge1
1University of Wisconsin-Madison, Madison, Wisconsin; 2Abbvie Inc., North Chicago, IL

Antikropp-klyvning-över-och-under-hinge

Interview with Valegh Faid at LFB Biotechnologies in France

 

Unique enzymatic digestions in study of antibody disulphides

 

Valegh Faid and colleagues at LFB Biotechnologies in France have developed and published an assay to study antibody disulphide bonds using middle-up LC-MS (Faid et al., 2017). The combination of FabRICATOR® for digestion below the hinge and FabALACTICA™ for digestion above the hinge, generated three fragments from a human IgG1 antibody; the hinge peptide, Fab and Fc/2 fragments. These fragments were resolved using RP-HPLC and mass spectrometry and enabled analysis of antibody disulphide bridges and other quality attributes.

 

 

Interview with Valegh Faid, Scientist at LFB and first author of the paper:

 

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.

 

How did you come up with the idea to combine FabRICATOR (IdeS) and FabALACTICA (IgdE)?

 

We have been using IdeS for many years in order to cleave IgG’s below the hinge; following DTT reduction, more amenable fragments for RP-HPLC/MS analysis are generated as previously published by our laboratory (Chevreux et al., 2011). This 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.

In this context, IgdE is an interesting enzyme that cleaves specifically IgGs above the hinge and without requiring reducing conditions as papain do. The combination of IdeS and IgdE 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 the gold standard to investigate disulphide bonding of biotherapeutics. However, data interpretation is time consuming even if dedicated software to improve the treatment of data has largely improved. Although being slightly less informative than peptide mapping, this combined IdeS/IgdE middle-up approach increases the throughput for the investigation of free thiols and disulphide scrambling. Considering that other CQAs can also be monitored in the same experiment, it should be more applicable to routine use in process optimization, formulation screening and stability studies.

 

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.

 

How are you implementing this assay at LFB Biotechnologies?

 

This assay is integrated in our portfolio of analytical approaches for the analysis of mAbs currently in development, for process optimisation, batch characterization and stability studies.

 

 

Read more about FabALACTICA and FabRICATOR.

 

References:

 

Chevreux, G. et al., 2011. Fast analysis of recombinant monoclonal antibodies using IdeS proteolytic digestion and electrospray mass spectrometry. Anal Biochem. 15;415(2): pp. 212-4.

 

Faid, V. et al., 2017. Middle-up analysis of monoclonal antibodies after combined IgdE and IdeS hinge proteolysis: Investigation of free sulfhydryls. Journal of Pharmaceutical and Biomedical Analysis, 149, pp.541–546.

 

 

KevinCook-social-media-linkedin

Meet Our New Colleague Kevin Cook

May 17, 2018 | Genovis Team |

 

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

 

Tell us a bit about yourself.
Even though a Focused Generalist does not make perfect sense, I think it fits since I have a broad life sciences discovery background with a specificity towards LC-MS and orthogonal technology.

 

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

Curious.

 

Tell us a bit about your previous working life.

My on-the-job learning experiences, with small and large companies, started with DNA/RNA focused laboratory work, moved into LC-MS to support small molecule pharmacokinetics and drug metabolism, and has migrated into a focus on providing biologics sample preparation technology for LC-MS analysis.

 

What will your main focus be here at Genovis?

On behalf of the Genovis team, I have the pleasure of building relationships with scientists in the western US. I am excited to learn more about the challenges my colleagues in the industry are facing and how our team can help.

 

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

Genovis has built a customer centric reputation that shows in current products and formats, and how LC-MS scientists are utilizing these tools. Genovis has demonstrated the ability to rapidly respond to customer requests and I am looking forward to helping promote current products while building for the future through customer feedback and collaboratory efforts.

 

4 Quick Questions:

Coffee or tea? 

Coffee, lots of coffee…

 

Aerosmith or Depeche Mode?

At the moment, Eric Church.

 

Ice cream or candy?

Chocolate chip cookies.

 

Soccer or ice hockey?

American football though staying with the question, ice hockey – Chicago Blackhawks.

FragIT-digestion

Study on Glycoform Heterogeneity using Enzymatic Digestion and Native Mass Spectrometry

In a study by Wohlschlager et al. (2018), FragIT™ kit was used to digest the Fc-fusion protein etanercept, and the resulting fragments were analyzed using high-resolution native mass spectrometry (MS). Native MS offers a higher spatial resolution at a lower charge state, enabling studies of glycan heterogeneity, and FragIT digestion reduces sample complexity, enabling a detailed annotation of glycoforms on complex compounds.

 

A detailed knowledge about structure and post-translational modifications (PTMs) is required for biopharmaceuticals to be approved for clinical use, and an important quality attribute that may affect both the efficacy and safety of biopharmaceuticals is glycosylation.

 

Etanercept is a highly glycosylated Fc-fusion protein that is used to treat autoimmune diseases such as rheumatoid arthritis, and it consists of the TNF-𝛼 receptor domain fused to the Fc domain of human IgG1. FragIT – an immobilized version of the FabRICATOR® (IdeS) enzyme – digests IgG from several species and subclasses at a specific site below the hinge region. The resulting fragments are easily purified using the Fc-specific affinity resin that, together with FragIT, comprises the FragIT kit.

 

In this study, the researchers analyzed the glycosylation of etanercept on both the intact level, and on the middle-down level after FragIT digestion. By combining native MS analysis with enzymatic remodelling of etanercept, a detailed annotation of glycoforms could be achieved and transferred from subunit to whole protein level.

 

The authors end by concluding:

Comprehensive information on glycoform heterogeneity, fast analysis with minimal sample preparation and product-characteristic fingerprints render our method highly attractive for the quality control of biologics as well as for comparability studies following changes in the manufacturing process.”(Wohlschalger et al. 2018).

 

Read more about FragIT and FabRICATOR

 

References:

 

GlycOCATCH-launch

Genovis Launches GlycOCATCH™

April 26, 2018 | Applications, Products |

Please welcome GlycOCATCH™ to the Genovis SmartEnzymes™ family!

 

GlycOCATCH is an enrichment resin for affinity purification of O-glycosylated proteins and peptides. The resin is designed to bind proteins and peptides carrying O-glycans with high affinity, and it is provided in convenient spin columns to allow easy-to-use O-glycoprotein enrichment.

 

The applications of GlycOCATCH involve glycomics, specific enrichment or removal of O-glycoproteins and peptides, studies of O-glycosylation in complex samples and characterization of biopharmaceuticals.

 

The GlycOCATCH product is available for purchasing today.

 

Read more about GlycOCATCH here

mAb Deamidation Study using FabRICATOR® Digestion and HIC Separation

FabRICATOR-horisontell

Hydrophobic interaction chromatography (HIC) is often used in characterization of therapeutic antibody products due to its ability to separate direct or indirect structural changes in the studied protein. Scientists at Alexion have published a study where FabRICATOR (IdeS) was used to generate Fc and F(ab’)2 fragments of an antibody to study conformational changes of a monoclonal antibody (King et al. 2018).

 

Separation of the intact antibody on HIC reveled two major peaks that were collected and subjected to FabRICATOR digestion. After digestion, the Fc and F(ab’)2 fragments separated well, and the heterogeneity was localized to the F(ab’)2 domain. Variations in the Fc were observed and attributed to oxidation modifications. Peptide mapping of the domains were carried out and a 1 Da difference was localized , indicating deamidation of Asn to either Asp or isoAsp in the complementarity-determining region (CDR) of the light chain. The observed difference in HIC separation pattern was also linked to changes in antigen binding, since the deamidation of the Asn residues reduced the binding of the antibody to its target antigen.

 

Taken together, this paper indicates that a single deamidation in the light chain changed the hydrophobicity profile of the antibody and impacted the antigen binding. The use of FabRICATOR (IdeS) digestion and HIC separation could serve as a quick screening assay to study deamidation changes in the F(ab’)2 domain.

 

 

King, C. et al., 2018. Characterization of recombinant monoclonal antibody variants detected by hydrophobic interaction chromatography and imaged capillary isoelectric focusing electrophoresis. Journal of Chromatography B, 1085, pp.96–103.


 

A New Assay to Study IgG Galactosylation in Serum

March 21, 2018 | Applications, Products |

IgGZERO-glycation

In a study by Vanderschaeghe et al. (2018), a new assay to measure IgG galactosylation in serum has been developed. The setup includes hydrolysis of IgG Fc glycans using the IgG-specific endoglycosidase IgGZERO® (EndoS).

 

A reduced level of IgG galactosylation in serum is a promising biomarker to evaluate the severity, determine the treatment and assess the efficacy of the treatment of autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and Crohn’s disease.

 

Traditionally, it has been difficult to study IgG galactosylation in serum because of the requirement to purify the antibodies, a procedure that is both complex and time-consuming. However, Vanderschaeghe et al. demonstrate a new assay where IgGZERO is used to efficiently hydrolyze serum IgG Fc glycans before analyzing galactosylation on high-throughput DNA sequencers. IgGZERO works on natively folded IgG, meaning that the assay can be performed on complex serum without first needing to purify the IgG, a feature that renders the assay both fast and simple.

 

The authors conclude by describing their new IgGZERO-based assay:

“… an important breakthrough towards the clinical implementation of the proposed biomarker” (Vanderschaeghe et al. 2018).

 

Read more about IgGZERO

 

Find the full text of the paper here:

Vanderschaeghe et al., 2018. Clinical assay for direct assessment of IgG galactosylation in serum using endoglycosidase S. BioRxiv.

 

Antibody Glycation Study using Intact LC-MS

IgGZERO_Logo_Gubbe

A new study from Janssen by Mo et al, demonstrates the use of intact mass spectrometry to determine the levels of glycation on therapeutic antibodies. To perform the assay, the authors used IgGZERO for rapid removal of the Fc-glycans.

 

Glycation occurs when reducing sugars such as glucose, galactose or fructose, reacts with protein amino acids through the Maillard reaction, and results in attachment of sugars to the protein. For therapeutic antibodies, glycation not only increases the heterogeneity of the drug but may also affect safety and efficacy.

 

To study the level of glycation on antibodies, the authors used both intact mass of the reduced antibody and peptide mapping to find the +162 Da mass shift indicating an addition of a hexose sugar. The Fc-glycan of an antibody contain 0, 1 or 2 galactose sugars that also gives a mass shift of 162 Da. To specifically remove the Fc-glycans, the scientist used IgGZERO (EndoS) from Genovis. Using this enzymatic pretreatment, the authors could determine glycation levels using intact mass spectrometry.

 

The authors found the peptide mapping and the intact LC-MS to give correlating results but conclude: “intact LC- MS is a quicker and simpler method to quantitate the total glycation levels and is more useful for routine testing”(Mo et al. 2018).

 

 

Find the full text of the paper here:

Mo, J. et al., 2018. Quantitative analysis of glycation and its impact on antigen binding. mAbs, 154, pp.1–10.