Articles in the Category ”Conferences”

Digested-guide-to-ASMS-2019 2

The Digested Guide to ASMS 2019

Many of Genovis’ customers have submitted abstracts for ASMS 2019 in Atlanta, in which the SmartEnzymes have been used. Below is a digested guide to these abstracts so that you can plan your days at ASMS.

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

 

Sunday, June 2

New Enzymatic Workflows for Analysis of O-Glycosylated Biopharmaceuticals

Andreas Nägeli

Thermo Fisher Scientific User Meeting

Pharma/BioPharma Breakout Session 9:30 AM – 12.15 PM

Marquis Ballroom C

Thermo-user-meeting-blog

Monday, June 3

Improved middle-down characterization of antibodies using multiple ion activation techniques and Proton Transfer Reaction on a modified Orbitrap mass spectrometer 

Romain Huguet

MP 676, Poster Session 10.30 AM – 2.30 PM

FabRICATOR® and GingisKHAN®

 

Direct Determination of Antibody Chain Pairing by Top-Down Mass Spectrometry using Electron Capture Dissociation and Ultraviolet Photodissociation 

Jared Shaw

Room B302-305, Oral Presentation 3.50 PM – 4.10 PM

FabRICATOR® and GingisKHAN®

 

Unraveling a complex immunoprotein profile in multiple myeloma with middle-down de novo sequencing and native mass spectrometry 

Valerie J Winton

Room A411-412, Oral Presentation 9.30 AM – 9.50 AM

FabRICATOR®

 

Middle Down Approach for the Characterization of Monoclonal Antibodies After Ides Digestion and ETD Fragmentation

Colin Wynne

MP 782, Poster Session 10.30 AM – 2.30 PM

FabRICATOR®

 

Tuesday, June 4

Analysis of Zika Viral Polyprotein N- and O-glycosylation Using a Novel Lectin-chemoenzymatic Enrichment 

Shuang Yang

TP 655, Poster Session 10.30 AM – 2.30 PM

OpeRATOR® and GlycOCATCH™

 

Comprehensive Characterization of Antibody Drug Conjugates Enabled by Top-down and Middle-down Mass Spectrometry Strategies

Eli J Larson

TP 601, Poster Session 10.30 AM – 2.30 PM

GlycINATOR®FabRICATOR® and GingisKHAN®

 

Wednesday, June 5

Analysis of O-glycosylated Biopharmaceuticals using an O-glycan dependent Endoprotease and LC-MS

Andreas Nägeli

WP 334, Poster Session 10.30 AM – 2.30 PM

OpeRATOR® and OglyZOR®

 

MALDI-in-source decay FT-ICR MS for top-down and middle-down characterization of monoclonal antibodies 

Simone Nicolardi

WP 032, Poster Session 10.30 AM – 2.30 PM

FabRICATOR® and GingisKHAN®

 

Application of a label-free and domain-specific free thiol method in monoclonal antibody characterization

Yi Pu

WP 040, Poster Session 10.30 AM – 2.30 PM

FabALACTICA® and FabRICATOR®

 

Collision induced unfolding experiments to decipher the structural regions of a hybrid monoclonal antibody

Thomas Botzanowski

WP 481, Poster Session 10.30 AM – 2.30 PM

IgGZERO®

 

Thursday, June 6

Intact and Subunit Mass Analysis Using Native Ion Exchange Chromatography Coupled to an Orbitrap Mass Spectrometer 

Qian Liu

ThP 663, Poster Session 10.30 AM – 2.30 PM

FabRICATOR®

 

Ultra-Fast Analysis of Intact Proteins Using SPE- TOF 

Kevin McCann

ThP 149, Poster Session 10.30 AM – 2.30 PM

FabRICATOR®

 

Isomeric linkage determination of Sialic acid on O-glycopeptides using O-protease and LC-MS/MS 

Jieqiang Zhong

ThP 071, Poster Session 10.30 AM – 2.30 PM

OpeRATOR®

Poster Presentations at PEGS Europe 2018

IMG_0337 2
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.

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