14 research outputs found
Combination of intact, middle-up and bottom-up levels to characterize 7 therapeutic monoclonal antibodies by capillary electrophoresis â Mass spectrometry
Significant growth of biopharmaceuticals requires powerful analytical methods to better understand their structure by establishing a complete characterization. To this end, a combination of bottom-up, middle-up and intact molecule levels with a capillary electrophoresis-mass spectrometry coupling has been performed to have a comprehensive picture of monoclonal antibodies. In this study, 7 worldwide health authorities approved mAbs have been analyzed to get information about their charge heterogeneity, the identification of post translational modifications (PTMs), their location and relative quantitation. Intact mAbs isoforms have been partially separated in less than 12 minutes and enabled to have a global illustration of mAbs heterogeneity and high masses PTMs characterization notably major N-glycosylation forms. Particularly, 2X-glycosylated and 1X-glycosylated forms have been partially separated. To deepen characterize PTMs carried by the backbone structure, advanced investigations at a middle-up level have been performed. Limited IdeS proteolysis allowed to study independently Fc/2 and F(ab)â2 fragments. Following the same separation conditions, isoforms of these fragments have been separated and data interpretation allowed to disclose additional PTMs as K-clip, oxidations or deamidations. A second intermediate level has been examined by adding a reduction step to establish a more precise assessment of PTMs and isoforms from the F(ab)â2 fragment. This reduction step released the light chains from the Fd fragment to get only 25 kDa fragments to analyze. CE-ESI-MS coupling allowed to get more information particularly about low masses PTMs. The precise location and relative quantitation of each PTM has been investigated at the peptidic level induced by a tryptic digestion of the studied mAbs. The concordance of the results shows the efficiency of the CE-ESI-MS coupling to characterize mAbs and highlight the need of the multi-level combination to get a comprehensive characterization of biotherapeutics
Intact monoclonal antibodies separation and analysis by sheathless capillary electrophoresis-mass spectrometry
Capillary electrophoresis mass spectrometry coupling (CE-MS) is a growing technique in biopharmaceutics characterization. Assessment of monoclonal antibodies (mAbs) is well known at middle-up and bottom-up levels to obtain information about the sequence, post-translational modifications (PTMs) and degradation products. Intact protein analysis is an actual challenge to be closer to the real protein structure. At this level, actual techniques are time consuming or cumbersome processes. In this work, a 20 minutes separation method has been developed to optimize characterization of intact mAbs. Thus, separation have been done on a positively-charged coated capillary (PEI) with optimized volatile background electrolyte (BGE) and sample buffer (SB). A sheathless interface allowed to hyphenate CE to a quadrupole-time-of-flight mass spectrometer (Q-TOF) which parameters has been tuned to improve the high masses detection and identification of intact mAbs. Three world-wide health authorities approved mAbs have been used to set up a rapid and ease of use method. Intact trastuzumab, rituximab and palivizumab isoforms have been partially separated with this method in less than 20 minutes under denaturing conditions. For each mAb, 2X-glycosylated and 1X-glycosylated structures have been identified and separated. Concerning basic and acidic variants potential Iso-Asp modification and Asn deamidation have been observed. Accurate mass determination for high-mass molecular species remains a challenge, but the progress in intact mAbs separation appears very promising for biopharmaceutics characterization
Structural characterization of antibody drug conjugate by a combination of intact, middle-up and bottom-up techniques using sheathless capillary electrophoresis â Tandem mass spectrometry as nanoESI infusion platform and separation method
Antibody-drug conjugates (ADCs) represent a fast growing class of biotherapeutic products. Their production leads to a distribution of species exhibiting different number of conjugated drugs overlaying the inherent com-plexity resulting from the monoclonal antibody format, such as glycoforms. ADCs require an additional level of characterization compared to first generation of biotherapeutics obtained through multiple analytical tech-niques for complete structure assessment. We report the development of complementary approaches imple-menting sheathless capillary electrophoresis-mass spectrometry (sheathless CE-MS) to characterize the differ-ent aspects defining the structure of brentuximab vedotin. Native MS using sheathless CE-MS instrument as a nanoESI infusion platform enabled accurate mass measurements and estimation of the average drug to anti-body ratio alongside to drug load distribution. Middle-up analysis performed after limited IdeS proteolysis allowed to study independently the light chain, Fab and F(abâ)2 subunits incorporating 1, 0 to 4 and 0 to 8 pay-loads respectively. Finally, a CZE-ESI-MS/MS methodology was developed in order to be compatible with hy-drophobic drug composing ADCs. From a single injection, complete sequence coverage could be achieved. Using the same dataset, glycosylation and drug-loaded peptides could be simultaneously identified revealing robust information regarding their respective localization and abundance. Drug-loaded peptide fragmentation mass spectra study demonstrated drug specific fragments reinforcing identification confidence, undescribed so far. Results reveal the method ability to characterize ADCs primary structure in a comprehensive manner while reducing tremendously the number of experiments required. Data generated showed that sheathless CZE-ESI-MS/MS characteristics position the methodology developed as a relevant alternative for comprehensive multi-level characterization of these complex biomolecule
Novel sheathless CE-MS interface as an original and powerful infusion platform for nanoESI study: from intact proteins to high molecular mass noncovalent complexes.:
Development of nano-electrospray (nanoESI) sources allowed to increase significantly the sensitivity which is often lacking when studying biological noncovalent assemblies. However, the flow rate used to infuse the sample into the mass spectrometer cannot be precisely controlled with nanoESI and the robustness of the system could represent an issue. In this study, we have used a sheathless capillary electrophoresis-mass spectrometry (CESI) prototype as a nanoESI infusion device. The hydrodynamic mobilization of the capillary content was characterized and the ability of the system to generate a stable electrospray under controlled flow rate conditions ranging from 4 up to 900 nL/min was demonstrated. The effect of the infusing flow rate on the detection of an intact model protein analyzed under native conditions was investigated. Results demonstrated a significant increase in sensitivity of 46-fold and a signal-to-noise ratio improvement of nearly 5-fold when using an infusing flow rate from 456.9 down to 13.7 nL/min. The CESI prototype was further used to detect successfully the ÎČ ring homodimer in its native conformation. Obtained results were compared with those achieved with conventional ESI. Intensity signals were increased by a factor of 5, while sample consumption decreased 80 times. ÎČ ring complexed with the P14 peptide was also studied. Finally, the CESI interface was used to observe the quaternary structure of native hemocyanins from Carcinus maenas crabs; this high molecular complex coexisting under various degrees of complexation and resulting in masses ranging from 445 kDa to 1.34 MD
Monoclonal antibodies biosimilarity assessment using transient isotachophoresis capillary zone electrophoresis-tandem mass spectrometry
Out of all categories, monoclonal antibody (mAb) therapeutics attract the most interest due to their strong therapeutic potency and specificity. Six of the ten top-selling drugs are antibody-based therapeutics that will lose patent protection soon. The European Medicines Agency has pioneered the regulatory framework for approval of biosimilar products and approved the first biosimilar antibodies by the end of 2013. As highly complex glycoproteins with a wide range of micro-variants, mAbs require extensive characterization through multiple analytical methods for structure assessment rendering manufacturing control and biosimilarity studies particularly product and time-consuming. Here, capillary zone electrophoresis coupled to mass spectrometry by a sheathless interface (CESI-MS) was used to characterize marketed reference mAbs and their respective biosimilar candidate simultaneously over different facets of their primary structure. CESI-MS/MS data were compared between approved mAbs and their biosimilar candidates to prove/disconfirm biosimilarity regarding recent regulation directives. Using only a single sample injection of 200 fmol, CESI-MS/MS data enabled 100% amino acids (AA) sequence characterization, which allows a difference of even one AA between two samples to be distinguished precisely. Simultaneously glycoforms were characterized regarding their structures and position through fragmentation spectra and glycoforms semiquantitative analysis was established, showing the capacity of the developed methodology to detect up to 16 different glycans. Other posttranslational modifications hotspots were characterized while their relative occurrence levels were estimated and compared to biosimilars. These results proved the value of using CESI-MS because the separation selectivity and ionization efficiency provided by the system allowed substantial improvement in the characterization workflow robustness and accuracy. Biosimilarity assessment could be performed routinely with a single injection of each candidate enabling improvements in the biosimilar development pipeline
Rapid and multi-level characterization of trastuzumab using sheathless capillary electrophoresis-tandem mass spectrometry
Monoclonal antibodies (mAbs) are highly complex proteins that display a wide range of microheterogeneity that requires multiple analytical methods for full structure assessment and
quality control. As a consequence, the characterization of mAbs on different levels is particularly product - and time - consuming. This work presents the characterization of trastuzumab sequence using sheathless capillary electrophoresis (referred as CESI) â tandem mass spectrometry (CESIMS/MS). Using this bottom-up proteomic-like approach, CESI-MS/MS provided 100% sequence coverage for both heavy and light chain via peptide fragment fingerprinting (PFF) identification. The result was accomplished in a single shot, corresponding to the analysis of 100 fmoles of digest. The same analysis also enabled precise characterization of the post-translational hot spots of trastuzumab, used as a representative widely marketed therapeutic mAb, including the structural confirmation of the five major N-glycoforms
NIST Interlaboratory Study on Glycosylation Analysis of Monoclonal Antibodies: Comparison of Results from Diverse Analytical Methods
Glycosylation is a topic of intense current interest in the
development of biopharmaceuticals because it is related
to drug safety and efficacy. This work describes results of
an interlaboratory study on the glycosylation of the Primary
Sample (PS) of NISTmAb, a monoclonal antibody
reference material. Seventy-six laboratories from industry,
university, research, government, and hospital sectors
in Europe, North America, Asia, and Australia submit-
Avenue, Silver Spring, Maryland 20993; 22Glycoscience Research Laboratory, Genos, Borongajska cesta 83h, 10 000 Zagreb, Croatia;
23Faculty of Pharmacy and Biochemistry, University of Zagreb, A. KovacË icÂŽ a 1, 10 000 Zagreb, Croatia; 24Department of Chemistry, Georgia
State University, 100 Piedmont Avenue, Atlanta, Georgia 30303; 25glyXera GmbH, Brenneckestrasse 20 * ZENIT / 39120 Magdeburg, Germany;
26Health Products and Foods Branch, Health Canada, AL 2201E, 251 Sir Frederick Banting Driveway, Ottawa, Ontario, K1A 0K9 Canada;
27Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama Higashi-Hiroshima 739â8530 Japan; 28ImmunoGen,
830 Winter Street, Waltham, Massachusetts 02451; 29Department of Medical Physiology, Jagiellonian University Medical College,
ul. Michalowskiego 12, 31â126 Krakow, Poland; 30Department of Pathology, Johns Hopkins University, 400 N. Broadway Street Baltimore,
Maryland 21287; 31Mass Spec Core Facility, KBI Biopharma, 1101 Hamlin Road Durham, North Carolina 27704; 32Division of Mass
Spectrometry, Korea Basic Science Institute, 162 YeonGuDanji-Ro, Ochang-eup, Cheongwon-gu, Cheongju Chungbuk, 363â883 Korea
(South); 33Advanced Therapy Products Research Division, Korea National Institute of Food and Drug Safety, 187 Osongsaengmyeong 2-ro
Osong-eup, Heungdeok-gu, Cheongju-si, Chungcheongbuk-do, 363â700, Korea (South); 34Center for Proteomics and Metabolomics, Leiden
University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands; 35Ludger Limited, Culham Science Centre, Abingdon,
Oxfordshire, OX14 3EB, United Kingdom; 36Biomolecular Discovery and Design Research Centre and ARC Centre of Excellence for Nanoscale
BioPhotonics (CNBP), Macquarie University, North Ryde, Australia; 37Proteomics, Central European Institute for Technology, Masaryk
University, Kamenice 5, A26, 625 00 BRNO, Czech Republic; 38Max Planck Institute for Dynamics of Complex Technical Systems, Sandtorstrasse
1, 39106 Magdeburg, Germany; 39Department of Biomolecular Sciences, Max Planck Institute of Colloids and Interfaces, 14424
Potsdam, Germany; 40AstraZeneca, Granta Park, Cambridgeshire, CB21 6GH United Kingdom; 41Merck, 2015 Galloping Hill Rd, Kenilworth,
New Jersey 07033; 42Analytical R&D, MilliporeSigma, 2909 Laclede Ave. St. Louis, Missouri 63103; 43MS Bioworks, LLC, 3950 Varsity Drive
Ann Arbor, Michigan 48108; 44MSD, Molenstraat 110, 5342 CC Oss, The Netherlands; 45Exploratory Research Center on Life and Living
Systems (ExCELLS), National Institutes of Natural Sciences, 5â1 Higashiyama, Myodaiji, Okazaki 444â8787 Japan; 46Graduate School of
Pharmaceutical Sciences, Nagoya City University, 3â1 Tanabe-dori, Mizuhoku, Nagoya 467â8603 Japan; 47Medical & Biological Laboratories
Co., Ltd, 2-22-8 Chikusa, Chikusa-ku, Nagoya 464â0858 Japan; 48National Institute for Biological Standards and Control, Blanche Lane, South
Mimms, Potters Bar, Hertfordshire EN6 3QG United Kingdom; 49Division of Biological Chemistry & Biologicals, National Institute of Health
Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158â8501 Japan; 50New England Biolabs, Inc., 240 County Road, Ipswich, Massachusetts
01938; 51New York University, 100 Washington Square East New York City, New York 10003; 52Target Discovery Institute, Nuffield Department
of Medicine, University of Oxford, Roosevelt Drive, Oxford, OX3 7FZ, United Kingdom; 53GlycoScience Group, The National Institute for
Bioprocessing Research and Training, Fosters Avenue, Mount Merrion, Blackrock, Co. Dublin, Ireland; 54Department of Chemistry, North
Carolina State University, 2620 Yarborough Drive Raleigh, North Carolina 27695; 55Pantheon, 201 College Road East Princeton, New Jersey
08540; 56Pfizer Inc., 1 Burtt Road Andover, Massachusetts 01810; 57Proteodynamics, ZI La Varenne 20â22 rue Henri et Gilberte Goudier 63200
RIOM, France; 58ProZyme, Inc., 3832 Bay Center Place Hayward, California 94545; 59Koichi Tanaka Mass Spectrometry Research Laboratory,
Shimadzu Corporation, 1 Nishinokyo Kuwabara-cho Nakagyo-ku, Kyoto, 604 8511 Japan; 60Childrenâs GMP LLC, St. Jude Childrenâs
Research Hospital, 262 Danny Thomas Place Memphis, Tennessee 38105; 61Sumitomo Bakelite Co., Ltd., 1â5 Muromati 1-Chome, Nishiku,
Kobe, 651â2241 Japan; 62Synthon Biopharmaceuticals, Microweg 22 P.O. Box 7071, 6503 GN Nijmegen, The Netherlands; 63Takeda
Pharmaceuticals International Co., 40 Landsdowne Street Cambridge, Massachusetts 02139; 64Department of Chemistry and Biochemistry,
Texas Tech University, 2500 Broadway, Lubbock, Texas 79409; 65Thermo Fisher Scientific, 1214 Oakmead Parkway Sunnyvale, California
94085; 66United States Pharmacopeia India Pvt. Ltd. IKP Knowledge Park, Genome Valley, Shamirpet, Turkapally Village, Medchal District,
Hyderabad 500 101 Telangana, India; 67Alberta Glycomics Centre, University of Alberta, Edmonton, Alberta T6G 2G2 Canada; 68Department
of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2 Canada; 69Department of Chemistry, University of California, One Shields Ave,
Davis, California 95616; 70HorvaÂŽ th Csaba Memorial Laboratory for Bioseparation Sciences, Research Center for Molecular Medicine, Doctoral
School of Molecular Medicine, Faculty of Medicine, University of Debrecen, Debrecen, Egyetem ter 1, Hungary; 71Translational Glycomics
Research Group, Research Institute of Biomolecular and Chemical Engineering, University of Pannonia, Veszprem, Egyetem ut 10, Hungary;
72Delaware Biotechnology Institute, University of Delaware, 15 Innovation Way Newark, Delaware 19711; 73Proteomics Core Facility, University
of Gothenburg, Medicinaregatan 1G SE 41390 Gothenburg, Sweden; 74Department of Medical Biochemistry and Cell Biology, University of
Gothenburg, Institute of Biomedicine, Sahlgrenska Academy, Medicinaregatan 9A, Box 440, 405 30, Gothenburg, Sweden; 75Department of
Clinical Chemistry and Transfusion Medicine, Sahlgrenska Academy at the University of Gothenburg, Bruna Straket 16, 41345 Gothenburg,
Sweden; 76Department of Chemistry, University of Hamburg, Martin Luther King Pl. 6 20146 Hamburg, Germany; 77Department of Chemistry,
University of Manitoba, 144 Dysart Road, Winnipeg, Manitoba, Canada R3T 2N2; 78Laboratory of Mass Spectrometry of Interactions and
Systems, University of Strasbourg, UMR Unistra-CNRS 7140, France; 79Natural and Medical Sciences Institute, University of Tuš bingen,
Markwiesenstrae 55, 72770 Reutlingen, Germany; 80Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical
Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands; 81Division of Bioanalytical Chemistry, Amsterdam Institute for
Molecules, Medicines and Systems, Vrije Universiteit Amsterdam, de Boelelaan 1085, 1081 HV Amsterdam, The Netherlands; 82Department
of Chemistry, Waters Corporation, 34 Maple Street Milford, Massachusetts 01757; 83Zoetis, 333 Portage St. Kalamazoo, Michigan 49007
Authorâs ChoiceâFinal version open access under the terms of the Creative Commons CC-BY license.
Received July 24, 2019, and in revised form, August 26, 2019
Published, MCP Papers in Press, October 7, 2019, DOI 10.1074/mcp.RA119.001677
ER: NISTmAb Glycosylation Interlaboratory Study
12 Molecular & Cellular Proteomics 19.1
Downloaded from https://www.mcponline.org by guest on January 20, 2020
ted a total of 103 reports on glycan distributions. The
principal objective of this study was to report and compare
results for the full range of analytical methods presently
used in the glycosylation analysis of mAbs. Therefore,
participation was unrestricted, with laboratories
choosing their own measurement techniques. Protein glycosylation
was determined in various ways, including at
the level of intact mAb, protein fragments, glycopeptides,
or released glycans, using a wide variety of methods for
derivatization, separation, identification, and quantification.
Consequently, the diversity of results was enormous,
with the number of glycan compositions identified by
each laboratory ranging from 4 to 48. In total, one hundred
sixteen glycan compositions were reported, of which 57
compositions could be assigned consensus abundance
values. These consensus medians provide communityderived
values for NISTmAb PS. Agreement with the consensus
medians did not depend on the specific method or
laboratory type. The study provides a view of the current
state-of-the-art for biologic glycosylation measurement
and suggests a clear need for harmonization of glycosylation
analysis methods. Molecular & Cellular Proteomics
19: 11â30, 2020. DOI: 10.1074/mcp.RA119.001677.L
Independent highly sensitive characterization of asparagine deamidation and aspartic acid isomerization by sheathless CZE-ESI-MS/MS: Characterization of Asn and Asp PTMs by CZE-ESI-MS
Amino acids residues are commonly submitted to various physicochemical modifications occurring at physiological pH and temperature. Post-translational modifications (PTMs) require comprehensive characterization due to their major influence on protein structure and involvement in numerous in vivo process or signaling. Mass spectrometry (MS) has gradually became an analytical tool of choice to characterize PTMs, however some modifications are still challenging due to sample faint modification levels or difficulty to separate an intact peptide from modified counterparts before their transfer to the ionization source. Here we report the implementation of capillary zone electrophoresis coupled to electrospray ionization tandem mass spectrometry (CZE-ESI-MS/MS) by the intermediate of a sheathless interfacing for independent and highly sensitive characterization of asparagine deamidation (deaN) and aspartic acid isomerization (isoD). CZE selectivity regarding deaN and isoD was studied extensively using different sets of synthetic peptides based on actual tryptic peptides. Results demonstrated CZE ability to separate the unmodified peptide from modified homologous exhibiting deaN, isoD or both independently with a resolution systematically superior to 1.29. Developed CZE-ESI-MS/MS method was applied to the characterization of monoclonal antibodies and complex protein mixture. Conserved CZE selectivity could be demonstrated even for complex samples and foremost results obtained showed that CZE selectivity is similar regardless of the composition of the peptide. Separation of modified peptides prior to the MS analysis allowed to characterize and estimate modification levels of the sample independently for deaN and isoD even for peptides affected by both modifications and as a consequence enables to distinguish the formation of L-aspartic acid or D-aspartic acid generated from deaN. Separation based on peptide modification allowed, supported by the ESI ionization efficiency provided by CZE-ESI-MS properties, enabled to characterize and estimate studied PTMs with an unprecedented sensitivity and proved the relevance of implementing an electrophoretic driven separation for MS based peptide analysis
Analysis of monoclonal antibody by a novel CE-UV/MALDI-MS interface: CE and CEC
Monoclonal antibodies (mAb) are highly complex proteins that advertise a wide range of microheterogeneity that requires multiple analytical methods for full structure assessment and quality control. As a consequence, the characterization of mAbs on different levels is particularly product - and time - consuming. Capillary Electrophoresis-Mass Spectrometry (CE-MS) couplings, especially to matrix-assisted laser desorption/ionization (MALDI) appear really attractive methods for the characterization of biological samples. In this work, we report on the last instrumental development and performance of the first totally automated off-line CE-UV/MALDI-MS/MS. This interface is based on the removal of the original UV cell of the CE apparatus, the modification of the spotting device geometry and the creation of an integrated delivery matrix system. The performance of the method was evaluated with separations of 5 intact proteins and a tryptic digest mixture of 9 proteins. Intact proteins application shows the acquisition of electropherograms with high resolution and high repeatability. In the peptide mapping approach, a total number of 154 unique identified peptides were characterized using MS/MS spectra corresponding to average sequence coverage of 64.1%. Comparison with NanoLC/MALDI-MS/MS showed complementarity at the peptide level with an increase by 42% when using CE/MALDI-MS coupling. Finally, this work represents the first analysis of intact mAb charge variants by capillary zone electrophoresis using a MS detection. Moreover, using a peptide mapping approach CE-UV/MALDI-MS/MS fragmentation allowed 100% sequence coverage of the light chain and 92 % on the heavy chain and the separation of the 4 major glycosylated peptides and their structural characterizatio