Forced Degradation Testing as Complementary Tool for Biosimilarity Assessment

Oxidation of monoclonal antibodies (mAbs) can impact their efficacy and may therefore represent critical quality attributes (CQA) that require evaluation. To complement classical CQA, bevacizumab and infliximab were subjected to oxidative stress by H2O2 for 24, 48, or 72 h to probe their oxidation susceptibility. For investigation, a middle-up approach was used utilizing liquid chromatography hyphenated with mass spectrometry (LC-QTOF-MS). In both mAbs, the Fc/2 subunit was completely oxidized. Additional oxidations were found in the light chain (LC) and in the Fd’ subunit of infliximab, but not in bevacizumab. By direct comparison of methionine positions, the oxidized residues in infliximab were assigned to M55 in LC and M18 in Fd’. The forced oxidation approach was further exploited for comparison of respective biosimilar products. Both for bevacizumab and infliximab, comparison of posttranslational modification profiles demonstrated high similarity of the unstressed reference product (RP) and the biosimilar (BS). However, for bevacizumab, comparison after forced oxidation revealed a higher susceptibility of the BS compared to the RP. It may thus be considered a useful tool for biopharmaceutical engineering, biosimilarity assessment, as well as for quality control of protein drugs

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. Kovacˇ ic´ 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 739–8530 Japan; 28ImmunoGen, 830 Winter Street, Waltham, Massachusetts 02451; 29Department of Medical Physiology, Jagiellonian University Medical College, ul. Michalowskiego 12, 31–126 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, 363–883 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, 363–700, 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, 5–1 Higashiyama, Myodaiji, Okazaki 444–8787 Japan; 46Graduate School of Pharmaceutical Sciences, Nagoya City University, 3–1 Tanabe-dori, Mizuhoku, Nagoya 467–8603 Japan; 47Medical & Biological Laboratories Co., Ltd, 2-22-8 Chikusa, Chikusa-ku, Nagoya 464–0858 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 158–8501 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 20–22 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; 60Children’s GMP LLC, St. Jude Children’s Research Hospital, 262 Danny Thomas Place Memphis, Tennessee 38105; 61Sumitomo Bakelite Co., Ltd., 1–5 Muromati 1-Chome, Nishiku, Kobe, 651–2241 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; 70Horva´ 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 Tu¨ 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 Author’s Choice—Final 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: 11–30, 2020. DOI: 10.1074/mcp.RA119.001677.L

ANALYSIS OF THE FIR CYANAMIDE SPECTRUM BY THE "MULTIMOLECULE" RITZ PROGRAM

Author Institution: Dipartimento di Fisica dell' Universit\`{a} di Pisa, Piazza Torricelli; Physikalisch-Chemisches Institut der Justus-Liebig-Universit\""{a}t, Heinrich-Buff-Ring 58The multi-molecule version of the "Ritz" program, presented at the last Columbus Symposium, has been used for the investigation of the ground small-amplitude vibrational state of $H_{2}NCN$. The absorption spectrum between 20 and 985 $cm^{-1}$ has been measured on the Bruker Fourier transform spectometer at the Physikalisch-Chemisches Institut of the Justus Liebig University, Giessen. The resolution of the spectra was 0.00166 $cm^{-1}$, so that the spectrum is fully resolved. The sample was vaporized at 100 C in a slow flow through a three meter sample cell maintained at 130-140 C for the duration of the measurements. At the moment, the assignment database contains more than 18800 assigned lines, corresponding to transitions between more than 3900 energy levels of the inversion states with the quantum number $n = 0$ and 1. The cyanamide molecule has two identical H atoms in the $NH_{2}$ group. The total nuclear spin of the two H atoms can thus be either $I = 1$ or $I = 0$. This is reflected over the symmetry of the complete molecular wavefunction, which must be antisymmetrical under exchange of the two protons. Since radiation-induced transitions cannot change the nuclear-spin wavefunction, the levels of cyanamide belong to two different species, labeled by the nuclear spin. The same behavior is observed also in other molecules with two equivalent hydrogens, notably in water. The two species are called para (lower spin multiplicity, 1, corresponding to $I = 0$) and ortho (higher spin multiplicity, 3, corresponding to $I = 1$), in analogy to the case of the hydrogen molecule. Transitions between the two species are forbidden. The levels assigned for the ortho species include levels with the quantum number K ranging from 0 to 12 for $n = 0$, and from 0 to 8 and 11 for n = 1. For the para species, the assigned levels have K ranging from 0 to 11 for n = 0, and from 0 to 9 and 10 for $n = 1$. The level sequences (n = 1, K = 10) of the ortho species, and $(n = 1, K = 9)$ of the para species, probably affected by strong perturbations, are still under investigation. Important improvements have been introduced into the line-analysis routine, on which a new peak-finder program is based

THE CARBODIIMIDE SPECTRUM IN THE WAVENUMBER RANGE OF 450−1000cm−1450-1000 cm^{-1} AND IN THE THz REGION

$^{a}$V. Wagener, M. Winnewisser, M. Bellini, J. Mol. Sepctrosc, 170, 323 (1995)Author Institution: Physikalisch- Chemisches Institut, Justus-Liebig-Universit\""{a}t; I. Physikalisches Institut, Universit\""{a}t zu K\""{o}lnCarbodiimide, HNCNH, is an accidently nearly symmetric top molecule ($\approx= -0.99999537$) and it undergoes internal rotation hindered by a cis and a trans energy barrier of nearly equal height of about $2070 cm^{-1}$. This internal motion leads to torsional splittings of the rotational energy levels, which shows an unusual dependence on the $K_{a}$ rotational quantum number. The strong coupling between the torsional and the HNC bending motion leads to rotationally dependent contributions to the effective torsional potential function. To extend our knowledge about these complex internal motions and the structure of the molecule we recorded the spectrum of the equilibrium mixture of carbodiimide and cyanamide, $H_{2}NCN$, with a Bruker IFS 120HR in the range from 450 to $1000 cm^{-1}$. Unfortunately the strong dominance of cyanamide (approximately 115:1 at $110^{\circ} C$) makes it very difficult to identify the torsional fundamental vibration. However, the antisymmetric HNC-bending fundamental could be analysed. To support the assignment procedure ab - initio calculations using the CCSD(T) method had been carried out. Spectra of the terahertz region offer the opportunity to study another remarkable property of the molecule, namely the recently observed inversion of the $K_{a} = 2$ energy $levels^{a}$ and the shift of the $K_{a} = 1$ levels due to a centrifugal distortion interaction with $\Delta K_{a} =4$. We have measured and analysed the $^{r}Q_{1}$ branch at 1.1 THz. The analysis of the terahertz data and the infrared data in the light of the recent ab-initio calculations will be presente

Susceptibilities of Human Cytomegalovirus Clinical Isolates to BAY38-4766, BAY43-9695, and Ganciclovir

BAY38-4766 and BAY43-9695 are nonnucleosidic compounds with activities against human cytomegalovirus (HCMV). Two phenotypic assays were used to determine the drug susceptibilities of 36 HCMV clinical isolates to the BAY compounds and ganciclovir. Using either assay, both BAY compounds at a concentration of approximately 1 μM inhibited the replication of all 36 HCMV clinical isolates, including 11 ganciclovir-resistant clinical isolates, by 50%

Detection of proteoforms using top-down mass spectrometry and diagnostic ions

Characterization of protein structure modifications is an important field in mass spectrometry (MS)-based proteomics. Here, we describe a process to quickly and reliably identify a mass change in a targeted protein sequence by top-down mass spectrometry (TD MS) using electron transfer dissociation (ETD). The step-by-step procedure describes how to develop a TD MS method for data acquisition as well as the data analysis process. The described TD MS workflow utilizes diagnostic ions to characterize an unknown sample in a few hours

Rotation and Rotation–Vibration Spectroscopy of the 0⁺–0^– Inversion Doublet in Deuterated Cyanamide

The pure rotation spectrum of deuterated cyanamide was recorded at frequencies from 118 to 649 GHz, which was complemented by measurement of its high-resolution rotation-vibration spectrum at 8–350 cm^–1. For D₂NCN the analysis revealed considerable perturbations between the lowest <i>K</i>_<i>a</i> rotational energy levels in the 0⁺ and 0^– substates of the lowest inversion doublet. The final data set for D₂NCN exceeded 3000 measured transitions and was successfully fitted with a Hamiltonian accounting for the 0⁺ ↔ 0^– coupling. A smaller data set, consisting only of pure rotation and rotation-vibration lines observed with microwave techniques was obtained for HDNCN, and additional transitions of this type were also measured for H₂NCN. The spectroscopic data for all three isotopic species were fitted with a unified, robust Hamiltonian allowing confident prediction of spectra well into the terahertz frequency region, which is of interest to contemporary radioastronomy. The isotopic dependence of the determined inversion splitting, Δ<i>E</i> = 16.4964789(8), 32.089173(3), and 49.567770(6) cm^–1, for D₂NCN, HDNCN, and H₂NCN, respectively, is found to be in good agreement with estimates from a simple reduced quartic-quadratic double minimum potential

Rotation and Rotation–Vibration Spectroscopy of the 0⁺–0^– Inversion Doublet in Deuterated Cyanamide

The pure rotation spectrum of deuterated cyanamide was recorded at frequencies from 118 to 649 GHz, which was complemented by measurement of its high-resolution rotation-vibration spectrum at 8–350 cm^–1. For D₂NCN the analysis revealed considerable perturbations between the lowest <i>K</i>_<i>a</i> rotational energy levels in the 0⁺ and 0^– substates of the lowest inversion doublet. The final data set for D₂NCN exceeded 3000 measured transitions and was successfully fitted with a Hamiltonian accounting for the 0⁺ ↔ 0^– coupling. A smaller data set, consisting only of pure rotation and rotation-vibration lines observed with microwave techniques was obtained for HDNCN, and additional transitions of this type were also measured for H₂NCN. The spectroscopic data for all three isotopic species were fitted with a unified, robust Hamiltonian allowing confident prediction of spectra well into the terahertz frequency region, which is of interest to contemporary radioastronomy. The isotopic dependence of the determined inversion splitting, Δ<i>E</i> = 16.4964789(8), 32.089173(3), and 49.567770(6) cm^–1, for D₂NCN, HDNCN, and H₂NCN, respectively, is found to be in good agreement with estimates from a simple reduced quartic-quadratic double minimum potential

Rotation and Rotation–Vibration Spectroscopy of the 0⁺–0^– Inversion Doublet in Deuterated Cyanamide

The pure rotation spectrum of deuterated cyanamide was recorded at frequencies from 118 to 649 GHz, which was complemented by measurement of its high-resolution rotation-vibration spectrum at 8–350 cm^–1. For D₂NCN the analysis revealed considerable perturbations between the lowest <i>K</i>_<i>a</i> rotational energy levels in the 0⁺ and 0^– substates of the lowest inversion doublet. The final data set for D₂NCN exceeded 3000 measured transitions and was successfully fitted with a Hamiltonian accounting for the 0⁺ ↔ 0^– coupling. A smaller data set, consisting only of pure rotation and rotation-vibration lines observed with microwave techniques was obtained for HDNCN, and additional transitions of this type were also measured for H₂NCN. The spectroscopic data for all three isotopic species were fitted with a unified, robust Hamiltonian allowing confident prediction of spectra well into the terahertz frequency region, which is of interest to contemporary radioastronomy. The isotopic dependence of the determined inversion splitting, Δ<i>E</i> = 16.4964789(8), 32.089173(3), and 49.567770(6) cm^–1, for D₂NCN, HDNCN, and H₂NCN, respectively, is found to be in good agreement with estimates from a simple reduced quartic-quadratic double minimum potential