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A broad-based interlaboratory study of glycosylation profiles of a reference and modified IgG antibody involving 103 reports from 76 laboratories., Graphical Abstract Highlights A broad-based interlaboratory study of the glycosylation of a reference antibody: NISTmAb. 103 reports were received from 76 diverse laboratories worldwide. Analysis involved two samples, the NISTmAb and an enzymatically modified sample, enabling within-lab separation of random and systematic errors using the “Youden two-sample” method. Consensus values were derived and similar performance across all experimental methods was noted., 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 submitted 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 community-derived 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.

Background Biological diagnosis of hemoglobin disorders is a complex process relying on the combination of several analytical techniques to identify Hb variants in a particular sample. Currently, hematology laboratories usually use high-performance liquid chromatography (HPLC), capillary electrophoresis and gel-based methods to characterize Hb variants. Co-elution and co-migration may represent major issues for precise identification of Hb variants, even for the most common ones such as Hb S and C. Methods We adapted a top-down selected reaction monitoring (SRM) electron transfer dissociation (ETD) mass spectrometry (MS) method to fit with a clinical laboratory environment. An automated analytical process with semi-automated data analysis compatible with a clinical practice was developed. A comparative study between a reference HPLC method and the MS assay was performed on 152 patient samples. Results The developed workflow allowed to identify with high specificity and selectivity the most common Hb variants (Hb S and Hb C). Concordance of the MS-based approach with HPLC was 71/71 (100%) for Hb S and 11/11 (100%) for Hb C. Conclusions This top-down SRM ETD method can be used in a clinical environment to detect Hb S and Hb C.

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&rsquo, 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&rsquo, 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.

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.

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.

The regulatory bodies request full sequence data assessment both for innovator and biosimilar monoclonal antibodies (mAbs). Full sequence coverage is typically used to verify the integrity of the analytical data obtained following the combination of multiple LC-MS/MS datasets from orthogonal protease digests (so called “bottom-up” approaches). Top-down or middle-down mass spectrometric approaches have the potential to minimize artifacts, reduce overall analysis time and provide orthogonality to this traditional approach. In this work we report a new combined approach involving middle-up LC-QTOF and middle-down LC-MALDI in-source decay (ISD) mass spectrometry. This was applied to cetuximab, panitumumab and natalizumab, selected as representative US Food and Drug Administration- and European Medicines Agency-approved mAbs. The goal was to unambiguously confirm their reference sequences and examine the general applicability of this approach. Furthermore, a new measure for assessing the integrity and validity of results from middle-down approaches is introduced – the “Sequence Validation Percentage.” Full sequence data assessment of the 3 antibodies was achieved enabling all 3 sequences to be fully validated by a combination of middle-up molecular weight determination and middle-down protein sequencing. Three errors in the reference amino acid sequence of natalizumab, causing a cumulative mass shift of only −2 Da in the natalizumab Fd domain, were corrected as a result of this work.

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 Ka 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, ΔE = 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.

In-depth site-specific investigations of protein glycosylation are the basis for understanding the biological function of glycoproteins. Mass spectrometry-based N- and O-glycopeptide analyses enable determination of the glycosylation site, site occupancy, as well as glycan varieties present on a particular site. However, the depth of information is highly dependent on the applied analytical tools, including glycopeptide fragmentation regimes and automated data analysis. Here, we used a small set of synthetic disialylated, biantennary N-glycopeptides to systematically tune Q-TOF instrument parameters towards optimal energy stepping collision induced dissociation (CID) of glycopeptides. A linear dependency of m/z-ratio and optimal fragmentation energy was found, showing that with increasing m/z-ratio, more energy is required for glycopeptide fragmentation. Based on these optimized fragmentation parameters, a method combining lower- and higher-energy CID was developed, allowing the online acquisition of glycan and peptide-specific fragments within a single tandem MS experiment. We validated this method analyzing a set of human immunoglobulins (IgA1+2, sIgA, IgG1+2, IgE, IgD, IgM) as well as bovine fetuin. These optimized fragmentation parameters also enabled software-assisted glycopeptide assignment of both N- and O-glycopeptides including information about the most abundant glycan compositions, peptide sequence and putative structures. Twenty-six out of 30 N-glycopeptides and four out of five O-glycopeptides carrying >110 different glycoforms could be identified by this optimized LC-ESI tandem MS method with minimal user input. The Q-TOF based glycopeptide analysis platform presented here opens the way to a range of different applications in glycoproteomics research as well as biopharmaceutical development and quality control. Graphical Abstract ᅟ Electronic supplementary material The online version of this article (doi:10.1007/s13361-015-1308-6) contains supplementary material, which is available to authorized users.

The rotational spectrum of cyanamide recorded for deuterated cyanamide at frequencies from 118 to 649 GHz, and more limited spectra for isotopically enriched H2N13CN and H 2 15 N C15N were subjected to a detailed analysis using Loomis–Wood type graphical assignment techniques. Transitions in the lowest inversion doublet of 0+ and 0− substates were assigned for six new isotopic species: D2N13CN, HDN13CN, HD15NCN, HDNC15N, H2N13CN, and H 2 15 N C15N. The data for four other species, D 2 15 N CN, D2NC15N, H 2 15 N CN, and H2NC15N have been considerably extended. Spectroscopic constants and the inversion splitting have been determined for all of these species from a coupled fit of the two substates. Rotational constants for all known isotopic species of cyanamide have been combined with vibration–rotation constants obtained from an ab initio calculation in an evaluation of the r e SE geometry of this molecule.

Proteins are an important class of biologics. Their higher-order structures and therefore their functions are fundamentally determined by the correct formation of disulfide bonds (DSBs), making DSB analysis a central part of their development and production. Mass spectrometry-based bottom-up approaches are most widely used and are further classified according to different methods applied for DSB cleavage. Despite the importance of DSB analysis and the wide range of available methodologies, it is often a challenging and time consuming task. However, due to the current increase in biosimilar development in which animal and clinical trials can be reduced by extensive analytical comparability studies, increased efforts are being made to simplify DSB analysis. As an example of these developments, a matrix-assisted laser desorption/ionization time-of-flight (TOF)/TOF workflow for the automated profiling and identification of DSBs is presented. Furthermore, mass spectrometry based methodologies, which do not identify DSBs directly but measure their influence on the higher-order structure, are also considered.

The ground state rotational spectra and internal dynamics in five isotopomers 14N2–IH, 14N2–ID, 15N2–IH, 14N15N–IH, and 15N14N–IH of the nitrogen–hydrogen iodine dimer have been investigated by pulsed-nozzle, Fourier-transform microwave spectroscopy. Analysis of the recorded spectra yields rotational, centrifugal distortion, nuclear quadrupole and spin-rotation coupling constants for the five isotopomers. The spectroscopic constants are interpreted in terms of a ground-state NN–IH structure using the parameters rcm, kσ, and the oscillation angles θN2 and θHI of the N2 and IH moieties. For 14N2–IH, the values rcm=4.197 872(67) A, kσ=1.461 52(32) N/m, θN2=25.61(22)°, and θHI=23.454 6(91)° are obtained. The rotational transitions of 14N2–IH, 14N2–ID, and 15N2–IH show a doubling, which is a consequence of tunneling associated with the interchange of the two nitrogen nuclei. The experimental findings are compared with results from ab initio calculations.

An experimentally determined r s -type structure of HNCNH is reported: r NH = 1.0074 A, r CN = 1.2242 A, ∠HNC = 118.63°, ∠NCN = 170.63°, ∠HN ··· NH = 88.99°. The number of digits quoted allow for errors with two significant figures. In order to obtain these values we recorded rotational—torsional spectra of HN13CNH, H15NC15NH and DNCND, by using isotopically enriched cyanamide. A chemical equilibrium exists between carbodiimide, HNCNH, and the more stable isomer cyanamide, H2NCN, which strongly favours cyanamide (approximately 1:115 at 110 °C). The expensive C- and N-substituted isotopomers could only be investigated in the millimetre wave region, while for DNCND the far infrared spectrum between 10–350 cm−1 was also recorded. Rotational constants of the three isotopomers, as well as of the parent species, were determined by fitting the assigned spectral transitions to the Watson Hamiltonian in S reduction. Using fitting programs written by Schwendeman and Rudolph, r o, rs and rρ m structures of HNCNH wer...

The molecular parameters of carbodiimide (HNCNH) have been determined in large-scale ab initio calculations using the coupled-cluster method (CCSD(T)) and the correlation-consistent basis sets of double- through quadruple-zeta quality. The effect of core-electron correlation on the structural parameters was investigated. The equilibrium structural parameters of the molecule were estimated to be r (CN)=1.2242 A, r (NH)=1.0092 A, ∠(NCN)=170.62°, ∠(CNH)=117.84°, and ∠(HN…NH)=89.18°. The potential energy barriers for the trans and cis conformers were determined to be nearly equal, with the values 2636 and 2556 cm −1 , respectively. The calculated molecular parameters were found to be in good agreement with experiment.

The "Ritz" program, originally written for the analysis of the Fourier transform spectra of the methanol isotopomers and presented in previous papers, has been extended in order to analyze the spectra of other molecules. This program evaluates the term values involved in the assigned transitions by the Rydberg-Ritz combination principle, and can tackle such perturbations as Fermi-type resonances or Coriolis interactions. As a first application of the extended version, we present an investigation of the Fourier transform spectrum of cyanamide between 25 and 980 cm-1. More than 16 000 lines have been assigned. Our Ritz database now comprises a list of more than 19 000 assigned lines (including of the microwave and FIR lines available in the JPL database) and more than 3900 term values. All of the lines presented in this paper correspond to transitions within the ground and first excited inversion levels of the ground vibrational state of the small-amplitude modes. Copyright 1998 Academic Press.

The r Q 1 branch of the skew-chain molecule HNCNH has been measured at 1.1 THz using a frequency and phase stabilized and continuously tunable backward wave oscillator with a frequency accuracy better than 100 kHz for unblended lines. The newly assigned transitions were added to the existing data base and then fitted to constants of the Watson Hamiltonian in S -reduction. The r Q 1 -branch spectrum is complex because it consists of the superposition of four subbranches resulting from an internal torsional motion through a trans - and a cis -torsional potential energy barrier of nearly equal height (∼2070 cm −1 ) and the resolved K a doubling. As discovered previously, the K a =2 energy levels in HNCNH are inverted, as in the H 2 S 2 and D 2 S 2 molecules. This is a consequence of the dominance of the centrifugal-distortion splitting over the pure inertial asymmetry splitting in these accidentally nearly symmetric tops. The contributions of the two effects to the energy levels and to the r Q 1 -branch transitions have been analysed as well as the J dependence of the torsional splitting.

One of the principal goals of glycoprotein research is to correlate glycan structure and function. Such correlation is necessary in order for one to understand the mechanisms whereby glycoprotein structure elaborates the functions of myriad proteins. The accurate comparison of glycoforms and quantification of glycosites are essential steps in this direction. Mass spectrometry has emerged as a powerful analytical technique in the field of glycoprotein characterization. Its sensitivity, high dynamic range, and mass accuracy provide both quantitative and sequence/structural information. As part of the 2012 ABRF Glycoprotein Research Group study, we explored the use of mass spectrometry and ancillary methodologies to characterize the glycoforms of two sources of human prostate specific antigen (PSA). PSA is used as a tumor marker for prostate cancer, with increasing blood levels used to distinguish between normal and cancer states. The glycans on PSA are believed to be biantennary N-linked, and it has been observed that prostate cancer tissues and cell lines contain more antennae than their benign counterparts. Thus, the ability to quantify differences in glycosylation associated with cancer has the potential to positively impact the use of PSA as a biomarker. We studied standard peptide-based proteomics/glycomics methodologies, including LC-MS/MS for peptide/glycopeptide sequencing and label-free approaches for differential quantification. We performed an interlaboratory study to determine the ability of different laboratories to correctly characterize the differences between glycoforms from two different sources using mass spectrometry methods. We used clustering analysis and ancillary statistical data treatment on the data sets submitted by participating laboratories to obtain a consensus of the glycoforms and abundances. The results demonstrate the relative strengths and weaknesses of top-down glycoproteomics, bottom-up glycoproteomics, and glycomics methods.

The European Medicines Agency received recently the first marketing authorization application for a biosimilar monoclonal antibody (mAb) and adopted the final guidelines on biosimilar mAbs and Fc-fusion proteins. The agency requires high similarity between biosimilar and reference products for approval. Specifically, the amino acid sequences must be identical. The glycosylation pattern of the antibody is also often considered to be a very important quality attribute due to its strong effect on quality, safety, immunogenicity, pharmacokinetics and potency. Here, we describe a case study of cetuximab, which has been marketed since 2004. Biosimilar versions of the product are now in the pipelines of numerous therapeutic antibody biosimilar developers. We applied a combination of intact, middle-down, middle-up and bottom-up electrospray ionization and matrix assisted laser desorption ionization mass spectrometry techniques to characterize the amino acid sequence and major post-translational modifications of the marketed cetuximab product, with special emphasis on glycosylation. Our results revealed a sequence error in the reported sequence of the light chain in databases and in publications, thus highlighting the potency of mass spectrometry to establish correct antibody sequences. We were also able to achieve a comprehensive identification of cetuximab's glycoforms and glycosylation profile assessment on both Fab and Fc domains. Taken together, the reported approaches and data form a solid framework for the comparability of antibodies and their biosimilar candidates that could be further applied to routine structural assessments of these and other antibody-based products.

The rotational spectrum of the 15 N-substituted quasilinear molecule fulminic acid, HCNO, has been measured in the frequency range from 90 to 440 GHz covering rotational transitions up to J = 19↔18. Rotational transitions arising from HC 15 NO molecules in 52 vibrational levels were detected and assigned. Additional information was obtained by measuring the direct l-type transitions for v 5 = 1 and 3 and for v 4 = 1 in the frequency range from 18 to 40 GHz. With the information from the rotational spectrum the analysis of the far and mid-infrared spectra (170-1300 cm −1 ), which was started by Wagner et al. [J. Mol. Spectrosc. 162, 82 (1993)], could be extended so that the vibrational term values of HC 15 NO were determined for almost every state below 2500 cm −1 . The assignment of the 114 subbands involved allowed in turn the assignment of 25 subbands in the mid-infrared from the data of Quapp et al. [J. Mol. Spectrosc. 160, 540 (1993)]. The infrared spectra were recorded with a Bruker IFS 120 HR interferometer at resolutions between 0.0019 and 0.0034 cm −1 . For the vibrational states (v 1 v 2 v 3 v 4 v 5 ) = (00000), (00001), (00002), (00003), (00004), (00010), (00011) an (00100) the spectroscopic constants of an effective Hamiltonian for linear molecules could be improved by combining the already published infrared data with the millimeter wave, microwave and newly assigned infrared data presented in this work. For the states (00013) and (00101) these constants were also calculated. A calibration problem with the infrared data became obvious and important. The effects of a Fermi-type resonance between the vibrational levels (00004) Oe and (00100) Oe and a Coriolis resonance between the states (00002) and (00010) on the rotational and rovibrational spectra were observed and analysed. The difficulties encountered in determining the Coriolis resonance parameters are discussed

We present a novel and compact frequency multiplier design for broad-band spectroscopy up to 1.38 THz. The multiplier generates harmonics of a fundamental frequency between 110 and 240 GHz. The key element of the multiplier is a newly developed planar Schottky diode. Harmonic power generation is optimized with the aid of three tuning elements. The performance of the multiplier was checked spectroscopically.

The reactions of titanium(IV) halogen compounds with the monoalkali and dialkali metal salts of L (+)-ascorbic acid and 5,6-O-isopropylidene- L -ascorbic acid, respectively, in EtOH or THF as solvent, generally result in the formation of complexes of the appropriate dianions. Whereas the ascorbate dianion exclusively forms an ate-complex, this is not observed in the case of the 5,6,O-isopropylidene- L -ascorbate dianion. The results of our spectroscopic studies show the ene-diolate group to be the coordination site for both of the dianions. However, for the 5,6-O-isopropylidene- L -ascorbate dianion an additional weak interaction between one of the O atoms of its dioxolane ring and Ti(IV) cannot be excluded.

(Microwave spectra of the four isotopologue/isotopomers, HI-(12)C(16)O(2), HI-(12)C(18)O(2), HI-(12)C(18)O(16)O, and HI-(12)C(16)O(18)O, have been recorded using pulsed-nozzle Fourier transform microwave spectroscopy. In the last two isotopomers, the heavy oxygen atom tilted toward and away from the HI moiety, respectively. Only b-type Ka = 1

Polynuclear chromium ascorbate complexes were isolated and physicochemically analyzed in a comparative manner with their mononuclear analog (1). Characterization by elemental analysis, electronic, vibrational, 13C-n.m.r and mass spectroscopies, and variable temperature magnetic susceptibility studies, allowed structural proposals for the binuclear, [Cr2(μ-OH)2(H2O)(C6H7O6)3(OH)] · 4H2O (2), and trinuclear, [Cr3(μ-O)3(H2O)6(C6H7O6)3] · 4 H2O (3) complexes. The pseudo-octahedral Cr III centers were suggested to be connected through hydroxo bridges in (2) and in (3) by oxo bridges forming a hexocyclic ring.

Die Reaktion des Titelkomplexes (I) mit einem grosen Uberschus an Pyridin fuhrt zum Pyridin-Derivat (IIa), das bei langerem Stehen oder THF-Extraktion einen Pyridin-Liganden unter reversibler Bildung des funffach-koordinierten Komplexes (IIb) abspaltet.