The conserved region (Fc) of IgG antibodies dictates the interactions with designated receptors thus defining the immunological effector functions of IgG. Amino acid sequence variations in the Fc,... Show moreThe conserved region (Fc) of IgG antibodies dictates the interactions with designated receptors thus defining the immunological effector functions of IgG. Amino acid sequence variations in the Fc, recognized as subclasses and allotypes, as well as post-translational modifications (PTMs) modulate these interactions. Yet, the high similarity of Fc sequences hinders allotype-specific PTM analysis by state-of-the-art bottom-up methods and current subunit approaches lack sensitivity and face co-elution of near-isobaric allotypes.To circumvent these shortcomings, we present a nanoscale reversed-phase (RP) HPLC-MS workflow of intact Fc subunits for comprehensive characterization of Fc proteoforms in an allotype- and subclass-specific manner. Polyclonal IgGs were purified from individuals followed by enzymatic digestion releasing single chain Fc subunits (Fc/2) that were directly subjected to analysis. Chromatographic conditions were optimized to separate Fc/2 subunits of near-isobaric allotypes and subclasses allowing allotype and proteoform identification and quantification across all four IgG subclasses. The workflow was complemented by a semi-automated data analysis pipeline based on the open-source software Skyline followed by post-processing in R. The approach revealed pronounced differences in Fc glycosylation between donors, besides inter-subclass and inter-allotype variability within donors. Notably, partial occupancy of the N-glycosylation site in the CH3 domain of IgG3 was observed that is generally neglected by established approaches. The described method was benchmarked across several hundred runs and showed good precision and robustness.This methodology represents a first mature Fc subunit profiling approach allowing truly subclass- and allotype-specific Fc proteoform characterization beyond established approaches. The comprehensive information obtained paired with the high sensitivity provided by the miniaturization of the approach guarantees applicability to a broad range of research questions including clinically relevant (auto)antibody characterization or pharmacokinetics assessment of therapeutic IgGs. Show less
Marco, F. di; Blümel, G.; Blöchl, C.; Wuhrer, M.; Huber, C.G. 2023
BackgroundGonadotropins are a class of heavily glycosylated protein hormones, thus extremely challenging to characterize by mass spectrometry. As biopharmaceuticals, gonadotropins are prescribed... Show moreBackgroundGonadotropins are a class of heavily glycosylated protein hormones, thus extremely challenging to characterize by mass spectrometry. As biopharmaceuticals, gonadotropins are prescribed for the treatment of infertility and are derived from different sources: either from pooled urine of pregnant women or upon production in genetically modified Chinese Hamster Ovary cells. Human chorionic gonadotropin (hCG) is sold as a biopharmaceutical under the name Pregnyl® (urinary hCG, u-hCG) and Ovitrelle® (recombinant hCG, r-hCG), and recombinant human follicle stimulating hormone (r-hFSH) is marketed as Gonal-f®. Recently, we reported the exhaustive characterization of r-hCG at different structural levels.ResultsWe implement size exclusion (SE) HPLC-MS to automatize the acquisition of native mass spectra of r-hCG dimer, but also u-hCG and r-hFSH, comparing the drug products up to intact heterodimer level. A hybrid HPLC-MS approach was employed for the characterization of r-hCG, u-hCG and r-hFSH drug products at different structural levels. Released glycans were analyzed by porous graphitized carbon (PGC)-HPLC-MS/MS, glycopeptides by reversed-phase (RP)-HPLC-MS/MS, subunits by RP-HPLC-MS and finally the intact native heterodimers by semi-automated online buffer exchange SE-HPLC-MS. The data were integrated using bioinformatic tools, to finally unravel the composition of 1481 co-existing dimeric glycoforms for r-hCG, 1167 glycoforms for u-hCG, and 1440 glycoforms for r-hFSH, and to compare critical quality attributes of the different drug products such as their degree of sialylation and O-glycosylation.Significance and noveltyThe strong alliance of bioanalytics and bioinformatics data integration at the different structural levels allowed the identification of more than thousand different glycoforms of r-hCG, u-hCG, and r-hFSH. The results showed that these biopharmaceuticals differ considerably in their glycosylation patterns and highlight the importance of in-depth characterization of biopharmaceuticals for quality control. Show less
Schaick, G. van; Wuhrer, M.; Domínguez-Vega, E. 2023
Proteins carry a plethora of post-translational modifications (PTMs), such as glycosylation or phosphorylation, which may affect stability and activity. Analytical strategies are needed to... Show moreProteins carry a plethora of post-translational modifications (PTMs), such as glycosylation or phosphorylation, which may affect stability and activity. Analytical strategies are needed to investigate these PTMs in their native state to determine the link between structure and function. The coupling of native separation techniques with mass spectrometry (MS) has emerged as a powerful tool for in-depth protein characterization. Yet obtaining high ionization efficiency still can be challenging. Here, we explored the potential of dopant-enriched nitrogen (DEN) gas to improve nano-electrospray ionization (nano-ESI)-MS of native proteins after anion exchange chromatography. The dopant gas was enriched with different dopants (acetonitrile, methanol, and isopropanol) and the effects were compared with the use of solely nitrogen gas for six proteins covering a wide range of physicochemical properties. The use of DEN gas resulted generally in lower charge states, independent of the selected dopant. Moreover, less adduct formation was observed, particularly for the acetonitrile-enriched nitrogen gas. Importantly, striking differences in MS signal intensity and spectral quality were observed for extensively glycosylated proteins, where isopropanol- and methanol-enriched nitrogen appeared to be most beneficial. Altogether, the use of DEN gas improved nano-ESI of native glycoproteins and increased spectral quality for highly glycosylated proteins that normally suffer from low ionization efficiency. Show less
Sample preparation is a labor-intensive and time-consuming procedure, especially for the bioanalysis of small-volume samples with low-abundant analytes. To minimize losses and dilution, sample... Show moreSample preparation is a labor-intensive and time-consuming procedure, especially for the bioanalysis of small-volume samples with low-abundant analytes. To minimize losses and dilution, sample preparation should ideally be hyphenated to downstream on-line analysis such as liquid chromatography-mass spectrometry (LC-MS). In this study, an automated three-phase electro-extraction (EE) method coupled to machine vision was developed, integrated with a robotic autosampler hyphenated to LC-MS. Eight model compounds, i.e. amitriptyline, clemastine, clomipramine, haloperidol, loperamide, propranolol, oxeladin, and verapamil were utilized for the optimization and evaluation of the automated EE setup. The stability of automated EE was evaluated by monitoring the acceptor droplet size by machine vision and recording the current during EE. A Design of Experiment approach (Box-Behnken design) was utilized to optimize the critical parameters of the EE method, i. e., the ratio of formic acid in the sample to acceptor phase, extraction voltage, and extraction time. The developed quadratic models showed good fitness (p < 0.001, R-2 > 0.95). Automated EE could be achieved in less than 2 min with enrichment factors (EF) up to 387 and extraction recoveries (ER) up to 97% for academic samples. Finally, the optimized EE method was successfully applied to both spiked human urine and plasma samples with low-concentration (50 ng mL(-1)) analytes and a low starting sample volume of 20 mu L of plasma and urine in 10-fold diluted samples. The developed automated EE setup is easy to operate, provides a fast extraction method for analytes from volume-limited biological samples, and is hyphenated with on-line LC-MS analysis. Therefore, this method can provide fast and automated sample preparation to solve bottlenecks in high-throughput bioanalysis workflows. Show less
He, B.S.; X.Y. di; Guled, F.; Harder, A.V.E.; Maagdenberg, A.M.J.M. van den; Terwindt, G.M.; ... ; Hankemeier, T. 2022
The endocannabinoid system (ECS) is implicated in various brain disorders. Changes in the composition of the cerebrospinal fluid (CSF) may be associated with ECS-related pathologies.... Show moreThe endocannabinoid system (ECS) is implicated in various brain disorders. Changes in the composition of the cerebrospinal fluid (CSF) may be associated with ECS-related pathologies. Endocannabinoids (eCBs) and their analogues are present at low concentrations in human CSF, which hampered the investigation of the ECS in this body fluid. In this study, we developed a highly sensitive and selective micro-flow liquid chromatographytandem mass spectrometry (micro-LC-MS/MS) method for the analysis of eCBs and eCB analogues in human CSF. The developed method allowed for the quantitative analysis of 16 eCBs and their analogues in human CSF. Micro-LC-MS/MS analyses were performed at a flow-rate of 4 mu L min(-1) with a 0.3-mm inner diameter column. A minor modification of a novel spray needle was carried out to improve the robustness of our method. By using an injection volume of 3 mu L, our method reached limits of detection in the range from 0.6 to 1293.4 pM and limits of quantification in range from 2.0 to 4311.3 pM while intra- and interday precisions were below 13.7%. The developed workflow was successfully used for the determination of eCBs in 288 human CSF samples. It is anticipated that the proposed approach will contribute to a deeper understanding of the role of ECS in various brain disorders. Show less
Sample preparation is a challenge for high-throughput analysis, especially for volume-limited samples with low-abundant analytes. Ideally, sample preparation enriches the analytes of interest while... Show moreSample preparation is a challenge for high-throughput analysis, especially for volume-limited samples with low-abundant analytes. Ideally, sample preparation enriches the analytes of interest while removing the interferents to reduce the matrix effect and improve both sensitivity and quantification. In this study, a three-phase electroextraction (EE) method hyphenated to fast online liquid chromatography -mass spectrometry (LC-MS) was developed. Four model acidic drugs of relevance for drug monitoring in plasma, i.e. naproxen, fenoprofen, flurbiprofen, and ibuprofen, were utilized for the optimization and evaluation of the method. A Design of Experiment approach (Box-Behnken design) was used to optimize the critical parameters of the method, i.e., the type of organic solvent, pH of the sample and acceptor phase, and the extraction voltage and time. Good fitness (P < 0.02, R2 > 0.95) was observed for the developed quadratic model. Extraction could be achieved in less than 2 min (115 s) with enrichment factors (EF) up to 190 and extraction recoveries (ER) up to 38% for academic samples. Additionally, the optimized three-phase EE method was successfully applied to spiked plasma samples with lowabundant (50 ng mL-1) analytes and a low sample volume of 15 mL plasma in 10-fold diluted samples. Finally, two crucial contributors to the matrix effect of three-phase EE application on plasma samples were determined. Specifically, the ion-suppression effect in the MS source was reduced by the fast LC separation, and the matrix effect during extraction was negligible for the diluted protein-precipitated plasma samples. The developed three-phase EE method is easy to operate and provides fast and online extraction of trace-level acidic analytes from volume-limited biological samples. Therefore, this method can provide a potential solution for sample-preparation bottlenecks in high-throughput bioanalysis workflows. (c) 2021 Published by Elsevier B.V. Show less
Lippold, S.; Thavarajah, R.; Reusch, D.; Wuhrer, M.; Nicolardi, S. 2021
Recombinant human erythropoietin (EPO) is a complex therapeutic glycoprotein with three N- and one O-glycosylation sites. Glycosylation of EPO influences its safety and efficacy and is defined as a... Show moreRecombinant human erythropoietin (EPO) is a complex therapeutic glycoprotein with three N- and one O-glycosylation sites. Glycosylation of EPO influences its safety and efficacy and is defined as a critical quality attribute. Thus, analytical methods for profiling EPO glycosylation are highly demanded. Owing to the complexity of the intact protein, information about EPO glycosylation is commonly derived from released glycan and glycopeptide analysis using mass spectrometry (MS). Alternatively, comprehensive insights into the glycoform heterogeneity of intact EPO are obtained using ESI MS-based methods with or without upfront separation of EPO glycoforms. MALDI MS, typically performed with TOF mass analyzers, has been also used for the analysis of intact EPO but, due to the poor glycoform resolution, has only provided limited glycoform information. Here, we present a MALDI FT-ICR MS method for the glycosylation profiling of intact EPO with improved glycoform resolution and without loss of sialic acid residues commonly observed in MALDI analysis. Three EPO variants were characterized in-depth and up to 199 glycoform compositions were assigned from the evaluation of doubly-charged ions, without any deconvolution of the mass spectra. Key glycosylation features such as sialylation, acetylation, and N- acetyllactosamine repeats were determined and found to agree with previously reported data obtained from orthogonal analyses. The developed method allowed for a fast and straightforward data acquisition and evaluation and can be potentially used for the high-throughput comparison of EPO samples throughout its manufacturing process. (c) 2021 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Show less
Pot, S.; Gstottner, C.; Heinrich, K.; Hoelterhoff, S.; Grunert, I.; Leiss, M.; ... ; Dominguez Vega, E. 2021
Characterization of post-translational modifications (PTMs) of therapeutic antibodies is commonly performed by bottom-up approaches, involving sample preparation and peptide analysis by liquid... Show moreCharacterization of post-translational modifications (PTMs) of therapeutic antibodies is commonly performed by bottom-up approaches, involving sample preparation and peptide analysis by liquid chromatography-mass spectrometry (LC-MS). Conventional sample preparation requires extensive hands-on time and can increase the risk of inducing artificial modifications as many off-line steps - denaturation, disulfide-reduction, alkylation and tryptic digestion - are performed. In this study, we developed an on-line multidimensional (mD)-LC-MS bottom-up approach for fast sample preparation and analysis of (formulated) monoclonal antibodies and antibody-derived therapeutics. This approach allows on-column reduction, tryptic digestion and subsequent peptide analysis by RP-MS. Optimization of the 1D -and 2D flow and temperature improved the trapping of small polar peptides during on-line peptide mapping analysis. These adaptations increased the sequence coverage (95-98% versus 86-94% for off-line approaches) and allowed identification of various PTMs (i.e. deamidation of asparagine, methionine oxidation and lysine glycation) within a single analysis. This workflow enables a fast (<2 h) characterization of antibody heterogeneities within a single run and a low amount of protein (10 mu g). Importantly, the new mD-LC-MS bottom-up method was able to detect the polar, fast-eluting peptides: Fc oxidation at Hc-Met-252 and the Fc N-glycosylation at Hc-Asn-297, which can be challenging using mD-LC-MS. Moreover, the method showed good comparability across the different measurements (RSD of retention time in the range of 0.2-1.8% for polar peptides). The LC system was controlled by only a standard commercial software package which makes implementation for fast characterization of quality attributes relatively easy. (C) 2021 The Author(s). Published by Elsevier B.V. Show less
Sample preparation is often reported as the main bottleneck of analytical processes. To meet the requirements of both high-throughput and high sensitivity, improved sample-preparation methods... Show moreSample preparation is often reported as the main bottleneck of analytical processes. To meet the requirements of both high-throughput and high sensitivity, improved sample-preparation methods capable of fast analyte preconcentration are urgently needed. To this end, a new three-phase electroextraction (EE) method is presented that allows for ultrafast electroextraction hyphenated to flow-injection analysis mass spectrometry (FIA-MS). Four model compounds, i.e., propranolol, amitriptyline, bupivacaine, and oxeladin, were used to optimize and evaluate the method. Within only 30 s extraction time, enrichment factors (EF) of 105-569 and extraction recoveries (ER) of 10.2%-55.7% were achieved for these analytes, with limits of detection (LODs) ranging from 0.36 to 3.21 ng mL(-1), good linear response function (R-2 > 0.99), low relative standard deviation (0.6%-17.8%) and acceptable accuracy (73-112%). Finally, the optimized three-phase EE method was successfully applied to human urine and plasma samples. Our three-phase electroextraction method is simple to construct and offers ultrafast, online extraction of trace amounts of analytes from biological samples, and therefore has great potential for high-throughput analysis. (C) 2021 The Authors. Published by Elsevier B.V. Show less
Sample preparation is often reported as the main bottleneck of analytical processes. To meet the requirements of both high-throughput and high sensitivity, improved sample-preparation methods... Show moreSample preparation is often reported as the main bottleneck of analytical processes. To meet the requirements of both high-throughput and high sensitivity, improved sample-preparation methods capable of fast analyte preconcentration are urgently needed. To this end, a new three-phase electroextraction (EE) method is presented that allows for ultrafast electroextraction hyphenated to flow-injection analysis mass spectrometry (FIA-MS). Four model compounds, i.e., propranolol, amitriptyline, bupivacaine, and oxeladin, were used to optimize and evaluate the method. Within only 30 s extraction time, enrichment factors (EF) of 105-569 and extraction recoveries (ER) of 10.2%-55.7% were achieved for these analytes, with limits of detection (LODs) ranging from 0.36 to 3.21 ng mL(-1), good linear response function (R-2 > 0.99), low relative standard deviation (0.6%-17.8%) and acceptable accuracy (73-112%). Finally, the optimized three-phase EE method was successfully applied to human urine and plasma samples. Our three-phase electroextraction method is simple to construct and offers ultrafast, online extraction of trace amounts of analytes from biological samples, and therefore has great potential for high-throughput analysis. (C) 2021 The Authors. Published by Elsevier B.V. Show less
Schaick, G. van; Gstottner, C.; Buttner, A.; Reusch, D.; Wuhrer, M.; Dominguez Vega, E. 2021
Assessment of critical quality attributes of the biopharmaceutical erythropoietin (EPO) prior to product release requires the use of several analytical methods. We developed an MS-compatible anion... Show moreAssessment of critical quality attributes of the biopharmaceutical erythropoietin (EPO) prior to product release requires the use of several analytical methods. We developed an MS-compatible anion exchange (AEX) method for monitoring multiple quality attributes of EPO biopharmaceuticals. AEX was performed using a stationary phase with quaternary ammonium functional groups and a pH gradient for elution. Baseline separation of charge variants and high-quality MS data were achieved using 30 mM ammonium formate pH 5.5 and 30 mM formic acid pH 2.5 as mobile phases. In a single experiment, assessment of critical quality attributes, such as charge heterogeneity, sialic acid content and number of N-ace-tyllactosamine units, was possible while providing additional information on other modifications such as O-acetylation and deamidation. In addition, good repeatability and robustness for the relative areas of the individual glycoforms and average number of Neu5Ac per EPO molecule were observed. The results were comparable to common pharmacopeia and standard methods with the advantage of requiring fewer analytical methods and less sample treatment saving time and costs. (C) 2020 The Authors. Published by Elsevier B.V. Show less
Laan, T. van der; Elfrink, H.L.; Azadi-Chegeni, F.; Dubbelman, A.C.; Harms, A.C.; Jacobs, D.M.; ... ; Hankemeier, T. 2020
The unambiguous identification of unknown compounds is of utmost importance in the field of metabolomics. However, current identification workflows often suffer from error-sensitive methodologies,... Show moreThe unambiguous identification of unknown compounds is of utmost importance in the field of metabolomics. However, current identification workflows often suffer from error-sensitive methodologies, which may lead to incorrect structure annotations of small molecules. Therefore, we have developed a comprehensive identification workflow including two highly complementary techniques, i.e. liquid chromatography (LC) combined with mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR), and used it to identify five taste-related retention time and m/z features in soy sauce. An off-line directed two-dimensional separation was performed in order to purify the features prior to the identification. Fractions collected during the first dimension separation (reversed phase low pH) were evaluated for the presence of remaining impurities next to the features of interest. Based on the separation between the feature and impurities, the most orthogonal second dimension chromatography (hydrophilic interaction chromatography or reversed phase high pH) was selected for further purification. Unknown compounds down to tens of micromolar concentrations were tentatively annotated by MS and structurally confirmed by MS and NMR. The mass (0.4-4.2 mu g) and purity of the isolated compounds were sufficient for the acquisition of one and two-dimensional NMR spectra. The use of a directed two-dimensional chromatography allowed for a fractionation that was tailored to each feature and remaining impurities. This makes the fractionation more widely applicable to different sample matrices than one-dimensional or fixed two-dimensional chromatography. Five proline-based 2,5-diketopiperazines were successfully identified in soy sauce. These cyclic dipeptides might contribute to taste by giving a bitter flavour or indirectly enhancing umami flavour. (C) 2020 The Author(s). Published by Elsevier B.V. Show less
Gstottner, C.; Nicolardi, S.; Haberger, M.; Reusch, D.; Wuhrer, M.; Dominguez Vega, E. 2020
Bispecific antibodies (BsAb) are next-generation, antibody-based pharmaceuticals which come with a great functional versatility and often a vast structural heterogeneity. Although engineering of... Show moreBispecific antibodies (BsAb) are next-generation, antibody-based pharmaceuticals which come with a great functional versatility and often a vast structural heterogeneity. Although engineering of the primary sequence of BsAbs guides the proper pairing of the different chains, several side products can often be observed contributing to the macroheterogeneity of these products. Furthermore, changes in the amino acid sequence can result in different protein modifications which can affect the properties of the antibody and further increase the structural complexity. A multi-methods approach can be used for the characterization of their heterogeneity but new analytical strategies are needed for a more accurate and in-depth analysis.Here, we present a combination of intact antibody and subunit-specific mass measurements using sheathless capillary electrophoresis-mass spectrometry for assessing the macro- and microheterogeneity of BsAbs. Two homologous BsAbs with the same bispecificity but slightly different amino acid sequences were analyzed. Intact measurements were performed using a positively coated capillary and a background electrolyte (BGE) consisting of 3% acetic acid. For intact BsAbs, the separation permitted the characterization of free light chains, homo- and heterodimers as well as incomplete assemblies. For subunit-specific measurements, BsAbs were hinge region cleaved using two different enzymes (SpeB and IdeS) followed by disulfide-bond reduction. The six different subunits (Lc1, Lc2, Fd'1, Fd'2, (Fc/2)1 and (Fc/2)2) were separated using the same positively-coated capillary and a BGE consisting of 20% acetic acid and 10% methanol. Mass measurements of hinge region cleaved antibodies were performed at isotopic resolution (resolving power 140000 at m/z 1100) for a more confident analysis of low abundance proteoforms. For both BsAbs several proteoforms with e.g. pyroglutamic acid (Pyro-Glu) or glycation which could not be properly assigned at the intact level, were accurately determined in the subunits showing the complementarity of both approaches. (C) 2020 Elsevier B.V. All rights reserved. Show less
Gargano, A.F.G.; Schouten, O.; Schaick, G. van; Roca, L.S.; Berg-Verleg, J.H. van den; Haselberg, R.; ... ; Somsen, G.W. 2020
Many industrial enzymes exhibit macro- and micro-heterogeneity due to co-occurring post-translational modifications. The resulting proteoforms may have different activity and stability and,... Show moreMany industrial enzymes exhibit macro- and micro-heterogeneity due to co-occurring post-translational modifications. The resulting proteoforms may have different activity and stability and, therefore, the characterization of their distributions is of interest in the development and monitoring of enzyme products. Protein glycosylation may play a critical role as it can influence the expression, physical and biochemical properties of an enzyme.We report the use of hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) to profile intact glycoform distributions of high mannose-type N-glycosylated proteins, using an industrially produced fungal lipase for the food industry as an example. We compared these results with conventional reversed phase LC-MS (RPLC-MS) and sodium dodecyl sulfate-polyacrylamide gel-electrophoresis (SDS-PAGE). HILIC appeared superior in resolving lipase heterogeneity, facilitating mass assignment of N-glycoforms and sequence variants. In order to understand the glycoform selectivity provided by HILIC, fractions from the four main HILIC elution bands for lipase were taken and subjected to SDS-PAGE and bottom-up proteomic analysis. These analyses enabled the identification of the most abundant glycosylation sites present in each fraction and corroborated the capacity of HILIC to separate protein glycoforms based on the number of glycosylation sites occupied.Compared to RPLC-MS, HILIC-MS reducted the sample complexity delivered to the mass spectrometer, facilitating the assignment of the masses of glycoforms and sequence variants as well as increasing the number of glycoforms detected (69 more proteoforms, 177% increase). The HILIC-MS method required relatively short analysis time (<30 min), in which over 100 glycoforms were distinguished.We suggest that HILIC(-MS) can be a valuable tool in characterizing bioengineering processes aimed at steering protein glycoform expression as well as to check the consistency of product batches. (C) 2020 Elsevier B.V. All rights reserved. Show less
Acker, T. van; Buckle, T.; Malderen, S.J.M. van; Willigen, D.M. van; Unen, V. van; Leeuwen, F.W.B. van; Vanhaecke, F. 2019
Over the past few years, loss of patent protection for blockbuster monoclonal antibody (mAb) drugs has caused a significant shift in the pharmaceutical industry towards the development of... Show moreOver the past few years, loss of patent protection for blockbuster monoclonal antibody (mAb) drugs has caused a significant shift in the pharmaceutical industry towards the development of biosimilar products. As a result, multiple biosimilar mAbs are becoming available for a single originator drug. As opposed to small-molecular drugs, protein biopharmaceuticals do not have fully defined and reproducible structures, making it impossible to create identical copies. Therefore, regulators demand biosimilar sponsors to demonstrate similarity with the reference product to prevent safety and efficacy issues with the proposed product. Protein glycosylation is considered a crucially important quality attribute, because of its major role in immunogenicity and clinical efficacy of therapeutic proteins. However, the intrinsic biological variability of glycan structures creates a significant challenge for the current analytical platforms. In this review, we discuss the importance of glycan characterization on therapeutic proteins, with a particular focus on the analytical techniques applied for glycan profiling of biosimilar mAb products. In addition, we present a case study on infliximab biosimilars to illustrate the potential clinical implications of differences in glycan profile between originator and biosimilar mAb products. Show less
Antibody-based pharmaceuticals often encompass a complex structural heterogeneity requiring enhanced analytical methods for reliable characterization of variants and degradation products. We have... Show moreAntibody-based pharmaceuticals often encompass a complex structural heterogeneity requiring enhanced analytical methods for reliable characterization of variants and degradation products. We have explored the capabilities of low-flow sheathless capillary electrophoresis-mass spectrometry (CE-MS) for the high-resolution and sensitive profiling of antibody therapeutics. Near-zero electroosmotic flow was achieved by employing a novel neutral capillary coating that also prevents protein adsorption. CE-MS analysis of intact model proteins using an acidic background electrolyte demonstrated satisfactory performance, with overall migration-time RSDs below 2.2% from three different capillaries tested. For system evaluation, three nanobody preparations, including mono- and bivalent forms, and three monoclonal antibodies (mAbs) were analyzed. Intact nanobodies were resolved from their degradation products, which could be assigned to deamidated, cleaved, and truncated forms at the C-terminal tag. Excellent resolution of isomeric deamidated products was obtained. The mAbs were analyzed intact and after digestion by the endoproteinase IdeS (middle-up approach). CE-MS of intact mAbs provided resolution of clipped species (e.g. light chain and light chain-heavy chain fragments) from the native protein. Moreover, glycoforms containing sialic acids were resolved from their non-sialylated counterparts. For IdeS-digested, F (ab)(2) and Fc/2 portions where efficiently resolved for the three mAbs. Whereas the migration time of the Fc/2 fragments was fairly similar, the migration time of the F (ab)(2) part was strongly varied among the mAbs. For all mAbs, separation of Fc/2 charge variants - including sialylated glycoforms and other post-translational modifications, such as loss of C-terminal lysine or asparagine deamidation - was achieved. This allowed a detailed and reliable assessment of the Fc/2 heterogeneity (18-33 proteoforms) of the three analyzed mAbs. (C) 2018 The Authors. Published by Elsevier B.V. Show less
Giera, M.; Kaisar, M.M.M.; Derks, R.J.E.; Steenvoorden, E.; Kruize, Y.C.M.; Hokke, C.H.; ... ; Everts, B. 2018
