97 results on '"Haselberg R"'
Search Results
2. Hydrophilic interaction liquid chromatography-mass spectrometry for the characterization of glycoproteins at the glycan, peptide, subunit, and intact level
- Author
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Gargano, A.F.G., Haselberg, R., Somsen, G.W., El Rassi, Z., Supramolecular Separations (HIMS, FNWI), BioAnalytical Chemistry, AIMMS, and El Rassi, Ziad
- Subjects
chemistry.chemical_classification ,Glycans ,Glycan ,Glycosylation ,Chromatography ,Hydrophilic interaction liquid chromatography ,Mass spectrometry ,biology ,Chemistry ,Protein subunit ,Hydrophilic interaction chromatography ,Glycopeptides ,Peptide ,Context (language use) ,chemistry.chemical_compound ,HILIC-MS ,biology.protein ,Glycoprotein ,Glycoproteins - Abstract
This chapter presents a methodical overview of recent research literature dealing with the characterization of glycoproteins by hydrophilic interaction chromatography separations coupled to mass spectrometry (HILIC-MS). This includes the analysis of intact glycosylated proteins as well as released glycans, glycopeptides, and protein subunits obtained after enzymatic digestion. The chapter provides basic background information on HILIC and glycoproteins, and the molecular levels at which glycosylation can be characterized. Next, essential HILIC parameters such as (the choice of) stationary phase type, mobile phase conditions, injection volume, and column temperature are reviewed. Herein focus is on the effect that these parameters have on both HILIC separation and MS detection. This is discussed in the context of the respective levels (glycan/peptide/subunit/intact) of glycoprotein analysis and current trends are highlighted. In the final part of this chapter, these developments are put in perspective by treating several applications of HILIC-MS for glycoprotein analysis in more detail, particularly in biopharmaceutical and clinical fields.
- Published
- 2021
3. Capillary electrophoresis-mass spectrometry using noncovalently coated capillaries for the analysis of biopharmaceuticals
- Author
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Haselberg, R., Brinks, V., Hawe, A., de Jong, G. J., and Somsen, G. W.
- Published
- 2011
- Full Text
- View/download PDF
4. Profiling of a high mannose-type N-glycosylated lipase using hydrophilic interaction chromatography-mass spectrometry
- Author
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Gargano, A. F.G., Schouten, O., van Schaick, G., Roca, L. S., van den Berg-Verleg, J. H., Haselberg, R., Akeroyd, M., Abello, N., Somsen, G. W., Gargano, A. F.G., Schouten, O., van Schaick, G., Roca, L. S., van den Berg-Verleg, J. H., Haselberg, R., Akeroyd, M., Abello, N., and Somsen, G. W.
- Abstract
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, 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
- Published
- 2020
- Full Text
- View/download PDF
5. Profiling of a high mannose-type N-glycosylated lipase using hydrophilic interaction chromatography-mass spectrometry
- Author
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Gargano, A.F.G., primary, Schouten, O., additional, van Schaick, G., additional, Roca, L.S., additional, van den Berg-Verleg, J.H., additional, Haselberg, R., additional, Akeroyd, M., additional, Abello, N., additional, and Somsen, G.W., additional
- Published
- 2020
- Full Text
- View/download PDF
6. Clinical Metabolomics: Expanding the Metabolome Coverage Using Advanced Analytical Techniques
- Author
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Haselberg, R., Pirok, B.W.J., Gargano, A.F.G., Kohler, I., and Supramolecular Separations (HIMS, FNWI)
- Abstract
Leiden Academic Center for Drug Research, Leiden University, Division of Systems Biomedicine and Pharmacology, Leiden, The Netherlands Metabolomics, the comprehensive analysis of all metabolites and intermediate products of reactions present within a biological system, is a promising field to enable precision medicine. Clinical metabolomics faces two main challenges at the bioanalytical level. The first is the need for high resolution to obtain maximum metabolome coverage. This is exemplified by the latest version of the Human Metabolome Database (HMDB), which reports more than 110,000 metabolites and endogenous compounds. The second is the high-throughput needed to enable the analysis of a large number of samples typically encountered in large-scale cohort studies. Reversed-phase liquid chromatography (LC)--at regular or ultrahigh pressures--combined with high-resolution mass spectrometry (HRMS) has long been considered the "gold standard" in metabolomics. However, these conventional reversed-phase LC-MS approaches are no longer sufficient to analyze the vast variety of polar compounds, as well as discriminate closely related compounds such as isomers or enantiomers. This review article discusses the novel separation and detection strategies that are considered promising in clinical metabolomics to enhance the metabolome coverage. It includes hydrophilic interaction chromatography (HILIC), supercritical fluid chromatography (SFC), multidimensional LC approaches, as well as ion-mobility mass spectrometry (IM-MS) and data-independent acquisition (DIA) analysis methods.
- Published
- 2019
7. Middle-Up Characterization of the Monoclonal Antibody Infliximab by Capillary Zone Electrophoresis-Mass Spectrometry
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Michalikova, K., Dominguez-Vega, E., Somsen, G.W., and Haselberg, R.
- Abstract
Capillary zone electrophoresis-electrospray ionization time-of-flight mass spectrometry (CZE-ESI-TOF-MS) for the characterization of the monoclonal antibody (mAb) infliximab and its variants is presented. Infliximab was analyzed using a middle-up approach involving either reduction or digestion with the enzyme IdeS. A multilayer capillary coating of Polybrene-dextran sulfate-Polybrene (PB-DS-PB) in combination with a background electrolyte of 40% acetic acid provided efficient separation of the obtained antibody fragments, that is, light chain (LC) and heavy chain (HC), as well as F(ab')2 and Fc/2 parts. C-terminal lysine variants were also resolved. Recorded mass spectra of HC and Fc/2 fragments permitted assignment of 13 glycoforms and provided a quantitative profile, with GOF the most abundant glycoform (similar to 50%). CZE-ESI-TOF-MS represents an efficient means for the straightforward analysis of a monoclonal antibody and its proteoforms.
- Published
- 2019
8. Heterogeneity assessment of antibody-derived therapeutics at the intact and middle-up level by low-flow sheathless capillary electrophoresis-mass spectrometry
- Author
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Haselberg, R., Vijlder, T. de, Heukers, R., Smit, M.J., Romijn, E.P., Somsen, G.W., and Dominguez-Vega, E.
- Subjects
Low-flow CE ,Sheathless CE-MS ,Intact protein analysis ,Nanobodies ,Middle-up analysis ,Monoclonal antibodies - Abstract
Antibody-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.
- Published
- 2018
9. Evanescent-Wave Cavity Enhanced Spectroscopy as a Tool in Label-Free Biosensing
- Author
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van der Sneppen, L, Ritchie, G, Hancock, G, Ariese, F, Gooijer, C, Ubachs, W, Haselberg, R, Somsen, G, de Jong, G, and IEEE
- Abstract
A variety of evanescent-wave cavity-enhanced techniques is used in studying interfacial kinetics as well as the performance of anti-biofouling coatings, demonstrating the potential of these techniques in label-free biosensing. © 2010 OSA.
- Published
- 2016
10. Intact protein analysis by capillary electrophoresis - mass spectrometry
- Author
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Haselberg, R., Somsen, G.W., De Jong, G.J., BioAnalytical Chemistry, and AIMMS
- Published
- 2016
11. Developments in interfacing designs for CE–MS: towards enabling tools for proteomics and metabolomics
- Author
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Lindenburg, P.W., Haselberg, R., Rozing, G., Ramautar, R., Lindenburg, P.W., Haselberg, R., Rozing, G., and Ramautar, R.
- Abstract
Capillary electrophoresis–mass spectrometry (CE–MS) can be considered a useful analytical technique for the analysis of charged compounds in the fields of proteomics and metabolomics. Currently, the commercially available co-axial sheath–liquid interface is generally employed for coupling CE to MS in most application areas. Although it has proven to be rather robust for various proteomics, glycomics and metabolomics studies, the intrinsically low-flow separation property of CE is not effectively utilized in this set-up. In this type of interfacing the sheath–liquid (typical flow-rate between 1 and 10 µL/min) dilutes the CE effluent (flow-rate between 20 and 100 nL/min), thereby reducing the detection sensitivity. Over the past few years some significant developments that aim to overcome this limitation have been made in interfacing techniques for CE–MS, which resulted in an increased interest of CE–MS for proteomics and metabolomics. This paper provides an overview of these developments and the utility of CE–MS employing the new interfacing techniques is demonstrated by representative examples in the fields of proteomics, glycomics and metabolomics. Finally, general conclusions and perspectives are provided.
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- 2015
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12. Quality of Original and Biosimilar Epoetin Products
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Brinks, V., Hawe, A., Basmeleh, A.H.H., Joachin-Rodriguez, L., Haselberg, R., Somsen, G.W., Jiskoot, W., Schellekens, H., Advanced drug delivery systems/drug targeting, Biomolecular Analysis, Innovation Studies, Farmaceutische Analyse, Pharmaceutics, Sub Biotechnological drugs, Sub Biomolecular analysis, Dep Farmaceutische wetenschappen, BioAnalytical Chemistry, AIMMS, Advanced drug delivery systems/drug targeting, Biomolecular Analysis, Innovation Studies, Farmaceutische Analyse, Pharmaceutics, Sub Biotechnological drugs, Sub Biomolecular analysis, and Dep Farmaceutische wetenschappen
- Subjects
protein characterization ,Drugmisbruik en verslaving ,Therapeutic equivalency ,media_common.quotation_subject ,Pharmacology toxicology ,Pharmaceutical Science ,Enzyme-Linked Immunosorbent Assay ,Pharmacology ,immunogenicity ,Biomedische technologie en medicijnen ,Farmacie/Biofarmaceutische wetenschappen (FARM) ,Mice ,Pharmaceutical technology ,hemic and lymphatic diseases ,Medical technology ,Animals ,Protein Isoforms ,Medicine ,Potency ,Quality (business) ,Pharmacology (medical) ,Bescherming en bevordering van de menselijke gezondheid ,recombinant human erythropoietin ,Erythropoietin ,media_common ,Biological Products ,business.industry ,Organic Chemistry ,Farmacie(FARM) ,Electrophoresis, Capillary ,Biosimilar ,Recombinant Proteins ,Therapeutic Equivalency ,Chromatography, Gel ,Molecular Medicine ,Electrophoresis, Polyacrylamide Gel ,Public Health ,biosimilar ,business ,Analytical chemistry ,Research Paper ,medicine.drug ,Biotechnology - Abstract
Purpose To compare the quality of therapeutic erythropoietin (EPO) products, including two biosimilars, with respect to content, aggregation, isoform profile and potency. Methods Two original products, Eprex (epoetin alfa) and Dynepo (epoetin delta), and two biosimilar products, Binocrit (epoetin alfa) and Retacrit (epoetin zeta), were compared using (1) high performance size exclusion chromatography, (2) ELISA, (3) SDS-PAGE, (4) capillary zone electrophoresis and (5) in-vivo potency. Results Tested EPO products differed in content, isoform composition, and potency. Conclusion Of the tested products, the biosimilars have the same or even better quality as the originals. Especially, the potency of originals may significantly differ from the value on the label.
- Published
- 2011
13. Capillary electrophoresis – mass spectrometry using noncovalently coated capillaries for the analysis of biopharmaceuticals
- Author
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Haselberg, R., Biomolecular Analysis, Farmaceutische Analyse, Sub Biomolecular analysis, de Jong, Ad, Somsen, Govert, and University Utrecht
- Subjects
Drugmisbruik en verslaving ,Medical technology ,Farmacie(FARM) ,Bescherming en bevordering van de menselijke gezondheid ,Public Health ,Biomedische technologie en medicijnen ,Analytical chemistry - Abstract
With efficient methodologies available in biotechnology today, increasing numbers of recombinantly manufactured pharmaceutical peptides and proteins are being commercialized. The assessment of biopharmaceutical quality in terms of identity, content and purity is an important issue during manufacturing. The biotechnological production process may show variability, which can introduce product diversity, isoforms and closely-related degradation products. Clearly, there is an increasing demand for suitable analytical methods that allow not only protein and peptide identification, but also the separation and quantification of impurities and possible degradation products Capillary electrophoresis-mass spectrometry (CE-MS) provides the high separation efficiency and mass-selective detection that can be very useful for biopharmaceutical product characterization. Many protein modifications, like glycosylation and deamidation, may involve changes of net charge of a protein and, thus, also of its electrophoretic mobility. MS detection of high mass accuracy and resolution, such as provided by time-of-flight (TOF) instruments can yield accurate information on the molecular weight of analyzed proteins species. In this thesis, the potential of CE-TOF-MS for the analysis of biopharmaceuticals has been explored. Emphasis was on development of CE methods for basic proteins, paying particular attention to the prevention of protein adsorption, which deteriorates the separation efficiency. A positively charged Polybrene-dextran sulfate-Polybrene (PB-DS-PB) coating was applied for this purpose. These coatings were prepared by successive flushes with solutions of the charged polymers, leading to formation of electrostatically adsorbed layers. The performance and stability of the triple-layer coating was evaluated for basic proteins analyzed at medium and low pH. The suitability of the triple-layer coating for CE-TOF-MS was studied, paying attention to compatibility. CE-MS coupling was achieved via a sheath-liquid interface, and optimum interfacing conditions were investigated. A disadvantage of this type of interfacing is that the sheath liquid dilutes the CE effluent leading to reduced detection sensitivity. In order to avoid this drawback, the performance of a prototype sheathless interface for intact protein analysis by CE-MS was investigated and compared to sheath-liquid CE-MS. The potential of the developed CE-TOF-MS systems was evaluated by analyzing various pharmaceutical proteins, addressing issues such as purity, stability, heterogeneity, and product composition.
- Published
- 2010
14. Simultaneous Assessment of Protein Heterogeneity and Affinity by Capillary Electrophoresis–Mass Spectrometry
- Author
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Domínguez-Vega, E., primary, Haselberg, R., additional, Somsen, G. W., additional, and de Jong, G. J., additional
- Published
- 2015
- Full Text
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15. A simple capillary electrophoresis-mass spectrometry method for complex glycan analysis using a flow-through microvial interface
- Author
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Jayo, R.G., Thaysen-Andersen, M., Lindenburg, P.W., Haselberg, R., Hankemeijer, T., Ramautar, R., Chen, D.D.Y., Jayo, R.G., Thaysen-Andersen, M., Lindenburg, P.W., Haselberg, R., Hankemeijer, T., Ramautar, R., and Chen, D.D.Y.
- Abstract
A flow-through microvial is used to interface capillary electrophoresis and mass spectrometry (CE-MS) to develop a method for simultaneous profiling both neutral and sialylated glycans without derivatization or labeling. The CE separation was performed at near-zero electroosmotic flow in a capillary with neutral, hydrophilic coating, using 50 mM ammonium acetate in 20% methanol (pH 3.1) as the background electrolyte. The method was optimized with reversed CE polarity and negative ion ESI-MS. Enzymatically released N-glycans from human immunoglobulin G (IgG) were used as the test sample. The approach was also used to study the more complex N-glycans from recombinant human erythropoietin (rHuEPO) expressed in Chinese hamster ovary (CHO) cells. Glycoscreening of rHuEPO was performed using a triple quadrupole MS and an ultrahigh resolution TOF-MS. The high sensitivity and high mass accuracy of the TOF-MS revealed the presence of more than 70 glycans. Three mono-and di-sialylated tetra-antennary N-glycans and one mono-sialylated tri-antennary N-glycan of rHuEPO are reported for the first time. Further glycan heterogeneity was identified of the highly sialylated N-glycans of rHuEPO by extensive acetylation, Neu5Ac/Neu5Gc variation and the presence of N-acetyl-lactosamine repeats. For comparative purposes, porous graphitic carbon-based LC-MS/MS was also used to glycoprofile rHuEPO. This work demonstrates the potential of CE-MS to provide a comprehensive glycosylation profile with detailed features of the secondary glycan modifications. The CE-MS based method eliminates the need to label the N-glycans, as well as the requirement to desialylate before analysis, and could complement other established techniques for glycan characterization of therapeutic glycoproteins. © 2014 American Chemical Society.
- Published
- 2014
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16. Targeting hepatocyte growth factor receptor (Met) positive tumor cells using internalizing nanobody-decorated albumin nanoparticles
- Author
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Heukers, R., Altintas, I., Raghoenath, S., De Zan, E., Pepermans, R., Roovers, R.C., Haselberg, R., Hennink, W.E., Schiffelers, R.M., Kok, R.J., van Bergen en Henegouwen, P.M.P., Heukers, R., Altintas, I., Raghoenath, S., De Zan, E., Pepermans, R., Roovers, R.C., Haselberg, R., Hennink, W.E., Schiffelers, R.M., Kok, R.J., and van Bergen en Henegouwen, P.M.P.
- Abstract
The hepatocyte growth factor receptor (HGFR, c-Met or Met) is a receptor tyrosine kinase that is involved in embryogenesis, tissue regeneration and wound healing. Abnormal activation of this proto-oncogene product is implicated in the development, progression and metastasis of many cancers. Current therapies directed against Met, such as ligand- or, dimerization-blocking antibodies or kinase inhibitors, reduce tumor growth but hardly eradicate the tumor. In order to improve anti-Met therapy, we have designed a drug delivery system consisting of crosslinked albumin nanoparticles decorated with newly selected anti-Met nanobodies (anti-Met-NANAPs). The anti-Met NANAPs bound specifically to and were specifically taken up by Met-expressing cells and transported to lysosomes for degradation. Treatment of tumor cells with anti-Met NANAPs also resulted in downregulation of the total Met protein. This study shows that anti-Met NANAPs offer a potential system for lysosomal delivery of drugs into Met-positive tumor cells. © 2013 Elsevier Ltd.
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- 2014
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17. Microperoxidase Analysis Using Liquid Chromatography, Post-Column Substrate Conversion and Fluorescence Detection
- Author
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Haselberg, R., Hempen, C.M., van Leeuwen, S.M., Vogel, M., and Karst, U.
- Subjects
METIS-226795 - Published
- 2005
18. Quality of Original and Biosimilar Epoetin Products
- Author
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Advanced drug delivery systems/drug targeting, Biomolecular Analysis, Innovation Studies, Farmaceutische Analyse, Pharmaceutics, Sub Biotechnological drugs, Sub Biomolecular analysis, Dep Farmaceutische wetenschappen, Brinks, V., Hawe, A., Basmeleh, A.H.H., Joachin-Rodriguez, L., Haselberg, R., Somsen, G.W., Jiskoot, W., Schellekens, H., Advanced drug delivery systems/drug targeting, Biomolecular Analysis, Innovation Studies, Farmaceutische Analyse, Pharmaceutics, Sub Biotechnological drugs, Sub Biomolecular analysis, Dep Farmaceutische wetenschappen, Brinks, V., Hawe, A., Basmeleh, A.H.H., Joachin-Rodriguez, L., Haselberg, R., Somsen, G.W., Jiskoot, W., and Schellekens, H.
- Published
- 2011
19. Capillary electrophoresis – mass spectrometry using noncovalently coated capillaries for the analysis of biopharmaceuticals
- Author
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Biomolecular Analysis, Farmaceutische Analyse, Sub Biomolecular analysis, de Jong, Ad, Somsen, Govert, Haselberg, R., Biomolecular Analysis, Farmaceutische Analyse, Sub Biomolecular analysis, de Jong, Ad, Somsen, Govert, and Haselberg, R.
- Published
- 2010
20. 121 Tumor-targeted Nanobullets for Anti-cancer Combination Therapy
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van der Meel, R., primary, Oliveira, S., additional, Altintas, I., additional, Haselberg, R., additional, Roovers, R.C., additional, van Bergen en Henegouwen, P.M.P., additional, Hennink, W.E., additional, Storm, W.E., additional, Schiffelers, R.M., additional, and Kok, R.J., additional
- Published
- 2012
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21. Evanescent-Wave Cavity Enhanced Spectroscopy as a Tool in Label-Free Biosensing
- Author
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van der Sneppen, L., primary, Ritchie, G., additional, Hancock, G., additional, Ariese, F., additional, Gooijer, C., additional, Ubachs, W., additional, Haselberg, R., additional, Somsen, G.W., additional, and de Jong, G.J., additional
- Published
- 2010
- Full Text
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22. Analysis of microperoxidases using liquid chromatography, post-column substrate conversion and fluorescence detection
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HASELBERG, R, primary, HEMPEN, C, additional, VANLEEUWEN, S, additional, VOGEL, M, additional, and KARST, U, additional
- Published
- 2006
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23. Evanescent-wave cavity enhanced spectroscopy as a tool in label-free biosensing.
- Author
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van der Sneppen, L., Ritchie, G., Hancock, G., Ariese, F., Gooijer, C., Ubachs, W., Haselberg, R., Somsen, G.W., and de Jong, G.J.
- Published
- 2010
24. A novel nanospray liquid junction interface for versatile CE-MS
- Author
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Křenková, Jana, Haselberg, R., Somsen, G. W., and Foret, František
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liquid junction interface ,mass spectrometry ,capillary electrophoresis - Abstract
In this work we present a new polyimide-based liquid junction nanospray interface, combining the advantages of sheath flow and sheathless designs. The liquid junction interface was prepared as a hybrid microfabricated plastic liquid junction sprayer allowing to perform analysis in standard (30-100 micro m ID) separation capillaries. The interface incorporates a self-aligning port for coupling of the separation capillary as well as ports for automated flushing of both the liquid junction and the separation capillary. The interface permits performing CE both with and without electroosmotic flow. The use of large bore capillaries allows injection and preconcentration of larger sample volumes, favoring detection sensitivity. Different compositions of the spray liquid and BGE can be used when needed.
- Published
- 2016
25. A monodispersed metal-complexing peptide-based polymer for mass cytometry enabling spectral applications.
- Author
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Verhoeff J, van Asten S, Kuijper L, van den Braber M, Amstalden-van Hove E, Haselberg R, Kalay H, and Garcia-Vallejo JJ
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- Sulfhydryl Compounds chemistry, Peptides chemistry, Metals chemistry, Chelating Agents chemistry, Antibodies, Polymers chemistry, Coordination Complexes, Maleimides
- Abstract
We report the synthesis of a novel class of metal-complexing peptide-based polymers, which we name HyperMAPs (Hyper-loaded MetAl-complexed Polymers). The controlled solid-phase synthesis of HyperMAPs' scaffold peptide provides our polymer with a well-defined molecular structure that allows for an accurate on-design assembly of a wide variety of metals. The peptide-scaffold features a handle for direct conjugation to antibodies or any other biomolecules by means of a thiol-maleimide-click or aldehyde-oxime reaction, a fluorogenic moiety for biomolecule conjugation tracking, and a well-defined number of functional groups for direct incorporation of metal-chelator complexes. Since metal-chelator complexes are prepared in a separate reaction prior to incorporation to the peptide scaffold, polymers can be designed to contain specific ratios of metal isotopes, providing each polymer with a unique CyTOF spectral fingerprint. We demonstrate the complexing of 21 different metals using two different chelators and provide evidence of the application of HyperMAPs on a 13 parameter CyTOF panel and compare its performance to monoisotopic metal-conjugated antibodies., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)
- Published
- 2024
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26. Investigating direct current potentials that affect native protein conformation during trapped ion mobility spectrometry-mass spectrometry.
- Author
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Voeten RLC, Majeed HA, Bos TS, Somsen GW, and Haselberg R
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- Animals, Cattle, Mass Spectrometry methods, Protein Conformation, Ions, Ion Mobility Spectrometry methods, Proteins chemistry
- Abstract
Trapped ion mobility spectrometry-time-of-flight mass spectrometry (TIMS-TOFMS) has emerged as a tool to study protein conformational states. In TIMS, gas-phase ions are guided across the IM stages by applying direct current (DC) potentials (D1-6), which, however, might induce changes in protein structures through collisional activation. To define conditions for native protein analysis, we evaluated the influence of these DC potentials using the metalloenzyme bovine carbonic anhydrase (BCA) as primary test compound. The variation of DC potentials did not change BCA-ion charge and heme content but affected (relative) charge-state intensities and adduct retention. Constructed extracted-ion mobilograms and corresponding collisional cross-section (CCS) profiles gave useful insights in (alterations of) protein conformational state. For BCA, the D3 and D6 potential (which are applied between the deflection transfer and funnel 1 [F1] and the accumulation exit and the start of the ramp, respectively) had most profound effects, showing multimodal CCS distributions at higher potentials indicating gradual unfolding. The other DC potentials only marginally altered the CCS profiles of BCA. To allow for more general conclusions, five additional proteins of diverse molecular weight and conformational stability were analyzed, and for the main protein charge states, CCS profiles were constructed. Principal component analysis (PCA) of the obtained data showed that D1 and D3 exhibit the highest degree of correlation with the ratio of folded and unfolded protein (F/U) as extracted from the mobilograms obtained per set D potential. The correlation of D6 with F/U and protein charge were similar, and D2, D4, and D5 showed an inverse correlation with F/U but were correlated with protein charge. Although DC boundary values for induced conformational changes appeared protein dependent, a set of DC values could be determined, which assured native analysis of most proteins., (© 2024 The Authors. Journal of Mass Spectrometry published by John Wiley & Sons Ltd.)
- Published
- 2024
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27. Online multimethod platform for comprehensive characterization of monoclonal antibodies in cell culture fluid from a single sample injection - Intact protein workflow.
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Sadighi R, de Kleijne V, Wouters S, Lubbers K, Somsen GW, Gargano AFG, and Haselberg R
- Subjects
- Workflow, Chromatography, Reverse-Phase, Glycosylation, Cell Culture Techniques, Antibodies, Monoclonal
- Abstract
Background: Therapeutic monoclonal antibodies (mAbs) comprise a large structural variability with respect to charge, size and post-translational modifications. These critical quality attributes (CQAs) need to be assessed during and after the production of mAbs. This normally requires off-line purification and sample preparation as well as several chromatographic selectivities, which makes the whole process time-consuming and error-prone. To improve on this, we developed an integrated and automated multi-dimensional analytical platform for the simultaneous assessment of multiple CQAs of mAbs in cell culture fluid (CCF) from upstream processes., Results: The on-line system allows mAb characterization at the intact level, combining protein A affinity chromatography (ProtA) with size-exclusion, ion-exchange, and reversed-phase liquid chromatographic modes with UV and mass spectrometric detection. Multiple heart cuts of a single mAb elution band from ProtA are stored in 20-μL loops and successively sent to the multimethod options in the second dimension. ProtA loading and elution conditions and their compatibility with second-dimension LC modes were studied and optimized. Subsequently, heart-cutting and valve-switching schemes were investigated to achieve effective and reproducible analyses. The applicability of the developed workflow was demonstrated by the direct analysis (i.e. not requiring off-line sample preparation) of a therapeutic mAb in CCF, obtaining useful information on accurate molecular mass, glycosylation, and charge and size variants of the mAb product at the same time and in just over 1 h., Significance: The developed multidimensional platform is the first system that allows for multiple fractions from a single ProtA band to be characterized using different chromatographic selectivities in a single run allowing direct correlation between CQAs. The performance of the system is comparable to established off-line methods, fully compatible with upstream process samples, and provides a significant time-reduction of the characterization procedure., Competing Interests: Declaration of competing interest The authors declare the following financial interests/ which may be considered as potential competing interests: Govert W. Somsen reports equipment was provided by Agilent Technologies Inc., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)
- Published
- 2024
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28. Corrigendum to "Micro-flow size-exclusion chromatography for enhanced native mass spectrometry of proteins and protein complexes" [Anal. Chim. Acta 1266 (2023) 341324].
- Author
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Ventouri IK, Veelders S, Passamonti M, Endres P, Roemling R, Schoenmakers PJ, Somsen GW, Haselberg R, and Gargano AFG
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- 2023
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29. Micro-flow size-exclusion chromatography for enhanced native mass spectrometry of proteins and protein complexes.
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Ventouri IK, Veelders S, Passamonti M, Endres P, Roemling R, Schoenmakers PJ, Somsen GW, Haselberg R, and Gargano AFG
- Subjects
- Mass Spectrometry methods, Chromatography, Gel, Chromatography, High Pressure Liquid methods, Salts, Proteins analysis
- Abstract
Size-exclusion chromatography (SEC) employing aqueous mobile phases with volatile salts at neutral pH combined with native mass spectrometry (nMS) is a valuable tool to characterize proteins and protein aggregates in their native state. However, the liquid-phase conditions (high salt concentrations) frequently used in SEC-nMS hinder the analysis of labile protein complexes in the gas phase, necessitating higher desolvation-gas flow and source temperature, leading to protein fragmentation/dissociation. To overcome this issue, we investigated narrow SEC columns (1.0 mm internal diameter, I.D.) operated at 15-μL/min flow rates and their coupling to nMS for the characterization of proteins, protein complexes and higher-order structures (HOS). The reduced flow rate resulted in a significant increase in the protein-ionization efficiency, facilitating the detection of low-abundant impurities and HOS up to 230 kDa (i.e., the upper limit of the Orbitrap-MS instrument used). More-efficient solvent evaporation and lower desolvation energies allowed for softer ionization conditions (e.g., lower gas temperatures), ensuring little or no structural alterations of proteins and their HOS during transfer into the gas phase. Furthermore, ionization suppression by eluent salts was decreased, permitting the use of volatile-salt concentrations up to 400 mM. Band broadening and loss of resolution resulting from the introduction of injection volumes exceeding 3% of the column volume could be circumvented by incorporating an online trap-column containing a mixed-bed ion-exchange (IEX) material. The online IEX-based solid-phase extraction (SPE) or "trap-and-elute" set-up provided on-column focusing (sample preconcentration). This allowed the injection of large sample volumes on the 1-mm I.D. SEC column without compromising the separation. The enhanced sensitivity attained by the micro-flow SEC-MS, along with the on-column focusing achieved by the IEX precolumn, provided picogram detection limits for proteins., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)
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- 2023
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30. Characterizing Non-covalent Protein Complexes Using Asymmetrical Flow Field-Flow Fractionation On-Line Coupled to Native Mass Spectrometry.
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Ventouri IK, Chang W, Meier F, Drexel R, Somsen GW, Schoenmakers PJ, de Spiegeleer B, Haselberg R, and Astefanei A
- Subjects
- Sodium Hydroxide, Mass Spectrometry, Refractometry, Asparaginase, Proteins, Fractionation, Field Flow methods
- Abstract
We report an online analytical platform based on the coupling of asymmetrical flow field-flow fractionation (AF4) and native mass spectrometry (nMS) in parallel with UV-absorbance, multi-angle light scattering (MALS), and differential-refractive-index (UV-MALS-dRI) detectors to elucidate labile higher-order structures (HOS) of protein biotherapeutics. The technical aspects of coupling AF4 with nMS and the UV-MALS-dRI multi-detection system are discussed. The "slot-outlet" technique was used to reduce sample dilution and split the AF4 effluent between the MS and UV-MALS-dRI detectors. The stability, HOS, and dissociation pathways of the tetrameric biotherapeutic enzyme (anticancer agent) l-asparaginase (ASNase) were studied. ASNase is a 140 kDa homo-tetramer, but the presence of intact octamers and degradation products with lower molecular weights was indicated by AF4-MALS/nMS. Exposing ASNase to 10 mM NaOH disturbed the equilibrium between the different non-covalent species and led to HOS dissociation. Correlation of the information obtained by AF4-MALS (liquid phase) and AF4-nMS (gas phase) revealed the formation of monomeric, tetrameric, and pentameric species. High-resolution MS revealed deamidation of the main intact tetramer upon exposure of ASNase to high pH (NaOH and ammonium bicarbonate). The particular information retrieved from ASNase with the developed platform in a single run demonstrates that the newly developed platform can be highly useful for aggregation and stability studies of protein biopharmaceuticals.
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- 2023
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31. Binding Properties 2022.
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Haselberg R and Stein M
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- 2022
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32. Studying protein structure and function by native separation-mass spectrometry.
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van Schaick G, Haselberg R, Somsen GW, Wuhrer M, and Domínguez-Vega E
- Subjects
- Mass Spectrometry methods, Proteins analysis, Biological Products
- Abstract
Alterations in protein structure may have profound effects on biological function. Analytical techniques that permit characterization of proteins while maintaining their conformational and functional state are crucial for studying changes in the higher order structure of proteins and for establishing structure-function relationships. Coupling of native protein separations with mass spectrometry is emerging rapidly as a powerful approach to study these aspects in a reliable, fast and straightforward way. This Review presents the available native separation modes for proteins, covers practical considerations on the hyphenation of these separations with mass spectrometry and highlights the involvement of affinity-based separations to simultaneously obtain structural and functional information of proteins. The impact of these approaches is emphasized by selected applications addressing biomedical and biopharmaceutical research questions., (© 2022. Springer Nature Limited.)
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- 2022
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33. Assessment of Macro- and Microheterogeneity of Monoclonal Antibodies Using Capillary Zone Electrophoresis Hyphenated with Mass Spectrometry.
- Author
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Gstöttner C, Haselberg R, Wuhrer M, Somsen GW, and Domínguez-Vega E
- Subjects
- Electrophoresis, Capillary methods, Mass Spectrometry methods, Protein Processing, Post-Translational, Spectrometry, Mass, Electrospray Ionization methods, Antibodies, Monoclonal chemistry, Antineoplastic Agents, Immunological
- Abstract
This chapter focuses on the application of capillary zone electrophoresis hyphenated with mass spectrometry (CZE-MS) for the characterization of monoclonal antibodies (mAbs). mAbs are complex molecules comprising different glycoforms and many other posttranslational modifications. In addition to this inherent microheterogeneity, misassembling of antibodies can take place during production contributing to their macroheterogeneity. CZE-MS is a versatile and powerful technique which has demonstrated high potential for the assessment of both micro- and macroheterogeneity of mAbs. In this chapter, technical and practical considerations for the characterization of mAbs by CZE-MS are described. CE-MS interfacing, capillary coatings for the prevention of mAb adsorption, and sample preparation considerations are covered in detail. The assessment of the macro- and microheterogeneity is discussed and exemplified through three different approaches involving analysis of intact, enzymatically digested, and reduced antibodies. The examples also illustrate the use of two commercially available interfacing techniques (i.e., sheath liquid and sheathless) as well as different types of capillary coatings (positively charged and neutral coatings)., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)
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- 2022
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34. Perceptions about Research Participation among Individuals at Risk and Individuals with Premanifest Huntington's Disease: A Survey Conducted by the European Huntington Association.
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Júlio F, Blanco R, Casanova JP, D'Alessio B, De Schepper B, De Sousa D, De Sousa P, Ferreira C, Gommans H, Haselberg R, Hermant E, Lis D, Maffi S, Olsen SO, Papantoniou M, Squitieri F, Tretyakova M, Umakhanova Z, Václavík V, Winkelmann M, Arnesen A, and On Behalf Of The European Huntington Association
- Abstract
There has been great progress in Huntington's disease (HD) research. Yet, effective treatments to halt disease before the onset of disabling symptoms are still unavailable. Scientific breakthroughs require an active and lasting commitment from families. However, they are traditionally less involved and heard in studies. Accordingly, the European Huntington Association (EHA) surveyed individuals at risk (HDRisk) and with premanifest HD (PreHD) to determine which factors affect their willingness to participate in research. Questions assessed research experience and knowledge, information sources, reasons for involvement and noninvolvement, and factors preventing and facilitating participation. The survey included 525 individuals, of which 68.8% never participated in studies and 38.6% reported limited research knowledge. Furthermore, 52% trusted patient organizations to get research information. Reasons for involvement were altruistic and more important than reasons for noninvolvement, which were related to negative emotions. Obstacles included time/financial constraints and invasive procedures, while professional support was seen as a facilitator. PreHD individuals reported less obstacles to research participation than HDRisk individuals. Overall, a high motivation to participate in research was noted, despite limited experience and literacy. This motivation is influenced by subjective and objective factors and, importantly, by HD status. Patient organizations have a key role in fostering motivation through education and support.
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- 2021
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35. Microfluidic ion stripper for removal of trifluoroacetic acid from mobile phases used in HILIC-MS of intact proteins.
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Wouters S, Eeltink S, Haselberg R, Somsen GW, and Gargano AFG
- Subjects
- Animals, Cattle, Chickens, Chromatography, Liquid, Equipment Design, Horses, Hydrophobic and Hydrophilic Interactions, Spectrometry, Mass, Electrospray Ionization, Lab-On-A-Chip Devices, Proteins analysis, Trifluoroacetic Acid isolation & purification
- Abstract
Trifluoroacetic acid (TFA) is commonly used as mobile phase additive to improve retention and peak shape characteristics in hydrophilic interaction liquid chromatography (HILIC) of intact proteins. However, when using electrospray ionization-mass spectrometry (ESI-MS) detection, TFA may cause ionization suppression and adduct formation, leading to reduced analyte sensitivity. To address this, we describe a membrane-based microfluidic chip with multiple parallel channels for the selective post-column removal of TFA anions from HILIC. An anion-exchange membrane was used to physically separate the column effluent from a stripper flow solution comprising acetonitrile, formic acid, and propionic acid. The exchange of ions allowed the post-column removal of TFA used during HILIC separation of model proteins. The multichannel design of the device allows the use of flow rates of 0.2 mL/min without the need for a flow splitter, using mobile phases containing 0.1% TFA (13 mM). Separation selectivity and efficiency were maintained (with minor band broadening effects) while increasing the signal intensity and peak areas by improving ionization and reducing TFA adduct formation.
- Published
- 2021
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36. Limited Lactosylation of Beta-Lactoglobulin from Cow's Milk Exerts Strong Influence on Antigenicity and Degranulation of Mast Cells.
- Author
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Bosman GP, Oliveira S, Simons PJ, Sastre Torano J, Somsen GW, Knippels LMJ, Haselberg R, Pieters RJ, Garssen J, and Knipping K
- Subjects
- Allergens analysis, Animals, Cattle, Female, Humans, Immunoglobulin E analysis, Immunoglobulin G, Lactose analysis, Maillard Reaction, Mast Cells, Milk Proteins analysis, Whey, Whey Proteins analysis, Lactoglobulins analysis, Milk chemistry, Milk Hypersensitivity
- Abstract
Background: beta-lactoglobulin (BLG) is one of the major cow's milk proteins and the most abundant allergen in whey. Heating is a common technologic treatment applied during milk transformational processes. Maillardation of BLG in the presence of reducing sugars and elevated temperatures may influence its antigenicity and allergenicity., Primary Objective: to analyze and identify lactosylation sites by capillary electrophoresis mass spectrometry (CE-MS)., Secondary Objective: to assess the effect of lactosylated BLG on antigenicity and degranulation of mast cells., Methods: BLG was lactosylated at pH 7, a water activity (aw) of 0.43, and a temperature of 65 °C using a molar ratio BLG:lactose of 1:1 by incubating for 0, 3, 8, 16 or 24 h. For the determination of the effect on antibody-binding capacity of lactosylated BLG, an ELISA was performed. For the assessment of degranulation of the cell-line RBL-hεIa-2B12 transfected with the human α-chain, Fcε receptor type 1 (FcεRI) was used., Results: BLG showed saturated lactosylation between 8 and 16 incubation hours in our experimental setup. Initial stage lactosylation sites L1 (N-terminus)-K47, K60, K75, K77, K91, K138 and K141-have been identified using CE-MS. Lactosylated BLG showed a significant reduction of both the IgG binding ( p = 0.0001) as well as degranulation of anti-BLG IgE-sensitized RBL-hεIa-2B12 cells ( p < 0.0001)., Conclusions and Clinical Relevance: this study shows that lactosylation of BLG decreases both the antigenicity and degranulation of mast cells and can therefore be a promising approach for reducing allergenicity of cow's milk allergens provided that the process is well-controlled.
- Published
- 2021
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37. Probing Polyester Branching by Hybrid Trapped Ion-Mobility Spectrometry-Tandem Mass Spectrometry.
- Author
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Voeten RLC, van de Put B, Jordens J, Mengerink Y, Peters RAH, Haselberg R, and Somsen GW
- Abstract
Trapped ion-mobility spectrometry combined with quadrupole time-of-flight mass spectrometry (TIMS-QTOFMS) was evaluated as a tool for resolving linear and branched isomeric polyester oligomers. Solutions of polyester samples were infused directly into the ion source employing electrospray ionization (ESI). TIMS-MS provides both mobility and m / z data on the formed ions, allowing construction of extracted-ion mobilograms (EIMs). EIMs of polyester molecules showed multimodal patterns, indicating conformational differences among isomers. Subsequent TIMS-MS/MS experiments indicated mobility differences to be caused by (degree of) branching. These assignments were supported by liquid chromatography-TIMS-MS/MS analysis, confirming that direct TIMS-MS provided fast (500 ms/scan) distinction between linear and branched small oligomers. Observing larger oligomers (up to 3000 Da) using TIMS required additional molecular charging to ensure ion entrapment within the mobility window. Molecular supercharging was achieved using m -nitrobenzyl alcohol (NBA). The additional charges on the oligomer structures enhanced mobility separation of isomeric species but also added to the complexity of the obtained fragmentation mass spectra. This complexity could be partly reduced by post-TIMS analyte-decharging applying collision-induced dissociation (CID) prior to Q1 with subsequent isolation of the singly charged ions for further fragmentation. The as-obtained EIM profiles were still quite complex as larger molecules possess more possible structural isomers. Nevertheless, distinguishing between linear and symmetrically branched oligomers was possible based on measured differences in collisional cross sections (CCSs). The established TIMS-QTOFMS approach reliably allows branching information on isomeric polyester molecules up to 3000 Da to be obtained in less than 1 min analysis time.
- Published
- 2021
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38. Asymmetrical flow field-flow fractionation to probe the dynamic association equilibria of β-D-galactosidase.
- Author
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Ventouri IK, Astefanei A, Kaal ER, Haselberg R, Somsen GW, and Schoenmakers PJ
- Subjects
- Chromatography, Gel, Dynamic Light Scattering, Ions chemistry, Magnesium chemistry, Polymers chemistry, Fractionation, Field Flow, beta-Galactosidase chemistry
- Abstract
Protein dynamics play a significant role in many aspects of enzyme activity. Monitoring of structural changes and aggregation of biotechnological enzymes under native conditions is important to safeguard their properties and function. In this work, the potential of asymmetrical flow field-flow fractionation (AF4) to study the dynamic association equilibria of the enzyme β-D-galactosidase (β-D-Gal) was evaluated. Three commercial products of β-D-Gal were investigated using carrier liquids containing sodium chloride or ammonium acetate, and the effect of adding magnesium (II) chloride to the carrier liquid was assessed. Preservation of protein structural integrity during AF4 analysis was essential and the influence of several parameters, such as the focusing step (including use of frit-inlet), cross flow, and injected amount, was studied. Size-exclusion chromatography (SEC) and dynamic light scattering (DLS) were used to corroborate the in-solution enzyme oligomerization observed with AF4. In contrast to SEC, AF4 provided sufficiently mild separation conditions to monitor protein conformations without disturbing the dynamic association equilibria. AF4 analysis showed that ammonium acetate concentrations above 40 mM led to further association of the dimers ("tetramerization") of β-D-Gal. Magnesium ions, which are needed to activate β-D-Gal, appeared to induce dimer association, raising justifiable questions about the role of divalent metal ions in protein oligomerization and on whether tetramers or dimers are the most active form of β-D-Gal., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)
- Published
- 2021
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39. CE-MS for Proteomics and Intact Protein Analysis.
- Author
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Kuzyk VO, Somsen GW, and Haselberg R
- Subjects
- Mass Spectrometry, Peptides, Proteins, Electrophoresis, Capillary, Proteomics
- Abstract
This chapter aims to explore various parameters involved in achieving high-end capillary electrophoresis hyphenated to mass spectrometry (CE-MS) analysis of proteins, peptides, and their posttranslational modifications. The structure of the topics discussed in this book chapter is conveniently mapped on the scheme of the CE-MS system itself, starting from sample preconcentration and injection techniques and finishing with mass analyzer considerations. After going through the technical considerations, a variety of relevant applications for this analytical approach are presented, including posttranslational modifications analysis, clinical biomarker discovery, and its growing use in the biotechnological industry., (© 2021. Springer Nature Switzerland AG.)
- Published
- 2021
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40. Probing Protein Denaturation during Size-Exclusion Chromatography Using Native Mass Spectrometry.
- Author
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Ventouri IK, Malheiro DBA, Voeten RLC, Kok S, Honing M, Somsen GW, and Haselberg R
- Subjects
- Animals, Heart, Horses, Hydrogen-Ion Concentration, Protein Denaturation, Spectrometry, Mass, Electrospray Ionization, Chromatography, Gel, Myoglobin analysis
- Abstract
Size-exclusion chromatography employing aqueous mobile phases with volatile salts at neutral pH combined with electrospray-ionization mass spectrometry (SEC-ESI-MS) is a useful tool to study proteins in their native state. However, whether the applied eluent conditions actually prevent protein-stationary phase interactions, and/or protein denaturation, often is not assessed. In this study, the effects of volatile mobile phase additives on SEC retention and ESI of proteins were thoroughly investigated. Myoglobin was used as the main model protein, and eluents of varying ionic strength and pH were applied. The degree of interaction between protein and stationary phase was evaluated by calculating the SEC distribution coefficient. Protein-ion charge state distributions obtained during offline and online native ESI-MS were used to monitor alterations in protein structure. Interestingly, most of the supposedly mild eluent compositions induced nonideal SEC behavior and/or protein unfolding. SEC experiments revealed that the nature, ionic strength, and pH of the eluent affected protein retention. Protein-stationary phase interactions were effectively avoided using ammonium acetate at ionic strengths above 0.1 M. Direct-infusion ESI-MS showed that the tested volatile eluent salts seem to follow the Hofmeister series: no denaturation was induced using ammonium acetate (kosmotropic), whereas ammonium formate and bicarbonate (both chaotropic) caused structural changes. Using a mobile phase of 0.2 M ammonium acetate (pH 6.9), several proteins (i.e., myoglobin, carbonic anhydrase, and cytochrome c) could be analyzed by SEC-ESI-MS using different column chemistries without compromising their native state. Overall, with SEC-ESI-MS, the effect of nonspecific interactions between protein and stationary phase on the protein structure can be studied, even revealing gradual structural differences along a peak.
- Published
- 2020
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41. NIST Interlaboratory Study on Glycosylation Analysis of Monoclonal Antibodies: Comparison of Results from Diverse Analytical Methods.
- Author
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De Leoz MLA, Duewer DL, Fung A, Liu L, Yau HK, Potter O, Staples GO, Furuki K, Frenkel R, Hu Y, Sosic Z, Zhang P, Altmann F, Grunwald-Grube C, Shao C, Zaia J, Evers W, Pengelley S, Suckau D, Wiechmann A, Resemann A, Jabs W, Beck A, Froehlich JW, Huang C, Li Y, Liu Y, Sun S, Wang Y, Seo Y, An HJ, Reichardt NC, Ruiz JE, Archer-Hartmann S, Azadi P, Bell L, Lakos Z, An Y, Cipollo JF, Pucic-Bakovic M, Štambuk J, Lauc G, Li X, Wang PG, Bock A, Hennig R, Rapp E, Creskey M, Cyr TD, Nakano M, Sugiyama T, Leung PA, Link-Lenczowski P, Jaworek J, Yang S, Zhang H, Kelly T, Klapoetke S, Cao R, Kim JY, Lee HK, Lee JY, Yoo JS, Kim SR, Suh SK, de Haan N, Falck D, Lageveen-Kammeijer GSM, Wuhrer M, Emery RJ, Kozak RP, Liew LP, Royle L, Urbanowicz PA, Packer NH, Song X, Everest-Dass A, Lattová E, Cajic S, Alagesan K, Kolarich D, Kasali T, Lindo V, Chen Y, Goswami K, Gau B, Amunugama R, Jones R, Stroop CJM, Kato K, Yagi H, Kondo S, Yuen CT, Harazono A, Shi X, Magnelli PE, Kasper BT, Mahal L, Harvey DJ, O'Flaherty R, Rudd PM, Saldova R, Hecht ES, Muddiman DC, Kang J, Bhoskar P, Menard D, Saati A, Merle C, Mast S, Tep S, Truong J, Nishikaze T, Sekiya S, Shafer A, Funaoka S, Toyoda M, de Vreugd P, Caron C, Pradhan P, Tan NC, Mechref Y, Patil S, Rohrer JS, Chakrabarti R, Dadke D, Lahori M, Zou C, Cairo C, Reiz B, Whittal RM, Lebrilla CB, Wu L, Guttman A, Szigeti M, Kremkow BG, Lee KH, Sihlbom C, Adamczyk B, Jin C, Karlsson NG, Örnros J, Larson G, Nilsson J, Meyer B, Wiegandt A, Komatsu E, Perreault H, Bodnar ED, Said N, Francois YN, Leize-Wagner E, Maier S, Zeck A, Heck AJR, Yang Y, Haselberg R, Yu YQ, Alley W, Leone JW, Yuan H, and Stein SE
- Subjects
- Antibodies, Monoclonal metabolism, Glycomics methods, Glycopeptides metabolism, Glycosylation, Humans, Laboratories, Polysaccharides metabolism, Protein Processing, Post-Translational, Proteomics methods, Antibodies, Monoclonal chemistry, Biological Products, Biopharmaceutics methods
- Abstract
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., (© 2020 De Leoz et al.)
- Published
- 2020
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42. Computer-aided gradient optimization of hydrophilic interaction liquid chromatographic separations of intact proteins and protein glycoforms.
- Author
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van Schaick G, Pirok BWJ, Haselberg R, Somsen GW, and Gargano AFG
- Subjects
- Acetonitriles chemistry, Adsorption, Amides chemistry, Chromatography, Reverse-Phase, Hydrophobic and Hydrophilic Interactions, Software, Solvents chemistry, Tandem Mass Spectrometry, Chemistry Techniques, Analytical methods, Chromatography, Liquid, Glycoproteins isolation & purification, Proteins isolation & purification
- Abstract
Protein glycosylation is one of the most common and critical post-translational modification, which results from covalent attachment of carbohydrates to protein backbones. Glycosylation affects the physicochemical properties of proteins and potentially their function. Therefore it is important to establish analytical methods which can resolve glycoforms of glycoproteins. Recently, hydrophilic-interaction liquid chromatography (HILIC)-mass spectrometry has demonstrated to be a useful tool for the efficient separation and characterization of intact protein glycoforms. In particular, amide-based stationary phases in combination with acetonitrile-water gradients containing ion-pairing agents, have been used for the characterization of glycoproteins. However, finding the optimum gradient conditions for glycoform resolution can be quite tedious as shallow gradients (small decrease of acetonitrile percentage in the elution solvent over a long time) are required. In the present study, the retention mechanism and peak capacity of HILIC for non-glycosylated and glycosylated proteins were investigated and compared to reversed-phase liquid chromatography (RPLC). For both LC modes, ln k vs. φ plots of a series of test proteins were calculated using linear solvent strength (LSS) analysis. For RPLC, the plots were spread over a wider φ range than for HILIC, suggesting that HILIC methods require shallower gradients to resolve intact proteins. Next, the usefulness of computer-aided method development for the optimization of the separation of intact glycoform by HILIC was examined. Five retention models including LSS, adsorption, and mixed-mode, were tested to describe and predict glycoprotein retention under gradient conditions. The adsorption model appeared most suited and was applied to the gradient prediction for the separation of the glycoforms of six glycoproteins (Ides-digested trastuzumab, alpha-acid glycoprotein, ovalbumin, fetuin and thyroglobulin) employing the program PIOTR. Based on the results of three scouting gradients, conditions for high-efficiency separations of protein glycoforms varying in the degree and complexity of glycosylation was achieved, thereby significantly reducing the time needed for method optimization., (Copyright © 2019 Elsevier B.V. All rights reserved.)
- Published
- 2019
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43. Experimental design and measurement uncertainty in ligand binding studies by affinity capillary electrophoresis.
- Author
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Stein M, Haselberg R, Mozafari-Torshizi M, and Wätzig H
- Subjects
- Algorithms, Models, Statistical, Monte Carlo Method, Research Design, Electrophoresis, Capillary methods, Electrophoresis, Capillary statistics & numerical data, Ligands, Uncertainty
- Abstract
In all life sciences ligand binding assays (LBAs) play a crucial role. Unfortunately these are very error prone. One part of this uncertainty results from the unavoidable random measurement uncertainty, another part can be attributed to the experimental design. To investigate the latter, uncertainty propagation was evaluated as a function of the given experimental design. A design space including the normalized maximum response range (nMRR), the data point position (DPP), the data point range (DPR) and the number of data points (NoDP) was defined. Based on ten measured ms ACE source data sets 20 specific parameter sets were selected by Design of Experiments. Monte Carlo simulations using 100 000 repeats for every parameter set were employed. The resulting measurement uncertainty propagation factors (measurement uncertainty multiplier: MUM) were used to describe the whole design space by polynomial regression. The resulting 5-dimensional response surface was investigated to evaluate the design parameter's influence and to find the minimal uncertainty propagation. It could be shown, that the nMRR is of highest importance, followed by DPP and DPR. Interestingly, the NoDP is less relevant. However, the interactions of the four parameters need to be carefully considered during design optimization. Using at least five data points which cover over 40% of the upper part of the binding hyperbola (DPP > 0.57) the MUM will be minimized (MUM approximately 1.5) when the nMRR is appropriate. It is possible to reduce the measurement uncertainty propagation more than one order of magnitude., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2019
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44. Heterogeneity assessment of antibody-derived therapeutics at the intact and middle-up level by low-flow sheathless capillary electrophoresis-mass spectrometry.
- Author
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Haselberg R, De Vijlder T, Heukers R, Smit MJ, Romijn EP, Somsen GW, and Domínguez-Vega E
- Subjects
- Antibodies, Monoclonal pharmacology, Antibodies, Monoclonal therapeutic use, Immunoglobulin Fc Fragments chemistry, Sialic Acids chemistry, Antibodies, Monoclonal analysis, Antibodies, Monoclonal chemistry, Electrophoresis, Capillary methods, Mass Spectrometry methods
- Abstract
Antibody-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., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)- Published
- 2018
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45. Affinity profiling of monoclonal antibody and antibody-drug-conjugate preparations by coupled liquid chromatography-surface plasmon resonance biosensing.
- Author
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Lakayan D, Haselberg R, Gahoual R, Somsen GW, and Kool J
- Subjects
- Ado-Trastuzumab Emtansine, Biosensing Techniques methods, Chromatography, Liquid methods, Drug Compounding, Mass Spectrometry, Maytansine analogs & derivatives, Maytansine chemistry, Particle Size, Surface Plasmon Resonance methods, Trastuzumab chemistry, Antibodies, Monoclonal chemistry, Antibody Affinity
- Abstract
Monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) are highly potent biopharmaceuticals designed for targeted cancer therapies. mAbs and ADCs can undergo modifications during production and storage which may affect binding to target receptors, potentially altering drug efficacy. In this work, liquid chromatography was coupled online to surface plasmon resonance (LC-SPR) to allow label-free affinity evaluation of mAb and ADC sample constituents (size and charge variants), under near-native conditions. Trastuzumab and its ADC trastuzumab emtansine (T-DM1) were used as a test sample and were analyzed by aqueous size-exclusion chromatography (SEC)-SPR before and after exposure to aggregate-inducing conditions. SEC-SPR allowed separation of the formed aggregates and measurement of their affinity towards the ligand-binding domain of the human epidermal growth factor receptor 2 (HER2) receptor immobilized on the surface of the SPR sensor chip. The monomer and aggregates of the mAb and ADC were shown to have similar antigen affinity. Conjugation of drugs to trastuzumab appeared to accelerate the aggregate formation. In addition, cation-exchange chromatography (CEX) was coupled to SPR enabling monitoring the maximum ligand-analyte binding capacity (R
max ) of individual charge variants present in mAbs. Deamidated species and lysine variants in trastuzumab sample were separated but did not show different binding affinities to the immobilized HER2-binding domain. In order to allow protein variant assignment, parallel MS detection was added to the LC-SPR setup using a column effluent split. The feasibility of the LC-MS/SPR system was demonstrated by analysis of trastuzumab and T-DM1 providing information on antibody glycoforms and/or determination of the drug-to-antibody ratio (DAR), while simultaneously monitoring binding of eluting species to HER2. Graphical abstract ᅟ.- Published
- 2018
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46. High-resolution glycoform profiling of intact therapeutic proteins by hydrophilic interaction chromatography-mass spectrometry.
- Author
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Domínguez-Vega E, Tengattini S, Peintner C, van Angeren J, Temporini C, Haselberg R, Massolini G, and Somsen GW
- Subjects
- Chromatography, High Pressure Liquid, Glycosylation, Humans, Hydrophobic and Hydrophilic Interactions, Mass Spectrometry, Polysaccharides analysis, Recombinant Proteins analysis, Erythropoietin analysis, Interferon beta-1a analysis
- Abstract
Glycosylation is considered a critical quality attribute of therapeutic proteins. Protein heterogeneity introduced by glycosylation includes differences in the nature, number and position of the glycans. Whereas analysis of released glycans and glycopeptides provides information about the composition and/or position of the glycan, intact glycoprotein analysis allows assignment of individual proteoforms and co-occurring modifications. Yet, resolving protein glycoforms at the intact level is challenging. We have explored the capacity of hydrophilic liquid chromatography-mass spectrometry (HILIC-MS) for assessing glycosylation patterns of intact pharmaceutical proteins by analyzing the complex glycoproteins interferon-beta-1a (rhIFN-β - 1a) and recombinant human erythropoietin (rhEPO). Efficient glycoform separation was achieved using a superficially-porous amide HILIC stationary phase and trifluoroacetic acid (TFA) as eluent additive. In-source collision-induced dissociation proved to be very useful to minimize protein-signal suppression effects by TFA. Direct injection of therapeutic proteins in aqueous formulation was possible without causing extra band dispersion, provided that the sample injection volume was not larger than 2 μL. HILIC-MS of rhIFN-β - 1a and rhEPO allowed the assignment of, respectively, 15 and 51 glycoform compositions, next to a variety of posttranslational modifications, such as succinimide, oxidation and N-terminal methionine-loss products. MS-based assignments showed that neutral glycan units significantly contributed to glycoform separation, whereas terminal sialic acids only had a marginal effect on HILIC retention. Comparisons of HILIC-MS with the selectivity provided by capillary electrophoresis-MS for the same glycoproteins, revealed a remarkable complementarity of the techniques. Finally it was demonstrated that by replacing TFA for difluoroacetic acid, peak resolution somewhat decreased, but rhEPO glycoforms with relative abundances below 1% could be detected by HILIC-MS, increasing the overall rhEPO glycoform coverage to 72., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)
- Published
- 2018
- Full Text
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47. Development of a capillary zone electrophoresis method to quantify E. colil-asparaginase and its acidic variants.
- Author
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Yao H, Vandenbossche J, Sänger-van de Griend CE, Janssens Y, Fernández CS, Xu X, Wynendaele E, Somsen GW, Haselberg R, and De Spiegeleer B
- Subjects
- Asparaginase chemistry, Bacterial Proteins chemistry, Boric Acids chemistry, Electrolytes chemistry, Electrophoresis, Capillary standards, Enzyme Assays, Enzyme Stability, Escherichia coli chemistry, Factor Analysis, Statistical, Hydrogen-Ion Concentration, Isoenzymes chemistry, Isoenzymes isolation & purification, Limit of Detection, Tromethamine chemistry, Ultraviolet Rays, Asparaginase isolation & purification, Bacterial Proteins isolation & purification, Electrophoresis, Capillary methods, Escherichia coli enzymology
- Abstract
A capillary zone electrophoresis (CZE) method with UV detection was developed for the quantification of the E.colil-asparaginase (l-ASNase) and its acidic variants. During the initial method development, a variety of experimental conditions were screened. Subsequently, a Design of Experiments (DoE) was used to optimize the pH and concentration of the selected background electrolyte (BGE) containing both TRIS and boric acid. Optimization was performed taking into account both the separation efficiency of l-ASNase and its acidic variants as well as overall method robustness. A repeatable separation between E.colil-ASNase and its acidic variants was achieved on a bare fused silica capillary in combination with a BGE consisting of both 400 mM TRIS and boric acid. The method was validated for linearity, accuracy, precision, LOD, LOQ and robustness. The recovery for l-ASNase was 97.9-104.4% with a precision RSD of 1.5-3.2%, while the recovery of impurities was 92.1-109.8% with a RSD of 1.7-4.6%. The quantification limit was 1.9% (m/m). Moreover, the CZE-UV method was applied to determine the degradation rate in the presence of ammonium bicarbonate, confirming the suitability of the method. The degraded, partially charged l-ASNase was evaluated for its in-vitro enzymatic activity showing an insignificant different enzyme activity compared to the unmodified sample., (Copyright © 2018 Elsevier B.V. All rights reserved.)
- Published
- 2018
- Full Text
- View/download PDF
48. Capillary Electrophoresis: Trends and Recent Advances.
- Author
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Voeten RLC, Ventouri IK, Haselberg R, and Somsen GW
- Published
- 2018
- Full Text
- View/download PDF
49. Fully compatible and ultra-sensitive micellar electrokinetic chromatography-tandem mass spectrometry using sheathless porous-tip interfacing.
- Author
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Moreno-González D, Haselberg R, Gámiz-Gracia L, García-Campaña AM, de Jong GJ, and Somsen GW
- Subjects
- Caprylates chemistry, Carbamates analysis, Chemistry Techniques, Analytical instrumentation, Environmental Monitoring instrumentation, Fluorocarbons chemistry, Limit of Detection, Mass Spectrometry methods, Reproducibility of Results, Signal-To-Noise Ratio, Surface-Active Agents chemistry, Chemistry Techniques, Analytical methods, Chromatography, Micellar Electrokinetic Capillary, Environmental Monitoring methods, Tandem Mass Spectrometry, Water Pollutants, Chemical analysis
- Abstract
The on-line coupling of micellar electrokinetic chromatography and mass spectrometry (MEKC-MS) is often hampered by incompatibility problems leading to reduced separation performance and unfavorable limits of detection (LODs). Here we propose a new selective and highly sensitive MEKC-MS/MS method employing a sheathless porous-tip interface in combination with a micellar phase comprised of semi-volatile surfactant molecules. Carbamate pesticides (CRBs) were selected as representative model compounds being neutral toxic pollutants potentially present at trace levels in environmental water samples. A background electrolyte of 75mM perfluorooctanoic acid adjusted to pH 9.0 with ammonium hydroxide allowed efficient separation of 15 CRBs and appeared fully compatible with electrospray ionization (ESI)-MS. Interfacing parameters, such as the distance between the capillary tip and mass-spectrometer inlet, ESI voltage, and dry gas temperature and flow were optimized in order to attain good spray stability and high analyte signal-to-noise ratios. For CRBs the LODs ranged from 0.2 to 3.9ngL
-1 (13nL injected, i.e., 2% of capillary volume), representing an improvement for certain CRBs of more than 300-fold when compared with conventional sheath-liquid interfacing. Good linearity (R2 >0.99) and satisfactory reproducibility were obtained for all CRBs with interday RSD values for peak area and migration time of 4.0-11.3% and below 1.5%, respectively. Analysis of spiked mineral water showed that the new MEKC-MS/MS method allows selective and quantitative determination of CRB concentrations below the maximum residue limit of 100ngL-1 without the need for sample preconcentration., (Copyright © 2017 Elsevier B.V. All rights reserved.)- Published
- 2017
- Full Text
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50. A Novel Platinum(II)-Based Bifunctional ADC Linker Benchmarked Using 89Zr-Desferal and Auristatin F-Conjugated Trastuzumab.
- Author
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Sijbrandi NJ, Merkul E, Muns JA, Waalboer DC, Adamzek K, Bolijn M, Montserrat V, Somsen GW, Haselberg R, Steverink PJ, Houthoff HJ, and van Dongen GA
- Subjects
- Aminobenzoates, Animals, Antineoplastic Agents chemistry, Cell Line, Tumor, Deferoxamine, Drug Design, Humans, Immunoconjugates chemistry, Mice, Oligopeptides, Platinum Compounds, Radioactive Tracers, Stomach Neoplasms pathology, Trastuzumab chemistry, Xenograft Model Antitumor Assays, Zirconium, Antibodies, Monoclonal pharmacology, Antineoplastic Agents pharmacology, Immunoconjugates pharmacology, Trastuzumab pharmacology
- Abstract
Greater control is desirable in the stochastic conjugation technology used to synthesize antibody-drug conjugates (ADC). We have shown recently that a fluorescent dye can be stably conjugated to a mAb using a bifunctional platinum(II) linker. Here, we describe the general applicability of this novel linker technology for the preparation of stable and efficacious ADCs. The ethylenediamine platinum(II) moiety, herein called Lx, was coordinated to Desferal (DFO) or auristatin F (AF) to provide storable "semifinal" products, which were directly conjugated to unmodified mAbs. Conjugation resulted in ADCs with unimpaired mAb-binding characteristics, DAR in the range of 2.5 to 2.7 and approximately 85% payload bound to the Fc region, presumably to histidine residues. To evaluate the in vivo stability of Lx and its effect on pharmacokinetics and tumor targeting of an ADC, Lx-DFO was conjugated to the HER2 mAb trastuzumab, followed by radiolabeling with
89 Zr. Trastuzumab-Lx-DFO-89 Zr was stable in vivo and exhibited pharmacokinetic and tumor-targeting properties similar to parental trastuzumab. In a xenograft mouse model of gastric cancer (NCI-N87) or an ado-trastuzumab emtansine-resistant breast cancer (JIMT-1), a single dose of trastuzumab-Lx-AF outperformed its maleimide benchmark trastuzumab-Mal-AF and FDA-approved ado-trastuzumab emtansine. Overall, our findings show the potential of the Lx technology as a robust conjugation platform for the preparation of anticancer ADCs. Cancer Res; 77(2); 257-67. ©2016 AACR., (©2016 American Association for Cancer Research.)- Published
- 2017
- Full Text
- View/download PDF
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