4 results on '"Luckau L"'
Search Results
2. Reference Protocol to Assess Analytical Performance of Higher Order Structural Analysis Measurements:Results from an Interlaboratory Comparison
- Author
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Groves, K., Ashcroft, A.E., Cryar, A., Sula, A., Wallace, B.A., Stocks, B.B., Burns, C., Cooper-Shepherd, D., De Lorenzi, E., Rodriguez, E., Zhang, H., Ault, J.R., Ferguson, J., Phillips, J.J., Pacholarz, K., Thalassinos, K., Luckau, L., Ashton, L., Durrant, O., Barran, P., Dalby, P., Vicedo, P., Colombo, R., Davis, R., Parakra, R., Upton, R., Hill, S., Wood, V., Soloviev, Z., Quaglia, M., Groves, K., Ashcroft, A.E., Cryar, A., Sula, A., Wallace, B.A., Stocks, B.B., Burns, C., Cooper-Shepherd, D., De Lorenzi, E., Rodriguez, E., Zhang, H., Ault, J.R., Ferguson, J., Phillips, J.J., Pacholarz, K., Thalassinos, K., Luckau, L., Ashton, L., Durrant, O., Barran, P., Dalby, P., Vicedo, P., Colombo, R., Davis, R., Parakra, R., Upton, R., Hill, S., Wood, V., Soloviev, Z., and Quaglia, M.
- Abstract
Measurements of protein higher order structure (HOS) provide important information on stability, potency, efficacy, immunogenicity, and biosimilarity of biopharmaceuticals, with a significant number of techniques and methods available to perform these measurements. The comparison of the analytical performance of HOS methods and the standardization of the results is, however, not a trivial task, due to the lack of reference protocols and reference measurement procedures. Here, we developed a protocol to structurally alter and compare samples of somatropin, a recombinant biotherapeutic, and describe the results obtained by using a number of techniques, methods and in different laboratories. This, with the final aim to provide tools and generate a pool of data to compare and benchmark analytical platforms and define method sensitivity to structural changes. Changes in somatropin HOS, induced by the presence of zinc at increasing concentrations, were observed, both globally and at more localized resolution, across many of the methods utilized in this study and with different sensitivities, suggesting the suitability of the protocol to improve understanding of inter- and cross-platform measurement comparability and assess analytical performance as appropriate. © 2021 The Authors. Published by American Chemical Society.
- Published
- 2021
3. Impact of Bioconjugation on Structure and Function of Antibodies for Use in Immunoassay by Hydrogen-Deuterium Exchange Mass Spectrometry.
- Author
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Luckau L, Groves K, Blencowe C, Scrimshaw S, Dent A, and Quaglia M
- Abstract
Monoclonal antibodies (mAbs) are widely used as analytical components in immunoassays to detect target molecules in applications such as clinical diagnostics, food analysis and drug discovery. Functional groups are often conjugated to lysine or cysteine residues to aid immobilization of mAbs or to enable their detection in an antibody antigen complex. Good assay performance depends on the affinity and specificity of the mAbs for the antigen. The conjugation reaction however can cause higher order structural (HOS) changes and ultimately affect the assay performance. In this study, four differently conjugated mAbs were selected as model systems and characterized by mass spectrometry. Particularly, intact protein analysis by liquid-chromatography mass-spectrometry (LC-MS) was performed to determine the amount and distribution of conjugation. Hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments were carried out for the structural characterization of the conjugated mAbs. Immunoassay experiments were performed to monitor the effects of conjugation on the binding properties of the antibodies selected. Good agreement between the mass spectrometry and binding experiment results was found. Particularly, it was noted that the overall structural flexibility of the antibodies increases upon cysteine conjugation and decreases for lysine conjugation. The conjugation of mAbs with bulky functional groups tends to decrease the deuterium uptake kinetics due to induced steric effects. Overall, this study shows correlations between conjugation, structure and function of immunoassay antibodies and the benefits of mass spectrometry to improve understanding of the conjugation reaction and provide insights that can predict immunoassay performance., Competing Interests: CB, SS, and AD were employed by the Company Fleet Bioprocessing Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Luckau, Groves, Blencowe, Scrimshaw, Dent and Quaglia.)
- Published
- 2022
- Full Text
- View/download PDF
4. Biomacromolecules as tools and objects in nanometrology-current challenges and perspectives.
- Author
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Hashemi P, Luckau L, Mischnick P, Schmidt S, Stosch R, and Wünsch B
- Subjects
- Animals, Biomarkers analysis, Humans, Indicator Dilution Techniques, Mass Spectrometry methods, Microscopy, Fluorescence methods, Nanotechnology methods, Spectrum Analysis, Raman methods, DNA analysis, Polysaccharides analysis, Proteins analysis
- Abstract
Nucleic acids, proteins, and polysaccharides are the most important classes of biopolymers. The inherent properties of biomacromolecules are contrary to those of well-defined small molecules consequently raising a number of specific challenges which become particularly apparent if biomacromolecules are treated as objects in quantitative analysis. At the same time, their specific functional ability of molecular recognition and self-organization (e.g., enzymes, antibodies, DNA) enables us to make biomacromolecules serving as molecular tools in biochemistry and molecular biology, or as precisely controllable dimensional platforms for nanometrological applications. Given the complexity of biomacromolecules, quantitative analysis is not limited to the measurement of their concentration but also involves the determination of numerous descriptors related to structure, interaction, activity, and function. Among the biomacromolecules, glycans set examples that quantitative characterization is not necessarily directed to the measurement of amount-of-substance concentration but instead involves the determination of relative proportions (molar ratios) of structural features for comparison with theoretical models. This article addresses current activities to combine optical techniques such as Raman spectroscopy with isotope dilution approaches to realize reference measurement procedures for the quantification of protein biomarkers as an alternative to mass spectrometry-based techniques. Furthermore, it is explored how established ID-MS protocols are being modified to make them applicable for quantifying virus proteins to measure the HIV viral load in blood samples. As an example from the class of carbohydrates, the challenges in accurate determination of substitution patterns are outlined and discussed. Finally, it is presented that biomacromolecules can also serve as tools in quantitative measurements of dimensions with an example of DNA origami to generate defined dimensional standards to be used for calibration in super-resolution fluorescence microscopy. Graphical abstract Quantitative analysis of biomacromolecules is accompanied with special challenges different from low molecular weight compounds. In addition, they are not only objects but also tools applicable for quantitative measurements.
- Published
- 2017
- Full Text
- View/download PDF
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