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High-throughput profiling of N-linked oligosaccharides in therapeutic antibodies using a microfluidic CD platform and MALDI-MS
Stockholm University, Faculty of Science, Department of Analytical Chemistry.
Stockholm University, Faculty of Science, Department of Analytical Chemistry.
2011 (English)In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 399, no 4, 1601-1611 p.Article in journal (Refereed) Published
Abstract [en]

Recombinant therapeutic antibodies have shown a great potential in the treatment of several severe medical conditions such as cancer and autoimmune diseases. Glycosylation plays a critical role in biological activity and immunogenic properties of these compounds. The analysis of glycan profiles is therefore necessary in many steps of the development and manufacturing process from early development to quality control of the final product. In this paper, a fast, parallel, and robust sample preparation platform for glycosylation profiling using a microfluidic compact disc (CD) is presented. A sequential process including selective capture of antibody from a crude cell supernatant using protein A beads, enzymatic release of glycans, purification with a graphitized carbon black column, and crystallisation for MALDI-TOF analysis were performed on the CD. Glycosylation profiles of an antibody intended for therapeutic use produced in two different cell lines were compared.

Place, publisher, year, edition, pages
2011. Vol. 399, no 4, 1601-1611 p.
Keyword [en]
Microfluidics/Microfabrication, Microfluidic CD, Glycosylation profiling, Recombinant therapeutic antibodies, MALDI-MS, Biotechnological products
National Category
Analytical Chemistry
Identifiers
URN: urn:nbn:se:su:diva-67492DOI: 10.1007/s00216-010-4469-yISI: 000286599200021OAI: oai:DiVA.org:su-67492DiVA: diva2:470514
Note

authorCount :3

Available from: 2011-12-29 Created: 2011-12-28 Last updated: 2017-12-08Bibliographically approved
In thesis
1. Compact-disc microfluidic methods for characterization of therapeutic antibodies: Analysis of post-translational modifications
Open this publication in new window or tab >>Compact-disc microfluidic methods for characterization of therapeutic antibodies: Analysis of post-translational modifications
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Characterization of post-translational modifications (PTMs) of therapeutic proteins is very important during the bioprocess development to maintain desired product quality and during the submission process to regulatory authorities for product approval. Monitoring glycosylation in pharmacokinetic studies can be useful to evaluate the dependence of clearance rates on different glycoforms. The cost and efficiency of characterization affect the speed to market of biopharmaceutical proteins. A reduction in the number of manual processing steps, cost of reagents and consumption of sample, as well as the time required for chemical analysis, is therefore necessary.

The research presented in this thesis is focused on the potential of using microfluidic discs for automated, miniaturized, parallel and rapid sample preparation for PTM characterization of therapeutic monoclonal antibodies. Paper I describes the method development for N-linked glycosylation profiling. Several sample preparation steps have been performed in an integrated process in the microfluidic compact disc (CD). Paper II demonstrates the use of the method presented in paper I in combination with multivariate statistics for discrimination of glycosylation profiles of different therapeutic antibodies and simulation of a real case of quality control. Paper III is focused on a method for monitoring changes in glycosylation profiles of therapeutic antibodies in serum over time by incubation with an exoglycosidase enzyme. Paper IV describes the method for peptide mapping of therapeutic antibodies. In addition, recent work (unpublished results) assesses the potential of this method for methionine oxidation detection.

The developed methods were fast, robust with low sample/reagent consumption. Generation of glycosylation profile data for one sample was established in approximately 2 h. The amount of samples and antigens loaded into the CD platform for one replicate was less than 0.3 μg and approximately 0.06 μg, respectively. Furthermore, considering the parallel function of the CD, conducting the analysis for 54 samples can be completed within a day.

Place, publisher, year, edition, pages
Stockholm: Department of Analytical Chemistry, Stockholm University, 2012. 120 p.
Keyword
Microfluidic CD, therapeutic antibodies, post-translational modifications, glycosylation profiling, multivariate statistical analysis, MALDI-MS
National Category
Chemical Sciences
Research subject
Analytical Chemistry
Identifiers
urn:nbn:se:su:diva-83355 (URN)978-91-7447-607-1 (ISBN)
Public defence
2013-01-18, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.

Available from: 2012-12-27 Created: 2012-12-10 Last updated: 2017-11-16Bibliographically approved

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