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Discrimination between glycosylation patterns of therapeutic antibodies using a microfluidic platform, MALDI-MS and multivariate statistics
Stockholm University, Faculty of Science, Department of Analytical Chemistry.
Stockholm University, Faculty of Science, Department of Analytical Chemistry.
Stockholm University, Faculty of Science, Department of Analytical Chemistry.
Stockholm University, Faculty of Science, Department of Analytical Chemistry.
2012 (English)In: Journal of Pharmaceutical and Biomedical Analysis, ISSN 0731-7085, E-ISSN 1873-264X, Vol. 70, 47-52 p.Article in journal (Refereed) Published
Abstract [en]

Optimal glycosylation with respect to the efficacy, serum half-life time, and immunogenic properties is essential in the generation of therapeutic antibodies. The glycosylation pattern can be affected by several different parameters during the manufacture of antibodies and may change significantly over cultivation time. Fast and robust methods for determination of the glycosylation patterns of therapeutic antibodies are therefore needed. We have recently presented an efficient method for the determination of glycans on therapeutic antibodies using a microfluidic CD platform for sample preparation prior to matrix-assisted laser-desorption mass spectrometry analysis. In the present work, this method is applied to analyse the glycosylation patterns of three commercially available therapeutic antibodies and one intended for therapeutic use. Two of the antibodies produced in mouse myeloma cell line (SP2/0) and one produced in Chinese hamster ovary (CHO) cells exhibited similar glycosylation patterns but could still be readily differentiated from each other using multivariate statistical methods. The two antibodies with most similar glycosylation patterns were also studied in an assessment of the method's applicability for quality control of therapeutic antibodies. The method presented in this paper is highly automated and rapid. It can therefore efficiently generate data that helps to keep a production process within the desired design space or assess that an identical product is being produced after changes to the process.

Place, publisher, year, edition, pages
2012. Vol. 70, 47-52 p.
Keyword [en]
Microfluidic CD, MALDI-TOF-MS, Recombinant antibodies, Glycosylation, Multivariate statistical analysis
National Category
Analytical Chemistry Pharmacology and Toxicology
Research subject
Analytical Chemistry
Identifiers
URN: urn:nbn:se:su:diva-83031DOI: 10.1016/j.jpba.2012.05.020ISI: 000309314600007OAI: oai:DiVA.org:su-83031DiVA: diva2:574406
Note

AuthorCount:4;

Available from: 2012-12-05 Created: 2012-12-03 Last updated: 2017-12-07Bibliographically 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
2. Data analysis of non-targeted mass spectrometry experiments
Open this publication in new window or tab >>Data analysis of non-targeted mass spectrometry experiments
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Data processing tools are valuable to the analytical chemist as they can speed up the analysis, and sometimes solve problems that are not feasible to solve in a traditional manner. However, the complexity of many data processing tools can make their use daunting for the inexperienced user.

This thesis includes two applications and two tools for data processing. The first application focuses on minimizing the manual input, reducing the time required for a simple task. The second application required more manual input, in the form of parameter selection, but process far more data.  The data processing tools both include features that simplify the manual work required. The first by including visual diagnostics tools that helps in setting the parameters. The second via internal validation that makes the tool’s process more robust and reliable, and thereby less sensitive to small changes in the parameters.

No matter how good or precise a data processing tool is, if it is so cumbersome that it is not used by the analytical chemists that need it, it is useless. Therefore, the main focus of this thesis is to make data processing easier.

Place, publisher, year, edition, pages
Stockholm: Department of Environmental Science and Analytical Chemistry, Stockholm University, 2015
Keyword
Analytical chemistry, Chemometrics, mass spectrometry, HPLC, metabolomics
National Category
Analytical Chemistry
Research subject
Analytical Chemistry
Identifiers
urn:nbn:se:su:diva-116820 (URN)978-91-7649-175-1 (ISBN)
Public defence
2015-06-08, Magnelisalen, 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 4: Submitted.

Available from: 2015-05-15 Created: 2015-04-28 Last updated: 2015-05-25Bibliographically approved

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