Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Glycosylation profiling of therapeutic antibodies in serum samples 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.
(English)Manuscript (preprint) (Other academic)
Abstract [en]

The serum clearance rate of therapeutic antibodies is important as it affects the clinical efficacy, required dose and dose frequency. The glycosylation of antibodies has in some studies been shown to have an impact on the elimination rates in vivo. Monitoring changes to the glycan profiles in pharmacokinetics studies can reveal whether the clearance rates of the therapeutic antibodies depend on the different glycoforms, thereby providing useful information for improvement of the drugs. In this paper a novel method for glycosylation analysis of therapeutic antibodies in serum samples is presented. A microfluidic CD platform in combination with MALDI-MS was used to monitor changes to the glycosylation profiles of samples incubated in vitro. Antibodies were selectively purified from serum using immunoaffinity capture on immobilized target antigens. The glycans were enzymatically released, purified and finally analyzed by MALDI-TOF-MS. To simulate changes to glycan profiles after administration in vivo, a therapeutic antibody was incubated in serum with the enzyme α1-2,3 mannosidase to artificially reduce the amount of the high mannose glycoforms. Glycan profiles were monitored at specific intervals during the incubation. The relative abundance of the high mannose 5 glycoform was clearly found to decrease and simultaneously, that of high mannose 4 increased over the incubation period. The method can be performed in a rapid, parallel and automated fashion for glycosylation profiling consuming low amounts of samples and reagents. This can contribute to less labor work and reduced cost of the studies of therapeutic antibodies glycosylation in vitro and in vivo.Keywords:

Keyword [en]
Microfluidic CD, MALDI-TOF-MS, serum clearance rate, glycosylation, therapeutic antibodies
National Category
Natural Sciences
Research subject
Analytical Chemistry
Identifiers
URN: urn:nbn:se:su:diva-83525OAI: oai:DiVA.org:su-83525DiVA: diva2:576141
Available from: 2012-12-12 Created: 2012-12-12 Last updated: 2012-12-13Bibliographically 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

Open Access in DiVA

No full text

Search in DiVA

By author/editor
Tran, ThuyThorsén, Gunnar
By organisation
Department of Analytical Chemistry
Natural Sciences

Search outside of DiVA

GoogleGoogle Scholar

urn-nbn

Altmetric score

urn-nbn
Total: 38 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf