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
Parallel sample preparation of proteins, from crude samples to crystals ready for MALDI-MS, in an integrated microfluidic system
Stockholm University, Faculty of Science, Department of Analytical Chemistry.
Stockholm University, Faculty of Science, Department of Analytical Chemistry.
2010 (English)In: Journal of chromatography. B, ISSN 1570-0232, E-ISSN 1873-376X, Vol. 878, no 28, 2803-2810 p.Article in journal (Refereed) Published
Abstract [en]

A microfluidic structure is presented where selective capture of proteins in complex samples, followed by clean-up, enzymatic processing, and MALDI-MS sample preparation of peptides generated, can be performed. The structure uses an affinity column to capture the protein while all other components in the sample are disposed of. The protein of interest is then eluted from the affinity column and captured on a second column on which the enzymatic processing is performed. Salts and hydrophilic contaminants are then removed before the products from the enzymatic reaction are eluted together with a suitable MALDI matrix and the solvent evaporated in a designated MALDI target structure. All steps can be performed automatically in 54 parallel microstructures on a microfluidic compact disc. The process is demonstrated by the selective capture and tryptic digest of recombinant IgG molecules from samples containing other proteins: an excess of bovine serum albumin or spent cell culture media.

Place, publisher, year, edition, pages
2010. Vol. 878, no 28, 2803-2810 p.
Keyword [en]
Microfluidics, Microfluidic CD, MALDI-MS, Recombinant therapeutic antibodies, Tryptic digestion, Peptide mapping
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:su:diva-51445DOI: 10.1016/j.jchromb.2010.08.031ISI: 000283687200021OAI: oai:DiVA.org:su-51445DiVA: diva2:385677
Note
authorCount :4Available from: 2011-01-12 Created: 2011-01-10 Last updated: 2017-12-11Bibliographically 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

Other links

Publisher's full text

Search in DiVA

By author/editor
Thuy, Tran ThiThorsén, Gunnar
By organisation
Department of Analytical Chemistry
In the same journal
Journal of chromatography. B
Natural Sciences

Search outside of DiVA

GoogleGoogle Scholar

doi
urn-nbn

Altmetric score

doi
urn-nbn
Total: 85 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