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Compact-disc microfluidic methods for characterization of therapeutic antibodies: Analysis of post-translational modifications
Stockholm University, Faculty of Science, Department of Analytical Chemistry.
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 In addition, recent work (unpublished results) assesses the potential of this method for methionine oxidation detection.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 [en]
Microfluidic CD, therapeutic antibodies, post-translational modifications, glycosylation profiling, multivariate statistical analysis, MALDI-MS
National Category
Chemical Sciences
Research subject
Analytical Chemistry
Identifiers
URN: urn:nbn:se:su:diva-83355ISBN: 978-91-7447-607-1 (print)OAI: oai:DiVA.org:su-83355DiVA: diva2:575375
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
List of papers
1. High-throughput profiling of N-linked oligosaccharides in therapeutic antibodies using a microfluidic CD platform and MALDI-MS
Open this publication in new window or tab >>High-throughput profiling of N-linked oligosaccharides in therapeutic antibodies using a microfluidic CD platform and MALDI-MS
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.

Keyword
Microfluidics/Microfabrication, Microfluidic CD, Glycosylation profiling, Recombinant therapeutic antibodies, MALDI-MS, Biotechnological products
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:su:diva-67492 (URN)10.1007/s00216-010-4469-y (DOI)000286599200021 ()
Note

authorCount :3

Available from: 2011-12-29 Created: 2011-12-28 Last updated: 2017-12-08Bibliographically approved
2. Discrimination between glycosylation patterns of therapeutic antibodies using a microfluidic platform, MALDI-MS and multivariate statistics
Open this publication in new window or tab >>Discrimination between glycosylation patterns of therapeutic antibodies using a microfluidic platform, MALDI-MS and multivariate statistics
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.

Keyword
Microfluidic CD, MALDI-TOF-MS, Recombinant antibodies, Glycosylation, Multivariate statistical analysis
National Category
Analytical Chemistry Pharmacology and Toxicology
Research subject
Analytical Chemistry
Identifiers
urn:nbn:se:su:diva-83031 (URN)10.1016/j.jpba.2012.05.020 (DOI)000309314600007 ()
Note

AuthorCount:4;

Available from: 2012-12-05 Created: 2012-12-03 Last updated: 2017-12-07Bibliographically approved
3. Glycosylation profiling of therapeutic antibodies in serum samples using a microfluidic CD platform and MALDI-MS
Open this publication in new window or tab >>Glycosylation profiling of therapeutic antibodies in serum samples using a microfluidic CD platform and MALDI-MS
(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
Microfluidic CD, MALDI-TOF-MS, serum clearance rate, glycosylation, therapeutic antibodies
National Category
Natural Sciences
Research subject
Analytical Chemistry
Identifiers
urn:nbn:se:su:diva-83525 (URN)
Available from: 2012-12-12 Created: 2012-12-12 Last updated: 2012-12-13Bibliographically approved
4. Parallel sample preparation of proteins, from crude samples to crystals ready for MALDI-MS, in an integrated microfluidic system
Open this publication in new window or tab >>Parallel sample preparation of proteins, from crude samples to crystals ready for MALDI-MS, in an integrated microfluidic system
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.

Keyword
Microfluidics, Microfluidic CD, MALDI-MS, Recombinant therapeutic antibodies, Tryptic digestion, Peptide mapping
National Category
Natural Sciences
Identifiers
urn:nbn:se:su:diva-51445 (URN)10.1016/j.jchromb.2010.08.031 (DOI)000283687200021 ()
Note
authorCount :4Available from: 2011-01-12 Created: 2011-01-10 Last updated: 2012-12-10Bibliographically approved

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