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Methods for structural studies of an antibody, screening metabolites in rat urine and analysis of spent cell cultivation media using LC/ESI-MS and chemometrics
Stockholm University, Faculty of Science, Department of Analytical Chemistry.
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis describes bioanalytical methods for generating fingerprints of biological systems for extracting relevant information with (protein) drugs in focus. Similarities and differences between samples can reveal the hidden relevant information, which can be used to optimize the production and facilitate the quality control of such protein drugs during their development and manufacture. Metabolic fingerprinting and multivariate data analysis (MVDA) can also facilitate early diagnosis of diseases and the effects and toxicity of drugs.

Currently, several protein drugs are available on the global market. Nevertheless, despite, the success of such biotherapeutics significant challenges remain to be overcome in maintaining their stability and efficacity throughout their production cycle and long-term storage. The native structure and functional activity of therapeutic proteins is affected by many variables from production to delivery, incl. variables assoc. with conditions in bioreactors, purification, storage and delivery. Thus, part of the work underlying this thesis focused on structural analysis of a protein drug using chemical labeling, peptide mapping, and evaluation of the charge state distributions of the whole protein generated by ESI. The other part focuses on non-targeted metabolomics with a view to optimizing the cell cultivation process and assessment of the drug’s toxicity. A combination of appropriate analytical methods and MVDA is needed to find markers that can facilitate optimization of the cultivation system and expression of the target proteins in early stages of process development. Rapid methods for characterizing the protein drugs in different stages of the process are also required for quality control.

In order to obtain high quality fingerprints analytical separation techniques with high resolution (such as HPLC or UHPLC) and sensitive analytical detection techniques (such as ESI, quadrupole or TOF MS) have been used, singly or in combination.

Place, publisher, year, edition, pages
Stockholm University , 2009. , 74 p.
Keyword [en]
Screening, Fingerprinting, metabolomics, Protein drugs, Mammalian Cell lines, LC/ESI-MS, chemometrics
National Category
Analytical Chemistry
Identifiers
URN: urn:nbn:se:kth:diva-87121ISBN: 978-91-7155-897-8 (print)OAI: oai:DiVA.org:kth-87121DiVA: diva2:501408
Note
QC 20120215Available from: 2012-02-15 Created: 2012-02-14 Last updated: 2012-02-15Bibliographically approved
List of papers
1. Metabolic fingerprinting of rat urine by LC/MS.: Part1. Analysis by hydrophilic interaction liquid chromatography-electrospray ionization mass spectrometry
Open this publication in new window or tab >>Metabolic fingerprinting of rat urine by LC/MS.: Part1. Analysis by hydrophilic interaction liquid chromatography-electrospray ionization mass spectrometry
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2005 (English)In: Journal of Chromatography B: Biomedical Sciences and Applications, ISSN 1387-2273, E-ISSN 1878-5603, Vol. 828, no 1-2, 9-13 p.Article in journal (Refereed) Published
Abstract [en]

Complex biological samples, such as urine, contain a very large number of endogenous metabolites reflecting the metabolic state of an organism. Metabolite patterns can provide a comprehensive signature of the physiological state of an organism as well as insights into specific biochemical processes. Although the metabolites excreted in urine are commonly highly polar, the samples are generally analyzed using reversed-phase liquid chromatography mass spectrometry (RP-LC/MS). In Part I of this work, a method for detecting highly polar metabolites by hydrophilic interaction liquid chromatography-electrospray ionization mass spectrometry (HILIC/ESI-MS) is described as a complement to RP-LC/ESI-MS. In addition, in an accompanying paper (Part 2), different multivariate approaches to extracting information from the resulting complex data are described to enable metabolic fingerprints to be obtained. The coverage of the method for the screening of as many metabolites as possible is highly improved by analyzing the urine samples using both a C-18 column and a ZIC (R)-HILIC column. The latter was found to be a good alternative when analyzing highly polar compounds, e.g., hydroxyproline and creatinine, to columns typically used for reversed-phase liquid chromatography. (c) 2005 Elsevier B.V. All rights reserved.

National Category
Analytical Chemistry
Identifiers
urn:nbn:se:kth:diva-87083 (URN)10.1016/j.jchromb.2005.07.031 (DOI)000233944100002 ()
Note
QC 20120219Available from: 2012-02-14 Created: 2012-02-14 Last updated: 2017-12-07Bibliographically approved
2. Conformational studies of a monoclonal antibody, IgG1, by chemical oxidation: Structural analysis by ultrahigh-pressure LC-electrospray ionization time-of-flight MS and multivariate data analysis
Open this publication in new window or tab >>Conformational studies of a monoclonal antibody, IgG1, by chemical oxidation: Structural analysis by ultrahigh-pressure LC-electrospray ionization time-of-flight MS and multivariate data analysis
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2008 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 380, no 2, 155-163 p.Article in journal (Refereed) Published
Abstract [en]

We describe the development of a method in which protein oxidation by H2O2 followed by ultrahigh-pressure liquid chromatography (UHPLC) coupled with electrospray ionization time-of-flight mass spectrometry (ESI-ToFMS) and multivariate analysis are used to detect alterations in conformational states of proteins. In the study reported here, an IgG1 monoclonal antibody in native and denatured conformational states was oxidized by treatment with hydrogen peroxide. Peptide fragments generated by tryptic digestion were then analyzed by UHPLC-ESI-ToFMS. After reducing noise and extracting peaks from the LC-MS data using MzExplorer, software developed in-house and based on Matlab, we were able to distinguish peptides arising from the native and denatured states of the oxidized protein by principal component analysis. Peptides containing residues, which are inclined to undergo oxidation, such as methionine, are founded to be particularly important in this approach. We believe that the methodology could facilitate attempts to characterize the conformational states of recombinant monoclonal antibodies and other proteins.

Keyword
Mass spectrometry, Methionine, Peptide mapping, Principal component analysis, Protein conformation, Recombinant monoclonal antibody, Ultrahigh-pressure liquid chromatography
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:kth:diva-87062 (URN)10.1016/j.ab.2008.05.054 (DOI)000258313100001 ()18577369 (PubMedID)
Note
QC 20120214Available from: 2012-02-14 Created: 2012-02-14 Last updated: 2017-12-07Bibliographically approved
3. Extracellular metabolic fingerprinting of spent mammalian cell culture medium analysis in relation to the quality of an expressed recombinant protein
Open this publication in new window or tab >>Extracellular metabolic fingerprinting of spent mammalian cell culture medium analysis in relation to the quality of an expressed recombinant protein
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(English)Manuscript (preprint) (Other academic)
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:kth:diva-87115 (URN)
Note
QC 20120302Available from: 2012-02-14 Created: 2012-02-14 Last updated: 2012-03-02Bibliographically approved
4. Discrimination among IgG1-Κ monoclonal antibodies produced by two cell lines using charge state distributions in nanoESI-TOF mass spectra
Open this publication in new window or tab >>Discrimination among IgG1-Κ monoclonal antibodies produced by two cell lines using charge state distributions in nanoESI-TOF mass spectra
2009 (English)In: Journal of the American Society for Mass Spectrometry, ISSN 1044-0305, E-ISSN 1879-1123, Vol. 20, no 6, 1030-1036 p.Article in journal (Refereed) Published
Abstract [en]

Charge state distributions (CSDs) of proteins in nanoESI mass spectra are affected by the instrumental settings and experimental conditions, in addition to the conformations of the proteins in the analyzed solutions. In the presented study, instrumental and experimental parameters-the desolvation gas flow rate, temperature, pH, buffer (ammonium acetate), and organic modifier (methanol) concentrations-were optimized according to a reduced central composite face experimental design to maximize the separation of CSDs of monoclonal IgG1-kappa antibodies produced by two production systems (CHO and GS-NS0 cell lines). Principal component analysis and Fisher linear discriminant analysis were then used to reduce the dimensions of the acquired dataset and quantify the separation of the protein classes, respectively. The results show that the IgG1-kappa molecules produced by the two production systems can be clearly distinguished using the described approach, which could be readily applied to other proteins and production systems.

National Category
Analytical Chemistry
Identifiers
urn:nbn:se:kth:diva-87076 (URN)10.1016/j.jasms.2009.01.008 (DOI)000266466600015 ()
Note
QC 20120214Available from: 2012-02-14 Created: 2012-02-14 Last updated: 2017-12-07Bibliographically approved

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