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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: Department of Analytical Chemistry, Stockholm University , 2009. , 74 p.
Keyword [en]
Screening, Fingerprinting, metabolomics, Protein drugs, Mammalian Cell lines, LC/ESI-MS, chemometrics
National Category
Analytical Chemistry
Research subject
Analytical Chemistry
Identifiers
URN: urn:nbn:se:su:diva-28921ISBN: 978-91-7155-897-8 (print)OAI: oai:DiVA.org:su-28921DiVA: diva2:227887
Public defence
2009-09-16, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript.Available from: 2009-08-25 Created: 2009-07-20 Last updated: 2009-08-27Bibliographically approved
List of papers
1. Metabolic fingerprinting of rat urine by LC/MS. Part 1. 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. Part 1. Analysis by hydrophilic interaction liquid chromatography-electrospray ionization mass spectrometry
Show others...
2005 (English)In: Journal of chromatography. B, ISSN 1570-0232, E-ISSN 1873-376X, Vol. 828, no 1-2, 9-13 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:su:diva-24518 (URN)10.1016/j.jchromb.2005.07.031 (DOI)
Available from: 2007-11-01 Created: 2007-10-16 Last updated: 2017-12-13Bibliographically approved
2. Conformational studies of a monoclonal antibody, IgG1, by chemical oxidation:Structural analysis by ultrahigh-pressure LC–electrospray ionizationtime-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 ionizationtime-of-flight MS and multivariate data analysis
Show others...
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 ultrahighpressure 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.

Identifiers
urn:nbn:se:su:diva-28891 (URN)10.1016/j.ab.2008.05.054 (DOI)000258313100001 ()
Available from: 2009-07-21 Created: 2009-07-16 Last updated: 2017-12-13Bibliographically approved
3. Extracellular metabolite fingerprinting of spent mammalian cell culture medium in relation to the quality of an expressed recombinant protein
Open this publication in new window or tab >>Extracellular metabolite fingerprinting of spent mammalian cell culture medium in relation to the quality of an expressed recombinant protein
(English)Manuscript (preprint) (Other academic)
Abstract [en]

This study demonstrates methodology for predicting the quality (in terms, for instance, of specific activities) of recombinant proteins expressed by mammalian cell cultures, at upstream process stages, using metabolic fingerprint analysis. Metabolites extracted from samples taken from a culture of a transfected Chinese Hamster Ovary (CHO) cell line expressing a recombinant protein in a bioreactor at various days of the cell culture process were analyzed by ultra high-pressure liquid chromatography-electrospray ionization-time of flight mass spectrometry (UHPLC-ESI-TOFMS). The LC-MS data were processed and the extracted information was correlated with the concentration of the active protein by partial least squares (PLS) regression, which revealed strong correlations between the LC-MS results and the concentration of active protein (R

2 = 0.99). The correlations between the LC-MS data and other parameters (glucose concentration, lactate concentration and the number of viable cells) were also studied. To obtain an overview of the data, Principal Component Analysis (PCA) was applied to the LC-MS data obtained from the samples to observe clustering or separation in the sample set. The PCA indicated that the LC-MS data obtained from samples from different days were significantly separated in temporal order from day 7 to day 28, according to the first Principal Component.

Identifiers
urn:nbn:se:su:diva-28900 (URN)
Available from: 2009-07-21 Created: 2009-07-16 Last updated: 2010-01-14Bibliographically 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-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-_ 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.

Identifiers
urn:nbn:se:su:diva-28893 (URN)10.1016/j.jasms.2009.01.008 (DOI)000266466600015 ()
Available from: 2009-07-21 Created: 2009-07-16 Last updated: 2017-12-13Bibliographically approved

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