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Chemical characterisation of different separation media based on agarose by static time-of-flight secondary ion mass spectrometry
2004 (English)In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1023, no 1, 49-56 p.Article in journal (Refereed) Published
Abstract [en]

In this paper, the novel application of time-of-flight secondary ion mass spectrometry (TOF-SIMS) for qualitative and semi-quantitative investigation of the surface chemistry of separation media based on beaded agarose is reported. Five different media were studied: DEAE Sepharose Fast Flow, Q Sepharose Fast Flow, SP Sepharose Fast Flow, Phenyl Sepharose Fast Flow at ligand densities between 7 and 33% (w/w) and the base matrix Sepharose 6 Fast Flow. The obtained TOF-SIMS spectra reveal significant chemical information regarding the ligands (DEAE, Q, SP and Phenyl) which are covalently attached to the agarose-based matrix Sepharose 6 Fast Flow. For the anion-exchange media (DEAE and Q Sepharose Fast Flow), the positive TOF-SIMS spectra yielded several strong characteristic fragment peaks from the amine ligands. Structural information was obtained, e.g. from the peak at m/z 173.20, originating from the ion structure [(C2H5)2NCH2CH2NH(C2H5)2l+, which shows that the ligand in DEAE Sepharose Fast Flow is composed of both tertiary and quaternary amines. The positive spectrum of Phenyl Sepharose Fast Flow contained major fragments both from the base matrix and the ligand. The cation-exchanger (SP Sepharose Fast Flow) gave rise to a positive spectrum resembling that of the base matrix (Sepharose 6 Fast Flow) but with a different intensity pattern of the matrix fragments. In addition, peaks with low intensity at m/z 109.94, 125.94 and 139.95 corresponding to Na2SO2+, Na2SO3+ and Na2SO3CH2+, respectively, were observed. The positive TOF-SIMS spectrum of Sepharose 6 Fast Flow contains a large number of fragments in the mass range up to m/z 200 identified as CxHyOz and CxHy structures. The results clearly show that positive TOF-SIMS spectra of different media based on Sepharose 6 Fast Flow are strongly influenced by the ligand coupled to the matrix. The negative TOF-SIMS spectra contained several ligand-specific, characteristic peaks for the cation-exchanger, having sulphonate as the ion-exchange group. Negative fragments such as S-, SO-, SO2-, SO3-, C2H3SO3-, C3H5SO3- and OC3H5SO3- were observed. Phenyl Sepharose Fast Flow, which has an uncharged group (Phenyl) coupled to the agarose matrix yielded one ligand-related peak corresponding to the C6H5O- fragment. DEAE and Q ligands could only be identified by the appearance of the fragments CN- and CNO- in the negative spectrum. However, a strong peak corresponding to the counter ion (Cl-) was observed. TOF-SIMS analysis can also be used for the investigation of residues from the coupling procedure that bonds the ligands to the matrix. One example is the observation of bromine peaks in the negative spectrum of Q Sepharose Fast Flow. Furthermore, it has also been shown that different ligand concentrations of Phenyl Sepharose Fast Flow can easily be detected by TOF-SIMS analysis. Information regarding the difference between the ligand density on the surface of the beads and in the bulk can also be obtained. However, spectra registered on the outermost surface and on the pore surface (crushed beads) of DEAE Sepharose Fast Flow clearly show that the agarose and the DEAE groups are homogeneously distributed in the beads.

Place, publisher, year, edition, pages
2004. Vol. 1023, no 1, 49-56 p.
National Category
Analytical Chemistry
URN: urn:nbn:se:uu:diva-234613DOI: 10.1016/j.chroma.2003.10.008PubMedID: 14760849OAI: oai:DiVA.org:uu-234613DiVA: diva2:757863
Available from: 2014-10-23 Created: 2014-10-21 Last updated: 2015-02-03Bibliographically approved
In thesis
1. Characterisation of Chromatography Media Aimed for Purification of Biomolecules
Open this publication in new window or tab >>Characterisation of Chromatography Media Aimed for Purification of Biomolecules
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Chromatography media (resins) are very important for and widely used by the biopharma industry in large scale production of biopharmaceuticals, e.g. monoclonal antibodies. Today there are several hundred biopharmaceuticals released globally on the healthcare market. This thesis discusses various strategies and methods for the characterisation of chemical and functional stability of chromatography media. In addition, various analytical techniques used in these areas were evaluated and applied. Further, more specific physical and chemical characterisation methods were evaluated and applied to explore different properties of various chromatography media.

In Papers I-III, established methodologies for performing chemical and functional stability studies were used. Mainly agarose-based chromatography media were investigated. For fast screening of the chemical stability, the total organic carbon analysis technique was evaluated and applied. This technique that measures the carbon leakage from the chromatography media at different conditions, proved to be very suitable and robust. For detection and/or identification of leakage compounds responsible for or for part of the measured carbon leakage, different methods such as (high performance) liquid chromatography and gas chromatography mass spectrometry were used.

In Papers IV-VII, different properties (i.e. functional performance, ligand content and surface chemistry) were evaluated for different agarose-based chromatography media. Standard chromatographic methods (ion exchange chromatography) and spectroscopic methods (e.g. Fourier transform infrared spectroscopy and time-of-flight secondary ion mass spectrometry) were evaluated and applied. Chemometric methods were used for efficient evaluation of data.

Information of chemical, functional and leakage data of chromatography media are valuable and important for the biopharmaceutical companies to be able to fulfil the regulatory requirements of biopharmaceuticals. In addition, information of various chemical, functional and physical properties of chromatography media is likewise important during development and set up of new biopharmaceutical processes.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. 73 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1202
Chromatography, Media, Characterisation, Stability
National Category
Analytical Chemistry
Research subject
Chemistry with specialization in Analytical Chemistry
urn:nbn:se:uu:diva-234743 (URN)978-91-554-9102-4 (ISBN)
Public defence
2014-12-19, B22, BMC, Uppsala, 13:15 (English)
Available from: 2014-11-28 Created: 2014-10-23 Last updated: 2015-02-03

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