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The Influence of the Degree of Cross-linking, Type of Ligand and Support on the Chemical Stability of Chromatography Media Intended for protein Purification
1998 (English)In: Process Biochemistry, ISSN 1359-5113, E-ISSN 1873-3298, Vol. 33, no 1, 47-55 p.Article in journal (Refereed) Published
Abstract [en]

The release of organic compounds from different liquid chromatography media in static conditions has been analysed with a total organic carbon (TOC) analyser. TOC results show that chemical stability increases with the degree of cross-linking in agarose beaded chromatography media and thus extend the working pH-range of the media. Of the unsubstituted chromatography media investigated, Sepharose® 6B, Sepharose CL-6B, Sepharose 4 Fast Flow, Sepharose 6 Fast Flow and Sepharose High Performance, the latter was the most stable medium. Sepharose High Performance releases only about 0·06% of its total carbon content after 1 week in 0·01 m HCl. Agarose beads are more stable to basic conditions (pH 14) compared with acidic conditions (pH 2). From UV spectroscopic and gel filtration results it was found that all Sepharose media release low amounts of 5-(hydroxymethyl)-2-furaldehyde and agarose fragments in acidic conditions. To investigate the effect of different ligands on chemical stability Q Sepharose 6 Fast Flow, DEAE Sepharose 6 Fast Flow, SP Sepharose 6 Fast Flow, CM Sepharose 6 Fast Flow, Phenyl Sepharose 6 Fast Flow, Octyl Sepharose 4 Fast Flow media were also studied under static conditions. In basic conditions it was found that all these chromatography media release carbon compounds to a higher extent than the unsubstituted Sepharose support. In addition, Hofmann elimination of Q and DEAE groups contributes to the decrease in the carbon content of the corresponding anion exchangers. During exposure to acidic conditions (pH 2) the release of carbon compounds was lower than the release from the support to which the ligands were coupled. The exceptions are Octyl Sepharose 4 Fast Flow and SP Sepharose 6 Fast Flow. In the case of Octyl Sepharose 4 Fast Flow, the ligand did not seem to influence chemical stability, whereas the SP group increases the degradation of the Sepharose support. In the case of SP Sepharose 6 Fast Flow the stability in acidic conditions can be improved by increasing the ionic strength. Anion exchangers based on different support polymers (agarose-, polystyrene-, methacrylate- and polyvinyl-based matrixes) were studied under static conditions. Agarose-based anion exchanger was the most stable in basic conditions (pH 14). In acidic conditions (pH 2) the chemical stability was about the same for many different anion exchangers.

Place, publisher, year, edition, pages
1998. Vol. 33, no 1, 47-55 p.
National Category
Analytical Chemistry
URN: urn:nbn:se:uu:diva-234604DOI: 10.1016/S0032-9592(97)00068-XOAI: oai:DiVA.org:uu-234604DiVA: diva2:757848
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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