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Characterisation of Chromatography Media Aimed for Purification of Biomolecules
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. (Analytisk kemi)
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.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1202
Keyword [en]
Chromatography, Media, Characterisation, Stability
National Category
Analytical Chemistry
Research subject
Chemistry with specialization in Analytical Chemistry
Identifiers
URN: urn:nbn:se:uu:diva-234743ISBN: 978-91-554-9102-4 (print)OAI: oai:DiVA.org:uu-234743DiVA: diva2:759759
Public defence
2014-12-19, B22, BMC, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2014-11-28 Created: 2014-10-23 Last updated: 2015-02-03
List of papers
1. The Influence of the Degree of Cross-linking, Type of Ligand and Support on the Chemical Stability of Chromatography Media Intended for protein Purification
Open this publication in new window or tab >>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.

National Category
Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-234604 (URN)10.1016/S0032-9592(97)00068-X (DOI)
Available from: 2014-10-23 Created: 2014-10-21 Last updated: 2017-12-05Bibliographically approved
2. Characterization of the Chemical and Functional Stability of DEAE Sepharose Fast Flow
Open this publication in new window or tab >>Characterization of the Chemical and Functional Stability of DEAE Sepharose Fast Flow
1993 (English)In: Process Biochemistry, ISSN 1359-5113, E-ISSN 1873-3298, Vol. 28, no 4, 223-230 p.Article in journal (Refereed) Published
Abstract [en]

The release of amines from the ion-exchange groups in DEAE Sepharose® Fast Flow has been studied under static and column conditions. The leakage compounds have been identified and quantified by gas chromatography—mass spectrometry. It was shown that the main leakage product under acidic (pH 1) and basic conditions (pH 14) was N,N,N′,N′-tetraethylethylenediamine. Three other amines were also identified, namely N,N,N′-triethylethylenediamine, diethylaminoethanol and diethylamine. The leakage of amines from DEAE Sepharose Fast Flow treated at pH 1 or 14 for 672 h at 40°C corresponds to a reduction of only 1% of the total ion-exchange capacity.

The functional stability of DEAE Sepharose Fast Flow was also studied by separation of a protein mixture during repeated cleaning-in-place treatments with 0·10 m HCl or 1·0 m NaOH. The separation behaviour was unaltered after the gel had been treated for a total contact time of 672 h with 0·10 m HCl or 1·0 m NaOH.

The clearance of ethanol from a DEAE Sepharose Fast Flow column stored in a 20% ethanol aqueous solution was also studied.

National Category
Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-234606 (URN)10.1016/0032-9592(93)80038-I (DOI)
Available from: 2014-10-23 Created: 2014-10-21 Last updated: 2017-12-05Bibliographically approved
3. A Systematic Approach to Screening Ion-Exchange Chromatography Media for Process Development
Open this publication in new window or tab >>A Systematic Approach to Screening Ion-Exchange Chromatography Media for Process Development
1996 (English)In: Biopharm, ISSN 1040-8304, Vol. 9, no 8, 42-45 p.Article in journal (Refereed) Published
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-234607 (URN)
Available from: 2014-10-23 Created: 2014-10-21 Last updated: 2017-12-05Bibliographically approved
4. Evaluation of Several Anion-exchange Media for process Separations Using a Variety of Proteins and Aromatic Acids
Open this publication in new window or tab >>Evaluation of Several Anion-exchange Media for process Separations Using a Variety of Proteins and Aromatic Acids
2001 (English)In: International Journal of Bio-Chromatography, ISSN 1068-0659, Vol. 6, 285-301 p.Article in journal (Refereed) Published
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-234609 (URN)
Available from: 2014-10-23 Created: 2014-10-21 Last updated: 2015-02-03Bibliographically approved
5. The multivariate use of vibrational spectroscopy for chemical characterisation of chromatography media
Open this publication in new window or tab >>The multivariate use of vibrational spectroscopy for chemical characterisation of chromatography media
2002 (English)In: Vibrational Spectroscopy, ISSN 0924-2031, E-ISSN 1873-3697, Vol. 29, 133-138 p.Article in journal (Refereed) Published
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-234611 (URN)
Available from: 2014-10-23 Created: 2014-10-21 Last updated: 2017-12-05Bibliographically approved
6. Surface Chemical Analysis of Carbohydrate Materials Used for Chromatography Media by Time-of-Flight Secondary Ion Mass Spectrometry
Open this publication in new window or tab >>Surface Chemical Analysis of Carbohydrate Materials Used for Chromatography Media by Time-of-Flight Secondary Ion Mass Spectrometry
2004 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 76, no 7, 1857-1864 p.Article in journal (Refereed) Published
Abstract [en]

The surface chemical structure of two raw materials (agarose and dextran) and four base matrixes used in the manufacture of chromatography media were analyzed using time-of-flight secondary ion mass spectrometry (TOF-SIMS). The results show that the small differences in molecular structure between these materials result in significant differences in the TOF-SIMS spectra and that these differences can be identified and quantified using either of two different approaches. In a novel approach, fragment ion distributions were extracted from the TOF-SIMS spectra for each material, providing an immediate and systematic overview of the spectral features. Difference fragment distributions were used to highlight spectral differences between the materials. The results of the fragment ion distribution analysis, in terms of identification and quantification of spectral variations between different materials, were found to be in agreement with the results from a principal component analysis using the same set of data. Both methods were found capable of (i) distinguishing between agarose and dextran and (ii) detecting and quantifying the degree of cross-linking present in the four base matrix materials. In addition, using a deuterated chemical cross-linker, it was possible to identify spectral features specifically connected to the cross-link molecular structure.

National Category
Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-234612 (URN)10.1021/ac035457g (DOI)15053644 (PubMedID)
Available from: 2014-10-23 Created: 2014-10-21 Last updated: 2017-12-05Bibliographically approved
7. Chemical characterisation of different separation media based on agarose by static time-of-flight secondary ion mass spectrometry
Open this publication in new window or tab >>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.

National Category
Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-234613 (URN)10.1016/j.chroma.2003.10.008 (DOI)14760849 (PubMedID)
Available from: 2014-10-23 Created: 2014-10-21 Last updated: 2017-12-05Bibliographically approved

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