Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Purification Processes for Complex Biomacromolecules
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Physical Organic Chemistry.
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis details various techniques and considerations for the purification of complex biomacromolecules.

 

Initially an α-mannosidase from babaco fruit was purified using anion exchange-, lectin affinity- and size exclusion chromatography.  The enzyme was approximately 260-280 kDa in size with an apparent an unusual octagonal stoichiometry and displayed properties similar to other known plant α-mannosidases.

 

Mucins were fractionated by ion exchange and size exclusion chromatography to assess the properties that govern the mucin surface coating interactions in biomaterial research.  Commercially available mucins, of bovine and porcine origin, as wells as crude human mucin were tested. All showed to consist of a population of molecules which differ in size, charge and composition.

 

The third part of the thesis concerns different aspects of plasmid DNA purification processes.

A two-step method for analysis of plasmid DNA consisting of size exclusion followed by thiophilic adsorption chromatography was evaluated. It allowed determination of the supercoiled plasmid DNA concentration in all process steps without requirement for extensive sample preparation. This method was shown to be fully comparable in terms of accuracy to capillary gel electrophoresis, considered as the industry standard.

Purification of plasmid DNA generally involves bacterial cell alkaline lysis, which creates a solution with flocculate material which needs to be removed prior to further processing. The addition of ammonium hydrogen carbonate to the suspension was evaluated to clarify the solution. The released carbon dioxide and ammonium lifts the flocculate to the surface and allows draining of a clear solution. The method is fully scalable, does not affect the plasmid DNA quality and requires no special equipment.

Thiophilic adsorption chromatography was evaluated for simplification of an existing commercial large scale purification process and was shown to increase both product purity and yields of several tested plasmids. Also, implementation of this step significantly reduced overall production process time.

 

 

 

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2012. , 61 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 928
Keyword [en]
Chromatography, enzymes, mucins, fractionation, plasmid DNA, analysis, clarification, process design
National Category
Chemical Sciences
Research subject
Chemistry with specialization in Surface Biotechnology
Identifiers
URN: urn:nbn:se:uu:diva-172892ISBN: 978-91-554-8353-1 (print)OAI: oai:DiVA.org:uu-172892DiVA: diva2:515987
Public defence
2012-05-25, BMC B7:101, Husargatan 3, Uppsala, 13:15 (English)
Supervisors
Available from: 2012-05-04 Created: 2012-04-17 Last updated: 2012-08-01Bibliographically approved
List of papers
1. Purification and Characterization of an alpha-Mannosidase from the Tropical Fruit Babaco (Vasconcellea x Heilbornii Cv. Babaco)
Open this publication in new window or tab >>Purification and Characterization of an alpha-Mannosidase from the Tropical Fruit Babaco (Vasconcellea x Heilbornii Cv. Babaco)
2008 (English)In: Journal of Agricultural and Food Chemistry, ISSN 0021-8561, E-ISSN 1520-5118, Vol. 56, no 22, 10872-10878 p.Article in journal (Refereed) Published
Abstract [en]

An alpha-mannosidase (EC 3.2.1.24) present in the lyophilized latex of babaco (Vasconcellea heilbornii) has been purified to apparent homogeneity by native PAGE. The purification involves a three-step procedure with successive anion exchange with 0 Sepharose HP, lectin affinity chromatography using ConA Sepharose 4B, and gel filtration using Superdex 200 prep grade. The molecular mass was determined to be in the range of 260-280 kDa by Superdex 200 prep grade gel filtration, and isoelectric focusing showed a pI range between 5.85 and 6.55, suggesting different glycosylated isoforms. The optimal temperature for the alpha-mannosidase was determined to lie between 50 and 60 degrees C, and the optimal pH was 4.5 at 50 degrees C. The K-m value for p-nitrophenyl alpha-mannopyranoside (pNPM) was found to be 1.25 mM and the V-max, 2.4 mu kat mg(-1) at 50 degrees C and 1.94 mu kat mg(-1) at 40 degrees C. The pure alpha-mannosidase was specific for mannose and did not display activity for any other tested synthetic substrates.

Keyword
alpha-Mannosidase, babaco, latex, purification, characterization, subunit composition
National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-107031 (URN)10.1021/jf800857k (DOI)000261056700065 ()
Available from: 2009-07-15 Created: 2009-07-15 Last updated: 2017-12-13Bibliographically approved
2. Potential use of mucins as biomaterial coatings. I. Fractionation, characterization, and model adsorption of bovine, porcine, and human mucins
Open this publication in new window or tab >>Potential use of mucins as biomaterial coatings. I. Fractionation, characterization, and model adsorption of bovine, porcine, and human mucins
2009 (English)In: Journal of Biomedical Materials Research Part A, ISSN (printed): 1549-3296. (electronic): 1552-4965., Vol. 91A, no 3, 762-772 p.Article in journal (Refereed) Published
Abstract [en]

Previously, we presented evidence that mucins have potential as   biomaterial coatings. Here, we reveal substantial batch-to-batch   variations for a frequently used commercial bovine salivary mucin   preparation (BSM) and stress the importance of standardizing mucins   intended for comparative purposes. "Mild" fractionation strategies,   aiming at preserving natural mucin functions, were used to prepare two   more defined BSM fractions as well as three mucin fractions from   porcine gastric (PGM) and human salivary (MG1) sources. While the BSM   and PGM were highly purified and mainly adopted random coil   conformations in solution, the MG1 contained mucin-bound components   (1.6 wt% albumin) and appeared compact. Average molar masses and   root-mean-square radii for the predominant BSM, PGM, and MG1 species   spanned 0.8-4.2 MDa and 46-86 nm, respectively. An ellipsometric   evaluation, using hydrophilic and hydrophobic silica, showed the mucin   adsorption to be slow and related to mucin charge, size, conformation,   and compositional complexity. The mass uptakes on hydrophobic silica   averaged 2.6, 2.6, and 5.0 mg/m(2), for BSM, PGM, and MG1,   respectively. Finally, we find that stable mucin coatings can be formed   on polymers of different wettability. The reported mucin preparations   serve as platforms for a series of studies on the biocompatibility of  mucin coatings.

Keyword
mucin fractionation, mucin-associated components, size-exclusion chromatograpliy-multiangle light scattering-refractometry (SEC-MALS-RI), biomaterial coating, MUC5B
National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-97899 (URN)10.1002/jbm.a.32266 (DOI)000271588800014 ()19051309 (PubMedID)
Available from: 2008-11-28 Created: 2008-11-28 Last updated: 2012-08-01Bibliographically approved
3. A chromatographic method for determination of supercoiled plasmid DNA concentration in complex solutions.
Open this publication in new window or tab >>A chromatographic method for determination of supercoiled plasmid DNA concentration in complex solutions.
2009 (English)In: Journal of chromatography. B, ISSN 1570-0232, E-ISSN 1873-376X, Vol. 877, no 24, 2530-6 p.Article in journal (Refereed) Published
Abstract [en]

A method for determination of the plasmid DNA concentration with subsequent analysis of the ratio supercoiled to open circular form is presented. The method is suitable for samples from all steps of the manufacturing process, from fermentation to final product. The analysis consists of size exclusion chromatography, followed by analytical thiophilic aromatic chromatography. In the first step, the plasmid DNA concentration is determined by group separation, including removal of RNA and other impurities, within less than 2 min. The limit of detection and quantification was 0.28 and 0.83 microg/ml, respectively. The precision of the method is high, providing a coefficient of variation as low as below 2%. In the second step, the ratio of open circular to supercoiled plasmid DNA is determined following separation of the two plasmid DNA isoforms with a linear salt gradient. The precision of the second step was evaluated using serial injections of aliquots of a sample stock solution. In comparison with the two most commonly used methods, the developed analysis was found to be significantly more accurate than agarose gel electrophoresis and equivalent to capillary gel electrophoresis. The combined methods for quantification and control of homogeneity of plasmid DNA presented here enable reliable and precise analysis at all steps of the manufacturing process.

Keyword
Concentration determination, Supercoiled plasmid DNA, Analytical group separation, Thiophilic aromatic chromatography
National Category
Chemical Sciences
Research subject
Chemistry with specialization in Surface Biotechnology
Identifiers
urn:nbn:se:uu:diva-172792 (URN)10.1016/j.jchromb.2009.06.037 (DOI)19616488 (PubMedID)
Available from: 2012-04-16 Created: 2012-04-16 Last updated: 2017-12-07
4. Flocculate removal after alkaline lysis in plasmid DNA production.
Open this publication in new window or tab >>Flocculate removal after alkaline lysis in plasmid DNA production.
2010 (English)In: Vaccine, ISSN 0264-410X, E-ISSN 1873-2518, Vol. 29, no 1, 6-10 p.Article in journal (Refereed) Published
Abstract [en]

Alkaline lysis is the most commonly used method following harvest of bacterial cells for production of plasmid DNA. The method was originally developed for laboratory scale experiments and has shown to be challenging at larger scales. A major problem prior to further downstream processing is the risk of filter clogging without efficient removal of the flocculate that occurs after neutralization. For this purpose we here present a scalable method where the clarification of alkaline lysate is greatly simplified. Through a rapid procedure, involving the addition of ammonium hydrogen carbonate to the neutralized alkaline lysate, the flocculate is lifted to the surface of the solution by the released carbon dioxide and ammonium. After this step a clarified solution can be drained from the bottom of the vessel. The procedure does not impact pH, plasmid DNA concentration or the ratio of open circular to supercoiled plasmid DNA in the solution.

Keyword
Alkaline lysis, Plasmid DNA, Flocculate removal
National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-172791 (URN)10.1016/j.vaccine.2010.10.021 (DOI)20974301 (PubMedID)
Available from: 2012-04-16 Created: 2012-04-16 Last updated: 2017-12-07
5. Scale Up of a Plasmid DNA Purification Process: Use of a Commercial Resin to Produce GMP-Grade pDNA for Clinical Studies
Open this publication in new window or tab >>Scale Up of a Plasmid DNA Purification Process: Use of a Commercial Resin to Produce GMP-Grade pDNA for Clinical Studies
Show others...
2010 (English)In: BioProcess International, ISSN 1542-6319, Vol. 8, no 11, 46-54 p.Article in journal (Refereed) Published
Keyword
Chromatography, plasmid DNA
National Category
Chemical Sciences
Research subject
Chemistry with specialization in Surface Biotechnology
Identifiers
urn:nbn:se:uu:diva-172889 (URN)
Available from: 2012-04-17 Created: 2012-04-17 Last updated: 2012-08-01Bibliographically approved

Open Access in DiVA

fulltext(3857 kB)941 downloads
File information
File name FULLTEXT01.pdfFile size 3857 kBChecksum SHA-512
3b008165bcf64692c67fbf5aa4039f15b8d1b8cb6ca1dcdb62e337fb27a85d869ce0f3b083a4417d69487d72520efc86c6b1f833ae77c6d5eb909b0d56632b42
Type fulltextMimetype application/pdf
Buy this publication >>

Search in DiVA

By author/editor
Blom, Hans
By organisation
Physical Organic Chemistry
Chemical Sciences

Search outside of DiVA

GoogleGoogle Scholar
Total: 941 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

isbn
urn-nbn

Altmetric score

isbn
urn-nbn
Total: 920 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf