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.
| Identifer | oai:union.ndltd.org:UPSALLA1/oai:DiVA.org:uu-172892 |
| Date | January 2012 |
| Creators | Blom, Hans |
| Publisher | Uppsala universitet, Fysikalisk-organisk kemi, Uppsala |
| Source Sets | DiVA Archive at Upsalla University |
| Language | English |
| Detected Language | English |
| Type | Doctoral thesis, comprehensive summary, info:eu-repo/semantics/doctoralThesis, text |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, 1651-6214 ; 928 |
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