Development of rapid chromatographic technologies for complex biofermentation sample analysis.
Alwael, Hassan (2012) Development of rapid chromatographic technologies for complex biofermentation sample analysis. PhD thesis, Dublin City University.
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The recent growth in the biopharmaceutical industry is remarkable due to the introduction of many new therapeutic proteins for the treatment of different diseases. The production processes of the biotherapeutics are complicated and have to be maintained under strict and regulated conditions. Therefore, the development of rapid, sensitive and cost-effective analytical assays is highly demanded for monitoring the biofermentation processes and the key parameters that could affect the final product quality and production consistency.
The overall aim of this research is to develop sensitive and selective analytical methods for the determination of raw material components in highly complex samples supplied from the biopharma industry based on solid phase extraction (SPE) and rapid resolution liquid chromatography (RRLC). This development includes the use of narrow-bore columns packed with sub-2 μm silica particles or made of monolithic materials. The developed methods were applied for the qualitative and quantitative analysis of common monosaccharides, including sialic acid, and cysteine/cystine ratio in a range of biopharmaceutical production samples such as raw material yeast extract powders, fermentation feedstocks, chemically defined media and in-process fermentation broth samples in which they were taken from different lots in order to estimate lot-to-lot variability. For evaluation purposes, standard analytical performance criteria were examined for all the developed methods. In addition, a novel solid phase microextraction in a pipette tip for selective enrichment of galactosylated proteins is presented. The extraction device is fabricated by in-situ photopolymerisation of ethylene dimethacrylate porous polymer monolith within the confines of 20 μL polypropylene pipette tip. Then the surface of the monolith was significantly enhanced by immobilising gold nano-particles (AuNPs) which was functionalised with Erythrina cristagalli lectin (ECL) afterwards. The ECLmodified tip was successfully applied for the enrichment of galactosylated proteins versus non-galactosylated proteins from different sample matrices including Escherichia coli cell lysate. Reversed-phase capillary LC was used to validate the efficiency and selectivity of the developed extraction device which resulted in an increase in extraction recovery of ~95% due to the AuNPs enhanced surface area.
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