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Graphical frameworks for facilitating the study of chromatographic separations

Ngiam, Sheau Huey; (2003) Graphical frameworks for facilitating the study of chromatographic separations. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

Chromatographic separations are employed for their excellent resolving power in order to achieve the purity required for today's bio-therapeutics, diagnostics, and other biological products. However, existing methods for the prediction of chromatographic sequence performance are limited and time-consuming given the highly interactive nature of such steps. This PhD study proposes a graphical framework for the rapid representation of key process trade-offs that need to be made during chromatographic separation. The output of the framework developed in this thesis reflects the degree of trade-off between levels of yield and purity and between levels of yield and the degree of contamination achieved and provides a fast and precise prediction on the sample fraction collection strategy needed to meet a desired process specification. The graphical methodology is able to accommodate difficulties posed by situations where the product and impurity content are measured by different assay techniques and are expressed in different units. Importantly by the use of cubic-spline methods, situations where the quality of process information is limited by the small number of fractions collected can be dealt with. The thesis details the development of the theoretical basis behind fractionation, purification factor- yield and contamination index-yield diagrams and the mathematical algorithms used to connect chromatographic data with these diagrams. To demonstrate the practicality and usefulness of the approach proposed a range of chromatographic separation data were employed as case studies as follows. For a single step chromatographic operation, the approach is demonstrated by four different separation systems: simulation results from a verified size exclusion model separating three globular proteins (i.e. γ-globulin, ovalbumin and Ribonuclease A); experimental results from the separation of a labile enzyme alcohol dehydrogenase (ADH) from Bakers yeast homogenate on a hydrophobic interaction STREAMLINE™ Phenyl matrix operated in an expanded bed; industrial data for the removal of endotoxin from DNA by ion exchange chromatography, and industrial data for the purification of recombinant protein from host cell protein contaminants by size exclusion chromatography. The approach is further demonstrated by another simulated separation operation involving chromatographic sequences; the separation of a hypothetical three-component protein system by hydrophobic interaction and size exclusion chromatography. I he studies successfully show how the analysis of chromatographic performance can be carried out more easily and in a straightforward fashion using the resultant graphical diagrams compared to the use of conventional chromatograms. The methodology proposed serves as a useful tool for identifying the process trade-offs during a chromatographic separation and indicating the impact on further processing of the cut-point decisions that are made.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Graphical frameworks for facilitating the study of chromatographic separations
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Pure sciences; Chromatographic separation
URI: https://discovery.ucl.ac.uk/id/eprint/10097542
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