New methods to evaluate the effects of fouling on process chromatography

This thesis examines new approaches to evaluate the effects of fouling on process chromatography. Fouling can have a serious, negative impact on the performance of chromatography and considerable effort is normally spent to prevent fouling species reaching the column, or in developing clean-in-place (CIP) protocols of ever increasing complexity to mitigate their effects. Despite this, the knowledge of chromatographic fouling often seems anecdotal, with only a few systematic investigations currently reported in literature. Furthermore, conventional approaches to investigate chromatographic fouling only provide an overall indication of their state. New approaches to investigate fouling at increasingly fine detail are studied in this thesis and provide valuable insights to the mechanism of fouling. At the whole-column level, the method of frontal analysis was used to determine the effects of fouling a packed bed column (DEAE Sepharose FF) with yeast homogenate. The shape and position of breakthrough curves generated by frontal analysis were used to quantitatively assess the impact of fouling on binding capacity and to qualitatively infer the overall changes in mass transfer properties. In particular, the effects of solids particulate and different modes of applying the fouling stream to the column were examined. Breakthrough curve analysis was also used to investigate the effectiveness of a rigorous CIP procedure in restoring the characteristics of a fouled column. An extended reverse-flow technique using an acetone tracer was then developed to quantify the dispersive effects of fouling on defined axial sections within a packed bed column, giving more than an overall indication of the fouling condition. The influence of column diameter, bed length and two different header designs on the extent of fouling were examined. The technique allows the band broadening effects due to reversible macroscopic factors, such as flow maldistribution in the flow distributor and inside the packed bed caused by packing heterogeneity, to be separated from irreversible microscopic factors, such as intraparticle diffusion, external fluid film mass transfer and interparticle axial dispersion. It was shown to be a simple, nondestructive method for investigating chromatographic fouling at an intra-column level. Finally, confocal scanning laser microscopy (CSLM) was proven to be a powerful technique to directly visualise fouling at a single-bead level. A particularly aggressive fouling stream of partially clarified E. coli homogenate was used to challenge an anion exchange resin (Q Sepharose FF) in a finite bath, and subsequently in a packed structure under flow conditions. The fouling caused by the material was visualised by fluorescently labelling DNA and host cell proteins in the fouling stream and by measuring the binding capacity and uptake rate for a fluorescently-labelled test protein, BSA. The use of CSLM also allowed the applications of various CIP procedures to be visually followed. The competitive adsorption of whole cells or cell debris and DNA to Q Sepharose FF has also been visualised. Confocal images obtained provide insights to the spatial distribution of key foulant types within a single bead. This thesis concludes with recommendations for future work which will seek to extend the analysis to situations where fouling occurs in a flow situation by the design of appropriate flow cells and methods of analysis