Downstream processing of biologicals: Industrial-scale chromatography and protein refolding methods for beta-galactosidase.

摘要

The field of biotechnology presents many opportunities for chemical engineers to solve complex problems by applying engineering fundamentals. My background has given me the opportunity to investigate problems in both diagnostics, as illustrated in the characterization of large-scale chromatographic columns, and application, as illustrated in the improvement of existing methods for refolding beta-galactosidase. The first part of my thesis will focus on analyzing the performance of industrial-scale chromatographic columns. There has been a strong interest in understanding the factors that contribute to deviations from idealized linear chromatographic models. The two most important of these factors are dispersion due to packing heterogeneity and non-uniform flow from the column flow distributors. A reverse-flow technique is employed to separate the reversible effects of flow maldistribution from unavoidable irreversible microscopic band broadening processes. By performing the reverse-flow study over different bed depths, this technique decouples the maldistribution in the flow distributors from the maldistribution from heterogeneous packing.; The next part of my thesis will focus on the recovery of biologically active proteins from inclusion bodies. Using inclusion bodies has many economic benefits; however, the bottleneck in their recovery is the refolding of the denatured polypeptides. For this research, the refolding of a substrate protein, beta-galactosidase, was examined. Initial experiments were designed to explore the enhancement in the yield of biologically active protein by the addition of a molecular chaperone, GroEL, to the refolding buffer. Recombinant DNA technology was employed to develop a cell line capable of overexpressing GroEL. After the expression and purification, the kinetics of beta-galactosidase refolding was investigated in both the presence and absence of the chaperone. The addition of the molecular chaperone produced a substantial improvement in yield over refolding by dilution, but still only gave modest yields in the recovery of biologically active protein. To further understand this process, the refolding was monitored by dynamic light scattering. These experiments indicated that aggregation is very rapid, with large aggregates being formed within minutes. This motivated the use of denatured ion-exchange chromatography, which showed marked improvement over chaperone-assisted refolding by dilution.