Continuous methods of protein chromatography

Timothy Kenneth Nadler, Purdue University

Abstract

Growth in the biotechnology industry is increasing the demand for high throughput protein purifications. Chromatography, which separates proteins based on their preferential interaction with sorbent surfaces, is limited by packed column methods which are difficult to incorporate into automated production processes. Existing continuous methods are limited by problems with filtering devices, complexity and sorbent transport. Two new continuous protein chromatography systems have been developed to address these problems. The first system, which was inspired by an examination of Taylor vortex flow, mixes sorbent between two concentric cylinders (8.2 cm O.D. in 10.2 cm I.D.) in which discrete mixing chambers have been installed along the axis (0.85 cm dia.). A cross-current flow is used to wash or desorb protein from the sorbent in each chamber, while axial flow moves the sorbent through the chambers. The sorbent is retained in the chambers by an array of fluidized beds allowing the purified protein solution to be removed. In comparison to the cross-current flow, the counter-current flow arrangement resulted in the greatest purity and recovery. Hemoglobin was separated from a mixture of hemoglobin and lysozyme with 95% purity and a recovery of 99%, demonstrating the efficacy of the novel, filterless, vortex based separation system for simplified sorbent transport in continuous protein separations. The second system, an automated continuous selective non-adsorption preparative (SNAP) chromatography procedure was also developed to make efficient use of column volume. SNAP chromatography was optimized for the purification of IgG from hybridoma culture media and bovine serum. This procedure purified IgG from bovine serum at rate of 1 mg of serum protein processed each minute per milliliter column volume, which represents one of the most efficient uses of column volume reported. Automated process monitoring systems were developed that utilized a single high performance liquid chromatograph (HPLC) to monitor monoclonal antibody and $\gamma$-interferon production in cell cultures. Furthermore, an automated two-dimensional method for determination of IgG dimers in complex formulations was developed which used a single HPLC. These methods were designed for monitoring both upstream and downstream processes in recombinant protein production.

Degree

Ph.D.

Advisors

Regnier, Purdue University.

Subject Area

Biochemistry|Analytical chemistry

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