(205c) Dual Mode Precipitation for the Recovery of Recombinant Proteins From E. Coli

The volumetric productivity of therapeutic proteins using bacterial expression has risen to up to 2 g/L through cell engineering, media optimisation and better process control (Andersen and Reilly, 2004). However, the increased titres pose a challenge in downstream processing since the capacity of packed bed chromatography operations does not change the cost, the benefits of improved titre are not passed down the process. Some of the alternative purification techniques that could potentially benefit from increased titres include crystalisation, precipitation and aqueous two-phase separations. Precipitation of either the product or the contaminants can be used to assist the chromatographic purification if not replace (Przybycien et al., 2004).

Combining one or more precipitating agents termed dual mode precipitation can be expected to alter the solubility profile of the product through synergistic or antagonistic effects. Typical process variables involved in a precipitation process include, ionic strength, precipitant type, pH, temperature and concentration of the feed. We used a high throughput microscale solubility studies for rapid identification of the precipitation conditions and propose an alternative purification process using dual mode precipitation for the recovery of antibody fragment product Fab' from E. coli homogenates. The proposed process route resulted in 30% increase in the amount of product extracted while improving the clarification of the homogenates by 4 fold simultaneously. At an ionic strength of 7.2M, calculated theoretically considering the concentration and valency of each ion involved, dual mode precipitation was found to result in 98% recovery of Fab' compared to only 67% by single salt precipitation. Approximately 40% of the soluble protein contaminants were removed simultaneously.

References

Andersen DC, Reilly DE. Production technologies for monoclonal antibodies and their fragments. Current Opinion in Biotechnology. 2004;15(5):456-62.

Przybycien TM, Pujar NS, Steele LM. Alternative bioseparation operations: life beyond packed-bed chromatography. Current Opinion in Biotechnology. 2004;15(5):469-78.