Human chymotrypsinogen B production from Pichia pastoris by integrated development of fermentation and downstream processing. Part 2. Protein recovery

The purification of human chymotrypsinogen B (hCTRB) after expression and s ecretion by the yeast Pichia pastoris is described based on two different a pproaches using integrated initial recovery. Extraction employing aqueous t wo-phase systems (ATPS) from poly(ethylene glycol) and sodium sulfate allow s direct processing of cell containing yeast suspensions of 50% wet weight. The target protein is obtained partially purified in the top phase while c ells and cell debris are partitioned to the bottom phase of the system. hCT RB is further purified by adsorption from the top phase to the cation excha nger SP Sepharose Big Beads and elution in a salt step. The single step iso lation of hCTRB is possible by expanded bed adsorption (EBA) using a fluidi zed cation exchanger (Streamline SP XL). A design strategy is shown taking both target protein binding and stable fluidization of the stationary phase in cell containing suspensions into consideration. For the example of hCTR B isolation from cell containing P. pastoris suspensions, a successful use of this strategy is demonstrated. Both initial recovery strategies deliver a product that can be further purified and formulated by ultrafiltration/di afiltration followed by lyophilization, resulting in a homogeneous product. Scale-up to 30-90 L of culture suspension was shown for both methods, resu lting in a product of similar quality. Comparing both strategies reveals th at the two-step ATPS route is better suited for high cell density cultures, while the single step EBA method is preferred for cultures of moderate cel l density. This is due to the fact that application of EBA is restricted to suspensions of 10-12.5% wet weight cell concentration, thus necessitating dilution of the original broth prior to sample application. The data presen ted show that integrated recovery operations are a valuable alternative to traditional processing for systems that are problematic during initial soli d-liquid separation.