A combined pH and temperature precipitation step facilitates the purification of tobacco-derived recombinant proteins that are sensitive to extremes of either parameter

Abstract

Incubation at pH 4.0 or blanching at ∼65°C facilitates the purification of biopharmaceutical proteins from plants by precipitating most of the host cell proteins (HCPs) before chromatography. However, both methods are compatible only with pH or thermostable target proteins whereas many target proteins may irreversibly denature, e.g., at pH < 4.0. Here, we developed a combined pH/temperature treatment for clarified tobacco extracts and intact leaves. The latter were subjected to a blanching procedure, i.e., the submersion into a hot buffer. Using a design of experiments approach we identified conditions that remove ∼70% of HCPs at ∼55°C, using the thermosensitive antibody 2G12 and the pH-sensitive DsRed as model proteins. We found that pH and temperature exerted a combined effect during the precipitation of HCPs in the pH range 5.0–7.0 at 35°C–60°C. For clarified extracts, the temperature required to achieve a DsRed purity threshold of 20% total soluble protein (TSP) increased from 54°C to 63°C when the pH was increased from 6.4 to 7.3. The pH-stable antibody 2G12 was less responsive to the combined treatment, but the purity of 1% TSP was achieved at 35°C instead of 44°C when the pH was reduced from 6.3 to 5.8. When blanching intact leaves, product losses were not exacerbated at pH 4.0. Indeed, the highest DsRed purity (58% TSP) was achieved at this pH, combined with a temperature of 60°C and an incubation time of 30 min. In contrast, the highest 2G12 purity (0.7% TSP) was achieved at pH 5.1 and 40°C with an incubation time of 20 min. Our data suggest that a combined pH/temperature regime can avoid extreme values of either parameter; therefore, broadening the applicability of these simple purification techniques to other recombinant proteins.