Formulation development is one of the critical steps in developing a protein as a therapeutic product. Maintaining the integrity of purified protein during pharmaceutical processing, storage, handling, and delivery to patient is a major challenge. There are many solution conditions (e.g. buffer pH, ionic charge, concentration, etc.) and pharmaceutical excipients which one can choose and combine to achieve the best condition for a solubilized protein. This process is often times very time consuming and costly.

Soluble Biotech’s OptiPharma kit provides high throughput screening method to identify key pharmaceutical solution conditions and ingredients to solubilize and stabilize a protein of interest. OptiPharma Protein Solubility Screening Kit contains a combination of 10 types of buffers (pH varies from 3 to 8.5) and 13 pharmaceutical excipients (salts, amino acids, sugars, surfactants, preservatives). A total of 93 different formulations in a 96-well format can be tested in single, label-free experimental setup. The remaining 3 wells are for experiment controls. All ingredients contained in the OptiPharma kit are proven pharmaceutical excipients and buffers.

OptiPharma applies a filtration method to separate soluble protein molecules from aggregated protein molecules. Protein is detected in formulations that promote solubility after passing through a specific molecular weight cut-off filter. While aggregated protein, due to sub-optimal formulations, is retained on the filter. Figure 1 provides an overview of experimental steps. Detailed method can be found in OptiPharma Quickstart Guide and OptiPharma Manual.

Figure 1: General experimental steps to formulate and solubilize protein using OptiPharma kit.

There are four types of OptiPharma kit. Each type is specific to a range of molecular weight cut-off filter. Here, we used OptiPharma II for solubilization study of α-chymotrypsin (from bovine pancreas, Mw. ca. 25kDa). Protein was mixed with each formulation and incubated for 4 hours at room temperature. The mixtures were then transferred to 96-well format filter plate and centrifuged. Protein was detected at the wavelength of 280 nm using a plate reader.

Figure 2: Percent elution of α-chymotrypsin in various buffers in the presence of pharmaceutical excipients. Percent elution is a ratio of protein amount in filtrate and original amount of protein before filtration. Note the values larger than 100% are due to experimental errors.

Data in Figure 2 provide several insights into solution properties that are amenable to solubilizing α-chymotrypsin. Sodium citrate buffer pH 6.5, potassium phosphate buffer pH 7.0 and Na/K buffer pH 7.5 are likely good buffers for stabilizing α-chymotrypsin in a solution. On the other hand, one may want to stay away from buffers with pH less than 6.5. The result suggests that Arg-Glu 50/50 could be key excipient in protein solubilization. In buffers pH 6.5 to 7.5, most excipients are not hindering protein solubility, except poloxamer 188 and sodium bisulfate.

In conclusion, OptiPharma kit provides high-throughput assessment of protein behavior in multiple solution conditions in a short period of time. Using an OptiPharma kit, a formulation scientist can reduce several weeks of trial-and-error experimental work into a few hours of work combined with stress period. The OptiPharma kit is designed as an initial formulation screen. The experimenter is expected to follow up this initial screen with a characterization of biophysical properties in the solutions to confirm the findings.