Keywords: Protein Solubilization, Pharmaceutical Formulation, Formulation Development, Protein Aggregation, Protein Physical Stability

Our previous blog describes the use of OptiPharma kit on α-chymotrypsin, commercially available protein. Here we are presenting a formulation study on a proprietary pharmaceutical protein (protein X, molecular weight of 70 kDa) using the OptiPharma Kit III. Figure 1 shows an overview of experimental procedure using an OptiPharma kit. In this experiment, a protein solution was mixed with each OptiPharma formulation and incubated for 4 hours at room temperature. The mixtures were then transferred to 96-well format filter plate and centrifuged. The amount of protein in the filtrates was detected at the wavelength of 280 nm using a UV/Vis plate reader. 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.

Figure 2 shows the result from OptiPharma Kit III for protein X. The plots were generated using OptiPharma Dashboard. This Dashboard is available with the purchased kit. Data in Figure 2 suggests that buffers at pH 6.0 to 7.5 (except Histidine and Tris buffers) may be good buffers for the protein. This result confirms an independent study that Tris buffer is detrimental to protein X. Comparing the performance of additives in various buffers, poloxamer 188 and sodium bisulfate may hinder protein solubility in certain buffers. Amino acids and sugars seem to support protein X solubility in various buffers. Figure 3 shows the ‘positive’ effect of Arg-Glu 50/50 and ‘negative’ effect of poloxamer 188 in buffers of different pH. There are several additive-buffer combinations that would be good candidates for solubilizing protein X. Arg-Glu 50/50 in buffers pH 6.0-6.5 (preferably citrate buffer) would be good starting formulation for protein X.

Table 1 lists nine best additives out of 40 candidate additives for formulating Protein X in solutions using HSCTM Technology. HSCTM Technology, Soluble Biotech’s core formulation technology, is a high-throughput protein formulation method based on self-interaction chromatography. Self-interaction chromatography allows the measurement of protein-protein interactions in the form of second virial coefficient, B22. High B22 values indicate enhanced protein solubility, while low B22 values indicate propensity to protein aggregation in a solution. The data obtained from OptiPharma kit are in agreement with data obtained using HSC method. From HSC method, the B22 values for the two poor performing additives, i.e. poloxamer 188 and sodium bisulfate, are 1.0 and 0.3 B units, respectively, compared to a range of 1.6 to 2.6 for the best performing additives.

Figure 2: Percent elution of Protein X 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 value less than 0% or more than 100% is experimental errors.

Figure 3: Percent elution of Protein X in buffers of different pH in the presence of Arg-Glu 50/50 (left) and Poloxamer 188 (right).

Table 1: Best additives for formulating protein X as suggested from HSCTM Method

AdditiveMeasured B22 Value (mol * mL / g2 * 10-4)
1. Citric acid2.6
2. Arg/Glu 50/502.5
3. NaCl2.1
4. Dextrose2.0
5. Sucrose2.0
6. EDTA1.8
7. Arginine-HCl1.7
8. Glycine1.6
9. Succinic acid1.6

Note: B22 values calculated for solutions containing individual 40 additives ranged from 0.1 to 2.6.

In conclusion, OptiPharma Kit provides a rough estimate of best additive and/or best pH/buffer for first order of formulation development. Using an OptiPharma kit, a formulation scientist can reduce several weeks of 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.