Application of a PEG precipitation method for solubility screening: a tool for developing high protein concentration formulations

Abstract

Previous publications demonstrated that the extrapolated solubility by polyethylene glycol (PEG) precipitation method (Middaugh et al., J Biol Chem 1979; 254:367-370; Juckes, Biochim Biophys Acta 1971; 229:535-546; Foster et al., Biochim Biophys Acta 1973; 317:505; Mahadevan and Hall, AIChE J 1990; 36:1517-1528; Stevenson and Hageman, Pharm Res 1995; 12:1671-1676) has a strong correlation to experimentally measured solubility of proteins. Here, we explored the utility of extrapolated solubility as a method to compare multiple protein drug candidates when nonideality of a highly soluble protein prohibits accurate quantitative solubility prediction. To achieve high efficiency and reduce the amount of protein required, the method is miniaturized to microwell plate format for high-throughput screening application. In this simplified version of the method, comparative solubility of proteins can be obtained without the need of concentration measurement of the supernatant following the precipitation step in the conventional method. The monoclonal antibodies with the lowest apparent solubilities determined by this method are the most difficult to be concentrated, indicating a good correlation between the prediction and empirical observations. This study also shows that the PEG precipitation method gives results for opalescence prediction that favorably compares to experimentally determined opalescence levels at high concentration. This approach may be useful in detecting proteins with potential solubility and opalescence problems prior to the time-consuming and expensive development process of high concentration formulation.

Keywords: high-throughput screen; monoclonal antibody; polyethylene glycol; solubility; volume exclusion.

Figure 1

Apparent solubility of mAb1 by PEG 10k. Measurements were conducted at 10 mg/mL in 50 mM histidine pH 6 at 20°C. Data are shown as mean ± SD (n = 3).

Figure 2

Microplate screen of antibody apparent solubility by the PEG precipitation method. Concentration of all proteins is 10 mg/mL in 50 mM histidine, pH 6.

Figure 3

Correlation of opalescence of mAbs at 90 mg/mL with apparent solubilities measured by the PEG precipitation method.

Figure 4

Opalescence levels and apparent solubilities by PEG precipitation method of mAb7 at different purification steps. The material from early development stage has the same apparent solubility read out (C, top) as material from more refined purification process (C, bottom), despite its higher opalescence (A and B) due to the presence of residual host cell protein/DNA. A: 100 mg/mL mAb7 purified by single-column purification process; B: 100 mg/mL mAb7 purified by two-column purification process; C: apparent solubility of 10 mg/mL single-column purified (top) and two-column purified (bottom) mAb7 by the microplate PEG precipitation method.