A major hurdle for biologics development is the determination of a formulation that keeps a protein soluble. Soluble Biotech OptiSol Protein Solubilization Screening Kit (96-well format) contains a systematically varied array of buffers (from pH 3 to pH 10) and a series of solubility enhancers (salts, amino acids, sugars, polyols, reducing agents). Parallel filtration of the protein through multimer-excluding MWCO filters enables rapid identification of solubilizing formulations by a simple comparison of elution concentrations. A total of 90 different formulations with solubility enhancers are tested in a single, label-free experiment within 4 hours or less.
Here we explore the utility of OptiSol kit for formulating L-glutamic dehydrogenase (LGDH) in solution. Experimental steps were performed as described in Figure 1. Depending on study goals, one can choose to stress the sample using various methods (i.e. temperature, shear force, chemical exposure, etc.). In this case, we incubated the protein-reagent mixtures at room temperature for several hours. Four types of Optisol Kits are available for four different ranges of protein molecular weights. Solubilized protein molecules will be found in the filtrates. Formulations that are detrimental to protein solubilization will result in aggregated protein being retained on the filter membrane. Various detection assays can be employed to qualitatively or quantitatively analyze the amount of protein in the filtrates. In this case study, UV280 absorbance using a plate reader was employed.
Optisol IV kit was utilized to screen LGDH (MW: ~310 kDa). Figure 2 shows percent elutions of LGDH in Optisol formulations ranked from maximum elution to minimum elution. The top best formulations are tabulated in Table 1, in comparison to storage formulation (SB). The result suggests that polysorbate 20, glycerol, and TMAO may be suitable additives that promote LGDH solubility in solution. Data also show that various buffers and pHs can be used to solubilize LGDH.
Figure 2: Percent elution of LGDH with respect to reagents in Optisol IV kit. Percent elution is a ratio of protein amount in filtrate and original amount of protein before filtration. Data were from triplicate experiments.
Table 1: Top formulations for LGDH and Dynamic Light Scattering (DLS) data of LGDH in respective formulations
This study shows that Optisol kit can provide high-throughput analysis of multiple formulations in a short period of time. This is very advantageous at various steps of biopharmaceutical developments in identifying key pharmaceutical excipients to go forward with or to avoid.
Table 2: B22 values of LGDH in selected formulations
We further analyzed the properties of LGDH in top formulations using Dynamic Light Scattering (DLS) and self-interaction chromatography (SIC) methods. DLS provides insights into molecular properties of protein in solutions, i.e. hydrodynamic radius (Rh), aggregation behavior, etc. Table 1 shows that some positive formulations resulted in protein instability after a period of 1 week. We applied SIC2 method to obtain B22 (second virial coefficient) values of LGDH in some top formulations. Positive values indicate better solubility, while negative values indicate poor solubility. Data in Table 2 shows that the selected formulations performed better than storage buffer in solubilizing LGDH in solution. The addition of 1 %w/v of polysorbate into storage buffer formulation resulted in improved formulation for LGDH. Our results show that OptiSol kit provides a reliable high-throughput method for identifying solubilizing protein formulations.
1. OptiSolTM Protein Solubility Screening Kit Application Manual. SolubleBioScience Inc.
2. Johnson, D. H, et al. High-Throughput Self-Interaction Chromatography: Applications in Protein Formulation Prediction. Pharmaceutical Research 26 (2): 296-301 (2009).