Chemical denaturation as a tool in the formulation optimization of biologics

Abstract

Biologics have become the fastest growing segment in the pharmaceutical industry. As is the case with all proteins, biologics are susceptible to denature or to aggregate; conditions that, if present, preclude their use as pharmaceuticals. Identifying the solvent conditions that maximize their structural stability is crucial during development. Since the structural stability of a protein is susceptible to different chemical and physical conditions, the use of several complementary techniques can be expected to provide the best answers. Stability measurements that rely on temperature or chemical [urea or guanidine hydrochloride (GuHCl)] denaturation have been the preferred ones in research laboratories and together provide a thorough evaluation of protein stability. In this review, we will discuss chemical denaturation as a tool in the optimization of formulation conditions for biologics, and how chemical denaturation complements the role of thermal denaturation for this purpose.

FIGURE 1

The temperature dependence of the Gibbs energy of protein stability calculated for typical parameters obtained for globular proteins [5,6]. Panel A illustrates the influence of ΔC_p on the curvature of the temperature dependence. Both proteins have a T_m of 60°C but the red one a ΔC_p of 2.5 kcal/K mol compared to 1.5 kcal/K mol for the blue curve. Panel B illustrates the reversal of the stability rank order. In this case the blue protein has a lower T_m (55°C) but a higher stability at room temperature and below. In this case also ΔC_p is 2.5 kcal/K mol for the red curve compared to 1.5 kcal/K mol for the blue curve. In all cases ΔH at T_m was assumed to be 120 kcal/mol.

FIGURE 2

Urea denaturation of cytochrome c at pH 5. The experiments were performed with an AVIA 2304 Automated Protein Denaturation System using intrinsic fluorescence detection (excitation wavelength 280 nm, emission wavelength 356 nm). The final concentration of cytochrome c was 0.025 mg/mL. The AVIA instrument automatically generated a gradient of 36 different urea concentrations equally spaced between 0 and 9 m. Each of the 36 unique solutions was generated with identical protein concentrations and identical buffer concentrations. This group of solutions at pH 5.00 was one of a larger group of automatically prepared solutions, each group set to one specific pH value. The top (green) and bottom (red) straight lines correspond to the calculated characteristic values for the native and denaturated states. The solid line through the points is the best non-linear least squares fit to the data (see text for details).

FIGURE 3

Correlation between m values and ΔC_p. Both values are correlated with changes in solvent accessible surface area upon unfolding. Data taken from Myers et al. [16].

FIGURE 4

Experimental temperature (measured by DSC, left panel) and urea denaturation at 25°C (measured by fluorescence spectroscopy, right panel) of anti-EGFR monoclonal antibody. Experiments were performed in phosphate buffer saline pH 7.4 (Roche Diagnostics GmbH, Mannheim, Germany). DSC experiments were performed in a Microcal VP-DSC instrument (GE Healthcare, Northampton, MA) at a protein concentration of 1 mg/mL. Fluorescence measurements were performed with the AVIA 2304 Automated Protein Denaturation System (excitation wavelength = 280 nm; emission wavelength collected between 300–500 nm) at a protein concentration of 5.5 μg/mL. In the DSC experiment, reversibility was tested using the standard procedure of rescanning the sample (blue line = first scan; red line = rescan). To test reversibility in the chemical denaturation experiment, the monoclonal antibody was dissolved into PBS, pH 7.4 at a concentration of 0.4 mg/mL and divided into two aliquots. Urea was added to a final concentration of 9 m to one of the aliquots and the same volume of buffer was added to the other and both samples were incubated for 24 hours at 25°C. The samples were then dialyzed against several changes of PBS for three days at 4°C and finally concentrated to the same concentration (blue line = first denaturation; red line = second denaturation). Abbreviations: DSC: differential scanning calorimetry; EGFR: epidermal growth factor receptor.

FIGURE 5

Four independent urea denaturation curves for cytochrome c at pH 7.0. The figure illustrates the level of reproducibility obtained by the AVIA 2304 Automated Protein Denaturation System. All samples were prepared automatically by the instrument starting from stock basic and acid buffer components and a stock protein solution. Analysis of the data yields the following thermodynamic parameters: ΔG° = 10.09 ± 0.12 kcal/mol; m = 1.4 ± 0.08 kcal/mol m; C_1/2 = 6.3 ± 0.07 m.

FIGURE 6

Urea denaturation of cytochrome c at pH 5.00, 6.00, 7.00 and 8.00 (left to right). The figure shows the normalized curves as described by Eqn 3. The experiments were performed with an AVIA 2304 Automated Protein Denaturation System using intrinsic fluorescence detection (excitation wavelength 280 nm, emission wavelength 356 nm). Different pH solutions were generated by mixing 10 mm succinic acid, 10 mm phosphoric acid, and 10 mm l-histidine at pH 4.0 with different proportions of 10 mm Na₂-succinate, 10 mm Na₂H-phosphate, and 10 mm l-histidine at pH 8.0. For each pH, cytochrome c was dispensed in aliquots of 10 μL into 24 wells of a microplate containing 130 μL buffer with a gradient of 0.0–9.0 m urea. The inset shows the variation of ΔG° (green in kcal/mol), m (red in kcal/mol*M) and C_1/2 (blue in m urea).

FIGURE 7

Urea denaturation of carbonic anhydrase in the absence and presence of 5 μm of the inhibitor trifluoromethanesulfonamide (TFMSA). Carbonic anhydrase, isoform I, from human erythrocytes was dissolved in 25 mm MES, pH 6.0 at a concentration of 10 μm (0.3 mg/mL). Two sets of 17 test tubes were prepared containing 180 μL of MES with a gradient of 0.0–8.0 m urea (final concentration). DMSO (2 μL) was added to one of the series of test tubes and 2 μL of 500 μm TFMSA dissolved in DMSO was added to the other series for a final concentration of 5 μm TFMSA. The protein was dispensed in aliquots of 20 μL into the test tubes and incubated for four hours at 25°C. The final concentration of protein was 1 μm (0.03 mg/mL). The excitation wavelength was 280 nm and the emission wavelength 355 nm. Fluorescence measurements were performed manually in a Cary Eclipse spectrofluorometer (Varian, Agilent).