Study on the effect of solution conditions on heat induced-aggregation of human alpha interferon

Abstract

A major problem in the formulation of therapeutic proteins is the irreversible protein aggregation. Recombinant human interferon alpha2b (rhIFNα2b) has poor stability and undergoes physical degradation. The aim of this study was to investigate the effect of solution conditions on the heat-induced aggregation of rhIFNα2b. The protein was incubated for 1 h at 40-70 °C and for up to 240 h at 50 °C and its aggregation tendency was then studied using optical density (at 350 nm), SE-HPLC, dynamic light scattering and SDS-PAGE methods. The effect of various pH (5, 6 and 7) and buffer concentrations (10, 55 and 100 mM) on the aggregation of protein following incubation at 50 °C for 72 h was also evaluated. The results obtained for samples incubated at 50 °C for up to 240 h showed that OD350 and the amount of higher molecular weight aggregates (HMW) increased and the monomer content decreased significantly (p<0.05) as the incubation time increased. Following incubation at various temperatures, a significant increase in OD350, drop in monomer content and increase in the amount of HMW aggregates were observed (p<0.05). Data obtained from incubation of samples at 50 °C for 72 h confirmed that regardless of the buffer concentration, the percentage of monomer at pH 6 was significantly higher than that at pH 7 and pH 5 (p<0.05). At constant pH, although not significant, the same trend was observed when the buffer concentration increased to 100 mM. In conclusion, the change in solution conditions can influence the aggregation extent of rhIFNα2b.

Keywords: Interferon alpha2b; Optical density; SDS-PAGE; SE-HPLC; Thermal aggregation.

Figure 1

Optical density of rhIFNα2b solutions (100 μg/mL) following incubation at various temperatures for 1 h (mean ± SD; n = 3).

Figure 2

SE-HPLC chromatograms of non-incubated rhIFNα2b and a sample incubated at 50 °C for 240 h.

Figure 3

A) Plots of percent of monomer and B) percent of high molecular weight aggregates, following incubation of rhIFNα2b solutions (100 μg/mL) for 1 h at various temperatures (mean ± SD; n = 3).

Figure 4

Plots of A) percent of monomer, B) percent of high molecular weight aggregates, and C) optical density as a function of time, following incubation of rhIFNα2b solutions (100 μg/mL) at 50 °C (mean ± SD; n = 3).

Figure 5

SDS–PAGE results of 1) non-incubated rhIFNα2b, and 2) incubated rhIFNα2b at 50 °C for 240 h, under non-reducing (A) and reducing (B) conditions

Figure 6

Size distribution of A) non-incubated and B) incubated rhIFNα2b at 50 °C for 240 h.