Determining suitable surfactant concentration ranges to avoid protein unfolding in pharmaceutical formulations using UV analysis

Abstract

Protein stability is fundamental to maintain pharmaceutical efficacy in the nascent field of biologics. One particular property that is essential for therapeutic effect is retention of the folded 3-dimensional conformation, i.e. once unfolding has occurred the biologic is often rendered inactive. In this work we propose a modified form of a recently published UV spectroscopic method that identifies protein unfolding. In this study we determine concentration limits to avoid protein unfolding of two model surfactants, namely polysorbate 20 and polysorbate 80, by correlating surfactant concentration with percentage 'unfolded' for three model proteins. For each scenario two distinct regions were observed, firstly surfactant concentrations at which no unfolding had occurred, followed by a second region whereby unfolding steadily increased with surfactant concentration. In general for the combinations analysed in this study, this second region began to appear around ten times below the critical micellar concentration of each surfactant, regardless of the protein or polysorbate chosen. It is therefore proposed that this adapted method could be used by researchers in the early stages of formulation development as a convenient and simple screening tool to confirm the 'onset of unfolding' concentration for protein-surfactant formulations, thus helping to optimise surfactant concentration selection in pharmaceutical formulations to maintain the benefits of surfactants yet avoid inadvertent unfolding.

Keywords: Biocompatibility; Critical micellar concentration; Pharmaceutical; Polysorbate; Protein; Spectroscopy; Tween; UV.

Graphical abstract

Fig. 1

Absorbance spectra at the three specified wavelengths for IgG with Tween 20 (top left), Super Refined Polysorbate 20 (top right), Tween 80 (bottom left) and Super Refined Polysorbate 80 (bottom right). For each surfactant a series of concentrations were measured relative to each CMC: 0.01x (dark blue), 0.02x (orange), 0.1x (grey), 1x (yellow) and 10 x (light blue). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Absorbance spectra at the three specified wavelengths for HSA with Tween 20 (top left), Super Refined Polysorbate 20 (top right), Tween 80 (bottom left) and Super Refined Polysorbate 80 (bottom right). For each surfactant a series of concentrations were measured relative to each CMC: 0.01x (dark blue), 0.02x (orange), 0.1x (grey), 1x (yellow) and 10 x (light blue). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Absorbance spectra at the three specified wavelengths for β-Ig with Tween 20 (top left), Super Refined Polysorbate 20 (top right), Tween 80 (bottom left) and Super Refined Polysorbate 80 (bottom right). For each surfactant a series of concentrations were measured relative to each CMC: 0.01x (dark blue), 0.02x (orange), 0.1x (grey), 1x (yellow) and 10 x (light blue). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Percentage of IgG remaining folded (compared with native sample) with polysorbate concentration (relative to each CMC). (n ≥ 3, error = ±SD).

Fig. 5

Percentage of HSA remaining folded (compared with native sample) with polysorbate concentration (relative to each CMC). (n ≥ 3, error = ±SD).

Fig. 6

Percentage of β-Ig remaining folded (compared with native sample) with polysorbate concentration (relative to each CMC). (n ≥ 3, error = ±SD).