Polysorbates, Peroxides, Protein Aggregation, and Immunogenicity – A growing Concern

Abstract

Aggregation underlies a number of deleterious effects for biotherapeutics including loss of efficacy, induction of unwanted immunogenicity, altered pharmacokinetics, and reduced shelf life. Aggregation is ameliorated by the inclusion of surfactants in biotherapeutics formulations -- typically non-ionic polymeric ether surfactants. The most commonly used examples are Tween 20 (Polysorbate 20) and Tween 80 (Polysorbate 80). Others include Triton X-100, Pluronic F-68, Pluronic F-88, Pluoronic F-127 (poloxamers), and Brij 35 (polyoxyethylene alkyl ether). The usefulness of polysorbates in particular in preventing protein aggregation in biotherapeutic formulations is well accepted. However, polysorbates contain ether linkages and unsaturated alkyl chains that have been shown to auto-oxidize in aqueous solution to protein-damaging peroxides and reactive aldehydes including formaldehyde and acetaldehyde. The peroxides principally affect methionine and tryptophan moieties. The aldehydes react with primary amino groups on proteins and are known to induce immunogenicity of proteins in the absence of aggregation or adjuvants. Detection of protein aggregation and prevention of aggregation using polysorbates is relatively straightforward using light scatter or size exclusion chromatography methods. Detection of oxidative damage to amino acyl moieties or increased immunogenicity resulting from reaction of biotherapeutic with the degradation products of polysorbates is considerably more difficult and has generally been ignored in the scientific literature. As an increasing number of biotherapeutic agents, come into use in common clinical practice, both innovator and biosimilar products, these latter issues will come under increased scrutiny. Substitution of non-ionic non-ether-based surfactants could offer significant improvements in stability, reduced immunogenicity, and shelf life and represents a significant unmet need in the field of biotherapeutics formulation.