Cis/Trans Isomerization of Unsaturated Fatty Acids in Polysorbate 80 During Light Exposure of a Monoclonal Antibody–Containing Formulation

Abstract

Light exposure of a monoclonal antibody formulation containing polysorbate 80 (PS80) leads to cis/trans isomerization of monounsaturated and polyunsaturated fatty acids. This cis/trans isomerization was monitored by positive electrospray ionization mass spectrometry of intact PS80 components as well as by negative ion electrospray ionization mass spectrometry analysis of free fatty acids generated via esterase-catalyzed hydrolysis. The light-induced cis/trans isomerization of unsaturated fatty acids in PS80 required the presence of the monoclonal antibody, or, at a minimum (for mechanistic studies), a combination of N-acetyltryptophan amide and glutathione disulfide, suggesting the involvement of thiyl radicals generated by photoinduced electron transfer from Trp to the disulfide. Product analysis confirmed the conversion of PS80-bound oleic acid to elaidic acid; furthermore, together with linoleic acid, we detected conjugated linoleic acids in PS80, which underwent light-induced cis/trans isomerization.

Near UV and Visible Light Photo-Degradation Mechanisms in Citrate Buffer: One-Electron Reduction of Peptide and Protein Disulfides promotes Oxidation and Cis/Trans Isomerization of Unsaturated Fatty Acids of Polysorbate 80

“…56 We previously demonstrated that photo-irradiation of a monoclonal antibody-containing formulation in acetate buffer with low doses (< 40 Wh/m 2 ) of UV-B and UV-C light caused cis/trans isomerization of oleic and linoleic acid in their respective POE sorbitan esters. 57 Under these conditions, thiyl radicals were generated via photo-induced electron transfer from protein Trp resides to disulfide bonds. The following experiments will show that peptide or protein disulfide reduction by • CO 2 ─ , generated from near UV or visible light exposure of citrate buffer in the presence of Fe 3+ , will similarly lead to cis/trans isomerization of unsaturated fatty acids.…”

“…73 We had previously discussed some potential consequences of trans fatty acid formation in PScontaining formulations. 57 In brief, the formation of trans fatty acids in PS may lead to changes in micelle structure, 74,75 but the extent of these changes will depend on the yields of trans fatty acids.…”

Photo-Oxidation of Therapeutic Protein Formulations: From Radical Formation to Analytical Techniques

“…Thus, SEC-UV has Since polysorbates (PS) (Figure 9) are used in pharmaceutical industry as surfactants of protein formulations, investigations on the photo-degradation are currently of high interest. Light exposure to PS solutions can result in structural changes of the PS molecule itself [413] via peroxide formation [191,414,415]. The formation of ROS species such as peroxides can result in oxidation of polysorbates [413]and of subsequent amino acid residues of proteins.…”

“…Light exposure to PS solutions can result in structural changes of the PS molecule itself [413] via peroxide formation [191,414,415]. The formation of ROS species such as peroxides can result in oxidation of polysorbates [413]and of subsequent amino acid residues of proteins. However, the detailed mechanism and its consequences to proteins are not well understood yet [416].…”

Reductive Stress of Sulfur-Containing Amino Acids within Proteins and Implication of Tandem Protein–Lipid Damage

“…A recent study of protein-lipid tandem damage showed that other amino acids of the molecular structure can trigger the formation of thiyl radicals by reductive stress and induce tandem damages. This is the case of a monoclonal antibody containing tryptophan moieties that, exposed to photoirradiation at 300 nm wavelength in the presence of Polysorbate-80, produces a tryptophan radical cation and a hydrated electron, which in its turn reduces a protein disulfide bond thus forming thiyl radicals and creating the lipid isomerization catalyst [ 67 ].…”