All-in-one stability indicating polysorbate 20 degradation root-cause analytics via UPLC-QDa

Abstract

Polysorbates (PS) are the most frequently used surfactants to stabilize biologicals. Ironically, these excellent stabilizing non-ionic surfactants have inherent structural properties, which lead to instabilities of their own. Such PS degradation can be triggered by multiple root-causes, like chemical and enzymatic hydrolysis or oxidative degradation. This can on the one hand reduce the concentration of surface-active PS and on the other hand lead to the formation of unfavorable degradants, like poorly soluble free fatty acids (FFA), which may phase separate and form visible FFA particles. Due to the potential criticality of PS degradation in biopharmaceutical formulations, various analytics have been established in recent years not only to monitor the PS content but also to evaluate specific PS markers and crucial degradants. However, in most cases sample preparations and several analytical assays have to be conducted to obtain a comprehensive picture of potential PS degradation root-causes. Here we show a novel approach for PS degradation UPLC-QDa based root-cause analytics, which utilizes previously established analytics for (i) most relevant polysorbate 20 (PS20) esters, (ii) PS20 free fatty acids and (iii) a newly developed method for the evaluation of PS20 specific oxidation markers. Thereby, this triad of analytical methods uses the same sample preparation and detector, which reduces the overall necessary effort, time investment and sample volume. Furthermore, the innovative PS20 oxidation marker method allows to quantify specific concentrations of the determined markers by external calibration and possible perception of oxidative degradation processes prior to relevant losses of PS20 esters, which could serve as an early indication during formulation development. The applicability of this method set was verified using several PS20 containing stress samples, which cover the most relevant root-causes, including acidic and alkaline hydrolysis, enzyme mediated hydrolysis, oxidative AAPH stress and Fe²⁺/H₂O₂ mediated degradation as well as autoxidation via long-term storage at elevated temperatures. Overall, this analytical setup has shown to deliver in-depth data about PS20 degradation, which can be used to narrow down the causative stress without the necessity of fundamentally different methods. Therefore, it can be seen as all-in-one solution during sometimes troublesome development of biopharmaceutical formulations, that supports the elucidation of the PS degradation mechanism(s) and thus establish mitigation strategies.

Keywords: Ester hydrolysis; Esters; Fatty acids; Label-free quantification; Oxidation; Polysorbate; QDa; Selective mode; UPLC.