Protein Formulations Containing Polysorbates: Are Metal Chelators Needed at All?

Abstract

Proteins are prone to post-translational modifications at specific sites, which can affect their physicochemical properties, and consequently also their safety and efficacy. Sources of post-translational modifications include oxygen and reactive oxygen species. Additionally, catalytic amounts of Fe(II) or Cu(I) can promote increased activities of reactive oxygen species, and thus catalyse the production of particularly reactive hydroxyl radicals. When oxidative post-translational modifications are detected in the biopharmaceutical industry, it is common practice to add chelators to the formulation. However, the resultant complexes with metals can be even more damaging. Indeed, this is supported here using an ascorbate redox system assay and peptide mapping. Ethylenediaminetetraacetic acid (EDTA) addition strongly accelerated the formation of hydroxyl radicals in an iron-ascorbate system, while diethylenetriaminepentaacetic acid (DTPA) addition did not. When Fe(III) was substituted with Cu(II), EDTA addition almost stopped hydroxyl radical production, whereas DTPA addition showed continued production, but at a reduced rate. Further, EDTA accelerated metal-catalysed oxidation of proteins, and thus did not protect them from Fe-mediated oxidative damage. As every formulation is unique, justification for EDTA or DTPA addition should be based on experimental data and not common practice.

Oxidation of polysorbates – An underestimated degradation pathway?

“…As described above, EDTA was reported to prevent iron-induced polysorbate oxidation ( Doyle et al, 2019 ; Kranz et al, 2019 ; Gopalrathnam et al, 2018 ; Bensaid et al, 2022 ; Kranz et al, 2020 ; Yarbrough et al, 2019 ), however, it was also claimed to drive protein oxidation under certain circumstances with ascorbate and Fe 3+ ( Stadtman, 1990 ). Similar behavior was demonstrated by Brovč and colleagues (2020), stressing proteins and polysorbate with Fe 3+ and Cu 2+ in the presence and absence of ascorbate as well as EDTA/DTPA ( Brovč et al, 2020c ). A dependency of OH • formation on chelator and on metal ions could be observed.…”

“…Another key characteristic of oxidative degradation is that PS polyesters are significantly more affected by oxidation than monoesters as reported by various stress tests with either H 2 O 2 , AAPH, temperature, or iron ( Borisov et al, 2015 ; Lippold et al, 2017 ; Liu et al, 2022a ; Zhang et al, 2017 ; Kranz et al, 2019 ; Bensaid et al, 2022 ; Zhang et al, 2018 ; Kranz et al, 2020 ; Brovč et al, 2020c ). Via CAD, Kranz and colleagues (2019) showed that higher-order esters of PS20 HP completely disappear after treatment with 10 ppb Fe 2+ at 40 °C after seven days.…”

“…After examination of the oxidative processes in polysorbate in detail, it remains to be answered how oxidative polysorbate degradation can be reduced or prevented. Various approaches are being pursued for this, like the addition of chelators, antioxidants, or by reducing the impurities such as metals ( Doshi et al, 2020a ; Doyle et al, 2019 ; Schmidt et al, 2020 ; Knepp et al, 1996 ; Mcclements and Decker, 2000 ; Waraho et al, 2009 ; Yarbrough et al, 2019 ; Kranz et al, 2019 ; Gopalrathnam et al, 2018 ; Schröter et al, 2021 ; Bensaid et al, 2022 ; Doshi et al, 2021a ; Kranz et al, 2020 ; Brovč et al, 2020c ; Doshi et al, 2021b ). Alternatively, production processes or environmental conditions can be adapted, as for instance exposure to stainless steel/iron has been extensively examined as critical impact for oxidative polysorbate degradation ( Liu et al, 2022a ; Doyle et al, 2019 ; Kranz et al, 2019 ; Gopalrathnam et al, 2018 ; Schröter et al, 2021 ; Bensaid et al, 2022 ; Kranz et al, 2020 ; Klair et al, 2021 ; Prajapati et al, 2022 ).…”

Theoretical Study of the Iron Complexes with Aminoguanidine: Investigating Secondary Antioxidant Activity

“…Nevertheless, to the best of our knowledge, besides a recent paper from our group [ 24 ], this same type of theoretical investigation has not been carried out for the analogous Fe(III)/Fe(II) reduction, and no comparison has been made to the Cu(II)/Cu(I) system for the same set of antioxidants. But this is a relevant topic which has been explored experimentally for various compounds [ 50 , 51 , 52 , 53 , 54 ].…”

Theoretical Study of the Iron Complexes with Lipoic and Dihydrolipoic Acids: Exploring Secondary Antioxidant Activity

“…While a few theoretical studies have evaluated the secondary antioxidant activity (with rate constant calculations) of various organic ligands with respect to the Cu(II)/Cu(I) reduction with and ascorbate, [ 10 , 11 , 26 , 27 , 28 ] to the best of our knowledge, theoretical kinetic studies of secondary antioxidant activity relative to the Fe(III)/Fe(II) reduction, and their comparison with the Cu(II)/Cu(I) redox system, have not been previously reported for any antioxidant. However, this is a topic of relevance that has been studied experimentally for a variety of substrates [ 29 , 30 , 31 , 32 , 33 ].…”