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

Area of Science:

• Biochemistry
• Protein Chemistry
• Pharmaceutical Science

Background:

• Post-translational modifications (PTMs) alter protein properties, impacting safety and efficacy.
• Oxidative PTMs, often catalyzed by metal ions like iron and copper, are a concern in biopharmaceuticals.
• Chelators are commonly added to mitigate oxidative damage, but their effects can be complex.

Purpose of the Study:

• To investigate the impact of chelators ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA) on metal-catalyzed hydroxyl radical production.
• To evaluate the protective effects of EDTA and DTPA against oxidative damage in protein formulations.
• To determine the efficacy of EDTA and DTPA in iron- and copper-mediated oxidative systems.

Main Methods:

• Utilized an ascorbate redox system assay to measure hydroxyl radical formation.
• Employed peptide mapping to assess protein oxidation.
• Compared the effects of EDTA and DTPA in iron-ascorbate and copper-ascorbate systems.

Main Results:

• EDTA significantly accelerated hydroxyl radical formation in an iron-ascorbate system.
• DTPA did not accelerate hydroxyl radical formation in the iron-ascorbate system.
• EDTA inhibited hydroxyl radical production in a copper-ascorbate system, while DTPA reduced it.
• EDTA accelerated metal-catalyzed protein oxidation, failing to protect against iron-mediated damage.

Conclusions:

• The addition of EDTA can exacerbate oxidative damage to proteins by enhancing hydroxyl radical production in the presence of iron.
• DTPA showed a more favorable profile in mitigating oxidative damage compared to EDTA in the tested systems.
• The choice of chelator for biopharmaceutical formulations requires specific experimental validation due to unique formulation characteristics.