Mechanistic insight into the oxidative degradation of monoclonal antibodies: relevance to developability and the design of stable pharmaceutical formulations

Abstract

Oxidation represents a key pathway for the chemical degradation of therapeutic monoclonal antibodies (mAbs), and chemical liabilities such as amino acid residue oxidation are an integral part of developability studies. Mechanistically, oxidation reactions in formulations of therapeutic mAbs are frequently not well understood, as critical information such as the nature of the oxidant(s) is frequently lacking. This brief review summarizes recent screening and developability studies of therapeutic proteins specifically focusing on oxidative liabilities and discusses these data in view of mechanistic and complementary analytical information on the oxidation of amino acid residues in specific protein sequences.

Introduction

Chemical stability is one important parameter for the design of safe and efficacious formulations of therapeutic proteins. A major degradation pathway in these formulations is oxidation, though mechanistically this pathway is significantly less understood compared with other degradation routes such as, e.g., hydrolysis. There are various reasons for this, chief among them the facts that (i) the nature of oxidants in pharmaceutical formulations is frequently unknown, (ii) the nature of oxidants can change depending on the formulation composition, the identity and quantity of impurities, and the type of stresses the formulation is exposed to, and (iii) all amino acid residues of a protein are susceptible to oxidation, though preferential targets are the aromatic and sulfur-containing amino acids. Despite the complexity of reactions, it is possible to distinguish to some extent between individual oxidation pathways through careful analysis of mechanisms and reaction products. This entails the use of complementary analytical methods, including high-resolution mass spectrometry (MS) and nuclear magnetic resonance spectroscopy. Individual oxidants may be monitored by specific tests (e.g., the Amplex Red assay for hydrogen peroxide [1,2]) or, if free radicals are involved, by spin-trapping in combination with either electron spin resonance spectroscopy or mass spectrometry [3].

Protein oxidation reactions can be mediated by reactive oxygen species (ROS) generated in the bulk solution/environment and/or by site-specific processes. The latter confine the oxidation reactions(s) to select protein domains where specific factors such as amino acid composition, peptide sequence, and/or geometry allow for efficient oxidant generation and reactivity. Site-specific processes are significant for metal-catalyzed protein oxidation supported by the preferential binding of metals to select protein domains [4,5]. Critical to site-specific oxidation is the binding of a redox-active metal, which is in or can be converted to a reduced oxidation state (either through a reductant or light) and is available for reaction with either oxygen or peroxides within the metal-binding site (importantly, peroxides can function as reductants for redox-active metals [6]). This is schematically illustrated in Figure 1, where L denotes metal-binding ligands located on the protein.

Such conditions will be relevant for the case study on FeII-dependent polyreactivity, presented below. Protein oxidation can also be mediated by light. Here, the nature and intensity of the incident light will play a major role with respect to the nature and yield of photo-oxidation products. The incident light may directly interact with chromophores of the protein but also with chromophores present through excipients and impurities, a situation that may lead to a variety of reactive intermediates originating from both protein and excipients [7]. Potential chromophores on the protein include tryptophan (Trp), as further discussed below, and protein degradation products (e.g., Trp oxidation products, age-related glycation end products, thiolato-cobalamine adducts, etc.) [7,8]. Potential chromophores on excipients include iron complexes of buffers such as citrate, His, and aspartate (Asp), where exposure to light can lead to the generation of a variety of ROS as well as a powerful reductant, the carbon dioxide radical anion (•CO2−) [9–14]. Formulations of therapeutic proteins will be exposed predominantly to visible and near UV light, and a longstanding question is how visible light exposure can trigger protein oxidation despite the fact that individual amino acids in proteins do not significantly absorb visible light. A potential answer to this question will be provided below.

This review will focus predominantly on monoclonal antibodies (mAbs). Typically, the excipients in commercial formulations of mAbs encompass combinations of buffers (predominantly histidine (His), phosphate, citrate, and/or acetate), cryo- or lyoprotectants (e.g., sucrose or trehalose), surfactants (e.g., polysorbate or poloxamer), amino acids (e.g., arginine (Arg), glycine (Gly), proline (Pro), or lysine (Lys)), and tonicity modifiers [15,16]. The pH values of monoclonal antibody formulations predominantly cover the range of pH 5–7 [15]. Typical excipient concentrations vary depending on whether the products are designed as liquid versus lyophilized or low- versus high-antibody-concentration formulations [15,16]. Especially the presence of surfactants at concentrations above the critical micelle concentration will create biphasic environments where oxidation reactions may proceed more efficiently within the lipid cores of surfactant micelles [17–19]. While proteins will not be in contact with these micellar lipid cores, oxidation processes within these cores can create ROS (e.g., lipid hydroperoxides), which may subsequently translocate to micellar surfaces (peroxidized lipids are more surface-active than non-peroxidized lipids and lower the interfacial tension of a hexadecane/water bilayer [20]). These ROS can react with the proteins at the micelle surface, in the bulk solution or at other interfaces present in the formulation (e.g., the air-water interface).

Mechanistic details could be of great assistance for the design of stable formulations through lead optimization, the interpretation of developability assessment, and mitigation strategies. Therefore, this brief review will focus on mechanistic aspects of important oxidation processes in pharmaceutical formulations and their impact on design, manufacturing, storage, and patient administration. Specific emphasis will be placed on the developability of therapeutic mAbs, mechanistic details on the oxidation of Trp and methionine (Met), and the oxidative degradation of antibody–drug conjugates (ADCs).

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Christian Schöneich; Mechanistic insight into the oxidative degradation of monoclonal antibodies: relevance to developability and the design of stable pharmaceutical formulations. Biochem Soc Trans 27 May 2026; 54 (5): 523–533. doi: https://doi.org/10.1042/BST20250134

Read also our introduction article on Monoclonal Antibodies here:

• A perspective on high-concentration spray-dried monoclonal antibody suspensions for subcutaneous delivery
• High-dose biologics and bioconjugates delivery: Integrating molecular optimization with device design and routes of administration
• Feasibility and stability evaluation of parabens as an alternative preservative for liquid monoclonal antibody formulations