Christian Schöneich

Oxidation represents an important chemical degradation process in formulations of therapeutic peptides and proteins. Potential consequences of peptide and protein oxidation are loss of efficacy, aggregation and/or immunogenicity. Depending on the oxidant(s) present or generated in pharmaceutical formulations, oxidation can target any amino acid, though preferred oxidation targets are the aromatic and sulfur-containing amino acids. The rational development of stable peptide and protein formulations would be greatly assisted by a detailed mechanistic understanding of protein oxidation processes in these formulations. This review will provide an overview about oxidation mechanisms relevant for pharmaceutical peptide and protein formulations. It will cover oxidation mechanisms of peptide and protein amino acids as well as processes which may lead to the formation of oxidants in pharmaceutical formulations, together with a mechanistic analysis of potential mitigation strategies.

Light exposure of therapeutic proteins can affect chemical and physical stability and compromise safety and efficacy. In order to develop stable pharmaceutical formulations without undesired side effects, it is essential to understand both product formation and reaction mechanisms which lead to light-induced protein degradation. We recently reported that visible and near UV light induce site-specific fragmentation and photo-conversion of a heavy chain Thr residue in IgG1-Fc, full length IgG1 and IgG1-based fusion proteins formulated in Fe(III)-containing histidine buffer. These post-translational modifications manifest differently in each modality, with the photo-conversion of Thr to Ala being more prominent in IgG1-Fc. Mechanistically, these photo-degradation pathways occur within a Fe(III)-binding pocket present in the CH2 domain. Here, we investigate the role of individual metal-binding residues located in the Fe(III)-binding pocket in the photo-degradation of IgG1-Fc. The single mutants of Asp25 to Ala, Thr32 to Ala, and Glu34 to Gln in IgG1-Fc completely prevent the site-specific photo-degradation of Thr32, while the mutation of Pro33 to Ala had only a relatively small effect. A comparison of crystal structures reveals that the replacement of Pro33 by Ala does not significantly affect the structure of the Fe(III)-binding pocket. Our results indicate that a single amino acid mutation can prevent the Fe(III)-catalyzed photo-degradation, suggesting that single point mutations may be a future pathway towards photo-stable pharmaceutical formulations.

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.

Near-UV and visible-light photodegradation is a concern for the development of stable and efficacious protein therapeutics, but mechanistic information on the underlying processes is scarce. We previously reported on the photoconversion and photofragmentation of IgG1-Fc, IgG1, and an IgG1-based fusion protein in the CH₂ domain. Here, we report a novel site-specific photodegradation mechanism, converting Thr into Ala catalyzed by Fe(III). Site-directed mutagenesis and spin-trapping in conjunction with high pressure liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis reveal that this novel photoconversion proceeds via the generation of intermediary protein glycyl and methyl (^•CH₃) radicals analogous to metal-catalyzed cross-coupling reactions observed in synthetic organic processes.

In previous studies, we discovered that the exposure of IgG4-Fc and IgG1 to UV light resulted in the side chain cleavage of specific Tyr and Trp residues, converting these amino acids into a series of products, including Gly [Haywood, J. Mol. Pharmaceutics 2013, 10(3), 1146-1150; Kang, H. Mol. Pharmaceutics 2019, 16, 258-272]. In order to evaluate the physicochemical consequences of such photochemical transformations, we prepared a series of IgG4-Fc mutants, in which Trp and Tyr residues were replaced by Gly, i.e., Y300G, Y373G, Y436G, and W381G, for biophysical studies. As the expression yields of W381G IgG4-Fc were low, we also prepared W381A to achieve higher yields required for some of the studies. Among these mutants, Y373G displayed significantly lower melting temperatures compared to wild-type IgG4-Fc, as analyzed by differential scanning calorimetry and fluorescence spectroscopy, indicating a decrease in thermal stability of both the CH₂ and CH₃ domains. In contrast, W381A showed no thermal transitions, indicating a significant loss of overall thermal stability. As for binding affinity to FcγRIIIA, Y300G and Y436G displayed ca. 10-fold reduction compared to wild-type IgG4-Fc. Interestingly, W381A and W381G IgG4-Fc did not only contain N-linked glycans but also high levels of O-mannose (>60%) at Ser³⁷⁵. Therefore, we prepared additional mutants, S375A and S375A/W381A IgG4-Fc, to evaluate the effect of N- vs O-glycosylation on thermal stability and the susceptibility of Tyr and Trp residues to undergo photodegradation. The removal of N-glycans specifically destabilized the CH₂ domain of IgG4-Fc. The removal of O-glycans did not affect the overall thermal stability of S375A IgG4-Fc, and S371A/W381A shows no thermal transitions, suggesting that the W381A mutation alone is sufficient to perturb the protein structure irrespective of O-glycosylation. As for photostability, we observed a 2.6-13.5 fold reduction of yields of Tyr side chain fragmentation products for W381A IgG4-Fc, potentially due to a lower probability for electron transfer between Tyr and oxidized Trp, and photodegradation of alternative Trp residues in W381A IgG4-Fc.

Purpose Evaluation of the stability of an IgG2 in citrate buffer, upon exposure to near-UV and visible light and in the presence of relevant trace amounts of iron. Methods We monitored the oxidation of amino acid residues, the formation of protein aggregates, fragments and charge variants by SDS-PAGE and two-dimensional gel electrophoresis (2-DIGE). Degradation products in individual gel spots were isolated and characterized by HPLC–MS/MS. Results We detected an increase in the formation of DOPA, protein aggregates, and fragments with increasing concentrations of added Fe³⁺. Light exposure resulted in the generation of more acidic products, evident from a shift of the protein pI detected by 2-DIGE. Oxidation products of Tyr, His, Cys, and Trp were detected. The addition of EDTA or DTPA showed a significant protection against degradation. Conclusions IgG2 was significantly modified by the photo-Fenton reaction in citrate buffer. We demonstrated the oxidation of Tyr, His, Cys, and Trp residues, the formation of aggregation and degradation products, as well as the formation of different charge variants.

Polysorbate 80 (PS80) is a commonly used surfactant for stabilizing biotherapeutics by preventing protein adsorption at the air-liquid interface. However, PS80 is susceptible to oxidative degradation during manufacturing and storage. We show here that light exposure combined with the presence of metals can result in byproduct formation and potentially decrease the surfactant's ability to prevent protein adsorption to the air-liquid interface. PS80 formulated in citrate buffer can undergo cis/trans isomerization of unsaturated fatty acids in the presence of disulfides and iron (Prajapati et al., 2022). This work investigates novel surface activity aspects of polysorbate formulations before and after exposure to UV-A light. Polysorbate samples of different grades were formulated in citrate buffer containing iron and glutathione disulfide (GSSG; as a surrogate for peptide and protein disulfides), and a Langmuir trough was used to monitor the surface pressure during adsorption to the air-solution interface. Our results showed significant changes in the polysorbate surface activity after photoirradiation: all-oleate PS80 exhibited a 3-fold increase in the apparent critical micelle concentration (CMC), and the presence of both cis and trans fatty acids was confirmed. Also, the impact of photoirradiation on surface pressure depended on the surfactant concentration during irradiation, suggesting that the presence of micelles can alter the degradation pathway and byproduct formation.

Alpha-galactosylation, galactose-α-1,3-galactose (α-Gal), is always regarded as a critical quality attribute due to its potential to provoke immunogenic responses in patients. Consequently, monitoring alpha-galactosylation in therapeutic proteins is essential, but current analytical techniques fall short in terms of identification sensitivity and quantification accuracy. Specifically, the released glycan assay by hydrophilic interaction liquid chromatography-fluorescence-mass spectrometry, the gold standard for glycan separation/identification, faces challenges due to ambiguities with isomeric glycan structures. To address these challenges, we developed a comprehensive analytical method that enhances both the identification sensitivity and quantification accuracy for α-Gal. We developed an integrated workflow that combines an advanced mass spectrometry technique - parallel reaction monitoring triple-stage mass spectrometry - with exoglycosidase sample treatment. This approach generates structurally specific signature ions, enhances identification sensitivity, enables the separation of α-Gal from its isomers, and improves quantification accuracy. By employing this more sensitive analytical approach without ambiguity in assignment for common glycan structures found in monoclonal antibodies, the safety of therapeutic proteins can be better assured, effectively minimizing the risk of α-Gal-induced immunogenicity.