Near UV Photodegradation Mechanisms of Amino Acid Excipients: Formation of the Carbon Dioxide Radical Anion from Aspartate and Fe(III).

Carbon dioxide radical anion (•CO2-) is a powerful reducing agent that can reduce protein disulfide bonds and convert molecular oxygen to superoxide. Therefore, the generation of •CO2- can be detrimental to pharmaceutical formulations. Iron is among the most prevalent impurities in formulations, where Fe(III) chelates of histidine (His) can produce •CO2- upon exposure to near-UV light (Zhang and Schöneich, Eur. J. Pharm. Biopharm. 2023, 190, 231-241). Here, we monitor by spin-trapping in combination with electron paramagnetic resonance spectroscopy and/or high-performance liquid chromatography-mass spectrometry analysis the photochemical formation of •CO2- for a series of common amino acid excipients, including arginine (Arg), methionine (Met), proline (Pro), glutamic acid (Glu), glycine (Gly), aspartic acid (Asp), and lysine (Lys). Our results indicate that in the presence of Fe(III), Asp, and Glu produce significant yields of •CO2- under photoirradiation with near-UV light. Notably, Asp demonstrates the highest efficiency of •CO2- generation compared with that of the other amino acid excipients. Stable isotope labeling indicates that •CO2- exclusively originates from the α-carboxyl group of Asp. Mechanistic studies reveal two possible pathways for •CO2- formation, which involve either a ß-carboxyl radical or an amino radical cation intermediate.

Near UV and Visible Light-Induced Degradation of Bovine Serum Albumin and a Monoclonal Antibody Mediated by Citrate Buffer and Fe(III): Reduction vs Oxidation Pathways.

Light exposure during manufacturing, storage, and administration can lead to the photodegradation of therapeutic proteins. This photodegradation can be promoted by pharmaceutical buffers or impurities. Our laboratory has previously demonstrated that citrate-Fe(III) complexes generate the •CO2- radical anion when photoirradiated under near UV (λ = 320-400 nm) and visible light (λ = 400-800 nm) [Subelzu, N.; Schöneich, C. Mol. Pharmaceutics 2020, 17 (11), 4163-4179; Zhang, Y. Mol. Pharmaceutics 2022, 19 (11), 4026-4042]. Here, we evaluated the impact of citrate-Fe(III) on the photostability and degradation mechanisms of disulfide-containing proteins (bovine serum albumin (BSA) and NISTmAb) under pharmaceutically relevant conditions. We monitored and localized competitive disulfide reduction and protein oxidation by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis depending on the reaction conditions. These competitive pathways were affected by multiple factors, including light dose, Fe(III) concentration, protein concentration, the presence of oxygen, and light intensity.

Near UV and Visible Light Photodegradation in Solid Formulations: Generation of Carbon Dioxide Radical Anions from Citrate Buffer and Fe(III).

Near UV and visible light photodegradation can target therapeutic proteins during manufacturing and storage. While the underlying photodegradation pathways are frequently not well-understood, one important aspect of consideration is the formulation, specifically the formulation buffer. Citrate is a common buffer for biopharmaceutical formulations, which can complex with transition metals, such as Fe(III). In an aqueous solution, the exposure of such complexes to light leads to the formation of the carbon dioxide radical anion (•CO2-), a powerful reductant. However, few studies have characterized such processes in solid formulations. Here, we show that solid citrate formulations containing Fe(III) lead to the photochemical formation of •CO2-, identified through DMPO spin trapping and HPLC-MS/MS analysis. Factors such as buffers, the availability of oxygen, excipients, and manufacturing processes of solid formulations were evaluated for their effect on the formation of •CO2- and other radicals such as •OH.

Coarse-Grained Molecular Dynamics Simulation of Heterogeneous Polysorbate 80 Surfactants and their Interactions with Small Molecules and Proteins.

Polysorbate 80 (PS80) is widely used in pharmaceutical formulations, and its commercial grades exhibit certain levels of structural heterogeneity. The objective of this study was to apply coarse-grained molecular dynamics simulations to better understand the effect of PS80 heterogeneity on micelle self-assembly, the loading of hydrophobic small molecules into the micelle core, and the interactions between PS80 and a protein, bovine serum albumin (BSA). Four representative PS80 variants with different head and tail structures were studied. Our simulations found that PS80 structural heterogeneity could affect blank micelle properties such as solvent-accessible surface area, aggregation number, and micelle aspect ratio. It was also found that hydrophobic small molecules such as ethinyl estradiol preferentially partitioned into the PS80 micelle core and PS80 dioleates formed a more hydrophobic core compared to PS80 monooleates. Furthermore, multiple PS80 molecules could bind to BSA, and PS80 heterogeneity profoundly changed the binding ratio as well as the surfactant-protein contact area.

Molecular Dynamics Simulation of an Iron(III) Binding Site on the Fc Domain of IgG1 Relevant for Visible Light-Induced Protein Fragmentation.

Molecular dynamics simulations were employed to investigate the interaction between Fe(III) and an iron-binding site composed of THR259, ASP252, and GLU261 on the Fc domain of an IgG1. The goal was to provide microscopic mechanistic information for the photochemical, iron-dependent site-specific oxidative fragmentation of IgG1 at THR259 reported in our previous paper. The distance between Fe(III) and residues of interest as well as the binding pocket size was examined for both protonated and deprotonated THR259. The Fe(III) binding free energy (ΔG) was estimated by using an umbrella sampling approach. The pKa shift of the THR259 hydroxyl group caused by the presence of nearby Fe(III) was estimated based on a thermodynamic cycle. The simulation results show that Fe(III) resides inside the proposed binding pocket and profoundly changes the pocket configuration. The ΔG values indicate that the pocket possesses a strong binding affinity for Fe(III). Furthermore, Fe(III) profoundly lowers the pKa value of the THR259 hydroxyl group by 5.4 pKa units.

In-Depth Characterization of Acidic Variants Induced by Metal-Catalyzed Oxidation in a Recombinant Monoclonal Antibody.

Characterization of antibody charge heterogeneity is an important task for antibody drug development. Recently, a correlation between acidic charge heterogeneity and metal-catalyzed oxidation has been observed for antibody drugs. However, to date, the acidic variants induced by metal-catalyzed oxidation have not been elucidated. In addition, it is challenging to satisfactorily explain the induced acidic charge heterogeneity, as the existing analytical workflows, which relied on either untargeted or targeted peptide mapping analysis, could lead to incomplete identification of the acidic variants. In this work, we present a new characterization workflow by combining untargeted and targeted analyses to thoroughly identify and characterize the induced acidic variants in a highly oxidized IgG1 antibody. As a part of this workflow, a tryptic peptide mapping method was also developed for accurate determination of the relative extent of site-specific carbonylation, where a new hydrazone reduction procedure was established to minimize under-quantitation artifacts caused by incomplete reduction of hydrazones during sample preparation. In summary, we identified 28 site-specific oxidation products, which are located on 26 residues and of 11 different modification types, as the sources of the induced acidic charge heterogeneity. Many of the oxidation products were reported for the first time in antibody drugs. More importantly, this study provides new insights to understanding acidic charge heterogeneity of antibody drugs in the biotechnology industry. Additionally, the characterization workflow presented in this study can be applied as a platform approach in the biotechnology industry to better address the need for in-depth characterization of antibody charge variants.

Visible Light Induces Site-Specific Oxidative Heavy Chain Fragmentation of a Monoclonal Antibody (IgG1) Mediated by an Iron(III)-Containing Histidine Buffer.

Fragmentation of therapeutic monoclonal antibodies represents a critical quality attribute. Here, we report a novel visible light-induced heavy chain fragmentation of IgG1 mediated by an Fe(III)-containing histidine (His) buffer. Based on non-reducing sodium dodecylsulfate-polyacrylamide gel electrophoresis and mass spectrometry analysis, IgG1 fragments with apparent molecular weights of ∼130, ∼110, and ∼22 kDa were detected in photo-irradiated samples and were mechanistically rationalized with an oxidative cleavage at Thr259. Specifically, the reactions are proposed to involve the generation of an intermediary alkoxyl radical, which undergoes ß-cleavage to yield a glycyl radical. The latter either converts into Gly or adds oxygen and follows a peroxyl radical chemistry. The cleavage process requires the presence of His, while only negligible yields of cleavage products are formed when His is replaced by acetate, succinate, or phosphate buffer. Importantly, the fragmentation can be prevented by ethylenediaminetetraacetic acid (EDTA) only when the EDTA concentrations are in significant excess over the concentrations of Fe(III) and proteins, suggesting a strong binding between Fe(III) and IgG1.

Near-UV and Visible Light Degradation of Iron (III)-Containing Citrate Buffer: Formation of Carbon Dioxide Radical Anion via Fragmentation of a Sterically Hindered Alkoxyl Radical.

Citrate is a commonly used buffer in pharmaceutical formulations which forms complexes with adventitious metals such as Fe3+. Fe3+-citrate complexes can act as potent photosensitizers under near-UV and visible light exposure, and recent studies reported evidence for the photo-production of a powerful reductant, carbon dioxide radical anion (•CO2-), from Fe3+-citrate complexes (Subelzu, N.; Schöneich, N., Mol. Pharm. 2020, 17, 4163-4179). The mechanisms of •CO2- formation are currently unknown but must be established to devise strategies against •CO2- formation in pharmaceutical formulations which rely on the use of citrate buffer. In this study, we first established complementary evidence for the photolytic generation of •CO2- from Fe3+-citrate through spin trapping and electron paramagnetic resonance (EPR) spectroscopy, and subsequently used spin trapping in conjunction with tandem mass spectrometry (MS/MS) for mechanistic studies on the pathways of •CO2- formation. Experiments with stable isotope-labeled citrate suggest that the central carboxylate group of citrate is the major source of •CO2-. Competition studies with various inhibitors (alcohols and dimethyl sulfoxide) reveal two mechanisms of •CO2- formation, where one pathway involves ß-cleavage of a sterically hindered alkoxyl radical generated from the hydroxyl group of citrate.

Advanced Oxidation Processes in Pharmaceutical Formulations: Photo-Fenton Degradation of Peptides and Proteins.

Formulations of therapeutic proteins are sensitive to photo-degradation by near UV and visible light. Mechanistically, especially the processes leading to protein modification under visible light exposure are not understood. Potentially, these processes may be triggered by a ligand to metal charge transfer in excipient-metal complexes. This article summarizes recent analytical and mechanistic work on such reactions under experimental conditions relevant to pharmaceutical formulations.

Analysis of N15-rat growth hormone after incubation with rat subcutaneous tissue and immune cells using ultra-pressure chromatography-mass spectrometry.

Therapeutic proteins (TPs) are exposed to various immune cells like macrophages and neutrophils, especially after subcutaneous (SC) administration. It is well known that the immune cells can generate reactive oxygen species (ROS) and this may lead to oxidation of TPs. The oxidation can occur in the SC tissue after SC administration, during distribution to the immune organs like lymph nodes and spleen, and even in the blood circulation. The oxidation can lead to alteration of their pharmacokinetics and efficacy. Therefore, it is important to study the oxidation of TPs in the biological matrices using ultra-pressure chromatography-mass spectrometry. Rat growth hormone (rGH) was selected as a test protein due to its similarity with human growth hormone (hGH), which is widely used for treatment of growth hormone deficiency. In this manuscript, we have summarized sample processing strategy and ultra-pressure chromatography-mass spectrometry methodology to identify rGH and its degradation products after ex-vivo incubation with rat SC tissue, and in vitro incubation with rat splenocytes and canine peripheral blood mononuclear cells (cPBMCs) as a model foreign host species. We did not observe oxidation of rGH in these biological matrices. This could be due to very minor yields of oxidation products, lack of sensitivity of the mass spectrometry method, loss of protein during sample processing, rapid turnover of oxidized protein or a combination of all factors.

Visible Light Degradation of a Monoclonal Antibody in a High-Concentration Formulation: Characterization of a Tryptophan-Derived Chromophoric Photo-product by Comparison to Photo-degradation of N-Acetyl-l-tryptophan Amide.

We investigated the discoloration of a highly concentrated monoclonal antibody (mAbZ) in sodium acetate (NaAc) and histidine/lysine (His/Lys) buffer after exposure to visible light. The color change of the mAbZ formulation was significantly more intense in NaAc buffer and developed a characteristic absorbance with a λmax of ca. 450 nm. We characterized this photo-chemically generated chromophore by comparison with visible light photo-degradation of a concentrated solution of a model compound for protein Trp residues, N-acetyl-l-tryptophan amide (NATA). The photo-degradation of NATA generated a chromophoric product with a λmax of ca. 450 nm and UV-vis spectroscopic properties identical to those of the product generated from mAbZ. This product was isolated and analyzed by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) and 1H, 13C, and 1H-13C heteronuclear single-quantum correlation NMR spectroscopy. MS/MS analysis reveals a product characterized by the loss of 33 Da from NATA, referred to as NATA-33. Together, the NMR data suggest that this product may be N-(2,4-dihydrocyclopenta[b]indol-2-yl)acetamide (structure P3a) or a tautomer (P3b-d).

Pharmaceutical Excipients Enhance Iron-Dependent Photo-Degradation in Pharmaceutical Buffers by near UV and Visible Light: Tyrosine Modification by Reactions of the Antioxidant Methionine in Citrate Buffer.

PURPOSE: To evaluate the effect of excipients, including sugars and amino acids, on photo-degradation reactions in pharmaceutical buffers induced by near UV and visible light. METHODS: Solutions of citrate or acetate buffers, containing 1 or 50 µM Fe3+, the model peptides methionine enkephalin (MEn), leucine enkephalin (LEn) or proctolin peptide (ProP), in the presence of commonly used amino acids or sugars, were photo-irradiated with near UV or visible light. The oxidation products were analyzed by reverse-phase HPLC and HPLC-MS/MS. RESULTS: The sugars mannitol, sucrose and trehalose, and the amino acids Arg, Lys, and His significantly promote the oxidation of peptide Met to peptide Met sulfoxide. These excipients do not increase the yields of hydrogen peroxide, suggesting that other oxidants such as peroxyl radicals are responsible for the oxidation of peptide Met. The addition of free Met reduces the oxidation of peptide Met, but, in citrate buffer, causes the addition of Met oxidation products to Tyr residues of the target peptides. CONCLUSIONS: Commonly used excipients enhance the light-induced oxidation of amino acids in model peptides.

Light-Induced Histidine Adducts to an IgG1 Molecule Via Oxidized Histidine Residue and the Potential Impact of Polysorbate-20 Concentration.

PURPOSE: Histidine (His) undergoes light-induced reactions such as oxidation, crosslinking and addition. These reactions are initiated by singlet oxygen (1O2) to generate His photo-oxidation products, which are subject to nucleophilic attack by a non-oxidized His residue from another protein or by nucleophilic buffer components such as Tris and His. This report aims to identify light-induced His-adducts to a monoclonal antibody (mAb-1) due to the reaction of His molecules in the buffer with the photooxidized His residues under ICH light conditions. Since polysorbate-20 (PS-20) is a commonly used excipient in biotherapeutics formulation, it is also important to study the impact of PS-20 concentration on protein photostability. RESULTS: We identified and characterized light-induced His-adducts of mAb-1 by LC-MS/MS. We showed that the levels of light-induced His-adducts generally correlate with the solvent accessibility of His residues in the protein. In addition, the presence of PS-20 at concentrations commonly used in protein drug formulations can significantly increase the levels of light-induced His-adducts. CONCLUSIONS: Since His residues are present in a conserved region in the Fc domain, and may be present in the complementarity-determining region (CDR), the impact on the biological functions of the His-adducts observed here should be further studied to evaluate the risk of their presence.

Near UV and Visible Light Induce Iron-Dependent Photodegradation Reactions in Pharmaceutical Buffers: Mechanistic and Product Studies.

Near UV (λ = 320-400 nm) and visible light (λ = 400-800 nm) can lead to the oxidation of pharmaceutical proteins, which can affect efficiency and promote immunogenicity. However, no concise mechanism has been established for the photo-oxidation of pharmaceutical proteins under near UV and visible light. Here, we show that carboxylic acid buffer-Fe3+ complexes can function as photosensitizers, causing peptide degradation via the formation of various radicals and oxidants. Three pharmaceutical relevant carboxylic acid buffers (citrate, acetate, and succinate) were tested under near UV and visible light. Oxidation reactions were monitored for model peptides containing readily oxidizable amino acids, such as methionine- or leucine-enkephalin and proctolin peptide. Oxidation products were evaluated by RP-HPLC coupled to UV or fluorescent detection and RP-HPLC-MS/MS. Specifically for citrate buffer, the light-induced formation of H2O2, •OH, •CO2-, and formaldehyde was demonstrated. The peptides displayed oxidation of Met, hydroxylation of Tyr and Phe, as well as the formation of novel products from Tyr. Experiments with 18O2 resulted in the incorporation of 18O into various reaction products, consistent with a metal-catalyzed activation of O2 into reactive oxygen species. The addition of EDTA and DTPA did not prevent the oxidation of the peptides and, in some cases, enhanced the oxidation. Our results demonstrate that pharmaceutical buffer-Fe3+ complexes, exposed to UV and visible light, can promote various pathways of oxidation reactions in pharmaceutical formulations.

Water Distribution and Clustering on the Lyophilized IgG1 Surface: Insight from Molecular Dynamics Simulations.

Water has a critical role in the stability of the higher-order structure of proteins. In addition, it is considered to be a major destabilization factor for the physical and chemical stability of freeze-dried proteins and peptides. Physical and chemical aspects of protein/water relationships are commonly studied with the use of water vapor sorption isotherms for amorphous lyophilized proteins, which, in turn, are commonly analyzed using the Brunauer-Emmett-Teller (BET) equation to obtain the parameters, Wm and CB. The parameter Wm is generally referred to as the "monolayer limit of adsorption" and has a narrow range of 6-8% for most proteins. In this study, the water distribution on an IgG1 surface is investigated by molecular dynamics (MD) simulations at different water contents. The monolayer of water molecules was found to have limited coverage of the protein surface, and the true monolayer coverage of the protein globule actually occurs at a hydration level above 30%. The distribution of water molecules on the IgG1 surface is also highly heterogeneous, and the heterogeneity is not considered in the BET theory. In this study, a mechanistic model has been developed to describe the water vapor sorption isotherm. This model is based on the analysis of the hydrogen bonding network extracted from the MD simulations. The model is consistent with the experimental Type-II isotherm, which is usually observed for proteins. The physical meaning of the BET monolayer was redefined as the onset of water cluster formation. A simple model to calculate the onset water level, Wm, is proposed based on the hydration of different amino acids, as determined from the MD simulations.

Probing Protein Conformation Destabilization in Sterile Liquid Formulations through the Formation of 3,4-Dihydroxyphenylalanine.

This work demonstrates the use of a fluorescent probe to screen protein conformational changes in mixtures of monoclonal antibodies and determine the region of where such changes may originate through a footprinting mass spectrometry approach. The oxidative stress of mixtures of two different immunoglobulins (IgG1, IgG2, or IgG4) performed in the presence of 2,2'-azobis(2-amidinopropane dihydrochloride) results in sequence-specific tyrosine oxidation reactions depending on the time of incubation of the IgG molecules and the nature of the excipients present in the formulation. The combination of a fluorescence assay, based on the detection of 3,4-dihydroxyphenylalanine (DOPA) and mass spectrometry analyses, permits the identification of protein conformation changes. In a mixture of IgG2 and IgG4, a destabilization of IgG4 in the presence of IgG2 is observed. The destabilized region involves the Fab region of IgG4 between Tyr63 and Tyr81 and potentially multiple regions of IgG2.

Novel Formaldehyde-Induced Modifications of Lysine Residue Pairs in Peptides and Proteins: Identification and Relevance to Vaccine Development.

Formaldehyde-inactivated toxoid vaccines have been in use for almost a century. Despite formaldehyde's deceptively simple structure, its reactions with proteins are complex. Treatment of immunogenic proteins with aqueous formaldehyde results in heterogenous mixtures due to a variety of adducts and cross-links. In this study, we aimed to further elucidate the reaction products of formaldehyde reaction with proteins and report unique modifications in formaldehyde-treated cytochrome c and corresponding synthetic peptides. Synthetic peptides (Ac-GDVEKGAK and Ac-GDVEKGKK) were treated with isotopically labeled formaldehyde (13CH2O or CD2O) followed by purification of the two main reaction products. This allowed for their structural elucidation by (2D)-nuclear magnetic resonance and nanoscale liquid chromatography-coupled mass spectrometry analysis. We observed modifications resulting from (i) formaldehyde-induced deamination and formation of α,ß-unsaturated aldehydes and methylation on two adjacent lysine residues and (ii) formaldehyde-induced methylation and formylation of two adjacent lysine residues. These products react further to form intramolecular cross-links between the two lysine residues. At higher peptide concentrations, these two main reaction products were also found to subsequently cross-link to lysine residues in other peptides, forming dimers and trimers. The accurate identification and quantification of formaldehyde-induced modifications improves our knowledge of formaldehyde-inactivated vaccine products, potentially aiding the development and registration of new vaccines.

Photo-Degradation of Therapeutic Proteins: Mechanistic Aspects.

PURPOSE: Therapeutic proteins are sensitive to photo-degradation by UV A and visible light. As none of the essential amino acids exhibits significant absorption in the UV A and visible light regions, the underlying mechanisms of photo-degradation induced by UV A and visible light are not well understood. This review addresses potential mechanisms, by which protein structure, oxidative modifications or impurities can promote the photo-degradation of therapeutic proteins during the exposure to UV A and visible light.

Photoinduced Tyrosine Side Chain Fragmentation in IgG4-Fc: Mechanisms and Solvent Isotope Effects.

Immunoglobulin gamma (IgG) monoclonal antibodies (mAbs) are glycoproteins that have emerged as powerful and promising protein therapeutics. During the process of production, storage and transportation, exposure to ambient light is inevitable, which can cause protein physical and chemical degradation. For mechanistic studies of photodegradation, we have exposed IgG4-Fc to UV light. The photoirradiation of IgG4-Fc with monochromatic UVC light at λ = 254 nm and UVB light with λmax = 305 nm in air-saturated solutions revealed multiple photoproducts originating from tyrosine side chain fragmentation at Tyr300, Tyr373, and Tyr436. Tyr side chain fragmentation yielded either Gly or various backbone cleavage products, including glyoxal amide derivatives. A mechanism is proposed involving intermediate Tyr radical cation formation, either through direct light absorption of Tyr or through electron transfer to an initial Trp radical cation, followed by elimination of quinone methide. Product formation showed either no (cleavage of Tyr373) or significant (cleavage of Tyr436) inverse product solvent isotope effects (SIEs), indicating a role for proton transfer in the cleavage mechanism of Tyr436. The role of electron transfer in the cleavage of Tyr436 was further investigated through mutation of an adjacent Trp381. This is the first observation of a photoinduced Tyr side chain cleavage reactions in a protein.

Thiyl Radical Reactions in the Chemical Degradation of Pharmaceutical Proteins.

Free radical pathways play a major role in the degradation of protein pharmaceuticals. Inspired by biochemical reactions carried out by thiyl radicals in various enzymatic processes, this review focuses on the role of thiyl radicals in pharmaceutical protein degradation through hydrogen atom transfer, electron transfer, and addition reactions. These processes can lead to the epimerization of amino acids, as well as the formation of various cleavage products and cross-links. Examples are presented for human insulin, human and mouse growth hormone, and monoclonal antibodies.