Islatravir Dimer: Crystal Form Dependence of a Radiation-Induced Degradation Product.

A rare example of crystal form-dependent, gamma radiation-induced degradation is presented. Islatravir is known to exist in several polymorphic forms, but only one of these forms shows the generation of a specific dimer degradation product under gamma irradiation. Extended gamma irradiation studies demonstrated that only one of the known crystalline forms shows an appreciable rate of dimer formation. Additionally, this dimer is not observed to form under other forced stress conditions. We present the structural elucidation of this dimer impurity and rationalize its form-dependent generation based on the analysis of the underlying crystal structure.

Absolute Quantitation of Proteins by Coulometric Mass Spectrometry.

Accurate quantification is essential in the fields of proteomics, clinical assay, and biomarker discovery. Popular methods for absolute protein quantitation by mass spectrometry (MS) involve the digestion of target protein and employ isotope-labeled peptide internal standards to quantify chosen surrogate peptides. Although these methods have gained success, syntheses of isotope-labeled peptides are time-consuming and costly. To eliminate the need for using standards or calibration curves, herein we present a coulometric mass spectrometric (CMS) approach for absolute protein quantitation, based on the electrochemical oxidation of a surrogate peptide combined with mass spectrometric measurement of the oxidation yield. To demonstrate the utility of this method, several proteins were analyzed such as model proteins ß-casein, and apomyoglobin as well as circadian clock protein KaiB isolated from Escherichia coli. In our experiment, tyrosine-containing peptides were selected as surrogate peptides for quantitation, considering the oxidizable nature of tyrosine. Our data showed that the results for surrogate peptide quantity measured by our method and by traditional isotope dilution method are in excellent agreement, with the discrepancy of 0.3-3%, validating our CMS method for absolute quantitation. Furthermore, therapeutic monoclonal antibody (mAb) could be quantified by our method as well. Due to the high specificity and sensitivity of MS and no need to use isotope-labeled peptide standards, our CMS method would be of high value for the absolute proteomic quantification.

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.

Fragmentation of a Monoclonal Antibody by Peroxotungstate.

PURPOSE: Tungsten and tungsten oxide leachates found in glass pre-filled syringes were identified to initiate protein precipitation and aggregation. Here, we tested the possibility of tungsten and tungsten oxide to induce the chemical degradation of proteins via reaction with hydrogen peroxide, a possible impurity present in protein formulations, to yield peroxotungstate. METHODS: A monoclonal antibody (mAb) was incubated with various concentrations of peroxotungstate and the reaction mixtures analyzed by SDS-PAGE and mass spectrometry. RESULTS: Exposure of a mAb to 1.07-1070 ppm peroxotungstate (based on tungsten content) at temperatures of 4°C and 22°C (pH 5-7) induced protein fragmentation. The extent of fragmentation increased with higher temperatures, lower pH and higher peroxotungstate concentrations. The mAb fragments were identified to contain different combinations of heavy chains (H) and light chains (L). Analogous mAb fragments were generated when the protein was exposed to H2O2 and orthotungstate at levels as low as 5 ppm. In addition, extracts from tungsten pins used to manufacture glass pre-filled syringes, in combination with H2O2 caused comparable fragmentation of the mAb. Mass spectrometric identification of the fragments suggests fragment generation by oxidative disulfide bond cleavage between the heavy and light chains, confirmed by mass spectrometry data on product formation. The mechanism of oxidative fragmentation was separately confirmed with insulin. CONCLUSION: Fragmentation of the mAb by peroxotungstate is proposed to occur through inter-chain disulfide bond oxidation to form thiosulfinate (CyS(═O)SCy) and thiosulfonate [CyS(═O)2SCy], followed by hydrolysis.

Identification of D-Amino Acids in Light Exposed mAb Formulations.

PURPOSE: We previously demonstrated that D-amino acids can form as a result of photo-irradiation of a monoclonal antibody (mAb) at both λ = 254 nm and λ > 295 nm (λmax = 305 nm), likely via reversible hydrogen transfer reactions of intermediary thiyl radicals. Here, we investigate the role of various excipients (sucrose, glucose, L-Arg, L-Met and L-Leu) on D-amino acid formation, and specifically the distribution of D-amino acids in mAb monomers and aggregates present after light exposure. METHODS: The mAb-containing formulations were photo-irradiated at λ = 254 nm and λmax = 305 nm, followed by fractionation of aggregate and monomer fractions using size exclusion chromatography. These aggregate and monomer fractions were subjected to hydrolysis and subsequent amino acid analysis. RESULTS: Both aggregate and monomer fractions collected from all formulations showed the formation of D-Glu and D-Val, whereas the formation of D-Ala was limited to the aggregate fraction collected from an L-Arg-containing formulation. Interestingly, quantitative analysis revealed higher yields of D-amino acids in the L-Arg-containing formulation. CONCLUSIONS: Generally, D-amino acids accumulated to similar extents in monomers and aggregates.

Dual Effect of Histidine on Polysorbate 20 Stability: Mechanistic Studies.

PURPOSE: L-Histidine (L-His) and polysorbate 20 (PS20) are two excipients frequently included in parenteral products to stabilize biotherapeutics. The objective of the current work was to investigate the impact of L-His on PS20 stability in aqueous solutions when subjected to forced oxidation and accelerated stability testing. METHODS: The stability of PS20 in L-His buffer was compared with that in acetate buffer. Forced oxidation of PS20 in these two buffer systems was initiated by a free radical generator, 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH), while accelerated stability tests were carried out at 40°C. Ultra-performance liquid chromatography mass spectrometry was utilized to monitor intact PS20 and to analyze degradation products. RESULTS: Our results demonstrate a dual effect of L-His on PS20 stability. During exposure to AAPH, L-His protects PS20 from oxidation. Stable isotope labeling of L-His with 13C was employed for mechanistic investigations. The protection of L-His was abrogated when acetate was added to L-His buffer, implying that the anti-oxidative activity of L-His may be compromised by specific counter ions. The replacement of L-His by various His derivatives led to significant changes in the protection of PS20 against AAPH-induced degradation. In contrast to forced degradation, the addition of L-His promoted oxidative PS20 degradation during accelerated storage at 40°C in solution, generating mainly short chain POE-laurates. CONCLUSION: L-His exhibits a dual effect on the stability profile of PS20, protecting against AAPH-induced oxidation but promoting oxidative degradation during accelerated stability testing.

An Efficient and Rapid Method to Monitor the Oxidative Degradation of Protein Pharmaceuticals: Probing Tyrosine Oxidation with Fluorogenic Derivatization.

PURPOSE: The loss of potency of protein therapeutics can be linked to the oxidation of specific amino acid residues leading to a great variety of oxidative modifications. The comprehensive identification of these oxidative modifications requires high-resolution mass spectrometry analysis, which requires time and expensive resources. Here, we propose a fluorogenic derivatization method of oxidized Tyr and Phe yielding benzoxazole derivatives, as an orthogonal technique for the rapid screening of protein oxidation. METHODS: Four model proteins, IgG1, human growth hormone (hGH), insulin and bovine serum albumin (BSA) were exposed to oxidation via peroxyl radicals and metal-catalyzed reactions and efficiently screened by fluorogenic derivatization of Tyr and Phe oxidation products. Complementary LC-MS analysis was done to identify the extent of methionine oxidation in oxidized proteins. RESULTS: The Fluorogenic derivatization technique can easily be adapted to a 96-well plate, in which several protein formulations can be screened in short time. Representatively for hGH, we show that the formation of benzoxazole parallels the oxidation of Met to methionine sulfoxide which enables estimation of Met oxidation by just recording the fluorescence. CONCLUSIONS: Our rapid fluorescence based screening allows for the fast comparison of the stability of multiple formulations.

Degradation Mechanisms of Polysorbate 20 Differentiated by 18O-labeling and Mass Spectrometry.

PURPOSE: To investigate the mechanisms of polysorbate (PS) degradation with the added objective of differentiating the hydrolysis and oxidation pathways. METHODS: Ultra-performance liquid chromatography mass spectrometry (UPLC-MS) was utilized to characterize all-laurate polysorbate 20 (PS20) and its degradants. 18O stable isotope labeling was implemented to produce 18O-labeled degradation products of all-laurate PS20 in H218O, with subsequent UPLC-MS analysis for location of the cleavage site on the fatty acid-containing side chain of PS20. RESULTS: The analysis reveals that hydrolysis of all-laurate PS20 leads to a breakdown of the ester linkage to liberate free lauric acid, showing a distinct dependence on pH. Using a hydrophilic free radical initiator, 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH) to study the oxidative degradation of all-laurate PS20, we demonstrate that free lauric acid and polyoxyethylene (POE) laurate are two major decomposition products. Measurement of 18O incorporation into free lauric acid indicated that hydrolysis primarily led to 18O incorporation into free lauric acid via "acyl-cleavage" of the fatty acid ester bond. In contrast, AAPH-exposure of all-laurate PS20 produced free lauric acid without 18O-incorporation. CONCLUSIONS: The 18O-labeling technique and unique degradant patterns of all-laurate PS20 described here provide a direct approach to differentiate the types of PS degradation.

Photo-oxidation of IgG1 and Model Peptides: Detection and Analysis of Triply Oxidized His and Trp Side Chain Cleavage Products.

PURPOSE: Triply oxidized histidine in an IgG1 monoclonal antibody was noticed when exposed to ICH light conditions. In order to understand the role of light source, irradiation wavelengths and primary sequence, specifically those of a nearby tryptophan, we synthesized and exposed several peptides to ICH light conditions and analyzed the products using LC-MS analysis. METHODS: Protein and peptide samples were photo-irradiated under ICH conditions as well as with monochromatic light at λ = 254 nm and analyzed using either LTQ Orbitrap or a LTQ-FT ion cyclotron resonance mass spectrometer respectively. RESULTS: A triply oxidized His residue was detected along with a second doubly oxidized His residue in an IgG1. Both of these oxidized His residues are located near Trp residues. In order to investigate the role of Trp photosensitization in His oxidation we synthesized model peptides and Ala mutants. Peptides exposed to ICH light stress conditions revealed a small percent of triply oxidized His in the Trp-containing peptide sequences but not in their corresponding Ala mutants. CONCLUSIONS: The differences in product formation under different photo-irradiation conditions underline the importance of light source, irradiation wavelengths and primary sequence in the photosensitivity of proteins.

Photodegradation Pathways of Protein Disulfides: Human Growth Hormone.

PURPOSE: Comprehensive product characterization was performed for the photodegradation of protein disulfides, representatively of human growth hormone (somatotropin; hGH), in order to provide a product database, which will be useful for the general analysis of protein stability. METHODS: HGH was photo-irradiated at λ = 254 and λ > 295 nm and tryptic digests were analyzed by HPLC-MS to investigate light-induced disulfide degradation pathways. RESULTS: A total of 60 products were detected, and structures/tentative structures were assigned to the products by MS2 and MS3 analysis. The main products were reduced Cys residues, dithiohemiacetal, thioether and disulfide scrambling products. In addition, we detected Cys degradation products such as Cys thioaldehyde, dehydroalanine (Dha), Ala, Ser semialdehyde, Ser, S-sulfocysteine, and Gly. Frequently, the tryptic fragments contained more than one modification, i.e. a Cys degradation product in close proximity to a dehydrated amino acid. Several novel cross-links were detected between Cys and Tyr, Cys, Ser and Phe, Cys and Trp, and Trp and Tyr. Photo-induced protein fragmentation was detected specifically at or in close proximity to the disulfide bond between T6 and T16. An in-house packed 75 cm nano-column enabled us to resolve various isomers/diastereomers of the photo-degradation products. CONCLUSION: A comprehensive analysis of photodegradation products revealed a variety of novel photo-products, including cross-links, originating from disulfide degradation. The mechanisms of product formation are discussed.

Profiling the Photochemical-Induced Degradation of Rat Growth Hormone with Extreme Ultra-pressure Chromatography-Mass Spectrometry Utilizing Meter-Long Microcapillary Columns Packed with Sub-2-µm Particles.

In recent years protein therapeutics have seen increasing use in the therapeutic arena. As with traditional small molecule drug substances, one is obligated to ensure purity and stability of the various dosage forms. With these higher molecular weight therapeutics a common approach for analytical characterization is enzymatic digestion followed by gradient elution liquid chromatography with mass spectrometry detection to create a peptide map (bottom-up protein analysis). Due to the difficult to separate mixtures frequently encountered, there is the need for advanced chromatographic systems featuring increased resolution and/or peak capacity that can be operated in the gradient elution format. Presently we describe an extreme ultra-pressure liquid chromatography (XUPLC) system that has been implemented as an in-house add-on to a commercial ultra-pressure chromatography system. This add-on allows operation at the 38 Kspi range, accommodates the use of capillary columns in excess of one meter packed with sub-2 µm particles and can be operated in the gradient elution format. To evaluate the utility of this system, rat growth hormone was used as a model protein and was exposed to light (λ 254 nm) to create a stress environment. When enzymatic digests of control and stressed protein were analyzed with the XUPLC system using MS detection, greater than 92% peptide coverage was achieved, including the identification some peptides where pre-oxidation of Met residues had occurred, as well as chemistry specifically related to the photolysis of protein disulfide linkages. When the same samples were analyzed by commercial UPLC and compared to the XUPLC results, the utility of the increased peak capacity available with the XUPLC was apparent as previously co-eluting peaks were now well resolved. In particular one specific degradation route was identified where a pair of isobaric cis/trans diastereomerically related peptides were well resolved by XUPLC while they were unresolved by UPLC. Clearly the use of this system operating at the higher pressure regime with long capillary columns is and will be useful in continued investigations of protein stability, especially in cases where only subtle differences in the amino acid residues have occurred during degradation.

Site-Specific Hydrolysis Reaction C-Terminal of Methionine in Met-His during Metal-Catalyzed Oxidation of IgG-1.

The metal-catalyzed oxidation by [Fe(II)(EDTA)](2-)/H2O2 of IgG-1 leads to the site-specific hydrolysis of peptide bonds in the Fc region. The major hydrolytic cleavage occurs between Met428 and His429, consistent with a mechanism reported for the site-specific hydrolysis of parathyroid hormone (1-34) between Met8 and His9 (Mozziconacci, O.; et al. Mol. Pharmaceutics 2013, 10 (2), 739-755). In IgG-1, to a lesser extent, we also observe hydrolysis reactions between Met252 and Ile253. After 2 h of oxidation (at pH 5.8, 37 °C) approximately 5% of the protein is cleaved between Met428 and His429. For comparison, after 2 h of oxidation, the amount of tryptic peptides containing a Met sulfoxide residue represents less than 0.1% of the protein. The effect of this site-specific hydrolysis on the conformational stability and aggregation propensity of the antibody was also examined. No noticeable differences in structural integrity and conformational stability were observed between control and oxidized IgG-1 samples as measured by circular dichroism (CD), fluorescence spectroscopy, and static light scattering (SLS). Small amounts of soluble and insoluble aggregates (3-6%) were, however, observed in the oxidized samples by UV-visible absorbance spectroscopy and size exclusion chromatography (SEC). Over the course of metal-catalyzed oxidation, increasing amounts of fragments were also observed by SEC. An increase in the concentration of subvisible particles was detected by microflow imaging (MFI).

Sarcoendoplasmic reticulum Ca(2+) ATPase. A critical target in chlorine inhalation-induced cardiotoxicity.

Autopsy specimens from human victims or experimental animals that die due to acute chlorine gas exposure present features of cardiovascular pathology. We demonstrate acute chlorine inhalation-induced reduction in heart rate and oxygen saturation in rats. Chlorine inhalation elevated chlorine reactants, such as chlorotyrosine and chloramine, in blood plasma. Using heart tissue and primary cardiomyocytes, we demonstrated that acute high-concentration chlorine exposure in vivo (500 ppm for 30 min) caused decreased total ATP content and loss of sarcoendoplasmic reticulum calcium ATPase (SERCA) activity. Loss of SERCA activity was attributed to chlorination of tyrosine residues and oxidation of an important cysteine residue, cysteine-674, in SERCA, as demonstrated by immunoblots and mass spectrometry. Using cardiomyocytes, we found that chlorine-induced cell death and damage to SERCA could be decreased by thiocyanate, an important biological antioxidant, and by genetic SERCA2 overexpression. We also investigated a U.S. Food and Drug Administration-approved drug, ranolazine, used in treatment of cardiac diseases, and previously shown to stabilize SERCA in animal models of ischemia-reperfusion. Pretreatment with ranolazine or istaroxime, another SERCA activator, prevented chlorine-induced cardiomyocyte death. Further investigation of responsible mechanisms showed that ranolazine- and istaroxime-treated cells preserved mitochondrial membrane potential and ATP after chlorine exposure. Thus, these studies demonstrate a novel critical target for chlorine in the heart and identify potentially useful therapies to mitigate toxicity of acute chlorine exposure.

Low-temperature NMR characterization of reaction of sodium pyruvate with hydrogen peroxide.

It was proposed that the reaction of sodium pyruvate and H2O2 generates the intermediate 2-hydroperoxy-2-hydroxypropanoate, which converts into acetate, CO2, and H2O ( Aleksankin et al. Kernenergie 1962 , 5 , 362 - 365 ). These conclusions were based on the products generated in (18)O-enriched water and H2O2 reacting with pyruvic acid at room temperature; however, the lifetime of 2-hydroperoxy-2-hydroxypropanoate at room temperature is too short for direct spectroscopic observation. Therefore, we applied the combination of low-temperature and (13)C NMR techniques to verify, for the first time, the formation of 2-deuteroperoxy-2-deuteroxypropanoate in mixtures of D2O and methanol-d4 and to monitor directly each species involved in the reaction between D2O2 and (13)C-enriched pyruvate. Our NMR results confirm the formation of 2-deuteroperoxy-2-deuteroxypropanoate, where the respective chemical shifts are supported by density functional theory (DFT) calculations. At near-neutral apparent pD (pD*) and -35 °C, the formation of 2-deuteroperoxy-2-deuteroxypropanoate occurred with k = 2.43 × 10(-3) dm(3)·mol(-1)·s(-1). The subsequent decomposition of 2-deuteroperoxy-2-deuteroxypropanoate into acetate, CO2, and D2O occurred with k = 2.58 × 10(-4) s(-1) at -35 °C. In order to provide a full kinetic analysis, we also monitored the equilibrium of pyruvate and methanol with the hemiacetal (2-deuteroxy-2-methoxypropanoate). The kinetics for the reaction of sodium pyruvate and D2O2 were fitted by taking into account all these equilibria and species.

Sequence-specific formation of d-amino acids in a monoclonal antibody during light exposure.

The photoirradiation of a monoclonal antibody 1 (mAb1) at λ = 254 nm and λmax = 305 nm resulted in the sequence-specific generation of d-Val, d-Tyr, and potentially d-Ala and d-Arg, in the heavy chain sequence [95-101] YCARVVY. d-Amino acid formation is most likely the product of reversible intermediary carbon-centered radical formation at the (α)C-positions of the respective amino acids ((α)C(•) radicals) through the action of Cys thiyl radicals (CysS(•)). The latter can be generated photochemically either through direct homolysis of cystine or through photoinduced electron transfer from Trp and/or Tyr residues. The potential of mAb1 sequences to undergo epimerization was first evaluated through covalent H/D exchange during photoirradiation in D2O, and proteolytic peptides exhibiting deuterium incorporation were monitored by HPLC-MS/MS analysis. Subsequently, mAb1 was photoirradiated in H2O, and peptides, for which deuterium incorporation in D2O had been documented, were purified by HPLC and subjected to hydrolysis and amino acid analysis. Importantly, not all peptide sequences which incorporated deuterium during photoirradiation in D2O also exhibited photoinduced d-amino acid formation. For example, the heavy chain sequence [12-18] VQPGGSL showed significant deuterium incorporation during photoirradiation in D2O, but no photoinduced formation of d-amino acids was detected. Instead this sequence contained ca. 22% d-Val in both a photoirradiated and a control sample. This observation could indicate that d-Val may have been generated either during production and/or storage or during sample preparation. While sample preparation did not lead to the formation of d-Val or other d-amino acids in the control sample for the heavy chain sequence [95-101] YCARVVY, we may have to consider that during hydrolysis N-terminal residues (such as in VQPGGSL) may be more prone to epimerization. We conclude that the photoinduced, radical-dependent formation of d-amino acids requires not only the intermediary formation of a (α)C(•) radical but also sufficient flexibility of the protein domain to allow both pro-chiral faces of the (α)C(•) radical to accept a hydrogen atom.

Effect of conformation on the photodegradation of Trp- and cystine-containing cyclic peptides: octreotide and somatostatin.

The synthetic cyclic octapeptide octreotide is less stable under UV and fluorescent lights than its naturally occurring derivative somatostatin. Upon irradiation at λ > 290 nm, and in the presence of oxygen, octreotide is quickly transformed into a photoproduct showing an increase of mass of 16 Da. The increase of 16 Da to the mass of octreotide is related to a complex transformation where (i) the side chain of d-Trp is transferred onto the side chain of the Lys residue and (ii) the former d-Trp residue is transformed into hydroxyglycine, which ultimately fragments. The complex sequence of phototransformation is orchestrated by the presence of d-Trp and the unusual orientation of its carbonyl group toward the disulfide bond. The phototransformation of octreotide is illustrated by product A.

Metal-catalyzed oxidation of protein methionine residues in human parathyroid hormone (1-34): formation of homocysteine and a novel methionine-dependent hydrolysis reaction.

The oxidation of PTH(1-34) catalyzed by ferrous ethylenediaminetetraacetic acid (EDTA) is site-specific. The oxidation of PTH(1-34) is localized primarily to the residues Met[8] and His[9]. Beyond the transformation of Met[8] and His[9] into methionine sulfoxide and 2-oxo-histidine, respectively, we observed a hydrolytic cleavage between Met[8] and His[9]. This hydrolysis requires the presence of Fe(II) and oxygen and can be prevented by diethylenetriaminepentaacetic acid (DTPA) and phosphate buffer. Conditions leading to this site-specific hydrolysis also promote the transformation of Met[8] into homocysteine, indicating that the hydrolysis and transformation of homocysteine may proceed through a common intermediate.

Light-induced conversion of Trp to Gly and Gly hydroperoxide in IgG1.

The exposure of IgG1 in aqueous solution to light with λ = 254 nm or λ > 295 nm yields products consistent with Trp radical cation formation followed by (α)C-(ß)C cleavage of the Trp side chain. The resulting glycyl radicals either are reduced to Gly or add oxygen prior to reduction to Gly hydroperoxide. Photoirradiation at λ = 254 nm targets Trp at positions 191 (light chain), 309 and 377 (heavy chain) while photoirradiation at λ > 295 nm targets Trp at position 309 (heavy chain). Mechanistically, the formation of Trp radical cations likely proceeds via photoinduced electron or hydrogen transfer to disulfide bonds, yielding thiyl radicals and thiols, where thiols may serve as reductants for the intermediary glycyl or glycylperoxyl radicals.

Fluorogenic tagging methodology applied to characterize oxidized tyrosine and phenylalanine in an immunoglobulin monoclonal antibody.

PURPOSE: Metal-catalyzed oxidation (MCO) of proteins is of primary concern in the development of biotherapeutics as it represents a prominent degradation pathway with potential undesired biological and biotherapeutic consequences. METHODS: We developed a fluorogenic derivatization methodology to study the MCO of IgG1 using a model oxidation system, CuCl2/L-ascorbic acid. RESULTS: Besides the oxidation of Met, Trp and His residues, we detected significant oxidation of Phe and Tyr in IgG1. CONCLUSION: The fluorogenic derivatization method provides an alternative approach for the rapid detection of oxidized Tyr and Phe as their benzoxazole derivatives by fluorescence spectrometry and size exclusion chromatography coupled to fluorescence detection.

The photolysis of disulfide bonds in IgG1 and IgG2 leads to selective intramolecular hydrogen transfer reactions of cysteine Thiyl radicals, probed by covalent H/D exchange and RPLC-MS/MS analysis.

PURPOSE: The evaluation of photo-instability of biotherapeutic products is mandated by regulatory agencies. Photo-irradiation can induce oxidative modifications in proteins, which may lead to undesired biological and therapeutic consequences. Among the modifications, epimerization of amino acid residues can occur upon photo-irradiation of IgGs. METHODS: We show here, that UV irradiation (λ = 253.7 nm) of IgG1 and IgG2 leads to the formation of intermediary carbon-centered radicals, validated by covalent incorporation of deuterium into the protein primary sequence. RESULTS: By MS/MS analysis we identified the sites of deuterium incorporation, such as the sequence QD [303:304, HC], present in the peptide of VVSVLTVVHQDWLNGK [294:309, HC] in both IgG1 and IgG2, and V [111, LC] and K [116, LC], present in the peptide VTVLGQPK [109:116, LC] in IgG2. Both peptides are in the proximity of intrachain disulfide bonds. CONCLUSIONS: The exposure of IgG1 and IgG2 to UV-light (λ = 253.7 nm) generates specific carbon-centered radicals. The latter were evidenced by a covalent H-D exchange reaction that likely occurred through a hydrogen atom transfer reaction between cysteine thiyl radical and C-H bond.