Tryptophan Oxidation of a Monoclonal Antibody Under Diverse Oxidative Stress Conditions: Distinct Oxidative Pathways Favor Specific Tryptophan Residues.

Tryptophan oxidation can play an important role in selecting therapeutic monoclonal antibodies for commercialization. Monoclonal antibodies that harbor particularly sensitive tryptophan residues are typically discarded in favor of oxidation resistant antibodies. The susceptibility of any individual tryptophan residue to oxidation is typically evaluated through forced degradation studies during the molecule development process. We compared the results of several common forced degradation "stress tests" for each tryptophan residue in a monoclonal antibody and found that high-stress oxidation conditions consistently provide a different ranking of oxidative sensitivity across the individual tryptophan residues compared to long-term thermal stability or low-stress conditions. We subsequently determined that this difference in ranking is largely due to an overabundance of double oxidation (i.e. detected as +32 Da) of specific tryptophan residues under high stress conditions compared to single oxidation (i.e. +16 Da). We posit that this double oxidation is in fact mechanistically distinct from the observed single oxidation and that high stress conditions favor the double oxidation mechanism (and double oxidation sensitive tryptophan residues) while single oxidation appears to be the primary mode of oxidation under H2O2 stress and long-term thermal stability and favors different tryptophan residues which are more susceptible to the single oxidation mechanism.

A novel approach for quantitation of nonderivatized sialic acid in protein therapeutics using hydrophilic interaction chromatographic separation and nano quantity analyte detection.

This paper describes a novel approach for the quantitation of nonderivatized sialic acid in glycoproteins, separated by hydrophilic interaction chromatography, and detection by Nano Quantity Analyte Detector (NQAD). The detection technique of NQAD is based on measuring change in the size of dry aerosol and converting the particle count rate into chromatographic output signal. NQAD detector is suitable for the detection of sialic acid, which lacks sufficiently active chromophore or fluorophore. The water condensation particle counting technology allows the analyte to be enlarged using water vapor to provide highest sensitivity. Derivatization-free analysis of glycoproteins using HPLC/NQAD method with PolyGLYCOPLEX™ amide column is well correlated with HPLC method with precolumn derivatization using 1, 2-diamino-4, 5-methylenedioxybenzene (DMB) as well as the Dionex-based high-pH anion-exchange chromatography (or ion chromatography) with pulsed amperometric detection (HPAEC-PAD). With the elimination of derivatization step, HPLC/NQAD method is more efficient than HPLC/DMB method. HPLC/NQAD method is more reproducible than HPAEC-PAD method as HPAEC-PAD method suffers high variability because of electrode fouling during analysis. Overall, HPLC/NQAD method offers broad linear dynamic range as well as excellent precision, accuracy, repeatability, reliability, and ease of use, with acceptable comparability to the commonly used HPAEC-PAD and HPLC/DMB methods.

Glycation of human erythrocyte glutathione peroxidase: effect on the physical and kinetic properties.

BACKGROUND: Glutathione peroxidase (GPx) is a significant antioxidant enzyme that plays a key role in protecting the body from reactive oxygen species (ROS) and their toxicity. As a biocatalyst, the enzyme has been shown to reduce hydrogen peroxide to water and lipid hydroperoxides to their respective alcohols. The increased levels of ROS in patients with diabetes have been speculated to arise, in part, from alterations in the activity of glutathione antioxidant enzymes, perhaps, by mechanisms such as the glycation of the protein, in vivo. METHODS: Under physiological conditions of temperature and pH, we investigated the susceptibility of human glutathione peroxidase to glycation, determined the effects of glycation on the physical and kinetic properties of the enzyme, and identified the protein's vulnerable amino acid sites of glycation. RESULTS: Circular dichroism, UV and mass spectrometry studies revealed that methylglyoxal and DL-glyceraldehyde are potent glycators of glutathione peroxidase; destabilizing its structure, altering its pH activity and stability profiles and increasing its Km value. CONCLUSIONS: In comparison to DL-glyceraldehyde, methylglyxol was a more potent glycator of the enzyme and was found to nonenzymatically condense with Arg-177, located near the glutathione binding site of GPx.

Monitoring nonenzymatic glycation of human immunoglobulin G by methylglyoxal and glyoxal: A spectroscopic study.

The accumulation of dicarbonyl compounds, methylglyoxal (MG) and glyoxal (G), has been observed in diabetic conditions. They are formed from nonoxidative mechanisms in anaerobic glycolysis and lipid peroxidation, and they act as advanced glycation endproduct (AGE) precursors. The objective of this study was to monitor and characterize the AGE formation of human immunoglobulin G (hIgG) by MG and G using ultraviolet (UV) and fluorescence spectroscopy, circular dichroism (CD), and matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). hIgG was incubated over time with MG and G at different concentrations. Formation of AGE was monitored by UV and fluorescence spectroscopy. The effect of AGE formation on secondary structure of hIgG was studied by CD. Comparison of AGE profile for MG and G was performed by MALDI-MS. Both MG and G formed AGE, with MG being nearly twice as reactive as G. The combination of these techniques is a convenient method for evaluating and characterizing the AGE proteins.