Identification of plasma protease derived metabolites of glucagon and their formation under typical laboratory sample handling conditions

Copyright © 2014 John Wiley & Sons, Ltd. RATIONALE Glucagon modulates glucose production, and it is also a biomarker for several pathologies. It is known to be unstable in human plasma, and consequently stabilisers are often added to samples, although these are not particularly effective. Despite this, there have not been any studies to identify in vitro plasma protease derived metabolites; such a study is described here. Knowledge of metabolism should allow the development of more effective sample stabilisation strategies. METHODS Several novel metabolites resulting from the incubation of glucagon in human plasma were identified using high-resolution mass spectrometry with positive electrospray ionisation. Tandem mass spectrometric (MS/MS) scans were acquired for additional confirmation using a QTRAP. Separation was performed using reversed-phase ultra-high-performance liquid chromatography. The formation of these metabolites was investigated during a time-course experiment and under specific stress conditions representative of typical laboratory handling conditions. Clinical samples were also screened for metabolites. RESULTS Glucagon 3-29 and [pGlu] 3 glucagon 3-29 were the major metabolites detected, both of which were also present in clinical samples. We also identified two oxidised forms of [pGlu] 3 glucagon 3-29 as well as glucagon 19-29 , or 'miniglucagon', along with the novel metabolites glucagon 20-29 and glucagon 21-29 . The relative levels of these metabolites varied throughout the time-course experiment, and under the application of the different sample handling conditions. Aprotinin stabilisation of samples had negligible effect on metabolite formation. CONCLUSIONS Novel plasma protease metabolites of glucagon have been confirmed, and their formation characterised over a time-course experiment and under typical laboratory handling conditions. These metabolites could be monitored to assess the effectiveness of new sample stabilisation strategies, and further investigations into their formation could suggest specific enzyme inhibitors to use to increase sample stability. In addition the potential of the metabolites to affect immunochemistry-based assays as a result of cross-reactivity could be investigated

Physicochemical stability and effector function of IgG4-Fc: impact of photo-induced chemical modification and glycosylation

Immunoglobulin gamma monoclonal antibodies 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 photo-degradation, 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 photo-products 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. 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 et al. Mol. Pharm. 2013, 10, 1146-1150; Kang et al. Mol. Pharm. 2019, 16, 258-272). In order to evaluate the physico-chemical consequences of such photochemical transformations, we prepared a series of IgG4-Fc mutants, in which Trp and Tyr residues were mutated to Gly, i.e., Y300G, Y373G, Y436G, W381G, and W381A for biophysical 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 of thermal stability of both the CH2 and CH3 domains. In contrast, W381G and W381A showed no thermal transitions, indicating a significant loss of overall thermal stability. Both, W381A and Y300G IgG4-Fc displayed ca. 10-fold reduction of binding affinity to FcγRIIIA as 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. Furthermore, a series of well-defined N-glycosylated IgG4-Fc variants were utilized as model to investigate the effect of glycan structure on the physico-chemical properties (conformational stability and photostability) and interaction with FcɣRIIIA. The size of glycans at Asn297 affects the yields of light-induced Tyr side chain fragmentation products, where the yields decreased in the following order: N297Q GlcNAc1 Man5 HM. These yields correlate with the thermal stability of the glycoforms. The presence of HM and Man5 reveals increased affinity for FcɣRIIIA by at least 14.7-fold, as compared to GlcNAc1 IgG4-Fc. N297Q does not present a detectable affinity to FcɣRIIIA

The development of mass spectrometry-based methodologies for the high throughput quantitation of peptides in biological matrices

The aim of this research was the development of mass spectrometry-based methodologies for the high-throughput quantitation of peptides in biological matrices. Glucagon and GLP-1, which are of interest as biomarkers and in the development of therapeutics, were chosen as model peptides. Immunoassays that are traditionally used to quantify these often perform poorly; therefore, necessitating the development of alternative methodologies. Application of mass spectrometry-based methodologies to these analytes has, however, been limited, primarily due to sensitivity challenges, but also due to analytical challenges associated with their endogenous nature and instability in biological matrices. Chapter 2 describes the development and qualification of the first liquid-chromatography coupled tandem mass spectrometry (LC-MS/MS) method for the quantitation of endogenous glucagon from human plasma. A novel 2D extraction procedure was developed to ensure robustness and sensitivity, whilst a novel surrogate matrix quantitation strategy took into account the endogenous nature of the analyte. A lower limit of quantitation (LLOQ) of 25 pg/mL was qualified, which was a considerable improvement over that previously reported in the literature (250 pg/mL) for a LC-MS/MS method. Clinical samples were cross-validated against a conventional radioimmunoassay (RIA), and similar pharmacokinetic (PK) profiles resulted, demonstrating that the methods were complementary. In Chapter 2 glucagon instability in biological matrix was noted. To characterise this further, in Chapter 3 in vitro glucagon metabolites were identified using high-resolution mass spectrometry (HRMS). Metabolites observed by others (glucagon19-29, glucagon3 29 and [pGlu]3glucagon3 29) in alternative matrices were identified, alongside novel metabolites (glucagon20-29 and glucagon21-29). Cross-interference of these metabolites in immunoassays may help to explain their poor performance, whilst knowledge of metabolism may also aid the development of future stabilisation strategies. The method developed in Chapter 2 was refined in Chapter 4 to improve sensitivity, robustness and throughput, and to add GLP-1 as a secondary analyte. The sensitivity achieved (glucagon: 15 pg/mL LLOQ, GLP-1: 25 pg/mL LLOQ) is the highest reported for both peptides for an extraction avoiding immunoenrichment. Specificity of endogenous glucagon quantitation was assured using a novel approach with a supercharging mobile phase additive to access a sensitive qualifier transition. A cross-validation against established immunoassays using physiological study samples demonstrated some similarities between the methods. Differences between the immunoassay results exemplified the need to develop alternative methodologies. The resulting LC-MS/MS method is considered a viable alternative to immunoassays, for the quantitation of endogenous glucagon, dosed glucagon and/or dosed GLP-1 in human plasma