Determination of Deamidation in Adjuvanted Vaccine Antigens through Isoaspartic Acid Quantification.

Deamidation is a post-translational chemical modification that occurs within proteins and can be influenced by many factors, including temperature and pH. In vaccines, deamidation is considered undesirable as it may lead to changes in structure, function, stability, and immunogenicity. Detecting deamidation in vaccines, especially adjuvanted vaccines, can be challenging due to the lack of simple quantitative techniques. In this study, the quantification of isoaspartic acid (isoAsp) was used to assess deamidation in model antigens in the presence and absence of common vaccine adjuvants. This study shows that the detection of isoAsp was possible in the presence of various types of adjuvants with little to no interference. High levels of isoAsp were detected in thermally and pH-stressed adjuvanted vaccines, suggesting significant deamidation and highlighting the stability-indicating capabilities of the assay. The quantification of isoAsp in stability programs of a vaccine drug product could possibly find applications in product shelf-life determination, using thermal kinetic modeling to predict deamidation over time. The ability to detect deamidation early in vaccine development enhances process improvements and ultimately improves the vaccine's stability. To summarize, this paper describes a rapid and simple method to determine deamidation in adjuvanted vaccines. This method could be applicable to formulation development, stability assessment, or shelf-life determination.

Probabilistic model applied to ion abundances in product-ion spectra: quantitative analysis of aspartic acid isomerization in peptides.

Evaluation of post-translational modifications of protein molecules is important for both basic and applied biomedical research. Mass spectrometric quantitative studies of modifications, which do not change the mass of the protein, such as isomerization of aspartic acid, do not necessarily require the use of isotope-labelled standards. However, the accurate solution of this problem requires a deep understanding of the relationship between the mole fractions of the isomers and the peak intensities in the mass spectra. In previous studies on the isomerization of aspartic acid in short beta-amyloid fragments, it has been shown that calibration curves used for such quantitative studies often have a non-linear form. The reason for the deviation in the shape of the calibration curves from linearity has not yet been established. Here, we propose an explanation for this phenomenon based on a probabilistic model of the fragmentation process and present a general approach for the selection of fragments that can be used for quantitative studies of the degree of isomerization. Graphical Abstract.

Leveraging Electron Transfer Dissociation for Site Selective Radical Generation: Applications for Peptide Epimer Analysis.

Traditional electron-transfer dissociation (ETD) experiments operate through a complex combination of hydrogen abundant and hydrogen deficient fragmentation pathways, yielding c and z ions, side-chain losses, and disulfide bond scission. Herein, a novel dissociation pathway is reported, yielding homolytic cleavage of carbon-iodine bonds via electronic excitation. This observation is very similar to photodissociation experiments where homolytic cleavage of carbon-iodine bonds has been utilized previously, but ETD activation can be performed without addition of a laser to the mass spectrometer. Both loss of iodine and loss of hydrogen iodide are observed, with the abundance of the latter product being greatly enhanced for some peptides after additional collisional activation. These observations suggest a novel ETD fragmentation pathway involving temporary storage of the electron in a charge-reduced arginine side chain. Subsequent collisional activation of the peptide radical produced by loss of HI yields spectra dominated by radical-directed dissociation, which can be usefully employed for identification of peptide isomers, including epimers. Graphical Abstract ᅟ.

Improved Reversed Phase Chromatography of Hydrophilic Peptides from Spatial and Temporal Changes in Column Temperature.

Reversed phase chromatography is an established method for peptide separation and frequently coupled to electrospray ionization-mass spectrometry for proteomic analysis. Column temperature is one parameter that influences peptide retention and elution, but it is often overlooked as its implementation requires additional equipment and method optimization. An apparatus that allows temperature manipulation in three areas of a two-column setup was evaluated for improvements in chromatography. Using commercially available standards, we demonstrate that a low column temperature (0 °C) during sample loading enhances the peak shape of several bovine serum albumin hydrophilic peptides. For digested HeLa lysates, approximately 15% more peptide identifications were obtained by increasing the precolumn temperature to 50 °C after the 500 ng sample was loaded at a low temperature. This method also identified additional early eluting peptides with grand average of hydropathicity values less than -2. We also investigated the effect of cooler column temperatures on peptides with post-translational modifications. It was possible to minimize the coelution of an isoaspartylated peptide and its unmodified version when the analytical column temperature was decreased to 5 °C. Aside from demonstrating the utility of lower temperatures for improved chromatography, its application at specific locations and time points is critical for peptide detection and separation.

Distinguishing Aspartic and Isoaspartic Acids in Peptides by Several Mass Spectrometric Fragmentation Methods.

Six ion fragmentation techniques that can distinguish aspartic acid from its isomer, isoaspartic acid, were compared. MALDI post-source decay (PSD), MALDI 157 nm photodissociation, tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP) charge tagging in PSD and photodissociation, ESI collision-induced dissociation (CID), electron transfer dissociation (ETD), and free-radical initiated peptide sequencing (FRIPS) with CID were applied to peptides containing either aspartic or isoaspartic acid. Diagnostic ions, such as the y-46 and b+H2O, are present in PSD, photodissociation, and charge tagging. c•+57 and z-57 ions are observed in ETD and FRIPS experiments. For some molecules, aspartic and isoaspartic acid yield ion fragments with significantly different intensities. ETD and charge tagging appear to be most effective at distinguishing these residues. Graphical Abstract ᅟ.

Mildly acidic conditions eliminate deamidation artifact during proteolysis: digestion with endoprotease Glu-C at pH 4.5.

Common yet often overlooked, deamidation of peptidyl asparagine (Asn or N) generates aspartic acid (Asp or D) or isoaspartic acid (isoAsp or isoD). Being a spontaneous, non-enzymatic protein post-translational modification, deamidation artifact can be easily introduced during sample preparation, especially proteolysis where higher-order structures are removed. This artifact not only complicates the analysis of bona fide deamidation but also affects a wide range of chemical and enzymatic processes; for instance, the newly generated Asp and isoAsp residues may block or introduce new proteolytic sites, and also convert one Asn peptide into multiple species that affect quantification. While the neutral to mildly basic conditions for common proteolysis favor deamidation, mildly acidic conditions markedly slow down the process. Unlike other commonly used endoproteases, Glu-C remains active under mildly acid conditions. As such, as demonstrated herein, deamidation artifact during proteolysis was effectively eliminated by simply performing Glu-C digestion at pH 4.5 in ammonium acetate, a volatile buffer that is compatible with mass spectrometry. Moreover, nearly identical sequence specificity was observed at both pH's (8.0 for ammonium bicarbonate), rendering Glu-C as effective at pH 4.5. In summary, this method is generally applicable for protein analysis as it requires minimal sample preparation and uses the readily available Glu-C protease.

Detection and quantitation of succinimide in intact protein via hydrazine trapping and chemical derivatization.

The formation of aspartyl succinimide is a common post-translational modification of protein pharmaceuticals under acidic conditions. We present a method to detect and quantitate succinimide in intact protein via hydrazine trapping and chemical derivatization. Succinimide, which is labile under typical analytical conditions, is first trapped with hydrazine to form stable hydrazide and can be directly analyzed by mass spectrometry. The resulting aspartyl hydrazide can be selectively derivatized by various tags, such as fluorescent rhodamine sulfonyl chloride that absorbs strongly in the visible region (570 nm). Our tagging strategy allows the labeled protein to be analyzed by orthogonal methods, including HPLC-UV-Vis, liquid chromatography mass spectrometry (LC-MS), and SDS-PAGE coupled with fluorescence imaging. A unique advantage of our method is that variants containing succinimide, after derivatization, can be readily resolved via either affinity enrichment or chromatographic separation. This allows further investigation of individual factors in a complex protein mixture that affect succinimide formation. Some additional advantages are imparted by fluorescence labeling including the facile detection of the intact protein without proteolytic digestion to peptides; and high sensitivity, for example, without optimization, 0.41% succinimide was readily detected. As such, our method should be useful for rapid screening, optimization of formulation conditions, and related processes relevant to protein pharmaceuticals.

Assessing analytical methods to monitor isoAsp formation in monoclonal antibodies.

A ubiquitous post-translational modification observed in proteins is isomerization of aspartic acid to isoaspartic acid (isoAsp). This non-enzymatic post-translational modification occurs spontaneously in proteins and plays a role in aging, autoimmune response, cancer, neurodegeneration, and other diseases. Formation of isoAsp is also a significant issue for recombinant monoclonal antibody based protein therapeutics particularly when isomerization occurs in a complementarity-determining region due to potential impact to the clinical efficacy. Here, we present and compare three analytical methods to monitor and/or quantify isoAsp formation in a monoclonal antibody. The methods include two peptide map based technologies with quantitation from either UV integration or total ion peak areas, as well as an alternative approach using IdeS digestion to generate Fc/2 and Fab'2 regions, followed by hydrophobic interaction chromatography (HIC) to separate the population of Fab'2 containing an isoAsp. The level of isoAsp detected by the peptide map and the digested-HIC methods presented here show similar trends although sample throughput varies by method.