Stability-indicative and conformation-specific enzyme linked immunosorbent assay for analysis of recombinant human gamma interferon.

Recombinant human interferon gamma (rhIFN-γ) is a promising molecule for the treatment of several diseases. A pair of conformation-specific monoclonal antibodies (mAbs) against rhIFN-γ was selected from generated hybridoma cell lines to design a sensitive, stability-indicative, sandwich-type ELISA. The main assay parameters were optimized by the checkerboard method for the highest signal-to-noise ratio: assay buffer composition, coating buffer pH and composition, coating temperature-incubation time parameters, and coating mAb concentration and conjugate dilution. Detection and quantification limits were estimated between 0.019 and 0.078 ng/mL, respectively, and recovery values were from 92.03% to 98.40%. The coefficient of variation of intra-assay precision parameters ranged from 2.32% to 9.21% while the inter-analyst variation was between 4.70% and 10.63%, supporting the method's repeatability. The ELISA was specific for correctly folded and non-aggregated molecular species, as compared to intrinsic Trp fluorescence (chemical denaturation) and optical density at 340 nm (thermal aggregation), respectively. However, the method was not sensitive to the small C-terminal degradation of full-length rhIFN-γ1-144 (losses of 6-12 amino acid residues) as compared to results with mass spectrometry and gel electrophoresis. ELISA showed good correlation with rhIFN-γ antiviral biological activity. This method was applied to the stability evaluation of rhIFN-γ in physiological buffer at low concentrations using polypropylene and glass vials also in the presence of adsorption protectant excipients. Furthermore, ELISA could be adapted to other applications such as quantification of IFN-γ in serum samples, Mycobacterium tuberculosis diagnosis, etc.

Chimeric Antigen by the Fusion of SARS-CoV-2 Receptor Binding Domain with the Extracellular Domain of Human CD154: A Promising Improved Vaccine Candidate.

COVID-19 is a respiratory viral disease caused by a new coronavirus called SARS-CoV-2. This disease has spread rapidly worldwide with a high rate of morbidity and mortality. The receptor-binding domain (RBD) of protein spike (S) mediates the attachment of the virus to the host's cellular receptor. The RBD domain constitutes a very attractive target for subunit vaccine development due to its ability to induce a neutralizing antibody response against the virus. With the aim of boosting the immunogenicity of RBD, it was fused to the extracellular domain of CD154, an immune system modulator molecule. To obtain the chimeric protein, stable transduction of HEK-293 was carried out with recombinant lentivirus and polyclonal populations and cell clones were obtained. RBD-CD was purified from culture supernatant and further characterized by several techniques. RBD-CD immunogenicity evaluated in mice and non-human primates (NHP) indicated that recombinant protein was able to induce a specific and high IgG response after two doses. NHP sera also neutralize SARS-CoV-2 infection of Vero E6 cells. RBD-CD could improve the current vaccines against COVID-19, based in the enhancement of the host humoral and cellular response. Further experiments are necessary to confirm the utility of RBD-CD as a prophylactic vaccine and/or booster purpose.

In-solution buffer-free digestion allows full-sequence coverage and complete characterization of post-translational modifications of the receptor-binding domain of SARS-CoV-2 in a single ESI-MS spectrum.

Subunit vaccines based on the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 provide one of the most promising strategies to fight the COVID-19 pandemic. The detailed characterization of the protein primary structure by mass spectrometry (MS) is mandatory, as described in ICHQ6B guidelines. In this work, several recombinant RBD proteins produced in five expression systems were characterized using a non-conventional protocol known as in-solution buffer-free digestion (BFD). In a single ESI-MS spectrum, BFD allowed very high sequence coverage (≥ 99%) and the detection of highly hydrophilic regions, including very short and hydrophilic peptides (2-8 amino acids), and the His6-tagged C-terminal peptide carrying several post-translational modifications at Cys538 such as cysteinylation, homocysteinylation, glutathionylation, truncated glutathionylation, and cyanylation, among others. The analysis using the conventional digestion protocol allowed lower sequence coverage (80-90%) and did not detect peptides carrying most of the above-mentioned PTMs. The two C-terminal peptides of a dimer [RBD(319-541)-(His)6]2 linked by an intermolecular disulfide bond (Cys538-Cys538) with twelve histidine residues were only detected by BFD. This protocol allows the detection of the four disulfide bonds present in the native RBD, low-abundance scrambling variants, free cysteine residues, O-glycoforms, and incomplete processing of the N-terminal end, if present. Artifacts generated by the in-solution BFD protocol were also characterized. BFD can be easily implemented; it has been applied to the characterization of the active pharmaceutical ingredient of two RBD-based vaccines, and we foresee that it can be also helpful to the characterization of mutated RBDs.

Synthesis, LC-MS/MS analysis, and biological evaluation of two vaccine candidates against ticks based on the antigenic P0 peptide from R. sanguineus linked to the p64K carrier protein from Neisseria meningitidis.

A peptide from the P0 acidic ribosomal protein (pP0) of ticks conjugated to keyhole limpet hemocyanin from Megathura crenulata has shown to be effective against different tick species when used in host vaccination. Turning this peptide into a commercial anti-tick vaccine will depend on finding the appropriate, technically and economically feasible way to present it to the host immune system. Two conjugates (p64K-Cys1pP0 and p64K-ßAla1pP0) were synthesized using the p64K carrier protein from Neisseria meningitidis produced in Escherichia coli, the same cross-linking reagent, and two analogues of pP0. The SDS-PAGE analysis of p64K-Cys1pP0 showed a heterogeneous conjugate compared to p64K-ßAla1pP0 that was detected as a protein band at 91kDa. The pP0/p64K ratio determined by MALDI-MS for p64K-Cys1pP0 ranged from 1 to 8, being 3-5 the predominant ratio, while in the case of p64K-ßAla1pP0 this ratio was 5-7. Cys1pP0 was partially linked to 35 out of 39 Lys residues and the N-terminal end, while ßAla1pP0 was mostly linked to the six free cysteine residues, to the N-terminal end, and, in a lesser extent, to Lys residues. The assignment of the conjugation sites and side reactions were based on the identification of type 2 peptides. Rabbit immunizations showed the best anti-pP0 titers and the highest efficacy against Rhipicephalus sanguineus ticks when the p64K-Cys1pP0 was used as vaccine antigen. The presence of high molecular mass aggregates observed in the SDS-PAGE analysis of p64K-Cys1pP0 could be responsible for a better immune response against pP0 and consequently for its better efficacy as an anti-tick vaccine. Graphical abstract.

Mass spectrometric and kinetics characterization of modified species of Growth Hormone Releasing Hexapeptide generated under thermal stress in different pH and buffers.

Growth Hormone Releasing Peptide-6 (GHRP-6) is a promising molecule (H-His1-d-Trp- Ala-Trp-d-Phe-Lys6-NH2) for the treatment of several diseases. Studies on the degradation pathways of this molecule under stressed conditions are needed to develop appropriate formulations. Degradation products (DPs) of GHRP-6, generated by heating in the dark at 60 °C with pH ranging from 3.0 to 8.0 and in presence of common buffers, were isolated by RP-HPLC and characterized by ESI-MS/MS. C-terminal deamidation of GHRP-6 was generated preferentially at pH 3.0 and 8.0. Hydrolysis and head-to-tail cyclization were favored at pH ranging from 6.0 to 7.0 in phosphate containing buffers. A DP with +12 Da molecular mass was presumably originated by the reaction with formaldehyde derived from some of the additives and/or elastomeric closures. Certain DPs derived from the acylation reaction of the tri- and di-carboxylic buffering species were favored at pH 3.0-6.0 and indicate that buffer components, including those "Generally Recognized as Safe", may potentially introduce chemical modifications and product heterogeneity. Nano LC-MS/MS analysis revealed GHRP-6 was also detected as a low-abundance species with Trp oxidized to 5-hydroxy, kynurenine, and N-formylkynurenine. The kinetics for the formation of the major degradation products was also studied by RP-HPLC.

Characterization of low-abundance species in the active pharmaceutical ingredient of CIGB-300: A clinical-grade anticancer synthetic peptide.

CIGB-300 is a first-in-class synthetic peptide-based drug of 25 amino acids currently undergoing clinical trials in cancer patients. It contains an amidated disulfide cyclic undecapeptide fused to the TAT cell-penetrating peptide through a beta-alanine spacer. CIGB-300 inhibits the CK2-mediated phosphorylation leading to apoptosis of tumor cells in vitro, and in vivo in cancer patients. Despite the clinical development of CIGB-300, the characterization of peptide-related impurities present in the active pharmaceutical ingredient has not been reported earlier. In the decision tree of ICHQ3A(R2) guidelines, the daily doses intake, the abundance, and the identity of the peptide-related species are pivotal nodes that define actions to be taken (reporting, identification, and qualification). For this, purity was first assessed by reverse-phase chromatography (>97%) and low-abundance impurities (≤0.27%) were collected and identified by mass spectrometry. Most of the impurities were generated during peptide synthesis, the spontaneous air oxidation of the reduced peptide, and the lyophilization step. The most abundant impurity, with no biological activity, was the full-length peptide containing Met17 transformed into a sulfoxide residue. Interestingly, parallel and antiparallel dimers of CIGB-300 linked by 2 intermolecular disulfide bonds exhibited a higher antiproliferative activity than the CIGB-300 monomer. Likewise, very low abundance trimers and tetramers of CIGB-300 linked by disulfide bonds (≤0.01%) were also detected. Here we describe for the first time the presence of active dimeric species whose feasibility as novel CIGB-300 derived entities merits further investigation.

Protein content of the Hylesia metabus egg nest setae (Cramer [1775]) (Lepidoptera: Saturniidae) and its association with the parental investment for the reproductive success and lepidopterism.

Hylesia metabus is a neotropical moth possessing toxic setae, which once in contact with the skin cause a severe dermatitis to humans known as lepidopterism. The only known function of the setae in the life cycle is to provide protection during the mating and egg-hatching stages. Approximately 65% of the protein content of the setae is a cluster of five proteases (28-45kDa) showing sequence homology to other S1A serine proteases. The N-glycans of a 40kDa protease are a mixture of neutral and sulfated G0F structures. The sulfated N-glycans have an important role in triggering the inflammatory response typical of lepidopterism while the proteolytic activity may promote the erosion of blood vessels and tissues causing focal hemorrhages. The presence of Chitinase and a 30kDa lipoprotein is probably related to the antifungal defense. In addition, chitin digestion of the setae may potentiate the inflammatory reaction caused by the toxins due to the formation of chitin adjuvants fragments. The combined effect of proteases and a chitinase may dissuade predating arthropods, by damaging their exoskeletons. Vitellogenin, a bacteriostatic protein, is able to recognize pathogen-associated patterns, which suggests its possible role in protecting the embryonated eggs from pathogenic microorganisms. SIGNIFICANCE: The present study is the first report describing the different protein species present in the urticating egg nest setae of the neotropical moth Hylesia metabus - the most harmful of the Hylesia moths - causing a severe urticating dermatitis in humans known as lepidopterism. A distinctive feature of the venom is the presence of five different S1A serine proteases probably used to guarantee a more efficient degradation of a wider number of protein substrates. This work confirms that the presence of sulfated N-glycans is not an isolated finding since its presence has been demonstrated in two different proteases affirming that this PTM is of importance for the activation of the inflammatory response typical of lepidopterism. Additionally, this study gives useful information on the defense mechanisms used for protection of its progeny vs. vertebrate predators, fungus, bacteria or other arthropods such as ants. The proteins detected in the egg nest should be seen as an extended parental effort made by the females in order to achieve an optimal reproductive success, thus compensating for the considerable loss of progeny during the larval stages that seriously limits the number of sexually mature adults reaching the reproductive phase.