Post-translational modifications and glycoprofiling of palivizumab by UHPLC-RPLC/HILIC and mass spectrometry.

Viral infections are progressively becoming a global health burden, as witnessed in the ongoing COVID-19 pandemic. Respiratory Syncytial Virus (RSV) is another highly contagious negative-sense RNA virus that causes lower respiratory tract infections and high mortality in infants. Palivizumab (Synagis®) is the only humanized monoclonal antibody (mAb) approved by the FDA against RSV. The virus neutralization efficacy often depends on the nature and abundance of the glycoforms in therapeutic mAbs. Therefore, a thorough estimation of their PTM profile, especially glycosylation, is relevant. Here, we describe the intact and released glycan analysis of palivizumab (Synagis®) using HILIC chromatography and mass spectrometry. We detected five glycoforms (Man5/G0FB, G0F/G1F, G1F/G1F, G0FB/G0FB, and G2F/G2F) in deconvoluted MS spectra of intact glycosylated palivizumab. The mapping of the peptide and glycopeptides using LC-ESI-MS led to the detection of associated PTMs and the direct identification of a glycopeptide, GlcNAc3Man2. EEQYNSTYR, derived from the heavy chain of palivizumab.Release glycan analysis using UHPLC-HILIC revealed a typical glycan profile consisting of major glycans, G0F (33.94%), G1F (35.50%), G2F (17.24%) also reported previously and minor G1F' (5.81%), Man5 (3.96%) and G0FB (2.26%) forms with the superior resolution of isomeric G1F/G1F'. Next, we provide the first experimental evidence of Neu5Gc in the commercial palivizumab formulation using DMB labelling. The estimated monosaccharide composition was consistent with previous studies. The findings of the study highlight the efficiency of the release glycan method in providing a correct measure of the total palivizumab glycan pool compared to the intact glycoprotein/glycopeptide approach. The UHPLC-RPLC/HILIC and MS combinations provide a more comprehensive glycoprofile assessment due to the parallel use of fluorescent labels for the analysis of the release of N-glycan, sialic acid, and monosaccharide composition. This approach is suitable for quick quality testing and market surveillance of therapeutic mAbs. Alongside a well-perceived need for cost-effective immunoprophylaxis and the ongoing fast-paced development of next-generation variants of palivizumab, such as MEDI8897, the study reiterates glycosylation as a critical parameter that needs monitoring for drug characterization and quality control. Supplementary Information: The online version contains supplementary material available at 10.1007/s42485-022-00086-1.

Purification, characterization and functional properties of exopolysaccharide from a novel halophilic Natronotalea sambharensis sp. nov.

An exopolysaccharide producing strain AK103T was isolated from Sambharlake, Rajasthan, India. Based on polyphasic taxonomy, the strain AK103T was found to be a novel species for which the name Natronotalea sambharensis sp. nov. is proposed. The strain was able to secrete (1.2 gL-1) EPS under optimum conditions. The FT-IR spectroscopy revealed that EPS had carboxyl, hydroxyl, carbonyl and sulfate ester groups. The high performance liquid chromatography (HPLC), gel permeation chromatography (GPC) and NMR revealed that the exopolysaccharide was composed of mannose, glucose and glucuronic acid with a molecular weight of 4.6 × 106 KDa. This novel EPS was designated as Nat-103. Interestingly, the EPS was found to have antioxidant activity and in vitro antioxidant studies showed that DPPH (2,2-diphenyl-1-picrylhydrazyl) activity increased in a dose dependent manner. Furthermore, EPS Nat-103 was able to produce gold nanoparticles which were further characterized by FT-IR, DLS and Zeta potential (ZP).

Structural characterization and antioxidant potential of a novel anionic exopolysaccharide produced by marine Microbacterium aurantiacum FSW-25.

An exopolysaccharide (EPS) producing strain FSW-25 was isolated from the Rasthakaadu beach Kanyakumari, Tamil Nadu India. Based on polyphasic taxonomy, the strain FSW-25 was assigned to the genus Microbacterium and found to be the closest relative of the species aurantiacum. Large quantity of EPS (7.81 g/l) was secreted by the strain upon fermentation using Reasoner's 2A medium enriched with 2.5% glucose and was designated as Mi-25. FT-IR spectrum revealed presence of hydroxyl, carbonyl, carboxyl, methyl and sulfate functional groups in purified EPS. The EPS Mi-25 has a molecular weight of 7.0 × 106 Da and mainly comprises of glucuronic acid followed by glucose, mannose and fucose. Rheological study revealed that Mi-25 possesses significant viscosity with pseudoplastic nature. Interestingly, it was observed that the EPS Mi-25 has higher antioxidant activity as compared to xanthan. The characteristics of EPS Mi-25 suggested that, it can be used as a potential antioxidant with viscosifier properties in diverse industrial sectors.

Production, characterization and bio-emulsifying activity of a novel thermostable exopolysaccharide produced by a marine strain of Rhodobacter johrii CDR-SL 7Cii.

An exopolysaccharide (EPS) producing bacterial strain was isolated from the surface of marine macroalgae (Padina sp.). Based on polyphasic taxonomy, the strain CDR-SL 7Cii was assigned to the genus Rhodobacter and found to be the closest relative of the species Rhodobacter johrii. The strain was able to produce 6.2 g/l of EPS upon fermentation using R2A medium enriched with 2.5% glucose. FT-IR analysis revealed the presence of hydroxyl, carboxyl and diacyl-ester functional groups in the purified EPS. Further Chromatographic study revealed that R. johrii synthesized a high molecular weight anionic exopolysaccharide composed of glucose, glucuronic acid, rhamnose and galactose in a molar ratio of 3:1.5:0.25:0.25. The 1D and 2D NMR spectroscopy (COSY/HSQC) analysis revealed the presence of 1,6 linked-α-d-Glcp, 1,4 linked-ß-d-Glcp, 1,3 linked-ß-d-GlcA, 1,3 linked-ß-d-Galp, 1,6 linked-ß-d-Galf and 3-α-l-Rhmp residues. Moreover, the purified EPS has shown stability towards elevated temperature and also acted as a bio-emulsifier to create a high pH and temperature stable emulsion of hydrocarbon/water indicating its industrial potential. This is the first report of EPS production by a strain of Rhodobacter johrii.