New Opportunities in Glycan Engineering for Therapeutic Proteins

Glycans as sugar polymers are important metabolic, structural, and physiological regulators for cellular and biological functions. They are often classified as critical quality attributes to antibodies and recombinant fusion proteins, given their impacts on the efficacy and safety of biologics drugs. Recent reports on the conjugates of N-acetyl-galactosamine and mannose-6-phosphate for lysosomal degradation, Fab glycans for antibody diversification, as well as sialylation therapeutic modulations and O-linked applications, have been fueling the continued interest in glycoengineering. The current advancements of the human glycome and the development of a comprehensive network in glycosylation pathways have presented new opportunities in designing next-generation therapeutic proteins.

Challenges with development of a pharmacokinetics assay to measure a variably glycosylated fusion protein

Aim: Development of recombinant fusion proteins as drugs poses unique challenges for bioanalysis. This paper describes a case study of a glycosylated fusion protein, where variable glycosylation, matrix interference and high sensitivity needs posed unique challenges. Results: Six different assay configurations, across four different platforms were evaluated for measurement of drug concentrations. Two platforms that achieved the assay requirements were Simoa HD-1 and immune-capture LC–MS/MS-based assay. Conclusion: Both, Simoa HD-1 and the mass spectrometry-based methods were able to detect total drug by providing the adequate matrix tolerance, required sensitivity and detection of all the various glycosylated fusion proteins to support clinical sample analysis. The mass spectrometry-based method was selected due to robustness and ease of method transfer.

Solid-phase synthesis of imprinted nanoparticles as artificial antibodies against the C-terminus of the cannabinoid CB1 receptor: exploring a viable alternative for bioanalysis

The production of artificial anti-CB1 antibodies in nanoparticle format is described using the solid-phase imprinting approach. Instead of whole protein imprinting, a linear C-terminus sequence of the receptor comprising 15 amino acids (458-KVTMSVSTDTSAEAL-472) has been used as template, in accordance with the epitope imprinting approach. This sequence is located intracellularly, and it is involved in coupling to Gi/o proteins, being responsible for CB1 receptor desensitisation and internalisation. Developed molecularly imprinted materials were found to be in the nanometre scale, with a particle size of 126.4 ± 10.5 nm at pH 3 (25 ºC) and spherical shape. It was also observed that the size was sensible to temperature changes being reduced to 106.3 ± 15.2 nm at 35 °C. Lower critical solution temperature of this polymer was found to be ≈ 33.4 °C. The affinity and selectivity of the artificial antibody were assessed through dot blot and Western blot experiments. For the latter, recombinant fusion proteins GST-CB1414-472 and GST-CB1414-442 were produced to work respectively as target and negative control proteins. The control protein did not carry the target epitope for being devoid of last 30 amino acids at the C-terminus. The results demonstrated that the anti-CB1 material recognised selectively the target protein, thanks to the presence of the 15-amino acid sequence selected as epitope, which revealed that binding occurred at the C-terminus of the receptor itself. The methodology presented may pave the way for the development of novel imprinted nanomaterials for other proteins included in the superfamily of the G-protein-coupled receptors (GPCR). Graphical abstract

DEVELOPMENT OF THE VACCINE BASED ON THE RECOMBINANT ANTIGENS OF PSEUDOMONAS AERUGINOSA

Aim. The aim is obtaining, investigation and selection of recombinant antigens for inclusion theirs into the against Pseudomonas vaccine. Materials and methods. The genes encoding of the outer membrane proteins F, L and I and Exotoxin A were synthesized by PCR with the genomic DNA of Pseudomonas aeruginosa. The amplified sequences were cloned into plasmid vectors for expression in cells of Escherichia coli. As the result of expression were the synthesized recombinant proteins that were purified in columns with a nickel-activated sorbent. The authenticity of the recombinant antigens was assessed by electrophoresis and immunoblotting. For assessing the immunogenicity of the recombinant proteins,they were sorbed on aluminum hydroxide and used for intraperitoneal immunization of mice. After a course of immunization, mice were injected intraperitoneally with a live virulent culture or еxotoxin A. Results. The obtained recombinant outer membrane proteins OprF, OprL and OprI, as well as the deletion variant of еxotoxin A (toxoid) stimulated immune reactions and protected the experimental animals from the virulent culture of P. aeruginosa. Using of the complexes of the recombinant proteins, as well as immunization with the fusion proteins consisting from sequences of two or three recombinant antigens, produced an additive increase in protective effects. The combination of the recombinant OprF protein and the recombinant toxoid (efficiency index of protective properties (EI 3.0) and two recombinant fusion proteins (EI 3.5) were the most effective. The first recombinant fusion protein (OprF-aTox-OprI) consisted from fused polypeptide sequences of OprF, toxoid and OprI. The second recombinant fusion protein (OprF-OprI) consisted from fused polypeptide sequences of OprF and OprI. Conclusion. The data obtained showed the fundamental possibility of using recombinant fusion proteins OprF-aTox-OprI and OprF-OprI as well as the complex of the recombinant OprF protein and the recombinant toxoid as the candidated vaccines against Pseudomonas aeruginosa.