Poly(glutamic acid)-Based Viscosity Reducers for Concentrated Formulations of a Monoclonal IgG Antibody.

Above a concentration threshold, the viscosity of solutions of proteins increases abruptly, which hampers the injectability of therapeutic formulations. Concentrations above 200 g/L are an ideal goal for subcutaneous application of antibodies. Molecular additives, such as amino acids (e.g., arginine) help decrease the viscosity, but they are used at concentrations as high as about 200 mmol/L. We addressed the question of whether poly(amino acids) could be more efficient than small molecular additives. We observed marked fluidification of a model therapeutic monoclonal antibody (mAb) solution by poly(d,l-glutamic acid) and poly(l-glutamic acid) derivatives added at concentrations of <6.5 g/L (i.e., a mAb/polymer chain molar ratio between 4:1 and 1:1 mol/mol). The bare poly(glutamate) parent chains were compared with polyethylene glycol-grafted chains as PEGylation is a common way to enhance stability. Viscosity could be decreased to ∼20 mPa s as compared to values of ∼100 mPa s in the absence of polymers at 200 g/L mAb. Formation of complexes between the mAb and the polyglutamates was characterized by capillary electrophoresis analysis in dilute solutions (1 g/L mAb) and by observation of phase separation at higher concentrations, suggesting tight association at about 2:1 mol/mol mAb/polymer. Altogether, these results show that polyglutamate derivatives hold an untapped potential as an excipient for fluidification of concentrated protein solutions.

Salting-Out Approach Is Worthy of Comparison with Ultracentrifugation for Extracellular Vesicle Isolation from Tumor and Healthy Models.

The role of extracellular vesicles (EVs) has been completely re-evaluated in the recent decades, and EVs are currently considered to be among the main players in intercellular communication. Beyond their functional aspects, there is strong interest in the development of faster and less expensive isolation protocols that are as reliable for post-isolation characterisations as already-established methods. Therefore, the identification of easy and accessible EV isolation techniques with a low price/performance ratio is of paramount importance. We isolated EVs from a wide spectrum of samples of biological and clinical interest by choosing two isolation techniques, based on their wide use and affordability: ultracentrifugation and salting-out. We collected EVs from human cancer and healthy cell culture media, yeast, bacteria and Drosophila culture media and human fluids (plasma, urine and saliva). The size distribution and concentration of EVs were measured by nanoparticle tracking analysis and dynamic light scattering, and protein depletion was measured by a colorimetric nanoplasmonic assay. Finally, the EVs were characterised by flow cytometry. Our results showed that the salting-out method had a good efficiency in EV separation and was more efficient in protein depletion than ultracentrifugation. Thus, salting-out may represent a good alternative to ultracentrifugation.

Direct Exposure of Dry Enzymes to Atmospheric Pressure Non-Equilibrium Plasmas: The Case of Tyrosinase.

The direct interaction of atmospheric pressure non-equilibrium plasmas with tyrosinase (Tyr) was investigated under typical conditions used in surface processing. Specifically, Tyr dry deposits were exposed to dielectric barrier discharges (DBDs) fed with helium, helium/oxygen, and helium/ethylene mixtures, and effects on enzyme functionality were evaluated. First of all, results show that DBDs have a measurable impact on Tyr only when experiments were carried out using very low enzyme amounts. An appreciable decrease in Tyr activity was observed upon exposure to oxygen-containing DBD. Nevertheless, the combined use of X-ray photoelectron spectroscopy and white-light vertical scanning interferometry revealed that, in this reactive environment, Tyr deposits displayed remarkable etching resistance, reasonably conferred by plasma-induced changes in their surface chemical composition as well as by their coffee-ring structure. Ethylene-containing DBDs were used to coat tyrosinase with a hydrocarbon polymer film, in order to obtain its immobilization. In particular, it was found that Tyr activity can be fully retained by properly adjusting thin film deposition conditions. All these findings enlighten a high stability of dry enzymes in various plasma environments and open new opportunities for the use of atmospheric pressure non-equilibrium plasmas in enzyme immobilization strategies.