The use of germicidal ultraviolet light, vaporised hydrogen peroxide and dry heat to decontaminate face masks and filtering respirators contaminated with a SARS-CoV-2 surrogate virus

peer reviewe

Study of insulin aggregation by SEC and CGE

Introduction: Insulin is a widely used antidiabetic drug, which regulates carbohydrate and fat metabolism of human body. This hormone is mostly formulated in hexamer by addition of zinc as an excipient but only the monomeric form is active once dissociated in the bloodstream. Insulin is prone to unfold when submitted to denaturating factors as temperature, ionic strength, agitation and pH. An accumulation of unfolded proteins results in a high tendency to aggregate and form amyloid fibrils. A deposit of those fibrils in the subcutaneous tissue leads to a complication called “insulin-derived amyloidosis”. Moreover, during its production, insulin is often subjected to extreme conditions making lack of aggregates an important parameter to be controlled during its quality control. United States and European Pharmacopoeias use both size exclusion chromatography (SEC) to assess the level of covalent high molecular weight species. This technique is reproducible, and easy to use but shows many drawbacks including possible changes in the aggregates composition by dilution into the HPLC system or adsorption of sample onto the stationary phase. For those reasons other techniques have been considered in the literature for studying aggregation of insulin. Optical microscopy, electron microscopy, dynamic light scattering, turbidimetry, Fourier Transform infrared spectroscopy, Raman spectroscopy, thioflavin T fluorescence and circular dichroism spectroscopy are some of them. In any cases, the use of orthogonal techniques is essential to assess the relevance of the results. Results: In this study, insulin aggregates were generated after optimization of incubation conditions (pH, temperature, agitation…). Those aggregates were then analyzed by SEC and capillary electrophoresis (CE). CE shows many advantages in terms of sample and solvent consumption and enables analysis of samples under their native form. We showed that capillary gel electrophoresis (CGE) is a promising technique to analyze covalent aggregates of insulin due to the fact that it separates the aggregates according to their size and not to their size/charge ratio. The use of a laser-induced fluorescence detector was also found attractive to enhance the sensitivity of the method

Insulin aggregation assessment by size-exclusion chromatography and capillary gel electrophoresis

Size-exclusion chromatography (SEC) is the method of choice for the analysis of protein aggregates in biopharmaceuticals. Aggregates could be formed during production and storage of formulations and may lead to complications in insulin therapy. For that reason, they are important parameters to investigate for insulin quality control. The United States and European Pharmacopoeias currently use a SEC method with acidic mobile phase for the quantification of aggregates in insulin formulations. However, changes in aggregates assessment have been reported when the mobile phase composition differs from the sample dissolution medium. [1] To investigate the impact of mobile phase on human insulin aggregates analysis, the aggregated human insulin samples were analyzed by SEC using neutral (nSEC) or acidic mobile phases (aSEC). The aggregated samples were obtained by dissolving human insulin in acidic media, followed by agitation for 8, 16, 24, 32, 40 and 48 hours respectively. During SEC method development, the impact of arginine and acetonitrile addition to the mobile phase was pointed out. Additionally, an orthogonal capillary-gel electrophoresis method (CGE) for the assessment of insulin aggregates was developed. The optimal pH 8.1 CGE buffer was formulated without SDS in order to preserve the non-covalent aggregates. After the optimization of the methods, human insulin and aggregated samples were analyzed using nSEC, aSEC and CGE. A similar increase of dimers percentage with incubation time was noticed by both nSEC and CGE, while no significant increase of dimers content was observed by aSEC. However, an insulin polymeric complex was detectable for some samples with aSEC. The three methods were used to analyze an insulin formulation and a similar tendency was observed. The results obtained emphasize the importance of mobile phase choice in SEC. The good correlation between dimers percentage obtained by nSEC and CGE suggests that these technics most probably enable the detection of the species initially present in the sample and do not change the composition of the sample during analysis. The developed CGE method is a fast and reliable tool for the study of the complex process of insulin aggregation. Furthermore, the CGE method could be easily applied to other proteins since it proved to be highly reproducible and has the advantages of low sample consumption, and no expensive column or organic solvents are required

Challenges in the determination of amyloid oligomeric species by two electrophoretic techniques

Parkinson’s disease is a frequent degenerative disorder, and for the moment the diagnosis is mainly clinical. When the first symptoms appear, loss of more than 70% of the dopaminergic cells already occurred. Knowing that, it is of high interest to have one (or more) reliable biomarker(s) at our disposal to diagnose Parkinson before the first symptoms appear. Alpha-synuclein (aSyn) is a protein physiologically expressed at high level by neuronal cells, under a monomeric form. This protein would play a critical role in the development of the disease because under certain conditions, aSyn is capable of self-assembly to form fibrils like those found in Lewy bodies. Other intermediate soluble forms like dimers and oligomers are also formed. As these forms seems to be the toxic species, they are the center of many attentions. The quantification of each form would be a great help, but for the moment only the total forms (of monomeric or oligomeric) can be quantified. In this study, aSyn oligomers were generated after optimization of incubation conditions (pH, temperature, agitation, …). Then, different approaches were investigated to detect and follow the different species formed during the aggregation. We analyzed the oligomers by capillary gel electrophoresis (CGE) and SDS-PAGE. We found that capillary gel electrophoresis is a promising automated technique to analyze aSyn oligomers, due to the fact that it separates the aggregates according to their size, like the SDS-PAGE, but with more advantages. To gain sensitivity and selectivity by CGE, we used a laser-induced fluorescence detector. As aSyn do not have a native fluorescence, we derivatized it. After careful screening and optimization of various derivatization reagents, we could quantify with high sensitivity aSyn oligomers by CGE-LIF. We realized different calibration curves, and we had promising results that will allow us to quantify the different aSyn oligomeric forms in biological fluids

Quality control and aggregation follow-up of insulin formulations

The prevalence of diabetes is increasing every year making insulin formulations widely prescribed medicines. Fast and efficient methods to assess the quality of such biopharmaceutical products are thus required, more particularly methods to assess the content of API and potential aggregates. We started our study on insulin aggregation using the European Pharmacopeia method. Since methods to assess aggregate content are often contradictory, we also developed original and orthogonal methods using size-exclusion chromatography (SEC) and capillary gel electrophoresis (CGE). It was demonstrated that methods under neutral conditions (SEC and CGE) yield to similar aggregate content contrary to pharmacopeia SEC method that works under acidic conditions. Ion-mobility Q-TOF mass spectrometer was also used to confirm the presence and the identity of insulin dimers. Then, we applied the three methods to the analysis of an insulin formulation and similar results to those obtained for human insulin as raw material were observed. We also used the CGE method to study the stability of human insulin under different storage conditions. Finally, we used UHPLC and mass spectrometry to quantify insulin formulation from different supply chains. We demonstrated that all the analyzed formulations had a potency between 95.0 % and 105.0 % of the potency stated on the label. This was useful to dispel doubts regarding issues in insulin cold chain supply recently described in the litterature