Targeting the Structural Integrity of Extracellular Vesicles via Nano Electrospray Gas-Phase Electrophoretic Mobility Molecular Analysis (nES GEMMA).

Extracellular vesicles (EVs) are in the scientific spotlight due to their potential application in the medical field, ranging from medical diagnosis to therapy. These applications rely on EV stability during isolation and purification-ideally, these steps should not impact vesicle integrity. In this context, we investigated EV stability and particle numbers via nano electrospray gas-phase electrophoretic mobility molecular analysis (nES GEMMA) and nanoparticle tracking analysis (NTA). In nES GEMMA, native, surface-dry analytes are separated in the gas-phase according to the particle size. Besides information on size and particle heterogeneity, particle number concentrations are obtained in accordance with recommendations of the European Commission for nanoparticle characterization (2011/696/EU, 18 October 2011). Likewise, and in contrast to NTA, nES GEMMA enables detection of co-purified proteins. On the other hand, NTA, yielding data on hydrodynamic size distributions, is able to relate particle concentrations, omitting electrolyte exchange (and resulting EV loss), which is prerequisite for nES GEMMA. Focusing on EVs of different origin, we compared vesicles concentrations and stability, especially after electrolyte exchange and size exclusion chromatography (SEC). Co-isolated proteins were detected in most samples, and the vesicle amount varied in dependence on the EV source. We found that depletion of co-purified proteins was achievable via SEC, but was associated with a loss of EVs and-most importantly-with decreased vesicle stability, as detected via a reduced nES GEMMA measurement repeatability. Ultimately, we propose the repeatability of nES GEMMA to yield information on EV stability, and, as a result, we propose that nES GEMMA can yield additional valuable information in EV research.

A possible role of gas-phase electrophoretic mobility molecular analysis (nES GEMMA) in extracellular vesicle research.

The emerging role of extracellular vesicles (EVs) as biomarkers and their envisioned therapeutic use require advanced techniques for their detailed characterization. In this context, we investigated gas-phase electrophoresis on a nano electrospray gas-phase electrophoretic mobility molecular analyzer (nES GEMMA, aka nES differential mobility analyzer, nES DMA) as an alternative to standard analytical techniques. In gas-phase electrophoresis, single-charged, surface-dry, native, polydisperse, and aerosolized analytes, e.g., proteins or bio-nanoparticles, are separated according to their electrophoretic mobility diameter, i.e., globular size. Subsequently, monodisperse particles are counted after a nucleation step in a supersaturated atmosphere as they pass a focused laser beam. Hence, particle number concentrations are obtained in accordance with recommendations of the European Commission for nanoparticle characterization (2011/696/EU from October 18th, 2011). Smaller sample constituents (e.g., co-purified proteins) can be detected next to larger ones (e.g., vesicles). Focusing on platelet-derived EVs, we compared different vesicle isolation techniques. In all cases, nanoparticle tracking analysis (NTA) confirmed the presence of vesicles. However, nES GEMMA often revealed a significant co-purification of proteins from the sample matrix, precluding gas-phase electrophoresis of less-diluted samples containing higher vesicle concentrations. Therefore, mainly peaks in the protein size range were detected. Mass spectrometry revealed that these main contaminants belonged to the group of globulins and coagulation-related components. An additional size exclusion chromatography (SEC) step enabled the depletion of co-purified, proteinaceous matrix components, while a label-free quantitative proteomics approach revealed no significant differences in the detected EV core proteome. Hence, the future in-depth analysis of EVs via gas-phase electrophoresis appears feasible. Platelet-derived extracellular vesicles (EVs)with/without additional size exclusion chromatographic (SEC) purification were subjected to nanoparticle tracking analysis (NTA) and gas-phase electrophoresis (nES GEMMA). The latter revealed presence of co-purified proteins, targetable via mass spectrometry (MS). MS also revealed that SEC did not influence EV protein content. To conclude, nES GEMMA is a valuable tool for quality control of EV-containing samples under native conditions allowing for detection of co-purified proteins from complex matrices.

Comparative Analysis of Platelet-Derived Extracellular Vesicles Using Flow Cytometry and Nanoparticle Tracking Analysis.

Growing interest in extracellular vesicles (EVs) has prompted the advancements of protocols for improved EV characterization. As a high-throughput, multi-parameter, and single particle technique, flow cytometry is widely used for EV characterization. The comparison of data on EV concentration, however, is hindered by the lack of standardization between different protocols and instruments. Here, we quantified EV counts of platelet-derived EVs, using two flow cytometers (Gallios and CytoFLEX LX) and nanoparticle tracking analysis (NTA). Phosphatidylserine-exposing EVs were identified by labelling with lactadherin (LA). Calibration with silica-based fluorescent beads showed detection limits of 300 nm and 150 nm for Gallios and CytoFLEX LX, respectively. Accordingly, CytoFLEX LX yielded 40-fold higher EV counts and 13-fold higher counts of LA+CD41+ EVs compared to Gallios. NTA in fluorescence mode (F-NTA) demonstrated that only 9.5% of all vesicles detected in scatter mode exposed phosphatidylserine, resulting in good agreement of LA+ EVs for CytoFLEX LX and F-NTA. Since certain functional characteristics, such as the exposure of pro-coagulant phosphatidylserine, are not equally displayed across the entire EV size range, our study highlights the necessity of indicating the size range of EVs detected with a given approach along with the EV concentration to support the comparability between different studies.

Quality-Related Properties of Equine Immunoglobulins Purified by Different Approaches.

Whole IgG antivenoms are prepared from hyperimmune animal plasma by various refinement strategies. The ones most commonly used at industrial scale are precipitation by sodium or ammonium sulphate (ASP), and caprylic acid precipitation (CAP) of non-immunoglobulin proteins. The additional procedures, which have so far been used for experimental purposes only, are anion-exchange (AEX) and cation-exchange chromatography (CEX), as well as affinity chromatography (AC) using IgG's Fc-binding ligands. These protocols extract the whole IgG fraction from plasma, which contains both venom-specific and therapeutically irrelevant antibodies. Such preparations represent a complex mixture of various IgG subclasses whose functional and/or structural properties, as well as relative distribution, might be affected differently, depending on employed purification procedure. The aim of this work was to compare the influence of aforementioned refinement strategies on the IgG subclass distribution, venom-specific protective efficacy, thermal stability, aggregate formation and retained impurity profile of the final products. A unique sample of Vipera ammodytes ammodytes specific hyperimmune horse plasma was used as a starting material, enabling direct comparison of five purification approaches. The highest purity was achieved by CAP and AC (above 90% in a single step), while the lowest aggregate content was present in samples from AEX processing. Albumin was the main contaminant in IgG preparations obtained by ASP and CEX, while transferrin dominantly contaminated IgG sample from AEX processing. Alpha-1B-glycoprotein was present in CAP IgG fraction, as well as in those from ASP- and AEX-based procedures. AC approach induced the highest loss of IgG(T) subclass. CEX and AEX showed the same tendency, while CAP and ASP had almost no impact on subclass distribution. The shift in IgG subclass composition influenced the specific protective efficacy of the respective final preparation as measured in vivo. AC and CEX remarkably affected drug's venom-neutralization activity, in contrary to the CAP procedure, that preserved protective efficacy of the IgG fraction. Presented data might improve the process of designing and establishing novel downstream processing strategies and give guidance for optimization of the current ones by providing information on potency-protecting and purity-increasing properties of each purification principle.

Streamlined downstream process for efficient and sustainable (Fab')2 antivenom preparation.

BACKGROUND: Antivenoms are the only validated treatment against snakebite envenoming. Numerous drawbacks pertaining to their availability, safety and efficacy are becoming increasingly evident due to low sustainability of current productions. Technological innovation of procedures generating therapeutics of higher purity and better physicochemical characteristics at acceptable cost is necessary. The objective was to develop at laboratory scale a compact, feasible and economically viable platform for preparation of equine F(ab')2 antivenom against Vipera ammodytes ammodytes venom and to support it with efficiency data, to enable estimation of the process cost-effectiveness. METHODS: The principle of simultaneous caprylic acid precipitation and pepsin digestion has been implemented into plasma downstream processing. Balance between incomplete IgG breakdown, F(ab')2 over-digestion and loss of the active drug's protective efficacy was achieved by adjusting pepsin to a 1:30 substrate ratio (w/w) and setting pH at 3.2. Precipitation and digestion co-performance required 2 h-long incubation at 21 °C. Final polishing was accomplished by a combination of diafiltration and flow-through chromatography. In vivo neutralization potency of the F(ab')2 product against the venom's lethal toxicity was determined. RESULTS: Only three consecutive steps, performed under finely tuned conditions, were sufficient for preservation of the highest process recovery with the overall yield of 74%, comparing favorably to others. At the same time, regulatory requirements were met. Final product was aggregate- and pepsin-free. Its composition profile was analyzed by mass spectrometry as a quality control check. Impurities, present in minor traces, were identified mostly as IgG/IgM fragments, contributing to active drug. Specific activity of the F(ab')2 preparation with respect to the plasma was increased 3.9-fold. CONCLUSION: A highly streamlined mode for production of equine F(ab')2 antivenom was engineered. In addition to preservation of the highest process yield and fulfillment of the regulatory demands, performance simplicity and rapidity in the laboratory setting were demonstrated. Suitability for large-scale manufacturing appears promising.

Streamlined downstream process for efficient and sustainable (Fab)2 antivenom preparation

Antivenoms are the only validated treatment against snakebite envenoming. Numerous drawbacks pertaining to their availability, safety and efficacy are becoming increasingly evident due to low sustainability of current productions. Technological innovation of procedures generating therapeutics of higher purity and better physicochemical characteristics at acceptable cost is necessary. The objective was to develop at laboratory scale a compact, feasible and economically viable platform for preparation of equine F(ab')2 antivenom against Vipera ammodytes ammodytes venom and to support it with efficiency data, to enable estimation of the process cost-effectiveness. Methods: The principle of simultaneous caprylic acid precipitation and pepsin digestion has been implemented into plasma downstream processing. Balance between incomplete IgG breakdown, F(ab')2 over-digestion and loss of the active drug's protective efficacy was achieved by adjusting pepsin to a 1:30 substrate ratio (w/w) and setting pH at 3.2. Precipitation and digestion co-performance required 2 h-long incubation at 21 °C. Final polishing was accomplished by a combination of diafiltration and flow-through chromatography. In vivo neutralization potency of the F(ab')2 product against the venom's lethal toxicity was determined. Results: Only three consecutive steps, performed under finely tuned conditions, were sufficient for preservation of the highest process recovery with the overall yield of 74%, comparing favorably to others. At the same time, regulatory requirements were met. Final product was aggregate- and pepsin-free. Its composition profile was analyzed by mass spectrometry as a quality control check. Impurities, present in minor traces, were identified mostly as IgG/IgM fragments, contributing to active drug. Specific activity of the F(ab')2 preparation with respect to the plasma was increased 3.9-fold. Conclusion: A highly streamlined mode for production of equine F(ab')2 antivenom was engineered. In addition to preservation of the highest process yield and fulfillment of the regulatory demands, performance simplicity and rapidity in the laboratory setting were demonstrated. Suitability for large-scale manufacturing appears promising.(AU)