Two-dimensional sequential selective comprehensive chiral×reversed-phase liquid chromatography of synthetic phosphorothioate oligonucleotide diastereomers.

In recent years, many nucleic acid-based pharmaceuticals have been approved and entered the market, and even a larger number are in late stage clinical trials. Conventional oligonucleotides are facing issues in vivo like fast renal clearance and nuclease degradation. Therefore, to increase their stability, phosphorothioation is a frequent modification of therapeutic oligonucleotides (ONs) which also leads to improved binding affinity facilitating cell internalization and intracellular distribution. At the same time, by replacing a phosphodiester linkage with a phosphorothioate group, a phosphorous stereogenic center is generated which causes the formation of Rp- and Sp-diastereomers. It increases the structural diversity. For example, with 15 of those phosphorothioate (PS) linkages, 32,768 different diastereomers are expected. Since the phosphorothioate is introduced non-stereoselectively, the molecular complexity of the resultant phosphorothioate ON products is tremendously increased impeding the chromatographic separation in the course of quality control. Since distinct phosphorothioate diastereomers have different bioactivities and pharmacological properties, there is increasing interest in implications of stereoisomerism of phosphorothiate oligonucleotides. From a quality and regulatory viewpoint, batch-to-batch reproducibility of the diastereomer profile may be of significant concern. In order to address this issue, this study investigates the stereoselectivity of LC methods for two phosphorothioate oligonucleotide (PSO) compounds differing in their molecular size and numbers of PS linkages. Diastereoselectivity of ion-pairing reversed-phase liquid chromatography (IP-RPLC), RPLC without ion-pairing agents and LC with chiral polysaccharide-based column were evaluated for model PSOs and an active pharmaceutical ingredient (API) of PSO with trivalent N-acetylgalactosamine (GalNAc) conjugate. Due to the structural complexity of PSOs, the separation power for the diastereomer mixture was increased by using sequential selective comprehensive two-dimensional chromatography with an amylose tris(α-methylbenzylcarbamate)-immobilized chiral stationary phase (CSP) in the first dimension and ion-pair RPLC with ethylammonium acetate in the second dimension. Improved diastereomer selectivity was obtained and a larger number of peaks could be separated.

Pharmacodynamics, pharmacokinetics and CYP3A4 interaction potential of the selective P2X3 receptor antagonist filapixant: A randomized multiple ascending-dose study in healthy young men.

AIMS: We report on investigations exploring the P2X3-receptor antagonist filapixant's effect on taste perception and cough-reflex sensitivity and describe its pharmacokinetics, including its CYP3A4-interaction potential. METHODS: In a randomized, placebo-controlled, double-blind study, 3 × 12 healthy men (18-45 years) were assigned (3:1) to filapixant (20, 80 or 250 mg by mouth) or placebo twice daily over 2 weeks. A single dose of midazolam (1 mg), a CYP3A4 substrate, was administered with and without filapixant. Assessments included a taste-strips test, a taste questionnaire, cough challenge with adenosine triphosphate, adverse event reports and standard safety assessments. RESULTS: Taste disturbances were observed mainly in the 250-mg group: six of nine participants (67%) in this group reported hypo- or dysgeusia in the questionnaire; eight participants (89%) reported taste-related adverse events. Five participants (56%) had a decrease in overall taste-strips-test scores ≥2 points (point estimate -1.1 points, 90% confidence interval [-3.3; 1.1]). Cough counts increased with adenosine triphosphate concentration but without major differences between treatments. Filapixant exposure increased proportionally to dose. Co-administration of filapixant had no clinically relevant effect on midazolam pharmacokinetics. Area under the concentration-time curve ratios and 90% confidence intervals were within 80-125%. No serious or severe adverse events were reported. CONCLUSIONS: Overall, filapixant was safe and well tolerated, apart from mild, transient taste disturbances. Such disturbances occurred more frequently than expected based on (in vitro) receptor-selectivity data, suggesting that other factors than P2X3:P2X2/3 selectivity might also play an important role in this context. The cough-challenge test showed no clear treatment effect. Filapixant has no clinically relevant CYP3A4 interaction potential.

Tackling Issues Observed during the Development of a Liquid Chromatography Method for Small Molecule Quantification in Antibody-Chelator Conjugate.

In the context of targeted radionuclide therapy, antibody-chelator conjugates (ACCs) are an evolving class of antibody-related drugs with promising applications as tumor-targeted pharmaceuticals. Generally, a typical ACC consists of a recombinant monoclonal antibody (mAb) coupled to radionuclide via a chelating agent. Characterizing the ACC structure represents an analytical challenge since various impurities must be constantly monitored in the presence of formulation components during the quality control (QC) process. In this contribution, a reliable method devoted to the monitoring of an ACC sample, and its small molecule-related synthesis impurities, has been developed via liquid chromatography (LC). A problem-solving approach of common analytical issues was used to highlight some major issues encountered during method development. This included separation of poorly retained impurities (issue #1); interferences from the formulation components (issue #2); analysis of impurities in presence of ACC at high concentration (issue #3); and recovery of impurities during the whole analytical procedure (issue #4). To the best of our knowledge, this is the first time that a chromatographic method for the analysis of ACC synthesis impurities is presented. In addition, the developed approach has the potential to be more widely applied to the characterization of similar ACCs and other antibody-related drugs.

Feedback from the Science Café at the 7th European Bioanalysis Forum Young Scientist Symposium.

The 7th Young Scientist Symposium, a meeting again organized as a hybrid online event by young scientists for young scientists under the umbrella of the European Bioanalysis Forum and in collaboration with the Universities of Bologna and Ghent, included a variety of interesting presentations on cutting-edge bioanalytical science and processes. On the morning of day 2, the meeting hosted their traditional Science Café around the theme: 'How has COVID-19 changed our future?' in which the Young Scientist Symposium organizing committee engaged with the delegates on how the COVID-19 pandemic has impacted the careers of young scientists working in a bioanalytical (industry or academic) laboratory, that is, things they lost, for good or for bad - things they gained, wanted or unwanted, things they learned about themselves and their industry. This manuscript provides feedback from those discussions.

Feedback from the Sixth European Bioanalysis Forum Young Scientist Symposium.

As part of the European Bioanalysis Forum mission to provide development opportunities for scientists, a Young Scientist Symposium has been organized every year since 2014. The meetings, organized by and for young scientists, aim at immersing talent from industry and academia in the scientific and process challenges important for their (future) professional environment. In an ideal world, the setting of an interactive symposium in stimulating auditorium sets the foundation for long lasting peer scientific relationship. This year, a pandemic has descended across all continents, changing the dynamics of the meeting. This manuscript summarizes the discussions at the Sixth EBF Young Scientist Symposium, originally planned as a face-to face event in March 2020 in Bologna, Italy but finally executed as a hybrid meeting in Cyberspace and on location in a few regions across Europe between 24-25 September 2020.

Feedback from the Science Café from the Sixth European Bioanalysis Forum Young Scientist Symposium.

The 6th Young Scientist Symposium, a meeting organized by young scientists for young scientists under the umbrella of the European Bioanalysis Forum vzw and in collaboration with the Universities of Bologna and Ghent, included a variety of interesting presentations on cutting edge bioanalytical science and processes. Integrated in the meeting, an interactive round table session, the Science Café, discussed the challenges related to sustainability for bioanalytical lab activities. This manuscript reflects conclusions from these discussions. They can provide our community a compass for future business practices to embrace more sustainable laboratory activities considerate of smarter use of a wide array of resources and laboratory tools, resulting in increased wellbeing for our next generations and our planet.

Pharmacokinetics, excretion, distribution, and metabolism of 60-kDa polyethylene glycol used in BAY 94-9027 in rats and its value for human prediction.

The covalent binding of proteins with polyethylene glycol (PEG) molecules is a valuable tool to extend the half-life of many biotherapeutics, including factor VIII (FVIII) products to treat patients with haemophilia A. Although PEG has low toxicity, accumulation of large PEG molecules (>20-30 kDa) with long-term exposure is a potential concern. Thus, it is important to determine whether sufficient excretion processes exist for PEG molecules used in biotherapeutics. BAY 94-9027 is an extended-half-life FVIII product modified through addition of a 60-kDa (branched: dual 30-kDa) PEG molecule. BAY 1025662 is the 60-kDa PEG moiety used for PEGylation of BAY 94-9027. This study investigated the pharmacokinetic (PK) properties, distribution, and excretion of BAY 1025662 in rats in order to predict estimated 60-kDa PEG PK properties in patients. Plasma concentrations in male rats after a single 11-mg/kg intravenous dose of BAY 1025662 (approximating the cumulative PEG-60 exposure in patients during 30 years of BAY 94-9027 treatment) decreased with an initial half-life of 119 h (5 days) in the interval of 114-336 h post administration. Single-dose mass balance studies using radiolabeled BAY 1025662 ([prop-14C]BAY 1025662) showed that 30.4% of radioactivity was excreted within 1 week and 79.1% by Day 168 (primarily in urine). The terminal half-life of radioactivity elimination was approximately 24 days in blood and plasma and was 31-68 days in the majority of other organs up to Day 168. Elimination was nearly complete at the end of the experiment on Day 168; only ~4% of residual radioactivity was present in the animal body. There was no irreversible binding of radioactivity to any tissues and no penetration of the blood-brain barrier. Based on these results, very low steady-state concentrations of 60-kDa PEG were predicted in patients treated with BAY 94-9027, and the validity of these predictions was supported by clinical studies in which almost all 179 patients receiving BAY 94-9027 for prophylaxis had undetectable PEG in plasma for up to >5 years; those with detectable PEG levels demonstrated concentrations within the predicted range. These combined preclinical and clinical observations suggest that excretion processes are in place for high-molecular-weight PEGs such as the PEG-60 moiety used in BAY 94-9027.

Characterization of peak capacity of microbore liquid chromatography columns using gradient kinetic plots.

The performance of micro-liquid chromatography columns with an inner diameter of 0.3mm was investigated on a dedicated micro-LC system for gradient elution. Core-shell as well as fully porous particle packed columns were compared on the basis of peak capacity and gradient kinetic plot limits. The results for peak capacity showed the superior performance of columns packed with sub-2µm fully porous particles compared to 3.0µm fully porous and 2.7µm core-shell particles within a range of different gradient time to column void time ratios. For ultra-fast chromatography a maximum peak capacity of 16 can be obtained using a 30s gradient for the sub-2µm fully porous particle packed column. A maximum peak capacity of 121 can be achieved using a 5min gradient. In addition, the influence of an alternative detector cell on the basis of optical waveguide technology and contributing less to system variance was investigated showing an increased peak capacity for all applied gradient time/column void time ratios. Finally, the influence of pressure was evaluated indicating increased peak capacity for maximum performance whereas a limited benefit for ultra-fast chromatography with gradient times below 30s was observed.

Micro-liquid chromatography mass spectrometry for the analysis of antineoplastic drugs from wipe samples.

A fast quantification method for the determination of 11 antineoplastic drugs from wipe samples was developed using micro-scale liquid chromatography in combination with tandem mass spectrometry. The extraction efficiency from the wipes has been investigated using different extraction solvents. The results indicate that a mixture of 70/30 water/isopropanol (v/v) acidified with 0.1 % formic acid is suitable to desorb the antineoplastic drugs with sufficient recovery between 80 and 120 %. Compared to conventional liquid chromatography, the total analysis time can be reduced to 2.25 min using a 50 × 0.3 mm column at a flow rate of 25 µL min-1. Ion source parameters as well as the injection volume were optimized to ensure the highest sensitivity. The results of method validation showed an instrumental limit of quantification between 0.0068 and 0.0488 ng mL-1 using an injection volume of 4.25 µL estimated by the signal to noise ratio. Moreover, the retention time repeatability was determined with a maximum relative standard deviation of 0.4 %. Graphical abstract Micro-LC-MS/MS separation of 11 antineoplastic drugs from wipe samples.

Characterization of the efficiency of microbore liquid chromatography columns by van Deemter and kinetic plot analysis.

The efficiency of miniaturized liquid chromatography columns with inner diameters between 200 and 300 µm has been investigated using a dedicated micro-liquid chromatography system. Fully porous, core-shell and monolithic commercially available stationary phases were compared applying van Deemter and kinetic plot analysis. The sub-2 µm fully porous as well as the 2.7 µm core-shell particle packed columns showed superior efficiency and similar values for the minimum reduced plate heights (2.56-2.69) before correction for extra-column contribution compared to normal-bore columns. Moreover, the influence of extra-column contribution was investigated to demonstrate the difference between apparent and intrinsic efficiency by replacing the column by a zero dead volume union to determine the band spreading caused by the system. It was demonstrated that 72% of the intrinsic efficiency could be reached. The results of the kinetic plot analysis indicate the superior performance of the sub-2 µm fully porous particle packed column for ultra-fast liquid chromatography.

Selectivity screening and subsequent data evaluation strategies in liquid chromatography: the example of 12 antineoplastic drugs.

Optimization of the chromatographic selectivity is the most important parameter if a separation is needed for the hyphenation of liquid chromatography with mass spectrometry. In mass spectrometry, this is necessary if the investigated analytes have identical mass transitions, like isomers or epimers. For the separation of the 12 most important antineoplastic drugs, a selectivity screening was performed using 20 columns and two organic modifiers and temperatures to find a suitable phase system in order to separate critical peak pairs. Therefore, an evaluation strategy was applied in form of a principal component analysis (PCA), selectivity factor, and overall selectivity comparison to find a suitable phase system. Some boundary conditions were defined to consider the specific requirements of tandem mass spectrometry. The results clearly indicated that the selectivity factor of the critical peak pairs increased using methanol at higher temperature.

Online and splitless NanoLC × CapillaryLC with quadrupole/time-of-flight mass spectrometric detection for comprehensive screening analysis of complex samples.

A novel multidimensional separation system based on online comprehensive two-dimensional liquid chromatography and hybrid high-resolution mass spectrometry has been developed for the qualitative screening analysis and characterization of complex samples. The core of the system is a consistently miniaturized two-dimensional liquid chromatography that makes the rapid second dimension compatible with mass spectrometry without the need for any flow split. Elevated temperature, ultrahigh pressure, and a superficially porous sub-3-µm stationary phase provide a fast second dimension separation and a sufficient sampling frequency without a first dimension flow stop. A highly loadable porous graphitic carbon stationary phase is employed in the first dimension to implement large volume injections that help countervailing dilution caused by the sampling process between the two dimensions. Exemplarily, separations of a 99-component standard mixture and a complex wastewater sample were used to demonstrate the performance of the dual-gradient system. In the second dimension, 30 s gradients at a cycle time of 1 min were employed. One multidimensional separation took 80-90 min (~120 min including extended hold and re-equilibration in the first dimension). This approach represents a cost-efficient alternative to online LC × LC strategies working with conventionally sized columns in the rapid second dimension, as solvent consumption is drastically decreased and analytes still are detectable at environmentally relevant concentrations.