High-throughput untargeted screening of biotherapeutic macromolecules in equine plasma by UHPLC-HRMS/MS: Application to monoclonal antibodies and Fc-fusion proteins for doping control.

Many innovative biotherapeutics have been marketed in the last decade. Monoclonal antibodies (mAbs) and Fc-fusion proteins (Fc-proteins) have been developed for the treatment of diverse diseases (cancer, autoimmune diseases, and inflammatory disorders) and now represent an important part of targeted therapies. However, the ready availability of such biomolecules, sometimes characterized by their anabolic, anti-inflammatory, or erythropoiesis-stimulating properties, raises concerns about their potential misuse as performance enhancers for human and animal athletes. In equine doping control laboratories, a method has been reported to detect the administration of a specific human biotherapeutic in equine plasma; but no high-throughput method has been described for the screening without any a priori knowledge of human or murine biotherapeutic. In this context, a new broad-spectrum screening method involving UHPLC-HRMS/MS has been developed for the untargeted analysis of murine or human mAbs and related macromolecules in equine plasma. This approach, consisting of a "pellet digestion" strategy performed in a 96-well plate, demonstrates reliable performances at low concentrations (pmol/mL range) with high-throughput capability (≈100 samples/day). Targeting species-specific proteotypic peptides located within the constant parts of mAbs enables the "universal" detection of human biotherapeutics only by monitoring 10 peptides. As proof of principle, this strategy successfully detected different biotherapeutics in spiked plasma samples, and allowed, for the first time, the detection of a human mAb up to 10 days after a 0.12 mg/kg administration to a horse. This development will expand the analytical capabilities of horse doping control laboratories towards protein-based biotherapeutics with adequate sensitivity, throughput, and cost-effectiveness.

High-Throughput Equine Doping Controls on a Trapped Ion Mobility Quadrupole-Time-of-Flight Mass Spectrometer: Technical Considerations of dia/slice/prmPASEF Applied to the Long-Term Detection of Monoclonal Antibodies.

Data-independent acquisition (DIA) methods employing a scanning quadrupole were recently described across multiple platforms. These strategies display remarkable performances in untargeted proteomics studies thanks to rapid duty cycles, leading to ultrashort liquid chromatography gradients while maintaining enough data points per peaks when coupled to fast-scanning mass analyzer. In this article, we perform the evaluation of three data acquisition strategies named diaPASEF,slicePASEF, and prmPASEF on a trapped ion mobility spectrometry quadrupole-time-of-flight (TIMS-Q-TOF) mass spectrometer for high-throughput doping control screening analyses. We report that slicePASEF outperforms diaPASEF and is almost as sensitive as prmPASEF in detecting humanized monoclonal antibodies for several weeks in equine plasma after administration. We observed that diaPASEF is still providing the best performances in untargeted proteomics studies employing high amounts of input materials, which is linked with the high complexity of slicePASEF data and current processing algorithms.

A sDOE (Simple Design-of-Experiment) Approach for Parameter Optimization in Mass Spectrometry. Part 1. Parameter Selection and Interference Effects in Top-Down ETD Fragmentation of Proteins in a UHR-QTOF Instrument.

Design-of-experiment (DOE) approaches, originally conceived by Fischer, are widely applied in industry, particularly in the context of production for which they have been greatly expended. In a research and development context, DOE can be of great use for method development. Specifically, DOE can greatly speed up instrument parameter optimization by first identifying parameters that are critical to a given outcome, showing parameter interdependency where it occurs and accelerating optimization of said parameters using matrices of experimental conditions. While DOE approaches have been applied in mass spectrometry experiments, they have so far failed to gain widespread adoption. This could be attributed to the fact that DOE can get quite complex and daunting to the everyday user. Here we make the case that a subset of DOE tools, hereafter called SimpleDOE (sDOE), can make DOE accessible and useful to the Mass Spectrometry community at large. We illustrate the progressive gains from a purely manual approach to sDOE through a stepwise optimization of parameters affecting the efficiency of top-down ETD fragmentation of proteins on a high-resolution Q-TOF mass spectrometer, where the aim is to maximize sequence coverage of fragmentation events.

An innovative derivatization-free IC-MS/MS method for the detection of bisphosphonates in horse plasma.

Bisphosphonates are prohibited drugs according to Article 6 of the International Agreement on Breeding, Racing and Wagering of the International Federation of Horseracing Authorities (IFHA) and the International Equestrian Federation (FEI). These compounds are used for the treatment of lameness, navicular and bone diseases in horses and are divided into two groups: non-nitrogen-containing bisphosphonate drugs (e.g. clodronic acid) and nitrogen-containing bisphosphonate drugs (e.g. zoledronic acid). Their hydrophilic properties and the high affinity for the bone matrix make the control of their use quite difficult. Current analytical strategies to detect such compounds often rely on a solid phase extraction (SPE) followed by detection by means of UHPLC-MS/MS after methylation with chemical reagents. To improve the analysis throughput and to eliminate the need for chemical derivatization, an innovative 96-well SPE followed by ion chromatography-mass spectrometry was developed. Analyses are conducted on an ICS-6000 HPIC system coupled to a TSQ Altis™ (Thermo Scientific™). The use of a 96-well SPE allowed 5-fold sample increase and a 6-fold throughput improvement. While preliminary results conducted on horse plasma exhibited similar performances to the method for the detection of non-nitrogen-containing bisphosphonates, the analytical performances of nitrogen-containing bisphosphonates were greatly improved.