Projets de recherche

Code: 18B04AM 

The increasing number of biosimilars of the first generation EPOs (epoetin alfa and beta) produced all over the world raise the question of their detection. Due to small structural changes, for some EPO Biosimilars the identification criteria edicted by WADA for rEPO may not always be reached, which would render their use by athletes undetectable. Preliminary results have identified Hemax and Epotin authorized in Algeria and Jimaixin authorized in China as rEPOs with apparent molecular weights lower than the original epoetin alfa (Eprex) and closer to endogenous EPO. Thus identification using SDS/SAR-PAGE method could be problematic while their IEF profiles always remain very basic and distinct from endogenous EPO. To go further, evaluating detection from samples resulting of a real administration in healthy subjects is needed.

The objectives of this project are:
- to analyze blood and urine samples using IEF and SAR-PAGE methods according to the TD2014EPO and to compare identification capacities and establish the window of detection for each rEPO tested.
- to test complementary strategies to improve detection of EPO biosimilars: neuraminidase treatment that has been shown to improve the migration distance between rEPO and endogenous EPO as well as a 2-D separation gel approach (mixing IEF and SDS separation) will be tested by AFLD on samples that show a problematic rEPO identification.

In addition specific glycosylations of each Biosimilar will be characterized by LC-MS coupled to fluorescence.

Main Findings: 

Recombinant erythropoietin (rEPO) biosimilars are generic epoetin drugs developed following an expired patent. All licensed EPO biosimilars shall demonstrate the same saferty and efficacy for therapeutic use as the original drug but small structural differences compared to the reference product due to some variations in the production process can be accepted. After analyzing various EPO-biosimilars, three different kinds were selected for further characterization due to their slightly lower apparent molecular weight (MW) compared to the original epoetin alpha drug Eprex®.  Jimaixin™ authorized in China, and Hemax® and Epotin™ authorized in Algeria.

The aims of this research were:
i) to study the electrophorectic profiles obtained by IEF and SDS-PAGE of the three Biosimilars spiked in urine and plasma and to evaluate their identification following WADA's criterai (TD2014EPO),
ii) to test complementary strategies to improve detection of EPO biosimilars using a two-dimensional electrophoresis approach (SDS separation following IEF) and a neuraminidase treatment of the sample shown to increase the separation between recombinant and endogenous EPO by SDS-PAGE,
iii) to evaluate by mass spectrometry the specific N-glycosylation pattern of each biosimilar and compare with the original rEPO Eprex®. 

Experiments with spiked urine and plasma samples showed that samples spiked witht he Biosimilars were more challenging to identify for rEPO compared to Eprex in particular at low amount. Differences were seen according to the method of identification used and the biosimilar spiked. Epotin and Jimaixin were more difficult to identify by IEF compared to SDS-PAGE while it was the opposite for Hemax. The SDS-PAGE method applied to urine samples had the higher identificaiton rate considering the three biosimilars. Two-dimensional electrophoresis experiments did not improve the detection. This analysis proved complex to perform and no clear criteria could be used to identify rEPO.

Samples pre-treated with Neuraminidase gave promising results. Using the SDS-PAGE, the EPO bands were slightly broader compard to untreated samples. Neuraminidase-treated Dynepo could be used as a sparation marker between endogoenous and exogenous signals and this could improve the identification of low doses of biosimilars. By IEF, an interesting pattern for the biosimilars treated with neuraminidase was found. Using a 2-10 pH gradient gel was necessary to detect three thin additional bands inserted between the main EPO isoforms. This characteristic was observed for the three biosimilars while these bands were totally absent in non-spiked samples.

N-glycosylations of Eprex® and the biosimilars were identified by MALDI-TOF and adundance of the various glycan forms were compared.
All three biosimilars were enriched in bi and tri antennae forms while a decrease was observed in particular for the main glycan forms of Eprex® (tetraantennae tri- and tetra-sialylated forms). Hemax had the glycan profile the closest to Eprex® while Epotin™ and Jimaixin™ presented more loss of sialic acids. These results on N-glycan were in good agreement with the presence of additional basic isoforms in their IEF-PAGE profile.

In conclusion, even if these biosimilars may present some challenges at low concentration, current methods used in the anti-doping laboratories and if necessary an easy-to-implement neuraminidase pretreatment of the samples can assure detection of these compounds

Code: ISF17SC01KS

Serum samples from 35 individuals (25 males, 10 females) who were administered Human Growth Hormone (hGH) or placebo for 3 weeks and followed for a total of 4 weeks pre-administration, the 3-week administration period and a further 6 weeks post-administration will be analyzed on the SOMAscan biomarker discovery platform for the detection of potentially novel biomarkers of hGH abuse.

Deliverables. The primary aims of this study were the following:

• Identify proteins that respond to hGH treatment.

• Identify proteins that distinguish the hGH treated samples from the untreated samples.

a. The Deliverables provided to Client will include:

i. Normalized and, if applicable, calibrated data, measured in Relative Fluorescence Units (RFU), for each of the analytes organized on a per-sample basis, delivered in an Excel-compatible format;

ii. For each marker that reaches significance threshold: boxplots with longitudinal profiles (line plots) for individuals

iii. Spreadsheet (CSV) containing model statistics for all markers tested ordered by significance of fixed effect for time.

The following analyses are proposed:

Analysis 1: Identify protein biomarkers that show a time- and dose-dependent change in protein signal in response to hGH treatment. Baseline, During and Post treatment logRFU will be fit with mixed models to account for the correlated structure of the time series data and to evaluate the fixed effects of time. Pair-wise comparisons (to baseline) based on difference in least squares means within significant fixed effects will be performed to identify specific time-dependent differences.

Additional Analysis to be Provided:

Building of classification models for selection of proteins that exhibit “stable” differential expression between treatment groups and over time of study protocol.

Main findings

We reported quantitative blood circulating levels for over 1,300 proteins in serum from 35 volunteers, sampled at 22 time points. The study includes controls and three levels of doping, and samples were collected at baseline, during the doping phase and in a follow-up period. We identified 66 proteins that displayed strong association with doping. We characterized these proteins with respect to the influence of diurnal variation, sex, and genetic background of the study participants and computed multiple ROC curves for each protein, depending on the targeted period and dosage. We then built multi-variate classification models and showed that models with an AUC of up to 93.3% can be constructed using a Random Forest approach with 66 proteins, and 82.2% using glmnet.lasso with stability selection based on five protein markers alone. Furthermore, we evaluated the 66 proteins on their potential as a doping biomarker based on the requirement that they do not identify any false positive, using the percentage of doped individuals that have been identified at least once as a quality criterion. Finally, we tested the performance of selected sets of composite markers. Using 5 markers and aiming at a zero false positive rate, around 25-35% of all doped samples could be identified in a real-world scenario. In a second multi-marker analysis we evaluated the performance of six multi-marker models under more realistic conditions. Table 4 provides the top performing models, as a function of the selection criteria. The provided supporting data and plots can now be used to further develop and select the most suitable candidate markers for development of targeted markers.

Code: ISF17A15ZG

our objectives are:

- to reproduce an ABT experiment similar to what athletes are susceptible to perform (small volume of blood: 200ml) and to compare in the same assay reinfusion of refrigerated blood (stored less than one month) or reinfusion of cryopreserved blood. A group of subjects that will donate blood but will not be reinfused will also be integrated as controls.

- to focus on the characterization of small cells (RBC) and microparticles before and after ABT to identify morphological and cell surface markers changes, as well as changes in protein content by proteomic analyses of microparticles.

- to analyse the structural and antigenic properties of band 3, the major protein of the red cell membrane.

We will :

(i) determine if these techniques can efficiently identify an ABT of small volume. And if similar modifications are identified when blood was conserved refrigerated or frozen.

(ii) determine the window of detection of an ABT of small volume with these tests..

Code: ISF17A32DE

To explore the detection capability of rEPO administration from a dried blood spot.  Primarily, the immature reticulocyte surface protein CD71 will be analyzed in a longitudinal manner to evaluate changes following EPO administration.  Secondarily, we will seek to optimize direct detection of rEPO from a single or multiple dried blood spots using conventional SAR-PAGE techniques.  Importantly, the ability to detect ESA abuse from a dried blood spot, whether directly or via longitudinal means (i.e. CD71+ cells), would be critical for the future of doping control testing. 

Code: 17A20LM 

Urine samples collected worldwide for anti-doping testing are not always shipped in refrigerated conditions from collection sites to WADA accredited laboratories, in particular when several transport days are required. Commensal urethral microbiota, urinary pathogens and environmental bacteria may contaminate urine and the enzymatic activity by microbial contamination can cause severe modifications to the excreted compounds, in particular to doping-relevant peptides such as hormones and growth factors. Since delays are inevitable and sample refrigeration is not always possible and effective, sound methods for preserving and stabilizing urine samples are desirable.
This Project involves the use of Volumetric Absorptive Microsampling (VAMSTM) and Dried Urine Spot (DUS) strategies for the collection of dried microsamples processed by means of streamlined workflows to be developed ad hoc, for high-throughput LC-MS/MS analysis of several peptide hormones and growth factors (i.e. GHRP-1, GHRP-2, GHRP-6, hexarelin, alexamorelin, triptorelin, AOD9064, CJC-1293, desmopressin, TB-500, hCG and ACTH). DUS and VAMS approaches represent promising alternatives to the procedures currently performed by anti-doping laboratories, allowing analyte stabilization by water loss and the consequent broadening of their detection window. This innovative sampling produces logistics savings due to the small transported volume (by air shipments and through customs) and to the possibility to keep the specimens at room temperature, with significant implications on overall analysis cost. 
The ultimate goal of this project is to establish and validate feasible but reliable protocols for the collection of urine microvolumes, stably storable and shippable with no particular precautions. As a proof of concept, instrumental analytical methods will be developed, validated and applied for the stability assessment of several peptide hormones and grow factors in dried urine microsamples stored at different conditions. In parallel, stability will be assessed in classic fluid urine stored at temperature-controlled conditions and thorough comparisons with dried microsamples will be carried out.

Main Findings: 

•    Innovative microsampling and pretreatment procedures based on dried matrices (DUS and urine VAMS) were optimised for the application to urine specimens for anti-doping purposes. Important experimental parameters were studied and specifically optimised. 
•    Original MS, HRMS and FT-IR methods were used for peptide chemical identity confirmation; then LC-MS/MS and LC-HRMS methods were developed for the simultaneous analysis of the peptides of interest in dried urine microsamples. After suitable study of the experimental conditions, the final methods provided good, solid performance within relatively short run times.
•    The LC-MS/MS and LC-HRMS methods were validated according to current guidelines, with good results for all assays and all analytes, and with similar or better performances than those of comparable published methods.
•    The microsampling, pretreatment and analysis workflow was successfully applied to the study of peptide stability in dried matrices.
•    Mid-term stability assays were carried out, both on dried and fluid urine-based matrices, with outstanding results. In fact, DUS and urine VAMS were remarkably stable, even though they were kept at RT, with all studied peptides recovered in the 80-95% range at the end of the study period (3 months). The stability of the chosen peptides, which are known to be prone to degradation under common storage conditions, was greatly enhanced in comparison to fluid urine stored at freezing (-20°C) and ultra-freezing (-80°C) temperatures. Analyte loss in fluid urine at -80°C was regularly 5-10% larger at study end than the loss in dried matrices, with widely worse losses (up to 35% more) for fluid urine kept at -20°C.
The use of innovative microsampling media offers significantly interesting perspectives towards the development of engineered, highly reliable devices.

Code: ISF17D20AM

Our objectives are:
- to reproduce an ABT experiment similar to what athletes are susceptible to perform (small volume of blood: 200ml) and to compare in the same assay reinfusion of refrigerated blood (stored less than one month) or reinfusion of cryopreserved blood. A group of subjects that will donate blood but will not be reinfused will also be integrated as controls. 
- to focus on the characterization of microparticles before and after ABT:  to identify morphological and cell surface markers changes, as well as changes in protein content by proteomic analyses of microparticles. 

We will :
(i) determine if these techniques can efficiently identify an ABT of small volume. And if similar modifications are identified when blood was conserved refrigerated or frozen. 
(ii) determine the window of detection of an ABT of small volume with these tests..

Code: ISF17E07FB 

The rationale of our proposal is based on the hypothesis that gene transfer with AAVs will leave an immunological and genetic footprint in any human subject clearly distinguishable from a naturally occurring AAV infection with the wild-type virus. In the context of the detection of gene doping, the main objective of this project is to develop a two-step method for the detection of AAV-mediated gene doping based on the combination of immunological and molecular tools. This general objective can be divided in the following specific aims, describing the two-step approach proposed for the identification of subjects who underwent AAV-based gene doping. Specifically: 2.1. Profiling of the anti-AAV antibody responses. A sensitive, pan-AAV antibody assay will be developed, validated, and streamlined to distinguish humoral responses deriving from natural exposure to AAV from gene doping with AAV vectors. Thus, this assay will permit to establish a baseline to be used as reference value to discriminate between subjects positive or negative for AAV-gene doping. 2.2. Detection of AAV genomes in blood/PBMCs by means of target capture NGS. To this end, a panel will be designed containing at least 4 million capture probes specific for at least 100 genome elements, such as viral ITs, regulatory sequences and different doping genes involved in improvement of muscle strength and performance.

Main Findings

The main findings are not available due to the sensitivity of the information and results developed in this project.

Code: T17R03DC 

Athletes are required to provide urine samples, as part of an anti-doping programme, in a doping control station and the sample collection is witnessed by a Doping Control Officer. As part of the process, a Doping Control Form (example attached) is completed and the samples are transported to the Drug Control Centre (DCC) at King's College London. The part of the Doping Control Form received by the DCC contains no information about the athlete (e.g. name, address). It contains information about sample volume, specific gravity, event code, and any medication declared to have been used by the athlete. A box on the form indicates whether the athlete has consented for his/her samples to be used for the research purposes. Once the athlete’s sample arrives in the DCC, it is split and undergoes the sample preparation steps (e.g. solid
phase extraction) prior to analysis. The usual method for sample analysis is liquid chromatography-high resolution mass spectrometry (LC-HRMS) (please see Musenga and Cowan, Use of ultra-high pressure liquid chromatography coupled to high resolution mass spectrometry for fast screening in high throughput doping control. J. Chromatography A 2013;1288:82-95). In our research project, funded by the World Anti-Doping Agency (WADA), we would like to investigate the potential application of supercritical fluid chromatography-mass spectrometry (SFCMS) in the analysis of doping control samples. We aim to compare the performance of SFC-MS with LC-HRMS. In order to do our research, once the samples have been analysed by LC-HRMS, we will re-analyse them by SFCMS and compare two methods by results obtained. We plan to do our research with approximately 1000 samples and our research refers to secondary data analysis. The DCC is required by WADA to analyse all samples received by LC- HRMS no matter if the athlete had given research consent or not. However, we will only analyse samples by SFC-MS with the research consent provided by the athlete on the Doping Control Form. As a laboratory accredited by the WADA, we have to comply with strict criteria in the use of athlete samples for research. These include: waiting at least 3 months after the anti-doping analytical report has been issued; transference of the sample from the coded bottle to a separate container so that it is impossible for the identity of the athlete who provided the sample to be discoverable by anyone.

Main Findings:

This study clearly demonstrated the applicability of SFC-MS for routine antidoping analysis. We analysed approximately 3,000 samples (3 x 1,000) in total using SFC-MS equipment from three different vendors to investigate whether this technology is applicable for routine anti-doping analysis. Our work demonstrated SFC-MS to be a robust analytical technique. It was demonstrated that it may be considered as a complementary technique to LC-MS, which is readily available in anti-doping laboratories. Three SFC-MS instrument manufacturers (Agilent Technologies, Shimadzu UK Ltd and Waters Corporation) generously contributed to our study to investigate the routine applicability of SFC-MS in doping control. All three vendors dedicated instrument time and resource for the project. A large number of anti-doping samples and QCs were delivered to each manufacturer. We worked closely with each manufacturer in agreeing the method of operation and helped with the analysis of the samples, data review and undertook the independent statistical analysis of the data.
All three instruments appeared to meet the requirement for robustness needed for routine use. Most of our compounds showed excellent chromatography. There were only a few examples of co-elution due to interference with endogenous compounds that could benefit from modification of the chromatographic conditions. Retention times were generally remarkably stable over the analysis time. We demonstrated some software features that would facilitate data review especially with the software from one manufacturer. Despite using a “dilute and inject” approach, the SFC columns were found to be stable over more than 1,200 injections of samples, standards and quality controls on three different systems. In general, SFC-MS was found to be similar to LC-MS for routine use but with the advantage of complementarity providing additional information of the chromatographic properties of the analyte thereby further confirming the identity of a doping agent.

Code: T17R02TK

Ultra-high performance supercritical fluid chromatography-mass spectrometry (UHPSFC-MS) could represent in the near future an orthogonal technique to LC-MS and GC-MS for routine doping analysis. This technique now benefits from a broader recognition and interest, thanks to new technological improvements and the recent commercialization of new platforms.

The aim of this project is to evaluate the potential of UHPSFC-MS/MS for screening and confirmation purposes in routine anti-doping analysis. This aim will be achieved through a comprehensive robustness study (different columns chemistries, column batches, instruments and laboratories) by selecting representative compounds of major classes of prohibited substances (approx. 50 compounds) fortified in urine samples. Various aspects will be evaluated such as the stability of retention times and the inter-batch variability of SFC columns. Then, an inter-laboratory study as well as an inter-instrument study will be performed with other academic and/or industrial laboratories equipped with the same brand of SFC instrument (Waters Acquity UPC² system) or equipped with other brands of UHPSFC instruments, including Agilent and Shimadzu, to evaluate the ruggedness of the SFC-MS/MS method.

Main Findings

The aim of this study was to assess the interlaboratory reproducibility of ultra-high performance supercritical fluid chromatography coupled with tandem mass spectrometry method for routine antidoping analyses.To do so, a set of 21 doping agents, spiked in urine and analyzed after dilute and shoot treatment, was used to assess the variability of their retention times between four different laboratories, all equipped with the same chromatographic system and with the same ultra-high performance supercritical fluid chromatography stationary phase chemistry. The average relative standard deviations (RSD%) demonstrated a good reproducibility of the retention times for 19 out of 21 analytes, with RSD% values below 3.0%. Only for two substances, namely fenbutrazate and niketamide, the retention was not repeatable between laboratories, with RSD% of approximately 15% in both cases. This behaviour was associated with (a) the low organic modifier percentage (around 2-4%) in the mobile phase at the corresponding retention times, and (b) the influence of the system volume on poorly retained analytes. An analysis on seven “blind” urines was subsequently carried out in the same four laboratories. In these blind samples, either one, two, or none of the 21 doping agents previously analyzed were present at an unknown concentration. Each laboratory had to perform the identification of the compounds in the samples and estimate their concentrations. All laboratories assigned all target analytes correctly in all blind urine samples and provide a comparable estimation of their concentrations.

Code: 17D07MM

Azole compounds were introduced into the therapy of fungal infections of humans in the early 1970s. The primary target of azole compounds is the CYP51, the enzyme involved in the synthesis of ergosterol from lanosterol in the fungal cell membranes. However, azoles have a potential to interact also with other cytochrome P-450-dependent enzymes, leading to toxicologically relevant changes in the liver and endocrine system. Indeed, depending on their effects on specific enzymes inhibited they can cause (i) a reduced formation of either androgens or estrogens, and/or (ii) alterations in the elimination rate of xenobiotics. CYP19 (aromatase) is one of the cytochrome P450 enzyme inhibited by azoles. Several azole fungicides disrupt normal aromatase function leading to a decrease in estrogens formation, and for this reason these agents are not prescribed in pregnancy and are instead used in the management of advanced estrogen-responsive breast tumors in postmenopausal women. Reduction of estrogen levels by CYP19 inhibition is the working principle of anti-aromatase agents. These agents together with other anti-oestrogenic compounds, are included in the section S4 “Hormone and Metabolic Modulators” of the WADA prohibited List. Azole antifungals are instead included in the WADA TDEAAS as confounding factors. In a previous WADA-funded project, focused on the evaluation of the alterations provoked by the administration of azole antifungals on the basal levels of the parameters that are part of the steroid profile, we have reported that both miconazole and fluconazole is capable of altering the levels of the endogenous steroids in a different manner. In this project we aim to extend the study by increasing the number of measurements, including more individual, different ethnicities, route of administration, doses, range of age and by evaluating whether similar effects can be caused also by other azole antifungals.