Olivier Heudi

Towards absolute quantification of therapeutic monoclonal antibody in serum by LC-MS/MS using isotope-labeled antibody standard and protein cleavage isotope dilution mass spectrometry.

Although LC-MS methods are increasingly used for the absolute quantification of proteins, the lack of appropriate internal standard (IS) hinders the development of rapid and standardized analytical methods for both in vitro and in vivo studies. Here, we have developed a novel method for the absolute quantification of a therapeutic protein, which is monoclonal antibody (mAb). The method combines liquid chromatography tandem mass spectrometry (LC-MS/MS) and protein cleavage isotope dilution mass spectrometry with the isotope-labeled mAb as IS. The latter was identical to the analyzed mAb with the exception that each threonine contains four (13)C atoms and one (15)N atom. Serum samples were spiked with IS prior to the overnight trypsin digestion and subsequent sample cleanup. Sample extracts were analyzed on a C18 ACE column (150 mm x 4.6 mm) using an LC gradient time of 11 min. Endogenous mAb concentrations were determined by calculating the peak height ratio of its signature peptide to the corresponding isotope-labeled peptide. The linear dynamic range was established between 5.00 and 1000 microg/mL mAb with accuracy and precision within +/-15% at all concentrations and below +/-20% at the LLOQ (lower limit of quantification). The overall method recovery in terms of mAb was 14%. The losses due to sample preparation (digestion and purification) were 72% from which about 32% was due to the first step of the method, the sample digestion. This huge loss during sample preparation strongly emphasizes the necessity to employ an IS right from the beginning. Our method was successfully applied to the mAb quantification in marmoset serum study samples, and the precision obtained on duplicate samples was, in most cases, below 20%. The comparison with enzyme-linked immunosorbent assay (ELISA) showed higher exposure in terms of AUC and Cmax with the LC-MS/MS method. Possible reasons for this discrepancy are discussed in this study. The results of this study indicate that our LC-MS/MS method is a simple, rapid, and precise approach for the therapeutic mAb quantification to support preclinical and clinical studies.

New microfluidic-based sampling procedure for overcoming the hematocrit problem associated with dried blood spot analysis.

Hematocrit (Hct) is one of the most critical issues associated with the bioanalytical methods used for dried blood spot (DBS) sample analysis. Because Hct determines the viscosity of blood, it may affect the spreading of blood onto the filter paper. Hence, accurate quantitative data can only be obtained if the size of the paper filter extracted contains a fixed blood volume. We describe for the first time a microfluidic-based sampling procedure to enable accurate blood volume collection on commercially available DBS cards. The system allows the collection of a controlled volume of blood (e.g., 5 or 10 μL) within several seconds. Reproducibility of the sampling volume was examined in vivo on capillary blood by quantifying caffeine and paraxanthine on 5 different extracted DBS spots at two different time points and in vitro with a test compound, Mavoglurant, on 10 different spots at two Hct levels. Entire spots were extracted. In addition, the accuracy and precision (n = 3) data for the Mavoglurant quantitation in blood with Hct levels between 26% and 62% were evaluated. The interspot precision data were below 9.0%, which was equivalent to that of a manually spotted volume with a pipet. No Hct effect was observed in the quantitative results obtained for Hct levels from 26% to 62%. These data indicate that our microfluidic-based sampling procedure is accurate and precise and that the analysis of Mavoglurant is not affected by the Hct values. This provides a simple procedure for DBS sampling with a fixed volume of capillary blood, which could eliminate the recurrent Hct issue linked to DBS sample analysis.

Laser diode thermal desorption-positive mode atmospheric pressure chemical ionization tandem mass spectrometry for the ultra-fast quantification of a pharmaceutical compound in human plasma.

An ultra-fast, reliable and sensitive analytical method enabling high-throughput quantitative analysis of pharmaceutical compounds in human plasma is described. The quantitative work was performed on one of our compound currently under clinical trial by employing a deuterated internal standard (IS). Plasma samples were treated on solid phase micro-extraction (SPME) plates prior their analysis by laser diode thermal desorption and atmospheric pressure chemical ionization coupled to tandem mass spectrometry (LDTD/APCI-MS/MS) in positive mode. The sample analysis run time was 10s as compared to the 7 min obtained for the validated LC-MS/MS method. The limit of quantification (LOQ) of the method was estimated at 1 ng/mL. The calibration graphs were linear with a regression coefficient R(2) > 0.997. The data of the partial validation show that LDTD/APCI-MS/MS assay was highly reproducible and selective. In addition, the deviations for intra and inter assay accuracy and precision data were within 15% at all quality control levels. The LDTD/APCI-MS/MS method was successfully applied to the analysis of clinical samples and the data obtained were consistent with those found with a validated LC-MS/MS assay. This work demonstrates that LDTD/APCI-MS/MS could be used for the ultra-fast and reliable quantitative analysis of pharmaceutical compounds in human plasma without using the separation step commonly associated with the LC-MS/MS assay.

Validation of an on-line solid-phase extraction method coupled to liquid chromatography–tandem mass spectrometry detection for the determination of Indacaterol in human serum

Indacaterol has been recently approved in Europe for the treatment of chronic obstructive pulmonary disease (COPD). In the present study, we have developed and validated a rapid and sensitive on-line solid phase extraction (SPE) method coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) detection for the determination of Indacaterol in human serum. The sample preparation involves the serum dilution with a 0.2% acetic acid solution prior to the on-line SPE on a mixed-mode cationic (MCX) polymer based sorbent. The samples were then eluted on a reversed phase column with a mobile phase made of acidified water and methanol and detection was performed by MS using electrospay ionization in positive mode. The analysis time between 2 samples was 7.0 min. Standard curves were linear over the range of 10.0 pg/mL (LLOQ) to 1000 pg/mL with correlation coefficient (r(2)) greater than 0.990. The method specificity was demonstrated in six different batches of human serum. Intra-run and inter-run precision and accuracy within ± 20% (at the LLOQ) and ± 15% (other levels) were achieved during a 3-run validation for quality control samples (QCs). The stability at room temperature (38 h) was determined and reported. In addition, the comparison between an off-line SPE procedure and our method gave equivalent results. The results of the present work demonstrated that our on-line SPE-LC-MS/MS method is rapid, sensitive, specific and could be applied to the quantitative analysis of Indacaterol in human serum samples. Our method effectively eliminated the tedious conditioning and rinsing steps associated with conventional off-line SPE and reduced the analysis time. The on-line SPE approach appears attractive for supporting the analysis of several hundreds of clinical samples.

Biodistribution and Metabolism Studies of Lipid Nanoparticle–Formulated Internally [3H]-Labeled siRNA in Mice

Absorption, distribution, metabolism, and excretion properties of a small interfering RNA (siRNA) formulated in a lipid nanoparticle (LNP) vehicle were determined in male CD-1 mice following a single intravenous administration of LNP-formulated [3H]-SSB siRNA, at a target dose of 2.5 mg/kg. Tissue distribution of the [3H]-SSB siRNA was determined using quantitative whole-body autoradiography, and the biostability was determined by both liquid chromatography mass spectrometry (LC-MS) with radiodetection and reverse-transcriptase polymerase chain reaction techniques. Furthermore, the pharmacokinetics and distribution of the cationic lipid (one of the main excipients of the LNP vehicle) were investigated by LC-MS and matrix-assisted laser desorption ionization mass spectrometry imaging techniques, respectively. Following i.v. administration of [3H]-SSB siRNA in the LNP vehicle, the concentration of parent guide strand could be determined up to 168 hours p.d. (post dose), which was ascribed to the use of the vehicle. This was significantly longer than what was observed after i.v. administration of the unformulated [3H]-SSB siRNA, where no intact parent guide strand could be observed 5 minutes post dosing. The disposition of the siRNA was determined by the pharmacokinetics of the formulated LNP vehicle itself. In this study, the radioactivity was widely distributed throughout the body, and the total radioactivity concentration was determined in selected tissues. The highest concentrations of radioactivity were found in the spleen, liver, esophagus, stomach, adrenal, and seminal vesicle wall. In conclusion, the LNP vehicle was found to drive the kinetics and biodistribution of the SSB siRNA. The renal clearance was significantly reduced and its exposure in plasma significantly increased compared with the unformulated [3H]-SSB siRNA.

Fast simultaneous quantitative analysis of FTY720 and its metabolite FTY720-P in human blood by on-line solid phase extraction coupled with liquid chromatography-tandem mass spectrometry.

Fingolimod (Gilenya; FTY720), has been recently approved for the treatment of multiple sclerosis in Europe and in the USA. In the present study, we have developed and validated a rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to simultaneously quantify FTY720 and FTY720-P in human blood. The sample preparation involves the sample dilution with a solution made of dimethylhexylamine (DMHA), ortho-phosphoric acid and methanol prior to the on-line solid phase extraction (SPE) on a C(18) cartridge. The samples were then eluted on a C(18) column with a gradient elution of DMHA solution and acetonitrile and analyzed by LC-MS/MS using electrospay ionization in positive mode. The analysis time between 2 samples was 7.5 min. Standard curves were linear over the ranges of 0.0800 ng/mL (LLOQ) to 16.0 ng/mL for FTY720 and 0.100 ng/mL (LLOQ) to 20.0 ng/mL for FTY720-P with correlation coefficient (r(2)) greater than 0.997. The method selectivities for FTY720 and FTY720-P were demonstrated in six different batches of human blood. Intra-run and inter-run precision and accuracy within ± 20% (at the LLOQ) and ± 15% (other levels) were achieved during a 3-run validation for quality control samples (QCs). In addition, stability data obtained during freeze-thaw (3 cycles), at room temperature (24h), and in an auto-sampler were determined and reported. The method robustness was demonstrated by the consistent data obtained by reanalyzing human blood samples for several clinical studies. In addition comparative data for FTY720 and FTY720-P were obtained between our current method and those of two available separate LC-MS/MS assays. The results of the present work demonstrated that our bioanalytical LC-MS/MS method is rapid, sensitive, specific and reliable for the simultaneous quantitative analysis of FTY720 and FTY720-P in human blood.

Quantitative analysis of maytansinoid (DM1) in human serum by on-line solid phase extraction coupled with liquid chromatography tandem mass spectrometry - Method validation and its application to clinical samples

A sensitive and specific method was developed and validated for the quantitation of maytansinoid (DM1) in human serum using on-line solid phase extraction (SPE)-liquid chromatography-tandem mass spectrometry (LC-MS/MS). Because DM1 contains a free thiol moiety, likely to readily dimerize or react with other thiol-containing molecules in serum, samples were pre-treated with a reducing agent [tris (2-carboxyethyl) phosphine] (TCEP) and further blocked with N-ethylmaleimide (NEM). The resulting samples were diluted with acetonitrile prior to the on-line solid phase extraction (SPE) on a C18 cartridge. A C18 (150×4.6mm ID 3μm particle size) column was used for chromatographic separation with a 10.0min HPLC gradient and DM1-NEM was detected in the selected reaction monitoring mode of a triple quadrupole mass spectrometer. DM1 concentrations were back-calculated from DM1-NEM amount found in the human serum samples. The quantitation range of the method was 0.200-200ng/mL when using 0.25mL serum. Within-run day precisions (n=6) were 0.9-4.4% and between-run day (3 days runs; n=18) precisions 2.5-5.6%. Method biases were between 3.5-14.5% across the whole calibration range. DM1-NEM exhibited sufficiently stability under all relevant analytical conditions and no DM1 losses from the ADC were observed. Finally, the assay was used for DM1 determination in human serum concentration after the intravenous administration of an investigational antibody drug conjugate (ADC) containing DM1 as payload.

Generic Hybrid Ligand Binding Assay Liquid Chromatography High-Resolution Mass Spectrometry-Based Workflow for Multiplexed Human Immunoglobulin G1 Quantification at the Intact Protein Level: Application to Preclinical Pharmacokinetic Studies

The quantitative analysis of human immunoglobulin G1 (hIgG1) by mass spectrometry is commonly performed using surrogate peptides after enzymatic digestion. Since some limitations are associated with this approach, a novel workflow is presented by hybridizing ligand binding assay (LBA) with liquid chromatography-high-resolution mass spectrometry (LC-HRMS) for hIgG1 quantification directly at the intact protein level. Different hIgG1s, including a [13C]-labeled version used as internal standard, were immuno-enriched from rat serum with a fully automated platform based on streptavidin coated tips and a biotinylated mouse anti-hIgG capture antibody targeting the fragment crystallizable region followed by overnight deglycosylation prior to LC-HRMS analysis. The proposed quantitative workflow utilized extracted ion chromatograms (XICs) from the nondeconvoluted full-scan MS spectrum. The assay was validated in terms of selectivity, sensitivity, accuracy/precision, carry-over, dilution linearity, and reproducibility. Consistent data between the conventional approach based on surrogate peptide analysis and our proposed workflow were obtained in vitro and in vivo with the advantage of a less extensive sample pretreatment. Multiplexing capabilities for simultaneous quantification of different hIgG1s within the same spiked sample were also exemplified. Altogether our results pave the way not only for the thorough application of intact hIgG1 quantification by LBA-LC-HRMS but also as a generic quantitative analytical method for other hIgG isotypes or next generation biotherapeutics.

Tetrazole-based deoxyamodiaquines: Synthesis, ADME/PK profiling and pharmacological evaluation as potential antimalarial agents

A series of new deoxyamodiaquine-based compounds was synthesized via the modified TMSN3-Ugi multi-component reaction and evaluated in vitro for antiplasmodial activity. The most potent compounds, 6b, 6c and 6j, showed IC50 values in the range of 6-77nM against chloroquine-resistant K1- and W2-strains of Plasmodium falciparum. In vitro ADME characterization of frontrunner compounds 6b and 6c indicates that these two compounds are rapidly metabolized and have a high clearance rate in human and rat liver microsomes. This result correlated well with an in vivo pharmacokinetics study, which showed low bioavailability of 6c in rats. Tentative metabolite identification was determined by LC-MS and suggested metabolic lability of groups attached to the tertiary nitrogen. Preliminary studies on 6b and 6c suggested strong inhibitory activity against the major CYP450 enzymes. In silico docking studies were used to rationalize strong inhibition of CYP3A4 by 6c. Full characterization and biological evaluation of the metabolites is currently underway in our laboratories.

The flexibility of a generic LC–MS/MS method for the quantitative analysis of therapeutic proteins based on human immunoglobulin G and related constructs in animal studies

An increasing demand of new analytical methods is associated with the growing number of biotherapeutic programs being prosecuted in the pharmaceutical industry. Whilst immunoassay has been the standard method for decades, a great interest in assays based on liquid chromatography tandem mass spectrometry (LC-MS/MS) is evolving. In this present work, the development of a generic method for the quantitative analysis of therapeutic proteins based on human immunoglobulin G (hIgG) in rat serum is reported. The method is based on four generic peptides GPSVFPLAPSSK (GPS), TTPPVLDSDGSFFLYSK (TTP), VVSVLTVLHQDWLNGK (VVS) and FNWYVDGVEVHNAK (FNW) originating from different parts of the fraction crystallizable (Fc) region of a reference hIgG1 (hIgG1A). A tryptic pellet digestion of rat serum spiked with hIgG1A and a stable isotope labeled protein (hIgG1B) used as internal standard (ISTD) was applied prior LC-MS/MS analysis. The upper limit of quantification was at 1000μg/mL. The lower limit of quantitation was for GPS, TTP and VVS at 1.00μg/mL whereas for FNW at 5.00μg/mL. Accuracy and precision data met acceptance over three days. The presented method was further successfully applied to the quantitative analysis of other hIgG1s (hIgG1C and hIgG1D) and hIgG4-based therapeutic proteins on spiked quality control (QC) samples in monkey and rat serum using calibration standards (Cs) prepared with hIgG1A in rat serum. In order to extend the applicability of our generic approach, a bispecific-bivalent hIgG1 (bb-hIgG1) and two lysine conjugated antibody-drug conjugates (ADC1 and ADC2) were incorporated as well. The observed values on spiked QC samples in monkey serum were satisfactory with GPS for the determination of bb-hIgG1 whereas the FNW and TTP peptides were suitable for the ADCs. Moreover, comparable mean concentration-time profiles were obtained from monkeys previously dosed intravenously with ADC2 measured against Cs samples prepared either with hIgG1A in rat serum (presented approach) or with the actual ADC2 in monkey serum (conventional approach). The results of this study highlight the great flexibility of our newly developed generic approach and that the choice of the surrogate peptide still remains critical when dealing with different matrix types or modalities.