An integrated outsourcing practice of nonclinical LC-MS bioanalysis and toxicokinetics at Novartis small molecule drug development.

With decommissioning of internal regulated bioanalytical (BA) and toxicokinetic (TK) capabilities, Novartis has relied on external service providers (ESPs) for all nonclinical LC-MS BA and majority of the associated TK work since 2017. This paper outlines an integrated outsourcing practice of the Novartis nonclinical LC-MS BA/TK group, which covers the roles and responsibilities of Novartis nonclinical LC-MS BA/TK expert scientific monitors, selection of ESPs for Novartis nonclinical LC-MS BA/TK studies, qualification of BA/TK ESPs, study conduct and completion, ESP oversight and evaluation, issue mitigation, and future perspectives.

Dixon's Q-test and Student's t-test to assess analog internal standard response in nonregulated LC-MS/MS bioanalysis.

Aim: In bioanalytical assays, analyte response is normalized to an internal standard response. When the internal standard works well, it compensates for processing and detection variability. However, in case the internal standard introduces additional variability, due to addition errors or other issues, scientists need to identify this. Results: A new method, using a Q-test for outliers and a t-test to compare internal standard response from different sample types, is applied to 15 cases. The results show that the Q-test/t-test, which uses confidence level rather than arbitrary cut-points, is more discerning of deviations compared with widely used methods. Conclusion: This work may improve the quality of and rationale for the internal standard response monitoring method.

Bioanalytical Challenges in Support of Complex Modalities of Antibody-Based Therapeutics.

Antibody-based therapeutic classes are evolving from monoclonal antibodies to antibody derivatives with complex structures to achieve advanced therapeutic effect. These antibody derivatives may contain multiple functional domains and are often vulnerable to in vivo biotransformation. Understanding the pharmacokinetics of these antibody derivatives requires a sophisticated bioanalytical approach to carefully characterize the whole drug and each functional domain with respect to quantity, functionality enabled by biotransformation, and corresponding immune responses. Ligand binding assays and liquid chromatography-mass spectrometry assays are predominantly used in bioanalytical support of monoclonal antibodies and are continuously used for antibody derivatives such as antibody drug conjugate and bispecific antibodies. However, they become increasingly cumbersome in coping with increased complexity of drug modality and associated biotransformation. In this mini-review, we examined the current pharmacokinetic assays in the literature for antibody drug conjugate and bispecific antibodies, and presented our view of promising bioanalytical technologies to address the distinct bioanalytical needs of complex modalities.

Development and validation of an LC-MS/MS method for the quantitative analysis of the adenosine A2a receptor antagonist NIR178 and its monohydroxy metabolite in human plasma: Application to clinical pharmacokinetics.

We report a selective LC-MS/MS method for the simultaneous quantitative determinations of the adenosine A2a receptor antagonist NIR178 (NIR178) and its major metabolite NJI765 in human plasma. Sample preparation steps involved protein precipitation, sample evaporation and reconstitution using a plasma sample volume of 0.1 ml plasma. Separation was achieved in 10 min on an Acquity UPLC BEH C18 1.7 µm, 2.1 × 50 mm column heated at 60°C with a gradient elution at 0.6 ml/min mobile phase made of water and acetonitrile both acidified with 0.1% formic acid. The detection was performed in positive ion mode and quantification based on multiple reaction monitoring. The linear response range was 1.00-1,000 ng/ml using a 1/x2 weighting factor. The intra- and inter-day accuracies (bias %) and intra- and inter-day precisions (CV, %) obtained for NIR178 and NJI765 were within the acceptance criteria. The normalized NIR178 and NJI765 matrix factor calculated from six lots of normal, lipemic and hemolyzed plasmas ranged from 0.97 to 1.05. The normalized recoveries of both NIR178 and NJI765 compared with their internal standards were consistent and reproducible with a CV ≤8.0. This method was successfully applied to support pharmacokinetic studies in adult patients with cancer.

Application of tail vein serial microsampling for plasma or dried plasma spots in toxicokinetic assessment in rats using acetaminophen as the model compound.

In the current study, two groups of rats (five per group) were administered a single oral dose of 500 mg/kg acetaminophen. For toxicokinetic assessment, the Group 1 animals were bled via conventional sparse (two animals/time point) sublingual vein bleeding (~0.5 ml) with anesthesia, while the Group 2 animals were bled via serial tail vein microsampling (~0.075 ml) without anesthesia. All collected blood was processed for plasma. Each Group 2 plasma sample (~30 µl) was divided into 'wet' and 'dried' (dried plasma spots). All plasma samples were analyzed by LC-MS/MS for acetaminophen and its major metabolites acetaminophen glucuronide and acetaminophen sulfate. In addition, plasma and urine samples were collected for analysis of corticosterone and creatinine to assess stress levels. Comparable plasma exposure to acetaminophen and its two metabolites was observed in the plasma obtained via conventional sparse sublingual vein bleeding and serial tail vein microsampling and between the 'wet' and 'dried' plasma obtained by the latter. Furthermore, comparable corticosterone levels or corticosterone/creatinine ratios between the two groups suggested that serial microsampling without anesthesia did not increase the levels of stress as compared with conventional sampling with anesthesia, confirming the utility of microsampling for plasma or dried plasma spots in rodent toxicokinetic assessment.

Evaluation, identification and impact assessment of abnormal internal standard response variability in regulated LC-MS bioanalysis.

Internal standard (IS) plays an important role in LC-MS bioanalysis by compensating for the variability of the analyte of interest in bioanalytical workflow. Due to the complexity of biological sample compositions and bioanalytical processes, a certain level of IS response variability across a run or a study is anticipated. However, an extensive variability may raise doubts to the accuracy of the measured results and also suggest nonoptimal analytical method. In this current paper, recent publications and guidelines regarding IS response in LC-MS bioanalysis were thoroughly reviewed with focus on the evaluation, identification and impact assessment of 'abnormal' IS response variability. A systematic decision tree was proposed to facilitate investigation into abnormal IS response variability after each run.

Highly selective and sensitive LC-MS/MS quantification of a therapeutic protein in human serum using immunoaffinity capture enrichment.

We report the development, validation and application of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) bioanalytical method for the determination of a recombinant protein drug candidate, NVS001, in human serum. A unique surrogate peptide, IPAETTIYNR (IPA), was identified to distinguish NVS001 from its endogenous counterpart, i.e., the full length and the C-terminal of protein X in LC-MS/MS. The selection of IPA for the LC-MS/MS determination of NVS001 was supported by the absence of peak responses due to endogenous components in the LC-MS/MS chromatograms of the extracted blank human serum samples. The optimal chromatographic separation of IPA from the extracted matrix components was achieved on a Waters Cortecs C18 (100 × 2.1 mm, 2.7 µm) column using gradient elution with a run cycle time of approximately 7.5 min. The mobile phases were water containing 0.1% formic acid (mobile phase A) and acetonitrile containing 0.1% formic acid (mobile phase B). The method was validated for specificity, sensitivity, matrix effect, recovery, linearity, accuracy and precision, dilution integrity, and stability. The validated assay dynamic range was 10.0 to 1000 ng/mL using a 50 µL sample volume. The accuracy and precision for the LLOQ (10.0 ng/mL) sample results were within ±9.2% bias and ≤6.0% CV, respectively. From the intra-day and inter-day assay performance evaluations, the precision of the QC sample (30, 500 and 750 ng/mL) results were ≤3.5% CV and the accuracy within ±3.3% bias, respectively. An additional assessment of incurred sample reanalysis (ISR) was conducted to demonstrate the ruggedness and robustness of the assay method. The validated method was successfully implemented in support of a first-in-human study.

Quantitative analysis of clofazimine (Lamprene®), an antileprosy agent, in human dried blood spots using liquid chromatography-tandem mass spectrometry.

An LC-MS/MS method was developed and validated for bioanalysis of clofazimine in human dried blood spot (DBS) samples in support of a clinical study on multidrug-resistant tuberculosis in developing countries. The validated assay dynamic range was from 10.0 to 2000 ng/mL using a 1/8 inch DBS punch. The accuracy and precision of the assay were ±11.0% (bias) and ≤13.5% (CV) for the LLOQs (10.0 ng/mL) and ±15% (bias) and ≤15% (CV) for all other QC levels. The assay was proved to be free from the possible impact owing to spot size and storage temperature (e.g. at 60°C, ≤ - 60°C). The validated assay is well suited for the intended clinical study where conventional pharmacokinetic sample collection is not feasible.

ICP-MS determination of serum aluminum in monkeys subcutaneously administered an alhydrogel-formulated drug candidate.

AIM: To develop and validate an inductively coupled plasma-mass spectrometry method for quantitative bioanalysis of aluminum (Al) in monkey serum in support of a GLP TOX study with alhydrogel-formulated drug candidate. METHODS & RESULTS: The method was linear over a dynamic range of 10-1000 ng/ml using a 50-µl sample volume. The intra-/inter-run precision (%CV) of the quality control sample results were ≤7.9% (CV) and the accuracy (%bias) within ±11.0% across all quality control concentrations evaluated. Other validation parameters, including stability under various conditions, extraction recovery and matrix effect, all met the acceptance criteria. CONCLUSION: The validated method was successfully implemented for the quantitative analysis of Al in monkey serum to assess the systemic exposure to Al.

Combined Falling Drop/Open Port Sampling Interface System for Automated Flow Injection Mass Spectrometry.

The aim of this work was to demonstrate and to evaluate the analytical performance of a combined falling drop/open port sampling interface (OPSI) system as a simple noncontact, no-carryover, automated system for flow injection analysis with mass spectrometry. The falling sample drops were introduced into the OPSI using a widely available autosampler platform utilizing low cost disposable pipet tips and conventional disposable microtiter well plates. The volume of the drops that fell onto the OPSI was in the 7-15 µL range with an injected sample volume of several hundred nanoliters. Sample drop height, positioning of the internal capillary on the sampling end of the probe, and carrier solvent flow rate were optimized for maximum signal. Sample throughput, signal reproducibility, matrix effects, and quantitative analysis capability of the system were established using the drug molecule propranolol and its isotope labeled internal standard in water, unprocessed river water and two commercially available buffer matrices. A sample-to-sample throughput of ∼45 s with a ∼4.5 s base-to-base flow injection peak profile was obtained in these experiments. In addition, quantitation with minimally processed rat plasma samples was demonstrated with three different statin drugs (atorvastatin, rosuvastatin, and fluvastatin). Direct characterization capability of unprocessed samples was demonstrated by the analysis of neat vegetable oils. Employing the autosampler system for spatially resolved liquid extraction surface sampling exemplified by the analysis of propranolol and its hydroxypropranolol glucuronide phase II metabolites from a rat thin tissue section was also illustrated.

LC-MS/MS determination of a human mAb drug candidate in rat serum using an isotopically labeled universal mAb internal standard.

We report the application of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) bioanalytical method for the determination of a recombinant human immunoglobulin G1 (hIgG1), NVSMAb-1, in rat serum. A stable isotopically labeled universal monoclonal antibody (SILuMab), instead of stable isotopically labeled surrogate peptide, was employed as the internal standard. The internal standard was added to the sample matrix in the first step of the sample preparation process, which involved protein precipitation and pellet digestion. The digestion of the resulting pellet with trypsin was performed prior to analysis of surrogate peptides of both NVSMAb-1 and SILuMab using LC-MS/MS. Precipitation reagents (1% TCA in IPA, 75% MeOH and 14% PEG) and digestion conditions (50°C for 2h and 60°C for 0.5h) were evaluated by monitoring LC-MS/MS responses of GPS and VVS in the resulting sample extracts. Overall, the use of 1% TCA in IPA appeared to be more effective as compared to 75% methanol in protein precipitation and removal of unwanted matrix components, e.g., albumin, and more appealing than 14% PEG as it avoided additional steps that are necessary to remove PEG or reduce PEG to a negligible level. The yield (LC-MS/MS response) of GPS is less sensitive than VVS to the changes of digestion conditions (time and temperature). The results obtained using SILuMab over SIL surrogate peptide as the internal standard appeared unaffected by the suboptimal sample processing method. For the current assay, surrogate peptide GPSVFPLAPSSK (GPS) was selected as surrogate peptide over VVSVLTVLHQDWLNGK (VVS) for quantitative analysis of NVSMAb-1. The optimal chromatographic separation was achieved on a Waters Cortecs C18 (100×2.1mm, 2.7µm) column using gradient elution with a total cycle time of approximately 8min. The mobile phases were water containing 0.1% formic acid (mobile phase A) and acetonitrile containing 0.1% formic acid (mobile phase B). The current method was validated for specificity, sensitivity, matrix effect, recovery, linearity, accuracy and precision, dilution integrity, and stability. The validated assay dynamic range was 10-5000µg/mL using 20µL of rat serum. The accuracy and precision for the LLOQs (10µg/mL) were within ±6.0% bias and ≤6.5% CV, respectively. From the intra-day and inter-day assay performance evaluations, the precision of the other QC sample (30, 300, 2500 and 3750µg/mL) results were ≤6.8% CV and the accuracy within ±4.8% bias, respectively. Additional assessment of incurred sample reanalysis (ISR) was conducted to demonstrate the ruggedness and robustness of the assay method. The validated method was successfully implemented in support of a toxicity study in rats administered 30, 150 and 750mg/kg/week intravenous infusion and 150mg/kg/week subcutaneous injection of NVSMAb-1.

Pharmacokinetics, Distribution, Metabolism, and Excretion of Omadacycline following a Single Intravenous or Oral Dose of 14C-Omadacycline in Rats.

The absorption, distribution, metabolism, and excretion (ADME) of omadacycline, a first-in-class aminomethylcycline antibiotic with a broad spectrum of activity against Gram-positive, Gram-negative, anaerobic, and atypical bacteria, were evaluated in rats. Tissue distribution was investigated by quantitative whole-body autoradiography in male Long-Evans Hooded (LEH) rats. Following an intravenous (i.v.) dose of 5 mg/kg of body weight, radioactivity widely and rapidly distributed into most tissues. The highest tissue-to-blood concentration ratios (t/b) were observed in bone mineral, thyroid gland, and Harderian gland at 24 h post-i.v. dose. There was no evidence of stable accumulation in uveal tract tissue, suggesting the absence of a stable binding interaction with melanin. Following a 90 mg/kg oral dose in LEH rats, the highest t/b were observed in bone mineral, Harderian gland, liver, spleen, and salivary gland. The plasma protein binding levels were 26% in the rat and 15% to 21% in other species. Omadacycline plasma clearance was 1.2 liters/h/kg, and its half-life was 4.6 h; the steady-state volume of distribution (Vss) was 6.89 liters/kg. Major circulating components in plasma were intact omadacycline and its epimer. Consistent with observations in human, approximately 80% of the dose was excreted into the feces as unchanged omadacycline after i.v. administration. Fecal excretion was primarily the result of biliary excretion (∼40%) and direct gastrointestinal secretion (∼30%). However, urinary excretion (∼30%) was equally prominent after i.v. dosing.

Clinical disposition, metabolism and in vitro drug-drug interaction properties of omadacycline.

1. Absorption, distribution, metabolism, transport and elimination properties of omadacycline, an aminomethylcycline antibiotic, were investigated in vitro and in a study in healthy male subjects. 2. Omadacycline was metabolically stable in human liver microsomes and hepatocytes and did not inhibit or induce any of the nine cytochrome P450 or five transporters tested. Omadacycline was a substrate of P-glycoprotein, but not of the other transporters. 3. Omadacycline metabolic stability was confirmed in six healthy male subjects who received a single 300 mg oral dose of [14C]-omadacycline (36.6 µCi). Absorption was rapid with peak radioactivity (∼610 ngEq/mL) between 1-4 h in plasma or blood. The AUClast of plasma radioactivity (only quantifiable to 8 h due to low radioactivity) was 3096 ngEq h/mL and apparent terminal half-life was 11.1 h. Unchanged omadacycline reached peak plasma concentrations (∼563 ng/mL) between 1-4 h. Apparent plasma half-life was 17.6 h with biphasic elimination. Plasma exposure (AUCinf) averaged 9418 ng h/mL, with high clearance (CL/F, 32.8 L/h) and volume of distribution (Vz/F 828 L). No plasma metabolites were observed. 4. Radioactivity recovery of the administered dose in excreta was complete (>95%); renal and fecal elimination were 14.4% and 81.1%, respectively. No metabolites were observed in urine or feces, only the omadacycline C4-epimer.

Compound Property Optimization in Drug Discovery Using Quantitative Surface Sampling Micro Liquid Chromatography with Tandem Mass Spectrometry.

Surface sampling micro liquid chromatography tandem mass spectrometry (SSµLC-MS/MS) was explored as a quantitative tissue distribution technique for probing compound properties in drug discovery. A method was developed for creating standard curves using surrogate tissue sections from blank tissue homogenate spiked with compounds. The resulting standard curves showed good linearity and high sensitivity. The accuracy and precision of standards met acceptance criteria of ±30%. A new approach was proposed based on an experimental and mathematical method for tissue extraction efficiency evaluation by means of consecutively sampling a location on tissue twice by SSµLC-MS/MS. The observed extraction efficiency ranged from 69% to 82% with acceptable variation for the test compounds. Good agreement in extraction efficiency was observed between surrogate tissue sections and incurred tissue sections. This method was successfully applied to two case studies in which tissue distribution was instrumental in advancing project teams' understanding of compound properties.

Utilization of Stable Isotope Labeling to Facilitate the Identification of Polar Metabolites of KAF156, an Antimalarial Agent.

Identification of polar metabolites of drug candidates during development is often challenging. Several prominent polar metabolites of 2-amino-1-(2-(4-fluorophenyl)-3-((4-fluorophenyl)amino)-8,8-dimethyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)ethanone ([(14)C]KAF156), an antimalarial agent, were detected in rat urine from an absorption, distribution, metabolism, and excretion study but could not be characterized by liquid chromatography-tandem mass spectrometry (LC-MS/MS) because of low ionization efficiency. In such instances, a strategy often chosen by investigators is to use a radiolabeled compound with high specific activity, having an isotopic mass ratio (i.e., [(12)C]/[(14)C]) and mass difference that serve as the basis for a mass filter using accurate mass spectrometry. Unfortunately, [(14)C]KAF156-1 was uniformly labeled (n = 1-6) with the mass ratio of ∼0.1. This ratio was insufficient to be useful as a mass filter despite the high specific activity (120 µCi/mg). At this stage in development, stable isotope labeled [(13)C6]KAF156-1 was available as the internal standard for the quantification of KAF156. We were thus able to design an oral dose as a mixture of [(14)C]KAF156-1 (specific activity 3.65 µCi/mg) and [(13)C6]KAF156-1 with a mass ratio of [(12)C]/[(13)C6] as 0.9 and the mass difference as 6.0202. By using this mass filter strategy, four polar metabolites were successfully identified in rat urine. Subsequently, using a similar dual labeling approach, [(14)C]KAF156-2 and [(13)C2]KAF156-2 were synthesized to allow the detection of any putative polar metabolites that may have lost labeling during biotransformations using the previous [(14)C]KAF156-1. Three polar metabolites were thereby identified and M43, a less polar metabolite, was proposed as the key intermediate metabolite leading to the formation of a total of seven polar metabolites. Overall this dual labeling approach proved practical and valuable for the identification of polar metabolites by LC-MS/MS.

Disposition and metabolism of [(14)C] Sacubitril/Valsartan (formerly LCZ696) an angiotensin receptor neprilysin inhibitor, in healthy subjects.

1. Sacubitril/valsartan (LCZ696) is an angiotensin receptor neprilysin inhibitor (ARNI) providing simultaneous inhibition of neprilysin (neutral endopeptidase 24.11; NEP) and blockade of the angiotensin II type-1 (AT1) receptor. 2. Following oral administration, [(14)C]LCZ696 delivers systemic exposure to valsartan and AHU377 (sacubitril), which is rapidly metabolized to LBQ657 (M1), the biologically active neprilysin inhibitor. Peak sacubitril plasma concentrations were reached within 0.5-1 h. The mean terminal half-lives of sacubitril, LBQ657 and valsartan were ∼1.3, ∼12 and ∼21 h, respectively. 3. Renal excretion was the dominant route of elimination of radioactivity in human. Urine accounted for 51.7-67.8% and feces for 36.9 to 48.3 % of the total radioactivity. The majority of the drug was excreted as the active metabolite LBQ657 in urine and feces, total accounting for ∼85.5% of the total dose. 4. Based upon in vitro studies, the potential for LCZ696 to inhibit or induce cytochrome P450 (CYP) enzymes and cause CYP-mediated drug interactions clinically was found to be low.

Differential Mobility Spectrometry Coupled with Multiple Ion Monitoring in Regulated LC-MS/MS Bioanalysis of a Therapeutic Cyclic Peptide in Human Plasma.

A differential mobility spectrometry (DMS) in combination with a multiple ion monitoring (MIM) method was developed and validated for quantitative LC-MS/MS bioanalysis of pasireotide (SOM230) in human plasma. Pasireotide, a therapeutic cyclic peptide, exhibits poor collision-induced dissociation (CID) efficiency for multiple reaction monitoring (MRM) detection. Therefore, in an effort to increase the overall sensitivity of the assay, a DMS-MIM approach was explored. By selecting the most abundant doubly charged precursor ion in both the Q1 and Q3 of the mass analyzer in MIM and combining the DMS capability to significantly reduce the high matrix/chemical background noise, this new LC-DMS-MIM method overcomes the sensitivity challenge in the typical MRM method due to poor CID fragmentation of the analyte. Human plasma was spiked with pasireotide with concentrations in the range 0.01-50 ng/mL. Weak cation-exchange solid-phase extraction was employed for sample preparation. The sample extracts were analyzed with a SCIEX QTRAP 6500 system equipped with an ESI source and DMS device. The separation voltage and compensation voltage of the DMS and other parameters of the MS system were optimized to maximize signal responses. The performance of the LC-DMS-MIM assay for quantitative analysis of pasireotide in human plasma was evaluated and compared to those obtained via LC-MRM and LC-MIM without DMS. Overall, the assay sensitivity with DMS-MIM was approximately 5-fold better than that observed in MRM or MIM without DMS. The assay was validated with accuracy (% bias) and precision (% CV) of the QC results at eight concentration levels (0.01, 0.02, 0.05, 0.15, 0.3, 1.5, 15, and 37.5 ng/mL) evaluated ranging from -4.8 to 5.0% bias and 0.7 to 8.6% CV for the intraday and interday runs. The current LC-DMS-MIM workflow can be expanded to quantitative analysis of other molecules that have poor fragmentation efficiency in CID.

Quantitative analysis of factor P (Properdin) in monkey serum using immunoaffinity capturing in combination with LC-MS/MS.

AIM: Factor P (Properdin), an endogenous glycoprotein, plays a key role in innate immune defense. Its quantification is important for understanding the pharmacodynamics (PD) of drug candidate(s). RESULTS: In the present work, an immunoaffinity capturing LC-MS/MS method has been developed and validated for the first time for the quantification of factor P in monkey serum with a dynamic range of 125 to 25,000 ng/ml using the calibration standards and QCs prepared in factor P depleted monkey serum. The intra- and inter-run precision was ≤7.2% (CV) and accuracy within ±16.8% (%Bias) across all QC levels evaluated. Results of other evaluations (e.g., stability) all met the acceptance criteria. CONCLUSION: The validated method was robust and implemented in support of a preclinical PK/PD study.