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

Practical Approaches to Incurred Sample LC-MS/MS Reanalysis: Confirming Unexpected Results

Incurred sample reanalysis (ISR) is an important step in assuring the quality of an LC-MS/MS bioanalytical assay and the integrity of bioanalysis conduct. A conventional ISR involves analysis of at least 20 samples taken from an in vivo study a second time using the method that was described in pre-study validation and employed in generating the initial study sample results. However, this practice is sometimes inadequate to confirm bioanalytical results that are unexpected. The present report discuss several additional exploratory activities that were performed to confirm the unexpected plasma concentration-time results of NVP-1, an investigational drug candidate, observed in the plasma samples collected from patients in a phase II trial. These approaches include (1) LC-MS/MS reanalysis of the study samples after multiple freeze/thaw cycles followed by a short term bench top storage, (2) evaluation of additional MS/MS transitions in LC-MS/MS, (3) employment of different sample preparation procedure in LC-MS/MS and (4) study sample dilution using plasma samples from healthy volunteers. These procedures are practical and can be readily implemented to provide confirmatory LC-MS/MS bioanalysis of other small molecules

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

LC-MS/MS determination of Pasireotide (SOM230), a cyclic peptide, in monkey plasma

A novel liquid chromatographic method with tandem mass spectrometric detection (LC-MS/MS) for the determination of Pasireotide (SOM230) was developed and validated with dynamic ranges of 0.500 to 250 ng/mL using 0.0500 mL of monkey plasma. SOM230 and the internal standard, [M+6]SOM230, were extracted from monkey plasma via µElution SPE. The acidified sample matrix was loaded onto the preconditioned Waters WCX plate for further processing. The analyte was eluted from the SPE plate using freshly prepared elution solvent, followed by dilution and LC-MS/MS analysis. The optimal chromatographic separation was achieved on an Atlantis dC18 (50 x 2.1 mm, 5 µm particle size) column using gradient elution. The total analysis cycle time is approximately 3.5 min per injection. Trifluoroacetic acid (TFA) (0.05%, v/v) and acetic acid (0.5%, v/v) were incorporated into the mobile phases to improve the chromatographic performance and to minimize the carryover due to nonspecific binding of the analyte to the walls of 96-well plate. The current method was validated for sensitivity, selectivity, linearity, reproducibility, stability and recovery. The accuracy and precision for the LLOQs (0.500 ng/mL) were within ±5.6% bias and ≤7.8 % CV, respectively. During the intra-day and inter-day evaluations the QC sample (1.50, 7.50, 75.0 and 190 ng/mL) precision ranged from 2.7 to 4.9% CV and the accuracy (% bias) from -1.3 to 7.3%, respectively. Additional assessments of incurred sample reanalysis (ISR) were conducted to demonstrate the ruggedness and robustness of the assay method. The validated method was successfully used to support a toxicity study in monkeys administered with 5 and 30 mg of SOM230 in a single intramuscular injection of a long acting release (LAR) formulation

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

An industry perspective on tiered approach to the investigation of metabolites in drug development

Background: A tiered approach to drug metabolite measurement and identification is often used industry wide to fulfill regulatory requirements specified in recent US FDA and European Medicines Agency guidance. Although this strategy is structured in its intent it can be customized to address unique challenges which may arise during early and late drug development activities. These unconventional methods can be applied at any stage to facilitate metabolite characterization. Results: Two case studies are described NVS 1 and 2. NVS 1: plasma concentrations, measured using a radiolabeled MS-response factor exploratory method, were comparable to those from a validated bioanalytical method. The NVS 2 example showed how in vitro analysis helped to characterize an unexpectedly abundant circulating plasma metabolite M3. Conclusion: A tiered approach incorporating many aspects of conventional and flexible analytical methodologies can be pulled together to address regulatory questions surrounding drug metabolite characterization. © 2014 Future Science Ltd

ICP-MS determination of total serum aluminum in monkeys following subcutaneous administration of a drug candidate formulated with Alhydrogel

Background: To develop and validate an inductively coupled plasma MS (ICP-MS) method for quantitative bioanalysis of aluminum in monkey serum in support of a GLP TOX study with Alhydrogel formulated drug candidate. Methods and results: The method was linear over a dynamic range of 10.0 to 1000 ng/mL using a 50 µL sample volume. The intra-/inter-run precision (%CV) of the QC sample results were ≤ 7.9% (CV) and the accuracy (%Bias) within ±11.0% across all QC 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 aluminum in monkey serum to assess the systemic exposure to aluminum. Key words: •ICP-MS: Inductively coupled plasma MS is a technique to quantify one or simultaneously many elements after ionization and mass separation with a mass spectrometer •Alhydrogel: A compound with many biomedical applications: as a gastric antacid, an antiperspirant, in dentifrices, as an emulsifier, as an adjuvant in bacterins and vaccines, in water purification, etc

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

The pharmacokinetics, absorption, distribution, metabolism and excretion 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 (QWBA) in male Long Evans Hooded (LEH) rats. Following a 5 mg/kg IV dose, radioactivity widely and rapidly distributed into most tissues. The highest tissue-to-blood ratio was observed in bone mineral, thyroid gland, and harderian gland at 24 h post IV dose. There was no evidence of stable accumulation in uveal tract tissue suggesting no stable binding interaction with melanin. Following a 90 mg/kg oral dose in LEH rats, the highest tissue-to-blood ratios (t/b) were observed in bone mineral, harderian gland, liver, spleen, and salivary gland. Plasma protein binding was moderate in rat (26%) and other tested species (15% - 21%). Omadacycline plasma clearance and half-life was estimated to be moderate (1.2 L/h/kg and 4.6 h); steady-state volume of distribution (Vss) was estimated to be large (6.89 L/kg). Major circulating components in plasma were intact omadacycline and its epimer. Consistent with observations in human, fecal and urine excretion was the main route of elimination in rat . Approximately 80% of the dose was excreted into the feces as unchanged omadacycline after intravenous administration. Fecal excretion was primarily the result of biliary excretion (~40%) and direct gastrointestinal secretion (~30%). However, urinary excretion (~30%) was equally prominent after IV dosing. Omadacycline can be categorized as a preliminary Biopharmaceutics Drug Disposition Classification System (BDDCS) III drug given its extensive excretion in vivo, low absorption, low lipophilicity and high aqueous solubility

A semi-automated high performance liquid chromatography-tandem mass spectrometric method for determination of LCI699, an 11-hyroxylase inhibitor, in human plasma

A liquid chromatographic method with tandem mass spectrometric detection (LC-MS/MS) for the determination of LCI699 was developed and validated with concentration ranges of 0.0500-50.0 ng/ml and 1.00-1000 ng/ml using 0.05 ml and 0.1 ml, respectively, of human plasma. LCI699 and the internal standard, [M+6]LCI699, were extracted from fortified human plasma via protein precipitation. After transfer or dilution of the supernatant followed by solvent evaporation and reconstitution, the extract was injected onto the LC-MS/MS system. Optimal chromatographic separation was achieved on an ACE C18 (50 x 4.6 mm, 3 µm) column with 30% aqueous methanol (containing 0.5% acetic acid and 0.05% TFA) as the mobile phase run in isocratic at a flow rate of 1.0 ml/min. The total analysis cycle time is approximately 3.5 min per injection. The addition of an ion-pair reagent, 0.05% TFA, to the mobile phases significantly improved the chromatographic retention and resolution of the analyte on silica based reversed phase column. Although addition of TFA to the mobile phase suppresses the ESI signals of the analyte due to its ion-pairing characteristics in the gas phase of MS source, this negative impact was effectively alleviated by adding 0.5% acetic acid to the mobile phase. The current method was validated for sensitivity, selectivity, linearity, reproducibility, stability and recovery. Additional assessments of incurred sample stability (ISS) and incurred sample reanalysis (ISR) were conducted to demonstrate the ruggedness and robustness of the assay method. Lack of adverse matrix effect and carryover was also demonstrated. The overall precision and accuracy of the quality control samples were 15% (CV) and within 15 % (bias), respectively. The validated method was successfully used in support of rapid turnaround time pharmacokinetic studies in huma