Determination of five positive control drugs in hERG external solution (buffer) by LC-MS/MS to support in vitro hERG assay as recommended by ICH S7B.

ICH S7B recommends screening for hERG channel block using patch clamp recordings to assess a drug's proarrhythmic risk. Block of the hERG channel has been associated with clinical QTC prolongation as well as the rare, but potentially fatal ventricular tachyarrhythmia Torsade de Pointes (TdP). During recording, drug concentrations perfused to the cells can deviate from nominal concentrations due to molecule-specific properties (such as non-specific binding), thereby introducing error when assessing drug potency. To account for this potential source of error, both the original ICH S7B and the newly released ICH E14/S7B Q&As guidelines call for verifying drug solutions' concentrations. Dofetilide, cisapride, terfenadine, sotalol and E-4031 are hERG blockers commonly used as positive controls to illustrate hERG assay sensitivity. The first four compounds are also clinical drugs associated with high TdP risk; therefore, their safety margins may be useful comparators to better understand an investigational product's TdP risk. Having analytical methods to quantify these five compounds in the hERG external solution that will be used for patch clamp recordings is important from a regulatory science research perspective. However, a literature search revealed no analytical methods or stability information for these molecules in the high salt, serum-free matrix that constitutes the hERG external solution. This study was conducted to develop and validate LC-MS/MS methods to quantify these 5 molecules in hERG external solution. The bioanalytical methods for these positive controls were validated as per the FDA's bioanalytical method validation guidance along with various stabilities.

hERG block potencies for 5 positive control drugs obtained per ICH E14/S7B Q&As best practices: Impact of recording temperature and drug loss.

According to the ICH S7B guideline, drug candidates are screened for hERG block prior to first-in-human testing to predict the likelihood of delayed repolarization associated with a rare, but life-threatening, ventricular tachyarrhythmia. The new ICH E14 Q&As guideline allows hERG results to be used in later clinical development for decision-making (Q&As 5.1 and 6.1). To pursue this path, the hERG assay should be conducted following the new ICH S7B Q&A 2.1 guideline, which calls for best practice considerations of the recording temperature, voltage protocol, stimulation frequency, recording/data quality, and concentration verification. This study investigated hERG block by cisapride, dofetilide, terfenadine, sotalol, and E-4031 - positive controls commonly used to demonstrate assay sensitivity - using the manual whole cell patch clamp method and an action potential-like voltage protocol presented at 0.2 Hz. Recordings were conducted at room and near physiological temperature. Drug concentrations were measured using samples collected during real patch clamp experiments and satellite experiments. Results showed temperature effects for E-4031, terfenadine, and sotalol, but not cisapride and dofetilide. Cisapride and terfenadine showed substantial concentration losses, largely due to nonspecific binding to the perfusion apparatus. Using concentrations measured from the real and satellite experiments to assess block potencies yielded comparable results, indicating that satellite sample collection may be viable for drugs with nonspecific binding concerns only. In summary, this study provides block potencies for 5 hERG positive controls, and serves as a case study for hERG assays conducted, and results illustrated in accordance with the new ICH E14/S7B Q&As.

Intact quantitative bioanalytical method development and fit-for-purpose validation of a monoclonal antibody and its related fab fragment in human vitreous and aqueous humor using LC-HRMS.

Ranibizumab is an FDA-approved drug used to treat wet age-related macular degeneration (AMD), diabetic retinopathy, macular edema, and myopic choroidal neovascularization. Bevacizumab is another drug often used off-label to treat wet AMD. In order to reduce unwanted angiogenesis, ranibizumab and bevacizumab target circulating VEGF-A in the eye. Concentration levels in human vitreous and aqueous humor can be used to provide valuable efficacy information. However, vitreous and aqueous humor's aqueous environment, and vitreous humor's viscosity, as well as the stickiness of the analytes can provide bioanalytical challenges. In this manuscript, we describe the development, optimization, and fit-for-purpose validation of an LC-HRMS method designed for intact quantitative bioanalysis of ranibizumab and bevacizumab in human vitreous and aqueous humor following intravitreal administration. In order to fully develop this method, evaluations were conducted to optimize the conditions, including the data processing model (extracted ion chromatograms (XICs) vs deconvolution), carryover mitigation, sample preparation scheme optimization for surrogate and primary matrices, use of internal standard/immunocapture/deglycosylation, and optimization of the extraction and dilution procedure, as well as optimization of the liquid chromatography and mass spectrometry conditions. Once the method was fully optimized, a fit-for-purpose validation was conducted, including matrix parallelism, with a linear calibration range of 10 to 200 µg/mL. The development of this intact quantitative method using LC-HRMS provides a proof-of-concept template for challenging, but valuable new and exciting bioanalytical techniques.

New science, drug regulation, and emergent public health issues: The work of FDA's division of applied regulatory science.

The U.S. Food and Drug Administration (FDA) Division of Applied Regulatory Science (DARS) moves new science into the drug review process and addresses emergent regulatory and public health questions for the Agency. By forming interdisciplinary teams, DARS conducts mission-critical research to provide answers to scientific questions and solutions to regulatory challenges. Staffed by experts across the translational research spectrum, DARS forms synergies by pulling together scientists and experts from diverse backgrounds to collaborate in tackling some of the most complex challenges facing FDA. This includes (but is not limited to) assessing the systemic absorption of sunscreens, evaluating whether certain drugs can convert to carcinogens in people, studying drug interactions with opioids, optimizing opioid antagonist dosing in community settings, removing barriers to biosimilar and generic drug development, and advancing therapeutic development for rare diseases. FDA tasks DARS with wide ranging issues that encompass regulatory science; DARS, in turn, helps the Agency solve these challenges. The impact of DARS research is felt by patients, the pharmaceutical industry, and fellow regulators. This article reviews applied research projects and initiatives led by DARS and conducts a deeper dive into select examples illustrating the impactful work of the Division.

Multifaceted Bioanalytical Methods for the Comprehensive Pharmacokinetic and Catabolic Assessment of MEDI3726, an Anti-Prostate-Specific Membrane Antigen Pyrrolobenzodiazepine Antibody-Drug Conjugate.

Complex biotherapeutic modalities, such as antibody-drug conjugates (ADC), present significant challenges for the comprehensive bioanalytical characterization of their pharmacokinetics (PK) and catabolism in both preclinical and clinical settings. Thus, the bioanalytical strategy for ADCs must be designed to address the specific structural elements of the protein scaffold, linker, and warhead. A typical bioanalytical strategy for ADCs involves quantification of the Total ADC, Total IgG, and Free Warhead concentrations. Herein, we present bioanalytical characterization of the PK and catabolism of a novel ADC. MEDI3726 targets prostate-specific membrane antigen (PMSA) and is comprised of a humanized IgG1 antibody site-specifically conjugated to tesirine (SG3249). The MEDI3726 protein scaffold lacks interchain disulfide bonds and has an average drug to antibody ratio (DAR) of 2. Based on the structural characteristics of MEDI3726, an array of 4 bioanalytical assays detecting 6 different surrogate analyte classes representing at least 14 unique species was developed, validated, and employed in support of a first-in-human clinical trial (NCT02991911). MEDI3726 requires the combination of heavy-light chain structure and conjugated warhead to selectively deliver the warhead to the target cells. Therefore, both heavy-light chain dissociation and the deconjugation of the warhead will affect the activity of MEDI3726. The concentration-time profiles of subjects dosed with MEDI3726 revealed catabolism of the protein scaffold manifested by the more rapid clearance of the Active ADC, while exhibiting minimal deconjugation of the pyrrolobenzodiazepine (PBD) warhead (SG3199).

Development and optimization of on-line 2-dimensional chromatographic approaches for eliminating matrix effects and improving bioanalysis of peptides in human plasma using UHPLC-MS/MS.

Online 2-dimensional chromatographic approaches for eliminating matrix effects and optimizing bioanalysis of peptides using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) were studied. Three therapeutic peptides (octreotide, desmopressin, and vasopressin) were selected as model analytes. Human plasma was precipitated with acetonitrile; peptides were analyzed on C(8), C(18), Phenyl and HILIC ACQUITY UPLC columns. For simpler online clean-up applications, a C(18) pre-column was coupled to the analytical column via a switching valve. For more complex heart-cutting applications, two analytical columns were used with optional online dilution to refocus the analyte peaks prior to the second dimension separation. This allows the use of MS incompatible mobile phases, such as TFA, in the first dimension separation. Online clean-up effectiveness was investigated by monitoring phospholipids. Flushing direction, mobile phase composition, flow rate and transfer window were evaluated. Phospholipids were readily retained on reversed-phase columns, and the peptides were reproducibly transferred, individually or as a group, to the second column using appropriate transfer windows. The best peak shapes were obtained when the second dimension column was more retentive (e.g. C(18) vs. C(8)). However, C(8) to HILIC gave broad unresolved peaks due to mobile phase mismatch. Trapped phospholipids were efficiently removed from either guard columns or first dimensional columns by forward- or back-flushing at high flows; however, back-flushing was more efficient with lower flow rates on larger columns.