Physiological-Temperature hERG Assays by Manual Patch Clamp
We assess human hERG/IKr inhibition using manual whole-cell patch clamp at a controlled temperature of 35-37 °C. The study is designed with the relevant ICH E14/S7B Q&A best-practice considerations in mind. Recording at near-physiological temperature supports characterization of channel kinetics and IC50 under conditions relevant to early compound selection and risk assessment; it does not by itself demonstrate clinical safety or regulatory compliance.
Assay Workflow
The standard service workflow includes:
- Client sample preparation
- Sample receipt and registration, including physicochemical information, vehicle and DMSO percentage
- Manual patch-clamp recording of hERG currents at 35-37 °C
- Data analysis and interpretation, including IC50 where supported, mechanistic considerations and uncertainty
- Report delivery
Why Assess hERG at Physiological Temperature?
- IKr contributes to cardiac repolarization and is relevant to QT/QTc prolongation and arrhythmia risk. Early inhibition studies help identify liabilities for further evaluation, without guaranteeing a reduction in clinical risk.
- Channel gating and drug effects can be temperature-sensitive. Recording at 35-37 °C allows IC50, kinetics and, where included in the protocol, frequency- or state-dependent effects to be assessed under near-physiological conditions. These findings still require exposure-aware interpretation.
- Room-temperature results should not simply be extrapolated to physiological temperature. Direct measurement, documentation of actual recording temperature and attention to compound exposure and positive-control performance improve interpretability.
Deliverables
- A complete experimental PDF report covering background, protocol, data and interpretation, together with Excel-format data.
The study report specifies recording conditions, controls, analysis assumptions and limitations. Concentration-response fitting is reported only when supported by the data. The scope of exposure verification, additional mechanistic experiments and any regulatory-use requirements must be defined in the study plan rather than assumed from the assay temperature.
Physiological Temperature in the hERG Assessment Framework
The ICH E14/S7B Questions and Answers places in vitro hERG/IKr assessment within an integrated evaluation of delayed repolarization and QT/QTc risk. Physiological-temperature recording is one element of that framework, alongside exposure characterization, controls and relevant nonclinical and clinical evidence.
Temperature and Voltage Protocol
The best-practice recommendations specify near-physiological recording at 35-37 °C for cells overexpressing cardiac ion channels, including hERG, CaV1.2 and NaV1.5. Voltage protocols should approximate the relevant elements of a ventricular action potential. For hERG, a stimulation frequency of 0.2-1 Hz is recommended. The chosen protocol, temperature and frequency are documented; mentioning other channels here describes the guidance, not an additional assay included in this hERG service.
Exposure Verification and Controls
Actual cell exposure should be checked, for example by quantifying compound concentrations in the perfusion solution. Nominal and measured concentrations should be reported; when they differ significantly, the measured concentrations should inform the IC50 concentration-response analysis. A suitable reference positive control, such as dofetilide, should demonstrate reproducible responses across the 20-80% inhibition range using sufficient replicates and at least two concentrations. Unexpected control results require investigation before the study is relied on for the relevant risk-assessment decisions.
Integrated Risk Assessment
Where appropriate and subject to the guidance's conditions, nonclinical hERG results, in vivo QT findings and clinical ECG data can be considered together, including situations in which clinical study design is constrained. The adequacy of the nonclinical methods and coverage of high clinical exposure are important to that interpretation.
Physiological-temperature recording is a recommended best-practice condition, not a stand-alone certificate of compliance. Temperature control, exposure verification, positive controls and integrated interpretation must be considered together; an early discovery assay does not replace the complete evidence package needed for a regulatory decision.
