A compliant hERG assay measures block of the IKr current carried by the hERG channel and delivers a documented IC50 with quality-control metrics, under recording conditions that regulators can trace and repeat. That means physiological temperature control, a defined voltage protocol, concentration verification, and pass/fail thresholds for seal and series resistance, all captured in the study report alongside the safety margin calculation used to judge cardiac risk.

TL;DR:

• The hERG assay measures IKr channel block at physiological temperature using verified compound concentrations to generate IC50 values with confidence intervals, supporting clinical risk assessments.
• Proper cell health, stable expression, and use of the FDA’s recommended voltage protocol are essential for reproducible and regulator-acceptable results.
• Seal resistance, series resistance, and minimal run-down are critical quality controls, with raw data and QC filters documented for review.
• Verifying actual drug concentrations with LC-MS/MS is necessary, especially for insoluble compounds, to ensure accurate exposure and safety margin calculations.
• Cross-platform validation and standardization of SOPs, temperature control, and compound handling reduce variability and strengthen data reliability for regulatory submissions.

Table of Contents

• What the hERG assay measures and why regulators care
• Core experimental requirements: cell systems, solutions, temperature, and voltage protocol
• Data quality and acceptance criteria for seals, resistance, and run down
• Concentration handling and verification for accurate exposure
• Dose-response design, IC50 fitting, and the safety margin calculation
• Automated versus manual patch clamp: validation and common pitfalls
• Reporting, GLP considerations, and fitting hERG data into risk assessment
• How Mayflower Bioscience supports hERG assay setup and validation
• Building a regulator-ready hERG workflow
• Getting hERG assay support from Mayflower Bioscience
• Sources
• FAQ

What the hERG assay measures and why regulators care

The hERG channel, encoded by the gene KCNH2, carries the rapid delayed rectifier potassium current known as IKr. That current drives the final phase of ventricular repolarization, and when a compound blocks it, the action potential lengthens, showing up on an ECG as QT prolongation. A prolonged QT interval raises the risk of torsade de pointes, a polymorphic ventricular arrhythmia that can be fatal, so hERG block has become the single most tested cardiac liability in early drug discovery.

Regulators do not treat the hERG assay as a standalone gatekeeper. Under ICH S7B, the in vitro hERG result sits inside an integrated nonclinical assessment that also weighs in vivo QT data, structure-activity relationships, and, later, clinical exposure. The E14/S7B Q&A document, finalized at Step 4 in 2022, extended that logic into the clinic: when nonclinical data (including a well-characterized hERG IC50 and an adequate safety margin) meet defined criteria, sponsors may reduce or waive the dedicated thorough QT study and rely on ECG data collected during standard clinical trials instead.

That shift raised the bar on assay quality rather than lowering it. A hERG result that is going to substitute for a piece of clinical testing has to be defensible on its own: clean traces, verified concentrations, and a margin calculation that a reviewer can reconstruct from the report.

The practical implications for a bench scientist come down to a few recurring requirements:

• Record IKr block at a temperature and voltage protocol consistent with published recommendations, not whatever is convenient for the rig.
• Verify that the concentrations cells were exposed to correspond closely to the nominal concentrations in the plate, as accurately as feasible.
• Report IC50 with a confidence interval, not a point estimate alone.
• Tie the IC50 to a clinical exposure metric so the safety margin means something to a reviewer.

Core experimental requirements: cell systems, solutions, temperature, and voltage protocol

Most labs express hERG in a recombinant HEK293 or CHO cell line, using either constitutive or inducible expression systems. Inducible lines let you control expression timing and density, which helps keep current amplitude consistent from one plate to the next, a real advantage when you are trying to compare IC50 values across weeks of testing. Before recording, check that cells are healthy, uniformly expressing, and not overgrown or over-passaged, since expression drift is one of the quieter sources of inter-run variability.

The FDA’s recommended voltage protocol for hERG uses a ramp-down design: cells are held near a physiological resting potential, stepped to a depolarizing voltage to activate the channel, then stepped down to around minus 80 millivolts before returning to holding, which produces the characteristic hERG tail current used to quantify block. A full cycle typically runs on the order of five seconds, repeated at a fixed interval throughout drug application so the tail current can be tracked as it stabilizes.

External and internal solutions matter as much as the voltage steps. The FDA protocol documentation specifies example recipes for both, and any deviation should be justified and recorded, because ionic composition affects current kinetics and reversal potential. Liquid junction potential correction is a detail that is easy to skip and hard to defend later: uncorrected junction potentials shift the effective voltage the cell experiences, which can bias apparent potency.

Recording temperature deserves its own line item. Physiological temperature, roughly 35 to 37 degrees Celsius, is the recommended condition because hERG kinetics and drug binding are temperature-dependent, and IC50 values measured at room temperature can differ meaningfully from those measured at physiological temperature. A protocol-dependent difference preprint documents how stimulation frequency, waveform, and temperature produce predictable shifts in measured potency, which is exactly why the voltage protocol and temperature need to be fixed and reported, not left to individual operator preference.

A basic setup sequence looks like this:

1. Confirm cell line identity, passage number, and expression status before seeding for recording.
2. Prepare external and internal solutions fresh, matching the documented recipe, and correct for liquid junction potential.
3. Equilibrate the recording chamber or plate to the target temperature before establishing seals.
4. Apply the standardized voltage protocol and collect a stable baseline before any compound addition.
5. Select at least four test concentrations bracketing the expected IC50, aiming for inhibition spanning roughly 20% to 80%.
6. Include a reference compound run (commonly a well-characterized hERG blocker) in every assay session to confirm system sensitivity.

Pro Tip: Run your reference compound at the start and end of every plate or session, not just once a week: a shift in its IC50 is often the first sign that solution prep, temperature control, or cell health has drifted.

Data quality and acceptance criteria for seals, resistance, and run down

A hERG recording is only as trustworthy as the seal it was taken through. A gigaseal, meaning seal resistance at or above 1 gigaohm, is the baseline most protocols require before a cell is accepted into the dataset, because a lower resistance seal lets current leak around the membrane rather than through it, which distorts the measured current and biases the apparent block.

A validated hERG protocol from the FDA specifies seal resistance, series resistance, and holding current stability as core acceptance criteria for IC50 determination, and labs should log the actual value for every cell, not just note pass or fail, so a reviewer can see the distribution behind the accepted dataset (FDA protocol).

Series resistance and membrane resistance are the next filters. A common working range is series resistance below roughly 10 megohms, kept stable throughout the recording, and membrane resistance above roughly 500 megohms, since a leaky or low-resistance membrane produces noisy, unreliable current traces. Series resistance compensation should be applied and documented, since uncompensated series resistance introduces voltage errors that grow with current amplitude, exactly the range where you are trying to measure block most precisely.

Run down, the gradual decline in current amplitude over time that is unrelated to drug effect, is one of the most common reasons for excluding a cell. A minimum tail current amplitude, often around 250 picoamperes, is also worth setting as a floor, since very small currents produce fractional block estimates that are mostly noise.

The rest of the acceptance framework covers replicate strategy and reporting:

• Plan for a practical number of replicate cells per concentration, adjusting upward for compounds with variable responses.
• Decide up front whether you will apply multiple concentrations to a single cell (cumulative design) or a single concentration per cell, and hold that choice constant within a study.
• Report QC-filtered raw traces alongside the filtering rules applied, so a reviewer can see what was excluded and why.
• Present per-cell fractional block values in a table, not just the aggregated IC50, so outliers are visible rather than buried in a summary statistic.

Documenting exclusions this way is what turns a bench dataset into something a GLP reviewer can actually evaluate, rather than a black box that produced a single number.

Concentration handling and verification for accurate exposure

The IC50 you calculate is only meaningful if the cells were actually exposed to the concentrations written on the plate map. That sounds obvious, but compound adsorption to plastic, poor solubility, and slow equilibration routinely create a gap between nominal and actual concentration, especially at the higher end of a dose-response curve where lipophilic compounds are most likely to precipitate or stick to tubing.

Plate and stock preparation should follow a few operational rules:

1. Prepare compound dilutions immediately before the experiment rather than storing diluted working solutions, since stability in aqueous buffer is often shorter than in the original stock solvent.
2. Use glass inserts or low-binding consumables when a compound is known or suspected to adsorb to standard plastics.
3. Filter stock and working solutions through a 0.2 micron filter and check for light scattering or visible turbidity as a quick solubility flag before running the plate.
4. Prepare satellite samples alongside the recording plate so you have material to verify concentration without disturbing the cells under test.

Pro Tip: If a compound shows an unexpectedly flat or shallow dose-response curve, check solubility before you question the biology: a precipitating compound often looks like weak block simply because the free concentration never reached the nominal target.

For poorly soluble compounds, bioanalytical verification stops being optional. The FDA’s guidance on solubility handling for hERG describes validated LC-MS/MS workflows developed for compounds like lopinavir and ritonavir, along with sample handling steps designed to avoid measuring insoluble particulate rather than solubilized drug interacting with the channel. Real-time or near-real-time sampling from the recording chamber, paired with a validated bioanalytical method, lets you confirm that the concentration driving the observed block matches the concentration you intended to test.

When measured concentrations differ from nominal ones, report the IC50 against the measured values, not the nominal plate concentrations. That single choice can materially change a safety margin calculation, and a reviewer comparing your data to reference literature will expect to see which concentration basis you used.

Dose-response design, IC50 fitting, and the safety margin calculation

A robust IC50 estimate starts with the concentration range.

Fractional block per cell is typically calculated by averaging several consecutive tail-current traces (commonly around five) at steady state for each concentration, comparing the drug-exposed amplitude to the pre-drug baseline for that same cell. Aggregating those per-cell fractional block values across replicates, rather than pooling raw currents across cells of different baseline amplitude, keeps the analysis from being skewed by naturally larger or smaller cells.

The aggregated concentration-response data are then fit to the Hill equation, which returns the IC50 and the Hill coefficient describing the steepness of the curve. Both values should be reported with their 95% confidence intervals: IC50 with a defined confidence interval is the primary deliverable ICH training materials point to for in vitro hERG assay evaluation, since a point estimate alone gives a reviewer no sense of how precisely the assay pinned down potency.

From there, the safety margin calculation is what actually connects the assay to clinical risk. The standard approach normalizes the hERG IC50 to the free (protein-unbound) maximum plasma concentration, or Cmax, expected at steady-state clinical exposure:

• Calculate free Cmax by multiplying total Cmax by the unbound fraction measured in plasma protein binding studies.
• Divide the hERG IC50 by that free Cmax to produce the safety margin.
• When protein binding is uncertain or cannot be reliably measured below a floor value, a conservative fallback is to assume 1% unbound if the measured unbound fraction is below 1%, which avoids overstating the margin from an unreliable low-binding measurement.
• Present a sensitivity analysis showing how the margin shifts across a plausible range of unbound fraction and measured-versus-nominal concentration assumptions.

Reporting should pair the per-cell data table and the IC50 with confidence interval with that sensitivity analysis, so a reviewer can see not just the headline margin but how much it depends on assumptions that carry their own uncertainty.

Automated versus manual patch clamp: validation and common pitfalls

Automated patch clamp platforms have become the workhorse for hERG screening because they process far more cells per day than manual patch clamp, but throughput does not substitute for validation. Peer-reviewed methods work on early hERG liability screening describes how automated platforms must translate manual QC criteria and be benchmarked against manual patch clamp before their data can be trusted for decision-making (automated patch clamp methods, 2014).

The comparison should cover the same metrics used to qualify manual recordings: membrane resistance, series resistance, initial tail current amplitude, and, most importantly, IC50 values for a shared set of reference compounds run on both systems. If a platform’s reference-drug IC50 consistently sits outside the range reported in manual patch clamp literature, that gap needs to be understood and documented before the platform generates data for a submission.

A few artifacts show up disproportionately on automated systems and are worth watching for:

• Compound adsorption to plastic consumables, which is more pronounced in automated fluidics than in glass-based manual setups and can flatten dose-response curves for lipophilic compounds.
• Shorter average recording lifetimes per cell, which limits the number of concentrations that can be tested cumulatively on a single cell.
• Liquid handling variability between wells or plates, which shows up as noisier replicate-to-replicate IC50 estimates even for a well-behaved reference compound.

The most useful validation design runs the same reference compound panel across both platforms, computes a system-specific safety margin for each, and includes concentration verification on the automated side where adsorption is a known risk. Documenting equivalence in a report means more than a paragraph asserting the platforms agree: a statistical comparison, a ratio metric or Bland-Altman style plot of paired IC50 values, and a repeatability measure across runs give a reviewer something concrete to evaluate.

Reporting, GLP considerations, and fitting hERG data into risk assessment

Whether a hERG study needs to run under Good Laboratory Practice depends on where it sits in the development timeline and what regulatory decision it is meant to support; a screening assay used to rank early candidates generally does not need GLP status, while a definitive study intended to support an integrated risk assessment or influence thorough QT study waiver decisions typically does.

A GLP-ready report should include a defined minimum set of contents:

• The full protocol as executed, including any deviations from the standard voltage waveform, solutions, or temperature, with a rationale for each.
• Raw traces alongside the QC filters applied, so excluded cells and the reason for exclusion are visible.
• Concentration verification data, especially for poorly soluble compounds, showing measured versus nominal values.
• A description or reference to the curve-fitting method and software used to derive IC50 and Hill coefficient.
• Reference compound results for that run, positioned against the laboratory’s own historical range for that compound.

Laboratory-specific safety margin thresholds are worth establishing and stating explicitly in the report, since a “sufficient” margin is a judgment call informed by the compound class and intended clinical population, not a fixed universal number.

Some compounds warrant follow-up beyond the parent hERG assay. Testing relevant metabolites is appropriate when a metabolite is expected to reach meaningful plasma concentrations, and additional ion current assays, such as ICaL or INa, become relevant when the compound’s structure-activity profile or clinical ECG signal suggests effects beyond IKr alone. A multi-laboratory comparison study published in 2025 found that even standardized protocols following ICH S7B Q&A best practices still produce measurable inter-laboratory variability in IC50 values, which is a reminder to document your own lab’s historical variability rather than assume any single result stands alone.

Before submission, a short internal checklist helps: confirm the dataset is complete for every planned concentration, confirm statistical notes accompany the IC50 and margin, and confirm the sensitivity analysis for protein binding assumptions is attached.

How Mayflower Bioscience supports hERG assay setup and validation

Meeting the parameters above starts with the cell line. Mayflower Bioscience supplies an inducible HEK 293 hERG cell line and an inducible CHO hERG cell line, both built for the reproducible, controllable expression that consistent IC50 estimates depend on across recording sessions.

For labs that need bench capacity or platform validation rather than another cell line, Mayflower’s ion channel services cover patch clamp testing, and the broader ion channel cell line catalog includes related consumables suited to low-adsorption compound handling, the kind of detail that matters most when concentration verification is on the table.

Building a regulator-ready hERG workflow

The single highest-value fix I would make in most labs is temperature control, followed closely by concentration verification. Both are easy to underinvest in because they do not show up as a failed seal or a bad trace; they show up as a subtly wrong IC50 that looks perfectly clean.

Standardizing SOPs for solution prep, running a reference compound on every session, and cross-checking results between operators catch drift long before it reaches a report. When a compound’s margin is borderline, or the intended use is to support a thorough QT study waiver, that is the point to escalate to a formal GLP study or complementary in vivo QT testing rather than stretching a screening-grade dataset to do a job it was not built for.

— Alina

Getting hERG assay support from Mayflower Bioscience

Running a compliant hERG study is less about any single step and more about having the right cell line, consumables, and bench capacity lined up before the first plate goes on the rig. Mayflower Bioscience’s ion channel testing services cover patch clamp work for labs that need testing capacity, GLP-aligned documentation, or a second system to validate against.

Before reaching out, have your compound identity, whether the study needs to be GLP-compliant, and your target sample timeline ready, since those details shape which service tier and turnaround fit your project. Reach the team through the ion channel testing and patch clamp services page to request a quote or a technical consultation, or browse the inducible hERG cell lines directly if sourcing material is the immediate need.

Sources

• S7B nonclinical evaluation of the potential for delayed ventricular repolarization (QT interval prolongation)
• ICH E14/S7B Q&A step 4 (2022)
• Early identification of hERG liability in drug discovery programs by automated patch clamp (Frontiers / PMC)

For teams also managing broader inter-lab quality processes, this central lab oversight overview from an industry partner covers standardization practices relevant beyond hERG alone.

This article is general information, not a substitute for advice from a qualified doctor. Consult a qualified healthcare professional about your own circumstances before acting on anything here.

FAQ

What does the hERG assay measure?

The hERG assay measures block of the IKr potassium current carried by the hERG channel, which drives ventricular repolarization. It is used in nonclinical safety testing to flag compounds that may prolong the QT interval and increase the risk of torsade de pointes, as described in ICH S7B.

Is hERG the same as KCNH2?

KCNH2 is the gene that encodes the hERG channel protein, so the two terms refer to the same channel from different angles: KCNH2 is the gene, hERG is the channel and current it produces. Assay reports typically use “hERG” for the functional current and “KCNH2” when referring to genetic identity or cell line characterization.

What is the hERG patch clamp assay used for?

The hERG patch clamp assay is used to determine a compound’s IC50 for blocking the IKr current, a key input into the cardiac safety margin calculation described in ICH E14/S7B Q&A. It is run using either manual or automated patch clamp platforms, following a standardized voltage protocol and physiological recording temperature.

What does hERG stand for?

hERG stands for the human Ether-a-go-go-Related Gene, the gene family name originally applied to the channel before it was formally designated KCNH2. Both names are still used interchangeably in cardiac safety literature and regulatory guidance.

What is a typical hERG safety margin calculation?

A typical safety margin divides the hERG IC50 by the free, protein-unbound maximum plasma concentration expected at clinical exposure, giving a ratio that regulators use alongside other nonclinical and clinical data under ICH S7B. When protein binding is uncertain, a conservative fallback assumes 1% unbound fraction if the measured value is below that threshold.

Recommended

• hERG Cell Line (HEK 293) inducible expression
• hERG Cell Line (HEK 293) constitutive expression
• hERG Cell Line (CHO) inducible expression
• How to Analyze Assay Data for Accurate and Reproducible Results