About Sophion Technologies
Sophion is dedicated to providing state of the art products and integrated solutions for automated patch clamping. The patch clamp technique is a laboratory technique in electrophysiology that allows the study of single or multiple ion channels in cells.
Find out more below about the patch clamp technique and our chosen technological path to secure reliable ion channel recordings. Please see below.
Sophion provides high tech ion channel screening solutions. You can read more about ion channels below.
Ion channels consist of proteins and are embedded in all cell membranes of all living organisms. They are responsible for electrical signaling within cells and between cells. The signal is carried by ions (i.e., ‘salt’) that normally cannot pass the cell membrane but can diffuse passively (no energy expenditure) through ion channels, when they open in response to certain conditions. This electrical signaling is crucial for all physiological processes.
Examples of such processes are:
- Generation of electrical activity in nerves
- Control of contractile activity in the heart and muscles
- Nutrient uptake
- Hormone secretion
There are many ion channels
A typical cell has 100-1000 ion channels of different types. Most ion channels allow only one specific ion to permeate through them. This is reflected in the common names of the ion channels, e.g., K+, Na+, Ca2+ and Cl– ion channels. The sequencing of the human genome has recently led to the identification of more than 400 putative ion channels. Only about 100 of these have been cloned and functionally tested so far.
Ion channels play a role in many diseases
A number of human diseases, including pathological pain conditions, epilepsy, cystic fibrosis and a variety of neural and muscular disorders, are caused by defects in the function of ion channels.
Many drugs affect ion channels
The large number of physiological processes regulated by ion channels and their role in many diseases make ion channels highly interesting as targets for new drugs. Today, about 20% of all registered drugs target ion channels. The drugs modulate specific ion channels, resulting in altered cellular behavior.
Ion channels are difficult to explore
Ion channels are important targets in drug discovery, but they are also difficult to study. Despite significant research efforts, there is still limited knowledge about ion channel function and how ion channels are related to specific diseases. This is mainly a result of the limitations and complexity in the technologies that are available to ion channel research. Ion channels are therefore considered to be largely unexploited compared to other target classes.
Methods to explore ion channels
There are two main methods to investigate ion channels: direct and indirect methods. The only direct method is called ‘patch clamp’. In brief, patch clamp is very accurate, but also very time consuming (i.e., has low throughput), while the indirect methods are less accurate, but typically much faster.
The patch clamp technique is considered the gold standard in ion channel research. The technique was developed in the 1970’s by Erwin Neher and Bert Sakmann, who received the Nobel Prize in Physiology and Medicine (1991) for their work. In a traditional patch clamp experiment, a tiny glass pipette containing an electrode is attached or ‘sealed’ tightly to the cell membrane. The cell membrane is ruptured manually by applying suction through the glass pipette. The tiny currents (10-12-10-9 amperes) through the ion channels can be measured via the electrode, which is connected to an amplifier. The typical throughput is three to ten successful patch clamp experiments per day and requires patience and high-level insight in electrophysiology.
Indirect methods to study ion channels
There are several indirect methods for studying ion channels. The most important indirect methods are based on fluorescent dyes that are loaded into cells and then analyzed using specialized plate readers. The readers detect a change in the concentration of certain ions that is the result of ion channel activity or a change in the voltage across the cell membrane, also caused by the flow of ion through the channel. The main benefits of these methods are their high throughput and low cost per data point. The main limitations of the methods are low accuracy and sensitivity, and lack of control of the voltage across the cell membrane, making it possible to study only certain types of ion channels and rather simple ways of drugs interacting with the ion channels.
Automated patch clamp technologies
QPatch is Sophion’s product family for medium-throughput automated patch clamp, and Qube is the newest high-throughput automated patch clamp system. With the QPatch and Qube, patch clamp experiments are done on a planar ‘glass’ surface that creates a connection between the cell and the amplifiers that are very similar to the manual ‘gold standard’ patch clamping. QPatch and Qube operate automatically and these systems increase patch clamp throughput due to the high degree of parallelism.
QPatch and Qube therefore combine the accuracy of traditional patch clamp with the high throughput of indirect methods.
Pharmaceutical companies developing ion channel-based drugs have a great demand for automated patch clamp systems that can quickly deliver accurate results.
The drug discovery process in short
The drug discovery process in pharmaceutical companies can begin either with a certain target, e.g. a specific ion channel, or a certain disease and a search for relevant therapeutic targets to modulate. One important class of targets comprises ion channels. After a target validation phase, a test system (an assay) is developed to measure interactions with the target. Pharmaceutical companies have huge libraries of chemical compounds, the largest of which contain up to a couple of million compounds. One or more of these compounds may be identified as so-called hits with a desired effect on a specific target. From this hit, chemical modifications can make the identified molecule suitable as a drug. This process is iterative and called lead optimization. For every modification, new patch clamp tests are needed to check that the desired effect is preserved or improved. Further tests in disease relevant animal-models and safety test are carried out before the development in the clinical trials (test in humans) can lead to an approval by health authorities to market a new drug.
Finding the hits is like finding a needle in a haystack! For the last 20 years it has been routine to test either the entire compound library, or part of it, with indirect methods in a so-called High Throughput Screening (HTS) department in the pharmaceutical company that identifies the initial hits.
With Qube and downstream—The drug discovery revolution
The very low throughput of traditional patch clamp technology means that this technology is currently only used in the initial target identification and validation phases and in the final steps of lead optimization.
The process of ion channel drug discovery changes dramatically when automated patch clamp systems, such as QPatch and Qube, are employed systematically. Qube uses the 384-well format, which makes it suitable for the HTS segment, and QPatch has the high flexibility that makes it ideal for lead optimization. Qube performs direct electrophysiological tests and hence identifies more interesting hits with higher validity than the indirect methods. As a result, the hit validation can be shortened significantly or even eliminated and still identify lead compounds of higher quality. The result may be the development of more and also more efficient ion channel-based drugs, with a 20–50% shorter pre-clinical development timeline.
More and better leads—faster. That’s why we say that the combination of Qube and QPatch is leading a revolution in drug discovery.
QPatch and Qube are an automated patch clamp system for high-throughput electrophysiology measurements on ion channels. You can read more about ion channels below.
Ion channels are targets for selective drugs
Ion channels constitute a large and diverse group of membrane proteins that function as electrical signal transducers, and they govern the electrical properties of all living cells. For example, the coordinated activity of several ion channels underlies action potentials in excitable cells, such as those in the heart and brain.
Ion channels are generally heteromultimeric membrane proteins that constitute water filled passageways for ions across the phospholipid bilayer membrane. The physical pore is shaped by an assembly of several subunits, and the pore is lined with hydrophilic amino acid residues. A narrow region of the pore constitutes a ‘selectivity-filter’ that determines which ions can pass through the pore.
Ion channels may open and close in response to e.g. membrane potential (voltage-gated ion channels) or chemical (ligand-gated ion channels) stimuli.
Classification of Ion Channels
Each ion channel is characterized by its ion selectivity sequence. It may be highly specific for a single ion, or it may be less specific, conducting a few or several different ions. The selectivity is reflected in the common classification of the channels:
- K+ channels
- Na+ channels
- Ca2+ channels
- Cl– channels
- non-selective cation channels
Functionally, ion channels are broadly divided into voltage- and ligand-gated channels, referring to the type of physiological stimulus that activates the channel.
Diseases linked to Ion Channels
A multitude of human and animal diseases are caused by ion-channel dysfunction. This may be genetic, i.e. caused directly by mutations in genes coding for ion channels. Such diseases are called ‘channelopathies’. Examples of channelopathies are cystic fibrosis, epilepsy, and cardiac arrhythmias, e.g. the long QT syndrome. Diseases may also result from defects caused by mutations in genes coding for proteins that regulate ion channels.
Alternatively, ion channels may be involved in non-genetic diseases, e.g. diarrhea, which is mediated by toxicological effects on ion channel function.
Ion channels as drug targets
The search for new, potent and selective drugs that interact with specific ion channels is strongly technology driven and focused on high-throughput screening. Active substances from these high-throughput screens are further analyzed in functional studies, such as patch clamp. This development towards screening at the molecular level has been enabled primarily by: 1) the cloning and expression of relevant ion channels in cell lines, and 2) novel biological high-throughput screening techniques.
Technologies for ion channel characterization
The only direct way of validating the effect of a chemical entity on an ion channel is to measure the ionic current through the channel and determine whether the compound causes a change in this current. The patch clamp technique
has proven to be extremely useful in revealing many aspects of ion channel function. However, the traditional patch clamp technique has serious shortcomings in pharmaceutical discovery and screening, because the throughput is low, and it requires highly specialized personnel.
With the advent of automated patch clamp equipment these shortcomings are largely eliminated. QPatch and the Qube allow an operator without prior electrophysiological knowledge to conduct experiments. You can be a patch clamper too, with the help of the highly skilled application scientists at Sophion, or a skilled electrophysiologist in your staff!
The Patch Clamp Technique
Since its introduction by Professors Bert Sakmann and Erwin Neher in the mid 1970’s, patch clamp has been the classic method for studying ion channel function. It allows direct measurement of single channel currents as well as of the total current across the entire cell membrane. A glass micro-pipette, containing an electrolyte and an electrode, is pressed against the cell membrane, and a piece of membrane (the ‘patch’) is positioned within the pipette orifice. A tight seal of gigaohm electrical resistance (a ‘gigaseal’) is formed between the pipette rim and the cell membrane. If the patch contains ion channels then movement of ions through these channels is measured as tiny (picoampere) currents. The leak current across the seal is insignificant due to the high resistance of the gigaseal.
Specifically, five configurations may be employed:
- Cell-attached (on-cell): the pipette makes a gigaseal with the intact cell, allowing measurements of single-channel currents.
- Inside-out: cell-attached configuration is achieved and the pipette is withdrawn while the gigaseal is maintained, rupturing the cell. The inside (cytoplasmic) side of the membrane faces the bath fluid. This configuration is used for single-channel recordings with the ability to change the ‘intracellular’ solution.
- Whole-cell: the cell-attached configuration is achieved and vigorous suction is applied to the pipette, causing the patch to break. The cytoplasm and the pipette solution are subsequently in direct contact. After a short time, diffusion of cytoplasmic constituents (molecules and cell organelles) leads to identical (unphysiological) chemical composition of the fluids in the cell and in the pipette. The activity of all membrane ion channels is measured in this configuration.
- Outside-out: the whole-cell configuration is achieved and the pipette is gently withdrawn. This causes the membrane to break outside the sealing zone. The membrane fragments subsequently flip over, reseal, and constitute an inverted membrane patch exposing the extracellular side to the bath fluid. This configuration is used for single-channel recordings.
- Perforated whole-cell: the cell-attached configuration is achieved with pore-forming compounds (e.g. amphotericin B, nystatin) in the pipette solution. This causes perforation of the patch allowing small molecules and ions, but not larger compounds, to cross the patch. Consequently, larger molecules and cell organelles remain within the cell. The sum of all ion channel currents is measured, as with conventional whole-cell patch clamp.
Patch clamp has proven particularly powerful In combination with molecular biological techniques: specific ion channels may be expressed in cultured cell lines allowing a thorough characterization of their biophysical and pharmacological properties by patch clamp.
Patch clamp technologies for high-thoroughput screening
Unfortunately patch clamp is a low throughput technique. It is time-consuming and demands a skilled operator. The QPatch family and the Qube systems enable automated parallel ion channel screening with throughputs of hundreds or thousands of data points per day, enabling faster, and more accurate drug discovery.
The sophisticated automated patch clamp systems Sophion provides for high throughput electrophysiology measurements on ion channels is based on three components:
- Patch hole micro technology
- Patch clamp amplifier
- Assay software
These are critical to obtaining high-quality results, under control and with high throughput. You can read more about these technologies below.
Patch hole technology
We develop and manufacture the consumables for our instruments: QPlate, for use with QPatch, and QChip 384, for use with Qube. The heart of the consumables is the silicon-based patch hole, which replaces conventional glass pipettes. This is an advanced and exclusive technology developed and owned by Sophion. The patch hole enables the routine formation of genuine gigaseals, ensuring uncompromised data quality.
A unique feature of QPlate and QChip 384 is the compound application principle, which is based on an advanced microfluidic flow channel that leads compounds and buffers to the cell. Each site has its own flow channel that enables both fast and reproducible liquid exchange, effectively increasing throughput and reducing the cost per data point.
Furthermore, the measurement site is based on a sophisticated combination of glass and silicon that avoids problems with ‘sticky’ compounds. This makes our consumables ideal for experiments with both ligand-gated and voltage-gated ion channels.
Finally, the embedded silver-chloride electrodes ensure superior voltage control, reduce risk of cross contamination, and completely eliminate the need for maintenance.
Patch clamp amplifier
Sophion develops custom-made patch clamp amplifiers in-house. These feature automatic compensation for series resistance (Rs), cell capacitance (Cslow), and up to 300 nA maximal current measured, depending on amplifier type. This specification contributes to very high quality recordings that are regarded as a hallmark of Sophion products. We believe that amplifiers are so vital in obtaining reliable and reproducible ion channel recordings that we need full control, by in-house development utilizing the accumulated experience of our experts. We are convinced that our in-depth understanding of how amplifiers should be integrated into the patch clamp instrument means that we can offer the best amplifier solution on the market.
From the platform of the screening station, a ‘bed-of-nails’ connects the patch clamp electrodes directly with the amplifier front ends. This distance is critical and minimized to reduce any noise pick-up from the surroundings.
Several parallel patch clamp amplifiers are implemented on a standard printed circuit board, enabling all amplifiers to be positioned near the patch clamp electrodes. Due to their small size, these amplifiers are ideally suited for extensive parallelism and provide considerable scalability.
Sophion products contain a large amount of software, including robotic control software, embedded software and user-facing application software. Our software is developed by a dedicated team of software developers who are experts in their respective fields, such as signal processing, statistical data analysis, robotics, and user interfaces, and also have an in-depth knowledge of the application field – automated patch clamping. Through daily collaboration with our customers, we have gained valuable insights to the research, protocols, quality parameters, work flows and procedures used in electrophysiology groups and HTS labs. Sophion software is under continuous development, always with the final user in mind.
Advanced data analysis
Large patch clamp data sets are easily obtained with our instruments, necessitating rapid and automated data handling. Our Assay Software leads in the industry as it provides advanced data analysis on hundreds or thousands of experiments simultaneously, and maintains the high information content characteristic of patch clamp experiments.
Biolin Scientific, a CiPA partner, has a long-standing interest and extensive experience in cardiac ion channels. Our instruments are used for in vitro safety screening by major pharmaceutical companies and CRO’s worldwide and we are ready to assist you in setting up your CiPA assays.
Brochure: All this talk about CiPA
Article: CiPA assays on QPatch
Article: MICE Model in Drug safety using QPatch
The Comprehensive in vitro Proarrhythmia Assay (CiPA) is a proposal by the FDA, HESI, CSRC and SPS ultimately aimed at revising the non-clinical ICH-S7B and replacing the clinical ICH-E14 guidelines. The S7B and E14 guidelines were implemented 2005 in response to reports of certain drugs causing TdP and these guidelines have been successful in the respect that no drugs have been withdrawn from market for being proarrhythmic after 2005. However, the connection between proarrhythmia and QTc prolongation is complex and depends on several other factors in addition to drugs blocking the hERG channel and while QTc prolongation is a sensitive marker for proarrhythmia it is also moderately specific (i.e. torsadogenic compounds prolong QTc but not all QTc prolonging drugs are torsadogenic).
The intention of the CiPA proposal is to increase the efficacy of the drug development process by 1) moving the evaluation of proarrhythmic risk to an earlier stage in the drug development process, and 2) enabling compounds with properties that today are considered as problematic to be further developed and 3) provide a stronger scientific foundation for improved future drug labeling. The CiPA proposal is based on a mechanistic understanding of proarrhythmic risk and is built around a three-component process:
- candidate drugs are tested in multiple and standardized ion channels assays using overexpressing cell lines. This proposal includes Nav5 (peak and late currents), Kv4.3 (Ito), hERG (IKr), KvLTQ1/mink (IKs) and Kir2.1 (IK1),
- the data from the ion channel assays are used in a computational model of a cardiomyocyte action potential model to see if the compound yields proarrhythmic markers. This model is calibrated using data from well characterized reference compounds,
- the results from the in silico simulations are verified using iPS derived cardiomyocytes
The FDA has proposed a revision of the S7B by June 2016 but there are still many questions regarding protocols, validation, translation and more that still awaits answers. and it is difficult to say when it is finally implemented.
Biolin Scientific is a CiPA partner and has a long-standing interest and extensive experience in cardiac ion channels. Our instruments are used for in vitro safety screening by major pharmaceutical companies and CRO’s worldwide and we are ready to assist you in setting up your CiPA assays.
CSRC Cardiac Safety Research Consortium
FDA Food and Drug Administration
HESI Health and Environmental Science Institute
SPS safety Pharmacology Society
TdP torsade de points
Please download our CiPA brochure CiPA Brochure.