PIC50 PERSONAL BIO-ASSAY SYSTEM

Screening Bottlenecks On average the Medicinal Chemist in a large pharma waits between 5-10 days for potency data on their compounds. During this time they have no feedback on how well the compounds have addressed the target. From a work flow perspective, this means that if the compounds take a week to finalise, then the iterative process is slowed to at most, one increment per week. By the use of genapta PIC50 rapid assessment tool, potency data can be available in a few hours and thus the Med. Chem. can formulate the next iteration based on real potency data. This has the following advantages:
FORMULATE COMPOUND FORMULATE COMPOUND 1-2 HOURS
FORMULATE RELATED COMPOUNDS

POTENCY SCREEN

1-2 WEEKS POTENCY SCREEN BASIC TOX

(A)

(B)

Able to shave months off discovery

phase.

Increase number of iterations from

tens to hundreds per annum.

Formulate all compounds based on Less of a reluctance to drop series

which may have problems in later stage.
The genapta PIC50 assay is shown alongside its pumping system. The system is designed to independantly control all reagents down to an accuracy of a few nano-litres per minute (Photo courtesy Cambridge University, Department of Chemistry).

Panel (A) shows the current process flow in the medicinal chemistry laboratory, where a compound is formulated, then past to the screening group for assay. At best this process takes ~1-2 weeks, other related compounds may be formulated in an attempt to increase the chances of a hit, or an improvement over previous iterations. This leads to ~20-30 iterations per annum, per investigator being possible. In contrast panel (B) shows what is possible with the genapta PIC50 system. Here basic IC50 data is generated in less than a couple of hours leading to the chemist being able to formulate the next compound based on real potency data. It is also possible to run basic p450 in the machine which means the next compound in the series maybe both optimised for potency and TOX. Thus the possible number and quality of iterations rises dramatically.
10-490nl/Min
COMPOUND

feedback from previous. As such the need for library formulation is reduced.

Possible to look at simple CYP450 assay using same methodology. Methodology For a competitive IC50 experiment, the method of continuous dilution-mixing on a microfluidic chip is used. Using state of the art nano-flow pumping system, the compound would be diluted by introducing it to and mixing it with a stream of buffer. Here the buffer is pumped over a 50X gradient in opposition to a 50X gradient on the compound. In this way the compound is diluted over a ~2.5K range with a constant overall flow. In turn this is introduced to the enzyme-substrate reaction mix. In all the concentration of the compound is dependent on the initial starting concentration of the compound solution. As all the mixing takes place on the chip, the systems equillibriates quickly allowing the measument to take place soon afterward. The optical measurement system is coupled directly into the chip. It can look at any of the standard fluorescent read-out techniques (see facing panel) and the system uses minor modification of standard reagent systems. This leads to the system being complementary to plate based assays.

Microfluidic Mixing The device uses real-time mixing in 20M microfluidic channels in order to promote the biochemical reactions. In these contstrained dimensions interdiffusional mixing occurs on a short timescale. As a result it is able to measure the binding reactions of an enzyme and substrate in response to a varying background of compound. In this way both static and dynamic measurement modes are possible, the latter allowing the evolution of the binding process to be monitored. Confocal Fluorescence Spectroscopy Using genapta patented fiber optic based detection module, sub-picolitre volumes can be probed. Furthermore the multicolour capability allows a number of different aspects of the reaction to be investigated. The following mesurement methods are available: Fluorescence Resonant Energy Transfer (FRET) Dual channel Fluorescence Polarisation (DFP) Time resolved, sub-picosecond fluorescent intensity decay
50 45 40 35 30 25 20 15 10 5 0 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 [Fluorescein] (nM) 100 80 60 40 20 0 FP Par 530 nm Fluorescence (KHz)

490-10nl/Min
BUFFER SOLUTION

250nl/Min
ENZYME

500nl/Min

250nl/Min
SUBSTRATE

1l/Min

OPTICAL MEASUREMENT

REAGENT WASTE

About genapta Limited genapta Limited is a spin-out from Cambridge University. Founded by a group of engineers and biologists, the company aims to apply breakthrough technologies in science and engineering to the field of life sciences. genapta has a portfolio of projects and works predominantly with university and large pharma to bring new technologies into the laboratory.

Comparison to conventional methods In comparison to conventional plate based methodologies, the genapta screening system offers a number of advantages; The system is small enough to sit on the lab bench and as such is accessible to the biologist or medicinal chemist on an as needed basis. Due to the more controllable nature of microfluidic mixing the results are more consistant than plate based assays. In particular by its very nature the system does not suffer from 'carry-over' in the same way as a plate assay may do, plus the fast diffusional mixing is more effective at ensuring that all the measurements occur with the same overall conditions. Compound and protein usage is reduced. In the current system each data point is obtained using ~20nl of sample mixture. In contrast a 1536 well plate based reader will use ~5-10L of sample per measured data point. When losses in sample handling and flushing are taken into account, the device uses less than 1% of the material to obtain an equivelant data sets. Compounds do not have to await re-plating before they are introduced, they are directly injected into the system. As a consequence of the above, a whole level of laboratory automation is peeled away that is currently needed for replating samples, sealing plates, handling plates into and out of the readers and then finally re-sealing and disposing of the plates. As the conditions are dynamically configured by the pumping rates it is relatively straightforward to perform repeat measurements if interesting results are obtained.

FP (mP)

For more information contact;

genapta Limited William James House Cowley Road Cambridge, CB4 0WX United Kingdom

400 MOLECULES
Material Usage As an illustration of the sensitivity of the device the above shows the signal to noise performance of the device in FP mode. Here the concentration of the fluorophore is being reduced and the mP signal is being mesured. From the graph its evident that the signal is consistant to sub nM concentrations, which given the sub-picolitre probe volumes corresponds to ~1000 fluorophores being measured at any one time. This sensitivity is one of the major contributing factors to why the system is able to use >99% less material for the equivelant measurements

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