pH stat titration for efficient enzyme screening

Hoffmann-La Roche with headquarters in Basle, Switzerland, is one of the leading companies in the world among those firms carrying out health sector research in the fields of pharmaceuticals, diagnostics and vitamins.

The innovative products and services provided by Roche cover the prevention, diagnosis and therapy of illnesses and thus make a contribution to strengthening the feeling of well-being and to improving the quality of life. Introduction.

The complexity of the molecular structure of active substances has increased continually in recent times; at the same time the demands placed on new medicaments by the approval authorities have also increased.

Today new active substances must have an exactly defined spatial configuration of their atoms; these substances often contain stereocenters and are optically active.

The preparative use of enzymes - highly selective biocatalysts whose particular characteristic is to react only with compounds showing a strictly defined three-dimensional structure ) - represents one possible way of fulfilling these high synthetic requirements.

Automated screening of enzymes and reaction conditions for the efficient development of new biotransformations.

The pharmaceutical research laboratory has the task of determining the most suitable enzyme for a particular biotransformation and of subsequently optimising the reaction conditions.This initially requires laboratory tests for selecting the active enzymes from the large number of biocatalysts available.

In order to achieve throughputs that are as high as possible these tests have been automated with the help of modern robot systems.

A simple and rapid test used for the selection of active hydrolases - the most frequently used enzymes - is based on the measurement of the color change of a pH indicator that is caused by acids formed during the hydrolysis reaction The technical feasibility of an enzymatic synthesis is often only achieved by making the correct choice of the reaction parameters. It is therefore necessary to further investigate the selected active enzymes and measure their catalytic performance under varying reaction conditions.

This is carried out by titrating the acid formed during the enzyme reaction with a completely automated pH stat system.

The two primary characteristics of the enzymatic synthesis, the activity (conversion per unit of time) and the selectivity of the particular enzyme, are obtained from the titration curve and by determining the spatial configuration of the product formed.

The target compound is then synthesised for research purposes on a laboratory scale under the conditions that have previously been determined and the optimised enzyme reaction is transferred to the process development division.

The automated pH stat titration system in detail.

The Metrohm system used for the automatic determination of the enzyme activities consists of a 730 Sample Changer, a 736 GP Titrino with 1ml exchange unit, two 729 Dosimat Interfaces, a total of eight 700 Dosinos (pipetting the enzyme samples, adding the substrate solution and various auxiliary solutions, etc) and a PC.

The Metrodata TiNet 2 software controls the system.

On the first tower of the sample changer a 759 Swing Head with pipetting tip is mounted.

The titration head of the second workstation is equipped with a combined pH glass electrode, a temperature sensor and a 722 Rod Stirrer with PTFE propeller.

It also carries the buret and dosing tips, the rinsing nozzles and an aspiration tip. Two different sample racks are used for the enzyme studies.

In order to verify the results of the pH indicator test a renewed screening of the selected enzymes is first carried out.

This is done using a standard rack, containing 126x15ml sample vessels and two 250ml beakers (titration and rinsing vessels).

For the subsequent optimization of the reaction parameters a special sample rack has been developed.

This provides space for five titration vessels, a rinsing beaker (cleaning the titration head) and two 75ml glass vessels containing solvents (cleaning the pipetting tip).

The rack also accommodates 24 test tubes containing the enzymes to be investigated as well as additives, eg salt solutions or organic solvents.

How the analysis works. The active enzymes to be investigated are weighed into the sample vessels in the form of aliquots of the solid substances and these are placed in the sample changer rack.

The subsequent sample preparation, the sample transfer, the pH stat titration including the evaluation and calculation of the results as well as the rinsing and cleaning processes then take place fully automatically.

The enzyme sample is first dissolved by adding a buffer solution.

A defined volume is removed from the sample vessel and transferred to the titration vessel using the pipetting equipment consisting of a 700 Dosino with Dosing Unit, a 10ml transfer tube and a pipetting tip.

Afterwards buffer solution is added and the initial pH value is adjusted, then the reaction is started by the addition of a defined volume of substrate solution (0.1-1.0ml).

In the reaction phase the pH value is held constant under vigorous stirring by adding sodium hydroxide solution (pH stat titration) and the NaOH consumption is recorded as a function of time.

The reaction is terminated when either a given conversion (NaOH consumption) has been achieved, if the reaction rate (NaOH consumption per unit of time) is too small or after the maximum permitted reaction time has elapsed.

This is done by lowering the pH value of the reaction solution and/or adding a deactivating solvent.

If the result is satisfactory, ie, if the enzyme shows a sufficiently high activity, then an aliquot of the reaction solution is removed and transferred to a clean vessel on the sample rack for further analysis.

Thorough cleaning of the sensors, propeller stirrer, tubing and burette and dosing tips is then carried out. The titration vessel is rinsed out and emptied several times, after which the system is ready for the next analysis.Using the standard rack it is possible to investigate up to 120 enzymes in a continuous run with the automated pH stat system; the time required per enzyme sample varies between one and ten hours.

In a similar way the selected enzyme reactions are then optimized by systematically varying the chemical and physical reaction conditions (pH value, volume of substrate solution used, addition of organic solvents or salt solutions).

The special rack with five titration vessels is used for this.

Results and summary.

The combination of the simple color test with the automated pH stat titration produces new biotransformations for the synthesis of optically active target substances very rapidly and efficiently. The pH indicator test allows the complete screening of all biocatalysts in one to two days.

With the help of the automated titration system the active enzymes selected in this way can then be subjected to further systematic investigations.

By deliberately varying the chemical and physical reaction parameters it is possible to successfully optimise the enzyme reaction for technical use relatively quickly.

Whereas before the introduction of the automated system a limited, rationally selected number of enzymes was tested, today the systematic screening of all available biocatalysts is carried out; this is rewarded by the unexpected discovery of successful 'outsider' enzymes.