AU626315B2

AU626315B2 - Process and apparatus for fully automatic simultaneous synthesis of a plurality of different polypeptides

Info

Publication number: AU626315B2
Application number: AU40130/89A
Authority: AU
Inventors: Wilhelm Knapp; Gerd Schnorrenberg
Original assignee: Boehringer Ingelheim International GmbH
Current assignee: Boehringer Ingelheim International GmbH
Priority date: 1988-08-23
Filing date: 1989-08-22
Publication date: 1992-07-30
Anticipated expiration: 2009-08-22
Also published as: EP0355582B1; DK173633B1; DK412789A; DE3828576A1; JPH02167297A; ES2036309T3; EP0355582A3; DE58902869D1; ATE82983T1; DE3828576C2; AU4013089A; DK412789D0; GR3006611T3; DE8816749U1; EP0355582A2

Abstract

Process and apparatus for the completely automatic simultaneous synthesis of several polypeptides, where up to 96 different polypeptides are synthesised by the solid-phase synthetic method in a robot pipetter.

Description

I I S F Ref: 104147 FORM COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952 i COMPLETE SPECIFICATION

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Class Int Class Complete Specification Lodged: Accepted: Published: Priority: Related Art: Name and Address of Applicant: .4.

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9 D-6507 Ingelheim am Rhein FEDERAL REPUBLIC OF GERMANY Address for Service: Spruson Ferguson, Patent Attorneys Level 33 St Martins Tower, 31 Market Street Sydney, New South Wales, 2000, Australia Complete Specification for the invention entitled: Process and Apparatus for Fully Automatic Simultaneous Synthesis of a Plurality of Different Polypeptides The following statement is a full description of this invention, including the best method of performing it known to me/us 5845/3 1 Abstract Process and apparatus for the fully automatic simultaneous synthesis of different polypeptides, in which up to 96 different polypeptides are synthesised in a pipetting robot by the solid phase synthesis method.

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S 0 55 05 00 S 55 0 00 S 00 5S 0 C SO 0 CC 2 Rapid evaluation of structure-activity relationships of biologically active peptides by means of receptor binding studies and rapid epitope determination for immunology in peptides and proteins require relatively small amounts (less than 20 mg) of a number of peptides.

These peptides are conveniently prepared by solid phase peptide synthesis. This synthesis is based on the method developed by R.B. Merrifield Barany, R.B.

Merrifield in The Peptides, Analysis, Synthesis, Biology, Vol. 2, 3-284 (1980), published by Gross, Meienhofer Academic Press, New York), in which the peptide chain is built up step by step. The steps of synthesis can be summarised as follows: binding the first amino acid of the peptide chain via an anchor group to a polymeric support, 0*S@ b) stepwise addition by condensation of the other amino acids of the peptide chain, c) intermediate steps between the individual condensations consisting of washing, cleaving of s* Sprotective groups and neutralisation, d) if desired, acylation of terminal amino groups, e) cleaving of the peptide from the support.

This peptide synthesis will take up to 18 hours, usually up to 4 hours for each amino acid. (The individual condensation processes generally take 1 to 2 hours' reaction time; between the condensation processes, 10 intermediate steps are usually required, each of which will take about 2 to 15 minutes). Thus, the preparation of peptides consisting of a large number of amino acids is a very time consuming, labour intensive and expensive process.

-3 For solid phase synthesis of analogous peptides a method has been proposed by R.A. Houghten (Proc. Natl. Acad. Sci, USA, Vol. 82, pp.

5131-5135, August 1985, Immunology). The polymeric carrier for the synthesis is then transferred into small porous polypropylene bags in batches of 50-100 mg and the bags are sealed by fusion. The intermediate steps common to all the syntheses (washing, neutralisation, cleaving of protecting groups) are carried out on all the bags simultaneously in a single reaction vessel and the individual condensation processes are performed separately. The method mray be carried out manually or semiautomatically using a peptide synthesizer.

The disadvantage of the method described is that the bags are somewhat awkward to handle, they cannot be re-used, they have to be separated from each other for condensation of the various peptides and no samples can be taken for monitoring during the entire synthesis.

The aim of the invention is to provide a process and an apparatus which makes it possible to syntheses a plurality of different polypeptides simultaneously by an automated method and which avoids the disadvantages described above.

This is achieved by modifying the solid phase synthesis method described above so that it can be carried out using a suitably adapted pipetting robot.

According to a first embodiment of this invention, there is provided a process for fully automatic simultaneous synthesis of a plurality of different polypeptides by the solid phase synthesis method 25 using a pipetting robot, according to which polymeric support, which may be charged with the C-terminal amino acid or with the initial portion of the desired peptide, is placed in the wells of a microtiter plate, then using the solid-phase synthesis method which is known per se the peptides are synthesised in the wells and if desired free amino groups and/or hydroxy groups of the peptides are acylated and/or subsequently the peptides are cleaved from their polymeric support, the reagents or washing liquids required for the individual steps being introduced into the wells by means of one or more robot arms with a cannula from the corresponding storage vessels and after the required retention time of the reagents or washing liquids the liquids contained in the wells and those lying above the carrier material are sucked up by means of a robot "EH/0880Z 4 3A arm with a cannula, the individual steps of the process being controlled by the program in the computer which is connected to the robot.

According to a second embodiment of this invention, there is provided a cannula for a pipetting robot, wherein the upper end of the cannula is connected to the metering means of the robot arm, and wherein the cannula is provided with a rinsing device the upper end of which is connected with the robot arm and the lower end of which extends to just underneath the lower end of the cannula, the opening of said rinsing device being arranged so that the lower end of the cannula can be rinsed.

Pipetting robots have been used up till now for series analysis.

For example, a pipetting robot made by Messrs. TECAN, RSP 5052, may be used.

Pipetting robots have the following external components: at least one arm with a metering pipette, a clamp with storage vessels and a microtiter plate which may contain up to 96 wells. The robot arm brings the reagents from the storage vessels and transfers them into the associated wells in the microtiter plate and if necessary sucks liquids up out of the wells. The cannula of the metering pipette can also be constructed eQ

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EH/0880Z 4 so that it is divided into two parts by a partition wall extending from top to bottom. (With this divided cannula it is possible to supply two different substances, or one supply and one removal, with the same arm). The operation of the apparatus is controlled by a computer program.

According to the invention, solid phase peptide synthesis is carried out using a pipetting robot of this kind as follows: Polymeric support (preferably granulated resin) is placed in the wells of a microtiter plate.

The carrier material may be charged with the initial part of the desired peptide. The liquids required for .6 *e the reactions and washing steps are held in readiness in the storage vessels of the apparatus. If the peptide is to be removed from the carrier at the end of synthesis oroo and/or if free amino groups are to be acylated, the reagents required for these reactions are also kept in readiness in the storage vessels. The reaction times necessary show that it is convenient to use a microtiter plate containing not more than 96 wells. Accordingly, not more than 96 different polypeptides can be synthesized in a single program. In accordance with the program, which has been adapted to the synthesis of these peptides, the robot places the reagents and washing liquids in the individual wells and, after the required retention time, sucks up the liquid lying over the support.

Using the two-armed pipetting robot RSP 5052 made by Messrs. TECAN the process and the necessary adaptation of the apparatus to the process will be explained more fully. However, the application of the process is not restricted to this apparatus. Pipetting robots of other kinds, particularly one- or multi-armed robots may also be adapted to the process according to the invention.

A microtiter plate with 96 wells is used. One well contains for example 10 mg of resin which may be charged with an amino acid and rather more than 300 p1 of liquid. This quantity of resin corresponds to about gmol of amino acid or is suitable for the preparation of about 5 Amol of peptide. It is possible to use conventional carrier materials based on polystyrene or polyacrylamide. It is expedient to synthesise peptides containing not more than 20 amino acids. The reagent solutions and washing liquids required for the process are kept in readiness in the storage vessels provided for this purpose. Arm 1 of the apparatus is equipped with a metering pipette and arm 2 with a suction cannula 00e provided with rinsing means. The rinsing means are 2 preferably connected to a separate storage vessel for the solvent used. Synthesis is carried out in accordance with the program in the attached PC.

With the arm 1, all the reagent solutions are added, taken from open storage vessels. Before the metering pipette changes from one reagent solution to another, the metering pipette is rinsed in a special rinsing position with solvent. With the arm 2, the o, reagent and washing liquids are sucked up through a cannula provided with a filter. To prevent resin losses .0 and contaminations of adjacent wells, the outside of this cannula is washed after each suction process with solvent, by means of a feed line mounted on the side of the cannula. At the same time the next washing process is begun with this solvent. The cannula is then rinsed Soff in a cannula rinsing position.

In order to cleave the peptide from the resin, trifluoroacetic acid for example is introduced into the wells by means of the arm 1.

After cleavage has been carried out, the solution is sucked up by means of the suction cannula and transferred into a second microtiter plate from where it is worked up.

As mentioned hereinbefore, the suction cannula may 09 a Qa 4: 0o 16 4. 'o 6 be fitted with a filter. This filter is provided at the lower end of the cannula and prevents resin from being sucked up. Expediently, the filter will consist of stainless steel gauze secured, for example, with a ring of solder. Another possibility is to fit a sintered metal plate or sintered ceramic plate into the opening in the cannula. If a cannula is used without a filter, the sucking up of resin can be largely prevented by carrying out the suction process slowly. However, the time taken for the suction process will be increased substantially as a result.

The outside of the suction cannula is rinsed with solvent after each suction process. This is expediently carried out by feeding the solvent through a feed line (hose or tube) extending along the longitudinal side of the cannula and mounted in such a way that its opening is just above the lower end of the cannula. It is important that the lower end of the cannula should be rinsed right round with solvent.

The arm 2 of the apparatus may be equipped with a comb instead of the suction cannula. This comb will have a plurality of suction cannulae (usually 4-12 cannulae) of the type described above. With a comb of this kind, several wells can be served at the same time., which results in a considerable time saving.

Usually, DMF or N-methylpyrrolidone is used as solvent. Accordingly, the wells and any rinsing means provided must be made from solvent-resistant material such as polypropylene or Teflon. In standard commercial equipment, the metering pipettes and suction cannulae are made of stainless steel. This material is suitable for the process according to the invention.

If it is desired to use a larger quantity of resin 50 mg of resin) for each peptide in this method of synthesis, microtiter plates with larger wells have to be used. These microtiter plates are standardised and commercially available. They will then have 7 correspondingly fewer wells.

Instead of a two-armed pipetting robot it is also possible to use a one-armed apparatus. In this case, the suction cannula must have a subdivision in its free space. One part will then be used to suck up the washing and reagent solutions whilst the other part of the cannula performs the function of the arm 1 of the two-armed apparatus, i.e. it serves to supply the reagent solutions. The sucking up of resin will be prevented in the same way as in the two-armed apparatus.

The cannula will also be rinsed as described above.

The process is carried out for example with the o ofollowing substances (quantity specified is for each well): the starting material is 10 mg of resin charged ***ewith Fmoc-amino acids (particle size 200-400 mesh); Fmoc-protected amino acids are used in an excess which Soo: may be up to a 10 fold excess for each individual coupling step, i.e. 200 gl of a DMF solution of 50 gmol of Fmoc-amino acid and 50 .mol of l-hydroxybenzotriazole and 100 gl of a DMF solution of 75 gmol of N,N-

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0 "SC dicyclohexylcarbodiimide are added; the coupling time is about 1 hour. The Fmoc protecting group is split off using 300 il of a 40% solution of piperidine in DMF.

The time taken is about 20 minutes. The washing steps are carried out with 300 g.l of DMF each time.

00 .The separation of the finished peptide from the 0" support may be carried out in the wells by the manual or automatic addition of 300 gl of trifluoroacetic acid minutes' reaction time). The acylation of free groups

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2 OH) can be effected analogously by the addition of suitable acid anhydrides, e.g. acetic anhydride, aid pyridine. After these reactions have ended, the solution is removed by suction and taken for working up.

As will become apparent from the above explanation, all the steps of the peptide synthesis are carried out in open vessels. Nevertheless, because of the method of synthesis according to the invention, the peptides 8 obtained are extremely pure.

Figure 1 shows an example of a cannula for a onearmed pipetting robot: a cannula; and a feed side and suction side of the cannula; a tube for supplying the solvent for rinsincg a filter.

The Example which follows illustrates the process of the method, describing the synthesis of a peptide in a well. The purity of the product should be noted.

Example: 44 different undecapeptides are synthesised using the Tecan pipetting robot RSP 5052. In each case the starting material is a tetrapeptide bound to polystyrene via a linker, namely Fmoc-Arg(Mtr)-Gln- Arg(Mtr)-Tyr(tBu)-linker polystyrene (charge approximately 0.5 mmol/g of resin). 44 times 10 mg of this resin were introduced into the wells of a o** microtitre plate (5 pmol of peptide) and synthesis was begun. By way of an example, the synthesis of Ac-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr-NHz is described. The other 44 peptides were synthesised analogously.

Synthesis cycle: 300 il of piperidine in DMF (5 min), 300 Al of piperidine in DMF (20 min), washing 10 times with 300 gl of DMF (2 min), 200 il of a DMF solution containing 50 imol of Fmoc-Thr(tBu)-OH and 50 Umol of HOBt, 100 L1 of a DMF solution containing 75 imol of DCC (lh), washing 10 times with 300 /1l of DMF (2 min), In the same way, equally concentrated solutions of Fmoc-Ile-OH Fmoc-Leu-OH Fmoc-Asn-OH goof

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Fmoc-Ile-OH Fmoc-Tyr(tBu)-OH Fmoc-His(Trt)-OH were used in identical synthesis cycles. The final acetylation-was carried out after Fmoc cleaving in the usual way with 300 pl of a DMF solution containinq 40 umol of acetic anhydride and pyridine. Cleavage from the resin was achieved using 300 p1 of trifluoroacetic acid/5 anisole for minutes, washing 5 times with 300 il of trifluoroacetic acid. The combined trifluoroacetic acid solutions were left to stand for 1 hour at 50*C with the exclusion of moisture and the trifluoroacetic acid was distilled off in vacuo. The residue was treated with ether in an ultrasonic bath, the ether was decanted off, the residue was taken up in water and freeze-dried. HPLC chromatogram (RP18 column) (Fig. 2).

Eluant A water/acetonitrile/trifluoroacetic acid 95/5/0.2 Eluant B water/acetonitrile/trifluoroacetic acid 20/80/0.2 FAB mass spectrogram: M+H 1518 0 0Sr

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Claims

1. Process for fully automatic simultaneous synthesis of a plurality of different polypeptides by the solid phase synthesis method using a pipetting robot, according to which polymeric support, which may be charged with the C-terminal amino acid or with the initial portion of the desired peptide, is placed in the wells of a microtiter plate, then using the solid-phase synthesis method which is known per se the peptides are synthesised in the wells and if desired free amino groups and/or hydroxy groups of the peptides are acylated and/or subsequently the peptides are cieaved from their polymeric support, the reagents or washing liquids required for the individual steps being introduced into the wells by means of one or more robot arms with a cannula from the corresponding storage vessels and after the required retention time of the reagents or washing liquids the liquids contained in the wells and those lying above the carrier material are sucked up by means of a robot arm with a cannula, the individual steps of the process being controlled by the program in the computer which is connected to the robot. Process according to claim 1, in which the outside of the .cannula is rinsed between the sucking up of the liquid and the addition 20 of the reagents. Process according to claim 1 or 2, in which the reagents are used in an excess of up to 20 times the stoichiometric amount.

4. A cannula for a pipetting robot, wherein the upper end of the cannula is connected to the metering means of the robot arm, and wherein 25 the cannula is provided with a rinsing device the upper end of which is connected with the robot arm and the lower end of which extends to just underneath the lower end of the cannula, the opening of said rinsing device being arranged so that the lower end of the cannula can be rinsed. The cannula according to Claim 4, wherein the lower end of the cannula is provided with a filter.

6. The cannula according to Claim 4 or 5, wherein the bore of the cannula is divided into two portions by a partition wall extending from top to bottom.

7. A cannula comb member composed of a plurality of cannulae according to any one of Claims 4 to 6. KEH/088Z KEH/0880Z i 1 0 11

8. Use of a pipetting robot for the fully automatic simultaneous synthesis of a plurality of polypeptides following the solid-phase synthesis technique.

9. The product of the process of any one of claims 1 to 3.

10. Process for fully automatic simultaneous synthesis of a plurality of different polypeptides by the solid phase synthesis method using a pipetting robot, substantially as hereinbefore described with reference to the Example.

11. Cannula for a pipetting robot, substantially as hereinbefore described with reference to Figure 1 of the accompanying drawings. DATED this FOURTH day of MAY 1992 Boehringer Ingelheim International GmbH Patent Attorneys for the Applicant SPRUSON FERGUSON