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DK173633B1 - Method and cannula for pipetting robot for fully automatic simultaneous synthesis of multiple polypeptides - Google Patents

Method and cannula for pipetting robot for fully automatic simultaneous synthesis of multiple polypeptides Download PDF

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DK173633B1
DK173633B1 DK198904127A DK412789A DK173633B1 DK 173633 B1 DK173633 B1 DK 173633B1 DK 198904127 A DK198904127 A DK 198904127A DK 412789 A DK412789 A DK 412789A DK 173633 B1 DK173633 B1 DK 173633B1
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cannula
wells
peptides
robot
reagents
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DK198904127A
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DK412789A (en
DK412789D0 (en
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Gerd Schnorrenberg
Wilhelm Knapp
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Boehringer Ingelheim Kg
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K1/00General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C07K1/045General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers using devices to improve synthesis, e.g. reactors, special vessels
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277Apparatus
    • B01J2219/00279Features relating to reactor vessels
    • B01J2219/00306Reactor vessels in a multiple arrangement
    • B01J2219/00313Reactor vessels in a multiple arrangement the reactor vessels being formed by arrays of wells in blocks
    • B01J2219/00315Microtiter plates
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277Apparatus
    • B01J2219/00351Means for dispensing and evacuation of reagents
    • B01J2219/00364Pipettes
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277Apparatus
    • B01J2219/00351Means for dispensing and evacuation of reagents
    • B01J2219/00423Means for dispensing and evacuation of reagents using filtration, e.g. through porous frits
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277Apparatus
    • B01J2219/00497Features relating to the solid phase supports
    • B01J2219/005Beads
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583Features relative to the processes being carried out
    • B01J2219/00585Parallel processes
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583Features relative to the processes being carried out
    • B01J2219/0059Sequential processes
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583Features relative to the processes being carried out
    • B01J2219/00596Solid-phase processes
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/0068Means for controlling the apparatus of the process
    • B01J2219/00686Automatic
    • B01J2219/00691Automatic using robots
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718Type of compounds synthesised
    • B01J2219/0072Organic compounds
    • B01J2219/00725Peptides
    • CCHEMISTRY; METALLURGY
    • C40COMBINATORIAL TECHNOLOGY
    • C40BCOMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00Libraries per se, e.g. arrays, mixtures
    • C40B40/04Libraries containing only organic compounds
    • C40B40/10Libraries containing peptides or polypeptides, or derivatives thereof
    • CCHEMISTRY; METALLURGY
    • C40COMBINATORIAL TECHNOLOGY
    • C40BCOMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B60/00Apparatus specially adapted for use in combinatorial chemistry or with libraries
    • C40B60/14Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Analytical Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Automatic Analysis And Handling Materials Therefor (AREA)

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 DK 173633 B1in DK 173633 B1

Opfindelsen angår en fremgangsmåde til fuldautomatisk simultan syntese af flere polypeptider ifølge faststofsyntesemetoden samt en kanyle til pipetteringsrobot.The invention relates to a method for fully automatic simultaneous synthesis of multiple polypeptides according to the solid synthesis method as well as a pipetting robot needle.

5 Til hurtig bedømmelse af struktur-virkningsfor- hold af biologisk aktive peptider ved receptor-bindingsundersøgelser og hurtig epitop-undersøgelse for immunologien ved peptider og proteiner anvendes relativt små mængder (mindre end 20 mg af hver) af mange 10 forskellige peptider. Fremstillingen af disse peptider sker hensigtsmæssigt ifølge fastfasepeptidsyntesen.5 For rapid assessment of structure-effect relationships of biologically active peptides in receptor binding studies and rapid epitope study for the immunology of peptides and proteins, relatively small amounts (less than 20 mg of each) of many 10 different peptides are used. The preparation of these peptides is conveniently done according to the solid phase peptide synthesis.

Denne syntese er baseret på den af R.B. Merrifield udviklede metode (G. Barany, R.B. Merrifield i The Peptides, Analysis, Synthesis, Biology, vol. 2, 3-284 15 (1980), udgivet af Gross, Meienhofer Academic Press,This synthesis is based on that of R.B. Merrifield developed method (G. Barany, R.B. Merrifield in The Peptides, Analysis, Synthesis, Biology, vol. 2, 3-284 15 (1980), published by Gross, Meienhofer Academic Press,

New York), hvorved peptidkæden opbygges trinvis. Syntesetrinene kan sammenfattes som følger: a) Binding af den første aminosyre i peptidkæden 20 til et polymert bæremateriale via en ankergruppe, b) trinvis tilkondensering af peptidkædens øvrige aminosyrer, 25 c) mellemliggende trin mellem de enkelte kondensationer bestående af vask, fraspaltning af beskyttelsesgrupper og neutralisering, 30d) eventuelt acylering af endestillede aminogrup- per, og e) fraspaltning af peptidet fra bæreren.New York), which builds up the peptide chain in stages. The synthesis steps can be summarized as follows: a) Binding of the first amino acid in peptide chain 20 to a polymeric carrier via an anchor group, b) stepwise condensation of the other amino acids of the peptide chain, c) intermediate steps between the individual condensations consisting of washing, cleavage of protecting groups and neutralization, 30d) optionally acylation of terminated amino groups, and e) cleavage of the peptide from the carrier.

2 DK 173633 B12 DK 173633 B1

Ved denne peptidsyntese skal man regne med en syntesetid på op til 18 timer,, for det meste op til 4 timer pr. aminosyre. (De enkelte kondensationer behøver for det meste 1 til 2 timers reaktionstid; mellem 5 kondensationerne kræves som regel ca. 10 mellemliggende trin, hvortil man skal påregne ca. 2 til 15 minutter pr. trin). Fremstillingen af peptider med et større antal aminosyrer er således meget tidsrøvende, arbejdskrævende og dyr.For this peptide synthesis, a synthesis time of up to 18 hours, usually up to 4 hours per day, is to be expected. amino acid. (The individual condensations usually require 1 to 2 hours of reaction time; between the 5 condensations usually require about 10 intermediate steps, to which should be estimated about 2 to 15 minutes per step). Thus, the production of peptides with a greater number of amino acids is very time consuming, labor intensive and expensive.

10 Til fastfasesyntesen af analoge peptider har R.A. Houghten (Proc. Natl. Acad. Sci., USA, Vol. 82, side 5131-5135, august 1985, Immunology) beskrevet en metode. Ifølge denne fyldes polymert bæremateriale til syntesen i små porøse polypropylenposer i portioner på 15 50-100 mg, poserne smeltes sammen, de fælles mellemtrin ved synteserne (vask, neutralisering og fraspaltning af beskyttelsesgrupper) udføres samtidig på alle poser i en samlet reaktionsbeholder, og de enkelte kondensationer udføres adskilt. Metoden kan udføres manuelt eller 20 delvis automatisk ved anvendelse af et peptidsynteseap-parat.10 For the solid phase synthesis of analogous peptides, R.A. Houghten (Proc. Natl. Acad. Sci., USA, Vol. 82, pages 5131-5135, August 1985, Immunology) described a method. According to this, polymeric carrier material for the synthesis is filled into small porous polypropylene bags in portions of 50-100 mg, the bags are fused together, the common intermediate steps of the syntheses (washing, neutralization and cleavage of protecting groups) are carried out simultaneously on all bags in a single reaction vessel, and the individual condensations are carried out separately. The method can be performed manually or partially automatically using a peptide synthesizer.

Ulempen ved den beskrevne metode ligger i, at håndteringen af poserne er en smule omstændig, at poserne ikke kan genanvendes, at poserne skal sorteres 25 til kondensation af de forskellige peptider, og at der ikke kan tages kontrolprøver under syntesen.The disadvantage of the method described is that the handling of the bags is a bit complicated, that the bags cannot be reused, that the bags have to be sorted for condensation of the various peptides and that no control samples can be taken during the synthesis.

Opfindelsens formål er at tilvejebringe en fremgangsmåde og en kanyle, der muliggør automatisk simultan syntese af flere polypeptider og undgår de oven-30 nævnte ulemper.The object of the invention is to provide a method and a cannula which enables automatic simultaneous synthesis of multiple polypeptides and avoids the above-mentioned disadvantages.

Opgaven løses ved, at den i forvejen nævnte fastfasesyntesemetode varieres således, at den kan udføres ved hjælp af en tilsvarende tilpasset pipette- DK 173633 B1 3 ringsrobot. Pipetteringsrobotter er hidtil anvende til serieanalyser. Eksempelvis kan pipetteringsrobotten, RSP 5052 fra firmaet TECAN, anvendes.The task is solved by varying the aforementioned solid phase synthesis method so that it can be carried out by means of a correspondingly adapted pipette-robot robot. Pipetting robots have so far been used for serial analysis. For example, the pipetting robot, RSP 5052 from the company TECAN, can be used.

En pipetteringsrobot har følgende ydre bestand-5 dele: Mindst én arm med doseringspipette, én holder med lagerbeholdere samt én mikrotiterplade, der kan indeholde op til 96 brønde. Robotarmen bringer reagenserne fra beholderne ud i mikrotiterpladens enkelte brønde og suger, når det er nødvendigt, væsker ud af 10 brøndene. Doseringspipettens kanyle kan også være fremstillet således, at den er adskilt i to dele af en fra oven og nedefter løbende skillevæg. (Ved denne delte kanyle er det muligt at udføre to forskellige doseringer eller en dosering og en frasugning med en 15 arm). Apparatets arbejdsforløb styres af et computerprogram.A pipetting robot has the following outer components: At least one arm with metering pipette, one holder with storage containers, and one microtiter plate that can hold up to 96 wells. The robot arm brings the reagents from the containers into the individual wells of the microtiter plate and, when necessary, sucks liquids out of the 10 wells. The metering pipette cannula may also be made so that it is separated into two parts by a dividing wall running from above and below. (With this divided cannula it is possible to perform two different dosages or one dosing and one suction with a 15 arm). The workflow of the device is controlled by a computer program.

Fastfasepeptidsyntesen ifølge opfindelsen foregår i en sådan pipetteringsrobot som følger: I mikrotiterpladens brønde anbringes bæremateri-20 ale (fortrinsvis granuleret bæremateriale). Bærematerialet kan være påhæftet med begyndelsesdelen af de enkelte ønskede peptider. De til reaktionerne og vasketrinene nødvendige væsker hældes på lagerbeholderne.The solid phase peptide synthesis of the invention is carried out in such a pipetting robot as follows: In the wells of the microtiter plate, support materials (preferably granulated support material) are placed. The carrier material may be attached to the starting portion of the individual desired peptides. The fluids needed for the reactions and washing steps are poured into the storage containers.

Hvis peptidet skal skilles fra bærematerialet efter 25 endt syntese, og/eller hvis frie aminogrupper skal acy-leres, skal de til disse reaktioner krævede reagenser også forberedes i lagerbeholderne. Ud fra de krævede reaktionstider ses det, at det er hensigtsmæssigt at anvende en mikrotiterplade, der ikke indeholder mere 30 end 96 brønde. Således kan der i et programforløb maksimalt syntetiseres 96 forskellige polypeptider. Ifølge programmet, der er tilpasset disse peptiders syntese, bringer robotten reagenserne og vaskevæskerne til 4 DK 173633 B1 de enkelte brønde og frasuger efter passende opholdstider den over bærematerialet stående væske.If the peptide is to be separated from the carrier after synthesis is completed and / or if free amino groups are to be acylated, the reagents required for these reactions must also be prepared in the storage containers. From the required reaction times, it is seen that it is appropriate to use a microtiter plate containing no more than 30 wells. Thus, in a program run, a maximum of 96 different polypeptides can be synthesized. According to the program adapted to the synthesis of these peptides, the robot brings the reagents and washing liquids to the individual wells and suction the liquid over the carrier material after appropriate residence times.

Fremgangsmåden og den nødvendige tilpasning af apparatet til fremgangsmåden forklares nærmere på bag-5 grund af den toarmede pipetteringsrobot RSP 5052 fra firmaet Tecan. Anvendelsen af fremgangsmåden er dog ikke begrænset til dette apparat. .Andre typer pipetteringsrobotter, især også en- eller flerarmede robotter, kan tilpasses fremgangsmåden ifølge nærværende opfin-10 dele.The method and the necessary adaptation of the apparatus to the method are explained in more detail on the basis of the two-armed pipetting robot RSP 5052 from the company Tecan. However, the application of the method is not limited to this apparatus. Other types of pipetting robots, especially also single or multi-armed robots, can be adapted to the method of the present invention.

Der vælges en mikrotiterplade med 96 brønde. En brønd rummer f.eks. 10 mg harpiks, der kan være påført med en aminosyre, og lidt mere end 300 μΐ væske. Harpiksmængden svarer ca. til 5 ymol aminosyre eller er 15 egnet til fremstillingen af ca. 5 ymol peptid. Sædvanlige bærematerialer på polystyren- eller polyacrylamid-basis er anvendelige. Det er hensigtsmæssigt at opbygge peptider, der maksimalt indeholder 20 aminosyrer.A 96-well microtiter plate is selected. For example, a well contains. 10 mg of resin that can be applied with an amino acid and slightly more than 300 μΐ fluid. The amount of resin corresponds to approx. to 5 µmol of amino acid or is suitable for the preparation of ca. 5 µmol peptide. Usual carrier materials on a polystyrene or polyacrylamide basis are useful. It is convenient to construct peptides containing a maximum of 20 amino acids.

De dertil anvendte reagensopløsninger og vaskevæsker 20 tilberedes i de dertil indrettede lagerbeholdere. Ap-paratets arm 1 er udstyret med en doseringspipette, arm 2 med en sugekanyle med skylleanordning. Denne skylleanordning er fortrinsvis forbundet med en separat stående lagerbeholder til det anvendte opløsningsmid-25 del. Syntesen sker efter det i den tilsluttede PC programmerede program.The reagent solutions and washing liquids 20 used for this purpose are prepared in the storage containers provided for this purpose. The apparatus 1 arm 1 is equipped with a metering pipette, arm 2 with a suction cannula with flushing device. This flushing device is preferably connected to a separate standing storage container for the solvent used. The synthesis is done according to the program programmed in the connected PC.

Tildoseringen af samtlige reagensopløsninger, der tages fra åbne lagerbeholdere, sker med arm 1.The dosage of all reagent solutions taken from open storage containers is done with arm 1.

Før doseringspipetten skifter fra en reagensopløsning 30 til en anden, skylles doseringspipetten med opløsningsmiddel i en speciel skylleposition. Frasugning af reagenser og vaskevæsker sker med arm 2 gennem en med et filter udstyret kanyle. For at forhindre harpikstab og DK 173633 B1 5 kontaminering af nabobrøndene skylles kanylens yderside med opløsningsmiddel af en på kanylens yderside anbragt tilledning efter hver frasugningsprocedure. Næste vaskeprocedure startes samtidig med dette opløsningsmid-5 del. Herefter skylles kanylen i en kanyleskylleposi-tion.Before changing the dosing pipette from one reagent solution 30 to another, the dosing pipette is rinsed with solvent in a special rinsing position. Extraction of reagents and washing fluids takes place with arm 2 through a filter-equipped cannula. To prevent resin loss and contamination of the neighboring wells, the outside of the cannula is rinsed with solvent by a feed located on the outside of the cannula after each suction procedure. The next washing procedure is started at the same time as this solvent. Thereafter, the cannula is rinsed in a cannula flush position.

Til adskillelse af peptidet fra harpiksen tilføres brøndene f.eks. trifluoreddikesyre igennem arm 1.For separating the peptide from the resin, the wells are fed e.g. trifluoroacetic acid through arm 1.

Efter fraspaltning frasuges opløsningen med su-10 gekanylen og overføres til en anden mikrotiterplade, hvorfra oparbejdningen sker.After cleavage, the solution is suctioned with the suction cannula and transferred to another microtiter plate from which the processing takes place.

Som nævnt ovenfor, kan sugekanylen udstyres med et filter. Dette filter er anbragt i kanylens nedre ende og forhindrer frasugning og medrivning af harpiks.As mentioned above, the suction cannula can be fitted with a filter. This filter is located at the lower end of the cannula and prevents suction and entrainment of resin.

15 Piltret kan hensigtsmæssigt bestå af et net af rustfrit stål, der f.eks. fastgøres med en påloddet ring. En anden mulighed består i at tilpasse en metalsinterpla-de eller keramiksinterplade i kanylens åbning. Anvendes en kanyle uden filter, kan medrivningen og opsugningen 20 af harpiks undgås i stor udstrækning ved langsom opsugning. Derved forlænges tiden for opsugningen dog væsentligt.The gate may conveniently consist of a stainless steel mesh which e.g. fastened with a soldered ring. Another option is to fit a metal interplate or ceramic interplate into the opening of the cannula. If a cannula is used without a filter, the entrainment and aspiration of resin 20 can be largely avoided by slow aspiration. This, however, significantly prolongs the time of aspiration.

Sugekanylens yderside skylles med opløsningsmiddel efter hver sugeprocedure. Det sker hensigtsmæssigt 25 ved tilledning af opløsningsmidlet i en ledning (slange eller rør), der løber langs kanylen og er således anbragt, at åbningen ligger knapt ovenfor kanylens neder-ste ende. Det er vigtigt, at opløsningsmidlet skyller hele omkredsen af kanylens nederste ende.Rinse the outside of the suction cannula with solvent after each suction procedure. Conveniently, this is effected by feeding the solvent into a conduit (hose or tube) running along the cannula and positioned so that the aperture is barely above the lower end of the cannula. It is important that the solvent rinse the entire circumference of the lower end of the cannula.

30 Apparatets arm 2 kan være udstyret med en kam i stedet for sugekanylen. En sådan kam omfatter flere sugekanyler (for det meste 4-12 kanyler) af den ovenfor beskrevne udførelsesform. Ved hjælp af en sådan kam DK 173633 B1 6 kan flere brønde betjenes samtidigt, hvilket fører til en væsentlig tidsbesparelse.The device arm 2 may be provided with a comb instead of the suction cannula. Such a comb comprises several suction cannulas (usually 4-12 needles) of the embodiment described above. By means of such comb DK 173633 B1 6, several wells can be operated simultaneously, leading to a significant time saving.

Sædvanligvis anvendes DMF eller N-methylpyrroli-don som opløsningsmiddel. Brøndene og den eventuelt 5 dertil indrettede skylleanordning skal ligeledes være fremstillet af opløsningsmiddelbestandigt materiale, såsom polypropylen eller teflon. I de i handlen tilgængelige apparater er doseringspipetterne og sugekanylerne fremstillet af rustfrit stål. Dette materialer 10 er egnet til fremgangsmåden ifølge opfindelsen.Usually, DMF or N-methylpyrrolidone is used as the solvent. The wells and the optional flushing device, if any, must also be made of solvent-resistant material such as polypropylene or teflon. In the commercially available appliances, the dosing pipettes and suction cannulas are made of stainless steel. This material 10 is suitable for the process of the invention.

Hvis man ved denne syntese vil anvende en større mængde harpiks pr. peptid (f.eks. 50 mg harpiks), skal der anvendes mikrotiterplader med større brønde. Disse mikrotiterplader er standardiserede og tilgængelige i 15 handlen. De indeholder så et tilsvarende lavere antal brønde.In this synthesis, if a greater amount of resin is used per peptide (e.g., 50 mg of resin), larger well microtiter plates should be used. These microtiter plates are standardized and commercially available. They then contain a correspondingly lower number of wells.

I stedet for en pipetteringsrobot med to arme kan der også anvendes et étarms-apparat. I dette tilfælde skal sugekanylen have en opdeling i frie volumi-20 ner. Med den ene del sker frasugning af vaske- og reagensopløsninger, den anden del opfylder arm l's funktion fra toarms-apparatet, dvs. udfører tildosering af reagensopløsningerne. Beskyttelsen mod opsugning af harpiks sker som beskrevet ved toarm-apparatet. Også 25 denne kanyle skylles som beskrevet ovenfor.Instead of a two-arm pipetting robot, a one-arm apparatus can also be used. In this case, the suction cannula must have a division into free volumes. With one part suction of washing and reagent solutions takes place, the other part fulfills the function of arm 1 from the two-arm apparatus, ie. performs dosing of the reagent solutions. The protection against resin aspiration is as described by the two-arm apparatus. Also, this cannula is flushed as described above.

Fremgangsmåden udføres eksempelvis med de følgende stoffer (mængdeangivelse pr. brønd): Udgangsmateriale er 10 mg med Fmoc-aminosyrer påhæftet harpiks (kornstørrelse 200-400 mesh); Fmoc-beskyttede aminosy-30 rer anvendes i op til ti gange af overskud for hver enkelt koblingstrin, dvs. der tilsættes 200 \il af en DMF-opløsning af 50 ymol Fmoc-aminosyre og 50 ymol 1-hydroxybenzotriazol og 100 yl af en DMF-opløsning af DK 173633 B1 7 75 ymol Ν,Ν-dicyclohexylcarbodiimid; koblingstiden er ca. en time. Fraspaltning af Fmoc-beskyttelsesgruppen sker ved hjælp af 300 μΐ af en 40%'s opløsning af pipe-ridin i DMF. Fraspaltningstiden er ca. 20 minutter.The process is carried out, for example, with the following substances (quantity declaration per well): Starting material is 10 mg with Fmoc amino acids resin attached (grain size 200-400 mesh); Fmoc-protected amino acids are used in up to ten times the excess for each coupling step, ie. 200 µl of a DMF solution of 50 µmol of Fmoc amino acid and 50 µmol of 1-hydroxybenzotriazole and 100 µl of a DMF solution of 75 µmol of Ν, Ν-dicyclohexylcarbodiimide are added; the switching time is approx. one hour. Cleavage of the Fmoc protecting group is done by using 300 μΐ of a 40% solution of pipe-ridin in DMF. The cleavage time is approx. 20 minutes.

5 Vasketrinene udføres med 300 yl DMF.5 The washing steps are performed with 300 µl DMF.

Fraspaltningen af det færdige peptid fra bærematerialet kan ske i brøndene ved manuel eller automatisk tilsætning af 300 yl trifluoreddikesyre (20 minutters reaktionstid). Acyleringen af frie grupper (NH2,OH) 10 kan ske analogt ved tilsætning af egnede syreanhydri-der, f.eks. eddikesyreanhydrid og pyridin. Efter endt omsætning frasuges opløsningen og ledes til oparbejdning.The final peptide cleavage from the carrier can be effected in the wells by manual or automatic addition of 300 µl trifluoroacetic acid (20 minutes reaction time). The acylation of free groups (NH 2, OH) 10 can be done analogously by the addition of suitable acid anhydrides, e.g. acetic anhydride and pyridine. After completion of the reaction, the solution is sucked off and led to reprocessing.

Som det ses i ovennævnte forklaring, udføres al-15 le trin i peptidsyntesen i åbne beholdere. Ved udførelsen af syntesen ifølge opfindelsen opnås peptiderne alligevel i meget høj renhed.As seen in the above explanation, all steps of peptide synthesis are performed in open containers. Nevertheless, in carrying out the synthesis of the invention, the peptides are obtained in very high purity.

Fig. 1 viser et eksempel på en kanyle til en pipetteringsrobot med en arm: (1) kanyle; (2) og (3) 20 kanylens tildoseringsside og sugeside; (4) slange til tilledning af opløsningsmiddel til skylning; (5) filter.FIG. 1 shows an example of a needle for a pipetting robot with an arm: (1) needle; (2) and (3) the needle and suction side of the needle; (4) hose for rinsing solvent for rinsing; (5) filter.

Det følgende eksempel viser fremgangsmådens forløb, idet der beskrives opbygning af et peptid i en 25 brønd. Der lægges mærke til produktets renhed.The following example illustrates the process of the process, describing the build-up of a peptide in a well. The purity of the product is noticed.

EksempelExample

Der syntetiseredes 44 forskellige undecapeptider 30 ved hjælp af Tecan-pipetteringsrobotten RSP 5052. Udgangsmateriale var i alle tilfælde et over en linker til polystyren bundet tetrapeptid Fmoc-Arg(Mtr)-Gin-Arg- (Mtr) -Tyr (tBu)-linker-polystyrol (påhæftet ca. 0,5 DK 173633 B1 8 mmol/g harpiks). 44 gange indførtes 10 mg af denne harpiks i brøndene på en mikrotiterplade (5 ymol peptid), og syntesen startedes. Som eksempel beskrives syntesen af: 544 different undecapeptides 30 were synthesized by the Tecan pipetting robot RSP 5052. In any case, starting material was a tetrapeptide Fmoc-Arg (Mtr) -Gin-Arg- (Mtr) -Tyr (tBu) -linker-linked tetrapeptide linker. polystyrene (attached about 0.5 DK 8 mmol / g resin). 44 times, 10 mg of this resin was introduced into the wells of a microtiter plate (5 µmol peptide) and the synthesis started. By way of example, the synthesis of: 5

Ac-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr-NH2Ac-His-Tyr-Ile-Leu-Asn-Ile-Thr-Arg-Gln-Arg-Tyr-NH 2

Syntesen af de øvrige 4 peptider forløb analogt .The synthesis of the other 4 peptides proceeded analogously.

10 Syntesecyklus: 300 yl piperidin (40%) i DMF (5 minutter) 300 yl piperidin (40%) i DMF (20 minutter) 15 10 gange vask hver gang med 300 yl DMF (2 minutter) 200 yl af en DMF-opløsning, der er 50 ymolær med hensyn til Fmoc-Thr (tBu)-OH og 5 0 ymolær med hensyn til HOBt 20 og 100 yl af en DMF-opløsning, der er 75 ymolær med hensyn til DCC (1 time) 25 10 gange vask med 300 yl DMF (2 minutter).10 Synthesis cycle: 300 µl piperidine (40%) in DMF (5 minutes) 300 µl piperidine (40%) in DMF (20 minutes) 10 times washing each time with 300 µl DMF (2 minutes) 200 µl of a DMF solution which is 50 ymolar with respect to Fmoc-Thr (tBu) -OH and 50 ymolar with respect to HOBt 20 and 100 µl of a DMF solution 75 ymolar with respect to DCC (1 hour) 25 times wash with 300 µl DMF (2 minutes).

DK 173633 B1 9 I identiske syntesecykler anvendtes tilsvarende opløsninger af:In identical synthesis cycles, similar solutions of:

Fmoc-Ile-OHFmoc-Ile-OH

55

Fmoc-Leu-OHFmoc-Leu-OH

Fmoc-Asn-OHFmoc-Asn-OH

10 Fmoc-Ile-OHFmoc-Ile-OH

Fmoc-Tyr(tBU)-OHFmoc-Tyr (tBu) -OH

Fmoc-His(Trt)-OH.Fmoc-His (Trt) -OH.

15 med samme koncentrationer. Den afsluttende acetylering gennemførtes som sædvanlig med 300 μΐ af en DMF-opløs-ning, der er 40 ymolær med hensyn til eddikesyreanhy-drid og pyridin, efter Fmoc-spaltning.15 with the same concentrations. The final acetylation was carried out as usual with 300 µl of a DMF solution 40 ymolar in acetic anhydride and pyridine after Fmoc digestion.

20 Fraspaltningen fra harpiks skete med 300 yl trifluoreddikesyre/1% anisol i 30 minutter, vask 5 gange med 300 yl trifluoreddikesyre. De samlede trifluor-eddikesyreopløsninger opbevaredes under fugtig-hedsudelukkelse i en 1 time ved 50°C, og trifluoreddi-25 kesyren afdestilleredes under vakuum. Resten behandledes med ether i ultralydsbad, etheren dekanteredes, resten opløstes i vand og frysetørredes. HPLC-chromato-gram (RPl8-søjle) (fig. 2).The resin cleavage occurred with 300 µl trifluoroacetic acid / 1% anisole for 30 minutes, washing 5 times with 300 µl trifluoroacetic acid. The combined trifluoroacetic acid solutions were stored under moisture exclusion for 1 hour at 50 ° C and the trifluoroacetic acid was distilled off under vacuum. The residue was treated with ether in an ultrasonic bath, the ether was decanted, the residue dissolved in water and lyophilized. HPLC chromatogram (RP18 column) (Fig. 2).

10 DK 173633 B1 Løbemiddel A Vand/acetonitril/trifluoreddikesyre 95/5/0,2 Løbemiddel B Vand/acetonitril/trifluoreddikesyre 5 20/80/0,2 FAB-massespektrogram: M+H: 1518.10 DK 173633 B1 Running agent A Water / acetonitrile / trifluoroacetic acid 95/5 / 0.2 Running agent B Water / acetonitrile / trifluoroacetic acid 5 20/80 / 0.2 FAB mass spectrogram: M + H: 1518.

Claims (6)

1. Fremgangsmåde til fuldautomatisk simultan syntese af flere polypeptider ifølge fastfasesynteseme-toden, kendetegnet ved, at man anvender en pipetteringsrobot, hvorved polymert bæremateriale, der 5 kan være påhæftet med de ønskede peptiders begyndelsesdele, anbringes i brønde på mikrotiterplader, hvorefter peptiderne opbygges i brøndene efter den i og for sig kendte fastfasesyntesemetode, og peptidernes eventuelle frie aminogrupper og/eller hydroxygrupper acyleres, 10 og/eller peptiderne derefter adskilles fra bærematerialet, idet de til de enkelte trin krævede reagenser eller vaskevæsker tilføres brøndene fra de tilsvarende lagerbeholdere via en eller flere robotarme med kanyler, og idet de over bærematerialet stående reagenser 15 eller vaskevæsker, der er til stede i brøndene, frasu-ges med en robotarm med kanyle efter den tilstrækkelige opholdstid, idet de enkelte trin i fremgangsmåden styres af det i computeren, der er tilsluttet til robotten, programmerede program.A method for fully automatic simultaneous synthesis of multiple polypeptides according to the solid phase synthesis method, characterized in that a pipetting robot is used, whereby polymeric carrier material which may be attached to the starting portions of the desired peptides is placed in wells on microtiter plates, after which the peptides are built into wells. according to the known solid phase synthesis method, and any free amino groups and / or hydroxy groups of the peptides are acylated, and / or the peptides are then separated from the carrier, the reagents or washing liquids required for the individual stages being supplied to the wells of the corresponding storage containers via one or more robotic arms with cannulas, and the reagents or washing liquids present in the wells present in the wells are aspirated with a robotic arm with cannula after the sufficient residence time, the individual steps of the process being controlled by that of the computer connected for the robot, programmed p rogram. 2. Fremgangsmåde ifølge krav 1, kende tegnet ved, at kanylen skylles på ydersiden hver gang mellem opsugning af væsken og tilsætning af reagenser.2. A method according to claim 1, characterized in that the cannula is rinsed on the outside each time between aspirating the liquid and adding reagents. 3. Fremgangsmåde ifølge krav 1 eller 2, k e n -25detegnet ved, at reagenserne anvendes i op til 20 ganges overskud af de støkiometrisk nødvendige mængder.3. A process according to claim 1 or 2, characterized in that the reagents are used for up to 20 times the excess of the stoichiometric amounts required. 4. Kanyle til pipetteringsrobot, kendetegnet ved, at dens øvre ende er sluttet til ro- 30 botarmens doseringsanordning, og hvis nedre ende er udstyret med et filter og/eller en skylleanordning, hvis DK 173633 B1 øvre ende er tilsluttet robotarmen.., og hvis nedre ende når ned til knapt over den nedre ende af kanylen, og hvis åbning er udført således, at den nedre ende af kanylen kan skylles.4. Pipette robot needle, characterized in that its upper end is connected to the robot arm's metering device and the lower end of which is equipped with a filter and / or a flushing device, whose DK 173633 B1 upper end is connected to the robot arm .., and the lower end of which reaches down to barely above the lower end of the cannula, and whose opening is made such that the lower end of the cannula can be flushed. 5. Kanyle ifølge krav 4, kendetegnet ved, at hulrummet er delt i to dele af en fra oven og nedefter løbende skillevæg.A cannula according to claim 4, characterized in that the cavity is divided into two parts by a dividing wall running from above and below. 6. Kanylekam, kendetegnet ved, at den består af flere kanyler ifølge krav 4 eller 5.A cannula comb, characterized in that it consists of several needles according to claim 4 or 5.
DK198904127A 1988-08-23 1989-08-22 Method and cannula for pipetting robot for fully automatic simultaneous synthesis of multiple polypeptides DK173633B1 (en)

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US5702672A (en) * 1992-10-08 1997-12-30 Warner-Lambert Company Apparatus and method for multiple simultaneous synthesis
US5714127A (en) * 1992-10-08 1998-02-03 Warner-Lambert Company System for multiple simultaneous synthesis
US5324483B1 (en) * 1992-10-08 1996-09-24 Warner Lambert Co Apparatus for multiple simultaneous synthesis
US5567391A (en) * 1992-10-08 1996-10-22 Warner-Lambert Company Apparatus for multiple simultaneous synthesis
JPH06220084A (en) * 1993-01-23 1994-08-09 Shimadzu Corp Peptide synthesizer
AU4424496A (en) * 1995-03-27 1996-10-16 Warner-Lambert Company A method for the synthesis of mixtures of compounds
DE19844988A1 (en) * 1998-09-30 2000-04-13 Stefan Seeger Parallel solid phase synthesis
AU3191900A (en) * 1999-03-15 2000-10-04 Sankyo Company Limited Method for synthesizing peptide
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