Chemistry: analytical and immunological testing – Food or dairy products – Dairy product
Reexamination Certificate
1999-11-30
2003-02-11
McKane, Joseph K. (Department: 1627)
Chemistry: analytical and immunological testing
Food or dairy products
Dairy product
C422S131000, C422S187000, C422S236000, C422S238000, C422S239000, C435S007100
Reexamination Certificate
active
06518067
ABSTRACT:
The synthesis of polymeric biomolecules, such as oligonucleotides, peptides or non-natural analogues thereof, according to the principle of solid phase synthesis is an established technique (2).
Although multiple parallel synthesis of peptides in open reaction systems is part of the prior art (14, 15), oligonucleotides, for example, tend to be prepared individually. The principal problem in oligonucleotide synthesis lies in the extreme sensitivity to water of the phosphoramidite chemistry of the prior art (2). Automated oligosynthesis apparatuses are therefore closed systems and operate under protective gas. A published apparatus for the parallel synthesis of up to 96 oligonucleotides uses a reaction system that likewise is closed and, for the dispensing of reagents, a large number of valves (16). Conventional supports and manual work-up procedures are used therein.
The synthesis system according to the invention is therefore based on the idea of configuring synthesis, supports, anchor groups and work-up procedure for the simultaneous, fully automatic preparation of biomolecules. With the reagents' being distributed by means of a pipetting robot, the reaction columns can be arranged in a format that is suitable for further processing. In order also to be able to carry out a water- and air-sensitive synthesis protocol using a pipetting robot, it is necessary to take certain measures in terms of design. As an example of a possible solution, there will be described hereinbelow the principle of operation of the automated apparatus and the course of synthesis.
The automated apparatus can operate using conventional supports and reagents, but handling is simplified by specially modified, newly developed supports and anchor groups.
By adding on a special method for simultaneous purification and aliquotting, the quality of the products is improved and use is simplified.
The invention relates especially to the following embodiments: apparatus for automated simultaneous chemical synthesis and purification of a large number of products on solid phase, as well as support material and chemical building blocks for solid phase synthesis, characterised in that
1. a large number (from 10 to 1000, preferably 48 and a multiple thereof, preferably 400) of separate reaction vessels, which are open at the top and at the bottom, are provided in the form of channels or small columns, which are arranged in parallel in a block (
FIG. 6
) and which are removable either together or separately; the support material for the synthesis (solid phase) is placed in the channels/columns, either being arranged between two inert porous frit plates or, preferably, being itself in the form of a chemically modified frit or filter plate (FIG.
7
), so that liquid media added from above are held in the reactor solely as a result of surface tension and wetting of the material;
2. the reactor/reactor block according to 1. is mounted on a trough connected to a vacuum pump by means of a switchable valve, and so the liquid media can be aspirated simultaneously from the reactors and the support materials contained therein;
3. the upper inlets to the reaction columns in the reactor block according to 1., which are covered by a perforated screen (baffle plate) mounted above them, can be flooded with inert gas (e.g. nitrogen, argon) and the flow of inert gas is optionally increased considerably during the aspirating procedure according to 2.; alternatively, the space above the reaction columns/channels can be selectively closed off by means of a second, displaceable perforated screen so that the reagents are blown out of the reaction columns/channels by pressurised inert gas;
4. chemical building blocks, reagents and solvents are distributed to the reaction vessels by an xyz-pipetting robot by means of electronically controllable dispensing syringes (dilutors) having one or more dispensing needles and optionally, in addition, one or more dispensing manifolds so that each reactor can be addressed individually;
5. the dispensing needle according to 4. is equipped with a plurality (at least two) of internal channels, which are connected to separate dispensing syringes, that is to say which can be filled separately, the ends of which channels meet only shortly before the outlet (
FIG. 8
) and so, when a plurality of reagents are being dispensed simultaneously, mixing occurs in the tip of the dispensing needle shortly before delivery, it being possible for a channel to be connected to the inert gas supply also and so the mixed volume can be expelled by means of a pulse of inert gas;
6. the dispensing needle according to 4. and 5. is mounted so as to be resilient along the longitudinal axis, so that it can set down on the support material or top frits in the reactor channels without damage and so can reliably deposit even extremely small volumes down to 1 nanoliter;
7. a large number (from two to one hundred, preferably 24) of chemical building blocks and reagents, where appropriate dissolved in suitable solvents, are provided in vessels which are sealed by means of septa and which are arranged in a reagent block separate from the reaction block;
8. the septum-sealed necks of the vessels in the reactor block according to 7., which are covered by a perforated screen (baffle plate) mounted above them, can be flooded with inert gas (e.g. nitrogen, argon);
9. using the dispensing needle according to 4. and 5., reagents can also be withdrawn from transfer ports, which are connected to storage bottles either directly or by means of switchable valves (
FIG. 6
) and the storage bottles are slightly pressurised with inert gas;
10. solvents and reagents can be distributed from solvent bottles by means of dispensing syringes or by pressurised inert gas and also by way of one or more dispensing manifolds according to 4. simultaneously to a plurality of reactors row by row;
11. the support material according to 1. forms a layer in the reactor channel, through which an even flow of the reagents and solvents applied from above passes solely under the action of gravity. According to the principle of solid phase synthesis (FIG.
2
), which is part of the prior art, the individual products of each reactor are covalently bonded on the surface of the support material and are built up in parallel in steps by means of a succession of pipetting operations. During all the steps of synthesis (build-up reactions, repetitive protecting group removals and washing operations), the products remain covalently linked to the support material and are removed from the support material, and brought into solution, only in one or more final reaction steps;
12. the chemical building blocks (monomers) used for building up the products are coded as ASCII characters and so the products are described as a sequence of build-up reactions (monomer incorporation reactions) by means of ASCII words; the totality of all products for a synthesis program is consequently a list of ASCII words, which are converted by suitable software on a control computer into valve-switching operations, dispensing-syringe movement operations and robot arm movement operations, it being possible for each monomer incorporation to consist of a succession of several reaction steps and switching operations;
13. linking building blocks (linkers) suitable for covalently linking the products to the support material according to 11. are provided, which allow final removal of the products to take place selectively under mild conditions (FIG.
1
). For the synthesis of oligomeric compounds, such as oligonucleotides, peptides etc., it is preferable to provide a “universal” linker, to which there can also be linked the building blocks of the first build-up reaction of the same chemical reaction type as that used for the further build-up reactions, as a result of which only one type of building block is needed for the entire synthesis of a class of compounds (e.g. only nucleoside-3′-phosphoramidites for the synthesis of 3′-OH-oligonucleotides);
14. in the final removal reactions, the linker accordin
Frank Ronald
Gausepohl Heinrich
Matysiak Stefan
Rosenthal Andre
Schreuer Olaf
Friend Tomas
Gesellschaft fuer Biotechnologische Forschung mbH (GBF)
McKane Joseph K.
Santucci Ronald R.
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