The multi-stage synthesis of an organic molecule usually involves numerous steps for the isolation of intermediates. These intermediates often require purification to remove excess reagents and reaction by-products. Purification involves procedures such as precipitation, filtration, bi-phase solvent extraction, solid-phase extraction, crystallization and chromatography.

If the reaction chemistry is well defined many of the isolation procedures used in solution phase synthesis are avoided by reversibly attaching the target molecule to a solid support in a way analogous to the use of a protecting group in a traditional synthesis.

Excess reagents can be removed by filtration and washing of the solid support. Providing that the reactions are efficient and no solid support is lost the target molecule may be recovered in more or less quantitative yield, an objective rarely achieved in solution phase synthesis. In addition, the time required to perform operations on a solid support is generally accepted to be approximately one fifth of that required to carry out the equivalent stage in a solution phase synthesis. Another advantage of the solid-phase approach is that the whole assembly is carried out in a single reactor.

Solid-phase synthesis of peptides and other organic molecules is normally carried out on microporous supports in batch-wise reactors. Conversely, oligonucleotides are normally assembled on macroporous supports in fixed volume continuous flow reactors.

Microporous supports have a low level of crosslinker and are therefore readily solvate in an appropriate solvent to form a gel. Macroporous supports are rigid due to high levels of crosslinker and contain very large pores. In macroporous supports the chemistry is carried out on the surface of the polymer only. The rigidity of macroporous polymers allows them to be packed into a fixed volume column and operated under continuous flow.

Microporous supports have a number of advantages in comparison to macroporous supports including significantly higher loading capacities and a greater accessibility to reactive sites in the polymer. However these resins swell in certain solvents (such as DCM), and shrink in other solvents (such as methanol). The solid-phase volume is also dependant on the nature of the chemical moiety pendant on the polymer backbone and, for example, will change with the growth of a pendant peptide chain. This variation in bed volume means that microporous supports are not really suitable for use with continuous flow reactors since as the solid-phase bed volume changes flow characteristics are affected. If the support shrinks during continuous flow based operation a'dead space'can be formed in the column resulting in poor mixing and poor flow distribution. If the support swells in a fixed bed column the pressure will increase, hindering flow and potentially bursting the column or seals.

In any chemical reaction it is essential that the progress of the reaction is monitored, preferably by taking and analysing samples. This is achieved by removing a sample of solid support from the reactor. This can have two possible effects. Firstly, there is a physical change in the total amount of solid support and secondly if the sample is taken from a point away from the surface of the support then the physical act of doing this will lead to disruption of the column volume. Thus, in addition to the change in bed volume seen with microporous column systems the bed column of any system can be affected as samples are taken, again making the use of continuous flow based reaction systems difficult.

Continuous flow based reaction systems have advantages over batch-wise reactors because they are readily automated and, if good laminar flow is obtained, the amount of solvent used in wash cycles can be reduced dramatically. This can result in significant cost-saving in large scale commercial processes.

In this invention a method for solid-phase synthesis has been developed using a variable volume reactor designed to compensate for the changing volumes of microporous and other supports and so enable them to be used in sampled continuous flow systems.

According to the present invention there is provided a method for carrying out solid-phase synthesis in a reactor wherein the reactor comprises, a reactor column containing a solid-phase support and having a first sealed end and a second sealed end where at least one of the sealed ends is displaceable within the reactor column, a drive means able to drive the displaceable sealed end (s) and a detector which detects changes in pressure and/or volume within the reactor column and is in communication with the drive means so that as the volume of the solid-phase support changes during the course of a solid-phase synthesis the detector detects the resultant changes in pressure and/or volume and the drive means drives the displaceable end (s) within the reactor column so as to maintain a predetermined axial pressure on the surface of the solid-phase support.

The method of the present invention may be applied to any chemical synthesis able to be carried out on a solid-phase support. Preferably it is a method for solid-phase peptide synthesis or solid-phase oligonucleotide synthesis.

The oligonucleotide may be an oligodeoxyribonucleotide or oligoribonucleotide or any analogue that would be known to one skilled in the art. Thus, the term oligonucleotide encompasses synthetic analogues such as oligonucleotide phosphoramidites and oligonucleotide phosphorothioates.

The reactor column may be any column suitable for carrying out solid-phase synthesis, typically the reactor column will be of cylindrical cross-section and constructed from plastic, glass or metal.

The solid-phase support used in the reactor will vary depending on the nature of the reaction to be carried out. However, the solid-phase support is preferably based on a polystyrene or polydimethylacrylamide polymer. More preferably the support is a copolymer of styrene with about 0.5 to 2% divinyl benzene as a cross-linking agent or a

polydimethylacrylamide polymer comprising N, N-dimethylacrylamide, N, N- bisacryloylethylenediamine and acryloylsarcosine methyl ester monomers. Details of these preferred supports and other suitable supports may be found in Chan and White "Fmoc Solid-phase Peptide Synthesis"Oxford University Press, solid supports of which are incorporated herein by reference.

The sealed ends should form watertight seals within the reactor column and may be formed of glass, ceramic, metal or plastic material or a mixture thereof and comprise one or more components or members. Preferably the sealed ends comprise a sintered material.

The drive means may comprise any suitable device able to drive the displaceable end (s) including a spring, a stepper motor, a rotary motor, a hydraulic pressure device or any combination thereof. Preferably the drive means comprises a motor, more preferably the drive means comprises a rotary motor.

Any detector may be used which is able to monitor changes in the pressure and/or volume within the reactor column. Preferably the detector is a pressure sensor. The pressure sensor is preferably positioned so as to monitor the force created by the complete system (resin and fluid) rather than on an outlet pipe which would only indicate the hydrodynamic pressure. The pressure sensor may advantageously be positioned in one of the end fittings or incorporated into the pressure sensor of a hydraulic piston used to drive the displaceable sealed end. Pressure sensors suitable for use in the reactor of the present invention may be obtained from Keller (UK) Ltd.

Changes in the pressure and/or volume within the reactor column may be communicated to the drive means by any, preferably electronic, means. In a preferred embodiment a pressure sensor is connected to a trip amplifier. Thus, when the pressure sensor detects a change in pressure the trip amplifier sends a signal to the drive means which then moves the displaceable sealed end (s) a corresponding amount to compensate for the change in volume in the solid-phase support within the reactor column. As the displaceable sealed end (s) move (s) the pressure detected by the pressure sensor changes and when the pressure reaches a desired value the trip amplifier signals the drive means to stop movement.

The communication system is preferably designed to minimise movement of the drive means due to variations in system pressure caused by a change in fluid flow rate.

Thus, in another preferred embodiment the drive means is in communication with, and controlled by, both the reactor column pressure and/or volume detector and a fluid flow pressure detector.

The predetermined axial pressure on the surface of the solid-phase support will vary depending on the reaction conditions, type of synthesis and particularly the nature of the solid-phase support. The axial pressure is that which enables there to be a minimum head volume on the top of the solid-phase support yet does not compress the solid-phase support, which can lead to column failure.

The method of the present invention reliably corrects for changes in the volume of a solid-phase support and allows microporous supports to be used in continuous flow based reaction systems.

According to a second aspect of the invention there is provided a sampling device for use with a solid-phase reactor column or chromatography column containing a solid- phase support, that comprises a column connector, a sample collection member, a sample trap member, a sample collection port and a sample drive wherein the solid- phase support within the column is sampled by means of the sample drive, reversibly moving the sample collection member through the column and so collecting a sample of a solid-phase support which, when the sample collection member is in association with the sample trap may be removed for analysis via the sample collection port.

The column connector may permanently fix the sampling device to the column or allow the sampling device to be reversibly attached, optionally in different positions, to the column.

The end of the sample collection member that passes through the solid-phase support is preferably adapted to collect a sample of the solid-phase support, it is also preferably adapted to allow the solid-phase support to be easily removed when it is aligned with the sample collection port. In a preferred embodiment the sample collection member has a concave end able to collect solid-phase resin as it passes through the column.

In another preferred embodiment the end of the sample collection member is in communication with a hole that passes through the body of the sample collection member, so that the collected solid-phase support is forced from the end of the sample collection member to the hole which when in alignment with the sample collection port allows the solid-phase support to be washed from the sample collection member into the sample collection port.

An especially preferred embodiment provides a sample collection member with a concave end that is in communication with a hole that passes through the sample collection member.

The sample trap, which defines the final position of the sample collection member with respect to the sample collection port, is preferably associated with a resilient means.

This allows the sample trap member to be forced by the sample collection member to a fixed final position, i. e. aligned with the sample collection port, wherein the sample trap member is resiliently biased against the sample collection member. This forces the sample trap member back to its starting position when the sample collection member is withdrawn.

This resilient means is preferably a spring.

The sample collection port may be any opening or collection point into which solid- phase support sample may be transferred from the sample collection member. Sample collected via the sample collection port is taken for chemical analysis.

The sample drive may be manually driven or utilise any suitable device such as a spring, a stepper motor, a rotary motor or a hydraulic pressure device or combination thereof. Preferably the sample drive comprises a motor, more preferably a stepper motor.

In a preferred embodiment the sample drive further comprises guiding means for ensuring a reproducible straight linear movement of the sample collection member.

It is also preferred that the sample drive member comprises means to ensure that the sample collection member is correctly aligned with the sample collection port after passage through the column. In a preferred embodiment this is achieved by means of limit switches that detect movement associated with the sample collection member.

According to a third aspect of the invention there is provided a solid-phase reactor or chromatography column provided with a sampling device as described in the second aspect of the invention.

The solid-phase reactor or chromatography column according to the third aspect of the invention may be either empty or may contain a solid-phase support or chromatography resin.

According to a fourth aspect of the invention there is provided a solid-phase reactor or chromatography column which comprises a column able to hold a solid-phase resin and having a first sealed end and second sealed end, where at least one of the sealed ends is displaceable within the reactor column, a drive means able to drive the displaceable sealed end (s), a detector which detects changes in pressure and/or volume within the reactor column and is in communication with the drive means and a sampling device connected to the reactor column able to remove samples of solid-phase resin, for analysis, from the body of the reactor.

The solid-phase reactor according to the third aspect of the invention may be used for any chemical or chromatographic process that involves a solid-phase able to change its volume during the course of the process.

The solid-phase reactor according to the third aspect of the invention is preferably used for solid-phase peptide synthesis or oligonucleotide synthesis.

Preferred embodiments for the reactor column, solid-phase support, sealed ends, drive means, detector and communication systems are as set out with respect to the first aspect of the invention.

Preferably the sampling device is as described in the second aspect of the invention.

The invention will now be illustrated by way of example and with reference to the accompanying drawings in which: Figures 1 shows a cross section of a variable volume reactor (without the sampling device attached).

Figure 2 shows a cross section of a variable volume reactor with the sampling device attached.

Figure 3 is a side view showing the sampling device attached to a column and linked to a motor drive and motor.

Figure 4 is a side view schematic showing a breakdown of the main components of the sampling device.

Figure 5 is a side view cross-section of the sampling device attached to a column showing the sample device moving from its first position (a) to its second position (b).

Figure 6 is a side view cross-section showing the end of the sample collection member aligning with the sample trap member and sample collection port.

Figure 7 is a cross section through the main part of the sampling system.

The variable volume reactor (1) comprises a cylinder shaped housing (2) containing a solid-phase support (3) and having a static sinter at one end (4) and a moveable sinter at the other end (5) each having an associated support (6,7). The sinters are held in the column by end pieces that are screwed on to the column (8,9). The moveable sinter has an associated piston (10). The reactor further comprises a solvent inlet (12) and solvent outlet (11). A pressure sensor (13) is positioned in the static sinter support (6). The piston (10) is in threaded connection (14,15) with a drive ring (16) held in place with bearings (17,18) by the end piece (9).

The pressure sensor is in communication via a trip amplifier equipped with a display and a drive card, with a rotary motor (not shown) that is connected to the drive ring (16) by a drive belt (not shown) that fits in a groove (19).

When the pressure sensor detects a change in pressure the trip amplifier sends a signal to the motor. The motor drives the drive belt which results in the piston (10) moving in towards the solid-phase support or away from it, via the threaded connection, depending on the direction of movement of the drive belt. As the moveable sinter (5) moves the pressure detected by the monitor changes and as it reaches its desired value the trip amplifier signals the motor to stop movement.

The sampling device (20) comprises a sampler body (21) and a sleeve (22) for attaching the body to the reactor column (1) and a vessel connector ring (23) whereby the body and sleeve are held in place on the column. The sample body, which is held horizontal to the reactor column, comprises a sample collection member (24) which is held within a drive seal plug (25). When in its resting position the sample collection member (24) is substantially coincident with the surface of and is sealed in the wall of the column by a seal (30). As shown in Figure 6 in a preferred embodiment the sample collection member has a recess at its first end (27) and a vertical hole (28) at a predetermined distance from the first end, which is connected to the recess.

On the opposite side of the column and aligned with the first end of the sample collection member is a sample trap member (29) which in its resting position has a first end which is substantially coincident with the surface of the column and is sealed in the wall of the column by a seal (31). The sample trap member is secured by and passes

through a spring seal inner plug (32), with an associated seal (33), and through another seal (34) into a spring seal outer plug (35). The sample trap member is retained in the spring seal outer plug by a retaining flange (36) part way along the sample trap member.

From the retaining flange to the second end (37) of the sample trap member the sample trap member passes through a return spring (38). In the first resting position the end of the sample trap member rests on the spring plug (39) and is aligned with a hole (40) in the spring plug.

A substantially vertically sample collection port (41) passes through the main body (21) and inner spring seal (32). In the resting first position the sample trap (29) member blocks the sample collect port (41).

The sample collection member (24) is actuated by a worm gear (42), driven by a linear stepper motor (43). The motor is attached to a mounting plate (44). The mounting plate is connected to the main sampler body (21) by four guide rails (45). These also provide mechanical support for the motor. An alignment guide (46) travels along the rails.

The guide keeps the sample collection member (24) correctly aligned with the main body of the sampler body (21), and prevents rotation of the worm-gear drive screw.

Figure 5 shows the sample collection member (24) starting to move from its resting first position through the solid-phase resin (3) towards the sample trap member (29).

In the preferred embodiment shown in Figure 6 then as the sample collection member passes through the column the solid support is forced into its concave first end (27) and then into the hole (28) which passes through the sample collection member (24).

Figure 5 (b) shows the sample device in its second position with the hole in the sample collection member (28) substantially aligned with the sample collection port (41) and resiliently biased against the first end of the sample trap member through compression of the return spring (38) by the retaining flange (36). In this second position the second end of the sample trap member is forced into the aligned hole (40) in the spring plug (39). In the second position, sample, collected by the sample collection member when passing through the solid support, may be dislodged by pulse of fluid (gas or a suitable solvent) from the hole in the sample collection member (28) through the sample collection port (41) and into a sample collection point. This could be to either a vial on a carousel for collection, or a reaction vessel for real time sample preparation.

As the sample collector member is moved back to its original position the sample trap member is pushed back into its first position by the action of the return spring (38) on the retaining flange (36). The return spring should provide sufficient force so that the sample trap member seals the reaction column whilst the actuated sample collection member is withdrawn. To do this, it must overcome the friction resulting mainly from the seals, and the internal pressure of the reactor.

The seals (30,31, 34 are"piston"type o-ring seals, having to seal against a moving element (Cal-rez, 2.9 mm ; 1.78 mm x-section). One additional seal (Cal-rez, 15 mm ; 1.78 mm x-section) (33) is required to prevent solvent loss past the spring seal inner plug.

The seals are held in place by locating plugs (one on the drive side (25), two plugs on the spring side (32) and (35) ). These plugs also act as guides for the sample collection member (24) and sample collection member (29). The spring seal inner plug retains two o-rings.

A motor controller board provides the drive sequence to the stepper motor. To function, the controller board requires additional, control logic inputs, and a 12 V supply. A built in clock can be used to control the motor speed. The clock frequency (motor speed) can be adjusted by altering a potentiometer.

The actuator should be precisely controlled to ensure that the sample collection member is correctly aligned with the exit port after insertion, to allow removal of the sample, and so that when the sample collector is withdrawn from the process, it is aligned with the sidewall of the reactor column. The position of the sample collection member is sensed by having optical slotted switches set the limit positions. A brass strip, attached to the alignment guide, breaks the beam, providing a motor stop signal. The optical sensors are positioned at either end of the guide rails (45). The limit switches are used to produce input signals for the control logic board. In addition to the two limit switches, there is a direction (in/out) switch, and a start/stop switch. Additional digital logic circuitry ensures that when a limit switch is triggered, the actuator is not be able to drive the sample collection member past the limit position, but when the direction is reversed, it allows the sample collection member to move away from this limit position. The controller uses the limit switch, direction switch, and start/stop switch, to produce a motor enable signal for the motor controller board.

The sample is flushed from the tip of the sample collection member preferably by using a fixed volume of solvent. A sample loop is loaded with solvent, and then used to dispense a precise quantity of solvent to the sampling device. Approximately 1.6 ml of solvent may be used to flush the sample into a collection vial.

Repeated flushing has shown that there are around 5-10 beads in a second flush and none in subsequent flushes. Thus sample collection should be followed by a single flush to prevent cross contamination.

The invention will now be illustrated, without limitation, by way of Example.

Example 1 To demonstrate that the ability of the variable volume as shown in Figure 1 to respond to the changes in resin volume encountered in a typical solid-phase synthesis the following experiment was carried out.

Swollen DMA resin was loaded into a variable volume reactor of the type shown in Figure 1, which was based on a 17mm internal diameter High Pressure Chromatography Column as supplied by Omnifit.

N, N'-dimethylformamide (DMF) was then pumped through the reactor, which was pressure tested to 4 barg without any sign of leakage or other problems. This resulted in

the plunger moving to a maximum displacement. Ethyl ether was pumped through the reactor which caused to the beads to shrink. The resin bed volume collapsed within a few minutes. As the bed volume decreased the change in the pressure exerted by the beads was detected by a pressure sensor and the upper plunger was driven downward in response to this change until it reached a position of minimum displacement. DMF was then pumped back through the reactor and as the pressure readings increased as the beads swelled and plunger moved back up the column to the original position of maximum displacement.