WO1997007126A1 - Appareil et procede de synthese en plusieurs etapes de molecules organiques a chaine longue en phase solide - Google Patents

Appareil et procede de synthese en plusieurs etapes de molecules organiques a chaine longue en phase solide Download PDF

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Publication number
WO1997007126A1
WO1997007126A1 PCT/US1996/011448 US9611448W WO9707126A1 WO 1997007126 A1 WO1997007126 A1 WO 1997007126A1 US 9611448 W US9611448 W US 9611448W WO 9707126 A1 WO9707126 A1 WO 9707126A1
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Prior art keywords
fluid
bed
monitoring
reservoir
flow
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PCT/US1996/011448
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English (en)
Inventor
Stephen Allen
Hubert KÖSTER
Edward Ashare
Donald W. Euwart
Jennifer Fernandes
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Hybridon, Inc.
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Publication date
Priority claimed from US08/608,702 external-priority patent/US5807525A/en
Application filed by Hybridon, Inc. filed Critical Hybridon, Inc.
Priority to AU64564/96A priority Critical patent/AU6456496A/en
Publication of WO1997007126A1 publication Critical patent/WO1997007126A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0006—Controlling or regulating processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C07K1/045—General 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00279—Features relating to reactor vessels
    • B01J2219/00281—Individual reactor vessels
    • B01J2219/00286—Reactor vessels with top and bottom openings
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00351—Means for dispensing and evacuation of reagents
    • B01J2219/00389—Feeding through valves
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00497—Features relating to the solid phase supports
    • B01J2219/005—Beads
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/0059—Sequential processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00596—Solid-phase processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/0068—Means for controlling the apparatus of the process
    • B01J2219/00686—Automatic
    • B01J2219/00689—Automatic using computers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/0068—Means for controlling the apparatus of the process
    • B01J2219/00698—Measurement and control of process parameters
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00722—Nucleotides
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B60/00—Apparatus specially adapted for use in combinatorial chemistry or with libraries
    • C40B60/14—Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries

Definitions

  • the present invention relates generally to improvements in the preparation of long-chained polymers (i.e. peptides, polysaccharides, nucleic acids, and the like) , and more particularly to an improved apparatus and process for the solid-phase synthesis of oligonucleotides.
  • long-chained polymers i.e. peptides, polysaccharides, nucleic acids, and the like
  • Solid-phase, multi-stage synthesis of complex organic molecules using controlled fluid flow across a fixed bed is well-known in the art. This general technique has been successfully applied to the synthesis of peptides, oligonucleotides and similar long-chained organic substances.
  • a good description of Merrifield' s well-known work on solid- phase peptide synthesis appears in John M. Stewart and Janis D. Young's book “Solid Phase Peptide Synthesis” (2nd ed. 1984) published by Pierce Chemical Company, which book is incorporated herein by reference.
  • a description of early work on solid-phase oligonucleotide synthesis appears in R.L. Letsinger and V.
  • antisense oligonucleotide technology provides a novel approach to the inhibition of viral expression, and hence, to the treatment or prevention of various viral- associated diseases such as chronic and acute hepatitis, AIDS, hepatocellular carcinoma, and others.
  • oligonucleotides useful in such applications are typically composed of deoxyribonucleotides, ribonucleotides, modified oligonucleotides such as 2-O-methyl-ribonucleotides, or some combination thereof, generally comprising at least 6 nucleotides in length, preferably 12-50 nucleotides long, with 15 to 30mers being the most common.
  • antisense oligonucleotides are able to inhibit splicing and translation of RNA, and transcription and replication of genomic DNA. In this way, antisense oligonucleotides are able to inhibit gene expression and protein translation.
  • a typical precursor for oligonucleotide synthesis is an amidite
  • a typical polymeric support comprises beads of controlled- pore glass (CPG) loaded with a suitably-protected nucleoside and contained in a column.
  • CPG controlled- pore glass
  • the first reaction step of a synthesis operation is to deblock this first building block, typically accomplished by passing acid solution through the column containing the CPG beads and the precursor until the deblocking is substantially completed. The column must then be flushed with an appropriate washing liquid to remove any remaining acid and by-products formed during the deblocking reaction.
  • Acetonitrile is commonly used as a wash in oligonucleotide synthesis.
  • the second reaction step the coupling step
  • the coupling step of the synthesis can begin.
  • typically phosphoramidites are used as building blocks and are added to the free, reactive end of the first nucleoside anchored to the solid support using a suitable activator to promote the desired coupling reaction.
  • suitable activators include tetrazole in an acetonitrile solution.
  • the amidite becomes relatively unstable once mixed with activator and must therefore be mixed just prior to addition to the reaction vessel. It will be appreciated that the failure to thoroughly flush acid and by-products from the system before beginning the coupling step could result in promoting undesired reactions which could adversely affect purity and yield as well as wasting relatively expensive raw materials.
  • the coupling reaction is substantially completed, the system must undergo another acetonitrile wash step to remove unreacted amidite and activator.
  • the third reaction step an oxidation step
  • oxygen, sulfur, or other oxidizing or sulfurizing reagent is introduced into the system to stabilize the newly-created phosphitetriester linkage.
  • an aqueous iodine solution may be utilized as an oxidant for the oxidation step of the process.
  • still another acetonitrile wash step is used to remove any residual oxidant or solvent from the system in preparation for the fourth step of the synthesis.
  • the final reaction step the capping step
  • the capping step materials such as acetic anhydride, N-methyl imidazole, and pyridine or mixtures thereof in predetermined ratios are added to the system to cap active sites at the free ends of incomplete nucleotide chains, i.e. chains that failed to complete the earlier coupling step.
  • materials such as acetic anhydride, N-methyl imidazole, and pyridine or mixtures thereof in predetermined ratios are added to the system to cap active sites at the free ends of incomplete nucleotide chains, i.e. chains that failed to complete the earlier coupling step.
  • the capping reagents are relatively unstable and must be mixed just prior to addition to the reaction chamber.
  • This capping step is followed by still another wash step, after which the process returns to reaction step 1 to begin a new cycle for the addition of the next set of amidite molecules to the growing oligonucleotide chains.
  • This arduous and time-consuming multi-step cycle must be repeated each time the oligonucleotide chains are extended, twenty, thirty, forty times or more to produce the longer-chain oligonucleotides that have been found to have such important therapeutic applications.
  • a reagent such as ammonium hydroxide may be used to cleave the raw oligonucleotides from the polymeric support.
  • the oligonucleotides thus obtained are separated, deprotected and purified by routine downstream processing to produce a final product.
  • a principal object of this invention is to provide an apparatus and process for commercial (gram) -scale production of oligonucleotides.
  • Another object of this invention is to provide an integrated system for oligonucleotide synthesis in which fluid control valves are activated for initiating or terminating the feed of various reagents to a reaction chamber in pre ⁇ determined sequence in feedback-controlled response to signals generated by monitoring equipment, such as optical scanners, located at one or more locations throughout the system to monitor the contents of associated fluid streams.
  • monitoring equipment such as optical scanners
  • optical monitoring such as an ultraviolet or visible light detector
  • Still other objects of this invention include providing various specifically-adapted components in an integrated, automated, optimized, commercial-scale oligonucleotide production operation designed to enhance product purity and yield.
  • the invention accordingly comprises the apparatus and process, involving the various components and the several steps, and the relation and order of one or more of such components or steps with respect to each of the others and to the apparatus, as exemplified in the following detailed disclosure and as illustrated by the drawings.
  • the oligonucleotide synthesis apparatus of this invention generally comprises a plurality of reagent reservoirs, each associated with a two- or three-way diaphragm valve, and interconnected by a system of inert fluid conduits to a reaction chamber containing a polymeric support and starting material for building a desired nucleotide chain.
  • the apparatus comprises multiple pumps and flowmeters situated to combine otherwise unstable reagents in precise proportions directly before addition to the reaction chamber.
  • the apparatus further comprises at least one monitoring device, such as an optical scanner, located to continuously monitor the chemical composition of the outlet fluid from the reaction chamber thereby to determine when each of the various reaction and wash steps are substantially completed.
  • the process of this invention comprises feedback control of an integrated oligonucleotide synthesis by detecting and monitoring the compositions of feed streams and/or effluent streams during both reaction and wash cycles. Optimization of process parameters is thereby achieved facilitating commercial-scale production of the valuable oligonucleotide products.
  • Fig. 1 is a process flow chart illustrating a typical single cycle in building a nucleotide chain.
  • Fig. 2 is a schematic illustration of one representative embodiment of the overall oligonucleotide production system of this invention.
  • the multi-step nucleotide chain-building synthesis can be scaled-up for commercial production designed to produce several grams or more of product utilizing an optimized, automated system as shown in Fig. 2. It will be understood by those skilled in the art that the exact sequence of steps as illustrated in Fig. 1 may vary depending on the recipe being used to synthesize a particular end product. Thus, in some applications, step 4 (capping) might come before step 3 (oxidation) , and so forth. In Fig.
  • reaction vessel 88 is first loaded with a suitable starting material, such as a protected nucleoside or other building blocks, fixed at one end of the chain to a suitable polymeric support such as CPG beads.
  • Reaction vessel 88 is then connected to a set of reagent reservoirs by an interconnected system of fluid conduits (not numbered) that are made of a material that is substantially inert with respect to all of the various reagents used in the process.
  • tubing for use in this system may be advantageously fashioned from a fluorocarbon polymer plastic or high-quality stainless steel.
  • Perfluoroalkane (PFA) tubing is especially preferred in this application because it is readily available commercially and combines a high level of inertness with somewhat better resilience than a tetrafluoroethylene resin like Teflon.
  • reaction step 1 (see Fig. 1) of a chain-building cycle, a flowstream of a suitable deblocking compound, such as a solution of dichloro acetic acid (DCA) in methylene chloride, is withdrawn from fluid reservoir 144 through three-way valves 126 and 128 and routed through the fluid conduit containing pump 80, flowmeter 82, and one or more valves, such as three- way valves 86 and 87, to the bottom of reaction vessel 88.
  • the deblocking solution is then passed upward through vessel 88 contacting the protected nucleoside or oligonucleotide fixed to the CPG beads resulting in unblocking or activating those functional groups.
  • a flowstream comprising reaction by ⁇ products and excess deblocking solution is withdrawn as effluent from the top of vessel 88, passed through one or more control valves, such as three-way valves 90 and 91, and directed through or past a monitoring device, such as optical scanner 92, which continuously monitors the chemical composition of the flowstream exiting vessel 88.
  • Optical scanner 92 preferably comprises an ultraviolet (UV) or visible light detector capable of monitoring at least two wavelengths.
  • UV ultraviolet
  • visible light detector capable of monitoring at least two wavelengths.
  • Such scanners are well-known in the art and relatively inexpensive. Still better, although somewhat more expensive, are full spectrum UV/visible scanners, which are also well-known and commercially available.
  • Other types of optical scanners, such as infrared (IR) scanners are also considered to be within the scope of this invention.
  • optical scanner 92 For optical scanner 92 to operate, of course, the portion of the outlet fluid conduit to which it is adjacent must contain an optically transparent window, such as a quartz window, to permit relatively unimpeded and undistorted entry and exit of the UV or other light used for monitoring. In addition, as they become more reliable and cost-effective, it is anticipated that other monitoring devices, for example density monitors, could be substituted for or used in conjunction with one or more of the optical scanners used herein. Either based on calibration or calculation, optical scanner 92 can be used to determine when the deblocking step has been substantially completed and the flow of deblocking solution through vessel 88 should therefore be discontinued.
  • an optically transparent window such as a quartz window
  • electrical signals from optical scanner 92 can be fed to a computer unit 100 programmed to evaluate the incoming signals, to determine from the scanning data the substantial completion of the deblocking (or any other) step, and to effectuate the transition from one process step to the next by electronically closing one set of system valves and opening another.
  • This computerized embodiment of the invention represents a highly automated, highly optimized, and extremely efficient oligonucleotide synthesis that can process vastly larger quantities of product in much shorter times than anything that has ever before been achieved. For example, whereas conventional oligonucleotide synthesis, equipment and processes have a maximum realizable output of about . 3 millimoles
  • the computerized embodiment of this invention has an expected output of at least about 100 millimoles (typically several grams) of the same product over about a twelve hour period. This dramatic and wholly unexpected improvement in production turns an essentially small-scale laboratory process into a viable commercial enterprise.
  • valve 126 is closed to stop the flow of deblocking solution from reservoir 144.
  • two-way valve 110 and three-way valves 112, 114, 122, 124, 126 and 128 are positioned to permit the flow of washing fluid, typically acetonitrile, from wash reservoir 20 through the connecting fluid passage to the bottom of vessel 88.
  • washing fluid typically acetonitrile
  • the wash fluid would flush the fluid passageway of residual deblocking solution.
  • the effluent wash stream passes scanner 92, where it is continuously monitored, and is withdrawn at valve 94 as waste stream 96.
  • optical or other monitoring of the effluent wash stream can be used to determine relatively precisely when the wash step has been substantially completed.
  • signals from the optical scanner can be relayed to computer 100, which is programmed to automatically switch the system from the wash step to the next reaction step when the wash step is substantially completed.
  • computer 100 which is programmed to automatically switch the system from the wash step to the next reaction step when the wash step is substantially completed.
  • Valves 110 and 128 are closed to stop the flow of acetonitrile wash fluid, and, instead, valves associated with one of the several amidite reservoirs are opened.
  • each amidite reservoir has associated therewith a three-way valve, namely valves 50, 52, 54, 56, 58, 60, 62 and 64 respectively.
  • the apparatus as shown in Fig. 2 may be utilized for the synthesis of a nucleotide chain having fewer than eight different amidite components by simply leaving the extra reservoirs empty and the associated valves closed and deactivated.
  • amidite By selectively activating and opening one of the valves 50, 52, etc., amidite can be withdrawn from any one of the amidite reservoirs and directed through three-way valve 66 and flowmeter 68 on the way to reaction vessel 88, while the other seven valves remain closed and their associated amidite reservoirs stay off-line.
  • Activator compound is stored in reservoir 72 and is also fed into the system during the coupling reaction step (step 2) via three-way valve 74 and pump 76.
  • a preferred activator for oligonucleotide synthesis is a solution of tetrazole in acetonitrile.
  • the reagents are ratio fed to the reaction vessel 88 in order to maintain optimum proportions of the reagents.
  • the flow meters 68 and 82 monitor the different stream flowrates. These flowrates are utilized by the process controller to adjust the pumps 80 and 76 to provide activator and amidite through the three-way mixing valve 70 to the reaction chamber 88 in the optimum predetermined proportions.
  • another monitoring device such as optical scanner 78, may be located along the flow path between valves 70 and 128 and used to confirm the identity of the amidite being added.
  • a third monitoring device such as optical scanner 84, may be located along the flow path between flowmeter 82 and three-way valve 86 to monitor the composition of the feed stream to vessel 88, for example following the addition of a recycle stream 95 of effluent from vessel 88.
  • monitoring device 92 is an indispensable element of this invention, however, the use of additional monitoring devices, such as scanners 78 and 84, at other locations in the system is optional and not required for the operability of the invention.
  • valves in the line containing valves 74, 70 and 128 would be positioned to permit the flow of wash fluid through this line to flush out residual activator.
  • the wash fluid would also eventually be channeled through vessel 88 to flush out activator, unreacted amidite, and coupling reaction by- products.
  • the effluent stream from vessel 88 during this second wash step would pass through optical scanner 92 for continuous monitoring to determine substantial completion of this wash step and, preferably, with computer-actuated valve switching at the proper time based on the scanner output .
  • Valves are closed to stop the flow of wash fluid, and, instead, three- way valves 124, 126 and 128 are opened to begin the flow of oxidizing or sulfurizing solution from oxidation reservoir 142 to vessel 88.
  • effluent from reactor vessel 88 is passed through scanner 92, where it is continuously monitored to determine when the oxidation reaction is substantially completed. Signals from scanner 92 can be relayed to computer unit 100 for automatically switching the system to the next wash step when the oxidation step is completed.
  • some portion of the effluent stream from vessel 88, containing unreacted oxidant may be advantageously recycled to vessel 88 via recycle stream 95.
  • valve 124 When the oxidation reaction is substantially completed, valve 124 is closed to stop the flow of oxidant from reservoir 142. Valves 110, 112, 114, 122, 124, 126 and 128 are positioned, however, to permit wash fluid from reservoir 20 to flush the line that had carried the oxidation solution, as well as to flush residual oxidation solution and by-products from the oxidation reaction from vessel 88.
  • the effluent stream from vessel 88 during this third wash step passes through scanner 92 for continuous monitoring to determine substantial completion of this wash step, preferably with computer-actuated valve switching at the appropriate time based on the scanner output.
  • Valves are closed to stop the flow of wash fluid, and, instead, two- way valves 132 and/or 140, together with valves 122, 124, 126 and 128, are positioned to begin the flow of capping compound to vessel 88.
  • a ratioed mixture of two different capping compounds may be utilized in this step.
  • reservoir 130 contains a first capping material, such as acetic anhydride, while reservoir 138 contains a second capping material, such as a base N-methyl imidazole.
  • An optimum mass ratio of the two capping materials being fed to vessel 88 may be calculated or determined by routine experimentation, and that optimum ratio can then be established and maintained using pump 134 and flowmeter 136 to control the mass flow of material from reservoir 130.
  • effluent from reactor vessel 88 is passed through scanner 92, where it is continuously monitored to determine when the capping reaction is substantially completed. Signals from scanner 92 can be relayed to computer unit 100 for automatically switching the system to the next wash step when the capping step is completed.
  • some portion of the effluent stream from vessel 88, containing unreacted capping compounds, may be advantageously recycled to vessel 88 via recycle stream 95.
  • valves 132 and 140 are closed to stop the flow of capping materials from reservoirs 130 and 138 respectively.
  • Valves 110, 112, 114, 122, 124, 126 and 128 are positioned, however, to permit wash fluid from reservoir 20 to flush the line that carried the capping fluid mixture, as well as to flush residual capping materials and by-products from the capping reactions from vessel 88.
  • the effluent stream from vessel 88 during this fourth wash step passes through scanner 92 for continuous monitoring to determine substantial completion of this final wash step, preferably with computer-actuated valve switching at the appropriate time based on the scanner output.
  • valve 114 can be opened to begin the flow of the cleavage compound, typically ammonium hydroxide, from reservoir 120 through the system to vessel 88.
  • the effect of the cleavage compound is to separate the completed nucleotide chains from the CPG polymer support in vessel 88.
  • the effluent from vessel 88 in this step of the process contains the raw oligonucleotide product, which is recovered by conventional means for further deprotection and purification.
  • scanner 92 may be utilized to determine when the cleavage reaction is substantially completed and all of the oligonucleotide product recovered from vessel 88.
  • Diaphragm-type valves minimize wetted interior surfaces subject to fluid corrosion as well as providing an interior configuration which facilitates complete sweeping with the wash fluid during the wash steps.
  • Diaphragm-type valves suitable for use in this invention which may be operated automatically in response to computer-generated electrical signals, are known in the art and available commercially.
  • Another useful enhancement of the basic apparatus and process of this invention is the use of one or more flowmeters in the fluid conduits, for example flowmeter 82, which is capable of monitoring fluid density as well as measuring and regulating mass flowrates.
  • flowmeter 82 which is capable of monitoring fluid density as well as measuring and regulating mass flowrates.
  • fluid density measures at one or more points in the system over time can be useful in monitoring the on-going process and in providing data for making mid-course adjustments as necessary.
  • density monitoring can supplement or, in some cases, substitute for one or more of the optical scanners.
  • means are also provided for monitoring the moisture content of any feed streams entering reaction vessel 88. Because the presence of water can adversely affect one or more of the various reactions being carried out in vessel 88, particularly the coupling and capping steps, it is normally desirable to maintain low or substantially zero tolerance for moisture.
  • a moisture monitoring device 77 located at valve 79, where recycle stream 95 is mixed with a fresh reagent stream from valve 128, can monitor moisture levels in both the fresh reagent and the recycle stream. Signals from moisture monitor 77 can be relayed to computer unit 100 for automatically controlling valves 79 and 94.
  • valves 79 and 94 can be automatically switched so as to divert the reagent stream to waste stream 96 and thereby prevent contamination of the contents of column 88.
  • fluid conduits leading to and from reaction vessel 88 can be arranged to facilitate reverse (downward) flow through the column.
  • valves 86, 87, 90 and 91 fluid flow can be directed from valve 86 to valve 90 (as shown by the dotted lines), then into the top of column 88.
  • the effluent stream is withdrawn from the bottom of column 88, and directed from valve 87 to valve 91 (as shown by the dotted lines) , where it rejoins the main fluid conduit system.
  • Reverse flow through column 88 may be utilized periodically to optimize distribution in the reactor and to complete utilization of all reactive sites in the bed.
  • Still another useful enhancement in the apparatus and process of this invention is the use of a Triplex pump, for example pump 80 in Fig. 2.
  • the advantages of the Triplex pump used for pump 80 are that it has a wide range of fluid flow capabilities and pressures, and the smooth pumping action of this design helps to avoid unduly disturbing the bed of reaction vessel 88.
  • Such pumps are known in the art and available commercially.
  • Programmable logic-controlling (PLC) computer units capable of receiving continuous electrical inputs from an optical scanner, as well as data on valve positions, flowrates, densities, pressures, etc., and processing that data according to certain predetermined algorithms, and generating electrical outputs based on that data for selectively activating one or more valves in a system such as that shown in Fig. 2, are well- known in the art.
  • Programming the computer software to carry out the monitoring, optimizing, feedback control, and automation functions as described herein is within the purview of those of ordinary skill in this art.
  • Software will utilize custom-tailored recipes for each particular oligonucleotide synthesis depending on the particular reagents used and other desired process parameters.
  • This invention leads to optimization of reaction and wash times, as well as optimization of the quantities of reagents.
  • This invention makes possible economical, commercial-scale production of these increasingly valuable and important complex organic materials.
  • This invention also leads to optimum yields of a purer product, at lower cost, and in less time than has heretofore been possible.

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  • Saccharide Compounds (AREA)

Abstract

L'invention porte sur un appareil et un procédé permettant d'optimiser les étapes répétitives de la synthèse en phase solide d'oligonucléotide par balayage optique en continu d'un courant d'effluent depuis le module de réaction et par traitement informatique et mise en application des données de balayage.
PCT/US1996/011448 1995-08-17 1996-07-09 Appareil et procede de synthese en plusieurs etapes de molecules organiques a chaine longue en phase solide WO1997007126A1 (fr)

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US51627595A 1995-08-17 1995-08-17
US08/516,275 1995-08-17
US08/608,702 US5807525A (en) 1995-08-17 1996-02-29 Apparatus and process for multi stage solid phase synthesis of long chained organic molecules
US08/608,702 1996-02-29

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WO1999030817A1 (fr) * 1997-12-15 1999-06-24 Kalibrant Limited Procede et dispositif de synthese chimique
US5961925A (en) * 1997-09-22 1999-10-05 Bristol-Myers Squibb Company Apparatus for synthesis of multiple organic compounds with pinch valve block
GB2336327A (en) * 1997-12-15 1999-10-20 Kalibrant Limited Method and apparatus for chemical synthesis
US6274094B1 (en) 1997-01-13 2001-08-14 Weller, Iii Harold Norris Nestable, modular apparatus for synthesis of multiple organic compounds
EP1714695A1 (fr) * 2005-04-18 2006-10-25 TechniKrom, Inc. Appareil industriel automatisé pour synthèse de composés biologiquement actifs et procédé
EP1638989B1 (fr) * 2003-06-25 2008-07-30 Peregrine Pharmaceuticals, Inc. Procede et appareil de radiomarquage continu a grande echelle de proteines
US9034798B2 (en) 2003-01-16 2015-05-19 Caprotec Bioanalytics Gmbh Capture compounds, collections thereof and methods for analyzing the proteome and complex compositions
SE2151135A1 (en) * 2021-09-16 2023-03-17 Peptisystems Ab Processes and systems for flow-through oligonucleotide synthesis

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US5233044A (en) * 1989-06-08 1993-08-03 Millipore Corporation Active esters for solid phase peptide synthesis
WO1992015867A1 (fr) * 1991-03-06 1992-09-17 Rhone-Poulenc Rorer S.A. Procede et appareil de synthese en phase heterogene de macromolecules telles que des peptides, des polynucleotides ou des oligosaccharides
EP0541340A2 (fr) * 1991-11-05 1993-05-12 The Perkin-Elmer Corporation Appareillage et procédé pour le synthèse des biopolymères
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WO1994001214A1 (fr) * 1992-07-06 1994-01-20 Beckman Instruments, Inc. Procede de surveillance de la reaction et de l'ecoulement d'un processus en ligne

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6274094B1 (en) 1997-01-13 2001-08-14 Weller, Iii Harold Norris Nestable, modular apparatus for synthesis of multiple organic compounds
US5961925A (en) * 1997-09-22 1999-10-05 Bristol-Myers Squibb Company Apparatus for synthesis of multiple organic compounds with pinch valve block
US6267930B1 (en) 1997-09-22 2001-07-31 Waldemar Ruediger Apparatus for synthesis of multiple organic compounds with pinch valve block
WO1999030817A1 (fr) * 1997-12-15 1999-06-24 Kalibrant Limited Procede et dispositif de synthese chimique
GB2336327A (en) * 1997-12-15 1999-10-20 Kalibrant Limited Method and apparatus for chemical synthesis
GB2336327B (en) * 1997-12-15 2000-03-08 Kalibrant Limited Method and apparatus for chemical synthesis
US6290915B1 (en) 1997-12-15 2001-09-18 Kalibrant Limited Apparatus having multiple inputs for solid-phase chemical synthesis
US9034798B2 (en) 2003-01-16 2015-05-19 Caprotec Bioanalytics Gmbh Capture compounds, collections thereof and methods for analyzing the proteome and complex compositions
EP1638989B1 (fr) * 2003-06-25 2008-07-30 Peregrine Pharmaceuticals, Inc. Procede et appareil de radiomarquage continu a grande echelle de proteines
EP1964847A3 (fr) * 2003-06-25 2008-09-17 Peregrine Pharmaceuticals, Inc. Procédé et appareil de radiomarquage à grande échelle continu de protéines
AU2004253924B2 (en) * 2003-06-25 2009-07-30 Peregrine Pharmaceuticals, Inc. Method and apparatus for continuous large-scale radiolabeling of proteins
US7591953B2 (en) 2003-06-25 2009-09-22 Peregrine Pharmaceuticals, Inc. Methods and apparatus for continuous large-scale radiolabeling of proteins
US8137540B2 (en) 2003-06-25 2012-03-20 Peregrine Pharmaceuticals, Inc. Methods and apparatus for continuous large-scale radiolabeling
EP1714695A1 (fr) * 2005-04-18 2006-10-25 TechniKrom, Inc. Appareil industriel automatisé pour synthèse de composés biologiquement actifs et procédé
SE2151135A1 (en) * 2021-09-16 2023-03-17 Peptisystems Ab Processes and systems for flow-through oligonucleotide synthesis
WO2023043359A1 (fr) * 2021-09-16 2023-03-23 Peptisystems Ab Procédés et systèmes de synthèse continue d'oligonucléotides
SE546008C2 (en) * 2021-09-16 2024-04-09 Peptisystems Ab Processes and systems for flow-through oligonucleotide synthesis

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