Process for the removal of trifluoroacetic acid from a cleavage spray precipitation condensation system

Abstract

The present application relates to a kind of cleavage spray precipitation condensing system and the method for removing trifluoroacetic acid, mainly solve the technical problem that the method for generally removing trifluoroacetic acid is not safe, dangerous, inefficient, very slow speed.The trifluoroacetic acid and cleavage mixed solution are transported to cleavage container by solvent input system, and polypeptide resin is added to cleavage container, and stirring device mixes trifluoroacetic acid / cleavage mixed solution and polypeptide resin clockwise or counterclockwise at suitable polypeptide temperature, after cleavage, trifluoroacetic acid / cleavage mixed solution and polypeptide resin are filtered by container filter plate and are transported by solvent delivery pipe to the nozzle of spray precipitation container, nozzle is heated by pressurized hot nitrogen, trifluoroacetic acid / cleavage mixed solution / polypeptide resin and nitrogen are sprayed at the end of nozzle, gasified trifluoroacetic acid is sprayed, residual concentrated pulp-like polypeptide resin is precipitated in spray precipitator, and trifluoroacetic acid gas is condensed after passing through condenser and is input into trifluoroacetic acid collector.

Description

TECHNICAL FIELD

[0001] The present invention relates to cleavage systems, in particular to a cleavage spray precipitation condensation system (CSPCS) that improves the efficiency of cleaving a polypeptide from a resin to which the polypeptide is bonded and the removal of protecting groups on certain side chains of the polypeptide after solid phase polypeptide synthesis. BACKGROUND

[0002] Solid phase polypeptide synthesis (SPPS) involves the assembly of amino acids into a polypeptide of a desired sequence while one end of the chain is anchored to an insoluble resin. After the entire polypeptide is assembled onto the resin, a reagent (e.g., trifluoroacetic acid and an appropriate cleavage agent) is used to cut the polypeptide from the resin and protecting groups on certain side chains. The primary reagent used for cleavage is trifluoroacetic acid. Trifluoroacetic acid cleavage is typically performed at low temperature for a short period of time or at room temperature. This typically effectively cleaves the polypeptide from the resin and removes all side chain protecting groups. The trifluoroacetic acid is then removed, for example, after the cleaved polypeptide and trifluoroacetic acid cleavage solution is separated from the resin, the cleaved solution is sprayed and hot nitrogen gas is passed through the spray precipitation condenser and the vaporized trifluoroacetic acid is exchanged from the gas to the solution and collected in a condensing collector with an appropriate vacuum. A common problem in the production process is that the inefficiency of removing the trifluoroacetic acid causes the polypeptide to be in contact with the trifluoroacetic acid for an extended period of time, which produces unwanted byproducts. Notably, this problem is more pronounced in large scale cleavage when large amounts of trifluoroacetic acid cannot be quickly removed. In addition, the above-mentioned side reactions also result in a lower purity of the intended polypeptide, thus a lower yield of the pure polypeptide recovered. The inability to effectively remove the trifluoroacetic acid, leaving more trifluoroacetic acid in the crude peptide solution also results in a less efficient downstream process, using more organic solvents (diethyl ether or methyl tert-butyl ether, etc.) than necessary. As described in U.S. Patents 5,380,495, 5,453,487 and 6,320,025, after the coupling of the desired protected polypeptide on the resin is completed using an automated polypeptide synthesizer, the resin-bound polypeptide is transferred to a cleavage vessel where trifluoroacetic acid and a cleavage cocktail are used to cleave the polypeptide from the resin and to block groups on certain side chains to release the free peptide. The resin-bound polypeptide is treated with trifluoroacetic acid at a cleavage temperature of about -5°C to about 40°C for about a half hour to about 4 hours to substantially completely cleave the polypeptide from the resin. After the polypeptide is cleaved, the resulting mixed solution contains the resin, the cleaved polypeptide, the trifluoroacetic acid, and the cleavage cocktail. The cleavage cocktail solution is quickly filtered to remove the resin, leaving the trifluoroacetic acid cocktail and the peptide in the filtrate.

[0003] Therefore, it is highly desirable to develop a cleavage spray precipitation condensation system that effectively removes the trifluoroacetic acid, reduces or eliminates side reactions, improves the efficiency of the downstream process, reduces the use of solvents, and improves the safety of the process. SUMMARY

[0004] The present application aims to provide a cleavage spray precipitation condensing system and a method for removing trifluoroacetic acid, mainly solving the technical problems that the methods of removing trifluoroacetic acid from a polypeptide resin / trifluoroacetic acid mixed solution after polypeptide synthesis using a rotary evaporator, a vacuum centrifuge or a distillation device are unsafe, dangerous, inefficient and slow.

[0005] The technical scheme of the present application is as follows: a cleavage spray precipitation condensing system, comprising a cleavage system and a spray precipitation system, wherein the cleavage system and the spray precipitation system comprise a cleavage container and a spray precipitation container, both of which are provided with stirring devices, the stirring device is a motor connected with stirring blades through a stirring shaft, and a container filter plate is fixed at the tail end of the stirring shaft; the bottom of the cleavage container is connected with the top of the spray precipitator through a solvent conveying pipe, the top of the cleavage container and the top of the spray precipitation container are both provided with a thermometer and a pressure gauge, the top of the cleavage container is provided with a gas input pipe with a two-way valve, the top of the cleavage container is provided with a solvent input system, the solvent input system has one of the following structures, one is that a plurality of two-way valve pipelines are connected with a solvent input pipeline, and then connected with a spray head in the cleavage container, and the solvent input pipeline is connected with a pump and a two-way valve in sequence; the other is that a plurality of two-way valve pipelines are connected with a solvent input pipeline, and then connected with a spray head in the cleavage container, and a pumping pipeline is arranged at the top of the cleavage container, and a two-way valve and a pump are connected in sequence on the pumping pipeline; the bottom of the spray precipitation container is provided with a solvent output pipeline, and a two-way valve, a pump and a three-way valve are connected in sequence on the solvent output pipeline; the top of the spray precipitation container is provided with a gas input pipe, and a heating device is arranged on the gas input pipe, and a two-way valve, a pump and a cooling device are connected in sequence on the solvent conveying pipe or a two-way valve and a cooling device are connected in sequence; the solvent pipe and the gas input pipe are connected with a nozzle after entering the spray precipitation container, one kind of nozzle is a sleeve barrel structure, the solvent pipe is connected with an inner barrel, and the gas input pipe is connected with the sleeve gap; the other kind of nozzle is a double-channel structure, and the solvent pipe and the gas input pipe are connected with a double-channel nozzle; the top of the spray precipitation container is provided with a gas output pipeline, the gas output pipeline passes through a condenser and is connected with a plurality of trifluoroacetic acid collectors through a two-way valve after being branched, a gas pipe filter is arranged at the inlet end and the outlet end of the condenser, an exhaust pipe is connected with the outlet end of the condenser, a three-way valve is arranged at the tail end of the exhaust pipe, and a vacuum pump is arranged on one of the three-way valves.

[0006] A method for removing trifluoroacetic acid from a polypeptide mixture solution using a lytic spray precipitation condensation system, comprising the steps of: delivering trifluoroacetic acid and a cleavage mixture solution to a cleavage vessel through a solvent delivery system, and adding a polypeptide resin to the cleavage vessel. An agitation device mixes the trifluoroacetic acid / cleavage mixture solution and the polypeptide resin clockwise or counterclockwise at a temperature suitable for the polypeptide resin. After cleavage, the trifluoroacetic acid / cleavage mixture solution and the polypeptide resin are filtered through a vessel filter plate and delivered through a solvent delivery tube to a nozzle of a spray precipitator, the nozzle is heated by pressurized hot nitrogen, the trifluoroacetic acid / cleavage mixture solution / polypeptide resin and nitrogen gas are ejected at the end of the nozzle to vaporize the trifluoroacetic acid, the remaining concentrated slurry of polypeptide resin is precipitated in the spray precipitator, and the trifluoroacetic acid gas is condensed by a condenser and delivered to a trifluoroacetic acid collector.

[0007] The present invention has the following advantages: The trifluoroacetic acid and cleavage mixture solution used to cleave polypeptides from a polypeptide resin can be safely performed. The present invention reduces the use of large amounts of diethyl ether or methyl tert-butyl ether in the process. It is expected to reduce the use of diethyl ether or methyl tert-butyl ether by at least 5 times. The present invention quickly and effectively removes trifluoroacetic acid, reducing the contact time of polypeptides with trifluoroacetic acid, thus minimizing or eliminating side reactions, resulting in lower expected purity of polypeptides and lower yield of pure peptides. BRIEF DESCRIPTION OF DRAWINGS

[0008] Figure 1 The structure diagram of Example 1 of the present invention.

[0009] Figure 2 The structure diagram of Example 2 of the present invention.

[0010] Figure 3 The structure diagram of nozzle a of the present invention.

[0011] Figure 4 The structure diagram of nozzle b of the present invention.

[0012] In the figure: 100 - cleavage system, 101 - two-way valve a, 102 - two-way valve b, 103 - two-way valve c, 104 - two-way valve d, 105 - pump a, 106 - two-way valve e, 107 - solvent input line, 111 - two-way valve f, 112 - gas input line a, 113 - thermometer a, 114 - pressure gauge a, 121 - motor a, 122 - stirring shaft a, 123 - stirring blade a, 124 - vessel filter plate a, 125 - cleavage vessel, 126 - cleavage vessel top, 127 - cleavage vessel bottom, 131 - two-way valve g, 132 - solvent delivery line a, 133 - pump b, 200 - spray precipitation system, 201 - cooling device, 202 - solvent line, 203 - nozzle, 211 - heating device, 212 - gas input line b, 213 - thermometer b, 214 - pressure gauge b, 221 - motor b, 222 - stirring shaft b, 223 - stirring blade b, 224 - vessel filter plate b, 225 - spray precipitation vessel, 226 - spray precipitation vessel top, 227 - spray precipitation vessel bottom, 231 - two-way valve h, 232 - solvent delivery line b, 233 - pump c, 234 - three-way valve a, 241 - gas output line, 242 - gas line filter, 251 - condenser, 252 - three-way valve b, 253 - vacuum pump, 254 - two-way valve i, 255 - two-way valve j, 256 - trifluoroacetic acid collector a, 257 - trifluoroacetic acid collector b, 258 - exhaust line. DETAILED DESCRIPTION

[0013] Example 1, Reference Figure 14, a pyrolysis spray precipitation condensation system, comprising a pyrolysis system 100 and a spray precipitation system 200, the pyrolysis system 100 and the spray precipitation system 200 comprising a pyrolysis container 125 and a spray precipitation container 225, the spray precipitation container 225 is provided with stirring device, the pyrolysis container 125 stirring device is motor a121, stirring shaft a122 is connected with stirring blade a123 through stirring shaft a122, and the tail end of stirring shaft a122 is fixed with container filter plate a124;The stirring device of the spray precipitation container 225 is motor b221, stirring shaft b222 is connected with stirring blade b223 through stirring shaft b222, and the tail end of stirring shaft b222 is fixed with container filter plate b224;The bottom of the pyrolysis container 125 is connected with the top 226 of the spray precipitation container through the solvent conveying pipe 107, the top 126 of the pyrolysis container is provided with thermometer a113 and pressure gauge a114, the top 126 of the pyrolysis container is provided with gas input pipe a112 with two-way valve f111, the top 226 of the spray precipitation container is provided with thermometer b213 and pressure gauge b214, the top 126 of the pyrolysis container is provided with a solvent input system, the solvent input system is connected with the spray head in the pyrolysis container through two-way valve a101, two-way valve b102, two-way valve c103, two-way valve d104 pipeline confluence, solvent input pipeline 107, pump a105 and two-way valve e106 in sequence;The bottom 227 of the spray precipitation container is provided with solvent conveying pipe b232, the solvent conveying pipe b232 is connected with two-way valve h231, pump c233 and three-way valve a234 in sequence;The top 226 of the spray precipitation container is provided with gas input pipe b212, the gas input pipe b212 is provided with heating device 211, the solvent conveying pipe a132 is connected with two-way valve g131, pump b133 and cooling device 201 in sequence;Solvent pipe 202 and gas input pipe b212 enter the spray precipitation container 225 and are connected with nozzle 203 through spray nozzle 203, the nozzle 203 is a double-channel structure, and the solvent pipe 202 and the gas input pipe b212 are connected with the double-channel nozzle 203;The top 226 of the spray precipitation container is provided with gas output pipeline 241, the gas output pipeline 241 passes through the condenser 251 and is branched to two-way valve i254 and two-way valve j255 respectively, and is connected with trifluoroacetic acid collector a256 and trifluoroacetic acid collector b257 respectively, gas pipe filters 242 are additionally installed at the inlet end and the outlet end of the condenser 251, an exhaust pipe 258 is connected at the outlet end of the condenser, a three-way valve b252 is additionally installed at the tail end of the exhaust pipe 258, and a vacuum pump 253 is additionally installed on one of the roads of the three-way valve b252.

[0014] Oxytocin polypeptide resin: Cys(Trt)-Tyr(tBu)-Ile-Gln(Trt)-Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-NH2

[0015] The cleavage vessel 125 is cleaved with trifluoroacetic acid at 20-30 °C for 2 hours. At the end of the cleavage, the trifluoroacetic acid / cleavage mixture and the polypeptide resin are filtered through the vessel filter plate a 124 and delivered through the solvent delivery tube a 132 to the nozzle 203 of the spray precipitation vessel 225, which provides hot nitrogen gas under pressure. The trifluoroacetic acid gas is collected under vacuum and condensed in a condenser. Chilled diethyl ether or methyl tert-butyl ether is pumped into the polypeptide resin / trifluoroacetic acid cleavage mixture in the spray precipitation vessel 225. A faint white solid suspension is deposited in the spray precipitation vessel 225. The trifluoroacetic acid liquid is collected in the trifluoroacetic acid collector a 256 and the trifluoroacetic acid collector b 257. The total crude polypeptide resin recovered from the trifluoroacetic acid filtrate is 95% and more than 75% of the trifluoroacetic acid is removed.

[0016] Example 2, with reference Figure 24. A pyrolysis spray precipitation condensation system, comprising a pyrolysis system 100 and a spray precipitation system 200, the pyrolysis system 100 and the spray precipitation system 200 comprising a pyrolysis container 125 and a spray precipitation container 225, the pyrolysis container 125 and the spray precipitation container 225 each being provided with a stirring device, the stirring device of the pyrolysis container 125 comprising a motor a121 connected to stirring blades a123 through a stirring shaft a122, the tail end of the stirring shaft a122 being fixed with a container filter plate a124; the stirring device of the spray precipitation container 225 comprising a motor b221 connected to stirring blades b223 through a stirring shaft b222, the tail end of the stirring shaft b222 being fixed with a container filter plate b224; the bottom of the pyrolysis container 125 being connected to the top 226 of the spray precipitation container through a solvent delivery pipe 107, the top 126 of the pyrolysis container being provided with a thermometer a113 and a pressure gauge a114, the top 126 of the pyrolysis container being provided with a gas input pipe a112 provided with a two-way valve f111, the top 226 of the spray precipitation container being provided with a thermometer b213 and a pressure gauge b214, the top 126 of the pyrolysis container being provided with a solvent input system, the solvent input system comprising a two-way valve a101, a two-way valve b102, a two-way valve c103, a two-way valve d104, a solvent input pipe 107, a nozzle in the pyrolysis container, a suction pipe provided at the top 126 of the pyrolysis container, the suction pipe being connected to a two-way valve e106 and a pump a105 in sequence; the bottom 227 of the spray precipitation container being provided with a solvent delivery pipe b232, the solvent delivery pipe b232 being connected to a two-way valve h231, a pump c233 and a three-way valve a234 in sequence; the top 226 of the spray precipitation container being provided with a gas input pipe b212, the gas input pipe b212 being provided with a heating device 211, the solvent delivery pipe a132 being connected to a two-way valve g131 and a cooling device 201 in sequence; a solvent pipe 202 and the gas input pipe b212 being connected to a double-channel nozzle 203; the top 226 of the spray precipitation container being provided with a gas output pipe 241, the gas output pipe 241 passing through a condenser 251 and being branched to a three-way valve i254 and a three-way valve j255, the three-way valve i254 and the three-way valve j255 being connected to a trifluoroacetic acid collector a256 and a trifluoroacetic acid collector b257 respectively, a gas pipe filter 242 being provided at the inlet end and the outlet end of the condenser 251, an exhaust pipe 258 being connected to the outlet end of the condenser 251, a three-way valve b252 being provided at the tail end of the exhaust pipe 258, a vacuum pump 253 being provided at one of the branches of the three-way valve b252.

[0017] Exenatide polypeptide resin: His(Trt)-Gly-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)- Ser(tBu)-Asp(OtBu)-Leu-Ser(tBu)-Lys(Boc)-Gln(Trt)-Met-Glu(OtBu)-Glu(OtBu)- Glu(OtBu)-Ala-Val-Arg(Pbf)-Leu-Phe-lle-Glu(OtBu)-Trp(Boc)-Leu-Lys(Boc)- Asn(Trt)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-NH2

[0018] The cleavage vessel is filtered at the end of the cleavage with trifluoroacetic acid at 20-30°C. The trifluoroacetic acid / cleavage mixture and the polypeptide resin are filtered through the vessel filter plate a 124 and delivered through the solvent delivery tube a 132 to the nozzle 203 of the spray precipitation vessel 225, which provides hot nitrogen gas under pressure. The trifluoroacetic acid gas is collected under vacuum and condensed in a condenser. Chilled diethyl ether or methyl tert-butyl ether is pumped into the polypeptide resin / trifluoroacetic acid cleavage mixture in the spray precipitation vessel 225. A faint white solid suspension is deposited in the spray precipitation vessel 225. The trifluoroacetic acid liquid is collected in the trifluoroacetic acid collector a 256 and the trifluoroacetic acid collector b 257. The total crude polypeptide recovered from the trifluoroacetic acid filtrate is 96% and more than 75% of the trifluoroacetic acid is removed.

[0019] Example 3, with reference to Figure 3 The nozzle 203 is a sleeve barrel configuration with the solvent tube 202 attached to the inner barrel and the gas input tube b 212 communicating with the sleeve gap; the remainder is as in Example 1.

Claims

1. A pyrolysis spray precipitation condensation system characterized by: The system comprises a cracking system and a spray precipitation system, the cracking system and the spray precipitation system comprise a cracking container and a spray precipitation container, the cracking container and the spray precipitation container are both provided with stirring devices, the bottom of the cracking container is connected with the top of the spray precipitator through a solvent conveying pipe, the top of the cracking container and the top of the spray precipitation container are both provided with a thermometer and a pressure gauge, the top of the cracking container is provided with a gas input pipe with a two-way valve, the top of the cracking container is provided with a solvent input system, the bottom of the spray precipitation container is provided with a solvent output pipeline, and the solvent output pipeline is sequentially connected with a two-way valve, a pump and a three-way valve; the solvent conveying pipe at the bottom of the cracking container is sequentially connected with a two-way valve and a cooling device, or a pump is further arranged between the two-way valve and the cooling device; the top of the spray precipitation container is provided with a gas input pipe, the gas input pipe is provided with a heating device, and the solvent pipe and the gas input pipe are connected with a nozzle after entering the spray precipitation container, the top of the spray precipitation container is provided with a gas output pipeline, the gas output pipeline passes through a condenser and is branched to be connected with a plurality of trifluoroacetic acid collectors through a two-way valve, gas pipe filters are arranged at the inlet end and the outlet end of the condenser, an exhaust pipe is connected with the outlet end of the condenser, and a three-way valve is arranged at the tail end of the exhaust pipe, and a vacuum pump is arranged on one of the paths of the three-way valve.

2. The lytic spray precipitation condensation system of claim 1, wherein: The solvent input system is a plurality of two-way valve pipelines converging, then connected with a solvent input pipeline, and then connected with a spray head in the cracking container, and the solvent input pipeline is sequentially connected with a pump and a two-way valve.

3. The lytic spray precipitation condensation system of claim 1, wherein: The solvent input system is a plurality of two-way valve pipelines converging, then connected with a solvent input pipeline, and then connected with a spray head in the cracking container, and the solvent input pipeline is sequentially connected with a pump and a two-way valve.

4. The lytic spray precipitation condensation system of claim 1, wherein: The stirring device is a motor connected with stirring blades through a stirring shaft, and a container filter plate is fixed at the tail end of the stirring shaft.

5. The lytic spray precipitation condensation system of claim 1, wherein: The nozzle is a sleeve barrel structure, the solvent pipe is connected with an inner barrel, and the gas input pipe is communicated with the gap of the sleeve.

6. The lytic spray precipitation condensation system of claim 1, wherein: The nozzle is a double-channel structure, and the solvent pipe and the gas input pipe are connected with a double-channel nozzle.

7. A method for removing trifluoroacetic acid using the cleaving spray precipitation condensation system of claim 1, characterized by: The system comprises the following steps: The trifluoroacetic acid and the cracking mixed solution are conveyed into the cracking container through the solvent input system, the polypeptide resin is added into the cracking container, the stirring device mixes the trifluoroacetic acid / cracking mixed solution and the polypeptide resin clockwise or counterclockwise at a suitable temperature of the polypeptide resin, after cracking, the trifluoroacetic acid / cracking mixed solution and the polypeptide resin are filtered through the container filter plate and conveyed into the nozzle of the spray precipitation container through the solvent conveying pipe, the nozzle is heated by pressurized hot nitrogen, the trifluoroacetic acid / cracking mixed solution / polypeptide resin and nitrogen gas are sprayed at the end of the nozzle to gasify trifluoroacetic acid, the residual concentrated slurry polypeptide resin is precipitated in the spray precipitator, and the trifluoroacetic acid gas is condensed through the condenser and then input into the trifluoroacetic acid collector.

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