CN110003306B - Apparatus and method for solid phase synthesis of polypeptides - Google Patents
Apparatus and method for solid phase synthesis of polypeptides Download PDFInfo
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- CN110003306B CN110003306B CN201910260208.XA CN201910260208A CN110003306B CN 110003306 B CN110003306 B CN 110003306B CN 201910260208 A CN201910260208 A CN 201910260208A CN 110003306 B CN110003306 B CN 110003306B
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Classifications
-
- 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
-
- 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
- C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a device for solid phase synthesis of polypeptide, which comprises: at least one amino acid reservoir; at least two condensing agent reservoirs; a multi-channel switching device; a peristaltic pump; a liquid measuring device; a transfer device; a reactor; and a deprotecting agent reservoir. The invention also discloses a method for synthesizing the polypeptide by using the equipment. The equipment of the invention fundamentally solves the problem of pipeline blockage, has compact design, greatly reduces the occupied space and the volume, and saves the cost.
Description
Technical Field
The invention relates to the field of polypeptide synthesis, in particular to equipment and a method for solid-phase synthesis of polypeptides.
Background
Polypeptides are compounds formed by dehydration condensation of alpha-amino acids followed by peptide bonds. Currently, polypeptide synthesis techniques including solid phase synthesis have been developed.
In the solid phase synthesis method, natural amino acids in which the relevant functional groups are protected with specific protecting groups are selected as raw materials. During synthesis, the reaction starts on the solid support. The steps of deprotection, activation, crosslinking and the like are repeatedly carried out, so that peptide bonds are formed between amino acids, and the amino acids are connected to a solid-phase carrier one by one to form a polypeptide chain.
In existing devices for solid phase synthesis of polypeptides, a gas, such as nitrogen, is typically used as the liquid transfer driving substance. In the apparatus having such a structure, when the reaction in the reaction vessel is waited for, a part of the raw material amino acid solution is retained in the pipe after being fed, and the problem of precipitation of the amino acid solution in the pipe occurs over a long period of time. Amino acid precipitation can clog the pipeline, and if not cleaned in time, the reaction can be stopped unexpectedly, so that the efficiency of polypeptide synthesis is greatly affected.
Thus, there is a need for an apparatus for solid phase synthesis of polypeptides that prevents the occurrence of amino acid precipitation.
Disclosure of Invention
In one aspect, the present invention provides an apparatus for solid phase synthesis of a polypeptide, the apparatus comprising:
at least one amino acid reservoir for storing an amino acid solution;
at least two condensing agent reservoirs for storing condensing agents;
a multi-channel switching device having a plurality of inlets and one outlet, and allowing a fluid channel to be formed between any one of the plurality of inlets and the outlet while no fluid channel is formed between the other inlet and the outlet;
a peristaltic pump;
a liquid measuring device;
a transfer device;
a reactor; and
a deprotecting agent reservoir for storing a deprotecting agent,
wherein,
each of the at least one amino acid reservoir and each of the at least two condensing agent reservoirs are in fluid communication with an inlet of the multi-channel switching device,
the outlet of the multichannel switching device is in fluid communication with one port of the peristaltic pump,
the other port of the peristaltic pump is in fluid communication with the inlet of the liquid metering device,
the outlet of the liquid metering device is in fluid communication with the inlet of the transfer device,
the outlet of the transfer device is in fluid communication with the reactor, and
the deprotecting agent reservoir is in fluid communication with the reactor.
Optionally, the multi-channel switching device is a multi-channel switching valve.
Optionally, the multi-channel switching device is a plurality of multi-channel switching valves connected in series.
Optionally, the apparatus further comprises at least one cleaning fluid reservoir, each of the at least one cleaning fluid reservoir being in fluid communication with an upstream-most inlet of the multi-channel switching device, respectively.
Optionally, the liquid measuring device is provided with a liquid level sensor.
Optionally, the reactor is a two-pass reactor.
Optionally, the apparatus further comprises a motor configured to cause the reactor to turn over.
Optionally, the apparatus further comprises a cutting fluid reservoir in fluid communication with the reactor.
In another aspect, the present invention provides a method for synthesizing a polypeptide using the above apparatus, the method comprising:
setting the multi-channel switching device such that a fluid channel is formed between one of the at least one amino acid reservoir and the peristaltic pump;
operating a peristaltic pump in a forward direction to deliver the amino acid solution stored in the one amino acid reservoir to the solution metering device;
conveying the amino acid solution in the liquid measuring device to the transfer device;
reversing the peristaltic pump to deliver the amino acid solution retained in the tubing between the peristaltic pump and the liquid metering device back to the amino acid reservoir;
setting the multi-channel switching device so that a fluid channel is formed between one condensing agent reservoir of the at least two condensing agent reservoirs and the peristaltic pump;
operating a peristaltic pump in a forward direction to deliver a first condensing agent stored in said one condensing agent reservoir to said liquid metering device;
reversing the peristaltic pump to deliver the first condensing agent retained in the tubing between the peristaltic pump and the metering device back to the one condensing agent reservoir;
conveying the condensing agent in the liquid measuring device to the transfer device;
setting the multi-channel switching device so that a fluid channel is formed between the peristaltic pump and the other condensing agent reservoir of the at least two condensing agent reservoirs;
operating the peristaltic pump in a forward direction to deliver the second condensing agent stored in the other condensing agent reservoir to the liquid metering device;
conveying the second condensing agent in the liquid measuring device to the transfer device;
reacting a mixture comprising the amino acid solution, the first condensing agent, and the second condensing agent in the transfer device to activate the amino acid;
transferring the activated amino acid from the transfer device to the reactor and reacting on a solid support placed in the reactor to form a peptide chain; and
delivering a deprotecting agent stored in the deprotecting agent reservoir to the reactor to deprotect the peptide chain.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are required to be used in the description of the embodiments will be briefly described below. It is evident that the drawings in the following description are only some embodiments of the present invention and that other drawings may be obtained from these drawings without inventive effort for a person of ordinary skill in the art.
FIG. 1 shows a schematic diagram of an apparatus for solid phase synthesis of polypeptides according to an embodiment of the invention.
FIG. 2 shows a schematic representation of an apparatus for solid phase synthesis of polypeptides according to an embodiment of the invention based on FIG. 1.
Figure 3 shows a schematic diagram of a more specific apparatus for solid phase synthesis of polypeptides according to an embodiment of the invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to fall within the scope of the invention.
FIG. 1 shows a schematic diagram of an apparatus for solid phase synthesis of polypeptides according to an embodiment of the invention. It comprises the following steps: at least one amino acid reservoir 8, at least two condensing agent reservoirs 6, a multi-channel switching device 3, a peristaltic pump 5, a dosing device 12, a transfer device 14, a reactor 4, and a deprotecting agent reservoir 21.
The amino acid reservoir 8 and condensing agent reservoir 6 are used to store synthetic raw materials for the polypeptide reaction and may be referred to as raw material reservoirs. The number of amino acid reservoirs 8 is at least one, but may be plural, but is represented by a square in FIG. 1. In the present invention, the number of amino acid reservoirs is not particularly limited, and may be 1, 2, 3, 4, 5 or even more, for example, up to 24. Multiple amino acid reservoirs 8 can be used to store different types of amino acid solutions to synthesize polypeptides comprising multiple amino acid monomers. At least two condensing agent reservoirs 6 are provided, but a plurality of condensing agent reservoirs may be provided, but are indicated by a square in fig. 1. Since at least two condensing agents are typically required for polypeptide synthesis, there are at least two condensing agent reservoirs in the system. In the present invention, the number of condensing agent reservoirs is not particularly limited. It may be 2 and used to store two different condensing agents, namely a first condensing agent and a second condensing agent, respectively.
Polypeptide synthesis typically requires the sequential use of two condensing agents to accomplish the amino acid condensation. One example of a first condensing agent and a second condensing agent in one system is DIC and oxama. The reaction scheme is schematically shown below.
By the procedure shown above, polypeptides were synthesized from amino acid solid phases. Other examples of first/second condensing agent systems may also be HCTU/DIEA, HATU/DIEA, etc. HATU and HCTU are structurally similar, in principle, but HATU is more active.
The raw material reservoirs are all connected with one end of a peristaltic pump 5 through a multichannel switching device 3. The multi-channel switching device 3 has a plurality of inlets and one outlet, and allows a fluid channel to be formed between any one of the plurality of inlets and the outlet while no fluid channel is formed between the other inlets and the outlet. The multi-channel switching device 3 may be a multi-channel switching valve, for example a 6-channel switching valve. When there are more reservoirs of raw materials, then a plurality of serially connected multi-channel switching valves may be used to form the multi-channel switching device 3, the outlet of the upstream multi-channel switching valve being connected to one inlet of its downstream multi-channel switching valve, and wherein the outlet of the downstream-most one of the multi-channel switching valves is directly connected to the peristaltic pump 5. In fig. 1, the multi-channel switching device 3 is represented by a block. In the present invention, the number of the multi-channel switching valves is not particularly limited. The multi-channel switching device 3 ensures that only one raw material reservoir is in fluid communication with the peristaltic pump 5 at the same time. It should be noted that when a plurality of multi-channel switching valves connected in series are used to form the multi-channel switching device 3, each multi-channel switching valve has an upstream-downstream division in the flow path due to the presence of a pipe connecting each multi-channel switching valve in series. The liquid flowing in from the most upstream multi-channel switching valve will flow through all the series pipes. The individual inlets are equivalent for the same multi-channel switching valve.
One port of the peristaltic pump 5 is in fluid communication with the outlet of the multichannel switching device 3 and the other port thereof is in fluid communication with the liquid metering device 12. Typically, the further port is in fluid communication with the top or upper portion of the dosing device 12, so that liquid may fall under gravity into the dosing device and collect at the bottom. Peristaltic pumps are well known to enable bi-directional delivery of fluids. Thus, peristaltic pump 5 may either pump liquid from the reservoir to metering device 12 or pump the reaction liquid retained in the line between metering device 12 and peristaltic pump 5 back into its reservoir. The direction in which peristaltic pump 5 delivers liquid to liquid delivery device 12 is referred to herein as forward, and the direction in which peristaltic pump 5 delivers liquid to the feedstock reservoir is referred to as reverse.
In the present invention, fluid communication means that there is a fluid passage, and a fluid control member such as a valve, a pump, or the like may be provided in the fluid passage.
The dosing device 12 is used for dosing the required raw liquid. Which may be a conventional container. The liquid metering device 12 may optionally be equipped with a liquid level sensor. For example, it may be equipped with a photosensor 13 and when the liquid level in the liquid measuring device 12 reaches a desired height, a signal is emitted indicating that the liquid amount is sufficient. Further, the signal may be used as a trigger to stop the peristaltic pump 5 from running in the forward direction and begin running in the reverse direction, thereby automatically controlling the feeding process.
The outlet of the dosing device 12 is in fluid communication with the transfer device 14. Typically, the level of the dosing device 12 is higher than the level of the transfer device 14 and is in fluid communication with the top or upper portion of the transfer device 14 so that liquid may flow from the dosing device 12 into the transfer device 14 by gravity. The transfer device 14 is used to pre-mix the reaction feed liquid sufficiently to activate the amino acids before entering the reactor.
The transfer device 14 is in fluid communication with the reactor 4 so that the mixed reaction solution can be fed to the reactor 4 and reacted. Typically, the mixed reaction solution is transferred from transfer device 14 to reactor 4 using the line pressure of peristaltic pump operation.
The reactor 4 may be any reactor suitable for solid phase synthesis of polypeptides. In the reaction, a solid carrier of the polypeptide is put into the reaction vessel. Moreover, the reactor 4 may also assist the reaction by mechanical movement. For example, the reactor 4 may be mounted on a motor and turned upside down continuously during the reaction by the motor so that the activated amino acid and resin are sufficiently mixed and reacted.
The apparatus for solid phase synthesis of polypeptides of the invention may also comprise other conventional components required in polypeptide synthesis.
The apparatus for solid phase synthesis of polypeptides should also comprise a storage and feed system for the deprotection agent. The apparatus includes at least a deprotecting agent reservoir in fluid communication with the reactor. The deprotecting agent may be, for example, piperidine. The storage and feeding system of the deprotection agent may be similar to the raw material storage and feeding system using peristaltic pumps.
The apparatus for solid phase synthesis of a polypeptide may further comprise a waste liquid treatment system connected to the reactor. The waste treatment system may include a pump, a waste collector, and the like. The apparatus for solid phase synthesis of a polypeptide may further comprise a product collection system connected to the reactor. The product collection system may include a pump, a product collector, and the like. The waste treatment system and the product collection system may share a common conduit.
The apparatus for solid phase synthesis of polypeptides may also include the required automated feedback and control devices.
The apparatus for solid phase synthesis of polypeptides of the invention may include any suitable valves, pumps, sensors and flow path designs. The pumps of the present invention are preferably peristaltic pumps and thus do not include any means for driving the flow of liquid using gas.
The apparatus for solid-phase synthesis of polypeptides according to the invention is characterized in that by feeding with a combination of peristaltic pumps and multichannel valves, the reaction liquid, in particular the amino acid reaction liquid, retained in the tubing between the pumps and the liquid-measuring device can be conveyed back into its reservoir, thus avoiding precipitation of the amino acid solution in the tubing and thus clogging of the tubing. In addition, compared with the mode of using gas to drive fluid in the prior art, the feeding mode is compact in design, saves cost and greatly reduces occupied space.
Optionally, the apparatus for solid phase synthesis of polypeptides of the present invention further comprises a wash liquid reservoir 9 connected to the most upstream inlet of the multi-channel switch 3. When a polypeptide is synthesized using a plurality of amino acids in the present invention, after each amino acid is attached, peristaltic pump 5, liquid measuring device 12, transfer device 14, reactor 4 and resin carrier therein need to be washed for further synthesis or cleavage. At this time, the cleaning liquid is caused to flow through the entire flow path by providing the multi-channel switching device 3, so that the cleaning is performed. The most upstream is provided to enable cleaning of the entire flow path. When the multi-channel switching device 3 is only one multi-channel switching valve, either inlet is the most upstream inlet. When the multi-channel switching device 3 is a multi-channel switching valve connected in series, the most upstream inlet is any one of the inlets of the most upstream multi-channel switching valve. The cleaning solution used to prepare for the next synthesis and the cleaning solution used to prepare for cutting may be different cleaning solutions. The cleaning liquid reservoir 9 is at least one, but may be a plurality, but is represented by a square in fig. 1.
Optionally, the apparatus for solid phase synthesis of polypeptides of the invention further comprises a cutting fluid storage and feeding system. The cutting fluid is used for cutting the finally synthesized polypeptide from the solid carrier. The storage and feeding system for the cutting fluid may be similar to the raw material storage and feeding system using peristaltic pumps. However, since the cutting fluid does not have the problem of clogging the pipe, other feeding means are also contemplated.
Optionally, the apparatus for solid phase synthesis of a polypeptide of the present invention further comprises a product wash liquid reservoir for storing a product wash liquid. After cleavage is completed, the cleavage liquid containing the product is discharged from the reactor, but some reaction products containing the polypeptide remain in the reactor. The product cleaning liquid is used for cleaning the reactor and the resin so as to avoid waste of the product.
FIG. 2 shows a schematic representation of an apparatus for solid phase synthesis of polypeptides according to an embodiment of the invention based on FIG. 1.
Wherein it comprises a three amino acid reservoir 8 1 、8 2 、8 3 The method comprises the steps of carrying out a first treatment on the surface of the Two condensing agent reservoirs 6 1 、6 2 The method comprises the steps of carrying out a first treatment on the surface of the A cleaning liquid reservoir 9 connected to the multi-channel valve 3; a photoelectric liquid level sensor 13 provided in the liquid measuring device 12; a deprotecting agent reservoir 21 in fluid communication with reactor 4; a motor 17 for turning the reactor 4 upside down; a waste liquid collector 23 for storing waste liquid; and a cutting fluid reservoir 25 storing cutting fluid.
In this document, the subscripts of the numerical designations of the components are used to distinguish the same type of components. For example, 8 1 、8 2 、8 3 Is used to denote first, second and third amino acid reservoirs.
The device is operated in the following mode: first switching the multi-channel valve 3 to and from the amino acid reservoir 8 storing the first amino acid 1 Communication, the peristaltic pump 5 is operated in a forward direction, thereby delivering the first amino acid to the fluid delivery device 12. When the first amino acid amount in the liquid equivalent device 12 reaches a preset value, the first amino acid amount is detected by a photoelectric liquid level sensor, and the peristaltic pump is controlled to reversely run, so that the amino acid retained between the peristaltic pump 5 and the liquid equivalent device 12The solution is returned to the amino acid reservoir 8 1 Is a kind of medium. A metered amount of the amino acid solution is transferred from the dosing device 12 to the transfer device 14. Subsequently, the multi-channel valve 3 is switched to the condensing agent reservoir 6 storing the first condensing agent 1 Communication, the peristaltic pump 5 is operated in a forward direction, thereby delivering the first condensing agent to the liquid-dispensing device 12. When the amount of the first condensing agent in the liquid metering device 12 reaches a preset value, the first condensing agent is detected by the photoelectric liquid level sensor and is controlled to reversely run, and the first condensing agent retained between the peristaltic pump 5 and the liquid metering device 12 is sent to the condensing agent reservoir 6 1 Is a kind of medium. A metered amount of the first condensing agent is transferred from the liquid-metering device 12 to the transfer device 14. Subsequently, the multi-channel valve 3 is switched to the condensing agent reservoir 6 for storing the second condensing agent 2 And in communication, the peristaltic pump 5 is operated in a forward direction, thereby delivering the second condensing agent to the liquid-dispensing device 12. When the amount of the second condensing agent in the liquid metering device 12 reaches a preset value, the second condensing agent is detected by the photoelectric liquid level sensor and is controlled to reversely run, and the second condensing agent retained between the peristaltic pump 5 and the liquid metering device 12 is sent to the condensing agent reservoir 6 2 Is a kind of medium. In this case, the peristaltic pump may not be operated in reverse, i.e. the second condensing agent which is retained is not fed back into the condensing agent reservoir 6 2 But rather waits directly for the flow path cleaning described below. However, from a reagent-saving standpoint, the second condensing agent to be retained is selected to be delivered to the condensing agent reservoir 6 2 Is a kind of medium. A metered amount of the second condensing agent is transferred from the liquid metering device 12 to the transfer device 14. It should be noted that in the above process, the sequence of steps of reversing the peristaltic pump and delivering the liquid from the liquid metering device 12 to the transfer device 14 may be arbitrary. That is, the liquid from which the dosing is completed may be transferred from the dosing device to the transfer device before, after or simultaneously with the transfer of the retained liquid back to the raw material reservoir. The present invention is not limited in this order. The first amino acid, the first condensing agent, and the second condensing agent are thoroughly mixed in the transfer device 14 to form a mixture that reacts to activate the amino acid. The mixture is conveyed from the transfer device 14 into the reactor 4. The motor 17 is actuated to turn the reactor 4 up and down to react the mixture with the solid support therein. After the reaction, the waste liquid is discharged to the wasteA liquid collector 23. Subsequently, the multi-channel valve 3 is switched to be communicated with the cleaning liquid reservoir 9, so that the peristaltic pump 5 is operated in the forward direction, and the flow path is cleaned fully. The washed waste liquid is discharged to the waste liquid collector 23. Thus, the synthesis of the first amino acid in the polypeptide is completed. Subsequently, a deprotection agent is fed from a deprotection agent reservoir 21 to the reactor to deprotect the carboxyl groups of the existing polypeptide chain. The cleaning process is then repeated.
The above steps are repeated except that a liquid reservoir 8 from an amino acid is used 2 To complete the synthesis of the second amino acid in the polypeptide. The above steps are repeated again, except that a solution from the amino acid reservoir 8 is used 3 To complete the synthesis of the third amino acid in the polypeptide.
Finally, the polypeptide is cleaved from the solid support using the cleavage liquid from the cleavage liquid reservoir 25 to yield a crude peptide solution. The crude peptide is withdrawn from the reactor 4 and subjected to subsequent treatments.
As described above, the combination of the equipment for solid-phase synthesis of polypeptide of the invention utilizes peristaltic pump and multi-channel switching valve, which solves the problem that the amino acid retention blocks the pipeline in the existing device for solid-phase synthesis of polypeptide. In addition, the device of the invention has compact design, does not need to use a gas driving system, and greatly saves space and cost. At the same time, peristaltic pumps can also provide higher feed accuracy.
Figure 3 shows a schematic diagram of a more specific apparatus for solid phase synthesis of polypeptides according to an embodiment of the invention.
In fig. 3, a 24 amino acid reservoir 8 is provided 1 To 8 24 They are connected in batches to a multi-channel switching valve 3 2 To 3 6 . The multi-channel switching valve 3 here 6 To 3 2 Sequentially connected in series. I.e. multichannel switching valve 3 6 Is connected to the multi-channel switching valve 3 5 Is a multi-channel switching valve 3 5 Is connected to the multi-channel switching valve 3 4 And so on. Multi-channel switching valve 3 2 In turn connected to the multi-channel switching valve 3 1 Is provided. Multiple onesChannel switching valve 3 1 To 3 6 All are 6-channel switching valves which together form a multi-channel switching device. Two cleaning liquid reservoirs 9 are also provided 1 And 9 2 For storing DMF and DCM respectively, and is connected to the most upstream multi-channel switching valve 3 6 . In addition, five condensing agent reservoirs 6 1 To 6 5 Connected to the multi-channel switching valve 3 1 Respectively, can be used for storing DIC, oxyma, HATU, HCTU, DIEA. It will be appreciated that the connection of the reservoir to the multi-channel switch valve described above may vary. Due to the serial relationship of the six-way switching valves, only one reservoir can be in fluid communication with peristaltic pump 5 at a time by their switching gear positions.
Multi-channel switching valve 3 1 Is in fluid communication with one port of peristaltic pump 5 and the other port of peristaltic pump 5 is in fluid communication with liquid metering device 12.
The deprotecting agent reservoir 21, waste liquid collector 23, and cutting fluid reservoir 25 of fig. 3, etc. are described above. In fig. 3, suitable valves v and pumps p are provided in their flow paths to deliver the liquid flow.
In fig. 3 there are two reactors 4 1 And 4 2 Also known as a two-pass reactor. The advantage of a double reactor is that when one reactor is reacting, the other reactor is being cleaned. When the reaction is completed in the reactor where the reaction is performed, the other reactor may be subjected to the reaction when the step of washing is performed. This cross-reaction approach reduces standby time and improves reaction efficiency. More reactors may also be provided.
Also shown in fig. 3 is a reactant collector 27 for storing the crude peptide. Which shares the same reactor outlet as the waste liquid collector 23 and switches the flow path with a switching valve as needed.
Also shown in fig. 3 is a product cleaning fluid reservoir 29.
The operation of the device of FIG. 3 will be described below using the single condensation-deprotection and cleavage of a single polypeptide reaction as an example.
The peristaltic pump 5 is started, and six 6-channel switching valves are switched to enable the peristaltic pump 5 to contain glycerolAmino acid reservoir 8 of amino acid (Gly) 1 And in a connected state, an amino acid transmission channel is formed. Amino acid reservoir 8 1 Is fed into the dosing device 12 by means of the peristaltic pump 5. When the liquid level position reaches the preset position of the photoelectric sensor 13, the sensor 13 responds and then sends a command to the peristaltic pump 5, so that the motor in the peristaltic pump 5 rotates reversely. The peristaltic pump 5 returns the glycine liquid retained in the pipeline to the glycine storage bottle after reversing. At the same time, a signal is sent to a valve below the dosing device 12. The valve is opened so that the liquid in the liquid measuring device 12 falls into the transfer device 14 by the action of gravity. The multi-channel switching valve 3 is then used 1 Switching to make condensing agent reservoir 6 1 Is in communication with peristaltic pump 5. Peristaltic pump 5 is turned on to deliver the first condensing agent DIC (N, N' -diisopropylcarbodiimide) to the dosing device 12, and feedback after dosing the volume resembles the previous glycine. Peristaltic pump 5 inverts the excess fluid from the discharge tube. With the valve open, the first condensing agent is transferred to the transfer device 14. The multi-channel switching valve 3 is then used 1 Switching to make condensing agent reservoir 6 2 Is in communication with peristaltic pump 5. The same procedure as above is repeated, and a quantitative volume of the second condensing agent Oxyma (2-oxime cyanoacetate) is measured into the relay device 14. A mixture of glycine and condensing agent is formed in the transfer device 14.
Standing for 30s to activate amino acid, opening valve and pump below transfer device 14, transferring the mixture of activated glycine and condensing agent into reactor 4 as required 1 And 4 2 Any one of them. Reactor 4 1 And 4 2 Two reactor vials of a two-channel reactor are possible. After the mixture is transferred into, a motor at the rear part continuously turns up and down, and the activated amino acid and the resin are fully mixed for reaction. After the reaction is completed, a valve and a pump below the reactor are opened to enable the redundant reaction liquid to enter a waste liquid barrel. Subsequently switching the multi-channel switching valve to bring the cleaning liquid reservoir 9 1 In fluid communication with peristaltic pump 5, the cleaning solution N, N-dimethyl-formamide (DMF) is delivered to a dosing device 12, to a transfer device 14, and to a reaction flask. Starting the motor and reversing up and downTransfer the cleaning resin and the reaction flask. After a period of time, the waste liquid is introduced into the waste liquid collector by opening the valve and the pump.
Deprotection is performed after the coupling is completed. The deprotecting agent piperidine was transferred to a reaction flask. Peristaltic pumps, liquid metering devices, sensors, and the like may be provided in the flow path. After transfer, the motor is started, after the mixture is turned upside down and mixed for a period of time, the reacted liquid is discharged into a waste liquid collector, and then the DMF cleaning step is repeated, so that the reaction bottle and the resin in the reaction bottle are cleaned.
Subsequently, the whole procedure described above can be repeated from the amino acid reservoir 8 2 The next amino acid is transported for polypeptide synthesis. For example, alanine (Ala), leucine (Leu), isoleucine (Ile), valine (Val), proline (Pro), phenylalanine (Phe), egg (methylthio) amino acid (Met), tryptophan (Trp), serine (Ser), glutamine (gin), threonine (Thr), cysteine (Cys), asparagine (Asn), tyrosine (Tyr), aspartic acid (Asp), glutamic acid (Glu), lysine (Lys), arginine (Arg), histidine (His), and the like may be stored in the amino acid tank. Other condensing agents may be used as desired. Such as 2- (7-benzotriazol) -N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HATU), 6-chlorobenzotriazol-1, 3-tetramethyluronium Hexafluorophosphate (HCTU), diisopropylethylamine (DIEA), 1-hydroxy-7-azobenzotriazol (HOAt), and the like.
After the polypeptide synthesis is completed and before cleavage is performed, the multi-channel switching valve is switched to a wash liquid reservoir 9 which is made to hold Dichloromethane (DCM) 2 Is in communication with peristaltic pump 5. After the peristaltic pump 5 is turned on, the cleaning solution is delivered to the reactor. And after the washing is turned upside down, transferring the waste liquid into the waste liquid. Repeating for 3-4 times, and ensuring that the redundant DMF in the reactor is removed. Two or more cleaning fluids may be used for different cleaning objects, for example, may be selected based on the solubility of the cleaning objects. Subsequently, the cleavage reagent is transferred from the cleavage liquid reservoir to the reaction flask, and the turnover is started to start the online cleavage of the polypeptide. After 2h, the valve and pump were turned on and the reactant was transferred to reactant collector 27. Finally, clear from the productThe washing liquid reservoir 29 is used for supplying trichloroacetic acid (TFA) to the reaction flask for washing. After the resin is washed, the washing liquid is also collected in the reactant collector 27. And (5) carrying out post-treatment on the collected product to obtain a crude product of the polypeptide.
The foregoing describes specific embodiments of the present invention. The peristaltic pump and the multi-channel valve are combined for feeding, so that the reaction liquid, particularly the amino acid reaction liquid, retained in the pipeline between the pump and the liquid measuring device can be conveyed back to the liquid storage device, and the problem that the amino acid solution stays in the pipeline to be separated out and then cause pipeline blockage is avoided. In addition, compared with the mode of using gas to drive fluid in the prior art, the feeding mode is compact in design, saves cost and greatly reduces occupied space.
It will be apparent to those skilled in the art that, for convenience and brevity of description, only the above-described division of the functional modules is illustrated, and in practical application, the above-described functional allocation may be performed by different functional modules according to needs, i.e. the internal structure of the apparatus is divided into different functional modules to perform all or part of the functions described above.
The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the invention thereto, but to limit the invention thereto, and any modifications, equivalents, improvements and equivalents thereof may be made without departing from the spirit and principles of the invention.
Claims (9)
1. An apparatus for solid phase synthesis of a polypeptide, the apparatus comprising:
at least one amino acid reservoir for storing an amino acid solution;
at least two condensing agent reservoirs for storing condensing agents;
a multi-channel switching device having a plurality of inlets and one outlet, and allowing a fluid channel to be formed between any one of the plurality of inlets and the outlet while no fluid channel is formed between the other inlet and the outlet;
a peristaltic pump;
a liquid measuring device;
a transfer device;
a reactor; and
a deprotecting agent reservoir for storing a deprotecting agent,
wherein,
each of the at least one amino acid reservoir and each of the at least two condensing agent reservoirs are in fluid communication with an inlet of the multi-channel switching device,
the outlet of the multichannel switching device is in fluid communication with one port of the peristaltic pump,
the other port of the peristaltic pump is in fluid communication with the inlet of the metering device, the other port being in fluid communication with the top or upper portion of the metering device so that liquid may fall under gravity into the metering device and collect at the bottom,
the outlet of the liquid metering device is in fluid communication with the inlet of the transfer device,
the outlet of the transfer device is in fluid communication with the reactor, and
the deprotecting agent reservoir is in fluid communication with the reactor.
2. The apparatus of claim 1, wherein the device comprises a plurality of sensors,
the multi-channel switching device is a multi-channel switching valve.
3. The apparatus of claim 1, wherein the device comprises a plurality of sensors,
the multi-channel switching device is a plurality of multi-channel switching valves connected in series.
4. The apparatus of claim 1, further comprising at least one cleaning fluid reservoir, each of the at least one cleaning fluid reservoir being in fluid communication with an upstream-most inlet of the multi-channel switching device, respectively.
5. The apparatus of claim 1, wherein the liquid metering device has a liquid level sensor.
6. The apparatus of claim 1, wherein the reactor is a two-pass reactor.
7. The apparatus of claim 1, further comprising a motor configured to cause the reactor to turn over.
8. The apparatus of claim 1, further comprising a cutting fluid reservoir in fluid communication with the reactor.
9. A method of synthesizing a polypeptide using the apparatus of claim 1, the method comprising:
setting the multi-channel switching device such that a fluid channel is formed between one of the at least one amino acid reservoir and the peristaltic pump;
operating a peristaltic pump in a forward direction to deliver the amino acid solution stored in the one amino acid reservoir to the solution metering device;
conveying the amino acid solution in the liquid measuring device to the transfer device;
reversing the peristaltic pump to deliver the amino acid solution retained in the tubing between the peristaltic pump and the liquid metering device back to the amino acid reservoir;
setting the multi-channel switching device so that a fluid channel is formed between one condensing agent reservoir of the at least two condensing agent reservoirs and the peristaltic pump;
operating a peristaltic pump in a forward direction to deliver a first condensing agent stored in said one condensing agent reservoir to said liquid metering device;
reversing the peristaltic pump to deliver the first condensing agent retained in the tubing between the peristaltic pump and the metering device back to the one condensing agent reservoir;
conveying the condensing agent in the liquid measuring device to the transfer device;
setting the multi-channel switching device so that a fluid channel is formed between the peristaltic pump and the other condensing agent reservoir of the at least two condensing agent reservoirs;
operating the peristaltic pump in a forward direction to deliver the second condensing agent stored in the other condensing agent reservoir to the liquid metering device;
conveying the second condensing agent in the liquid measuring device to the transfer device;
reacting a mixture comprising the amino acid solution, the first condensing agent, and the second condensing agent in the transfer device to activate the amino acid;
transferring the activated amino acid from the transfer device to the reactor and reacting on a solid support placed in the reactor to form a peptide chain; and
delivering a deprotecting agent stored in the deprotecting agent reservoir to the reactor to deprotect the peptide chain.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106317163A (en) * | 2016-08-23 | 2017-01-11 | 安徽天达谱申生物科技有限公司 | High-efficiency automatic polypeptide synthesis method |
| CN107522769A (en) * | 2017-10-23 | 2017-12-29 | 希施生物科技（上海）有限公司 | Polypeptide heats solid phase synthesis process |
| CN108264536A (en) * | 2018-03-27 | 2018-07-10 | 润辉生物技术（威海）有限公司 | A kind of continuous high-flux polypeptide synthesizer and its application method |
| CN210176770U (en) * | 2019-04-01 | 2020-03-24 | 中国科学技术大学 | Apparatus for solid phase synthesis of polypeptides |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106317163A (en) * | 2016-08-23 | 2017-01-11 | 安徽天达谱申生物科技有限公司 | High-efficiency automatic polypeptide synthesis method |
| CN107522769A (en) * | 2017-10-23 | 2017-12-29 | 希施生物科技（上海）有限公司 | Polypeptide heats solid phase synthesis process |
| CN108264536A (en) * | 2018-03-27 | 2018-07-10 | 润辉生物技术（威海）有限公司 | A kind of continuous high-flux polypeptide synthesizer and its application method |
| CN210176770U (en) * | 2019-04-01 | 2020-03-24 | 中国科学技术大学 | Apparatus for solid phase synthesis of polypeptides |
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