Online replacement system, control method and chromatographic system matched with online replacement system

Abstract

The invention provides a line replacement system, a control method and a matched chromatographic system. The line replacement system comprises a first input pipeline and a second input pipeline, a first feeding pump and a second feeding pump are respectively arranged on the first input pipeline and the second input pipeline; the output end of the first input pipeline and the output end of the second input pipeline are connected with the inlet end of a mixer through pipelines, the output end of the mixer is connected with the inlet end of a tangential flow filter through a pipeline, the cut-off end of the tangential flow filter is connected with a first output pipeline, and a first discharging pump is arranged on the first output pipeline; the permeation end of the tangential flow filter is connected with a permeate pipeline; when in use, the three pumps are controlled to realize online replacement and chromatography to reach a steady state; the device has continuous concentration and replacement, and the efficiency is improved; the cost is reduced; and the flow required by a replacement system and a chromatographic system can be met.

Description

Technical Field

[0001] The present invention relates to buffer replacement and separation and purification in the downstream of biomedicine, and in particular to an online replacement system, a control method and a matching chromatography system. Background Art

[0002] Chromatography technology is widely used in many fields such as biology, medicine, and chemistry. Before loading the product, the pH and concentration of the product need to be controlled. Only after the loading conditions are met can the subsequent separation and purification steps be carried out. Therefore, the current existing processes are to concentrate and replace the product first, collect it in the circulation tank, and then load it on the chromatographic column for separation and purification. Common product concentration and replacement methods mostly use single pump mode. Figure 1 As shown, the bottom outlet of the circulation tank is connected to the inlet of the tangential flow filter through the feed pump, the reflux end of the tangential flow filter is connected to the top inlet of the circulation tank, and the permeate end of the tangential flow filter is connected to the permeate pipe. When in use, the sample to be processed is filtered through the tangential flow filter and the buffer is added to the retained species at the same time, achieving the purpose of concentration and buffer replacement, so that the pH and concentration of the sample to be processed meet the conditions for chromatographic loading. Therefore, it is divided into two separate process steps. Its disadvantages are: (1) the tangential flow filtration of a single pump is low in efficiency, takes a long time, and requires more filter membranes; (2) the tangential flow of a single pump requires a circulation tank, and the circulation requires a weight sensor, a stirrer, and a nozzle; (3) the two separate process steps require two separate process equipment, which not only takes a lot of time to concentrate and replace the sample before chromatographic loading, but also requires two sets of equipment, and requires space for equipment use.

[0003] Displacement chromatography is performed by connecting a chromatographic column to a direct tangential flow system, without using the recirculation structure of the tangential flow system. However, this method suffers from the inability to coordinate the flow rates required by the tangential flow system and the column. Therefore, it is technically difficult to achieve a direct connection between a tangential flow system and a chromatographic column without using a recirculation structure. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, the present invention provides an online displacement system, control method, and associated chromatography system. These systems enable continuous concentration and displacement, improving efficiency; require less time and fewer membranes; and eliminate the need for circulation tanks, weight sensors, stirring devices, or spray nozzles, reducing equipment footprint and costs. They can also meet the flow rates required by both the displacement and chromatography systems.

[0005] In response to the above technical problems, the first aspect of the present invention discloses an online replacement system, comprising a first input pipeline and a second input pipeline; the first input pipeline and the second input pipeline are respectively provided with a first feed pump and a second feed pump; the output ends of the first input pipeline and the second input pipeline are connected to the inlet end of the mixer through a pipeline, the output end of the mixer is connected to the inlet end of the tangential flow filter through a pipeline, the intercepting end of the tangential flow filter is connected to the first output pipeline, and the first output pipeline is provided with a first discharge pump; the permeate end of the tangential flow filter is connected to the permeate pipeline.

[0006] In some embodiments, flow sensors are respectively provided on the first input pipe and / or the second input pipe and / or the first output pipe.

[0007] In some embodiments, the flow rate ratio between the first input conduit and the second input conduit is used to control the concentration ratio of the product to the buffer.

[0008] In some embodiments, the flow ratio between the first input pipeline and the second input pipeline is used to control the percolation ratio.

[0009] In some embodiments, the flow rate ratio between the first input channel and the second input channel is used to control the buffer exchange ratio.

[0010] In some embodiments, the buffer is replaced when the flow rate of the first input pipeline or the second input pipeline is equal to the flow rate of the first output pipeline.

[0011] In response to the above technical problems, the second aspect of the present disclosure proposes a control method based on an online replacement system, the steps of which are as follows:

[0012] Step 1: The flow rate of the first input pipeline is F1, and the flow rate of the second input pipeline is F2; the first input pipeline and the second input pipeline respectively deliver buffer solution, and the ratio of F1 to F2 is controlled;

[0013] Step 2: Control the flow rates of the first input pipeline and the second input pipeline so that F1 and F2 reach the target flow rates; at the same time, control the flow rate F3 in the first output pipeline so that F3 is at zero flow or low flow rate;

[0014] Step 3: When F1 and F2 reach the target flow rate, control the flow rate in the first output pipe so that F3 reaches the target flow rate;

[0015] Step 4: When the flow rate F1 in the first input pipe is equal to the flow rate F3 in the first output pipe, the first input pipe is connected to the product tank to deliver the product to be processed, so that the flow rate F1 adjusts itself and stabilizes again;

[0016] Step 5: Continuously and stably control the flow of the first input pipeline, the second input pipeline, and the first output pipeline.

[0017] In response to the above technical problems, the third aspect of the present disclosure proposes a chromatography system based on an online replacement system, including the above-mentioned online replacement system, wherein the first output pipe of the online replacement system is connected to the inlet end of the chromatography column through the third input pipe, and the outlet end of the chromatography column is respectively connected to the collection pipe and the waste pipe through the second output pipe, and the inlet ends of the collection pipe and the waste pipe are respectively provided with valves.

[0018] In some embodiments, a valve is provided at the outlet end of the first output pipe of the online replacement system for adjusting the resistance of sample loading.

[0019] In some embodiments, a side branch pipeline is connected between the third input pipeline at the inlet end of the chromatography column and the second output pipeline at the outlet end of the chromatography column. The third input pipeline is provided with a valve, and the valve is located between the inlet end of the chromatography column and the connection part between one end of the side branch pipeline and the second output pipeline; the second output pipeline is provided with two valves, and the two valves are respectively located on both sides of the connection part between the other end of the side branch pipeline and the second output pipeline.

[0020] Beneficial effects of the present invention:

[0021] (1) The present invention adopts flow control of three pumps, which significantly improves efficiency by 30% compared with the existing UF / DF, and controls flow instead of pressure.

[0022] (2) No need to rely on the batch operation of traditional TFF, the sample to be processed can be loaded by continuous online buffer exchange during chromatography.

[0023] (3) With sufficient membrane area, one or half a PFR can achieve the ideal state.

[0024] (4) The tangential buffer flow displacement system composed of three pump systems does not require a circulation tank, a weight sensor, a stirring device, or a nozzle, thereby reducing the equipment footprint and reducing costs.

[0025] Figures in the specification

[0026] Figure 1 It is a UF / DF system of the prior art.

[0027] Figure 2 The following is a schematic diagram of the replacement system structure.

[0028] Figure 3 Schematic diagram of the structure of the online displacement chromatography system.

[0029] Figure 4 Schematic diagram of protein concentration at the inlet and cut-off ends of a tangential flow filter.

[0030] Figure 5 Schematic diagram of salt concentration at the inlet and interception ends of a tangential flow filter. DETAILED DESCRIPTION

[0031] The technical content of the present invention is described below using specific embodiments. Those skilled in the art will readily understand the other advantages and effects of the present invention from the disclosure herein. The present invention may also be implemented or applied through other different specific embodiments. Those skilled in the art may make various modifications and alterations without departing from the spirit of the present invention.

[0032] Before introducing the specific embodiments of the present disclosure in detail, some terms used in the present disclosure are first explained.

[0033] Unless otherwise defined hereinafter, the meaning of all technical terms and scientific terms used herein is intended to be the same as those generally understood by those skilled in the art. Mention that the technology used in this article is intended to refer to the technology generally understood in the art, including those changes in technology that are obvious to those skilled in the art or the replacement of equivalent technologies. Although it is believed that the following terms are well understood by those skilled in the art, the following definitions are still set forth to better explain the present invention. When a top-grade product name appears in this article, it is intended to refer to its corresponding top-grade product. All patents, published patent applications and publications cited herein are incorporated herein by reference.

[0034] As used herein, the terms “connect,” “connected,” “coupled,” or similar terms are not limited to direct connections but also include indirect connections.

[0035] As used herein, a "sample" is a biomolecule, including proteins, nucleic acids, lipids, carbohydrates, small nucleotides, amino acids, and their derivatives.

[0036] As used herein, the term "online monitoring" or "real-time monitoring" refers to the real-time detection of certain parameters or properties of a buffer, reaction fluid, or fluid flowing out of a flow reactor, such as pH, pressure, flow rate, conductivity, etc., during the use of a chromatography system. Unlike offline detection or analysis, online monitoring or real-time monitoring can provide real-time feedback of the detection results.

[0037] For example, the positional relationship words "up", "down", "left", "right", "front", "back", etc. involved in this article are determined according to the layout direction of the drawings in the specification. They are only used to express relative positional relationships. When the absolute position of the described object changes, the relative positional relationship may also change accordingly.

[0038] As mentioned in the article, UF / DF, UF (ultrafiltration or concentration) refers to the separation of components based on molecular weight and size to isolate the biological agent of interest. DF (filtration or buffer exchange) refers to the addition of a buffer to replace the liquid surrounding the target with a liquid that is more conducive to storage stability.

[0039] As referred to herein, PFR refers to a tubular reactor.

[0040] Reference Figure 2 As shown, the online replacement system includes a first input pipeline 1 and a second input pipeline 2; a first feed pump 3 is provided on the first input pipeline 1, and a first flow sensor 4 is provided on the first input pipeline 1 at the outlet position of the first feed pump 3, a second feed pump 5 is provided on the second input pipeline 2, and a second flow sensor 6 is provided on the second input pipeline 2 at the outlet position of the second feed pump 5; the output ends of the first input pipeline 1 and the second input pipeline 2 are respectively connected to the inlet end of a mixer 7, the outlet end of the mixer 7 is connected to the inlet end of a tangential flow filter 8 through a pipeline, the intercepting end of the tangential flow filter 8 is connected to a first output pipeline 9, a first discharge pump 10 is provided on the first output pipeline 9, and a third flow sensor 11 is provided on the first output pipeline 9 at the outlet end of the first discharge pump 10; the permeate end of the tangential flow filter 8 is connected to a permeate pipeline 12.

[0041] The flow rates of the first feed pump 3 on the first input pipeline 1 and the second feed pump 5 on the second input pipeline 2 control the transmembrane pressure on the tangential flow filter 8 .

[0042] The flow rate of the first discharge pump 10 on the first output pipe 9 controls the tangential flow rate of the fluid.

[0043] The first input pipeline 1 and the second input pipeline 2 are used to transport different fluids; when the first input pipeline 1 transports the product to be processed, the second input pipeline 2 is used to input the buffer solution; when the first input pipeline 1 transports the buffer solution, the second input pipeline 2 is used to input the product to be processed.

[0044] During use, the first and second input conduits 1 and 2 are connected to a buffer solution, respectively. The flow rate of the first input conduit 1 under the action of the first feed pump 3 is F1, and the flow rate of the second input conduit 2 under the action of the second feed pump 5 is F2. The ratio of F1 to F2 is controlled. This ratio is used to control the diafiltration ratio; the ratio of F1 to F2 is used to control the buffer exchange ratio.

[0045] The first feed pump 3 and the second feed pump 5 are controlled so that the flow rates of the first input pipeline 1 and the second input pipeline 2 reach the target flow rates; at the same time, the first discharge pump 10 does not work, and the flow rate F3 in the first output pipeline 9 is at zero flow or low flow rate.

[0046] When F1 and F2 reach the target flow rate, the first discharge pump 10 is started to control the flow rate in the first output pipe 9 so that F3 reaches the target flow rate.

[0047] When the flow rate F1 in the first input pipe 1 equals the flow rate F3 in the first output pipe 9, the first input pipe 1 is connected to the product tank and the product to be processed is introduced, causing the flow rate F1 to automatically adjust and stabilize. When the flow rates in the first input pipe 1 and the first output pipe 9 are equal, the buffer solution is replaced.

[0048] The first feed pump 3 , the second feed pump 5 and the first discharge pump 10 are continuously and stably operated to control the flow rates of the first input pipeline 1 , the second input pipeline 2 and the first output pipeline 9 .

[0049] Reference Figure 3 As shown, the chromatography system based on the online replacement system includes a first input pipeline 21 and a second input pipeline 22. The first input pipeline 21 is provided with a first feed pump 23, and a first flow sensor 24 is provided on the first input pipeline 21 at the outlet position of the first feed pump 23. The second input pipeline 22 is provided with a second feed pump 25, and a second flow sensor 26 is provided on the second input pipeline 22 at the outlet position of the second feed pump 25; the output ends of the first input pipeline 21 and the second input pipeline 22 are respectively connected to the inlet ends of the mixer 27, the outlet end of the mixer 27 is connected to the inlet end of the tangential flow filter 28 through a pipeline, the intercepting end of the tangential flow filter 28 is connected to the first output pipeline 29, the first output pipeline 29 is provided with a first discharge pump 210, and the first output pipeline 29 at the outlet end of the first discharge pump 210 is provided with a third flow sensor 211; the permeate end of the tangential flow filter 28 is connected to the permeate pipeline 212. The outlet of first output pipeline 29 is connected to the inlet of third input pipeline 214 via first valve 213. The outlet of third input pipeline 214 is connected to the inlet of chromatography column 215, which is in turn connected to the inlet of second output pipeline 216. The outlet of second output pipeline 216 is connected to the inlet of collection pipeline 217 and waste discharge pipeline 218, respectively. A bypass pipeline 219 is connected between third input pipeline 214 and second output pipeline 216. A second valve 220 is provided on third input pipeline 214 between one end of bypass pipeline 219 and the connection point between the third input pipeline 214 and the inlet of chromatography column 215. A third valve 221 and a fourth valve 222 are provided on second output pipeline 216 on either side of the connection point between the other end of bypass pipeline 219 and second output pipeline 216, respectively. A fifth valve 223 is also provided on bypass pipeline 219.

[0050] The inlet ends of the collecting pipe 217 and the waste discharge pipe 218 are respectively provided with a sixth valve 224 and a seventh valve 225 .

[0051] A UV sensor 226 , a conductivity transmitter 227 and a pH sensor 228 are respectively provided on the second output pipe 216 between one side of the fourth valve 222 and the outlet end of the second output pipe 216 .

[0052] The first valve 213 at the outlet end of the first output pipe 29 is installed based on the size of the sample loading resistance. If the resistance is large, the first valve 213 is not required. If the resistance is small, the first valve 213 needs to be installed to adjust the sample loading resistance.

[0053] During use, the first input conduit 21 and the second input conduit 22 are connected to the buffer solution, respectively. The flow rate of the first input conduit 21 under the action of the first feed pump 23 is F1, and the flow rate of the second input conduit 22 under the action of the second feed pump 25 is F2. The ratio of F1 to F2 is controlled. This ratio is used to control the diafiltration ratio; the ratio of F1 to F2 is used to control the buffer exchange ratio.

[0054] The first feed pump 23 and the second feed pump 25 are controlled so that the flow rates of the first input pipeline 21 and the second input pipeline 22 reach the target flow rates; at the same time, the first discharge pump 210 does not work, and the flow rate F3 in the first output pipeline 29 is at zero flow or low flow.

[0055] When the flow rates of F1 and F2 reach the target, the first discharge pump 210 is activated to control the flow rate in the first output pipe 29 so that F3 reaches the target flow rate. The fifth valve 223, the fourth valve 222, and the seventh valve 225 of the bypass pipe 219 are opened, and the buffer solution flows through the bypass pipe 219 and the second output pipe 216 into the waste pipe 218.

[0056] When F1 and F2 reach the target flow rate, the first discharge pump 210 is started to control the flow rate in the first output pipe 29 so that F3 reaches the target flow rate.

[0057] When the flow rate F1 in the first input pipeline 21 equals the flow rate F3 in the first output pipeline 29, the first input pipeline 21 is connected to the product tank to introduce the product to be processed, allowing the flow rate F1 to automatically adjust and stabilize. When the flow rates in the first input pipeline 1 and the first output pipeline 9 are equal, the buffer is replaced. At this point, the fifth valve 223 and the seventh valve 225 of the bypass pipeline 219 are closed; the second valve 220, the third valve 221, and the sixth valve 224 are opened. The sample to be processed, after being loaded into the chromatography column 215, flows through the second output pipeline 216 and into the collection pipeline 217.

[0058] The first feed pump 23 , the second feed pump 25 and the first discharge pump 210 are continuously and stably operated to control the flow rates of the first input pipeline 21 , the second input pipeline 22 and the first output pipeline 29 .

[0059] Reference Figure 4 As shown, the first input pipeline 1 and the second input pipeline 2 are connected to the product liquid and the buffer solution respectively, and the first feed pump 3 and the second feed pump 5 are controlled so that the flow of the first input pipeline 1 and the second input pipeline 2 reaches the target flow; at the same time, the first discharge pump 10 does not work, and the flow F3 in the first output pipeline 9 is at zero flow or low flow. At this time, the protein concentration at the inlet end of the tangential flow filter 8 remains unchanged, and the protein concentration at the cutoff end of the tangential flow filter 8 is zero or low. When F1 and F2 reach the target flow, the first discharge pump 10 is started to work, and the flow in the first output pipeline 9 is controlled so that F3 reaches the target flow. When the flow F1 in the first input pipeline 1 is equal to the flow F3 in the first output pipeline 9, the protein concentration at the inlet end of the tangential flow filter 8 is equal to the protein concentration at the cutoff end of the tangential flow filter 8.

[0060] Reference Figure 5 As shown, the first input pipeline 1 and the second input pipeline 2 are connected to the product liquid and the buffer solution, respectively. The first feed pump 3 and the second feed pump 5 are controlled so that the flow rates of the first input pipeline 1 and the second input pipeline 2 reach the target flow rates. At the same time, the first discharge pump 10 is not operating, and the flow rate F3 in the first output pipeline 9 is at zero flow or low flow. At this time, the salt concentration at the inlet of the tangential flow filter 8 remains unchanged, and the salt concentration at the permeate end of the tangential flow filter 8 is lower than the salt concentration at the inlet of the tangential flow filter 8. When F1 and F2 reach the target flow rates, the first discharge pump 10 is activated and the flow rate in the first output pipeline 9 is controlled so that F3 reaches the target flow rate. When the flow rate F1 in the first input pipeline 1 equals the flow rate F3 in the first output pipeline 9, the salt concentration at the permeate end of the tangential flow filter 8 remains unchanged.

Claims

1. An online replacement system, characterized in that: The invention comprises a first input pipeline and a second input pipeline; the first input pipeline and the second input pipeline are respectively provided with a first feed pump and a second feed pump; the output ends of the first input pipeline and the second input pipeline are connected to the inlet end of the mixer through pipelines, the output end of the mixer is connected to the inlet end of the tangential flow filter through a pipeline, the intercepting end of the tangential flow filter is connected to the first output pipeline, and the first output pipeline is provided with a first discharge pump; the permeate end of the tangential flow filter is connected to the permeate pipeline.

2. The online replacement system according to claim 1, characterized in that: The first input pipe and / or the second input pipe and / or the first output pipe are respectively provided with flow sensors.

3. The online replacement system according to claim 1, characterized in that: The flow rate ratio between the first input conduit and the second input conduit is used to control the concentration ratio of the product to the buffer solution.

4. The online replacement system according to claim 1, characterized in that: The flow ratio between the first input pipeline and the second input pipeline is used to control the percolation ratio.

5. The online replacement system according to claim 1, characterized in that: The flow rate ratio between the first input channel and the second input channel is used to control the buffer exchange ratio.

6. The online replacement system according to claim 1, characterized in that: The buffer solution is replaced when the flow rates of the first input pipeline or the second input pipeline are equal to the flow rate of the first output pipeline.

7. A control method for an online replacement system according to any one of claims 1 to 6, characterized in that: The steps are: Step 1: The flow rate of the first input pipeline is F1, and the flow rate of the second input pipeline is F2; the first input pipeline and the second input pipeline respectively deliver buffer solution, and the ratio of F1 to F2 is controlled; Step 2: Control the flow rates of the first input pipeline and the second input pipeline so that F1 and F2 reach the target flow rates; at the same time, control the flow rate F3 in the first output pipeline so that F3 is at zero flow or low flow rate; Step 3: When F1 and F2 reach the target flow rate, control the flow rate in the first output pipe so that F3 reaches the target flow rate; Step 4: When the flow rate F1 in the first input pipe is equal to the flow rate F3 in the first output pipe, the first input pipe is connected to the product tank to deliver the product to be processed, so that the flow rate F1 adjusts itself and stabilizes again; Step 5: Continuously and stably control the flow of the first input pipeline, the second input pipeline, and the first output pipeline.

8. Chromatographic system based on online displacement system, characterized in that The online replacement system comprises the one of claims 1 to 6, wherein the first output pipe of the online replacement system is connected to the inlet end of the chromatography column through the third input pipe, the outlet end of the chromatography column is connected to the collection pipe and the waste pipe respectively through the second output pipe, and the inlet ends of the collection pipe and the waste pipe are respectively provided with valves.

9. The chromatography system based on the online displacement system according to claim 8, characterized in that: The outlet end of the first output pipeline of the online replacement system is provided with a valve for adjusting the resistance of sample loading.

10. The chromatography system based on the online displacement system according to claim 8, characterized in that: A side branch pipeline is connected between the third input pipeline at the inlet end of the chromatography column and the second output pipeline at the outlet end of the chromatography column. The third input pipeline is provided with a valve, and the valve is located between the inlet end of the chromatography column and the connection part between one end of the side branch pipeline and the second output pipeline; two valves are provided on the second output pipeline, and the two valves are respectively located on both sides of the connection part between the other end of the side branch pipeline and the second output pipeline.

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