Conjugation device and method for producing conjugates

Abstract

To provide a conjugation device and a method for producing conjugates using the conjugation device.SOLUTION: A conjugation device includes at least one flow reactor, a fluid delivery unit, and a fluid collection unit. The flow reactor has an inlet and an outlet and is completely filled with a support such as a matrix, the support including: 1) chromatography beads, fibers, or membranes, and 2) a biologic catalyzer, namely the enzyme ligase, which is immobilized onto this support. The fluid delivery unit is in fluid communication with the inlet of the flow reactor and is configured to continuously provide the flow reactor with at least one kind of a reaction fluid such as antibody and linker-payload according to stages of the conjugation process. The fluid collection unit is in fluid communication with the outlet of the flow reactor and is configured to control collection of fluid flowing out of the outlet of the flow reactor according to the stages of the conjugation process.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present disclosure generally relates to the fields of biotechnology and pharmaceutical production, and more specifically, to a conjugation device and a method for generating a conjugate using the conjugation device.

Background Art

[0002] Conjugation is the linking of one molecule to another via a specific linker by biological or chemical means. The demand for high-quality conjugates, particularly bioconjugates for bioscience research, diagnostic or therapeutic purposes, etc., is increasing day by day. Bioconjugates, such as antibody-drug conjugates (ADCs), antibody-immunagonist conjugates, antibody-cytokine conjugates, and antibody-radionuclide conjugates, are formed by linking a targeting molecule to a payload via a linker.

[0003] Taking ADC as an example, the main components of ADC include an antibody, a linker, and a small molecule compound. The antibody is mainly used to deliver the small molecule compound to a specific target, and the small molecule compound (including but not limited to cytotoxins) brings a therapeutic effect to the target. Currently, the nine ADC drugs approved by the FDA are formed by chemical conjugation methods, that is, the cytotoxins are randomly conjugated onto lysine or cysteine residues of the antibody framework.

[0004] In the chemical conjugation method, it is necessary to generate a monoclonal antibody stock solution in the upstream and downstream processes using antibody raw materials before the conjugation process. Subsequently, in the conjugation process, the following steps are performed. The antibody is pretreated (e.g., chemically reduced), and then a linker is conjugated, and the dissociable linker is removed by UF / DF and / or chromatography. Then, a small molecule compound is conjugated, and aggregates are removed by cationic and / or anionic and / or hydrophobic chromatography. Finally, the dissociable small molecule compound is removed by UF / DF and / or chromatography.

Summary of the Invention

[0005] In chemical conjugation, there are various problems, for example, the conjugation site is random, the product is heterogeneous, the centralized control is complex, and the scale-up of the yield is limited.

[0006] First of all, the product of chemical conjugation is generally a mixture of non-uniform structures and components. Since the conjugation of the linker and the antibody is highly random, the number of small molecule compounds conjugated to the antibody varies, and the conjugation sites of the antibody also vary. As a result, bioconjugates such as ADC drugs may have different drug / antibody ratios (DAR). As a result, the bioconjugate exhibits high heterogeneity, which increases the batch-to-batch difference, narrows the therapeutic window, and further poses challenges to drug production and quality control.

[0007] Secondly, as described above, the production process of chemical conjugation involves a considerable number of steps including multiple upstream and downstream purification steps, so it is time-consuming and labor-intensive.

[0008] Furthermore, in some cases, it is necessary for the operator to regularly sample during chemical conjugation, and the DAR of the sample is calculated. If the calculation result meets the internal quality standard The following operations are aborted until they become consistent. Therefore, in such cases, the calculation of DAR takes time, the cost is relatively high, and the risk of human error increases.

[0009] In addition, conventional reactors are generally employed for chemical conjugation. The scalability is limited by the volume of the reactor. At the same time, an organic phase is required in conjugation. Therefore, it is difficult to accurately control the conjugation process.

[0010] In view of the above technical problems, a first aspect of the present disclosure provides a conjugation device. The conjugation device includes at least one flow reactor, a fluid transport unit, and a fluid collection unit. The flow reactor has an inlet and an outlet and is completely filled with a support in the form of a matrix. The support includes chromatography beads, fibers or membranes, and a biocatalyst, i.e., an enzyme ligase immobilized on the support. The fluid transport unit is in fluid communication with the inlet of the flow reactor and is configured to continuously supply at least one reaction fluid (such as an antibody, a linker-toxin, a mixed antibody and a linker-toxin, etc.) to the flow reactor according to the stages of the conjugation process. The at least one process fluid contains a first part and a second part of the conjugate to be produced. The fluid collection unit is in fluid communication with the outlet of the flow reactor and is configured to control the collection of the fluid flowing out of the outlet of the flow reactor according to the stages of the conjugation process. While continuously passing the at least one reaction fluid through the flow reactor, the first part and the second part undergo a conjugation reaction under the catalysis of the ligase to produce a conjugate.

[0011] In the conjugation device provided in the first aspect of the present disclosure, the ligase is directionally immobilized on a support and filled in a flow reactor, whereby two portions of the conjugate to be generated contained in the reaction fluid are continuously and stably conjugated while the reaction fluid passes through the fluid reactor. Compared with chemical conjugation, the conjugation device significantly reduces the process steps, remarkably reduces the complexity, and is particularly suitable for saving the soaring manufacturing costs. Further, the flow reactor enables a linear scale-up of the conjugation process to meet the industrial demand for a larger scale, shortens the unit time for conjugation, and reduces the occupied space in the manufacturing area. By using the conjugation device to generate a bioconjugate, site-specific conjugation of the payload-linker and the targeting molecule is realized, the homogeneity is improved, and further the therapeutic window is expanded. Further, the conjugation process can be integrated with the production procedure of biomolecules such as monoclonal antibodies. For example, the conjugation may be completed at the production stage of the monoclonal antibody intermediate and the monoclonal antibody stock solution. Therefore, the process has high flexibility and excellent consistency.

[0012] In some embodiments, the at least one reaction fluid includes a first reaction fluid and a second reaction fluid. The first reaction fluid contains the first portion, and the second reaction fluid contains the second portion.

[0013] In some embodiments, the conjugation process includes the steps of equilibration before reaction, conjugation reaction, post-reaction, and flushing after post-reaction in this order. Further, the fluid transport unit is further set to continuously supply a buffer solution to the flow reactor at the stages of equilibration before reaction, post-reaction, and flushing after post-reaction, and to continuously supply the first reaction fluid and the second reaction fluid to the flow reactor simultaneously during the conjugation reaction.

[0014] In some embodiments, the buffer solution, the first reaction fluid, and the second reaction fluid are stored in a first container, a second container, and a third container, respectively. The fluid transport unit includes a first transport pump and a second transport pump. The first container and the second container are connected to the first transport pump via a first container outlet pipe and a second container outlet pipe, and the third container is connected to the second transport pump via a third container outlet pipe. The first transport pump and the second transport pump are connected to an inlet main pipe via a first inlet branch pipe and a second inlet branch pipe, respectively, and the inlet main pipe is connected to the inlet of the flow reactor. Further, in the stages of pre-reaction equilibration, post-reaction, and post-reaction flushing, the buffer solution in the first container is pumped into the inlet main pipe by the first transport pump, and in the stage of the conjugation reaction, the first reaction fluid in the second container is pumped into the inlet main pipe by the first transport pump, and the second reaction fluid in the third container is pumped into the inlet main pipe by the second transport pump.

[0015] In some embodiments, the fluid transport unit further includes a first valve, a second valve, a third valve, and a fourth valve. The first valve, the second valve, and the third valve are respectively disposed on the first container outlet pipe, the second container outlet pipe, and the third container outlet pipe, and are for controlling the flow path of the fluid in the first container outlet pipe, the second container outlet pipe, and the third container outlet pipe, respectively. The fourth valve is disposed on the first inlet branch pipe and is for controlling the flow path of the fluid in the first inlet branch pipe.

[0016] In some embodiments, in the stages of pre-reaction equilibration, post-reaction, and post-reaction flushing, the first valve and the fourth valve are open, and the second valve and the third valve are closed. In the stage of the conjugation reaction, the first valve is closed, and the second valve, the third valve, and the fourth valve are open.

[0017] In some embodiments, the first container outlet pipe, the second container outlet pipe, the third container outlet pipe, the first inlet branch pipe, the second inlet branch pipe, and the inlet main pipe are disposable or non-disposable, and are each manufactured from one of stainless steel, titanium, and silicone. The first container, the second container, and the third container are each selected from a disposable liquid storage bag, a disposable liquid storage bottle, a stainless steel container, and a glass or plastic container that can be disposable or non-disposable.

[0018] In some embodiments, the fluid collection unit is further configured to collect the fluid flowing out of the outlet of the flow reactor in the pre-reaction equilibration and post-reaction flushing stages into a fourth container, and to collect the fluid flowing out of the outlet of the flow reactor in the conjugation reaction and post-reaction stages into a fifth container.

[0019] In some embodiments, the fourth container and the fifth container are each connected to an outlet main pipe that is connected to the outlet of each flow reactor via a fourth container inlet pipe and a fifth container inlet pipe. Also, the fluid collection unit includes a fifth valve and a sixth valve that are respectively disposed on the fourth container inlet pipe and the fifth container inlet pipe to control the flow path of the fluid in the fourth container inlet pipe and the fifth container inlet pipe.

[0020] In some embodiments, in the pre-reaction equilibration and post-reaction flushing stages, the fifth valve is open and the sixth valve is closed. In the conjugation reaction and post-reaction stages, the fifth valve is closed and the sixth valve is open.

[0021] In some embodiments, the fourth container inlet pipe and the fifth container inlet pipe are disposable or non-disposable, and are each manufactured from one of stainless steel, titanium, and silicone. The fourth container and the fifth container are each selected from a disposable liquid storage bag, a disposable liquid storage bottle, a stainless steel container, and a disposable or non-disposable glass or plastic container.

[0022] In some embodiments, the conjugation device further comprises a temperature control unit configured to control the temperature of the fluid flowing into the inlet of the flow reactor and the fluid flowing out of the flow reactor in the conjugation process.

[0023] In some embodiments, the temperature control unit includes a heating module disposed at the inlet of the flow reactor for heating the fluid flowing into the inlet, and a cooling module disposed at the outlet of the flow reactor for cooling the fluid flowing out of the outlet.

[0024] In some embodiments, the conjugation device further comprises a sampling detection unit in fluid communication with the outlet of the flow reactor, and the sampling detection unit is configured to collect a sample fluid from the fluid flowing out of the outlet of the flow reactor according to a preset sampling time, and detect a conjugate in the sample fluid to obtain a detection result indicating whether the conjugate meets a predefined criterion.

[0025] In some embodiments, the sampling detection unit includes a sampling pump, a first switching valve, an elution pump, at least one analytical column, and a detector. The sampling pump is connected to the outlet of the flow reactor through a sampling pipe, a sample loop is arranged on the first switching valve, and the first switching valve can switch between a first state and a second state according to the preset sampling time. When the first switching valve is in the first state, the sampling pump is in fluid communication with the sample loop, and the sampling pipe collects the sample fluid from the fluid flowing out of the outlet of the flow reactor and pumps the sample fluid into the sample loop. When the first switching valve is in the second state, the elution pump, the sample loop, the at least one analytical column, and the detector are in fluid communication through a detection pipe, and the elution pump pumps an eluent into the detection pipe and passes the eluent through the sample loop, so that the sample fluid in the sample loop passes through one of the at least one analytical column before entering the detector.

[0026] In some embodiments, two analytical columns are arranged, and the sampling detection unit further includes a second switching valve that can switch between two states and a washing pump. When the second switching valve is in any of the states, the sample loop and the detector are in fluid communication with one of the two analytical columns, the sample fluid in the sample loop flows into one analytical column by the eluent, the washing pump is in fluid communication with the other analytical column, and pumps a buffer solution into the other analytical column for equilibration.

[0027] In some embodiments, the first switching valve is a six-way valve, the second switching valve is a ten-way valve, and the elution pump is a quaternary pump.

[0028] In some embodiments, the conjugation device further comprises a recycling unit disposed between the inlet and the outlet of the flow reactor. In the case of a detection result indicating that the conjugate does not meet the predefined criteria, the fluid collection unit is set to stop collecting the fluid flowing out of the outlet of the flow reactor, and the recycling unit is set to control the fluid flowing out of the outlet of the flow reactor to re-enter the inlet for performing a re-conjugation reaction in the flow reactor.

[0029] In some embodiments, the recycling unit includes a seventh valve disposed on a recycling pipe. The recycling pipe is connected between the inlet and the outlet of the flow reactor, and a recycling container is disposed on the recycling pipe. In the case of a detection result indicating that the conjugate does not meet the predefined criteria, the seventh valve is open, and the fluid flowing out of the outlet of the flow reactor flows into the inlet after passing through the recycling pipe and the recycling container.

[0030] In some embodiments, the flow reactor is a conjugation column.

[0031] In some embodiments, the first portion includes one of a ligase receptor substrate recognition motif and a ligase donor substrate recognition motif, and the second portion includes the other of the ligase receptor substrate recognition motif and the ligase donor substrate recognition motif.

[0032] In some embodiments, the conjugation device further comprises at least one of a pressure sensing module, a flow rate measuring module, a pH measuring module, a conductivity measuring module, and a UV detection module disposed at the inlet and / or the outlet respectively.

[0033] The second aspect of the present disclosure provides a method for generating a conjugate, including the step of preparing at least one reaction fluid containing a first part and a second part of the conjugate to be generated, and the step of generating the conjugate using the conjugation device described in the above embodiment.

[0034] In the method for generating a conjugate provided by the second aspect of the present disclosure, the ligase is directionally immobilized on a support and filled in a flow reactor, whereby the two parts of the conjugate to be generated contained in the reaction fluid are continuously and stably conjugated while the reaction fluid passes through the fluid reactor. Compared with chemical conjugation, the conjugation method significantly reduces the process steps, remarkably reduces the complexity, and is particularly suitable for saving the soaring manufacturing costs. In addition, the flow reactor enables the linear scale-up of the conjugation process to meet the industrial demand for a larger scale, shortens the unit time for conjugation, and reduces the occupied space in the manufacturing area. By generating a conjugate using the conjugation method, site-specific conjugation of the payload-linker and the targeting molecule is realized, the homogeneity is improved, and the therapeutic window is further expanded. In addition, the conjugation process can be integrated with the production procedure of biomolecules such as monoclonal antibodies. For example, the conjugation may be completed at the production stage of the monoclonal antibody intermediate and the monoclonal antibody stock solution. Therefore, the process has high flexibility and excellent consistency.

[0035] The features, advantages, and other aspects of each embodiment in the present disclosure will become clearer by referring to the drawings and in connection with the following detailed description. Some embodiments of the present disclosure are described illustratively rather than restrictively. The description of the drawings is set forth below.

Brief Description of the Drawings

[0036]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Mode for Carrying Out the Invention

[0037] Hereinafter, the technical content of the present invention will be described with specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention may be implemented or applied in other different specific embodiments. Those skilled in the art can make various modifications and changes without departing from the spirit of the present invention.

[0038] Before explaining the specific embodiments of the present disclosure in detail, some terms used in the present disclosure will be explained first.

[0039] General terms and definitions Unless otherwise defined herein, all technical and scientific terms used in this specification are the same as those generally understood by those skilled in the art. The technologies mentioned in this specification are intended to mean technologies generally understood in the art and include obvious technical changes or equivalent technology substitutions for those skilled in the art. Despite the terms in this specification being considered well understood by those skilled in the art, their definitions are described below to better explain the present invention. The product names described in this specification refer to the corresponding products. All patents, published patent applications, and publications cited in this specification are incorporated herein by reference.

[0040] Unless otherwise specified, singular forms such as "one" and "the" include plural forms. The expressions "one or more" or "at least one" may represent 1, 2, 3, 4, 5, 6, 7, 8, and 9 or more.

[0041] As used herein, the term "simultaneously" means that one or more events described herein occur at the same time.

[0042] Terms such as "comprising", "containing", and "including" and similar terms used herein are open terms, i.e., terms of "including / including but not limited to", meaning that other contents may also be included. The term "based on" means "at least partially based on". The term "one embodiment" represents "at least one embodiment". The term "another embodiment" represents "at least one another embodiment".

[0043] As used herein, the term "and / or" (e.g., in an expression such as A and / or B) is intended to include "A and B", "A or B", "A", and "B". Similarly, as used herein, "and / or" (e.g., in an expression such as "A, B, and / or C") is intended to include any of the implementations of A, B, and C, A, B, or C, A or C, A or B, B or C, A and C, A and B, B and C, A (alone), B (alone), and C (alone).

[0044] As used herein, terms such as "connect", "connection", "couple", or "coupled" and other similar words are not limited to direct connection and may include indirect connection.

[0045] As used herein, the definition of "biomolecule" includes proteins, nucleic acids, lipids, carbohydrates, small nucleic acids, amino acids, and their derivatives.

[0046] As used herein, the term "ligase" refers to an enzyme capable of catalyzing a covalent bond between two or more molecules. The ligase may specifically catalyze the conjugation of a first part containing a ligase donor substrate recognition motif and a second part containing a ligase acceptor substrate recognition motif to generate a target conjugate.

[0047] As used herein, the term "conjugation" refers to a covalent linkage of at least both (e.g., at least two molecules or at least two termini of the same molecule).

[0048] As used herein, the term "conjugate" refers to something that can be prepared by covalent linkage of at least both (e.g., at least two molecules or at least two termini of the same molecule).

[0049] As used herein, the term "bioconjugate" refers to a conjugate in which at least one conjugation participant is a biomolecule. Examples of the bioconjugate include, but are not limited to, antibody-drug conjugates, antibody-immune agonist conjugates, antibody-cytokine conjugates, antibody-radionuclide conjugates, etc.

[0050] As used herein, the term "linker" refers to any chemical moiety capable of conjugating a payload to a targeting molecule in a stable covalent manner.

[0051] As used herein, the term "payload" refers to the functional part contained in the conjugate linked by the linker. Examples of the payload include, but are not limited to, small molecule compounds (also called small molecule drugs, e.g., inhibitors and toxic drugs (e.g., cytotoxic drugs)), radionuclides, glycans, nucleic acids and their analogs, tracer molecules, etc. The payload and the linker are covalently linked by an active group to obtain a linker-loaded intermediate.

[0052] As used herein, the term "targeted molecule" refers to a molecule having an affinity for a specific target (e.g., receptor, cell surface protein, cytokine, etc.). A targeted molecule can deliver a payload to a specific site in vivo in a targeted manner. A targeted molecule may recognize one or more targets, and its specific target site is defined by the recognized target. For example, a targeted molecule targeting a receptor can deliver a cytotoxic drug to a site containing a large amount of the receptor. Examples of the targeted molecule include, but are not limited to, antibodies, antibody fragments, binding proteins of a given antigen, antibody mimetics, scaffold proteins having an affinity for a given target, ligands, etc.

[0053] As used herein, the term "antibody-drug conjugate (ADC)" refers to a conjugate comprising an antibody or antibody fragment covalently conjugated to a payload.

[0054] As used herein, the term "small molecule compound" refers to a molecule of a size comparable to that of organic molecules commonly used in pharmaceuticals. The term does not include biopolymers (e.g., proteins, nucleic acids, etc.), but includes low molecular weight peptides such as dipeptides, tripeptides, tetrapeptides, pentapeptides, etc. or derivatives thereof. Typically, the molecular weight of the small molecule compound may be, for example, about 100 Da to about 2000 Da, about 200 Da to about 1000 Da, about 200 Da to about 900 Da, about 200 Da to about 800 Da, about 200 Da to about 700 Da, about 200 Da to about 600 Da, about 200 Da to about 500 Da.

[0055] As used herein, the term "cytotoxin" refers to a substance that inhibits or blocks the expression activity and cell function of cells and / or causes cell destruction. In some cases, the cytotoxins commonly used in ADCs may be more toxic than those commonly used in chemotherapeutic drugs. Examples of cytotoxins include, but are not limited to, drugs targeting the microtubule cytoskeleton, DNA, RNA, kinesin-mediated protein transport, and regulation of apoptosis.

[0056] As used herein, the term "continuously conjugated" or "continuous conjugation process" refers to one or more types required for the conjugation process when a conjugation reaction occurs and at least one conjugate is produced. of the reaction fluid is continuously added into the conjugation device. The generated conjugate can be continuously collected from the conjugation reaction during the progress of the conjugation reaction.

[0057] As used herein, the term "flow reactor" refers to any reaction vessel for continuously performing a chemical reaction (such as conjugation). The flow reactor may be manufactured from stainless steel, glass, polymer and other materials, and is generally tubular. The flowing reaction fluid enters the flow reactor, continuously performs a chemical reaction in the flow reactor, and then flows out of the flow reactor.

[0058] As used herein, the term "support" refers to a water-insoluble substance separable in solid or semi-solid form from a reaction mixture, such as a surface, gel, polymer, matrix, particle, resin, bead or membrane.

[0059] As used herein, the term "conjugation column" is a type of flow reactor and refers to a tubular or columnar reaction vessel for continuously performing a conjugation reaction.

[0060] As used herein, the term "online monitoring" or "real-time monitoring" refers to detecting specific parameters or characteristics of a buffer solution, a reaction fluid and a fluid flowing out of a flow reactor, such as pH, pressure, flow rate, conductivity of a portion and conjugation conditions, in real time during the use of a conjugation device. Different from offline detection or analysis, the online monitoring or real-time monitoring can provide real-time feedback of detection results.

[0061] Hereinafter, one embodiment of the present disclosure will be described with reference to FIGS. 1 and 2. FIG. 1 shows a flow path diagram of a conjugation device according to one embodiment of the present disclosure. FIG. 2 shows a conjugation process of the conjugation device in FIG. 1. For illustrative purposes, in FIG. 1, each component of the conjugation device 100 is connected via a tube, and five containers for storing fluid are connected. The ligase is directionally immobilized on a support such as a matrix and filled into the flow reactor 10. The support and the ligase may be selected according to actual needs. For example, the support may include, but is not limited to, fillers such as silica gel, agarose, polyacrylic, synthetic fiber, cellulose acetate membrane, and polyethersulfone membrane. The ligase can be transpeptidase or glycosidase. The flow reactor 10 has an inlet 101 and an outlet 102. Fluid continuously flows into the inlet 101 of the flow reactor 10, passes through the flow reactor 10, and continuously flows out from the outlet 102. In this embodiment, the flow reactor 10 is a conjugation column, but in another embodiment, the flow reactor 10 may be any other similar reaction vessel.

[0062] The fluid transport unit 11 is in fluid communication with the inlet 101 on the inlet 101 side of the flow reactor 10. The fluid transport unit 11 continuously supplies the first reaction fluid, the second reaction fluid, or the buffer solution to the flow reactor 10 according to different stages of the conjugation process. The fluid collection unit 12 is in fluid communication with the outlet 102 on the outlet 102 side of the flow reactor 10. The fluid collection unit 12 controls the collection of the fluid flowing out from the outlet 102 of the flow reactor 10 according to different stages of the conjugation process. While continuously passing the first reaction fluid and the second reaction fluid through the flow reactor 10, the first part contained in the first reaction fluid and the second part contained in the second reaction fluid undergo a conjugation reaction under the catalytic action of the ligase to generate a conjugate. The conjugate is at the outlet of the flow reactor 10 It is contained in the fluid flowing out from 102.

[0063] Specifically, as shown in FIG. 1, the inlet 101 of the flow reactor 10 is connected to an inlet main pipe 131. Two branch pipes, namely, a first inlet branch pipe 132 and a second inlet branch pipe 133 are arranged in parallel and connected to the inlet main pipe 131 via a fluid connector (e.g., a T-junction). The openings of the first container 141, the second container 142, and the third container 143 are respectively connected to a first container outlet pipe 134, a second container outlet pipe 135, and a third container outlet pipe 136. The fluid transport unit 11 includes a first valve 161, a second valve 162, a third valve 163, a fourth valve 164, a first transport pump 171, and a second transport pump 172. The first container outlet pipe 134 and the second container outlet pipe 135 are arranged in parallel, integrally connected via a fluid connector, and connected to the first transport pump 171. The third container outlet pipe 13 is connected to the second transport pump 172. The first transport pump 171 and the second transport pump 172 are respectively connected to the inlet main pipe 131 via the first inlet branch pipe 132 and the second inlet branch pipe 133.

[0064] The first pinch valve 151 and the first valve 161 are arranged on the first container outlet pipe 134. Similarly, the second pinch valve 152, the second valve 162, the third pinch valve 153 and the third valve 163 are arranged on the second container outlet pipe 135 and the third container outlet pipe 136 respectively. The first pinch valve 151, the second pinch valve 152 and the third pinch valve 153 are all manual pinch valves and are used to control the outflow of the fluid from the first container 141, the second container 142 and the third container 143. During the preparation of conjugation, the buffer solution, the first reaction fluid and the second reaction fluid are respectively pumped into the corresponding containers by a peristaltic pump. The pinch valves 151 to 153 are open during conjugation. Therefore, the buffer solution, the first reaction fluid and the second reaction fluid flow out from the corresponding containers. The first valve 161, the second valve 162 and the third valve 163 are respectively used to control the flow path of the fluid in the first container outlet pipe 134, the second container outlet pipe 135 and the third container outlet pipe 136. The fourth valve 164 is arranged on the first inlet branch pipe 132 and is used to control the flow path of the fluid in the first inlet branch pipe 132.

[0065] As the excitation force of the fluid, the first transport pump 171 and the second transport pump 172 may be any type of pump, including, but not limited to, injection pumps, plunger pumps, peristaltic pumps and diaphragm pumps, and can provide different flow rate ranges. For example, some injection pumps can be precision syringe pumps of 1 ml, 10 ml and 50 ml and can provide a flow rate range of 0.01 - 50 ml / min. Some peristaltic pumps can provide a flow rate range of 5 - 200 ml / min. Some diaphragm pumps can provide a flow rate range of 40 - 400 ml / min.

[0066] In this embodiment, the first to fourth valves 161 to 164 for controlling the flow path of the buffer solution or the reaction fluid in the corresponding pipe are shown. However, in another embodiment, the fluid transport unit 11 may include only the first transport pump 171 and the second transport pump 172, and the first to fourth valves 161 to 164 may be unnecessary. For example, when the first transport pump 171 and the second transport pump 172 are plunger pumps or diaphragm pumps, the first to fourth valves 161 to 164 are unnecessary.

[0067] Continuing to refer to FIG. 1, the outlet 102 of the flow reactor 10 is connected to the outlet main pipe 137. The openings of the fourth container 144 and the fifth container 145 are connected to the fourth container inlet pipe 138 and the fifth container inlet pipe 139, respectively. The fluid collection unit 12 includes a fifth valve 165 and a sixth valve 166. The fifth valve 165 and the sixth valve 166 are disposed on the fourth container inlet pipe 138 and the fifth container inlet pipe 139, respectively. Also, a fourth pinch valve 154 is disposed on the fourth container inlet pipe 138. Similarly, a fifth pinch valve 155 is disposed on the fifth container inlet pipe 139. Similar to the first to fourth pinch valves 151 to 153 described above, both the fourth pinch valve 154 and the fifth pinch valve 155 are manual pinch valves for controlling the outflow of fluid from the fourth container 144 and the fifth container 145. The fifth valve 165 and the sixth valve 166 are for controlling the flow path of the fluid in the fourth container inlet pipe 138 and the fifth container inlet pipe 139, respectively. The fourth container inlet pipe 138 and the fifth container inlet pipe 139 are connected to the outlet main pipe 137 via fluid connectors. 4 pinch valve 154 is disposed on the fourth container inlet pipe 138. Similarly, a fifth pinch valve 155 is disposed on the fifth container inlet pipe 139. Similar to the first to fourth pinch valves 151 to 153 described above, both the fourth pinch valve 154 and the fifth pinch valve 155 are manual pinch valves for controlling the outflow of fluid from the fourth container 144 and the fifth container 145. The fifth valve 165 and the sixth valve 166 are for controlling the flow path of the fluid in the fourth container inlet pipe 138 and the fifth container inlet pipe 139, respectively. The fourth container inlet pipe 138 and the fifth container inlet pipe 139 are connected to the outlet main pipe 137 via fluid connectors.

[0068] The first to sixth valves 161 to 166 may be valves of any type, such as pneumatic valves, electric valves, and hydraulic valves. Preferably, the first to sixth valves 161 to 166 are electromagnetic valves.

[0069] The forms, materials, and capacities of the first to fifth containers 141 to 145 may be selected according to actual needs. For example, disposable liquid storage bags, disposable liquid storage bottles, stainless steel containers, and disposable and non-disposable glass or plastic containers of different specifications may be selected. On the other hand, the materials and specifications (such as inner diameter) of the pipes connected between the components of the conjugation device 100 may be selected according to actual needs. The pipes may be made of silicone, titanium, stainless steel, or any other suitable material. For example, the first to third container outlet pipes 134 to 136, the fourth and fifth container inlet pipes 138 and 139, and the outlet pipe 137 are ordinary-thickness silicone tubes, while the inlet main pipe 131 and the first and second inlet branch pipes 132 and 133 are disposable silicone tubes with increased thickness. Therefore, they can withstand higher flow rates and pressures. Preferably, the first to fifth containers 141 to 145 and the respective pipes are a set of disposable silicone tubes and disposable liquid storage bags, and can be directly used as sterilized (e.g., by gamma-ray irradiation) tubes and bags. The set of disposable silicone tubes and liquid storage bags can be realized in a plug-and-play manner on the conjugation device 100, avoiding frequent cleaning and being easy to use. Therefore, it can be easily matched with the fully closed system required for drug production, and can protect drugs from external contamination during the production process. In another embodiment, the first to fifth containers 141 to 145 and the pipes connected between the components of the conjugation device 100 are made of stainless steel and can be repeatedly used by implementing cleaning validation. In these embodiments, the production cost can be reduced, and the service life of the containers and the pipes can be extended.

[0070] In addition, the conjugation device 100 shown in FIG. 1 further includes a temperature control unit 18. In the present embodiment, the temperature control unit 18 includes a heating module 181 disposed on the inlet main pipe 131 and a cooling module 182 disposed on the outlet main pipe 137. The heating module 181 and the cooling module 182 are for heating and cooling the inlet main pipe 131 and the outlet main pipe 137, respectively. The heating module 181 heats the fluid flowing into the inlet 101 of the flow reactor 10 to an appropriate reaction temperature (for example, 37° C.), and the cooling module 182 cools the fluid flowing out of the flow reactor 10 to an appropriate temperature (for example, room temperature). The temperature control range, flow rate, and material of the heating module 181 and the cooling module 182 may be selected according to actual needs. For example, the temperature control range of the heating module 181 is 20 to 60° C., the temperature control range of the cooling module 182 is 10 to 30° C., and both the heating module 181 and the cooling module 182 are made of sanitary-grade stainless steel or disposable materials. In another embodiment, the temperature control unit 18 may be in other forms, including, but not limited to, air heating temperature control, water bath temperature control (that is, the flow reactor 10 is placed in a water bath), jacket water bath temperature control (that is, a jacket is fitted on the outer part of the flow reactor 10, and water at a constant temperature circulates in the jacket), and coil winding temperature control, etc. It includes, but is not limited to, a jacket that is fitted, and water at a constant temperature circulates in the jacket.

[0071] In the conjugation device 100 in FIG. 1, the buffer solution is stored in the first container 141, and the first reaction fluid and the second reaction fluid are stored in the second container 142 and the third container 143 respectively. However, in another embodiment, the reaction fluid and the buffer solution may be stored in the container in another manner according to actual needs. The number of the containers for storing the reaction fluid or the buffer solution can also be increased according to actual needs. For example, more containers, container outlet pipes, pinch valves and valves increase in parallel with the first container 141, the second container 142 and / or the third container 143. Accordingly, the reaction fluid or the buffer solution that needs to be pumped is selected by the first transport pump 171 and the second transport pump 172 according to the process procedure. In another embodiment, the number of transfer pumps may be selected according to actual needs. For example, each container is equipped with a corresponding transfer pump.

[0072] The first reaction fluid contains the first part of the conjugate to be generated, and the second reaction fluid contains the second part of the conjugate. The reaction fluid may be liquid or gas. The first part contains one of a ligase receptor substrate recognition motif and a ligase donor substrate recognition motif, and the second part contains the other of the ligase receptor substrate recognition motif and the ligase donor substrate recognition motif. In this embodiment, the first part further includes a targeting molecule such as an antibody, an antibody fragment, an antigen-specific binding protein, and an artificial antibody. The second part further includes a linker-payload intermediate formed by coupling a molecule such as a cytokine, a small molecule toxin, and a radionuclide with a linker, and the generated conjugate is a bioconjugate. In another embodiment, the first part and the second part may include other types of molecules as long as one molecule has a ligase receptor substrate recognition motif and the other molecule has a ligase donor substrate recognition motif.

[0073] In one embodiment, the ligase is a transpeptidase. In one embodiment, the ligase is selected from the group consisting of natural transpeptidases, unnatural transpeptidases, variants thereof, and combinations thereof. Unnatural transpeptidase enzymes may be obtained by engineering natural transpeptidases, but are not limited thereto. In a preferred embodiment, the ligase is selected from the group consisting of natural sortases, unnatural sortases, and combinations thereof. Types of natural sortases include sortase A, sortase B, sortase C, sortase D, sortase L. plantarum, etc. (see US20110321183A1). Since the type of ligase corresponds to the ligase recognition motif, it is used to achieve specific coupling between different molecules or structural fragments. In one embodiment, the ligase receptor substrate recognition motif is selected from the group consisting of oligomeric glycine, oligomeric alanine, and mixtures of oligomeric glycine / alanine with a degree of polymerization of 3 to 10. In a particular embodiment, the ligase receptor substrate recognition motif is Gn, where G is glycine (Gly) and n is an integer from 3 to 10. In another particular embodiment, the ligase is sortase A derived from Staphylococcus aureus. Accordingly, the ligase recognition motif can be the typical recognition motif LPXTG of the enzyme. In yet another particular embodiment, the ligase donor substrate recognition motif is LPXTGJ and the ligase receptor substrate recognition motif is G n where X may be any natural or unnatural single amino acid, and J is present or not, or is an amino acid fragment containing 1 to 10 amino acids that is optionally labeled. In one embodiment, J is absent. In yet another embodiment, J is an amino acid fragment containing 1 to 10 amino acids, where each amino acid is independently any natural or unnatural amino acid. In another embodiment, J is G m where where m is an integer from 1 to 10. In yet another specific embodiment, the ligase donor substrate recognition motif is LPETG. In another specific embodiment, the ligase donor substrate recognition motif is LPETGG. In one embodiment, the ligase is sortase B derived from Staphylococcus aureus, and the corresponding donor substrate recognition motif may be NPQTN. In another embodiment, the ligase is sortase B derived from Bacillus anthracis, and the corresponding donor substrate recognition motif may be NPKTG. In yet another embodiment, the ligase is sortase A derived from Streptococcus pyogenes, and the corresponding donor substrate recognition motif may be LPXTGJ, where J is as defined above. In another embodiment, the ligase is sortase subfamily 5 derived from Streptomyces cellicola, and the corresponding donor substrate recognition motif may be LAXTG. In yet another embodiment, the ligase is sortase A derived from Lactobacillus plantarum, and the corresponding donor substrate recognition motif may be LPQTSEQ. The ligase recognition motif may further be other artificially designed recognition sequences for transpeptidases optimized by manual screening.

[0074] In another embodiment, the first reaction fluid and the second reaction fluid may be mixed before being conjugated in the conjugation device 100. In such an embodiment, the mixture of the first reaction fluid and the second reaction fluid is pumped into the second container 142 or the third container 143 during the conjugation preparation process. During the conjugation process, only the container storing the mixture and its associated pipes and components are used. Alternatively, the conjugation device 100 may include only one of the second container 142 and the third container 143 and its associated pipes and components.

[0075] Next, with reference to FIGS. 1 and 2, the conjugation process 200 of the conjugation apparatus 100 will be described. In the conjugation process 200 of FIG. 2, step 201 includes performing pre-reaction equilibration on the flow reactor 10 to discharge the waste fluid. At this stage, the first valve 161, the fourth valve 164, and the fifth valve 165 are all in the open state, and all other valves are closed. The buffer solution in the first container 141 continuously flows out of the first container 141 under the control of the first transfer pump 171, enters the inlet main pipe 131 after passing through the first container outlet pipe 134 and the first inlet branch pipe 132. The outflow rate and duration of the buffer solution may be determined by presetting the pump speed and operating time of the first transfer pump 171. During the inflow into the inlet main pipe 131, the buffer solution flows into the heating module 181 and is preheated by the heating module 181. After flowing out of the heating module 181, the buffer solution flows into the inlet 101 of the flow reactor 10 to equilibrate the flow reactor 10. Then, the buffer solution flows out of the outlet 102 of the flow reactor 10, passes through the outlet main pipe 137, flows into the cooling module 182 for post-cooling, and is discharged into the fourth container 144 through the fourth container inlet pipe 138 to collect the waste fluid. By equilibrating the flow reactor 10 to supply the first reaction fluid and the second reaction fluid, a reaction environment with an appropriate pH and ionic strength can be provided for the flow reactor 10.

[0076] Subsequently, in step 102, a conjugation reaction is carried out to collect the fluid flowing out of the flow reactor 10. At this stage, the first valve 161 and the fifth valve 165 are switched to the closed state, the fourth valve 164 is maintained in the open state, and the second valve 162, the third valve 163 and the sixth valve 166 are switched to the open state. The first reaction fluid 132 in the second container continuously flows out of the second container 142 under the control of the first transport pump 171 and passes through the second container outlet pipe 134 and the first inlet branch pipe 132. The second reaction fluid 143 in the third container continuously flows out of the third container 143 under the control of the second transport pump 172 and passes through the third container outlet pipe 136 and the second inlet branch pipe 133. The flow rates of the first reaction fluid and the second reaction fluid are determined by presetting the pump speeds of the first transport pump 171 and the second transport pump 172 respectively. According to different process requirements, the conjugation flow rate and the flow rates of various reaction fluids (for example, 1 / 2 conjugation flow rate) are calculated by dividing the reaction volume by the residence time of the reaction fluid in the flow reactor 10. The flow rates of the first reaction fluid and the second reaction fluid are determined by presetting the pump speeds of the first transport pump 171 and the second transport pump 172 respectively. According to different process requirements, the conjugation flow rate and the flow rates of various reaction fluids (for example, 1 / 2 conjugation flow rate) are calculated by dividing the reaction volume by the residence time of the reaction fluid in the flow reactor 10.

[0077] Thereafter, the two types of reaction fluids merge into the inlet main pipe 131, are preheated in the heating module 181, and then flow into the inlet 101 of the flow reactor 10. In the flow reactor 10, the first portion contained in the first reaction fluid and the second portion contained in the second reaction fluid undergo a conjugation reaction under the catalytic action of the ligase, and the conjugate is generated. On the other hand, the post-reaction fluid containing the conjugate continuously flows out from the outlet 102 of the flow reactor 10. The fluid flowing out from the outlet 102 of the flow reactor 10 is post-cooled in the cooling module 182 and then flows into the fifth container 135 through the fifth container inlet pipe 139 to collect the conjugate. Note that since the first reaction fluid and the second reaction fluid are continuously supplied to the flow reactor 10, it should be noted that the above conjugation reaction is a continuous conjugation reaction. The theoretical conjugation time may be calculated by dividing a relatively small volume of the first reaction fluid and the second reaction fluid by the flow rate of the reaction fluid. The operating times of the first transfer pump 171 and the second transfer pump 172 are set longer than the theoretical conjugation time in advance.

[0078] After the conjugation reaction, since a very small amount of unreacted and reacting reaction fluid still remains in each tube and in the flow reactor 10, the tube and the flow reactor 10 should be flushed, whereby the unreacted and reacting reaction fluid can react and be collected as much as possible. Therefore, step 103 includes performing a post-reaction, and the fluid flowing out of the flow reactor 10 is collected. At this stage, the first valve 161 is switched to the open state, the second valve 162 and the third valve 163 are switched to the closed state, the fourth valve 164 and the sixth valve 166 are maintained in the open state, and the fifth valve 165 is maintained in the closed state. The buffer solution in the first container 141 continuously flows out of the first container 141 under the control of the first transfer pump 171, passes through the first container outlet pipe 134 and the first inlet branch pipe 132, and enters the inlet main pipe 131. Similarly, the outflow rate and duration of the buffer solution may be determined by presetting the pump speed and operating time of the first transfer pump 171. Thereafter, the buffer solution flows into the heating module 181 and is preheated by the heating module 181. After flowing out of the heating module 181, the buffer solution flows into the inlet 101 of the flow reactor 10 to flush the flow reactor 10. Subsequently, the buffer solution flows out of the outlet 102 of the flow reactor 10, flows into the cooling module 182 through the outlet main pipe 137 for post-cooling, and flows into the fifth container 135 through the fourth container inlet pipe 138 to continue collecting the conjugate. By performing a post-reaction with the buffer solution after the conjugation reaction is completed, the residual reaction fluid in the tube and the flow reactor can be fully utilized, so the yield increases.

[0079] After step 103, post-reaction flushing is performed in step 104, and the waste fluid is discharged. At this stage, both the first valve 161 and the fourth valve 164 are maintained in the open state, the fifth valve 165 is switched to the open state, the sixth valve 166 is switched to the closed state, and the states of the second valve 162 and the third valve 163 are maintained in the closed state. The buffer solution in the first container 141 continues to flow out of the first container 141 under the control of the first transfer pump 171, passes through the first container outlet pipe 134 and the first inlet branch pipe 132, and then enters the inlet main pipe 131. Similarly, the The outflow rate and duration of the buffer solution may be determined by presetting the pump speed and operating time of the first transfer pump 171. Thereafter, the buffer solution is preheated in the heating module 181, enters the inlet 101 of the flow reactor 10, and continues to flush the flow reactor 10. Subsequently, the buffer solution flows out of the outlet 102 of the flow reactor 10, flows into the cooling module 182 through the outlet main pipe 137 for post-cooling, and is discharged into the fourth container 144 through the fourth container inlet pipe 138 to collect the waste fluid. By supplying the buffer solution after the stage of post-reaction flushing, the residues in the pipe and the flow reactor can be completely flushed away.

[0080] The above-described first to sixth valves 161 to 166, the first transfer pump 171, and the second transfer pump 172 can cooperate with each other because they can be controlled by a control signal. In the present embodiment, the control unit or processing unit of the above components is communicatively coupled to a computing device. The computing device and the conjugation device form an integrated conjugation system physically. In another embodiment, the computing device may be positioned away from the conjugation device, such as a remote computing device. The computing device is communicatively coupled to the above components via an analog, digital, or analog / digital combination bus, or a wireless communication link or network, and may be any type of computing device, such as a server, a workstation, or a portable computing device (e.g., a laptop, a tablet computer, and a mobile phone). The computing device and each component store a plurality of applications that they themselves execute. The computing device receives signals or other information regarding the components from each component and executes a control application. The control application makes a control decision and generates one or more control signals based on the received information. Then, the control application transmits the one or more control signals to each component via the communication link or network, thereby controlling the operation of the components. By using the setup application in the computing device, an operator can create or change setup parameters in the control application, such as the start and end times of each stage in the conjugation process and the flow rate of the fluid, via a user interface before the conjugation process starts. The setup parameters may be determined by the operator based on the instance of the reaction fluid in a specific process design and the conditions required for conjugation. The viewer application in the computing device receives data from the control application and displays the data to the operator via a user interface.For example, the data may include the current flow rate, the state of each valve, the pump speed of each transport pump, the temperature of the inlet fluid, the temperature of the outlet fluid, etc. Thereby, in the conjugation process 200, the production staff can monitor the real-time state of the conjugation device. The data historian application in the computer device receives data from the setting application and the control application, and stores historical data including the operator history operations, historical parameters in the conjugation process 200, and the conjugation results in the conjugation process 200 in the storage device of the computer device. The steps 201 to 204 in the conjugation process 200 can be automatically and continuously executed without human intervention.

[0081] In the above embodiment, the ligase is directionally immobilized on the support and filled in the flow reactor, whereby the two parts of the conjugate to be generated contained in the reaction fluid are continuously and stably conjugated while the reaction fluid passes through the fluid reactor. Compared with chemical conjugation, the conjugation method has significantly reduced process steps, significantly reduced complexity, and is particularly suitable for saving soaring manufacturing costs. In addition, the flow reactor enables the linear scale-up of the conjugation process to meet the industrial demand for a larger scale, shortens the unit time for conjugation, reduces the occupied space in the manufacturing area. By using the conjugation method to generate a conjugate, site-specific conjugation of the payload-linker and the targeting molecule is realized, the homogeneity is improved, and the therapeutic window is further expanded. In addition, the conjugation process can be integrated with the production procedure of biomolecules such as monoclonal antibodies. For example, the conjugation may be completed at the production stage of the monoclonal antibody intermediate and the monoclonal antibody stock solution. Therefore, the process has high flexibility and excellent consistency.

[0082] In the following, another embodiment of the present disclosure will be described with reference to FIGS. 3 and 4. FIG. 3 shows a flow path diagram of the conjugation device in another embodiment of the present disclosure. FIG. 4 shows the conjugation process of the conjugation device in FIG. 3. In FIG. 3, the same reference numerals as in FIG. 1 identify the same features as those described with reference to FIG. 1. Compared with FIG. 1, the flow path diagram of the conjugation device 300 in FIG. 3 further includes a sampling detection flow path, while the conjugation flow path in FIG. 3 is the same as that in FIG. 1 and the description thereof will be omitted.

[0083] The conjugation device 300 in FIG. 3 includes a sampling and detection unit 30 that is in fluid communication with the outlet 102 of the flow reactor 10. The sampling and detection unit 30 samples the fluid flowing out from the outlet 102 of the flow reactor 10 according to a preset sampling time, detects the conjugate in the sample fluid, and obtains a detection result indicating whether the conjugate meets a predefined standard. Specifically, in the present embodiment, the sampling and detection unit 30 includes a sampling pump 321, a first switching valve 322, an elution pump 323, a cleaning pump 325, a first analytical column 326, a second analytical column 327, and a detector 328. The sampling pump 321 is connected to the outlet pipe 137 through a sampling pipe 31. The first switching valve 322 is connected to the sampling pump 321. In the present embodiment, the first switching valve 322 is a hexagonal valve in which a sample loop is arranged. The first switching valve 322 is switched between two states for sample injection and sample transportation. The first switching valve 322 is connected to the elution pump 323 and the second switching valve 324 through a detection pipe 33. In the present embodiment, the second switching valve 324 is a decagonal valve. The first analytical column 326 and the second analytical column 327 are connected in parallel to form a dual-column sample injection mode. By switching the second switching valve 324 between two states, one of the first analytical column 326 and the second analytical column 327 is selected so that the sample fluid can pass through. The cleaning pump 325 pumps the buffer solution when the sample fluid passes through the selected analytical column, whereby the buffer solution passes through the other analytical column to equilibrate the other analytical column.

[0084] As shown in FIG. 3, the first switching valve 322 includes six ports 1 to 6, and a sample injection state and a sample transport state. The second switching valve 324 includes ten ports 1 to 10, and an equilibrium state and a detection state. The connection of each port of the first switching valve 322 is as follows. Port 1 and port 4 are connected by connecting an external sample injection loop, port 2 is connected to the elution pump 323, port 3 is connected to port 4 of the second switching valve 324, port 5 is connected to the sampling pump 321, and port 6 is connected to a waste discharge pipe. The connection of each port of the second switching valve 324 is as follows. Ports 1 and 8 are respectively connected to both ends of the first analysis column 326, port 2 is connected to the cleaning pump 325, ports 3 and 6 are respectively connected to both ends of the second analysis column 327, port 4 is connected to port 3 of the first switching valve 322, port 5 is connected to port 10, port 7 is connected to the waste discharge pipe, and port 9 is connected to the inlet of the detector 328.

[0085] Referring to FIG. 4, compared with the conjugation method 200 in FIG. 2, steps 401 and 403 to 404 in the conjugation method 400 are the same as steps 201 and 203 to 204 in FIG. 2 respectively, and the difference between FIGS. 2 and 4 lies only in step 402. Therefore, only step 402 will be described with reference to FIG. 3, and the descriptions of steps 401 and 403 to 404 will be omitted.

[0086] ​Step 402 includes performing a conjugation reaction and performing online monitoring of the generated conjugate to determine whether the generated conjugate meets a predefined criterion. In the conjugation reaction, the sampling pump 321 samples a predetermined amount of sample fluid from the outlet main pipe 137 according to the preset sampling time. At this time, the first switching valve 322 is in the first state (sample injection state). In this state, port 5 and port 4 of the first switching valve 322 communicate with each other, port 1 communicates with port 6, and port 2 communicates with port 3. Since port 1 communicates with port 4 via the sample loop, the flow path of the sample fluid is like port 5 - port 4 - sample loop - port 1 - port 6. In this way, the sample fluid is pumped into the sample loop and stored therein, and the excess sample fluid is discharged from port 6, thereby completing the sampling. The specifications of the sample loop may be selected based on different detection methods and the volume of sample fluid required for each sampling, such as 5 μL, 10 μL, 20 μL, and 30 μL. On the other hand, the elution pump 323 pumps the buffer solution through the detection tube 33 to port 2 of the first switching valve 322, the buffer solution flows out from port 3, and then flows into port 4 of the second switching valve 324. At this time, the second switching valve 324 is in the first state, port 4 of the second switching valve 324 communicates with port 5, port 8 communicates with port 9, port 10 communicates with port 1, and port 2 communicates with port 3. The flow path of the inflowing buffer solution in the second switching valve 324 is like port 4 - port 5 - port 10 - port 1 - the first analytical column 326 - port 8 - port 9 - detector 328 to pre-equilibrate the first analytical column 326. By the inlet pipe of the washing pump 325 receiving the equilibration buffer solution, the equilibration buffer solution is pumped into port 2 of the second switching valve 324. The flow path of the equilibration buffer solution in the second switching valve 324 is like port 2 - port 3 - the second analytical column 327 - port 6 - port 7 - waste discharge to equilibrate the second analytical column 327.

[0087] The first switching valve 322 is switched to the second state (i.e., the sample transport state) after sampling. In the second state, port 1 of the first switching valve 322 communicates with port 2, port 3 communicates with port 4, and port 5 communicates with port 6. Therefore, the flow path of the sample fluid is like this: elution pump 323 - port 2 - port 1 - sample loop - port 4 - port 3 - port 4 of the second switching valve 324. After entering the second switching valve 324, the flow path of the sample fluid is like this: port 4 - port 5 - port 10 - port 1 - first analysis column 326 - port 8 - port 9 - detector 328. In this embodiment, the elution pump 323 is a quaternary pump, which controls the ratio of the four eluents and pumps the eluent into the detection tube 33. Thus, the eluent passes through the sample loop between port 1 and port 4 of the first switching valve 322, and further the sample fluid in the sample loop enters the second switching valve 324. The sample fluid is eluted into the first analysis column 326 by controlling the gradient of the eluent by the elution pump 323, and the fluid flowing out of the first analysis column 326 enters the detector 328 to detect the conjugate.

[0088] The valve position of the second switching valve 324 is switched for the next sample injection, that is, port 1 communicates with port 2, port 3 communicates with port 4, port 5 communicates with port 6, port 7 communicates with port 8, and port 9 communicates with port 10. At this valve position, the fluid flowing from port 4 of the second switching valve 324 passes through port 3, and the fluid enters the second analysis column 327, and then flows out of the second analysis column 327, passing through port 6, port 5, port 10 and port 9 in sequence and entering the detector 328. On the other hand, the first analysis column 326 is equilibrated with the buffer solution pumped by the washing pump 325.

[0089] The detection result obtained by the detector 328 indicates whether the conjugate meets a predefined criterion. In the conjugation reaction, the sampling pump 321 samples the sample fluid according to the preset sampling time (e.g., at regular time intervals), and detects the conjugate contained in the sample fluid in real time, thereby monitoring the conjugate generated in the entire conjugation process online. When the ADC drug is generated by the conjugation device 300 in FIG. 3, the average DAR value of the ADC is detected to obtain the average number of small molecule toxins conjugated to each antibody molecule. Thereby, the quality of the conjugate can be evaluated. The detection result may further be transmitted to the control units of the fifth valve 165 and the sixth valve 166, whereby the opening and closing of the fifth valve 165 and the sixth valve 166 can be controlled according to the detection result. In this case, when the flow reactor 10 is balanced and the conjugation reaction has just started, the fifth valve 165 is maintained in an open state, and the sixth valve 166 is maintained in a closed state. The sampling pump 321 samples the sample fluid from the fluid in the outlet main pipe 137, and the conjugate in the sample fluid is detected. When the conjugate meets the predefined criterion, the fifth valve 165 is switched to a closed state, the sixth valve 166 is switched to an open state, and the fluid in the outlet main pipe 137 is collected in the fifth container 145.

[0090] The above-described first to sixth valves 161 to 166, the first transport pump 171, and the second transport pump 172 are the same as those in the conjugation device 100 in FIG. 1. The control unit or processing unit of each component is communicatively coupled to one computing device. The device also enables the control and integration of the sampling pump 321. The control units or processing units of the first switching valve 322, the elution pump 323, the second switching valve 324, and the detector 328 are communicatively coupled to the same computing device so that they can cooperate with each other. In addition to the above-described content, in the present embodiment, the detector 328 transmits the acquired detection result to the control application in the computing device, and the control application determines whether the conjugate meets a predefined criterion based on the detection result. The setup parameters in the control application may further include the sampling start time, a certain sampling interval, the pump speed of the sampling pump, the pump speed of the elution pump, the pump speed of the cleaning pump, the valve position switching time of each switching valve, the eluent gradient, the detection time, the predefined criterion of the conjugate (for example, a preset DAR value), etc. The viewer application in the computing device further displays the real-time data of the above parameters received from the control application via the user interface.

[0091] In this embodiment, the detector 328 is a UV detector (ultraviolet absorption detector). Both the first analytical column 326 and the second analytical column 327 are HIC (hydrophobic interaction chromatography) analytical columns. The efficiency of the conjugate is detected by an HIC-HPLC (high performance liquid chromatography) detection method. In another embodiment, the efficiency of the conjugate may be detected by other detection methods such as RP-HPLC, SEC-HPLC, and Protein A-HPLC. Alternatively, the detector 328 may be a mass spectrometry detector, a fluorescence detector, or the like. In this embodiment, dual-column detection is employed, and the flow paths in the two analytical columns are switched by the second switching valve 324. In another embodiment, other numbers of analytical columns may be used. For example, only one analytical column is arranged, and in this case, the second switching valve 324 is not required. ysis column is arranged, and in this case, the second switching valve 324 is not required.

[0092] In this embodiment, the sampling pump 321 performs active sampling from the outlet main pipe 137 through the sampling pipe 31. The sampling pump 321 may be a peristaltic pump or an injection pump so that the sampling volume can be accurately controlled and reduced, thereby increasing the product yield. In another embodiment, instead of the sampling pump 321, one valve is arranged in the sampling pipe 31. By switching the valve position of the valve, the fluid in the outlet main pipe 137 flows into the sampling pipe 31 and enters the sample loop of the first switching valve 322.

[0093] By adding the sampling detection unit to the conjugation device 300, it is possible to online monitor whether the conjugate generated in the flow reactor meets a predefined standard, thereby contributing to the collection of the post-reaction fluid that meets the standard, reducing the processing time and cost, and improving the product consistency. Also, the manual sampling by the operator in the conjugation process becomes unnecessary, thereby increasing the process convenience, reducing the operation complexity, and avoiding possible human errors.

[0094] In the above embodiment, the ligase is directionally immobilized on the support and filled in the flow reactor, whereby the two parts of the conjugate to be generated contained in the reaction fluid are continuously and stably conjugated while the reaction fluid passes through the fluid reactor. Compared with chemical conjugation, the conjugation device has significantly reduced process steps, significantly reduced complexity, and is particularly suitable for saving the soaring manufacturing cost. Also, the flow reactor enables the linear scale-up of the conjugation process to meet the industrial demand for a larger scale, shortens the unit time for conjugation, and reduces the occupied space in the manufacturing area. By using the conjugation device to generate the conjugate, site-specific conjugation of the payload-linker and the targeting molecule is realized, the homogeneity is improved, and furthermore, the therapeutic window is expanded. Also, the conjugation process can be integrated with the production procedure of biomolecules such as monoclonal antibodies. For example, the conjugation may be completed at the production stages of the monoclonal antibody intermediate and the monoclonal antibody stock solution. Therefore, the process has high flexibility and excellent consistency.

[0095] In the following, with reference to FIG. 5, yet another embodiment of the present disclosure will be described. FIG. 5 shows a flow path diagram of the conjugation device in a further embodiment of the present disclosure. In FIG. 5, the same reference numerals as in FIGS. 1 and 3 identify the same features as those described with reference to FIGS. 1 and 3.

[0096] Compared with FIG. 3, the flow path diagram of the conjugation device 500 in FIG. 5 further includes a recycle flow path. In the flow path diagram of FIG. 5, a recycle unit 50 is arranged between the inlet 101 and the outlet 102 of the flow reactor 50. In the conjugation reaction, when the detection result obtained by the detector 328 indicates that the conjugate does not meet a predefined criterion, the fluid collection unit 12 stops collecting the fluid flowing out from the outlet 102 of the flow reactor 10. Also, the recycle unit 50 controls the fluid flowing out from the outlet 102 of the flow reactor 10 to re-enter the inlet 101 so that the fluid performs a circulating conjugation reaction in the flow reactor 10.

[0097] Specifically, in FIG. 5, in addition to being connected to the sampling tube 31, the outlet main pipe 137 is also connected to one end of a circulation pipe 51. The other end of the circulation pipe 51 and both the third container outlet pipe 136 are connected to the second inlet pipe 133 via fluid connectors. The circulation A recycle container 53, a sixth pinch valve 521, a seventh pinch valve 522, a seventh valve 523, and an eighth valve 524 are arranged on the circulation pipe 51.

[0098] The recycle container 53 is for storing reaction fluid that does not meet a predefined criterion. Similar to the first to fifth containers 141 to 145, the form, material, and capacity of the recycle container 53 may be selected according to actual needs. For example, disposable liquid storage bags, disposable liquid storage bottles, stainless steel containers, and disposable and non-disposable glass or plastic containers of different specifications may be selected. The material and specifications (such as inner diameter) of the circulation pipe 51 may be selected according to actual needs, such as a disposable silicone tube of normal thickness. Preferably, the recycle container 53, the first to fifth containers 131 to 135, and each pipe are a set of the disposable silicone tube and the liquid storage bag.

[0099] Both the seventh pinch valve 521 and the eighth pinch valve 522 are manual pinch valves for controlling the inflow and outflow of fluid in the recycling container 53. The seventh valve 523 and the eighth valve 524 are for controlling the flow path of fluid in the circulation pipe 52 in the circulation conjugation process. Similar to the first to sixth valves 161 - 166, the seventh valve 523 and the eighth valve 524 may be any type of valve, such as a pneumatic valve, an electric valve, and a hydraulic valve. Preferably, the seventh valve 523 and the eighth valve 524 are solenoid valves.

[0100] In the conjugation reaction, the sampling pump 321 collects a predetermined amount of sample fluid from the outlet main pipe 137 according to a preset sampling time. The sample fluid enters the second switching valve 324 through the cooperation of the elution pump 323 and the first switching valve 322, passes through one of the analysis columns 326 and 327, and flows into the detector 328. The detector 328 detects the sample fluid, and the detection result indicates whether the conjugate contained in the sample fluid meets a predefined criterion. Since the detailed detection process is the same as that in Figure 3, the description is omitted here. If the conjugate contained in the sample fluid does not meet the predefined criterion, all of the first to sixth valves 161 - 166 are switched to the closed state, and the seventh valve 523 and the eighth valve 524 are open. The fluid flowing out of the outlet main pipe 137 flows into the circulation pipe 51 and is temporarily stored in the recycling container 53. Then, the temporarily stored fluid passes through the eighth valve 524 under the control of the second transport pump 172, flows into the inlet pipe 131, and is re - conjugated in the flow reactor 10.

[0101] When the conjugate contained in the sample fluid sampled after reconjugation meets a predefined criterion, the sixth valve 166 is switched to the open state, the seventh valve 523 is switched to the closed state, and the fluid flowing out from the outlet main pipe 137 is collected in the fifth container 135. When the conjugate contained in the sample fluid sampled after reconjugation still does not meet the predefined criterion, the first to sixth valves 161 - 166 are maintained in the closed state, the seventh valve 523 and the eighth valve 524 are maintained in the open state, and the reconjugated fluid continues to flow into the circulation pipe 51 for reconjugation until the generated conjugate meets the predefined criterion. In the circulation conjugation process, the fluid performing the conjugation reaction in the flow reactor 10 continues to be the fluid transported by the circulation pipe 51 until the fluid in the recycle container 53 and the circulation pipe 51 is substantially discharged. The flow velocity of the fluid may be determined by presetting the pump speed of the second transport pump 172. The flow velocity of the circulation conjugation is calculated by dividing the holding volume of the recycle container 53 (for example, 1 / 3 of the reaction volume of the flow reactor 10) by the holding time according to different process requirements. When the fluid in the recycle container 53 and the circulation pipe 51 is substantially discharged, the second valve 162, the third valve 163, and the fourth valve 164 are switched to the open state, and the seventh valve 523 and the eighth valve 524 are switched to the closed state, whereby the first reaction fluid 142 in the second container and the second reaction fluid 143 in the third container continuously flow into the flow reactor 10 to perform the conjugation reaction. Since the conjugation process of the conjugation device 500 is the same as the conjugation process 400 of the conjugation device 300 in FIG. 3, the description is omitted. The flow velocity of the circulation conjugation is calculated by dividing the holding volume of the recycle container 53 (for example, 1 / 3 of the reaction volume of the flow reactor 10) by the holding time according to different process requirements. When the fluid in the recycle container 53 and the circulation pipe 51 is substantially discharged, the second valve 162, the third valve 163, and the fourth valve 164 are switched to the open state, and the seventh valve 523 and the eighth valve 524 are switched to the closed state, whereby the first reaction fluid 142 in the second container and the second reaction fluid 143 in the third container continuously flow into the flow reactor 10 to perform the conjugation reaction. Since the conjugation process of the conjugation device 500 is the same as the conjugation process 400 of the conjugation device 300 in FIG. 3, the description is omitted.

[0102] As described above, the conjugation conditions of the portion contained in the reaction fluid can be monitored online. By adding a circulation flow path to the conjugation device 500, when the detection result of the conjugate satisfies the standard, the fluid flowing out of the flow reactor is automatically collected. When the detection result of the conjugate does not satisfy the standard, the fluid flowing out of the flow reactor is recycled into the flow reactor for re-conjugation until it satisfies the standard. The entire production process of the conjugate is automatically completed without manual sampling by the operator in the production process, thereby increasing the process convenience, reducing the complexity of the operator's operation, and avoiding possible human errors.

[0103] Also, as shown in FIG. 5, in the present embodiment, a pressure sensing module 525 and a flow rate measuring module 526 are respectively arranged on the inlet main pipe 131 of the conjugation flow path. Therefore, the fluid entering the inlet main pipe 131 enters the heating module 181 after passing through the pressure sensing module 525 and the flow rate measuring module 526. A pressure threshold for the pressure sensing module 525 may be set in advance. When the fluid pressure measured by the pressure sensing module 525 exceeds the pressure threshold, the conjugation device 500 issues an alarm and automatically stops temporarily to avoid rupture of the flow reactor 10 due to extremely high pressure. The flow rate measuring module 526 can monitor the flow rate of the fluid flowing through the inlet main pipe 131 in real time.

[0104] In another embodiment, the pressure sensing module 525 and / or the flow rate measurement module 526 may be disposed on the outlet main pipe 137 to monitor the pressure and flow rate of the fluid flowing out from the outlet 102 of the flow reactor 10 in real time. Also, in another embodiment, other measuring devices, such as a conductivity measurement module, a pH measurement module, and a UV detection module, may be disposed on the inlet main pipe 131 and / or the outlet main pipe 137 to monitor parameters such as the conductivity, pH, and UV value of the conjugation buffer, the reaction fluid, and / or the fluid flowing out from the outlet 102 of the flow reactor 10 in real time. In another embodiment, an automatic collector may be disposed on the outlet main pipe 137 to collect the fluid flowing out from the outlet 102 of the flow reactor 10 section by section.

[0105] The first to sixth valves 161 to 166, the first transport pump 171, the second transport pump 172, the sampling pump 321, the first switching valve 322, the elution pump 323, the second switching valve 324, and the detector 328 described above are the same as those in the conjugation device 300 in FIG. 3. The control unit or processing unit of each component is communicatively coupled to one computing device. The control units or processing units of the pressure sensing module 525, the flow rate measurement module 526, and the seventh and eighth valves 523 and 524 can cooperate with each other by being communicatively coupled to the same computing device. In addition to the above-described content, in the present embodiment, the pressure sensing module 525 and the flow rate measurement module 526 transmit measurement results to a control application in the computing device. The control application makes a control decision based on these measurement results and transmits a control signal to a related component. The setup parameters in the control application may further include a pressure threshold, a flow rate threshold, a flow velocity during cyclic conjugation, and the like. The viewer application in the computing device displays real-time data of the above parameters received from the control application via a user interface.

[0106] In the above embodiment, the ligase is directionally immobilized on a support and filled in a flow reactor, whereby two parts of the conjugate to be generated contained in the reaction fluid are continuously and stably conjugated while the reaction fluid passes through the fluid reactor. Compared with chemical conjugation, the conjugation device significantly reduces the process steps, remarkably reduces the complexity, and is particularly suitable for saving soaring manufacturing costs. In addition, the flow reactor enables the linear scale-up of the conjugation process to meet the industrial demand for a larger scale, shortens the unit time for conjugation, and reduces the occupied space in the manufacturing area. By generating a conjugate using the conjugation device, site-specific conjugation of the payload-linker and the targeting molecule is realized, the homogeneity is improved, and the therapeutic window is further expanded. In addition, the conjugation process can be integrated with the production procedure of biomolecules such as monoclonal antibodies. For example, the conjugation may be completed at the production stage of the monoclonal antibody intermediate and the monoclonal antibody stock solution. Therefore, the process has high flexibility and excellent consistency.

[0107] In yet another embodiment of the present disclosure, a method for generating a conjugate is provided. The method includes the steps of preparing at least one reaction fluid containing a first part and a second part of the conjugate to be generated, and generating the conjugate using any of the conjugation devices in the above embodiment.

[0108] The above description is only an alternative embodiment of the present disclosure and does not limit the embodiments of the present disclosure. Those skilled in the art can make various modifications and changes to the embodiments of the present disclosure.

[0109] The claims shall be construed in the broadest manner and shall thereby encompass all modifications, equivalent structures, and functions. Modifications, equivalent forms, and improvements made within the spirit and principles of the embodiments of the present disclosure shall all be included in the claims of the present disclosure.

Claims

1. at least one flow reactor, each filled with a support and having an inlet and an outlet, wherein a ligase is immobilized on the support, wherein the ligase is a transpeptidase or a glycosidase; a fluid transport unit in fluid communication with the inlet of the flow reactor and configured to continuously supply at least one reaction fluid to the flow reactor according to a stage of a conjugation process, the at least one reaction fluid comprising a first portion and a second portion of a bioconjugate comprising a targeting molecule to be produced; a fluid collection unit in fluid communication with the outlet of the flow reactor and configured to control collection of fluids exiting the outlet of the flow reactor according to a stage of the conjugation process; a sampling and detection unit in fluid communication with the outlet of the flow reactor, while continuously passing the at least one reaction fluid through the flow reactor, the first portion and the second portion undergo a conjugation reaction catalyzed by the ligase to produce a bioconjugate comprising a targeting molecule; and The sampling detection unit includes: Collecting a sample fluid from the fluid exiting the outlet of the flow reactor according to a preset sampling time; and The device is configured to detect a bioconjugate comprising a targeting molecule in the sample fluid and obtain a detection result indicating whether the bioconjugate comprising the targeting molecule meets a predefined criterion.

2. The at least one reactant fluid includes a first reactant fluid and a second reactant fluid, and the first reactant fluid 10. The conjugation device of claim 1, wherein a fluid contains the first portion and the second reaction fluid contains the second portion.

3. The conjugation process includes the steps of pre-reaction equilibration, conjugation reaction, post-reaction, and post-reaction washing in this order, and the fluid transport unit further comprises: continuously supplying a buffer solution to the flow reactor during the pre-reaction equilibration, post-reaction, and post-reaction flushing steps; and 3. The conjugation apparatus of claim 2 configured to continuously and simultaneously supply the first and second reactant fluids to the flow reactor during a conjugation reaction.

4. the buffer solution, the first reaction fluid, and the second reaction fluid are stored in a first container, a second container, and a third container, respectively; the fluid transport unit includes a first transport pump and a second transport pump, the first container and the second container are connected to the first transport pump via a first container outlet pipe and a second container outlet pipe, and the third container is connected to the second transport pump via a third container outlet pipe; The first transport pump and the second transport pump are connected to an inlet main pipe via a first inlet branch pipe and a second inlet branch pipe, respectively, and the inlet main pipe is connected to the inlet of the flow reactor; During the pre-reaction equilibration, post-reaction, and post-reaction washout stages, the buffer solution in the first container is pumped into the main inlet pipe by the first transfer pump; and 4. The conjugation apparatus of claim 3, wherein during the conjugation reaction, the first reaction fluid in the second container is pumped into the main inlet pipe by the first transport pump, and the second reaction fluid in the third container is pumped into the main inlet pipe by the second transport pump.

5. the fluid transport unit further includes a first valve, a second valve, a third valve, and a fourth valve; the first valve, the second valve, and the third valve are disposed on the first container outlet pipe, the second container outlet pipe, and the third container outlet pipe, respectively, for controlling a fluid flow path within the first container outlet pipe, the second container outlet pipe, and the third container outlet pipe, respectively; and 5. The conjugation device of claim 4, wherein the fourth valve is disposed on the first inlet branch for controlling a fluid flow path within the first inlet branch.

6. During the pre-reaction equilibration, post-reaction, and post-reaction flushing stages, the first valve and the fourth valve are open, and the second valve and the third valve are closed; and The conjugation apparatus according to claim 5 , wherein the first valve is closed and the second valve, the third valve, and the fourth valve are open during the conjugation reaction.

7. the first vessel outlet pipe, the second vessel outlet pipe, the third vessel outlet pipe, the first inlet branch pipe, the second inlet branch pipe, and the main inlet pipe are disposable or non-disposable and are each fabricated from one of stainless steel, titanium, and silicone; and 5. The conjugation device of claim 4, wherein the first container, the second container, and the third container are each selected from one of a disposable liquid storage bag, a disposable liquid storage bottle, a stainless steel container, and a disposable and non-disposable glass or plastic container.

8. The fluid collection unit further comprises: collecting fluids exiting the outlet of at least one of the flow reactors during the pre-reaction equilibration and post-reaction flushing stages in a fourth vessel; and 4. The conjugation apparatus of claim 3, configured to collect fluids exiting the outlet of at least one of the flow reactors during the conjugation reaction and post-reaction stages in a fifth vessel.

9. the fourth vessel and the fifth vessel are connected to a main outlet pipe via a fourth vessel inlet pipe and a fifth vessel inlet pipe, respectively, and the main outlet pipe is connected to the outlet of at least one of the flow reactors; and 9. The conjugation device of claim 8, wherein the fluid collection unit includes a fifth valve and a sixth valve disposed on the fourth container inlet pipe and the fifth container inlet pipe, respectively, for controlling the flow path of fluid within the fourth container inlet pipe and the fifth container inlet pipe.

10. During the pre-reaction equilibration and post-reaction flushing steps, the fifth valve is open and the sixth valve is closed; and The conjugation device according to claim 9 , wherein the fifth valve is closed and the sixth valve is open during the conjugation reaction and post-reaction stages.

11. the fourth vessel inlet pipe, the fifth vessel inlet pipe, and the main outlet pipe are disposable or non-disposable and are each fabricated from one of stainless steel, titanium, and silicone; and 10. The conjugation device of claim 9, wherein the fourth container and the fifth container are each selected from one of a disposable liquid storage bag, a disposable liquid storage bottle, a stainless steel container, and a disposable or non-disposable glass or plastic container.

12. The conjugation device of claim 1 , wherein the sampling and detection unit comprises at least one analytical column and a detector.

13. the sampling detection unit includes a sampling pump, a first switching valve, an elution pump, at least one analytical column, and a detector, the sampling pump is connected to the outlet of at least one of the flow reactors through a sampling pipe, a sample loop is disposed on the first switching valve, and the first switching valve can switch between a first state and a second state according to the preset sampling time; and When the first switching valve is in the first state, the sampling pump is in fluid communication with the sample loop, and collects the sample fluid from the fluid flowing out of the outlet of at least one of the flow reactors through the sampling tube and pumps the sample fluid into the sample loop; 2. The conjugation device of claim 1, wherein when the first switching valve is in the second state, the elution pump, the sample loop, the at least one analytical column, and the detector are fluidly connected via a detection tube, and the elution pump pumps an eluent into the detection tube to pass the eluent through the sample loop, thereby passing the sample fluid in the sample loop through one of the at least one analytical column before entering the detector.

14. two analytical columns are arranged, and the sampling and detection unit further includes a second switching valve that can be switched between two states and a washing pump; When the second switching valve is in one of the states, the sample loop and the detector are in fluid communication with one of the two analytical columns, the eluent causes the sample fluid in the sample loop to flow into the one analytical column, and the wash pump is in fluid communication with the other analytical column and pumps a buffer into the other analytical column for equilibration. The conjugation device of claim 13.

15. further comprising a recycle unit disposed between the inlet and the outlet of at least one of the flow reactors; 2. The conjugation device of claim 1, wherein, in the event of a detection result indicating that the bioconjugate does not meet the predefined criteria, the fluid collection unit stops collecting fluid flowing out from the outlet of at least one of the flow reactors, and the recycling unit controls the fluid flowing out from the outlet of at least one of the flow reactors to re-enter the inlet for a reconjugation reaction within the at least one flow reactor.

16. the recycle unit further includes a seventh valve disposed on a recycle pipe connected between the inlet and the outlet of the flow reactor, and a recycle container disposed on the recycle pipe; 16. The conjugation device of claim 15, wherein in the event of a detection result that the bioconjugate does not meet the predefined criteria, the seventh valve is open and fluid flowing out of the outlet of the flow reactor passes through the recycle pipe, the recycle vessel, and then flows into the inlet.

17. 10. The conjugation apparatus of claim 1, wherein the flow reactor is a conjugation column.

18. 2. The conjugation device of claim 1, wherein the first portion comprises one of a ligase acceptor substrate recognition motif and a ligase donor substrate recognition motif, and the second portion comprises the other of the ligase acceptor substrate recognition motif and the ligase donor substrate recognition motif.

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