WO2022085767A1

WO2022085767A1 - Method of producing antibody-drug conjugate

Info

Publication number: WO2022085767A1
Application number: PCT/JP2021/039001
Authority: WO
Inventors: 豊松田; 祐一中原; ブライアンアランメンデルソン
Original assignee: 味の素株式会社
Priority date: 2020-10-22
Filing date: 2021-10-21
Publication date: 2022-04-28

Abstract

The present invention provides a method that can speed up the production of an antibody-drug conjugate (ADC). More particularly, the present invention provides a method of producing an ADC, said method comprising: (1) preparing a first liquid mixture that contains a solution containing a starting antibody having a specific cleavable site and a solution containing a cleaving agent capable of cleaving the specific cleavable site; (2) passing the first liquid mixture through a first reaction channel to give a solution that contains an antibody derivative having a specific reaction site and the cleaving agent; (3) by mixing in a collision type micromixer, preparing a second liquid mixture that contains the antibody derivative, the cleaving agent and a drug; and (4) passing the second liquid mixture through a second reaction channel to give the ADC. In this method, the treatments (1) to (4) are continuously performed in a flow microreactor and the representative diameter ratio of the collision type micromixer to the first reaction channel (collision type micromixer/first reaction channel) is not more than 0.95.

Description

Method for producing antibody drug conjugate

The present invention relates to a method for producing an antibody drug conjugate.

In the reaction of multiple molecules in a liquid phase system, different solutions containing different molecules are mixed before the reaction. The mixing time required to obtain a uniform solution depends on the diffusion distance of the molecules in the solution. Mixing in the batch method (eg, mixing with a stir bar in a flask) requires at least a few seconds to obtain a uniform solution by mixing different solutions due to the large diffusion distance of the molecules. Therefore, in the batch method, it is difficult to react a plurality of molecules in a short time such as less than a second order because it takes time to mix. In order to react multiple molecules in a short time, rapid mixing of multiple components is required.

The flow microreactor (FMR) is a flow type reactor that reacts in a microchannel, which is also simply called a microreactor. FMR is mainly used for organic synthesis of small molecule organic compounds. In FMR, a micromixer having a microstructure is utilized in order to enable mixing and reaction of a plurality of components in a microenvironment.

Mixing of solutions by a micromixer in FMR is mixing in a microchannel, and the diffusion distance of molecules is small. Therefore, in FMR, a uniform solution can be obtained quickly by speeding up mixing with a micromixer, and a plurality of components can be reacted in a short time such as less than a second order.

Various mixed types can be used in the micromixer in FMR. Examples of such a mixed type include a sheath flow type in which a plurality of solutions flowing in the forward direction merge, a static type in which mixing is promoted by a structure in the flow path after the merging of a plurality of solutions, and a three-dimensional spiral. Examples thereof include various mixed type micromixers such as a Helix type in which mixing is performed by formation, and a multi-layer flow type in which a plurality of channels are merged so that a plurality of solutions alternately flow at short intervals. The micromixer has characteristics according to its mixed type. For example, in the sheath flow type, typically, by inserting the tube of the second solution into the tube through which the first solution flows, the first and second solutions flowing in the forward direction merge. The sheath flow type having such characteristics is suitable for mixing a plurality of solutions having greatly different flow rates, but the mixing rate is not high. The Static type tends to avoid problems such as blockage of the flow path, but the degree of mixing of a plurality of solutions at the time of contact is not high.

Recently, FMR has been used not only for the synthesis of small molecule organic compounds but also for the synthesis of antibody drug conjugates (ADCs).

For example, Patent Document 1 describes a method for synthesizing an ADC, which comprises the following treatment, which comprises using an inhibitor against a reducing agent for the purpose of controlling the value of the drug-antibody ratio (DAR) of the ADC. Has been (see example):
(1) Partial reduction of IgG antibody by mixing IgG antibody and reducing agent (antibody derivatization reaction); and (2) (A) (a1) reducing agent and (a2) partial reduction. By mixing a solution containing an IgG antibody reduced to (a3) and a linker containing a drug (a3) and a solution containing an inhibitor against the reducing agent (B), the antibody and the drug are linked via the linker. That (conjugation reaction).
For the above purposes, in the examples of Patent Document 1, the batch method (Examples 1 and 6), the microreactor method (Examples 2, 3, 5, 7 to 9), and the analysis of DAT (Example 4) are performed. It is done. Regarding the mixed type of components in the microreactor method in Patent Document 1, the sheath flow described in Patent Document 2 describes that the flow path of the first raw material having a high flow rate is branched into a plurality of channels and then merges with the flow path of the second raw material having a low flow rate. In addition to the mold (Examples 2, 3, 5, 7, 9), the Patent mold (Example 8) adopted in the YMC microreactor Spica is used. The reactions include a reduction reaction at 35 ° C. for 15 minutes (antibody derivatization reaction), a conjugation reaction at 35 ° C. for 15 minutes (Examples 2 and 3), and a reduction reaction at room temperature for 10 seconds to 2 minutes (antibody reaction). Since the derivatization reaction) and the conjugation reaction (Examples 5 and 7 to 9) are carried out at room temperature for 20 minutes to 35 minutes, it takes 20 minutes or more to synthesize the ADC.

Patent Document 3 is characterized by using single-pass tangential flow filtration (SPTFF) for ADC enrichment and removal of unreacted products (particularly unreacted drugs). A method for preparing an ADC composition comprising continuously performing the following treatments is described:
(1) Derivatizing a drug so that it can react with an amino group in the side chain of a lysine residue in an antibody (drug derivatization reaction);
(2) A drug derivatized so as to be capable of reacting with an amino group in the lysine residue side chain in the antibody is reacted with the antibody via ADC (amino group in the lysine residue side chain in the antibody). Generating an antibody-drug conjugate (conjugation reaction); and (3) purifying the ADC by single-pass tangential flow filtration (SPTFF).
In the examples of Patent Document 3, when the above method is performed by the microreactor method, the conjugation reaction requires a long time (at least about 1 hour or more).

Patent Document 4 and Non-Patent Document 1 describe a method for synthesizing an ADC, which comprises performing the following processing using a microreactor (see Examples):
(1) A drug derivatized in advance so as to be able to react with an amino group in the lysine residue side chain in the antibody is reacted with the antibody via an ADC (amino group in the lysine residue side chain in the antibody). (Amino acid and drug conjugated) (drug derivatization reaction).

International Publication No. 2020/07518 International Publication No. 2017/179353 U.S. Patent Application Publication No. 2019/0270769 International Publication No. 2019/016067

An object of the present invention is a method capable of accelerating the production of ADC in a method for producing a drug antibody complex (ADC) including (a) antibody derivatization reaction and (b) antibody-drug conjugation reaction using FMR. Is to provide. Further, an object of the present invention is to provide a method for rapidly producing an ADC having a good DAT in the method for producing an ADC including the above two reactions.

As a result of diligent studies, the present inventors selected a cleavage reaction as the antibody derivatization reaction, and then obtained (a') an antibody cleavage reaction (antibody derivatization reaction) and (b') the cleavage reaction. It has been found that a high-quality ADC can be rapidly produced by carrying out a specific ADC production method including an antibody derivative having a specific reactive site and a drug reaction (conjugation reaction) in a specific FMR format. That is, an antibody derivative having a specific reactive site obtained after mixing and reacting a solution containing a raw material antibody having a specific cleaving site and a solution containing a cleaving agent having a cleaving ability of a specific cleaving site. Can be rapidly mixed and reacted with a solution containing a drug in a collision-type micromixer to rapidly produce an ADC having a good DAT. The collision type micromixer is a micromixer that promotes mixing by generating a mixing vortex at the time of contact of a plurality of solutions. The present inventors also found that, according to the method as described above, the cleavage reaction can be carried out at a relatively high temperature, so that the production of the ADC can be further accelerated, and the ADC with a higher DAR can be produced. I found it. The present inventors have further found that the generation of unwanted by-products (aggregates and decomposition products) in the production of ADC can be reduced by the above-mentioned method, and have completed the present invention.

That is, the present invention is as follows.
[1] A method for producing an antibody-drug conjugate.
(1) A solution containing a raw material antibody having a specific cleaving site and a solution containing a cutting agent having a cleaving ability of the specific cleaving site are mixed with an arbitrary micromixer, and the raw material antibody and the cleaving site are mixed. Producing a first mixture containing the agent;
(2) An antibody derivative having a specific reactive site and the above by passing the first mixed solution through the first reaction flow path and reacting the raw material antibody and the cleavage agent in the first reaction flow path. It is to generate a solution containing a cleaving agent, wherein the antibody derivative having the specific reactive site is a raw material by cleaving the specific cleaving site with the cleaving agent in the reaction. It is produced from antibodies;
(3) Mixing the antibody derivative, the solution containing the cleavage agent, and the solution containing the drug with a collision-type micromixer to produce a second mixed solution containing the antibody derivative, the cleavage agent, and the drug; (4) The antibody drug conjugate is produced by passing the second mixed solution through the second reaction flow path and reacting the antibody derivative and the drug in the second reaction flow path.
The processes (1) to (4) are continuously performed in the flow microreactor, and the representative diameter ratio between the collision type micromixer and the first reaction flow path (collision type micromixer / first reaction flow path) is determined. A method that is 0.95 or less.
[2] The method of [1], wherein the total value of the flow rate of the solution containing the antibody derivative and the cleavage agent and the flow rate of the solution containing the drug is 2.0 mL / min or more.
[3] The method of [1] or [2], wherein the solution containing the drug is introduced into the collision type micromixer at a flow rate of 0.4 mL / min or more.
[4] The method according to any one of [1] to [3], wherein the collision type micromixer has a representative diameter of 0.8 mm or less.
[5] The method according to any one of [1] to [4], wherein the collision type micromixer is a T-shaped micromixer.
[6] The method according to any one of [1] to [5], wherein the arbitrary micromixer is a collision type micromixer.
[7] The method according to any one of [1] to [6], wherein the reaction in the first reaction flow path is carried out at 30 to 60 ° C.
[8] The method according to any one of [1] to [7], wherein the reaction in the first reaction flow path is carried out at 37 to 50 ° C.
[9] In the second reaction flow path, the additional reaction of the raw material antibody and the cleavage agent, and the reaction of the antibody derivative and the drug produced by the additional reaction proceed in parallel, according to [1] to [8]. Either way.
[10] The method according to any one of [1] to [9], wherein the residence time of the first mixed solution in the first reaction flow path is less than 5 minutes.
[11] The method according to any one of [1] to [9], wherein the residence time of the second mixed solution in the second reaction flow path is less than 15 minutes.
[12] The method according to any one of [1] to [11], wherein the residence time of the second mixed solution in the second reaction flow path is less than 5 minutes.
[13] The flow rate of the solution containing the drug introduced into the collision type micromixer includes the flow rate of the solution containing the raw material antibody introduced into any micromixer, and the cutting agent introduced into any micromixer. The method of any of [1] to [12], which is faster than any of the flow rates of the solution.
[14] The time required from the arrival of the solution containing the raw material antibody and the solution containing the cleavage agent to any micromixer to the passage through the second reaction flow path is less than 20 minutes, [1] to [13]. ] Any method.
[15] The method according to any one of [1] to [14], wherein the specific reactive site is a thiol group.
[16] The method according to any one of [1] to [15], wherein the raw material antibody having the specific cleavage site is an antibody having a disulfide group.
[17] The method according to any one of [1] to [16], wherein the antibody having a disulfide group is an unmodified antibody having a disulfide group.
[18] The method of [16], wherein the raw material antibody is an antibody having four disulfide bonds between chains.
[19] The method according to any one of [1] to [18], wherein the raw material antibody is IgG.
[20] The method of any of [1] to [19], wherein the drug has a maleimide group and / or a disulfide group, or is derivatized to have a maleimide group and / or a disulfide group.
[21] The method according to any one of [1] to [20], wherein the antibody-drug conjugate has a drug-antibody ratio of 2.0 or more.
[22] The method according to any one of [1] to [21], wherein the monomer ratio in the antibody drug conjugate analyzed by size exclusion chromatography is 98% or more.
[23] The method according to any one of [1] to [22], wherein the drug is a drug, a labeling substance, or a stabilizer.

According to the method of the present invention, an ADC having a good DAR can be produced in a short time. Further, according to the method of the present invention, since the derivatization reaction (cutting reaction) of the antibody can be carried out at a relatively high temperature, the production of the ADC can be further accelerated, and the ADC having a higher DAT can be obtained. Can be manufactured. Furthermore, according to the method of the present invention, it is possible to reduce the generation of unwanted by-products (aggregates and decomposition products) in the production of ADC.

FIG. 1 is a diagram showing an example of an FMR configuration that can be used by the method of the present invention. FIG. 2 is a diagram showing the analysis results of an antibody drug conjugate (trastuzumab-MMAE) by reverse phase high performance liquid chromatography (RP-HPLC) (Example 1). FIG. 3 is a diagram showing the results of analysis of an antibody drug conjugate (trastuzumab-DM1) by RP-HPLC (Example 4). FIG. 4 is a diagram showing the results of analysis of an antibody drug conjugate (trastuzumab-MMAF) by RP-HPLC (Example 5). FIG. 5 is a diagram showing the results of analysis of an antibody drug conjugate (trastuzumab-PEG) by RP-HPLC (Example 11). FIG. 6 is a diagram showing the results of analysis of an antibody drug conjugate (trastuzumab-MMAE) by size exclusion chromatography (SEC) (Example 13). FIG. 7 is a diagram showing the results of analysis of an antibody drug conjugate (trastuzumab-MMAE) by SEC (Comparative Example 1).

The present invention provides a method for producing an antibody drug conjugate. The method of the present invention includes the following treatments (1) to (4).
(1) A solution containing a raw material antibody having a specific cleaving site and a solution containing a cutting agent having a cleaving ability of the specific cleaving site are mixed with an arbitrary micromixer, and the raw material antibody and the cleaving site are mixed. Producing a first mixture containing the agent;
(2) An antibody derivative having a specific reactive site and the above by passing the first mixed solution through the first reaction flow path and reacting the raw material antibody and the cleavage agent in the first reaction flow path. It is to generate a solution containing a cleaving agent, wherein the antibody derivative having the specific reactive site is a raw material by cleaving the specific cleaving site with the cleaving agent in the reaction. It is produced from antibodies;
(3) Mixing the antibody derivative, the solution containing the cleavage agent, and the solution containing the drug with a collision-type micromixer to produce a second mixed solution containing the antibody derivative, the cleavage agent, and the drug; (4) The antibody drug conjugate is produced by passing the second mixed solution through the second reaction flow path and reacting the antibody derivative and the drug in the second reaction flow path.
In addition, the above processes (1) to (4) are continuously performed in the flow microreactor (FMR), and the representative diameter ratio between the collision type micromixer and the first reaction flow path (collision type micromixer). / 1st reaction flow path) is 0.95 or less.

Hereinafter, the details of the processes (1) to (4) will be described in order.

Processing (1)
In the treatment of (1), a solution containing a raw material antibody having a specific cleavage site is introduced into the first introduction flow path, and a solution containing a cleavage agent having a cleavage ability of the specific cleavage site is introduced into the second introduction flow. It can be introduced into the path so that both solutions are mixed by an arbitrary micromixer at the confluence of the first introduction flow path and the second introduction flow path. Such mixing produces a first mixed solution containing a solution containing a raw material antibody and a solution containing a cleavage agent. The introduction of the solution into the first and second introduction channels is performed, for example, by sending liquid from the reservoir or passing the liquid from the upstream channel (eg, the confluence of the first upstream channel and the second upstream channel). It can be done by passing liquid from the road). For example, the liquid feeding can be performed using a pump. With regard to liquid flow, for example, by sending liquid from the upstream reservoir to the upstream flow path using a pump, liquid flow from the upstream flow path can be promoted.

The raw material antibody used in the treatment (1) is the reaction of the treatment (2) (that is, the derivative of the antibody) for producing the antibody derivative used in the reaction of the treatment (4) (that is, the conjugation reaction of the antibody and the drug). It is an antibody used for (derivatization reaction). Therefore, the raw material antibody is not particularly limited as long as it is an antibody used for such a derivatization reaction, and may be an unmodified antibody or a modified antibody. When the raw material antibody is an unmodified antibody, a solution containing the unmodified antibody can be introduced from the reservoir into the first introduction channel. When the raw material antibody is a modified antibody, a solution containing the modified antibody may be introduced from the reservoir into the first introduction channel, or a modified antibody production system existing upstream of the first introduction channel (eg, non-modified antibody). A first upstream introduction channel for introducing a modified antibody, a second upstream introduction channel containing a modification reagent for an unmodified antibody, a micromixer at the confluence of these channels, and an unmodified antibody and a modifying reagent. A solution containing the modified antibody may be introduced from the outflow channel of the flow path system including the upstream reaction channel that reacts to generate the modified antibody.

The term "antibody" in the raw material antibody, the antibody derivative produced from the raw material antibody, and the antibody drug conjugate is as follows.

The origin of the antibody is not particularly limited, and may be derived from an animal such as a mammal, a bird (eg, a chicken), for example. Preferably, the immunoglobulin unit is derived from a mammal. Such mammals include, for example, primates (eg, humans, monkeys, chimpanzees), rodents (eg, mice, rats, guinea pigs, hamsters, rabbits), pet animals (eg, dogs, cats), domestic animals. (Eg, cows, pigs, goats), servants (eg, horses, sheep), preferably primates or rodents, more preferably humans.

The type of antibody may be polyclonal antibody or monoclonal antibody. The antibody may also be a divalent antibody (eg, IgG, IgD, IgE) or a tetravalent or higher antibody (eg, IgA antibody, IgM antibody). Preferably, the antibody is a monoclonal antibody. The monoclonal antibody is modified to have, for example, a chimeric antibody, a humanized antibody, a human antibody, or an antibody to which a predetermined sugar chain is added (eg, a sugar chain binding consensus sequence such as an N-type sugar chain binding consensus sequence). Antibodies), bispecific antibodies, Fc region proteins, Fc fusion proteins. Isotypes of monoclonal antibodies include, for example, IgG (eg, IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD, IgE, and IgY. In the present invention, a full-length antibody or an antibody fragment containing a variable region and CH1 domain and CH2 domain can be used as the monoclonal antibody, but a full-length antibody is preferable. The antibody is preferably an IgG monoclonal antibody, more preferably an IgG full-length monoclonal antibody.

Any antigen can be used as the antigen of the antibody. For example, such antigens include proteins [oligopeptides, polypeptides. It may be a protein modified with a biomolecule such as sugar (eg, glycoprotein)], sugar chains, nucleic acids, small molecule compounds. Preferably, the antibody may be an antibody that uses a protein as an antigen. Examples of proteins include cell membrane receptors, cell membrane proteins other than cell membrane receptors (eg, extracellular matrix proteins, channel proteins, transporter proteins), ligands, and soluble receptors.

More specifically, the protein that is the antigen of the antibody may be a disease target protein. Examples of disease target proteins include the following.

(1) Oncology area PD-L1, GD2, PDGFRα (platelet-derived growth factor receptor), CD22, HER2, phosphatidylserine (PS), EpCAM, fibronectin, PD-1, VEGFR-2, CD33, HGF, gpNMB, CD27, DEC-205, Folic Acid Receptor, CD37, CD19, Trop2, CEACAM5, S1P, HER3, IGF-1R, DLL4, TNT-1 / B, CPAAs, PSMA, CD20, CD105 (Endoglin), ICAM-1, CD30, CD16A, CD38, MUC1, EGFR, KIR2DL1,2, NKG2A, tenascin-C, IGF (Insulin-like growth factor), CTLA-4, meshothelin, CD138, c-Met, Ang2, VEGF-A, CD79 ENPD3, Folic Acid Receptor α, TEM-1, GM2, Glypican 3, macrophage inhibitory factor, CD74, Notch1, Notch2, Notch3, CD37, TLR-2, CD3, CSF-1R, FGFR2b, HLA-DR, GM-CSF EphA3, B7-H3, CD123, gpA33, Frizzled7 receptor, DLL4, VEGF, RSPO, LIV-1, SLITRK6, Nectin-4, CD70, CD40, CD19, SEMA4D (CD100), CD25, MET, Tissue Factor, IL- 8, EGFR, cMet, KIR3DL2, Bst1 (CD157), P-cadherin, CEA, GITR, TAM (tumor associated macropage), CEA, DLL4, Ang2, CD73, FGFR2, CXCR4, LAG-3, GI -1, Angioceptin 2, CSF-1R, FGFR3, OX40, BCMA, ErbB3, CD137 (4-1BB), PTK7, EFNA4, FAP, DR5, CEA, Ly6E, CA6, CEACAM5, LAMP1, this B7-H3, FGFR4, FGFR2, α2-PI, A33, GDF15, CAIX, CD166, ROR1, GITR, BCMA, TBA, LAG-3, EphA2, TIM-3, CD-200, EGFRvIII, CD16A, CD32B, PIGF, Axl, MICA / B, Thomasen-Friendenrich, CD39, CD37, CD73, CLEC12A, Lgr3, transferrin receptor, TGFβ, IL-17, 5T4, RTK, Immune Suppressor Protein, NaPi2b, Lewis blood group B antigen, A34, Lysyl-Oxid -1, TROP-2, α9 integrin, TAG-72 (CA72-4), CD70

(2) Autoimmune diseases / inflammatory diseases IL-17, IL-6R, IL-17R, INF-α, IL-5R, IL-13, IL-23, IL-6, ActRIIB, β7-Integrin, IL- 4αR, HAS, Eotaxin-1, CD3, CD19, TNF-α, IL-15, CD3ε, Fibronectin, IL-1β, IL-1α, IL-17, TSLP (Thymic stromal Integrin), LAMP (Alpha4 Integrin) , IL-23, GM-CSFR, TSLP, CD28, CD40, TLR-3, BAFF-R, MAdCAM, IL-31R, IL-33, CD74, CD32B, CD79B, IgE (immunoglobulin E), IL-17A, IL-17F, C5, FcRn, CD28, TLR4, MCAM, B7RP1, CXCR1, 2 Lands, IL-21, Cadherin-11, CX3CL1, CCL20, IL-36R, IL-10R, CD86, TNF-α, IL-7R , Kv1.3, α9 integrin, LIFT

(3) Neurological disorders CGRP, CD20, β-amyloid, β-amyloid protofibrin, Calcitonin Gene-Related Peptide Receptor, LINGO (Ig Domain Contining 1), α-synuclein, α-synuclein, extracellular tau, CD52, insulin receptor T, , SOD1, TauC3, JC virus

(4) Infectious diseases Clostridium difficile toxin B, cytomegalovirus, RS virus, LPS, S. Aureus Alpha-toxin, M2e protein, Psl, PcrV, S. et al. Aureus toxin, influenza A, Alginate, Staphylococcus aureus, PD-L1, influenza B, Acinetobacter, F-protein, Env, CD3, pathogenic Escherichia coli, Klebsiella, Streptococcus pneumoniae

(5) Hereditary and rare diseases Amyloid AL, SEMA4D (CD100), insulin receptor, ANGPTL3, IL4, IL13, FGF23, corticostimulatory hormone, transthyretin, huntingtin

(6) Eye diseases Factor D, IGF-1R, PGDFR, Ang2, VEGF-A, CD-105 (Endoglin), IGF-1R, β-amyloid

(7) Bone and Orthopedic Surgery Area Sklerostin, Myostatin, Dickkopf-1, GDF8, RNAKL, HAS, Siglec-15

(8) Blood diseases vWF, Factor IXa, Factor X, IFNγ, C5, BMP-6, Ferroportin, TFPI

(9) Other diseases BAFF (B cell activating factor), IL-1β, PCSK9, NGF, CD45, TLR-2, GLP-1, TNFR1, C5, CD40, LPA, prolactin receptor, VEGFR-1, CB1, Endoglin, PTH1R, CXCL1, CXCL8, IL-1β, AT2-R, IAPP

Specific examples of monoclonal antibodies include specific chimeric antibodies (eg, rituximab, baciliximab, infliximab, cetuximab, siltuximab, dinutuximab, altertoximab), specific humanized antibodies (eg, dacrizumab, paribizmab, trastuzumab, trussumab, allenzumab). , Efarizumab, Bebashizumab, Natarizumab (IgG4), Toshirizumab, Ekurizumab (IgG2), Mogamurizumab, Pertsuzumab, Obinutsuzumab, Bedrizumab, Penproridumab (IgG4), Mepolidumab, Erotsumub Human antibodies (eg, adalimumab (IgG1), panitumumab, golimumab, ustequinumab, canaquinumab, ofatumumab, denosmab (IgG2), ipilimumab, berimumab, rapiximab, lambsilmab, nibolumab, dupilumab (IgG4) Cetuximab, Brodalmab (IgG2), Oralatumab) can be mentioned (if the IgG subtype is not mentioned, it indicates IgG1).

In one embodiment, the raw material antibody may be an unmodified antibody. Unmodified antibodies are antibodies that have not been modified by genetic engineering and organic chemistry techniques. Modifications by genetic engineering techniques include, for example, introduction of mutations into the antibody chain gene for modification of the amino acid sequence of the antibody chain. Modifications by organic chemistry techniques include, for example, the introduction of specific cleavage sites for the side chains of amino acid residues present in antibody chains (particularly the constant regions of heavy and / or light chains).

In another embodiment, the source antibody may be a modified antibody. Examples of the modification in the modified antibody include the above-mentioned genetic engineering technique and modification by an organic chemical technique. By introducing a specific cleavage site by such a method, a raw material antibody having a specific cleavage site can be obtained. Amino acid residues in the antibody utilized when introducing a specific cleavage site into the raw material antibody include, for example, lysine residue, tyrosine residue, serine residue, and threonine residue. For example, in human IgG such as human IgG1, the following amino acid residues existing in the heavy chain constant region can be exposed on the antibody surface, and these amino acid residues can be used for introduction of a specific cleavage site (for example). The position of the amino acid residue is according to EU numbering; see http: //www.imgt.org/IMGTScientificCart/Numering/Hu_IGHGnber.html).
(1) Exposed lysine residue CH2 domain (eg, 246th, 248th, 274th, 288th, 290th, 317th, 320th, 322nd)
CH3 domain (eg, 360th, 414th, 439th)
(2) Exposed tyrosine residue CH2 domain (eg, 278th, 296th, 300th)
CH3 domain (eg, 436th)
(3) Exposed serine residue CH2 domain (eg, 254th, 267th, 298th)
CH3 domain (eg, 400th, 415th, 440th)
(4) Exposed threonine residue CH2 domain (eg, positions 256 and 289)
CH3 domain (eg, 335th and 359th)

When the raw material antibody is a modified antibody, the modified antibody may be a regioselectively modified antibody. In such a case, as the antibody derivative, an antibody derivative having a specific reactive site regioselectively is produced. Regioselective modifications of antibodies are well known in the art and can be performed, for example, by organic chemistry or genetic engineering techniques. Alternatively, the modified antibody may be a position-nonselectively modified antibody. In such a case, as the antibody derivative, an antibody derivative having a specific reactive site non-regioselectively is produced.

When the raw material antibody is a modified antibody, the modified antibody may be a modified antibody containing a modified site having a specific cleavable site. The specific cleavage site may be a cleavage site that produces a bioorthogonal functional group on the antibody side by cleavage of the specific cleavage site using a cleavage agent. Bioorthogonal functional groups are those that do not react with biological constituents (eg, amino acids, nucleic acids, lipids, sugars, phosphates) or that react slowly with biological constituents, but with respect to components other than biological constituents. A group that reacts selectively. Bioorthogonal functional groups are well known in the art (eg, Sharpless KB et al., Angelw. Chem. Int. Ed. 40, 2004 (2015); Bertozzi CR et al., See Science 291 and 357 (2001); Bertozzi CR et al., Nature Chemical Biology 1, 13 (2005)). In the present invention, a bioorthogonal functional group containing a thiol group can be utilized. The combination of the cleavable site that generates a bioorthogonal functional group on the antibody side by cleaving with a cleaving agent and the bioorthogonal functional group is, for example, as follows.

Preferably, the cleavage site is a disulfide group or a thioester group capable of producing a thiol group on the antibody side by cleavage using a cleavage agent. When a modified antibody containing a modified site having such a specific cleaving site (preferably a disulfide group or a thioester group) is used, the reaction in the first reaction flow path causes a bioorthogonal functional group (preferably, preferably). An antibody derivative having a thiol group) is produced as an antibody derivative having a specific reaction site. Next, by utilizing a drug having a functional group that specifically reacts with the bioorthogonal functional group in the antibody derivative, the antibody derivative is linked to the drug by the reaction in the second reaction flow path to form an antibody drug complex. Can be generated.

In a particular embodiment, the raw material antibody is an antibody having a disulfide group.

In a preferred embodiment, the antibody having a disulfide group may be a modified antibody containing a modification site having a disulfide group. In this case, the reaction in the first reaction channel produces a modified antibody derivative containing a modified site having a thiol group.

In another preferred embodiment, the antibody having a disulfide group may be an unmodified antibody having a disulfide group. The unmodified antibody has a disulfide group because the heavy chain and the heavy chain / light chain are linked by a disulfide bond. For example, an IgG antibody composed of two heavy chains and two light chains has four disulfide groups because the heavy chains and the heavy and light chains are linked by four disulfide bonds. Have. Therefore, when the raw material antibody is an unmodified antibody having a disulfide group, the reaction in the first reaction flow path produces an unmodified antibody derivative having a thiol group. For example, when all four disulfide bonds are cleaved by the reaction in the first reaction flow path, an unmodified IgG antibody derivative having eight thiol groups is produced. By the way, even if all four interchain disulfide bonds are reduced, the heavy chain and the light chain of the antibody are not dissociated. This is because each chain can maintain the higher-order structure of the antibody by non-covalent bonding. This non-covalent bond also maintains antibody properties (target and binding strength). For example, trastuzumab derkustecan, known as Enhertz®, is a DA = 8 ADC in which all four interchain disulfide bonds have been reduced (cleaved) and then the drug has been attached. Such ADCs are known to act as antigen-specific agents by maintaining their antibody properties.

The concentration of the raw material antibody in the solution is not particularly limited as long as it can sufficiently react with the cleavage agent, and may be, for example, 0.1 to 30 mg / mL. The concentration may be preferably 0.2 mg / mL or more, more preferably 0.3 mg / mL or more, still more preferably 0.4 mg / mL or more, and particularly preferably 0.5 mg / mL or more. The concentration may also be 25 mg / mL or less, 20 mg / mL or less, 18 mg / mL or less, 16 mg / mL or less, or 14 mg / mL or less.

The cleavage agent used in the treatment (1) is a substance having the ability to cleave a specific cleavage site in the raw material antibody. Examples of the cleaving agent include reducing agents (eg, tricarboxyethylphosphine (TCEP), cysteine, dithiothreitol, reduced glutathione, β-mercaptoethanol), acidic substances (eg, inorganic acidic substances such as hydrochloric acid and sulfuric acid, etc.). And organic acidic substances such as acetic acid and citric acid), basic substances (eg, inorganic basic substances such as sodium hydroxide and potassium hydroxide, and organic basic substances such as hydroxylamine and triethylamine), oxidizing agents (eg,) Sodium periodate, oxidized glutathione), and enzymes. Preferably, the cutting agent is a reducing agent.

In a preferred embodiment, the cleavage agent is a reducing agent having the ability to cleave a disulfide bond. Examples of such reducing agents include tricarboxyethylphosphine (TCEP), cysteine, dithiothreitol, reduced glutathione, and β-mercaptoethanol. Preferably, the reducing agent is TCEP. When such a reducing agent is used, the disulfide group is cleaved by the reaction in the first reaction flow path to generate an antibody derivative having a thiol group, and then the thiol group is generated by the reaction in the second reaction flow path. By utilizing a functional group that reacts with, an antibody derivative can be linked to a drug to form an antibody-drug complex.

The concentration of the cleavage agent in the solution is not particularly limited as long as it can sufficiently react with the antibody, and may be, for example, 0.1 to 50 mM. The concentration may be preferably 0.3 mM or more, more preferably 0.5 mM or more, still more preferably 0.8 mM or more, and particularly preferably 1.0 mM or more. The concentration may also be 40 mM or less, 30 mM or less, 20 mM or less, 10 mM or less, or 5 mM or less. The concentration of the cleavage agent may also be defined as an equivalent to the antibody. Therefore, the concentration of the cleavage agent is, for example, 1 to 100 molar equivalents, preferably 1 to 50 molar equivalents (or 2 to 50 molar equivalents), more preferably 1 to 30 molar equivalents (or 3 to 30 molar equivalents), relative to the antibody. Equivalents), even more preferably 1-20 molar equivalents (or 4-20 molar equivalents), particularly preferably 1-15 molar equivalents (or 5-15 molar equivalents).

An aqueous solution can be used as a solution such as a solution containing a raw material antibody and a solution containing a cutting agent. Examples of the aqueous solution include water (eg, distilled water, sterile distilled water, purified water, physiological saline), buffer solution (eg, phosphoric acid aqueous solution, Tris-hydrochloride buffer solution, carbonic acid-dicarbonate buffer solution, boric acid aqueous solution). , Glycin-sodium hydroxide buffer, citrate buffer), but a buffer is preferred. The pH of the solution is, for example, 5.0 to 9.0, preferably 5.5 to 8.5. The aqueous solution may contain other components. Examples of such other components include arbitrary components such as chelating agents and organic solvents (eg, alcohol).

The first and second introduction channels can be designed to be the same or different channels. For example, the length, representative diameter, shape and material of the first and second introduction channels are the confluence of the first introduction channel and the second introduction channel, respectively, for the solution containing the raw material antibody and the solution containing the cutting agent. It is not particularly limited as long as it can be introduced into. The length of the first and second introduction channels is, for example, 0.1 to 10 meters, preferably 0.1 to 5 meters, more preferably 0.2 to 3 meters, still more preferably 0.2 to 3 meters. be. The representative diameters of the first and second introduction channels are, for example, 0.05 to 3.0 mm, preferably 0.10 to 0.75 mm, and more preferably 0.25 to 0.5 mm. The "representative diameter" means the diameter of a circular tube equivalent to the cross section of the flow path. Therefore, when the shape of the flow path cross section has a circular cross section, the representative diameter is the inner diameter. On the other hand, when the shape of the flow path cross section has a non-circular cross section having the same or different width and depth, the representative diameter is the diameter of a circular pipe having a cross section equivalent to the cross section obtained by the product of the width and the depth. Is. The shapes of the first and second introduction channels may be straight or non-straight. Materials of the first and second introduction channels include, for example, metal materials [eg, stainless steel (SUS), Hasteroy®, Inconel], resins [eg, polytetrafluoroethylene (PTFE), polyethersalkane. Examples include phon (PES), polyetheretherketone (PEEK), polydimethylsiloxane (PDMS), tetrafluoroethylene-perfluoroalkyl vinyl ether copolymer (PFA)], and glass.

The flow rates of the solutions in the first introduction channel and the second introduction channel may be the same or different, and may be, for example, 0.05 to 30 mL / min. The flow rate may be preferably 0.1 mL / min or more, more preferably 0.2 mL / min or more, still more preferably 0.4 mL / min or more, and particularly preferably 0.5 mL / min or more. The flow rate is also 20 mL / min or less, 15 mL / min or less, 10 mL / min or less, 8 mL / min or less, 6 mL / min or less, 5 mL / min or less, 4 mL / min or less, 3 mL / min or less, or 2 mL / min or less. There may be. More specifically, the flow rate is preferably 0.1 to 20 mL / min, more preferably 0.2 to 15 mL / min, even more preferably 0.4 to 10 mL / min, and particularly preferably 0.5 to 8 mL. It may be / minute.

Any micromixer can be used at the confluence of the first introduction flow path and the second introduction flow path. Examples of such a micromixer include various mixed type micromixers such as collision type (eg, T-shaped), sheath flow type, Static type, Helix type, and multi-layer flow type. The representative diameter of the micromixer at the confluence of the first introduction flow path and the second introduction flow path is not particularly limited, but is preferably equal to or smaller than the representative diameter of the first introduction flow path and / or the second introduction flow path. Typical diameters of such micromixers are, for example, 1.0 mm or less, 0.9 mm or less, 0.8 mm or less, 0.7 mm or less, 0.6 mm or less, 0.5 mm or less, 0.4 mm or less, 0.3 mm. It may be less than or equal to 0.25 mm or less. The representative diameter of the micromixer may also be 0.05 mm or more, or 0.1 mm or more. The shape of the flow path cross section of the confluence in the micromixer may have a non-circular cross section having the same or different width and depth, or may have a circular cross section. The confluence of the first introduction flow path and the second introduction flow path may be single or a plurality (when there are a plurality of first introduction flow paths and / or a plurality of second introduction flow paths). From the viewpoint of easy design and manufacture of FMR, a single unit is preferable. Materials for the micromixer include, for example, metal materials [eg, stainless steel (SUS), Hasteroy®, Inconel], resins [eg, polytetrafluoroethylene (PTFE), polyethersulfone (PES), poly. Ether ether ketone (PEEK), polydimethylsiloxane (PDMS), tetrafluoroethylene-perfluoroalkyl vinyl ether copolymer (PFA)], and glass.

Preferably, the micromixer used at the confluence of the first introduction flow path and the second introduction flow path is a collision type micromixer used at the confluence of the first reaction flow path and the third introduction flow path, which will be described later. It may be the same or different collision type micromixer.

In a particular embodiment, the representative diameter ratio of the micromixer to the first introduction channel (micromixer / first introduction channel) and / or the micromixer / first at the confluence of the first introduction channel and the second introduction channel. 2 The representative diameter ratio of the introduction flow path (micromixer / second introduction flow path) may be less than 1.0. According to such a representative diameter ratio, the solution is accelerated at the confluence to generate finer solution units, so that a uniform solution can be produced more quickly. Such representative diameter ratios are, for example, 0.95 or less, 0.90 or less, 0.85 or less, 0.80 or less, 0.75 or less, 0.70 or less, 0.65 or less, 0.60 or less, It may be 0.55 or less, 0.50 or less, 0.45 or less, 0.40 or less, 0.35 or less, 0.30 or less, or 0.25 or less. The smaller the representative diameter ratio, the faster the uniform solution can be produced. Such a representative diameter ratio may also be 0.05 or more, or 0.1 or more.

In a specific embodiment, the micromixer used at the confluence of the first introduction flow path and the second introduction flow path may have a representative diameter of 0.8 mm or less. The representative diameter of the micromixer is preferably 0.7 mm or less, more preferably 0.6 mm or less, still more preferably 0.5 mm or less, particularly preferably 0.4 mm or less, 0.3 mm or less, or 0.25 mm or less. There may be. The representative diameter of the micromixer may also be 0.05 mm or more, or 0.1 mm or more.

Processing (2)
When the first mixed solution produced as described above is passed through the first reaction flow path, the raw material antibody having a specific cleavage site and the cleavage agent having a cleavage ability of the specific cleavage site become the first. 1 React in the reaction flow path. This produces a solution containing an antibody derivative and a cleavage agent having a specific reactive site.

Since the first reaction flow path controls the reaction time in the reaction between the raw material antibody and the cleavage agent, the first reaction flow path can be designed so as to achieve the desired residence time of the first mixed solution in the first reaction flow path. Such residence time is not particularly limited, but may be, for example, less than 15 minutes, preferably less than 10 minutes, more preferably less than 8 minutes, and even more preferably less than 6 minutes. The residence time of the first mixed solution in the first reaction flow path adjusts, for example, the flow rate of the solutions in the first introduction flow path and the second introduction flow path, and the length and representative diameter of the first reaction flow path. It can be controlled by this.

Since the reaction between the raw material antibody and the cleavage agent can be shortened by setting the reaction temperature in the first reaction flow path to a relatively high temperature, the first reaction flow path is designed to achieve a shorter residence time. You may. Such a short residence time is, for example, less than 5 minutes, preferably less than 4 minutes, more preferably less than 3 minutes.

The reaction temperature in the first reaction flow path can be easily controlled. In FMR, which has a large surface area per unit volume, heat transfer occurs at high speed, so that the temperature can be controlled precisely and quickly. Controlling the reaction temperature is described, for example, by using a temperature controller mounted outside the reaction flow path, or by using a bath (eg, a water bath) capable of immersing the reaction flow path, or by a pre-temperature control mechanism (eg, Examples). It can be done by using a coil retention tube). The reaction in the first reaction flow path can be carried out at an arbitrary reaction temperature under mild conditions described later. Alternatively, the reaction temperature may be set to a relatively high temperature in order to shorten the reaction time. Therefore, the reaction temperature may be, for example, 30 to 60 ° C, preferably 35 to 55 ° C, and more preferably 37 to 50 ° C.

The first reaction flow path is not particularly limited as long as it is configured so as to achieve the residence time of the first mixed solution as described above, and for example, the length, representative diameter, shape and material of the first reaction flow path may be used. , May be set as follows. The length of the first reaction flow path may be, for example, 1 to 30 meters, preferably 2 to 20 meters, more preferably 3 to 15 meters, and even more preferably 4 to 10 meters. The representative diameter of the first reaction flow path is, for example, 0.5 to 3 mm, preferably 0.6 to 2.5 mm, more preferably 0.7 to 2.0 mm, and even more preferably 0.8 to 1.5 mm. There may be. The shape of the first reaction flow path may be a straight line or a non-straight line [eg, a shape having one or more curved portions and a straight portion, a circular shape (eg, a coil shape, a spiral shape)]. As the material of the first reaction flow path, the same materials as those of the first and second introduction flow paths can be used.

The reaction in the first reaction channel can be carried out under mild conditions that cannot cause denaturation / degradation of the antibody (eg, cleavage of the amide bond) (eg, GJL Bernardes et al., Chem. Rev., 115, 2174 (2015); GG Bernardes et al., Chem. Asian.J., 4,630 (2009); BG Devices et al., Nat. Commun. , 5, 4740 (2014); see A. Wagner et al., Bioconjugate. Chem., 25, 825 (2014)). The reaction can be complete or partial. The degree of reaction can be controlled, for example, by adjusting conditions such as the concentrations of the raw material antibody and the cleavage agent, the residence time of the first mixed solution in the first reaction flow path, and the reaction temperature in the first reaction flow path. can.

Processing (3)
In the treatment of (3), the outflow solution from the first reaction flow path is mixed with the solution containing the drug introduced through the third introduction flow path and the confluence of the first reaction flow path and the third introduction flow path. This can be done by mixing with a collision type micromixer. Such mixing produces a second mixture containing the antibody derivative, cleavage agent and drug.

The drug used in the present invention is not particularly limited as long as it is a substance that imparts an arbitrary function to the antibody, and examples thereof include pharmaceuticals, labeling substances, and stabilizers. The drug may also be a single drug or a substance in which two or more drugs are linked.

The medicine may be a medicine for any disease. Such diseases include, for example, cancer (eg, lung cancer, gastric cancer, colon cancer, pancreatic cancer, kidney cancer, liver cancer, thyroid cancer, prostate cancer, bladder cancer, ovarian cancer, uterine cancer, bone cancer, skin cancer, etc. Brain tumor, melanoma), autoimmune and inflammatory diseases (eg, allergic disease, rheumatoid arthritis, systemic erythematosus), neurological diseases (eg, cerebral infarction, Alzheimer's disease, Parkinson's disease, muscular atrophic lateral sclerosis), Infectious diseases (eg, bacterial infections, viral infections), hereditary and rare diseases (eg, hereditary globular erythema, non-dystrophy myotension), eye diseases (eg, age-related luteal degeneration, diabetic retinopathy, etc.) Retinal pigment degeneration), diseases in the field of bone and orthopedic surgery (eg, osteoarthritis), blood diseases (eg, leukemia, purpura), and other diseases (eg, diabetes, hyperlipidemia, etc.) , Liver disease, kidney disease, lung disease, cardiovascular disease, digestive system disease). The medicine may be a preventive or therapeutic drug for a disease, or a palliative drug for side effects.

More specifically, the drug is an anticancer drug. Anti-cancer agents include, for example, chemotherapeutic agents, toxins, radioisotopes or substances containing them. Examples of the chemotherapeutic agent include DNA damaging agents, metabolic antagonists, enzyme inhibitors, DNA intercalating agents, DNA cleavage agents, topoisomerase inhibitors, DNA binding inhibitors, tubularin binding inhibitors, cytotoxic nucleosides, and the like. Examples include platinum compounds. Examples of toxins include bacterial toxins (eg, diphtheria toxins) and plant toxins (eg, ricin). Radioisotopes include, for example, radioisotopes of hydrogen atoms (eg, ^3H ), radioisotopes of carbon atoms (eg, ^14C ), radioisotopes of phosphorus atoms (eg, ^32P ), and sulfur atoms. Radioisotopes (eg, 35 ^S ), yttrium radioisotopes (eg ⁹⁰ Y), technetium radioisotopes (eg ^99m Tc), indium radioisotopes (eg ¹¹¹ In), iodine atom radioactivity Isotopes (eg ¹²³ I, ¹²⁵ I, ¹²⁹ I, ¹³¹ I), samarium radioisotopes (eg ¹⁵³ Sm), renium radioisotopes (eg ¹⁸⁶ Re), asstatin radioisotopes (eg 156 Re). ²¹¹ At), a radioisotope of bismuth (eg, ²¹² Bi). More specifically, as pharmaceuticals, auristatin (MMAE, MMAF), maytancin (DM1, DM4), PBD (pyrrolobenzodiazepine), IGN, camptothecin analog, calicheamicin, duocarminine, eribulin, anthracycline, dmDNA31, tubricin. Can be mentioned.

The labeling substance is a substance that enables the detection of targets (eg, tissues, cells, substances). Labeling substances include, for example, enzymes (eg, peroxidase, alkaline phosphatase, luciferase, β-galactosidase), affinity substances (eg, streptavidin, biotin, digoxygenin, aptamer), fluorescent substances (eg, fluorescein, fluorescein isothiocyanate, rhodomin). , Green fluorescent protein, red fluorescent protein), luminescent substances (eg, luciferin, equolin, acridinium ester, tris (2,2'-bipyridyl) ruthenium, luminol), radioactive isotopes (eg, those mentioned above), or Examples include substances containing it.

The stabilizer is a substance that enables the stabilization of the antibody. Stabilizers include, for example, high molecular weight compounds (eg polyethylene glycol (PEG)), diols, glycerin, nonionic surfactants, anionic surfactants, natural surfactants, saccharides, and polyols. Can be mentioned.

Drugs are also peptides, proteins (eg, antibodies), nucleic acids (eg, DNA, RNA, and artificial nucleic acids), small molecule organic compounds (eg, small molecule organic compounds described below), chelators, sugar chains, lipids, polymers. It may be a compound, a metal (eg, gold).

When the drug has a functional group that easily reacts with the bioorthogonal functional group, the functional group of the drug can be appropriately reacted with the bioorthogonal functional group in the antibody derivative. The functional group that easily reacts with the bioorthogonal functional group may also differ depending on the specific type of the bioorthogonal functional group. Those skilled in the art can appropriately select an appropriate functional group as a functional group that easily reacts with a bioorthogonal functional group (eg, Boutureira et al., Chem. Rev., 2015, 115, 2174-2195). ). Examples of the functional group that easily reacts with the bioorthogonal functional group include, but are not limited to, maleimide residues and disulfide residues when the bioorthogonal functional group is a thiol residue.

If the drug does not have a functional group that is likely to react with a bioorthogonal functional group (eg, a thiol group) in the antibody derivative, the drug may be derivatized to have such a functional group. Derivatization is a common technical practice in the art (eg, International Publication No. 2004/010957, US Patent Application Publication No. 2006/0074008, US Patent Application Publication No. 2005/0238649). For example, derivatization may be carried out using any cross-linking agent. Alternatively, the derivatization may be carried out using a specific linker having the desired functional group. For example, such a linker may be one in which the drug and the antibody can be separated by cleavage of the linker in an appropriate environment (eg, intracellular or extracellular). Such linkers include, for example, peptidyl linkers that are degraded by specific proteases [eg, intracellular proteases (eg, proteases present in lysosomes or endosomes), extracellular proteases (eg, secretory proteases)]. For example, US Pat. No. 6,214,345; Dubowchik et al., Pharma. Therapeutics 83: 67-123 (1999), a linker that can be cleaved at a locally acidic site present in vivo (eg, US Pat. No. 5, US Pat. No. 5). , 622,929, 5,122,368; 5,824,805). The linker may be self-immolative (eg, WO 02/083180, WO 04/043493, WO 05/1192919). In the present invention, derivatized drugs are also simply referred to as "drugs".

In certain embodiments, the drug has a maleimide group and / or a disulfide group (preferably a maleimide group) or is derivatized to have a maleimide group and / or a disulfide group (preferably a maleimide group). It is preferable to have.

The concentration of the drug in the solution is not particularly limited as long as it can sufficiently react with the antibody derivative, and may be, for example, 0.1 to 50 mM. The concentration may be preferably 0.15 mM or more, more preferably 0.2 mM or more, still more preferably 0.25 mM or more, and particularly preferably 0.3 mM or more. The concentration may also be 40 mM or less, 30 mM or less, 20 mM or less, 10 mM or less, or 5 mM or less. The concentration of the drug may also be defined as an equivalent relative to the antibody derivative. Therefore, the concentration of the drug is, for example, 1 to 100 molar equivalents, preferably 1 to 50 molar equivalents, more preferably 1 to 30 molar equivalents, even more preferably 1 to 20 molar equivalents, particularly preferably 1 to 20 molar equivalents, relative to the antibody derivative. It may be 1 to 15 molar equivalents.

The introduction of the solution into the third introduction channel can be performed in the same manner as the introduction of the solution into the first and second introduction channels. For example, the length, representative diameter, shape and material of the third introduction channel, and the flow rate of the solution in the third introduction channel may be the same as those of the first and second introduction channels.

In a particular embodiment, the total flow rate of the solution containing the antibody derivative and the cleavage agent and the flow rate of the solution containing the drug may be 2.0 mL / min or more. By flowing the solution having such a total flow rate into the collision type micromixer, strong collision is possible and rapid mixing can be achieved. Such a total value is preferably 2.5 mL / min or more, more preferably 3.0 mL / min or more, even more preferably 3.5 mL / min or more, and particularly preferably 4.0 mL / min or more. good. Such total values may also be 60 mL / min or less, 50 mL / min or less, 40 mL / min or less, 30 mL / min or less, 20 mL / min or less, or 10 mL / min or less. More specifically, such total values are preferably 2.0 to 60 mL / min, more preferably 2.5 to 50 mL / min, even more preferably 3.0 to 40 mL / min, and particularly preferably 4 It may be 0.0 to 30 mL / min, 4.0 to 20 mL / min, or 4.0 to 10 mL / min.

In a particular embodiment, the flow rate of the solution in the first reaction channel may be 0.2 mL / min or higher. The flow rate of the solution in the first reaction flow path can be indirectly adjusted by adjusting the flow rate of the solution in the first introduction flow path and the second introduction flow path. The flow rate of the solution in the first reaction channel is preferably 0.5 mL / min or more, more preferably 1.0 mL / min or more, even more preferably 1.5 mL / min or more, and particularly preferably 2.0 mL / min. It may be the above. Such flow rates may also be 40 mL / min or less, 30 mL / min or less, 20 mL / min or less, or 10 mL / min or less. More specifically, the flow rate is preferably 0.2-40 mL / min, more preferably 0.5-30 mL / min, even more preferably 1.0-20 mL / min, and particularly preferably 1.5-10 mL. It may be / min, or 2.0 mL to 10 mL / min.

In certain embodiments, the flow rate of the solution in the third introduction channel is greater than either (a) the flow rate of the solution in the first introduction channel and (b) the flow rate of the solution in the second introduction channel. It may be fast. By adopting such a flow rate in the third introduction flow path, the solutions collide strongly at the confluence to generate finer solution units, so that a uniform solution can be generated more quickly. The flow rate of the solution into the third introduction flow path is, for example, 1.2 times or more, 1.4 times or more, 1.6 times or more, 1.8 times or more, or the flow rate of (a) or (b). It may be 2.0 times or more. The flow rate of the solution in the third introduction channel may also be 0.4 mL / min or higher (eg 0.4-30 mL / min). The flow rate may be preferably 0.6 mL / min or more, more preferably 0.8 mL / min or more, still more preferably 1.0 mL / min or more, and particularly preferably 1.5 mL / min or more. The flow rate is also 20 mL / min or less, 15 mL / min or less, 10 mL / min or less, 8 mL / min or less, 6 mL / min or less, 5 mL / min or less, 4 mL / min or less, 3 mL / min or less, or 2 mL / min or less. There may be. More specifically, the flow rate is preferably 0.6 to 20 mL / min, more preferably 0.8 to 15 mL / min, even more preferably 1.0 to 10 mL / min, and particularly preferably 1.5 to 8 mL. It may be / minute.

A collision type micromixer is used at the confluence of the first reaction flow path and the third introduction flow path as described above. In the present invention, the collision type micromixer refers to a micromixer that promotes mixing by generating a mixing vortex when a plurality of solutions are in contact with each other. In the present invention, as a collision-type micromixer, a first solution (eg, a solution containing an antibody derivative having a specific reaction site and a cleavage agent) and a second solution (eg, a drug) containing different components to be reacted with each other are used. A micromixer provided with a confluence of multiple solutions from multiple inflow channels, including at least two inflow paths arranged in a positional relationship that allows a mixed vortex to be generated upon contact of the containing solution). Is used. Here, as the positional relationship, the two inflow paths (solution inflow directions) are in a facing position, or a constant angle (angles X and Y, respectively) on the outflow path side with respect to the facing position. The relationship is in a position where it may be tilted (Table B). In the above two inflow paths, the angle X set with respect to the first inflow path (eg, at least one first reaction flow path) is an angle tilted toward the outflow side with respect to the facing position, and is the second angle. The angle Y set with respect to the inflow path (eg, at least one third introduction channel) is an angle tilted toward the outflow path with respect to the facing position. In such a case, two solutions in at least two inflow paths (first inflow path and second inflow path) can collide with each other in a flow completely or substantially opposite to each other, and the collision force is significantly increased. Even if collision is not possible due to a completely opposite flow, strong collision is possible because the collision force does not escape by colliding with the flow path wall (solid phase) in the micromixer. The angles X and Y are the same or different, respectively, within 30 °, preferably within 25 °, more preferably within 20 °, even more preferably within 15 °, and particularly preferably within 10 °. Within 9 °, within 8 °, within 7 °, within 6 °, within 5 °, within 4 °, within 3 °, within 2 °, or within 1 °. The smaller the angle, the stronger the collision force of the solutions from the two inflow paths, so that a uniform solution can be produced more quickly. Such a collision type micromixer used in the present invention is different from the Static type micromixer in which mixing is promoted by a structure in the flow path after merging a plurality of solutions. In the collision type micromixer used in the present invention, both the first solution and the second solution containing different components to be reacted with each other flow into the micromixer in a forward positional relationship and flow out into the outflow path. Sheath-flow type micromixer (for example, at least one inflow solution flows into the micromixer in the forward direction with the outflow solution and flows out into the outflow path to reduce the collision force). It is different from the sheath flow type micromixer described in Patent Document 2 which is easy to miss. Preferably, the collision-type micromixer comprises two inflow channels (eg, a solution containing an antibody derivative having a specific reaction site and a cleavage agent) through a first solution and a second solution containing different components to be reacted with each other. It is a T-shaped micromixer including a combined flow path in which a first reaction flow path through which a drug is passed and a third introduction flow path through which a solution containing a drug is passed are facing each other, and two inflow paths and one outflow path are orthogonal to each other.

In (a), the microreactor in which the outflow path is flush with the first inflow path and the second inflow path is a T-shaped microreactor.

The representative diameter of the collision type micromixer is not particularly limited, but is preferably equal to or smaller than the representative diameter of the first reaction flow path and / or the third introduction flow path. Typical diameters of such micromixers are, for example, 1.0 mm or less, 0.9 mm or less, 0.8 mm or less, 0.7 mm or less, 0.6 mm or less, 0.5 mm or less, 0.4 mm or less, 0.3 mm. It may be less than or equal to 0.25 mm or less. The representative diameter of the micromixer may also be 0.05 mm or more, or 0.1 mm or more.

The shape of the flow path cross section of the confluence in the collision type micromixer may have a non-circular cross section having the same or different width and depth, or may have a circular cross section. The confluence of the first reaction flow path and the third introduction flow path may be single or plural (when there are a plurality of confluence portions of the first reaction flow path and the third introduction flow path). , Single is preferable from the viewpoint of easy design / manufacturing of FMR. Materials for collision-type micromixers include, for example, metal materials [eg, stainless steel (SUS), Hasteroy®, Inconel], resins [eg, polytetrafluoroethylene (PTFE), polyethersulfone (PES)). , Polyetheretherketone (PEEK), polydimethylsiloxane (PDMS), tetrafluoroethylene-perfluoroalkyl vinyl ether copolymer (PFA)], and glass.

In a particular embodiment, the representative diameter ratio of the collision type micromixer and the first reaction flow path (collision type micromixer / first reaction flow path) and / or at the confluence of the first reaction flow path and the third introduction flow path. The representative diameter ratio (collision type micromixer / second introduction flow path) between the collision type micromixer and the third introduction flow path may be 0.95 or less. With such a representative diameter ratio, the solution is significantly accelerated at the confluence to produce smaller solution units, so that a uniform solution can be produced very quickly. Such a representative diameter ratio is preferably 0.90 or less, more preferably 0.85 or less, still more preferably 0.80 or less, particularly preferably 0.75 or less, 0.70 or less, 0.65 or less, It may be 0.60 or less, 0.55 or less, 0.50 or less, 0.45 or less, 0.40 or less, 0.35 or less, 0.30 or less, or 0.25 or less. The smaller the representative diameter ratio, the faster the uniform solution can be produced. Such a representative diameter ratio may also be 0.05 or more, or 0.1 or more.

In a specific embodiment, the collision type micromixer preferably has a representative diameter of 0.8 mm or less among the above-mentioned representative diameters. By using a collision type micromixer having such a narrow representative diameter, the solution is further accelerated at the confluence to generate finer solution units, so that a uniform solution can be produced more quickly.

In certain embodiments, the flow rate of the drug-containing solution introduced into the impact micromixer is faster than either the flow rate of the solution in the first introduction channel and the flow rate of the solution in the second introduction channel. May be. By using a collision type micromixer having such a narrow representative diameter, the solution is further accelerated at the confluence to generate finer solution units, so that a uniform solution can be produced more quickly.

Processing (4)
When the second mixed solution produced as described above is passed through the second reaction flow path, the antibody derivative having a specific reactive site and the drug react in the second reaction flow path. This produces an antibody drug conjugate.

Since the second reaction flow path controls the reaction time in the reaction between the antibody derivative and the drug, the second reaction flow path can be designed so as to achieve the desired residence time of the second mixed solution in the second reaction flow path. Such residence time is not particularly limited, but may be, for example, less than 30 minutes, preferably less than 20 minutes, more preferably less than 15 minutes, still more preferably less than 10 minutes, and particularly preferably less than 5 minutes. Such residence time may also be 30 seconds or longer, preferably 40 seconds or longer, more preferably 50 seconds or longer, and even more preferably 1 minute or longer. The residence time of the second mixed solution in the second reaction flow path adjusts, for example, the flow rate of the solution in the first, second and third introduction flow paths, and the length and representative diameter of the second reaction flow path. It can be controlled by this.

The reaction in the second reaction flow path can be carried out under the above-mentioned mild conditions that cannot cause denaturation / decomposition of the antibody (eg, cleavage of the amide bond). The reaction in the second reaction flow path may be, for example, room temperature, preferably 10 to 30 ° C. The reaction temperature in the second reaction flow path can be easily controlled in the same manner as the reaction temperature in the first reaction flow path.

The second reaction flow path is not particularly limited as long as it is configured so as to achieve the residence time of the second mixed solution as described above. For example, the length, representative diameter, shape and material of the second reaction channel may be set as follows.

The length of the second reaction flow path may be, for example, 1 to 30 meters, preferably 2 to 20 meters, more preferably 3 to 15 meters, and even more preferably 4 to 10 meters. Alternatively, the length of the second reaction flow path may be set so as to adjust the relationship between the residence time in the second reaction flow path and the residence time in the first reaction flow path. For example, the length of the second reaction flow path may be set so that the residence time of the second mixed solution in the second reaction flow path is equal to or less than the residence time of the first mixed solution in the first reaction flow path. can. In this case, the length of the second reaction flow path may be set to be equal to or less than the length of the first reaction flow path (for example, 3/4 length, preferably 1/2 length).

The representative diameter of the second reaction flow path is, for example, 0.5 to 3 mm, preferably 0.6 to 2.5 mm, more preferably 0.7 to 2.0 mm, and even more preferably 0.8 to 1.5 mm. There may be. Alternatively, the representative diameter of the second reaction flow path may be set so as to adjust the relationship between the residence time in the second reaction flow path and the residence time in the first reaction flow path. For example, the representative diameter of the second reaction flow path may be set so that the residence time of the second mixed solution in the second reaction flow path is equal to or less than the residence time of the first mixed solution in the first reaction flow path. can. In this case, the representative diameter of the second reaction flow path may be set to be equal to or less than the representative diameter of the first reaction flow path (for example, a representative diameter of 3/4 or less, preferably a representative diameter of 1/2 or less).

The shape and material of the second reaction flow path are the same as those of the first reaction flow path.

In the second mixed solution in the second reaction flow path, the raw material antibody and the cleavage agent in the first mixed solution are not completely consumed by the reaction in the first reaction flow path, and are described in Patent Document 1. When the inflow path of the inhibitor for the cutting agent is not arranged upstream of the second reaction flow path, the unreacted raw material antibody and the cutting agent can be contained in addition to the antibody derivative and the drug. In this case, not only the reaction between the antibody derivative and the drug but also the reaction between the raw material antibody and the cleavage agent proceed in parallel in the second reaction flow path. That is, an antibody derivative produced by the reaction of the raw material antibody and the cleavage agent, which progresses in parallel, can also be used for the reaction with the drug. Therefore, the raw material antibody and the cleavage agent contained in the first mixed solution in the first reaction flow path do not need to completely react in the first reaction flow path. Therefore, the present invention has the above-mentioned feature that the reaction temperature in the first reaction flow path can be set to a relatively high temperature, and that the raw material antibody and the cleavage agent can be reacted also in the second reaction flow path. Combined with further features, the residence time of the first mixed solution in the first reaction flow path can be set short. Therefore, in a particular embodiment, the present invention is that the raw antibody and cleavage agent in the first mixture are not completely consumed by the reaction in the first reaction flow path, and the inflow path of the inhibitor to the cleavage agent is the first. 2 It may be performed in an FMR that is not located upstream of the reaction flow path (eg, upstream of the confluence of the first reaction flow path and the third introduction path). Alternatively, in another embodiment, the invention is an inhibitor of an inhibitor against a cleavage agent [eg, an inhibitor of a reducing agent (eg, TCEP), to allow further control of the drug-antibody binding ratio of the drug-antibody complex. And pH neutralizers (eg, acidic or basic substances)] may be performed in the FMR located upstream of the second reaction channel (see, eg, Patent Document 1).

In the present invention, the residence time of the first mixed solution in the first reaction flow path can be set short, and the residence time of the second mixed solution in the second reaction flow path can be set to be short in the first reaction flow path. Since the residence time of the first mixed solution can be set to be less than or equal to that of the first mixed solution, the antibody-drug conjugate can be produced in a short time.

In certain embodiments, the time required to produce an antibody-drug conjugate is from the arrival of the solution containing the raw material antibody and the solution containing the cleavage agent to any micromixer to the passage through the second reaction channel. It can be specified by time. In light of the fact that mixing with a micromixer can be performed instantaneously, the time required for producing the antibody-drug conjugate is mainly the residence time of the first mixed solution in the first reaction flow path and the residence time in the second reaction flow path. It can be determined according to the residence time of the second mixed solution of. Thus, in certain embodiments, the time required to produce an antibody-drug conjugate is, for example, less than 20 minutes, preferably less than 15 minutes, less than 14 minutes, less than 13 minutes, less than 12 minutes, less than 11 minutes, less than 10 minutes. , Less than 9 minutes, less than 8 minutes, less than 7 minutes, less than 6 minutes, or less than 5 minutes.

According to the present invention, an antibody drug conjugate having a good drug-antibody ratio (DAR) of 2.0 or more can be produced. The DAT will vary depending on the type of source antibody used in the method of the invention (eg, the number of heavy and light chains). Therefore, in the present invention, DAT can be defined as the number per immunoglobulin unit having two heavy chains and two light chains. The DAT may be preferably 2.5 or more, more preferably 3.0 or more, still more preferably 3.5 or more, and particularly preferably 4.0 or more. The DAT may also be 8.0 or less, 6.0 or less, or 4.0 or less. The DAT can be measured by reverse phase high performance liquid chromatography (RP-HPLC) according to the previous report (Anal. Chem., 2019, 91, 20, 12724-12732). DA is the sum of the number of drug bonds of each peak in RP-HPLC (for example, in the case of IgG, typically DA = 0, 2, 4, 6, 8) and each value obtained by multiplying the peak area. Can be determined by dividing by 100.

According to the present invention, it is possible to reduce the generation of unwanted by-products (aggregates and decomposition products) in the production of antibody-drug conjugates. Therefore, the antibody-drug conjugate produced by the method of the present invention can be defined by its purity. The purity of an antibody-drug conjugate can be evaluated by the ratio of monomers (units containing two light chains and two heavy chains) in the antibody-drug conjugate. The monomer ratio in the antibody-drug conjugate may be, for example, 98% or more, preferably 98.5% or more, more preferably 99% or more, and even more preferably 99.5% or more. In the present invention, the measurement of the monomer ratio in the antibody drug conjugate can be performed by size exclusion chromatography (SEC) according to the previously reported (ACS Omega 2020, 5, 7193-7200).

The antibody drug conjugate produced by the reaction in the second reaction flow path can be appropriately recovered and purified. For example, recovery can be performed in a container (eg, fraction collector) located at the outlet of the second reaction channel. The drug antibody complex may be purified. For example, for purification of the drug-antibody complex, the recovered drug-antibody complex is chromatographed (eg, gel filtration chromatography, ion exchange chromatography, reverse phase column chromatography, high performance liquid chromatography, affinity chromatography) or the like. It can be done by attaching to any method of. Purification of the drug-antibody complex may also be performed continuously in the FMR. For example, in such a case, a purification channel for the antibody-drug conjugate (eg, single-pass tangential flow filtration as described in Patent Document 3) can be arranged downstream of the second reaction channel.

The drug-antibody complex produced in the present invention may be provided in the form of a salt. Examples of such a salt include a salt with an inorganic acid, a salt with an organic acid, a salt with an inorganic base, a salt with an organic base, and a salt with an amino acid. Examples of the salt with an inorganic acid include salts with hydrogen chloride, hydrogen bromide, phosphoric acid, sulfuric acid, and nitric acid. Examples of salts with organic acids include formic acid, acetic acid, trifluoroacetic acid, lactic acid, tartaric acid, fumaric acid, oxalic acid, maleic acid, citric acid, succinic acid, malic acid, benzenesulfonic acid, and p-toluenesulfonic acid. Salt is mentioned. Salts with inorganic bases include, for example, alkali metals (eg, sodium, potassium), alkaline earth metals (eg, calcium, magnesium), and other metals such as zinc, aluminum, and salts with ammonium. .. Examples of the salt with an organic base include salts with trimethylamine, triethylamine, propylenediamine, ethylenediamine, pyridine, ethanolamine, monoalkylethanolamine, dialkylethanolamine, diethanolamine, and triethanolamine. Examples of salts with amino acids include salts with basic amino acids (eg, arginine, histidine, lysine, ornithine) and acidic amino acids (eg, aspartic acid, glutamic acid). The salt is preferably a salt with an inorganic acid (eg, hydrogen chloride) or a salt with an organic acid (eg, trifluoroacetic acid). Of course, the drug-antibody complex produced in the present invention may be provided in a non-salt form.

The present invention will be described in detail with reference to the following examples, but the present invention is not limited to the following examples.

Outline of Flow Microreactor (FMR) FIG. 1 shows an outline of the configuration of the FMR used in the following examples.
In the examples, the flow path, the micromixer, and the reaction tube used had a circular cross section. Therefore, in the following, the expression "inner diameter" is used as the representative diameter.
1) Reservoir First reservoir containing antibody solution (1)
Second reservoir containing reducing agent solution (2)
Third reservoir containing payload solution (3)
2) Flow path from the reservoir to the micromixer The first introduction flow path (4) for introducing the antibody solution, which connects the first reservoir (1) to the first micromixer (M1).
A second introduction flow path (5) for introducing a reducing agent solution, which connects the second reservoir (2) to the first micromixer (M1).
A third introduction channel (6) for the introduction of the payload solution, which connects from the third reservoir (3) to the second micromixer (M2).
3) The first pump (7) provided in the first pump flow path (4).
A second pump (8) provided in the second flow path (5)
A third pump (9) provided in the third flow path (6)
4) Micromixer A first micromixer (M1) for producing a first mixed solution of an antibody solution and a reducing agent solution.
A second micromixer (M2) for producing a second mixture of the first reaction solution and the payload solution.
As the first and second micromixers, a T-shaped micromixer that merges the first flow path and the second flow path facing each other was used.
Table 1 shows the inner diameters and shapes of the flow paths in the first micromixer (M1) and the second micromixer (M2). The inner diameter of the flow path indicates the flow path width of the mixed portion of the two kinds of solutions.
5) Reaction tube The first reaction tube (R1) for reacting the antibody and the reducing agent in the mixed solution of the antibody solution and the reducing agent solution.
A second reaction tube (R2) for reacting the reducing antibody and the payload in the mixed solution of the first reaction solution and the payload solution.
6) Fraction collector Fraction collector (10) that collects the second reaction solution from the second reaction tube.

(Temperature adjustment)
For temperature control, the first reaction tube (R1) and the second reaction tube (R2) were designed so that a coil retention tube for pre-temperature control and a water bath for temperature control could be used as needed.

(Standards for flow paths, micromixers and reaction tubes)
The length of the first, second and third introduction channels is 1.0 m. The inner diameter of the first introduction flow path is 1.0 mm, the inner diameter of the second introduction flow path is 1.0 mm, and the inner diameter of the third introduction flow path is 1.0 mm.
Table 1 shows the inner diameters and shapes of the flow paths in the first micromixer (M1) and the second micromixer (M2).
Table 2 shows the inner diameters and lengths of the flow paths of the first reaction tube (R1) and the second reaction tube (R2).

In the following examples, the FMR apparatus constructed in this manner is used to perform (A) partial reduction of the interchain disulfide bond of the antibody and (B) conjugation of the partially reduced antibody and the payload, and the residence time. The antibody drug conjugate (ADC) was synthesized within 10 minutes. More specifically, the antibody solution and the reducing agent solution are pumped, mixed in the first micromixer, the mixed solution is passed through the first reaction tube, and the payload solution is sent thereto. The ADC was prepared by mixing in a second micromixer, passing the liquid through a second reaction tube, and collecting the solution with a fraction collector. When changing the mixing temperature, the micromixer was immersed in a water bath. Unless otherwise specified, the mixing temperature was set to 25 ° C.

In the following examples, the measurement of DA by RP-HPLC (pretreatment and measurement conditions) was in accordance with the previous report (Anal. Chem., 2019, 91, 20, 12724-12732). More specifically, the conditions are as follows.
(Pretreatment conditions before measurement by RP-HPLC)
To ADC (1 mg / mL), add 8 M guanidine hydrochloride solution, 500 mM Tris buffer (pH 7.5), and 1 M DTT solution to make a 0.6 mg / mL ADC solution. The ADC solution is heated at 80 ° C. for 5 minutes to cleave all disulfide bonds of the antibody.
(Measurement conditions by RP-HPLC)
A 0.1% TFA-containing aqueous solution was used as the eluent A, and a 0.1% TFA-containing acetonitrile solution was used as the eluent B. 20 μL of ADC (0.6 mg / mL) after the above reduction treatment was injected into HPLC and measured. The column used was AdvantageBio RP-mAb Biphenyl (manufactured by Agilent), and the column temperature was 70 ° C. As the gradient condition, the condition of 70% eluent A and 30% eluent B was used, and the condition of increasing the ratio of eluent B from 30% to 48% over 2 minutes and 22 minutes was used. Finally, the column was washed for 3 minutes at a ratio of 95% eluent B.

[Example 1] Synthesis of trastuzumab-MMAE The effect of the conjugation reaction time on the drug-antibody ratio (DAR) of an antibody-drug conjugate (ADC) was investigated.

In the first reservoir (1) of FMR (Fig. 1), anti-HER2 IgG antibody trastuzumab (Chugai Pharmaceutical) 10 mg / mL PBSE buffer (50 mM phosphate buffered saline (PBS), 10 mM ethylenediaminetetraacid (EDTA), pH = 7. ) Was put in. In the second reservoir (2), a PBSE solution of 1.3 mM tris (2-carboxyethyl) phosphate chloride (TCEP; reducing agent) was placed. The payload MC-VC-MMAE (Compound 1) was placed in a third reservoir (3) as a 0.25 mM solution with a conjugation buffer (PBSE: DMA = 9: 1). The antibody solution is introduced at a flow rate of 1.0 mL / min and the TCEP solution is introduced at a flow rate of 1.0 mL / min, mixed with the first micromixer (M1), and reduced in the first reaction tube (R1) for 3 minutes. I let you. Subsequently, the reaction solution flowing in the first reaction tube (R1) at a flow rate of 2.0 mL / min is mixed with the payload solution introduced at a flow rate of 2.0 mL / min with a second micromixer (M2). Then, it was subjected to a conjugation reaction in the second reaction tube (R2). The length of the second reaction tube (R2) and the conjugation reaction time defined by the length of the reaction tube are as shown in Table 3. The solution after the conjugation reaction in the second reaction tube (R2) was collected with a fraction collector pre-added with an excess amount of N-acetylcysteine (NAC). NAC is used to stop the conjugation reaction after collection of fraction collectors in order to accurately evaluate the DAT of the ADC generated by the conjugation reaction in the liquid through the second reaction tube (R2). The ADC DAT contained in the collected fractions was then measured by RP-HPLC. The results are shown in Table 3.

R1 indicates the length of the flow path of the first reaction tube.
R2 indicates the length of the flow path of the second reaction tube.
The reduction reaction time corresponds to the time during which the mixed solution of the antibody and the reducing agent stays in the first reaction tube (R1).
The conjugation reaction time corresponds to the time that the partially reduced antibody and payload mixture stays in the second reaction tube (R2).

The relationship between the observed retention time and the antibody chain was as follows (Table 3, Condition 2).
6.3 minutes: unreacted antibody light chain 9.8 minutes: antibody light chain with one payload (MMAE) 10.4 minutes: unreacted antibody heavy chain 12.3 minutes: antibody heavy chain 15.4 minutes with one payload added: 2 payloads added to the antibody heavy chain 17.8 minutes: 3 payloads added to the antibody heavy chain The calculation of DAT is shown in Example 3 below. I went like that.
From these results, the average DAT of the ADC obtained in the reaction was calculated to be 3.2.

From the above, ADC with a DAT of 3.0 or more could be obtained within a total residence time of 10 minutes by high-speed mixing using a T-shaped micromixer in FMR. Further, as shown in Table 3, by setting a flow path of an appropriate length in the second reaction tube (R2) and adjusting the conjugation reaction time, it is possible to stably obtain an ADC having a DAT of 3.2. did it.

[Example 2] Synthesis of trastuzumab-MMAE under aging conditions Payload for anti-HER2 IgG antibody trastuzumab (Chugai Pharmaceutical Co., Ltd.) using the reaction conditions of FMR (Fig. 1) and Table 3 (Condition 2) of Example 1. MC-VC-MMAE (Compound 1) was reacted. NAC was not added in advance to the fraction collector receiving the reaction solution, and each fraction was aged at room temperature for 30 minutes. After 30 minutes, NAC was added and aged for another 15 minutes, and then DAR was measured by RP-HPLC. As a result, the DAT was calculated to be 3.2.

From the results of Examples 1 and 2, the degree of conjugation remains at DA 3.2 regardless of the progress or cessation of the conjugation reaction depending on the presence or absence of NAC. Therefore, the conjugation reaction is the second reaction tube of FMR. It was shown to be completed in 0.75 minutes, which is the residence time in (R2).

[Example 3] Synthesis of trastuzumab-MMAE The effects of the concentration of the reducing agent and the temperature of the reduction reaction on the DAT of the ADC were investigated.
Anti-HER2 IgG antibody trastuzumab (Chugai Pharmaceutical) 10 mg / mL PBSE buffer (50 mM phosphate buffered saline (PBS), 10 mM EDTA, pH = 7.4) was placed in the first reservoir (1) of FMR (FIG. 1). .. A PBSE solution of a reducing agent (TCEP) was placed in the second reservoir (2). The concentration of the reducing agent was changed as shown in Table 4. The payload MC-VC-MMAE (Compound 1) was placed in a third reservoir (3) as a 0.25 mM solution with a conjugation buffer (PBSE: DMA = 9: 1). The antibody solution was introduced at a flow rate of 1.0 mL / min and the TCEP solution was introduced at a flow rate of 1.0 mL / min, mixed with the first micromixer (M1), and reduced in the first reaction tube (R1). .. The length of the first reaction tube (R1) and the reduction reaction time defined by the length are as shown in Table 4. Subsequently, the reaction solution flowing in the first reaction tube (R1) at a flow rate of 2.0 mL / min is mixed with the payload solution introduced at a flow rate of 2.0 mL / min with a second micromixer (M2). Then, it was subjected to the conjugation reaction for 1.5 minutes in the second reaction tube (R2). The solution after the conjugation reaction in the second reaction tube (R2) was collected with a fraction collector pre-added with an excess amount of N-acetylcysteine (NAC). The ADC DAT contained in the collected fractions was measured by RP-HPLC. The results are shown in Table 4.

The total residence time is the sum of the reduction reaction time (1.5 minutes or 3.0 minutes) and the conjugation reaction time (1.5 minutes).

The RP-HPLC results in Table 4 (Condition 6) are shown in FIG. The relationship between the observed retention time and the antibody chain was as follows.
6.8 minutes: Unreacted antibody light chain 9.7 minutes: Antibody light chain with one payload (MMAE) added 10.4 minutes: Unreacted antibody heavy chain 12.4 minutes: Antibody heavy chain One payload added 15.6 minutes: Two payloads added to the antibody heavy chain 17.7 minutes: Three payloads added to the antibody heavy chain

The calculation of DAT was performed as follows.
For the antibody light chain, the ratio of the area% of HPLC was the unreacted light chain (59.4%) and the antibody light chain with one payload (MMAE) added (40.6%). As a result, it can be calculated as 0 × 0.594 + 1 × 0.406 = 0.406, and the average DA is 0.41.
For antibody heavy chains, the ratio of HPLC area% is unreacted heavy chains (12.5%), antibody heavy chains with one payload (MMAE) added (35.5%), and antibody heavy chains. Two payloads (MMAE) were added to the antibody (23.6%), and three payloads (MMAE) were added to the antibody heavy chain (28.4%). As a result, the average DA can be calculated as 0 × 0.125 + 1 × 0.355 + 2 × 0.236 + 3 × 0.284 = 1.679, and the average DA is 1.68.
The sum of the DAs of the heavy chain and the light chain is 0.41 + 1.68 = 2.09, and when this is converted into the whole IgG antibody (two heavy chains and two light chain molecules), 2.09 × 2 = 4. It becomes 18.
From these results, the average DR of the ADC obtained in the reaction was calculated to be 4.2 (Condition 6).

From the above, it was possible to obtain an ADC having a DA of 2.0 or more by high-speed mixing using a T-shaped micromixer in FMR and control of the reduction reaction conditions of the antibody. Further, by carrying out the reduction reaction at a high temperature (50 ° C.), an ADC having a high DAT (4.2) could be obtained.

[Example 4] Synthesis of trastuzumab-DM1 SMCC which is a payload against anti-HER2 IgG antibody trastuzumab (Chugai Pharmaceutical Co., Ltd.) using the reaction conditions of FMR (FIG. 1) and Table 4 (condition 6) of Example 3. -DM1 (Compound 2) was reacted. After the reaction, DAR was measured by RP-HPLC.

The result of RP-HPLC is shown in FIG. The relationship between the observed retention time and the antibody chain was as follows.
6.5 minutes: unreacted antibody light chain 8.0 minutes: antibody light chain with one payload added 10.2 minutes: unreacted antibody heavy chain 11.1 minutes: antibody heavy chain with one payload 12.5 minutes: 2 payloads added to the antibody heavy chain 14.0 minutes: 3 payloads added to the antibody heavy chain From these results, the DA was calculated to be 3.3. ..

[Example 5] Synthesis of trastuzumab-MMAF MC which is a payload against anti-HER2 IgG antibody trastuzumab (Chugai Pharmaceutical Co., Ltd.) using the reaction conditions of FMR (FIG. 1) and Table 4 (condition 6) of Example 3. -MMAF (Compound 3) was reacted. After the reaction, DAR was measured by RP-HPLC.

The result of RP-HPLC is shown in FIG. The relationship between the observed retention time and the antibody chain was as follows.
6.7 minutes: Unreacted antibody light chain 9.6 minutes: One payload added to the antibody light chain 10.6 minutes: Unreacted antibody heavy chain 12.0 minutes: One payload to the antibody heavy chain 14.6 minutes: 2 payloads added to the antibody heavy chain 17.2 minutes: 3 payloads added to the antibody heavy chain From these results, the DA was calculated to be 3.4. ..

[Example 6] Synthesis of rituximab-MMAE MC which is a payload against the anti-CD20 IgG antibody rituximab (Roche) using the reaction conditions of FMR (FIG. 1) and Table 4 (condition 6) of Example 3. -MMAE (Compound 1) was reacted. After the reaction, DAR was measured by RP-HPLC.

As a result, the relationship between the observed retention time and the antibody chain was as follows.
5.0 minutes: Unreacted antibody light chain 8.8 minutes: One payload added to the antibody light chain 9.3 minutes: Unreacted antibody heavy chain 11.4 minutes: One payload to the antibody heavy chain 14.4 minutes: 2 payloads added to the antibody heavy chain 16.8 minutes: 3 payloads added to the antibody heavy chain From these results, the DA was calculated to be 3.3. ..

[Example 7] Synthesis of rituximab-DM1 MC which is a payload against anti-CD20 IgG antibody rituximab (Roche) using the reaction conditions of FMR (FIG. 1) and Table 4 (condition 6) of Example 3. -DM1 (Compound 2) was reacted. After the reaction, DAR was measured by RP-HPLC.

As a result, the relationship between the observed retention time and the antibody chain was as follows.
4.5 minutes: unreacted antibody light chain 7.2 minutes: antibody light chain with one payload added 9.6 minutes: unreacted antibody heavy chain 10.5 minutes: antibody heavy chain with one payload 12.5 minutes: 2 payloads added to the antibody heavy chain 13.7 minutes: 3 payloads added to the antibody heavy chain From these results, the DA was calculated to be 3.8. ..

[Example 8] Synthesis of rituximab-MMAF MC which is a payload against anti-CD20 IgG antibody rituximab (Roche) using the reaction conditions of FMR (FIG. 1) and Table 4 (condition 6) of Example 3. -MMAF (Compound 3) was reacted. After the reaction, DAR was measured by RP-HPLC.

As a result, the relationship between the observed retention time and the antibody chain was as follows.
5.7 minutes: Unreacted antibody light chain 8.6 minutes: One payload added to the antibody light chain 9.8 minutes: Unreacted antibody heavy chain 11.2 minutes: One payload to the antibody heavy chain 14.3 minutes: antibody heavy chain with two payloads 16.3 minutes: antibody heavy chain with three payloads DAT was calculated to be 3.3 from these results. ..

[Example 9] Synthesis of infliximab-MMAE Using the reaction conditions of FMR (Fig. 1) and Table 4 (Condition 6) of Example 3, the anti-TNF-α IgG antibody rituximab (Centocor) was used as a payload. A certain MC-MMAE (Compound 1) was reacted. After the reaction, DAR was measured by RP-HPLC.

As a result, the relationship between the observed retention time and the antibody chain was as follows.
8.6 minutes: unreacted antibody light chain 10.95 minutes: unreacted antibody heavy chain 10.96 minutes: antibody light chain with one payload added 12.6 minutes: antibody heavy chain with one payload 15.6 minutes: 2 payloads added to the antibody heavy chain 18.1 minutes: 3 payloads added to the antibody heavy chain From these results, the DA was calculated to be 3.7. ..

[Example 10] Synthesis of infliximab-DM1 Using the reaction conditions of FMR (FIG. 1) and Table 4 (condition 6) of Example 3, the anti-TNF-α IgG antibody infliximab (Centocor) was used as a payload. A certain MC-DM1 (Compound 2) was reacted. After the reaction, DAR was measured by RP-HPLC.

As a result, the relationship between the observed retention time and the antibody chain was as follows.
8.2 minutes: unreacted antibody light chain 10.3 minutes: unreacted antibody heavy chain 10.4 minutes: antibody light chain with one payload added 12.2 minutes: antibody heavy chain with one payload 14.2 minutes: 2 payloads added to the antibody heavy chain 16.7 minutes: 3 payloads added to the antibody heavy chain From these results, the DA was calculated to be 3.8. ..

[Example 11] Synthesis of trastuzumab-PEG Using the reaction conditions of FMR (FIG. 1) and Table 4 (condition 6) of Example 3, m-dPEG- was used against the anti-HER2 IgG antibody trastuzumab (Chugai Pharmaceutical Co., Ltd.). Maleimide (Compound 4 manufactured by quanta bodysign) was reacted. After the reaction, the molecular species was measured by Q-TOFMS.

ESI-Q-TOFMS was measured under the reduction conditions according to the previous report (International Publication No. 2019/240288). The analysis result is shown in FIG.

As a result, the relationship between the observed mass peak and the antibody chain was as follows.
23441: Unreacted antibody light chain 24681: One compound 4 introduced into the light chain 50600: Unreacted antibody heavy chain 51840: One compound 4 introduced into the heavy chain 53081: 2 in the heavy chain One with one compound 4 introduced 54315: One with three compounds 4 introduced into the heavy chain

[Example 13] Synthesis of trastuzumab-MMAE using a T-shaped micromixer with a flow path inner diameter of 0.5 mm The first and second micromixers (M1, M2) in the FMR (FIG. 1) are used as a T-shaped micromixer (flow path). The inner diameter was changed to 0.5 mm) and ADC synthesis was performed. Other conditions were in accordance with the reaction conditions in Table 4 (Condition 6) of Example 3. After the reaction, RP-HPLC was measured and the DA was calculated to be 3.1.

[Comparative Example 1] Synthesis of trastuzumab-MMAE using a T-shaped micromixer with a flow path inner diameter of 1 mm The first and second micromixers (M1 and M2) in the FMR (FIG. 1) are T-shaped micromixers (flow path inner diameter 1 mm). ) Was changed to ADC synthesis. In this case, the ratio (collision type micromixer / first reaction flow path) between the representative diameter (1 mm) of the T-shaped micromixer, which is a collision type micromixer, and the representative diameter (1 mm) of the first reaction flow path is 1. be.
The antibody solution was introduced at a flow rate of 1.0 mL / min and the TCEP solution was introduced at a flow rate of 1.0 mL / min, mixed with the first micromixer (M1), and reduced in the first reaction tube (R1). .. The length of the first reaction tube (R1) and the reduction reaction time defined by the length are as shown in Table 5. Subsequently, the reaction solution was mixed with the payload solution introduced at a flow rate of 2.0 mL / min with a second micromixer (M2), and subjected to a conjugation reaction in the second reaction tube (R2) for 1.5 minutes. bottom. The solution after the conjugation reaction in the second reaction tube (R2) was collected by a fraction collector to which an excess amount of NAC was previously added. The ADC DAT contained in the collected fractions was measured by RP-HPLC. The results are shown in Table 5.

The total residence time is the sum of the reduction reaction time (3.0 minutes) and the conjugation reaction time (1.5 minutes).

[Example 13] Monomer ratio analysis of trastuzumab-MMAE For trastuzumab-MMAE synthesized in Table 4 (Condition 6) of Example 3, size exclusion chromatography was performed according to the previous report (ACS Omega 2020, 5, 7193-7200). (SEC) analysis was performed. More specifically, the analysis of the monomer (unit containing two light chains and two heavy chains of IgG) ratio in the antibody drug conjugate by SEC is as follows. AdvantageBio SEC 300 Å (manufactured by Agilent) was used for the column, and 100 mM NaHPO ₄ / NaH ₂ PO ₄ , 250 mM NaCl, 10% v / v isopropanol, pH 6.8 was used as the eluent. 40 μL of ADC sample (1 mg / mL) dissolved in buffer was injected into HPLC and eluted for 15 minutes.
As a result, the monomer ratio exceeded 99% even though the antibody derivatization reaction (disulfide reduction reaction) was carried out at 50 ° C. (FIG. 6).

[Example 14] Monomer ratio analysis of trastuzumab-DM1 The trastuzumab-DM1 synthesized in Example 4 was subjected to size exclusion chromatography (SEC) analysis according to the above-mentioned report. Although the antibody derivatization reaction (disulfide reduction reaction) was carried out at 50 ° C., the monomer ratio exceeded 99%.

[Example 15] Monomer ratio analysis of trastuzumab-MMAF The trastuzumab-MMAF synthesized in Example 5 was subjected to size exclusion chromatography (SEC) analysis according to the above-mentioned report. Although the antibody derivatization reaction (disulfide reduction reaction) was carried out at 50 ° C., the monomer ratio exceeded 99%.

[Example 16] Monomer ratio analysis of rituximab-MMAE The rituximab-MMAE synthesized in Example 6 was subjected to size exclusion chromatography (SEC) analysis according to the above-mentioned report. Although the antibody derivatization reaction (disulfide reduction reaction) was carried out at 50 ° C., the monomer ratio exceeded 98%.

[Example 17] Monomer ratio analysis of rituximab-DM1 The rituximab-DM1 synthesized in Example 7 was subjected to size exclusion chromatography (SEC) analysis according to the above-mentioned report. Although the antibody derivatization reaction (disulfide reduction reaction) was carried out at 50 ° C., the monomer ratio exceeded 99%.

[Example 18] Monomer ratio analysis of rituximab-MMAF The rituximab-MMAF synthesized in Example 8 was subjected to size exclusion chromatography (SEC) analysis according to the above-mentioned report. Although the antibody derivatization reaction (disulfide reduction reaction) was carried out at 50 ° C., the monomer ratio exceeded 99%.

[Example 19] Monomer ratio analysis of infliximab-MMAE The infliximab-MMAE synthesized in Example 9 was subjected to size exclusion chromatography (SEC) analysis according to the above-mentioned report. Although the antibody derivatization reaction (disulfide reduction reaction) was carried out at 50 ° C., the monomer ratio exceeded 98%.

[Example 20] Monomer ratio analysis of infliximab-DM1 The infliximab-MMAE synthesized in Example 8 was subjected to size exclusion chromatography (SEC) analysis according to the above-mentioned report. Although the antibody derivatization reaction (disulfide reduction reaction) was carried out at 50 ° C., the monomer ratio exceeded 98%.

From the results of Examples 12 to 19, the method of the present invention reduces the generation of aggregates even when the antibody derivatization reaction (disulfide reduction reaction) is carried out at a high temperature (50 ° C.). It has been shown that ADCs showing good DAT can be synthesized.

[Comparative Example 2] Batch synthesis and monomer ratio analysis of trastuzumab-MMAE With reference to the previous report (ACS Omega 2020, 5, 7193-7200), only the reaction temperature of the reduction step using TCEP was changed to 50 ° C., and trastuzumab was used in a batch method. -MMAE was synthesized. The obtained ADC was analyzed by RP-HPLC, and a peak was confirmed at the same retention time as in the previous report (Anal. Chem. 2019, 91, 20, 12724-12732) and FIG.

The trastuzumab-MMAE synthesized by the batch method was analyzed by size exclusion chromatography (SEC) according to the previous report (ACS Omega 2020, 5, 7193-7200). As a result, the monomer ratio was 89.7%, and aggregates (6.2%) and decomposition products (4.1%) were confirmed (FIG. 7).

1 1st reservoir 2 2nd reservoir 3 3rd reservoir 4 1st introduction flow path 5 2nd introduction flow path 6 3rd introduction flow path 7 1st pump 8 2nd pump 9 3rd pump 10 Fraction collector M1 1st micromixer M2 2nd micromixer R1 1st reaction tube R2 2nd reaction tube

Claims

A method for producing an antibody-drug conjugate,
(1) A solution containing a raw material antibody having a specific cleaving site and a solution containing a cutting agent having a cleaving ability of the specific cleaving site are mixed with an arbitrary micromixer, and the raw material antibody and the cleaving site are mixed. Producing a first mixture containing the agent;
(2) An antibody derivative having a specific reactive site and the above by passing the first mixed solution through the first reaction flow path and reacting the raw material antibody and the cleavage agent in the first reaction flow path. It is to generate a solution containing a cleaving agent, wherein the antibody derivative having the specific reactive site is a raw material by cleaving the specific cleaving site with the cleaving agent in the reaction. It is produced from antibodies;
(3) Mixing the antibody derivative, the solution containing the cleavage agent, and the solution containing the drug with a collision-type micromixer to produce a second mixed solution containing the antibody derivative, the cleavage agent, and the drug; (4) The antibody drug conjugate is produced by passing the second mixed solution through the second reaction flow path and reacting the antibody derivative and the drug in the second reaction flow path.
The processes (1) to (4) are continuously performed in the flow microreactor, and the representative diameter ratio between the collision type micromixer and the first reaction flow path (collision type micromixer / first reaction flow path) is determined. A method that is 0.95 or less.
The method according to claim 1, wherein the total value of the flow rate of the solution containing the antibody derivative and the cleavage agent and the flow rate of the solution containing the drug is 2.0 mL / min or more.
The method according to claim 1 or 2, wherein the solution containing the drug is introduced into the collision type micromixer at a flow rate of 0.4 mL / min or more.
The method according to any one of claims 1 to 3, wherein the collision type micromixer has a representative diameter of 0.8 mm or less.
The method according to any one of claims 1 to 4, wherein the collision type micromixer is a T-shaped micromixer.
The method according to any one of claims 1 to 5, wherein the arbitrary micromixer is a collision type micromixer.
The method according to any one of claims 1 to 6, wherein the reaction in the first reaction flow path is carried out at 30 to 60 ° C.
The method according to any one of claims 1 to 7, wherein the reaction in the first reaction flow path is carried out at 37 to 50 ° C.
The invention according to any one of claims 1 to 8, wherein the additional reaction of the raw material antibody and the cleavage agent and the reaction of the antibody derivative and the drug produced by the additional reaction proceed in parallel in the second reaction flow path. the method of.
The method according to any one of claims 1 to 9, wherein the residence time of the first mixed solution in the first reaction flow path is less than 5 minutes.
The method according to any one of claims 1 to 9, wherein the residence time of the second mixed solution in the second reaction flow path is less than 15 minutes.
The method according to any one of claims 1 to 11, wherein the residence time of the second mixed solution in the second reaction flow path is less than 5 minutes.
The flow rate of the solution containing the drug introduced into the collision type micromixer is the flow rate of the solution containing the raw material antibody introduced into any micromixer, and the flow rate of the solution containing the cutting agent introduced into any micromixer. The method according to any one of claims 1 to 12, which is faster than any of the above.
Any one of claims 1 to 13, wherein the time required from the arrival of the solution containing the raw material antibody and the solution containing the cleavage agent to any micromixer to the passage through the second reaction flow path is less than 20 minutes. The method described in the section.
The method according to any one of claims 1 to 14, wherein the specific reactive site is a thiol group.
The method according to any one of claims 1 to 15, wherein the raw material antibody having the specific cleavage site is an antibody having a disulfide group.
The method according to any one of claims 1 to 16, wherein the antibody having a disulfide group is an unmodified antibody having a disulfide group.
The method according to claim 16, wherein the raw material antibody is an antibody having four disulfide bonds between chains.
The method according to any one of claims 1 to 18, wherein the raw material antibody is IgG.
The method according to any one of claims 1 to 19, wherein the drug has a maleimide group and / or a disulfide group, or is derivatized so as to have a maleimide group and / or a disulfide group.
The method according to any one of claims 1 to 20, wherein the antibody-drug conjugate has a drug-antibody ratio of 2.0 or more.
The method according to any one of claims 1 to 21, wherein the monomer ratio in the antibody drug conjugate analyzed by size exclusion chromatography is 98% or more.
The method according to any one of claims 1 to 22, wherein the drug is a drug, a labeling substance, or a stabilizer.