WO2022085767A1 - Procédé de production d'un conjugué anticorps-médicament - Google Patents

Procédé de production d'un conjugué anticorps-médicament Download PDF

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WO2022085767A1
WO2022085767A1 PCT/JP2021/039001 JP2021039001W WO2022085767A1 WO 2022085767 A1 WO2022085767 A1 WO 2022085767A1 JP 2021039001 W JP2021039001 W JP 2021039001W WO 2022085767 A1 WO2022085767 A1 WO 2022085767A1
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antibody
reaction
flow path
drug
micromixer
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PCT/JP2021/039001
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English (en)
Japanese (ja)
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豊 松田
祐一 中原
ブライアン アラン メンデルソン
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味の素株式会社
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment

Definitions

  • the present invention relates to a method for producing an antibody drug conjugate.
  • the flow microreactor 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.
  • a micromixer having a microstructure is utilized in order to enable mixing and reaction of a plurality of components in a microenvironment.
  • 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.
  • 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.
  • FMR antibody drug conjugates
  • 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.
  • DAR drug-antibody ratio
  • 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.
  • 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.
  • the Patent mold (Example 8) adopted in the YMC microreactor Spica is used.
  • the reactions include a reduction reaction at 35 ° C.
  • Patent Document 3 is characterized by using single-pass tangential flow filtration (SPTFF) for ADC enrichment and removal of unreacted products (particularly unreacted drugs).
  • SPTFF single-pass tangential flow filtration
  • conjugation reaction Generating an antibody-drug conjugate (conjugation reaction); and (3) purifying the ADC by single-pass tangential flow filtration (SPTFF).
  • 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).
  • 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.
  • ADC drug antibody complex
  • a cleavage reaction as the antibody derivatization reaction, and then obtained (a') an antibody cleavage reaction (antibody derivatization reaction) and (b') the cleavage reaction.
  • 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.
  • 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.
  • the present invention is as follows.
  • 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.
  • 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.
  • 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.
  • 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 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 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.
  • [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.
  • the drug is a drug, a labeling substance, or a stabilizer.
  • 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).
  • 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.
  • 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.
  • 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 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.
  • 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).
  • the liquid feeding can be performed using a pump.
  • 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.
  • 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.
  • 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.
  • the immunoglobulin unit is derived from a mammal.
  • 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).
  • 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.
  • 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.
  • 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.
  • the antibody may be an antibody that uses a protein as an antigen.
  • 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.
  • the protein that is the antigen of the antibody may be a disease target protein.
  • diseases target proteins include the following.
  • Amyloid AL Hereditary and rare diseases Amyloid AL, SEMA4D (CD100), insulin receptor, ANGPTL3, IL4, IL13, FGF23, corticostimulatory hormone, transthyretin, huntingtin
  • monoclonal antibodies include specific chimeric antibodies (eg, rituximab, baciliximab, infliximab, cetuximab, siltuximab, dinutuximab, altertoximab), specific humanized antibodies (eg, dacrizumab, paribizmab, trastuzumab, trussumab, allenzumab).
  • specific chimeric antibodies eg, rituximab, baciliximab, infliximab, cetuximab, siltuximab, dinutuximab, altertoximab
  • specific humanized antibodies eg, dacrizumab, paribizmab, trastuzumab, trussumab, allenzumab.
  • Efarizumab 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
  • 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).
  • the source antibody may be a modified antibody.
  • the modification in the modified antibody include the above-mentioned genetic engineering technique and modification by an organic chemical technique.
  • 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.
  • 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 modified antibody may be a regioselectively modified antibody.
  • 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.
  • the modified antibody may be a position-nonselectively modified antibody.
  • an antibody derivative having a specific reactive site non-regioselectively is produced.
  • 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.
  • 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.
  • 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.
  • a modified antibody containing a modified site having such a specific cleaving site preferably a disulfide group or a thioester group
  • 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.
  • 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.
  • the raw material antibody is an antibody having a disulfide group.
  • the antibody having a disulfide group may be a modified antibody containing a modification site having a disulfide group.
  • the reaction in the first reaction channel produces a modified antibody derivative containing a modified site having a thiol group.
  • 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.
  • 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.
  • the reaction in the first reaction flow path produces an unmodified antibody derivative having a thiol group.
  • 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.
  • the cleaving agent include reducing agents (eg, tricarboxyethylphosphine (TCEP), cysteine, dithiothreitol, reduced glutathione, ⁇ -mercaptoethanol), acidic substances (eg, inorganic acidic substances such as hydrochloric acid and sulfuric acid, etc.).
  • 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
  • enzymes e.g, the cutting agent is a reducing agent.
  • the cleavage agent is a reducing agent having the ability to cleave a disulfide bond.
  • reducing agents include tricarboxyethylphosphine (TCEP), cysteine, dithiothreitol, reduced glutathione, and ⁇ -mercaptoethanol.
  • the reducing agent is TCEP.
  • 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.
  • 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.
  • 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.
  • 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.
  • metal materials eg, stainless steel (SUS), Hasteroy®, Inconel
  • resins eg, polytetrafluoroethylene (PTFE), polyethersalkane.
  • PTFE polytetrafluoroethylene
  • PES polytetrafluoroethylene
  • PEEK polyetheretherketone
  • PDMS polydimethylsiloxane
  • PFA tetrafluoroethylene-perfluoroalkyl vinyl ether copolymer
  • 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.
  • 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.
  • 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.
  • metal materials eg, stainless steel (SUS), Hasteroy®, Inconel
  • resins eg, polytetrafluoroethylene (PTFE), polyethersulfone (PES), poly. Ether ether ketone (P
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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)].
  • 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)].
  • 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.
  • 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.
  • 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.
  • the drug is an anticancer drug.
  • Anti-cancer agents include, for example, chemotherapeutic agents, toxins, radioisotopes or substances containing them.
  • 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.
  • 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 of hydrogen atoms eg, 3H
  • radioisotopes of carbon atoms eg, 14C
  • radioisotopes of phosphorus atoms eg, 32P
  • sulfur atoms 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).
  • auristatin MMAE, MMAF
  • maytancin DM1, DM4
  • PBD pyrrolobenzodiazepine
  • IGN camptothecin analog
  • calicheamicin duocarminine
  • eribulin anthracycline
  • dmDNA31 tubricin.
  • 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, ⁇ -galactosidase), affinity substances (eg, streptavidin, biotin, digoxygenin, aptamer), fluorescent substances (eg, fluorescein, fluorescein isothiocyanate, rhodomin).
  • Green fluorescent protein 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
  • 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).
  • 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.
  • 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).
  • derivatization may be carried out using any cross-linking agent.
  • the derivatization may be carried out using a specific linker having the desired functional group.
  • 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).
  • 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)].
  • specific proteases eg, intracellular proteases (eg, proteases present in lysosomes or endosomes), extracellular proteases (eg, secretory proteases)
  • proteases eg, intracellular proteases (eg, proteases present in lysosomes or endosomes), extracellular proteases (eg, secretory proteases)
  • US Pat. No. 6,214,345 Dubowchik et al., Pharma. Therapeutics 83: 67-123 (1999)
  • the linker may be self-immolative (eg, WO 02/083180, WO 04/043493, WO 05/1192919).
  • derivatized drugs are also simply referred to as "drugs".
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a first solution eg, a solution containing an antibody derivative having a specific reaction site and a cleavage agent
  • a second solution eg, a drug
  • 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).
  • the two inflow paths 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).
  • the angle X set with respect to the first inflow path eg, at least one first reaction flow path
  • the angle Y set with respect to the inflow path is an angle tilted toward the outflow path with respect to the facing position.
  • two solutions in at least two inflow paths 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 °.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the collision type micromixer preferably has a representative diameter of 0.8 mm or less among the above-mentioned representative diameters.
  • the solution is further accelerated at the confluence to generate finer solution units, so that a uniform solution can be produced more quickly.
  • 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.
  • the solution is further accelerated at the confluence to generate finer solution units, so that a uniform solution can be produced more quickly.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the reaction between the antibody derivative and the drug 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.
  • 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.
  • 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.
  • pH neutralizers eg, acidic or basic substances
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the salt with an inorganic acid include salts with hydrogen chloride, hydrogen bromide, phosphoric acid, sulfuric acid, and nitric acid.
  • 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).
  • the drug-antibody complex produced in the present invention may be provided in a non-salt form.
  • FIG. 1 shows an outline of the configuration of the FMR used in the following 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • Fraction collector Fraction collector (10) that collects the second reaction solution from the second reaction tube.
  • 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.
  • 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
  • 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).
  • 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.
  • the measurement of DA by RP-HPLC 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.
  • TCEP phosphate buffered saline
  • TCEP reducing agent
  • 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.
  • NAC N-acetylcysteine
  • 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).
  • 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.
  • 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).
  • 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.
  • 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).
  • M2 second micromixer
  • 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 N-acetylcysteine
  • 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 calculation of DAT was performed as follows.
  • 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%).
  • 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.
  • 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.
  • 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.
  • 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- ⁇ 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.
  • 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- ⁇ 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ (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 ⁇ L 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.
  • SEC size exclusion chromatography
  • 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.
  • SEC size exclusion chromatography
  • Example 16 Monomer ratio analysis of rituximab-MMAE
  • SEC size exclusion chromatography
  • 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.
  • SEC size exclusion chromatography
  • 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.
  • SEC size exclusion chromatography
  • Example 19 Monomer ratio analysis of infliximab-MMAE
  • SEC size exclusion chromatography
  • 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.
  • SEC size exclusion chromatography
  • 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.
  • 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).

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Abstract

La présente invention concerne un procédé qui peut accélérer la production d'un conjugué anticorps-médicament (ADC). Plus particulièrement, la présente invention concerne un procédé de production d'un ADC, ledit procédé consistant à : (1) préparer un premier mélange liquide qui contient une solution contenant un anticorps de départ comprenant un site clivable spécifique et une solution contenant un agent de clivage capable de cliver le site clivable spécifique ; (2) faire passer le premier mélange liquide à travers un premier canal de réaction pour obtenir une solution qui contient un dérivé d'anticorps comprenant un site de réaction spécifique et l'agent de clivage ; (3) par mélange dans un micromélangeur de type collision, préparer un second mélange liquide qui contient le dérivé d'anticorps, l'agent de clivage et un médicament ; et (4) faire passer le second mélange liquide à travers un second canal de réaction pour obtenir l'ADC. Dans ce procédé, les traitements (1) à (4) sont exécutés en continu dans un microréacteur d'écoulement, et le rapport de diamètre type du micromélangeur de type collision au premier canal de réaction (micromélangeur de type collision/premier canal de réaction) n'est pas supérieur à 0,95.
PCT/JP2021/039001 2020-10-22 2021-10-21 Procédé de production d'un conjugué anticorps-médicament WO2022085767A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005288254A (ja) * 2004-03-31 2005-10-20 Ube Ind Ltd マイクロデバイスおよび流体の合流方法
WO2020075817A1 (fr) * 2018-10-10 2020-04-16 武田薬品工業株式会社 Procédé de production d'un conjugué anticorps-médicament
WO2020095894A1 (fr) * 2018-11-05 2020-05-14 味の素株式会社 Procédé de production d'une protéine repliée à l'aide d'un microréacteur à flux, et appareil de repliement de protéine

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005288254A (ja) * 2004-03-31 2005-10-20 Ube Ind Ltd マイクロデバイスおよび流体の合流方法
WO2020075817A1 (fr) * 2018-10-10 2020-04-16 武田薬品工業株式会社 Procédé de production d'un conjugué anticorps-médicament
WO2020095894A1 (fr) * 2018-11-05 2020-05-14 味の素株式会社 Procédé de production d'une protéine repliée à l'aide d'un microréacteur à flux, et appareil de repliement de protéine

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