Ultrafiltration / percolation purification method

Abstract

Disclosed herein is a purification process comprising an organic ultrafiltration / diafiltration process in which a crude mixture containing a conjugate compound to be purified is filtered through a semipermeable membrane and the filtrate from the crude mixture is replaced with an exchange medium containing an organic solvent and a buffer system.

Description

Background Technology

[0001] Antibody-drug conjugates (ADCs) have emerged as a new class of biotherapeutic agents in fields such as oncology, infectious diseases, and immunology. ADCs typically comprise an antigen-targeting monoclonal antibody (mAb) covalently attached via chemical bonds (i.e., linkers) and at least one payload (e.g., a drug or other active agent). Most ADCs rely on chemical methods to attach the linker and payload (e.g., a drug or other active agent) to residues of the antibody (such as lysine or cysteine ​​groups). The chemical processes involved in ADC production generate a mixture of reagents, solvents, reaction byproducts, side products, and other small molecule impurities that are generally unrelated to conventional mAb production. Some impurities, such as residual free drug, residual drug linkers, and residual linkers, are known to be particularly difficult to remove from ADCs effectively using various techniques, such as conventional ultrafiltration / difiltration (UF / DF) processes.

[0002] UF / DF is a common method for purifying and exchanging buffers in biopharmaceutical products. For example... Figure 1 and Figure 2 As shown, the UF / DF process typically involves continuously filtering the crude product contained in an aqueous buffer solution through a membrane while adding an exchange medium in a manner that allows the desired product to be retained by the membrane in the osmotic chamber, while smaller compounds (e.g., unwanted solvents, reaction intermediates, residual reagents, byproducts, side products, and / or other impurities) are removed in the filtrate.

[0003] While UF / DF has proven effective in removing smaller compounds or impurities from certain classes of biopharmaceutical products, it is not particularly effective in removing certain impurities in some cases. For example, as mentioned above, conventional UF / DF techniques may struggle to remove some residual free drug, residual drug-linkers, residual linkers, byproducts, and / or side products associated with the synthesis of ADCs and other conjugates. Theoretically, conventional UF / DF is ineffective in removing certain impurities due to the formation of non-covalent bonds and / or other non-covalent interactions that may occur between the impurity and the desired product—thus preventing these impurities from being filtered out of the desired product. This technical challenge can be particularly problematic when UF / DF is used to purify some antibody-drug conjugates (ADCs), such as camptothecin-based ADCs and camptothecin derivative ADCs, such as exatecan-based ADCs.

[0004] There is a need to discover new separation methods for removing small molecule impurities (such as residual free drug, residual drug-linkers, residual linkers, and / or byproducts associated with the synthesis of the conjugate compound) from antibody-drug conjugates and other conjugates. Surprisingly, the UF / DF method disclosed herein addresses this need. Summary of the Invention

[0005] This article provides purification methods that are highly effective in removing organic impurities (e.g., residual free drug compounds, residual drug-connector compounds, residual connector compounds, reaction byproducts, residual side products, and / or combinations thereof), including product-bound impurities that are difficult to remove using conventional aqueous UF / DF processes.

[0006] In one embodiment, the present invention provides a purification method comprising:

[0007] (a) subjecting a sample containing at least one conjugate compound and at least one impurity to an organic ultrafiltration / difiltration (UF / DF) process, such that the sample is filtered through a semipermeable membrane having a pore size that allows at least one impurity to pass through while retaining at least one conjugate compound as a effluent, wherein the organic UF / DF process uses a first exchange medium and produces a effluent containing at least one conjugate compound; and

[0008] (b) subjecting the effluent product of (a) to an aqueous UF / DF process, such that it is filtered through a semi-permeable membrane having a pore size that allows at least one impurity to pass through while retaining the conjugate compound in the effluent, wherein the aqueous UF / DF process uses a second exchange medium and results in a effluent containing at least one conjugate compound.

[0009] in:

[0010] At least one impurity includes residual free drug compound, residual drug-connector compound, residual connector compound, residual by-product compound, residual side-product compound, or any combination thereof;

[0011] The first exchange medium comprises at least one organic solvent and a first buffer system; and

[0012] The first and second switching media are different.

[0013] In some embodiments, a purification method is provided comprising the following steps: (a) combining a first exchange medium with a crude composition to obtain a crude mixture, wherein the crude composition comprises a conjugated compound and at least one impurity; (b) subjecting the crude mixture to an organic UF / DF process, such that the crude mixture is filtered through a first semipermeable membrane having a first pore size that allows the impurity to pass through the membrane into a first filtrate while retaining the conjugated compound in a first filtrate, and the volume of liquid filtered from the crude mixture is at least partially replaced in the first filtrate by an additional first exchange medium to obtain a separated composition; and (c) subjecting the separated composition to aqueous ultrafiltration / diafiltration. The filtration process involves passing the separated composition through a second semi-permeable membrane having a second pore size that allows impurities to pass through the membrane while retaining the conjugate compound in the second effluent, and at least partially replacing the liquid volume filtered from the separated composition with a second exchange medium comprising a second buffer system to obtain a purified composition in the second effluent, wherein the first exchange medium comprises at least one organic solvent and the first buffer system, and wherein the first exchange medium and the second exchange medium are different, and wherein the impurities may include residual free drug compounds, residual drug-connector compounds, residual connector compounds, residual byproduct compounds, residual side-product compounds, or any combination thereof.

[0014] In some embodiments, the conjugate compound comprises an antibody-drug conjugate (ADC). In other embodiments, the conjugate compound comprises a conjugate other than an ADC. Therefore, the present invention can also be applied to non-antibody-drug conjugates.

[0015] Surprisingly, it has been found that adding at least one organic solvent to the first exchange medium enables the method of the present invention to more effectively remove small molecule impurities (e.g., residual free drug compounds, residual drug-connector compounds, residual connector compounds, residual byproduct compounds, etc.) that cannot be effectively removed using conventional UF / DF and / or TFF processes. Therefore, the first exchange medium used in the present invention will contain at least one organic solvent. As illustrated in Examples 1-6, the method of the present invention can be used to effectively remove small molecule impurities, such as residual drug compounds and drug-connector compounds, from ADCs and other conjugates.

[0016] In some embodiments, the isolated composition is substantially free of small molecule impurities, such as product-bound impurities. In some embodiments, the purified composition is substantially free of small molecule impurities (e.g., product-bound impurities) and organic solvents. In some embodiments, the isolated composition produced from the purification method of the present invention lacks the free linker form of the linker present in the ADC. In some embodiments, the purified drug conjugate will contain no impurities or only impurities at levels insignificant to the administration to the subject.

[0017] In some embodiments, a method is provided for preparing purified conjugate compounds using separately prepared drug-connector compounds, comprising the steps of: (i) reducing a target agent by contacting it with a reducing agent to obtain a crude reduced target agent; (ii) conjugating the reduced target agent with a drug-connector compound to obtain a crude composition comprising the conjugate compound; (iii) optionally adding a quencher to the crude composition; and (iv) purifying the crude composition using the UF / DF method of this disclosure, wherein a first exchange medium comprises at least one organic solvent to obtain the purified conjugate compound. In some embodiments, the target agent is an antibody. In some embodiments, the target agent comprises a non-antibody scaffold.

[0018] In some embodiments, a method is provided for preparing purified conjugate compounds using separately prepared drug-connector compounds, comprising the following steps:

[0019] (i) Reduce the target agent by contacting it with a reducing agent to obtain a reduced target agent;

[0020] (ii) conjugating the reduced target agent with a drug-connector compound to obtain a crude composition comprising a conjugated compound containing the conjugate; and

[0021] (iii) The crude composition is purified using the UF / DF method of this disclosure, wherein the first exchange medium comprises at least one organic solvent. In some embodiments, the target agent is an antibody. In some embodiments, the target agent comprises a non-antibody scaffold.

[0022] In some embodiments, a method is provided for preparing a purified conjugate compound by sequential attachment of a linker group and a pharmaceutical compound, comprising the steps of: (i) reducing a target agent by contacting it with a reducing agent to obtain a crude reduced target agent; (ii) attaching at least one linker group to the reduced target agent to obtain a crude composition comprising a target agent-linker intermediate; (iii) attaching at least one pharmaceutical compound to a linker of the target agent-linker intermediate to obtain a crude composition comprising a conjugate compound; (iv) optionally adding a quencher to the crude composition; and (v) purifying the crude composition using the UF / DF method of this disclosure, wherein a first exchange medium comprises at least one organic solvent to obtain the purified conjugate compound. In some embodiments, the target agent is an antibody. In some embodiments, the target agent comprises a non-antibody scaffold.

[0023] In some embodiments, the target is an antibody or antigen-binding fragment, and the purified conjugate compound is a purified antibody-drug conjugate (ADC). In other embodiments, the target comprises a non-antibody scaffold, and the purified conjugate compound is a purified non-ADC conjugate.

[0024] In some embodiments, any method of the present invention may further include formulating the obtained purified product containing the drug conjugate with a pharmaceutical carrier or excipient to obtain a pharmaceutical composition. Attached Figure Description

[0025] The implementation scheme is shown in all the figures by way of example rather than by way of limitation, wherein:

[0026] Figure 1 The basic process of conventional ultrafiltration / difiltration (UF / DF) using an exemplary device is shown;

[0027] Figure 2 This is a schematic diagram of one embodiment of a UF / DF apparatus that can be used to perform the UF / DF process of this disclosure;

[0028] Figure 3 The graph shows the residual free drug versus percolation volume in UF / DF studies 4.1-4.3 of Example 4; and

[0029] Figure 4 This is a flowchart summarizing one embodiment of the synthesis process for preparing ADC1 (including the UF / DF method of this disclosure). Detailed Implementation

[0030] This article provides a highly efficient purification method for removing small molecule impurities (e.g., residual free drug compounds, residual drug-connector compounds, and / or residual connector compounds) that are typically difficult to remove using conventional UF / DF or TFF processes. Surprisingly, it has been found that adding at least one organic solvent to the first exchange medium enables the method of the present invention to effectively remove small molecule impurities that cannot be effectively removed using conventional UF / DF or TFF processes.

[0031] definition

[0032] For convenience, certain terms in the specification, embodiments, and claims are defined herein. Unless otherwise stated or implied by context, the following terms and phrases have the meanings provided below. These definitions are provided to aid in describing particular embodiments and are not intended to limit the claimed invention, as the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0033] As used herein, unless otherwise stated, the terms “a” and “an” are understood to mean “one,” “at least one,” or “one or more.” Unless the context requires otherwise, singular terms used herein shall include the plural, and plural terms shall include the singular.

[0034] Except as indicated in the examples or otherwise, all figures used herein to express quantities of components or process conditions should be understood to be modified by the term “about” in all cases. Ranges and quantities may be expressed as “about” a specific value or range. The term “about” also includes an exact quantity. Thus, the phrase “about 30%” means “about 30%” and also “30%”. In some embodiments, “about” means within ±10% of the value (e.g., the phrase “about 30%” means 27-33%). In some embodiments, “about” means within ±5% of the value. In some embodiments, “about” means within 4% of the value. In some embodiments, “about” means within 3% of the value. In some embodiments, “about” means within 2% of the value. In some embodiments, “about” means within 1% of the value. Generally, the term “about” includes quantities expected to be within experimental error.

[0035] As used herein, unless otherwise specified, the word “or” means “any / or”, but is not limited to “any / or”. Alternatively, “or” may also mean “and / or”.

[0036] Unless the context explicitly requires otherwise, throughout the specification and claims, the terms “comprise”, “comprising”, etc., should be interpreted as inclusive, as opposed to exclusive or exhaustive; that is, they should be interpreted as “including but not limited to”.

[0037] As used herein, the term "consisting essentially of" refers to the elements required for a given implementation scheme. This term allows for the presence of elements that do not substantially affect the fundamental, new, or functional characteristics of the implementation scheme. Where the implementation scheme is described herein using inclusive language, implementation schemes that are essentially composed of the described content are also provided.

[0038] As used herein, the term "consisting of" refers to compositions, methods, and their corresponding components as described herein, excluding any elements not described in the description of the embodiments. Where embodiments are described herein using inclusive language, embodiments consisting of the described content are also provided.

[0039] In the context of numerical values, the terms “decreased,” “reduce,” “reduced,” “reduction,” “decrease,” and “inhibit” are generally used here to refer to a statistically significant reduction relative to a reference.

[0040] The term "plurality" means "two or more" unless otherwise explicitly stated. For example, "plurality" can simply refer to multiple injections into the esophageal tissue.

[0041] The terms "statistically significant" or "significantly" refer to statistical significance and generally mean a difference of two standard deviations (2SD) above or below a reference value.

[0042] The term "statistically free" means that the referred component is not contained in the composition, or is contained in the composition in a proportion of less than 10% by weight or volume, or less than 5% by weight or volume, or less than 3% by weight or volume, or less than 2% by weight or volume, or less than 1% by weight or volume, or less than 0.5% by weight or volume relative to the total weight or volume of the composition.

[0043] As used in this article, the term “targeting agent” refers to an antibody (including antigen-binding fragments) or other non-antibody scaffold that specifically binds to target molecules such as proteins, carbohydrates, glycoproteins, etc.

[0044] As used herein, the term "conjugate compound" refers to an ADC or other conjugate that contains an antibody or non-antibody scaffold as a target agent, which is covalently linked to at least one drug moiety via at least one linker group (connector).

[0045] As used herein, the term “conjugation reaction” refers to the chemical process of attaching an antibody or other non-antibody protein scaffold to at least one connector and at least one drug to form a conjugated compound.

[0046] As used herein, the term "conjugation mixture" refers to a mixture of crude products produced by a conjugation reaction.

[0047] As used herein, the term “antibody drug conjugate” or “ADC” refers to a conjugate compound containing a targeting antibody covalently linked to at least one drug moiety via at least one linker group.

[0048] As used herein, the term “non-ADC conjugate” or “conjugate other than ADC” refers to a conjugate compound comprising at least one antibody-free target agent (i.e., the target moiety) covalently linked to at least one payload moiety (such as a drug payload) via at least one linking group (connector).

[0049] As used herein, the term "drug-linker compound" refers to a compound comprising a drug moiety and a linker moiety covalently linked to that drug moiety, wherein the drug-linker compound is capable of covalently linking to a protein (e.g., an antibody or other binder) or other target. As used herein, the term "binding agent" refers to a protein comprising an antibody or non-antibody scaffold that can be covalently linked to a drug-linker compound. The term antibody also includes antigen-binding antibody fragments.

[0050] As used herein, the term "drug moiety" refers to a drug, prodrug, or other active agent attached to a linker of a drug-linker compound or conjugate compound.

[0051] As used herein, the term "protein-linker compound" refers to a compound comprising a protein moiety and a linker moiety covalently attached to the protein moiety, wherein the protein-linker compound is covalently linked to a drug compound.

[0052] As used herein, the term “drug-antibody ratio” or “DAR” refers to the average number of drugs or drug portions attached to an antibody or other target. For example, a DAR of 1:4 means an average of 4 drugs or drug portions per antibody or other target.

[0053] As used herein, the term "small molecule impurities" includes impurities from the conjugation reaction that are still present at the termination of the conjugation reaction, including residual free drug compound, residual drug linker compound, residual linker compound, reaction byproducts and / or side products.

[0054] As used herein, the term "product-bound impurities" refers to small molecule impurities from conjugation reactions that tend to associate with the conjugate compound due to non-covalent bonds and other non-covalent interactions or associations that can form between the impurity and the conjugate. Product-bound impurities can include residual free drug compounds, residual drug-connector compounds, and / or residual connector compounds. Product-bound impurities can also be protein-bound impurities that tend to associate with the protein moiety of an ADC or other conjugate.

[0055] As used herein, the term "organic solvent" refers to a non-reactive organic compound containing carbon, at least partially soluble in water, and facilitating the removal of small molecule impurities from crude compositions. As used herein, the term "non-reactive" means that the organic solvent does not react with, denature, or decompose the ADC or other conjugate compound at temperatures below 50°C when the concentration of the organic solvent is equal to or less than 30% (w / v) relative to the total volume of the exchange medium containing the organic solvent. In some embodiments, the organic solvent comprises carbon and at least one atom selected from oxygen, nitrogen, and sulfur. In some embodiments, the organic solvent is miscible with water. In some embodiments, the term "organic solvent" is used according to its common meaning in the chemical field. Suitable organic solvents may include, for example, alcohols, polar aprotic compounds, and combinations thereof. In some embodiments, suitable organic solvents do not include sugar alcohols.

[0056] As used herein, the term "alcohol compounds" refers to a nonreactive carbon-containing organic compound containing at least one hydroxyl (-OH) group. Alcohol compounds can include monohydric alcohols (containing one OH group), dihydric alcohols (containing two OH groups), and polyhydric alcohols (containing three or more OH groups).

[0057] "Monohydric alcohols" can include, for example, aliphatic monohydric alcohols (e.g., paraffinic alcohols, olefinic alcohols) which can be straight-chain, branched and / or cyclic; aromatic monohydric alcohols (e.g., phenolic alcohols, benzyl alcohols); heterocyclic monohydric alcohols having 1 to 20 carbon atoms (e.g., furfuryl alcohols); polycyclic monocyclic alcohols (e.g., sterols), etc., or combinations thereof.

[0058] "Monolithic alcohols" may include, for example, methanol, ethanol, n-propanol, isopropanol, 1-butanol, 2-butanol, tert-butanol, isobutanol, 1-pentanol, 2-pentanol, 3-pentanol, isopentanol, tert-pentanol, hexanol (straight-chain, branched and / or cyclic), ethylene glycol monomethyl ether, cis-3-hexen-1-ol, trans-2-hexen-1-ol, 5-hexen-1-ol, phenol, benzyl alcohol, etc., or combinations thereof.

[0059] "Dihydric alcohols" can include, for example, 1,2-ethylene glycol, 1,2-propanediol, 1,3-propanediol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 1,2-pentanediol, 1,5-pentanediol, 2,2-dimethylpropane-1,3-diol, 2-butyl-2-ethylpropane-1,3-diol, 2-methyl-2,4-pentanediol, 2-ethyl-1,3-hexanediol, 2-methyl-1,3-propanediol, 1,2-hexanediol, 1,5-hexanediol, 1,6-hexanediol, 1,8-octanediol, 1,10-decanediol, 2,2,4,4 Tetramethylcyclobutane-1,3-diol, 1,3-cyclopentanediol, 1,2-cyclohexanediol, 1,3-cyclohexanediol, 1,4-cyclohexanediol, 1,2-cyclohexanediethanol, 1,3-cyclohexanediethanol, 1,4-cyclohexanediethanol, 1,4-cyclohexanediethanol, isosorbide, monoglyceride, monoglyceride, trimethylolpropane monoester, trimethylolpropane monoether, pentaerythritol diester, pentaerythritol diether, dipropylene glycol, diethylene glycol, triethylene glycol, 2-methyl-1,3-propanediol, 2,2-dimethyl-1,3-propanediol, neopentyl glycol, xylylene glycol), bis(p-hydroxy)diphenyl, bis(p-hydroxy)diphenylpropane, 2,2'-bis[4-(2-hydroxyethoxy)phenyl]propane, bis[4-(2-hydroxyethoxy)phenyl]sulfone, 1,1-bis[4-(2-hydroxyethoxy)phenyl]cyclohexane, or combinations thereof.

[0060] "Polyhydric alcohols" can include, for example, aldosterones, 2-ethyl-2-hydroxymethyl-1,3-propanediol, 1,2,4-butanetriol, 1,2,5-pentanetriol, 1,2,6-hexanetriol, 1,2,3,6-hexanetetrol, glycerol (also known as glycerol), diglycerol, triglycerol, tetraglycerol, pentaglycerol, hexaglycerol, triethanolamine, trimethylolethane, trimethylolpropane, bis(trimethylolpropane), tri-trimethylolpropane, 2-methylpropanetriol, 2-methyl-1,2,4-butanetriol, pentaerythritol, dipentaerythritol, tripentaerythritol, methylglucoside, sorbitol, mannitol, and sucrose. 1,3,5-Trihydroxybenzene, 1,2,4-Trihydroxybenzene, (poly)oxyethylene glycerol, (poly)oxypropylene glycerol, (poly)oxyethylene diglycerol, (poly)oxypropylene diglycerol, (poly)oxyethylene trimethylolpropane, (poly)oxypropylene trimethylolpropane, (poly)oxyethylene bis(trimethylolpropane), (poly)oxypropylene bis(trimethylolpropane), erythritol, inositol, threitol, arabinitol, xylitol, ribitol, galactitol, mannitol, sorbitol, (poly)oxyethylene pentaerythritol, (poly)oxypropylene pentaerythritol, (poly)oxyethylene dipentaerythritol, (poly)oxypropylene dipentaerythritol, etc., and combinations thereof.

[0061] "Sugar alcohols" are generally derived from carbohydrates and include at least one hydroxyl group (-OH) or a hydroxyl-derived ether group (-O-) attached to each carbon atom. Sugar alcohols can include, for example, arabinitol, erythritol, fructose, galactitol, glucose, idutitol, isomaltitol, lactitol, lactose, mannitol, maltitol, maltose, maltotriose, inositol, perseitol, ribitol, sorbitol, sucrose, threitol, trehalose, volemitol, xylitol, xylose, and combinations thereof.

[0062] The term "polar aprotic compound" refers to an organic compound that has a high dielectric constant and a high dipole moment and does not contain acidic hydrogen. Polar aprotic compounds can include, for example, acetone (ACE), acetonitrile (I), 4-acetylmorpholine, N-cyclohexyl-2-pyrrolidone (CHP), 1,2-dimethoxyether (DME), N,N-dimethylacetamide (DMAc), N,N-diethylacetamide, dimethylformamide (DMF), diethylformamide, N,N-dimethylpropionamide, 3-methoxy-N,N-dimethylpropionamide, 3-methoxy-N,N-diethylpropionamide, 3-methoxy-N,N-methylethylpropionamide, 3-ethoxy-N,N-dimethylpropionamide, 1,3-dimethyl-2-imidazolium ketone (DMI), dimethyl sulfoxide (DMSO), 1,4-dioxane, and 1,3-dioxolane. (DN), N,N'-dimethylacrylurea (DMPU), hexamethylphosphoramide (HMPA), N-methyl-2-pyrrolidone (NMP), N-ethyl-2-pyrrolidone, N-methyl-ε-caprolactam, 2-methyltetrahydrofuran (Me-THF), 2,5-dimethyltetrahydrofuran, 4-propionylmorpholine, sulfolane, tetrahydrofuran (THF), tris(N,N-tetramethylene)phosphotriamide, alcohol ethoxylates, diethyl ester dimethylammonium chloride, linear alkylbenzene sulfonates, and combinations thereof.

[0063] As used herein, the term "miscible" refers to the property that two substances (i.e., liquids) form a homogeneous mixture when mixed together at room temperature. For example, if an organic solvent forms a homogeneous mixture with water at room temperature (without phase separation), then the organic solvent is miscible with water.

[0064] As used herein, the term “partially soluble” means that at least 1% by mass of the solute is dissolved in the solvent, or that the two substances form a homogeneous mixture (without phase separation) only when the mixture is heated to at least 30°C.

[0065] As used herein, the term "polarity index" refers to the Burdick & Jackson polarity index (see, for example, Przybitek (1980) "High Purity Solvent Guide," Burdick and Jackson Laboratories, Inc.). Polarity indices for various solvents are available in "High Purity Solvent Guide," Burdick and Jackson Laboratories, Inc., published by American Scientific Products.

[0066] In this disclosure, the value “logP” means that it is determined by the distribution of small molecule impurities between the first exchange medium and the first buffer system at 25 °C, referring to the “logP” value described in Dearden et al. (Molecular Informatics, Vol 7(3), page 133-134, 1988). “P” represents the partition coefficient.

[0067] As used herein, the term "residual free drug compound" refers to the drug compound (e.g., active load) remaining in the conjugation reaction used to form an ADC or other conjugate.

[0068] As used herein, the term "residual drug-linker compound" refers to the drug-linker compound remaining after a conjugation reaction used to form an ADC or other conjugate.

[0069] As used herein, the term "residual linker compound" refers to the linker compound remaining after a conjugation reaction is used to form an ADC or other conjugate.

[0070] As used herein, the term "residual reagent" refers to the reagent (e.g., reducing agent and / or quencher) remaining in the crude ADC composition after the conjugation reaction used to form the ADC.

[0071] As used herein, the term “residual byproduct” refers to the product of a desired conjugation reaction (e.g., a reaction used to form an ADC or other conjugated compound), but is not the desired product of the desired reaction (i.e., the conjugated compound).

[0072] The term "residual side product" refers to a product generated by a side reaction that is not the primary or desired reaction (i.e., a conjugation reaction).

[0073] As used herein, the description of the numerical range of a variable is intended to convey that this disclosure can be practiced with respect to any value within that range. Thus, for a variable that is inherently discrete, it can be equal to any integer value within the numerical range, including the endpoints of that range. Similarly, for a variable that is inherently continuous, it can be equal to any real value within the numerical range, including the endpoints of that range. As an example and without restriction, if a variable is inherently discrete, a variable described as having values ​​between 0 and 2 can take the values ​​0, 1, or 2, and if a variable is inherently continuous, it can take the values ​​0.0, 0.1, 0.01, 0.001, or any other real value ≥0 and ≤2.

[0074] Purification methods

[0075] In one embodiment, the present invention provides a purification method comprising:

[0076] (a) subjecting a sample containing at least one conjugate compound and at least one impurity to an organic ultrafiltration / difiltration (UF / DF) process, such that the sample is filtered through a semipermeable membrane having a pore size that allows at least one impurity to pass through the membrane while retaining at least one conjugate compound as a effluent, wherein the organic UF / DF process uses a first exchange medium and produces a effluent containing at least one conjugate compound; and

[0077] (b) subjecting the effluent product of (a) to an aqueous UF / DF process, such that it is filtered through a semi-permeable membrane having a pore size that allows at least one impurity to pass through while retaining the conjugate compound in the effluent, wherein the aqueous UF / DF process uses a second exchange medium and results in a effluent containing at least one conjugate compound.

[0078] in:

[0079] At least one impurity includes residual free drug compound, residual drug-connector compound, residual connector compound, residual by-product compound, residual side-product compound, or any combination thereof;

[0080] The first exchange medium comprises at least one organic solvent and a first buffer system; and

[0081] The first and second exchange media are different. In some embodiments, the sample is formed prior to (a) by combining the drug conjugate with the first exchange media.

[0082] In some embodiments, a purification method is provided comprising the following steps: (a) combining a first exchange medium with a crude composition containing a conjugated compound and at least one impurity to obtain a crude mixture; (b) subjecting the crude mixture to an organic UF / DF process, such that the crude mixture is filtered through a first semi-permeable membrane having a first pore size that allows the impurity to pass through the membrane into a first filtrate while retaining the conjugated compound in a first filtrate, and the volume of liquid filtered from the crude mixture is at least partially replaced in the first filtrate with an additional first exchange medium to obtain a separated composition; and (c) subjecting the separated composition to aqueous ultrafiltration / diafiltration. The filtration process involves passing the separated composition through a second semi-permeable membrane having a second pore size that allows impurities to pass through the membrane while retaining the conjugate compound in the second residue, and at least partially replacing the liquid volume filtered from the separated composition with a second exchange medium comprising a second buffer system to obtain a purified composition in the second residue, wherein: the impurities include residual free drug compounds, residual drug-connector compounds, residual connector compounds, residual byproduct compounds, residual side-product compounds, or any combination thereof; the first exchange medium comprises at least one organic solvent and a first buffer system; and the first and second exchange media are different.

[0083] In some embodiments, the isolated composition is substantially free of product-bound impurities. In some embodiments, the isolated composition is substantially free of small molecule impurities. In some embodiments, the isolated composition is substantially free of residual free drug compounds, residual drug-connector compounds, residual connector compounds, residual byproduct compounds, or any combination thereof. In some embodiments, the purified composition is substantially free of organic solvents.

[0084] As shown in the experimental section, it was surprisingly found that the presence of at least one organic solvent in the first exchange medium enabled the disclosed method to effectively separate small molecule impurities from crude compositions containing conjugated compounds. For example, as illustrated in Table 5, the presence of an organic solvent (even in relatively small amounts) can reduce the amount of residual free drug, drug-connector compounds, and / or residual connectors in the purified composition by a factor of twenty or more. In some embodiments, the organic solvent may be in solid form before being added to the first buffer system. In some embodiments, the first exchange medium is aqueous. The separation of impurities in the first exchange medium is better than in the organic solvent alone, and better than in the first buffer system alone.

[0085] In some embodiments, the organic solvent comprises an alcohol compound, a polar aprotic compound, or a combination thereof. In some embodiments, the organic solvent comprises an alcohol compound. In some embodiments, the organic solvent comprises a polar aprotic compound. In some embodiments, the organic solvent comprises both an alcohol compound and a polar aprotic compound. The alcohol compound may include monohydric alcohols, dihydric alcohols, polyhydric alcohols, or combinations thereof. In some embodiments, the organic solvent comprises a monohydric alcohol. In some embodiments, the organic solvent comprises a dihydric alcohol. In some embodiments, the organic solvent comprises at least two of monohydric alcohols, dihydric alcohols, and polyhydric alcohols.

[0086] In some embodiments, the organic solvent comprises a monohydric alcohol selected from the following: methanol, ethanol, n-propanol, isopropanol, 1-butanol, 2-butanol, tert-butanol, isobutanol, 1-pentanol, 2-pentanol, 3-pentanol, isopentanol, tert-pentanol, hexanol (straight-chain, branched, and / or cyclic), ethylene glycol monomethyl ether, cis-3-hexen-1-ol, trans-2-hexen-1-ol, 5-hexen-1-ol, phenol, benzyl alcohol, etc., and combinations thereof. For example, in some embodiments, the organic solvent is selected from lower alcohols, such as methanol, ethanol, n-propanol, isopropanol, 1-butanol, and / or 2-butanol.

[0087] In some embodiments, the organic solvent comprises a monohydric alcohol selected from methanol, ethanol, n-propanol, isopropanol, n-butanol, sec-butanol, tert-butanol, and combinations thereof.

[0088] In some embodiments, the organic solvent comprises a monohydric alcohol, which is methanol, ethanol, or a combination thereof.

[0089] In some embodiments, the organic solvent comprises at least one monohydric alcohol and a combination of at least one of the dihydric alcohols, polyhydric alcohols and / or polar aprotic compounds described herein.

[0090] In some embodiments, the organic solvent comprises a diol selected from the following: 1,2-ethylene glycol, 1,2-propanediol, 1,3-propanediol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 1,2-pentanediol, 1,5-pentanediol, 2,2-dimethylpropane-1,3-diol, 2-butyl-2-ethylpropane-1,3-diol, 2-methyl-2,4-pentanediol, 2-ethyl-1,3-hexanediol, 2-methyl-1,3-propanediol, 1,2-hexanediol, 1,5-hexanediol, 1,6-hexanediol, 1,8-octanediol, 1,10-decanediol, 2,2,4,4-tetramethylcyclobutane-1,3-diol, 1,3-cyclopentanediol, 1,2-cyclohexanediol, 1,3-cyclohexanediol, etc. Alcohols, 1,4-cyclohexanediol, 1,2-cyclohexanediethanol, 1,3-cyclohexanediethanol, 1,4-cyclohexanediethanol, 1,4-cyclohexanediethanol, isosorbide, monoglycerides, monoglycerides, trimethylolpropane monoesters, trimethylolpropane monoethers, pentaerythritol diesters, pentaerythritol diethers, dipropylene glycol, diethylene glycol, triethylene glycol, 2-methyl-1,3-propanediol, 2,2-dimethyl-1,3-propanediol, neopentyl glycol, benzyl glycol, bis(p-hydroxy)diphenyl, bis(p-hydroxy)diphenylpropane, 2,2'-bis[4-(2-hydroxyethoxy)phenyl]propane, bis[4-(2-hydroxyethoxy)phenyl]sulfone, 1,1-bis[4-(2-hydroxyethoxy)phenyl]cyclohexane, and combinations thereof. For example, in some embodiments, the organic solvent is selected from lower molecular weight diols, such as 1,2-ethylene glycol, 1,2-propanediol, 1,3-propanediol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 1,2-pentanediol, 1,5-pentanediol, 2,2-dimethylpropane-1,3-diol, 2-butyl-2-ethylpropane-1,3-diol, 2-methyl-2,4-pentanediol, 2-ethyl-1,3-hexanediol, 2-methyl-1,3-propanediol, 1,2-hexanediol, 1,5-hexanediol, and / or 1,6-hexanediol.

[0091] In some embodiments, the organic solvent comprises a diol selected from the following: 1,2-ethylene glycol, 1,2-propanediol, 1,3-propanediol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 1,2-pentanediol, 1,5-pentanediol, and any combination thereof.

[0092] In some embodiments, the organic solvent comprises a diol selected from the group consisting of 1,2-ethylene glycol, 1,2-propanediol, 1,3-propanediol, and any combination thereof. In some embodiments, the organic solvent comprises 1,2-propanediol.

[0093] In some embodiments, the organic solvent comprises at least one diol and a combination of at least one of the monohydric alcohols, polyhydric alcohols and / or polar aprotic compounds described herein.

[0094] In some embodiments, the organic solvent comprises a polyol selected from the group consisting of: alditol, 2-ethyl-2-hydroxymethyl-1,3-propanediol, 1,2,4-butanetriol, 1,2,5-pentanetriol, 1,2,6-hexanetriol, 1,2,3,6-hexanetetraol, glycerol (also known as glycerol), diglycerol, triglycerol, tetraglycerol, pentaglycerol, hexaglycerol, triethanolamine, trimethylolethane, and trimethylolpropane. The organic solvent is selected from low molecular weight polyols such as bis(trimethylolpropane), tri-trimethylolpropane, 2-methylpropanetriol, 2-methyl-1,2,4-butanetriol, pentaerythritol, dipentaerythritol, tripentaerythritol, methyl glucoside, sorbitol, mannitol, sucrose, 1,3,5-trihydroxybenzene, 1,2,4-trihydroxybenzene, erythritol, inositol, threitol, arabinitol, xylitol, ribitol, galactitol, mannitol, sorbitol, and combinations thereof. For example, in some embodiments, the organic solvent is selected from low molecular weight polyols such as alditol, 1,2,3-propanetriol, 2-ethyl-2-hydroxymethyl-1,3-propanediol, 1,2,4-butanetriol, 1,2,5-pentanetriol, 1,2,6-hexanetriol, 1,2,3,6-hexanetriol, glycerol (also known as glycerol), and / or diglycerol.

[0095] In some embodiments, the organic solvent comprises a polyol selected from the following: glycerol (also known as glycerol), diglycerol, triethanolamine, trimethylolethane, trimethylolpropane, bis(trimethylolpropane), tri-trimethylolpropane, 2-methylglycerol, 2-methyl-1,2,4-butanetriol, and any combination thereof.

[0096] In some embodiments, the organic solvent comprises a polyol selected from the following: glycerol (also known as glycerol), diglycerol, trimethylolethane, trimethylolpropane, and any combination thereof.

[0097] In some embodiments, the organic solvent comprises at least one polyol and a combination of at least one of the monohydric alcohols, dihydric alcohols and / or polar aprotic compounds described herein.

[0098] In some embodiments, the organic solvent does not include sugar alcohols. In some embodiments, the organic solvent does not include one or any combination of the following sugar alcohols: arabinitol, erythritol, fructose, galactitol, glucose, idutitol, isomaltitol, lactitol, lactose, mannitol, maltitol, maltose, maltotriose, inositol, perseitol, ribitol, sorbitol, sucrose, threitol, trehalose, volemitol, xylitol, xylitose, etc. In some embodiments, the organic solvent does not include glycerol.

[0099] In some embodiments, the organic solvent includes a polar aprotic compound selected from: acetone (ACE), acetonitrile (ACN), 4-acetylmorpholine, N-cyclohexyl-2-pyrrolidone (CHP), 1,2-dimethoxyether (DME), N,N-dimethylacetamide (DMA), N,N-diethylacetamide, dimethylformamide (DMF), diethylformamide, N,N-dimethylpropionamide, 3-methoxy-N,N-dimethylpropionamide, 3-methoxy-N,N-diethylpropionamide, 3-methoxy-N,N-methylethylpropionamide, 3-ethoxy-N,N-dimethylpropionamide, 1,3-dimethyl... -2-Imidazolidinone (DMI), dimethyl sulfoxide (DMSO), 1,4-dioxane, 1,3-dioxolane (DN), N,N'-dimethylacrylurea (DMPU), hexamethylphosphoramide (HMPA), N-methyl-2-pyrrolidone (NMP), N-ethyl-2-pyrrolidone, N-methyl-ε-caprolactam, 2-methyltetrahydrofuran (Me-THF), 2,5-dimethyltetrahydrofuran, 4-propionylmorpholine, sulfolane, tetrahydrofuran (THF), tris(N,N-tetramethylene)phosphotriamide, alcohol ethoxylates, diethyl ester dimethylammonium chloride, linear alkylbenzene sulfonates, and combinations thereof. For example, in some embodiments, the organic solvent is selected from acetone (ACE), acetonitrile (ACN), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), N,N'-dimethylacrylurea (DMPU), hexamethylphosphoramide (HMPA), N-methyl-2-pyrrolidone (NMP) and / or tetrahydrofuran (THF).

[0100] In some embodiments, the organic solvent comprises a polar aprotic compound selected from the following: acetone (ACE), acetonitrile (ACN), 1,2-dimethoxy ether (DME), N,N-dimethylacetamide (DMA), N,N-diethylacetamide, dimethylformamide (DMF), N,N-dimethylpropionamide, 3-methoxy-N,N-dimethylpropionamide, dimethyl sulfoxide (DMSO), N,N'-dimethylacrylamide (DMPU), hexamethylphosphoramide (HMPA), N-methyl-2-pyrrolidone (NMP), N-ethyl-2-pyrrolidone, N-methyl-ε-caprolactam, tetrahydrofuran (THF), and any combination thereof.

[0101] In some embodiments, the organic solvent comprises a polar aprotic compound selected from the following: acetone (ACE), acetonitrile (ACN), N,N-dimethylacetamide (DMA), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), N,N'-dimethylacrylurea (DMPU), hexamethylphosphoramide (HMPA), N-methyl-2-pyrrolidone (NMP), tetrahydrofuran (THF), and any combination thereof.

[0102] In some embodiments, the organic solvent comprises a combination of at least one polar aprotic compound and at least one of the monohydric alcohols, dihydric alcohols and / or polyhydric alcohols described herein.

[0103] In some embodiments, the organic solvent is selected from methanol (MeOH), ethanol (EtOH), 1,2-propanediol (PG), glycerol, acetone (ACE), acetonitrile (ACN), N,N-dimethylacetamide (DMA), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), N,N'-dimethylacrylurea (DMPU), hexamethylphosphoramide (HMPA), N-methyl-2-pyrrolidone (NMP), tetrahydrofuran (THF), and combinations thereof. For example, in some embodiments, the organic solvent is selected from DMSO, DMA, PG, and any combination thereof.

[0104] In some embodiments, the first exchange medium used is one of those media listed in Table 5 that contain at least one organic solvent. In one embodiment, the at least one organic solvent used is at least one of DMA, acetone, acetonitrile, THF, ethanol, and 1,2-propylene glycol. In one embodiment, the at least one organic solvent is acetonitrile. In one embodiment, it is 15% to 25% acetonitrile. In another embodiment, it is about 20% acetonitrile. In one embodiment, the at least one organic solvent is THF. In one embodiment, the at least one organic solvent is 10% to 15% THF. In one embodiment, the at least one organic solvent is about 15% THF. In one embodiment, the at least one organic solvent is 15% to 25% ethanol. In one embodiment, it is about 20% ethanol. In one embodiment, a combination of acetonitrile and 1,2-propylene glycol is used as the organic solvent. In one embodiment, the buffer used with such amounts of organic solvent comprises about 50 mM PBS at pH 7.2. In another embodiment, the buffer used is 20 mM histidine at pH 5.5. In some embodiments, the first exchange medium used is one of those media listed in Table 6. In some implementations, the first exchange medium used is one of those listed in Table 7.

[0105] In some embodiments, the amount of impurities in the purified product is less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%. In some embodiments, the amount of impurities in the purified product is 0.1%-5%, 0.1%-4%, 0.1%-3%, 0.1%-2%, or 0.1%-1%.

[0106] In some embodiments, the organic solvent is miscible with water at room temperature. In some embodiments, the organic solvent becomes miscible with water when the resulting mixture is heated to at least 30°C. In some embodiments, the organic solvent becomes miscible with water when the resulting mixture is cooled to a temperature between 5°C and 15°C. In some embodiments, when used in combination, at least two organic solvents are miscible with water. In some embodiments, when used in combination and when the resulting mixture is heated to at least 30°C, at least two organic solvents are miscible with water. In some embodiments, when used in combination and when the resulting mixture is cooled to a temperature in the range of 5°C to 15°C, at least two organic solvents are miscible with water. In some embodiments, the organic solvent is partially soluble in water. In some embodiments, the organic solvent is partially soluble in water when the resulting mixture is heated to at least 30°C. In some embodiments, the organic solvent is partially soluble in water when the resulting mixture is heated to at least 30°C.

[0107] In some embodiments, the organic solvent has a boiling point of at least 150°C. In some embodiments, the organic solvent has a boiling point of at least 160°C. In some embodiments, the organic solvent has a boiling point of at least 170°C. In some embodiments, the organic solvent has a boiling point in the range of about 50°C to about 300°C, about 80°C to about 275°C, about 100°C to about 250°C, or about 150°C to about 225°C. In some embodiments, the organic solvent is a solid in its pure form at room temperature.

[0108] In some embodiments, the organic solvent has a concentration of at least 0.7 g / cm³. 3 The density. In some embodiments, the organic solvent has a density of at least 0.8 g / cm³. 3 The density. In some embodiments, the organic solvent has a density of at least 0.9 g / cm³. 3 The density. In some embodiments, the organic solvent has a density of at least 1.0 g / cm³. 3 The density. In some embodiments, the organic solvent has a density of at least 1.1 g / cm³. 3 The density. In some embodiments, the organic solvent has a density of at least 1.2 g / cm³. 3 The density. In some embodiments, the organic solvent has a density in the range of about 0.6 g / cm³. 3 To approximately 1.5 g / cm 3 Or approximately 0.7 g / cm 3 Approximately 1.4 g / cm 3 or approximately 0.8 g / cm 3 Approximately 1.3 g / cm 3 Or approximately 0.9 g / cm 3 Approximately 1.3 g / cm 3 Or approximately 1.0 g / cm³ 3 Approximately 1.3 g / cm 3 The density.

[0109] In some embodiments, the organic solvent has a polarity index of at least 3.0. In some embodiments, the organic solvent has a polarity index of at least 4.0. In some embodiments, the organic solvent has a polarity index of at least 5.0. In some embodiments, the organic solvent has a polarity index of at least 6.0. In some embodiments, the organic solvent has a polarity index of at least 7.0. In some embodiments, the organic solvent has a polarity index of at least 8.0. In some embodiments, the organic solvent has a polarity index ranging from about 3.0 to about 9.0, or about 4.0 to about 8.5, or about 4.5 to about 8.5, or about 5.0 to about 8.5, or about 5.0 to about 8.0, or about 5.5 to about 8.0.

[0110] In some embodiments, the organic solvent has a logP of less than 0, as measured at 25°C. In some embodiments, the organic solvent has a logP of less than -0.1, as measured at 25°C. In other embodiments, the organic solvent has a logP of about -0.04 to -1.5, as measured at 25°C.

[0111] In some embodiments, the first exchange medium comprises at least two organic solvents. In some embodiments, the first exchange medium comprises at least three organic solvents. In some embodiments, the first exchange medium comprises an alcohol compound and a polar aprotic compound. In some embodiments, the first exchange medium comprises a monohydric alcohol and a polar aprotic compound. In some embodiments, the first exchange medium comprises a dihydric alcohol and a polar aprotic compound. In some embodiments, the first exchange medium comprises a polyhydric alcohol and a polar aprotic compound. In some embodiments, the first exchange medium comprises multiple alcohol compounds. In some embodiments, the first exchange medium comprises multiple polar aprotic compounds.

[0112] In some embodiments, the proportion of organic solvent in the first exchange medium ranges from about 1% to about 30% based on the mass of organic solvent relative to the total volume of the first exchange medium. In some embodiments, the proportion of organic solvent in the first exchange medium ranges from about 1% to about 30%, or about 2% to about 28%, or about 3% to about 27%, or about 4% to about 26%, or about 5% to about 25%, or about 6% to about 24%, or about 7% to about 23%, or about 8% to about 22%, or about 9% to about 21%, or about 10% to about 20%. In some embodiments, the proportion of organic solvent in the first exchange medium ranges from about 5% to about 20% based on the mass of organic solvent relative to the total volume of the first exchange medium. In some embodiments, the first exchange medium comprises at least two organic solvents, such that the total proportion of the at least two organic solvents in the first exchange medium ranges from about 1% to about 30% based on the total mass of the at least two organic solvents relative to the total volume of the first exchange medium. In some embodiments, the total proportion of at least one organic solvent in the first exchange medium is less than about 30%, based on the total mass of at least one organic solvent relative to the total volume of the first exchange medium.

[0113] In some embodiments, the first and second buffer systems are independently selected from: acetate buffer systems, ADA ((N-(2-acetamino)iminoacetic acid) buffer systems, ACES (N-(2-acetamino)-2-aminoethanesulfonic acid) buffer systems, AMPD (2-amino-2-methyl-1,3-propanediol) buffer systems, AMPSO (3-(1,1-dimethyl-2-hydroxyethyl)amino-2-hydroxypropanesulfonic acid) buffer systems, BES (N,N-bis-(2-hydroxyethyl)-2-aminoethanesulfonic acid) buffer systems, bicarbonate buffer systems, bicine (N,N-bis(2-hydroxyethyl)glycine) buffer systems, bis-tris(2-bis(2-hydroxyethyl)amino-2-(hydroxymethyl)-1,3-propanediol) buffer systems, BTP (1,3-bis(tris(hydroxymethyl)methylamino)propane) buffer systems, CABS (4-(cyclohexylamino)-1-butyric acid) buffer systems, CAPS (N-cyclohexyl-3-aminopropanesulfonic acid) buffer system, CAPSO (3-(cyclohexylamino)-2-hydroxypropanesulfonic acid) buffer system, citrate buffer system, DIPSO (3-(N,N-bis[2-hydroxyethyl]amino)-2-hydroxypropanesulfonic acid) buffer system, EDTA (ethylenediaminetetraacetic acid) buffer system, Gly-Gly buffer system, His-Glu buffer system, HEPBS (N-(2-hydroxyethyl)piperazine-N'-(4-butyric acid)) buffer system, HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid) buffer system, HEPPSO ((2-hydroxyethyl)piperazine-N-2-hydroxypropanesulfonic acid) buffer system, histidine buffer system, MES (2-(N-morpholino)ethanesulfonic acid) buffer system, MOBS (4-(4-morpholino)butyric acid) buffer system, MOPS (3-(N-morpholino)propanesulfonic acid) buffer system, MOPSO The following buffer systems are available: (3-morpholino-2-hydroxypropanesulfonic acid) buffer system, PB (phosphate buffer) buffer system, PBS (phosphate buffered saline) buffer system, PIPES (piperazine-N,N′-bis(2-ethanesulfonic acid)) buffer system, POPSO (piperazine-N,N′-bis(2-hydroxypropanesulfonic acid)) buffer system, succinate buffer system, TES (2-{[1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl]amino}ethane-1-sulfonic acid) buffer system, TAPSO (N-[tris(hydroxymethyl)methyl]-3-amino-2-hydroxypropanesulfonic acid) buffer system, TAE (triacetate-EDTA) buffer system, Tricine (N-(tris(hydroxymethyl)methyl)glycine) buffer system, and TAPS ([tris(hydroxymethyl)methylamino]propanesulfonic acid) buffer system.

[0114] In some implementations, the first and second buffer systems are independently selected from: ADA buffer system, acetate buffer system, bicarbonate buffer system, Bis-Tris buffer system, CABS buffer system, citrate buffer system, EDTA buffer system, Gly-Gly buffer system, HIS (histidine) buffer system, His-Glu buffer system, His-Gly buffer system, PB (phosphate buffer) buffer system, PBS (phosphate-buffered saline) buffer system, succinate buffer system, and Tricine buffer system.

[0115] In some implementations, the first and second buffer systems are independently selected from: acetate buffer systems, bicarbonate buffer systems, citrate buffer systems, Gly-Gly buffer systems, HIS (histidine) buffer systems, PB (phosphate buffer) buffer systems, PBS (phosphate buffered saline) buffer systems, and succinate buffer systems.

[0116] In some embodiments, the first buffer system and the second buffer system are the same buffer. In other embodiments, the first buffer system and the second buffer system are different buffers. In some embodiments, the first exchange medium and the second exchange medium differ only in that the first exchange medium contains at least one organic solvent not present in the second exchange medium. In other embodiments, the two exchange media differ in other respects.

[0117] In some embodiments, the concentrations of the first aqueous buffer and the second aqueous buffer are the same. In other embodiments, the concentrations of the first aqueous buffer and the second aqueous buffer are different.

[0118] In some embodiments, organic UF / DF and aqueous UF / DF are carried out in the same residue volume, e.g., in the same apparatus. In other embodiments, organic UF / DF and aqueous UF / DF are carried out in different residue volumes, e.g., in different apparatuses. In some embodiments, the organic UF / DF process and the aqueous UF / DF process are carried out as tangential flow filtration processes.

[0119] In some embodiments, the pH ranges of the first and second exchange media are independently from about 4.0 to about 10.0 or about 4.5 to about 9.5, about 5.0 to about 9.5, about 5.0 to about 9.0, about 5.0 to about 8.5, about 5.0 to about 8.0, about 5.5 to about 8.0, or about 5.5 to about 7.5. In some embodiments, the first and second exchange media have the same pH. In other embodiments, the first and second exchange media have different pH values.

[0120] In some implementations, organic UF / DF and aqueous UF / DF are used independently in the range of about 50 L / hr / m 2 Approximately 800 L / hr / m 2 or approximately 100 L / hr / m 2 Approximately 700 L / hr / m 2 or approximately 150 L / hr / m 2 Approximately 600 L / hr / m 2 or approximately 200L / hr / m 2 Approximately 600 L / hr / m 2 or approximately 200 L / hr / m 2 Approximately 550 L / hr / m 2 or approximately 200 L / hr / m 2 Approximately 500 L / hr / m 2 or approximately 250 L / hr / m 2 Approximately 500 L / hr / m 2 or approximately 250 L / hr / m 2 Approximately 450 L / hr / m 2 or approximately 250 L / hr / m 2 Approximately 400 L / hr / m 2 The flux rate is constant. In some embodiments, organic UF / DF and aqueous UF / DF are processed at the same flux rate. In other embodiments, organic UF / DF and aqueous UF / DF are processed at different flux rates.

[0121] In some embodiments, the organic UF / DF and aqueous UF / DF are induced independently at transmembrane pressures ranging from about 2 psi to about 50 psi, or about 5 psi to about 40 psi, or about 5 psi to about 30 psi, or about 10 psi to about 30 psi, or about 10 psi to about 25 psi, or about 15 psi to about 25 psi. In some embodiments, the organic UF / DF and aqueous UF / DF are induced at the same transmembrane pressure. In other embodiments, the organic UF / DF and aqueous UF / DF are induced at different transmembrane pressures.

[0122] In some embodiments, the organic UF / DF and aqueous UF / DF are carried out independently, such that the temperature range in the residue chamber containing the compound is from about 5°C to about 60°C, or from about 5°C to about 55°C, or from about 5°C to about 50°C, or from about 5°C to about 45°C, or from about 5°C to about 40°C, or from about 5°C to about 35°C, or from about 5°C to about 30°C, or from about 5°C to about 25°C, or from about 5°C to about 20°C, or from about 5°C to about 15°C, or from about 5°C to about 10°C. In some embodiments, the organic UF / DF and aqueous UF / DF are carried out at the same temperature in the residue chamber. In other embodiments, the organic UF / DF and aqueous UF / DF are carried out at different temperatures in the residue chamber.

[0123] In some embodiments, the percolation volume (DV) ranges independently for organic UF / DF and aqueous UF / DF as about 2 to about 50, about 2 to about 35, about 2 to about 30, about 2 to about 25, about 2 to about 20, about 2 to about 18, about 2 to about 17, about 2 to about 16, about 2 to about 15, about 2 to about 14, about 2 to about 13, about 2 to about 12, about 2 to about 11, about 2 to about 10, about 2 to about 9, about 2 to about 8, about 2 to about 7, or about 2 to about 6. In some embodiments, the DV is at least 5. In some embodiments, the DV of organic UF / DF and aqueous UF / DF are the same. In other embodiments, the DV of organic UF / DF and aqueous UF / DF are different. In some embodiments, the DV range of organic UF / DF is about 12 to 16, about 12 to 15, or about 12 to 14.

[0124] In some embodiments, the first and second semipermeable membranes are independently selected from polyolefins, polystyrene, polysulfone, polyesters, polyamides, polyacrylates, polycarbonates, and copolymers and combinations thereof. In some embodiments, the first and second semipermeable membranes are independently selected from polyethersulfone (PES), polytetrafluoroethylene (PTFE), polypropylene, sulfonated polysulfone, polyvinylidene fluoride (PVDF), polyacrylonitrile (PAN), cellulose polymers, polyimide, polyetherimide (PEI), aliphatic polyamide, polyetheretherketone (PEEK), polyphenylene ether (PPO), polysulfone (PSf), and mixtures and copolymers thereof. In some embodiments, the first and second semipermeable membranes are the same semipermeable membrane. In other embodiments, the first and second semipermeable membranes are different semipermeable membranes. In some embodiments, both the first and second semipermeable membranes are uncharged neutral membranes. In other embodiments, only one of the first and second semipermeable membranes is an uncharged neutral membrane.

[0125] In some embodiments, the first aperture and the second aperture range independently are about 0.2 kDa MWCO to about 500 kDa MWCO, or about 0.2 kDa MWCO to about 1 kDa MWCO, or about 1 kDa MWCO to about 10 kDa MWCO, or about 10 kDa MWCO to about 50 kDa MWCO, or about 50 kDa MWCO to about 100 kDa MWCO, or about 75 kDa MWCO to about 125 kDa MWCO, or about 100 kDa MWCO to about 200 kDa MWCO, or about 200 kDa MWCO to about 500 kDa MWCO.

[0126] In some implementations, the organic UF / DF process and the aqueous UF / DF process are performed as tangential flow filtration (TFF) processes.

[0127] In some embodiments, the concentration of the ADC or other conjugate compound in the crude composition ranges from about 0.5 mg / mL to about 500 mg / mL, or about 0.5 mg / mL to about 200 mg / mL, or about 1 mg / mL to about 200 mg / mL, or about 2 mg / mL to about 150 mg / mL, or about 1 mg / mL to about 100 mg / mL, or about 5 mg / mL to about 100 mg / mL, or about 5 mg / mL to about 50 mg / mL, or about 5 mg / mL to about 25 mg / mL.

[0128] In some embodiments, the amount of ADC (or other conjugate compound) in the crude composition to be purified ranges from about 1 gram to about 1 kilogram. In some embodiments, the amount of ADC (or other conjugate compound) in the crude composition ranges from about 10 grams to about 1 kilogram. In some embodiments, the amount of ADC (or other conjugate compound) in the crude composition ranges from about 10 grams to about 1 kilogram. In some embodiments, the amount of ADC (or other conjugate compound) in the crude composition ranges from about 100 grams to about 1 kilogram. In some embodiments, the amount of ADC (or other conjugate compound) in the crude composition ranges from about 100 grams to about 10 kilograms. In some embodiments, the amount of ADC (or other conjugate compound) in the crude composition ranges from about 1 kilogram to about 10 kilograms.

[0129] In some embodiments, in addition to ADC or conjugates other than ADC, the crude mixture also contains at least one of residual free drug compound, residual drug-connector compound, and / or residual connector compound. In some embodiments, in addition to ADC or conjugates other than ADC, the crude mixture also contains at least one of residual free drug compound, residual drug-connector compound, residual connector compound, and / or residual byproduct.

[0130] In some embodiments, the impurities comprise pharmaceutical compounds selected from: topoisomerase I (TOP1) inhibitors, topoisomerase II (TOP2) inhibitors, microtubule disruptors, DNA damaging agents (such as alkylating agents), protein degrading agents, TLR7 agonists, TLR8 agonists, STING agonists, XPO1 inhibitors, and derivatives thereof. For example, the crude mixture may comprise at least one pharmaceutical compound selected from: eczema, DXd, camptothecin, SN-38 (7-ethyl-10-hydroxycamptothecin), microtubule disruptors such as eribulin, DM1 (maytansine), DM1 derivatives, DM4 (maytansine), MMAE (monomethylaurestatin E), monomethylaurestatin F (MMAF), DNA intercalating agents such as pyrrolobenzodiazepine (PBD), DNA alkylating agents such as duocarmycin, and others such as leptomysin B and derivatives thereof. In some embodiments, the crude mixture may contain eczema or its derivatives.

[0131] In some embodiments, the impurity comprises a residual connector compound, such as the connector or a derivative thereof described in PCT / CN2021 / 104174 (Publication No. WO 2022 / 217022) and PCT / CN2024 / 071901 (Publication No. WO2024149345A1), all of which are incorporated herein by reference in their entirety. In some embodiments, the connector compound has the following structure:

[0132] .

[0133] In some embodiments, the connector compound has the following structure:

[0134] .

[0135] In some embodiments, the impurity comprises a drug-connector compound containing at least one pharmaceutical moiety derived from one of the drug compounds listed above as a payload. In some embodiments, the impurity comprises a drug-connector compound containing at least one connector moiety derived from one of the aforementioned connector compounds. In some embodiments, the drug-connector compound in its free form is one of the drug-connector compounds or derivatives thereof described in PCT / CN2021 / 104174 (Publication No. WO 2022 / 217022) and PCT / CN2024 / 071901 (Publication No. WO2024149345A1), all of which are incorporated herein by reference in their entirety. In some embodiments, the drug-connector compound is the LD038 compound provided in Example 1 of this disclosure and has the following structure:

[0136] .

[0137] In some embodiments, the drug-connector compound is an LD343 compound and has the following structure:

[0138] .

[0139] In some embodiments, the crude mixture contains an ADC. In some embodiments, the ADC contains a humanized antibody, a fully human antibody, a chimeric antibody, a bispecific antibody, a trispecific antibody, or a multispecific antibody. In some embodiments, the ADC contains a monoclonal antibody, Fab, Fab', F(ab'), Fv, a disulfide-linked Fc, scFv, a single-domain antibody, a bispecific antibody, a bispecific antibody, or a multispecific antibody. In some embodiments, the ADC contains scFv1-ScFv2, scFv12-Fc-scFv22, IgG-scFv, DVD-Ig, triomab / quadroma, two-in-one IgG, scFv2-Fc, TandAb, and scFv-HSA-scFv.

[0140] In some embodiments, the ADC comprises a drug-linker moiety in the form of a covalently bound antibody, such as the drug-linker moiety or derivative described in WO2022 / 217022 and WO2024 / 092067, all of which are incorporated herein by reference in their entirety. In some embodiments, the drug-linker moiety is a derivative of the LD038 compound provided in Example 1 of this disclosure. In some embodiments, the drug-linker moiety is a portion formed based on a linker (such as mc-VC-PAB, CL2, CL2A, or (succinimide-3-yl-N)-(CH2)nC(=O)-Gly-Gly-Phe-Gly-NH-CH2-O-CH2-(C=O)-) and a drug (such as camptothecin or a camptothecin derivative, such as eczema).

[0141] In some embodiments, the crude mixture contains conjugates other than the ADC. In some embodiments, the conjugates other than the ADC contain non-antibody protein scaffolds. Such non-antibody scaffolds include, for example, affibody, affilin, anticalin, attrimer, avimer, bicyclic peptides, cys-knot, DARPin, FN3 scaffolds (e.g., adnectin, centyrin, pronectin, and Tn3), fynomer, Kunitz domains, and OBody. (See, for example, Vazquez-Lombardi et al., Drug Discovery Today 20(10):1271 (2015) and references cited therein.) Such non-antibody protein scaffolds include, for example, affibody, affilin, anticalin, attrimer, avimer, bicyclic peptides, cys-knot, DARPin, FN3 scaffolds (e.g., adnectin, centyrin, pronectin, and Tn3), fynomer, Kunitz domains, and OBody. (See, for example, Vazquez-Lombardi et al., Drug Discovery Today 20(10):1271 (2015) and the references cited therein)

[0142] In some embodiments, the ADC (or conjugates other than ADC) has a molecular weight of at least 25 kilodaltons (kDa), at least 50 kDa, at least 75 kDa, or at least 100 kDa.

[0143] In some embodiments, the crude mixture comprises an antibody-drug conjugate (ADC), such as those described in PCT / CN2021 / 104174 (Publication No. WO 2022 / 217022), PCT / US2023 / 077814 (Publication No. WO 2024 / 092067), and CN202310035642.4 (Publication No. CN116036303A), all of which are incorporated herein by reference in their entirety. In some embodiments, the crude mixture comprises an antibody-drug conjugate (ADC) comprising at least one pharmaceutical moiety of a pharmaceutical compound derived from the group consisting of: eczema, camptothecin, eribulin, SN-38 (7-ethyl-10-hydroxy-camptothecin), DM1 (matansine), leptomysin B, and MMAE (monomethylaurestatin E). In some implementations, the pharmaceutical component is camptothecin selected from the group consisting of: 10-hydroxycamptothecin, topotecan, irinotecan, 9-aminocamptothecin, 9-nitrocamptothecin, SN-38, lurtotecan, BMS422461, CMMD-Gly, morpholino-CPT, ixotecan, belotecan, DB-67, calentecin, ST1481, and chimmitecan.

[0144] In some implementations, the ADC has the following structure: . In some embodiments, n is 8 and Ab represents antibody F131 comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region comprises a complementarity-determining region HCDR1 having the amino acid sequence of SEQ ID NO: 1, HCDR2 having the amino acid sequence of SEQ ID NO: 2, and HCDR3 having the amino acid sequence of SEQ ID NO: 3, and the VL region comprises an LCDR1 having the amino acid sequence of SEQ ID NO: 4, an LCDR2 having the amino acid sequence of SEQ ID NO: 5, and an LCDR3 having the amino acid sequence of SEQ ID NO: 6. In some embodiments, n is 8 and Ab represents antibody 2E7 comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region comprises a complementarity-determining region HCDR1 having the amino acid sequence of SEQ ID NO: 7, HCDR2 having the amino acid sequence of SEQ ID NO: 8, and HCDR3 having the amino acid sequence of SEQ ID NO: 9, and the VL region comprises LCDR1 having the amino acid sequence of SEQ ID NO: 10, LCDR2 having the amino acid sequence of SEQ ID NO: 11, and LCDR3 having the amino acid sequence of SEQ ID NO: 12. In some embodiments, n is 8 and Ab represents trastuzumab.

[0145] In some embodiments, the conjugate compound has an ADC with a drug-antibody ratio ranging from about 1:1 to about 30:1, or about 1:1 to about 25:1, or about 1:1 to about 24:1, or about 1:1 to about 20:1, or about 1:1 to about 16:1, or about 1:1 to about 10:1, or about 1:1 to about 9:1, or about 1:1 to about 8:1, or about 1:1 to about 7:1, or about 1:1 to about 6:1, or about 1:1 to about 5:1, or about 1:1 to about 4:1.

[0146] In some embodiments, the crude composition is a conjugated mixture comprising an antibody-drug conjugate (ADC) (or a conjugate other than an ADC) and at least one of a residual free drug compound, a residual adapter compound, a residual free drug-adaptor compound, and / or a residual byproduct. In some embodiments, based on the total volume of the purified conjugated compound, the concentration of the residual free drug compound, the residual adapter compound, the residual drug-adaptor compound, or any combination thereof in the purified composition is less than 200 μg / mL, less than 150 μg / mL, less than 100 μg / mL, less than 75 μg / mL, less than 50 μg / mL, or less than 40 μg / mL. The purified conjugate compounds described herein are present in concentrations ranging from 1 mg / mL to 200 mg / mL, 2 mg / mL to 150 mg / mL, 3 mg / mL to 120 mg / mL, 4 mg / mL to 80 mg / mL, 5 mg / mL to 50 mg / mL, 8 mg / mL to 40 mg / mL, 10 mg / mL to 35 mg / mL, or 12 mg / mL to 30 mg / mL. In some embodiments, the purified conjugate compounds described herein are present in concentrations of 15 mg / mL, 18 mg / mL, 20 mg / mL, 25 mg / mL, or 30 mg / mL.

[0147] Methods for preparing antibody-drug conjugates (ADCs) and conjugates other than ADCs

[0148] In some embodiments, a method is provided for preparing a purified conjugate compound using at least one separately prepared drug-connector compound, comprising the steps of: (i) reducing a target agent by contacting it with a reducing agent to obtain a crude reduced target agent; (ii) conjugating the reduced target agent with a drug-connector compound to obtain a crude composition comprising the conjugate compound; (iii) optionally adding a quencher to the crude composition; and (iv) purifying the crude composition using the UF / DF method of this disclosure, wherein the first exchange medium comprises at least one organic solvent.

[0149] In some embodiments, a method is provided for preparing a purified conjugate compound by sequential attachment of a linker group and a pharmaceutical compound, comprising the steps of: (i) reducing a target agent by contacting it with a reducing agent to obtain a crude reduced target agent; (ii) attaching at least one linker group to the reduced target agent to obtain a crude composition comprising a target agent-linker intermediate; (iii) attaching at least one pharmaceutical compound to a linker of the target agent-linker intermediate to obtain a crude composition comprising a conjugate compound; (iv) optionally adding a quencher to the crude composition; and (v) purifying the crude composition using the UF / DF method of this disclosure, wherein a first exchange medium comprises at least one organic solvent to obtain the purified conjugate compound.

[0150] In some implementations, the conjugation reaction is carried out as generally described in WO 2023 / 280227, WO 2005 / 081711, WO 2005 / 077090, WO 2012 / 135517, WO 2006 / 086733, WO 2006 / 034488 and WO 2013 / 055993.

[0151] In some embodiments, the target is an antibody or antigen-binding fragment, and the purified conjugate compound is a purified antibody-drug conjugate (ADC). In other embodiments, the target is an antibody-free target, and the purified conjugate compound is a purified conjugate other than an ADC.

[0152] In some embodiments, suitable reducing agents include sulfide reducing agents that break disulfide bonds, such as tris(2-carboxyethyl)phosphine (TCEP), 2-mercaptoethanol (BME), dithiothreitol (DTT), dithioerythritol (DTE), sodium borohydride, sodium cyanoborohydride, 3,3',3''-phosphanetriyltris(benzenesulfonic acid) trisodium (TPPTS), cysteine ​​hydrochloride, and cysteine. In some embodiments, the reducing agent is TCEP.

[0153] In some implementations, suitable quenchers include cysteine, TCEP, ketone quenchers such as acetone, alcohol quenchers such as tert-butanol, amine quenchers such as ethanolamine, azide quenchers such as penta-PEG azide (e.g., Kantner et al., ACS Omega (2017), 2, 5785-5791), and maleimide or haloacetamide quenchers such as 4-maleimidebutyric acid, 3-maleimidepropionic acid, N-ethylmaleimide, iodoacetamide, or iodoacetamidepropionic acid, or combinations thereof.

[0154] In some embodiments, the method for preparing the purified conjugate compound further includes purifying the crude target agent by performing an initial aqueous UF / DF process on the crude reduced target agent before converting it into the conjugate compound. The initial aqueous UF / DF process can be performed in a manner similar to step (c) aqueous UF / DF of the method disclosed herein, or the initial aqueous UF / DF process can be performed using a conventional UF / DF process.

[0155] In some embodiments, the conjugate compound comprises at least one pharmaceutical moiety derived from a pharmaceutical compound selected from the group consisting of: topoisomerase I (TOP1) inhibitors, topoisomerase II (TOP2) inhibitors, microtubule disruptors, DNA damaging agents, DNA intercalating agents, protein degrading agents, TLR7 agonists, TLR8 agonists, STING agonists, and XPO1 inhibitors. In some embodiments, the pharmaceutical compound is selected from eczema, iribulin, DXd, camptothecin, SN-38 (7-ethyl-10-hydroxycamptothecin), DM1 (matansine), DM1 derivatives, DM4 (matansine), MMAE (monomethylaurestatin E), MMAF, pyrrolobenzodiazepine (PBD), and leptomysin B.

[0156] In some implementations, the pharmaceutical component is camptothecin selected from the group consisting of: 10-hydroxycamptothecin, topotecan, irinotecan, 9-aminocamptothecin, 9-nitrocamptothecin, SN-38, lurtotecan, BMS422461, CMMD-Gly, morpholino-CPT, ixotecan, belotecan, DB-67, calentecin, ST1481, and chimmitecan.

[0157] Method for preparing pharmaceutical compositions

[0158] Any method of the present invention may involve the step of formulating a purified pharmaceutical conjugate with a pharmaceutical carrier or excipient to obtain a pharmaceutical composition. Therefore, the present invention also provides a method for producing a pharmaceutical composition comprising performing the purification method of the present invention and then formulating the purified product with a pharmaceutical carrier or excipient to obtain a pharmaceutical composition comprising the pharmaceutical conjugate. Examples of suitable formulations can be found at Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, lipid-containing (cationic or anionic lipids) vesicles (such as LIPOFECTIN). TM DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsion carbowax (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al., "Compendium of excipients for parenteral formulations," PDA (1998), J Pharm SciTechnol 52:238-311.

[0159] Implementation Plan

[0160] The description of embodiments of this disclosure is not intended to be exhaustive or to limit this disclosure to the precise form disclosed. Although specific embodiments and examples of this disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of this disclosure, as will be recognized by those skilled in the art. The teachings of this disclosure can be suitably applied to other procedures or methods. The various embodiments described herein can be combined to provide further embodiments. If desired, aspects of this disclosure can be modified to incorporate the composition, function, and concept of the foregoing references and applications to provide even further embodiments of this disclosure. These and other changes may be made to this disclosure in consideration of specific implementations.

[0161] Specific elements of any of the foregoing embodiments may be combined with or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of this disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments must exhibit such advantages to fall within the scope of this disclosure.

[0162] All identified patents and such publications are expressly incorporated herein by reference for the purpose of describing and disclosing methods described in other publications, for example, that may be used in conjunction with this invention. These disclosures are provided solely because of their publication prior to the filing date of this application. In this respect, nothing should be construed as an admission that the inventor has no right to claim prior disclosure by virtue of a prior invention or for any other reason. All statements regarding the dates or contents of these documents are based on information available to the applicant and do not constitute any admission of the accuracy of the dates or contents of these documents.

[0163] The following numbered embodiments represent further embodiments of the present invention:

[0164] 1. Purification methods, including:

[0165] (a) Combining a first exchange medium with a crude composition to obtain a crude mixture, wherein the crude composition comprises a conjugated compound and at least one impurity;

[0166] (b) subjecting the crude mixture to an organic ultrafiltration / difiltration (UF / DF) process, such that the crude mixture is filtered through a first semi-permeable membrane having a first pore size that allows impurities to pass through the first semi-permeable membrane into the first filtrate while retaining the conjugate compound in the first filtrate, and the liquid volume filtered from the crude mixture is at least partially replaced in the first filtrate by an additional first exchange medium to obtain a separated composition; and

[0167] (c) The separated composition is subjected to an aqueous UF / DF process, wherein the separated composition is filtered through a second semi-permeable membrane having a second pore size that allows impurities to pass through while retaining the conjugate compound in the second residue, and the liquid volume filtered from the separated composition is at least partially replaced by a second exchange medium comprising a second buffer system to obtain a purified composition in the second residue.

[0168] in:

[0169] Impurities include residual free drug compounds, residual drug-connector compounds, residual connector compounds, residual by-product compounds, residual side-product compounds, or any combination thereof;

[0170] The first exchange medium comprises at least one organic solvent and a first buffer system; and

[0171] The first and second switching media are different.

[0172] 2. According to the method of 1, wherein the separated composition is substantially free of product-bound impurities.

[0173] 3. The method according to 1 or 2, wherein the purified composition is substantially free of organic solvents.

[0174] 4. The method according to any one of 1-3, wherein the organic solvent includes an alcohol compound, a polar aprotic compound, or a combination thereof.

[0175] 5. The method according to any one of 1-4, wherein the organic solvent includes a monohydric alcohol, a dihydric alcohol, a polyhydric alcohol, or a combination thereof.

[0176] 6. The method according to any one of 1-5, wherein the organic solvent comprises a monohydric alcohol selected from the group consisting of: methanol, ethanol, n-propanol, isopropanol, 1-butanol, 2-butanol, tert-butanol, isobutanol, 1-pentanol, 2-pentanol, 3-pentanol, isopentanol, tert-pentanol, hexanol (straight-chain, branched and / or cyclic), ethylene glycol monomethyl ether, cis-3-hexen-1-ol, trans-2-hexen-1-ol, 5-hexen-1-ol, phenol, benzyl alcohol, etc., and combinations thereof.

[0177] 7. The method according to any one of 1-6, wherein the organic solvent comprises a diol selected from the group consisting of: 1,2-ethylene glycol, 1,2-propanediol, 1,3-propanediol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 1,2-pentanediol, 1,5-pentanediol, 2,2-dimethylpropane-1,3-diol, 2-butyl-2-ethylpropane-1,3-diol, 2-methyl-2,4-pentanediol, 2-ethyl-1,3-hexanediol, 2-methyl-1,3-propanediol, 1,2-hexanediol, 1,5-hexanediol, 1,6-hexanediol, 1,8-octanediol, 1,10-decanediol, 2,2,4,4-tetramethylcyclobutane-1,3-diol, 1,3-cyclopentanediol, 1,2-cyclohexanediol 1,3-Cyclohexanediol, 1,4-Cyclohexanediol, 1,2-Cyclohexanediethanol, 1,3-Cyclohexanediethanol, 1,4-Cyclohexanediethanol, 1,4-Cyclohexanediethanol, isosorbide, monoglyceride, monoglyceride, trimethylolpropane monoester, trimethylolpropane monoether, pentaerythritol diester, pentaerythritol diether, dipropylene glycol, diethylene glycol, triethylene glycol, 2-methyl-1,3-propanediol, 2,2-dimethyl-1,3-propanediol, neopentyl glycol, benzyl glycol, bis(p-hydroxy)diphenyl, bis(p-hydroxy)diphenylpropane, 2,2'-bis[4-(2-hydroxyethoxy)phenyl]propane, bis[4-(2-hydroxyethoxy)phenyl]sulfone, 1,1-bis[4-(2-hydroxyethoxy)phenyl]cyclohexane, and combinations thereof.

[0178] 8. The method according to any one of 1-7, wherein the organic solvent comprises a polyol selected from the group consisting of: alditol, 1,2,3-propanetriol, 2-ethyl-2-hydroxymethyl-1,3-propanediol, 1,2,4-butanetriol, 1,2,5-pentanetriol, 1,2,6-hexanetriol, 1,2,3,6-hexanetetraol, glycerol (also known as glycerol), diglycerol, triglycerol, tetraglycerol, pentaglycerol, hexaglycerol, triethanolamine. Trimethylolethane, trimethylolpropane, bis(trimethylolpropane), tri-trimethylolpropane, 2-methylpropanetriol, 2-methyl-1,2,4-butanetriol, pentaerythritol, dipentaerythritol, tripentaerythritol, methyl glucoside, sorbitol, mannitol, sucrose, 1,3,5-trihydroxybenzene, 1,2,4-trihydroxybenzene, erythritol, inositol, threitol, arabinitol, xylitol, ribitol, galactitol, and combinations thereof.

[0179] 9. The method according to any one of 1-8, wherein the organic solvent comprises a polar aprotic compound selected from the group consisting of: acetone (ACE), acetonitrile (ACN), 4-acetylmorpholine, N-cyclohexyl-2-pyrrolidone (CHP), 1,2-dimethoxyether (DME), N,N-dimethylacetamide (DMA), N,N-diethylacetamide, dimethylformamide (DMF), diethylformamide, N,N-dimethylpropionamide, 3-methoxy-N,N-dimethylpropionamide, 3-methoxy-N,N-diethylpropionamide, 3-methoxy-N,N-methylethylpropionamide, 3-ethoxy-N,N-dimethylpropionamide 1,3-Dimethyl-2-imidazolium ketone (DMI), dimethyl sulfoxide (DMSO), 1,4-dioxane, 1,3-dioxolane (DN), N,N'-dimethylacrylurea (DMPU), hexamethylphosphoramide (HMPA), N-methyl-2-pyrrolidone (NMP), N-ethyl-2-pyrrolidone, N-methyl-ε-caprolactam, 2-methyltetrahydrofuran (Me-THF), 2,5-dimethyltetrahydrofuran, 4-propionylmorpholine, sulfolane, tetrahydrofuran (THF), tris(N,N-tetramethylene)phosphotriamide, alcohol ethoxylates, diethyl ester dimethylammonium chloride, linear alkylbenzene sulfonates, and combinations thereof.

[0180] 10. The method according to any one of 1-9, wherein the organic solvent is selected from the group consisting of: methanol, ethanol, 1,2-propanediol, glycerol, acetone (ACE), acetonitrile (ACN), N,N-dimethylacetamide (DMA), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), N,N'-dimethylacrylurea (DMPU), hexamethylphosphoramide (HMPA), N-methyl-2-pyrrolidone (NMP), tetrahydrofuran (THF), and combinations thereof.

[0181] 11. The method according to any one of 1-10, wherein the organic solvent is not a sugar alcohol.

[0182] 12. The method according to any one of 1-11, wherein the organic solvent is miscible with water.

[0183] 13. The method according to any one of 1-12, wherein the organic solvent has a boiling point of at least 150°C.

[0184] 14. The method according to any one of 1-13, wherein the organic solvent has a concentration of at least 0.9 g / cm³. 3 The density.

[0185] 15. The method according to any one of 1-14, wherein the organic solvent has a polarity index of at least 6.0.

[0186] 16. The method according to any one of 1-15, wherein the organic solvent has a logP less than 0, as measured at 25°C.

[0187] 17. The method according to any one of 1-16, wherein the organic solvent has a logP in the range of about -0.04 to -1.5, as measured at 25°C.

[0188] 18. The method according to any one of 1-17, wherein the first exchange medium comprises at least two organic solvents.

[0189] 19. The method according to any one of 1-18, wherein the organic solvent includes a diol and a polar aprotic compound.

[0190] 20. The method according to any one of 1-19, wherein the mass of the organic solvent based on the total volume is relative to the first exchange medium, and the proportion of the organic solvent in the first exchange medium ranges from about 1% to about 30%.

[0191] 21. The method according to any one of 1-19, wherein the total proportion of at least one organic solvent in the first exchange medium is less than about 30% based on the total mass of at least one organic solvent relative to the total volume of the first exchange medium.

[0192] 22. According to the method of any one of 1-21, wherein the first buffer system and the second buffer system are independently selected from the group consisting of: acetate buffer system, ADA ((N-(2-acetamido)iminoacetic acid) buffer system, ACES (N-(2-acetamido)-2-aminoethanesulfonic acid) buffer system, AMPD (2-amino-2-methyl-1,3-propanediol) buffer system, AMPSO (3-(1,1-dimethyl-2-hydroxyethyl)amino-2-hydroxypropanesulfonic acid) buffer system, BES (N,N-bis-(2-hydroxyethyl)-2-aminoethanesulfonic acid) buffer system, bicarbonate buffer system, bicine (N,N-bis(2-hydroxyethyl)glycine) buffer system, bis-tris(2-bis(2-hydroxyethyl)amino-2-(hydroxymethyl)-1,3-propanediol) buffer system, BTP (1,3-bis(tris(hydroxymethyl)methylamino)propane) buffer system, CABS (4-(cyclohexylamino)-1-butyric acid) buffer system, CAPS (N-cyclohexyl-3-aminopropanesulfonic acid) buffer system, CAPSO (3-(cyclohexylamino)-2-hydroxypropanesulfonic acid) buffer system, citrate buffer system, DIPSO (3-(N,N-bis[2-hydroxyethyl]amino)-2-hydroxypropanesulfonic acid) buffer system, EDTA (ethylenediaminetetraacetic acid) buffer system, Gly-Gly buffer system, His-Glu buffer system, HEPBS (N-(2-hydroxyethyl)piperazine-N'-(4-butyric acid)) buffer system, HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid) buffer system, HEPPSO ((2-hydroxyethyl)-piperazine-N-2-hydroxypropanesulfonic acid) buffer system, histidine buffer system, MES (2-(N-morpholino)ethanesulfonic acid) buffer system, MOBS (4-(4-morpholino)butyric acid) buffer system, MOPS (3-(N-morpholino)propanesulfonic acid) buffer system, MOPSO (3-morpholino-2-hydroxypropanesulfonic acid) buffer system, PB (phosphate buffer) buffer system, PBS (phosphate buffered saline) buffer system, PIPES (piperazine-N,N′-bis(2-ethanesulfonic acid)) buffer system, POPSO (piperazine-N,N′-bis(2-hydroxypropanesulfonic acid)) buffer system, succinate buffer system, TES (2-{[1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl]amino}ethane-1-sulfonic acid) buffer system, TAPSO (N-[tris(hydroxymethyl)methyl]-3-amino-2-hydroxypropanesulfonic acid) buffer system, TAE (triacetate-EDTA) buffer system, tricine (N-(tris(hydroxymethyl)methyl)glycine) buffer system and TAPS ([tris(hydroxymethyl)methylamino]propanesulfonic acid) buffer system.

[0193] 23. The method according to any one of 1-22, wherein the pH range of the first exchange medium is from about 5.0 to about 8.0.

[0194] 24. The method according to any one of 1-23, wherein the pH range of the second exchange medium is from about 5.0 to about 8.0.

[0195] 25. According to any one of 1-24, wherein the organic UF / DF process is carried out at a rate of approximately 200 L / hr / m 2 Approximately 500 L / hr / m 2 The flux rate is determined.

[0196] 26. According to any one of 1-25, wherein the aqueous UF / DF process is in the range of approximately 200 L / hr / m 2 Approximately 500 L / hr / m 2 The flux rate is determined.

[0197] 27. The method according to any one of 1-26, wherein the organic UF / DF process is carried out at a transmembrane pressure ranging from about 5 psi to about 30 psi.

[0198] 28. The method according to any one of 1-27, wherein the aqueous UF / DF process is carried out at a transmembrane pressure ranging from about 5 psi to about 30 psi.

[0199] 29. The method according to any one of 1-28, wherein the organic UF / DF process is carried out such that the temperature in the residue chamber containing the conjugate compound ranges from about 5°C to about 40°C.

[0200] 30. The method according to any one of 1-29, wherein the aqueous UF / DF process is carried out such that the temperature in the residue chamber containing the conjugate compound ranges from about 5°C to about 40°C.

[0201] 31. The method according to any one of 1-30, wherein the percolation volume of the organic UF / DF process ranges from about 2 to about 20.

[0202] 32. The method according to any one of 1-31, wherein the percolation volume of the aqueous UF / DF process ranges from about 2 to about 20.

[0203] 33. The method according to any one of 1-32, wherein the first semipermeable membrane and the second semipermeable membrane are independently selected from the group consisting of: polyolefins, polystyrene, polysulfone, polyesters, polyamides, polyacrylates, polycarbonates, and mixtures and copolymers thereof.

[0204] 34. The method according to any one of 1-33, wherein the first semipermeable membrane and the second semipermeable membrane are independently selected from the group consisting of: polyethersulfone (PES), polytetrafluoroethylene (PTFE), polypropylene, sulfonated polysulfone, polyvinylidene fluoride (PVDF), polyacrylonitrile (PAN), cellulose polymers, polyimide, polyetherimide (PEI), aliphatic polyamide, polyetheretherketone (PEEK), polyphenylene ether (PPO), polysulfone (PSf), and mixtures and copolymers thereof.

[0205] 35. The method according to any one of 1-34, wherein the first semipermeable membrane and the second semipermeable membrane are the same semipermeable membrane.

[0206] 36. The method according to any one of 1-35, wherein both the first semipermeable membrane and the second semipermeable membrane are neutral membranes without charge.

[0207] 37. The method according to any one of 1-6, wherein the organic UF / DF process and the aqueous UF / DF process are carried out as tangential flow filtration (TFF) processes.

[0208] 38. The method according to any one of 1-37, wherein the first aperture and the second aperture range independently from about 0.2 kDa MWCO to about 500 kDa MWCO.

[0209] 39. The method according to any one of 1-38, wherein the conjugate compound includes an antibody-drug conjugate (ADC).

[0210] 40. The method according to any one of 1-38, wherein the conjugate compound comprises conjugates other than antibody-drug conjugates (ADCs).

[0211] 41. The method according to any one of 1-40, wherein the concentration of the conjugated compound in the crude composition ranges from about 1 mg / mL to about 100 mg / mL.

[0212] 42. The method according to any one of 1-41, wherein the impurity includes residual free drug compound, residual drug-connector compound or combination thereof.

[0213] 43. The method according to any one of 1-38, wherein the impurities include residual free drug compounds.

[0214] 44. According to the method of 43, the residual free drug compound is selected from the group consisting of: topoisomerase I (TOP1) inhibitors, topoisomerase II (TOP2) inhibitors, microtubule disruptors, DNA damage agents, DNA intercalation agents, protein degraders, TLR7 agonists, TLR8 agonists, STING agonists, XPO1 inhibitors, and any combination thereof.

[0215] 45. According to the method of 43 or 44, wherein the residual free drug compound is selected from the group consisting of: eczema, DXd, camptothecin, SN-38 (7-ethyl-10-hydroxy-camptothecin), DM1 (mertansine), DM1 derivatives, DM4 (mertansine), MMAE (monomethylaurestatin E), MMAF (monomethylaurestatin F), leptomysin B, and any combination thereof.

[0216] 46. ​​The method according to any one of 1-45, wherein the impurities include residual drug-connector compounds.

[0217] 47. The method of 46, wherein the residual drug-connector compound comprises at least one drug moiety derived from a drug compound selected from the group consisting of: topoisomerase I (TOP1) inhibitors, topoisomerase II (TOP2) inhibitors, microtubule disruptors, DNA damaging agents, DNA intercalating agents, protein degrading agents, TLR7 agonists, TLR8 agonists, STING agonists, XPO1 inhibitors, and any combination thereof.

[0218] 48. According to the method of 47, wherein the pharmaceutical compound is selected from eczema, DXd, camptothecin, SN-38 (7-ethyl-10-hydroxycamptothecin), DM1 (matansine), DM1 derivatives, DM4 (matansine), MMAE (monomethylaurestatin E), MMAF, DNA alkylating agents, pyrrolobenzodiazepine (PBD), leptomysin B, and any combination thereof.

[0219] 49. The method according to any one of 1-48, wherein the conjugate compound includes an antibody-drug conjugate (ADC).

[0220] 50. The method of 49, wherein the ADC comprises at least one pharmaceutical portion derived from a pharmaceutical compound selected from the group consisting of: topoisomerase I (TOP1) inhibitors, topoisomerase II (TOP2) inhibitors, microtubule disruptors, DNA damage agents, DNA intercalators, protein degraders, TLR7 agonists, TLR8 agonists, STING agonists, XPO1 inhibitors, and any combination thereof.

[0221] 51. According to the method of 50, the pharmaceutical compound is selected from eczema, DXd, camptothecin, SN-38 (7-ethyl-10-hydroxy-camptothecin), DM1 (matansine), DM1 derivatives, DM4 (matansine), MMAE (monomethylaurestatin E), MMAF, pyrrolobenzodiazepine (PBD), leptomysin B, and any combination thereof.

[0222] 52. The method according to any one of 49-51, wherein the drug-antibody ratio of the antibody-drug conjugate (ADC) is about 1:1 to about 10:1.

[0223] 53. The method according to any one of 1-52, wherein the crude mixture is a conjugate mixture.

[0224] 54. The method according to any one of 1-53, wherein the concentration of at least one impurity in the purified composition is less than 50 μg / mL based on the total volume of the purified composition.

[0225] 55. A method for preparing purified conjugate compounds, the method comprising:

[0226] (i) The target agent is reduced by contacting it with a reducing agent to obtain a crude reduced target agent;

[0227] (ii) The reduced target agent is conjugated with a drug-connector compound to obtain a crude composition containing the conjugated compound;

[0228] (iii) Optionally, a quencher may be added to the crude composition; and

[0229] (iv) Purify the crude composition using any one of the methods in 1-54 to obtain the purified conjugate compound.

[0230] 56. A method for preparing purified conjugate compounds, the method comprising:

[0231] (i) The target agent is reduced by contacting it with a reducing agent to obtain a crude reduced target agent;

[0232] (ii) Attaching at least one linking group to a reduced target agent to obtain a crude composition comprising a target agent-connector intermediate;

[0233] (iii) Attaching at least one pharmaceutical compound to a connector of a target-connector intermediate to obtain a crude composition comprising the conjugate compound;

[0234] (iv) Optionally, a quencher may be added to the crude composition; and

[0235] (v) Purify the crude composition using the UF / DF method according to any one of 1-54 to obtain the purified conjugate compound.

[0236] 57. According to the method in 55 or 56, wherein the target is an antibody or antigen-binding fragment, and the purified conjugate compound is a purified ADC.

[0237] 58. According to the method of 55 or 56, wherein the target agent is an antibody-free target agent, and the purified conjugate compound is a purified conjugate other than the ADC.

[0238] 59. The method according to any one of 55-58, further comprising purifying the crude reduced target agent by an initial aqueous UF / DF process before conjugating the reduced target agent with a drug-connector compound to obtain a conjugated compound.

[0239] 60. The method according to any one of 55-59, wherein the conjugate compound comprises at least one pharmaceutical moiety derived from a pharmaceutical compound selected from the group consisting of: topoisomerase I (TOP1) inhibitors, topoisomerase II (TOP2) inhibitors, microtubule disruptors, DNA damaging agents, DNA intercalation agents, protein degrading agents, TLR7 agonists, TLR8 agonists, STING agonists, XPO1 inhibitors, and any combination thereof.

[0240] 61. According to the method of 60, the pharmaceutical compound is selected from eczema, DXd, camptothecin, SN-38 (7-ethyl-10-hydroxy-camptothecin), DM1 (matansine), DM1 derivatives, DM4 (matansine), MMAE (monomethylaurestatin E), MMAF, pyrrolobenzodiazepine (PBD), leptomysin B, and any combination thereof.

[0241] 62. The method according to any one of 55-61, wherein, based on the total volume of the purified conjugate compound, the concentration of residual free drug compound, residual adapter compound, residual drug-adaptor compound or any combination thereof in the purified conjugate compound is less than 200 μg / mL.

[0242] The present disclosure is illustrated by the following examples, and these embodiments are not intended to be limiting.

[0243] Example

[0244] A. Abbreviations

[0245] Ab: Antibody;

[0246] ACE: Acetone;

[0247] ACN: Acetonitrile;

[0248] ADC1: An antibody-drug conjugate formed from a drug-connector compound (LD038);

[0249] DAD: Diode Array Detector;

[0250] DAR: Drug-Antibody Ratio;

[0251] DCM: Dichloromethane;

[0252] DIPEA: N,N-diisopropylethylamine;

[0253] DMA: Dimethylacetamide;

[0254] DMF: Dimethylformamide;

[0255] DMPU: N,N′-Dimethylpropylidene urea;

[0256] DMSO: Dimethyl sulfoxide;

[0257] DV: Percolation volume;

[0258] EDCI: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide;

[0259] EDTA: Ethylenediaminetetraacetic acid;

[0260] EtOH: Ethanol;

[0261] GLY: Glycerin;

[0262] HATU: 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate;

[0263] HIS: Histidine Buffer;

[0264] HMPA: Hexamethylphosphamide;

[0265] HOSu: N-hydroxysuccinimide

[0266] HPLC: High-performance liquid chromatography;

[0267] LCMS: Liquid chromatography-mass spectrometry;

[0268] LMH: L / hr / m 2 ;

[0269] MeOH: Methanol;

[0270] MWCO: Molecular weight cutoff

[0271] MWD: Multiwavelength detector

[0272] NMP: N-methyl-2-pyrrolidone;

[0273] LD038: The drug-connector compound prepared in Example 1;

[0274] PBS: Phosphate-buffered saline;

[0275] PD: 1,2-Propanediol;

[0276] RCF: Relative centrifugal force;

[0277] RP: Inverted;

[0278] TCEP: Tris(2-carboxyethyl)phosphine

[0279] TFA: Trifluoroacetic acid;

[0280] THF: Tetrahydrofuran;

[0281] TMP: Transmembrane pressure;

[0282] UPLC: Ultra-high performance liquid chromatography.

[0283] B. Equipment and Instruments

[0284] Table 1 below summarizes the equipment and instruments used in the following studies.

[0285] Table 1 Equipment and instruments

[0286]

[0287] C. HPLC grade reagents and solvents

[0288] Table 2 below summarizes the HPLC-grade reagents and solvents used in the following studies.

[0289] Table 2 HPLC grade reagents and solvents

[0290]

[0291] D. Chromatographic standards

[0292] Table 3 below summarizes the chromatographic standards used in the following studies.

[0293] Table 3 Chromatographic standards

[0294]

[0295] E. Preparation of chromatographic solutions

[0296] The volumes of the chromatographic solutions described below can be scaled proportionally as needed.

[0297] Preparation of protein precipitants:

[0298] Weigh 0.50 ± 0.1 g NaCl into 50 mL MeOH, mix well, and let the solution stand for at least 1 hour before use. Use the supernatant as a protein precipitant. Expiry date: 3 months when stored at 10 - 30℃.

[0299] Preparation of diluent:

[0300] Mix 3 mL of the above protein precipitant with 1 mL of Milli-Q water thoroughly.

[0301] Preparation of mobile phase A: 0.1% TFA / water:

[0302] Add 1 mL of TFA to 1000 mL of Milli-Q water and mix by inverting. Transfer the resulting mixture to a labeled 1 L solvent bottle. For detailed instructions on preparing this solution, see Preparation of Commonly Used Solutions in Quality Control Laboratories. Expiry Date: 14 days when stored at room temperature.

[0303] Preparation of mobile phase B: 0.1% TFA / CAN:

[0304] Add 1 mL of TFA to 1000 mL of ACN water and mix by inverting. Transfer the resulting mixture to a labeled 1 L solvent bottle. For detailed instructions on preparing this solution, see Preparation of Commonly Used Solutions in Quality Control Laboratories. Expiry Date: 1 month when stored at room temperature.

[0305] F. Chromatographic Analysis Procedures

[0306] Preparation of blank control:

[0307] Transfer at least 1000 μL of the above diluent to an HPLC vial as a blank control, injecting 10 μL each time.

[0308] Preparation of stock solutions for the eczema standard curve:

[0309] Weigh 50±5 mg of eczemab mesylate into a 50 mL volumetric flask, then add 40 mL of 30% acetonitrile aqueous solution to completely dissolve the eczemab mesylate. Adjust the volume to 50 mL with more 30% acetonitrile solution to prepare a stock solution of eczemab mesylate. Aliquot this stock solution of eczemab mesylate into 250 µL vials as stock solutions. Expiry date: Six months when stored at -80°C. Ecinotecan concentration.

[0310] The stock solution of methanesulfonate can be calculated using the following equation (I):

[0311] [Ecinotecan Mesylate Stock Solution] = Weight / Volume of Ecinotecan Mesylate × Determination Value of Ecinotecan Mesylate in CoA × 435.46 ÷ 531.51 (I)

[0312] Preparation of ADC1 standard curve stock solution:

[0313] Weigh 50±5 mg LD038·TFA into a 50 mL volumetric flask, then add 40 mL of 30% acetonitrile solution to completely dissolve eczemab mesylate. Adjust the volume to 50 mL with 30% acetonitrile solution. Divide the LD038 solution into 250 µL vials as stock solutions. Expiry date: Twelve months when stored at -80°C. The concentration of the LD038 stock solution can be calculated using the following equation (II):

[0314] [LDLD038 stock solution] = LDLD038·TFA / net volume of solution LD038·TFA COA determination result ÷ 2204.34×2090.32 (II)

[0315] Preparation of standard curve solutions:

[0316] A mixed standard curve solution of eciletidine mesylate and LD038 was prepared such that the concentrations of eciletidine ranged from 0.5 µg / mL to 4.8 µg / mL and the concentrations of LD038 ranged from 1.0 µg / mL to 20.0 µg / mL. The samples were thoroughly mixed and transferred to HPLC vials to prepare individual samples for each concentration level.

[0317] Preparation of sample solution:

[0318] Add 100 μL of the ADC sample to a 1.5 mL Eppendorf tube, followed by 300 μL of the aforementioned protein precipitant. Vortex the mixture at 2000 rpm for 5 min at 10–30 °C. Centrifuge the resulting solution at 20,000 RCF for 30 min at 4 °C, maintaining a temperature ≤15 °C. Immediately transfer the resulting supernatant to an HPLC vial. The sample solution can be stored in an autosampler at 2–8 °C for 24 hours prior to chromatography.

[0319] Table 4 below summarizes the chromatographic parameters used in the chromatographic analyses described in the following studies.

[0320] Table 4 chromatographic parameters

[0321]

[0322] Calibration curve:

[0323] The calibration curves for eczema and LD038 were generated by plotting the total peak area of ​​each peak along the y-axis and the sample concentration along the x-axis—making the linear relationship based on the following equation (III), where k is the slope of the line and B is the intercept of the line with the y-axis.

[0324] (III)

[0325] Normalization of measured concentrations based on dilution factor:

[0326] The normalized concentrations of eczemacon and LD038 were calculated using the following equations (IV) and (V) based on the relevant dilution factors, where “C 依喜替康 "C" represents the normalized concentration (μg / mL) of eczema in the original sample. 计算的依喜替康 "The measurement of eczema concentration is based on the relevant calibration curve ("C") LD038 "and "C 计算的LD038 "" is the corresponding parameter of LD038), and "dilution factor" is based on the dilution during the preparation of the relevant sample solution.

[0327] C 依喜替康 = C 计算的依喜替康 ×Dilution factor

[0328] C LD038 = C 计算的LD038 ×Dilution factor (V)

[0329] G. Preparation of drug-connector compounds and ADCs

[0330] Example 1 Preparation of drug-connector compound LD038 containing PEG units and a cleavable connector attached to eczema.

[0331]

[0332] The preparation of the drug-connector compound LD038 is also described below.

[0333] Step 1 :

[0334]

[0335] A solution of compound 38-1 (650 mg, 0.774 mmol) and HOSu (177.98 mg, 1.548 mmol) in anhydrous DCM (8 mL) was stirred at room temperature, and then EDCI (296.69 mg, 1.548 mmol) was added. The resulting solution was stirred at room temperature for another 1 hour until LCMS indicated that all starting amines had disappeared and the desired product was detected. The resulting solution was washed with water, the organic layer was collected, and the aqueous phase was then subjected to DCM (10 mL). 2) Extraction. The combined organic layers were dried over sodium sulfate, filtered, and concentrated to dryness to give compound 38-2 (552 mg, 0.589 mmol, 76.12%) as a colorless oil, which was used unchanged in the next step. LCMS: m / z = 959.4 (M+Na) + .

[0336] Step 2 :

[0337]

[0338] A solution of compound 38-2 (300 mg, 0.357 mmol) and DIPEA (138.22 mg, 1.071 mmol) in anhydrous DMF (2 mL) was stirred at room temperature, and then compound 38-3 (87.97 mg, 0.357 mmol) was added, suspending the starting amine in the solution. The resulting mixture was stirred at room temperature for another 6 hours. During this time, the starting amine gradually dissolved, and the suspension became a clear, pale yellow solution. The reaction solution was terminated and purified directly by reversed-phase liquid chromatography (40 g C18 column, eluted over 15 minutes with an aqueous solution of 0-100% acetonitrile containing 0.01% TFA) to give compound 38-4 (260 mg, 0.243 mmol, 68.14%) as a pale yellow oil, LCMS ((M-100) / 2+H). + = 484.9.

[0339] Step 3 :

[0340]

[0341] A solution of compound 38-4 (260 mg, 0.243 mmol) in acetonitrile (1.8 mL) was stirred at rt, and anhydrous diethylamine (0.2 mL, 1.941 mmol) was added. The resulting solution was stirred at rt for 2 h until LCMS showed that most of the starting material had been consumed. The solution was then concentrated to dryness, and the residue was purified by reversed-phase column chromatography (12 g C18 column, eluted with an aqueous solution of 0-50% acetonitrile containing 0.01% TFA) to give the expected fraction of compound 38-5 (170 mg, 0.201 mmol, 82.54%) as a pale yellow oil. LCMS, ESI m / z = 846.6 (M+H) + Retention time (0.01% TFA) = 1.451 min; No UV.

[0342] Step 4 :

[0343]

[0344] A clear reaction solution of 38-5 (170 mg, 0.201 mmol), D-glucose (217.08 mg, 1.206 mmol), and acetic acid (1.21 mg, 0.020 mmol) in methanol (5 mL) was heated at 50 °C for 30 min, followed by the addition of NaCNBH3 (75.98 mg, 1.206 mmol). The resulting solution was stirred at 50 °C under N2 for 4 hr. Additional NaCNBH3 (75.98 mg, 1.206 mmol) and D-glucose (217.08 mg, 1.206 mmol) were then added, and the mixture was stirred overnight at 50 °C. After stirring for 20 hr, LCMS indicated the reaction was complete. The solvent was evaporated, and the residue was purified by C18 reversed-phase chromatography to give the desired product 38-6 (106 mg, 0.090 mmol, 44.92%). LCMS, ESI m / z = 537.9 ((M-100) / 2+H) + .

[0345] Step 5 :

[0346]

[0347] A solution of compound 38-6 (250 mg, 0.213 mmol), HATU (121.45 mg, 0.319 mmol), and DIPEA (82.41 mg, 0.639 mmol) in anhydrous DMF (2 mL) was stirred at room temperature for 5 min, followed by the addition of compound 38-7 (178.88 mg, 0.213 mmol). The resulting solution was stirred at room temperature for another 2 hr until LCMS indicated complete reaction. The reaction solution was purified directly by reversed-phase liquid chromatography (40 g C18 column, eluted over 15 min with an aqueous solution of 0-70% acetonitrile containing 0.01% TFA) to give compound 38-8 (270 mg, 0.135 mmol, 63.48%) as a white solid. LCMS, ESI m / z = 666.6 (M / 3+H) + 999.2 (M / 2+H) + .

[0348] Step 6 :

[0349]

[0350] A solution of compound 38-8 (120 mg, 0.060 mmol) in TFA (2 mL) was stirred at rt for 1 h. LCMS of the mixture showed that the reaction was complete, all starting material was consumed, and the desired product (m / z = 633 = 1896 / 3 + H, RT 1.501 min) and the glycoesterified product (TFA condensed with the hydroxyl group in the sugar unit, monoester m / z = (1896 + 96) / 2 + H = 665, RT 1.58 min) were formed. The completed reaction solution was concentrated to dryness and then dissolved again in THF (4 mL) and water (2 mL), and treated with saturated aqueous sodium carbonate solution to adjust the pH to 8-9. The resulting solution was stirred at room temperature for 1 h to achieve complete hydrolysis. The solution was then neutralized with diluted TFA and concentrated. The residue was purified by reversed-phase liquid chromatography (C18 column, eluted for 15 min with an aqueous solution of 0-25% acetonitrile containing 0.01% TFA), and lyophilized to give the expected product 38-9 (80 mg, 0.042 mmol, 70.19%) as a white solid. LCMS, ESI m / z = 633.2 (M / 3+H) + , 949.2 (M / 2+H).

[0351] Step 7 :

[0352]

[0353] A solution of compound 38-9 (20 mg, 0.011 mmol) and DIPEA (4.08 mg, 0.032 mmol) in anhydrous DMF (1 mL) was stirred at room temperature for 5 min, and then a solution of compound 38-10 (4.88 mg, 0.016 mmol) in anhydrous DMF (1 mL) was added dropwise over 2 min using a syringe. The resulting solution was stirred at room temperature for another 4 h until all the starting amine disappeared and the mass of the desired product was detected. The resulting solution was neutralized with formic acid to adjust the pH to 6–7. The reaction solution was then purified by preparative HPLC (eluting over 20 min with a gradient containing 0.01% TFA) to give LD038 (11 mg, 0.005 mmol, 49.91%) as a white solid (TFA salt). LCMS, m / z = 697.7 (M / 3+H) + ; 1HNMR(400MHz, DMSO-d6): δ 10.03 (s, 1H), 8.19-8.11 (m, 2H), 8.07 (d, J = 8.8 Hz,1H), 7.96 (d, J = 7.6 Hz, 1H), 7.82-7.77 (m, 2H), 7.66 (d, J = 8.4 Hz, 1H),7.60 (d, J = 8.4 Hz, 2H), 7.36 (d, J = 8.0 Hz, 2H), 7.32 (s, 1H),7.00 (s,2H), 6.53 (s, 1H), 5.99 (t, J = 5.6 Hz, 1H), 5.45-5.43 (m, 6H), 5.30-5.24 (m,3H), 5.08 (s,2H), 4.84-4.74 (m, 2H), 4.65-4.49 (m, 4H), 4.45-4.35 (m, 3H),4.27-4.17 (m, 2H), 4.04-3.95 (m, 2H), 3.80-3.77 (m, 2H), 3.71-3.67 (m, 2H), 3.62-3.55 (m, 9H), 3.53-3.43 (m, 44H), 3.27-3.21 (m, 2H), 3.16-3.07 (m,2H),3.07-2.93 (m,6H), 2.38 (s,3H), 2.29 (t, J= 6.4 Hz, 2H), 2.23-2.13 (m, 2H),2.13-2.08 (m, 2H), 2.00-1.82 (m, 4H), 1.73-1.54(m, 4H), 1.54-1.40 (m, 7H),1.40-1.30 (m, 4H), 1.30-1.14 (m, 5H), 0.90-0.81 (m, 9H) ppm.

[0354] Example 2 Preparation of crude antibody-drug conjugate 1 (ADC1) formed from mAb and drug-connector compound LD038

[0355]

[0356] A solution of 2 mL of antibody (10 mg / mL) in 50 mM sodium phosphate buffer containing 5 mM EDTA (pH = 6.9) was added to an aqueous solution of 10 mM TCEP HCl at a molar ratio of TCEP:mAb = 8.0. The resulting reduction reaction was carried out at 25 °C for 2 hours to obtain crude reduced mAb. Excess TCEP and its byproducts were removed by ultrafiltration using 50 mM sodium phosphate buffer (pH = 6.9) as the exchange medium. LD038 (a TFA salt from Example 1) was dissolved in water at a concentration of 20 mg / mL, and the resulting solution was added to the reduced mAb at a molar ratio of 7.7 (LD038:mAb). The coupling reaction was stirred at 25 °C for 2 hours. Excess LD038 and its impurities were removed by ultrafiltration using 50 mM sodium phosphate buffer. The resulting crude ADC was stored in a 20 mM histidine buffer containing 6% sucrose and 0.02% (w / v) Tween 20. The purity of the crude ADC (“ADC1”) was determined to be 97.5% by HPLC, and the DAR value was determined to be 7.6 by LC-MS using the chromatographic analysis procedure described below. The crude ADC1 was stored in different buffer systems for subsequent analysis—standard solutions in the following buffer systems were prepared.

[0357] 50 mM PBS (pH = 7.2)

[0358] 20 mM HIS (pH = 5.7)

[0359] 10 mM HIS (pH = 5.6)

[0360] Example 3 Effects of different organic solvents on the UF / DF process (free drug-linker (DL) / total DL)

[0361] A series of studies were conducted using the crude ADC1 prepared in Example 2, wherein different organic solvents were used during the first UF / DF in studies (Std.) 3.1–3.16 compared to comparative studies (Comp. Std.) 3.1 and 3.2 conducted without organic solvents. The same UF / DF apparatus was used in each study (see [link to study 3.1-3.16]). Figure 2 The membrane filter used is the Amicon Ultra-4 (UFC500396) with the process parameters shown below.

[0362] Crude ADC1 and the different buffers listed in Table 5 were added to Amicon Ultra-4 tubes. For studies 3.1–3.15 of this invention, UF / DF was performed using 8 DV of a first exchange medium containing an organic solvent (or a mixture thereof). For comparative studies 3.1 and 3.2, the first exchange medium did not contain an organic solvent. Aqueous UF / DF was then performed twice using 8 DV of an exchange medium containing the same buffer system as in the first exchange medium. The resulting purified ADC1 retained in the Amicon Ultra-4 tubes was then analyzed to determine the molar ratio of free DL to total DL. Free DL in this context includes residual drug-adaptor compounds, and total DL includes both residual drug-adaptor compounds and drug-adaptor compounds from the ADC1.

[0363] Table 5 summarizes the experimental data from Example 3.

[0364] Table 5 Effects of different solvents on free DL / total DL

[0365]

[0366] The data in Table 5 show that the presence of organic solvents significantly reduced the level of free DL in ADC1 samples purified using the process of this invention, as shown in Studies 3.1–3.16. Specifically, with the same initial free DL / total DL level of 10.10%, all samples using DMSO (Std. 3.1), DMA (Std. 3.2), acetone (Std. 3.3), acetonitrile (Std. 3.4, 3.10–3.12), DMF (Std. 3.5), THF (Std. 3.6), PD (1,2-propanediol) (Std. 3.7, 3.13, 3.15), methanol (Std. 3.8), ethanol (Std. 3.9), and combinations of organic solvents (Std. 3.13, 3.14, 3.16) in the UF / DF process showed a reduction in the free DL / total DL level after 3 or 5 cycles.

[0367] These three UF / DF speeds are classified according to the duration of the UF / DF process. Generally, the "normal" speed is well-known to those skilled in the art as the generally accepted speed for performing UF / DF; therefore, the duration corresponding to the "slow" speed is approximately twice the duration corresponding to the "normal" speed; and the duration corresponding to the "very slow" speed is more than twice the duration corresponding to the "slow" speed. It should also be noted that the "very slow" speed may damage the UF / DF tube.

[0368] Example 4Investigating the effect of percolation volume (DV) on the organic UF / DF step.

[0369] A series of studies were conducted using the crude ADC1 prepared in Example 2, in which the UF / DF process of the present invention was performed using three different first exchange media to understand how the percolation volume (DV) of the organic UF / DF step affects the amount of residual free drug (LD038 and / or eczema) in the percolate. The same UF / DF apparatus was used in each study (see [link to study]). Figure 2 The membrane filter used is the Amicon Ultra-4 (UFC500396) with the process parameters shown below.

[0370] Feed throughput: 300 LMH (throughput value, average flow rate, L / hr / m³) 2 )

[0371] TMP: 10-20 PSI

[0372] ADC concentration: 20 mg / mL

[0373] Loading capacity: 300 g / cm³ 2

[0374] Table 6 below summarizes the UF / DF process conditions of studies (Std.) 4.1–4.3, in which three different first exchange media were used to perform the organic UF / DF steps, and the organic UF / DF steps were performed from 0 DV to 16 DV, during which the amount of residual free drug in the exudate was continuously measured.

[0375] Table 6 Summary of the UF / DF process conditions studied in sections 5.1-5.3

[0376]

[0377] Table 7 below summarizes the residual free drug data measured at different time points throughout the organic UF / DF process—corresponding to percolation volumes (DV) from 0 to 16. A graph of this data is shown below. Figure 3 As shown.

[0378] Table 7 Residual free drug concentration as a function of percolation volume (DV)

[0379]

[0380] like Figure 3As illustrated, for each of Studies 4.1–4.3, it was observed that the amount of residual free drug in the osmate during organic UF / DF (UF / DF-1) appeared to stabilize after approximately DV 14. It was also observed that, in the first exchange medium of Study 4.1, the use of 30% PG in 10 nM HIS (pH = 5.6) enabled slightly more efficient removal of residual free drug from crude ADC1.

[0381] Example 5 : ADC1 amplification and purification

[0382] A series of studies were conducted using the crude ADC1 prepared in Example 2, where three different first exchange media were used to perform the UF / DF process of the present invention on a larger scale. Each study used the same UF / DF device (see [link]). Figure 2 The membrane filter used is the Amicon Ultra-4 (UFC500396) with the process parameters shown below.

[0383] Feed throughput: 300 LMH (throughput value, average flow rate, L / hr / m³) 2 )

[0384] TMP: 10-20 PSI

[0385] Crude ADC1 concentration: 20 mg / mL

[0386] The concentration of residual free drug in crude ADC1 is approximately 120-140 μg / mL.

[0387] Loading capacity: 300 g / cm³ 2

[0388] DV = 16 for UF / DF-1

[0389] DV = 8 for UF / DF-2

[0390] Table 8 below summarizes the experimental results of the scale-up purification of ADC1 from studies 5.1–5.3. Each purified ADC1 product was analyzed to determine the final concentration of ADC1 (mg / mL); the drug-antibody ratio (measured using both UV and MS detection); the percentages of high molecular weight (HMW%), low molecular weight (LMW%), and monomer (monomer%) based on size exclusion chromatography (SEC); and the concentration of residual free drug (LD038 and / or eczemab) in the residue.

[0391] Table 8 Summary of data from the scale-up and purification of ADC1

[0392]

[0393] The data in Table 8 illustrate that the UF / DF process of the present invention can be used to perform scale-up purification of ADC1 in a manner that significantly reduces the concentration of residual free drug without causing significant degradation or denaturation of ADC1.

[0394] Example 6 Synthesis of purified ADC1

[0395] use Figure 4 The synthesis process summarized in the present invention prepared purified ADC1, which includes the UF / DF method of the present invention (in... Figure 4 (The image is labeled "UFDF-2 purification"). The purified ADC1 had a concentration of 20 g / L and a pH of 6.0.

[0396] sequence list

[0397] SEQ ID NO: 1 F131 HCDR1

[0398] SYGMH

[0399] SEQ ID NO: 2 F131 HCDR2

[0400] VISYDGSNKYYADSVKG

[0401] SEQ ID NO: 3 F131 HCDR3

[0402] PRAYYGAYGSSFDY

[0403] SEQ ID NO: 4 F131 LCDR1

[0404] RASQGISSWLA

[0405] SEQ ID NO: 5 F131 LCDR2

[0406] AASSLQS

[0407] SEQ ID NO: 6 F131 LCDR3

[0408] QQSYSTPLT

[0409] SEQ ID NO: 7 2E7 HCDR1

[0410] SSDYYYWS

[0411] SEQ ID NO: 8 2E7 HCDR2

[0412] YIYYSGSTNYNPSLKS

[0413] SEQ ID NO: 9 2E7 HCDR3

[0414] GDGDFLGVCFDY

[0415] SEQ ID NO: 10 2E7 LCDR1

[0416] RASQSVSSYLA

[0417] SEQ ID NO: 11 2E7 LCDR2

[0418] DASNRAT

[0419] SEQ ID NO: 12 2E7 LCDR3

[0420] QQRSNWPLT

Claims

1. Purification methods, including: (a) subjecting a sample containing at least one conjugate compound and at least one impurity to an organic ultrafiltration / difiltration (UF / DF) process, such that the sample is filtered through a semipermeable membrane having a pore size that allows the at least one impurity to pass through the membrane while retaining the at least one conjugate compound as a effluent, wherein the organic UF / DF process uses a first exchange medium and produces a effluent containing the at least one conjugate compound; and (b) subjecting the effluent product of (a) to an aqueous UF / DF process, such that it is filtered through a semi-permeable membrane having a pore size that allows the at least one impurity to pass through while retaining the conjugate compound in the effluent, wherein the aqueous UF / DF process uses a second exchange medium and results in a effluent containing the at least one conjugate compound. in: The at least one impurity includes residual free drug compound, residual drug-connector compound, residual connector compound, residual by-product compound, residual side-product compound, or any combination thereof; The first exchange medium comprises at least one organic solvent and a first buffer system; and The first switching medium and the second switching medium are different.

2. The method of claim 1, wherein prior to step (a), the method further comprises combining a crude composition comprising the at least one conjugated compound and at least one impurity with the first exchange medium to produce a sample for step (a).

3. The method according to claim 1 or 2, wherein step (b) further comprises adding a first exchange medium to the permeate in (a) prior to performing the aqueous UF / DF process in (b) to replace at least a portion of the volume lost in step (a).

4. The method according to any one of claims 1 to 3, wherein the second exchange medium: (i) does not contain the at least one organic solvent present in the first exchange medium; or (ii) does not contain any organic solvent.

5. The method according to any one of claims 1 to 4, wherein step (a) and / or (b) is performed more than once.

6. The method of claim 5, wherein step (b) is performed at least three times.

7. The method according to any one of claims 1 to 6, wherein step (b) is performed at least 5 times.

8. The method according to any one of claims 1-7, wherein the separated composition is substantially free of product-bound impurities.

9. The method according to any one of claims 1-8, wherein the purified composition is substantially free of the organic solvent.

10. The method according to any one of claims 1-9, wherein the organic solvent comprises an alcohol compound, a polar aprotic compound, or a combination thereof.

11. The method according to any one of claims 1-10, wherein the organic solvent comprises a monohydric alcohol, a dihydric alcohol, a polyhydric alcohol, or a combination thereof.

12. The method according to any one of claims 1-11, wherein the organic solvent comprises a monohydric alcohol selected from the group consisting of methanol, ethanol, n-propanol, isopropanol, 1-butanol, 2-butanol, tert-butanol, isobutanol, 1-pentanol, 2-pentanol, 3-pentanol, isopentanol, tert-pentanol, hexanol (straight-chain, branched and / or cyclic), ethylene glycol monomethyl ether, cis-3-hexen-1-ol, trans-2-hexen-1-ol, 5-hexen-1-ol, phenol, benzyl alcohol, and combinations thereof.

13. The method according to any one of claims 1-12, wherein the organic solvent comprises a diol selected from the group consisting of: 1,2-ethylene glycol, 1,2-propanediol, 1,3-propanediol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 1,2-pentanediol, 1,5-pentanediol, 2,2-dimethylpropane-1,3-diol, 2-butyl-2-ethylpropane-1,3-diol, 2-methyl-2,4-pentanediol, 2-ethyl-1,3-hexanediol, 2-methyl-1,3-propanediol, 1,2-hexanediol, 1,5-hexanediol, 1,6-hexanediol, 1,8-octanediol, 1,10-decanediol, 2,2,4,4-tetramethylcyclobutane-1,3-diol, 1,3-cyclopentanediol, 1,2-cyclohexanediol, etc. Diols, 1,3-cyclohexanediol, 1,4-cyclohexanediol, 1,2-cyclohexanediethanol, 1,3-cyclohexanediethanol, 1,4-cyclohexanediethanol, 1,4-cyclohexanediethanol, isosorbide, monoglyceride, monoglyceride, trimethylolpropane monoester, trimethylolpropane monoether, pentaerythritol diester, pentaerythritol diether, dipropylene glycol, diethylene glycol, triethylene glycol, 2-methyl-1,3-propanediol, 2,2-dimethyl-1,3-propanediol, neopentyl glycol, benzyl glycol, bis(p-hydroxy)diphenyl, bis(p-hydroxy)diphenylpropane, 2,2'-bis[4-(2-hydroxyethoxy)phenyl]propane, bis[4-(2-hydroxyethoxy)phenyl]sulfone, 1,1-bis[4-(2-hydroxyethoxy)phenyl]cyclohexane, and combinations thereof.

14. The method according to any one of claims 1-13, wherein the organic solvent comprises a polyol selected from the group consisting of: alditol, 1,2,3-propanetriol, 2-ethyl-2-hydroxymethyl-1,3-propanediol, 1,2,4-butanetriol, 1,2,5-pentanetriol, 1,2,6-hexanetriol, 1,2,3,6-hexanetetraol, glycerol (also known as glycerol), diglycerol, triglycerol, tetraglycerol, pentaglycerol, hexaglycerol, triethylglycerol, triethylglycerol, triglyceride ... Alkylamines, trimethylolethane, trimethylolpropane, bis(trimethylolpropane), tri-trimethylolpropane, 2-methylpropanetriol, 2-methyl-1,2,4-butanetriol, pentaerythritol, dipentaerythritol, tripentaerythritol, methyl glucoside, sorbitol, mannitol, sucrose, 1,3,5-trihydroxybenzene, 1,2,4-trihydroxybenzene, erythritol, inositol, threitol, arabinitol, xylitol, ribitol, galactitol, and combinations thereof.

15. The method according to any one of claims 1-14, wherein the organic solvent comprises a polar aprotic compound selected from the group consisting of: acetone (ACE), acetonitrile (ACN), 4-acetylmorpholine, N-cyclohexyl-2-pyrrolidone (CHP), 1,2-dimethoxy ether (DME), N,N-dimethylacetamide (DMA), N,N-diethylacetamide, dimethylformamide (DMF), diethylformamide, N,N-dimethylpropionamide, 3-methoxy-N,N-dimethylpropionamide, 3-methoxy-N,N-diethylpropionamide, 3-methoxy-N,N-dimethylethylpropionamide, 3-ethoxy-N,N-dimethylpropionamide, and 3-ethoxy-N,N-dimethylpropionamide. Propionamide, 1,3-dimethyl-2-imidazolium ketone (DMI), dimethyl sulfoxide (DMSO), 1,4-dioxane, 1,3-dioxolane (DN), N,N'-dimethylacrylurea (DMPU), hexamethylphosphoramide (HMPA), N-methyl-2-pyrrolidone (NMP), N-ethyl-2-pyrrolidone, N-methyl-ε-caprolactam, 2-methyltetrahydrofuran (Me-THF), 2,5-dimethyltetrahydrofuran, 4-propionylmorpholine, sulfolane, tetrahydrofuran (THF), tris(N,N-tetramethylene)phosphotriamide, alcohol ethoxylates, diethyl ester dimethylammonium chloride, linear alkylbenzene sulfonates, and combinations thereof.

16. The method according to any one of claims 1-15, wherein the organic solvent is selected from the group consisting of methanol, ethanol, 1,2-propanediol, glycerol, acetone (ACE), acetonitrile (ACN), N,N-dimethylacetamide (DMA), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), N,N'-dimethylacrylurea (DMPU), hexamethylphosphoramide (HMPA), N-methyl-2-pyrrolidone (NMP), tetrahydrofuran (THF), and combinations thereof.

17. The method according to any one of claims 1-16, wherein the organic solvent is not a sugar alcohol.

18. The method according to any one of claims 1-17, wherein the organic solvent is miscible with water.

19. The method according to any one of claims 1-18, wherein the organic solvent has a boiling point of at least 150°C.

20. The method according to any one of claims 1-19, wherein the organic solvent has a concentration of at least 0.9 g / cm³. 3 The density.

21. The method according to any one of claims 1-20, wherein the organic solvent has a polarity index of at least 6.

0.

22. The method according to any one of claims 1-21, wherein the organic solvent has a logP of less than 0, as measured at 25°C.

23. The method according to any one of claims 1-22, wherein the organic solvent has a logP in the range of about -0.04 to -1.5, as measured at 25°C.

24. The method according to any one of claims 1-23, wherein the first exchange medium comprises at least two organic solvents.

25. The method according to any one of claims 1-24, wherein the organic solvent comprises a diol and a polar aprotic compound.

26. The method according to any one of claims 1-25, wherein the proportion of the organic solvent in the first exchange medium ranges from about 1% to about 30% based on the mass of the organic solvent relative to the total volume of the first exchange medium.

27. The method according to any one of claims 1-26, wherein the total proportion of the at least one organic solvent in the first exchange medium is less than about 30% based on the total mass of the at least one organic solvent relative to the total volume of the first exchange medium.

28. The method according to any one of claims 1-27, wherein the buffer system of the first exchange medium and the buffer system of the second exchange medium are independently selected from the group consisting of: acetate buffer system, ADA ((N-(2-acetamido)iminoacetic acid) buffer system, ACES (N-(2-acetamido)-2-aminoethanesulfonic acid) buffer system, AMPD (2-amino-2-methyl-1,3-propanediol) buffer system, AMPSO (3-(1,1-dimethyl-2-hydroxyethyl)amino-2-hydroxypropanesulfonic acid) buffer system, BES (N,N-bis-(2-hydroxyethyl)-2-aminoethanesulfonic acid) buffer system, bicarbonate buffer system, bicine (N,N-bis(2-hydroxyethyl)glycine) buffer system, bis-tris(2-bis(2-hydroxyethyl)amino-2-(hydroxymethyl)-1,3-propanediol) buffer system, BTP (1,3-bis(tris(hydroxymethyl)methylamino)propane) buffer system, CABS (4-(cyclohexylamino)-1-butyric acid) buffer system, CAPS (N-cyclohexyl-3-aminopropanesulfonic acid) buffer system, CAPSO (3-(cyclohexylamino)-2-hydroxypropanesulfonic acid) buffer system, citrate buffer system, DIPSO (3-(N,N-bis[2-hydroxyethyl]amino)-2-hydroxypropanesulfonic acid) buffer system, EDTA (ethylenediaminetetraacetic acid) buffer system, Gly-Gly buffer system, His-Glu buffer system, HEPBS (N-(2-hydroxyethyl)piperazine-N'-(4-butyric acid)) buffer system, HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid) buffer system, HEPPSO ((2-hydroxyethyl)-piperazine-N-2-hydroxypropanesulfonic acid) buffer system, histidine buffer system, MES (2-(N-morpholino)ethanesulfonic acid) buffer system, MOBS (4-(4-morpholino)butyric acid) buffer system, MOPS (3-(N-morpholino)propanesulfonic acid) buffer system, MOPSO (3-morpholino-2-hydroxypropanesulfonic acid) buffer system, PB (phosphate buffer) buffer system, PBS (phosphate buffered saline) buffer system, PIPES (piperazine-N,N′-bis(2-ethanesulfonic acid)) buffer system, POPSO (piperazine-N,N′-bis(2-hydroxypropanesulfonic acid)) buffer system, succinate buffer system, TES (2-{[1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl]amino}ethane-1-sulfonic acid) buffer system, TAPSO (N-[tris(hydroxymethyl)methyl]-3-amino-2-hydroxypropanesulfonic acid) buffer system, TAE (triacetate-EDTA) buffer system, tricine (N-(tris(hydroxymethyl)methyl)glycine) buffer system and TAPS ([tris(hydroxymethyl)methylamino]propanesulfonic acid) buffer system.

29. The method according to any one of claims 1-28, wherein the buffer system of the first exchange medium and the buffer system of the second exchange medium are: (i) the same; or (ii) different.

30. The method according to any one of claims 1-29, wherein the pH range of the first exchange medium is from about 5.0 to about 8.

0.

31. The method according to any one of claims 1-30, wherein the pH range of the second exchange medium is from about 5.0 to about 8.

0.

32. The method according to any one of claims 1-31, wherein the organic UF / DF process of (a) and / or the aqueous UF / DF process of (b) are carried out in a continuous flux system.

33. The method according to any one of claims 1-32, wherein the organic UF / DF process is carried out at a rate of approximately 200 L / hr / m 2 Approximately 500 L / hr / m 2 The flux rate is determined.

34. The method according to any one of claims 1-33, wherein the aqueous UF / DF process is in the range of about 200 L / hr / m 2 Approximately 500 L / hr / m 2 The flux rate is determined.

35. The method according to any one of claims 1-34, wherein the organic UF / DF process is carried out at a transmembrane pressure ranging from about 5 psi to about 30 psi.

36. The method according to any one of claims 1-35, wherein the aqueous UF / DF process is carried out at a transmembrane pressure ranging from about 5 psi to about 30 psi.

37. The method according to any one of claims 1-36, wherein the organic UF / DF process is carried out such that the temperature in the residue chamber containing the conjugate compound ranges from about 5°C to about 40°C.

38. The method according to any one of claims 1-37, wherein the aqueous UF / DF process is carried out such that the temperature in the residue chamber containing the conjugate compound ranges from about 5°C to about 40°C.

39. The method according to any one of claims 1-38, wherein the percolation volume of the organic UF / DF process ranges from about 2 to about 20.

40. The method according to any one of claims 1-39, wherein the percolation volume of the aqueous UF / DF process ranges from about 2 to about 20.

41. The method according to any one of claims 1-40, wherein the first semipermeable membrane and the second semipermeable membrane are independently selected from the group consisting of polyolefins, polystyrene, polysulfone, polyesters, polyamides, polyacrylates, polycarbonates, and mixtures and copolymers thereof.

42. The method according to any one of claims 1-41, wherein the first semipermeable membrane and the second semipermeable membrane are independently selected from the group consisting of: polyethersulfone (PES), polytetrafluoroethylene (PTFE), polypropylene, sulfonated polysulfone, polyvinylidene fluoride (PVDF), polyacrylonitrile (PAN), cellulose polymers, polyimide, polyetherimide (PEI), aliphatic polyamide, polyetheretherketone (PEEK), polyphenylene ether (PPO), polysulfone (PSf), and mixtures and copolymers thereof.

43. The method according to any one of claims 1-42, wherein the first semipermeable membrane and the second semipermeable membrane are the same semipermeable membrane.

44. The method according to any one of claims 1-43, wherein the first semipermeable membrane and the second semipermeable membrane are both neutral membranes without charge.

45. The method according to any one of claims 1-44, wherein the organic UF / DF process and the aqueous UF / DF process are performed as a tangential flow filtration (TFF) process.

46. ​​The method according to any one of claims 1-45, wherein the first aperture and the second aperture range independently from about 0.2 kDa MWCO to about 500 kDa MWCO.

47. The method according to any one of claims 1-46, wherein the conjugate compound comprises an antibody-drug conjugate (ADC).

48. The method according to any one of claims 1-47, wherein the conjugate compound comprises conjugates other than antibody-drug conjugates (ADCs).

49. The method according to any one of claims 1-48, wherein the concentration of the conjugated compound in the crude composition ranges from about 1 mg / mL to about 100 mg / mL.

50. The method according to any one of claims 1-49, wherein the at least one impurity comprises the residual free drug compound, the residual drug-connector compound, or a combination thereof.

51. The method according to any one of claims 1-50, wherein the at least one impurity comprises the residual free drug compound.

52. The method of claim 51, wherein the residual free drug compound is selected from the group consisting of: topoisomerase I (TOP1) inhibitors, topoisomerase II (TOP2) inhibitors, microtubule disruptors, DNA damage agents, DNA intercalating agents, protein degraders, TLR7 agonists, TLR8 agonists, STING agonists, XPO1 inhibitors, and any combination thereof.

53. The method according to claim 51 or 52, wherein the residual free drug compound is selected from the group consisting of: eczema, DXd, camptothecin, SN-38 (7-ethyl-10-hydroxy-camptothecin), DM1 (matansine), DM1 derivatives, DM4 (matansine), MMAE (monomethylaurestatin E), MMAF (monomethylaurestatin F), leptomysin B, and any combination thereof.

54. The method according to any one of claims 1-53, wherein the impurity comprises the residual drug-connector compound.

55. The method of claim 54, wherein the residual drug-connector compound comprises at least one pharmaceutical moiety derived from a pharmaceutical compound selected from the group consisting of: topoisomerase I (TOP1) inhibitors, topoisomerase II (TOP2) inhibitors, microtubule disruptors, DNA damage agents, DNA intercalators, protein degraders, TLR7 agonists, TLR8 agonists, STING agonists, XPO1 inhibitors, and any combination thereof.

56. The method according to claim 55, wherein the pharmaceutical compound is selected from eczema, DXd, camptothecin, SN-38 (7-ethyl-10-hydroxycamptothecin), DM1 (matansine), DM1 derivatives, DM4 (matansine), MMAE (monomethylaurestatin E), MMAF, DNA alkylating agents, pyrrolobenzodiazepine (PBD), leptomysin B, and any combination thereof.

57. The method according to any one of claims 1-56, wherein the conjugate compound comprises an antibody-drug conjugate (ADC).

58. The method of claim 57, wherein the ADC comprises at least one pharmaceutical portion derived from a pharmaceutical compound selected from the group consisting of: topoisomerase I (TOP1) inhibitors, topoisomerase II (TOP2) inhibitors, microtubule disruptors, DNA damage agents, DNA intercalators, protein degraders, TLR7 agonists, TLR8 agonists, STING agonists, XPO1 inhibitors, and any combination thereof.

59. The method according to claim 58, wherein the pharmaceutical compound is selected from eczema, DXd, camptothecin, SN-38 (7-ethyl-10-hydroxycamptothecin), DM1 (matansine), DM1 derivatives, DM4 (matansine), MMAE (monomethylaurestatin E), MMAF, pyrrolobenzodiazepine (PBD), leptomysin B, and any combination thereof.

60. The method according to any one of claims 57-59, wherein the drug-antibody ratio of the antibody-drug conjugate (ADC) ranges from about 1:1 to about 10:

1.

61. The method according to any one of claims 1-60, wherein the sample is a conjugated mixture.

62. The method according to any one of claims 1-61, wherein the concentration of the at least one impurity in the purified composition is less than 50 μg / mL based on the total volume of the purified composition.

63. The method according to any one of claims 1-62, wherein the method further comprises: (c) Formulate the residual product of step (b) to produce a pharmaceutical composition suitable for administration to a subject, optionally a human subject.

64. A method for preparing a purified conjugate compound, the method comprising: (i) The target agent is reduced by contacting it with a reducing agent to obtain a crude reduced target agent; (ii) The reduced target agent is conjugated with a drug-connector compound to obtain a crude composition containing the conjugated compound; (iii) Optionally, a quencher is added to the crude composition to obtain a sample; and (iv) Purify the sample of (iii) using the method according to any one of claims 1-62 to obtain the purified conjugate compound.

65. A method for preparing a purified conjugate compound, the method comprising: (i) The target agent is reduced by contacting it with a reducing agent to obtain a crude reduced target agent; (ii) Attaching at least one linker group to the reduced target agent to obtain a crude composition comprising a target agent-connector intermediate; (iii) Attaching at least one pharmaceutical compound to the connector of the target-connector intermediate to obtain a sample containing the conjugate compound; (iv) Optionally, a quencher may be added to the sample; and (v) Purify the sample using the UF / DF process according to any one of claims 1-62 to obtain the purified conjugate compound.

66. The method of claim 64 or 65, wherein the targeting agent is an antibody or an antigen-binding fragment, and the purified conjugate compound is a purified ADC.

67. The method of claim 64 or 65, wherein the target agent is an antibody-free target agent, and the purified conjugate compound is a purified conjugate other than an ADC.

68. The method according to any one of claims 64-67, further comprising purifying the crude reduced target agent by performing an initial aqueous UF / DF process on the crude reduced target agent before conjugating the reduced target agent with the drug-connector compound to obtain the conjugated compound.

69. The method according to any one of claims 64-68, wherein the conjugate compound comprises at least one pharmaceutical moiety derived from a pharmaceutical compound selected from the group consisting of: topoisomerase I (TOP1) inhibitors, topoisomerase II (TOP2) inhibitors, microtubule disruptors, DNA damaging agents, DNA intercalation agents, protein degrading agents, TLR7 agonists, TLR8 agonists, STING agonists, XPO1 inhibitors, and any combination thereof.

70. The method according to claim 69, wherein the pharmaceutical compound is selected from eczema, DXd, camptothecin, SN-38 (7-ethyl-10-hydroxycamptothecin), DM1 (matansine), DM1 derivatives, DM4 (matansine), MMAE (monomethylaurestatin E), MMAF, pyrrolobenzodiazepine (PBD), leptomysin B, and any combination thereof.

71. The method according to any one of claims 64-70, wherein, based on the total volume of the purified conjugate compound, the concentration of residual free drug compound, residual adapter compound, residual drug-adaptor compound, or any combination thereof in the purified conjugate compound is less than 200 μg / mL.

72. A method for producing a pharmaceutical composition, comprising: (a) Performing the method according to any one of claims 64 to 71; and (b) Formulate the purified conjugate compound produced in (a) to obtain a pharmaceutical composition comprising the purified conjugate compound and a pharmaceutically acceptable carrier or excipient.

73. A purified conjugate compound obtainable by any one of claims 1-62 or 64 to 71.

74. A pharmaceutical composition that can be obtained by the method according to claim 63 or 72.

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