Flow-through cation exchange chromatography purification method for antibody drug conjugates

Abstract

The present invention relates to a method for developing a purification process for antibody drug conjugates using cation exchange chromatography in flow-through mode that utilizes the purification conditions of an antibody intermediate without altering the critical quality attributes (CQAs) of the ADC.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 236,170, filed August 23, 2021, which is incorporated by reference in its entirety.

[0002] FIELD OF THEINVENTION In general, the present invention relates to a method for developing a purification method for antibody drug conjugates using cation exchange chromatography in flow-through mode. In particular, the present invention relates to a purification method for cysteine-targeted antibody drug conjugates using cation exchange chromatography in flow-through mode. More particularly, the present invention relates to a purification method for cysteine-targeted antibody drug conjugates using cation exchange chromatography in flow-through mode that utilizes purification conditions for antibody intermediates. [Background technology]

[0003] 2. Background of the Invention Antibody molecules, as part of the protein pharmaceutical group, are highly susceptible to physical and chemical degradation. Chemical degradation includes any process involving modification of proteins through bond formation or cleavage, resulting in new chemical compounds. Various chemical reactions are known to affect proteins. These reactions may include hydrolysis, including peptide bond cleavage, as well as deamidation, isomerization, oxidation and degradation. Physical degradation refers to higher order structural changes and includes denaturation, adsorption to surfaces, aggregation and precipitation. Protein stability is influenced by the characteristics of the protein itself, e.g., amino acid sequence, glycosylation pattern, and by external influences such as temperature, solvent, pH, excipients, interfaces, or shear rate.

[0004] Antibody-drug conjugates (ADCs) are targeted anti-cancer therapeutics designed to reduce non-specific toxicity and increase efficacy compared to traditional small molecule and antibody cancer chemotherapy. They use the powerful targeting ability of monoclonal antibodies to specifically deliver highly potent conjugated small molecule therapeutics to cancer cells. ADCs consist of a potent small molecule drug conjugated to an antibody, allowing targeted delivery to tumor cells. The conjugation process involves chemical reaction of the antibody and cytotoxic drug to achieve the desired drug-to-antibody ratio (DAR). (RVJChari,MLMiller,WCWiddison,Antibody-Drug Conjugates:An Emerging Concept in Cancer Therapy,Ange.Chem.Int.Ed.53(2014)3796-3827;P.Polakis,Pharmacological Reviews(2016),3-19).DAR needs to be tightly controlled as it directly impacts both safety and efficacy. DAR also needs to be controlled to appropriately narrow specifications to ensure product consistency.

[0005] The chemical reaction steps required to form the antibody-drug conjugate may require reaction conditions such as long hold times, high pH, ​​and solvent background that may result in protein aggregation. As a final drug substance, the level of aggregates in the conjugate must be controlled within the required specifications. In addition to the total level of aggregates, it may be necessary to specifically focus on product multimers larger than dimers, often referred to as very high molecular weight species (vHMWS). Due to the increased risk of immunogenicity from protein aggregates, especially for ultra-high molecular weight species (vHMWS), there has been a concerted effort to reduce the formation of this particular aggregate species (W. Wang, S.K. Singh, N. Li, M.R. Toler, K.R. King, S. Nema, Immunogenicity of protein aggregates--concerns and realities, Int J Pharm. 431 (2012) 1-11).

[0006] The starting monoclonal antibody (mAb) intermediate is manufactured and purified to achieve product quality similar to standard biotherapeutics (AA Shukla, B. Hubbard, T. Tressel, S. Guhan S, D. Low, Downstream processing of monoclonal antibodies--application of platform approaches, J Chromatogr B Analyt Technol Biomed Life Sci. 848 (2007) 28-39; P. Gronemeyer, R. Ditz, J. Strube. Trends in Upstream and Downstream Process Development for Antibody Manufacturing. Bioengineering (Basel) 1 (2014) 188-212).

[0007] Antibody purification is typically performed using bind-elute or flow-through chromatography. Weak partitioning chromatography (Kelley, BD et al., 2008 Biotechnol Bioeng 101(3):553-566; US Patent Publication No. 2007 / 0060741) and overloading chromatography (PCT / US2011 / 037977) have been used to enhance antibody purification on anion exchange resins (AEX) and cation exchange resins (CEX), respectively.

[0008] Purification methods utilizing platform processes and HTS methods are typically used to develop antibody purification using cation exchange chromatography (CEX), which is commonly used to remove aggregates and impurities in antibody purification processes. CEX is typically operated in bind-elute mode with a relatively low target loading density (HF Liu, J. Ma, C. Winter, R. Bayer, Recovery and purification process development for monoclonal antibody production, mAbs, 2 (2010) 480-499).

[0009] Bind-elute chromatography: In bind-elute chromatography, the product is usually loaded to maximize the dynamic binding capacity (DBC) on the chromatographic material, and then the washing and elution conditions are specified to achieve maximum product purity in the eluate. The limitation of bind-elute chromatography is the restriction of the loading density to the actual resin DBC. Therefore, bind-elute chromatography purification requires a larger column size due to the low loading density. The purification process in bind-elute mode is more complicated to develop and implement at the manufacturing stage. The pooling criteria of the bind-elute purification process can be a critical parameter and can result in reduced yields and equipment fit challenges.

[0010] Flow-through chromatography: Flow-through chromatography is used to identify loading conditions where impurities bind strongly to the chromatographic material while the product flows through. Flow-through chromatography allows for high loading densities of standard antibodies.

[0011] Overloaded chromatography: In this mode of chromatography, the product of interest is loaded beyond the dynamic binding capacity of the chromatographic material for the product, hence referred to as overloaded. The mode of operation has been demonstrated to provide antibody purification using cation exchange (CEX) media, particularly membranes. However, a limitation of this technique is that the resin can result in low yields due to the absence of an elution phase. An additional challenge of overloaded chromatography is the appropriate critical process parameters, including equipment and appropriate loading conditions for high titers.

[0012] High-throughput screening (HTS) robotic devices are typically used for standard monoclonal antibody purification development. However, such high-throughput screening (HTS) robotic devices may not be suitable or safe for handling cytotoxic compounds (JL Coffman, JF Kramarczyk, BD Kelley, High-throughput screening of chromatographic separations: I. Method development and column modeling. Biotechnol. Bioeng., 100 (2008) 605-618; MI Hensgen, B. Stump, Safe Handling of Cytotoxic Compounds in a Biopharmaceutical Environment. In: Ducry L. (ed.), Antibody-Drug Conjugates. Methods in Molecular Biology (Methods and Protocols), 1045 (2013); Humana Press, Totowa, NJ. 2013, pp. 130-142). This poses challenges when using HTS for the purification of ADCs.

[0013] If the formation of aggregates cannot be reliably controlled during the conjugation reaction to form the ADC, a purification step must be performed after conjugation to achieve the drug substance aggregate specifications. Purification must be performed without compromising the safety requirements of handling potent compounds and without affecting the quality of the desired ADC product, such as DAR and drug loading distribution. ADC purification techniques also use hydrophobic interaction chromatography (HIC). HIC is a useful tool for separating molecules based on their hydrophobicity. Typically, sample molecules in a high salt buffer are loaded onto a HIC column. The salt in the buffer interacts with water molecules to reduce solvation of the molecules in solution, thereby exposing hydrophobic regions in the sample molecules, which then adsorb to the HIC column. The more hydrophobic the molecule, the less salt is needed to promote binding. Typically, a decreasing salt gradient is used to elute the sample from the column. As the ionic strength decreases, the exposure of the hydrophilic regions of the molecules increases, and molecules elute from the column in order of increasing hydrophobicity. Elution of the sample may also be achieved by adding mild organic modifiers or surfactants to the elution buffer. HIC is reviewed in Protein Purification, 2nd Edition, Springer-Verlag, New York, pp. 176-179 (1988).

[0014] However, HIC techniques, which are typically performed at near-neutral pH in the presence of high salt concentrations, can pose safety issues in handling potent compounds due to precipitation of ADCs as well as contamination of filters. (Becker CL, Duffy RJ, Gandarilla J., Richter SM (2020) Purification of ADCs by Hydrophobic Interaction Chromatography. In: Tumey L. (ed.) Antibody-Drug Conjugates. Methods in Molecular Biology, vol 2078. Humana, New York, NY. https: / / doi.org / 10.1007 / 978-1-4939-9929-3_19)

[0015] In general, the development of ADC purification is more challenging due to the safety requirements of handling cytotoxic compounds. Large-scale, cost-effective purification of ADCs to sufficient purity for use as human therapeutics remains a challenging task.

[0016] Various ADC purification techniques have been described in the literature. For example, CN104208719 describes elution and overloading for ADC purification. However, CN104208719 does not provide any teaching on purification in flow-through mode. Furthermore, CN104208719 does not discuss antibody purification, nor does it provide any teaching on utilizing the purification conditions developed during antibody intermediate purification for the purification of ADC. In another example, US Patent Publication No. 2013245139 uses a CEX membrane for antibody aggregate purification. However, US Patent Publication No. 2013245139 does not provide any teaching on the purification of ADC, particularly on utilizing the purification conditions developed during antibody intermediate purification for the purification of ADC.

[0017] It is therefore an object of the present invention to provide a method for developing a purification process for antibody drug conjugates using cation exchange chromatography in flow-through mode that exploits the purification conditions of the antibody intermediate.

[0018] It is also an object of the present invention to identify conditions during purification to remove aggregates that also do not affect the critical quality attributes (CQAs) of the ADC product, such as DAR and drug loading distribution.

[0019] Another object of the present invention is to provide for the development of a rapid and robust purification process for antibody drug conjugates using cation exchange chromatography in flow-through mode.

[0020] It is a further object of the present invention to provide a low-cost, robust purification process for antibody drug conjugates using cation exchange chromatography in flow-through mode.

[0021] It is also an object of the present invention to specifically remove vHMWS.

[0022] The simplified ADC purification method and process significantly reduced vHMWS, achieved consistently high yields, and did not alter the critical quality attributes (CQAs) of the ADC product. This purification approach may also be used to develop purification methods for vHMWS removal for ADCs with minimal development. Summary of the Invention

[0023] overview The present invention provides methods for utilizing purification conditions developed during antibody intermediate purification for the purification of ADCs.

[0024] In one aspect, the invention provides an improved method for reducing the concentration of very high molecular weight species (vHMWS) in a cysteine-directed antibody drug conjugate (ADC), the method comprising: a. performing a first purification of the antibody on a cation exchange chromatography material using a first set of purification conditions to obtain a purified antibody intermediate; b. conjugating the purified antibody intermediate with a cytotoxic drug to form a crude preparation comprising a cys ADC and protein aggregates; and c. performing a second purification of the crude preparation by cation exchange chromatography material in flow-through mode using the first set of purification conditions to produce a purified cys ADC. Including, The method is provided wherein the set of purification conditions includes loading density, buffer species, pH and conductivity of the buffer system.

[0025] In another aspect, the invention provides for reducing the concentration of vHMWS in an eluate by at least 85% relative to the concentration of protein aggregates in a crude mixture of cysADC and protein aggregates without altering the critical quality attributes (CQAs) of the cysADC.

[0026] In yet another aspect, the invention provides a method of reducing vHMWS in an ADC to less than 0.1%.

[0027] In yet another embodiment, the present invention provides a method, wherein the resin used in the cation exchange column is selected from POROS 50HS, POROS XS, and SPFF resins.

[0028] In a further aspect, the invention provides a method for purifying a cysteine-directed antibody drug conjugate (cys ADC), the method comprising: a. performing a first purification of the antibody on a cation exchange chromatography material using a first set of purification conditions to obtain a purified antibody intermediate; b. conjugating the purified antibody intermediate with a cytotoxic drug to form a crude preparation of a cys ADC; and c. performing a second purification of the crude preparation by cation exchange chromatography material in flow-through mode using the first set of purification conditions to produce a purified cys ADC. Including, The method is provided wherein the set of purification conditions includes loading density, buffer species, pH and conductivity of the buffer system.

[0029] In one embodiment of the method of purifying a cys ADC, the cytotoxic agent is selected from the group consisting of an auristatin, a maytansinoid, and a DNA damaging agent.

[0030] In another embodiment of the method for purifying a Cys ADC, the method of claim 23, wherein the DNA damaging agent is a derivative selected from the group consisting of calicheamicin, anthracyclines, and pyrrolobenzodiazepines.

[0031] In yet a further embodiment of the invention, the antibodies used to form the cys ADC are purified in a bind-elute mode.

[0032] In another embodiment of the invention, the first purification to obtain a purified antibody intermediate comprises step elution.

[0033] In yet another embodiment of the invention, the first purification to obtain a purified antibody intermediate comprises gradient elution.

[0034] In another embodiment of the method for purifying an antibody, the screening method for determining the binding behavior of the antibody is a high throughput screening (HTS).

[0035] In another embodiment of the invention, HTS used in the purification of antibodies is used to map the binding behavior of antibodies as a function of pH and counterion concentration.

[0036] In another embodiment, the results of antibody HTS used to map the binding behavior of antibodies are leveraged to identify flow-through conditions for ADC purification.

[0037] In a further embodiment of the invention, the protein aggregate species removed during purification of an antibody or cys ADC include very high molecular weight species (vHMWS) and high molecular weight species (HMWS) of the antibody or cys ADC.

[0038] In yet a further embodiment of the invention, the protein aggregate species removed during purification are very high molecular weight species (vHMWS).

[0039] In another embodiment of the invention, cys ADCs are selected from engineered cysteines targeting native cysteines and site-directed conjugates via interchain cysteine ​​conjugates.

[0040] In another embodiment, the cys ADC is a site-specific conjugate via an engineered cysteine.

[0041] In yet another embodiment, the cys ADC is an interchain cysteine ​​conjugate that targets a native cysteine.

[0042] In yet a further embodiment of the invention, the method for developing the purification method comprises thiomab antibiotic antibody conjugate (AAC).

[0043] In another embodiment of the invention, the pooling standard for cys ADCs is 0.5 OD to 0.5 OD. [Brief description of the drawings]

[0044] [Figure 1] Figure 1 shows an example of a chromatogram of protein impurities analyzed by SEC-HPLC using a TSKgel G3000SWxL column (7.8 × 300 mm, Tosoh Bioscience, Tokyo, Japan). Peaks were separated by isocratic separation using a mobile phase of 15% IPA and 85% 0.2 M potassium phosphate, 0.25 M potassium chloride, pH 6.95. The flow rate was maintained at 0.5 mL / min at ambient temperature with UV detection at 280 nm. The two main aggregate species detected include vHMWS and HMWS. HMWS are protein dimers of antibody-drug conjugates, and vHMWS are oligomers of antibody-drug conjugates.

[0045] [Diagram 2] FIG. 2 shows examples of the mean DAR and drug loading distribution determined using an analytical hydrophobic interaction chromatography (HIC) method of interchain cysteine ​​conjugates.

[0046] [Diagram 3] FIG. 3 shows examples of mean DAR and drug loading distribution determined using analytical hydrophobic interaction chromatography (HIC) methodology of site-specific conjugates.

[0047] [Figure 4] FIG. 4 shows an example of batch binding contour plots comparing the binding behavior for an antibody on a CEX resin and its corresponding cysteine-directed antibody drug conjugate (cys ADC).

[0048] [Diagram 5] FIG. 5 shows an example of aggregated species (vHMWS and HMWS) breakthrough of cysteine-directed antibody-drug conjugates for a CEX column with a conjugate loading density of 500 g / Lr. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0049] Detailed Description of the Invention The present invention relates to methods for developing purification of cysteine-directed antibody drug conjugates, including purifying antibodies and exploiting the binding behavior of antibody intermediate aggregate species for ADC purification.

[0050] For the purposes of this specification and claims, unless otherwise indicated, all numbers expressing amounts of ingredients, proportions or ratios of materials, reaction conditions, and other numerical values ​​used in this specification and claims, whether expressly indicated or not, should be understood to be modified in all cases by the term "about". The term "about" generally refers to a range of numbers that is considered equivalent to the recited value. In many cases, the term "about" may include numbers that are rounded to the nearest significant figure. Moreover, all ranges disclosed herein should be understood to include all subranges subsumed therein.

[0051] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

[0052] Before describing the invention in more detail, certain terms are defined. The use of these terms does not limit the scope of the invention, but serves only to facilitate the description of the invention. Definition:

[0053] The term "antibody" is used in the broadest sense and specifically includes intact monoclonal antibodies (mAbs), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.

[0054] An "antibody fragment" comprises a portion of an intact antibody, generally the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0055] As used herein, "antibody intermediate" refers to a purified antibody that has been purified by a first purification using a first set of purification conditions, which is used to conjugate with a cytotoxic drug to produce an antibody drug conjugate.

[0056] As used in this specification and the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise.

[0057] As used herein, "binding behavior" refers to the binding or non-binding of an antibody, antibody intermediate or ADC to a resin under particular conditions, including pH and counterion concentration.

[0058] As used herein, "buffer" refers to a buffer solution that resists changes in pH due to the action of its acid-base complex components. Buffers of the CEX chromatography embodiment of the invention have a pH in the range of about 4.5 to 6.5, preferably about 5.3 to 5.7. Examples of buffers that adjust the pH to within this range include phosphate, acetate, citrate or ammonium buffers, or two or more buffers. Preferred such buffers are acetate, citrate and ammonium buffers, most preferably sodium acetate buffer. A "loading buffer" is one that is used to load the mixture of ADC and impurities / contaminants onto the CEX column, and an "equilibration / washing buffer" is one that is used to wash the ADC from the column to recover the antibody while the impurities / contaminants are retained on the column. Often, the loading buffer and equilibration / washing buffer have the same pH and / or conductivity conditions.

[0059] The term "sequential" as used herein with respect to chromatography refers to having a first chromatography followed by a second chromatography. Additional steps may be included between the first and second chromatography.

[0060] The term "continuous" as used herein with respect to chromatography refers to having a first chromatographic material and a second chromatographic material that are directly connected or connected via some other mechanism that allows for continuous flow between the two chromatographic materials.

[0061] The term engineered cysteine ​​as used herein refers to an antibody that has an engineered reactive cysteine ​​residue for site-specific conjugation and exhibits homogeneous conjugation.

[0062] As used herein, the term cysteine-directed antibody drug conjugate (cys ADC) refers to a conjugate of an antibody that has a cysteine ​​residue available for conjugation with a cysteine ​​drug.

[0063] The term native cysteine ​​as used herein refers to interchain disulfide bonds, which are generated by partial reduction resulting in a heterogeneous complex consisting of 0-, 2-, 4-, 6-, and 8-DAR forms.

[0064] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cell function and / or causes cell death or destruction.Cytotoxic agents include, but are not limited to, DNA damaging agents, including auristatins, maytansinoids, and calicheamicin, anthracyclines and pyrrolobenzodiazepines, radioisotopes; chemotherapeutic agents or drugs (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitors; enzymes such as nucleases and fragments thereof; antibiotics; toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, such as fragments and / or variants thereof; and various antitumor or anticancer agents.

[0065] As used herein, the term "DAR" is the mean drug-antibody ratio. The DAR directly affects the safety and efficacy of the ADC and is directly controlled during the ADC manufacturing process.

[0066] The term "drug loading distribution" as used herein refers to the number of drugs conjugated to an antibody.

[0067] The term critical quality attribute (CQA) includes the average drug-to-antibody ratio (DAR) and drug loading distribution, which determine the amount of cytotoxic drug that can be delivered by the ADC.

[0068] The "dynamic binding capacity" of a chromatographic material is the amount of product, e.g., polypeptide, that the material will bind under practical flow conditions before significant breakthrough of unbound product occurs.

[0069] As used herein, "partition coefficient," K p refers to the molar concentration of a product, e.g., a polypeptide, in the stationary phase divided by the molar concentration of the product in the mobile phase.

[0070] "Loading density" refers to the amount (e.g., grams) of a composition contacted with a volume (e.g., liters) of a chromatographic material. In some examples, loading density is expressed in g / L r It is expressed as:

[0071] As used herein, the terms "ion exchange" and "ion exchange chromatography" refer to a chromatographic method in which an antibody or antibody-drug conjugate of interest interacts or does not interact with a charged compound linked to a solid-phase ion exchange material, such that impurities or aggregates in the mixture elute from a column of ion exchange material faster or slower than the antibody or antibody-drug conjugate of interest and are bound to or excluded from the resin relative to the impurities or aggregates. "Ion exchange chromatography" specifically includes cation exchange (CEX) chromatography, anion exchange chromatography, and mixed-mode chromatography.

[0072] As used herein, the term "anion exchange resin" or "AEX" refers to a positively charged solid phase, having one or more positively charged ligands, such as, for example, quaternary amino groups, attached thereto. Commercially available anion exchange resins include DEAE cellulose, QAE SEPHADEX™, and FAST Q SEPHAROSE™ (Pharmacia). Anion exchange chromatography can bind and subsequently elute target molecules, or can primarily bind impurities while target antibodies or antibody-drug conjugates pass through the column.

[0073] A cation exchange chromatography material is a negatively charged solid phase that has free anions to exchange with cations in an aqueous solution (such as a composition containing an antibody and impurities) that passes through the solid phase. In some embodiments of any of the methods described herein, the cation exchange material can be a membrane, a monolith, or a resin. In some embodiments, the cation exchange material can be a resin. The cation exchange material can include carboxylic acid or sulfonic acid functional groups, for example, but not limited to, sulfonate, carboxyl, carboxymethylsulfonic acid, sulfoisobutyl, sulfoethyl, carboxyl, sulfopropyl, sulfonyl, sulfoxyethyl, or orthophosphate. In some embodiments described above, the cation exchange chromatography material is a cation exchange chromatography column. In some embodiments described above, the cation exchange chromatography material is a cation exchange chromatography membrane. Examples of cation exchange materials, including resins, are known in the art and include, but are not limited to, Mustang® S, Sartobind® S, S03Monolith (e.g., CIM®, CIMmultus®, and CIMac® S03), S Ceramic HyperD®, Poros® XS, Poros® HS 50, Poros® HS 20, sulphopyl-Sepharose® Fast Flow (SPSFF), SP-Sepharose® XL (SPXL), CM Sepharose® Fast Flow, Capto™ S, Fractogel® EMD Se Hicap, Fractogel® EMD S03, or Fractogel® EMD COO. In some embodiments, the cation exchange chromatography is performed in a "bind-elute" mode. In some embodiments, the cation exchange chromatography is performed in a "flow-through" mode. In some embodiments above, the cation exchange chromatography material is in a column. In some embodiments above, the cation exchange chromatography material is in a membrane.

[0074] "Impurities" refer to substances that are different from the desired polypeptide product. Impurities may refer to product-specific polypeptides, such as one-armed antibodies and misassembled antibodies, antibody variants, including basic and acidic variants, and aggregates. Other impurities include, but are not limited to, process-specific impurities, including host cell materials, such as host cell proteins (HCPs); leached protein A; nucleic acids; other polypeptides; endotoxins; viral contaminants; cell culture media components, and the like. In some examples, impurities may be, but are not limited to, HCPs from bacterial cells, such as E. coli cells (ECPs), insect cells, prokaryotic cells, eukaryotic cells, yeast cells, mammalian cells, avian cells, fungal cells. In some examples, impurities may be HCPs from mammalian cells, such as CHO cells, i.e., CHO cell proteins (CHOPs). Impurities may refer to accessory proteins used to facilitate expression, folding, or assembly of multispecific antibodies; for example, prokaryotic chaperones, such as FkpA, DsbA, and DsbC, and the like.

[0075] As used herein, high molecular weight substance (HMWS) refers to a protein dimer of an ADC or a protein dimer of an antibody.

[0076] As used herein, ultrahigh molecular weight agents refer to oligomers of ADCs or oligomers of antibodies.

[0077] As used herein, the term protein includes antibodies and ADCs.

[0078] Purity is a relative term and does not necessarily mean absolute purity. The terms "purification", "separation" or "isolation" used interchangeably herein refer to increasing the purity of a desired molecule from a composition or sample that contains the desired molecule and one or more impurities. Typically, the degree of purity of a desired molecule is increased by removing (completely or partially) at least one impurity from the composition.

[0079] Purification conditions are also relative terms and these conditions may vary from one purification method to another. Purification conditions may include loading density of the buffer system, buffer species, pH and conductivity.

[0080] material and method A drug conjugate

[0081] Conjugation is a multi-step process for modifying proteins that may differ based on conjugate design. Two types of conjugates were investigated for purification: site-specific conjugation via engineered cysteines targeting native cysteines and interchain cysteine ​​conjugates.

[0082] For site-specific conjugation, the purified intermediate is incubated overnight with a reducing agent to completely reduce the antibody's native and engineered cysteines and remove any cysteine ​​or glutathione cap from the engineered cysteines. The reduced antibody is buffer exchanged to remove residual reductant and cap species. The interchain disulfide bonds are reformed via a reoxidation step, making the engineered cysteines available for conjugation with the linker-drug. An excess of linker-drug is added to ensure complete conjugation with all free thiols (J. Junutula, H. Raab, S. Clark, et al., Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index, Nat Biotechnol 26 (2008) 925-932). Depending on the linker-drug, conjugation is quenched or stopped by lowering the pH of the reaction. Finally, residual free drug is removed.

[0083] In the case of interchain cysteine ​​conjugates, the native cysteines of the antibody intermediate are partially reduced with a predefined amount of reducing agent prior to conjugation with the linker drug (MMC Sun, KS Beam, CG Cerveny, KJ Hamblett, RS Blackmore, MY Torgov, FGM Handley, NC Ihle, PD Center, SC Alley, Reduction-Alkylation Strategies for the Modification of Specific Monoclonal Antibody Disulfides, Bioconjugate Chemistry 16 (2005) 1282-1290). Excess linker drug is quenched and residual free drug is removed.

[0084] b. Complex column purification Column chromatography experiments were performed using an AKTA Explorer 100 equipped with columns of various sizes packed with cation exchange resin. A calibrated load was applied to the equilibrated column at various loading densities and the flow-through was collected. After the loading step, the column was washed with equilibration buffer to increase the recovery of ADC. The pool was terminated at the end of the wash step or when the OD was less than 0.5. The column was regenerated with 0.5N NaCl, cleaned with 0.5N NaOH and stored in 0.1N NaOH.

[0085] c. Antibody intermediate purification development High-throughput screening (HTS) was performed on the antibody intermediates using known methods (P. McDonald, B. Tran, CR Williams, M. Wong, T. Zhao, BD Kelley, P. Lester, The rapid identification of elution conditions for therapeutic antibodies from cation-exchange chromatography resins using high-throughput screening, J Chromatogr A. 1433 (2016) 66-74). HTS maps the binding behavior of the antibodies as a function of pH and buffer concentration (JL Coffman, JF Kramarczyk, BD Kelley, High-throughput screening of chromatographic separations: I. Method development and column modeling. Biotechnol. Bioeng., 100 (2008) 605-618). To develop the binding and elution conditions on the cation-exchange chromatography resins, HTS equipped with a Tecan Robotic liquid handling system or a 96-well filter plate using a multichannel pipette was used for batch binding experiments. A packed bed laboratory scale column was used to verify and optimize conditions.

[0086] The antibody intermediate purification method includes CEX to remove aggregates and host cell impurities, and is carried out at 100 g / L rThe column is operated in bind-elute mode with a loading density of less than 1000 μg / ml (HF Liu, B. McCooey, T. Duarte, DE Myers, T. Hudson, A. Amanullah, R. van Reis, BD Kelley, Exploration of overloaded cation exchange chromatography for monoclonal antibody purification, J Chromatogr A. 1218 (2011) 6943-52). After loading the product and washing the column, the monomer was eluted with elution buffer. The binding behavior of antibody intermediate aggregate species was exploited for ADC purification development.

[0087] d.Analysis method Protein concentrations were quantified by UV-vis spectrophotometry (Agilent 8453). Protein concentrations were determined by absorbance at 280 nm minus absorbance at either 320 nm or 400 nm to correct for light scattering. The extinction coefficient ε of the sample was used in the following equation, where: TIFF2024532238000001.tif6170 is the sample path length, A 280 and A 320 are the measured absorbance values ​​at 280 nm and 320 nm, respectively. TIFF2024532238000002.tif17170

[0088] Protein impurities were analyzed by SEC-HPLC using a TSKgel G3000SWxL column (7.8 × 300 mm, Tosoh Bioscience, Tokyo, Japan). Peaks were separated by isocratic separation using a mobile phase of 15% IPA and 85% 0.2 M potassium phosphate, 0.25 M potassium chloride, pH 6.95. The flow rate was maintained at 0.5 mL / min at ambient temperature with UV detection at 280 nm. Example chromatograms are shown in Figure 1 and are representative of both complex types. The two main aggregate species detected include vHMWS and HMWS. HMWS are protein dimers and vHMWS are oligomers of antibodies / ADCs.

[0089] The average DAR and drug loading distribution were measured using an analytical hydrophobic interaction chromatography (HIC) method, as shown in Figure 2 and Figure 3. Samples were injected onto a Tosoh Biosciences Butyl-NPR column (4.6 mm x 3.5 cm, 2.5 µm) and eluted over a linear gradient with solvent B at a flow rate of 0.8 mL / min and absorbance was monitored at 280 nm. The gradient and solvent B for the individual conjugates are shown in Table 1. [Table 1]

[0090] Purification methods for the ADCs were developed based on the development of their respective antibody intermediates. Each of the antibody intermediates underwent independent purification method development based on their properties (e.g., pI, binding properties, etc.), resulting in slightly different purification processes and operating modes (Table 2). Based on the different antibody purification steps, different approaches were used to develop flow-through purification conditions for the conjugate molecules described herein.

[0091] ADC-1: Antibody-1 utilized gradient elution, so manual resin screening was performed with ADC-1 and compared to the HTS results for Antibody-1. ADC-1 strongly binds aggregates but does not bind monomer as monomer flows through the column, with a LogK between 0.75 and 1.25. p Promising conditions were selected, including conditions with (antibody) and pH / conductivity conditions. Packed bed column experiments were used to test the promising conditions to determine optimal flow-through conditions.

[0092] ADC-2: HTS was not performed on the ADC, instead developmental HTS results for antibody-2 were utilized. Antibody mid-step elution conditions were developed such that monomers would elute from the column while retaining aggregates. These conditions were applied to the conjugate such that the column loading material would result in product flow-through while removing aggregated species. The robustness of the loading conditions relative to purification capacity was assessed using packed bed column experiments to evaluate performance at manufacturing scale.

[0093] ADC-3: The theory that antibody purification conditions can be applied to ADC was tested with a third product. As with Antibody-2, HTS was not performed on ADC-3, but the step elution conditions from Antibody-3 were applied to the ADC to remove aggregated species in the flow-through. A single packed column experiment was performed to confirm the purification potential of the conjugate. [Table 2]

[0094] Unless otherwise stated, a small-scale column with 0.66 cm internal diameter (ID) × 1.0 cm bed height (BH) was used for the ADC purification run. Also, equilibration / wash buffer conditions were adjusted to match the loading conditions. Although the focus of the ADC purification was the removal of vHMWS, HMWS removal was also monitored. Furthermore, it was not desired to alter the DAR or drug loading distribution over the purification steps.

[0095] Example 1: ADC-1 purification method

[0096] To determine the flow-through conditions for ADC-1, a manual batch binding screen was performed with CEX resin using a multichannel pipette and a 96-well plate. The results of the conjugate screen were comparable between antibodies and conjugates with similar binding behavior (Figure 4). A LogK of approximately 1. p Loading conditions of 214 mM sodium acetate, pH 5.5, corresponding to the value, were selected as the target conditions to allow monomer to flow through while aggregates remained bound to the resin.

[0097] The selected target purification conditions were applied to ADC-1 so that the purification step could be carried out in flow-through mode. The ADC load was titrated to the target conductivity and pH and was 500 g / L. r The column was loaded to a loading density of 1000 μg / ml. The column was washed with 10 CV of equilibration buffer to recover ADC-1. The loads and pools were analyzed by SEC-HPLC for aggregates and by HIC-HPLC for effect on DAR (Table 3). [Table 3]

[0098] Flow-through purification conditions were successful in reducing vHMWS to 0.10% for ADC-1. A yield of 89% was achieved with no impact on mean DAR or drug distribution. Although antibody-1 did not use step elution for purification, this study demonstrated that antibody HTS can be leveraged for ADC purification development, eliminating the need for manual batch binding screening with conjugates. To further test the theory that antibody HTS results can be leveraged to identify flow-through conditions for ADC purification, two additional products were evaluated.

[0099] Example 2: ADC-2 purification method i. High-throughput screening of antibody 2 The development of the CEX step for antibody-2 was performed using HTS. These experiments supported the identification of promising step elution conditions that were predicted to elute the monomer while the aggregated impurities remained bound to the resin. p Contour plots were generated. Based on these data, a target elution buffer was selected for antibody-2, and subsequent robustness studies confirmed the robustness of the step elution purification. ii. Purification and development of ADC-2 b. Antibody-2 process elution conditions were utilized during the development of the ADC-2 CEX process to identify conditions such that aggregates bind to the resin and are removed while the desired product (monomer) flows through. To accomplish this, the column equilibration, loading and wash phases were adjusted to match the elution conditions (pH and conductivity) used for the Antibody-2 process. c. After demonstrating successful aggregate removal at the target conditions, the robustness of the process near the target was tested at various pH and conductivity conditions (Table 4). "Low" conditions were selected to represent stronger binding conditions for the CEX column due to decreased pH and decreased conductivity. Conversely, "High" loading conditions were identified that likely not only reduced product binding but also reduced aggregate removal. Multiple "High" loading conditions were tested at various pH and conductivity ranges to explore the robustness of operation near worst-case conditions with respect to aggregate removal. The complex load was adjusted to the desired conductivity and pH using appropriate buffer species. Equilibration and wash buffers were adjusted to fit the various conditions of loading. Loading density was 220-300 g / L. r The range was. [Table 4] d. The "Low" loading condition showed initial binding to the resin and subsequent breakthrough, similar to the overloading mechanism of purification, and HMWS was also significantly reduced at this loading condition (HF Liu, B. McCooey, T. Duarte, DE Myers, T. Hudson, A. Amanullah, R. van Reis, BD Kelley, Exploration of overloaded cation exchange chromatography for monoclonal antibody purification, J Chromatogr A. 1218 (2011) 6943-52). Higher mass loading density improved yields. The "High++" loading condition identified failure points and did not remove vHMWS. However, the range of loading conditions was demonstrated to be robust (±0.1 pH, ±0.7 mS / cm) around the target loading conditions between the "Low" and "High+" conditions. Loading and buffer specifications were set to ensure robust implementation into manufacturing. e. After an acceptable operating range has been demonstrated, a loading density of 500 g / L is achieved using a 0.66 cm ID x 20 cm BH column. r The conjugated pool was adjusted to the target loading pH and conductivity and loaded onto the column at 500 g / L. r The load was up to . f. The yield from the high loading density experiment was 96% and vHMWS was reduced to 0.02% in the pool (Table 5). Furthermore, the average DAR was not affected by purification. The elution conditions for the antibody-2CEX step were 500 g / L. r The composite was effectively applied in flow-through mode at a loading density of 0.01 μg / cm2. [Table 5]

[0100] The ADC-2 purification process was successfully scaled up to manufacturing scale using a 14 cm ID x 15 cm BH column. Three runs at the target conditions were performed, with the column producing approximately 260 g / L per run. r The drug substance results showed no detectable vHMWS and all other product quality attributes were met (Table 6). Furthermore, the chromatograms of the three runs were consistent and there was no pressure increase during the loading step. [Table 6] a. Abbreviations: ND = not detected a Results are for the final drug substance. In-process pooling analysis was not performed in the large scale run.

[0101] Example 3: ADC-3 purification development

[0102] Similar to Antibody-2, the purification method for Antibody-3 used a step-elution CEX step to remove aggregates and impurities. Without performing the development of the ADC purification, the Antibody-3 step-elution conditions were utilized for the ADC-3 purification. The antibody step-elution conditions were applied to the ADC such that aggregates bound to the resin and were removed while the desired product (monomer) flowed through.

[0103] The pH and conductivity of the conjugate ADC-3 was adjusted to target antibody-3 elution buffer conditions to achieve product flow-through. The adjusted conjugate load was 500 g / L on the CEX column. r The column was washed with 10 CV of equilibration buffer and fractions were diluted to a loading density of 50 g / L.r Fractions were pooled and the results compared to loading (Table 7). [Table 7]

[0104] 300g / L r Starting with a loading density of 500 g / L, a small breakthrough of vHMWS was observed (Figure 5). To mitigate vHMWS breakthrough, lower loading densities or slightly stronger binding conditions were used. However, the final level of vHMWS in the purified pool was 500 g / L. r Even when loaded to a load density of 1000, the load was reduced to an acceptable level.

[0105] These results demonstrate that the antibody intermediate purification step can be leveraged for ADC purification. Development of ADC purification was not performed since the antibody-3 step elution conditions were successfully implemented for the conjugate run in flow-through mode. The flow-through purification conditions successfully reduced the vHMWS to 0.39%-0.03% for ADC-3 with a yield of 98%. Furthermore, the mean DAR and drug distribution were not affected.

[0106] conclusion Antibody purification development data may be used to streamline the development of a simple flow-through purification step for each conjugate. Antibody stage elution conditions (pH and conductivity) are converted to ADC to enable flow-through purification. In the absence of antibody stage elution conditions, antibody HTS may be leveraged to identify ideal ADC flow-through conditions. ADC Purification Steps: ● vHMWS significantly reduced ● Consistently achieved high yields, but ● There was no change in mean DAR or drug load distribution.

[0107] The above disclosure may encompass multiple separate inventions with independent utility. Numerous variations are possible, and the specific embodiments disclosed and illustrated herein should not be considered in a limiting sense. The following claims particularly point out certain combinations and subcombinations that are deemed novel and non-obvious. Inventions embodied in other combinations and subcombinations of features, functions, elements, and / or properties may be claimed in applications claiming priority from this or related applications. Such claims are considered to be within the subject matter of the invention taught herein, whether directed to different or the same invention, and whether broader, narrower, equal, or different in scope than the original claims.

Claims

1. 1. A method for reducing the concentration of protein aggregates in a cysteine-directed antibody drug conjugate (cys ADC), said method comprising: a. performing a first purification of the antibody on a cation exchange chromatography material using a first set of purification conditions to obtain a purified antibody intermediate; b. conjugating the purified antibody intermediate with a cytotoxic drug to form a crude preparation comprising a cys ADC and protein aggregates; and c. performing a second purification of said crude preparation by cation exchange chromatography material in flow-through mode using said first set of purification conditions to produce a purified cys ADC. Including, The method, wherein the first set of purification conditions comprises loading density, buffer species, pH and conductivity of a buffer system.

2. 10. The method of claim 1, further comprising the additional step of washing the cation exchange chromatography material to recover purified cys ADC.

3. 3. The method of claim 2, wherein the yield of the purified cys ADC is greater than 98% by weight.

4. 3. The method of claim 2, wherein the concentration of protein aggregates in the purified cys ADC is reduced by at least 85% relative to the concentration of protein aggregates in a crude mixture of cys ADC and protein aggregates without altering the critical quality attributes (CQAs) of the cys ADC.

5. 2. The method of claim 1, wherein the protein aggregates are selected from the group consisting of very high molecular weight species (vHMWS) and high molecular weight species (HMWS).

6. The method of claim 5, wherein the protein aggregate is vHMWS.

7. 7. The method of claim 6, wherein the vHMWS is an oligomer.

8. 3. The method of claim 2, wherein the vHMWS in the purified cys ADC is reduced to less than 0.1%.

9. 3. The method of claim 2, wherein the vHMWS in the cys ADC is reduced to less than 0.02%.

10. 1. A method for purifying a cysteine-directed antibody drug conjugate (cys ADC), the method comprising: a. performing a first purification of the antibody on a cation exchange chromatography material using a first set of purification conditions to obtain a purified antibody intermediate; b. conjugating the purified antibody intermediate with a cytotoxic drug to form a crude preparation of a cys ADC; and c. performing a second purification of said crude preparation by cation exchange chromatography material in flow-through mode using said first set of purification conditions to produce a purified cys ADC. Including, The method, wherein the first set of purification conditions comprises loading density, buffer species, pH and conductivity of a buffer system.

11. 11. The method of claim 10, further comprising the additional step of washing the cation exchange chromatography material to recover purified cys ADC.

12. 12. The method of claim 11, wherein the yield of the purified cys ADC is greater than 98% by weight.

13. 11. The method of claim 1 or claim 10, wherein the cys ADC is selected from the group consisting of engineered cysteines targeting native cysteines and site-directed conjugates via interchain cysteine ​​conjugates.

14. 11. The method of claim 1 or claim 10, wherein the cation exchange material is a resin.

15. 15. The method of claim 14, wherein the resin is selected from the group consisting of POROS 50HS, POROS XS, and SPFF resins.

16. The loading density in the second purification of the crude mixture of cys ADC and protein aggregates is 100 g / L. ｒ ~1000g / L ｒ The method of claim 1 or claim 10, wherein

17. the loading density in the second purification of the crude mixture of cys ADC and protein aggregates is 500 g / L; ｒ 17. The method of claim 16, wherein:

18. 11. The method of claim 1 or claim 10, wherein the first purification of the antibody using a first set of purification conditions to obtain a purified antibody intermediate is performed in bind-elute mode.

19. 11. The method of claim 1 or claim 10, wherein the cytotoxic agent is selected from the group consisting of an auristatin, a maytansinoid, and a DNA damaging agent.

20. 20. The method of claim 19, wherein the DNA damaging agent is a derivative selected from the group consisting of calicheamicin, anthracyclines, and pyrrolobenzodiazepines.

21. 21. The method of claim 20, wherein the cytotoxic agent is a pyrrolobenzodiazepine monoamide or MMAE (monomethylauristatin E).

22. 11. The method of claim 1 or claim 10, wherein the cytotoxic drug forms a linker-drug conjugate.

23. 23. The method of claim 22, wherein the linker-drug conjugate is vcMMAE (monomethyl auristatin E, a cytotoxin with a valine-citrulline (vc-) linker).

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