Methods for improving antibody drug conjugate quality

Abstract

The present disclosure relates to a method for improving the quality of glycosylated antibody drug conjugates (DAR distribution, stability, etc.) using activated carbon. Specifically, the present disclosure employs hydrophobic interaction chromatography (HIC) column, protein A chromatography, activated carbon stirring, and buffer exchange, alone or in series, to remove free glycosyltransferases. As a result, the purity of the glycosylated antibody drug conjugates is improved, and the degradation of the antibody drug conjugates is prevented. Ultimately, the drug substance (DS) of OBI-902 (anti-TROP2 antibody drug conjugate) and OBI-904 (anti-Nectin-4 antibody drug conjugate) is further determined.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 621,166, filed January 16, 2024, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] This invention relates to a method for improving the quality of glycosylated antibodies and / or antibody-drug complexes (ADCs) using activated carbon, including improving properties such as DAR distribution and stability. Background Technology

[0004] Protein purification involves separating target proteins from impurities using a combination of different column chromatography techniques. For example, separation can be based on characteristics such as charge, hydrophilicity, and molecular size. Especially when the target protein is an antibody, Protein A affinity chromatography or Protein G affinity chromatography is often used to purify the antibody. Purification is achieved by utilizing the binding properties of Protein A or Protein G to specific regions of the antibody (such as the Fc chain) (see PCT Publication No.: WO2009 / 009523A2).

[0005] Antibody-drug complexes (ADCs), hailed as "magic bullets" in the therapeutic field, consist of antibodies bound to drug molecules. These antibodies can be in small protein formats (such as single-chain antibody fragments scFv, Fab fragments, designed ankyrin repeats, affibodies, nanoantibodies, etc.), but are typically based on monoclonal antibodies (mAbs) due to their high selectivity and affinity for specific antigens, long circulating half-life, and low immunogenicity. Therefore, as protein ligands targeting specific biological receptors, monoclonal antibodies provide an ideal platform for selectively delivering drugs to target cells. For example, a monoclonal antibody known to specifically bind to a specific cancer-associated antigen can be used to deliver bound cytotoxic agents to tumor cells, achieving therapeutic effects through binding, internalization, intracellular processing, and ultimately the release of active metabolites. These cytotoxic drugs may be small molecule toxins, protein toxins, or other forms such as nucleotides. Therefore, targeted delivery of active drugs to specific cellular sites has become a powerful strategy for treating various diseases, exhibiting many significant advantages over systemic drug administration. Summary of the Invention

[0006] As stated above, the objective of this invention is to disclose a manufacturing process for active pharmaceutical ingredients (DS) of an anti-TROP2 antibody drug complex (also known as OBI-902) and an anti-Nectin-4 antibody drug complex (also known as OBI-904), and to further evaluate methods for improving their purity and / or quality.

[0007] Another object of the present invention is to provide purified antibody-drug complexes (ADCs) for evaluating and completing the preparation of pharmaceutical compositions.

[0008] The present invention also provides a method for evaluating the quality of OBI-902, comprising: (a) glycosylation of R4702 antibody; (b) hydrophobic interaction chromatography (HIC) and activated carbon purification; (c) buffer replacement of R4702-(NSCT-2N3)2; (d) biocoupling between glycosylated antibody and linker-loaded drug; and (e) buffer replacement and purification of antibody-drug complex (ADC).

[0009] Preferably, the HIC is performed at a flow rate of 0.1 to 10.0 mL / min.

[0010] Preferably, the tubing used in this HIC is an agar tubing.

[0011] Preferably, the HIC column is filled with agar beads modified with phenyl, ethyl, butyl, and benzyl groups via non-electrolyzed, chemically stable diethyl ether.

[0012] Preferably, the HIC column is a HiScreen Phenyl HP or HiScreen Butyl HP column.

[0013] The present invention also provides a method for evaluating the quality of OBI-904, comprising: (a) glycosylation of 10K06 antibody; (b) Protein A chromatography and activated carbon purification; (c) buffer replacement of 10K06-(NSCT-4N3)2; (d) bio-coupling between glycosylated antibody and linker-loaded drug; (e) second activated carbon purification; and (f) buffer replacement and purification of antibody-drug complex (ADC).

[0014] Preferably, the Protein A chromatography is performed at a flow rate of 0.1 to 10.0 mL / min.

[0015] Preferably, the column used for Protein A is an agar-based column.

[0016] Preferably, the Protein A chromatography column contains agar beads, polyacrylamide, sepharose, or other polymers.

[0017] More preferably, the Protein A chromatography column is a Mabselect PrismA. TM Protein A column.

[0018] In this invention, "impurities" may include chemical substances, glycosides, enzymes, host cell proteins (HCPs), protein-derived polymers, protein-derived degradation products, protein-derived modifications due to denaturation, removal of glycosylated components, oxidation, deamidation, or nucleic acids; preferably, they include chemical substances, glycosides, enzymes, host cell proteins, protein-derived polymers, protein-derived degradation products, or nucleic acids.

[0019] In one embodiment, the present invention provides an example of "enzyme removal" or "residual enzyme analysis" related to glycosyl processing enzymes, wherein the enzyme is a glycosynthase, glycosidase, or glycosyltransferase; preferably, the enzyme is a glycosynthase, glycosidase, or glycosyltransferase.

[0020] In one embodiment, the present invention provides examples of "liquid chromatography", including Protein A chromatography, liquid-solid chromatography (LSC), reverse phase chromatography (RPC), ion exchange chromatography (IEC), molecular sieve chromatography (SEC), high performance liquid chromatography (HPLC), affinity chromatography or hydrophobic interaction chromatography (HIC); preferably, the liquid chromatography is hydrophobic interaction chromatography (HIC).

[0021] In one embodiment, the present invention provides an example of "glycosylation", including N-chain glycosylation or O-chain glycosylation.

[0022] In one embodiment, the present invention provides an "improved" example, which is achieved by using a His Tag ELISA detection kit to analyze residual enzymes, with enzyme residue ratios reaching 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or approximately 500 times. This ratio is calculated as follows:

[0023] Raw crude R4702-(NSCT-2N3)2 / R4702-(NSCT-2N3)2 after activated carbon treatment;

[0024] Or raw 10K06-(NSCT-4N3)2 crude product / 10K06-(NSCT-4N3)2 after activated carbon treatment. Attached Figure Description

[0025] Figure 1Results of HIC column purification of OBI-902 using a GE ÄKTA FPLC™ Fast Protein Liquid Chromatograph (FPLC).

[0026] Figure 2 (a) Marker, (b) R4702, (c) R4702-GlcNAc, (d) R4702-(NSCT-2N3)2 (crude) and (e) R4702-(NSCT-2N3)2 (purified) SDS-PAGE images.

[0027] Figure 3A and 3B Chromatographic images of the intermediate product N-PM-0022 under illumination. Figure 3A shows the dark control group, and Figure 3B shows the illuminated group.

[0028] Figure 4A and 4B HIC-UV tomography images of OBI-902 under 0.5x illumination (Fig. 4A) and 1x illumination (Fig. 4B).

[0029] Figure 5A and 5B SEC-UV tomography of OBI-902 under 0.5x illumination (Fig. 5A) and 1x illumination (Fig. 5B).

[0030] Figure 6 OBI-902's TROP2 antigen-binding activity after light exposure.

[0031] Figure 7 The cytotoxicity of OBI-902 on human pancreatic cancer BxPC3 cells after light exposure.

[0032] Figure 8 Results of Protein A column purification of OBI-904 using a GE ÄKTA FPLC™ Fast Protein Liquid Chromatograph (FPLC). Detailed Implementation

[0033] The manufacturing process of the anti-TROP2 antibody-drug complex (OBI-902) active pharmaceutical ingredient (DS) comprises five steps, including: (a) glycosylation of the R4702 antibody; (b) hydrophobic interaction chromatography (HIC) and activated carbon purification; (c) buffer replacement of the glycosylated antibody; (d) biocoupling between the glycosylated antibody and the linker-loaded drug; and (e) buffer replacement and purification of the ADC.

[0034] The manufacturing process of the anti-Nectin-4 antibody-drug complex (OBI-904) active pharmaceutical ingredient (DS) comprises six steps, including: (a) glycosylation of the 10K06 antibody; (b) Protein A chromatography and activated carbon purification; (c) buffer replacement of the glycosylated antibody; (d) bio-coupling between the glycosylated antibody and the linker-loaded drug; (e) secondary activated carbon purification; and (f) buffer replacement and purification of the ADC.

[0035] The elution can be carried out at an appropriate flow rate depending on the operating conditions. However, in a preferred embodiment, the elution is carried out at a flow rate of 0.1 to 10.0 mL / min.

[0036] In some embodiments, the antibody is an anti-TROP2 antibody selected from hRS7, Hu2G10, hu4D3, MAAP-9001a, Pr1E11, Apt20s2, MHB036A, R4702, datopotamb, or sacituzumab. In some preferred embodiments, the antibody is R4702. R4702 is provided by Bio-Incident Technologies and is described in PCT Patent Publication No. WO2022222992A1, the entire contents of which are incorporated herein by reference.

[0037] In some embodiments, the antibody is an anti-Nectin-4 antibody selected from 5A12.2, 15A17.5, N4MU01, MW282, LY4052031, 08B04, 10K06, 14I08, 05O04, 12E03, 02P14, 11O23, 14B21, 08C24, 13C24, or enfortumab. In some embodiments, the antibody is 10K06 manufactured by OBI Pharma Inc., Taiwan.

[0038] In some embodiments, the glycosyl synthase variants are EndoSd-D232M and EndoSz-D234M. Exemplary EndoSd-D232M and EndoSz-D234M are described in PCT Patent Publication No. WO2020006176A1, the contents of which are incorporated herein by reference in their entirety.

[0039] abbreviation

[0040] ACN: Acetonitrile; ADC: Antibody-drug conjugate; DAR: Drug-to-antibody ratio; DMSO: Dimethyl sulfoxide; GlcNAc: N-acetylglycosamine; HIC: Hydrophobic interaction chromatography; HPLC: High performance liquid chromatography; IEC: Ion exchange chromatography; LSC: Liquid solid chromatography; mAb: Monoclonal antibody; NaOAc: Sodium acetate; NaOH: Sodium hydroxide; NSCT: Sialylated complex type N-glycan; PBS: Phosphate buffered saline; RPC: Reversed phase chromatography. Chromatography); SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis; SEC: size exclusion chromatography.

[0041] Example

[0042] Example 1: Manufacturing process of OBI-902 active pharmaceutical ingredient (DS)

[0043] The manufacturing process of the TROP2 antibody-drug complex (OBI-902) active pharmaceutical ingredient (DS) involves five steps. The detailed manufacturing process is described below:

[0044] Step 1: Antibody Glycation

[0045] (a) Reaction buffer: 50 mM histidine, 250 mM Tris buffer, pH 7.1.

[0046] (b) Add anti-TROP2 monoclonal antibody R4702 (400 mg), reaction buffer (2.62 mL), EndoSz-D234M glycosyl synthase (6.69 mg), Endo H glycosidase (40 μL) and H2O (3.66 mL) to the reactor at 25 ± 5°C.

[0047] (c) After mixing, incubate at 37±3°C for at least 8 hours.

[0048] (d) Cool the reaction to 25±5°C.

[0049] (e) Add NSCT-N3 polysaccharide (56 mg; OBI Pharma Inc., Taiwan), reaction buffer (5.31 mL) and H2O (5.56 mL) at 25±5°C.

[0050] (f) After mixing, incubate at 25±5°C for at least 2 hours.

[0051] (g) Add 10.5 mL of 5 M NaCl solution and adjust the conductivity at 25±5°C.

[0052] Step 2: Hydrophobic Interaction Chromatography (HIC) and Activated Carbon Purification

[0053] (a) Instrument: GE ÄKTA FPLC™ Rapid Protein Chromatography System.

[0054] (b) Buffer preparation:

[0055] Buffer A: PBS, 3 M NaCl, pH 7.2.

[0056] Buffer B: 20 ​​mM sodium phosphate, pH 7.0, containing 20% ​​isopropanol.

[0057] (c) R4702-(NSCT-2N3)2 solution was added to a HiScreen Phenyl HP column (column volume: 4.7 mL; product number: 28-9505-16; GE Healthcare Bio-Sciences AB) at a flow rate of 4 mL / min (120 cm / h). The gradient conditions were: A / B = 85 / 15 × 10 column volumes (CV), increasing the gradient to A / B = 50 / 50 over 3 CV, and then maintaining A / B = 50 / 50 for 5 CV. The flushing fluid with an absorbance >30 mAu at wavelength A280 nm was collected.

[0058] (d) Mix the desired embankment fraction with activated carbon (800 mg; product number: 0320-0460; Showa Chemical Co., Ltd.) and stir at 25±5°C for at least 1 hour.

[0059] (e) The resulting solution was filtered through 5 μm and 0.22 μm filters to remove activated carbon.

[0060] Figure 1 The chromatograms show the results of HIC column purification using a GE ÄKTA FPLC™ rapid protein chromatography system. The damming peaks appear in fractions 14 through 19, each 14 mL, with the highest absorbance at fraction 16 being 661.57 mAu.

[0061] Step 3: Buffer replacement of R4702-(NSCT-2N3)2 monoclonal antibody

[0062] The purified R4702-(NSCT-2N3)2 monoclonal antibody was dialyzed through 7 volumes of storage buffer (20 mM sodium acetate buffer, pH 5.0) and concentrated to 15 ± 2 mg / mL. The resulting solution was then filtered through a 0.2 μm sterile filter, placed in sterilized vials, and stored at 2–8°C.

[0063] Figure 2 shows SDS-PAGE images of various R4702 antibodies. To confirm the effectiveness of HIC and activated carbon purification, the original R4702 monoclonal antibody and R4702 antibodies with different glycosylation (R4702-GlcNAc and R4702-(NSCT-2N3)2) were analyzed. The bands from (b) to (e) gradually weaken at a molecular weight (MW) of 115 kDa, indicating that unbound glycosyl synthases and glycosidases were successfully removed. In addition, the bands from (b) to (e) gradually thicken at a molecular weight (MW) of 50 kDa, indicating that the purity of the crude ADC was improved.

[0064] Step 4: Bioconjugation of R4702-(NSCT-2N3)2 monoclonal antibody with linker-loaded drug

[0065] Preparation of the linker-loaded drug solution: Dissolve 20 ± 0.5 mg / mL of the N-PM-0022 intermediate (OBI Pharma Inc., Taiwan) in DMSO. Add six equivalents of N-PM-0022 / DMSO to an R4702-(NSCT-2N3)2 solution and stir at 25 ± 2°C for at least 4 hours.

[0066] Step 5: Buffer replacement and purification of ADC

[0067] The crude ADC was buffer-displaced using a tangential flow filtration (TFF) system (Satorious AG). 40 volumes of storage buffer (20 mM sodium acetate buffer, pH 5.0) were circulated per unit volume for replacement. The crude ADC was eventually concentrated to 15 ± 1 mg / mL, and samples were taken to determine the concentration and residual free N-PM-0022 intermediate. The ADC concentration was then adjusted to 10 ± 1 mg / mL with storage buffer, filtered through a 0.2 μm sterile filter, and samples were taken to determine the DAR value and purity. The qualified ADC solution was bottled and stored at below -65°C.

[0068] Example 2: ELISA analysis of residual enzymes in OBI-902

[0069] This assay was performed using the His Tag ELISA kit (GenScript, catalog number: L00436). This kit is designed for rapid and high-throughput detection of His-tagged proteins. The reagents and assay procedure are shown below:

[0070] reagents

[0071] (a) His label tray (8 holes × 12 stripes):

[0072] A His-tagged protein with a molecular weight of 12.7 kDa was coated.

[0073] (b) Test sample:

[0074] R4702-(NSCT-2N3)2 crude product.

[0075] R4702-(NSCT-2N3)2 was purified by HIC.

[0076] R4702-(NSCT-2N3)2 after being treated with activated carbon.

[0077] (c) Standards: His-labeled standards (0, 1, 3, 9, 27, 81, 243, 729 ng / mL).

[0078] (d) Anti-His monoclonal antibody (GenScript, catalog number: A00186).

[0079] (e) Premix (primary antibody):

[0080] Add 60 µL of sequentially diluted standard, test sample and anti-His monoclonal antibody for serial dilution.

[0081] Stir at 750 rpm and 25°C for 30 minutes.

[0082] (f) Antibody tracking agent (secondary antibody):

[0083] Horseradish peroxidase (HRP)-coupled goat anti-mouse IgG.

[0084] Testing process

[0085] (a) Add 100 µL of premix to the reagent pan wells and incubate at 25°C for 30 minutes.

[0086] (b) Clean the plate holes four times with 200 µL of washing solution.

[0087] (c) Add 100 µL of antibody tracking agent (secondary antibody) and incubate at 25°C for 30 minutes.

[0088] (d) Clean the plate holes four times again with 200 µL of washing solution.

[0089] (e) Add 100 µL of TMB matrix and incubate at 25°C for 15 minutes.

[0090] (f) Finally, add 50 µL of stop solution and measure the OD. 450nm value.

[0091] Table 1 shows the efficiency of HIC and activated carbon purification in the removal of residual enzymes.

[0092] In the crude R4702-(NSCT-2N3)2, the original content of residual enzymes exceeded 10,000 ppm. After HIC purification, the residual enzyme content could be reduced to approximately 1,000 ppm. However, further activated carbon treatment reduced the residual enzyme content to below 5 ppm. These results demonstrate that activated carbon purification is the most effective method for removing residual enzymes.

[0093] Table 1. Results of residual enzyme determination in each purification step of OBI-902

[0094]

[0095] Example 3: Photostability Test of N-PM-0022 Intermediate and OBI-902

[0096] 3-1. Stability of Connector-Loaded Drugs in Bio-Coupling Processes

[0097] Materials and Instruments

[0098] (a) Bio-coupling buffer: 20 mM sodium acetate, pH 5.0.

[0099] (b) Illuminance meter: T&D model TR-74Ui.

[0100] (c) Light source: Liquid observation lamp, illuminance range 2000 to 3000 lux / hour

[0101] Sample preparation

[0102] The intermediate N-PM-0022 (OBI Pharma, Inc.) was dissolved in DMSO to prepare a stock solution of 20 mg / mL.

[0103] Take 50 µL of N-PM-0022 stock solution, add 1000 µL of bio-coupling buffer (in a 4 mL glass vial), and mix thoroughly.

[0104] The final concentration of N-PM-0022 was approximately 0.95 mg / mL.

[0105] Light exposure design

[0106] One sample was left unprotected and directly exposed to light (light exposure group), while the other sample was wrapped in aluminum foil to protect it from light (dark control group). Samples were exposed to 2147 lux of light per hour for 18 hours, and samples were taken at 0, 2, and 18 hours for quality analysis.

[0107] Analytical methods

[0108] Purity determination was performed using a C18 reversed-phase liquid chromatography-ultraviolet (RPLC-UV) method. Analysis was conducted using a Chromanik SunShell C18 UHPLC column (90 Å, 2.6 µm, 3 mm x 150 mm) with an acetonitrile / methanol gradient. The relative content of the samples was quantified using eczema standards, while purity and impurities were assessed as a percentage of the relevant peak area.

[0109] result

[0110] Table 2 summarizes the purity and impurities of the N-PM-0022 intermediate. The chromatograms at various time points, and the trends in relative content and purity with exposure time, are shown in Figures 3A and 3B.

[0111] Table 2. Purity and Impurities of N-PM-0022 Intermediate

[0112]

[0113] N-PM-0022 maintained good quality under light-protected conditions, exhibiting high relative content and purity. In the dark control group, the purity difference between 0 and 18 hours of exposure was only 3.72% (decreasing from 97.20% to 93.48%). This indicates that N-PM-0022 underwent only slight degradation in bio-coupling buffer (20 mM sodium acetate, pH 5.0) at room temperature. Therefore, N-PM-0022 possesses sufficient stability during ADC coupling under light-protected conditions.

[0114] In contrast, the quality of the samples treated with light changed significantly. After 2 hours of light exposure, the purity of the N-PM-0022 intermediate dropped sharply from 97.35% to 43.16%, and further decreased to 0.35% after 18 hours. These results confirm that the N-PM-0022 intermediate is unstable and decomposes under light conditions.

[0115] In summary, during the bio-coupling process of ADC preparation, the dissolved N-PM-0022 intermediate should be protected from light. The use of light-resistant containers (such as stainless steel bioreactors or amber glass vials) is essential. Furthermore, using low-intensity light sources (such as red light) during manufacturing helps protect the connector-loaded drug and ADC from degradation and decomposition.

[0116] 3-2. Study on photostability of OBI-902

[0117] instrument

[0118] (a) UV / Vis stability test chamber: MMM Medcenter model FC-B2V / FC222.

[0119] (b) Illuminance meter: T&D model TR-74Ui.

[0120] Light exposure design

[0121] Measurements were performed to achieve the required illuminance levels in the illumination test chamber, resulting in a visible illuminance of 15.49 klux and a near-ultraviolet (UV) energy of 13.70 Wh / m². According to the ICH Q1B guideline illumination standards, one level of illumination exposure (1.2 million lux·h and 200 Wh / m²) requires 79 hours of visible light exposure and 15 hours of UV exposure. This study tested two different illuminance levels: 0.5 times and 1 times the ICH standard illumination conditions.

[0122] 0.5 times the visible light: total irradiation 0.612 million lux·hour, irradiation time 39.5 hours.

[0123] 1 times the visible light: total irradiance 1.224 million lux·hour, irradiation time 79 hours.

[0124] 0.5 times ultraviolet light: total irradiation dose 102.75 Wh / m², irradiation time 7.5 hours.

[0125] 1x UV light: Total irradiation dose 205.5 Wh / m², irradiation time 15 hours.

[0126] Sample preparation

[0127] A total of 24 sample vials were prepared for this study, each containing 2 mL of OBI-902 in a 4 mL glass vial. Three different packaging methods were tested: unprotected transparent vials, directly exposed to light; vials wrapped in aluminum foil to avoid light exposure; and vials wrapped in cardboard boxes, which did not completely block light but reduced direct light exposure. Detailed settings are shown in Table 3. Samples 1 and 5 were exposed to 0.5 and 1 times the visible light, respectively, without light protection. Samples 2 and 6 were exposed to 0.5 and 1 times the ultraviolet light, respectively, without light protection. Samples 3 and 7 were wrapped in aluminum foil as a light protection control group and exposed to 0.5 and 1 times the visible and ultraviolet light, respectively. Samples 4 and 8 were not wrapped but placed in cardboard boxes and exposed to 0.5 and 1 times the visible and ultraviolet light, respectively. After the exposure period ended, samples were taken for analysis to assess the chemical degradation of the antibody-drug complex (ADC) (such as impurity formation or aggregation) and the reduction in potency (such as antigen-binding activity or cytotoxicity).

[0128] Table 3. Sample Information and Photostability Test Plan

[0129]

[0130] result

[0131] The results are summarized in Table 4. Unprotected samples 1, 2, 5, and 6 were used to evaluate the photosensitivity and photostability of the antibody-drug complex (ADC). Samples 4 and 8 were placed in a cardboard box to assess whether this packaging could protect OBI-902 from light and maintain its stability. Samples 3 and 7, wrapped in aluminum foil, served as the dark control group.

[0132] Table 4. Summary of photostability study results for OBI-902

[0133]

[0134] HIC-UV analysis (Figures 4A and 4B) revealed changes in the peak shape of the OBI-902 spectrum, particularly after UV irradiation. For unprotected samples (samples 1, 2, 5, and 6), the peaks in the HIC-UV analysis became difficult to discern. Furthermore, aggregation was observed in SEC-UV analysis after irradiation (Figures 5A and 5B). After irradiation with 0.5x and 1x visible light, the monomer content decreased to 94.07% (sample 1) and 89.46% (sample 5), respectively; however, after UV irradiation, the monomer content decreased even more significantly to 46.63% (sample 6). Additionally, multiple high molecular weight aggregates (HMWS) were detected in sample 6.

[0135] In efficacy tests of OBI-902, unprotected OBI-902 showed decreased TROP2 antigen-binding activity and reduced cytotoxicity in human pancreatic cancer BxPC3 cells. After 0.5x and 1x visible light irradiation, EC... 50 The levels rose to 58.63 ng / mL (sample 1) and 71.02 ng / mL (sample 5), respectively; however, after UV irradiation, EC... 50 The concentration significantly increased to 300.1 ng / mL (sample 6) (Figure 6). A similar trend was also observed in the BxPC3 cytotoxicity test (Figure 7). After UV irradiation, the IC50 concentration increased significantly. 50 After 0.5x and 1x UV treatment, the concentrations increased to 1.78 nM (sample 2) and 10.15 nM (sample 6), respectively.

[0136] The results showed that OBI-902 is a photosensitive and photostability-dependent ADC, and its properties are altered by both ultraviolet and visible light irradiation. Light exposure affects the quality of OBI-902, leading to aggregation, changes in charge variation distribution, and, most importantly, a significant decrease in its binding activity and cytotoxicity. Compared to visible light, ultraviolet light irradiation causes more severe degradation and irreversible damage to OBI-902. All analytical indicators showed a decline in quality, with the degradation caused by ultraviolet light irradiation being particularly pronounced.

[0137] Example 4: Manufacturing process of OBI-904 active pharmaceutical ingredient (DS)

[0138] The manufacturing process of OBI-904 active pharmaceutical ingredient (DS) involves six steps, detailed as follows:

[0139] Step 1: Antibody Glycation

[0140] (a) Reaction buffer: 100 mM sodium phosphate buffer (pH 7.0).

[0141] (b) Reaction composition:

[0142] Anti-Nectin-4 monoclonal antibody 10K06 (600 mg).

[0143] EndoSz-D234M Glycosyl Synthase (2.0 mg) (EndoSz-D234M to monoclonal antibody ratio is 1:300)

[0144] Endo H glycosidase (50 U / mg monoclonal antibody).

[0145] (c) After mixing, incubate at 37 °C for 42 hours.

[0146] (d) Cool the reaction to 15 °C.

[0147] (e) Add Oxazoline-NSCT-tetra-N3 (oxa-NSCT-4N3) (8 equivalents; OBI Pharma, Inc.) and reaction buffer.

[0148] (f) After mixing, incubate at 15°C for 8 hours.

[0149] Step 2: Protein A chromatography and activated carbon purification

[0150] (a) Instrument: GE ÄKTA FPLC™ Rapid Protein Liquid Chromatography System.

[0151] (b) Buffer preparation:

[0152] Buffer A1: PBS + 3M NaCl, pH 7.2 (3 column volumes, CV).

[0153] Buffer A2: 100 mM sodium citrate buffer (NaCi), pH 6.0 (5 CV).

[0154] Buffer B1: 100 mM sodium citrate buffer (NaCi), pH 5.5 (5 CV).

[0155] Buffer B2: 50 mM sodium citrate buffer (NaCi) + 150 mM NaCl, pH 3.0 (5 CV).

[0156] (c) Load 10KO6-(NSCT-4N3)2 solution into pre-equilibrated Mabselect PrismA TM Protein A chromatography column (Cytiva, model 17549803). Collect the flushing fraction with absorbance >30 mAu at A280nm. Wash for unbound impurities: 3CV PBS + 3M NaCl buffer (pH 7.2), 5CV 100 mM sodium citrate buffer (pH 6.0), 5CV 100 mM sodium citrate buffer (pH 5.5). Flushing 10K06-(NSCT-4N3)2: 5CV 50 mM sodium citrate buffer + 150 mM NaCl (pH 3.0), immediately neutralize the flushing fraction to natural pH with 1 M Tris-HCl (pH 9.0).

[0157] (d) Combine the target portion with activated carbon (5:1 weight ratio relative to the monoclonal antibody) (SHOWA Chemical Co. Ltd., model 0320-0460) and stir at 25 °C for 2 hours.

[0158] (e) Use 5 µm and 0.22 µm filters to remove activated carbon impurities.

[0159] Figure 8 shows the chromatographic purification curve of Protein A, with the damming peak appearing in tubes 39 to 43, each containing approximately 10.5 mL.

[0160] Step 3: Buffer replacement of 10K06-(NSCT-4N3)2 monoclonal antibody

[0161] The purified 10K06-(NSCT-4N3)2 monoclonal antibody was dialyzed to replace the storage buffer: 20 mM sodium acetate buffer (pH 5.0) and concentrated to 10 mg / mL. The solution was then aseptically filtered through a 0.2 µm filter, bottled, and stored at 2–8 °C.

[0162] Step 4: Biocoupling reaction of 10K06-(NSCT-4N3)2 monoclonal antibody with linker-loaded drug

[0163] Connector-loaded drug solution preparation: N-PM-0022 intermediate (OBI Pharma, Inc.) was dissolved in DMSO. 12 equivalents of N-PM-0022 / DMSO were added to 20 mM NaOAc (pH 5.0) buffer, mixed with 10K06-(NSCT-4N3)2 monoclonal antibody, and stirred at 20°C for 18 hours.

[0164] Step 5: ADC purification and buffer replacement

[0165] Activated carbon (0.5:1 weight ratio relative to the ADC stock solution) was added after the coupling reaction, and the mixture was stirred at 25 °C for 2 hours. The activated carbon was removed by filtration using 5 µm and 0.22 µm filters. The ADC stock solution was sampled to test for residual free N-PM-0022 intermediate. The purified ADC was loaded into a TFF reservoir and concentrated 2-fold (volume reduced to half its original volume) using a tangential flow filtration system (TFF, Sartorius AG). The solution was then dialyzed using 10-fold volume of storage buffer (10 mM histidine, 250 mM sucrose, pH 6.2). The purified ADC was sampled for concentration and testing for residual free N-PM-0022 intermediate. The ADC concentration was adjusted to 10 mg / mL and aseptically filtered using a Millex-GP syringe filter (PES, 0.22 µm).

[0166] Example 5: ELISA analysis of residual enzymes in OBI-904

[0167] This study used a His-tagged ELISA assay kit (GenScript; catalog number L00436) for analysis. This kit is suitable for rapid and high-throughput detection of His-tagged proteins. The reagent and assay procedure are shown below:

[0168] reagents

[0169] (a) His-tag tray (8 holes × 12 rows):

[0170] His-tagged protein (molecular weight 12.7 kDa) was coated.

[0171] (b) Test sample:

[0172] 10K06-(NSCT-4N3)2 monoclonal antibody crude product / 10K06-(NSCT-4N3)2 monoclonal antibody (after Protein A chromatography) / 10K06-(NSCT-4N3)2 monoclonal antibody (after activated carbon purification) / 10K06-(NSCT-4N3)2 monoclonal antibody / OBI-904.

[0173] (c) Standard: EndoSz-D234M reference standard (concentration range 600 to 1.17 ng / mL).

[0174] (d) Anti-His monoclonal antibody (GenScript; catalog number A00186).

[0175] (e) Antibody tracer (secondary antibody): horseradish peroxidase (HRP)-labeled goat anti-mouse IgG.

[0176] (f) Buffer solution: 20 mM sodium acetate (pH 5.0).

[0177] Testing process

[0178] (a) Replace the buffer solution of the test sample with sodium acetate buffer solution and dilute the sample by an appropriate factor.

[0179] (b) Add 55 µL of anti-His monoclonal antibody to each well, add 55 µL of sample, and incubate at 25°C in the dark, shaking at 750 rpm for 30 minutes.

[0180] (c) Transfer 100 µL of the reaction mixture to the His tag well and incubate at 25 °C for 30 minutes.

[0181] (d) Wash 4 times with 200 µL of washing solution.

[0182] (e) Add 100 µL of HRP-labeled secondary antibody to each well and incubate at 25 °C for 30 minutes.

[0183] (f) Wash 4 times with 200 µL of washing solution.

[0184] (g) Add 100 µL of TMB matrix to each well and incubate at 25 °C for 15 minutes.

[0185] (h) Add 50 µL of stop solution to each well and read the OD. 450nm Absorbance value.

[0186] Table 5 shows that Protein A and activated carbon purification are effective methods for removing residual enzymes. The crude sample in 10K06-(NSCT-4N3)2 contained >2000 ppm of residual enzymes. After Protein A purification, the residual enzyme content was reduced to <300 ppm. However, after activated carbon purification, the residual enzyme content could be further reduced to <5 ppm. This proves that activated carbon purification is the most effective process.

[0187] Table 5. Results of residual enzyme determination in each purification step of OBI-904

[0188]

[0189] Unless otherwise defined, all technical and scientific terms used herein, as well as any prefixes and abbreviations, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although any compositions, methods, kits, and information transmission devices similar to or equivalent to those described herein may be used to practice this invention, only preferred compositions, methods, kits, and information transmission devices are described herein.

[0190] All references cited in this paper are incorporated herein by way of citation to the fullest extent permitted by law. The discussion of these references is solely for the purpose of summarizing the arguments of their authors. This paper does not acknowledge any reference (or any part thereof) as relevant prior art. The applicant reserves the right to challenge the accuracy and relevance of any cited references.

Claims

1. A method for evaluating the quality of an anti-TROP2 antibody-drug complex (ADC), comprising: (a) Glycosylation of antibodies; (b) Hydrophobic interaction chromatography (HIC) and activated carbon purification; (c) Buffer replacement of glycosylated antibodies; (d) Biocoupler between glycosylated antibodies and linker-loaded drugs; and (e) Buffer replacement and purification of anti-TROP2 ADC.

2. The method of claim 1, wherein the HIC column is filled with agar beads modified with phenyl, ethyl, butyl and benzyl groups bonded to non-electrolyzed, chemically stable diethyl ether.

3. The method of claim 1, wherein the HIC is performed at a flow rate of 0.1 to 10.0 mL / min.

4. The method of claim 1, wherein the antibody against TROP2 ADC is an anti-TROP2 antibody selected from hRS7, Hu2G10, hu4D3, MAAP-9001a, Pr1E11, Apt20s2, MHB036A, R4702, datopotamab, or sacituzumab.

5. The antibody-drug complex of claim 1, wherein the linker-loaded drug or anti-TROP2 ADC shall be stored in a light-proof container.

6. A method for evaluating the quality of an anti-Nectin-4 antibody-drug complex (ADC), comprising: (a) Glycosylation of antibodies; (b) Protein A chromatography and activated carbon purification; (c) Buffer replacement of glycosylated antibodies; (d) Biocoupler between glycosylated antibodies and linker-loaded drugs; (e) Second activated carbon purification; and (f) Buffer replacement and purification of anti-Nectin-4 ADC.

7. The method of claim 6, wherein the Protein A chromatography column is filled with agar beads, polyacrylamide, sepharose or other polymers.

8. The method of claim 6, wherein the Protein A chromatography is performed at a flow rate of 0.1 to 10.0 mL / min.

9. The method of claim 6, wherein the anti-Nectin-4 ADC antibody is an anti-Nectin-4 antibody selected from 5A12.2, 15A17.5, N4MU01, MW282, LY4052031, 08B04, 10K06, 14I08, 05O04, 12E03, 02P14, 11O23, 14B21, 08C24, 13C24 or enfortumab.

10. An ADC manufactured by the following process, which improves DAR distribution and stability: (a) Hydrophobic interaction chromatography (HIC) or Protein A chromatography; and / or (b) Activated carbon purification.

11. The ADC of claim 10, wherein the improvement is measured by ELISA (enzyme immunosorbent assay) or SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis).

12. The ADC of claim 10, wherein the improvement is measured by detecting residual enzymes.

13. The ADC of claim 12, wherein the enzyme is a glycosynthase, glycosidase, or glycosyltransferase.

14. The ADC of claim 13, wherein the improvement is a 2 to 500-fold reduction in enzyme residue ratio.

15. The ADC of claim 10, wherein the ADC is OBI-902 or OBI-904.

16. A method for purifying glycosylated antibodies from a sample containing antibodies and impurities, comprising: (a) Antibody glycosylation; (b) Liquid chromatography; and (c) Activated carbon purification, in, The impurities include chemical substances, polysaccharides, enzymes, host cell proteins, protein-derived polymers, protein-derived degradation products, or nucleic acids.

17. The method of claim 16, wherein the liquid chromatography is selected from Protein A chromatography, liquid-solid chromatography (LSC), reverse phase chromatography (RPC), ion exchange chromatography (IEC), molecular sieve chromatography (SEC), high performance liquid chromatography (HPLC), affinity chromatography, or hydrophobic interaction chromatography (HIC).

18. The method of claim 16, wherein the glycosylated antibody is an antibody or antigen-binding portion thereof that can bind to TROP2, Nectin-4, or one or more tumor-associated antigens or cell surface receptors.

19. The method of claim 16, wherein the enzyme is a glycosynthase, glycosidase, or glycosyltransferase.

20. The method of claim 16, wherein the glycosylation is N-linked glycosylation or O-linked glycosylation.

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