Method for antibody-drug conjugate quality improvement

Abstract

The present disclosure relates to a method for enhancing the quality (DAR distribution, stability and etc.) of glycosylated antibody-drug conjugates (ADCs) using active charcoal. In particular, the present disclosure adopts hydrophobic interaction chromatography (HIC) column, Protein A chromatography, active charcoal stirs and buffer exchange either alone or in tandem in order to exclude free glycosynthase enzymes. Therefore, the purity of glycosylated ADCs could be raised and ADC degradation could be prevented. Finally, the quality of OBI-902 (anti-TROP2 ADC) and OBI-904 (anti-Nectin-4 ADC) drug substance (DS) can be further determined.

Description

METHOD FOR ANTIBODY-DRUG CONJUGATE QUALITY IMPROVEMENTCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority of U.S. Provisional Patent Applications No. 63 / 621,166 (filed on January716, 2024). The entirety7of the aforementioned application is incorporated herein by reference.FIELD

[0002] The present invention relates to a method for enhancing the quality (DAR distribution, stability7and etc.) of glycosylated antibody and / or antibody-drug conjugates (ADCs) using activated charcoal.BACKGROUND

[0003] Protein purification is carried out by using combinations of different modes of column chromatography to separate the protein of interest from impurities. For example, based on charge, hydrophilicity7, molecular size or the hke. In particular, when the protein of interest is an antibody, Protein A affinity chromatography or Protein G affinity' chromatography' is used as one of chromatography for purifying the antibody, by using binding property7of Protein A or Protein G to the specific region of antibody such as Fc chain (please refer to PCT publication: W02009 / 009523A2).

[0004] Antibody-Drug Conjugates (ADCs), considered as magic bullets in therapy, are comprised of an antibody to which a pharmaceutical agent is attached. The antibodies can be small protein formats (scFv’s, Fab fragments, designed ankyrin repeat proteins, Affibodies, Nanobody, etc.) but are generally monoclonal antibodies (mAbs) which have been selected based on their selectivity' and affinity for a given antigen, their long circulating half-lives, and little to no immunogenicity. Thus, mAbs as protein ligands for a carefully selected biological receptor provide an ideal platform for delivery7of selective pharmaceutical drugs to the target cells. For example, a monoclonal antibody known to bind selectively with a specific cancer- associated antigen can be used for delivery of conjugated cytotoxic agent to the tumor, via binding, internalization, intracellular processing and finally release of active catabolite. The cytotoxic agent may be a small molecule toxin, a protein toxin or in other formats, like oligonucleotides. Hence, the concept of targeted delivery of an active pharmaceutical drug toa specific cellular location of choice is a powerful approach for the treatment of a wide range of diseases, with many beneficial aspects versus systemic delivery of the same drug.SUMMARY

[0005] In light of the foregoing, the objective of the present invention is to disclose an anti- TROP2 antibody-drug conjugate (also termed as OBI-902) and anti-Nectin-4 antibody-drug conjugate (also termed as OBI-904) drug substance (DS) manufacturing process and the methods to improve the purity and / or quality thereof can be further evaluated.

[0006] Another objectives of the present invention is to provide purified ADCs so that the pharmaceutical composition can be evaluated and fulfilled.

[0007] The present invention more provides a method for evaluating the quality’ of OBI-902, including: (a) R4702 antibody glycosylation; (b) hydrophobic interaction chromatography (HIC) and activated charcoal purification; (c) buffer exchange for R4702-(NSCT-2Ns)2; (d) bioconjugation between the glycosylated antibody and a linker-payload; and (e) buffer exchange and purification of the ADC.

[0008] Preferably, said HIC is conducted at a flow rate of 0. 1 to 10.0 mL / min.

[0009] Preferably, a column used in the HIC is an agarose column.

[0010] Preferably, said HIC column is filled in agarose beads modified with phenyl, ethyl, butyl, and benzyl groups via uncharged, chemically stable ether linkages.

[0011] More preferably, said HIC column is a HiScreen Phenyl HP or HiScreen Butyl HP column.

[0012] The present invention also provides a method for evaluating the quality' of OBI-904, including: (a) 10K06 antibody glycosylation; (b) Protein A chromatography and activated charcoal purification; (c) buffer exchange for 10K06-(NSCT-4N3)2; (d) bioconjugation between the glycosylated antibody and a linker-payload; (e) second activated charcoal purification; and (f) buffer exchange and purification of the ADC.

[0013] Preferably, said Protein A chromatography is conducted at a flow rate of 0. 1 to 10.0 mL / min.

[0014] Preferably, said Protein A chromatography column is an agarose column.

[0015] Preferably, said Protein A chromatography column includes agarose beads, polyacrylamide, sepharose, or other polymers.

[0016] More preferably, said Protein A chromatography column is a Mabselect PrismA™ Protein A column.

[0017] In the present invention, the '‘impurity7” may include chemical, glycan, enzyme, hostcell protein (HCP), protein-derived polymer, protein-derived degradation, protein-derived modification resulting from denaturation, removal of sugar chain component, oxidation, deamidation or nucleic acid, and preferably chemical, glycan, enzyme, host cell protein, protein-derived polymer, protein-derived degradation, or nucleic acid.

[0018] In one embodiment, the present invention provides an example of “enzy me remove” or “residual enzyme analysis” is relating to glycan processing enzyme, wherein the enzyme is glycosynthase, glycosidase, or glycosyltransferase. Preferably, said enzyme is glycosynthase, glycosidase, or glycosyltransferase.

[0019] In one embodiment, the present invention provides an example of “liquid chromatography” which includes Protein A chromatography, liquid solid chromatography (LSC), reversed phase chromatography (RPC), ion exchange chromatography (IEC), size exclusion chromatography (SEC), high performance liquid chromatography (HPLC), affinity chromatography, or hydrophobic interaction chromatography (HIC). Preferably, said liquid chromatography is hydrophobic interaction chromatography (HIC).

[0020] In one embodiment, the present invention provides an example of “Glycosylation” which includes N-linked glycosylation or O-linked glycosylation.

[0021] In one embodiment, the present invention provides for an example of “improvement”, which is accomplished by residual enzyme analysis using His Tag ELISA detection Kit having 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold. 80-fold, 90-fold. 100-fold. 200-fold. 300-fold. 400- fold, or about 500-fold of enzyme residual ratio. It is calculated by: original R4702-(NSCT-2N3)2 crude / R4702-(NSCT-2Ns)2 after activated charcoal treatment; or original 10K06-(NSCT-4N3)2 crude / 10K06-(NSCT-4N3)2 after activated charcoal treatment.BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG. 1. HIC column purification result using GE AKTA FPLC™ Fast Protein Liquid Chromatograph of OBI-902.

[0023] FIG. 2. SDS PEGE image of (a) Marker, (b) R4702, (c) R4702-GlcNAc. (d) R4702- (NSCT-2N.)2(crude) and (e) R4702-(NSCT-2N3)2(purified).

[0024] FIGs. 3A and 3B. Chromatograms of N-PM-0022 intermediated under light exposure. FIG. 3A is Dark control and FIG. 3B is Lighted-exposed.

[0025] FIGs. 4 A and 4B. HIC-UV chromatograms of OBI-902 under 0.5-fold illumination (FIG. 4A) and 1 -fold illumination exposure (FIG. 4B).

[0026] FIGs. 5 A and 5B. SEC-UV chromatograms of OBI-902 under 0.5-fold illumination (FIG. 5A) and 1-fold illumination exposure (FIG. 5B).

[0027] FIG. 6. TROP2 antigen binding activity for OBI-902 after light exposure

[0028] FIG. 7. Human pancreatic cancer BxPC3 cell cytotoxicity results for OBI-902 after light exposure.

[0029] FIG. 8. Protein A column purification result using GE AKTA FPLC™ Fast Protein Liquid Chromatograph of OBI-904.DETAILED DESCRIPTION

[0030] The manufacturing process of an anti-TROP2 antibody-drug conjugate (OBI-902) drug substance (DS) manufacturing process involves five steps. The method includes: (a) R4702 antibody glycosylation; (b) hydrophobic interaction chromatography (HIC) and activated charcoal purification; (c) buffer exchange for glycosylated antibody; (d) bioconjugation between the glycosylated antibody and a linker-payload; and (e) buffer exchange and purification of the ADC.

[0031] The manufacturing process of an anti-Nectin-4 antibody-drug conjugate (OBI-904) drug substance (DS) manufacturing process involves six steps. The method includes: (a) 10K06 antibody glycosylation; (b) Protein A chromatography and activated charcoal purification; (c) buffer exchange for glycosylated antibody; (d) bioconjugation between the glycosylated antibody and a linker-payload; (e) second activated charcoal purification; and (f) buffer exchange and purification of the ADC.

[0032] Said eluting can be conducted by a suitable flow rate depending on the conditions of the operation. Nevertheless, in a preferable embodiment, said eluting is conducted at a flow rate of 0.1 to 10.0 mL / min.

[0033] In certain embodiments, the antibody is an anti-TROP2 antibody selected from hRS7, Hu2G10, hu4D3, MAAP-9001a, PrlEl l, Apt20s2, MHB036A, R4702, datopotamb, or sacituzumab. In certain preferred embodiments, the antibody is R4702. R4702 is obtained from Biosion Inc. and described in PCT patent publication No. WO2022222992A1, the content of which is incorporated herein by reference in its entirety.

[0034] In certain embodiments, the antibody is an anti-Nectin-4 antibody selected from 5A12.2, 15A17.5, N4MU01, MW282, LY4052031, 08B04, 10K06, 14108, 05004, 12E03, 02P14, 11023, 14B21, 08C24, 13C24, or enfortumab. In certain embodiment, the antibody is 10K06 produced by OBI Pharma, Inc.

[0035] In certain embodiment, the glycosynthase variants are EndoSd-D232M and EndoSz- D234M. Exemplary EndoSd-D232M and EndoSz-D234M are as described in PCT patent publication (W02020006176A1), patent applications, the contents of which are incorporated by reference in its entirety.Abbreviations

[0036] ACN: acetonitrile; ADC: Antibody-drug conjugate; DAR: drug-to-antibody ratio; DMSO: dimethyl sulfoxide; GlcNAc: N-acetylglycosamine; HIC: hydrophilic 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; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis; SEC: size exclusion chromatography.EXAMPLESExample 1: OBI-902 drug substance (DS) manufacturing process

[0037] Anti-TROP2 antibody-drug conjugate (OBI-902) drug substance (DS) is manufactured in five steps. The detailed manufacturing process is described in the section below:

[0038] Step 1 : Antibody glycosylation(a) Reaction buffer: 50 mM Histidine, 250 mM Tris buffer, pH 7. 1.(b) Anti-TROP2 mAb R4702 (400 mg), reaction buffer (2.62 mL), EndoSz-D234M glycosynthase (6.69 mg), Endo H glycosidase (40 pE), and H2O (3.66 mL) were added to the reactor at 25±5 °C.(c) After mixing, the mixture was incubated at 37±3 °C at least eight hours.(d) Cooled down the reaction to 25±5 °C.(e) NSCT-N3 Glycan (56 mg; OBI Pharma, Inc.), reaction buffer (5.31 mL), and H2O (5.56 mL) were added to the reactor at 25±5 °C.(f) After mixing, incubated the reaction at 25±5 °C at least two hours.(g) Added 10.5 mL, 5 M NaCl solution to adjust the conductivity at 25±5 °C.

[0039] Step 2: Hydrophobic interaction chromatography (HIC) and activated charcoal purification(a) Instrument: GE AKTA FPLC™ Fast Protein Liquid Chromatograph(b) Prepare buffer:Buffer A: PBS. 3 M NaCl, pH 7.2.Buffer B: 20 mM Sodium Phosphate, pH 7.0 with 20% isopropanol.(c) The resulting solution of R4702-(NSCT-2Ns)2 was loaded to HiScreen Phenyl HP column (Quantity: 4.7 mL; Cat. No.: 28-9505-16; GE Healthcare Bio-Sciences AB), flow rate 4 mL / min (120 cm / hour). Gradient: A / B=85 / 15 10CV (column volume), lift the gradient to A / B=50 / 50 within 3CV, then A / B=50 / 50 5CV. Collect the eluted fractions with UV absorbance >30mAu at wavelength A280nm).(d) Combined the desired fractions with activated charcoal (800 mg; Cat. No.: 0320-0460; SHOWA Chemical Co. Ltd.), and then stirred at 25±5 °C at least one hour.(e) The resulting solution was filtered through 5 pm and 0.22 pm filters to remove activated charcoal.

[0040] FIG. 1 indicated the HIC column purification profile by using GE KTA FPLC™ fast protein liquid chromatograph. The elution peak was observed from fraction 14 to 19, 14 mL / per fraction and the maximum absorbance was 661.57 mAu at fraction 16.

[0041] Step 3: Buffer exchange for R4702-(NSCT-2N?)2 mAb

[0042] The purified R4702-(NSCT-2Ns)2 mAb underwent diafiltration with 7-fold volumes of storage buffer (20 mM Sodium Acetate buffer, pH 5.0), and then was concentrated to 15±2 mg / mL. The resulting solution was filtered with 0.2 pm sterile filter, and then filled into a sterilized bottle and stored at 2~8 °C.

[0043] FIG. 2 indicated the SDS PAGE image of several R4702 antibodies. In order to confirm the efficacy of HIC and activated charcoal purification, original R4702 mAb and different glycosylated R4702 antibodies (R4702-GlcNAc and R4702-(NSCT-2N3)2) were loaded. The bends from (b) to (e) was fading at the site of molecular weight (MW) 115 kDa and it indicated the non-bind glycosynthase and glycosidase enzymes was removed successfully. Furthermore, the bends from (b) to (e) was thickening at the site of molecular weight (MW) 50 kDa and it indicated the purity w as raised of crude ADC.

[0044] Step 4: Bioconjugation between R4702-(NSCT-2N3)2 mAb and linker-payload

[0045] Linker-payload solution preparation: 20±0.5 mg / mL of N-PM-0022 intermediate (OBI Pharma, Inc.) was dissolved in DMSO. Added six equivalent of N-PM-0022 / DMSO into the solution of R4702-(NSCT-2Ns)2 and stirred at 25±2 °C at least four hours.

[0046] Step 5: Buffer exchange and purification of ADC

[0047] The tangential flow filtration (TFF) system (Satorious AG) was employed to exchange buffer of ADC crude. Total 40-fold volume of storage buffer (20 mM SodiumAcetate buffer, pH 5.0) was exchanged circulating for every single volume. Finally the ADC crude was concentrated to 15±1 mg / mL. Sampled the purified ADC for concentration and residual free N-PM-0022 intermediate testing. Adjusted the concentration ADC to 10±l mg / mL by storage buffer. The solution of ADC was filtered with a 0.2 pm sterile filter and sampled for DAR value, and urity testing. The qualified ADC solution was filled into a sterilized bottle and stored at below -65 °C.Example 2: Residual enzyme analysis of OBI-902 by ELISA

[0048] This assay was conducted by using His Tag ELISA detection Kit (GenScript; Cat. No.: L00436). The kit is used for rapid and high throughput detection of His-tagged proteins. The reagent and assay process were listed as follows:

[0049] Reagent(a) His-tag plate (8 wellsxl2 strips):Coated with His-tagged protein with the molecular weight of 12.7 kDa.(b) Test sample:R4702-(NSCT-2N3)2crude / R4702-(NSCT-2N3)2after HIC / R4702-(NSCT-2N3)2after activated charcoal(c) Standard: His Tag Standards (0, 1, 3, 9, 'll, 81, 243, 729 ng / mL)(d) Anti-His monoclonal antibody (GenScript; Cat. No.: A00186)(e) Pre-mixture (primary antibody): Added 60 pL serial diluted standard, test sample and Anti-His monoclonal antibody together for serial dilution. Stirred at 750 rpm, 25 °C for 30 minutes.(f) Antibody tracer (secondary antibody): horseradish peroxidase (HRP) conjugated Goat anti mouse IgG

[0050] Assay process(a) Added 100 pL of Pre-mixture to the wells of kit plated. Incubated at 25 °C for 30 minutes.(b) Washed the plate with 200 pL of Wash Solution four times.(c) Added 100 pL of Antibody tracer and incubated at 25 °C for 30 minutes.(d) Washed the plate again with 200 pL of Wash Solution four times.(e) Added 100 pL of TMB Substrate and incubated at 25 °C for 15 minutes.(f) Finally added 50 pL of Stop solution and read the ODrsonm value.

[0051] Table 1 indicated the efficiency of residual enzyme remove between HIC and activated charcoal purification. The original amount of residual enzyme was >10000 ppm inR4702-(NSCT-2N3)2 crude. After HIC process, the amount of residual enzyme could be decreased to about 1000 ppm. However, the amount of residual enzyme could be decreased to less than 5 ppm by further treating with activated charcoal. It could demonstrate that “activated charcoal” is the most effective process.Table 1. Results of residual enzyme in each process steps of OBI-902.Example 3: Photostability test of linker-payload (N-PM-0022 intermediate) and OB 1-902

[0052] 3-1. Stability7of linker-payload during the bioconjugation process

[0053] Material and Instrument(a) Bioconjugation buffer: 20 mM sodium acetate, pH 5.0(b) Illumination photometer: T&D type TR-74Ui(c) Light source: liquid viewer with illumination 2000 to 3000 lux per hour

[0054] Sample preparationN-PM-0022 intermediate (OBI Pharma, Inc.) was dissolved in DMSO to form 20 mg / mL stock solution. A 50 pL aliquot of N-PM-0022 stock solution was then pipetted into 1000 pL of bioconjugation buffer in a 4 mL glass vial and mix well. This resulted in a final concentration of approximately 0.95 mg / mL of N-PM-0022.

[0055] Light exposure designOne solution was left unprotected and directly exposed to light (Light-exposed), while the other was wrapped in aluminum foil to protect it from light (Dark control). The samples were exposed to 2147 lux per hour for eighteen hours at room temperature and sampled at 0, 2 and 18 hours for quality analysis.

[0056] Analytical methodThe purity assay was determined using the Cl 8 RPLC-UV method. A Chromanik SunShell C18 UHPLC analytical column (90 A. 2.6 pm, 3 mm x 150 mm) with an ACN / methanol gradient was used for the analysis. The relative assay of the samples was quantified using an exatecan standard, while purity and impurities were assessed based on the percentage of related peak areas.

[0057] ResultTable 2 summarized the purity’ and impurities of N-PM-0022 intermediate. The chromatograms for each time point, along with the trends in relative content and purity over the exposure time, were presented in FIGs. 3A and 3B.Table 2. The purity and impurity of N-PM-0022 intermediate.

[0058] N-PM-0022 maintained its quality-, exhibiting high relative content and purity when protected from light exposure. The difference in purity' between 0 and eighteen hours of exposure for the dark controls was only 3.72% (from 97.20% to 93.48%). This indicates that N-PM-0022 experienced only a slight decrease in the bioconjugation buffer (20 mM sodium acetate, pH 5.0) at room temperature over eighteen hours. Therefore. N-PM-0022 appeared to be stable enough throughout the bioconjugation process for ADC formation when protected from light exposure.

[0059] In contrast, the quality attributes of the light-exposed sample were significantly different. The purity of the light-exposed N-PM-0022 intermediate dramatically dropped from 97.35% to 43.16% after two hours of exposure, and further to 0.35% after eighteen hours of exposure. These results confirmed that N-PM-0022 intermediate was unstable and decomposed under light exposure.

[0060] In conclusion, the dissolved N-PM-0022 intermediate should be protected from light exposure during the bioconjugation process of ADC preparation. Using light-resistant containers, such as stainless steel bioreactors or amber glass bottles, was necessary. Additionally, employing low-intensity light, such as red light, during the manufacturing process could help protect the linker pay load and ADC from degradation and decomposition.

[0061] 3-2. Photostability study of OBI-902

[0062] Instrument(a) UV / visible light stability' test chamber: MMM Medcenter type FC-B2V / FC222(b) Illumination photometer: T&D ty pe TR-74Ui

[0063] Light exposure designTo achieve the required illumination levels in the light test chamber, measurements were taken, revealing an illumination of 15.49 klux and a near-UV energy of 13.70 Wh / m2To meet the illumination criteria specified by ICH Q1B guideline, 79 hours of visible light exposure and 15 hours of UV exposure were needed for a 1-fold light exposure (1.2 million lux hours and 200 Wh / m2). In this study, two different illumination levels were tested: 0.5-fold and 1-fold of the ICH requirements.0.5-fold Visible light: illumination exposure 0.612 million lux hours exposing 39.5 hours 1-fold Visible light: illumination exposure 1.224 million lux hours exposing 79 hours 0.5-fold UV light: illumination exposure 102.75 Wh / m2exposing 7.5 hours 1-fold UV light: illumination exposure 205.5 Wh / m2exposing 15 hours

[0064] Sample preparationA total of twenty-four vials were prepared for this study, with 2 mL of OBI-902 aliquoted into each 4 mL glass bottle. Three different types of packaging were tested in this study: one was an unprotected transparent bottle directly exposed to light, another was wrapped in aluminum foil, and the third was enclosed in a paper box. The details were provided in Table 3. Sample 1 and Sample 5 were exposed to 0.5-fold and 1-fold visible light without any light protection. Sample 2 and Sample 6 were exposed to 0.5-fold and 1-fold UV light without any light protection. Sample 3 and Sample 7 were wrapped in aluminum foil to prevent light exposure and were exposed to 0.5-fold and 1-fold visible and UV light as the control samples. Sample 4 and Sample 8 were not covered but placed in a paper box and exposed to 0.5-fold and 1-fold visible and UV light. At the end of exposure period, ADCs were sampled and examined for chemical degradation (e.g., formation of impurities or aggregations) and potency decrease (e.g. antigen binding activity or cell cytotoxicity).Table 3. Sample information and photostability plan.

[0065] Result

[0066] The results were summarized in Table 4. Unprotected Samples 1, 2, 5, and 6 were used to evaluate the photosensitivity and photostability of ADC. Sample 4 and Sample 8 were placed in a paper box to assess whether the packaging could protect OBI-902 from light exposure and maintain stability. Samples 3 and 7, wrapped in aluminum foil, served as dark controls.Table 4. Summary results of OBI-902 for photostability study.

[0067] It was observed in the profde change of OBI-902 from HIC-UV analysis (FIGs. 4A and 4B), especially after UV exposure. The peaks were hard to identify of the unprotected samples (Samples 1, 2, 5 and 6) in the HIC-UV analysis. Furthermore, aggregations were also observed by SEC-UV following light exposure (FIGs. 5A and 5B). The percentage of monomer decreased to 94.07% (Sample 1) and 89.46% (Sample 5) after 0.5-fold and 1-fold visible light exposure, respectively, with a more significant decrease (decreased to 46.63%; Sample 6) found after UV light exposure. Additionally, several high molecular weight species (HMWS) were identified in Sample 6.

[0068] Regarding potency assay of OBI-902, both the TROP2 antigen binding activity and human pancreatic cancer BxPC3 cell cytotoxicity' of the unprotected OBI-902 were reduced. The EC so increased to 58.63 ng / mL (Sample 1) and 71.02 ng / mL (Sample 5) after 0.5-fold and 1-fold visible light exposure, respectively, with a more significant increase (increased to 300. 1ng / mL; Sample 6) found after UV light exposure. (FIG. 6). A similar trend was observed in BxPC3 cell cytotoxicity (FIG. 7). After UV light exposure, the ICso increased to 1.78 nM (Sample 2) and 10. 15 nM (Sample 6) after 0.5-fold and 1-fold UV light treatment, respectively.

[0069] OBI-902 was a photosensitive and photolabile ADC. Both UV and visible light exposure induced changes in OBI-902. Light exposure affected the quality of OBI-902, leading to aggregation formation, alterations in charge variant distribution, and. most importantly, a loss of binding activity and cytotoxic potency. UV light exposure caused more severe degradation and irreversible damage to OBI-902 compared to visible light exposure. Each analytical attribute showed a decline in quality, with UV exposure resulting in obvious deterioration.Example 4: OBI-904 drug substance (DS) manufacturing process

[0070] Anti-Nectin-4 Antibody-Drug Conjugate (OBI-904) drug substance (DS) is manufactured in six steps. The detailed manufacturing process is described in the section below:

[0071] Step 1 : Antibody glycosylation(a) Reaction buffer: 100 mM Sodium phosphate buffer, pH 7.0.(b) Anti-Nectin-4 mAb 10K06 (600 mg), EndoSz-D234M glycosynthase (2.0 mg) [EndoSz- D234M: mAh is 1:300] and Endo H glycosidase (50U / mg mAb).(c) After mixing, the mixture was incubated at 37°C for 42 hours.(d) Cooled down the reaction to 15 °C.(e) Oxazoline-NSCT-tetra-Ns (oxa-NSCT-4N3) (8 equivalents; OBI Pharma, Inc.) and reaction buffer.(f) After mixing, incubated the reaction at 15°C at for 8 hours.

[0072] Step 2: Protein A chromatography and activated charcoal purification(a) Instrument: GE AKTA FPLC™ fast protein liquid chromatography(b) Prepare buffer:Buffer Al: 3CV of PBS+3M NaCl, pH 7.2Buffer A2: 100 mM sodium citrate buffer (NaCi), pH 6.0Buffer Bl: 100 mM sodium citrate buffer (NaCi). pH 5.5Buffer B2: 50 mM sodium citrate buffer (NaCi)+ 150 mM NaCi, pH 3.0.(c) The resulting solution of 10K06-(NSCT-4Ns)2 was loaded to pre-equilibrated Mabselect PrisrnA™ Protein A column (Cat.No.: 17549803; Cytiva). Collect the eluted fractions with UV absorbance >30mAu at wavelength A280nm). The non-bound contaminations were washed by3CV of lxPBS+3 M NaCl buffer, pH 7.2, 5CV of 100 mM sodium citrate buffer, pH 6.0, and 5CV of 100 mM sodium citrate buffer, pH 5.5. 10K06-(NSCT-4N3)2 was eluted with 5CV of 50 M sodium citrate buffer+150 mM NaCl, pH 3.0. and the eluted fractions were immediately neutralized with 1 M Tris-HCl pH 9.0 to natural pH.(d) Combined the desired fractions with activated charcoal at 5:1 weight ratio relative to mAb (Cat. No.: 0320-0460; SHOWA Chemical Co. Ltd.), and then stirred at 25°C for two hours.(e) The resulting solution was filtered through 5 pm and 0.22 pm filters to remove activated charcoal.

[0073] FIG. 8 indicated the Protein A column purification profile by using GE AKTA FPLC™ fast protein liquid chromatography. The elution peak was observed from fraction 39 to 43, 10.5 mL / per fraction.Step 3: Buffer exchange for 10K06-(NSCT-4Ns)2mAb

[0074] The purified 10K06-(NSCT-4N3)2 mAb underwent diafiltration of storage buffer (20 mM Sodium Acetate buffer, pH 5.0), and then was concentrated to 10 mg / mL. The resulting solution was filtered with 0.2 pm sterile filter, and then filled into a sterilized botle and stored at 2-8 °C.

[0075] Step 4: Bioconjugation between 10K06-(NSCT-4N3)2 mAb and linker-payload

[0076] Linker-payload solution preparation: N-PM-0022 intermediate (OBI Pharma, Inc.) was dissolved in DMSO. Added twelve equivalent of N-PM-0022 / DMSO into 20 mM NaOAc, pH 5.0 buffer of 10K06-(NSCT-4N3)2 mAb and stirred at 20 °C for 18 hours.

[0077] Step 5: Purification and buffer exchange of ADC

[0078] After conjugation, activated charcoal were added to the ADC crude at 0.5: 1 weight ratio relative to ADC crude, and then stirred at 25 °C for two hours. The resulting solution was filtered through 5 pm and 0.22 pm filters to remove activated charcoal. Sampled the purified ADC for residual free N-PM-0022 intermediate testing. The tangential flow filtration (TFF) system (Satorious AG) was employed to exchange buffer of purified ADC. The purified ADC was loaded to TFF sample tank, and the concentration of purified ADC was increased 2-fold by reducing the total volume to half of the original. The resulting solution then underwent diafiltration with 10-fold volumes of storage buffer (10 mM Histidine. 250 mM Sucrose, pH 6.2) relative to the concentrated sample. Sampled the purified ADC for concentration and residual free N-PM-0022 intermediate testing. Adjusted the concentration ADC to 10 mg / mL and sterilized by passing through Millex-GP Syringe Filter (PES, 0.22 pm).Example 5: Residual enzyme analysis of OBI-904 by ELISA

[0079] This assay was conducted by using His Tag ELISA detection Kit (GenScript; Cat. No.: L00436). The kit was used for rapid and high throughput detection of His-tagged proteins. The reagent and assay process were listed as follows:

[0080] Reagent(a) His-tag plate (8 wellsxl2 strips):Coated with His-tagged protein with the molecular weight of 12.7 kDa.(b) Test sample:10K06-(NSCT-4N3)2 mAb crude / 10K06-(NSCT-4N3)2 mAb after Protein A purificationI 10K06-(NSCT-4N3)2 mAb after activated charcoal purification / 10K06-(NSCT-4N3)2 mAb / OBI-904(c) Standard: EndoSz-D234M Reference Standard [600 to 1.17 ng / mL and blank (assay diluent)](d) Anti-His monoclonal antibody (GenScript; Cat. No.: A00186)(e) Antibody tracer (secondary antibody): horseradish peroxidase (HRP) conjugated Goat anti-mouse IgG(1) 20 mM Sodium acetate, pH 5.0

[0081] Assay process(a) Replaced the buffer of test samples by sodium acetate buffer. Diluted the samples with assay diluent with suitable factors.(b) Dispensed 55 pL of Anti-His Monoclonal Antibody into each well. Add 55 pL of samples. Incubated at 25 °C in dark with 750 rpm shaking for 30 minutes.(c) Transferred 100 pL of mixture to His Tag strip wells and incubated at 25 °C for 30 minutes.(d) Washed the plate with 200 pL of Wash Solution four times.(e) Dispensed 100 pL of Antibody Tracer into each well and incubated at 25 °C for 30 minutes.(1) Washed the plate with 200 pL of Wash Solution four times.(g) Added 100 pL of TMB Substrate and incubated at 25 °C for 15 minutes.(h) Finally added 50 pL of Stop solution and read the OD45onm value.

[0082] Table 5 indicated the efficiency of residual enzyme remove between Protein A and activated charcoal purification. The original amount of residual enzyme was >2000 ppm in 10K06-(NSCT-4N3)2 crude. After Protein A purification, the amount of residual enzyme could be decreased to less than 300 ppm. However, the amount of residual enzyme could be decreasedto less than 5 ppm by further treating with activated charcoal. It could demonstrate that “activated charcoal'’ is the most effective process.Table 5. Results of residual enzyme in each process steps of OBI-904.

[0083] Unless defined otherwise, all technical and scientific terms and any acronyms used herein have the same meanings as commonly understood by one of ordinary skill in the art in the field of this invention. Although any compositions, methods, kits, and means for communicating information similar or equivalent to those described herein can be used to practice this invention, the preferred compositions, methods, kits, and means for communicating information are described herein.

[0084] All references cited herein are incorporated herein by reference to the full extent allowed by law. The discussion of those references is intended merely to summarize the assertions made by their authors. No admission is made that any reference (or a portion of any reference) is relevant prior art. Applicants reserve the right to challenge the accuracy and pertinence of any cited reference.

Claims

CLAIMSWhat is claimed is:

1. A method for qualifying of an anti-TROP2 antibody-drug conjugate (ADC), comprising:(a) antibody glycosylation;(b) hydrophobic interaction chromatography (HIC) and activated charcoal purification;(c) buffer exchange for glycosylated antibody;(d) bioconjugation between the glycosylated antibody and a linker-payload; and(e) buffer exchange and purification of the anti-TROP2 ADC.

2. The method of claim 1, wherein a column used in the HIC comprises agarose beads modified with phenyl, ethyl, butyl, and benzyl groups via uncharged, chemically stable ether linkages.

3. The method of claim 1, wherein the HIC is conducted at a flow rate of 0. 1 to 10.0 mL / min.

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

5. The method of claim 1, wherein the linker-payload or the anti-TROP2 ADC should be stored within a light-resistant container.

6. A method for qualifying of an anti-Nectin-4 antibody-drug conjugate (ADC), comprising:(a) antibody glycosylation;(b) Protein A chromatography and activated charcoal purification;(c) buffer exchange for glycosylated antibody;(d) bioconjugation between the glycosylated antibody and a linker-payload;(e) second activated charcoal purification; and(1) buffer exchange and purification of the anti-Nectin-4 ADC.

7. The method of claim 6, wherein the Protein A chromatography column comprises agarose beads, polyacrylamide, sepharose, or other polymers.

8. The method of claim 6, wherein the Protein A chromatography is conducted at a flow rate of 0. 1 to 10.0 mL / min.

9. The method of claim 6. wherein an antibody of the anti-Nectin-4 ADC is an anti-Nectin- 4 antibody selected from 5A12.2, 15A17.5, N4MU01, MW282, LY4052031, 08B04, 10K06, 14108, 05004, 12E03, 02P14, 11023, 14B21, 08C24, 13C24, or enfortumab.

10. An ADC with improved DAR distribution and stability, which is produced by the steps of:(a) hydrophobic interaction chromatography (HIC) or Protein A chromatography, and / or(b) activated charcoal purification.

11. The ADC of claim 10, wherein the improvement is measured by ELISA or SDS-PAGE.

12. The ADC of claim 10, wherein the improvement is measured by residual enzyme detection.

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

14. The ADC of claim 13, wherein the improvement is 2 to 500 folds ratio of enzyme residual.

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

16. A method for purifying a glycosylated antibody from a sample comprising an antibody and an impurity, comprising:(a) antibody glycosylation;(b) liquid chromatography; and(c) activated charcoal purification, wherein the impurity comprises chemical, glycan, enzyme, host cell protein, protein- derived polymer, protein degradation, or nucleic acid.

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

18. The method of claim 1 , wherein the glycosylated antibody is an antibody or an antigenbinding portion thereof which binds TROP2, Nectin-4, or which binds to one or more tumor- associated antigens or cell-surface receptors.

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

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

Images