Method for preparing conjugate by site-specific glycan coupling

Abstract

The present invention relates to a method for preparing protein molecules having an Fc region with site-specifically remodeled homogeneous glycan chains, comprising the following steps: a) providing a liquid comprising protein molecules having an Fc region with heterogeneous glycan chains expressed by eukaryotic cells before purification, wherein preferably, the protein molecule having the Fc region with the heterogeneous glycan chains is a Y-type antibody, an Fc fusion protein, or a heavy chain antibody; for example, the liquid comprising the protein molecules having the Fc region with the heterogeneous glycan chains expressed by the eukaryotic cells before purification is a cell supernatant; b) adding a disaccharide linker represented by formula I; and c) adding an endoglycosidase having an activity of specifically hydrolyzing heterogeneous glycan chains in glycoproteins and catalyzing a transglycosylation reaction to carry out a reaction, thereby obtaining a solution comprising protein molecules having an Fc region with site-specifically remodeled homogeneous glycan chains. The present invention further relates to a two-step method and a one-step method for preparing a protein molecule-drug conjugate having an Fc region with site-specifically remodeled homogeneous glycan chains using a liquid comprising the protein molecules having the Fc region with the heterogeneous glycan chains expressed by the eukaryotic cells before purification. The methods can be used in antibody glycoengineering and / or in the preparation of an antibody-drug conjugate.

Description

Methods for preparing conjugates by site-specific coupling of sugars TECHNICAL FIELD

[0001] The present invention relates to the field of biotechnology. Specifically, the present invention relates to methods for preparing protein molecules with site-specifically remodeled homogeneous sugar chains of Fc region from protein molecules with heterogeneous sugar chains of Fc region (e.g., cell supernatant) by enzymatic reaction, and two-step method and one-step method for preparing conjugated drugs of protein molecules with site-specifically remodeled homogeneous sugar chains of Fc region from protein molecules with heterogeneous sugar chains of Fc region (e.g., cell supernatant). The methods of the present invention can be used in the glycoengineering of antibodies or in the preparation of antibody-drug conjugates. BACKGROUND

[0002] With the continuous development of antibody drug conjugates (ADC, also known as antibody drug conjugates), there is a need in the art for ADCs with higher degree of homogeneity. Site-specific glycosylation technology is the most popular one because of its good versatility and excellent clinical data. However, site-specific glycosylation of ADCs requires the introduction of enzymes to remove and modify the original glycosylation groups on the Fc of the antibody, and therefore requires the use of chromatography steps to remove the introduced glycosidases. The increased chromatography steps increase the difficulty, time and cost of the process.

[0003] In the prior art, first, the antibody needs to be purified by a classical antibody purification step to obtain an antibody that meets the quality requirements. The purified antibody that meets the quality requirements is subjected to a glycosyl modification reaction with a disaccharide substrate under the action of an endoglycosidase, and the site-specific glycosylation product needs to be subjected to affinity chromatography, anion / cation chromatography to remove the introduced glycosidases and other impurities, and further conjugated with a small molecule drug to obtain an ADC drug. This process requires multiple chromatography and ultrafiltration / diafiltration (UF / DF) steps, which not only takes a long time, has a high cost of multiple chromatography steps, but also has a complex process, requires more parameters to be controlled, and has a high degree of difficulty.

[0004] There is a need in the art to provide a method for preparing ADC drugs that saves time, improves yield, and does not affect the conjugation effect. SUMMARY

[0005] The present application develops a new production process, which uses the antibody before purification, for example, uses the cell culture supernatant, directly carries out the site-specific glyco-reconstruction, and then carries out the coupling with the small molecule drug after the conventional purification step to obtain the purified site-specific glyco-reconstruction product; or uses the antibody before purification, for example, uses the cell culture supernatant, directly in the presence of the glycoside endoglycosidase and the disaccharide-small molecule drug conjugate to prepare the antibody conjugate drug. The quality of the antibody conjugate drug obtained by the method of the present application is consistent with the DAR value of the prior art, and the yield is obviously improved, the time is greatly shortened, and the production cost is reduced, and the present application has great application prospect in the industrialized production of the antibody conjugate drug.

[0006] In the first aspect, the present application provides a method for preparing a protein molecule having an Fc region with site-specifically reconstructed homogeneous sugar chains, comprising the following steps:

[0007] a) providing a liquid containing a protein molecule having an Fc region with heterogeneous sugar chains expressed by a eukaryotic cell before purification;

[0008] b) adding a disaccharide linker, for example, a disaccharide linker represented by formula I;

[0009] In formula I, the G ring represents a monosaccharide molecule selected from galactose, N-acetyl galactose, mannose and glucose, which is connected to the 4-position of the 1,2-cyclic N-acetyl-D-glucosamine through a glycosidic bond, and the glycosidic bond is a 1,4 glycosidic bond, a 2,4 glycosidic bond or a 3,4 glycosidic bond;

[0010] Z-Y-X- represents a substituent on the G ring, and the substitution position of Z-Y-X- is any position except the 1-position of the G ring of the monosaccharide molecule, for example, the substitution position of Z-Y-X- is the 2-position, the 3-position, the 4-position, the 5-position or the 6-position of the G ring of the monosaccharide molecule, preferably the 6-position;

[0011] c) adding a glycoside endoglycosidase with specific hydrolysis of the heterogeneous sugar chains in the glycoprotein and with catalytic transglycosylation activity to react;

[0012] Thus, a solution containing a protein molecule having an Fc region with site-specifically reconstructed homogeneous sugar chains is obtained.

[0013] In some embodiments, the method of the present application for preparing a protein molecule with Fc region of homogenous glycoforms with site-directed remodeling further comprises a step d) purifying the solution of the protein molecule with Fc region of homogenous glycoforms with site-directed remodeling, for example, affinity chromatography (e.g., protein A affinity chromatography, protein G affinity chromatography or protein L affinity chromatography); or, affinity chromatography (e.g., protein A affinity chromatography, protein G affinity chromatography or protein L affinity chromatography) and anion chromatography and / or cation chromatography; or, affinity chromatography (e.g., protein A affinity chromatography, protein G affinity chromatography or protein L affinity chromatography) and low pH incubation, anion chromatography, cation chromatography, nanofiltration and / or ultrafiltration / diafiltration; for example, purifying in the following order: affinity chromatography, low pH incubation, anion chromatography, cation chromatography, nanofiltration and ultrafiltration / diafiltration; or affinity chromatography, low pH incubation, nanofiltration, anion chromatography, cation chromatography and ultrafiltration / diafiltration; or affinity chromatography, low pH incubation, anion chromatography, nanofiltration, cation chromatography and ultrafiltration / diafiltration.

[0014] In a second aspect, the present application provides a two-step method for preparing a protein molecule with Fc region of homogenous glycoforms with site-directed remodeling conjugated drug, comprising:

[0015] Step (I): performing the method of the first aspect of the present application to obtain the purified protein molecule with Fc region of homogenous glycoforms with site-directed remodeling;

[0016] Step (II): adding the small molecule drug-linker of Formula IV to the purified protein molecule with Fc region of homogenous glycoforms with site-directed remodeling obtained in Step (I) to obtain the protein molecule with Fc region of homogenous glycoforms with site-directed remodeling conjugated drug;

[0017] E-L-D (Formula IV)

[0018] E is a corresponding group that undergoes orthogonal reaction with azido group, which is selected from linear alkynyl, DBCO-like structure, BCN-like structure,

[0019] L represents a linker,

[0020] D represents a cytotoxic or cytostatic drug.

[0021] Optionally, the protein molecule with Fc region of homogenous glycoforms with site-directed remodeling conjugated drug is purified, for example, by ultrafiltration / diafiltration (UF / DF).

[0022] In a third aspect, the present application provides a one-step method for preparing a protein molecule with Fc region of homogenous glycoforms with site-directed remodeling conjugated drug, comprising:

[0023] a) providing a liquid comprising a eukaryotic cell-expressed Fc region- containing protein molecule with heterogeneous sugar chains before purification; preferably, the Fc region-containing protein molecule with heterogeneous sugar chains is a Y-type antibody, an Fc fusion protein or a heavy chain antibody; for example, the liquid comprising a eukaryotic cell-expressed Fc region-containing protein molecule with heterogeneous sugar chains before purification is a cell supernatant;

[0024] b) adding a disaccharide-small molecule drug conjugate of Formula V

[0025] In Formula V, G ring, X, Y, L, D are as defined in Formula I and Formula IV,

[0026] Z' is independently -(CH2)p- wherein p is an integer from 1 to 5, or Z' is absent, or is a group that can react with the Z group in Formula I;

[0027] c) adding a glycoside hydrolase enzyme with specific hydrolysis of the heterogeneous sugar chains in the glycoprotein and with catalytic transglycosylation activity to react,

[0028] thereby obtaining a solution of the Fc region-containing protein molecule drug conjugate with homogeneous sugar chains of site-directed remodeling.

[0029] In some embodiments, the one-step method for preparing the Fc region-containing protein molecule drug conjugate with homogeneous sugar chains of site-directed remodeling of the present application further comprises a step d) purifying the solution of the Fc region-containing protein molecule drug conjugate with homogeneous sugar chains of site-directed remodeling, for example, affinity chromatography (e.g., protein A affinity chromatography, protein G affinity chromatography or protein L affinity chromatography); or, affinity chromatography (e.g., protein A affinity chromatography, protein G affinity chromatography or protein L affinity chromatography) and anion chromatography and / or cation chromatography; or, affinity chromatography (e.g., protein A affinity chromatography, protein G affinity chromatography or protein L affinity chromatography) and low pH incubation, anion chromatography, cation chromatography, nanofiltration and / or ultrafiltration / diafiltration; for example, purifying in the following order: affinity chromatography, low pH incubation, anion chromatography, cation chromatography, nanofiltration and ultrafiltration / diafiltration; or affinity chromatography, low pH incubation, nanofiltration, anion chromatography, cation chromatography and ultrafiltration / diafiltration; or affinity chromatography, low pH incubation, anion chromatography, nanofiltration, cation chromatography and ultrafiltration / diafiltration.

[0030] In a fourth aspect, the present application provides a Fc region-containing protein molecule with homogeneous sugar chains of site-directed remodeling prepared according to the method of the first aspect of the present application.

[0031] In a fifth aspect, the present application provides a Fc region-containing protein molecule drug conjugate with homogeneous sugar chains of site-directed remodeling prepared according to the method of the second aspect or the third aspect of the present application.

[0032] In some embodiments, the protein molecule having an Fc region with heterogeneous sugar chains used in the method according to the second or third aspect of the present application is an antibody having a glycosylation site,

[0033] Preferably, the antibody is an antibody having an N-glycosylation site,

[0034] More preferably, the antibody is a dual-antenna antibody having an N-glycosylation site,

[0035] Most preferably, the antibody is an IgG having a conservative N-glycosylation site at N297 of the Fc region,

[0036] For example, the antibody is a monoclonal antibody, a polyclonal antibody, a diabody, a triabody, a nanobody fused with an Fc domain, a therapeutic antibody or a functional antibody of different species origin,

[0037] For example, the antibody is a human antibody, a murine antibody or a chimeric antibody;

[0038] For example, the antibody is an IgG1, IgG2, IgG4;

[0039] For example, the target point of the antibody is selected from the group consisting of HER2, Claudin 18.2, EGFR, TROP2, c-Met, NECTIN4, CD276, HER3, CD3, FOLR1, BCMA, CD20, DLL3, MUC1, PD-L1, ROR1, TF, CD19, CD22, CD30, CD70, CD79B, FGFs, MSLN, NT5E, TNFα, CD147, CD24, CD38, CD47, CDH3, CDK4, CDK6, CEACAM5, CLDN6, CTLA4, DDR1, DR5, FAPα, FGFR3, GPRC5D, GR, HLA-DR, ICAM1, IL2R, MELTF, ROR2, TPBG(5T4), VTCN1, ZIP6, CD33, CD25, RSV, VEGF, RANKL, VEGFR2, CTLA-4, CD52, CD319, PD-1, CD274, IgE, IL-6, IL-12, IL-2, C5, IL-17A, CD25, SLAMF7, F10, factor IXa, HAb18G, PCSK9, BLyS, IL23, α4β7, IL-4R-α, HAE, FGF23 and IL6R; preferably, the target point of the antibody is selected from the group consisting of HER2, CD20, TROP2, EGFR, PD-1.

[0040] In particular, the antibody includes bevacizumab, trastuzumab, rituximab, pertuzumab, panitumumab, tremelimumab, tremelimumab, nivolumab, visilizumab, and sacituzumab.

[0041] In some embodiments, in the method of the second or third aspect of the present application, the reaction is performed using a protein molecule having a heterogeneous sugar chain of Fc region at a final concentration of > 1 mg / mL, preferably, the protein molecule having a heterogeneous sugar chain of Fc region at a final concentration of about 1 to 50 mg / mL, more preferably, the protein molecule having a heterogeneous sugar chain of Fc region at a final concentration of about 1 to 20 mg / mL, most preferably, the protein molecule having a heterogeneous sugar chain of Fc region at a final concentration of about 1 to 10 mg / mL, for example, about 2, about 3, about 4, about 5, about 6, about 7, about 8, or about 9 mg / mL.

[0042] In some embodiments, in the method of the second or third aspect of the present application, the reaction is performed using a disaccharide linker (e.g., diAz-LacNAc-ox) at an equivalent ratio of > about 20 to the protein molecule having a heterogeneous sugar chain of Fc region; preferably, the reaction is performed using a disaccharide linker (e.g., diAz-LacNAc-ox) at an equivalent ratio of about 20 to 300 to the protein molecule having a heterogeneous sugar chain of Fc region, more preferably, the reaction is performed using a disaccharide linker (e.g., diAz-LacNAc-ox) at an equivalent ratio of about 20 to 200 to the protein molecule having a heterogeneous sugar chain of Fc region, most preferably, the reaction is performed using a disaccharide linker (e.g., diAz-LacNAc-ox) at an equivalent ratio of about 20 to 160 to the protein molecule having a heterogeneous sugar chain of Fc region, for example, at an equivalent ratio of about 40, about 60, about 80, about 100, about 120, or about 140.

[0043] In some embodiments, in the method of the second or third aspect of the present application, the reaction is performed using an endoglycosidase at an added amount of > about 5 μg / mg, preferably, the reaction is performed using an endoglycosidase at an added amount of about 5 to 150 μg / mg, more preferably, the reaction is performed using an endoglycosidase at an added amount of about 10 to 80 μg / mg, most preferably, the reaction is performed using an endoglycosidase at an added amount of about 10 to 40 μg / mg, for example, the reaction is performed using an endoglycosidase at an added amount of about 15, about 20, about 25, about 30, or about 40 μg / mg.

[0044] In some embodiments, the method of the second aspect or the third aspect of the present application, the endoglycosidase reaction is performed at a pH of about 4.0 to 8.0, preferably at a pH of about 4.5 to 7.7, more preferably at a pH of about 5.0 to 7.5, most preferably at a pH of about 5.3 to 7.2, for example, at a pH of about 6, about 6.5, or about 7.0.

[0045] In some embodiments, the method of the second aspect or the third aspect of the present application, the endoglycosidase reaction is performed at a reaction temperature of about 5°C to 50°C, preferably at a reaction temperature of about 6°C to 45°C, more preferably at a reaction temperature of about 15°C to 40°C, most preferably at a reaction temperature of about 20°C to 37°C, for example, at a reaction temperature of about 22°C, about 25°C, about 30°C, or about 35°C.

[0046] In some embodiments, the method of the second aspect or the third aspect of the present application, the endoglycosidase reaction is performed at a reaction time of about 0.1 hour to 30 hours, preferably at a reaction time of about 0.2 hour to 25 hours, more preferably at a reaction time of about 0.5 hour to 8 hours, most preferably at a reaction time of about 0.75 hour to 4 hours, for example, at a reaction time of about 0.8 hour, about 1 hour, about 1.5 hour, or about 2 hours.

[0047] The present application reduces the difficulty of the process, shortens the process time, and reduces the production cost by integrating and optimizing the ADC coupling process and the downstream antibody purification process, thereby promoting the development of glyco-site coupling technology. BRIEF DESCRIPTION OF DRAWINGS

[0048] Figure 1 illustrates a production process for preparing an antibody with a homogenous glycan chain of site-specific remodeling (also referred to herein as “glycoengineering of antibodies”), wherein “original production process flow” represents the process flow in the prior art; “new production process flow” represents the process flow of the present application.

[0049] Figure 2 illustrates a two-step production process for preparing an antibody drug with a homogenous glycan chain of site-specific remodeling, wherein “original production process flow” represents the process flow in the prior art; “new production process flow” represents the process flow of the present application.

[0050] Figure 3 shows the RP-HPLC detection of DAR values (i.e., RP-DAR detection profile) and SEC-HPLC detection of antibody monomer purity (i.e., SEC detection profile) of ADCs prepared using cell supernatant of bevacizumab, with the two-step production process of the present application (referred to as "New" in the figure) and the two-step production process of the prior art (referred to as "Ori" or "original process" in the figure).

[0051] Figure 4 shows the RP-DAR detection and SEC detection results of ADCs prepared with the two-step production process of the present application (referred to as "New process" in the figure) and the two-step production process of the prior art (referred to as "original process" in the figure).

[0052] Figure 5 shows the yield and process time of ADCs prepared with the two-step production process of the present application (referred to as "New process" in the figure) and the two-step production process of the prior art (referred to as "original process" in the figure).

[0053] Figure 6 shows the RP-HPLC detection of DAR values (i.e., RP-DAR detection profile) and SEC-HPLC detection of antibody monomer purity (i.e., SEC detection profile) of ADCs prepared with the two-step production process of the present application using different endoglycosidases, Endo S2 and Endo S2M3, respectively.

[0054] Figure 7 shows the RP-DAR results of ADCs prepared after purification of samples taken at different times during the glycosylation reaction (i.e., preparation of antibodies with site-specific remodeled homogeneous sugar chains) using endoglycosidases Endo S2 or Endo S2M3 in the two-step production process of the present application.

[0055] Figure 8 shows the effect of antibody reaction concentration used in the two-step production process of the present application on the DAR values of the prepared ADCs.

[0056] Figure 9 shows the RP-HPLC detection of DAR values of ADCs prepared using cell supernatant of trastuzumab, with the two-step production process of the present application (referred to as "New" in the figure) and the two-step production process of the prior art (referred to as "Ori" in the figure).

[0057] Figure 10 shows the SEC-HPLC detection of antibody monomer purity of ADCs prepared using cell supernatant of trastuzumab, with the two-step production process of the present application (referred to as "New" in the figure) and the two-step production process of the prior art (referred to as "Ori" in the figure).

[0058] Figure 11 shows the effect of the equivalent ratio of disaccharide substrate to antibody used in the two-step production process of the present application on the DAR values of the prepared ADCs.

[0059] Figure 12 shows the effect of the amount of endoglycosidase added in the two-step production process of the present application on the DAR values of the prepared ADCs.

[0060] Figure 13 shows the effect of the pH value of the endoglycosidase reaction in the two-step production process of the present application on the DAR values of the prepared ADCs.

[0061] Figure 14 shows the effect of the temperature of the endoglycosidase reaction in the two-step production process of the present application on the DAR values of the prepared ADCs.

[0062] Figure 15 shows the effect of the reaction time of the endoglycosidase addition in the two-step production process of the present application on the DAR values of the prepared ADCs.

[0063] Figure 16 shows the profile of the DAR values of the ADCs prepared using the cell supernatant of bevacizumab, employing the one-step production process of the present application (referred to as "New" in the figure) and the one-step production process of the prior art (referred to as "Ori" in the figure) as detected by RP-HPLC.

[0064] Figure 17 shows the profile of the antibody monomer purity of the ADCs prepared using the cell supernatant of bevacizumab, employing the one-step production process of the present application (referred to as "New" in the figure) and the one-step production process of the prior art (referred to as "Ori" in the figure) as detected by SEC-HPLC.

[0065] Figure 18 shows the profile of the DAR values of the cysteine conjugated samples prepared by the new process and the original process as detected by RP-HPLC.

[0066] Figure 19 illustrates the one-step production process for preparing antibody conjugated drugs with homogenous sugar chains with site-directed remodeling, wherein "original production process flow" indicates the process flow of the prior art; and "new production process flow" indicates the process flow of the present application.

[0067] Figure 20 shows the transglycosylation activity of the endoglycosidase when the endoglycosidase Endo-S2M3 or Endo-S2 mutant 1 is used in the one-step preparation of site-directed ADC compounds by endoglycosidase.

[0068] Figure 21 shows the antibody transglycosylation activity of the endoglycosidases Endo-S2 and Endo-S2M3 enzymes on Az-LacNAc-ox substrate.

[0069] Figure 22 shows the hydrolytic activity of the endoglycosidase Endo-S2 and Endo-S2M3 on transglycosylated glycoengineered antibodies at various enzyme amounts for 2 hours.

[0070] Figure 23 shows the hydrolytic activity of the endoglycosidase Endo-S2 and Endo-S2M3 on transglycosylated glycoengineered antibodies at the same enzyme amount in each panel over increasing reaction time. DETAILED DESCRIPTION

[0071] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting. Other features, objects, and advantages of the application will be apparent from the description and drawings, and from the claims.

[0072] I. DEFINITIONS

[0073] As used herein, the term "about," when used in conjunction with a numerical value, is intended to encompass numerical values falling within 5% of the indicated numerical value, both ways. The term is also intended to encompass values within ±1%, ±0.5%, or ±0.1% of the indicated numerical value.

[0074] As used herein, the expression "and / or" is used to indicate any one of the stated conditions alone, or any and all possible combinations of the stated conditions.

[0075] As used herein, the term "comprising" or "including," means including the recited elements, integers, or steps, or groups thereof, but does not exclude other elements, integers, or steps, or groups thereof. As used herein, when the term "comprising" or "including" is used, it is also intended to cover the case where the recited elements, integers, or steps are the only ones, unless otherwise stated. For example, when referring to a polypeptide / protein "comprising" a certain specific sequence, it is also intended to cover a polypeptide / protein consisting of the certain specific sequence.

[0076] In the present context, the term "DAR" refers to the ratio of other molecules (e.g., disaccharide molecules or small molecule drugs) coupled to the Fc region of a protein molecule (e.g., an antibody) having site-specifically reconfigured homogeneous glycochains as described herein to the Fc region of a protein molecule (e.g., an antibody) having site-specifically reconfigured homogeneous glycochains. In some embodiments of preparing antibodies having site-specifically reconfigured homogeneous glycochains, the DAR value refers to the ratio of disaccharide molecules coupled to the Fc region of a protein molecule (e.g., an antibody) having site-specifically reconfigured homogeneous glycochains as described herein to the molecules of a protein molecule (e.g., an antibody) having site-specifically reconfigured homogeneous glycochains. In embodiments of antibody conjugate drugs (ADCs), the DAR value refers to the ratio of small molecule drug moieties coupled to the Fc region of a protein molecule (e.g., an antibody) having site-specifically reconfigured homogeneous glycochains as described herein to the Fc region of a protein molecule (e.g., an antibody) having site-specifically reconfigured homogeneous glycochains. In some embodiments, the DAR is calculated as the average DAR of the population of molecules in the product, i.e., the total ratio of disaccharide molecules or small molecule drugs coupled to, e.g., an antibody moiety as described herein to the antibody moiety in the product as measured by detection methods (e.g., by conventional methods such as mass spectrometry, ELISA assays, electrophoresis, and / or HPLC), which is referred to herein as the average DAR. In some embodiments, the average DAR value of the conjugates of the present application is, e.g., 1.0-8.0, 2.0-6.0, e.g., 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, or 8, and ranges with two of these values as endpoints.

[0077] In some embodiments, the DAR value is calculated as follows:

[0078] Drug loading distribution (%) = [peak area of heavy (light) chain with n drugs / sum of all peak areas of heavy (light) chain] x 100 (%)

[0079] where n is the number of drugs carried

[0080] Weighted peak area of heavy (light) chain = n x drug loading distribution %

[0081] DAR = 2 x [∑(weighted peak area of heavy chain) + ∑(weighted peak area of light chain)]

[0082] For example, the DAR value in FIG. 11 is a DAR value calculated using this method.

[0083] In some embodiments, the DAR value is calculated as follows:

[0084] Drug loading distribution (%) = (peak area of drug loaded / sum of peak areas) x 100 (%)

[0085] where n is the number of drugs loaded

[0086] Weighted peak area = number of drugs loaded n x drug loading distribution %

[0087] DAR = ∑(weighted peak area)

[0088] As used herein, the term "antibody" (Ab) includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity. The term "immunoglobulin" (Ig) is used interchangeably with "antibody" herein. As used herein, "human antibody" refers to an antibody, a functional fragment thereof, or a humanized antibody that naturally occurs in the human body, i.e., a genetically engineered antibody in which a portion (e.g., a framework region or an Fc region) is derived from a naturally occurring human antibody.

[0089] The term "heterogeneous sugar chains in glycoproteins" refers to a post-translational modification of proteins by glycosylation in eukaryotic cells, in which a sugar chain reacts with an active group on the side chain of an amino acid of a protein, thereby allowing the sugar chain to be linked to the protein through a glycosidic bond to form a glycoprotein. Due to the variety of compositions and linkage modes of sugar chains, glycoproteins have highly structurally heterogeneous sugar chains.

[0090] The terms "disaccharide linker" or "disaccharide substrate" are used interchangeably herein to refer to a disaccharide molecule having an oxazoline-ylated GlcNAc at the reducing end of its sugar chain. When a disaccharide linker is used for sugar remodeling for the purpose of drug discovery, it is preferable to use a disaccharide linker having a human sugar chain or a human-compatible sugar chain, which causes few problems when applied to humans. Such a sugar chain is a sugar chain known to exhibit no antigenicity in the human body.

[0091] The term "small molecule drug" refers to a low molecular weight organic compound capable of modulating a biological process. A "small molecule" is defined as a molecule having a molecular weight of less than 10 kD, typically less than 2 kD, and preferably less than 1 kD. Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimetics, and antibody mimetics. As therapeutic agents, small molecules can be more able to penetrate cells, less susceptible to degradation, and less likely to elicit an immune response than macromolecules.

[0092] The term "cell supernatant" or "supernatant" refers to the clear liquid obtained by centrifugation and / or filtration of a harvest containing a protein molecule having an Fc region, cells, and other impurities (cell debris, gels, etc.) from a eukaryotic cell culture, such as a shake flask or reactor, for the production of a protein molecule having an Fc region (e.g., an antibody) known in the art.

[0093] In some embodiments, a cell supernatant is obtained by culturing a eukaryotic cell expressing an antibody, for example, by cell resuscitation, shake flask expansion of the cells, fed-batch culturing of the cells, harvesting of the cell culture supernatant at the end of the culturing, centrifugation and / or filtration in small scale antibody production, or separation harvest processing, such as depth filtration, in industrial large scale antibody production. In laboratory scale, separation of the cell culture supernatant from cells and cell debris can be accomplished by simple batch centrifugation, but large scale antibody production primarily employs continuous separation methods, including, for example, depth filtration, tangential flow filtration, and continuous flow centrifugation. Depth filtration is effective in separating cells, cell debris, and other particulate or gel-like material from the harvest. In this context, the step of obtaining a cell supernatant is not included in the purification steps of the antibody production process.

[0094] In some embodiments, a CHO cell expressing an antibody is cultured, shake flask expansion is performed using a basal medium, and the cells are passaged to prepare a suitable seed volume and maintain a cell viability of greater than 90%. The cells are then transferred to a 3L-50L reactor for fed-batch culturing, during which the nutritional needs of the cells are maintained and antibody expression is promoted by the addition of feed medium, glucose, and the like. At the end of the culturing, the cell culture is harvested, centrifuged, and sterile filtered, for example, at 5000-15000 rpm for 30 min or more, using a 0.02-18 um sterile filter, to collect the supernatant containing the antibody. In another example, the cell culture is centrifuged at 9500 rpm for 30 min, and the supernatant containing the antibody is collected using a 0.22 μιη microfiltration membrane.

[0095] In some embodiments, the antibody-expressing CHO cells are cultured, and the shake flask amplification culture is performed using a basal medium, the seed volume is prepared by step-by-step passage, and the cell viability is maintained at more than 90%, and then the cells are transferred to a 3L-50L reactor for fed-batch culture, during which the nutritional requirements of the cells are maintained and antibody expression is promoted by supplementing with a feed medium, glucose solution, etc., and after the culture ends, the cell culture solution is collected and subjected to depth filtration to obtain supernatant containing the antibody.

[0096] The type of cell culture medium is not limited. The basal medium can be, for example, CHOMAX D, Merck Millipore, MB1104.201; Ex-cell 302, SAFC, item number 24326-100L6428; CD07 medium, Opiyo, P081307-001; Dynamis AGT medium, Gibco, item number A2617502. The feed medium can be, for example, CHOMAX FA, Merck Millipore, MB1201.202; CHOMAX FB, Merck Millipore, MB1202.202; CD Efficient feed C medium, Gibco, item number A13275-01; Cell Boost 7a, Cytiva, item number SH31026.03; Cell Boost 7b, Cytiva, SH31027.02CN; Feed B, Gibco, item number A12456-01.

[0097] The term "reversed-phase high-performance liquid chromatography (RP-HPLC)" is high-performance liquid chromatography in which the polarity of the mobile phase is greater than that of the stationary phase. Molecules passing through such reversed-phase chromatography columns move according to their polarity, with greater polarity resulting in faster movement.

[0098] The term "size exclusion high performance liquid chromatography (SEC-HPLC)" is a method used for monoclonal antibody standardization and quality control. This method separates molecules based on differences in their size or hydrodynamic radius. By SEC-HPLC, antibodies can be separated into three main forms: high molecular weight species (HMMS), main peak (mainly antibody monomer), and low molecular weight species (LMMS). Antibody purity can be calculated as the percentage of the main peak area over the sum of all peak areas on the chromatogram. By SEC-HPLC, the percentage of antibody monomer in a formulation product can be measured, giving information on the content of soluble aggregates and shear species. Further description of the SEC-HPLC method can be found, for example, in J. Pharm. Scien., 83: 1645-1650, (1994); Pharm. Res., 11 :485 (1994); J. Pharm. Bio. Anal., 15: 1928 (1997); J. Pharm. Bio. Anal., 14: 1133-1140 (1986). Further, see, for example, R. Yang et al., High resolution separation of recombinant monoclonal antibodies by size exclusion ultra-high performance liquid chromatography (SE-UHPLC), Journal of Pharmaceutical and Biomedical Analysis (2015), http: / / dx.doi.org / 10.1016 / j.jpba.2015.02.032; and Alexandre Goyon et al., Protocols for the analytical characterization of therapeutic monoclonal antibodies. I-Non-denaturing chromatographic techniques, Journal of Chromatography, http: / / dx.doi.org / 10.1016 / j.jchromb.2017.05.010.

[0099] II. Purification process of protein molecules with Fc region

[0100] The aim of the purification of protein molecules with Fc region (e.g., antibodies) is to separate the antibodies from process-related impurities and product-related impurities, ultimately obtaining a protein molecule with Fc region (e.g., antibody) of high purity and low potential hazard for use as a drug.

[0101] In the purification of protein molecules with Fc region (e.g., antibodies), process-related impurities that need to be removed include cells, cell debris, host cell proteins, host cell nucleic acids, and media and feed components; product-related impurities that need to be removed mainly include antibody fragments and antibody aggregates. The purification process also needs to have sufficient viral inactivation removal capacity to remove endogenous virus-like particles expressed by the host cells and exogenous viruses that are not discovered in time. Low-pH incubation and nanofiltration in the purification process are steps for viral inactivation removal.

[0102] Most antibody purification processes use affinity chromatography (e.g., protein A affinity chromatography, protein G affinity chromatography, or protein L affinity chromatography) to capture antibodies. The presence of Fc fragments enables protein A affinity chromatography to serve as a universal antibody capture step,

[0103] Protein molecules with Fc region (e.g., antibodies) need to be further purified (hereinafter referred to as “refined purification”) after affinity chromatography (e.g., protein A affinity chromatography, protein G affinity chromatography, or protein L affinity chromatography) in order to reduce trace impurities to a level that meets product safety requirements.

[0104] Chromatography commonly used in the refined purification of antibodies includes cation exchange (CEX), anion exchange (AEX), hydrophobic interaction (HIC), and hydroxyapatite (HA), etc. In addition, immobilized metal-chelate affinity chromatography (IMAC) and size-exclusion chromatography (SEC) can also be used. In some embodiments, two of the above-mentioned four common chromatography are selected as the refined purification step after affinity chromatography (e.g., protein A affinity chromatography, protein G affinity chromatography, or protein L affinity chromatography).

[0105] In the prior art, the method for recovering protein molecules with Fc region (e.g., antibodies) from cell culture generally includes the following steps:

[0106] a) obtaining cell supernatant;

[0107] b) capturing protein molecules with Fc region (e.g., antibodies) in the cell supernatant by affinity chromatography;

[0108] c) low pH incubation (e.g., adjusting the pH to 3.4-3.7) for viral inactivation;

[0109] d) purification by cation chromatography;

[0110] e) performing anion chromatography;

[0111] f) removing viruses by nanofiltration;

[0112] g) ultrafiltration / diafiltration (UF / DF).

[0113] Processes for the purification of protein molecules with Fc regions are also disclosed in the prior art, see e.g. CN105358572A, WO2012135415A1, WO201 1090719A2, CN101981056A, CN102239177A, CN1504482A, CN102574911A.

[0114] In some embodiments, the protein molecules with Fc regions (e.g., antibodies) are purified to a purity of greater than 95% or 99%, as determined by, e.g., electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis, CE-SDS) or chromatography (e.g., size exclusion chromatography or ion exchange or reverse phase HPLC). For an overview of methods for assessing, e.g., antibody purity, see Flatman, S. et al., J. Chrom. B 848 (2007) 79-87.

[0115] III. Two-step process for the preparation of protein molecules with Fc regions of homogeneous glycoforms coupled drugs

[0116] The present application provides a method which shortens the process time and improves the yield of protein molecules with Fc regions of homogeneous glycoforms (e.g., antibodies) relative to prior art processes.

[0117] In some embodiments, the present application provides a two-step process for the preparation of protein molecules with Fc regions of homogeneous glycoforms coupled drugs, comprising:

[0118] Step (I.1 ): site-specific glycoremodification of protein molecules with Fc regions of heterogeneous glycoforms in cell culture supernatant

[0119] The cell supernatant containing the protein molecule having the Fc region with the heterogeneous sugar chain is added to a buffer (e.g., His-HCl buffer, phosphate buffer) (e.g., pH 4 to 8, preferably pH 4.5 to 7.7), a disaccharide linker (e.g., the disaccharide linker represented by Formula I) is added, a glycoside hydrolase having specific hydrolysis of the heterogeneous sugar chain in the glycoprotein and having catalytic transglycosylation activity (e.g., the glycoside hydrolase S2 (Endo-S2) represented by SEQ ID NO: 1 or an amino acid sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity thereto, the glycoside hydrolase S2M3 (Endo-S2M3) represented by SEQ ID NO: 2 or an amino acid sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity thereto, the glycoside hydrolase Endo-S2 mutant 1 represented by SEQ ID NO: 3 or an amino acid sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity thereto) is added (herein, the order of addition of the cell supernatant, the disaccharide linker, and the glycoside hydrolase is not limited), and incubated for a certain period of time (e.g., at a temperature of 6 to 37°C, for 0.25 hours to 22 hours) to obtain a reaction solution A containing the protein molecule having the Fc region with the homogeneous sugar chain with site-directed remodeling (e.g., an antibody containing the homogeneous sugar chain with site-directed remodeling).

[0120] Amino acid sequence of glycoside hydrolase S2 (Endo-S2) (SEQ ID NO: 1)

[0121] Amino acid sequence of glycoside hydrolase S2M3 (Endo-S2M3) (SEQ ID NO: 2)

[0122] Amino acid sequence of glycoside hydrolase Endo-S2 mutant 1 (SEQ ID NO: 3)

[0123] The "endoglycosidase" used in the preparation method of the present application can be any endoglycosidase having the ability to specifically hydrolyze a heterogeneous sugar chain in a glycoprotein and having the ability to catalyze transglycosylation reaction, including but not limited to the endoglycosidases disclosed in Sjogren, Biochem J. 2013 Oct 1; 455(1): 107-18, CN 108026518A. In some specific embodiments, the present application uses the endoglycosidase S2 (Endo-S2) shown in SEQ ID NO: 1, the endoglycosidase S2M3 (Endo-S2M3) shown in SEQ ID NO: 2, and the endoglycosidase Endo-S2 mutant 1 shown in SEQ ID NO: 3 as the endoglycosidase in the preparation method of the present application.

[0124] In some embodiments, the endoglycosidase S2M3 (Endo-S2M3) shown in SEQ ID NO: 2 is used, which has excellent ability to specifically hydrolyze a heterogeneous sugar chain in a glycoprotein and has the ability to catalyze transglycosylation reaction.

[0125] In some embodiments, the endoglycosidase S2 (Endo-S2) shown in SEQ ID NO: 1 is used, which has excellent ability to specifically hydrolyze a heterogeneous sugar chain in a glycoprotein and has the ability to catalyze transglycosylation reaction.

[0126] In some embodiments, the endoglycosidase Endo-S2 mutant 1 shown in SEQ ID NO: 3 is used, which has the ability to specifically hydrolyze a heterogeneous sugar chain in a glycoprotein and has the ability to catalyze transglycosylation reaction.

[0127] The disaccharide linker used in the preparation method of the present application is a disaccharide linker comprising an oxazoline-modified GlcNAc structure. In some embodiments, the disaccharide linker has a structure shown in Formula I:

[0128] In Formula I, the G ring represents a monosaccharide molecule selected from galactose, N-acetyl galactose, mannose, and glucose, which is linked to the 4-position of 1,2-cyclic N-acetyl-D-glucosamine by a glycosidic bond, and the glycosidic bond is a 1,4 glycosidic bond, a 2,4 glycosidic bond, or a 3,4 glycosidic bond;

[0129] Z-Y-X- represents a substituent on the G ring, and the substitution position of Z-Y-X- is any position other than the 1-position of the G ring of the monosaccharide molecule, for example, the substitution position of Z-Y-X- is the 2-position, the 3-position, the 4-position, the 5-position, or the 6-position of the G ring of the monosaccharide molecule, preferably the 6-position;

[0130] wherein, in the structure Z-Y-X-, Z-Y- can be present or absent,

[0131] X is an aldehyde group, -NH2, -CH2-NH2, -COOH, -N3, -CH2-N3 when Z-Y- is not present;

[0132] X is selected from the group consisting of -CH2-, -CH2-O-, -CH2-Se-, -CO-NH-, -ON=CH-, -CONH-N=CH-, -NHCH2-, -CH=CH- or is not present, and the following structures:

[0133] Y is a bivalent or multivalent linker connecting X and Z,

[0134] Preferably, Y is selected from the group consisting of -(CH2)m-(CH-w)n-, -(CH2-CH2-O)m-(CH-w)n-, -NH-,

[0135] wherein m and n are independently selected from an integer between 0 and 30, and w is a hydrogen atom or a polyethylene glycol structure of varying length;

[0136] Z is selected from the group consisting of an azide residue, an aldehyde residue, a thiol residue, an alkyne residue, an alkene residue, a halogen residue, a tetrazine residue, a nitrone residue, a hydroxylamine residue, a nitrile residue, a hydrazine residue, a ketone residue, a boronic acid residue, a cyano benzothiazole residue, an allyl residue, a phosphine residue, a maleimide residue, a disulfide residue, a thioester residue, an alpha-halo carbonyl residue, an isonitrile residue, a stilbene ketone residue, a conjugated diene residue, a cycloalkyne residue, and a cycloalkene residue;

[0137] Alternatively, Z is selected from the group consisting of:

[0138] wherein n is an integer from 1 to 30, and m1, m2 each independently represent an integer from 1 to 12, and R1and R2are each independently selected from H, -CH3, -CH2CH3, cyclopropyl, or cyclobutyl.

[0139] In some embodiments, when G in Formula I is galactose, then X in Formula I is selected from the group consisting of:

[0140] wherein l, m, n are each independently an integer from 0 to 30, and m1, m2 each independently represent an integer from 1 to 12.

[0141] wherein n is an integer from 0 to 30.

[0142] In some embodiments, when G in Formula I is galactose, then X in Formula I is selected from the group consisting of: ​is selected from the group consisting of:

[0143] wherein m is an integer from 0 to 30, for example, m is an integer from 0 to 25;

[0144] l is an integer from 0 to 30, for example, l is an integer from 0 to 25;

[0145] n is an integer from 0 to 30, for example, n is an integer from 0 to 25.

[0146] For example,

[0147] In some embodiments, when G in Formula I is galactose, then in Formula I is selected from the group consisting of:

[0148] wherein m is an integer from 0 to 30, for example, m is an integer from 0 to 25;

[0149] l is an integer from 0 to 30, for example, l is an integer from 0 to 25;

[0150] n is an integer from 0 to 30, for example, n is an integer from 0 to 25.

[0151] For example,

[0152] In some embodiments, when G in Formula I is galactose, then in Formula I is selected from the group consisting of:

[0153] Thus, the disaccharide linker of Formula I is

[0154] In some embodiments, when G in Formula I is galactose, then in Formula I is selected from the group consisting of:

[0155] wherein m is an integer from 0 to 30, for example, m is an integer from 0 to 25;

[0156] l is an integer from 0 to 30, for example, l is an integer from 0 to 25;

[0157] n is an integer from 0 to 30, for example, n is an integer from 0 to 25.

[0158] For example,

[0159] In some embodiments, when G in Formula I is galactose, then in Formula I is selected from the group consisting of:

[0160] wherein m1, m2 each represents an integer from 1 to 12.

[0161] For example,

[0162] Thus, the disaccharide linker of Formula I is

[0163] In some embodiments, when G in Formula I is galactose, then in Formula I the structure is selected from the group consisting of:

[0164] wherein m is an integer from 0 to 30, for example, m is an integer from 0 to 25;

[0165] l is an integer from 0 to 30, for example, l is an integer from 0 to 25;

[0166] n is an integer from 0 to 30, for example, n is an integer from 0 to 25.

[0167] For example,

[0168] For example,

[0169] wherein l, m, n are 0;

[0170] or

[0171] wherein l, m, n are 0

[0172] In some embodiments, when G in Formula I is galactose, then in Formula I the structure is:

[0173] In some embodiments, when G in Formula I is galactose, then in Formula I the structure is selected from the group consisting of:

[0174] wherein R is aryl, the term "aryl" refers to a monocyclic or bicyclic aromatic hydrocarbon group having 6 to 10 carbon atoms in the ring portion. Representative examples of aryl groups are phenyl or naphthyl.

[0175] In some embodiments, when G in Formula I is galactose, then in Formula I the structure is selected from the group consisting of:

[0176] wherein n is an integer from 0 to 30, for example, n is an integer from 0 to 25, preferably n is 1.

[0177] In some embodiments, when G in formula I is galactose, G in formula I is the structure is selected from the group consisting of:

[0178] In some embodiments, when G in formula I is mannose, G is attached to the 4 position of the 1,2-cyclo ring N-acetyl-D-glucosamine via a 1,4 glycosidic bond, and the substituent Z-Y-X- at the 6 position of the G ring is as defined above, except that G is replaced with mannose.

[0179] In some embodiments, when G in formula I is mannose, G in formula I is the structure is selected from the group consisting of:

[0180] wherein, I, m, n are independently an integer from 0 to 30, m1, m2 represent an integer from 1 to 12;

[0181] n is an integer from 0 to 30, for example, n is an integer from 0 to 10.

[0182] Preferably, wherein when G in formula I is mannose, G in formula I is the structure is selected from the group consisting of:

[0183] Preferably, when G in formula I is mannose, G in formula I is the structure is selected from the group consisting of:

[0184] wherein, m is an integer from 0 to 30, for example, m is an integer from 0 to 25;

[0185] 1 is an integer from 0 to 30, for example, I is an integer from 0 to 25;

[0186] n is an integer from 0 to 30, for example, n is an integer from 0 to 25;

[0187] for example,

[0188] In some embodiments, when G in formula I is glucose, G is attached to the 4 position of the 1,2-cyclo ring N-acetyl-D-glucosamine via a 1,4 glycosidic bond, and the substituent Z-Y-X- at the 6 position of the G ring is as defined in G is galactose, except that G is replaced with glucose.

[0189] In some embodiments, when G in formula I is glucose, G in formula I is the structure is selected from the group consisting of:

[0190] wherein, I, m, n are independently an integer from 0 to 30, m1, m2 represent independently an integer from 1 to 12;

[0191] n is an integer from 0 to 30, for example, n is an integer from 0 to 10;

[0192] Preferably, wherein when G in formula I is glucose, then the structure is selected from:

[0193] Preferably, wherein when G in formula I is glucose, then the structure is selected from:

[0194] wherein, m is an integer from 0 to 30, for example, m is an integer from 0 to 25;

[0195] I is an integer from 0 to 30, for example, I is an integer from 0 to 25;

[0196] n is an integer from 0 to 30, for example, n is an integer from 0 to 25;

[0197] for example,

[0198] Step (I.2): purification of the reaction solution A obtained in step (I.1)

[0199] In some embodiments, the purification is performed using antibody purification processes known in the art, including affinity chromatography (e.g., protein A affinity chromatography, protein G affinity chromatography, or protein L affinity chromatography) and other separation and purification steps, for example, anion chromatography, cation chromatography, nanofiltration, ultrafiltration / diafiltration, etc., with the steps and order adjusted according to known in the art, such as changing the order of steps, omitting a certain step.

[0200] In some embodiments, in the above step (I.1), the harvest solution after subjecting the cell supernatant to affinity chromatography (e.g., protein A affinity chromatography, protein G affinity chromatography, or protein L affinity chromatography) is used for site-specific glycoengineering of the Fc region protein molecule having heterogeneous sugar chains, and then in step (I.2), the reaction solution obtained in step (I.1) is subjected to other separation and purification steps, for example, anion chromatography, cation chromatography, nanofiltration, ultrafiltration / diafiltration, etc., with the steps and order adjusted according to known in the art, such as changing the order of steps, omitting a certain step.

[0201] Since the method of the present application enables site-specific glycoengineering of the protein molecule having Fc region with heterogeneous sugar chains in the above step (I.1) using cell supernatant, site-specific glycoengineering of the protein molecule having Fc region with heterogeneous sugar chains in the above step (I.1) using the harvest obtained from any step before the end of the purification step is also contemplated.

[0202] Step (II): Preparation of the drug conjugate of the protein molecule having Fc region with site-specifically engineered homogeneous sugar chains

[0203] The protein molecule having Fc region with site-specifically engineered homogeneous sugar chains prepared in step (I.2) (e.g., an antibody comprising site-specifically engineered homogeneous sugar chains) is added as an intermediate I to a buffer (e.g., His-HCl buffer, phosphate buffer, Tris buffer) (pH 5.5-7.5, e.g., about 6.5), an organic solvent and a small molecule drug are added to obtain a reaction solution B, and purification is performed.

[0204] Step (II) can be performed using conventional methods known in the art for conjugating an antibody to a small molecule drug.

[0205] In some embodiments, the small molecule drug is a small molecule drug-linker of the following Formula IV:

[0206] E-L-D (Formula IV)

[0207] E is a corresponding group that orthogonally reacts with an azido group, which is selected from a linear alkyne group, a DBCO-like structure, a BCN-like structure,

[0208] L represents a linker,

[0209] L is preferably selected from -(CH2)a-(OCH2 CH2)b-(NHCO)n-(CH2)c-, or from the following groups:

[0210] wherein V and W are bifunctional linkers comprising a structure of bifunctional linker with lysine, propargyl glycine, for example, L is selected from:

[0211] wherein a, b, c, d and e are each independently selected from an integer between 0-30, m, n are 0 or 1, R3and R4are each independently selected from CH3-, (CH 3)2 CH-, PhCH2, NH2(CH2)4-, NH2 CONH(CH2)3-, R is selected from an azidizable monosaccharide, disaccharide, oligosaccharide or a PEG structure of different length of azido group or a combination of PEG and chain or cyclic monosaccharide, disaccharide, oligosaccharide, wherein the oligosaccharide includes branched oligosaccharide chains;

[0212] representing a linkage site;

[0213] For example, the L is -Lysm-PEGn-GGFG-; or Lysm-PEGn-VC-PAB-, m, n are independently selected from an integer between 0-30; the L is -PEG4-vc-PAB- or -PEG4-GGFG-;

[0214] D represents a cytotoxic or cytostatic drug,

[0215] For example, (1) microtubule inhibitors / disruptors: for example, but not limited to, auristatin class (e.g., MMAE or MMAF), maytansine derivatives (e.g., DM1, DM2, DM4), Tubulysins, Cryptomycins, anti-mitotic EG5 inhibitors (e.g., KSP inhibitors of the kinesin spindle protein);

[0216] (2) DNA damaging agents: for example, but not limited to, Pyrrolobenzodiazepines (e.g., pyrrolo[2,1-c][1,4]benzodiazepine (PBD)), Indolinobenzodiazepine; Duocarmycins; Calicheamicin class;

[0217] (3) topoisomerase inhibitors: for example, but not limited to, camptothecin class (e.g., exatecan and its derivatives Dxd);

[0218] (4) others: apoptosis inducers (Bcl-xL inhibitors), thailanstatin and its analogues, amatoxins, nicotinamide phosphoribosyltransferase (NAMPT) inhibitors, Kameycin.

[0219] In some embodiments, the small molecule drug is a small molecule drug-linker selected from the group consisting of:

[0220] In some embodiments, the small molecule drug is a small molecule drug-linker selected from the group consisting of:

[0221] wherein, m is an integer from 0-5; n is an integer from 0-30 (Note: "P" in the numbering P1-P30 is the abbreviation of "payload").

[0222] Optionally, the protein molecule conjugate drug with Fc region of homogenous glycochain of site-directed remodeling is purified, for example, by ultrafiltration / diafiltration (UF / DF).

[0223] In some embodiments, the protein molecule conjugate drug with Fc region of homogenous glycochain of site-directed remodeling is prepared using the following general operation A as a two-step production process of the present application.

[0224] General operation A: a method for preparing a protein molecule conjugate drug with Fc region of homogenous glycochain of site-directed remodeling, comprising:

[0225] Step (I.1): site-directed glyco-reconstruction of the protein molecule with Fc region of heterogenous glycochain in cell culture supernatant

[0226] The cell supernatant containing the protein molecule with Fc region of heterogenous glycochain (for example, the cell supernatant containing the antibody with N297-linked heterogenous glycochain) is added into a buffer (for example, His-HCl buffer, phosphate buffer) (pH 4.5-7.7, for example, about 6.5), a disaccharide linker (for example, the disaccharide linker shown in formula I, preferably diAz-LacNAc-ox shown in formula II or Az-LacNAc-ox shown in formula III) is added, an endoglycosidase (for example, endoglycosidase S2 (Endo-S2) shown in SEQ ID NO: 1, endoglycosidase S2M3 (Endo-S2M3) shown in SEQ ID NO: 2, endoglycosidase Endo-S2 mutant 1 shown in SEQ ID NO: 3) is added, and incubated for a period of time (for example, at a temperature of 6-37℃, incubated for 0.25 hours-22 hours) to obtain reaction solution A containing the protein molecule with Fc region of homogenous glycochain of site-directed remodeling (for example, the antibody containing the homogenous glycochain of site-directed remodeling).

[0227] Step (I.2): purification of reaction solution A obtained in step (I.1)

[0228] In some embodiments, the purification of reaction solution A obtained in step (I.1) is an operation called "purification X". Purification X includes affinity chromatography, low-pH incubation, anion chromatography, cation chromatography, nanofiltration, ultrafiltration / diafiltration, and bulk preparation of the protein molecule with Fc region of homogenous glycochain of site-directed remodeling (for example, the antibody containing the homogenous glycochain of site-directed remodeling).

[0229] The basic operation of affinity chromatography generally includes 5 steps, namely, equilibrating the chromatography column, sample loading, washing impurities, sample elution, and regeneration of the chromatography column. The column load is usually ≤50 g / L.

[0230] In some embodiments, the protein A affinity chromatography process of step (I.2) is as follows: the chromatography column is rinsed with water for 1-2 column volumes (CV), treated with 0.1 M sodium hydroxide solution for ≥40 min; equilibrated with affinity equilibration buffer until the effluent pH and conductivity are similar to those of the equilibration buffer; the sample containing the target antibody is loaded, with a loading capacity of ≤50 g / L, and after loading is complete, the column is rinsed with affinity equilibration buffer for 2-4 CV, and with affinity wash buffer for 2-4 CV; elution is performed with affinity elution buffer, and the eluate is collected, which is the affinity chromatography collection, and a sample is taken to detect the protein concentration, and the yield is calculated: yield = (amount of protein in the affinity chromatography collection) / (volume of cell culture supernatant x protein expression amount / ml) x 100%, and the collection is promptly subjected to low-pH incubation.

[0231] In some embodiments, the low-pH incubation process in step (I.2) is as follows: the affinity chromatography collection is diluted to less than 20 g / L, 1 M citric acid is used to adjust the pH to 3.6±0.1, and incubation is performed at room temperature for 60±10 min; after incubation is complete, 1 M Tris buffer is used to adjust the pH to be similar to that of the anion equilibration buffer, the conductivity is detected, the sample is diluted to <5 mS / cm using water for injection, and then filtered through a 0.22 μm filter, which is the low-pH incubation collection. The yield is calculated: yield = (total amount of protein in the low-pH incubation collection) / (total amount of protein in the affinity chromatography collection) x 100%.

[0232] In some embodiments, the anion chromatography process in step (I.2) is as follows: treated with 0.5 M sodium hydroxide solution for ≥40 min; equilibrated with equilibration buffer until the effluent pH is similar to that of the equilibration buffer; the low-pH collection is loaded, with a loading capacity of ≤40 g / L, and after loading is complete, the column is rinsed with equilibration buffer, and the flow-through is collected during loading and rinsing; the pH of the collection is adjusted to be similar to that of the cation equilibration buffer using 1 M citric acid buffer, the conductivity is adjusted to <5 mS / cm using cation equilibration buffer, and 0.22 μm filtration is performed, which is the anion chromatography collection, and a sample is taken to detect the protein concentration. The yield is calculated: yield = (total amount of protein in the anion chromatography collection) / (total amount of protein in the low-pH incubation collection) x 100%. After collection is complete, the column is treated with 1 M sodium chloride solution, 0.5 M sodium hydroxide solution, and water in sequence, and is stored after being rinsed with 0.01 M sodium hydroxide solution.

[0233] In some embodiments, the cation exchange process in step (I.2) is as follows: treat with 0.5M sodium hydroxide solution for ≥ 40 min; equilibrate to a pH close to the elution buffer with cation equilibration buffer; load the anion exchange pool, keeping the load ≤ 40 g / L; after loading, rinse with equilibration buffer; elute with cation elution buffer and collect samples; adjust the pH of the samples to a value close to the ultrafiltration / diafiltration buffer with 1M Tris buffer, 0.22 μm filter, and the cation exchange pool is obtained. Measure the protein concentration of the samples, and calculate the yield: yield = total protein amount of the cation exchange pool / total protein amount of the anion exchange pool x 100%. After elution, treat the column with 1M sodium chloride solution, 0.5M sodium hydroxide solution, and water in sequence; rinse the column with 0.01M sodium hydroxide solution and store.

[0234] In some embodiments, the nanofiltration process in step (I.2) is as follows: rinse the prefilter and nanofilter with cation elution buffer; filter the cation exchange pool, and then rinse the nanofilter with cation elution buffer, and collect the filtrate, which is the nanofiltration pool. Measure the protein concentration of the samples. Calculate the yield: yield = total protein amount of the nanofiltration pool / total protein amount of the cation exchange pool x 100%. After filtration, rinse the system with water, and treat the system with 0.5M sodium hydroxide solution for at least 30 min; rinse with water, and store with 20% ethanol.

[0235] In some embodiments, buffer exchange is performed. Buffer exchange is a process that changes or removes the solvent or buffer environment of a biological product, which includes two independent steps, namely ultrafiltration (UF) and diafiltration (DF), referred to as UF / DF. Ultrafiltration (UF) aims to achieve preliminary concentration by reducing or removing the solvent that is not needed, and then achieve the purpose of buffer exchange by adding new buffer, which is implemented by diafiltration (DF). The goal of UF / DF is to achieve a suitable target product concentration (such as antibodies, etc.) and buffer composition. In some embodiments, the UF / DF process in step (I.2) is as follows: treat the ultrafiltration system and membrane package with 0.5M sodium hydroxide solution for at least 40 min; rinse the system and membrane package with water and ultrafiltration buffer in sequence until the pH of the permeate is close to the pH of the ultrafiltration buffer. Use a flow rate of 3-6 L / min / m 2The tangential flow rate, transmembrane pressure (TMP) 0.2-0.8 bar, and the volume of the sample is 8-10 times the volume of the sample. After the washing, the sample is concentrated and collected. The system is washed with the ultrafiltration buffer, and the washing liquid is collected, which is the ultrafiltration collection liquid, and the yield is calculated: yield = total protein amount of the ultrafiltration collection liquid / total protein amount of the nanofiltration collection liquid x 100%. After collection, the system and the membrane package are washed with the ultrafiltration buffer, 0.5M sodium hydroxide solution, and water, and the membrane package is washed with 0.1M sodium hydroxide solution and stored.

[0236] The total yield of the purification stage is calculated: yield = total protein amount of the sample / total protein amount of the cell culture supernatant x 100%.

[0237] Step (II.1): Preparation of protein molecule conjugated drugs with Fc region of homogenous glycan chains with site-directed remodeling

[0238] The protein molecule with Fc region of homogenous glycan chains with site-directed remodeling prepared in step (I.2) (e.g., an antibody comprising homogenous glycan chains with site-directed remodeling) is taken as intermediate I and added to a buffer (e.g., His-HCl buffer, phosphate buffer, Tris buffer) (pH 5.5-7.5, e.g., about 6.5), the final concentration of the antibody is adjusted (e.g., the final concentration of the antibody is not less than 3.0 mg / mL), an organic solvent (e.g., DMSO, DMA, or DMF) and a small molecule drug are added, and reaction liquid B is obtained. The organic solvent is not particularly limited as long as it can dissolve the small molecule drug used.

[0239] Step (II.2): Purification of protein molecule conjugated drugs with Fc region of homogenous glycan chains with site-directed remodeling

[0240] Reaction liquid B of step (II.1) is subjected to ultrafiltration / diafiltration (UF / DF), and the preparation of the protein molecule conjugated drug original liquid with Fc region of homogenous glycan chains with site-directed remodeling is carried out, for example, using a 30 kDa ultrafiltration tube or membrane package ultrafiltration, and using 5-20 times the volume of ultrafiltration replacement liquid.

[0241] The yield is calculated: yield = total protein amount of the ultrafiltration collection liquid / total protein amount of the sample x 100%.

[0242] The total yield of the purification stage is calculated: yield = total protein amount of the sample / total protein amount of the cell culture supernatant x 100%.

[0243] In some embodiments, the experimental conditions for site-directed glycan remodeling in step (I.1) are as follows:

[0244] Table 1 Antibody site-directed glycan remodeling reaction conditions In some embodiments, the experimental conditions for site-directed glycan remodeling in step (I.1) are as follows:

[0244] Table 1 Antibody site-directed glycan remodeling reaction conditions In some embodiments, the experimental conditions for site-directed glycan remodeling in step (I.1) are as follows:

[0244] Table 1 Antibody site-directed glycan remodeling reaction conditions In some embodiments, the experimental conditions for site-directed glycan remodeling in step (I.1) are as follows:

[0244] Table 1 Antibody site-directed glycan remodeling reaction conditions In some embodiments, the experimental conditions for site-directed glycan remodeling in step (I.1) are as follows:

[0244] Table 1 Antibody site-directed glycan remodeling reaction conditions In some embodiments, the experimental conditions for site-directed glycan remodeling in step (I.1) are as follows:

[0244] Table 1 Antibody site-directed glycan remodeling reaction conditions In some embodiments, the experimental conditions for site-directed glycan remodeling in step (I.1) are as follows:

[0244] Table 1 Antibody site-directed glycan remodeling reaction conditions In some embodiments, the experimental conditions for site-directed glycan remodeling in step (I.1) are as follows:

[0244] Table 1 Antibody site-directed glycan remodeling reaction conditions In some embodiments, the experimental conditions for site-directed glycan remodeling in step (I.1) are as follows:

[0244] Table 1 Antibody site-directed glycan remodeling reaction conditions In some embodiments, the experimental conditions for site-directed glycan remodeling in step (I.1) are as follows:

[0244] Table 1 Antibody site-directed glycan remodeling reaction conditions In some embodiments, the experimental conditions for site-directed glycan remodeling in step (I.1) are as follows:

[0244] Table 1 Antibody site-directed glycan remodeling reaction conditions In some embodiments, the experimental conditions for site-directed glycan remodeling in step (I.1) are as follows:

[0244] Table 1 Antibody site-directed glycan remodeling reaction conditions In some embodiments, the experimental conditions for site-directed glycan remodeling in step (I.1) are as follows:

[0244] Table 1 Antibody site-directed glycan remodeling reaction conditions In some embodiments, the experimental conditions for site-directed glycan remodeling in step (I.1) are as follows:

[0244] Table 1 Antibody site-directed glycan remodeling reaction conditions In some embodiments, the experimental conditions for site-directed glycan remodeling in step (I.1) are as follows:

[0244] Table 1 Antibody site-directed glycan remodeling reaction conditions In some embodiments, the experimental conditions for site-directed glycan remodeling in step (I.1) are as follows:

[0244] Table 1 Antibody site-directed glycan remodeling reaction conditions In some embodiments, the experimental conditions for site-directed glycan remodeling in step (I.1) are as follows:

[0244] Table 1 Antibody site-directed glycan remodeling reaction conditions In some embodiments, the experimental conditions for site-directed glycan remodeling in step (I.1) are as follows:

[0244] Table 1 Antibody site-directed glycan remodeling reaction conditions In some embodiments, the experimental conditions for site-directed glycan remodeling in step (I.1) are as follows:

[0244] Table 1 Antibody site-directed glycan remodeling reaction conditions In some embodiments, the experimental conditions for site-directed glycan remodeling in step (I.1) are as follows:

[0244] Table 1 Antibody site-directed glycan remodeling reaction conditions In some embodiments, the experimental conditions for site-directed glycan remodeling in step (I.1) are as follows:

[0244] Table 1 Antibody site-directed glycan remodeling reaction conditions In some embodiments, the experimental conditions for site-directed glycan remodeling in step (I.1) are as follows:

[0244] Table 1 Antibody site-directed glycan remodeling reaction conditions In some embodiments, the experimental conditions for site-directed glycan remodeling in step (I.1) are as follows:

[0244] Table 1 Antibody site-directed glycan remodeling reaction conditions In some embodiments, the experimental conditions for site-directed glycan remodeling in step (I.1) are as follows:

[0244] Table 1 Antibody site-directed glycan remodeling reaction conditions In some embodiments, the experimental conditions for site-directed glycan remodeling in step (I.1) are as follows:

[0245] In some embodiments, the experimental conditions for the site-directed glycoremodification in step (I.1) are:

[0246] Table 2 Site-directed glycoremodification reaction conditions for antibodies

[0247] In some embodiments, the experimental conditions for the site-directed glycoremodification in step (I.1) are:

[0248] In some embodiments, the experimental conditions for the site-directed glycoremodification in step (I.1) are:

[0249] In some embodiments, the experimental conditions for the site-directed glycoremodification in step (I.1) are:

[0250] In some embodiments, the experimental conditions for the site-directed glycoremodification in step (I.1) are:

[0251] In some embodiments, the experimental conditions for the site-directed glycoremodification in step (I.1) are:

[0252] Step (II.1): Preparation of protein molecules with site-directed glycoremodified Fc region conjugated drugs

[0253] Take the protein molecules with site-directed glycoremodified Fc region prepared in step (I.2) (for example, an antibody containing site-directed glycoremodified homogeneous sugar chains) stock solution as intermediate I, add it to a buffer (for example, His-HCl buffer, phosphate buffer, Tris buffer) (pH 5.5-7.5, for example, about 6.5), adjust the final concentration of the antibody reaction (for example, the final concentration of the antibody reaction is not less than 3.0 mg / mL), add an organic solvent (for example, 0-30% DMSO, DMA or DMF) and an appropriate amount of small molecule drug (for example, sufficient amount, or small molecule / antibody equivalent ≥2, for example, DBCO-PEG4-vc-PAB-MMAE, or DBCO-PEG4-GGFG-DXd), incubate (for example, temperature at 4-37°C, preferably 15-30h, 20-30h, 22-25h, time ≥0.5h, preferably 1-16h, 2-8h), to obtain reaction liquid B.

[0254] Step (II.2): Purification of protein molecules with site-directed glycoremodified Fc region conjugated drugs

[0255] The unconjugated small molecule drug was removed by 30 kDa ultrafiltration tube or membrane, and the DAR value of the sample was analyzed and identified by RP-HPLC or HIC or LC-MS.

[0256] In some embodiments, the experimental conditions for carrying out the conjugation in step (II.1) are as follows:

[0257] Table 3 Small molecule drug conjugation reaction conditions

[0258] In some embodiments, antibody intermediate I is added into PBS buffer (pH 6.8), followed by adding 8% (v / v) DMSO and 12 equivalents of small molecule (DBCO-PEG4-vc-PAB-MMAE) in sequence, and incubating at 22°C for 2 hours.

[0259] Experimental results: DAR value detected by RP-HPLC: 3.68.

[0260] In some embodiments, antibody intermediate I is added into PBS buffer (pH 6.5), followed by adding 5% (v / v) DMSO and 10 equivalents of small molecule (DBCO-PEG4-vc-PAB-MMAE) in sequence, and incubating at 25°C for 3 hours.

[0261] Experimental results: DAR value detected by RP-HPLC: 3.74.

[0262] In some embodiments, antibody intermediate I is added into His-HCl buffer (pH 6.5), followed by adding 12% (v / v) DMSO and 10 equivalents of small molecule (DBCO-PEG4-vc-PAB-MMAE) in sequence, and incubating at 28°C for 2 hours. The DAR value of the obtained ADC detected by RP-HPLC is 3.69.

[0263] In some embodiments, antibody intermediate I is added into Tris-HCl buffer (pH 6.5), followed by adding 5% (v / v) DMSO and 10 equivalents of small molecule (DBCO-PEG4-vc-PAB-MMAE) in sequence, and incubating at 25°C for 2 hours. The DAR value of the obtained ADC detected by RP-HPLC is 3.62.

[0264] In some embodiments, antibody intermediate I is added into His-HCl buffer (pH 6.5), followed by adding 10% (v / v) DMSO and 10 equivalents of small molecule (DBCO-PEG4-vc-PAB-MMAE) in sequence, and incubating at 25°C for 2 hours. The DAR value of the obtained ADC detected by RP-HPLC is 3.74.

[0265] The two-step production process in the prior art as a control, referred to herein as "General Operation B", is implemented as follows.

[0266] General Operation B: (Production process in the prior art) A method for preparing a protein molecule conjugate drug with Fc region of homogeneous glycan chain with site-directed reconstitution, comprising:

[0267] Step (I.1): Purification of protein molecules with Fc region of heterogeneous glycan chain in cell culture supernatant

[0268] The cell supernatant containing protein molecules with Fc region of heterogeneous glycan chain (e.g., cell supernatant containing antibodies with N297-linked heterogeneous glycan chain) is subjected to purification Y to obtain purified protein molecules with Fc region of heterogeneous glycan chain (e.g., purified antibodies with N297-linked heterogeneous glycan chain);

[0269] Purification Y includes the following operations: affinity chromatography, low-pH incubation, anion chromatography, cation chromatography, nanofiltration, ultrafiltration / diafiltration, and preparation of purified protein molecules with Fc region of heterogeneous glycan chain (e.g., purified antibodies with N297-linked heterogeneous glycan chain) stock solution.

[0270] In some embodiments, the affinity chromatography process of step (I.1) is as follows: the chromatography column is rinsed with water for 1-2 column volumes (CV), treated with 0.1M sodium hydroxide solution for ≥40 min; equilibrated with affinity equilibration buffer until the effluent pH and conductivity are similar to those of the equilibration buffer; the sample containing the target antibody is loaded, with a loading capacity ≤50 g / L; after loading is complete, the column is rinsed with 2-4 CV of affinity equilibration buffer, and then with 2-4 CV of affinity wash buffer; elution is performed with affinity elution buffer, and the eluate is collected as the affinity chromatography collection, which is sampled to detect protein concentration and calculate yield: yield = protein amount in affinity chromatography collection / (cell culture supernatant volume x protein expression amount / ml) x 100%, and the collection is promptly subjected to low-pH incubation.

[0271] In some embodiments, the low-pH incubation process in step (I.1) is as follows: the affinity chromatography collection is diluted with ultrapure water to less than 20 g / L, and after dilution, 1M citric acid buffer is added to adjust the pH to 3.6±0.1, with gentle stirring during the process, and incubation at room temperature (18-26°C) for 60±10 min; after incubation is complete, the pH is adjusted to be similar to that of the anion equilibration buffer with 1M Tris buffer, the conductivity is detected, the sample is diluted with water for injection to <5 mS / cm, and then filtered through a 0.22 μm filter to obtain the low-pH incubation collection. Calculate the yield: yield = total protein amount in low-pH incubation collection / total protein amount in affinity chromatography collection x 100%.

[0272] In some embodiments, the anion chromatography process in step (I.1) is as follows: treat with 0.5M sodium hydroxide solution for > 40 min; equilibrate with the buffer to a pH of the effluent close to that of the equilibration buffer; take the low pH incubation collection for loading, with a loading capacity of < 40 g / L; after loading, continue to rinse the column with the equilibration buffer; collect the flow-through during loading and rinsing; adjust the pH of the collection to a value close to that of the cationic equilibration buffer with 1M citric acid buffer; adjust the conductivity to < 5 mS / cm with the cationic equilibration buffer; 0.22 μm filtration; the anion chromatography collection is obtained; take samples to detect the protein concentration. Calculate the yield: yield = total amount of protein in the anion chromatography collection / total amount of protein in the low pH incubation collection x 100%. After the collection, treat the column with 1M sodium chloride solution, 0.5M sodium hydroxide solution, and water in sequence; rinse the column with 0.01M sodium hydroxide solution and then store.

[0273] In some embodiments, the cation chromatography process in step (I.1) is as follows: treat with 0.5M sodium hydroxide solution for > 40 min; equilibrate with the buffer to a pH of the effluent close to that of the equilibration buffer; take the anion chromatography collection for loading, with a loading capacity of < 40 g / L; after loading, rinse with the equilibration buffer; elute with the cation elution buffer and collect samples; adjust the pH of the samples to a value close to that of the ultrafiltration / diafiltration buffer with 1M Tris buffer; 0.22 μm filtration; the cation chromatography collection is obtained. Take samples to detect the protein concentration, and calculate the yield: yield = total amount of protein in the cation chromatography collection / total amount of protein in the anion chromatography collection x 100%. After elution, treat the column with 1M sodium chloride solution, 0.5M sodium hydroxide solution, and water in sequence; rinse the column with 0.01M sodium hydroxide solution and then store.

[0274] In some embodiments, the nanofiltration process in step (I.1) is as follows: rinse the pre-filter and nanofilter with the cation elution buffer; filter the cation chromatography collection, and then rinse the nanofilter with the cation elution buffer; collect the filtrate, which is the nanofiltration collection. Take samples to detect the protein concentration. Calculate the yield: yield = total amount of protein in the nanofiltration collection / total amount of protein in the cation chromatography collection x 100%. After filtration, rinse with water, treat the system with 0.5M sodium hydroxide solution for at least 30 min; rinse with water and then store with 20% ethanol.

[0275] In some embodiments, the UF / DF process in step (I.1) is as follows: ultrafiltration through a 30 kDa ultrafiltration tube or membrane package. Treat the ultrafiltration system and membrane package with 0.5M sodium hydroxide solution for at least 40 min; rinse the system and membrane package with water for injection and ultrafiltration buffer in sequence until the pH of the permeate is close to that of the ultrafiltration buffer. Ultrafiltrate at 3-6 L / min / m 2The tangential flow velocity is 0.2-0.8 bar, the transmembrane pressure (TMP) is 0.2-0.8 bar, the sample is washed with the ultrafiltration buffer at a volume of 8-10 times the sample volume, and the sample is concentrated and collected after the washing is completed. The system is rinsed with the ultrafiltration buffer, and the rinse is collected as the ultrafiltration collection.

[0276] The total yield of the purification stage is calculated as follows: yield = total amount of protein in the original solution / total amount of protein in the cell culture supernatant x 100%.

[0277] Step (I.2): Site-directed glyco-reconstitution of the purified protein molecule with Fc region of heterogeneous glycan (e.g., purified antibody with N297-linked heterogeneous glycan)

[0278] The purified protein molecule with Fc region of heterogeneous glycan (e.g., purified antibody with N297-linked heterogeneous glycan) obtained in step (I.1) is taken as Intermediate I, added to His-HCl buffer (pH 6.5), added with a disaccharide linker (e.g., a disaccharide linker represented by Formula I, preferably diAz-LacNAc-ox represented by Formula II or Az-LacNAc-ox represented by Formula III), endoglycosidase (e.g., endoglycosidase S2M3), and incubated at 25°C for a period of time to obtain reaction solution A of the protein molecule with Fc region of site-reconstituted homogeneous glycan (e.g., antibody with site-reconstituted homogeneous glycan).

[0279] In some embodiments, the experimental conditions for site-directed glyco-reconstitution in step (I.2) are the same as those for site-directed glyco-reconstitution in General Operation A described above.

[0280] Step (I.3): Purification Z of reaction solution A obtained in step (I.2)

[0281] Purification Z includes the following operations: affinity chromatography, anion / cation chromatography (e.g., cation chromatography), ultrafiltration / diafiltration. The specific operations are the same as those in affinity chromatography, anion / cation chromatography (e.g., cation chromatography), ultrafiltration / diafiltration in step (I.1).

[0282] The reaction solution A obtained in step (I.2) is subjected to the purification Z to obtain the purified protein molecule with Fc region of site-reconstituted homogeneous glycan (e.g., antibody with site-reconstituted homogeneous glycan) as Intermediate II.

[0283] Step (II.1): Preparation of a protein molecule with Fc region of site-reconstituted homogeneous glycan conjugated with a drug

[0284] The protein molecule (e.g., an antibody) having an Fc region with homogenous glycoforms of site-directed remodeling prepared in step (I.3) is added to His-HCl buffer (pH 6.5) as intermediate II, and the final concentration of the antibody is controlled at 5.0 mg / mL, 10% (v / v) DMSO and 10 equivalents of a small molecule drug are added to obtain reaction solution B.

[0285] Step (II.2): Purification of the protein molecule having an Fc region with homogenous glycoforms of site-directed remodeling conjugated with a drug

[0286] The reaction solution B of step (II.1) is subjected to ultrafiltration / diafiltration (UF / DF), and the purified product is subjected to preparation of a stock solution of the protein molecule having an Fc region with homogenous glycoforms of site-directed remodeling conjugated with a drug.

[0287] In some embodiments, the UF / DF process in step (II.2) is as follows: the ultrafiltration system and membrane package are treated with 0.5 M sodium hydroxide solution for at least 40 min; the system and membrane package are sequentially rinsed with water for injection, ultrafiltration buffer until the pH of the permeate is close to that of the ultrafiltration buffer. The ultrafiltration buffer is used for washing at a tangential flow rate of 3-6 L / min / m 2 The system is rinsed with the ultrafiltration buffer, and the rinse is collected as the ultrafiltration collection solution, and the yield is calculated: yield = total protein amount of the ultrafiltration collection solution / total protein amount of the sample x 100%. After collection, the system and membrane package are sequentially rinsed with the ultrafiltration buffer, 0.5 M sodium hydroxide solution, water, and the membrane package is rinsed with 0.1 M sodium hydroxide solution and stored.

[0288] The total yield of the purification stage is calculated: yield = total protein amount of the stock solution of the protein molecule having an Fc region with homogenous glycoforms of site-directed remodeling conjugated with a drug / total protein amount of the cell culture supernatant x 100%.

[0289] III. One-step method for preparing a protein molecule having an Fc region with homogenous glycoforms of site-directed remodeling conjugated with a drug

[0290] The present application also provides a one-step method for preparing a protein molecule having an Fc region with homogenous glycoforms of site-directed remodeling conjugated with a drug.

[0291] In some embodiments, the one-step preparation method of the present application comprises: adding a disaccharide-small molecule drug conjugate (e.g., a disaccharide-small molecule drug conjugate represented by Formula V) into a cell culture supernatant, adding a glycoside endonuclease having a specific hydrolysis of heterogeneous sugar chains in a glycoprotein and having a catalytic transglycosylation activity (e.g., a glycoside endonuclease S2 (Endo-S2) represented by SEQ ID NO: 1 or an amino acid sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity thereto, a glycoside endonuclease S2M3 (Endo-S2M3) represented by SEQ ID NO: 2 or an amino acid sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity thereto, a glycoside endonuclease Endo-S2 mutant 1 represented by SEQ ID NO: 3 or an amino acid sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity thereto) (herein, the order of addition of the cell supernatant, the disaccharide-small molecule drug conjugate, and the glycoside endonuclease is not limited), incubating for a period of time (at a temperature of 15-37°C), to obtain a reaction solution A of a protein molecule conjugate drug comprising an Fc region having a site-directed reconfiguration of homogeneous sugar chains, and then purifying the reaction solution A.

[0292] In some embodiments, in the one-step preparation method of the present application, the cell culture supernatant is replaced with a harvest solution obtained from the cell culture supernatant after affinity chromatography (e.g., protein A affinity chromatography, protein G affinity chromatography, or protein L affinity chromatography). Specifically, the harvest solution obtained from the cell culture supernatant after affinity chromatography (e.g., protein A affinity chromatography, protein G affinity chromatography, or protein L affinity chromatography) is added with a disaccharide-small molecule drug conjugate (e.g., a disaccharide-small molecule drug conjugate represented by Formula V), added with a glycoside hydrolase having the activity of specifically hydrolyzing heterogeneous sugar chains in a glycoprotein (e.g., Endo-S2 represented by SEQ ID NO: 1 or an amino acid sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity thereto, Endo-S2M3 represented by SEQ ID NO: 2 or an amino acid sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity thereto, Endo-S2 mutant 1 represented by SEQ ID NO: 3 or an amino acid sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity thereto) (herein, the order of addition of the cell culture supernatant, the disaccharide-small molecule drug conjugate, and the glycoside hydrolase is not limited), incubated for a certain period of time (at a temperature of 15 to 37°C), to obtain a reaction solution of a protein molecule-drug conjugate having an Fc region with a homogenous sugar chain reconstituted at a specific site, and then the reaction solution is optionally purified, for example, by ultrafiltration / diafiltration (UF / DF).

[0293] Since the protein molecule-drug conjugate having an Fc region with a homogenous sugar chain reconstituted at a specific site can be obtained using the cell culture supernatant in the one-step preparation method of the present application, it is also contemplated that the one-step preparation method of the present application is performed using a harvest solution obtained from any step before the step of purifying the cell culture supernatant, instead of the cell culture supernatant, to prepare the protein molecule-drug conjugate having an Fc region with a homogenous sugar chain reconstituted at a specific site.

[0294] In Formula V, G ring, X, Y, L, and D are as defined in Formula I and Formula IV,

[0295] Z' is independently -(CH2)p- where p is an integer from 1 to 5, or Z' is absent, or is a group that can react with the Z group in claim 1,

[0296] For example, Z' is selected from the following groups:

[0297] wherein R1and R2are each independently selected from H, -CH3, -CH2CH3, cyclopropyl, or cyclobutyl; represents a point of attachment.

[0298] In some embodiments, the disaccharide-small molecule drug conjugate is selected from

[0299] (Note: "SP" in the numbers SP1-SP10 is an abbreviation of "substrate-payload")

[0300] In some specific embodiments, the protein molecule conjugate drug with Fc region of homogenous glycochain of site-directed remodeling is prepared using the following general operation C as a one-step production process of the present application, comprising the following steps:

[0301] General operation C: (Production process of the present application) A method for preparing a protein molecule conjugate drug with Fc region of homogenous glycochain of site-directed remodeling, comprising:

[0302] Step (I.1): Preparation of a protein molecule conjugate drug with Fc region of homogenous glycochain of site-directed remodeling from a protein molecule with Fc region of heterogeneous glycochain in cell culture supernatant

[0303] The cell supernatant containing the protein molecule with Fc region of heterogeneous glycochain (for example, the cell supernatant containing the antibody with N297-linked heterogeneous glycochain) is added to a buffer (for example, His-HCl buffer, phosphate buffer) (for example, pH 4-8, preferably pH 4.5-7.7), a disaccharide-small molecule drug conjugate (for example, the disaccharide-small molecule drug conjugate represented by formula V), an endoglycosidase (for example, endoglycosidase S2 (Endo-S2) represented by SEQ ID NO: 1, endoglycosidase S2M3 (Endo-S2M3) represented by SEQ ID NO: 2, endoglycosidase Endo-S2 mutant 1 represented by SEQ ID NO: 3) is added, and incubated for a period of time (incubated for 0.25 hours-22 hours at a temperature of 6-37°C) to obtain reaction solution A containing the protein molecule conjugate drug with Fc region of homogenous glycochain of site-directed remodeling (for example, the antibody conjugate drug with Fc region of homogenous glycochain of site-directed remodeling).

[0304] Step (I.2): Purification X of reaction solution A obtained in step (I.1)

[0305] Purification X is carried out in the same way as purification X in the two-step general operation A, except that reaction solution A is different.

[0306] The following one-step production process in the prior art, referred to herein as "General Operation D", is implemented as a control.

[0307] General Operation D: (Production process in the prior art) A method for preparing a protein molecule conjugate drug with an Fc region of homogenous glycan chains with site-directed remodeling, comprising:

[0308] Step (I.1): Purification of protein molecules with Fc regions of heterogeneous glycan chains in cell culture supernatant

[0309] The same as step (I.1) in the two-step general operation B.

[0310] Step (I.2): Preparation of protein molecule conjugate drugs with Fc regions of homogenous glycan chains with site-directed remodeling from purified protein molecules with Fc regions of heterogeneous glycan chains

[0311] Take the purified protein molecules with Fc regions of heterogeneous glycan chains (e.g., purified antibodies with N297-linked heterogeneous glycan chains) of step (I.1) as intermediate I, add a disaccharide-small molecule drug conjugate (e.g., a disaccharide-small molecule drug conjugate represented by formula V), an endoglycosidase (e.g., endoglycosidase S2M3) to a His-HCl buffer (pH 6.5), incubate at 25°C for a period of time, to obtain reaction solution A containing protein molecule conjugate drugs with Fc regions of homogenous glycan chains with site-directed remodeling (e.g., antibody conjugate drugs containing antibodies with site-directed remodeling of homogenous glycan chains).

[0312] Step (I.3): Purification Z on reaction solution A obtained in step (I.2)

[0313] Purification Z is implemented the same as purification Z in the two-step general operation B, except that reaction solution A is different.

[0314] Examples

[0315] The technical solutions in the embodiments of the present application will be described below in a clear and complete manner. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present application. Unless otherwise specified, various reaction reagents involved in the embodiments can be purchased through commercial channels. Unless otherwise specified, the small molecule compounds described herein are obtained from the patent CN115949236A or WO2022226420A9, or commercially available (e.g., purchased from Shanghai Haoyuan Pharmaceutical Co., Ltd.).

[0316] Example 1 Preparation of antibody conjugate drug with homogeneous glycan chains of site-directed remodeling by two-step method

[0317] In this example, an antibody conjugate drug (ADC) with homogeneous glycan chains of site-directed remodeling was prepared by two-step method using the production process of the present application (right small graph of FIG. 1 and right small graph of FIG. 2).

[0318] Specifically, the VEGF antibody bevacizumab was prepared as described in CN 1191276C, CN 100480629C, and the VEGF antibody containing N297-linked heterogeneous glycan chains was obtained in the cell culture supernatant.

[0319] The cell culture supernatant containing the VEGF antibody bevacizumab was prepared as follows. The CHO cells producing bevacizumab were taken, and a shake flask expansion culture was performed using a basic medium (commercially available, OPM-CHO CD07 DPM, Opmic, product number M120067), and the cells were passaged step by step to 450 mL and maintained at a cell viability of 90% or more, and then transferred to a 3L reactor for fed-batch culture, during which the cell nutritional requirements were maintained by supplementing a feed medium (commercially available, CDF1-SDY DPM, PFF2-SDY DPM, Opmic, product numbers M120001, M120007), glucose solution, etc., and after the culture was completed, the cell culture liquid was collected, centrifuged at 9500 rpm for 30 min, and 0.22 μm microporous filter membrane was used to collect the supernatant containing the antibody;

[0320] The VEGF antibody conjugate drug with homogeneous glycan chains of site-directed remodeling was prepared using the following steps.

[0321] Step (I.1): Site-directed glycan remodeling of VEGF antibody with heterogeneous glycan chains in cell culture supernatant

[0322] The cell supernatant containing 500 mg of VEGF antibody with N297-linked heterogeneous glycan chains was added to His-HCl buffer (pH 6.5), and 80 equivalents of diAz-LacNAc-ox represented by formula II, 10 mg of endoglycosidase S2M3 (i.e., 20 μg of enzyme was added per mg of antibody), were added, and incubated at 25°C for 1 h to obtain reaction liquid A containing VEGF antibody with homogeneous glycan chains of site-directed remodeling.

[0323] Step (I.2): The reaction liquid A obtained in step (I.1) was purified according to the purification X described in the general operation A of the two-step method to obtain intermediate I.

[0324] Step (II.1): Preparation of VEGF antibody conjugate drug with homogeneous glycan chains of site-directed remodeling

[0325] The intermediate prepared in step (I.2) was added to His-HCl buffer (pH 6.5) to give a final concentration of 5.0 mg / mL of VEGF antibody, 10% (v / v) DMSO and 10 equivalents of DBCO-PEG4-vc-PAB-MMAE were added, and the mixture was incubated at 25°C for 2 hours to obtain reaction solution B.

[0326] Step (II.2) for DBCO-PEG4-vc-PAB-MMAE: purification of VEGF antibody-MMAE conjugate with homogenous glycoforms with site-directed remodeling

[0327] Reaction solution B from step (II.1) was subjected to ultrafiltration / diafiltration (UF / DF) according to the procedure described in step (II.2) of General Procedure A for two-step method to obtain the purified product, thereby preparing a stock solution of VEGF antibody-MMAE conjugate with homogenous glycoforms with site-directed remodeling.

[0328] As a comparison, a VEGF antibody conjugated drug with homogenous glycoforms with site-directed remodeling was prepared by two-step method using the production process in the prior art (left panel of FIG. 1 and left panel of FIG. 2).

[0329] Specifically, the VEGF antibody bevacizumab was produced using CHO cells, and the cell culture supernatant obtained contained VEGF antibody with heterogeneous glycoforms linked to N297.

[0330] The VEGF antibody conjugated drug with homogenous glycoforms with site-directed remodeling was prepared using the following steps.

[0331] Step (I.1): purification of VEGF antibody with heterogeneous glycoforms in cell culture supernatant

[0332] The cell supernatant containing 500 mg of VEGF antibody with heterogeneous glycoforms linked to N297 was purified according to the procedure described in step (I.1) of General Procedure B for two-step method to obtain purified VEGF antibody with heterogeneous glycoforms linked to N297;

[0333] Step (I.2): site-directed glyco remodeling of the purified antibody with heterogeneous glycoforms linked to N297

[0334] The purified VEGF antibody with heterogeneous glycoforms linked to N297 from step (I.1) was added to His-HCl buffer (pH 6.5), diAz-LacNAc-ox represented by Formula II, endoglycosidase S2M3 were added, and the mixture was incubated at 25°C for a period of time to obtain reaction solution A of VEGF antibody with homogenous glycoforms with site-directed remodeling;

[0335] Step (I.3): Purification Z of reaction liquid A obtained in step (I.2)

[0336] The reaction liquid A obtained in step (I.2) was purified according to the purification Z described in step (I.3) of the general operation B of the two-step method to obtain a purified VEGF antibody stock solution with site-specifically remodeled homogeneous sugar chains;

[0337] Step (II.1): Preparation of VEGF antibody-MMAE conjugate with site-specifically remodeled homogeneous sugar chains

[0338] The VEGF antibody stock solution with site-specifically remodeled homogeneous sugar chains prepared in step (I.3) was added to His-HCl buffer (pH 6.5) to give a final concentration of 5.0 mg / mL VEGF antibody, 10% (v / v) DMSO and 10 equivalents of DBCO-PEG4-vc-PAB-MMAE were added to obtain reaction liquid B.

[0339] Step (II.2): Purification of VEGF antibody-MMAE conjugate with site-specifically remodeled homogeneous sugar chains

[0340] Reaction liquid B of step (II.1) was subjected to ultrafiltration / diafiltration (UF / DF) according to step (II.2) of the general operation B of the two-step method, and the purified product was obtained by ultrafiltration through a 30 kDa membrane package, thereby preparing a VEGF antibody-MMAE conjugate stock solution with site-specifically remodeled homogeneous sugar chains.

[0341] The ADCs prepared by the two-step method of the present application and the two-step method in the prior art were subjected to RP-HPLC and SEC-HPLC, and the results are shown in Figure 3 and Table 4: the ADCs prepared by the two-step method in the prior art and the two-step method of the present application both reached the target DAR value, and the conjugation results and purity were similar, as shown in Figure 3 and Table 4, the DAR values were 3.76 and 3.82, respectively, the conjugation efficiency was more than 90%, and the SEC purity was 98.10% and 98.15%, respectively.

[0342] Table 4: Comparison of experimental results of the two-step method of the present application and the two-step method in the prior art

[0343] Table 5: Data summary of the two-step method of the present application and the two-step method in the prior art

[0344] Note: NA means "not applicable"; UF / DF1 means first ultrafiltration / diafiltration, UF / DF2 means second ultrafiltration / diafiltration, UF / DF3 means third ultrafiltration / diafiltration; Total 1 is the result of process time, antibody amount and yield at the end of Step I; Total 2 is the result of process time, antibody amount and yield at the end of Step II; the calculation method of yield: the yield under the "Total 1" and "Total 2" entries is the total yield, which is the ratio of the antibody amount at the end of Step I, at the end of Step II, respectively, to the starting antibody amount 500 mg, and the yield under other entries is the relative yield of the next step / the previous step. L0 means light chain with 0 payload; H0 is heavy chain with 0 payload; H2 is heavy chain with 2 payloads; since the double azido substrate, diAz-LacNAc-ox, is used, 1 payload corresponds to a DAR value of 2; 2 payloads correspond to a DAR value of 4.

[0345] Results:

[0346] As shown in Table 5, at the end of Step I, when using the prior art method, the process time required to obtain purified antibody with site-specific remodeled homogeneous sugar chains was about 25.13 hours, the antibody amount was about 343.13 mg, and the yield was about 68.83%; when using the method of the present application, the process time required to obtain purified antibody with site-specific remodeled homogeneous sugar chains was about 16.47 hours, the antibody amount was about 382.12 mg, and the yield was about 76.42%. At the end of Step II, when using the prior art method, the process time required to obtain purified ADC was about 30.53 hours, the antibody amount was about 357.06 mg, and the yield was about 71.41%; when using the method of the present application, the process time required to obtain purified ADC was about 21.75 hours, the antibody amount was about 403.37 mg, and the yield was about 80.67%. Compared with the prior art, the two-step production process of the present application has obvious advantages in process, yield and process time (Figure 5, Table 4). First, compared with the two-step production process of the prior art, the two-step production process of the present application omits the step of purifying the cell culture supernatant before site-specific sugar remodeling of the antibody and omits the step of purifying the site-specific sugar remodeling product, and the process is simpler, and the omitted steps can reduce the amount of chromatographic filler, membrane package and the like used in the entire process, thereby reducing the cost to some extent; second, at a 500 mg antibody scale, the final yield of the two-step production process of the present application was 80.67%, and the final yield of the two-step production process of the prior art was 71.41%, and the two-step production process of the present application was about 9.26% higher than the two-step production process of the prior art in terms of yield; in terms of process time, the two-step production process of the present application was 9 hours shorter than the two-step production process of the prior art at a 500 mg antibody scale, and the process time was shortened by about 28.8%. As shown in Table 5, the yield and the time used in some of the same sub-processes of the two-step production process of the present application and the two-step production process of the prior art are similar, so human operation errors can be excluded, thereby indicating that the more simplified overall process facilitates a significant increase in yield and a significant shortening of process time.

[0347] Example 2 Study on the applicability of the two-step production process of the present application

[0348] Example 2.1. Study on the applicability of different endoglycosidases used in Step (I.1) of the two-step production process of the present application

[0349] Example 2.1.1 Wild-type endoglycosidase Endo S2

[0350] A) Study on the applicability of the enzyme to the process of the present application

[0351] Based on the suitability of the mutant enzyme Endo S2M3 in the two-step production process of the present application proved in Example 1, the suitability of the wild-type glycoside endo-enzyme Endo S2 in the two-step production process of the present application was studied. With the bevacizumab cell supernatant as the raw material, the antibody conjugate drug (ADC) with homogenous sugar chains of site-specific remodeling was prepared by the production process of the present application through a two-step method. The research method was the same as that in Example 1, except that different glycoside endo-enzymes were used, i.e. Endo S2 or S2M3 was used to catalyze the site-specific remodeling of the homogenous sugar chains of bevacizumab with heterogeneous sugar chains, and the bevacizumab conjugate ADC with a theoretical DAR value of 4 was prepared.

[0352] Experimental results:

[0353] The ADC prepared by the two-step method of the present application using Endo S2 or S2M3 catalysis was subjected to RP-HPLC and SEC-HPLC analysis, and the results are shown in Figure 6 and Table 6. After Endo S2 or S2M3 catalysis, the SEC detection results of the obtained ADC were similar, and the monomer purity was about 98%; the DAR values of the antibody conjugate drugs prepared by Endo S2 and Endo S2M3 catalysis were 3.75 and 3.78, respectively, and the conjugation efficiency was more than 90%, indicating that the wild-type and the modified glycoside endo-enzymes are both suitable for the process of the present application.

[0354] Table 6 Detection results of the suitability study of glycoside endo-enzymes (Endo S2 and Endo S2M3)

[0355] B) Stability of enzymes to the process of the present application

[0356] Experimental method: With the bevacizumab cell supernatant in Example 1, the general operation A was carried out according to Example 1, step (I), and the reaction was carried out under the catalysis of Endo-S2 or S2M3 for 22 h. Samples were taken at different times of the glycosylation reaction (i.e. at 1 h, 2 h, 4 h, 8 h, 22 h) and purified, and after purification, small molecule conjugation experiments were carried out and the DAR value was detected by RP-HPLC (also referred to as "RP-DAR" herein). The results are shown in Figure 7 and Table 7.

[0357] Experimental results: As can be seen from Figure 7 and Table 7, when the sample was taken at 1 h of the glycosylation reaction for subsequent operation, the DAR values of the products obtained by Endo-S2 or S2M3 catalysis were similar. With the extension of time, under the catalysis of Endo-S2, the DAR value decreased by 0.27 from the peak value of 3.83 at 8 h, and under the catalysis of S2M3, the DAR value decreased by 0.15 from the peak value of 3.82 at 8 h, indicating that Endo-S2M3 makes the process more stable.

[0358] Table 7 Influence of reaction time of glycoside endo-enzyme Endo S2 or Endo S2 M3 on DAR value

[0359] Example 2.1.2 Stability study of glycoside endo-enzyme Endo-S2 mutant 1 on the process of the present application

[0360] Experimental method: Using the bevacizumab cell supernatant in Example 1, the general operation A was implemented according to Example 1, wherein step (I) was catalyzed by glycoside endo-enzyme Endo-S2 mutant 1 (the amino acid sequence of which is shown as SEQ ID NO: 3), and the disaccharide used was diAz-LacNAc-ox, and the small molecule was DBCO-PEG4-vc-PAB-MMAE antibody, and ADC-11 was prepared.

[0361] Experimental results: The DAR value was 3.79, and the yield was 76.3%.

[0362] Example 2.1.3 Stability study of glycoside endo-enzyme Endo-S2 mutant 1 on the process of the present application

[0363] Experimental method: Using the bevacizumab cell supernatant in Example 1, the general operation A was implemented according to Example 1, wherein step (I) was catalyzed by glycoside endo-enzyme Endo-S2 mutant 1 (the amino acid sequence of which is shown as SEQ ID NO: 3), and the disaccharide used was Az-LacNAc-ox, and the small molecule was DBCO-PEG4-vc-PAB-MMAE antibody, and ADC-12 was prepared.

[0364] Experimental results: The DAR value was 1.68, and the yield was 77.1%.

[0365] Example 2.2. Applicability study of different disaccharide linkers used in step (I.1) of the two-step production process of the present application

[0366] Example 2.2.1 When G in formula I is galactose:

[0367] Example 2.2.1.1 When G in formula I is galactose: The structure is:

[0368] Experimental method: Using the bevacizumab cell supernatant in Example 1, the general operation A was implemented according to Example 1, wherein step (I) was catalyzed by glycoside endo-enzyme Endo-S2 M3, and the disaccharide used was Az-LacNAc-ox, and VEGF antibody Ab-1 with homogenous glycan chains with site-directed remodeling was prepared.

[0369] Experimental results: DAR value: 1.74; yield: 78.1% by LC-MS detection analysis. The detection method is LC-MS, same below.

[0370] Example 2.2.1.2. When the structure of formula I is:

[0371] A)

[0372] Experimental method: using the bevacizumab cell supernatant in Example 1, the implementation of General Operation A refers to Example 1, wherein step (I) is catalyzed by glycoside endonuclease Endo-S2M3, and the disaccharide used is as shown above, to prepare VEGF antibody Ab-2 with site-specific restructured homogeneous sugar chains.

[0373] Experimental results: DAR value: 1.92; yield: 77.2% by LC-MS detection analysis.

[0374] B)

[0375] Experimental method: using the bevacizumab cell supernatant in Example 1, the implementation of General Operation A refers to Example 1, wherein step (I) is catalyzed by glycoside endonuclease Endo-S2M3, and the disaccharide used is as shown above, to prepare VEGF antibody Ab-3 with site-specific restructured homogeneous sugar chains.

[0376] Experimental results: DAR value: 1.71; yield: 76.5% by LC-MS detection analysis.

[0377] Example 2.2.1.3. When the structure of formula I is:

[0378] A)

[0379] Experimental method: using the bevacizumab cell supernatant in Example 1, the implementation of General Operation A refers to Example 1, wherein step (I) is catalyzed by glycoside endonuclease Endo-S2M3, and the disaccharide used is as shown above, to prepare VEGF antibody Ab-4 with site-specific restructured homogeneous sugar chains.

[0380] Experimental results: DAR value: 1.83; yield: 77.1% by LC-MS detection analysis.

[0381] B)

[0382] ​​Experimental method: using the bevacizumab cell supernatant in example 1, the general operation A of the implementation reference example 1, wherein step (I) is catalyzed by glycoside endonuclease Endo-S2M3, and the disaccharide used is as shown above, to prepare VEGF antibody Ab-5 with site-specific restructured homogeneous sugar chains.

[0383] Experimental results: DAR value: 1.74; yield: 76.3%.

[0384] Example 2.2.1.4. Effect of azido group in disaccharide linker

[0385] The applicability of disaccharide linker diAz-LacNAc-ox in the two-step production process of the present application has been proved, on this basis, the coupling of disaccharide linker Az-LacNAc-ox shown in formula III in the two-step production process of the present application and the two-step production process in the prior art is studied. Using bevacizumab cell supernatant as raw material, using disaccharide linker Az-LacNAc-ox and glycoside endonuclease S2M3 to incubate at 25℃ for 4h, to prepare bevacizumab with site-specific restructured homogeneous sugar chains, other parameters are the same as general operation A and general operation B of example 1 two-step method. In the small molecule drug coupling step, the equivalent ratio of small molecule DBCO-PEG4-vc-PAB-MMAE in the two-step production process of the present application and the two-step production process in the prior art is 6, and other parameters are the same as general operation A and general operation B of example 1 two-step method.

[0386] Experimental results:

[0387] The ADC prepared by the two-step production process of the present application and the two-step production process in the prior art is analyzed by RP-HPLC and SEC-HPLC, and the results are shown in table 8. In the two-step production process of the present application and the two-step production process in the prior art, the antibody is modified by site-specific homogeneous sugar chain with disaccharide linker Az-LacNAc-ox, the DAR value of the antibody conjugate prepared by the two processes is 1.83, the coupling efficiency is more than 90%, and the SEC-HPLC detection results are consistent. The results show that disaccharide linker Az-LacNAc-ox also has good applicability in the two-step production process of the present application.

[0388] Table 8 detection results of disaccharide linker (Az-LacNAc-ox) applicability study

[0389] Example 2.2.1.5. Effect of alkyne group in disaccharide linker

[0390] The disaccharide linker of formula I is prepared. The structure is: ​

[0391] Experimental method: The bevacizumab cell supernatant from Example 1 was used. The general operation A was performed as described in Example 1, wherein step (I) was catalyzed by the glycoside endonuclease Endo-S2M3, and the disaccharide used was as shown above, to prepare VEGF antibodies Ab-6 and Ab-7 with site-specific reconstructed homogeneous sugar chains.

[0392] Experimental results: The yields were 77.7% and 76.4% respectively; the DAR values ​​were 1.78 and 1.83 respectively.

[0393] Example 2.2.1.6. Effect of aldehyde group in disaccharide linker

[0394] The formula I was prepared The structure is The disaccharide linker. See G2 of patent CN 114949236 A.

[0395] Experimental method: The bevacizumab cell supernatant from Example 1 was used. The general operation A was performed as described in Example 1, wherein step (I) was catalyzed by the glycoside endonuclease Endo-S2M3, and the disaccharide used was as shown above, to prepare VEGF antibody Ab-8 with site-specific reconstructed homogeneous sugar chains.

[0396] Experimental results: Yield: 78.3%; DAR value: 1.84

[0397] Example 2.2.1.7. Effect of other disaccharide linkers

[0398] A) Prepared The structure is The disaccharide linker. See G11 of patent CN 114949236A.

[0399] Experimental method: The bevacizumab cell supernatant from Example 1 was used. The general operation A was performed as described in Example 1, wherein step (I) was catalyzed by the glycoside endonuclease Endo-S2M3, and the disaccharide used was as shown above, to prepare VEGF antibody Ab-9 with site-specific reconstructed homogeneous sugar chains.

[0400] Experimental results: Yield: 77.0%; DAR value: 1.67.

[0401] B) prepared The structure is The disaccharide linker. See G13 of patent CN 114949236A.

[0402] Experimental method: Using the bevacizumab cell supernatant in Example 1, the general operation A is implemented according to Example 1, wherein step (I) is catalyzed by glycoside endonuclease Endo-S2M3, and the disaccharide used is as shown above, to prepare VEGF antibody Ab-10 with site-specific restructured homogeneous sugar chains.

[0403] Experimental results: Yield: 78.1%; DAR value: 1.73.

[0404] Example 2.2.2. When G in formula I is mannose:

[0405] Example 2.2.2.1 When G in formula I is the structure is:

[0406] The disaccharide linker (compound 6 in the literature) is synthesized according to the literature Bioconjugate Chem. 2022, 33, 1179-1191.

[0407] Experimental method: Using the bevacizumab cell supernatant in Example 1, the general operation A is implemented according to Example 1, wherein step (I) is catalyzed by glycoside endonuclease Endo-S2M3, and the disaccharide used is as shown above, to prepare VEGF antibody Ab-15 with site-specific restructured homogeneous sugar chains.

[0408] Experimental results: Yield: 78.3%; DAR value: 1.78.

[0409] Example 2.2.3. When G in formula I is glucose:

[0410] When G in formula I is the structure is:

[0411] The disaccharide linker (compound 14 in the literature) is synthesized according to the literature Bioconjugate Chem. 2022, 33, 1179-1191.

[0412] Experimental method: Using the bevacizumab cell supernatant in Example 1, the general operation A is implemented according to Example 1, wherein step (I) is catalyzed by glycoside endonuclease Endo-S2M3, and the disaccharide used is as shown above, to prepare VEGF antibody Ab-17 with site-specific restructured homogeneous sugar chains.

[0413] Experimental results: Yield: 77.6%; DAR value: 1.72.

[0414] Example 2.3. Applicability of different small molecule drugs in step (II.1) of the two-step production process of the application

[0415] The above experiments have proved the applicability of DBCO-PEG4-vc-PAB-MMAE as a small molecule drug in the two-step production process of the present application. On this basis, using bevacizumab cell supernatant as raw material, the applicability of other small molecule drugs in the two-step production process of the present application is studied.

[0416] Example 2.3.1 Azido-based coupling using small molecule drugs in Example 2.2

[0417] A) Coupling with small molecule P16

[0418] Experimental method: Using bevacizumab cell supernatant in Example 1, the implementation of General Procedure A is referred to Example 1, wherein step (I) is catalyzed by glycoside endonuclease Endo-S2M3, and the disaccharide used is diAz-LacNAc-ox; coupling with small molecule P16 (i.e. DBCO-Lys-PEG24-MMAE), wherein the coupling and UF / DF steps and parameters are implemented according to General Procedure A, to prepare ADC-1.

[0419] Experimental results: Yield: 99.8%; DAR value: 3.86. The above yield is the yield calculation before and after the coupling of intermediate I with the small molecule.

[0420] B) Coupling with small molecule P22

[0421] Experimental method: Using bevacizumab cell supernatant in Example 1, the implementation of General Procedure A is referred to Example 1, wherein step (I) is catalyzed by glycoside endonuclease Endo-S2M3, and the disaccharide used is diAz-LacNAc-ox; coupling with small molecule P22, wherein the coupling and UF / DF steps and parameters are implemented according to General Procedure A, to prepare ADC-2.

[0422] Experimental results: Yield: 98.9%; DAR value: 3.72. The above yield is the yield calculation before and after the coupling of intermediate I with the small molecule.

[0423] C) Coupling with small molecule P19

[0424] Experimental method: Using bevacizumab cell supernatant in Example 1, the implementation of General Procedure A is referred to Example 1, wherein step (I) is catalyzed by glycoside endonuclease Endo-S2M3, and the disaccharide used is diAz-LacNAc-ox; coupling with small molecule P19, reaction conditions: homogeneous glycan chain VEGF antibody reaction concentration 5 mg / ml, 6 mM CuSO4, 30 mM BTTAA, 90 mM sodium ascorbate solution, 37°C reaction for 4h, coupling and UF / DF steps and parameters are implemented according to General Procedure A, to prepare ADC-3.

[0425] Experimental results: yield: 99.0%; DAR value: 3.74. The above yield is the yield calculation before and after the coupling of intermediate I with the small molecule.

[0426] Example 2.3.2 Coupling based on aldehyde group in Example 2.2 using small molecule drugs

[0427] In this example, the VEGF antibody Ab-8 with homogeneous sugar chains prepared using the disaccharide linker of formula I Structure is will be coupled with small molecule drugs.

[0428] A) Coupling with small molecule P5

[0429] Experimental method: the VEGF antibody Ab-8 with homogeneous sugar chains prepared in Example 2.2.1.6 was coupled with small molecule drug P5, wherein the coupling and UF / DF steps and parameters were carried out according to the general operation A of Example 1, to prepare ADC-4.

[0430] Experimental results: yield: 99.4%; DAR value: 1.70. The above yield is the yield calculation before and after the coupling of intermediate I with the small molecule.

[0431] B) Coupling with small molecule P18

[0432] Experimental method: the VEGF antibody Ab-8 with homogeneous sugar chains prepared in Example 2.2.1.6 was coupled with small molecule drug P18, wherein the coupling and UF / DF steps and parameters were carried out according to the general operation A of Example 1, to prepare ADC-5.

[0433] Experimental results: yield: 99.3%; DAR value: 1.67. The above yield is the yield calculation before and after the coupling of intermediate I with the small molecule.

[0434] Example 2.3.3 Comparative study using different small molecule drugs

[0435] The specific experimental process is as follows: through the two-step production process of the application, using diAz-LacNAc-ox and endoglycosidase S2M3 to obtain a bevacizumab stock solution with site-specific restructured homogeneous sugar chains, which is divided into two equal parts, and is coupled with DBCO-PEG4-vc-PAB-MMAE (P28) and DBCO-PEG4-GGFG-DXD (P27) shown by the following formula in step (II.1), respectively. The coupling process and related parameters are the same as step (II.1) of general operation A of two-step method of Example 1, except that the small molecule drugs used are different. Bevacizumab conjugated ADC with a theoretical DAR value of 4 is prepared.

[0436] Results of experiments:

[0437] The ADC prepared by conjugating DBCO-PEG4-vc-PAB-MMAE and DBCO-PEG4-GGFG-DXD with bevacizumab was subjected to MS-DAR and SEC-HPLC analysis, and the results are shown in Table 9. The DAR values of the antibody-drug conjugates prepared by conjugating the small molecules DBCO-PEG4-vc-PAB-MMAE and DBCO-PEG4-GGFG-DXD were 3.92 and 3.86, respectively, and the conjugation efficiencies were both above 90%, indicating that the small molecule drug DBCO-PEG4-GGFG-DXD is also suitable for the two-step production process of the present application.

[0438] Table 9. Results of detection of applicability of small molecule drugs

[0439] Example 2.4. Feasibility study of further simplification of the purification of X in step (I.2) of the two-step production process of the present application

[0440] The above experiments have demonstrated that the purification of X includes the following operations: affinity chromatography, low-pH incubation, anion chromatography, cation chromatography, nanofiltration, ultrafiltration / diafiltration, and the applicability of the preparation of a protein molecule (e.g., an antibody comprising an Fc region with homogenous glycan chains with site-directed remodeling) having a site-directed remodeling of homogenous glycan chains in the two-step production process of the present application,

[0441] On this basis, the feasibility of further simplifying the operations in the purification of X was studied. Affinity chromatography, anion chromatography or cation chromatography, or nanofiltration was omitted in the operations in the purification of X, respectively, and the other steps, parameters, etc. were the same as those in the general operation A for preparing ADC using the two-step method.

[0442] Results of experiments:

[0443] The prepared ADC was subjected to RP-HPLC and SEC-HPLC analysis, and the results showed that it was not feasible to omit affinity chromatography, anion chromatography or cation chromatography, or nanofiltration in the operations in the purification of X in the two-step production process of the present application.

[0444] Example 2.5. Applicability study of changing the order of operations in the purification of X in step (I.2) of the two-step production process of the present application

[0445] The above experiments have proved that the purification X includes the following operations: affinity chromatography, low pH incubation, anion chromatography, cation chromatography, nanofiltration, ultrafiltration / diafiltration, and the applicability of the protein molecule (e.g., an antibody comprising a homogeneous glycan chain with site-specific remodeling) in the preparation of a stock solution with a homogeneous glycan chain with site-specific remodeling in the two-step production process of the present application,

[0446] On this basis, the feasibility of changing the operation sequence in the purification X was studied. The nanofiltration was respectively implemented before the anion chromatography or before the cation chromatography in the operation of the purification X, and the other steps, parameters, etc. were the same as the general operation A for preparing the ADC using the two-step method.

[0447] Experimental results:

[0448] The prepared ADC was analyzed by RP-HPLC and SEC-HPLC, and the results showed that it was feasible to change the operation sequence in the purification X and implement the nanofiltration before the anion chromatography or before the cation chromatography in the two-step production process of the present application.

[0449] Example 2.6. Antibody concentration in the cell supernatant used in step (I.1) of the two-step production process of the present application

[0450] The cell supernatant containing the antibody was added into His-HCl buffer (pH 6.5) to make the final concentration of the antibody 1-8 mg / mL, and then diAz-LacNAc-ox and endoglycosidase (Endo S2M3) were sequentially added and incubated at 25°C for 1 hour. The design of the specific experimental conditions is shown in Table 10, and the other operations of step (I.1) and step (I.2) were the same as the general operation A using the two-step method.

[0451] Table 10. Screening experimental conditions for antibody concentration in cell supernatant

[0452] The design of the specific experimental conditions of step (II.1) is shown in Table 11, and the other operations of step (II.1) and step (II.2) were the same as the general operation A using the two-step method, and the small molecule drug used was DBCO-PEG4-vc-PAB-MMAE.

[0453] Table 11. Small molecule coupling experimental conditions

[0454] The coupling reaction solution was subjected to a 30 kDa ultrafiltration tube to remove the uncoupled small molecule drug, and the sample DAR value was analyzed and identified by RP-HPLC.

[0455] Experimental results:

[0456] As shown in Figure 8, the DAR value increases with the increase of antibody reaction concentration between 1-4 mg / mL, when the antibody reaction concentration reaches 3 mg / mL, the DAR value is 3.63, the reaction efficiency is greater than 90%, and when the antibody reaction concentration is ≥4 mg / mL, it reaches a plateau.

[0457] Therefore, the antibody concentration in the cell supernatant used in step (I.1) of the two-step production process of the present application can be ≥1 mg / mL of antibody concentration.

[0458] Example 2.7. Yield of ADC prepared by the two-step production process of the present application

[0459] As in Example 1, the same batch of cell supernatant was divided into two parts, each 500 ml, and ADC was prepared using the two-step production process of the present application and using the two-step production process in the prior art.

[0460] Experimental results: when ADC is prepared using the two-step production process of the present application and using the two-step production process in the prior art, the yield of the process of the present application using cell supernatant is higher.

[0461] Example 2.8. Study on the applicability of different antibodies in the two-step production process of the present application

[0462] The above experiments have all been carried out using cell supernatant of bevacizumab, which fully proves the applicability of bevacizumab in the two-step production process of the present application. In order to prove that the process is also feasible in other antibodies, further studies were carried out using cell culture supernatant of other monoclonal antibodies.

[0463] A) Trastuzumab

[0464] Further studies were carried out using cell supernatant for producing trastuzumab. The trastuzumab cell supernatant was prepared into antibody conjugate drugs with a theoretical DAR value of 4 by General Operation A and General Operation B, respectively. The method for obtaining the cell supernatant of trastuzumab is the same as the method for culturing and preparing the cell supernatant of bevacizumab in Example 1, except that the CHO cells used are CHO cells expressing trastuzumab.

[0465] Experimental method: as in Example 1, except that cell supernatant producing different antibodies was used. The ADC prepared by conjugation using the two-step production process in the prior art and the two-step production process of the present application, respectively, was subjected to RP-HPLC and SEC-HPLC analysis, and the results are shown in Figures 9, 10 and Table 12.

[0466] Experimental results: the ADC conjugation results and SEC detection results of trastuzumab prepared by the two-step production process of the application and the two-step production process in the prior art are similar, the DAR values are all ≥3.60, which are 3.81 and 3.89 respectively, the conjugation efficiencies are all more than 90%, and the SEC purities are 98.83% and 98.72% respectively, indicating that trastuzumab is also suitable for the two-step production process of the application.

[0467] Table 12 Experimental results of trastuzumab suitability study

[0468] B) Veltuzumab

[0469] Experimental method: the cell supernatant for producing veltuzumab was used for research, and the antibody conjugated drug with a theoretical DAR value of 4 was prepared by the general operation A. The method for obtaining the cell supernatant of veltuzumab is the same as the culture and preparation method of the cell supernatant of bevacizumab in Example 1, except that the CHO cells used are CHO cells expressing veltuzumab. Veltuzumab is an anti-HER2 antibody, which is the antibody part of the ADC drug (trade name: Herceptin) marketed by Rongchang. The ADC prepared by the two-step production process of the application was analyzed by RP-HPLC and SEC-HPLC.

[0470] Experimental results: DAR value: 3.72; yield: 78.0%.

[0471] C) Sacituzumab

[0472] Experimental method: the cell supernatant for producing sacituzumab was used for research, and the antibody conjugated drug with a theoretical DAR value of 4 was prepared by the general operation A. The method for obtaining the cell supernatant of sacituzumab is the same as the culture and preparation method of the cell supernatant of bevacizumab in Example 1, except that the CHO cells used are CHO cells expressing sacituzumab. Sacituzumab is an anti-TROP2 antibody. The ADC prepared by the two-step production process of the application was analyzed by RP-HPLC and SEC-HPLC.

[0473] Experimental results: DAR value: 3.69; yield: 76.7%.

[0474] Example 2.9. Stability study of glycosylation modification product

[0475] Experimental materials: cell culture supernatant of bevacizumab, disaccharide linker substrate diAz-LacNAc-ox, small molecule drug DBCO-PEG4-vc-PAB-MMAE

[0476] Experimental method: The steps (I.1) and (I.2) in Example 1 were adopted, and the site-directed glycosylation reconstruction was carried out using the existing process and the original process. The reaction liquid B obtained after the enzymatic reaction was allowed to stand for 0 h, 8 h and 16 h, respectively, and then coupled with DBCO-PEG4-vc-PAB-MMAE. The small molecule coupling step was the same as step (II.1) in Example 1. After the coupling was completed, RP-DAR detection was carried out.

[0477] Table 13 Glycosylation modification sample stability study

[0478] The stability results at 8 h and 16 h after the reaction showed that the DAR value of the existing process was reduced by 0.16 and 0.55, respectively. The DAR value of the process of the application was reduced by 0.13 and 0.40, respectively, under the same conditions. The DAR value reduction of the process of the application was significantly smaller than that of the existing process, indicating that the antibody glycosylation modification product obtained by the process of the application was more stable.

[0479] Example 2.10 Screening of equivalent ratio of disaccharide substrate in site-directed reconstruction homogeneous glycan modification reaction

[0480] The antibody-containing cell supernatant was added to His-HCl buffer (pH 6.5), 20-140 equivalents of diAz-LacNAc-ox were added, endoglycosidase (Endo S2M3) was added, and incubation was carried out at 25°C for 1 hour. The enzyme and substrate in the reaction liquid were removed by protein A affinity chromatography to obtain an antibody with site-directed reconstruction of homogeneous glycan. The specific design is shown in Table 14:

[0481] Table 14 Experimental conditions for screening of equivalent ratio of disaccharide substrate in site-directed reconstruction homogeneous glycan modification reaction

[0482] The antibody with site-directed reconstruction of homogeneous glycan was added to His-HCl buffer (pH 6.5), and then 10% (v / v) DMSO and 10 equivalents of small molecules (DBCO-PEG4-vc-PAB-MMAE) were added in sequence. Incubation was carried out at 25°C for 2 hours. The experimental conditions and parameters were the same as those in Table 11. The coupling reaction liquid was subjected to ultrafiltration with a 30 kDa ultrafiltration tube to remove uncoupled small molecule drugs. RP-HPLC was used to analyze and identify the DAR value of the sample.

[0483] Experimental results:

[0484] As shown in Figure 11, the DAR value increased with the increase of the amount of diAz-LacNAc-ox added, when the equivalent ratio of diAz-LacNAc-ox was between 20 and 40. When the equivalent ratio was 40, the DAR value was 3.61, and the reaction efficiency reached 90%. With the increase of the substrate concentration, the DAR value further increased until it was stabilized at above 3.8. Therefore, the equivalent ratio of the diAz-LacNAc-ox in the site-directed remodeling of homogeneous glycan chain modification reaction was preferably above 20, more preferably 40-140, and even more preferably 80-140.

[0485] Example 2.11 Enzyme amount in site-directed remodeling of homogeneous glycan chain modification reaction

[0486] The same as Example 1 was implemented, except that the supernatant of cells producing different antibodies was used. The supernatant of cells containing antibodies was added to His-HCl buffer, then diAz-LacNAc-ox was added, and finally different proportions of endoglycosidase (Endo S2M3) were added, respectively, and incubated at 25°C for 1 hour. The enzymes and substrates in the reaction solution were removed by protein A affinity chromatography to obtain antibodies with site-directed remodeling of homogeneous glycan chains. The specific design is shown in Table 15:

[0487] Table 15 Experimental conditions for screening enzyme amount in site-directed remodeling of homogeneous glycan chain modification reaction

[0488] The antibodies with site-directed remodeling of homogeneous glycan chains were added to His-HCl buffer (pH 6.5), and then 10% (v / v) DMSO and 10 equivalents of small molecules (DBCO-PEG4-vc-PAB-MMAE) were added in sequence, and incubated at 25°C for 2 hours. The experimental conditions and parameters were the same as those in Table 11. The coupling reaction solution was passed through a 30 kDa ultrafiltration tube to remove uncoupled small molecule drugs, and the DAR value of the sample was analyzed and identified by RP-HPLC.

[0489] Experimental results:

[0490] As shown in Figure 12, in the site-directed remodeling of homogeneous glycan chain modification reaction, the DAR increased with the increase of the enzyme amount when the enzyme amount was 5-20 μg / mg. When the enzyme amount was 10 μg / mg, the DAR reached about 3.70. With the gradual increase of the enzyme amount to 80 μg / mg, the DAR value was stably increased and maintained at about 3.80, keeping a plateau. Therefore, the enzyme amount in the site-directed remodeling of homogeneous glycan chain modification reaction was preferably greater than 5 μg / mg.

[0491] Example 2.12 pH in site-directed remodeling of homogeneous glycan chain modification reaction

[0492] The same as Example 1 was implemented, except that the cell supernatant producing different antibodies was used. The antibody-containing cell supernatant was added to His-HCl buffer, then diAz-LacNAc-ox was added, the pH was adjusted to the corresponding value in the table below, and finally the endoglycosidase (Endo S2M3) was added, and incubated at 25°C for 1 hour. The enzyme and substrate in the reaction solution were removed by protein A affinity chromatography to obtain an antibody with site-specifically remodeled homogeneous sugar chains. The specific design is shown in Table 16:

[0493] Table 16 Experimental conditions for pH screening of site-specifically remodeled homogeneous sugar chain modification reaction

[0494] The antibody with site-specifically remodeled homogeneous sugar chains was added to His-HCl buffer (pH 6.5), then 10% (v / v) DMSO and 10 equivalents of small molecules (DBCO-PEG4-vc-PAB-MMAE) were added in sequence, and incubated at 25°C for 2 hours. The experimental conditions and parameters are the same as those in Table 11. The coupling reaction solution was passed through a 30kDa ultrafiltration tube to remove uncoupled small molecule drugs, and the DAR value of the sample was analyzed and identified using RP-HPLC.

[0495] Experimental results:

[0496] As shown in Figure 13, between pH 5.3 and 6.2, the DAR value gradually increased, and when the pH was greater than 6.8, the DAR decreased with the increase of pH; when the pH was in the range of 5.30-7.20, the DAR value was >3.60, and the reaction efficiency was >90%. Therefore, the reaction pH is in the range of 4.8-7.7, preferably 5.3-7.2, and more preferably 6.2-6.8.

[0497] Example 2.13 Reaction temperature of site-specifically remodeled homogeneous sugar chain modification reaction

[0498] The same as Example 1 was implemented, except that the cell supernatant producing different antibodies was used. The antibody-containing cell supernatant was added to His-HCl buffer, then diAz-LacNAc-ox and endoglycosidase (S2M3) were added, and incubated at different temperatures for 1 hour. The enzyme and substrate in the reaction solution were removed by protein A affinity chromatography to obtain an antibody with site-specifically remodeled homogeneous sugar chains. The specific design is shown in Table 17:

[0499] Table 17 Experimental conditions for temperature screening of site-specifically remodeled homogeneous sugar chain modification reaction

[0500] The antibody with the homogenous glycoengineered glycan was added to His-HCl buffer (pH 6.5), followed by the addition of 10% (v / v) DMSO and 10 equivalents of the small molecule (DBCO-PEG4-vc-PAB-MMAE) at a final concentration, and incubation at 25°C for 2 hours. The experimental conditions and parameters were the same as those in Table 11. The coupling reaction solution was subjected to a 30-kDa ultrafiltration tube to remove uncoupled small molecule drugs, and RP-HPLC was used to analyze and identify the DAR value of the sample.

[0501] Experimental results:

[0502] As shown in FIG. 14, the reaction temperature was between 6 and 22°C, and as the temperature increased, the DAR value increased. When the temperature was greater than 37°C, the DAR decreased as the temperature increased. When the reaction temperature was 45°C, the DAR value was low, and the coupling effect was not ideal. Therefore, the reaction temperature was in the range of 6-37°C, preferably 15-37°C, which could achieve a target DAR value of more than 3.60, and the reaction efficiency was greater than 90%.

[0503] Example 2.14 Reaction time of homogenous glycoengineered glycan modification

[0504] The cell supernatant containing the antibody was added to His-HCl buffer, followed by the addition of diAz-LacNAc-ox and endoglycosidase (S2M3), and incubation at 25°C for different times. The enzyme and substrate in the reaction solution were removed by protein A affinity chromatography to obtain the antibody with the homogenous glycoengineered glycan. The specific design is shown in Table 18:

[0505] Table 18 Experimental conditions for screening reaction time of homogenous glycoengineered glycan modification

[0506] The antibody with the homogenous glycoengineered glycan was added to His-HCl buffer (pH 6.5), followed by the addition of 10% (v / v) DMSO and 10 equivalents of the small molecule (DBCO-PEG4-vc-PAB-MMAE) at a final concentration, and incubation at 25°C for 2 hours. The experimental conditions and parameters were the same as those in Table 11. The coupling reaction solution was subjected to a 30-kDa ultrafiltration tube to remove uncoupled small molecule drugs, and RP-HPLC was used to analyze and identify the DAR value of the sample.

[0507] Experimental results:

[0508] As shown in FIG. 15, the reaction time was 0.25-0.75 h, and the DAR value increased as the time increased. When the reaction time was greater than 4 h, the DAR value decreased, and when the time was extended to 22 h, the DAR value was still 3.10. Therefore, the reaction time could be 0.25-22 h, preferably 0.50-4 h.

[0509] Example 2.15 One of the studies of changing multiple variables in the method of the present application

[0510] The above examples are studies of changing a single variable in the method of the present application. In order to achieve a DAR value of, for example, 1.7, preferably 2.0, when using a mono-azide and a DAR value of, for example, 3.6, preferably 4.0, when using a bis-azide for the disaccharide linker, studies of changing multiple variables were performed on the method of the present application.

[0511] The antibody-containing cell supernatant was added to His-HCl buffer, followed by diAz-LacNAc-ox and endoglycosidase (Endo S2M3) in sequence, and glycosylation modification was performed according to the reaction conditions in the table below. The enzymes and substrates in the reaction solution were removed by protein A affinity chromatography to obtain antibody intermediate I. The specific experimental conditions are designed as follows:

[0512] Antibody intermediate I was added to His-HCl buffer (pH 6.5), followed by 10% (v / v) DMSO and 10 equivalents of small molecules (DBCO-PEG4-vc-PAB-MMAE) in sequence, and incubated at 25°C for 2 hours.

[0513] Experimental results: DAR value: 3.64

[0514] Example 2.16 Second of the studies of changing multiple variables in the method of the present application

[0515] The antibody-containing cell supernatant was added to His-HCl buffer, followed by diAz-LacNAc-ox and endoglycosidase (Endo S2M3) in sequence, and glycosylation modification was performed according to the reaction conditions in the table below. The enzymes and substrates in the reaction solution were removed by protein A affinity chromatography to obtain antibody intermediate I. The specific experimental conditions are designed as follows:

[0516] Antibody intermediate I was added to His-HCl buffer (pH 6.5), followed by 10% (v / v) DMSO and 10 equivalents of small molecules (DBCO-PEG4-vc-PAB-MMAE) in sequence, and incubated at 25°C for 2 hours.

[0517] Experimental results: DAR value: 3.76

[0518] Example 2.17 Third of the studies of changing multiple variables in the method of the present application

[0519] The antibody-containing cell supernatant was added to His-HCl buffer, followed by diAz-LacNAc-ox, endoglycosidase (Endo S2M3) in sequence, and glycosylation modification was performed according to the reaction conditions in the table below. The enzymes and substrates in the reaction solution were removed by protein A affinity chromatography to obtain antibody intermediate I. The specific experimental conditions are designed as follows:

[0520] Antibody intermediate I was added to His-HCl buffer (pH 6.5), followed by 10% (v / v) DMSO and 10 equivalents of small molecules (DBCO-PEG4-vc-PAB-MMAE) in sequence, and incubated at 25°C for 2 hours.

[0521] Experimental results: DAR value: 3.65

[0522] Example 2.18 Study IV of changing multiple variables in the method of the application

[0523] The antibody-containing cell supernatant was added to His-HCl buffer, followed by diAz-LacNAc-ox, endoglycosidase (Endo S2M3) in sequence, and glycosylation modification was performed according to the reaction conditions in the table below. The enzymes and substrates in the reaction solution were removed by protein A affinity chromatography to obtain antibody intermediate I. The specific experimental conditions are designed as follows:

[0524] Antibody intermediate I was added to His-HCl buffer (pH 6.5), followed by 10% (v / v) DMSO and 10 equivalents of small molecules (DBCO-PEG4-vc-PAB-MMAE) in sequence, and incubated at 25°C for 2 hours.

[0525] Experimental results: DAR value: 3.79

[0526] Example 2.19 Study V of changing multiple variables in the method of the application

[0527] The antibody-containing cell supernatant was added to His-HCl buffer, followed by diAz-LacNAc-ox, endoglycosidase (Endo S2M3) in sequence, and glycosylation modification was performed according to the reaction conditions in the table below. The enzymes and substrates in the reaction solution were removed by protein A affinity chromatography to obtain antibody intermediate I. The specific experimental conditions are designed as follows:

[0528] Antibody intermediate I was added to His-HCl buffer (pH 6.5), followed by 10% (v / v) DMSO and 10 equivalents of small molecules (DBCO-PEG4-vc-PAB-MMAE) in sequence, and incubated at 25°C for 2 hours.

[0529] Experimental results: DAR value: 3.81

[0530] Example 2.20 Sixth study of changing multiple variables in the method of the present application

[0531] The antibody-containing cell supernatant was added to His-HCl buffer, and then diAz-LacNAc-ox and endoglycosidase (Endo S2M3) were sequentially added to perform glycosylation modification according to the reaction conditions in the following table. The enzymes and substrates in the reaction solution were removed by protein A affinity chromatography to obtain antibody intermediate I. The specific experimental conditions are designed as follows:

[0532] The antibody intermediate I was added to PBS buffer (pH 6.8), and then 8% (v / v) DMSO and 12 equivalents of small molecules (DBCO-PEG4-vc-PAB-MMAE) were sequentially added to incubate at 22°C for 2 hours.

[0533] Experimental results: DAR value: 3.68

[0534] The antibody intermediate I was added to PBS buffer (pH 6.5), and then 5% (v / v) DMSO and 10 equivalents of small molecules (DBCO-PEG4-vc-PAB-MMAE) were sequentially added to incubate at 25°C for 3 hours.

[0535] Experimental results: DAR value: 3.74

[0536] The antibody intermediate I was added to His-HCl buffer (pH 6.5), and then 12% (v / v) DMSO and 10 equivalents of small molecules (DBCO-PEG4-vc-PAB-MMAE) were sequentially added to incubate at 28°C for 2 hours.

[0537] Experimental results: DAR value: 3.69

[0538] The antibody intermediate I was added to Tris-HCL buffer (pH 6.5), and then 5% (v / v) DMSO and 10 equivalents of small molecules (DBCO-PEG4-vc-PAB-MMAE) were sequentially added to incubate at 25°C for 2 hours.

[0539] Experimental results: DAR value: 3.62

[0540] The antibody intermediate I was added to His-HCl buffer (pH 6.5), and then 10% (v / v) DMSO and 10 equivalents of small molecules (DBCO-PEG4-vc-PAB-MMAE) were sequentially added to incubate at 25°C for 2 hours.

[0541] Experimental results: DAR value: 3.74

[0542] Example 3 Preparation of antibody conjugate drug with homogeneous sugar chain of site-directed remodeling by one-step method

[0543] In this example, an antibody conjugate drug (ADC) with homogeneous sugar chain of site-directed remodeling was prepared by one-step method using the production process of the present application.

[0544] Specifically, VEGF antibody bevacizumab was produced using CHO cells, and the VEGF antibody containing heterogeneous sugar chains linked to N297 was obtained in the cell culture supernatant.

[0545] The VEGF antibody conjugate drug with homogeneous sugar chain of site-directed remodeling was prepared as follows.

[0546] Step (I.1): Preparation of VEGF antibody conjugate drug with homogeneous sugar chain of site-directed remodeling from VEGF antibody with heterogeneous sugar chain in cell culture supernatant

[0547] The cell supernatant containing VEGF antibody with heterogeneous sugar chains was added to His-HCl buffer (pH 6.5), and the disaccharide-small molecule drug conjugate MMAE-PAB-VC-NH-LacNAc-ox represented by Formula V, endoglycosidase S2M3 were added, and incubated at 25°C for 4h to obtain reaction solution A containing VEGF antibody conjugate drug with homogeneous sugar chain of site-directed remodeling.

[0548] Step (I.2): Purification X of reaction solution A obtained in step (I.1)

[0549] Purification X was performed in the same manner as in Purification X in two-step general operation A, except that reaction solution A was different.

[0550] As a comparison, an antibody conjugate drug with homogeneous sugar chain of site-directed remodeling was prepared by one-step method using the production process in the prior art.

[0551] Specifically, VEGF antibody bevacizumab was produced using CHO cells, and the VEGF antibody containing heterogeneous sugar chains linked to N297 was obtained in the cell culture supernatant.

[0552] The VEGF antibody conjugate drug with homogeneous sugar chain of site-directed remodeling was prepared as follows.

[0553] Step (I.1): Purification of VEGF antibody with heterogeneous sugar chain in cell culture supernatant

[0554] It was performed in the same manner as step (I.1) in two-step general operation B.

[0555] Step (I.2): Preparation of VEGF antibody conjugate drug with site-specifically remodeled homogeneous sugar chains from the purified VEGF antibody with heterogeneous sugar chains

[0556] The purified VEGF antibody with heterogeneous sugar chains from step (I.1) was taken as intermediate I and added to His-HCl buffer (pH 6.5), and a disaccharide-small molecule drug conjugate MMAE-PAB-VC-NH-LacNAc-ox represented by formula V, endoglycosidase S2M3 were added, and incubated at 25°C for 4h to obtain a VEGF antibody conjugate drug containing site-specifically remodeled homogeneous sugar chains.

[0557] Step (I.3): Purification Z of reaction solution A obtained in step (I.2)

[0558] Purification Z was performed in the same manner as purification Z in the two-step method general operation B, except that reaction solution A was different.

[0559] Table 19 Experimental conditions used in the one-step method conjugation applicability study

[0560] Experimental results:

[0561] RP-HPLC and SEC-HPLC were performed on the ADC prepared by the one-step method production process shown in Figure 19 of the present application and the one-step method production process in the prior art, and the results are shown in Figures 16, 17 and Table 20. The DAR values of the ADC prepared by the process of the present application and the process of the prior art were 1.78 and 1.86, respectively, both reaching the target DAR value; the SEC purities were 98.29% and 98.34%, respectively, and the conjugation results and SEC purities were basically consistent, indicating that direct conjugation of the small molecule MMAE-PAB-VC-NH-LacNAc-ox with the antibody by the one-step method is also applicable to the one-step method production process of the present application.

[0562] Table 20 Experimental results of the one-step method conjugation applicability study

[0563] Example 4 Applicability study of different disaccharide-small molecule drugs in the one-step method production process of the present application

[0564] A) One of the disaccharide-small molecule drugs

[0565] Experimental method: Using the bevacizumab cell supernatant in Example 1, referring to the one-step method conjugation new process in Example 3, using endoglycosidase Endo S2M3, disaccharide-small molecule drug SP2, ADC-8 was prepared.

[0566] Experimental results: yield: 99.8%; DAR value: 1.84, the yield is the yield of intermediate I before and after coupling with small molecules, the same below.

[0567] B) disaccharide-small molecule drug two

[0568] Experimental method: using the cell supernatant of bevacizumab in example 1, referring to the one-step coupling new process of example 3, using glycoside endonuclease Endo S2M3, disaccharide-small molecule drug SP4, ADC-9 was prepared.

[0569] Experimental results: yield: 98.9%; DAR value: 1.78

[0570] C) disaccharide-small molecule drug three

[0571] Experimental method: using the cell supernatant of bevacizumab in example 1, referring to the one-step coupling new process of example 3, using glycoside endonuclease Endo S2M3, disaccharide-small molecule drug SP10 (see patent CN 114949236 A dDG-1), ADC-10 was prepared.

[0572] Experimental results: yield: 99.9; DAR value: 3.64

[0573] Example 5 Hydrolysis and transglycosylation activity of different glycoside endonucleases based on commercially available or purified antibodies

[0574] Example 5.1 One-step preparation of glycoside endonuclease for site-directed ADC compounds

[0575] MMAE-PAB-VC-NH-LacNAc-ox (0.5 mM) was added to phosphate buffer (pH 6.5), and the following commercially available or purified antibody solutions were added to the above reaction system: wild type herceptin (5 mg / mL), adding glycoside endonuclease (80 μg / mg, Endo-S2M3, Endo-S2 mutant 1 respectively), 30℃ incubation for 2 hours. The results are shown in Figure 20.

[0576] Endo-S2M3 has the best transglycosylation activity, and Endo-S2 mutant 1 has certain transglycosylation activity.

[0577] Example 5.2 Antibody transglycosylation activity test of Endo-S2 and Endo-S2M3 enzyme amount on Az-LacNAc-ox substrate

[0578] Az-LacNAc-ox (0.5 mM, structure shown) was added to phosphate buffer (pH 6.5), and the following antibody solution was added to the above reaction system: trastuzumab (5 mg / mL), and endoglycosidase (Endo-S2 or Endo-S2M3, 0.02 - 2.4 mg / mL) was added. Incubation was performed at 30°C for 2 hours, and LC-MS analysis was performed for identification.

[0579] The specific design is shown in Table 21:

[0580] Table 21. Antibody transglycosylation activity of Endo-S2 and Endo-S2M3 enzyme amount on Az-LacNAc-ox substrate reaction conditions

[0581] As shown in Table 21, the antibody transglycosylation activity of Endo-S2 or Endo-S2M3 enzyme amount of 4, 20, 40, 80, 160, 240, 320 and 480 μg / mg (enzyme amount / antibody amount) was tested, wherein the trastuzumab concentration was 5 mg / mL, the sugar substrate Az-LacNAc-ox concentration was 0.5 mM, the reaction was performed in a 50 mM phosphate, pH 6.5 buffer system, and LC-MS analysis was performed after 2 hours of reaction at 30°C, as shown in Figure 21.

[0582] The results show that under low enzyme amount conditions (about < 50 μg / mg), both wild-type Endo-S2 and mutant enzyme Endo-S2M3 have a positive promoting effect on the DAR value. Under higher enzyme amount conditions, Endo-S2 shows a downward trend in DAR value with increasing enzyme amount, while Endo-S2M3 shows a longer plateau period, and can always maintain a high DAR value in a wide range (about 100-500 μg / mg), which indicates that the mutant enzyme Endo-S2M3 has more stable transglycosylation activity than Endo-S2.

[0583] Example 5.3. Antibody transglycosylation activity test of Endo-S2 and Endo-S2M3 enzyme amount on diAz-LacNAc-ox substrate

[0584] diAz-LacNAc-ox (0.5 mM, structure shown) was added to phosphate-HCl buffer (pH 6.5), and the following antibody solution was added to the above reaction system: trastuzumab (5 mg / mL), and endoglycosidase (0.02 - 2.4 mg / mL) was added. Incubation was performed at 30°C, and LC-MS analysis was performed for identification at 2 hours.

[0585] The specific design is shown in Table 22:

[0586] Table 22 Antibody transglycosylation activity of Endo-S2 and Endo-S2M3 enzymes on diAz-LacNAc-ox substrate under different enzyme amounts

[0587] As shown in Table 22, the antibody transglycosylation activity of Endo-S2 or Endo-S2M3 enzymes was tested at enzyme amounts of 4, 20, 40, 80, 160, 240, 320 and 480 pg / mg (enzyme amount / antibody amount), with trastuzumab concentration of 5 mg / mL, sugar substrate diAz-LacNAc-ox concentration of 0.5 mM, reaction in 50 mM phosphate, pH 6.5 buffer system, reaction at 30 °C, 2-hour time point sampling for LC-MS analysis, as shown in Figure 22.

[0588] The results show that with the increase of enzyme amount, the mutant enzyme Endo-S2M3 shows lower and slower hydrolysis activity than Endo-S2, which is more beneficial to process production.

[0589] Summary: Based on the results of Examples 5.1-5.3, it can be seen that compared with Endo-S2, the mutant enzyme Endo-S2M3 shows significant superiority in antibody transglycosylation reactions on different sugar substrates. The reaction curve of the mutant enzyme Endo-S2M3 has obvious enzymatic reaction characteristics, and the plateau remains stable, maintaining a high DAR value of the product in a wide range of enzyme amounts. This indicates that after the mutant enzyme Endo-S2M3 hydrolyzes the Fc sugar chain of the antibody, it has high transglycosylation activity to transfer the Az-LacNAc-ox sugar substrate to the antibody, and has no hydrolysis activity on the transglycosylated sugar-engineered antibody. This high and stable transglycosylation activity makes the mutant enzyme have the characteristics of enzyme amount tolerance in the preparation process of antibody conjugates. On the one hand, the longer plateau weakens the influence of enzyme amount on the DAR value of the product, and on the other hand, the decrease of substrate hydrolysis activity highlights the enhancement of the mutant enzyme transfer activity. In summary, the mutant enzyme has more superior practical application value in the process.

[0590] Example 5.4 Antibody transglycosylation activity of Endo-S2 and Endo-S2M3 enzymes on diAz-LacNAc-ox substrate under the same enzyme amount and different reaction times

[0591] The operation is the same as Example 6, and specific reference is made to Table 23 below to investigate the antibody transglycosylation activity of Endo-S2 and Endo-S2M3 enzymes on diAz-LacNAc-ox substrate under the same enzyme amount and different reaction times.

[0592] Table 23 Antibody transglycosylation activity of Endo-S2 and Endo-S2M3 enzymes on diAz-LacNAc-ox substrate under different reaction conditions

[0593] The results are shown in Figure 23. As can be seen from Figure 23, with the extension of reaction time, the mutant enzyme Endo-S2M3 shows lower and slower hydrolysis activity than Endo-S2 under various enzyme amounts, which is more conducive to process production.

[0594] Note: In theory, the target product with a DAR value of 4 is usually obtained by using diAz-LacNAc-ox. In Figure 22 and Figure 23, the DAR value can be recorded as 2 when normalized with respect to the use of diAz-LacNAc-ox.

[0595] Comparative Example 1 Preparation of antibody conjugate drug with site-specific remodeling using MTG enzyme

[0596] Examples 1-3 have demonstrated the applicability of Endo S2 enzyme and Endo S2M3 enzyme in the process of the present application. On this basis, in the present example, the applicability of other types of enzymes in the process of the present application is studied.

[0597] Specifically, using the production process of the present application and the production process in the prior art described in Example 3, respectively, using bevacizumab cell supernatant directly, or using purified bevacizumab cell supernatant as raw material, and in order to avoid the steric hindrance of N297 site, first, Endo S2 is added to the reaction system to remove the glycosylation side chain of the site. Then, glutamine transaminase (MTG, Zedira, hereinafter also referred to as "MTG" enzyme) and small molecule drug (small molecule drug replaced with "Gly3-VC-PAB-MMAE", commercially available, Haoyuan Pharmaceutical, Bioconjugate Chem. 2024, 35, 465) are added to the reaction system at the same time to prepare antibody conjugate drug (ADC) with site-specific remodeling, reference Bioconjugate Chem. 2020, 31, 1070.

[0598] Using the prior art process, using bevacizumab cell supernatant as raw material, bevacizumab is obtained by purifying Y, first, Endo S2 is added to the reaction system to react, then MTG enzyme and small molecule are added. Endo S2 is used to remove the glycosylation side chain of the site, and the antibody with the removed sugar side chain is then coupled with small molecule Gly3-VC-PAB-MMAE at Q295 site under the catalysis of MTG enzyme. The specific reaction conditions are as follows, and the ADC sample coupled by the prior art process is prepared by UF / DF.

[0599] In the process of the application, the bevacizumab cell supernatant is directly used, Endo S2 is added in the reaction system to remove the glycosylation side chain of the site, then MTG enzyme and small molecules are added, and the antibody with the sugar side chain removed is coupled with small molecules Gly3-VC-PAB-MMAE at the Q295 site under the catalysis of MTG enzyme. The reaction conditions are as follows, and the ADC sample coupled by the process of the application is prepared after the reaction solution is purified by X.

[0600] Table 24 Experimental conditions of MTG enzyme-mediated coupling reaction

[0601] Experimental results: the coupled ADC samples prepared by the new process and the original process are analyzed by RP-HPLC, and the results are as follows. The DAR value of the coupled sample prepared by the original process (antibody after purification for coupling) is 1.88, while the DAR value of the sample prepared by the new process (cell supernatant directly coupled) is 1.27. Under the same MTG enzyme reaction conditions, the coupling efficiency catalyzed by MTG enzyme in the new process is obviously lower than that in the original process. Therefore, the coupling efficiency of the site-coupled drug prepared by MTG enzyme in the new process is not up to the requirements, and therefore, the antibody-coupled drug based on the glycoside endo-enzyme Endo-S2 and its mutants in the application has obvious specific advantages, the DAR value is uniform, and the coupling efficiency has no obvious difference compared with the original process.

[0602] Application of the new process in chemical coupling (cysteine coupling) in Comparative Example 2

[0603] The bevacizumab cell supernatant is used as the raw material, the cell supernatant is purified by Y in the original process to obtain bevacizumab, the purified bevacizumab is reduced by TCEP at 25°C for 2h, DMSO and small molecules VC-MMAE are added after reduction, and the coupling reaction is carried out at 25°C for 2h, then the small molecules are removed by UF / DF to prepare the cysteine coupling sample of the original process. In the new process, the cell supernatant is directly subjected to TCEP reduction reaction and small molecule VC-MMAE coupling reaction, and the reduction and coupling reaction conditions are the same as those of the original process. After the reaction is completed, the cysteine coupling sample prepared by the new process is obtained after the purification step X. The specific experimental conditions are shown in Tables 25 and 26.

[0604] Table 25 Antibody reduction reaction experimental conditions

[0605] Table 26 Antibody coupling reaction experimental conditions

[0606] Experimental results: the cysteine conjugated samples prepared by the two processes were analyzed by RP-HPLC, and the results are shown in Figure 18. The DAR value of the cysteine conjugated sample prepared by the original process (antibody after purification for conjugation) was 3.53, while the DAR value of the sample prepared by the new process (direct conjugation of cell supernatant) was 0.02, and almost no small molecules were conjugated, indicating that the new process is not suitable for cysteine conjugation.

[0607] The foregoing describes exemplary embodiments of the present application, and those skilled in the art should understand that these disclosures are only exemplary, and various other substitutions, adaptations and modifications can be made within the scope of the present application. Therefore, the present application is not limited to the specific embodiments listed herein.

Claims

1. A method for preparing a protein molecule having an Fc region with homogenous sugar chains with site-directed remodeling, comprising the following steps: a) providing a liquid containing a protein molecule having an Fc region with heterogeneous sugar chains expressed by a eukaryotic cell before purification; b) adding a disaccharide linker; c) adding a glycoside hydrolase having a specific activity of hydrolyzing the heterogeneous sugar chains in the glycoprotein and catalyzing a transglycosylation reaction, and reacting; thereby obtaining a solution containing a protein molecule having an Fc region with homogenous sugar chains with site-directed remodeling.

2. A method for preparing a protein molecule having an Fc region with homogenous sugar chains with site-directed remodeling, comprising the following steps: a) providing a liquid containing a protein molecule having an Fc region with heterogeneous sugar chains expressed by a eukaryotic cell before purification; preferably, the protein molecule having an Fc region with heterogeneous sugar chains is a Y-type antibody, an Fc fusion protein or a heavy chain antibody; for example, the liquid containing a protein molecule having an Fc region with heterogeneous sugar chains expressed by a eukaryotic cell before purification is a cell supernatant; b) adding a disaccharide linker; c) adding a glycoside hydrolase having a specific activity of hydrolyzing the heterogeneous sugar chains in the glycoprotein and catalyzing a transglycosylation reaction, and reacting; thereby obtaining a solution containing a protein molecule having an Fc region with homogenous sugar chains with site-directed remodeling. In formula I, G ring represents a monosaccharide molecule selected from galactose, N-acetyl galactose, mannose and glucose, which is connected to the 4 position of 1,2-cyclic N-acetyl-D-glucosamine by a glycosidic bond, the glycosidic bond being a 1,4 glycosidic bond, a 2,4 glycosidic bond or a 3,4 glycosidic bond; Z-Y-X- represents a substituent on the G ring, the substitution position of Z-Y-X- being any position other than the 1 position of the G ring of the monosaccharide molecule, for example, the substitution position of Z-Y-X- is the 2 position, the 3 position, the 4 position, the 5 position or the 6 position of the G ring of the monosaccharide molecule, preferably the 6 position; wherein, in structure Z-Y-X-, Z-Y- can be present or absent, when Z-Y- is absent, X is an aldehyde group, -NH2, -CH2-NH2, -COOH, -N3, -CH2-N3; Y is a divalent linker or a multivalent linker connecting X and Z, b) adding a disaccharide linker of the formula I; preferably, Y is selected from the following groups: -(CH2)m-(CH-w)n-, -(CH2-CH2-O)m-(CH-w)n-, -NH-, wherein m and n are independently selected from integers between 0-30, and w is a hydrogen atom or a polyethylene glycol structure of different lengths; Z is selected from the following reactive groups: azide residue, aldehyde residue, thiol residue, alkyne residue, alkene residue, halogen residue, tetrazine residue, nitroso compound residue, hydroxylamine residue, nitrile residue, hydrazine residue, ketone residue, boronic acid residue, cyanobenzothiazole residue, allyl residue, phosphine residue, maleimide residue, disulfide residue, thioester residue, alpha-halo carbonyl residue, isonitrile residue, staudinger ketone residue, conjugated diene residue, cycloalkyne residue and cycloalkene residue; wherein n is an integer from 1 to 30, m1, m2 each represents an integer from 1 to 12, and R1 and R2 are each independently selected from H, -CH3, -CH2CH3, cyclopropyl or cyclobutyl; X is selected from the group consisting of -CH2-, -CH2-O-, -CH2-Se-, -CO-NH-, -ON=CH-, -CONH-N=CH-, -NHCH2-, -CH=CH- or nothing, and the following structures: c) adding a glycoside hydrolase having a specific activity of hydrolyzing the heterogeneous sugar chains in the glycoprotein and catalyzing a transglycosylation reaction, and reacting, thereby obtaining a solution containing a protein molecule having an Fc region with homogenous sugar chains with site-directed remodeling. ​ ​ Alternatively, Z is selected from the following groups: ​ ​ ​ 3. The method according to claim 1 or 2, further comprising: d) purifying the solution of protein molecules comprising Fc regions with homogeneous sugar chains with site-directed remodeling, e.g., affinity chromatography (e.g., protein A affinity chromatography, protein G affinity chromatography, or protein L affinity chromatography); or, affinity chromatography (e.g., protein A affinity chromatography, protein G affinity chromatography, or protein L affinity chromatography) and anion chromatography and / or cation chromatography; or, affinity chromatography (e.g., protein A affinity chromatography, protein G affinity chromatography, or protein L affinity chromatography) and low pH incubation, anion chromatography, cation chromatography, nanofiltration, and / or ultrafiltration / diafiltration; e.g., purifying in the following order: affinity chromatography, low pH incubation, anion chromatography, cation chromatography, nanofiltration, and ultrafiltration / diafiltration; or affinity chromatography, low pH incubation, nanofiltration, anion chromatography, cation chromatography, and ultrafiltration / diafiltration; or affinity chromatography, low pH incubation, anion chromatography, nanofiltration, cation chromatography, and ultrafiltration / diafiltration.

4. The method of claim 1 or 2, wherein when G in Formula I is galactose, then in Formula I the structure is selected from: wherein, l, m, n are each independently an integer from 0 to 30, ml, m2 each represent an integer from 1 to 12; wherein n is an integer from 0 to 30; i) preferably, when G in formula I is galactose, then the structure is selected from: wherein m is an integer from 0 to 30, e.g., m is an integer from 0 to 25; l is an integer from 0 to 30, e.g., l is an integer from 0 to 25; n is an integer from 0 to 30, e.g., n is an integer from 0 to 25; For example, m = 0, l + n = 2; Preferably, when G in formula I is galactose, then in formula I the structure is selected from: wherein m is an integer from 0 to 30, e.g., m is an integer from 0 to 25; l is an integer from 0 to 30, e.g., l is an integer from 0 to 25; n is an integer from 0 to 30, e.g., n is an integer from 0 to 25; For example, More preferably, when G in formula I is galactose, then in formula I the structure is: Thus, the disaccharide linker of Formula I is Alternatively, preferably when G in formula I is galactose, then in formula I the structure is selected from: wherein m is an integer from 0 to 30, e.g., m is an integer from 0 to 25; l is an integer from 0 to 30, e.g., l is an integer from 0 to 25; n is an integer from 0 to 30, e.g., n is an integer from 0 to 25; For example, l = 0, m = 0, n = 0 ii) preferably, when G in formula I is galactose, then in formula I the structure is selected from: wherein m1, m2 each represents an integer from 1 to 12; For example, Thus, the disaccharide linker of Formula I is iii) preferably, when G in formula I is galactose, then in formula I the structure is selected from: wherein m is an integer from 0 to 30, e.g., m is an integer from 0 to 25; l is an integer from 0 to 30, e.g., l is an integer from 0 to 25; n is an integer from 0 to 30, e.g., n is an integer from 0 to 25; For example, e.g., wherein l, m, n are 0; or wherein l, m, n are 0; iv) preferably, when G in formula I is galactose, then The structure is: v) preferably, when G in formula I is galactose, then in formula I the structure is selected from: wherein R is aryl, e.g., phenyl or naphthyl; Preferably, when G in formula I is galactose, then in formula I the structure is selected from: wherein n is an integer from 0 to 30, e.g., n is an integer from 0 to 25, preferably n is 1; Preferably, when G in formula I is galactose, then in formula I the structure is selected from:

5. The method according to claim 1 or 2, wherein when G in formula I is mannose, G is attached to the 4-position of 1,2-cyclo-N-acetyl-D-glucosamine via a 1,4 glycosidic bond, and the substituent Z-Y-X- on the 6-position of the G ring is as defined in claim 4, except that G is replaced with mannose; For example, when G in Formula I is mannose, then the structure is selected from: wherein, l, m, n are each independently an integer from 0 to 30, ml, m2 each represent an integer from 1 to 12; n is an integer from 0 to 30, e.g., n is an integer from 0 to 10; Preferably, wherein when G in Formula I is mannose, then in Formula I the structure is selected from: Preferably, when G in formula I is mannose, then in formula I the structure is selected from: wherein m is an integer from 0 to 30, e.g., m is an integer from 0 to 25; l is an integer from 0 to 30, e.g., l is an integer from 0 to 25; n is an integer from 0 to 30, e.g., n is an integer from 0 to 25; For example, 6. The method according to claim 1 or 2, wherein when G in Formula I is glucose, G is connected to the 4-position of the 1,2-cyclic N-acetyl-D-glucosamine through a 1,4 glycosidic bond, the substituent Z-Y-X- on the 6-position of the G ring is as defined in claim 4, except that G is replaced by glucose; For example, when G in Formula I is glucose, then the structure is selected from: wherein l, m, n are each independently an integer from 0 to 30, ml, m2 each represent an integer from 1 to 12; n is an integer from 0 to 30, for example, n is an integer from 0 to 10; Preferably, wherein when G in formula I is glucose, then in formula I the structure is selected from: Preferably, when G in formula I is glucose, then in formula I the structure is selected from: wherein, m is an integer from 0 to 30, for example, m is an integer from 0 to 25; l is an integer from 0 to 30, for example, l is an integer from 0 to 25; n is an integer from 0 to 30, for example, n is an integer from 0 to 25; For example, 7. The method according to any one of claims 2-6, wherein the cell supernatant is a cell supernatant of eukaryotic cells cultured (e.g., fed-batch cultured) to secrete a protein molecule having an Fc region with heterogeneous sugar chains, and the cell culture fluid is collected after the end of the culture, and the cell supernatant containing the protein molecule having the Fc region with heterogeneous sugar chains is collected after centrifugation and / or filtration.

8. The method according to claim 1 or 2, wherein the endoglycosidase is an endoglycosidase S2 (Endo-S2) as shown in SEQ ID NO: 1, an endoglycosidase S2M3 (Endo-S2M3) as shown in SEQ ID NO: 2, or an endoglycosidase Endo-S2 mutant 1 as shown in SEQ ID NO:

3.

9. A two-step method for preparing a protein molecule having an Fc region with a homogenous sugar chain with site-directed remodeling conjugated drug, comprising: Step (I): performing the method according to any one of claims 2-8 to obtain a purified protein molecule having an Fc region with a homogenous sugar chain with site-directed remodeling; Step (II): adding a small molecule drug-linker of Formula IV to the purified protein molecule having an Fc region with a homogenous sugar chain with site-directed remodeling obtained in step (I) to obtain a protein molecule having an Fc region with a homogenous sugar chain with site-directed remodeling conjugated drug; E-L-D (Formula IV) E is a corresponding group that undergoes orthogonal reaction with an azido group, which is selected from linear alkynyl, DBCO-like structure, BCN-like structure, L represents a linker, L is preferably selected from -(CH2)a-(OCH2CH2)b-(NHCO)n-(CH2)c-, or from the following groups: wherein V and W are bifunctional linkers, including structures with lysine, propargyl glycine bifunctional linkers, for example, L is selected from: wherein a, b, c, d and e are each independently selected from an integer between 0-30, m, n are 0 or 1, R3and R4are each independently selected from CH3-, (CH 3)2 CH-, PhCH2, NH2(CH2)4-, NH2CONH(CH2)3-, R is selected from an azidizable monosaccharide, disaccharide, oligosaccharide or different length of azido-PEG structure or combination of PEG and chain or cyclic monosaccharide, disaccharide, oligosaccharide, wherein the oligosaccharide includes branched oligosaccharide chains; represents a linking site; For example, the L is -Lysm-PEGn-GGFG-; or Lysm-PEGn-VC-PAB-, m, n are independently selected from an integer between 0-30; the L is -PEG4-vc-PAB- or -PEG4-GGFG-; D represents a cytotoxic or cytostatic drug, For example, (1) microtubule inhibitors / disruptors: such as, but not limited to, auristatins (e.g., MMAE or MMAF), maytansine derivatives (e.g., DM1, DM2, DM4), Tubulysins, Cryptomycins, anti-mitotic EG5 inhibitors (e.g., Kinesin Spindle Protein (KSP) inhibitors); (2) DNA damaging agents: for example, but not limited to, Pyrrolobenzodiazepines (e.g., pyrrolo[2,1-c][1,4]benzodiazepine (PBD)), Duocarmycin, Indolinobenzodiazepine; Duocarmycins; Calicheamicin; (3) topoisomerase inhibitors: for example, but not limited to, camptothecins (e.g., irinotecan and its derivative Dxd); (4) Others: Apoptosis inducers (Bcl-xL inhibitors), thailanstatin and its analogues, amatoxins, nicotinamide phosphoribosyltransferase (NAMPT) inhibitors, Kameycin; Preferably, the small molecule drug is a small molecule drug-linker selected from the group consisting of: Preferably, the small molecule drug is a small molecule drug-linker selected from the group consisting of: wherein m is an integer from 0 to 5; n is an integer from 0 to 30.

10. The method of claim 9, further comprising purifying the solution of the protein molecule conjugate drug comprising Fc region with homogeneous glycan having site-directed remodeling, for example, by ultrafiltration / diafiltration.

11. A one-step method for preparing a protein molecule conjugate drug comprising Fc region with homogeneous glycan having site-directed remodeling, comprising: a) providing a liquid comprising eukaryotic cell-expressed protein molecule comprising Fc region with heterogeneous glycan before purification; preferably, the protein molecule comprising Fc region with heterogeneous glycan is a Y-type antibody, an Fc fusion protein or a heavy chain antibody; for example, the liquid comprising eukaryotic cell-expressed protein molecule comprising Fc region with heterogeneous glycan before purification is a cell supernatant; for example, the cell supernatant is a cell culture supernatant of eukaryotic cells secreting-expressed protein molecule comprising Fc region with heterogeneous glycan, which is collected after centrifugation and / or filtration of the cell culture supernatant after the end of the culture; b) adding a disaccharide-small molecule drug conjugate of Formula V in formula V, G ring, X, Y, L, D are as defined in formula I and formula IV, Z' is independently -(CH2)p- wherein p is an integer from 1 to 5, or Z' is absent, or is a group that can react with the Z group in claim 1, For example, Z' is selected from the following groups: wherein R1 and R2 are each independently selected from H, -CH3, -CH2CH3, cyclopropyl or cyclobutyl; represents the connection position, Preferably, the disaccharide-small molecule drug conjugate is selected from the group consisting of: c) adding a glycoside endo-enzyme having the activity of specifically hydrolyzing the heterogeneous glycan in the glycoprotein and catalyzing transglycosylation reaction to react, thereby obtaining a solution of the protein molecule conjugate drug comprising Fc region with homogeneous glycan having site-directed remodeling.

12. The method of claim 11, further comprising purifying the solution of the protein molecule conjugate drug comprising Fc region with homogeneous glycan having site-directed remodeling, for example, affinity chromatography, low pH incubation, anion chromatography, cation chromatography, nanofiltration and / or ultrafiltration / diafiltration; preferably, the solution is purified in the following order: affinity chromatography, low pH incubation, anion chromatography, cation chromatography, nanofiltration and ultrafiltration / diafiltration; or affinity chromatography, low pH incubation, nanofiltration, anion chromatography, cation chromatography and ultrafiltration / diafiltration; or affinity chromatography, low pH incubation, anion chromatography, nanofiltration, cation chromatography and ultrafiltration / diafiltration.

13. The method of claim 11 or 12, wherein the glycoside endo-enzyme is the glycoside endo-enzyme S2 (Endo-S2) as shown in SEQ ID NO: 1, the glycoside endo-enzyme S2M3 (Endo-S2M3) as shown in SEQ ID NO: 2 or the glycoside endo-enzyme Endo-S2 mutant 1 as shown in SEQ ID NO:

3.

14. The method according to any one of claims 1-13, wherein the protein molecule having an Fc region with heterogeneous sugar chains is an antibody having a glycosylation site, Preferably, the antibody is an antibody having an N-glycosylation site, More preferably, the antibody is a dual-antenna antibody having an N-glycosylation site, Most preferably, the antibody is an IgG having a conserved N-glycosylation site at N297 of the Fc region, For example, the antibody is a monoclonal antibody, a polyclonal antibody, a bifunctional antibody, a trifunctional antibody, a nanobody fused with an Fc domain, a therapeutic antibody or a functional antibody of different species of origin, For example, the antibody is a human antibody, a murine antibody or a chimeric antibody; For example, the antibody is an IgG1, IgG2, IgG4; For example, the target point of the antibody is selected from the group consisting of HER2, Claudin 18.2, EGFR, TROP2, c-Met, NECTIN4, CD276, HER3, CD3, FOLR1, BCMA, CD20, DLL3, MUC1, PD-L1, ROR1, TF, CD19, CD22, CD30, CD70, CD79B, FGFs, MSLN, NT5E, TNFα, CD147, CD24, CD38, CD47, CDH3, CDK4, CDK6, CEACAM5, CLDN6, CTLA4, DDR1, DR5, FAPα, FGFR3, GPRC5D, GR, HLA-DR, ICAM1, IL2R, MELTF, ROR2, TPBG(5T4), VTCN1, ZIP6, CD33, CD25, RSV, VEGF, RANKL, VEGFR2, CTLA-4, CD52, CD319, PD-1, CD274, IgE, IL-6, IL-12, IL-2, C5, IL-17A, CD25, SLAMF7, F10, factor IXa, HAb18G, PCSK9, BLyS, IL23, α4β7, IL-4R-α, HAE, FGF23 and IL6R; preferably, the target point of the antibody is selected from the group consisting of HER2, CD20, TROP2, EGFR, PD-1; In particular, the antibody includes bevacizumab, trastuzumab, rituximab, pertuzumab, panitumumab, tremelimumab, tremelimumab, nivolumab, visilizumab, satralizumab.

15. The method of any one of claims 1-14, wherein the reaction is performed using a protein molecule having a heterogeneous glycochain of an Fc region at a final concentration of > 1 mg / mL, preferably the protein molecule having a heterogeneous glycochain of an Fc region is at a final concentration of about 1-50 mg / mL, more preferably the protein molecule having a heterogeneous glycochain of an Fc region is at a final concentration of about 1-20 mg / mL, most preferably the protein molecule having a heterogeneous glycochain of an Fc region is at a final concentration of about 1-10 mg / mL, e.g., about 2, about 3, about 4, about 5, about 6, about 7, about 8, or about 9 mg / mL.

16. The method of any one of claims 1-15, wherein the reaction is performed using a disaccharide linker (e.g., diAz-LacNAc-ox) at an equivalent ratio to the protein molecule having a heterogeneous glycochain of an Fc region of > about 20; preferably the reaction is performed using a disaccharide linker (e.g., diAz-LacNAc-ox) at an equivalent ratio to the protein molecule having a heterogeneous glycochain of an Fc region of about 20-300, more preferably the reaction is performed using a disaccharide linker (e.g., diAz-LacNAc-ox) at an equivalent ratio to the protein molecule having a heterogeneous glycochain of an Fc region of about 20-200, most preferably the reaction is performed using a disaccharide linker (e.g., diAz-LacNAc-ox) at an equivalent ratio to the protein molecule having a heterogeneous glycochain of an Fc region of about 20-160, e.g., at an equivalent ratio of about 40, about 60, about 80, about 100, about 120, or about 140.

17. The method of any one of claims 1-16, wherein the reaction is performed using an endoglycosidase at an added amount of > about 5 pg / mg, preferably the reaction is performed using an endoglycosidase at an added amount of about 5-150 pg / mg, more preferably the reaction is performed using an endoglycosidase at an added amount of about 10-80 pg / mg, most preferably the reaction is performed using an endoglycosidase at an added amount of about 10-40 pg / mg, e.g., at an added amount of about 15, about 20, about 25, about 30, or about 40 pg / mg.

18. The method of any one of claims 1-17, wherein the reaction of the endoglycosidase is performed at a pH of about 4.0-8.0, preferably the reaction of the endoglycosidase is performed at a pH of about 4.5-7.7, more preferably the reaction of the endoglycosidase is performed at a pH of about 5.0-7.5, most preferably the reaction of the endoglycosidase is performed at a pH of about 5.3-7.2, e.g., at a pH of about 6, about 6.5, or about 7.

0.

19. The method according to any one of claims 1-18, wherein the reaction of the endoglycosidase is performed at a reaction temperature of about 5°C to about 50°C, preferably at a reaction temperature of about 6°C to about 45°C, more preferably at a reaction temperature of about 15°C to about 40°C, most preferably at a reaction temperature of about 20°C to about 37°C, for example at a reaction temperature of about 22°C, about 25°C, about 30°C, or about 35°C.

20. The method according to any one of claims 1-19, wherein the reaction of the endoglycosidase is performed at a reaction time of about 0.1 hour to about 30 hours, preferably at a reaction time of about 0.2 hour to about 25 hours, more preferably at a reaction time of about 0.5 hour to about 8 hours, most preferably at a reaction time of about 0.75 hour to about 4 hours, for example at a reaction time of about 0.8 hour, about 1 hour, about 1.5 hour, or about 2 hours.

21. The method according to any one of claims 1-20, for use in the glycoengineering of an antibody or for use in the preparation of an antibody-drug conjugate.

22. A protein molecule having a Fc region with site-directed remodeling of homogeneous glycochains or a solution comprising a protein molecule having a Fc region with site-directed remodeling of homogeneous glycochains, prepared according to the method of any one of claims 1-8.

23. A protein molecule having a Fc region with site-directed remodeling of homogeneous glycochains conjugated to a drug or a solution comprising a protein molecule having a Fc region with site-directed remodeling of homogeneous glycochains conjugated to a drug, prepared according to the method of any one of claims 9-22.

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