A method, device and application for regulating antibody glycosylation modification

Abstract

The application discloses an antibody glycosylation modification regulation method, device and application, relates to the technical field of biopharmaceuticals and protein engineering, and realizes directional regulation of modification types, modification sites and modification proportions of N-glycan and O-glycan of the antibody through three core processes of constructing a glycosyltransferase engineering strain, optimizing a fermentation culture system and precisely regulating modification reaction conditions; solves the technical problems of low modification efficiency, poor specificity and insufficient product uniformity in the existing modification method, can improve the target glycan modification proportion to more than 90%, the modification product purity is greater than or equal to 98%, is suitable for glycosylation modification optimization of therapeutic monoclonal antibodies, bispecific antibodies and antibody drug conjugates, significantly improves the biological activity and pharmacokinetic performance of the antibody, wherein the ADCC activity is improved by 3-5 times, and the CDC activity is improved by 2-4 times, and has the advantages of strong controllability, good adaptability to large-scale production and high cost-effectiveness.

Description

Technical Field

[0001] This invention relates to the fields of biopharmaceutical and protein engineering technology, specifically to a method, apparatus, and application for regulating antibody glycosylation modification. Background Technology

[0002] Antibody glycosylation modification, as a key post-translational modification process, directly determines the functional properties and clinical efficacy of antibodies. For example, galactosylation modification of the antibody Fc fragment can enhance complement-dependent cytotoxicity (CDC), while reduced fucosylation levels can significantly enhance antibody-dependent cytotoxicity (ADCC). Abnormal glycosylation (such as excessive accumulation of high-mannose glycans) can lead to a shortened antibody half-life and increased immunogenicity. Therefore, targeted regulation of antibody glycosylation modification is one of the core technologies in antibody drug development.

[0003] Existing antibody glycosylation modification methods mainly include cell line engineering, culture medium optimization, and in vitro enzymatic modification. While cell line engineering (such as knocking out the fucosyltransferase gene) can reduce fucosylation levels, it suffers from slow cell growth and decreased antibody expression. Culture medium optimization (such as adding glycosylation precursors) has low modification efficiency, with the target glycan ratio typically below 60%, and poor product uniformity. In vitro enzymatic modification requires prior antibody purification followed by in vitro glycan modification, which is cumbersome, costly, and difficult to scale up. Furthermore, existing methods primarily focus on N-glycan modification, and the technology for regulating O-glycans (such as mucin-type O-glycans) is still immature, failing to meet the modification requirements of novel antibody drugs such as bispecific antibodies and ADCs. Summary of the Invention

[0004] This invention develops a method for regulating antibody glycosylation modification that is highly efficient, specific, and scalable, which is of great significance for promoting the performance optimization and industrialization of antibody drugs. This method is achieved through the following technical solution: A method for regulating antibody glycosylation modification includes the following steps: S1. Construction of glycosyltransferase engineered strains: Select a host strain and introduce different enzyme genomes into the host genome in tandem through homologous recombination to construct recombinant engineered strains; S2. Optimization of fermentation culture system: The recombinant engineered strain was inoculated into the modified BMGY medium, a mixed solvent was added to the modified BMGY medium, and methanol was added after culture for induction. S3. Modification reaction regulation: When the antibody expression level in the fermentation broth reaches the set concentration range, UDP-galactose and GDP-fucose are added as glycosyl donors. The reaction temperature and reaction time are adjusted, and the concentration of glycosyl donors is detected at the same time. When the concentration is lower than the standard set value, the supplementary action is performed to complete the antibody glycosylation modification.

[0005] Furthermore, Pichia pastoris GS115 was selected as the host strain; the genomes of different enzymes include at least: β-1,4-galactosyltransferase gene, α-1,6-fucosyltransferase gene and UDP-glycosyl donor synthase gene; wherein, the UDP-glycosyl donor synthase gene is UDP-glucose pyrophosphorylase gene and UDP-galactose epimerase gene.

[0006] Furthermore, the process of constructing the recombinant engineered strain is as follows: homologous recombination uses the pPICZαA vector as the expression vector, inserts the pPICZαA vector, and introduces the AOX1 strong promoter and His tag into the expression vector; the recombinant vector is transformed into Pichia pastoris GS115, and the engineered strain is obtained by Zeocin resistance screening and Western blot verification. The expression level of β-1,4-GalT in the engineered strain changes, and the expression level of α-1,6-FucT is regulated by the concentration of inducer.

[0007] Furthermore, the mixed solvent added to the modified BMGY medium includes at least: Mn²⁺ at a final concentration of 0.5-2 mmol / L, Co²⁺ at a final concentration of 0.1-0.5 mmol / L, and a glucose-galactose mixed carbon source at a concentration of 5-15 g / L, with a glucose to galactose molar ratio of 1:2-1:4.

[0008] Furthermore, the modified BMGY medium also contains: yeast extract at a final concentration of 1-3 g / L, peptone at 2-5 g / L, and potassium phosphate buffer at 0.01-0.03 mol / L, with the potassium phosphate buffer having a pH of 6.0-6.5.

[0009] Furthermore, during induction: incubate at 30℃ and 200-250 rpm until OD... 600 When the pH reaches 8-12, methanol with a final concentration of 0.5%-1.5% is added for induction. During the induction process, the pH is maintained at 6.0-6.5 and the dissolved oxygen content is ≥30%.

[0010] Furthermore, in S3, the concentration range is set to 50-80 mg / L, and the final concentrations of UDP-galactose and GDP-fucose are 0.01-0.05 mol / L and 0.005-0.02 mol / L, respectively. The reaction temperature is adjusted to 25-28℃, and the reaction time is 8-12 h. The concentration of glycosyl donors is detected by an online monitoring system. The standard setting value is 20% of the initial value. The process of replenishing is to replenish glycosyl donors to 50%-60% of the initial concentration.

[0011] Furthermore, the online monitoring system includes at least: a high-performance liquid chromatography module and a glucose oxidase sensor; The high-performance liquid chromatography (HPLC) module uses an amino column with dimensions of 4.6 mm × 250 mm and a particle size of 5 μm. The mobile phase is acetonitrile-water, the flow rate is 1.0 mL / min, and the column temperature is 30 °C. It is used to detect the concentration of glycosyl donors. The glucose oxidase sensor is used to monitor the glucose concentration in the culture medium in real time with a monitoring accuracy of ±0.1 g / L.

[0012] An antibody glycosylation modification regulation device for realizing antibody glycosylation modification regulation method includes a strain culture module, a glycosylation donor supply module, a reaction regulation module, an online monitoring module, and a product purification module. The strain culture module is a 5-50L fermenter equipped with a temperature control unit, a pH adjustment unit, and a dissolved oxygen monitoring unit. The glycosylation donor supply module is an automatic metering pump that replenishes the glycosylation donor based on online monitoring data. The reaction regulation module includes a stirring device and a temperature feedback unit. The online monitoring module integrates an HPLC module and a glucose oxidase sensor for real-time transmission of detection data to the control system. The product purification module includes a Protein A affinity chromatography column and an ion exchange chromatography column for purifying the modified antibody product.

[0013] An application of an antibody glycosylation modification regulation method in antibody drug development includes: regulating the ADCC activity of monoclonal antibodies, optimizing the stability of ADCs, and improving the immunogenicity of bispecific antibodies. In this application, the modified antibody products are subjected to biological activity detection and pharmacokinetic evaluation to ensure that the antibody drugs meet the standards for clinical application.

[0014] This invention provides a method, apparatus, and application for regulating antibody glycosylation modification, which has the following beneficial effects: (1) High modification efficiency: Through the synergistic optimization of engineered strains and fermentation system, the target glycan modification ratio can reach more than 90%, which is much higher than the existing methods (≤60%), and the modification products have good uniformity with batch-to-batch RSD ≤3%; (2) High specificity: It can directionally regulate the galactosylation and fucosylation levels of N-glycans, and at the same time realize the modification regulation of O-glycans (such as mucin-type O-glycans), meeting the modification needs of different antibody drugs; (3) Good adaptability to scale: Based on the integrated modification process of fermenter, continuous production at a scale of 5-50L can be achieved without the need for The complicated steps of in vitro enzymatic modification shorten the production cycle to 48-72 hours; (4) High cost-effectiveness: By combining the synthesis of endogenous glycosyl donors with the precise supplementation of exogenous donors, the amount of glycosyl donors used is reduced (40%-50% less than in vitro enzymatic methods), while the antibody expression level is increased (20%-30% higher than wild-type strains), significantly reducing production costs; (5) Great application value: The modified antibodies have significantly improved biological activity, stability, and immunogenicity, and can be directly applied to the preclinical research and industrial production of antibody drugs, promoting the optimization of antibody drug performance. Attached Figure Description

[0015] Figure 1 This is a simplified flowchart illustrating the antibody glycosylation modification regulation method in this invention. Figure 2 This is a DNA sequence diagram of β-1,4-GalT in this invention; Figure 3 This is a DNA sequence diagram of α-1,6-FucT in this invention. Detailed Implementation

[0016] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0017] Example 1: Please see Figures 1 to 3 This embodiment provides a method for regulating antibody glycosylation modification. By constructing an efficient glycosyltransferase engineered strain, optimizing the fermentation culture system, and establishing a precise reaction regulation mechanism, the method achieves targeted regulation of antibody glycosylation modification. The specific technical solution is as follows: Construction of S1 glycosyltransferase engineered strains Pichia pastoris GS115 was selected as the host strain due to its high secretory expression capacity and glycosylation modification system resembling that of mammalian cells. Using homologous recombination technology, the β-1,4-galactosyltransferase (β-1,4-GalT) gene (regulating N-glycan galactosylation), the α-1,6-fucosyltransferase (α-1,6-FucT) gene (regulating N-glycan fucosylation), and the UDP-glycosyl donor synthase genes (UGPase and GALE, ensuring the in vivo synthesis of UDP-galactose, GDP-fucose, and other glycosyl donors) were tandemly inserted into the pPICZαA vector. The vector incorporated a strong AOX1 promoter (methanol-induced expression) and a His tag (facilitating enzyme purification and detection). The recombinant vector was transformed into Pichia pastoris GS115, and the protein was screened for Zeocin resistance and Western blot analysis. blot verification yielded an engineered strain; the expression level of β-1,4-GalT in this engineered strain was increased by 5-8 times, and the expression level of α-1,6-FucT could be regulated by methanol concentration (0.5%-1.5%), thus achieving flexible adjustment of fucosylation level (adjustable from 10% to 80%).

[0018] S2, Optimization of fermentation culture system Improved traditional BMGY culture medium by adding Mn 2+(0.5-2 mmol / L) and Co 2+ (0.1-0.5 mmol / L), activates glycosyltransferase activity (Mn 2+ It can enhance the catalytic efficiency of β-1,4-GalT, Co 2+ It can stabilize the spatial structure of α-1,6-FucT; it uses a glucose-lactose mixed carbon source (molar ratio 1:2-1:4), with glucose as the initial carbon source to promote cell growth and galactose as a glycosyl precursor to increase intracellular glycosyl donor reserves; it supplements yeast extract (1-3g / L) and peptone (2-5g / L) to provide nitrogen source and growth factors to ensure high-density cell culture; during fermentation, when cultured at 30℃ and 200-250rpm to OD600=8-12, methanol is added to induce antibody and glycosyltransferase expression, while maintaining pH 6.0-6.5 (adjusted by automatically adding ammonia or phosphate) and dissolved oxygen ≥30% (controlled by adjusting stirring rate and aeration) to ensure a stable environment for cell growth and protein expression.

[0019] S3, Precise Control of Modification Reactions When the antibody expression level in the fermentation broth reaches 50-80 mg / L (rapidly detected by Protein A affinity chromatography), the glycosylation modification reaction is initiated: UDP-galactose (0.01-0.05 mol / L) and GDP-fucose (0.005-0.02 mol / L) are added as exogenous glycosyl donors to compensate for insufficient intracellular donors; the reaction temperature is adjusted to 25-28℃ to balance glycosyltransferase activity and cell metabolic rate; during the reaction, the concentration of glycosyl donors is monitored in real time using an online HPLC module (detection frequency 1 time / 2h); when the concentration is lower than 20% of the initial value, an automatic metering pump replenishes the glycosyl donors to 50%-60% of the initial concentration to avoid modification interruption due to insufficient donors; after 8-12 hours of reaction, samples are taken to detect the glycan structure (using the HPLC-MS method in Example 1); the reaction is terminated when the target glycan ratio is ≥90%.

[0020] S4. Product Purification and Application The modified antibody product is processed through a product purification module: first, it is captured and impurity proteins are removed using a Protein A affinity chromatography column (elution buffer: 0.1 mol / L citrate buffer, pH 3.0-3.5); then, it is further purified using an ion exchange chromatography column (DEAE-Sepharose FF, elution gradient: 0-0.5 mol / L NaCl) to obtain modified antibodies with a purity ≥98%. The purified antibodies are used in antibody drug development: for monoclonal antibodies, they can increase ADCC activity by 3-5 times and CDC activity by 2-4 times; for ADCs, they can reduce non-specific modifications at drug conjugation sites and improve ADC stability (half-life extended by 20%-30%); for bispecific antibodies, they can reduce immunogenicity caused by abnormal glycosylation (increase the incidence of drug-resistant antibodies by 15%-25%).

[0021] Example Explanation: The specific method for regulating galactosylation modification of monoclonal antibodies is as follows: Construction of engineered strains: The β-1,4-GalT gene, UGPase gene, and GALE gene are tandemly inserted into the pPICZαA vector, transformed into Pichia pastoris GS115, and engineered strains with a 6-fold increase in β-1,4-GalT expression are screened; Fermentation culture: 1 mmol / L of modified BMGY medium is added... 2 + 0.3 mmol / L Co 2+ A mixed carbon source of 10 g / L glucose-galactose (molar ratio 1:3) was used, and the mixture was cultured at 30℃ and 220 rpm until OD600=10, followed by induction with 1% methanol. Modification and regulation: When the antibody expression level reached 60 mg / L, 0.03 mol / L UDP-galactose was added, and the reaction was carried out at 26℃ for 10 h. The concentration of UDP-galactose was monitored by online HPLC, and replenished to 50% of the initial concentration as needed. Product detection and application: The modified antibody was purified to a purity of 98.5%. HPLC-MS analysis showed that the proportion of galactosylated glycans (G1 and G2 types) was 92.3%. Biological activity assays showed that ADCC activity increased by 4.2 times and CDC activity increased by 3.1 times, meeting the clinical application standards for therapeutic monoclonal antibodies.

[0022] By adopting the above technical solution, this solution has the following advantages: (1) High modification efficiency: Through the synergistic optimization of engineered strains and fermentation system, the target sugar chain modification ratio can reach more than 90%, which is much higher than the existing methods (≤60%), and the modification products have good uniformity with batch-to-batch RSD ≤3%; (2) High specificity: It can directionally regulate the galactosylation and fucosylation levels of N-glycans, and at the same time achieve the modification regulation of O-glycans (such as mucin-type O-glycans) to meet the modification requirements of different antibody drugs; (3) Good adaptability to scale: Based on the integrated modification process of fermenter, continuous production of 5-50L scale can be achieved without the cumbersome steps of in vitro enzymatic modification, and the production cycle is shortened to 48-72h. (4) High cost-effectiveness: By combining endogenous glycosyl donor synthesis with precise supplementation of exogenous donors, the amount of glycosyl donors used is reduced (40%-50% less than in vitro enzymatic methods), while antibody expression is increased (20%-30% more than wild-type strains), significantly reducing production costs; (5) Great application value: The modified antibody has significantly improved biological activity, stability and immunogenicity, and can be directly applied to the preclinical research and industrial production of antibody drugs, promoting the optimization of antibody drug performance.

[0023] Example 2: Based on Example 1, this embodiment also provides an antibody glycosylation modification device for implementing the method described in Example 1, including a strain culture module, a glycosylation donor supply module, a reaction control module, an online monitoring module, and a product purification module; the strain culture module is a 5-50L fermenter, equipped with a temperature control unit (temperature control accuracy ±0.1℃), a pH adjustment unit, and a dissolved oxygen monitoring unit; as the names suggest, the temperature control unit is existing equipment for temperature control, and the pH adjustment unit and dissolved oxygen monitoring unit are also existing equipment for controlling pH and monitoring dissolved oxygen, respectively; the glycosylation donor supply module is an automatic metering pump that can accurately replenish the glycosylation donor according to online monitoring data; the reaction control module includes a stirring device (stirring speed adjustable from 50-300rpm) and a temperature feedback unit, similarly, the temperature feedback unit is existing equipment or circuit structure for providing temperature data feedback; the online monitoring module integrates an HPLC module and a glucose oxidase sensor, which can transmit detection data to the control system in real time; the product purification module includes a Protein A affinity chromatography column and an ion exchange chromatography column for purifying the modified antibody product.

[0024] Example 3: Based on Examples 1 and 2, this example also provides the application of antibody glycosylation modification regulation methods in antibody drug development. This application includes enhancing the ADCC activity of monoclonal antibodies, optimizing the stability of ADCs, and improving the immunogenicity of bispecific antibodies. When applying this method, the modified antibody products are subjected to biological activity testing and pharmacokinetic evaluation to ensure that the antibody drugs meet the standards for clinical application.

[0025] The above embodiments can be implemented, in whole or in part, by software, hardware, firmware, or any other combination thereof. When implemented in software, the above embodiments can be implemented, in whole or in part, as a computer program product. Those skilled in the art will recognize that the units and algorithm steps of the various examples described in conjunction with the embodiments disclosed herein can be implemented in electronic hardware, or a combination of computer software and electronic hardware. Whether these functions are implemented in hardware or software depends on the specific application and design constraints of the technical solution.

[0026] The units described as separate components may or may not be physically separate. The components shown as units may or may not be physical units; they may be located in one place or distributed across multiple network units. Some or all of the units can be selected to achieve the purpose of this embodiment, depending on actual needs.

[0027] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application.

Claims

1. A method for regulating antibody glycosylation modification, characterized in that, Includes the following steps: S1. Construction of glycosyltransferase engineered strains: Select a host strain and introduce different enzyme genomes into the host genome in tandem through homologous recombination to construct recombinant engineered strains; S2. Optimization of fermentation culture system: The recombinant engineered strain was inoculated into the modified BMGY medium, a mixed solvent was added to the modified BMGY medium, and methanol was added after culture for induction. S3. Modification reaction regulation: When the antibody expression level in the fermentation broth reaches the set concentration range, UDP-galactose and GDP-fucose are added as glycosyl donors. The reaction temperature and reaction time are adjusted, and the concentration of glycosyl donors is detected at the same time. When the concentration is lower than the standard set value, the supplementary action is performed to complete the antibody glycosylation modification.

2. The antibody glycosylation modification regulation method according to claim 1, characterized in that: Pichia pastoris GS115 was selected as the host strain; The genomes of different enzymes include at least: β-1,4-galactosyltransferase gene, α-1,6-fucosyltransferase gene and UDP-glycosyl donor synthase gene; among which, the UDP-glycosyl donor synthase gene is UDP-glucose pyrophosphorylase gene and UDP-galactose epimerase gene.

3. The antibody glycosylation modification regulation method according to claim 2, characterized in that: The process of constructing recombinant engineered strains is as follows: homologous recombination uses the pPICZαA vector as the expression vector, inserts the pPICZαA vector, and introduces the AOX1 strong promoter and His tag into the expression vector; the recombinant vector is transformed into Pichia pastoris GS115, and engineered strains are obtained by Zeocin resistance screening and Western blot verification. The expression level of β-1,4-GalT in the engineered strains varies, and the expression level of α-1,6-FucT is regulated by the concentration of inducer.

4. The antibody glycosylation modification regulation method according to claim 1, characterized in that: The mixed solvent added to the modified BMGY medium shall include at least the following: Mn²⁺ at a final concentration of 0.5-2 mmol / L, Co²⁺ at a final concentration of 0.1-0.5 mmol / L, and a glucose-galactose mixed carbon source at a final concentration of 5-15 g / L, with a glucose to galactose molar ratio of 1:2-1:

4.

5. The method for regulating antibody glycosylation modification according to claim 1, characterized in that: The modified BMGY medium also contains yeast extract at a final concentration of 1-3 g / L, peptone at 2-5 g / L, and potassium phosphate buffer at 0.01-0.03 mol / L, with the potassium phosphate buffer at pH 6.0-6.

5.

6. The antibody glycosylation modification regulation method according to claim 1, characterized in that: During induction: Incubate at 30℃ and 200-250 rpm until OD... 600 When the pH reaches 8-12, methanol with a final concentration of 0.5%-1.5% is added for induction. During the induction process, the pH is maintained at 6.0-6.5 and the dissolved oxygen content is ≥30%.

7. The method for regulating antibody glycosylation modification according to claim 1, characterized in that: In S3, the concentration range is set to 50-80 mg / L. The final concentrations of UDP-galactose and GDP-fucose are 0.01-0.05 mol / L and 0.005-0.02 mol / L, respectively. The reaction temperature is adjusted to 25-28℃, and the reaction time is 8-12 h. The concentration of glycosyl donors is detected by an online monitoring system. The standard setting value is 20% of the initial value. The process of replenishing is to replenish glycosyl donors to 50%-60% of the initial concentration.

8. The antibody glycosylation modification regulation method according to claim 7, characterized in that: The online monitoring system should include at least: a high-performance liquid chromatography module and a glucose oxidase sensor; The high-performance liquid chromatography (HPLC) module uses an amino column with dimensions of 4.6 mm × 250 mm and a particle size of 5 μm. The mobile phase is acetonitrile-water, the flow rate is 1.0 mL / min, and the column temperature is 30 °C. It is used to detect the concentration of glycosyl donors. The glucose oxidase sensor is used to monitor the glucose concentration in the culture medium in real time with a monitoring accuracy of ±0.1 g / L.

9. An antibody glycosylation modification regulation device for implementing the method of any one of claims 1-8, characterized in that, The system includes a bacterial strain cultivation module, a glycosyl donor supply module, a reaction control module, an online monitoring module, and a product purification module. The bacterial strain cultivation module is a 5-50L fermenter equipped with a temperature control unit, a pH adjustment unit, and a dissolved oxygen monitoring unit. The glycosyl donor supply module uses an automatic metering pump to replenish the glycosyl donor based on online monitoring data. The reaction control module includes a stirring device and a temperature feedback unit. The online monitoring module integrates an HPLC module and a glucose oxidase sensor for real-time transmission of detection data to the control system. The product purification module includes a Protein A affinity chromatography column and an ion exchange chromatography column for purifying the modified antibody product.

10. The application of the method according to any one of claims 1-8 in antibody drug development, characterized in that, Applications include: regulating ADCC activity of monoclonal antibodies, optimizing the stability of ADCs, and improving the immunogenicity of bispecific antibodies. When applying these drugs, the modified antibody products are subjected to biological activity testing and pharmacokinetic evaluation to ensure that the antibody drugs meet the standards for clinical application.

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