Method for preparing bispecific antibodies

Abstract

The invention provides a method for preparing a bispecific antibody, and relates to the technical field of biology. The method comprises the following steps: culturing host cells expressing a bispecific antibody by adopting a perfusion culture process, in the culture process of the perfusion culture process, the culture temperature of 0-6 days is 35-36.5 DEG C, the culture temperature after 6 days is 33-35 DEG C, and the perfusion rate is 0.8-1.5 RV / day; a culture medium used in the perfusion culture process comprises an excipient composition; the excipient composition comprises the following components: 30 to 50 mmol / L of trehalose, 30 to 50 mmol / L of cane sugar, 4 to 8 g / L of glucose and 15 to 25 mmol / L of mannitol. The method shows a remarkable effect on reducing the polymer level of the bispecific antibody, has no obvious negative influence on cell growth and motility rate, has small influence on antibody quality, can help to improve the antibody expression quantity of host cells, and has wide adaptability.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for preparing bispecific antibodies. Background Technology

[0002] The following statements are provided only as background information in relation to the present invention and do not necessarily constitute prior art.

[0003] Bispecific antibodies (BsAbs), or simply bispecific antibodies, are antibody molecules that can simultaneously and specifically bind to two different antigen molecules or two different antigenic epitopes on the same antigen. Traditional monoclonal antibodies can only bind to one antigen at a time, while bispecific antibodies can target multiple pathways simultaneously, thus achieving a wider range of therapeutic effects.

[0004] Because bispecific antibodies have a more complex molecular structure than monoclonal antibodies, they are more prone to forming protein aggregates. These aggregates vary in size, reversibility, solubility, covalent / non-covalent interactions, and native / non-native conformations. These structural changes can lead to the loss of the biological activity of the intact protein. Furthermore, aggregates and misfolding can induce the presentation of novel epitopes, potentially posing unknown risks to human immune responses. Aggregates are also commonly considered a critical quality attribute (CQA) in antibody drug production and must therefore be removed during the manufacturing process.

[0005] The main methods for removing aggregates generated by bispecific antibodies include: 1. Reduce the aggregation of bispecific antibodies by modifying the molecular structure. For example, replace hydrophobic residues with hydrophilic or charged amino acids through single-point mutation to reduce antibody aggregation caused by hydrophobic driving effects; or increase the net charge on the antibody surface to reduce intermolecular interactions and introduce glycosylation sites by using electrostatic repulsion; or introduce new N-glycosylation sites near the highly hydrophobic regions on the antibody surface to cover or shield potential hydrophobic regions through glycans.

[0006] 2. CHO cell line development optimization strategies, such as optimizing plasmid transfection ratios, can reduce the large amount of homodimers and antibody aggregates generated due to the unbalanced expression of different chains of bispecific antibodies.

[0007] 3. Change the process parameters in cell culture, such as adjusting the pH control range, feed concentration, or carbon source concentration during cell culture.

[0008] 4. Remove bispecific antibody aggregates through protein purification processes, such as affinity chromatography or hydrophobic chromatography.

[0009] However, the above method has the following drawbacks: 1. Methods to reduce polymers through molecular modification and cell line engineering: On the one hand, this method is time-consuming and difficult, and is not suitable for drugs with tight development time. Moreover, for molecules that have already progressed to the process development stage, it is necessary to go back to construct cell lines, resulting in a large waste of time and resources. On the other hand, it may involve related gene technology patent barriers.

[0010] 2. Regulation of process parameters in cell culture: Reducing the aggregation of bispecific antibodies by changing cell culture process parameters is not a universal regulatory method. It may vary greatly among different types of bispecific antibodies and different cell lines, and may not be applicable to multiple bispecific antibody molecules or cell lines. The regulatory effect is uncertain, and changing the process parameters in cell culture may affect cell growth and expression, and change other quality properties of bispecific antibodies.

[0011] 3. Removal of bispecific antibody aggregates through protein purification processes: If the proportion of aggregates in the bispecific antibody product is too high, removal through purification processes will lead to a decrease in the total protein yield, increase costs, and require the development of different purification methods for different bispecific antibodies, thus prolonging the process development time.

[0012] In view of this, the present invention is hereby proposed. Summary of the Invention

[0013] The purpose of this invention is to provide a method for preparing bispecific antibodies, so as to alleviate the defects in the preparation process of bispecific antibodies in the prior art, which are prone to the formation of aggregates and the loss of total protein yield during purification.

[0014] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for preparing bispecific antibodies, comprising culturing host cells expressing bispecific antibodies using a perfusion culture process; The culture medium used in the perfusion culture process contains an excipient composition; the excipient composition includes: 30-50 mmol / L trehalose, 30-50 mmol / L sucrose, 4-8 g / L glucose and 15-25 mmol / L mannitol; During the perfusion culture process, the culture temperature is 35-36.5℃ from day 0 to day 6, and the culture temperature is 33-35℃ after day 6. The perfusion rate is 0.8-1.5 RV / day.

[0015] As a further technical solution, the excipient composition comprises: 32 mmol / L trehalose, 50 mmol / L sucrose, 8 g / L glucose and 25 mmol / L mannitol.

[0016] As a further technical solution, the culture medium is ExcelProCHO Production Medium 01, a perfusion culture medium containing an excipient composition.

[0017] As a further technical solution, the bispecific antibody includes IgG antibody.

[0018] As a further technical solution, the bispecific antibody includes: anti-CD3 × Claudin 18.2 bispecific antibody or anti-BAFF × APRIL bispecific antibody.

[0019] As a further technical solution, the host cell includes mammalian cells.

[0020] As a further technical solution, the host cell includes CHO cells.

[0021] As a further technical solution, the method also includes the step of separating and purifying the bispecific antibody.

[0022] Compared with the prior art, the present invention has the following beneficial effects: This invention offers a novel strategy to address the problem of high aggregation of bispecific antibodies by adding an excipient composition containing trehalose, sucrose, glucose, and mannitol as a culture medium additive to the culture medium of host cells expressing bispecific antibodies. Specifically, it reduces and controls antibody aggregation during the upstream cell culture stage. The inventors have found that using a culture medium containing the excipient composition provided by this invention and employing a specific perfusion culture process to culture host cells containing bispecific antibodies significantly reduces aggregation levels without significantly negatively impacting cell growth and viability. It also has minimal impact on other antibody quality properties (such as charge isomers and glycoforms) and can help increase antibody expression levels in host cells, thus contributing to lower unit antibody production costs and improving antibody drug production efficiency. Attached Figure Description

[0023] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0024] Figure 1 Cell growth curves during culture in a 3L reactor; Figure 2 The cell viability curve is shown in the 3L reactor. Figure 3The curve shows the antibody expression level during culture in a 3L reactor. Detailed Implementation

[0025] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. 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 skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] In this document, the terms “comprising” or “including” are open-ended expressions used in this disclosure to mean the phrase “including but not limited to”, and are used interchangeably with it, meaning that they include the contents specified in this disclosure, but do not exclude other contents.

[0027] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may or may not occur as described below, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0028] In this document, “and / or” is used to indicate that one or both of the situations described may occur, for example, A and / or B includes (A and B) and (A or B).

[0029] In this document, unless otherwise stated, any numbering is used to distinguish one entity or behavior from another, and is not required to require or imply any actual relationship, order, or importance between these entities or behaviors, such as numbering i, ii; first, second, etc.

[0030] In this document, the terms “comprising” or “including” mean that the stated elements, integers or steps are included, but do not exclude any other elements, integers or steps.

[0031] In this document, unless otherwise stated, the various reactions or operational steps may be performed sequentially or not in sequence. Preferably, the reaction methods described herein are performed sequentially.

[0032] In a first aspect, the present invention provides a method for preparing bispecific antibodies, comprising culturing host cells expressing bispecific antibodies using a perfusion culture process; The culture medium used in the perfusion culture process contains an excipient composition; The excipient composition comprises: 30-50 mmol / L trehalose, 30-50 mmol / L sucrose, 4-8 g / L glucose, and 15-25 mmol / L mannitol. The concentration of trehalose can be, for example, but not limited to, 30 mmol / L, 40 mmol / L, or 50 mmol / L; the concentration of sucrose can be, for example, but not limited to, 30 mmol / L, 40 mmol / L, or 50 mmol / L; the concentration of glucose can be, for example, but not limited to, 4 g / L, 6 g / L, or 8 g / L; and the concentration of mannitol can be, for example, but not limited to, 15 mmol / L, 20 mmol / L, or 25 mmol / L. It is understood that the culture medium may also contain components known in the art, optionally for cell culture, such as components for constituting a basal culture medium, and the present invention does not limit this.

[0033] During the perfusion culture process, the culture temperature is 35-36.5℃ from day 0 to day 6, and the culture temperature is 33-35℃ after day 6. The perfusion rate is 0.8-1.5 RV / day.

[0034] The inventors have discovered that culturing host cells containing bispecific antibodies in a culture medium containing the excipient composition of this invention and then culturing host cells expressing bispecific antibodies using a perfusion culture process has a significant effect on reducing aggregate levels and can also help increase the antibody expression level of host cells.

[0035] The preparation method provided by this invention is simple, easy to operate, stable and reliable, has no significant negative impact on cell growth and viability, has little impact on other quality properties of the antibody, has a short process development cycle, can significantly reduce the aggregation of bispecific antibodies in cell culture harvest broth, and can be successfully scaled up to production scale.

[0036] In a preferred embodiment, the excipient composition comprises: 32 mmol / L trehalose, 50 mmol / L sucrose, 8 g / L glucose and 25 mmol / L mannitol.

[0037] By further optimizing and adjusting the concentrations of each component of the excipient, the aggregate level can be further reduced when used in the preparation of bispecific antibodies.

[0038] In some alternative embodiments, the culture medium is ExcelPro CHO Production Medium 01, a perfusion culture medium containing an excipient composition.

[0039] In some alternative implementations, the bispecific antibody includes an IgG antibody.

[0040] In some alternative embodiments, the bispecific antibody includes, but is not limited to, anti-CD3 × Claudin18.2 bispecific antibody and anti-BAFF × APRIL bispecific antibody.

[0041] In some alternative implementations, the host cell includes a mammalian cell.

[0042] In some alternative implementations, the host cells derived from mammals include, but are not limited to, human, mouse, rat, dog, monkey, pig, goat, cow, horse, and hamster cells, including but not limited to Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, young hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, and HEK-293 cells.

[0043] In some alternative implementations, the host cell is a CHO cell.

[0044] In some alternative implementations, a step of separating and purifying the bispecific antibody is also included.

[0045] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0046] The experimental materials used in the following examples are as follows: 1. CHO cell lines stably expressing anti-CD3 × Claudin 18.2 bispecific antibodies (CHOK1SV, Lonsa, USA), GS selection system; CHO cell lines stably expressing anti-APRIL monoclonal antibodies (CHOK1SV, Lonsa, USA), GS selection system; CHO cell lines stably expressing anti-BAFF × APRIL bispecific antibodies (CHOK1SV, Lonsa, USA), GS selection system.

[0047] 2. Perfusion culture medium ExcelPro CHO Production Medium 01 (from Hangzhou Yian Jishi Biopharmaceutical Co., Ltd.); 3. Trehalose and sucrose (Pfanstiehl, USA), glucose and mannitol (Merck, USA).

[0048] The detection method in the following embodiments includes: 1. Live cell density assay: Live cell density assay was performed using Vi-CELL (Beckman, Germany).

[0049] 2. Cell metabolic biochemical value detection: Cell metabolic biochemical values ​​such as glucose, lactate, IgG, etc. are detected during the culture process using the M-100 biosensor (Silman, China) or Cedex Bio (Roche, Switzerland).

[0050] 3. Antibody content detection during culture: The antibody content during the culture process was detected using Cedex Bio (Roche, Switzerland).

[0051] 4. Aggregate Level Detection: Size exclusion high-performance liquid chromatography (SEC-HPLC) was used to detect the aggregate level of the antibody at a wavelength of 280 nm. The peak preceding the main peak was defined as the high molecule weight (HMW) peak, representing the aggregate content of the antibody.

[0052] 5. Antibody Fragmentation Level Detection: The antibody fragmentation level was analyzed using non-reducing capillary gel electrophoresis (NR CE-SDS) at a detection wavelength of 220 nm. The peak preceding the main peak was defined as the low molecular weight (LMW) peak, representing the antibody fragmentation level.

[0053] 6. Detection of antibody charge isomer levels: The distribution of antibody charge isomers was detected using imaged capillary isoelectric focusing (iCIEF) electrophoresis. The left side of the main peak represents the acidic variant peak, and the right side represents the basic variant peak.

[0054] Example 1: Examples of different combinations of excipient concentrations: Experimental methods: 1. Seed cell resuscitation and passage CHO seed cells were retrieved from the cell bank and thawed and revived at a concentration of (0.30 ± 0.05) × 10⁻⁶. 6 Cells were seeded at a density of 60 mL in 250 mL shake flasks, with a seed volume of 60 mL. The shake flasks were placed in a CO2 shaker incubator at a temperature of 36.5℃, a CO2 concentration of 5%, a humidity of 80%, and a rotation speed of 110 rpm. Seed cells were passaged every 3 days, with the seed density at passage being controlled to achieve a viable cell density of (0.30 ± 0.05) × 10⁻⁶ cells / mL. 6 cells / mL.

[0055] 2. Cell perfusion culture in shake tubes When the viable cell density of CHO seed cells is greater than 3.0 × 10⁻⁶ 6 After centrifuging the seed cells and discarding the supernatant, resuspend the cells in perfusion medium and seed them into 50 mL TPP® TubeSpin bioreactors (hereinafter referred to as shakers). The seeding density is 0.5~2.0 × 10⁶ cells / mL. 6 Cells / mL, initial working volume 10 mL, place the shaker in a shaker at 35℃, 5% CO2, 80% humidity, and 190 rpm, with the shaker tilted at an angle of 10–20°. Take 0.5 mL samples daily for cell counting and metabolic parameter detection. When the viable cell density is greater than 4.0 × 10⁶ cells / mL... 6 After reaching a cell count of [number] cells / mL, perfusion medium was introduced, followed by centrifugation and medium replacement at a perfusion rate of 1.0 RV / day. Appropriate amounts of glucose were added based on metabolic parameters to maintain normal cell growth and metabolism. On day 4, the perfusion medium was replaced with one containing excipients (Table 1). Exfiltration was performed as needed to control maximum viable cell density. The collected supernatant was stored at -80°C for subsequent antibody expression and aggregate level detection. Perfusion culture was terminated on day 14 or when cell viability fell below 80%. The excipient concentrations added in each group and the results of the examples are shown in Table 2.

[0056] Table 1. Excipient components and concentration range

[0057] Table 2. Summary of Excipient Concentration Combination Examples and Results

[0058] The results show that within the above-mentioned excipient concentration range, all excipients can significantly reduce the aggregate level of bispecific antibodies without significantly affecting protein expression. Among them, the third group of excipients showed the best effect.

[0059] Example 2: Optimization of Irrigation Process Parameters 1. Experimental Methods: Same as Example 1, except that the culture temperature and perfusion rate settings are shown in Table 3 below, and the excipient concentration combination used is 32 mmol·L⁻¹. -1 Trehalose, 50 mmol·L -1 Sucrose, 8 g·L -1 Glucose, 25 mmol·L -1 Mannitol was used, while the control group received no excipients. The results are shown in Table 3.

[0060] Table 3. Summary of Irrigation Process Parameter Examples and Results

[0061] The results show that a certain culture temperature does not significantly affect the aggregate level of bispecific antibodies, but it does significantly affect protein expression levels. Compared to 35℃ (#14), setting the culture temperature during the expression phase (after day 6 of culture) to 36.5℃ (#13) and 33℃ (#15) resulted in a significant decrease in protein expression levels. Overall, a culture temperature of 33℃~35℃ during the expression phase is more favorable for aggregate levels and protein expression levels. During the growth phase (days 0-6 of culture), with culture temperatures of 36.5℃ and 35℃, the aggregate levels and protein expression levels in the 35℃ (#16) group were not significantly different from those in the 36.5℃ (#14) group. The perfusion rate has a significant impact on the aggregate level and protein expression level of the bispecific antibody. When the perfusion rate increases from 0.5 RV / day to 2.0 RV / day, the aggregate level first decreases and then increases, while the protein expression level first increases and then decreases. When the perfusion rate is set to 0.8 RV / day to 1.5 RV / day, both the aggregate level and the protein expression level are at a relatively good level.

[0062] Example 3: Monoclonal Antibody Comparative Example 1. Experimental Methods: The method was the same as in Example 1, except that another CHO cell line expressing anti-APRIL monoclonal antibody was selected for culture, and a culture without excipients was set up as a control. The results are shown in Table 4.

[0063] Table 4. Monoclonal antibody comparative design and results summary

[0064] The results show that applying the technical solution of this application to monoclonal antibodies has no effect on reducing the aggregate level, indicating that the technical solution of this application is a specific and effective solution for bispecific antibodies.

[0065] Example 4: Scale-up production in a 3L reactor The effect of optimal process on bispecific antibody polymers was investigated in a 3L bioreactor (Thermo-Finesse G3lab, USA). Two reactors were set up for the experiment: one reactor used excipient-free culture as a control group, and the other reactor used the optimal process conditions in Example 2 as the experimental group. The reactor culture experimental design is shown in Table 5.

[0066] Table 5 Design scheme for reactor culture experiment

[0067] The results are shown in Tables 6-8. Figures 1-3 As shown.

[0068] Depend on Figure 1 Cell growth curves showed that the cell densities of the experimental group and the control group remained basically the same, with the highest viable cell density in the control group reaching 72.7 × 10⁻⁶. 6 The highest viable cell density in the experimental group was 68.4 × 10⁶ cells / mL. 6 The cell density was not significantly different between the two processes (cells / mL). Figure 2 The cell viability curves show that the viability of the experimental group was significantly better than that of the control group. Figure 3 The antibody expression curves show that the antibody expression level in the experimental group was significantly higher than that in the control group.

[0069] Table 6 shows the results of polymer detection in the reactor culture. It can be found that the polymer level in the experimental group was significantly lower than that in the control group, while the proportion of the main peak was significantly higher.

[0070] Table 6 Summary of SEC results in 3L reactor culture

[0071] Table 7 shows the results of antibody fragmentation detection in the reactor culture. It can be found that the fragmentation level of the experimental group was significantly lower than that of the control group, while the proportion of the main peak was significantly increased.

[0072] Table 7 Summary of NR CE results during culturing in a 3L reactor

[0073] Table 8 shows the results of antibody charge isomer detection during reactor culture. It can be found that the process in the experimental group does not affect the antibody charge isomer distribution.

[0074] Table 8 Summary of iCIEF results in 3L reactor culture

[0075] Example 5: Application to other bispecific antibodies 1. Experimental Methods: The method was the same as in Example 1. Another CHO cell line expressing anti-BAFF × APRIL bispecific antibody was selected for culture, and a culture without excipients was set up as a control. The results are shown in Table 9.

[0076] Table 9 Summary of other examples and results of bispecific antibodies

[0077] The results show that applying the technical solution of this application to the anti-BAFF × APRIL bispecific antibody also significantly reduced the aggregation level of the bispecific antibody molecule, indicating that the technical solution of this application has a certain degree of versatility on different bispecific antibody molecules.

[0078] The experimental results above show that excipients can reduce polymer content, increase monomer content, and effectively reduce antibody fragmentation level. They have no effect on the distribution of acid-base isomers, and can significantly improve cell viability. In addition, they can also significantly increase antibody yield.

[0079] In summary, this invention effectively reduces the aggregation level of bispecific antibodies by combining excipients and perfusion technology during cell culture. It also improves the overall performance in terms of cell growth, antibody expression efficiency, and antibody fragmentation quality, achieving the development goals. Furthermore, it exhibits excellent scalability in reactors, demonstrating that the method is robust, mature, reliable, and has great industrialization potential.

[0080] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method of making a bispecific antibody, characterized in that, The application relates to a method for culturing host cells expressing a bispecific antibody by using a perfusion culture process; The culture medium used in the perfusion culture process comprises an excipient composition; the excipient composition comprises 30-50 mmol / L trehalose, 30-50 mmol / L sucrose, 4-8 g / L glucose and 15-25 mmol / L mannitol; During the perfusion culture process, the culture temperature is 35-36.5 DEG C in the first 0-6 days and is 33-35 DEG C after 6 days, and the perfusion rate is 0.8-1.5 RV / day.

2. The method of claim 1, wherein, The excipient composition comprises 32 mmol / L trehalose, 50 mmol / L sucrose, 8 g / L glucose and 25 mmol / L mannitol.

3. The method of claim 1, wherein, The culture medium is a perfusion culture medium ExcelPro CHO Production Medium 01 containing the excipient composition.

4. The method of claim 1, wherein, The bispecific antibody comprises an IgG antibody.

5. The method of claim 1, wherein, The bispecific antibody comprises an anti-CD3 x Claudin 18.2 bispecific antibody or an anti-BAFF x APRIL bispecific antibody.

6. The method of claim 1, wherein, The host cell comprises a mammalian cell.

7. The method of claim 6, wherein, The host cell comprises a CHO cell.

8. The method of claim 1, wherein, The method further comprises a step of isolating and purifying the bispecific antibody.

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