Perfusion culture preparation method and application of recombinant humanized anti-rabies virus monoclonal antibody R71

Abstract

The invention provides a perfusion culture preparation method and application of a recombinant human anti-rabies virus monoclonal antibody R71, and relates to the technical field of biological medicines. According to the method, key process parameters in the culture process of the engineering cell strain expressing the R71 antibody are optimized, the key processes comprise a culture medium, a rotating speed, pH, an ATF P-flow set value and a dissolved oxygen (DO) range, and experimental results show that the receiving amount of R71 in each batch of the optimized perfusion culture method can reach more than 200g and is 5-8 times that of fed-batch culture under the same culture scale.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a perfusion culture preparation method and application of recombinant human anti-rabies virus monoclonal antibody R71. Background Technology

[0002] In recent years, the large-scale culture and production of therapeutic protein drugs using animal cells has become a research hotspot. Chinese hamster ovary cells (CHO cells) possess post-translational modification capabilities, and drugs produced from them exhibit excellent safety profiles, thus they are widely used for large-scale commercial production of therapeutic protein drugs. There are three main animal cell culture methods: batch culture, fed-batch culture, and perfusion culture. Feed-batch and perfusion culture methods are commonly used in production. Perfusion culture allows for the timely removal of culture products, preventing the degradation of some unstable proteins, significantly extending culture time, reducing reaction volume, and decreasing plant space and equipment investment.

[0003] Chinese patent CN117771365A discloses a formulation containing the anti-rabies virus combination monoclonal antibody R71. This formulation has a low dosage, high affinity, strong neutralizing activity, and good thermal stability. Currently, there is an urgent need to develop a commercially viable large-scale production method for this antibody. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a perfusion culture preparation method for recombinant human anti-rabies virus monoclonal antibody R71 and its application.

[0005] On one hand, the present invention provides a perfusion culture preparation method for recombinant human anti-rabies virus monoclonal antibody R71, comprising the following steps: (1) Prepare engineered cells that stably express R71; (2) Inoculate the engineered cells into the reactor and adjust the key process parameters for cultivation; The key process parameters include: rotation speed, pH, temperature, ATF P-flow, and dissolved oxygen content.

[0006] Specifically, the engineered cells include prokaryotic cells and eukaryotic cells.

[0007] More specifically, the prokaryotic cells include: Escherichia coli, Bacillus subtilis, lactic acid bacteria, Streptomyces, Proteus mirabilis, Corynebacterium glutamicum, thermophilic bacteria, cyanobacteria, halophilic bacteria, or Bacillus subtilis.

[0008] More specifically, the eukaryotic cells include: yeast, filamentous fungi, insect cells, mammalian cells, or plant cells.

[0009] Preferably, the yeast includes: Pichia pastoris, Saccharomyces cerevisiae, Schizosomalidomyces, Kluyveromyces lactis, or Hansenula polymorpha.

[0010] Preferably, the filamentous fungi include: Trichoderma, Aspergillus niger, or Trichoderma reesei.

[0011] Preferably, the insect cells include: Sf9, Sf21 or High-5 cell lines.

[0012] Preferably, the mammalian cells include: CHO, COS, BHK, NIH3T3 or SP2 / 0 cell lines.

[0013] Specifically, among the key process parameters, the rotational speed ranges from 150 to 270 r / min.

[0014] Specifically, among the key process parameters, the pH range is 7.05-7.15.

[0015] Specifically, among the key process parameters, the temperature range is 33-37℃.

[0016] Specifically, among the key process parameters, the set value range of ATF P-flow is 2.7-3.1.

[0017] More specifically, the ATF P-flow setting range is 2.8.

[0018] Specifically, among the key process parameters, the dissolved oxygen value ranges from 30% to 80%.

[0019] More specifically, the dissolved oxygen value is set at 50%.

[0020] Specifically, the culture process uses Balan CD CHO Growth A medium.

[0021] On the other hand, the present invention provides the application of the above-mentioned perfusion culture preparation method in the production of R71.

[0022] Compared with the prior art, the present invention has the following advantages: The perfusion culture method developed in this application produces R71 antibodies at a yield 5-8 times higher than that of flow-through culture at the same scale. The method provided by this invention can achieve higher antibody protein yields with fewer culture batches, while also reducing factory investment costs for enterprises. Attached Figure Description

[0023] Figure 1 The live cell density of three batches of R71 antibody perfusion culture.

[0024] Figure 2 Cell viability for three batches of R71 antibody perfusion culture.

[0025] Figure 3 The glucose concentration for three batches of R71 antibody perfusion culture.

[0026] Figure 4 The lactate concentration for three batches of R71 antibody perfusion culture.

[0027] Figure 5 NH3 cultured for three batches of R71 antibody perfusion 4+ concentration.

[0028] Figure 6 The osmotic pressure for three batches of R71 antibody perfusion culture.

[0029] Figure 7 The antibody protein yield from three batches of R71 antibody perfusion culture. Detailed Implementation

[0030] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0031] Example 1: Recombinant CHO cell line expressing R71 antibody After codon optimization of the R71 antibody sequence, the target gene was synthesized and inserted into an empty plasmid containing the GS selection gene to construct a stable expression vector. After sequencing confirmed that the constructed vector was consistent with the design, the linearized enzyme-digested expression plasmid was introduced into CHO-K1 host cells by electroporation, and engineered cell lines that stably express the R71 antibody were obtained through screening.

[0032] 2.2 Main Reagents and Instruments Balan CD CHO Growth A culture medium was purchased from Irvine Scientific, USA; glucose from Sigma-Aldrich, USA; cell culture shake flasks from Thermo Fisher Scientific, Inc.; ISF1-XC temperature-controlled culture shaker from KNAI, Switzerland; Countstar Altair cell analyzer from Shanghai Ruiyu Biotechnology Co., Ltd.; Advanced 3250 osmometer from Advanced, USA; XDS-1B digital inverted microscope from Chongqing Optoelectronic Instrument Co., Ltd.; SORVALL ST8R centrifuge from Thermo Fisher Scientific (China) Co., Ltd.; Cedex Bio multi-parameter biochemical analyzer from Elitelife Science Co., Ltd.; 15 L bioreactor from Guangzhou Aibetai Biotechnology Co., Ltd.; BioWelder TC aseptic connector from Sartorius Group, Germany; and ATF4 cell retention device from Elitelife Science Co., Ltd.

[0033] 2.3 Indicator Testing 2.3.1 Cell detection Take 20 μL of the culture medium taken from the reactor into a 0.5 mL centrifuge tube, add 20 μL of trypan blue staining solution and mix well. Take 20 μL of the mixed sample and add it to the test plate. Measure the cell density and cell viability on a cell counter.

[0034] 2.3.2 Biochemical indicators and osmolarity detection Transfer 1 mL of culture medium to a 1.5 mL centrifuge tube, centrifuge at 300 × g for 5 min, and collect the supernatant. Transfer 500 μL of the supernatant to a new 1.5 mL tube and analyze the glucose concentration, lactate concentration, NH4+ concentration, and antibody protein titer using a biochemical analyzer. Separately, take 200 μL of the supernatant and analyze the osmotic pressure using an osmometer.

[0035] 2.4 Cell perfusion culture 2.4.1 Seed cell resuscitation and expansion One cell line from the R71 antibody working library was taken from the liquid nitrogen tank, thawed in a 37°C water bath, centrifuged at 300×g for 5 min, and the supernatant was discarded in a clean bench. The cells were resuspended in 1 mL of preheated Balan CD CHO Growth A medium (37°C) and transferred to a 125 mL shake flask. The medium volume was added to 30 mL, and the flask was placed in a temperature-controlled shaker for cell culture. The culture conditions were: 125 rpm; 37°C; 80% humidity; and 5.0% CO2. When the cell viability reached 90%, the cells were cultured at 0.3-3.0×10⁻⁶ cells / mL. 6 Subculture amplification at an inoculation density of 10 cells / mL.

[0036] 2.4.2 Cell culture in the reactor Once the cell count in the shake flask reaches the required level for reactor inoculation, add 0.3-1.0 × 10⁻⁶ cells. 6 Inoculation was performed in the reactor at a density of [number] cells / mL for perfusion culture. In a 15L bioreactor, with a working volume of 11L, three experiments were designed based on key process parameters. The experiment numbers and culture conditions are shown in Table 1. Table 1. Experiment Numbers and Culture Conditions

[0037] In this embodiment, the minimum rotation speed is the initial culture setting value. For each additional day of culture, the rotation speed setting value is increased by 10 r / min until the maximum value is reached. The initial culture temperature is 37℃, and culture is continued until the cell density >10 × 10⁻⁶. 6 After reaching cells / mL, adjust the temperature setting to the lowest value in each of the different temperature ranges and continue fermentation until completion.

[0038] 3 Results and Analysis 3.1 Cell density and viability Results from three consecutive perfusion culture experiments showed that the cell growth trend was consistent over a 29-day culture period, with the peak viable cell density and time being: R71-1, 19.3 × 10⁻⁶. 6 Cells / mL (10d); R71-2, 20.3×10 6 Cells / mL (8d); R71-3, 20.9×10 6 Cells / mL (10d); see Figure 1 There was no significant difference in cell viability among the three batches of perfusion culture; cell viability remained above 70% throughout the entire culture period. Figure 2 .

[0039] 3.2 Cellular Metabolism 3.2.1 Changes in glucose concentration Results from three consecutive perfusion culture experiments showed that the glucose concentration trends were consistent across different batches. From the start of perfusion culture on day 3 until the end of the culture, the glucose concentration remained at a relatively stable level. Figure 3 .

[0040] 3.2.2 Changes in lactic acid concentration Results from three consecutive perfusion culture experiments showed that the lactic acid concentration trends were consistent across different batches, exhibiting a pattern of accumulation followed by consumption. In the mid-to-late stages of culture, the lactic acid concentration in each batch remained generally below 0.5 g / L. Figure 4 .

[0041] 3.2.3 NH4+ Concentration change The results of three consecutive batches of perfusion culture experiments showed that different batches of NH 4+ The concentration change trends were also consistent, but throughout the entire culture period, NH4+... 4+ The concentration remained consistently below 4 mM, which is within an acceptable range. (See attached image) Figure 5 .

[0042] 3.3 Osmotic pressure Results from three consecutive batches of perfusion culture experiments showed that the osmotic pressure changes of different batches were consistent, with the osmotic pressure of each batch remaining relatively stable within the range of 290-350 mOsm / kg, which is within an acceptable range. Figure 6 .

[0043] 3.4 Antibody protein yield The antibody protein yields of three consecutive batches of perfusion culture experiments were: R71-1: 316g; R71-2: 262g; R71-3: 239g; while the antibody protein yields of three consecutive batches of fed-batch culture were: 45g, 44g, and 41g, respectively. Figure 7 .

[0044] 4. Conclusion Data from three consecutive batches of perfusion culture experiments of R71 antibody showed that the perfusion culture process was stable. In a 15L reactor, the culture time could be maintained for approximately 30 days, with stable trends in cell growth, cell metabolism, and osmotic pressure changes. The antibody protein yield per batch reached over 200g, which is 6-8 times that of fed-batch culture at the same scale. The highest antibody protein yield was achieved when the key culture process parameters were set as shown in Group R71-1 in Table 2.

[0045] Table 2 Comparison of different process parameter settings and total antibody protein yield in R71 antibody perfusion culture

[0046] The perfusion culture of R71 antibody using the above culture conditions is reliable, which can reduce the number of culture batches and reduce the investment cost of the plant required for the production of the antibody after it is launched on the market.

[0047] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing recombinant human anti-rabies virus monoclonal antibody R71 by perfusion culture, characterized in that, Includes the following steps: (1) Prepare engineered cells that stably express R71; (2) Inoculate the engineered cells into the reactor and adjust the key process parameters for cultivation; The key process parameters include: rotation speed, pH, temperature, ATF P-flow, and dissolved oxygen content.

2. The perfusion culture preparation method according to claim 1, characterized in that, The engineered cells include prokaryotic cells and eukaryotic cells.

3. The perfusion culture preparation method according to claim 2, characterized in that, The prokaryotic cells include: Escherichia coli, Bacillus subtilis, lactic acid bacteria, Streptomyces, Proteus mirabilis, Corynebacterium glutamicum, thermophilic bacteria, cyanobacteria, halophilic bacteria, or Bacillus subtilis.

4. The perfusion culture preparation method according to claim 2, characterized in that, The eukaryotic cells mentioned include: yeast, filamentous fungi, insect cells, mammalian cells, or plant cells.

5. The perfusion culture preparation method according to claim 4, characterized in that, The yeasts mentioned include: Pichia pastoris, Saccharomyces cerevisiae, Schizosomalidomyces, Kluyveromyces lactis, or Hansenula polymorpha.

6. The perfusion culture preparation method according to claim 4, characterized in that, The filamentous fungi mentioned include: Trichoderma, Aspergillus niger, or Trichoderma reesei.

7. The perfusion culture preparation method according to claim 4, characterized in that, The insect cells mentioned include: Sf9, Sf21 or High-5 cell lines.

8. The perfusion culture preparation method according to claim 4, characterized in that, The mammalian cells mentioned include: CHO, COS, BHK, NIH3T3 or SP2 / 0 cell lines.

9. The perfusion culture preparation method according to claim 1, characterized in that, Among the key process parameters, the rotational speed ranges from 150 to 270 r / min.

10. The perfusion culture preparation method according to claim 1, characterized in that, Among the key process parameters, the pH range is 7.05-7.

15.

11. The perfusion culture preparation method according to claim 1, characterized in that, Among the key process parameters, the temperature range is 33-37℃.

12. The perfusion culture preparation method according to claim 1, characterized in that, Among the key process parameters, the ATFP-flow setting is 2.7-3.

1.

13. The perfusion culture preparation method according to claim 12, characterized in that, Among the key process parameters, the ATFP-flow setting is 2.

8.

14. The perfusion culture preparation method according to claim 1, characterized in that, Among the key process parameters, the dissolved oxygen value ranges from 30% to 80%.

15. The perfusion culture preparation method according to claim 14, characterized in that, Among the key process parameters, the dissolved oxygen value is set to 50%.

16. The perfusion culture preparation method according to claim 1, characterized in that, The culture process uses BalanCD CHO Growth A medium.

17. The application of the perfusion culture preparation method according to any one of claims 1-14 in the production of R71.

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