HK40117190A - A cell culture method for production of human bone morphogenetic protein - Google Patents

Description

A cell culture method for producing human bone morphogenetic proteins

Technical Field

This invention belongs to the field of pharmaceutical technology, specifically relating to a cell culture method for producing human bone morphogenetic proteins.

Background Technology

Bone morphogenetic protein (BMP) is a member of the transforming growth factor-β (TGF-β) superfamily. It promotes the differentiation of mesenchymal stem cells (MSCs) into chondrocytes and osteoblasts by binding to receptors on the surface of MSCs. Recent studies have shown that BMP plays an important role in bone and soft tissue regeneration. Local application of BMP can support the formation of new bone, cartilage, and ligaments. Products containing BMP2, a member of the BMP family, have been approved for marketing in the United States, the European Union, and China for promoting spinal fusion in degenerative disc diseases. Examples include INFUSE™ BoneGraft/LT-CAGE™ (approved as a medical device in the United States in 2002) and INFUSE BONE GRAFT (approved in China in 2023). Products containing BMP7 have been approved for marketing in the United States and the European Union, such as OP-1™ Putty (approved as a medical device in the United States in 2004) and (approved in the European Union in 2009), but were withdrawn from the market in 2015 for commercial reasons. BMP6, also a member of the BMP family, promotes bone formation and accelerates bone healing. A candidate drug containing recombinant human bone morphogenetic protein 6 (rhBMP6) for promoting spinal fusion is currently in phase III clinical trials.

To support Phase III clinical trials and the commercial production of the candidate drug after approval, it is crucial to adopt a culture method that is highly efficient, process-stable, meets regulatory requirements, and is suitable for large-scale production. In recent years, mainstream cell culture methods in the industrial production of recombinant proteins have included batch culture, fed-batch culture, semi-continuous culture, continuous culture, and perfusion culture. Batch culture involves placing cells in a closed reactor with a fixed amount of culture medium and culturing them for a period of time without replenishing fresh medium or removing metabolic waste. During batch culture, the composition of cells and the culture medium changes over time. The cell environment changes significantly during culture, making it impossible to maintain optimal conditions consistently. In later stages, the accumulation of metabolic byproducts can affect cell growth, leading to reduced cell yield. Fed-batch culture starts with a fixed amount of culture medium, and nutrients are continuously added to the system as cells grow and products form, allowing cells to continue growing and metabolizing until the end of the culture. Semi-continuous culture is based on the batch culture principle, periodically removing a portion of the culture medium and replenishing it with an equal amount of fresh medium. Continuous culture continuously adds fresh nutrient solution to a non-closed system, removes inhibitory factors, optimizes the growth environment, and collects culture products. Perfusion culture involves continuously removing part of the conditioned medium and filling it with fresh medium while most of the cells remain in the reactor.

Achieving batch-to-batch stability in the expression of recombinant proteins using mammalian cells presents numerous challenges. Variations in cell culture conditions such as temperature, pH, and dissolved oxygen levels can lead to batch-to-batch differences, significantly impacting cell metabolism and recombinant protein expression. During prolonged cell culture, the accumulation of metabolites can affect cell health and protein expression levels, leading to unpredictable consequences for product quality. Furthermore, scaling up from laboratory to industrial production presents the problem of scale-up effects. Processes feasible in the laboratory may become complex in large-scale production; for example, issues such as controlling reaction conditions, managing heat, and improving mixing efficiency become more pronounced at increased scale. Differences exist between laboratory and industrial equipment, making the selection of suitable industrial equipment for transferring laboratory processes to industrial production a challenge. Additionally, the differences between small-scale laboratory production and large-scale industrial production can affect the stability of cell growth and protein expression, making it difficult to ensure consistent quality and properties for each batch in large-scale production.

Semi-continuous, continuous, and perfusion cultures are favored because they stably provide fresh nutrients and remove waste, not only replenishing nutrients but also alleviating product inhibition and reducing the accumulation of metabolic byproducts. This improves the culture environment, helps maintain stable cell viability, and promotes continued product synthesis. For example, continuous culture methods have been shown to optimize growth conditions and enhance the production of secretory recombinant therapeutic drugs (CN114127260A). Perfusion culture, by continuously injecting fresh culture medium and removing waste medium, solves the problems of unstable protein levels and low expression, improving production efficiency (CN112592948B). Semi-continuous, continuous, and perfusion cultures have advantages in increasing recombinant protein yield, providing a more stable culture environment for cells, reducing batch-to-batch variability, and improving product consistency and quality, making them the main large-scale production processes for recombinant protein drugs and antibody drugs.

Although semi-continuous and perfusion culture methods are widely recognized as more efficient and are preferred for application, in actual cultivation, these two methods suffer from batch-to-batch instability and low protein expression levels when used for rhBMP6 production. Therefore, an industrial-scale rhBMP6 production culture method that meets the requirements for batch-to-batch stability, protein expression levels, and expression efficiency is still lacking.

Summary of the Invention

Due to the aforementioned drawbacks of semi-continuous and perfusion culture for rhBMP6 production, which cannot meet the demands of industrial-scale production, a superior culture method needs to be explored. During this process, the inventors of this application unexpectedly discovered that batch culture is actually an effective method suitable for the industrial production of rhBMP6. Specifically, by batch culturing engineered cell lines in a closed reactor under appropriate culture conditions, high cell viability can be maintained, high protein yields can be obtained, and good batch-to-batch stability can still be maintained even when scaled up to 2000 liters. This gives batch culture a particular advantage in the industrial production of rhBMP6, providing an efficient and scalable production method.

Therefore, this invention provides a comprehensive method for producing recombinant human bone morphogenetic protein 6 (rhBMP6) through batch culture in a controlled cell culture system. This method covers all stages of cell culture, including cell resuscitation, seed culture, production culture, harvesting, and protein purification.

Wherein, the rhBMP6 includes SEQ ID NO: 1: SASSRRRQQSRNRSTQSQDVARVSSASDYNSSELKTACRKHELYVSFQDLGWQDWIIAPKG YAANYCDGECSFPLNAHMNATNHAIVQTLVHLMNPEYVPKPCCAPTKLNAISVLYFDDNS NVILKKYRNMVVRACGCH.

This invention provides a method for producing recombinant human bone morphogenetic protein 6 (rhBMP6), wherein the method is a batch culture method.

In some embodiments, the method for producing recombinant human bone morphogenetic protein 6 (rhBMP6) according to the present invention is a batch culture method; during the culture process, the yield of rhBMP6 protein is not less than 7.6 mg/L, preferably not less than 7.7 mg/L, more preferably not less than 8.3 mg/L, more preferably not less than 8.5 mg/L, more preferably not less than 8.8 mg/L, and more preferably not less than 9.1 mg/L.

In some embodiments, the method includes seed culture: inoculating cells expressing recombinant human bone morphogenetic protein 6 into a cell culture medium to obtain a seed culture solution.

In some implementations, the method includes production culture: inoculating seed culture medium with seed culture medium, culturing for 5-15 days or until cell viability decreases, and harvesting the cell culture medium.

In some implementations, the seed culture is a single-stage or multi-stage seed culture.

In some implementations, the production cultivation process includes a cooling step; the cooling step includes cooling treatment after 1-4 days of cultivation.

In some implementations, the cooling step includes cooling the culture for 1-4 days by 3-7°C.

In some implementations, the cooling step includes: setting the initial temperature to 36-38°C, culturing for 1-4 days, and then cooling the temperature to 31-33°C.

In some implementations, the pH during the production culture is 6 to 8.

In some implementations, the dissolved oxygen (DO) in the production culture is 10-100%.

In some implementations, the cells expressing recombinant human bone morphogenetic protein 6 are Chinese hamster ovary (CHO) cells.

In some embodiments, the culture volume of the seed culture is selected from one or more of 250 mL, 500 mL, 1 L, 2 L, 15 L, 50 L, 200 L, and 500 L.

In some implementations, the seed culture time is 24-96 hours, more preferably, the seed culture time is 24-72 hours.

In some embodiments, the culture volume of the production culture is selected from one or more of 50L, 200L, 500L, and 2000L.

In some embodiments, the yield of the recombinant human bone morphogenetic protein 6 is not less than 7.6 mg/L, 7.7 mg/L, more preferably not less than 8.3 mg/L, more preferably not less than 8.5 mg/L, more preferably not less than 8.8 mg/L, and more preferably not less than 9.1 mg/L.

In some implementations, the inoculation density for production culture is 0.50 × ^10⁶ - 0.90 × ^10⁶ cells/ml; preferably 0.70 × ^10⁶ cells/ml.

In some implementations, the temperature is lowered after 3-4 days of incubation; more preferably, the temperature is lowered after 3 days of incubation.

In some embodiments, the temperature difference between the initial temperature and the cooled temperature is 3.5-6.0℃, more preferably 3.8-5.8℃.

In some embodiments, the initial temperature is 36.3-37.3°C, and more preferably, the cooling temperature is 36.8°C.

In some embodiments, the cooled temperature is 31.5-32.5°C, and more preferably, the cooled temperature is 32°C.

In some embodiments, the pH is 6.65-7.15, more preferably 6.90.

In some embodiments, the dissolved oxygen (DO) is 20-80, more preferably 20-70, more preferably 20-60, and even more preferably 40.

In preliminary research and laboratory-scale production, a stable and efficient production system was established through a series of optimization measures and process improvements. Building on these foundations, this invention further successfully applied these optimized process conditions to larger-scale production. By precisely controlling key parameters such as temperature, _CO2 level, dissolved oxygen, and stirring speed in the bioreactor, a seamless transition from laboratory-scale to industrial-scale production was achieved.

In some implementations, the primary or multi-stage seed culture is carried out in shake flasks or bioreactors that are scaled up in stages.

In some implementation schemes, scale-up cultivation is achieved by adjusting parameters during the seed culture stage.

In some implementations, the stirring speed is adjusted according to the volume of the bioreactor or shake flask to maintain similar shear conditions.

In some embodiments, when the culture volume is 2L, the stirring speed is in the range of 240-260rpm, more preferably 250rpm.

In some embodiments, when the culture volume is 500L, the stirring speed is in the range of 65-75rpm, more preferably 70rpm.

In some embodiments, when the culture volume is 2000L, the stirring speed is in the range of 40-60rpm, more preferably 50rpm.

In some implementations, the _partial pressure of CO2 is maintained in the range of 20-80 mmHg, more preferably in the range of 30-60 mmHg.

In some embodiments, the cell culture medium used for seed culture and/or production culture contains sodium dextran sulfate.

In some embodiments, the concentration of the sodium dextran sulfate (DSS) is 2 mg/L to 2 g/L.

In some embodiments, the concentration of the sodium dextran sulfate (DSS) is 2 mg/L to 20 mg/L.

In some embodiments, the concentration of the sodium dextran sulfate is 2 mg/L-2 g/L, preferably 2 mg/L-20 mg/L.

In some embodiments, the cell culture medium used in the seed culture and/or production culture process comprises one or more basal media selected from DMEM, RPMI-1640, or other cell culture media specifically designed for CHO cell culture.

In some implementations, the cell culture medium specifically designed for CHO cell culture is selected from one or more of the following: ProCHO ^™ -5AGT, CHO, ActiCHO P, BalanCD CHO, CD OptiCHO, or Ex-Cell CDCHO.

In some implementations, the cell culture medium specifically designed for CHO cell culture is ProCHO ^™ -5AGT.

In some embodiments, the cell culture medium used in the seed culture and/or production culture process further comprises one or more of the following components: growth factors, buffers, pH adjusters, DNA synthesis promoters, or surfactants;

In some embodiments, the growth factor is selected from insulin-like growth factor, and more preferably, the growth factor is recombinant human insulin.

In some embodiments, the buffer is selected from buffer salts, HEPES, or MOPS, and more preferably, the buffer is selected from sodium bicarbonate or HEPES.

In some embodiments, the pH adjuster is selected from sodium hydroxide or potassium hydroxide, and more preferably, the pH adjuster is sodium hydroxide.

In some embodiments, the DNA synthesis promoter is selected from purine compounds or nucleotides, more preferably, the DNA synthesis promoter is selected from hypoxanthine or thymidine.

In some embodiments, the surfactant is selected from nonionic surfactants, and more preferably, the surfactant is poloxamer 188.

Another aspect of the present invention provides a cell culture medium for culturing cells expressing recombinant human bone morphogenetic protein 6 (rhBMP6).

In some embodiments, the cell culture medium is used to culture cells expressing recombinant human bone morphogenetic protein 6, and the cell culture medium is used to carry out the above-described method.

The recombinant human bone morphogenetic protein 6 contains the amino acid sequence shown in SEQ ID NO:1.

In some embodiments, the pH range of the cell culture medium is from pH 6 to 8, preferably from pH 6.65 to 7.15, and more preferably from pH 6.90.

In some embodiments, the culture medium comprises a basal medium selected from DMEM, RPMI-1640, or other commercial cell culture media specifically designed for CHO cell culture.

In some implementations, the commercial cell culture medium specifically designed for CHO cell culture is selected from ProCHO ^™ -5AGT (Lonza), CHO (Merck), ActiCHO P (GE Healthcare LifeSciences), BalanCD CHO (Irvine Scientific), CD OptiCHO, or Ex-Cell CD CHO.

In some implementations, the commercial cell culture medium specifically designed for CHO cell culture is ProCHO ^™ -5AGT.

In some embodiments, the cell culture medium has an optimized amount for the production of rhBMP6.

In some embodiments, the concentration of the basal culture medium is 10-50 g/L, more preferably 20 g/L.

In some embodiments, the concentration of the growth factor is 2-20 mg/L, more preferably 10 mg/L.

In some embodiments, the concentration of the buffer is 1-5 g/L, more preferably 2 g/L.

In some embodiments, the concentration of the pH adjuster is 1-10 g/L, more preferably 2 g/L.

In some embodiments, the concentration of the DNA synthesis promoter is 0.05-0.2 mM, more preferably 0.1 mM.

In some embodiments, the concentration of the nucleotide is 0.01-0.05 mM, more preferably 0.02 mM.

In some embodiments, the concentration of the surfactant is 0.1-2 g/L, more preferably 1 g/L.

In some embodiments, the pH range of the cell culture medium is 6.8 to 7.0, more preferably 6.9.

In cell culture, pH control is crucial for cell growth and protein expression. Different pH values significantly affect cellular metabolic activities, nutrient absorption, and the quality and yield of final products. To ensure the optimal culture environment, this invention specifically focuses on the systematic optimization of pH.

Studies have shown that a cell culture medium pH range of 6.65 to 7.15 provides a stable environment conducive to cell growth. pH values that are too low or too high can adversely affect cellular metabolic activities, thereby reducing protein yield and quality. Through experimental data and practical production experience, this invention has determined that a pH range of 6.65 to 7.15 is the most suitable condition. Experimental results show that within a pH range of 6.65 to 7.15, cell growth curves and protein expression levels exhibit optimal performance. Within the range of 6.8 to 7.0, especially 6.9, cell density and viability remain stable throughout the culture period, and the accumulation of metabolites (such as lactic acid) is effectively controlled. This optimization significantly improves the efficiency of the production process and the consistency of the product, providing a solid foundation for industrial-scale production.

In some embodiments, the cell culture medium further comprises an additive for enhancing protein expression, the additive being selected from heparin, chondroitin sulfate, hyaluronic acid, polyethylene glycol (PEG), Ficoll, agar, or sodium dextran sulfate (DSS).

In some implementations, the cell resuscitation step in the method includes:

Thaw the engineered cell lines in a water bath at 36.5-37.5℃;

Add the thawed cell suspension to the preheated basal culture medium;

After centrifugation to remove the supernatant, the cells were resuspended in preheated culture medium on a shaker;

Transfer the suspension to a shake flask containing culture medium and incubate at 36.5-37.5°C for 24-72 hours.

In some implementations, the seed culture step in the method includes:

Seed cells into shake flasks or cell culture bags at a density of 0.50× ^10⁶ to 0.90× ^10⁶ cells/ml;

Incubate at 36.3-37.3℃, 5% _CO2 , 80% humidity, and 120 rpm for 24-72 hours;

When the cell viability is below 90%, centrifugation is performed for propagation.

In cell culture, the control of dissolved oxygen (DO) is crucial. To ensure an optimal cell growth environment, this invention precisely controls and optimizes the dissolved oxygen (DO) parameter. Through experimental data analysis, the optimal control range for dissolved oxygen was determined to be 10-100, preferably 20-80, more preferably 20-70, even more preferably 20-60, and even more preferably 40. In particular, maintaining DO (%) between 20-60, with an optimal value of 40, achieves the best cell growth and protein expression results.

In some implementations, the production cultivation in the method includes the following steps:

i. The initial temperature is 36.3-37.3℃, preferably 36.5-37.0℃, more preferably 36.8℃; incubate for 3 days;

ii. After 3 days, adjust the temperature to 31.5-32.5℃, preferably 32℃, and continue culturing until 7-8 days or when the cell viability drops below 75%.

The method of this invention has achieved significant progress in realizing batch-to-batch stability, protein expression levels, and expression efficiency of rhBMP6 in industrial and large-scale production, particularly by employing appropriate batch culture techniques to improve the reproducibility and scalability of rhBMP6 production. This method addresses key challenges faced in the research and development and production of a class of innovative drugs with rhBMP6 as the main active ingredient, especially in the biopharmaceutical field, where process stability and the ability to scale up operations efficiently are crucial for obtaining regulatory approval and successful new drug development. The following is a detailed description of the various advancements achieved by this technical solution.

The method of the present invention exhibits the following innovations and improvements in the following aspects:

This invention successfully established an effective method suitable for the industrial production of rhBMP6, solving the problems of poor batch-to-batch stability and relatively low expression levels in the large-scale production of rhBMP6. It fills the technological gap that makes this protein difficult to produce industrially, achieving unexpected technical results. Furthermore, by progressively scaling up production and employing reasonable batch culture conditions (such as dissolved oxygen, temperature, and pH), this invention optimizes cell metabolism and production efficiency, ensuring that cells reach optimal growth and protein expression conditions at key stages during large-scale production. This improves the batch-to-batch stability of protein expression, ensures stable product quality, and achieves stability of cell metabolism and recombinant protein expression from laboratory to industrial scale, meeting the requirements of drug regulatory approval. Currently, the batch culture production of rhBMP6 has completed Phase II clinical trials and is in Phase III clinical trials.

Furthermore, by adding an appropriate amount of sodium dextran sulfate (DSS) to the culture system, protein yield was significantly increased and high cell viability was maintained in the later stages of culture, ensuring that cells remained under optimal growth conditions throughout the scale-up process. This achieved increased protein yield without complicating the culture process. By altering the temperature conditions and lowering the temperature during protein expression, the production of metabolic byproducts such as lactic acid was reduced, resulting in an effective improvement in cell viability and yield.

Simultaneously, this invention improves the cost efficiency of batch culture operations, reducing overall production costs by enhancing the robustness and yield of each batch. Significant progress has been made in improving the reproducibility and scalability of batch cultures. This approach not only meets the stringent standards of biopharmaceutical manufacturing but also provides the industry with a practical and efficient solution for optimizing its bioprocessing operations. Reproducibility ensures that each batch meets the same high quality and high efficiency standards, while improved scalability allows for a seamless transition from research to commercial-scale production.

Attached Figure Description

Figure 1 shows the relationship between the density of live cells in batch culture and time at a 2-liter scale.

Figure 2 shows the change in cell viability over time in batch culture at a 2-liter scale.

Figure 3 shows the relationship between live cell density and time in batch cultures at 500L and 2000L scales.

Figure 4 shows the change in cell viability over time in batch cultures at 500L and 2000L scales.

Figure 5 shows the pH change over time during batch culture.

Figure 6 shows the change of oxygen partial pressure ( _pO2 ) over time during batch culture.

Figure 7 shows the cell growth curves for the first two batches of semi-continuous culture.

Figure 8 shows the cell growth curves for four batches after semi-continuous culture.

Figure 9 shows the batch protein expression levels at different concentrations of sodium dextran sulfate (DSS) during batch culture.

Figure 10 shows cell growth curves at different concentrations of sodium dextran sulfate (DSS) in batch culture.

Figure 11 shows cell growth curves under different cooling strategies in batch culture.

Figure 12 shows the changes in lactate concentration under different cooling strategies during batch culture.

Detailed Implementation

Definitions and Explanations

To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used in this invention have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. It should be understood that the terminology used in this invention is for descriptive purposes only and is not intended to be limiting.

Unless otherwise stated, the following terms used in this invention have the following meanings:

As used herein, the term "cell culture medium" refers to a variety of liquid or gel formulations used to support the growth, maintenance, and proliferation of cells in vitro. In some embodiments, the culture medium is a commercially available medium, including but not limited to DMEM, RPMI-1640, or other commercially available cell cultures specifically designed for CHO cell culture, selected from ProCHO ^™ -5AGT, CHO, ActiCHO P, BalanCD CHO, CDOptiCHO, or Ex-Cell CD CHO; in some embodiments, the culture medium is a custom formulation for enhancing rhBMP6 production.

As used herein, the term "Pro CHO 5 medium" refers to ProCHO ^™ -5AGT, a proprietary medium for Chinese hamster ovary (CHO) cells designed specifically for protein production applications. ^ProCHO™ -5AGT, manufactured by Lonza BioScience, is a protein-free and animal-free medium, particularly suitable for high-density suspension culture of CHO cells and the production of recombinant proteins.

As used in this article, the term "batch culture" refers to a cell culture method in which the cell culture medium is used to inoculate cells and provide all nutrients at the beginning of the culture period, and no nutrients are added during the entire process until the end.

As used herein, the term "cell resuscitation" refers to the process of restoring a cryopreserved cell line to an active growth state. This includes thawing the frozen cell suspension, removing the cryoprotectant, and initiating cell growth in a controlled environment.

As used herein, the term "seed culture" refers to the initial stage of cell culture to expand its quantity. This step involves inoculating cells in appropriate culture containers and maintaining optimal growth conditions in preparation for large-scale production.

As used in this article, the term "production culture" refers to the stage in which cells are transferred to a bioreactor for large-scale cultivation. During this stage, cells grow under controlled conditions, with parameters such as temperature, pH, and dissolved oxygen precisely regulated to maximize cell density and productivity.

As used herein, the term "harvest" refers to the process of collecting cell culture medium after the production phase. This includes separating the cells from the culture medium to obtain a liquid containing the target protein.

As used herein, the term "sample pretreatment and virus inactivation" refers to the steps taken to purify the harvested culture medium. This includes removing impurities, clarifying the liquid, and inactivating any potential viral contaminants to ensure the safety and quality of the final product.

As used herein, the term "product purification" refers to the process of separating and purifying a target protein from a pretreated culture medium. This includes using techniques such as affinity chromatography to achieve a high level of purity suitable for the intended application.

As used in this article, "total protein yield determination" refers to the quantification of the total protein content in the purified eluent during a chromatography step. It is typically performed using ultraviolet spectrophotometry, which provides a measure of protein concentration, reflecting the efficiency and success of the production process.

As used herein, the term “semi-continuous culture” refers to a culture method that involves repeatedly harvesting portions of the cell culture medium and intermittently adding fresh culture medium throughout the culture period.

As used herein, the term "perfusion culture" refers to a cell culture method in which cells are continuously or semi-continuously supplied with fresh culture medium while metabolic waste and product-containing culture medium are removed.

As used in this article, the term “reproducibility” refers to the consistency and reliability of producing recombinant proteins (such as rhBMP6) across different batches and bioreactor scales, ensuring minimal variation in production results.

As used in this article, the term "scalability" refers to the ability to maintain consistency in production parameters and results, such as protein yield and cell viability, as production volumes are increased from smaller to larger bioreactor scales.

As used in this article, the terms “scalable,” “scale-up,” or “expansion” refer to the process of increasing cell culture volume from a laboratory or pilot scale to a larger industrial scale while maintaining consistent protein expression and yield.

As used in this article, the term "industrial scale" refers to large-capacity production systems, typically exceeding 500 liters, used for the commercial manufacture of recombinant proteins such as rhBMP6.

As used in this article, the term “Viable Cell Density (VCD)” or “cell density” refers to the concentration of live cells in a culture system, usually expressed as cells per milliliter (cells/mL), which is a key parameter for optimizing protein production.

As used in this article, the term “viability (Via)” refers to the percentage of live, healthy cells in a culture system, which is essential for efficient recombinant protein production.

As used in this article, the term "partial pressure of oxygen ( _pO2 )" refers to the partial pressure of oxygen dissolved in a liquid (such as a culture medium). Oxygen partial pressure represents the portion of pressure occupied by oxygen in a gas mixture and is an important parameter for measuring the oxygen content in a gas. In cell culture, oxygen partial pressure is usually expressed in millimeters of mercury (mmHg) or kilopascals (kPa), reflecting the distribution of oxygen between the gas and liquid phases.

As used herein, the term "dissolved oxygen (DO)" refers to the concentration of oxygen dissolved in a liquid (such as a culture medium). Dissolved oxygen is the amount of oxygen that can dissolve in a given volume of liquid, usually expressed in milligrams per liter (mg/L) or as a percentage (%). Dissolved oxygen concentration has a significant impact on cell growth and metabolism; too low a concentration can lead to cell hypoxia, while too high a concentration can cause oxygen toxicity.

During cell culture, _pO2 and DO (%) are correlated. By controlling the DO (%) level and monitoring pO2, the optimal conditions of the cell culture environment can be ensured, thereby improving cell viability and protein expression levels.

As used herein, the term "protein titer" refers to the concentration of purified recombinant human bone morphogenetic protein 6 (rhBMP6), measured in milligrams (mg/L) per liter of culture medium. In some embodiments of the invention, the protein titer represents the concentration of rhBMP6 quantified by UV spectrophotometry after affinity purification. This method provides a direct measurement of the purified rhBMP6 concentration, reflecting the efficiency of the protein production process.

As used herein, the term "batch protein expression level" refers to the concentration of a specific protein (rhBMP6 in this example) measured in batches of culture samples, expressed in milligrams (mg/L) per liter of culture medium. Unlike the extensive assessments in previous examples, the batch protein expression level in Example 6 was determined using an ELISA method. The term "ELISA" refers to enzyme-linked immunosorbent assay, a plate-based detection technique used to detect and quantify soluble substances such as proteins, antibodies, and hormones. In this application, the ELISA was specifically used to quantify rhBMP6 produced in the culture medium.

As used herein, the term "protein yield" refers to the concentration of recombinant human bone morphogenetic protein 6 (rhBMP6) produced per liter of culture medium, expressed in milligrams (mg/L), reflecting the efficiency and optimization of the overall process. Specifically, protein yield reflects both the expression level and the final purified protein titer. Protein titer, a component of protein yield, represents the concentration of purified rhBMP6 and is measured using ultraviolet spectrophotometry. Protein yield is used to comprehensively evaluate the overall performance and production capacity at each stage of the process, from laboratory to industrial-scale production.

As used in this article, the term "inoculation" refers to the process of adding cells to a cell culture medium in which the cells will adhere, grow, and multiply.

As used in this article, the term “harvest” refers to the process of collecting cells or cell products (such as recombinant proteins) from a culture medium after a defined growth cycle.

As used herein, the term "dextran sulfate sodium salt (DSS)" refers to a sulfated dextran polymer that possesses unique biological and chemical properties due to its high negative charge.

Example

Materials and Methods

The present invention is further illustrated by specific embodiments. It should be understood that these embodiments are intended to illustrate the application and advantages of the invention, but are not intended to limit the scope of the invention. Various modifications and variations will be apparent to those skilled in the art, and the scope of the invention is intended to cover all such modifications and variations falling within the spirit and scope of the appended claims.

A method for producing recombinant human bone morphogenetic protein 6 (rhBMP6) using recombinant DNA technology in Chinese hamster ovary (CHO) cells is described below. The amino acid sequence of the rhBMP6 monomer is shown in SEQ ID NO:1, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO:2. Exemplarily, the present invention clones the rhBMP6 encoding gene into the commercial expression vector pCHO1.0 to construct the recombinant expression plasmid pCHO1.0-rhBMP6. The constructed pCHO1.0-rhBMP6 recombinant expression plasmid is transfected into CHO suspension cells. After transfection, stable transfected CHO cell lines for expressing rhBMP6 are obtained through screening.

SEQ ID NO:2:TCAGCCTCCAGCCGGCGCCG ACAACAGAGT CGTAATCGCT CTACCCAGTCCCAGGACGTG GCGCGGGTCT CCAGTGCTTC AGATTACAAC AGCAGT GAAT TGAAAACAGC CTGCAGGAAGCATGAGCTGT ATGTGAGTTT CCAAGACCTG GGATGGCAGG ACTGGATCAT TGCACCCAAG GGCTATGCTGCCAATTACTG TG ATGGAGAATGCTCCTTCC CACTCAACGC ACACATGAAT GCAACCAACC AGCCGATTGTGCAGACCTTG GTTCACCTTATGAACCCCGA GTATGTCCCC AAACCGTGCT GTGCGCCAAC TAAGCTAAATGCCATCTCGG TTCTTTACTT TGATGACAAC TCCAATGTCATTCTGAAAAAATACAGGAAT ATGGTTGTAAGAGCTTGTGG ATGCCAC.

Preparation of culture medium

The culture medium used in Examples 1-5 of this invention is a composite culture medium, which includes components that support CHO cell growth and rhBMP6 production:

ProCHO ^TM -5AGT (ProCHO-5): 10-50g/L;

Recombinant human insulin: 2-20 mg/L;

Sodium bicarbonate: 1-5 g/L;

Sodium dextran sulfate: 2-20 mg/L;

Sodium hydroxide: 1-10 g/L;

HEPES: 1-5g/L;

Hypoxanthine: 0.05-0.2 mM;

Thymidine powder: 0.01-0.05mM;

Poloxamer 188 (P188): 0.1-2 g/L.

The culture media in Examples 6 and 7 contained ProCHO ^™ -5AGT commercial medium with the appropriate doses of sodium dextran sulfate added (concentrations are shown in Examples 6 and 7).

Example 1: Production of recombinant human bone morphogenetic protein 6 (rhBMP6)

The method for producing rhBMP6 in CHO cells using recombinant DNA technology includes the following steps: cell resuscitation, seed culture, production culture, harvesting, and product purification.

1. Cell resuscitation:

Thaw the engineered cell lines in a 37°C water bath. Add the thawed cell suspension to 14 ml of preheated basal medium, centrifuge to remove the supernatant, and then resuspend in 5 ml of preheated medium on a shaker. Transfer this suspension to a shake flask containing 15 ml of medium, for a final volume of 20 ± 2 ml, and incubate at 37°C on a shaker for 24–72 hours.

2. Seed culture:

Seed cells at a density of 0.50 × ^10⁶ - 0.90 × ^10⁶ cells/ml into shake flasks or cell culture bags and culture for 24-72 hours at 36.3-37.3℃, 5% _CO₂ , 80% humidity, and 120 rpm, 50 mm amplitude. Expand the seeding gradually as needed. If cell viability is below 90%, centrifugation is required for further propagation.

3. Production and cultivation:

Seed cell culture was inoculated into the reactor at an inoculation density of 0.50 × ^10⁶ - 0.90 × ^10⁶ cells/ml. After culturing for 3 days at a controlled temperature of 36.3-37.3℃, a pH of 6.65-7.15, a DO (%) of 20-60, and a _pCO₂ (mmHg) of 20-80, the temperature was adjusted to 31.5-32.5℃, and culture continued until day 7-8 or when cell viability dropped below 75%. The culture medium was then collected by centrifugation. During the culture process, the trends of cell density, viability, diameter, glucose, lactate concentration, DO (%), and pH were monitored daily.

Example 2: Expanded cell culture for rhBMP6 production using batch culture method

This study aimed to scale up the production of recombinant human bone morphogenetic protein 6 (rhBMP6) using CHO cells at different production volumes (specifically 2L, 500L, and 2000L) and to evaluate the reproducibility, scalability, and protein yield at these scales.

Operating steps:

1.2L production culture:

Cell culture was conducted in 2L shake flasks, with seed cell culture inoculated at a seeding density of 0.50 × ^10⁶ - 0.90 × ^10⁶ cells/ml. The cells were cultured for 3 days at 36.3-37.3℃, pH 6.65-7.15, and DO (%) 20-60. The temperature was then lowered to 31.5-32.5℃, and cultured for another 7-8 days or until cell viability was below 75%. The rotation speed was set to 250 rpm, maintaining _pCO₂ (mmHg) at 20-80. Cell density, viability, diameter, glucose, lactate concentration, DO (%), and pH were monitored daily during culture.

2.500L production culture:

Cell culture was conducted in a 500L reactor, with seed cell culture inoculated into 500L cell culture bags at a seeding density of 0.50× ^10⁶ –0.90× ^10⁶ cells/ml. Cells were cultured for 3 days at 36.3–37.3℃, pH 6.65–7.15, and DO (%) 20–60. The temperature was then lowered to 31.5–32.5℃, and cultured for another 7–8 days or until cell viability was below 75%. The air rotation speed was set to 70 rpm, maintaining _pCO₂ (mmHg) at 20–80. Cell density, viability, diameter, glucose, lactate concentration, DO (%), and pH were monitored daily during culture.

3.2000L production culture:

Cell culture was conducted in a 2000L reactor, with seed cell culture inoculated into 2000L cell culture bags at a seeding density of 0.50× ^10⁶ –0.90× ^10⁶ cells/ml. Cells were cultured for 3 days at 36.3–37.3℃, pH 6.65–7.15, and DO (%) 20–60. The temperature was then lowered to 31.5–32.5℃, and cultured for another 7–8 days or until cell viability was below 75%. The rotation speed was set to 50 rpm, maintaining _pCO₂ (mmHg) at 20–80. Cell density, viability, diameter, glucose, lactate concentration, DO (%), and pH were monitored daily during culture.

Example 3: Analysis of rhBMP6 production using batch culture method

This embodiment aims to analyze the final product and various process parameters of rhBMP6 under different bioreactor volumes. Based on the cell culture process established in Example 2, key process parameters such as cell density, viability, and metabolic characteristics are monitored. The stability of batch culture is assessed by measuring protein yield and quality, verifying its suitability for industrial-scale production.

After harvesting the culture medium and performing affinity purification, the total protein yield was determined.

The total protein content after purification was determined by ultraviolet spectrophotometer according to the Warburg-Christian method, and the total protein content harvested per liter of culture medium (mg total protein/L culture medium) was calculated.

Batch details are shown in Table 1 below.

Table 1. Batch Details for Each Group

serial number scale R1-01 2L R1-02 2L R1-03 2L R2-01 500L R3-01 2000L R3-02 2000L

Results and Discussion:

1. Protein titer

Protein titer analysis was conducted to determine the concentration of rhBMP6 produced in bioreactors of different sizes (specifically 2L, 500L, and 2000L). Protein yield data are shown in Table 2 below, displaying the concentration (mg/L) of rhBMP6 in the culture medium for each group.

Table 2. rhBMP6 concentration (mg/L) in each batch of batch culture

Note: The protein titer refers to the concentration of purified rhBMP6 obtained by UV spectrophotometry after affinity purification.

Protein titer data showed that stable rhBMP6 production was achieved across different bioreactor scales. At the 2L scale, protein titers ranged from 7.6 to 8.3 mg/L, with an average titer of 7.9 mg/L. Protein yield increased with scale increases to 500L and 2000L, with the highest recorded titer of 9.1 mg/L at the 2000L scale.

The average protein titer across all scales was 8.3 mg/L with a standard deviation of 0.6, demonstrating high reproducibility of the production process. Slightly higher protein titers observed at larger scales (500 L and 2000 L) indicate high stability of protein expression under appropriate cell culture and expression processes.

The total protein titer was measured at 8.8 mg/L for the 500L batch and 8.5 mg/L and 9.1 mg/L for the 2000L batch, respectively. The average yield was 8.8 mg/L, with a standard deviation of 0.3, indicating that the variation was not statistically significant.

The results show that the optimized culture and purification process enables the batch culture method to achieve high batch-to-batch stability, high batch reproducibility and scalability at different scales, and effectively maintains high protein expression levels during the scale-up process, ensuring the effective transfer from laboratory level to commercial production scale.

2. Cell density and viability trends:

Figure 1 shows the relationship between viable cell density (VCD) and time at a 2L scale, indicating a rapid increase in cell density during the initial few days, peaking around day 3-4, then stabilizing and gradually decreasing. Figure 2 shows the change in cell viability (Via) over time during 2L culture, showing a high cell viability that consistently remained above 80%, with only a slight decrease towards the end of the culture period.

Figure 3 shows the relationship between viable cell density and time at 500L (R2-01) and 2000L (R3-01 and R3-02) scales. Similar to the 2L batch, the cell density in these larger batches increased rapidly, peaking around day 4, and then plateaued, indicating effective scale-up of the cell culture process. Figure 4 shows the change in cell viability over time in 500L and 2000L cultures, maintaining high cell viability comparable to the 2L batch, and gradually declining after day 6.

The data in Figures 1 and 3 show that consistency was maintained across different scales, indicating that the process used for cell culture is not only scalable but also highly reproducible. Viable cell density peaked within the same timeframe in 2L and larger batches (500L and 2000L), demonstrating that culture conditions optimized for small-scale operations were effectively transferred to larger volumes without loss of cell growth efficiency.

The survival trends in Figures 2 and 4 show that high survival rates were maintained across all scales throughout the culture period. Slight decreases in survival rates were observed later in the culture phase in the 2L, 500L, and 2000L batches, reflecting a normal progression as nutrients are consumed or metabolic waste accumulates.

The results support the superior performance of the employed method in terms of high batch-to-batch stability, high batch reproducibility, and scalability. The consistent trends in cell density and viability across different scales confirm the robustness of the cell culture process for producing rhBMP6, maintaining cell health and productivity during scale-up. This robustness is crucial for commercial production, as process predictability and reliability are key to meeting production demands and quality standards.

3. Optimization of pH and dissolved oxygen (DO) (%)

During the production culture phase, the cell density, cell viability, cell diameter, online pH, _pCO2 , _pO2 , glucose concentration, and lactate concentration were systematically monitored. Figure 5 shows the pH change over time at 500L and 2000L scales, and Figure 6 shows the oxygen partial pressure ( _pO2 ) change over time at 500L and 2000L scales.

Maintaining a suitable pH level is crucial for cell growth and metabolism during cell culture. The pH of the cell culture medium affects enzyme activity, metabolic rate, nutrient absorption, and waste removal. As shown in Figure 5, the online pH change over time maintained a value of approximately 6.9 throughout the culture process, with an error margin of about ±0.1, exhibiting slight fluctuations but remaining within a controllable range. The consistent pH changes across batches indicate that the cell culture environment was relatively stable within this range, which is conducive to cell growth and protein expression. Experimental verification showed that cell viability and metabolic characteristics remained good within the pH range of 6.65 to 7.15, with no significant adverse effects observed. In particular, cell growth and protein yield were optimal at a pH close to 6.9.

Dissolved oxygen (DO) is another crucial parameter in cell culture, directly affecting cellular respiration and metabolic activities. Both excessively high and low DO values negatively impact normal cell growth and metabolism. As shown in Figure 6, the partial pressure of oxygen ( _pO2 ) changes over time. _pO2 decreases rapidly in the early stages of culture and then stabilizes at a low level. In the 2000L (R3-02) batch, because the DO was below the set value before sampling, oxygen was supplemented during sampling, resulting in a momentary spike in pO2.

During the experiment, by adjusting the oxygen supply, it was found that maintaining a DO (%) value within the range of 10-100 maintained normal cell growth. However, to optimize cell metabolism and protein expression, the DO (%) value was maintained between 20-80, more preferably between 20-70, and even more preferably between 20-60, especially close to 40, where cell performance was optimal. DO (%) values within this range effectively prevented oxidative stress caused by excessive oxygen and also prevented metabolic disorders caused by insufficient oxygen.

Example 4: Semi-continuous culture production of rhBMP6

This embodiment aims to compare the semi-continuous culture method of the present invention with an optimized batch culture method. The study focuses on evaluating the stability and productivity of the semi-continuous method, particularly its impact on cell growth, protein expression, and overall culture efficiency. Culture conditions were adaptively adjusted to achieve better culture results, even with differences in culture methods and containers.

Operating steps:

Cell resuscitation and initial expansion:

The engineered cell lines were revived according to the method described in Example 1. After revival, they were cultured in basal medium for 48-96 hours or until the cell density reached 1.0× ^10⁶ -3.0× ^10⁶ cells/mL.

Inoculation and Wave bioreactor culture:

Once the cell seeding meets the requirements, inoculate the cells into the Wave bioreactor at a density of 0.2 × ^10⁶ to 0.5 × ^10⁶ cells/mL. Culture conditions are set at 36.3–37.3 °C, a rotation speed of 10 rpm, an angle of 8°, and an aeration rate of 300 ccm (air + 3% _CO₂ ). Adjust the rotation speed, angle, and aeration strategy promptly based on cell growth, pH, and dissolved oxygen (DO) trends. The initial culture volume is 1.5 L, with fresh culture medium added every 2–5 days according to cell growth status until the cell density reaches 2.5 × ^10⁶ –4.0 × ^10⁶ cells/mL or the culture volume reaches 3–5 L.

Expanding to a 50L Wave Bioreactor: Once the above-mentioned density is reached, transfer the cells to a 50L Wave bioreactor using the same seeding density. Culture conditions are set at 36.3–37.3°C, 10 rpm, 8°C, and 300 ccm of gas (air + 3% _CO₂ ). Adjust the rotation speed, angle, and aeration strategy according to cell growth, pH, and dissolved oxygen (DO) trends. This step marks the beginning of a complete cycle of growth, protein expression, and medium replenishment. The culture process requires 5–10 cycles. Add fresh medium every 1–3 days, depending on cell growth, until the cell density reaches 3.0 × ^10⁶ –5.0 × ^10⁶ cells/mL.

Protein expression and harvest:

When the cell density reaches 3.0 × ^10⁶ - 5.0 × ^10⁶ cells/mL, lower the temperature to 31.5 - 32.5℃ to promote protein expression. Continue culturing for 2 - 5 days, then harvest. After each harvest, add fresh culture medium and raise the temperature to 36.3 - 37.3℃ to begin another culture and expression cycle.

Daily monitoring and final results:

Throughout the culture process, monitor cell density, cell viability, glucose, and lactate concentrations daily. If cell viability falls below 70%, stop the culture or harvest the cells after 5-10 cycles.

Protein expression measurement:

rhBMP6 was purified from the culture medium of the cultured cells and measured using the same method as in Example 3.

Results and Discussion:

1. Protein titer

In the semi-continuous culture model of Example 4, six batches of cell culture were harvested and affinity purified. Table 3 lists the detailed results of the harvest:

Table 3. rhBMP6 concentration (mg/L) in semi-continuous culture

serial number Protein titer (mg/L) SC01 4.3 SC02 1.6 SC03 1.7 SC04 3.1 SC05 2.4 SC06 2.0 Mean 2.5 SD 1.0

Note: The protein titer refers to the concentration of purified rhBMP6 obtained by UV spectrophotometry after affinity purification.

The results showed that the protein titers of the six batches of harvested semi-continuous culture medium ranged from 1.6 mg/L to 4.3 mg/L, with an average protein titer of 2.5 mg/L and a standard deviation of 1.0, representing 40% of the average. In contrast, the protein titers of the batch culture method (Example 3) ranged from 7.6 to 9.1 mg/L, with an average protein titer of 8.3 mg/L and a standard deviation of 0.6, representing 6.6% to 7.9% of the average.

Comparison reveals that for semi-continuous culture, with a culture scale of 50L, the deviation in protein titers among the six harvested batches is relatively large. However, for batch culture, with a culture scale range of 2L-2000L, the deviation in protein titers among the six harvested batches is smaller, indicating low batch-to-batch variability. Therefore, even with the same culture scale in semi-continuous culture, while exhibiting high batch-to-batch stability and reproducibility in conventional recombinant protein production, it is less effective than batch culture with a significantly larger culture scale range when used for rhBMP6 production. In terms of average protein titer, batch culture yields 3.3 times that of semi-continuous culture, resulting in a significantly higher average protein yield.

While semi-continuous culture is generally considered to offer advantages over batch culture in terms of nutrient supplementation, product inhibition, and reduced accumulation of metabolic byproducts, significant differences in protein titers between batches were observed during rhBMP6 production, indicating instability and poor reproducibility, thus failing to meet the requirements for further scale-up production. Surprisingly, batch culture showed higher inter-batch stability and higher protein titers, particularly between the three batches at 500L and 2000L, meeting the process stability requirements for GMP validation and drug regulatory approval, making it more suitable for industrial-scale production.

2. Cell density and viability trends:

Figure 7 shows the cell growth curves for two batches of semi-continuous culture. A peak cell count of 3.09 × ^10⁶ cells/mL was observed on day 3, followed by a gradual decline in viability. A portion of the culture medium was harvested on day 7, fresh medium was added, and culture continued until all cells were harvested on day 15. In the later stages of culture, cell count and viability were lower than before the first harvest. Similarly, Figure 8 shows the cell growth curves for four batches of semi-continuous culture, with continuous culture for 24 days.

In Figures 7 and 8, cell density and cell viability peaked and then began to decline and fluctuate, indicating that cells face potential stress and instability in the semi-continuous culture system. This reflects the difficulty of maintaining stable growth conditions and cell health during extended cycles. The high variability in cell growth and viability leads to inconsistent product quality and yield in subsequent downstream processing, failing to meet the process stability requirements for GMP process validation and drug regulatory approval, making it unsuitable for industrial-scale production. In contrast, batch culture (as discussed in Example 3) shows a more stable growth and viability trend, which is conducive to industrial-scale production.

Example 5: Production of rhBMP6 by perfusion culture

This embodiment aims to compare the batch culture method and the perfusion culture method described in Embodiments 2 and 3. While the culture methods and containers differed, the culture conditions were adaptively adjusted to obtain better culture results.

Operating steps:

Cell resuscitation and inoculation:

The engineered cell line was revived according to the method described in Example 1. The shake-flask-adapted cells were seeded at a density of 0.40 × ^10⁶ - 0.60 × ^10⁶ cells/mL into a 2L bioreactor equipped with an ATF2 perfusion system.

Cultivation conditions:

The temperature was controlled within the range of 36.3-37.3℃, the pH within the range of 6.65-7.15, and the DO (%) within the range of 20-60. After 7 days of culture, the temperature was adjusted to 31.5-32.5℃, and the growth and expression of cells at different cell densities and perfusion rates were observed. Culture was terminated when the cell viability was below 70%. During the culture process, the trends of cell density, viability, diameter, glucose, lactate concentration, DO (%), and pH were monitored daily.

Harvesting and purification:

After 14 days of culture, the cell culture medium was collected and divided into two parts: the culture medium inside the reaction vessel and the perfusion culture medium after being filtered through hollow fiber. The perfusion culture medium (outside the vessel) was further divided into two parts to obtain harvest liquid 1 and harvest liquid 2. The culture medium inside the vessel, harvest liquid 1 and harvest liquid 2 were purified by affinity chromatography.

Protein quantification:

The purified solution containing rhBMP6 was quantified using ultraviolet spectrophotometry to calculate the total protein content (mg total protein/L) per liter of culture medium.

Results and Discussion:

In the optimized perfusion culture of Example 5, the observed protein titers were significantly lower than those of the batch culture method, especially in the bioreactor and the harvested liquid. Table 4 shows the measured protein titers:

Table 4. rhBMP6 concentration (mg/L) during perfusion culture

Protein titer (mg/L) Inside the reaction vessel 0.9 Harvest fluid 1 0.1 Harvest fluid 2 0.2

Note: The protein titer refers to the concentration of purified rhBMP6 obtained by UV spectrophotometry after affinity purification.

The protein titers in the perfusion culture were significantly lower than those in the batch culture method of this invention. Specifically, the yield recorded in the bioreactor was 0.9 mg/L, while the yields recorded in the harvest liquid were even lower, at 0.1 mg/L and 0.2 mg/L, respectively. This contrasts sharply with the average protein titer of 8.3 mg/L achieved in the batch culture method of Example 3.

The batch culture methods used in Examples 2 and 3 provided higher and more stable protein yields. In contrast, while the continuous flow and dynamic conditions in perfusion culture theoretically enhance nutrient supply and metabolite removal, perfusion culture is not suitable for rhBMP6 production, as the protein may adsorb onto the hollow fibers of the ATF system, leading to reduced protein yield. Furthermore, the complexity of managing perfusion culture, such as the need for sophisticated equipment and the risk of protein adsorption, can complicate these systems and increase production costs.

summary:

In Examples 3 to 5, the applicability of various culture methods to industrial production was analyzed, with a focus on the stability, reproducibility, and efficiency of protein yield.

Example 3 shows that batch culture was scaled up to 500L and 2000L while maintaining high reproducibility and ease of operation. Data indicate that protein yields were consistently high across different scales (2L, 500L, and 2000L), highlighting the advantages of batch culture in achieving stable and enhanced protein expression. By optimizing culture medium composition and process parameters (such as pH and dissolved oxygen levels), batch culture ensured high batch-to-batch stability, high batch reproducibility, and high yields at different scales. This consistency and ease of operation make batch culture particularly suitable for commercial-scale scaling, where predictability and high yields are crucial.

In Example 4, semi-continuous culture was evaluated. Significant batch-to-batch variation in protein expression was observed across six harvests, reflecting unstable production results and poor batch-to-batch reproducibility. This variability suggests that semi-continuous culture may not be suitable for industrial-scale production of rhBMP6, as it cannot guarantee consistent product quality and yield. Furthermore, semi-continuous culture is more complex in process control and management, increasing operational difficulty and cost.

Example 5 explored perfusion culture and found that protein yields in both the bioreactor and the harvest broth were lower than in batch culture. Further analysis indicated that the hollow fibers of the ATF system may have adsorbed a large amount of the target product, thereby reducing yield. This issue, coupled with the complexity of perfusion system management and operation, suggests that perfusion culture may not be suitable for industrial-scale production of rhBMP6; industrial production tends to favor more efficient and simpler processes.

In summary, while semi-continuous and perfusion cultures offer certain advantages in terms of extended culture periods and potentially enhanced cell viability, the results of Examples 3 through 5 demonstrate that batch culture is a more reliable and efficient method for industrial production of rhBMP6. By optimizing culture media and process parameters, the batch culture method not only improves production efficiency but also ensures high batch-to-batch stability and high batch-to-batch reproducibility in large-scale production. Its high protein yield, extremely high reproducibility, and ease of operation make it the preferred method for scaling up in commercial bioprocessing environments.

Building upon Examples 3 to 5, a batch culture method exhibiting higher inter-batch stability and meeting the requirements for industrial production of rhBPM6 protein was unexpectedly developed. Inter-batch stability is a key indicator for evaluating process stability during GMP process validation and is also an important factor considered in drug regulatory approval. The following examples explore the optimization of protein expression in batch culture.

Example 6: Optimization of protein expression in batch culture

This embodiment aims to investigate the effects of different concentrations of sodium dextran sulfate (DSS) on protein expression in a batch culture environment.

Operating steps:

Cells were seeded at a density of 0.5 × ^10⁶ cells/mL in Pro CHO 5 medium during the logarithmic growth phase. The initial culture volume was set at 20 mL using TPP culture vessels. Culture was conducted at 37°C, 5.0% _CO₂ , 80% humidity, and a shaking speed of 250 rpm. Dextran sulfate sodium salt was added at seeding concentrations of 0.5 g/L, 1 g/L, and 2 g/L, respectively. Cell viability, cell density, glucose, and lactate concentrations were monitored daily, and culture was terminated when cell viability fell below 70%. Protein expression in the culture supernatant was quantified using ELISA.

Results and Discussion:

The experiment explored the effects of different concentrations of sodium dextran sulfate on protein expression in a batch culture environment. Table 6 and Figure 9 show the batch protein expression levels obtained under different conditions:

Table 5. Effects of sodium dextran sulfate on protein expression levels in batches

serial number condition Batch protein expression level (mg/L) control NA 13.4 A6 0.5g/L DSS 22.9 A7 1g/L DSS 21.6 A8 2g/L DSS 22.6

Note: Batch protein expression level refers to the concentration of rhBMP6 in each liter of culture medium measured by ELISA, expressed in mg/L.

Compared to the control group without sodium dextran sulfate (DSS), the addition of different concentrations of DSS significantly increased protein production. In the 0.5 g/L DSS (A6) group, the batch protein expression level increased to 22.9 mg/L, an increase of approximately 70.9% compared to the control group. In the 1 g/L DSS (A7) group, the batch protein expression level reached 21.6 mg/L, an increase of 61.2% compared to the control group. In the 2 g/L DSS (A8) group, the batch protein expression level was 22.6 mg/L, an increase of 68.7% compared to the control group. These data indicate that DSS has a significant promoting effect on protein expression, and the effect of DSS concentration changes on protein expression enhancement is similar.

Figure 9 shows the cell growth curves under different concentrations of sodium dextran sulfate (DSS), illustrating the changes in viable cell density (VCD) and cell viability (Via) over culture time at different DSS concentrations. In the initial stage (0 to 3 days), cell density increased rapidly under all conditions, reaching approximately 2.5 × ^10⁶ cells/mL. In the middle stage (4 to 5 days), cell density stabilized, with viability remaining above 90%. In the later stage (6 to 8 days), cell density decreased slightly but remained at a high level overall; viability remained high under DSS supplementation, while it decreased to below 70% in the control group. The results indicate that the addition of DSS significantly improved both cell density and viability, especially in the later stages of culture, where the presence of DSS helped maintain high cell viability and prevent rapid cell decline.

In summary, the addition of sodium dextran sulfate (DSS) helps cells maintain high viability and significantly increases protein expression. By studying the effect of different DSS concentration gradients on the production of rhBMP6 in batch cultures, it was found that changes in DSS concentration had similar effects on protein expression enhancement. This finding suggests that in the optimization of batch culture conditions, to further optimize protein expression, if other culture conditions or auxiliary reagents are needed, the study can be expanded to higher or lower DSS concentration ranges.

Example 7: Optimization of cooling strategy in batch culture

The purpose of this embodiment is to optimize the cooling strategy in batch culture to evaluate its impact on cell growth and metabolic activity.

Operating steps:

Cells were seeded at a density of 0.5 × ^10⁶ cells/mL in ProCHO 5 medium supplemented with 1 g/L dextran sulfate sodium (DSS). Initial culture temperature was maintained within the range of 36.3–37.3 °C at 5.0% _CO₂ and 80% humidity, with shaking at 250 rpm. Temperature was lowered to 31.5–32.5 °C at different days post-inoculation (no cooling, 2 days, 3 days, and 4 days), corresponding to experimental groups F1, F2, F3, and F4, respectively. Cell viability, density, and lactate concentration were monitored daily throughout the 7-day culture period.

Results and Discussion:

Figure 9 shows cell growth curves under different cooling strategies, illustrating the effect of different cooling initiation times on viable cell density (VCD) and cell viability (Via) during the 7-day culture period. In the control group (F1) without cooling, viability decreased significantly after day 5, reaching a low viability (78.2%) at harvest. Conversely, the groups that underwent cooling (F2, F3, F4) showed more stable cell density and significantly higher viability, all remaining above 90% at harvest. The most significant improvement was seen in group F2, where cooling was initiated on day 2, maintaining high cell viability and density throughout the culture period.

Figure 10 illustrates the changes in lactate concentration under different cooling strategies. The lactate production curves show that lactate levels remained stable in the cooling groups (F2, F3, F4), while the control group (F1) maintained consistently high lactate levels throughout the culture period. This persistently high lactate level in the control group may lead to culture medium acidification, which is detrimental to maintaining cell health.

These findings indicate that cooling strategies play a crucial role in optimizing batch culture conditions. Cooling cells after a period of culture can improve cell viability and stabilize metabolic processes. This suggests that, in optimizing batch culture conditions, the most suitable cooling strategies can be implemented and explored in conjunction with protein expression requirements to further optimize protein expression.

In the rhBMP6 production process described in the above embodiments, the batch culture method has consistently demonstrated superiority over semi-continuous and perfusion cultures in terms of simplicity, scalability, and reproducibility. Experimental results confirm the following advantages of batch culture:

Efficiency of batch culture: Batch culture methods have higher batch stability and higher protein yield, especially the three batches of 500L and 2000L, which show higher batch stability. This characteristic is one of the key indicators for evaluating process stability in GMP process validation and an important factor considered in the drug regulatory approval process, making it more suitable for industrial-scale production.

Effects of sodium dextran sulfate (DSS): Adding different concentrations of DSS to batch cultures significantly increased cell density and viability, especially in the later stages of culture. The presence of DSS helped maintain high cell viability and prevented rapid cell decline. The results showed that the presence of DSS stably enhanced protein expression levels. Notably, the observed DSS concentrations had similar effects on protein expression enhancement. This finding suggests that in optimizing batch culture conditions, further optimization of protein expression could be achieved by exploring other culture conditions or auxiliary reagents, and the study could be expanded to higher or lower DSS concentration ranges.

Optimization of cooling strategies: Experiments on optimizing cooling strategies showed a significant impact on cell viability and lactate metabolism. The cooled group exhibited significantly improved cell viability at harvest, consistently maintaining a viability value above 90%. In contrast, the viability of the uncooled group declined rapidly, reaching only 78.2% at harvest. This finding suggests that during the optimization of batch culture conditions, to further optimize protein expression, the most suitable cooling strategy can be implemented and explored based on protein expression requirements.

The techniques applied to the batch culture framework in this invention have proven effective in enhancing the overall efficiency of bioprocessing. By incorporating sodium dextran sulfate and optimizing cooling strategies, the robustness and efficiency of batch culture have been significantly improved. These improvements not only increase yield but also ensure greater consistency and easier scalability, which are crucial for the success of commercial bioproduction. These optimizations provide tangible benefits, highlighting the potential of batch culture as a mainstream method in the biotechnology and pharmaceutical industries.

Claims (10)

1. A method for producing recombinant human bone morphogenetic protein 6, characterized in that the method comprises the following steps: Seed culture: Cells expressing recombinant human bone morphogenetic protein 6 were inoculated into cell culture medium to obtain seed culture medium; Production culture: Inoculate the seed culture solution into the cell culture medium, culture for 5-15 days or until the cell viability decreases, then harvest the cell culture solution; The seed culture is a single-stage or multi-stage seed culture; The method described is a batch culture method. 2. The method according to claim 1, characterized in that the production cultivation includes a cooling step; the cooling step includes cooling treatment after culturing for 1-4 days; Preferably, the cooling step includes: cooling down by 3-7°C after culturing for 1-4 days; Preferably, the cooling step includes: setting the initial temperature to 36-38℃, culturing for 1-4 days, and then performing a cooling treatment, setting the temperature after cooling to 31-33℃; Preferably, the pH during the production culture is 6 to 8; Preferably, in the production culture, the dissolved oxygen (DO) is 10-100%. 3. The method according to claim 1 or 2, wherein the cells expressing recombinant human bone morphogenetic protein 6 are Chinese hamster ovary (CHO) cells. 4. The method according to any one of claims 1 to 3, wherein the culture volume of the seed culture is selected from one or more of 250 mL, 500 mL, 1 L, 2 L, 15 L, 50 L, 200 L, and 500 L; Preferably, the production culture volume is selected from one or more of 50L, 200L, 500L, and 2000L; Preferably, the seed culture time is 24-96 hours, more preferably, the seed culture time is 24-72 hours; Preferably, the yield of recombinant human bone morphogenetic protein 6 is not less than 7.6 mg/L, more preferably not less than 7.7 mg/L, more preferably not less than 8.3 mg/L, more preferably not less than 8.5 mg/L, more preferably not less than 8.8 mg/L, and more preferably not less than 9.1 mg/L. Preferably, the inoculation density for production culture is 0.50× ^10⁶ - 0.90× ^10⁶ cells/ml; more preferably 0.70× ^10⁶ cells/ml. 5. The method according to any one of claims 1 to 4, wherein the temperature is lowered after culturing for 3-4 days, more preferably, the temperature is lowered after culturing for 3 days; Preferably, the temperature difference between the initial temperature and the cooled temperature is 3.5-6.0℃, more preferably 3.8-5.8℃; Preferably, the initial temperature is 36.3-37.3°C; more preferably, the initial temperature is 36.8°C. Preferably, the temperature after cooling is 31.5-32.5℃, more preferably, the temperature after cooling is 32℃; Preferably, the pH is 6.65-7.15, more preferably 6.90; Preferably, the dissolved oxygen (DO) is 20-80, more preferably 20-70, more preferably 20-60, and even more preferably 40. 6. The method according to any one of claims 1-5, wherein the bioreactor volume is scaled up by adjusting parameters during the production and cultivation phase; Preferably, the stirring speed is adjusted according to the volume of the bioreactor or shake flask to maintain similar shear conditions; More preferably, when the culture volume is 2L, the stirring speed is in the range of 240-260rpm, more preferably 250rpm; More preferably, when the culture volume is 500L, the stirring speed is in the range of 65-75rpm, more preferably 70rpm; More preferably, when the culture volume is 2000L, the stirring speed is in the range of 40-60rpm, more preferably 50rpm; Preferably, the partial pressure _{of CO2} is maintained in the range of 20-80 mmHg, more preferably in the range of 30-60 mmHg. 7. The method according to any one of claims 1-6, wherein the cell culture medium used for seed culture and/or production culture contains sodium dextran sulfate; Preferably, the concentration of the sodium dextran sulfate is 2 mg/L-2 g/L, more preferably 2-20 mg/L. 8. The method according to any one of claims 1-7, wherein the cell culture medium used for seed culture and/or production culture comprises one or more basal media selected from the following: DMEM, RPMI-1640, or other cell culture media specifically designed for CHO cell culture; Preferably, the cell culture medium specifically designed for CHO cell culture is selected from one or more of the following: ProCHO ^™ -5AGT, CHO, ActiCHO P, BalanCD CHO, CD OptiCHO, or Ex-Cell CD CHO; More preferably, the cell culture medium specifically designed for CHO cell culture is ProCHO ^™ -5 AGT. 9. The method according to any one of claims 1-8, wherein the cell culture medium used for seed culture and/or production culture further comprises one or more of the following components: growth factors, buffers, pH adjusters, DNA synthesis promoters, or surfactants; Preferably, the growth factor is selected from insulin-like growth factor; more preferably, the growth factor is recombinant human insulin. Preferably, the buffer is selected from buffer salts, HEPES, or MOPS; more preferably, the buffer is selected from sodium bicarbonate or HEPES. Preferably, the pH adjuster is selected from sodium hydroxide or potassium hydroxide, and more preferably, the pH adjuster is sodium hydroxide; Preferably, the DNA synthesis promoter is selected from purine compounds or nucleotides; more preferably, the DNA synthesis promoter is selected from hypoxanthine or thymidine. Preferably, the surfactant is selected from nonionic surfactants, and more preferably, the surfactant is poloxamer 188. 10. A cell culture medium, characterized in that the cell culture medium is used to culture cells expressing recombinant human bone morphogenetic protein 6, wherein the cell culture medium is the cell culture medium as described in any one of claims 7 to 9; preferably, the culture medium is used in the method described in any one of claims 1 to 9.