CN115404198B

CN115404198B - Application of pine needle extract in preparation of CHO cell culture solution and CHO cell culture method

Info

Publication number: CN115404198B
Application number: CN202211341840.5A
Authority: CN
Inventors: 张鼎岳; 牛庆田; 张晓莹; 刘兵; 林康; 卜纪斌; 罗顺; 阚子义; 仇金树
Original assignee: Shanghai Jianshibai Biotechnology Co ltd
Current assignee: Aosikang Biology Nantong Co ltd; Jianshun Biosciences Co ltd; Jianshun Biotechnology Nantong Co ltd; Shanghai Aosikang Biopharmaceutical Co ltd; Shanghai Jianshibai Biotechnology Co ltd
Priority date: 2022-10-31
Filing date: 2022-10-31
Publication date: 2023-01-20
Anticipated expiration: 2042-10-31
Also published as: CN115404198A

Abstract

The application discloses an application of a pine needle extract in preparation of a CHO cell culture solution and a CHO cell culture method. The application creatively adopts the pine needle extract to optimize the chemically defined CHO culture medium, and finds that different pine needle extracts can delay the death of CHO cells in the late culture period and improve the survival rate of the CHO cells; under a certain concentration, the pine needle extract can reduce the acidic species variants of the monoclonal antibody, increase the basic species variants of the monoclonal antibody, and is beneficial to the function and effectiveness of antibody protein; different influences are generated on the glycosylation of the monoclonal antibodies with different glycoforms, and the glycosylation of proteins is controlled, so that a specific oligosaccharide structure is targeted better; the low-concentration pine needle extract can increase the EC50 value of the monoclonal antibody, and improve the safety of the medicine to a certain extent.

Description

Application of pine needle extract in preparation of CHO cell culture solution and CHO cell culture method

Technical Field

The application relates to the technical field of biology, in particular to application of a pine needle extract in preparation of a CHO cell culture solution and a CHO cell culture method.

Background

Cell engineering plays an important role in the biological pharmaceutical industry, and according to statistics, 50% of medicine products in the world come from cell engineering pharmacy, so that a technical operation basis is provided for new medicine development, and the medicine has wide application in the aspects of treating immune diseases, improving the curative effect of treating diseases, innovating medicines and the like. Nowadays, biopharmaceuticals and cell engineering are closely linked, and with the widespread application of cell engineering technology in biopharmaceutical production, the biopharmaceutical industry develops rapidly and achieves huge economic benefits.

The recombinant protein medicine is prepared by modifying engineering bacteria or engineering cells by using genetic engineering technology to express human functional protein or mutant thereof in batches, is used for making up for the deletion of corresponding functional protein in vivo caused by congenital gene defect or acquired diseases of organisms and mainly comprises polypeptide hormone, cell factors, recombinase and other multiple subdivided fields. Since the first approvals of recombinant insulin and human growth hormone in the early 80's of the 20 th century, a variety of recombinant protein therapeutics have been put to use, nearly 70% of which are produced in Chinese Hamster Ovary (CHO) cells.

In vitro cell culture adopts a critical ring for expressing recombinant protein therapeutic drugs by adopting a cell engineering technology, a cell culture medium is an external environment which is suitable for the growth of cells and is created for the cells in the in vitro cell culture technology, various substances and energy required in the cell growth and metabolism process are provided, and the yield, the quality and the safety of metabolites of the cells are directly influenced. There have been studies to date that cell culture media and supplement formulations can affect cell culture performance, particularly glycosylation and charge variants.

The traditional cell culture mode is that the cells grow in an adherent way in a culture medium containing serum, and the serum provides hormones, growth factors, transfer proteins and other nutrients required by growth and proliferation for the cells, and causes a plurality of adverse effects on the large-scale culture process of the animal cells due to the complex components and the quality difference between different production places and batches. In addition, serum has been a major obstacle to isolation and purification during the process of obtaining products by cell culture. Therefore, serum-free media and chemically defined protein-free media have been the development of mammalian cell culture technology.

The formula of a Chemical Defined Medium (CDM) is optimized, so that the culture performance of the CHO cell in vitro culture and the protein quality secreted by the cell can be improved. For example, ganoderma lucidum contains abundant bioactive components in vivo, wherein the bioactive components can be mainly divided into 10 types such as polysaccharides, nucleosides, triterpenes, fats, sterols, mineral elements and the like, and the main effective components which are widely researched comprise polysaccharides with the function of enhancing immunity and triterpenes with the function of resisting cancers can act through various cell metabolic pathways. Research shows that Ganoderma spore powder with certain concentration in the culture medium has positive correlation with CHO cell growth.

However, the prior CHO cell culture medium optimizing formula has limited effect on improving the cell culture performance and the protein quality, and how to optimize the CHO cell culture medium formula is a difficult point for improving the CHO cell culture performance and the protein secretion thereof.

Disclosure of Invention

In order to solve the above problems, a first objective of the present application is to provide an application of a pine needle extract in preparation of a CHO cell culture solution, so as to optimize a chemically defined CHO medium formula and improve the culture performance of CHO cells and the quality of protein secreted by the CHO cells.

In one embodiment, the pine needle extract comprises at least one of an ethanol extract of pine needles, a water extract of pine needles, and a water extract of pine needle polysaccharides.

In one embodiment, the concentration of the pine needle extract is 5 mg/mL-1000 mg/mL.

In one embodiment, when the pine needle extract is a pine needle ethanol extract or a pine needle water extract, the concentration of the pine needle extract is 5 mg/L-15 mg/L.

In one embodiment, when the pine needle extract is a pine needle polysaccharide water extract, the concentration of the pine needle extract is 50 mg/L-1000 mg/L.

A second object of the present application is to provide a CHO cell culture broth comprising a chemically defined CHO medium containing the pine needle extract used for the above application.

In one embodiment, when the pine needle extract is a pine needle polysaccharide water extract, the concentration of the pine needle extract is 5-15 mg/L.

In one embodiment, the CHO cell culture solution further comprises a first supplement and a second supplement, wherein the volume of the first supplement is 3.5-4.5% of the volume of the chemically defined CHO culture medium, and the volume of the second supplement is 0.35-0.45% of the volume of the chemically defined CHO culture medium.

The third objective of the present application is to provide a CHO cell culture method, wherein a CHO cell culture solution is used to culture CHO cells.

In one embodiment, the culture is initiated by mixing chemically defined CHO medium containing pine needle extract and CHO cells for cell culture.

In one embodiment, the cell culture is a fed-batch culture.

In one embodiment, the fed-batch culture specifically comprises:

adding a first supplement and a second supplement in batches during the culture period, wherein the volume of the first supplement is 3.5-4.5% of the volume of the chemically defined CHO culture medium, and the volume of the second supplement is 0.35-0.45% of the volume of the chemically defined CHO culture medium.

In one embodiment, the fed-batch culture is carried out for 14 to 16 days.

The application creatively adopts the pine needle extract to optimize the chemically defined CHO culture medium, and finds that different pine needle extracts can delay the death of CHO cells in the late culture period and improve the survival rate of the CHO cells; the pine needle extract can reduce the acidic species variants of the monoclonal antibody, increase the alkaline species variants of the monoclonal antibody, and is beneficial to the function and effectiveness of antibody protein; different influences are generated on the glycosylation of the monoclonal antibody with different glycoforms, and the glycosylation of protein is controlled, so that a specific oligosaccharide structure is targeted better; the low-concentration pine needle extract can increase the EC50 value of the monoclonal antibody, and improve the safety of the medicine to a certain extent.

Drawings

In order to more clearly illustrate the detailed description of the present application or the technical solutions in the prior art, the drawings used in the detailed description or the prior art description will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 shows the effect of PNWE on CHO viable cell density in example 1 of the present application;

FIG. 2 shows the effect of PNEE on CHO viable cell density in example 1 of the present application;

FIG. 3 shows the effect of PNPWE on CHO viable cell density in example 1 of the present application;

FIG. 4 shows the effect of PNWE on CHO cell viability in example 1 of the present application;

FIG. 5 shows the effect of PNEE on CHO cell viability in example 1 of the present application;

FIG. 6 shows the effect of PNPWE on CHO cell viability in example 1 of the present application;

FIG. 7 shows the effect of pine needle extract on the potency of monoclonal antibody produced by CHO cells in example 2 of the present application;

FIG. 8 shows the effect of pine needle extract on the charge variation of CHO cell-produced monoclonal antibody in example 4 of the present application;

FIG. 9 shows the results of five glycosylation effects of the pine needle extract on the monoclonal antibodies produced by CHO cells in example 5 of the present application;

FIG. 10 shows the effect of the pine needle extract on five additional glycosylation of CHO cell-produced monoclonal antibody in example 5 of the present application;

FIG. 11 is a four-parameter fit regression curve of the activity analysis of the monoclonal antibody samples of the PNWE experimental group in example 6 of the present application;

FIG. 12 is a four-parameter regression curve of the activity analysis of the monoclonal antibody samples of the PNEE experimental group in example 6 of the present application;

FIG. 13 is a four-parameter regression curve of the activity analysis of the monoclonal antibody samples of the PNPWE experimental group in example 6 of the present application;

FIG. 14 shows the effect of different pine needle extracts on the activity of CHO cells producing mAbs in example 6 of the present application.

Detailed Description

Reference now will be made in detail to embodiments of the present application, one or more examples of which are described below. Each example is provided by way of explanation and not limitation of the present application. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made in the present application without departing from the scope or spirit of the application. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.

It is therefore intended that the present application cover such modifications and variations as fall within the scope of the appended claims and their equivalents. Other objects, features and aspects of the present application are disclosed in or are apparent from the following detailed description. It is to be understood by one of ordinary skill in the art that the present discussion is a description of exemplary embodiments only, and is not intended as limiting the broader aspects of the present application.

As above, how to develop more optimized culture medium formulations is a difficult point to improve the culture performance of CHO cells and the quality of the protein secreted by the CHO cells.

In order to at least partially solve at least one of the above technical problems, the first aspect of the present application provides a use of a pine needle extract in preparation of a CHO cell culture solution, so as to optimize a chemically defined CHO medium formula and improve CHO cell culture performance and protein quality.

Specifically, the pine needle is leaf of plant of Pinus of Pinaceae, also called pine hair, and the main source is Pinus massoniana, pinus armandi, pinus pumila, pinus thunbergii, pinus tabulaeformis, pinus koraiensis, etc. Pine needles are a natural renewable resource, are one of the main byproducts of pine plants, and have the advantages of high regeneration speed, wide distribution, large natural accumulation and sustainable utilization, and can be harvested all the year round. Pine needles contain a large number of active volatile substances as well as rich nutritional components, such as amino acids, terpenes, volatile oils, abscisic acid, a large number of water-soluble vitamin B, sugars, carotenes, chlorophylls, flavonoids, more than ten trace elements, crude proteins, etc., and the pine needle extract can be obtained by a relatively simple extraction method using water, ethanol, etc. as a solvent.

Chemically Defined Media (CDM), also known as defined chemical composition media, refers to media in which all the components are defined, and which does not contain animal proteins, does not add plant hydrolysates, and only uses some small molecule compounds with known structure and function, such as short peptides, plant hormones, etc.

In some embodiments, the CHO cell culture media of the present application use pinus massoniana needle extract crop supplements to optimize the cell culture performance and protein quality produced by chemically defined CHO media.

In some embodiments, the pine needle extract comprises at least one of an ethanol extract of pine needles, a water extract of pine needles, and a water extract of pine needle polysaccharides. The pine needle ethanol extract is prepared from pine needles by an extraction method using ethanol as a solvent. The pine needle water extract is prepared from pine needles by an extraction method using water as a solvent. The pine needle polysaccharide water extract is a pine needle extract obtained by an extraction method using polysaccharide water as a solvent.

In some embodiments, the concentration of the pine needle extract is 5mg/mL to 1000mg/mL, further 5mg/L to 15mg/L, further 5mg/L to 10mg/L, or 50mg/L to 1000mg/L, further 500mg/L to 1000mg/L. It should be noted that, for the pine needle extracts with different concentrations, the influence of the pine needle extracts on the culture performance and the protein quality of the CHO cells is different along with the change of the concentrations. Wherein, when the pine needle extract is a pine needle ethanol extract or a pine needle water extract, the concentration of the pine needle extract is 5 mg/L-15 mg/L, and further 5 mg/L-10 mg/L; when the pine needle extract is a pine needle polysaccharide water extract, the concentration of the pine needle extract is 50 mg/L-1000 mg/L, and further 500 mg/L-1000 mg/L.

The application creatively adopts the pine needle extract to optimize the chemically defined CHO culture medium, and finds that different pine needle extracts can delay the death of CHO cells in the late culture period and improve the survival rate of the CHO cells; the pine needle extract can also reduce the acidic species variants of the monoclonal antibody, increase the basic species variants of the monoclonal antibody, and is beneficial to the function and effectiveness of antibody protein; different influences are generated on the glycosylation of the monoclonal antibody with different glycoforms, and the glycosylation of protein is controlled, so that a specific oligosaccharide structure is targeted better; the low-concentration pine needle extract can increase the EC50 value of the monoclonal antibody, and improve the safety of the medicine to a certain extent.

According to the application, the influence of the pine needle extract on the CHO cell culture is researched, a novel chemically defined CHO culture medium is developed, and the CHO cell culture performance and the protein quality are improved through aspects such as cell viability, charge variation, oligosaccharide structure and safety.

Accordingly, in a second aspect of the present application, there is provided a CHO cell culture broth comprising a chemically defined CHO medium containing an extract from pine needles used in the above applications.

Specifically, the chemically defined CHO medium refers to a medium in which all components in the medium are clear, and some small molecule compounds with known structures and functions, such as short peptides, plant hormones and the like, do not contain animal proteins, and are more favorable for analyzing the secretion products of CHO cells. According to the application, the pine needle extract is creatively adopted to optimize the chemically defined CHO culture medium, and the fact that the pine needle extracts extracted by different methods can delay the death of CHO cells in the late culture period, the survival rate of the CHO cells is improved, and the yield of CHO cell protein is further improved.

It should be noted that, the pine needle extract is creatively added into a chemically defined CHO culture medium, and the content changes of glucose, glutamine, lactic acid, ammonium salt and glutamic acid in the culture medium are detected after the cells are cultured by the culture medium, so as to evaluate the influence of the additive on other culture medium components. Experiments show that different pine needle extracts used in the method slightly affect the consumption of glucose, but do not affect the consumption of glutamine, lactic acid, ammonium salt and glutamic acid, the death of CHO cells can be delayed, and the survival rate of the CHO cells can be improved in the later culture period.

As described above, the pine needle extract contained in the chemically defined medium includes at least one of a pine needle ethanol extract, a pine needle water extract and a pine needle polysaccharide water extract, specifically, the concentration of the pine needle extract is 5mg/mL to 1000mg/mL, further, when the pine needle extract is the pine needle polysaccharide water extract, the concentration of the pine needle extract is 5mg/L to 15mg/L, further, 5mg/L to 10mg/L, and when the pine needle extract is the pine needle polysaccharide water extract, the concentration of the pine needle extract is 50mg/L to 1000mg/L, further, 500mg/L to 1000mg/L.

In some embodiments, when the CHO cells are cultured by fed batch, the CHO cell culture fluid further comprises a first supplement and a second supplement, the volume of the first supplement being 4% of the volume of the chemically defined CHO medium, and the volume of the second supplement being 0.4% of the volume of the chemically defined CHO medium, for providing nutrients to the CHO cells during the culturing process.

Therefore, the third aspect of the present application provides a CHO cell culture method, wherein a CHO cell culture solution is used for cell culture of CHO cells, so as to improve the culture performance and protein quality of the CHO cells by increasing the cell survival rate, reducing the acidic charge variants, optimizing the oligosaccharide structure, improving the safety and the like.

In some embodiments, the cell culture of the CHO cells using the CHO cell culture solution specifically comprises: mixing a chemically defined CHO medium containing a pine needle extract and CHO cells to perform cell culture at the beginning of the culture, wherein the concentration of the pine needle extract is 5 mg/mL-1000 mg/mL at the beginning of the culture, further, the concentration of the pine needle extract is 5 mg/L-15 mg/L, further 5 mg/L-10 mg/L when the pine needle extract is a pine needle polysaccharide water extract, and the concentration of the pine needle extract is 50 mg/L-1000 mg/L, further 500 mg/L-1000 mg/L when the pine needle extract is a pine needle polysaccharide water extract.

Further, the cell culture is a fed-batch culture. In some embodiments, the fed-batch culture specifically comprises: adding a first supplement and a second supplement in batches during the culture period, wherein the volume of the first supplement is 3.5-4.5% of the volume of the chemically defined CHO culture medium, and the volume of the second supplement is 0.35-0.45% of the volume of the chemically defined CHO culture medium.

In some embodiments, in order to achieve better culture performance and obtain better protein quality, the fed-batch culture time is not less than 14 days, so that the life of the CHO cells is prolonged, the cell survival rate is increased, and the culture performance and the protein quality of the CHO cells are further improved.

According to the application, the pine needle extract is added into the chemically defined CHO cell culture solution, and the unique influence of the pine needle extract on the CHO cell culture is creatively discovered under the condition of having statistical significance. From the perspective of cell growth, the pine needle extract can delay cell death of cells in the late stage of Fed-batch culture, so that the cell density and the life span are kept at a higher level. As the pine needle extract maintains high-activity cells, the yield of the target protein is improved to the maximum extent. Therefore, the pine needle extract can improve the growth density and the lifespan of the cells to achieve the increase of the yield of the target protein.

In terms of the produced monoclonal antibody, in the CHO cell culture product, the pine needle extract reduces the acidic species of the monoclonal antibody on the whole, mostly increases the basic species, and increases a small amount of main species. The pine needle extract used in the present application does not increase major species to a greater extent, but does not sacrifice other process properties, e.g., does not significantly affect binding activity and potency, but delays cell senescence, increasing recombinant protein production.

Glycosylation characteristics represent one of the most important key product quality attributes, and affect glycosylation in cell lines, cell culture media and supplements, and under a variety of conditions during culture. CHO cells produce different types of mabs and their glycosylation requirements will vary, and will achieve the appropriate glycosylation profile depending on the desired mechanism of action and other characteristics, such as antibody glycoform ratios. In the field of biosimilar, the glycosylation characteristics of the produced monoclonal antibody need to be matched with those of the original drug, so that the glycosylation characteristics of the biosimilar need to be adjusted. In the aspect of glycosylation of the monoclonal antibody, the pine needle extract can adjust the glycosylation of the monoclonal antibody, target a specific oligosaccharide structure, such as reducing the high mannose ratio of the monoclonal antibody and the like, and simplify the method for adjusting the glycosylation characteristics, thereby reducing the development cost.

In the aspect of the activity of the monoclonal antibody, the pine needle extract does not cause great activity reduction, and simultaneously increases the EC50, which indicates that the combination of the produced recombinant protein and the antigen is not influenced by the addition of the pine needle extract, but the safety of the recombinant protein can be improved.

Embodiments of the present application will be described in detail with reference to examples.

Dimethyl sulfoxide (DMSO) and glucose dry powder used in the examples of this application were purchased from Sigma-Aldrich (USA). Phosphate Buffered Saline (PBS) was purchased from Chemicals, inc. of the national drug group, complete chemically defined Medium CD CHO 050, and supplement CD Feed were provided by Jianshun Biotechnology, inc., under product names CD Feed008 and CD Feed 2 and CD Feed009, respectively, of CD Feed 1. The cell line used in this example was a stable recombinant CHO K1 cell line expressing IgG monoclonal antibodies, and was provided by Ausmelt biopharmaceutical Co., ltd. The pine needle extract is purchased from solid powder obtained by water extraction and alcohol precipitation operations on the market. Pine Needle Water Extract (PNWE) and Pine Needle Ethanol Extract (PNEE) were purchased from Shanxi Sinot Biotechnology GmbH; pine Needle Polysaccharide Water Extract (PNPWE) was purchased from Woltrees Biotech, inc., lanzhou.

Example 1 Effect of pine needle extract on CHO viable cell Density and cell viability

Fed-batch was performed after the same CHO K1 cell line was subcultured, as described below.

Preparing solutions with certain concentrations from the three pine needle extracts according to experimental requirements, fully dissolving the solutions with purified water, filtering the solutions in a biological safety cabinet by using a 0.22 mu m filter respectively to ensure that the solutions are in an aseptic state, and storing the solutions at the temperature of 2-8 ℃. Then, the PNWE group and the PNEE group are respectively provided with three concentration gradients of 5mg/L, 10mg/L and 15mg/L, and the PNPWE group is provided with three concentration gradients of 50mg/L,500mg/L and 1000mg/L. Three pine needle extracts were added at the beginning of the culture and the culture was harvested on day 14.

Specifically, thawing the original cells and then reviving them, keeping them at a concentration of 5% CO ₂ In a carbon dioxide shaking incubator (Kuhner, switzerland) at 37 ℃ with a humidity of 80%, the rotation speed was 200rpm. Cells were then passaged every 3 days at 1.0X 10 ⁶ The individual viable cells/mL were inoculated into fresh medium. Passage was stopped when the number of cells was sufficient for subsequent experiments. Set up 9 experimental groups plus a blank control group. Each experimental group was set up with 3 parallel experiments. Independent cultures were performed in 50mL cell culture tubes (TPP, switzerland). A total of 30 cell culture tubes, each with a cell volume of 25mL, and a seeding density of 1.0X 10 ⁶ Viable cells/mL.

CD Feed was supplemented from the third day, and CD Feed was added on days 3/5/7/9/11/13, respectively. The two types of CD Feed 1 and CD Feed 2 added simultaneously account for 4% and 0.4% of the initial culture volume, respectively. The glucose concentration was kept at a lower concentration of 2g/L at all times based on the daily Nova test. Viable Cell Density (VCD) in excess of 10X 10 ⁶ For each viable cell/mL, the cell density of the sample was diluted one time with PBS for measurement in order to ensure measurement accuracy. According to the process design, the temperature of the culture is reduced collectively on the 7 th day of culture. The temperature was reduced from 37 ℃ to 32 ℃. After the 14 th day of incubation, the samples were centrifuged with a high-speed refrigerated centrifuge (Beckman coulter, USA) at 20 ℃ and 4500rpm for 30 minutes, and the supernatant was taken as a fed-batch culture. Cultures were tested for Viable Cell Density (VCD) and cell Viability (Viability) using a Vi-cell XR (Beckman Coulter, USA). Glucose, glutamine, lactic acid, ammonium, glutamic acid, pH and osmolality in the culture were determined with BioProfile 400 (Nova BioMedical, USA).

The results of the determination of Viable Cell Density (VCD) and cell Viability (Viability) of the three experimental groups and the control group are shown in FIGS. 1 to 6, compared with the control group. The PNWE experimental group has different decreases of viable cell density compared with the control group under the conditions of the concentration of 10mg/L and 15mg/L, and the details are shown in figure 1. FIG. 1 shows that 10mg/L and 15mg/L of pine needle extract significantly inhibited the growth of CHO cells. The density of the living cells of the culture medium with the concentration of 5mg/L is not obviously different from that of the control group in the initial culture period, the density of the living cells in the later culture period is obviously higher than that of the control group, and the result has statistical significance, which indicates that PNWE has the effect of delaying CHO cell death under the concentration, and the expression in the cell survival rate is particularly obvious, and is shown in fig. 4.

In the later culture period, the CHO cell survival rates of the PNWE experimental groups with three concentrations are higher than those of the control group and have statistical significance. The PNEE group showed very similar results to the PNWE group, and the inhibition of cell growth was observed at concentrations of 10mg/L and 15mg/L, as shown in FIG. 2, and the cell death was delayed in the late stage of culture, as shown in FIG. 5. As shown in FIGS. 3 and 6, the PNPWE experimental group showed good proliferation and growth of viable cell density compared with the control group throughout the Fed-batch culture, and the PNPWE experimental group with a concentration of 50mg/L reached the maximum viable cell density of 16.37X 10 at day 7 of culture ⁶ cells/mL, and the highest viable cell density of the experimental group at both 500mg/L and 1000mg/L concentrations also exceeded the control group.

At the end of the 14 th day culture, the PNPWE three experimental groups maintain higher viable cell density and viable rate, and the viable cell density is 12 multiplied by 10 ⁶ About cells/mL, and about 82% of cell viability. The control group has viable cell density of 10.23 × 10 ⁶ cells/mL, activity 79.3%, P compared to control<0.05, has statistical significance. The glucose consumption in the PNWE and PNEE test groups was about the same as that in the control group except that the glucose consumption was small at the concentrations of 10mg/L and 15mg/L during the culture period. The contents of glutamine, lactic acid, ammonium salt and glutamic acid are not greatly different from those of the control group.

Example 2 Effect of pine needle extract on the titer of monoclonal antibodies produced by CHO cells

After the Fed-batch culture was completed, cell supernatants were collected and assayed for monoclonal antibody titer by CHO cells by HPLC, as shown in FIG. 7. From the results, it can be seen that the potency of the monoclonal antibody in the PNWE experimental group is improved to different degrees compared with the control group, and the results have statistical significance, but the potency of the monoclonal antibody is decreased with the increase of the concentration of the added pine needle extract. The potency of the monoclonal antibody of the PNEE experimental group is higher than that of the control group and has statistical significance under the condition of 5mg/L concentration, and the potency of the monoclonal antibody of the PNEE experimental group is lower than that of the control group under the concentrations of 10mg/L and 15mg/L, wherein the potency of the monoclonal antibody of the 15mg/L concentration experimental group is obviously lower than that of the control group, and similar to that of the PNWE experimental group, and the potency is reduced along with the increase of the concentration. The PNPWE experimental group is different from the PNPWE experimental group and the PNPWE experimental group, compared with the control group, the titer of the monoclonal antibody is obviously improved along with the increase of the concentration of the added extract, the titer is also obviously increased, a dose dependence relationship is formed, and in the aspect of statistics, the P values of three groups of data are less than 0.001 and have extremely significant difference.

Example 3 Effect of pine needle extract on the purity of monoclonal antibodies produced by CHO cells

After the end of the Fed-batch culture, the cell supernatant was first filtered through a 0.22 μm filter to remove cell residues from the supernatant. The experiment was performed by affinity chromatography using a 1mL gravity column to purify the monoclonal antibody sample from the culture. The purified monoclonal antibody samples were stored at-80 ℃ for subsequent further analysis of the spatial geometry of the antibody, charge variants of the antibody, antibody glycosylation, and antibody binding activity. In this example, the influence of the pine needle extract on the purity of the monoclonal antibody is observed from the spatial geometry level by size exclusion chromatography, specifically, TSKgel G3000SWxl Molecular size exclusion chromatography (TOSOH, JPN) is used, an HPLC system of Thermo Science Vanquish (Thermo Fisher, USA) is used to analyze the culture, and the influence of the pine needle extract on the spatial geometry level of the monoclonal antibody is monitored, and the specific detection results are shown in table 1, wherein Main peak represents Main peak, and High Molecular Weight represents High Molecular Weight; low Molecular Weight means Low Molecular Weight.

TABLE 1

The control of the purity of lgG produced by CHO cells is a very important part of the whole CHO cell culture process. The factors influencing the purity of lgG are mainly upstream Fed-batch culture and downstream supernatant purification process. The addition of the pine needle extract changes the material composition of the CHO cell growth environment, and it can be known from Table 1 that the purity of the monoclonal antibody decreases to a certain extent when the concentration of the PNPWE group increases, but on the whole, the pine needle extract has no great influence on the molecular size of the monoclonal antibody, and the relative percentage of the SEC main peak of all experimental groups is above 99%, which indicates that the purity of the monoclonal antibody cannot be adversely affected in the molecular size when the pine needle extract is added into the culture medium as a compound.

Example 4 Effect of pine needle extract on the Charge variants of monoclonal antibodies produced by CHO cells

This example examined the effect of pine needle extract on the charge variants of the mab produced by CHO cells by strong cation exchange chromatography (SCX-HPLC), specifically, the purified mab sample was analyzed using an HPLC system of Thermo Science Vanquish equipped with a Proteomix SCX-NP 5 μm 4.6 × 150mm analytical column (Sepax Technologies, inc), the peak eluted before the main peak was called "acidic peak", the peak eluted after the main peak was called "basic peak", and the amount of charge variation was determined by calculating the peak area percentage, and the specific examination results are shown in fig. 8.

According to the analysis results in fig. 8, the acidic species of the monoclonal antibody are decreased compared with the control group in the PNWE experimental group, the acidic species of the monoclonal antibody are decreased to a greater extent and the basic species are increased with higher PNWE concentration, and the ratio of the basic species is increased with the increase of the PNWE concentration, and the results of the two groups are very significantly statistically different compared with the control group. While the proportion of the major species of the mAb increases at 5mg/L and 10mg/L, and decreases at 15mg/L, but only a nominal increase or decrease, without statistical significance. In the PNEE experimental group, similar results to those of the PNWE experimental group were shown, and the acidic species decreased more with increasing concentration of the additive. The ratio of the alkaline substance is increased with the increase of the addition concentration. In addition to the statistical differences in the P-value <0.05 with the addition of 5mg/L concentration in alkaline species, the other experimental groups showed a very significant statistical difference in the P-value <0.001, while the main substances only showed a nominal increase in 5mg/L and 10 mg/L. The PNPWE experimental group had a reduction in acid species with the greatest reduction in acid species at 500mg/L and the results were very statistically different. The proportion of the basic species increases with increasing concentration. The main species of antibody was reduced in the proportion at 1000mg/L, and increased, but only nominally, at the concentrations of 50mg/L and 500 mg/L.

The combined results show that the three pine needle extracts can reduce the acidic species variant of the protein produced by the CHO cells under a certain concentration, but can not obviously increase the main species, but increase the alkaline species. The presence of acidic species may have some effect on the effectiveness and function of the protein produced. Therefore, in the production process of the monoclonal antibody, the ratio of the acidic species is controlled to be relatively low, the level of the main species is improved, the function and the effectiveness of the protein are facilitated, and the method is an important target in the protein manufacturing process. In the embodiment, the pine needle extract can remarkably reduce the generation of acidic species, and can generate certain benefits on the function of the monoclonal antibody in a certain aspect.

Example 5 Effect of pine needle extract on glycosylation of monoclonal antibodies produced by CHO cells

In this example, the glycosylation content of purified monoclonal antibody samples of each experimental group was analyzed by ultra high performance liquid chromatography fluorescence detection (UPLC-FLD), specifically using ACQUITY UPLC glycine BEH Amide,130 a, 1.7 μm,2.1x150mm chromatography column (Waters, USA) to obtain glycosylation pattern. The purified monoclonal antibody samples were desugarized and labeled using GlycoWorks RapidFluor-MS N-saccharide assay kit (Waters, USA) according to the saccharide assay kit instructions. The antibody protein was calibrated for the polysaccharide species according to the known glycosylation pattern, and the glycosylation change was analyzed as normalized peak area (percentage of peak area to the sum of all peak areas), as shown in fig. 9 and 10. Wherein G0, G2F, G1F GlcNAc, man5, G0 minus GlcNAc, G0F, G1Fa, G1Fb, G0F minus GlcNAc, man6, and the like indicate that different glycoforms are used for the designation by the IgG glycoform designation system.

As can be seen from fig. 9 and 10, glycosylation of the ten glycoforms was observed in the purified mabs of each experimental group, wherein the content of glycosylation in the highest glycoform was G0F, which was about 70%, while the addition of the pine needle extract reduced the level of G0F, and in the PNPWE experimental group, the decrease of G0F was more significant with the increase of the addition concentration, which was 8.73% at most compared with the control group. Similarly, the levels of G0 minus GlcNAc and G0F minus GlcNAc also decreased with increasing concentration of the extract from pine needles. The three sugar forms of G1Fa, G1Fb and G2F show the phenomenon that the level is increased along with the increase of the concentration of the added pine needle extract. In the PNPWE experimental group, G1Fa increased by 0.56%,4.41%,6.7% at increasing concentrations of three species. The G1Fb is increased by 0.96%,2.07% and 2.91%. While G2F is added to the pine needles at a level of only 0.22%, the levels increased by 0.1%,0.57%,1.01%, almost 3.6 times and 5.6 times at concentrations of 500mg/L and 1000mg/L. G0 showed an increase in level with increasing concentration in the PNWE and PNEE experimental groups. In the PNPWE group, G0 and G1F GlcNAc reached the highest levels at a concentration of 500mg/L, and thereafter showed a tendency that their G0 glycoform levels were decreased at high concentrations. Whereas Man5 and Man6 reached the highest levels at a concentration of 50 mg/L.

The sugars in the medium serve to meet the energy requirements of the CHO cells and are also a source of carbohydrates that allow the protein glycosylation pathway to proceed. Most of the cell-dependent saccharides are glucose, and according to the detection result of the glycosylation content, the saccharides and other unknown substances in the pine needle extract can influence the glycosylation of proteins and control the product quality. Therefore, by changing the components of the CHO cell culture medium and adding the pine needle extract with a certain concentration, the glycosylation of the protein can be controlled, and a specific oligosaccharide structure can be better targeted.

Example 6 Effect of pine needle extract on binding Activity of monoclonal antibodies produced by CHO cells

In this embodiment, enzyme-linked immunosorbent assay (ELISA) is used to determine the binding activity of the pine needle extract to the purified mab sample, and a four-parameter fitting regression model in SoftMax Pro computer software is used to plot a curve according to the detection result, where the regression curve for mab sample activity analysis in the PNWE experimental group is shown in fig. 11, the regression curve for mab sample activity analysis in the PNEE experimental group is shown in fig. 12, the regression curve for mab sample activity analysis in the PNPWE experimental group is shown in fig. 13, the abscissa in the curve represents the mab sample concentration, the ordinate represents the detected a450 value, the parameter C in the curve is the corresponding concentration (EC 50) at 50% of maximum benefit, and the relative activity is reported by calculating the EC50 ratio between the control group and the experimental group, specifically shown in fig. 11 and table 2, where R2 represents the coefficient for determining the regression curve for mab sample activity analysis in each experimental group.

TABLE 2

As can be seen from fig. 14 and table 2, the addition of the low-concentration pine needle extract reduced the lgG activity of the monoclonal antibody sample, and as the concentration increased, the activity also increased, and the lgG activity was concentration-dependent. Except for the PNEE experimental group, the activity of the monoclonal antibodies in the other groups is lower than that of the control group. According to the lgG binding activity requirement of the CHO K1 cell culture used in the embodiment, the lgG binding activity after adding different pine needle compounds is within an acceptable range, and the pine needle extract has little influence on the lgG binding activity secreted by the CHO.

In terms of safety index of the drug, the half maximal effect concentration (EC 50) of the mab produced by CHO cells was increased overall, and specifically as shown in fig. 11 and table 2, the EC50 of the PNWE experimental group and the PNEE experimental group decreased with the increase of the concentration of the pine needle extract, and approached to the control group. The EC50 values of the PNPWE experimental group at the two concentrations of 50mg/L and 500mg/L are close and are higher than those of the control group. The EC50 at a concentration of 1000mg/L was close to that of the control group. Therefore, the low concentration of the pine needle extract can increase the EC50 value of the monoclonal antibody produced by the CHO cells in the embodiment, and the safety of the medicine is improved to a certain extent.

All possible combinations of the technical features in the above embodiments may not be described for the sake of brevity, but should be considered as being within the scope of the present disclosure as long as there is no contradiction between the combinations of the technical features.

The above examples only express several embodiments of the present application, and the description thereof is more specific and detailed, but not to be construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the concept of the present application, which falls within the scope of protection of the present application. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. Use of a pine needle extract in the preparation of a CHO cell culture broth comprising a chemically defined CHO medium containing the pine needle extract;

the pine needle extract is a masson pine needle extract;

the pine needle extract comprises at least one of a pine needle ethanol extract, a pine needle water extract and a pine needle polysaccharide water extract;

the concentration of the pine needle ethanol extract or the pine needle water extract is 5 mg/L-15 mg/L;

the concentration of the pine needle polysaccharide water extract is 50 mg/L-1000 mg/L.

2. A CHO cell culture fluid, characterized by comprising a chemically defined CHO culture medium, wherein the chemically defined culture medium contains pine needle extract;

the pine needle extract is a masson pine needle extract;

the pine needle extract comprises at least one of pine needle ethanol extract, pine needle water extract and pine needle polysaccharide water extract;

3. The CHO cell broth of claim 2, further comprising a first supplement in a volume of 3.5% to 4.5% of the volume of the chemically defined CHO medium.

4. The CHO cell culture solution of claim 3, further comprising a second supplement in a volume ranging from 0.35% to 0.45% of the volume of the chemically defined CHO medium.

5. A CHO cell culture method characterized in that a CHO cell culture solution according to any one of claims 2 to 4 is used for cell culture of CHO cells.

6. The culture method according to claim 5, wherein the cell culture satisfies at least one of the following characteristics:

(1) Mixing a chemically defined CHO culture medium containing the pine needle extract and CHO cells at the beginning of culture to perform cell culture;

(2) The cell culture is fed-batch culture;

(3) The culture time is 14-16 days.

7. The culture method according to claim 6, wherein the fed-batch culture specifically comprises:

adding a first supplement and a second supplement in portions during the cell culture period, wherein the volume of the first supplement is 3.5% -4.5% of the volume of the chemically defined CHO culture medium, and the volume of the second supplement is 0.35% -0.45% of the volume of the chemically defined CHO culture medium.