Cell nutrition composition, cell nutrition additive, method for producing virus based on host cell, and method for producing vaccine

Abstract

The invention relates to a cell nutrition composition, a cell nutrition additive, a method for producing viruses based on host cells and a vaccine production method in the technical field of biological products. The cell nutrition composition comprises glucose, glutamine, methionine, lysine, valine, isoleucine, phenylalanine, leucine, tryptophan, threonine, sodium hypoxanthate, adenine and thymine according to a proper ratio. In the process of producing viruses based on host cells, the cell nutrition composition with a proper formula is selected and added, so that high-density culture of the cells is realized, and the virus titer is greatly improved after the viruses are inoculated.

Description

Technical Field

[0001] The present application relates to the technical field of biological products, and in particular to a cell nutrition composition, a cell nutrition additive, a method for producing viruses based on host cells, and a method for producing vaccines. Background Art

[0002] Avian adenovirus belongs to the genus Avian adenovirus of the family Adenoviridae. According to the difference in specific antigens, it is divided into three groups: I, II and III. Group I is mainly divided into 5 species (AE), including 12 serotypes. In recent years, avian adenovirus type 4 of group I serotype (FAdV-4) has continued to spread, causing serious economic losses to the poultry industry. FAdV-4 infection mainly causes symptoms such as pericardial effusion and inclusion body hepatitis in infected chickens. Vaccine immunization can effectively prevent and control FAdV-4 infection. The FAdV-4 antigen used in the preparation of the vaccine can be obtained by culturing FAdV-4 in chicken embryos, primary chicken embryo liver cells or chicken hepatoma cell lines (LMH). Studies have shown that FAdV-4 has a better proliferation effect on LMH cells.

[0003] LMH cells were first cultivated in 1981 by T Kawaguchi et al. using white Leghorn chicken liver cells suffering from hepatocellular carcinoma and induced by diethylnitrosamine mutagenesis. LMH is an anchorage-dependent cell. Large-scale culture usually uses roller bottles, cell factories or microcarrier suspension culture, which limits the rapid expansion of production scale. LMH suspension cell culture can be rapidly expanded. In traditional technology, the production of adenovirus by LMH cells is limited by low cell density and low cell toxin production, resulting in low yield. The main reason for the low cell density is that cell culture is affected by nutrient supply, oxygen, and metabolism, making it difficult to culture LMH cells at high density. The growth of infected cells and normal cells, the competition for nutrients, and the state of infected cells lead to low toxin production. Although regular addition of fresh culture medium to maintain the nutrient environment can improve cell growth, this growth is limited. Summary of the invention

[0004] Based on this, one or more embodiments of the present application provide a cell nutrition composition, a cell nutrition additive, a method for producing viruses based on host cells, and a method for producing vaccines. The technical solutions include the following:

[0005] One or more embodiments of the present application provide a cell nutrient composition, which includes (100g-150g): (7.3g-14.6g): (3.5g-4.6g): (6.6g-8.2g): (4.9g-6.2g): (4.8g-5.9g): (4.5g-5.4g): (7.2g-8.4g): (2.2g-4.4g): (6.5g-7.4g): (0.325g-0.478g): (0.0365g-0.0453g), (0.0653g-0.0767g) of glucose, glutamine, methionine, lysine, valine, isoleucine, phenylalanine, leucine, tryptophan, threonine, sodium hypoxanthine, adenine and thymine.

[0006] In some embodiments of the present application, the cell nutrition composition includes the cell nutrition composition including (120g-130g): (10.5g-11.5g): (3.8g-4.4g): (7g-8g): (5g-6g): (5.3g-5.7g): (4.5g-5.4g): (7.2g-8.4g): (3g-3.5g): (6.5g-7.4g): (0.4g-0.45g): (0.039g-0.041g), (0.07g-0.072g) of glucose, glutamine, methionine, lysine, valine, isoleucine, phenylalanine, leucine, tryptophan, threonine, sodium hypoxanthine, adenine and thymine.

[0007] In some embodiments of the present application, the cell nutrition composition is a serum-free cell nutrition composition.

[0008] One or more embodiments of the present application also provide a cell nutrition additive, the cell nutrition additive comprising 100g / L-150g / L glucose, 7.3g / L-14.6g / L glutamine, 3.5g / L-4.6g / L methionine, 6.6g / L-8.2g / L lysine, 4.9g / L-6.2g / L valine, 4.8g / L-5.9g / L isoleucine, amino acid, 4.5g / L-5.4g / L phenylalanine, 7.2g / L-8.4g / L leucine, 2.2g / L-4.4g / L tryptophan, 6.5g / L-7.4g / L threonine, 0.325g / L-0.478g / L sodium hypoxanthine, 0.0365g / L-0.0453g / L adenine and 0.0653g / L-0.0767g / L thymine.

[0009] In some embodiments of the present application, the cell nutrient additive includes 120g / L-130g / L glucose, 10.5g / L-11.5g / L glutamine, 3.8g / L-4.4g / L methionine, 7g / L-8g / L lysine, 5g / L-6g / L valine, 5.3g / L-5.7g / L isoleucine, 4.5g / L-5.4g / L phenylalanine, 7.2g / L-8.4g / L leucine, 3g / L-3.5g / L tryptophan, 6.5g / L-7.4g / L threonine, 0.4g / L-0.45g / L sodium hypoxanthine, 0.039g / L-0.041g / L adenine and 0.07g / L-0.072g / L thymine.

[0010] In some embodiments of the present application, the cell nutrition additive is a serum-free cell nutrition additive.

[0011] One or more embodiments of the present application further provide a cell culture product, the cell culture product comprising:

[0012] Cell culture medium; and,

[0013] The cell nutrition composition or the cell nutrition additive.

[0014] In some embodiments of the present application, the basic cell culture comprises a serum-free medium.

[0015] One or more embodiments of the present application also provide a method for producing viruses based on host cells, wherein the method comprises adding the cell nutrient composition or the cell nutrient additive during the process of producing the viruses.

[0016] In some embodiments of the present application, the method comprises the following steps:

[0017] Cultivating host cells, inoculating viruses, adding the cell nutrition composition or the cell nutrition additive, continuing to culture until the cell viability reaches a preset level, and harvesting the viruses;

[0018] Optionally, 5 mL to 50 mL of the cell nutrient additive is added to every 1 L of cell culture medium.

[0019] In some embodiments of the present application, the method adopts a perfusion culture method.

[0020] In some embodiments of the present application, the perfusion culture method satisfies one or more of the following conditions:

[0021] (1) The cell density of the host cells is 4×10 6 cells / mL-5×10 6cells / mL, and the perfusion was started at a rate of 0.5VVD-1VVD; the host cells were cultured to a cell density of 10×10 6 cells / mL-15×10 6 cells / mL, and the perfusion rate was adjusted to 1.5VVD-2VVD;

[0022] (2) after adding the cell nutrition composition or the cell nutrition additive, stopping the perfusion for 1 hour to 5 hours and then restarting the perfusion, the perfusion rate is 1VVD to 3VVD; and,

[0023] (3) culturing until the cell viability reaches the preset level, and stopping the perfusion; and,

[0024] (4) Perfusion culture was performed at 36.5-36.5°C, pH 7.0-7.2, stirring speed 30 rpm-300 rpm and dissolved oxygen concentration 30%-80%.

[0025] In some embodiments of the present application, the inoculated virus satisfies one or more of the following conditions:

[0026] 2) The virus inoculation MOI is 10 -2 -10; and,

[0027] 3) The cell density of the host cells is 15×10 6 cells / mL-20×10 6 The virus was inoculated under the condition of 1:1 cells / mL.

[0028] In some embodiments of the present application, the method satisfies one or more of the following conditions:

[0029] (A) The host cell comprises a LMH cell; optionally, the LMH cell comprises a LMH suspension cell;

[0030] (B) the virus comprises an avian adenovirus; optionally, the avian adenovirus comprises a group I serotype 4 avian adenovirus;

[0031] (C) the cell culture medium used includes serum-free medium;

[0032] (D) The preset level includes a cell viability lower than 80%; optionally, the cell viability is 80%-90%.

[0033] One or more embodiments of the present application further provide a method for producing a vaccine, the method comprising: producing a virus using the method; and,

[0034] A vaccine is prepared using the virus.

[0035] The details of one or more embodiments of the present application are set forth in the description which follows, and other features, objects, and advantages of the present application will be apparent from the description and its claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0037] Figure 1 This is a comparison chart of the highest density of LMH cells in perfusion culture and batch culture. DETAILED DESCRIPTION

[0038] The present application will be further described in detail below in conjunction with the accompanying drawings, embodiments and examples. It should be understood that these embodiments and examples are only used to illustrate the present application and are not used to limit the scope of the present application. The purpose of providing these embodiments and examples is to make the understanding of the disclosure of the present application more thorough and comprehensive. It should also be understood that the present application can be implemented in many different forms, is not limited to the embodiments and examples described herein, and those skilled in the art can make various changes or modifications without violating the connotation of the present application, and the equivalent form obtained also falls within the protection scope of the present application. In addition, in the description below, a large number of specific details are given in order to provide a more comprehensive understanding of the present application, and it should be understood that the present application can be implemented without one or more of these details.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing implementation modes and embodiments and are not intended to limit this application.

[0040] the term

[0041] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0042] The terms "and / or", "or / and", and "and / or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0043] In the present application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a number greater than 2 or equal to 2. For example, "one or more" means one or greater than or equal to two.

[0044] As used herein, "combination thereof", "any combination thereof", "any combination thereof" etc. include all suitable combinations of any two or more of the listed items.

[0045] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc., shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0046] Herein, “preferred”, “better”, “more preferred” and “suitable” are merely used to describe implementation methods or examples with better effects, and it should be understood that they do not constitute limitations on the scope of protection of this application.

[0047] In the present application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present application.

[0048] In this application, "optionally", "optional", and "optional" mean optional or dispensable, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0049] In the present application, the terms "first", "second", "third", "fourth", etc. in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0050] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0051] In the present application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all sub-ranges included therein.

[0052] The temperature parameters in this application, unless otherwise specified, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0053] In the present application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0054] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the application purpose and / or technical solution of this application, the cited documents involved in this application are cited with all contents and all purposes. When the cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the cited documents are involved in this application, the examples and preferred methods of the cited relevant technical features can also be incorporated into this application as references, but are limited to the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0055] According to a first aspect of an embodiment of the present application, a cell nutrition composition is provided, comprising (100g-150g): (7.3g-14.6g): (3.5g-4.6g): (6.6g-8.2g): (4.9g-6.2g): (4.8g-5.9g): (4.5g-5.4g): (7.2g-8.4g): (2.2g-4.4g): (6.5g-7.4g): (0.325g-0.478g): (0.0365g-0.0453g), (0.0653g-0.0767g) of glucose, glutamine, methionine, lysine, valine, isoleucine, phenylalanine, leucine, tryptophan, threonine, sodium hypoxanthine, adenine and thymine.

[0056] The cell nutrition composition of the present application, the dosage ratio is the weight ratio during use, and can also be referred to as the working weight ratio. The cell nutrition composition of the present application can be a mixture of the above-mentioned ingredients, or each component can be packaged independently, or some components can be packaged independently, and then mixed in proportion during use. That is, the cell nutrition composition of the present application can be ready-to-use or ready-to-match. In the process of producing viruses based on host cells, the present application selects the cell nutrition composition of adding a suitable formula, which not only realizes high-density cell culture, but also greatly improves the virus titer after inoculation of the virus.

[0057] In some examples, the cell nutrient composition includes the cell nutrient composition including (120g-130g): (10.5g-11.5g): (3.8g-4.4g): (7g-8g): (5g-6g): (5.3g-5.7g): (4.5g-5.4g): (7.2g-8.4g): (3g-3.5g): (6.5g-7.4g): (0.4g-0.45g): (0.039g-0.041g), (0.07g-0.072g) of glucose, glutamine, methionine, lysine, valine, isoleucine, phenylalanine, leucine, tryptophan, threonine, sodium hypoxanthine, adenine and thymine.

[0058] In some examples, the cell nutritional composition is a serum-free cell nutritional composition.

[0059] In a second aspect of the embodiment of the present application, a cell nutrition additive is provided, wherein the cell nutrition additive comprises 100 g / L-150 g / L glucose, 7.3 g / L-14.6 g / L glutamine, 3.5 g / L-4.6 g / L methionine, 6.6 g / L-8.2 g / L lysine, 4.9 g / L-6.2 g / L valine, 4.8 g / L-5.9 g / L isoleucine, Acid, 4.5g / L-5.4g / L phenylalanine, 7.2g / L-8.4g / L leucine, 2.2g / L-4.4g / L tryptophan, 6.5g / L-7.4g / L threonine, 0.325g / L-0.478g / L sodium hypoxanthine, 0.0365g / L-0.0453g / L adenine and 0.0653g / L-0.0767g / L thymine.

[0060] The amount used in the cell nutrition additive of the present application is measured in the addition state. It is understandable that when the concentration is high, the addition amount can be appropriately reduced, and when the concentration is low, the addition amount can be appropriately increased. It is understandable that the cell nutrition additive of the present application can be ready-to-use or ready-to-mix. If it is ready-to-mix, each component can be packaged independently. It is understandable that, for ease of addition, the cell nutrition additive of the present application is liquid, and the solvent is water. In some examples, the cell nutrient additives include 120g / L-130g / L glucose, 10.5g / L-11.5g / L glutamine, 3.8g / L-4.4g / L methionine, 7g / L-8g / L lysine, 5g / L-6g / L valine, 5.3g / L-5.7g / L isoleucine, 4.5g / L-5.4g / L phenylalanine, 7.2g / L-8.4g / L leucine, 3g / L-3.5g / L tryptophan, 6.5g / L-7.4g / L threonine, 0.4g / L-0.45g / L sodium hypoxanthine, 0.039g / L-0.041g / L adenine and 0.07g / L-0.072g / L thymine.

[0061] In some examples, the cell nutritional supplement is a serum-free cell nutritional supplement.

[0062] In a third aspect of the embodiments of the present application, a cell culture product is provided, the cell culture product comprising:

[0063] Cell culture medium; and,

[0064] The cell nutrition composition or the cell nutrition additive.

[0065] The cell culture product in the present application may be a packaged product, including a cell culture medium for cell culture and a cell nutrition composition or cell nutrition additive that needs to be added during the culture process. The definitions of the cell nutrition composition and the cell nutrition additive refer to the definitions in the first aspect and the second aspect, respectively.

[0066] The present application does not specifically limit the type of cell culture medium, such as serum-free culture medium. The cell culture medium of the present application is any known culture medium suitable for host cell growth and target virus reproduction, including but not limited to the following specific examples of Jianshun Bio LMH serum-free suspension medium LMH SFM 1516 (Cat. No.: 11301-1516), or LMH SFM 777 (Cat. No.: 11301-777).

[0067] A fourth aspect of the embodiments of the present application provides a method for producing viruses based on host cells, wherein the method comprises adding the cell nutrient composition or the cell nutrient additive during the process of producing the viruses.

[0068] In some examples, the method includes the following steps:

[0069] Cultivating host cells, inoculating viruses, adding the cell nutrition composition or the cell nutrition additive, continuing to culture until the cell viability reaches a preset level, and harvesting the viruses;

[0070] Optionally, 5 mL-50 mL (e.g., 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 mL) of the cell nutrient additive is added to every 1 L of cell culture medium.

[0071] In some examples, the method adopts a perfusion culture method. The present application does not specifically limit the equipment used for the perfusion culture method, including using a continuous perfusion device that uses an alternating tangential flow cell retention system.

[0072] In some examples, the perfusion culture method satisfies one or more of the following conditions:

[0073] (1) The cell density of the host cells is 4×10 6 cells / mL-5×10 6 cells / mL(4×10 6 cells / mL、4.2×10 6 cells / mL、4.4×10 6 cells / mL、4.6×10 6 cells / mL、4.8×10 6 cells / mL、5×10 6 cells / mL), and the perfusion rate is 0.5VVD-1VVD (for example, 0.5, 0.6, 0.7, 0.8, 0.9, 1VVD); the host cells are cultured to a cell density of 10×10 6 cells / mL-15×10 6 cells / mL(10×10 6 cells / mL、11×10 6 cells / mL、12×10 6 cells / mL、13×10 6 cells / mL、14×10 6 cells / mL、15×10 6cells / mL), and the perfusion rate was adjusted to 1.5VVD-2VVD (e.g., 1.5, 1.6, 1.7, 1.8, 1.9, 2VVD);

[0074] (2) after adding the cell nutrition composition or the cell nutrition additive, stop perfusion for 1 h-5 h (for example, 1, 2, 3, 4, 5 h) and then restart perfusion, the perfusion rate is 1 VVD-3 VVD (for example, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3 VVD);

[0075] (3) culturing until the cell viability reaches the preset level, and stopping the perfusion; and,

[0076] (4) perfusion culture at 34.5-36.5°C (e.g., 34.5, 34.6, 34.7, 34.8, 34.9, 35, 35.1, 35.2, 35.3, 35.4, 35.5, 35.6, 35.7, 35.8, 35.9, 36, 36.1, 36.2, 36.3, 36.4, 36.5°C), pH 7.0-7.2, stirring speed of 30 rpm-300 rpm (e.g., 30, 40, 50, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300 rpm, in a specific embodiment, the preferred stirring speed of the above culture can be 200 rpm) and dissolved oxygen concentration of 30%-80%.

[0077] It should be understood that before starting the perfusion culture, the host cells of the present application can be cultured in a conventional batch culture method, including: 6The host cells are inoculated into a bioreactor at a cell density of cells / mL and cultured at a temperature of 36.5-37.5°C (e.g., 36.5, 36.6, 36.7, 36.8, 36.9, 37.0, 37.1, 37.2, 37.3, 37.4, 37.5°C) and a pH of 6.7-7.5 (e.g., 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4; optionally, 6.8-7.2 ), a stirring speed of 30rpm-300rpm (such as 30, 40, 50, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300rpm. In a specific embodiment, the preferred stirring speed of the above culture can be 200rpm), and a dissolved oxygen concentration of 20%-80% (for example, 20%, 30%, 40%, 50%, 60%, 70%, 80%).

[0078] In some embodiments of the present application, the inoculated virus satisfies one or more of the following conditions:

[0079] 2) The virus inoculation MOI is 10 -2 -10 (e.g. 10 -2 , 10 -1 , 1, 10); and,

[0080] 3) The cell density of the host cells is 15×10 6 cells / mL-20×10 6 cells / mL (e.g. 15×10 6 cells / mL、16×10 6 cells / mL、17×10 6 cells / mL、18×10 6 cells / mL、19×10 6 cells / mL、20×10 6 The virus was inoculated under the condition of 1:1 cells / mL.

[0081] The present application does not specifically limit the type of host cells, for example, they may be LMH cells, which may be grown in adherent culture or in suspension culture. In some examples, they are LMH suspension cells.

[0082] In some embodiments of the present application, the method satisfies one or more of the following conditions:

[0083] (A) The host cell comprises a LMH cell; optionally, the LMH cell comprises a LMH suspension cell;

[0084] (B) the virus comprises an avian adenovirus; optionally, the avian adenovirus comprises a group I serotype 4 avian adenovirus;

[0085] (C) the cell culture medium used includes serum-free medium;

[0086] (E) The preset level includes a cell viability lower than 80%; optionally, the cell viability is 80%-90%.

[0087] In a fifth aspect of the embodiments of the present application, a method for producing a vaccine is provided, the method comprising: producing a virus using the method; and,

[0088] A vaccine is prepared using the virus.

[0089] The present application does not particularly limit the specific operation of preparing the vaccine using the virus, for example, including the steps of virus inactivation (for example: inactivation in formaldehyde solution at 37° C. for 24 hours) and emulsification.

[0090] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0091] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0092] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0093] Some experimental material information used in each embodiment of this application can be as follows:

[0094] Cells: LMH suspension cell strain, domesticated cell strain according to Example 1 in CN114480286A;

[0095] LMH suspension cell serum-free medium: Jianshun Bio LMH SFM 1516 (Cat. No.: 11301-1516);

[0096] 2L bioreactor: Applikon;

[0097] ATF: Repligen;

[0098] Counter: Beckman.

[0099] 1. Cultivation of Host Cells

[0100] 1.1 Initial Preparation

[0101] After LMH cells were recovered, they were expanded at a rate of 1.5 × 10 6 The host cells were inoculated into a 2 L bioreactor at a cell density of cells / mL, the culture volume was 1 L, the temperature was 37° C., the pH value was 6.8-7.2, the dissolved oxygen concentration was 40%, and the rotation speed was 200 rpm for cultivation.

[0102] 1.2.1 Perfusion process

[0103] (1) Carry out perfusion culture under the first perfusion condition as shown below:

[0104] In the first culture stage, at a cell density of 4 × 10 6 cells / mL-5×10 6 cells / mL, and the perfusion rate was 0.1 nL / cell / day for culture.

[0105] In the second culture stage, at a cell density of 8×10 6 cells / mL-10×10 6 cells / mL, the perfusion rate was adjusted to 0.2 nL / cell / day for culture;

[0106] In the third culture stage, at a cell density of 12×10 6 cells / mL-15×10 6 cells / mL, the perfusion rate was adjusted to 0.3 nL / cell / day for culture;

[0107] In the fourth culture stage, at a cell density of 16 × 10 6 cells / mL-19×10 6 cells / mL, the perfusion rate was adjusted to 0.4 nL / cell / day for culture;

[0108] In the fifth culture stage, at a cell density of 20×10 6 cells / mL-25×10 6 cells / mL, the perfusion rate was adjusted to 0.5 nL / cell / day for culture;

[0109] At the 6th culture stage, when the cell density was 28×10 6 cells / mL-35×10 6Under the condition of 10 cells / mL, the perfusion rate was adjusted to 0.7 nL / cell / day for culture.

[0110] (2) Performing perfusion culture under the second perfusion condition as shown below:

[0111] In the first culture stage, the cell density was 4×10 6 cells / mL-5×10 6 cells / mL, and the perfusion rate was 1VVD;

[0112] In the second culture stage, the cell density was 10×10 6 cells / mL-12×10 6 cells / mL, the perfusion rate was adjusted to 2VVD for culture;

[0113] In the third culture stage, at a cell density of 25×10 6 cells / mL-30×10 6 cells / mL, the perfusion rate was adjusted to 3VVD for culture.

[0114] The initial preparation and perfusion culture were carried out for a total of 11 days, and the viable cell concentration and cell viability were counted every day.

[0115] Table 1. Density and cell viability of LMH cells cultured under two perfusion conditions

[0116]

[0117]

[0118] Considering the complexity of process operation, production cost, cell density and viability, it is better to culture LMH cells under the second perfusion condition.

[0119] 1.2.2 Conventional batch culture

[0120] Different from the perfusion culture in 1.2.1, this example also sets up a conventional batch culture group. Specifically, the culture is continued for 11 days according to the conditions in 1.1, and the cell density and cell viability are recorded every day. The results are shown in Figure 1 "Batch culture cell density" and "Batch cell viability" in Figure 1 The "perfusion culture cell density" and "perfusion culture cell viability" in the above table are the results obtained under the second perfusion culture condition in 1.2.1. Figure 1 Comparing LMH cell batch culture with perfusion culture, it is obvious that perfusion process is superior to conventional batch culture process, and the cell density is increased from 15.4×10 6 cells / mL increased to 34.21×10 6cells / mL.

[0121] 2. Drug

[0122] In the early stage, the LMH suspension cell perfusion culture was carried out under the second perfusion culture condition in 1.2.1, at 10×10 6 cells / mL-14×10 6 cells / mL (D6, first cell density), 15×10 6 cells / mL-20×10 6 cells / mL (D7, second cell density) and 21×10 6 cells / mL-25×10 6 cells / mL (D8, the third cell density) were inoculated with the virus, and the MOI was 10 -1 After infection, perfusion was stopped for 1 hour and then restarted. The perfusion rate was 2VVD, the temperature was adjusted to 35°C, the pH was 7.0-7.2, the dissolved oxygen concentration was 40%, and the rotation speed was 200rpm. The culture was terminated when the cell viability dropped below 80%. The virus TCID50 was tested according to the half-cell infection dose. The test results are shown in Table 2.

[0123] Table 2. Comparison of virus production at different cell densities

[0124] Drug intake time First cell density Second cell density The third cell density Infection density <![CDATA[12.96×10 6 cells / mL]]> <![CDATA[16.42×10 6 cells / mL]]> <![CDATA[24.38×10 6 cells / mL]]> TCID50 <![CDATA[10 9.8 TCID50 / mL]]> <![CDATA[10 10.4 TCID50 / mL]]> <![CDATA[10 10.0 TCID50 / mL]]>

[0125] The above test results show that the LMH perfusion cell density is lower than 15×10 6 cells / mL-20×10 6 cells / mL. After inoculation, the virus TCID50 increased with the increase of density. The LMH perfusion cell density was higher than 15×10 6 cells / mL-20×10 6 cells / mL. After inoculation, the virus TCID50 decreased with the increase of density.

[0126] Furthermore, based on the above comparison, the LMH suspension cell perfusion culture was performed under the second perfusion culture condition in 1.2.1. 6 cells / mL-20×10 6 cells / mL (D7, the second cell density), and the MOI was 10 -1After infection, perfusion was stopped for 1 hour and then restarted. The perfusion rates were 1VVD (first perfusion rate), 2VVD (second perfusion rate) and 3VVD (third perfusion rate). The temperature was adjusted to 35°C, pH to 7.0-7.2, and dissolved oxygen concentration to 40%. The culture was terminated when the cell viability dropped below 80%. The virus TCID50 was tested according to the half-cell infection dose. The test results are shown in Table 3.

[0127] Table 3. Results of cell toxin production at different perfusion rates after inoculation

[0128] Perfusion rate 1VVD 2VVD 3VVD TCID50 <![CDATA[10 9.87 TCID50 / mL]]> <![CDATA[10 10.2 TCID50 / mL]]> <![CDATA[10 10.23 TCID50 / mL]]>

[0129] The above test results show that after infection, there is no significant difference in the toxin-producing ability of perfused cells at perfusion rates of 2VVD and 3VVD. Considering the production cost, the second perfusion rate of 2VVD can be used.

[0130] In the early stage, the LMH suspension cell perfusion culture was carried out under the second perfusion culture condition in 1.2.1, and the cell density was 15×10 6 cells / mL-20×10 6 cells / mL (D7, the second cell density) were inoculated with the virus, the MOI of the virus inoculation was 10-1, the perfusion was stopped for 1 hour after the virus inoculation and then restarted, the perfusion rate was 2VVD (the second perfusion rate), the temperatures were adjusted to 33°C (the first perfusion temperature), 35°C (the second perfusion temperature) and 37°C (the third perfusion temperature), the pH was 7.0-7.2, the dissolved oxygen concentration was 40%, the rotation speed was 200rpm, the culture was terminated when the cell viability dropped below 80%, and the virus TCID50 was tested according to the half cell infection dose. The test results are shown in Table 4.

[0131] Table 4. Results of cell toxin production at different temperatures after inoculation

[0132] temperature 33℃ (first perfusion temperature) 35℃ (second perfusion temperature) 37℃ (third perfusion temperature) TCID50 <![CDATA[10 10 TCID50 / mL]]> <![CDATA[10 10.23 TCID50 / mL]]> <![CDATA[10 9.5 TCID50 / mL]]>

[0133] The above test results show that the cells perfused at 35°C after infection have the greatest toxin production capacity.

[0134] In the early stage, the LMH suspension cell perfusion culture was carried out under the second perfusion culture condition in 1.2.1, and the cell density was 15×10 6 cells / mL-20×10 6cells / mL (D7, the second cell density) were inoculated with the virus, the MOI of the virus inoculation was 10-1, the perfusion was stopped for 1 hour after the virus inoculation and then restarted, the perfusion rate was 2VVD (the second perfusion rate), the temperature was adjusted to 35°C (the second perfusion temperature), the pH was 6.8-7.0 (the first perfusion pH value), 7.0-7.2 (the second perfusion pH value) and 6.8-7.2 (the third perfusion pH value), the dissolved oxygen concentration was 40%, the rotation speed was 200 rpm, the culture was terminated when the cell viability dropped below 80%, and the virus TCID50 was tested according to the half cell infection dose. The test results are shown in Table 5.

[0135] Table 5. Results of cell toxin production under different pH control after inoculation

[0136] pH 6.8-7.0 (first perfusion pH) 7.0-7.2 (second perfusion pH) 6.8-7.2 (third perfusion pH) TCID50 <![CDATA[10 9 TCID50 / mL]]> <![CDATA[10 10.23 TCID50 / mL]]> <![CDATA[10 10.0 TCID50 / mL]]>

[0137] The above test results show that the toxin production capacity of perfusion cultured cells reaches the maximum at pH 7.0-7.2 after infection.

[0138] After the above comparison, the method for preparing avian adenovirus can be determined to include the following steps:

[0139] Step 1: After LMH cells were revived, they were expanded at a rate of 1.5×10 6 The host cells were inoculated into a 2 L bioreactor at a cell density of cells / mL, the culture volume was 1 L, the temperature was 37° C., the pH value was 6.8-7.2, the dissolved oxygen concentration was 40%, and the rotation speed was 200 rpm for cultivation.

[0140] Step 2: Perfusion culture:

[0141] In the first culture stage, the cell density was 4×10 6 cells / mL-5×10 6 cells / mL, and the perfusion rate was 1VVD;

[0142] In the second culture stage, the cell density was 10×10 6 cells / mL-12×10 6 cells / mL, the perfusion rate was adjusted to 2VVD for culture; the cell density was cultured to 15×10 6 cells / mL-20×10 6 cells / mL.

[0143] Step 3: Take the poison

[0144] After infection, stop perfusion for 1 hour and then restart perfusion, the perfusion rate is 2VVD (the second perfusion rate), the temperature is adjusted to 35°C (the second perfusion temperature), the pH is 7.0-7.2 (the second perfusion pH value), the dissolved oxygen concentration is 40%, the rotation speed is 200rpm, and the culture is terminated when the cell viability drops below 80%.

[0145] The culture medium used in items 1 and 2 was LMH SFM 1516.

[0146] 3. Perfusion Additives

[0147] The preparation method of avian adenovirus determined under item 2 was adopted. After inoculation, serum-free additives of different amounts and different formulations (see Table 6 below) were added respectively. The perfusion was stopped for 1 hour and then restarted. The perfusion rate was 2VVD (the second perfusion rate). The temperature was adjusted to 35°C (the second perfusion temperature), pH was 7.0-7.2 (the second perfusion pH value), dissolved oxygen concentration was 40%, and the rotation speed was 200 rpm. The culture was terminated when the cell viability dropped below 80%. The virus TCID50 was tested according to the half cell infection dose. The test results are shown in Table 7.

[0148] Table 6. Chemical composition-defined serum-free supplement formula (unit: g / L)

[0149] Element Recipe 1 Recipe 2 Recipe 3 Recipe 4 Recipe 5 glucose 100 150 125 100 150 Glutamine 7.3 14.6 10.95 14.6 7.3 Methionine 3.5 3.5 4.1 4.6 4.6 Lysine 6.6 8.2 7.4 8.2 6.6 Valine 4.9 6.2 5.6 6.2 6.2 Isoleucine 4.8 4.8 5.5 4.8 4.8 Phenylalanine 4.5 4.5 4.9 5.4 4.5 Leucine 7.2 8.4 7.7 7.2 7.2 Tryptophan 2.2 4.4 3.3 4.4 2.2 Threonine 6.5 7.4 6.9 6.5 7.4 Sodium hypoxanthine 0.325 0.478 0.425 0.325 0.478 Adenine 0.0365 0.0365 0.0393 0.0365 0.0365 Thymine 0.0653 0.0767 0.0717 0.0653 0.0767

[0150] Table 7. Comparison of different formulations and different addition amounts of serum-free additives with defined chemical composition

[0151] Addition amount 0mL 10mL 20mL Recipe 1 <![CDATA[10 10.25 TCID50 / mL]]> <![CDATA[10 10.75 TCID50 / mL]]> <![CDATA[10 10.60 TCID50 / mL <!-- 10 -->]]> Recipe 2 <![CDATA[10 10.25 TCID50 / mL]]> <![CDATA[10 10.75 TCID50 / mL]]> <![CDATA[10 10.50 TCID50 / mL]]> Recipe 3 <![CDATA[10 10.25 TCID50 / mL]]> <![CDATA[10 11.00 TCID50 / mL]]> <![CDATA[10 10.75 TCID50 / mL]]> Recipe 4 <![CDATA[10 10.25 TCID50 / mL]]> <![CDATA[10 10.75 TCID50 / mL]]> <![CDATA[10 10.60 TCID50 / mL]]> Recipe 5 <![CDATA[10 10.25 TCID50 / mL]]> <![CDATA[10 10.75 TCID50 / mL]]> <![CDATA[10 10.75 TCID50 / mL]]>

[0152] The results in the table above show that when 10 mL of Formula 3 is added, the virus yield is significantly increased.

[0153] Referring to the preparation method of avian adenovirus recorded under this item, after inoculation, different amounts and different control formulations (formulation 3-D1 to formulation 3-D6) of serum-free additives were added, and the perfusion was restarted after stopping the perfusion for 1 hour. The perfusion rate was 2VVD (the second perfusion rate), the temperature was adjusted to 35°C (the second perfusion temperature), the pH was 7.0-7.2 (the second perfusion pH value), and the dissolved oxygen concentration was 40%. The culture was terminated when the cell viability dropped below 80%, and the virus TCID50 was tested according to the half-cell infection dose. The test results are shown in Table 8.

[0154] The only difference between the additives in Formula 3-D1 and those in Formula 3 in Table 6 is that flavin adenine dinucleotide disodium salt is used instead of hypoxanthine sodium.

[0155] The only difference between Formulation 3-D2 and the additives shown in Formulation 3 in Table 6 is that flavin adenine dinucleotide disodium salt is used instead of adenine.

[0156] The only difference between Formulation 3-D3 and the additives shown in Formulation 3 in Table 6 is that thymidine is used instead of thymine.

[0157] The only difference between the additives in Formula 3-D4 and those in Formula 3 in Table 6 is that the concentrations of sodium hypoxanthine, adenine, and thymine are 0.3 g / L, 0.05 g / L, and 0.05 g / L, respectively.

[0158] Table 8

[0159] Addition amount 0mL 10mL 20mL Formula 3-D1 <![CDATA[10 10.25 TCID50 / mL]]> <![CDATA[10 10.50 TCID50 / mL]]> <![CDATA[10 10.40 TCID50 / mL]]> Formula 3-D2 <![CDATA[10 10.25 TCID50 / mL]]> <![CDATA[10 10.50 TCID50 / mL]]> <![CDATA[10 10.40 TCID50 / mL]]> Formula 3-D3 <![CDATA[10 10.25 TCID50 / mL]]> <![CDATA[10 10.60 TCID50 / mL]]> <![CDATA[10 10.50 TCID50 / mL]]> Formula 3-D4 <![CDATA[10 10.25 TCID50 / mL]]> <![CDATA[10 10.60 TCID50 / mL]]> <![CDATA[10 10.50 TCID50 / mL]]>

[0160] The above embodiments of the present application provide a technical method for high-density culture of avian adenovirus host cells (such as LMH cells) through a perfusion culture process. The selected culture conditions and perfusion conditions are suitable for the growth of adenovirus host cells (such as LMH cells). Through this technical method, a higher cell density and virus infection than batch and conventional perfusion processes can be obtained. Through perfusion culture and interception devices, fresh culture medium can be continuously added to the reactor to take away metabolic waste in real time, achieving high-density cell culture, and greatly improving the virus titer after inoculation of avian adenovirus. The technical solution provided by the present application realizes the high-density growth of avian adenovirus host cells (such as LMH cells) while increasing the cell yield of the virus after infection.

[0161] The technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. To make the description concise, not all possible combinations of the technical features in the above-mentioned implementation modes and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0162] The above-described embodiments only express several implementation methods of the present application, which is convenient for understanding the technical solution of the present application in detail, but it cannot be understood as a limitation on the scope of protection of the patent application. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several deformations and improvements can be made, which all belong to the protection scope of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, the technicians in this field can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the protection scope of the present application. It should also be understood that the technical solutions obtained by the technicians in this field through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the protection scope of the claims attached to the present application. Therefore, the protection scope of the patent of the present application shall be based on the content of the attached claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. A cell nutrition composition, characterized in that The cell nutrient composition includes (100g-150g): (7.3g-14.6g): (3.5g-4.6g): (6.6g-8.2g): (4.9g-6.2g): (4.8g-5.9g): (4.5g-5.4g): (7.2g-8.4g): (2.2g-4.4g): (6.5g-7.4g): (0.325g-0.478g): (0.0365g-0.0453g), (0.0653g-0.0767g) of glucose, glutamine, methionine, lysine, valine, isoleucine, phenylalanine, leucine, tryptophan, threonine, hypoxanthine sodium, adenine and thymine.

2. The cell nutrition composition according to claim 1, characterized in that The cell nutrition composition includes the following: (120g-130g): (10.5g-11.5g): (3.8g-4.4g): (7g-8g): (5g-6g): (5.3g-5.7g): (4.5g-5.4g): (7.2g-8.4g): (3g-3.5g): (6.5g-7.4g): (0.4g-0.45g): (0.039g-0.041g), (0.07g-0.072g) of glucose, glutamine, methionine, lysine, valine, isoleucine, phenylalanine, leucine, tryptophan, threonine, sodium hypoxanthine, adenine and thymine.

3. The cell nutrition composition according to any one of claims 1 to 2, characterized in that The cell nutrition composition is a serum-free cell nutrition composition.

4. A cell nutrition additive, characterized in that: The cell nutrient additive includes 100g / L-150g / L glucose, 7.3g / L-14.6g / L glutamine, 3.5g / L-4.6g / L methionine, 6.6g / L-8.2g / L lysine, 4.9g / L-6.2g / L valine, 4.8g / L-5.9g / L isoleucine, 4.5g / L-5.4g / L phenylalanine, 7.2g / L-8.4g / L leucine, 2.2g / L-4.4g / L tryptophan, 6.5g / L-7.4g / L threonine, 0.325g / L-0.478g / L sodium hypoxanthine, 0.0365g / L-0.0453g / L adenine and 0.0653g / L-0.0767g / L thymine.

5. The cell nutrition additive according to claim 4, characterized in that The cell nutrient additives include 120g / L-130g / L glucose, 10.5g / L-11.5g / L glutamine, 3.8g / L-4.4g / L methionine, 7g / L-8g / L lysine, 5g / L-6g / L valine, 5.3g / L-5.7g / L isoleucine, 4.5g / L-5.4g / L phenylalanine, 7.2g / L-8.4g / L leucine, 3g / L-3.5g / L tryptophan, 6.5g / L-7.4g / L threonine, 0.4g / L-0.45g / L sodium hypoxanthine, 0.039g / L-0.041g / L adenine and 0.07g / L-0.072g / L thymine.

6. The cell nutrition additive according to any one of claims 4 to 5, characterized in that The cell nutrition additive is a serum-free cell nutrition additive.

7. A cell culture product, characterized in that: The cell culture products include: Cell culture medium; and, The cell nutrition composition according to any one of claims 1 to 3, or the cell nutrition additive according to any one of claims 4 to 6.

8. The cell culture product according to claim 7, characterized in that The cell-based culture includes serum-free medium.

9. A method for producing viruses based on host cells, characterized in that: The method comprises adding the cell nutrition composition according to any one of claims 1 to 3 or the cell nutrition additive according to any one of claims 4 to 6 during the virus production process.

10. The method for producing virus based on host cells according to claim 9, characterized in that: The method comprises the following steps: Cultivating host cells, inoculating viruses, adding the cell nutrition composition or the cell nutrition additive, continuing to culture until the cell viability reaches a preset level, and harvesting the viruses; Optionally, 5 mL to 50 mL of the cell nutrient additive is added to every 1 L of cell culture medium.

11. The method for producing virus based on host cells according to claim 10, characterized in that: The method adopts a perfusion culture method; Optionally, the perfusion culture method satisfies one or more of the following conditions: (1) The cell density of the host cells is 4×10 6 cells / mL-5×10 6 cells / mL, and the perfusion was started at a rate of 0.5VVD-1VVD; the host cells were cultured to a cell density of 10×10 6 cells / mL-15×10 6 cells / mL, and the perfusion rate was adjusted to 1.5VVD-2VVD; (2) after adding the cell nutrition composition or the cell nutrition additive, stopping the perfusion for 1 hour to 5 hours and then restarting the perfusion, the perfusion rate is 1VVD to 3VVD; and, (3) culturing until the cell viability reaches the preset level, and stopping the perfusion; and, (4) Perfusion culture was performed at 34.5°C-36.5°C, pH 7.0-7.2, stirring speed 30 rpm-300 rpm and dissolved oxygen concentration 30%-80%.

12. The method for producing virus based on host cells according to claim 10, characterized in that: The inoculated virus meets one or more of the following conditions: 2) The virus inoculation MOI is 10 -2 -10; and, 3) The cell density of the host cells is 15×10 6 cells / mL-20×10 6 The virus was inoculated under the condition of 10 cells / mL.

13. The method for producing virus based on host cells according to any one of claims 10 to 12, characterized in that: The method satisfies one or more of the following conditions: (A) The host cell comprises a LMH cell; optionally, the LMH cell comprises a LMH suspension cell; (B) the virus comprises an avian adenovirus; optionally, the avian adenovirus comprises a group I serotype 4 avian adenovirus; (C) the cell culture medium used includes a serum-free medium; and, (D) The preset level includes a cell viability lower than 80%; optionally, the cell viability is 80%-90%.

14. A method for producing a vaccine, characterized in that: The production method comprises: producing a virus using the method of any one of claims 9 to 13; and A vaccine is prepared using the virus.

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