A production process for producing a recombinant antibody protein by hybridoma cell suspension culture

Abstract

The application discloses a production process of recombinant antibody protein produced by hybridoma cell suspension culture, and comprises the following steps: 1) resuscitation culture, 2) static expansion culture, and 3) suspension domestication and expansion culture. Compared with the semi-adherent culture method of hybridoma cells, the hybridoma cell suspension culture method has the following advantages: no trypsin digestion is needed during cell passage; the cell survival rate is higher, and the culture is easy to enlarge.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of hybridoma cell production of recombinant antibody protein, and particularly relates to a production process of hybridoma cell suspension culture for producing recombinant antibody protein. BACKGROUND

[0002] Hybridoma cells are formed by fusing myeloma cells with mouse B cells under specific conditions. Hybridoma cells retain the characteristics of both parents, and can both secrete antibodies and proliferate indefinitely.

[0003] At present, hybridoma cells are obtained by fusing mouse spleen cells with myeloma cells, and then purified monoclonal cells are obtained by further screening. The cells are cultured to produce antibodies, and the produced antibodies are monoclonal antibodies. Monoclonal antibody technology has always been the basis for the progress of human immune-related experiments. By culturing hybridoma cells, a large amount of antibodies with uniform specificities, sensitivities, etc. can be obtained. Monoclonal antibodies have good batch stability, stable product characteristics, low late-stage investment, short cycle, can quickly obtain a large amount of antibodies, and are suitable for large-scale production, etc. Therefore, monoclonal antibodies have always been an indispensable part of antibody preparation technology.

[0004] During the culture of hybridoma cells, the culture medium plays a key role. If the nutrition of the culture medium is insufficient, it is easy to cause the hybridoma cells to be in a poor state, reduce the activity, or even die, which ultimately affects the yield of antibodies. The current hybridoma culture medium can be used to culture hybridoma cells in a good state, but for hybridoma cells in a poor state and with low activity, the culture effect is obviously poor. The main reason for this problem is that the existing culture medium is not nutritious enough, and cannot improve the state and activity of the hybridoma cells. The usual way to solve this problem is to increase the concentration of serum in the culture medium or replace it with better serum to improve the nutrition of the culture medium and improve the state and activity of the cells. However, in this way, the consumption of serum will increase, and the cost will greatly increase. Moreover, some imported fetal bovine serum is good, but the blood source is tight, and even there is no market. The most important thing is that the addition of a high proportion of serum brings exogenous antibodies that interfere with the production of monoclonal antibodies, which is not conducive to the production of monoclonal antibodies.

[0005] Since hybridoma cells are semi-adherent cells, they need to be immobilized in order to increase their culture density. At present, the hybridoma cell suspension domestication technology on the market is not mature, and a new suspension culture method needs to be tried for high-density large-scale culture of hybridoma cells, so as to improve the production scale of antibodies. At the same time, this suspension domestication technology also greatly improves the survival rate of hybridoma cells. SUMMARY

[0006] The application provides a production process of hybridoma cell suspension culture for producing recombinant antibody protein to solve the above problems in the prior art.

[0007] The application provides the following scheme:

[0008] The application provides a production process of hybridoma cell suspension culture for producing recombinant antibody protein, comprising the following steps:

[0009] 1) resuscitation culture, the frozen hybridoma cells are inoculated in RPMI 1640 containing 5% FBS and placed in a carbon dioxide incubator for resuscitation culture;

[0010] 2) static amplification culture, the hybridoma cells in good condition after resuscitation are digested with 0.05% trypsin at 37 DEG C for 45s, then terminated with RPMI 1640 containing 5% FBS, the cell suspension is collected and centrifuged at 600 rpm / min for 5 min, the supernatant is discarded, and the cells are resuspended in Hybrid 01 serum-free medium and inoculated in a T75 cell culture bottle at a ratio of 1:5, and subsequent static amplification passage is carried out at a ratio of 1:5;

[0011] 3) suspension domestication and expansion culture, the hybridoma cells cultured in the T75 bottle are gently tapped to make the cells suspended, the cells are collected at 600 rpm / min for 5 min, the supernatant is discarded, 3 mL of Hybrid 01 serum-free medium for hybridoma cells is added to resuspend the cells, and the cells are inoculated in a 125 mL shaking flask at a cell density of 1.0*105-3.0*105 cells / mL and a volume of 15-20 mL, and the cells are cultured in suspension at Tm: 37 DEG C, CO2 concentration: 5%, shaking speed: 90-120 rpm and amplitude: 50 mM, the cells are sampled and counted and the viability is analyzed every day, and the expression amount is determined on the fifth day. 5 cells / mL cell density and a volume of 15-20 mL, and the cells are cultured in suspension at Tm: 37 DEG C, CO2 concentration: 5%, shaking speed: 90-120 rpm and amplitude: 50 mM, the cells are sampled and counted and the viability is analyzed every day, and the expression amount is determined on the fifth day.

[0012] As a preferred technical feature, the culture condition of step 2) is 37 DEG C, the CO2 concentration is 5%, the shaking speed is 120 rpm, and the culture is carried out for 24 h.

[0013] As a preferred technical feature, the culture condition of step 3) is 37 DEG C, the CO2 concentration is 5%, and the shaking speed is 120 rpm.

[0014] The application also discloses a self-developed Hybrid 01 serum-free medium for hybridoma cells, wherein the Hybrid 01 medium comprises compound amino acids, vitamins, trace elements and supplement factors.

[0015] As a preferred technical feature, the components and concentration contents of the self-developed serum-free and animal-origin component-free basic medium for hybridoma cells are as follows:

[0016] The lipid component comprises: 0.000008 g / L linoleic acid, 1.0 g / L cholesterol;

[0017] The basic component comprises:

[0018] The inorganic salt component: 1.6 g / L potassium chloride, 0.6 g / L calcium chloride, 3.0 g / L sodium dihydrogen phosphate, 1.0 g / L disodium hydrogen phosphate, 6.5 g / L sodium chloride, 1.0 g / L magnesium sulfate, 3.0 g / L P188;

[0019] The amino acid and sugar component: 1.2 g / L asparagine, 0.2 g / L methionine, 0.3 g / L histidine, 0.9 g / L aspartic acid, 0.2 g / L serine, 0.9 g / L glycine, 1.3 g / L arginine, 0.3 g / L isoleucine, 1.3 g / L leucine, 1.2 g / L glutamic acid, 1.4 g / L valine, 1.0 g / L lysine, 0.5 g / L phenylalanine, 0.2 g / L proline, 0.1 g / L threonine, 1.4 g / L glutamine, 0.4 g / L tyrosine, 0.5 g / L alanine, 6 g / L glucose;

[0020] The vitamin component: 0.0001 g / L vitamin B6, 0.00003 g / L vitamin B1, 0.00008 g / L biotin, 0.00004 g / L vitamin B12, 0.000001 g / L pantothenic acid, 0.00004 g / L folic acid, 0.0008 g / L nicotinic acid, 0.00004 g / L riboflavin, 0.004 g / L choline chloride, 0.00004 g / L inositol;

[0021] The trace element component: 0.00004 g / L copper chloride, 0.0001 g / L zinc chloride, 0.0004 g / L ferrous sulfate, 0.000001 g / L ammonium molybdate, 0.00002 g / L manganese chloride.

[0022] Advantages of the application:

[0023] The hybridoma cell suspension culture method of the application has more advantages over the hybridoma cell adherent culture method, including no need for trypsin digestion and dispersion during subculture, avoiding enzyme, EDTA and mechanical damage; high cell yield, and online continuous direct detection of cell density; using the Hybrid 01 medium derived from the self-developed serum-free hybridoma culture medium enables the hybridoma cells to grow rapidly under suspension culture conditions without adaptation, the cell density and cell activity can be comparable to those of serum-containing medium, which can meet the hybridoma cell culture requirements, effectively avoid many adverse factors caused by the use of serum, reduce production cost, and completely replace the serum-containing medium for high-density large-scale culture of hybridoma cells. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 Figure of cell viable cell density analysis data table of Hybrid 01 base medium suspension culture and 5% FBS + RPMI 1640 medium adherent culture hybridoma cells after 5 days of culture in the embodiments of the present application;

[0025] Figure 2 Figure of cell viability analysis data table of Hybrid 01 base medium suspension culture and 5% FBS + RPMI 1640 medium adherent culture hybridoma cells after 5 days of culture in the embodiments of the present application;

[0026] Figure 3 Figure of cell diameter analysis data table of Hybrid 01 base medium suspension culture and 5% FBS + RPMI 1640 medium adherent culture hybridoma cells after 5 days of culture in the embodiments of the present application;

[0027] Figure 4 Figure of expression analysis data table of Hybrid 01 base medium suspension culture and 5% FBS + RPMI 1640 medium adherent culture hybridoma cells after 5 days of culture. DETAILED DESCRIPTION

[0028] A hybridoma cell suspension culture production process for producing recombinant antibody proteins, comprising the following steps:

[0029] 1) Resuscitation culture, after resuscitation of the frozen hybridoma cells, inoculate in RPMI 1640 containing 5% FBS and place in a carbon dioxide incubator for resuscitation culture;

[0030] 2) Static expansion culture, after resuscitation, well hybridoma cells are digested with 0.05% trypsin at 37°C for 45s, then terminated with RPMI 1640 containing 5% FBS, the cell suspension is collected and centrifuged at 600 rpm / min for 5 min, the supernatant is discarded and the cells are resuspended with Hybrid 01 serum-free medium according to a 1:5 ratio, inoculated in a T75 cell culture flask, and subsequent static expansion passage is carried out at a ratio of 1:5;

[0031] 3) Suspension acclimation and expansion culture, gently tap the hybridoma cells cultured in the T75 bottle to make the cells suspended, collect the cells at 600 rpm / min for 5 min, discard the supernatant, add 3mL of hybridoma cell serum-free medium Hybrid 01 to resuspend, inoculate in a 125mL shake flask at a cell density of 1.0×105~3.0×105 cells / mL according to a volume of 15~20mL, cultivate at Tm: 37°C, CO2 concentration: 5%, shaking speed: 90~120rpm, amplitude: 50mM, carry out cell suspension culture, sample and count every day for 5 days, and analyze the viability, and determine the expression on the fifth day. 5 cells / mL cell density according to a volume of 15~20mL, cultivate at Tm: 37°C, CO2 concentration: 5%, shaking speed: 90~120rpm, amplitude: 50mM, carry out cell suspension culture, sample and count every day for 5 days, and analyze the viability, and determine the expression on the fifth day.

[0032] The culture conditions of step 2) are temperature 37℃, CO2 concentration 5%, shaking speed 120 rpm, and culture for 24 h.

[0033] The culture conditions of step 3) are temperature 37℃, CO2 concentration 5%, and shaking speed 120 rpm.

[0034] The culture medium RPMI 1640 medium is from Gibco, FBS (fetal bovine serum) is from Duoning, and Hybrid 01 medium is from a self-developed serum-free hybridoma culture medium.

[0035] In order to make the technical means, creative features, purposes and effects achieved by the present application easy to understand, the present application is further described below in combination with specific examples.

[0036] Example 1:

[0037] Recovery culture: after the recovery of the frozen hybridoma cells, the cells were inoculated in RPMI 1640 containing 5% FBS and placed in a carbon dioxide incubator for recovery culture; static expansion culture: after the recovery of the hybridoma cells in good condition, the cells were digested with 0.05% trypsin at 37℃ for 45 s, then terminated with RPMI 1640 containing 5% FBS, the cell suspension was collected and centrifuged at 600 rpm / min for 5 min, the supernatant was discarded and the cells were resuspended with Hybrid 01 serum-free medium, inoculated in a T75 cell culture bottle at a ratio of 1:5, and the subsequent static expansion passage was carried out at a ratio of 1:5; suspension domestication and expansion culture: the hybridoma cells cultured in the T75 bottle were gently tapped to make the cells suspended, the cells were collected at 600 rpm / min for 5 min, the supernatant was discarded, 3 mL of Hybrid 01 serum-free hybridoma cell culture medium was added for resuspension, and the cells were inoculated in a 125 mL shake flask at a cell density of 2.0 x 10 5 cells / mL at a volume of 15 mL, and the cells were inoculated in a 125 mL shake flask for suspension culture at Tm: 37℃, CO2 concentration: 5%, shaking speed: 90 rpm, and amplitude: 50 mM, the cells were cultured for 5 days, sampling and counting were carried out every day, and the expression amount was determined on the fifth day.

[0038] The viable cell density, viability and diameter are shown in Table 1, Table 2 and Table 3, respectively. Figure 1 、 2 、3; the expression amount is shown in Table 4. Figure 4 .

[0039] Comparative Example 1:

[0040] Recovery culture, after the recovery of the frozen hybridoma cells, inoculate in RPMI 1640 containing 5% FBS and place in the carbon dioxide incubator for recovery culture; static expansion culture, after the recovery of the hybridoma cells in good condition, use 0.05% trypsin to digest at 37℃ for 45s, then terminate with RPMI 1640 containing 5% FBS, collect the cell suspension and centrifuge at 600rpm / min for 5min, discard the supernatant, resuspend with RPMI 1640 containing 5% FBS, inoculate in T75 cell culture flask at a ratio of 1:5, inoculate in T75 cell culture flask at a ratio of 1:5 for subsequent static expansion passage; expansion culture, after the recovery of the hybridoma cells in good condition, use 0.05% trypsin to digest at 37℃ for 45s, then terminate with RPMI 1640 containing 5% FBS, collect the cell suspension and centrifuge at 600rpm / min for 5min, discard the supernatant, resuspend with RPMI 1640 containing 5% FBS, inoculate in T75 cell culture flask at a ratio of 2.0x10 5 cells / mL, 15mL in T75 cell culture flask, culture for 5 days, resuspend and count by tapping every day, analyze the viability, and determine the expression on the fifth day.

[0041] The above shows and describes the basic principles, main features and advantages of the present application. It should be understood by those skilled in the art that the present application is not limited by the above examples, and the above examples and descriptions in the specification are only to illustrate the principles of the present application. Without departing from the spirit and scope of the present application, various changes and improvements can be made to the present application, and these changes and improvements all fall within the scope of the present application. The scope of protection of the present application is defined by the appended claims and their equivalents.

Claims

1. A production process of a recombinant antibody protein produced by a hybridoma cell suspension culture, characterized by, Comprising the following steps: 1) recovery culture, the frozen hybridoma cells are inoculated in RPMI 1640 containing 5% FBS and placed in a carbon dioxide incubator for recovery culture; 2) static expansion culture, the well recovered hybridoma cells are digested with 0.05% trypsin at 37℃ for 45s, then terminated with RPMI 1640 containing 5% FBS, the cell suspension is collected and centrifuged at 600rpm / min for 5min, the supernatant is discarded and directly resuspended with Hybrid 01 serum-free medium, inoculated in T75 cell culture flask at a ratio of 1:5, and the subsequent static expansion passage is carried out at a ratio of 1:5; The Hybrid 01 serum-free medium is a self-developed serum-free and animal-derived component-free basic medium for hybridoma cells, the lipid group in the self-developed serum-free and animal-derived component-free basic medium for hybridoma cells is 0.000008g / L linoleic acid and 1.0g / L cholesterol; the inorganic salt component in the self-developed serum-free and animal-derived component-free basic medium for hybridoma cells is 1.6g / L potassium chloride, 0.6g / L calcium chloride, 3.0g / L sodium phosphate, 1.0g / L disodium hydrogen phosphate, 6.5g / L sodium chloride, 1.0g / L magnesium sulfate, and 3.0g / L P188; the amino acid and sugar component in the self-developed serum-free and animal-derived component-free basic medium for hybridoma cells is 1.2g / L asparagine, 0.2g / L methionine, 0.3g / L histidine, 0.9g / L aspartic acid, 0.2g / L serine, 0.9g / L glycine, 1.3g / L arginine, 0.3g / L isoleucine, 1.3g / L leucine, 1.2g / L glutamic acid, 1.4g / L valine, 1.0g / L lysine, 0.5g / L phenylalanine, 0.2g / L proline, 0.1g / L threonine, 1.4g / L glutamine, 0.4g / L tyrosine, 0.5g / L alanine, and 6g / L glucose; the vitamin component in the self-developed serum-free and animal-derived component-free basic medium for hybridoma cells is 0.0001g / L vitamin B6, 0.00003g / L vitamin B1, 0.00008g / L biotin, 0.00004g / L vitamin B12, 0.000001g / L pantothenic acid, 0.00004g / L folic acid, 0.0008g / L nicotinic acid, 0.00004g / L riboflavin, 0.004g / L choline chloride, and 0.00004g / L inositol; the trace element component in the self-developed serum-free and animal-derived component-free basic medium for hybridoma cells is 0.00004g / L copper chloride, 0.0001g / L zinc chloride, 0.0004g / L ferrous sulfate, 0.000001g / L ammonium molybdate, and 0.00002g / L manganese chloride; 3) Suspension domestication and scale-up culture, gently tap the hybridoma cells cultured in T75 bottles to suspend the cells, collect the cells at 600 rpm / min for 5 min, discard the supernatant, add 3 mL of hybridoma cell serum-free medium Hybrid 01 to resuspend, inoculate at a cell density of 1.0 x 105~3.0 x 105 cells / mL to a volume of 15~20 mL in a 125 mL shake flask, and perform cell suspension culture at Tm: 37°C, CO2 concentration: 5%, shaking speed: 90~120 rpm, and shaking amplitude: 50 mM. Sample and count every day and analyze the viability for 5 days, and perform expression determination on the fifth day. 5 cells / mL cell density to a volume of 15~20 mL in a 125 mL shake flask, and perform cell suspension culture at Tm: 37°C, CO2 concentration: 5%, shaking speed: 90~120 rpm, and shaking amplitude: 50 mM. Sample and count every day and analyze the viability for 5 days, and perform expression determination on the fifth day.

2. The production process of a recombinant antibody protein produced by a hybridoma cell suspension culture according to claim 1, characterized by: The culture conditions of steps 1) and 2) are temperature 37℃ and CO2 concentration 5%, placed in a carbon dioxide incubator.

3. The process for producing recombinant antibody protein by hybridoma cell suspension culture according to claim 1, characterized by: The culture condition of step 3) is temperature 37℃, CO2 concentration 5%, and shaking speed 120 rpm. The culture condition of step 3) is temperature 37℃, CO2 concentration 5%, and shaking speed 120 rpm.

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