SP2 / 0 Cell Culture Medium and Method for Producing Recombinant Monoclonal Antibodies Against Canine Parvovirus

Abstract

The present invention belongs to the technical field of cell culture media, and relates to an SP2 / 0 cell culture medium and a method for producing recombinant canine parvovirus monoclonal antibodies. The culture medium includes additives such as amino acids, vitamins, lipids, sugars, trace elements, inorganic salts, cytokines, hypoxanthine, thymidine, cholesterol, complex soy hydrolyzate, and polyamines. The SP2 / 0 cell culture medium of the present invention does not contain serum and has no animal-derived components. It has the characteristics of low cost, high safety, low pollution risk, simple process operation, facilitating the purification of products, and stability of production batches. It is suitable for large-scale production and is applied to the continuous perfusion culture of SP2 / 0 cells, which can meet the needs of large-scale industrial production of recombinant canine parvovirus monoclonal antibodies and has great application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell culture media, and particularly relates to a serum-free and chemically defined SP2 / 0 cell culture medium, and a method for producing recombinant canine parvovirus monoclonal antibody using this culture medium. Background Art

[0002] Canine Parvovirus (CPV) infection is an acute infectious disease of dogs. Clinically, infected dogs show hemorrhagic enteritis or non-suppurative myocarditis, mostly occurring in puppies, with a fatality rate of 10% - 50%. In addition, this virus infection also threatens experimental dogs. Currently, the main clinical treatment is symptomatic treatment, combined with hyperimmune serum and monoclonal antibody treatment.

[0003] The currently more advanced method for preparing canine parvovirus monoclonal antibody (CPV McAb) is: through cell fusion technology, splenocytes from BALB / c mice immunized with canine parvovirus are fused with SP2 / 0 myeloma cells to prepare a hybridoma cell line that can secrete anti-canine parvovirus monoclonal antibody. Specific hybridoma cells are screened out, inoculated into a bioreactor for culture, and the culture medium is purified to prepare specific antibodies.

[0004] In terms of culture methods, the existing technologies applicable to animal cell culture include adherent culture, suspension culture, and perfusion culture. In industrial production, the amplification principle and process control of suspension culture process parameters are easier to understand and master than other culture systems, and it has the advantages of uniform culture conditions, high cell yield, high protein production, and continuous on-line detection. Therefore, suspension cell culture process is mostly selected in large-scale animal cell culture production. However, the main disadvantage of suspension culture is that cells grow in suspension and free, with a large cell culture volume, high equipment investment, and relatively low cell density in production.

[0005] Compared with traditional suspension culture, perfusion culture has lower requirements for equipment, smaller space, and is easier to scale up and industrialize. Especially in the continuous perfusion culture process, fresh culture medium is continuously added to continuously provide nutrients to cells, and the culture supernatant can be harvested intermittently or continuously, maintaining a good growth environment for cells, increasing the cell density in the bioreactor, reducing the accumulation of harmful metabolites, extending the cell expression time, and thus increasing the cell yield.

[0006] Due to the complexity of in vitro culture of animal cells, serum-containing media are mostly used in the prior art to ensure high-density cell growth and achieve high yields. Chinese Patent CN102120768B mentions a method for producing therapeutic canine parvovirus monoclonal antibodies using a bioreactor. From seed resuscitation to cell culture, an 85% DMEM medium and 15% calf serum are used throughout the process. The produced canine parvovirus monoclonal antibodies have a relatively high purity, representing a significant improvement compared to the traditional method of preparing monoclonal antibodies by immunizing mice. However, since the medium contains animal serum, there are potential risks, and the cell culture time is relatively short, resulting in a still not-high batch yield. Moreover, the hemagglutination inhibition titer of the harvested culture fluid in this patent is only ≥256.

[0007] Since adding serum to the medium has the potential risk of animal virus infection and is not conducive to purification, perfusion culture using serum-free media has become a major trend in the field of cell culture today. Using serum-free culture can not only reduce costs and simplify the separation and purification steps but also avoid the hazards caused by virus contamination.

[0008] In addition, various existing commercial serum-free media not only have unclear compositions but also have different adaptabilities to cell lines for producing different products, lacking broad generality. Even when using a stable commercial serum-free medium, the culture process still needs to be optimized according to the growth and metabolic characteristics of each type of cell, and this cell domestication process often takes a long time. Currently, there are few methods for perfusion culture of SP2 / 0 cells using a serum-free and chemically defined medium to achieve a high cell density for efficient production of recombinant canine parvovirus monoclonal antibodies.

[0009] Chinese Patent Application CN103773732A mentions that a chemically defined medium can increase cell density and the yield of the target product. Using this medium to culture SP2 / 0 cells in a vial reaches a peak cell density of 5-6×10 6 cells / mL, and the culture duration is 6-7 days. This medium has a low cell density, low yield, and high cost.

[0010] In summary, there is an urgent need in the fields of scientific research and medical applications for a safe, efficient, serum-free, and chemically defined medium for perfusion culture of SP2 / 0 cells, as well as a method for high-yield production of recombinant canine parvovirus monoclonal antibodies. Summary of the Invention

[0011] In view of the numerous problems existing in the existing cultivation methods of canine parvovirus monoclonal antibodies, such as the culture medium containing serum, low expression in existing serum-free media, short expression time, and low batch products, the present invention provides a serum-free and chemically defined culture medium and a method for producing canine parvovirus monoclonal antibodies by perfusion culture using this culture medium to solve the existing problems of the culture medium or culture process.

[0012] To achieve the above object, the present invention provides a serum-free SP2 / 0 cell perfusion culture medium, and the components of the culture medium include: amino acids, vitamins, lipids, sugars, trace elements, inorganic salts, compound soy hydrolysate, polyamines, hypoxanthine, thymidine, cholesterol, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, P188 (i.e., poloxamer 188), and malic acid.

[0013] Preferably, the amino acids include glutamine.

[0014] Preferably, the culture medium includes a cytokine, and the cytokine is recombinant human insulin.

[0015] Preferably, the cholesterol is cholesterol of non-animal origin.

[0016] Preferably, the amino acids include:

[0017] 300 - 500 mg / L of L-arginine, 100 - 300 mg / L of L-arginine hydrochloride, 800 - 1000 mg / L of L-asparagine, 300 - 500 mg / L of L-aspartic acid, 600 - 800 mg / L of hydroxyproline, 300 - 500 mg / L of L-isoleucine, 200 - 300 mg / L of L-cysteine, 200 - 400 mg / L of L-cysteine-2-hydrochloride, 200 - 400 mg / L of L-methionine, 300 - 500 mg / L of L-glutamic acid, 50 - 80 mg / L of glycine, 30 - 50 mg / L of taurine, 350 - 500 mg / L of L-histidine hydrochloride, 400 - 600 mg / L of L-leucine, 400 - 600 mg / L of L-lysine monohydrate, 200 - 400 mg / L of L-proline, 300 - 500 mg / L of L-serine, 200 - 400 mg / L of L-threonine, 100 - 300 mg / L of L-tryptophan, 100 - 300 mg / L of tyrosine, 5 - 10 mg / L of L-α-amino-n-butyric acid, 100 - 300 mg / L of cystine-2-hydrochloride, 100 - 300 mg / L of L-phenylalanine, 200 - 400 mg / L of L-tyrosine disodium dihydrate, 200 - 400 mg / L of L-valine, 3 - 5 mg / L of reduced glutathione, 1 - 4 g / L of glutamine;

[0018] Preferably, the vitamins include:

[0019] Biotin at 0.1 - 0.3 mg / L, choline chloride at 50 - 70 mg / L, vitamin B12 at 2 - 4 mg / L, folic acid at 10 - 15 mg / L, inositol at 100 - 150 mg / L, D-calcium pantothenate at 18 - 25 mg / L, pyridoxine hydrochloride at 15 - 20 mg / L, riboflavin at 1 - 3 mg / L, thiamine hydrochloride at 12 - 15 mg / L, nicotinamide at 0.5 - 1 mg / L, DL-α-lipoic acid at 0.4 - 1 mg / L, tocopherol at 0.0005 - 0.001 mg / L;

[0020] Preferably, the lipids include: linoleic acid at 0.04 - 0.1 mg / L;

[0021] Preferably, the sugars include:

[0022] Sucrose at 3000 - 5000 mg / L, galactose at 1000 - 2000 mg / L, maltose monohydrate at 2000 - 4000 mg / L, glucose at 1000 - 3000 mg / L, sodium pyruvate at 100 - 140 mg / L;

[0023] Preferably, the trace elements include:

[0024] Calcium chloride at 60 - 100 mg / L, ammonium ferric citrate at 300 - 500 mg / L, magnesium chloride at 30 - 50 mg / L, magnesium sulfate at 30 - 50 mg / L, potassium chloride at 800 - 1000 mg / L, ammonium metavanadate at 0.006 - 0.008 mg / L, ammonium molybdate tetrahydrate at 0.005 - 0.008 mg / L, copper sulfate pentahydrate at 0.05 - 0.1 mg / L, sodium selenite at 0.001 - 0.003 mg / L, manganese chloride tetrahydrate at 0.001 - 0.003 mg / L, manganese sulfate monohydrate at 0.004 - 0.006 mg / L, nickel sulfate at 0.001 - 0.003 mg / L, sodium metavanadate nonahydrate at 0.004 - 0.006 mg / L, sodium metasilicate nonahydrate at 0.5 - 1.5 mg / L, stannous chloride dihydrate at 0.001 - 0.002 mg / L, ferrous sulfate heptahydrate at 0.2 - 0.5 mg / L, potassium nitrate at 0.03 - 0.05 mg / L, zinc sulfate heptahydrate at 2 - 10 mg / L, ethanolamine hydrochloride at 10 - 15 mg / L;

[0025] Preferably, the inorganic salts include:

[0026] Sodium dihydrogen phosphate at 800 - 1000 mg / L, disodium hydrogen phosphate monohydrate at 100 - 150 mg / L, sodium chloride at 2000 - 3000 mg / L;

[0027] Preferably, the inosine content is 1 - 5 mg / L, the thymidine content is 1 - 5 mg / L, the 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid content is 50 - 100 mg / L, the P188 content is 1000 - 5000 mg / L, the malic acid content is 300 - 500 mg / L, and the cholesterol content is 0.5 - 1 mg / L;

[0028] Preferably, the recombinant human insulin content is 1 - 10 mg / L;

[0029] Preferably, the content of the composite soy hydrolysate is 1 - 5 g / L; the polyamine is putrescine, and the content is 50 - 80 mg / L.

[0030] The present invention also provides a method for culturing SP2 / 0 cells by perfusion method using the above medium to produce recombinant canine parvovirus monoclonal antibody, and the method comprises the following steps:

[0031] 1) After resuscitating and activating SP2 / 0 cells, use the above medium for cell activation and amplification culture to obtain a seed solution;

[0032] 2) Inoculate the seed solution into the reactor, and perform perfusion culture when the glucose content is less than or equal to 2 g / L;

[0033] 3) When the cells in the reactor reach a steady state, start collecting the culture solution produced by perfusion culture; stop collecting the culture solution when the cell viability is lower than 80%;

[0034] 4) Filter, concentrate, purify, and quantify the collected culture solution to obtain the finished product of recombinant canine parvovirus monoclonal antibody.

[0035] Preferably, the cell density of the solution after inoculation in step 2) is 0.3 - 1.0×10 6 / mL.

[0036] Preferably, during the perfusion culture, the glucose content in the culture solution is greater than or equal to 1 g / L and less than or equal to 2 g / L.

[0037] Preferably, during the perfusion culture, the cell-specific perfusion rate of the perfusion culture is 40 - 60 p·L / (c·d), maintaining the cell density at 2 - 4×10 7 cells / mL, preferably the cell-specific perfusion rate is 50 p·L / (c·d), maintaining the cell density at 3.0 - 3.5×10 7 cells / mL.

[0038] Preferably, after the seed solution is inoculated into the reactor, the culture conditions in the reactor are as follows: the culture temperature is 33-37°C, the pH is 6.8-7.1, the dissolved oxygen is 30%-60%, and the rotational speed of the reactor is 180-250 rpm; preferably, the culture temperature is 34°C, the pH value is 7.0, the dissolved oxygen is 40%, and the rotational speed of the reactor is 200 rpm.

[0039] The hollow fiber column connected to the reactor is equipped with a cell retention device, which can filter the expression products and metabolic wastes in the culture solution. At the same time, the hollow fiber column has a reverse flushing function, which can avoid fiber blockage caused by long-term cell retention, improve the cell density, and extend the production cycle of the cells.

[0040] The present invention also provides a recombinant canine parvovirus monoclonal antibody prepared by the above method.

[0041] Compared with the prior art, the technical effects achieved by the present invention are as follows:

[0042] (1) The SP2 / 0 cell culture medium of the present invention does not contain serum and has no animal-derived components. It has the characteristics of low cost, high safety, low pollution risk, simple process operation, facilitating product purification, and stable production batches, and is suitable for large-scale production.

[0043] (2) By adding composite soy hydrolyzate, recombinant human insulin, and glutamine to the SP2 / 0 cell culture medium provided by the present invention, the SP2 / 0 cell density, cell viability, and hemagglutination inhibition titer of canine parvovirus are significantly increased.

[0044] (3) The present invention also clarifies the production process for perfusion culture using the SP2 / 0 cell culture medium, and defines parameters such as cell seeding density, cell culture maintenance density, glucose control points during cell culture, culture temperature, pH value, dissolved oxygen, and rotational speed, and determines the production process most suitable for the continuous perfusion culture of SP2 / 0 cells using this culture medium, fully meeting the requirements for the large-scale industrial production of high-yield and high-performance recombinant canine parvovirus monoclonal antibody. Description of the Drawings

[0045] Figure 1 It is a graph showing the change of cell density over time in Example 1;

[0046] Figure 2 It is a graph showing the change of cell viability over time in Example 1;

[0047] Figure 3 It is the titer result of cells obtained by culturing in different culture media in Example 1;

[0048] Figure 4 It is a graph showing the change of cell density and viability over time in Example 2;

[0049] Figure 5 Graph of the change of cell density and viability over time for Example 3;

[0050] Figure 6 Graph of the change of cell density and viability over time for Example 4;

[0051] Figure 7 Graph of the change of cell density and viability over time for Example 5. Detailed implementation mode

[0052] The technical solutions in the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0053] The instruments, consumables, and reagents used in the following embodiments are all conventional instruments, consumables, and reagents in the art. If the sources are not specifically indicated, those skilled in the art can obtain them through commercial channels. The composite soy hydrolyzate used in the present invention is purchased from Kerry Company, USA (product name: sheff-CHO PF ACF).

[0054] The inspection standard for recombinant canine parvovirus is to conduct inspections in accordance with the appendix of the "Pharmacopoeia of the People's Republic of China" (2015 Edition). The hemagglutination inhibition titer of monoclonal antibodies against canine parvovirus in the harvested culture medium ≥ 256 is considered qualified.

[0055] For the finished product obtained after concentrating and ultrafiltrating the harvested cell culture medium, the finished product is inspected in accordance with the appendix of the "Pharmacopoeia of the People's Republic of China" (2015 Edition). The hemagglutination inhibition titer of monoclonal antibodies against canine parvovirus in the finished product ≥ 1024 is considered qualified.

[0056] Example 1 Flask culture test of different culture media

[0057] (1) Cell resuscitation

[0058] Take out a vial of recombinant canine parvovirus monoclonal antibody-expressing SP2 / 0 cells from the liquid nitrogen tank. After thawing in a 37°C water bath, transfer the cells into a 15 mL sterile centrifuge tube, add 5 mL of pre-warmed (37°C) medium, which is CDM4MAb (brand: HyClone, produced by Cytiva, USA, serum-free), gently pipette to mix, centrifuge at 800 rpm for 5 min, discard the supernatant, re-suspend with 5 mL of fresh CDM4MAb medium, transfer the cell suspension into a 125 mL vented shake flask, supplement with 15 mL of medium, and culture at 37°C, 120 rpm, 8% CO2, and 70% - 90% humidity. Sample and count every other day, supplement the medium, and maintain the cell density at 2 - 3×10 6 cells / mL with a cell viability greater than 95%. After continuous passage 2 - 3 times, set aside for use.

[0059] (2) Shake flask culture

[0060] Take the reserved cells and inoculate them into CDM4MAb, MAbMaxA, MAbMaxA1, MAbMaxA2, and MAbMaxA3 media (the differences in each medium are shown in Table 1) at a density of 5×10 5 cells / mL in 125 mL cell culture flasks with an inoculated volume of 30 mL. The culture temperature is 36.5°C, the rotation speed is 120 rpm, and the humidity is 70 - 90%. Sample and count daily, and monitor the glucose content (when the glucose content is lower than 4 g / L, supplement it to 6 g / L). Stop culturing when the cell viability is lower than 80%. Take the supernatant and measure the hemagglutination inhibition titer of canine parvovirus (the results are shown in Table 3). The cell density, viability, and the titer results of the cells obtained from culturing in different media are respectively as Figure 1 、 2 、shown in Figure 3.

[0061] Table 1 Information of the tested media

[0062] Name Main Ingredients CDM4MAb Unknown MAbMaxA See Table 2 MAb MaxA1 Add 2 g / L of compound soy hydrolyzate based on MAbMaxA MAb MaxA2 Add 1 g / L of glutamine based on MAb MaxA1 MAb MaxA3 Add 4 mg / L of recombinant human insulin based on MAb MaxA2

[0063] Table 2 Composition and concentration of MAbMaxA medium

[0064]

[0065]

[0066]

[0067] Table 3 Titer results of shake flask culture in different media

[0068] Name Potency CDM4MAb 128 MAbMaxA 32 MAb MaxA1 128 MAb MaxA2 512 MAb MaxA3 1024

[0069] Example 2 Production of Recombinant Canine Parvovirus Monoclonal Antibody Using Commercial Medium CDM4Mab

[0070] (1) Cell Resuscitation

[0071] Take out a vial of SP2 / 0 cells expressing recombinant canine parvovirus monoclonal antibody from the liquid nitrogen tank. After thawing in a 37°C water bath, transfer the cells into a 15 mL sterile centrifuge tube, add 5 mL of pre-warmed medium at 37°C. The medium is CDM4MAb (brand: hyclone, produced by Cytiva, USA, serum-free). Gently pipette to mix, centrifuge at 800 rpm for 5 min, discard the supernatant, re-suspend with 5 mL of fresh CDM4MAb medium, transfer the cell suspension into a 125 mL vented shake flask, supplement with 15 mL of medium, and culture at 37°C, 120 rpm, 8% CO2, and 70% - 90% humidity. Sample and count every other day, supplement the medium, and maintain the cell density at 3 - 4×10 6 cells / mL. The volume of the shake flask is gradually increased from 125 mL, 250 mL, 500 mL, to 1000 mL. After 5 - 7 days of amplification, the seed volume reaches 200 mL and the density is 3.6×10 6 cells / mL.

[0072] (2) Inoculation of Reactor

[0073] The volume after inoculation in the bioreactor is 1.8 L, the cell density is 4.5×10 5 cells / mL, the culture temperature is 36.5°C, the rotation speed is 200 rpm, the dissolved oxygen is 40%, and the pH value is 7.0. Sample and count daily, and monitor glucose.

[0074] (3) Perfusion Culture

[0075] When the glucose content is lower than 2 g / L, start perfusion culture. Open the liquid exchange pipeline and clarified liquid collection pipeline of the hollow fiber retention device, close the cleaning pipeline and reverse flushing pipeline. After the collection of the clarified liquid is completed, start reverse flushing. After the flushing is completed, close the pipeline, and run the cleaning pipeline for continuous cleaning of the main body. The perfusion volume increases with the increase of cell density. The cell specific perfusion rate (CSPR) is 50 p·L(c·d) -1 , maintain the cell density at 3.0 - 3.5×10 7 cells / mL. Remove the excess cells using a peristaltic pump. When the cells are in a stable state, lower the culture temperature to 34°C, the rotation speed is 200 rpm, the dissolved oxygen is 40%, and the pH value is 7.0. Continuously perfuse and start collecting the liquid, maintain the glucose content greater than or equal to 1 g / L and less than or equal to 2 g / L; stop collecting the liquid until the cell viability is lower than 80%.

[0076] The entire perfusion culture was maintained for 14 days starting from the inoculation reactor. The cell density and cell viability were as Figure 4 shown. A total of 20 L of liquid was collected, which was 11 times the culture volume. The hemagglutination inhibition titer of canine parvovirus monoclonal antibody in the harvested culture broth was ≥512.

[0077] According to the titer of the therapeutic monoclonal antibody in the cell culture broth, the cell culture broth was concentrated and ultrafiltered using an ultrafiltration system, and the cell debris in the harvested liquid was removed using a filtration system. The filtered liquid harvested was stored at a temperature below -20°C. The concentrated liquid was detected according to the appendix of the Chinese Veterinary Pharmacopoeia (2015 Edition), and there was no growth of bacteria, molds, and mycoplasmas. The hemagglutination inhibition titer of the canine cell virus monoclonal antibody in the concentrated liquid was ≥4096. After the purified therapeutic monoclonal antibody was diluted, it was aseptically and quantitatively dispensed into sterile containers. After placing a sterilized stopper and crimping the lid, the finished product was obtained and stored at a temperature below -20°C.

[0078] The relevant results are shown in Table 5.

[0079] Example 3 Production of Recombinant Canine Parvovirus Monoclonal Antibody Using Medium MAb MaxA4

[0080] (1) Preparation of MAb MaxA4 Medium

[0081] The components and dosages of the medium in Example 3 (MAb MaxA4 medium) are shown in Table 4.

[0082] Table 4 Components and Contents of the Medium

[0083]

[0084]

[0085]

[0086] The preparation method of the dry powder medium is as follows:

[0087] 1) Weigh the above raw materials according to the volume ratio and mix the raw materials evenly to obtain a mixed raw material;

[0088] 2) For the materials that cannot be directly weighed, prepare a concentrated solution, drip it into substances such as sodium chloride or glucose, air-dry overnight in a biosafety cabinet, and then add it to the mixed raw material;

[0089] 3) Grinding: Grind the mixed raw material using a stainless steel grinding jar. The grinding balls are made of zirconia. The volume ratio of the dry powder medium: zirconia ball volume: remaining tank volume = 1:1:1; Grinding parameters: 300 rpm, 20 min / time; Use a standard sieve to test the particle size of the dry powder after grinding, and the particle size control range is a passing rate of more than 50% through a 200-mesh standard sieve.

[0090] 4) Separate the ball powder, subpackage the dry powder and label it;

[0091] 5) Store it at 2 - 8°C in the dark.

[0092] Preparation of liquid medium:

[0093] 1) Measure 90% of the final volume of the ultrapure water (18 - 25°C) for preparing the liquid medium into a designated container, slowly add 19 - 30 g / L of the above dry powder medium, and stir vigorously for 10 - 30 min until the dry powder is completely dissolved into a suspension;

[0094] 2) Slowly add 2.26 mL / L of 5 mol / L sodium hydroxide solution and stir for 10 - 30 min;

[0095] 3) Add sodium bicarbonate powder in an amount of 2.33 g / L, stir for 10 - 20 min until all components are completely dissolved;

[0096] 4) Use 12 mol / L concentrated hydrochloric acid to adjust the pH value of the medium to 6.7 - 6.8 and stir for 3 - 5 min;

[0097] 5) Make up the volume to 1 L with ultrapure water and continue to stir for 5 - 10 min;

[0098] 6) Measure the final pH value (6.7 - 7.4) and osmotic pressure (280 - 340 mOsmol / kg) of the medium;

[0099] 7) Filter through a 0.22 - μm membrane;

[0100] 8) Store it at 2 - 8°C in the dark for standby.

[0101] (2) Cell resuscitation

[0102] The cell resuscitation method is the same as that in Example 2, except that MAb MaxA4 liquid medium is used.

[0103] (3) Inoculating the reactor and (4) Perfusion culture

[0104] The processes of inoculating the reactor and perfusion culture are the same as those in Example 2; the difference is that: the whole perfusion culture lasted for 20 days starting from inoculating the reactor, and the cell density and cell viability are as Figure 5 shown. A total of 35 L of liquid was collected, which is 19 times the culture volume, and the hemagglutination inhibition titer of the harvested culture fluid against canine parvovirus is ≥2048.

[0105] According to the titer of the therapeutic monoclonal antibody in the cell culture fluid, use an ultrafiltration system to concentrate and ultrafilter the cell culture fluid, use a filtration system to remove cell debris from the harvested fluid, and store the filtered fluid at below - 20°C.

[0106] The concentrated solution was detected according to the appendix of the "Pharmacopoeia of Veterinary Drugs of the People's Republic of China" (2015 edition), and no bacteria, molds, or mycoplasmas grew. The hemagglutination inhibition titer of the canine cytovirus in the concentrated solution was ≥20480.

[0107] The purified therapeutic monoclonal antibody was diluted, aseptically and quantitatively dispensed into sterile containers, and after placing a sterilized stopper and crimping the lid, the finished product was obtained and stored at -20°C or below.

[0108] Example 4 Production of recombinant canine parvovirus monoclonal antibody using medium MAb MaxA5

[0109] (1) Preparation of MAb MaxA5 medium

[0110] The components and dosages of the MAb MaxA5 medium are shown in Table 4. The preparation methods of the dry powder and liquid media are the same as those in Step of Example 3 and will not be elaborated here.

[0111] (2) Cell resuscitation

[0112] The cell resuscitation method was the same as that in Example 2, except that MAb MaxA5 liquid medium was used.

[0113] (3) Inoculating the bioreactor and (4) Perfusion culture

[0114] The processes of inoculating the bioreactor and perfusion culture were the same as those in Example 2; the difference was that the entire perfusion culture was maintained for 24 days starting from inoculating the bioreactor. The cell density and cell viability were as Figure 6 shown. A total of 45 L of liquid was collected, which was 25 times the culture volume. The hemagglutination inhibition titer of the canine parvovirus in the harvested culture broth was ≥4096.

[0115] According to the titer of the therapeutic monoclonal antibody in the cell culture broth, the cell culture broth was concentrated and ultrafiltered using an ultrafiltration system, and the cell debris in the harvested solution was removed using a filtration system. The filtered solution obtained was stored at -20°C or below.

[0116] The concentrated solution was detected according to the appendix of the "Pharmacopoeia of Veterinary Drugs of the People's Republic of China" (2015 edition), and no bacteria, molds, or mycoplasmas grew. The hemagglutination inhibition titer of the canine cytovirus in the concentrated solution was ≥40960.

[0117] The purified therapeutic monoclonal antibody was diluted, aseptically and quantitatively dispensed into sterile containers, and after placing a sterilized stopper and crimping the lid, the finished product was obtained and stored at -20°C or below.

[0118] Example 5 Production of recombinant canine parvovirus monoclonal antibody using medium MAb MaxA6

[0119] (1) Preparation of MAb MaxA6 medium

[0120] The components and dosages of the MAb MaxA6 medium are shown in Table 4. The preparation methods of the dry powder and liquid media are the same as those in Step of Example 3, and will not be repeated here.

[0121] (2) Cell resuscitation

[0122] The cell resuscitation method is the same as that in Example 2, except that the MAb MaxA6 liquid medium is used.

[0123] (3) Inoculating the bioreactor and (4) perfusion culture

[0124] The processes of inoculating the bioreactor and perfusion culture are the same as those in Example 2; the differences are as follows: the entire perfusion culture lasted for 21 days starting from inoculating the bioreactor, and the cell density and cell viability are as Figure 7 shown. A total of 38 L of liquid was collected, which is 21 times the culture volume. The hemagglutination inhibition titer of canine parvovirus in the harvested culture broth was ≥2048.

[0125] According to the titer of the therapeutic monoclonal antibody in the cell culture broth, the cell culture broth was concentrated and ultrafiltered using an ultrafiltration system, and the cell debris in the harvested solution was removed using a filtration system. The filtered solution was stored at a temperature below -20°C.

[0126] The concentrated solution was detected according to the appendix of the Chinese Veterinary Pharmacopoeia (2015 Edition). There was no growth of bacteria, molds, and mycoplasmas, and the hemagglutination inhibition titer of canine cell virus in the concentrated solution was ≥20480.

[0127] After diluting the purified therapeutic monoclonal antibody, it was aseptically and quantitatively dispensed into sterile containers. After placing a sterilized stopper and crimping the lid, the finished product was obtained and stored at a temperature below -20°C.

[0128] Table 5 Culture effects of different media

[0129]

[0130]

[0131] In summary, the serum-free medium disclosed in the present invention is low in cost and suitable for industrial production. The hollow fiber column externally connected to the bioreactor for continuous perfusion culture is equipped with a cell retention device, which can filter the expression products and metabolic wastes in the culture broth. At the same time, the hollow fiber column has a function of reverse flushing, which can avoid fiber blockage caused by long-term cell retention, improve the cell density, and has high utilization value.

[0132] The embodiments of this article are intended to illustrate the principle of use and application effect of the present invention, rather than limiting the present invention. Those skilled in the art can make appropriate modifications without departing from the spirit and scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. A method for producing recombinant canine parvovirus monoclonal antibody, characterized in that, It includes the following steps: 1) After resuscitating and activating the SP2 / 0 cells expressing recombinant canine parvovirus monoclonal antibody, use SP2 / 0 medium for cell activation and amplification culture to obtain a seed solution; 2) Inoculate the seed solution into a reactor. When the glucose content is less than or equal to 2 g / L, perform perfusion culture to produce a culture solution; 3) After the cells in the reactor reach a steady state, start collecting the culture solution produced by the perfusion culture; stop collecting the culture solution when the cell viability is lower than 80%; 4) Filter, concentrate, purify, and quantify the collected culture solution to obtain the finished product of recombinant canine parvovirus monoclonal antibody; The SP2 / 0 medium consists of the following components: amino acids, vitamins, lipids, sugars, trace elements, inorganic salts, compound soy hydrolysate, polyamines, hypoxanthine, thymidine, cholesterol, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, P188, malic acid, glutamine, and recombinant human insulin; the compound soy hydrolysate is SHEFF-CHO PF ACF; The amino acids include: 400 mg / L of L-arginine, 200 mg / L of L-arginine hydrochloride, 9000 mg / L of L-asparagine, 400 mg / L of L-aspartic acid, 700 mg / L of hydroxyproline, 400 mg / L of L-isoleucine, 250 mg / L of L-cysteine, 300 mg / L of L-cysteine-2-hydrochloride, 300 mg / L of L-methionine, 400 mg / L of L-glutamic acid, 70 mg / L of glycine, 40 mg / L of taurine, 380 mg / L of L-histidine hydrochloride, 500 mg / L of L-leucine, 500 mg / L of L-lysine monohydrate, 300 mg / L of L-proline, 400 mg / L of L-serine, 300 mg / L of L-threonine, 200 mg / L of L-tryptophan, 200 mg / L of tyrosine, 8 mg / L of L-α-amino-n-butyric acid, 200 mg / L of cystine-2-hydrochloride, 200 mg / L of L-phenylalanine, 300 mg / L of L-tyrosine disodium dihydrate, 300 mg / L of L-valine, 4 mg / L of reduced glutathione, 2 g / L of glutamine; The vitamins include: 0.2 mg / L of biotin, 60 mg / L of choline chloride, 3 mg / L of vitamin B12, 13 mg / L of folic acid, 130 mg / L of inositol, 21 mg / L of D-calcium pantothenate, 18 mg / L of pyridoxine hydrochloride, 2 mg / L of riboflavin, 13.5 mg / L of thiamine hydrochloride, 0.8 mg / L of nicotinamide, 0.75 mg / L of DL-α-lipoic acid, 0.0008 mg / L of tocopherol; The lipids include: 0.07 mg / L of linoleic acid; The sugars include: 4000 mg / L of sucrose, 1500 mg / L of galactose, 3000 mg / L of maltose monohydrate, 3000 mg / L of glucose, 125 mg / L of sodium pyruvate; The trace elements include: calcium chloride at 80 mg / L, ammonium ferric citrate at 400 mg / L, magnesium chloride at 40 mg / L, magnesium sulfate at 40 mg / L, potassium chloride at 900 mg / L, ammonium metavanadate at 0.007 mg / L, ammonium molybdate tetrahydrate at 0.007 mg / L, copper sulfate pentahydrate at 0.0751 mg / L, sodium selenite at 0.002 mg / L, manganese chloride tetrahydrate at 0.002 mg / L, manganese sulfate monohydrate at 0.005 mg / L, nickel sulfate at 0.002 mg / L, sodium metavanadate nonahydrate at 0.005 mg / L, sodium metasilicate nonahydrate at 1 mg / L, stannous chloride dihydrate at 0.0016 mg / L, ferrous sulfate heptahydrate at 0.35 mg / L, potassium nitrate at 0.04 mg / L, zinc sulfate heptahydrate at 8 mg / L, ethanolamine hydrochloride at 13 mg / L; The inorganic salts include: sodium dihydrogen phosphate at 900 mg / L, disodium hydrogen phosphate monohydrate at 135 mg / L, sodium chloride at 2500 mg / L; The polyamine is putrescine, with a content of 68 mg / L; The content of hypoxanthine is 3 mg / L, the content of thymidine is 3 mg / L, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid is 80 mg / L, the content of P188 is 3000 mg / L, malic acid is 400 mg / L, cholesterol is 0.8 mg / L; the content of the composite soy hydrolysate is 3 g / L; The content of recombinant human insulin is 5 mg / L.

2. The method for producing a recombinant canine parvovirus monoclonal antibody according to claim 1, characterized in that: The cell density in the inoculated solution in step 2) is 0.3 - 1.0×10 6 cells / mL.

3. The method for producing recombinant canine parvovirus monoclonal antibody according to claim 1, wherein: During the perfusion culture process, the glucose content in the culture solution is greater than or equal to 1 g / L and less than or equal to 2 g / L.

4. The method for producing recombinant canine parvovirus monoclonal antibody according to claim 1, characterized in that: During the perfusion culture process, the cell-specific perfusion rate of the perfusion culture is 40-60 p·L / (c·d), and the maintained cell density is 3.0-3.5×10 7 cells / mL.

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