Method for perfusion culture of cells by using cell culture medium waste liquid

Abstract

The invention relates to a method for performing perfusion culture on cells by utilizing cell culture medium waste liquid. The method comprises the following steps: carrying out perfusion culture on cells in a bioreactor by adopting a serum-free cell culture medium; separating to obtain a cell culture medium waste liquid, and then adding a non-animal source protein hydrolysate into the cell culture medium waste liquid to obtain a recycled cell culture medium; carrying out perfusion culture on the cells by using a recovered cell culture medium; repeating the steps until the culture is finished, and harvesting cells; wherein the non-animal source protein hydrolysate comprises three or more than three of a high free amino acid yeast hydrolysate, a high glutathione yeast hydrolysate, a small molecule peptide type yeast hydrolysate and a soybean protein hydrolysate. According to the method, the culture medium waste liquid can be recycled, normal growth, protein expression and cell differentiation of cells can be maintained, and the raw material cost of cell culture is remarkably reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of cell culture, and particularly to a method for culturing cells by perfusion with waste liquid of a cell culture medium. Background Art

[0002] Currently, the largest cost in the field of animal cell culture is still the cell culture medium, and the main costs are concentrated in serum and growth factors. The addition of serum has problems such as unstable components between batches of the culture medium, easy occurrence of pathogen contamination, high cost, and being unfavorable to animal welfare. In order to replace serum and significantly reduce the cost of cell culture, non-animal-derived hydrolysates have been widely used in the large-scale culture of cells (such as CHO, HEK293, etc.). In the process of continuous and large-scale serum-free cell culture, the problem of high cost is further amplified.

[0003] In the emerging field of cultured meat, one of the main reasons restricting its development is the high cost in the process of large-scale cell culture. Currently, the serum-free culture medium with protein hydrolysate as the core raw material can reduce the overall cost of the culture medium by up to 30%. However, in the industrialization process, a 30% reduction in the overall cost of the culture medium is still far from enough. In long-term, continuous, and large-scale cell culture, it is urgent to improve the overall utilization rate of the culture medium, seek a serum-free culture solution with lower cost, and filter waste from the cell culture medium and recycle the culture medium for cell culture.

[0004] Chinese Patent CN114703126A discloses a method for in vitro isolation and culture of bovine muscle stem cells, a culture medium and its application. The characteristic of its novel hydrolysate system is to add non-animal-derived hydrolysate to replace serum in the muscle stem cell culture medium, which can promote the proliferation of muscle stem cells in vitro and keep the in vitro proliferated muscle stem cells in a stem state. However, this invention does not recycle the cell culture medium, and the production cost is still relatively high.

[0005] Chinese Patent CN114891733A discloses a serum-free culture method for inducing myosatellite cells to differentiate into myotubes. The characteristic is that a differentiation rate as high as 46.2% can be obtained by using this method, and myosatellite cells can be efficiently induced to differentiate into myotubes. Similarly, this invention does not recycle the cell culture medium, and there is also the problem of high production cost. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a method for culturing cells by perfusion with waste liquid of a cell culture medium, which can recycle the waste liquid of the cell culture medium, improve the overall utilization rate of the cell culture medium, and reduce the cost in the process of cell culture.

[0007] To achieve the above object, the present invention provides the following technical solutions: Technical Solution 1: A method for culturing cells by perfusion with waste liquid of cell culture medium. In this method, the following steps are included: (1) Perfuse and culture cells in a bioreactor using a serum-free cell culture medium; (2) Separate the cell culture medium from the bioreactor through a separation system to obtain waste liquid of cell culture medium, and retain the cells in the bioreactor; (3) Add non-animal source protein hydrolysate to the waste liquid of cell culture medium obtained in step (2) to obtain recycled cell culture medium; (4) Perfuse and culture cells in the bioreactor using the recycled cell culture medium obtained in step (3); (5) Repeat steps (2)-(4) until the culture ends, and harvest the cells; Among them, the non-animal source protein hydrolysate in step (3) includes: high free amino acid yeast hydrolysate, high glutathione yeast hydrolysate, and small peptide type yeast hydrolysate; high glutathione yeast hydrolysate, small peptide type yeast hydrolysate, and soy protein hydrolysate; high free amino acid yeast hydrolysate, high glutathione yeast hydrolysate, and soy protein hydrolysate; or high free amino acid yeast hydrolysate, high glutathione yeast hydrolysate, small peptide type yeast hydrolysate, and soy protein hydrolysate; Among them, for the high free amino acid yeast hydrolysate, calculated by its weight, its free amino acid content is ≥50%; For the high glutathione yeast hydrolysate, calculated by its weight, its glutathione content is >10%; For the small peptide type yeast hydrolysate, calculated by its weight, the mass percentage of peptide segments with a molecular weight less than or equal to 1000 daltons in the total peptide segments of the small peptide type yeast hydrolysate is ≥96%; For the soy protein hydrolysate, the mass percentage of peptide segments with a molecular weight greater than 1000 daltons in the total peptide segments of the soy protein hydrolysate is ≥7%.

[0008] Technical Solution 2: The method according to Technical Solution 1, wherein, relative to each liter of waste liquid of cell culture medium, the addition amount of the non-animal source protein hydrolysate in step (3) is 1-20 g / L.

[0009] Technical Solution 3: The method according to Technical Solution 1 or 2, wherein, relative to each liter of waste liquid of cell culture medium, the addition amount of the non-animal source protein hydrolysate in step (3) is 10-20 g / L.

[0010] Technical solution 4: According to the method described in any one of technical solutions 1-3, wherein, by weight, the free amino acid content in the high free amino acid yeast hydrolysate is 50-65%; the glutathione content in the high glutathione yeast hydrolysate is 10-20%; the mass percentage of peptide segments with a molecular weight less than or equal to 1000 Dalton in the small molecular peptide type yeast hydrolysate accounts for 96-99% of the total peptide segments in the small molecular peptide type yeast hydrolysate; the mass percentage of peptide segments with a molecular weight greater than 1000 Dalton in the soy protein hydrolysate accounts for 7-17% of the total peptide segments in the high soy protein hydrolysate.

[0011] Technical solution 5: According to the method described in any one of technical solutions 1-4, wherein the glutathione content in the high free amino acid yeast hydrolysate is 0.6-1.5%; and / or, the free amino acid content in the high glutathione yeast hydrolysate is 8.14-14.21%; and / or, the free amino acid content in the small molecular peptide type yeast hydrolysate is 20-48%, and the glutathione content is 0.6-2%; and / or, the free amino acid content in the soy protein hydrolysate is 6.25-11.85%.

[0012] Technical solution 6: According to the method described in any one of technical solutions 1-5, wherein the non-animal source protein hydrolysate includes the high glutathione yeast hydrolysate and two or more substances selected from the group consisting of the high free amino acid yeast hydrolysate, the small molecular peptide type yeast hydrolysate, and the soy protein hydrolysate.

[0013] Technical solution 7: According to the method described in technical solution 6, wherein the weight ratio of the high glutathione yeast hydrolysate to two or more substances selected from the group consisting of the high free amino acid yeast hydrolysate, the small molecular peptide type yeast hydrolysate, and the soy protein hydrolysate is 1-4:4-7.

[0014] Technical solution 8: According to the method described in any one of technical solutions 1-7, wherein step (2) further includes a step of detecting the content of nutrient components in the cell culture waste liquid.

[0015] Technical solution 9: According to the method described in any one of technical solutions 1-8, wherein step (2) further includes a step of purifying the cell culture waste liquid using cation resin and / or anion resin.

[0016] Technical solution 10: According to the method described in any one of technical solutions 1-9, wherein the cell includes mammalian cells.

[0017] Technical solution 11: The method according to technical solution 10, wherein the mammalian cells include one or two of CHO-1 cells, CHO-S cells or MDCK cells.

[0018] Technical solution 12: The method according to any one of technical solutions 1-11, wherein the mammalian cells are CHO-1 cells, and the non-animal-derived protein hydrolysate includes high free amino acid yeast hydrolysate, high glutathione yeast hydrolysate and small molecule peptide type yeast hydrolysate.

[0019] Technical solution 13: The method according to technical solution 12, wherein the weight ratio of the small molecule peptide type yeast hydrolysate, the high free amino acid yeast hydrolysate and the high glutathione yeast hydrolysate is 1.5-2.5:1.5-2.5:3.5-4.5.

[0020] Technical solution 14: The method according to any one of technical solutions 1-11, wherein the mammalian cells are CHO-S, and the non-animal-derived protein hydrolysate includes high free amino acid yeast hydrolysate, high glutathione yeast hydrolysate and soybean protein hydrolysate.

[0021] Technical solution 15: The method according to technical solution 14, wherein the weight ratio of the high free amino acid yeast hydrolysate, the high glutathione yeast hydrolysate and the soybean protein hydrolysate is 0.5-1.5:3.5-4.5:2.5-3.5.

[0022] Technical solution 16: The method according to any one of technical solutions 1-11, wherein the mammalian cells are MDCK cells, and the non-animal-derived protein hydrolysate includes small molecule peptide type yeast hydrolysate, high glutathione yeast hydrolysate and soybean protein hydrolysate.

[0023] Technical solution 17: The method according to technical solution 16, wherein the weight ratio of the small molecule peptide type yeast hydrolysate, the high glutathione yeast hydrolysate and the soybean protein hydrolysate is 0.5-1.5:0.5-1.5:5.5-6.5.

[0024] Advantages of the present invention: After cell culture, the cell culture medium of the present invention can be recycled by recovering the cultured medium and adding non-animal-derived protein hydrolysate for reuse. It can maintain the normal growth, protein expression and cell differentiation of cells. Moreover, this process can significantly improve the cell utilization rate of all components of the medium, while significantly reducing the raw material cost of cell culture, which can be reduced by 40 - 80%. And the recycled cell culture medium obtained by this method can be applied to cell culture to reach or even exceed the level of fresh cell culture medium, and can be widely applied to the fields that require continuous and large-scale culture of animal cells. Brief Description of the Drawings

[0025] Figure 1 It is the result of the change of viable cell density and cell viability over time in the continuous perfusion culture of CHO-K1 cells in Example 1.

[0026] Figure 2 It is the result of the change of viable cell density and cell viability over time in the continuous perfusion culture of CHO-S cells in Example 2.

[0027] Figure 3 It is the result of the change of viable cell density and cell viability over time in the continuous perfusion culture of MDCK cells in Example 3. Detailed Embodiments

[0028] To make the objectives, technical solutions and technical effects of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely. The following described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Combining the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

[0029] The interval ranges defined in this application all include the endpoint values. For example, 10 - 20% includes the endpoint values 10% and 20%.

[0030] Non-animal-derived protein hydrolysate is a natural mixture rich in protein, peptides with different molecular weights, trace elements, vitamins, lipids, cholesterol, functional peptides, nucleotides, amino acids, etc., which is prepared by using plant protein or microbial whole cells as raw materials and carrying out directional enzymatic hydrolysis by using its own endogenous enzyme system or adding exogenous enzymes. It can provide various essential nutritional components such as amino acids, nucleotides, vitamins, sugars, etc. for the growth of animal cells. Protein hydrolysates of certain special processes contain components such as growth factors and serum-like substances, which can meet the special nutritional needs of animal cell growth.

[0031] Currently, in the field of serum-free animal cell culture, adding non-animal-derived protein hydrolysates to cell culture media can not only reduce the cost of serum-free media but also improve cell culture effects to a certain extent.

[0032] Through intensive research, the inventors of this application have discovered a method for culturing cells by perfusion with cell culture media waste liquid. This method can be applied to the continuous and large-scale culture process of animal cells. After recovering the cell culture media waste liquid, it can be recycled as a culture medium by supplementing various types of non-animal-derived protein hydrolysates and then used for continuous perfusion. The non-animal-derived protein hydrolysates in this method are used on the one hand to replace serum and on the other hand as nutritional supplements to be added to the cell culture media waste liquid, formulated into a culture medium that can continue to be used for continuous perfusion, thus realizing the recycling of cell culture media waste liquid.

[0033] In a specific embodiment of this application, a method for culturing cells by perfusion with cell culture media waste liquid is provided, and the method includes the following steps: (1) Perfusion culture of cells in a bioreactor using a serum-free cell culture medium; (2) Separating the cell culture medium from the bioreactor through a separation system to obtain cell culture media waste liquid, and retaining the cells in the bioreactor; (3) Adding non-animal-derived protein hydrolysates to the cell culture media waste liquid obtained in step (2) to obtain recycled cell culture medium; (4) Perfusion culture of cells in a bioreactor using the recycled cell culture medium obtained in step (3); (5) Repeating steps (2)-(4) until the end of the culture, and harvesting the cells; Among them, the non-animal-derived protein hydrolysates in step (3) include: high free amino acid yeast hydrolysate, high glutathione yeast hydrolysate, and small peptide type yeast hydrolysate; high glutathione yeast hydrolysate, small peptide type yeast hydrolysate, and soy protein hydrolysate; high free amino acid yeast hydrolysate, high glutathione yeast hydrolysate, and soy protein hydrolysate; or high free amino acid yeast hydrolysate, high glutathione yeast hydrolysate, small peptide type yeast hydrolysate, and soy protein hydrolysate; Among them, for the high free amino acid yeast hydrolysate, calculated by its weight, its free amino acid content is ≥50%; For the high glutathione yeast hydrolysate, calculated by its weight, its glutathione content is >10%; For the small peptide type yeast hydrolysate, the mass percentage of peptide segments with a molecular weight less than or equal to 1000 daltons in the total peptide segments of the small peptide type yeast hydrolysate is ≥96%; For the soy protein hydrolyzate, the mass percentage of peptide segments with a molecular weight greater than 1000 Da in the total peptide segments of the soy protein hydrolyzate is ≥7%.

[0034] In some embodiments of the present application, relative to each liter of the cell culture medium waste liquid, the addition amount of the non-animal-derived protein hydrolyzate in step (3) is 1-20 g / L, preferably 5-15 g / L.

[0035] In some embodiments of the present application, relative to each liter of the cell culture medium waste liquid, the addition amount of the non-animal-derived protein hydrolyzate in step (3) can be 1-20 g / L, 5-20 g / L, 10-20 g / L, 1-15 g / L, 1-10 g / L, 5-15 g / L, 5-10 g / L or 15-20 g / L. In some embodiments, relative to each liter of the cell culture medium waste liquid, the addition amount of the non-animal-derived protein hydrolyzate in step (3) can be 1 g / L, 1.2 g / L, 1.5 g / L, 1.8 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 7.5 g / L, 7.9 g / L, 8 g / L, 8.1 g / L, 8.2 g / L, 8.5 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L, 20 g / L or a concentration within the numerical range formed by any two of the above specific values as endpoints. It should be understood that in specific embodiments, any of the above ranges can be combined with any other range.

[0036] In some embodiments of the present application, for the high free amino acid yeast hydrolyzate, calculated by its weight, its free amino acid content is ≥55%; it should be noted that in the present invention, any commercially available or self-made high free amino acid yeast hydrolyzate can be used in the present invention. Specifically, the inventors have found through research that as long as the free amino acid content in the commercially available or self-made high free amino acid yeast hydrolyzate is ≥55%, it can be used in the present invention, and the free amino acid content is preferably 55%-65%.

[0037] More specifically, the inventors have found through research that using a high free amino acid yeast hydrolyzate containing various amino acids that meet the following conditions has a better culture effect for the recycling of waste cell culture medium.

[0038] Based on the weight of the high-free amino acid yeast hydrolysate, the free amino acids are aspartic acid 2-3%, threonine 2-3%, serine 2-3%, glutamic acid 6.5-7.4%, glycine 4-5%, alanine 13-15%, cystine 0.5-0.6%, valine 3-4%, methionine 0.5-1%, isoleucine 2-3%, leucine 4-5%, tyrosine 1-1.5%, phenylalanine 2-3%, lysine 2-3%, histidine 1-1.5%, arginine 4-5% and proline 0.5-1%.

[0039] In some specific embodiments, in order to better recycle the waste cell culture medium, the high-free amino acid yeast hydrolysate may further contain the following components: Based on the weight of the high-free amino acid yeast hydrolysate, the total nitrogen content in the high-free amino acid yeast hydrolysate is 11-12%. In some embodiments, the ammonia nitrogen content is 5-6%. In some embodiments, the glutathione content is 0.6-1.5%. In some embodiments, the oxidized glutathione content is 0.5-1% and the reduced glutathione content is 0.1-0.5%.

[0040] Based on the weight of the high-free amino acid yeast hydrolysate, the mass percentage of peptide segments with a molecular weight greater than or equal to 2000 daltons in the total peptide segments of the high-free amino acid yeast hydrolysate is 3-5%, the mass percentage of peptide segments with a molecular weight greater than 1000 and less than 2000 daltons in the total peptide segments of the high-free amino acid yeast hydrolysate is 1-3%, the mass percentage of peptide segments with a molecular weight greater than 400 and less than or equal to 1000 daltons in the total peptide segments of the high-free amino acid yeast hydrolysate is 10-15%, the mass percentage of peptide segments with a molecular weight greater than 180 and less than or equal to 400 daltons in the total peptide segments of the high-free amino acid yeast hydrolysate is 40-45%, and the mass percentage of peptide segments with a molecular weight less than or equal to 180 daltons in the total peptide segments of the high-free amino acid yeast hydrolysate is 35-40%.

[0041] Based on the weight of the high-free amino acid yeast hydrolysate, the free nucleotide content in the high-free amino acid yeast hydrolysate is 0.21-0.58%. Among them, based on the weight of the high-free amino acid yeast hydrolysate, the free nucleotides include uridine monophosphate (UMP) 0.05-0.15%, adenosine monophosphate (AMP) 0.1-0.3%, guanosine monophosphate (GMP) 0.01-0.03% and cytidine monophosphate (CMP) 0.05-0.1%.

[0042] In some specific embodiments, the high-glutathione yeast hydrolysate, based on its weight, its The glutathione content is > 10%; it should be noted that any commercially available high-glutathione yeast hydrolysate can be used in the present invention. Specifically, the inventor of the present invention has found through research that as long as the glutathione content in the commercially available or self-made high-glutathione yeast hydrolysate is > 10%, it can be used in the present invention In the present invention, the glutathione content is preferably 10% - 20%, where this range does not include the case of being equal to 10%.

[0043] More specifically, the inventor of the present invention has found that using a high-glutathione yeast hydrolysate with the type of glutathione satisfying the following conditions has a better culture effect for the recycling of waste cell culture medium.

[0044] Based on the weight of the high-glutathione yeast hydrolysate, glutathione includes 0.5 - 5% of oxidized glutathione and 9.5 - 15% of reduced glutathione.

[0045] In some specific embodiments, in order to obtain a better cell culture effect, the high-glutathione yeast hydrolysate may further contain the following components: Based on the weight of the high-glutathione yeast hydrolysate, the total nitrogen content in the high-glutathione yeast hydrolysate is 8 - 10%, and in some embodiments, the ammonia nitrogen content is 2 - 5%.

[0046] The mass percentage of peptide segments with a molecular weight greater than or equal to 2000 daltons in the high-glutathione yeast hydrolysate accounts for 3 - 5% of the total peptide segments in the high-glutathione yeast hydrolysate, the mass percentage of peptide segments with a molecular weight greater than 1000 and less than 2000 daltons accounts for 10 - 12% of the total peptide segments in the high-glutathione yeast hydrolysate, the mass percentage of peptide segments with a molecular weight greater than 400 and less than or equal to 1000 daltons accounts for 20 - 23% of the total peptide segments in the high-glutathione yeast hydrolysate, the mass percentage of peptide segments with a molecular weight greater than 180 and less than or equal to 400 daltons accounts for 23 - 26% of the total peptide segments in the high-glutathione yeast hydrolysate, and the mass percentage of peptide segments with a molecular weight less than or equal to 180 daltons accounts for 35 - 40% of the total peptide segments in the high-glutathione yeast hydrolysate.

[0047] Based on the weight of the high glutathione yeast hydrolysate, the content of free nucleotides in the high glutathione yeast hydrolysate is 1-2%. Among them, based on the weight of the high glutathione yeast hydrolysate, the free nucleotides include uridine monophosphate (UMP) 0.1-0.3%, adenosine monophosphate (AMP) 0.6-1.0%, inosine monophosphate (IMP) 0.1-0.3%, guanosine monophosphate (GMP) 0.1-0.2% and cytidine monophosphate (CMP) 0.1-0.2%.

[0048] Based on the weight of the high glutathione yeast hydrolysate, the content of free amino acids in the high glutathione yeast hydrolysate is 8.14-14.21%. Among them, based on the weight of the high glutathione yeast hydrolysate, the free amino acids are aspartic acid 0.3-0.4%, threonine 0.5-0.6%, serine 0.3-0.4%, glutamic acid 4-5%, glycine 0.1-0.6%, alanine 0.8-1.0%, cystine 0.6-1.0%, valine 0.1-0.3%, methionine 0.01-0.05%, isoleucine 0.1-0.5%, leucine 0.1-0.5%, tyrosine 0.1-0.5%, phenylalanine 0.01-0.1%, lysine 0.1-0.5%, histidine 0.01-1.2%, arginine 1-1.5% and proline 0.01-0.06%.

[0049] In some specific embodiments, for the small molecule peptide type yeast hydrolysate, the mass percentage of the peptide segments with a molecular weight less than or equal to 1000 daltons in the total peptide segments of the small molecule peptide type yeast hydrolysate is ≥96%. It should be noted that any commercially available small molecule peptide type yeast hydrolysate can be used in the present invention. Specifically, the inventors have found through research that as long as the mass percentage of the peptide segments with a molecular weight less than or equal to 1000 daltons in the total peptide segments of the commercially available or self-made small molecule peptide type yeast hydrolysate is ≥96%, it can be used in the present invention, and preferably it is 96%-99%.

[0050] More specifically, the inventors have found through research that the small molecule peptide type yeast hydrolysate with the following peptide relative molecular weight distribution has a better cultivation effect when used for the recovery of cell culture medium waste liquid.

[0051] Based on the weight of the small molecule peptide type yeast hydrolysate, the mass percentage of peptide segments with a molecular weight greater than or equal to 2000 Daltons in the total peptide segments of the small molecule peptide type yeast hydrolysate is 0 to 0.1%, the mass percentage of peptide segments with a molecular weight greater than 1000 and less than 2000 Daltons in the total peptide segments of the small molecule peptide type yeast hydrolysate is 1 to 3%, the mass percentage of peptide segments with a molecular weight greater than 400 and less than or equal to 1000 Daltons in the total peptide segments of the small molecule peptide type yeast hydrolysate is 15 to 20%, the mass percentage of peptide segments with a molecular weight greater than 180 and less than or equal to 400 Daltons in the total peptide segments of the small molecule peptide type yeast hydrolysate is 46 to 50%, and the mass percentage of peptide segments with a molecular weight less than or equal to 180 Daltons in the total peptide segments of the small molecule peptide type yeast hydrolysate is 35 to 40%.

[0052] In some specific embodiments, in order to obtain better cell culture effects, the small molecule peptide type yeast hydrolysate may further contain the following components: Based on its weight, the free amino acid content of the small molecule peptide type yeast hydrolysate is 20 to 48%, and the glutathione content is 0.5 to 2%; it should be noted that in the present invention, any commercially available or self-made small molecule peptide type yeast hydrolysate can be used in the present invention. Specifically, the inventors have found through research that as long as the free amino acid content in the commercially available or self-made small molecule peptide type yeast hydrolysate is 20 to 48% and the glutathione content is 0.5 to 2%, it can be used in the present invention.

[0053] More specifically, the inventors have found through research that using a small molecule peptide type yeast hydrolysate with the content of various amino acids meeting the following conditions has better culture effects for recycling waste cell culture media.

[0054] Based on the weight of the small molecule peptide type yeast hydrolysate, the free amino acid content in the small molecule peptide type yeast hydrolysate is 24.6 to 43.3%. Among them, the free amino acids are 1 to 1.9% aspartic acid, 1 to 1.9% threonine, 2 to 3% serine, 4 to 6% glutamic acid, 1 to 2% glycine, 5 to 10% alanine, 0.1 to 0.5% cystine, 2 to 3% valine, 0.5 to 1% methionine, 1 to 2% isoleucine, 2 to 3% leucine, 1 to 1.5% tyrosine, 1 to 1.5% phenylalanine, 1 to 2% lysine, 0.5 to 1.0% histidine, 1 to 2% arginine, and 0.5 to 1% proline.

[0055] In some specific embodiments, in order to obtain better cell culture effects, the small molecule peptide type yeast hydrolysate may further contain the following components: Based on the weight of the small-molecule peptide-type yeast hydrolysate, the total nitrogen content in the small-molecule peptide-type yeast hydrolysate is 11-12%. In some embodiments, the ammonia nitrogen content is 5-6%. In some embodiments, the glutathione content is 0.6-2%. In some specific embodiments, the content of oxidized glutathione is 0.5-1.5% and the content of reduced glutathione is 0.1-0.5%.

[0056] Based on the weight of the small-molecule peptide-type yeast hydrolysate, the free nucleotide content in the small-molecule peptide-type yeast hydrolysate is 0.1-0.3%. Among them, based on the weight of the small-molecule peptide-type yeast hydrolysate, the free nucleotides include uridine monophosphate (UMP) 0.01-0.05%, adenosine monophosphate (AMP) 0.03-0.1%, guanosine monophosphate (GMP) 0.01-0.05%, and cytidine monophosphate (CMP) 0.05-0.1%.

[0057] In some specific embodiments, for the soy protein hydrolysate, the mass percentage of peptide segments with a molecular weight greater than 1000 daltons in the total peptide segments of the soy protein hydrolysate is ≥7%; it should be noted that any commercially available soy protein hydrolysate can be used in the present invention. Specifically, the inventor of the present invention has found through research that as long as the mass percentage of peptide segments with a molecular weight greater than 1000 daltons in the total peptide segments of the commercially available or self-made soy protein hydrolysate is ≥7%, it can be used in the present invention, preferably 7%-17%.

[0058] More specifically, the inventor of the present invention has found that using a soy protein hydrolysate with the following peptide relative molecular weight distribution for the recovery of cell culture waste liquid has a better cultivation effect.

[0059] The mass percentage of peptide segments with a molecular weight greater than or equal to 2000 daltons in the total peptide segments of the soy protein hydrolysate is 2-7%, the mass percentage of peptide segments with a molecular weight greater than 1000 and less than 2000 daltons in the total peptide segments of the soy protein hydrolysate is 5-10%, the mass percentage of peptide segments with a molecular weight greater than 400 and less than or equal to 1000 daltons in the total peptide segments of the soy protein hydrolysate is 17-22%, the mass percentage of peptide segments with a molecular weight greater than 180 and less than or equal to 400 daltons in the total peptide segments of the soy protein hydrolysate is 27-32%, and the mass percentage of peptide segments with a molecular weight less than or equal to 180 daltons in the total peptide segments of the soy protein hydrolysate is 35-40%.

[0060] In some specific embodiments, in order to obtain a better cell culture effect, the soy protein hydrolysate may further contain the following components: Based on the weight of the soy protein hydrolyzate, the total nitrogen content in the soy protein hydrolyzate is 10-15% and the ammonia nitrogen content is 1-5%. Preferably, the total nitrogen content is 10.5-15% and the ammonia nitrogen content is 1-1.95%.

[0061] Based on the weight of the soy protein hydrolyzate, the free amino acid content in the soy protein hydrolyzate is 6.25-13.85%. Among them, based on the weight of the soy protein hydrolyzate, the free amino acids are aspartic acid 0.1-0.5%, threonine 0.1-0.5%, serine 0.1-0.5%, glutamic acid 0.4-0.7%, glycine 0.1-0.5%, alanine 0.1-0.5%, cystine 0.1-0.5%, valine 0.5-1.0%, methionine 0.05-0.15%, isoleucine 0.5-1%, leucine 1-1.5%, tyrosine 0.5-1.5%, phenylalanine 1-1.5%, lysine 0.5-1%, histidine 0.1-0.5%, arginine 1-1.5% and proline 0.1-0.5%. In some embodiments of the present application, the non-animal-derived protein hydrolyzate includes high glutathione yeast hydrolyzate and two or more substances selected from the group consisting of high free amino acid yeast hydrolyzate, small molecule peptide-type yeast hydrolyzate and soy protein hydrolyzate.

[0062] In some embodiments of the present application, the weight ratio of the high glutathione yeast hydrolyzate to two or more substances selected from the group consisting of high free amino acid yeast hydrolyzate, small molecule peptide-type yeast hydrolyzate and soy protein hydrolyzate is 1-4:4-5.

[0063] In some embodiments of the present application, the cells include mammalian cells; preferably, the mammalian cells include one or two of CHO-1 cells, CHO-S cells or MDCK cells.

[0064] In some embodiments of the present application, the mammalian cells are CHO-1 cells, and the non-animal-derived protein hydrolyzate includes small molecule peptide-type yeast hydrolyzate, high free amino acid yeast hydrolyzate and high glutathione yeast hydrolyzate. Preferably, the weight ratio of small molecule peptide-type yeast hydrolyzate, high free amino acid yeast hydrolyzate and high glutathione yeast hydrolyzate is 1.5-2.5:1.5-2.5:3.5-4.5.

[0065] In some embodiments of the present application, the mammalian cell is CHO-S, and the non-animal-derived protein hydrolysate includes high-free-amino-acid yeast hydrolysate, high-glutathione yeast hydrolysate, and soy protein hydrolysate. Preferably, the weight ratio of high-free-amino-acid yeast hydrolysate, high-glutathione yeast hydrolysate, and soy protein hydrolysate is 0.5-1.5:3.5-4.5:2.5-3.5.

[0066] In some embodiments of the present application, the mammalian cell is MDCK cell, and the non-animal-derived protein hydrolysate includes small-peptide-type yeast hydrolysate, high-glutathione yeast hydrolysate, and soy protein hydrolysate. Preferably, the weight ratio of small-peptide-type yeast hydrolysate, high-glutathione yeast hydrolysate, and soy protein hydrolysate is 0.5-1.5:0.5-1.5:5.5-6.5.

[0067] In some embodiments of the present application, the serum-free cell culture medium in step (1) is selected from the commonly used cell culture media on the market, and there is no particular limitation as long as it can meet the needs of cell growth. In some embodiments, the serum-free cell culture medium is selected from one or more of HyCell CHO Medium, EX-CELL® CD CHO, or EX-CELL® MDCK.

[0068] In some embodiments of the present application, step (2) further includes a step of separating the waste liquid of the cell culture medium. Preferably, the separation is selected from centrifugation or filtration. By separation, the residual cell debris in the culture medium waste liquid can be removed, and the purification effect of the culture medium waste liquid can be improved.

[0069] In some embodiments of the present application, step (2) further includes a step of detecting the content of nutrient components in the waste liquid of the cell culture medium.

[0070] In some embodiments of the present application, step (2) further includes a step of purifying the waste liquid of the cell culture medium using cation resin and / or anion resin. In some embodiments, the purification further includes a membrane filtration step, wherein the pore size of the membrane is 0.1-10 um.

[0071] In some embodiments of the present application, before the perfusion culture of the cells in step (1), there is a step of batch culturing the cells in a bioreactor using a serum-free cell culture medium. Preferably, the seeding density of the cells is 1-10×10 5 cells / mL; more preferably, when the glucose consumption in the cell culture medium is greater than 30%, the perfusion culture is started.

[0072] It should be noted that the batch culture described in the present application refers to a culture process in which the culture medium is not replaced, and the cells grow and metabolize in a fixed nutrient environment.

[0073] In some embodiments of the present application, the separation system described in step (2) is an alternating tangential flow (ATF) cell retention system.

[0074] In some embodiments of the present application, the perfusion rate of the perfusion culture described in step (1) and step (4) is 0.5 - 1.5 VVD.

[0075] VVD refers to the vessel volume per day, that is, the ratio of the volume of the culture medium removed from the bioreactor per day to the total volume of the reactor.

[0076] The beneficial effects of the present invention will be further illustrated by specific examples below.

[0077] The raw materials or reagents used in the present invention are all purchased from mainstream manufacturers in the market. Those without indicating the manufacturer or concentration are raw materials or reagents of analytical purity grade that can be obtained conventionally. As long as they can play the expected role, there is no special limitation.

[0078] In this embodiment, for those without specifying the specific technology or conditions, the technology or conditions described in the literature in this field or according to the product specification are followed.

[0079] Hereinafter, the present invention will be more specifically described using examples and comparative examples, but the technical scope of the present invention is not limited to these examples. It should be noted that unless otherwise specifically stated, all percentages, parts, and ratios used in the present invention are based on mass.

[0080] The sources of the various reagents and instruments used in the following examples are shown in Table 1.

[0081] Table 1 Information table of raw materials used in the examples Table 1 (continued) Information table of raw materials used in the examples It should be noted that the inventor has found through a large number of experiments that although there are other undetected components in the high free amino acid yeast hydrolysate, high glutathione yeast hydrolysate, small molecule peptide type yeast hydrolysate, and soy protein hydrolysate, these other undetected components have been proven through a large number of experiments not to affect the experimental effect of culturing stem cells in the present invention. That is to say, the components in the high free amino acid yeast hydrolysate, high glutathione yeast hydrolysate, small molecule peptide type yeast hydrolysate, and soy protein hydrolysate that affect cell culture are specifically as follows: (1) High free amino acid yeast hydrolysate (Angel yeast extract CM05 (powdered) for cell culture), based on the weight of the high free amino acid yeast hydrolysate, its free amino acid content is 57.1%. Among them, based on the weight of the high-free amino acid yeast hydrolysate, the free amino acids are aspartic acid 2.77%, threonine 2.47%, serine 2.78%, glutamic acid 6.92%, glycine 4.64%, alanine 13.98%, cystine 0.56%, valine 3.44%, methionine 0.96%, isoleucine 2.12%, leucine 4.13%, tyrosine 1.37%, phenylalanine 2.04%, lysine 2.79%, histidine 1.01%, arginine 4.17% and proline 0.95%.

[0082] Based on the weight of the high-free amino acid yeast hydrolysate, the total nitrogen content in the high-free amino acid yeast hydrolysate is 11.6%, the ammonia nitrogen content is 5.8%, the oxidized glutathione content is 0.7% and the reduced glutathione content is 0.2%. Based on the weight of the high-free amino acid yeast hydrolysate, the mass percentage of peptide segments with a molecular weight greater than or equal to 2000 daltons in the total peptide segments of the high-free amino acid yeast hydrolysate is 3.17%, the mass percentage of peptide segments with a molecular weight greater than 1000 and less than 2000 daltons in the total peptide segments of the high-free amino acid yeast hydrolysate is 2.03%, the mass percentage of peptide segments with a molecular weight greater than 400 and less than or equal to 1000 daltons in the total peptide segments of the high-free amino acid yeast hydrolysate is 13.37%, the mass percentage of peptide segments with a molecular weight greater than 180 and less than or equal to 400 daltons in the total peptide segments of the high-free amino acid yeast hydrolysate is 42.61%, and the mass percentage of peptide segments with a molecular weight less than or equal to 180 daltons in the total peptide segments of the high-free amino acid yeast hydrolysate is 38.83%.

[0083] Based on the weight of the high-free amino acid yeast hydrolysate, the free nucleotide content in the high-free amino acid yeast hydrolysate is 0.41%. Among them, based on the weight of the high-free amino acid yeast hydrolysate, the free nucleotides are uridine monophosphate (UMP) 0.10%, adenosine monophosphate (AMP) 0.20%, inosine monophosphate (IMP) 0.00%, guanosine monophosphate (GMP) 0.02% and cytidine monophosphate (CMP) 0.09%.

[0084] (2) High-glutathione yeast hydrolysate (Angel yeast extract CM07 (powdered) for cell culture), with a glutathione content of 11.2%. Among them, based on the weight of the high-glutathione yeast hydrolysate, the glutathione includes 0.7% oxidized glutathione and 10.5% reduced glutathione.

[0085] Based on the weight of the high-glutathione yeast hydrolysate, the total nitrogen content in the high-glutathione yeast hydrolysate is 9.2% and the ammonia nitrogen content is 3.0%.

[0086] The mass percentage of peptide segments with a molecular weight greater than or equal to 2000 Daltons in the high glutathione yeast hydrolysate to the total peptide segments in the high glutathione yeast hydrolysate is 4.58%, the mass percentage of peptide segments with a molecular weight greater than 1000 and less than 2000 Daltons to the total peptide segments in the high glutathione yeast hydrolysate is 11.39%, the mass percentage of peptide segments with a molecular weight greater than 400 and less than or equal to 1000 Daltons to the total peptide segments in the high glutathione yeast hydrolysate is 21.24%, the mass percentage of peptide segments with a molecular weight greater than 180 and less than or equal to 400 Daltons to the total peptide segments in the high glutathione yeast hydrolysate is 25.04%, and the mass percentage of peptide segments with a molecular weight less than or equal to 180 Daltons to the total peptide segments in the high glutathione yeast hydrolysate is 37.76%.

[0087] Based on the weight of the high glutathione yeast hydrolysate, the content of free nucleotides in the high glutathione yeast hydrolysate is 1.4%. Among them, based on the weight of the high glutathione yeast hydrolysate, the free nucleotides include 0.20% uridine monophosphate (UMP), 0.70% adenosine monophosphate (AMP), 0.20% inosine monophosphate (IMP), 0.20% guanosine monophosphate (GMP), and 0.10% cytidine monophosphate (CMP).

[0088] Based on the weight of the high glutathione yeast hydrolysate, the content of free amino acids in the high glutathione yeast hydrolysate is 9.96%. Among them, based on the weight of the high glutathione yeast hydrolysate, the free amino acids are 0.38% aspartic acid, 0.54% threonine, 0.31% serine, 4.22% glutamic acid, 0.28% glycine, 0.96% alanine, 0.62% cystine, 0.18% valine, 0.04% methionine, 0.14% isoleucine, 0.23% leucine, 0.31% tyrosine, 0.07% phenylalanine, 0.24% lysine, 0.09% histidine, 1.31% arginine, and 0.04% proline.

[0089] (3) Small molecule peptide type yeast hydrolysate (Angel yeast extract CM01 (powder) for cell culture) Based on the weight of the small molecule peptide type yeast hydrolysate, its free amino acid content is 32.34%. Among them, calculated based on the weight of the small molecule peptide type yeast hydrolysate, the free amino acids are aspartic acid 1.66%, threonine 1.56%, serine 2.17%, glutamic acid 4.56%, glycine 1.01%, alanine 7.31%, cystine 0.16%, valine 2.38%, methionine 0.57%, isoleucine 1.32%, leucine 2.26%, tyrosine 1.18%, phenylalanine 1.11%, lysine 1.9%, histidine 0.57%, arginine 1.85% and proline 0.77%.

[0090] Calculated based on the weight of the small molecule peptide type yeast hydrolysate, the total nitrogen content in the small molecule peptide type yeast hydrolysate is 11.2%, the ammonia nitrogen content is 5.5%, the oxidized glutathione content is 0.9% and the reduced glutathione content is 0.2%.

[0091] Calculated based on the weight of the small molecule peptide type yeast hydrolysate, the mass percentage of peptide segments with a molecular weight greater than or equal to 2000 daltons in the total peptide segments of the small molecule peptide type yeast hydrolysate is 0.01%, the mass percentage of peptide segments with a molecular weight greater than 1000 and less than 2000 daltons in the total peptide segments of the small molecule peptide type yeast hydrolysate is 2.21%, the mass percentage of peptide segments with a molecular weight greater than 400 and less than or equal to 1000 daltons in the total peptide segments of the small molecule peptide type yeast hydrolysate is 15.29%, the mass percentage of peptide segments with a molecular weight greater than 180 and less than or equal to 400 daltons in the total peptide segments of the small molecule peptide type yeast hydrolysate is 47.04%, and the mass percentage of peptide segments with a molecular weight less than or equal to 180 daltons in the total peptide segments of the small molecule peptide type yeast hydrolysate is 35.46%.

[0092] Calculated based on the weight of the small molecule peptide type yeast hydrolysate, the free nucleotide content in the small molecule peptide type yeast hydrolysate is 0.19%. Among them, calculated based on the weight of the small molecule peptide type yeast hydrolysate, the free nucleotides are uridine monophosphate (UMP) 0.04%, adenosine monophosphate (AMP) 0.08%, inosine monophosphate (IMP) 0.00%, guanosine monophosphate (GMP) 0.01% and cytidine monophosphate (CMP) 0.06%.

[0093] (4) Soybean protein hydrolysate (Angel soybean protein hydrolysate PU041 (powdered)) Calculated based on the weight of the soybean protein hydrolysate, the total nitrogen content in the soybean protein hydrolysate is 10.65% and the ammonia nitrogen content is 1.91%.

[0094] The mass percentage of peptide segments with a molecular weight greater than or equal to 2000 Daltons in the soy protein hydrolysate accounts for 5.4% of the total peptide segments in the soy protein hydrolysate, the mass percentage of peptide segments with a molecular weight greater than 1000 and less than 2000 Daltons accounts for 7.36% of the total peptide segments in the soy protein hydrolysate, the mass percentage of peptide segments with a molecular weight greater than 400 and less than or equal to 1000 Daltons accounts for 20.29% of the total peptide segments in the soy protein hydrolysate, the mass percentage of peptide segments with a molecular weight greater than 180 and less than or equal to 400 Daltons accounts for 30.02% of the total peptide segments in the soy protein hydrolysate, and the mass percentage of peptide segments with a molecular weight less than or equal to 180 Daltons accounts for 36.92% of the total peptide segments in the soy protein hydrolysate.

[0095] Based on the weight of the soy protein hydrolysate, the content of free amino acids in the soy protein hydrolysate is 9.04%. Among them, based on the weight of the soy protein hydrolysate, the free amino acids are 0.12% aspartic acid, 0.42% threonine, 0.31% serine, 0.58% glutamic acid, 0.11% glycine, 0.29% alanine, 0.14% cystine, 0.6% valine, 0.1% methionine, 0.62% isoleucine, 1.41% leucine, 0.97% tyrosine, 1.21% phenylalanine, 0.63% lysine, 0.25% histidine, 1.15% arginine and 0.13% proline.

[0096] The determination methods for the content of free amino acids, total nitrogen content, ammonia nitrogen content, peptide relative molecular weight distribution, free nucleotide content, oxidized glutathione content and reduced glutathione content in the above protein hydrolysate are specifically as follows: (1)Determination of free amino acid content Weigh 0.5 g - 1 g of the test sample (accurate to 0.001 g) and place it in a 50 mL volumetric flask. Add 20 mL of sulfosalicylic acid and ultrasonically dissolve it until it is completely dissolved, then make up the volume to the 50 mL mark, mix well, let it stand for 1 h, and then accurately pipette 1 mL of the supernatant into a 25 mL volumetric flask. Add 0.02 mol / L hydrochloric acid solution or sodium citrate buffer to make up the volume to the mark, mix well, filter through a 0.22 μm microporous filter membrane into an injection bottle for testing. According to the requirements of the amino acid analyzer instruction manual, make the concentration of the mixed amino acid standard solution within the optimal detection range of the instrument and use it as an external standard for on-machine determination.

[0097] The content of amino acids in the test sample solution is calculated according to formula (1): (1) In the formula: C i : The content of amino acid i in the test sample solution, in units of nmol / mL; Ai : The peak area of amino acid i in the sample determination solution; A s : The peak area of amino acid s in the amino acid standard working solution; c s : The content of amino acid s in the amino acid standard working solution, in nmol / mL.

[0098] The content of each amino acid in the sample is calculated according to formula (2): (2) In the formula: X i : The content of amino acid i in the sample, in %; c i : The content of amino acid i in the sample solution, in nmol / mL; F: Dilution factor; V: The volume of the sample fixed volume, in mL; M: The molar mass of amino acid i, in g / mol; m: The sample weighing amount, in g.

[0099] (2) Determination of total nitrogen: The Kjeldahl method in 6.4 of the national standard GB / T 23530-2009 is adopted: Take the sample (equivalent to 30,440 mg of total nitrogen), under the action of 5 g of mixed catalyst a (potassium sulfate and anhydrous copper sulfate are mixed in a ratio of 97:3) and 2.5 g of catalyst b (selenium powder and potassium sulfate are mixed in a ratio of 0.1:100), add 20 mL of concentrated sulfuric acid for digestion; then carry out distillation, absorb the product ammonia with boric acid; then titrate with 0.1 mol / L hydrochloric acid, read the data, and calculate the total nitrogen content.

[0100] (3) Determination of ammonia nitrogen (amino acid nitrogen) content: The detection method of amino acid nitrogen in 6.5 of the national standard GB / T 23530-2009 is adopted: Take 5 g of the sample, dilute it and titrate it with 0.5 mol / L sodium hydroxide solution to pH 8.2 and keep it for 1 min. Slowly add 10 mL of 36% formaldehyde solution to react with the non-dissociated amino groups in neutral amino acids to form monohydroxymethyl and dihydroxymethyl derivatives, and this reaction proceeds quantitatively. The hydrogen ions released at this time are titrated with the above sodium hydroxide, and the content of amino acid nitrogen is calculated according to the consumption of the alkali solution.

[0101] (4) Determination of the relative molecular weight distribution of peptides Determination of the relative molecular weight distribution of peptides: Modify the standard product in the determination method of the relative molecular weight distribution of peptides in Appendix A of GB / T 22492-2008 Soy Peptide Powder. The specific operation steps are as follows: Weigh 10 mg of the sample into a 10 mL volumetric flask, add a little mobile phase, and ultrasonically oscillate to fully dissolve and mix the sample. Dilute to the scale with the mobile phase, filter through a 0.45 μm organic membrane, and wait for injection analysis. Weigh 5 mg of each of the standards (cytochrome C, bacitracin, glycine-glycine-tyrosine-arginine, glycine-glycine-glycine) into a 10 mL volumetric flask, dissolve and make up the volume with the mobile phase, filter through a 0.45 μm organic phase membrane for injection, and obtain the chromatogram of the standards. Plot the logarithm of the relative molecular mass against the retention time to obtain the calibration curve and its equation.

[0102] X = A / A 总 × 100% In the formula: X—the mass fraction of a peptide segment with a certain relative molecular mass in the peptide segments in the sample, % A—the peak area of a peptide segment with a certain relative molecular mass; A 总 —the sum of the peak areas of peptide segments with various relative molecular masses.

[0103] (5)Determination of the content of free nucleotides Determination is carried out according to Appendix H of GB / T 20886.2—2021.

[0104] (6)Determination of the content of oxidized glutathione and the content of reduced glutathione Determination is carried out according to the method in Appendix A of GB / T 35882-2018.

[0105] In the examples of this application, the test method for viable cell density and cell viability is as follows: Take 100 μL of cell suspension and mix it evenly with 100 μL of 0.4% trypan blue staining solution. Aspirate the mixed solution into a cell counting chamber, and use a cell counter to measure the viable cell density and cell viability. The measurements of viable cell density and cell viability both need to be measured three times and the average value is taken.

[0106] Example 1 Recycling of cell culture medium for perfusion culture of CHO-K1 cells 1. Serum-free cell culture medium culture Add 800 mL of HyCell CHO Medium serum-free cell culture medium into a 2 L bioreactor. Set the reactor to a temperature control of 37 °C, a rotation speed of 100 rpm, and turn on the air control system. After the temperature reaches 37 °C and the dissolved oxygen is stable, calibrate the dissolved oxygen, and then quickly inject the seed cells into the bioreactor. Among them, the seed cells are CHO-K1 cells, and the inoculation density is 8×10 5cells / mL, turn on the pH setting to 7.2, associate with CO2 and adjust the pH using 1 M NaOH. Set the DO (dissolved oxygen) to 50% and associate with air. Detect its cell density, cell viability, and biochemical indicators such as glucose, lactate, and ammonium ions in the culture medium every 24 hours. When the glucose consumption is higher than 30%, prepare to start perfusion culture.

[0107] 2. Recovery of cell culture medium waste liquid 2L bioreactor continuous perfusion culture system: Connect the feed pump and the discharge pump, and start the ATF interception system. Continuously and uniformly pump the HyCell CHO serum-free cell culture medium into the reactor through the feed pump for 24 hours. At this time, the discharge pump continuously pumps out the culture medium waste liquid through the ATF interception system. Set the perfusion rate and the recovery rate to be equal, both 1VVD (Vessel Volume per Day), that is, replace 1 total culture volume of the culture medium in one day to ensure the dynamic balance of the total volume of the bioreactor. The discharge pump is connected to the waste liquid bottle. When the perfusion culture lasts for 24 hours, the volume of the waste liquid collected in the waste liquid bottle reaches about 800 mL. Centrifuge the waste liquid using a centrifuge to obtain the supernatant as the culture medium waste liquid. Among them, the centrifugal force for centrifugal separation is 3000×g, the rotation speed is 4000 revolutions per minute, and the centrifugation time is 10 minutes; then detect the accumulation of lactate and ammonium ions in the culture medium waste liquid. Among them, the ammonium ion content in the culture medium waste liquid is 8.1 mM, and the lactate content is 3.2 mM. Pump the culture medium waste liquid into the anion exchange resin column to adsorb and remove most of the lactate; after adjusting the pH, pump the culture medium waste liquid into the cation exchange resin column to adsorb and remove most of the ammonium ions. The culture medium waste liquid after passing through the ion exchange resin column is adjusted to a pH of 6.8 - 7.1, and then filtered through a 0.22 µm filter membrane. The filtrate at this time is the culture medium filtrate A.

[0108] 3. Detection and analysis of the main components of the culture medium before and after culturing cells and preparation of the recovered cell culture medium 1) Take the serum-free cell culture medium before culture and the culture medium filtrate A, and quantitatively detect the main nutrient components such as amino acids, nucleotides, vitamins, and sugars in them by liquid chromatography-mass spectrometry (LC-MS).

[0109] 2) Take the serum-free cell culture medium before culture and the culture medium filtrate A, and quantitatively detect lactate and ammonium ions by a biochemical analyzer. The detection data are shown in Table 2. Table 2 Detection data Amino acid consumption Nucleotide consumption Vitamin consumption Carbohydrate consumption Lactic acid content Ammonium ion content Culture medium filtrate A 45.8% 13.7% 44% 55.6% 1.5 mM 2.8 mM 3) Based on the volume of the culture medium filtrate A, add 10 g / L of non-animal-derived protein hydrolysate to the culture medium filtrate A to obtain the recycled cell culture medium B. Among them, the non-animal-derived protein hydrolysate is prepared from the following raw materials in the following weight ratio: small molecule peptide-type yeast hydrolysate CM01: high free amino acid yeast hydrolysate CM05: high glutathione yeast hydrolysate CM07 = 2:2:4.

[0110] 4. Use the recycled cell culture medium B for perfusion culture According to the method for recycling the waste liquid of the cell culture medium in step 2, replace the HyCell CHO serum-free cell culture medium with the recycled cell culture medium B. Continuously pump the recycled cell culture medium B into the reactor through a feed pump, and the discharge pump continuously pumps out the waste liquid of the culture medium through the ATF interception system. Set the perfusion rate equal to the recycling rate to ensure the dynamic balance of the total volume of the bioreactor. The discharge pump is connected to a waste liquid bottle. When the perfusion culture is carried out for 24 h, the volume of the waste liquid collected in the waste liquid bottle reaches about 800 mL. Centrifuge the waste liquid using a centrifuge to obtain the supernatant as the waste liquid of the culture medium. Among them, the centrifugal force for centrifugation is 3000×g, the rotation speed is 4000 revolutions per minute, and the centrifugation time is 10 min; then detect the accumulation of lactic acid and ammonium ions in the waste liquid of the culture medium. If the lactic acid content in the waste liquid of the culture medium is greater than or equal to 3 mM, pump the waste liquid of the culture medium into an anion exchange resin column to adsorb and remove most of the lactic acid and adjust the pH; if the ammonium ion content in the waste liquid of the culture medium is greater than or equal to 8 mM, then pump the waste liquid of the culture medium into a cation exchange resin column again to adsorb and remove most of the ammonium ions. Adjust the pH of the waste liquid of the culture medium after passing through the ion exchange resin column to 6.8 - 7.1, and then filter it through a 0.22 µm filter membrane. The filtrate at this time is the culture medium filtrate A.

[0111] Prepare the recycled cell culture medium B according to the method for detecting and analyzing the main components of the culture medium before and after culturing cells in step 3 and preparing the recycled cell culture medium (wherein, if the ammonium ion content in the waste liquid of the culture medium is less than 8 mM and the lactic acid content is less than 3 mM, directly prepare the recycled cell culture medium with the waste liquid of the culture medium; if the ammonium ion content in the waste liquid of the culture medium is greater than or equal to 8 mM and / or the lactic acid content is greater than or equal to 3 mM, then prepare the recycled cell culture medium with the filtrate).

[0112] Repeat the above steps and perform perfusion culture on the cells using the recycled cell culture medium B. If the previous recycled cell culture medium is used up and the next recycled cell culture medium has not been prepared yet, then use the fresh HyCell CHO serum-free cell culture medium for perfusion culture to ensure the continuity of the perfusion culture. When the next recycled culture medium is prepared, replace the HyCell CHO serum-free cell culture medium with the prepared recycled cell culture medium B and continue the perfusion culture.

[0113] After 12 days of perfusion, the viable cell density reached 2.57×10 7 cells / mL, entering the plateau phase. The cell viability began to decrease significantly. The test results are as Figure 1 shown. Perfusion culture was terminated on the 13th day, and the cells were harvested.

[0114] As a control, fresh serum-free cell culture medium HyCell CHO Medium and culture medium filtrate A were used for perfusion culture respectively to compare and test cell growth.

[0115] The specific method is as follows: 1) Use fresh serum-free cell culture medium HyCell CHO Medium for perfusion culture Add 800 mL of HyCell CHO Medium serum-free cell culture medium to a 2 L bioreactor. Set the reactor to a temperature control of 37°C, a rotation speed of 100 rpm, and turn on the air control system. After the temperature reaches 37°C and the dissolved oxygen is stable, calibrate the dissolved oxygen. Then quickly inject the seed cells into the bioreactor. Among them, the seed cells are CHO-K1 cells, and the inoculation density is 8×10 5 cells / mL. Set the pH to 7.2, associate CO2 and use 1 M NaOH to adjust the pH. Set the DO (dissolved oxygen) to 50% and associate air. Detect its cell density, cell viability, and biochemical indexes such as glucose, lactic acid, and ammonium ions in the culture medium every 24 h. When the pH shows a rapid downward trend and the glucose consumption is higher than 30%, prepare to start perfusion culture. Connect a feed pump and a discharge pump to the 2 L bioreactor, and start the ATF interception system. Continuously and uniformly pump the HyCell CHO serum-free cell culture medium into the reactor through the feed pump for 24 h. At this time, the discharge pump continuously pumps out the culture medium waste liquid through the ATF interception system. Set the perfusion speed and the recovery speed to be equal, both 1 VVD (Vessel Volume per Day), that is, replace 1 culture total volume of the culture medium in one day to ensure the dynamic balance of the total volume of the bioreactor. The discharge pump is connected to a waste liquid bottle.

[0116] After 12 days of perfusion, the viable cell density reached 2.63×10 7 cells / mL, and then entered the plateau phase. The cell viability began to decrease significantly. The test results are as Figure 1 shown. Perfusion culture was terminated on the 13th day, and the cells were harvested.

[0117] 2) Use culture medium filtrate A for perfusion culture a. Serum-free cell culture medium culture Add 800 mL of HyCell CHO Medium serum-free cell culture medium to a 2L bioreactor. Set the bioreactor to a temperature of 37°C, a rotation speed of 100 rpm, and turn on the air control system. After the temperature reaches 37°C and the dissolved oxygen is stable, calibrate the dissolved oxygen. Then quickly inject the seed cells into the bioreactor. The seed cells are CHO-K1 cells, and the inoculation density is 8×10 5 cells / mL. Set the pH to 7.2, associate CO2 and use 1 M NaOH to adjust the pH. Set the DO (dissolved oxygen) to 50% and associate air. Detect the cell density, cell viability, and biochemical indicators such as glucose, lactic acid, and ammonium ions in the culture medium every 24 hours. When the glucose consumption is higher than 30%, prepare to start perfusion culture.

[0118] b. Recovery of cell culture waste liquid 2L bioreactor continuous perfusion culture system: Connect the feed pump and the discharge pump, and start the ATF interception system. Continuously and uniformly pump the HyCell CHO serum-free cell culture medium into the reactor through the feed pump for 24 hours. At this time, the discharge pump continuously pumps out the culture waste liquid through the ATF interception system. Set the perfusion rate and the recovery rate to be equal, both 1VVD (Vessel Volume per Day), that is, replace 1 total culture volume of the medium in one day to ensure the dynamic balance of the total volume of the bioreactor. The discharge pump is connected to a waste liquid bottle. When the perfusion culture lasts for 24 hours, the volume of the waste liquid collected in the waste liquid bottle reaches about 800 mL. Centrifuge the waste liquid using a centrifuge. Take the supernatant to obtain the culture waste liquid. Among them, the centrifugal force for centrifugation is 3000×g, the rotation speed is 4000 revolutions per minute, and the centrifugation time is 10 minutes. Then detect the accumulation of lactic acid and ammonium ions in the culture waste liquid. If the lactic acid content in the culture waste liquid is greater than or equal to 3 mM, pump the culture waste liquid into an anion exchange resin column to adsorb and remove most of the lactic acid and adjust the pH. If the ammonium ion content in the culture waste liquid is greater than or equal to 8 mM, then pump the culture waste liquid into a cation exchange resin column to adsorb and remove most of the ammonium ions. Adjust the pH of the culture waste liquid after passing through the ion exchange resin column to 6.8 - 7.1, and then filter it through a 0.22 µm filter membrane. The filtrate at this time is culture filtrate A.

[0119] c. Use culture filtrate A or culture waste liquid for perfusion culture According to the method for recycling the waste liquid of cell culture medium in step b, use the culture medium filtrate A or the waste liquid of the culture medium to replace the HyCell CHO serum-free cell culture medium. Continuously pump the culture medium filtrate A or the waste liquid of the culture medium into the reactor through a feed pump, and the discharge pump continuously pumps out the waste liquid of the culture medium through the ATF interception system. Set the perfusion rate equal to the recycling rate to ensure the dynamic balance of the total volume of the bioreactor. The discharge pump is connected to a waste liquid bottle. When the perfusion culture is carried out for 24 hours, the volume of the waste liquid collected in the waste liquid bottle reaches about 800 mL. Centrifuge the waste liquid using a centrifuge to obtain the supernatant as the waste liquid of the culture medium. Among them, the centrifugal force for centrifugal separation is 3000×g, the rotation speed is 4000 revolutions per minute, and the centrifugation time is 10 minutes; then detect the accumulation of lactic acid and ammonium ions in the waste liquid of the culture medium. If the lactic acid content exceeds 3 mM, pump the waste liquid of the culture medium into an anion exchange resin column to adsorb and remove most of the lactic acid and adjust the pH; if the ammonium ion content in the waste liquid of the culture medium exceeds 8 mM, then pump the waste liquid of the culture medium into a cation exchange resin column again to adsorb and remove most of the ammonium ions. The waste liquid of the culture medium after passing through the ion exchange resin column is adjusted to a pH of 6.8 - 7.1 and then filtered through a 0.22 µm filter membrane. The filtrate at this time is the culture medium filtrate A.

[0120] Repeat the above steps to perform perfusion culture on the cells using the culture medium filtrate A or the waste liquid of the culture medium. If the previous culture medium filtrate A is used up and the next culture medium filtrate A is not yet processed, use fresh HyCell CHO serum-free cell culture medium for perfusion culture to ensure the continuity of perfusion culture. When the next culture medium filtrate A is processed, replace the HyCell CHO serum-free cell culture medium with the prepared culture medium filtrate A and continue the perfusion culture.

[0121] After 8 days of perfusion, the viable cell density reaches 0.56×10 7 cells / mL, and then enters the plateau phase. The cell viability begins to decline significantly. The test results are as Figure 1 shown. Terminate the perfusion culture on the 9th day and harvest the cells.

[0122] It can be seen from Figure 1 that during the perfusion culture of CHO-K1 cells, the use of non-animal-derived protein hydrolysates in continuous perfusion culture can recycle the waste liquid of the cell culture medium and maintain the normal growth of cells. The maximum viable cell density can exceed 2.5×10 7 cells / mL.

[0123] The cell growth operation results of perfusion using three different culture media are shown in Table 3. Table 3 Cell Growth Operation Results Fresh culture medium Fresh culture medium + cell culture filtrate A Fresh culture medium + recycled culture medium B Maximum viable cell density <![CDATA[2.63×10 7 cells / mL]]> <![CDATA[0.56×10 7 cells / mL]]> <![CDATA[2.57×10 7 cells / mL]]> Example 2: Recycling of CHO-S Cell Culture Medium for Perfusion Culture 1. Serum-free Cell Culture Medium Culture Add 800 mL of EX-CELL® CD CHO serum-free cell culture medium to a 2 L bioreactor. Set the reactor to a temperature control of 37 °C, a rotation speed of 100 rpm, and turn on the air control system. After the temperature reaches 37 °C and the dissolved oxygen is stable, calibrate the dissolved oxygen. Then quickly inject the seed cells into the bioreactor. The seed cells are CHO-S cells, and the inoculation density is 8×10 5 cells / mL. Set the pH to 7.2, associate CO2, and use 1 M NaOH to adjust the pH. Set the DO to 50% and associate air. Detect the cell density, cell viability, and biochemical indicators such as glucose, lactate, and ammonium ions in the medium every 24 hours. When the pH shows a rapid downward trend and the glucose consumption is higher than 30%, prepare to start perfusion culture.

[0124] 2. Recycling of Cell Culture Medium Waste Liquid 2 L bioreactor continuous perfusion culture system: Connect the feed pump and the discharge pump, and start the ATF interception system. Continuously and uniformly pump the EX-CELL® CD CHO serum-free cell culture medium into the reactor through the feed pump for 24 hours. At this time, the discharge pump continuously pumps out the medium waste liquid through the ATF interception system. Set the perfusion speed and the recovery speed to be equal, both 1 VVD (Vessel Volume per Day), to ensure the dynamic balance of the total volume of the bioreactor. The discharge pump is connected to a waste liquid bottle. When the perfusion culture lasts for 24 hours, the volume of the waste liquid collected in the waste liquid bottle reaches about 800 mL. Centrifuge the waste liquid using a centrifuge. Take the supernatant to obtain the medium waste liquid. Among them, the centrifugal separation centrifugal force is 3000×g, the rotation speed is 4000 revolutions per minute, and the centrifugation time is 10 minutes. Then detect the accumulation of lactate and ammonium ions in the medium waste liquid. Among them, the ammonium ion content in the medium waste liquid is 4 mM, and the lactate content is 3.2 mM. Pump the medium waste liquid into an anion exchange resin column to adsorb and remove most of the lactate. For the medium waste liquid after passing through the ion exchange resin column, adjust the pH to 6.8 - 7.1, and then filter it through a 0.22 µm filter membrane. The filtrate at this time is the medium filtrate C.

[0125] 3. Detection and Analysis of the Main Components of the Culture Medium before and after Culturing Cells and Preparation of the Recycled Cell Culture Medium 1) Take an appropriate amount of the serum-free cell culture medium and the medium filtrate C before culturing, and quantitatively detect the main nutrient components such as amino acids, nucleotides, vitamins, and sugars in them by liquid chromatography-mass spectrometry (LC-MS).

[0126] 2) Take appropriate amounts of the serum-free cell culture medium before culturing and the culture medium filtrate C, and quantitatively detect lactic acid and ammonium ions through a biochemical analyzer. The detection data are shown in Table 4. Table 4 Detection Data Amino acid consumption Nucleotide consumption Vitamin consumption Carbohydrate consumption Lactic acid content Ammonium ion content Culture medium filtrate C 46.6% 16.6% 49.87% 48% 2.6 mM 1.9 mM 3) Based on the volume of the culture medium filtrate C, add 10 g / L of non-animal-derived protein hydrolysate to the culture medium filtrate C to obtain the recycled cell culture medium D. The non-animal-derived protein hydrolysate is prepared from the following raw materials in a weight ratio: high free amino acid yeast hydrolysate CM05: high glutathione yeast hydrolysate CM07: soybean protein hydrolysate PU041 = 1:4:3.

[0127] 4. Use the recycled cell culture medium D for perfusion culture According to the method for recycling the waste liquid of the cell culture medium in step 2, replace the EX-CELL® CDCHO serum-free cell culture medium with the recycled cell culture medium D. Continuously pump the recycled cell culture medium D into the reactor through a feed pump, and continuously pump out the waste liquid of the culture medium through an ATF interception system by a discharge pump. Set the perfusion rate equal to the recycling rate to ensure the dynamic balance of the total volume of the bioreactor. The discharge pump is connected to a waste liquid bottle. When the perfusion culture lasts for 24 h, the volume of the waste liquid collected in the waste liquid bottle reaches about 800 mL. Centrifuge the waste liquid using a centrifuge to obtain the supernatant as the waste liquid of the culture medium. Among them, the centrifugal force for centrifugation is 3000×g, the rotation speed is 4000 revolutions per minute, and the centrifugation time is 10 min; then detect the accumulation of lactic acid and ammonium ions in the waste liquid of the culture medium. If the lactic acid content in the waste liquid of the culture medium exceeds 3 mM, pump the waste liquid of the culture medium into an anion exchange resin column to adsorb and remove most of the lactic acid and adjust the pH; if the ammonium ion content in the waste liquid of the culture medium is greater than or equal to 8 mM, then pump the waste liquid of the culture medium into a cation exchange resin column to adsorb and remove most of the ammonium ions. Adjust the pH of the waste liquid of the culture medium after passing through the ion exchange resin column to 6.8 - 7.1, and then filter it through a 0.22 µm filter membrane. The filtrate at this time is the culture medium filtrate C.

[0128] Prepare the recycled cell culture medium D according to the method for detecting and analyzing the main components of the culture medium before and after culturing cells and preparing the recycled cell culture medium in step 3 (wherein, if the ammonium ion content in the waste liquid of the culture medium is less than 8 mM and the lactic acid content is less than 3 mM, directly prepare the recycled cell culture medium with the waste liquid of the culture medium; if the ammonium ion content in the waste liquid of the culture medium is greater than or equal to 8 mM and / or the lactic acid content is greater than or equal to 3 mM, then prepare the recycled cell culture medium with the filtrate).

[0129] Repeat the above steps to perform perfusion culture on the cells using the recycled cell culture medium D. If the recycled cell culture medium from the previous time has been used up and the recycled cell culture medium for the next time has not been prepared yet, use fresh EX-CELL® CD CHO serum-free cell culture medium for perfusion culture to ensure the continuity of perfusion culture. When the recycled cell culture medium for the next time is prepared, replace the EX-CELL® CD CHO serum-free cell culture medium with the prepared recycled cell culture medium D and continue perfusion culture.

[0130] After 10 days of perfusion, the viable cell density reaches 1.96×10 7 cells / mL and enters the plateau phase, and the viability begins to decrease significantly. The test results are as Figure 2 shown. Perfusion culture is terminated on the 11th day, and the cells are harvested.

[0131] As a control, fresh serum-free cell culture medium EX-CELL® CD CHO and culture medium filtrate C are used for perfusion culture respectively to compare and test cell growth.

[0132] The specific method is as follows: 1) Use fresh serum-free cell culture medium EX-CELL® CD CHO for perfusion culture Add 800 mL of EX-CELL® CD CHO serum-free cell culture medium to a 2 L bioreactor. Set the reactor to a temperature control of 37°C, a rotation speed of 100 rpm, and turn on the air control system. After the temperature reaches 37°C and the dissolved oxygen is stable, calibrate the dissolved oxygen. Then quickly inject the seed cells into the bioreactor. Among them, the seed cells are CHO-K1 cells, and the inoculation density is 8×10 5 cells / mL. Set the pH to 7.2, associate CO2 and use 1 M NaOH to adjust the pH. Set the DO (dissolved oxygen) to 50% and associate air. Detect its cell density, cell viability, and biochemical indexes such as glucose, lactate, and ammonium ions in the culture medium every 24 hours. When the pH shows a rapid downward trend and the glucose consumption is higher than 30%, prepare to start perfusion culture. Connect a feed pump and a discharge pump to the 2 L bioreactor and start the ATF interception system. Continuously and uniformly pump the EX-CELL® CD CHO serum-free cell culture medium into the reactor through the feed pump for 24 hours. At this time, the discharge pump continuously pumps out the culture medium waste liquid through the ATF interception system. Set the perfusion rate and the recovery rate to be equal, both 1 VVD (Vessel Volume per Day), that is, replace the culture medium of 1 total culture volume in one day to ensure the dynamic balance of the total volume of the bioreactor. The discharge pump is connected to a waste liquid bottle.

[0133] After 10 days of perfusion, the viable cell density reaches 1.89×10 7cells / mL, and then entered the plateau phase. The cell viability began to decline significantly. The test results are as Figure 2 shown. Perfusion culture was terminated on the 11th day, and the cells were harvested.

[0134] 2) Use the culture medium filtrate D for perfusion culture a. Serum-free cell culture medium culture Add 800 mL of EX-CELL® CD CHO serum-free cell culture medium to a 2 L bioreactor. Set the reactor to a temperature of 37 °C, a rotation speed of 100 rpm, and turn on the air control system. After the temperature reaches 37 °C and the dissolved oxygen is stable, calibrate the dissolved oxygen. Then quickly inject the seed cells into the bioreactor. Among them, the seed cells are CHO-S cells, and the inoculation density is 8×10 5 cells / mL. Turn on the pH setting to 7.2, associate CO2 and use 1 M NaOH to adjust the pH. Set the DO to 50% and associate air. Detect its cell density, cell viability, and biochemical indexes such as glucose, lactic acid, and ammonium ions in the culture medium every 24 h. When the pH shows a rapid downward trend and the glucose consumption is higher than 30%, prepare to start perfusion culture.

[0135] b. Recovery of cell culture medium waste liquid 2 L bioreactor continuous perfusion culture system: Connect the feed pump and the discharge pump, and start the ATF interception system. Continuously and uniformly pump the EX-CELL® CD CHO serum-free cell culture medium into the reactor through the feed pump for 24 h. At this time, the discharge pump continuously pumps out the culture medium waste liquid through the ATF interception system. Set the perfusion rate and the recovery rate to be equal, both 1 VVD (Vessel Volume per Day) to ensure the dynamic balance of the total volume of the bioreactor. The discharge pump is connected to the waste liquid bottle. When the perfusion culture lasts for 24 h, the volume of the waste liquid collected in the waste liquid bottle reaches about 800 mL. Centrifuge the waste liquid using a centrifuge. Take the supernatant to obtain the culture medium waste liquid. Among them, the centrifugal force for centrifugation is 3000×g, the rotation speed is 4000 revolutions per minute, and the centrifugation time is 10 min. Then detect the accumulation of lactic acid and ammonium ions in the culture medium waste liquid. If the ammonium ion content in the culture medium waste liquid exceeds 8 mM and / or the lactic acid content exceeds 3 mM, pump the culture medium waste liquid into the anion exchange resin column to adsorb and remove most of the lactic acid. After adjusting the pH, pump the culture medium waste liquid into the cation exchange resin column to adsorb and remove most of the ammonium ions. The culture medium waste liquid after passing through the ion exchange resin column is adjusted to a pH of 6.8 - 7.1 and then filtered through a 0.22 µm filter membrane. The filtrate at this time is the culture medium filtrate C.

[0136] c. Use the culture medium filtrate C or the culture medium waste liquid for perfusion culture According to the method for recycling the waste liquid of cell culture medium in step b, the culture medium filtrate C or the waste liquid of the culture medium is used to replace the EX-CELL® CD CHO serum-free cell culture medium. The culture medium filtrate C or the waste liquid of the culture medium is continuously pumped into the reactor through a feed pump, and the waste liquid of the culture medium is continuously pumped out by a discharge pump through an ATF interception system. The perfusion rate is set to be equal to the recycling rate to ensure the dynamic balance of the total volume of the bioreactor. The discharge pump is connected to a waste liquid bottle. When the perfusion culture is carried out for 24 hours, the volume of the waste liquid collected in the waste liquid bottle reaches about 800 mL. The waste liquid is centrifuged by a centrifuge to obtain the supernatant as the waste liquid of the culture medium. Among them, the centrifugal force for centrifugation is 3000×g, the rotation speed is 4000 revolutions per minute, and the centrifugation time is 10 minutes; then, the accumulation of lactic acid and ammonium ions in the waste liquid of the culture medium is detected. If the lactic acid content in the waste liquid of the culture medium is greater than or equal to 3 mM, the waste liquid of the culture medium is pumped into an anion exchange resin column to adsorb and remove most of the lactic acid and adjust the pH; if the ammonium ion content in the waste liquid of the culture medium is greater than or equal to 8 mM, the waste liquid of the culture medium is pumped into a cation exchange resin column again to adsorb and remove most of the ammonium ions. The waste liquid of the culture medium after passing through the ion exchange resin column is adjusted to a pH of 6.8 - 7.1 and then filtered through a 0.22 µm filter membrane. The filtrate at this time is the culture medium filtrate C.

[0137] Repeat the above steps to perform perfusion culture on the cells using the culture medium filtrate C or the waste liquid of the culture medium. If the previous culture medium filtrate C is used up and the next culture medium filtrate C is not yet processed, fresh EX-CELL® CD CHO serum-free cell culture medium is used for perfusion culture to ensure the continuity of perfusion culture. When the next culture medium filtrate C is processed, the prepared culture medium filtrate C is used to replace the EX-CELL® CD CHO serum-free cell culture medium and continue the perfusion culture.

[0138] After 8 days of perfusion, the viable cell density reaches 0.44×10 7 cells / mL, and then enters the plateau phase. The cell viability begins to decrease significantly. The test results are as Figure 2 shown. The perfusion culture is terminated on the 9th day, and the cells are harvested.

[0139] It can be seen from Figure 2 that during the perfusion culture of CHO-S cells, the use of nutritional supplements in continuous perfusion culture can recycle the waste liquid of the cell culture medium and maintain the normal growth of cells. The maximum viable cell density can exceed 1.9×10 7 cells / mL.

[0140] The cell growth operation results of perfusion using three different culture media are shown in Table 5, Table 5 Cell Growth Operation Results Fresh culture medium Fresh culture medium + cell culture filtrate C Fresh culture medium + recycled culture medium D Maximum viable cell density <![CDATA[1.89×10 7 cells / mL]]> <![CDATA[0.44×10 7 cells / mL]]> <![CDATA[1.96×10 7 cells / mL]]> Example 3 Recycling of MDCK Cell Culture Medium for Perfusion Culture 1. Serum-free Cell Culture Medium Culture Add 800 mL of EX-CELL® MDCK serum-free cell culture medium to a 2 L bioreactor. Set the reactor to a temperature control of 37 °C, a rotation speed of 100 rpm, and turn on the air control system. After the temperature reaches 37 °C and the dissolved oxygen is stable, calibrate the dissolved oxygen. Then quickly inject the seed cells into the bioreactor. Among them, the seed cells are MDCK cells, and the inoculation density is 8×10 5 cells / mL. Turn on the pH setting to 7.1, associate CO2 and use 1 M NaOH to adjust the pH. Set the DO to 40% and associate air. Detect its cell density, cell viability, and biochemical indicators such as glucose, lactate, and ammonium ions in the culture medium every 24 hours. When the viable cell density reaches 10×10 5 cells / mL and the glucose consumption is higher than 30%, prepare to start perfusion culture.

[0141] 2. Recycling of Cell Culture Medium Waste Liquid 2 L bioreactor continuous perfusion culture system: Connect the feed pump and the discharge pump, and start the ATF interception system. Continuously and uniformly pump the EX-CELL® MDCK serum-free cell culture medium into the reactor through the feed pump for 24 hours. At this time, the discharge pump continuously pumps out the culture medium waste liquid through the ATF interception system. Set the perfusion speed and the recovery speed to be equal, both 1 VVD (Vessel Volume per Day), that is, replace 1 culture total volume of the culture medium in one day to ensure the dynamic balance of the total volume of the bioreactor. The discharge pump is connected to a waste liquid bottle. When the perfusion culture is carried out for 24 hours, the volume of the waste liquid collected in the waste liquid bottle reaches about 800 mL. Centrifuge the waste liquid using a centrifuge to obtain the supernatant as the culture medium waste liquid. Among them, the centrifugal separation centrifugal force is 3000×g, the rotation speed is 4000 revolutions per minute, and the centrifugation time is 10 minutes; then detect the accumulation of lactate and ammonium ions in the culture medium waste liquid. Among them, the ammonium ion content in the culture medium waste liquid is 9.1 mM, and the lactate content is 3.3 mM. Pump the culture medium waste liquid into an anion exchange resin column to adsorb and remove most of the lactate; after adjusting the pH, pump the culture medium waste liquid into a cation exchange resin column to adsorb and remove most of the ammonium ions. The culture medium waste liquid after passing through the ion exchange resin column is adjusted to a pH of 7.0 - 7.2 and then filtered through a 0.22 µm filter membrane. The filtrate at this time is the culture medium filtrate E.

[0142] 3. Detection and Analysis of the Main Components of the Culture Medium before and after Culturing Cells and Preparation of the Recycled Cell Culture Medium 1) Take appropriate amounts of the serum-free cell culture medium before culturing and the culture medium filtrate E, and quantitatively detect the main nutrient components such as amino acids, nucleotides, vitamins, and sugars in them by liquid chromatography-mass spectrometry (LC-MS).

[0143] 2) Take appropriate amounts of the serum-free cell culture medium before culturing and the culture medium filtrate E, and quantitatively detect lactic acid and ammonium ions by a biochemical analyzer. The detection data are shown in Table 6. Table 6 Detection Data Amino acid consumption Nucleotide consumption Vitamin consumption Carbohydrate consumption Lactic acid content Ammonium ion content Culture medium filtrate E 50.62% 8.69% 39.54% 39% 1.8 mM 1.5 mM 3) Based on the volume of the culture medium filtrate E, add 20 g / L of non-animal-derived protein hydrolysate to the culture medium filtrate E to obtain the recycled cell culture medium F. The non-animal-derived protein hydrolysate is prepared from the following raw materials in a weight ratio: small molecule peptide type yeast hydrolysate CM01: high glutathione yeast hydrolysate CM07: soybean protein hydrolysate PU041 = 1:1:6.

[0144] 4. Use the recycled cell culture medium F for perfusion culture According to the method for recycling the waste liquid of the cell culture medium in step 2, replace the EX-CELL® MDCK serum-free cell culture medium with the recycled cell culture medium F. Continuously pump the recycled cell culture medium F into the reactor through a feed pump, and continuously pump out the waste liquid of the culture medium through an ATF interception system by a discharge pump. Set the perfusion rate equal to the recycling rate to ensure the dynamic balance of the total volume of the bioreactor. The discharge pump is connected to a waste liquid bottle. When the perfusion culture is carried out for 24 h, the volume of the waste liquid collected in the waste liquid bottle reaches about 800 mL. Centrifuge the waste liquid using a centrifuge to obtain the supernatant as the waste liquid of the culture medium. Among them, the centrifugal force for centrifugation is 3000×g, the rotation speed is 4000 revolutions per minute, and the centrifugation time is 10 min; then detect the accumulation of lactic acid and ammonium ions in the waste liquid of the culture medium. If the lactic acid content in the waste liquid of the culture medium is greater than or equal to 3 mM, pump the waste liquid of the culture medium into an anion exchange resin column to adsorb and remove most of the lactic acid and adjust the pH; if the ammonium ion content in the waste liquid of the culture medium is greater than or equal to 8 mM, then pump the waste liquid of the culture medium into a cation exchange resin column to adsorb and remove most of the ammonium ions. Adjust the pH of the waste liquid of the culture medium after passing through the ion exchange resin column to 6.8 - 7.1, and then filter it through a 0.22 µm filter membrane. The filtrate at this time is the culture medium filtrate E.

[0145] Prepare the recycled cell culture medium F according to the method for detecting and analyzing the main components of the culture medium before and after culturing cells and preparing the recycled cell culture medium in step 3 (wherein, if the ammonium ion content in the waste liquid of the culture medium is less than 8 mM and the lactic acid content is less than 3 mM, directly prepare the recycled cell culture medium with the waste liquid of the culture medium; if the ammonium ion content in the waste liquid of the culture medium is greater than or equal to 8 mM and / or the lactic acid content is greater than or equal to 3 mM, then prepare the recycled cell culture medium with the filtrate).

[0146] Repeat the above steps to perform perfusion culture on the cells using the recycled cell culture medium F. If the previous recycled cell culture medium is used up and the subsequent recycled cell culture medium has not been prepared yet, use fresh EX-CELL® MDCK serum-free cell culture medium for perfusion culture to ensure the continuity of perfusion culture. When the subsequent recycled culture medium is prepared, replace the EX-CELL® MDCK serum-free cell culture medium with the prepared recycled cell culture medium B and continue the perfusion culture.

[0147] After 13 days of perfusion, when the viable cell density exceeds 3×10 7 cells / mL, it enters the plateau phase and the viability begins to decline significantly. The test results are as Figure 3 shown. Perfusion culture is terminated on the 14th day and the cells are harvested.

[0148] As a control, fresh serum-free cell culture medium EX-CELL® MDCK and culture medium filtrate E are used respectively to compare and test cell growth.

[0149] The specific method is as follows: 1) Use fresh serum-free cell culture medium EX-CELL® MDCK for perfusion culture Add 800 mL of EX-CELL® MDCK serum-free cell culture medium to a 2 L bioreactor. Set the reactor to a temperature control of 37 °C, a rotation speed of 100 rpm, and turn on the air control system. After the temperature reaches 37 °C and the dissolved oxygen is stable, calibrate the dissolved oxygen. Then quickly inject the seed cells into the bioreactor. Among them, the seed cells are CHO-K1 cells, and the inoculation density is 8×10 5 cells / mL. Set the pH to 7.1, associate CO2 and use 1 M NaOH to adjust the pH. Set the DO (dissolved oxygen) to 50% and associate air. Detect its cell density, cell viability, and biochemical indexes such as glucose, lactic acid, and ammonium ions in the culture medium every 24 h. When the viable cell density reaches 10×10 5 cells / mL and the pH shows a rapid downward trend and the glucose consumption is higher than 30%, then prepare to start perfusion culture. Connect a feed pump and a discharge pump to the 2 L bioreactor and start the ATF interception system. Continuously and uniformly pump the EX-CELL® CD CHO serum-free cell culture medium into the reactor through the feed pump for 24 h. At this time, the discharge pump continuously pumps out the culture medium waste liquid through the ATF interception system. Set the perfusion speed and the recycling speed to be equal, both 1 VVD (Vessel Volume per Day), that is, replace 1 total culture volume of the medium in one day to ensure the dynamic balance of the total volume of the bioreactor. The discharge pump is connected to a waste liquid bottle.

[0150] After 13 days of perfusion, the viable cell density reached 2.76×10 7 cells / mL, and then entered the plateau phase. The cell viability began to decline significantly. The test results are as Figure 3 shown. Perfusion culture was terminated on the 14th day, and the cells were harvested.

[0151] 2) Use culture medium filtrate E for perfusion culture a. Serum-free cell culture medium culture Add 800 mL of EX-CELL® MDCK serum-free cell culture medium to a 2 L bioreactor. Set the reactor to a temperature control of 37°C, a rotation speed of 100 rpm, and turn on the air control system. After the temperature reaches 37°C and the dissolved oxygen is stable, calibrate the dissolved oxygen. Then quickly inject the seed cells into the bioreactor. Among them, the seed cells are CHO-S cells, and the inoculation density is 8×10 5 cells / mL. Open the pH setting to 7.1, associate CO2 and use 1 M NaOH to adjust the pH. Set the DO to 50% and associate air. Detect its cell density, cell viability, and biochemical indicators such as glucose, lactate, and ammonium ions in the culture medium every 24 h. When the pH shows a rapid downward trend and the glucose consumption is higher than 30%, prepare to start perfusion culture.

[0152] b. Recovery of cell culture medium waste liquid 2 L bioreactor continuous perfusion culture system: Connect the feed pump and the discharge pump, and start the ATF interception system. Continuously and uniformly pump the EX-CELL® MDCK serum-free cell culture medium into the reactor through the feed pump for 24 h. At this time, the discharge pump continuously pumps out the culture medium waste liquid through the ATF interception system. Set the perfusion speed and the recovery speed to be equal, both 1 VVD (Vessel Volume per Day), to ensure the dynamic balance of the total volume of the bioreactor. The discharge pump is connected to the waste liquid bottle. When the perfusion culture lasts for 24 h, the volume of the waste liquid collected in the waste liquid bottle reaches about 800 mL. Centrifuge the waste liquid using a centrifuge. Take the supernatant to obtain the culture medium waste liquid. Among them, the centrifugal force for centrifugation is 3000×g, the rotation speed is 4000 revolutions per minute, and the centrifugation time is 10 min; then detect the accumulation of lactate and ammonium ions in the culture medium waste liquid. If the lactate content in the culture medium waste liquid exceeds 3 mM, pump the culture medium waste liquid into an anion exchange resin column to adsorb and remove most of the lactate and adjust the pH; if the ammonium ion content in the culture medium waste liquid exceeds 8 mM, then pump the culture medium waste liquid into a cation exchange resin column to adsorb and remove most of the ammonium ions. Adjust the pH of the culture medium waste liquid after passing through the ion exchange resin column to 6.8 - 7.1, and then filter it through a 0.22 µm filter membrane. The filtrate at this time is culture medium filtrate E.

[0153] c. Using the culture medium filtrate E or the waste culture medium for perfusion culture According to the method for recycling the waste cell culture medium in step b, use the culture medium filtrate E or the waste culture medium to replace the EX-CELL® MDCK serum-free cell culture medium. Continuously pump the culture medium filtrate E or the waste culture medium into the reactor through a feed pump, and the discharge pump continuously pumps out the waste culture medium through the ATF interception system. Set the perfusion rate equal to the recycling rate to ensure the dynamic balance of the total volume of the bioreactor. The discharge pump is connected to a waste liquid bottle. When the perfusion culture is carried out for 24 h, the volume of the waste liquid collected in the waste liquid bottle reaches about 800 mL. Centrifuge the waste liquid using a centrifuge to obtain the supernatant as the waste culture medium. Among them, the centrifugal force for centrifugation is 3000×g, the rotation speed is 4000 revolutions per minute, and the centrifugation time is 10 min; then detect the accumulation of lactic acid and ammonium ions in the waste culture medium. If the lactic acid content in the waste culture medium is greater than or equal to 3 mM, pump the waste culture medium into an anion exchange resin column to adsorb and remove most of the lactic acid and adjust the pH; if the ammonium ion content in the waste culture medium is greater than or equal to 8 mM, then pump the waste culture medium into a cation exchange resin column again to adsorb and remove most of the ammonium ions. Adjust the pH of the waste culture medium after passing through the ion exchange resin column to 6.8 - 7.1, and then filter it through a 0.22 µm filter membrane. The filtrate at this time is the culture medium filtrate E.

[0154] Repeat the above steps to perform perfusion culture on the cells using the culture medium filtrate E or the waste culture medium. If the previous culture medium filtrate E is used up and the next culture medium filtrate E is not yet processed, use fresh EX-CELL® MDCK serum-free cell culture medium for perfusion culture to ensure the continuity of perfusion culture. When the next culture medium filtrate E is processed, replace the EX-CELL® MDCK serum-free cell culture medium with the prepared culture medium filtrate E and continue the perfusion culture.

[0155] After 8 days of perfusion, the viable cell density reaches 0.46×10 7 cells / mL and enters the plateau phase. The cell viability begins to decline significantly. The test results are as Figure 3 shown. Terminate the perfusion culture on the 9th day and harvest the cells.

[0156] It can be seen from Figure 3 that during the perfusion culture of MDCK cells, the use of nutritional supplements in continuous perfusion culture can recycle the waste cell culture medium and maintain the normal growth of cells. The maximum viable cell density can exceed 3.09×10 7 cells / mL.

[0157] The cell growth operation results of perfusion using three different culture media are shown in Table 7 Table 7 Cell growth operation results Fresh culture medium Fresh culture medium + cell culture filtrate E Fresh culture medium + recycled culture medium F Maximum viable cell density <![CDATA[2.76×10 7 cells / mL]]> <![CDATA[0.46×10 7 cells / mL]]> <![CDATA[3.09×10 7 cells / mL]]> In summary, the present application recycles the waste liquid of the cell culture medium after perfusion, and adds a variety of different types of non-animal-derived protein hydrolysates. The multiple active components synergistically promote cell growth, improve the culture effect of the waste liquid of the cell culture medium, realize the recycling of the waste liquid of the culture medium, and reduce the raw material cost of cell culture.

[0158] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A method for culturing cells by perfusion with spent cell culture medium, characterized in that, The method includes the following steps: (1) Perfusion culture of cells in a bioreactor using a serum-free cell culture medium; (2) Separating the cell culture medium from the bioreactor through a separation system to obtain cell culture medium waste liquid, and retaining the cells in the bioreactor; (3) Adding a non-animal-derived protein hydrolyzate to the cell culture medium waste liquid obtained in step (2) to obtain a recycled cell culture medium; (4) Perfusion culture of cells in a bioreactor using the recycled cell culture medium obtained in step (3); (5) Repeating steps (2)-(4) until the end of the culture, and harvesting the cells; Among them, the non-animal-derived protein hydrolyzate in step (3) includes: high free amino acid yeast hydrolyzate, high glutathione yeast hydrolyzate, and small peptide type yeast hydrolyzate; high glutathione yeast hydrolyzate, small peptide type yeast hydrolyzate, and soy protein hydrolyzate; high free amino acid yeast hydrolyzate, high glutathione yeast hydrolyzate, and soy protein hydrolyzate; or high free amino acid yeast hydrolyzate, high glutathione yeast hydrolyzate, small peptide type yeast hydrolyzate, and soy protein hydrolyzate; Among them, for the high free amino acid yeast hydrolyzate, by weight, its free amino acid content ≥ 50%; For the high glutathione yeast hydrolyzate, by weight, its glutathione content > 10%; For the small peptide type yeast hydrolyzate, the mass percentage of peptide segments with a molecular weight less than or equal to 1000 daltons in the total peptide segments of the small peptide type yeast hydrolyzate is ≥ 96%; For the soy protein hydrolyzate, the mass percentage of peptide segments with a molecular weight greater than 1000 daltons in the total peptide segments of the soy protein hydrolyzate is ≥ 7%.

2. The method according to claim 1, wherein Relative to each liter of cell culture medium waste liquid, the addition amount of the non-animal-derived protein hydrolyzate in step (3) is 1-20 g / L.

3. The method according to claim 1, wherein Relative to each liter of cell culture medium waste liquid, the addition amount of the non-animal-derived protein hydrolyzate in step (3) is 10-20 g / L.

4. The method according to claim 1, wherein By weight, the free amino acid content in the high free amino acid yeast hydrolyzate is 50-65%; the glutathione content in the high glutathione yeast hydrolyzate is 10-20%; the mass percentage of peptide segments with a molecular weight less than or equal to 1000 daltons in the total peptide segments of the small peptide type yeast hydrolyzate is 96-99%; the mass percentage of peptide segments with a molecular weight greater than 1000 daltons in the total peptide segments of the soy protein hydrolyzate is 7-17%.

5. The method according to claim 4, wherein The glutathione content in the high free amino acid yeast hydrolyzate is 0.6-1.5%; and / or, The free amino acid content in the high glutathione yeast hydrolyzate is 8.14-14.21%; and / or, The free amino acid content in the small peptide type yeast hydrolyzate is 20-48%, and the glutathione content is 0.6-2%; and / or, The free amino acid content in the soy protein hydrolyzate is 6.25-13.85%.

6. The method according to claim 1, wherein The non-animal-derived protein hydrolysate includes high glutathione yeast hydrolysate and two or more substances selected from the group consisting of high free amino acid yeast hydrolysate, small molecule peptide type yeast hydrolysate, and soy protein hydrolysate.

7. The method according to claim 6, wherein The weight ratio of the high glutathione yeast hydrolysate to two or more substances selected from the group consisting of high free amino acid yeast hydrolysate, small molecule peptide type yeast hydrolysate, and soy protein hydrolysate is 1-4:4-7.

8. The method according to claim 1, characterized in that, Step (2) further includes a step of detecting the content of nutrient components in the waste liquid of the cell culture medium.

9. The method according to claim 1, characterized in that, Step (2) further includes a step of purifying the waste liquid of the cell culture medium using cation resin and / or anion resin.

10. The method according to any one of claims 1-9, characterized in that, The cell includes mammalian cells.

11. The method according to claim 10, characterized in that, The mammalian cells include one or two of CHO-K1 cells, CHO-S cells, or MDCK cells.

12. The method according to claim 10, characterized in that, The mammalian cell is CHO-K1 cell, and the non-animal-derived protein hydrolysate includes high free amino acid yeast hydrolysate, high glutathione yeast hydrolysate, and small molecule peptide type yeast hydrolysate.

13. The method according to claim 12, characterized in that, The weight ratio of the small molecule peptide type yeast hydrolysate, the high free amino acid yeast hydrolysate, and the high glutathione yeast hydrolysate is 1.5-2.5:1.5-2.5:3.5-4.

5.

14. The method according to claim 10, wherein The mammalian cell is CHO-S, and the non-animal-derived protein hydrolysate includes high free amino acid yeast hydrolysate, high glutathione yeast hydrolysate, and soy protein hydrolysate.

15. The method according to claim 14, characterized in that, The weight ratio of the high free amino acid yeast hydrolysate, the high glutathione yeast hydrolysate, and the soy protein hydrolysate is 0.5-1.5:3.5-4.5:2.5-3.

5.

16. The method according to claim 10, characterized in that, The mammalian cell is MDCK cell, and the non-animal-derived protein hydrolysate includes small molecule peptide type yeast hydrolysate, high glutathione yeast hydrolysate, and soy protein hydrolysate.

17. The method according to claim 16, wherein The weight ratio of the small molecule peptide type yeast hydrolysate, the high glutathione yeast hydrolysate, and the soy protein hydrolysate is 0.5-1.5:0.5-1.5:5.5-6.5.

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