Sugar control method for balancing lactic acid and complete sugar chain antibody production and application thereof

Abstract

The invention provides a sugar control method for balancing lactic acid and complete sugar chain antibody production and application thereof, and the sugar control method comprises the following steps: inoculating CHO1 cells to a production medium, and carrying out first-stage culture D0-D7 and second-stage culture D8-11 in sequence; by controlling glucose deletion time in the second-stage culture process, lactic acid consumption in a cell culture system is promoted, and antibody glycosylation modification deletion is avoided; a control strategy is selected from any one of the following: D8-11 days of single sugar deficiency each day: the residual sugar concentration is maintained at 0 g / L, and the duration time is 3-5 hours; d8-11 days of intermittent sugar deficiency every day: maintaining the residual sugar concentration at 0 g / L for 3-5 hours, supplementing glucose consumption for 2-14 hours, and then maintaining the residual sugar concentration at 0 g / L for 3-5 hours; d8-11 days are lack of sugar once every day, GlcNAc and Mannose are supplemented, the concentration of residual sugar is maintained to be 0 g / L, and the duration time is 5-12 h.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceuticals, especially the field of batch fed culture technology for mammalian cells, and specifically relates to a method for controlling glucose in the production of antibodies that balance lactate and intact glycan chains, and its application. Background Technology

[0002] Chinese hamster ovary (CHO) cells, as a core production platform in the biopharmaceutical industry, have become the gold standard for recombinant protein drug production due to their high protein expression capabilities, suspension culture characteristics, and near-human post-translational modification capabilities. Currently, over 70% of approved therapeutic antibodies and recombinant protein drugs (such as monoclonal antibodies and coagulation factors) rely on CHO cell production.

[0003] In CHO cell culture, lactate is a major byproduct of glycolysis. Early low concentrations of lactate can promote cell proliferation, but as it accumulates (often reaching 10-20 mM), the resulting decrease in culture medium pH inhibits cell growth, reduces product expression, and even alters protein glycosylation patterns. Furthermore, a high lactate environment may suppress mitochondrial function, forcing cells to rely on less efficient glycolysis for energy. To alleviate this problem, industry has used optimized feeding strategies (such as dynamic glucose control) and additives to reduce lactate production. However, the effectiveness of lactate control often varies from clone to clone, lacking a universally applicable control method. Glucose, as the primary carbon source, directly affects cell growth, antibody yield, and quality. Its metabolism not only provides ATP to cells but also generates glycosylation precursors such as UDP-GlcNAc (uridine diphosphate-N-acetylglucosamine), GDP-Mannose (guanosine diphosphate-mannose), and UDP-Glucose (uridine diphosphate-glucose) through the hexosamine pathway. These are key substrates for N-glycan synthesis.

[0004] Studies have shown that prolonged glucose deficiency can affect the synthesis of the N-glycosylated 14-glycan core (GlcNAc2Man9Glc3), thereby impacting the synthesis of complex glycoforms. Furthermore, antibody functions related to N-glycosylation, such as ADCC and CDC, may be affected by glycan deletion.

[0005] Therefore, the problem of missing glycosylation modifications that accompanies the consumption of lactic acid in traditional sugar-deficient processes urgently needs to be addressed. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a method for controlling the production of balanced lactate and intact glycan antibodies, and its application. This invention drives lactate consumption by controlling the amount of glucose added and prevents the depletion of glycosylation precursors (such as UDP-GlcNAc and UDP-Mannose) to avoid loss of glycosylation modification.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a method for controlling glucose in the production of antibodies that balance lactate and intact glycan chains. The method includes: seeding CHO1 cells into a production culture medium and sequentially performing a first-stage culture (D0-D7) and a second-stage culture (D8-11); by controlling the glucose deficiency time during the second-stage culture process, promoting lactate consumption in the cell culture system, and avoiding the loss of antibody glycosylation modification; the method for controlling the glucose deficiency time is selected from any of the following:

[0009] (1) During the cultivation process, on days 8-11, sugar deficiency is performed once a day: maintain the residual sugar concentration at 0 g / L for 3-5 h, for example, 3 h, 4 h or 5 h, etc.

[0010] (2) During the cultivation process, from day 8 to day 11, intermittent glucose deficiency is required every day: maintain the residual glucose concentration at 0 g / L for 3-5 hours, for example, 3 hours, 4 hours or 5 hours, and supplement the glucose consumption for 2-14 hours (for example, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours or 14 hours), and then maintain the residual glucose concentration at 0 g / L for 3-5 hours, for example, 3 hours, 4 hours or 5 hours.

[0011] (3) During the cultivation process, from day 8 to day 11, sugar deficiency is performed once a day and GlcNAc and Mannose are supplemented: the residual sugar concentration is maintained at 0 g / L for 5-12 h, for example, 5 h, 7 h, 9 h, 10 h or 12 h.

[0012] This invention effectively promotes lactate consumption in cell culture systems by controlling the duration of a single glucose deficiency, and avoids the problem of NGHC (glycosylation modification loss), thus avoiding the quality changes of NGHC caused by a single long-term glucose deficiency in the traditional sense.

[0013] This invention effectively avoids changes in NGHC caused by prolonged sugar deficiency by repeatedly controlling sugar deficiency, or by adding precursors of GlcNAc and Mannose, and effectively drives lactic acid consumption.

[0014] Preferably, the calculation method for the amount of GlcNAc and Mannose added is as follows: the sum of the moles of GlcNAc and Mannose added = the theoretical glucose consumption moles during the glucose deficiency period × (0.8-2) × safety factor; the safety factor is 1.1; wherein, the mole ratio of GlcNAc and Mannose is (0.4:0.6)-(0.6:0.4).

[0015] In one specific embodiment, the amount of GlcNAc added is 2.78 mM, and the addition volume = working volume × target concentration (2.78 mM) / mother liquor concentration; the amount of Mannose added is 2.78 mM, and the addition volume = working volume × target concentration (2.78 mM) / mother liquor concentration.

[0016] In this invention, GlcNAc is phosphorylated intracellularly and then reacts with UTP to generate UDP-GlcNAc; Mannose is converted intracellularly to GDP-Mannose. These are among the initial precursors for the N-glycosylation synthesis of the oligosaccharide core structure (GlcNAc2Man9Glc3). In conventional culture, these precursors can be synthesized through the glucose metabolism pathway. In glucose-deficient culture, the addition of GlcNAc and Mannose can rapidly restore the sharply decreased concentrations of UDP-GlcNAc and GDP-Mannose in the endoplasmic reticulum and Golgi apparatus, thereby allowing the interrupted glycan assembly to continue.

[0017] Preferably, the seeding density of the CHO1 cells is (0.40±0.10)×10⁻⁶. 6 cell / mL, for example, could be 0.30 × 10⁻⁶. 6 cell / mL, 0.40×10 6 cell / mL or 0.50×10 6 cell / mL, etc.

[0018] Preferably, the production culture medium comprises ActiPro and 0-5 g / L NaHCO3, for example, it can be 0 g / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L or 5 g / L, etc., preferably ActiPro and 3.3 g / L NaHCO3.

[0019] Preferably, the conditions for the first stage of culture are: an initial culture temperature of 36-36.5℃, and a cell density reaching (9.00-11.00) × 10⁻⁶. 6 The cell / mL temperature was lowered to 32.5-33.0℃ and cultured for the second stage. The conditions for culturing D8-11 were: culture temperature 32.5-33.0℃.

[0020] Preferably, the cooling temperature is 33.0°C.

[0021] Preferably, the first and second stage cultures are fed with a feeding medium, wherein the feeding medium is CB7a and CB7b in a volume ratio of 10:1.

[0022] In one specific embodiment, the feeding strategy is as follows: the volume ratio of the feeding medium supplemented by D3-D4-D5-D6-D7-D8-D9-D10 is 4%-0%-5%-0%-1%-1%-2%-2%.

[0023] Preferably, the sugar supplementation strategy during the first stage of cultivation from D0 to D7 is to maintain the residual sugar concentration at 0.1-10 g / L, for example, 0.1 g / L, 0.5 g / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, or 10 g / L.

[0024] The above-mentioned maintenance of residual sugar concentration of 0.1-10 g / L means that there is no sugar deficiency during culture.

[0025] As a preferred embodiment of the present invention, the method for controlling glucose production by balancing lactic acid and intact glycan antibodies includes:

[0026] CHO1 cells were seeded into the production culture medium at a seeding density of (0.40 ± 0.40) × 10⁻⁶ cells. 6 cell / mL; the production culture medium includes: ActiPro and 0-5 g / L NaHCO3;

[0027] The cells were cultured in two phases sequentially: Phase 1 (D0-D7) and Phase 2 (D8-11). The conditions for Phase 1 culture were: initial culture temperature of 36-36.5℃, and cell density reaching (9.00-11.00) × 10⁻⁶ cells / year. 6 The cell / mL temperature was lowered to 32.5-33.0℃ and cultured for the second stage; the conditions for culturing D8-11 were: culture temperature 32.5-33.0℃;

[0028] In the first stage of culture D0-D7 and the second stage of culture D8-11, supplemental culture medium was used for feeding, and the supplemental culture medium was CB7a and CB7b with a volume ratio of 10:1;

[0029] The sugar replenishment strategy during the first stage of cultivation, from D0 to D7, is to maintain a residual sugar concentration of 0.1-10 g / L.

[0030] The glucose supplementation strategy during the second stage of D8-11 cultivation is selected from any of the following:

[0031] (1) During the cultivation process, from day 8 to day 11, sugar deficiency was performed once a day to maintain the residual sugar concentration at 0 g / L for 3-5 hours.

[0032] (2) During the cultivation process, from day 8 to day 11, intermittent sugar deficiency was carried out daily: the residual sugar concentration was maintained at 0 g / L for 3-5 h, the glucose consumption was supplemented for 2-14 h, and then the residual sugar concentration was maintained at 0 g / L for 3-5 h.

[0033] (3) During the cultivation process, from day 8 to day 11, sugar deficiency was performed once a day and GlcNAc and Mannose were supplemented: the residual sugar concentration was maintained at 0 g / L for 5-12 h. The calculation method for the amount of GlcNAc and Mannose added is: the sum of the moles of GlcNAc and Mannose added = the theoretical glucose consumption moles during the sugar deficiency period × (0.8-2) × safety factor; the safety factor is 1.1; where the mole ratio of GlcNAc and Mannose is (0.4:0.6)-(0.6:0.4).

[0034] In this invention, the conditions for harvesting antibodies are: when the activity rate is below 60%, or when lactate is below 0.5 g / L under a certain condition, or when D11 is reached, whichever occurs first.

[0035] The sugar deficiency strategy adopted in this invention not only has the advantage of universally reducing lactic acid in traditional operations, but also avoids the quality problem of missing glycosylation modification by: (1) single or multiple intermittent short-term (3-5 h) sugar deficiency; (2) long-term sugar deficiency (5-12 h) and the addition of precursor substances.

[0036] This invention solves the problem of missing glycosylation modification that accompanies the forced consumption of lactic acid in traditional sugar-deficient processes; the method of this invention provides a feasible way to promote lactic acid consumption when unexplained lactic acid rise occurs during cell culture, thus ensuring safe production.

[0037] Secondly, the present invention provides the application of the sugar control method for producing antibodies that balance lactate and intact sugar chains as described in the first aspect in the production of glycosylated modified antibodies.

[0038] The numerical range described in this invention includes not only the point values ​​listed above, but also any point values ​​within the numerical ranges not listed above. Due to space limitations and for the sake of brevity, this invention will not exhaustively list all the specific point values ​​included in the range.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] (1) A single glucose deficiency period of 3-5 h effectively promotes lactate consumption in the cell culture system and avoids the problem of glycosylation modification loss (NGHC); it can avoid the NGHC quality changes caused by a single long-term glucose deficiency in the traditional sense.

[0041] (2) Multiple intermittent glucose deficiency control (maintaining residual glucose concentration = 0 g / L for 3-5 h for 2 times and supplementing glucose consumption for 2-14 h between the two times), or maintaining residual glucose concentration = 0 g / L for 5-12 h, and adding GlcNAc and Mannose can effectively avoid NGHC changes caused by long-term glucose deficiency and effectively drive lactic acid consumption. Attached Figure Description

[0042] Figure 1 This is a graph showing the cell growth and metabolic results at different glucose deficiency times.

[0043] Figure 2 These are SDS CE_R spectra after four treatments with different sugar deficiency times.

[0044] Figure 3 This is a diagram showing the cell growth and metabolic results of the strategy to alleviate the lack of glycosylation modification.

[0045] Figure 4 This is the SDS CE_R spectrum after four treatments using the mitigation strategy. Detailed Implementation

[0046] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0047] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0048] The equipment information used in the following specific implementations is shown in Table 1.

[0049] Table 1

[0050]

[0051] The reagent information used in the following specific implementation methods is shown in Table 2.

[0052] Table 2

[0053]

[0054] In the following specific implementation, the Kuhner shaker parameter settings are shown in Table 3.

[0055] Table 3

[0056]

[0057] Example 1

[0058] This embodiment provides a method for controlling glucose production by balancing lactic acid and intact glycan antibody production.

[0059] This study uses the CHO1 cell line (which produces IgG1 monoclonal antibodies) as an example, culturing it in a 250 mL Nice shake flask to produce antibodies.

[0060] CHO1 cells were seeded into the production culture medium at a seeding density of 0.40 × 10⁻⁶ cells / year. 6 The production culture medium comprises ActiPro and 3.3 g / L NaHCO3.

[0061] The cells were cultured in two phases sequentially: Phase 1 (D0-D7) and Phase 2 (D8-11). The conditions for Phase 1 culture were: initial culture temperature of 36.5℃, and culture time until the cell density reached 10 × 10⁻⁶ cells / day. 6 The cell / mL temperature was lowered to 33.0℃ and cultured for the second stage. The culture conditions for D8-11 were: culture temperature 33.0℃.

[0062] In the first stage of culture (D0-D7) and the second stage of culture (D8-11), supplemental feeding medium is used, which is CB7a and CB7b in a volume ratio of 10:1. The feeding strategy is as follows: the volume ratio of supplemental feeding medium added from D3 to D4 to D5 to D6 to D7 to D8 to D9 to D10 is 4%-0%-5%-0%-1%-1%-2%-2%.

[0063] The sugar replenishment strategy during the first stage of cultivation from D0 to D7 is to maintain a residual sugar concentration >0.5 g / L (less than 10 g / L).

[0064] The sugar supplementation strategy during the second stage of cultivation, D8-11, is as follows: during D8-11 of the cultivation process, sugar is deficient once a day to maintain the residual sugar concentration at 0 g / L for 4 hours.

[0065] Antibodies were harvested at day 11 after the culture was completed.

[0066] Example 2

[0067] This embodiment provides a method for controlling glucose production to balance lactate and intact glycan antibody production. The only difference between this embodiment and Embodiment 1 is that the glucose supplementation strategy during the second stage of culture (D8-11) is as follows: During days D8-11 of the culture process, glucose is intermittently deprived daily: the residual glucose concentration is maintained at 0 g / L for 4 hours, glucose consumption is supplemented for 2 hours, and then the residual glucose concentration is maintained at 0 g / L for another 4 hours.

[0068] Example 3

[0069] This embodiment provides a method for controlling glucose production to balance lactate and intact glycan antibody production. The only difference between this embodiment and Embodiment 1 is that the glucose supplementation strategy during the second stage of culture (D8-11) is as follows: during days D8-11 of the culture process, glucose is deprived once a day and supplemented with GlcNAc and Mannose to maintain the residual glucose concentration at 0 g / L for 8 hours; the amount of GlcNAc added is 2.78 mM; the amount of Mannose added is 2.78 mM.

[0070] Example 4

[0071] This embodiment investigates the effect of different sugar deficiency times on glycosylation modification.

[0072] This study uses the CHO1 cell line (which produces IgG1 monoclonal antibodies) as an example, culturing it in a 250 mL Nice shake flask to produce antibodies.

[0073] 1. Experimental steps.

[0074] CHO1 cells were seeded into the production culture medium at a seeding density of 0.40 × 10⁻⁶ cells / year. 6 The production culture medium comprises ActiPro and 3.3 g / L NaHCO3.

[0075] The cells were cultured in two phases sequentially: Phase 1 (D0-D7) and Phase 2 (D8-11). The conditions for Phase 1 culture were: initial culture temperature of 36.5℃, and culture time until the cell density reached 10 × 10⁻⁶ cells / day. 6 The cell / mL temperature was lowered to 33.0℃ and cultured for the second stage. The culture conditions for D8-11 were: culture temperature 33.0℃.

[0076] In the first stage of culture (D0-D7) and the second stage of culture (D8-11), supplemental feeding medium is used, which is CB7a and CB7b in a volume ratio of 10:1. The feeding strategy is as follows: the volume ratio of supplemental feeding medium added from D3 to D4 to D5 to D6 to D7 to D8 to D9 to D10 is 4%-0%-5%-0%-1%-1%-2%-2%.

[0077] The sugar replenishment strategy during the first stage of cultivation from D0 to D7 is to maintain a residual sugar concentration >0.5 g / L (less than 10 g / L).

[0078] The glucose supplementation strategy during the second stage of D8-11 cultivation is as follows:

[0079] SF01: During days 8-11 of the culture process, maintain a residual sugar concentration >0.5 g / L daily.

[0080] SF02: During days 8-11 of the culture process, maintain a residual sugar concentration of 0 g / L for 4 hours each day.

[0081] SF03: During days 8-11 of the culture process, maintain a residual sugar concentration of 0 g / L for 8 hours each day.

[0082] SF04: During days 8-11 of the culture process, maintain a residual sugar concentration of 0 g / L for 12 hours each day.

[0083] After culture, antibodies were harvested at day 11, and the loss of N-glycosylation modification and yield of the antibodies were detected. The method for detecting the loss of N-glycosylation modification was SDS-CEL, and the method for detecting protein yield was Cedex titrator.

[0084] 2. Experimental results.

[0085] Figure 1 The graph shows the cell growth and metabolic results at different glucose deficiency times.

[0086] from Figure 1 It was found that under glucose deficiency conditions of 0, 4, 8, and 12 hours / day, lactic acid was consumed more rapidly with longer durations of glucose deficiency. Meanwhile, cell growth, viability, and yield were comparable across the four experimental conditions, showing no significant impact from glucose deficiency. Therefore, inhibiting lactic acid growth through glucose deficiency is feasible.

[0087] Figure 2 SDS-CER spectra after four treatments with different glucose deficiency times. Figure 2 As can be seen, the SDS CE_R method can effectively separate heavy chains, light chains, and unglycosylated heavy chains (NGHC), and the target peak 2 (NGHC) of different samples is also clearly distinguishable. See Table 4 for specific peak values.

[0088] Table 4 shows the Cedex_titer and SDS CE_R detection results for D7, D9, and D11 in experiments SF01-SF04.

[0089] Table 4

[0090]

[0091] In Table 4, NGHC represents Non-glycosylation heavy chain.

[0092] As shown in Table 4, the proportion of N-glycosylation modification missing in the shake flasks without sugar deficiency treatment remained at 1.0 ± 0.1 g / L. The longer the duration of a single sugar deficiency treatment (SF02 vs. SF03 vs. SF04), the higher the proportion of N-glycosylation modification missing.

[0093] Example 5

[0094] This embodiment investigates the mitigation of glycosylation modification deficiency.

[0095] This study uses the CHO1 cell line (which produces IgG1 monoclonal antibodies) as an example, culturing it in a 250 mL Nice shake flask to produce antibodies.

[0096] 1. Experimental steps.

[0097] CHO1 cells were seeded into the production culture medium at a seeding density of 0.40 × 10⁻⁶ cells / year. 6 The production culture medium comprises ActiPro and 3.3 g / L NaHCO3.

[0098] The cells were cultured in two phases sequentially: Phase 1 (D0-D7) and Phase 2 (D8-11). The conditions for Phase 1 culture were: initial culture temperature of 36.5℃, and culture time until the cell density reached 10 × 10⁻⁶ cells / day. 6 The cell / mL temperature was lowered to 33.0℃ and cultured for the second stage. The culture conditions for D8-11 were: culture temperature 33.0℃.

[0099] In the first stage of culture (D0-D7) and the second stage of culture (D8-11), supplemental feeding medium is used, which is CB7a and CB7b in a volume ratio of 10:1. The feeding strategy is as follows: the volume ratio of supplemental feeding medium added from D3 to D4 to D5 to D6 to D7 to D8 to D9 to D10 is 4%-0%-5%-0%-1%-1%-2%-2%.

[0100] The sugar replenishment strategy during the first stage of cultivation from D0 to D7 is to maintain a residual sugar concentration >0.5 g / L (less than 10 g / L).

[0101] The glucose supplementation strategy during the second stage of D8-11 cultivation is as follows:

[0102] SF05: During days 8-11 of the culture process, maintain a residual sugar concentration >0.5 g / L daily.

[0103] SF06: During days 8-11 of the culture process, maintain a residual sugar concentration of 0 g / L for 4 hours each day.

[0104] SF07: During days D8-11 of the culture process, intermittent glucose deficiency was performed daily: the residual glucose concentration was maintained at 0 g / L for 4 hours, glucose consumption was replenished for 2 hours, and then the residual glucose concentration was maintained at 0 g / L for 4 hours.

[0105] SF08: During the cultivation process, from day 8 to day 11, maintain a residual sugar concentration of 0 g / L for 8 hours each day.

[0106] SF09: During days 8-11 of the culture process, glucose was deprived once a day and supplemented with GlcNAc and Mannose: the residual glucose concentration was maintained at 0 g / L for 8 h; the amount of GlcNAc added was 2.78 mM; the amount of Mannose added was 2.78 mM.

[0107] After culture, antibodies were harvested at day 11, and the loss of N-glycosylation modification and yield of the antibodies were detected. The method for detecting the loss of N-glycosylation modification was SDS-CEL, and the method for detecting protein yield was Cedex titrator.

[0108] 2. Experimental results.

[0109] Figure 3 Figure showing cell growth and metabolic results for strategies to mitigate the loss of glycosylation modification.

[0110] from Figure 3 It was found that under a single 4-hour / day glucose deficiency condition, lactate accumulation in SF06 cells showed a decreasing trend compared to the control group SF05 cells without glucose deficiency. The lactate reduction level in shake-flask SF07 cells subjected to two short-term (4-hour / day) glucose deficiency treatments was comparable to that under a single 8-hour / day glucose deficiency condition. The experimental group SF09 cells supplemented with GlcNAc and Mannose showed a significant decrease in lactate. Meanwhile, cell growth, viability, and yield were comparable across the five experimental conditions. Therefore, all three methods—short-term glucose deficiency, multiple intermittent glucose deficiency, or long-term glucose deficiency with the addition of GlcNAc and Mannose—can reduce lactate accumulation.

[0111] Figure 4 The SDS CE_R spectrum after four treatments using the mitigation strategy. From... Figure 4 As can be seen, the SDS CE_R method can effectively separate heavy chains, light chains, and unglycosylated heavy chains (NGHC), and the target peak 2 (NGHC) of different samples is also clearly distinguishable. See Table 5 for specific peak values.

[0112] Table 5 shows the Cedex_titer and SDS CE_R detection results for D7, D9, and D11 in experiments SF05-SF09.

[0113] Table 5

[0114]

[0115] In Table 5, NGHC represents Non-glycosylation heavy chain.

[0116] Table 5 shows that after four experimental conditions, the increase in NGHC ratio in shake flask SF06 under short-term glucose deprivation was extremely low. Shake flask SF07, which underwent multiple short-term glucose deprivation treatments, showed a much lower proportion of N-glycosylation modification loss compared to a single 8 h / day treatment (SF08). During 8 h / day glucose deprivation: adding GlcNAc and Mannose during the glucose deprivation period effectively and completely prevented the generation of antibodies with additional glycosylation modification loss (SF09).

[0117] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A method for controlling glucose production by balancing lactic acid and intact glycan antibodies, characterized in that, The glucose control method includes: seeding CHO1 cells into a production medium and sequentially performing a first-stage culture (D0-D7) and a second-stage culture (D8-11); by controlling the glucose deficiency time during the second-stage culture process, lactate consumption in the cell culture system is promoted, thus avoiding the loss of antibody glycosylation modification; the method for controlling the glucose deficiency time is selected from any of the following: (1) During the cultivation process, from day 8 to day 11, sugar deficiency was performed once a day to maintain the residual sugar concentration at 0 g / L for 3-5 hours. (2) During the cultivation process, from day 8 to day 11, intermittent sugar deficiency was carried out daily: the residual sugar concentration was maintained at 0 g / L for 3-5 h, the glucose consumption was supplemented for 2-14 h, and then the residual sugar concentration was maintained at 0 g / L for 3-5 h. (3) During the cultivation process, from day 8 to day 11, sugar deficiency was performed once a day and supplemented with GlcNAc and Mannose: the residual sugar concentration was maintained at 0 g / L for 5-12 h.

2. The method for controlling glucose production by balancing lactic acid and intact glycan antibody according to claim 1, characterized in that, The calculation method for the amount of GlcNAc and Mannose added is as follows: the sum of the moles of GlcNAc and Mannose added = the theoretical glucose consumption moles during the glucose deficiency period × (0.8-2) × safety factor; the safety factor is 1.1; wherein, the mole ratio of GlcNAc and Mannose is (0.4:0.6)-(0.6:0.4).

3. The method for controlling glucose production by balancing lactate and intact glycan antibody according to claim 1 or 2, characterized in that, The seeding density of the CHO1 cells was (0.40±0.10)×10⁻⁶. 6 cell / mL.

4. The method for controlling glucose production of antibodies that balance lactate and intact glycans according to any one of claims 1-3, characterized in that, The production culture medium includes ActiPro and 0-5 g / L NaHCO3.

5. The method for controlling glucose production of antibodies that balance lactate and intact glycans according to any one of claims 1-4, characterized in that, The conditions for the first stage of culture were: an initial culture temperature of 36-36.5℃, and a cell density of (9.00-11.00) × 10⁻⁶. 6 The cell / mL temperature was lowered to 32.5-33.0℃ and cultured for the second stage. The conditions for culturing D8-11 were: culture temperature 32.5-33.0℃.

6. The method for controlling glucose production by balancing lactic acid and intact glycan antibody according to claim 5, characterized in that, The cooling temperature is 33.0℃.

7. The method for controlling glucose production of antibodies that balance lactic acid and intact glycans according to any one of claims 1-6, characterized in that, In the first and second stage cultures, feed medium is used for feeding, and the feed medium is CB7a and CB7b with a volume ratio of 10:

1.

8. The method for controlling glucose production of antibodies that balance lactate and intact glycans according to any one of claims 1-7, characterized in that, The sugar replenishment strategy during the first stage of cultivation (D0-D7) is to maintain the residual sugar concentration at 0.1-10 g / L.

9. The method for controlling glucose production of antibodies that balance lactate and intact glycans according to any one of claims 1-8, characterized in that, The blood sugar control method includes: CHO1 cells were seeded into the production medium at a density of (0.40±0.10)×10⁻⁶ cells / mL. 6 cell / ml; the production culture medium includes: ActiPro and 3.3 g / L NaHCO3; The cells were cultured in two phases sequentially: Phase 1 (D0-D7) and Phase 2 (D8-11). The conditions for Phase 1 culture were: initial culture temperature of 36-36.5℃, and cell density reaching (9.00-11.00) × 10⁻⁶ cells / year. 6 The cell / mL temperature was lowered to 32.5-33.0℃ and cultured for the second stage; the conditions for culturing D8-11 were: culture temperature 32.5-33.0℃; In the first stage of culture D0-D7 and the second stage of culture D8-11, supplemental culture medium was used for feeding, and the supplemental culture medium was CB7a and CB7b with a volume ratio of 10:1; The sugar replenishment strategy during the first stage of cultivation, from D0 to D7, is to maintain a residual sugar concentration of 0.1-10 g / L. The glucose supplementation strategy during the second stage of D8-11 cultivation is selected from any of the following: (1) During the cultivation process, from day 8 to day 11, sugar deficiency was performed once a day to maintain the residual sugar concentration at 0 g / L for 3-5 hours. (2) During the cultivation process, from day 8 to day 11, intermittent sugar deficiency was carried out daily: the residual sugar concentration was maintained at 0 g / L for 3-5 h, the glucose consumption was supplemented for 2-14 h, and then the residual sugar concentration was maintained at 0 g / L for 3-5 h. (3) During the cultivation process, from day 8 to day 11, sugar deficiency was performed once a day and GlcNAc and Mannose were supplemented: the residual sugar concentration was maintained at 0 g / L for 5-12 h. The calculation method for the amount of GlcNAc and Mannose added is: the sum of the moles of GlcNAc and Mannose added = the number of moles of theoretical glucose consumption during the sugar deficiency period × (0.8-2); where the ratio of the moles of GlcNAc and Mannose is (0.4:0.6)-(0.6:0.4).

10. The method for controlling the production of antibodies by balancing lactate and intact glycans according to any one of claims 1-9 is used in the production of glycosylated modified antibodies.

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