CN112608906B - 一种cho细胞组合糖类补料培养基 - Google Patents
一种cho细胞组合糖类补料培养基 Download PDFInfo
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Abstract
一种用于CHO细胞培养的组合糖类补料培养基以及鉴于温度变化CHO细胞的培养基添加方法,培养过程中采用补料培养基在不同培养温度节点添加,温度节点依次为30℃‑34℃‑37℃‑34℃‑32℃,补料培养基1‑5的糖类浓度和组合经过优化选择,所述方法可有效提高外源蛋白的提高。
Description
技术领域
本发明涉及生物技术领域,具体属于CHO细胞培养基领域。
背景技术
中国仓鼠卵巢细胞(CHO)已被广泛的用于治疗性抗体的商业生产,随着对治疗性抗体需求的不断激增,CHO细胞使用的普遍性会持续存在。通过动物细胞培养产生基因工程外源蛋白是获得治疗目的的单克隆抗体的常用方法,但是由于其表达量低于原核和酵母表达***,因此提高外源基因在CHO细胞中的表达效率十分重要。优化培养基是提高细胞活性,激活表达通路的重要方式。
当前针对 CHO 细胞无血清培养基的研究,主要方法是在基础培养基如 DMEM/F12的基础上,添加蛋白水解物,胰岛素、转铁蛋白、生长因子、维生素、脂类、微量元素等。现有技术中已有多种培养基优化的研究基础,以优化培养基组分、添加剂、补料、浓度等为几个主要研究方向,其中,培养温度变化是提高外源蛋白表达的重要方式,US20170356022A1发现在指数生长极端之后将生产培养物冷却到30-33℃的温度T生产,低于细胞系通常培养的温度,可以提高目的蛋白的稳定性;CN107974432A,发现在培养CHO细胞株表达猪瘟E2蛋白时,可在逐级放大培养过程中,初期采用常规温度,恒定期后开始温度和pH下调,可提高蛋白产率;VN10008813000B发现在培养液达到最高细胞密度的20-80%时降低温度,可降低CHO细胞培养液中乳酸和铵的浓度;US5,705,364描述了在链烷酸存在下控制温度和渗透压控制哺乳动物细胞CHO中产生的糖蛋白中唾液酸含量的方法;US20030190710A1提供了一种使用CHO细胞表达单克隆抗体中利用温度和渗透压变化控制IgG非糖基化重链NGHC的方法。然而上述现有技术中,均没有体现温度变化和糖浓度的关系,现有技术中对于糖浓度变化主要在于营养补充剂中一个简单的提高性补偿,容易产生较多副产物不利于细胞状态。基于温度变化节点与不同糖类组合的关联性尚未见有关报道。
发明内容
根据现有技术存在的问题,发明人意在提供一种随温度变化在不同节点添加不同糖类的培养基和添加方法。
对于糖的选择,现有技术中一般而言以葡萄糖为主,偶有采用其他糖类,但也没有与表达量变化产生明显关联,而温度变化也仅限于一个生产温度和细胞培养温度的两类温度的转变关系。然而,发明人意外发现温度变化与不同的糖浓度变化存在关联性,由于不同种类的糖可参与外源蛋白表达通路的激活,这种关联性可以映射到外源蛋白表达量的提高,尤其是提高抗体的浓度以及提高其活性,并提高蛋白稳定性。培养温度随着CHO细胞培养时间的推移,按一定速率进行阶段性升温,以及一定速率阶段性下降,配合不同糖类的添加,是本发明的主要构思。
将现有技术中已知的糖根据分子大小进行分类:
单糖:葡萄糖,半乳糖,核糖,果糖,木糖
二糖:麦芽糖,麦芽酮糖,异麦芽糖,甘露二糖,纤维二糖,黑曲霉二糖
多糖:葡聚糖,纤维素,淀粉,糖原,支链淀粉,凝胶多糖,香菇多糖,酵母多糖,右旋糖酐。
培养温度对于细胞的生长有显著的影响,较低的培养温度不利于细胞增殖,细胞的比生长速度随着培养温度的降低而降低,特别是低于30℃时细胞生长受到较大的抑制,然而较低的培养温度对细胞维持和外源蛋白的稳定性维持有利,也能够明显延长培养周期。因此发明人根据现有技术选择CHO细胞培养温度范围:30℃-37℃,并意外发现随着温度的一定速度的阶段性提升后再进行阶段性降低维持,对于外源蛋白表达有着明显的影响,并且,伴随每个温度变化节点添加糖类组合,可达到协同增效的作用。
根据温度变化阶段需要,设计补料1-5五套营养补料培养基,分别于特定的温度节点变化时添加,用于表达外源蛋白的工程化载体转染后进行营养补料。
通过大量基于响应面、全因子实验、Box-Behnken等多因素实验设计,获得培养基补料最优温度、添加时间变化模趋势如表1所示,其中从30℃提升到33℃的提升方式为:30℃12h,31℃12h,32℃12h,33℃12h;从34℃提升到36℃的提升方式为:34℃ 12h,35℃ 10h,36℃ 10h;从37℃降低到35℃的下降方式为:37℃ 13h,36℃ 13h,35℃ 14h;从34℃降低到33℃的下降方式为:34℃ 10h,33℃ 10h。
表1 补料培养基添加
培养基 | 添加时培养温度调整 | 添加时间 | 培养时段 |
补料1 | 30℃ | 0-48h | 启动期 |
补料2 | 34℃ | 48-80h | 对数增长期 |
补料3 | 37℃ | 80-120h | 对数增长期 |
补料4 | 34℃ | 120h-140h | 恒定期 |
补料5 | 32℃ | 140h | 代谢产物积累期 |
根据单糖、二糖、多糖中多种糖类进行组合,以外源蛋白表达量、抗体活性等为筛选标准,获得糖类以温度为变化标准的补料培养基成份。
表2 葡萄糖组合补料培养基(单位mg/L)
补料1 | 补料2 | 补料3 | 补料4 | 补料5 | |
对比例1 | 30℃ | 34℃ | 37℃ | 34℃ | 32℃ |
葡萄糖 | 1000 | 3000 | 8000 | 3000 | 2000 |
半乳糖 | 200 | 500 | 3000 | 800 | 600 |
麦芽糖 | 500 | 1000 | 2000 | 1600 | 1500 |
甘露二糖 | 500 | 500 | 500 | 800 | 1000 |
葡聚糖 | 1500 | 1800 | 2000 | 2000 | 2000 |
淀粉 | 800 | 700 | 600 | 500 | 400 |
对比例2 | |||||
葡萄糖 | 1000 | 3000 | 8000 | 3000 | 2000 |
核糖 | 200 | 500 | 1000 | 800 | 800 |
麦芽糖 | 500 | 1000 | 2000 | 1600 | 1500 |
纤维二糖 | 200 | 300 | 400 | 300 | 200 |
葡聚糖 | 1500 | 1800 | 2000 | 2000 | 2000 |
纤维素 | 500 | 500 | 400 | 400 | 300 |
对比例3 | |||||
半乳糖 | 200 | 500 | 3000 | 800 | 600 |
果糖 | 2000 | 1800 | 1600 | 1500 | 1400 |
麦芽酮糖 | 100 | 400 | 600 | 500 | 500 |
甘露二糖 | 500 | 500 | 500 | 800 | 1000 |
纤维素 | 500 | 500 | 400 | 400 | 300 |
糖原 | 2000 | 1800 | 1600 | 1500 | 1400 |
对比例4 | |||||
核糖 | 200 | 500 | 1000 | 800 | 800 |
果糖 | 2000 | 1800 | 1600 | 1500 | 1400 |
异麦芽糖 | 1000 | 1000 | 1000 | 800 | 600 |
纤维二糖 | 200 | 300 | 400 | 300 | 200 |
支链淀粉 | 700 | 800 | 900 | 800 | 700 |
纤维素 | 500 | 500 | 400 | 400 | 300 |
对比例5 | |||||
果糖 | 2000 | 1800 | 1600 | 1500 | 1400 |
木糖 | 1000 | 2000 | 3000 | 4000 | 3000 |
麦芽酮糖 | 100 | 400 | 600 | 500 | 500 |
纤维二糖 | 200 | 300 | 400 | 300 | 200 |
葡聚糖 | 1500 | 1800 | 2000 | 2000 | 2000 |
凝胶多糖 | 100 | 200 | 300 | 400 | 500 |
(注:发明人还设计了大量的多组糖类组合实验,鉴于篇幅限制,在此舍弃了部分获得外源蛋白含量没有明显变化以及甚至有外源蛋白表达相比对照组有所下降的组,上述对比例1-5为效果最优的五组)。
进一步的,培养基中还包含其他成分。
表3 组合培养基其他成分(单位mg/L)
补料1 | 补料2 | 补料3 | 补料4 | 补料5 | |
亚麻酸 | 0.04-0.12 | 0.04-0.12 | 0.04-0.12 | 0.04-0.12 | 0.04-0.12 |
亚油酸 | 0.08-0.27 | 0.08-0.27 | 0.08-0.27 | 0.08-0.27 | 0.08-0.27 |
胆固醇 | 1.5-7.5 | 1.5-7.5 | 1.5-7.5 | 1.5-7.5 | 1.5-7.5 |
花生四烯酸 | 0.001-0.007 | 0.001-0.007 | 0.001-0.007 | 0.001-0.007 | 0.001-0.007 |
孕酮 | 0.02-0.15 | 0.02-0.15 | 0.02-0.15 | 0.02-0.15 | 0.02-0.15 |
棕榈酸 | 0.15-0.30 | 0.15-0.30 | 0.15-0.30 | 0.15-0.30 | 0.15-0.30 |
胰岛素 | 0.01-0.05 | 0.01-0.05 | 0.01-0.05 | 0.01-0.05 | 0.01-0.05 |
甘氨酸 | 28-84 | 28-84 | 28-84 | 28-84 | 28-84 |
L-异亮氨酸 | 80-250 | 80-250 | 80-250 | 80-250 | 80-250 |
L-亮氨酸 | 85-270 | 85-270 | 85-270 | 85-270 | 85-270 |
L-组氨酸 | 45-135 | 45-135 | 45-135 | 45-135 | 45-135 |
L-赖氨酸 | 135-400 | 135-400 | 135-400 | 135-400 | 135-400 |
L-蛋氨酸 | 35-115 | 35-115 | 35-115 | 35-115 | 35-115 |
L-苯丙氨酸 | 60-150 | 60-150 | 60-150 | 60-150 | 60-150 |
L-苏氨酸 | 100-200 | 100-200 | 100-200 | 100-200 | 100-200 |
L-色氨酸 | 30-90 | 30-90 | 30-90 | 30-90 | 30-90 |
L-缬氨酸 | 80-220 | 80-220 | 80-220 | 80-220 | 80-220 |
L-丙氨酸 | 6.75-27.5 | 6.75-27.5 | 6.75-27.5 | 6.75-27.5 | 6.75-27.5 |
L-谷氨酰胺 | 800-3200 | 800-3200 | 800-3200 | 800-3200 | 800-3200 |
L-精氨酸 | 420-850 | 420-850 | 420-850 | 420-850 | 420-850 |
L-天冬氨酸 | 20-80 | 20-80 | 20-80 | 20-80 | 20-80 |
L-天冬酰胺 | 100-450 | 100-450 | 100-450 | 100-450 | 100-450 |
L-半胱氨酸 | 20-75 | 20-75 | 20-75 | 20-75 | 20-75 |
L-胱氨酸 | 45-150 | 45-150 | 45-150 | 45-150 | 45-150 |
L-谷氨酸 | 4-45 | 4-45 | 4-45 | 4-45 | 4-45 |
L-脯氨酸 | 30-150 | 30-150 | 30-150 | 30-150 | 30-150 |
L-丝氨酸 | 50-150 | 50-150 | 50-150 | 50-150 | 50-150 |
L-酪氨酸 | 80-240 | 80-240 | 80-240 | 80-240 | 80-240 |
叶酸 | 8-25 | 8-25 | 8-25 | 8-25 | 8-25 |
维生素A | 0.8-2 | 0.8-2 | 0.8-2 | 0.8-2 | 0.8-2 |
维生素C | 1-4 | 1-4 | 1-4 | 1-4 | 1-4 |
维生素B1 | 0.5-3 | 0.5-3 | 0.5-3 | 0.5-3 | 0.5-3 |
维生素B2 | 0.3-0.8 | 0.3-0.8 | 0.3-0.8 | 0.3-0.8 | 0.3-0.8 |
维生素B6 | 0.5-3 | 0.5-3 | 0.5-3 | 0.5-3 | 0.5-3 |
维生素B12 | 0.45-1.35 | 0.45-1.35 | 0.45-1.35 | 0.45-1.35 | 0.45-1.35 |
维生素K | 5-12 | 5-12 | 5-12 | 5-12 | 5-12 |
维生素E | 2.5-7.5 | 2.5-7.5 | 2.5-7.5 | 2.5-7.5 | 2.5-7.5 |
维生素H | 0.005-0.4 | 0.005-0.4 | 0.005-0.4 | 0.005-0.4 | 0.005-0.4 |
硫辛酸 | 0.2-0.5 | 0.2-0.5 | 0.2-0.5 | 0.2-0.5 | 0.2-0.5 |
D-泛酸钙 | 5-30 | 5-30 | 5-30 | 5-30 | 5-30 |
烟酰胺 | 5-28 | 5-28 | 5-28 | 5-28 | 5-28 |
NH4VO3 | 0-0.02 | 0-0.02 | 0-0.02 | 0-0.02 | 0-0.02 |
Na2SeO3 | 0.01-0.07 | 0.01-0.07 | 0.01-0.07 | 0.01-0.07 | 0.01-0.07 |
MnCl2·4H2O | 0.06-0.1 | 0.06-0.1 | 0.06-0.1 | 0.06-0.1 | 0.06-0.1 |
CuSO4·5H2O | 0.08-0.18 | 0.08-0.18 | 0.08-0.18 | 0.08-0.18 | 0.08-0.18 |
ZnCl2 | 0.3-1.0 | 0.3-1.0 | 0.3-1.0 | 0.3-1.0 | 0.3-1.0 |
MgCl2 | 0.2-0.5 | 0.2-0.5 | 0.2-0.5 | 0.2-0.5 | 0.2-0.5 |
K2SO4 | 10-25 | 10-25 | 10-25 | 10-25 | 10-25 |
AlCl3 | 0.01-0.05 | 0.01-0.05 | 0.01-0.05 | 0.01-0.05 | 0.01-0.05 |
CuSO4·5H2O | 0.001-0.005 | 0.001-0.005 | 0.001-0.005 | 0.001-0.005 | 0.001-0.005 |
NaCl | 0.1-0.3 | 0.1-0.3 | 0.1-0.3 | 0.1-0.3 | 0.1-0.3 |
NaHCO3 | 0.02-0.08 | 0.02-0.08 | 0.02-0.08 | 0.02-0.08 | 0.02-0.08 |
CaCl2 | 0.1-0.5 | 0.1-0.5 | 0.1-0.5 | 0.1-0.5 | 0.1-0.5 |
有益效果
发明人意外发现在CHO细胞培养表达外源蛋白过程中,相较于现有技术的同类培养基的补料,使用分批次不同成分的补料培养基,配合一定的培养时间和培养温度变化、不同温度节点添加不同的糖类组合,可意外的提高外源蛋白表达效率,尤其是增强抗体表达的产量与活性,并且可维持较好的蛋白稳定性,可起到协同增效的效果。
附图说明
图1 CHO活细胞密度培养变化曲线。
图2 细胞形态学照片。
图3 不同补料培养基的PD1抗体表达量比较。
图4 不同补料培养基的VEGF抗体表达量比较。
图5 不同补料培养基的AGR2抗体表达量比较。
图6 不同补料培养基的狂犬病毒抗体表达量比较。
具体实施方式
实施例1 培养基配置组合梯度培养基-高浓度培养基-市售培养基配置
按表格比例配置10个对比例的培养基,以去离子水补齐培养基体积,并过滤除菌。
表4-1 对比例1(单位mg/L)
补料1 | 补料2 | 补料3 | 补料4 | 补料5 | |
30℃ | 34℃ | 37℃ | 34℃ | 32℃ | |
葡萄糖 | 1000 | 3000 | 8000 | 3000 | 2000 |
半乳糖 | 200 | 500 | 3000 | 800 | 600 |
麦芽糖 | 500 | 1000 | 2000 | 1600 | 1500 |
甘露二糖 | 500 | 500 | 500 | 800 | 1000 |
葡聚糖 | 1500 | 1800 | 2000 | 2000 | 2000 |
淀粉 | 800 | 700 | 600 | 500 | 400 |
亚麻酸 | 0.08 | 0.08 | 0.08 | 0.08 | 0.08 |
亚油酸 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 |
胆固醇 | 1.8 | 1.8 | 1.8 | 1.8 | 1.8 |
花生四烯酸 | 0.005 | 0.005 | 0.005 | 0.005 | 0.005 |
孕酮 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 |
棕榈酸 | 0.18 | 0.18 | 0.18 | 0.18 | 0.18 |
胰岛素 | 0.03 | 0.03 | 0.03 | 0.03 | 0.03 |
甘氨酸 | 50 | 50 | 50 | 50 | 50 |
L-异亮氨酸 | 120 | 120 | 120 | 120 | 120 |
L-亮氨酸 | 130 | 130 | 130 | 130 | 130 |
L-组氨酸 | 78 | 78 | 78 | 78 | 78 |
L-赖氨酸 | 256 | 256 | 256 | 256 | 256 |
L-蛋氨酸 | 108 | 108 | 108 | 108 | 108 |
L-苯丙氨酸 | 68 | 68 | 68 | 68 | 68 |
L-苏氨酸 | 123 | 123 | 123 | 123 | 123 |
L-色氨酸 | 45 | 45 | 45 | 45 | 45 |
L-缬氨酸 | 160 | 160 | 160 | 160 | 160 |
L-丙氨酸 | 20.5 | 20.5 | 20.5 | 20.5 | 20.5 |
L-谷氨酰胺 | 1600 | 1600 | 1600 | 1600 | 1600 |
L-精氨酸 | 560 | 560 | 560 | 560 | 560 |
L-天冬氨酸 | 56 | 56 | 56 | 56 | 56 |
L-天冬酰胺 | 230 | 230 | 230 | 230 | 230 |
L-半胱氨酸 | 48 | 48 | 48 | 48 | 48 |
L-胱氨酸 | 99 | 99 | 99 | 99 | 99 |
L-谷氨酸 | 32 | 32 | 32 | 32 | 32 |
L-脯氨酸 | 78 | 78 | 78 | 78 | 78 |
L-丝氨酸 | 95 | 95 | 95 | 95 | 95 |
L-酪氨酸 | 224 | 224 | 224 | 224 | 224 |
叶酸 | 12 | 12 | 12 | 12 | 12 |
维生素A | 1.5 | 1.5 | 1.5 | 1.5 | 1.5 |
维生素C | 3 | 3 | 3 | 3 | 3 |
维生素B1 | 2.5 | 2.5 | 2.5 | 2.5 | 2.5 |
维生素B2 | 0.59 | 0.59 | 0.59 | 0.59 | 0.59 |
维生素B6 | 2.58 | 2.58 | 2.58 | 2.58 | 2.58 |
维生素B12 | 1.06 | 1.06 | 1.06 | 1.06 | 1.06 |
维生素K | 11 | 11 | 11 | 11 | 11 |
维生素E | 5.8 | 5.8 | 5.8 | 5.8 | 5.8 |
维生素H | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 |
硫辛酸 | 0.35 | 0.35 | 0.35 | 0.35 | 0.35 |
D-泛酸钙 | 25 | 25 | 25 | 25 | 25 |
烟酰胺 | 18 | 18 | 18 | 18 | 18 |
NH4VO3 | 0.01 | 0.01 | 0.01 | 0.01 | 0.01 |
Na2SeO3 | 0.03 | 0.03 | 0.03 | 0.03 | 0.03 |
MnCl2·4H2O | 0.08 | 0.08 | 0.08 | 0.08 | 0.08 |
CuSO4·5H2O | 0.12 | 0.12 | 0.12 | 0.12 | 0.12 |
ZnCl2 | 0.89 | 0.89 | 0.89 | 0.89 | 0.89 |
MgCl2 | 0.35 | 0.35 | 0.35 | 0.35 | 0.35 |
K2SO4 | 22 | 22 | 22 | 22 | 22 |
AlCl3 | 0.03 | 0.03 | 0.03 | 0.03 | 0.03 |
CuSO4·5H2O | 0.003 | 0.003 | 0.003 | 0.003 | 0.003 |
NaCl | 0.28 | 0.28 | 0.28 | 0.28 | 0.28 |
NaHCO3 | 0.06 | 0.06 | 0.06 | 0.06 | 0.06 |
CaCl2 | 0.45 | 0.45 | 0.45 | 0.45 | 0.45 |
表4-2 对比例2对比例2(单位mg/L)
补料1 | 补料2 | 补料3 | 补料4 | 补料5 | |
30℃ | 34℃ | 37℃ | 34℃ | 32℃ | |
葡萄糖 | 1000 | 3000 | 8000 | 3000 | 2000 |
核糖 | 200 | 500 | 1000 | 800 | 800 |
麦芽糖 | 500 | 1000 | 2000 | 1600 | 1500 |
纤维二糖 | 200 | 300 | 400 | 300 | 200 |
葡聚糖 | 1500 | 1800 | 2000 | 2000 | 2000 |
纤维素 | 500 | 500 | 400 | 400 | 300 |
亚麻酸 | 0.08 | 0.08 | 0.08 | 0.08 | 0.08 |
亚油酸 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 |
胆固醇 | 1.8 | 1.8 | 1.8 | 1.8 | 1.8 |
花生四烯酸 | 0.005 | 0.005 | 0.005 | 0.005 | 0.005 |
孕酮 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 |
棕榈酸 | 0.18 | 0.18 | 0.18 | 0.18 | 0.18 |
胰岛素 | 0.03 | 0.03 | 0.03 | 0.03 | 0.03 |
甘氨酸 | 50 | 50 | 50 | 50 | 50 |
L-异亮氨酸 | 120 | 120 | 120 | 120 | 120 |
L-亮氨酸 | 130 | 130 | 130 | 130 | 130 |
L-组氨酸 | 78 | 78 | 78 | 78 | 78 |
L-赖氨酸 | 256 | 256 | 256 | 256 | 256 |
L-蛋氨酸 | 108 | 108 | 108 | 108 | 108 |
L-苯丙氨酸 | 68 | 68 | 68 | 68 | 68 |
L-苏氨酸 | 123 | 123 | 123 | 123 | 123 |
L-色氨酸 | 45 | 45 | 45 | 45 | 45 |
L-缬氨酸 | 160 | 160 | 160 | 160 | 160 |
L-丙氨酸 | 20.5 | 20.5 | 20.5 | 20.5 | 20.5 |
L-谷氨酰胺 | 1600 | 1600 | 1600 | 1600 | 1600 |
L-精氨酸 | 560 | 560 | 560 | 560 | 560 |
L-天冬氨酸 | 56 | 56 | 56 | 56 | 56 |
L-天冬酰胺 | 230 | 230 | 230 | 230 | 230 |
L-半胱氨酸 | 48 | 48 | 48 | 48 | 48 |
L-胱氨酸 | 99 | 99 | 99 | 99 | 99 |
L-谷氨酸 | 32 | 32 | 32 | 32 | 32 |
L-脯氨酸 | 78 | 78 | 78 | 78 | 78 |
L-丝氨酸 | 95 | 95 | 95 | 95 | 95 |
L-酪氨酸 | 224 | 224 | 224 | 224 | 224 |
叶酸 | 12 | 12 | 12 | 12 | 12 |
维生素A | 1.5 | 1.5 | 1.5 | 1.5 | 1.5 |
维生素C | 3 | 3 | 3 | 3 | 3 |
维生素B1 | 2.5 | 2.5 | 2.5 | 2.5 | 2.5 |
维生素B2 | 0.59 | 0.59 | 0.59 | 0.59 | 0.59 |
维生素B6 | 2.58 | 2.58 | 2.58 | 2.58 | 2.58 |
维生素B12 | 1.06 | 1.06 | 1.06 | 1.06 | 1.06 |
维生素K | 11 | 11 | 11 | 11 | 11 |
维生素E | 5.8 | 5.8 | 5.8 | 5.8 | 5.8 |
维生素H | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 |
硫辛酸 | 0.35 | 0.35 | 0.35 | 0.35 | 0.35 |
D-泛酸钙 | 25 | 25 | 25 | 25 | 25 |
烟酰胺 | 18 | 18 | 18 | 18 | 18 |
NH4VO3 | 0.01 | 0.01 | 0.01 | 0.01 | 0.01 |
Na2SeO3 | 0.03 | 0.03 | 0.03 | 0.03 | 0.03 |
MnCl2·4H2O | 0.08 | 0.08 | 0.08 | 0.08 | 0.08 |
CuSO4·5H2O | 0.12 | 0.12 | 0.12 | 0.12 | 0.12 |
ZnCl2 | 0.89 | 0.89 | 0.89 | 0.89 | 0.89 |
MgCl2 | 0.35 | 0.35 | 0.35 | 0.35 | 0.35 |
K2SO4 | 22 | 22 | 22 | 22 | 22 |
AlCl3 | 0.03 | 0.03 | 0.03 | 0.03 | 0.03 |
CuSO4·5H2O | 0.003 | 0.003 | 0.003 | 0.003 | 0.003 |
NaCl | 0.28 | 0.28 | 0.28 | 0.28 | 0.28 |
NaHCO3 | 0.06 | 0.06 | 0.06 | 0.06 | 0.06 |
CaCl2 | 0.45 | 0.45 | 0.45 | 0.45 | 0.45 |
表4-3 对比例3(单位mg/L)
补料1 | 补料2 | 补料3 | 补料4 | 补料5 | |
30℃ | 34℃ | 37℃ | 34℃ | 32℃ | |
半乳糖 | 200 | 500 | 3000 | 800 | 600 |
果糖 | 2000 | 1800 | 1600 | 1500 | 1400 |
麦芽酮糖 | 100 | 400 | 600 | 500 | 500 |
甘露二糖 | 500 | 500 | 500 | 800 | 1000 |
纤维素 | 500 | 500 | 400 | 400 | 300 |
糖原 | 2000 | 1800 | 1600 | 1500 | 1400 |
亚麻酸 | 0.08 | 0.08 | 0.08 | 0.08 | 0.08 |
亚油酸 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 |
胆固醇 | 1.8 | 1.8 | 1.8 | 1.8 | 1.8 |
花生四烯酸 | 0.005 | 0.005 | 0.005 | 0.005 | 0.005 |
孕酮 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 |
棕榈酸 | 0.18 | 0.18 | 0.18 | 0.18 | 0.18 |
胰岛素 | 0.03 | 0.03 | 0.03 | 0.03 | 0.03 |
甘氨酸 | 50 | 50 | 50 | 50 | 50 |
L-异亮氨酸 | 120 | 120 | 120 | 120 | 120 |
L-亮氨酸 | 130 | 130 | 130 | 130 | 130 |
L-组氨酸 | 78 | 78 | 78 | 78 | 78 |
L-赖氨酸 | 256 | 256 | 256 | 256 | 256 |
L-蛋氨酸 | 108 | 108 | 108 | 108 | 108 |
L-苯丙氨酸 | 68 | 68 | 68 | 68 | 68 |
L-苏氨酸 | 123 | 123 | 123 | 123 | 123 |
L-色氨酸 | 45 | 45 | 45 | 45 | 45 |
L-缬氨酸 | 160 | 160 | 160 | 160 | 160 |
L-丙氨酸 | 20.5 | 20.5 | 20.5 | 20.5 | 20.5 |
L-谷氨酰胺 | 1600 | 1600 | 1600 | 1600 | 1600 |
L-精氨酸 | 560 | 560 | 560 | 560 | 560 |
L-天冬氨酸 | 56 | 56 | 56 | 56 | 56 |
L-天冬酰胺 | 230 | 230 | 230 | 230 | 230 |
L-半胱氨酸 | 48 | 48 | 48 | 48 | 48 |
L-胱氨酸 | 99 | 99 | 99 | 99 | 99 |
L-谷氨酸 | 32 | 32 | 32 | 32 | 32 |
L-脯氨酸 | 78 | 78 | 78 | 78 | 78 |
L-丝氨酸 | 95 | 95 | 95 | 95 | 95 |
L-酪氨酸 | 224 | 224 | 224 | 224 | 224 |
叶酸 | 12 | 12 | 12 | 12 | 12 |
维生素A | 1.5 | 1.5 | 1.5 | 1.5 | 1.5 |
维生素C | 3 | 3 | 3 | 3 | 3 |
维生素B1 | 2.5 | 2.5 | 2.5 | 2.5 | 2.5 |
维生素B2 | 0.59 | 0.59 | 0.59 | 0.59 | 0.59 |
维生素B6 | 2.58 | 2.58 | 2.58 | 2.58 | 2.58 |
维生素B12 | 1.06 | 1.06 | 1.06 | 1.06 | 1.06 |
维生素K | 11 | 11 | 11 | 11 | 11 |
维生素E | 5.8 | 5.8 | 5.8 | 5.8 | 5.8 |
维生素H | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 |
硫辛酸 | 0.35 | 0.35 | 0.35 | 0.35 | 0.35 |
D-泛酸钙 | 25 | 25 | 25 | 25 | 25 |
烟酰胺 | 18 | 18 | 18 | 18 | 18 |
NH4VO3 | 0.01 | 0.01 | 0.01 | 0.01 | 0.01 |
Na2SeO3 | 0.03 | 0.03 | 0.03 | 0.03 | 0.03 |
MnCl2·4H2O | 0.08 | 0.08 | 0.08 | 0.08 | 0.08 |
CuSO4·5H2O | 0.12 | 0.12 | 0.12 | 0.12 | 0.12 |
ZnCl2 | 0.89 | 0.89 | 0.89 | 0.89 | 0.89 |
MgCl2 | 0.35 | 0.35 | 0.35 | 0.35 | 0.35 |
K2SO4 | 22 | 22 | 22 | 22 | 22 |
AlCl3 | 0.03 | 0.03 | 0.03 | 0.03 | 0.03 |
CuSO4·5H2O | 0.003 | 0.003 | 0.003 | 0.003 | 0.003 |
NaCl | 0.28 | 0.28 | 0.28 | 0.28 | 0.28 |
NaHCO3 | 0.06 | 0.06 | 0.06 | 0.06 | 0.06 |
CaCl2 | 0.45 | 0.45 | 0.45 | 0.45 | 0.45 |
表4-4 对比例4(单位mg/L)
补料1 | 补料2 | 补料3 | 补料4 | 补料5 | |
30℃ | 34℃ | 37℃ | 34℃ | 32℃ | |
核糖 | 200 | 500 | 1000 | 800 | 800 |
果糖 | 2000 | 1800 | 1600 | 1500 | 1400 |
异麦芽糖 | 1000 | 1000 | 1000 | 800 | 600 |
纤维二糖 | 200 | 300 | 400 | 300 | 200 |
支链淀粉 | 700 | 800 | 900 | 800 | 700 |
纤维素 | 500 | 500 | 400 | 400 | 300 |
亚麻酸 | 0.08 | 0.08 | 0.08 | 0.08 | 0.08 |
亚油酸 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 |
胆固醇 | 1.8 | 1.8 | 1.8 | 1.8 | 1.8 |
花生四烯酸 | 0.005 | 0.005 | 0.005 | 0.005 | 0.005 |
孕酮 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 |
棕榈酸 | 0.18 | 0.18 | 0.18 | 0.18 | 0.18 |
胰岛素 | 0.03 | 0.03 | 0.03 | 0.03 | 0.03 |
甘氨酸 | 50 | 50 | 50 | 50 | 50 |
L-异亮氨酸 | 120 | 120 | 120 | 120 | 120 |
L-亮氨酸 | 130 | 130 | 130 | 130 | 130 |
L-组氨酸 | 78 | 78 | 78 | 78 | 78 |
L-赖氨酸 | 256 | 256 | 256 | 256 | 256 |
L-蛋氨酸 | 108 | 108 | 108 | 108 | 108 |
L-苯丙氨酸 | 68 | 68 | 68 | 68 | 68 |
L-苏氨酸 | 123 | 123 | 123 | 123 | 123 |
L-色氨酸 | 45 | 45 | 45 | 45 | 45 |
L-缬氨酸 | 160 | 160 | 160 | 160 | 160 |
L-丙氨酸 | 20.5 | 20.5 | 20.5 | 20.5 | 20.5 |
L-谷氨酰胺 | 1600 | 1600 | 1600 | 1600 | 1600 |
L-精氨酸 | 560 | 560 | 560 | 560 | 560 |
L-天冬氨酸 | 56 | 56 | 56 | 56 | 56 |
L-天冬酰胺 | 230 | 230 | 230 | 230 | 230 |
L-半胱氨酸 | 48 | 48 | 48 | 48 | 48 |
L-胱氨酸 | 99 | 99 | 99 | 99 | 99 |
L-谷氨酸 | 32 | 32 | 32 | 32 | 32 |
L-脯氨酸 | 78 | 78 | 78 | 78 | 78 |
L-丝氨酸 | 95 | 95 | 95 | 95 | 95 |
L-酪氨酸 | 224 | 224 | 224 | 224 | 224 |
叶酸 | 12 | 12 | 12 | 12 | 12 |
维生素A | 1.5 | 1.5 | 1.5 | 1.5 | 1.5 |
维生素C | 3 | 3 | 3 | 3 | 3 |
维生素B1 | 2.5 | 2.5 | 2.5 | 2.5 | 2.5 |
维生素B2 | 0.59 | 0.59 | 0.59 | 0.59 | 0.59 |
维生素B6 | 2.58 | 2.58 | 2.58 | 2.58 | 2.58 |
维生素B12 | 1.06 | 1.06 | 1.06 | 1.06 | 1.06 |
维生素K | 11 | 11 | 11 | 11 | 11 |
维生素E | 5.8 | 5.8 | 5.8 | 5.8 | 5.8 |
维生素H | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 |
硫辛酸 | 0.35 | 0.35 | 0.35 | 0.35 | 0.35 |
D-泛酸钙 | 25 | 25 | 25 | 25 | 25 |
烟酰胺 | 18 | 18 | 18 | 18 | 18 |
NH4VO3 | 0.01 | 0.01 | 0.01 | 0.01 | 0.01 |
Na2SeO3 | 0.03 | 0.03 | 0.03 | 0.03 | 0.03 |
MnCl2·4H2O | 0.08 | 0.08 | 0.08 | 0.08 | 0.08 |
CuSO4·5H2O | 0.12 | 0.12 | 0.12 | 0.12 | 0.12 |
ZnCl2 | 0.89 | 0.89 | 0.89 | 0.89 | 0.89 |
MgCl2 | 0.35 | 0.35 | 0.35 | 0.35 | 0.35 |
K2SO4 | 22 | 22 | 22 | 22 | 22 |
AlCl3 | 0.03 | 0.03 | 0.03 | 0.03 | 0.03 |
CuSO4·5H2O | 0.003 | 0.003 | 0.003 | 0.003 | 0.003 |
NaCl | 0.28 | 0.28 | 0.28 | 0.28 | 0.28 |
NaHCO3 | 0.06 | 0.06 | 0.06 | 0.06 | 0.06 |
CaCl2 | 0.45 | 0.45 | 0.45 | 0.45 | 0.45 |
表4-5 对比例5(单位mg/L)
补料1 | 补料2 | 补料3 | 补料4 | 补料5 | |
30℃ | 34℃ | 37℃ | 34℃ | 32℃ | |
果糖 | 2000 | 1800 | 1600 | 1500 | 1400 |
木糖 | 1000 | 2000 | 3000 | 4000 | 3000 |
麦芽酮糖 | 100 | 400 | 600 | 500 | 500 |
纤维二糖 | 200 | 300 | 400 | 300 | 200 |
葡聚糖 | 1500 | 1800 | 2000 | 2000 | 2000 |
凝胶多糖 | 100 | 200 | 300 | 400 | 500 |
亚麻酸 | 0.08 | 0.08 | 0.08 | 0.08 | 0.08 |
亚油酸 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 |
胆固醇 | 1.8 | 1.8 | 1.8 | 1.8 | 1.8 |
花生四烯酸 | 0.005 | 0.005 | 0.005 | 0.005 | 0.005 |
孕酮 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 |
棕榈酸 | 0.18 | 0.18 | 0.18 | 0.18 | 0.18 |
胰岛素 | 0.03 | 0.03 | 0.03 | 0.03 | 0.03 |
甘氨酸 | 50 | 50 | 50 | 50 | 50 |
L-异亮氨酸 | 120 | 120 | 120 | 120 | 120 |
L-亮氨酸 | 130 | 130 | 130 | 130 | 130 |
L-组氨酸 | 78 | 78 | 78 | 78 | 78 |
L-赖氨酸 | 256 | 256 | 256 | 256 | 256 |
L-蛋氨酸 | 108 | 108 | 108 | 108 | 108 |
L-苯丙氨酸 | 68 | 68 | 68 | 68 | 68 |
L-苏氨酸 | 123 | 123 | 123 | 123 | 123 |
L-色氨酸 | 45 | 45 | 45 | 45 | 45 |
L-缬氨酸 | 160 | 160 | 160 | 160 | 160 |
L-丙氨酸 | 20.5 | 20.5 | 20.5 | 20.5 | 20.5 |
L-谷氨酰胺 | 1600 | 1600 | 1600 | 1600 | 1600 |
L-精氨酸 | 560 | 560 | 560 | 560 | 560 |
L-天冬氨酸 | 56 | 56 | 56 | 56 | 56 |
L-天冬酰胺 | 230 | 230 | 230 | 230 | 230 |
L-半胱氨酸 | 48 | 48 | 48 | 48 | 48 |
L-胱氨酸 | 99 | 99 | 99 | 99 | 99 |
L-谷氨酸 | 32 | 32 | 32 | 32 | 32 |
L-脯氨酸 | 78 | 78 | 78 | 78 | 78 |
L-丝氨酸 | 95 | 95 | 95 | 95 | 95 |
L-酪氨酸 | 224 | 224 | 224 | 224 | 224 |
叶酸 | 12 | 12 | 12 | 12 | 12 |
维生素A | 1.5 | 1.5 | 1.5 | 1.5 | 1.5 |
维生素C | 3 | 3 | 3 | 3 | 3 |
维生素B1 | 2.5 | 2.5 | 2.5 | 2.5 | 2.5 |
维生素B2 | 0.59 | 0.59 | 0.59 | 0.59 | 0.59 |
维生素B6 | 2.58 | 2.58 | 2.58 | 2.58 | 2.58 |
维生素B12 | 1.06 | 1.06 | 1.06 | 1.06 | 1.06 |
维生素K | 11 | 11 | 11 | 11 | 11 |
维生素E | 5.8 | 5.8 | 5.8 | 5.8 | 5.8 |
维生素H | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 |
硫辛酸 | 0.35 | 0.35 | 0.35 | 0.35 | 0.35 |
D-泛酸钙 | 25 | 25 | 25 | 25 | 25 |
烟酰胺 | 18 | 18 | 18 | 18 | 18 |
NH4VO<sub>3</sub> | 0.01 | 0.01 | 0.01 | 0.01 | 0.01 |
Na2SeO<sub>3</sub> | 0.03 | 0.03 | 0.03 | 0.03 | 0.03 |
MnCl<sub>2</sub>·4H<sub>2</sub>O | 0.08 | 0.08 | 0.08 | 0.08 | 0.08 |
CuSO<sub>4</sub>·5H<sub>2</sub>O | 0.12 | 0.12 | 0.12 | 0.12 | 0.12 |
ZnCl2 | 0.89 | 0.89 | 0.89 | 0.89 | 0.89 |
MgCl2 | 0.35 | 0.35 | 0.35 | 0.35 | 0.35 |
K2SO4 | 22 | 22 | 22 | 22 | 22 |
AlCl3 | 0.03 | 0.03 | 0.03 | 0.03 | 0.03 |
CuSO4·5H2O | 0.003 | 0.003 | 0.003 | 0.003 | 0.003 |
NaCl | 0.28 | 0.28 | 0.28 | 0.28 | 0.28 |
NaHCO3 | 0.06 | 0.06 | 0.06 | 0.06 | 0.06 |
CaCl2 | 0.45 | 0.45 | 0.45 | 0.45 | 0.45 |
表4-6 对比例6(单位mg/L)
补料1 | 补料2 | 补料3 | 补料4 | 补料5 | |
37℃ | 37℃ | 37℃ | 37℃ | 37℃ | |
葡萄糖 | 1000 | 3000 | 8000 | 3000 | 2000 |
半乳糖 | 200 | 500 | 3000 | 800 | 600 |
麦芽糖 | 500 | 1000 | 2000 | 1600 | 1500 |
甘露二糖 | 500 | 500 | 500 | 800 | 1000 |
葡聚糖 | 1500 | 1800 | 2000 | 2000 | 2000 |
淀粉 | 800 | 700 | 600 | 500 | 400 |
亚麻酸 | 0.08 | 0.08 | 0.08 | 0.08 | 0.08 |
亚油酸 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 |
胆固醇 | 1.8 | 1.8 | 1.8 | 1.8 | 1.8 |
花生四烯酸 | 0.005 | 0.005 | 0.005 | 0.005 | 0.005 |
孕酮 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 |
棕榈酸 | 0.18 | 0.18 | 0.18 | 0.18 | 0.18 |
胰岛素 | 0.03 | 0.03 | 0.03 | 0.03 | 0.03 |
甘氨酸 | 50 | 50 | 50 | 50 | 50 |
L-异亮氨酸 | 120 | 120 | 120 | 120 | 120 |
L-亮氨酸 | 130 | 130 | 130 | 130 | 130 |
L-组氨酸 | 78 | 78 | 78 | 78 | 78 |
L-赖氨酸 | 256 | 256 | 256 | 256 | 256 |
L-蛋氨酸 | 108 | 108 | 108 | 108 | 108 |
L-苯丙氨酸 | 68 | 68 | 68 | 68 | 68 |
L-苏氨酸 | 123 | 123 | 123 | 123 | 123 |
L-色氨酸 | 45 | 45 | 45 | 45 | 45 |
L-缬氨酸 | 160 | 160 | 160 | 160 | 160 |
L-丙氨酸 | 20.5 | 20.5 | 20.5 | 20.5 | 20.5 |
L-谷氨酰胺 | 1600 | 1600 | 1600 | 1600 | 1600 |
L-精氨酸 | 560 | 560 | 560 | 560 | 560 |
L-天冬氨酸 | 56 | 56 | 56 | 56 | 56 |
L-天冬酰胺 | 230 | 230 | 230 | 230 | 230 |
L-半胱氨酸 | 48 | 48 | 48 | 48 | 48 |
L-胱氨酸 | 99 | 99 | 99 | 99 | 99 |
L-谷氨酸 | 32 | 32 | 32 | 32 | 32 |
L-脯氨酸 | 78 | 78 | 78 | 78 | 78 |
L-丝氨酸 | 95 | 95 | 95 | 95 | 95 |
L-酪氨酸 | 224 | 224 | 224 | 224 | 224 |
叶酸 | 12 | 12 | 12 | 12 | 12 |
维生素A | 1.5 | 1.5 | 1.5 | 1.5 | 1.5 |
维生素C | 3 | 3 | 3 | 3 | 3 |
维生素B1 | 2.5 | 2.5 | 2.5 | 2.5 | 2.5 |
维生素B2 | 0.59 | 0.59 | 0.59 | 0.59 | 0.59 |
维生素B6 | 2.58 | 2.58 | 2.58 | 2.58 | 2.58 |
维生素B12 | 1.06 | 1.06 | 1.06 | 1.06 | 1.06 |
维生素K | 11 | 11 | 11 | 11 | 11 |
维生素E | 5.8 | 5.8 | 5.8 | 5.8 | 5.8 |
维生素H | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 |
硫辛酸 | 0.35 | 0.35 | 0.35 | 0.35 | 0.35 |
D-泛酸钙 | 25 | 25 | 25 | 25 | 25 |
烟酰胺 | 18 | 18 | 18 | 18 | 18 |
NH4VO3 | 0.01 | 0.01 | 0.01 | 0.01 | 0.01 |
Na2SeO3 | 0.03 | 0.03 | 0.03 | 0.03 | 0.03 |
MnCl2·4H2O | 0.08 | 0.08 | 0.08 | 0.08 | 0.08 |
CuSO4·5H2O | 0.12 | 0.12 | 0.12 | 0.12 | 0.12 |
ZnCl2 | 0.89 | 0.89 | 0.89 | 0.89 | 0.89 |
MgCl2 | 0.35 | 0.35 | 0.35 | 0.35 | 0.35 |
K2SO4 | 22 | 22 | 22 | 22 | 22 |
AlCl3 | 0.03 | 0.03 | 0.03 | 0.03 | 0.03 |
CuSO4·5H2O | 0.003 | 0.003 | 0.003 | 0.003 | 0.003 |
NaCl | 0.28 | 0.28 | 0.28 | 0.28 | 0.28 |
NaHCO3 | 0.06 | 0.06 | 0.06 | 0.06 | 0.06 |
CaCl2 | 0.45 | 0.45 | 0.45 | 0.45 | 0.45 |
表4-7 对比例7(单位mg/L)
补料1 | 补料2 | 补料3 | 补料4 | 补料5 | |
30℃ | 34℃ | 37℃ | 34℃ | 32℃ | |
葡萄糖 | 1000 | 3000 | 8000 | 3000 | 2000 |
亚麻酸 | 0.08 | 0.08 | 0.08 | 0.08 | 0.08 |
亚油酸 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 |
胆固醇 | 1.8 | 1.8 | 1.8 | 1.8 | 1.8 |
花生四烯酸 | 0.005 | 0.005 | 0.005 | 0.005 | 0.005 |
孕酮 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 |
棕榈酸 | 0.18 | 0.18 | 0.18 | 0.18 | 0.18 |
胰岛素 | 0.03 | 0.03 | 0.03 | 0.03 | 0.03 |
甘氨酸 | 50 | 50 | 50 | 50 | 50 |
L-异亮氨酸 | 120 | 120 | 120 | 120 | 120 |
L-亮氨酸 | 130 | 130 | 130 | 130 | 130 |
L-组氨酸 | 78 | 78 | 78 | 78 | 78 |
L-赖氨酸 | 256 | 256 | 256 | 256 | 256 |
L-蛋氨酸 | 108 | 108 | 108 | 108 | 108 |
L-苯丙氨酸 | 68 | 68 | 68 | 68 | 68 |
L-苏氨酸 | 123 | 123 | 123 | 123 | 123 |
L-色氨酸 | 45 | 45 | 45 | 45 | 45 |
L-缬氨酸 | 160 | 160 | 160 | 160 | 160 |
L-丙氨酸 | 20.5 | 20.5 | 20.5 | 20.5 | 20.5 |
L-谷氨酰胺 | 1600 | 1600 | 1600 | 1600 | 1600 |
L-精氨酸 | 560 | 560 | 560 | 560 | 560 |
L-天冬氨酸 | 56 | 56 | 56 | 56 | 56 |
L-天冬酰胺 | 230 | 230 | 230 | 230 | 230 |
L-半胱氨酸 | 48 | 48 | 48 | 48 | 48 |
L-胱氨酸 | 99 | 99 | 99 | 99 | 99 |
L-谷氨酸 | 32 | 32 | 32 | 32 | 32 |
L-脯氨酸 | 78 | 78 | 78 | 78 | 78 |
L-丝氨酸 | 95 | 95 | 95 | 95 | 95 |
L-酪氨酸 | 224 | 224 | 224 | 224 | 224 |
叶酸 | 12 | 12 | 12 | 12 | 12 |
维生素A | 1.5 | 1.5 | 1.5 | 1.5 | 1.5 |
维生素C | 3 | 3 | 3 | 3 | 3 |
维生素B1 | 2.5 | 2.5 | 2.5 | 2.5 | 2.5 |
维生素B2 | 0.59 | 0.59 | 0.59 | 0.59 | 0.59 |
维生素B6 | 2.58 | 2.58 | 2.58 | 2.58 | 2.58 |
维生素B12 | 1.06 | 1.06 | 1.06 | 1.06 | 1.06 |
维生素K | 11 | 11 | 11 | 11 | 11 |
维生素E | 5.8 | 5.8 | 5.8 | 5.8 | 5.8 |
维生素H | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 |
硫辛酸 | 0.35 | 0.35 | 0.35 | 0.35 | 0.35 |
D-泛酸钙 | 25 | 25 | 25 | 25 | 25 |
烟酰胺 | 18 | 18 | 18 | 18 | 18 |
NH4VO3 | 0.01 | 0.01 | 0.01 | 0.01 | 0.01 |
Na2SeO3 | 0.03 | 0.03 | 0.03 | 0.03 | 0.03 |
MnCl2·4H2O | 0.08 | 0.08 | 0.08 | 0.08 | 0.08 |
CuSO4·5H2O | 0.12 | 0.12 | 0.12 | 0.12 | 0.12 |
ZnCl2 | 0.89 | 0.89 | 0.89 | 0.89 | 0.89 |
MgCl2 | 0.35 | 0.35 | 0.35 | 0.35 | 0.35 |
K2SO4 | 22 | 22 | 22 | 22 | 22 |
AlCl3 | 0.03 | 0.03 | 0.03 | 0.03 | 0.03 |
CuSO4·5H2O | 0.003 | 0.003 | 0.003 | 0.003 | 0.003 |
NaCl | 0.28 | 0.28 | 0.28 | 0.28 | 0.28 |
NaHCO3 | 0.06 | 0.06 | 0.06 | 0.06 | 0.06 |
CaCl2 | 0.45 | 0.45 | 0.45 | 0.45 | 0.45 |
对比例8 Merck公司培养基Cellvento Feed-200。
对比例9 SIGMA公司培养基 Immediate advantage F#1。
对比例10 HYCLONE公司培养基 Cell boost6 。
实施例2
取工程CHO细胞株在37℃水浴中解冻,移到含有商用复苏培养基的离心管中混匀后,1000rpm离心5min小心弃去上清,取30-50ml传代基础培养基重悬后在CO2培养箱中摇床复苏培养3-5天,取样检测活细胞密度以及细胞活率,到细胞密度达到5×105并且细胞活率大于85%时达到接种条件,可将其接入放大培养容器中。抗PD-1特异性抗体的基因产物克隆到哺乳动物表达载体pTT5中,并在CHO细胞中瞬时表达(Raymond C.等Methods. 55(1):44-51(2011))。
采用对比例1-10培养基补料1-5的组分,进行按照表5以及附注的培养基添加方式培养CHO细胞(除对比例6),第12d培养结束后,收集培养液离心获得上清液,使用蛋白A盒(POROSA20柱,Invitrogen,Grand Island, NY,部件号2-1001-00,2.1mmD x 30mmH,104μL)的600/717/996 HPLC***(Waters Corporation,Milford,MA)进行上清液中的抗体定量。培养过程中每24h取样监测活细胞密度和细胞活率。
细胞计数方法:取样约1.5ml置于24孔板,吹打均匀后加入一定体积的0.4%台盼蓝染色,使用血球计数板计数,每个样品计数三次,取平均值。
表5 对比例1-5、7-10补料培养基添加
培养基 | 添加时培养温度调整 | 添加时间 | 培养时段 |
补料1 | 30℃ | 0-48h | 启动期 |
补料2 | 34℃ | 48-80h | 对数增长期 |
补料3 | 37℃ | 80-120h | 对数增长期 |
补料4 | 34℃ | 120h-140h | 恒定期 |
补料5 | 32℃ | 140h-336h | 代谢产物积累期 |
附注:从30℃提升到33℃的提升方式为:30℃12h,31℃12h,32℃12h,33℃12h;从34℃提升到36℃的提升方式为:34℃ 12h,35℃ 10h ,36℃ 10h;从37℃降低到35℃的下降方式为:37℃ 13h,36℃ 13h,35℃ 14h;从34℃降低到33℃的下降方式为:34℃ 10h,33℃10h。
其中对比例6补料1-5添加方式为37℃恒温。
实验结果
1 CHO活细胞密度检测结果。
表6 CHO活细胞密度培养活细胞密度 单位(× 105cells/ml)
0d | 1d | 2d | 3d | 4d | 5d | 6d | 7d | 8d | 9d | 10d | 11d | 12d | |
对比例1 | 0 | 20 | 38 | 57 | 86 | 154 | 200 | 236 | 256 | 209 | 193 | 178 | 156 |
对比例2 | 0 | 19 | 37 | 53 | 85 | 139 | 185 | 228 | 238 | 222 | 185 | 145 | 135 |
对比例3 | 0 | 18 | 36 | 51 | 83 | 125 | 178 | 215 | 227 | 202 | 178 | 136 | 124 |
对比例4 | 0 | 20 | 35 | 50 | 82 | 113 | 169 | 203 | 195 | 165 | 168 | 125 | 112 |
对比例5 | 0 | 19 | 36 | 48 | 79 | 108 | 158 | 196 | 178 | 155 | 144 | 111 | 100 |
对比例6 | 0 | 16 | 33 | 47 | 78 | 96 | 144 | 187 | 175 | 162 | 148 | 103 | 91 |
对比例7 | 0 | 18 | 32 | 46 | 75 | 82 | 130 | 151 | 168 | 144 | 124 | 95 | 83 |
对比例8 | 0 | 20 | 35 | 44 | 76 | 78 | 125 | 142 | 156 | 128 | 98 | 88 | 75 |
对比例9 | 0 | 19 | 36 | 43 | 77 | 78 | 120 | 136 | 142 | 115 | 85 | 75 | 63 |
对比例10 | 0 | 18 | 37 | 42 | 68 | 69 | 113 | 125 | 138 | 102 | 86 | 74 | 52 |
结果见表6、图1显示,对比例1-10获得曲线趋势近似,但对比例1可达到最高活细胞密度,并且在恒定期和衰退期也可维持较高的活细胞密度,超过商用培养基、恒温培养组、单独葡萄糖组以及其他糖组合对比组。
2 形态学观察
通过显微镜观察,CHO细胞悬浮培养状态良好,边缘光滑,无明显污染,图2为对比例1第7d时细胞图片。
3 抗体PD1表达检测
表7 不同补料培养基的PD1抗体表达量比较(单位:g/L)
培养基种类 | 抗体浓度 |
对比例1 | 4.82 |
对比例2 | 4.25 |
对比例3 | 4.35 |
对比例4 | 3.78 |
对比例5 | 4.01 |
对比例6 | 3.94 |
对比例7 | 3.86 |
对比例8 | 3.56 |
对比例9 | 3.98 |
对比例10 | 4.02 |
实施例3:其他多种抗体表达
培养方法与实施例2基本相同,用于表达VEGF人源化单克隆抗体、AGR2人源化单克隆抗体、狂犬病毒抗体,结果参见图3-6,结果显示,对比例1-5均可获得较高的外源蛋白表达,并且可高于商用培养基、恒温组、葡萄糖组对照组,具有显著性差异,证明本发明设计的组合糖类补料培养基取得了优良的技术效果。
表8 不同补料培养基的VEGF抗体表达量比较(单位:g/L)
培养基种类 | 抗体浓度 |
对比例1 | 4.06 |
对比例2 | 4.77 |
对比例3 | 4.26 |
对比例4 | 4.18 |
对比例5 | 4.06 |
对比例6 | 3.65 |
对比例7 | 3.77 |
对比例8 | 3.89 |
对比例9 | 3.58 |
对比例10 | 4.01 |
表9 不同补料培养基的AGR2抗体表达量比较(单位:g/L)
培养基种类 | 抗体浓度 |
对比例1 | 3.91 |
对比例2 | 4.25 |
对比例3 | 4.26 |
对比例4 | 4.08 |
对比例5 | 3.88 |
对比例6 | 3.01 |
对比例7 | 3.24 |
对比例8 | 3.18 |
对比例9 | 2.99 |
对比例10 | 2.89 |
表10 不同补料培养基的狂犬病毒抗体表达量比较(单位:g/L)
培养基种类 | 抗体浓度 |
对比例1 | 3.78 |
对比例2 | 3.75 |
对比例3 | 3.22 |
对比例4 | 3.45 |
对比例5 | 3.66 |
对比例6 | 3.01 |
对比例7 | 2.98 |
对比例8 | 3.00 |
对比例9 | 3.08 |
对比例10 | 3.18 |
Claims (10)
1.一种CHO细胞培养方法,其特征在于:培养过程中采用补料培养基1-5在不同培养温度节点添加,所述补料培养基1-5添加的对应温度节点依次为30℃-34℃-37℃-34℃-32℃,以mg/L为单位,其中补料培养基中的糖分选自下表中的对比例1-5中的一种:
。
2.根据权利要求1所述的CHO细胞培养方法,所述培养过程的温度变化节点位于特定的培养时间,0-48h 培养温度为30℃,48-80h培养温度为34℃,80-120h培养温度为37℃,120h-140h培养温度为34℃,140h-336h培养温度为32℃。
3.根据权利要求2所述的CHO细胞培养方法,所述的温度变化为,从30℃提升到33℃的提升方式为:30℃12h,31℃12h,32℃12h,33℃12h;从34℃提升到36℃的提升方式为:34℃12h,35℃ 10h,36℃ 10h;从37℃降低到35℃的下降方式为:37℃ 13h,36℃ 13h,35℃14h;从34℃降低到33℃的下降方式为:34℃ 10h,33℃ 10h。
4.根据权利要求1-3之一所述的CHO细胞培养方法,所述的补料培养基中还进一步包括脂类、氨基酸、维生素、无机盐。
5.根据权利要求4所述的CHO细胞培养方法,以mg/L为单位,所述的补料培养基中还包括组合培养基成分:
。
6.一种用于CHO细胞培养的培养基,所述培养基为在CHO细胞不同培养温度节点添加的补料培养基1-5,所述补料培养基1-5添加的对应温度节点依次为30℃-34℃-37℃-34℃-32℃,以mg/L为单位,其中补料培养基中的糖分选自下表中的对比例1-5中的一种:
。
7.根据权利要求6所述的用于CHO细胞培养的培养基,所述培养基进一步包括脂类、氨基酸、维生素、无机盐。
8.根据权利要求7所述的用于CHO细胞培养的培养基,以mg/L为单位,所述的补料培养基中还包括组合培养基成分:
。
9.权利要求1-5所述的培养方法或权利要求6-8所述的CHO细胞培养基,其特征在于,用于CHO细胞的外源蛋白表达。
10.根据权利要求9所述的培养方法或培养基,所述外源蛋白为单克隆抗体。
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