Study on the Optimal Scale-down Model for Cell Growth and Mab-A Production of Sp2/0 Cells

Guo-qiang WANG,Jian-ping LIU,Hang ZHOU

China Biotechnology ›› 2018, Vol. 38 ›› Issue (6) : 63-69.

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China Biotechnology ›› 2018, Vol. 38 ›› Issue (6) : 63-69. DOI: 10.13523/j.cb.20180609

Study on the Optimal Scale-down Model for Cell Growth and Mab-A Production of Sp2/0 Cells

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Abstract

Five different kinds of basal media were screened in batch mode to find out the optimal basal medium for cell growth of Sp2/0. The peak viable cell density in batch mode was 13.12×10 6cells/ml, and the culture duration was 7 days. Various cell culture conditions including different shaking speeds, concentrations of carbon dioxide, glutamine replaced by GlutaMAX TM, addition of trace element and different culture temperatures were studied in batch mode as well. Few different cell growth were found with these conditions except for culture temperatures, which leaded to different peak viable cell concentrations, different viabilites and then different culture durations. Fourteen kinds of combinations of feed media were screened in fed-batch mode. The peak viable cell densities were up to 30×10 6cells/ml, the culture duration were around 9 days, and the highest daily Mab-A production was 27.20mg/L. Batch re-feed mode was used in the third part of the study. The peak viable cell density was 50.42×10 6cells/ml, the culture duration was 14 days, and the highest daily Mab-A production (141.10mg/L) was 5.19 fold greater than in fed-batch mode. These studies suggest batch re-feed mode is the optimal scale-down mode for cell growth and Mab-A production of Sp2/0 cells.

Key words

Sp2/0 / Scale-down mode / Batch re-feed

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Guo-qiang WANG, Jian-ping LIU, Hang ZHOU. Study on the Optimal Scale-down Model for Cell Growth and Mab-A Production of Sp2/0 Cells[J]. China Biotechnology, 2018, 38(6): 63-69 https://doi.org/10.13523/j.cb.20180609
Sp2/0-Ag14 细胞株于上世纪70年代因特定抗体生产需求建立起来[1]。随后的几十年中,国际制药巨头应用Sp2/0细胞生产出Cetuximab(Erbitux®,百时美施贵宝),Basiliximab(Simulect®,诺华),Canakinumab(Ilaris®,诺华),Abciximab(Reopro®,Centocor Ortho Biotech Inc.,礼来)等多种单抗药物[2],这些单抗药物每年创造了上百亿美元的利润。国内生物大分子药物在最近几十年发展迅速,但多数为仿制药。Sp2/0细胞培养方法因为国际制药公司技术壁垒等原因,极少见诸于报。另外一方面,国内有部分关于Sp2/0细胞的研究,但研究更多的是针对如何抑制Sp2/0细胞的生长[3,4,5],其目的是为各药物在多发性骨髓瘤治疗中的应用提供理论依据。掌握Sp2/0细胞大规模培养关键技术对于国内生物制药公司而言非常必要。一个良好的缩小(scale-down)模型是大规模生产运行的基础。本文就Sp2/0细胞缩小模型培养体系的优化开展研究工作。

1 材料与方法

1.1 细胞株

实验所用为表达单克隆抗体-A(Mab-A)的Sp2/0-Ag14,购买于ATCC®。

1.2 培养基

本研究所用培养基均为GE,ThermoFisher等培养基生产商生产的商业化培养基,具体信息详见表1。所有基础培养基在使用前均将谷氨酰胺含量调整为8mmol/L。
Table 1 Medium information

表1 培养基信息表

Medium code Vendor Category
A,B ThermoFisher Basal medium
C,D,E GE
F,G ThermoFisher Feed medium
H,I,J,K GE
L Merck
M Kerry
N,O Lonza
P,Q Kerry Hydrolysate
R Merck Additive
Dexamethasone Merck
GlutaMAXTM ThermoFisher

1.3 培养方法

从液氮罐中取出冻存的种子细胞,37℃水浴锅中进行孵化,离心去上清(200g,5min),使用新鲜的培养基A进行细胞重悬,以0.3×106cells/ml的密度接种于250ml的摇瓶内,置于培养箱内培养。培养箱条件为转速125r/min,6%的CO2,36.5℃及80%的湿度。每隔48h,种子细胞悬液以0.3×106 cells/ml的接种密度,使用新鲜培养基A进行稀释传代。摇瓶内的批次培养实验:取处于对数生长期的种子细胞悬液,以0.3×106cells/ml的密度接种于125ml的摇瓶内,添加实验所用新鲜培养基(表2),工作体积为30ml,置于培养箱内培养。
Table 2 Conditions for batch culture

表2 批次培养条件

Culture No. Medium Code Condition
SF1,5 A+C(50%+50%) Control
SF2,6 D
SF3,7 B
SF4,8 E
SF9 A+C(50%+50%) 160 RPM
SF10 D
SF11 B
SF12 E
SF13 A+C(50%+50%) 10% CO2
SF14 D
SF15 B
SF16 E
SF17 A+C(50%+50%) 33℃
SF19 E
SF18 A+C(50%+50%) 35℃
SF20 E
SF21 A+C(50%+50%) Trace element
SF22 E
SF23 A+C(50%+50%) GlutaMAXTM
SF24 D
SF25 B
SF26 E
控制组SF1-8培养箱条件为转速125r/min,6%的CO2,36.5℃及80%的湿度。控制组每个条件独立重复两次。实验组SF9-20的培养条件除转速、CO2和培养温度依次同控制组不相同外,其它培养参数同控制组相同;实验组SF21、22在基础培养基中添加0.2ml/L浓度的微量元素;实验组SF23-26将每天添加的谷氨酰胺替换成GlutaMAXTM;条件设计详情见表2。所有条件每隔24h取样计数,测生化。谷氨酰胺或GlutaMAXTM低于8mmol/L补到8mmol/L,葡萄糖低于3g/L补到5g/L。待细胞活性降至50%以下时,结束批培养。摇瓶内的流加培养实验:取处于对数生长期的种子细胞悬液,以0.3×106cells/ml的密度接种于125ml的摇瓶内,添加新鲜培养基D,工作体积为30ml,置于培养箱内培养。培养箱条件为转速125r/min,6%的CO2,80%的湿度。实验组SF27-39依次按第3、7天流加1.5ml,第4、6天流加2.5ml,第5天流加3ml的策略对各种流加培养基进行添加;培养温度为35℃。实验组SF40依次按第2、6天流加1.5ml,第3、5天流加2.5ml,第4天流加3ml的策略对流加培养基进行添加;起始培养温度为36.5℃,第4天降温至33℃。条件设计详情见表3。所有条件每隔24h取样计数,测生化。谷氨酰胺低于8mmol/L补到8mmol/L,葡萄糖低于3g/L补到5g/L。待细胞活性降至50%以下时,结束批培养。
Table 3 Conditions for fed-batch culture

表3 流加培养条件

Culture No. Medium Code Conditon
SF27 99% F+1% L Feed medium screen
SF28 99% G+1% L
SF29 49.5% F+49.5% G+1% L
SF30 99% H+1% L
SF31 49.5% H+49.5% I+1% L
SF32 99% J+1% L
SF33 80% J+1% L+19% water
SF34 99% K+1% L
SF35 99% J (with 15 g/L P)+1% L
SF36 99% J (with 15 g/L Q)+1% L
SF37 99% N+1% L
SF38 99% M+1% L
SF39 99% O+1% L
SF40 99% J+1% L 36.5℃ shift to
33℃ on day 4
摇管内的批次-反复流加培养实验:取处于对数生长期的种子细胞悬液,以0.3×106cells/ml的密度接种于50ml的TTP内,添加新鲜培养基D,工作体积为20ml,置于培养箱内培养。
培养箱条件为转速225r/min,6%的CO2,80%的湿度;起始培养温度为36.5℃,在活细胞数达到10×106cells/ml时降温至33℃。控制组ST1和实验组ST2、ST3均按以下策略每天离心(200g,5min)换液:第2天更换新鲜培养基和工作体积的体积比(fresh medium volume/working volume per day, VVD)为0.5的新鲜培养基D;第3天0.8VVD的新鲜培养基D;第4天起0.8 VVD的混合培养基(D:J:L=88.2:9.8:2.0)。实验组ST2从第4天起每天添加0.5μmol/L的地塞米松,ST3从第4天起每天添加0.1μmol/L的地塞米松。所有条件每隔24h取样计数,测生化。谷氨酰胺低于8mmol/L补到8mmol/L,葡萄糖低于3g/L补到5g/L。待细胞活性降至50%以下时,结束批培养。

1.4 分析方法

1.4.1 细胞计数 使用细胞计数仪(Vi-CELL XR,Beckman Coulter)测量细胞密度,细胞活率和细胞直径,其计数原理为台盼蓝染色法。
1.4.2 生化及抗体浓度的检测 使用Cedex(Cedex Bio HT,Roche)测量葡萄糖,谷氨酰胺和GlutaMAXTM,其原理为分光光度法;同时,Mab-A浓度也是由Cedex测得,其原理为比浊法。

2 结果与讨论

2.1 不同培养条件、微量元素和GlutaMAXTM在批次培养中对Sp2/0细胞生长的影响

控制组使用了5种不同的培养基或培养基组合进行了实验。由图1可以看出,每个条件的独立重复结果非常一致,因此后续实验不再设立重复。控制组中,培养基D、E可以达到最高的峰值细胞,其浓度为13.12×106cells/ml,培养天数为7天。使用更高摇床转速的实验组SF9-SF12结果如表4所示,其峰值细胞数(12.69×106cells/ml)和培养天数(7天)同控制组相比并没有显著性差异。同样,使用10%的二氧化碳的实验组SF13-SF16,并没有比控制组使用6%的二氧化碳获得更高的峰值细胞数和更长的培养天数;其中,使用基础培养基E的SF16其峰值细胞数反而下降为9.90×106cells/ml。温度对Sp2/0细胞生长和活率的影响见图2。由图上可以看出,Sp2/0细胞在培养基E中,其在36.5℃,35℃,33℃下的峰值细胞分别为12.32,11.03和8.09×106cells/ml,而相应的培养天数为7天,8天和9天,即培养温度越高,其峰值细胞数越高,但培养天数越短。同样的结果在培养基A+C(50%+50%)也观察到了。添加微量元素的实验条件SF21、22结果如表4所示,其峰值细胞数和培养天数与控制组SF1、4相比均未有显著性差异。同样,以GlutaMAXTM替换谷氨酰胺的实验组其结果与控制组相比也未有显著性差异。
Fig.1 Cell growth (a) and viability (b) in various basal media

图1 Sp2/0在不同基础培养基中的生长及活率曲线

Full size|PPT slide

Table 4 Influence of shaking speed, CO2, trace element and GlutaMAXTM on cells’ performance

表4 批次培养条件(摇床转速、CO2浓度、微量元素、GlutaMAXTM运用)对Sp2/0细胞生长影响

SF9 SF10 SF11 SF12 SF13 SF14 SF15 SF16 SF21 SF22 SF23 SF24 SF25 SF26
Peak VCC (106 cells/ml) 7.81 12.44 3.56 12.69 8.50 12.39 4.02 9.90 6.71 12.45 8.77 13.44 3.46 12.85
Culture duration (days) 5 7 4 7 5 7 4 7 5 7 5 7 4 7
Fig.2 Cell growth (a) and viability (b) under different culture temperatures

图2 温度对Sp2/0细胞生长和活率的影响

Full size|PPT slide

动物细胞生长均需要合适的溶氧浓度和二氧化碳浓度,在批次培养模式下,当摇床转速从125r/min提高到160r/min,二氧化碳浓度从6%提高到10%时,Sp2/0细胞并没有生长得更高,活率下降也并没有更缓慢,说明之前的培养条件已不是限制Sp2/0细胞生长的限制性条件。更低的培养温度往往可以获得更好的细胞活率,进而获得更长的细胞培养周期。本实验中,Sp2/0也随3个不同的培养温度呈现了3个不同的培养周期,但更低的培养温度同时也导致了更低的细胞密度。为了平衡细胞密度和培养周期,下一个实验将在35℃培养条件下进行。有文献报道,锌、胰岛素[6]、胆固醇[7]、磷[8]对杂交瘤细胞生长起关键作用,但如表4所示,在本文研究范围内,这些添加剂并未促进细胞生长或延长细胞培养周期。同样,谷氨酰胺是Sp2/0生长的关键氨基酸,但由消耗谷氨酰胺所累积的氨却会在一定浓度下抑制细胞生长;GlutaMAXTM是L-谷氨酰胺和L-丙氨酸的二肽化合物,而细胞可逐渐释放氨肽酶,水解二肽,因此L-谷氨酰胺和L-丙氨酸可缓慢释放至培养基中。原设计是希望谷氨酰胺的理论缓释迫使Sp2/0利用葡萄糖作为碳源和能源,进而最少地累积氨。但结果表明,实验组SF23-26的氨的累积量同控制组相比小于等于1mmol/L(数据并未展示)。其可能的原因是:Sp2/0对于谷氨酰胺的需求较为强烈,而GlutaMAXTM并未达到缓释谷氨酰胺的作用。
批次培养模型下进行的基础培养基筛选,培养基D、E获得了比其它条件更高的细胞密度和更长的培养周期。但13.12×106cells/ml的峰值细胞数和7天的培养天数对于单抗工业化生产来说太低,批次培养模型不能成为Sp2/0细胞生长的最适缩小模型。因此实验下一步进行流加培养,考察流加培养能否成为单克隆抗体Mab-A生产细胞Sp2/0的最适培养模型。

2.2 不同流加培养基在流加培养中对Sp2/0细胞生长、单克隆抗体Mab-A表达的影响

14种补料组合的筛选结果见表5。补料的添加大幅提高了细胞密度,与批次实验的SF2、6相比,增幅最大的条件SF35增加了183%,为37.08×106cells/ml;培养天数并没有因为补料的添加产生显著性差别,最长培养天数为9天(SF35)。SF40所用补料同SF32相同,两者培养条件差异为培养温度。SF40细胞峰值比SF32仅低了2.74×106cells/ml,SF40培养天数比SF32也仅多了一天,但SF40单抗产量却是SF32的1.57倍,单细胞产量(qMab-A)是SF32的1.26倍。拥有最高峰值细胞数和最长培养天数的SF35其单抗产量和单细胞产量均显著性低于条件SF40。其它补料组合其单抗产量和/或单细胞产量也显著性低于条件SF40。
Table 5 Cells’ performance in various feed media

表5 Sp2/0在不同补料培养基中的表现

Culture
No.
Peak VCC
(106
cells/ml)
Culture
duration
(days)
Titer
(mg/L)
qMab-A,
(pg/cell/
day)
SF27 25.65 7 73.73 0.99
SF28 27.56 7 35.40 0.46
SF29 26.57 7 46.93 0.63
SF30 20.58 7 39.69 0.68
SF31 21.49 7 41.39 0.74
SF32 23.26 7 138.58 1.97
SF33 20.48 8 99.14 1.28
SF34 22.96 6 41.74 0.87
SF35 37.08 9 165.92 1.23
SF36 20.29 8 157.20 1.83
SF37 25.63 6 42.94 0.83
SF38 22.57 6 47.85 1.02
SF39 22.91 6 56.72 1.13
SF40 20.52 8 217.59 2.48
批次培养虽然简单且节约成本,但没能获得较高的细胞密度,且培养天数也较短,不能很好地成为一个缩小模型。流加培养虽然略复杂,但可以多次提供营养成分,现已发展成为最常见的单抗商业化生产细胞培养模型。在市面上,有部分培养基生产商如Corning®等会专门为杂交瘤细胞生长设计相应的基础培养基[9],也有公司 或学者就补料培养基进行了研究,其中包括化学限定培养基[10]和植物蛋白水解物[11]等。本研究就将市面上的12种补料培养基进行表3中的组合后,进行Sp2/0细胞培养。同时有文献报道,先利用较高的温度生长细胞,然后降温培养,将达到高产蛋白的目的[12]。结合批次实验中更高的培养温度获得了更高的细胞密度,更低的温度获得了更长的培养周期,实验SF40即使用了同SF32相同的流加培养基,但不同的培养温度的策略。结果表明,先利用较高的温度生长细胞,然后降温培养以达到高产蛋白的方式对Sp2/0细胞同样适用。
由于本实验是研究Mab-A生产细胞Sp2/0的最适缩小培养模型,所以评价更多的是基于细胞生长,培养周期和蛋白产量。由于条件SF40拥有最高的单细胞产量,因此后续实验将以该条件为基础展开。而培养周期8天,蛋白217.59mg/L的产量对于药物剂量本来就较大的单抗来说,依然有较大提升空间。而流加培养一方面不能持续地提供营养,另外一方面也不能及时地排出代谢副产物,这可能是造成Sp2/0细胞培养周期不长、蛋白产率较低的原因。因此,实验第三部分将采用第三种培养模型,批次-反复流加培养来进行研究。

2.3 批次-反复流加培养对Sp2/0细胞生长、蛋白表达的影响

批次-反复流加培养实验结果如表6所示,控制组ST1其细胞数为流加培养条件SF40的2.46倍,为批次培养条件的3.84倍;ST1平均每天蛋白产量(141.10mg/L)是流加培养(SF40,27.20mg/L)的5.19倍。添加地塞米松为0.1μmol/L的ST3其平均每天蛋白产量高出控制组(ST1)15%;当地塞米松添加量为每天0.5μmol/L时,Sp2/0细胞受到了一定程度的影响,峰值细胞数、平均每天蛋白产量和单细胞产量均低于控制组。
Table 6 Cells’ performance in batch re-feed mode

表6 Sp2/0在批次-反复流加培养中的表现

Culture
No.
Peak VCC
(106
cells/ml)
Culture
duration
(days)
Titer
(mg/L)1)
qMab-A,
(pg/cell/
day)
ST1 50.42 14 141.10 2.98
ST2 42.23 14 115.38 2.77
ST3 53.01 14 162.56 3.19
1) Average dialy titer from day 7 to day 14
灌流培养可以持续地提供营养的同时及时地排出代谢副产物[13],但如果研究直接在反应器上进行,相应的成本将会较高[14]。为此,Villiger-Oberbek等[15]利用揺管建立了一个模拟反应器灌流培养的平台,并声称该平台可以很好地用于克隆挑选和培养基筛选。因此,本研究尝试将该模式应用于Sp2/0细胞。另外,Zhao等[16]发现地塞米松有保护细胞免于凋亡的作用。而摇瓶剪切力小,造成细胞坏死的可能性小,Sp2/0细胞活率在培养后期迅速下降很有可能是因为细胞发生了凋亡。为此,本研究也添加了地塞米松,以观察Sp2/0细胞表现。结果表明,地塞米松在添加量为每天0.1μmol/L时提升细胞数2.59×106cells/ml,提升平均每天蛋白产量15%,培养天数相对于控制组并没有提高;而与此同时,更换模型提升细胞数为2.46倍,提升平均每天蛋白产量5.19倍。这说明,ST1-3较高的蛋白产量更多的是因为培养模型的改变。而ST1-3其培养天数均为14天,说明地塞米松维持活率、延长培养的效果并没有体现出来。
在批次-反复流加培养模型下,细胞数在50×106cells/ml左右,培养天数为14天,单克隆抗体产量在1g/L左右,这样的缩小培养模型对于前期进行Sp2/0细胞培养基筛选,克隆筛选,甚至部分培养工艺条件筛选都是适合的。虽然Sp2/0细胞有一些特异性,例如它更多的利用谷氨酰胺作为碳源,但在其它动物细胞培养实验下得到的经验和结论依旧可以在此细胞上部分地得到验证或重现。

3 结 论

生物制品从制备之初起就因为其特异性强、疗效显著、毒性低等巨大的经济价值而被药物市场所关注。而想要将一个药物顺利地送入临床及市场,一个良好的工艺研究平台必不可少。本文就Sp2/0细胞生长和蛋白表达进行了批次培养和流加培养对比,发现Sp2/0在培养后期其活率下降迅速,每天下降超过10%,且蛋白产量较低。为了在工艺开发早期有一个良好的平台可以进行克隆挑选和培养基筛选,批次-流加培养模型因为其每天更换新鲜培养基、每天将代谢副产物排出体系而比流加培养和批次培养更适合于Sp2/0细胞。但这个缩小模型其每天最高只能更换1VVD的新鲜培养基,且每天只能进行一次代谢副产物的排出,这与反应器上的连续培养基更换还是很不一样的[17,18],因此该模型针对Sp2/0细胞培养的放大可行性还有待考察。另外,虽然本文就一些培养条件、微量元素等进行了研究,但决定细胞健康生长的因素非常多,如渗透压[19,20]、pH[21]、DO[22]等,这些可以在以后的研究中测试Sp2/0细胞对其的反应。还有更多的添加剂,有的报导可以提高蛋白表达,如氯化锂[23]、维生素[24]、氨基酸[24]、纳巴霉素[25]等,有的报导可以更好地维持细胞活率,如氢化可的松[26],白介素6[27],以及部分氨基酸[28],这些研究都将对Sp2/0抗体生产工业化产生重要的意义。

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Steady state metabolic parameters for hybridoma cell line H22 were determined over a wide range of cell densities and specific growth rates in a filtration based homogeneous perfusion reactor. Operating the reactor at perfusion rates of 0.75, 2.0, and 2.9 day-1(each at four different specific growth rates), viable cell densities as high as 2 107 cells/mL were obtained. For the cell line under investigation, the specific monoclonal antibody production rate was found to be a strong function of the viable cell density, increasing with increasing cell density. In contrast, most of the substrate consumption and product formation rates were strong functions of the specific growth rate. Substrate metabolism became more efficient at high cell densities and low specific growth rates. The Specific rates of metabolite formation and the apparent yields of lactate from glucose and ammonia from glutamine decreased at low specific growth rates and high cell densities. While the specific oxygen consumption rate was independent of the specific growth rate and cell density, ATP production was more oxidative at lower specific growth rate and higher cell density. These observed shifts are strong indications of the production potential of high-density perfusion culture. ? 1995 John Wiley & Sons, Inc.
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Process intensification in biomanufacturing has attracted a great deal of interest in recent years. Manufacturing platform improvements leading to higher cell density and bioreactor productivity have been pursued. Here we evaluated a variety of intensified mammalian cell culture processes for producing monoclonal antibodies. Cell culture operational modes including fed‐batch (normal seeding density or high seeding density with N‐1 perfusion), perfusion, and concentrated fed‐batch (CFB) were assessed using the same media set with the same Chinese Hamster Ovary (CHO) cell line. Limited media modification was done to quickly fit the media set to different operational modes. Perfusion and CFB processes were developed using an alternating tangential flow filtration device. Independent of the operational modes, comparable cell specific productivity (fed‐batch: 29.4 pg/cell/day; fed‐batch with N‐1 perfusion: 32.0 pg/cell/day; perfusion: 31.0 pg/cell/day; CFB: 20.1 – 45.1 pg/cell/day) was reached with similar media conditions. Continuous media exchange enabled much higher bioreactor productivity in the perfusion (up to 2.29 g/L/day) and CFB processes (up to 2.04 g/L/day), compared with that in the fed‐batch processes (ranging from 0.39 to 0.49 g/L/day), largely due to the higher cell density maintained. Furthermore, media cost per gram of antibody produced from perfusion was found to be highly comparable with that from fed‐batch; and the media cost for CFB was the highest due to the short batch duration. Our experimental data supports the argument that media cost for perfusion process could be even lower than that in a fed‐batch process, as long as sufficient bioreactor productivity is achieved. This article is protected by copyright. All rights reserved.
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A high-throughput (HT) cell culture model has been established for the support of perfusion-based cell culture processes operating at high cell densities. To mimic perfusion, the developed platform takes advantage of shake tubes and operates them in a batch-refeed mode with daily medium exchange to supply the cultures with nutrients and remove toxic byproducts. By adjusting the shaking parameters, such as the speed and setting angle, we have adapted the shake tubes to a semi-continuous production of a recombinant enzyme in a perfusion-like mode. We have demonstrated that the developed model can be used to select clones and cell culture media ahead of process optimization studies in bioreactors and confirmed the applicability of shake tubes to a perfusion-like cell culture reaching 50E6 viable cells/mL. Furthermore, through regular cell mass removal and periodic medium exchange we have successfully maintained satellite cultures of bench-top perfusion bioreactors, achieving a sustainable cell culture performance at 30E6 viable cells/mL and viabilities >80% for over 58 days. The established HT model is a unique and powerful tool that can be used for the development and screening of media formulations, or for testing selected process parameters during both process optimization and manufacturing support campaigns.
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Glucocorticoids are effective for the treatment of acute-on-chronic pre-liver failure, severe chronic hepatitis B and acute liver failure; however, the mechanism underlying the effects of treatment by glucocorticoids remains to be fully elucidated. The role and detailed mechanism of how glucocorticoids prevent liver disease progression can be elucidated by investigating the apoptosis of hepatocytes following glucocorticoid treatment. Pglycoproteins (Pgps) also confer resistance to apoptosis induced by a diverse range of stimuli. Glucocorticoids, particularly dexamethasone (DEX), upregulate the expression of Pgp in several tissues. In the present study, the normal human L02 liver cell line was used, and techniques, including immunocytochemistry, western blot analysis, flow cytometry and reverse transcriptionquantitative polymerase chain reaction analysis were used for determining the expression levels of Pgps, and for evaluating the effect of DEX pretreatment on the expression of Pgps. DEX (110 microM) was added to the cell culture media and incubated for 2472 h. The results revealed that DEX upregulated the mRNA and protein levels of Pgp in a dose and timedependent manner. Subsequently, tumor necrosis factorrelated apoptosisinducing ligand (TRAIL) was used for the induction of apoptosis in the cells, followed by a terminal deoxynucleotidyl transferase dUTP nick end labeling assay to assess the apoptotic stages. The results demonstrated that apoptosis in the group of cells, which were pretreated with DEX was significantly lower than that in the control group. Treatment with tariquidar, a Pgp inhibitor, reduced the antiapoptotic effects of DEX. These results established that DEX protects normal human liver cells from TRAILinduced apoptosis by upregulating the expression of P-gp. These observations may be useful for elucidating the mechanism of DEX for preventing the progression of liver disease.
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This review focuses on cultivation of mammalian cells in a suspended perfusion mode. The major technological limitation in the scaling-up of these systems is the need for robust retention devices to enable perfusion of medium as needed. For this, cell retention techniques available to date are presented, namely, cross-flow filters, hollow fibers, controlled-shear filters, vortex-flow filters, spin-filters, gravity settlers, centrifuges, acoustic settlers, and hydrocyclones. These retention techniques are compared and evaluated for their respective advantages and potential for large-scale utilization in the context of industrial manufacturing processes. This analysis shows certain techniques have a limited range of perfusion rate where they can be implemented (most microfiltration techniques). On the other hand, techniques were identified that have shown high perfusion capacity (centrifuges and spin-filters), or have a good potential for scale-up (acoustic settlers and inclined settlers). The literature clearly shows that reasonable solutions exist to develop large-scale perfusion processes. ? 2003 Wiley Periodicals, Inc. Biotechol Bioeng 82: 751-765, 2003.
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Mechanisms for increased antibody production in batch cultures of murine hybridoma cells in response to hyperosmotic stress were investigated. The rates of immunoglobulin transcription and protein translation and posttranslational processing were determined in control and hyperosmotic cultures. Changes in immunoglobulin transcription played a minor role in the increase in antibody production in response to hyperosmotic stress. In contrast, protein translation increased substantially in response to osmotic stress. However, the antibody translation rate remained relatively constant after correcting for the overall increase in protein translation. Cell size and intracellular antibody pool also increased in response to hyperosmolarity. The intracellular antibody pool increased proportionately with the increase in cell size, indicating that hyperosmotic cultures do not selectively increase their intracellular antibody population. Changes in cell cycle distribution in response to osmotic stress and the relationship between the cell cycle and antibody production were also evaluated. Hyperosmotic stress altered the cell cycle distribution, increasing the fraction of the cells in S-phase. However, this change was uncorrelated with the increase in antibody production rate. Immunoglobulin degradation was relatively low (15%) and remained largely unchanged in response to hyperosmotic stress. There was no apparent increase in immunoglobulin stability as a result of osmotic stress. Antibody secretion rates increased approximately 50% in response to osmotic stress, with a commensurate increase in the antibody assembly rate. The rate of transit through the entire posttranslational processing apparatus increased, particularly for immunoglobulin light chains. The levels of endoplasmic reticulum chaperones did not increase as a fraction of the total cellular protein but were increased on a per cell basis as the result of an increase in total cellular protein. A difference in the interactions between the immunoglobulin heavy chains and BiP/GRP78 was observed in response to hyperosmotic conditions. This change in interaction may be correlated with the decrease in transit time through the posttranslational pathways. The increase in the posttranslational processing rate appears to be commensurate with the increase in antibody production in response to hyperosmotic stress.
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To investigate the effect of hyperosmotic medium on production and aggregation of the variant of Angiopoietin-1 (Ang1), cartilage oligomeric matrix protein (COMP)–Ang1, in recombinant Chinese hamster ovary (CHO) cells, CHO cells were cultivated in shaking flasks. NaCl and/or sorbitol were used to raise medium osmolality in the range of 300–450 mOsm/kg. The specific productivity of COMP–Ang1, q COMP–Ang1, increased as medium osmolality increased. At NaCl-450 mOsm/kg, the q COMP–Ang1 was 7.7-fold higher than that at NaCl-300 mOsm/kg, while, at sorbitol-450 mOsm/kg, it was 2.9-fold higher than that at sorbitol-300 mOsm/kg. This can be attributed to the increased relative mRNA level of COMP–Ang1 at NaCl-450 mOsm/kg which was approximately 2.4-fold higher than that at sorbitol-450 mOsm/kg. Western blot analysis showed that COMP–Ang1 aggregates started to occur in the late-exponential phase of cell growth. When sorbitol was used to raise the medium osmolality, a severe aggregation of COMP–Ang1 was observed. On the other hand, when NaCl was used, the aggregation of COMP–Ang1 was drastically reduced at NaCl-400 mOsm/kg. At NaCl-450 mOsm/kg, the aggregation of COMP–Ang1 was hardly observed. This suggests that environmental conditions are critical for the aggregation of COMP–Ang1. Taken together, the use of NaCl-induced hyperosmotic medium to cell culture process turns out to be an efficient strategy for enhancing COMP–Ang1 production and reducing COMP–Ang1 aggregation.
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Abstract BACKGROUND Changes in glycosylation profiles of monoclonal antibodies can have significant impact upon the product quality. Control of critical process parameters in order to ensure consistent product quality is one of the core requirements of the FDA's QbD and PAT initiatives. The effect of operating conditions upon cell metabolism and the glycosylation profile of monoclonal antibody produced using hybridoma cell culture is investigated in this report. RESULTS PCA analysis of online process data and amino acid concentration profiles reflecting the cell metabolism indicates significant dependence on the operating conditions, particularly DO and pH. PLS models predicting product titre based on amino acid concentration and those predicting the glycosylation profile either based on product titre and glucose concentration or on amino acid concentrations show that both titre and glycosylation can be predicted with satisfactory accuracy for a range of operating conditions. CONCLUSIONS Accurate prediction of product titre and glycosylation profile based on amino acid concentration and process variables easily measured in real time opens up the opportunity to control the product quantity and quality during cultivation using critical process parameters, such as pH and DO. 2014 Society of Chemical Industry
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Lithium chloride (LiCl), which is a specific inhibitor of glycogen synthase kinase-3 , is known to induce cell cycle arrest at the G2/M phase and to regulate apoptosis. To determine the potential of LiCl as a chemical additive to enhance specific productivity (q p) of recombinant Chinese hamster ovary (rCHO) cells through cell cycle arrest at G2/M phase, rCHO cells producing Fc-fusion protein were cultivated in serum-free media with LiCl concentrations ranging from 0 to 20 mM. The addition of LiCl induced cell cycle arrest at G2/M phase and thereby decreased the specific cell growth rate. However, LiCl increased q p in a dose-dependent manner. The beneficial effect of LiCl on q p outweighed its detrimental effect on , resulting in improved maximum Fc-fusion protein concentration (MFPC) at 10 mM LiCl. The q p and MFPC in the bioreactor culture with 10 mM LiCl were 5.0 and 2.1 times higher than those without LiCl, respectively. In addition, the presence of LiCl at 10 mM did not significantly affect either intracellular 2,3-ST or extracellular sialidase activity. LiCl also inhibited apoptosis of cells in the decline phase of growth by increasing Bcl-2 expression. Taken together, the results obtained in this study demonstrate the potential of LiCl as a q p-enhancing additive in CHO cell culture for improved recombinant protein production.
[24]
Ducommun P, Ruffieux P A, Stockar U V , et al. The role of vitamins and amino acids on hybridoma growth and monoclonal antibody production. Cytotechnology, 2001,37(2):65-73.
A balanced supplementation method was applied to develop a serum and protein- free medium supporting hybridoma cell batch culture. The aim was to improve systematically the initial formulation of the medium to prevent limitations due to unbalanced concentrations of vitamins and amino acids. In a first step, supplementation of the basal formulation with 13 amino acids, led to an increase of the specific IgA production rate from 0.60 to 1.07 pg cell(-1) h(-1). The specific growth rate remained unchanged, but the supplementation enabled maintenance of high cell viability during the stationary phase of batch cultures for some 70 h. Since IgA production was not growth- related, this resulted in an approximately4-fold increase in the final IgA concentration, from 26.6 to 100.2 mgl(-1). In a second step, the liposoluble vitamins E and K(3) were added to the medium formulation. Although this induced a slightly higher maximal cell concentration, it was followed by a sharp decline phase with the specific IgA production rate falling to 0.47 pg cell(-1) h(-1). However, by applying a second cycle of balanced supplementation with amino acids this decline phase could be reduced and a high cell viability maintained for over 300 h of culture. In this vitamin- and amino acid- supplemented medium, the specific IgA production rate reached a value of 1.10 pg cell(-1)h(-1) with a final IgA concentration of 129.8 mgl(-1). The latter represents an increase of approximately5-fold compared to the non- supplemented basal medium.
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Balcarcel R R, Stephanopoulos G . Rapamycin reduces hybridoma cell death and enhances monoclonal antibody production. Biotechnol Bioeng, 2001,76(1):1-10.
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Rouiller Y , Pe'rilleux A, Marsaut M, et al. Effect of hydrocortisone on the production and glycosylation of an Fc-Fusion protein in CHO cell cultures. Biotechnol Prog, 2012,28(3):803-813.
Glucocorticoids are known to modulate various cellular functions such as cell proliferation, metabolism, glycosylation, and secretion of many proteins. We tested the effect of hydrocortisone (HC) on cell growth, viability, metabolism, protein production, and glycosylation of an Fc-protein expressing Chinese hamster ovary (CHO) cell culture. HC extended cell viability but impaired cell growth. The inhibitory effect on cell growth was dose-dependent and decreased when the glucocorticoid addition was delayed. When HC was added after 2 or 3 days of culture, an increase in glutamate consumption was observed, which was reversed by the glucocorticoid receptor antagonist mifepristone (Mif). Titer and specific productivity increased in the presence of HC. The increase in titer was only slightly reversed by Mif. On the other hand, Mif by itself induced an increase in titer to a level comparable to or higher than HC. Protein glycosylation was altered by the glucocorticoid in a dose- and time-dependent manner, with a shift to more acidic bands, which correlated with an increase in sialic acid moieties. This increase, which was not linked to a decrease in extracellular sialidase activity in HC-treated cultures, was reversed by Mif. Predictive models based on design of experiments enabled the definition of optimal conditions for process performance in terms of viability and titer and for the quality of the Fc-fusion protein in terms of glycosylation. The data obtained suggest a use of glucocorticoids for commercial production of Fc-fusion proteins expressed in CHO cells. 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012
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Chung J D, Zabel C, Sinskey A J , et al. Extension of Sp2/0 hybridoma cell viability through interleukin-6 supplementation. Biotechnol Bioeng, 1997,55(2):439-446.
Abstract Sp2/0 hybridoma cells die principally by apoptosis in batch culture. We have found that cultures of the Sp2/0 hybridoma exhibit increased viability in response to interleukin 6 (IL-6) supplementation relative to control cultures during serum shiftdown experiments. When shifted from a medium containing 10% fetal bovine serum (FBS) to a medium with 1% FBS, IL-6 supplemented cultures displayed viabilities and viable cell densities similar to control cultures containing 10% FBS. The degree of the survival response induced varied in accordance with the severity of the shiftdown, as cells resuspended in a high serum medium showed little observable enhancement in viability. The extension in culture viability was not accompanied by an observable decrease in growth relative to control cultures, indicating that the effect was not a consequence of growth inhibition. These results suggest the existence of serum components with behavior functionally similar to IL-6, with respect to enhancing cell survival, and that under certain experimental conditions IL-6 serves as a survival factor. In contrast to the extended viability displayed by cultures supplemented with IL-6, Sp2/0 cultures transfected with IL-6 cDNA expression vectors displayed a growth inhibitory response relative to control cultures. This inhibitory response was characterized by an extended lag phase following inoculation, and a decrease in batch culture cell yield. The depression in cell yield varied with serum concentration, with the largest depression occurring at high serum concentrations. We conclude that interactions between components in serum, presumably growth factors, and cytokines play an important role in altering the behavior of industrially relevant cell lines in culture. 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 439 446, 1997.
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Simpson N H, Singh R P, Perani A , et al. In hybridoma cultures, deprivation of any single amino acid leads to apoptotic death, which is suppressed by the expression of the bcl-2 gene. Biotechnol Bioeng, 1998,59(1):90-98.

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The authors have declared that no competing interests exist.

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