pH对重组CHO细胞生长、单抗表达及质量的影响北大核心CSCD

在1 L反应器中探究p H对CHO细胞生长、单抗表达及质量的影响。在1 L反应器中对p H进行探究,实验研究表明当p H为7.05时最适合CHO细胞生长和抗体表达,在此条件下培养时第9天获得最高细胞密度1.54×107cells/m L,在第11天获得最高抗体浓度1 355.71 mg/L。p H对p CO2、乳酸积累、抗体的单体含量、电荷异质性和糖基化也有较大影响,当p H在6.95至7.25之间时,高p H培养能够降低乳酸积累和p CO2,但是会导致抗体的碱性峰增加。p H两项培养能够降低细胞活力,从而提高抗体表达量。     

pH对重组CHO细胞生长、单抗表达及质量的影响

在1 L反应器中探究p H对CHO细胞生长、单抗表达及质量的影响。在1 L反应器中对p H进行探究,实验研究表明当p H为7.05时最适合CHO细胞生长和抗体表达,在此条件下培养时第9天获得最高细胞密度1.54×107cells/m L,在第11天获得最高抗体浓度1 355.71 mg/L。p H对p CO2、乳酸积累、抗体的单体含量、电荷异质性和糖基化也有较大影响,当p H在6.95至7.25之间时,高p H培养能够降低乳酸积累和p CO2,但是会导致抗体的碱性峰增加。p H两项培养能够降低细胞活力,从而提高抗体表达量。     

连续灌流培养杂交瘤细胞生产单克隆抗体

自 2 0世纪 70年代以来 ,工程抗体在基础医学研究、临床诊断和治疗 ,以及免疫预防等领域中的广泛应用 ,大大促进了其产业化的进程。目前工业化生产单克隆抗体的主要方法是通过发酵罐、中空纤维和固定床等生物反应器培养系统 ,以微载体、微包囊法在体外大规模高密度培养杂交瘤细胞 ,再通过相关的纯化手段浓缩纯化制备抗体[1 ,2 ] 。就操作方式而言 ,一般采用两个基本策略 :①大容量高密度的悬浮培养 ,最多采用的是搅拌式气升式生物反应器 ,通过微载体依托细胞相对固定化 ,降低了搅拌培养时对细胞的剪切力 ,提高细胞的密度和稳定性及生产率。…     

表达内皮抑素的rCHO细胞的微囊化培养

微囊化重组基因细胞移植治疗肿瘤是一种新兴的肿瘤基因治疗方法,如果将此技术应用到临床研究,就需要制备大量的细胞活性良好、重组蛋白表达量高的生物微胶囊。种子细胞是生物微胶囊治疗作用的执行者, 是构建微囊微反应器的基本元素。如何获得大量高活性的种子细胞已经成为规模化制备生物微胶囊所面临的最关键的限制因素。本实验考察了搅拌式生物反应器内扩增的重组CHO细胞进行包囊及微囊化细胞在生物反应器内规模化培养的可行性。实验结果显示:重组CHO细胞在生物反应器内可以快速生长,并且对数期细胞包囊,微囊化细胞活性良好。制备的微囊化细胞可以在生物反应器内培养,与培养板培养比较细胞生长较快、内皮抑素表达量较高。应用生物反应器培养技术能够在体外快速、大量扩增重组CHO细胞,满足微囊化细胞制备对种子细胞量与质的要求,微囊化细胞可以在生物反应器内培养。     

短期连续剪切对光生物反应器内海带配子体细胞生长及其恢复能力的影响

以不同搅拌速率(0~1000r/min)对海带(Laminaria japonica)配子体细胞施加短期(0~60h)连续剪切,卸载剪切力之后细胞静止恢复23.5d,在此期间研究不同搅拌速率分别在连续剪切和恢复期间对细胞生长及其恢复能力的影响。研究结果表明,连续剪切期间,90r/min下细胞叶绿素浓度积累达到最大值2.36mg/L;中高速搅拌速率(270~1000r/min)下叶绿素浓度迅速下降,胞内氮磷池释放,1000r/min下细胞损伤率为静止对照样的18倍。恢复期间,所有组别细胞均呈现较强的恢复能力。此外,在连续剪切实验中,海带配子体细胞内叶绿素浓度比干重更能有效的表达生物量浓度,除细胞损伤率外,磷源释放可以作为判别细胞受损的参考指标之一。     

表达重组抗CD52单克隆抗体CHO细胞灌流培养基的筛选

目的筛选重组抗CD52单克隆抗体CHO细胞株培养和连续灌流表达用培养基,以提高抗体表达量。方法通过调整原有批培养用培养基中谷氨酰胺和植物水解蛋白,获得5种培养基配比。使用模拟灌注方式进行细胞培养,分析细胞密度、活细胞比率和目标蛋白表达,筛选连续灌流细胞培养和表达用培养基。最后在7 L反应器中采用灌注培养方式对筛选获得的培养基进行验证。结果使用50 mL细胞培养管进行模拟灌注培养时,活细胞比率较高,达到90%以上;CHO细胞在添加谷氨酰胺至4.0 mmol/L和植物水解蛋白至5.0 g/L的批培养用培养基中生长速度最快;在基础培养基中抗体表达量比优化前高15%。20 d培养周期内,优化的培养基在7 L反应器中可以维持CHO细胞密度在(2 727±253)万个/mL,活细胞比率在95%以上。结论通过模拟灌注培养,筛选获得了一种在7 L反应器灌流培养中适宜于重组抗CD52单克隆抗体CHO细胞表达的培养基。     

短期连续剪切对光生物反应器内海带配子体细胞生长及其恢复能力的影响

以不同搅拌速率（0～1000r/min）对海带（Laminaria japonica）配子体细胞施加短期（0～60h）连续剪切,卸载剪切力之后细胞静止恢复23.5d,在此期间研究不同搅拌速率分别在连续剪切和恢复期间对细胞生长及其恢复能力的影响。研究结果表明,连续剪切期间,90r/min下细胞叶绿素浓度积累达到最大值2.36mg/L;中高速搅拌速率（270～1000r/min）下叶绿素浓度迅速下降,胞内氮磷池释放,1000r/min下细胞损伤率为静止对照样的18倍。恢复期间,所有组别细胞均呈现较强的恢复能力。此外,在连续剪切实验中,海带配子体细胞内叶绿素浓度比干重更能有效的表达生物量浓度,除细胞损伤率外,磷源释放可以作为判别细胞受损的参考指标之一。     

抗汉滩病毒鼠/人嵌合抗体工程细胞的驯化、抗体表达及鉴定

目的:通过对稳定表达抗汉滩病毒鼠/人嵌合抗体的CHO细胞进行驯化,检测其抗体的表达情况,并对所表达抗体的生物学活性进行初步鉴定。方法:将本课题组前期筛选得到的稳定高表达细胞株1-D9进行无血清驯化,使其适合悬浮培养后接种到摇瓶,通过调整不同的培养条件,每天检测细胞的活率和抗体表达量,并对表达的抗体进行生物学活性鉴定。结果:通过驯化得到了适合悬浮培养的CHO细胞株,其抗体表达量高,生产周期短;并优化了其悬浮培养条件,表达的抗体经鉴定其生物学活性稳定。结论:优化了抗汉滩病毒鼠/人嵌合抗体表达条件,为下一步工业化生产提供了实验依据。     

多孔微球固定化CHO工程细胞生产rt-PA的研究

以Cytopore多孔微球固定产重组组织型纤溶酶原激活剂(rtPA)CHO工程细胞株4B3,在2L搅拌式生物反应器用无血清培养基DF5S连续灌流培养。4B3细胞的最大活细胞密度和rtPA生产水平分别达到8.83×10⁶/mL和12473 IU/mL。含rtPA的4B3细胞培养上清经MPG吸附层析和Lysine-sepharose 4B亲和层析两步纯化,rt-PA的纯度达到98%。     

分泌重组抗体的CHO细胞体外培养条件的优化

目的：对生物反应器细胞培养时培养时培养基组成进行优化。方法：以稳定转染了抗CD3人源化抗体的CHO细胞为模型,以无血清培养基经生物反应器高密度细胞培养后分子量小于50kDR的超滤液为基础培养基,在细胞培养板中考察添加氨基酸、丁酸钠、柠檬酸铁等多种成分对细胞生长状态和蛋白表达量的影响、结果：2mmol/L丁酸钠可以有效地诱导蛋白的表达,丁酸钠和柠檬酸铁对于促蛋白表达有协同作用,适量添加培养过程中消耗较快的氨基酸可提高细胞数和蛋白的表达量。结论：利用所述方法可快速优化培养基成分,显著提高生物反应器中细胞的蛋白表达量。     

A comprehensive comparison of mixing, mass transfer, Chinese hamster ovary cell growth, and antibody production using Rushton turbine and marine impellers

Large scale production of monoclonal antibodies has been accomplished using bioreactors with different length to diameter ratios, and diverse impeller and sparger designs. The differences in these physical attributes often result in dissimilar mass transfer, mechanical stresses due to turbulence and mixing inside the bioreactor that may lead to disparities in cell growth and antibody production. A rational analysis of impeller design parameters on cell growth, protein expression levels and subsequent antibody production is needed to understand such differences. The purpose of this study was to examine the impact of Rushton turbine and marine impeller designs on Chinese hamster ovary (CHO) cell growth and metabolism, and antibody production and quality. Experiments to evaluate mass transfer and mixing characteristics were conducted to determine if the nutrient requirements of the culture would be met. The analysis of mixing times indicated significant differences between marine and Rushton turbine impellers at the same power input per unit volume of liquid (P/V). However, no significant differences were observed between the two impellers at constant P/V with respect to oxygen and carbon dioxide mass transfer properties. Experiments were conducted with CHO cells to determine the impact of different flow patterns arising from the use of different impellers on cell growth, metabolism and antibody production. The analysis of cell culture data did not indicate any significant differences in any of the measured or calculated variables between marine and Rushton turbine impellers. More importantly, this study was able to demonstrate that the quality of the antibody was not altered with a change in the impeller geometry.     

Performance of mammalian cell culture bioreactor with a new impeller design

To improve the oxygen transfer in a mammalian cell bioreactor, a new type of impeller consisting of a double-screen concentric cylindrical cage impeller (annular cage impeller in short) was designed and its mass transfer rate evaluated. This new impeller design increases the specific screen area, and the convective mass transfer rate through the annular cage was significantly increased. The oxygen transfer rates with the new impeller and the commercially available cell-lift impeller (CelliGen by New Brunswick Scientific Co.) were evaluated and their performance compared at various rates of aeration and agitation. The results showed that with the new impeller, the oxygen transfer rate was increased by 19% in water and 21% in cell-free culture medium supplemented with 10% horse serum, the total hybridoma cell concentration was increased to 3.4 x 10(7) cells/mL, and the IgG(1) subtype monoclonal antibody (MAb) product concentration was also increased to 512 mg/L in perfusion culture of murine hybridoma cell line 62'D3. These improvements in oxygen transfer rate, cell concentration, and MAb product concentration are all very significant. The mass transfer resistance in the cell-lift impeller system was found to be mainly due to the surface area of the single-screen cage impeller. The new annular cage impeller not only provided the increased surface area for convective oxygen transfer but also protected cells from hydrodynamic shear damage, thereby achieving a significant bioprocess improvement in terms of higher viable cell concentration, higher product concentration, and higher oxygen transfer rate in the mammalian cell bioreactor system.     

Shear stress effects on cell growth and <Emphasis Type="SmallCaps">L</Emphasis>-DOPA production by suspension culture of <Emphasis Type="Italic">Stizolobium hassjoo</Emphasis> cells in an agitated bioreactor

Suspension cultures of Stizolobium hassjoo cells were cultivated in a 7l bioreactor. The growth rate and intracellular L-DOPA content of the cells using two different turbine impellers were compared. There were distinct differences in growth behavior and L-DOPA productivity in the range of 100 to 500 rpm for flat-blade turbine impeller. Disk turbine retarded significantly the cell growth but not so significantly for L-DOPA production in the range of 200 to 300 rpm. The shear force intensity of the two impellers at various rotational rates was compared with shear force index (SFI), and power input per unit mass and eddy length scale. There was good consistency among the three indexes for shear force intensity. Thus with SFI the shear force intensity of bioreactor can be indirectly estimated. A critical shear stress that may cause sublytic effect in cells was identified for flat-blade turbine operated at 400 rpm. The common effect between the shear stress and the proton elicitation in the bioreactor was elucidated with a hypothesis of signal transduction by second messenger, H⁺. Our results suggested that H⁺ transduced the signal to protoplast when S. hassjoo cells were stimulated by shear stress. This resulted in an increase of H⁺ which triggered a similar reaction to the pH control of culture broth and enhanced the L-DOPA production.     

Evaluation of mixing effect and shear stress of different impeller combinations on nemadectin fermentation

Mass transfer and shear force have significant effects on nemadectin production by Streptomyces cyaneogriseus ssp. noncyanogenus. They are always the conflict-ridden problems in nemadectin fermentation process. In this study, the flow field characteristics under different impeller combinations were quantitatively evaluated in 5 L stirred-tank bioreactor through the laser particle image velocimetry (PIV) system. Results demonstrated that the radial-axial impeller combinations with the time average velocity at 0.38-0.54 U_tip, the turbulent kinetic energy dissipation rate at 6.4–10.6 ε/N³D², and the shear stress rate was 40-150 s⁻¹, were more conductive to cell growth, nemadectin biosynthesis, cell’s activity, respiratory metabolism than other combinations. The highest nemadectin yield was evaluated up to 1543.3 ± 18.5 μg/mL, which was 31.68 % higher than that of the radial flow impeller combinations. This study provided the important guideline for the selection impeller combinations’ on large-scale nemadectin production.     

A novel centrifugal impeller bioreactor. I. Fluid circulation, mixing, and liquid velocity profiles

A novel centrifugal impeller bioreactor for shear-sensitive biological systems was designed by installing a centrifugal-pumplike impeller in a stirred vessel. The fluid circulation, mixing, and liquid velocity profiles in the new bioreactor (5-L) were assessed as functions of the principal impeller designing and bioreactor operating parameters. The performances of the centrifugal impeller bioreactor were compared with those of a widely used cell-lift bioreactor. The newly developed bioreactor showed higher liquid lift capacity and shorter mixing time than the cell lift with comparable dimensions. Furthermore, the experiments of the liquid velocity profiles around an impeller region indicated that the centrifugal impeller bioreactor produced lower shear stress than the cell lift. This conclusion was also supported by evaluating the changes in size distributions of granulated agar particles that were sheared with those two types of impeller.     

High density and scale-up cultivation of recombinant CHO cell line and hybridomas with porous microcarrier Cytopore

Using porous microcarrier Cytopore and a low-serum medium supplement BIGBEF-3, we have successfully cultivated recombinant CHO cell line CL-11G producing prourokinase and hybridomas producing anti-prourokinase monoclonal antibody in Celligen 1.5 or 5 L bioreactor. The cell density obtained ranged from 1 to 2 × 107 cells mL-1. The yields of prourokinase and monoclonal antibody increased with increasing cell density. As the cells could spontaneously release from and reattach to porous microcarriers, it was very easy to scale-up the cultivation. Thus the bead to bead cell transfer method has been used to scale up the cultivation of CL-11G cells to a 20 L reactor-scale for the pilot production of prourokinase, and also to scale-up the culture of hybridomas for the production of monoclonal antibody for the purification of prourokinase. This revised version was published online in July 2006 with corrections to the Cover Date.     

Efficient and reproducible mammalian cell bioprocesses without probes and controllers?

Bioprocesses for recombinant protein production with mammalian cells are typically controlled for several physicochemical parameters including the pH and dissolved oxygen concentration (DO) of the culture medium. Here we studied whether these controls are necessary for efficient and reproducible bioprocesses in an orbitally shaken bioreactor (OSR). Mixing, gas transfer, and volumetric power consumption (P(V)) were determined in both a 5-L OSR and a 3-L stirred-tank bioreactor (STR). The two cultivation systems had a similar mixing intensity, but the STR had a lower volumetric mass transfer coefficient of oxygen (k(L)a) and a higher P(V) than the OSR. Recombinant CHO cell lines expressing either tumor necrosis factor receptor as an Fc fusion protein (TNFR:Fc) or an anti-RhesusD monoclonal antibody were cultivated in the two systems. The 5-L OSR was operated in an incubator shaker with 5% CO(2) in the gas environment but without pH and DO control whereas the STR was operated with or without pH and DO control. Higher cell densities and recombinant protein titers were obtained in the OSR as compared to both the controlled and the non-controlled STRs. To test the reproducibility of a bioprocess in a non-controlled OSR, the two CHO cell lines were each cultivated in parallel in six 5-L OSRs. Similar cell densities, cell viabilities, and recombinant protein titers along with similar pH and DO profiles were achieved in each group of replicates. Our study demonstrated that bioprocesses can be performed in OSRs without pH or DO control in a highly reproducible manner, at least at the scale of operation studied here.     

Understanding the effect of high gas entrance velocity on Chinese hamster ovary (CHO) cell culture performance and its implications on bioreactor scale-up and sparger design

There are three main potential sources for cell shear damage existing in stirred tank bioreactors. One is the potential high energy dissipation in the immediate impeller zones; another from small gas bubble burst; and third is from high gas entrance velocity (GEV) emitting from the sparger. While the first two have been thoroughly addressed for the scale-up of Chinese hamster ovary (CHO) cell culture knowing that a wide tolerable agitation range with non-damaging energy dissipation exists and the use of shear protectants like Pluronic F68 guard against cell damage caused by bubble burst, GEV remains a potential scale-up problem across scales for the drilled hole or open pipe sparger designs. GEV as high as 170 m/s due to high gas flow rates and relatively small sparger hole diameters was observed to be significantly detrimental to cell culture performance in a 12,000 L bioreactor when compared to a satellite 2 L bioreactor run with GEV of <1 m/s. Small scale study of GEV as high as 265 m/s confirmed this. Based on the results of this study, a critical GEV of >60 m/s for CHO cells is proposed, whereas previously 30 m/s has been reported for NS0 cells by Zhu, Cuenca, Zhou, and Varma (2008. Biotechnol. Bioeng., 101, 751–760). Implementation of new large scale spargers with larger diameter and more holes lowered GEV and helped improve the cell culture performance, closing the scale-up gap. Design of such new spargers was even more critical when hole plugging was discovered during large scale cultivation hence exacerbating the GEV impact. Furthermore, development of a scale down model based on mimicry of the large scale GEV profile as a function of time was proven to be beneficial for reproducing large scale results.     

Plant cell bioreactor for the production of protoberberine alkaloids from immobilized Thalictrum rugosum cultures

Cultured Thalictrum rugosum cells were immobilized using a glass fiber substratum previously shown to provide optimum immobilization efficiency based on spontaneous adhesion mechanisms. When cultivated in shake flasks, immobilized cells exhibited decreased growth and protoberberine alkaloid production rates in comparison to freely suspended cells. Since alkaloid production is growth associated in T. rugosum, the decreased specific production rate was a function of the slower growth rate. Cells immobilized on glass fiber mats appear to be amenable for extended culture periods. Maximum biomass and protoberberine alkaloid levels were maintained for at least 14 days in immobilized cultures. In contrast, fresh weight, dry weight, and total alkaloid content decreased in suspension cultures following the linear growth phase.Glass fiber mats were incorporated in to a 4.5-L plant cell bioreactor as horizontal disks supported on a central rod. Mixing in the reactor was provided by the combined actions of a magnetic impeller and a cylindrical sparging colum. fThe magnetic impeller and a cylindrical sparging column. The entire inoculum biomass of T. rougosum, introduced as suspension, was spontaneously immobilized with in 8h. During liner phase, the growth rate of bioreactor cultivated immobilized cells (mu = 0.06 day(-1)) was 50% that immobilized cell viability in both systems was determined to be similar. The increase in specific production of protoberberine alklodis was initially similar in bioreactor-and culture period. The increase in specific production of protoberberine alkaloids was initially similar in bioreactor-and shake-flask-cultivated immobilized cells. However, the maximum specific production of bioreactor grown cultures was lower. The scale up potential of an immobilization strategy based on the spontaneous adhesion of immobilization strategy based on the spontaneous adhesion of cultured plant cells to glass fiber is demonstrated.     

A quantitative analysis of shear effects on cell suspension and cell culture of perilla frutescens in bioreactors

The short-time effects of shear on suspended cells of Perilla frutescens were quantitatively analyzed by exposing the cells to a well-defined flow field in a rotating drum reactor. It was found that both shear rate and shearing time significantly affected cell viability. The quantitative effects of shear on cell growth and the production of anthocyanin, a secondary metabolite, by the cell cultures were further investigated in a series of batch cultivations using a 5-L plant cell bioreactor with a marine impeller. The results indicated that there was an optimum range of shear rate; i.e., an average shear rate of 20 to 30 s(-1) or an impeller tip speed of 5 to 8 dm/s, which maximized all the values of the following parameters: the specific growth rate, the maximum cell concentration, the (specific) production and productivity of anthocyanin, and the cell and anthocyanin yields. (c) 1994 John Wiley & Sons, Inc.