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。     

用填充床生物反应器连续灌流培养CHO细胞生产HBsAg

为了获得重组CHO细胞持续高效表达HBsAg的最佳条件 ,应用填充床生物反应器和聚酯片 ,连续灌流培养分泌HBsAg的重组CHO细胞 ,考察灌流培养基中葡萄糖浓度对细胞代谢和HBsAg生产的影响。连续培养 60天 ,细胞密度可达 5 0× 1 0 6 ml。灌流培养液中葡萄糖浓度从5 0g L增加到 7 6g L时 ,葡萄糖消耗速率和乳酸浓度均随之上升。当葡萄糖浓度继续增加至9 3g L时 ,葡萄糖消耗速率和乳酸浓度呈下降趋势 ;当将葡萄糖浓度降回至 7 6g L后又开始回升。培养过程中最高抗原滴度达 1∶5 1 2 ,出现在以含 5 0g L葡萄糖的培养液灌流阶段的末期 ,即第 2 6天 ,维持 1 0天左右即迅速下降至 1∶1 2 8水平。共收液 335L ,纯化后获得抗原蛋白2 1 3 0 4mg ,平均产率为 635 94μg L ,较传统转瓶工艺 ( 4 1 4μg L)提高 5 3 6%。表明CHO细胞较长时间处于高乳酸水平下 ( >30mmol L)会严重影响产物的表达 ,应控制灌流培养液中的葡萄糖浓度在较低水平 ,或通过适当提高灌流速率使培养基中乳酸水平维持在较低水平 ,从而有利于HBsAg的高效稳定表达     

狂犬病毒单克隆抗体CHO细胞灌流培养中细胞特异性灌流速率的研究

目的 研究灌流培养中,不同的细胞特异性灌流速率(cell specific perfusion rate, CSPR)对狂犬病毒单克隆抗体CHO细胞生长及抗体蛋白表达的影响,摸索适合本细胞株灌流培养的CSPR。方法 在标号为CSPR 1～5[CSPR1;0.02 nL/(细胞·d)、CSPR2:0.03 nL/(细胞·d)、CSPR3:0.04 nL/(细胞·d)、CSPR4:0.05 nL/(细胞·d)、CSPR5:0.06 nL/(细胞·d),每组3个重复]的15个TPP管中按100万个/mL的初始密度接种相同的CHO种子细胞;摇床中,以转速225 r/min、CO₂浓度5.0%、湿度80%和温度37℃的条件培养细胞;以后每天取细胞样品,分别检测活细胞密度(viable cell density, VCD)、细胞活率、葡萄糖浓度、乳酸浓度和渗透压。接种3 d起,每天分别从CSPR 1～5的各管中,按0.02～0.06 nL/(细胞·d)的CSPR计算所需要更换的细胞悬液体积,将各管中的细胞悬液离心,取出相应体积的上清并补入相同体积的新鲜培养液重悬细胞后,继续培...     

枸杞原生质体培养及高效成株体系的建立

由枸杞髓部组织诱导出胚性愈伤组织,并由此愈伤组织建立起稳定的细胞悬浮系。从悬浮细胞游离的原生质体在改良KM培养基(1.5 mg/L 6_BA,0.5 mg/L NAA和0.5 mg/L 2,4_D)中进行液体浅层培养,3～4 d后出现第一次分裂,第7 d统计分裂频率为50.3%,15 d左右可形成细胞团,3～4周后形成肉眼可见的愈伤组织,愈伤组织植板率为1.25%。将细胞团转移到液体分化培养基(MS+6_BA 1.5 mg/L+2,4_D 0.2 mg/L) 8～10 d可形成大量胚状体,及时将胚性愈伤组织块转移到固体分化培养基上(MS+6_BA 0.2 mg/L),可形成大量绿芽,分化率54.17%。绿芽在生根培养基（MS+NAA 0.2 mg/L）可形成完整植株,移栽后成活良好。     

枸杞原生质体培养及高效成株体系的建立

由枸杞髓部组织诱导出胚性愈伤组织,并由此愈伤组织建立起稳定的细胞悬浮系,从悬浮细胞游离的原生质体在改良KM培养基（1．5mg/L 6-BA,0.5mg/L NAA和0．5mg/L2,4-D)中进行液体浅层培养,3－4d后出现第一次分裂,第7d统计分裂频率为50．3％,15d左右可形成细胞团,3－4周后形成肉眼可见的愈伤组织,愈伤组织植板率为1．25％,将细胞团转移到液体分化培养基（MS＋6－BA 1．5mg/L 2,4-D 0.2mg/L)8-10d可形成大量胚状体,及时将胚性愈伤组织块转移到固体分化培养基上（MS 6-BA 0.2mg/L)可形成大量绿芽,分化率54．17％。绿芽在生根培养基（MS＋NAA 0．2mg/L)可形成完整植株,移栽后成活良好。     

应用生物反应器连续培养基因重组CHO细胞的研究

应用5L生物反应器悬浮培养乙肝重组DNA转化的CHO细胞B43株生产HBsAg。试验了细胞接种量、微载体的加入方式、培养方法(半流加培养和连续灌流名养)对细胞生长形态和HBsAg分泌的影响,初步建立了5L生物反应器的生产工艺,在5L生物反应器最适合连续灌流培养的条件是pH 7．30—7．40,Do 25～3;％,T 36．5℃,微载体6～8g／L,灌流速度5．5—7．5L,24h细胞连续培养60天,细胞密度维持在5．O×10　6～1．O×10　7／ml之间,收获细胞液的RPHA效价为l：512—1：1024．HlBsAg含量为3－5mg／I。     

‘正午’牡丹微繁殖体系的建立

本研究以‘正午’牡丹腋芽为外植体,建立了高效的牡丹微繁殖体系。腋芽的初始培养基为WPM+0.5mg/L BA+0.2mg/L GA3,培养50d后,一个丛生芽平均可切分为13个繁殖体用于增殖培养;增殖培养基为WPM[1668mg/L Ca(NO3)2·4H2O]+0.5mg/L BA+0.2mg/L GA3,以35d为一个继代培养周期,增殖率为3.0,共继代培养7次;生根培养时,先将无根苗在复壮培养基[1/2MS(296mg/L CaCl2)+0.5g/L活性炭]上培养20d,再转入根诱导培养基[1/2 MS(296 mg/L CaCl2)+1.0 mg/L腐胺+1.0 mg/L IBA]培养30d,最后转入根形成培养基[1/2 MS(296mg/L CaCl2)+4.0g/L活性炭]培养20d,其生根率达77.2%;驯化与移栽基质为珍珠岩∶蛭石∶草炭土=1∶1∶1,组培苗移栽成活率高达92.1%。这表明以‘正午’牡丹腋芽建立的微繁殖体系具备规模化商业生产的价值。     

紫花苞舌兰类原球茎诱导及植株再生

为建立紫花苞舌兰类原球茎离体再生体系,本研究以其腋芽为外植体,研究了不同激素浓度配比、添加剂对其类原球茎诱导、分化及生根诱导的影响。结果表明,外植体在1/2 MS+6-BA2.0 mg/L+NAA0.2 mg/L+AC(活性炭)0.2 g/L+CW(椰汁)10%培养基上培养35 d后,获得无菌苗,其成活率达80%以上;无菌苗的顶端分生组织在1/2 MS+10 mg/L Picloram培养基上的类原球茎的诱导率最高,达75%;类原球茎在1/2 MS+6-BA 2.0 mg/L+IBA 0.2 mg/L培养基上培养45 d后分化出小苗,分化率为54%;小苗在1/2 MS+6-BA 0.5 mg/L+IBA 1.0 mg/L+AC 0.2 g/L培养基上生根培养35 d后,生根率达85%以上;小苗驯化移栽成活率达95%,长势良好。     

牛肾细胞在两种不同载体中培养效果的比较

目的通过牛肾细胞在两种不同载体中培养效果的比较,为牛肾细胞在细胞工厂中规模化生产提供真实的、有力的支持。方法不同代次牛肾细胞在两种载体中经过相同培养条件进行培养。结果实验中原代牛肾细胞在细胞工厂接种密度为5.5×104/cm2左右,在15 L转瓶接种密度为9.0×104/cm2左右。一代牛肾细胞在细胞工厂接种密度为6.5×104/cm2左右,在15 L转瓶接种密度为10×104/cm2左右。二代牛肾细胞在细胞工厂接种密度为7.0×104/cm2左右,在15 L转瓶接种密度为14×104/cm2左右。两种载体中牛肾细胞生长状况均能达到培养要求。结论细胞工厂能在有限的空间内利用最大限度的培养表面培养牛肾细胞,不仅节约了传代前的细胞用量,而且提高了培养后的细胞产量。     

利用高通量生物反应器优化一种CHO细胞无血清培养基

研究以DMEM/F12(1:1 V/V)培养基为基础,添加不同添加剂优化一种适宜CHO DG44细胞生长的廉价培养基。以细胞密度和细胞活率为主要指标,对DMEM/F12(1:1 V/V)培养基进行了优化。通过正交试验和单因素试验筛选出了CHO DG44细胞生长的最佳培养基。正交试验结果表明添加8mg/L Insulin、10mg/L Transferrin、12mM Glutamine、9mg/L Ethanolamine、9mg/L Sodium selenite、0.5×Lipids、0.5×Vitamin,对细胞生长有较好促进作用,细胞密度从0.6×106 cells/mL上升到1.8×106 cells/mL。在此基础上添加2.5g/L Malt Peptone和2.5g/L YeastExtract可使细胞密度达到2.65×106 cells/mL,基本上达到商业培养基的培养效果,而成本降低了约60%。     

Perfusion culture of hybridoma cells for hyperproduction of IgG(2a) monoclonal antibody in a wave bioreactor-perfusion culture system

A novel wave bioreactor-perfusion culture system was developed for highly efficient production of monoclonal antibody IgG2a (mAb) by hybridoma cells. The system consists of a wave bioreactor, a floating membrane cell-retention filter, and a weight-based perfusion controller. A polyethylene membrane filter with a pore size of 7 microm was floating on the surface of the culture broth for cell retention, eliminating the need for traditional pump around flow loops and external cell separators. A weight-based perfusion controller was designed to balance the medium renewal rate and the harvest rate during perfusion culture. BD Cell mAb Medium (BD Biosciences, CA) was identified to be the optimal basal medium for mAb production during batch culture. A control strategy for perfusion rate (volume of fresh medium/working volume of reactor/day, vvd) was identified as a key factor affecting cell growth and mAb accumulation during perfusion culture, and the optimal control strategy was increasing perfusion rate by 0.15 vvd per day. Average specific mAb production rate was linearly corrected with increasing perfusion rate within the range of investigation. The maximum viable cell density reached 22.3 x 105 and 200.5 x 105 cells/mL in the batch and perfusion culture, respectively, while the corresponding maximum mAb concentration reached 182.4 and 463.6 mg/L and the corresponding maximum total mAb amount was 182.4 and 1406.5 mg, respectively. Not only the yield of viable cell per liter of medium (32.9 x 105 cells/mL per liter medium) and the mAb yield per liter of medium (230.6 mg/L medium) but also the mAb volumetric productivity (33.1 mg/L.day) in perfusion culture were much higher than those (i.e., 22.3 x 105 cells/mL per liter medium, 182.4 mg/L medium, and 20.3 mg/L.day) in batch culture. Relatively fast cell growth and the perfusion culture approach warrant that high biomass and mAb productivity may be obtained in such a novel perfusion culture system (1 L working volume), which offers an alternative approach for producing gram quantity of proteins from industrial cell lines in a liter-size cell culture. The fundamental information obtained in this study may be useful for perfusion culture of hybridoma cells on a large scale.     

用改装的Ｓuper—Spinner搅拌瓶连续灌流培养产凝血酶原ＣＨＯ工程细胞

在动物细胞培养过程中对培养体系实施培基连续灌流能及时地补充细胞生长所需的营养物质、控制细胞代谢产物对细胞的影响 ,实现细胞的高密度长期培养 ,提高目的产品的生产效率[1,2 ] 。细胞连续灌流培养的前提是在实施培基连续灌流的同时培养体系能有效地截留细胞[3] 。这一前提增加了细胞培养装置的复杂程度 ,使之特化为价格昂贵的生物反应器 ,限制了细胞连续灌流培养的应用。如能通过对普通的细胞搅拌培养瓶进行改进 ,使之能用于细胞的连续灌流培养 ,则有利于细胞连续灌流培养的推广应用。1　材料和方法1 1　细胞产人重组凝血酶原ＣＨＯ工…     

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。…     

Long-term stable production of monocyte-colony inhibition factor (M-CIF) from CHO microcarrier perfusion cultures

Monocyte-colony inhibition factor (M-CIF) was produced in microcarrier perfusion cultures from engineered Chinese hamster ovary (CHO) cells. Three and fifteen liter microcarrier perfusion bioreactors equipped with internal spin filters were operated for over two months. Approximately 60 L and 300 L of culture filtrate were harvested from the 3L and 15L microcarrier perfusion bioreactors respectively. During the perfusion operation, cell density reached 2–6 × 10⁶ cells/ml. Importantly, stable expression of M-CIF from the CHO cells under non-selection condition was maintained at a level of 4–10 mg/L. Specific productivity was maintained at 1.8–3.4 mg/billion cells/day. The ability of the recombinant CHO cells to migrate from microcarrier to microcarrier under our proprietary HGS-CHO-3 medium greatly facilitated microcarrier culture scale-up and microcarrier replenishment. Future directions for microcarrier perfusion system scale-up and process development are highlighted. This revised version was published online in August 2006 with corrections to the Cover Date.     

Modulating and optimizing Pluronic F-68 concentrations and feeding for intensified perfusion Chinese hamster ovary cell cultures

Perfusion culture is often performed with micro-sparger to fulfill the high oxygen demand from the densified cells. Protective additive Pluronic F-68 (PF-68) is widely used to mitigate the adverse effect in cell viability from micro-sparging. In this study, different PF-68 retention ratio in alternating tangential filtration (ATF) columns was found to be crucial for cell performance of different perfusion culture modes. The PF-68 in the perfusion medium was found retained inside the bioreactor when exchanged through ATF hollow fibers with a small pore size (50 kD). The accumulated PF-68 could provide sufficient protection for cells under micro-sparging. On the other hand, with large-pore-size (0.2 μm) hollow fibers, PF-68 could pass through the ATF filtration membranes with little retention, and consequently led to compromised cell growth. To overcome the defect, a PF-68 feeding strategy was designed and successfully verified on promoting cell growth with different Chinese hamster ovary (CHO) cell lines. With PF-68 feeding, enhancements were observed in both viable cell densities (20%–30%) and productivity (~30%). A threshold PF-68 concentration of 5 g/L for high-density cell culture (up to 100 × 10⁶ cells/mL) was also proposed and verified. The additional PF-68 feeding was not observed to affect product qualities. By designing the PF-68 concentration of perfusion medium to or higher than the threshold level, a similar cell growth enhancement was also achieved. This study systematically investigated the protecting role of PF-68 in intensified CHO cell cultures, shedding a light on the optimization of perfusion cultures through the control of protective additives.     

Perfusion culture process plus H2O2 stimulation for efficient astaxanthin production by Xanthophyllomyces dendrorhous

A semicontinuous perfusion culture process (repeated medium renewal with cell retention) was evaluated together with batch and repeated fed-batch processes for astaxanthin production in shake-flask cultures of Xanthophyllomyces dendrorhous. The perfusion process with 25% medium renewal every 12 h for 10 days achieved a biomass density of 65.6 g/L, a volumetric astaxanthin yield of 52.5 mg/L, and an astaxanthin productivity of 4.38 mg/L-d, which were 8.4-fold, 5.6-fold, and 2.3-fold of those in the batch process, 7.8 g/L, 9.4 mg/L, and 1.88 mg/L-d, respectively. The incorporation of hydrogen peroxide (H(2)O(2)) stimulation of astaxanthin biosynthesis into the perfusion process further increased the astaxanthin yield to 58.3 mg/L and the productivity to 4.86 mg/L-d. The repeated fed-batch process with 8 g/L glucose and 4 g/L corn steep liquor fed every 12 h achieved 42.2 g/L biomass density, 36.5 mg/L astaxanthin yield, and 3.04 mg/L-d astaxanthin productivity. The lower biomass and astaxanthin productivity in the repeated fed-batch than in the perfusion process may be mostly attributed to the accumulation of inhibitory metabolites such as ethanol and acetic acid in the culture. The study shows that perfusion process plus H(2)O(2) stimulation is an effective strategy for enhanced astaxanthin production in X. dendrorhous cultures.     

Perfusion strategy for rosmarinic acid production by Anchusa officinalis

The production of an intracellular secondary metabolite rosmarinic acid (RA) by plant cell suspensions of Anchusa officinalis cultivated with intermittent medium exchange is investigated. Initially, a two-stage perfusion culture method was employed. After being cultured in the batch mode for ca. 6 days in B5 medium plus 3% sucrose, 1 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.1 mg/L kinetin (2,4-D B5 medium), Anchusa culture was cultivated to high cell density by perfusion during the growth stage using a hormone-free Gamborg B5 medium supplemented with 6% sucrose. This was followed by a production stage, in which a complete medium exchange into B5 medium plus 3% sucrose and 0.25 mg/L naphthleneacetic acid (NAA) was conducted. The two-stage perfusion culture had a higher maximum culture RA concentration but a lower RA content per cell than the batch stock culture maintained in the 2,4-D B5 medium. Higher culture RA concentration was due primarily to high cell density. The high packed cell volume, however, seemed to reduce the synergistic effect of NAA on RA synthesis. Subsequently, a single-stage perfusion culture method was investigated. The best result was obtained by growing the culture in the batch mode for ca. 10 days using B5 medium supplemented with 3% sucrose and 0.25 mg/L NAA, followed by perfusing the culture with B5 medium plus 6% sucrose and 0.25 mg/L NAA at a constant perfusion rate of 0.1/day. A maximum cell dry weight of 35 g/L and a RA concentration of almost 4 g/L were achieved. This is the highest RA concentration ever reported in the Anchusa culture. (c) 1993 John Wiley & Sons, Inc.     

A perfusion culture system using a stirred ceramic membrane reactor for hyperproduction of IgG2a monoclonal antibody by hybridoma cells

A novel perfusion culture system for efficient production of IgG2a monoclonal antibody (mAb) by hybridoma cells was developed. A ceramic membrane module was constructed and used as a cell retention device installed in a conventional stirred-tank reactor during the perfusion culture. Furthermore, the significance of the control strategy of perfusion rate (volume of fresh medium/working volume of reactor/day, vvd) was investigated. With the highest increasing rate (deltaD, vvd per day, vvdd) of perfusion rate, the maximal viable cell density of 3.5 x 10(7) cells/mL was obtained within 6 days without any limitation and the cell viability was maintained above 95%. At lower deltaD's, the cell growth became limited. Under nutrient-limited condition, the specific cell growth rate (mu) was regulated by deltaD. During the nonlimited growth phase, the specific mAb production rate (qmAb) remained constant at 0.26 +/- 0.02 pg/cell x h in all runs. During the cell growth-limited phase, qmAb was regulated by deltaD within the range of 0.25-0.65 vvdd. Under optimal conditions, qmAb of 0.80 and 2.15 pg/cell x h was obtained during the growth-limited phase and stationary phase, respectively. The overall productivity and yield were 690 mg/L x day and 340 mg/L x medium, respectively. This study demonstrated that this novel perfusion culture system for suspension mammalian cells can support high cell density and efficient mAb production and that deltaD is an important control parameter to regulate and achieve high mAb production.     

High productivity of human recombinant beta-interferon from a low-temperature perfusion culture

Recombinant human interferon-beta (β-IFN), used in the therapeutic treatment of multiple sclerosis (MS), can be produced on a large-scale from genetically engineered Chinese hamster ovary (CHO) cells. However, its hydrophobicity causes non-reversible, molecular aggregation in culture. The parameters affecting aggregation were determined to be concentration, culture residence time, temperature and glycosylation. Although the protein can be produced in Escherichia coli in a non-glycosylated form, the addition of glycans confers a reduced rate of aggregation as well as a 10-fold higher bioactivity. We report on the application of a low temperature perfusion culture designed to control the parameters that cause aggregation. In this three-phase culture system there is a transition to a low temperature (32°C) in a batch mode prior to implementing perfusion at 1 volume/day using an acoustic cell separator. Perfusion at the low temperature resulted in a 3.5-fold increase in specific productivity and a 7-fold increase in volumetric productivity compared to the batch culture at 37°C. The percentage aggregation of β-IFN was reduced from a maximum of 43% in batch culture to a minimum of 5% toward the end of the perfusion phase. The glycosylation profile of all samples showed predominantly sialylated biantennary fucosylated structures. The extent of sialylation, which is important for bioactivity, was enhanced significantly in the perfusion culture, compared to the batch culture.     

Two-stage depth filter perfusion culture for recombinant antibody production by recombinant Chinese hamster ovary cell

A rCHO cell line of DUKX origin 26*-320, producing recombinant antibody against the human platelet, was cultivated in a two-stage depth filter perfusion system (DFPS) for 20 days in order to attain high recombinant antibody concentration. The productivity of the first stage DFPS bioreactor reached 53 times that of the batch culture in a controlled stirred tank reactor and was showed 12.1 mg/L antibody concentration at a perfusion rate of 6.0 d⁻¹. Glucose concentration in the first DFPS was maintained at 1.5 g/L to avoid cell damage in the perfusion culture. A second stage DFPS system was attached to the first DFPS, which resulted in a low glucose concentration of 0.02 g/L and a high antibody concentration of 23.9 mg/L. The two-stage depth filter perfusion culture yielded 60% higher product concentration than the batch and 49-fold higher productivity of 69.3 mg/L/d in comparison with that (1.4 mg/L/d) in a batch system. Furthermore, antibody concentration of the second stage was 97% higher than that of the first stage, and the antibody productivities were comparable to that of the first stage. This two-stage DFPS system also showed potential for higher titer production of recombinant antibody and high volumetric productivity for long-term culture of bio-pharmaceutical substances.