不同细胞培养工艺生物反应器产率和培养基成本比较
发布时间：
2022-11-16
作者：
用于重组蛋白和单克隆抗体(mAb)生产的细胞培养工艺有不同的方式。补料分批(Feb-Batch)工艺由于操作简单，且较易规模放大，被临床和商业化生产广泛采用，目前的技术发展已可在18天内获得20-30x10^6cells/mL的细胞密度，同时获得>10g/L的滴度水平。
灌流工艺以往更多用于生产不稳定的产品，如血液凝集因子和酶类产品，但也有用于生产 mAb产品，如Remicade(英利昔单抗)。在灌流培养中，通过培养基置换，降低产物在反应器内的滞留时间，而灌流速率取决于特异性的产物和/或工艺需求。
近几年，在上游工艺中，基于灌流的工艺强化获得了极大的发展，驱动力主要来自于对降低成本和占地的需求，以及提高设备灵活性。随着细胞系、培养基和细胞截留设备的发展，现在的灌流工艺已可获得较高的细胞密度和产量，使其成为一个非常有吸引力的选择，包括mAb的生产。例如，在mAb生产中，结合2vvd的培养基置换速率，通常可达到50-60x10^6cells/mL的稳态细胞密度，以及高达4g/L/day的生物反应器产率。此外，浓缩补料分批(CFB)也可以通过培养基置换，维持高细胞密度，而将产物截留在生物反应器内。
灌流和CFB的差异在于所用的中空纤维膜的孔径。对于抗体，使用Per.C6细胞系，可在12-13天内，达到21.4g/L的终产物滴度(峰细胞密度>150x10^6cells/mL)，而使用CHO细胞系时，可在16天内达到25.3g/L的滴度，峰细胞密度>180x10^6cells/mL。随着生物反应器产率的提高，可使用占地更小、成本更低的一次性设备，来替代大规模的不锈钢设备(10,000-25,000L)，通过增加设备轮转或连续工艺，生产等量的产物。
尽管灌流工艺可使用基于过滤的细胞截留设备，如TFF和ATF，在生物反应器内获得并维持高细胞密度，但通常会要求使用较高的培养基置换速率，以将高密度细胞的活性维持在可接受的水平。与不同工艺相关的培养基成本是评估其生产等量产物时经济性的关键因素。而即使单位培养基成本适当，较高的培养基置换速率也会显著影响生产产品成本(CoG)，亦即，上游操作成本与培养基成本紧密相关。
生产单位产品的总生产CoG和上/下游成本的比重会随产物滴度和设备尺寸的变化而变化。在分析CoG的所有输入值中，一旦工艺确定，培养基用量及其成本是固定的，不管设备、设施等是否发生改变。细胞培养工程师的一个主要目标是降低培养基成本，同时获得高产量。本文使用相同的基础(basal)和补料(feed)培养基，稍作优化，开发了具有高生物反应器产率的不同细胞培养工艺(补料分批、灌流和CFB)，并比较了不同操作模式的生物反应器产率及其相关的培养基成本。
实验
实验使用生产单克隆抗体的重组CHO细胞系，不同工艺使用相同的3L生物反应器，培养基使用专利的基础(basal)和补液(feed)培养基，后者又分为两种补液-A和补液-B，均富含葡萄糖、氨基酸、维他命等。详细细胞系和种子扩增、生物反应器操作信息请参看原文。
对于补料分批培养，反应器起始工作体积1.5L，接种密度为0.5或2x10^6cells/mL，后者通过3天的N-1灌流来达到目标密度。生物反应器补液以每日葡萄糖水平为基础进行。
对于CFB工艺，使用50kD PS中空纤维过滤器的灌流设备，对于灌流，使用0.2μm PES中空纤维过滤器的灌流设备。接种密度1x10^6cells/mL，工作体积1.3L，一般第2天开始培养基置换，最大置换速率1vvd。灌流培养在第8天开始进行细胞废弃(cell bleeding)，以维持所需细胞密度和活性。
细胞培养每日取样分析，详细分析内容和方法，请参考原文。
讨论
不同操作模式的细胞培养性能
实验测试操作模式包括：补料分批、灌流和CFB，使用相同的3L生物反应器规格以及基础和补料培养基组合，以便比较细胞/生物反应器产率和培养基成本。
补料分批模式 对于补料分批模式，接种密度为0.5或2x10^6cells/mL，后者通过N-1灌流，可使对数生长期降低2天，所以8天就可达到峰密度，而前者需要10天。两种条件达到的峰细胞密度范围均为20.2-26.2x10^6cells/mL。两种接种密度在第14天分别达到5.4±0.1g/L和6.8±0.2g/L的滴度。生物反应器单位体积产率(VPR)按最终生物反应器滴度除以培养周期计算。2x10^6cells/mL接种密度条件，相比0.5x10^6cells/mL，可获得更高的VPR(0.49±0.01g/L/day vs. 0.39±0.01g/L/day)，主要是由于前者降低了起始生长阶段的时间，延长了生产期。
灌流模式 在灌流培养中，使用了2种不同的培养基组成：1种只使用基础培养基，另一种为基础加补液-A。在培养过程中，通过合适的cell bleeding，维持较高的活性>85%。只使用基础培养基时，平均细胞密度为44±4.1x10^6cells/mL，从第8天至32天的日产量为0.7±0.04g/L/day。在基础+补液条件中，随细胞密度的增加，补液-A作为培养基置换的一部分，逐渐引入，而总培养基置换率保持为1vvd，平均细胞密度增加至73.9±5.4x10^6cells/mL，日产量增加至2.29±0.28g/L/day。细胞特异性产率从16.0±1.2pg/cell/day增加至30.1±2.3pg/cell/day，从而使反应器产量增加~230%。
浓缩补料分批模式(CFB) 与灌流相似，评估了只使用基础培养基和使用基础+补液培养基的条件。与灌流工艺相比，CFB不需要进行cellbleeding，细胞质累积至更高的水平。当只使用基础培养基时，在第18天达到峰细胞密度72.0±9.6x10^6cells/mL，上清液滴度为12.2±0.6g/L。使用基础+6%补液-A+2%补液-B时，峰细胞密度为117.4x10^6cells/mL，第18天上清液滴度为21.4g/L，使用基础+8% 补液-A +8% Feed-B时，峰细胞密度为83.4x10^6cells/mL，第18天上清液滴度为36.7g/L。可见，增加补液-A和补液-B的量，可显著提高细胞特异性产率至45.1pg/cell/day。
细胞特异性产率、生物反应器产率和产物质量
当只使用基础培养基时，批次、灌流和CFB工艺可达到相似的qP，范围为14.7-17.1pg/cell/day。在此条件下，累积的细胞数量会直接影响产物滴度和单位体积产率。正如预期，批次培养的VPR显著较低，仅为0.08g/L/day，而灌流和CFB工艺由于可维持更高的细胞密度，可获得相当的VPR，0.68-0.70g/L/day。
浓缩补液培养基通常用于补料分批工艺，以提高细胞生长和细胞特异性产率。在此研究中，补加补液培养基，可显著提高qP和VPR。对于补料分批培养，qP提高至29.4-32.0pg/cell/day，VPR达到0.39g/L/day(接种密度0.5x10^6cells/mL)或0.49g/L/day(接种密度2x10^6cells/mL)。N-1灌流获得的更高的接种密度可提高VPR，因为缩短了生长期的时间，延长了生产期，提高产量。但是，即使与只使用基础培养基的灌流和CFB相比，补料分批培养的VPR仍较低，因为细胞密度差别显著。
相比补料分批工艺，只使用基础培养基以1vvd的速率进行培养基置换时，可轻松地将细胞密度提高2-3倍。而与只使用基础培养基的条件相比，在灌流培养中补充10%补液-A可使VPR提高~230%，qP提高~90%。相似的，在CFB工艺中，补充不同比例的补液-A和补液-B可将VPR提高至1.19-2.04g/L/day。
最近有报道显示，长寿命的人浆细胞可在体外维持120pg/cell/day的IgG分泌率，对于基因工程哺乳动物细胞，最高生产速率估计为~100pg/cell/day。qP的提高将来自于细胞系和培养基的优化。所以，理论上，在灌流工艺中，如稳态细胞密度维持为100x10^6cells/mL时，每日产量可高达10g/L/day。
实验同时评估了不同操作模式的产物质量特征，结果显示，CFB会形成更高水平的HMW和稍高的酸性异构体，主要是由于产物所暴露的细胞培养环境。在补料分批和浓缩补料分批中，产物滞留时间为整个培养周期。此外，在仅使用基础培养基的CFB工艺中，HMW最高，说明培养基组成可能在HMW形成中扮演了重要的角色。但是，产生的HMW仍低于5%，且大部分可在纯化步骤中去除。另一方面，即使是相同的高细胞密度环境和相似的培养基组成，灌流培养的酸性异构体和HMW更低，可能是由于产物在罐内更低的滞留时间。
培养基成本分析
由于细胞系或培养基组成的变化会显著影响产物滴度/产率，所以对不同操作模式的比较需使用相同的细胞系和培养基条件才有意义。本文使用从小规模生物反应器获得的细胞培养性能，来比较不同操作模式的培养基成本，并假定在规模放大时，不同工艺没有显著的产率下降。需要指出的是，实验中的灌流速率没有在对数生长期，以细胞特异性为基础，进行良好的优化。相反，在整个培养周期中，将灌流速率固定为1vvd。在不同的培养阶段，对细胞特异性灌流速率进行精细调节，应可进一步降低培养基用量和成本。
当只使用基础培养基时，生产每克抗体的培养基成本在批量和灌流工艺中都很高。加入适量的补料培养基，可降低每克mAb的培养基成本，且即使补料培养基相对较贵，细胞密度和qP的增加相比培养基成本的增加更加显著。
使用N-1灌流的补料分批的培养基成本比常规补料分批工艺低，N-1灌流需要3x基础培养基置换，但因接种密度的提高，继而获得的滴度的增加，抵消了培养基用量的增加。N-1灌流的补料分批和灌流的培养基成本相当，~$10/g mAb。这说明，虽然往常认为由于较高的灌流速率，灌流的培养基用量更高，继而培养基成本更高，但只需要生物反应器产率达到一定的阈值，从培养基成本上来看，还是相当有竞争力的。
CFB工艺的培养基成本与其它操作模式的趋势不同。在只使用基础培养基的条件中，成本与批量和灌流工艺相当，但CFB培养基成本会随补料培养基的使用而增加，其相对较高的培养基成本(>$17/g)可能是因为需要较长的细胞生长时间，在培养中，直到第10天，细胞密度达到峰水平，才开始出现显著的产物滴度增加。降低CFB培养基成本的一种方法是优化细胞寿命，延长批次时间，但更长的罐内滞留时间，可能会影响产物质量属性，或是进一步优化培养基，如替换昂贵的成分和优化其滴度。
总生产COG
除了培养基成本的不同，使用诸如灌流和CFB之类的工艺，结合一次性设备，在小规模上进行生物制品生产，可显著降低成本投入，从而获得更加灵活的生产策略，当产品需求增加时，可以快速地进行规模扩展(scale out)，而不是规模放大(ScaleuP)。与传统不锈钢设备相关的固定成本，可以转变为“可变”的成本结构。基于此处的案例，灌流工艺的培养基成本实际上低于补料分批工艺。
进行总成本分析时，如下游均以批量模式进行，且认为不同工艺的劳动力成本相当，则本文建模分析结果显示，N-1灌流的补料分批和灌流工艺的下游CoG/g相当，分别为$63/g和$59/g，而标准补料分批和CFB工艺的下游CoG/g稍高，分别为$71/g和$81/g。对于mAb和不稳定的产品，基于灌流的连续工艺都可以提供显著的经济优势。
总结
在本研究中，比较了不同操作模式下，生物反应器的产率，包括补料分批、灌流和CFB工艺。对于研究的细胞系，qP高度取决于所用的培养基，不管采用哪种操作模式，这使得累积细胞密度成为决定产物滴度和生物反应器产率的主要因素。结果显示，补料分批培养生物反应器产率最低(0.39-0.49g/L/day)，而基于灌流的培养方式，由于可维持更高的细胞密度，产率相对较高，灌流为2.29g/L/day，CFB为1.19-2.04g/L/day。灌流的一个显著优势是可以达到并维持极高的细胞密度，用于产物形成。
灌流工艺一个经常观察到的缺点是培养基用量较高，因为需要进行连续的培养基置换，以维持所需的高活细胞密度。这里的研究显示，高产率灌流培养的培养基成本实际上低于补料分批工艺。CFB工艺的培养基成本最高，虽然在18天内达到了36.7g/L的极高滴度，为降低CFB工艺的培养基成本，建议可以精调培养基置换率，以在起始的生长阶段获得更好的培养基利用，或通过培养基优化，提高细胞特异性产率。
Introduction to the
Cell culture techniques used to produce recombinant proteins and monoclonal antibodies (mAb) have different ways. Fill material Batch (Feb - Batch) process because of its simple operation, and is easy to scale, are widely used in clinical and commercial production, the current technology has been available in 18 days for 20-30 x10 ^ 6 cells/mL cell density, at the same time get > 10 g/L drop degree level.
Irrigation has traditionally been used to produce more unstable products, such as blood coagulation factors and enzymes, but also mAb products, such as Remicade. In perfusion culture, the retention time of the product in the reactor is reduced by medium replacement, and the perfusion rate depends on the specific product and/or process requirements.
In recent years, the process intensification based on irrigation has been greatly developed in the upstream process, mainly driven by the need to reduce costs and land use, and improve the flexibility of equipment. With the development of cell lines, culture medium and cellular intercept equipment, now perfusion technique has high cell density and yield can be obtained, making it a very attractive option, including the production of the mAb. In the mAb production, for example, in combination with 2 VVD medium of exchange rate, usually can reach 50 to 60 x10 ^ 6 cells/mL steady cell density, and up to 4 g/L/day production rate of the bioreactor. In addition, enrichment batch (CFB) can also maintain high cell density through medium replacement and retain the product in the bioreactor.
The difference between irrigation and CFB is the pore size of the hollow fiber membrane used. For antibody, use Per C6 cell line, can be in 12-13 days, 21.4 g/L of end product drop degree peak cell density (> 150 x10 ^ 6 cells/mL), and using a CHO cell line, can reach 25.3 g/L within 16 days the drop degree, peak cell density > 180 x10 ^ 6 cells/mL. With the improvement of production rate bioreactor, can use covers an area of smaller, cheaper disposable equipment, to replace the mass (10000-25000 L) stainless steel equipment, by increasing the rotary or continuous process equipment, production of the same amount of product.
Although cellular intercept perfusion technique can be used based on filtering equipment, such as TFF and ATF, in order to make and maintain a high cell density in a bioreactor, but usually requires the use of higher medium displacement rate, to maintain the activity of high density cells at an acceptable level. The cost of culture medium associated with different processes is the key factor to evaluate the economy of the same product. However, even if the cost per unit of culture medium is appropriate, the higher medium replacement rate will significantly affect the cost of production products (CoG), that is, the upstream operating cost is closely related to the cost of culture medium.
The proportion of cocog and upper/lower cost per unit of production varies with product titer and equipment size. In the analysis of all the input values of CoG, once the process is determined, the amount and cost of the medium are fixed, regardless of whether the equipment, facilities, etc have changed. One of the main goals of cell culture engineers is to reduce the cost of the medium and to achieve high yields. In this paper, using the same base (basal hominins) and feed (feed) medium, slightly optimization, developed bioreactor with high yield of different cell culture processes (filling material in batches, the perfusion and CFB), and compare the different operating modes of bioreactor production rate and its related cost of medium.
The experiment
Experiment using the production of monoclonal antibodies, recombinant CHO cell line different process using the same 3 l bioreactor, medium using patent (basal hominins) and rehydration (feed) the basis of the culture medium, the latter is divided into two kinds of rehydration - A and rehydration - B, are rich in glucose, amino acid, vitamin, etc. For more information on cell lines and seed amplification and bioreactor operations, see the original article.
Starting work to fill material batch cultivation, reactor volume 1.5 L, inoculation density of 0.5 or 2 x10 ^ 6 cells/mL, the latter by perfusion of N - 1 to 3 days to target density. Bioreactor rehydration is based on daily glucose levels.
For CFB process, the irrigation equipment of 50kD PS hollow fiber filter is used, and for irrigation flow, the irrigation equipment of 0.2%chevrons hollow fiber filter is used. Inoculation density 1 x10 ^ 6 cells/mL, working volume of 1.3 L, 2 days commonly medium displacement, maximum displacement rate 1 VVD. The perfusion culture began on day 8 with cell bleaching to maintain the required cell density and activity.
Cell culture daily sampling analysis, detailed analysis content and methods, please refer to the original text.
discuss
Cell culture properties of different operating modes
Laboratory testing operation mode including: filling material in batches, the perfusion and CFB, use the same 3 l bioreactor specifications as well as the foundation and the combination of feeding medium, in order to compare cell/production rate of bioreactor and medium cost.
Fill material batch mode for filling material batch mode, inoculation density of 0.5 or 2 x10 ^ 6 cells/mL, the latter by N - 1 perfusion, can lower the logarithmic phase 2 days, 8 days so can reach the peak density, while the former need to 10 days. Two kinds of conditions to achieve peak cell density range to 20.2-26.2 x10 ^ 6 cells/mL. The two inoculation densities reached 5.4 + - 0.1g/L and 6.8-0.2g/L respectively on day 14. The yield per unit volume (VPR) of the bioreactor is calculated according to the final bioreactor titer divided by the culture period. 2 x 10 ^ 6 cells/mL inoculation density condition, compared to 0.5 x 10 ^ 6 cells/mL, can obtain higher VPR (0.49 + 0.01 g/L/day vs. 0.39 + 0.01 g/L/day), is mainly due to the former reduces the initial growth stage time, extend the production period.
In the perfusion culture, two different media are used: one is only using the basic medium, and the other is supplemented with -a. During the culture process, high > activity was maintained 85% through appropriate cell bleaching. Only use the basic culture medium, the average cell density was 44 + / - 4.1 x10 ^ 6 cells/mL, from 8 to 32 days of daily output is 0.7 + 0.04 g/L/day. In base + rehydration conditions, with the increase of cell density, rehydration -a as part of the culture medium displacement, introduced gradually, and always keep 1 VVD medium replacement rate, the average cell density increased to 73.9 + / - 5.4 x10 ^ 6 cells/mL, daily output increased to 2.29 + / - 0.28 g/L/day. The cell specific yield increased from 16.0 + - 1.2pg/cell/day to 30.1-2.3pg/cell/day, thus increasing the reactor yield by ~230%.
The enrichment batch mode (CFB) is similar to the irrigation flow, and the conditions of using only the basal medium and the basal + supplementary medium are evaluated. Compared with the irrigation process, CFB does not need cellbleeding, and the cytoplasm accumulates to a higher level. When using only basic medium, cell density in 18 days reach peak of 72.0 + / - 9.6 x10 ^ 6 cells/mL, supernatant liquid droplets was 12.2 + / - 0.6 g/L. Use basic + 6% rehydration - A + 2% rehydration - B, peak cell density of 117.4 x10 ^ 6 cells/mL, clear sky droplet 18 degrees of 21.4 g/L, using basic + 8% rehydration - A + 8% Feed - B, peak cell density of 83.4 x10 ^ 6 cells/mL, clear sky droplet 18 degrees is 36.7 g/L. It can be seen that increasing the amount of rehydration -a and rehydration -b can significantly increase the cell specific production rate to 45.1pg/cell/day.
Cell specific yield, bioreactor yield and product quality
When only the basic medium was used, batch, irrigation and CFB processes could achieve similar qP, ranging from 14.7-17.1pg/cell/day. Under these conditions, the accumulated cell number will directly influence the product titration and yield per unit volume. As expected, VPR of the batch culture was significantly lower, only 0.08g/L/day, while the irrigation and CFB process could obtain a considerable VPR, 0.68-0.70g/L/day, because it could maintain a higher cell density.
Concentrated rehydration medium is usually used in batch feeding process to improve cell growth and cell specific yield. In this study, qP and VPR could be significantly improved by adding supplementary media. For filling material batch cultivation, qP increase to 29.4-32.0 pg/cell/day, VPR is 0.39 g/L/day (inoculation density 0.5 x10 ^ 6 cells/mL) or 0.49 g/L/day (2 x10 inoculation density ^ 6 cells/mL). The higher inoculation density obtained by n-1 irrigation can increase VPR, because it shortens the growth period, lengthens the production period and increases the yield. However, VPR was lower in batch culture even compared with irrigation and CFB using only basic media, because of the significant difference in cell density.
The cell density can be easily increased by 2-3 times when the substrate is replaced at a rate of 1vvvd, compared with the batch feeding process. Compared with the condition of using only basic medium, the addition of 10% supplement -A in irrigation culture can increase VPR by ~230% and qP by ~90%. Similarly, in CFB process, VPR can be increased to 1.19-2.04g/L/day by adding different proportions of rehydration -a and rehydration -b.
Recently, it has been reported that the IgG secretion rate of long-lived human plasma cells can maintain 120pg/cell/day in vitro, and the maximum production rate for genetically engineered mammalian cells is estimated to be ~100pg/cell/day. The improvement in qP will come from the optimization of cell lines and media. So, in theory, in the perfusion technique, such as steady cell density to maintain 100 x10 ^ 6 cells/mL, the daily output of up to 10 g/L/day.
Experiment and to evaluate the product quality characteristics of the different operating modes, the results show that the CFB can form a higher level of HMW and slightly higher acid isomer, products is mainly due to the exposure of the cell culture environment. In the feeding batch and concentrated feeding batch, the product retention time is the whole culture period. In addition, HMW is the highest in the CFB process using only basic media, indicating that medium composition may play an important role in the formation of HMW. However, the resulting HMW is still less than 5%, and most can be removed in the purification process. On the other hand, even if is the same environment and the high cell density culture medium composition, similar to that of perfusion culture acid isomer and HMW lower, may be due to the product of lower retention time on the interior of the tank.
Cost analysis of culture medium
Because changes in cell line or medium composition can significantly affect product titer/yield, it is meaningful to compare different operating modes using the same cell line and medium conditions. This article USES the small scale bioreactors for cell culture performance, cost, to compare different operating modes of medium and assumption in scale, different process without a significant decline in production rate. It needs to be pointed out that the irrigation rate in the experiment is not well optimized in logarithmic growth period, based on cell specificity. In contrast, the irrigation rate was fixed at 1vvd throughout the culture period. Careful regulation of the cell-specific perfusion rate at different stages of culture should further reduce the amount and cost of culture medium.
When only the basic medium is used, the cost of producing each gram of antibody is high in both batch and irrigation processes. Adding suitable amount of filling material medium, which can reduce the cost of mAb per gram of culture medium, and relatively expensive, even feeding medium of cell density and qP increase compared to the increased cost of medium even more significant.
Using N - 1 perfusion of partial medium filling material cost is lower than conventional filling material batch process, N - 1 perfusion based medium replacement need 3 x, but because of the inoculation density increase, then the drop degree increase, offset by an increase in the amount of medium. The feeding batch of n-1 irrigation is equivalent to the medium cost of irrigation, ~$10/g mAb. Think this shows that although the usual due to the high rate of perfusion, the perfusion medium dosage is higher, then medium cost is higher, but only need bioreactor yield reaches a certain threshold, from medium cost point of view, is quite competitive.
The culture medium cost of CFB process is different from other operating modes. , under the condition of using only basic medium cost and batch and perfusion technique, but the CFB medium will cost increases with the use of feeding medium, its relatively high cost of medium (> $17 / g) might be due to need long time of cell growth, in training, until the 10th day, cell density reached peak level, began to appear product drop degree increased significantly. One way to decrease the cost of CFB medium is optimization of cell life, extend the time of batch, but longer tank retention time, may affect product quality attributes, or to further optimize the culture medium, such as replacing expensive ingredient and optimize its degree.
Total production COG
Costs in addition to the culture medium is different, the use of such as perfusion and CFB technology, combined with the disposable devices, biological products in small-scale production, can significantly reduce costs, thereby gaining a more flexible production strategy, when the product demand, can quickly to scale (scale out), rather than the scale (ScaleuP). Fixed costs associated with conventional stainless steel equipment can be transformed into "variable" cost structures. Based on the case here, the media cost of the irrigation process is actually lower than that of the batch feeding process.
Total cost analysis, if the downstream all carried out in batch mode, and think that labor cost is of different process, the modeling analysis results showed that N - 1 perfusion filling material batch and perfusion process of downstream CoG/g, respectively, $63 and $59 / g/g, while the standard fill material batch and CFB technology of downstream CoG/g is a bit high, respectively, for $71 and $81 / g/g. For mAb and unstable products, continuous process based on irrigation can provide significant economic advantages.
conclusion
In this study, the yield of bioreactors under different operating modes was compared, including feeding batch, irrigation flow and CFB process. For the study of cell lines, qP highly depends on the medium used, no matter adopt what kind of operation mode, this makes the cumulative cell density decided to product drop degree is the main factor of yield and biological reactor. Results show that the filling material batch culture bioreactor yield the lowest (0.39 0.49 g/L/day), and based on the cultivation of the perfusion method, because can maintain a higher cell density, relatively high production rate of perfusion is 2.29 g/L/day, CFB is 1.19 2.04 g/L/day. One of the notable advantages of irrigation is the ability to achieve and maintain high cell density for product formation.
One oft-observed disadvantage of irrigation is that the amount of the medium is high, as continuous replacement of the medium is required to maintain the required high cell density. The research here shows that the cost of culture medium for high yield perfusion is actually lower than that for batch feeding. CFB technology medium cost is highest, although reached 36.7 g/L in 18 days of high degree, to reduce the cost of the CFB technology medium Suggestions can fine adjustment medium displacement rate, in the initial stages of growth for better use of culture medium, or through a medium optimization, improve the production rate of cell specificity.