對于CFB工藝，使用50kD PS中空纖維過濾器的灌流設備，對于灌流，使用0.2μm PES中空纖維過濾器的灌流設備。接種密度1x10^6cells/mL，工作體積1.3L，一般第2天開始培養基置換，最大置換速率1vvd。灌流培養在第8天開始進行細胞廢棄(cell bleeding)，以維持所需細胞密度和活性。
補料分批模式 對于補料分批模式，接種密度為0.5或2x10^6cells/mL，后者通過N-1灌流，可使對數生長期降低2天，所以8天就可達到峰密度，而前者需要10天。兩種條件達到的峰細胞密度范圍均為20.2-26.2x10^6cells/mL。兩種接種密度在第14天分別達到5.4±0.1g/L和6.8±0.2g/L的滴度。生物反應器單位體積產率(VPR)按最終生物反應器滴度除以培養周期計算。2x10^6cells/mL接種密度條件，相比0.5x10^6cells/mL，可獲得更高的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長階段的時間，延長了生產期。
灌流模式 在灌流培養中，使用了2種不同的培養基組成：1種只使用基礎培養基，另一種為基礎加補液-A。在培養過程中，通過合適的cell bleeding，維持較高的活性>85%。只使用基礎培養基時，平均細胞密度為44±4.1x10^6cells/mL，從第8天至32天的日產量為0.7±0.04g/L/day。在基礎+補液條件中，隨細胞密度的增加，補液-A作為培養基置換的一部分，逐漸引入，而總培養基置換率保持為1vvd，平均細胞密度增加至73.9±5.4x10^6cells/mL，日產量增加至2.29±0.28g/L/day。細胞特異性產率從16.0±1.2pg/cell/day增加至30.1±2.3pg/cell/day，從而使反應器產量增加~230%。
濃縮補料分批模式(CFB) 與灌流相似，評估了只使用基礎培養基和使用基礎+補液培養基的條件。與灌流工藝相比，CFB不需要進行cellbleeding，細胞質累積至更高的水平。當只使用基礎培養基時，在第18天達到峰細胞密度72.0±9.6x10^6cells/mL，上清液滴度為12.2±0.6g/L。使用基礎+6%補液-A+2%補液-B時，峰細胞密度為117.4x10^6cells/mL，第18天上清液滴度為21.4g/L，使用基礎+8% 補液-A +8% Feed-B時，峰細胞密度為83.4x10^6cells/mL，第18天上清液滴度為36.7g/L。可見，增加補液-A和補液-B的量，可顯著提高細胞特異性產率至45.1pg/cell/day。
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.
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.
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.
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.