不同細胞培養(yǎng)工藝生物反應器產(chǎn)率和培養(yǎng)基成本比較
發(fā)布時間：
2022-11-16
作者：
用于重組蛋白和單克隆抗體(mAb)生產(chǎn)的細胞培養(yǎng)工藝有不同的方式。補料分批(Feb-Batch)工藝由于操作簡單，且較易規(guī)模放大，被臨床和商業(yè)化生產(chǎn)廣泛采用，目前的技術(shù)發(fā)展已可在18天內(nèi)獲得20-30x10^6cells/mL的細胞密度，同時獲得>10g/L的滴度水平。
灌流工藝以往更多用于生產(chǎn)不穩(wěn)定的產(chǎn)品，如血液凝集因子和酶類產(chǎn)品，但也有用于生產(chǎn) mAb產(chǎn)品，如Remicade(英利昔單抗)。在灌流培養(yǎng)中，通過培養(yǎng)基置換，降低產(chǎn)物在反應器內(nèi)的滯留時間，而灌流速率取決于特異性的產(chǎn)物和/或工藝需求。
近幾年，在上游工藝中，基于灌流的工藝強化獲得了極大的發(fā)展，驅(qū)動力主要來自于對降低成本和占地的需求，以及提高設備靈活性。隨著細胞系、培養(yǎng)基和細胞截留設備的發(fā)展，現(xiàn)在的灌流工藝已可獲得較高的細胞密度和產(chǎn)量，使其成為一個非常有吸引力的選擇，包括mAb的生產(chǎn)。例如，在mAb生產(chǎn)中，結(jié)合2vvd的培養(yǎng)基置換速率，通常可達到50-60x10^6cells/mL的穩(wěn)態(tài)細胞密度，以及高達4g/L/day的生物反應器產(chǎn)率。此外，濃縮補料分批(CFB)也可以通過培養(yǎng)基置換，維持高細胞密度，而將產(chǎn)物截留在生物反應器內(nèi)。
灌流和CFB的差異在于所用的中空纖維膜的孔徑。對于抗體，使用Per.C6細胞系，可在12-13天內(nèi)，達到21.4g/L的終產(chǎn)物滴度(峰細胞密度>150x10^6cells/mL)，而使用CHO細胞系時，可在16天內(nèi)達到25.3g/L的滴度，峰細胞密度>180x10^6cells/mL。隨著生物反應器產(chǎn)率的提高，可使用占地更小、成本更低的一次性設備，來替代大規(guī)模的不銹鋼設備(10,000-25,000L)，通過增加設備輪轉(zhuǎn)或連續(xù)工藝，生產(chǎn)等量的產(chǎn)物。
盡管灌流工藝可使用基于過濾的細胞截留設備，如TFF和ATF，在生物反應器內(nèi)獲得并維持高細胞密度，但通常會要求使用較高的培養(yǎng)基置換速率，以將高密度細胞的活性維持在可接受的水平。與不同工藝相關(guān)的培養(yǎng)基成本是評估其生產(chǎn)等量產(chǎn)物時經(jīng)濟性的關(guān)鍵因素。而即使單位培養(yǎng)基成本適當，較高的培養(yǎng)基置換速率也會顯著影響生產(chǎn)產(chǎn)品成本(CoG)，亦即，上游操作成本與培養(yǎng)基成本緊密相關(guān)。
生產(chǎn)單位產(chǎn)品的總生產(chǎn)CoG和上/下游成本的比重會隨產(chǎn)物滴度和設備尺寸的變化而變化。在分析CoG的所有輸入值中，一旦工藝確定，培養(yǎng)基用量及其成本是固定的，不管設備、設施等是否發(fā)生改變。細胞培養(yǎng)工程師的一個主要目標是降低培養(yǎng)基成本，同時獲得高產(chǎn)量。本文使用相同的基礎(basal)和補料(feed)培養(yǎng)基，稍作優(yōu)化，開發(fā)了具有高生物反應器產(chǎn)率的不同細胞培養(yǎng)工藝(補料分批、灌流和CFB)，并比較了不同操作模式的生物反應器產(chǎn)率及其相關(guān)的培養(yǎng)基成本。
實驗
實驗使用生產(chǎn)單克隆抗體的重組CHO細胞系，不同工藝使用相同的3L生物反應器，培養(yǎng)基使用專利的基礎(basal)和補液(feed)培養(yǎng)基，后者又分為兩種補液-A和補液-B，均富含葡萄糖、氨基酸、維他命等。詳細細胞系和種子擴增、生物反應器操作信息請參看原文。
對于補料分批培養(yǎng)，反應器起始工作體積1.5L，接種密度為0.5或2x10^6cells/mL，后者通過3天的N-1灌流來達到目標密度。生物反應器補液以每日葡萄糖水平為基礎進行。
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(yǎng)基置換，最大置換速率1vvd。灌流培養(yǎng)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。
細胞培養(yǎng)每日取樣分析，詳細分析內(nèi)容和方法，請參考原文。
討論
不同操作模式的細胞培養(yǎng)性能
實驗測試操作模式包括：補料分批、灌流和CFB，使用相同的3L生物反應器規(guī)格以及基礎和補料培養(yǎng)基組合，以便比較細胞/生物反應器產(chǎn)率和培養(yǎng)基成本。
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(shù)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(chǎn)率(VPR)按最終生物反應器滴度除以培養(yǎng)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(chǎn)期。
灌流模式 在灌流培養(yǎng)中，使用了2種不同的培養(yǎng)基組成：1種只使用基礎培養(yǎng)基，另一種為基礎加補液-A。在培養(yǎng)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(yǎng)基時，平均細胞密度為44±4.1x10^6cells/mL，從第8天至32天的日產(chǎn)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(yǎng)基置換的一部分，逐漸引入，而總培養(yǎng)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(chǎn)量增加至2.29±0.28g/L/day。細胞特異性產(chǎn)率從16.0±1.2pg/cell/day增加至30.1±2.3pg/cell/day，從而使反應器產(chǎn)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(yǎng)基和使用基礎+補液培養(yǎng)基的條件。與灌流工藝相比，CFB不需要進行cellbleeding，細胞質(zhì)累積至更高的水平。當只使用基礎培養(yǎng)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(chǎn)率至45.1pg/cell/day。
細胞特異性產(chǎn)率、生物反應器產(chǎn)率和產(chǎn)物質(zhì)量
當只使用基礎培養(yǎng)基時，批次、灌流和CFB工藝可達到相似的qP，范圍為14.7-17.1pg/cell/day。在此條件下，累積的細胞數(shù)量會直接影響產(chǎn)物滴度和單位體積產(chǎn)率。正如預期，批次培養(yǎng)的VPR顯著較低，僅為0.08g/L/day，而灌流和CFB工藝由于可維持更高的細胞密度，可獲得相當?shù)腣PR，0.68-0.70g/L/day。
濃縮補液培養(yǎng)基通常用于補料分批工藝，以提高細胞生長和細胞特異性產(chǎn)率。在此研究中，補加補液培養(yǎng)基，可顯著提高qP和VPR。對于補料分批培養(yǎng)，qP提高至29.4-32.0pg/cell/day，VPR達到0.39g/L/day(接種密度0.5x10^6cells/mL)或0.49g/L/day(接種密度2x10^6cells/mL)。N-1灌流獲得的更高的接種密度可提高VPR，因為縮短了生長期的時間，延長了生產(chǎn)期，提高產(chǎn)量。但是，即使與只使用基礎培養(yǎng)基的灌流和CFB相比，補料分批培養(yǎng)的VPR仍較低，因為細胞密度差別顯著。
相比補料分批工藝，只使用基礎培養(yǎng)基以1vvd的速率進行培養(yǎng)基置換時，可輕松地將細胞密度提高2-3倍。而與只使用基礎培養(yǎng)基的條件相比，在灌流培養(yǎng)中補充10%補液-A可使VPR提高~230%，qP提高~90%。相似的，在CFB工藝中，補充不同比例的補液-A和補液-B可將VPR提高至1.19-2.04g/L/day。
最近有報道顯示，長壽命的人漿細胞可在體外維持120pg/cell/day的IgG分泌率，對于基因工程哺乳動物細胞，最高生產(chǎn)速率估計為~100pg/cell/day。qP的提高將來自于細胞系和培養(yǎng)基的優(yōu)化。所以，理論上，在灌流工藝中，如穩(wěn)態(tài)細胞密度維持為100x10^6cells/mL時，每日產(chǎn)量可高達10g/L/day。
實驗同時評估了不同操作模式的產(chǎn)物質(zhì)量特征，結(jié)果顯示，CFB會形成更高水平的HMW和稍高的酸性異構(gòu)體，主要是由于產(chǎn)物所暴露的細胞培養(yǎng)環(huán)境。在補料分批和濃縮補料分批中，產(chǎn)物滯留時間為整個培養(yǎng)周期。此外，在僅使用基礎培養(yǎng)基的CFB工藝中，HMW最高，說明培養(yǎng)基組成可能在HMW形成中扮演了重要的角色。但是，產(chǎn)生的HMW仍低于5%，且大部分可在純化步驟中去除。另一方面，即使是相同的高細胞密度環(huán)境和相似的培養(yǎng)基組成，灌流培養(yǎng)的酸性異構(gòu)體和HMW更低，可能是由于產(chǎn)物在罐內(nèi)更低的滯留時間。
培養(yǎng)基成本分析
由于細胞系或培養(yǎng)基組成的變化會顯著影響產(chǎn)物滴度/產(chǎn)率，所以對不同操作模式的比較需使用相同的細胞系和培養(yǎng)基條件才有意義。本文使用從小規(guī)模生物反應器獲得的細胞培養(yǎng)性能，來比較不同操作模式的培養(yǎng)基成本，并假定在規(guī)模放大時，不同工藝沒有顯著的產(chǎn)率下降。需要指出的是，實驗中的灌流速率沒有在對數(shù)生長期，以細胞特異性為基礎，進行良好的優(yōu)化。相反，在整個培養(yǎng)周期中，將灌流速率固定為1vvd。在不同的培養(yǎng)階段，對細胞特異性灌流速率進行精細調(diào)節(jié)，應可進一步降低培養(yǎng)基用量和成本。
當只使用基礎培養(yǎng)基時，生產(chǎn)每克抗體的培養(yǎng)基成本在批量和灌流工藝中都很高。加入適量的補料培養(yǎng)基，可降低每克mAb的培養(yǎng)基成本，且即使補料培養(yǎng)基相對較貴，細胞密度和qP的增加相比培養(yǎng)基成本的增加更加顯著。
使用N-1灌流的補料分批的培養(yǎng)基成本比常規(guī)補料分批工藝低，N-1灌流需要3x基礎培養(yǎng)基置換，但因接種密度的提高，繼而獲得的滴度的增加，抵消了培養(yǎng)基用量的增加。N-1灌流的補料分批和灌流的培養(yǎng)基成本相當，~$10/g mAb。這說明，雖然往常認為由于較高的灌流速率，灌流的培養(yǎng)基用量更高，繼而培養(yǎng)基成本更高，但只需要生物反應器產(chǎn)率達到一定的閾值，從培養(yǎng)基成本上來看，還是相當有競爭力的。
CFB工藝的培養(yǎng)基成本與其它操作模式的趨勢不同。在只使用基礎培養(yǎng)基的條件中，成本與批量和灌流工藝相當，但CFB培養(yǎng)基成本會隨補料培養(yǎng)基的使用而增加，其相對較高的培養(yǎng)基成本(>$17/g)可能是因為需要較長的細胞生長時間，在培養(yǎng)中，直到第10天，細胞密度達到峰水平，才開始出現(xiàn)顯著的產(chǎn)物滴度增加。降低CFB培養(yǎng)基成本的一種方法是優(yōu)化細胞壽命，延長批次時間，但更長的罐內(nèi)滯留時間，可能會影響產(chǎn)物質(zhì)量屬性，或是進一步優(yōu)化培養(yǎng)基，如替換昂貴的成分和優(yōu)化其滴度。
總生產(chǎn)COG
除了培養(yǎng)基成本的不同，使用諸如灌流和CFB之類的工藝，結(jié)合一次性設備，在小規(guī)模上進行生物制品生產(chǎn)，可顯著降低成本投入，從而獲得更加靈活的生產(chǎn)策略，當產(chǎn)品需求增加時，可以快速地進行規(guī)模擴展(scale out)，而不是規(guī)模放大(ScaleuP)。與傳統(tǒng)不銹鋼設備相關(guān)的固定成本，可以轉(zhuǎn)變?yōu)?ldquo;可變”的成本結(jié)構(gòu)。基于此處的案例，灌流工藝的培養(yǎng)基成本實際上低于補料分批工藝。
進行總成本分析時，如下游均以批量模式進行，且認為不同工藝的勞動力成本相當，則本文建模分析結(jié)果顯示，N-1灌流的補料分批和灌流工藝的下游CoG/g相當，分別為$63/g和$59/g，而標準補料分批和CFB工藝的下游CoG/g稍高，分別為$71/g和$81/g。對于mAb和不穩(wěn)定的產(chǎn)品，基于灌流的連續(xù)工藝都可以提供顯著的經(jīng)濟優(yōu)勢。
總結(jié)
在本研究中，比較了不同操作模式下，生物反應器的產(chǎn)率，包括補料分批、灌流和CFB工藝。對于研究的細胞系，qP高度取決于所用的培養(yǎng)基，不管采用哪種操作模式，這使得累積細胞密度成為決定產(chǎn)物滴度和生物反應器產(chǎn)率的主要因素。結(jié)果顯示，補料分批培養(yǎng)生物反應器產(chǎn)率最低(0.39-0.49g/L/day)，而基于灌流的培養(yǎng)方式，由于可維持更高的細胞密度，產(chǎn)率相對較高，灌流為2.29g/L/day，CFB為1.19-2.04g/L/day。灌流的一個顯著優(yōu)勢是可以達到并維持極高的細胞密度，用于產(chǎn)物形成。
灌流工藝一個經(jīng)常觀察到的缺點是培養(yǎng)基用量較高，因為需要進行連續(xù)的培養(yǎng)基置換，以維持所需的高活細胞密度。這里的研究顯示，高產(chǎn)率灌流培養(yǎng)的培養(yǎng)基成本實際上低于補料分批工藝。CFB工藝的培養(yǎng)基成本最高，雖然在18天內(nèi)達到了36.7g/L的極高滴度，為降低CFB工藝的培養(yǎng)基成本，建議可以精調(diào)培養(yǎng)基置換率，以在起始的生長階段獲得更好的培養(yǎng)基利用，或通過培養(yǎng)基優(yōu)化，提高細胞特異性產(chǎn)率。
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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.