In the future, with the investment of pharmaceutical enterprises in the field of biological preparations, it is expected that the global biological medicine market will grow at a compound annual value-added rate of 19% by 2021, and will reach 20 billion US dollars by 2021 based on the market micro scale. Due to the small base, the growth space and growth rate of China's biopharmaceutical market will be larger and stronger. It is estimated that the market scale of China's biopharmaceutical market will reach 9.2 billion yuan in 2021. With the successive listing of domestic biopharmaceutical analogues, it is expected to show explosive growth.
CHO cells are widely used in the production of therapeutic proteins in the field of Biopharmaceutics because they can express a variety of recombinant proteins rapidly and at a high level.
In the development of cell culture process, the materials used should meet the nutritional requirements of cells, fully comply with the provisions of cGMP, select the culture medium without the limitation of chemical components of animal origin, and meet the culture conditions of genotype expansion and corresponding screening system. Commonly used medium can be divided into subculture medium, production medium and supplement medium. Compared with the production medium, the subculture medium is rich in nutrients, which is conducive to the recovery of cell activity. The supplemented medium is usually a compound of amino acids, which is added separately according to the difference of isoelectric point of amino acids to avoid precipitation. In addition to the culture medium, we need to prepare the corresponding carbon supplement, generally glucose. In addition, the universality and operability of large-scale culture should be considered when selecting materials.
In the small-scale test stage, the selection of the shaker should take into account the demand of cells for OTR and evaporation. Usually, when transferring cell culture process, there will be problems that the shakers of both sides are inconsistent, resulting in deviation of OTR. A simple conversion of centripetal force can be made according to the orbital radius of the shaker, and then verified by experiments.
F: Centripetal force - N; m: mass - G; N: rotational speed - rpm; R: track radius - mm
In the process of cell production and enlargement, the order and scale of seed tank are usually determined according to different scales. Take the pilot scale of 200 L as an example, the first level 50L seed tank is usually used. At this time, the process parameters in the amplification process need to be considered, which can be achieved through KLA, P / V, tip speed, Como micro scale and C respectively The model of O2 escape speed and mixing time are considered comprehensively. The rotation speed, bottom ventilation type and flow, surface ventilation flow of seed tank and production tank are set respectively, as well as the regulation of oxygen, air and carbon dioxide in the cultivation process. In general, KLA can achieve high-density cell culture at 5-12 H-1; KLA is usually adjusted and determined on the basis of P / V; when mammalian cells are cultured, the T IP speed is usually controlled below 2 m / s, which reduces the injury of cells by shear force; the micro scale of Como should be greater than 50 times of cell diameter, so as to reduce the injury of shear force to cells.
Based on the above principles, an amplification test is carried out as follows:
Cell resuscitation and flask expansion
1.1 put the subculture medium (cdcho) at room temperature, make it return to room temperature, and then transfer it to 37.0 ℃ shaking table to preheat for 30 min;
1.2 use the pipette to suck out the seed cell liquid from the cryopreservation tube and put it into the shaker with preheating medium. The shaker parameters are set as follows:
1.3 after resuscitation, the cell density was 0.31 * 10e6 cells / ml, and the activity rate was 92.4%;
1.4 n-2 ~ n-1 is expanded by wave reactor, and the specifications of reaction bags used are 20 L and 50 L respectively. The experimental results of amplification stage are as follows:
The dilution ratio of N-5 to n-3 was 14, the activity rate increased from 97.2% to 99.5%, the cell growth was relatively stable, the specific growth rate was 0.86 to 0.88 day-1.
In the N-2 ~ n-1 stage, the specific growth rate of the readytoprocess wave 25 reactor was 0.88 day-1 and 0.87 day-1, respectively. Compared with the shaker, it was stable, and there was no change in the specific growth rate due to the replacement of the reactor and container. The readytoprocess wave 25 parameters are set as follows:
Production of xdr-200 disposable bioreactor
2.1 before production, the pH (4.01 and 7.00), do (0 and 100%) and temperature electrode were corrected at two points;
2.2 put the production medium actipro into the XDR 200 L reaction bag and preheat it to 37 ℃ for inoculation. After inoculation, the living cell density is 0.45 * 10e6 cells / ml;
2.3 the concentration of glucose and lactate were measured every day. When the sum of glucose and lactate concentration was less than 4 g / L, the concentration of glucose was 4 g / L in the culture medium;
2.4 on the third day, the fifth day, the seventh day and the ninth day, respectively, 3% of the supplemented medium cell boost 7a and 0.3% of cell boost 7b were added to the culture medium to supplement amino acids.
The change of cell density is shown in Figure 1. In the 200L experiment, when the cell density was 17 * 10e6 cells / ml on the fifth day, the temperature was adjusted to 31.0 ℃, which was lower than the growth rate, reaching the highest value of 24.87 * 10e6 cells / ml on the seventh day, and then stabilized between 22 * 10e6 cells / ml and 24 * 10e6 cells / ml.
Fig. 1 change of cell density
Fig. 2 change of cell viability
The changes of glucose and lactate concentration are shown in Fig. 3 and Fig. 4. Glucose is rapidly utilized during the exponential growth period of cells, resulting in a large amount of lactate, reaching a peak value of 2.03 g / L on the 4th day. Since then, due to the optimization of sugar supplement strategy, the concentration of lactate has gradually decreased, but it still contains 0.38 g / L on the day of harvest, so the culture process needs to be further optimized. Due to the influence of lactate, the cell viability gradually decreased after the 4th day, and it was 95.4% by the day of harvest.
Figure 3 glucose concentration change
Fig. 4 change of lactate concentration
The cell density was 23.59 * 10e6 cells / ml and IgG production was 4.3 g / L, which was consistent with 15 l experimental results. The parameters of XDR bioreactor are as follows:
Bioreactor is still in the initial stage of large-scale application in China. With the development of modern biotechnology, using bioreactor to produce biological products is the inevitable trend of industry development. It is the core technology of biological products to produce antigens and antibodies by suspension culture of cells in bioreactor. Combined with screening and domestication of high expression cell lines and fine amplification process control, it can effectively improve production efficiency, product quality, reduce cost and improve product competitiveness.