Introduction: Recombinant human interferon β-1a is a protein product that is obtained by the recombinant method and is used in the treatment of condyloma acuminatum and relapsing forms of multiple sclerosis (MS disease) and reduces the frequency of disease recurrence and relieves the patient's weakness and disability. In order to produce rh-IFN β-1a protein, Chinese hamster ovary cells are transformed with human beta interferon gene and used in the industrial production process. IFN β-1a is expressed by the host in a perfusion production system using serum-free culture medium in a stirred bioreactor and secreted in the culture medium. In the early stages, there were many challenges such as the lack of cell growth and viability, clogging of the spin filter, the challenge of increasing the amount of host DNA. To solve this problems, various optimizations were performed to select optimal conditions for cell growth and protein expression. In this work, the perfusion cell line of producing rh-IFN β-1a was studied to overcome challenges mentioned.
Methods: 1- Lack of proper cell growth in the logarithmic phase
In the early stages of setting up, one of the challenges ahead was the lack of proper cell growth and the doubling time was longer than expected, so that the maximum cell density obtained was about 6 million cells per cc. With this cell density, the amount of the final product was less than expected, and the cost of the finished product was higher than expected. To solve this problem, various experiments were conducted with various culture mediums to select the optimal culture medium for growth.
2- Temperature shift in order to changing in cell growth phase and enhance protein expression
Various parameters have been used to slow down cell growth. Mild hypothermia is probably the most well-known environmental factor that can improve the performance of mammalian cell culture processes. Temperature performance between 30 and 34 degrees Celsius has shown that by stopping the cell in the G0/G1 phase, it affects the cell cycle, cell growth is inhibited, and the amount of glucose or amino acid consumption tends to decrease. One of the most widely used strategies is to reduce the culture temperature in order to change the behavior of the cell so that instead of cell proliferation, the target protein has a higher expression.
3- Clogging of the perfusion system during the production process due to the adhesion of the host cell to the spin filter:
In cell culture, perfusion is a process that uses a method to keep cells in a bioreactor while continuously exchanging culture medium. Fresh medium replenishes nutrients and carbon sources, while cellular waste and medium depleted of nutrients are removed. In the perfusion system, spin filters are used as a fixed substrate for cell maintenance. The physical parameters used for cell maintenance are particle size and density. In spite of the advantages of perfusion system, there are several problems that which occurred in these systems. The increase in titers and live cell density, makes the downstream purification steps more challenging because with the increase of the titer, the amount of DNA released in the environment increases. The sedimentation of cell DNA in the cell holding system (spin filter) occurred frequently.
Results: 1- Optimization of cell growth
With the changes made on the culture medium, the results showed that the cell density can be increased more than twice by choosing the optimized medium. The changes applied in the formulation were not only in the basic culture medium, but also with changes in the amount of glutamine and glucose, as well as the addition of poloxamer in order to reduce shear stress on the cell and helped the cell growth to show an acceptable doubling time.
2- Temperature shift optimization to enhance protein expression
In the production process, by lowering the temperature from 37 to 32 degrees, it was observed that the percentage of cell survival dropped suddenly, so that after about 48 hours, the survival rate reached less than 40%. On the other hand, lowering the temperature to higher temperatures such as 33 and 34 degrees did not have the expected effect on increasing the expression of interferon β-1a. In order to overcome this challenge, we used the strategy of reducing the temperature step by step. This step change in temperature significantly increased the percentage of survival at 32 degrees and helped to prolong the period of cell stagnation and in other words, the period of product production by the cell became wider.
3- Modification of spin filter to prevent clogging
The reliable and efficient spin filter not only prevents the cells from leaving the reactor during the harvest process, but also fulfills some of the important requirements of the system's performance, including avoiding product retention, preventing shear stress, sufficient aeration to the cells, and preventing sedimentation and clogging.
Conclusion: In conclusion to solve the problems mentioned above, various optimizations were performed to select optimal conditions for cell growth and protein expression. The changes applied on the culture medium and supplements, such as changes in the amount of glucose, adding poloxamer in order to reduce the shear stress on the cells, helped the cell growth to show an acceptable doubling time. In the other hand, the modifications applied in structure of the spin filter fulfill the requirements of the system's performance, including avoiding product retention, preventing shear stress, sufficient aeration, and prevent of spin filter clogging. In addition, step by step changes in temperature shift increased the percentage of cell viability and changed cell growth phase to expression phase significantly.
Keywords: Interferon β-1a, Perfusion bioreactor challenge, CHO cells, Temperature shift, spin filter clogging