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Knowledge Sharing] Tangential flow filtration technology applied to fermentation clarification process

2020-12-01

In recent years, with the rapid development of biotechnology and fermentation engineering, more and more recombinant proteins have been developed and applied. Currently, the main way of obtaining recombinant proteins is fermentation production using bacterial, fungal and animal cell expression systems. The downstream purification of recombinant proteins produced by fermentation process faces the problems of excessive product volume, many types of impurities, and high ion concentration.

Clarification of fermentation broths is usually achieved by continuous flow centrifugation, with tube centrifuges and disc centrifuges being the commonly used continuous flow centrifugation equipment. Traditional centrifuges used in process production tend to operate in batch mode, which has many drawbacks. Centrifugal equipment has high manufacturing and operating costs, and the limited rotational speeds they can achieve do not meet the requirements of modern purification processes for high-resolution purification. In addition, centrifuges are very noisy, which is not conducive to personnel health. The batch mode requires periodic slag draining, which can lead to an increase in labor and material resources and cause disruption of the process.

Additionally some of the products secreted during fermentation of the target product (or some non-target products) will inhibit the growth of the bacterium, affect the expression of the target product or the quality of the product of the target product, which will make it difficult to increase the expression and quality of the target product to meet the increasing demand.

Solution

To solve the above pain points in fermentation broth clarification, we designed a solution to replace centrifugation with tangential flow filtration. Instead of centrifugation, we designed a tangential flow filtration program. Tangential flow filtration is used to retain the bacteria or break the liquid to achieve clarification. This solution can realize the continuous automation of the process, reduce human error and avoid problems caused by personnel operation. It can reduce labor and material resources, and lower operating costs. The tangential flow filtration device can be integrated with the fermenter into a perfusion fermenter to realize the perfusion culture of the bacterium, and the real-time supplementation of the medium and the discharge of the expression product or non-target product during the cultivation process can effectively reduce its impact on the bacterium or the target product, so as to improve the expression of the target product as well as the quality, etc., and also reduce the pressure of downstream purification.

Experimental data

Experimental Record I:

The following record shows the results of an experiment in which a recombinant protein product was clarified in place of centrifugation by tangential flow filtration through an MF filter membrane after yeast fermentation. This experiment simulates the perfusion culture process, which improves the expression of the target product and at the same time replaces the centrifugal clarification operation.

Experiment 2:

The following record shows the experimental results of a recombinant protein product, the fermentation broth after bacterial fermentation was clarified by tangential flow filtration through MF filtration membrane, the purpose of this experiment is to replace the tube centrifuge to remove the bacteria and collect the fermentation clarified broth.

Conclusion

From the above data, it is clear that the filtration flux of the membrane is relatively stable when clarifying the fermentation broth (fermentation stage or harvested fermentation broth) by tangential flow filtration. By analyzing the test data, it can be seen that the clarification scheme by tangential flow filtration has a collection effect on the target products. The recovery of the membrane can be more than 90% after the experiment through the standard cleaning process, and the recovery is good. Therefore, the bacteria can be directly trapped by tangential flow filtration mode to achieve the purpose of fermentation broth clarification.

Equipment used for the experiment