An Efficient And Low-cost Process For Clarification And Ultrafiltration Of Yeast Expressing Recombinant Proteins
Yeast expression of exogenous genes can not only produce recombinant proteins and peptides, antibodies and vaccines, cytokines, growth factors, hormones and other bioactive substances, but also can be used to produce industrial enzymes, functional foods and biological materials, which has broad application prospects.
The yeast expression system includes two commonly used expression systems, that is, saccharomyces cerevisiae expression system and methanotrophic yeast expression system. The methanol yeast expression system is the most widely used yeast expression system at present, and Pichia is the most commonly used, which can use methanol as carbon and energy. In addition, there are Hansen's yeast, Sphaeroides and so on.
No matter what kind of yeast expression system, its downstream separation and purification processes are similar, and the specific process flow takes yeast expression of recombinant proteins and polypeptide drugs as an example.
Guidling microfiltration membrane cassettes adopts open flow channel and tangential flow filtration mode, which can effectively process complex feed liquid such as yeast fermentation liquid and E. coli cracking liquid at high speed, and can ensure linear amplification from small test to production scale. Compact modular design, simple pipeline connection, independence and readability of the system can protect operators and prevent biological pollution. And no maintenance and cleaning verification. Therefore, it is very suitable for the clarification step of early downstream processing.
The main purpose of the UF/DF process is to remove impurities and displace buffers. Guidling ultrafiltration membrane cassette has good homogeneity of membrane pore size and strong hydrophilicity, which can give the membrane higher flux and lower protein adsorption, and optimized flow channel design, with smaller pressure drop, easy material transfer and significantly shorten filtration time. Moreover, the product series is complete and flexible to meet the needs of various applications of different scales. Therefore, it is very suitable for the UF/DF step of downstream processing.
The following is the application case of Guidling membrane cassette in yeast expression of recombinant proteins.
Microfiltration clarification
We used 0.11 square meters 0.22μm pore membrane cassette to clarify 1.5L yeast fermentation liquid (target protein molecular weight 42-55 KD) by microfiltration. In the whole process of microfiltration, the recovery rate of target protein, turbidity change and membrane flux depletion were investigated.
0-17min is the concentration process, 17-32min is the washing and filtration process. During the whole microfiltration process, TMP gradually increased, the liquid inlet speed remained unchanged, and the average material flux was 45LMH.
The turbidity of the liquid was 600NTU before microfiltration and 8NTU after microfiltration, and the turbidity changed obviously.
The results showed that the yield of target protein was > 90%. Guidling Technology 0.22μm membrane cassette is stable and suitable for this application scenario.
Ultrafiltration concentration
Ultrafiltration concentration of 1.5L clarified liquid (target protein molecular weight 42-55kd) was performed using 0.11 square meters of 10kd membrane cassette. In the whole process of ultrafiltration, the recovery rate of target protein, membrane flux depletion and water flux recovery rate were investigated.
Before and after use, the recovery rate of the membrane can reach 90%.
0-15min is the concentration process, 15-18min is the liquid exchange process. During the whole ultrafiltration process, TMP gradually increased, the liquid inlet speed remained unchanged, and the average material flux was 50LMH.
The results showed that the target protein was completely trapped. Guidling Technology 10kd membrane cassettes can run stably and is suitable for this application scenario.
Most of the recombinant proteins expressed by yeast were not highly expressed. The turbidity of the filtrated liquid was obviously reduced and the protein recovery rate was higher. Because the subsequent chromatographic time is too long, it will cause adverse effects on the protein, in order to shorten the chromatographic sampling time, one step of ultrafiltration concentration can be added, and the target protein can be concentrated quickly after the sample, which can achieve the purpose of saving time, reducing the filler volume and thus reducing the production cost.
Guidling Technology microfiltration membrane cassettes can generally reduce the turbidity of the feed liquid to less than 20NTU, fully meeting the needs of subsequent ultrafiltration and chromatography. Ultrafiltration recovery rate of ultrafiltration membrane cassettes will vary due to different materials, the general recovery rate can reach 90-95%.
Guidling Technology welcomes you to request test kits for related evaluation.
About Guidling
Guidling Technology is a national high-tech enterprise focusing on biopharmaceuticals, cell culture, purification and concentration of biomedicine, diagnosis and industrial fluids. We have successfully developed centrifugal filter devices, ultrafiltration & microfiltration cassettes, virus filter, TFF system, depth filter, hollow fiber, etc. Which fully meet the application scenarios of biopharmaceuticals, cell culture, and so on. Our membranes and membrane filters are widely used in concentration, extraction and separation of pre-filtration, microfiltration, ultrafiltration and nanofiltration. Our many product lines, from small, single-use laboratory filtration to production filtration systems, sterility testing, fermentation, cell culture and more, meet the needs of testing and production. Guidling Technology is looking forward to cooperating with you!
