Research data of hGH raw liquid production process
Part I: Fermentation Process Research
Recombinant hGH fermentation process research points: laboratory shake flask research and canning process research. The main purpose of the laboratory shake flask study is to determine the basic process conditions for the upper tank fermentation by optimizing the expression experiment of rhGH. Then the process is continuously optimized and verified by the fermenter level (5L). Finally, the stable fermentation process of the engineering bacteria was determined, and the expression level of rhGH could reach nearly 2g/L per liter of culture liquid. The process was continuously multi-stage pilot scale (5L volume), confirming the process stability.
1. Laboratory study of rhGH expression process
Under the condition that the conditions of the fermenter are as close as possible, a certain amount of experimental data is obtained by using a small shake to provide a certain data reference for the late-stage test. The 500ml Erlenmeyer flask is mainly used for individual experiments. 2 times within the batch, 1 batch between batches. The subjects were selected to screen the most stable and highly expressed strain EB0200901.
1.1 Optimal induction of pH
Method: Take a single clone, inoculate it into BMGY first-class seed solution, and culture for 17-20 hr; inoculate in a 500 ml Erlenmeyer flask of 50 ml medium in a ratio of 1:10 (each tube has a secondary tube), and culture for about 24 hr. , 1% methanol induction, the pH of the induction phase should be adjusted according to the following table (because it is BMGY, all the pH is directly adjusted in BMMY medium). Samples were induced for 24 hr, OD and pH were determined, and induction was continued with 1% methanol. A total of 48 hrs were induced. Samples were tested for SDS-PAGE before induction and at different times.
1: shake flask supernatant before induction
2, 3, 5, 6, 7: Induction of 24 hr shake flask supernatant (pH 1.0 / point) under the conditions of pH 3~7
8,9,10: Induction of 48 hr shake flask supernatant (pH 1.0/point) under the conditions of pH 3~5
Figure 1. Shake flask pH optimized electropherogram
Conclusion: Since the culture medium used in the shake flask experiment is different in the upper tank, and the pH control in the experiment is not very accurate, it is considered that HGH is better at pH=4.0.
1.2 Type and concentration of protective agent
METHODS: Monoclonal cells were inoculated into BMGY primary seed solution for 17-20 hrs; secondary inoculation in 50 ml low-salt medium (pH 6.0 adjusted with ammonia water) in a ratio of 1:10, cultured in a 500 ml Erlenmeyer flask. About 24 hr, 1% methanol induction. Different protective agents were added according to the following table, and 24 hr samples were taken, and OD and pH were measured for 48 hr. Samples were tested for SDS-PAGE before induction and at different times.
Figure 2. Optimization of electrophoresis of shake flask feeding
Conclusion: First, the Yeast extract has better effects in the three protective agents, and the 1% concentration is also better than the 0.5% concentration. In addition, the induction of 48 hours can increase the expression level, which can be used as a guide for fermentation of the upper tank.
2. Research on fermentation process of rhGH fermenter (pilot study)
Initially completed the pilot test, and obtained certain experimental data, which provided a certain data reference for the stable high expression process of the later pilot test, and provided the basis for further optimization of the pilot process. With a 5L fermenter, the working volume is 3L. The subjects were selected to be the most stable and highly expressed strain EB0200901.
2.1 Preliminary identification of classical methods for Pichia fermentation
METHODS: Monoclonal samples were taken and inoculated into BMGY primary seed solution for 17-20 hrs; inoculated in 250 ml BMGY 1 L Erlenmeyer flask at a ratio of 1:10, cultured for 4-8 hrs, fermented in cans, pH 5. 0 (adjusted with ammonia water), temperature 30 ° C, DO > 35%, after the dissolved oxygen rises, add 10 ml of 50% glycerol at 10-15 rpm, and then start to induce with 100% methanol at a rotation 1 after the dissolved oxygen rises again. Gradually increase the speed, and after the induction, the sample was centrifuged at -20 ° C every 4 hr, and induced to end at 36 hr. The sample was tested for SDS-PAGE and protein content.
1: Fermentation supernatant before induction
2~10: Fermentation supernatants in different induction times (up to 36hr)
Figure 3. Electrophoresis pattern before optimization of fermentation parameters
Conclusion: Through the classical method, we found that Juvetrope HGH can have a certain expression, but the expression level is low, indicating that the fermentation process of this project still needs to continuously explore, optimize the fermentation process route and improve the expression level.
2.2 Optimal pH
METHODS: Monoclonal cells were inoculated into BMGY primary seed solution for 17-20 hrs; inoculated in 3 bottles of 250 ml BMGY 1 L Erlenmeyer flask at a ratio of 1:10, cultured for 4-8 hrs, fermented in cans, The initial pH is 5.0, the pH is induced (as shown in the following table), the temperature is 30 ° C, and the DO is 35%. After the dissolved oxygen rises, 300 ml of 50% glycerol is added at 10-15 rpm, and the dissolved oxygen is raised again and then 100% methanol is used. The degree of rotation 1 began to induce, and the speed was gradually increased. After the induction, the sample was centrifuged at -20 ° C every 4 hr, and the induction was terminated at 48 hr. The sample was tested for SDS-PAGE and protein content.
Conclusion: From the results of electrophoresis, the difference from the shake flask results is: optimal induction of pH = 3.5. Based on literature and experience, it may be possible to use a lower pH (3.0) to achieve better expression levels, and the next step is to use a lower pH for comparison.
2.3 Inducing optimal pH verification
METHODS: Monoclonal cells were inoculated into BMGY primary seed solution for 17-20 hrs; secondary inoculation into 2 bottles of 250 ml BMGY 1 L conical flask at a ratio of 1:10, cultured for 4-8 hrs, initial fermentation of the upper tank pH5.0, induced (as shown in the table below), temperature 30 ° C, DO > 35%, after the dissolved oxygen rise, add 10% glycerol 300ml at 10~15 rotation, and then use 100% methanol to rotate after dissolved oxygen rises again. 1 Start the induction, gradually increase the speed, and then freeze the sample at -20 ° C every 4 hr after induction, and induce the end of 48 hr. The sample was tested for SDS-PAGE and protein content.
Figure 5. Optimized electropherogram of fermentation pH parameters
Conclusion: From the electrophoresis results, the induced pH3.0 expression level is significantly better than the pH 3.5, indicating that the lower pH induction is completely feasible and correct. In addition, the main problem solved by the protective agent and the induced pH selection is to prevent the degradation of the target protein, and we have reached a higher expression level only by changing the induced pH, fully satisfying the process requirements, taking this and cost into account. For reasons of this, the protective agent experiment originally prepared for exploration may not be carried out.
2.4 Fermentation process stability verification:
METHODS: Monoclonal cells were inoculated into BMGY primary seed solution for 17-20 hr; inoculated in 250 ml BMGY 1 L conical flask at a ratio of 1:10, cultured for 4-8 hrs, and the initial fermentation pH of the upper tank was 5. 0, induced pH (the following table), temperature 30 ° C, DO> 35%, after the dissolved oxygen rises, 10% glycerol is added at 10-15 rotations, and after the dissolved oxygen rises again, start with 100% methanol at a rotation of 1. Induction, gradually increase the speed, and after each induction, the sample was centrifuged at -20 ° C every 4 hr, and the induction was terminated at about 48 hr. The sample was tested for SDS-PAGE and protein content.
Through this experiment, the fermentation process of hGH was basically determined and verified. Under the conditions of this process, the expression level of hGH was stable and the expression level was high. The final sample scanning showed that the expression level of the target protein was above 60%, the total amount reached 3g/L fermentation broth, and there was no obvious degradation band. Both provide great convenience for purification. Therefore, it can be said that the current fermentation process is stable, mature and high level.
Part II: Research on rhGH purification process
Mature human growth hormone is a non-glycosylated protein consisting of 191 amino acids. hGH contains two intramolecular disulfide bonds and four cysteines. These two intramolecular secondary bonds form the correct globularity. The conformation plays an important role. Its molecular weight is 22kD.
1. Purification laboratory research
If CM-Serpharose FF (Pharmacia) is selected as the chromatographic medium, the pH of the sample is kept below the isoelectric point of rhGH. The experiment found that the sample loading is very low and the yield of the target protein is very low. The use of heparin and phenyl media, both above and below the isoelectric point, results in low loading and low protein yield.
If Q-Serpharose FF or DEAE-Serpharose (Pharmacia) is selected as the chromatographic medium, the pH of the sample is adjusted to above the isoelectric point of rhGH 5.0, which increases the amount of rhGH loaded and the recovery rate of the target protein is also higher. . Comparing the two anionic media, it was found that Q is more advantageous in terms of loading.
Since it has been reported in the literature that rhGH is prone to amidation above pH 8.0, it is not conducive to the activity of the protein under Ph3.0. Q-sepharose FF was selected as the chromatographic medium, and the download amount was high in the Ph6.0-7.5 PB buffer system, and the separation effect was good.
1.2 1ml pre-packed column, 10-30ml packed column small test
Based on the results of the tube test, a 1ml prepacked column and a 10-30ml packed column were used to further explore the Q and determine the optimal chromatographic conditions (buffer concentration, flow rate, loading conditions, loading, elution conditions). . Column chromatography experiments were performed using a 1 ml prepacked column to investigate the effect of different pH and buffer on the binding of the target protein. The results showed that the buffer system was 10 mM PB, and the binding effect of protein was the best when Ph7.5 was used. The elution of 0.1M NaCl could better wash the target protein and improve the purity of the target protein.
Through experiments we selected the best first-step chromatographic conditions: medium was Q- Serpharose FF, buffer system was 10 mM PB, Ph7.5, eluted with 10% Solution B stage.
Through the small test, we further explored some basic conditions of the second step of chromatography:
The sample containing the target protein eluted by the first step chromatography was linearly eluted by Q-sepharose FF column. From the chromatogram and electropherogram, there was no obvious separation effect between the impurity and the target protein, mainly concentrated in one wash. Out of the peak.
However, after the sample containing the target protein eluted by the first step chromatography was separated by a phenyl column, a target protein having a purity of 98% or more was obtained.
Through the above test results, some basic conditions were initially determined: when the 3K ultrafiltered sample was subjected to the first step chromatography, Q-sepharose FF was selected as the chromatographic medium, 10 mM PB, and Ph7.5 was used as the loading buffer. The target protein was eluted in the 10% Solution B phase; in the second step chromatography, phenyl-sepharose FF was used as the chromatographic medium, 10 mM PB+1M(NH4)2SO4, Ph7.5 was used as the loading buffer, and 10 mM PB, Ph7 was used. .5 As the elution buffer, the purity of rhGH after the two-step chromatography reached 98% or more.
2. rhGH purification pilot test:
Drawing on the results of laboratory research, the amplification and optimization of chromatographic conditions, preliminary determination of the process of pilot research