CN113461804A - Method for reducing pigment in fermentation process of recombinant human serum albumin - Google Patents
Method for reducing pigment in fermentation process of recombinant human serum albumin Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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- C07K—PEPTIDES
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
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Abstract
The invention belongs to the field of biological medicine, and particularly relates to a method for reducing pigment in a fermentation process of recombinant human serum albumin. The method for reducing the pigment in the fermentation process of the recombinant human serum albumin comprises the step of adding sodium caprylate and/or cysteine into fermentation liquor. The invention adds cysteine and sodium caprylate in the fermentation process of the recombinant human serum albumin, can effectively reduce the pigment level in the fermentation liquor, is beneficial to the downstream purification process to reach the level of the pharmaceutical-grade gene recombinant human serum albumin, and simultaneously can stabilize the half-life period of the target protein in the fermentation liquor and protect the albumin from being degraded by protease.
Description
Technical Field
The invention belongs to the field of biological medicine, and particularly relates to a method for reducing pigment in a fermentation process of recombinant human serum albumin.
Background
Human serum albumin HSA is a single chain protein consisting of 585 amino acid residues, aspartic acid at the N-terminus and leucine at the C-terminus. mRNA consists of 2078 nucleotides. The amino acid sequence of the mature HSA molecule has 17 disulfide bonds, one cysteine site and one tryptophan site, and the spatial structure is similar to an ellipse.
Human Serum albumin, hsa, (human Serum albumin) serves as the most abundant protein in human plasma, and plays important roles in maintaining plasma osmolality, transport nutrition, and other factors. The traditional Chinese medicine composition is clinically used for treating shock, burn, trauma, hypoproteinemia or acute hypovolemia, the dosage of the traditional Chinese medicine composition is generally up to 10g, is tens of thousands to millions times of that of other biological factor products, and accounts for the first of the international market blood preparation sales.
In the prior art, the human serum albumin is expressed by utilizing the recombination of microorganisms, but the recombinant albumin obtained by the method contains more exogenous substances, and trace antigenic substances can cause anaphylactic reaction and even harm the life safety of a patient due to the large injection dosage of the human serum albumin, so the requirement on the purity of the recombinant human serum albumin in genetic engineering is higher, and the purification technology is more difficult.
The methanol yeast fermentation process often produces a large amount of pigment, which is very disadvantageous for downstream purification. Currently, there are two main factors that limit albumin to pharmaceutical grade levels: one is the removal of pigment; the other is the removal of HCP. In the existing recombinant human serum albumin production process, after fermentation is completed, a step of removing pigment is added in the purification process, the process is complicated, and the purification effect is not ideal.
Disclosure of Invention
The invention aims to provide a method for reducing pigment in a fermentation process of recombinant human serum albumin.
A method for reducing pigments in a fermentation process of recombinant human serum albumin according to an embodiment of the present invention comprises the step of adding sodium caprylate and/or cysteine to the fermentation broth.
During the fermentation process of the gene recombinant human serum albumin, sodium caprylate and cysteine or a composition of the sodium caprylate and the cysteine are added to achieve the effect of reducing the pigment concentration of the fermentation liquor.
Wherein the dosage of the sodium caprylate is 1-16 mu M, and the dosage of the cysteine is 1-16 mu M; specifically, the amount of sodium caprylate added is 1. mu.M, 2. mu.M, 3. mu.M, 4. mu.M, 5. mu.M, 6. mu.M, 7. mu.M, 8. mu.M, 9. mu.M, 10. mu.M, 11. mu.M, 12. mu.M, 13. mu.M, 14. mu.M, 15. mu.M, 16. mu.M, and the amount of sodium caprylate may be adjusted to a non-integer amount, such as 1.5. mu.M, 2.5. mu.M, 3.5. mu.M, 4.5. mu.M, 5.5. mu.M, 6.5. mu.M, 7.5. mu.M, 8.5. mu.M, 9.5. mu.M, 10.5. mu.M, 11.5. mu.M, 12.5. mu.M, 13.5. mu.M, 14.5. mu.M, 15.5. mu.M, depending on the actual production;
similarly, the amount of cysteine added is 1. mu.M, 2. mu.M, 3. mu.M, 4. mu.M, 5. mu.M, 6. mu.M, 7. mu.M, 8. mu.M, 9. mu.M, 10. mu.M, 11. mu.M, 12. mu.M, 13. mu.M, 14. mu.M, 15. mu.M, 16. mu.M, and the amount of cysteine may be adjusted to a non-integer amount, such as 1.5. mu.M, 2.5. mu.M, 3.5. mu.M, 4.5. mu.M, 5.5. mu.M, 6.5. mu.M, 7.5. mu.M, 8.5. mu.M, 9.5. mu.M, 10.5. mu.M, 11.5. mu.M, 12.5. mu.M, 13.5. mu.M, 14.5. mu.M, 15.5. mu.M, depending on the actual production.
Preferably, the dosage of the sodium caprylate is 1-3 mu M, and the dosage of the cysteine is 1-3 mu M; when 2 μ M sodium caprylate and 2 μ M cysteine were added, the removal of pigments in the fermentation broth was most significant.
The inventor finds that in addition to sodium caprylate and cysteine, agents such as glutathione, DTT, mercaptoethanol and acetyltryptophan also have the effect of reducing the pigment concentration in the fermentation process, and the dosage of the agents is 1-16 mu M.
The sodium caprylate and/or cysteine are added at the same time of beginning of methanol induction, before the beginning of methanol induction or during the methanol induction, wherein the sodium and/or cysteine are added at the same time of beginning of methanol induction, and the effect of pigment reduction is most obvious.
A method for reducing pigments in a fermentation process of recombinant human serum albumin according to an embodiment of the present invention comprises the steps of:
(1) inoculating the seed liquid into a first-stage seed tank for culture;
(2) transferring the fermentation liquor in the first-stage seeding tank to a second-stage seeding tank, and continuing to culture;
(3) transferring the fermentation liquor in the secondary seed tank to a fermentation tank for methanol induction, wherein sodium caprylate and/or cysteine are added while the methanol induction is carried out.
In the method, the seed liquid is inoculated into a first-stage seed tank after being prepared, the YPD culture medium is contained in the first-stage seed tank, the capacity of the first-stage seed tank can be 30L, the first-stage seed tank is generally cultured for 12 hours, and the first-stage seed tank is transferred into a second-stage seed tank when the dissolved oxygen of the fermentation liquid begins to rise again.
The secondary seed tank contains YPD culture medium, the capacity of the secondary seed tank can be 300L, the culture is carried out for about 8 hours, and the secondary seed tank is transferred into a fermentation tank when dissolved oxygen begins to rise again;
the fermentation tank contains organic synthetic culture medium F1, phosphoric acid, potassium hydroxide and glycerol, and vitamin C and synthetic p1 supplementary solution are added before transferring into the fermentation liquid.
Wherein YPD medium comprises glucose, yeast extract powder, and tryptone, and can be mixed at a certain ratio or purchased as a commercial product. In the fermentation tank, 21.6kg of organic synthetic medium F1, 18690mL of phosphoric acid, 2.891kg of potassium hydroxide and 28kg of glycerol were taken. 1L of ascorbic acid and 0.5L of a make-up solution of synthetic p1 were added before the seed transfer in the fermenter, and the pH was adjusted to 5.8 with ammonia.
In the actual fermentation process, the dosage of the reagent can be adjusted according to the volume of the fermentation tank and the like.
According to the method for reducing the pigment in the fermentation process of the recombinant human serum albumin, in the step (3), the dosage of the sodium caprylate is 1-16 mu M, and the dosage of the cysteine is 1-16 mu M.
According to the method for reducing pigment in the fermentation process of the recombinant human serum albumin, in the step (3), the fermentation liquor is transferred to a fermentation tank containing vitamin C and synthetic P1 supplementary liquor.
According to the method for reducing the pigment in the fermentation process of the recombinant human serum albumin, in the step (3), when the wet weight ratio (the ratio of the fermentation liquor to the thalli) of the fermentation liquor is more than 26, methanol induction is carried out.
The invention has the beneficial effects that:
cysteine and sodium caprylate are added in the fermentation process of the recombinant human serum albumin, so that the pigment level in the fermentation liquid can be effectively reduced, the downstream purification process can be facilitated to reach the level of the pharmaceutical-grade gene recombinant human serum albumin, and meanwhile, the content of HCP can be effectively reduced by adding the cysteine and the sodium caprylate in the fermentation process.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 shows a calibration curve during the determination of HCP content.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.
Example 1 Shake flask cultivation phase
1. And (3) a shake flask culture stage: FM21 medium (25mL/250mL Erlenmeyer flask) was prepared and sterilized, and recombinant human serum albumin yeast strains were inoculated uniformly into each prepared FM21 medium, and pH was adjusted to 5.8. Sealing with gauze, placing into shaking table, culturing at 31 deg.C and 220RPM for 24 h.
2. Methanol induction: preparing and preparing FM21 (without glycerol) culture medium (25mL/250mL triangular flask), centrifuging the cultured bacteria liquid for 5min at 3000rpm by using a centrifuge, removing supernatant to retain yeast cells, transferring the yeast cells into FM21 (without glycerol) culture medium, adding 4% methanol, and adjusting the pH to 5.8. The cells were placed on a shaker at 30 ℃ and 260RPM for further cultivation. Methanol was added every 24 hours, 4% methanol each time (1 mL of 100% methanol was added, calculated as 25 mL). Methanol was induced 4 times in total to stop.
1.1 investigating the effect of cysteine in removing pigments and HCPs from fermentation broths
At the same time as the methanol induction started, 5 flasks were taken, each flask being dosed with a different amount of cysteine: 1. mu.M, 2. mu.M, 4. mu.M, 8. mu.M, 16. mu.M. Cysteine was added only once at the beginning of induction per flask. After induction, sampling and removing yeast cells by centrifugation (8000rpm, 10min), taking supernatant to detect the pigment level of the fermentation liquor under different dosages: different ratios of A280, A350, A450 and A600 verify the decoloring effect of cysteine on fermentation liquor, and simultaneously detect the content of HCP.
Using Cary60 uv-vis spectrophotometer, the detection procedure was turned on and the desired wavelengths a280nm, a350nm, a450nm, a600nm were set. A Zero-click blank (purified water) was placed in the sample chamber for reading, and the sample was added for measurement after calibration was completed. Three measurements per sample were averaged.
TABLE 1 decolorization of fermentation broths by cysteine at various doses
Note: the measured data of each group are average values obtained by measuring three times
As shown in Table 1, the decolorization effect was exhibited to some extent when cysteine was added to the fermentation broth, wherein the amount of A350/A280 was only 0.126 at a cysteine amount of 2. mu.M, which was significantly lower than that of the other groups, and thus the pigment removal effect was the best at a cysteine amount of 2. mu.M.
Establishment of a standard curve for HCP content:
sample dilution: after the sample was equilibrated at room temperature for about 30 minutes, the test protein concentration was diluted to 50mg/mL with HCP diluent.
The antigen-coated plate was removed and 25. mu.L of standard (1ng/mL, 4ng/mL, 20ng/mL, 75ng/mL, 250ng/mL), negative control (0ng/mL) and test article were added to the plate well using a pipette gun. Each sample was provided with 2 wells as replicates.
100 μ L of horseradish peroxidase-labeled antibody was added to each well. The coated plate was covered with a sealing plate film and incubated at room temperature (24 ℃ C. + -4 ℃ C.) for 3 hours on an orbital shaker at 400 rpm.
The washing solution is diluted while the coated plate is incubated, and the concentrated washing solution needs to be diluted by 20 times before being used. 19mL of purified water is measured and placed in a 50mL beaker, 1mL of concentrated washing solution is measured by a pipette, added into the beaker, shaken well and placed into a liquid adding groove for standby.
Pouring the liquid in the coating plate into a waste liquid tank, and gently but with certain force, beating on absorbent paper to remove most of the residual liquid. Add 280. mu.L of diluted wash solution to the well with a pipette, decant the solution again and tap. The plate washing was repeated 4 times. The residual liquid on the bottom and outside of the coated plate was wiped off. The lotion is not left in the wells for too long a time, and the wells are not allowed to dry before the substrate is applied. 100 μ of LTMB substrate was added to each well.
Cover plate gel and incubate in the dark at room temperature for 30 min without shaking.
Add 100. mu.L of stop buffer to each well and shake gently.
And opening the microplate reader 30 minutes in advance, preheating, slightly putting the coated plate into the microplate reader, and measuring the absorbance at the wavelength of 450nm and 650 nm.
The standard (1ng/mL, 4ng/mL, 20ng/mL, 75ng/mL, 250ng/mL) had corresponding A450nm absorbance values of 0.186, 0.160, 0.190, 0.412, 1.060, 2.624, and the results are shown in FIG. 1.
The absorbance of the standard solution was plotted against the corresponding concentration to obtain a four-parameter curve, and the absorbance of the test solution was plotted on the four-parameter curve to obtain the corresponding mycoprotein content, the results of which are shown in Table 2.
TABLE 2 Effect of different dosages of cysteine on HCP content of fermentation broths
As shown in Table 2, the addition of cysteine to the fermentation broth resulted in a certain degree of host protein removal, wherein the HCP content was 109189.703ng/mL at a cysteine content of 2. mu.M, which was significantly lower than that of the other groups, and thus the impurity removal effect was the best at a cysteine content of 2. mu.M.
1.2 examine the effect of sodium caprylate on removing pigments and HCP from the fermentation broth.
According to the fermentation method, 5 shake flasks were each added with different doses of sodium caprylate at the same time as the methanol induction was started: 1 μ M, 2 μ M, 4 μ M, 8 μ M, 16 μ M, sodium caprylate was added only once at the beginning of induction per flask. After induction, sampling and removing yeast cells by centrifugation (8000rpm, 10min), taking supernatant to detect the pigment level of the fermentation liquor under different dosages: different ratios of A280, A350, A450 and A600 verify the decoloring effect of the fermentation liquor, and simultaneously detect the content of HCP.
TABLE 3 decolorization of fermentation broths by sodium caprylate at various doses
Note: the measured data of each group are average values obtained by measuring three times
As shown in Table 3, the decolorization effect was achieved to some extent when sodium caprylate was added to the fermentation broth, wherein the A350/A280 was only 0.1311 at a sodium caprylate dose of 2. mu.M, which was significantly lower than that of the other groups, and thus the pigment removal effect was the best at a sodium caprylate dose of 2. mu.M.
TABLE 4 Effect of different doses of sodium caprylate on HCP content of fermentation broths
As shown in Table 4, the sodium caprylate added to the fermentation broth has a certain effect of removing host proteins, wherein the HCP content of 105100.935ng/mL was significantly lower than that of the other groups at a sodium caprylate dose of 2. mu.M, and thus the impurity removal effect was the best at a sodium caprylate dose of 2. mu.M.
1.3 investigating the effect of sodium caprylate and cysteine in combination on removing pigments and HCP in the fermentation broth
According to the fermentation method described above, 5 flasks were each charged with different doses of sodium caprylate and cysteine (combined in amounts of 1. mu.M, 2. mu.M, 4. mu.M, 8. mu.M, 16. mu.M, respectively) at the beginning of the methanol induction, and sodium caprylate was added only once at the beginning of the induction. After induction, sampling and removing yeast cells by centrifugation (8000rpm, 10min), taking supernatant to detect the pigment level of the fermentation liquor under different dosages: different ratios of A280, A350, A450 and A600 verify the decoloring effect of the fermentation liquor, and simultaneously detect the content of HCP.
TABLE 5 decolorization of fermentation broths by sodium caprylate and cysteine combination
As shown in Table 3, the decolorization effect to a certain extent can be achieved by adding cysteine and sodium caprylate to the fermentation broth, wherein, when the amounts of sodium caprylate and cysteine agent are both 2 μ M, the A350/A280 value is only 0.068, which is significantly lower than that of the other groups, and when the amounts of sodium caprylate and cysteine agent are added singly, the A350/A280 value of the fermentation broth is also significantly lower than that of the fermentation broth singly added with 2 μ M of sodium caprylate, 2 μ M of cysteine, sodium caprylate and cysteine agent, so that the pigment removal effect is the best, and the synergistic decolorization effect can be achieved when the amounts of sodium caprylate and cysteine agent are 2 μ M.
TABLE 6 Effect of sodium caprylate and cysteine combination on HCP content of fermentation broths
The results are shown in table 6, and the effect of removing host protein is achieved to some extent after the cysteine and the sodium caprylate are added into the fermentation broth at the same time, wherein, when the amounts of the sodium caprylate and the cysteine agent are both 2 μ M, the HCP content is 50572.818mg/mL, which is significantly lower than that of the other groups, and the HCP content is also significantly lower than that of the 2 μ M cysteine and 2 μ M sodium caprylate added singly. Therefore, when the amount of sodium caprylate and cysteine agent is 2 μ M, the impurity removal effect is the best, and a synergistic effect can be obtained.
Example 2 Process verification
After the seed solution is prepared, a 30L first-class seed tank (YPD culture medium) is inoculated, the seed solution is generally cultured for 12 hours, the dissolved oxygen is recovered, the seed solution is transferred into a 300L second-class seed tank (YPD culture medium), the seed solution is cultured for about 8 hours, the pH is recovered to 0.05, the seed solution is transferred into a 2000L fermentation tank (organic synthetic culture medium F1, phosphoric acid, potassium hydroxide and glycerol), vitamin C and synthetic p1 supplementary solution are added before the seed is transferred into the 2000L fermentation tank, then the pH is adjusted to 5.8 by ammonia water, the supplement of the glycerol-supplemented mixed solution is started when the dissolved oxygen is recovered for the first time, and the methanol induction is started when the wet weight reaches more than 26.
Experimental groups: 2 μ M sodium caprylate and 2 μ M cysteine were added in one portion at the same time as the methanol induction. Methanol was continuously fed, and after the completion of the fermenter culture, a sample was taken and the yeast cells were removed by centrifugation (8000rpm, 10min), and the supernatant was taken.
Control group: the difference from the experimental group was that 2. mu.M sodium caprylate and 2. mu.M cysteine were not added at the time of methanol induction.
Detecting the pigment level of the fermentation liquid under different dosages: different ratios of A280, A350, A450 and A600 verify the decoloring effect of cysteine on fermentation liquor, and simultaneously detect the stability of albumin and the content of HCP.
TABLE 7 Effect of the addition of cysteine and sodium caprylate on fermentation broth pigment
| Group of | Control group | Experimental group |
| A350nm | 1.728 | 0.667 |
| A450nm | 0.876 | 0.375 |
| A600nm | 0.287 | 0.104 |
| A280nm | 3.829 | 3.798 |
| A350/A280 | 0.451 | 0.175 |
| A450/A280 | 0.229 | 0.099 |
| A600/A280 | 0.075 | 0.027 |
The results are shown in Table 7, when the amounts of sodium caprylate and cysteine agent added to the fermentation broth are both 2 μ M, the A350/A280 value is 0.175, which is obviously lower than the value of the control group, which indicates that the decolorization effect of the fermentation broth is best when the amounts of sodium caprylate and cysteine agent added to the fermentation broth are 2 μ M.
TABLE 8 Effect of the addition of cysteine and sodium caprylate on the quality of the fermentation broths
The results are shown in Table 8, when the amounts of sodium caprylate and cysteine agent added to the fermentation broth are both 2 μ M, the HCP content value is 120168.004ng/mL, which is significantly lower than the control value, indicating that the impurity removal effect of the fermentation broth is optimal when the amounts of sodium caprylate and cysteine agent added to the fermentation broth are 2 μ M.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.
Claims (8)
1. A method for reducing pigments in a fermentation process of recombinant human serum albumin, comprising the step of adding sodium caprylate and/or cysteine to the fermentation broth.
2. The method of claim 1, wherein the amount of sodium caprylate is 1-16 μ M and the amount of cysteine is 1-16 μ M.
3. The method for reducing pigment in fermentation process of recombinant human serum albumin as claimed in claim 1 or 2, wherein sodium caprylate and/or cysteine are added during methanol induction.
4. The method of claim 3, wherein the amount of sodium caprylate is 1-3 μ M and the amount of cysteine is 1-3 μ M.
5. The method for reducing pigment in fermentation process of recombinant human serum albumin as claimed in claim 1, characterized in that, the method comprises the following steps:
(1) inoculating the seed liquid into a first-stage seed tank for culture;
(2) transferring the fermentation liquor in the first-stage seeding tank to a second-stage seeding tank, and continuing to culture;
(3) transferring the fermentation liquor in the secondary seed tank to a fermentation tank for methanol induction, wherein sodium caprylate and/or cysteine are added while the methanol induction is carried out.
6. The method for reducing pigment in fermentation process of recombinant human serum albumin as claimed in claim 1, wherein in step (3), the amount of sodium caprylate is 1-16 μ M and the amount of cysteine is 1-16 μ M.
7. The method for reducing pigment in fermentation process of recombinant human serum albumin as claimed in claim 1, wherein in step (3), the fermentation broth is transferred to a fermenter containing vitamin C and synthetic P1 supplement.
8. The method for reducing pigment in fermentation process of recombinant human serum albumin as claimed in claim 1, wherein in step (3), methanol induction is performed when the wet weight ratio of the fermentation broth is more than 26.
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| CN1496993A (en) * | 2002-05-15 | 2004-05-19 | ������ҩ�����������ι�˾ | Albumin purifaction |
| CN102070714A (en) * | 2010-11-26 | 2011-05-25 | 大连理工大学 | Method for separating and purifying recombined proserum |
| US20170349645A1 (en) * | 2015-01-01 | 2017-12-07 | Navya Biologicals Pvt Ltd. | Novel method for efficient purification of human serum albumin |
| CN109988803A (en) * | 2019-05-16 | 2019-07-09 | 齐智 | A kind of fermentation process of efficient production recombination human serum albumin |
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| CN1496993A (en) * | 2002-05-15 | 2004-05-19 | ������ҩ�����������ι�˾ | Albumin purifaction |
| CN102070714A (en) * | 2010-11-26 | 2011-05-25 | 大连理工大学 | Method for separating and purifying recombined proserum |
| US20170349645A1 (en) * | 2015-01-01 | 2017-12-07 | Navya Biologicals Pvt Ltd. | Novel method for efficient purification of human serum albumin |
| CN109988803A (en) * | 2019-05-16 | 2019-07-09 | 齐智 | A kind of fermentation process of efficient production recombination human serum albumin |
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| CN116082490A (en) * | 2023-04-10 | 2023-05-09 | 健通(济南)生物科技有限公司 | Method for removing pigment in recombinant human serum albumin purification process |
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