CN102838655A - Solid-liquid separation method for Pichia pastoris fermentation broth - Google Patents
Solid-liquid separation method for Pichia pastoris fermentation broth Download PDFInfo
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Abstract
The invention discloses a solid-liquid separation method for Pichia pastoris fermentation broth. The method comprises the following steps: uniformly mixing chitosan solution and Pichia pastoris fermentation broth, adjusting pH and stirring, subjecting chitosan to flocculation in the Pichia pastoris fermentation broth, and finally removing strains and macromolecule impurity through centrifugation or filtering to obtain solution comprising recombinant protein. By adopting the method disclosed by the invention, the removal rate of Pichia pastoris strains and the recovery rate of recombinant protein are both significantly improved, the time for separation is short, the cost for separation is low, the obtained solution has high clarity, and the method is more suitable for industrial production.
Description
Technical field
The invention belongs to bioengineering field, be specifically related to the separation purification method of Pichia anomala expression recombinant protein fermentation liquor.
Background technology
Pichia yeast expression system is a kind of novel exogenous protein expression system that grows up the phase at the beginning of the eighties in last century, have operation simple and easy, be easy to that cultivation, fast growth, expression amount are high, low cost and other advantages.This system has become the production that is widely used in recombinant protein at present, like recombinant human serum albumin and fusion rotein thereof, industrial enzymes etc.
Maturation and development along with pichia spp high density fermentation technology; Improve fermentation density and become a kind of Critical policies that improves expressing quantity; Pichia spp zymophyte body burden can reach 40~50% of total fermented liquid at present; Even higher, this has higher requirement for the solid-liquid separation of the total fermented liquid of pichia spp, particularly in commercial process.Pichia spp solid-liquid separating method commonly used at present mainly contains filter press method, membrane separation process, expanding bed chromatography, centrifuging etc.The filter press method mainly adopts plate-and-frame filter press; Reduce the method that filtration resistance realizes roughing out through adding flocculating aids; Have recovery height, easy and simple to handle, advantage such as cost is low; But this method equipment used floor space is big, clarity is low, filter effect is unstable, be difficult for " locked in " operation, and needs to add the flocculating aids of complicated ingredient, is unfavorable for Control of Impurities; Membrane separation process is a new technology that occurs, after the sixties, emerges rapidly in 20 beginnings of the century, carries out the pichia spp roughing out with this method, and clarity is higher, and is easy to operate; Be easy to robotization, but need be with operation behind 3~4 times of the diluted samples, the running time is long, and shearing force is bigger than normal; Be not suitable for separation [Guo Zhenyou etc., the application of tubular fibre millipore filtration (PVDF) in fermentation industry is produced, the Chinese biological engineering magazine of labile protein; 2004,24 (4), 81~85]; The expanding bed chromatography is as pichia spp initial gross separation method; Can replace solid-liquid separation, concentrate and three steps operation such as preliminary purification; Have raising the efficiency, shorten advantages such as running time;, the industrialization of pichia spp is applied in producing at present; But this method is high to equipment requirements, investment is big, cost high [Sumi A.et al Purification of recombinant human serum albumin efficient purification using STREAMLINE, Bioseparation, 1999; 8 (1-5): 195~200]; Centrifuging is the most frequently used solid-liquid separating method of present pichia spp, can satisfy the separation requirement of various scales with different centrifugation apparatus, but this method energy consumption is bigger than normal, the cycle is long, and the recovery is low, separates not thoroughly, often need dilute or repeatedly centrifugal.Thereby press for a kind of novel solid-liquid separating method, be used for suitability for industrialized production.
The flocculation separation technology is through adding flocculation agent; Make the particle aggregation in the solution, utilize neutralizing effect, bridging effect, the throw out net of compression and the electric charge of electrostatic double layer to catch mechanism such as effect and remove small colloid, particle and suspended substance in the solution and isolating technology [Hao Shaoli etc., the application of precipitated separation technology aspect the protein processing; Grain and foodstuffs industry; 2007,14 (1), 20~22].Flocculation agent mainly is divided into inorganic flocculating agent and polymeric flocculant, and iron commonly used, aluminium inorganic flocculating agent and be that the organic polymer class flocculation agent of representative exists problems such as effect is bad or toxic with the SEPIGEL 305 are so should not adopt.Chitosan is the deacetylation product of chitin, extensively is present in the cell walls of insect, shrimp, crab shell animal shell and fungi, algae.The chemical name of chitosan is 1, and 4-2-amido-2-deoxidation-B-D-glucose is not because of material and preparing method's different relative molecular weights wait from hundreds thousand of to millions of.Containing a large amount of free amine groups in the chitosan molecule, under proper condition, can show cationic polyelectrolyte, therefore have the intensive throwing out, is a kind of rising natural macromolecule flocculating agent.Because of it has characteristics such as natural, nontoxic, tasteless, chitosan is widely used in clarification of drinking water purification, heavy metal recovery, molasses and fruit juice etc.Aspect the leavened prod flocculation; Accidental employing chitosan carries out the report of microbial fermentation small molecules product solid-liquid separation as flocculation agent; But because flocculate with chitosan has certain adsorption for protein, so this method is not used widely in the roughing out of pichia spp fermented liquid.
Zhang Mingzheng etc. disclose a kind of chitosan that adopts to the method that the bacillus thuringiensis fermented liquid flocculates, and add 0.0025% chitosan, pH5.0~6.0; During 30~35 ℃ of temperature, 550rpm stirs 5min, can remove most of water and foreign pigment in the fermented liquid; And make insecticidal proteins crystal and gemma in the fermented liquid obtain concentrating well that [Zhang Mingzheng etc., chitosan are to the throwing out of BT fermented liquid, the Chinese biological control; 2009,25 (2), 133~137].
Liu Bingtao etc. disclose a kind of method that adopts treatment with chitosan to contain proteinic wastewater, add 0.01~0.02% chitosan, and pH is 6.0 o'clock, flocculates, and can effectively remove the protein in the proteinic wastewater, and clearance can reach more than 95%.[Liu Bingtao etc., chitosan are to the throwing out of BT fermented liquid, and Chinese biological is prevented and treated, 2009,25 (2), 133~137].
Wang Qiujing etc. disclose a kind of method that adopts chitosan to remove the thalline in the Sunlover 10 fermented liquid, and the result shows that chitosan is that 4 o'clock flocculating effects are best in the pH value, and the soybean polypeptide concentration in the fermented liquid does not significantly reduce simultaneously.[Wang Qiujing etc., chitosan are to the Study on Flocculated of Sunlover 10 fermented liquid, and soybean is circulated a notice of, and 2007,1 (86), 21-24].
It is flocculation agent that the present invention adopts chitosan; Through controlling solution temperature and pH value, flocculation agent addition, hydrophilic organic solvent addition, formed a kind of solid-liquid separating method of the pichia spp fermented liquid that is applicable to suitability for industrialized production of uniqueness, improved flocculation efficiency; Greatly reduce separating difficulty; Reduced running time and cost, method of the present invention is when guaranteeing the target protein high-recovery simultaneously, and proteic removal also has effect preferably for macromolecule impurity.
Summary of the invention
The invention provides a kind of solid-liquid separating method of brand-new pichia spp fermented liquid, can effectively solve in the pichia spp high density fermentation liquid thalline and remove problems such as difficulty, solid-liquid separation cost height.
The present invention addresses the above problem through following technical scheme:
A kind of solid-liquid separating method of pichia spp fermented liquid may further comprise the steps:
1) a kind of solution that is dissolved with chitosan is provided;
2) solution with step 1) mixes with the pichia spp fermented liquid that contains the recombinant protein tunning;
3) with alkaline matter regulating step 2) pH value to 6~12 of gained mixed solution, stir and make flocculate with chitosan;
4), obtain to contain the solution of recombinant protein with step 3) gained solution centrifugal or filtration.
Wherein, Can adopt and well known to a person skilled in the art method preparation steps 1) the described solution that is dissolved with chitosan; For example, chitosan is dissolved in the hydrochloric acid soln of the chitosan that forms behind hydrochloric acid or the acetic acid or the acetum of chitosan, perhaps with the solution of the direct water-soluble formation of water-soluble chitosan.
Wherein, step 1) further comprises:
A) a kind of hydrophilic organic solvent is joined in the described solution that is dissolved with chitosan of step 1).
The volume ratio of hydrophilic organic solvent and chitosan solution is 1: 9~1: 99, and hydrophilic organic solvent is selected from ethanol, n-propyl alcohol, Virahol, or their mixture, preferred alcohol.
Step 2) in the gained mixed solution content of chitosan be 0.04%~1% (w/v, g/ml).
The described alkaline matter of step 3) is a sodium hydroxide, yellow soda ash, sodium hydrogencarbonate, or their mixture.PH value preferred 8~10.This step is at 2 ℃~25 ℃, is preferably to carry out under 5 ℃~15 ℃ the temperature.Make flocculate with chitosan through stirring, the chitosan crosslinked thalline of flocculation forms larger particles, through filtering or centrifugal removal particle, obtains to contain the solution of recombinant protein at last.
Said recombinant protein is recombinant human serum albumin and fusion rotein thereof, preferred recombinant human serum albumin/il-1 fusion rotein.
Advantage of the present invention is, through the throwing out of chitosan, makes thalline form larger particles, more is prone to realize solid-liquid separation, simultaneously, adds a small amount of hydrophilic organic solvent and can increase the thalline dispersity, improves protein recovery.Compare with existing stripping technique, method disengaging time of the present invention is short, separation costs is low, and the operation controlled range is wide, and last gained clarity of solution is high, is more suitable for suitability for industrialized production.In addition, the present invention has tangible removal effect for some proteic macromolecule impurities, can reduce the downstream purification operation pressure.
Description of drawings
Fig. 1 is the HPLC analytical results of embodiment 1 gained supernatant.
Fig. 2 is the HPLC analytical results of embodiment 4 gained supernatants.
Fig. 3 is the HPLC analytical results of embodiment 7 gained samples.
Fig. 4 is under different chitosan content, thallus removing rate and recombinant protein recovery comparison diagram, and wherein, X-coordinate is represented chitosan content, and ordinate zou is represented per-cent, " ◆-" represent the recombinant protein recovery, " ■-" represents thallus removing rate.
Embodiment
Cell concentration p measuring method among the embodiment: get the centrifugal 15min of 1ml fermented liquid 12000rpm, carry out HPLC behind the supernatant liquid filtering and analyze, confirm that protein content is C
1, other gets the 500ul fermented liquid, accurately adds after the 500ul purified water mixes, and the centrifugal 15min of 12000rpm carries out HPLC and analyzes behind the supernatant liquid filtering, confirm that protein content is C
2, cell concentration p=(C
1-2C
2)/(C
1-C
2) * 100%.
The Tot Prot method of calculation are among the embodiment: C
1* V * (1-p), wherein V is a fermentating liquid volume.
The protein recovery method of calculation are among the embodiment: Tot Prot * 100% in Tot Prot/fermented liquid in the supernatant after the solid-liquid separation.
Thallus removing rate measure to adopt spectrophotometry, under 600nm, measures the OD value of supernatant after fermented liquid and the solid-liquid separation respectively, and thallus removing rate is the ratio of the difference of the OD value of fermented liquid and supernatant and fermented liquid OD value.
Adopt Agilent 1200 performance liquid chromatography to carry out the macromolecule impurity assay among the embodiment, method is with reference to " three appendix VI of Chinese pharmacopoeia version in 2010 Q rHSA polymer measuring method.
Embodiment 1 (Comparative Examples)
Adopt own bacterial classification and fermentation technique in the 100L fermentor tank, to carry out recombinant human serum albumin production, the OD600 that measures fermented liquid is 421, and target protein content C in the supernatant is detected in centrifugal back
1Be 9.5g/L, C is detected in the dilution back
2Be 3.5g/L, calculating cell concentration p is 41.7%.
Get 400ml recombinant human serum albumin fermented liquid (the calculating Tot Prot is 2.2g) after the fermentation ends, place Centrifuge Cup, under 4 ℃ in the Beckman whizzer the centrifugal 5min of 10000rpm; Obtain supernatant 210ml; Protein content is 8.1g/L (Tot Prot is 1.70g) in the detection supernatant, and macromolecule impurity content is 7.5% (RT=8.853 and 9.451 two impurity peaks in the accompanying drawing 1), and centrifugal secondary fermentation liquid OD600 is 22.3; Calculating thallus removing rate is 94.7%, and protein recovery is 77.3%.
Embodiment 2
The 0.176g chitosan is dissolved in the acetum of 39.6ml 1%, then adds the 0.4ml absolute ethyl alcohol, stir; Join then in the fermented liquid of 400ml recombinant human serum albumin described in the embodiment 1; 15 ℃ of red-tape operati temperature stir 30min, add sodium hydrogencarbonate; Regulate pH to 9.5, stir and make flocculate with chitosan.
Above-mentioned mixed solution is placed Centrifuge Cup; Under 4 ℃ in the Beckman whizzer the centrifugal 5min of 10000rpm, obtain supernatant 250ml, detect that protein content is 7.06g/L (Tot Prot is 1.765g) in the supernatant; Macromolecule impurity content is 3.0%; Centrifugal secondary fermentation liquid OD600 is 2.8, and calculating thallus removing rate is 99.3%, and protein recovery is 80.2%.
Embodiment 3
The 0.44g chitosan is dissolved in the acetum of 40ml 1%, joins then in the fermented liquid of 400ml recombinant human serum albumin described in the embodiment 1,15 ℃ of red-tape operati temperature stir 30min, add yellow soda ash, regulate pH to 8.5, stir and make flocculate with chitosan.
Above-mentioned mixed solution is placed Centrifuge Cup; Under 4 ℃ in the Beckman whizzer the centrifugal 5min of 10000rpm, obtain supernatant 250ml, detect that protein content is 7.2g/L (Tot Prot is 1.8g) in the supernatant; Macromolecule impurity content is 3.3%; Centrifugal secondary fermentation liquid OD600 is 1.1, and calculating thallus removing rate is 99.7%, and protein recovery is 81.8%.
Embodiment 4
The 0.44g chitosan is joined in the acetum of 38ml 1%, then add the 2ml absolute ethyl alcohol, stirring and evenly mixing; Join then in the fermented liquid of 400ml recombinant human serum albumin described in the embodiment 1; 15 ℃ of red-tape operati temperature stir 30min, add yellow soda ash; Regulate pH to 8.5, stir and make flocculate with chitosan.
Above-mentioned mixed solution is placed Centrifuge Cup; Under 4 ℃ in the Beckman whizzer the centrifugal 5min of 10000rpm, obtain supernatant 250ml, detect that protein content is 7.5g/L (Tot Prot is 1.875g) in the supernatant; Macromolecule impurity content is 3.1% (accompanying drawing 2RT=8.866 and 9.447 two impurity peaks); Centrifugal secondary fermentation liquid OD600 is 1.2, and calculating thallus removing rate is 99.7%, and protein recovery is 85.2%.
The 2.2g chitosan is dissolved in the acetum of 36ml 1%, then adds the 4ml Virahol, stirring and evenly mixing; Join then in the fermented liquid of 400ml recombinant human serum albumin described in the embodiment 1; 5 ℃ of red-tape operati temperature stir 30min, add yellow soda ash; Regulate pH to 8.0, stir and make flocculate with chitosan.
Above-mentioned mixed solution is placed Centrifuge Cup; Under 4 ℃ in the Beckman whizzer the centrifugal 5min of 10000rpm, obtain supernatant 245ml, detect that protein content is 7.8g/L (Tot Prot is 1.91g) in the supernatant; Macromolecule impurity content is 2.8%; Centrifugal secondary fermentation liquid OD600 is 1.0, and calculating thallus removing rate is 99.8%, and protein recovery is 86.9%.
Embodiment 6
The 4.4g chitosan is dissolved in the hydrochloric acid soln of 38ml 0.5%, then adds the 2ml absolute ethyl alcohol, stirring and evenly mixing; Join then in the fermented liquid of 400ml recombinant human serum albumin described in the embodiment 1; 10 ℃ of red-tape operati temperature stir 30min, add yellow soda ash; Regulate pH to 10, stir and make flocculate with chitosan.
Above-mentioned mixed solution is placed Centrifuge Cup; Under 4 ℃ in the Beckman whizzer the centrifugal 5min of 10000rpm, obtain supernatant 240ml, detect that protein content is 8.0g/L (Tot Prot is 1.92g) in the supernatant; Macromolecule impurity content is 2.7%; Centrifugal secondary fermentation liquid OD600 is 1.0, and calculating thallus removing rate is 99.8%, and protein recovery is 87.3%.
Embodiment 7
The supernatant that contains recombinant human serum albumin that embodiment 5 is obtained carries out purification process according to the CN201010154987.4 disclosed method; Obtain the recombinant human serum albumin liquid concentrator; Adopting its purity of HPLC methods analyst is 99.6% (accompanying drawing 3; RT=14.808), host protein<0.01ppm analyzes with the sulfuric acid phynol method that chitosan is residual not to be higher than 0.2%.
The result shows that the present invention can effectively remove thalline, and through the downstream purification operation, can obtain high-quality recombinant protein, and the chitosan molecule of adding also is easy to remove.
Embodiment 8
The recombination yeast of construction expression recombinant human serum albumin/il-1 fusion rotein is produced bacterial strain; In the 100L fermentor tank, ferment; Acquisition contains the fermented liquid of recombinant human serum albumin/il-1 fusion rotein; The OD600 that measures fermented liquid is 330, and macromolecule impurity content is 6%, target protein content C in the supernatant
1Be 0.2g/L, protein content C is detected in the dilution back
2Be 0.082g/L, calculating cell concentration is 30.5%.
The 2.2g water-soluble chitosan is dissolved in the 38ml purified water, then adds the 2ml absolute ethyl alcohol, stirring and evenly mixing; Joining above-mentioned 400ml then contains in the fermented liquid of recombinant human serum albumin/il-1 fusion rotein (target protein total amount 55.6mg); 15 ℃ of red-tape operati temperature stir 30min, add yellow soda ash; Regulate pH to 8.5, stir and make flocculate with chitosan.
Above-mentioned mixed solution is placed Centrifuge Cup; Under 4 ℃ in the Beckman whizzer the centrifugal 5min of 10000rpm, obtain supernatant 275ml, detect that protein content is 0.17g/L (Tot Prot is 46.75mg) in the supernatant; Macromolecule impurity content is 2.0%; Centrifugal secondary fermentation liquid OD600 is 1.2, and calculating thallus removing rate is 99.6%, and protein recovery is 84.1%.
Embodiment 9
Adopt own bacterial classification and fermentation technique in the 5000L fermentor tank, to carry out recombinant human serum albumin production, obtain fermented liquid 3800L after the fermentation ends, the OD600 that measures fermented liquid is 450, and macromolecule impurity content is 8.4%, target protein content C in the supernatant
1Be 9.8g/L, C is detected in the dilution back
2Be 3.6g/L, cell concentration is 41.9% in the calculating fermented liquid, and the target protein total amount is 21.64kg in the fermented liquid.
The 41.8kg chitosan is dissolved in the acetum of 361L 1%, then adds the 19L absolute ethyl alcohol, stirring and evenly mixing; The 3800L chitosan solution is changed in the above-mentioned recombinant human serum albumin fermented liquid; 15 ℃ of control fermentation jar temperatures stir 30min, add sodium hydrogencarbonate; Regulate pH to 9.5, stir and make flocculate with chitosan.
Above-mentioned mixed solution is added 1% zeyssatite, mix, open transferpump, fermented liquid is pumped into remove thalline in the plate-and-frame filter press, filter the back feeding pressurized air that finishes the debris in the sheet frame is dried up, keep filter pressure to be no more than 0.5MPa.Filter the back and amount to acquisition supernatant 2100L; Protein content is 8.6g/L (Tot Prot is 18.06kg) in the detection supernatant, and macromolecule impurity content is 2.5%, and centrifugal secondary fermentation liquid OD600 is 3.6; Calculating thallus removing rate is 99.2%, and protein recovery is 83.5%.
The parameters detection architecture sees attached list 1 among embodiment 1-6 and the embodiment 8,9.
Subordinate list 1
Can know from the detected result of subordinate list 1,, can effectively improve the recovery of thallus removing rate and target protein, reduce the content of macromolecule impurity through in the pichia spp fermented liquid, adding the agent chitosan of wadding a quilt with cotton with fixed attention.Embodiment 3 shows that with the comparative result of embodiment 4 adding a small amount of hydrophilic organic solvent can improve protein recovery.
Claims (11)
1. the solid-liquid separating method of a pichia spp fermented liquid may further comprise the steps:
1) a kind of solution that is dissolved with chitosan is provided;
2) solution with step 1) mixes with the pichia spp fermented liquid that contains the recombinant protein tunning;
3) with alkaline matter regulating step 2) pH value to 6~12 of gained mixed solution, stir and make flocculate with chitosan;
4), obtain to contain the solution of recombinant protein with step 3) gained solution centrifugal or filtration.
2. the solid-liquid separating method of a kind of pichia spp fermented liquid according to claim 1; It is characterized in that; The described solution that is dissolved with chitosan of step 1) is selected from: the hydrochloric acid soln of chitosan, the acetum of chitosan is perhaps with the solution of the direct water-soluble formation of water-soluble chitosan.
3. the solid-liquid separating method of a kind of pichia spp fermented liquid according to claim 1 is characterized in that, step 1) further comprises:
A) a kind of hydrophilic organic solvent is joined in the described solution that is dissolved with chitosan of step 1).
4. the solid-liquid separating method of a kind of pichia spp fermented liquid according to claim 3 is characterized in that, the volume ratio of said hydrophilic organic solvent and chitosan solution is 1: 9~1: 99.
5. according to the solid-liquid separating method of claim 3 or 4 described a kind of pichia spp fermented liquids, it is characterized in that said hydrophilic organic solvent is selected from ethanol, n-propyl alcohol, Virahol, or their mixture, preferred alcohol.
6. the solid-liquid separating method of a kind of pichia spp fermented liquid according to claim 1 is characterized in that step 2) in the gained mixed solution content of chitosan be 0.04%~1% (w/v, g/ml).
7. the solid-liquid separating method of a kind of pichia spp fermented liquid according to claim 1 is characterized in that, described alkaline matter is selected from sodium hydroxide, yellow soda ash, sodium hydrogencarbonate, or their mixture.
8. the solid-liquid separating method of a kind of pichia spp fermented liquid according to claim 1 is characterized in that, the said pH value of step 3) is 8~10.
9. the solid-liquid separating method of a kind of pichia spp fermented liquid according to claim 1 is characterized in that, step 3) is at 2 ℃~25 ℃, is preferably to carry out under 5 ℃~15 ℃ the temperature.
10. the solid-liquid separating method of a kind of pichia spp fermented liquid according to claim 1 is characterized in that, said recombinant protein is recombinant human serum albumin and fusion rotein thereof.
11. the solid-liquid separating method of a kind of pichia spp fermented liquid according to claim 10 is characterized in that, said recombinant human serum albumin and fusion rotein thereof are recombinant human serum albumin/il-1 fusion rotein.
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CN112375749A (en) * | 2020-11-17 | 2021-02-19 | 河北省微生物研究所 | Solid-liquid separation method for pichia pastoris fermentation liquor for producing glucose oxidase |
CN115554978A (en) * | 2022-09-26 | 2023-01-03 | 武汉新华扬生物股份有限公司 | Compound for post-treatment of fermentation liquor and post-treatment method of fermentation liquor |
CN117024567A (en) * | 2023-10-09 | 2023-11-10 | 健通(济南)生物科技有限公司 | Process for improving centrifugal yield of recombinant human serum albumin fermentation liquor |
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CN109097313A (en) * | 2018-10-10 | 2018-12-28 | 上海芳甸生物科技有限公司 | A kind of fermentation process of bacillus and application |
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CN112301006A (en) * | 2020-11-17 | 2021-02-02 | 武汉新华扬生物股份有限公司 | Method for accelerating filtering speed of pichia pastoris fermentation liquor |
CN112375749A (en) * | 2020-11-17 | 2021-02-19 | 河北省微生物研究所 | Solid-liquid separation method for pichia pastoris fermentation liquor for producing glucose oxidase |
CN112301006B (en) * | 2020-11-17 | 2023-08-08 | 武汉新华扬生物股份有限公司 | Method for accelerating filtration speed of pichia pastoris fermentation broth |
CN115554978A (en) * | 2022-09-26 | 2023-01-03 | 武汉新华扬生物股份有限公司 | Compound for post-treatment of fermentation liquor and post-treatment method of fermentation liquor |
CN117024567A (en) * | 2023-10-09 | 2023-11-10 | 健通(济南)生物科技有限公司 | Process for improving centrifugal yield of recombinant human serum albumin fermentation liquor |
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