CN101735303B - Method for separating recombinant protein fermentation liquid - Google Patents
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- CN101735303B CN101735303B CN 200810079823 CN200810079823A CN101735303B CN 101735303 B CN101735303 B CN 101735303B CN 200810079823 CN200810079823 CN 200810079823 CN 200810079823 A CN200810079823 A CN 200810079823A CN 101735303 B CN101735303 B CN 101735303B
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Abstract
The invention provides a method for performing solid-liquid separation on recombinant protein fermentation liquid prepared by microbial fermentation through a ceramic membrane, which comprises the following steps of: pressurizing the fermentation liquid to 0.1 to 0.6MPa at the temperature of between 0 and 30 DEG C; and then entering a ceramic membrane filtering system, so that the microorganisms and suspended solids in the fermentation liquid are intercepted on the feeding side of the membrane and the permeating liquid is solution of a recombinant protein product. The method effectively separates the macromolecular protein products from other solid impurities in the recombinant protein fermentation liquid and has the advantages of stable process, good clarity of the permeating liquid after being filtered, contribution to the subsequent chromatographic treatment, easy realization of industrialization and the like.
Description
Technical field
The present invention relates to a kind of biological fermentation liquid separating method, particularly a kind of separation method of the recombinant protein fermentation liquid to microbial fermentation.
Background technology
At the bio-fermented liquid separation field, the separation method of recombinant protein fermentation liquid mainly comprises centrifugation technique, Plate Filtration technology and membrane separation technique at present.
Centrifugation technique is to be subject to a kind of isolation technique that the behavior of the centrifugal force of an extroversion grows up when making uniform circular motion according to particle.Utilize this technology to carry out solid-liquid separation in the widespread use of a plurality of fields, such as the throw out of isolating after chemical reaction, natural biomacromolecule, inorganics, organism are commonly used to collecting cell, organoid and biomacromolecule material in biological chemistry and other field of biology.Progress along with technology, feed liquid for different states, the multiple centrifugal means that comprise disk stack centrifuge and continuous flow centrifuge have been developed, but these centrifugation techniques exist its intrinsic shortcoming that is difficult to overcome, and comprise the operation power cost is high, be difficult to process a large amount of high solid content feed liquid etc.
The Plate Filtration technology is used for the solid-liquid separation that recombinant protein drug is produced, and its advantage is that equipment investment is few, and cost is low, its shortcoming be need to be larger human input, have a large amount of manual operations, thereby be difficult to realize good batch of repeatability; Sealing difficulty completely, product yield is low; Microbiological contamination feed liquid difficult treatment is difficult to guarantee different feed liquid clarity consistent (Chinese biological engineering magazine, 2004,24 (4): 81-85).
In membrane separation technique, can be divided into polymeric membrane, mineral membrane and liquid membrane according to the material of film used.Commonly used is polymeric membrane, is secondly mineral membrane.Macromolecule member material has cellulose family, polyamide-based, aromatic heterocycle class, polysulfones, polyolefins, silicone rubber kinds, fluoro containing polymers class etc.The polymeric membrane advantage is that kind is many, and alternative large, price is low, and membrane module structure is simpler etc.; Shortcoming is chemical stability and poor heat stability, is subject to microbiological deterioration, easy densification etc.Mineral membrane is made mainly with metal, metal oxide, pottery etc., can high temperature resistant, high pressure, and stable chemical nature, the life-span is long, and the material flux is large, allows to use harsh cleaning condition; Shortcoming is that film is crisp, and is frangible, installation cost high (membrane separation technique, Chemical Industry Press, 1998).That polymeric membrane membrane module structure pattern commonly used mainly contains is flat, 4 kinds of tubular type, rolling and tubular fibre formulas.Plate type membrane assembly is commonly used the plate and frame structure, and its advantage is that membrane washing and replacing are easy, and the feed liquid actual internal area is large, is difficult for stopping up; Shortcoming is that sealing difficulty is large, and the component processing accuracy requirement is high, and the feed liquid flow process is short, needs repeatedly circulation for reaching certain degree of enrichment, is usually used in small-sized the choosing in the film experiment.The tubular membrane component structural principle is similar to tubular heat exchanger, and its advantage is that the feed velocity variable range is large, can process the higher feed liquid of suspended sediment concentration, easy to clean; Shortcoming is that the unit volume membrane area is less, and cost is high.Rolled membrane module structure and spiral-plate heat exchanger are similar, and the unit volume media area is large; But Pressure Drop is large, easily stops up between rete, cleans difficulty.The external diameter of hollow-fibre membrane is 0.5～2mm, can be in membrane module self-supported, membrane module unit volume Intimal area is large, Pressure Drop is little; But easily stop up, feed liquid needs pre-treatment (Hebei University of Science and Technology's journal, 2002,23 (3): 21-26).
Ceramic membrane relates in the application of biological chemical field that cell removes, the clarification of sterilized water production and low molecule organic matter and biofilm reactor etc.At present, the application of ceramic membrane in pharmaceutical industries relates generally to the solid-liquid separation of microbiological pharmacy small molecular bio-fermented liquid.Cai Yueming etc. disclose the method (CN1245247C, 2006.3.15) of small molecules bio-fermented liquids such as utilizing ceramic membrane separation lactic acid, alcohol.(the J CleanerProd such as Kim, 1997,5 (4): be 263～267) 50000 inorganic ceramic membrane ultrafiltration stillage with the relative molecular mass that dams, the macromole that dams and solid residue are pressed into grouts after whizzer is processed, and the main component of penetrating fluid is water and small molecules reducing sugar.
The recombinant protein fermentation liquid of microbial fermentation, for example, the Escherichia coli fermentation cellular lysate liquid such as yeast fermentation broth and the apoptosis induction ligand related fermentation thalline of the recombinant tumor necrosis factor lysates such as recombinant human serum albumin fermented liquid, recombinant human Transferrins,iron complexes fermented liquid, the SNDH fermentation thalline lysate of recombinating etc., the material to be separated of such fermented liquid is tens thousand of daltonian high molecular weight protein products, but also contains the impurity such as somatic cells and suspended solids.For the separation method of above-mentioned recombinant protein fermentation liquid, those skilled in the art are badly in need of a kind of effective solution.
Summary of the invention
The contriver utilizes the ceramic membrane to have carried out a large amount of experiments to multiple recombinant protein fermentation liquid by research for many years, has successfully realized effectively separating of macro-molecular protein product and the impurity such as somatic cells, suspended solids.
Particularly, the present invention relates to:
(1) a kind of ceramic membrane that utilizes carries out the method for solid-liquid separation to recombinant protein fermentation liquid, it is characterized in that, fermented liquid is entered the ceramic membrane filter system after being pressurized to 0.1～0.6Mpa at the temperature of 0～30 ℃, somatic cells in fermented liquid and suspended solids are trapped within charging one side of film, and the penetrating fluid of outflow is recombinant protein product solution.
(2) according to the described separation method of item (1), ceramic membrane aperture wherein is 0.01～2 micron, is preferably 0.2～1.2 micron, is particularly preferably 0.5～0.8 micron.
(3) according to item (1) or (2) described separation method, wherein the fermented liquid temperature is 8～29 ℃, is preferably 10～26 ℃.
(4) according to item (1) or (2) described separation method, wherein to enter the pressure before the ceramic membrane filter system be 0.2～0.5Mpa to recombinant protein fermentation liquid, is preferably 0.3Mpa.
(5) according to item (1) or (2) described separation method, wherein the pH value of recombinant protein fermentation liquid is 5.0～8.0, is preferably 6.0.
(6) according to item (1) or (2) described separation method, wherein the circulation of recombinant protein fermentation liquid in the ceramic membrane filter system is 30～100 liters/meter
2Hour, be preferably 75 liters/meter
2Hour.
(7) according to item (1) or (2) described separation method, wherein the molecular weight of recombinant protein is 10,000～80,000 dalton.
(8) according to item (1) or (2) described separation method, wherein the ceramic membrane filter system is comprised of one or more unit membrane system.
(9) according to item (1) or (2) described separation method, wherein the cultivation host of recombinant protein is eukaryotic cell or prokaryotic cell prokaryocyte.
(10) according to item (1) or (2) described separation method, wherein recombinant protein fermentation liquid is the recombinant protein fermentation liquid of microbial fermentation.
(11) according to item (1) or (2) described separation method, wherein recombinant protein fermentation liquid is selected from one of following: recombinant human serum albumin fermented liquid, recombinant human Transferrins,iron complexes fermented liquid, recombinant tumor necrosis factor apoptosis induction ligand related fermentation thalline lysate or restructuring SNDH fermentation thalline lysate.
The present invention uses ceramic membrane filter system can be comprised of single or multiple ceramic membranes, ceramic membrane wherein (available from of a specified duration my Gao Ke limited-liability company in Jiangsu) aperture is 0.01～2 micron, be preferably 0.2～1.2 micron, be particularly preferably 0.5～0.8 micron.Described ceramic membrane filter system also can be comprised of one or more unit membrane system, and unit membrane system wherein is comprised of a plurality of ceramic membranes.The composing method of above-mentioned ceramic membrane filter system or unit membrane system is the usual means of this area, gets final product according to the shop instruction operation.It will be appreciated by those skilled in the art that, be to satisfy the requirement to recombinant protein product solution different output, described ceramic membrane filter system can use ceramic membrane or the unit membrane system of different quantities.
Recombinant protein in the present invention utilizes the expressed protein of fermentative Production goal gene for utilizing molecular biology method that goal gene is imported in protokaryon or eukaryotic cell, and cultivating the host is the eukaryotic cells such as yeast, can be also prokaryotic cell prokaryocyte.As an example, recombinant protein of the present invention can be microbial fermentation recombinant human serum albumin, recombinant human Transferrins,iron complexes, recombinant tumor necrosis factor is apoptosis induction ligand related or the restructuring SNDH.
Further, it is 0.1～0.6Mpa that recombinant protein fermentation liquid enters the front pressure of ceramic membrane filter system, is preferably 0.2～0.5Mpa, is particularly preferably 0.3Mpa.The fermented liquid temperature is 0～30 ℃, is preferably 8～29 ℃, is particularly preferably 10～26 ℃, and according to the character of protein, it can be constant temperature that temperature is controlled, and can be also alternating temperature.Fermented liquid pH value is 5.0～8.0, is preferably 6.0.The circulation of fermented liquid in the ceramic membrane filter system is 30～100 liters/meter
2Hour, be preferably 75 liters/meter
2Hour.
Compare with traditional separating technologies such as adopting continuous flow centrifuge or hollow-fibre membrane, the separation method of recombinant protein fermentation liquid of the present invention, except chemical stability with ceramic membrane good (acidproof, alkaline-resisting, resistance to oxidation, organic solvent-resistant), high temperature resistant, physical strength is large, wear resistance is good, outside the advantages such as separation accuracy high (can reach nanofiltration) and easy cleaning, also have process stabilizing, with low cost, separation accuracy is high, required manpower is few, can reach better with follow-up Image processing and be connected, be highly susceptible to the advantages such as industrialization.
Particularly, the parameters such as the temperature of the present invention by controlling recombinant protein fermentation liquid in the ceramic membrane filter system, pressure, circulation and ceramic membrane aperture, the good clarity that can realize feed liquid separates, reduce the upper column pressure of chromatography, improve chromatography column and process flow velocity, realized being connected with the better of follow-up Image processing, improved working efficiency.In addition, method provided by the invention can adopt determined processing parameter in small scale experiments in scale operation, as ceramic membrane flow channel length and caliber, only need quantitative linear increasing, be highly susceptible to industrialization and amplify, have high commercial application and be worth.
Description of drawings
Fig. 1 is ceramic membrane filter system stream schematic diagram.
Fig. 2 is recombinant human serum albumin fermented liquid SDS-PAGE electrophorogram, and wherein swimming lane A is centrifugal rear supernatant, and swimming lane B is through supernatant after ceramic membrane filter, and swimming lane C is that protein molecular weight standard is (97400,66200,43000,31000) from big to small.
Fig. 3 is restructuring tumor necrosin relative death inducing ligand fermented liquid SDS-PAGE electrophorogram, wherein swimming lane A is centrifugal rear supernatant, swimming lane B is through supernatant after ceramic membrane filter, swimming lane C is that protein molecular weight standard is (97400 from big to small, 66200,43000,31000,20100,14400).
Fig. 4 is restructuring SNDH fermented liquid SDS-PAGE electrophorogram, and wherein swimming lane a is centrifugal rear supernatant PBAD/SND, and swimming lane b is that protein molecular weight standard is (94000,67000,43000,30000) from big to small.
The flux decline curve that Fig. 5 is the recombinant human serum albumin fermented liquid in the ceramic membrane filter system.
Fig. 6 is the flux decline curve of restructuring SNDH fermentation thalline lysate in the ceramic membrane filter system.
Embodiment
Following embodiment specifically sets forth the separation method of recombinant protein fermentation liquid of the present invention, only understands better the present invention for those skilled in the art, should not be construed as limitation of the present invention.
Embodiment 1 utilizes ceramic membrane filter recombinant human serum albumin fermented liquid
Use genetic engineering bacterium and production method acquisition recombinant human serum albumin fermented liquid that own patent application technology (CN1854301A and CN1854306A) builds, its cell concentration is 40%, and protein concn 16g/L, protein molecular weight are 66KD.
Under 24-30 ℃, above-mentioned fermented liquid (the pH value is 6.0) is pressurized to the ceramic membrane filter system of directly entering after 0.3Mpa (ceramic membrane aperture be 0.5 micron).Yeast cell in fermented liquid and suspended solids etc. are trapped within charging one side of film, get back to head tank by current return circuit, and penetrating fluid is recombinant protein product solution.When weight in wet base reaches 60% (charging 50L, penetrating fluid are 21L), begin to carry out stream by purge path and add the water operation, stream adds water 20L altogether, weight in wet base 70% in the filter residue liquid that backflow detected, filter residue liquid supernatant protein concn stop during lower than 1g/L collecting, and obtain altogether supernatant liquor 45L.The flux decline curve of fermented liquid in the ceramic membrane filter system seen accompanying drawing 5.The fermented liquid electrophoresis result is seen Fig. 2.Concrete experimental data sees the following form.
Embodiment 2 utilizes ceramic membrane filter recombinant human Transferrins,iron complexes fermented liquid
Using the saccharomyces cerevisiae engineered yeast of the expression human transferrin of own technique construction, is 20% through the cell concentration of the fermentation fermented liquid that obtains, and protein concn 0.1g/L, protein molecular weight are 75KD.Under 24-26 ℃, above-mentioned fermented liquid (the pH value is 6.0) is pressurized to the ceramic membrane filter system of directly entering after 0.2Mpa (ceramic membrane aperture be 1.2 microns), yeast cell in fermented liquid, suspended solids etc. are trapped within charging one side of film, get back to head tank by current return circuit, and penetrating fluid is recombinant protein product solution.When weight in wet base reaches 60% (charging 30L, penetrating fluid is 15L) time, begin to carry out stream by purge path and add the water operation, stream adds water 12L altogether, weight in wet base 70% in the filter residue liquid that backflow detected, filter residue liquid supernatant protein concn stops during lower than 0.005g/L collecting, and obtains supernatant liquor 15L, and accumulative total obtains supernatant liquor 30L.Concrete experimental data sees the following form.
Embodiment 3 utilizes the apoptosis induction ligand related fermentation thalline of ceramic membrane filter recombinant tumor necrosis factor lysate
Using the apoptosis induction ligand related colibacillus engineering of expression recombinant tumor necrosis factor of own technique construction, is 10% through the cell concentration of the fermentation fermented liquid that obtains, and target protein concentration is 5g/L, and protein molecular weight is 18KD.At 4 ℃ of lower ultrasonic degradation thalline, (the pH value is 7.0 with above-mentioned lysate, solid concentration is 7%, centrifugal weighting method is measured) directly enter ceramic membrane filter system (ceramic membrane aperture be 0.2 micron) after being pressurized to 0.5Mpa under 10-15 ℃, microbial cells fragment in lysate, suspended solids etc. are trapped within charging one side of film, get back to head tank by current return circuit, and penetrating fluid is recombinant protein product solution.When weight in wet base reaches 50% (charging 30L, penetrating fluid is 25L) time, begin to carry out stream by purge path and add the water operation, stream adds water 10L altogether, weight in wet base 70% in the filter residue liquid that backflow detected, filter residue liquid supernatant protein concn stops during lower than 0.1g/L collecting, and obtains supernatant liquor 11L, and accumulative total obtains supernatant liquor 36L.The fermented liquid electrophoresis result as shown in Figure 3.Concrete experimental data sees the following form.
Embodiment 4 utilizes ceramic membrane filter restructuring SNDH fermentation thalline lysate
The genetic engineering bacterium that utilizes own patented technology (ZL200310116831.7) to build ferments, and the cell concentration of the escherichia coli fermented broth that obtains is 4%, and protein concn 2g/L, protein molecular weight are 45KD,
After fermented liquid was collected, under 5000r/min, centrifugal 5min collected thalline, with the Na of pH8.0
2HPO
4-NaH
2PO
4Damping fluid washing 3 times is dissolved in the thalline after centrifugal in ultrasonic damping fluid (physiological saline of precooling or the Tris-HCl of pH8.0) 500w, 10s/10s fragmentation 20 minutes according to the ratio of 1:10.Under 4 ℃, above-mentioned ultrasonic degradation liquid (the pH value is 8.0) is pressurized to the ceramic membrane filter system of directly entering after 0.3Mpa (ceramic membrane aperture be 0.5 micron), suspended solids in lysate is trapped within (comprising cell debris and inclusion body) charging one side of film, get back to head tank by current return circuit, and penetrating fluid is recombinant protein product solution.When weight in wet base reaches 30%, begin to carry out stream by purge path and add water operation, stream adds water 25L altogether, stops collecting during lower than 0.05g/L when the filter residue liquid supernatant protein concn that backflow detected, obtains altogether supernatant liquor 61L.The fermented liquid electrophoresis result as shown in Figure 4.The flux decline curve of lysate in the ceramic membrane filter system seen accompanying drawing 6.Concrete experimental data sees the following form.
Embodiment 5 utilizes ceramic membrane filter, realizes that better chromatography is connected
The recombinant human serum albumin fermented liquid is carried out respectively centrifugal (15 ℃, 5000 rev/mins, 30 minutes) and ceramic membrane filter (embodiment 1) is processed, the solid suspension of trace is arranged in centrifugal rear supernatant, the clarity of ceramic permeate is obviously higher; Above-mentioned centrifugal rear cleer and peaceful ceramic membrane is seen through liquid, and (applicant has patented technology EP1504031B1 by oneself with same purifying process, chromatography column adopts BPG140) process, on the chromatography of pottery permeate, column pressure is lower (under same linear rate of flow, the upper column pressure of centrifugal rear supernatant is 0.15MPa, and the upper column pressure of ceramic permeate is 0.09Mpa).Under same upper prop pressure condition, the upper column flow rate of pottery permeate is higher, and (the upper column flow rate of centrifugal rear supernatant is 80cm/h, the upper column flow rate 120cm/h of pottery permeate), the loading time, more centrifugal rear supernatant shortened 50%, improved the chromatography working efficiency, thereby realized that better technique is connected.
The industrialization scale-up of embodiment 6 ceramic membrane filters
Utilize the recombinant human serum albumin fermented liquid to carry out the experiment of three phases.Fs utilizes the single ceramic film to complete, and the design parameter of operation is: the ceramic membrane aperture is 0.5 micron, and fermentation liquid circulation filtering system temperature is kept 25 ℃, pump-in pressure 0.3MPa.When weight in wet base reaches 60%, begin to carry out stream by the scavenging solution path and add water operation, it is 0.5 times of initial fermentating liquid volume that stream adds volume of water, weight in wet base 70% in the filter residue liquid that backflow detected, filter residue liquid supernatant protein concn stops collecting during lower than 1g/L.Subordinate phase and phase III utilize respectively systematize integrated 16 and 288 ceramic membranes (membrane area is respectively 3.5 square metres and 63 square metres) to carry out the experiment of pilot scale and production level, and experiment parameter is identical with the fs.
Fermented liquid quality after the separation that the contrast three phases is gathered in the crops, clarity is suitable, and on chromatography, column pressure maintains 0.09MPa, and upper column flow rate all reaches 120cm/h.
Claims (15)
1. one kind is utilized ceramic membrane recombinant protein fermentation liquid to be carried out the method for solid-liquid separation, it is characterized in that, fermented liquid is entered the ceramic membrane filter system after being pressurized to 0.1～0.6Mpa at the temperature of 0～30 ℃, somatic cells in fermented liquid and suspended solids are trapped within charging one side of film, the penetrating fluid that flows out is recombinant protein product solution, the pH value of wherein said fermented liquid is 6.0-8.0, and the aperture of described ceramic membrane is 0.2～1.2 micron.
2. separation method according to claim 1, ceramic membrane aperture wherein is 0.5～0.8 micron.
3. separation method according to claim 1 and 2, wherein the fermented liquid temperature is 8～29 ℃.
4. separation method according to claim 1 and 2, wherein the fermented liquid temperature is 10～26 ℃.
5. separation method according to claim 1 and 2, wherein to enter the pressure before the ceramic membrane filter system be 0.2～0.5Mpa to recombinant protein fermentation liquid.
6. separation method according to claim 1 and 2, wherein to enter the pressure before the ceramic membrane filter system be 0.3Mpa to recombinant protein fermentation liquid.
7. separation method according to claim 1 and 2, wherein the pH value of recombinant protein fermentation liquid is 6.0.
8. separation method according to claim 1 and 2, wherein the circulation of recombinant protein fermentation liquid in the ceramic membrane filter system is 30～100 liters/meter
2Hour.
9. separation method according to claim 1 and 2, wherein the circulation of recombinant protein fermentation liquid in the ceramic membrane filter system is 75 liters/meter
2Hour.
10. separation method according to claim 1 and 2, wherein the molecular weight of recombinant protein is 10,000～80,000 dalton.
11. separation method according to claim 1 and 2, wherein the ceramic membrane filter system is comprised of one or more unit membrane system.
12. separation method according to claim 1 and 2, wherein the cultivation host of recombinant protein is eukaryotic cell or prokaryotic cell prokaryocyte.
13. separation method according to claim 1 and 2, wherein recombinant protein fermentation liquid is the recombinant protein fermentation liquid of microbial fermentation.
14. separation method according to claim 1 and 2, wherein recombinant protein fermentation liquid is selected from one of following: recombinant human serum albumin fermented liquid, recombinant human Transferrins,iron complexes fermented liquid.
15. separation method according to claim 1 and 2, described recombinant protein fermentation liquid is replaced with recombinant tumor necrosis factor apoptosis induction ligand related fermentation thalline lysate or restructuring SNDH fermentation thalline lysate, wherein said fermentation thalline lysate comprises microbial cells fragment, suspended solids and soluble recombinant protein matter product, and its thalline by fermented liquid obtains through ultrasonic degradation.
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| Chew Tin Lee et al.Combined in-fermenter extraction and cross-flow microfiltration for improved inclusion body processing.《Biotechnology and Bioengineering》.2003,第85卷(第1期),103-113. * |
| 郭善辉等.陶瓷微滤膜及其在食品与发酵行业中的应用.《食品与发酵工业》.2008,第34卷(第3期),96-100. * |
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