CN116082490B

CN116082490B - Method for removing pigment in recombinant human serum albumin purification process

Info

Publication number: CN116082490B
Application number: CN202310368838.5A
Authority: CN
Inventors: 艾云鹏; 柏凯; 高重; 赵娜
Original assignee: Jiantong Jinan Biotechnology Co ltd
Current assignee: Jiantong Jinan Biotechnology Co ltd
Priority date: 2023-04-10
Filing date: 2023-04-10
Publication date: 2023-07-04
Anticipated expiration: 2043-04-10
Also published as: CN116082490A

Abstract

The application discloses a method for removing pigment in the purification process of recombinant human serum albumin, and relates to the technical field of biology. The method comprises the following steps: (1) Centrifuging a fermentation liquor containing recombinant human serum albumin, filtering and concentrating, and regulating the pH value of the fermentation liquor to 5.0-5.5; (2) Sequentially passing the fermentation liquor after the pH adjustment in the step (1) through an anion exchange chromatographic column and a reversed phase polymer filler chromatographic column to perform pigment adsorption; (3) L-cysteine, sodium octoate and EDTA are added into the chromatographic product, and then the chromatographic product is decolored by heating. Through the mode that anion exchange chromatography and reversed phase polymer filler chromatography combine together, carry negative charge and polarity weak pigment impurity in the specificity absorption fermentation liquor to further add L-cysteine, sodium octoate and EDTA and heat decoloration in order to get rid of the pigment that combines in human serum albumin inside, the method that this application provided is when guaranteeing the product yield, still effectively gets rid of pigment impurity, can improve the security of product in clinical use.

Description

Method for removing pigment in recombinant human serum albumin purification process

Technical Field

The application relates to the field of biotechnology, in particular to a method for removing pigment in the purification process of recombinant human serum albumin.

Background

Human serum albumin (Human Serum Albumin, HSA) is a single-chain non-glycosylated soluble polypeptide consisting of 585 amino acids, has a molecular weight of 66.6kDa and accounts for about 60% of total protein in blood plasma, and HSA plays an important role in maintaining blood pressure and anticoagulation of blood plasma as the protein with the most abundant content in the blood plasma, can protect and stabilize immunoglobulin in blood, and has a great clinical demand. With the development of genetic recombination techniques, obtaining recombinant human serum albumin (recombinant Human Serum Albumin, rHSA) by genetic engineering such as yeast (USP 5330901, JP11-509525, JP 6-100592) and further purifying to obtain HSA has become a popular field today.

However, the recombinant human serum albumin fermentation broth obtained by genetic engineering has more impurities, and main impurity components comprise hetero protein, nucleic acid, fatty acid, pigment, polysaccharide, various proteases and the like, and especially the recombinant human serum albumin obtained from pichia pastoris is wrapped with a plurality of pigments which are difficult to remove, so that the albumin is yellow, even dark yellow, rather than colorless or light yellow when the concentration of the common albumin is high, and considering that the dosage of the human serum albumin is large in clinic, the dosage of most protein medicines such as cytokines and hormones is only ug or ng level, the purity requirement of the human serum albumin is more strict than that of other protein medicines, and therefore, a method for effectively removing various pigments in the process of separating and purifying the recombinant human serum albumin is needed.

In the prior art, macroporous adsorption resin or macroporous anion decolorizing resin is mostly used, and pretreated fermentation liquor circularly flows through a decolorizing column filled with resin for decolorizing, or anion chromatography is used for removing pigment. On one hand, the washing regeneration period of the resin material is long, the decolorization is long, so the operation is inconvenient, and the problems of abnormal rise of pH and absorption of target protein by the resin also exist in the decolorization process of the resin, so that the yield of the protein is low; on the other hand, no matter the decolorization resin or the anion chromatography is used for removing pigment, the problem that a small amount of pigment impurities can not be removed still exists in the obtained human serum albumin product, the purity requirement on the human serum albumin used in clinical treatment is very strict, and the yield of the product human serum albumin in the decolorization process in the prior art is also required to be improved.

Disclosure of Invention

In order to solve the problems, the invention aims to provide a method for removing pigment in the purification process of recombinant human serum albumin, which can ensure the yield of the recombinant human serum albumin and effectively remove pigment impurities which are difficult to remove in the product.

In order to achieve the above purpose of the present invention, the present invention adopts the following technical scheme:

The invention provides a method for removing pigment in the purification process of recombinant human serum albumin, which comprises the following steps:

(1) Centrifuging a fermentation liquor containing recombinant human serum albumin, filtering and concentrating, and regulating the pH value of the fermentation liquor to 5.0-5.5;

(2) Sequentially passing the fermentation liquor after the pH adjustment in the step (1) through an anion exchange chromatography column and a reversed-phase polymer filler chromatography column for pigment adsorption, wherein the anion exchange chromatography is a flow-through chromatography, and the reversed-phase polymer filler chromatography is a flow-through chromatography;

(3) After L-cysteine, sodium octoate and EDTA are added into the chromatographic product, the chromatographic product is decolored by heating, and the L-cysteine is used for entering the interior of a human serum albumin molecule to change the tertiary structure of the human serum albumin, so that the human serum albumin is unfolded into a two-dimensional structure.

Most of the dye impurities with small molecular weight and some protein impurities with large molecular weight in the fermentation liquor have isoelectric points lower than 5.0, the isoelectric points of the human serum albumin are between 4.7 and 4.9, the pH of the fermentation liquor is adjusted to 5.0 to 5.5, negative-charge colored substances in the fermentation liquor can be effectively removed by matching anion exchange chromatography, the isoelectric points of the human serum albumin are very close to 5.0 to 5.5, so that the dye impurities adsorbed by a chromatography medium can be avoided to flow through, the dye impurities with negative charges in the fermentation liquor can be effectively removed, then the dye impurities with weak polarity in the fermentation liquor are further removed by matching reversed-phase polymer filler chromatography, and the two chromatographs are matched in sequence, so that most of the dye impurities in the fermentation liquor can be removed, and only a small part of dye impurities which are difficult to remove remain.

Preferably, the pH of the buffer solution is 5.0, the negative charge of the recombinant human serum albumin is correspondingly increased as the pH is higher, the adsorption capacity of the filler to the recombinant human serum albumin is enhanced, the sample penetration is not facilitated, and when the pH of the buffer solution is 5.0, the pigment removal effect is obvious, and the highest sample yield can be ensured.

By adding L-cysteine, sodium octoate and EDTA into the chromatographic product and then heating for decoloring, pigment impurities which are tightly combined with the human serum albumin, especially combined in the human serum albumin, can be further removed, so that the human serum albumin product with higher purity is obtained, and the safety in clinical treatment is improved.

Preferably, in the step (2), the particle size of the anion exchange chromatography medium is 100-200 mu m, and the ligand of the anion exchange chromatography medium is diethyl aminoethyl, secondary amine or tertiary amine; the buffer solution for leaching is sodium chloride and acetic acid buffer solution, wherein the concentration of sodium chloride in the buffer solution is 50-200 mM, and the concentration of acetic acid is 10-100 mM;

preferably, the ligand of the anion exchange chromatography medium is tertiary amine, and as a strong anionic group, the ligand has a stronger ability to adsorb pigment molecules containing negative charges, so that pigment impurities can be effectively and specifically adsorbed.

Preferably, the concentration of sodium chloride in the buffer solution is 100+/-10 mM, the concentration of acetic acid is 50+/-10 mM, and the pH value is 5.0-5.5;

preferably, the anion exchange chromatography medium is NMgel-200Q; because the sample concentration after fermentation liquor pretreatment is higher, and the contained impurities are complex, the anion exchange chromatography medium of the monodisperse uniform particle size polymer microsphere is used for replacing macroporous resin used in the conventional technology, so that the back pressure is smaller and the sample treatment capacity is large and fast under the condition of a certain sample flow rate, and the large-scale production can be realized.

Preferably, the column height of the anion exchange chromatography column is 370+/-10 mm, the column plate (N/M) is more than or equal to 1000, the larger the particle size of the filler is, the smaller the theoretical column plate number is, the column effect is measured after the NMgel-200Q filler is loaded for a plurality of times by using an electric compression chromatography column with the diameter of 450mm, and the column plate (N/M) is all stabilized above 1000.

The anion exchange chromatography adopts a flow-through chromatography method, and is matched with proper ligand, buffer solution and chromatography medium, so that pigment impurities with negative charges can be adsorbed, and target human serum albumin flows through, meanwhile, a large-particle-size chromatography medium with the size of 100-200 mu m is selected, so that the high efficiency of chromatography speed can be ensured, the adsorption effect on the pigment impurities can be ensured, the particle size is too small, the filtering effect of the chromatography medium is stronger, the service life of the chromatography medium is short, the chromatography speed is slow, the adsorption effect of the chromatography medium on the pigment impurities is not ideal if the particle size is too large, and the adsorption effect and the operation difficulty of pigments can be balanced by selecting proper particle size of the chromatography medium.

Preferably, in the step (2), the particle size of the reversed-phase polymer filler is 100-200 mu m, the ligand of the reversed-phase polymer chromatographic filler is butyl or phenyl, the buffer solution for leaching is sodium chloride and PB buffer solution, the concentration of the sodium chloride in the buffer solution is 50-200 mM, and the PB concentration is 50+/-20 mM;

preferably, the ligand of the reversed-phase polymer chromatographic packing is phenyl, so that the capacity of adsorbing hydrophobic impurities or weak-polarity pigment components is higher, and pigment impurities can be adsorbed more specifically.

Preferably, the concentration of sodium chloride in the buffer solution is 100+/-10 mM, the PB concentration is 50+/-10 mM, and the pH value is 6.5-7.0;

preferably, the inverse polymeric filler is NM200-100;

preferably, the reverse phase polymer packing chromatographic column is 370+/-10 mm in height, and the column plate (N/M) is more than or equal to 1000.

The reverse phase filler is usually a bonding phase with polystyrene as a matrix and relatively weak polar functional groups bonded on the surface; the mobile phase used for the reverse phase packing is relatively polar, typically a mixture of water, buffer and ethanol. The sequence of the samples flowing out of the chromatographic column is that the combination with stronger polarity is flushed out, the components with weaker polarity can be reserved on the chromatographic column more strongly, meanwhile, the fillers have stronger hydrophobicity, macroporous polymer fillers are very effective for separating the samples such as protein and the like, NM200-100 fillers with non-monodisperse particle sizes are used, the sample processing capacity is large, the yield is stable, and the large-scale production can be realized.

Preferably, in the step (3), the dosage of the sodium octoate is 0.5-2 mmol/g recombinant human serum albumin, the dosage of the L-cysteine is 0.5-2 mmol/g recombinant human serum albumin, and the dosage of the EDTA is 0.5-2 mmol/g recombinant human serum albumin;

preferably, the sodium octoate is used in an amount of 1 + -0.2 mmol/g recombinant human serum albumin, the L-cysteine is used in an amount of 1 + -0.2 mmol/g recombinant human serum albumin, and the EDTA is used in an amount of 1 + -0.2 mmol/g recombinant human serum albumin.

Preferably, the heating temperature in the step (3) is 55-65 ℃ and the heating time is 0.3-2 h; preferably, the heating temperature is 60-65 ℃ and the heating time is 0.5-1 h;

preferably, after heating and decoloring, cooling to 10-15 ℃, filtering by adopting a hollow fiber membrane, and carrying out liquid exchange and concentration by adopting a flat membrane package.

The material liquid flows at a certain flow speed on the surface of the inner side membrane layer of the membrane tube under the pressure drive of a rotor pump, substances with small particles such as water, small organic molecules, inorganic ions and the like in the material liquid can be transferred to the other side of the membrane through micropores on the wall of the hollow fiber, and large-size substances such as suspended substances, colloid, particles, organic macromolecules and the like in the material liquid cannot be intercepted through the wall of the hollow fiber, thereby realizing component filtration and separation and purification.

Meanwhile, the chelate generated by EDTA and heavy metal pigment ions can be subjected to dialysis replacement by using the flat membrane to pack the replacement liquid for concentration, so that pigment impurities are separated from fermentation liquid. The flat membrane package is also a tangential flow filtration mode, however, unlike the hollow fiber, the mode of liquid flow in the membrane package belongs to turbulence, under the action of a rotor pump, the liquid flows on the surface of the membrane on the inner side of the membrane package at a set flow rate, water, small molecular organic matters, inorganic ions and the like in the liquid are transferred to the other side of the membrane through the membrane holes on the surface of the membrane package, and macromolecular substances such as protein, nucleic acid and the like in a sample cannot be trapped through the membrane package hole diameter, so that the purposes of certain liquid exchange, concentration, separation and purification are achieved. The 30K ultrafiltration flat plate membrane package used in the embodiment of the application can simultaneously replace residual L-cysteine, EDTA and sodium octoate, and the opened structure of the recombinant human serum albumin can be reduced and folded into a tertiary-quaternary structure.

Preferably, the concentration of the recombinant human serum albumin in the fermentation liquid is diluted to 10-15 mg/mL before L-cysteine, sodium octoate and EDTA are added in the step (3), the concentration of the human serum albumin is not too high, and the yield of the human serum albumin is reduced when the human serum albumin is heated.

Preferably, after step (2) and before step (3), the fermentation broth after reverse phase polymer filler chromatography is sequentially: cationic SP chromatography, hydrophobic butyl chromatography and DEAE chromatography.

The anion exchange chromatography and the reversed-phase polymer filler chromatography are further refined and purified, so that the recombinant human serum albumin can be further purified, other impurities except pigment impurities are removed, the concentrated human serum albumin fermentation broth is obtained, the fermentation broth containing the human serum albumin with higher purity of liquid phase detection monomers is obtained, and then the L-cysteine, EDTA and sodium octoate are added for heating, so that the pigment impurities combined in the recombinant human serum albumin can be more specifically and more effectively removed.

Preferably, the cation SP chromatography is elution chromatography, the SP filler is balanced by acetic acid solution, the mixture is firstly washed by sodium chloride and acetic acid buffer solution after sample loading, and then eluted by sodium chloride and PB buffer solution, and eluent is collected;

preferably, the concentration of the acetic acid solution for balancing is 20+/-5 mM, and the pH value is 4.5+/-0.05;

preferably, the concentration of sodium chloride in the buffer solution for washing impurities is 0.1-0.3M, the concentration of acetic acid is 20+ -3 mM, and the pH value is 4.5+ -0.05;

Preferably, the concentration of sodium chloride in the buffer solution for elution is 0.5-0.6M, and the PB concentration is 20+ -3 mM, pH 7.5+ -0.05.

Preferably, the hydrophobic butyl chromatography is a flow-through chromatography, the butyl filler is balanced by sodium chloride and PB buffer solution, and after sample loading, the butyl filler is washed by the same sodium chloride and PB buffer solution and collected to obtain a flow-through liquid;

preferably, the concentration of sodium chloride in the sodium chloride and PB buffer solution is 0.4-0.5M, the concentration of PB is 20+ -3 mM, and the pH value is 7.0+ -0.05.

Preferably, the DEAE chromatography is elution chromatography, the DEAE filler is balanced by Tris-HCl buffer solution, the mixture is washed by sodium chloride and Tris-HCl buffer solution after the sample is loaded, and the eluent is collected after the mixture is eluted by sodium chloride and Tris-HCl buffer solution;

preferably, the concentration of the Tris-HCl buffer for balancing is 20+/-3 mM, and the pH value is 8.0+/-0.05;

preferably, the concentration of sodium chloride in the washing sodium chloride and Tris-HCl buffer solution is 35+/-3 mM, the concentration of Tris-HCl is 20+/-3 mM, and the pH value is 8.0+/-0.05;

preferably, the concentration of sodium chloride in the buffer solution of the sodium chloride and the Tris-HCl for elution is 160+/-10 mM, the concentration of the Tris-HCl is 20+/-3 mM, and the pH value is 8.0+/-0.05.

According to the method, firstly, negative charge and weak-polarity pigments in fermentation liquor are specifically adsorbed through anion exchange chromatography and reversed-phase polymer filler chromatography, so that fermentation liquor containing recombinant human serum albumin flows through, most pigments which are easy to remove in the fermentation liquor can be adsorbed and removed, meanwhile, the product protein can be prevented from being adsorbed through a flow-through chromatography method, so that the product yield is effectively ensured, few pigments which are difficult to remove in tight combination with recombinant human serum albumin products are further heated and decolored through adding L-cysteine, sodium octoate and EDTA, wherein the L-cysteine can open the tertiary structure of the recombinant human serum albumin, the pigments which are combined in the tertiary structure are exposed, and therefore, a small amount of pigments which are combined with the tight interior of the recombinant human serum albumin can be effectively removed.

In the process of separating and purifying recombinant human serum albumin, in order to mainly remove pigments by anion exchange chromatography and reverse polymer chromatography in the early stage of separation and purification and adding L-cysteine, sodium octoate and EDTA in the later stage of separation and purification for heating and decoloring, the yield of the recombinant human serum albumin can be ensured as much as possible by chromatography in the two flow-through modes in the early stage, most of pigment impurities in fermentation liquor can be removed, the fermentation liquor containing the recombinant human serum albumin with high concentration of liquid phase detection monomers is treated in the later stage of separation and purification, the pigment impurities combined in the recombinant human serum albumin can be removed more effectively and pertinently, the waste of L-cysteine, sodium octoate and EDTA is avoided, the pigment removal effect can be ensured, the whole process is reasonable by adopting a staged treatment mode aiming at pigment types from outside to inside, the operation is simple, the yield of the human serum albumin can be ensured, and most of all, the pigment which is difficult to remove in a small amount in the interior of the human serum albumin can be removed, and the recombinant human serum albumin product with extremely high purity is obtained.

The beneficial effects of this application include:

1. the anion dye and the dye with weak polarity in most fermentation liquor can be removed under the condition of ensuring the yield of the target protein by adopting anion exchange chromatography and reversed phase polymer filler chromatography, optimizing the chromatography conditions and adopting a target protein flow-through mode.

2. In the later period of protein purification, the metal pigment impurities combined in the target protein are removed in a heating decoloration mode after L-cysteine, sodium octoate and EDTA are added, so that the purity of the product is further improved, and the safety in clinical use is improved.

3. In anion exchange chromatography and reversed phase polymer filler chromatography, adopt 100~200 mu m's big particle diameter medium, can guarantee good decortication effect, can also reduce the decoloration time spent, the operation is more convenient, uses the chromatography medium of big particle diameter moreover, and the life of chromatography medium is longer, can effectively reduce cost.

4. After the secondary structure is confirmed by circular dichroism and the tertiary structure is verified by disulfide bond pairing, the secondary structure of the recombinant human serum albumin obtained by adopting the method for removing pigments is completely consistent with the secondary structure of the blood product human serum albumin, the tertiary structure of the recombinant human serum albumin obtained after renaturation is also completely consistent with the tertiary structure of the blood product human serum albumin, and the recombinant human serum albumin product obtained after being treated by adopting the method for removing pigments can be applied to clinical treatment.

Drawings

The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiments of the application and together with the description serve to explain the application and do not constitute an undue limitation to the application. In the drawings:

fig. 1 is a schematic view of the effect of removing color after anion exchange chromatography according to example 1 of the present application: in the figure, a group 1 is fermentation liquor before anion chromatography, a group 2 is collection liquor after anion chromatography, a group 3 is pigment and impurities after first elution of an anion chromatography column, and a group 4 is a small amount of impurities after re-elution of the anion chromatography column;

FIG. 2 is a schematic representation of the effect of color removal after chromatography of reverse polymer fillers according to example 1 of the present application: in the figure, a group 1 is fermentation liquor before reverse polymer filler chromatography, a group 2 is collection liquor after reverse polymer filler chromatography, a group 3 is pigment and impurities after first elution of a reverse polymer seasoning chromatographic column, and a group 4 is a small amount of impurities after re-elution of the reverse polymer filler chromatographic column;

FIG. 3 is a mass spectrum identification chart of SS1 peptide fragment of test Y20210405 in disulfide bond pairing verification tertiary structure according to experimental example 4 of the present application;

FIG. 4 is a mass spectrum identification chart of SS2-3 peptide fragment of test Y20210405 in disulfide bond pairing verification tertiary structure according to experimental example 4 of the present application;

FIG. 5 is a mass spectrum identification chart of SS4-5 peptide fragment of test Y20210405 in disulfide bond pairing verification tertiary structure according to experimental example 4 of the present application;

FIG. 6 is a mass spectrum identification chart of SS6-7 peptide fragment of test Y20210405 in disulfide bond pairing verification tertiary structure according to experimental example 4 of the present application;

FIG. 7 is a mass spectrum identification chart of SS8-9 peptide fragment of test Y20210405 in disulfide bond pairing verification tertiary structure according to experimental example 4 of the present application;

FIG. 8 is a mass spectrum identification chart of SS10-11 peptide fragment of test Y20210405 in disulfide bond pairing verification tertiary structure according to experimental example 4 of the present application;

FIG. 9 is a mass spectrum identification chart of SS12-13-14-15 peptide fragments of test Y20210405 in disulfide bond pairing verification tertiary structure according to experimental example 4 of the present application;

FIG. 10 is a mass spectrum identification chart of SS16-17 peptide fragment of test Y20210405 in disulfide bond pairing verification tertiary structure according to experimental example 4 of the present application;

FIG. 11 is a diagram showing the information of a sample for round two chromatography in the secondary structure of Experimental example 3 of the present application;

FIG. 12 is a graph showing the results of the test sample secondary structure in the round dichroism spectrum of Experimental example 3;

FIG. 13 is a diagram showing the information of a test sample for disulfide bond pairing in the tertiary structure of disulfide bond pairing verification in Experimental example 4 of the present application;

FIG. 14 shows the gradient of liquid phase analysis in disulfide bond pair-verified tertiary structure of Experimental example 4 of the present application.

Detailed Description

In order to more clearly illustrate the general concepts of the present application, a detailed description is provided below by way of example in connection with the accompanying drawings.

The recombinant human serum albumin (rHSA) as referred to herein refers to Human Serum Albumin (HSA) produced by a gene recombination technique, i.e., a gene sequence encoding HSA is introduced into a suitable host cell, the host cell is cultured under suitable conditions, and the gene of HSA is expressed in the host cell, thereby producing rHSA. The host cell is selected from bacteria such as E.coli and Bacillus subtilis, yeasts such as Saccharomyces cerevisiae, pichia pastoris, and preferably the microorganism is Pichia pastoris.

The unit mM is mmol/L, the unit M is mol/L, and the unit mmol/g is mmol/g of human serum albumin.

TL-01, TL-02, TL-03, TL-04, TL-05, as used in this application, are all available from Suzhou Nami micro-tech Co., ltd.

The inventor develops a method with obvious pigment removal effect in the rHSA separation and purification process through extensive experimental research.

The inventor found that in the prior art, in the process of decoloring by using macroporous resin, the phenomenon of abnormal rise of pH and absorption of rHSA exists, which results in low protein yield, and the cleaning and regeneration cycle of the macroporous resin is long, especially the macroporous anion resin, and the condition that pH flushing cannot be carried out exists.

The inventor selects anion exchange chromatography media with a large grain size of 100-200 mu m, most of pigments in the obtained fermentation liquor are negatively charged, so that a large amount of pigments in the fermentation liquor can be removed, an electric axial compression chromatography column is used for replacing a stainless steel chromatography column, an APPS chromatography system is used for replacing a centrifugal pump, on-line detection and sample accurate collection control are realized through electric conduction, ultraviolet absorption and an on-line pH meter for sample treatment, meanwhile, an inverted polymer filler with a polarity of 100-200 mu m is matched with the anion exchange chromatography, functional groups with a polarity of relatively weak are bonded on the surface of the inverted polymer filler and are used for further absorbing pigment impurities with a polarity of relatively weak, finally, strong base and ethanol are used for removing absorbed pigment components, a further decolorization effect is achieved, and rHSA products are enabled to flow through due to the adoption of an rHSA flow-through chromatography mode and targeted absorption of pigment impurities through optimization of chromatography conditions, and therefore the yield of rHSA after the decolorization is very high.

The inventors have further found that, regardless of how the conditions of the above-described chromatography are improved or the step of removing the color is optimized, a small amount of pigment still cannot be removed by means of chromatography. The inventors therefore hypothesize that the possible reasons for this are that the above-described modified method used only uses decolorizing fillers to remove pigments, and therefore only the pigment substances outside rHSA are removed, and pigment impurities bound to rHSA itself, especially to the inside, cannot be removed, and that serum albumin can transport fatty acids, bile pigments, amino acids, steroid hormones, metal ions, etc. in body fluids, indeed has a high affinity for certain ions and compounds, thus making it difficult to remove the pigment impurities bound to rHSA inside.

The inventor has found through experimental verification that, because the rHSA molecule is relatively large and has a relatively loose structure, the hydrophobic cavity is in a free state, L-cysteine is added, L-cysteine is utilized to enter the rHSA molecule, the three-level structure of the hydrophobic cavity is disturbed, rHSA can be unfolded into a peptide chain with a two-dimensional structure, pigment and heavy metal ions combined in the rHSA are exposed, EDTA and nonferrous metal ions are chelated into a stable water-soluble substance, sodium octoate is added, then other pigment impurities combined in the rHSA are removed by heating, finally, 750K hollow fiber membranes are used for filtration and concentration, the released free pigment impurities are removed, the residual L-cysteine is replaced by a 30K ultrafiltration membrane package, and the recombinant human serum albumin can be automatically subjected to renaturation reduction and folding into a three-four-level structure.

The inventor has verified the decoloring effect of various amino acids and short peptides through extensive experiments, and found that adding L-cysteine can effectively open the tertiary structure of rHSA, and the effect of removing pigments is best, wherein glutathione is a tripeptide containing gamma-amide bond and sulfhydryl group and consists of glutamic acid, cysteine and glycine, and the inventor has found that glutathione has a certain decoloring promoting effect in the comparative experiment of heating and decoloring glutathione and cysteine, but glutathione and protein are combined to open the tertiary structure of rHSA to a certain extent, but the glutathione has two problems: firstly, glutathione has poor effect of opening rHSA tertiary structure, which results in poor effect of removing pigment, secondly, glutathione has larger molecular weight, which results in that the transmittance of the combined glutathione and rHSA combination is obviously reduced when 750KD hollow filtration clarification is carried out, so that the yield of the glutathione is much lower than that of L-cysteine in the filtration step after decolorization, and the inventor finally combines factors of the color removal effect and the yield to select L-cysteine, and the scheme is specifically described in the following specific examples and the results thereof.

Example 1

Pretreatment of fermentation broth

The GS115 Pichia pastoris strain (from ATCC), a pPIC9K polyclonal site is inserted into a full-length HSA cDNA fragment, an rHSA expression plasmid formed by Sal I enzyme linearization is used for transforming the GS115 strain by an electroporation method, the transformed Pichia pastoris containing rHSA genes is subjected to fermentation induction for 100 hours to obtain a fermentation liquor 1500L with the expression quantity of 7.8g/L, the stirring rotation speed of a fermentation liquor collecting tank is set to be 350rpm, and centrifugal control parameters are set: the temperature is 15 ℃, the rotating speed is 11500rpm, the feeding flow rate is regulated, so that the solid content of concentrated liquid is 60% -80%, the centrifugal clear liquid enters a centrifugal clear liquid collecting tank, a 750KD hollow fiber system is opened, the pump frequency is regulated to be 35Hz, the clarification filtration is carried out, a 30KD hollow fiber system is opened, the pump frequency is regulated to be 20Hz, the clear liquid is concentrated to be lower than 2400L, 12mM sodium octoate and 10% Vol ethanol are added, the temperature is reduced to 15 ℃ after heating and heat preservation for 1h at 68 ℃, the 750KD hollow fiber system is opened again to filter and clarify the heated raw liquid, and meanwhile, the 30KD ultrafiltration system is opened to carry out dialysis concentration to be 20mg/mL, so that a pretreated sample (a) is obtained.

Anion exchange chromatography

The electric compression chromatographic column (450 x 1000 mM) is filled with ion exchange chromatographic medium TL-01 (NMgel-200Q), the column filling height is 370mM, the column plate (N/M) is more than or equal to 1000, 100L of 1% glacial acetic acid solution is used for flushing the chromatographic column, then 100mM sodium chloride, 50mM glacial acetic acid and buffer solution with pH of 5.5 are used for balancing, and a pretreated sample (a) is loaded, wherein the volume of the pretreated sample (a) is 114L, and the rHSA concentration is 26.3055g/L; after elution with 100mM sodium chloride and 50mM glacial acetic acid buffer, collection of (b) samples, which had a volume of 290L, rHSA concentration of 10.0745g/L and a yield of 97.42%, was started when rHSA-containing flow-through appeared, and the color removal effect was as shown in FIG. 1, and it was found from the results of FIG. 1 that group 3 contained a large amount of eluted impurities and group 2 collected liquid contained only a small amount of light yellowish green impurities, although the figure was a gray scale, as compared with the gray scale of FIG. 1, the group 2 collected liquid after anion exchange chromatography did indeed have removed a large amount of pigment.

Reversed phase polymeric filler chromatography

The electrokinetic compression column (450 x 1000 mM) was packed with reverse phase polymer packing TL-02 (NM 200-100), the column height was 370mM, the column plate (N/M) was not less than 1000, the column was rinsed with 100L 1% glacial acetic acid solution, equilibrated with 100mM sodium chloride, 50mM PB, pH7.0 buffer, 290L of sample (b) was concentrated by 30KD flat membrane pack, sample (c) was collected, its volume was 150L, rHSA concentration was 18.9673g/L, after loading onto the second reverse phase polymeric packing NMGel-200Q column, rinsing with 100mM sodium chloride, 50mM PB buffer was started, after rHSA-containing flow-through was present, sample collection (d) was started, its volume was 165L, rHSA concentration was 16.9512g/L, yield was 98.31%, the results of FIG. 2 showed that group 3 contained a large amount of removed pigment, and the substantial clear pigment removal was not observed by visual observation in group 2.

Fine purification

Sample (d) was subjected to ultrafiltration with a solution of 20mM acetic acid, pH4.5, and then loaded into SP packed column TL-03 (NanoGel 50 SP) equilibrated with a solution of 20mM acetic acid, washed with a buffer solution of 0.2M sodium chloride, 20mM acetic acid, pH4.5, and then eluted with a buffer solution of 0.55M sodium chloride, 20mM PB, pH7.6, while collecting the eluate to obtain sample (e) having a volume of 163L, rHSA concentration of 15.0460 g/L, and liquid phase monomer detection purity of 80.5317%;

Sample (e) was subjected to ultrafiltration with 0.45M sodium chloride, 20mM PB, pH7.0 buffer solution, and then loaded onto Butyl packed column TL-04 (UniHR Butyl 80L) equilibrated with 0.45M sodium chloride, 20mM PB, pH7.0 buffer solution, and washed with 0.45M sodium chloride, 20mM PB, pH7.0 buffer solution, and then collected to give sample (f) having a volume of 235L, rHSA concentration of 8.9917 g/L, and liquid phase monomer detection purity of 94.1076%;

sample (f) was subjected to ultrafiltration with a buffer of 20mM Tris-HCl, pH8.0, and then loaded onto a DEAE-packed column TL-05 (Unigel-30 DEAE) equilibrated with a buffer of 20mM Tris-HCl, pH8.0, washed with a buffer of 35mM NaCl, 20mM Tris-HCl, pH8.0, and eluted with a buffer of 158mM NaCl, 20mM Tris-HCl, pH8.0, while collecting the eluate to obtain sample (g) having a volume of 101L, rHSA concentration of 18.6697 g/L and a liquid phase monomer detection purity of 98.5738%.

Adding L-cysteine, sodium octoate and EDTA, and decolorizing under heating

1mmol/g sodium octoate, 1mmol/g L-cysteine and 1mmol/g EDTA are respectively weighed into a beaker, 1L of water for injection is respectively added under a super clean bench and stirred until the solution is dissolved, the dissolved L-cysteine, sodium octoate and EDTA solution are filtered into a 10L feed supplement bottle through a 0.22um sterile filter cup, the prepared buffer solution is added into a mobile product tank containing a finely purified recombinant albumin stock solution (g) sample through a peristaltic pump, stirring is started, the stirring rotation speed is adjusted to be 100rpm, the temperature is 60 ℃ and heated for 1 hour for decoloration reaction, then the solution is clarified through 750KD hollow fibers, and a 30KD flat membrane package is subjected to liquid change concentration to obtain (h) sample, wherein the volume of the sample is 98L, the rHSA concentration is 12.6225g/L, and the yield is 92.27%.

Example 2

Pretreatment of fermentation broth

The GS115 Pichia pastoris strain (from ATCC), a pPIC9K polyclonal site is inserted into a full-length HSA cDNA fragment, an rHSA expression plasmid formed by Sal I enzyme linearization is used for transforming the GS115 strain by an electroporation method, and after the transformed Pichia pastoris containing rHSA genes is subjected to fermentation induction for 100 hours, fermentation broth 1500L with the expression level of 7.8g/L is obtained, the stirring rotation speed of a fermentation broth collecting tank is set to be 350rpm, and the centrifugation control parameters are set: the temperature is 15 ℃, the rotating speed is 11500rpm, the feeding flow rate is regulated, so that the solid content of concentrated liquid is 60% -80%, the centrifugal clear liquid enters a centrifugal clear liquid collecting tank, a 750KD hollow fiber system is opened, the pump frequency is regulated to be 25Hz, the clarification filtration is carried out, a 30KD hollow fiber system is opened, the pump frequency is regulated to be 25Hz, the clear liquid is concentrated to be lower than 2400L, 10mM sodium octoate and 10% Vol ethanol are added, the temperature is reduced to 15 ℃ after heating and heat preservation for 1h at 68 ℃, the 750KD hollow fiber system is opened again to filter and clarify the heated raw liquid, and meanwhile, the 30KD ultrafiltration system is opened to carry out dialysis concentration to be 30mg/mL, so that a pretreated sample (a) is obtained.

Anion exchange chromatography

The electric compression chromatographic column (450 x 1000 mM) is filled with ion exchange chromatographic medium TL-01 (NMgel-200Q), the column filling height is 370mM, the column plate (N/M) is more than or equal to 1000, 100L of 1% glacial acetic acid solution is used for flushing the chromatographic column, then 100mM sodium chloride, 50mM glacial acetic acid and buffer solution with pH of 5.0 are used for balancing, and a pretreated sample (a) is loaded, wherein the volume of the pretreated sample (a) is 134L, and the rHSA concentration is 23.6313g/L; eluting with 100mM sodium chloride, 50mM glacial acetic acid buffer, pH5.0, and collecting (b) sample after rHSA-containing flow-through appears, wherein the sample is 360L, the rHSA concentration is 8.2437g/L, and the yield is 93.72%.

Reversed phase polymeric filler chromatography

The electric compression chromatographic column (450 x 1000 mM) is filled with reverse phase polymer filler TL-02 (NM 200-100), the column loading height is 370mM, the column plate (N/M) is more than or equal to 1000, 100L of 1% glacial acetic acid solution is used for flushing the chromatographic column, then 100mM sodium chloride, 50mM PB and buffer solution with pH of 6.5 are used for balancing, 290L of sample (b) is concentrated by a 30KD flat membrane package, the sample (c) is collected, the volume of the sample (c) is 149L, the rHSA concentration is 19.5157g/L, after the sample is loaded on a second reverse phase polymerization filler NMgel-200Q chromatographic column, the sample (d) is collected after rHSA-containing flow-through liquid appears, the sample is started to be collected, the volume of the sample (d) is 194L, the rHSA concentration is 14.2609g/L, and the yield is 94.45%.

Fine purification

Sample (d) was subjected to ultrafiltration with a solution of 20mM acetic acid, pH4.2, and then loaded into SP packed column TL-03 (NanoGel 50 SP) equilibrated with a solution of 20mM acetic acid, washed with a buffer solution of 0.2M sodium chloride, 20mM acetic acid, pH4.2, and then eluted with a buffer solution of 0.55M sodium chloride, 20mM PB, pH7.0, while collecting the eluate to obtain sample (e) having a volume of 206L, rHSA concentration of 12.0366g/L, and liquid phase monomer detection purity of 89.62%;

Sample (e) was subjected to ultrafiltration with 0.45M sodium chloride, 20mM PB, pH6.5 buffer solution, and then loaded onto Butyl packed column TL-04 (UniHR Butyl 80L) equilibrated with 0.45M sodium chloride, 20mM PB, pH6.5 buffer solution, washed with 0.45M sodium chloride, 20mM PB, pH6.5 buffer solution, and then collected to obtain sample (f) having a volume of 325L, rHSA concentration of 6.6920 g/L, and liquid phase monomer detection purity of 85.40%;

sample (f) was subjected to ultrafiltration with a buffer of 20mM Tris-HCl, pH7.5, and then loaded onto a DEAE-packed chromatography column TL-05 (Unigel-30 DEAE) equilibrated with a buffer of 20mM Tris-HCl, pH7.5, washed with a buffer of 35mM NaCl, 20mM Tris-HCl, pH7.5, and then eluted with a buffer of 158mM NaCl, 20mM Tris-HCl, pH7.5, while collecting the eluate to obtain sample (g) having a volume of 87L, rHSA concentration of 19.6855g/L, and liquid phase monomer detection purity of 80.22%.

Adding L-cysteine, sodium octoate and EDTA, and decolorizing under heating

1mmol/g sodium octoate, 1mmol/g L-cysteine and 1mmol/g EDTA are respectively weighed into a beaker, 1L of water for injection is respectively added under a super clean bench and stirred until the solution is dissolved, the dissolved L-cysteine, sodium octoate and EDTA solution are filtered into a 10L feed supplement bottle through a 0.22um sterile filter cup, the prepared buffer solution is added into a mobile product tank containing a finely purified recombinant albumin stock solution (g) sample through a peristaltic pump, stirring is started, the stirring rotation speed is adjusted to be 100rpm, the temperature is 60 ℃ and heated for 1 hour for decoloration reaction, then the solution is clarified through 750KD hollow fibers, and a 30KD flat membrane package is subjected to liquid change concentration to obtain (h) sample, wherein the volume of the sample is 97L, the rHSA concentration is 14.4708g/L, and the yield is 95.78%.

Example 3

Pretreatment of fermentation broth

The GS115 Pichia pastoris strain (from ATCC), a pPIC9K polyclonal site is inserted into a full-length HSA cDNA fragment, an rHSA expression plasmid formed by Sal I enzyme linearization is used for transforming the GS115 strain by an electroporation method, and after the transformed Pichia pastoris containing rHSA genes is subjected to fermentation induction for 100 hours, fermentation broth 1500L with the expression level of 7.8g/L is obtained, the stirring rotation speed of a fermentation broth collecting tank is set to be 350rpm, and the centrifugation control parameters are set: the temperature is 15 ℃, the rotating speed is 11500rpm, the feeding flow rate is regulated, so that the solid content of concentrated liquid is 60% -80%, the centrifugal clear liquid enters a centrifugal clear liquid collecting tank, a 750KD hollow fiber system is opened, the pump frequency is regulated to be 30Hz, the clarification filtration is carried out, the 30KD hollow fiber system is opened, the pump frequency is regulated to be 30Hz, the clear liquid is concentrated to be lower than 2400L, 15mM sodium octoate and 10% Vol ethanol are added, the temperature is reduced to 15 ℃ after heating and heat preservation for 1h at 68 ℃, the 750KD hollow fiber system is opened again to filter and clarify the heated raw liquid, and meanwhile, the 30KD ultrafiltration system is opened to carry out dialysis concentration to be 25mg/mL, so that the pretreated sample (a) is obtained.

Anion exchange chromatography

An electric compression chromatographic column (450 x 1000 mM) is filled with an ion exchange chromatographic medium TL-01 (NMgel-200Q), the column filling height is 370mM, the column plate (N/M) is more than or equal to 1000, 100L of 1% glacial acetic acid solution is used for flushing the chromatographic column, 200mM sodium chloride, 100mM glacial acetic acid buffer solution with pH of 5.5 is used for balancing, and a pretreated sample (a) is loaded, wherein the volume of the pretreated sample (a) is 134L, and the rHSA concentration is 23.6313g/L; eluting with 200mM sodium chloride, 100mM glacial acetic acid buffer, pH5.5, and when rHSA-containing flow-through appears, starting to collect (b) sample, volume 310L, rHSA concentration 9.9920g/L, yield 92.46%.

Reversed phase polymeric filler chromatography

The electrokinetic compression column (450 x 1000 mM) was packed with reverse phase polymer packing TL-02 (NM 200-100), the column packing height was 370mM, the column plates (N/M) was not less than 1000, the column plates were washed with 100L of 1% glacial acetic acid solution, equilibrated with 200mM sodium chloride, 70mM PB, pH7.0 buffer, 310L of sample (b) was concentrated by 30KD flat membrane pack exchange, sample (c) was collected at a volume of 151L with rHSA concentration of 19.7514g/L, after loading onto the second reverse phase polymeric packing NMGel-200Q column, elution with 200mM sodium chloride, 70mM PB, pH7.0 buffer was started, and after the flow through containing rHSA was present, sample (d) was collected at a volume of 168L with rHSA concentration of 17.7653g/L, yield of 100.07%.

Fine purification

Sample (d) was loaded onto SP packed column TL-03 (NanoGel 50 SP) equilibrated with 25mM acetic acid, pH4.5 solution after ultrafiltration exchange with 25mM acetic acid, pH4.5 solution, washed with 0.3M sodium chloride, 25mM acetic acid, pH4.5 buffer solution, eluted with 0.6M sodium chloride, 25mM PB, pH7.6 buffer solution, and the eluate was collected to give sample (e) having a volume of 189L, rHSA concentration of 13.1692g/L, and liquid phase monomer detection purity of 88.71%;

Sample (e) was subjected to ultrafiltration with 0.5M sodium chloride, 25mM PB, pH7.0 buffer solution, and then loaded into Butyl packed column TL-04 (UniHR Butyl 80L) equilibrated with 0.5M sodium chloride, 25mM PB, pH7.0 buffer solution, and washed with 0.5M sodium chloride, 25mM PB, pH7.0 buffer solution to obtain sample (f) having a volume of 290L, rHSA concentration of 7.3447g/L, and liquid phase monomer detection purity of 85.70%;

sample (f) was subjected to ultrafiltration with 25mM Tris-HCl, pH8.0 buffer, and then loaded onto DEAE-packed chromatography column TL-05 (Unigel-30 DEAE) equilibrated with 25mM Tris-HCl, pH8.0 buffer, washed with 38mM NaCl, 25mM Tris-HCl, pH8.0 buffer, and then eluted with 170mM NaCl, 25mM, pH8.0Tris-HCl buffer, and the eluate was collected to obtain sample (g) having a volume of 184L, rHSA concentration of 11.4565g/L and liquid phase monomer detection purity of 98.97%.

Adding L-cysteine, sodium octoate and EDTA, and decolorizing under heating

1mmol/g sodium octoate, 1mmol/g L-cysteine and 1mmol/g EDTA are respectively weighed into a beaker, 1L of water for injection is respectively added under a super clean bench and stirred until the solution is dissolved, the dissolved L-cysteine, sodium octoate and EDTA solution are filtered into a 10L feed supplement bottle through a 0.22um sterile filter cup, the prepared buffer solution is added into a mobile product tank containing a finely purified recombinant albumin stock solution (g) sample through a peristaltic pump, stirring is started, the stirring rotation speed is adjusted to be 100rpm, the temperature is 60 ℃ and heated for 1 hour for decoloration reaction, then the solution is clarified through 750KD hollow fibers, and a 30KD flat membrane package is subjected to liquid change concentration to obtain (h) sample, wherein the volume of the sample is 100L, the rHSA concentration is 15.4403g/L, and the yield is 91.92%.

Experimental example 1 detection A350nm/A280nm

Samples (a), (b), (d), (g) and (h) of examples 1 to 3 were diluted to 4mg/mL with cooled injection water, and were subjected to colorimetric analysis and detection of A350nm/A280nm values, with the results shown in Table 1 below.

In the colorimetric analysis, an Agilent Cary60 ultraviolet-visible spectrophotometer is adopted, rHSA samples are diluted to 3-5 mg/mL by using cooled water for injection, the cooled water for injection is used as a blank, a Simple Reads icon is double-clicked by a mouse in a Cary WinUV main window, and a Simple reading control program page is entered. The software can measure the absorbance of the sample at fixed wavelength simply, and can calculate a plurality of wavelength measuring points simply by using a User collector option, thus obtaining the A350nm/A280nm value.

TABLE 1

(data in Table 1 are A350nm/A280 nm)

As is clear from the analysis of the results of the color analysis A350nm/A280nm in Table 1, in examples 1 to 3, it is clear that the sample (b) and the sample (d) can remove most of the pigment impurities in rHSA fermentation broth by the combination of anion exchange chromatography and reversed phase polymerization packing chromatography as compared with the A350nm/A280nm value of the sample (a), and that the pigment impurities are adsorbed by the flow-through chromatography and the selective chromatography conditions at the early stage of fermentation broth treatment, and rHSA product is flowed through, so that not only most of the pigment impurities are removed, but also the rHSA yield is high and the loss of the target protein is small.

A small amount of pigment impurities in a fermentation broth sample (g) subjected to a series of fine purification processes cannot be removed, and compared with the A350nm/A280nm value of a sample (h), the fermentation broth sample (g) is proved to have obvious decolorization effect by adding L-cysteine, sodium octoate and EDTA (ethylene diamine tetraacetic acid) and then heating and decolorizing, wherein the A350nm/A280nm of the fermentation broth sample (g) in the embodiment 1 is as low as 0.0010.

Experimental example 2 influence of different conditions of L-cysteine, sodium octoate and EDTA thermal discoloration

The sample (g) obtained in example 1 was subjected to an experiment according to the procedure of "adding L-cysteine, sodium octoate and EDTA for thermal discoloration" in example 1, to obtain samples 1# to 8# and comparative samples D1# to D5#, the specific differences being shown in Table 2, and other conditions were the same as in example 1, and A350nm/A280nm values and rHSA yields were examined, and the results were shown in Table 2 below.

TABLE 2

As is clear from the analysis of the results in table 2, the D1# result shows that the normal color removal method of adding sodium octoate and heating cannot continue to remove the residual pigment impurities, the D2# result shows that the addition of L-cysteine has a weak color removal effect on the basis of heating by adding sodium octoate, the D3# result shows that the color removal effect of EDTA on the basis of heating by adding sodium octoate is not obvious, the D4# result shows that the addition of L-cysteine and EDTA has a certain color removal effect under the condition of not containing sodium octoate but can lead to a rapid decrease in the yield of the target protein, and the D5# result shows that the addition of sodium octoate, L-cysteine and EDTA has a certain effect of removing the pigment impurities after stirring at normal temperature, but the effect of EDTA complexing the pigment impurities at normal temperature is poor due to lower temperature.

Experimental example 3, round two chromatography confirm the secondary structure

Circular dichroism scanning analysis of proteins and polypeptides is very significant for studying their secondary and higher structures, and there are several methods for studying the secondary and higher structures of proteins and polypeptides, such as X-ray crystal diffraction technique, nuclear magnetic resonance technique, circular dichroism technique, etc. The round two-chromatographic technique is a simpler and effective technique, provides a convenient means for confirming the advanced structure of protein products expressed by genetic engineering, and is a rapid, simple and accurate method for researching protein conformation in dilute solution. The method can be used for measuring in a solution state, is closer to the physiological state, is quick and simple, is sensitive to conformational change, is one of the main means for researching the secondary structure of the protein at present, and is widely applied to conformational research of the protein. In the experimental example, the secondary structure of a protein test sample is analyzed by collecting Circular Dichroism (CD) absorption maps of the protein test sample in far ultraviolet (190-260 nm) and near ultraviolet (250-340 nm) and software.

The experimental principle is that one incident plane polarized light can be regarded as polarized light composed of two beams of left and right polarized light with the same amplitude and opposite phases, after the polarized light passes through an optical active molecule, the absorption of the optical active molecule to left and right circularly polarized light is different, so that the amplitude of the left and right circularly polarized light is changed, the left and right circularly polarized light is transmitted to form elliptical polarized light, the vibration plane deviates from the incident vibration plane, and a certain included angle is formed, and the phenomenon is called circular dichroism. Circular dichroism scanning is to analyze the structure of proteins by utilizing the difference of absorption of circular dichroism and asymmetric molecules to left and right circular polarized light. Proteins are biological macromolecules of a specific structure formed by amino acids linked by peptide bonds. The main photoactive groups in proteins or polypeptides are peptide bonds, aromatic amino acid residues, disulfide bonds, etc. in the backbone of the peptide chain. When the absorption of the planar circularly polarized light is not the same, an absorption difference is generated. Due to the presence of such an absorption difference, an amplitude difference of the polarized light vector is caused, and the circularly polarized light becomes elliptically polarized light, which is circular dichroism of proteins.

The information of three groups of test products is shown in fig. 11, the three groups of test products are recombinant human serum albumin stock solution obtained after the color removal by adopting the method for removing pigments in the application, a chitosan PLUS V100 is adopted as an experimental instrument, and the reagents used in the method comprise: 1) NaH (NaH) ₂ PO ₄ (national medicine, cat. Number: 10020318, lot number: 20210104), 2) Na ₂ HPO ₄ (national medicine, cat# 20040718, lot # 20210524), 3) 10KD ultrafiltration tube (Sartorius, cat#: NV01H02, lot number: 212400607).

Preparation of test article: since salt content in the sample buffer may affect the circular dichroism analysis, the sample is concentrated and buffer-replaced by using a 10KD ultrafiltration tube, the sample is dissolved in 20mM PB, the sample concentration for near-UV scanning is adjusted to about 1.0mg/mL, and the sample concentration for far-UV scanning is adjusted to about 0.2mg/mL.

The standard substance is scanned by far and near ultraviolet: wherein, the scanning wavelength is set to 180-340nm for background test and blank buffer test, and then the round two-color far and near ultraviolet absorption of 1mg/mLCSA standard solution in the range of 180-340nm is collected.

Sample near ultraviolet scanning: colorimetric blood with 2MHNO ₃ Soaking overnight, washing with deionized water, air drying, collecting background, collecting blank buffer solution, adding appropriate amount of sample into cuvette, performing near ultraviolet scanning at 250-340nm, and collecting data.

Sample far ultraviolet scanning: cuvette with 2MHNO ₃ Soaking overnight, washing with deionized water, air drying, collecting background, collecting blank buffer solution, adding appropriate amount of sample into cuvette, performing 190-260nm far ultraviolet scanning, and collecting data.

Scanning map processing: and subtract baseline and smoothing treatment is carried out on all the scanned maps by using software Pro-Data Viewer, and the ratio of the peak and trough CD values of the standard product is calculated, wherein the effective ratio range is 2.08+/-0.06.

According to the far and near circular dichroism scan patterns of the sample and the prediction result of the detailed secondary structure in fig. 12, the circular dichroism scan structure of each batch of sample is basically consistent and accords with the secondary structure of human serum albumin.

Experimental example 4, disulfide bond pairing verifies tertiary structure

Disulfide bonds are important post-translational modification forms of proteins, and are also a relatively special modification, proteins are linked together through various inter-and intra-chain cysteines, which are critical for maintaining the correct higher structure of protein molecules and maintaining the necessary biological activity, so disulfide bonds have been a focus in biochemical analysis of protein drugs. In this experimental example, the disulfide bond pairing modes of the test sample are analyzed by combining non-reductive enzymolysis of protein molecules in solution and LC-MS/MS detection, so that various intra-chain and inter-chain disulfide bond pairing modes of the test sample are defined, and experimental evidence for determining the disulfide bond pairing modes is provided, including primary and secondary mass spectrum data and the like.

The information of the adopted test samples is shown in figure 13, and all three groups of test samples are recombinant human serum albumin stock solution obtained after the color removal by adopting the method for removing pigments.

Enzymolysis: diluting 5uL of the sample to 10 g/mu L, adding 38uL8M UA (pH is adjusted to 5.0 by acetic acid/sodium acetate) into 20 mu L of the diluted sample, adding 2uL 300mM NEM, uniformly mixing, carrying out light-proof reaction at 37 ℃ for 1h, displacing to 1.6MUA buffer solution, adding 2 mu g of Lys-C, carrying out digestion for 2h, adding 2ug of Trypsin, and carrying out digestion for 18h. After the enzyme digestion is finished, 10% of FA is added for acidification to terminate the enzymolysis reaction.

And (3) liquid quality detection: and separating the enzymolysis sample by adopting a UPLC liquid phase system. The solution A is 0.1% formic acid aqueous solution, and the solution B is 0.1% acetonitrile formate solution. Flow rate: 0.3mL/min; ultraviolet detection wavelength 214nm; the column temperature is 55 ℃, the enzymolysis peptide Jing Sepu is subjected to column gradient elution and separation, and the chromatographic gradient is shown in figure 14.

And the enzymolysis peptide segment is separated by liquid chromatography and then enters a mass spectrometer for detection and analysis. The detection mode is as follows: positive ion, parent ion scan range: 50-2500m/z.

Mass spectrum data processing: the mass spectrum acquisition data adopts UNIFIV1.9.4 (Waters) software to carry out theoretical disulfide peptide fragment data retrieval and comparison, verification and analysis before and after reduction.

As shown in FIG. 3-10, according to the disulfide bond pairing verification result, the recombinant human serum albumin stock solutions (batch No. Y20210405, Y20210507 and Y20210609) are subjected to non-reductive enzymolysis and liquid quality detection analysis, 17 disulfide bonds conforming to the theoretical pairing mode are identified, the specific connection forms are C53-C62, C75-C91, C90-C101, C124-C169, C168-C177, C200-C246, C245C253, C265-C279, C278-C289, C316-C361, C360-C369, C392-C438, C437-C448, C461.C477, C476-C487, C514-C559 and C558-C567, and the disulfide bond detection results of the test samples of each batch are basically consistent, so that the three-level structure of the human serum albumin is consistent.

In the scheme, after L-cysteine and EDTA are added, sodium octoate is added and heated, the L-cysteine can open the tertiary structure of rHSA and expose pigment impurities combined in the rHSA, and the heating operation can promote rHSA to change the tertiary structure and simultaneously enable the pigment impurities to be separated from the state combined with rHSA more easily, wherein sodium octoate not only serves as a stabilizer, sodium ions in sodium octoate can also provide a certain sodium ion concentration, and the existence of sodium ions can promote heavy metal pigment ions to be separated from the state combined with rHSA and then be complexed with EDTA. When the amount of L-cysteine, EDTA or sodium octoate is too low, the effect of removing the pigment impurities is reduced, and when the rHSA dilution concentration before heating is too high, the yield of the target protein is remarkably reduced.

In summary, the scheme of removing pigment in the invention sequentially passes through anion exchange chromatography and reversed-phase polymer filler chromatography, most of pigment impurities in fermentation liquor can be removed through the synergistic compounding effect of ligand, filler and buffer solution in the chromatography process, meanwhile, the high yield of product protein is ensured, and finally, a very obvious color removing effect can be obtained on a small amount of pigment impurities which are difficult to remove in the fermentation liquor finally by adding L-cysteine, sodium octoate and EDTA and heating and decoloring, so that the method can provide recombinant human serum albumin products with higher purity, and has great significance on purification and clinical medication of recombinant human serum albumin.

In this specification, each embodiment is described in a progressive manner, and identical and similar parts of each embodiment are all referred to each other, and each embodiment mainly describes differences from other embodiments. In particular, for system embodiments, since they are substantially similar to method embodiments, the description is relatively simple, as relevant to see a section of the description of method embodiments.

The foregoing is merely exemplary of the present application and is not intended to limit the present application. Various modifications and changes may be made to the present application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc. which are within the spirit and principles of the present application are intended to be included within the scope of the claims of the present application.

Claims

1. A method for removing pigments during purification of recombinant human serum albumin, comprising:

(3) And (3) sequentially carrying out reverse-phase polymer filling chromatography on the fermentation liquor: cation SP chromatography, hydrophobic butyl chromatography and DEAE chromatography to obtain chromatography products;

(4) Adding L-cysteine, sodium octoate and EDTA into the chromatographic product, and then heating for decoloring, wherein the L-cysteine is used for entering the interior of a human serum albumin molecule to change the tertiary structure of the human serum albumin, so that the human serum albumin is unfolded into a two-dimensional structure;

in the step (2), the particle size of the anion exchange chromatography medium is 100-200 mu m, and the ligand of the anion exchange chromatography medium is diethyl aminoethyl, secondary amine or tertiary amine; the buffer solution for leaching is sodium chloride and acetic acid buffer solution, wherein the concentration of sodium chloride in the buffer solution is 50-200 mM, and the concentration of acetic acid is 10-100 mM;

in the step (2), the particle size of the reversed-phase polymer filler is 100-200 mu m, and the ligand of the reversed-phase polymer chromatographic filler is butyl or phenyl; the buffer solution for leaching is sodium chloride and PB buffer solution, wherein the concentration of sodium chloride in the buffer solution is 50-200 mM, and the concentration of PB is 50+ -20 mM;

the consumption of the sodium octoate in the step (4) is 0.5-2 mmol/g recombinant human serum albumin, the consumption of the L-cysteine is 0.5-2 mmol/g recombinant human serum albumin, the consumption of the EDTA is 0.5-2 mmol/g recombinant human serum albumin, the heating temperature in the step (4) is 55-65 ℃ and the heating time is 0.3-2 h, and the concentration of the recombinant human serum albumin in the fermentation broth is diluted to 10-15 mg/mL before the L-cysteine, the sodium octoate and the EDTA are added in the step (4).

2. The method according to claim 1, wherein the cation SP chromatography is elution chromatography, the SP packing is equilibrated with acetic acid solution, the eluting is performed by eluting with sodium chloride and acetic acid buffer solution, eluting with sodium chloride and PB buffer solution, and collecting the eluate.

3. The method according to claim 1, wherein the hydrophobic butyl chromatography is flow-through chromatography, the butyl packing is equilibrated with sodium chloride and PB buffer solution, and after loading, the sample is rinsed with the same sodium chloride and PB buffer solution, and the flow-through solution is collected.

4. The method according to claim 1, wherein the DEAE chromatography is elution chromatography, the DEAE packing is equilibrated with Tris-HCl buffer, washed with sodium chloride, tris-HCl buffer, eluted with sodium chloride, tris-HCl buffer, and collected to obtain an eluate.