CN117126227A - Application of surfactant in improving recovery rate of protein purified by hydrophobic chromatography - Google Patents

Application of surfactant in improving recovery rate of protein purified by hydrophobic chromatography Download PDF

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CN117126227A
CN117126227A CN202210552428.1A CN202210552428A CN117126227A CN 117126227 A CN117126227 A CN 117126227A CN 202210552428 A CN202210552428 A CN 202210552428A CN 117126227 A CN117126227 A CN 117126227A
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eluent
polysorbate
protein
column
antibody
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余剑平
李瑶
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Foshan Hanteng Biotechnology Co ltd
Foshan Pu Jin Bioisystech Co ltd
Cantonbio Co ltd
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Foshan Hanteng Biotechnology Co ltd
Foshan Pu Jin Bioisystech Co ltd
Cantonbio Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/18—Ion-exchange chromatography

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Abstract

The invention provides application of a surfactant in improving recovery rate in protein purification by hydrophobic chromatography. The application can obviously reduce the impurity content of Host Cell Proteins (HCP) and the like while improving the recovery rate of the proteins, so that the content of the HCP in the proteins is reduced to tens of ppm, even a few ppm, and the high-purity and high-quality proteins are prepared.

Description

Application of surfactant in improving recovery rate of protein purified by hydrophobic chromatography
Technical Field
The invention belongs to the technical field of biological medicine, and particularly relates to application of a surfactant in improving recovery rate of protein purified by hydrophobic chromatography.
Background
Hydrophobic chromatography, also known as chromatography under hydrophobic interaction (hydrophobic interaction chromatography HIC), also belongs to the class of adsorption chromatography from the point of view of the mechanism of separation and purification of living matter. The principle of hydrophobic chromatography is as follows: the protein surface is generally provided with hydrophobic and hydrophilic groups, and the hydrophobic chromatography is to combine a carrier with the hydrophobicity with the protein surface at a high salt concentration by utilizing the hydrophobicity of a certain part of the protein surface. In the elution, the salt concentration is gradually reduced, and the proteins are sequentially eluted and purified due to different hydrophobicity, so that the method can be used for separating and purifying the proteins, especially the proteins which are difficult to separate and purify by other methods such as salting out, isoelectric precipitation and the like.
In the purification of proteins, many proteins are subjected to affinity chromatography using a protein a column. However, the price of protein a resin is about 30 times higher than other commonly used ion exchange resins, increasing the cost of purifying the protein. Thus, antibodies are generally purified using non-affinity chromatography purification methods.
The non-affinity chromatography method comprises capturing with cations and then performing anion chromatography. After chromatography in these two steps, the Host Cell Protein (HCP) of the antibody is still greater than 100ppm, rendering the antibody undesirable for quality. Hydrophobic chromatography plays a role in removing HCP and reducing the polymer during the purification of antibodies. However, the inventors found that when CD20 antibody and dolapride were purified by hydrophobic chromatography, the elution effect of the eluent was poor, and recovery rates of CD20 antibody and dolapride were particularly low. Therefore, in the process of purifying proteins by hydrophobic chromatography, there is a need for a method of purifying proteins having strong hydrophobicity to increase the recovery rate of the target protein, and HCP impurities satisfy the quality requirements.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention aims to provide an application of a surfactant in improving the recovery rate of a protein antibody purified by hydrophobic chromatography so as to improve the recovery rate of the protein.
To achieve the purpose, the invention provides the application of the surfactant in improving the recovery rate of the protein purified by hydrophobic chromatography.
In some embodiments of the invention, the protein is selected from the group consisting of a nucleoprotein, glycoprotein, lipoprotein, chromoprotein, phosphoprotein, or recombinant protein.
In some embodiments of the invention, the recombinant protein is a fusion protein.
In some embodiments of the invention, the fusion protein is selected from an immunoglobulin fusion protein, a parathyroid hormone fusion protein or a cytokine recombinant fusion protein.
In some embodiments of the invention, the fusion protein is selected from GLP-1 or a variant thereof.
In some embodiments of the invention, the fusion protein is dolapride.
In some embodiments of the invention, the protein is selected from antibodies.
In some embodiments of the invention, the antibody is selected from IgM, igD, igG, igA or IgE, or an antigen-binding fragment thereof, or a recombinant antibody thereof, respectively.
In some embodiments of the invention, the recombinant antibody is selected from a chimeric antibody, a humanized antibody, a fully humanized antibody, a small molecule antibody or a bispecific antibody.
In some embodiments of the invention, the recombinant antibody is an anti-rabies virus antibody.
In some embodiments of the invention, the recombinant antibody is a recombinant mab or a recombinant diabody.
In some embodiments of the invention, the recombinant antibody is selected from at least one of CD1-CD 166.
In some embodiments of the invention, the recombinant monoclonal antibody is an anti-CD 20 antibody.
In some embodiments of the invention, the anti-CD 20 antibody is selected from at least one of rituximab, ibritumomab tiuxetan, ofatuzumab, tositumomab, oreuzumab, veltuzumab, pertuzumab, or atouzumab.
In some embodiments of the invention, the CD20 antibody is rituximab.
In some embodiments of the invention, the first eluent used for hydrophobic chromatography contains a surfactant.
In some embodiments of the invention, the surfactant is selected from anionic surfactants, cationic surfactants, zwitterionic surfactants, or nonionic surfactants.
In some embodiments of the invention, the nonionic surfactant is polysorbate.
In some embodiments of the invention, the polysorbate is selected from at least one of polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, and polysorbate 80.
In some embodiments of the invention, the first eluent used for hydrophobic chromatography comprises polysorbate 80.
In some embodiments of the invention, the concentration of polysorbate 80 in the first eluate is 0.5-2.5w/w%.
In some embodiments of the invention, the concentration of polysorbate 80 in the first eluate is 0.5-1.95w/w%.
In some embodiments of the invention, the concentration of polysorbate 80 in the first eluate is 1.95-2.05w/w%.
In some embodiments of the invention, the concentration of polysorbate 80 in the first eluate is 2.05-2.1w/w%.
In some embodiments of the invention, the concentration of polysorbate 80 in the first eluate is 2.1-2.5w/w%.
In some embodiments of the invention, the concentration of polysorbate 80 in the first eluate is 1-2w/w%.
In some embodiments of the invention, the first eluent comprises a first eluent a and a first eluent B;
the first eluent A contains 30-100mM phosphate-citric acid and 0.3-0.6M ammonium sulfate, and the pH value is 6-8;
the first eluent B contains the polysorbate 80 and 30-100mM phosphate-citric acid, and has a pH value of 6-8.
In some embodiments of the invention, the first eluent a contains 50mM phosphate-citric acid and 0.5M ammonium sulfate, and has a pH of 6.5;
the first eluent B contains the polysorbate 80 and 30-100mM phosphate-citric acid, and has a pH value of 6.5.
In some embodiments of the invention, the loading of the first eluent is 5-20mg/mL.
In some embodiments of the invention, the loading of the first eluent is 10mg/mL.
In some embodiments of the invention, the hydrophobic chromatography purified protein comprises the steps of:
equilibrating the hydrophobic chromatography column with a first equilibration buffer, loading a sample containing CD20 antibody into the hydrophobic chromatography column;
eluting the target protein in the hydrophobic chromatographic column by using the first eluent, and collecting the eluent;
the first balance buffer contains 50-100mM phosphate-citric acid, 0.5-1.5M ammonium sulfate and pH value is 6.0-8.0.
In some embodiments of the invention, the elution mode of hydrophobic chromatography is linear elution.
In some embodiments of the invention, the volume of the first eluent a decreases linearly from 100% to 0%, while the volume of the first eluent B increases linearly from 0% to 100%.
In some embodiments of the invention, the collection range of the eluent is 100mAu before the peak of the protein of interest, and 100mAu after the peak of the protein of interest.
In some embodiments of the invention, the packing within the hydrophobic chromatography column comprises butyl, phenyl, octyl, or polypropylene glycol groups.
In some embodiments of the invention, the hydrophobic chromatography column is Capto Phenyl Impres.
In some embodiments of the invention, the first eluent is used in an amount of 5 to 40 column volumes.
In some embodiments of the invention, the first eluent is used in an amount of 10 to 20 column volumes.
In some embodiments of the invention, the first eluent is used in an amount of 10 to 15 column volumes.
In some embodiments of the invention, the first eluent is used in an amount of 15 column volumes.
In some embodiments of the invention, the sample containing the target protein is a sample obtained by cation exchange chromatography or a sample obtained by anion exchange chromatography after cation exchange chromatography.
In some embodiments of the invention, the sample containing the protein of interest is a sample obtained by cation exchange chromatography.
In some embodiments of the invention, the sample containing the protein of interest is a sample obtained by cation exchange chromatography followed by anion exchange chromatography.
In some embodiments of the invention, the cation exchange chromatography comprises the steps of:
balancing a cation exchange chromatographic column with a second balancing buffer, loading a sample containing a protein of interest into the cation exchange chromatographic column;
washing the cation exchange chromatography column with a second equilibration buffer;
eluting the target protein in the cation exchange chromatographic column by using a second eluent, and collecting the eluent to obtain a sample containing the target protein;
the second equilibration buffer contains 50mM phosphate-citrate and 50mM sodium chloride, pH 4.5;
the second eluent contains a buffer A and a buffer B; the volume ratio of the buffer solution A to the buffer solution B is 20-40:80-60;
the buffer A contains 50mM phosphate-citric acid and 50mM sodium chloride, and has a pH value of 4.5;
the buffer B contained 50mM phosphate-citrate and 50mM sodium chloride, pH 8.0.
In some embodiments of the invention, the second equilibration buffer is the buffer a.
In some embodiments of the invention, the packing of the cation exchange chromatography column comprises sulfonic acid functionality or carboxylic acid functionality.
In some embodiments of the invention, the cation exchange chromatography column is Poros XS.
In some embodiments of the invention, the second eluent is used in an amount of 5 to 40 column volumes.
In some embodiments of the invention, the second eluent is used in an amount of 10 to 20 column volumes.
In some embodiments of the invention, the anion exchange chromatography comprises the steps of:
balancing an anion exchange chromatographic column by using a third balancing buffer solution, and then loading the eluent collected by cation exchange chromatography into the anion exchange chromatographic column;
eluting the target protein in the anion exchange chromatographic column by using a third eluent;
the third balance buffer contains 50mM phosphate-citric acid, and the pH value is 6-7.5;
the third eluent contains 50mM phosphate-citric acid and has a pH value of 6-7.5.
In some embodiments of the invention, the anion exchange chromatography column comprises a tertiary amine ion functionality, a quaternary ammonium ion functionality, a polyamine functionality, or a diethylaminoethyl functionality.
In some embodiments of the invention, the anion exchange chromatography column is a Capto sphere.
In some embodiments of the invention, the third eluent is used in an amount of 8 to 20 column volumes.
In some embodiments of the invention, the third eluent is used in an amount of 10 to 15 column volumes.
The application of the surfactant provided by the invention in improving the recovery rate in the protein purification by hydrophobic chromatography can improve the recovery rate of the protein, so that the recovery rate is more than 93%, even more than 95%. The application can obviously reduce the impurity content of Host Cell Proteins (HCP) and the like while improving the recovery rate of the proteins, so that the content of the HCP in the proteins is reduced to tens of ppm, even a few ppm, and the high-purity and high-quality proteins are prepared.
Drawings
FIG. 1 is a chromatogram of the eluate of the hydrophobic chromatography of example 1 containing 2w/w% PS80. The peak indicated by arrow 1 in the figure represents rituximab, arrow 2 represents conductivity, and arrow 3 represents volume percent of eluent B.
FIG. 2 is a chromatogram of the eluent of the comparative hydrophobic chromatography of example 1 without additives. The peak indicated by arrow 1 in the figure represents rituximab, arrow 2 represents conductivity, and arrow 3 represents volume percent of eluent B.
FIG. 3 is a chromatogram of the comparative hydrophobic chromatography of example 1, wherein the eluent contains 10w/w% glycerol. The peak indicated by arrow 1 in the figure represents rituximab, arrow 2 represents conductivity, and arrow 3 represents volume percent of eluent B.
FIG. 4 is a chromatogram of the eluate of the hydrophobic chromatography of example 1 containing 10w/w% propylene glycol. Peaks indicated by arrows in the figure represent rituximab.
FIG. 5 is a flow-through chromatographic profile of the hydrophobic chromatography of example 2. The peak indicated by arrow 1 in the figure represents rituximab and arrow 2 represents conductivity.
FIG. 6 is a flow-through chromatographic profile of the comparative hydrophobic chromatography of example 2. The peak indicated by arrow 1 in the figure represents rituximab and arrow 2 represents conductivity.
FIG. 7 is a chromatogram of the eluate of the hydrophobic chromatography of example 3 containing 2w/w% PS80. The peak indicated by arrow 1 in the figure represents the degree of radlutide, arrow 2 represents the conductivity, and arrow 3 represents the volume percentage of eluent B.
FIG. 8 is a flow-through chromatographic profile of the comparative hydrophobic chromatography of example 3. The peak indicated by arrow 1 in the figure represents the degree of radlutide, arrow 2 represents the conductivity, and arrow 3 represents the volume percentage of eluent B.
Detailed Description
The technical scheme of the invention is further described below by the specific embodiments with reference to the accompanying drawings. It should be apparent to those skilled in the art that the detailed description is merely provided to aid in understanding the invention and should not be taken as limiting the invention in any way.
Definition of the definition
The term "nucleoprotein" herein refers to a complex protein formed by the binding of a protein and a nucleic acid.
The term "glycoprotein" as used herein refers to a complex carbohydrate comprising a branched oligosaccharide chain covalently linked to a polypeptide chain, the backbone being shorter, in most cases the amount of carbohydrate being less than the protein. Meanwhile, glycoprotein is also a binding protein, and glycoprotein is a molecule formed by covalently linking a short oligosaccharide chain with a protein.
The term "lipoprotein" as used herein refers to a class of spherical particles composed of a hydrophobic inner core rich in sterols, triglycerides and an outer shell composed of proteins, phospholipids, cholesterol, etc.
The term "chromoprotein" herein refers to a protein that consists of a protein and a pigment.
The term "Phosphoprotein" as used herein refers to a protein that is covalently bound to a phosphate or phosphate-derived group.
The term "recombinant protein" herein refers to a protein obtained by applying recombinant DNA or recombinant RNA techniques.
The term "fusion protein" herein refers to an expression product after two gene recombination obtained by a DNA recombination technique.
Immunoglobulin (Ig) fusion proteins, parathyroid hormone (PTH) fusion proteins or cytokine recombinant fusion proteins.
The term "immunoglobulin fusion protein" herein also referred to as Ig fusion protein refers to a recombinant protein having the two-part domains described above, which is expressed in eukaryotic or prokaryotic cells by linking a gene of interest to an Ig-part fragment gene at the gene level. Proteins of interest can be divided into two major classes, depending on their linkage to different fragments of Ig: one class is Fab (Fv) fusion proteins; the other class is Fc fusion proteins.
The term "parathyroid hormone fusion protein" herein also referred to as PTH fusion protein refers to the use of genetic engineering methods to link and express the corresponding protein fusion product to gene sequences encoding parathyroid hormone and other proteins having a specific function. Such as human serum albumin and human parathyroid hormone fusion protein.
The term "cytokine recombinant fusion protein" as used herein refers to a fusion product of a corresponding protein by genetic engineering techniques to link and express the gene sequences encoding cytokines and other proteins of specific function. The structure is characterized in that the functional active domain of the cell factor is fused with the active domains of other molecules, and each component can play a synergistic effect, so that the biological activity of the fusion protein is greatly enhanced compared with that of each monomer.
The term "dolapride" herein also called "dolapride" is obtained by fusing two GLP-1 analogues with DPP-4 inhibition and a human immunoglobulin heavy chain IgG4-Fc fragment, has activity similar to endogenous GLP-1, has a half-life of 5 days, and can effectively delay the clearance of kidneys.
The term "antibody" as used herein refers to an immunoglobulin that is a tetrapeptide chain structure formed by joining two identical heavy chains and two identical light chains via interchain disulfide bonds. The immunoglobulin heavy chain constant region differs in amino acid composition and sequence, and thus, in antigenicity. Accordingly, immunoglobulins can be assigned to five classes, or different types of immunoglobulins, namely IgM, igD, igG, igA and IgE, the heavy chain constant regions corresponding to the different classes of immunoglobulins being referred to as a, d, e, g, and m, respectively. IgG represents the most important class of immunoglobulins, which can be divided into 4 subclasses again due to differences in chemical structure and biological function: igG1, igG2, igG3 and IgG4. Light chains are classified as either kappa or lambda chains by the difference in constant regions. Subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.
The term "antigen binding fragment" as used herein refers to Fab fragments, fab 'fragments, F (ab') 2 Fragments, or single Fv fragments. Fv antibodies contain antibody heavy chain variable regions, light chain variable regions, but no constant regions, and have a minimal antibody fragment of the entire antigen binding site. Fv antibodies also typically comprise a polypeptide linker between the VH and VL domains and are capable of forming the structures required for antigen binding.
The term "recombinant antibody" herein also referred to as "genetically engineered antibody" refers to an antibody obtained by transforming and recombining the corresponding gene sequences of the antibody as required by a DNA recombination technique, constructing on plasmids, and transfecting/transforming the plasmids constructed by a protein exogenous expression technique into a suitable host cell for expression.
The term "bispecific antibody" as used herein is an antibody having two specific antigen binding sites that bind to two antigens simultaneously, e.g., to target cells (cancer cells) and effector cells (T cells) simultaneously, with the directional mediation of killing of the target cells by the effector cells.
Examples
The cation exchange resin POROS XS is available from Thermo Scientific and the anion exchange resin Capto addition is available from Cytiva; hydrophobic chromatography column Capto Phenyl ImpRes was purchased from Cytiva.
EXAMPLE 1 purification of rituximab
Culturing CHO-K1 cells expressing rituximab, collecting cell culture solution, centrifuging at 4000rpm for 20min, and keeping supernatant for later use.
1. Cation exchange Chromatography (CEX)
Cation exchange resin: poros XS, column volume 19mL.
Sample preparation: the supernatant was collected, adjusted to pH 4.5 with 1M citric acid solution, centrifuged at 2500rpm for 5min, filtered with a filter having a pore size of 0.22 μm, and 128mL of the filtrate was collected.
Buffer solution preparation:
buffer a:50mM disodium hydrogen phosphate-citric acid+50 mM sodium chloride, pH=4.5.
Buffer B:50mM disodium hydrogen phosphate-citric acid+50 mM sodium chloride, pH=8.0.
Chromatography: the Poros XS cation exchange chromatography column was equilibrated, loaded, washed and eluted sequentially, and the eluate was collected under the chromatographic conditions shown in table 1 below:
TABLE 1
Column volume 19mL
Balancing Buffer A, flow rate 3.2mL/min
Loading sample 80mg sample/mL resin, 3.2mL/min flow rate
Washing Buffer A, flow rate 3.2mL/min
Elution Pre-eluting: 40% buffer A+60% buffer B flow rate 3.2mL/min
Collecting peak parameters 100-Peak-200 mAU
Collecting a sample of rituximab from the eluate collected by the cation exchange chromatography: 79.4mL, 11.0mg/mL, and recovery rate of 86.4%.
2、HIC
And (3) filling: capto Phenyl ImpRes.
And taking the eluent collected after the first step of cation exchange chromatography to perform the second step of hydrophobic chromatography, and examining the influence of different additives on the recovery rate of the hydrophobic chromatography.
Sample treatment: 19.2g of CEX sample was added with 19.3g of 1M ammonium sulfate, and the pH was adjusted to 6.5 with 1M Tris at a concentration of 5.5mg/mL.
The eluate contained 2w/w% PS80, PS80 being polysorbate 80.
Chromatography column: capto Phenyl Impres, cv=4.2 mL.
Balance: 50mM Na 2 HPO 4 Citric acid, 0.5M ammonium sulphate, pH6.5.
Load: 10mg/mL.
Eluting:
the eluent consists of eluent A and eluent B.
Eluent a:50mM Na2HPO 4 Citric acid, 0.5M ammonium sulphate pH6.5.
Eluent B:50mM Na2HPO 4 Citric acid, pH6.5, containing 2w/w% PS80.
Elution mode: and (5) linear elution.
Specifically, the volume of the eluent A is linearly reduced from 100% to 0%, and the volume of the eluent B is linearly increased from 0% to 100%.
Elution volume: 15 column volumes, sample collection range: 100 mAu-peak-100 mAu.
Collecting a sample: 15mL, concentration 2.6mg/mL, recovery 93%.
The chromatographic profile of the eluate of the hydrophobic chromatography containing 2w/w% PS80 is shown in FIG. 1.
Comparative example rituximab purification
Culturing CHO-K1 cells expressing rituximab, collecting cell culture solution, centrifuging at 4000rpm for 20min, and keeping supernatant for later use.
1. Cation exchange Chromatography (CEX)
Cation exchange resin: poros XS, column volume 19mL.
Sample preparation: the supernatant was collected, adjusted to pH 4.5 with 1M citric acid solution, centrifuged at 2500rpm for 5min, filtered with a filter having a pore size of 0.22 μm, and 128mL of the filtrate was collected.
Buffer solution preparation:
buffer a:50mM disodium hydrogen phosphate-citric acid+50 mM sodium chloride, pH=4.5.
Buffer B:50mM disodium hydrogen phosphate-citric acid+50 mM sodium chloride, pH=8.0.
Chromatography: the Poros XS cation exchange chromatography column was equilibrated, loaded, washed and eluted sequentially, and the eluate was collected under the chromatographic conditions shown in Table 1.
Collecting a sample of rituximab from the eluate collected by the cation exchange chromatography: 79.4mL, 11.0mg/mL, and recovery rate of 86.4%.
2、HIC
And (3) filling: capto Phenyl ImpRes.
And taking the eluent collected after the first step of cation exchange chromatography to perform the second step of hydrophobic chromatography, and examining the influence of different additives on the recovery rate of the hydrophobic chromatography.
Sample treatment: 19.2g of CEX sample was added with 19.3g of 1M ammonium sulfate, and the pH was adjusted to 6.5 with 1M Tris at a concentration of 5.5mg/mL.
1) No additive in the eluent:
chromatography column packing: capto Phenyl Impres, cv=4.2 mL. CV represents column volume.
Balance: 50mM Na 2 HPO 4 Citric acid, 0.5M ammonium sulphate, pH6.5.
Load: 10mg/mL.
Eluting:
the eluent consists of eluent A and eluent B.
Eluent a:50mM Na 2 HPO 4 Citric acid, 0.5M ammonium sulphate pH6.5.
Eluent B:50mM Na 2 HPO 4 Citric acid, ph6.5.
Elution mode: and (5) linear elution.
Specifically, the volume of the eluent A is linearly reduced from 100% to 0%, and the volume of the eluent B is linearly increased from 0% to 100%.
Elution volume: 15 column volumes, sample collection range: 100 mAu-peak-100 mAu.
Collecting a sample: 29.2mL, 0.8mg/mL, and a recovery rate of 55.6%.
The chromatographic profile of the eluate of the hydrophobic chromatography without additives is shown in figure 2.
2) The eluent contains 10w/w% glycerol
Chromatography column: capto Phenyl Impres, cv=4.2 mL.
Balance: 50mM Na 2 HPO 4 Citric acid, 0.5M ammonium sulphate pH6.5.
Load: 10mg/mL.
Eluting:
the eluent consists of eluent A and eluent B.
Eluent a:50mM Na 2 HPO 4 Citric acid, 0.5M ammonium sulphate pH6.5.
Eluent B:50mM Na 2 HPO 4 Citric acid, pH6.5, containing 10w/w% glycerol.
Elution mode: and (5) linear elution.
Specifically, the volume of the eluent A is linearly reduced from 100% to 0%, and the volume of the eluent B is linearly increased from 0% to 100%.
Elution volume: 15 column volumes, sample collection range: 100 mAu-peak-100 mAu.
Collecting a sample: 29.3mL, 1.0mg/mL, and recovery rate was 69.8%.
The chromatographic chart of the eluent containing 10% glycerol from the hydrophobic chromatography is shown in FIG. 3.
3) The eluent contains 10w/w% propylene glycol
Chromatography column: capto Phenyl Impres, cv=4.2 mL.
Balance: 50mM Na 2 HPO 4 Citric acid, 0.5M ammonium sulphate, pH6.5.
Load: 10mg/mL.
Eluting:
the eluent consists of eluent A and eluent B.
Eluent a:50mM Na 2 HPO 4 Citric acid, 0.5M ammonium sulphate pH6.5.
Eluent B:50mM Na 2 HPO 4 Citric acid, pH6.5, containing 10w/w% propylene glycol.
Elution mode: and (5) linear elution.
Specifically, the volume of the eluent A is linearly reduced from 100% to 0%, and the volume of the eluent B is linearly increased from 0% to 100%.
Elution volume: 15 column volumes, sample collection range: 100 mAu-peak-100 mAu.
Collecting a sample: 26.9mL, 1.2mg/mL, and recovery rate was 76.9%.
The chromatographic profile of the eluate of the hydrophobic chromatography containing 10w/w% propylene glycol is shown in FIG. 4.
From the results of example 1 and comparative example, it was found that during hydrophobic chromatography, the recovery rate of rituximab was 94% by adding 2w/w% PS80 to the eluate, which was significantly higher than that of the eluate without the additive, 10w/w% glycerol, and 10w/w% propylene glycol.
EXAMPLE 2CEX-AEX-HIC three-step purification of rituximab
1. Cation exchange Chromatography (CEX)
The method of cation exchange chromatography was the same as that of the cation exchange chromatography in example 1, please refer to the cation exchange chromatography in example 1.
2. Anion exchange chromatography (AEX)
Chromatographic column: capto sphere, cv=4.7 mL.
Sample treatment: the eluate collected by cation exchange chromatography (containing rituximab) was mixed with 1M citric acid and water, pH: 6.4, conductivity: 5.7mS/cm, sample concentration: 5.7mg/mL. The experimental parameters are detailed in table 2. The results are shown in Table 3.
TABLE 2
TABLE 3 Table 3
3. Hydrophobic chromatography (HIC)
Chromatographic column: phenyl ImpRes, cv=4.2 mL.
Sample treatment: 22.9g of anion exchange collected flow-through solution was added with 1.0M (NH) 4 ) 2 SO 4 Mixing evenly, and pH value: 6.5, conductivity: 76.2mS/cm, concentration: 2.1mg/mL.
The experimental methods are detailed in table 4:
TABLE 4 Table 4
The chromatographic profile of the hydrophobic chromatography elution is shown in FIG. 5.
As shown in table 5, the collection range of each section of eluent: h01:50mAU-950mAU peak, H02:950mAU peak front-peak 2762mAU-1000mAU peak rear, H03:1000mAU peak-485 mAU peak.
TABLE 5
Thus, it was found that rituximab, a target protein, was eluted efficiently in the presence of 2w/w% PS80, the SEC purity was increased from 91.5% to 94.5%, the Host Cell Protein (HCP) was decreased from 564ppm to 77ppm, and the recovery rate in the H02 fraction was 78.2%. The SEC purity data shows that the hydrophobic chromatography not only improves rituximab recovery, but also reduces polymer and fragment impurities.
Comparative example of example 2
This comparative example differs from example 2 only in that the eluent used for hydrophobic chromatography was not PS80 added. The results are detailed in Table 6 and FIG. 6. The source of the loading solution was the same as that of HIC loading solution in example 2. Eluent collection range:
h04:50mAU peak-200 mAU peak-front;
h05:200mAU peak-648 mAU peak-200 mAU peak-after.
TABLE 6
From the peak height responses of fig. 5 and 6, with PS80 as an additive, the highest response reached 2762mAU, and the response without PS80 was only 648mAU.
From the above data, it is clear that the recovery rate of rituximab purified by hydrophobic chromatography is significantly improved after PS80 is added to the eluate of hydrophobic chromatography, and HCP can be controlled within 100 ppm. Therefore, the HIC hydrophobic chromatography purified rituximab can remarkably improve the removal effect of HCP, and the recovery rate is over 95 percent.
The above examples show that rituximab is difficult to elute by hydrophobic chromatography, and PS80 is used as an additive to solve the technical problem of difficult elution. Similarly, when the protein is purified by hydrophobic chromatography, PS80 can be used as an additive for any target protein which is difficult to elute, so that the technical problem that the target protein is difficult to elute by hydrophobic chromatography is solved.
EXAMPLE 3 purification of degree La Lu Taidu Laruptan
CHO cells expressing dolapril were cultured, cell culture medium was collected, centrifuged at 4000rpm for 20min, and supernatant was kept ready for use.
The supernatant was subjected to Affinity Chromatography (AC) and anion exchange chromatography (AEX) purification, and the purified sample was collected.
Hydrophobic chromatography (HIC)
Sample treatment: 20g of the collected purified sample was added with 1.9g of 1M ammonium sulfate, pH6.5 was adjusted with 0.5M citric acid, conductivity 41.8ms/cm, concentration: 4.1mg/ml.
The eluent contained 2w/w% PS80.
Chromatography column: capto Phenyl Impres, cv=4.2 mL.
Balance: 50mM disodium hydrogen phosphate-citric acid, 0.3M ammonium sulfate, pH6.5.
Loading: 5.5ml; load: 5.4mg/mL.
Eluting:
the eluent consists of eluent A and eluent B.
Eluent a:50mM disodium hydrogen phosphate-citric acid, 0.3M ammonium sulfate pH6.5.
Eluent B:50mM disodium hydrogen phosphate- -citric acid, 2w/w% PS80, pH6.5.
Elution mode: and (5) linear elution.
Specifically, the volume of the eluent A is linearly reduced from 100% to 0%, and the volume of the eluent B is linearly increased from 0% to 100%.
Elution volume: 10 column volumes, sample collection range: 100 mAu-peak-100 mAu.
Collecting a sample: 12.2mL, 1.65mg/mL, and recovery of 89.1%. Adding
The chromatographic profile of the eluate of the hydrophobic chromatography containing 2w/w% PS80 is shown in FIG. 7, and the duloxetine is detected. It can be seen that the addition of 2w/w% PS80 eluent is capable of eluting dolapride, and that the recovery of dolapride is high.
Comparative example 3
CHO cells expressing dolapril were cultured, cell culture medium was collected, centrifuged at 4000rpm for 20min, and supernatant was kept ready for use.
The supernatant was subjected to Affinity Chromatography (AC) and anion exchange chromatography (AEX) purification, and the purified sample was collected.
Hydrophobic chromatography (HIC)
Sample treatment: 20g of the collected purified sample was added with 1.9g of 1M ammonium sulfate, pH was adjusted to 6.5 with 0.5M citric acid, conductivity was 41.8ms/cm, and the concentration was: 4.1mg/ml.
No additives are added in the eluent.
Chromatography column: capto Phenyl Impres, cv=4.2 mL.
Balance: 50mM disodium hydrogen phosphate-citric acid, 0.3M ammonium sulfate, pH6.5.
Loading: 5.5ml; load: 5.4mg/mL.
Eluting:
the eluent consists of eluent A and eluent B.
Eluent a:50mM disodium hydrogen phosphate-citric acid, 0.3M ammonium sulfate, pH6.5.
Eluent B:50mM disodium hydrogen phosphate- -citric acid, pH6.5.
Elution mode: and (5) linear elution.
Specifically, the volume of the eluent A is linearly reduced from 100% to 0%, and the volume of the eluent B is linearly increased from 0% to 100%.
Elution volume: 10 column volumes, sample collection range: 100 mAu-peak-100 mAu.
The chromatographic chart of the eluent without the additive of the hydrophobic chromatography is shown in figure 8, and the target egg white glycopeptide can not be detected. It can be seen that the eluent without additives did not elute the duloxetine.
From the results of example 3 and the comparative examples, it is understood that the surfactant provided by the present invention can be applied to the purification of CD20 antibody by hydrophobic chromatography to improve the recovery rate of dolapride.
While the invention has been described in detail in the foregoing general description, embodiments and experiments, it will be apparent to those skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims (10)

1. The use of surfactants to improve recovery in the purification of proteins by hydrophobic chromatography.
2. The use according to claim 1, wherein the first eluent used for hydrophobic chromatography contains a surfactant;
preferably, the protein is selected from the group consisting of nucleoprotein, glycoprotein, lipoprotein, chromoprotein, phosphoprotein or recombinant protein;
preferably, the recombinant protein is a fusion protein;
preferably, the fusion protein is selected from an immunoglobulin fusion protein, a parathyroid hormone fusion protein or a cytokine recombinant fusion protein;
preferably, the fusion protein is selected from GLP-1 or variants thereof;
preferably, the fusion protein is dolapride;
preferably, the protein is selected from antibodies;
preferably, the antibody is selected from IgM, igD, igG, igA or IgE, or an antigen-binding fragment thereof, or a recombinant antibody thereof, respectively;
preferably, the recombinant antibody is an anti-rabies virus antibody;
preferably, the recombinant antibody is a recombinant monoclonal antibody or a recombinant diabody;
preferably, the recombinant antibody is selected from at least one of CD1-CD 166;
preferably, the recombinant monoclonal antibody is an anti-CD 20 antibody;
preferably, the anti-CD 20 antibody is selected from at least one of rituximab, ibritumomab, ofatuzumab, tositumomab, oreuzumab, veltuzumab, pertuzumab, or atouzumab;
more preferably, the CD20 antibody is rituximab;
preferably, the surfactant is selected from anionic, cationic, zwitterionic or nonionic surfactants;
preferably, the nonionic surfactant is polysorbate;
more preferably, the polysorbate is selected from at least one of polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, and polysorbate 80;
more preferably, the polysorbate is polysorbate 80.
3. The use according to claim 2, wherein the non-ionic surfactant is polysorbate 80 and the concentration of polysorbate 80 in the first eluent is 0.5-2.5w/w%.
4. The use according to claim 2, wherein the concentration of polysorbate 80 in the first eluent is 0.5-1.95w/w%,1.95-2.05w/w%,2.05-2.1w/w% or 2.1-2.5w/w%;
preferably, the concentration of polysorbate 80 in the first eluent is 1-2w/w%.
5. The use according to any one of claims 2-4, wherein the first eluent comprises a first eluent a and a first eluent B;
the first eluent A contains 30-100mM phosphate-citric acid and 0.3-0.6M ammonium sulfate, and the pH value is 6-8;
the first eluent B contains the polysorbate 80 and 30-100mM phosphate-citric acid, and the pH value is 6-8;
preferably, the first eluent a contains 50mM phosphate-citric acid and 0.5M ammonium sulfate, and has a pH of 6.5;
the first eluent B contains the polysorbate 80 and 30-100mM phosphate-citric acid, and has a pH value of 6.5.
6. The use according to claim 5, wherein the hydrophobic chromatography purification of the protein comprises the steps of:
balancing the hydrophobic chromatography column by using a first balancing buffer solution, and loading a sample containing target proteins into the hydrophobic chromatography column;
eluting target proteins in the hydrophobic chromatographic column by using the first eluent, and collecting the eluent;
the first balance buffer solution contains 50-100mM phosphate-citric acid, 0.5-1.5M ammonium sulfate and pH value of 6.0-8.0;
preferably, the elution mode of the hydrophobic chromatography is linear elution;
more preferably, the volume of the first eluent a is linearly reduced from 100% to 0%, while the volume of the first eluent B is linearly increased from 0% to 100%;
preferably, the first eluent is used in an amount of 5 to 40 column volumes, preferably 10 to 20 column volumes; preferably 10-15 column volumes.
7. The use according to claim 6, wherein the packing in the hydrophobic chromatography column comprises butyl, phenyl, octyl or polypropylene glycol groups;
preferably, the hydrophobic chromatography column is Capto Phenyl Impres.
8. The use according to claim 6, wherein the sample containing the target protein is a sample obtained by cation exchange chromatography or a sample obtained by anion exchange chromatography after cation exchange chromatography.
9. The use according to claim 8, wherein the cation exchange chromatography comprises the steps of:
balancing a cation exchange chromatographic column with a second balancing buffer, loading a sample containing a protein of interest into the cation exchange chromatographic column;
washing the cation exchange chromatography column with a second equilibration buffer;
eluting the target protein in the cation exchange chromatographic column by using a second eluent, and collecting the eluent to obtain a sample containing the target protein;
the second equilibration buffer contains 50mM phosphate-citrate and 50mM sodium chloride, pH 4.5;
the second eluent contains a buffer A and a buffer B; the volume ratio of the buffer solution A to the buffer solution B is 20-40:80-60;
the buffer A contains 50mM phosphate-citric acid and 50mM sodium chloride, and has a pH value of 4.5;
the buffer B contains 50mM disodium hydrogen phosphate-citric acid and 50mM sodium chloride, and has a pH of 8.0;
preferably, the second equilibration buffer is the buffer a;
preferably, the packing of the cation exchange chromatography column comprises sulfonic acid functionality or carboxylic acid functionality;
preferably, the cation exchange chromatography column is Poros XS;
preferably, the second eluent is used in an amount of 5 to 40 column volumes, preferably 10 to 20 column volumes.
10. The use according to claim 9, wherein the anion exchange chromatography comprises the steps of:
balancing an anion exchange chromatographic column by using a third balancing buffer solution, and then loading the eluent collected by cation exchange chromatography into the anion exchange chromatographic column;
eluting the target protein in the anion exchange chromatographic column by using a third eluent;
the third balance buffer contains 50mM phosphate-citric acid, and the pH value is 6-7.5;
the third eluent contains 50mM phosphate-citric acid, and the pH value is 6-7.5;
preferably, the anion exchange chromatography column comprises a tertiary amine ion functionality, a quaternary ammonium ion functionality, a polyamine functionality or a diethylaminoethyl functionality;
preferably, the anion exchange chromatography column is Capto sphere;
preferably, the third eluent is used in an amount of 8 to 20 column volumes, preferably 10 to 15 column volumes.
CN202210552428.1A 2022-05-20 2022-05-20 Application of surfactant in improving recovery rate of protein purified by hydrophobic chromatography Pending CN117126227A (en)

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