CN111153993A

CN111153993A - Preparation method of anti-TNF- α monoclonal antibody

Info

Publication number: CN111153993A
Application number: CN201911005798.8A
Authority: CN
Inventors: 谢一龙; 尚战强; 陆建胜; 苏贤德; 胡伟伟; 陈强; 陈玄; 刘倩倩
Original assignee: Chia Tai Tianqing Pharmaceutical Group Co Ltd; Nanjing Shunxin Pharmaceutical Co Ltd
Current assignee: Chia Tai Tianqing Pharmaceutical Group Co Ltd; Nanjing Shunxin Pharmaceutical Co Ltd
Priority date: 2018-11-07
Filing date: 2019-10-22
Publication date: 2020-05-15
Anticipated expiration: 2039-10-22
Also published as: CN111153993B

Abstract

The invention relates to a preparation method of an anti-TNF- α monoclonal antibody, which comprises the steps of affinity chromatography, virus inactivation, deep filtration, anion exchange chromatography, cation exchange chromatography and the like.

Description

Preparation method of anti-TNF- α monoclonal antibody

Technical Field

The invention belongs to the field of protein purification, and particularly relates to a preparation or purification method of an anti-TNF- α monoclonal antibody.

Background

However, the long-term high level of TNF- α is closely related to the occurrence of various autoimmune diseases and inflammatory diseases, such as rheumatoid arthritis, psoriasis, Crohn's disease, ankylosing spondylitis and ulcerative colitis, TNF- α monoclonal antibody can specifically bind to TNF- α, competitively blocks the binding of TNF- α to its receptor, prevents the regulation of immune response and the induction of inflammation, has the characteristics of strong targeting, low toxicity and high efficiency, and therefore plays an important role in clinical medicine.

Monoclonal antibody drugs are typically expressed using mammalian cells, such as CHO, BHK cells, etc., that are genetically engineered to produce the protein of interest, including introduction of the gene of interest into the cell line, to ensure the desired folding and glycosylation. In the process of mammalian Cell culture, a culture medium containing various components such as amino acids, salts, growth factors and the like, Host Protein (HCP) and nucleic acid produced by Host cells and the like are added, so that the components of Cell culture products are complicated; and the monoclonal antibody has large molecular weight and complex structure, which brings great difficulty to the manufacturing process. However, in order to ensure the safety of the antibody drug to human body, it is necessary to remove the pollutants accumulated in the production process and ensure the antibody drug to achieve higher purity. The downstream processes for antibody drug manufacturing are complex and any imperfections can cause significant losses, and therefore it is essential to establish efficient downstream purification processes to increase the yield and purity of monoclonal antibodies. The purification means commonly used in the downstream process at present include affinity chromatography, cation exchange chromatography, anion exchange chromatography, hydrophobic chromatography and the like, and although these chromatography methods are usually adopted in the production, the differences of specific conditions, parameters and filler selection of the chromatography can produce completely different results.

Affinity chromatography is an important means of chromatographic separation of monoclonal antibodies and is commonly used to capture products in cell culture. Because the affinity chromatography material can specifically adsorb the antibody, when the antibody carrying capacity is appropriate, the product of the cell culture solution after affinity chromatography can often reach higher monomer purity, and meanwhile, higher recovery rate can be ensured. If the affinity chromatography step can remove HCP and nucleic acid pollutants as much as possible, will reduce the subsequent chromatographic and depth filtration pressure, and then reduce the antibody production cost.

Cation exchange chromatography is generally used for antibody purification, and the main function of the cation exchange chromatography is to remove high molecular weight impurities of polymers, and simultaneously further remove HCP and nucleic acid, and improve the purity of monoclonal antibodies. It is a common practice in the art to select a suitable concentration of salt to compete for binding to the cation exchange material after loading a low concentration of salt, thereby eluting the antibody for separation. For the separation of an antibody or an antibody drug, it is necessary not only to reduce the content of HCP, nucleic acid, high molecular weight impurities of a polymer, and the like as much as possible, but also to separate charge isomers. However, conventional cation exchange chromatography is less effective for separating acidic and basic analogues having a small difference in charge from the target protein. In order to solve the above problems, it is necessary to develop a new method for purifying an antibody, which improves the purity and quality of the antibody.

Disclosure of Invention

In one aspect, the present invention is directed to an antibody purification process that can improve the purity of a monoclonal antibody preparation.

In another aspect, the present invention also aims to provide a method for purifying a protein, comprising the steps of:

(a) deep filtering the cell culture solution;

(b) affinity chromatography;

(c) inactivating viruses;

(d) secondary deep filtration;

(e) anion exchange chromatography;

(f) cation exchange chromatography;

(g) virus filtration and/or ultrafiltration.

In some embodiments, the steps (a) - (g) are performed sequentially.

In some embodiments, affinity chromatography may employ protein a affinity chromatography packing. In some embodiments, affinity chromatography includes, but is not limited to, steps of sterilization, equilibration, loading, three rinses, elution, and the like. In some embodiments, the elution step of affinity chromatography comprises an increase in the conductivity of the elution buffer. For example, the elution may be performed with a 50mM Tris-acetate buffer containing 150mM sodium chloride, followed by a 50mM sodium acetate-acetate buffer containing 1M sodium chloride, and finally with a 50mM sodium acetate-acetate buffer. In some embodiments, the affinity chromatography is performed using a low pH buffer for elution of the antibody, e.g., using a pH 3-4 buffer, preferably a pH 3.5 elution buffer.

In some embodiments, affinity chromatography is performed using Protein a affinity chromatography packing comprising ① equilibration with 150mM sodium chloride in 50mM Tris-acetate buffer at pH 7.4, ② load, ③ wash 1, first wash with 150mM sodium chloride in 50mM Tris-acetate buffer at pH 7.4, ④ wash 2, second wash with 1M sodium chloride in 50mM sodium acetate-acetate buffer at pH 5.0, ⑤ wash 3, third wash with 50mM sodium acetate-acetate buffer at pH 5.0, ⑥ elution with 50mM sodium acetate-acetate buffer at pH 3.5.

In some embodiments, the eluate of the affinity chromatography is subjected to low pH viral inactivation. In one embodiment, virus inactivation may be achieved by adjusting the pH of the sample to about 3.0-4.0 by citric acid or Tris solution and allowing the sample to stand at 18-26 ℃ for 2-4 hours.

In some embodiments, the virus-inactivated solution is subjected to a second depth filtration. In some embodiments, the pH of the sample is adjusted to about 5.6 prior to the second depth filtration.

In some embodiments, anion exchange chromatography may employ various fillers, either conventional or commercially available, for example, POROS 50HQ anion fillers. In some embodiments, the anion exchange chromatography step includes, but is not limited to, pre-equilibration, loading, equilibration, regeneration, equilibration, storage, wherein the pre-equilibration solution may be selected from 10-100mM sodium acetate-acetic acid buffer containing 0.5-2.5M sodium chloride, preferably 50mM sodium acetate-acetic acid buffer containing 1M sodium chloride, and the equilibration solution may be selected from 10-100mM sodium acetate-acetic acid buffer, preferably 50mM sodium acetate-acetic acid buffer.

In some embodiments, cation exchange chromatography may employ conventional packing or commercial packing, for example, POROS XS cationic packing. In some embodiments, the cation exchange chromatography step includes, but is not limited to, pre-equilibration, loading, washing, elution, and the like. In some embodiments, the washing comprises 1-3 washes, e.g., 1 wash, 2 washes, or 3 washes, preferably 3 washes. In some embodiments, the washing step of cationic chromatography comprises a change in the pH of the washing buffer, such as a rise or a fall in pH. In some embodiments, the product is loaded into the cation exchange material after equilibration of the column, the composition is at a first pH, a first elution is performed with an equilibration buffer (also known as a first elution buffer) (elution 1), a second elution is performed with a second elution buffer having a second pH value that is higher than the first pH (elution 2), a third elution is performed with a third elution buffer having a third pH value that is lower than the second pH (elution 3), and finally the column is washed with an eluent having a conductivity greater than the conductivities of the first, second, and third elution buffers.

In some embodiments, the second pH of the cation exchange chromatography is at least about 1.5 or greater above the first pH. In some embodiments, the second pH is at least about 1.5, at least about 1.6, at least about 1.7, at least about 1.8, at least about 1.9, at least about 2.0, at least about 2.1, at least about 2.2, at least about 2.3, at least about 2.4, at least about 2.5, at least about 2.6, at least about 2.7, at least about 2.8, at least about 2.9, at least about 3.0, at least about 3.1, at least about 3.2, at least about 3.3, at least about 3.4, at least about 3.5, at least about 3.6, at least about 3.7, at least about 3.8, at least about 3.9, or at least about 4.0 or more higher than the first pH. In some embodiments of the invention, the second pH is at least about 1.5 or greater above the first pH. In some embodiments, the pH of the second elution of the cation exchange chromatography is at least about 1.5, at least about 1.6, at least about 1.7, at least about 1.8, at least about 1.9, at least about 2.0, at least about 2.1, at least about 2.2, at least about 2.3, at least about 2.4, at least about 2.5, at least about 2.6, at least about 2.7, at least about 2.8, at least about 2.9, at least about 3.0, at least about 3.1, at least about 3.2, at least about 3.3, at least about 3.4, at least about 3.5, at least about 3.6, at least about 3.7, at least about 3.8, at least about 3.9, or at least about 4.0 or more higher than the pH of the equilibrium, loading and/or first elution.

In some embodiments, the third pH of the cation exchange chromatography is at least about 2.0 or more lower than the second pH. In some embodiments, the third pH of the cation exchange chromatography is at least about 2.0, at least about 2.1, at least about 2.2, at least about 2.3, at least about 2.4, at least about 2.5, at least about 2.6, at least about 2.7, at least about 2.8, at least about 2.9, at least about 3.0, at least about 3.1, at least about 3.2, at least about 3.3, at least about 3.4, at least about 3.5, at least about 3.6, at least about 3.7, at least about 3.8, at least about 3.9, at least about 4.0, at least about 4.1, at least about 4.2, at least about 4.3, at least about 4.4, or at least about 4.5, or more, lower than the second pH. In some embodiments of the invention, the third pH is at least about 2.0 or greater below the second pH. In some embodiments, the pH of the third elution of the cation exchange chromatography is at least about 2.0, at least about 2.1, at least about 2.2, at least about 2.3, at least about 2.4, at least about 2.5, at least about 2.6, at least about 2.7, at least about 2.8, at least about 2.9, at least about 3.0, at least about 3.1, at least about 3.2, at least about 3.3, at least about 3.4, at least about 3.5, at least about 3.6, at least about 3.7, at least about 3.8, at least about 3.9, at least about 4.0, at least about 4.1, at least about 4.2, at least about 4.3, at least about 4.4, or at least about 4.5, or more lower than the pH of the second elution.

In some embodiments, the pH of the equilibration, loading, first wash buffer is the same or different for cation exchange chromatography.

In some embodiments, the equilibration buffer, loading buffer, and/or first elution buffer employ a 50mM sodium acetate-acetate buffer. In some embodiments, the equilibration buffer, loading buffer and/or first wash buffer has a pH of 4 to 7, preferably 5 to 6, for example 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0.

In some embodiments, the salt in the second elution buffer is a potassium phosphate salt. In some embodiments, the pH of the second wash buffer may be 6.5-9.0, preferably 7.0-8.5, more preferably 7.5-8.5, for example 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4 or 8.5. In some embodiments, the second elution buffer is a 20mM dipotassium phosphate-potassium dihydrogen phosphate buffer.

In some embodiments, the third elution buffer employs a 50mM sodium acetate-acetate buffer. In some embodiments, the third elution buffer has a pH of 4 to 7, preferably 4.5 to 5.5, for example 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, or 5.5.

In some embodiments, the antibody is eluted using a high conductivity buffer, preferably 50mM sodium acetate-acetic acid buffer containing 0.3M sodium chloride. In some embodiments, the pH of the eluent is 4 to 7, preferably 4.5 to 5.5, for example 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, or 5.5.

In this context, the elution buffer in cation chromatography has a relatively high conductivity, so that the desired antibody product is eluted from the cation exchange material. Preferably, the conductivity of the elution buffer is greater than the conductivity of the equilibration buffer, first, second, and third elution buffers.

In some embodiments, the cation exchange chromatography eluate is subjected to viral filtration, preferably nanofiltration, and/or ultrafiltration.

In some specific embodiments, the acidic variants of monoclonal antibodies produced by the present invention are less than 15%, preferably less than 13%, and more preferably less than 11%.

In some specific embodiments, the monoclonal antibodies produced by the present invention have a monomer content of greater than 99.0%, more preferably greater than 99.5%.

The invention also provides an integrated process for protein purification, which can ensure that the total recovery rate of the protein is 70% or higher, and the method comprises the following steps:

(a) carrying out deep filtration on the cell culture solution, and collecting filtrate;

(b) subjecting the filtrate collected in (a) to affinity chromatography;

(c) performing virus inactivation on the sample obtained in the step (b), and performing deep filtration on the sample subjected to virus inactivation;

(d) subjecting the sample obtained in (c) to anion exchange chromatography;

(e) subjecting the sample obtained in (d) to cation exchange chromatography;

(f) subjecting the sample obtained in (e) to virus filtration and/or ultrafiltration.

In some specific embodiments, the monoclonal antibodies produced by the invention have a final overall recovery of 70% or greater.

In some specific embodiments, the protein is a monoclonal antibody, optionally a monoclonal antibody that binds human TNF- α, including but not limited to adalimumab, infliximab, golimumab, certolizumab ozogamicin, and the like.

In some specific embodiments, the protein is a monoclonal antibody, optionally, a human IgG 1-type antibody.

In a solution, current flows by ion transport, so the conductivity of the solution can be changed by changing the ion concentration therein. For example, the concentration of the buffer and/or the concentration of a salt (e.g., sodium chloride, sodium acetate, or potassium chloride) in the solution can be varied to achieve a desired conductivity. Preferably, the salt concentration of the various buffers is modified to achieve the desired conductivity.

In some embodiments, the cell culture fluid is derived from a mammalian cell culture fluid, such as a CHO cell culture fluid.

In yet another aspect, the invention provides a process for the preparation of a pharmaceutical composition, said process comprising purifying a protein using the purification method provided by the invention.

The method of the invention has at least the following beneficial effects: the conductivity of the buffer solution is increased in the affinity chromatography elution process, and the pH of the buffer solution is changed or greatly changed in the cation exchange chromatography elution process, so that the purity of the monoclonal antibody is improved; meanwhile, the whole preparation method can lead the monoclonal antibody to reach the total recovery rate of 70 percent or higher.

Interpretation of terms

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Where there are multiple definitions of a term of the present invention, the definitions used in this section should be used unless otherwise indicated.

"treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Subjects in need of treatment include subjects already suffering from the disorder as well as subjects in whom the disorder is to be prevented.

The autoimmune disease in the "autoimmune disease and inflammatory disease" refers to a disease caused by the body's immune reaction to self-antigen to cause self-tissue damage, including but not limited to rheumatoid arthritis, psoriasis, etc.; the autoinflammatory diseases are caused by autoimmune disorders to cause systemic inflammatory reaction, including but not limited to gout, Crohn's disease and the like.

"antibody" is used in the broadest sense and includes a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), fusion proteins, immunoconjugates and any antibody fragments known in the art so long as they can exhibit the desired antigen binding activity.

"genetic engineering" includes but is not limited to obtaining target gene fragment by molecular biology method, constructing target gene expression vector, introducing target gene into cell line, and making the cell line able to stably express target protein for long term.

Folding in "folding and glycosylation" refers to the process by which a loosely structured polypeptide chain folds through interaction forces into a protein molecule with a native spatial structure; glycosylation refers to the process of forming glycosidic bonds between sugars and amino acid residues on proteins under the action of enzymes. Proper folding and glycosylation are important for the biological function of a protein.

"host protein (HCP)" refers to a protein component derived from a cell line producing an expressed antibody (e.g., CHO cells), including but not limited to proteins secreted by the cell line, apoptotic, metabolically produced structural proteins. The safety of HCPs to antibody drugs is at risk and requires stringent control and detection.

"contaminants" refers to substances that are different from the desired antibody product. Contaminants include, but are not limited to: host cell material, host cell protein; a nucleic acid; a variant, fragment, aggregate or derivative of the desired antibody; other polypeptides; an endotoxin; viral contaminants; cell culture media components.

"high molecular weight impurities" refers to the collective term for impurities having a molecular weight greater than the desired antibody product.

"Charge isomer" refers to an isomer that directly or indirectly changes the charge of an antibody molecule through glycosylation, deamidation, oxidation, isomerization, and the like. These isomers are generally classified as either acidic or basic isomers. When charge isomers are analyzed by weak cation exchange Chromatography (CEX), an acid peak (acidic isomer) elutes earlier than the main peak, and a base peak (basic isomer) elutes later than the main peak.

"loading" herein refers to the content of the composition loaded onto the chromatography column. Wherein the column is pre-equilibrated and equilibrated prior to loading the composition to be purified to remove residual impurities from the column.

"elution" means the application of a solution to a chromatographic material to remove non-specifically bound polypeptide and non-polypeptide compounds from the chromatographic material, particularly components such as host cell proteins, host cell DNA and high molecular weight impurities of the polymer. The term "eluting" generally does not include substantial elution of bound antibody from the chromatography material.

The "elution buffer" is used to elute the antibody of interest bound to the solid phase.

By "increasing conductivity" is meant increasing the ability of an aqueous solution to conduct current between two electrodes.

"Virus inactivation" includes the inactivation of viruses contained in a mixture or the removal of viruses from a mixture to be purified. The virus may originate from antibody production, downstream processing steps, or the manufacturing environment. Methods for inactivating or removing viruses include heat inactivation, pH inactivation, chemical inactivators, and the like.

Unless otherwise stated, "about" in the context of the present invention means within + -5%, preferably within + -2%, more preferably within + -1% of the specified numerical range given. For example, a pH of about 5.5 means a pH of 5.5. + -. 5%, preferably a pH of 5.5. + -. 2%, more preferably a pH of 5.5. + -. 1%.

Drawings

FIG. 1 CE-SDS electrophoretogram of TNF- α monoclonal antibody, including CE-SDS non-reduced electrophoretogram (FIG. 1a) and CE-SDS reduced electrophoretogram (FIG. 1b)

FIG. 2CEX analysis of Charge heterogeneity chromatograms of TNF- α monoclonal antibody

FIG. 3SEC analysis of the chromatogram of the TNF- α monoclonal antibody

Detailed Description

The invention will now be further described with reference to specific examples, which are, however, intended to be illustrative only and not to limit the scope of the invention. Likewise, the present invention is not limited to any particular preferred embodiment described herein. It will be appreciated by those skilled in the art that equivalent substitutions for the features of the invention, or corresponding modifications, may be made without departing from the scope of the invention. The reagents used in the following examples are commercially available products, and the solutions can be prepared by techniques conventional in the art, except where otherwise specified.

EXAMPLE 1 upstream Process for antibody production

To express the antibody or antibody portion of the present invention, DNA encoding part or all of the antibody is cloned into an expression vector, which is then transfected into a host cell, such that the gene is transcribed and translated. "transfection" is intended to encompass a variety of techniques commonly used to introduce foreign DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. The antibodies of the invention may be expressed in eukaryotic cells, preferably mammalian cells, most preferably CHO cells as host cells. In one embodiment, the seed cells are subjected to subculture amplification by using a conventional culture process until the cell density reaches 3.0-5.5 × 10⁶cells/mL, inoculating into a primary cell reactor containing culture medium for culture. When the cell density reaches 2.0-4.0X 10⁶cells/mL were transferred to another cell reactor. Culture parameters, temperature: 36-37 ℃, preferably 36.5 ℃, rotation speed: 26-30rpm, pH: 6.9. + -. 0.2 dissolved oxygen (pO)₂): 40 percent. When the cells are cultured to the 5 th day, the culture temperature is reduced to 33 ℃, and other culture conditions are not changed;when the cells were cultured to day 8, the culture temperature was lowered to 31 ℃ and other culture conditions were not changed until the end of 14 days of cell culture, at which time the cell viability was less than 80%.

EXAMPLE 2 determination of Charge isomer content

Weak Cation Exchange Chromatography (CEX) is a method for separating charge isomers in monoclonal antibodies by separating them based on differences in the number of charges carried by analytes. The sample is charged with positive charges and adsorbed on a chromatographic column, elution is carried out by adopting proper salt concentration and/or pH gradient, and various charge isomer components are washed out in sequence and detected by ultraviolet. And determining the content of an acidic peak and the content of a lysine variant in the sample by a peak area normalization method. Performing high performance liquid chromatography test by weak cation chromatography, balancing the chromatographic column with initial proportion mobile phase until the base line is stable, wherein the ratio of the mobile phase A: 10mM disodium hydrogen phosphate, ph7.5, mobile phase B: 10mM disodium hydrogen phosphate, 500mM sodium chloride, pH 5.5. The specific parameters are shown in table 1 below:

TABLE 1 Weak cation exchange chromatography liquid phase analysis parameters

Flow rate of flow	1-1.3mL/min
		Column temperature	25-30℃
Temperature control of automatic sample injector	2-8℃
		Sample loading volume	100-130μL
UV wavelength	280nm
		Run time	40-45min

EXAMPLE 3 determination of the content of Polymer and degradation product fragments

Protein polymer high molecular weight impurities and degradant fragment low molecular weight impurities were analyzed by Size Exclusion Chromatography (SEC), which separates the components of the analyte according to their molecular weight differences. The sample enters the chromatographic column, and the small molecular weight substance enters the gel hole, so that the retention time is longer; the high molecular weight substance can not enter the pores of the gel and can be eluted earlier, and the components are washed out in sequence from high molecular weight to low molecular weight and detected by ultraviolet. And determining the contents of immunoglobulin monomer, polymer high molecular weight impurity and degradation product low molecular weight impurity fragment in the sample by a peak area normalization method. Testing by high performance liquid chromatography, measuring by using molecular exclusion chromatography, balancing a chromatographic column by using a mobile phase until a base line is balanced, wherein the mobile phase: 50mM phosphate, 300mM sodium chloride, pH 7.0. + -. 0.1. The specific parameters are shown in table 2 below:

TABLE 2 parameters of size exclusion chromatography liquid phase analysis

Flow rate of flow	1.0-1.1mL/min
		Column temperature	25-30℃
Temperature control of automatic sample injector	2-8℃
		Sample loading volume	10μL
UV wavelength	280nm
		Gradient type	Equal degree
Run time	20-25min

EXAMPLE 4 isolation and purification of anti-TNF- α humanized monoclonal antibody

For example, a first wash with 50mM Tris-acetate buffer containing 150mM sodium chloride, followed by a wash with 50mM sodium acetate-acetate buffer containing 1M sodium chloride, followed by a wash with 50mM sodium acetate-acetate buffer containing 50mM sodium chloride, followed by a wash with 50mM sodium acetate-acetate buffer, wherein the cation chromatographic wash process comprises a change in buffer pH, equilibrating the column, loading the sample comprising the antibody onto the cation exchange material, the composition being at a first pH, performing a first wash (wash 1) with the equilibration buffer (also known as the first wash buffer), followed by a second wash (wash 2) with a second wash buffer having a pH at least 2.0 greater than the first pH, followed by a third wash (wash 3) with a third wash buffer having a pH at least 2.5 less than the second wash buffer.

(a) Deep filtration: collecting cell culture solution, performing deep filtration, and collecting filtrate;

(b) ①, using 50mM Tris-acetate buffer solution containing 150mM sodium chloride to carry out equilibration, the pH is 7.4, ② to load, ③ to wash 1, using 50mM Tris-acetate buffer solution containing 150mM sodium chloride to carry out pH 7.4, ④ to wash 2, using 50mM sodium acetate-acetate buffer solution containing 1M sodium chloride to carry out second washing solution, the pH is 5.0, ⑤ to wash 3, using 50mM sodium acetate-acetate buffer solution to carry out third washing solution, the pH is 5.0, using ⑥ to elute, using 50mM sodium acetate-acetate buffer solution to carry out elution, the pH is 3.5;

(c) virus inactivation: adjusting the pH value of the eluted sample to 3.5 +/-0.2 by using citric acid or Tris solution, and standing for 2-4 hours at the temperature of 18-26 ℃ for virus inactivation;

(d) secondary deep filtration: adjusting the pH value of the sample after virus inactivation to about 5.6, performing secondary deep filtration, and collecting filtrate;

(e) anion exchange chromatography: adopting POROS 50HQ anion filler, which comprises the steps of pre-balancing, sampling, balancing, regenerating, balancing and storing, wherein the pre-balancing liquid: 50mM sodium acetate-acetic acid buffer containing 1M sodium chloride, pH 5.0, equilibration: 50mM sodium acetate-acetic acid buffer, pH 5.7;

(f) ① pre-equilibrium, wherein the pre-equilibrium solution adopts 50mM sodium acetate-acetic acid buffer solution containing 1M sodium chloride, the pH is 5.0, ② balances and loads, the equilibrium solution adopts 50mM sodium acetate-acetic acid buffer solution, the pH is 5.5, ③ washes 1, the first washing solution adopts 50mM sodium acetate-acetic acid buffer solution, the pH is 5.5, ④ washes 2, the second washing solution adopts 20mM dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer solution, the pH is 7.9, ⑤ washes 3, the third washing solution adopts 50mM sodium acetate-acetic acid buffer solution, the pH is 5.0, ⑥ elutes, and the eluent contains 50mM sodium acetate-acetic acid buffer solution containing 0.3M sodium chloride, the pH is 5.0;

(g) virus filtration and/or ultrafiltration: and removing viruses by adopting a nanofiltration method, and then carrying out ultrafiltration liquid exchange on the sample to obtain the composition containing the monoclonal antibody.

The purity of the antibody product was determined by the detection method of sodium dodecyl sulfate capillary electrophoresis (CE-SDS) under reducing and non-reducing conditions, based on the difference in migration velocity due to the difference in molecular weight FIG. 1 is a CE-SDS electropherogram of the purified TNF- α humanized monoclonal antibody according to the present invention.

Calculating the total recovery rate: total recovery (%). the amount of stock solution protein/amount of cell harvest solution protein was obtained 100%

Table 3 shows the results of CEX-HPLC and SEC-HPLC detection of TNF- α humanized mAb after two separation and purification experiments.

TABLE 3 isolation and purification results of the humanized monoclonal antibody TNF- α

The results show that by increasing the conductivity of the buffer during the elution of the affinity chromatography and/or by changing the pH of the buffer greatly during the elution of the cation exchange chromatography, the content of the charge isomers is greatly reduced (see fig. 2), the content of high molecular weight impurities of the polymer and low molecular weight impurity fragments of the degradation products is reduced (see fig. 3), and the purity of the monoclonal antibody is improved. Meanwhile, the whole preparation method can lead the monoclonal antibody to reach the total recovery rate of 70 percent or higher.

While the compositions and methods of this invention have been described in terms of preferred embodiments in light of the present disclosure, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention.

The disclosures of all documents cited herein are incorporated by reference herein, to the extent that they provide exemplary, procedural and other details supplementary to those set forth herein.

Claims

1. A method of purifying a protein, the method comprising the steps of:

(a) affinity chromatography;

(b) inactivating viruses;

(c) deep filtering;

(d) anion exchange chromatography;

(e) cation exchange chromatography.

2. The method of claim 1, wherein the elution step of the affinity chromatography comprises an increase in the conductivity of the elution buffer, such as by washing with a 50mM Tris-acetate buffer containing 150mM sodium chloride, followed by a 50mM sodium acetate-acetate buffer containing 1M sodium chloride, followed by a final elution with a 50mM sodium acetate-acetate buffer, and the affinity chromatography further comprises eluting the protein of interest with a lower pH buffer.

3. The method of any one of claims 1-2, wherein the cation exchange chromatography step comprises equilibration, loading, washing three times, elution, and the like.

4. The method of any one of claims 1-3, wherein the cation exchange chromatography washing process comprises a change in buffer pH.

5. The method of any one of claims 1-4, wherein the cation exchange chromatography comprises:

1) loading the product into a cation exchange material, bringing the composition to a first pH,

2) performing a first elution with a first elution buffer to continue the composition at the first pH,

3) performing a second elution with a second elution buffer, the second elution buffer having a second pH,

4) performing a third elution with a third elution buffer, the third elution buffer having a third pH.

6. The method of claim 5, wherein the second pH is at least about 1.5 or greater above the first pH.

7. The method of any one of claims 5-6, wherein the third pH is at least about 2.0 or more lower than the second pH.

8. The method of any one of claims 1-5, further comprising washing the protein of interest after the elution with an eluent having a conductivity greater than the first, second, and third elution buffers.

9. The method according to any one of claims 1 to 8, wherein the protein is an antibody, preferably a monoclonal antibody that specifically binds TNF- α, more preferably adalimumab.

10. A process for the preparation of a pharmaceutical composition, said process comprising purifying a protein by a method according to any one of claims 1 to 9.