CN111153993B

CN111153993B - Preparation method of anti-TNF-alpha monoclonal antibody

Info

Publication number: CN111153993B
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: 2024-06-28
Anticipated expiration: 2039-10-22
Also published as: CN111153993A

Abstract

The invention relates to a preparation method of an anti-TNF-alpha monoclonal antibody, which comprises the steps of affinity chromatography, virus inactivation, depth filtration, anion exchange chromatography, cation exchange chromatography and the like. The invention increases the conductivity of the buffer solution in the process of affinity chromatography leaching and changes the pH value of the buffer solution in the process of cation exchange chromatography leaching, thereby improving the purity of the product and obtaining higher total recovery rate.

Description

Preparation method of anti-TNF-alpha 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-alpha monoclonal antibody.

Background

Tumor necrosis factor alpha (Tumor Necrosis Factor alpha, TNF-alpha) is a pro-inflammatory cytokine, which is secreted mainly by macrophages, and can promote infiltration of lymphocytes and accelerate activation of T cells, and it plays an anti-infective and anti-invasive role in normal immune response. However, long-term high levels of TNF- α are closely associated with the occurrence of a variety of autoimmune and inflammatory diseases, such as rheumatoid arthritis, psoriasis, crohn's disease, ankylosing spondylitis, and ulcerative colitis. The TNF-alpha monoclonal antibody can specifically bind to TNF-alpha, competitively block the binding of TNF-alpha and a receptor thereof, prevent the regulation and control of immune response and the induction of inflammation, and has the characteristics of strong targeting, low toxic and side effects, high efficiency and the like, thereby playing an important role in clinical medical treatment. Currently, a variety of TNF- α monoclonal antibody drugs have been approved for the treatment of autoimmune and inflammatory diseases, including infliximab, golimumab (golimumab), adalimumab (adalimumab), cetuzumab (certolizumab pegol).

Monoclonal antibody drugs are typically expressed using genetically engineered mammalian cells, such as CHO, BHK cells, etc., that produce the protein of interest, to ensure that the desired folding and glycosylation is achieved, including the introduction of the gene of interest into the cell line. The addition of a culture medium comprising a plurality of components such as amino acids, salts, growth factors, and the like during mammalian cell culture, host proteins (Host Cell Protein, HCP) and nucleic acids produced by the host cells themselves, and the like, results in complex cell culture product components; and the monoclonal antibody medicine has large molecular weight and complex structure, and brings great difficulty to the manufacturing process. However, in order to ensure the safety of the antibody drug to human body, the accumulated pollutants in the production process must be removed, so as to ensure that the antibody drug reaches higher purity. Downstream processes for antibody drug manufacturing are complex and any imperfection may cause significant losses, so it is highly necessary to establish an efficient downstream purification process to increase the yield and purity of monoclonal antibodies. 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 the chromatographic methods are commonly adopted in production, the specific conditions, parameters and filler selection of the chromatography can produce completely different results.

Affinity chromatography is often used to capture products in cell culture fluid as an important means for chromatographic separation of monoclonal antibodies. Because the affinity chromatography material can specifically adsorb the antibody, when the antibody load is proper, the product of the cell culture fluid after the affinity chromatography can always reach higher monomer purity, and simultaneously, higher recovery rate can be ensured. If contaminants such as HCP and nucleic acid can be removed as much as possible in the affinity chromatography step, the pressure of subsequent chromatography and depth filtration will be reduced, thereby reducing the production cost of the antibody.

Cation exchange chromatography is generally used for the purification of antibodies, and has the main function of removing high molecular weight impurities of polymers, and simultaneously, HCP and nucleic acid can be further removed, so that the purity of monoclonal antibodies is improved. It is conventional in the art to select a salt of a suitable concentration to compete for binding to the cation exchange material after loading the low concentration salt, thereby eluting the antibody for separation purposes. For the separation of antibodies or antibody drugs, it is necessary not only to reduce the content of HCP, nucleic acid, polymer high molecular weight impurities and the like as much as possible, but also to separate the charge isomers. However, conventional cation exchange chromatography is less effective for separating acid-base analogues that have a smaller difference in charge from the target protein. In order to solve the above problems, development of a new antibody purification method is required to improve the purity and quality of the antibody.

Disclosure of Invention

In one aspect, the invention provides 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, the method comprising the steps of:

(a) Deep filtration of cell culture fluid;

(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, each of 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 times of rinsing, elution, and the like. In some embodiments, the step of eluting the affinity chromatography comprises eluting the increase in buffer conductivity. For example, washing may be performed with 50mM Tris-acetate buffer containing 150mM sodium chloride, washing with 50mM sodium acetate-acetate buffer containing 1M sodium chloride, and washing with 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 using a ph=3.5 elution buffer.

In some embodiments, the affinity chromatography uses a Protein a affinity chromatography packing comprising the steps of: ① Equilibration with 50mM Tris-acetate buffer containing 150mM sodium chloride, pH=7.4; ② loading; ③ Eluting 1, wherein the first eluting solution adopts a buffer solution containing 150mM sodium chloride and 50mM Tris-acetic acid, and the pH=7.4; ④ Eluting 2, wherein the second eluting solution adopts 50mM sodium acetate-acetic acid buffer solution containing 1M sodium chloride, and the pH value is=5.0; ⑤ Eluting 3, wherein the third eluting solution adopts 50mM sodium acetate-acetic acid buffer solution, and the pH=5.0; ⑥ Elution, eluent was 50mM sodium acetate-acetic acid buffer, 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 with citric acid or Tris solution and allowing it to stand at 18-26℃for 2-4 hours.

In some embodiments, the solution that has been subjected to viral inactivation as described above is subjected to secondary depth filtration. In some embodiments, it is desirable to adjust the pH of the sample to about 5.6 prior to secondary depth filtration.

In some embodiments, anion exchange chromatography may employ various conventional or commercial packing materials, such as POROS 50HQ anion packing. 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 or commercial packing, such as POROS XS cationic packing. In some embodiments, the cation exchange chromatography step includes, but is not limited to, pre-equilibration, loading, leaching, elution, and the like. In some embodiments, the rinsing comprises 1-3 rinsing, e.g. 1 rinsing, 2 rinsing, or 3 rinsing, preferably 3 rinsing. In some embodiments, the rinsing step of the cationic chromatography comprises a change in the pH of the rinsing buffer, such as an increase or decrease 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, the column is rinsed with equilibration buffer (also known as a first rinse buffer) a first time (rinse 1), a second time (rinse 2) with a second rinse buffer having a second pH higher than the first pH, a third time (rinse 3) with a third rinse buffer having a third pH lower than the second pH, and finally the column is rinsed with an eluent having a conductivity greater than the conductivities of the first, second, and third rinse buffers.

In some embodiments, the second pH of the cation exchange chromatography is at least about 1.5 or more higher than 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 than the first pH. In some aspects of the invention, the second pH is at least about 1.5 greater than 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 than equilibrium.

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. In some aspects of the invention, the third pH is at least about 2.0 or more lower than 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.

In some embodiments, the balance of cation exchange chromatography, loading, and pH of the first elution buffer are the same or different.

In some embodiments, the equilibration buffer, loading buffer, and/or first elution buffer employs 50mM sodium acetate-acetic acid buffer. In some embodiments, the equilibration buffer, loading buffer and/or first wash buffer have a pH of 4 to 7, preferably 5 to 6, such as 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 leaching buffer is a potassium phosphate salt. In some embodiments, the pH of the second wash buffer may be from 6.5 to 9.0, preferably from 7.0 to 8.5, more preferably from 7.5 to 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 wash buffer uses 20mM dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer.

In some embodiments, the third elution buffer employs 50mM sodium acetate-acetic acid buffer. In some embodiments, the third wash 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 with a high conductivity buffer, preferably 50mM sodium acetate-acetic acid buffer containing 0.3M sodium chloride. In some embodiments, the pH of the eluate is from 4 to 7, preferably from 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 such 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, the first elution buffer, the second elution buffer, and the third elution buffer.

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

In some specific embodiments, the monoclonal antibody acidic variants prepared according to the invention are less than 15%, preferably less than 13%, 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 overall process for purifying a protein, which ensures that the overall recovery of the protein is 70% or higher, the process steps being as follows:

(a) Deep filtering the cell culture solution, and collecting filtrate;

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

(c) Inactivating the virus of the sample obtained in the step (b), and performing depth filtration on the sample after 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) And (3) carrying out virus filtration and/or ultrafiltration on the sample obtained in the step (e).

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, cetuzumab, and the like.

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

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

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

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 buffer solution conductivity is increased in the affinity chromatography leaching process, and the pH value of the buffer solution is changed or greatly changed in the cation exchange chromatography leaching process, so that the purity of the monoclonal antibody is improved; meanwhile, the whole preparation method can ensure that the monoclonal antibody can reach 70 percent or higher total recovery rate.

Interpretation of the terms

Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For the purposes of this invention where there are multiple definitions of a term, 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 who have a disorder as early as possible and subjects who are to be prevented from a disorder.

Autoimmune diseases in the terms of "autoimmune diseases and inflammatory diseases" refer to diseases caused by damage to self tissues due to immune reaction of an organism to self antigens, and include, but are not limited to, rheumatoid arthritis, psoriasis, etc.; while auto-inflammatory diseases are systemic inflammatory reactions due to autoimmune disorders, including but not limited to gout, crohn's disease, and the like.

"Antibody" is used in its 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 fragment known in the art so long as they can exhibit the desired antigen-binding activity.

"Genetic engineering" includes, but is not limited to, obtaining a fragment of a gene of interest using molecular biological methods, constructing an expression vector for the gene of interest, and introducing the gene of interest into a cell line such that the cell line stably expresses the protein of interest for a long period of time.

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

"Host protein (HCP)" refers to a protein component from a cell line (e.g., CHO cells) producing an expressed antibody, including but not limited to proteins secreted by the cell line, apoptosis, metabolically produced structural proteins. HCP presents a risk to the safety of an antibody drug, requiring strict control and detection thereof.

"Contaminant" refers to a substance that is different from the desired antibody product. Contaminants include, but are not limited to: host cell material, host cell protein; a nucleic acid; variants, fragments, aggregates or derivatives of the desired antibodies; other polypeptides; endotoxins; viral contaminants; cell culture medium composition.

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

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

"Rinsing" means applying a solution to a chromatographic material to remove non-specifically bound polypeptides and non-polypeptide compounds, particularly host cell proteins, host cell DNA, and polymeric high molecular weight impurities, from the chromatographic material. The term "rinsing" generally does not include substantial elution of bound antibodies from the chromatographic material.

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

"Increasing conductivity" refers to increasing the ability of an aqueous solution to conduct current between two electrodes.

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

Unless specifically stated otherwise, "about" in the present invention means that the range of the specified numerical value ranges is fluctuating within + -5%, preferably within + -2%, more preferably within + -1%. 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 electrophoresis patterns of TNF- α monoclonal antibodies, including CE-SDS non-reducing electrophoresis patterns (FIG. 1 a) and CE-SDS reducing electrophoresis patterns (FIG. 1 b)

FIG. 2CEX analysis of charge heterogeneity chromatograms of TNF-alpha monoclonal antibodies

FIG. 3SEC analysis of TNF-alpha monoclonal antibody chromatograms

Detailed Description

The invention will be further described with reference to specific examples which are, however, only intended to illustrate and not limit the scope of the invention. Also, the invention is not limited to any particular preferred embodiment described herein. It should be understood by those skilled in the art that equivalent substitutions and corresponding modifications of the technical features of the present invention are included in the scope of the present invention. The reagents used in the examples below are commercially available products, and the solutions may be formulated using techniques conventional in the art, unless otherwise specified.

Example 1 upstream Process for antibody production

To express an antibody or antibody portion of the invention, the DNA encoding part or all of the antibody is cloned into an expression vector, which is then transfected into a host cell, allowing the gene to be transcribed and translated. "transfection" is intended to encompass a variety of techniques commonly used to introduce exogenous 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, seed cells are cultured by passaging expansion using conventional culture techniques, and when the seed cells reach a cell density of 3.0-5.5X10 ⁶ cells/mL, they are inoculated into a primary cell reactor containing a culture medium for culture. When the cell density reached 2.0-4.0X10 ⁶ cells/mL, it was transferred to another cell reactor. Culture parameters, temperature: 36-37 ℃, preferably 36.5 ℃, rotational speed: 26-30rpm, pH: 6.9+ -0.2, dissolved oxygen (pO ₂): 40%. When the cells are cultured to the 5 th day, the culture temperature is reduced to 33 ℃, and other culture conditions are unchanged; when the cells were cultured to day 8, the culture temperature was lowered to 31℃and the other culture conditions were unchanged until the end of the 14-day cell culture, at which time the cell viability was lower than 80%.

Example 2 determination of charge isomer content

Weak cation exchange chromatography (Weak Cation Exchange Chromatography, CEX) is a separation based on the difference in charge numbers of analytes, which enables separation of charge isomers in monoclonal antibodies. The sample is positively charged and adsorbed on a chromatographic column, elution is carried out by adopting proper salt concentration and/or pH gradient, and all the charge isomer components are sequentially eluted and detected by ultraviolet. And determining the content of acidic peaks and lysine variants in the sample by a peak area normalization method. According to the high performance liquid chromatography test, weak cation chromatography is adopted for measurement, the chromatographic column is balanced by the mobile phase with the initial proportion until the base line is stable, and 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 analysis parameters

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

Example 3 determination of Polymer and degradation fragment content

The content of high molecular weight impurities of the protein polymer and low molecular weight impurities of the degradation product fragments are analyzed by using a molecular exclusion chromatograph (Size Exclusion Chromatography, SEC), and the method is to separate according to the molecular weight difference of each component in the analyte. The sample enters a chromatographic column, and the substances with small molecular weight enter a gel hole, so that the retention time is long; the large molecular weight substances can not enter the gel holes and can be eluted earlier, and the components are sequentially eluted according to the sequence of the large molecular weight and the small molecular weight and are detected by ultraviolet. And determining the contents of immunoglobulin monomers, high molecular weight impurities of polymers and low molecular weight impurity fragments of degradation products in the sample by a peak area normalization method. According to high performance liquid chromatography, the molecular exclusion chromatography is adopted to measure, the chromatographic column is balanced by the mobile phase until the baseline is balanced, and the mobile phase is: 50mM phosphate, 300mM sodium chloride, pH=7.0.+ -. 0.1. The specific parameters are shown in Table 2 below:

TABLE 2 size exclusion chromatography liquid analysis parameters

Flow rate	1.0-1.1mL/min
		Column temperature	25-30℃
Temperature control of automatic sampler	2-8℃
		Sample application volume	10μL
UV wavelength	280nm
		Gradient type	Isocratic of
Run time	20-25min

EXAMPLE 4 isolation and purification of anti-TNF-alpha humanized monoclonal antibodies

The anti-TNF-alpha humanized monoclonal antibody is isolated and purified by the following steps, wherein the eluting step of the affinity chromatography comprises an increase in the conductivity of the eluting buffer. For example, washing may be performed with 50mM Tris-acetate buffer containing 150mM sodium chloride, followed by washing with 50mM sodium acetate-acetate buffer containing 1M sodium chloride, and then washing with 50mM sodium acetate-acetate buffer. Wherein the cationic chromatographic elution process comprises a change in buffer pH, loading a sample comprising antibodies onto the cation exchange material after equilibration of the chromatographic column, said composition being at a first pH, performing a first elution with equilibration buffer (also known as a first elution buffer) (elution 1), subsequently performing a second elution with a second elution buffer having a pH at least 2.0 above said first pH (elution 2), and performing a third elution with a third elution buffer having a pH at least 2.5 below said second elution buffer (elution 3). The specific method comprises the following steps:

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

(b) Affinity chromatography: adopts Protein A affinity chromatography packing, and comprises the following steps: ① Equilibration with 50mM Tris-acetate buffer containing 150mM sodium chloride, pH=7.4; ② loading; ③ Eluting 1, wherein the first eluting solution adopts 50mM Tris-acetic acid buffer solution containing 150mM sodium chloride, and the pH=7.4; ④ Eluting 2, wherein the second eluting solution adopts 50mM sodium acetate-acetic acid buffer solution containing 1M sodium chloride, and the pH value is=5.0; ⑤ Eluting 3, wherein the third eluting solution adopts 50mM sodium acetate-acetic acid buffer solution, and the pH=5.0; ⑥ Eluting with 50mM sodium acetate-acetic acid buffer, ph=3.5;

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

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

(e) Anion exchange chromatography: the method adopts POROS 50HQ anion filler, and comprises the steps of pre-balancing, loading, balancing, regenerating, balancing and preserving, wherein the pre-balancing liquid is as follows: 50mM sodium acetate-acetic acid buffer with 1M sodium chloride, ph=5.0, equilibration: 50mM sodium acetate-acetate buffer, ph=5.7;

(f) Cation exchange chromatography: the method adopts POROS XS cation material, and comprises the following steps: ① Pre-equilibration, pre-equilibration with 50mM sodium acetate-acetic acid buffer with 1M sodium chloride, ph=5.0; ② Equilibration and loading, equilibration solution using 50mM sodium acetate-acetic acid buffer, ph=5.5; ③ Leaching 1, wherein the first leaching solution adopts 50mM sodium acetate-acetic acid buffer solution, and the pH=5.5; ④ Leaching 2, wherein the second leaching solution adopts 20mM dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer solution, and the pH=7.9; ⑤ Eluting 3, wherein the third eluting solution adopts 50mM sodium acetate-acetic acid buffer solution, and the pH=5.0; ⑥ Eluting, wherein the eluent contains 50mM sodium acetate-acetic acid buffer solution of 0.3M sodium chloride, and the pH=5.0;

(g) Virus filtration and/or ultrafiltration: removing virus by nanofiltration, and performing ultrafiltration liquid exchange on the sample to obtain the composition containing the monoclonal antibody.

The purity of the antibody product is detected by adopting a detection method of sodium dodecyl sulfate capillary electrophoresis (CE-SDS) under the conditions of reduction and non-reduction according to the difference of migration speeds caused by the difference of molecular weights. FIG. 1 is a CE-SDS electrophoretogram of a humanized TNF- α monoclonal antibody purified by the process of the present invention.

Calculating the total recovery rate: total recovery (%) = obtained stock protein amount/cell harvest protein amount x 100%

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

TABLE 3 separation and purification results of TNF-alpha humanized monoclonal antibody

The results show that the buffer conductivity is increased in the affinity chromatography leaching process, and/or the pH value of the buffer is greatly changed in the cation exchange chromatography leaching process, so that the content of charge isomers is greatly reduced (see figure 2), the content of polymer high molecular weight impurities and degradation product low molecular weight impurity fragments is reduced (see figure 3), and the purity of the monoclonal antibody is improved. Meanwhile, the whole preparation method can ensure that the monoclonal antibody can reach 70 percent or higher total recovery rate.

In accordance with the present disclosure, while the compositions and methods of this invention have been described in terms of preferred embodiments, 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 hereby incorporated by reference 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, wherein the elution step of the affinity chromatography comprises eluting with 50mM Tris-acetate buffer containing 150mM sodium chloride and pH 7.4, eluting with 50mM sodium acetate-acetate buffer containing 1M sodium chloride and pH 5.0, eluting with 50mM sodium acetate-acetate buffer and pH 5.0, and eluting with 50mM sodium acetate-acetate buffer and pH 3.5;

(b) Adjusting the pH value of the affinity chromatography eluent to 3.0-4.0, and inactivating viruses;

(c) Deep filtration;

(d) Anion exchange chromatography, wherein the balance of the anion exchange chromatography is 50mM sodium acetate-acetic acid buffer solution with pH of 5.7;

(e) The leaching step of the cation exchange chromatography comprises the steps of firstly leaching with a first leaching buffer, then leaching with a second leaching buffer, and finally leaching with a third leaching buffer, wherein the first leaching buffer is 50mM sodium acetate-acetic acid buffer with the pH of 5.5, the second leaching buffer is 20mM dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer with the pH of 7.9, the third leaching buffer is 50mM sodium acetate-acetic acid buffer with the pH of 5.0, and eluting with 50mM sodium acetate-acetic acid buffer with the pH of 5.0 and containing 0.3M sodium chloride after leaching;

the protein is adalimumab.

2. A process for preparing a pharmaceutical composition, said process comprising purifying a protein using the method of claim 1.