CN113621063A

CN113621063A - Affinity purification method for reducing content of host cell protein in monoclonal antibody production

Info

Publication number: CN113621063A
Application number: CN202110882251.7A
Authority: CN
Inventors: 戴长松; 李帅; 郭斌; 黄文俊; 戴璐; 裘浙伟; 王逸尘; 何永梅; 吴亦亮
Original assignee: Jiangsu Saifushi Biotechnology Co ltd; Jiangsu Qyuns Therapeutics Co Ltd
Current assignee: Jiangsu Saifushi Biotechnology Co ltd; Jiangsu Qyuns Therapeutics Co Ltd
Priority date: 2021-08-02
Filing date: 2021-08-02
Publication date: 2021-11-09
Also published as: CN114057878A; CN114057878B

Abstract

The invention discloses an affinity purification method for reducing the content of host cell protein in the production of a monoclonal antibody, which comprises the following steps: balancing the affinity chromatography medium by using a first balancing buffer solution to obtain a well-balanced affinity chromatography medium, and combining the monoclonal antibody fermentation liquor with the well-balanced affinity chromatography medium; then carrying out intermediate pre-elution and elution, and then carrying out final elution to remove host cell protein to obtain a monoclonal antibody; the monoclonal antibody is an isolated anti-human interferon alpha receptor 1(IFNAR1) monoclonal antibody comprising three heavy chain complementarity determining regions (CDR-H1, CDR-H2 and CDR-H3) and three light chain complementarity determining regions (CDR-L1, CDR-L2 and CDR-L3). The method is simple and feasible, can carry out amplification purification production, the cell fermentation supernatant does not need pre-treatment, the yield of an elution sample is higher, and meanwhile, the HCP residual quantity is kept at a lower level (the residual control quantity is not higher than 0.1 percent), thereby relieving the pressure of removing HCP in the subsequent purification step.

Description

Affinity purification method for reducing content of host cell protein in monoclonal antibody production

Technical Field

The invention relates to the field of biotechnology, in particular to an affinity purification method for reducing the content of host cell protein in the production of an anti-human interferon alpha receptor 1 monoclonal antibody.

Background

The affinity purification in the production process of antibody drugs is a very critical process step, and the process captures and concentrates the antibody in the fermentation liquor to realize the first step of crude purification of the antibody. During the large-scale fermentation process of genetic engineering cell strains such as Chinese Hamster Ovary (CHO) cells and the like, the cells are subjected to apoptosis and lysis in different physiological cycles, and Host Cell Proteins (HCPs) are released. HCP refers to protein components derived from host cells, including host cell structural proteins and transforming proteins (cell secreted growth promoting proteins). The HCP not only can possibly induce the organism to generate anti-HCP antibody to cause anaphylactic reaction, but also can possibly cause the organism to generate antibody to protein medicine by 'adjuvant effect' to influence the treatment effect of the medicine, and the quantitative determination of the HCP remained in the genetic engineering medicine is an important means of quality control, and is helpful to keep the effectiveness and consistency of the purification process. In the antibody affinity purification, HCP generated by engineering cells in the fermentation process needs to be effectively removed while high-efficiency antibody recovery rate is ensured. Therefore, an economically feasible antibody affinity purification process which can be widely applied to large-scale fermentation of the antibody is researched, and the method is very significant for further industrialized popularization of antibody medicines. At present, many methods for removing HCP residues are available, and each method has the following characteristics: 1) the chromatography mode is as follows: the method comprises Protein A affinity chromatography, anion and cation chromatography, and removal capability to HCP in three chromatography processes, wherein the Protein A affinity chromatography is used as a basis, the removal capability is strong, and the method is a main step for removing HCP, and the anion and cation chromatography is mainly used as a further removal process of subsequent HCP. The process for removing HCP by chromatography is a main means for removing HCP, and various chromatography processes continuously put forward and apply actual production in the continuous improvement process; 2) tangential flow ultrafiltration mode: the removal capability of HCP is limited, the residual quantity is difficult to control, the process control coefficient is not high, and the HCP can only be used as an auxiliary process for removing HCP; 3) polymer precipitation mode: polymers such as PEG, polyacrylic acid and the like have positive charges in a wider pH range, are combined with antibodies to form precipitates through charge action, and HCP is not easy to precipitate due to lower isoelectric point. The HCP content of the precipitated antibody samples was significantly reduced, but the process was not suitable for process scale-up, and the precipitation may have some effect on the activity of the antibody. Therefore, many processes for removing HCPs are required, and a comprehensive consideration and selection process is required due to the difference in the sample fermentation process and the properties of the antibody of each purpose.

Disclosure of Invention

In order to solve the problem of Host Cell Protein (HCP) residue in antibody production, the invention provides a novel method for effectively reducing CHO Host Cell Protein (HCP) in antibody purification production, which can be widely applied to an antibody affinity purification process, and the used material has low cost and price and is easy to amplify the process. In the invention, the HCP content is effectively reduced mainly by intermediate pre-elution and elution of antibody affinity chromatography, the requirement of large-scale high-quality purification preparation of the antibody drug is met, and the safety of clinical use of the antibody drug is ensured.

The specific technical scheme of the invention is as follows:

1. an affinity purification process for reducing the protein content of a host cell in the production of a monoclonal antibody comprising the steps of:

balancing the affinity chromatography medium by using a first balancing buffer solution to obtain a well-balanced affinity chromatography medium, and combining the monoclonal antibody fermentation liquor with the well-balanced affinity chromatography medium;

then carrying out intermediate pre-elution and elution, and then carrying out final elution to remove host cell protein to obtain a monoclonal antibody;

the monoclonal antibody is an isolated anti-human interferon alpha receptor 1(IFNAR1) monoclonal antibody comprising three heavy chain complementarity determining regions (CDR-H1, CDR-H2 and CDR-H3) and three light chain complementarity determining regions (CDR-L1, CDR-L2 and CDR-L3), wherein:

(a) the amino acid sequence of CDR-H1 is shown in SEQ ID NO:1 is shown in the specification;

(b) the amino acid sequence of CDR-H2 is shown in SEQ ID NO:2 is shown in the specification;

(c) the amino acid sequence of CDR-H3 is shown in SEQ ID NO:3 is shown in the specification;

(d) the amino acid sequence of CDR-L1 is shown in SEQ ID NO:4 is shown in the specification;

(e) the amino acid sequence of CDR-L2 is shown in SEQ ID NO:5 is shown in the specification; and is

(f) The amino acid sequence of CDR-L3 is shown in SEQ ID NO: and 6.

2. The affinity purification method according to claim 1, wherein, the anti human interferon alpha receptor 1(IFNAR1) monoclonal antibody contains heavy chain variable region and light chain variable region, wherein,

the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 7 is shown in the specification; and the number of the first and second electrodes,

the amino acid sequence of the light chain variable region is shown as SEQ ID NO: shown in fig. 8.

3. The affinity purification method according to claim 2, wherein the amino acid sequence of the heavy chain of the anti-human interferon alpha receptor 1(IFNAR1) monoclonal antibody is as shown in SEQ ID NO: 10 is shown in the figure; the amino acid sequence of the light chain is shown as SEQ ID NO: shown at 11.

4. The affinity purification method according to any one of claims 1 to 3, wherein the affinity chromatography medium is a chromatography medium in which ligands are crosslinked to agarose, polyvinyl ether, hydroxylated polyether resin, polyacrylic resin, polystyrene divinyl phenyl resin, polymethacrylic resin, polystyrene resin, hydroxyapatite or glass matrix, preferably, the affinity chromatography medium is a chromatography medium in which ligands are crosslinked to polyvinyl ether;

preferably, the ligand is Protein A, Protein G or Protein L, preferably Protein A.

5. The affinity purification process according to any one of claims 1 to 4, wherein the first equilibration buffer is a phosphate buffer, a Tris-HCl buffer or a boric acid-borax buffer, the salt concentration in the first equilibration buffer is between 5mM and 0.25M, and the pH is between 5.5 and 8.0.

6. The affinity purification method according to any one of claims 1 to 5, wherein the intermediate pre-elution buffer is a neutral buffer and/or an acidic buffer; preferably, the neutral buffer solution is a phosphate buffer solution, a tris buffer solution or a glycine buffer solution; the acidic buffer solution is a citric acid-disodium hydrogen phosphate buffer solution, an acetic acid-sodium acetate buffer solution or a citric acid-trisodium citrate buffer solution.

7. The affinity purification method according to item 6, wherein the pH of the intermediate pre-elution buffer is 5.0 to 7.5.

8. The affinity purification method according to item 6, wherein a pre-elution active agent is added to the intermediate pre-elution buffer, preferably the pre-elution active agent is guanidine hydrochloride, polysorbate 80 or sodium chloride, further preferably the pre-elution active agent is guanidine hydrochloride.

9. The affinity purification method according to claim 8, wherein the concentration of guanidine hydrochloride is 0.01-1M.

10. The affinity purification method according to any one of claims 1 to 9, wherein after the intermediate pre-elution rinse, before the final elution, the method further comprises the following steps:

the equilibration is performed using a second equilibration buffer, preferably a phosphate buffer, a Tris-HCl buffer, or a boric acid-borax buffer.

11. The affinity purification method according to any one of claims 1 to 10, wherein the final elution buffer is one or more selected from the group consisting of a citric acid-disodium hydrogen phosphate buffer, an acetic acid buffer, a glycine-HCl buffer, and a citric acid-sodium citrate buffer, and preferably is a citric acid-disodium hydrogen phosphate buffer.

12. The affinity purification method according to claim 11, wherein the pH of the citric acid-disodium phosphate buffer is 2.0 to 7.0.

ADVANTAGEOUS EFFECTS OF INVENTION

The affinity process is simple and feasible, can carry out amplification purification production, the cell fermentation supernatant does not need pre-treatment, the yield of an elution sample is higher, and meanwhile, the HCP residual quantity is kept at a lower level (the residual control quantity is not higher than 0.1 percent), so that the pressure for removing HCP in the subsequent purification step is reduced, and the HCP residual quantity of the final antibody sample is ensured to be at an extremely low level. Meanwhile, the affinity purification process of the fermentation supernatants of different batches is verified, so that the affinity purification process has good stability.

Compared with the existing human interferon alpha receptor 1 monoclonal antibody (Aniflumab), the anti-human interferon alpha receptor 1(IFNAR1) monoclonal antibody has the advantages that the binding affinity of the anti-human interferon alpha receptor 1 monoclonal antibody to IFNAR1 is equivalent, and the neutralizing activity at a cellular level is equivalent to that of the Aniflumab.

The monoclonal antibody shows a neutralizing activity at a cellular level comparable to that of Aniflumab (prepared by expression of a patent publication sequence), and is expected to exhibit a good clinical effect in the prevention and treatment of related diseases.

Drawings

FIG. 1 is a diagram showing the results of nucleic acid electrophoresis for constructing transient expression plasmids of HZD 1203-45. Wherein, M: marker; strip 1: PCR product 362VH-Hu 6; strip 2: pHZDCH, HindIII/NheI; the strip 3: PCR product 362VK-Hu 20; the strip 4: pHZDCK, HindIII/BsiWI.

Fig. 2 is a transient expression flow diagram.

FIG. 3 is an electrophoretic image of QX006N (HZD 1203-45-IgG4.1).

Detailed Description

The present invention is described in detail below with reference to the attached drawing figures, wherein like numerals represent like features throughout. While specific embodiments of the invention are shown in the drawings, it should be understood that the invention may be embodied in various forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

It should be noted that certain terms are used throughout the description and claims to refer to particular components. As one skilled in the art will appreciate, various names may be used to refer to a component. This specification and claims do not intend to distinguish between components that differ in name but not function. In the following description and in the claims, the terms "include" and "comprise" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. The description which follows is a preferred embodiment of the invention, however, the description is given for the purpose of illustrating the general principles of the invention and not for the purpose of limiting the scope of the invention. The scope of the present invention is defined by the appended claims.

The invention provides an affinity purification method for reducing the content of host cell protein in the production of a monoclonal antibody, which comprises the following steps:

(a) the amino acid sequence of CDR-H1 (CDR-H1 in this specification represents the heavy chain CDR1) is as shown in SEQ ID NO:1 is shown in the specification;

(b) the amino acid sequence of CDR-H2 (CDR-H2 in this specification represents the heavy chain CDR2) is as shown in SEQ ID NO:2 is shown in the specification;

(c) the amino acid sequence of CDR-H3 (CDR-H3 in this specification represents the heavy chain CDR3) is as shown in SEQ ID NO:3 is shown in the specification;

(d) the amino acid sequence of CDR-L1 (CDR-L1 in this specification represents the light chain CDR1) is set forth in SEQ ID NO:4 is shown in the specification;

(e) the amino acid sequence of CDR-L2 (CDR-L2 in this specification represents the light chain CDR2) is set forth in SEQ ID NO:5 is shown in the specification; and is

(f) The amino acid sequence of CDR-L3 (CDR-L3 in this specification represents the light chain CDR3) is set forth in SEQ ID NO: and 6.

Wherein, SEQ ID NO:1 is as follows:

SYYMT

SEQ ID NO:2 is as follows:

VINVYGGTYYASWAKG

SEQ ID NO:3 is as follows:

EDVAVYMAIDL

SEQ ID NO:4 is as follows:

QASQSISNQLS

SEQ ID NO:5 has the following amino acid sequence:

DASSLAS

SEQ ID NO:6 is as follows:

LGIYGDGADDGIA

the monoclonal antibodies represent antibodies from a population of substantially homologous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, with such variants typically being present in minor amounts, except for possible variant antibodies (e.g., containing naturally occurring mutations or produced during the production of monoclonal antibody preparations). Unlike polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention can be prepared by a variety of techniques including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods using transgenic animals comprising all or part of a human immunoglobulin locus, such methods and other exemplary methods of preparing monoclonal antibodies being described herein.

The host cell refers to a cell into which an exogenous nucleic acid has been introduced, including progeny of such a cell. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom (regardless of the number of passages). Progeny may not be identical to the parent cell in terms of nucleic acid content, but may contain mutations. Progeny of mutants screened or selected for the originally transformed cell to have the same function or biological activity are included in the present specification.

The Host Cell Protein (HCP) is a process-related impurity that is expressed by host cells used for the production of biopharmaceutical proteins. During the purification process, most of the HCPs were removed (> 99%), but the amount of residual HCPs remained in the distributed product, such as monoclonal antibodies (mabs), antibody-drug conjugates (ADCs), therapeutic proteins, vaccines, and other protein-based biopharmaceuticals.

The anti-human interferon alpha receptor 1(IFNAR1) monoclonal antibody represents such a monoclonal antibody: which is capable of binding to human interferon alpha receptor 1 with sufficient affinity such that the monoclonal antibody is useful as a diagnostic and/or therapeutic agent targeting human interferon alpha receptor 1.

The anti-human interferon alpha receptor 1(IFNAR1) monoclonal antibody of the invention does not bind to target-independent proteins. Here, "irrelevant protein" means a protein other than human interferon alpha receptor 1 as a target; here, "not to bind" means: the invention of the anti human interferon alpha receptor 1(IFNAR1) monoclonal antibodies and its target human interferon alpha receptor 1 binding capacity as 100%, the invention of the anti human interferon alpha receptor 1 monoclonal antibodies and the unrelated protein binding capacity of less than 10%, such as 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0.

The monoclonal antibody of the human interferon alpha receptor 1(IFNAR1) can not be combined with the interferon alpha receptor 1 of other animal species. Here, "other animal species" refers to other animal species than humans, such as marmoset, cynomolgus, pig, dog, rabbit, rat, mouse, guinea pig, and the like; here, "not to bind" means: the invention of the anti human interferon alpha receptor 1(IFNAR1) monoclonal antibodies with other animal species of interferon alpha receptor 1 binding capacity of less than 10%, such as 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0 under the condition of the invention of the human interferon alpha receptor 1(IFNAR1) monoclonal antibodies and its target of human interferon alpha receptor 1 binding capacity as 100%.

The anti-human interferon alpha receptor 1 monoclonal antibody has an equilibrium dissociation constant (KD) of less than or equal to 1 mu M, less than or equal to 100nM, less than or equal to 50nM and less than or equal to 40 nM.

The Human Interferon alpha Receptor 1(Human Interferon alpha/beta Receptor 1, IFNAR1) represents a membrane protein derived from Human, and the amino acid sequence of the extracellular region of the membrane protein is shown as SEQ ID NO: 9, wherein the underlined part indicates the signal peptide.

SEQ ID NO：9：

MMVVLLGATTLVLVAVAPWVLSAAAGGKNLKSPQKVEVDIIDDNFILRWNRSDESVGNVTFSFDYQKTGMDNWIKLSGCQNITSTKCNFSSLKLNVYEEIKLRIRAEKENTSSWYEVDSFTPFRKAQIGPPEVHLEAEDKAIVIHISPGTKDSVMWALDGLSFTYSLVIWKNSSGVEERIENIYSRHKIYKLSPETTYCLKVKAALLTSWKIGVYSPVHCIKTTVENELPPPENIEVSVQNQNYVLKWDYTYANMTFQVQWLHAFLKRNPGNHLYKWKQIPDCENVKTTQCVFPQNVFQKGIYLLRVQASDGNNTSFWSEEIKFDTEIQAFLLPPVFNIRSLSDSFHIYIGAPKQSGNTPVIQDYPLIYEIIFWENTSNAERKIIEKKTDVTVPNLKPLTVYCVKARAHTMDEKLNKSSVFSDAVCEKTKPGNTSK

The present invention enables the residual amount of host cell proteins of monoclonal antibodies to be at an extremely low level by affinity purification using the above-described method.

In one embodiment, the anti-human interferon alpha receptor 1(IFNAR1) monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein,

Wherein, SEQ ID NO: 7 has the following amino acid sequence:

EVQLVESGGGLVQPGGSLRLSCAASGFSLSSYYMTWVRQAPGKGLEWVSVINVYGGTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREDVAVYMAIDLWGQGTLVTVSS

SEQ ID NO: the amino acid sequence of 8 is as follows:

AIQMTQSPSSLSASVGDRVTITCQASQSISNQLSWYQQKPGKAPKLLIYDASSLASGVPSRFSGSRSGTKFTLTISSLQPEDFATYYCLGIYGDGADDGIAFGGGTKVEIK

in one embodiment, the amino acid sequence of the heavy chain of the anti-human interferon alpha receptor 1(IFNAR1) monoclonal antibody is as set forth in SEQ ID NO: 10 is shown in the figure; the amino acid sequence of the light chain is shown as SEQ ID NO: shown at 11.

Wherein, SEQ ID NO: 10 is as follows:

EVQLVESGGGLVQPGGSLRLSCAASGFSLSSYYMTWVRQAPGKGLEWVSVINVYGGTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREDVAVYMAIDLWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

SEQ ID NO: 11 is as follows:

AIQMTQSPSSLSASVGDRVTITCQASQSISNQLSWYQQKPGKAPKLLIYDASSLASGVPSRFSGSRSGTKFTLTISSLQPEDFATYYCLGIYGDGADDGIAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

wherein, SEQ ID NO: 10 and 11 are both humanized sequences.

In one embodiment, the affinity chromatography medium is a chromatography medium in which ligands are crosslinked to agarose, polyvinyl ether, hydroxylated polyether resin, polyacrylic resin, polystyrene divinyl phenyl resin, polymethacrylic resin, polystyrene resin, hydroxyapatite or glass matrix, preferably, the affinity chromatography medium is a chromatography medium in which ligands are crosslinked to polyvinyl ether;

The ligand can specifically bind to a monoclonal antibody.

The affinity filler is not limited in the present invention and can be confirmed according to the needs of those skilled in the art, for example, the affinity filler can be selected from Mabselect, Mabselect Sure, of GE healthcare, Protein A Diamond, of Borglon Biotechnology, Inc., and MERCK

A。

In one embodiment, the first equilibration buffer is a phosphate buffer, a Tris-HCl buffer, or a boric acid-borax buffer.

The salt concentration in the first equilibration buffer is not limiting in the present invention and can be selected by the skilled person as desired, for example the salt concentration in the first equilibration buffer is 5mM-0.25M and the pH is 5.5-8.0.

For example, the salt concentration in the first equilibration buffer may be 5mM, 10mM, 20mM, 50mM, 0.1M, 0.15M, 0.2M, 0.25M, etc.; the pH may be 5.5, 6, 7, 8, etc.

Preferably, the first equilibration buffer is phosphate buffer or Tris-hydrochloric acid buffer, and more preferably, NaCl or Na is added into the first equilibration buffer₂SO₄To reduce non-specific adsorption between the non-antibody protein and the filler.

Preferably, the salt concentration in the first equilibration buffer is between 5mM and 0.15M, preferably between 10mM and 50mM, more preferably between 20 mM.

The pH of the first equilibration buffer is not subject to any restriction of the invention and can be selected as desired by the person skilled in the art, for example the pH of the first equilibration buffer is 6.5 to 7.5, preferably 6.9.

For NaCl or Na₂SO₄In the presence of NaCl or Na, which can be determined according to the requirements of the person skilled in the art₂SO₄The concentration of (A) can be selected by those skilled in the art as desired, for example, NaCl or Na₂SO₄The concentration of (B) may be 0-250mM, preferably 150 mM.

Preferably, the phosphate buffer may be, for example, a buffer of disodium hydrogen phosphate and sodium dihydrogen phosphate.

In one embodiment, a first equilibration buffer is used for equilibration when binding the monoclonal antibody fermentation broth to the equilibrated affinity chromatography medium.

In one embodiment, the intermediate pre-elution buffer is a neutral buffer and/or an acidic buffer; preferably, the neutral buffer solution is a phosphate buffer solution, a tris buffer solution or a glycine buffer solution; the acidic buffer solution is a citric acid-disodium hydrogen phosphate buffer solution, an acetic acid-sodium acetate buffer solution or a citric acid-trisodium citrate buffer solution.

The pH of the intermediate pre-elution buffer is not subject to any restriction and can be selected by the skilled person as desired, for example, the pH of the intermediate pre-elution buffer is 5.0 to 7.5, preferably 5.8.

For example, the pH of the intermediate pre-elution buffer may be 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and the like.

In one embodiment, a pre-elution active agent is added to the intermediate pre-elution buffer, preferably the pre-elution active agent is guanidine hydrochloride, polysorbate 80 or sodium chloride, further preferably the pre-elution active agent is guanidine hydrochloride.

The concentration of guanidine hydrochloride is not limited in the present invention, and can be selected as desired by those skilled in the art, for example, the concentration of guanidine hydrochloride is 0.01 to 1M, preferably 0.05 to 0.15M, and more preferably 0.1M.

For example, the concentration of guanidine hydrochloride can be 0.01M, 0.05M, 0.1M, 0.5M, 1M.

The salt concentration in the intermediate pre-elution buffer is not subject to any limitation of the present invention and can be selected by the skilled person as desired, for example, in one embodiment the salt concentration in the intermediate pre-elution buffer is 0-0.5M, preferably 0.1M.

For example, the salt concentration in the intermediate pre-elution buffer may be 0, 0.1M, 0.2M, 0.3M, 0.4M, 0.5M, etc.

In one embodiment, after the intermediate pre-elution rinse, the following steps are further included before the final elution is performed:

Preferably, the second equilibration buffer is phosphate buffer or Tris-HCl buffer, and NaCl or Na is preferably added into the second equilibration buffer₂SO₄To maintain conductance while retaining some of the buffering capacity.

For the salt concentration in the second equilibration buffer, the pH of the second equilibration buffer and NaCl or Na₂SO₄The concentration of (b) is not limited in any way by the present invention and can be selected by the skilled person as desired, for example, in one embodiment, the salt concentration in the second equilibration buffer is between 5mM and 0.15M, preferably between 10mM and 50mM, and more preferably between 20 mM; preferably, the pH is 5.5 to 8.0, preferably 6.5 to 7.5, and more preferably 7.2; preferably, NaCl or Na₂SO₄Is 0-250mM, preferably 10 mM.

The phosphate buffer may be, for example, a buffer of disodium hydrogen phosphate and sodium dihydrogen phosphate.

In one embodiment, the final elution buffer is selected from one or more of a citric acid-disodium hydrogen phosphate buffer, an acetic acid buffer, a glycine-HCl buffer, and a citric acid-sodium citrate buffer, and is preferably a citric acid-disodium hydrogen phosphate buffer.

The pH of the final elution buffer is not subject to any limitation of the present invention and can be selected as desired by the skilled person, for example, in one embodiment, the pH of the final elution buffer is 2.0 to 7.0, preferably 3.0 to 4.0.

For example, the final elution buffer can have a pH of 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, and the like.

The present invention is not limited in any way with respect to the salt concentration in the final elution buffer, which can be selected by the skilled person as desired, for example, in one embodiment the salt concentration in the final elution buffer is between 5 and 100mM, preferably between 10 and 50 mM.

For example, the salt concentration in the final elution buffer can be 5mM, 10mM, 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, and the like.

In one embodiment, elution is performed followed by regeneration of the affinity medium, preferably, regeneration buffers include, but are not limited to, citric acid-disodium hydrogen phosphate buffer, hydrochloric acid, glycine, NaOH, preferably citric acid-disodium hydrogen phosphate buffer and NaOH solution.

The invention uses Protein A affinity purification technology, which is simple and easy to operate, can carry out amplification purification production, the cell fermentation supernatant does not need pre-treatment, the yield of the elution sample is higher, and the HCP residual quantity is also kept at a lower level (the residual control quantity is not higher than 0.1 percent), thereby reducing the pressure of removing HCP in the subsequent purification step, and ensuring that the HCP residual quantity of the final sample of the antibody is at an extremely low level. Meanwhile, the affinity purification process of the fermentation supernatants of different batches is verified, so that the affinity purification process has good stability.

In one embodiment, the monoclonal antibody is produced by in vitro fermentation using mammals including, but not limited to, various hybridoma cells, chinese hamster ovary Cells (CHO), preferably CHO cells, currently in use.

Examples

The invention is described generally and/or specifically for the materials used in the tests and the test methods, in the following examples,% means wt%, i.e. percent by weight, unless otherwise specified. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.

Example 1 preparation of anti-human Interferon alpha receptor 1 monoclonal antibody QX006N

Human interferon alpha receptor 1(IFNAR1) is purchased from Shanghai near-shore science and technology Limited company, is used for immunizing New Zealand rabbits, and an antigen binding specificity antibody clone is obtained by applying a B cell cloning technology, so that a monoclonal antibody which is bound with human IFNAR1 and has human IFNAR1 inhibitory activity is screened. First, cell supernatants were examined by Binding ELISA, and clones Binding to human IFNAR1 were selected; then HEK Blue IFN alpha/beta reporter gene cell method is used for detection, and the clone with human IFNAR1 inhibitory activity is selected. The immunization and screening process is entrusted to a commercial company for completion.

37 clones were selected in sequence for recombinant expression and sequencing. 362# and 1203# were determined to have the best cell neutralizing activity and the sequences of the two clones were very similar. Therefore, 362# was humanized and transformed, and when 1203# was obtained by screening and 1203# activity was found to be better, 1203# clone was humanized and transformed on the basis of 362# humanization. Carrying out homology alignment of human IgG germ line sequences (Germine) by using NCBI Igblast, selecting IGHV3-66 a 01 as a heavy chain CDR grafting template, and grafting CDR regions (namely CDR-H1(SEQ ID No:1), CDR-H2(SEQ ID No:2) and CDR-H3(SEQ ID No:3)) of 1203# clone heavy chain into framework regions of IGHV3-66 a 01; selecting IGKV 1-6X 01 as light chain CDR grafting template, grafting the CDR regions of 1203# clone light chain (namely CDR-L1(SEQ ID No:4), CDR-L2(SEQ ID No:5) and CDR-L3(SEQ ID No:6)) into the framework region of IGKV 1-6X 01; and (3) carrying out back mutation on a specific site of the framework region to obtain the variable region of the monoclonal antibody QX 006N. Finally, the humanized heavy chain variable region sequence is shown in SEQ ID NO: 7 is shown in the specification; the amino acid sequence of the humanized light chain variable region is shown as SEQ ID NO: shown in fig. 8.

The gene for the heavy chain variable region (SEQ ID NO: 7) and the gene for the light chain variable region (SEQ ID NO: 8) were obtained by PCR amplification using the gene sequence of the 362# humanized antibody as a template. The HindIII and NheI are used for double enzyme digestion of the heavy chain expression plasmid pHZDCH; HindIII and BsiWI are used for double digestion of the light chain expression plasmid pHZDCK; the PCR amplified genes were inserted into the corresponding expression plasmids using Infusion recombinase, respectively, to construct a heavy chain expression plasmid pHZDDCH-362 VH-Hu6 and a light chain expression plasmid pHZDCK-362VK-Hu 20. In the humanization modification process, the gene of the 1203# humanized antibody was numbered 362 and the protein was numbered 1203.

The results of double restriction by electrophoresis of nucleic acids are shown in FIG. 1. As can be seen from the results shown in FIG. 1, the PCR amplification results of the heavy chain variable region and the light chain variable region of the antibody and the results of double digestion of the heavy chain and light chain expression plasmids are shown, wherein the plasmid size of the heavy chain and the light chain is about 10000bp, the plasmid size of the light chain variable region is about 447bp, and the plasmid size of the heavy chain variable region is about 471 bp.

And obtaining a humanized antibody HZD1203-45 by performing humanized transformation on 1203 #. In order to reduce the ADCC effect of the antibody, the human IgG1 constant region of HZD1203-45 heavy chain expression plasmid pHZDCH-362VH-Hu6 was replaced by human IgG4 to obtain the heavy chain expression plasmid pHZDCH-362VH-Hu 6-IgG4.1.

ExpicHO-S cells were co-transfected with the correct sequence of the heavy chain expression plasmid pHZDDCH-362 VH-Hu6-IgG4.1 and the light chain expression plasmid pHZDCK-362VK-Hu 20. One day before transfection, ExpCHO-S cells were diluted to 3X 10⁶Individual cells/ml were passaged before transfection. On the day of transfection, cell density was diluted to 6X 10⁶Individual cells/ml, 125ml shake flasks with 25ml cells, waiting for transfection. The transfection and expression process is shown in FIG. 2.

Culture supernatants were harvested 4-8 days after transfection and purified in one step with ProteinA. The purified antibody was detected by SDS-PAGE and designated as QX006N (HZD1203-45-IgG4.1), and the results of detection of the antibody by protein electrophoresis are shown in FIG. 3. The protein electrophoresis was performed using denatured reducing gel, and the result of FIG. 3 shows two bands having sizes of about 50kDa and 25kDa, respectively, which are consistent with the theoretical molecular weights of the heavy chain (48.9kDa) and the light chain (23.4 kDa).

Example 2 equilibrium dissociation constant (K)_D) Measurement of (2)

BiacoreT200 was used to detect the affinity of QX006N (HZD1203-45-IgG4.1) to human IFNAR1, all at 25 ℃. Adopting commercialized Protein A chip, and performing capture methodAn appropriate amount of antibody was fixed such that Rmax was around 50RU and the capture flow rate was 10. mu.l/min. The antigen is subjected to gradient dilution, the flow rate of the instrument is switched to 30 mul/min, the antigen sequentially flows through a reference channel and a channel for fixing the antibody according to the sequence of the concentration from low to high, and the antigen flows through a buffer solution to serve as a negative control. After each binding and dissociation, the chip was regenerated with glycine of pH 1.5. Selecting a 1:1 binding model in Kinetics options by using self-contained analysis software of an instrument for fitting, and calculating a binding rate constant k of the antibody_aDissociation rate constant k_dAnd dissociation equilibrium constant K_DThe value is obtained.

In addition, QX006N (HZD1203-45-IgG4.1) was compared with the affinity of a monoclonal antibody against human IFNAR1, that is, Anifrolumab, which has entered clinical stage III, and the detection method for the known antibody was the same as that for QX006N, and the results are shown in Table 1. Wherein the Anifrolumab is obtained by constructing an expression plasmid according to a 9D4 sequence provided by WO2009100309A2 and transforming an ExpicHO-S cell.

TABLE 1 affinity of antibodies for binding to human IFNAR1

Sample name	k_a(10⁵M^-1S^-1)	k_d(10^-5S^-1)	K_D(10^-10M)
				HZD1203-45-IgG4.1	3.47	3.76	1.08
Anifrolumab	18.67	12.40	0.67

The data in the table are: each sample was tested in triplicate and the data for the mean was calculated.

Example 3QX006N and Anifrolumab neutralize human Interferon-induced HEK Blue IFN α/β cell STAT1/2 phosphorylation Activity

HEK Blue IFN alpha/beta reporter cell line is used for measuring the phosphorylation activity of intracellular signaling molecule STAT1/2 mediated by IFNAR1 of QX006N antagonistic interferon: cells in culture were plated at 4X 10 per well⁴Cells were added to 96 wells, followed by incubation at 37 ℃ and 5% CO₂Incubated under conditions overnight. To the cells were added serial dilutions of antibody in the range of 0 to 5. mu.g/ml, and 0.2ng/ml IFN α.2b. Then at 37 ℃ and 5% CO₂Culturing for 24 hr, collecting cell culture supernatant, adding 10% QUANTI-Blue^TMThe detection reagent is at 37 ℃ and 5% CO₂The reaction is carried out for 1 hour under the condition, then the OD630nm value is detected and a dose-effect curve is drawn, and then the antagonistic activity of the antibody is analyzed, and the experimental result shows that QX006N can inhibit STAT1/2 phosphorylation in interferon-induced HEK Blue IFN alpha/beta cells and inhibit IC of 1/2 phosphorylation activity in interferon-induced HEK Blue IFN alpha/beta cells₅₀5.23ng/ml, while Aniflumab inhibited the IC of 1/2 phosphorylation activity in interferon-induced HEK Blue IFN alpha/beta cells₅₀It was 4.43 ng/ml.

Example 4QX006N and Anifrolumab Activity to neutralize human Interferon to inhibit Daudi cell proliferation

Determination of the cell proliferation activity of QX006N antagonistic interferon induced by IFNAR1 using Daudi human lymphoma cell line: cells in culture were plated at 4X 10 per well⁴Cells were added to 96 wells, followed by incubation at 37 ℃ and 5% CO₂Incubated under conditions overnight. Addition of antibody concentrations ranging from 0 to 20. mu.g/ml to cellsDilute and add 0.8ng/ml IFN alpha 2 b. Then at 37 ℃ and 5% CO₂Culturing for 72 hours under the condition, collecting cell culture, detecting cell proliferation condition by adopting CellTiter-Glo, drawing a dose effect curve, further analyzing antagonistic activity of the antibody, and the experimental result shows that QX006N can inhibit interferon-induced Daudi cell proliferation and inhibit EC of interferon-induced Daudi cell proliferation activity₅₀29.9ng/ml, and the EC of Aniflumab for inhibiting interferon-induced cell proliferation activity of Daudi₅₀31.7 ng/ml.

Example 5QX006N and Anifrolumab neutralize human interferon to induce CXCL10/IP10 activity in whole blood.

Determination of the activity of QX006N in antagonizing interferon release by IFNAR1 at CXCL10/IP10 using human whole blood: whole blood was added to a 96-well plate at 100. mu.l/well and stored temporarily at 37 ℃ and 5% CO₂Under these conditions, serial dilutions of antibody ranging in concentration from 0 to 40. mu.g/ml were added to whole blood, with IFN α, 2b, 40ng/ml TNF α being added. Then at 37 ℃ and 5% CO₂Culturing for 48 hours under the condition, collecting cell culture supernatant, detecting the expression of CXCL10/IP10 in the supernatant by adopting a sandwich ELISA method, drawing a dose effect curve, and further analyzing the antagonistic activity of the antibody, wherein the experimental result shows that QX006N can inhibit interferon-induced whole blood from releasing CXCL10/IP10 and inhibit interferon-induced whole blood from releasing CXCL10/IP10 active IC₅₀698ng/ml, while Aniflumab inhibited interferon-induced whole blood release of CXCL10/IP10 Activity IC₅₀562 ng/ml.

Example 6 comparison of the Effect of different Pre-elution buffers on HCP clearance from recombinant humanized anti-IFNAR 1 monoclonal antibody (QX006N) fermentation broth

The antibody QX006N obtained in example 1 was produced using CHO cells as host cells and Dynamis as a fermentation basal medium. Culturing cells by conventional cell culture process, starting harvesting when the cell viability is lower than 80% or culturing to 18 days, performing depth filtration on the harvest by using a primary filter MD0HC10FS1 and a secondary filter MX0HC10FS1, and collecting clarified cell culture supernatant, wherein the clarified cell culture supernatant is marked as a fermentation broth intermediate. Carrying out affinity chromatography on the fermentation liquor intermediate by the following method:

using a first equilibration buffer (12mmol/L Na)₂HPO₄、8mmol/L NaH₂PO₄And 0.15mol/L NaCl) equilibrium Protein A chromatography column (Merck)

A, 0.15L) and the QX006N broth intermediate was loaded onto the equilibrated Protein a chromatography column to bind thereto and then equilibrated with a first equilibration buffer until the broth completely flowed through the column;

then, an intermediate pre-elution buffer was applied, the composition of the intermediate pre-elution buffer is shown in Table 2, and a second equilibration buffer (6mmol/L Na) was applied after elution₂HPO₄、4mmol/L NaH₂PO₄pH7.2) to balance;

then, final elution is carried out to collect samples, and the collected samples are subjected to antibody concentration and host protein (HCP) residual content determination, wherein the final elution buffer is 5mmol/L Na₂HPO₄6.5mmol/L citric acid, pH 3.6, and regenerating the column with regeneration buffer 100mmol/L NaOH, 1mol/L NaCl.

The antibody concentration was determined as follows:

1. the wavelength of the spectrophotometer was adjusted to 280nm and the second equilibration buffer was used as a control for zero calibration.

2. Diluting the sample to be tested by using a second balance buffer solution, measuring the absorbance value of the sample at 280nm (the absorbance value is ensured to be between 0.5 and 1.5), and calculating the sample concentration according to the following formula (the extinction coefficient of QX006N is 1.469).

The results obtained are shown in table 3.

HCP content determination method:

1. sample dilution: the choice of dilution factor is generally made based on the estimated value of HCP in the sample, so that the final HCP concentration falls within the range of the standard curve (typically 10ng/ml to 80 ng/ml). The dilution factor of the sample in one step is not more than 10 times, and the minimum sampling quantity is not less than 5 mul.

2. To the removal of the strip per well add Anti-CHO HRP 100. mu.l.

3. Loading: adding standard sample, and standard sample (two wells for standard sample, no wells for standard sample), 50 μ l/well, and sealing. The mixture was placed on a horizontal shaker at room temperature for 2 hours at 180rpm in the dark.

4. Washing the plate: discarding the liquid in the hole, adding the washing liquid 300 mul/hole by using a multichannel pipettor, standing for 30 seconds, then throwing off the liquid, patting on absorbent paper, and washing the plate for 4 times. After the last plate washing is finished, residual washing liquid in the holes needs to be patted dry as much as possible.

5. Color development and end reading: TMB reagent (TMB Substrate) was added at 100. mu.l/well, and the mixture was allowed to stand for 30 minutes to develop color, protected from light. After 30 minutes, 100. mu.l/well of Stop Solution (Stop Solution) was added, and the reading was taken by a microplate reader at 450nm, with 650nm as reference.

6. And selecting analysis software to analyze data, and drawing a four-parameter standard curve by taking the OD value of the standard substance as a vertical coordinate and the concentration as a horizontal coordinate. And substituting the OD value measured by the sample into the standard curve to obtain the actual measurement value of the added sample HCP.

CHO cell protein residual amount (%). The average measured value (ng/ml). times.dilution factor/undiluted sample protein content (mg/ml) amount^-4(%), the results are shown in Table 3.

TABLE 2 composition of intermediate Pre-elution buffer

Experiment of	Pre-elution buffer
		1	0.1mol/L sodium citrate, 0.1mol/L guanidine hydrochloride, 11mmol/L citric acid, pH5.8
2	12mmol/L Na₂HPO₄，8mmol/L NaH₂PO₄，0.15mol/L NaCl，0.5％Tween 80，pH7.0
		3	0.1mol/L sodium citrate, 0.5mol/L sodium chloride, 7mmol/L citric acid, pH5.8

TABLE 3QX006N concentration and HCP residual content results

Experiment of	HCP content% (w/w) of cell culture supernatant	Yield%	HCP residual amount after affinity chromatography (w/w)
				1	18.25	97.5	0.089
2	18.53	97.7	0.543
				3	18.26	97.4	0.456

The results in table 3 show that the HCP residue in the fermentation broth intermediate is greater than 15%, and the yields are greater than 95% after the treatment of the affinity purification process; for the removal of HCP, the method is adopted, the HCP residue of the sample after affinity chromatography is lower than 0.1%, the load of the subsequent purification process step for removing HCP is obviously reduced, the pH value of the sample is adjusted, and when the sample is loaded in the subsequent chromatography process step (anion exchange chromatography), the sample is kept extremely clear and can be directly injected without other treatment, so that the simplicity of the process is enhanced, and the process time is saved.

Example 7 comparison of the Effect of HCP clearance by fermentation broths of different batches of recombinant humanized anti-IFNAR 1 monoclonal antibody (QX006N)

The preparation and affinity chromatography methods of the fermentation broth intermediate were the same as in example 6, three batches of the fermentation broth intermediate were prepared, and the intermediate pre-elution and elution both used a pre-elution buffer containing guanidine hydrochloride: 0.1mol/L sodium citrate, 0.1mol/L guanidine hydrochloride and 11mmol/L citric acid, and the pH value is 5.8. The affinity process yield and HCP residual content were measured as described in example 6, and the results are shown in table 4.

TABLE 4 different batches of QX006N concentration and HCP residual content results

Batches of	HCP content% (w/w) of cell culture supernatant	Yield%	HCP residual amount after affinity chromatography (w/w)
				1	18.35	97.8	0.078
2	17.34	97.6	0.086
				3	18.12	96.7	0.075

The results in table 4 show that the HCP residues of fermentation broth intermediate QX006N in different batches are all above 15%, and the cell culture process is stable; the yield of the affinity process of the three batches of samples is more than 95 percent, and the yield meets the process requirement; the HCP residues of the samples after the affinity chromatography are all lower than 0.1 percent, and the removal of the HCP is kept stable by adopting guanidine hydrochloride to carry out pre-elution.

In conclusion, the present invention adopts the affinity purification method described above, the residual amount of HCP in the obtained monoclonal antibody is kept at a low level, the residual control amount is not higher than 0.1%, thereby reducing the pressure for removing HCP in the subsequent purification step, and the affinity purification process described in the present invention has good stability.

The foregoing is directed to preferred embodiments of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention are within the protection scope of the technical solution of the present invention.

Sequence listing

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Jiangsu Saifu Shi Biotechnology Co., Ltd.

<120> affinity purification method for reducing host cell protein content in monoclonal antibody production

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Claims

(f) The amino acid sequence of CDR-L3 is shown in SEQ ID NO: and 6.

3. The affinity purification method according to claim 2, wherein the amino acid sequence of the heavy chain of the anti-human interferon alpha receptor 1(IFNAR1) monoclonal antibody is as set forth in SEQ ID NO: 10 is shown in the figure; the amino acid sequence of the light chain is shown as SEQ ID NO: shown at 11.

4. The affinity purification method according to any one of claims 1 to 3, wherein the affinity chromatography medium is a chromatography medium in which ligands are cross-linked to agarose, polyvinyl ether, hydroxylated polyether resin, polyacrylic resin, polystyrene divinyl phenyl resin, polymethacrylic resin, polystyrene resin, hydroxyapatite or glass matrix, preferably the affinity chromatography medium is a chromatography medium in which ligands are cross-linked to polyvinyl ether;

6. The affinity purification method according to any one of claims 1 to 5, wherein the intermediate pre-elution buffer is a neutral buffer and/or an acidic buffer; preferably, the neutral buffer solution is a phosphate buffer solution, a tris buffer solution or a glycine buffer solution; the acidic buffer solution is a citric acid-disodium hydrogen phosphate buffer solution, an acetic acid-sodium acetate buffer solution or a citric acid-trisodium citrate buffer solution;

preferably, the pH of the intermediate pre-elution buffer is between 5.0 and 7.5.

7. The affinity purification method according to claim 6, wherein a pre-elution active agent is added to the intermediate pre-elution buffer, preferably the pre-elution active agent is guanidine hydrochloride, polysorbate 80 or sodium chloride, further preferably the pre-elution active agent is guanidine hydrochloride.

8. The affinity purification process according to claim 7, wherein the concentration of guanidine hydrochloride is 0.01-1M.

9. Affinity purification method according to any one of claims 1-8, wherein after the intermediate pre-elution rinse, before performing the final elution further comprises the following steps:

10. The affinity purification method according to any one of claims 1 to 9, wherein the final elution buffer is selected from one or more of a citric acid-disodium hydrogen phosphate buffer, an acetic acid buffer, a glycine-HCl buffer, and a citric acid-sodium citrate buffer, preferably a citric acid-disodium hydrogen phosphate buffer;

preferably, the pH of the citric acid-disodium hydrogen phosphate buffer is 2.0-7.0.