CN116063491A - Method for enhancing stability of aqueous antibody preparation and application thereof - Google Patents

Abstract

The invention analyzes the influence factors of the component stability and clinical safety of the aqueous antibody Deshumaumab preparation, and reduces the content of HCP (host cell protein) and the esterase activity thereof in the purification process of the recombinant antibody, and eliminates the degradation of polysorbate, thereby enhancing the component stability of the aqueous antibody preparation. The method of the invention can reduce the formation of undesired degradation products of aqueous antibody formulations and enhance the storage stability of the antibody formulations.

Description

Method for enhancing stability of aqueous antibody preparation and application thereof

Technical Field

The invention relates to the field of medicine preparation, in particular to an affinity purification process method for reducing the content of Host Cell Protein (HCP) in monoclonal antibody production.

Background

Biomedical technology is the core strength of the development of the current medical technology. Biological products typified by monoclonal antibody drugs are important drug classes for the treatment of malignant tumors, autoimmune diseases, viral infections, and the like. The purification process is critical in antibody drug production, and the fermentation product usually contains a large amount of impurities, wherein the removal of HCP is the most central. In the process of mass fermentation of genetically engineered cell lines such as chinese hamster ovary Cells (CHO), cells undergo apoptosis and lysis in different physiological cycles, releasing host cell proteins (Host cell protein, HCP). HCP refers to protein components derived from host cells, including host cell structural proteins and transforming proteins (cell secreted growth-promoting proteins). The HCP may induce the body to produce anti-HCP antibody to induce allergic reaction, and may also cause the body to produce antibody to protein medicine to affect the medicine treating effect.

We have previously found that trace amounts of HCPs can lead to degradation of polysorbate (also known as Tween) in pharmaceutical formulations. Polysorbate, such as Polysorbate 20 and 80, are surfactants commonly used in biopharmaceuticals to improve the stability of protein pharmaceutical formulations and prevent aggregation and denaturation of proteins during long-term storage and transport thereof, thereby preventing degradation of the pharmaceutical quality of the protein formulations. However, polysorbate is easily hydrolyzed under certain conditions, which causes aggregation of protein and causes degradation of drug quality. The hydrolysis mechanism of polysorbate has been widely studied by the industry, and trace amounts of HCP remaining in protein formulations in addition to acid and base induced hydrolysis are the most predominant cause of polysorbate hydrolysis. Therefore, the purification process of protein drugs puts higher demands on HCP removal. For the reasons stated above, it has become extremely important to develop a purification process that can reduce HCP levels below 1ppm under large scale and economically viable process conditions, while requiring removal of a class of HCPs that lead to polysorbate degradation.

The main purpose of antibody medicine production is to obtain monoclonal antibody molecules with high purity, and through protein purification method, the purity is improved while the recovery rate of monoclonal antibody molecules is improved. For example, the antibody purification method disclosed in patent CN105017418A includes: 1) Affinity chromatography; 2) Adjusting the pH value of the affinity chromatography eluent to 3.3-3.8, and inactivating viruses; 3) Regulating pH value to weak acidity, and performing deep filtration; 4) Anion exchange chromatography; 5) Cation exchange chromatography. The patent CN107793469a provides an affinity purification process for removing HCP content, which changes the pH of the leaching step 2, and adds at least one of sodium chloride, calcium chloride and arginine hydrochloride as an additive in the leaching step 2 to reduce the interaction among HCP, target protein and filler matrix, thereby playing a role in removing HCP.

The prior art aims at removing HCP and obtaining high-purity antibody molecules, and cannot effectively solve the contradiction between the purity and the recovery rate of the antibody molecules in industrial production. The invention analyzes the steps and specific operation methods of Protein A affinity chromatography, low ph incubation and deep filtration, anion chromatography, cation chromatography, nanofiltration and ultrafiltration of the current general antibody purification process. The removal capacity of HCP is based on Protein A affinity chromatography and anion chromatography, the removal capacity is high, the method is a main step of HCP removal, low ph incubation and deep filtration can effectively reduce esterase activity in the HCP, cation chromatography is mainly used as a further removal process of the subsequent HCP, and the removal capacity of nanofiltration and ultrafiltration on the HCP is limited. Based on the general purification process, the invention fully researches the residual biological activity of HCP and the influence on antibody preparation, adjusts control parameters by optimizing process conditions, further reduces the content of HCP, and simultaneously optimizes and selects the HCP activity capable of degrading polysorbate in antibody preparation by taking esterase activity as an index.

Disclosure of Invention

In order to solve the technical problem that the purification method taking the purity of the antibody as an index is difficult to industrialize in the prior art, the invention analyzes aiming at factors influencing the components and clinical safety of the aqueous antibody preparation, and performs method optimization and parameter selection by taking two indexes of esterase activity and antibody purity as the basis. The stability of the components of the aqueous antibody formulation is enhanced by removing the esterase activity of HCP and eliminating degradation of polysorbate during purification of the recombinant antibody. The method of the invention can reduce the formation of undesired degradation products of aqueous antibody formulations and enhance the storage stability of the antibody formulations.

Specifically:

in one aspect, the present invention provides a method for enhancing the stability of an aqueous antibody preparation, which is characterized in that in the method for purifying a target antibody molecule by using a recombinant cell fermentation broth containing the target antibody molecule as a target, the HCP content and the esterase activity thereof are reduced, and the degradation of polysorbate by the HCP content and the esterase activity thereof is eliminated.

Further, the method for enhancing the stability of an aqueous antibody preparation according to the present invention is characterized in that the HCP content of the purified target antibody molecule is not higher than 1ppm, and the esterase activity of the HCP is inactivated by low pH incubation.

Further, the method for enhancing the stability of an aqueous antibody preparation of the present invention is characterized in that the method for purifying an antibody molecule of interest comprises: affinity chromatography with intermediate elution step, low pH incubation inactivation, anion exchange chromatography, cation exchange chromatography.

Further, the method for enhancing the stability of the aqueous antibody preparation is characterized in that an intermediate washing solution containing 0.2M arginine hydrochloride is adopted in the affinity chromatography process.

Further, the method for enhancing the stability of the aqueous antibody preparation is characterized in that in the affinity chromatography method, column equilibrium liquid with pH of 6.8, equilibrium liquid after sample loading and intermediate washing liquid are adopted, and eluent with pH of 3.1 is adopted.

Further, the method for enhancing the stability of the aqueous antibody preparation is characterized in that the low pH incubation inactivation comprises adjusting the pH of the affinity chromatography eluent to 3.3-3.5 by using 1M citric acid.

Further, the method for enhancing the stability of the aqueous antibody preparation is characterized in that the anion exchange chromatography is carried out, the loading pH is 8.0+/-0.2, the Cond value is 2-5mS/cm, and the loading capacity is not more than 60mg/ml.

Further, the method for enhancing the stability of an aqueous antibody preparation according to the present invention is characterized in that, in the affinity chromatography method,

the column balancing liquid is as follows: 0.15M NaCl,0.02M phosphate buffer, pH6.8;

the equilibrium liquid after sample loading is: 0.15M NaCl,0.02M phosphate buffer, pH6.8;

the intermediate washing liquid is as follows: 0.15M NaCl,0.02M phosphate, 0.2M arginine hydrochloride buffer, pH6.8;

the eluent is as follows: 0.0175M Na2HPO 4-citrate buffer, pH3.1.

Further, the method for enhancing the stability of the aqueous antibody preparation is characterized in that the low pH incubation and inactivation are performed, and the virus and esterase inactivation is performed by incubation for 60-90min at room temperature; adjusting pH to weak acidity (pH 5.3-5.5), and filtering with deep layer filtering membrane to remove precipitate.

Further, the method for enhancing the stability of an aqueous antibody preparation according to the present invention is characterized in that, in the anion exchange chromatography method,

the chromatographic column flushing liquid is as follows: 0.02M Tris-HCl,1M NaCl buffer, pH 8.0;

the pH of the sample is 8.0+/-0.2, the Cond value is 2-5mS/cm, and the loading capacity is not more than 60mg/ml.

The equilibrium liquid after chromatographic column loading is: 0.02M Tris-HCl,0.04M NaCl buffer, pH 8.0.

In another aspect, the invention provides a method of enhancing the stability of an aqueous antibody formulation, characterized in that the antibody is desquamation.

In another aspect, the invention provides the use of a method of enhancing the stability of an aqueous antibody formulation in the preparation of an antibody medicament.

For a better understanding of the invention, some terms are first defined. Other definitions are set forth throughout the detailed description.

The term "Deshumab" is a monoclonal antibody (IgG 2) to the fully human anti-nuclear factor-kappa B receptor activator ligand (ligand of receptor activator of nuclear factor-kappa B, RANKL), an IgG2 antibody consisting of 2 light chains (kappa chains) and 2 heavy chains (gamma 2 chains), the whole molecular weight being about 147.7kDa. Each light chain has 215 amino acid residues and each heavy chain has 448 amino acid residues.

The term "host cell" encompasses plant cells and animal cells. Animal cells encompass invertebrates, non-mammalian vertebrates (e.g., birds, reptiles, and amphibians), and mammalian cells. Examples of invertebrate cells include the following insect cells: spodoptera frugiperda (Spodoptera frugiperda) (caterpillars), aedes aegypti (mosquitoes), aedes albopictus (mosquitoes), drosophila melanogaster (Drosophila melanogaster) (fruit flies), and Bombyx mori (silkworm) (see, e.g., luckow, V.A. et al, bio/Technology 6 (1988) 47-55; miller, D.W. et al, genetic Engineering, setlow, J.K. et al (eds.), volume 8, plenum Publishing (1986), pages 277-298; and Maeda, S. Et al, nature 315 (1985) 592-594).

The term "antibody" is intended to include full length antibodies and any antigen-binding fragment (i.e., antigen-binding portion) or single chain thereof. Full length antibodies are glycoproteins comprising at least two heavy (H) chains and two light (L) chains, the heavy and light chains being linked by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2 and CH3. Each light chain is composed of a light chain variable region (VL) and a light chain constant region. The light chain constant region is composed of one domain CL. VH and VL regions can also be divided into hypervariable regions called Complementarity Determining Regions (CDRs) which are separated by more conserved Framework Regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The variable regions of the heavy and light chains comprise binding domains that interact with antigens. The constant region of an antibody may mediate the binding of an immunoglobulin to host tissues or factors, including various immune system cells (e.g., effector cells) and the first component of the traditional complement system (C1 q).

The term "monoclonal antibody" or "mab" or "monoclonal antibody composition" refers to a preparation of antibody molecules of single molecular composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope.

The term "antigen-binding fragment" of an antibody (or simply an antibody portion) as used herein refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been demonstrated that the antigen binding function of an antibody can be performed by fragments of full length antibodies. Examples of binding fragments contained in the "antigen-binding portion" of an antibody include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH 1; (ii) A F (ab') 2 fragment, a bivalent fragment comprising two Fab fragments disulfide-bridged at the hinge region; (iii) an Fd fragment consisting of VH and CH 1; (iv) Fv fragments consisting of single arm VL and VH of the antibody; (v) dAb fragments consisting of VH (Ward et al., (1989) Nature 341:544-546); (vi) an isolated Complementarity Determining Region (CDR); and (vii) nanobodies, a heavy chain variable region comprising a single variable domain and two constant domains. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined by recombinant methods via a synthetic linker that makes both single protein chains, in which the VL and VH regions pair to form a monovalent molecule (known as a single chain Fc (scFv); see, e.g., bird et al, (1988) Science 242:423-426;and Huston et al, (1988) Proc.Natl. Acad. Sci. USA 85:5879-5883). These single chain antibodies are also included in the term meaning. These antibody fragments can be obtained by common techniques known to those skilled in the art, and the fragments can be functionally screened in the same manner as the whole antibody.

Antigen binding fragments of the invention include those capable of specifically binding to an antigen. Examples of antibody binding fragments include, for example, but are not limited to, fab ', F (ab') ₂ Fv fragments, single chain Fv (scFv) fragments and single domain fragments.

The Fab fragment contains the constant domain of the light chain and the first constant domain of the heavy chain (CH 1). Fab' fragments differ from Fab fragments in the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab 'fragments are generated by cleavage of disulfide bonds at the hinge cysteines of the F (ab') 2 pepsin digestion products. Additional chemical coupling of antibody fragments is known to those of ordinary skill in the art. Fab and F (ab') 2 fragments lack the fragment crystallizable (Fc) region of intact antibodies, clear more rapidly from the circulation of animals, and may have less non-specific tissue binding than intact antibodies (see, e.g., wahl et al, 1983, j. Nucleic. Med. 24:316).

As generally understood in the art, an "Fc" region is a fragment of an antibody that does not comprise an antigen-specific binding region, a crystallizable constant region. In IgG, igA and IgD antibody isotypes, the Fc region consists of two identical protein fragments, the second and third constant domains (CH 2 and CH3 domains, respectively) derived from the two heavy chains of the antibody. IgM and IgE Fc regions contain three heavy chain constant domains (CH 2, CH3 and CH4 domains) in each polypeptide chain.

The term "affinity chromatography" refers to a chromatography method using "affinity chromatography material". In affinity chromatography, polypeptides are isolated based on their biological activity or chemical structure (which depends on the formation of electrostatic interactions, hydrophobic bonds and/or hydrogen bond formation with chromatographic functionalities). To recover specifically bound polypeptides from affinity chromatography materials, competing ligands are added or chromatographic conditions, such as pH, polarity or ionic strength of the buffer, are altered.

An "affinity chromatography material" is a chromatography material comprising complex chromatography functionalities that combine different individual chromatography functionalities to bind a specific type of polypeptide. The chromatographic material specifically binds to a particular type of polypeptide according to the specificity of its chromatographic functional group. Exemplary "affinity chromatography materials" are "metal chelating chromatography materials", such as Ni (II) -NTA or Cu (II) -NTA containing materials, for binding to fusion polypeptides containing hexahistidine tags, or polypeptides having a number of surface exposed histidine, cysteine and/or tryptophan residues, or "antibody binding chromatography materials", such as protein a, or "enzyme binding chromatography materials", such as chromatography materials comprising an enzyme substrate analog, enzyme cofactor or enzyme inhibitor as a chromatography functional group, or "lectin binding chromatography materials", such as chromatography materials comprising polysaccharides, cell surface receptors, glycoproteins or intact cells as a chromatography functional group.

The term "anion exchange chromatography" refers to chromatography in which the solid phase is positively charged (e.g., has one or more positively charged ligands attached thereto, such as quaternary ammonium groups). Commercially available anion exchange matrices include DEAE cellulose, QAE SEPHADEX, FAST Q SEPHAROSETM Capto Q and Capto Q expressers (GE Healthcare), unosphere and NuviaQ (BioRad), gigaCapQ (Tosoh), mustang Q XT (Pall), fractogel Q and Eshmuno Q (Merck Millipore) and anion exchange membrane adsorbers such as SartoBind Q (Sartorius), and monolith adsorbers such as QA monolith (Bia Separations).

The term "cation exchange chromatography" refers to a process in which a packing medium is negatively charged and has free cations that can exchange with cations in an aqueous solution that passes through or across a solid phase. The negative charge may be provided by attaching more than one charged ligand to the solid phase (e.g., by covalent attachment). Alternatively or additionally, the charge may be an inherent property of the solid phase (e.g., silica has an overall negative charge). The cation exchange matrix (e.g., CEX resin) may be placed or packed into a chromatographic column for purifying the protein. CEX is an effective step for removing High Molecular Weight (HMW) proteins as well as host cell proteins, DNA and residual protein A (Zeid et al (2008), biotechnology and Bioengineering, 971-976; YIgzaw, Y. Et al (2009), current Pharmaceutical Biotechnology, 421-426; gagnon, P., purification tools for monoclonal antibodies (means for purifying monoclonal antibodies), 1996:Validated Biosystems, inc., stein, A. And Kiesewter, A. (2007), journal ofChromatography B848, 151-158; staby, A. Et al (2006), journal of Chromatography 1118, 168-179). Typically, CEX operates in a bind-and-elute mode (BEM), wherein proteins bind to a CEX matrix (e.g., CEX resin) under low conductivity conditions at a pH below the pI of the target molecule. Elution of the binding protein is then typically achieved by increasing the conductivity and/or inducing a pH change. This can be done by a linear gradient or by stepwise elution to predetermined conditions. Impurities (particularly HMW species) are generally more tightly bound than mAb products and impurities can be separated from the main desired fraction by adjusting the elution conditions and pool collection criteria (Yigzaw, y. Et al (2009), supra; gagnon, P et al (1996), supra; pabst, t.m. et al (2009), journal of Chromatography 1216, 7950-7956).

The term "binding affinity" is used herein as a measure of the strength of non-covalent interactions between two molecules (e.g., an antibody or fragment thereof, and an antigen). The term "binding affinity" is used to describe monovalent interactions (intrinsic activity). The binding affinity between two molecules (e.g., an antibody or fragment thereof, and an antigen) via monovalent interactions can be quantified by determining the dissociation constant (KD). KD can then be determined by measurement of complex formation and dissociation kinetics, for example by SPR methods. The rate constants corresponding to the association and dissociation of the monovalent complex are referred to as association rate constant ka (or kon) and dissociation rate constant kd (or koff), respectively. KD is related to ka and KD by the equation kd=kd/ka. According to the definition above, binding affinities associated with different molecular interactions, e.g. comparison of binding affinities of different antibodies for a given antigen, can be compared by comparing KD values of the respective antibody/antigen complexes. Similarly, the specificity of an interaction can be assessed by determining and comparing the KD value for the interaction of interest (e.g., a specific interaction between an antibody and an antigen) to the KD value for the interaction of no interest. The value of the dissociation constant can be directly determined by well known methods, for example, standard assays for evaluating the binding capacity of a ligand (e.g., an antibody) to a target are known in the art and include, for example, ELISA, western blot, RIA, and flow cytometry analysis. The binding kinetics and binding affinity of the antibodies can also be assessed by standard assays known in the art, such as SPR. A competitive binding assay may be performed in which binding of an antibody to a target is compared to binding of an additional ligand (e.g., additional antibody) of the target to the target.

The invention aims to solve the problems of higher HCP content and stronger esterase activity in antibody production, and discloses a method for obviously reducing the HCP content in CHO in antibody purification production, and eliminating the esterase activity in micro HCP, which can be widely applied to an antibody purification process, and the used materials are low in cost and easy to process amplification. In the invention, the HCP content is effectively reduced mainly through the pre-elution in the middle of the antibody affinity chromatography, the esterase activity is reduced through low ph incubation, HCP is adsorbed by deep filtration under the acidic condition, HCP is further adsorbed through low-load and low-conductivity anion chromatography, and the like, thereby meeting the requirements of large-scale high-quality purification preparation of antibody medicaments, controlling the HCP content to be below 1ppm, and ensuring the safety, stability and effectiveness of the clinical use of the antibody medicaments.

In order to achieve the purpose of the invention, the following measures are mainly adopted:

1. affinity chromatography. The fermentation broth is subjected to affinity chromatography, and affinity media include, but are not limited to, chromatography media in which ligands capable of specifically binding to antibodies, such as ProteinA, proteinG, protein L, etc., are cross-linked to a matrix, such as agarose, hydroxylated polyether resin, polyacrylic resin, polystyrene diphenyl resin, polymethacrylic resin, polystyrene resin, hydroxyapatite, glass, etc., preferably the matrix is agarose. Preferred affinity fillers are Cytiva's MabSelect SuRe, mabSelect SuRe LX and Prism A, merck's EshmunoA, nanotechnology UniMabProteinA, and the like. Preferred affinity mediator ligands are Protein A or ligands that are structurally optimized on the basis of Protein A. The chromatography steps are as follows:

1.1 loading: the loading buffer comprises, but is not limited to, phosphate buffer, tris-HCl buffer and the like, preferably phosphate buffer or Tris-hydrochloric acid buffer, more preferably NaCl or Na2SO4 can be added in the buffer to reduce nonspecific adsorption between non-antibody protein and ProteinA filler, and the salt concentration of the phosphate buffer is between 5mM and 0.15M, preferably between 10mM and 50mM, more preferably 20mM; the pH is between 5.5 and 8.0; preferably between 6.5 and 7.5, more preferably 7.0, naCl or Na ₂ SO ₄ The concentration of (C) is preferably 0-250mM, more preferably 150mM

1.2 intermediate washes: intermediate wash buffers include, but are not limited to, one or a combination of several of the following buffers: 1) Neutral buffer systems, for example: phosphates, tris, glycine, and the like; 2) Acidic buffer system: citric acid-disodium hydrogen phosphate, acetic acid-sodium acetate, citric acid-trisodium citrate, and the like. The intermediate wash buffer is supplemented with an active agent including, but not limited to, arginine, polysorbate, urea, isopropyl alcohol, propylene glycol, ethylene glycol, tetramethylammonium chloride, and the like, preferably arginine hydrochloride, at a concentration of between 0.1 and 0.5M, preferably between 0.1 and 0.3M, and more preferably 0.2M. The pH of the intermediate pre-wash buffer is between 5.0 and 8.0, preferably 7.2.

1.3 final elution: elution buffers include, but are not limited to, one or a combination of several of the following buffers: citric acid, gly-hydrochloric acid, acetic acid buffer, etc., and the pH ranges from 2.0 to 7.0, preferably pH is 3.0 to 4.0. The concentration of the elution buffer is in the range of 5-100mM, preferably 20-50mM. The affinity medium may be regenerated after elution, and 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.

2. Incubation at low pH, and the method inactivates the virus while reducing esterase activity.

2.1 reducing the pH of the intermediate sample to 3.0-4.0; wherein the pH value of the low pH incubation is 3.0-4.0, and acidic solutions such as citric acid, acetic acid and hydrochloric acid are used for regulating the pH. The low pH incubation adjusts the pH to 3.1-3.8, preferably 3.2-3.6, more preferably 3.3+ -0.1, 3.4+ -0.1, 3.5+ -0.1, 3.6+ -0.1.

2.2 incubation period at low pH, said incubation at pH is carried out for a period of 0.5-2 hours, preferably 1-1.5 hours. And after the inactivation is finished, the pH of the sample is adjusted to 5-6 by using an alkaline solution, wherein the alkaline solution comprises a Tris solution, a sodium acetate solution and sodium citrate.

2.3 depth filtration: after incubation at low pH, the sample is subjected to deep filtration, wherein the deep membrane comprises merck X0HC, and the deep filtration membrane is washed with water for injection before filtration, and the washing volume is not less than 100L/m2. The filtration rate is not more than 10L/min, the pressure is not more than 2bar, the pH of the sample is adjusted to 8.0 after filtration, and the conductivity is not more than 5ms/cm.

3. Anion exchange chromatography

The retention time of the filler medium is Q Sepharose Fast Flow which is more than or equal to 4min. Washing the chromatographic column with a regeneration buffer solution for 1-2 times of column volume (0.02M Tris-HCl buffer solution+1M sodium chloride, pH 8.0), balancing the chromatographic column with a balancing buffer solution for 5-8 times of column volume (0.02M Tris-HCl,0.04M NaCl buffer solution, pH 8.0), loading (pH 8.0+ -0.2, cond value of 2-5mS/cm, loading no more than 60 mg/ml), adsorbing HCP by packing during loading, collecting a flow-through sample, and balancing the column volume for 2-4 times (0.02M Tris-HCl,0.04M NaCl buffer solution, pH 8.0). Another preferred example is a filler loading of no more than 60g/L.

4. Cation exchange chromatography

The principle is based on the mutual attraction between oppositely charged molecules, the packing type used in the cation exchange chromatography of the product is SP Sepharose Fast Flow (GE), and the functional groups are as follows: o-

CH2CHOHCH2OCH2CH2CH2SO3-, and the charge carried on the surface of the protein depends on the pI and the environmental pH, the isoelectric point of the product is 8.0, after anion exchange chromatography, the pH value of the sample is adjusted to 5.2-5.4 by using 1M citric acid solution, the pH value is lower than the pI, the target protein molecule is positively charged, and the target protein can be combined with negatively charged groups on cationic chromatography fillers during loading, SO that the target protein is separated from other HCPs which are not adsorbed by the fillers or can be separated in the subsequent leaching step.

Cationic chromatography (SP FastFlow), chromatography retention time 5min. Washing the chromatographic packing with 0.5M sodium hydroxide solution (contact time not less than 30 min), washing the chromatographic column 1-3 times the column volume (0.051M Na2HPO 4-citric acid, 0.5M NaCl, pH 5.1), balancing the chromatographic column 2-4 times the column volume (0.051M Na2HPO 4-citric acid, pH 5.3), loading (collecting sample after anion chromatography elution, adjusting pH to 5.2-5.4 with 1M citric acid solution, balancing 2-4 times the column volume (0.051M Na2HPO 4-citric acid, 0.5M NaCl, pH 5.1), eluting target protein with 5-7 times the column volume of 22% eluent 1 (0.051M Na2HPO 4-citric acid, pH 5.1), 78% eluent 2 (0.051M Na2HPO 4-citric acid, pH 5.3), starting collecting target component when the UV absorption at the A280 is increased to 0.1, and ending the UV absorption at the A280 decreased to 0.3.

Compared with the prior art, the technical scheme of the invention has the following advantages:

1. different from the traditional conception of condition optimization by taking the purity of the target antibody as an index, the invention analyzes the factors influencing the components and clinical safety of the aqueous antibody preparation, and performs method optimization and parameter selection by taking the two indexes of HCP content, esterase activity and antibody purity as the basis, and the optimized antibody preparation method can give consideration to the yield.

2. The invention provides a technical scheme capable of improving the stability of an antibody preparation based on the discovery that polysorbate is commonly contained in the antibody preparation, polysorbate degradation products are found in the antibody preparation stored for a long time and polysorbate degradation activity exists in a trace amount of HCPs, and the combination of a general antibody purification method in the prior art.

3. The antibody preparation obtained by the method for improving the antibody stable preparation has high batch-to-batch stability, eliminates the esterase activity of HCP, and further reduces the content of HCP to below 1 ppm.

Drawings

Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The drawings are only for purposes of illustrating the preferred embodiments and are not to be construed as limiting the invention. Also, like reference numerals are used to designate like parts throughout the figures. In the drawings:

fig. 1: HCP content higher than 10ppm, crude liquid obtained by conventional purification process, and detection result of polysorbate in Deshumab without esterase inactivation

Fig. 2: HCP content is lower than 1ppm, the stock solution is purified after optimization, and the detection result of polysorbate in Deshumab after esterase inactivation

Detailed Description

The technical scheme of the invention will be further described with reference to the attached drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be embodied in various forms and should not be 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 disclosure to those skilled in the art.

The implementation takes the conventional process of the Deshu monoclonal antibody in three batches and the optimization process of the patent in three batches as implementation cases. Deshumab is a monoclonal antibody (IgG 2) to the fully human antinuclear factor-kappa B receptor activator ligand (ligand of receptor activator of nuclear factor-kappa B, RANKL), an IgG2 antibody consisting of 2 light chains (kappa chains) and 2 heavy chains (gamma 2 chains), and has a complete molecular weight of about 147.7kDa. Each light chain has 215 amino acid residues and each heavy chain has 448 amino acid residues.

Cell seeds are subjected to resuscitating and shake flask amplification, WAVE amplification, seed tank amplification and production tank cell culture, and fermentation supernatant is obtained after centrifugation and depth filtration and is used as a starting sample of a purification process.

The reagents used in the present invention are shown in Table 1 below.

TABLE 1 reagents for production

Example 1 Deshu antigen solution obtained by conventional Process before optimization

And (3) taking three batches of Deshu monoclonal antibody fermentation supernatant, and purifying the obtained supernatant by using a conventional purification process including steps of affinity chromatography, anion exchange chromatography, cation exchange chromatography, virus removal filtration, ultrafiltration concentration, displacement, concentration filtration and the like to obtain a stock solution consistent with the requirements of a product prescription. The HCP content of each batch of stock solution was measured and is shown in table 2.

TABLE 2 HCP content in stock solution (ppm)

The HCP content in the stock solutions of the three batches obtained by the process is higher (more than or equal to 10 ppm), and the HCP is not inactivated by esterase.

And measuring the content of polysorbate in the Deshu monoclonal antibody sample in the batch 1 by a high performance liquid chromatograph, wherein the obtained map is shown in figure 1.

Example 2 Deshu antigen solution obtained by the optimized post-purification Process (the protection Process of this patent)

1 affinity chromatography

The model of the filler used for affinity chromatography of the product is MabSelectSuRe LX, and the retention time is more than or equal to 5min. The column was equilibrated with 2-4 column volumes of column equilibration solution (0.15M NaCl solution, 0.02M phosphate buffer, pH 6.8). And loading the Deshu monoclonal antibody fermentation liquor with the loading amount of 30-55g/L. After loading, column equilibration was carried out with 2-4 column volumes of column equilibration solution (0.15M NaCl solution, 0.02M phosphate buffer, pH 6.8). Intermediate washing with 4-6 times column volume of equilibration solution (0.15M NaCl,0.02M phosphate, 0.2M arginine hydrochloride buffer, pH 6.8), and adding elution buffer (0.0175M Na) ₂ HPO ₄ -citrate buffer, ph 3.1) eluting the protein of interest.

The target component starts to collect when the UV absorption at UV280 rises to 0.1AU and ends when the UV absorption at UV280 falls to 0.1 AU. The HCP content of each batch in the affinity chromatography eluate is shown in table 3.

TABLE 3 HCP content (ppm) in affinity chromatography eluate

2.2 Low pH incubation inactivation and depth filtration

Adjusting the pH value of the affinity chromatography eluent to 3.3-3.5 by using 1M citric acid, and carrying out virus inactivation after incubation for 60-90min at room temperature. The pH was adjusted to 5.3-5.5 using 2M Tris-HCl buffer (pH 9.5) for neutralization. After neutralization, the precipitate was removed by filtration through a MX0HC10FS1 deep filtration membrane pack and protein was retained in the filter by water for injection top flushing. The inlet pressure of filtration is controlled to be less than or equal to 2bar, and the filtered sample is filtered in a liquid storage bag by a 0.22 mu m filter. The HCP content of each batch after low pH incubation inactivation is shown in table 4.

TABLE 4 HCP content (ppm) after low pH incubation inactivation

	Lot number 4	Lot number 5	Lot number 6
				HCP	202	117	163

2.3 anion exchange chromatography

The packing used for anion exchange chromatography is model Q Sepharose Fast Flow (GE Healthcare), and the functional groups are: -O-CH2CHOHCH2OCH2CHOHCH2N+ (CH 3) 3, and the retention time of the anion exchange chromatography process is more than or equal to 4min. Washing the chromatographic column with 1-2 times of washing liquid (0.02M Tris-HCl buffer solution, 1M NaCl solution, pH 8.0), balancing the chromatographic column with 5-8 times of balancing liquid (0.02M Tris-HCl buffer solution, 0.04M NaCl solution, pH 8.0), and loading (pH 8.0+ -0.2, cond value of 2-5mS/cm, and load of no more than 60 mg/ml). After loading, the column was equilibrated again with 2-4 column volumes of column equilibration solution (0.02M Tris-HCl buffer, 0.04M NaCl solution, pH 8.0). The target fraction was collected starting when the UV absorption at A280 increased to 0.15AU and ending when the UV absorption at A280 decreased to 0.15 AU. The HCP content of each batch after anion exchange chromatography is shown in table 5.

TABLE 5 HCP content (ppm) after anion exchange chromatography

2.4 cation exchange chromatography

The cationic chromatography process was retained for 5min. 1-3 times of column volume of flushing liquid (0.051M Na) ₂ HPO ₄ Citric acid buffer, 0.5M NaCl solution, pH 5.1) washing the column, and using 2-4 column volumes of column equilibrium solution (0.051M Na ₂ HPO ₄ -citrate buffer, ph 5.3), equilibrium chromatography column. And (3) adjusting the pH value of the sample collected after anion chromatography elution to 5.2-5.4 by using a 1M citric acid solution, and loading the sample. Adopts 2-4 times of column volume of balance liquid (0.051M Na) ₂ HPO ₄ Citrate buffer, 0.5M NaCl solution, pH 5.1) was equilibrated after loading. The eluent (22% eluent 1 (0.051M Na) ₂ HPO ₄ Citric acid, 0.5M NaCl, pH 5.1), 78% eluent 2 (0.051M Na ₂ HPO ₄ -citric acid, ph 5.3)), eluting the protein of interest. The target fraction was collected starting when the UV absorption at A280 increased to 0.1AU and ending when the UV absorption at A280 decreased to 0.3 AU. The HCP content of each batch after cation exchange chromatography is shown in table 6.

TABLE 6 HCP content (ppm) after cation exchange chromatography

2.5 virus removal filtration

A prefilter membrane package (Merck Co., model MSPV10FS1, pore size 0.1 μm, membrane area 1.1m was used ² ) And nanofiltration membrane package (Millipore Co., model VPMG201NB1, membrane area 0.51 m) ² ) Filtration is performed with nanofiltration membrane loading not exceeding 2.72kg/m2. Pile filtering middle of series connection of prefilter film package and nanofiltration film packageAfter the product is produced, the membrane pile and the pipeline are pushed up by water for injection, and the pressure before the membrane is kept to be not more than 2bar during filtration.

2.6 Ultrafiltration concentration, displacement and concentration (including Polysorbate 20 addition)

The ultrafiltration membrane is made from Milpore company

2Maxi Cassette(P2B030A25)2.5 m ² (molecular weight cut-off 30 kD). The flow rate of the liquid is regulated to be less than or equal to 240LMH, TMP is controlled to be between 0.8 and 1.2bar, and the collected liquid after virus removal and filtration is concentrated. Concentrating the sample protein to 20-40 g/L. And after ultrafiltration and concentration, replacement is carried out, the flow rate of the feed liquor is regulated to be less than or equal to 240LMH, TMP is controlled to be between 0.8 and 1.2bar, and the volume of ultrafiltration exchange liquor is more than or equal to 7 times. After the displacement is finished, the sample in the ultrafiltration system is further concentrated and recovered, and the membrane package is washed by a displacement buffer solution, and the concentration of the recovered sample is controlled to be more than 70g/L.

Diluting the concentrated solution after ultrafiltration and replacement, and controlling the protein concentration of the concentrated solution after ultrafiltration and replacement to be 66.5-73.5g/L by using the concentrated solution of polysorbate 20 and a replacement buffer solution.

2.7 filtering to obtain antibody liquid

Adding 4% polysorbate 20 mother liquor into the concentrated solution after ultrafiltration replacement to ensure that the ultrafiltration sample contains 0.01% polysorbate 20 (w/v), diluting the sample to control the concentration to 66.5-73.5g/L, and filtering the sample after concentration determination to obtain an antibody stock solution by 0.22 mu m. HCP content in each batch of stock solution after purification of the fermentation supernatant of the Deshumab by the optimized process is shown in table 7.

TABLE 7 HCP content in stock solution (ppm)

The HCP content of the stock solution obtained by the purification process after optimization is lower than 1ppm, and the HCP is inactivated by esterase, so that the HCP content is obviously reduced compared with the process before optimization. The final product of batch 4 was subjected to polysorbate content determination by high performance liquid chromatography, and the obtained profile is shown in fig. 2.

The present invention is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present invention are intended to be included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims (12)

1. A method for enhancing the stability of an aqueous antibody preparation, characterized in that in the method for purifying a target antibody molecule by using a recombinant cell fermentation broth containing the target antibody molecule as a target, the esterase activity in a Host Cell Protein (HCP) is reduced and the degradation of polysorbate is eliminated.

2. The method of claim 1, wherein the HCP is present in the purified antibody molecule of interest in an amount of no more than 1ppm and wherein the esterase activity of the HCP is inactivated by low pH incubation.

3. The method of claim 1, wherein the method of purifying an antibody molecule of interest comprises: affinity chromatography with intermediate elution step, low pH incubation inactivation, anion exchange chromatography, cation exchange chromatography.

4. The method of claim 3, wherein an intermediate wash containing 0.2M arginine hydrochloride is used in the affinity chromatography process.

5. The method of claim 4, wherein the affinity chromatography method comprises a column equilibration solution at pH6.8, a post-loading equilibration solution and an intermediate wash solution, and wherein an eluent at pH3.1 is used.

6. A method of enhancing the stability of an aqueous antibody formulation according to claim 3, wherein the low pH incubation inactivation comprises adjusting the pH of the affinity chromatography eluate to 3.3-3.5 using 1M citric acid.

7. A method of enhancing the stability of an aqueous antibody formulation according to claim 3, wherein the anion exchange chromatography is carried out at a pH of 8.0±0.2 and a cond value of 2 to 5mS/cm with a loading of not more than 60mg/ml.

8. The method of claim 5, wherein the affinity chromatography method,

the column balancing liquid is as follows: 0.15M NaCl,0.02M phosphate buffer, pH6.8;

the equilibrium liquid after sample loading is: 0.15M NaCl,0.02M phosphate buffer, pH6.8;

the intermediate washing liquid is as follows: 0.15M NaCl,0.02M phosphate, 0.2M arginine hydrochloride buffer, pH6.8;

the eluent is as follows: 0.0175M Na ₂ HPO ₄ Citrate buffer, ph3.1.

9. The method of enhancing the stability of an aqueous antibody formulation of claim 6, wherein the low pH incubation for inactivation is performed at room temperature for 60-90min for virus and esterase inactivation; adjusting pH to weak acidity (pH 5.3-5.5), and filtering with deep layer filtering membrane to remove precipitate.

10. The method of claim 7, wherein in the anion exchange chromatography method,

the chromatographic column flushing liquid is as follows: 0.02M Tris-HCl,1M NaCl buffer, pH 8.0;

the pH of the sample is 8.0+/-0.2, the Cond value is 2-5mS/cm, and the loading capacity is not more than 60mg/ml.

The equilibrium liquid after chromatographic column loading is: 0.02M Tris-HCl,0.04M NaCl buffer, pH 8.0.

11. The method of enhancing the stability of an aqueous antibody formulation of any one of claims 1-10, wherein the antibody is desipramab.

12. Use of a method of enhancing the stability of an aqueous antibody formulation according to any one of claims 1-10 in the manufacture of an antibody medicament.

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