CN112675126A

CN112675126A - anti-CD 20 antibody preparation and application thereof

Info

Publication number: CN112675126A
Application number: CN201910991541.8A
Authority: CN
Inventors: 陈燕宇; 吴用; 周远清; 秦超; 萧翠珍; 吴志好; 黄丹丹; 刘育杰; 王盛武; 刘翠华; 李胜峰
Original assignee: Bio Thera Solutions Ltd
Current assignee: Bio Thera Solutions Ltd
Priority date: 2019-10-18
Filing date: 2019-10-18
Publication date: 2021-04-20

Abstract

The invention provides an anti-CD 20 antibody preparation and application thereof, belonging to the technical field of biological antibodies, wherein the antibody preparation comprises the following components in concentration: 15-80 mg/ml anti-CD 20 antibody, 10-30 mM buffer, 80-240 mM stabilizer, 0.1-0.4 mg/ml surfactant, and the pH value of the liquid preparation is 5.5-6.2. The antibody preparation of the invention has good stability.

Description

anti-CD 20 antibody preparation and application thereof

Technical Field

The invention belongs to the technical field of biological antibodies, and particularly relates to an anti-CD 20 antibody preparation and application thereof.

Background

The CD20 molecule is a hydrophobic transmembrane protein with a molecular weight of about 35kD, and is located on pre-B lymphocytes and mature lymphocytes. CD20 can be found on the surface of more than 90% of B cells derived from peripheral blood or lymphoid organs and is expressed during early pre-B cell development and remains until plasma cell differentiation. CD20 is present in both normal B cells and malignant cells, and CD20 is particularly expressed in more than 90% of B cell non-hodgkin's lymphomas.

Although the actual role of CD20 in promoting B cell proliferation and differentiation is uncertain, CD20 provides an important target for antibody-mediated therapy to control B cells involved in cancer and autoimmune diseases.

anti-CD 20 antibodies are larger and more complex than traditional organic and inorganic drugs. To maintain the biological activity of an antibody, the formulation needs to maintain the overall conformational integrity of the amino acid core sequence of the protein while preventing degradation of the various functional groups of the antibody.

Disclosure of Invention

Based on this, the invention provides anti-CD 20 antibody formulations and uses thereof.

In one aspect, the invention provides a liquid formulation comprising an anti-CD 20 antibody, comprising the following components in the concentrations: 10-120 mg/ml anti-CD 20 antibody, a buffering agent, a stabilizing agent, a surfactant and a liquid preparation, wherein the pH value of the liquid preparation is 5-7.

In some embodiments, the solvent of the antibody formulation is water. In some embodiments, the solvent of the antibody formulation is sterile water for injection.

In some embodiments, the liquid formulation comprises 10-120 mg/ml anti-CD 20 antibody, 10-30 mM buffer, 58-292 mM stabilizer, 0.1-0.5 mg/ml surfactant, and the pH of the liquid formulation is 5.5-6.5.

In some embodiments, the liquid formulation comprises 15-80 mg/ml anti-CD 20 antibody, 10-30 mM buffer, 80-240 mM stabilizer, 0.1-0.4 mg/ml surfactant, and the pH of the liquid formulation is 5.5-6.2.

In some embodiments, the buffer is selected from the group consisting of a succinic buffer, a citric buffer, a phosphoric buffer, a histidine buffer, and an acetic buffer.

In some embodiments, the stabilizing agent is selected from the group consisting of sucrose, trehalose, sorbitol, mannitol, and methionine; the surfactant is selected from polysorbate-80 and polysorbate-20.

In some embodiments, the succinic buffer comprises succinic acid and sodium succinate, the citric acid buffer comprises citric acid and sodium citrate, the histidine buffer comprises L-histidine and L-histidine hydrochloride, and the acetic acid buffer comprises acetic acid and sodium acetate.

In some embodiments, the concentration of the anti-CD 20 antibody is about 15mg/ml, 40mg/ml, 60mg/ml, 80mg/ml (80mg/ml is 8%), or a range between any two values (including endpoints); the concentration of the buffer is about 10mM, 15mM, 26mM, 30mM, or a range between any two values (including endpoint values); the concentration of the stabilizing agent is about 80mM, 140mM, 190mM, 240mM, or a range between any two values (including endpoint values); the surfactant concentration is about 0.1mg/ml, 0.3mg/ml, 0.4mg/ml (0.04% for 0.4 mg/ml), or a range between any two values (inclusive); the pH is about 5.5, 5.6, 5.7, 6.0, 6.2, or a range between any two values (inclusive).

In some embodiments, the anti-CD 20 antibody is selected from a monoclonal antibody and a fragment that binds to CD 20.

In some embodiments, the heavy chain of the anti-CD 20 antibody comprises the amino acid sequence set forth in SEQ ID No. 1 or an amino acid sequence having at least 90% identity to said SEQ ID No. 1 and the light chain of the anti-CD 20 antibody comprises the amino acid sequence set forth in SEQ ID No. 2 or an amino acid sequence having at least 90% identity to said SEQ ID No. 2.

In some embodiments, the liquid formulation comprises 20-50 mg/ml of an anti-CD 20 antibody; the concentration of the anti-CD 20 antibody is about 20mg/ml, 30mg/ml, 40mg/ml, 50mg/ml, or a range between any two values (inclusive).

In some embodiments, the liquid formulation comprises 18-22 mM histidine buffer; the concentration of histidine buffer is about 18mM, 19mM, 20mM, 22mM, or a range between any two values (inclusive).

In some embodiments, the molar concentration ratio of L-histidine to L-histidine hydrochloride in the histidine buffer is 1-2: 2.

In some embodiments, the liquid formulation comprises 158-225 mM trehalose; the trehalose concentration is about 158mM, 158.6mM, 170mM, 220mM, 224.6mM, 225mM (224.6mM is 7.68%), or a range between any two values, inclusive.

In some embodiments, the liquid formulation comprises 0.18-0.22 mg/ml polysorbate-80; polysorbate-80 is present at a concentration of about 0.18mg/ml, 0.19mg/ml, 0.2mg/ml, 0.22mg/ml (0.022% for 0.22 mg/ml), or a range between any two values (inclusive).

In some embodiments, the liquid formulation has a pH of 5.7 to 5.9; the pH is about 5.7, 5.8, 5.9, or a range between any two values (inclusive).

In some embodiments, the liquid formulation comprises the following concentrations of components: about 20mg/ml anti-CD 20 antibody, about 20mM histidine buffer, about 158.6mM trehalose (i.e., 54mg/ml trehalose), about 0.2mg/ml polysorbate-80, and a pH of the liquid formulation of about 5.8; the mass ratio of L-histidine to L-histidine hydrochloride in histidine buffer was 2:3 based on pH 5.8.

In some embodiments, the liquid formulation comprises the following concentrations of components: about 20mg/ml anti-CD 20 antibody, about 20mM histidine buffer, about 224.6mM trehalose (i.e., 76.8mg/ml trehalose), about 0.2mg/ml polysorbate-80, and a liquid formulation having a pH of about 5.8; the mass ratio of L-histidine to L-histidine hydrochloride in histidine buffer was 2:3 based on pH 5.8.

In some embodiments, the liquid formulation comprises the following concentrations of components: about 50mg/ml anti-CD 20 antibody, about 20mM histidine buffer, about 224.6mM trehalose (i.e., 76.8mg/ml trehalose), about 0.2mg/ml polysorbate-80, and a liquid formulation pH of 5.8; the mass ratio of L-histidine to L-histidine hydrochloride in histidine buffer was 2:3 based on pH 5.8.

In some embodiments, the liquid formulation is a liquid formulation formulated according to formula 1, formula 2, formula 3, or formula 4.

In some embodiments, the liquid formulation may be for injection, such as intravenous or subcutaneous injection.

In some embodiments, the liquid formulation formulated according to formula 1 is an intravenous formulation.

In some embodiments, the liquid formulation formulated according to prescription 2, 3 or 4 is a subcutaneous injection formulation.

In some embodiments, the present invention also provides an intravenous formulation comprising the above-described liquid formulation and a diluent solvent (e.g., water (e.g., sterile water for injection), isotonic solution (e.g., 0.9% NaCl solution for injection), etc.). In some embodiments, the ratio of the liquid formulation to the diluent solvent is 1:10 to 50, 1:20 to 40, or 1: 25-35.

A method of treating a disease involving cellular expression of CD20 in a mammal, comprising administering to a mammal in need thereof an effective amount of the liquid formulation described above.

In some embodiments, the mammal is a human.

In some embodiments, the mode of administration to the mammal is injection, such as intravenous or subcutaneous injection.

In some embodiments, when the mammal is administered by injection, the liquid formulation comprises the following concentrations of the components: about 20mg/ml anti-CD 20 antibody, about 20mM histidine buffer, about 158.6mM trehalose, about 0.2mg/ml polysorbate-80, and a pH of the liquid formulation of about 5.8.

In some embodiments, when the mammal is administered by injection, the liquid formulation comprises the following concentrations of the components: about 20mg/ml anti-CD 20 antibody, about 20mM histidine buffer, about 224.6mM trehalose, 0.2mg/ml polysorbate-80, and a pH of the liquid formulation of about 5.8.

In some embodiments, when the mammal is administered by injection, the liquid formulation comprises the following concentrations of the components: about 50mg/ml anti-CD 20 antibody, about 20mM histidine buffer, about 224.6mM trehalose, about 0.2mg/ml polysorbate-80, and a pH of the liquid formulation of about 5.8.

In some embodiments, the disease involving expression of CD20 by cells is selected from the group consisting of a neoplastic disease, an immune disease.

In some embodiments, the anti-CD 20 antibody is monoclonal antibody 1.

In some embodiments, the monoclonal antibody 1 is expressed by hamster ovary cells CHO.

In some embodiments, the monoclonal antibody 1 is expressed by an edited CHO host cell, which is deposited in the chinese culture collection center (CCTCC) with the deposit number CCTCC NO: c2017127, deposit date 2017.8.10, deposit address: china, wuhan university. The classification is named as: chinese hamster ovary cells CHO-BAT-KF fut8 (-/-).

In some embodiments, the glycosylation pattern of monoclonal antibody 1 is characterized by one or more of the following conditions:

the fucose content of the antibody is very low (0-5%);

the antibody has a low galactose content (less than or equal to 30%);

the antibody has a low mannose level (< 5%);

the antibody high mannose levels are lower (less than or equal to 5%);

the antibody G0 levels were higher.

In some embodiments, the neoplastic disease comprises a B cell lymphoma including precursor B cell lymphocytic leukemia/lymphoma and mature B cell tumors, lymphoplasmacytic lymphoma, Mantle Cell Lymphoma (MCL), low, intermediate and high follicular lymphoma, cutaneous follicular central lymphoma, MALT-type, nodal and splenic marginal zone B cell lymphoma, hairy cell leukemia, diffuse large B cell lymphoma, burkitt's lymphoma, plasmacytoma, plasma cell myeloma, post-transplant lymphoproliferative disorder, waldenstrom's macroglobulinemia and Anaplastic Large Cell Lymphoma (ALCL).

In some embodiments, the neoplastic disease comprises an immune disease comprising psoriasis, psoriatic arthritis, dermatitis, systemic scleroderma and sclerosis, Inflammatory Bowel Disease (IBD), Crohn's disease, ulcerative colitis, respiratory distress syndrome, meningitis, encephalitis, uveitis, glomerulonephritis, eczema, asthma, atherosclerosis, leukocyte adhesion deficiency, multiple sclerosis, Raynaud's syndrome, Sjogren's syndrome, juvenile onset diabetes, Lett's disease, Behcet's disease, immune complex nephritis, IgA nephropathy, IgM polyneuropathy, neuromyelitis optica, immune-mediated thrombocytopenia, hemolytic anemia, myasthenia gravis, lupus nephritis, systemic lupus erythematosus, Rheumatoid Arthritis (RA), atopic dermatitis, pemphigus, Graves' disease, hashimoto's thyroiditis, wegener's granulomatosis, Omenn's syndrome, chronic renal failure, acute infectious mononucleosis, chronic obstructive pulmonary disease.

The invention also provides application of the liquid preparation in preparing a medicament for treating diseases related to cell expression of CD20 in mammals, wherein the diseases related to cell expression of CD20 are selected from tumorigenic diseases and immune diseases.

In some embodiments, the mammal is a human.

During the development of the formulation, the pH is found to have a great influence on the stability of the formulation, so that it is necessary to maintain a specific pH range for the formulation, and the invention selects a suitable buffer system, such as a succinic acid buffer, a citric acid buffer, a phosphate buffer, a histidine buffer or an acetic acid buffer, and maintains the pH value at 5.5-6.2. The trehalose used as a stabilizer can reduce aggregation and degradation of antibody protein, and effectively protect the activity of active ingredients in the preparation. Polysorbate 80 plays an important role in preventing aggregation of antibody proteins as a surfactant. The antibody preparation of the invention has good stability.

Drawings

FIG. 1 shows the number of microparticles (pieces/mL) for samples containing different surfactants;

FIG. 2 shows turbidity after shaking of samples containing different surfactants;

fig. 3 shows microparticles after repeated 5 freeze-thaw cycles of samples containing different surfactants.

Term(s) for

Unless otherwise specified, each of the following terms shall have the meaning set forth below.

"about" refers to the conventional error range for corresponding numerical values as would be readily understood by one of ordinary skill in the relevant art. In some embodiments, reference herein to "about" refers to the numerical values recited and ranges of ± 10%, ± 5%, or ± 1% thereof.

"amino acid" refers to a carboxy alpha-amino acid, which may be encoded by a nucleic acid, either directly or in the form of a precursor. A single amino acid is encoded by a nucleic acid consisting of three nucleotides (so-called codons or base triplets). Each amino acid is encoded by at least one codon. The same amino acid is encoded by different codons and is referred to as "degeneracy of the genetic code". The term "amino acid" as used in the present application refers to naturally occurring carboxy alpha-amino acids, including alanine (three letter code: ala, one letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y) and valine (val, V).

The term "antibody" is used in its broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity.

An "antibody fragment" includes a portion of a full-length antibody (comprises), typically including the antigen-binding region thereof. Examples of antibody fragments include Fab, Fab ', F (ab')₂And Fv fragments; diabodies (diabodies); linear antibodies (linear antibodies); a single chain antibody molecule; and multispecific antibodies formed from antibody fragments.

A "monoclonal antibody" (mAb) is an antibody prepared from the same immune cell, which is all clones of a single parent cell. Monoclonal antibodies can have monovalent affinity because they bind the same epitope (the portion of the antigen that the antibody recognizes). In contrast, polyclonal antibodies bind to multiple epitopes and are usually secreted by several different plasma cells. Bispecific monoclonal antibodies can also be engineered by increasing the therapeutic target of one single monoclonal antibody to two epitopes. Monoclonal antibodies can be made by hybridoma, recombinant, transgenic, or other techniques known to those skilled in the art.

The amount of the buffer in the present invention means the total amount of the buffer pair in the buffer system constituting the buffer. In some embodiments, molarity is taken as a unit of amount of buffer, the value of which refers to the molarity of a buffer pair in the buffer system of the buffer. For example, where a histidine buffer consisting of L-histidine and L-histidine hydrochloride is used as the buffer, a given concentration of histidine buffer (e.g., 10mM) is the combined concentration of L-histidine and L-histidine hydrochloride (e.g., 5mM for L-histidine hydrochloride, or 6mM for L-histidine, 4mM for L-histidine hydrochloride, or 3.46mM for L-histidine, 6.54mM for L-histidine hydrochloride, etc.).

The preparation of the present invention may be formulated with the adjuvant or a hydrate thereof. For example, histidine hydrochloride, also known as histidine hydrochloride, can be anhydrous histidine hydrochloride, or a histidine hydrochloride hydrate, such as histidine hydrochloride monohydrate. As mentioned in the "5 mM histidine hydrochloride", 5mmol of histidine hydrochloride or histidine hydrochloride hydrate can be dissolved in a solvent to form a 1L solution; 958mg histidine hydrochloride, including 958mg histidine hydrochloride or a corresponding amount of hydrate.

The following are a few examples of the amount of partially anhydrous trehalose corresponding to trehalose hydrate: 1) trehalose 158.6mM means that 158.6mM trehalose anhydrous (158.6mM trehalose anhydrous corresponds to about 5.4% trehalose anhydrous) or 158.6mM trehalose dihydrate (158.6mM trehalose dihydrate corresponds to about 6% trehalose dihydrate) can be added; 2) trehalose 224.6mM means that either 224.6mM anhydrous trehalose (224.6mM anhydrous trehalose is equivalent to about 7.68% anhydrous trehalose) or 158.6mM trehalose dihydrate (224.6mM trehalose dihydrate is equivalent to about 8.5% trehalose dihydrate) can be added.

A "therapeutically effective amount" is an amount that can treat a disease or condition. The administration amount of the antibody preparation of the present invention to a human body varies depending on the age, body weight, sex, administration form, health condition and critical degree of disease of a patient.

In the present invention, "%" represents a mass-volume concentration in g/ml. For example, 0.9g of sodium chloride in 0.9% sodium chloride solution means that 0.9g of sodium chloride is dissolved in the solvent to form 100ml of solution, i.e., the solution contains 0.9g/100ml of sodium chloride.

Detailed Description

The technical solutions of the present invention are further illustrated by the following specific examples, which do not represent limitations to the scope of the present invention. Insubstantial modifications and adaptations of the present invention by others of the concepts fall within the scope of the invention.

Unless otherwise specified, the liquid formulation solvent in the examples below is water, such as water for injection.

Example 1

The preparation provided by the invention contains an anti-CD 20 antibody which is a monoclonal antibody 1, wherein the heavy chain of the monoclonal antibody is shown as SEQ ID NO. 1, and the light chain of the monoclonal antibody is shown as SEQ ID NO. 2;

EVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKG LEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTAL YYCAKDIQYGNYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPGSSKS TSGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK，SEQ ID NO:1；

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLL IYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITF GQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKS FNRGEC，SEQ ID NO:2。

pH Range screening

Selecting two buffer solutions with pH value covering the range of 5.0-6.5: 20mM histidine buffer (His) and 20mM acetate buffer (NaAc). The purified anti-CD 20 monoclonal antibody 1 (the heavy chain of the antibody is shown as SEQ ID NO:1, and the light chain of the antibody is shown as SEQ ID NO: 2) was subjected to ultrafiltration to prepare six groups of solution samples with pH of His5.5, His6.0, His6.5, NaAc5.0, NaAc5.5, and NaAc6.0, the antibody concentration was 18mg/mL, and the specific composition was shown in Table 1. Subpackaging and carrying out high temperature test (40 ℃).

The samples were split and placed at 40 ℃ for high temperature testing, and sampled at day 0, week 1, week 2 and week 3 for SEC-HPLC and IEC-HPLC tests, the results are shown in Table 2.

TABLE 1 buffer composition information

TABLE 240 ℃ Change over time of the SEC and IEC values for samples at different pH values

As shown in table 2, 1) from the SEC-HPLC monomer purity data, the SEC monomer purity of the six groups of samples all showed a decreasing trend, but the decreasing amplitudes of the groups were significantly different, and the decreasing amplitudes of his6.0 and his5.5 were the smallest and better than his6.5, naac5.5, naac5.0 and naac6.0, and the monomer purity stability was better when ph5.5 to ph6.0 in the His buffer solution; 2) as can be seen from the IEC-HPLC main peak content data, His6.0, His5.5 and NaAc6.0 are superior to NaAc5.5, NaAc5.0 and His6.5, which indicates that the antibody sample has good IEC main peak content stability in the buffer solution with the pH value of 5.5-6.0.

And by combining the results of SEC-HPLC and IEC-HPLC, the stability of the antibody sample is better when the pH range of the antibody sample is 5.5-6.0 at high temperature (40 ℃).

Buffer system screening

Under the condition of the pH5.5-pH6.0, the stability of the antibody is better; therefore, a buffer solution having a buffering capacity in the range of about pH5.5 to 6.0 was selected for the screening test. 5 different buffers were prepared: succinic acid-sodium succinate buffer (Sua), citric acid-sodium Citrate Buffer (CB), Phosphate Buffer (PB), histidine-histidine hydrochloride buffer (His), acetic acid-sodium acetate buffer (NaAc), see Table 3. The purified anti-CD 20 monoclonal antibody 1 sample was ultrafiltered and changed to the above 5 kinds of buffer solutions, the concentration was adjusted to be consistent (the concentration of the antibody was 20mg/mL), and then subpackaged, and subjected to a high temperature test at 40 ℃, and samples were taken at day 0, week 1, week 2 and week 3, and subjected to SEC-HPLC and IEC-HPLC tests, respectively, and the results are shown in Table 4.

TABLE 3 buffer composition information

TABLE 440 ℃ Change with time of antibody SEC, IEC in different buffers

As shown in table 4, 1) from SEC-HPLC monomer purity data, the change trends of five groups of samples are substantially consistent, but the decrease amplitudes are significantly different, the His buffer is the best, the PB buffer is relatively poor, and the other three groups are in the middle position and have little difference; 2) according to IEC-HPLC main peak content data, the antibody is unstable at the high temperature of 40 ℃, the main peak content of five groups of samples is reduced to different degrees along with the increase of time, and His buffer solution and NaAc buffer solution are better and better than those of other groups.

The results of SEC-HPLC and IEC-HPLC are combined to show that the stability of SEC-HPLC and IEC-HPLC is better than that of other groups of buffers when the antibody sample is in His buffer under the environment of high temperature (40 ℃).

pH value optimization screening

(1) pH value optimization screening 1

The stability of the antibody sample in histidine buffer solution with the pH value of 5.5-6.0 is better, the pH value optimization screening test is carried out on the antibody sample, the purified anti-CD 20 monoclonal antibody 1 sample is subjected to ultrafiltration liquid exchange, and a preparation formula with 5 pH gradients is prepared: histidine buffer (pH5.6), histidine buffer (pH5.8), histidine buffer (pH6.0), histidine buffer (pH6.2), and histidine buffer (pH 6.4). The antibody concentrations were adjusted to 20mg/mL, samples were subjected to a high temperature test at 40 ℃ and sampled at day 0, week 1, week 2 and week 3 for SEC-HPLC and IEC-HPLC tests, respectively, and the results are shown in Table 5.

TABLE 5 SEC-HPLC, IEC-HPLC changes with time at different pH

As shown in table 5, 1) the trend of the five samples was substantially consistent from the SEC-HPLC monomer purity data; 2) according to IEC-HPLC main peak content data, when the pH is 5.6-6.2, the IEC-HPLC main peak of the sample is slowly reduced, and the stability is good.

The results of SEC-HPLC and IEC-HPLC are combined to show that the stability of the antibody sample is better when the pH value of the buffer solution is 5.6-6.2.

(2) pH value optimization screening 2

As can be seen from the pH value optimization screening 1, the stability of the antibody sample is good when the pH value is 5.6-6.2, the IEC-HPLC performance is poor when the pH value exceeds 6.2, but the stability of the sample cannot be determined when the pH value is less than 5.6, so the pH screening test is performed again. Since the pH of the His buffer is greatly influenced by temperature, the pH of the buffer solution is changed by ultrafiltration by 6 samples of the anti-CD 20 monoclonal antibody 1 with different pH values, wherein the buffer solution is citric acid-PB (C-P): pH5.0, pH5.3, pH5.6, pH5.9, pH6.2, pH6.5. The antibody samples were subjected to a high temperature test at 40 ℃ and sampled at day 0 (D), week 1 (W), week 2 and week 3 for SEC-HPLC and IEC-HPLC tests, respectively, and the results are shown in Table 6.

TABLE 6 SEC-HPLC, IEC-HPLC changes with time at different pH

As shown in table 6, 1) from the SEC-HPLC monomer purity data, the trend of the six samples was substantially consistent: after the sample is placed for 4 weeks at a high temperature, the monomer purity of the sample is obviously reduced, and the monomer purity of the sample is poor when the pH value is less than 5.3; 2) according to IEC-HPLC main peak content data, antibody samples are unstable at high temperature, the main peak content of six groups of samples is reduced to different degrees along with the prolonging of time, and the IEC-HPLC main peak of the samples is reduced slowly and has good stability when the pH is 5.6-6.5.

The pH value is a key factor in the prescription of the liquid preparation, the antibody shows different stability in buffers with different pH values, but when the pH value fluctuates within a certain range, the quality of the antibody cannot be significantly influenced. Through the preliminary screening research and preliminary judgment of the pH fine screening, the stability of the antibody is good within the pH range of 5.5-6.2.

Stabilizer species screening

Preparing anti-CD 20 monoclonal antibody 1 solution containing seven stabilizers of sodium chloride, sucrose, trehalose, mannitol, sorbitol, arginine and methionine respectively, wherein the specific formula is shown in Table 7; tre (trehalose) in table 7 refers to trehalose dihydrate.

TABLE 7 formulation of different stabilizers

(1) High temperature test

The samples with the stabilizer and the samples without the stabilizer were subjected to a comparative test at a high temperature (40 ℃), the change of the sample quality was examined, and the samples at 1 week, 2 weeks, 3 weeks and 4 weeks were subjected to SEC-HPLC, and the results are shown in Table 8.

TABLE 8 SEC-HPLC behavior with time for different stabilizer samples

As shown in Table 8, from SEC-HPLC monomer purity data, the eight groups of samples showed substantially the same trend, but slightly different decline, and the three groups of samples, Tre, Sor and Suc, were the best.

(2) Light test

The samples added with the stabilizing agent are subjected to a comparative test under the illumination condition, the change of the sample quality is inspected, and samples are taken at 1 week and 2 weeks for SEC-HPLC and IEC-HPLC detection, and the results are shown in Table 9.

TABLE 9 SEC-HPLC, IEC-HPLC, variation with time for different stabilizer samples

As shown in table 9, 1) from the SEC-HPLC monomer purity data, the change trends of the eight groups of samples are substantially consistent, but the decrease amplitudes are slightly different, the decrease of the Met group of samples is the least, and the Tre, Suc and other groups of samples are followed; 2) from IEC-HPLC main peak content data, it is known that antibody protein is unstable under illumination conditions, and the main peak content of the eight groups of samples decreases to different degrees with the increase of time, and from the aspect of the decrease, Met and Arg are better, and next Tre, Suc, Sor and the like.

Combining the results of high temperature and light, trehalose performed well in both SEC-HPLC and IEC-HPLC.

Surfactant screening

Compatibility test with physiological saline

When the antibody preparation is administered by intravenous injection, it is clinically compatible with physiological saline, so that the compatibility test is performed. Antibody sample solutions containing different concentrations of different types of surfactants were prepared, and the specific formulation is shown in table 10. The concentration of the anti-CD 20 monoclonal antibody 1 in the sample solution is 30mg/mL, the sample solutions with the same volume and containing surfactants with different concentrations are respectively added into 0.9% sodium chloride solution according to the compatibility proportion (1mL sample: 29mL physiological saline), the characteristics of the solution are observed after the solution is slightly and fully mixed, the antibody solution after being placed for 2 hours is subjected to insoluble particle detection, and the detection result is shown in figure 1.

TABLE 10 antibody solution formulations

As shown in fig. 1, the number of microparticles in the blank group without surfactant was the highest, while the number of microparticles in the sample with surfactant (polysorbate-80 or polysorbate-20) was generally lower, and when the surfactant content was higher, the number of microparticles was slightly lower; however, when the surfactant concentration reaches a certain level, the number of fine particles in the sample increases. When the surfactant is polysorbate-80 at a concentration of 0.02%, the number of microparticles in the formulation is small.

Shock test

The sample inevitably vibrates in the transportation or transfer process, the vibration accelerates protein aggregation, and the surfactant is added into the preparation, so that the protein aggregation can be effectively prevented. anti-CD 20 monoclonal antibody 1 sample solutions were prepared containing different concentrations of different types of surfactants, the specific formulations are shown in Table 11. The sample was laid down on a shaker at 200rpm in a room temperature environment, and the antibody solution was subjected to a shaking test. Performing light detection and turbidity (OD340 value) detection on the solution at 0h, 2h, 4h, 8h, 24h, 48h and 72h, and examining the change of the quality of the antibody solution, wherein the result is shown in FIG. 2 (note: OD value is the value obtained by detecting the protein solution at UV340 nm); tre (trehalose) in table 11 refers to trehalose dihydrate.

TABLE 11 antibody solution formulations

As shown in fig. 2, after the sample is placed for 4 hours under the shaking condition, the blank sample and the sample added with only 6% Tre begin to be turbid, and the sample added with tween still keeps clear after shaking for 72 hours, which indicates that polysorbate-80 or polysorbate-20 has a certain protection effect on the sample under the shaking condition, can prevent protein aggregation from being turbid, and plays a role in solubilization; the turbidity was lowest in samples containing 0.02% and 0.03%, and relatively higher in samples containing 0.01% and 0.04%, regardless of polysorbate-80 or polysorbate-20, indicating that the polysorbate content should not be too low or too high; 0.02% polysorbate is preferred.

Repeated Freeze/thaw test 1

anti-CD 20 monoclonal antibody 1 samples with different polysorbate (i.e. Tween) contents and types are prepared to be subjected to repeated freeze-thaw tests (the specific formula is shown in Table 12, Tre (trehalose) in Table 12 refers to trehalose dihydrate), and after the sample solution is frozen at-80 ℃ for 24 hours, the sample solution is placed at 25 ℃ for 24 hours to be subjected to one freeze-thaw. Insoluble particle detection was performed on the samples after freeze-thawing and the results are shown in FIG. 3.

TABLE 12 antibody solution formulations

As shown in FIG. 3, after 5 times of repeated freeze thawing, the number of microparticles in the samples with Tween was significantly lower than that in the samples without Tween, indicating that Tween can effectively prevent protein aggregation during repeated freeze thawing.

Example 2

The invention provides a liquid preparation containing anti-CD 20 monoclonal antibody 1, which consists of the following components in concentration: about 15mg/ml anti-CD 20 antibody, about 10mM succinate buffer, about 80mM sucrose, about 0.1mg/ml polysorbate-20, and a pH of the liquid formulation of about 5.5.

Example 3

The invention provides a liquid preparation containing anti-CD 20 monoclonal antibody 1, which consists of the following components in concentration: about 40mg/ml anti-CD 20 antibody, about 15mM citrate buffer, about 140mM sorbitol, about 0.3mg/ml polysorbate-80, and a pH of the liquid formulation of about 5.6.

Example 4

The invention provides a liquid preparation containing anti-CD 20 monoclonal antibody 1, which consists of the following components in concentration: about 60mg/ml anti-CD 20 antibody, about 26mM acetate buffer, about 190mM sorbitol, about 0.35mg/ml polysorbate-20, and a pH of the liquid formulation of about 5.7.

Example 5

The invention provides a liquid preparation containing anti-CD 20 monoclonal antibody 1, which consists of the following components in concentration: about 70mg/ml anti-CD 20 antibody, about 26mM phosphate buffer, about 240mM methionine, about 0.38mg/ml polysorbate-20, and a pH of the liquid formulation of about 6.0.

Example 6

The invention provides a liquid preparation containing anti-CD 20 monoclonal antibody 1, which consists of the following components in concentration: about 80mg/ml anti-CD 20 antibody, about 30mM phosphate buffer, about 158mM mannitol, about 0.4mg/ml polysorbate-20, and a pH of the liquid formulation of about 6.2.

Example 7

The invention provides a liquid preparation containing anti-CD 20 monoclonal antibody 1, which consists of the following components in concentration: about 20mg/ml anti-CD 20 antibody, about 20mM histidine buffer, about 158.6mM trehalose, about 20mg/ml polysorbate-80, and a pH of the liquid formulation of about 5.8; the mass ratio of L-histidine to L-histidine hydrochloride in histidine buffer was 2:3 based on pH 5.8.

Example 8

The invention provides a liquid preparation containing anti-CD 20 monoclonal antibody 1, which consists of the following components in concentration: about 20mg/ml anti-CD 20 antibody, about 20mM histidine buffer, about 224.6mM trehalose, about 0.2mg/ml polysorbate-80, and a liquid formulation having a pH of about 5.8; the mass ratio of L-histidine to L-histidine hydrochloride in histidine buffer was 2:3 based on pH 5.8.

Example 9

The invention provides a liquid preparation containing anti-CD 20 monoclonal antibody 1, which consists of the following components in concentration: about 50mg/ml anti-CD 20 antibody, about 20mM histidine buffer, about 224.6mM trehalose, about 0.2mg/ml polysorbate-80, and a liquid formulation having a pH of about 5.8; the mass ratio of L-histidine to L-histidine hydrochloride in histidine buffer was 2:3 based on pH 5.8.

Example 10

The invention provides a liquid preparation containing anti-CD 20 monoclonal antibody 1, which consists of the following components in concentration: about 30mg/ml anti-CD 20 antibody, about 18mM histidine buffer, about 170mM trehalose, about 0.18mg/ml polysorbate-80, and a pH of the liquid formulation of about 5.7.

Example 11

The invention provides a liquid preparation containing anti-CD 20 monoclonal antibody 1, which consists of the following components in concentration: about 40mg/ml anti-CD 20 antibody, about 19mM histidine buffer, about 225mM trehalose, about 0.19mg/ml polysorbate-80, and a pH of the liquid formulation of about 5.9.

Example 12

The invention provides a liquid preparation containing anti-CD 20 monoclonal antibody 1, which consists of the following components in concentration: about 46mg/ml anti-CD 20 antibody, about 22mM histidine buffer, about 220mM trehalose, about 0.22mg/ml polysorbate-80, and a pH of the liquid formulation of about 6.0.

Comparison of stability

The formulations disclosed in CN101820912B patent (antibody concentration 20mg/mL, formulation a), comparative formulation (same as the formula adjuvant of CN101820912B, but the antibody is monoclonal antibody 1, formulation B), formulation 1 (example 6), formulation 2 (example 7), formulation 3 (example 8) and formulation 4 (the antibody is monoclonal antibody 1, antibody concentration 100mg/mL) were used as the subjects for stable detection, and are shown in table 13.

TABLE 13 formulation formula

1) Dissolution temperature, aggregation temperature and particle size

TABLE 14 dissolution temperature, aggregation temperature and particle size

As shown in table 14, 1) the anti-CD 20 antibody in the original prescription a was replaced with antibody 1, and there was substantially no difference between Tm1, Tagg, and DLS (particle size and uniformity) in the two preparations; 2) compared with the original prescription A, the Tm1 and Tagg of the preparation of the prescription 1 are both high, the PDI of the particle size is less than 0.25, the uniformity is good, and the stability of the preparation of the prescription 1 is good; 3) the Tm1, Tagg and DLS of the formulations of the prescription 2 and the prescription 3 are basically consistent with those of the formulation of the prescription 1 without changing the trehalose from 158.6mM to 224.6mM, and the stability is relatively better.

2) High temperature test at 40 deg.C

The formulations were dispensed and subjected to a high temperature test at 40 ℃ and samples were taken on day 0, week 1, week 2, week 3 and week 4 and subjected to SEC-HPLC, the results of which are shown in Table 15.

As shown in table 15, the monomer purity stability of the formulations of formula 1, formula 2 and formula 3 was better as seen from the SEC-HPLC monomer purity data; in particular, prescription 1 and prescription 2 are more stable than prescription B.

TABLE 15 monomer purity of the formulations as a function of time

3) Measurement by light test

The formulations were split and tested in the light, samples were taken on day 0, day 5, and day 10 and subjected to SEC-HPLC, the results of which are shown in Table 16.

As shown in table 16, the monomer purity stability of the formulations of formula 1, formula 2 and formula 3 was better as seen from the SEC-HPLC monomer purity data; in particular, prescription 1 and prescription 2 are more stable than prescription B.

TABLE 16 monomer purity of the formulations as a function of time

Compatibility testing

Through an accelerated stability test and a long-term stability test, the liquid preparation has good compatibility with the medicine bottle and meets the requirement of packaging.

Sequence listing

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Claims

1. A liquid formulation comprising an anti-CD 20 antibody, comprising the following components in the concentrations: 15-80 mg/ml anti-CD 20 antibody, 10-30 mM buffering agent, 80-240 mM stabilizer, 0.1-0.4 mg/ml surfactant, and the pH value of the liquid preparation is 5.5-6.2;

the buffer is selected from the group consisting of a succinic buffer, a citric buffer, a phosphoric buffer, a histidine buffer, and an acetic buffer;

the stabilizer is selected from sucrose, trehalose, sorbitol, mannitol and methionine;

the surfactant is selected from polysorbate-80 and polysorbate-20.

2. The liquid formulation of claim 1, wherein the anti-CD 20 antibody is selected from the group consisting of a monoclonal antibody and a fragment that binds to CD 20.

3. The liquid formulation of claim 2, wherein the heavy chain of the anti-CD 20 antibody comprises the amino acid sequence set forth in SEQ ID No. 1 or an amino acid sequence having at least 90% identity to said SEQ ID No. 1 and the light chain of the anti-CD 20 antibody comprises the amino acid sequence set forth in SEQ ID No. 2 or an amino acid sequence having at least 90% identity to said SEQ ID No. 2.

4. The liquid formulation of any one of claims 1 to 3, wherein the liquid formulation comprises 20 to 50mg/ml of the anti-CD 20 antibody.

5. The liquid formulation of any one of claims 1 to 3, wherein the liquid formulation comprises 18 to 22mM histidine buffer.

6. The liquid formulation of any one of claims 1 to 3, wherein the liquid formulation comprises 158 to 225mM trehalose.

7. The liquid formulation according to any one of claims 1 to 3, wherein the liquid formulation comprises 0.18 to 0.22mg/ml polysorbate-80.

8. The liquid preparation according to any one of claims 1 to 3, wherein the pH of the liquid preparation is 5.7 to 5.9.

9. The liquid formulation of claim 1, comprising the following concentrations of components: 20mg/ml anti-CD 20 antibody, 20mM histidine buffer, 158.6mM trehalose, 0.2mg/ml polysorbate-80, pH5.8 for the liquid formulation.

10. The liquid formulation of claim 1, comprising the following concentrations of components: 20mg/ml anti-CD 20 antibody, 20mM histidine buffer, 224.6mM trehalose, 0.2mg/ml polysorbate-80, pH5.8 for the liquid formulation.

11. The liquid formulation of claim 1, comprising the following concentrations of components: 50mg/ml anti-CD 20 antibody, 20mM histidine buffer, 224.6mM trehalose, 0.2mg/ml polysorbate-80, pH5.8 for the liquid formulation.

12. A method for treating a disease involving cellular expression of CD20 in a mammal comprising administering an effective amount of a liquid formulation according to any one of claims 1 to 11.

13. The method of claim 12, wherein the mode of administration to the mammal is intravenous or subcutaneous injection.

14. The method of claim 13, wherein when the mammal is administered by injection, the liquid formulation comprises the following concentrations of the components: 20mg/ml anti-CD 20 antibody, 20mM histidine buffer, 158.6mM trehalose, 0.2mg/ml polysorbate-80, pH5.8 for the liquid formulation.

15. The method of claim 13, wherein when the mammal is administered by injection, the liquid formulation comprises the following concentrations of the components: 20mg/ml anti-CD 20 antibody, 20mM histidine buffer, 224.6mM trehalose, 0.2mg/ml polysorbate-80, pH5.8 for the liquid formulation.

16. The method of claim 13, wherein when the mammal is administered by injection, the liquid formulation comprises the following concentrations of the components: 50mg/ml anti-CD 20 antibody, 20mM histidine buffer, 224.6mM trehalose, 0.2mg/ml polysorbate-80, pH5.8 for the liquid formulation.

17. The method of claims 12 to 16, wherein the disease involving expression of CD20 by cells is selected from the group consisting of a neoplastic disease, an immune disease.

18. The method of claim 17, wherein the neoplastic disease comprises B cell lymphoma, including hodgkin's lymphoma and non-hodgkin's lymphoma; the immune diseases include multiple sclerosis, immune thrombocytopenia, neuromyelitis optica, myasthenia gravis, rheumatoid arthritis, psoriasis and psoriatic arthritis.

19. Use of a liquid formulation according to any one of claims 1 to 11 for the manufacture of a medicament for the treatment of a disease involving cellular expression of CD20 in a mammal, wherein the disease involving cellular expression of CD20 is selected from the group consisting of a neoplastic disease, an immune disease.

20. The use of claim 19, wherein the neoplastic disease comprises B cell lymphoma, including hodgkin's lymphoma and non-hodgkin's lymphoma; the immune diseases include multiple sclerosis, immune thrombocytopenia, neuromyelitis optica, myasthenia gravis, rheumatoid arthritis, psoriasis and psoriatic arthritis.