Composition comprising antibody specifically binding to hepatitis b virus surface antigen (HBsAg)

Abstract

The present invention relates to a composition comprising an antibody specifically binding to a hepatitis B virus surface antigen (HBsAg), a buffer solution, a protective agent, a surfactant and an antioxidant. The composition has good stability under high concentration, is suitable for long-term storage, has proper osmotic pressure, and can be used as a subcutaneous injection preparation. In addition, the invention can be used for treating or preventing diseases or symptoms related to hepatitis B virus (HBV) infection.

Description

[0001] Cross - reference to related applications

[0002] This application claims the priority benefit of Chinese Application No. 202311870663.4, filed on December 29, 2023, the entire content of which is incorporated herein by reference. Technical field

[0003] The present invention relates to the field of antibody preparations, and particularly to pharmaceutical preparations comprising antibodies and / or antibody fragments that specifically bind to hepatitis B virus surface antigen (HBsAg). In addition, the present invention relates to the therapeutic use of these preparations. Background art

[0004] Hepatitis B virus (HBV) infection is a worldwide epidemic, but the prevalence intensity of HBV infection varies greatly in different regions. According to the World Health Organization, approximately 2 billion people worldwide have been infected with HBV, of which 240 million are chronic HBV infected patients. Each year, about 650,000 people die from liver failure, cirrhosis, and hepatocellular carcinoma (HCC) caused by HBV infection. Among patients with cirrhosis and HCC globally, the proportions caused by HBV infection are 30% and 45% respectively. Among patients with cirrhosis and HCC in China, the proportions caused by HBV infection are 60% and 80% respectively. China is a populous country with chronic hepatitis B. Epidemiological surveys show that currently more than 7% of the population are hepatitis B virus infected, with a total number close to 100 million. By the end of 2015, only 9% of hepatitis B virus infected patients had been tested and diagnosed; among the patients diagnosed with hepatitis B virus infection, only 8% had received treatment (data from the Chinese Foundation for Hepatitis Prevention and Control). They not only need long - term or even lifelong treatment, bringing a huge burden to families and society, but also have the risk of developing into cirrhosis and liver cancer. It is estimated that there are 28 million chronic hepatitis B patients in China, with nearly 1 million new cirrhotic patients and about 300,000 new liver cancer patients each year. Calculated according to the 5% consultation rate of chronic hepatitis patients and the 95% consultation rate of cirrhotic and liver cancer patients, the direct medical costs for treating hepatitis B - related diseases in China reach 80 - 120 billion yuan each year.

[0005] After antiviral treatment, although hepatitis B patients can reduce the viral load in the body and delay the progression of severe diseases, many people cannot completely eliminate the hepatitis B virus. Hepatitis B patients need to take medicine for a long time, which causes a heavy economic burden on them. According to the data dynamically released by the Chinese Center for Disease Control and Prevention, the average annual medical treatment cost of chronic hepatitis patients accounts for 56.24% of the average annual total income of the family, 81.53% for compensated cirrhosis, 157.21% for decompensated cirrhosis, and 96.76% for liver cancer. The heavy economic burden causes many patients to either give up treatment or lead to poverty due to illness in the family. In addition, looking at the evolution process of the medical treatment costs of liver diseases in the past ten-odd years, the medical treatment costs of liver diseases show an increasing trend year by year, with an annual increase of about 10% - 20%, and the individual share also increases accordingly.

[0006] The latest clinical research results show that for patients who have cleared viral DNA after nucleotide analogue treatment, if HBsAg is below a certain level (such as HBsAg < 1500 IU / mL), the probability of interferon clearing HBsAg can exceed 50%, which is much higher than the commonly believed interferon cure rate of only 5 - 10%.

[0007] However, due to the lack of effective drugs to reduce HBsAg at present, the HBsAg levels of the vast majority of patients (more than 90%) exceed this index, resulting in interferon being unable to cure these patients (this is the reason why the interferon cure rate is only 5 - 10%). These new clinical evidences have created a more realistic and urgent need for treatment methods to reduce HBsAg.

[0008] As is well known, antibody preparations must consider factors such as stability, administration method, concentration, etc. These factors will affect the physicochemical degradation of antibodies during manufacturing, storage, and delivery, and thus affect the risk to the recipient. Therefore, stable antibody preparations are necessary.

[0009] In addition, the current optimal administration method for antibody drugs is subcutaneous injection. Since the antibody dose required to exert an effect is relatively high, high-concentration antibody preparations usually need to be prepared. However, the increase in antibody concentration is accompanied by many defects. For example, it causes the viscosity of the antibody preparation to be too high, resulting in difficult injection, pain at the injection site, antibody residue in the container, and production difficulties. In addition, high-concentration antibodies are prone to aggregation and form microparticles, leading to formulation instability, increased immunogenicity, etc., which are difficult during storage and long-term storage. Therefore, there are still huge challenges in preparing high-concentration antibody preparations.

[0010] In summary, there is still a need in the art to develop an antibody that specifically binds to hepatitis B virus surface antigen (HBsAg) and a composition containing the same to meet the demand for a drug that can treat hepatitis B-related diseases and is stable and non-aggregating at a relatively high concentration for a long time. Summary of the Invention

[0011] The present invention meets the above needs by providing an antibody pharmaceutical composition specifically binding to hepatitis B virus surface antigen (HBsAg). This liquid preparation can stably hold the antibody and has an osmotic pressure close to isotonicity.

[0012] In one aspect, the present invention provides an antibody composition comprising

[0013] (i) an antibody specifically binding to HBsAg and its antigen-binding fragment;

[0014] (ii) a buffer;

[0015] (iii) a protectant;

[0016] (iv) a surfactant;

[0017] (v) an antioxidant;

[0018] The antibody specifically binding to hepatitis B virus surface antigen (HBsAg) and its antibody fragment comprise a heavy chain variable region (VH) sequence and a light chain variable region (VL) sequence, wherein:

[0019] The heavy chain variable region comprises the complementarity determining region (CDR) amino acid sequences shown below: HCDR1: GYTFTGYY,

[0020] HCDR2: INPNSGGT,

[0021] HCDR3: ARDLWX1X2X3X4X5DYGX6DV;

[0022] The light chain variable region comprises the CDR amino acid sequences shown below:

[0023] LCDR1: X7SIX8TX9,

[0024] LCDR2: AAS,

[0025] LCDR3: QQSYSTPLT;

[0026] Wherein:

[0027] X1 is selected from N, D, Y, I, S, V or M, X2 is selected from F, H, E, W, R, Q, M, P or D, X3 is selected from V, Q, P, R, H, T or Y, X4 is selected from S, T, D, L, Y, F or H, X5 is selected from D, Q or E, X6 is selected from M, I or L; X7 is selected from Q, S, H, Y, P, L, I or E, X8 is selected from S or D, X9 is selected from Y, F, D, T, S or V.

[0028] Those skilled in the art should be aware that each value of Xn (where n represents the integer subscript of each X in the general formula) in the general formula can be combined with each other. For example, in some embodiments, X1, X2, X3, X3, X4, X5, and X6 in HCDR3 are sequentially selected from any one of the following combinations: N, F, V, S, D, I; D, H, Q, S, Q, I; Y, F, P, S, E, M; I, E, R, T, E, M; S, W, H, D, E, M; D, R, Q, L, D, M; D, Q, T, Y, D, L; D, Q, T, F, D, L; V, M, Q, S, D, M; S, P, Y, S, D, M; M, E, R, T, D, M; S, D, Q, H, D, I. In some specific embodiments, HCDR3 is selected from ARDLWNFVSDDYGIDV (SEQ ID NO: 3); ARDLWDHQSQDYGIDV (SEQ ID NO: 4); ARDLWYFPSEDYGMDV (SEQ ID NO: 5); ARDLWIERTEDYGMDV (SEQ ID NO: 6); ARDLWSWHDEDYGMDV (SEQ ID NO: 7); ARDLWDRQLDDYGMDV (SEQ ID NO: 8); ARDLWDQTYDDYGLDV (SEQ ID NO: 9); ARDLWDQTFDDYGLDV (SEQ ID NO: 10); ARDLWVMQSDDYGMDV (SEQ ID NO: 11); ARDLWSPYSDDYGMDV (SEQ ID NO: 12); ARDLWMERTDDYGMDV (SEQ ID NO: 13); ARDLWSDQHDDYGIDV (SEQ ID NO: 14).

[0029] In LCDR1, X7, X8, and X9 are successively selected from any one of the following combinations: S, S, F; H, S, D; Y, S, D; S, S, D; P, S, T; Y, S, S; Q, S, V; Q, S, F; Y, S, T; L, S, Y; I, S, D; E, S, Y; S, D, D. In some specific embodiments, LCDR1 is selected from SSISTF (SEQ ID NO: 15); HSISTD (SEQ ID NO: 16); YSISTD (SEQ ID NO: 17); SSISTD (SEQ ID NO: 18); PSISTT (SEQ ID NO: 19); YSISTS (SEQ ID NO: 20); QSISTV (SEQ ID NO: 21); QSISTF (SEQ ID NO: 22); YSISTT (SEQ ID NO: 23); LSISTY (SEQ ID NO: 24); ISISTD (SEQ ID NO: 25); ESISTY (SEQ ID NO: 26); SSIDTD (SEQ ID NO: 27).

[0030] In some embodiments, the VH and VL sequences comprise complementarity determining region (CDR) amino acid sequences as shown below: HCDR3 as shown in SEQ ID NO:3, and LCDR1 (KW027-2-032) as shown in SEQ ID NO:15; or, HCDR3 as shown in SEQ ID NO:4, and LCDR1 (KW027-2-033) as shown in SEQ ID NO:16; or, HCDR3 as shown in SEQ ID NO:5, and LCDR1 (KW027-2-042) as shown in SEQ ID NO:17; or, HCDR3 as shown in SEQ ID NO:6, and LCDR1 (KW027-2-055) as shown in SEQ ID NO:18; or, HCDR3 as shown in SEQ ID NO:7, and LCDR1 (KW027-2-060) as shown in SEQ ID NO:19; or, HCDR3 as shown in SEQ ID NO:8, and LCDR1 (KW027-2-085) as shown in SEQ ID NO:20; or, HCDR3 as shown in SEQ ID NO:9, and LCDR1 (KW027-2-088) as shown in SEQ ID NO:21; or, HCDR3 as shown in SEQ ID NO:10, and LCDR1 (KW027-2-094) as shown in SEQ ID NO:22; or, HCDR3 as shown in SEQ ID NO:11, and LCDR1 (KW027-2-100) as shown in SEQ ID NO:23; or, HCDR3 as shown in SEQ ID NO:12, and LCDR1 (KW027-2-112) as shown in SEQ ID NO:24; or, HCDR3 as shown in SEQ ID NO:13, and LCDR1 (KW027-2-113) as shown in SEQ ID NO:25; or, HCDR3 as shown in SEQ ID NO:14, and LCDR1 (KW027-2-116) as shown in SEQ ID NO:26; HCDR3 as shown in SEQ ID NO:6, and LCDR1 (KW027-2-055-YD / HD) and amino acid sequences having at least 85% identity to the above VH or VL sequences.

[0031] The antibody and antibody fragments thereof that specifically bind to hepatitis B virus surface antigen (HBsAg), which further comprise a human consensus framework region (FR).

[0032] In some embodiments, VH comprises the FR amino acid sequence as shown below:

[0033] HFR1: QVQLVESGAEVKKPGASVKVSCKAS

[0034] HFR2: MHWVRQAPGQGLEWMGW

[0035] HFR3: NYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYC

[0036] HFR4: WGKGTLVTVSS

[0037] VL contains the FR amino acid sequences as shown below:

[0038] LFR1: DIQLTQSPSSLSASVGDRVTITCRAS

[0039] LFR2: LNWYQQKPGKAPKLLIY

[0040] LFR3: SLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC

[0041] LFR4: FGGGTKVDIKR.

[0042] In other embodiments, VH contains the FR amino acid sequences as shown below:

[0043] HFR1: QVQLVESGAEVKKPGASVKVSCKAS

[0044] HFR2: MHWVRQAPGQGLEWMGW

[0045] HFR3: YYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYC

[0046] HFR4: WGKGTLVTVSS

[0047] VL contains the FR amino acid sequences as shown below:

[0048] LFR1: DIQLTQSPSSLSASVGDRVTITCRAS

[0049] LFR2: LNWYQQKPGKAPKLLIY

[0050] LFR3: SLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC

[0051] LFR4: FGGGTKVDIKR.

[0052] In some further embodiments, VH comprises the FR amino acid sequences as shown below:

[0053] HFR1: QVQLVESGAEVKKPGASVKVSCKAS

[0054] HFR2: MHWVRQAPGQGLEWMGW

[0055] HFR3: HYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYC

[0056] HFR4: WGKGTLVTVSS

[0057] VL comprises the FR amino acid sequences as shown below:

[0058] LFR1: DIQLTQSPSSLSASVGDRVTITCRAS

[0059] LFR2: LNWYQQKPGKAPKLLIY

[0060] LFR3: SLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC

[0061] LFR4: FGGGTKVDIKR.

[0062] In some specific embodiments, the antibody of the present invention, wherein the VH sequence and the VL sequence are selected from the amino acid sequences of any one of the following groups:

[0063] 1) SEQ ID NO:30 and SEQ ID NO:45 (KW027-021),

[0064] 2) SEQ ID NO:31 and SEQ ID NO:46 (KW027-2-032),

[0065] 3) SEQ ID NO:32 and SEQ ID NO:47 (KW027-2-033),

[0066] 4) SEQ ID NO:33 and SEQ ID NO:48 (KW027-2-042),

[0067] 5) SEQ ID NO:34 and SEQ ID NO:49 (KW027-2-055),

[0068] 6) SEQ ID NO:35 and SEQ ID NO:50 (KW027-2-060),

[0069] 7) SEQ ID NO: 36 and SEQ ID NO: 51 (KW027-2-085),

[0070] 8) SEQ ID NO: 37 and SEQ ID NO: 52 (KW027-2-088),

[0071] 9) SEQ ID NO: 38 and SEQ ID NO: 53 (KW027-2-094),

[0072] 10) SEQ ID NO: 39 and SEQ ID NO: 54 (KW027-2-100),

[0073] 11) SEQ ID NO: 40 and SEQ ID NO: 55 (KW027-2-112),

[0074] 12) SEQ ID NO: 41 and SEQ ID NO: 56 (KW027-2-113),

[0075] 13) SEQ ID NO: 42 and SEQ ID NO: 57 (KW027-2-116),

[0076] 14) SEQ ID NO: 43 and SEQ ID NO: 58 (KW027-2-055-YD), or

[0077] 15) SEQ ID NO: 44 and SEQ ID NO: 58 (KW027-2-055-HD).

[0078] The amount of the antibody or its antigen-binding fragment contained in the antibody composition of the present invention can vary depending on the specific target properties of the composition, the specific environment, and the specific purpose of the preparation used. In one embodiment, the concentration of the antibody specifically binding to HBsAg in the antibody composition of the present invention is about 10 - 300 mg / ml. In another embodiment, the concentration of the antibody specifically binding to HBsAg in the antibody composition of the present invention is about 50 - 250 mg / ml. In other embodiments, the concentration of the antibody specifically binding to HBsAg in the antibody composition of the present invention is about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250 mg / ml. In a specific embodiment, the antibody concentration in the antibody composition of the present invention can be as high as about 200 mg / mL. For example, the antibody can stably exist under repeated freeze-thaw tests at about 200 mg / mL.

[0079] A buffer is a reagent that can maintain the pH of a solution within an acceptable range. In one embodiment, the concentration of the buffer in the antibody composition of the present invention is about 5 - 15 mM. In some embodiments, the concentration of the buffer in the antibody composition of the present invention is 5, 10, 15 mM. In some specific embodiments, the concentration of the buffer in the antibody composition of the present invention is about 10 mM.

[0080] In some embodiments, the buffer used in the composition of the present invention can control the pH of the composition of the present invention within a pH range of approximately 5.0 - 7.0, such as a pH value of about 6.0.

[0081] In some embodiments, the buffer is selected from citric acid - sodium citrate, histidine - histidine hydrochloride, sodium dihydrogen phosphate - disodium hydrogen phosphate, acetic acid - sodium acetate, sodium citrate. In one embodiment, the buffer used in the preparation of the present invention is a histidine - histidine hydrochloride composition at about 5 - 15 mmol / L. In one embodiment, the buffer used in the preparation of the present invention is a citric acid - sodium citrate composition at about 5 - 15 mmol / L. In one embodiment, the buffer used in the preparation of the present invention is an acetic acid - sodium acetate composition at about 5 - 15 mmol / L.

[0082] In one embodiment, the concentration of the cryoprotectant in the antibody composition of the present invention is about 5 - 30 mg / ml.

[0083] In one embodiment, the protective agent is selected from polyols (e.g., sorbitol), saccharides (e.g., sucrose), amino acids (e.g., arginine), and any combination thereof. In a specific embodiment, the stabilizer is a composition of mannitol at a concentration of 5 - 20 mg / ml and proline at a concentration of 5 - 20 mg / ml.

[0084] In some embodiments, the concentration of the surfactant in the antibody composition of the present invention is about 0.1 - 0.3 mg / ml, such as about 0.1, 0.2, 0.3 mg / ml.

[0085] In one embodiment, the surfactant is a non-ionic surfactant. In one embodiment, the surfactant is selected from polysorbate surfactants. In a specific embodiment, the surfactant in the antibody composition of the present invention is polysorbate-80 at 0.1 - 0.3 mg / ml.

[0086] In one embodiment, the concentration of the osmotic pressure regulator in the antibody composition of the present invention is about 0 - 20 mg / ml.

[0087] In one embodiment, the antibody composition of the present invention optionally further comprises an osmotic pressure regulator selected from glucose and sodium chloride. In a specific embodiment, the osmotic pressure regulator in the antibody composition of the present invention is sodium chloride.

[0088] In one embodiment, the antibody composition of the present invention comprises an antioxidant selected from any one or more of methionine or EDTA·2Na; in a specific embodiment, the surfactant in the antibody composition of the present invention is methionine at 01 - 10 mg / ml.

[0089] One embodiment of the present invention relates to a stable antibody composition, wherein the antibody composition contains:

[0090] (1) Antibodies and antigen-binding fragments thereof that specifically bind to HBsAg at a concentration of 20 - 200 mg / ml;

[0091] (2) A buffer at a concentration of 5 mM - 15 mM, the buffer being selected from citric acid - sodium citrate, histidine - histidine hydrochloride, sodium dihydrogen phosphate - disodium hydrogen phosphate, acetic acid - sodium acetate, sodium citrate;

[0092] (3) A protective agent at a concentration of 5 - 20 mg / ml, the protective agent being selected from one or more of sugars, alcohols, amino acids, and chlorides;

[0093] (4) A surfactant with a concentration of 0.1 mg / ml to 0.3 mg / ml, wherein the surfactant is selected from nonionic polymers, such as one or more selected from Tween 80, Tween 20, poloxamer, and polyethylene glycol;

[0094] (5) An osmotic pressure regulator with a concentration of 0 - 10 mg / ml, wherein the osmotic pressure regulator is selected from sodium chloride, sugars, or sugar alcohols;

[0095] (6) An antioxidant with a concentration of 0 - 10 mg / ml, wherein the antioxidant is selected from any one or more of methionine or EDTA·2Na, and the pH of the composition is 5.00 - 7.00;

[0096] In a specific embodiment, the antibody composition of the present invention comprises:

[0097] (1) An antibody specifically binding to HBsAg and its antigen-binding fragment with a concentration of 20 - 200 mg / ml;

[0098] (2) Acetic acid - sodium acetate with a concentration of 5 - 15 mmol / L;

[0099] (3) A composition of mannitol with a concentration of 5 - 20 mg / ml and proline with a concentration of 5 - 20 mg / ml;

[0100] (4) Tween 80 with a concentration of 0.1 - 0.3 mg / ml; and

[0101] (5) Methionine with a concentration of 0 - 10 mg / ml;

[0102] The pH of the composition is 5.00 - 7.00;

[0103] In one embodiment, the liquid composition is a pharmaceutical preparation, preferably an injection, such as a subcutaneous injection, intramuscular injection, intravenous injection, or intravenous infusion.

[0104] The liquid composition of the present invention can be stored stably for a long time, for example, at least 24 months or longer. In one embodiment, the liquid preparation of the present invention can be stored at about -80°C to about 45°C, such as -80°C, about -30°C, about -20°C, about 0°C, about 5°C, about 25°C, about 35°C, about 38°C, about 40°C, about 42°C, or about 45°C for at least 10 days, at least 20 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, or longer, and is stable.

[0105] In one embodiment, the liquid composition of the present invention can be stably stored for at least 24 months. In one embodiment, the liquid preparation of the present invention is stable at at least 40°C. In one embodiment, the liquid preparation of the present invention remains stable at about 2°C - 8°C for at least 3 months, preferably at least 12 months, more preferably at least 24 months. In one embodiment, the liquid preparation of the present invention remains stable at room temperature or, for example, at about 25°C for at least 2 months, preferably at least 3 months, more preferably at least 6 months. In yet another embodiment, the liquid preparation of the present invention remains stable at about 40°C for at least 2 weeks, preferably at least 1 month.

[0106] Method for Preparing Antibody Composition of the Present Invention

[0107] On the one hand, the present invention also provides a method for preparing the above-mentioned antibody preparation, which comprises the following steps:

[0108] (1) Dissolve the weighed buffer, stabilizer, and surfactant in water for injection;

[0109] (2) Adjust the liquid prepared in step (1) to a pH of 5 - 7 with an aqueous sodium hydroxide solution; preferably, the concentration of the aqueous sodium hydroxide solution is 1M;

[0110] (3) Filter the liquid prepared in step (2) into a sterile container; preferably, the pore size of the filter membrane is 0.22 μm, which is used to filter bacteria and fungi;

[0111] (4) Add the liquid prepared in step (3) to the antibody solution.

[0112] Stability Analysis of Antibody Composition of the Present Invention

[0113] In one embodiment, the stability of the preparation after storage can be indicated by detecting changes in the appearance, visible foreign matters, insoluble particles, protein content, turbidity, purity, and / or relative binding activity of the preparation.

[0114] In one embodiment, the stability of the liquid preparation of the present invention can be detected in a forced experiment under high-temperature stress, for example, after storing at 40°C ± 2°C for at least 1 week, 2 weeks, or preferably 1 month, or in an accelerated experiment, for example, after storing at 25°C ± 2°C for at least 1 month or 2 months, or in a long-term experiment, for example, after storing at 5°C ± 3°C for at least 2 months or 3 months, or after an oscillation experiment, or after a freeze-thaw experiment, or after a light experiment.

[0115] In one embodiment, after storage, the stability of the liquid preparation of the present invention is visually inspected, wherein the liquid preparation of the present invention remains lighter in color than BR-1 in appearance and has no foreign matters. In one embodiment, visually inspected under a clarity detector, there are no visible foreign matters in the preparation.

[0116] In one embodiment, after storage, the stability of the liquid preparation of the present invention is examined by measuring the change in protein content. For example, by ultraviolet spectrophotometry (UV), the rate of change in protein content does not exceed 20%, preferably does not exceed 10%, for example 7 - 8%, and more preferably does not exceed 5%, relative to the initial value on day 0 of storage.

[0117] In one embodiment, after storage, the stability of the liquid preparation of the present invention is examined by measuring the change in turbidity of the liquid preparation of the present invention. For example, detected by the OD350mm method, the change value does not exceed 0.06, preferably does not exceed 0.05, and more preferably does not exceed 0.04, relative to the initial value on day 0 of storage.

[0118] In one embodiment, after storage, the stability of the liquid preparation of the present invention is examined by measuring the change in purity of the liquid preparation of the present invention. By size exclusion high performance liquid chromatography (SEC - HPLC), the change value of monomer purity does not exceed 10%, for example does not exceed 5%, 4%, 3%, for example the change value does not exceed 1 - 2%, and preferably does not exceed 1%, relative to the initial value on day 0 of storage.

[0119] In one embodiment, after storage, the stability of the liquid preparation of the present invention is examined by measuring the change in purity of the liquid preparation of the present invention. By non - reducing and / or reducing sodium dodecyl sulfate capillary electrophoresis (CE - SDS) method, the decrease in the change value of monomer purity does not exceed 10%, for example does not exceed 5%, 4%, 3%.

[0120] In one embodiment, after storage, the stability of the liquid preparation of the present invention is detected by imaging capillary isoelectric focusing electrophoresis (iCIEF). The total change value of the charge variants (main component, acidic component, and basic component) of the antibody does not exceed 50%, for example does not exceed 40%, 30%, 20%, 10%, 5%, relative to the initial value on day 0 of storage.

[0121] In one embodiment, the preparation is stable after forced degradation tests, for example, after being repeatedly freeze - thawed 3 times at - 70°C ± 10°C / 5°C ± 3°C, and preferably has one or more of the following characteristics:

[0122] (i) Measured by ultraviolet spectrophotometry (UV), the rate of change in protein content does not exceed 10%, preferably does not exceed 5%, more preferably does not exceed 3%, does not exceed 2%, does not exceed 1%, does not exceed 0.5%, does not exceed 0.2%, and even shows substantially no degradation;

[0123] (ii) Measured by SEC - HPLC method, the preparation has a purity greater than 90%, preferably greater than 92%, 94%,

[0124] Purities of 96%, 98%, and 99%;

[0125] (iii) Measured by CEX-HPLC method;

[0126] (iv) Measured by reduced or non-reduced CE-SDS method, and the preparation has a purity greater than 90%, preferably greater than

[0127] 92%, 94%, 96%, 98%, 99%;

[0128] (v) Determining the relative binding activity of the antibody to the antigen by ELISA method.

[0129] Use of the Composition

[0130] The present invention also provides a method for treating HBV infection, which comprises administering a suitable dose of the aforementioned pharmaceutical composition to a patient. The suitable dose is adjusted according to different factors such as the age and body size of the subject, the target disease, symptoms, administration route, etc. When the pharmaceutical composition of the present invention is an antibody or its antigen-binding fragment specifically binding to HBsAg for treating various conditions and diseases related to hepatitis B virus infection in adults, the pharmaceutical composition can be administered by intravenous, subcutaneous or intramuscular injection. Usually, a single dose is administered at about 0.01 to about 20 mg of antibody per kilogram of body weight, such as about 0.1 to about 15, about 1 to about 10, about 3 to about 10 mg / kg body weight (mpk), about 12 mpk body weight. According to the severity of the condition, the frequency and duration of treatment can be adjusted. For example, the patient is administered once a week.

[0131] The pharmaceutical composition can be administered alone or in combination, and the symptoms of the patient are improved by reducing the load of hepatitis B virus surface antigen. In some embodiments, the administration is in combination, wherein the HBsAg antibody or its antigen-binding fragment is administered in combination with one or more therapeutic agents (or the second therapeutic agent). Co-administration and combination therapy are not limited to simultaneous administration, but also include treatment regimens in which the anti-HBsAg antibody or its antigen-binding fragment is administered at least once during a course of treatment involving administering at least one other therapeutic agent to the patient. The second therapeutic agent may be another HBV therapeutic drug, such as another antibody / antibody fragment, or a soluble cytokine receptor (such as interferon, interleukin, thymosin alpha 1, etc.), a nucleoside analogue (tenofovir, entecavir, adefovir, etc.).

[0132] Another aspect of the present invention relates to an article or a medicine box, comprising a container and a packaging insert, wherein the container contains the composition of the present invention, and the packaging insert carries the instructions for use of the drug. In a preferred embodiment, the article or the medicine box further comprises one or more containers which contain one or more other drugs for treating hepatitis B.

[0133] Suitable containers include, for example, ampoules, vials for injections, syringes, etc. The container can be formed of a variety of substances (such as glass or plastic), and the container contains or holds a small composition for treatment and can have a sterile access end (for example, the container can be an intravenous solution package or a bottle with a stopper that can be pierced by a subcutaneous injection needle). At least one active agent in the composition is the antibody or antigen-binding fragment of the present invention. The label or the medicine instructions indicate that the composition is used for treating the metabolic-related diseases, disorders or symptoms of an individual suffering from metabolic-related diseases, disorders or symptoms under the specific guidance of the dosage intervals of the antibody and any other drugs provided. The article can also include a second container, and the second container contains a pharmaceutical diluent buffer, bacteriostatic water for injection, phosphate buffer solution, Ringer's solution and glucose solution. The article can also include other substances that are required from a commercial and user perspective, including other buffers, diluents, filters, needles and syringes. The use of "medicine instructions" generally includes the instructions in the commercial packaging of a therapeutic product, which contain information such as indications, usage, dosage, administration, taboos, other therapeutic products combined with the packaged product and / or warnings regarding the use of these therapeutic products.

[0134] The article can also include other components, and each component of the article can be packaged in a single container and all the multiple containers can be placed in a single package.

[0135] Term definitions:

[0136] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0137] In this article, "antibody preparation" can be used interchangeably with "antibody composition", and refers to a preparation which is in a form that allows the biological activity of the antibody as an active ingredient to be effectively exerted and does not contain other components that have unacceptable toxicity to the subject to whom the preparation is to be administered.

[0138] As used herein, "stability" and "stable" mean that in a liquid preparation containing an antibody (including its antibody fragments), the antibody (including its antibody fragments) does not aggregate, degrade, or fragment, or only very rarely does so, under given production, preparation, transportation, and / or storage conditions. A "stable" preparation retains its biological activity under given production, preparation, transportation, and / or storage conditions. The degree of aggregation, degradation, or fragmentation of the preparation, which can be measured by techniques such as SEC-HPLC, IEC-HPLC, CE-SDS, etc., is used to evaluate the stability of the antibody (including its antibody fragments). Brief Description of the Drawings

[0139] Figure 1 Shows the binding activity of the antibody of the present invention to the antigen at different pH values.

[0140] Figure 2 Shows the effect of different antibodies of the present invention on the HBsAg content in the plasma of HBV mice.

[0141] Figure 3 Shows the effect of the antibodies KW027-021, KW027-2-055, KW027-2-055-HD, and KW027-2-055-YD of the present invention on the HBsAg content in the plasma of HBV mice. Detailed Description of the Invention

[0142] The technical solutions of the present invention will be described clearly and completely below. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention. The following non-limiting embodiments can enable those of ordinary skill in the art to more comprehensively understand the present invention, but do not limit the present invention in any way. The following content is only an exemplary description of the scope claimed in the present application. Those skilled in the art can make various changes to the invention of the present application based on the disclosed content, and it should also fall within the scope claimed in the present application.

[0143] Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the described content can be applied to the methods of the present invention. The preferred methods and materials described herein are for illustrative purposes only.

[0144] Description of Abbreviations

[0145] RH: Relative Humidity, relative humidity

[0146] SEC-HPLC: Size Exclusion - High Performance Liquid Chromatography

[0147] CE-SDS: Capillary Electrophoresis with Sodium Dodecyl Sulfate

[0148] NR CE-SDS: Non-Reductive Capillary Electrophoresis with Sodium Dodecyl Sulfate

[0149] R CE-SDS: Reductive Capillary Electrophoresis with Sodium Dodecyl Sulfate

[0150] CEX-HPLC: Weak Cation Exchange - High Performance Liquid Chromatography

[0151] Detection Methods

[0152] The following items were detected for the antibody preparation:

[0153] (i) Detect the appearance and the presence of visible foreign matters;

[0154] (ii) Detect the size and quantity of insoluble particles;

[0155] (iii) Determine the protein content in the preparation by ultraviolet method (UV method);

[0156] (iv) Determine the purity of the antibody preparation by size-exclusion chromatography high performance liquid chromatography (SEC-HPLC), expressed as the percentage of the area of the monomer in the sum of the areas of all peaks;

[0157] (v) Determine the charge variants in the antibody preparation by CEX-HPLC method, expressed as the percentages of the main component, acidic components and basic components;

[0158] (vi) Determine the purity of the antibody preparation by reductive sodium dodecyl sulfate capillary electrophoresis (reductive CE-SDS) and / or non-reductive sodium dodecyl sulfate capillary electrophoresis (non-reductive CE-SDS), expressed as the percentage of the area of the monomer in the sum of the areas of all peaks;

[0159] (vii) Determine the relative binding activity of the antibody to HBsAg antigen in the antibody preparation by immunoassay methods, such as direct ELISA method.

[0160] Among them, the general methods involved include:

[0161] 1 Detection of Visible Foreign Matters

[0162] Examine according to the "Examination Method for Visible Foreign Matters" in General Chapter <0904> of Chinese Pharmacopoeia 2015 Edition.

[0163] 2 Detection of Insoluble Particles

[0164] It was examined according to the "Insoluble Particles Inspection Method" in <General Principles 0903> of the Chinese Pharmacopoeia (2020 Edition).

[0165] 3 Protein Content Determination

[0166] The protein content in the sample was determined using an ultraviolet spectrophotometer (produced by Shimadzu, Japan, model UV-1900I).

[0167] 4 Purity (SEC-HPLC Method)

[0168] Separation was carried out using a size exclusion chromatography column (XBridge BEH 200 A SEC). The mobile phase was phosphate buffer solution. The injection volume was 50 μL, the flow rate was 0.8 mL / min, the running time was 25 min, the column temperature was 25 °C, the detection wavelength was 280 nm, and the sample tray temperature was 5 °C. The sample to be tested was diluted to 2 mg / mL with the mobile phase and transferred into a liquid phase vial as the test solution. The formulation buffer solution was diluted in the same way as above and used as the blank solution. The blank solution and the test solution were placed in the sample chamber of the instrument to start the detection.

[0169] 5 Charge Variant (CEX-HPLC Method)

[0170] It was detected by cation exchange chromatography (CEX-HPLC method). Separation was carried out using a ProPac ELite WCX weak cation exchange chromatography column. Mobile phase A was 20 mM ACES, pH 6.0 buffer solution; mobile phase B was 20 mM ACES, 500 mM NaCl, pH 6.0 buffer solution. The sample was diluted to 1.0 mg / mL with mobile phase A and transferred into a liquid phase vial as the test solution. The formulation buffer solution was diluted in the same way as above and used as the blank solution. The blank solution and the test solution were placed in the sample chamber of the liquid chromatograph. The flow rate of the mobile phase was 0.5 mL / min, the running time was 35 min, the column temperature was 30 °C, the detection wavelength was 280 nm, the sample tray temperature was 5 °C. The elution gradient is shown in the following table, and the injection volume was 20 μL. Inject for analysis, and calculate the contents of the main peak, acidic peak and basic peak according to the area normalization method.

[0171] 6 Purity (Reduced CE-SDS Method, Non-reduced CE-SDS Method)

[0172] 6.1 Reduced CE-SDS Method

[0173] Detection was carried out by capillary gel electrophoresis. The capillary was an uncoated capillary with an inner diameter of 50 μm, a total length of 30.2 cm, and an effective length of 20 cm. Before electrophoresis, the capillary column was rinsed with 0.1 N sodium hydroxide, 0.1 N hydrochloric acid, and ultrapure water under a pressure of 70 psi of electrophoresis gel. Take 50 μL of the test sample diluted to a concentration of about 2 mg / mL, add 50 μL of SDS Sample Buffer, 5 μL of β-mercaptoethanol, and 5 μL of 250 mM iodoacetamide solution. After thoroughly mixing the above solution by shaking, centrifuge the sample to the bottom of the tube, heat it at 90 °C for 5 min, cool it to room temperature, and centrifuge for 2 minutes at 6000 rpm. Take 90 μL of the supernatant and add it to the inner insert tube, and place the inner insert tube in the sample vial for testing. Take the same volume of the preparation buffer as the test sample and operate in the same manner as above to prepare a blank solution. Sample injection conditions: reversed-phase polar electrokinetic injection, 5 kV, 20 s; separation voltage: -15 kV, with a pressure of 20 psi maintained at both the inlet and outlet ends of the capillary, for 30 min. The capillary column temperature was controlled at 25 °C, and the detection wavelength was 214 nm.

[0174] 6.2 Non-reducing CE-SDS method

[0175] Detection was carried out by capillary gel electrophoresis. The capillary was an uncoated capillary with an inner diameter of 50 μm, a total length of 30.2 cm, and an effective length of 20 cm. Before electrophoresis, the capillary column was rinsed with 0.1 N sodium hydroxide, 0.1 N hydrochloric acid, and ultrapure water under a pressure of 70 psi of electrophoresis gel. Take 50 μL of the test sample diluted to a concentration of about 2 mg / mL, add 50 μL of SDS Sample Buffer and 5 μL of 250 mM iodoacetamide solution. After thoroughly mixing the above solution by shaking, centrifuge the sample to the bottom of the tube, heat it at 90 °C for 5 min, cool it to room temperature, and centrifuge for 2 minutes at 6000 rpm. Take 90 μL of the supernatant and add it to the inner insert tube, and place the inner insert tube in the sample vial for testing. Take the same volume of the preparation buffer as the test sample and operate in the same manner as above to prepare a blank solution. Sample injection conditions: reversed-phase polar electrokinetic injection, 5 kV, 20 s; separation voltage: -15 kV, with a pressure of 20 psi maintained at both the inlet and outlet ends of the capillary, for 40 min. The capillary column temperature was controlled at 25 °C, and the detection wavelength was 214 nm. 7 Relative binding activity (ELISA method)

[0176] Dilute the antigen to a concentration of 2μg / mL with coating buffer, add 100μL / well to the wells of the ELISA plate and incubate overnight at 4℃. Take out the ELISA plate, discard the residual reaction liquid in the well, add 300μL PBST to each well, and wash the plate 3 times. Add 200μL of blocking solution to each well, cover the sealing film to prevent water evaporation and foreign matter from falling into the wells, and place it horizontally at 25℃ for 1h. The standard / test sample diluted to 90.00μg / mL was further diluted 4 times, and a total of 8 concentration points were obtained, namely 90μg / mL, 22.5μg / mL, 5.625μg / mL, 1.406μg / mL, 0.352μg / mL, 0.088μg / mL, 0.022μg / mL, and 0.0KW027-2-055μg / mL. Take out the sealed ELISA plate, discard the residual reaction liquid in the well, add 300μl PBST to each well, and wash the plate 3 times. Add 100μL of the prepared standard / test solution to each well in parallel. Cover the plate with a sealing film and place it horizontally at 25℃ for 1h. Take out the ELISA plate, discard the residual reaction liquid in the well, add 300μL PBST to each well, and wash the plate 3 times. Dilute the HRP-labeled goat anti-human secondary antibody with diluent at 1:10000, add 100μL to each well, cover the plate with a sealing film, and place it horizontally at 25℃ for 1h. Color development: Take out the ELISA plate, discard the residual reaction liquid in the well, add 300μL PBST to each well, and wash the plate 3 times. Add TMB substrate to the ELISA plate at 100μL / well, and let it stand at 25℃ in the dark for 30±2min to develop color. Add 100μL / well of stop solution to the ELISA plate to stop the reaction. Place the plate in an ELISA reader and select 450nm wavelength to measure absorbance. Use the concentration as the horizontal axis and the average absorbance as the vertical axis. Use the analysis software that comes with the ELISA reader to fit, select the regression model of the four-parameter equation, and obtain an "S"-shaped standard curve. The software automatically generates the half-maximum effect concentration (EC50), the correlation coefficient of the four-parameter equation curve (R 2 ).

[0177] Example

[0178] Example 1: Construction of HBsAg antibody expression vector

[0179] The HindIII restriction site, Kozak sequence, secretion signal peptide gene and HBsAg antibody heavy chain encoding gene (including VH amino acid sequence encoding gene and human IgG1 constant region encoding gene), termination code and EcoRI encoding gene were sequentially fused in series, and the gene fragment was obtained by chemical synthesis. Through EcoRI and HindIII sites, the above fragment was inserted into the eukaryotic expression plasmid pCDNA3.1(+) (purchased from Invitrogen Corportation, catalog number V790-20) and sequenced to obtain the expression plasmid pCDNA3.1(+)-DH for HBsAg antibody heavy chain.

[0180] The HindIII restriction site, Kozak sequence, secretion signal peptide gene, and HBsAg light chain coding gene (including the coding gene for the VL amino acid sequence and the coding gene for the κ-chain constant region), stop code, and EcoRI coding gene were successively concatenated and fused, and a gene fragment was obtained by chemical synthesis. Through the EcoRI and HindIII sites, the above fragment was inserted into the eukaryotic expression plasmid pCDNA3.1(+) and verified by sequencing to obtain the expression plasmid pCDNA3.1(+)-DL for the antibody light chain.

[0181] Among the HBsAg antibodies tested in the examples of this application, the antibodies numbered KW027-021, KW027-2-032, KW027-2-033, KW027-2-042, KW027-2-055, KW027-2-060, KW027-2-085, KW027-2-088, KW027-2-094, KW027-2-100, KW027-2-112, KW027-2-113, KW027-2-116, KW027-2-055-YD, and KW027-2-055-HD, the specific VH and VL sequences are SEQ ID NO30-44 and SEQ ID NO: 45-58, respectively.

[0182] The heavy chain constant region of each antibody is as shown in SEQ ID NO:59, and the light chain constant region is as shown in SEQ ID NO:60.

[0183] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:59)

[0184] TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:60)

[0185] Example 2: Expression and purification of anti-HBsAg antibody

[0186] Using the expression plasmids pCDNA3.1(+)-DH and pCDNA3.1(+)-DL described in Example 1, the target antibody was expressed by eukaryotic expression cells ExpiCHO-S TM cells (ThermoFisher, A29127). According to the ExpiCHO-S Expression System operation manual, the cell density was adjusted to 1x10 6 cells / mL one day before plasmid transfection. On the day of plasmid transfection, the plasmids were combined according to the corresponding relationship between the heavy chain and the light chain (that is, the light chain and the heavy chain of the antibody with the same number were combined), mixed with the transfection reagent, and added to the cell culture medium. After culturing at 37°C and 8% CO2 for about 22 hours, feeding was carried out, and continuous culture was carried out at 32°C and 5% CO2 for 10 - 12 days, and the cell culture supernatant was collected for antibody purification.

[0187] The expression supernatant was filtered through a 0.22 μM filter membrane, and the expressed antibody was captured from the expression supernatant by Protein A affinity chromatography (purchased from Sepax, 65008). After equilibrating the chromatography column with the equilibration buffer (20 mM Tris + 100 mM NaCl, pH 7.5), it was passed through the affinity chromatography column, and eluted with the elution buffer (0.15 M glacial acetic acid, pH 3.0). The purified antibody was detected by SDS PAGE and SEC, and the purity was above 95%.

[0188] It can be seen that the amino acids selected for each Xn and each Zn in the antibodies used in the examples are different. Therefore, these antibodies can be used to test the change in the affinity of the antibody specifically binding to the antigen caused by the change in the values of the VH and VL sequence general formulas in the Xn and / or Zn parameters.

[0189] Example 3: Determination of antigen-binding activity at different pH values

[0190] Take an ELISA plate coated with HBsAg antigen (500 ng / mL, self-isolated by the applicant, such as SEQ ID NO: 59, which contains the amino acid sequence shown below). After blocking, add 50 μl of the antibody to be tested (concentration 2.5 μg / ml) to each well and incubate at room temperature for 2 h (two wells for each antibody). Subsequently, wash the plate 3 times with PBS at pH 7.4 for odd-numbered columns and at pH 6.0 for even-numbered columns, 3 min each time; then wash the plate 3 times with PBST at the corresponding pH, 3 min each time, and finally wash the whole plate 3 times with PBS at pH 7.4, 3 min each time. Add 50 μL of anti M13-HRP diluted 1:5000 and incubate at 37 °C for 30 min. Subsequently, wash the ELISA plate 4 times with PBS and add the substrate TMB solution. When the positive control well shows color after about 15 min, terminate the color reaction with H2SO4 and measure the reading at OD450.

[0191] The results showed that the binding activities of antibodies 032, 033, 042, KW027-2-055, 060, 085, 088, 094, 100, 112, 113, 116 to the antigen under neutral conditions (i.e., pH 7.4) were not weaker than those of the positive control antibody 021, and the difference in binding activities between neutral conditions and acidic conditions (i.e., pH 6.0) was not less than that of 021.

[0192] Take an ELISA plate coated with HBsAg (500 ng / mL, self-isolated by the applicant, which contains the amino acid sequence shown below) antigen. Add 50 μL of the blocked antibody to each well and incubate at room temperature for 2 h (two wells for each antibody). Subsequently, wash the plate 3 times with PBS at pH 7.4 for odd-numbered columns and at pH 6.0 for even-numbered columns, 3 min each time; then wash the plate 3 times with PBST at the corresponding pH, 3 min each time, and finally wash the whole plate 3 times with PBS at pH 7.4, 3 min each time. Add 50 μL of anti M13-HRP diluted 1:5000 and incubate at 37 °C for 30 min. Subsequently, wash the ELISA plate 4 times with PBS and add the substrate TMB solution. When the positive control well shows color after about 15 min, terminate the color reaction with H2SO4 and measure the reading at OD450.

[0193] The results showed that the binding activities of antibodies 032, 033, 042, KW027-2-055, 060, 085, 088, 094, 100, 112, 113, 116 to the antigen under neutral conditions (i.e., pH 7.4) were not weaker than those of the positive control antibody 021, and the difference in binding activities between neutral conditions and acidic conditions (i.e., pH 6.0) was not less than that of 021.

[0194] Example 4: In vivo pharmacodynamic experiment

[0195] In this experiment, the constant region of the antibody was selected as human IgG1.

[0196] Animals:

[0197] Male C57BL / 6 mice, 5 weeks old, specific pathogen-free, were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. and housed in individually ventilated cages. The breeding and use of the mice were approved by the experimental protocol of WuXi AppTec IACUC (IACUC#: ID01-013-2021v1.0). After a 4-day environmental adaptation period, the mice were injected with AAV / HBV virus (i.e., recombinant adeno-associated virus vector type 8 carrying 1.3 copies of hepatitis B virus rAAV8-1.3HBV, in this article, "AAV / HBV virus" and "rAAV8-1.3HBV" can be used interchangeably)

[0198] Solvent: PBS.

[0199] Dose of the test compound: 15 mpk.

[0200] Recombinant rAAV8-1.3HBV: rAAV8-1.3HBV (serotype D, ayw) was provided by WuXi AppTec, with the batch number ayw1-P4-200805, 1×10 12 viral genomes (v.g.) / mL. Before the experiment, it was diluted to 1×10 10 v.g. / 200 μL with sterile PBS. Each mouse was injected with 200 μL, that is, each mouse was injected with 1×10 10 v.g.

[0201] Test method:

[0202] Establishment of AAV / HBV mouse model

[0203] AAV / HBV injection. rAAV8-1.3HBV was pre-prepared with sterile PBS to a concentration of 1×10 11 1×10 10 v.g. / 200 μL solution. 33 mice were injected with 200 μL of rAAV8-1.3HBV solution via the tail vein.

[0204] Blood collection before grouping. On the 14th and 21st days after virus injection, all infected mice were bled ~60 μL from the submandibular vein for plasma collection. The collected venous blood was anticoagulated with K2-EDTA and centrifuged at 4°C, 7000 g / min for 10 minutes to collect plasma. Plasma was detected for HBsAg by ELISA. Plasma samples were stored at -80°C until sent to the in vitro laboratory of WuXi AppTec Biology Department for relevant item detection.

[0205] The first administration was recorded as day 0, which was also day 0 after infection. Mice were grouped according to the plasma HBsAg levels collected on days 14 and 21 after virus injection. Thirty-three mice were selected from 45 mice for the formal experiment and randomly divided into 11 groups, labeled as group 1 to group 11, with 3 mice in each group. All mice were injected with PBS or the test antibody via the tail vein at a dose of 15 mpk, once. All mice were weighed before administration.

[0206] Blood collection:

[0207] On days -14, -3, 1, 3, 5, 7, 10, 14, 17, 21, 24, and 28 after administration, all mice were bled via the submandibular vein to collect plasma for HBsAg detection.

[0208] Experimental endpoint: On day 28, all mice were first bled via the submandibular vein for ~60 μL to collect plasma for HBsAg detection, and then euthanized.

[0209] Body weight recording: During the in-vivo experiment, the status of the mice was observed daily, and the body weights of the mice were recorded on the day of administration and infection, as well as on the day of euthanasia.

[0210] Sample preservation and transfer. All plasma samples were stored at -80°C and transferred to the in-vitro laboratory of WuXi AppTec Biology Department using dry ice for corresponding detection.

[0211] ELISA was used to detect the content of HBsAg in mouse plasma. The experimental procedure referred to the instruction manual of the HBsAg ELISA (Antu Biological, CL0310) kit. Briefly, the plasma samples were diluted 20 or 600 times, added to the coated plate, incubated with the enzyme conjugate (37°C, 60 minutes), washed 5 times repeatedly, added with the luminescent substrate, reacted at room temperature in the dark for 10 minutes, and the luminescence intensity was detected using an enzyme-labeled instrument.

[0212] The inhibitory activity of each antibody against HBV replication in the AAV / HBV mouse model was evaluated by detecting the expression of HBsAg in mouse plasma. The results are as Figure 2 shown.

[0213] The results are as follows:

[0214] Effect of the test compound on plasma HBsAg in AAV / HBV mice

[0215] The HBsAg content in the plasma of mice in the solvent group (Group 1) remained relatively stable during the experiment, fluctuating between 4.27 - 4.73 log10 IU / mL (the fluctuation did not exceed 0.46 log10 IU / mL); compared with the solvent group, the HBsAg in the plasma of mice in Treatment Group 2 (Antibody 021, 15 mpk) decreased significantly on the 1st day after administration, with an average decrease of 1.03 log10 IU / mL (p < 0.01), and then showed a gradually increasing trend. By the 28th day, the HBsAg in the plasma of mice was 4.23 log10 IU / mL; the HBsAg in the plasma of mice in Treatment Group 3 (Antibody 033, 15 mpk) decreased significantly on the 1st day after administration, with an average decrease of 2.40 log10 IU / mL (p < 0.01), and then showed a gradually increasing trend. By the 28th day, the HBsAg in the plasma of mice was 4.23 log10 IU / mL; the HBsAg in the plasma of mice in Treatment Group 4 (Antibody KW027 - 2 - 055, 15 mpk) decreased significantly on the 1st day after administration, with an average decrease of 2.61 log10 IU / mL (p < 0.01), and then showed a gradually increasing trend. By the 28th day, the HBsAg in the plasma of mice was 2.97 log10 IU / mL; the HBsAg in the plasma of mice in Treatment Group 5 (Antibody 060, 15 mpk) decreased significantly on the 1st day after administration, with an average decrease of 1.00 log10 IU / mL (p < 0.01). By the 28th day, the HBsAg in the plasma of mice was 4.02 log10 IU / mL; the HBsAg in the plasma of mice in Treatment Group 6 (Antibody 085, 15 mpk) decreased significantly on the 1st day after administration, with an average decrease of 1.27 log10 IU / mL (p < 0.01). By the 28th day, the HBsAg in the plasma of mice was 4.12 log10 IU / mL; the HBsAg in the plasma of mice in Treatment Group 7 (Antibody 088, 15 mpk) decreased significantly on the 1st day after administration, with an average decrease of 1.98 log10 IU / mL (p < 0.01). By the 28th day, the HBsAg in the plasma of mice was 4.23 log10 IU / mL; the HBsAg in the plasma of mice in Treatment Group 8 (Antibody 094, 15 mpk) decreased significantly on the 1st day after administration, with an average decrease of 1.93 log10 IU / mL (p < 0.01). By the 28th day, the HBsAg in the plasma of mice was 3.75 log10 IU / mL; the HBsAg in the plasma of mice in Treatment Group 9 (Antibody 100, 15 mpk) decreased significantly on the 1st day after administration, with an average decrease of 2.46 log10 IU / mL (p < 0.01). By the 28th day, the HBsAg in the plasma of mice was 4.53 log10 IU / mL; the HBsAg in the plasma of mice in Treatment Group 10 (Antibody 113, 15 mpk) decreased significantly on the 1st day after administration, with an average decrease of 2.40 log10 IU / mL (p < 0.01), by day 28, the HBsAg in the plasma of the mice was 4.52 log10 IU / mL; in treatment group 11 (antibody 116, 15 mpk), the HBsAg in the plasma of the mice decreased significantly on the first day after dosing, with an average decrease of 1.05 log10 IU / mL (p<0.01), and by day 28, the HBsAg in the plasma of the mice was 4.04 log10 IU / mL. At the same time, compared with treatment group 2, all the test compounds could significantly reduce the content of HBsAg in the plasma. Among them, treatment group 4 (antibody KW027-2-055, 15 mpk) had the most significant effect. One day after dosing, the average decrease was 1.58 log10 IU / mL, and 28 days after dosing, the average decrease was 1.26 log10 IU / mL.

[0216] Example 5: In vivo activity assay of antibodies 021, KW027-2-055, KW027-2-055-YD, and KW027-2-055-HD

[0217] The experimental protocol was the same as that in Example 4. The mice were divided into 5 groups, with 4 mice in each group. The IgG1 used was murine IgG1. In groups G2 and G3, antibodies 021 and KW027-2-055 (dose 9 mpk) were injected into the tail veins. In groups G4 and G5, antibodies KW027-2-055-HD and KW027-2-055-YD (dose 9 mpk) were injected into the tail veins. Each group was dosed once, and the solvent group was injected with 15 mpk PBS. The HBsAg test results are shown in Figure 3 . The specific results are as follows:

[0218] The content of HBsAg in the plasma of the mice in each group.

[0219] The HBsAg content in the plasma of mice in the solvent group (Group 1) remained relatively stable during the experiment, fluctuating between 4.41–4.61 log10 IU / mL (the fluctuation did not exceed 0.20 log10 IU / mL); compared with the solvent group, the HBsAg in the plasma of mice in Treatment Group 2 (antibody 021m, 9 mpk) decreased significantly on the 1st day after administration, with an average decrease of 1.65 log10 IU / mL (p<0.01), and then showed a gradually increasing trend. By the 28th day, the HBsAg in the plasma of mice was 3.62 log10 IU / mL; the HBsAg in the plasma of mice in Treatment Group 3 (antibody KW027-2-055, 9 mpk) decreased significantly on the 1st day after administration, with an average decrease of 3.47 log10 IU / mL (p<0.01), and then showed a gradually increasing trend. By the 28th day, the HBsAg in the plasma of mice was 2.78 log10 IU / mL; the HBsAg in the plasma of mice in Treatment Group 4 (antibody KW027-2-055HD, 9 mpk) decreased significantly on the 1st day after administration, with an average decrease of 3.25 log10 IU / mL (p<0.01), and then showed a gradually increasing trend. On the 28th day, the HBsAg in the plasma of mice was 2.68 log10 IU / mL; the HBsAg in the plasma of mice in Treatment Group 5 (antibody KW027-2-055YD, 9 mpk) decreased significantly on the 1st day after administration, with an average decrease of 3.21 log10 IU / mL (p<0.01), and then showed a gradually increasing trend. By the 28th day, the HBsAg in the plasma of mice was 3.09 log10 IU / mL. At the same time, compared with Treatment Group 2, the test compounds could all significantly reduce the HBsAg content in the plasma. Among them, Treatment Group 3 (antibody KW027-2-055, 9 mpk) had the most significant effect. One day after administration, the average decrease was 1.82 log10 IU / mL, and 28 days after administration, the average decrease was 084 log10 IU / mL.

[0220] Example 6: Preparation of the stock solution of KW027-2-055 antibody protein

[0221] Prepare the fermentation broth of KW027-2-055 antibody according to Example 2, using Dynamis Medium supplemented with 0.2 g / L of DS as the basal medium, and Cell Boost TM 7a and Cell Boost TM 7b as the feeding medium. The initial culture temperature was 36.5 °C, and it was cooled to 33 °C on the 5th day, and cultured for a total of 14-16 days. The fermentation broth was subjected to Protein A affinity chromatography, low pH incubation, anion chromatography, cation chromatography and ultrafiltration. The final sample was stored in 10 mM histidine hydrochloride buffer, pH 5.9±0.2. This protein stock solution was used for the next formulation screening experiment.

[0222] 1) Protein A affinity chromatography

[0223] Diamond Protein AAT (Bogolong, XK26 / 40, column height 18.8 cm, CV = 99.76 mL). After the affinity chromatography elution peaks are combined after virus inactivation, the next chromatography is carried out, and the chromatography loading capacity is ≤ 40 mg antibody / ml packing material.

[0224] Equilibration buffer -1: 20 mM PB, 50 mM NaCl, pH 7.5 ± 0.2

[0225] Equilibration buffer -2: 20 mM PB, 500 mM NaCl, pH 6.0 ± 0.2

[0226] Elution buffer: 0.15 M acetic acid, pH 3.0 - 3.2

[0227] 2) Low pH incubation

[0228] Adjust the pH of the affinity chromatography elution peak to 3.5 ± 0.1 with 2 M acetic acid, incubate with the virus for 2 h, place at room temperature, adjust the pH back to 7.0 ± 0.2 with 1 M Tris, and dilute with water so that the conductivity ≤ 4 mS / cm.

[0229] 3) Anion exchange chromatography

[0230] Capto Q (GE Healthcare, XK26 / 20, column height 15.0 cm, CV = 79.6 mL), and the chromatography loading capacity is ≤ 80 mg antibody / ml packing material.

[0231] Equilibration buffer: 20 mM PB pH 7.0 ± 0.2

[0232] Adjust the pH of the Q chromatography breakthrough peak to 7.0 ± 0.2, and dilute so that the conductivity ≤ 4 mS / cm

[0233] 4) Cation exchange chromatography

[0234] POROS 50HS (Thermo Fisher, XK26 / 40, column height 17.7 cm, CV = 93.9 mL), and the chromatography loading capacity is ≤ 40 mg antibody / ml packing material.

[0235] Buffer A: 20 mM PB pH 7.0 ± 0.2

[0236] Buffer B: 20 mM PB + 150 mM NaCl pH 7.0 ± 0.2

[0237] 5) UF / DF

[0238] 30KD Membrane Package (Millipore, 50cm 2 ), loading capacity ≤ 600mg antibody / m 2 .

[0239] Displacement buffer: 10mM Histidine Hydrochloride Buffer, pH 5.9 ± 0.2

[0240] Example 7: Screening of Antibody Liquid Preparation Buffer System (Taking KW027-2-055 Antibody as an Example)

[0241] Material Information

[0242] Table 1 Material Information for Prescription Screening

[0243] Name Specification Manufacturer Batch Number Histidine 1 kg / bottle Merck K53464452 Histidine Hydrochloride 5 kg / bottle Pfanstiehl 04794A Sodium Citrate 5 kg / bottle Merck K93654132614 Citric Acid 5 kg / barrel Merck K52039842013 Sodium Dihydrogen Phosphate Monohydrate 1 kg / bag Hunan Jiudian T202007I06 Disodium Hydrogen Phosphate Anhydrous 1 kg / bag Hunan Jiudian T202107G02 Sodium Acetate Trihydrate 500 g / bottle Hunan Erkang 104520180605 Glacial Acetic Acid 500 ml / bottle Chengdu Huayi 20200316 Sucrose 500 g / bottle Guoyao Shanghai Style 20190508 Trehalose (for injection) 100 g / bottle Hayashibara 90220 Mannitol 2.5 kg / barrel AMRESCO 3406C501 Sorbitol 2.5 kg / bottle Merck MP19005397015 L-Arginine Hydrochloride 1 kg / bottle Merck K51914244010 Glycine 1 kg / bottle Merck V020042090037 Proline 100 g / bottle Guoyao Shanghai Style 20191209 Methionine N / A Tianjin Tianyao SMET130606 Sodium Chloride 1 kg / bag Jiangsu Qinfen 20200328 Disodium EDTA 500 g / bottle Nanjing Chemical 181114082C Polysorbate 80 500 g / bottle Nanjing Well Pharmaceutical Co., Ltd. 20181201-1 Polysorbate 20 500 g / bottle Nanjing Well Pharmaceutical Co., Ltd. 20190501-1K

[0244] Prepare KW027-2-055 antibody samples in citrate system, phosphate system, acetate and histidine salt system with different pH values. Through accelerated stability study at high temperature (40°C ± 2°C, 75% ± 5% RH), compare the effects of different pH values and different buffer solutions on the stability of KW027-2-055 antibody, so as to determine the buffer system and optimal pH of KW027-2-055 preparation prescription.

[0245] Sample Preparation

[0246] (1) Prepare mother liquor: According to Table 2, prepare mother liquor of preparation buffer in 6 different buffer systems.

[0247] (2) Membrane package buffer replacement: Prepare KW027-2-055 samples and replace the buffer in the membrane package to the corresponding target buffer according to Table 2.

[0248] (3) Preparation of final concentration samples: Use the mother liquor of the corresponding target buffer to dilute the samples after membrane package buffer replacement to the target concentration to obtain 6 groups of experimental group samples. At the same time, prepare sample blank control group samples with each mother liquor. (4) Sub-packaging: Sub-package the samples into vials at a specification of 0.5ml / vial, press the caps and store.

[0249] Table 2 First-round Preparation Prescription Screening of KW027-2-055 Antibody Injection Preparation Prescription

[0250]

[0251] Stability investigation: Through accelerated stability study at high temperature (40°C ± 2°C, 75% RH ± 5%RH), compare the effects of different pH values and different buffer solutions on the stability of KW027-2-055 antibody, so as to determine the buffer system and optimal pH of KW027-2-055 preparation prescription.

[0252] Appearance Detection Results and Discussion

[0253] After 4 weeks of high-temperature acceleration (40°C ± 2°C, 75% RH ± 5% RH), the appearance of the KW027-2-055 sample solution under different formulation buffer systems showed basically no change.

[0254] Protein content detection results and discussion

[0255] After 4 weeks of high-temperature acceleration (40°C ± 2°C, 75% RH ± 5% RH), the protein content of the KW027-2-055 sample solution under different formulation buffer systems showed basically no change (Table 3).

[0256] Table 3 Statistical table of protein content results in the high-temperature acceleration test for screening the formulation buffer system of KW027-2-055

[0257]

[0258] SEC-HPLC purity detection results

[0259] After 4 weeks of high-temperature acceleration (40°C ± 2°C, 75% RH ± 5% RH), under different formulation buffer systems and different pH values, the increase in SEC-HPLC aggregates did not exceed 0.03% per day; except that the decrease in SEC-HPLC monomer purity in the histidine salt buffer system at pH 6.5 was 0.40% per day, the decrease in SEC-HPLC monomer purity in other systems and pH values was less than 0.25% per day. This indicates that under pH 6.0 in the histidine salt buffer system, pH 6.0 in the acetate buffer system, pH 5.5 / pH 6.0 in the citrate buffer system, and pH 7.0 in the phosphate buffer system, the KW027-2-055 antibody can maintain a relatively stable structure and is not prone to aggregation (Table 4).

[0260] Table 4 Statistical table of SEC-HPLC results in the high-temperature acceleration test for screening the formulation buffer system of KW027-2-055

[0261]

[0262]

[0263] CEX-HPLC results

[0264] After 4 weeks of high-temperature acceleration (40°C ± 2°C, 75% RH ± 5% RH), under different formulation buffer systems and different pH values, the changes in the contents of each charge variant detected by CEX-HPLC were quite different. It was relatively stable under pH 6.0 in the histidine salt buffer system, pH 6.0 in the acetate buffer system, and pH 6.0 in the citrate buffer system, and the increase in acidic peaks was less than 1.00% per day (Table 5).

[0265] Table 5 Statistical Table of CEX-HPLC Test Results for Buffer System Screening of KW027-2-055 Preparation Prescription

[0266]

[0267]

[0268] CE-SDS Results

[0269] The results of non-reducing CE-SDS and reducing CE-SDS showed that after 4 weeks of high-temperature acceleration (40°C ± 2°C, 75% RH ± 5% RH), the samples were relatively stable under the phosphate buffer system at pH 7.0, the acetate buffer system at pH 6.0, and the citrate buffer system at pH 5.5 / pH 6.0. The reduction in the sum of the light and heavy chain contents of reducing CE-SDS was less than 0.15% per day, and the reduction in the main peak of non-reducing CE-SDS was less than 0.40% per day (Table 6).

[0270] Table 6 Statistical Table of CE-SDS Test Results for Buffer System Screening of KW027-2-055 Preparation Prescription

[0271]

[0272]

[0273] Summary

[0274] The high-temperature acceleration test results of the buffer system screening for KW027-2-055 preparation prescription comprehensively evaluated the stability of KW027-2-055 samples under each preparation prescription buffer system from the aspects of quality attributes such as appearance, aggregates, fragments, CE-SDS, and acidic peaks. From the appearance test results, the appearance of KW027-2-055 antibody showed no change and the particle size distribution was uniform. From the SEC-HPLC test results, except for the reduction in the monomer purity of histidine buffer system at pH 6.5 by 0.40% per day in SEC-HPLC, the reduction in monomer purity of other systems and pHSEC-HPLC was less than 0.25% per day. The CEX-HPLC results showed that the samples were relatively stable under the histidine buffer system at pH 6.0, the acetate buffer system at pH 6.0, and the citrate buffer system at pH 6.0, and the increase in acidic peaks was less than 1.00% per day. The CE-SDS results showed that except for histidine at pH 6.0 / pH 6.5, the reduction in the sum of the light and heavy chain contents of reducing CE-SDS in other groups was less than 0.15% per day,

[0275] Based on the above quality attributes, it can be determined that the KW027-2-055 antibody can achieve relatively good stability in different buffer systems. Among them, the stability is optimal in the pH 6.0 citrate buffer system and the pH 6.0 acetate buffer system. Therefore, the formulation development of the first round and the second stage of KW027-2-055 is carried out in the pH 6.0 citrate buffer system and the pH 6.0 acetate buffer system.

[0276] Example 8: Single-factor test of candidate components for formulation

[0277] On the basis of the determined formulation buffer system and the optimal pH, further single-factor screening of candidate components is carried out. Mainly investigate factors such as non-reducing disaccharides (sucrose, trehalose), sugar alcohols (sorbitol, mannitol), salts (sodium chloride), amino acid protectants (arginine hydrochloride, glycine, proline), antioxidants (methionine), chelating agents (EDTA·2Na), surfactants (polysorbate 80, polysorbate 20), etc. Through high-temperature acceleration (40°C ± 2°C, 75% RH ± 5%RH) stability studies, evaluate the effects of each candidate component on the stability of the KW027-2-055 antibody.

[0278] Sample preparation

[0279] (1) Prepare the stock solution: According to Table 7, prepare 16 groups of stock solutions of formulation buffers with different buffer systems. The concentration of each excipient in each group of stock solutions is determined by comprehensively considering the KW027-2-055 protein concentration after actual membrane buffer exchange, the final concentration of the excipient in each formulation, and the final protein concentration. It should be noted that the C1-F7 formulations contain arginine hydrochloride. After adding arginine hydrochloride, attention should be paid to the adjustment of pH to ensure that the solution pH meets the final pH requirements.

[0280] (2) Membrane buffer exchange: Prepare a high-concentration KW027-2-055 sample and exchange the membrane buffer into the target buffer salt solution (without any excipients).

[0281] (3) Preparation of samples with final concentrations: Use the corresponding target buffer stock solution to dilute the samples after membrane buffer exchange to the target protein concentration and target excipient concentration to obtain 16 groups of experimental group samples. At the same time, use each stock solution to prepare sample blank control group samples.

[0282] (4) Sub-packaging: Sub-package the samples into vials at a specification of 0.5 ml / vial, press the caps and store.

[0283] Table 7 Composition of single-factor screening formulations for candidate components of the formulation

[0284]

[0285]

[0286]

[0287] Stability Investigation and Detection

[0288] Test Conditions: 40°C ± 2°C, 75% RH ± 5% RH;

[0289] Stability Test Duration: 4 weeks

[0290] Sampling Points: Samples are taken for inspection at 0, 1, 2, and 4 weeks during the stability test;

[0291] Results and Discussion

[0292] Appearance: After 4 weeks of high-temperature acceleration (40°C ± 2°C, 75% RH ± 5% RH), under both buffer systems, there is no increase in visible foreign matter to the naked eye. The color changes from colorless to slightly yellowish, and the turbidity changes to varying degrees. The acetic acid system is better than the citric acid system; in the acetic acid buffer system, the turbidity results of the single-factor test show that the turbidity of the NaCl group in salts is more turbid than that of the control group; there is no difference in non-reducing disaccharides (sucrose, trehalose) and sugar alcohols (sorbitol, mannitol) compared to the control group; in the amino acid protectant group, there is no difference in glycine and proline compared to the control group, and the turbidity of L-arginine hydrochloride is more turbid than that of the control group; among the antioxidants, methionine performs better than the control group, and there is no difference in EDTA·2Na compared to the control group; among the surfactants, polysorbate 80 is better than polysorbate 20.

[0293] Aggregates: After 4 weeks of high-temperature acceleration (40°C ± 2°C, 75% RH ± 5% RH), the SEC-HPLC results show that there is no significant difference in the anti-aggregation ability of the acetic acid and citric acid buffer systems and each protectant, and the increase in aggregates does not exceed 0.10% per day (Table 8).

[0294] Table 8 Statistical Table of SEC-HPLC Results of Single-Factor Tests of Candidate Ingredients in the KW027-2-055 Formulation Prescription - High-Temperature Acceleration Test

[0295]

[0296]

[0297]

[0298] Acidic peak: After 4 weeks of high-temperature acceleration (40°C ± 2°C, 75% RH ± 5% RH), under the two buffer systems, the change in the increase of the acidic peak in the citric acid buffer system was relatively high. Except for the daily average change in the acidic peak of the C1-Cit-F7 arginine hydrochloride group being 0.64% / day, the daily average change in other groups was greater than 0.70% / day. The change in the increase of the acidic peak in the acetic acid buffer system was relatively small. Except for the daily average change in the acidic peak of the C1-Ac-F8 glycine group being 0.75% / day, it was less than 0.70% / day (Table 9).

[0299] Table 9 Statistical table of CEX-HPLC results of single-factor tests on candidate components of KW027-2-055 preparation prescription - High-temperature acceleration test

[0300]

[0301]

[0302]

[0303] Results of polysorbate 80 / polysorbate 20: After 4 weeks of high-temperature acceleration (40°C ± 2°C, 75% RH ± 5% RH), under the citric acid buffer system, there was no significant change in polysorbate 80 / polysorbate 20, all within 0.10 mg / ml - 0.30 mg / ml; under the acetic acid buffer system, the content of polysorbate 80 in C1-Ac-F3 at 0 h was 0.09 mg / ml, and the polysorbate 80 in C1-Ac-F3 at 4W was 0.02 mg / ml. The polysorbate 80 in C1-Ac-F3 might be abnormal during the sample preparation process, so it was discarded as an abnormal point; after 4W of high temperature, the content of polysorbate 80 in the C1-Ac-F4 sucrose group, C1-Ac-F5 sorbitol group, and C1-Ac-F8 glycine group was less than 0.10 (Table 10).

[0304] Table 10 Statistical table of results of polysorbate 80 / polysorbate 20 in single-factor tests on candidate components of KW027-2-055 preparation prescription - High-temperature acceleration test

[0305]

[0306]

[0307] Relative binding activity results: After 4 weeks of high-temperature acceleration (40°C ± 2°C, 75% RH ± 5% RH), under the two buffer systems, the relative binding activities of each single-factor test group were all between 70% and 135%, meeting the expectations (Table 11).

[0308] Table 11 Statistical table of relative binding activity results of single-factor tests on candidate components of KW027-2-055 preparation prescription - High-temperature acceleration test

[0309]

[0310]

[0311] Summary

[0312] NaCl is a commonly used ionic excipient in formulations. Adding NaCl to the KW027-2-055 formulation results in an increase in turbidity. Therefore, it is not suitable to add NaCl to the excipients of the KW027-2-055 formulation; sugar and sugar alcohol excipients can not only stabilize monoclonal antibodies under high-temperature conditions but also during the freeze-thaw process. In the single-factor experiment of the KW027-2-055 formulation prescription, the effects of trehalose, sucrose, sorbitol, and mannitol on the KW027-2-055 antibody were investigated. The results showed that compared with others, sucrose and sorbitol reduced the content of the surfactant polysorbate 80. There was basically no difference in the overall effect of trehalose and mannitol on the stability of KW027-2-055, but the daily increment of the inter-chain fragment of mannitol in R CE-SDS was lower, only 0.03% / day, making it more suitable as an excipient for the KW027-2-055 formulation; Amino acids can stabilize monoclonal antibodies through interactions or hydration with antibody proteins. Arginine hydrochloride increased turbidity, glycine reduced the content of the surfactant polysorbate 80, and proline showed the best performance and was more suitable as an excipient for the KW027-2-055 formulation; Methionine and EDTA·2Na are often used as reducing agents to reduce the oxidation reaction of monoclonal antibodies. Judging from the results, when adding the two to the KW027-2-055 formulation, the effect of EDTA·2Na was not obvious, while methionine significantly reduced turbidity and was more suitable as an excipient for the KW027-2-055 formulation; Polysorbate 20 and polysorbate 80 are common surfactants in monoclonal antibody drug formulations. The addition of surfactants can reduce viscosity, reduce protein aggregation during agitation, shaking, freeze-thaw, and lyophilization processes, and can prevent protein adsorption and subsequent loss on the product contact surface.

[0313] Summary of the First Round of Formulation Prescription Screening

[0314] Based on the experimental results of the formulation prescription buffer system screening in the first stage and the single-factor experiments of the candidate components of the formulation prescription in the second stage, it is summarized that for the KW027-2-055 sample, under the 10 mM sodium acetate - acetic acid pH 6.0 buffer system, the control of the acidic peak content and the clarity of the solution are better; compared with other sugars and sugar alcohols, mannitol can reduce the generation of inter-chain fragments of the KW027-2-055 antibody; Proline shows the best overall performance among the three amino acids; Among the reducing agents, methionine significantly reduces turbidity; For the surfactant, polysorbate 80 with a longer fatty acid side chain and better emulsifying ability is selected.

[0315] Based on the above, the formulation composition of KW027-2-055 preparation is determined as follows:

[0316] Buffer: 10 mM sodium acetate - acetic acid buffer system at pH 6.0;

[0317] Protectant: mannitol, proline;

[0318] Antioxidant: methionine;

[0319] Surfactant: polysorbate 80, concentration 0.2 mg / ml.

Claims

1. An antibody composition comprising (i) a therapeutically effective amount of an antibody or antigen-binding fragment thereof that specifically binds to hepatitis B virus surface antigen (HBsAg); (ii) a buffer, (iii) a protective agent, (iv) a surfactant, and (v) antioxidants, The antibody and antigen-binding fragment thereof that specifically bind to the hepatitis B virus surface antigen (HBsAg) comprises a heavy chain variable region (VH) sequence and a light chain variable region (VL) sequence, wherein: The heavy chain variable region comprises the complementary determining region (CDR) amino acid sequence shown below: HCDR1: GYTFTGYY, HCDR2:INPNSGGT, HCDR3:ARDLWX1X2X3X4X5DYGX6DV; The light chain variable region comprises the following CDR amino acid sequence: LCDR1: X7SIX8TX9, LCDR2: AAS, LCDR3:QQSYSTPLT; in: X1 is selected from N, D, Y, I, S, V or M, X2 is selected from F, H, E, W, R, Q, M, P or D, X3 is selected from V, Q, P, R, H, T or Y, X4 is selected from S, T, D, L, Y, F or H, X5 is selected from D, Q or E, X6 is selected from M, I or L; X7 is selected from Q, S, H, Y, P, L, I or E, X8 is selected from S or D, and X9 is selected from Y, F, D, T, S or V.

2. The antibody composition according to claim 1, characterized in that In the antibody composition, the antibody and antigen-binding fragment thereof that specifically bind to the hepatitis B virus surface antigen (HBsAg) wherein the VH and VL sequences comprise complementary determining region (CDR) amino acid sequences as follows: HCDR3 as shown in SEQ ID NO:3, and LCDR1 as shown in SEQ ID NO:15; or, HCDR3 as shown in SEQ ID NO:4, and LCDR1 as shown in SEQ ID NO:16; or, HCDR3 as shown in SEQ ID NO:5, and LCDR1 as shown in SEQ ID NO:17; or, HCDR3 as shown in SEQ ID NO:6, and LCDR1 as shown in SEQ ID NO:18; or, HCDR3 as shown in SEQ ID NO:7, and LCDR1 as shown in SEQ ID NO:19; or, HCDR3 as shown in SEQ ID NO:8, and LCDR1 as shown in SEQ ID NO:20; or, HCDR3 as shown in SEQ ID NO:9, and LCDR1 as shown in SEQ ID NO:21; or, HCDR3 as shown in SEQ ID NO:10, and LCDR1 as shown in SEQ ID NO:22; or, HCDR3 as shown in SEQ ID NO:11, and LCDR1 as shown in SEQ ID NO: or, HCDR3 as shown in SEQ ID NO:14 and LCDR1 as shown in SEQ ID NO:26; HCDR3 as shown in SEQ ID NO:6 and LCDR1 as shown in SEQ ID NO:27, and an amino acid sequence that is at least 85% identical to the above VH sequence or VL sequence.

3. The antibody composition according to claim 1, wherein: The buffer is selected from citric acid-sodium citrate, histidine-histidine hydrochloride, sodium dihydrogen phosphate-disodium hydrogen phosphate, acetic acid-sodium acetate, sodium citrate; The protective agent is selected from one or more of sugars, alcohols, amino acids and chloride salts; The surfactant is selected from non-ionic polymers, for example, one or more selected from polysorbate 80, polysorbate 20, poloxamer and polyethylene glycol; The antioxidant is selected from one or more of methionine and EDTA·2Na.

4. The antibody composition according to any one of claims 1 to 3, characterized in that The concentration of the antibody is 20-200 mg / mL, preferably, the concentration of the antibody is 100 mg / mL.

5. The antibody composition according to any one of claims 1 to 3, characterized in that The concentration of the buffer is 5 mM to 15 mM; preferably, the concentration of the buffer is 10 mM.

6. The antibody composition according to any one of claims 1 to 3, characterized in that The buffer used in the composition of the present invention can control the pH of the composition of the present invention within the pH range of 5.00-7.

00. Preferably, the composition has a pH of 6.

00.

7. The antibody composition according to any one of claims 1 to 3, characterized in that The concentration of the protective agent is 5-20 mg / ml, preferably 5-15 mg / ml.

8. The antibody composition according to any one of claims 1 to 3, characterized in that The concentration of the surfactant is 0.1 mg / ml to 0.3 mg / ml, preferably, the concentration of the surfactant is 0.2 mg / ml.

9. The antibody composition according to any one of claims 1 to 3, characterized in that The concentration of the antioxidant is 0-10 mg / ml.

10. The antibody composition according to any one of claims 1 to 9, wherein the antibody composition comprises: (1) antibodies and antigen-binding fragments thereof that specifically bind to HBsAg at a concentration of 20-200 mg / ml; (2) a buffer having a concentration of 5 mM to 15 mM, wherein the buffer is selected from citric acid-sodium citrate, histidine-histidine hydrochloride, sodium dihydrogen phosphate-disodium hydrogen phosphate, acetic acid-sodium acetate, and sodium citrate; (3) a protective agent at a concentration of 5-20 mg / ml, wherein the protective agent is selected from one or more of sugars, alcohols, amino acids and chloride salts; (4) a surfactant at a concentration of 0.1 mg / ml to 0.3 mg / ml, wherein the surfactant is selected from a non-ionic polymer, for example, one or more selected from Tween 80, Tween 20, poloxamer and polyethylene glycol; (5) an antioxidant at a concentration of 0-10 mg / ml, wherein the antioxidant is selected from any one or more of methionine or EDTA·2Na, The pH of the composition is 5.00 to 7.00; The antibody and antibody fragment thereof that specifically bind to the hepatitis B virus surface antigen (HBsAg) comprises a heavy chain variable region (VH) sequence and a light chain variable region (VL) sequence, wherein: 3, and LCDR1 as shown in SEQ ID NO:15; or, HCDR3 as shown in SEQ ID NO:4, and LCDR1 as shown in SEQ ID NO:16; or, HCDR3 as shown in SEQ ID NO:5, and LCDR1 as shown in SEQ ID NO:17; or, HCDR3 as shown in SEQ ID NO:6, and LCDR1 as shown in SEQ ID NO:18; or, HCDR3 as shown in SEQ ID NO:7, and LCDR1 as shown in SEQ ID NO:19; or, HCDR3 as shown in SEQ ID NO:8, and LCDR1 as shown in SEQ ID NO:20; or, HCDR3 as shown in SEQ ID NO:9, and LCDR1 as shown in SEQ ID NO:21; or, HCDR3 as shown in SEQ ID NO:10, and LCDR1 as shown in SEQ ID NO:22; or, HCDR3 as shown in SEQ ID NO:11, and LCDR1 as shown in SEQ ID NO:23; or, The invention relates to a HCDR3 as shown in SEQ ID NO:12, and a LCDR1 as shown in SEQ ID NO:24; or, a HCDR3 as shown in SEQ ID NO:13, and a LCDR1 as shown in SEQ ID NO:25; or, a HCDR3 as shown in SEQ ID NO:14, and a LCDR1 as shown in SEQ ID NO:26; a HCDR3 as shown in SEQ ID NO:6, and a LCDR as shown in SEQ ID NO:27, and an amino acid sequence that is at least 85% identical to the above-mentioned VH sequence or VL sequence.

11. The antibody composition according to claim 10, wherein the antibody composition comprises: (1) antibodies and antigen-binding fragments thereof that specifically bind to HBsAg at a concentration of 20-200 mg / ml; (2) Acetic acid-sodium acetate with a concentration of 5-15 mmol / L; (3) a combination of 5-20 mg / ml mannitol and 5-20 mg / ml proline; (4) Tween 80 at a concentration of 0.1-0.3 mg / ml; and (5) methionine at a concentration of 0-10 mg / ml; The pH of the composition is 5.00 to 7.00; The antibody and antibody fragment thereof that specifically bind to the hepatitis B virus surface antigen (HBsAg) comprises a heavy chain variable region (VH) sequence and a light chain variable region (VL) sequence, wherein: 3, and LCDR1 as shown in SEQ ID NO:15; or, HCDR3 as shown in SEQ ID NO:4, and LCDR1 as shown in SEQ ID NO:16; or, HCDR3 as shown in SEQ ID NO:5, and LCDR1 as shown in SEQ ID NO:17; or, HCDR3 as shown in SEQ ID NO:6, and LCDR1 as shown in SEQ ID NO:18; or, HCDR3 as shown in SEQ ID NO:7, and LCDR1 as shown in SEQ ID NO:19; or, HCDR3 as shown in SEQ ID NO:8, and LCDR1 as shown in SEQ ID NO:20; or, HCDR3 as shown in SEQ ID NO:9, and LCDR1 as shown in SEQ ID NO:21; or, HCDR3 as shown in SEQ ID NO:10, and LCDR1 as shown in SEQ ID NO:22; or, HCDR3 as shown in SEQ ID NO:11, and LCDR1 as shown in SEQ ID NO:23; or, The invention relates to a HCDR3 as shown in SEQ ID NO:12, and a LCDR1 as shown in SEQ ID NO:24; or, a HCDR3 as shown in SEQ ID NO:13, and a LCDR1 as shown in SEQ ID NO:25; or, a HCDR3 as shown in SEQ ID NO:14, and a LCDR1 as shown in SEQ ID NO:26; a HCDR3 as shown in SEQ ID NO:6, and a LCDR as shown in SEQ ID NO:27, and an amino acid sequence that is at least 85% identical to the above-mentioned VH sequence or VL sequence.

12. The antibody composition according to any one of claims 1 to 11, characterized in that The antibody composition is an injection preparation, preferably a subcutaneous injection preparation, an intravenous injection preparation, or an intramuscular injection preparation.

13. Use of the composition according to any one of claims 1 to 11 in the preparation of a medicament for treating or preventing hepatitis B virus (HBV) infection and / or alleviating symptoms of hepatitis B.

14. A container or a kit comprising the same, wherein the container comprises the antibody composition according to any one of claims 1 to 11.

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