Separated anti-NGF antibody preparation and application

Abstract

The anti-NGF antibody preparation provided by the invention has strong stability under the conditions of high temperature, illumination, oscillation, freeze thawing, acceleration and long term, can ensure that the preparation keeps good stability in preparation, transportation and storage processes, and ensures clinical medication safety and quality controllability.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a separated anti-NGF antibody preparation and application. Background Art

[0002] Nerve growth factor (NGF) was originally identified as a protein that promotes the growth of neurons in the developing chick embryo (Bueker 1948). It is now known that NGF belongs to the neurotrophin (NT) family, a group of structurally related proteins that includes brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 / 5 (NT-4 / 5). They are all secreted proteins that are essential for the normal development, patterning, and maintenance of the peripheral nervous system.

[0003] In healthy people, subcutaneous injection of NGF can cause local pain and hyperalgesia within a few minutes (Petty et al., 1994). Even low-dose systemic administration of NGF can cause myalgia. This indicates that NGF has an activating or sensitizing effect on nociceptors. In transgenic mice, after chronic contractile injury, in vivo overexpression of NGF driven by a neuroglial protein promoter can lead to enhanced neuropathic pain behavior and neuronal sprouting, which proves the connection between NGF and neuropathic pain and sympathetic nerve sprouting in dorsal root ganglia (DRG). NGF promotes the sprouting of sympathetic neurons and the formation of abnormal innervation of nociceptive neurons, which is thought to contribute to the induction and maintenance of chronic nociceptive / pain states (Ramer et al., 1998, 1999). Preclinical studies have found that NGF levels are locally increased in animals injected with CFA and carrageenan (Ma et al., 2000). NGF released by damaged tissues and its subsequent action in the periphery are thought to play a major role in inducing thermal hyperalgesia. This process is called "peripheral sensitization" (Mendell et al., 2002). After NGF binds to its high affinity receptor TrkA, is internalized and transported back to the nociceptive cell bodies of the DRG, it initiates the secretion of nociceptive neuropeptides (such as substance P, CGRP), and activates PKC in the dorsal horn of the spinal cord (Sah et al., 2003). This is a process related to "central sensitization". Anti-NGF antibodies are a method for treating diseases caused by increased NGF expression or increased sensitivity to NGF.

[0004] However, anti-NGF antibodies are large-molecule protein drugs. Compared with traditional small-molecule drugs, they are more prone to aggregation and degradation during storage, which can lead to adverse consequences such as increased differences between drug batches and changes in immunogenicity. Summary of the invention

[0005] The present invention provides an anti-NGF antibody preparation with excellent stability for treating NGF-mediated diseases and disorders.

[0006] In one aspect, the present invention provides an anti-NGF antibody preparation, which comprises an isolated anti-NGF antibody, a stabilizer, a surfactant, and a buffer, wherein the buffer is a histidine-histidine hydrochloride buffer, and the stabilizer is arginine hydrochloride.

[0007] In some embodiments, the antibody concentration is 5 mg / ml-50 mg / ml, preferably 8 mg / ml-40 mg / ml, more preferably 10 mg / ml-30 mg / ml. In some specific embodiments, the antibody concentration is 5 mg / ml, 8 mg / ml, 10 mg / ml, 15 mg / ml, 20 mg / ml, 25 mg / ml, 30 mg / ml, 35 mg / ml, 40 mg / ml, 45 mg / ml or 50 mg / ml.

[0008] In some embodiments, the concentration of the stabilizer is 80mM-180mM, preferably 100mM-160mM. In some specific embodiments, the concentration of the stabilizer is 80mM, 90mM, 100mM, 105mM, 110mM, 115mM, 120mM, 125mM, 130mM, 135mM, 140mM, 145mM, 150mM, 155mM, 160mM, 170mM or 180mM.

[0009] In some embodiments, the concentration of the buffer is 3mM-80mM; preferably, the concentration of the buffer is 5mM-50mM; more preferably, the concentration of the buffer is 10mM-30mM. In some specific embodiments, the concentration of the buffer is 3mM, 5mM, 8mM, 10mM, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 50mM, 60mM, 70mM or 80mM.

[0010] In some embodiments, the pH value of the formulation is 5.0-6.5, preferably 5.3-6.2, more preferably 5.5-6.0. In some specific embodiments, the pH value of the formulation is 5.0, 5.2, 5.3, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.2, 6.3 or 6.5.

[0011] In some embodiments, the surfactant is polysorbate; preferably, the polysorbate is Tween-20 or Tween-80.

[0012] In some embodiments, the concentration of the surfactant is 0.05mg / ml-1.0mg / ml; preferably, the concentration of the surfactant is 0.15mg / ml-0.45mg / ml. In some specific embodiments, the concentration of the surfactant is 0, 0.05mg / ml, 0.08mg / ml, 0.10mg / ml, 0.12mg / ml, 0.15mg / ml, 0.20mg / ml, 0.25mg / ml, 0.30mg / ml, 0.35mg / ml, 0.40mg / ml, 0.45mg / ml, 0.50mg / ml, 0.60mg / ml, 0.70mg / ml, 0.80mg / ml, 0.90mg / ml or 1.0mg / ml.

[0013] In some embodiments, the antibody preparation is any one of the following preparations:

[0014] (1) The antibody concentration is 10 mg / ml, 15 mg / ml, 20 mg / ml, 25 mg / ml or 30 mg / ml; the stabilizer is 100 mM-160 mM arginine hydrochloride; the buffer is 10 mM-30 mM histidine-histidine hydrochloride buffer; the pH value of the preparation is 5.5-6.0; the surfactant is 0.15 mg / ml-0.45 mg / ml Tween-20 and / or Tween-80;

[0015] (2) The antibody concentration is 10 mg / ml-30 mg / ml; the stabilizer is 100 mM, 105 mM, 110 mM, 115 mM, 120 mM, 125 mM, 130 mM, 135 mM, 140 mM, 145 mM, 150 mM, 155 mM or 160 mM arginine hydrochloride; the buffer is 10 mM-30 mM histidine-histidine hydrochloride buffer; the pH value of the preparation is 5.5-6.0; the surfactant is 0.15 mg / ml-0.45 mg / ml Tween-20 and / or Tween-80;

[0016] (3) The antibody concentration is 10 mg / ml-30 mg / ml; the stabilizer is 100 mM-160 mM arginine hydrochloride; the buffer is 10 mM, 15 mM, 20 mM, 25 mM or 30 mM histidine-histidine hydrochloride buffer; the pH value of the preparation is 5.5-6.0; the surfactant is 0.15 mg / ml-0.45 mg / ml Tween-20 and / or Tween-80;

[0017] (4) The antibody concentration is 10 mg / ml-30 mg / ml; the stabilizer is 100 mM-160 mM arginine hydrochloride; the buffer is 10 mM-30 mM histidine-histidine hydrochloride buffer; the pH value of the preparation is 5.5, 5.6, 5.7, 5.8, 5.9, 6.0; the surfactant is 0.15 mg / ml-0.45 mg / ml Tween-20 and / or Tween-80;

[0018] (5) The antibody concentration is 10 mg / ml-30 mg / ml; the stabilizer is 100 mM-160 mM arginine hydrochloride; the buffer is 10 mM-30 mM histidine-histidine hydrochloride buffer; the pH value of the preparation is 5.5-6.0; the surfactant is 0.15 mg / ml, 0.20 mg / ml, 0.25 mg / ml, 0.30 mg / ml, 0.35 mg / ml, 0.40 mg / ml, 0.45 mg / ml Tween-20 and / or Tween-80.

[0019] In some embodiments, the antibody preparation is any one of the following preparations:

[0020] (1) The antibody concentration is 30 mg / ml; the stabilizer is 100 mM arginine hydrochloride; the buffer is 10 mM histidine-histidine hydrochloride buffer; the pH value of the preparation is 5.5; the surfactant is 0.45 mg / ml Tween-20 and / or Tween-80;

[0021] (2) The antibody concentration is 20 mg / ml; the stabilizer is 140 mM arginine hydrochloride; the buffer is 20 mM histidine-histidine hydrochloride buffer; the pH value of the preparation is 5.7; the surfactant is 0.3 mg / ml Tween-20 and / or Tween-80;

[0022] (3) The antibody concentration is 10 mg / ml; the stabilizer is 160 mM arginine hydrochloride; the buffer is 30 mM histidine-histidine hydrochloride buffer; the pH value of the preparation is 6.0; and the surfactant is 0.15 mg / ml Tween-20 and / or Tween-80.

[0023] In some embodiments, the preparation may further include a preservative and / or an antioxidant. The preservative or antioxidant is a preservative or antioxidant commonly used in antibody preparations. In some embodiments, the preservative is ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA) or a combination thereof; the antioxidant is methionine.

[0024] In some embodiments, the concentration of the preservative is 0-0.2 mg / ml, preferably 0.02-0.1 mg / ml. In some specific embodiments, the concentration of the preservative is 0 mg / ml, 0.02 mg / ml, 0.04 mg / ml, 0.06 mg / ml, 0.08 mg / ml, 0.10 mg / ml, 0.12 mg / ml, 0.14 mg / ml, 0.16 mg / ml, 0.18 mg / ml or 0.20 mg / ml.

[0025] In some embodiments, the concentration of the antioxidant is 0-20 mM, the concentration of the antioxidant is 5 mM-15 mM. In some specific embodiments, the concentration of the antioxidant is 0, 5 mM, 8 mM, 10 mM, 12 mM, 15 mM, 18 mM or 20 mM.

[0026] In some embodiments, the antibody preparation described above is a liquid preparation or a powder for injection.

[0027] In some embodiments, as described above in any of the above-described isolated anti-NGF antibodies, the isolated anti-NGF antibody comprises an Fc fragment. In some embodiments, the isolated anti-NGF antibody is a full-length IgG antibody. In some embodiments, the isolated anti-NGF antibody is a full-length IgG1, IgG2, IgG3 or IgG4 antibody. In some embodiments, the isolated anti-NGF antibody is a chimeric, murine, fully human or humanized antibody. In some embodiments, the isolated anti-NGF antibody is an antigen-binding fragment selected from Fab, Fab', F(ab)'2, Fab'-SH, single-chain Fv (scFv), Fv fragment, dAb, Fd, nanobody, double-chain antibody (diabody) and linear antibody.

[0028] In some embodiments, the antibody comprises V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and V L , the V L It comprises: LC-CDR1, which comprises the amino acid sequence of SEQ ID NO:4, LC-CDR2, which comprises the amino acid sequence of SEQ ID NO:5, and LC-CDR3, which comprises the amino acid sequence of SEQ ID NO:6.

[0029] In some embodiments, the antibody comprises V H, which comprises the amino acid sequence SEQ ID NO:7, and V L , which comprises the amino acid sequence SEQ ID NO:8.

[0030] In some embodiments, the antibody comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10, and the light chain comprising the amino acid sequence of SEQ ID NO:11.

[0031] On the other hand, the present invention provides the use of any anti-NGF antibody as described above, or any anti-NGF antibody preparation as described above, in the preparation of a medicament for treating a disease; preferably, the disease or condition is caused by increased NGF expression or increased sensitivity to NGF; more preferably, the disease or condition is selected from one or more of inflammatory pain, postoperative incision pain, neuropathic pain, fracture pain, gouty joint pain, postherpetic neuralgia, pain caused by burns, cancer pain, osteoarthritis or rheumatoid arthritis pain, sciatica, pain associated with sickle cell crisis, and postherpetic neuralgia.

[0032] On the other hand, the present invention provides a method for treating a desired individual disease or condition, comprising administering to the individual an effective amount of any anti-NGF antibody or antibody preparation as described above. The administration methods include: intravenous injection, intra-arterial administration, intraperitoneal injection, intrapulmonary administration, oral administration, inhalation administration, intravascular administration, intramuscular injection, intratracheal administration, subcutaneous injection, intraocular administration, intrathecal administration, mucosal administration or transdermal administration. In some embodiments, the preparation is administered intravenously. In some embodiments, the preparation is administered subcutaneously. In some embodiments, the preparation is administered intramuscularly.

[0033] The "antibodies" of the present invention include full-length antibodies and antigen-binding fragments thereof. Full-length antibodies include two heavy chains and two light chains. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions in the two chains usually include three highly variable loops, known as complementary determining regions (CDRs) (light chain (LC) CDRs include LC-CDR1, LC-CDR2 and LC-CDR3, and heavy chain (HC) CDRs include HC-CDR1, HC-CDR2 and HC-CDR3). The CDR boundaries of the antibodies or antigen-binding fragments disclosed herein can be defined or identified by Kabat, Chothia or Al-Lazikani conventions (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). The three CDR regions of the heavy or light chain are inserted between flanking segments called framework regions (FRs), which are more conservative than the CDR regions and form a scaffold that supports the hypervariable loops. The constant regions of the heavy and light chains do not participate in antigen binding, but exhibit a variety of effector functions. Antibodies are classified based on the amino acid sequence of their heavy chain constant regions. The full-length antibodies of the present invention can be isotypes such as IgA, IgD, IgE, IgG and IgM, characterized by having α, δ, ε, γ and μ heavy chains, respectively. Further, the several major antibody classes described in the present invention can be further divided into subclasses, including: IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain) or IgA2 (α2 heavy chain).

[0034] The "antigen-binding fragment" described in the present invention includes antibody fragments, such as diabodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized double-chain antibodies (ds double-chain antibodies), single-chain antibodies (scFv), scFv dimers (divalent double-chain antibodies), multispecific antibodies composed of antibody fragments containing one or more CDRs, single-domain antibodies, nanobodies, domain antibodies, bivalent domain antibodies, or any other antibody fragments that can bind to an antigen but do not contain a complete antibody structure.

[0035] In the present invention, histidine-histidine hydrochloride is used as a buffer and arginine hydrochloride is used as a stabilizer, so that the anti-NGF antibody preparation has excellent stability under high temperature, light, oscillation, freeze-thaw, accelerated and long-term conditions, which can ensure the stability of the preparation during preparation, transportation and storage, and ensure the safety of clinical drug use and quality controllability. DETAILED DESCRIPTION

[0036] In order to make the technical problems to be solved, the technical solutions and advantages adopted by the present invention clearer, the present invention will be described in detail below in conjunction with specific embodiments. The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0037] Unless otherwise specified, the reagents used in the following examples were prepared by conventional methods or obtained from commercial sources; the experimental methods used were conventional methods unless otherwise specified; the materials, instruments, etc. used were obtained from commercial sources unless otherwise specified.

[0038] In the embodiments of the present invention, the anti-NGF antibody Ab4 and its mutants (hereinafter referred to as Ab4 mutants, whose heavy chain L234 and L235 (EU numbering) amino acid residues are mutated to A234 and A235) are used as examples. Among them, the amino acid sequences of Ab4 antibodies and their mutants are disclosed in the international patent application WO2021238886A1, and the disclosure thereof is incorporated herein by reference.

[0039] The amino acid sequences of the anti-NGF antibody Ab4 and Ab4 mutants used in the examples of the present invention are shown in Tables 1a and 1b:

[0040] Table 1a Amino acid sequences of anti-NGF antibody CDRs

[0041]

[0042] Table 1b. Amino acid sequences of anti-NGF antibodies

[0043]

[0044]

[0045] Example 1. Preparation of anti-NGF antibody preparation

[0046] The anti-NGF antibody preparation formulation is as follows:

[0047] Table 2 Anti-NGF antibody preparation prescription

[0048]

[0049] Preparations of anti-NGF antibody Ab4 and Ab4 mutant were prepared respectively with reference to the prescriptions in Table 2, wherein prescriptions 1-3 are the anti-NGF antibody preparation prescriptions of the present invention, and prescriptions 4-7 are comparative preparation prescriptions.

[0050] Example 2: Stability test of anti-NGF antibody preparation

[0051] The stability of the different antibody preparations prepared in Example 1 was investigated by setting conditions such as high temperature, illumination, oscillation, freeze-thaw, acceleration and long-term. The test items included visible foreign matter, concentration, turbidity, pH, osmotic pressure, viscosity, biological activity, thermal stability, insoluble particles, aggregation characteristics, degradation characteristics and charge heterogeneity of the antibody preparation. Among them, the polymer characteristics were detected by SEC method and NR-CE-SDS method, and the charge heterogeneity was detected by IEC method.

[0052] 1. Stability test under high temperature conditions

[0053] The stability of each formulation of each antibody was tested under high temperature conditions (40°C ± 2°C, 75% ± 5% RH (relative humidity), 0 days, 1 week, 2 weeks, 3 weeks).

[0054] (1) Table 3 shows the test results of each preparation of the exemplary antibody Ab4: the visible foreign matter, concentration, pH, osmotic pressure, viscosity, biological activity, thermal stability, and insoluble particles of each antibody preparation were basically unchanged. Although the aggregates in formulations 1-3 increased slightly and the main peak decreased slightly, they still showed a better stability effect than the comparative example: the increase in aggregates detected by SEC was about 0.1-0.2%, and the decrease in the main peak detected by NR-CE-SDS was about 2%. In comparison, the increase in aggregates detected by SEC in formulations 4-6 was 0.4-0.5%, the increase in aggregates detected by SEC in formulation 7 was 4.8%, and the decrease in the main peak detected by NR-CE-SDS in formulations 4-7 was 3.6-7.6%. The acid peak and the alkaline peak increased and the main peak slightly decreased after IEC detection: the acid peak of the preparations of prescriptions 1-3 increased by about 16-22%, the main peak decreased by about 23-30%, and the alkaline peak increased by about 8%; in contrast, the acid peak of the preparation in the comparative example increased by more than 28%, and the main peak decreased by more than 37%. The above results show that compared with the prescription of the comparative example, the preparation of the present invention has better stability under high temperature conditions.

[0055] Table 3 High temperature test results of antibody Ab4 preparation

[0056]

[0057]

[0058] (2) Each preparation of the Ab4 mutant also showed good stability under high temperature conditions: for prescriptions 1-3, within 3 weeks of high temperature, the visible foreign matter, concentration, turbidity, pH, osmotic pressure, viscosity, biological activity, thermal stability, and insoluble particles of the above two antibody preparations were basically unchanged compared to day 0. Although the aggregates in prescriptions 1-3 increased slightly and the main peak decreased slightly, they also showed better stability effects compared to the control group: the increase in aggregates detected by SEC was within 0.2%, and the decrease in the main peak detected by NR-CE-SDS was within 2%. The acid peak and alkaline peak increased according to IEC detection, and the main peak decreased slightly: the acid peak of the preparations of prescriptions 1-3 increased within 23%, the main peak decreased within 30%, and the alkaline peak increased within 10%. (Specific data not shown).

[0059] The above results indicate that the aggregation and degradation characteristics of the anti-NGF antibodies of the present invention (whether Ab4 or Ab4 mutant) in various preparations do not change significantly under high temperature conditions, indicating that each antibody preparation has excellent stability.

[0060] 2. Stability test under light conditions

[0061] Under lighting conditions (5℃±3℃, 4500±500lx, 5W / m 2 , illumination for 0 days, 5 days, 1 week, and 2 weeks) to detect the stability of the antibody preparation.

[0062] (1) Table 4 shows the test results of each preparation of the exemplary antibody Ab4: within 2 weeks of illumination, the visible foreign matter, concentration, pH, osmotic pressure, viscosity, biological activity, thermal stability, and insoluble particles of each antibody preparation were basically unchanged relative to day 0. Although the aggregates in formulations 1-3 increased slightly and the main peak decreased slightly, they still showed a better stability effect than the comparative example: the increase in aggregates detected by SEC was about 0.6-0.7%, and the decrease in the main peak detected by NR-CE-SDS was about 2.6-2.8%. In comparison, the increase in aggregates detected by SEC in formulations 4-6 was 1.1-1.3%, the increase in aggregates detected by SEC in formulation 7 was 4%, and the decrease in the main peak detected by NR-CE-SDS in formulation 4-7 was 5-7.7%. The IEC test showed that the acid peak and the alkaline peak increased, and the main peak decreased slightly: the acid peak of the preparations of prescriptions 1-3 increased by about 8-10%, the main peak decreased by about 19-23%, and the alkaline peak increased by about 11-13%; in contrast, the acid peak of the preparation in the comparative example increased by about 12-23%, the main peak decreased by about 26-39%, and the alkaline peak increased by about 15-18%. The above results show that compared with the prescription of the comparative example, the preparation of Ab4 antibody has better stability under high temperature conditions.

[0063] Table 4 Results of illumination test of Ab4 antibody preparation

[0064]

[0065] (2) Each formulation of the Ab4 mutant also showed good stability under light conditions: within 2 weeks of light exposure, the visible foreign matter, concentration, pH, osmotic pressure, viscosity, biological activity, thermal stability, and insoluble particles of the antibody formulations remained basically unchanged compared to day 0. Although the aggregates in formulations 1-3 increased slightly and the main peak decreased slightly, they also showed better stability than the comparative example: the increase in aggregates detected by SEC was about 0.5-0.7%, and the decrease in the main peak detected by NR-CE-SDS was about 2.4-2.9%. The acid peak and alkaline peak increased and the main peak decreased slightly by IEC detection: the acid peak of formulations 1-3 increased by about 9-12%, the main peak decreased by about 20-25%, and the alkaline peak increased by about 11-13%. Similar to formulations 1-3 of Ab4, formulations 1-3 of the Ab4 mutant had better stability under high temperature conditions than the comparative example. (Specific data not shown).

[0066] The above results indicate that the aggregation and degradation characteristics of the anti-NGF antibody (Ab4 or Ab4 mutant) preparations of the present invention do not change significantly under illumination conditions, indicating that each antibody preparation can remain basically stable.

[0067] 3. Stability test under oscillation conditions

[0068] The stability of the antibody preparation was tested under shaking conditions (5°C ± 3°C, 100 rpm shaking for 0 days, 3 days, and 5 days).

[0069] (1) Table 5 shows the test results of each preparation of the exemplary antibody Ab4: within 2 weeks of oscillation, relative to day 0, the visible foreign matter, concentration, turbidity, pH, osmotic pressure, viscosity, biological activity, thermal stability, and insoluble particles of each antibody preparation were basically unchanged; the change range of the aggregate detected by SEC was very small, all within 0.07%, especially the change range of the aggregates of the formulations 1-3 was within 0.01%; the change range of the main peak of NR-CE-SDS was within 0.5%, almost unchanged (specific values ​​not shown). The above results show the excellent stability of the Ab4 antibody preparation under oscillation conditions.

[0070] Table 5 Oscillation test results of Ab4 antibody preparation

[0071]

[0072] (2) The various preparations of the Ab4 mutants also showed excellent stability under oscillation conditions: within 2 weeks of oscillation, compared with day 0, there was basically no change in visible foreign matter, concentration, turbidity, pH, osmotic pressure, viscosity, biological activity, thermal stability, and insoluble particles. The change range of SEC detection of aggregates was very small, all within 0.05%, especially the change range of aggregates in formulations 1-3 was within 0.02%; the change range of the main peak of NR-CE-SDS was within 0.7%, with almost no change (specific values ​​not shown).

[0073] The results of the above-mentioned oscillation test show that the aggregation and degradation characteristics of the anti-NGF antibody of the present invention in each preparation are almost unchanged under oscillation conditions, indicating that each antibody preparation has excellent stability.

[0074] 4. Stability test under freeze-thaw conditions

[0075] The stability of the antibody preparation was tested under freeze-thaw conditions (freeze: -20°C ± 5°C; thaw: room temperature, freeze-thaw 0, 5, and 10 times).

[0076] (1) Table 6 shows the test results of each preparation of the exemplary antibody Ab4: within 5 freeze-thaw cycles, the visible foreign matter, concentration, turbidity, pH, osmotic pressure, viscosity, biological activity, thermal stability, and insoluble particles of each antibody preparation were basically unchanged compared to 0 freeze-thaw cycles; the change in aggregates detected by SEC was very small, all within 0.10%, especially the change in aggregates of formulations 1-3 was within 0.07%; the change in the main peak of NR-CE-SDS was within 1.0%, with almost no change (specific values ​​not shown). The above results show the excellent stability of the Ab4 antibody preparation under freeze-thaw conditions.

[0077] Table 6 Freeze-thaw test results of Ab4 antibody preparation

[0078]

[0079] (2) The various preparations of the Ab4 mutant also showed excellent stability under freeze-thaw conditions: within 5 freeze-thaw cycles, compared with 0 freeze-thaw cycles, there was basically no change in visible foreign matter, concentration, turbidity, pH, osmotic pressure, viscosity, biological activity, thermal stability, and insoluble particles; the SEC detection of aggregates had a very small change range, all within 0.10%, especially the aggregates of formulations 1-3 had a change range within 0.08%; the NR-CE-SDS main peak had a change range within 1.0%, with almost no change (specific values ​​not shown).

[0080] The results of the freeze-thaw test indicate that the aggregation and degradation characteristics of the anti-NGF antibody of the present invention in each preparation are almost unchanged under freeze-thaw conditions, indicating that each antibody preparation has excellent stability.

[0081] 5. Stability test under accelerated conditions

[0082] For Formulations 1-3, the stability of exemplary antibody Ab4 and Ab4 mutant formulations was tested under accelerated conditions (25°C ± 2°C, 60% ± 10% RH (relative humidity) accelerated for 0 days, 2 weeks, 1 month, 2 months).

[0083] (1) Table 7 shows the test results of various preparations of antibody Ab4. The results show that within 2 months of acceleration, the visible foreign matter, concentration, turbidity, pH, osmotic pressure, viscosity, activity, thermal stability and insoluble particles of various antibody preparations were basically unchanged compared with day 0. The aggregates were almost unchanged, and the main peak was slightly reduced: SEC detected almost no change in aggregates; the change range of the main peak detected by NR-CE-SDS was between 0.8%-0.9%; IEC detected a slight increase in acid peak and alkaline peak, and a slight decrease in the main peak: the acid peak increased by about 6-14%, the main peak decreased by about 12-16%, and the alkaline peak increased by about 2-6%. The above results show that the Ab4 antibody preparations of prescriptions 1-3 can basically remain stable under accelerated conditions.

[0084] Table 7 Accelerated test results of Ab4 formulation

[0085]

[0086] (2) The various preparations of antibody Ab4 mutants also showed excellent stability under accelerated conditions: within 2 months of acceleration, compared with day 0, the visible foreign matter, concentration, turbidity, pH, osmotic pressure, viscosity, activity, thermal stability and insoluble particles of the antibody preparations remained basically unchanged. The aggregates increased slightly and the main peak decreased slightly: the change range of aggregates detected by SEC was within 0.02%; the change range of the main peak detected by NR-CE-SDS was within 0.9%; the acid peak and alkaline peak increased slightly and the main peak decreased slightly by IEC: the change range of the acid peak was within 15%, the change range of the main peak was within 15%, and the change range of the alkaline peak was within 5% (specific data not shown).

[0087] The results of the accelerated test show that the aggregates and degradation characteristics of the anti-NGF antibody of the present invention in each preparation do not show significant changes under accelerated conditions, indicating that each antibody preparation has excellent stability.

[0088] 6. Stability test under long-term conditions

[0089] The stability of Ab4 antibody and Ab4 antibody mutant formulations 1-3 was tested under long-term conditions (5°C ± 3°C for 0 day, 2 weeks, 1 month, 3 months, and 6 months).

[0090] (1) Table 10 shows the test results of each preparation of the exemplary antibody Ab4: within 6 months under long-term conditions, relative to day 0, the visible foreign matter, concentration, turbidity, pH, osmotic pressure, viscosity, activity, thermal stability and insoluble particles of each antibody preparation were basically unchanged; the aggregates and main peaks were almost unchanged: the change range of aggregates detected by SEC was within 0.1%; the change range of the main peak detected by NR-CE-SDS was within 0.2%; the acid peak and alkaline peak slightly increased and the main peak slightly decreased by IEC: the acid peak changed within 0-0.7%, the main peak changed within 0.3-0.8%, and the alkaline peak changed within 0.1-0.4%. The above results show the excellent stability of the Ab4 antibody preparation under long-term conditions.

[0091] Table 10 Long-term stability results of Ab4 antibody formulation

[0092]

[0093] (2) The various preparations of the Ab4 mutants also showed excellent stability under long-term conditions: under long-term conditions for 6 months, the visible foreign matter, concentration, turbidity, pH, osmotic pressure, viscosity, activity, thermal stability and insoluble particles of the antibody preparations remained basically unchanged compared to day 0. The aggregates and main peaks were almost unchanged: the change range of aggregates detected by SEC was within 0.1%; the change range of the main peak detected by NR-CE-SDS was within 0.2%; the acid peak and alkaline peak slightly increased and the main peak slightly decreased by IEC: the acid peak changed within 0.6%, the main peak changed within 0.9%, and the alkaline peak changed within 0.5% (specific data not shown).

[0094] The results of the above-mentioned long-term test items show that the aggregation and degradation characteristics of the anti-NGF in each antibody preparation of the present invention are almost unchanged under long-term conditions, indicating that each antibody preparation has excellent stability.

[0095] In summary, the anti-NGF antibody preparation of the present invention has strong stability under high temperature, light, oscillation, freeze-thaw, accelerated and long-term conditions, which can ensure that the preparation maintains good stability during preparation, transportation and storage, and ensure clinical drug safety and quality controllability.

Claims

1. An anti-NGF antibody preparation, characterized in that The preparation comprises an isolated anti-NGF antibody, a stabilizer, a surfactant, and a buffer, wherein the buffer is a histidine-histidine hydrochloride buffer, and the stabilizer is arginine hydrochloride.

2. The preparation according to claim 1, characterized in that The antibody concentration is 5 mg / ml-50 mg / ml, preferably 8 mg / ml-40 mg / ml, and more preferably 10 mg / ml-30 mg / ml.

3. The preparation according to claim 1 or 2, characterized in that The concentration of the stabilizer is 80mM-180mM; preferably 100mM-160mM.

4. The preparation according to any one of claims 1 to 3, characterized in that The concentration of the buffer is 3mM-80mM; preferably, the concentration of the buffer is 5mM-50mM; more preferably, the concentration of the buffer is 10mM-30mM.

5. The preparation according to any one of claims 1 to 4, characterized in that The pH value of the preparation is 5.0-6.5; preferably 5.3-6.2, more preferably 5.5-6.

0.

6. The preparation according to any one of claims 1 to 5, characterized in that The surfactant is polysorbate; preferably, the polysorbate is Tween-20 or Tween-80.

7. The preparation according to any one of claims 1 to 6, characterized in that The concentration of the surfactant is 0.05 mg / ml-1.0 mg / ml; preferably, the concentration of the surfactant is 0.15 mg / ml-0.45 mg / ml.

8. The preparation according to any one of claims 1 to 7, characterized in that The formulation further comprises a preservative and / or an antioxidant.

9. The preparation according to claim 8, characterized in that The preservative is ethylenediaminetetraacetic acid and / or diethylenetriaminepentaacetic acid; and the antioxidant is methionine.

10. The preparation according to claim 8 or 9, characterized in that The concentration of the preservative is 0-0.2 mg / ml, preferably 0.02-0.1 mg / ml.

11. The preparation according to any one of claims 8 to 10, characterized in that The concentration of the antioxidant is 0-20 mM, preferably 5 mM-15 mM.

12. The preparation according to any one of claims 1 to 11, characterized in that The preparation is a liquid preparation or a powder for injection.

13. The preparation according to any one of claims 1 to 12, characterized in that The antibody comprises V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and V L , the V L It comprises: LC-CDR1, which comprises the amino acid sequence of SEQ ID NO:4, LC-CDR2, which comprises the amino acid sequence of SEQ ID NO:5, and LC-CDR3, which comprises the amino acid sequence of SEQ ID NO:

6.

14. The preparation according to any one of claims 1 to 13, characterized in that The antibody comprises V H , which comprises the amino acid sequence SEQ ID NO:7, and V L , which comprises the amino acid sequence SEQ ID NO:

8.

15. The preparation according to any one of claims 1 to 14, characterized in that The antibody comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10, and the light chain comprising the amino acid sequence of SEQ ID NO:

11.

16. Use of the preparation according to any one of claims 1-15 in the preparation of a medicament for treating a disease, preferably, the disease or condition is caused by increased NGF expression or increased sensitivity to NGF; more preferably, the disease or condition is selected from one or more of inflammatory pain, postoperative incision pain, neuropathic pain, fracture pain, gouty joint pain, postherpetic neuralgia, pain caused by burns, cancer pain, osteoarthritis or rheumatoid arthritis pain, sciatica, pain associated with sickle cell crisis and postherpetic neuralgia.

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