WO2023138499A1 - 一种特异性地识别粒细胞-巨噬细胞集落刺激因子受体的抗体制剂及其应用 - Google Patents

一种特异性地识别粒细胞-巨噬细胞集落刺激因子受体的抗体制剂及其应用 Download PDF

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WO2023138499A1
WO2023138499A1 PCT/CN2023/072069 CN2023072069W WO2023138499A1 WO 2023138499 A1 WO2023138499 A1 WO 2023138499A1 CN 2023072069 W CN2023072069 W CN 2023072069W WO 2023138499 A1 WO2023138499 A1 WO 2023138499A1
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buffer
antibody
sodium chloride
tween
concentration
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PCT/CN2023/072069
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English (en)
French (fr)
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廖川
海岗
张婷婷
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舒泰神（北京）生物制药股份有限公司
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Priority to CN202380009458.0A priority Critical patent/CN116887856A/zh
Publication of WO2023138499A1 publication Critical patent/WO2023138499A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • the invention belongs to the technical field of biomedicine, and in particular relates to an antibody preparation specifically recognizing granulocyte-macrophage colony-stimulating factor receptor alpha (GM-CSFR alpha) and application thereof.
  • GM-CSFR alpha granulocyte-macrophage colony-stimulating factor receptor alpha
  • Granulocyte-macrophage colony-stimulating factor is also known as colony-stimulating factor 2 (CSF2).
  • CSF2 colony-stimulating factor 2
  • GM-CSF is a type I pro-inflammatory cytokine that plays a role in exacerbating inflammatory, respiratory and autoimmune diseases.
  • GM-CSF receptor is a member of the hematopoietic receptor superfamily, which is a heterodimer composed of ⁇ and ⁇ subunits.
  • GM-CSF is capable of binding to the alpha subunit alone with relatively low affinity (Kd 1-5 nM), but not at all to the beta subunit alone.
  • International patent application WO2020/108423A1 discloses an isolated anti-GM-CSFR ⁇ antibody that can treat autoimmune diseases and/or inflammatory disorders or cancers (e.g., rheumatoid arthritis, asthma, myelogenous leukemia) characterized by high expression of GM-CSF and/or GM-CSFR ⁇ and/or abnormal function of GM-CSF/GM-CSFR ⁇ .
  • monoclonal antibody drugs are macromolecular protein drugs. Compared with traditional small molecule drugs, they are prone to aggregation and degradation during storage, which will cause adverse consequences such as increased differences between drug batches and changes in immunogenicity.
  • the present invention finally obtains an anti-GM-CSFR ⁇ antibody preparation with high stability through rational design of preparation prescription and detection.
  • the anti-GM-CSFR ⁇ antibody preparation of the present invention has strong stability, which can ensure good stability of the preparation during preparation, transportation and storage, as well as quality controllability and clinical drug safety.
  • the present invention provides an anti-GM-CSFR ⁇ antibody preparation, the antibody preparation comprising an anti-GM-CSFR ⁇ antibody, a stabilizer, a surfactant, and a buffer, the buffer being a phosphate buffer with a pH of 6.5-7.5, preferably 6.7-7.3, and in some specific embodiments, the pH is 6.7, 6.8, 6.9, 7.0, 7.1, 7.2 or 7.3; more preferably 7.0.
  • the concentration of the buffer is 10mM-30mM; in some specific embodiments, the concentration of the buffer is 10mM, 20mM or 30mM; more preferably 20mM.
  • the concentration of the antibody is 50mg/ml-200mg/ml, preferably, the concentration of the antibody is 50mg/ml-180mg/ml, more preferably 50mg/ml-150mg/ml, more preferably 100mg/m-150mg/ml. In some embodiments, the concentration of the antibody is 50 mg/ml, 75 mg/ml, 100 mg/ml, 125 mg/ml, 150 mg/ml or 180 mg/ml.
  • the stabilizer comprises sodium chloride.
  • the concentration of the sodium chloride is 70mM- 200mM, preferably 100mM-200mM, more preferably 100mM-150mM, further preferably 120mM-150mM.
  • the stabilizer further includes arginine hydrochloride.
  • the concentration of the arginine hydrochloride is 0-50mM, preferably 0-40mM, more preferably 15mM-40mM.
  • the concentration of the sodium chloride is 50mM-150mM, preferably 70mM-150mM; more preferably 70mM-120mM.
  • the stabilizer is:
  • the stabilizer is 100mM, 120mM, 150mM, 180mM or 200mM sodium chloride or arginine hydrochloride; or 15mM arginine hydrochloride and 120mM sodium chloride; or 25mM arginine hydrochloride and 100mM sodium chloride; or 40mM arginine hydrochloride and 70mM sodium chloride; mM sodium chloride; or 75 mM arginine hydrochloride and 75 mM sodium chloride; or 100 mM arginine hydrochloride and 50 mM sodium chloride, or 150 mM arginine hydrochloride and 150 mM sodium chloride.
  • the surfactant is polysorbate; preferably, the polysorbate is Tween-20 or Tween-80.
  • the concentration of the surfactant is 0.05 mg/ml-0.3 mg/ml; preferably, the concentration of the surfactant is 0.1 mg/ml-0.2 mg/ml.
  • the antibody preparation is any of the following preparations:
  • the antibody concentration is 50mg/ml, 75mg/ml, 100mg/ml, 125mg/ml, 150mg/ml or 180mg/ml;
  • the stabilizer is 50mM-150mM (preferably 70mM-120mM) sodium chloride and 0-50mM (preferably 15mM-40mM) arginine hydrochloride, or 100mM-200mM (preferably 120mM- 150mM) of sodium chloride;
  • the buffer is a phosphate buffer of 10mM-30mM; the pH of the buffer is 6.7-7.3;
  • the surfactant is Tween-20 and/or Tween-80 of 0.1mg/ml-0.2mg/ml;
  • the antibody concentration is 50mg/ml-180mg/ml;
  • the stabilizer is 15mM arginine hydrochloride and 120mM sodium chloride, or 25mM arginine hydrochloride and 100mM sodium chloride, or 40mM arginine hydrochloride and 70mM sodium chloride, or 50mM arginine hydrochloride and 100mM sodium chloride, or 100mM, 120mM, 150mM, 180mM or 200mM sodium chloride;
  • the buffer is 10mM-30mM phosphate buffer;
  • the pH of the buffer is 6.7-7.3;
  • the surfactant is Tween-20 and/or Tween-80 of 0.1mg/ml-0.2mg/ml;
  • the antibody concentration is 50mg/ml-180mg/ml;
  • the stabilizer is 50mM-150mM (preferably 70mM-120mM) sodium chloride and 0-50mM (preferably 15mM-40mM) arginine hydrochloride, or 100mM-200mM (preferably 120mM-150mM) sodium chloride;
  • the buffer is 10mM, 20mM, 30mM M phosphate buffer;
  • the pH value of the buffer is 6.7-7.3;
  • the surfactant is Tween-20 and/or Tween-80 of 0.1mg/ml-0.2mg/ml;
  • the antibody concentration is 50mg/ml-180mg/ml;
  • the stabilizer is 50mM-150mM (preferably 70mM-120mM) sodium chloride and 0-50mM (preferably 15mM-40mM) arginine hydrochloride, or 100mM-200mM (preferably 120mM-150mM) sodium chloride;
  • the buffer is 10mM-30mM phosphate buffer solution;
  • the pH value of the buffer is 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3;
  • the surfactant is Tween-20 and/or Tween-80 of 0.1mg/ml-0.2mg/ml;
  • the antibody concentration is 50mg/ml-180mg/ml; the stabilizer is 50mM-150mM (preferably 70mM-120mM) Sodium chloride and 0-50mM (preferably 15mM-40mM) arginine hydrochloride, or 100mM-200mM (preferably 120mM-150mM) sodium chloride; the buffer is 10mM-30mM phosphate buffer; the pH of the buffer is 6.7-7.3; the surfactant is 0.1mg/ml, 0.15mg/ml, 0.2mg/ml Tween-2 0 or Tween-80;
  • the antibody concentration is 50mg/ml-180mg/ml; the stabilizer is 100mM-200mM (preferably 120mM-150mM) sodium chloride; the buffer is 10mM-30mM phosphate buffer; the pH of the buffer is 6.7-7.3; the surfactant is 0.1mg/ml-0.2mg/ml Tween-20 or Tween-80;
  • the antibody concentration is 50mg/ml-180mg/ml; the stabilizer is 50mM-150mM (preferably 70mM-120mM) sodium chloride and 0-50mM (preferably 15mM-45mM) arginine hydrochloride; the buffer is 10mM-30mM phosphate buffer; the pH of the buffer is 6.7-7.3; the surfactant is 0.1mg/ml-0. 2 mg/ml of Tween-20 or Tween-80.
  • the antibody preparation is any of the following preparations:
  • the antibody concentration is 100mg/ml;
  • the stabilizer is 25mM arginine hydrochloride and 100mM sodium chloride;
  • the buffer is 20mM phosphate buffer;
  • the pH of the buffer is 6.7 or 7.0 or 7.3;
  • the surfactant is Tween-20 or Tween-80 of 0.1mg/ml or 0.2mg/ml;
  • the antibody concentration is 100mg/ml;
  • the stabilizer is 100mM or 120mM or 200mM sodium chloride;
  • the buffer is 20mM phosphate buffer;
  • the pH of the buffer is 7.0;
  • the surfactant is Tween-20 or Tween-80 of 0.1mg/ml or 0.2mg/ml;
  • the antibody concentration is 100mg/ml;
  • the stabilizer is 50mM arginine hydrochloride and 70mM sodium chloride;
  • the buffer is 20mM phosphate buffer;
  • the pH of the buffer is 7.0;
  • the surfactant is Tween-20 or Tween-80 of 0.1mg/ml or 0.2mg/ml;
  • the antibody concentration is 150mg/ml; the stabilizer is 15mM arginine hydrochloride and 120mM sodium chloride; the buffer is 20mM phosphate buffer; the pH of the buffer is 7.0; the surfactant is 0.1mg/ml or 0.2mg/ml of Tween-20 or Tween-80.
  • the antibody preparation may further comprise antioxidants and/or preservatives.
  • the antioxidant includes but not limited to ascorbic acid and/or methionine;
  • the preservative includes but not limited to octadecyl dimethyl benzyl ammonium chloride, hexamethyl ammonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butanol or benzyl alcohol, alkyl p-hydroxybenzoate, etc.
  • the antibody preparation is a liquid preparation or a powder for injection.
  • the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 or 2, and a light chain comprising the amino acid sequence of SEQ ID NO: 3.
  • the present invention discloses the use of the antibody preparation in the preparation of medicines for treating diseases.
  • the disease comprises an autoimmune disease, an inflammatory disorder, or cancer; preferably, the disease is rheumatoid arthritis, asthma, or myelogenous leukemia.
  • the anti-GM-CSFR ⁇ antibody preparation provided by the present invention has the following excellent effects:
  • the present invention screens out the best buffer system for anti-GM-CSFR ⁇ antibody. Compared with other buffer systems, the phosphate buffer system with pH6.5-7.5 The flushing solution can make the anti-GM-CSFR ⁇ antibody preparation have low turbidity, low viscosity, optimal thermal stability (Tm) and good uniformity.
  • the present invention uses sodium chloride and/or arginine hydrochloride as a stabilizer, especially sodium chloride combined with lower concentration of arginine hydrochloride (0-50mM) as a stabilizer or 100mM-200mM sodium chloride as a stabilizer, which can significantly reduce the formation of aggregates in antibody preparations, slow down the change of charge heterogeneity, prevent the formation and aggregation of acidic substances, and maintain good stability for antibody preparations.
  • the anti-GM-CSFR ⁇ antibody preparation of the present invention has strong stability, which can maintain good stability of the preparation during preparation, transportation and storage, and ensure quality controllability and clinical drug safety.
  • reagents used in the following examples were prepared by conventional methods or obtained from commercial sources; the experimental methods used, unless otherwise specified, were conventional methods; the materials and instruments used, if not otherwise specified, were obtained from commercial sources.
  • the anti-GM-CSFR ⁇ antibody is an antibody that binds to granulocyte-macrophage colony-stimulating factor receptor ⁇ (GM-CSFR ⁇ ).
  • GM-CSFR ⁇ granulocyte-macrophage colony-stimulating factor receptor ⁇
  • E35-1 used in the examples of the present invention is disclosed in the international patent application WO2020/108423A1, the disclosure of which is incorporated herein by reference.
  • Embodiment 1 screening the buffer system of anti-GM-CSFR ⁇ antibody preparation
  • the GM-CSFR ⁇ antibody E35-1 (without PS20) was dialyzed into the buffer system, and excipients (sodium chloride, mannitol, sucrose or sorbitol) and PS20 were added, the Tm value was detected by differential scanning fluorescence (DSF), and the turbidity was measured by nephelometry.
  • Tm value detection results are shown in Table 2. It can be seen from Table 2 that in buffer systems with different pH ranges, the Tm values of the tested antibodies are different. Antibody samples in phosphate buffer at pH 7.0 had a higher Tm (higher than 66°C), indicating that the anti-GM-CSFR ⁇ antibody had significantly higher thermal stability at this pH and buffer system.
  • the pH range of three different buffer systems (100mM histidine hydrochloride, 20mM phosphate and 200mM Tris hydrochloride) was adjusted within the range of 6.8-7.2, and the Tm value was further detected by DSF method.
  • the antibody sample in the phosphate buffer has a higher TM value compared to other commonly used cushioning systems (histidine and Tris hydrochloric acid).
  • the TM value of the antibody sample in the phosphate buffer in phosphate buffer is basically unchanged under different pH values.
  • Anti-GM-CSFR ⁇ antibody the pH value of about 6.8-7.2 phosphate buffer has a better stable effect than other commonly used buffer.
  • the GM-CSFR ⁇ antibody E35-1 (buffer system is 100mM histidine at pH 7.0 and 20mM phosphate buffer at pH 7.0) was concentrated to a high concentration (50mg/ml-150mg/ml), and its pH value and viscosity were measured.
  • the 20mM phosphate buffer system can well maintain the pH value at about 7.0; while in other buffer systems (for example, 100mM histidine buffer), the pH value decreases with the increase of the antibody concentration, and the pH value of the solution cannot be maintained stably.
  • the viscosity of the antibody at 50mg/ml and 100mg/ml was lower than 2.0cP, meeting the requirements for subcutaneous injection, but in contrast, the viscosity of the antibody in the 20mM phosphate buffer system was lower than that in the 100mM histidine hydrochloride buffer system.
  • the hydrodynamic radius of antibody molecules was examined using dynamic light scattering to assess the uniformity of antibody particle size.
  • the anti-GM-CSFR ⁇ antibody preparation in the 20mM phosphate buffer with a pH value of 7.0, compared with other buffer systems, the anti-GM-CSFR ⁇ antibody preparation has better turbidity and viscosity, and has the best thermal stability (Tm) and good uniformity.
  • Embodiment 2 the screening of tensio-active agent
  • Oscillating conditions 200rpm, at room temperature, and samples were collected on the 1st, 3rd, and 5th days to detect antibody concentration, turbidity, and aggregate characteristics.
  • the aggregate characteristics were detected by SEC method.
  • Microfluidic imager was used to quantify the particles below visible light.
  • the F1 sample had a relatively high number of particles (5145 particles/ml for >10 ⁇ m particles and 303 particles/ml for >25 ⁇ m particles) when placed in the vial on day 0, and the number increased beyond the detection range after 3 days of shaking; the samples of F2-F5 still maintained a low number of particles after shaking for 5 days (all particles >10 ⁇ m were below 642 particles/ml, and particles >25 ⁇ m less than 75/ml). There were no significant differences among groups F2-F5.
  • Antibody preparations were frozen at -70°C, thawed at room temperature, and mixed by pipette before thawing. Samples were collected after 3 and 5 cycles to detect antibody concentration, turbidity, and aggregate properties, which were detected by SEC.
  • the test results showed that after repeated freezing and thawing five times, the antibody concentration, turbidity and HWM% of the F1 group increased compared with those without freezing and thawing, and the antibody concentration, turbidity and HWM% of the F2-F5 group did not change compared with those without freezing and thawing, and there was no significant difference between the F2-F5 groups (results not shown).
  • PS20 and PS80 added at 0.01%-0.02% have good protective effects against GM-CSFR ⁇ antibody, and there is no significant difference between them.
  • the GM-CSFR ⁇ antibodies (E35-1 and E35-2) were dialyzed into pH7.3 and pH6.7 buffers, respectively, and corresponding adjuvants were added according to Table 7. Place it at 45°C, collect samples after 1, 2, 3, and 4 weeks, and detect the concentration, turbidity, aggregation characteristics, and charge heterogeneity of antibody preparations to evaluate the stability of different antibody preparations. Among them, the aggregation characteristics were detected by HPLC-SEC method, and the charge heterogeneity was detected by iIEF method.
  • the antibody concentration of each group had no significant change (results not shown), and the turbidity tended to increase slightly.
  • the F8 (arginine hydrochloride) group and the F9 (sodium chloride) group had the least increase in turbidity value compared to 0 weeks, only about 0.02, and the turbidity of the F8 (arginine hydrochloride) group and the F9 (sodium chloride) group was the lowest at 4 weeks, especially the turbidity value of the F9 group was only 0.297, indicating that the antibody stability of the F9 group was the highest.
  • the detection results of HPLC-SEC are shown in Table 8.
  • the aggregates in each group of preparations have an increasing trend, and the main peak has a decreasing trend.
  • the F8 (arginine hydrochloride) and F9 (sodium chloride) groups especially the F9 group aggregates had the slowest increase rate and the slowest main peak decrease rate.
  • the main peaks of both F8 and F9 were greater than 95%, in contrast, the proportions of the main peaks in the other groups had dropped below 95%.
  • the antibody concentration of each group had no significant change, and the turbidity showed a slight upward trend.
  • the F8 (arginine hydrochloride) group and the F9 (sodium chloride) group had the least increase in turbidity value compared with the 0-week period, which was only about 0.03.
  • the F8 (arginine hydrochloride) group and the F9 (sodium chloride) group had the lowest turbidity values, especially the F9 group. The turbidity value was only 0.269, indicating that the F9 group had the highest antibody stability.
  • the stabilizer is arginine hydrochloride and sodium chloride or the stabilizer is sodium chloride, which can significantly reduce the formation of aggregation, slow down the change of charge heterogeneity, and prevent the formation and aggregation of acidic substances.
  • the stabilizer is sodium chloride, and its stabilizing effect is more significant.
  • Embodiment 4 evaluate the combined effect of different stabilizers
  • samples were collected after 1, 2, 3 and 4 weeks, and the concentration, turbidity, aggregation characteristics, degradation characteristics, charge heterogeneity, and antigen binding ability of the E35-1 antibody preparation were detected.
  • the polymer characteristics were detected by HPLC-SEC method
  • the degradation characteristics were detected by NR-CE-SDS and R-CE-SDS methods
  • the charge heterogeneity was detected by iIEF method.
  • the osmotic pressure of each group of preparations is shown in Table 11, and the osmotic pressure of A2, A3, A5-A7 is between 300mOsm/kg-400mOsm/kg.
  • the result of turbidity test shows, when compared with 0 weeks, after 4 weeks, in the control group A1 and the A3 group that only added sodium chloride, the turbidity of each preparation has no obvious change, and the turbidity value of preparation in each group that adds arginine hydrochloride all increases to varying degrees: A5 and A7 groups (the addition amount of arginine hydrochloride is at 50mM-75mM) after 4 weeks, turbidity increases respectively 0.187 and 0.204, A2, A4, A6 (addition of arginine hydrochloride The amount is 100mM-150mM) group of preparations, after 4 weeks, the turbidity increase range is between 0.25-0.27.
  • Embodiment 5 Stability experiment of anti-GM-CSFR ⁇ antibody preparation
  • Table 16 shows the freeze-thaw results of each preparation of the E35-1 antibody. After 5 freeze-thaw cycles, the protein concentration was only slightly reduced (102.88mg/mL vs 100.25mg/mL) compared to the non-freeze-thaw preparation. However, the detection results of HPLC-SEC showed that the aggregate remained basically unchanged. The number of particles below visibility has been increased.
  • the E35-2 antibody preparation also showed good stability: after 5 times of freezing and thawing, the protein concentration was only slightly decreased compared to the non-freezing and thawing preparation (results not shown). However, the detection of aggregates by HPLC-SEC remained basically unchanged. Slightly increased particle count below visibility.
  • the antibody preparation of the T1 group (20mM PB, 25mM arginine hydrochloride, 100mM sodium chloride, pH 7) was filtered 5 times through a Meissner SteriLUX hydrophilic PVDF 0.2 filter, and the antibody concentration was detected after each filtration.
  • Table 17 shows the results of filtration experiments of various preparations of the E35-1 antibody. After 5 times of filtration, the concentration of the antibody did not change significantly compared to that without filtration.
  • the HPLC-SEC results are shown in Table 18. Compared with the 0th week, the aggregation degree of antibodies in each group showed a slight increase trend after 4 weeks. However, the main peaks in the T1-T3 and T5-T6 groups only decreased by 0.84%-1.29%, and the aggregates increased by 0.84%-1.29%; while the main peaks in the T4 and T7-T8 groups decreased by 1.39%-1.43%, and the aggregates increased by 1.34%-1.41%. In contrast, the stability of the antibody preparations in the T1-T3 group was better than that in the T4, T7-T8 group preparations.
  • the results of cGE are shown in Table 19.
  • the results showed that, compared with week 0, the main peak and fragments changed after 4 weeks, the main peak had a slight decreasing trend, and the fragments had a slight increasing trend.
  • the T1 non-reducing main peak decreased by 3.6%
  • the reducing main peak decreased by 1.4%
  • the T2-T3, T5-T6 non-reducing main peak decreased by 4.2%-4.5%
  • the T4, T7-T8 non-reducing main peak decreased by 4.8%-5.0%.
  • the above results further confirm that the stability of the antibody preparations in the T1-T3 group is better than that in the T4 and T7-T8 groups.
  • Table 20 shows the results of the binding ability of the antibody to the antigen. Compared with the 0 week, after 4 weeks of storage, the binding ability of all preparations to the antigen remained basically unchanged.
  • the T1 non-reducing main peak decreased by 3.4%
  • the reducing main peak decreased by 1.5%
  • the T2-T3, T5-T6 non-reducing main peak decreased by 4.2%-4.6%
  • the T4, T7-T8 non-reducing main peak decreased by more than 5.0%.
  • the stability of the antibody preparations of the T1-T3 group is better than that of the T5-T6 group preparations, and even better than that of the T4 and T7-T8 group preparations.
  • the anti-GM-CSFR ⁇ antibody preparation of the present invention is stable within 5 cycles of freezing and thawing; there is no obvious antibody loss after 5 times of filtration; the antibody is slightly degraded under high temperature conditions, but not obvious.
  • the combination of arginine hydrochloride and sodium chloride with a concentration lower than 50 mM as a stabilizer has a stronger protective effect on the antibody than sodium chloride alone as a stabilizer, and has a more excellent effect.
  • E35-1 and E35-2 antibody preparations T1 group and T4 group were prepared according to the recipe in Example 5.
  • the stability of the antibody preparation was tested under long-term storage conditions (5 ⁇ 3°C for 0 days, 1 month, 3 months, and 6 months).
  • Table 21 shows the stability results of E35-1 antibody preparations under long-term storage conditions. Compared with 0 months, after 6 months, the indicators of E35-1 antibody preparations in groups T1 and T4 remained basically unchanged.
  • E35-2 antibody preparations also showed good stability under long-term storage conditions. Compared with 0 months, after 6 months, the indicators of E35-2 antibody preparations in groups T1 and T4 remained basically unchanged.
  • E35-1 and E35-2 antibody preparations were tested under long-term accelerated conditions (25°C ⁇ 2°C for 0 days, 1 month, 3 months, and 6 months).
  • Table 22 shows the stability results of E35-1 antibody preparations under accelerated conditions. Compared with 0 months, at 6 months, the E35-1 antibody preparations only showed a slight decrease in the main peak, a slight increase in the acid peak, and other indicators remained basically unchanged.
  • the E35-2 antibody preparation also showed good stability under long-term accelerated conditions. Compared with 0 months, after 6 months, the E35-2 antibody preparation only showed a slight decrease in the main peak, a slight increase in the acid peak, and other indicators remained basically unchanged.
  • the anti-GM-CSFR ⁇ antibody preparation of the present invention has high stability under long-term storage and long-term accelerated conditions, which can ensure good stability of the preparation during preparation, transportation and storage, and ensure the safety and quality controllability of clinical medication.

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Abstract

一种抗GM-CSFRα抗体制剂，所述制剂包含抗GM-CSFRα抗体、稳定剂、表面活性剂、缓冲液，所述缓冲液为磷酸盐缓冲液，pH值为6.5-7.5。所述抗GM-CSFRα抗体制剂在制备、运输及存储过程中具有良好的稳定性，保证了质量可控性及临床用药安全性。

Description

一种特异性地识别粒细胞-巨噬细胞集落刺激因子受体的抗体制剂及其应用 技术领域
本发明属于生物医药技术领域，具体涉及一种特异性识别粒细胞-巨噬细胞集落刺激因子受体α(GM-CSFRα)的抗体制剂及其应用。
背景技术
粒细胞-巨噬细胞集落刺激因子(GM-CSF)也称为集落刺激因子2(CSF2)。GM-CSF是I型促炎细胞因子，其在加剧炎性、呼吸和自身免疫疾病方面起作用。GM-CSF受体是造血受体超家族成员之一，为异二聚体，由α和β亚单位组成。GM-CSF能够以相对低的亲和力与α亚单位单独结合(Kd 1-5nM)，但是完全不与β亚单位单独结合。而当α和β亚单位同时存在时，则会产生一个高亲和力的配体-受体复合物(Kd≈100pM)。因此，对GM-CSF与GM-CSFRα结合的中和作用，成为治疗GM-CSFRα介导的疾病和病症的治疗方法。
国际专利申请WO2020/108423A1公开了一种分离的抗GM-CSFRα抗体，该抗体可以治疗以高表达GM-CSF和/或GM-CSFRα和/或GM-CSF/GM-CSFRα功能异常为特征的自身免疫性疾病和/或炎性病症或癌症(例如，类风湿性关节炎、哮喘、骨髓性白血病)。但是单克隆抗体药物为大分子蛋白药物，与传统小分子药物相比，在储存过程中容易发生聚集和降解等现象，会造成药品批次间差异增大以及免疫原性改变等不良后果。
本发明通过合理设计制剂处方并检测，最终获得了一种稳定性高的抗GM-CSFRα抗体制剂。本发明的抗GM-CSFRα抗体制剂稳定性强，可保证该制剂在制备、运输及存储过程中保持良好的稳定性，保证质量可控性及临床用药安全性。
发明内容
在一个方面，本发明提供了一种抗GM-CSFRα抗体制剂，所述抗体制剂包含抗GM-CSFRα抗体、稳定剂、表面活性剂、缓冲液，所述缓冲液为磷酸盐缓冲液，pH值为6.5-7.5，优选为6.7-7.3，在一些具体实施方式中，pH为6.7、6.8、6.9、7.0、7.1、7.2或7.3；更优选为7.0。
在一些实施方式中，所述缓冲液的浓度为10mM-30mM；在一些具体实施方式中，所述缓冲液的浓度为10mM、20mM或30mM；更优选为20mM。
在一些实施方式中，所述抗体的浓度为50mg/ml-200mg/ml，优选地，所述抗体的浓度为50mg/ml-180mg/ml，进一步优选为50mg/ml-150mg/ml，更优选为100mg/m-150mg/ml。在一些具体实施方式中，抗体的浓度为50mg/ml、75mg/ml、100mg/ml、125mg/ml、150mg/ml或180mg/ml。
在一些实施方式中，所述稳定剂包含氯化钠。在一些实施方式中，所述氯化钠的浓度为70mM- 200mM，优选为100mM-200mM，更优选为100mM-150mM，进一步优选为120mM-150mM。
在一些实施方式中，所述稳定剂进一步包括精氨酸盐酸盐。在一些优选实施方式中，所述精氨酸盐酸盐的浓度为0-50mM，优选为0-40mM，更优选为15mM-40mM。在一些优选实施方式中，所述氯化钠的浓度为50mM-150mM，优选为70mM-150mM；更优选为70mM-120mM。
在一些实施方式中，所述稳定剂为：
(1)50mM-150mM(优选70mM-120mM)的氯化钠和0-50mM(优选15mM-40mM)的精氨酸盐酸盐；
(2)100mM-200mM(优选120mM-150mM)的氯化钠。在一些具体实施方式中，所述稳定剂为100mM、120mM、150mM、180mM或200mM的氯化钠或精氨酸盐酸盐；或15mM的精氨酸盐酸盐和120mM的氯化钠；或25mM的精氨酸盐酸盐和100mM的氯化钠；或40mM的精氨酸盐酸盐和70mM的氯化钠；或50mM的精氨酸盐酸盐和100mM的氯化钠；或75mM的精氨酸盐酸盐和75mM的氯化钠；或100mM的精氨酸盐酸盐和50mM的氯化钠，或150mM的精氨酸盐酸盐和150mM的氯化钠。
在一些实施方式中，所述表面活性剂是聚山梨醇酯；优选的，所述聚山梨醇酯为吐温-20或吐温-80。
在一些实施方式中，所述表面活性剂的浓度为0.05mg/ml-0.3mg/ml；优选地，所述表面活性剂的浓度为0.1mg/ml-0.2mg/ml。
在一些实施方式中，所述抗体制剂为下述制剂中的任一种：
(1)所述抗体浓度为50mg/ml、75mg/ml、100mg/ml、125mg/ml、150mg/ml或180mg/ml；所述稳定剂为50mM-150mM(优选70mM-120mM)的氯化钠和0-50mM(优选15mM-40mM)的精氨酸盐酸盐，或者100mM-200mM(优选120mM-150mM)的氯化钠；所述缓冲液为10mM-30mM的磷酸盐缓冲液；所述缓冲液的pH值为6.7-7.3；所述表面活性剂为0.1mg/ml-0.2mg/ml的吐温-20和/或吐温-80；
(2)所述抗体浓度为50mg/ml-180mg/ml；所述稳定剂为15mM的精氨酸盐酸盐和120mM氯化钠，或25mM的精氨酸盐酸盐和100mM氯化钠，或40mM的精氨酸盐酸盐和70mM氯化钠，或50mM的精氨酸盐酸盐和100mM氯化钠，或100mM、120mM、150mM、180mM或200mM氯化钠；所述缓冲液为10mM-30mM的磷酸盐缓冲液；所述缓冲液的pH值为6.7-7.3；所述表面活性剂为0.1mg/ml-0.2mg/ml的吐温-20和/或吐温-80；
(3)所述抗体浓度为50mg/ml-180mg/ml；所述稳定剂为50mM-150mM(优选70mM-120mM)的氯化钠和0-50mM(优选15mM-40mM)的精氨酸盐酸盐，或者100mM-200mM(优选120mM-150mM)的氯化钠；所述缓冲液为10mM、20mM、30mM的磷酸盐缓冲液；所述缓冲液的pH值为6.7-7.3；所述表面活性剂为0.1mg/ml-0.2mg/ml的吐温-20和/或吐温-80；
(4)所述抗体浓度为50mg/ml-180mg/ml；所述稳定剂为50mM-150mM(优选70mM-120mM)的氯化钠和0-50mM(优选15mM-40mM)的精氨酸盐酸盐，或者100mM-200mM(优选120mM-150mM)的氯化钠；所述缓冲液为10mM-30mM的磷酸盐缓冲液；所述缓冲液的pH值为6.7、6.8、6.9、7.0、7.1、7.2、7.3；所述表面活性剂为0.1mg/ml-0.2mg/ml的吐温-20和/或吐温-80；
(5)所述抗体浓度为50mg/ml-180mg/ml；所述稳定剂为50mM-150mM(优选70mM-120mM)的 氯化钠和0-50mM(优选15mM-40mM)的精氨酸盐酸盐，或者100mM-200mM(优选120mM-150mM)的氯化钠；所述缓冲液为10mM-30mM的磷酸盐缓冲液；所述缓冲液的pH值为6.7-7.3；所述表面活性剂为0.1mg/ml、0.15mg/ml、0.2mg/ml的吐温-20或吐温-80；
(6)所述抗体浓度为50mg/ml-180mg/ml；所述稳定剂为100mM-200mM(优选120mM-150mM)的氯化钠；所述缓冲液为10mM-30mM的磷酸盐缓冲液；所述缓冲液的pH值为6.7-7.3；所述表面活性剂为0.1mg/ml-0.2mg/ml的吐温-20或吐温-80；
(7)所述抗体浓度为50mg/ml-180mg/ml；所述稳定剂为50mM-150mM(优选70mM-120mM)的氯化钠和0-50mM(优选15mM-45mM)的精氨酸盐酸盐；所述缓冲液为10mM-30mM的磷酸盐缓冲液；所述缓冲液的pH值为6.7-7.3；所述表面活性剂为0.1mg/ml-0.2mg/ml的吐温-20或吐温-80。
在一些实施方式中，所述抗体制剂为下述制剂中的任一种：
(1)所述抗体浓度为100mg/ml；所述稳定剂为25mM精氨酸盐酸盐和100mM氯化钠；所述缓冲液为20mM的磷酸盐缓冲液；所述缓冲液的pH值为6.7或7.0或7.3；所述表面活性剂为0.1mg/ml或0.2mg/ml的吐温-20或吐温-80；
(2)所述抗体浓度为100mg/ml；所述稳定剂为100mM或120mM或200mM氯化钠；所述缓冲液为20mM的磷酸盐缓冲液；所述缓冲液的pH值为7.0；所述表面活性剂为0.1mg/ml或0.2mg/ml的吐温-20或吐温-80；
(3)所述抗体浓度为100mg/ml；所述稳定剂为50mM精氨酸盐酸盐和70mM氯化钠；所述缓冲液为20mM的磷酸盐缓冲液；所述缓冲液的pH值为7.0；所述表面活性剂为0.1mg/ml或0.2mg/ml的吐温-20或吐温-80；
(4)所述抗体浓度为150mg/ml；所述稳定剂为15mM精氨酸盐酸盐和120mM氯化钠；所述缓冲液为20mM的磷酸盐缓冲液；所述缓冲液的pH值为7.0；所述表面活性剂为0.1mg/ml或0.2mg/ml的吐温-20或吐温-80。
在一些实施方式中，所述抗体制剂还可包含抗氧化剂和/或防腐剂。所述抗氧化剂包括但不限于抗坏血酸和/或蛋氨酸；所述防腐剂包括但不限于十八烷基二甲基苄基氯化铵、六甲基氯化铵、苯扎氯铵、苄索氯铵、苯酚、丁醇或苄醇、对羟基苯甲酸烷基酯等。
在一些实施方式中，所述抗体制剂为液体制剂或注射用粉针剂。
在一些实施方式中，所述抗体包含重链，其包含氨基酸序列SEQ ID NO:1或2，以及轻链，其包含氨基酸序列SEQ ID NO:3。
在另一个方面，本发明公开了所述抗体制剂在制备治疗疾病的药物中的用途。在一些实施方式中，所述疾病包括自身免疫性疾病、炎性病症或癌症；优选地，所述疾病为类风湿性关节炎、哮喘或骨髓性白血病。
本发明提供的抗GM-CSFRα抗体制剂具有以下优异效果：
1、本发明筛选出抗GM-CSFRα抗体的最佳缓冲体系，相比于其他缓冲体系，pH6.5-7.5的磷酸盐缓 冲液可使抗GM-CSFRα抗体制剂的浊度低、粘度低，具有最佳的热稳定性(Tm)和良好的均一性。
2、本发明采用氯化钠和/或精氨酸盐酸盐作为稳定剂，尤其是氯化钠联合较低浓度的精氨酸盐酸盐(0-50mM)作为稳定剂或采用100mM-200mM氯化钠作为稳定剂，可以明显减少抗体制剂中聚集体的形成、减缓电荷异质性变化，防止酸性物质的形成和聚集，使抗体制剂保持良好的稳定性。
3、本发明的抗GM-CSFRα抗体制剂稳定性强，可使该制剂在制备、运输及存储过程中保持良好的稳定性，保证质量可控性及临床用药安全性。
具体实施方式
为使本发明要解决的技术问题、采用的技术方案和优点更加清楚，下面将结合具体实施例对本发明进行详细描述。以下实施例用于说明本发明，但不用来限制本发明的范围。
下述实施例中所使用的试剂，如无特别说明，均采用常规方法配制或者由商业途径得到；所使用的实验方法，如无特别说明，均为常规方法；所使用的材料、仪器等，如无特别说明，均由商业途径得到。
抗GM-CSFRα抗体是与粒细胞巨噬细胞集落刺激因子受体α(GM-CSFRα)结合的抗体，本发明实施例中采用的抗GM-CSFRα抗体E35-1披露于国际专利申请WO2020/108423A1中，其披露内容通过援引并入本文。
本发明实施例中采用的E35-1抗体和E35-2抗体的氨基酸序列如表1所示：
表1.E35-1抗体和E35-2抗体的氨基酸序列
实施例1、筛选抗GM-CSFRα抗体制剂的缓冲体系
1、通过采用差示扫描荧光法(DSF)和浊度法筛选缓冲体系
(1)筛选最佳pH值和缓冲体系
将GM-CSFRα抗体E35-1(不含PS20)透析到缓冲体系中，加入辅料(氯化钠、甘露醇、蔗糖或山梨醇)和PS20，采用差示扫描荧光法(DSF)检测Tm值，采用浊度法测定其浊度。
Tm值检测结果见表2。由表2可看出，在不同pH值范围的缓冲体系中，所测抗体的Tm值不同。在pH为7.0的磷酸盐缓冲液中的抗体样品具有更高的Tm(高于66℃)，说明在该pH值和缓冲体系下抗GM-CSFRα抗体具有明显更高的热稳定性。
表2.Tm的检测结果
浊度检测结果见表3。由表3可看出，在50℃下加热2小时后，在pH为5.0的醋酸盐缓冲液中和pH为7.0的磷酸盐缓冲液中，抗体样品的浊度值增加的幅度很低(仅为0.002-0.005左右)，表明抗体在上述缓冲体系下具有较好的稳定性。
表3.浊度的检测结果
结合Tm值检测结果及浊度检测结果可知，缓冲液pH为7.0左右是抗GM-CSFRα抗体的最佳缓冲pH范围，其效果明显优于其他的pH范围。
(2)确认pH范围内的缓冲体系
将三种不同的缓冲液体系(100mM组氨酸盐酸盐、20mM磷酸盐和200mM Tris盐酸)的pH值范围调节在6.8-7.2范围之内，并采用DSF方法进一步检测Tm值。
结果见表4，在pH 6.8-7.2范围内，磷酸盐缓冲液中的抗体样品与其他常用的缓冲体系(组氨酸和Tris盐酸)中的抗体样品相比具有更高的Tm值，而且，在不同的pH值下，磷酸盐缓冲液中抗体样品的Tm值基本维持不变，进一步验证了对于该抗GM-CSFRα抗体，pH值约为6.8-7.2范围内的磷酸盐缓冲液比起其他常用的缓冲液具有更优的稳定效果。
表4.DSF筛选缓冲体系的Tm检测结果
2、高浓度抗体条件下评估缓冲体系的效果
将GM-CSFRα抗体E35-1(缓冲体系分别为pH7.0的100mM组氨酸和pH7.0的20mM磷酸盐缓冲液)浓缩到高浓度(50mg/ml-150mg/ml)，测量其pH值和粘度。
结果见表5，抗体浓度在50mg/ml-150mg/ml范围内，20mM磷酸盐缓冲体系能够很好地将pH值维持在7.0左右；而在其他缓冲体系中(例如，100mM组氨酸缓冲液)，pH值随抗体浓度升高而降低，不能稳定地维持溶液的pH值。
两种缓冲体系中，抗体浓度在50mg/ml和100mg/ml时的抗体粘度均低于2.0cP，满足皮下注射的要求，但相比之下，20mM磷酸盐缓冲体系中的抗体粘度相比于100mM组氨酸盐酸盐缓冲体系中的抗体粘度更低。
表5.缓冲体系评估结果
3、动态光散射(Dynamic Light Scattering，DLS)
采用动态光散射方法检查抗体分子的流体力学半径，以评估抗体粒径的均一程度。
结果见表6，该结果表明该抗体在此条件下具有较小的多分散性，抗体均一性良好。
表6.DLS评估结果
综上可知，在pH值为7.0的20mM磷酸盐缓冲液中，相比起其他缓冲体系，抗GM-CSFRα抗体制剂具有更优的浊度、粘度，具有最佳的热稳定性(Tm)和良好的均一性。
实施例2、表面活性剂的筛选
在冻融、振荡条件下对不同的表面活性剂(PS20和PS80)及不同的添加量(0.01％和0.02％)进行筛选。
设置5组实验，每组含100mg/mL抗体E35-1，pH7的20mM PB，F1组不添加表面活性剂，F2组添加0.01％的PS20，F3组添加0.02％的PS20，F4组添加0.01％的PS80，F5组添加0.02％的PS80。
1、振荡实验
振荡条件：200rpm，室温下进行，并在第1、3、5天收集样品，检测抗体浓度、浊度、聚体特性，聚体特性采用SEC方法检测。
结果显示：振荡3天后F1样品变得浑浊，而F2-F5保持清晰。振荡5天后，相对于0天，各组样品浓度基本保持不变；F1样品的浊度从0.467增加到2.06，F2-F5样品的浊度基本保持不变；F1-F5的聚体在振荡3天后稍有增加，并在第5天基本保持不变。F2-F5各组之间没有显著差异。
使用微流控成像仪(MFI)对可见光以下的颗粒进行量化，F1样品在第0天放入小瓶时有较高的颗粒数(＞10μm的颗粒高达5145个/ml，＞25μm的颗粒高达303个/ml)，在振荡3天后数量增加到检测范围之外；F2-F5的样品在振荡5天后仍保持较低的颗粒数(＞10μm的颗粒均低于642个/ml，＞25μm的颗粒均低于75个/ml)。F2-F5各组之间没有明显的差异。
2、冻融实验
将抗体制剂在-70℃下冷冻，在室温下解冻，并在回冻前通过移液器混合。在3和5个循环后收集样品，检测抗体浓度、浊度、聚体特性，聚体特性采用SEC方法检测。
检测结果表明，反复冻融5次后，F1组的抗体浓度、浊度和HWM％相比于未冻融时有所增加，F2-F5组的抗体浓度、浊度和HWM％与未冻融时相比基本没有变化，F2-F5各组之间没有明显差异(结果未显示)。
综上所述，添加量在0.01％-0.02％的PS20和PS80对抗GM-CSFRα抗体均具有很好的保护作用，且相互之间没有明显差异。
实施例3、抗GM-CSFRα抗体制剂稳定剂的筛选
将GM-CSFRα抗体(E35-1和E35-2)分别透析至pH7.3和pH6.7缓冲液中，根据表7所示加入相应的辅料。45℃条件下放置，在1、2、3、4周后收集样品，检测抗体制剂的浓度、浊度、聚集特性、电荷异质性，以评估不同抗体制剂的稳定性。其中，聚集特性采用HPLC-SEC方法检测、电荷异质性采用iIEF方法检测。
表7.制剂处方设计

1、E35-1抗体制剂稳定剂的筛选结果
45℃条件下存放4周后，相比于0周时，各组抗体浓度均无明显变化(结果未显示)，浊度有轻微上升的趋势。然而，相比之下，F8(精氨酸盐酸盐)组和F9(氯化钠)组相比于0周时浊度值增加最少，仅为0.02左右，且4周时F8(精氨酸盐酸盐)组和F9(氯化钠)组浊度最低，尤其是F9组浊度值仅为0.297，表明F9组抗体稳定性最高。
HPLC-SEC的检测结果见表8，总体而言，随着时间推进，各组制剂中的聚体均具有增加的趋势，主峰具有下降的趋势。然而，在所有组别中，F8(精氨酸盐酸盐)和F9(氯化钠)组，尤其是F9组聚体的增加速度最慢，主峰下降速度也最慢。在第3周时，F8和F9的主峰均大于95％，相比之下，其他组别中的主峰比例已降至95％以下。
以上结果表明，相对于其他组，F8和F9组中的抗体更不容易发生聚集，稳定性更高。
表8.HPLC-SEC结果

注：ND表示未检出。
cIEF的检测结果见表9，在45℃条件下放置4周后，相对于第0天，各组中均具有酸性峰增加，而主峰减少的趋势，而碱性峰在第1周时开始增加，2-4周后逐渐减少。相比之下，F9(氯化钠)组的酸性峰增加的速度和主峰减少的速度最慢，表明F9组抗体的电荷异质性变化最小，稳定性最高。
表9.cIEF结果
2、E35-2抗体制剂稳定剂的筛选
E35-2抗体的各个制剂处方也表现出类似的结果：
45℃条件下存放4周后，相比于0周时，各组抗体浓度均无明显变化，浊度有轻微上升的趋势，相比之下，F8(精氨酸盐酸盐)组和F9(氯化钠)组相比于0周时浊度值增加最少，仅为0.03左右，且4周时F8(精氨酸盐酸盐)组和F9(氯化钠)组浊度最低，尤其是F9组浊度值仅为0.269，表明F9组抗体稳定性最高。
45℃条件下，各组制剂中的聚体均具有增加的趋势，主峰具有下降的趋势。在所有组别中，F8(精氨酸盐酸盐)和F9(氯化钠)组，尤其是F9组聚体的增加速度最慢(仅增加了1.93％)，主峰下降速度也最慢(仅降低了2.46％)。在第3周时，F8和F9的主峰均大于96％，相比之下，其他组别中的主峰比例已降至95％以下。
在45℃条件下放置4周后，相对于第0天，各组中均具有酸性峰增加，而主峰减少的趋势。相比之下，F9(氯化钠)组的酸性峰增加的速度(仅增加32.7％)和主峰减少的速度最慢(仅减少了33.6％)。
综上所述：对于本申请的抗GM-CSFRα抗体，稳定剂为精氨酸盐酸盐和氯化钠或稳定剂为氯化钠均能明显减少聚集的形成、减缓电荷异质性变化，防止酸性物质的形成和聚集，尤其是稳定剂为氯化钠，其稳定效果更加显著。
实施例4、评估不同稳定剂的组合效果
1、热稳定性实验
采用实验设计(DoE)的方法进行热稳定性研究。参照表10，制备不同浓度氯化钠和不同浓度精氨酸盐酸盐作为稳定剂组合的E35-1抗体制剂，目标渗透压为50到600(mOsm/kg H2O)，以评估不同含量和比例的氯化钠和精氨酸盐酸盐的组合对抗GM-CSFRα抗体的保护作用。
在40℃条件下放置，1、2、3和4周后收集样品，检测E35-1抗体制剂的浓度、浊度、聚集特性、降解特性、电荷异质性、与抗原结合力。其中，聚体特性采用HPLC-SEC方法检测、降解特性NR-CE-SDS和R-CE-SDS方法检测，电荷异质性采用iIEF方法检测。
表10.用DoE进行稳定剂优化设计
每组制剂的渗透压见表11，A2、A3、A5-A7的渗透压在300mOsm/kg-400mOsm/kg之间。
表11.渗透压结果
浓度检测的结果表明，相对于0周，4周后，各个制剂中的抗体浓度均未发生明显变化(结果未显示)。
浊度检测的结果表明，相对于0周时，4周后，对照组A1和仅添加氯化钠的A3组中，各个制剂的浊度没有明显变化，而添加精氨酸盐酸盐的各组中制剂的浊度值均不同程度地增加：A5和A7组(精氨酸盐酸盐的添加量在50mM-75mM)在4周后浊度分别增加0.187和0.204，A2、A4、A6(精氨酸盐酸盐的添加量为100mM-150mM)组制剂在4周后浊度增加幅度在0.25-0.27之间。上述检测结果表明A3组抗体更加稳定，其次是A5和A7组(精氨酸盐酸盐的添加量在50mM-75mM)，稳定性稍差的组为A2、A4、A6(精氨酸盐酸盐的添加量为100mM-150mM)。
HPLC-SEC结果见表12。与对照组A1相比，4周后A2-A7组的聚体相对于第0周时的增加程度较低，4周时的主峰比例均高于对照。相比之下，仅含氯化钠的制剂(A3)在第4周时显示出最低的聚体和最高的主峰，在所有组别中显示出了最优的稳定性效果。
表12.HPLC-SEC结果

注：ND表示未检出。
cIEF结果见表13。相对于0周，各组制剂中的酸性峰均出现增加的趋势，主峰均出现降低的趋势。与其他各组相比，在第4周时，仅含氯化钠的A3制剂组则显示出最高的主峰比例和最低的酸性峰比例，显示出了最优的稳定性。
表13.cIEF结果
cGE的结果见表14。相比之下，仅含氯化钠的A3组制剂在第4周时非还原性主峰和还原性主峰的比例最高，在各制剂组中显示出了最优的稳定性。
表14.cGE结果

综上结果表明，添加精氨酸盐酸盐和氯化钠对抗体均具有保护作用。但仅添加氯化钠组的抗体制剂的稳定性优于其他组，说明抗GM-CSFRα抗体制剂中仅添加氯化钠对抗体制剂的稳定作用优于仅添加精氨酸盐酸盐，也优于氯化钠和较高浓度精氨酸盐酸盐(50mM-150mM)的组合。
实施例5、抗GM-CSFRα抗体制剂的稳定性实验
参照表15制备E35-1和E35-2抗体制剂，在冻融条件和过滤条件下检测抗体制剂的稳定性。
表15.处方稳定性实验设计
1、冻融实验将T1组的抗体制剂(20mM PB，25mM精氨酸盐酸盐，100mM氯化钠，pH 7)在-70℃下冷冻，室温下解冻。在1、3和5次冻融后收集样品进行分析，检测抗体浓度、聚体特性和可见度以下的颗粒数。
(1)表16显示了E35-1抗体各个制剂的冻融结果，5次冻融后，相对于未冻融的制剂，蛋白质浓度仅略有降低(102.88mg/mL vs 100.25mg/mL)。而HPLC-SEC的检测结果显示聚体基本保持不变。可见度以下的颗粒数有所增加。
表16.冻融结果
(2)E35-2抗体制剂也表现出了良好的稳定性：5次冻融后，相对于未冻融的制剂，蛋白质浓度仅略有降低(结果未显示)。而HPLC-SEC的检测聚体基本保持不变。可见度以下的颗粒数略有增加。
由此可见，反复冻融对本发明的抗GM-CSFRα抗体制剂影响不大，抗体制剂整体上仍比较稳定。
2、过滤实验：
将T1组的抗体制剂(20mM PB，25mM精氨酸盐酸盐，100mM氯化钠，pH 7)通过Meissner SteriLUX亲水性PVDF 0.2过滤器过滤5次，每次过滤后检测抗体浓度。
(1)表17显示了E35-1抗体各个制剂的过滤实验结果，在5次过滤后，相对于未过滤时，抗体浓度没有明显变化。
表17.过滤结合实验
(2)E35-2抗体各个制剂，在5次过滤后，相对于未过滤时，抗体浓度也没有明显变化。
3、热稳定性实验
在40℃下放置4周，在1、2和4个周后收集样品进行分析，检测抗体制剂的浓度、浊度、聚集特性、降解特性、电荷异质性、与抗原结合力，以评估其稳定性。其中，聚体特性采用HPLC-SEC方法来检测，降解特性采用NR-CE-SDS和R-CE-SDS方法检测，电荷异质性采用iIEF方法检测。
(1)E35-1抗体制剂热稳定性结果
结果显示：相对于第0周时，4周后，所有组别的抗体浓度和浊度均未发生明显变化。相比之下，T1-T3组中的稳定效果(浊度仅增加0.02)优于仅含氯化钠作为稳定剂的T7-T8组(浊度增加了0.04)。
HPLC-SEC结果见表18，相比于第0周时，4周后各组别中抗体的聚集程度均有轻微增加的趋势。然而，T1-T3和T5-T6组中主峰仅降低了0.84％-1.29％，聚体增加了0.84％-1.29％；而T4、T7-T8组中主峰则降低1.39％-1.43％，聚体增加1.34％-1.41％，相比之下，T1-T3组的抗体制剂的稳定性优于T4、T7-T8组制剂。
表18.热稳定性实验的HPLC-SEC结果

注：ND表示未检出。
cGE的结果见表19，结果显示，相对于0周，主峰和片段在4周后发生了变化，主峰具有轻微降低的趋势，片段具有轻微增加的趋势。相比于0周，4周后，T1非还原性主峰下降3.6％，还原性主峰下降1.4％；T2-T3、T5-T6非还原性主峰下降4.2％-4.5％，T4、T7-T8非还原性主峰下降4.8％-5.0％。上述结果进一步证实T1-T3组的抗体制剂的稳定性优于T4、T7-T8组抗体制剂。
表19.热稳定性实验的cGE结果
表20为抗体与抗原的结合力结果，相对于0周，放置4周后，所有制剂与抗原的结合力基本保持不变。
表20.热稳定性实验的抗原结合结果
(2)E35-2抗体各个制剂也表现出良好的稳定性：
相对于第0周时，4周后，所有组别的抗体浓度和浊度均未发生明显变化；HPLC-SEC结果显示各组中抗体的聚集程度均有轻微增加的趋势，T1-T3和T5-T6组中主峰仅降低了0.78％-1.25％，聚体增加了0.79％-1.20％，相比之下，T4、T7-T8组中主峰降低了1.5％以上，聚体增加了1.4％以上；cGE结果显示，各组主峰具有轻微降低的趋势，片段具有轻微增加的趋势，T1非还原性主峰下降3.4％，还原性主峰下降1.5％，T2-T3、T5-T6非还原性主峰下降4.2％-4.6％，T4、T7-T8非还原性主峰下降了5.0％以上；放置4周后所有制剂与抗原的结合力基本保持不变。相比之下，T1-T3组的抗体制剂的稳定性优于T5-T6组制剂，更优于T4、T7-T8组制剂。
综上所述，本发明的抗GM-CSFRα抗体制剂在冻融5个周期内是稳定的；经过5次过滤没有明显的抗体损失；在高温条件下抗体稍有降解，但并不明显。相对而言，浓度低于50mM精氨酸盐酸盐和氯化钠的组合作为稳定剂比仅氯化钠作为稳定剂对抗体的保护作用更强，具有更优异的效果。
实施例6、长期稳定性实验
1、抗体制剂在长期存储条件下的稳定性
参照实施例5中的配方制备E35-1和E35-2抗体制剂T1组和T4组。在长期储存条件下(5±3℃放置0天、1月、3月、6月)检测抗体制剂的稳定性。
(1)表21显示了E35-1抗体制剂在长期储存条件下的稳定性结果，相比于0个月时，6个月后，T1组和T4组的E35-1抗体制剂各指标基本保持不变。
表21.长期储存条件下稳定性结果(5±3℃)

注：NR表示未检测。
(2)E35-2抗体制剂在长期储存条件下也表现出了良好的稳定性，相比于0个月时，6个月后，T1组和T4组的E35-2抗体制剂各指标基本保持不变。
2、抗体制剂在长期加速条件下的稳定性
在长期加速条件下(25℃±2℃加速0天、1月、3月、6月)检测E35-1和E35-2抗体制剂的稳定性。
(1)表22显示了E35-1抗体制剂在加速条件下的稳定性结果，相比于0个月时，6个月时，E35-1抗体制剂仅表现为主峰稍有减少，酸性峰稍有增加，其他指标均基本保持不变。
表22.长期加速条件下稳定性结果(25±2℃)

注：NR表示未检测。
(2)E35-2抗体制剂在长期加速条件下也表现出了良好的稳定性，相比于0个月时，6个月后，E35-2抗体制剂仅表现为主峰稍有减少，酸性峰稍有增加，其他指标均基本保持不变。
综上所述，本发明的抗GM-CSFRα抗体制剂在长期储存和长期加速条件下稳定性高，可保证该制剂在制备、运输及存储过程中保持良好的稳定性，保证临床用药安全性及质量可控性。

Claims (17)

  1. 一种抗GM-CSFRα抗体制剂，其特征在于，所述制剂包含抗GM-CSFRα抗体、稳定剂、表面活性剂、缓冲液，所述缓冲液为磷酸盐缓冲液，pH值为6.5-7.5，优选为6.7-7.3，更优选为7.0。
  2. 根据权利要求1所述的抗体制剂，其特征在于，所述缓冲液的浓度为10mM-30mM；优选地，所述缓冲液的浓度为20mM。
  3. 根据权利要求1或2所述的抗体制剂，其特征在于，所述抗体的浓度为50mg/ml-200mg/ml，优选为50mg/ml-180mg/ml，进一步优选为50mg/ml-150mg/ml，更优选为100mg/m-150mg/ml。
  4. 根据权利要求1-3任一项所述的抗体制剂，其特征在于，所述稳定剂包含氯化钠。
  5. 根据权利要求4所述的抗体制剂，其特征在于，所述氯化钠的浓度为70mM-200mM，优选为100mM-200mM，更优选为100mM-150mM，进一步优选为120mM-150mM。
  6. 根据权利要求4所述的抗体制剂，其特征在于，所述稳定剂进一步包括精氨酸盐酸盐；优选地，所述精氨酸盐酸盐的浓度为0-50mM，优选为0-40mM，更优选为15mM-40mM。
  7. 根据权利要求6所述的抗体制剂，其特征在于，所述氯化钠的浓度为50mM-150mM；优选为70mM-150mM；更优选为70mM-120mM。
  8. 根据权利要求1-5任一项所述的抗体制剂，其特征在于，所述稳定剂为：
    (1)50mM-150mM(优选70mM-120mM)的氯化钠和0-50mM(优选15mM-40mM)的精氨酸盐酸盐；
    (2)100mM-200mM(优选120mM-150mM)的氯化钠。
  9. 根据权利要求1-8任一项所述的抗体制剂，其特征在于，所述表面活性剂是聚山梨醇酯；优选地，所述聚山梨醇酯为吐温-20或吐温-80。
  10. 根据权利要求1-9任一项所述的抗体制剂，其特征在于，所述表面活性剂的浓度为0.05mg/ml-0.3mg/ml；优选地，所述表面活性剂的浓度为0.1mg/ml-0.2mg/ml。
  11. 根据权利要求1所述的抗体制剂，其特征在于，所述制剂为下述抗体制剂中的任一种：
    (1)所述抗体浓度为50mg/ml、75mg/ml、100mg/ml、125mg/ml、150mg/ml或180mg/ml；所述稳定剂为50mM-150mM(优选70mM-120mM)的氯化钠和0-50mM(优选15mM-40mM)的精氨酸盐酸盐，或者100mM-200mM(优选120mM-150mM)的氯化钠；所述缓冲液为10mM-30mM的磷酸盐缓冲液；所述缓冲液的pH值为6.7-7.3；所述表面活性剂为0.1mg/ml-0.2mg/ml的吐温-20和/或吐温-80；
    (2)所述抗体浓度为50mg/ml-180mg/ml；所述稳定剂为15mM的精氨酸盐酸盐和120mM的氯化钠，或25mM的精氨酸盐酸盐和100mM氯化钠，或40mM的精氨酸盐酸盐和70mM氯化钠，或50mM的精氨酸盐酸盐和100mM氯化钠，或100mM、120mM、150mM、180mM或200mM的氯化钠；所述缓冲液为10mM-30mM的磷酸盐缓冲液；所述缓冲液的pH值为6.7-7.3；所述表面活性剂为0.1mg/ml-0.2mg/ml的吐温-20和/或吐温-80；
    (3)所述抗体浓度为50mg/ml-180mg/ml；所述稳定剂为50mM-150mM(优选70mM-120mM)的 氯化钠和0-50mM(优选15mM-40mM)的精氨酸盐酸盐，或者100mM-200mM(优选120mM-150mM)的氯化钠；所述缓冲液为10mM、20mM、30mM的磷酸盐缓冲液；所述缓冲液的pH值为6.7-7.3；所述表面活性剂为0.1mg/ml-0.2mg/ml的吐温-20和/或吐温-80；
    (4)所述抗体浓度为50mg/ml-180mg/ml；所述稳定剂为50mM-150mM(优选70mM-120mM)的氯化钠和0-50mM(优选15mM-40mM)的精氨酸盐酸盐，或者100mM-200mM(优选120mM-150mM)的氯化钠；所述缓冲液为10mM-30mM的磷酸盐缓冲液；所述缓冲液的pH值为6.7、6.8、6.9、7.0、7.1、7.2、7.3；所述表面活性剂为0.1mg/ml-0.2mg/ml的吐温-20和/或吐温-80；
    (5)所述抗体浓度为50mg/ml-180mg/ml；所述稳定剂为50mM-150mM(优选70mM-120mM)的氯化钠和0-50mM(优选15mM-40mM)的精氨酸盐酸盐，或者100mM-200mM(优选120mM-150mM)的氯化钠；所述缓冲液为10mM-30mM的磷酸盐缓冲液；所述缓冲液的pH值为6.7-7.3；所述表面活性剂为0.1mg/ml、0.15mg/ml、0.2mg/ml的吐温-20或吐温-80；
    (6)所述抗体浓度为50mg/ml-180mg/ml；所述稳定剂为100mM-200mM(优选120mM-150mM)的氯化钠；所述缓冲液为10mM-30mM的磷酸盐缓冲液；所述缓冲液的pH值为6.7-7.3；所述表面活性剂为0.1mg/ml-0.2mg/ml的吐温-20或吐温-80；
    (7)所述抗体浓度为50mg/ml-180mg/ml；所述稳定剂为50mM-150mM(优选70mM-120mM)的氯化钠和0-50mM(优选15mM-45mM)的精氨酸盐酸盐；所述缓冲液为10mM-30mM的磷酸盐缓冲液；所述缓冲液的pH值为6.7-7.3；所述表面活性剂为0.1mg/ml-0.2mg/ml的吐温-20或吐温-80。
  12. 根据权利要求1所述的抗体制剂，其特征在于，所述抗体制剂为下述制剂中的任一种：
    (1)所述抗体浓度为100mg/ml；所述稳定剂为25mM精氨酸盐酸盐和100mM氯化钠；所述缓冲液为20mM的磷酸盐缓冲液；所述缓冲液的pH值为6.7或7.0或7.3；所述表面活性剂为0.1mg/ml或0.2mg/ml的吐温-20或吐温-80；
    (2)所述抗体浓度为100mg/ml；所述稳定剂为100mM或120mM或200mM氯化钠；所述缓冲液为20mM的磷酸盐缓冲液；所述缓冲液的pH值为7.0；所述表面活性剂为0.1mg/ml或0.2mg/ml的吐温-20或吐温-80；
    (3)所述抗体浓度为100mg/ml；所述稳定剂为40mM精氨酸盐酸盐和70mM氯化钠；所述缓冲液为20mM的磷酸盐缓冲液；所述缓冲液的pH值为7.0；所述表面活性剂为0.1mg/ml或0.2mg/ml的吐温-20或吐温-80；
    (4)所述抗体浓度为150mg/ml；所述稳定剂为15mM精氨酸盐酸盐和120mM氯化钠；所述缓冲液为20mM的磷酸盐缓冲液；所述缓冲液的pH值为7.0；所述表面活性剂为0.1mg/ml或0.2mg/ml的吐温-20或吐温-80。
  13. 根据权利要求1-12任一项所述的抗体制剂，其特征在于，所述抗体制剂还可包含抗氧化剂和/或防腐剂。
  14. 根据权利要求1-13任一项所述的抗体制剂，其特征在于，所述抗体制剂为液体制剂或注射用粉针剂。
  15. 根据权利要求1-14任一项所述的抗体制剂，其特征在于，所述抗体包含重链，其包含氨基酸序 列SEQ ID NO:1或2，以及轻链，其包含氨基酸序列SEQ ID NO:3。
  16. 权利要求1-15任一项所述的抗体制剂在制备治疗疾病的药物中的用途。
  17. 根据权利要求16所述的用途，其特征在于，所述疾病包括自身免疫性疾病、炎性病症或癌症；优选地，所述疾病为类风湿性关节炎、哮喘或骨髓性白血病。
PCT/CN2023/072069 2022-01-20 2023-01-13 一种特异性地识别粒细胞-巨噬细胞集落刺激因子受体的抗体制剂及其应用 WO2023138499A1 (zh)

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US20100215650A1 (en) * 2006-11-21 2010-08-26 Kalobios Pharmaceuticals, Inc. Methods of treating dementia using a gm-csf antagonist
CN103641915A (zh) * 2006-03-27 2014-03-19 医学免疫有限公司 Gm-csf受体结合元件
WO2019070680A2 (en) * 2017-10-02 2019-04-11 Humanigen, Inc. METHODS OF TREATING IMMUNOTHERAPY-RELATED TOXICITY USING GM-CSF ANTAGONIST
CN113164520A (zh) * 2018-09-10 2021-07-23 赫曼尼根公司 使用gm-csf拮抗剂治疗免疫疗法相关毒性的方法
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CN103641915A (zh) * 2006-03-27 2014-03-19 医学免疫有限公司 Gm-csf受体结合元件
CN101605547A (zh) * 2006-11-21 2009-12-16 卡罗拜奥斯制药公司 使用gm-csf拮抗剂治疗慢性炎症疾病的方法
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