WO2023138499A1 - Antibody preparation for specifically recognizing granulocyte-macrophage colony-stimulating factor receptor and use thereof - Google Patents

Abstract

An anti-GM-CSFRα antibody preparation. The preparation contains an anti-GM-CSFRα antibody, a stabilizer, a surfactant and a buffer, wherein the buffer is a phosphate buffer at a pH value of 6.5-7.5. The anti-GM-CSFRα antibody preparation has good stability during preparation, transportation and storage, and ensures quality controllability and clinical drug safety.

Description

An antibody preparation specifically recognizing granulocyte-macrophage colony-stimulating factor receptor and its application technical field

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.

Background technique

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is also known as colony-stimulating factor 2 (CSF2). 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 α and β 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. When both α and β subunits are present, a high-affinity ligand-receptor complex (Kd≈100pM) will be produced. Therefore, neutralization of GM-CSF binding to GM-CSFRα becomes a therapeutic approach for the treatment of GM-CSFRα-mediated diseases and conditions.

International patent application WO2020/108423A1 discloses an isolated anti-GM-CSFRα 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-CSFRα and/or abnormal function of GM-CSF/GM-CSFRα. However, 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-CSFRα antibody preparation with high stability through rational design of preparation prescription and detection. The anti-GM-CSFRα 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.

Contents of the invention

In one aspect, the present invention provides an anti-GM-CSFRα antibody preparation, the antibody preparation comprising an anti-GM-CSFRα 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, the stabilizer comprises sodium chloride. In some embodiments, the concentration of the sodium chloride is 70mM- 200mM, preferably 100mM-200mM, more preferably 100mM-150mM, further preferably 120mM-150mM.

In some embodiments, the stabilizer further includes arginine hydrochloride. In some preferred embodiments, the concentration of the arginine hydrochloride is 0-50mM, preferably 0-40mM, more preferably 15mM-40mM. In some preferred embodiments, the concentration of the sodium chloride is 50mM-150mM, preferably 70mM-150mM; more preferably 70mM-120mM.

In some embodiments, the stabilizer is:

(1) 50mM-150mM (preferably 70mM-120mM) sodium chloride and 0-50mM (preferably 15mM-40mM) arginine hydrochloride;

(2) 100mM-200mM (preferably 120mM-150mM) sodium chloride. In some specific embodiments, 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.

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

In some embodiments, 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.

In some embodiments, the antibody preparation is any of the following preparations:

(1) 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;

(2) 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;

(3) 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;

(4) 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;

(5) 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;

(6) 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;

(7) 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.

In some embodiments, the antibody preparation is any of the following preparations:

(1) 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;

(2) 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;

(3) 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;

(4) 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.

In some embodiments, 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.

In some embodiments, the antibody preparation is a liquid preparation or a powder for injection.

In some embodiments, 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.

In another aspect, the present invention discloses the use of the antibody preparation in the preparation of medicines for treating diseases. In some embodiments, the disease comprises an autoimmune disease, an inflammatory disorder, or cancer; preferably, the disease is rheumatoid arthritis, asthma, or myelogenous leukemia.

The anti-GM-CSFRα antibody preparation provided by the present invention has the following excellent effects:

1. The present invention screens out the best buffer system for anti-GM-CSFRα antibody. Compared with other buffer systems, the phosphate buffer system with pH6.5-7.5 The flushing solution can make the anti-GM-CSFRα antibody preparation have low turbidity, low viscosity, optimal thermal stability (Tm) and good uniformity.

2. 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.

3. The anti-GM-CSFRα 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.

Detailed ways

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

The reagents used in the following examples, unless otherwise specified, 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-CSFRα antibody is an antibody that binds to granulocyte-macrophage colony-stimulating factor receptor α (GM-CSFRα). The anti-GM-CSFRα antibody 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.

The amino acid sequences of the E35-1 antibody and the E35-2 antibody used in the examples of the present invention are shown in Table 1:

Table 1. Amino acid sequences of E35-1 antibody and E35-2 antibody

Embodiment 1, screening the buffer system of anti-GM-CSFRα antibody preparation

1. Screen the buffer system by using differential scanning fluorescence method (DSF) and turbidity method

(1) Screen the optimal pH value and buffer system

The GM-CSFRα 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.

The 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-CSFRα antibody had significantly higher thermal stability at this pH and buffer system.

Table 2. Tm detection results

The results of the turbidity test are shown in Table 3. It can be seen from Table 3 that after heating at 50°C for 2 hours, the increase in the turbidity value of the antibody sample was very low (only about 0.002-0.005) in the acetate buffer with a pH of 5.0 and in the phosphate buffer with a pH of 7.0, indicating that the antibody has good stability in the above buffer system.

Table 3. Test results of turbidity

Combined with the Tm value test results and turbidity test results, it can be seen that the pH of the buffer solution is around 7.0, which is the best buffer pH range for the anti-GM-CSFRα antibody, and its effect is obviously better than other pH ranges.

(2) Confirm the buffer system within the pH range

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.

As a result, in Table 4, in the range of pH 6.8-7.2, the antibody sample in the phosphate buffer has a higher TM value compared to other commonly used cushioning systems (histidine and Tris hydrochloric acid). In addition, the TM value of the antibody sample in the phosphate buffer in phosphate buffer is basically unchanged under different pH values. Anti-GM-CSFRα antibody, the pH value of about 6.8-7.2 phosphate buffer has a better stable effect than other commonly used buffer.

Table 4. Tm test results of DSF screening buffer system

2. Evaluate the effect of the buffer system under the condition of high concentration antibody

The GM-CSFRα 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 results are shown in Table 5. When the antibody concentration is in the range of 50mg/ml-150mg/ml, 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.

In the two buffer systems, 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.

Table 5. Buffer system evaluation results

3. Dynamic Light Scattering (DLS)

The hydrodynamic radius of antibody molecules was examined using dynamic light scattering to assess the uniformity of antibody particle size.

The results are shown in Table 6, which indicated that the antibody had a small polydispersity under this condition, and the antibody had good uniformity.

Table 6. DLS evaluation results

In summary, in the 20mM phosphate buffer with a pH value of 7.0, compared with other buffer systems, the anti-GM-CSFRα 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

Different surfactants (PS20 and PS80) and different addition amounts (0.01% and 0.02%) were screened under freeze-thaw and shaking conditions.

Set up 5 groups of experiments, each group containing 100mg/mL antibody E35-1, 20mM PB pH7, F1 group without surfactant, F2 group added 0.01% PS20, F3 group added 0.02% PS20, F4 group added 0.01% PS80, F5 group added 0.02% PS80.

1. Oscillation experiment

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.

The results showed that the F1 sample became turbid after shaking for 3 days, while the F2-F5 remained clear. After shaking for 5 days, compared with day 0, the concentration of samples in each group remained basically unchanged; the turbidity of F1 samples increased from 0.467 to 2.06, and the turbidity of F2-F5 samples remained basically unchanged; the aggregates of F1-F5 increased slightly after shaking for 3 days, and remained basically unchanged on day 5. There were no significant differences among groups F2-F5.

Microfluidic imager (MFI) was used to quantify the particles below visible light. The F1 sample had a relatively high number of particles (5145 particles/ml for >10 μm particles and 303 particles/ml for >25 μm 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 μm were below 642 particles/ml, and particles >25 μm less than 75/ml). There were no significant differences among groups F2-F5.

2. Freeze-thaw experiment

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).

In summary, PS20 and PS80 added at 0.01%-0.02% have good protective effects against GM-CSFRα antibody, and there is no significant difference between them.

Example 3, Screening of Anti-GM-CSFRα Antibody Preparation Stabilizers

The GM-CSFRα 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.

Table 7. Formulation formulation design

1. Screening results of E35-1 antibody preparation stabilizer

After storage at 45°C for 4 weeks, compared with that at 0 weeks, the antibody concentration of each group had no significant change (results not shown), and the turbidity tended to increase slightly. However, in contrast, 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. Generally speaking, as time progresses, the aggregates in each group of preparations have an increasing trend, and the main peak has a decreasing trend. However, among all the groups, 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. At week 3, 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 above results indicated that, compared with other groups, the antibodies in the F8 and F9 groups were less prone to aggregation and had higher stability.

Table 8. HPLC-SEC results

Note: ND means not detected.

The detection results of cIEF are shown in Table 9. After being stored at 45°C for 4 weeks, compared with the 0th day, the acidic peak increased and the main peak decreased in each group, while the alkaline peak began to increase at the first week and gradually decreased after 2-4 weeks. In contrast, the increase rate of the acidic peak and the decrease rate of the main peak in the F9 (sodium chloride) group were the slowest, indicating that the antibody in the F9 group had the least change in charge heterogeneity and the highest stability.

Table 9. cIEF results

2. Screening of stabilizers for E35-2 antibody preparations

The individual formulations of the E35-2 antibody also showed similar results:

After storage at 45°C for 4 weeks, compared with the 0-week period, the antibody concentration of each group had no significant change, and the turbidity showed a slight upward trend. In contrast, 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. At 4 weeks, 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.

At 45°C, the aggregates in each group of preparations tended to increase, and the main peaks tended to decrease. In all groups, F8 (arginine hydrochloride) and F9 (sodium chloride) groups, especially the increase rate of F9 group polymer is the slowest (only increased by 1.93%), and the main peak decline rate is also the slowest (only decreased by 2.46%). At week 3, the main peaks of both F8 and F9 were greater than 96%, in contrast, the proportions of the main peaks in the other groups had dropped below 95%.

After being stored at 45°C for 4 weeks, compared with day 0, the acid peak in each group tended to increase, while the main peak decreased. In contrast, the F9 (sodium chloride) group had the slowest rate of acid peak increase (only 32.7% increase) and main peak decrease (only 33.6% decrease).

To sum up: for the anti-GM-CSFRα antibody of the present application, 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. Especially the stabilizer is sodium chloride, and its stabilizing effect is more significant.

Embodiment 4, evaluate the combined effect of different stabilizers

1. Thermal stability experiment

The thermal stability study was carried out by design of experiments (DoE). Referring to Table 10, E35-1 antibody preparations with different concentrations of sodium chloride and different concentrations of arginine hydrochloride were prepared as stabilizer combinations, with a target osmotic pressure of 50 to 600 (mOsm/kg H2O), to evaluate the protective effect of combinations of sodium chloride and arginine hydrochloride in different contents and ratios against GM-CSFRα antibodies.

Placed at 40°C, 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. Among them, the polymer characteristics were detected by HPLC-SEC method, the degradation characteristics were detected by NR-CE-SDS and R-CE-SDS methods, and the charge heterogeneity was detected by iIEF method.

Table 10. Optimal design of stabilizers using DoE

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.

Table 11. Osmolality Results

The results of the concentration test showed that, compared with the 0 week, the antibody concentration in each preparation did not change significantly after 4 weeks (results not shown).

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. The above test results show that the antibodies in group A3 are more stable, followed by groups A5 and A7 (the addition amount of arginine hydrochloride is 50mM-75mM), and the groups with less stability are A2, A4, A6 (the addition amount of arginine hydrochloride is 100mM-150mM).

See Table 12 for HPLC-SEC results. Compared with the control group A1, after 4 weeks, the aggregates of groups A2-A7 had a lower increase relative to that at week 0, and the proportion of the main peak at 4 weeks was higher than that of the control. In contrast, the sodium chloride-only formulation (A3) showed the lowest aggregates and the highest main peak at week 4, showing the best stabilization effect among all groups.

Table 12. HPLC-SEC results

Note: ND means not detected.

See Table 13 for cIEF results. Compared with week 0, the acid peaks in each group of preparations showed an increasing trend, and the main peaks showed a decreasing trend. Compared with other groups, at the 4th week, the A3 preparation group containing only sodium chloride showed the highest main peak ratio and the lowest acidic peak ratio, showing the best stability.

Table 13. cIEF results

The results of cGE are shown in Table 14. In contrast, the preparations of group A3 containing only sodium chloride had the highest ratio of non-reducing main peaks and reducing main peaks at week 4, showing the best stability among the preparation groups.

Table 14. cGE results

In summary, the above results indicated that the addition of arginine hydrochloride and sodium chloride had protective effects on antibodies. However, the stability of the antibody preparation in the sodium chloride group was better than that of the other groups, indicating that the addition of sodium chloride alone in the anti-GM-CSFRα antibody preparation has a better stabilizing effect on the antibody preparation than that of arginine hydrochloride alone, and also better than the combination of sodium chloride and arginine hydrochloride at a higher concentration (50mM-150mM).

Embodiment 5, Stability experiment of anti-GM-CSFRα antibody preparation

Prepare E35-1 and E35-2 antibody preparations with reference to Table 15, and test the stability of the antibody preparations under freeze-thaw conditions and filtration conditions.

Table 15. Formulation Stability Experimental Design

1. Freeze-thaw experiment The antibody preparation (20mM PB, 25mM arginine hydrochloride, 100mM sodium chloride, pH 7) of the T1 group was frozen at -70°C and thawed at room temperature. Samples were collected for analysis after 1, 3, and 5 freeze-thaw cycles for antibody concentration, aggregate properties, and the number of particles below visibility.

(1) 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.

Table 16. Freeze-Thaw Results

(2) 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.

It can be seen that repeated freezing and thawing has little effect on the anti-GM-CSFRα antibody preparation of the present invention, and the antibody preparation is still relatively stable as a whole.

2. Filtration experiment:

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.

(1) 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.

Table 17. Filter Binding Experiments

(2) For each preparation of the E35-2 antibody, after 5 times of filtration, the antibody concentration did not change significantly compared to that without filtration.

3. Thermal stability experiment

After 4 weeks at 40°C, samples were collected for analysis after 1, 2 and 4 weeks, and the concentration, turbidity, aggregation characteristics, degradation characteristics, charge heterogeneity, and antigen binding ability of the antibody preparation were detected to evaluate its stability. Among them, the polymer characteristics were detected by HPLC-SEC method, the degradation characteristics were detected by NR-CE-SDS and R-CE-SDS methods, and the charge heterogeneity was detected by iIEF method.

(1) Thermal stability results of E35-1 antibody preparations

The results showed that compared with week 0, the antibody concentration and turbidity of all groups did not change significantly after 4 weeks. In contrast, the stabilizing effect in groups T1-T3 (increase in turbidity by only 0.02) was better than in groups T7-T8 (increase in turbidity by 0.04) containing only sodium chloride as a stabilizer.

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.

Table 18. HPLC-SEC results of thermal stability experiments

Note: ND means not detected.

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. Compared with week 0, after 4 weeks, the T1 non-reducing main peak decreased by 3.6%, and the reducing main peak decreased by 1.4%; the T2-T3, T5-T6 non-reducing main peak decreased by 4.2%-4.5%, and 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 19. cGE results of thermal stability experiments

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.

Table 20. Antigen binding results from thermostability experiments

(2) Each preparation of E35-2 antibody also showed good stability:

Compared with week 0, the antibody concentration and turbidity of all groups did not change significantly after 4 weeks; HPLC-SEC results showed that the aggregation degree of antibodies in each group had a slight increase trend, the main peak in T1-T3 and T5-T6 groups only decreased by 0.78%-1.25%, and the aggregates increased by 0.79%-1.20%. In contrast, the main peaks in the T4 and T7-T8 groups decreased by more than 1.5%, and the aggregates increased by 1. 4% or more; cGE results showed that the main peaks of each group had a slight decrease trend, and the fragments had a slight increase trend. 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%, and the T4, T7-T8 non-reducing main peak decreased by more than 5.0%. In contrast, 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.

In summary, the anti-GM-CSFRα 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. Relatively speaking, 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.

Embodiment 6, long-term stability experiment

1. Stability of antibody preparations under long-term storage conditions

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).

(1) 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.

Table 21. Stability results under long-term storage conditions (5±3°C)

Note: NR means not detected.

(2) 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.

2. Stability of antibody preparations under long-term accelerated conditions

The stability of E35-1 and E35-2 antibody preparations was tested under long-term accelerated conditions (25°C ± 2°C for 0 days, 1 month, 3 months, and 6 months).

(1) 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.

Table 22. Stability results under long-term accelerated conditions (25±2°C)

Note: NR means not detected.

(2) 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.

In summary, the anti-GM-CSFRα 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.

Claims (17)

An anti-GM-CSFRα antibody preparation, characterized in that the preparation comprises an anti-GM-CSFRα antibody, a stabilizer, a surfactant, and a buffer, and the buffer is a phosphate buffer with a pH value of 6.5-7.5, preferably 6.7-7.3, more preferably 7.0. The antibody preparation according to claim 1, wherein the concentration of the buffer is 10mM-30mM; preferably, the concentration of the buffer is 20mM. The antibody preparation according to claim 1 or 2, wherein the concentration of the antibody is 50mg/ml-200mg/ml, preferably 50mg/ml-180mg/ml, more preferably 50mg/ml-150mg/ml, more preferably 100mg/m-150mg/ml. The antibody preparation according to any one of claims 1-3, wherein the stabilizer comprises sodium chloride. The antibody preparation according to claim 4, wherein the concentration of the sodium chloride is 70mM-200mM, preferably 100mM-200mM, more preferably 100mM-150mM, further preferably 120mM-150mM. The antibody preparation according to claim 4, wherein the stabilizer further comprises arginine hydrochloride; preferably, the concentration of arginine hydrochloride is 0-50mM, preferably 0-40mM, more preferably 15mM-40mM. The antibody preparation according to claim 6, wherein the concentration of the sodium chloride is 50mM-150mM; preferably 70mM-150mM; more preferably 70mM-120mM. The antibody preparation according to any one of claims 1-5, wherein the stabilizer is: (1) 50mM-150mM (preferably 70mM-120mM) sodium chloride and 0-50mM (preferably 15mM-40mM) arginine hydrochloride; (2) 100mM-200mM (preferably 120mM-150mM) sodium chloride. The antibody preparation according to any one of claims 1-8, wherein the surfactant is polysorbate; preferably, the polysorbate is Tween-20 or Tween-80. The antibody preparation according to any one of claims 1-9, wherein 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 according to claim 1, wherein the preparation is any one of the following antibody preparations: (1) 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; (2) 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 M or 200mM 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; (3) 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 phosphate buffer; the pH of the buffer is 6.7-7.3; the surfactant is Tween-20 and/or 0.1mg/ml-0.2mg/ml or Tween-80; (4) 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; (5) 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 6.7-7.3; the surfactant is 0.1mg/ml, 0.15mg/ml, 0.2mg/ml Tween-20 or Tween-80; (6) 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; (7) 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. Tween-20 or Tween-80 at 2 mg/ml. The antibody preparation according to claim 1, wherein the antibody preparation is any one of the following preparations: (1) 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; (2) 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; (3) the antibody concentration is 100mg/ml; the stabilizer is 40mM 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; (4) 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 according to any one of claims 1-12, characterized in that, the antibody preparation may further comprise antioxidants and/or preservatives. The antibody preparation according to any one of claims 1-13, wherein the antibody preparation is a liquid preparation or a powder for injection. The antibody preparation according to any one of claims 1-14, wherein the antibody comprises a heavy chain comprising the amino acid sequence Column SEQ ID NO: 1 or 2, and a light chain comprising the amino acid sequence SEQ ID NO:3. Use of the antibody preparation according to any one of claims 1-15 in the preparation of medicines for treating diseases. The use according to claim 16, characterized in that the disease includes autoimmune disease, inflammatory disease or cancer; preferably, the disease is rheumatoid arthritis, asthma or myelogenous leukemia.