TW202333792A - Formulation of antibody specifically recognizing granulocyte macrophage-colony stimulating factor receptor and application thereof - Google Patents

Abstract

The present invention provides a formulation of anti-GM-CSFR[alpha] antibody comprising anti-GM-CSFR[alpha] antibody, stabilizer, surfactant, buffer which is phosphate buffer with a pH value of 6.5-7.5. The formulation of anti-GM-CSFR[alpha] antibody of the present invention exhibits excellent stability and maintains its quality and clinical medication safety in the process of preparation, transportation, and storage.

Description

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

The invention belongs to the field of biomedical technology, and specifically relates to an antibody preparation that specifically recognizes granulocyte-macrophage colony-stimulating factor receptor α (GM-CSFRα) and its application.

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. The GM-CSF receptor system is a member of the hematopoietic receptor superfamily and is a heterodimer composed of α and β subunits. GM-CSF is able to bind to the α subunit alone with relatively low affinity (Kd 1-5nM), but does not bind to the β subunit alone. When α and β subunits exist at the same time, a high-affinity ligand-receptor complex (Kd≈100pM) will be produced. Therefore, neutralization of the binding of GM-CSF to GM-CSFRα represents 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 autoimmunity characterized by high expression of GM-CSF and/or GM-CSFRα and/or GM-CSF/GM-CSFRα dysfunction. Disease and/or inflammatory conditions or cancer (e.g., rheumatoid arthritis, asthma, myeloid leukemia). However, single-selected antibody drugs are macromolecular protein drugs. Compared with traditional small molecule drugs, they are prone to aggregation and degradation during storage, which can lead to adverse consequences such as increased differences between drug batches and changes in immunogenicity.

By rationally designing the formulation and testing, the present invention finally obtains a highly stable anti-GM-CSFRα antibody preparation. The anti-GM-CSFRα antibody preparation of the present invention has strong stability, which can ensure that the preparation maintains good stability during preparation, transportation and storage, and ensures quality controllability and clinical drug safety.

In one aspect, the present invention provides an anti-GM-CSFRα antibody preparation. The antibody preparation includes an anti-GM-CSFRα antibody, a stabilizer, a surfactant, and a buffer. The buffer is a phosphate buffer with a pH value of 6.5- 7.5, ideally 6.7-7.3. In some embodiments, the pH is 6.7, 6.8, 6.9, 7.0, 7.1, 7.2 or 7.3; more ideally, it is 7.0.

In some embodiments, the concentration of the aforementioned buffer is 10mM-30mM; in some specific embodiments, the concentration of the aforementioned buffer is 10mM, 20mM or 30mM; more ideally, it is 20mM.

In some embodiments, the concentration of the aforementioned antibody is 50 mg/ml-200 mg/ml. Ideally, the concentration of the aforementioned antibody is 50 mg/ml-180 mg/ml, further preferably 50 mg/ml-150 mg/ml, and more preferably 100 mg. /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 aforementioned stabilizer includes sodium chloride. In some embodiments, the concentration of the aforementioned sodium chloride is 70mM-200mM, ideally 100mM-200mM, more preferably 100mM-150mM, further preferably 120mM-150mM.

In some embodiments, the aforementioned stabilizer further includes arginine hydrochloride. In some ideal embodiments, the concentration of the aforementioned spermine hydrochloride is 0-50mM, ideally 0-40mM, and more ideally 15mM-40mM. In some ideal embodiments, the concentration of the aforementioned sodium chloride is 50mM-150mM, ideally 70mM-150mM; more ideally, it is 70mM-120mM.

In some embodiments, the aforementioned stabilizer is: (1) 50mM-150mM (ideally 70mM-120mM) sodium chloride and 0-50mM (ideally 15mM-40mM) spermine hydrochloride; (2) 100mM-200mM (ideally 120mM-150mM) sodium chloride. In some specific embodiments, the aforementioned stabilizer is 100mM, 120mM, 150mM, 180mM or 200mM sodium chloride or spermine hydrochloride; or 15mM spermine hydrochloride and 120mM sodium chloride; or 25mM Spermine hydrochloride and 100mM sodium chloride; or 40mM spermine hydrochloride and 70mM sodium chloride; or 50mM spermine hydrochloride and 100mM sodium chloride; or 75mM spermine Amine hydrochloride and 75mM sodium chloride; or 100mM spermine hydrochloride and 50mM sodium chloride, or 150mM spermine hydrochloride and 150mM sodium chloride.

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

In some embodiments, the concentration of the aforementioned surfactant is 0.05 mg/ml-0.3 mg/ml; ideally, the concentration of the aforementioned surfactant is 0.1 mg/ml-0.2 mg/ml.

In some embodiments, the aforementioned antibody preparation is any one of the following preparations: (1) The aforementioned antibody concentration is 50mg/ml, 75mg/ml, 100mg/ml, 125mg/ml, 150mg/ml or 180mg/ml; the aforementioned stabilizer is 50mM-150mM (ideally 70mM-120mM) sodium chloride and 0-50mM (ideally 15mM-40mM) spermine hydrochloride, or 100mM-200mM (ideally 120mM-150mM) sodium chloride; the aforementioned buffer is 10mM-30mM phosphate buffer; the aforementioned buffer The pH value is 6.7-7.3; the aforementioned surfactant is 0.1mg/ml-0.2mg/ml Tween-20 and/or Tween-80; (2) The concentration of the aforementioned antibody is 50mg/ml-180mg/ml; the aforementioned stabilizer is 15mM spermine hydrochloride and 120mM sodium chloride, or 25mM spermine hydrochloride and 100mM sodium chloride, or 40mM Spermine hydrochloride and 70mM sodium chloride, or 50mM spermine hydrochloride and 100mM sodium chloride, or 100mM, 120mM, 150mM, 180mM or 200mM sodium chloride; the aforementioned buffer is 10mM-30mM Phosphate buffer; the pH value of the aforementioned buffer is 6.7-7.3; the aforementioned surfactant is 0.1mg/ml-0.2mg/ml Tween-20 and/or Tween-80; (3) The concentration of the aforementioned antibody is 50mg/ml-180mg/ml; the aforementioned stabilizer is 50mM-150mM (ideally 70mM-120mM) sodium chloride and 0-50mM (ideally 15mM-40mM) arginine hydrochloride salt, or 100mM-200mM (ideally 120mM-150mM) sodium chloride; the aforementioned buffer is 10mM, 20mM, 30mM phosphate buffer; the pH value of the aforementioned buffer is 6.7-7.3; the aforementioned surfactant is 0.1 mg/ml-0.2mg/ml of Tween-20 and/or Tween-80; (4) The concentration of the aforementioned antibody is 50mg/ml-180mg/ml; the aforementioned stabilizer is 50mM-150mM (ideally 70mM-120mM) sodium chloride and 0-50mM (ideally 15mM-40mM) arginine hydrochloride. salt, or 100mM-200mM (ideally 120mM-150mM) sodium chloride; the aforementioned buffer is 10mM-30mM phosphate buffer; the pH value of the aforementioned buffer is 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3; The aforementioned surfactant is 0.1mg/ml-0.2mg/ml Tween-20 and/or Tween-80; (5) The concentration of the aforementioned antibody is 50mg/ml-180mg/ml; the aforementioned stabilizer is 50mM-150mM (ideally 70mM-120mM) sodium chloride and 0-50mM (ideally 15mM-40mM) arginine hydrochloride salt, or 100mM-200mM (ideally 120mM-150mM) sodium chloride; the aforementioned buffer is 10mM-30mM phosphate buffer; the pH value of the aforementioned buffer is 6.7-7.3; the aforementioned surfactant is 0.1mg/ ml, 0.15mg/ml, 0.2mg/ml Tween-20 or Tween-80; (6) The concentration of the aforementioned antibody is 50mg/ml-180mg/ml; the aforementioned stabilizer is 100mM-200mM (ideally 120mM-150mM) sodium chloride; the aforementioned buffer is 10mM-30mM phosphate buffer; the aforementioned buffer is The pH value is 6.7-7.3; the aforementioned surfactant is 0.1mg/ml-0.2mg/ml Tween-20 or Tween-80; (7) The concentration of the aforementioned antibody is 50mg/ml-180mg/ml; the aforementioned stabilizer is 50mM-150mM (ideally 70mM-120mM) sodium chloride and 0-50mM (ideally 15mM-45mM) arginine hydrochloride. Salt; the aforementioned buffer is 10mM-30mM phosphate buffer; the pH value of the aforementioned buffer is 6.7-7.3; the aforementioned surfactant is 0.1mg/ml-0.2mg/ml Tween-20 or Tween-80 .

In some embodiments, the aforementioned antibody preparation is any one of the following preparations: (1) The concentration of the aforementioned antibody is 100mg/ml; the aforementioned stabilizer is 25mM spermine hydrochloride and 100mM sodium chloride; the aforementioned buffer is 20mM phosphate buffer; the pH value of the aforementioned buffer is 6.7 or 7.0 or 7.3; The aforementioned surfactant is 0.1mg/ml or 0.2mg/ml Tween-20 or Tween-80; (2) The concentration of the aforementioned antibody is 100mg/ml; the aforementioned stabilizer is 100mM or 120mM or 200mM sodium chloride; the aforementioned buffer is 20mM phosphate buffer; the pH value of the aforementioned buffer is 7.0; the aforementioned surfactant is 0.1 mg/ml or 0.2mg/ml of Tween-20 or Tween-80; (3) The concentration of the aforementioned antibody is 100mg/ml; the aforementioned stabilizer is 50mM spermine hydrochloride and 70mM sodium chloride; the aforementioned buffer is 20mM phosphate buffer; the pH value of the aforementioned buffer is 7.0; the aforementioned surface The active agent is 0.1mg/ml or 0.2mg/ml Tween-20 or Tween-80; (4) The concentration of the aforementioned antibody is 150mg/ml; the aforementioned stabilizer is 15mM spermine hydrochloride and 120mM sodium chloride; the aforementioned buffer is 20mM phosphate buffer; the pH value of the aforementioned buffer is 7.0; the aforementioned surface The active agent is Tween-20 or Tween-80 at 0.1 mg/ml or 0.2 mg/ml.

In some embodiments, the aforementioned antibody preparation may also include antioxidants and/or preservatives. The aforementioned antioxidants include, but are not limited to, ascorbic acid and/or methionine; the aforementioned preservatives include, but are not limited to, stearyldimethylbenzyl ammonium chloride, hexamethylammonium chloride, benzalkonium chloride, and benzethionate. Ammonium chloride, phenol, butanol or benzyl alcohol, alkyl parahydroxybenzoate, etc.

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

In some embodiments, the aforementioned antibody comprises a heavy chain comprising the amino acid sequence SEQ ID NO: 1 or 2, and a light chain comprising the amino acid sequence SEQ ID NO: 3.

In another aspect, the present invention discloses the use of the aforementioned antibody preparation in the preparation of medicaments for treating diseases. In some embodiments, the aforementioned disease includes an autoimmune disease, an inflammatory disorder, or cancer; ideally, the aforementioned disease is rheumatoid arthritis, asthma, or myeloid leukemia.

The anti-GM-CSFRα antibody preparation provided by the invention has the following excellent effects: 1. The present invention selects the best buffer system for anti-GM-CSFRα antibodies. Compared with other buffer systems, the phosphate buffer with pH 6.5-7.5 can make anti-GM-CSFRα antibody preparations have low turbidity and low viscosity. It has the best 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 a lower concentration of arginine hydrochloride (0-50mM) as a stabilizer or 100mM-200mM. As a stabilizer, sodium chloride can significantly reduce the formation of aggregates in antibody preparations, slow down changes in charge heterogeneity, prevent the formation and aggregation of acidic substances, and maintain good stability of antibody preparations. 3. The anti-GM-CSFRα antibody preparation of the present invention has strong stability, which can maintain good stability during preparation, transportation and storage, ensuring quality controllability and clinical drug safety.

In order to make the technical problems to be solved, the technical means adopted and the advantages of the present invention clearer, the present invention will be described in detail below with reference to 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, are all prepared using conventional methods or obtained from commercial sources; the experimental methods used, unless otherwise specified, are conventional methods; the materials, instruments, etc. used, Unless otherwise stated, all materials were obtained from commercial sources.

Anti-GM-CSFRα antibody system binds to granulocyte macrophage colony-stimulating factor receptor α (GM-CSFRα). The anti-GM-CSFRα antibody E35-1 used in the embodiments of the present invention is disclosed in international patent application WO2020/108423A1 , the disclosures thereof are incorporated into this specification by reference.

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

Table 1. Amino acid sequences of E35-1 antibody and E35-2 antibody sequence E35-1 heavy chain QMQLVQSGAEVKKPGASVKVSCKVSGYTLTELSIHWVRQAPGKGLEWMGGFDPEDGETIYAQKFQGRVMTTGDTSTDTAYLELSSLRSEDTALYYCATGRYTSLATTYGFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS NTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 1) E35-2 heavy chain QMQLVQSGAEVKKPGASVKVSCKVSGYTLTELSIHWVRQAPGKGLEWMGGFDPEDGETIYAQKFQGRVMTTGDTSTDTAYLELSSLRSEDTALYYCATGRYTSLATTYGFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS NTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 2) E35-1 and E35-2 light chains EIVMTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDNWPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC (SEQ ID NO: 3) [Example 1]

Screening for anti-GM-CSFRα Buffer systems for antibody preparations

1. Screen buffer systems by using differential scanning fluorescence (DSF) and turbidity methods

(1) Screen for the best pH value and buffer system

Dialyze GM-CSFRα antibody E35-1 (without PS20) into the buffer system, add excipients (sodium chloride, mannitol, sucrose or sorbitol) and PS20, and use differential scanning fluorescence (DSF) to detect the Tm value. , the turbidity was measured using turbidity method.

The Tm value detection results are shown in Table 2. It can be seen from Table 2 that in buffer systems with different pH value ranges, the Tm values of the tested antibodies are different. The antibody sample in phosphate buffer at pH 7.0 has a higher Tm (higher than 66°C), indicating that the anti-GM-CSFRα antibody has significantly higher thermal stability under this pH value and buffer system.

Table 2. Tm detection results Tm pH Control (without excipients) Excipients 20mM sodium chloride 1% mannitol 2% sucrose 2% sorbitol 20mM acetate 5.0 60.0 58.0 60.0 60.0 60.0 20mM Histamine HCl 5.5 59.4 58.0 58.8 58.7 60.1 6.0 62.5 62.0 63.0 63.0 63.0 20mM phosphate 7.0 66.2 66.1 66.5 66.6 66.4

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

Table 3. Turbidity test results Turbidity increase pH Control (without excipients) Excipients 20mM sodium chloride 1% mannitol 2% sucrose 2% sorbitol 20mM acetate 5.0 0.003 0.000 0.003 0.002 0.002 20mM Histamine HCl 5.5 0.002 0.059 0.022 0.091 0.014 6.0 0.012 0.046 0.032 0.131 0.018 20mM phosphate 7.0 0.001 0.003 0.005 0.003 0.002

Combining the Tm value detection results and turbidity detection results, it can be seen that the buffer pH of around 7.0 is the optimal buffer pH range for anti-GM-CSFRα antibodies, and its effect is significantly better than other pH ranges.

(2) Confirm the buffer system within the pH range

The pH range of three different buffer systems (100mM histamine hydrochloride, 20mM phosphate and 200mM Tris hydrochloric acid) was adjusted within the range of 6.8-7.2, and the DSF method was used to further detect the Tm value.

The results are shown in Table 4. In the pH range of 6.8-7.2, the antibody samples in phosphate buffer have a higher Tm value compared with the antibody samples in other commonly used buffer systems (histidine and Tris hydrochloric acid). Moreover, Under different pH values, the Tm value of the antibody sample in phosphate buffer remained basically unchanged, further verifying that for this anti-GM-CSFRα antibody, phosphate buffer with a pH value in the range of approximately 6.8-7.2 is better than other Commonly used buffers have better stabilizing effects.

Table 4. Tm test results of DSF screening buffer system Buffer pH Excipients control 20mM sodium chloride 2% trehalose 2% sucrose 2% sorbitol 1% mannitol 100mM Histamine HCl 6.8 65.0 65.0 65.9 65.3 65.3 65.1 7.0 65.9 65.6 66.2 66.0 66.2 66.1 20mM phosphate 6.8 66.1 66.0 66.3 66.4 66.4 66.3 7.0 66.2 66.1 66.5 66.5 66.7 66.3 7.2 66.1 66.1 66.5 66.5 66.6 66.2 200mM Tris hydrochloric acid 7.0 63.3 63.4 64.0 64.0 64.0 63.5 7.2 64.0 64.1 64.4 64.4 64.6 64.1

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

Concentrate GM-CSFRα antibody E35-1 (the buffer system is 100mM histidine at pH7.0 and 20mM phosphate buffer at pH7.0) to a high concentration (50mg/ml -150mg/ml), and measure its pH value and viscosity.

The results are shown in Table 5. The antibody concentration ranges from 50mg/ml to 150mg/ml. The 20mM phosphate buffer system can well maintain the pH value at around 7.0; while in other buffer systems (for example, 100mM histidine buffer ), the pH value decreases as the antibody concentration increases, and the pH value of the solution cannot be maintained stably.

In both buffer systems, the antibody viscosity at antibody concentrations of 50mg/ml and 100mg/ml was both lower than 2.0cP, meeting the requirements for subcutaneous injection. However, in comparison, the antibody viscosity in the 20mM phosphate buffer system was lower than Antibodies in 100mM histamine hydrochloride buffer system have lower viscosity.

Table 5. Buffer system evaluation results 100mM Histamine HCl pH 7.0 20mM phosphate pH 7.0 Concentration (mg/mL) Viscosity (cP) pH Concentration (mg/mL) Viscosity (cP) pH 150 2.65 6.78 150 2.37 6.94 100 1.90 6.81 100 1.75 6.94 50 1.34 6.83 50 1.22 6.93

3. Dynamic Light Scattering (DLS)

Dynamic light scattering was used to examine the hydrodynamic radius of the antibody molecules to assess the uniformity of the antibody particle size.

The results are shown in Table 6. The results indicate that the antibody has small polydispersity under these conditions and the antibody has good homogeneity.

Table 6. DLS evaluation results Preparation DLS Concentration (mg/ml) kD L/mg 0.5 1 2 4 8 16 20mM phosphate pH7.0 Radium (nm) 5.3 5.3 5.3 5.4 5.6 6.1 -9.88 %PD 1.5 0 0.8 0.6 1.6 1.9

In summary, it can be seen that in 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. [Example 2]

Screening of surfactants

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

Set up 5 groups of experiments, each group contains 100mg/mL antibody E35-1, 20mM PB at pH7, group F1 does not add surfactant, group F2 adds 0.01% PS20, group F3 adds 0.02% PS20, group F4 adds 0.01% PS80, F5 group adds 0.02% PS80.

1. Shock experiment

Shaking conditions: 200 rpm, at room temperature, and samples were collected on days 1, 3, and 5 to detect antibody concentration, turbidity, and aggregate properties. The aggregate properties were detected by SEC method.

The results showed that the F1 sample became turbid after 3 days of shaking, 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 sample increased from 0.467 to 2.06, and the turbidity of F2-F5 samples remained basically unchanged; the aggregates of F1-F5 were shaking It increased slightly after 3 days and remained basically unchanged on the 5th day. There were no significant differences between groups F2-F5.

Using a microflow imager (MFI) to quantify particles below visible light, the F1 sample had higher particle counts when placed in the vial on day 0 (up to 5145 particles/ml for >10 μm and 303 particles for >25 μm). /ml), the number increased beyond the detection range after 3 days of shaking; F2-F5 samples still maintained a low particle number after 5 days of shaking (>10 μm particles were all less than 642/ml, and >25 μm particles are less than 75/ml). There were no significant differences between groups F2-F5.

2. Freeze-thaw experiment

Antibody preparations were frozen at -70°C, thawed at room temperature, and mixed by pipetting before back-freezing. Samples were collected after 3 and 5 cycles to detect antibody concentration, turbidity, and aggregate properties. The aggregate properties were detected using the SEC method.

The test results showed that after repeated freezing and thawing for 5 times, the antibody concentration, turbidity and HWM% of the F1 group increased compared with those without freezing and thawing. There was basically no change compared with time, and there was no significant difference between the F2-F5 groups (results not shown).

In summary, PS20 and PS80 added in amounts of 0.01%-0.02% both have a good protective effect against anti-GM-CSFRα antibodies, and there is no obvious difference between them. [Example 3]

Anti-GM-CSFRα Screening of Stabilizers for Antibody Preparations

Dialyze GM-CSFRα antibodies (E35-1 and E35-2) into pH7.3 and pH6.7 buffers respectively, and add corresponding excipients as shown in Table 7. Place at 45°C and collect samples after 1, 2, 3, and 4 weeks to detect the concentration, turbidity, aggregation characteristics, and charge heterogeneity of the 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. Preparation formulation design Preparation Concentrationmg/mL pH Buffer Stabilizer Surfactant PS20 F1 100 7.0 20mMPB - 0.01% F2 100 6.7 20mMPB - 0.01% F3 100 7.3 20mMPB - 0.01% F4 100 7.0 20mMPB 2% sorbitol 0.01% F5 100 7.0 20mMPB 2% trehalose 0.01% F6 100 7.0 20mMPB 2% mannitol 0.01% F7 100 7.0 20mMPB 0.05mM EDTA 0.01% F8 100 7.0 20mMPB 50mM Arginine HCl 0.01% F9 100 7.0 20mMPB 150mM sodium chloride 0.01% F10 100 7.0 20mMPB 2% trehalose 0.01% F11 100 7.0 20mMPB 2% mannitol 0.01% F12 100 7.0 20mMPB - 0.01%

1. Screening results of stabilizers for E35-1 antibody preparations

After 4 weeks of storage at 45°C, the antibody concentrations in each group were lower than those at 0 weeks. There was no significant change (results not shown), and there was a slight upward trend in turbidity. However, in comparison, the F8 (spermine hydrochloride) and F9 (sodium chloride) groups had the smallest increase in turbidity value compared to 0 weeks, only about 0.02, and the F8 (spermine hydrochloride) group at 4 weeks Hydrochloride) group and F9 (sodium chloride) group have the lowest turbidity, especially the F9 group with a turbidity value of only 0.297, indicating that the F9 group has the highest antibody stability.

The HPLC-SEC test results are shown in Table 8. Generally speaking, as time goes by, the aggregates in each group of preparations have an increasing trend, and the main peak has a decreasing trend. However, among all groups, the F8 (arginine hydrochloride) and F9 (sodium chloride) groups, especially the F9 group, increased the slowest and the main peak decreased the slowest. In the third week, the main peaks of F8 and F9 were both greater than 95%. In contrast, the proportion of main peaks in other groups had dropped below 95%.

The above results show that compared with other groups, the antibodies in the F8 and F9 groups are less likely to aggregate and have higher stability.

Table 8. HPLC-SEC results Preparation 0 weeks 1 week 2 weeks 3 weeks 4 weeks Changes at 4 weeks compared to 0 weeks HMW% Main % LMW% HMW% Main % LMW% HMW% Main % LMW% HMW% Main % LMW% HMW% Main % LMW% ΔHMW% ΔMain % F1 1.45 98.55 ND 4.43 95.49 0.08 5.03 94.84 0.13 5.85 94.01 0.14 6.96 92.84 0.20 +5.51 -5.71 F2 2.18 97.82 ND 4.29 95.71 ND 4.97 94.94 0.09 5.69 94.16 0.15 6.61 93.26 0.13 +4.43 -4.56 F3 2.34 97.66 ND 4.55 95.35 0.10 5.29 94.58 0.13 6.10 93.73 0.17 7.10 92.68 0.22 +4.76 -4.98 F4 1.83 98.17 ND 4.33 95.58 0.08 5.24 94.63 0.13 6.05 93.80 0.15 7.17 92.66 0.17 +5.34 -5.51 F5 1.8 98.2 ND 4.20 95.72 0.08 4.93 94.96 0.10 5.73 94.12 0.15 6.68 93.13 0.19 +4.88 -5.07 F6 1.75 98.25 ND 4.26 95.74 ND 5.13 94.76 0.11 5.99 93.85 0.16 6.97 92.82 0.20 +5.22 -5.43 F7 1.82 98.18 ND 4.85 95.05 0.10 5.81 94.06 0.13 6.86 92.96 0.19 7.85 91.86 0.29 +6.03 -6.32 F8 2.66 97.37 ND 2.94 96.98 0.08 3.63 96.24 0.13 4.40 95.43 0.17 5.65 94.11 0.24 +2.99 -3.26 F9 2.71 97.29 ND 3.93 95.99 0.08 4.45 95.45 0.10 3.95 95.93 0.12 5.56 94.3 0.14 +2.85 -2.99 F10 2.76 97.24 ND 4.54 95.38 0.08 5.23 94.66 0.11 5.93 93.94 0.13 6.69 93.18 0.13 +3.93 -4.06 F11 2.76 97.24 ND 4.41 95.46 0.13 5.22 94.69 0.09 5.85 94.04 0.11 6.55 93.30 0.16 +3.79 -3.94 F12 2.73 97.27 ND 4.71 95.21 0.08 5.43 94.45 0.12 6.18 93.69 0.13 7.01 92.83 0.16 +4.28 -4.44 Note: ND means not detected.

The test results of cIEF are shown in Table 9. After being placed at 45°C for 4 weeks, compared with day 0, the acidic peak in each group showed a trend of increasing and the main peak decreasing, while the alkaline peak began to increase in the first week. , gradually decrease after 2-4 weeks. In contrast, the F9 (sodium chloride) group showed the slowest increase in the acidic peak and the slowest decrease in the main peak, indicating that the antibody in the F9 group had the smallest change in charge heterogeneity and the highest stability.

Table 9. cIEF results Preparation 0 weeks 1 week 2 weeks 3 weeks 4 weeks Change from week 4 to week 0 Acidic % Main % Basic % Acidic % Main % Basic % Acidic % Main % Basic % Acidic % Main % Basic % Acidic % Main % Basic % ΔAcidic % ΔMain % F1 23.7 52.3 24.0 41.5 32.0 26.5 51.0 25.0 24.0 59.3 20.8 19.9 66 13.6 20.4 +42.3 -38.7 F2 24.1 52.0 23.9 38.3 32.7 29.0 47.1 25.2 27.7 54.2 19.2 26.6 60.1 14.7 25.2 +36.0 -37.3 F3 24.1 51.9 24.0 44.8 28.8 26.4 56.8 22.2 21.0 66.7 12.6 20.7 73.5 9.3 17.2 +49.4 -42.6 F4 24.0 51.6 24.4 41.0 30.5 28.5 53.1 22.0 24.9 61.3 16.4 22.3 67.3 10.2 22.5 +43.3 -41.4 F5 24.0 51.9 24.1 42.2 30.3 27.5 52.2 23.9 23.9 60.2 18.1 21.7 66.2 12.8 21.0 +42.2 -39.1 F6 24.0 50.7 25.3 41.9 29.5 28.6 52.1 23.0 24.9 61.1 17.4 21.5 66.8 11.4 21.8 +42.8 -39.3 F7 24.2 51.1 24.7 43.2 29 27.8 53.1 21.6 25.3 60.8 17.3 21.9 66.8 12.4 20.8 +42.6 -38.7 F8 24.8 52.3 22.9 37.4 36.2 26.4 49.1 22.0 28.9 55.9 20.5 23.6 63.5 14.5 22.0 +38.7 -37.8 F9 24.5 52.7 22.8 36.1 37.3 26.6 47.6 23.4 29.0 52.7 21.8 25.5 59.1 17.9 23.0 +34.6 -34.8 F10 24.4 53.0 22.6 40.0 33.4 26.6 51.5 19.0 29.5 57.8 18.3 23.9 64.6 13.5 21.9 +40.2 -39.5 F11 24.5 52.7 22.8 41.1 32.2 26.7 52.2 19.3 28.5 58.3 17.8 23.9 64.2 15.3 20.5 +39.7 -37.4 F12 24.5 52.8 22.7 40.0 33.4 26.6 52.5 17.7 29.8 59.2 15.7 25.1 64.2 14.8 21.0 +39.7 -38.0

2. Screening of stabilizers for E35-2 antibody preparations

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

After 4 weeks of storage at 45°C, compared with 0 weeks, the antibody concentration in each group showed no significant change, and the turbidity showed a slight upward trend. In contrast, the F8 (spermine hydrochloride) group and F9 The (sodium chloride) group had the smallest increase in turbidity value compared to 0 weeks, only about 0.03, and the F8 (spermine hydrochloride) group and F9 (sodium chloride) group had the lowest turbidity at 4 weeks, especially The turbidity value of group F9 is only 0.269, indicating that the antibody stability of group F9 is the highest.

Under the condition of 45°C, the aggregates in each group of preparations have an increasing trend, and the main peak has a decreasing trend. Among all groups, the F8 (spermine hydrochloride) and F9 (sodium chloride) groups, especially the F9 group, had the slowest increase (only an increase of 1.93%), and the slowest decrease of the main peak (only an increase of 1.93%). decreased by 2.46%). In the third week, the main peaks of F8 and F9 were both greater than 96%. In contrast, the proportion of main peaks in other groups had dropped below 95%.

After being placed at 45°C for 4 weeks, compared with day 0, the acidic peaks in each group tended to increase while the main peaks decreased. In contrast, the acidic peak of the F9 (sodium chloride) group increased the fastest (only increased by 32.7%) and the main peak decreased the slowest (only decreased by 33.6%).

To sum up: for the anti-GM-CSFRα antibody of the present application, the stabilizer is spermine 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 when the stabilizer is sodium chloride, has a more significant stabilizing effect. [Example 4]

Evaluating the effectiveness of combinations of dissimilar stabilizers

1. Thermal stability experiment

The thermal stability study was conducted using the design of experiments (DoE) method. Referring to Table 10, prepare E35-1 antibody preparations with different concentrations of sodium chloride and different concentrations of spermine hydrochloride as stabilizer combinations. The target osmotic pressure is 50 to 600 (mOsm/kg H2O) to evaluate the difference. The combination of sodium chloride and spermine hydrochloride in amounts and proportions has a protective effect against GM-CSFRα antibodies.

Place at 40°C and collect samples after 1, 2, 3 and 4 weeks to detect the concentration, turbidity, aggregation characteristics, degradation characteristics, charge heterogeneity, and antigen binding capacity of the E35-1 antibody preparation. Among them, the polymer properties were detected by HPLC-SEC method, the degradation properties were detected by NR-CE-SDS and R-CE-SDS methods, and the charge heterogeneity was detected by iIEF method.

Table 10. Stabilizer optimization design using DoE Preparation Concentrationmg/mL pH Buffer surfactant Spermine hydrochloride sodium chloride A1 100 7.0 20Mm phosphate buffer PS20 0.01% - - A2 150mM - A3 - 150mM A4 150mM 150mM A5 75mM 75mM A6 100mM 50mM A7 50mM 100mM

The osmotic pressure of each group of preparations is shown in Table 11. The osmotic pressure of A2, A3, A5-A7 is between 300 mOsm/kg-400 mOsm/kg.

Table 11. Osmotic pressure results Preparation Osmotic pressure mOsm/kg A1 50 A2 317 A3 377 A4 604 A5 353 A6 342 A7 370

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

The results of the turbidity test showed that compared with 0 weeks, after 4 weeks, the turbidity of each preparation in the control group A1 and the A3 group with only sodium chloride added did not change significantly, while the turbidity of each preparation with the addition of spermine hydrochloride did not change significantly. The turbidity values of the preparations in the groups increased to varying degrees: the turbidity of groups A5 and A7 (the amount of spermine hydrochloride added was between 50mM and 75mM) increased by 0.187 and 0.204 respectively after 4 weeks, and that of A2, A4 and A6 (The added amount of spermine hydrochloride is 100mM-150mM) The turbidity increase of the preparation after 4 weeks is between 0.25-0.27. The above test results show that the A3 group of antibodies is more stable, followed by the A5 and A7 groups (the addition amount of arginine hydrochloride is 50mM-75mM), and the slightly less stable groups are A2, A4, and A6 (arginine hydrochloride). The amount of salt added is 100mM-150mM).

HPLC-SEC results are shown in Table 12. Compared with control group A1, the increase in aggregates in groups A2-A7 after 4 weeks was lower than that at week 0, and the proportions of the main peaks at 4 weeks were higher than those in the control. In contrast, the formulation containing only sodium chloride (A3) showed the lowest aggregates and the highest main peak at week 4, showing the best stability effect among all groups.

Table 12. HPLC-SEC results Preparation 0 weeks 1 week 2 weeks 3 weeks 4 weeks Change from week 4 to week 0 HMW% Main % LMW% HMW% Main % LMW% HMW% Main % LMW% HMW% Main % LMW% HMW% Main % LMW% ΔHMW% ΔMain % A1 3.08 96.92 ND 4.63 95.26 0.11 5.79 94.10 0.11 6.46 93.37 0.17 7.11 92.72 0.17 +4.03 -4.20 A2 2.72 97.28 ND 2.40 97.51 0.10 3.48 96.35 0.17 4.81 94.95 0.24 6.19 93.48 0.33 +3.47 -3.80 A3 2.88 97.12 ND 4.01 95.92 0.08 4.85 95.04 0.10 5.37 94.46 0.18 5.95 93.87 0.18 +3.07 -3.25 A4 2.67 97.33 ND 2.41 97.50 0.09 3.47 96.37 0.17 4.98 94.74 0.28 6.47 93.21 0.32 +3.80 -4.12 A5 2.68 97.32 ND 2.69 97.20 0.11 3.65 96.19 0.16 5.25 94.52 0.23 6.51 93.13 0.36 +3.83 -4.19 A6 2.69 97.31 ND 2.61 97.29 0.10 3.50 96.34 0.16 5.14 94.61 0.24 6.40 93.31 0.29 +3.71 -4.00 A7 2.73 97.27 ND 2.82 97.09 0.09 3.54 96.29 0.17 5.05 94.74 0.21 6.10 93.5 0.40 +3.37 -3.77 Note: ND means not detected.

The cIEF results are shown in Table 13. Compared with week 0, the acidic 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 optimal stability.

Table 13. cIEF results Preparation 0 weeks 1 week 2 weeks 3 weeks 4 weeks Change from week 4 to week 0 Acidic % Main % Basic % Acidic % Main % Basic % Acidic % Main % Basic % Acidic % Main % Basic % Acidic % Main % Basic % ΔAcidic % ΔMain % A1 23.8 53.1 23.1 40.5 28.9 30.6 53.8 20.3 25.9 62.0 15.4 22.6 64.6 24.2 11.2 +40.8 -28.9 A2 23.5 52.6 23.9 36.8 31.9 31.3 50.1 21.9 28.0 59.1 15.6 25.3 60.5 26.2 13.3 +37.0 -26.4 A3 23.5 52.3 24.2 37.5 32.0 30.5 48.7 24.7 26.6 56.7 18.9 24.4 59.8 27.0 13.2 +36.3 -25.3 A4 23.5 52.3 24.2 36.2 32.2 31.6 47.8 24.7 27.5 57.3 18.7 24.0 64.4 21.9 13.7 +40.9 -30.4 A5 23.5 52.8 23.7 37.6 31.4 31.0 50.0 22.9 27.1 58.7 17.1 24.2 61.7 25.2 13.1 +38.2 -27.6 A6 23.7 52.1 24.2 38.0 31.7 30.3 51.0 20.4 28.6 57.9 18.6 23.5 66.0 21.4 12.6 +42.3 -30.7 A7 23.8 52.2 24.0 39.7 30.8 29.5 48.7 24.2 27.1 58.0 18.2 23.8 60.6 25.8 13.6 +36.8 -26.4

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

Table 14. cGE results Preparation 0 2 weeks 4 weeks NR-SDS R-SDS NR-SDS R-SDS NR-SDS R-SDS Intact% Other % HC+LC% Other % Intact% Other % HC+LC% Other % Intact% Other % HC+LC% Other % A1 91.8 8.2 97.2 2.8 89.4 10.6 98.5 1.5 81.9 18.1 97.1 2.9 A2 - - - - 89.6 10.4 98.4 1.6 81.3 18.7 97.2 2.8 A3 - - - - 89.6 10.4 99.3 0.7 84.0 16.0 98.8 1.2 A4 - - - - 87.2 12.8 99.0 1.0 80.6 19.4 97.9 2.1 A5 - - - - 89.4 10.6 99.0 1.0 80.5 19.5 97.4 2.6 A6 - - - - 90.4 9.6 98.9 1.1 81.4 18.6 97.7 2.3 A7 - - - - 89.1 10.9 98.5 1.5 82.4 17.6 97.7 2.3

In summary, the results show that the addition of spermine hydrochloride and sodium chloride has a protective effect on antibodies. However, the stability of the antibody preparation in the sodium chloride-only group was better than that in the other groups, indicating that the stabilizing effect of sodium chloride alone on the anti-GM-CSFRα antibody preparation was better than that of spermine hydrochloride alone. In combination with sodium chloride and higher concentrations of spermine hydrochloride (50mM-150mM). [Example 5]

Anti-GM-CSFRα Stability testing of antibody preparations

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

Table 15. Formulation stability experimental design Preparation Concentrationmg/ml pH Buffer Stabilizer surfactant T1 100 7.0 20mMPB 25mM spermine hydrochloride, 100mM sodium chloride 0.01% PS20 T2 100 6.7 20mMPB 25mM spermine hydrochloride, 100mM sodium chloride 0.01% PS20 T3 100 7.3 20mMPB 25mM spermine hydrochloride, 100mM sodium chloride 0.01% PS20 T4 100 7.0 20mMPB 120mM sodium chloride 0.01% PS20 T5 100 7.0 10mMPB 40mM Arginine HCl, 70mM Sodium Chloride 0.02% PS20 T6 150 7.0 20mMPB 15mM spermine hydrochloride, 120mM sodium chloride 0.01% PS20 T7 180 7.0 30mMPB 200mM sodium chloride 0.01% PS80 T8 50 7.0 20mMPB 100mM sodium chloride 0.02%PS80

1. Freeze-thaw experiment: Freeze the antibody preparation of group T1 (20mM PB, 25mM spermine hydrochloride, 100mM sodium chloride, pH 7) at -70°C and thaw at room temperature. Samples were collected and analyzed after 1, 3, and 5 freeze-thaw cycles to detect antibody concentration, aggregate properties, and the number of particles below visibility.

(1) Table 16 shows the freeze-thaw results of each preparation of E35-1 antibody. After 5 freeze-thaws, the protein concentration was only slightly reduced (102.88 mg/mL vs 100.25 mg/mL) compared to the preparation that was not frozen and thawed. The HPLC-SEC detection results showed that the aggregates remained basically unchanged. The number of particles below visibility increases.

Table 16. Freeze-thaw results loop 0 1 time 3 times 5 times Concentrationmg/mL 102.88 102.63 101.39 100.25 HPLC-SEC HMW% 1.06 1.11 1.42 1.33 Main % 98.94 98.89 98.58 98.67 MFI Count/mL >10um 95±37 292±118 240±60 508±47 >25um 3±2 46±26 9±0 11±0

(2) The E35-2 antibody preparation also showed good stability: after 5 freezing and thawing times, the protein concentration was only slightly reduced compared to the preparation without freezing and thawing (results not shown). The detection aggregates of HPLC-SEC remained basically unchanged. The number of particles below visibility increases slightly.

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 overall antibody preparation is still relatively stable.

2. Filtration experiment:

The antibody preparation of group T1 (20mM PB, 25mM spermine hydrochloride, 100mM sodium chloride, pH 7) was filtered 5 times through 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 for various preparations of the E35-1 antibody. After 5 times of filtration, there was no significant change in the antibody concentration compared to that without filtration.

Table 17. Filter binding experiments Preparation Concentration (mg/mL) P0 100.71 P1 101.39 P2 100.91 P3 101.10 P4 100.96 P5 101.57

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

3. Thermal stability experiment

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

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

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 the T1-T3 group (turbidity increased by only 0.02) was better than that in the T7-T8 group containing only sodium chloride as a stabilizer (turbidity increased by 0.04).

The HPLC-SEC results are shown in Table 18. Compared with week 0, the degree of antibody aggregation in each group showed a slight increase trend after 4 weeks. However, the main peak 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 in the T4 and T7-T8 groups, the main peak 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 of the preparations in the T4 and T7-T8 groups.

Table 18. HPLC-SEC results of thermal stability experiments Preparation 0 weeks 1 week 2 weeks 4 weeks Change from week 4 to week 0 HMW% Main % LMW% HMW% Main % LMW% HMW% Main % LMW% HMW% Main % LMW% ΔHMW% ΔMain % T1 0.84 99.16 ND 1.15 98.85 ND 1.36 98.64 ND 1.90 98.05 0.05 +1.06 -1.11 T2 0.78 99.22 ND 1.08 98.92 ND 1.26 98.74 ND 1.62 98.38 ND +0.84 -0.84 T3 0.88 99.12 ND 1.18 98.82 ND 1.41 98.59 ND 2.04 97.89 0.07 +1.16 -1.23 T4 0.84 99.16 ND 1.50 98.50 ND 1.79 98.22 ND 2.23 97.77 ND +1.39 -1.39 T5 0.82 99.18 ND 1.22 98.78 ND 1.77 98.23 ND 2.11 97.89 ND +1.29 -1.29 T6 0.80 99.20 ND 1.11 98.89 ND 1.42 98.58 ND 2.09 97.91 ND +1.29 -1.29 T7 0.79 99.21 ND 1.09 98.91 ND 1.33 98.67 ND 2.20 97.78 0.02 +1.41 -1.43 T8 0.81 99.19 ND 1.21 98.79 ND 1.41 98.59 ND 2.15 97.79 0.06 +1.34 -1.40 Note: ND means not detected.

The results of cGE are shown in Table 19. The results show that compared to week 0, the main peak and fragments change after 4 weeks. The main peak has a slight decreasing trend and the fragments have a slight increasing trend. Compared with week 0, after 4 weeks, the non-reducing main peak of T1 decreased by 3.6% and the reducing main peak decreased by 1.4%; the non-reducing main peaks of T2-T3 and T5-T6 decreased by 4.2%-4.5%, and the non-reducing main peak of T4 and T7-T8 decreased by 4.2%-4.5%. The main reducing peak decreased by 4.8%-5.0%. The above results further confirmed that the stability of the antibody preparations in the T1-T3 group was better than that in the T4 and T7-T8 groups.

Table 19. cGE results of thermal stability experiments Preparation 0 1 week 2 weeks 4 weeks Change from week 4 to week 0 NR-SDS R-SDS NR-SDS R-SDS NR-SDS R-SDS NR-SDS R-SDS NR-SDS R-SDS Intact% Other % HC+LC% Other % Intact% Other % HC+LC% Other % Intact% Other % HC+LC% Other % Intact% Other % HC+LC% Other % ΔIntact % ΔHC +LC % T1 92.5 7.5 99.4 0.6 91.2 8.8 99.2 0.8 89.0 11 98.5 1.5 88.9 11.1 98.0 2 -3.6 -1.4 T2 92.1 7.9 99.1 0.9 90.3 9.7 98.9 1.1 89.9 10.1 98.6 1.4 87.9 12.1 97.5 2.5 -4.2 -1.6 T3 92.0 8.0 99.4 0.6 90.4 9.6 99.0 1 89.5 10.5 98.7 1.3 87.7 12.3 97.5 2.5 -4.3 -1.9 T4 91.4 8.6 99.8 0.2 90.9 9.1 98.9 1.1 89.4 10.6 98.7 1.3 86.6 13.4 97.8 2.2 -4.8 -2.0 T5 92.3 7.7 99.3 0.7 91.1 8.9 99.1 0.9 89.2 10.8 98.5 1.5 87.8 12.2 97.4 2.6 -4.5 -1.9 T6 92.1 7.9 99.5 0.5 91.0 9.0 99.4 0.6 89.5 10.5 98.6 1.4 88.0 12 97.6 2.4 -4.1 -1.9 T7 92.2 7.8 99.7 0.3 91.2 8.8 99.4 0.6 89.4 10.6 98.7 1.3 87.2 12.8 97.5 2.5 -5.0 -2.2 T8 92.4 7.6 99.2 0.8 91.2 8.8 99.0 1 89.0 11 98.1 1.9 87.4 12.6 97.1 2.9 -5.0 -2.1

Table 20 shows the results of the binding capacity of antibodies and antigens. Compared with week 0, after 4 weeks of storage, the binding capacity of all preparations and antigens remained basically unchanged.

Table 20. Antigen binding results of thermal stability experiments Preparation KdM ^-1 0 weeks 4 weeks T1 6.51E-10 6.70E-10 T2 6.68E-10 6.52E-10 T3 6.70E-10 6.38E-10 T4 6.70E-10 7.08E-10 T5 6.65E-10 6.59E-10 T6 6.77E-10 6.72E-10 T7 6.59E-10 6.46E-10 T8 6.67E-10 6.59E-10

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

Compared with week 0, after 4 weeks, the antibody concentration and turbidity of all groups did not change significantly; HPLC-SEC results showed that the degree of antibody aggregation in each group showed a slight increase trend, T1-T3 and T5 -The main peak in the T6 group 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 more than 1.4%; cGE results show , the main peak of each group has a slight decreasing trend, and the fragments have a slight increasing trend. The non-reducing main peak of T1 decreased by 3.4%, the reducing main peak decreased by 1.5%, and the non-reducing main peaks of T2-T3 and T5-T6 decreased by 4.2%-4.6%. , the non-reducing main peaks of T4 and T7-T8 decreased by more than 5.0%; after 4 weeks of storage, the binding force between all preparations and antigens remained basically unchanged. In contrast, the stability of the antibody preparations in the T1-T3 group was better than that in the T5-T6 group, and even better than that in the T4 and T7-T8 groups.

In summary, the anti-GM-CSFRα antibody preparation of the present invention is stable within 5 freeze-thaw cycles; there is no obvious antibody loss after 5 times of filtration; the antibody is slightly degraded under high temperature conditions, but it is not obvious. Relatively speaking, the combination of spermine hydrochloride and sodium chloride at a concentration lower than 50mM as a stabilizer has a stronger protective effect on antibodies than sodium chloride alone as a stabilizer, and has a more excellent effect. [Example 6]

Long term stability test

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

The E35-1 and E35-2 antibody preparations T1 group and T4 group were prepared with reference to the formula in Example 5. Test the stability of the antibody preparation under long-term storage conditions (5 ± 3°C for 0 days, January, March, and June).

(1) Table 21 shows the stability results of the E35-1 antibody preparation under long-term storage conditions. Compared with 0 months, after 6 months, the indicators of the E35-1 antibody preparations in the T1 group and T4 group basically remained unchanged. change.

Table 21. Stability results under long-term storage conditions (5±3℃) Detection indicators T1 group T4 group 0 month January March June 0 month January March June Visible particle analysis Particles≥10μm 140 NR NR NR 151 NR NR NR Particles≥25μm 0 NR NR NR 0 NR NR NR HPLC-SEC Main peak% 99.0 98.8 98.9 98.8 98.9 98.8 98.8 98.7 Polymer% 1.0 1.2 1.1 1.2 1.1 1.2 1.2 1.3 NR-SDS-CGE Main peak% 98 98 98 98 98 98 98 98 R-SDS-CGE HC+LC% 100 100 100 100 100 100 100 100 NGHC% 0 0 0 0 0 0 0 0 iEF Main peak% 61 64 63 61 60 62 62 62 Acidic peak% 14 14 14 16 15 15 16 16 Basic peak% 25 twenty two twenty three twenty three 25 twenty three twenty two twenty two Measurement of antibody concentration by ultraviolet spectroscopy 96 99 99 100 99 97 98 99 pH 7.1 NR NR 7.0 7.0 NR NR 7.1 Osmotic pressure mOsmol/kg 263 NR NR 274 280 NR NR 285 Bacterial endotoxin detection EU/mg <0.001 NR NR <0.002 <0.002 NR NR <0.002 Sterility test qualified NR NR NR qualified NR NR qualified Note: NR means not detected.

(2) The E35-2 antibody preparation also showed good stability under long-term storage conditions. Compared with 0 months, after 6 months, the indicators of the E35-2 antibody preparations in the T1 group and T4 group basically remained the same. change.

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

Test the stability of E35-1 and E35-2 antibody preparations under long-term accelerated conditions (accelerated at 25°C ± 2°C for 0 days, January, March, and June).

(1) Table 22 shows the stability results of the E35-1 antibody preparation under accelerated conditions. Compared with 0 months and 6 months, the E35-1 antibody preparation only showed a slight decrease in the main peak and a slight decrease in the acidic peak. There was an increase, while other indicators remained basically unchanged.

Table 22. Stability results under long-term acceleration conditions (25±2℃) Detection indicators T1 group T4 group 0 month January March June 0 month January March June Visible particle analysis Particles≥10μm 140 NR NR 247 151 NR NR 258 Particles≥25μm 0 NR NR NR 0 NR NR NR HPLC-SEC Main peak% 99.0 98.6 98.5 97.9 98.9 98.5 98.0 97.2 Polymer% 1.0 1.4 1.5 2.0 1.1 1.5 2.0 2.8 NR-SDS-CGE Main peak% 98 98 98 97 98 98 97 97 R-SDS-CGE HC+LC% 100 100 100 100 100 100 100 100 NGHC% 0 0 0 0 0 0 0 0 iEF Main peak% 61 61 61 45 60 59 58 40 Acidic peak% 14 19 twenty two 39 15 19 28 41 Basic peak% 25 20 16 17 25 twenty two 14 19 Measurement of antibody concentration by ultraviolet spectroscopy 96 99 99 98 99 97 98 99 pH 7.1 NR NR 7.1 7.0 NR NR 7.1 Osmotic pressure mOsmol/kg 263 NR NR 272 280 NR NR 285 Bacterial endotoxin detection EU/mg <0.001 NR NR NR <0.002 NR NR <0.002 Sterility test qualified NR NR NR qualified NR NR qualified 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 and the acidic peak. There was a slight increase, while 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 that the preparation maintains good stability during preparation, transportation and storage, and ensures the safety and safety of clinical medication. Quality controllability.

TW202333792A_112102840_SEQL.xml

Claims (17)

An anti-GM-CSFRα antibody preparation, characterized in that the preparation contains an anti-GM-CSFRα antibody, a stabilizer, a surfactant, and a buffer. The buffer is a phosphate buffer with a pH value of 6.5-7.5, ideally 6.7-7.3, more ideally 7.0. The antibody preparation according to claim 1, wherein the concentration of the buffer is 10mM-30mM; ideally, the concentration of the buffer is 20mM. The antibody preparation as described in claim 1 or 2, wherein the concentration of the antibody is 50 mg/ml-200 mg/ml, ideally 50 mg/ml-180 mg/ml, further preferably 50 mg/ml-150 mg/ml, more preferably 100mg/m-150mg/ml. The antibody preparation according to any one of claims 1 to 3, wherein the stabilizer includes sodium chloride. The antibody preparation as described in claim 4, wherein the concentration of sodium chloride is 70 mM-200mM, ideally 100 mM-200mM, more preferably 100 mM-150mM, further preferably 120 mM-150mM. The antibody preparation according to claim 4, wherein the stabilizer further comprises arginine hydrochloride; ideally, the concentration of arginine hydrochloride is 0-50mM, ideally 0-40mM, more ideally 15mM-40mM. The antibody preparation according to claim 6, wherein the concentration of sodium chloride is 50mM-150mM; ideally, it is 70mM-150mM; more preferably, it is 70mM-120mM. The antibody preparation according to any one of claims 1 to 5, wherein the stabilizer is: (1) 50mM-150mM (ideal 70mM-120mM) sodium chloride and 0-50mM (ideal 15mM-40mM) spermine hydrochloride; (2) 100mM-200mM (ideally 120mM-150mM) sodium chloride. The antibody preparation according to any one of claims 1 to 8, wherein the surfactant is polysorbate; ideally, the polysorbate is Tween-20 or Tween-80. The antibody preparation according to any one of claims 1 to 9, wherein the concentration of the surfactant is 0.05 mg/ml-0.3 mg/ml; ideally, the concentration of the surfactant is 0.1 mg/ml- 0.2mg/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 (ideally 70mM-120mM) sodium chloride and 0-50mM (ideally 15mM-40mM) spermine hydrochloride, or 100mM-200mM (ideally 120mM-150mM) sodium chloride; the buffer is 10mM-30mM phosphate buffer; the buffer The pH value is 6.7-7.3; the surfactant is 0.1mg/ml-0.2mg/ml Tween-20 and/or Tween-80; (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 spermine hydrochloride and 70mM sodium chloride, or 50mM spermine hydrochloride and 100mM sodium chloride, or 100mM, 120mM, 150mM, 180mM or 200mM sodium chloride; the buffer is 10mM- 30mM phosphate buffer; the pH value of the buffer is 6.7-7.3; the surfactant is 0.1mg/ml-0.2mg/ml Tween-20 and/or Tween-80; (3) The antibody concentration is 50mg/ml-180mg/ml; the stabilizer is 50mM-150mM (ideally 70mM-120mM) sodium chloride and 0-50mM (ideally 15mM-40mM) arginine hydrochloride Salt, or 100 mM -200mM (ideally 120mM - 150mM) sodium chloride; the buffer is 10mM, 20mM, 30mM phosphate buffer; the pH value of the buffer is 6.7-7.3; the surfactant It is 0.1mg/ml-0.2mg/ml Tween-20 and/or Tween-80; (4) The antibody concentration is 50mg/ml-180mg/ml; the stabilizer is 50mM-150mM (ideally 70mM-120mM) sodium chloride and 0-50mM (ideally 15mM-40mM) arginine hydrochloride Salt, or 100mM-200mM (ideally 120mM-150mM) sodium chloride; the buffer is 10mM-30mM phosphate buffer; the pH value of the buffer is 6.7, 6.8, 6.9, 7.0, 7.1, 7.2 , 7.3; The surfactant is 0.1mg/ml-0.2mg/ml Tween-20 and/or Tween-80; (5) The antibody concentration is 50mg/ml-180mg/ml; the stabilizer is 50mM-150mM (ideally 70mM-120mM) sodium chloride and 0-50mM (ideally 15mM-40mM) arginine hydrochloride Salt, or 100mM-200mM (ideally 120mM-150mM) sodium chloride; the buffer is 10mM-30mM phosphate buffer; 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 (ideally 120mM-150mM) sodium chloride; the buffer is 10mM-30mM phosphate buffer; the buffer The pH value 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 (ideally 70mM-120mM) sodium chloride and 0-50mM (ideally 15mM-45mM) arginine hydrochloride Salt; the buffer is 10mM-30mM phosphate buffer; the pH value of the buffer is 6.7-7.3; the surfactant is 0.1mg/ml-0.2mg/ml Tween-20 or Tween-80 . 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 spermine hydrochloride and 100mM sodium chloride; the buffer is 20mM phosphate buffer; the pH value of the buffer is 6.7 or 7.0 or 7.3; The surfactant is 0.1 mg/ml or 0.2 mg/ml Tween-20 or Tween-80; (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 value of the buffer is 7.0; the surfactant is 0.1 mg/ml or 0.2mg/ml of Tween-20 or Tween-80; (3) The antibody concentration is 100mg/ml; the stabilizer is 40mM spermine hydrochloride and 70mM sodium chloride; the buffer is 20mM phosphate buffer; the pH value of the buffer is 7.0; the surface The active agent is 0.1mg/ml or 0.2mg/ml Tween-20 or Tween-80; (4) The antibody concentration is 150mg/ml; the stabilizer is 15mM spermine hydrochloride and 120mM sodium chloride; the buffer is 20mM phosphate buffer; the pH value of the buffer is 7.0; the surface The active agent is Tween-20 or Tween-80 at 0.1 mg/ml or 0.2 mg/ml. The antibody preparation according to any one of claims 1 to 12, wherein the antibody preparation may also contain antioxidants and/or preservatives. The antibody preparation according to any one of claims 1 to 13, wherein the antibody preparation is a liquid preparation or an injection powder. The antibody preparation according to any one of claims 1 to 14, wherein the antibody comprises a heavy chain comprising the amino acid sequence SEQ ID NO: 1 or 2, and a light chain comprising the amino acid sequence SEQ ID NO: 3. The use of an antibody preparation as described in any one of claims 1 to 15 in the preparation of drugs for treating diseases. The use as claimed in claim 16, wherein the disease includes an autoimmune disease, an inflammatory disorder or cancer; ideally, the disease is rheumatoid arthritis, asthma or myeloid leukemia.