CN102552903A - Epidermal growth factor receptor (EGFR) resistance humanized monoclonal antibody preparation - Google Patents

Abstract

The invention discloses a liquid preparation of anti-EGFR humanized monoclonal antibody, which contains high-concentration anti-EGFR humanized monoclonal antibody, PEG, mannitol and buffer, and the preparation contains or does not contain solution-stabilized formulation, with or without an isotonicity adjusting agent. The preparation can significantly reduce the particles produced after long-term storage of high-concentration liquid preparations of anti-EGFR humanized monoclonal antibodies, and has good stability. .

Description

A kind of anti-EGFR humanized monoclonal antibody preparation

technical field

The invention relates to the field of biological preparations, in particular, the invention discloses an anti-EGFR humanized monoclonal antibody preparation.

Background technique

EGFR plays an important role in regulating the growth, repair and survival of tumor cells, angiogenesis, invasion and metastasis, and is expressed in a considerable part of human tumors. In many malignant tumors, the expression of EGFR is often associated with poor prognosis and lower survival rate. Therefore, many research institutions and researchers have conducted research on EGFR antibodies that can block EGFR activity. Among them, Imclone Company provides EGFR antibody drug Erbitux, but Erbitux is an anti-EGFR chimeric antibody, which is produced by mouse anti-epidermal The variable regions of growth factor antibodies are composed of immunoglobulins from the constant regions of the heavy and light chains of the human antibody G1, so when used, the patient develops a HAMA response (human anti-mouse antibody), which limits the antibody's access to its antigen The ability of the target to reduce the potency of the antibody. In order to overcome this defect, Shanghai Zhangjiang Biotechnology Co., Ltd. provided a recombinant anti-EGFR humanized monoclonal antibody (patent number: 200510024158.3 Chinese patent), but failed to provide a suitable recombinant anti-EGFR humanized monoclonal antibody preparation.

Although there are some preparations of anti-EGFR chimeric antibodies in the prior art, first of all, due to differences in the protein structure of anti-EGFR chimeric antibodies and anti-EGFR humanized monoclonal antibodies, the formulations of the preparations cannot be directly used; secondly, the current Anti-EGFR chimeric antibody preparations also have their own shortcomings. For example, Erbitux produced by Imclone is a liquid preparation consisting of recombinant antibody, sodium chloride, and phosphate buffer. Its pH is 7.2-7.4, and the preparation is prohibited from shaking. Or diluted, can not be used for intravenous injection, can only be injected into the body by infusion pump or syringe, has poor stability, and is extremely inconvenient to store and use; the Chinese patent application with application number 200610147279.1 provides a stable anti-EGFR chimeric antibody However, the concentration of the anti-EGFR chimeric antibody in the preparation is low, so when a large amount of anti-EGFR chimeric antibody needs to be injected, it will cause inconvenience in its use; the application number is 200780022178.4 and the application number is 200810137278.8 The Chinese patent application provides a lyophilized formulation of an anti-EGFR chimeric antibody, but since the lyophilized formulation needs to be redissolved before use, this leads to additional handling steps, and more importantly, it may also cause drug contamination and harm to patients. cause serious harm.

Therefore, it is necessary to provide a liquid formulation of recombinant anti-EGFR humanized monoclonal antibody which is suitable, especially stable at high concentration. For patients, if a stable, high-concentration liquid preparation of anti-EGFR humanized monoclonal antibody can be provided, the convenience of use can be improved, and the pain of use can be reduced (especially when large doses of anti-EGFR humanized monoclonal antibody are used). When cloning antibody drugs), it has important application value.

Contents of the invention

During the research, the applicant of the present invention accidentally discovered a liquid preparation of anti-EGFR humanized monoclonal antibody, which can significantly reduce the high concentration of anti-EGFR humanized monoclonal antibody liquid preparation after long-term storage. The particles are very stable, which saves the cumbersome steps of filtering the medicine before use, and at the same time, the medicine can be used more safely.

Therefore, the object of the present invention is to provide a stable, high-concentration liquid preparation of anti-EGFR humanized monoclonal antibody.

Therefore, the present invention discloses:

A liquid preparation of anti-EGFR humanized monoclonal antibody, which contains high-concentration anti-EGFR humanized monoclonal antibody, PEG, mannitol and buffer, said preparation contains or does not contain a solution stabilizer, said The formulations may or may not contain isotonicity adjusting agents.

According to a preferred embodiment of the present invention, the PEG in the liquid preparation is PEG 400-PEG8000, preferably PEG 1000-PEG 4000.

According to a preferred embodiment of the present invention, the buffer in the liquid preparation is one of phosphate buffer, Tris, and histidine buffer, preferably a phosphate buffer composed of disodium hydrogen phosphate and sodium dihydrogen phosphate .

According to a preferred embodiment of the present invention, the solution stabilizer in the liquid preparation is one of sucrose, trehalose, maltose or a combination thereof.

According to a preferred embodiment of the present invention, the isotonicity regulator in the liquid preparation is NaCl.

According to a more preferred embodiment of the present invention, the liquid preparation contains 20-100 mg/ml of humanized anti-EGFR monoclonal antibody, 0.01-5% PEG, 2-10 mg/ml of mannitol, and 0-10 mg of solution stabilizer /ml, buffer 2~15mmol/l, NaCl 0~10mg/ml.

According to a more preferred embodiment of the present invention, the liquid preparation contains anti-EGFR humanized monoclonal antibody 20mg/ml, PEG 0.01%, mannitol 2mg/ml, solution stabilizer 10mg/ml, buffer 2mmol/l Or contain anti-EGFR humanized monoclonal antibody 40mg/ml, PEG 2%, mannitol 5mg/ml, solution stabilizer 5mg/ml, buffer 5mmol/l or contain anti-EGFR humanized monoclonal antibody 60mg/ml, PEG 3%, mannitol 7mg/ml, solution stabilizer 8mg/ml, buffer 8mmol/l or containing anti-EGFR humanized monoclonal antibody 80mg/ml, PEG 1%, mannitol 6mg/ml, buffer 15mmol/l l or contain anti-EGFR humanized monoclonal antibody 100mg/ml, PEG 5%, mannitol 10mg/ml, solution stabilizer 3mg/ml, buffer 10mmol/l.

Detailed ways

The following examples and experimental examples only further illustrate the present invention, and should not be construed as limiting the present invention.

For the preparation of anti-EGFR humanized monoclonal antibody, please refer to the patent number: 200510024158.3, the Chinese patent of invention title: recombinant anti-EGFR humanized monoclonal antibody, the anti-EGFR humanized monoclonal antibody described in the present invention is not limited to this One, the anti-EGFR humanized monoclonal antibody obtained by those skilled in the art using general genetic engineering techniques is also included in the anti-EGFR humanized monoclonal antibody of the present invention.

In the following embodiments of the present invention, the PEG used is PEG 400-PEG 8000, preferably PEG1000-PEG 4000.

Other excipients are commercially available pharmaceutical grades unless otherwise specified.

In the following examples of the present invention, experimental example, although only used phosphate buffer, use other suitable buffer of pH, also can reach the object of the present invention, this is for those skilled in the art Obviously, therefore, it also belongs to the protection content of the present invention.

In the following embodiments of the present invention, the activity detection method refers to:

Dual-Agent Molecular Targeting of the Epidermal Growth Factor Receptor(EGFR)Combining Anti-EGFR Antibody with Tyrosine Kinase Inhibitor.ShyhminHuang, Eric A.Armstrong, Sergio Benavente, Prakash Chinnaiyan and Paul M.Harari.Cancerug. 15): 5355-62.

Combined Epidermal Growth Factor Receptor Targeting with the TyrosineKinase Inhibitor Gefitinib(ZD 1839) and the Monoclonal Antibody Cetuximab(IMC-C225).Superiority Over Single-Agent Receptor Targeting.Pablo Matar ¹ ，Federico Rojo ² ，Raúl Cassia-Bareno-Bureno ³ ，Gemaue Mo ³ , Serena Di Cosimo ⁴ , José Tabernero ¹ , Marta Guzmán ¹ , Sonia Rodriguez ¹ , Joaquín Arribas ¹ , José Palacios ³ and José Baselga ^1. Clin Cancer Res. 2004 Oct 1; 10(19): 6487-501.

The Effects of Cetuximab Alone and in Combination With Radiation and/or Chemotherapy in Lung Cancer. David Raben ¹ , Barb Helfrich ² , Daniel C. Chan ² , Fortunato Ciardiello ⁵ , LiMin Zhao ² , Wilbur Franklin ³ , Anna E. Barón ⁴ , Chan Zeng ⁴ , Tim K. Johnson ¹ and Paul A. Bunn, Jr ^2. Clin Cancer Res. 2005 Jan 15;11(2Pt1):795-805.

Embodiment 1-5, Preparation of Liquid Preparation of Anti-EGFR Humanized Monoclonal Antibody

The liquid preparations of the anti-EGFR humanized monoclonal antibody were respectively prepared according to the formulas in Table 1 to obtain semi-finished products.

Table 1. Liquid preparation formula of anti-EGFR humanized monoclonal antibody

Aseptically divide the semi-finished product into vials, cover with a rubber stopper and an aluminum-plastic cap to obtain a finished product.

Experimental Example 1: Stability Experiment

The product that obtains by above-mentioned embodiment 1-5 is preserved by the condition of table 2 respectively, and detects its activity (the product that each embodiment obtains has carried out three batches of experiments respectively, and the result in the table is the average of three batches Result), the activity test results are shown in Table 2, wherein, the product before preservation is used as a contrast, and the percentage value of the activity of the product after preservation and the activity of the product before preservation is defined as the activity of the product after preservation.

Table 2. In vitro biological activity results

Storage Conditions Example 1 Example 2 Example 3 Example 4 Example 5 4°C, dark, 3 years 101% 95% 102% 98% 96% 30℃, dark, 1 year 98% 92% 99% 97% 98%

According to visual inspection, the above-mentioned products remain clear after long-term storage. According to the results in Table 2, the activity of the above-mentioned products has no obvious change, therefore, they have good stability.

Example 6-10, Preparation and Stability Test of Liquid Preparation of Anti-EGFR Humanized Monoclonal Antibody Containing Isotonic Regulator

Add 2 to 10 mg/ml of NaCl as an isotonic regulator to the product of Examples 1-5, and preserve the product according to the method of Experimental Example 1 and detect product activity. The results show that the prepared above-mentioned anti-EGFR containing isotonic regulator The liquid preparation of the humanized monoclonal antibody is similar to the products of Examples 1-5, and remains clear after long-term storage, and has no obvious change in activity, and its stability is good.

According to the test results of in vitro biological activity, the preparation disclosed by the present invention has the advantage of good stability, can significantly reduce the particles produced after long-term storage of the liquid preparation of high-concentration anti-EGFR humanized monoclonal antibody, and has good stability and saves The cumbersome step of filtering before the use of the medicine is gone, and the medicine can be used more safely at the same time, and the purpose of the present invention has been achieved.

Claims (9)

1. a liquid preparation of anti-EGFR humanized monoclonal antibody, is characterized in that, contains high concentration anti-EGFR humanized monoclonal antibody, PEG, mannitol and damping fluid in the described preparation, contains or does not contain in the described preparation A solution stabilizer is contained, and the formulation may or may not contain an isotonicity adjusting agent. 2. liquid formulation as claimed in claim 1, is characterized in that, described PEG is PEG 400-PEG8000. 3. liquid formulation as claimed in claim 2, is characterized in that, described PEG is PEG 1000-PEG4000. 4. The liquid preparation according to claim 1, wherein the buffer is one of phosphate buffer, Tris, and histidine buffer. 5. The liquid preparation according to claim 4, wherein the buffer is a phosphate buffer composed of disodium hydrogen phosphate and sodium dihydrogen phosphate. 6. The liquid preparation according to claim 1, wherein the solution stabilizer is one of sucrose, trehalose, maltose or a combination thereof. 7. The liquid preparation according to claim 1, wherein the isotonicity regulator is NaCl. 8. The liquid preparation according to any one of claims 1-7, wherein the liquid preparation contains 20-100 mg/ml of anti-EGFR humanized monoclonal antibody, PEG 0.01-5%, mannitol 2~10mg/ml, solution stabilizer 0~10mg/ml, buffer 2~15mmol/l, NaCl 0~10mg/ml. 9. The liquid preparation according to claim 8, characterized in that, the liquid preparation contains anti-EGFR humanized monoclonal antibody 20mg/ml, PEG 0.01%, mannitol 2mg/ml, solution stabilizer 10mg/ml , buffer 2mmol/l, or contain anti-EGFR humanized monoclonal antibody 40mg/ml, PEG2%, mannitol 5mg/ml, solution stabilizer 5mg/ml, buffer 5mmol/l, or contain anti-EGFR humanized Monoclonal antibody 60mg/ml, PEG 3%, mannitol 7mg/ml, solution stabilizer 8mg/ml, buffer 8mmol/l, or anti-EGFR humanized monoclonal antibody 80mg/ml, PEG 1%, mannitol 6mg/ml, buffer 15mmol/l, or containing anti-EGFR humanized monoclonal antibody 100mg/ml, PEG 5%, mannitol 10mg/ml, solution stabilizer 3mg/ml, buffer 10mmol/l.