CN105012949A - Preparation of recombinant human anti-human TNF-alpha monoclonal antibody - Google Patents

Abstract

The invention discloses a preparation of a recombinant human anti-human TNF-alpha monoclonal antibody. The preparation is prepared from the components: a recombinant human anti-human TNF-alpha monoclonal antibody, an excipient, an osmotic pressure adjusting agent, a protective agent and a buffer salt, wherein the protein content of the recombinant human anti-human TNF-alpha monoclonal antibody in the preparation is 1-150 mg/mL; the content of the excipient in the preparation is 0-20%; the content of the osmotic pressure adjusting agent in the preparation is 1-20 g/L; the content of the protective agent in the preparation is 0.1-10 g/L; the concentration of the buffer salt in the preparation is 5-200 mmol/L; the pH value of the preparation is 4-8. The preparation has the advantages of high protein concentration, simpleness in preparation and good stability.

Description

The preparation of recombined human anti-human TNFa monoclonal antibody

Technical field

The present invention relates to a kind of monoclonal antibody preparation, particularly relate to a kind of preparation of stable recombined human anti-human TNFa monoclonal antibody.

Background technology

Tumor necrosis factor α (TNF α) is the cytokine produced by polytype cell, comprises mononuclear cell and macrophage, is at first to induce the ability of some murine tumor necrosis to identify according to them.Confirm TNF α and multiple mankind's disease association now, comprised sepsis, infected, autoimmune disease, transplant rejection etc.

Due to can in conjunction with and in and human tumour necrosis factor (hTNF α) thus suppress hTNF alpha active, anti-hTNF α becomes the means for the treatment of hTNF alpha associated disorders.Early stage hTNF α is mouse monoclonal antibody or the chimeric antibody (variable region of antibody chain comes from Mus) prepared with genetic engineering method or the humanized antibody (antibody chain hypervariable region comes from Mus) of hybridoma secretion, can not induce some people's effector function or can cause " people's anti-mouse antibody " reaction (HAMA) in human body.When considering that chronic administration is in chronic disease, during as rheumatoid arthritis, this defect seems more obvious.

The recombined human anti-human TNFa monoclonal antibody mentioned in this description refers to be prepared by recombination method, expresses, the people's antibody produced or be separated.Described antibody has the variable region and constant region that come from human germline immunoglobulin's sequence, and wherein some amino acid sites may receive in vitro mutagenesis.

Existing people anti-human TNFa monoclonal antibody preparation patent:

Abbott Lab Bermuda Ltd. H-J Barbara Krause, L Bao Site and the application of M Otto Dix " being used for the treatment of the preparation of people's antibody of TNF-α associated conditions " (application number is 03824272.9, Authorization Notice No. CN1688339B).Its advantage there are provided a kind of preparation, makes human TNF alpha monoclonal antibody have the stability of long period in higher concentrations, but shortcoming is that formula is complicated, and buffer system is made up of two kinds of buffer, and preparation is inconvenient.

Summary of the invention

The technical problem to be solved in the present invention is to provide a kind of preparation of recombined human anti-human TNFa monoclonal antibody.Said preparation has the advantages such as high protein concentration, preparation are simple, good stability.

For solving the problems of the technologies described above, the preparation of recombined human anti-human TNFa monoclonal antibody of the present invention, its composition comprises: recombined human anti-human TNFa monoclonal antibody, excipient, osmotic pressure regulator, protective agent and buffer salt;

Wherein, in described preparation, the protein content of recombined human anti-human TNFa monoclonal antibody is 1 ~ 150mg/mL;

The content of excipient in described preparation is 0 ~ 20%;

The content of osmotic pressure regulator in described preparation is 1 ~ 20g/L;

The content of protective agent in described preparation is 0.1 ~ 10g/L;

The concentration of buffer salt in described preparation is 5 ~ 200mmol/L;

The pH of described preparation is 4 ~ 8.

For above-mentioned preparation, the protein content of described recombined human anti-human TNFa monoclonal antibody is preferably 25 ~ 100mg/ml.

Described excipient comprises: saccharide or many alcohols, as comprised: one or more in sucrose, glucose, mannitol, sorbitol.Wherein, excipient content is in the formulation preferably 1 ~ 10%.

Described osmotic pressure regulator comprises: saccharide or salt, as comprised: one or more in sucrose, mannitol, sodium chloride.Wherein, the content of osmotic pressure regulator in described preparation is preferably 2 ~ 10g/L.

Described protective agent comprises: saccharide, alcohols or tween, as comprised: one or more in tween, mannitol, sucrose.Wherein, the content of protective agent in described preparation is preferably 0.5 ~ 2g/L.

Described buffer salt comprises: histidine buffer salt system (as comprise: the buffer system of histidine and histidine hydrochloride), wherein, the concentration of buffer salt in described preparation is 10 ~ 100mmol/L.

The pH of described preparation is preferably pH4 ~ 6.

In the present invention, sucrose, mannitol both can be used as protective agent, again as osmotic pressure regulator, also can be used as excipient.

In addition, about the preparation method of above-mentioned preparation, following methods can be enumerated.

As the preparation method of above-mentioned preparation, its step can comprise:

Prepare the Formulation Buffer of described preparation, comprising: 20mM histidine-histidine hydrochloride buffer, 50g/L sucrose, 2.93g/L sodium chloride, pH value 5.2;

By with ultrafilter membrane/pipe, recombined human anti-human TNFa monoclonal antibody good for separation and purification is added in above-mentioned Formulation Buffer, and with above-mentioned Formulation Buffer, recombined human anti-human TNFa monoclonal antibody concentration is adjusted to 50mg/mL; Then, adding polyoxyethylene sorbitan monoleate to its content is 1g/L;

With 0.22 μm of metre filter, and point to be filled in 2R cillin bottle.

Moreover the present invention also provides a kind of application of preparation of recombined human anti-human TNFa monoclonal antibody, its purposes is the medicine of harmful disease for the preparation for the treatment of wherein TNF alpha active, and the medicine wherein prepared is used by liquid injection form.

The actual amount of application (i.e. active component) of preparation of the present invention depends on many factors, as the seriousness of disease to be treated, the age for the treatment of target and relative health, the usefulness of antibody, route of administration and the form that use, and other factors.

Preparation of the present invention, its formula is simple, easy to prepare.Simultaneously, be compared to Abbott Lab Bermuda Ltd. H-J Barbara Krause, " being used for the treatment of the preparation of people's antibody of TNF-α associated conditions " patent (Authorization Notice No. CN1688339B) of L Bao Site and the application of M Otto Dix, invention formulation has stronger stability.

Detailed description of the invention

Describe content of the present invention by the following examples in detail, but these embodiments are not construed as limiting the invention.

The preparation of embodiment 1 preparation

Preparation of the present invention is prepared in accordance with the following methods.

Press listed constituent content obtain solution A, solution B and solution C in table 1,2 and 3 respectively.

Table 1 solution A

Reagent name	Consumption
		Histidine	7.8g/L
Sucrose	50g/L
		Sodium chloride	2.93g/L
Polyoxyethylene sorbitan monoleate	1g/L
		Ultra-pure water	Supply volume

Table 2 solution B

Reagent name	Consumption
		Histidine hydrochloride	9.6g/L
Sucrose	50g/L
		Sodium chloride	2.93g/L
Polyoxyethylene sorbitan monoleate	1g/L
		Ultra-pure water	Supply volume

Table 3 solution C

Reagent name	Consumption
		Polyoxyethylene sorbitan monoleate	1g/L
Ultra-pure water	Supply volume

The solution A prepared drops in solution B, drips pH value determination, until pH value is 5.2, and called after solution D.Solution C is extremely aseptic without in endotoxin receiving vessel with 0.22 μm of metre filter.

Change liquid with the recombined human anti-human TNFa monoclonal that ultrafilter membrane/pipe is good to separation and purification to concentrate, replace to above-mentioned buffer D.With solution D by change liquid concentrate after diluted sample be 50mg/mL to recombined human anti-human TNFa monoclonal antibody.Interpolation solution C is 1g/l to the content of polyoxyethylene sorbitan monoleate.Sample after dilution 0.22 μm of metre filter, divides and is filled in 2R cillin bottle, and 0.8mL/ manages, and by its formulation samples (preparation of the present invention) called after F1.

The comparative study of embodiment 2 stability

D2E7 in preparation prescription mentioned by " being used for the treatment of the preparation of people's antibody of TNF-α associated conditions " (the Authorization Notice No. CN1688339B) that apply for Abbott Lab Bermuda Ltd. replaces with recombined human anti-human TNFa monoclonal antibody of the present invention (this antibody used ultrafilter membrane/pipe separation and purification good), specifically consist of: the recombined human anti-human TNFa monoclonal antibody of 50mg/ml, the mannitol of 12mg/ml, anhydrous sorbitol polyoxyethylene (20) oleic acid ester of 1mg/ml, 1.3mg, the citric acid of ml, the sodium citrate of 0.3mg/ml, the two hypophosphite monohydrate disodium hydrogens of 1.5mg/ml, the two hypophosphite monohydrate sodium dihydrogens of 0.9mg/ml, the sodium chloride of 6.2mg/ml, pH value is 5.0, and by said preparation sample called after F2.

The stability of the preparation (F2) investigated by jolting study on the stability, 40 DEG C of high temperature accelerated stabilities and mention in 2 ~ 8 DEG C of study on the stability experiment preparation more of the present invention (F1 of embodiment) and Abbott Lab Bermuda Ltd.'s patent.Wherein, the preparation of two kinds of formulation samples all have employed Shanghai Yaoming Kangde New Medicine Development Co., Ltd and prepares and the recombined human anti-human TNFa monoclonal antibody provided.

Table 4 prescription 2 ~ 8 DEG C of results of stability

Table 5 prescription jolting results of stability

Table 6 prescription 40 DEG C of results of stability

As can be seen from table 4 and table 5, F1 and F2 two kinds of all Testing index of prescription sample show suitable stability under investigation condition.As can be seen from Table 6, under 40 DEG C of acceleration environments, F1 prescription sample shows the stability being better than F2 prescription sample in Key Quality Indicator SEC-HPLC purity and CEX-HPLC purity.Particularly, about SEC-HPLC purity, when the investigation time is 4 weeks, F1 prescription sample have dropped 4.8, F2 prescription sample and have dropped 6.9%; About CEX-HPLC purity, when the investigation time is 4 weeks, F1 prescription sample have dropped 18.0%, F2 prescription sample and have dropped 21.7%.

Claims (7)

1. a preparation for recombined human anti-human TNFa monoclonal antibody, is characterized in that, its composition comprises: recombined human anti-human TNFa monoclonal antibody, excipient, osmotic pressure regulator, protective agent and buffer salt;

Wherein, in described preparation, the protein content of recombined human anti-human TNFa monoclonal antibody is 1 ~ 150mg/mL;

The content of excipient in described preparation is 0 ~ 20%;

The content of osmotic pressure regulator in described preparation is 1 ~ 20g/L;

The content of protective agent in described preparation is 0.1 ~ 10g/L;

The concentration of buffer salt in described preparation is 5 ~ 200mmol/L;

The pH of described preparation is 4 ~ 8.

2. preparation as claimed in claim 1, is characterized in that: the protein content of described recombined human anti-human TNFa monoclonal antibody is 25 ~ 100mg/ml;

Described excipient comprises: saccharide or many alcohols;

Described osmotic pressure regulator comprises: saccharide or salt;

Described protective agent comprises: saccharide, alcohols or tween.

3. preparation as claimed in claim 2, is characterized in that: described excipient comprises: one or more in sucrose, glucose, mannitol, sorbitol; Wherein, excipient content is in the formulation 1 ~ 10%;

Described osmotic pressure regulator comprises: one or more in sucrose, mannitol, sodium chloride; Wherein, the content of osmotic pressure regulator in described preparation is 2 ~ 10g/L;

Described protective agent comprises: one or more in tween, mannitol, sucrose; Wherein, the content of protective agent in described preparation is 0.5 ~ 2g/L.

4. preparation as claimed in claim 1, is characterized in that: described buffer salt comprises: histidine buffer salt system;

Wherein, the concentration of buffer salt in described preparation is 10 ~ 100mmol/L;

The pH of described preparation is pH4 ~ 6.

5. preparation as claimed in claim 4, is characterized in that: described histidine buffer salt system comprises: the buffer system of histidine and histidine hydrochloride.

6. a preparation method for preparation as claimed in claim 1, is characterized in that, its step comprises:

Prepare the Formulation Buffer of described preparation, comprise 20mM histidine-histidine hydrochloride buffer, 50g/L sucrose, 2.93g/L sodium chloride, pH value 5.2;

Recombined human anti-human TNFa monoclonal antibody is added in above-mentioned Formulation Buffer, and with above-mentioned Formulation Buffer, recombined human anti-human TNFa monoclonal antibody concentration is adjusted to 50mg/mL; Then, adding polyoxyethylene sorbitan monoleate to its content is 1g/L;

With 0.22 μm of metre filter, and point to be filled in 2R cillin bottle.

7. an application for preparation as claimed in claim 1, is characterized in that: described preparation is the medicine of harmful disease for the preparation for the treatment of wherein TNF alpha active.