HK1263347A1 - Lyophilized preparation of infliximab monoclonal antibody - Google Patents

Description

Lyophilized preparation of infliximab

Technical Field

The invention relates to the field of pharmacy, in particular to a lyophilized preparation containing infliximab, which can be reconstituted to obtain an infliximab preparation suitable for subcutaneous administration.

Background

Infliximab (CAS #170277-31-3) is a human-mouse chimeric monoclonal antibody which specifically binds to TNF- α in a human body, prevents TNF- α from binding with a cell surface receptor thereof, blocks the biological activity of TNF- α, finally relieves inflammatory response and reduces osteoclast activation, and achieves the purpose of controlling and relieving symptoms.

Are on the market at presentThe injection preparation is a freeze-dried preparation, the concentration of infliximab in the system after being reconstituted by water is 10mg/mL, and the preparation is administrated by an intravenous injection (i.v.). Rheumatoid arthritis, psoriasis, ankylosing spondylitis, ulcerative colitis, Crohn's disease, etc. are all chronic diseases and require long-term or even lifetime administration to the patient. And the intravenous administration mode can only be carried out in the hospital environment, and usually 100-200ml products take one hour or more, which is very unfavorable for the use of patients, so the compliance of the preparation on the market is low at present.

The high concentration infliximab preparation can greatly shorten the administration time. Because of its ease of use, formulations of infliximab at high concentrations have become a focus of attention. However, increasing the concentration of the monoclonal antibody increases the viscosity of the formulation solution, making it difficult to prepare and administer the formulation.

Therefore, stable high concentration biologics suitable for subcutaneous administration have great market demand and prospects.

Disclosure of Invention

In one aspect, the present invention relates to a lyophilized formulation comprising infliximab, a histidine buffer system, a structure protecting agent, and a surfactant, wherein,

prior to lyophilization, infliximab is present in the formulation at a concentration of about 10-80mg/ml, histidine is present in a histidine buffer system at a concentration of about 1-20mM, a structure protecting agent is present at a concentration of about 5-100mg/ml, and a surfactant is present at a concentration of about 0.001-1 mg/ml; and the concentration of infliximab in the formulation after reconstitution is about 100-200 mg/ml.

In a preferred embodiment, the concentration of infliximab in the formulation prior to lyophilization may be about 25-60mg/ml, more preferably about 30-50mg/ml, and most preferably about 40 mg/ml.

In another preferred embodiment, histidine may be present in the formulation at a concentration of about 3-10mM, preferably about 5mM, prior to lyophilization.

In a further preferred embodiment, the structure protective agent in the formulation is selected from carbohydrates, amino acids and combinations thereof, preferably sucrose, trehalose or combinations thereof, more preferably sucrose. The concentration of the structure protectant may be about 5-75mg/ml, more preferably about 5-50mg/ml, and most preferably about 25mg/ml prior to lyophilization.

In yet another preferred embodiment, the surfactant in the formulation is selected from polysorbates, poloxamers, triton, sodium lauryl sulfate, sodium lauryl glycoside, lauryl-sulfobetaine, polyethylene glycol, polypropylene glycol, or mixtures thereof, preferably polysorbate 80. The surfactant concentration may be about 0.01-0.4mg/ml, more preferably about 0.025-0.1mg/ml, prior to lyophilization.

In one embodiment, the pH of the formulation prior to lyophilization is from about 4.0 to about 8.0, preferably from about 4.5 to about 6.0, and more preferably from about 5.0 to about 5.4.

In one embodiment, the lyophilized formulation, upon reconstitution, has at least one of the following properties:

a viscosity of about 40cP or less, an

The osmotic pressure was about 250-530 mOsmol/kg.

In one embodiment, the concentration of infliximab in the formulation after reconstitution may be about 130-190mg/ml, more preferably about 140-170mg/ml, 150-160 mg/ml.

In yet another aspect, the invention also relates to the use of the lyophilized formulation for the preparation of a medicament for the prevention or treatment of autoimmune related diseases in humans.

In yet another aspect, the invention also relates to a method of preventing or treating an autoimmune-related disease in a human, the method comprising administering to a subject in need thereof a lyophilized formulation of the invention.

In yet another aspect, the present invention also relates to a lyophilized formulation of the present invention for use in the prevention or treatment of an autoimmune-related disease in a human.

In one embodiment, the disease includes rheumatoid arthritis, ankylosing spondylitis, crohn's disease, ulcerative colitis, psoriatic arthritis, plaque psoriasis, and the like.

Detailed Description

General definitions and terms

Unless otherwise indicated, the terms and phrases used herein have the meanings set forth below. No particular term or phrase is to be construed as critical or unclear unless otherwise specifically defined, but rather construed according to meanings commonly understood by those skilled in the art. When a trade name appears herein, it is intended to refer to its corresponding commodity or its active ingredient.

The terms "about" and "approximately," when used in conjunction with a numerical variable, generally mean that the value of the variable and all values of the variable are within experimental error (e.g., within 95% confidence interval for the mean) or within ± 10% of the specified value, or more.

The expressions "comprising" or similar expressions "including", "containing" and "having" and the like which are synonymous are open-ended and do not exclude additional, unrecited elements, steps or components. The expression "consisting of …" excludes any element, step or ingredient not specified. The expression "consisting essentially of …" means that the scope is limited to the specified elements, steps or components, plus optional elements, steps or components that do not materially affect the basic and novel characteristics of the claimed subject matter. It is to be understood that the expression "comprising" covers the expressions "consisting essentially of …" and "consisting of …".

The terms "optionally" or "optionally" mean that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, the composition optionally comprising an antioxidant encompasses situations where an antioxidant is included or not included.

Percentages, parts, etc. herein are by weight unless otherwise indicated.

The term "histidine buffer" or "histidine buffer system" denotes a buffer system obtained by combining histidine with an optionally present acid for adjusting the pH, such as hydrochloric acid, acetic acid, sulfuric acid, and the like. In characterizing the histidine buffer system in a formulation, it is common to use the sum of the concentrations of histidine therein, including free and ionized forms thereof. Examples of histidine buffers include, but are not limited to, histidine/hydrochloric acid, histidine/acetic acid, histidine/sulfuric acid, and the like. The preferred histidine buffer of the present invention is a histidine/hydrochloric acid buffer.

The term "carbohydrate" includes monosaccharides, disaccharides, oligosaccharides and polysaccharides. Examples of carbohydrates include, but are not limited to, fructose, glucose, sucrose, trehalose, mannose, lactose, mannose, maltose, fructose, sorbose, dextran, dextrin, cyclodextrin, hydroxyethyl starch, or combinations thereof.

The term "amino acid" includes amino acids and/or pharmaceutically acceptable salts thereof. Examples of amino acids include, but are not limited to, alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, or combinations thereof.

"pharmaceutically acceptable salt" refers to a salt with a pharmaceutically acceptable non-toxic base or acid, including inorganic or organic bases and inorganic or organic acids. For example, salts with the following inorganic acids: such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, or nitric acid; or a salt with: such as formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, caproic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, nicotinic acid, pamoic acid, picric acid, dodecylsulfuric acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, alginic acid, maleic acid, fumaric acid, D-gluconic acid, mandelic acid, or ascorbic acid.

The term "lyophilization" refers to a drying technique in which a solution formulation is frozen at a relatively low temperature to a solid state, then the water therein is sublimated under vacuum directly to a gaseous state without passing through the liquid state, and finally the material is dehydrated. Lyophilization includes, but is not limited to, freezing, primary drying, and secondary drying.

In the present invention, a liquid composition is first prepared and then lyophilized to obtain a lyophilized formulation of the present invention. Accordingly, such liquid compositions are also referred to herein as "stock solutions". The term refers to the form of the lyophilized formulation prior to lyophilization. The preparation is usually carried out with an aqueous solution (usually water or physiological saline, e.g., water for injection).

The term "reconstitution" means that the components of the lyophilized formulation are dissolved with an aqueous solution (such as, but not limited to, ethanol, water, aqueous dextrose, or mixtures thereof, e.g., water for injection, physiological saline, etc.) to obtain a reconstituted pharmaceutical formulation. Reconstitution of the lyophilized formulation of the present invention can be carried out using techniques generally known to those skilled in the art. Thus, a reconstituted formulation of the invention can be obtained after reconstitution of a lyophilized formulation of the invention. The term refers to the reconstituted form of the lyophilized formulation.

The term "osmotic pressure" refers herein to the osmotic pressure of a lyophilized formulation after reconstitution. The osmotic pressure should be suitable for subcutaneous administration. The osmotic pressure of the lyophilized formulation of the present invention after reconstitution can be about 250-530mOsmol/kg, such as about 250-430mOsmol/kg, about 300-400 mOsmol/kg.

Infliximab composition, freeze-dried preparation and re-dissolved preparation

The development of high concentration infliximab formulations has the following problems:

first, increasing the concentration of the mab during the formulation of the stock solution prior to lyophilization increases the viscosity, making preparation and administration difficult. The stability of the system is difficult to maintain by sacrificing the concentration of other ingredients such as structure protectors to reduce the viscosity. Freeze-dried preparations of the prior art such asThe concentration of infliximab in the preparation after reconstitution can be higher through the adjustment of water in the reconstitution process, but the concentration of other components in the preparation such as sucrose can be increased, and accordingly, the osmotic pressure of the system is increased, and the drug cannot be administered.

Secondly, many detection methods such as protein quantification and the like are disturbed at high concentrations.

In addition, the formulation is more susceptible to denaturation, aggregation and precipitation under high concentration conditions. The products of these degradations can have a significant impact on biopharmaceutical safety. In particular, some protein aggregates can stimulate the immune response of human body, and the curative effect of biological medicines can be reduced in the mild case and even death of patients can be caused in the severe case. For antibody drugs of the infliximab class, it is not only necessary to obtain a high purity product at the time of production, but also to maintain a stable structure during transportation, storage and use. Therefore, how to maintain the stability of the system and have viscosity and osmotic pressure suitable for processing and administration under the condition of keeping the concentration of infliximab higher is the key of solving the problems. Suitable osmotic pressures for subcutaneous administration are, for example, about 250-530mOsmol/kg, about 250-430mOsmol/kg, and about 300-400 mOsmol/kg.

The invention relates to a freeze-dried preparation which comprises infliximab, a histidine buffer system, a structure protective agent and a surfactant.

In one embodiment, the concentration of infliximab in the stock solution prior to lyophilization is about 10-80mg/ml, preferably about 25-60mg/ml, more preferably about 30-50mg/ml, and most preferably about 40 mg/ml.

In the formulations of the present invention, examples of histidine buffer systems include, but are not limited to, histidine/hydrochloric acid, histidine/acetic acid, histidine/sulfuric acid, and the like, preferably histidine/hydrochloric acid buffer. In one embodiment, the concentration of histidine in the histidine buffer system in the stock solution prior to lyophilization is about 1-20mM, preferably about 3-10mM, more preferably about 5 mM.

The pharmaceutical formulation of the present invention may comprise a structure protecting agent. The structure protective agent can be used to stabilize the structure of the active ingredient in the pharmaceutical composition, providing protective properties at ambient temperature and in lyophilized state. Carbohydrates and amino acids are commonly used stabilizers for protein drugs. It also acts as a cryoprotectant and a lyoprotectant, protecting proteins from possible denaturation during lyophilization. In one embodiment, the structure protectant is a carbohydrate, amino acid, or combination thereof. In a preferred embodiment, the structure-protecting agent is selected from the group consisting of sucrose, trehalose, arginine hydrochloride, and combinations thereof, more preferably sucrose. Too low a concentration of the structure protectant may reduce the stability of the formulation. In a preferred embodiment, the concentration of the structure protective agent in the stock solution prior to lyophilization is from about 5 to 100mg/ml, preferably from about 5 to 75mg/ml, more preferably from about 5 to 50mg/ml, e.g., 10, 15, 20, 25, 30mg/ml, most preferably about 25 mg/ml.

The pharmaceutical formulation of the present invention may also comprise a surfactant. Surfactants are substances that alter (typically lower) the surface tension of a liquid or the interfacial tension between two phases. The surfactant has amphipathy and contains a hydrophilic group and a lipophilic group. Examples of surfactants include, but are not limited to, polysorbates, poloxamers, triton, sodium lauryl sulfate, sodium octyl glycoside, lauryl-sulfobetaine, polyethylene glycol, polypropylene glycol, or mixtures thereof. Examples of polysorbates are polysorbate 20, polysorbate 21, polysorbate 40, polysorbate 60, polysorbate 61, polysorbate 65, polysorbate 80, polysorbate 81, polysorbate 85 or mixtures thereof, preferably polysorbate 80. The concentration of surfactant in the stock solution prior to lyophilization is about 0.001-1mg/ml, preferably about 0.01-0.4mg/ml, more preferably about 0.025-0.1 mg/ml.

The pharmaceutical formulations of the present invention may optionally include antioxidants, which may inhibit oxidation of the pharmaceutical formulation, improve the stability of the drug and help prolong shelf life. Examples of antioxidants include, but are not limited to, ascorbic acid, tryptophan, methionine, glutathione, sodium thiosulfate, catalase, or combinations thereof.

The pharmaceutical formulations of the present invention may optionally include a preservative that inhibits the growth and reproduction of microorganisms, including, but not limited to, m-cresol, phenol, benzyl alcohol, benzalkonium chloride, phenoxyethanol, methylparaben, or a combination thereof.

The pharmaceutical formulations of the present invention may also contain one or more other ingredients known to those skilled in the art, such as the isotonic agent sodium chloride, the metal chelating agent EDTA and the like.

The choice of the pH is critical for the stability of the formulation and should be such that the corresponding active substance remains stable. Furthermore, the pH should be selected such that the other components of the stock solution retain their physical and chemical properties, e.g. do not degrade, prior to lyophilization. As a preferred embodiment, a suitable pH is from about 4.0 to about 8.0, preferably from about 4.5 to about 6.0, more preferably from about 5.0 to about 5.4, e.g. 5.2.

The lyophilized formulation of the present invention may be reconstituted to obtain a reconstituted formulation of the present invention.

The concentration of infliximab in the preparation after reconstitution is about 100-200mg/ml, preferably about 130-190mg/ml, more preferably about 140-170mg/ml and about 150-160 mg/ml.

The reconstituted formulation of the invention has at least one of the following properties:

1) viscosity: about 40cP or less, e.g., about 30cP or less, about 20cP or less, about 10cP or less, or about 5cP or less; and

2) osmotic pressure: the osmotic pressure is suitable for subcutaneous administration, or the osmotic pressure is about 250-530mOsmol/kg, such as about 250-430mOsmol/kg, about 300-400 mOsmol/kg.

Advantageous effects

The infliximab lyophilized preparation can be stably stored for a long time in a lyophilized state, can realize high concentration and low viscosity of the infliximab in use after reconstitution, and is suitable for subcutaneous administration. The stock solution, the freeze-dried preparation and the reconstituted preparation solve the problems of high viscosity and poor stability easily existing when the infliximab has high concentration, and have very wide market application prospect.

Examples

The present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the present invention is not limited by these examples.

Preparation of

According to the design scheme of the freeze-drying preparation, the using amount is calculated according to the concentration, the volume and the component concentration of the infliximab stock solution, and the injection water is used for preparing the drug solution to be freeze-dried. The fractions were mixed well and sterilized by 0.22 μm Millipore sterile filtration. The suspension was dispensed into 2ml western (West Pharmaceutical) penicillin bottles and visually inspected to give all products a clear or pale yellow appearance with clear liquid and no visible foreign matter. The product was lyophilized, the lyophilization process is shown in table 1. Filling or freeze-drying, and then adding a coated rubber plug and an aluminum plastic cover.

TABLE 1 Freeze-drying process of infliximab

The lyophilized formulation was subjected to high temperature and light tests, respectively.

High-temperature test: placing the sample in a thermostat with the temperature of 40 +/-2 ℃ and sampling and detecting on the 14 th day and the 28 th day respectively;

and (3) illumination test: the samples were placed in a light box (5 ℃ C. + -3 ℃ C., 4500 Lx. + -. 500Lx) and sampled after 10 days.

Reagent and detection instrument

Reagent:

infliximab, available from Zhejiang Haizian drug industry Co., Ltd

Histidine from JT Baker

Sucrose, purchased from Merck KGaA

Trehalose from Japanese forest

Arginine hydrochloride from JT Baker

Polysorbate 80 available from JT Baker

The instrument comprises the following steps:

agilent 1200 liquid chromatograph available from Agilent corporation, USA.

SEC-HPLC column: TSK-GEL G3000SWXL, 7.8X 300mm, manufactured by TOSOH corporation of Japan; mobile phase: 0.02mol/L sodium dihydrogen phosphate, 0.2mol/L sodium chloride buffer solution, 10% acetonitrile, pH 7.0; flow rate: 0.5 ml/min; column temperature: 30 ℃; operating time: 30 min; wavelength: 280nm

IEC-HPLC column: thermo MabPac-SCX-10BioLC^TM4X 250mm Analytical; detection wavelength: 214 nm; mobile phase A: 10mM sodium dihydrogen phosphate, pH 6.8; mobile phase B: 10mM sodium dihydrogen phosphate dihydrate, 0.1M sodium chloride, pH 6.8; flow rate: 0.8ml/min; column temperature: 30 ℃; sample introduction amount: 10 mu l of the mixture; gradient: 0min, 10% mobile phase B; 5min, 10% mobile phase B; 40min, 60% mobile phase B; 42min, 100% mobile phase B; 47min, 100% mobile phase B; 49min, 10% mobile phase B; 60min, 10% mobile phase B.

Broomfield DV2T viscometer: the viscosity measurements were made using methods described in the manufacturer's instructions, available from Broomfield, USA.

Vogel OM815 osmometer: purchased from LOSER, germany, the method of use of the osmolarity measurement was performed according to the manufacturer's instructions.

Example 1 Effect of different Structure protective Agents on the stability of infliximab

Each stock solution (the initial concentration of the monoclonal antibody is 40.0mg/ml) was prepared according to Table 2, and trehalose, sucrose, arginine hydrochloride and combinations thereof were selected as protecting agents. The stock solution was lyophilized and the lyophilized preparation was subjected to high temperature and light tests.

TABLE 2 content of each component in stock solution

After the test, the polymer content of the lyophilized preparation was measured by size exclusion chromatography (SEC-HPLC) and the charge isomeric main peak content was measured by ion exchange chromatography (IEC-HPLC), and the results are shown in tables 3 and 4. The polymer content is generally used to reflect the physical stability of the lyophilized formulation, and the charge isomerization main peak content is generally used to reflect the chemical stability of the lyophilized formulation.

From tables 3 and 4, it can be seen that the lyophilized preparation has increased polymer content and decreased charge isomerization main peak content after high temperature test. All freeze-dried preparations have small increase of polymer content and low decrease of main charge isomerization peak content after 10 days of illumination, namely, have relatively good physical and chemical stability. After the freeze-dried preparation is placed at the high temperature of 40 ℃ for 14 days and 28 days, the preparation 2, namely the freeze-dried preparation adopting the sucrose as the protective agent, has relatively low polymer content and relatively high charge isomerization main peak content, namely has better physical and chemical stability.

TABLE 3 Polymer content (%)

Serial number	At 0 time	14 days at 40 DEG C	28 days at 40 DEG C	Illuminating for 10 days
					Preparation 1	0.92±0.03	2.50±0.15	3.23±0.06	1.06±0.05
Preparation 2	0.91±0.00	1.88±0.09	2.52±0.03	1.01±0.01
					Preparation 3	0.99±0.01	3.43±0.09	4.45±0.04	1.31±0.02
Preparation 4	0.98±0.01	2.64±0.02	3.57±0.04	1.17±0.04
					Preparation 5	0.96±0.02	2.34±0.02	3.12±0.02	1.13±0.05
Preparation 6	0.95±0.05	2.49±0.21	3.41±0.03	1.12±0.03
					Preparation 7	0.90±0.03	2.16±0.05	2.89±0.05	1.08±0.01

TABLE 4 Charge isomerization main peak content (%)

Serial number	At 0 time	14 days at 40 DEG C	28 days at 40 DEG C	Illuminating for 10 days
					Preparation 1	68.63±0.38	64.17±1.80	62.30±0.15	67.24±0.21
Preparation 2	69.13±0.79	64.92±0.26	64.86±0.37	67.75±0.82
					Preparation 3	69.06±0.35	62.75±0.38	60.19±0.92	67.35±0.22
Preparation 4	69.31±0.31	63.79±0.67	62.96±0.60	67.49±0.15
					Preparation 5	69.65±0.54	64.14±0.86	63.30±0.81	67.10±0.27
Preparation 6	69.24±0.62	63.88±0.90	62.95±1.03	67.29±0.50
					Preparation 7	69.36±0.40	63.22±0.99	62.98±1.23	67.00±0.39

The lyophilized preparation was reconstituted with a small amount of water (1ml loading, reconstituted with 0.22ml water) and the concentration, viscosity and osmotic pressure of the mabs were measured, and the results are shown in table 5. The single antibody content in the preparation after redissolution is 136.20-143.89mg/ml, and the viscosity and osmotic pressure are suitable for subcutaneous administration.

TABLE 5 viscosity and osmotic pressure of the reconstituted formulation

Serial number	Infliximab (mg/ml)	Viscosity (CP)	Osmolarity (mOsmol/kg)
				Preparation 1	143.89	22.93	475
Preparation 2	143.76	23.91	451
				Preparation 3	143.76	34.56	397
Preparation 4	138.79	24.77	424
				Preparation 5	139.82	24.65	421
Preparation 6	136.20	22.5	438
				Preparation 7	139.19	22.81	439

Example 2 Effect of different concentrations of surfactant on the stability of infliximab

Each stock solution (original liquid concentration of infliximab 40mg/ml) was prepared as in table 6, and lyophilized, and the lyophilized preparation was subjected to high temperature and light test.

TABLE 6 content of each component in stock solution

The polymer content of the lyophilized preparation after the test was measured by size exclusion chromatography (SEC-HPLC) and the charge isomeric main peak content was measured by ion exchange chromatography (IEC-HPLC), and the results are shown in tables 7 and 8.

TABLE 7 Polymer content (%)

Serial number	At 0 time	14 days at 40 DEG C	28 days at 40 DEG C	Illuminating for 10 days
					Preparation 8	0.92±0.03	2.50±0.15	3.23±0.06	1.06±0.05
Preparation 9	0.92±0.02	2.47±0.06	3.37±0.02	1.12±0.01

TABLE 8 Charge isomerization main peak content (%)

Serial number	At 0 time	14 days at 40 DEG C	28 days at 40 DEG C	Illuminating for 10 days
					Preparation 8	68.63±0.38	64.17±1.80	62.30±0.15	67.24±0.21
Preparation 9	68.42±0.15	62.63±0.01	61.93±0.05	66.63±0.03

It can be seen from table 7 that different concentrations of surfactant had no significant effect on the increase in the multimeric content of infliximab. However, after the test at 40 ℃, the content reduction value of the main peak of the charge isomerism of the freeze-dried preparation containing 0.025mg/ml of polysorbate 80 is less than that of the freeze-dried preparation containing 0.1mg/ml of polysorbate 80, namely the chemical stability of the freeze-dried preparation is better when the content of polysorbate 80 is 0.025mg/ml under the high-temperature condition. In addition, the freeze-dried preparation containing 0.025mg/ml and 0.1mg/ml of polysorbate 80 has better charge isomerization main peak content maintenance, namely better chemical stability under the illumination condition.

The concentration, viscosity and osmotic pressure of the monoclonal antibody were measured after reconstitution of the lyophilized preparation with a small amount of water (1ml loading, reconstituted with 0.22ml water), and the results are shown in Table 9, wherein the content of the monoclonal antibody in the reconstituted preparation was between 141.57-143.89mg/ml, and both had a viscosity and osmotic pressure suitable for subcutaneous administration.

TABLE 9 viscosity and osmotic pressure of the formulations after reconstitution

Serial number	Infliximab (mg/ml)	Viscosity (CP)	Osmolarity (mOsmol/kg)
				Preparation 8	143.89	22.93	475
Preparation 9	141.57	22.83	471

Example 3 Effect of different mAb and Structure protectant concentrations and combinations with pH on stability of infliximab

Each stock solution was prepared as in table 10, lyophilized, and the lyophilized preparation was subjected to high temperature and light test.

TABLE 10 content of the components in the stock solution

The lyophilized preparation was subjected to high temperature and light test, and the polymer content of the lyophilized preparation was measured by size exclusion chromatography (SEC-HPLC) and the charge isomeric main peak content was measured by ion exchange chromatography (IEC-HPLC), and the results are shown in tables 11 and 12.

From Table 11, it can be seen that increasing the concentration of mAb increases the amount of polymer in the lyophilized formulation, but has no significant effect on the charge isomerization peak content. The increase of the sucrose concentration is beneficial to the reduction of the polymer content, but has no significant influence on the content of the main charge isomerization peak. pH 5.0 or 5.4 had no significant effect on the charge isomerization main peak content, but the polymer content was higher at pH 5.4 compared to pH 5.0 (formulations 10, 11 compared to or formulations 12, 13 compared to). That is, variations in the concentration of the monoclonal antibody, sucrose concentration and pH within a certain range may affect the stability, especially the physical stability, of the lyophilized preparation to some extent. The improvement of the concentration of the monoclonal antibody can reduce the physical stability of the freeze-dried preparation, and the improvement of the concentration of the sucrose can improve the physical stability of the freeze-dried preparation.

TABLE 11 Polymer content (%)

Serial number	At 0 time	14 days at 40 DEG C	28 days at 40 DEG C	Illuminating for 10 days
					Preparation 10	0.74±0.02	2.10±0.08	2.46±0.03	0.94±0.03
Preparation 11	0.83±0.01	2.30±0.10	2.48±0.00	1.01±0.01
					Preparation 12	0.80±0.00	2.68±0.02	2.96±0.03	1.02±0.02
Preparation 13	0.88±0.01	2.74±0.03	3.09±0.03	1.13±0.05
					Preparation 14	0.81±0.01	1.61±0.15	2.78±0.03	0.99±0.01
Formulation 15	0.88±0.01	2.42±0.06	3.56±0.08	1.13±0.01
					Preparation 16	0.81±0.00	1.99±0.01	2.80±0.03	1.00±0.01

TABLE 12 Charge isomerization main peak content (%)

Serial number	At 0 time	14 days at 40 DEG C	28 days at 40 DEG C	Illuminating for 10 days
					Preparation 10	72.65±1.71	65.86±0.66	64.65±0.71	70.40±0.98
Preparation 11	71.67±0.71	66.16±0.57	64.45±0.07	69.82±0.84
					Preparation 12	72.41±0.94	66.42±2.78	63.86±0.21	69.30±0.21
Preparation 13	71.97±0.55	65.70±1.43	64.13	69.73±0.98
					Preparation 14	70.41±0.44	66.57±0.18	64.47±0.33	70.25±1.14
Formulation 15	70.14±0.65	65.14±0.36	62.85±0.33	68.93±0.20
					Preparation 16	70.32±0.26	65.74±0.34	63.09±0.11	69.10±1.07

Less water was added (formulations 10-11 were reconstituted with 0.2ml water for 1ml, formulation 12 was reconstituted with 0.2ml and 0.25ml water for 1ml, formulation 13 was reconstituted with 0.25ml water for 1ml, and formulations 14-16 were reconstituted with 0.55ml water for 2.5 ml) to give a concentration of the mAb of about 120-190mg/ml in the reconstituted formulations, the resulting mAb concentrations, osmotic pressures and viscosity values are shown in Table 13, and the viscosity of each reconstituted formulation was low and had an osmotic pressure suitable for subcutaneous administration.

TABLE 13 viscosity and osmotic pressure of the formulations after reconstitution

	Infliximab (mg/ml)	Viscosity (cP)	Osmolarity (mOsmol/kg)
				Preparation 10	155.43	21.87	390
Preparation 11	157.65	21.03	502
				Formulation 12 (reconstituted with 0.2 ml)	186.11	27.04	534
Formulation 12 (reconstituted with 0.25 ml)	157.72	23.56	452
				Formulation 13 (reconstituted with 0.25 ml)	161.03	25.17	491
Preparation 14	142.15	14.48	392
				Formulation 15	165.50	28.27	419
Preparation 16	138.30	12.78	377

Example 4 feasibility analysis of the preparation of high concentrations of currently marketed infliximab formulations

Every bottle of the currently marketed infliximab lyophilized preparation contains 100mg of infliximab, 500mg of sucrose, 800.5 mg of polysorbate, 2.2mg of sodium dihydrogen phosphate and 4.86mg of disodium hydrogen phosphate. The stock solution is prepared according to the proportion and then is subjected to freeze-drying treatment, and after the stock solution is re-dissolved by using 10ml and 2.5ml of water respectively, the concentration, the viscosity and the osmotic pressure of the infliximab are measured. As can be seen from Table 14, the protein content was only about 9mg/ml when reconstituted with 10ml water, while the protein content increased to 31mg/ml when reconstituted with 2.5ml water, at which point the viscosity of the solution remained small, but the osmotic pressure (689.5mOsmol/kg) was well in excess of the range tolerated by humans, indicating that the preparation in the market place is not feasible for preparing highly concentrated formulations suitable for subcutaneous administration.

TABLE 14. infliximab concentration, viscosity and osmotic pressure in the currently marketed infliximab formulation (100mg standard) after reconstitution with different volumes of water

Volume of dilution	Infliximab (mg/ml)	Viscosity (cP)	Osmolarity (mOsmol/kg)
				10ml	8.91±0.08	1.34±0.03	156.5±0.5
2.5ml	30.71±2.42	4.61±0.04	689.5±7.5

Example 5 preparation of stock solutions of infliximab at different pH

Formulations 18-21 were designed and formulated, each containing a different pH (table 15).

TABLE 15 Infinimab stock solutions at different pH (before lyophilization)

Example 6 preparation of stock solutions of infliximab at different sucrose concentrations

Formulations 22-25 were designed and formulated to contain different sucrose concentrations, respectively (table 16).

TABLE 16 Infinimab stock solutions (before lyophilization) at different sucrose concentrations

Example 7 preparation of stock solutions of infliximab at different trehalose concentrations

Formulations 26-29 were designed and formulated to contain different trehalose concentrations, respectively (table 17).

TABLE 17 stock solutions of infliximab at different trehalose concentrations (before lyophilization)

Unless otherwise indicated, all numbers expressing quantities of ingredients, cell culture, processing conditions, and so forth used in the specification (including the claims) are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters are approximations and may vary depending upon the desired properties sought to be obtained by the present invention. The term "at least" preceding a series of elements is to be understood as referring to each element in the series, unless otherwise indicated. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The appended claims are intended to cover such equivalents.

It will be apparent to those skilled in the art that many modifications and variations of the present invention can be made without departing from its spirit and scope. The specific embodiments described herein are provided by way of example only and are not meant to be limiting in any way. The true scope and spirit of the invention is indicated by the appended claims, and the specification and examples are exemplary only.

Claims (10)

1. A lyophilized formulation comprising infliximab, a histidine buffer system, a structure protecting agent and a surfactant, wherein,

before freeze-drying, the concentration of infliximab in the preparation is 10-80mg/ml, the concentration of histidine in a histidine buffer system is 1-20mM, the concentration of a structure protective agent is 5-100mg/ml, and the concentration of a surfactant is 0.001-1 mg/ml; and is

The concentration of infliximab in the formulation after reconstitution was 100-200 mg/ml.

2. Lyophilized formulation according to claim 1, wherein the concentration of infliximab in the formulation prior to lyophilization is 25-60mg/ml, preferably 30-50mg/ml, more preferably 40 mg/ml.

3. Lyophilized formulation according to claim 1 or 2, wherein histidine is present in the formulation at a concentration of 3-10mM, preferably 5mM, prior to lyophilization.

4. Lyophilized formulation according to any of claims 1 to 3, wherein the structure protectant is selected from the group consisting of a carbohydrate, an amino acid and combinations thereof, preferably from the group consisting of sucrose, trehalose and combinations thereof, more preferably sucrose, and

the concentration of the structure-protecting agent prior to lyophilization is 5-75mg/ml, preferably 5-50mg/ml, more preferably 25 mg/ml.

5. Lyophilized formulation according to any of claims 1 to 4, wherein the surfactant is selected from polysorbates, poloxamers, triton, sodium lauryl sulfate, sodium octyl glycoside, lauryl-sulphobetaine, polyethylene glycol, polypropylene glycol or mixtures thereof, preferably polysorbate 80, and

the surfactant concentration is 0.01-0.4mg/ml, preferably 0.025-0.1mg/ml, prior to lyophilization.

6. Lyophilized formulation according to any of claims 1 to 5, wherein the pH of the formulation prior to lyophilization is 4.0-8.0, preferably 4.5-6.0, more preferably 5.0-5.4.

7. The lyophilized formulation according to any one of claims 1 to 6, wherein the formulation has at least one of the following properties after reconstitution:

a viscosity of 40cP or less, and

the osmotic pressure was 250-530 mOsmol/kg.

8. The lyophilized formulation according to any one of claims 1 to 7, wherein the concentration of infliximab in the formulation after reconstitution is 190mg/ml, more preferably 140 mg/ml, 170mg/ml, 150 mg/ml.

9. Use of the lyophilized formulation of any one of claims 1-8 in the manufacture of a medicament for the prevention or treatment of an autoimmune-related disease in a human.

10. Use according to claim 9, characterized in that said diseases comprise rheumatoid arthritis, ankylosing spondylitis, crohn's disease, ulcerative colitis, psoriatic arthritis and plaque psoriasis.