CN102988984B - Aqueous drug preparation of anti-TNF (tumor necrosis factor)-alpha human monoclonal antibody for strengthening stability - Google Patents
Aqueous drug preparation of anti-TNF (tumor necrosis factor)-alpha human monoclonal antibody for strengthening stability Download PDFInfo
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Abstract
The invention provides a new liquid aqueous drug preparation of an anti-TNF (tumor necrosis factor)-alpha human monoclonal antibody for strengthening the stability, wherein antibody solution is stabilized and osmotic pressure is regulated by sorbitol; disodium hydrogen phosphate/citric acid monohydrate or sodium acetate/acetic acid is taken as a buffer agent, and preferably, the disodium hydrogen phosphate/citric acid monohydrate is taken as the buffer agent; and tween-20 is taken as a surfactant, so that antibody preparation is stable, and long in retention time, meanwhile, preparation components are simplified, and the preparation is prepared simply and conveniently.
Description
Technical field
The invention belongs to biological technical field, relate to the liquid aqueous pharmaceutical preparation of human monoclonal antibodies's medicine of enhanced stability; Relate to the purposes applying liquid aqueous pharmaceutical preparation for treating relevant disease of the present invention.
Background technology
Huamn tumor necrosis factory alpha (TNF-α) has been proved relevant to various diseases, as the autoimmune disease such as rheumatoid arthritis, psoriasis.Multiple anti-tnf-alpha medicine is used for the treatment of the relevant autoimmune class disease of TNF-α, as adalimumab (Adalimumab, trade name Humira, Abbott) by U.S. FDA approval.
The adalimumab being applied to clinical treatment is liquid infusion preparation, and interior packaging material is precharging type syringe.Its preparation prescription comprises sodium chloride, sodium hydrogen phosphate, sodium dihydrogen phosphate, citric acid, sodium citrate, mannitol, Sorbitan polyoxyethylenated alcohol monoleate (Tween 80), water for injection (Humira description).Although said preparation existence and stability, but still be not enough to long-time stable antibody, estimate that finished product should not be placed for more time.This may be antioxidant because said preparation uses and osmotic pressure regulator---mannitol can not play excellent Stabilization.In addition, the surfactant that existing adalimumab uses is tween 80, although it can suppress and/or stop protein molecular at water with bad results such as being polymerized on Air Interface, interior packaging material contact surface, absorption thus stabilization of antibodies, but himself also can there is unsaturated bond oxidation and ester linkage hydrolyzing to a certain extent thus generation acid, aldehyde, hydrogen peroxide, thus have impact on the stability of antibody.Meanwhile, its prescription buffer solution composition redundancy is complicated, not only increases product cost, and makes the formulation operations of preparation loaded down with trivial details.
Summary of the invention
In order to solve the problem, the invention provides a kind of liquid aqueous pharmaceutical preparation of new anti-tnf-alpha human monoclonal antibodies, make antibody preparation more stable, the holding time is longer; Simplify formulation ingredients simultaneously, make the preparation of preparation more simple and convenient.
Aqueous medicament preparations provided by the invention comprises:
(a) anti-tnf-alpha human monoclonal antibodies;
(b) polyhydric alcohol;
(c) buffer agent;
(d) surfactant;
(e) sodium chloride;
(f) water;
Wherein, described polyhydric alcohol is sorbitol; Described buffer agent is sodium hydrogen phosphate/monohydrate potassium or sodium acetate/acetic acid, preferably phosphoric acid disodium hydrogen/monohydrate potassium; Described surfactant is polyoxyethylene sorbitan monolaurate (tween 20).
The pH of liquid aqueous pharmaceutical preparation of the present invention is 4-8, and conductance is 8-18mS/cm, and osmotic pressure is 250-400mOsm/kg.
Liquid aqueous pharmaceutical preparation of the present invention, it comprises 10-80g/L restructuring anti-tnf-alpha human monoclonal antibodies, 10-20g/L sorbitol, 0.01-3g/L tween 20,2-14g/L sodium chloride, and the buffer agent of sodium hydrogen phosphate/monohydrate potassium, its pH is 4-8, preferred pH 5-6, more preferably pH 5.0-5.4.
Liquid aqueous pharmaceutical preparation of the present invention, it comprises 10-80g/L restructuring anti-tnf-alpha human monoclonal antibodies, 10-20g/L sorbitol, 0.01-3g/L tween 20,2-14g/L sodium chloride, and the buffer agent of sodium acetate/acetic acid, and its pH is 4-8, preferred pH 5-6, more preferably pH 5.0-5.4.
Detailed description of the invention
For the ease of understanding the present invention, some term of the present invention is defined:
Term " pharmaceutical preparation " represents that its form makes the biologic activity of active component clearly effective, and it does not comprise other composition obviously poisonous to the experimenter using described preparation.
" stable " preparation is preparation when preserving, and antibody wherein can keep its physical stability and/or chemical stability and/or biological stability substantially.
" buffer solution ": when adding a certain amount of bronsted lowry acids and bases bronsted lowry in some solution, have the effect hindering pH value of solution change, be called cushioning effect, such solution is called buffer solution.
" buffer agent ": solution ph can be made to maintain substantially invariable material within the specific limits.
Within the scope of the invention, " the treatment effective dose " of antibody or " effective dose " represents the effective dose in prevention or disease therapy, and for the treatment of described disease, described antibody is effective.
Term " humanTNF-α " represents human cell factor, and it exists with the secreted form of 17kD and 26kD film associated forms, and biologic activity form comprises the trimer of covalently bound 17kD molecule.Its concrete structure sees Pennica, D., waits (1984) Nature 312:724-729; Davis, J.M., wait (1987) Biochemistry26:1322-1326; And Jones, E.Y., wait (1989) Nature 338:225-228.
Term " recombinant human antibody " is intended to the people's antibody comprising recombination method preparation, express, produce or be separated.
Stock solution: not containing the aqueous medicament preparations of tween.
Finished product: the aqueous medicament preparations adding tween according to quantity.
SEC-HPLC: Size Exclusion High Performance liquid chromatography.
CEX-HPLC (Acidic, Main, Basic): cation exchange-high performance liquid chromatography (acid peak, main peak, alkaline peak).
NR-CE-SDS: non-reduced high performance capillary electrophoresis.
Potency: biologic activity.
The invention provides aqueous medicament preparations, comprising:
(a) anti-tnf-alpha human monoclonal antibodies;
(b) polyhydric alcohol;
(c) buffer agent;
(d) surfactant;
(e) sodium chloride;
(f) water;
Wherein, described polyhydric alcohol is sorbitol; Described buffer agent is sodium hydrogen phosphate/monohydrate potassium or sodium acetate/acetic acid, preferably phosphoric acid disodium hydrogen/monohydrate potassium; Described surfactant is polyoxyethylene sorbitan monolaurate (tween 20).
The pH of liquid aqueous pharmaceutical preparation of the present invention is 4-8, and conductance is 8-18mS/cm, and osmotic pressure is 250-400mOsm/kg.
Aqueous medicament preparations of the present invention, the restructuring anti-tnf-alpha human monoclonal antibodies containing treatment effective dose, its concentration is 10-80g/L, preferred 20-70g/L, particularly preferably 50g/L.
Known polyhydric alcohol can play antioxidation, therefore can stabilization of antibodies.The present invention uses sorbitol, and concentration is 10-20g/L, preferred 12-18g/L, most preferably 15g/L, in embodiments of the invention, adopts in sorbitol more existing adalimumab formulation and uses mannitol can better stabilization of antibodies.
Aqueous medicament preparations pH of the present invention is 4-8, preferred 5-6, particularly preferably 5.2.Buffer agent sodium hydrogen phosphate/monohydrate potassium of the present invention or sodium acetate/acetic acid, be preferably sodium hydrogen phosphate/monohydrate potassium, the molar concentration scope of the buffer solution of its pH 5.2 is 10-40mM, preferred 21.5mM, i.e. sodium hydrogen phosphate 2g/L, monohydrate potassium 1.54g/L, and regulate final preparation pH 5.2 with sodium hydroxide.Use this buffer agent can make the buffer solution with each ion same concentrations in original adalimumab formulation, ensure that the change of phosphate radical, citrate, sodium ion is all within 5%, simplifies Formulation process, has saved cost.Buffer agent in one embodiment of the present of invention is sodium acetate/acetic acid, and the molar concentration scope of its pH 5.2 buffer solution is 10-40mM, preferred 18.5mM, i.e. sodium acetate 1.23g/L, acetic acid 0.2mL/L, and regulates final preparation pH 5.2 with acetic acid.
Inhibitory action is played in the polymerization of polyoxyethylene sorbitan fatty acid ester (tween) antagonist, and the tween 20 (polyoxyethylene sorbitan monolaurate) that the present invention uses unsaturated bond less is to reduce autoxidation thus the adverse effect of reduction antagonist stability.In an embodiment, the more original adalimumab formulation of tween 20 is adopted to adopt tween 80 (polyoxyethylene sorbitan monooleate dehydration) that pharmaceutical preparation can be made to have better stability.Especially, in stability (illumination experiment) embodiment, when using the buffer agent of sodium hydrogen phosphate/monohydrate potassium, tween 20 can significantly improve the stability of pharmaceutical preparation.Optional tween 20 concentration is 0.01-3g/L, preferred 1g/L.
Sodium chloride is osmotic pressure regulator, and in embodiment of the present invention, its concentration is 2-14g/L, preferred 3-12g/L, most preferably 6.16g/L.
The buffer solution of liquid aqueous pharmaceutical preparation of the present invention is sodium hydrogen phosphate/monohydrate potassium solution, pH 4-8, preferred pH 5-6, more preferably pH 5.0-5.4, conductance is 8-18mS/cm, preferred 11.1-14.1mS/cm, osmotic pressure is 250-400mOsm/kg, preferred 280-360mOsm/kg.
The buffer solution of liquid aqueous pharmaceutical preparation of the present invention is sodium acetate/acetic acid solution, and pH is 4-8, preferred pH 5-6, more preferably pH 5.0-5.4, conductance is 8-18mS/cm, preferred 9.6-13.0mS/cm, osmotic pressure is 250-400mOsm/kg, preferred 270-350mOsm/kg.
The invention provides a kind of liquid aqueous pharmaceutical preparation of new anti-tnf-alpha human monoclonal antibodies, make antibody preparation more stable, the holding time is longer; Simplify formulation ingredients simultaneously, make the preparation of preparation more simple and convenient.
Describe the present invention in detail referring to embodiment, should be understood that following embodiment is intended to illustrate, the present invention is not construed as limiting.
The IgG1 recombinant antibodies D2E7 (see Chinese patent ZL97193635.8) that the anti-tnf-alpha human monoclonal antibodies that the present invention uses is prepared for Genor Biopharma Co., Ltd.; Water is the self-produced water for injection of Genor Biopharma Co., Ltd.; Sorbitol is purchased from Shijiazhuang City, Hebei Province timely snow pharmaceutical Co. Ltd, and it meets Chinese Pharmacopoeia pharmaceutical grade; Other reagent is all purchased from J.T.Baker company, and it meets American Pharmacopeia pharmaceutical grade.
It is below INSTRUMENT MODEL used in the present invention: balance: Sartorius TE4101; PH and electrical conductivity integrated instrument: METTLER TOLEDO SevenMulti; Osmotic tester: Model3250Osmometer (ADVANCED INSTRUMENTS, INC.); HPLC:Waters2695-2487, wherein, SEC-HPLC analytical column used is Tosoh TSKgel G3000SW; CEX-HPLC analytical column used is Tosoh TSKgel CM-STAT; It is Beckman PA800plus that NR-CE-SDS measures instrument.
The preparation of embodiment 1. buffer solution
1.1 preparation 8L sodium hydrogen phosphate/monohydrate potassium ultrafiltration buffer solution (ultrafiltration buffer solution A)
Take 16g sodium hydrogen phosphate, 12.32g monohydrate potassium, 120g sorbitol, 49.28g sodium chloride respectively, add to 7.9L with water for injection, regulate pH 5.2 until dissolving completely, after mix homogeneously with 1M sodium hydroxide, be settled to 8.0L for subsequent use.
1.2 preparation 8L sodium hydrogen phosphate/sodium dihydrogen phosphate and sodium citrate/citric acid ultrafiltration buffer solution (ultrafiltration buffer solution B)
Take 6.88g bis-hypophosphite monohydrate sodium dihydrogen, 12.24g bis-hypophosphite monohydrate disodium hydrogen, 2.4g sodium citrate, 10.4g monohydrate potassium, 96g mannitol, 49.28g sodium chloride respectively, 7.9L is added to water for injection, regulate pH 5.2 until dissolving completely, after mix homogeneously with 1M sodium hydroxide, be settled to 8.0L for subsequent use.
1.3 preparation 8L sodium acetate/acetic acid ultrafiltration buffer solution (ultrafiltration buffer solution C)
Take 9.84g sodium acetate, 120g sorbitol, 49.28g sodium chloride respectively, water for injection adds to 7.9L, until dissolving completely, after mix homogeneously with second acid for adjusting pH 5.2, is settled to 8.0L for subsequent use.
Embodiment 2. antibody concentration is the preparation of the ultrafiltration and concentration liquid of 51g/L
2.1 buffer solution is the preparation (ultrafiltration and concentration liquid A) of the ultrafiltration and concentration liquid of sodium hydrogen phosphate/monohydrate potassium solution
Ultrafiltration instrument VIVA Flow 200 (producer Sartorius) cleans by manufacturer's recommended step 0.2M sodium hydroxide, comprise cleaning ultrafilter membrane, ultrafiltration cup, pipeline, outlet etc., then clean to remove sodium hydroxide with the ultrafiltration buffer solution A in 0.5L embodiment 1.1.0.48L anti-tnf-alpha human monoclonal antibodies solution (antibody concentration 25g/L, total antibody 12g) is added in ultrafiltration cup, starts equal-volume with ultrafiltration buffer solution A and change liquid, change liquid and amass as 4.8L.After changing liquid, antibody-solutions is concentrated into about 0.2L from 0.48L, reclaims antibody-solutions after depolarization, the antibody-solutions volume reclaimed after measured is 0.204L, and concentration is 52g/L (meet that volume is about 0.2L, concentration be greater than 51g/L and require).Add 4mL ultrafiltration buffer solution A by reclaim after antibody-solutions be diluted to 51g/L, with aseptic apyrogenic 0.22 μm of aseptic filtration membrane filtration, obtain filtering solution Preservation in sterile condition.
2.2 buffer solution are the preparation (ultrafiltration and concentration liquid B) of the ultrafiltration and concentration liquid of sodium hydrogen phosphate/sodium dihydrogen phosphate and sodium citrate/citric acid solution
Ultrafiltration instrument VIVA Flow 200 basic operation with described in embodiment 2.1, then is cleaned with the ultrafiltration buffer solution B in 0.5L embodiment 1.2 to remove sodium hydroxide.0.48L anti-tnf-alpha human monoclonal antibodies solution (antibody concentration 25g/L, total antibody 12g) is added in ultrafiltration cup, starts equal-volume with ultrafiltration buffer solution B and change liquid, change liquid and amass as 4.8L.After changing liquid, antibody-solutions is concentrated into about 0.2L from 0.48L, reclaims antibody-solutions after depolarization, the antibody-solutions volume reclaimed after measured is 0.209L, and concentration is 52g/L (meet that volume is about 0.2L, concentration be greater than 51g/L and require).Add 4mL ultrafiltration buffer solution B by reclaim after antibody-solutions be diluted to 51g/L, with aseptic apyrogenic 0.22 μm of aseptic filtration membrane filtration, obtain filtering solution Preservation in sterile condition.
2.3 buffer solution is the preparation (ultrafiltration and concentration liquid C) of the ultrafiltration and concentration liquid of sodium acetate/acetic acid solution
Ultrafiltration instrument VIVA Flow 200 basic operation with described in embodiment 2.1, then is cleaned with the ultrafiltration buffer solution C in 0.5L embodiment 1.3 to remove sodium hydroxide.0.48L anti-tnf-alpha human monoclonal antibodies solution (antibody concentration 25g/L, total antibody 12g) is added in ultrafiltration cup, starts equal-volume with ultrafiltration buffer solution C and change liquid, change liquid and amass as 4.8L.After changing liquid, antibody-solutions is concentrated into about 0.2L from 0.48L, reclaims antibody-solutions after depolarization, the antibody-solutions volume reclaimed after measured is 0.195L, and concentration is 54g/L (meet that volume is about 0.2L, concentration be greater than 51g/L and require).Add 11mL ultrafiltration buffer solution C by reclaim after antibody-solutions be diluted to 51g/L, with aseptic apyrogenic 0.22 μm of aseptic filtration membrane filtration, obtain filtering solution Preservation in sterile condition.
The preparation of embodiment 3. stock solution
3.1 preparation 0.9mL/ cillin bottle stock solution A (buffer solution is sodium hydrogen phosphate/monohydrate potassium, not containing the preparation of tween)
Under aseptic condition, get the ultrafiltration and concentration liquid A of 50mL embodiment 2.1, add the ultrafiltration buffer solution A of the 1mL embodiment 1.1 with aseptic apyrogenic 0.22 μm of aseptic filtration membrane filtration, abundant mix homogeneously, is stock solution A.Under aseptic condition, be sub-packed in the aseptic apyrogenic liquid infusion agent cillin bottle of 2mL, loading amount 0.9mL/ bottle, subpackage 54, after the Zha Gai that jumps a queue, 2-8 DEG C keeps in Dark Place stand-by.
Situation of often propping up illustrates: often prop up loading amount 0.9mL, labelled amount 0.8mL, it is composed as follows:
3.2 preparation 0.9mL/ cillin bottle stock solution B (buffer solution is sodium hydrogen phosphate/sodium dihydrogen phosphate and sodium citrate/citric acid solution, not containing the preparation of tween 80)
Under aseptic condition, get the ultrafiltration and concentration liquid B of 50mL embodiment 2.2, add the ultrafiltration buffer solution B of the 1mL embodiment 1.2 with aseptic apyrogenic 0.22 μm of aseptic filtration membrane filtration, abundant mix homogeneously, is stock solution B.Under aseptic condition, be sub-packed in the aseptic apyrogenic liquid infusion agent cillin bottle of 2mL, loading amount 0.9mL/ bottle, subpackage 54, after the Zha Gai that jumps a queue, 2-8 DEG C keeps in Dark Place stand-by.
Situation of often propping up illustrates: often prop up loading amount 0.9mL, labelled amount 0.8mL, it is composed as follows:
3.3 preparation 0.9mL/ cillin bottle stock solution C (buffer solution is sodium acetate/acetic acid solution, not containing the preparation of tween)
Under aseptic condition, get the ultrafiltration and concentration liquid C of 50mL embodiment 2.3, add the ultrafiltration buffer solution C of the 1mL embodiment 1.3 with aseptic apyrogenic 0.22 μm of aseptic filtration membrane filtration, abundant mix homogeneously, is stock solution C.Under aseptic condition, be sub-packed in the aseptic apyrogenic liquid infusion agent cillin bottle of 2mL, loading amount 0.9mL/ bottle, subpackage 54, after the Zha Gai that jumps a queue, 2-8 DEG C keeps in Dark Place stand-by.
Situation of often propping up illustrates: often prop up loading amount 0.9mL, labelled amount 0.8mL, it is composed as follows:
The preparation of embodiment 4. finished product
4.1 preparation 0.9mL/ cillin bottle finished product A (buffer solution is sodium hydrogen phosphate/monohydrate potassium, the preparation containing tween 20)
Take 1.00g tween 20, with water for injection standardize solution in 19.6mL, with aseptic apyrogenic 0.22 μm of aseptic filtration membrane filtration, get the 10-19mL Preservation in sterile condition after filtration for subsequent use, be 51 × polysorbas20 mother solution.
Under aseptic condition, get the ultrafiltration and concentration liquid A of 50mL embodiment 2.1, add 1mL 51 × polysorbas20 mother solution, abundant mix homogeneously, be finished product A.Under aseptic condition, be sub-packed in the aseptic apyrogenic liquid infusion agent cillin bottle of 2mL, loading amount 0.9mL/ bottle, subpackage 54, after the Zha Gai that jumps a queue, 2-8 DEG C keeps in Dark Place stand-by.
Situation of often propping up illustrates: often prop up loading amount 0.9mL, labelled amount 0.8mL, it is composed as follows:
4.2 preparations 0.9mL/ cillin bottle finished product B (buffer solution is sodium hydrogen phosphate/sodium dihydrogen phosphate and sodium citrate/citric acid solution, the preparation containing tween 80)
Take 1.00g Tween 80, with water for injection standardize solution in 19.6mL, with aseptic apyrogenic 0.22 μm of aseptic filtration membrane filtration, get the 10-19mL Preservation in sterile condition after filtration for subsequent use, be 51 × tween 80 mother solution.
Under aseptic condition, get the ultrafiltration and concentration liquid B of 50mL embodiment 2.2, add 1mL 51 × tween 80 mother solution, abundant mix homogeneously, be finished product B.Under aseptic condition, be sub-packed in the aseptic apyrogenic liquid infusion agent cillin bottle of 2mL, loading amount 0.9mL/ bottle, subpackage 54, after the Zha Gai that jumps a queue, 2-8 DEG C keeps in Dark Place stand-by.
Situation of often propping up illustrates: often prop up loading amount 0.9mL, labelled amount 0.8mL, it is composed as follows:
4.3 preparation 0.9mL/ cillin bottle finished product C (buffer solution is sodium acetate/acetic acid solution, the preparation containing tween 20)
Under aseptic condition, get the ultrafiltration and concentration liquid C of 50mL embodiment 2.3, add 51 × tween 20 mother solution obtained in 1mL embodiment 4.1, abundant mix homogeneously, is finished product C.Under aseptic condition, be sub-packed in the aseptic apyrogenic liquid infusion agent cillin bottle of 2mL, loading amount 0.9mL/ bottle, subpackage 54, after the Zha Gai that jumps a queue, 2-8 DEG C keeps in Dark Place stand-by.
Situation of often propping up illustrates: often prop up loading amount 0.9mL, labelled amount 0.8mL, it is composed as follows:
Embodiment 5. study on the stability
5.15 ± 3 DEG C of long-time stability experiments and 25 ± 2 DEG C of Acceleration study
Get the 0.9mL/ cillin bottle stock solution A in above-described embodiment, B, C and each 12 of finished product A, B, C respectively, be placed in the Scientific Revco cold storage refrigerator of 5 ± 3 DEG C, estimate respectively at the 0th day, the 5th day, the 15th day, the 30th day, the 3rd month, the 6th month sample analysis, often kind of each sample point of sample puts 2; Get the 0.9mL/ cillin bottle stock solution A in above-described embodiment, B, C and each 12 of finished product A, B, C respectively, be placed in the Climacell 222 constant temperature and humidity incubator of 25 ± 2 DEG C, respectively at the 0th day, the 5th day, the 15th day, the 30th day, the 3rd month, the 6th month sample analysis, often kind of each sample point of sample puts 2.Analytical method is the outward appearance of naked eyes detection immediately after sampling, then carries out the detection of SEC-HPLC, CEX-HPLC, NR-CE-SDS and biologic activity.In above-mentioned experimental design, if the result of the 30th day, the 6th month meets expection, then need not analyze the sample point of the 15th day, the 3rd month.Experimental result in table 1 to table 7.
Table 1. the 0th day testing result
Table 2.5 ± 3 DEG C, the 5th day long-time stability experimental result
Table 3.5 ± 3 DEG C, the 30th day long-time stability experimental result
Table 4.5 ± 3 DEG C, the 6th month long-time stability experimental result
Table 5.25 ± 2 DEG C, the 5th day Acceleration study result
Table 6.25 ± 2 DEG C, the 30th day Acceleration study result
Table 7.25 ± 2 DEG C, the 6th month Acceleration study result
Experimental result shows, in 5 ± 3 DEG C of groups, stock solution A, B, C and finished product A, B, C the 5th day, the 30th day, the 6th month all testing result all with the 0th day no significant difference, the stability using buffer agent sodium hydrogen phosphate/monohydrate potassium and tween 20 and use buffer agent sodium acetate/acetic acid and tween 20 can reach identical with the agent of A Da wood monoclonal antibody liquid infusion.
In 25 ± 2 DEG C of groups, stock solution A, B, C and finished product A, B, C was at the 5th day, all testing results of the 30th day all with the 0th day no significant difference, but the testing result of 6th month shows difference (as shown in table 7): by comparing discovery, the SEC-HPLC purity of finished product A is than finished product B, C high about 1% and the change at main peak oxytropism peak are than finished product B, the little about 7-8% of C, this illustrates that finished product A is better than finished product B and C, further, finished product A, B, the SEC-HPLC purity of C is respectively higher than its stock solution A, B, the purity about 1% of C, and the change at the less main peak oxytropism peak of corresponding generation, illustrate that tween has the effect of obvious stabilization of antibodies, particularly prevent the polymerization of antibody molecule, find that buffer agent sodium hydrogen phosphate/monohydrate potassium used in the present embodiment has better Stabilization than buffer agent sodium acetate/acetic acid antagonist by contrasting 3 stock solution groups, sorbitol used has better Stabilization than mannitol antagonist.
5.2 illumination experiment
Get the 0.9mL/ cillin bottle stock solution A in above-described embodiment, B, C and each 8 of finished product A, B, C respectively, lucifuge is wrapped up with aluminium foil, then be placed in the Climacell 404 constant temperature and humidity incubator of 40 ± 2 DEG C, respectively at the 0th day, the 5th day, the 15th day, the 30th day, two sampled respectively to often kind of sample and analyze; Get the 0.9mL/ cillin bottle stock solution A in above-described embodiment, B, C and each 8 of finished product A, B, C respectively, be placed on 40 ± 2 DEG C, in the Climacell 404 constant temperature and humidity incubator of illumination (intensity 4000-5000lx), respectively at the 0th day, the 5th day, the 15th day, the 30th day, two sampled respectively to often kind of sample and analyze.Analytical method is the outward appearance of naked eyes detection immediately after sampling, then carries out the detection of SEC-HPLC, CEX-HPLC, NR-CE-SDS and biologic activity.In above-mentioned experimental design, if the result of the 30th day meets expection, then need not analyze the sample point of the 15th day.Experimental result in table 8 to table 10.
Table 8.40 ± 2 DEG C, lucifuge 5 days influence factor's experimental results
Table 9.40 ± 2 DEG C, lucifuge 30 days influence factor's experimental results
Table 10.40 ± 2 DEG C, illumination 5 days influence factor's experimental results
Under aCEX detection, peak shape produces and moves to right, cannot accurate integration, therefore data are unlisted.
1) in 40 ± 2 DEG C of lucifuge groups, stock solution A, B, C and finished product A, B, C are showed no exception the 5th day, the 30th day outward appearance.Along with time lengthening; SEC-HPLC and NR-CE-SDS purity declines all to some extent; but stock solution A, C and finished product A, C purity fall are less than stock solution B and finished product B; illustrate that stock solution A, C and finished product A, C are better than stock solution B and finished product B, supposition may be that the sorbitol of higher concentration has better protective effect than mannitol antagonist molecule.Meanwhile, along with the prolongation of time, the main peak peak area of the antibody in each sample constantly reduces, and acid peak-to-peak area constantly increases, and part main peak is transformed into acid peak.In same time, it is more that main peak is transformed into acid peak, illustrate that antibody change itself is more, more unstable, further contrast 6 samples can find to be less than the increase by main peak oxytropism peak in finished product B and stock solution B by the increase at main peak oxytropism peak in finished product A, C and stock solution A, C, illustrate that finished product A, C and stock solution A, C are more conducive to the stable of antibody than finished product B and stock solution B.In whole experimental period, biologic activity does not detect decline.
2) 40 ± 2 DEG C of illumination (intensity 4000-5000lx) groups were at the 5th day outward appearance visible particulate material, SEC-HPLC and NR-CE-SDS purity all declines to a great extent, amplitude is about 20-35%, and CEX-HPLC cannot detect, and biologic activity sharply declines and loses analysis significance.The 10th day visible obviously solid-liquor separation phenomenon of outward appearance, the meaning therefore investigated sample and do not analyzed for the 15th day, 30 days.
Conclusion:
SEC-HPLC and NR-CE-SDS purity display order from excellent to bad of each stock solution, finished product is as shown in table 11:
Table 11.40 ± 2 DEG C illumination effect Factor Experiment result
Note: > > > represent significantly better than, comparatively the latter is high by 5% for the former purity; > > represents and is better than, and the former is purity comparatively the latter's height 2-5%; > represents and is slightly better than, and the former is purity comparatively the latter's height 1-2%;=expression is equal to, within both purity differences ± 1%.
Liquid infusion preparation provided by the invention as can be drawn from Table 11---buffer agent is sodium hydrogen phosphate-citric acid or sodium acetate/acetic acid, and surfactant is tween 20---in stability, be better than existing adalimumab liquid infusion preparation, preferred buffer system is sodium hydrogen phosphate-citric acid and surfactant is the liquid infusion preparation of tween 20, and its stability is significantly better than existing adalimumab liquid infusion preparation.
Claims (16)
1. a liquid aqueous pharmaceutical preparation, comprising:
The D2E7 anti-tnf-alpha human monoclonal antibodies of (a) 50g/L;
The sorbitol of (b) 10-20g/L;
(c) buffer agent;
The polyoxyethylene sorbitan monolaurate of (d) 0.01-3g/L;
The sodium chloride of (e) 2-14g/L;
(f) water;
Wherein, described buffer agent is sodium hydrogen phosphate/monohydrate potassium or sodium acetate/acetic acid;
The pH of described liquid aqueous pharmaceutical preparation is 5-6, conductance 8-18mS/cm, and osmotic pressure is 250-400mOsm/kg.
2. the liquid aqueous pharmaceutical preparation of claim 1, the concentration of wherein said sorbitol is 12-18g/L.
3. the liquid aqueous pharmaceutical preparation of claim 2, the concentration of wherein said sorbitol is 15g/L.
4. the liquid aqueous pharmaceutical preparation of claim 1, the concentration of wherein said polyoxyethylene sorbitan monolaurate is 1g/L.
5. the liquid aqueous pharmaceutical preparation of claim 1, wherein said buffer agent is sodium hydrogen phosphate/monohydrate potassium, pH 5.0-5.4.
6. the liquid aqueous pharmaceutical preparation of claim 5, wherein pH is 5.2, and its pH is the molar concentration scope of the buffer solution of 5.2 is 10-40mM, i.e. sodium hydrogen phosphate 2g/L, monohydrate potassium 1.54g/L, and regulates final preparation pH 5.2 with sodium hydroxide.
7. the liquid aqueous pharmaceutical preparation of claim 6, wherein pH is the molar concentration of the buffer solution of 5.2 is 21.5mM.
8. the liquid aqueous pharmaceutical preparation of claim 1, wherein said buffer agent is sodium acetate/acetic acid, pH 5.0-5.4.
9. the liquid aqueous pharmaceutical preparation of claim 8, wherein pH is 5.2, and the molar concentration scope of its pH 5.2 buffer solution is 10-40mM, i.e. sodium acetate 1.23g/L, acetic acid 0.2mL/L.
10. the liquid aqueous pharmaceutical preparation of claim 9, wherein the molar concentration of pH 5.2 buffer solution is 18.5mM.
The liquid aqueous pharmaceutical preparation of 11. any one of claim 1-10, the concentration of wherein said sodium chloride is 3-12g/L.
The liquid aqueous pharmaceutical preparation of 12. claim 11, the concentration of wherein said sodium chloride is 6.16g/L.
The liquid aqueous pharmaceutical preparation of 13. claim 11, the concentration of wherein said sorbitol is 12-18g/L; The concentration of described polyoxyethylene sorbitan monolaurate is 1g/L; Described buffer agent is the pH 5.0-5.4 of sodium hydrogen phosphate/monohydrate potassium or sodium acetate/acetic acid, described aqueous medicament preparations.
The liquid aqueous pharmaceutical preparation of 14. claim 13, the concentration of wherein said sorbitol is 15g/L; Described buffer agent is sodium hydrogen phosphate/monohydrate potassium, and the pH of described aqueous medicament preparations is 5.2.
The liquid aqueous pharmaceutical preparation of 15. claim 13, it is the aqueous solution of following material:
The liquid aqueous pharmaceutical preparation of 16. claim 13, it is the aqueous solution of following material:
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| CN105435221B (en) * | 2014-09-22 | 2021-09-28 | 正大天晴药业集团股份有限公司 | Pharmaceutical composition of humanized antibody for vascular endothelial growth factor |
| US11229702B1 (en) | 2015-10-28 | 2022-01-25 | Coherus Biosciences, Inc. | High concentration formulations of adalimumab |
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Effective date of registration: 20151216 Address after: 201203, room 1043, 602 Harley Road, Zhangjiang hi tech park, Shanghai, Pudong New Area Patentee after: Genor BioPharma Co., Ltd. Patentee after: Yuxi Jiahe Biotechnology Co., Ltd. Address before: 201203, room 1043, 602 Harley Road, Zhangjiang hi tech park, Shanghai, Pudong New Area Patentee before: Genor BioPharma Co., Ltd. |