CN106620690A - Stable antibody preparation - Google Patents

Abstract

The present invention relates to the technical field of biology, discloses a stable antibody preparation, specifically a high-concentration anti-EGFR antibody preparation containing hyaluronidase and cyclodextrin, wherein the preparation has good stability, is particularly suitable for subcutaneous injection, and can be used for treating human head and neck squamous cell carcinoma, colorectal cancer, pancreatic cancer, non-small cell lung cancer, bladder cancer, and other diseases.

Description

A kind of stable antibody preparation

Technical field

The present invention relates to biomedicine technical field, more specifically, a kind of the invention discloses stable preparation.

Background technology

EGF-R ELISA (EGFR) is the weight of a kind of epidermal growth factor cell propagation and signal transduction Acceptor is wanted, EGFR in combination with its part by producing effect.The ligands specific of EGFR is EGF and EGF Related polypeptide, including transforming growth factor α (TGF- α), amphiregulin and Heparin-binding EGF samples growth because Son.In order to activate EGFR, ligands, EGF (monomer) in combination with and connect two adjacent receptor chains, make The intracellular kinase local of acceptor mutual phosphorylation on multiple tyrosine, tyrosine kinase activity is improved so as to can To activate several signal path such as ras-MAPK paths, PI3 kinase pathway and JAK/STAT paths etc..EGFR Structure activates excessively EGFR caused by (not needing part) and autocrine stimulation after acceptor overexpression or mutation Function is related to kinds cancer generation.The tumorigenesis effect of EGFR include DNA synthesis start, promote cell growth, Invasion and attack and transfer, specific knockdown EGFR can cause cell cycle capture, Apoptosis and cancer cell Differentiation.Research finds, cancer that EGFR originates in various people's epithelial tissues (as bladder, mammary gland, uterine neck, Colon, neck, kidney, lung, pancreas and prostate etc.) in have the expression of varying level, EGFR is in knot Intestines carcinoma of the rectum expression rate is up to 25-77%, and related to the deterioration of tumour and poor prognosis.Just because of EGFR With These characteristics, the antibody drug with EGFR as target spot is continuously developed, and some products have been used for tumour The clinical practice for the treatment of：

(Erbitux, cetuximab, Cetuximab) is a kind of mouse of Imclone companies exploitation The monoclonal anti EGFR antibody that/people is fitted together to.It is a kind of aseptic, clear and bright, colourless liquid, pH 7.0 To 7.4, wherein easy visible, white, amorphous Cetuximab particulate in a small amount may be contained.Per bottle Erbitux Comprising：The sodium chloride of cetuximab, 8.48mg/mL of 2mg/mL, the phosphoric acid hydrogen two of 1.88mg/mL The water of sodium seven, 0.41mg/mL biphosphate sodium-hydrates, and water for injection (USP).Cetuximab can With with EGFR acceptors to be higher by the affinity of about 5 to 10 times of endogenous ligands and EGFR specific bonds, so as to competing The combination of striving property agonist ligand and EGFR, the combination for blocking acceptor and part suppresses ligand-mediated EGFR then The activation of EGFR-TK, so that various by EGFR in tumour cell or the stroma cell in tumor microenvironment The cell processes that signal path is adjusted are blocked, including EGFR is lowered, the suppression of Intracellular signals, cell week The suppression of phase, the induction of apoptosis, the suppression that DNA is repaired, the suppression of Angiogenesiss, Tumor Cell Migration, It is aggressive and it is metastatic suppress etc..U.S. FDA was ratified in 2 months 2004It is straight for tying The treatment of intestinal cancer, FDA in 2006 also ratifies it to be used to treat head and neck neoplasm.

(Victibix, panitumumab) is that the one kind developed by Amgen companies is used to control The full human monoclonal antibodies of anti-EGFR of metastatic colorectal carcinoma after chemotherapy failure are treated,It is a kind of Aseptic, colourless, pH 5.6 to 6.0 liquid preparation, there is tri- kinds of specifications of 5mL, 10mL, 20mL, wherein The single of 5mL specifications contains sodium chloride, the 34mg vinegar of panitumumab, 29mg of 100mg with bottle Sour sodium, and water for injection, USP；The single of 10mL specifications panitumumab of the bottle containing 200mg, The sodium chloride of 58mg, the sodium acetate of 68mg, and water for injection, USP；The single of 20mL specifications is with little Bottle Victibix containing 400mg, 117mg sodium chloride, 136mg sodium acetates and water for injection, USP. 2006It is approved by the fda in the United States for treatment transfer colon cancer.

201010148069.0 disclose a kind of monoclonal antibody against EGFR with high-affinity with it in system Purposes in standby antineoplastic；Wherein the amino acid sequence of weight chain variable district (VH) is SEQ IDNO：14, The amino acid sequence of light chain variable district (VL) is SEQ ID NO：6 or SEQ ID NO：12 antibody with The affinity of EGFR compares at least 10 times of raising with Cetuximab.

……

But, the preparation of existing these anti-egfr antibodies is the preparation of low concentration, and it is not suitable for Hypodermic injection, because the volume ratio intravenous infusion volume for hypodermic drug solution is much smaller, institute Require that antibody preparation concentration is typically up to the level of tens, even hundreds of mg/ml with it.And hypodermic injection is given Medicine using more convenient, safe and reliable, and is favorably improved patient's compared with intravenous injection administration Quality of life, the consultation time for reducing patient, have become at present following developing direction.But, high concentration Antibody-solutions it is more unstable for the preparation of low concentration, easily there is protein aggregation, and assemble easy The bioavailability for causing active antibodies is reduced, pharmacokinetics changes, unfavorable immunogenicity etc. is asked Topic generation (Frokjaer, S. and Otzen, D. Ε., Nat.Rev.Drug.Discov.4:298_306 (2005)；Jiskoot, W. and Crommelin, D.J.A., EJHP Practice 12：20-21(2006)). It is by adding excipient to protein solution, the stabilizer bag for often using to prevent from present assembling common strategy one Include sugar, salt, free amino acid, polyalcohol, polyethylene glycol (PEG) and protein-protein phase can be reduced The other polymers of interaction, such as polysorbate (polysorbates) or poloxamer (Nema, S.^A, PDAJournal of Pharmaceutical Science and Technology 51:166-171(1997))；Or it is logical Cross and add appropriate excipient (including freeze drying protectant) lyophilized to prevent protein aggregation.But, When these strategies are applied to the high concentration ejection preparation of more than mg/ml up to a hundred, effect is less desirable.

Therefore, how to develop a kind of stable antibody preparation of the high concentration for being suitable to hypodermic injection is still very It is necessary.

The content of the invention

Applicant of the present invention develops a kind of stable antibody system of high concentration through substantial amounts of research experiment Agent, said preparation includes hyaluronidase and cyclodextrin；Said preparation formula is particularly suitable for preparing the highly concentrated of injection Degree anti-egfr antibodies preparation.Specifically, the invention discloses：

1st, a kind of stable high concentration antibody preparation, it is included：Antibody, hyaluronidase and cyclodextrin.

2nd, the preparation described in above-mentioned 1, it also includes one or more stabilizer, nonionic surface active agent or slow Electuary.

3rd, a kind of stable high concentration antibody preparation, it is included：

A. antibody more than concentration 50mg/ml；

B.1 to 150mM buffers；

C.15 it is sugared to 250mM；

D.0.01 to 0.1% nonionic surface active agent；

E.40000 the hyaluronidase of individual more than U/ml；With

F.20% to 50% cyclodextrin.

4th, the arbitrary preparations of above-mentioned 1-3, it is characterised in that the hyaluronidase is rHuPH20.

5th, the arbitrary preparations of above-mentioned 1-4, the cyclodextrin is HP- beta cyclodextrins.

6th, the arbitrary preparations of above-mentioned 1-4, the sugar is 80 to 150mM sucrose.

7th, the arbitrary preparations of above-mentioned 1-6, the nonionic surface active agent is selected from the group：Polysorbate 20, Polyoxyethylene sorbitan monoleate and polyethylene-polypropylene copolymer.

8th, the nonionic surface active agent described in above-mentioned 7 is 0.02% to 0.08% polyoxyethylene sorbitan monoleate for concentration.

9th, the arbitrary preparations of above-mentioned 1-5, the antibody is the anti-egfr antibodies of concentration 100 to 250mg/ml.

10th, a kind of stable high concentration antibody preparation, it is included：

A. concentration 100 arrives the anti-egfr antibodies of 300mg/ml；

B.50 to the histidine buffer of 150mM；

C.100 to 150mM sucrose；

D.0.02% to 0.08% polyoxyethylene sorbitan monoleate；

E.40000 the rHuPH20 of individual U/ml to 70000 U/ml；With

F.15% to 40% HP- beta cyclodextrins.

Test result indicate that, the preparation of the present invention can effectively prevent high concentration antibody from sending out in prolonged storage Raw aggregation, so as to provide a kind of antibody preparation with good stability, even if said preparation is at normal temperatures With preferable stability, it is ensured that the preparation rational shelf-life.

The anti-egfr antibodies preparation of the present invention, it is straight that it can be used for the incidence squamous cell carcinoma for the treatment of people, colon Intestinal cancer, cancer of pancreas, non-small cell lung cancer, carcinoma of urinary bladder etc..Preferably can be used as injection aqueous solution formulation Use, more preferably solution can be filled and be used by hypodermic injection in precharging type syringe.

Specific embodiment

The following examples are in order to demonstrate the invention and help those skilled in the art to carry out and use this Invention, should not be understood as limitation of the present invention.

In following examples, anti-egfr antibodies are (with reference to the side disclosed in China Patent No. 201010148069.0 Method is prepared), other experiment materials are obtained by commercial channel, such as HP- beta cyclodextrins (purchase It is biological from source leaf), rHuPH20 (Halozyme), histidine (river Lay is biological), sucrose (promise occasion are biological), Polyoxyethylene sorbitan monoleate (containing the permanent wound in day) etc..

Any one of ordinary skill in the art will be understood that will be included in the composition various groups Point combination can complete in any suitable order, i.e. can add first, middle addition or Finally add buffer, and also adding first, in middle addition or in last addition surfactant Deng.Equally, one of ordinary skill in the art will be understood that these chemical substances in some combinations In some be incompatible, and therefore, they can easily by with similar quality but relevant mixed It is that compatible different chemical substance replaces in compound.

In addition to especially indicating, the content % ratio in preparation is quality concentration of volume percent (w/v), solution PH Can adjust as needed, preferred 4-8.

The hyaluronidase of embodiment 1 and cyclodextrin resist the impact of body preparation

25 DEG C of stability studies containing hyaluronidase and the anti-egfr antibodies preparation of cyclodextrin.The preparation is removed Can also contain outside hyaluronidase and cyclodextrin other according to the excipient gone out given in table 1.

Table 1：Anti-egfr antibodies pharmaceutical formulation

Above-mentioned sample formulation is incubated 60 days at 35 DEG C, then passes through in the 0th, 20,40,60 days time points respectively SDS-PAGE is analyzed.SDS-PAGE is used as a kind of analytical technology, can be according to molecular weight from natural egg Free and HMW species is isolated in white matter.Due in the antibody preparation of high concentration, macromolecular Antibody has high aggregation tendency, and non-reduced SDS-PAGE is used to evaluate covalent aggregation.

Table 2：Different formulations anti-egfr antibodies preparation aggregation SDS-PAGE analysis results

SDS-PAGE analysis results show that F1, F2 preparation for not adding hyaluronidase and cyclodextrin is more also easy to produce Antibody aggregates, added with preparation F5, F6, F7, F8 of two kinds of auxiliary materials of hyaluronidase and cyclodextrin aggregation is not almost produced, With more preferable stability.

The impact of the sugar antagonism body preparation of embodiment 2

Evaluate the impact of the sugar antagonism EGFR antibody preparation stability of varying level.We are positioned in sample - 20 DEG C, 8 DEG C and 45 DEG C, under conditions of 60% humidity and in different time points SE-HPLC methods are passed through The aggregation situation of antibody in detection anti-egfr antibodies preparation.

Table 3:Anti-egfr antibodies pharmaceutical formulation table

Sequence number	Composition	F9	F10	F11	F12
						1	Anti-egfr antibodies (mg/ml)	200	200	200	200
2	Sucrose (mM)	20	100	200	400
						3	Histidine buffer (mM)	150	150	150	150
4	Polyoxyethylene sorbitan monoleate (%)	0.02	0.02	0.02	0.02
						5	RHuPH20 (individual U/ml)	50000	50000	50000	50000
6	HP- beta cyclodextrins (%)	35	35	35	35

Table 4:The impact of sugar antagonism EGFR antibody preparation stability

As shown in Table 4 experimental result understands that sugar antagonism EGFR antibody preparation stability has certain impact, excellent Selection of land, when sucrose is 100-200mM, preparation stability is preferable.

The surfactant of embodiment 3 resists the impact of body preparation

From nonionic surfactant polysorbate (such as polysorbate 20 or 80) and Pa Luoshamu is (for example Pa Luoshamu 188 or 168) in filter out the optimal candidate in terms of stability.We are mainly by right Than analyzing the polysorbate 20 of different content and the anti-egfr antibodies preparation of Pa Luoshamu 188 in storage process In aggregation situation, so as to analyze impact of the different surfaces activating agent to preparation stability.We put in sample It is placed in 30 DEG C, anti-EGFR is detected by SE-HPLC methods under conditions of 60% humidity and in different time points The aggregation situation of antibody in antibody preparation.

Table 5:Anti-egfr antibodies pharmaceutical formulation table

Table 6：:Surfactant resists the impact of EGFR antibody preparation stability

From upper table 6, compared with the preparation containing the surfactants of Pa Luoshamu 188, polyoxyethylene sorbitan monoleate With less aggregation, it is preferable that the tool of the anti-egfr antibodies preparation containing 0.06% polyoxyethylene sorbitan monoleate There is more preferable stability.

The preparation and stability test of the anti-egfr antibodies preparation of embodiment 4

The anti-egfr antibodies preparation optimization formula of table 7

Anti-egfr antibodies	200mg/ml
		Sucrose	120mM
Histidine buffer	100mM
		Polyoxyethylene sorbitan monoleate	0.06%
rHuPH20	55000 U/ml
		HP- beta cyclodextrins	35%
PH	5.5-8

The pharmaceutical solutions compound method with reference to described in the ZL200610147281.9 prepare table 7 described in fill a prescription it is anti- EGFR antibody-solutions preparations.Then anti-egfr antibodies pharmaceutical solutions is stored at -20 DEG C, 5 DEG C and 25 DEG C to exist Storage starts, 1 month, 3 months and 6 months aggregation situations afterwards, anti-by SE-HPLC analyses The stability of EGFR antibody-solutions preparations.The results are shown in Table 8.

Table 8：Anti-egfr antibodies pharmaceutical solutions stability test result

(remarks：3 groups of parallel preparations of each temperature measuring are averaged)

In addition, we also by described in the table 9 for preparing fill a prescription anti-egfr antibodies pharmaceutical solutions in air layer Flow down with 0.2 μm of filter sterilised and store at 60 DEG C.Said preparation is analyzed in storage by SE-HPLC Deposit beginning, 10 days, 20 days, 30 days stability afterwards.The results are shown in Table 9.

Table 9:Anti-egfr antibodies pharmaceutical solutions stability test result

Determinea	0 day	10 days	20 days	30 days
					The aggregation % that SE-HPLC is determined	0.51	1.20	1.95	2.75

(remarks：3 groups of parallel preparations of each temperature measuring are averaged)

From above-mentioned table 8-9, the anti-egfr antibodies pharmaceutical solutions stability of the present invention is preferable, even if normal Still there is preferable stability in the case of temperature or higher temperature.

In addition, the researcher of the present invention is also anti-to other business-like cetuximab, panitumumab etc. EGFR antibody carries out above-mentioned 1-5 experiments, and preparation stability is similar with above-mentioned experimental result, is respectively provided with preferably Stability.

The anti-egfr antibodies preparation of the present invention, it is straight that it is used for the incidence squamous cell carcinoma for the treatment of people, colon Intestinal cancer, cancer of pancreas, non-small cell lung cancer, carcinoma of urinary bladder etc..Made preferably as injection aqueous solution With more preferably solution being filled and be used by hypodermic injection in precharging type syringe.

Claims (10)

1. a kind of stable high concentration antibody preparation, it is included：Antibody, hyaluronidase and cyclodextrin.

2. preparation according to claim 1, it also includes one or more stabilizer, nonionic surface active agent or buffer.

3. a kind of stable high concentration antibody preparation, it is included：

A. antibody more than concentration 50mg/ml；

B.1 to 150mM buffers；

C.15 it is sugared to 250mM；

D.0.01 to 0.1% nonionic surface active agent；

E.40000 the hyaluronidase of individual more than U/ml；With

F.20% to 50% cyclodextrin.

4. according to the arbitrary preparation of claim 1-3, it is characterised in that the hyaluronidase is rHuPH20.

5., according to the arbitrary preparation of claim 1-3, the cyclodextrin is HP- beta cyclodextrins.

6., according to the arbitrary preparation of claim 1-3, the sugar is 80 to 150mM sucrose.

7., according to the arbitrary preparation of claim 1-3, the nonionic surface active agent is selected from the group：It is polysorbate 20, poly- Sorb ester 80 and polyethylene-polypropylene copolymer.

8. nonionic surface active agent according to claim 7 is the polyoxyethylene sorbitan monoleate of concentration 0.02% to 0.08%.

9. a kind of stable high concentration antibody preparation, it is included：

A. concentration 100 arrives the anti-egfr antibodies of 300mg/ml；

B.50 to the histidine buffer of 150mM；

C.100 to 150mM sucrose；

D.0.02% to 0.08% polyoxyethylene sorbitan monoleate；

E.40000 the rHuPH20 of individual U/ml to 70000 U/ml；With

F.15% to 40% HP- beta cyclodextrins.

10. preparation according to claim 9, it is included：

A. anti-egfr antibodies of concentration 200mg/ml；

B.100mM histidine buffer；

C.120mM sucrose；

D.0.06% polyoxyethylene sorbitan monoleate；

E.55000 the rHuPH20 of individual U/ml；With

F.35% HP- beta cyclodextrins.