CN102552184A - Thrombopoietin peptide analogue freeze-dried preparation - Google Patents

Abstract

The invention provides a thrombopoietin peptide analogue freeze-dried preparation. The freeze-dried preparation is obtained by dissolving thrombopoietin and medicinal auxiliary materials into a medicinal buffering liquid of which the pH is 5-6.0, wherein the protein concentration of the thrombopoietin is 0.1-1 mg/ml; the medicinal auxiliary materials include mannite, glycine and tween 80; the mass concentration of the mannite is 2-8 percent; the mass concentration of the glycine is 1-5 percent; and the volume concentration of the tween 80 is 0.001-0.01 percent. The freeze-dried preparation containing a thrombopoietin peptide analogue has the advantages of high efficiency, easiness, convenience, low cost and suitability for industrial production.

Description

A kind of thrombopoietin is intended the peptide lyophilized formulations

Technical field

The present invention relates to a kind of freeze-dried powder that thrombopoietin is intended peptide that contains, and corresponding preparation technology.

Background technology

Chronic idiopathic thrombocytopenic purpura (ITP) is a kind of self property immune disease; Cause by causing thrombocytopenic a kind of autoimmune response; It is characterized in that platelet count reduces, its patient faces hemorrhage excessive risk, often causes the little angiorrbagia of patient; Symptom is green grass or young crops, epistaxis and the gingival hemorrhage of becoming silted up, and mortality gastrointestinal tract and intracerebral hemorrhage can appear in severe patient.At present the main means of treatment ITP are to use immunosuppressant class medicine, like corticosteroid, quiet notes immunoglobulin, anti-D immunoglobulin and Genentech Company products Rituximab, then are the row splenectomys to serious debilitating patient's last ditch.Wherein most of medicines had not experienced the investigation of placebo clinical trial at random, though all effective to different patient crowds, also can produce serious toxicity simultaneously, its mechanism of action is to stop platelet destruction, but not promotes platelet to produce.

Thrombopoietin (TPO) is a kind of glycoprotein, mainly results from liver, in megalokaryocyte and hematoblastic generation, plays important regulatory role.Its with can activate the downstream signal cascade reaction after c-Mpl combines, thereby cause Megakaryocytic differentiation and increment, and be accompanied by platelet levels and raise.Short platelet generated agent-glycosylation total length RhTPPO RHTPO (rhTPO) and Pegylation recombined human megakaryocyte growth and grew the factor (PEG-rHuMGDF) and comes out thereupon the 1st generation.It can promote that megalokaryocyte generates in health volunteer's body, and improves platelet counts, and has all obtained success in the research that is used for treating cancer patient, immunity thrombopoietin minimizing purpura patient and accepting the cancer patient of non-clear marrow property chemotherapy.Yet; The short platelet generation agent of the 1st generation may cause producing in the subject anti-TPO antibody; Thereby having the risk of bringing out thrombocytopenia, the exploitation of glycosylation total length RhTPPO RHTPO (rhTPO) and Pegylation recombined human megakaryocyte growth and the growth factor (PEG-rHuMGDF) is compelled to be stopped.After this research worker in polypeptide libraries, seek at random, the polypeptide of no dependency, attempt can stimulate the cell strain that depends on TPO under the situation that does not produce anti-TPO antibody; Polypeptide chain attempting screening is obtained is connected to various carrier molecules, to increase its half-life.Develop thus with thrombopoietin and intend the 2nd generation short platelet generation agent that peptide is representative.Thrombopoietin is intended the test of peptide in health volunteer and ITP patient and has all been obtained success, and does not also see the anti-TPO antibody of generation in the subject in the test.

It is a kind of novel recombinant protein that thrombopoietin is intended peptide, but specificity combines and activation thrombopoietin (TPO) receptor c-Mpl, thereby significantly improves platelet levels.Thrombopoietin is intended peptide and is contained two identical subunits, and each subunit is made up of an IgG1 Fc structural area and c-Mpl land covalent bond respectively, and does not have amino acid sequence homology with TPO, so can in medication person's body, not produce anti-TPO antibody.The production that thrombopoietin is intended peptide is to utilize technique for gene engineering to construct thrombopoietin to intend peptide prokaryotic expression bacterial strain; Adopt fermentation engineering to realize thrombopoietin plan peptide high density fermentation; Be purified into through protein renaturation and purification technique and meet other thrombopoietin of pharmaceutical grade and intend peptide stock solution, be mixed with preparation.Be mainly used in adult's immune thrombocytopenia of treatment splenectomy and non-splenectomy.

But the BA of pharmaceutical grade protein receives multiple Effect of Environmental, and multiple physics or chemical change possibly take place, phenomenons such as for example polymerization, oxidation and degraded, thereby loss of activity greatly.Therefore we have selected the dosage form, adjuvant, the production technology that are fit to, through controlling crucial process conditions, make system product kept very high active and stable.Pilot scale research shows that product manufacture is stable, and is simple to operate, and the cycle is short, and cost is low, is fit to industrialization production.

Summary of the invention

The objective of the invention is to preserve thrombopoietin plan peptide protein, and be suitable for the confused pin of lyophilizing that recombinant platelet generation element is intended peptide that contains of actual clinical use in order to provide a kind of ability to stablize.

For reaching goal of the invention, the technical scheme that the present invention adopts is:

A kind ofly contain the freeze-dried powder that thrombopoietin is intended peptide, said freeze-dried powder is intended in the buffer that peptide and pharmaceutical grade stability adjuvant be dissolved in pH5～6 by thrombopoietin and being obtained, and said thrombopoietin plan peptide protein concentration is 0.1～1mg/mL.

Thrombopoietin is intended peptide stock solution by my company's preparation, and its preparation method is an existing write up in 201110235281.5 at the number of patent application of my company's application, this no longer large fierce dog state.Said stable adjuvant can suitably add as required.Preferred stable adjuvant is mannitol, glycine and Tween 80 among the present invention, and preferred mannitol mass concentration is 2-8%, and more preferably the mannitol mass concentration is 5%.Preferred glycine mass concentration is l-5%, and more preferably the glycine mass concentration is 2.5%.Preferred Tween 80 volumetric concentration is 0.001%-0.01%, and more preferably warm 80 volumetric concentrations are 0.005%.

The protein concentration that said thrombopoietin is intended peptide is 0.1～1mg/mL.

Said buffer is phosphate buffer or citrate buffer solution or acetic acid one sodium-acetate buffer.Said buffer is preferably acetic acid one sodium-acetate buffer, and concentration is 5～50mmol/L.

Preferably; Said freeze-dried powder is to obtain in 5～50mmol/L acetic acid-sodium-acetate buffer by the concentration that thrombopoietin plan peptide and glycine are dissolved in pH5～6; It is 0.1～1mg/mL that said thrombopoietin is intended peptide protein concentration; Said mannitol mass concentration is 2-8%, and the glycine mass concentration is 1～4%, and the Tween 80 volumetric concentration is 0.001%-0.01%.

Preferred; Said freeze-dried powder is intended in acetic acid-sodium-acetate buffer that peptide protein and glycine be dissolved in pH5.5, concentration 20mmol/L by thrombopoietin and being obtained; It is 0.5mg/mL that said thrombopoietin is intended peptide protein concentration; Said mannitol mass concentration is 5%, and the glycine mass concentration is 2.5%, and the Tween 80 volumetric concentration is 0.005%.

The beneficial effect that contains thrombopoietin plan peptide freeze-dried powder pin of the present invention is mainly reflected in: provide a kind of ability the stable lyophilized formulations of preserving thrombopoietin plan peptide protein, and be suitable for industrialization production.

The specific embodiment

Contain the stability that thrombopoietin is intended the peptide medicament in order to improve; If having carried out deep research and discovery selects several kinds of adding preparations from proper supplementary material; And, then can effectively reach this purpose through selecting suitable pH and thrombopoietin to intend the protein concentration of peptide.

Intend in the peptide medicament stability study process at thrombopoietin, chosen the outward appearance detection, the SDS-PAGE electrophoresis detection, rp-hplc analysis, biological activity assays etc. are observed the situation of change that thrombopoietin is intended the peptide medicament as investigating index.

Below in conjunction with specific embodiment the present invention is described further, but protection scope of the present invention is not limited in this:

Embodiment 1: buffer system is intended the influence of stabilized peptide property to thrombopoietin

PH is a key factor that influences protein product stability in the injectable drug preparation.Through adding suitable buffering salt, keep preparation pH.As add phosphate, acetate, citrate etc.In order to investigate stability of formulation under the condition of different pH, carried out the influence experiment of condition of different pH to preparation stability by following condition:

Prepare acetic acid-sodium acetate buffer system respectively, citrate, phosphate buffer, 1ml/ bottle (protein concentration is 0.5mg/ml), pH is designed with 4.0,4.5, and 5.0,5.5,6.0,6.5,7.0,7.5,8.0 concentration are 20mmol/L, (seeing the following form 1).Buffer system through using SUPERDEX G-25 desalting column that thrombopoietin is intended peptide stock solution replaces to design team; And the thrombopoietin of above-mentioned different buffer systems is intended peptide solution be positioned over to deposit under 37 ℃ of conditions and carry out stability test, detect electrophoresis purity (seeing table 2) respectively at sampling in 7,14,28 and 60 days.Detection method; The non-reduced electrophoresis method of SDS-PAGE.

Table 1: the preparation of different buffer systems

Buffer 1	20mmol/L acetic acid-sodium acetate buffer system, pH4.0.
		Buffer 2	20mmol/L acetic acid-sodium acetate buffer system, pH4.5.
Buffer 3	20mmol/L acetic acid-sodium acetate buffer system, pH5.0.
		Buffer 4	20mmol/L acetic acid-sodium acetate buffer system, pH5.5.
Buffer 5	The 20mmol/L phosphate buffer, pH6.0.
		Buffer 6	The 20mmol/L phosphate buffer, pH6.5.
Buffer 7	The 20mmol/L phosphate buffer, pH7.0.
		Buffer 8	The 20mmol/L phosphate buffer, pH7.5.

Show 2:37 ℃ of condition and place the electrophoresis purity measurement result of TPO plan peptide under the different buffer systems in back

Specific activity

0 day

7 days

14 days

21 days

28 days

60 days

Buffer1

98.4

Separate out deposition

—

? —

—

—

Buffer2

99.1

?99.0

?98.0

Separate out deposition

—

—

Buffer3

98.2

?98.4

?98.1

?97.2

?95.7

Separate out deposition

Buffer4

99.3

?99.4

?99.2

?97.1

?96.4

Separate out deposition

Buffer5

98.5

?98.7

?98.8

?97.5

?96.7

Separate out deposition

Buffer6

98.3

?95.3

?80.3

Separate out deposition

—

—

Buffer7

99.5

Separate out deposition

—

—

—

—

Buffer8

98.1

Separate out deposition

—

—

—

? ?—

Conclusion: from the result shown in the last table, can find out; It is bigger that pH value is intended the influence of peptide stock solution purity to thrombopoietin, and stock solution is separated out deposition very soon under the pH4.0 condition, when pH5.0～6.0 scopes; It is more stable that thrombopoietin is intended peptide stock solution, so select the buffer system of pH5.5; The buffer capacity of acetic acid-sodium acetate buffer system is better under this pH value condition.Therefore, we confirm that metastable buffer system is acetic acid-sodium acetate buffer system of 20mmol/L pH5.5.

Embodiment 2: add different pharmaceutical adjuncts are intended stabilized peptide property to thrombopoietin influence

The protein freeze drying protectant is the emphasis of biological agent research always, also is that protein is as the problem of medicinal application to clinical preceding necessary solution.The adjuvant that we use protective agent commonly used in the present protein medicaments has albumin, saccharide, amino acids, surfactant etc.The adjuvant that the present invention has selected some to be suitable for human use screens.Though the human albumin has good protein protection effect, because its source is limited and have potential safety hazard, so do not investigate.

Be applicable to that polyol of the present invention can select mannitol and sorbitol, preferred mannitol is as excipient.

Be applicable to polypeptide of the present invention, aminoacid from following one group of material, to select and come: glycine, arginine, alanine and proline etc., the preferred glycine of the present invention.

In order to investigate the influence of different auxiliary material, different auxiliary material is screened by following condition to stability of formulation.The accessory formula that the present invention screened sees Table that percent concentration is a mass percent concentration in the 3(table).Take by weighing the adjuvant of aequum, or measure the dense adjuvant stock solution for preparing.Adjuvant solid or adjuvant stock solution are joined in the suitable buffer (acetic acid-sodium-acetate buffer of pH5.5); Add the high concentration thrombopoietin of joining then and intend peptide protein solution with suitable buffer (acetic acid-sodium-acetate buffer of pH5.5); Reuse lmol/1 HCL or 10%Na0H are adjusted to required pH value, and the acetic acid-sodium-acetate buffer, the protein concentration that contain 20mmol/L pH5.0 with the acquisition certain volume are the sample solution of 1mg/ml.The sample solution for preparing is divided into two batches, and every batch has 10 kinds of samples, each 10 bottles in every kind of sample, and the 0.5ml/ bottle carries out lyophilizing.A collection of as initial T=0 analysis, then in 4 ℃ of preservations.37 ℃ of preservations of another batch are investigated 4 months, do analyzing and testing every month.Analysis project is a sample appearance after the lyophilizing, and non-reduced electrophoresis of SDS-PAGE and RP-HPLC detect (result sees table 4).

Table 3: adjuvant is formed table

Group	Adjuvant	Prescription concentration
			l	Sucrose	1％
2	Sucrose	2.5％
			3	Sucrose	5%
4	Mannitol	5％
			5	Glycine l	1％
6	Glycine 2	2.5％
			7	Glycine 3	5％
8	Tween 80	0.005％
			9	Mannitol+glycine+Tween 80	5％，2.5%，0.005％
10	Mannitol+sucrose+Tween 80	5％，2.5%，0.005％

Below be different formulations stability experiment result:

The outward appearance of table 4 sample

Group	Sample appearance
		l	Against regulation
2	Up to specification
		3	Up to specification
4	Up to specification
		5	Against regulation
6	Up to specification
		7	Up to specification
8	Against regulation
		9	Up to specification
10	Up to specification

Sample after the biological product lyophilizing should be the loose body of white shell shape with reference to the relevant regulations in three ones of the Chinese Pharmacopoeias, adds to redissolve rapidly behind the labelled amount water for injection to be clear liquid.Table 4 shows that group 2,3,4,6,7,9,10 sample appearance are up to specification.

(1) electrophoresis impurity detects degraded and aggressiveness

Table 5: sample was deposited 4 months under 4 ℃ of conditions, detected with SDS-PAGE every other month

Group	0 month	January	February	March	April
						2	Do not have	Do not have	Do not have	Do not have	Do not have
3	Do not have	Do not have	Do not have	Do not have	Do not have
						4	Do not have	Do not have	Do not have	Do not have	Do not have
6	Do not have	Do not have	Do not have	Do not have	Do not have
						7	Do not have	Do not have	Do not have	Do not have	Do not have
9	Do not have	Do not have	Do not have	Do not have	Do not have
						10	Do not have	Do not have	Do not have	Do not have	Do not have

Table 6: sample was deposited 4 months under 37 ℃ of conditions, detected with SDS-PAGE every other month

Group	0 month	January	February	March	April
						2	Do not have	Have	Have	Have	Have
3	Do not have	Have	Have	Have	Have
						4	Do not have	Have	Have	Have	Have
6	Do not have	Have	Have	Have	Have
						7	Do not have	Have	Have	Have	Have
9	Do not have	Do not have	Do not have	Do not have	Have
						10	Do not have	Do not have	Do not have	Do not have	Have

Brief summary: we can find out from last table, and sample is deposited not degraded and aggressiveness generation in 4 months under 4 ℃ of conditions; Sample is under 37 ℃ of acceleration environments, and it is more stable relatively only to contain 9 and 10 sample, until degraded and aggressiveness generation were just arranged 4 months the time.

(3) RP-HPLC detects

Table 7: sample was deposited 4 months under 4 ℃ of conditions, detected with RP-HPLC every other month

Group	0 month	January	February	March	April
						2	99.5	98.7	97.4	96.3	95.5
3	99.3	99.2	97.5	97.5	96.6
						4	99.4	99.1	98.0	98.5	96.3
6	98.9	99.0	97.6	97.3	97.8
						7	99.5	98.7	98.2	98.5	96.1
9	99.3	99.0	98.9	98.1	99.2
						10	99.1	99.0	98.9	98.0	99.7

Table 8: sample was deposited 4 months under 37 ℃ of conditions, detected with RP-HPLC every other month

Group	0 month	January	February	March	April
						2	99.7	98.9	95.4	90.3	77.5
3	99.5	99.5	96.5	89.5	70.6
						4	99.3	99.2	96.1	90.5	75.6
6	98.9	99.0	94.6	87.3	79.8
						7	99.5	98.7	94.2	80.5	75.6
9	99.3	99.0	98.9	98.1	95.2
						10	99.1	99.0	98.9	98.0	95.7

We can find out from last table: sample is deposited 4 months internal stabilities under 4 ℃ of conditions better, and big variation does not take place sample purity; And under 37 ℃ of acceleration environments, have only the sample purity of the sample of group 9 and group 10 that big variation does not take place, the purity of other sample all has bigger reduction.

(4) BA (tiring)

Show that according to above studies result the sample that contains glycine or sucrose is the most stable, therefore only to the assessment of tiring of the sample of group 9 and group 10.Sample is deposited 4 months under 4 ℃ and 37 ℃ of conditions after respectively sampling carry out BA and detect.

Table 9: different prescription samples carry out BA at 4 ℃ after depositing 4 months with 37 ℃ of conditions and detect

Group	Storage temperature	% tires
			9	4℃	102.2
9	37℃	85.3
			10	4℃	105.2
10	37℃	89.5

Brief summary: we can find out from table 9: the tiring all in acceptability limit of all preparations.

Therefore select glycine+mannitol+Tween 80 or sucrose+mannitol+Tween 80 to intend the adjuvant of peptide lyophilized formulations as thrombopoietin.But sucrose does not have other authentication code of injection stage at home, can not be as the adjuvant of injection medicine, so the final adjuvant of selecting glycine+mannitol+Tween 80 to intend the peptide lyophilized formulations as thrombopoietin.

Comprehensive embodiment 1～2, it is ideal that following medicament is formed: the thrombopoietin of 0.1-1mg/ml is intended peptide protein, preferably 0.1-1mg/ml; The glycine of suitable concn, preferred concentrations are 2.5% (weight ratio).Selected buffer has phosphate buffer, Tris-Hcl buffer, acetic acid-sodium-acetate buffer, acetic acid-sodium-acetate buffer preferably, and concentration is 5-100mmol/L, and most preferred concentration is 20 mmol/L, and pH is between 5.0-6.0, and optimum is 5.5.The Tween 80 of suitable concn, preferred 0.005% (volume ratio).

Embodiment 3: contain the preparation that thrombopoietin is intended the lyophilized formulations of peptide

Get 500 milliliters of thrombopoietins and intend peptide stock solution; Protein concentration is 2 mg/ml; And contain 20mmol acetic acid-sodium-acetate buffer (pH 5.5); Take by weighing the 25g glycine and join in the stock solution dissolving fully, adding an amount of 20mmol acetic acid-sodium-acetate buffer (pH 5.5) dilution at last, to make the preparation final volume be 1000ml, said preparation carried out aseptic filtration and be sub-packed in the cillin bottle with the filter membrane of micron O.22 behind the mix homogeneously.Final preparation consists of: it is 1 mg/ml that thrombopoietin is intended peptide, and the concentration of acetic acid-sodium-acetate buffer is 20mmol/L (pH 5.5), and sweet oxygen acid content is 2.5% (weight ratio), and the content of Tween 80 is 0.005% (volume ratio).

Embodiment 4: contain the investigation that thrombopoietin is intended the lyophilized formulations stability of peptide.

According to embodiment 3 disclosed methods, prepare following several kinds of optimizing prescriptions:

Prescription 1: contain 0.5mg/ and prop up thrombopoietin plan peptide protein; Acetic acid-sodium-acetate buffer pH5.5 buffer; 2.5% glycine, the freeze-dried powder of 2.5% mannitol.

Prescription 2: contain 0.25mg/ and prop up thrombopoietin plan peptide protein; Acetic acid-sodium-acetate buffer pH5.5 buffer; 2.5% glycine, the freeze-dried powder of 2.5% mannitol.

Solution according to above-mentioned 2 kinds of optimizing prescriptions preparation; Carry out long term test reference " three ones of Chinese Pharmacopoeias " appendix " medicine stability test guideline " respectively at depositing 36 months under the 2-8 ℃ of condition; Took a sample once in per 3 months, respectively at 0 month, 3 months, 6 months, 9 months, 12 months, sampling in 18 months, 24 months, 36 months detected; With result and comparison in 0 month, the result is carried out statistical analysis.Detect (outward appearance of finished product, dissolubility, clarity, BA, pH value, electrophoresis purity, sterility test and moisture) by stable high spot reviews project, with result and comparison in 0 month.The result sees table 10 and table 11

Table 10: thrombopoietin is intended peptide 0.5mg/ and is propped up the long-term stable experiment result

Table 11: recombinant platelet generates the plain peptide 0.25mg/ that intends and props up the long-term stable experiment result

Conclusion: prescription 1 and prescription 2 reference " three ones of Chinese Pharmacopoeias " appendix " medicine stability test guideline " are respectively at depositing 36 months under the 2-8 ℃ of condition; Sampling in per 3 months once; Respectively at 0 month, 3 months, 6 months, 9 months, 12 months; Sampling in 18 months, 24 months, 36 months detects, and each item index all meets the requirements, and effect duration can fix tentatively 3 years.

Claims (6)

1. a thrombopoietin is intended the peptide lyophilized formulations, it is characterized in that: said lyophilized formulations is dissolved in by thrombopoietin and pharmaceutic adjuvant in the medicinal buffer of pH5-6.0 and obtains, and said thrombopoietin plan peptide protein concentration is 0.1-1mg/ml; Said pharmaceutic adjuvant is mannitol, glycine and Tween 80, and the mannitol mass concentration is 2-8%, and the glycine mass concentration is 1-5%, and the Tween 80 volumetric concentration is 0.001%-0.01%.

2. a kind of thrombopoietin as claimed in claim 1 is intended the peptide lyophilized formulations, it is characterized in that: it is 0.5 mg/ml that said thrombopoietin is intended peptide protein concentration.

3. a kind of thrombopoietin as claimed in claim 1 is intended the peptide lyophilized formulations, and it is characterized in that: said mannitol mass concentration is 5%, and the glycine mass concentration is 2.5%, and the Tween 80 volumetric concentration is 0.005%.

4. intend the peptide lyophilized formulations like the arbitrary described a kind of thrombopoietin of claim 1-3, it is characterized in that: said buffer is phosphate buffer or citrate buffer solution or acetic acid-sodium-acetate buffer.

5. a kind of thrombopoietin as claimed in claim 4 is intended the peptide lyophilized formulations, and it is characterized in that: said buffer is acetic acid-sodium-acetate buffer, and concentration is 5～50mmol/L, preferred 20 mmol/L.

6. a kind of thrombopoietin as claimed in claim 5 is intended the peptide lyophilized formulations, and it is characterized in that: said buffer is acetic acid-sodium-acetate buffer, and concentration is 20 mmol/L.