CN115429762A - Freeze-drying protective agent for high-concentration plasminogen preparation - Google Patents
Freeze-drying protective agent for high-concentration plasminogen preparation Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 17
- 102000013566 Plasminogen Human genes 0.000 title claims abstract description 11
- 108010051456 Plasminogen Proteins 0.000 title claims abstract description 11
- 238000004108 freeze drying Methods 0.000 title claims description 42
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims abstract description 50
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims abstract description 30
- 239000001632 sodium acetate Substances 0.000 claims abstract description 30
- 235000017281 sodium acetate Nutrition 0.000 claims abstract description 30
- 239000004471 Glycine Substances 0.000 claims abstract description 27
- 239000000243 solution Substances 0.000 claims abstract description 13
- 239000007974 sodium acetate buffer Substances 0.000 claims abstract description 11
- 239000004475 Arginine Substances 0.000 claims abstract description 10
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims abstract description 10
- 239000002904 solvent Substances 0.000 claims abstract description 5
- 101000605403 Homo sapiens Plasminogen Proteins 0.000 claims description 38
- 239000007853 buffer solution Substances 0.000 claims description 16
- 238000000502 dialysis Methods 0.000 claims description 12
- 238000009472 formulation Methods 0.000 claims description 12
- 239000000203 mixture Substances 0.000 claims description 12
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- 108090000623 proteins and genes Proteins 0.000 claims description 11
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- 238000000034 method Methods 0.000 claims description 9
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- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical group Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
- 125000000637 arginyl group Chemical group N[C@@H](CCCNC(N)=N)C(=O)* 0.000 claims description 6
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 claims description 6
- 238000010438 heat treatment Methods 0.000 claims description 6
- 238000007710 freezing Methods 0.000 claims description 2
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- 229960002449 glycine Drugs 0.000 description 21
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 description 20
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- 238000005457 optimization Methods 0.000 description 9
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- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
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- 238000000113 differential scanning calorimetry Methods 0.000 description 1
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- 238000004090 dissolution Methods 0.000 description 1
- 230000013020 embryo development Effects 0.000 description 1
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- 229940012356 eye drops Drugs 0.000 description 1
- 229950003499 fibrin Drugs 0.000 description 1
- 229940001501 fibrinolysin Drugs 0.000 description 1
- 239000003527 fibrinolytic agent Substances 0.000 description 1
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- 229960001269 glycine hydrochloride Drugs 0.000 description 1
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- 230000000241 respiratory effect Effects 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
- A61K38/482—Serine endopeptidases (3.4.21)
- A61K38/484—Plasmin (3.4.21.7)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
- A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
- A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21007—Plasmin (3.4.21.7), i.e. fibrinolysin
Abstract
The invention relates to a protective agent for a high-concentration plasminogen preparation (pg) freeze-dried product; the protective agent consists of the following components: (1) Arginine at a mass concentration of 1-9%, preferably at a concentration of 3-9%, most preferably 4%; or (2) glycine with a mass concentration of 0.4-3%, preferably 2-3%, most preferably 2.5%; (3) A sodium acetate buffer solution with the concentration of 2-30mmol/L, preferably, the content of sodium acetate is 2mmol/L; (4) a solvent and a necessary pH regulator.
Description
Technical Field
The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to a freeze-drying protective agent for a high-concentration plasminogen preparation.
Background
Plasminogen (Pg) is synthesized by the liver and circulates in the blood at a level of about 0.18g/L in plasma. Activated Pg is called fibrinolysin (Pm) and is an important component of a fibrinolytic system, and also participates in processes such as wound healing, cell transfer, tissue repair, angiogenesis, embryonic development and the like after being activated [1].
The Pg deficiency may be caused by gene mutation or asparaginase, etc., and includes type I and type II Pg deficiency. Pg deficiency type I is caused by autosomal abnormalities, is recessive and can cause severe clinical symptoms, mainly resulting in systemic mucosal fibrin deposition (Schott, 1998) [2], most commonly manifested as ocular lignified conjunctivitis. This symptom was discovered by Bouisson as early as 1847, and in 1933, borel [3] named lignified Conjunctivitis (Ligneeus Conjunctitis). At present, the disease can be removed only by means of surgical operation, but the symptoms still relapse because the fundamental cause is not solved. Type I Pg deficiencies occur at a rate of about 1.6/1,000,000 ([ 4] Tefs K, gueorguieva M, klammt J, allen CM, aktas D, anlar FY, aydogdu SD, brown D, ciftci E, contauruin P, dempfe CE, dostalek M, eisert S, gokbuget A, gunham O, hidayat AA, hugle B, isikoglu M, irkenc M, joss SK, keble S, knoppo C, kurtuus I, mehta PP, 2006 mek K, schnepentheim R, slreggard S, swerney E, rtswith S, veres G, schillee P, ziilu M, V. Type I: a series of 50properties, blood,108, 3021-3026), lignified conjunctivitis appears in 80% of patients [5] (Schuster V, 1999). Most patients with Pg deficiency are infants and children, with the first clinical symptoms appearing at approximately 10 months of age (Schuster, 2007 bateman 1986. Type i Pg deficiency also causes organ tissues such as ear, sinuses, tracheobronchial, urinary system, gum, and respiratory failure due to damage in the tracheobronchial region plus high viscosity secretions.
Mingers, et al, (1994) [6] used Lys-Pg concentrate for the first time to treat Pg congenital deficiency, but had no significant therapeutic effect due to the low dose. Schott et al (1998) [2] increased the dose by 2-10 fold, lignified conjunctivitis disappeared and both high-viscosity respiratory secretions and wound lesions healed.
Local injection of fresh plasma (containing Pg) or other eye drops containing Pg is effective in treating ocular lignified conjunctivitis (Heidemann et al 2003; watts et al 2002; pergantou et al 2011, and Tabbara et al 2004).
There is literature evaluating the effect of systemic administration of Pg. Kraft et al (2000) [7] Lys-Pg was injected daily to a child with severe Pg deficiency and as a result, dissolution of the conjunctival pseudomembrane was found. Schott et al (1998) [2] A single daily injection of Lys-Pg was given to an infant at the age of 6 months and a complete resolution of lignified conjunctivitis was found within 4 weeks.
The content of Pg antigen in the body of the Pg deficiency patient is about less than 0.1-0.9 g/L, the Pg activity is less than 1-50% [8] (Schuster et al, 2007). Pg activity remains in most patients, so Pg replacement therapy may be effective in treating Pg deficiency. Pg belongs to an endogenous protein and theoretically does not produce immunogenicity.
Several studies have shown that Pg concentrates are effective in treating or inhibiting disease recurrence [2] (Watts et al 2002, heidemann et al 2003, and Schott et al 1998), but since there is currently no purified Pg product available for topical or systemic treatment, the congenital deficiency of Pg remains unresolved.
The department develops a high-concentration pg freeze-dried preparation for local injection, the existing formula contains sucrose which is disaccharide and cannot be directly utilized by cells, the osmotic pressure of blood and body fluid is easy to change,
the present invention has been made based on this.
[1]Castellino,Francis J.,and Victoria A.Ploplis."Structure and function of the plasminogen/plasmin system."Thrombosis and haemostasis93.04(2005):647-654.
[2]Schott,Dorothee,et al."Therapy with a purified plasminogen concentrate in an infant with ligneous conjunctivitis and homozygous plasminogen deficiency."New England Journal of Medicine 339.23(1998):1679-1686.
[3]Borel MG.(1933)Un nouveau syndrome palpebral.Bull SocFr Ophthalmol,46,168-180.
[4]Tefs K,Gueorguieva M,Klammt J,Allen CM,Aktas D,Anlar FY,Aydogdu SD,Brown D,Ciftci E,Contaurni P,Dempfee CE,Dostalek M,Eisert S,Gokbuget A,Gunham O,Hidayat AA,Hugle B,Isikoglu M,Irkec M,Joss SK,Keble S,Kneppo C,Kurtulus I,Mehta PP,Ormek K,Schneppenheim R,Slregard S,Swerney E,TurtschiS,Veres G,Zeilee P,Zieplu M,Schusterv V.(2006)m olecular and clinical spectrum of type I plasminogen deficiency:a series of 50patients.Blood,108,3021-3026.
[5]Schuster V,Seidenspinner S,Zeitler P,Escher C,Pleyer U,Beznauer W,Stiehrn ER,Isenberg S,Olsson T,Mingers AM,Schambeck C,Kreth HW.(1999)Compound-heterozygous mutations in the plasminogen gene predispose to the development of ligneous conjunctivitis.Blood,93,3457-3466.
[6]Mingers AM,Philapitsch A,Zeitler P,Schuster V,Schwarz HP,Kreth HW.(1999)Homozigous type I plasminogen deficiency and ligneous conjunctivitis.APMIS,107,62-72.
[7]Kraft J,Lieb W,Zeitler P,Schuster V.(2000)Ligneous conjunctivitis in a girl with severe type I plasminogen deficiency.Graefes Arch Clin Exp Ophthalmol,238,797-800.
[8]Schuster,V.,B.Hügle,and K.Tefs."Plasminogen deficiency."Journal of Thrombosis and Haemostasis 5.12(2007):2315-2322.
Disclosure of Invention
The invention firstly relates to a protective agent for a high-concentration plasminogen preparation (pg) freeze-dried product; the protective agent consists of the following components:
(1) Arginine at a mass concentration of 1-9%, preferably at a concentration of 3-9%, most preferably 4%;
or (2) glycine with a mass concentration of 0.4-3%, preferably 2-3%, most preferably 2.5%;
(3) A sodium acetate buffer solution with the concentration of 2-30mmol/L, preferably, the content of sodium acetate is 2mmol/L;
(4) Solvent and necessary pH regulator.
Further, the solvent is water, and the pH regulator is hydrochloric acid.
Further, when the protective agent component is arginine, the pH value of a sodium acetate buffer solution is 5.2; when the protective agent component is glycine, the pH value of the sodium acetate buffer solution is 6.5;
further, the lyophilized preparation containing the protective agent plasminogen (pg) has a protein content of about: 15-20g/L, preferably about 20g/L.
The invention also relates to a preparation process of the high-concentration plasminogen preparation (pg) freeze-dried product, which comprises the following steps:
(1) Carrying out ultrafiltration dialysis on a human plasminogen sample before freeze-drying by using a buffer solution containing sodium acetate until the preset protein concentration is reached;
(2) Adding a freeze-drying protective agent;
(3) Filtering with 0.2 μm sterilizing filter membrane, and lyophilizing;
1) Pre-freezing to-45 deg.C for 1min, maintaining for 120min, and heating when the pressure reaches 13 Pa;
2) Heating to-15 deg.C for 30min, setting pressure at 15Pa, and maintaining for 900min;
3) Heating to 25 deg.C in 60min, setting pressure at 20Pa, maintaining for 270min, and then corking, deflating, and taking out;
wherein the lyoprotectant in step (2) is selected from:
1) Arginine at a mass concentration of 1-9%, preferably at a concentration of 3-9%, most preferably 4%;
or 2) glycine at a mass concentration of 0.4-3%, preferably 2-3%, most preferably 2.5%;
wherein the buffer solution of sodium acetate in the step (1) is:
1) The content of sodium acetate is 2-30mmol/L, preferably, the content of sodium acetate is 2mmol/L;
2) When the protective agent component is arginine, the pH value of the sodium acetate buffer solution is 5.2; when the protective agent component is glycine, the pH value of the sodium acetate buffer solution is 6.5.
Wherein, the disintegration temperature in the step (3) is preferably the higher temperature group, and the glass transition temperature is preferably the single group.
The beneficial effect of the invention is that,
providing a novel protective formulation for a protective agent for pg lyophilized products; the freeze-drying protective agent is characterized in that: does not contain animal-derived substances, can effectively protect the activity of the Pg in the freeze-drying process, and can ensure that the appearance and various detection indexes after redissolution meet the requirements of pharmacopoeia.
Drawings
FIG. 1 shows the results of stability tests for glycine formulations.
Figure 2, arginine hydrochloride prescription stability test results.
FIG. 3 shows that the protective agent (arginine hydrochloride) of the invention is applied to freeze-dry a human plasminogen product, which is a milky loose body after freeze-drying.
Fig. 4, glass transition temperature test curve diagram when the protective agent of the present invention is applied to the freeze-drying of human plasminogen product, 4A: a test curve diagram of the glass transition temperature of the arginine hydrochloride group; 4B: amino acid group glass transition temperature test curve.
Fig. 5 shows the results of the molecular size distribution detection after reconstitution of the lyophilized human plasminogen preparation prepared with the formula-optimized protective agent (arginine group).
Detailed Description
Reagents and materials
The purified human plasminogen sample is prepared by self-preparation, wherein the content of P201811002 protein is 6g/L, the content of P201903002 protein is 15g/L, the content of P201903003 protein is 20g/L, P201906007 is divided into three parts, the protein contents are respectively 6g/L, 15g/L and 20g/L, and the protein content of P201907008 is 20g/L.
Sodium acetate 20171113 for pharmaceutical use
Glycine 134004 for pharmaceutical use
Arginine hydrochloride, 17AH02M021, pharmaceutical
preparation of buffer solution at pH 6.5: sodium acetate and glycine are weighed, water for injection is added to constant volume and then stirred evenly, pH is measured, and the prepared solution is adjusted to pH6.5 by using 1M HCl or 6M sodium acetate.
preparing a buffer solution with the pH value of 5.2, weighing sodium acetate and arginine hydrochloride, adding water for injection to a constant volume, stirring uniformly, measuring the pH value, and adjusting the prepared solution to the pH value of 5.2 by using 1M HCl or 6M sodium acetate.
Example 1 preliminary optimization of the Freeze-drying protectant formulation (Glycine)
Taking the purified human plasminogen sample, carrying out ultrafiltration dialysis by using a buffer solution containing sodium acetate and having the pH value of 6.0-6.5, concentrating until the concentration of the human plasminogen is 5-20g/L, dividing the human plasminogen into four groups, respectively adding glycine to a target concentration range according to the following table, filtering by using a sterilizing filter membrane, and freeze-drying. The results of examining the titer recovery before and after lyophilization of human plasminogen are shown in the following table,
| concentration of Glycine | 0.4％ | 1.4％ | 2％ | 3％ |
| pH | 6.29 | 6.13 | 6.11 | 6.08 |
| Osmolarity (mOsmol/kg) | 222 | 345 | 432 | 522 |
| Titer recovery rate before and after human plasminogen freeze-drying | 85.66％ | 88.81％ | 97％ | 98.25％ |
As can be seen, the optimal concentration of glycine in the cryoprotectant formulation is 2-3%
Example 2 preliminary optimization of the Freeze-drying protectant formulation (arginine hydrochloride)
Taking the purified human plasminogen sample, carrying out ultrafiltration dialysis by using a buffer solution containing sodium acetate and having the pH value of 6.0-6.5, concentrating until the concentration of the human plasminogen is 5-20g/L, dividing the human plasminogen into four groups, respectively adding arginine hydrochloride to the target concentration range according to the following table, filtering by using a sterilizing filter membrane, and freeze-drying. The results of examining the titer recovery before and after lyophilization of human plasminogen are shown in the following table,
as can be seen, the optimal concentration of arginine hydrochloride in the formula of the freeze-drying protective agent is 3-9%.
Example 3 preliminary optimization of the Freeze-drying protectant formulation (sodium acetate)
Taking purified human plasminogen sample, carrying out ultrafiltration dialysis by using buffer solution (pH 7.0) containing 2-30mmol/L sodium acetate, concentrating until the concentration of human plasminogen is 5-20g/L, dividing into six groups, respectively adding glycine and arginine hydrochloride to the target concentrations of 2.5% and 4% according to the following table, filtering by using a sterilizing filter membrane, and freeze-drying. The results of examining the osmotic pressure (not less than 240 mOsmol/kg) and the titer recovery rate before and after human plasminogen lyophilization are shown in the following table,
as can be seen, in the prescription of the freeze-drying protective agent, the optimal content of sodium acetate is 2mmol/L.
EXAMPLE 4 further optimization of the Freeze-drying protectant formulation (arginine hydrochloride)
Taking a purified human plasminogen sample, carrying out ultrafiltration dialysis by using a buffer solution containing 2mmol/L sodium acetate and pH5.2, concentrating until the concentration of the human plasminogen is 5-20g/L, dividing into three groups, respectively adding arginine hydrochloride to a target concentration range according to the following table, filtering by using a sterilizing filter membrane, and freeze-drying. The results of examining the titer recovery rate, the water content (after freeze-drying, the water content is qualified within 3 percent) and the redissolution time before and after freeze-drying of human plasminogen are shown in the following table,
as can be seen, the optimal concentration of arginine hydrochloride in the prescription of the freeze-drying protective agent is 3-6%.
Example 5 further optimization of the lyoprotectant formulation (Glycine)
Taking a purified human plasminogen sample, carrying out ultrafiltration dialysis by using a buffer solution containing 2mmol/L sodium acetate and pH6.5, concentrating until the concentration of the human plasminogen is 5-20g/L, dividing into three groups, respectively adding glycine to a target concentration range according to the following table, filtering by using a sterilizing filter membrane, and freeze-drying. The results of examining the titer recovery rate, the water content (after freeze-drying, the water content is qualified within 3 percent) and the redissolution time before and after freeze-drying of human plasminogen are shown in the following table,
| concentration of |
2％ | 2.5％ | 3％ |
| Reconstitution time | ＜30s | ＜30s | ＜30s |
| Moisture content | 2.75％ | 2.61％ | 2.71％ |
| Titer recovery before and after freeze-drying | 98.25％ | 97.06％ | 92.54％ |
As can be seen, the optimal concentration of glycine in the lyoprotectant formulation is 2-3%.
Example 6 further optimization of the lyoprotectant formulation (protein content)
Taking a purified human plasminogen sample, carrying out ultrafiltration dialysis by using a buffer solution containing 2mmol/L sodium acetate, 4% arginine hydrochloride and pH5.2, concentrating until the concentration of the human plasminogen is 5-20g/L, dividing into four groups, respectively adding the human plasminogen sample to a target concentration range according to the following table, filtering by using a sterilizing filter membrane, and freeze-drying. The protein concentration range was examined.
Therefore, by using the formula of the freeze-drying protective agent, the titer recovery rate is better when the protein content is concentrated to 20g/L.
Example 7 accelerated stability test comparison
7.1 Glycine prescription
Taking a purified human plasminogen sample, carrying out ultrafiltration dialysis by using a buffer solution containing 2mmol/L sodium acetate, 2.5% glycine and pH6.5, concentrating until the concentration of the human plasminogen is 20g/L, filtering by using a sterilizing filter membrane, and carrying out accelerated stability test on a freeze-dried product. The results are shown in FIG. 1.
7.2 arginine prescription
Taking a purified human plasminogen sample, carrying out ultrafiltration dialysis by using a buffer solution containing 2mmol/L sodium acetate, 4% arginine hydrochloride and pH5.2, concentrating until the concentration of the human plasminogen is 20g/L, filtering by using a sterilizing filter membrane, and carrying out accelerated stability test on a freeze-dried product. The results are shown in FIG. 2.
It can be seen that the lyoprotectant containing arginine hydrochloride as a main component has a superior (time/temperature) stability effect compared to the lyoprotectant containing glycine as a main component.
Example 8 lyophilization Process optimization
Taking a purified human plasminogen sample, carrying out ultrafiltration subpackaging by using a buffer solution containing 2mmol/L sodium acetate, 4% arginine hydrochloride and pH5.2, then carrying out tests on disintegration temperature and glass transition temperature, and freeze-drying by setting freeze-drying parameters according to test results. The lyophilized product is shown in figure 3
8.1 disintegration temperature test
The results of the disintegration temperature test are shown in the table below under the condition that the quality of the freeze-dried product is guaranteed, and the formula of the group with higher disintegration temperature is preferred according to the results. The method specifically comprises the following steps: 2mmol/L of sodium acetate, 4% of arginine hydrochloride and 20g/L of pg protein.
And carrying out ultrafiltration dialysis on the purified human plasminogen sample by using a buffer solution containing 2mmol/L sodium acetate and pH6.5, concentrating until the concentration of the human plasminogen is set to be the set concentration, carrying out disintegration temperature test, setting freeze-drying parameters according to test results, and freeze-drying, wherein the results are shown in the following table, and the formula of the group with higher disintegration temperature is preferably selected according to the results. The method comprises the following specific steps: 2mmol/L of sodium acetate, 2-2.5% of glycine and 20g/L of pg protein.
| Glycine | Protein content | Disintegration temperature (. Degree.C.) | Appearance of Freeze drying | |
| 2％ | 20 | -34.5 | Complete cake shape | 1.800％ |
| 2.50％ | 20 | -39.7 | Complete cake shape | 1.300％ |
| 3％ | 20 | -44.8 | Complete cake shape | 1.400％ |
| 2％ | 15 | -49.6 | Complete cake shape, smooth surface and easy breaking | / |
| 2.50％ | 15 | -50 | Complete cake shape, smooth surface and easy breaking | 3.200％ |
| 3％ | 15 | Less than-60 | Complete cake-like, slight atrophy | / |
| 2％ | 6 | Less than-65 | Complete cake-like, slight atrophy | / |
| 2.50％ | 6 | Less than-65 | Honeycomb, bottom microfusion | / |
| 3％ | 6 | Less than-65 | Complete cake-like, slight atrophy | / |
8.2 glass transition temperature test
The final formulations of the arginine hydrochloride group and the glycine group after optimization are selected to carry out glass transition temperature test, and the results are shown in the following table.
The detection of the vitrification temperature adopts differential scanning calorimetry analysis, analyzes the crystallization, vitrification and melting temperature values of the tested product in the temperature reduction and temperature rise process, and preliminarily judges the types of substances contained in the product.
The glass transition temperature tests of the two groups are carried out, and the graphical display (figure 4) shows that the glass transition temperatures of the two groups of protective agents are not greatly different, but the arginine hydrochloride group has more single component.
The results of the above-described optimization of the lyophilization process show that,
(1) The freeze-drying result of the glycine group is ideal when the protein content is 20 g/L;
(2) The freeze-drying effect of the arginine hydrochloride group is influenced by the protein concentration and is lower than that of glycine.
The optimized protective agent (the formula is the same as the formula of the preparation for vitrification detection) is used for preparing the human plasminogen lyophilized preparation, and the detection result of the immunoturbidimetry method after redissolution is shown in the following table:
according to the detection result, the human plasminogen prepared by the invention only contains trace amounts of albumin, igG, igA and IgM, and other impurities are all lower than the detection range.
The detection result of the molecular size distribution of the human plasminogen lyophilized preparation prepared by using the optimized protective agent (arginine group) of the invention after redissolution is shown in fig. 5:
the human plasminogen of the test sample is analyzed by HPLC, and the purity of the sample is 99.709 percent by a peak area normalization method.
Finally, it should be noted that the above embodiments are only used to help those skilled in the art understand the essence of the present invention, and are not used to limit the protection scope of the present invention.
Claims (6)
1. A protectant for a lyophilized high-concentration plasminogen preparation (pg); the protective agent consists of the following components:
(1) Arginine at a mass concentration of 1-9%, preferably at a concentration of 3-9%, most preferably 4%;
or (2) glycine with a mass concentration of 0.4-3%, preferably 2-3%, most preferably 2.5%;
(3) A sodium acetate buffer solution with the concentration of 2-30mmol/L, preferably, the content of sodium acetate is 2mmol/L;
(4) Solvent and necessary pH regulator;
the protein content of the formulation prior to lyophilization is about: 15-20g/L, preferably about 20g/L.
2. The protective agent according to claim 1, wherein the solvent is water and the pH adjuster is hydrochloric acid.
3. The protective agent according to claim 1 or 2,
when the protective agent component is arginine, the pH value of the sodium acetate buffer solution is 5.2;
when the protective agent component is glycine, the pH value of the sodium acetate buffer solution is 6.5.
4. A method for preparing a high-concentration plasminogen preparation (pg) lyophilized product, comprising the steps of:
(1) Carrying out ultrafiltration dialysis on a human plasminogen sample before freeze-drying by using a buffer solution containing sodium acetate until the preset protein concentration is reached;
(2) Adding a freeze-drying protective agent;
(3) Filtering with 0.2 μm sterilizing filter membrane, and lyophilizing;
1) Pre-freezing to-45 deg.C for 1min, maintaining for 120min, and heating when the pressure reaches 13 Pa;
2) Heating to-15 deg.C for 30min, setting pressure at 15Pa, and maintaining for 900min;
3) Heating to 25 deg.C in 60min, setting pressure at 20Pa, maintaining for 270min, and then corking, deflating, and taking out;
wherein the lyoprotectant in step (2) is selected from:
1) Arginine at a mass concentration of 1-9%, preferably at a concentration of 3-9%, most preferably 4%;
or 2) glycine in a concentration of 0.4-3% by mass, preferably 2-3%, most preferably 2.5%.
5. The method according to claim 4, wherein the buffer solution of sodium acetate in step (1) is:
1) The content of sodium acetate is 2-30mmol/L, preferably, the content of sodium acetate is 2mmol/L;
2) When the protective agent component is arginine, the pH value of the sodium acetate buffer solution is 5.2; when the protective agent component is glycine, the pH value of the sodium acetate buffer solution is 6.5.
6. The method of claim 4 or 5, wherein the dialysis is performed to a protein concentration of 15-25g/L; preferably, 20-25g/L.
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