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Polyethylene glycol-integrated interferon variant lyophilized preparation

A freeze-dried preparation, polyethylene glycol technology, applied in freeze-dried delivery, medical preparations with non-active ingredients, medical preparations containing active ingredients, etc. Problems such as the high price of blood albumin

Active Publication Date: 2012-01-25
BEIJING KAWIN TECH SHARE HLDG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] At present, human serum albumin is usually used as the protective agent for most protein biological products for injection, but human serum albumin is expensive, resulting in high cost of the freeze-dried product, and human serum albumin has the risk of carrying viruses
In order to solve this problem, many people are working on the research of protein freeze-dried preparations, trying to replace human serum albumin with other substances as the freeze-drying protection agent, such as choosing dextran as the freeze-drying protection agent of recombinant interferon in the Chinese patent CN1260171A, Mannitol is also used as a freeze-drying protectant. When mannitol is used as a freeze-drying protectant, the problem of poor resolubility and affecting the protein effect often occurs. For some proteins, dextran is not conducive to its long-term storage. Preparation stability is relatively poor
[0011] Amino acids are known to act as protective agents for lyophilized protein products in certain states, and it has been reported that sodium glutamate and lysine salts have antifreeze effects on a protein, lactate dehydrogenase (Seguro, et al. al. , Cryobiology 27:70-79(1990)) and other types of molecules, including monosaccharides and disaccharides, such as lactose and trehalose, and polymers such as PVP, have also been reported as protective agents for lyophilized proteins, but for These disaccharides (lactose and trehalose) are expensive, and the cost of using them as cryoprotectants is relatively high
However, due to the differences in the physicochemical properties of different proteins, their utility for any given protein product is unpredictable
[0012] At present, there is still no polyethylene glycol-integrated interferon variant preparation on the market. Therefore, it is necessary to provide a preparation that is stable, convenient for clinical use, and suitable for storage

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Preparation of mPEG 20kDa -IFN-SA

[0038] IFN-SA solution (3.5mg / ml) is dissolved in sodium phosphate with a concentration of 100mM, pH6.0 contains 20mM NaCNBH 3 After cooling at 4°C, mix thoroughly, and add methoxypolyethylene glycol aldehyde (mPEG-propionaldehyde, average molecular weight 20kDa) in excess of 4 times the molar amount.

[0039] During the reaction, the modification degree of the protein was monitored by reversed-phase HPLC. After 10 hours, the reversed-phase HPLC analysis showed that 80% of the proteins with unblocked α-amino groups at the N-terminal were converted into mPEG-IFN-SA derivatives.

[0040] At the 10-hour time point, the reaction mixture was diluted 5-fold with water, and the purification of the mPEG-IFN-SA derivative was accomplished by ion-exchange chromatography on a Hiload 16 / 10 S Sepharose HP column buffered with 20 mM sodium acetate. The pH of the solution was balanced at 5.5, the reaction mixture was loaded into the colu...

Embodiment 2

[0041] Example 2 Prepare the freeze-dried stock solution made in the following proportions, and freeze-dry it into a finished product

[0042] Na 2 HPO 4 .12H 2 O, 2.58mg / ml, NaH 2 PO 4 .2H 2 O, 0.44mg / ml;

[0043] (1) 40mg / ml mannitol + 10mg / ml lysine hydrochloride;

[0044] (2) 35mg / ml mannitol + 12mg / ml arginine hydrochloride;

[0045] (3) 30mg / ml mannitol + 15mg / ml lysine hydrochloride;

[0046] (4) Mannitol of 40mg / ml;

[0047] Proportionally, take mannitol, or different proportions of mannitol-lysine hydrochloride, or mannitol-arginine hydrochloride, prepare it with a phosphate buffer solution with a pH of 6.5-7.5, filter and sterilize, and prepare different proportions of mPEG20 of Mannitol or Dexmannitol-Amino Acid Cryoprotectant kDa - IFN-SA solution containing mPEG per ml of solution 20kDa -IFN-SA 50-160μg / ml, mix well, aliquot into 1ml / bottle, and freeze-dry at -40°C for 4 hours, then vacuum-dry at -40°C to -7°C After 20 hours, the temperature was gradua...

Embodiment 3

[0052] Example 3 mPEG 20kDa - IFN-SA lyophilized preparation

[0053] mPEG 20kDa -IFN-SA 160 μg / ml

[0054] Na 2 HPO 4 .12H 2 O 2.58mg / ml

[0055] NaH 2 PO 4 .2H 2 O 0.44mg / ml

[0056] Mannitol 38.6mg / ml

[0057] Lysine Hydrochloride 10.2mg / ml

[0058] Preparation method and process:

[0059] Accurately prepare the diluent according to the following diluent formula, and use it to dilute the original solution after preparation. The utensils used must be treated without pyrogens, and the whole process is operated under sterile conditions. Diluent formula: mannitol 38.6g, lysine hydrochloride 10.2g, Na 2 HPO 4 12H 2 O 2.58 g, NaH 2 PO 4 2H 2 O 0.44 g, the volume for injection is determined to be 1 L, and the pH of the diluent is about 7.2

[0060] Take the qualified polyethylene glycol recombinant integrated interferon variant stock solution, accurately measure its volume, dilute with diluent to a protein concentration of 0.16 mg / ml, and filter aseptically to o...

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Abstract

The invention relates to polyethylene glycol-integrated interferon variant injection. Each 1ml of injection comprises 50 to 500 micrograms of polyethylene glycol-integrated interferon variant protein, a buffer system with the pH value of between 4.5 and 7.5 and a freeze-dried protective agent formed by a mannitol-alkaline amino acid mixed system. The polyethylene glycol-integrated interferon variant injection is stable and good in redissolution effect, and does not have the phenomenon of opalescence.

Description

technical field [0001] The invention relates to a polyethylene glycol-integrated interferon variant freeze-dried preparation, which belongs to the field of pharmaceutical preparations. Background technique [0002] As early as 1957, lssacs and others discovered that virus-infected cells produced a factor that could resist virus infection and interfere with virus replication, hence the name interferon (IFN). Interferon is a group of glycoproteins encoded by the genome of susceptible cells after inactivated or live viruses act on susceptible cells. Its activity and antigenicity depend on the protein in the molecule, and have nothing to do with its sugar group. According to its source and structure, IFN can be divided into IFN-α, IFN-β, IFN-γ, which are produced by leukocytes, fibroblasts and activated T cells, respectively. IFN-α is a polygene product with more than ten different subtypes, but their biological activities are basically the same. In addition to antiviral effe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K9/19A61K47/48A61K47/26A61K47/18A61K38/21A61P31/12A61P35/00A61P37/02A61P31/14A61P31/20
Inventor 侯建华周德胜张春丽
Owner BEIJING KAWIN TECH SHARE HLDG
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