CN112137967B - Recombinant human FGF21-Fc fusion protein freeze-dried preparation - Google Patents
Recombinant human FGF21-Fc fusion protein freeze-dried preparation Download PDFInfo
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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
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- 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
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/1825—Fibroblast growth factor [FGF]
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- 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
- A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
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- 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
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
Abstract
The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to a freeze-dried preparation of recombinant human FGF21-Fc fusion protein. The freeze-dried preparation of the FGF21-Fc fusion protein, which is good in stability, full in appearance and uniform in texture, is prepared by taking the povidone K30 and the vitamin C as stabilizers, is simple in preparation process, suitable for amplification, controllable in quality, good in drying effect, low in moisture content of the freeze-dried preparation, and convenient to store and transport.
Description
Technical Field
The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to a freeze-dried preparation of recombinant human FGF21-Fc fusion protein.
Background
Fibroblast growth factor is a polypeptide expressed in developing and adult tissues. The entire family where there are more than 20 fibroblast growth factors is called the FGF family. Three members of the FGF family, including FGF19, FGF21, and FGF23, form a subfamily that functions as endocrine factors involved in metabolic regulation.
FGF21 is a regulator of cellular metabolism that functions as an important metabolic regulator of glucose and lipid homeostasis. FGF21 promotes glucose uptake in adipocytes by up-regulating GLUT1 expression, a mechanism different from insulin. In diabetic rodents and monkeys, human FGF21 lowers fasting serum concentrations of glucose and lowers fasting serum concentrations of triglycerides, insulin and glucagon. Furthermore, in rodent models of diet-induced obesity, FGF21 administration resulted in a dose-dependent loss of cumulative body weight. FGF21 therefore has potential utility for the treatment of diabetes, obesity, dyslipidemia, metabolic syndrome.
Long-term research and clinical practice show that FGF21 has the defects of poor stability, short storage period and the like as other protein medicaments, and the treatment effect of the FGF21 is influenced. Patent CN107088224A discloses a freeze-dried preparation of human FGF21, which has improved stability, higher water content, and obvious changes in activity and purity when the preparation is placed at 37 ℃ for 3 months, and cannot fundamentally solve the problem of poor stability of FGF21 stored for a long time.
Natural FGF21 is difficult to be developed into a biological agent for clinical treatment, mainly for reasons including: 1. FGF21 protein has poor stability and is easily hydrolyzed or degraded enzymatically by protease; 2. the conformation of the natural FGF21 is very unstable and easy to aggregate, and the low stability also increases the difficulty of the FGF21 in large-scale production; 3. the half-life of natural FGF21 is very short, the half-life of human FGF21 in mice is only 0.5-1 hour, and the half-life in cynomolgus monkeys is 2-3 hours. Various protein long-lasting techniques have been reported to extend the in vivo half-life of recombinant FGF 21. For example, patents WO2005091944, WO2006050247, WO2008121563, WO2012066075 disclose that FGF21 is linked to a PEG molecule, increasing molecular weight, decreasing glomerular filtration rate, prolonging retention time in vivo; WO2010084169 and WO2012010553 disclose FGF21 fused to a fatty acid long chain (capable of binding to serum proteins); WO2011071783, WO2011130417, WO2012158704, WO2012170438 disclose that agonist antibodies capable of specifically binding to FGFR/β -klotho complex mimic the FGF21 mechanism of action to activate FGF/FGFR signaling pathway; WO2004110472, WO2005113606, WO2009149171, etc. disclose improving FGF half-life by fusion to an Fc fragment.
In the field of protein drug depuration technology, fc fusion technology is most widely used because it has fewer clinical side effects, e.g., it is not easy to induce allergic reaction or aggravate toxic effects of drugs due to prolonged half-life. The key points of developing FGF21-Fc long-acting fusion protein medicines are as follows: first, whether the biological activity of FGF21 is maintained. The C-terminus of FGF21 contains the binding site for beta-Klotho, so that Fc fusion to the C-terminus of FGF21 results in a substantial reduction in its activity, while fusion to the N-terminus maximizes its binding affinity for beta-Klotho. Therefore, most of Fc fragments in the prior art are fused with the N-terminal of FGF21 to form Fc-FGF21; secondly, after Fc is fused, the pharmacokinetic characteristic of FGF21 can be obviously improved, and the half-life period of the Fc is effectively prolonged so as to meet the clinical administration requirement of twice-weekly or even once-weekly administration; since FGF21 also contains a proteolytic site at the C-terminus, it is highly susceptible to degradation, and its in vitro activity to hydrolyze metabolites decreases nearly 200-fold. The half-life improvement of Fc-FGF21 was not significant as shown by pharmacokinetic studies, since FGF21 in the fusion protein was C-terminally exposed, not protected by Fc, and thus susceptible to degradation by protease. Thirdly, how to reduce the risk of the generation of immune response induced by the adaptor sequence or the introduction of mutation sites; and whether fusing Fc and/or introducing mutation sites can improve the stability of FGF21 and the polymerizability thereof in a high-concentration state. Therefore, the ideal FGF21-Fc fusion protein, on the one hand, has an enhanced resistance to proteolysis of the C-terminus of FGF21 and a significantly extended half-life; on the other hand, the binding affinity of the FGF 21C-terminal and the beta-Klotho is not obviously reduced due to the steric effect of Fc, so that the activity of the Fc is greatly reduced; meanwhile, the introduction of the fusion ligand and the connecting peptide does not increase the immunogenicity and can also improve the stability.
FGF21-Fc fusion proteins overcome a significant hurdle to the physiological instability associated with wild-type FGF 21. However, the recombinant human FGF21-Fc fusion protein has a tendency to aggregate easily. Freeze-dried formulations of FGF21-Fc fusion proteins are rarely reported. Factors that directly or indirectly cause protein drug instability are often subjected to various stresses throughout the lyophilization process of a biological lyophilized product. The excellent protein protective agent not only can play a good role in protecting protein medicaments in the freeze-drying process, but also can play a role in inhibiting the protein denaturation in the storage period of finished products. At present, no universal freeze-drying protective agent exists, and freeze-drying of any new drug biological preparation and selection of proper pharmaceutical excipients are extremely important works. Therefore, in view of the technical problem that the recombinant human FGF21-Fc fusion protein is easy to aggregate and unstable, a preparation of the stable FGF21-Fc fusion protein needs to be developed to meet the clinical medication requirement.
Disclosure of Invention
The invention aims to provide a stable freeze-dried preparation of recombinant human FGF21-Fc fusion protein, which is prepared by adopting povidone and vitamin C as stabilizers, can improve the glass transition temperature of the recombinant human FGF21-Fc fusion protein, and can inhibit crystallization of various components in the freeze-drying process, thereby improving the stability of the preparation.
The technical scheme of the invention is as follows:
a freeze-dried preparation of recombinant human FGF21-Fc fusion protein contains povidone K30, vitamin C and pharmaceutically acceptable auxiliary materials.
Preferably, the mass ratio of the povidone K30, the vitamin C and the recombinant FGF21-Fc fusion protein is 1-5; more preferably, the mass ratio of the povidone K30, the vitamin C and the recombinant FGF21-Fc fusion protein is 2-5.
Preferably, the acceptable excipients include excipients, solubilizers, and buffers.
Further preferably, the excipient is one or more selected from trehalose, sucrose, sorbitol and mannitol.
Further preferably, the solubilizer is tween 20 or tween 80, and more preferably tween 20.
Further preferably, the buffer is a Tris-hydrochloric acid buffer system, and the pH value is 7.5-8.5.
Preferably, the freeze-dried preparation of the recombinant FGF21-Fc fusion protein comprises the following components:
preferably, the freeze-dried preparation of the recombinant FGF21-Fc fusion protein comprises the following components:
in a preferred embodiment, the lyophilized formulation of recombinant FGF21-Fc fusion protein comprises the following components:
the recombinant FGF21-Fc fusion protein is prepared into a semi-finished product solution by adopting the following process:
weighing a prescription amount of Tris, dissolving the Tris with a proper amount of water for injection, adjusting the pH with hydrochloric acid, sampling, detecting the qualified pH and endotoxin, then accurately weighing the prescription amount of povidone K30, vitamin C, excipient, solubilizer and recombinant FGF21-Fc fusion protein stock solution, adding the povidone K30, vitamin C, excipient, solubilizer and recombinant FGF21-Fc fusion protein stock solution into a Tris/HCl buffer solution, uniformly mixing to obtain a semi-finished product solution, performing sterile filtration on the semi-finished product solution by using a 0.22 mu m filter membrane, filling into a penicillin bottle after detecting the qualified endotoxin, and freeze-drying to obtain the finished product.
Preferably, the freeze-drying process is freeze-drying by using the following freeze-drying process:
1) Pre-freezing: when the freeze-dried sample is pre-frozen for 0.5 to 1 hour at the temperature of between 0 and 5 ℃, the temperature is reduced to between 45 ℃ below zero and 40 ℃ below zero and is kept for 2 to 5 hours for freezing,
2) Sublimation drying: vacuumizing for 0.1-0.2mBar, heating to-35-20 deg.C, maintaining for 30-60h,
3) And (3) drying again: heating to 20-30 deg.C, maintaining for 8-10h, resolving, drying, and lyophilizing.
Preferably, the freeze-drying process of the freeze-dried preparation of the recombinant FGF21-Fc fusion protein comprises the following steps of:
1) Pre-freezing: placing a penicillin bottle filled with a recombinant FGF21-Fc fusion protein solution on a clapboard of a freeze dryer, pre-freezing at a set temperature of 5 ℃, keeping the temperature for 0.5-1h after setting for 10-30 min, cooling to-45 ℃, keeping the temperature for 0.5-1h until the temperature reaches the preset temperature, and freezing for 2-5 h;
2) Sublimation drying: heating the partition plate system to a set temperature of-30 to-20 ℃, keeping the preset temperature for 40-60h after a set time of 0.5-1h, and keeping the vacuum degree of 0.1-0.2mBar at the same time to complete sublimation drying;
3) And (3) drying again: heating the temperature of the clapboard system to 20-30 ℃, setting the time to reach the preset temperature for 0.5-1h, maintaining the preset temperature for 8-10h for drying, maintaining the vacuum degree of 0.1-0.3mBar, and finishing the freeze-drying.
The freeze-dried preparation of the FGF21-Fc fusion protein prepared by the invention has few auxiliary materials, further reduces the potential safety hazard of clinical medication, has low moisture content, good stability, full appearance, uniform texture and quick redissolution, and has multiple indexes of pH, clarity, color, purity, sterility and the like which accord with quality standards and are superior to the prior art. The FGF21-Fc fusion protein freeze-dried preparation has the advantages of few auxiliary materials, simple preparation process, stable and controllable quality, good drying effect, low water content of freeze-dried powder and convenience in storage and transportation, and is suitable for amplification.
Detailed Description
The invention is further illustrated by the following examples, which are intended as part of a formulation screening assay and are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions. In the examples, the stability test and the related biological test were carried out according to the specifications of the Chinese pharmacopoeia. The reagents described in the examples are all pharmaceutical grade and are commercially available.
Example 1:
(1) Prescription
(2) Preparation method
Weighing a prescription amount of Tris, dissolving the Tris with a proper amount of water for injection, adjusting the pH with hydrochloric acid, sampling, detecting the qualified pH and endotoxin, then accurately weighing the prescription amount of povidone K30, vitamin C, mannitol, tween 80 and recombinant FGF21-Fc fusion protein stock solution, adding the povidone K30, the vitamin C, the mannitol, the tween 80 and the recombinant FGF21-Fc fusion protein stock solution into a Tris/HCl buffer solution, uniformly mixing to obtain a semi-finished product solution, performing sterile filtration on the semi-finished product solution by using a 0.22 mu m filter membrane, filling into a penicillin bottle after detecting the qualified endotoxin, and freeze-drying according to the following freeze-drying curve to obtain the finished product.
And (3) freeze-drying process:
1) Pre-freezing: placing a penicillin bottle filled with a recombinant FGF21-Fc fusion protein solution on a clapboard of a freeze dryer, pre-freezing at a set temperature of 5 ℃, setting the temperature to reach the preset temperature for 10min, maintaining for 0.5h, cooling to-45 ℃, setting the temperature to reach the preset temperature for 0.5h, and maintaining for 4h for freezing;
2) Sublimation drying: heating the partition plate system to-25 deg.C, maintaining for 0.5h to reach the preset temperature and 60h, and maintaining the vacuum degree of 0.1-0.2mBar to complete sublimation drying;
3) And (3) drying again: and (3) heating the temperature of the clapboard system to 25 ℃, setting the temperature for 0.5h to reach the preset temperature, maintaining the preset temperature for 10h for drying, maintaining the vacuum degree of 0.1-0.3mBar, and finishing the freeze-drying.
Example 2
(1) Prescription
(2) Preparation method
Weighing a prescription amount of Tris, dissolving the Tris with a proper amount of water for injection, adjusting the pH with hydrochloric acid, sampling, detecting the qualified pH and endotoxin, then accurately weighing the prescription amount of povidone K30, vitamin C, sucrose, tween 80 and recombinant FGF21-Fc fusion protein stock solution, adding the povidone K30, the vitamin C, the sucrose, the tween 80 and the recombinant FGF21-Fc fusion protein stock solution into a Tris/HCl buffer solution, uniformly mixing to obtain a semi-finished product solution, performing sterile filtration on the semi-finished product solution by using a 0.22 mu m filter membrane, filling into a penicillin bottle after detecting the qualified endotoxin, and freeze-drying according to the following freeze-drying curve to obtain the finished product.
Freeze-drying curve:
1) Pre-freezing: placing a penicillin bottle filled with a recombinant FGF21-Fc fusion protein solution on a clapboard of a freeze dryer, pre-freezing at a set temperature of 0 ℃, keeping for 0.5h after the preset temperature is reached within a set time of 30min, cooling to-45 ℃, keeping for 2h after the preset temperature is reached within a set time of 1h, and freezing;
2) Sublimation drying: heating the partition plate system to-30 ℃ at a set temperature, maintaining the preset temperature for 1h and the vacuum degree for 40h, and maintaining 0.1-0.2mBar to complete sublimation drying;
3) And (3) drying again: and (3) heating the temperature of the clapboard system to 20 ℃, setting the temperature for 1h to reach the preset temperature, maintaining the vacuum degree for 8h for drying, and maintaining the vacuum degree for 0.1-0.3mBar, thus finishing the freeze-drying.
Example 3
(1) Prescription
(2) Preparation method
Weighing a prescription amount of Tris, dissolving the Tris with a proper amount of water for injection, adjusting the pH with hydrochloric acid, sampling, detecting the qualified pH and endotoxin, then accurately weighing the prescription amount of povidone K30, vitamin C, trehalose, tween 80 and recombinant FGF21-Fc fusion protein stock solution, adding the povidone K30, the vitamin C, the trehalose, the tween 80 and the recombinant FGF21-Fc fusion protein stock solution into a Tris/HCl buffer solution, uniformly mixing to obtain a semi-finished product solution, performing sterile filtration on the semi-finished product solution by using a 0.22 mu m filter membrane, filling into a penicillin bottle after detecting the qualified endotoxin, and freeze-drying according to the following freeze-drying curve to obtain the finished product.
Freeze-drying curve:
1) Pre-freezing: placing a penicillin bottle filled with a recombinant FGF21-Fc fusion protein solution on a clapboard of a freeze dryer, pre-freezing at a set temperature of 5 ℃, maintaining for 1h after the preset temperature is reached within 10min, cooling to-45 ℃, maintaining for 4h after the preset temperature is reached within 0.5h, and freezing;
2) Sublimation drying: heating the clapboard system to-20 deg.C for 0.5h, maintaining for 50h, and maintaining the vacuum degree of 0.1-0.2mBar to complete sublimation drying;
3) And (3) drying again: and (3) heating the temperature of the clapboard system to 30 ℃, setting the time for 0.5h to reach the preset temperature, maintaining the preset temperature for 10h for drying, maintaining the vacuum degree of 0.1-0.3mBar, and finishing the freeze-drying.
Example 4:
(1) Prescription
(2) Preparation method
Weighing a prescription amount of Tris, dissolving the Tris with a proper amount of water for injection, adjusting the pH with hydrochloric acid, sampling, detecting the qualified pH and endotoxin, then accurately weighing the prescription amount of povidone K30, vitamin C, sorbitol, tween 80 and recombinant FGF21-Fc fusion protein stock solution, adding the povidone K30, the vitamin C, the sorbitol, the tween 80 and the recombinant FGF21-Fc fusion protein stock solution into a Tris/HCl buffer solution, uniformly mixing to obtain a semi-finished product solution, performing sterile filtration on the semi-finished product solution by using a 0.22 mu m filter membrane, filling into a penicillin bottle after detecting the qualified endotoxin, and freeze-drying according to the freeze-drying curve of the embodiment 1 to obtain the finished product.
Example 5
(1) Prescription
(2) Preparation method
Weighing a prescription amount of Tris, dissolving the Tris with a proper amount of water for injection, adjusting the pH with hydrochloric acid, sampling, detecting the qualified pH and endotoxin, then accurately weighing the prescription amount of povidone K30, vitamin C, mannitol, tween 80 and recombinant FGF21-Fc fusion protein stock solution, adding the povidone K30, the vitamin C, the mannitol, the tween 80 and the recombinant FGF21-Fc fusion protein stock solution into a Tris/HCl buffer solution, uniformly mixing to obtain a semi-finished product solution, performing sterile filtration on the semi-finished product solution by using a 0.22 mu m filter membrane, filling into a penicillin bottle after detecting the qualified endotoxin, and freeze-drying according to the following freeze-drying curve to obtain the finished product.
Freeze-drying curve:
1) Pre-freezing: placing a penicillin bottle filled with a recombinant FGF21-Fc fusion protein solution on a clapboard of a freeze dryer, pre-freezing at a set temperature of 2 ℃, setting the temperature to reach the preset temperature for 10min, maintaining for 0.5h, cooling to-40 ℃, setting the temperature to reach the preset temperature for 0.5h, and maintaining for 4h for freezing;
2) Sublimation drying: heating the partition plate system to-30 deg.C, maintaining the temperature for 0.5h and the vacuum degree for 30h and 0.1-0.2mBar to complete sublimation drying;
3) And (3) drying again: heating the temperature of the clapboard system to 25 ℃, setting the temperature for 1h to reach the preset temperature, maintaining the temperature for 10h for drying, maintaining the vacuum degree of 0.1-0.3mBar, and finishing the freeze-drying.
Example 6
(1) Prescription
(2) Preparation method
Weighing a prescription amount of Tris, dissolving the Tris with a proper amount of water for injection, adjusting the pH with hydrochloric acid, sampling, detecting the pH and qualified endotoxin, then accurately weighing a prescription amount of povidone K30, vitamin C, trehalose, tween 80 and recombinant FGF21-Fc fusion protein stock solution, adding the povidone K30, the vitamin C, the trehalose, the Tween 80 and the recombinant FGF21-Fc fusion protein stock solution into a Tris/HCl buffer solution, uniformly mixing to obtain a semi-finished product solution, performing sterile filtration on the semi-finished product solution by using a 0.22 mu m filter membrane, filling into a penicillin bottle after qualified endotoxin detection, and performing freeze drying according to the freeze-drying curve of the embodiment 1 to obtain the finished product.
Example 7
(1) Prescription
(2) Preparation method
Weighing a prescription amount of Tris, dissolving the Tris with a proper amount of water for injection, adjusting the pH with hydrochloric acid, sampling, detecting the qualified pH and endotoxin, then accurately weighing the prescription amount of povidone K30, vitamin C, glucose, tween 80 and recombinant FGF21-Fc fusion protein stock solution, adding the povidone K30, the vitamin C, the glucose, the tween 80 and the recombinant FGF21-Fc fusion protein stock solution into a Tris/HCl buffer solution, uniformly mixing to obtain a semi-finished product solution, performing sterile filtration on the semi-finished product solution by using a 0.22 mu m filter membrane, filling into a penicillin bottle after detecting the qualified endotoxin, and freeze-drying according to the freeze-drying curve of the embodiment 1 to obtain the finished product.
Example 8
(1) Prescription
(2) Preparation method
Weighing a prescription amount of Tris, dissolving the Tris with a proper amount of water for injection, adjusting the pH with hydrochloric acid, sampling, detecting the qualified pH and endotoxin, then accurately weighing the prescription amount of povidone K30, vitamin C, sorbitol, tween 80 and recombinant FGF21-Fc fusion protein stock solution, adding the povidone K30, the vitamin C, the sorbitol, the tween 80 and the recombinant FGF21-Fc fusion protein stock solution into a Tris/HCl buffer solution, uniformly mixing to obtain a semi-finished product solution, performing sterile filtration on the semi-finished product solution by using a 0.22 mu m filter membrane, filling into a penicillin bottle after detecting the qualified endotoxin, and freeze-drying according to the freeze-drying curve of the embodiment 1 to obtain the finished product.
Example 9
(1) Prescription
(2) Preparation method
Weighing a prescription amount of Tris, dissolving the Tris with a proper amount of water for injection, adjusting the pH with hydrochloric acid, sampling, detecting the qualified pH and endotoxin, then accurately weighing the prescription amount of povidone K30, vitamin C, mannitol, tween 80 and recombinant FGF21-Fc fusion protein stock solution, adding the povidone K30, the vitamin C, the mannitol, the tween 80 and the recombinant FGF21-Fc fusion protein stock solution into a Tris/HCl buffer solution, uniformly mixing to obtain a semi-finished product solution, performing sterile filtration on the semi-finished product solution by using a 0.22 mu m filter membrane, filling into a penicillin bottle after detecting the qualified endotoxin, and freeze-drying according to the freeze-drying curve of the embodiment 1 to obtain the finished product.
Example 10
(1) Prescription
(2) Preparation method
Weighing a prescription amount of Tris, dissolving the Tris with a proper amount of water for injection, adjusting the pH with hydrochloric acid, sampling, detecting the qualified pH and endotoxin, then accurately weighing the prescription amount of povidone K30, vitamin C, mannitol, tween 80 and recombinant FGF21-Fc fusion protein stock solution, adding the povidone K30, the vitamin C, the mannitol, the tween 80 and the recombinant FGF21-Fc fusion protein stock solution into a Tris/HCl buffer solution, uniformly mixing to obtain a semi-finished product solution, performing sterile filtration on the semi-finished product solution by using a 0.22 mu m filter membrane, filling into a penicillin bottle after detecting the qualified endotoxin, and freeze-drying according to the freeze-drying curve of the embodiment 1 to obtain the finished product.
Example 11
(1) Prescription
(2) Preparation method
Weighing a prescription amount of Tris, dissolving the Tris with a proper amount of water for injection, adjusting the pH with hydrochloric acid, sampling, detecting the qualified pH and endotoxin, then accurately weighing the prescription amount of povidone K30, vitamin C, trehalose, tween 80 and recombinant FGF21-Fc fusion protein stock solution, adding the povidone K30, the vitamin C, the trehalose, the tween 80 and the recombinant FGF21-Fc fusion protein stock solution into a Tris/HCl buffer solution, uniformly mixing to obtain a semi-finished product solution, performing sterile filtration on the semi-finished product solution by using a 0.22 mu m filter membrane, filling into a penicillin bottle after detecting the qualified endotoxin, and freeze-drying according to the freeze-drying curve of the embodiment 1 to obtain the finished product.
Example 12
(1) Prescription
(2) Preparation method
Weighing a prescription amount of Tris, dissolving the Tris with a proper amount of water for injection, adjusting the pH with hydrochloric acid, sampling, detecting the pH and qualified endotoxin, then accurately weighing a prescription amount of povidone K30, vitamin C, sucrose, tween 80 and recombinant FGF21-Fc fusion protein stock solution, adding the povidone K30, the vitamin C, the sucrose, the Tween 80 and the recombinant FGF21-Fc fusion protein stock solution into a Tris/HCl buffer solution, uniformly mixing to obtain a semi-finished product solution, performing sterile filtration on the semi-finished product solution by using a 0.22 mu m filter membrane, filling into a penicillin bottle after qualified endotoxin detection, and performing freeze drying according to the following freeze-drying curve to obtain the finished product.
Freeze-drying curve:
a) Pre-freezing: placing a penicillin bottle filled with a recombinant FGF21-Fc fusion protein solution on a clapboard of a freeze dryer, pre-freezing at a set temperature of 0 ℃, keeping for 1h after a set time of 30min till the preset temperature is reached, then cooling to-50 ℃, keeping for 5min till the preset temperature is reached, keeping for 2h for freezing,
b) Sublimation drying: heating the partition plate system to-25 deg.C, maintaining for 50min for 50h while maintaining the vacuum degree of 0.1mBar, and sublimation drying;
c) And (3) drying again: and (3) heating the temperature of the clapboard system to 25 ℃, setting the temperature for 100min to reach the preset temperature, maintaining the preset temperature for 10h for analysis and drying, maintaining the vacuum degree of 0.2mBar, and finishing the freeze-drying.
Example 13
1) Prescription:
2) The preparation process comprises the following steps: the FGF21-Fc fusion protein was dialyzed against 20mM, pH7.4PB buffer, followed by centrifugation at 4 ℃ to 6mg/ml; dissolving and mixing the prescription auxiliary materials by using 20mM PB buffer solution, adjusting the pH value to 7.4, and then fixing the volume to make the concentration of the auxiliary materials be twice of the final concentration to obtain an auxiliary material stock solution. Then, carrying out sterile filtration on the FGF21-Fc fusion protein solution and the stock solution of the auxiliary materials by using a 0.22 mu m filter membrane; and mixing the protein solution and the auxiliary material stock solution according to the proportion of 1 to ensure that the protein concentration is 3mg/ml, measuring 1ml of solution after uniformly mixing, placing the solution in a 5ml penicillin bottle, and carrying out the mixing at the temperature of 2-8 ℃. Placing the half-plug rubber stopper of the penicillin bottle into a freeze dryer for freeze drying according to the following freeze drying conditions.
3) And (3) a freeze-drying process:
the vial was placed on the septum for 2 hours at room temperature and at a septum temperature of-40 ℃ and the pressure in the chamber was evacuated to 70mTorr, after which the septum temperature was raised from-40 ℃ to-20 ℃ at a rate of 2.5 ℃/min and this temperature was maintained for 30 hours for primary drying. After the first drying, the temperature of the partition board is raised from-20 ℃ to 24 ℃ at a rate of 0.3 ℃/min, and the temperature is maintained for 2 hours for second drying. After the second drying, the chamber was backfilled with dry nitrogen gas and then stoppered. And finally sealing the penicillin bottle by using an aluminum cover.
Comparative example 1
1) Prescription
2) The preparation process comprises the following steps: the FGF21-Fc fusion protein was dialyzed against 20mM, pH7.4PB buffer, followed by centrifugation at 4 ℃ to concentrate to 6mg/ml; dissolving and mixing the prescription auxiliary materials with 20mM PB buffer solution, adjusting the pH value to 7.4, and diluting to a constant volume to make the concentration of the auxiliary materials be twice of the final concentration to obtain an auxiliary material stock solution. Then, carrying out sterile filtration on the FGF21-Fc fusion protein solution and the stock solution of the auxiliary materials by using a 0.22 mu m filter membrane; and the protein solution and the auxiliary material stock solution are mixed according to the proportion of 1. And (3) placing the semi-plugged rubber stopper of the penicillin bottle into a freeze dryer for freeze drying.
The lyophilization profile was the same as in example 1.
Comparative example 2
1) Prescription:
2) The preparation process comprises the following steps: the FGF21-Fc fusion protein was dialyzed against 20mM, pH7.4PB buffer, followed by centrifugation at 4 ℃ to 6mg/ml; dissolving and mixing the prescription auxiliary materials by using 20mM PB buffer solution, adjusting the pH value to 7.4, and then fixing the volume to make the concentration of the auxiliary materials be twice of the final concentration to obtain an auxiliary material stock solution. Then, carrying out sterile filtration on the FGF21-Fc fusion protein solution and the stock solution of the auxiliary materials by using a 0.22 mu m filter membrane; and mixing the protein solution and the auxiliary material stock solution according to the proportion of 1 to ensure that the protein concentration is 3mg/ml, measuring 1ml of solution after uniformly mixing, placing the solution in a 5ml penicillin bottle, and carrying out the mixing at the temperature of 2-8 ℃. The penicillin bottle half-plug rubber stopper is placed in a freeze dryer to be freeze-dried according to the following freeze-drying conditions.
3) And (3) a freeze-drying process:
the vial was placed on the septum for 2 hours at room temperature and at a septum temperature of-40 ℃ and the pressure in the chamber was evacuated to 70mTorr, after which the septum temperature was raised from-40 ℃ to-20 ℃ at a rate of 2.5 ℃/min and maintained at this temperature for 30 hours for the first drying. After the first drying, the temperature of the partition board is raised from-20 ℃ to 24 ℃ at a rate of 0.3 ℃/min, and the temperature is maintained for 2 hours for second drying. After the second drying, the chamber was backfilled with dry nitrogen and then stoppered. And finally, sealing the penicillin bottle by using an aluminum cover.
Comparative example 3
(1) Prescription
(2) Preparation method
Weighing a prescription amount of Tris, dissolving the Tris with a proper amount of water for injection, adjusting the pH with hydrochloric acid, sampling, detecting the qualified pH and endotoxin, then accurately weighing the prescription amount of povidone K30, mannitol, tween 80 and recombinant FGF21-Fc fusion protein stock solution, adding the povidone K30, mannitol, tween 80 and recombinant FGF21-Fc fusion protein stock solution into a Tris/HCl buffer solution, uniformly mixing to obtain a semi-finished product solution, performing sterile filtration on the semi-finished product solution by using a 0.22 mu m filter membrane, filling into a penicillin bottle after detecting the qualified endotoxin, and performing freeze drying according to the freeze-drying curve of the embodiment 1 to obtain the finished product.
Comparative example 4
(1) Prescription
(2) Preparation method
Weighing a prescription amount of Tris, dissolving the prescription amount of Tris with a proper amount of water for injection, adjusting the pH with hydrochloric acid, sampling, detecting the qualified pH and endotoxin, then accurately weighing a prescription amount of vitamin C, mannitol, tween 80 and recombinant FGF21-Fc fusion protein stock solution, adding the prescription amount of the vitamin C, mannitol, tween 80 and recombinant FGF21-Fc fusion protein stock solution into a Tris/HCl buffer solution, uniformly mixing to obtain a semi-finished product solution, performing sterile filtration on the semi-finished product solution by using a 0.22 mu m filter membrane, filling into a penicillin bottle after detecting the qualified endotoxin, and performing freeze drying according to the freeze-drying curve of the embodiment 1 to obtain the finished product.
Comparative example 5
(1) Prescription
(2) Preparation method
Weighing a prescription amount of Tris, dissolving the Tris with a proper amount of water for injection, adjusting the pH with hydrochloric acid, sampling, detecting the pH and qualified endotoxin, then accurately weighing a prescription amount of povidone K30, methionine, mannitol, tween 80 and recombinant FGF21-Fc fusion protein stock solution, adding the weighed prescription amount of povidone K30, the methionine, the mannitol, the Tween 80 and the recombinant FGF21-Fc fusion protein stock solution into a Tris/HCl buffer solution, uniformly mixing to obtain a semi-finished product solution, performing sterile filtration on the semi-finished product solution by using a 0.22 mu m filter membrane, filling the semi-finished product solution into a penicillin bottle after qualified endotoxin detection, and performing freeze drying according to the freeze-drying curve of the embodiment 1 to obtain the finished product.
Comparative example 6
(1) Prescription
(2) Preparation method
Weighing a prescription amount of Tris, dissolving the Tris with a proper amount of water for injection, adjusting the pH with hydrochloric acid, sampling, detecting the qualified pH and endotoxin, then accurately weighing a prescription amount of povidone C30, vitamin C, mannitol, tween 80 and recombinant FGF21-Fc fusion protein stock solution, adding the povidone C, the vitamin C, the mannitol, the tween 80 and the recombinant FGF21-Fc fusion protein stock solution into a Tris/HCl buffer solution, uniformly mixing to obtain a semi-finished product solution, performing sterile filtration on the semi-finished product solution by using a 0.22 mu m filter membrane, filling into a penicillin bottle after detecting the qualified endotoxin, and freeze-drying according to the freeze-drying curve of the embodiment 1 to obtain the finished product.
Comparative example 7
(1) Prescription
(2) Preparation method
Weighing a prescription amount of Tris, dissolving the Tris with a proper amount of water for injection, adjusting the pH with hydrochloric acid, sampling, detecting the qualified pH and endotoxin, then accurately weighing the prescription amount of mannitol, tween 80 and recombinant FGF21-Fc fusion protein stock solution, adding the mixture into a Tris/HCl buffer solution, uniformly mixing to obtain a semi-finished product solution, performing sterile filtration on the semi-finished product solution by using a 0.22 mu m filter membrane, filling into a penicillin bottle after detecting the qualified endotoxin, and freeze-drying according to the freeze-drying curve of the embodiment 1 to obtain the injection.
Verification examples
1. Differential calorimetry (DSC)
The melting temperatures T of the semifinished solutions of the recombinant human FGF21-Fc fusion proteins of examples 1-13 and comparative examples 1-7, respectively, were determined using a MicroCalTM VP-Capillary DSC instrument at the temperature programmed in the instrument m The value is obtained. Diluting a sample by using a corresponding buffer solution until the concentration of the FGF21-Fc fusion protein is 1mg/mL, adding 350 mu L of the sample into a sample hole of a 96-well plate, adding 350 mu L of the corresponding buffer solution into a buffer hole, setting the scanning temperature to be 20-90 ℃, and setting the scanning speed to be 60 ℃/hr. Data analysis MicroCal VP-Capillary DSC automated analysis software was used and the results are given in Table 1.
TABLE 1T of recombinant human FGF21-Fc fusion protein m Value result
| Sample (I) | T m onset /℃ | T m1 /℃ | T m2 /℃ |
| Example 1 | 45.07 | 69.33 | 83.21 |
| Example 2 | 44.16 | 68.36 | 82.22 |
| Example 3 | 43.98 | 68.17 | 81.97 |
| Example 4 | 44.79 | 69.19 | 83.01 |
| Example 5 | 42.36 | 66.31 | 79.72 |
| Example 6 | 43.27 | 67.67 | 81.11 |
| Example 7 | 43.46 | 67.28 | 81.43 |
| Example 8 | 42.86 | 67.09 | 80.84 |
| Example 9 | 42.75 | 66.85 | 80.03 |
| Example 10 | 40.21 | 64.15 | 78.07 |
| Example 11 | 40.66 | 64.74 | 78.45 |
| Example 12 | 41.69 | 65.62 | 79.44 |
| Example 13 | 41.26 | 65.08 | 78.69 |
| Comparative example 1 | 34.27 | 58.24 | 74.03 |
| Comparative example 2 | 33.72 | 57.32 | 73.82 |
| Comparative example 3 | 35.39 | 59.77 | 75.25 |
| Comparative example 4 | 34.99 | 59.15 | 74.57 |
| Comparative example 5 | 37.26 | 61.36 | 76.43 |
| Comparative example 6 | 36.92 | 60.98 | 75.94 |
| Comparative example 7 | 33.06 | 55.63 | 72.99 |
2. Stability test
Three batches of samples were prepared according to examples 1-13 and comparative examples 1-7, respectively, with 60 bottles taken from each batch, and the storage stability was investigated using accelerated stability tests and long-term tests.
The long-term test is carried out under the condition of 2-8 ℃, and the test is carried out according to the stability key investigation items at the end of 0 month, 3 months, 6 months, 12 months and 24 months respectively; (the water content was measured by a Mettler-Zaliduo DL37 Karl Fischer titrator according to the coulometry method specified in the third general rule of the national pharmacopoeia 2015 edition, the purity was measured by the molecular array chromatography 0514 and the capillary electrophoresis 0542, and the activity was determined by the reporter gene method based on bioluminescence). The accelerated test was carried out at 25 ℃. + -. 2 ℃ for 12 months. The used equipment can control the temperature to +/-2 ℃ and monitor the actual temperature. Samples were taken at the end of 0, 3, 6, 12 months during the test period and examined according to stability emphasis. The results are shown in tables 2 and 3.
TABLE 2.2-8 ℃ long-term stability test results
TABLE 3.25 ℃ accelerated stability test results
Claims (2)
1. A freeze-dried preparation of recombinant human FGF21-Fc fusion protein is characterized by comprising the following components:
the excipient is selected from one or more of trehalose, sucrose, sorbitol and mannitol;
the solubilizer is Tween 20 or Tween 80;
the buffer is a Tris-hydrochloric acid buffer system.
2. A process for lyophilizing the formulation of claim 1, comprising the steps of:
1) Pre-freezing: after the freeze-dried sample is pre-frozen for 0.5 to 1 hour at the temperature of 0 to 5 ℃, the temperature is reduced to minus 45 to minus 40 ℃ and maintained for 2 to 5 hours for freezing,
2) Sublimation drying: vacuumizing for 0.1-0.2mBar, heating to-35 to-20 ℃, maintaining for 30-60h,
3) And (3) drying again: heating to 20-30 deg.C, maintaining for 8-10h, resolving, drying, and lyophilizing.
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| CN109821010A (en) * | 2019-01-25 | 2019-05-31 | 上海峰林生物科技有限公司 | A kind of freeze drying powder injection and its preparation method and application of anti-tumor protein TmSm |
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| CN107088224A (en) * | 2017-02-10 | 2017-08-25 | 温州医科大学 | People's FGF21 lyophilized formulations |
| CN109821010A (en) * | 2019-01-25 | 2019-05-31 | 上海峰林生物科技有限公司 | A kind of freeze drying powder injection and its preparation method and application of anti-tumor protein TmSm |
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