CN112137967A - 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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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 the regulation of metabolism.
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 decreased fasting serum concentrations of glucose and decreased fasting serum concentrations of triglycerides, insulin and glucagon. Furthermore, in rodent models of diet-induced obesity, administration of FGF21 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 human FGF21 lyophilized preparation, 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 long-term storage stability of FGF 21.
Natural FGF21 has been difficult to develop into a biological agent for clinical therapy, mainly for reasons including: 1. FGF21 protein is poorly stable and susceptible to proteolytic or enzymatic degradation; 2. the conformation of the natural FGF21 is very unstable, aggregation is easy to occur, and the difficulty of large-scale production of FGF21 is increased due to low stability; 3. the half-life of natural FGF21 is very short, and the half-life of human FGF21 is only 0.5-1 hour in mice and 2-3 hours in cynomolgus monkeys. Various protein-persistence 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, and prolonging in vivo retention time; WO2010084169 and WO2012010553 disclose the fusion of FGF21 with a fatty acid long chain (capable of binding to serum proteins); WO2011071783, WO2011130417, WO2012158704, WO2012170438 disclose that agonist antibodies capable of specific 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 to the development of FGF21-Fc long-acting fusion protein drugs lies in the following points: first, it retains the biological activity of FGF 21. The C-terminus of FGF21 contains a binding site for beta-Klotho, and thus the fusion of Fc to FGF 21C-terminus results in a substantial reduction in its activity, while the fusion at the N-terminus maximizes its binding affinity to beta-Klotho. Therefore, most of the Fc fragments in the prior art are fused with the FGF 21N-end to form Fc-FGF 21; secondly, the pharmacokinetic characteristic of FGF21 can be obviously improved after Fc fusion, and the half-life period of the Fc fusion drug can be effectively prolonged so as to meet the clinical drug administration requirement of twice weekly or even once weekly; since the C-terminus of FGF21 also contains a proteolytic site, degradation occurs very readily, and its in vitro activity to hydrolyze metabolites decreases nearly 200-fold. The half-life improvement is not significant as shown by the results of the pharmacokinetic studies of Fc-FGF21, because the C-terminal of FGF21 in the fusion protein is exposed, not protected by Fc, and thus susceptible to degradation by protease attack. 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 its ease of polymerization in a highly concentrated state. Therefore, the ideal FGF21-Fc fusion protein, on the one hand, has an enhanced resistance to proteolysis at the FGF 21C-terminus and a significantly prolonged half-life; on the other hand, the binding affinity of the FGF 21C-end and the beta-Klotho is not obviously reduced due to the steric hindrance 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, a universal freeze-drying protective agent does not exist, and freeze-drying of any new medicine biological preparation and selection of proper pharmaceutical excipients are extremely important work. Therefore, in view of the technical problem that the recombinant human FGF21-Fc fusion protein is easy to aggregate and unstable, a preparation of 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: 25; further preferably povidone K30, the mass ratio of the vitamin C to the recombinant FGF21-Fc fusion protein is 2-5: 1-3: 25.
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, an excipient, a solubilizer and a 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 the semi-finished product solution into a penicillin bottle after the qualified endotoxin is detected, 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: pre-freezing the freeze-dried sample at 0-5 ℃ for 0.5-1h, cooling to-45-40 ℃ for 2-5h, 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, the potential safety hazard of clinical medication is further reduced, the obtained freeze-dried preparation of the recombinant FGF21-Fc fusion protein has low water content, good stability, full appearance, uniform texture and quick redissolution, and multiple indexes of pH, clarity, color, purity, sterility and the like of the freeze-dried preparation meet the quality standard 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, suitability for amplification, stable and controllable quality, good drying effect, low water content of freeze-dried powder and convenience in storage and transportation.
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.
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 ℃, keeping for 0.5h after the preset temperature is reached within 10min, cooling to-45 ℃, keeping for 4h after the preset temperature is reached within 0.5 h;
2) sublimation drying: heating the clapboard system to-25 ℃ at a set temperature, keeping the preset temperature for 0.5h, keeping the preset temperature for 60h, and keeping the vacuum degree for 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 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 the qualified endotoxin is detected, 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 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 the qualified endotoxin is detected, 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 ℃, keeping for 1h after the preset temperature is reached within 10min, cooling to-45 ℃, keeping for 0.5h until the preset temperature is reached, and freezing for 4 h;
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 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 the qualified endotoxin is detected, and performing 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.
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 ℃, keeping for 0.5h after the preset temperature is reached within 10min, cooling to-40 ℃, keeping for 4h after the preset temperature is reached within 0.5 h;
2) sublimation drying: heating the clapboard system to-30 ℃ at a set temperature, keeping the preset temperature for 0.5h, keeping the vacuum degree for 30h, and keeping the vacuum degree for 0.1-0.2mBar, thereby completing sublimation drying;
3) and (3) drying again: and (3) heating the temperature of the clapboard system to 25 ℃, setting the temperature for 1h to reach the preset temperature, maintaining the vacuum degree for 0.1-0.3mBar, and drying after 10 h.
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 the prescription amount of povidone K30, vitamin C, trehalose, tween 80 and recombinant FGF21-Fc fusion protein stock solution, adding the 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 the qualified endotoxin is detected, 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 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 the qualified endotoxin is detected, and performing 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 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 the qualified endotoxin is detected, and performing 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.
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.
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 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 the qualified endotoxin is detected, and performing 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 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 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 the qualified endotoxin is detected, and 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 for 50min, maintaining 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, and then concentrated to 6mg/ml by centrifugation at 4 ℃; 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 mixing the protein solution and the auxiliary material stock solution in a ratio of 1:1 to ensure that the protein concentration is 3mg/ml, measuring 1ml of solution after uniformly mixing, placing 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) 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. When the first drying is finished, the temperature of the clapboard is increased from minus 20 ℃ to 24 ℃ at the speed 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 1
1) Prescription
2) The preparation process comprises the following steps: the FGF21-Fc fusion protein was dialyzed against 20mM, pH7.4PB buffer, and then concentrated to 6mg/ml by centrifugation at 4 ℃; 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 mixing the protein solution and the auxiliary material stock solution in a ratio of 1:1 to ensure that the protein concentration is 3mg/ml, measuring 1ml of solution after uniformly mixing, placing in a 5ml penicillin bottle, and carrying out the mixing at the temperature of 2-8 ℃. 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, and then concentrated to 6mg/ml by centrifugation at 4 ℃; 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 mixing the protein solution and the auxiliary material stock solution in a ratio of 1:1 to ensure that the protein concentration is 3mg/ml, measuring 1ml of solution after uniformly mixing, placing 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) 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. When the first drying is finished, the temperature of the clapboard is increased from minus 20 ℃ to 24 ℃ at the speed 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 freeze-drying according to the freeze-drying curve of the embodiment 1 to obtain the finished.
Comparative 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 a prescription amount of vitamin C, mannitol, Tween 80 and recombinant FGF21-Fc fusion protein stock solution, adding 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 injection.
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 qualified pH and endotoxin, then accurately weighing the 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, methionine, 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 freeze-drying according to the freeze-drying curve of the embodiment.
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 the prescription amount of povidone C30, vitamin C, 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 the semi-finished product solution into a penicillin bottle after the qualified endotoxin is detected, and performing 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 a 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 finished product.
Verification examples
1. Differential calorimetric scanning (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 instrument program-controlled temperaturemThe value is obtained. After a sample is diluted by using a corresponding buffer solution until the concentration of FGF21-Fc fusion protein is 1mg/mL, 350 mu L of the sample is added into a sample hole of a 96-well plate, 350 mu L of the corresponding buffer solution is added into a buffer hole, the scanning temperature is set to be 20-90 ℃, and the scanning speed is 60 ℃/hr. Data analysis was performed using MicroCal VP-Capillary DSC automated analysis software, with results shown in table 1.
TABLE 1T of recombinant human FGF21-Fc fusion proteinmValue result
| Sample (I) | Tm onset/℃ | Tm1/℃ | Tm2/℃ |
| 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 at the temperature 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 Provisions of the 2015 edition of Chinese pharmacopoeia, purity was measured by molecular sequencing chromatography according to the general rule 0514 and capillary electrophoresis according to the general rule 0542, and activity was determined by a 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, and 12 months during the test period and examined according to stability stress. The results are shown in tables 2 and 3.
TABLE 2.2-8 deg.C long-term stability test results
TABLE 3.25 ℃ accelerated stability test results
Claims (10)
1. A freeze-dried preparation of recombinant human FGF21-Fc fusion protein is characterized by comprising povidone K30, vitamin C and pharmaceutically acceptable auxiliary materials.
2. The lyophilized preparation according to claim 1, wherein the mass ratio of povidone K30, vitamin C and the recombinant FGF21-Fc fusion protein is 1-5: 25.
3. The lyophilized preparation according to claim 2, wherein the mass ratio of povidone K30, vitamin C and the recombinant FGF21-Fc fusion protein is 2-5: 1-3: 25.
4. The lyophilized formulation according to claim 1, wherein the acceptable excipients include an excipient, a solubilizer, and a buffer.
5. The lyophilized formulation according to claim 4, wherein the excipient is one or more selected from trehalose, sucrose, sorbitol and mannitol.
6. The lyophilized formulation according to claim 4, wherein the solubilizer is Tween 20 or Tween 80.
7. The lyophilized formulation according to claim 4, wherein the buffer is Tris-HCl buffer system.
8. The lyophilized formulation according to claim 4, characterized by comprising the following components:
9. the lyophilized formulation according to claim 8, characterized by comprising the following components:
10. a process for lyophilizing the formulation of claim 1, comprising the steps of:
1) pre-freezing: pre-freezing the freeze-dried sample at 0-5 ℃ for 0.5-1h, cooling to-45-40 ℃ for 2-5h, 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.
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| CN114893956A (en) * | 2022-06-01 | 2022-08-12 | 恺佧生物科技(上海)有限公司 | Freeze-drying method of Claudin18.2VLP product |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN85101301A (en) * | 1985-01-25 | 1986-07-23 | 武田药品工业株式会社 | The stable composition of interleukin II |
| CN107088224A (en) * | 2017-02-10 | 2017-08-25 | 温州医科大学 | People's FGF21 lyophilized formulations |
| US20180333493A1 (en) * | 2017-05-16 | 2018-11-22 | Bhami's Research Laboratory, Pvt. Ltd. | High concentration protein formulations with reduced viscosity |
| 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 |
-
2019
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN85101301A (en) * | 1985-01-25 | 1986-07-23 | 武田药品工业株式会社 | The stable composition of interleukin II |
| CN107088224A (en) * | 2017-02-10 | 2017-08-25 | 温州医科大学 | People's FGF21 lyophilized formulations |
| US20180333493A1 (en) * | 2017-05-16 | 2018-11-22 | Bhami's Research Laboratory, Pvt. Ltd. | High concentration protein formulations with reduced viscosity |
| 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 |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114893956A (en) * | 2022-06-01 | 2022-08-12 | 恺佧生物科技(上海)有限公司 | Freeze-drying method of Claudin18.2VLP product |
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