CN111228225B - Recombinant human tumor necrosis factor receptor-Fc fusion protein freeze-dried preparation - Google Patents
Recombinant human tumor necrosis factor receptor-Fc fusion protein freeze-dried preparation Download PDFInfo
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Abstract
The invention belongs to the technical field of protein and polypeptide medicines, and particularly relates to a recombinant human tumor necrosis factor receptor-Fc fusion protein freeze-dried powder preparation. The freeze-dried powder preparation comprises recombinant human tumor necrosis factor receptor-Fc fusion protein, amino acid, hydroxyethyl starch, a filler, a surfactant and a buffer. The recombinant human tumor necrosis factor receptor-Fc fusion protein obtained by freeze-drying through the formula and the freeze-drying process has high stability, is suitable for large-scale production, and is favorable for storage and transportation.
Description
Technical Field
The invention belongs to the technical field of protein and polypeptide medicines, and particularly relates to a recombinant human tumor necrosis factor receptor-Fc fusion protein freeze-dried powder preparation.
Background
Tumor Necrosis Factor (TNF) is a multifunctional cytokine, is closely related to various diseases and pathological reactions, plays a key role in autoimmune diseases such as inflammatory reaction, endotoxic shock, dyscrasia, rheumatoid arthritis and the like, and is one of the pathological mechanisms of various autoimmune diseases due to overexpression. Research on TNF receptors shows that the TNF receptors are widely distributed and have strong affinity with TNF. This soluble receptor neutralizes TNF and acts as a negative modulator of TNF activity in vivo. The extracellular region of the receptor is fused with the Fc segment gene of human immunoglobulin by using a receptor immunoglobulin fusion technology, and corresponding recombinant human tumor necrosis factor receptor (p75) -Fc fusion protein is expressed in vitro. It has the features of long in vivo half-life period, strong tissue penetrating power, etc. of immunoglobulin, and may be combined specifically with TNF to inhibit joint inflammation.
CN201480065341 discloses a liquid preparation of TNFR-Fc fusion protein, which comprises amino acids, a buffer solution and an isotonic agent, and although the stability of the TNFR-Fc fusion protein is improved to a certain extent, the phenomena of polymer content increase, activity reduction and the like can be caused after long-time storage.
CN201010613663 discloses a lyophilized powder for injection of recombinant human tumor necrosis factor receptor-Fc fusion protein, which contains sucrose, trehalose and other protective agents, and sodium acetate, tromethamine and other buffering agents, and the lyophilized recombinant human tumor necrosis factor receptor-Fc fusion protein has good stability, but the glass transition temperature of the protein still needs to be increased.
Since the freeze-drying process is a complex and variable process, there are many factors that affect the stability of proteins, thereby causing denaturation. The recombinant human tumor necrosis factor receptor-Fc fusion protein has a tendency of easy aggregation in the freeze-drying process, no matter what freeze-drying procedure, the pure protein is obviously damaged, and the problem to be solved is to select and add a proper protective agent to protect the activity of the recombinant human tumor necrosis factor receptor-Fc fusion protein.
Disclosure of Invention
The invention provides a freeze-dried preparation of recombinant human tumor necrosis factor receptor-Fc fusion protein, which has simple formula, stable protein system, convenient large-scale production, storage and transportation and convenient clinical administration. The inventor finds out a freeze-drying recombinant human tumor necrosis factor receptor-Fc fusion protein with a specific prescription by accident through a large number of creative experiments, develops a freeze-drying process suitable for the prescription, and the prepared recombinant human tumor necrosis factor receptor-Fc fusion protein preparation can improve the glass transition temperature of the protein and inhibit crystallization of various components in the freeze-drying process, thereby improving the stability of the recombinant human tumor necrosis factor receptor-Fc fusion protein.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a freeze-dried preparation of recombinant human tumor necrosis factor receptor-Fc fusion protein comprises the recombinant human tumor necrosis factor receptor-Fc fusion protein, amino acid, hydroxyethyl starch, a filling agent, a surfactant and a buffer.
The amino acid is one or more of arginine, cysteine, threonine and lysine.
The bulking agent is selected from one or more of sucrose, trehalose, mannitol and sorbitol.
The surfactant is tween 20.
The buffer is Tris/HCl.
The recombinant human tumor necrosis factor receptor-Fc fusion protein freeze-dried preparation comprises the following components:
preferably, the lyophilized preparation of recombinant human tumor necrosis factor receptor-Fc fusion protein comprises the following components:
the recombinant human tumor necrosis factor receptor-Fc fusion protein is prepared into semi-finished product liquid 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, accurately weighing the prescription amount of amino acid, a filling agent, hydroxyethyl starch, a surfactant and a recombinant human tumor necrosis factor receptor-Fc fusion protein stock solution, adding the amino acid, the filling agent, the hydroxyethyl starch, the surfactant and the recombinant human tumor necrosis factor receptor-Fc fusion protein stock solution into a Tris/HCl buffer solution, and uniformly mixing to obtain a semi-finished product solution; and (4) performing sterile filtration on the semi-finished product liquid by using a 0.22-micron filter membrane, and filling after detecting that the endotoxin is qualified.
A freeze-drying process of a recombinant human tumor necrosis factor receptor-Fc freeze-dried preparation adopts the following steps:
1) pre-freezing: pre-freezing the freeze-dried sample for 0.5-1h at 5 ℃, cooling to-60 ℃ to-40 ℃, keeping the temperature for 4-6h for freezing,
2) sublimation drying: maintaining the vacuum degree, setting the temperature to be between 25 ℃ below zero and 20 ℃ below zero for 10 hours, maintaining the vacuum degree for 20 to 30 hours,
3) and (3) drying again: maintaining the vacuum degree, heating to 30 deg.C, maintaining for 8-10h, resolving, drying, and lyophilizing.
Preferably, the freeze-drying process of the recombinant human tumor necrosis factor receptor-Fc freeze-dried preparation comprises the following steps:
1) pre-freezing: placing a penicillin bottle filled with a recombinant human tumor necrosis factor receptor-Fc fusion protein solution on a clapboard of a freeze dryer, pre-freezing at a set temperature of 5 ℃, keeping the preset temperature for 0.5-1h after setting for 10-30 min, cooling to-60-40 ℃, keeping the preset temperature for 5-10 min, and freezing for 4-6 h;
2) sublimation drying: heating the partition plate system to a set temperature of-25 ℃ to-20 ℃, keeping the temperature for 20-30h after 10h and keeping the vacuum degree for 0.2mBar, and finishing sublimation drying;
3) and (3) drying again: and (3) heating the temperature of the clapboard system to 30 ℃, setting the time to reach the preset temperature for 80-120 min, maintaining the drying time for 8-10h, maintaining the vacuum degree of 0.2mBar, and finishing the freeze-drying.
Compared with the prior art, the recombinant human tumor necrosis factor receptor-Fc fusion protein freeze-dried preparation has white and loose appearance, low product moisture content, quick redissolution, good stability and longer storage time, and is convenient for large-scale production, storage and transportation.
Detailed Description
The invention is further illustrated by the following examples. It should be properly understood that: the examples of the present invention are given solely for the purpose of illustration and not as limitations of the present invention, and therefore, simple modifications of the present invention in the context of the methods of the present invention are intended to fall within the scope of the claims.
Example 1
1) Prescription
2) The preparation process comprises the following steps: weighing a prescription amount of Tris, dissolving the Tris with a proper amount of water for injection, adjusting the pH to 8.0 by using hydrochloric acid, sampling, detecting the pH and detecting whether the endotoxin is qualified, accurately weighing the prescription amount of arginine, sucrose, hydroxyethyl starch, Tween 20 and recombinant human tumor necrosis factor receptor-Fc fusion protein stock solution, adding the mixture into a Tris/HCl buffer solution, and uniformly mixing to obtain a semi-finished product solution; and (4) performing sterile filtration on the semi-finished product liquid by using a 0.22-micron filter membrane, and filling after detecting that the endotoxin is qualified.
3) And (3) freeze-drying process:
a) pre-freezing: placing a penicillin bottle filled with a recombinant human tumor necrosis factor receptor-Fc fusion protein solution on a clapboard of a freeze dryer, pre-freezing at a set temperature of 5 ℃, keeping for 1h after a set time of 30min reaches a preset temperature, cooling to-60 ℃, keeping for 6h after a set time of 10min reaches the preset temperature, and freezing;
b) sublimation drying: heating the partition plate system to-25 ℃ at a set temperature, maintaining the preset temperature for 10h, maintaining the preset temperature for 25h, and maintaining the vacuum degree for 0.2mBar to complete sublimation drying;
c) and (3) drying again: and (3) heating the temperature of the clapboard system to 30 ℃, setting the temperature for 100min to reach the preset temperature, maintaining the vacuum degree for 0.2mBar, and drying after 10 h.
Example 2
1) Prescription
2) The preparation process comprises the following steps: weighing a prescription amount of Tris, dissolving the Tris with a proper amount of water for injection, adjusting the pH to 7.0 by using hydrochloric acid, sampling, detecting the pH and detecting whether the endotoxin is qualified, accurately weighing the prescription amount of cysteine, trehalose, hydroxyethyl starch, Tween 20 and recombinant human tumor necrosis factor receptor-Fc fusion protein stock solution, adding the prescription amount of cysteine, trehalose, hydroxyethyl starch, Tween 20 and recombinant human tumor necrosis factor receptor-Fc fusion protein stock solution into a Tris/HCl buffer solution, and uniformly mixing to obtain a semi-finished product solution; and (4) performing sterile filtration on the semi-finished product liquid by using a 0.22-micron filter membrane, and filling after detecting that the endotoxin is qualified.
3) And (3) freeze-drying process:
a) pre-freezing: placing a penicillin bottle filled with a recombinant human tumor necrosis factor receptor-Fc fusion protein solution on a clapboard of a freeze dryer, pre-freezing at a set temperature of 5 ℃, keeping the temperature for 0.5h after setting for 10min, cooling to-40 ℃, keeping the temperature for 4h after setting for 5min, and freezing;
b) sublimation drying: heating the partition plate system to-20 ℃ at a set temperature, maintaining the preset temperature for 10h, maintaining the vacuum degree for 20h, and maintaining the vacuum degree for 0.2mBar to complete sublimation drying;
c) and (3) drying again: and (3) heating the temperature of the clapboard system to 30 ℃, setting the temperature for 80min to reach the preset temperature, maintaining the vacuum degree for 8h for drying, and simultaneously maintaining the vacuum degree for 0.2mBar, thus finishing the freeze-drying.
Example 3
1) Prescription
2) The preparation process comprises the following steps: weighing a prescription amount of Tris, dissolving the Tris with a proper amount of water for injection, adjusting the pH to 8.5 by using hydrochloric acid, sampling, detecting the pH and detecting whether the endotoxin is qualified, accurately weighing the prescription amount of threonine, cysteine, mannitol, hydroxyethyl starch, Tween 20 and recombinant human tumor necrosis factor receptor-Fc fusion protein stock solution, adding the prescription amount of threonine, cysteine, mannitol, hydroxyethyl starch, Tween 20 and recombinant human tumor necrosis factor receptor-Fc fusion protein stock solution into a Tris/HCl buffer solution, and uniformly mixing to obtain a semi-finished product solution; and (4) performing sterile filtration on the semi-finished product liquid by using a 0.22-micron filter membrane, and filling after detecting that the endotoxin is qualified.
3) And (3) freeze-drying process:
a) pre-freezing: placing a penicillin bottle filled with a recombinant human tumor necrosis factor receptor-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 30min, maintaining for 1h, cooling to-60 ℃, setting the temperature to reach the preset temperature for 10min, and maintaining for 6h for freezing;
b) sublimation drying: heating the partition plate system to-25 ℃ at a set temperature, maintaining the preset temperature for 10h, maintaining the vacuum degree for 30h, and maintaining the vacuum degree for 0.2mBar to complete sublimation drying;
c) and (3) drying again: and (3) heating the temperature of the clapboard system to 30 ℃, setting the temperature for 120min to reach the preset temperature, maintaining the vacuum degree for 0.2mBar, and drying after 10 h.
Example 4
1) Prescription
2) The preparation process comprises the following steps: weighing a prescription amount of Tris, dissolving the Tris with a proper amount of water for injection, adjusting the pH to 8.0 by using hydrochloric acid, sampling, detecting the qualified pH and endotoxin, accurately weighing the prescription amount of lysine, trehalose, sorbitol, hydroxyethyl starch, Tween 20 and recombinant human tumor necrosis factor receptor-Fc fusion protein stock solution, adding the prescription amount of lysine, trehalose, sorbitol, hydroxyethyl starch, Tween 20 and recombinant human tumor necrosis factor receptor-Fc fusion protein stock solution into a Tris/HCl buffer solution, and uniformly mixing to obtain a semi-finished product solution; and (4) performing sterile filtration on the semi-finished product liquid by using a 0.22-micron filter membrane, and filling after detecting that the endotoxin is qualified.
3) And (3) freeze-drying process:
a) pre-freezing: placing a penicillin bottle filled with a recombinant human tumor necrosis factor receptor-Fc fusion protein solution on a clapboard of a freeze dryer, pre-freezing at a set temperature of 5 ℃, keeping the temperature for 0.8h after setting for 20min, cooling to-50 ℃, keeping the temperature for 5h after setting for 8min, and freezing;
b) sublimation drying: heating the partition plate system to-25 ℃ at a set temperature, maintaining the preset temperature for 10h, maintaining the preset temperature for 25h, and maintaining the vacuum degree for 0.2mBar to complete sublimation drying;
c) and (3) drying again: and (3) heating the temperature of the clapboard system to 30 ℃, setting the temperature for 100min to reach the preset temperature, maintaining the vacuum degree for 9h for drying, and simultaneously maintaining the vacuum degree for 0.2mBar, thus finishing the freeze-drying.
Comparative example 1
1) Prescription
2) The preparation process comprises the following steps: accurately measuring proline, glutamic acid, histidine and recombinant human tumor necrosis factor receptor-Fc fusion protein stock solution according to the prescription amount, adding the proline, the glutamic acid, the histidine and the recombinant human tumor necrosis factor receptor-Fc fusion protein stock solution into a citrate-phosphate buffer solution with the pH value of 6.3, and uniformly mixing to obtain a semi-finished product solution; and (4) performing sterile filtration on the semi-finished product liquid by using a 0.22-micron filter membrane, and filling after detecting that the endotoxin is qualified.
3) And (3) a freeze-drying process:
a) pre-freezing: placing a penicillin bottle filled with a recombinant human tumor necrosis factor receptor-Fc fusion protein solution on a clapboard of a freeze dryer, pre-freezing at a set temperature of 5 ℃, keeping for 1h after a set time of 30min reaches a preset temperature, cooling to-60 ℃, keeping for 6h after a set time of 10min reaches the preset temperature, and freezing;
b) sublimation drying: heating the partition plate system to-25 ℃ at a set temperature, maintaining the preset temperature for 10h, maintaining the preset temperature for 25h, and maintaining the vacuum degree for 0.2mBar to complete sublimation drying;
c) and (3) drying again: and (3) heating the temperature of the clapboard system to 30 ℃, setting the temperature for 100min to reach the preset temperature, maintaining the vacuum degree for 0.2mBar, and drying after 10 h.
Comparative example 2
1) Prescription
2) The preparation process comprises the following steps:
accurately weighing according to the prescription, adding water for injection, mixing well, and adjusting pH to 7.30 with hydrochloric acid. The medicinal liquid is filtered by 0.22 μm filter disc made of polyvinylidene fluoride under pressure. To obtain the semi-finished product of the recombinant human tumor necrosis factor receptor-Fc fusion protein. And (3) subpackaging the semi-finished product into penicillin bottles (1 ml/bottle), putting the penicillin bottles into a freeze dryer, and starting the freeze dryer, wherein the process comprises the following steps:
a) a pre-freezing stage: the pre-freezing temperature is-38 ℃, and the pre-freezing temperature is kept for 3 hours;
b) a sublimation drying stage: the vacuum degree is 3bar, the temperature is-32 ℃, and the temperature is kept for 12 hours;
c) and (3) an analysis drying stage: drying at-20 deg.C, -10 deg.C, -5 deg.C, 10 deg.C, 20 deg.C, 30 deg.C for 2 hr to obtain recombinant human tumor necrosis factor receptor-Fc fusion protein powder for injection.
Comparative example 3
1) Prescription
2) The preparation process comprises the following steps:
accurately weighing according to the prescription, adding water for injection, mixing well, and adjusting pH to 7.30 with hydrochloric acid. The medicinal liquid is filtered by 0.22 μm filter disc made of polyvinylidene fluoride under pressure. To obtain the semi-finished product of the recombinant human tumor necrosis factor receptor-Fc fusion protein. And (3) subpackaging the semi-finished product into penicillin bottles (1 mL/bottle), putting the penicillin bottles into a freeze dryer, and starting the freeze dryer, wherein the process comprises the following steps:
a) pre-freezing: placing a penicillin bottle filled with a recombinant human tumor necrosis factor receptor-Fc fusion protein solution on a clapboard of a freeze dryer, pre-freezing at a set temperature of 5 ℃, keeping for 1h after a set time of 30min reaches a preset temperature, cooling to-60 ℃, keeping for 6h after a set time of 10min reaches the preset temperature, and freezing;
b) sublimation drying: heating the partition plate system to-25 ℃ at the set temperature, keeping the set temperature for 10h, keeping the set temperature for 25h, and keeping the vacuum degree for 0.2mBar to complete sublimation drying;
c) and (3) drying again: and (3) heating the temperature of the clapboard system to 30 ℃, setting the temperature for 100min to reach the preset temperature, maintaining the vacuum degree for 0.2mBar, and drying after 10 h.
Comparative example 4
1) Prescription
2) The preparation process comprises the following steps: weighing a prescription amount of Tris, dissolving the Tris with a proper amount of water for injection, adjusting the pH to 8.0 by using hydrochloric acid, sampling, detecting the qualified pH and endotoxin, accurately weighing the prescription amount of sucrose, hydroxyethyl starch, Tween 20 and recombinant human tumor necrosis factor receptor-Fc fusion protein stock solution, adding the mixture into a Tris/HCl buffer solution, and uniformly mixing to obtain a semi-finished product solution; and (4) performing sterile filtration on the semi-finished product liquid by using a 0.22-micron filter membrane, and filling after detecting that the endotoxin is qualified.
3) And (3) freeze-drying process:
a) pre-freezing: placing a penicillin bottle filled with a recombinant human tumor necrosis factor receptor-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 30min, maintaining for 1h, cooling to-60 ℃, setting the temperature to reach the preset temperature for 10min, and maintaining for 6h for freezing;
b) sublimation drying: heating the partition plate system to-25 ℃ at a set temperature, maintaining the preset temperature for 10h, maintaining the preset temperature for 25h, and maintaining the vacuum degree for 0.2mBar to complete sublimation drying;
c) and (3) drying again: and (3) heating the temperature of the clapboard system to 30 ℃, setting the temperature for 100min to reach the preset temperature, maintaining the vacuum degree for 0.2mBar, and drying after 10 h.
Comparative example 5
1) Prescription
2) The preparation process comprises the following steps: weighing a prescription amount of Tris, dissolving the Tris with a proper amount of water for injection, adjusting the pH to 8.0 by using hydrochloric acid, sampling, detecting the qualified pH and endotoxin, accurately weighing the prescription amount of cysteine, sucrose, sorbitol, Tween 20 and recombinant human tumor necrosis factor receptor-Fc fusion protein stock solution, adding the obtained solution into a Tris/HCl buffer solution, and uniformly mixing to obtain a semi-finished product solution; and (4) performing sterile filtration on the semi-finished product liquid by using a 0.22-micron filter membrane, and filling after detecting that the endotoxin is qualified.
3) And (3) freeze-drying process:
a) pre-freezing: placing a penicillin bottle filled with a recombinant human tumor necrosis factor receptor-Fc fusion protein solution on a clapboard of a freeze dryer, pre-freezing at a set temperature of 5 ℃, keeping for 1h after a set time of 30min reaches a preset temperature, cooling to-60 ℃, keeping for 6h after a set time of 10min reaches the preset temperature, and freezing;
b) sublimation drying: heating the partition plate system to-25 ℃ at a set temperature, maintaining the preset temperature for 10h, maintaining the preset temperature for 25h, and maintaining the vacuum degree for 0.2mBar to complete sublimation drying;
c) and (3) drying again: and (3) heating the temperature of the clapboard system to 30 ℃, setting the temperature for 100min to reach the preset temperature, maintaining the vacuum degree for 0.2mBar, and drying after 10 h.
Comparative example 6
1) Prescription
2) The preparation process comprises the following steps: weighing a prescription amount of Tris, dissolving the Tris with a proper amount of water for injection, adjusting the pH to 8.0, sampling, detecting the pH and qualified endotoxin, accurately weighing the prescription amount of arginine, sucrose, polyethylene glycol and recombinant human tumor necrosis factor receptor-Fc fusion protein stock solution, adding the mixture into a Tris buffer solution, and uniformly mixing to obtain a semi-finished product solution; sterile filtering the semi-finished product liquid with 0.22 μm filter membrane, and packaging after endotoxin is detected to be qualified.
3) And (3) freeze-drying process:
a) pre-freezing: placing a penicillin bottle filled with a recombinant human tumor necrosis factor receptor-Fc fusion protein solution on a clapboard of a freeze dryer, pre-freezing at a set temperature of 5 ℃, keeping for 1h after a set time of 30min reaches a preset temperature, cooling to-60 ℃, keeping for 6h after a set time of 10min reaches the preset temperature, and freezing;
b) sublimation drying: heating the partition plate system to-25 ℃ at a set temperature, maintaining the preset temperature for 10h, maintaining the preset temperature for 25h, and maintaining the vacuum degree for 0.2mBar to complete sublimation drying;
c) and (3) drying again: and (3) heating the temperature of the clapboard system to 30 ℃, setting the temperature for 100min to reach the preset temperature, maintaining the vacuum degree for 0.2mBar, and drying after 10 h.
Comparative example 7
1) Prescription
2) The preparation process comprises the following steps: weighing disodium hydrogen phosphate and sodium dihydrogen phosphate according to a prescription amount, dissolving the disodium hydrogen phosphate and the sodium dihydrogen phosphate with a proper amount of water for injection, adjusting the pH to 6.0 by using hydrochloric acid, sampling and detecting the qualified pH and endotoxin, accurately weighing histidine, sucrose, hydroxyethyl starch, tween 20 and the recombinant human tumor necrosis factor receptor-Fc fusion protein stock solution according to the prescription amount, adding the histidine, sucrose, hydroxyethyl starch, tween 20 and the recombinant human tumor necrosis factor receptor-Fc fusion protein stock solution into a phosphoric acid buffer solution, and uniformly mixing to obtain a semi-finished product solution; and (4) performing sterile filtration on the semi-finished product liquid by using a 0.22-micron filter membrane, and filling after detecting that the endotoxin is qualified.
3) And (3) a freeze-drying process:
a) pre-freezing: placing a penicillin bottle filled with a recombinant human tumor necrosis factor receptor-Fc fusion protein solution on a clapboard of a freeze dryer, pre-freezing at a set temperature of 5 ℃, keeping for 1h after a set time of 30min reaches a preset temperature, cooling to-60 ℃, keeping for 6h after a set time of 10min reaches the preset temperature, and freezing;
b) sublimation drying: heating the partition plate system to-25 ℃ at a set temperature, maintaining the preset temperature for 10h, maintaining the preset temperature for 25h, and maintaining the vacuum degree for 0.2mBar to complete sublimation drying;
c) and (3) drying again: and (3) heating the temperature of the clapboard system to 30 ℃, setting the temperature for 100min to reach the preset temperature, maintaining the vacuum degree for 0.2mBar, and drying after 10 h.
Comparative example 8
1) Prescription
2) The preparation process comprises the following steps: weighing a prescription amount of Tris, dissolving the Tris with a proper amount of water for injection, adjusting the pH to 8.0 by using hydrochloric acid, sampling, detecting the qualified pH and endotoxin, accurately weighing the prescription amount of arginine, sucrose, hydroxyethyl starch and recombinant human tumor necrosis factor receptor-Fc fusion protein stock solution, adding the mixture into a Tris/HCl buffer solution, and uniformly mixing to obtain a semi-finished product solution; and (4) performing sterile filtration on the semi-finished product liquid by using a 0.22-micron filter membrane, and filling after detecting that the endotoxin is qualified.
3) And (3) freeze-drying process:
a) pre-freezing: placing a penicillin bottle filled with a recombinant human tumor necrosis factor receptor-Fc fusion protein solution on a clapboard of a freeze dryer, pre-freezing at a set temperature of 5 ℃, keeping for 1h after a set time of 30min reaches a preset temperature, cooling to-60 ℃, keeping for 6h after a set time of 10min reaches the preset temperature, and freezing;
b) sublimation drying: heating the partition plate system to-25 ℃ at a set temperature, maintaining the preset temperature for 10h, maintaining the preset temperature for 25h, and maintaining the vacuum degree for 0.2mBar to complete sublimation drying;
c) and (3) drying again: and (3) heating the temperature of the clapboard system to 30 ℃, setting the temperature for 100min to reach the preset temperature, maintaining the vacuum degree for 0.2mBar, and drying after 10 h.
Comparative example 9
1) Prescription
2) The preparation process comprises the following steps: weighing a prescription amount of Tris, dissolving the Tris with a proper amount of water for injection, adjusting the pH to 8.0 by using hydrochloric acid, sampling, detecting the pH and detecting whether the endotoxin is qualified, accurately weighing the prescription amount of arginine, sucrose, hydroxyethyl starch, Tween 20 and recombinant human tumor necrosis factor receptor-Fc fusion protein stock solution, adding the mixture into a Tris/HCl buffer solution, and uniformly mixing to obtain a semi-finished product solution; and (4) performing sterile filtration on the semi-finished product liquid by using a 0.22-micron filter membrane, and filling after detecting that the endotoxin is qualified.
3) And (3) a freeze-drying process:
a) pre-freezing: placing a penicillin bottle filled with a recombinant human tumor necrosis factor receptor-Fc fusion protein solution on a clapboard of a freeze dryer, pre-freezing at a set temperature of 5 ℃, keeping for 1h after a set time of 30min reaches a preset temperature, cooling to-60 ℃, keeping for 6h after a set time of 10min reaches the preset temperature, and freezing;
b) sublimation drying: heating the partition plate system to-25 ℃ at a set temperature, maintaining the preset temperature for 10h, maintaining the preset temperature for 25h, and maintaining the vacuum degree for 0.2mBar to complete sublimation drying;
c) and (3) drying again: and (3) heating the temperature of the clapboard system to 30 ℃, setting the temperature for 100min to reach the preset temperature, maintaining the vacuum degree for 10h for drying, and simultaneously maintaining the vacuum degree for 0.2mBar, thus finishing the freeze-drying.
Comparative example 10
1) Prescription
2) The preparation process comprises the following steps: weighing a prescription amount of Tris, dissolving the Tris with a proper amount of water for injection, adjusting the pH to 8.0 by using hydrochloric acid, sampling, detecting the pH and detecting whether the endotoxin is qualified, accurately weighing the prescription amount of arginine, sucrose, hydroxyethyl starch, Tween 20 and recombinant human tumor necrosis factor receptor-Fc fusion protein stock solution, adding the mixture into a Tris/HCl buffer solution, and uniformly mixing to obtain a semi-finished product solution; and (4) performing sterile filtration on the semi-finished product liquid by using a 0.22-micron filter membrane, and filling after detecting that the endotoxin is qualified.
3) And (3) freeze-drying process:
a) pre-freezing: placing a penicillin bottle filled with a recombinant human tumor necrosis factor receptor-Fc fusion protein solution on a clapboard of a freeze dryer, pre-freezing at a set temperature of 5 ℃, keeping for 1h after a set time of 10min reaches a preset temperature, cooling to-35 ℃, keeping for 2h after a set time of 10min reaches the preset temperature, and freezing;
b) sublimation drying: heating the partition plate system to-25 ℃ at a set temperature, maintaining the preset temperature for 10h, maintaining the vacuum degree for 0.2mBar, and completing sublimation drying;
c) and (3) drying again: and (3) heating the temperature of the clapboard system to 30 ℃, setting the temperature for 100min to reach the preset temperature, maintaining the vacuum degree for 5h for drying, and simultaneously maintaining the vacuum degree for 0.2mBar, thus finishing the freeze-drying.
Verification examples
1. Differential calorimetric scanning (DSC)
The melting temperatures T of the semi-finished solutions of the recombinant human TNF receptor-Fc fusion proteins of examples 1-4 and comparative examples 1-10 were determined using a MicroCalTM VP-Capillary DSC instrument at the instrument programmed temperature m The value is obtained. Diluting the sample with corresponding buffer solution until the concentration of the recombinant human tumor necrosis factor receptor-Fc fusion protein is 1mg/mL, adding 350 μ L of the sample into a sample well of a 96-well plate,and adding 350 mu L of corresponding buffer solution into the buffer hole, wherein the scanning temperature is set to be 20-90 ℃, and the scanning speed is 60 ℃/hr. Data analysis MicroCal VP-Capillary DSC automatic analysis software was used. The results are shown in Table 1.
TABLE 1T of recombinant human TNF receptor-Fc fusion protein m Value result
| Sample (I) | T m onset /℃ | T m1 /℃ | T m2 /℃ |
| Example 1 | 40.76 | 64.08 | 73.24 |
| Example 2 | 39.94 | 63.26 | 73.27 |
| Example 3 | 39.05 | 62.37 | 73.33 |
| Example 4 | 40.07 | 63.89 | 73.92 |
| Comparative example 1 | 30.37 | 56.69 | 72.92 |
| Comparative example 2 | 34.31 | 57.63 | 72.83 |
| Comparative example 3 | 34.31 | 57.63 | 73.53 |
| Comparative example 4 | 33.57 | 56.89 | 73.41 |
| Comparative example 5 | 32.06 | 55.38 | 73.53 |
| Comparative example 6 | 34.37 | 57.69 | 73.11 |
| Comparative example 7 | 36.41 | 59.73 | 73.14 |
| Comparative example 8 | 35.18 | 58.5 | 73.2 |
| Comparative example 9 | 36.69 | 60.01 | 73.79 |
| Comparative example 10 | 36.15 | 59.47 | 72.79 |
T m The value represents the midpoint temperature of the thermal transition of the protein, and there may be multiple Ts for a multidomain protein m The value, which is an important indicator of the thermal stability of a protein, the upward shift representing an increase in stability, can be used to assess the tendency of oligomers and aggregates to form. T is m onset Is the temperature at which unfolding starts, T m1 Is the first transition temperature, T m2 Is the second transition temperature. Comparative examples 1-3 are prior art formulations and the results in Table 1 show the highest T of the prior art m onset About 34.31 ℃ and T of the invention m onset The maximum temperature can reach 40.76 ℃, and is improved by 6.45 ℃ compared with the prior art. Comparative examples 4 to 10 all compare T of the present invention after changing the prescription m onset Low.
2. Stability test
Three batches of samples were prepared according to examples 1-4 and comparative examples 1-10, respectively, and 60 bottles were taken from each batch, and the storage stability was examined using accelerated stability tests and long-term tests.
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 long-term test is carried out at the temperature of 2-8 ℃, and the long-term test is carried out according to stability key examination items at the end of 0 month, 3 months, 6 months, 12 months and 24 months respectively; (the water content is measured by a Mettler-Zaliduo DL37 Karl Fischer titrator according to the coulomb method specified in the third general rule of 2015 edition of Chinese pharmacopoeia, and the purity is measured by a general rule 0514 molecular array chromatography and a general rule 0542 capillary electrophoresis), the result shows that no significant change exists in comparison with 0 month, the comparison ratio has more stable characteristics, and the result is shown in tables 2 and 3.
TABLE 2.2-8 deg.C long-term stability test results
TABLE 3.25 ℃ accelerated stability test results
The stability data results in tables 2 and 3 show that the lyophilized formulations of examples 1-4 are white loose bodies, clear and transparent after reconstitution, and slightly opalescent; the water content is less than 1.00%; the monomer content is not less than 98.6 percent after being stored for 24 months at the temperature of 2-8 ℃; the monomer content can still reach the effect of ≧ 95% when the product is stored for 12 months at 25 ℃. In the prior art, after the comparative examples 1 to 3 are stored at 2 to 8 ℃ for 24 months, the monomer contents are respectively 87.9%, 98.1% and 97.9%, and the monomer contents are respectively 86.7%, 93.5% and 92.0% after the comparative examples are stored at 25 ℃ for 12 months, the storage time under the accelerated experiment condition is far shorter than that of the invention, and the stability of the freeze-dried preparation is also far shorter than that of the invention after the formula freeze-drying process is changed compared with the comparative examples 4 to 10.
Claims (4)
1. A freeze-dried preparation of recombinant human tumor necrosis factor receptor-Fc fusion protein is characterized in that the freeze-dried preparation consists of the following components:
wherein the amino acid is one or more of arginine, cysteine, threonine and lysine; the buffer is Tris/HCl; the filler is one or more of sucrose, trehalose, mannitol and sorbitol; the surfactant is tween 20.
2. The lyophilized formulation of recombinant human tumor necrosis factor receptor-Fc fusion protein according to claim 1, wherein said lyophilized formulation comprises the following components:
3. a freeze-drying process of the recombinant human tumor necrosis factor receptor-Fc fusion protein freeze-dried preparation according to any one of claims 1-2, characterized in that the freeze-drying is carried out by adopting the following steps:
1) pre-freezing: pre-freezing the freeze-dried sample for 0.5-1h at 5 ℃, cooling to-60 ℃ to-40 ℃, keeping for 4-6h for freezing,
2) sublimation drying: maintaining the vacuum degree, setting the temperature to be between 25 ℃ below zero and 20 ℃ below zero for 10 hours, maintaining the temperature for 20 to 30 hours,
3) and (3) drying again: maintaining the vacuum degree, heating to 30 deg.C, maintaining for 8-10h, resolving, drying, and lyophilizing.
4. The process for lyophilizing a lyophilized preparation of recombinant human TNF-Fc fusion protein according to claim 3, wherein the lyophilization is carried out by the following steps:
1) pre-freezing: placing a penicillin bottle filled with a recombinant human tumor necrosis factor receptor-Fc fusion protein solution on a clapboard of a freeze dryer, pre-freezing at a set temperature of 5 ℃, keeping the preset temperature for 0.5-1h after setting for 10-30 min, cooling to-60 ℃ to-40 ℃, keeping the preset temperature for 5-10 min, and freezing for 4-6 h;
2) sublimation drying: heating the partition plate system to a set temperature of minus 25 ℃ to minus 20 ℃, keeping the temperature for 20-30h after a set time of 10h is up to the set temperature, and simultaneously keeping the vacuum degree of 0.2mBar, thereby completing sublimation drying;
3) and (3) drying again: and (3) heating the temperature of the clapboard system to 30 ℃, setting the time for 80-120 min to reach the preset temperature, maintaining for 8-10h for drying, maintaining the vacuum degree of 0.2mBar, and finishing the freeze-drying.
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