Non-final sterilization production process for improving stability of calcium folinate injection and application

Abstract

The application belongs to the technical field of pharmaceutical preparations, and particularly relates to a non-final sterilization production process for improving the stability of calcium folinate injection and application. The non-final sterilization production process for improving the stability of calcium folinate injection comprises the following steps: (1) dissolving calcium folinate in water for injection, adding sodium dihydrogen phosphate and disodium edetate, and adjusting the pH of the drug solution by using hydrochloric acid or sodium hydroxide solution; (2) filtering the drug solution through a 0.45-micron pre-filter filter core and a 0.22-micron terminal sterilization filter core in sequence; and (3) filling the drug solution into a container and sealing the container in an environment with a cleanliness of grade A. The process does not comprise a final sterilization step. The application reduces the risk of drug efficacy reduction and safety caused by drug degradation. Although the whole process does not comprise a final sterilization step, the sterility of the injection can be effectively guaranteed, and the risk of microbial contamination can be reduced. The production process is optimized, the production cost is reduced, and the application scenarios of the injection in clinical treatment are widened.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical preparation technology, specifically relating to a non-terminally sterilized production process and its application for improving the stability of calcium leucovorin injection. Background Technology

[0002] Leucovorin calcium, as a crucial adjuvant antitumor therapy drug in clinical applications, plays an indispensable role in enhancing the efficacy of fluorouracil chemotherapy drugs and detoxifying methotrexate overdose, especially in high-dose chemotherapy scenarios, where its position is irreplaceable.

[0003] However, the molecular structure of leucovorin calcium contains a p-aminobenzoyl functional group, which is chemically highly reactive. Many external factors, such as pH, temperature, light, and metal ions, can promote its hydrolysis or oxidation. This not only leads to the degradation of the active ingredient but also generates pteroic acid impurities. The formation of these impurities severely impacts the efficacy and safety of the drug, thus greatly limiting the widespread clinical application of leucovorin calcium. Improving the stability of leucovorin calcium preparations has become a key technical challenge that urgently needs to be overcome in this field.

[0004] In terms of formulation processes, traditional non-terminally sterilized processes, such as aseptic filling, while avoiding the degradation effects of high temperatures on drugs, rely entirely on the aseptic control of the production environment because they lack a terminal sterilization process. This places extremely high demands on the cleanroom classification, the sterilization effectiveness of equipment, and the stability of the filling process. Furthermore, compatibility issues between excipients and active pharmaceutical ingredients, as well as unavoidable temperature fluctuations during filling, can further exacerbate drug degradation or introduce the risk of microbial contamination, making it difficult to effectively control product quality. While existing technology (CN118059042A) discloses a non-terminally sterilized process, its pH range (6.5-8.5) is too wide; the degradation rate of calcium folinate increases sharply when pH > 6.7. Moreover, it fails to recognize the synergistic chelating effect of disodium edetate and sodium dihydrogen phosphate at pH 6.4-6.6; when the stabilizer cannot work synergistically, impurities exceed the standard by 50%.

[0005] In contrast, while terminal sterilization can kill microorganisms through high temperatures and provide reliable sterility for products, the heat-sensitive nature of calcium leucovorin makes it highly susceptible to degradation during high-temperature sterilization. This makes it difficult to ensure both sterilization effectiveness and drug stability in actual production, and the contradiction between the two urgently needs to be resolved.

[0006] Currently, there is an urgent clinical demand for leucovorin calcium in China. However, existing preparation processes generally suffer from poor stability, and no products have yet passed consistency evaluation. This means that there is a significant technological gap in the market for this product. Therefore, developing a process that can effectively control microbial contamination while maximizing drug stability has become a crucial technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0007] The purpose of this invention is to provide a non-terminally sterile manufacturing process and application for improving the stability of leucovorin calcium injection. Based on a deep understanding of this situation, this invention, through the synergistic optimization of precise pH control, excipient system selection, and low-temperature filling technology, significantly improves the stability of leucovorin calcium while ensuring aseptic safety, providing an innovative solution to meet the needs of high-quality clinical medication.

[0008] A non-terminally sterile manufacturing process for improving the stability of calcium leucovorin injection includes the following steps:

[0009] (1) Dissolve calcium folinate in water for injection, add 0.5-1.0 mg / ml sodium dihydrogen phosphate and 0.05-0.1 mg / ml disodium edetate, dissolve under nitrogen protection at 23-27℃, and adjust the pH to 6.4-6.6 with hydrochloric acid or sodium hydroxide; (2) Pass the drug solution sequentially through a 0.45 μm pre-filter cartridge made of PVDF material and a 0.22 μm terminal sterilization filter cartridge made of hydrophilic PES material, with a pressure difference of 0.1-0.3 MPa between the two stages of filtration; (3) At a temperature of 10-18℃, a pressure difference ≥10 Pa, and airborne bacteria ≤1 CFU / m³, the solution is purified. 3 In a Grade A environment with settling bacteria ≤1 CFU / plate, fill and seal at a filling speed of 5-8 ml / plate; the process does not include a final sterilization step, and steps (1)-(3) are carried out under nitrogen protection throughout.

[0010] Furthermore, the concentration of sodium dihydrogen phosphate in step (1) is 0.5-1.0 mg / ml, and the concentration of disodium edetate is 0.05-0.1 mg / ml.

[0011] Furthermore, the dissolution process described in step (1) is carried out under nitrogen protection, and the dissolution temperature is controlled at 23-27℃.

[0012] Furthermore, the 0.45μm pre-filter cartridge mentioned in step (2) is made of polyvinylidene fluoride (PVDF), and the 0.22μm terminal sterilization filter cartridge is made of hydrophilic polyethersulfone (PES). The pressure difference between the two filtration stages is controlled at 0.1-0.3MPa.

[0013] Furthermore, in step (3), the filling environment pressure difference is ≥10 Pa, and the airborne bacteria concentration is ≤1 CFU / m³. 3 Settling bacteria ≤1 CFU / plate, using Φ90mm culture plates, exposure time 4 hours.

[0014] Furthermore, after filling is completed in step (3), aluminum-plastic composite caps are immediately used for crimping and sealing. The sealing pressure is 80-120N and the crimping time is ≤5 seconds / bottle.

[0015] A calcium folinate injection solution, wherein the calcium folinate content is ≥99.5% and the total related substances are ≤0.65% when stored at 25℃±2℃ and RH60%±10% for 12 months; and the calcium folinate content is ≥98.5% and the total related substances are ≤0.85% when accelerated storage is carried out at 40℃±2℃ and RH75%±5% for 6 months.

[0016] The application of a calcium leucovorin injection in the preparation of a methotrexate antidote, wherein the administration regimen of the antidote is as follows: intravenous injection of calcium leucovorin 5-15 mg / m² within 24-48 hours after methotrexate administration. 2 Repeat every 6 hours until the serum methotrexate concentration is <0.05 μmol / L.

[0017] The application of a calcium leucovorin injection in the preparation of fluorouracil combined with chemotherapy drugs, wherein the dosage of fluorouracil in the application is 300-500 mg / m². 2 The dosage of calcium leucovorin is 20-500 mg / m². 2 Both are used in combination via intravenous infusion.

[0018] The application of a calcium leucovorin injection in the preparation of an antidote for folic acid antagonists, wherein the folic acid antagonist includes methotrexate, pyrimethamine, or trimethoprim, and when used as an antidote for methotrexate overdose, the calcium leucovorin is administered within 24-48 hours after methotrexate administration, at a dose of 5-15 mg / m². 2 Once every 6 hours.

[0019] The beneficial effects of this invention are as follows:

[0020] 1. Enhanced drug stability: By precisely controlling the pH of the drug solution to 6.3-6.7 and adding specific concentrations of sodium dihydrogen phosphate and disodium edetate, the hydrolysis and oxidation of leucovorin calcium are effectively inhibited. After 12 months of storage at 25℃±2℃ and RH 60%±10%, the leucovorin calcium content remains ≥99%, and the total related substances are ≤0.8%, ensuring the quality stability of the drug during long-term storage and reducing the risk of reduced efficacy and safety due to drug degradation.

[0021] 2. Ensuring sterility: A 0.45μm polyvinylidene fluoride (PVDF) pre-filter and a 0.22μm hydrophilic polyethersulfone (PES) terminal sterilization filter are used, with strict control of the pressure difference between the two stages at 0.1-0.3 MPa. This is combined with a temperature ≤18℃, cleanliness level A (pressure difference ≥10Pa, airborne bacterial concentration ≤1 CFU / m³). 3 The solution is filled in an environment with a settling bacteria count of ≤1 CFU / plate and a Φ90mm culture dish exposed for 4 hours. The solution is then immediately sealed with an aluminum-plastic composite cap under specific pressure (80-120N) and time (≤5 seconds / bottle). Although there is no final sterilization step in the whole process, it can effectively ensure the sterility of the injection solution and reduce the risk of microbial contamination.

[0022] 3. Optimized production process: This non-terminally sterilized production process avoids the damage to the stability of calcium folinate caused by high temperatures in the terminal sterilization process. At the same time, compared with the traditional non-terminally sterilized process, the parameters of each production link have been finely optimized. For example, the dissolution process is carried out under nitrogen protection and at 23-27℃, making the production process more scientific and reasonable, improving production efficiency and product quality stability. Moreover, the proposed batch sizes of different specifications (10ml:100mg specification 10,000 bottles / batch, 5ml:50mg specification 20,000 bottles / batch) are adapted to the needs of industrial production.

[0023] 4. Reduce production costs: On the one hand, avoiding high-temperature sterilization steps can save energy consumption and related equipment costs; on the other hand, stable production processes reduce scrap and rework costs caused by unstable product quality, improve production efficiency, and make products more cost-competitive in the market.

[0024] 5. Expanding Clinical Applications: The leucovorin calcium injection prepared using this process has clearly defined dosages and methods of application in fluorouracil-based chemotherapy and as an antidote for folic acid antagonists (such as methotrexate, pyrimethamine, or trimethoprim). In fluorouracil-based chemotherapy, a clearly defined dosage range (fluorouracil 300-500 mg / m²) is established. 2 Calcium folinate 20-500mg / m² 2 This provides clinicians with accurate medication guidance; in cases of methotrexate overdose detoxification, specific administration times (within 24-48 hours after administration) and dosages (5-15 mg / m²) are indicated. 2 (Every 6 hours), which broadens its application scenarios in clinical treatment and provides patients with more effective treatment methods.

[0025] 6. Improved medication safety: Stable drug quality and strict aseptic protection reduce adverse reactions caused by impurities generated by drug degradation and microbial contamination, significantly improving the safety of patients using this injection for adjuvant chemotherapy or detoxification treatment and reducing medication risks. Attached Figure Description

[0026] Figure 1 The process flow diagram for preparing calcium folinate injection.

[0027] Figure 2 The graph shows the results of validation for aseptic assurance, sealing, and filtration integrity. Detailed Implementation

[0028] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0029] Example 1

[0030] Preparation of 10ml:100mg leucovorin calcium injection

[0031] 1. Solution preparation process

[0032] First, prepare 80L of water for injection that has been filtered through a 0.22μm filter. Purge the water with nitrogen gas and bubble for 30 minutes to remove dissolved oxygen and create a low-oxygen environment for subsequent drug dissolution.

[0033] Next, accurately weigh 1.0 kg of calcium folinate, 50 g of sodium dihydrogen phosphate (concentration of 0.625 mg / ml) and 5 g of disodium edetate (concentration of 0.0625 mg / ml), and add them to the above-mentioned water for injection.

[0034] Under nitrogen protection, the mixture was stirred in an environment at 25℃±1℃ to ensure complete dissolution of all components. Then, the pH of the solution was precisely adjusted to 6.5 using 0.1M hydrochloric acid. This precise control of the pH range is crucial for maintaining the stability of calcium leucovorin.

[0035] 2. Filtration process

[0036] The prepared medicine solution passes through two filtration processes in sequence:

[0037] The particles first pass through a 0.45μm PVDF pre-filter, during which the pressure difference is controlled at 0.2MPa. This pre-filter can initially intercept larger particulate impurities, reducing the burden on subsequent sterilization filtration.

[0038] The solution was then passed through a 0.22μm hydrophilic PES sterilization filter cartridge, with the pressure differential controlled at 0.15MPa. This step ensured the solution met sterility requirements. After filtration, a sterile sample was taken for testing, showing a pH of 6.52 and passing the visible foreign matter test.

[0039] 3. Filling and sealing process

[0040] Filling operations shall be carried out under conditions that meet Class A environmental standards. The specific requirements for Class A environment are: temperature controlled between 16°C and 18°C, pressure differential maintained between 10 Pa and 15 Pa, and airborne bacteria content of 0 CFU / m³. 3 The settling bacteria content was 0 CFU / Φ90 dish-4h. Strict environmental control can effectively reduce the risk of microbial contamination.

[0041] The medication was dispensed into 10ml vials at a filling speed of 6ml / s, with a production batch of 10,000 vials.

[0042] Immediately after filling, the vials are crimped and sealed. An aluminum-plastic composite cap is used, with the sealing pressure controlled at 100N and the crimping time at 3 seconds per vial, ensuring a good seal and preventing external factors from affecting the medication.

[0043] Example 2

[0044] Preparation of 5ml:50mg leucovorin calcium injection

[0045] 1. Solution preparation: Weigh 0.5 kg of leucovorin calcium according to the required specifications to prepare 50 mg / 5 ml injection solution. The production batch is 20,000 bottles.

[0046] 2. Adjustment of excipients: Adjust the concentration of sodium dihydrogen phosphate to 0.8 mg / ml (i.e., 40 g / 50 L) and the concentration of disodium edetate to 0.08 mg / ml (i.e., 4 g / 50 L) to meet the excipient requirements of different specifications of injection solutions.

[0047] 3. Filling and Sealing: The filling speed was set to 7 ml / s, the capping pressure was adjusted to 90 N, and the remaining operating conditions were consistent with those in Example 1. This method was used to verify the feasibility and stability of different product specifications under similar processes.

[0048] See the process flow chart for the preparation of calcium leucovorin injection. Figure 1 .

[0049] Table 1: Stability Data

[0050] Test conditions time content Total amount of related substances 25℃±2℃ RH 60% ± 10% 0 months 100.3％ 0.10％ 25℃±2℃ RH 60% ± 10% 12 months 99.6％ 0.65％ 40℃±2℃ RH 75% ± 5% in June 98.5％ 0.85％

[0051] Application Example 1: Fluorouracil Combined with Chemotherapy

[0052] 1. Patient information: Colorectal cancer patients with a body surface area (BSA) of 1.8 m² were selected. 2 .

[0053] 2. Treatment regimen: A combination chemotherapy regimen of leucovorin calcium and fluorouracil was adopted, with a specific dosage of leucovorin calcium 400 mg / m². 2 (i.e., 720 mg) and fluorouracil 450 mg / m² 2 (i.e., 810 mg). Administered via intravenous infusion once weekly for four consecutive weeks. This application case validates the applicability and effectiveness of the product of this invention in a specific combination chemotherapy setting.

[0054] Application Example 2: Methotrexate Detoxification

[0055] 1. Timing of detoxification: Detoxification treatment should begin 30 hours after the patient has experienced a methotrexate overdose.

[0056] 2. Medication regimen: Administer leucovorin calcium as an antidote, at a dose of 10 mg / m². 2 (Patient's BSA = 1.6m) 2 The single dose is 16 mg, administered intravenously every 6 hours until the serum methotrexate concentration is <0.05 μmol / L. This application example demonstrates the practical effectiveness of the product of this invention in methotrexate detoxification.

[0057] Comparative Example 1

[0058] 1. Adjustment of the method: During the preparation process, the pH value was adjusted to 7.2, and other operations remained the same as in Example 1.

[0059] 2. Results Analysis: After 6 months of testing at 40℃, the results showed that the content decreased to 97.1% (below 98%), while related substances increased to 1.3% (above 1.0%). This demonstrates that exceeding the pH range protected in this application will have a significant adverse effect on the stability of the leucovorin calcium injection.

[0060] Comparative Example 2

[0061] 1. Adjustment of the plan: During the preparation of the solution, the solution is directly exposed to the air, and the nitrogen protection measures are cancelled.

[0062] 2. Results Analysis: Pre-filling testing revealed that the content of related substances reached 0.41%, while in Example 1 this value was only 0.10%. This indicates that nitrogen protection is crucial for inhibiting the formation of related substances and maintaining product stability.

[0063] Comparative Example 3

[0064] 1. Adjustment of the scheme: Increase the filling environment temperature to 25℃ (above 18℃), and the rest of the operation is the same as in Example 1.

[0065] 2. Results Analysis: Crystallization was observed 24 hours after filling. This indicates that the filling temperature exceeded the protection range of this application, which caused calcium leucovorin to crystallize in the injection solution, affecting product quality.

[0066] Comparative Example 4

[0067] 1. Solution adjustment: Change the terminal filter element from hydrophilic PES to hydrophobic PVDF, while other operating steps remain unchanged from Example 1.

[0068] 2. Results Analysis: Testing revealed that calcium folinate was lost due to adsorption on the hydrophobic PVDF filter element, with a loss rate of 9.2%, while the loss rate was less than 0.5% when using a PES filter element. This demonstrates the importance of specific filter materials in reducing drug loss and ensuring product quality.

[0069] Comparative Example 5

[0070] 1. Adjustment of the method: Disodium edetate was not added during the preparation process, and other conditions were the same as in Example 1.

[0071] 2. Results Analysis: After 12 months of testing at 25℃, the content of related substances reached 1.2% (higher than 0.8%). This indicates that disodium edetate is indispensable for inhibiting the formation of related substances and maintaining the stability of leucovorin calcium injection.

[0072] Key validation data

[0073] 1. Aseptic assurance: Validation was achieved through a culture medium filling test, with a total of 3 batches of 3000 bottles per batch. The test results showed a contamination rate of 0 / 9000, fully demonstrating the reliability of this process in ensuring aseptic performance.

[0074] 2. Sealing performance: The sealing performance of 300 samples was tested using the vacuum decay method. Under the pressure of 80-120N, the leakage rate was 0%, indicating that the product has good sealing performance and can effectively prevent the influence of external factors on product quality.

[0075] 3. Filtration Integrity: Testing of the PES filter element showed an inflated point >3.8 bar (standard requirement >3.5 bar), proving that the PES filter element maintains its integrity during filtration and can effectively perform its sterilization function. See the graph for the verification results of aseptic assurance, sealing performance, and filtration integrity. Figure 2 .

[0076] Comparative Example 6:

[0077] 1. Scheme adjustment: pH 7.0, no sodium dihydrogen phosphate / disodium edetate, no nitrogen gas throughout the process, the rest is the same as in Example 1.

[0078] 2. Results analysis: After 6 months of accelerated treatment at 40℃, the content was 97.0% and the impurities were 1.5%, which is far inferior to that of the present invention (98.5% / 0.85%).

[0079] The above description is merely a preferred embodiment of the present invention; however, the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and its improved concepts, should be covered within the scope of protection of the present invention.

Claims

1. A non-terminally sterilized production process for improving the stability of leucovorin calcium injection, characterized in that, Includes the following steps: (1) Dissolve calcium folinate in water for injection, add 0.5-1.0 mg / ml sodium dihydrogen phosphate and 0.05-0.1 mg / ml disodium edetate, dissolve under nitrogen protection at 23-27℃, and adjust the pH to 6.4-6.6 with hydrochloric acid or sodium hydroxide; (2) Pass the drug solution sequentially through a 0.45 μm pre-filter cartridge made of PVDF material and a 0.22 μm terminal sterilization filter cartridge made of hydrophilic PES material, with a pressure difference of 0.1-0.3 MPa between the two stages of filtration; (3) At a temperature of 10-18℃, a pressure difference ≥10 Pa, and airborne bacteria ≤1 CFU / m³, the solution is purified. 3 In a Grade A environment with settling bacteria ≤1 CFU / plate, fill and seal at a filling speed of 5-8 ml / plate; the process does not include a final sterilization step, and steps (1)-(3) are carried out under nitrogen protection throughout.

2. The non-terminally sterilized production process for improving the stability of leucovorin calcium injection according to claim 1, characterized in that, The concentration of sodium dihydrogen phosphate mentioned in step (1) is 0.625-0.8 mg / ml, and the concentration of disodium edetate is 0.0625-0.08 mg / ml.

3. The non-terminally sterilized production process for improving the stability of leucovorin calcium injection according to claim 1, characterized in that, The dissolution process described in step (1) is carried out under nitrogen protection, and the dissolution temperature is controlled at 25℃±1℃.

4. The non-terminally sterilized production process for improving the stability of leucovorin calcium injection according to claim 1, characterized in that, The 0.45μm pre-filter cartridge mentioned in step (2) is made of polyvinylidene fluoride (PVDF) material, with a pressure difference controlled at 0.2MPa. The 0.22μm terminal sterilization filter cartridge is made of hydrophilic polyethersulfone (PES) material, with a pressure difference controlled at 0.15MPa.

5. The non-terminally sterilized production process for improving the stability of leucovorin calcium injection according to claim 1, characterized in that, The filling environment pressure difference in step (3) is 10-15 Pa, and the airborne bacteria concentration is ≤1 CFU / m³. 3 Settling bacteria ≤1 CFU / plate, using Φ90mm culture plates, exposure time 4 hours.

6. The non-terminally sterilized production process for improving the stability of leucovorin calcium injection according to claim 1, characterized in that, After filling in step (3), immediately use an aluminum-plastic composite cap for crimping and sealing. The sealing pressure is 80-120N and the crimping time is ≤5 seconds / bottle.

7. The leucovorin calcium injection prepared using the non-terminally sterilized production process for improving the stability of leucovorin calcium injection according to any one of claims 1-6, characterized in that, The injection solution, when stored at 25℃±2℃ and RH60%±10% for 12 months, has a calcium folinate content ≥99.5% and a total related substances content ≤0.65%; when stored at 40℃±2℃ and RH75%±5% for 6 months, the calcium folinate content ≥98.5% and the total related substances content ≤0.85%.

Images