Stability-enhanced preparation method of ulinastatin freeze-dried powder injection

Abstract

The invention discloses a stability-enhanced preparation method of ulinastatin freeze-dried powder injection, which belongs to the technical field of freeze-dried powder injection preparation, and comprises the following steps: ulinastatin is dissolved in water for injection, an excipient system is added to form a prescription solution to be freeze-dried, the excipient system at least comprises a crystal form skeleton excipient and a glassy state protection excipient, and the freeze-dried prescription solution is freeze-dried. Adding a buffer system to regulate and control the pH value of the prescription solution to be freeze-dried in a target window, and regulating and controlling the ion strength to enable the absolute value of the pH value drift under the freezing enrichment condition to be not greater than 0.5 pH unit; the prescription solution to be freeze-dried is sterilized, filtered and aseptically filled into a penicillin bottle, and the penicillin bottle enters a freeze-drying cavity after being half plugged; by introducing synchronous nucleation control of pressure triggering or external ice crystal triggering in the freezing stage, the nucleation time of each penicillin bottle tends to be consistent with the ice crystal structure, so that the problems of pore structure difference and large intra-batch fluctuation caused by inter-bottle nucleation randomness in the existing freeze-drying process are solved.

Description

Technical Field

[0001] This invention relates to the field of lyophilized powder injection technology, and more specifically, to a method for enhancing the stability of ulinastatin lyophilized powder injection. Background Technology

[0002] Ulinastatin is a commonly used protein drug preparation in clinical practice. The existing lyophilized lyophilized powder injection of lyophilized ...

[0003] However, during the freeze-drying process, inconsistencies in the nucleation time between bottles and differences in ice crystal structure can easily occur during the freezing stage. This leads to different local concentrations of solute in the freeze enrichment zone and causes pH drift in the microenvironment. Consequently, inconsistencies between the pore structure and the crystallization state of the excipient are formed during the subsequent drying process. This further makes it difficult for residual water and water activity to stably enter the target window and amplifies the differences within the batch.

[0004] Meanwhile, existing freeze-drying processes mostly use preset shelf temperatures and chamber pressure programs for primary and secondary drying, lacking real-time closed-loop constraints and endpoint criterion control based on product temperature and chamber pressure signals. This can easily lead to local overheating or collapse, and cause protein aggregation or reduced activity.

[0005] In addition, after reconstitution, ulinastatin lyophilized products usually need to be further diluted with infusion medium and administered via infusion tubing. Existing processes and evaluations focus on conventional indicators such as the appearance and content of the finished product, and have failed to establish a stability control and verification pathway for the adsorption and shearing effects during the infusion stage after reconstitution. This may lead to risks such as activity decay, increased turbidity, or increased aggregates in the infusion solution under specified time and temperature conditions.

[0006] Therefore, how to quantitatively control the nucleation freezing events, solid phase structure and crystallization state, as well as the residual moisture and water activity at the drying endpoint throughout the freeze-drying process, and further incorporate the infusion stability after reconstitution into a verifiable index system, has become a key technical problem that urgently needs to be solved in the field of enhancing the stability of ulinastatin freeze-dried powder injection. Summary of the Invention

[0007] To address the problems mentioned in the background section, the present invention provides the following technical solution: A method for enhancing the stability of ulinastatin lyophilized powder for injection includes the following steps: S1, Ulinastatin is dissolved in water for injection, added to the excipient system to form a lyophilized formulation solution. The excipient system includes at least a crystal framework excipient and a glassy protective excipient. A buffer system is added to adjust the pH of the lyophilized formulation solution to the target window, and the absolute value of the pH drift under freezing enrichment conditions is not greater than 0.5 pH units by adjusting the ionic strength. S2, the freeze-dried prescription solution is sterilized and filtered and aseptically filled into vials, then partially stoppered and placed into the freeze-drying chamber; S3, the vial is pre-cooled to the nucleation trigger window, and pressure-triggered nucleation is used to achieve synchronous nucleation. After nucleation, the vial is further cooled to the final freezing temperature to form a uniform ice crystal structure and inhibit local excessive enrichment of solute. S4. After final freezing, a double annealing process is performed. The first annealing temperature window is used for ice crystal rearrangement and channel homogenization, and the second annealing temperature window is used for the full crystallization and crystal form locking of the crystal skeleton excipient. After double annealing, the temperature is lowered again to solidify the solid phase structure. S5 performs primary and secondary drying, and collects process monitoring signals, including at least product temperature and chamber pressure signals, in real time during the drying process. After comparing the process monitoring signals with preset thresholds, the shelf temperature and chamber pressure are dynamically adjusted to ensure that the product temperature is always lower than the collapse critical temperature or eutectic critical temperature of the freeze-dried formulation solution, and the residual moisture content and water activity of the finished product are controlled within the preset target window. S6, complete the plugging and sealing and obtain the lyophilized product. After reconstitution, the lyophilized product is further diluted with the infusion medium to form an infusion solution. The infusion solution meets the stability index threshold at the preset verification temperature and within the preset verification time. The stability index includes at least the activity retention rate not lower than the preset lower limit, visible foreign matter and turbidity meeting the limit, aggregate increment not higher than the preset upper limit, and pH change not exceeding the preset range. Furthermore, in step S3, pressure-triggered nucleation is used to achieve synchronous nucleation, specifically as follows: The partially stoppered vials were pre-cooled along the shelf to the nucleation trigger window temperature, which was -12°C to -4°C. Then, the pressure in the freeze-drying chamber was switched from the pre-cooling steady-state pressure to the nucleation trigger pressure and maintained for a preset nucleation holding time, which was 30 Pa to 200 Pa and 5 s to 60 s. After that, the chamber pressure was restored to the pre-cooling steady-state pressure and the temperature was further reduced to the final freezing temperature. The criterion for synchronous nucleation was that the temperature rebound amplitude caused by the release of latent heat in the temperature curve of each vial product was 0.5°C to 3.0°C and the maximum time difference between vials at the start of the rebound was no more than 20 s.

[0008] Further, the double annealing treatment in step S4 includes holding at the final freezing temperature for 10 to 60 minutes, then raising the shelf temperature to the first annealing temperature of -25°C to -15°C and holding for 0.5 to 4 hours to allow ice crystal rearrangement and ensure that the coefficient of variation of the pore size distribution of the freeze-dried block is no greater than 0.35. Subsequently, the shelf temperature is raised to the second annealing temperature of -12°C to -6°C and held for 0.25 to 2 hours to ensure that the crystallinity of the crystalline framework excipient is no less than 80% and the difference in crystallinity between bottles is no greater than 10%. After the double annealing is completed, the shelf temperature is lowered to no higher than -35°C to solidify the solid phase structure. The coefficient of variation of the pore size distribution is obtained by statistical analysis of the pore size of the cross-section of the freeze-dried block after annealing, and the crystallinity is determined by differential scanning calorimetry or powder X-ray diffraction.

[0009] Furthermore, in step S5, during the primary and secondary drying processes, a product temperature safety margin is first set based on the collapse critical temperature or eutectic critical temperature of the formulation solution to be freeze-dried. This safety margin ranges from 1°C to 5°C. The product temperature is constrained by the condition that it does not exceed the collapse critical temperature or eutectic critical temperature minus the safety margin. Subsequently, during the primary drying stage, the shelf temperature is gradually increased to the primary drying target temperature at a rate of 0.1°C per minute to 1.0°C per minute, while simultaneously controlling the chamber pressure between 10 Pa and 120 Pa to ensure the product... The temperature is kept close to the collapse critical temperature or eutectic critical temperature under temperature constraints for at least 20 minutes. The temperature fluctuation of the product within this holding time is no greater than 0.5℃ and the pressure fluctuation of the cavity is no greater than 5Pa, which is used as the steady-state criterion for the first drying stage. Then, the second drying stage is entered, and the shelf temperature is further increased to 15℃ to 35℃ and kept for 1h to 10h, so that the residual moisture content of the finished product is 0.3% to 1.5% and the water activity is 0.05 to 0.25. The residual moisture content is determined by Karl Fischer method and the water activity is determined by water activity meter.

[0010] Further, in step S5, the product temperature signal and the cavity pressure signal are synchronously acquired with a sampling period of 10s to 60s and closed-loop parameter tuning control is performed. The closed-loop parameter tuning control includes taking the product temperature meeting the temperature constraint condition as the first priority constraint. When the product temperature is detected to reach the upper limit boundary of the temperature constraint condition for 30s, the shelf temperature is reduced by 0.5℃ to 2℃ and the cavity pressure is increased by 5Pa to 30Pa. When the product temperature is detected to be more than 1℃ below the upper limit boundary for 10min and the cavity pressure fluctuation amplitude is not greater than 5Pa, the shelf temperature is restored at a heating slope of 0.1℃ per min to 0.5℃ per min and the cavity pressure is reduced by 5Pa to 30Pa. After the secondary drying stage is completed, the drying endpoint criterion verification is performed. The drying endpoint criterion verification includes closing the valve connected to the vacuum pump and recording the cavity pressure rise rate within 30s to 180s. When the cavity pressure rise rate is not greater than 0.5Pa per s, the secondary drying endpoint is determined to be established and the pressure plug sealing step is entered.

[0011] Furthermore, the verification of the reconstitution and infusion stability indicators of the lyophilized product in step S6 includes reconstitution of the lyophilized product with water for injection, with a reconstitution volume of 1 mL to 10 mL and a reconstitution time of no more than 120 s. After reconstitution, the product is diluted with an infusion medium to form an infusion solution. The infusion medium is 0.9% sodium chloride injection or 5% glucose injection. After the infusion solution is allowed to stand at 25°C for 4 to 8 hours, its activity retention rate is no less than 90% and the aggregate increment is no more than 2.0%. In addition, the pH change of the infusion solution does not exceed 0.2 pH units and the turbidity is no more than 3.0 NTU.

[0012] Furthermore, in step S6, the infusion solution is continuously infused through the infusion tubing at a flow rate of 1 mL / min to 10 mL / min for 30 min to 180 min, and then the adsorption and shear stability are verified. The relative difference in ulinastatin content before and after infusion is no more than 5% as the tubing adsorption limit, and the change in aggregate increment before and after infusion is no more than 0.5% as the shear stability limit. The ulinastatin content is determined by reversed-phase high-performance liquid chromatography or protein quantification method, and the aggregate increment is determined by size exclusion chromatography.

[0013] In summary, the present invention has the following beneficial effects: By introducing pressure-triggered or exogenous ice crystal-triggered synchronous nucleation control during the freezing stage, the nucleation time of each vial tends to be consistent with the ice crystal structure, thereby solving the problem of pore structure differences and large batch fluctuations caused by the randomness of inter-vial nucleation in the existing freeze-drying process. By performing double annealing after final freezing and using it for ice crystal rearrangement and crystal form framework excipient crystal locking respectively, the freeze-dried block pores are made uniform and the excipient crystal state is stable and consistent, thus solving the problems of unstable pore connectivity and crystal form drift causing decreased resolubility and stability decay in the prior art. By acquiring product temperature and chamber pressure signals in real time during the drying stage and implementing closed-loop parameter adjustment control, the product temperature is always kept below the collapse critical temperature or eutectic critical temperature, thus solving the problem that existing fixed-program drying is prone to local overheating and collapse, which can induce protein aggregation or reduced activity. By simultaneously controlling the residual moisture content and water activity within a preset target window, the moisture state of the freeze-dried solid phase is kept within a repeatable and stable range, thus solving the problem that existing technologies, which use only moisture or appearance as the endpoint, make it difficult to control the risk of chemical degradation during storage. By incorporating the activity retention rate, turbidity aggregate increment, and pH change of the reconstituted infusion solution at a preset verification temperature and within a preset verification time into the index verification, the stability evaluation covers actual clinical use scenarios, thereby solving the problem that existing processes only verify finished product indicators while ignoring stability during the infusion stage, leading to unpredictable quality risks during medication use. By establishing a limit criterion for the relative difference in content and the change in aggregate increment under infusion tubing conditions, the adsorption and shear-induced instability of the tubing can be quantified and constrained, thus solving the problem that the influence of infusion tubing on protein adsorption and shearing is difficult to evaluate and control in the existing technology. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0015] Figure 1 This is a schematic diagram of the pressure-triggered synchronous nucleation control process of the present invention; Figure 2 This is a schematic diagram of the process for homogenization of dual annealed channels and crystal form locking in this invention; Figure 3 This is a schematic flowchart of the signal-driven closed-loop drying and endpoint criterion diagram of the present invention; Figure 4 This is a schematic diagram of the reconstitution and infusion stability and pipeline risk verification process of the present invention. Detailed Implementation

[0016] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0017] The following examples use ulinastatin as an example, but the technical solution of the present invention is also applicable to the preparation of other protein or peptide lyophilized powder injection products, and has good universality and process portability.

[0018] Example 1: The following is in conjunction with the appendix Figure 1-4 The present invention will be described in further detail below.

[0019] Please see Figure 1-4 This invention provides a technical solution: a method for enhancing the stability of ulinastatin lyophilized powder for injection, such as... Figure 1-4 As shown, it includes the following steps: S1, Ulinastatin is dissolved in water for injection, added to the excipient system to form a lyophilized formulation solution. The excipient system includes at least a crystal framework excipient and a glassy protective excipient. A buffer system is added to adjust the pH of the lyophilized formulation solution to the target window, and the absolute value of the pH drift under freezing enrichment conditions is not greater than 0.5 pH units by adjusting the ionic strength. S2, the freeze-dried prescription solution is sterilized and filtered and aseptically filled into vials, then partially stoppered and placed into the freeze-drying chamber; S3, the vial is pre-cooled to the nucleation trigger window, and pressure-triggered nucleation is used to achieve synchronous nucleation. After nucleation, the vial is further cooled to the final freezing temperature to form a uniform ice crystal structure and inhibit local excessive enrichment of solute. S4. After final freezing, a double annealing process is performed. The first annealing temperature window is used for ice crystal rearrangement and channel homogenization, and the second annealing temperature window is used for the full crystallization and crystal form locking of the crystal skeleton excipient. After double annealing, the temperature is lowered again to solidify the solid phase structure. S5 performs primary and secondary drying, and collects process monitoring signals, including at least product temperature and chamber pressure signals, in real time during the drying process. After comparing the process monitoring signals with preset thresholds, the shelf temperature and chamber pressure are dynamically adjusted to ensure that the product temperature is always lower than the collapse critical temperature or eutectic critical temperature of the freeze-dried formulation solution, and the residual moisture content and water activity of the finished product are controlled within the preset target window. S6, complete the plugging and sealing and obtain the lyophilized product. After reconstitution, the lyophilized product is further diluted with the infusion medium to form an infusion solution. The infusion solution meets the stability index threshold at the preset verification temperature and within the preset verification time. The stability index includes at least the activity retention rate not lower than the preset lower limit, visible foreign matter and turbidity meeting the limit, aggregate increment not higher than the preset upper limit, and pH change not exceeding the preset range. This embodiment provides a method for enhancing the stability of ulinastatin lyophilized powder injection, thereby improving the consistency of the lyophilization process and reducing the risk of activity attenuation and aggregation during the infusion stage after reconstitution. The raw materials and excipients used in this embodiment include ulinastatin raw material, water for injection, mannitol as a crystal matrix excipient, trehalose as a glassy protective excipient, histidine as a buffer system component, and sodium chloride as an ionic strength regulating component. The main equipment used in this embodiment includes a lyophilizer with rapid chamber pressure switching function and equipped with product temperature acquisition channels and chamber pressure acquisition channels. The detection equipment includes a pH measuring device, a Karl Fischer moisture measuring device, a water activity measuring device, a turbidity measuring device, a size exclusion chromatography measuring device, and an ulinastatin activity measuring device. In the preparation of the prescription solution, the water for injection was pre-cooled to 2°C to 8°C, then ulinastatin was added and stirred to dissolve. Ulinastatin was added at a rate of 3.33 × 10⁻⁶ per 1 mL of prescription solution. 4 U, mannitol 30mg, trehalose 20mg, and histidine were added to make the buffer system concentration 10mmol / L, and sodium chloride was added to make the ionic strength range between 20mmol / L and 40mmol / L. The pH was adjusted to 6.0 using hydrochloric acid solution or sodium hydroxide solution. The mixture was stirred until the solution was clear and then allowed to stand to remove bubbles to obtain the prescription solution to be lyophilized. To meet the pH drift control requirements under freeze enrichment conditions, the same batch of prescription solutions to be freeze-dried were dispensed into vials of the same specification and frozen according to the freezing and annealing procedure of this embodiment. After freezing, the solutions were thawed to 25°C and the pH was measured. The absolute value of the difference between the pH after thawing and the pH before freezing was taken as the pH drift value. When the pH drift value was not greater than 0.5 pH units, step S1 was deemed to meet the requirements. The pH measurement was performed using a pH measuring device calibrated at two or three points. During the aseptic and filling process, the prescription solution to be lyophilized is filtered through a 0.22μm sterile filter membrane and aseptically filled into 10mL vials with a single vial filling volume of 3.0mL. After filling, the vials are partially stoppered and transferred into the lyophilization chamber. During loading, the vials are evenly arranged on the shelf, and a product temperature probe is placed on the representative vial to obtain the product temperature signal. During the controlled nucleation freezing process, the shelf cooling is initiated and the vials are pre-cooled to the nucleation trigger window temperature along with the shelf cooling. In this embodiment, the nucleation trigger window temperature is selected as −8℃ and held for 5 min. Then, pressure-triggered nucleation is performed, and the pressure in the freeze-drying chamber is switched from the pre-cooling steady-state pressure to the nucleation trigger pressure and held for the nucleation duration. In this embodiment, the pre-cooling steady-state pressure is selected as 90000 Pa, the nucleation trigger pressure is 100 Pa, and the nucleation duration is 20 s. After that, the chamber pressure is restored to the pre-cooling steady-state pressure and the temperature is further cooled to the final freezing temperature. In this embodiment, the final freezing temperature is selected as −40℃ and held for 30 min. The synchronous nucleation criterion is that the temperature curve of each vial product shows a temperature rebound caused by the release of latent heat, and the temperature rebound amplitude is 0.5℃ to 3.0℃, and the maximum time difference between vials at the start of the rebound is not greater than 20 s. During the double annealing process for crystal form locking, the double annealing treatment was performed after maintaining the final freezing temperature for 30 minutes. In the first annealing stage, the shelf temperature was raised to -20℃ and maintained for 2 hours to promote ice crystal rearrangement and achieve channel homogenization. In the second annealing stage, the shelf temperature was raised to -8℃ and maintained for 1 hour to promote the full crystallization of mannitol and achieve crystal form locking. After the double annealing, the shelf temperature was lowered to -40℃ and maintained for 20 minutes to solidify the solid phase structure. After annealing, freeze-dried blocks from different positions of vials were randomly selected for cross-sectional pore size statistics and the coefficient of variation of pore size distribution was calculated. When the coefficient of variation of pore size distribution was not greater than 0.35, the channel homogenization was deemed to meet the requirements. At the same time, differential scanning calorimetry or powder X-ray diffraction was performed on the freeze-dried blocks to obtain the crystallinity of mannitol. When the crystallinity was not less than 80% and the difference in crystallinity between vials was not greater than 10%, the crystal form locking was deemed to meet the requirements. In both the primary and secondary drying processes, the collapse critical temperature or eutectic critical temperature of the freeze-dried formulation solution is first determined using a freeze-drying microscope or differential scanning calorimetry. This temperature is then used as the upper limit of the product temperature constraint during the drying process. In this embodiment, the collapse critical temperature was measured to be −23℃. During the primary drying stage, the chamber pressure is controlled at 60 Pa, and the shelf temperature is increased from −30℃ to −15℃ at a rate of 0.3℃ per minute and maintained. During the drying process, the product temperature signal and the chamber pressure signal are simultaneously collected at a sampling period of 10 seconds and compared with a preset threshold. When the product temperature approaches −23℃, the process is initiated. Lower the shelf temperature or increase the chamber pressure to keep the product temperature below -23℃. When the product temperature is consistently below -23℃ and the chamber pressure fluctuation is no greater than 5Pa and is maintained for at least 20 minutes, proceed to the secondary drying stage. In the secondary drying stage, raise the shelf temperature to 25℃ and maintain it for 5 hours, while controlling the chamber pressure between 20Pa and 60Pa. After the secondary drying is completed, perform the drying endpoint verification. Close the valve connected to the vacuum pump and record the chamber pressure rise rate within 120 seconds. When the chamber pressure rise rate is no greater than 0.5Pa per second, the drying endpoint is determined to be established. After the drying endpoint is reached, the freeze-drying chamber is sealed by compression and the freeze-dried product is obtained by capping. The freeze-dried product is sampled and tested for residual moisture content and water activity. In this embodiment, the residual moisture content is determined by Karl Fischer method and controlled within 0.3% to 1.5%, and the water activity is determined by water activity meter and controlled within 0.05 to 0.25. When the residual moisture content and water activity are both within the corresponding target window, step S5 is deemed to meet the requirements. During the verification of reconstitution and infusion stability indicators, 5.0 mL of water for injection was added to each vial of the lyophilized product for reconstitution, and the solution was gently rotated to ensure complete reconstitution within 120 seconds. After the reconstituted solution was clear and free of visible foreign matter, it was diluted with 0.9% sodium chloride injection or 5% glucose injection to form an infusion solution, and then allowed to stand at 25°C for 6 hours. The activity retention rate, turbidity, aggregate increment, and pH change of the infusion solution were then measured. The activity retention rate was determined by the trypsin inhibition activity method or an equivalent method and was not less than 90%. The turbidity was measured by a turbidimeter and was not greater than 3.0 NTU. The aggregate increment was determined by size exclusion chromatography and was not greater than 2.0%. The pH change was the absolute value of the pH difference before and after the infusion solution had stood for 6 hours and was not greater than 0.2 pH units. When all the above stability indicators met the threshold requirements, step S6 was deemed to have met the requirements, proving that the method could achieve the enhanced stability of the lyophilized product under infusion conditions after reconstitution.

[0020] Example 2: like Figure 1-4 As shown, in step S3, pressure-triggered nucleation is used to achieve synchronous nucleation, specifically as follows: The partially stoppered vials were pre-cooled along the shelf to the nucleation trigger window temperature, which was -12°C to -4°C. Then, the pressure in the freeze-drying chamber was switched from the pre-cooling steady-state pressure to the nucleation trigger pressure and maintained for a preset nucleation holding time, which was 30 Pa to 200 Pa and 5 s to 60 s. After that, the chamber pressure was restored to the pre-cooling steady-state pressure and the temperature was further reduced to the final freezing temperature. The pre-cooling steady-state pressure was the chamber pressure used to maintain a stable gas environment during the pre-cooling stage before nucleation triggering. The criterion for synchronous nucleation was that the temperature rebound amplitude caused by the release of latent heat in the temperature curve of each vial product was 0.5°C to 3.0°C and the maximum time difference between vials at the start of the rebound was no more than 20 s.

[0021] In this embodiment: In order to further realize pressure-triggered synchronous nucleation control and improve the consistency of freezing events between different vials, this embodiment refines the controllable nucleation freezing process in step S3. The remaining steps S1, S2, S4, S5 and S6 can be executed as in the aforementioned embodiment, thereby ensuring that the conditions for verifying the stability of the prescription system, double annealing structure locking, signal-driven drying and reconstitution infusion are consistent, so as to attribute the stability improvement effect to the synchronous nucleation control strategy. Before entering the freezing stage, the partially stoppered vials are loaded onto the freeze dryer shelf and evenly arranged. At the same time, a product temperature probe is inserted into a representative vial to record the product temperature curve. The product temperature probe is preferably inserted below the liquid surface of the prescription solution and located in the center area of ​​the vial to reduce the influence of the wall heat transfer difference on the temperature measurement. Before freezing, the freeze drying chamber is evacuated to the pre-cooling steady-state pressure and maintained to allow each vial to be pre-cooled in the same gas environment. In this embodiment, the pre-cooling steady-state pressure is set to 90000 Pa. Subsequently, pre-cooling was performed, with the shelf temperature decreasing from 2°C to 8°C at a rate of 0.5°C to 2.0°C per minute to the nucleation trigger window temperature, and then maintained at this window temperature to ensure that the product temperature of each bottle tends to be consistent and enters a metastable supercooled state. In this embodiment, the nucleation trigger window temperature is selected as −12°C to −4°C, preferably −8°C, and maintained for 3 to 10 minutes. During this holding period, the product temperature of the representative bottle is continuously recorded to confirm that the product temperature is stable within the nucleation trigger window range and the temperature fluctuation amplitude does not exceed 0.5°C. When the nucleation triggering window temperature is reached, pressure-triggered nucleation is initiated. The pressure in the freeze-drying chamber is rapidly switched from the pre-cooling steady-state pressure to the nucleation triggering pressure and maintained for a preset nucleation holding time. In this embodiment, the nucleation triggering pressure is set to 30 Pa to 200 Pa, preferably 100 Pa, and the nucleation holding time is set to 5 s to 60 s, preferably 20 s. During the nucleation holding time, the product temperature signal is continuously collected and the product temperature curve change is recorded. After the nucleation triggering pressure ends, the chamber pressure is restored to the pre-cooling steady-state pressure and freezing cooling continues. The shelf temperature is reduced to the final freezing temperature at a cooling slope of 0.5 ℃ per min to 2.0 ℃ per min and maintained. In this embodiment, the final freezing temperature is set to −40 ℃ to −35 ℃, preferably −40 ℃, and maintained for 30 min to 60 min to allow the ice phase to fully form and complete the freezing and solidification. To determine whether synchronous nucleation is successful, this embodiment uses the temperature rebound caused by the release of latent heat in the product temperature curve as the marker of the nucleation event. Specifically, after the chamber pressure is switched, if the product temperature curves of multiple vials show a rebound process from a cooling trend to a short-term rise, the maximum temperature rise value of the rebound process is defined as the temperature rebound amplitude, and the rebound start time is defined as the moment when the slope of the product temperature curve changes from negative to positive. When the measured temperature rebound amplitude is between 0.5℃ and 3.0℃ and the maximum time difference between vials at the rebound start time is not greater than 20s, synchronous nucleation is determined to be successful. If the rebound amplitude is less than 0.5℃ or the maximum time difference between vials is greater than 20s, synchronous nucleation is determined to be unsatisfactory and the nucleation trigger window temperature or nucleation trigger pressure and its holding time need to be adjusted. To further ensure the repeatability of the synchronous nucleation process, this embodiment sets process correction rules when synchronous nucleation is not met. When the temperature rebound amplitude is less than 0.5℃, the nucleation trigger pressure is reduced by 10Pa to 50Pa or the nucleation holding time is extended by 5s to 20s to enhance the nucleation disturbance. When the maximum time difference between bottles is greater than 20s, the nucleation trigger window temperature is increased by 1℃ to 3℃ and the pre-cooling holding time is extended by 1min to 5min to reduce the difference in supercooling between bottles. After the parameter correction is completed, the above pressure-triggered nucleation steps are repeated until the synchronous nucleation criterion is met. By controlling the specific temperature, pressure, and time windows for pressure-triggered nucleation as described above, and combining the synchronous nucleation criteria of temperature rebound amplitude and inter-bottle time difference, the nucleation events during the freezing stage are kept highly consistent between bottles. This provides a consistent initial ice crystal structure basis for the channel homogenization and crystal form locking in the subsequent double annealing stage, and further improves the batch consistency of residual moisture content and water activity control in the subsequent drying stage. Ultimately, this helps to improve the stability index compliance rate of the reconstituted lyophilized product at the preset verification temperature and preset verification time.

[0022] Example 3: like Figure 1-4 As shown, the double annealing process in step S4 includes holding the freeze-dried block at the final freezing temperature for 10 to 60 minutes, then raising the shelf temperature to the first annealing temperature of -25°C to -15°C and holding it for 0.5 to 4 hours to allow ice crystal rearrangement and ensure that the coefficient of variation of the pore size distribution of the freeze-dried block is no greater than 0.35. Subsequently, the shelf temperature is raised to the second annealing temperature of -12°C to -6°C and held for 0.25 to 2 hours to ensure that the crystallinity of the crystalline framework excipient is no less than 80% and the difference in crystallinity between bottles is no greater than 10%. After the double annealing is completed, the shelf temperature is lowered to no higher than -35°C to solidify the solid phase structure. The coefficient of variation of the pore size distribution is obtained by statistical analysis of the pore size of the cross-section of the freeze-dried block after annealing, and the crystallinity is obtained by differential scanning calorimetry or powder X-ray diffraction. In this embodiment, the process, control parameters and criteria verification of step S4 are refined. The remaining steps S1, S2, S3, S5 and S6 can be performed as in embodiment 2 to ensure that the formulation system, synchronous nucleation basic conditions, drying closed-loop control and reconstitution infusion stability verification conditions are consistent, so as to facilitate attributing the subsequent stability improvement effect to the locking effect of the double annealing process on the solid phase structure. After final freezing, the shelf temperature is maintained at the final freezing temperature for 10 to 60 minutes to ensure complete stabilization of the ice phase inside each vial and to equalize the temperature gradient between vials. In this embodiment, the final freezing temperature is -40°C and maintained for 30 minutes. Then, the first annealing stage begins, with the shelf temperature increased at a rate of 0.1°C to 0.5°C per minute to the first annealing temperature and maintained for 0.5 to 4 hours. In this embodiment, the first annealing temperature is -20°C and maintained for 2 hours to promote ice crystal rearrangement and improve the connectivity and uniformity of the freeze-dried block's pores. After the initial annealing, the second annealing stage begins. The shelf temperature is increased to the second annealing temperature at a rate of 0.1°C to 0.5°C per minute and held for 0.25 to 2 hours. In this embodiment, the second annealing temperature is -8°C and held for 1 hour to promote the full crystallization of the mannitol excipient and to make the crystallization state more uniform between bottles. After the second annealing, the shelf temperature is reduced to no higher than -35°C and held for 20 to 60 minutes to solidify the solid structure. In this embodiment, the shelf temperature is reduced to -40°C and held for 20 minutes before proceeding to the subsequent primary and secondary drying steps. To verify the contribution of the first annealing stage to pore homogenization, in this embodiment, after freeze-drying, 10 bottles of finished product were extracted from different positions on the freeze-drying chamber shelf. For each bottle, a cross-sectional sample was cut along the axial direction of the freeze-dried block and subjected to microscopic characterization. After vacuum drying, the cross-sectional sample underwent surface conductivity treatment, and at least 5 field-of-view images were acquired at the same magnification. Image analysis software was used to identify and count at least 200 pore sizes in each field of view, and the average value and standard deviation of the pore size set were obtained. The ratio of the standard deviation to the average value was used as the pore size distribution variation coefficient. When the pore size distribution variation coefficient was not greater than 0.35, the pore homogenization was determined to meet the criterion requirements. If the pore size distribution variation coefficient was greater than 0.35, the first annealing holding time was increased within the range of 0.5h to 4h, or the first annealing temperature was increased within the range of −25℃ to −15℃, and the double annealing process was repeated until the criterion was met. To verify the contribution of the second annealing stage to the crystallinity locking of the crystal framework excipient, in this embodiment, lyophilized bulk samples were taken from the above 10 finished products in the same batch for crystallinity determination. Crystallinity determination was preferably performed using either differential scanning calorimetry (DSC) or powder X-ray diffraction (PXRD). When using DSC, the sample was scanned at a fixed heating rate under an inert atmosphere, and the enthalpy of melting of the mannitol melt endothermic peak was recorded. Simultaneously, the enthalpy of melting of a reference sample obtained after sufficient crystallization of the same batch of mannitol was used as a reference. As a whole crystallization reference, the crystallinity of the sample is taken as the ratio of the sample melting enthalpy to the melting enthalpy of the whole crystallization reference, expressed as a percentage. When the measured crystallinity is not less than 80% and the difference in crystallinity between different vials is not greater than 10%, the crystal form is considered to be locked and meets the criteria. If the crystallinity is less than 80% or the difference in crystallinity between vials is greater than 10%, the second annealing holding time is increased in the range of 0.25h to 2h or the second annealing temperature is increased in the range of −12℃ to −6℃ and the double annealing process is repeated until the criteria are met. By controlling the temperature and holding time windows of the above-mentioned double annealing steps, and verifying the criterion of pore size distribution variation coefficient, crystallinity, and inter-bottle differences, the ice phase structure and crystallization state of the crystal framework excipient after final freezing are repeatedly locked within the batch. This provides a consistent solid phase basis for the stable entry of residual moisture content and water activity into the target window in the subsequent drying stage, and further improves the stability of the finished product under the reconstituted infusion conditions.

[0023] Example 4: like Figure 1-4 As shown, in step S5, during the primary and secondary drying processes, a product temperature safety margin is first set based on the collapse critical temperature or eutectic critical temperature of the solution to be freeze-dried. This safety margin ranges from 1°C to 5°C. The product temperature is constrained by the condition that it does not exceed the collapse critical temperature or eutectic critical temperature minus the safety margin. Subsequently, during the primary drying stage, the shelf temperature is gradually increased to the target primary drying temperature at a rate of 0.1°C per minute to 1.0°C per minute, while simultaneously controlling the chamber pressure between 10 Pa and 120 Pa to ensure the product temperature... The temperature is kept close to the collapse critical temperature or eutectic critical temperature under temperature constraints for at least 20 minutes. The temperature fluctuation amplitude of the product is no greater than 0.5℃ and the pressure fluctuation amplitude of the cavity is no greater than 5Pa during this holding time as the steady-state criterion for the first drying stage. Then, the second drying stage is entered, and the shelf temperature is further increased to 15℃ to 35℃ and held for 1h to 10h, so that the residual moisture content of the finished product is 0.3% to 1.5% and the water activity is 0.05 to 0.25. The residual moisture content is determined by Karl Fischer method and the water activity is determined by water activity meter. In this embodiment, based on Example 3, in order to further realize the temperature constraint control of the primary and secondary drying processes and the entry of residual moisture content and water activity into the target window, this embodiment refines the operation parameters and criteria verification of step S5. The remaining steps S1, S2, S3, S4 and S6 can be executed as in Example 3 to ensure that the formulation system, the synchronous nucleation basis and the locking conditions of the double annealed solid phase structure are consistent, so as to facilitate attributing the stability improvement effect of the drying stage to the temperature constraint and endpoint window control strategy. Before entering the first drying stage, the collapse critical temperature or eutectic critical temperature of the prescription solution to be freeze-dried is determined. It is preferable to use a freeze-drying microscope to observe the frozen sample by heating to obtain the collapse critical temperature, or to use differential scanning calorimetry to obtain the eutectic initiation temperature and use it as the eutectic critical temperature. In this embodiment, the collapse critical temperature was measured to be −23℃. Then, the product temperature safety margin was set to 2℃. Accordingly, the product temperature constraint condition was set to the product temperature not exceeding −25℃. A product temperature probe was installed in a representative bottle in the freeze dryer. The product temperature probe was preferably inserted into the center area of ​​the frozen prescription solution in the bottle and the probe tip was located in the middle of the height inside the bottle to reduce the influence of the wall heat transfer difference on the temperature measurement. At the same time, the cavity pressure acquisition channel was opened to obtain the cavity pressure signal. In the first drying stage, the chamber pressure is controlled at 60 Pa, and the shelf temperature is set to -30°C as the starting point. Then, the temperature is gradually increased to -15°C at a rate of 0.3°C per minute. During the heating process, the product temperature signal and the chamber pressure signal are collected synchronously with a sampling period of 10 seconds and compared with the product temperature constraint. When the product temperature is detected to be close to -25°C and showing a continuous upward trend, the product temperature is reduced by lowering the shelf temperature setting or increasing the chamber pressure setting to make the product temperature drop and remain below -25°C. When the product temperature enters the stable section under the condition of below -25°C and is maintained for no less than 20 minutes, and the fluctuation amplitude of the product temperature is no greater than 0.5°C and the fluctuation amplitude of the chamber pressure is no greater than 5 Pa during this period, the first drying is determined to have reached the steady-state criterion and enters the second drying stage. In the secondary drying stage, the chamber pressure was controlled at 30 Pa, and the shelf temperature was increased from the end temperature of the first drying stage to 25°C at a rate of 0.2°C per minute and maintained for 5 hours. During the maintenance period, product temperature and chamber pressure signals were continuously collected for process recording. After the secondary drying was completed, samples were taken to test the residual moisture content and water activity. The residual moisture content was determined using the Karl Fischer method. Ten bottles were taken from the same batch of finished products, and freeze-dried blocks were taken from each sample for testing. The residual moisture content was controlled between 0.3% and 1.5%, and the water activity was measured using a water activity meter and controlled between 0.05 and 0.25. When both the residual moisture content and water activity fell within the above target window, step S5 was deemed to meet the control requirements, and a freeze-dried product meeting the conditions was provided for the subsequent reconstitution and infusion stability index verification in step S6.

[0024] Example 5: like Figure 1-4 As shown, in step S5, the product temperature signal and the cavity pressure signal are synchronously acquired with a sampling period of 10s to 60s and closed-loop parameter adjustment control is performed. The closed-loop parameter adjustment control includes taking the product temperature meeting the temperature constraint condition as the first priority constraint. When the product temperature is detected to reach the upper limit boundary of the temperature constraint condition for 30s, the shelf temperature is reduced by 0.5℃ to 2℃ and the cavity pressure is increased by 5Pa to 30Pa. When the product temperature is detected to be more than 1℃ below the upper limit boundary for 10min and the cavity pressure fluctuation amplitude is not greater than 5Pa, the shelf temperature is restored at a heating slope of 0.1℃ per min to 0.5℃ per min and the cavity pressure is reduced by 5Pa to 30Pa. After the secondary drying stage is completed, the drying endpoint criterion verification is performed. The drying endpoint criterion verification includes closing the valve connected to the vacuum pump and recording the cavity pressure rise rate within 30s to 180s. When the cavity pressure rise rate is not greater than 0.5Pa per s, the secondary drying endpoint is determined to be established and the pressure plug sealing step is entered. In this embodiment, based on embodiment 4, in order to further realize the synchronous acquisition of product temperature signal and cavity pressure signal and closed-loop parameter adjustment control, and to ensure that the secondary drying endpoint criterion can be repeatedly executed, this embodiment refines the signal acquisition method, criterion triggering condition, parameter adjustment action and endpoint verification process in step S5. The remaining steps S1, S2, S3, S4 and S6 can be executed as in embodiment 4, so as to keep the basic conditions of the formulation system and solid structure consistent, thereby making it easier to attribute the process consistency improvement effect to the closed-loop parameter adjustment control strategy. Before the start of a drying cycle, the product temperature acquisition channel and the chamber pressure acquisition channel of the freeze dryer are calibrated. The product temperature probe is a calibrated thermocouple or platinum resistance probe and is fixed in the center area of ​​the frozen body of the representative bottle. The chamber pressure is acquired by a capacitive thin-film vacuum gauge and zero-point calibration is performed at the same pressure point before the first drying cycle. Then, the sampling period of product temperature and chamber pressure is set to 10s, and the two signals are recorded synchronously using the same controller clock. At the same time, the product temperature constraint condition is set to the temperature constraint condition that is satisfied, which is the first priority constraint of closed-loop parameter tuning control. During a drying and heating process, the controller reads the product temperature and cavity pressure every 10 seconds and calculates the upper limit of the temperature constraint in real time. When the product temperature reaches the upper limit for 30 consecutive seconds, the first parameter adjustment action is executed. The first parameter adjustment action includes lowering the shelf temperature setting by 1℃ and raising the cavity pressure setting by 15Pa. The shelf temperature and cavity pressure setting values ​​are kept unchanged for 2 minutes after the first parameter adjustment action to avoid control oscillation. If the product temperature still reaches the upper limit for 30 consecutive seconds after 2 minutes, the first parameter adjustment action is repeated until the product temperature drops and falls below the upper limit again. During a drying process, when the product temperature is detected to be more than 1°C below the upper limit for 10 consecutive minutes and the fluctuation amplitude of the cavity pressure is no more than 5Pa, a second parameter adjustment action is performed. The second parameter adjustment action includes restoring the shelf temperature at a heating rate of 0.2°C per minute and lowering the cavity pressure setpoint by 15Pa. For 5 minutes after the second parameter adjustment action is performed, the heating rate is kept constant and the product temperature and cavity pressure are continuously monitored. If the product temperature is detected to be close to the upper limit again, the heating is stopped and the first parameter adjustment action is switched to ensure that the product temperature always meets the temperature constraint conditions. After the secondary drying stage is completed, the drying endpoint criterion verification is performed. In this embodiment, the valve closing pressure rise rate is used as the endpoint criterion. Specifically, under the condition of keeping the shelf temperature and cavity pressure set value unchanged, the valve connected to the vacuum pump is closed and the timing is started. Within 120 seconds, the cavity pressure data is collected at a recording period of 1 second and the pressure rise rate is calculated. The pressure rise rate is obtained by subtracting the pressure value at the 0th second after the valve is closed from the pressure value at the 120th second after the valve is closed and then dividing by 120 seconds. When the pressure rise rate is not greater than 0.5 Pa per second, the secondary drying endpoint is determined to be established and the plug sealing step is entered. If the pressure rise rate is greater than 0.5 Pa per second, the secondary drying endpoint is determined to be not reached and the secondary drying is extended by 1 hour. The above valve closing pressure rise rate verification is then performed again until the endpoint is established. By using the above-mentioned synchronous signal acquisition and closed-loop parameter adjustment control method, the product temperature is always controlled within the temperature constraint during the primary and secondary drying processes, reducing the risk of overheating between bottles. At the same time, the endpoint determination of the secondary drying endpoint can be repeated through the quantifiable valve closing pressure rise rate endpoint criterion, thereby providing a stable process control basis for the subsequent residual moisture content and water activity to enter the target window and for the stability indicators of the reconstituted infusion to meet the standards.

[0025] Example 6: like Figure 1-4 As shown, the verification of the reconstitution and infusion stability of the lyophilized product in step S6 includes reconstitution of the lyophilized product with water for injection, with a reconstitution volume of 1 mL to 10 mL and a reconstitution time of no more than 120 s. After reconstitution, the product is diluted with an infusion medium to form an infusion solution. The infusion medium is 0.9% sodium chloride injection or 5% glucose injection. After the infusion solution is allowed to stand at 25°C for 4 to 8 hours, its activity retention rate is no less than 90% and the aggregate increment is no more than 2.0%. Furthermore, the pH change of the infusion solution does not exceed 0.2 pH units and the turbidity is no more than 3.0 NTU. In this embodiment, the reconstitution operation, infusion solution preparation, standing conditions, sampling time and detection methods and judgment rules of various indicators in step S6 are refined. The remaining steps S1 to S5 can be performed as in Example 5 to ensure that the solid phase structure, residual moisture content and water activity of the freeze-dried block are under the same control basis. In the reconstitution step, 10 vials of the lyophilized product were placed in an environment of 20℃ to 25℃ for 30 minutes to equilibrate. Then, water for injection was added to each vial for reconstitution. The reconstitution volume was set to 5.0 mL and ranged from 1 mL to 10 mL. After adding water for injection, the product was mixed by slow rotation, without vigorous shaking. The time from the addition of water for injection until the lyophilized block completely disappeared and the solution became clear was recorded as the reconstitution time. The reconstitution time was controlled not to exceed 120 seconds. After reconstitution, an appearance inspection was performed. The absence of visible foreign matter and the clarity of the solution were used as the criteria for acceptance of the reconstitution appearance. The pH of the reconstituted solution was immediately measured as the initial value for subsequent pH changes. The pH was measured using a pH measuring device calibrated at two points at 25℃. In the infusion solution preparation step, the reconstituted solution is diluted with 0.9% sodium chloride injection and 5% glucose injection to form the infusion solution. The dilution method is to add 1.0 mL of the reconstituted solution to 9.0 mL of the infusion medium to obtain 10.0 mL of infusion solution, or add 5.0 mL of the reconstituted solution to 45.0 mL of the infusion medium to obtain 50.0 mL of infusion solution. Either of the above two preparation methods can be selected and kept consistent with the same batch of samples. The infusion solution preparation container is preferably a clean glass container or a clinically commonly used infusion container. After preparation, gently invert and mix, and immediately record it as the zero time sample. Then, place the infusion solution at 25°C for 6 hours and keep it within the range of 4 to 8 hours. During the standing period, avoid direct exposure to strong light and avoid mechanical vibration to ensure that the stability evaluation conditions can be repeated. In the activity retention rate determination, the activity of ulinastatin was determined for samples at time 0 and samples that had been left to stand for 6 hours. The activity determination method adopted the trypsin inhibitory activity method or the pharmacopoeia equivalent method. The specific operation was as follows: trypsin working solution was prepared and reacted with the sample to be tested at a specified volume ratio at 37°C for a preset reaction time. Then, the substrate was added and the absorbance change was measured at a specified wavelength. The number of active units of the sample was obtained by converting the standard curve. The ratio of the number of active units of the sample that had been left to stand for 6 hours to the number of active units of the sample that had been left to stand for 6 hours was multiplied by 100% to obtain the activity retention rate. When the activity retention rate was not less than 90%, the activity index was judged to meet the threshold requirement. In the determination of aggregate increment, size exclusion chromatography was performed on samples at time 0 and samples that had been standing for 6 hours to obtain the aggregate ratio. Size exclusion chromatography used a size exclusion column suitable for protein separation with phosphate buffer solution as the mobile phase. The flow rate was set to 0.5 mL / min to 1.0 mL / min, the detection wavelength was set to 214 nm or 280 nm, and the injection volume was set to 10 μL to 50 μL. The monomer peak area and the high molecular weight aggregate peak area were obtained by integration. The aggregate ratio was taken as the ratio of aggregate peak area to total peak area multiplied by 100%. The aggregate increment was obtained by subtracting the aggregate ratio of the sample at time 0 from the aggregate ratio of the sample that had been standing for 6 hours. When the aggregate increment was not higher than 2.0%, the aggregate index was determined to meet the threshold requirement. In the determination of pH change, the pH of the sample at zero time and the sample after standing for 6 hours are measured respectively. The absolute value of the difference between the two pH measurements is taken as the pH change. When the pH change does not exceed 0.2 pH units, the pH index is judged to meet the threshold requirement. The pH measurement is carried out at 25℃ and the pH measuring device is calibrated and confirmed before each measurement. In the turbidity determination, the turbidity of the samples at time 0 and the samples after standing for 6 hours were measured. The turbidity was measured using a scattering turbidimeter calibrated with formalin standard solution. The turbidity value of the sample was read and the turbidity value of the sample after standing for 6 hours was used as the judgment criterion. When the turbidity of the sample after standing for 6 hours was not greater than 3.0 NTU, the turbidity index was judged to meet the threshold requirement. At the same time, the absence of visible foreign matter was confirmed by visual inspection to ensure that the usability evaluation and stability index of the infusion fluid were consistent.

[0026] Example 7: like Figure 1-4 As shown, in step S6, the infusion solution is continuously infused through the infusion line at a flow rate of 1 mL / min to 10 mL / min for 30 min to 180 min, and then the adsorption and shear stability are verified. The relative difference of ulinastatin content before and after infusion is no more than 5% as the adsorption limit of the line, and the change in aggregate increment before and after infusion is no more than 0.5% as the shear stability limit. The ulinastatin content is determined by reversed-phase high-performance liquid chromatography or protein quantification method, and the aggregate increment is determined by size exclusion chromatography. In this embodiment, after the lyophilized product is reconstituted and diluted with the infusion medium to obtain the infusion solution, a commonly used clinical gravity infusion set or infusion pump tubing is selected as the infusion tubing. The infusion tubing is preferably made of medical-grade polyvinyl chloride or polyolefin material with an inner diameter of 2.5mm to 3.5mm and an effective length of 100cm to 200cm. An infusion pump is configured to provide a stable flow rate. The infusion solution is first used to pre-wet the tubing; specifically, the infusion medium or the same formulation infusion solution is introduced into the tubing at a flow rate of 2mL per min to 5mL per min for 5 to 10 minutes, after which the pre-wetting solution is discarded to reduce the risk of tubing drying. To mitigate the interference of initial adsorption on the results, the newly prepared infusion solution was used as the official sample for continuous infusion. The infusion flow rate was set at 5 mL / min and ranged from 1 mL / min to 10 mL / min, and the infusion duration was set at 60 min and ranged from 30 min to 180 min. During the infusion process, the ambient temperature was maintained at 25℃ and direct sunlight was avoided. The infusion solution was taken at the beginning of the infusion as the pre-infusion sample, and the effluent was collected from the end of the tubing at the end of the infusion as the post-infusion sample. The sampling volume was preferably 2 mL to 5 mL, and the sampling volume of each sample was kept consistent. In the pipeline adsorption limit verification, the ulinastatin content was determined in the pre-infusion and post-infusion samples. The content determination could be performed by reversed-phase high-performance liquid chromatography (RP-HPLC) or protein quantification. When using R-HPLC, a protein-suitable column was selected and gradient elution with an acidic aqueous solution and an organic phase was used. The detection wavelength was selected as 214 nm or 280 nm, and the content results were obtained by external standard method or area normalization method. The relative difference between the pre-infusion and post-infusion sample contents was used as the pipeline adsorption evaluation index. The relative difference was calculated by subtracting the post-infusion sample contents from the pre-infusion sample contents, dividing by the pre-infusion sample contents, and multiplying by 100%. When the relative difference was not greater than 5%, the pipeline adsorption limit was deemed to be met. In the shear stability limit verification, the aggregate ratio of the pre-infusion and post-infusion samples was determined by size exclusion chromatography. The preferred size exclusion chromatography conditions were a phosphate buffer solution as the mobile phase and a flow rate of 0.5 mL / min to 1.0 mL / min, with a detection wavelength of 214 nm or 280 nm. The monomer peak and high molecular weight aggregate peak were obtained by integration, and the aggregate ratio was calculated. The change in aggregate increment was obtained by subtracting the aggregate ratio of the pre-infusion sample from the aggregate ratio of the post-infusion sample. When the change in aggregate increment was not higher than 0.5%, the shear stability limit was deemed to be met. To eliminate the influence of settling time on the aggregates, the pre-infusion sample could be settling at 25°C for 60 min, the same as the infusion time, before size exclusion chromatography was performed as a control. This ensured that the change in aggregate increment mainly reflected the shear and interface effects introduced during the tubing infusion process.

[0027] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0028] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the present invention. Various changes and modifications can be made to the present invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed.

Claims

1. A method for enhancing the stability of ulinastatin lyophilized powder for injection, characterized in that, Includes the following steps: S1, Ulinastatin is dissolved in water for injection, added to the excipient system to form a lyophilized formulation solution. The excipient system includes at least a crystal framework excipient and a glassy protective excipient. A buffer system is added to adjust the pH of the lyophilized formulation solution to the target window, and the absolute value of the pH drift under freezing enrichment conditions is not greater than 0.5 pH units by adjusting the ionic strength. S2, the freeze-dried prescription solution is sterilized and filtered and aseptically filled into vials, then partially stoppered and placed into the freeze-drying chamber; S3, the vial is pre-cooled to the nucleation trigger window, and pressure-triggered nucleation is used to achieve synchronous nucleation. After nucleation, the vial is further cooled to the final freezing temperature to form a uniform ice crystal structure and inhibit local excessive enrichment of solute. S4. After final freezing, a double annealing process is performed. The first annealing temperature window is used for ice crystal rearrangement and channel homogenization, and the second annealing temperature window is used for the full crystallization and crystal form locking of the crystal skeleton excipient. After double annealing, the temperature is lowered again to solidify the solid phase structure. S5 performs primary and secondary drying, and collects process monitoring signals, including at least product temperature and chamber pressure signals, in real time during the drying process. After comparing the process monitoring signals with preset thresholds, the shelf temperature and chamber pressure are dynamically adjusted to ensure that the product temperature is always lower than the collapse critical temperature or eutectic critical temperature of the freeze-dried formulation solution, and the residual moisture content and water activity of the finished product are controlled within the preset target window. S6, complete the plugging and sealing and obtain the lyophilized product. After reconstitution, the lyophilized product is further diluted with the infusion medium to form an infusion solution. The infusion solution meets the stability index threshold at the preset verification temperature and within the preset verification time. The stability index includes at least the activity retention rate not lower than the preset lower limit, visible foreign matter and turbidity meeting the limits, the aggregate increment not higher than the preset upper limit, and the pH change not exceeding the preset range.

2. The method for enhancing the stability of ulinastatin lyophilized powder injection according to claim 1, characterized in that, In step S3, pressure-triggered nucleation is used to achieve synchronous nucleation, specifically as follows: The partially stoppered vials were pre-cooled along the shelf to the nucleation trigger window temperature, which was -12°C to -4°C. Then, the pressure in the freeze-drying chamber was switched from the pre-cooling steady-state pressure to the nucleation trigger pressure and maintained for a preset nucleation holding time, which was 30 Pa to 200 Pa and 5 s to 60 s. After that, the chamber pressure was restored to the pre-cooling steady-state pressure and the temperature was further reduced to the final freezing temperature. The criterion for synchronous nucleation was that the temperature rebound amplitude caused by the release of latent heat in the temperature curve of each vial product was 0.5°C to 3.0°C and the maximum time difference between vials at the start of the rebound was no more than 20 s.

3. The method for enhancing the stability of ulinastatin lyophilized powder injection according to claim 1, characterized in that, The double annealing process in step S4 includes holding the freeze-dried block at the final freezing temperature for 10 to 60 minutes, then raising the shelf temperature to the first annealing temperature of -25°C to -15°C and holding it for 0.5 to 4 hours to allow ice crystal rearrangement and ensure that the coefficient of variation of the pore size distribution of the freeze-dried block is no greater than 0.

35. Subsequently, the shelf temperature is raised to the second annealing temperature of -12°C to -6°C and held for 0.25 to 2 hours to ensure that the crystallinity of the crystalline framework excipient is no less than 80% and the difference in crystallinity between bottles is no greater than 10%. After the double annealing is completed, the shelf temperature is lowered to no higher than -35°C to solidify the solid phase structure. The coefficient of variation of the pore size distribution is obtained by statistical analysis of the pore size of the cross-section of the freeze-dried block after annealing, and the crystallinity is determined by differential scanning calorimetry or powder X-ray diffraction.

4. A method for enhancing the stability of ulinastatin lyophilized powder injection according to claim 1, characterized in that, In step S5, during the primary and secondary drying processes, a product temperature safety margin is first set based on the collapse critical temperature or eutectic critical temperature of the solution to be freeze-dried. This safety margin ranges from 1°C to 5°C. The product temperature is constrained by the condition that it does not exceed the collapse critical temperature or eutectic critical temperature minus the safety margin. Subsequently, during the primary drying stage, the shelf temperature is gradually increased to the target primary drying temperature at a rate of 0.1°C per minute to 1.0°C per minute, while simultaneously controlling the chamber pressure between 10 Pa and 120 Pa to ensure the product temperature remains within the target range. Under temperature constraints, the product is dried close to the collapse critical temperature or the eutectic critical temperature for at least 20 minutes. The temperature fluctuation of the product within this time is no more than 0.5℃ and the pressure fluctuation of the cavity is no more than 5Pa, which is used as the steady-state criterion for the first drying stage. Then, the product enters the second drying stage, and the shelf temperature is further increased to 15℃ to 35℃ and maintained for 1 hour to 10 hours, so that the residual moisture content of the finished product is 0.3% to 1.5% and the water activity is 0.05 to 0.

25. The residual moisture content is determined by Karl Fischer method and the water activity is determined by water activity meter.

5. A method for enhancing the stability of ulinastatin lyophilized powder for injection according to claim 1, characterized in that, In step S5, the product temperature signal and the cavity pressure signal are synchronously acquired with a sampling period of 10s to 60s and closed-loop parameter tuning control is performed. The closed-loop parameter tuning control includes taking the product temperature meeting the temperature constraint condition as the first priority constraint. When the product temperature is detected to reach the upper limit boundary of the temperature constraint condition for 30s, the shelf temperature is reduced by 0.5℃ to 2℃ and the cavity pressure is increased by 5Pa to 30Pa. When the product temperature is detected to be more than 1℃ below the upper limit boundary for 10min and the cavity pressure fluctuation amplitude is not greater than 5Pa, the shelf temperature is restored at a heating slope of 0.1℃ per min to 0.5℃ per min and the cavity pressure is reduced by 5Pa to 30Pa. After the secondary drying stage is completed, the drying endpoint criterion verification is performed. The drying endpoint criterion verification includes closing the valve connected to the vacuum pump and recording the cavity pressure rise rate within 30s to 180s. When the cavity pressure rise rate is not greater than 0.5Pa per s, the secondary drying endpoint is determined to be established and the pressure plug sealing step is entered.

6. A method for enhancing the stability of ulinastatin lyophilized powder for injection according to claim 1, characterized in that, The verification of the reconstitution and infusion stability of the lyophilized product in step S6 includes reconstitution of the lyophilized product with water for injection, with a reconstitution volume of 1 mL to 10 mL and a reconstitution time of no more than 120 s. After reconstitution, the product is diluted with an infusion medium to form an infusion solution. The infusion medium is 0.9% sodium chloride injection or 5% glucose injection. After the infusion solution is allowed to stand at 25°C for 4 to 8 hours, its activity retention rate is no less than 90% and the aggregate increment is no more than 2.0%. Furthermore, the pH change of the infusion solution does not exceed 0.2 pH units and the turbidity is no more than 3.0 NTU.

7. A method for enhancing the stability of ulinastatin lyophilized powder injection according to claim 1, characterized in that, In step S6, the infusion solution is continuously infused through the infusion tubing at a flow rate of 1 mL / min to 10 mL / min for 30 min to 180 min, and then the adsorption and shear stability are verified. The relative difference in ulinastatin content before and after infusion is no more than 5% as the tubing adsorption limit, and the change in aggregate increment before and after infusion is no more than 0.5% as the shear stability limit. The ulinastatin content is determined by reversed-phase high-performance liquid chromatography or protein quantification method, and the aggregate increment is determined by size exclusion chromatography.

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