A reduced scale model for development and characterization of a biological formulation freeze-thaw process and applications thereof

Abstract

This invention discloses a scaled-down model for the development and characterization of freeze-thaw processes for biological agents and its application. The model has a volume of 100-1000 mL. When placed vertically, the ratio of the maximum cross-sectional area in the horizontal direction to the length in the vertical direction is 1-10:1. The amount of biological agent is 20%-80% of its volume, and the ratio of the maximum cross-sectional area to the liquid level is 1-15:1. After the biological agent is loaded into the scaled-down model, it is transferred from room temperature to a freezer. After complete freezing, it is removed and thawed at room temperature. The freezing concentration factor is tested, and the duration of ice-water interface coexistence is recorded. The scaled-down model of this invention can obtain a freezing concentration factor greater than or close to that of the control model by comparing the freezing concentration factor with that of a large-volume freeze-thaw process. Multiple freeze-thaw cycles ensure that the ice-water interface coexistence time is not shorter than that of the large-volume container, which can be used for subsequent development and characterization of freeze-thaw processes for biological agents. The scaled-down model of this invention uses a small amount of biological agent and does not require temperature control equipment, which can significantly reduce research and development costs.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a scaled-down model for the development and characterization of freeze-thaw processes for biological agents and its applications. Background Technology

[0002] Biologics are an important part of the pharmaceutical industry today. Compared with traditional small molecule drugs, antibody drugs have higher selectivity and efficacy, and fewer toxic side effects. Therefore, antibody drugs have wide applications and a huge market in oncology, immunology, cardiovascular diseases, and other fields.

[0003] Freeze-thaw cycles are common processes in the manufacturing, storage, and transportation of biopharmaceuticals. For most antibody drugs, drug substance production and product filling are separate processes. Biopharmaceutical drug substances are typically batch-frozen before being transported to the filling plant and undergoing the final filling process. This process improves the storage stability of the drug substance and extends its shelf life by reducing reaction kinetics, limiting potential microbial growth, and minimizing potential interfacial stresses introduced during transportation. After the frozen drug substance is transported to the filling plant, it needs to be thawed before being filled into the final biopharmaceutical product.

[0004] While freeze-thaw processes are critical operational units in the filling / finishing process, they generate numerous stress conditions detrimental to proteins, potentially leading to aggregation or denaturation and impacting the quality of the final product. Among these, the freeze-concentration effect and the pressure at the ice-water interface are key considerations in freeze-thaw process research. As the stock solution gradually freezes into ice, the solute (proteins and excipients, etc.) gradually concentrates in the uncrystallized solution as ice crystals grow, increasing viscosity, decreasing diffusion coefficient, and ultimately forming a glassy state. This phenomenon is known as the freeze-concentration effect. The freeze-concentration effect affects the pH, ionic strength, permeate, and viscosity of the buffer solution; these physicochemical changes can lead to protein denaturation. Furthermore, proteins are subjected to pressure from the ice-water interface. During freezing, proteins interact with the ice surface, weakening their hydrophobic bonds and disrupting their native structure.

[0005] Because freeze-thaw processes can affect protein quality, freeze-thaw technology needs to be developed during drug development to reduce the negative impact of freeze-thaw cycles on product quality. In freeze-thaw process development, the degree of freeze concentration effect can be characterized by the freeze concentration factor. The freeze concentration factor represents the concentration change of a component in solution, generally referring to the factor by which the component is concentrated after a freeze-thaw process; specifically, it is the ratio of the component's current concentration to its initial concentration. For example, for protein molecules, the freeze concentration factor can be expressed as the ratio of the protein concentration in the sample to the initial protein concentration. If the freeze concentration factor of a protein molecule at a certain location is less than 1, it means that the protein concentration at that location is less than the protein concentration at that location before freeze-thaw. Conversely, if the freeze concentration factor of a protein at a certain location is greater than 1, it means that the protein concentration at that location is greater than the protein concentration at that location before freeze-thaw, meaning the protein has been "concentrated" after the freeze-thaw process. For electrolytes, such as sodium chloride, the freeze concentration factor at a certain location can be expressed as the ratio of the component's conductivity at that location to its original conductivity. For non-electrolytes, such as sucrose, the freeze-concentration factor at a given location can be expressed as the ratio of the osmotic pressure at that location to the original osmotic pressure. For crystallizable excipients, such as sodium chloride, as they gradually concentrate to their solubility limit during freeze-concentration, they crystallize out of the solution to form a eutectic, thus resulting in a relatively low freeze-concentration factor. For non-crystallizable excipients, such as sucrose, they are concentrated to a higher concentration during freeze-concentration, thus exhibiting a relatively higher freeze-concentration factor.

[0006] The pressure from the ice-water interface is often characterized by the ice-water interface duration. The ice-water interface duration refers to the total time the ice and water phases coexist during the freeze-thaw process, which can be further divided into the ice-water interface duration during freezing and the ice-water interface duration during thawing. In freeze-thaw process development, the total ice-water interface duration is generally the focus. In freeze-thaw process research, thermocouple temperature probes can be used to detect temperature changes throughout the solution system. To more accurately obtain the coexistence time of the ice and water phases, the ice-water interface duration can be defined as the duration for which the temperature value recorded by the temperature probe is within the range of +3°C to -3°C.

[0007] Due to differences in heat exchange area and resulting heat transfer efficiency between different sizes, the freeze-thaw process of biopharmaceuticals becomes more complex as the fill volume increases. Although the freeze-thaw process is closely related to the fill volume, in the early stages of biopharmaceutical development, biopharmaceutical materials are relatively scarce and expensive, making it difficult to obtain large volumes of biopharmaceutical materials for large-scale freeze-thaw process development. Therefore, the main strategy currently is to use a programmable temperature controller (PTC) to manually control the freezing and thawing rates, establishing a freeze-thaw scale-down model to simulate large-volume freeze-thaw processes. This approach of establishing a freeze-thaw scale-down model is "active" and relies on a PTC, thus requiring sophisticated equipment. Furthermore, the use of liquid nitrogen to cool samples during PTC operation often results in high overall operating costs. However, if a programmable temperature controller is not used, and only a quick-freezing cabinet or ultra-low temperature freezer is used, it is difficult to directly use a small volume of stock solution to simulate the freeze-thaw situation of a large volume of stock solution. For example, the freezing and concentration effect of a small volume of stock solution cannot reach the freezing and concentration effect of a large volume of stock solution, and the ice-water interface time of a small volume of stock solution cannot reach the ice-water interface time of a large volume of stock solution.

[0008] In summary, the development of freeze-thaw processes is a crucial part of biopharmaceutical R&D. Currently, the design and development strategies for scaled-down models are "proactive," relying heavily on programmed temperature controllers, which leads to high dependence on equipment and consequently high development costs and barriers to entry. Therefore, there is an urgent need for a new freeze-thaw scaled-down model design method and development strategy that can operate independently of programmed temperature controllers. Summary of the Invention

[0009] The technical problem to be solved by the present invention is to provide a scaled-down model for the development and characterization of freeze-thaw processes for biological agents and its application. The scaled-down model realizes small-volume freeze-thaw simulation of large-volume freeze-thaw processes. The freeze-thaw process developed based on the scaled-down model has high reliability, can save the amount of biological agents used, does not rely on programmed temperature control, and reduces the development cost of freeze-thaw processes. Therefore, it is easy to apply to the development of freeze-thaw processes.

[0010] To solve the above-mentioned technical problems, the first aspect of the present invention provides a scaled-down model for the development and characterization of the freeze-thaw process of biological agents, wherein when the scaled-down model is placed vertically, the ratio of the cross-sectional area of ​​its maximum horizontal section to its height in the vertical direction is 1-10:1.

[0011] The volume of the scaled-down model is 100-1000 mL;

[0012] The amount of biological agent contained in the miniature model is 20%-80% of its volume, and when the miniature model is placed vertically, the ratio of the maximum cross-sectional area of ​​its horizontal section to the height of the liquid surface is 1-15:1.

[0013] Preferably, the scaled-down model is a cylinder or a sphere.

[0014] In this invention, the volume of the scaled-down model is in the range of 100-1000 mL, which is suitable for small-volume freeze-thaw experiments. This volume of scaled-down model can significantly reduce the use of the freeze-thaw solution (i.e., the biological agent, including the simulated solution containing it, or the biological agent itself, i.e., the stock solution), thus saving development costs. The scaled-down model of this invention is generally a cylinder or a sphere. Cylinders include prisms, cylinders, elliptical cylinders, etc.; prisms include triangular prisms, cuboids, cubes, pentagonal prisms, etc.; spheres include spheres, etc. When the scaled-down model is a cylinder, its opening can be located at the end faces of both ends of the cylinder. It can also be located on its side, but it needs to meet the following requirements: when the opening is placed upwards (vertically), the ratio of the maximum cross-sectional area in the horizontal direction to the height in the vertical direction (referring to the height of the cavity) must be 1-10:1. When the freeze-thaw liquid is added, the amount of freeze-thaw liquid added must simultaneously meet the following requirements: accounting for 20%-80% of the volume of the reduced model, and when the opening of the reduced model is placed upwards (vertically), the ratio of the maximum cross-sectional area in the horizontal direction to the height of the liquid surface must be 1-15:1, so that the reduced model can meet the needs of simulating the actual large-volume freeze-thaw process.

[0015] It should be noted that, in this invention, "biological agent" refers to a product obtained using various biotechnologies or from various biological materials, such as biological drugs, selected from cytokines, erythropoietin, plasminogen activator, plasma factors, growth hormones, growth factors, insulin, immunoglobulins, antibodies (especially monoclonal antibodies), or vaccines (e.g., peptide, polypeptide, or protein vaccines); furthermore, the biological agent may contain or be composed of immunoglobulins; the "simulating solution" in this invention refers to a liquid used to simulate the freeze-thaw conditions of biological agents in actual production, and its formulation may be the same as or different from that of the biological agent, such as the biological product stock solution. The difference can be in the composition or in the concentration of components. For example, the simulated solution may be a combination of various candidate formulations of the biological agent; furthermore, the simulated solution may not contain one or more biological products contained in the biological agent in actual production; furthermore, the simulated solution is similar to a placebo in clinical trials, that is, the only difference between the simulated solution and the biological agent in actual production is that it does not contain biological products (e.g., proteins and monoclonal antibodies) as active ingredients.

[0016] In this invention, the scaled-down model can meet the needs of simulating actual large-volume freeze-thaw processes. It is mainly determined by the freezing concentration factor and the time required for coexistence of ice and water (including the freezing and thawing processes). Generally, the "worst-case analysis" is adopted, that is, the protein is subjected to greater pressure conditions in the scaled-down model than the various pressure conditions faced in actual large-volume production. If the quality properties of the biopharmaceutical do not change significantly in the scaled-down model, the biopharmaceutical will not experience a decrease in product quality in actual production. In this invention, when the freeze concentration factor of the scaled-down model is greater than or close to (for example, the maximum freeze concentration factor range of the large-volume freeze-thaw process is obtained, and the freeze concentration factor of the scaled-down model is within this range) the freeze concentration factor of the large-volume freeze-thaw process, the simulated liquid or stock solution will not affect the product quality (protein) in the scaled-down model, and will also not be affected in the large-volume freeze-thaw process. The coexistence time of the simulated liquid or stock solution at the ice-water interface in the scaled-down model can be simulated through multiple rounds of freeze-thaw processes. For example, after simulating several rounds, the total coexistence time of the ice-water interface is calculated, and the impact on the product quality (protein) is evaluated. When the coexistence time of the simulated liquid or stock solution at the ice-water interface in the large-volume freeze-thaw process is not longer than the total coexistence time of the ice-water interface in the scaled-down model, the impact on the product quality of the simulated liquid or stock solution in the large-volume freeze-thaw process is not greater than that in several rounds of freeze-thaw processes in the scaled-down model. It should be noted that in this invention, the freezing concentration factor is the change in concentration of a certain component in the solution, specifically the ratio of the concentration of that component at the sampling location to the initial concentration. The concentration of that component is measured at the time of sampling, while the initial concentration is the overall concentration of the solution before freezing and thawing. The freezing concentration factor also varies depending on the component. When the component is a protein, the freezing concentration factor is the ratio of the protein concentration before and after the freeze-thaw operation at the sampling location. When the component is an electrolyte (such as sodium chloride), the freezing concentration factor is the ratio of the conductivity value of the electrolyte before and after the freeze-thaw operation at the sampling location. When the component is a non-electrolyte (such as sucrose), the freezing concentration factor is the ratio of the osmotic pressure value of the non-electrolyte before and after the freeze-thaw operation at the sampling location.

[0017] To address the aforementioned technical problems, a second aspect of the present invention is to provide an application of a scaled-down model in the development and characterization of freeze-thaw processes for biopharmaceuticals. Understandably, since the scaled-down model provided by the present invention can meet the needs of simulating actual large-volume freeze-thaw processes, it can be applied to the development and characterization of freeze-thaw processes for biopharmaceuticals.

[0018] To address the aforementioned technical problems, a third aspect of the present invention is to provide a method for developing and characterizing the freeze-thaw process of biopharmaceuticals using a scaled-down model, characterized by comprising the following steps:

[0019] S1. Prepare the freeze-thaw solution;

[0020] S2. Fill the shrink model with the liquid to be frozen and thawed, the filling amount is 20%-80% of the shrink model volume, and after filling, the ratio of the cross-sectional area of ​​the maximum radial section of the shrink model to the liquid level height is 1-15:1.

[0021] S3. Place the miniature model containing the freeze-thaw solution into the freezer and freeze it. Record the time required for the ice-water interface to coexist. After freezing is complete, place it at room temperature to thaw until it is completely thawed. Record the time required for the ice-water interface to coexist. The freezing and thawing steps are repeated 1-10 times.

[0022] S4. After the freezing and thawing cycle is completed, samples are taken and tested to obtain the freezing concentration factor of the freeze-thaw solution and to obtain the effect of the freezing and thawing process on the quality of the freeze-thaw solution.

[0023] In this invention, the preparation of the freeze-thaw solution is consistent with that used in large-volume freeze-thaw processes, generally a simulated solution or a stock solution. Because a scaled-down model (e.g., 100-1000 mL) is used, the volume required is much lower than in large-volume freeze-thaw processes, significantly reducing costs. Preferably, the amount of freeze-thaw solution filled into the scaled-down model is 20%-80%, and after filling, the ratio of the maximum radial cross-sectional area of ​​the scaled-down model to the liquid level height is 1-15:1 to ensure simulation of the large-volume freeze-thaw process. The refrigerator used in this invention is, for example, a blast freezer or an ultra-low temperature freezer, the same type used in large-volume freeze-thaw processes, eliminating the need for temperature control equipment and further reducing simulation costs. The refrigerator is always kept at a set temperature, and the scaled-down model containing the freeze-thaw solution is directly placed inside. The small model can be placed in the freezer with its opening facing upwards for freezing. Generally, a thermocouple temperature probe needs to be inserted into the freeze-thaw mixture of the scaled-down model to monitor the temperature changes of the freeze-thaw mixture, especially the duration of the temperature at the ice-water interface. After the freeze-thaw mixture is completely frozen, it is removed from the freezer and allowed to thaw naturally at room temperature. During this process, the thermocouple temperature probe is used to monitor the temperature changes of the freeze-thaw mixture, especially the duration of the temperature at the ice-water interface. After complete thawing, it can be placed back in the freezer for the next round of freezing and thawing, and the total duration of the ice-water interface is recorded. It should be noted that the quality of the freeze-thaw mixture can be evaluated using conventional methods, which will not be elaborated here.

[0024] In one specific embodiment, the refrigerator temperature is maintained between -100°C and -5°C, or between -80°C and -20°C; complete freezing is defined as the temperature of the thawing solution dropping to between -100°C and -5°C, or between -80°C and -20°C; complete thawing is defined as the temperature of the thawing solution rising to 5°C to room temperature; and ice-water interface coexistence is defined as the time required for the thawing solution to maintain a temperature between -3°C and 3°C. In this invention, the temperature range for ice-water interface coexistence is defined as between -3°C and 3°C. Therefore, the duration of ice-water interface coexistence is the duration for which the temperature of the thawing solution detected by the thermocouple temperature detection device is within the temperature range of -3°C to 3°C, including the duration of the freezing process and the duration of the thawing process.

[0025] The method for developing and characterizing the freeze-thaw process of biological agents using a scaled-down model provided by this invention enables the simulation of a large-volume (e.g., 5L) freeze-thaw process using a scaled-down model (e.g., 100-1000mL), allowing for the development and characterization of the freeze-thaw process using a small amount of simulated solution or stock solution, and without the need for a temperature control device. Furthermore, by limiting the size of the scaled-down model and the sample volume, the freezing concentration factor of the freeze-thaw process using the scaled-down model is greater than or close to (for example, the maximum freezing concentration factor range of the large-volume freeze-thaw process is obtained, and the freezing concentration factor of the scaled-down model is within this range) the freezing concentration factor of the large-volume freeze-thaw process. Therefore, after multiple freeze-thaw cycles on a scaled-down scale (the total duration of ice-water interface coexistence is not less than the duration of ice-water interface coexistence in the large-volume freeze-thaw process), the quality evaluation of the freeze-thaw process is equivalent to or more stringent than that of the large-volume freeze-thaw process.

[0026] To address the aforementioned technical problems, a fourth aspect of the present invention provides a method for preparing a scaled-down model for the development and characterization of freeze-thaw processes of biological agents, comprising the following steps:

[0027] S1. Obtain the range of the comparative freezing concentration factor for the freeze-thaw solution in the container used in actual production, as well as the range of the total ice-water interface time during the freezing and thawing process.

[0028] S2. Determine the volume of the scaled-down model and design scaled-down models of various shapes;

[0029] S3. Use the scaled-down model designed in step S2 to perform freezing and thawing operations on the liquid to be frozen and thawed. After one cycle, take samples to test and obtain the freezing concentration factor of the liquid to be frozen and thawed in scaled-down models of different shapes. Select scaled-down models whose freezing concentration factor is within the range of the comparison freezing concentration factor or greater than the maximum value of the comparison freezing concentration factor for use, and discard scaled-down models whose freezing concentration factor is less than the minimum value of the comparison freezing concentration factor range.

[0030] S4. In the selected scaled-down model, change the amount of the freeze-thaw solution to be added, and perform freezing and thawing operations on the freeze-thaw solution. After one cycle, take samples to test and obtain the freeze concentration factor of the freeze-thaw solution in scaled-down models of different shapes. Select the amount of freeze-thaw solution to be added that has a freeze concentration factor within the range of the comparative freeze concentration factor or greater than the maximum value of the comparative freeze concentration factor.

[0031] The method for preparing a scaled-down model provided by this invention first screens the designed scaled-down models based on parameters of large-volume freeze-thaw cycles. This includes the shape of the scaled-down model, the ratio between the cross-sectional area of ​​the largest cross-section and its height (referring to the height of the containment cavity), and determining the ratio between the cross-sectional area of ​​the largest cross-section and the height of the liquid surface when the liquid to be frozen and thawed is in the scaled-down model. The final selected scaled-down model, after being frozen and thawed in the refrigerator used for large-volume freeze-thaw cycles, has a freezing concentration factor greater than or close to that of the large-volume freeze-thaw process, and after multiple freeze-thaw cycles, the total duration of ice-water interface coexistence is not less than the total duration of ice-water interface coexistence during the large-volume freeze-thaw process. The model includes cylinders and spheres, and the volume of the scaled-down model is in the range of 100-1000mL. If the volume is too large, it will consume too much freeze-thaw solution, and if the volume is too small, it will be difficult to simulate large-volume freeze-thaw cycles. When the selected scaled-down model is placed with the opening facing upward (vertically), the ratio of the maximum cross-sectional area in the horizontal direction to the height in the vertical direction (referring to the height of the containment cavity) is 1-10:1. When freeze-thaw solution is added, the amount of freeze-thaw solution added must simultaneously meet the following requirements: accounting for 20%-80% of the volume of the scaled-down model, and when the scaled-down model is placed with the opening facing upward (vertically), the ratio of the maximum cross-sectional area in the horizontal direction to the height of the liquid surface is 1-15:1. It should be noted that the total duration of ice-water interface coexistence during large-volume freeze-thaw cycles (including freezing and thawing processes) can be obtained through testing. However, generally, once the large-volume container is confirmed (mainly its capacity), the total duration of ice-water interface coexistence can be determined based on the actual amount of solution (biological agent) used in mass production. The type of biological agent has little impact on the total duration, so it can also be determined based on experience. Equipment manufacturers can also provide this information. Since the total duration of ice-water interface coexistence is basically determined once the large-volume container and the actual amount of biological agent used in production are determined, for a scaled-down model of a given volume, after loading a certain amount of biological agent and testing the total duration of ice-water interface coexistence during one freeze-thaw cycle, the number of freezing and thawing cycles can be determined to ensure that the total duration of ice-water interface coexistence after multiple cycles of the scaled-down model is not shorter than that of the large-volume container. Attached Figure Description

[0032] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the present invention 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.

[0033] Figure 1 This refers to the large-volume container and sampling location used in actual production as described in Example 1.

[0034] Figure 2 This is a temperature change graph of the freeze-thaw solution in a large-volume container used in actual production during the freezing process of the freeze-thawed liquid in Example 1, which shows the changes at different temperature test locations.

[0035] Figure 3 This is a temperature change graph of the thawing process of the liquid to be thawed in a large-volume container used in actual production, as shown in Example 1. The graph shows the changes at different temperature test locations.

[0036] Figure 4 These are the three views of container No. 1 in Embodiment 1;

[0037] Figure 5 These are the three views of container No. 2 in Embodiment 1;

[0038] Figure 6 These are the three views of container No. 3 in Example 1;

[0039] Figure 7 These are the three views of container number 4 in Example 1;

[0040] Figure 8 These are the three views of container No. 5 in Example 1;

[0041] Figure 9 This is a temperature change graph of the freeze-thawed liquid in five scaled-down models in Example 1;

[0042] Figure 10 This is a temperature change graph of the thawing process of the liquid to be thawed in five scaled-down models in Example 1;

[0043] Figure 11 This is a graph showing the temperature changes during the freezing process of the liquid to be thawed in container No. 2 with different amounts added, as shown in Example 1.

[0044] Figure 12 This is a temperature change graph of the freeze-thaw solution in container No. 2 at different addition levels in Example 1.

[0045] Figure 13 This is a schematic diagram of container number 6 in Example 2;

[0046] Figure 14This is a temperature change diagram of the freeze-thaw solution in container No. 2 during the freezing process in Example 2;

[0047] Figure 15 This is a temperature change graph showing the thawing process of the liquid to be thawed in container No. 2 in Example 2. Detailed Implementation

[0048] The technical solutions of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. 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.

[0049] In the embodiments of this invention, the ultra-low temperature freezer was purchased from Eppendorf (model: F740hi) or Thermofisher Scientific (model: FDE60086FV); the thermocouple temperature probe (model: 5SRTC series) and temperature detector (model: RDXL12SD) were purchased from OMEGA. The osmotic pressure analyzer (model: Osmotech Pro) was purchased from Advanced Instruments, Inc.; the multi-parameter measuring instrument (model: S470-B) was purchased from Mettler Toledo. The 5L polycarbonate bottle (model: 3405) was purchased from Thermofisher Scientific. Bovine serum albumin (BSA, catalog number: KY-01014) was purchased from Kangyuan Biotechnology Co., Ltd.

[0050] Example 1: Preparation of the scaled-down model

[0051] Step S1: Test the range of the freeze concentration factor for the freeze-thaw solution in the container used in actual production, as well as the range of the total ice-water interface time during the freezing and thawing process.

[0052] The simulated solution was formulated with 20 mM histidine buffer, 4% (w / v) sucrose, 70 mM sodium chloride, and 0.04% (w / v) polysorbate 80, pH 6.0. The 20 mM histidine buffer (pH 6.0) is a commonly used buffer system in commercially available biological agents. Sucrose and sodium chloride are not only common excipients in biological agents but also typical non-electrolytes and electrolytes, respectively, thus allowing for the study of the freeze-thaw concentration effect of non-electrolytes and electrolytes. Polysorbate 80 is also a commonly used surfactant in biological agents. The theoretical osmotic pressure of this simulated solution is approximately 287 mOsm / kg, classifying it as an isotonic solution. The measured osmotic pressure of the homogeneously mixed simulated solution was 275 mOsm / kg, and the conductivity was 7.49 mS / cm.

[0053] The large-volume containers used in actual production are 5L polycarbonate bottles, which are commonly used in reagent production.

[0054] To simulate the freeze-thaw process of large-volume biological agents, 4L of simulated solution was placed into 5L polycarbonate bottles, and the simulated solution was transferred from room temperature (18–24℃) to an ultra-low temperature freezer (-70±10℃, model: FDE60086FV). After it was completely frozen, the frozen 5L polycarbonate bottles containing the simulated solution were transferred from the -70±10℃ freezer to room temperature for static thawing. After the simulated solution returned to room temperature, samples were taken at six sampling locations for osmotic pressure and conductivity tests.

[0055] Six sampling locations were selected: the top center, the bottom corner and center. See details below. Figure 1 As shown, the freeze concentration factor was tested at six sampling locations (see Table 1). Thermocouple probes were used to detect temperature changes at position 1 (center of the solution) and 1 cm below position 1 (1 cm below the liquid surface) in the freeze-thaw process to determine the total duration of ice-water interface coexistence during the freeze-thaw process (including both freezing and thawing processes; temperature changes during freezing are shown in Table 1). Figure 2 The temperature changes during the thawing process are shown in the figure. Figure 3 The ice-water interface coexistence temperature was set from +3℃ to -3℃, and the resulting ice-water interface coexistence time is shown in Table 2.

[0056] Table 1

[0057]

[0058] As can be seen from Table 1, for the prepared simulated solution, the maximum freezing concentration factor is located at the bottom, and the maximum freezing concentration factor of sucrose is between 6 and 8, while that of sodium chloride is between 2 and 3.

[0059] Table 2

[0060]

[0061] It should be noted that, through Figure 2 and Figure 3 It can be seen that the temperature change is the same at the center of the solution and at 1 cm below the surface. Therefore, the choice of temperature measurement location has almost no effect on the temperature measurement of the system. Generally, the temperature is measured at 1 cm below the surface.

[0062] Step S2: Determine the volume of the scaled-down model and design scaled-down models of various shapes;

[0063] The volume of the scaled-down model is approximately 500 mL, and the designed shape of the scaled-down model is as follows: Figures 4-8 As shown (wall thickness is negligible, approximating the shape of the cavity), there are 5 types in total;

[0064] The first scaled-down model (container #1) has a rectangular body with the opening located on its end face (the end face is square, and the side length of the cavity is 7.5cm). When placed vertically, its cross-sectional area is 56.3cm². 2 The height of the receiving cavity is 9.3 cm, and the ratio of the maximum cross-sectional area to the height of the receiving cavity is 6.05 cm; when about 80% of the liquid to be frozen and thawed is placed in, the liquid level is 8 cm, and the ratio of the maximum cross-sectional area to the liquid level is 7.03 cm.

[0065] The second scaled-down model (container number 2) has a cylindrical body with the opening located on its end face (the end face is circular, and the diameter of the cavity is 6.4 cm). When placed vertically, its cross-sectional area is 32.2 cm². 2 The height of the containment cavity is 16.3 cm, and the ratio of the maximum cross-sectional area to the height of the containment cavity is 1.98 cm; when about 80% of the liquid to be frozen and thawed is placed in, the liquid level is 13.6 cm, and the ratio of the maximum cross-sectional area to the liquid level is 2.37 cm.

[0066] The third scaled-down model (container number 3) has a cylindrical body with the opening located on its end face (the end face is circular, and the diameter of the cavity is 7.5 cm). When placed vertically, its cross-sectional area is 44.2 cm². 2 The height of the receiving cavity is 11.6 cm, and the ratio of the maximum cross-sectional area to the height of the receiving cavity is 3.81 cm; when about 80% of the liquid to be frozen and thawed is placed in, the liquid level is 9.8 cm, and the ratio of the maximum cross-sectional area to the liquid level is 4.51 cm.

[0067] The fourth scaled-down model (container number 4) has a bottle body that is approximately a flattened cylinder (see reference). Figure 7 The three views shown indicate that when placed vertically, the side surface is a circle with a diameter of 11.5 cm, but the top and bottom are flat-cut into horizontal planes. The thickness of the receiving cavity (the distance between the front and rear end faces of the flattened cylindrical receiving cavity) is 6 cm. When placed vertically, the cross-sectional area of ​​the maximum cross-section (rectangle) is 69 cm². 2 The height of the receiving cavity is 10.6 cm, and the ratio of the maximum cross-sectional area to the height of the receiving cavity is 6.51 cm; when about 80% of the liquid to be frozen and thawed is placed in, the liquid level is 7.3 cm, and the ratio of the maximum cross-sectional area to the liquid level is 9.45 cm.

[0068] The fifth scaled-down model (container number 5) has a bottle body that is approximately spherical (see reference). Figure 8 The three views shown indicate that when placed vertically, the side surface is a circle with a diameter of 10cm, but the top and bottom are flat and horizontal; the largest cross-section in the receiving cavity is a circle with a diameter of 10cm, and its cross-sectional area is 78.5cm². 2The height of the receiving cavity is 9.4 cm; the ratio of the maximum cross-sectional area to the height of the receiving cavity is 8.35; when about 80% of the liquid to be frozen and thawed is placed in, the liquid level is 6.5 cm, and the ratio of the maximum cross-sectional area to the liquid level is 12.08 cm.

[0069] Step S3: Use the scaled-down model designed in step S2 to perform freezing and thawing operations on the liquid to be frozen and thawed. After one cycle, take samples and test to obtain the freezing concentration factor of the liquid to be frozen and thawed in scaled-down models of different shapes. Select scaled-down models whose freezing concentration factor is within the range of the comparison freezing concentration factor or greater than the maximum value of the comparison freezing concentration factor for use, and discard scaled-down models whose freezing concentration factor is less than the minimum value of the comparison freezing concentration factor range.

[0070] Specifically, five scaled-down models, each filled with approximately 80% simulated liquid, were transferred from room temperature (18–24°C) to an ultra-low temperature freezer (-70±10°C, model: FDE60086FV). After complete freezing, containers 1 through 5, containing the simulated liquid, were transferred from the -70±10°C freezer to room temperature for static thawing. Once the simulated liquid returned to room temperature, samples were taken and their osmotic pressure and conductivity were measured. The test results are shown in Table 3. The temperature changes during the freezing process of the five scaled-down models are shown in Table 3. Figure 9 The temperature changes during the thawing process are shown in the figure. Figure 10 Using +3℃ to -3℃ as the ice-water interface coexistence temperature, the ice-water interface coexistence time for one and four cycles is shown in Table 4 (the probe position is 1cm below the liquid surface).

[0071] Table 3

[0072]

[0073] As can be seen from Tables 2 and 3, the maximum freezing concentration factor of sucrose and the maximum freezing factor of sodium chloride in the five scaled-down models meet the requirements of worst-case analysis. It should be noted that the maximum osmotic pressure and maximum conductivity are the maximum values ​​measured after sampling from the bottom of the scaled-down model.

[0074] Table 4

[0075]

[0076] As can be seen from Tables 2 and 4, the ice-water interface duration of a single small-volume freeze-thaw cycle in the five designed scaled-down models is difficult to match that of a large-volume freeze-thaw cycle. However, after four freeze-thaw cycles, the total ice-water interface duration experienced by the biopharmaceutical in the small-volume container ranges from 36 to 43.6 hours, which is longer than the ice-water interface duration during a large-volume (5L polycarbonate bottle) freeze-thaw process. Therefore, the ice-water interface pressure faced by the biopharmaceutical in a large-volume freeze-thaw cycle can be simulated by increasing the number of freeze-thaw cycles in the scaled-down models.

[0077] Step S4: In the selected scaled-down model, change the amount of the freeze-thaw solution to be added, and perform freezing and thawing operations on the freeze-thaw solution. After one cycle, take samples to test and obtain the freeze concentration factor of the freeze-thaw solution in scaled-down models of different shapes. Select the amount of freeze-thaw solution to be added that is within the range of the freeze concentration factor used for comparison or greater than the maximum value of the freeze concentration factor used for comparison.

[0078] In this study, container No. 2 was used for freeze-thaw operations. 100 mL, 200 mL, 300 mL, and 400 mL of simulated solution were poured into container No. 2, resulting in liquid levels of 3.2 cm, 7 cm, 10.4 cm, and 13.7 cm, respectively. The simulated solution was transferred from room temperature (18–24°C) to an ultra-low temperature freezer (-70±10°C, model: FDE60086FV). After complete freezing, the frozen containers containing the simulated solution were transferred from the -70±10°C freezer to room temperature for static thawing. Samples were taken after the solution had recovered to normal temperature, and the osmotic pressure and conductivity were measured. The test results are shown in Table 5; the temperature changes during the freezing process are shown in [Table 5]. Figure 11 The temperature changes during the thawing process are shown in the figure. Figure 12 Using +3℃ to -3℃ as the ice-water interface coexistence temperature, the ice-water interface coexistence time for one round and multiple rounds is shown in Table 6 (the temperature probe is located 1 cm below the liquid surface).

[0079] Table 5

[0080]

[0081] Table 5 shows that the fill volume has a significant impact on the freeze-thaw concentration effect. In container No. 2, the larger the fill volume of the simulated liquid, the greater the maximum freeze-thaw concentration factor for sucrose and sodium chloride. When the fill volume reaches 300 mL, the maximum freeze-thaw concentration factors for sucrose and sodium chloride no longer increase significantly with increasing fill volume. Specifically, when the fill volumes are 200 mL, 300 mL, and 400 mL, the maximum freeze-thaw concentration factors achievable are similar to those achievable in large-volume freeze-thaw processes (the maximum freeze-thaw concentration factor for sucrose is approximately 6-8; the maximum freeze-thaw concentration factor for sodium chloride is approximately 2-3). Therefore, a fill volume of 20%-80% of the reduced model volume, with a maximum cross-sectional area to liquid level ratio between 2.4 and 10.1:1, can be used to simulate the freeze-thaw process of large-volume solutions.

[0082] Table 6

[0083]

[0084]

[0085] Table 6 shows that as the volume of the simulated liquid increases, the total ice-water interface time after one freeze-thaw cycle also increases. Therefore, the larger the volume of the simulated liquid, the fewer freeze-thaw cycles are required to achieve the total ice-water interface time in a large-volume freeze-thaw cycle (5L polycarbonate bottle). With a volume of 100mL, the total ice-water interface time is 4.2 hours, requiring 7 freeze-thaw cycles to be comparable to a large-volume freeze-thaw cycle; while with a volume of 400mL, the total ice-water interface time is 9.0 hours, requiring only 4 freeze-thaw cycles to be comparable to a large-volume freeze-thaw cycle. Therefore, the ice-water interface pressure faced by biological agents in large-volume freeze-thaw cycles can be simulated by changing the volume and the corresponding number of freeze-thaw cycles.

[0086] Example 2: The effect of reducing the model size on the freeze-thaw process

[0087] In this embodiment, the capacity of the scaled-down model is expanded to 1000mL, and it is determined to be container number 6, such as... Figure 13 As shown (when placed vertically, the horizontal cross-section is square). The simulated liquid in this embodiment is the same as in Embodiment 1, with the addition amount of simulated liquid being 80%. The simulated liquid was transferred from room temperature (18-24℃) to an ultra-low temperature freezer (-70±10℃, model: FDE60086FV). After it was completely frozen, the frozen container No. 6 containing the simulated liquid was transferred from the -70±10℃ freezer to room temperature for static thawing. After the simulated liquid returned to room temperature, samples were taken and osmotic pressure and conductivity were measured. Since the shape of container No. 6 is similar to that of container No. 1, the test data of the two were compared synchronously. The osmotic pressure and conductivity test data are shown in Table 7; the temperature changes during the freezing process are shown in Table 7. Figure 14 The temperature changes during the thawing process are shown in the figure. Figure 15 Using +3℃ to -3℃ as the ice-water interface coexistence temperature, the ice-water interface coexistence time for one round and multiple rounds is shown in Table 8, where the values ​​for container 1 are from Example 1.

[0088] Table 7

[0089]

[0090] As shown in Table 7, the larger the container size, the stronger the maximum freeze-concentration effect achievable by the simulated solution, especially for sucrose, where the maximum freeze-concentration factor increases. Since the maximum freeze-concentration factor of sodium chloride is relatively small overall, it is more susceptible to experimental errors. Therefore, the overall freeze-concentration effect strength can be judged based on the more sensitive maximum freeze-concentration factor of sucrose. The maximum freeze-concentration factors achievable by containers 1 and 6 examined here both reach the range of maximum freeze-concentration factors achievable in the large-volume solution freeze-thaw process of Example 1 (the maximum freeze-concentration factor of sucrose is approximately 6-8; the maximum freeze-concentration factor of sodium chloride is approximately 2-3), and therefore can be used to simulate the freeze-thaw process of large-volume solutions.

[0091] Table 8

[0092]

[0093] Table 8 shows that the total ice-water interface time for the simulated liquid in container 1 was 9.6 hours during one freeze-thaw cycle, while the total ice-water interface time for the simulated liquid in container 6 was 13.1 hours after one freeze-thaw cycle. After four or three freeze-thaw cycles, the total ice-water interface time for containers 1 and 6 was longer than that of the large-volume (5L polycarbonate bottle) freeze-thaw process. Therefore, the ice-water interface pressure faced by biological agents during large-volume freeze-thaw cycles can be simulated by increasing the number of freeze-thaw cycles in the scaled-down model.

[0094] Example 3: Reduced-scale model used for freeze-thaw process development and characterization of bovine serum albumin

[0095] Freeze-thaw solution (stock solution): 20 mg / mL BSA, 20 mM histidine buffer, 4% (w / v) sucrose, 70 mM NaCl, 0.04% PS80, pH 6.0.

[0096] Scaled-down model: Container No. 2 in Example 1;

[0097] In contrast, this embodiment uses a 5L polycarbonate bottle for actual production to simultaneously freeze and thaw the stock solution.

[0098] The stock solution was added to both the scaled-down model and the 5L polycarbonate bottle at 80% capacity, i.e., 400 mL for the scaled-down model and 4 L for the 5L polycarbonate bottle. The scaled-down model and 5L polycarbonate bottle containing the stock solution were then transferred from room temperature (18–24°C) to an ultra-low temperature freezer (-70±10°C, model: FDE60086FV). After complete freezing, the frozen container (No. 2) containing the simulated solution was transferred from the -70±10°C freezer to room temperature for thawing. Once the solution had recovered to normal temperature, samples were taken, and osmotic pressure, conductivity, and protein concentration were measured.

[0099] Investigation of the freeze-thaw concentration effect: The results (Table 9) show that the maximum protein concentration achievable by the freeze-thaw reduction model of this invention is 87.4 mg / mL, comparable to the maximum protein concentration of 91.8 mg / mL achievable by large-volume freeze-thaw. The maximum freeze-thaw concentration factor achievable by the reduction model for sucrose (11.18) and sodium chloride (2.32) is greater than that for large-volume freeze-thaw (10.33) and sodium chloride (2.19). This indicates that the reduction model based on container No. 2 can simulate the maximum freeze-thaw concentration effect faced by biopharmaceuticals during large-volume freeze-thaw.

[0100] Investigation of ice-water interface duration: The results show (Table 9) that after multiple (4) freeze-thaw cycles, the "passive" freeze-thaw shrinkage model can simulate the ice-water interface pressure faced by biological agents in large-volume freeze-thaw cycles. The data on the coexistence duration of the ice-water interface are referenced in Example 1.

[0101] Table 9

[0102]

[0103] Therefore, in summary, the freeze-thaw reduction model established by rationally designing the geometry, size, and internal volume of small-volume containers can simulate the large-volume freeze-thaw process during production without the need for temperature control devices, and can be used for the research and characterization of large-volume stock solution freeze-thaw.

[0104] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A scaled-down model for the development and characterization of freeze-thaw processes for biological agents, characterized in that, When the scaled-down model is placed vertically, the ratio of the cross-sectional area of ​​its maximum horizontal section to its length in the vertical direction is 1-10:

1. The volume of the scaled-down model is 100-1000 mL; The amount of biological agent contained in the miniature model is 20%-80% of its volume, and when the miniature model is placed vertically, the ratio of the maximum cross-sectional area of ​​its horizontal section to the height of the liquid surface is 1-15:

1. The reduced model is obtained through the following method: S1. Obtain the range of the comparative freezing concentration factor for the freeze-thaw solution in the container used in actual production, as well as the range of the total ice-water interface time during the freezing and thawing process. S2. Determine the volume of the scaled-down model and design scaled-down models of various shapes; S3. Use the scaled-down model designed in step S2 to perform freezing and thawing operations on the liquid to be frozen and thawed. After one cycle, take samples to test and obtain the freezing concentration factor of the liquid to be frozen and thawed in scaled-down models of different shapes. Select scaled-down models whose freezing concentration factor is within the range of the comparison freezing concentration factor or greater than the maximum value of the comparison freezing concentration factor for use, and discard scaled-down models whose freezing concentration factor is less than the minimum value of the comparison freezing concentration factor range. S4. In the selected scaled-down model, change the amount of the freeze-thaw solution to be added, and perform freezing and thawing operations on the freeze-thaw solution. After one cycle, take samples to test and obtain the freeze concentration factor of the freeze-thaw solution in scaled-down models of different shapes. Select the amount of freeze-thaw solution to be added that has a freeze concentration factor within the range of the comparative freeze concentration factor or greater than the maximum value of the comparative freeze concentration factor.

2. The scaled-down model as described in claim 1, characterized in that, The scaled-down model is a cylinder or a sphere.

3. The application of the scaled-down model as described in claim 1 or 2 in the development and characterization of freeze-thaw processes for biopharmaceuticals.

4. A method for developing and characterizing the freeze-thaw process of biopharmaceuticals using a scaled-down model as described in claim 1 or 2, characterized in that, Includes the following steps: S1. Prepare the freeze-thaw solution; S2. Fill the shrink model with the liquid to be frozen and thawed, the filling amount is 20%-80% of the shrink model volume, and after filling, the ratio of the cross-sectional area of ​​the maximum radial section of the shrink model to the liquid level height is 1-15:

1. S3. Place the miniature model containing the freeze-thaw solution into the freezer and freeze it. Record the time required for the ice-water interface to coexist. After freezing is complete, place it at room temperature to thaw until it is completely thawed. Record the time required for the ice-water interface to coexist. The freezing and thawing steps are repeated 1-10 times. S4. After the freezing and thawing cycle is completed, samples are taken and tested to obtain the freezing concentration factor of the freeze-thaw solution and to obtain the effect of the freezing and thawing process on the quality of the freeze-thaw solution.

5. The method for developing and characterizing the freeze-thaw process of biological agents using a scaled-down model as described in claim 4, characterized in that, The solution to be frozen and thawed is selected from either a simulated solution or the original solution.

6. The method for developing and characterizing the freeze-thaw process of biological agents using a scaled-down model as described in claim 4, characterized in that, The refrigerator temperature is maintained between -100°C and -5°C, or between -80°C and -20°C. Complete freezing occurs when the temperature of the thawed liquid drops to -100°C to -5°C, or -80°C to -20°C. Complete thawing means waiting for the temperature of the thawed liquid to rise to 5°C and then to room temperature. The coexistence of ice and water at the interface is determined as the time required for the freeze-thawed liquid to be maintained at -3°C to 3°C.

7. A method for preparing a scaled-down model for the development and characterization of the freeze-thaw process of a biological agent as described in claim 1 or 2, characterized in that, Includes the following steps: S1. Obtain the range of the comparative freezing concentration factor for the freeze-thaw solution in the container used in actual production, as well as the range of the total ice-water interface time during the freezing and thawing process. S2. Determine the volume of the scaled-down model and design scaled-down models of various shapes; S3. Use the scaled-down model designed in step S2 to perform freezing and thawing operations on the liquid to be frozen and thawed. After one cycle, take samples to test and obtain the freezing concentration factor of the liquid to be frozen and thawed in scaled-down models of different shapes. Select scaled-down models whose freezing concentration factor is within the range of the comparison freezing concentration factor or greater than the maximum value of the comparison freezing concentration factor for use, and discard scaled-down models whose freezing concentration factor is less than the minimum value of the comparison freezing concentration factor range. S4. In the selected scaled-down model, change the amount of the freeze-thaw solution to be added, and perform freezing and thawing operations on the freeze-thaw solution. After one cycle, take samples to test and obtain the freeze concentration factor of the freeze-thaw solution in scaled-down models of different shapes. Select the amount of freeze-thaw solution to be added that has a freeze concentration factor within the range of the comparative freeze concentration factor or greater than the maximum value of the comparative freeze concentration factor.

8. The preparation method according to claim 7, characterized in that, The solution to be frozen and thawed is selected from either a simulated solution or the original solution.

9. The preparation method according to claim 7, characterized in that, The scaled-down models come in various shapes, including cylindrical and spherical.

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