Miniaturized model for development and characterization of freezing and thawing process of biological agent and application thereof

Abstract

The invention discloses a shrinking model for developing and characterizing a biological agent freezing and thawing process and application of the shrinking model, the volume of the shrinking model is 100-1000mL, when the shrinking model is vertically placed, the ratio of the sectional area value of the maximum section in the horizontal direction to the length value in the vertical direction is (1-10): 1, the amount of a biological agent is 20-80% of the volume of the shrinking model, and the amount of the biological agent is 10-20% of the volume of the shrinking model. And the ratio of the sectional area value of the maximum section to the liquid level height value is (1-15): 1. After the biological agent is loaded into the reduced model, transferring into a refrigerator from room temperature, after complete freezing, taking out, thawing at room temperature, testing freeze concentration factors, and recording ice-water interface coexistence duration. According to the shrinking model, the freezing concentration factor is compared with the freezing concentration factor for comparison in the large-volume freezing and thawing process, the shrinking model with the freezing concentration factor larger than or similar to the freezing concentration factor for comparison is obtained, and the ice-water interface coexistence time is not shorter than that of a large-volume container due to multiple rounds of freezing and thawing; the method is used for subsequent biological agent freeze thawing process development and characterization. The small model is small in biological agent dosage, temperature control equipment does not need to be used, and the research and development cost can be greatly reduced.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a scaled-down model for freeze-thaw process development and characterization of biological preparations and application thereof. Background Art

[0002] Biological agents are an important part of today's pharmaceutical industry. Compared with traditional small molecule drugs, antibody drugs have higher selectivity and effectiveness, lower toxicity and side effects, so antibody drugs have wide applications and huge markets in oncology, immunology, cardiovascular diseases, etc.

[0003] Freeze-thaw is a common operation process for biologics in manufacturing, storage and transportation. For most antibody drugs, stock solution production and product filling are separated. Biologic stock solutions are usually frozen in batches before being transported to the filling plant and completing the final filling process. This improves the storage stability of the stock solution and extends the shelf life by reducing reaction kinetics, limiting potential microbial growth, and reducing potential interfacial pressure introduced during transportation. After the frozen stock solution is transported to the filling plant, it needs to be thawed before being filled into the final biologic product.

[0004] Although the freeze-thaw process is a key operating unit in the filling / finishing process, the freezing and thawing process will produce many unfavorable stress conditions for proteins, which may cause protein aggregation or denaturation and affect the quality of the final product. Among them, the pressure from the freeze-concentration effect and the ice-water interface are the key considerations in the study of freeze-thaw processes. When the stock solution is gradually frozen into ice, as the ice crystals continue to grow, the solutes (proteins and excipients and other substances) will gradually concentrate in the unfrozen solution, resulting in an increase in the viscosity of the solution, a decrease in the diffusion coefficient, and eventually the formation of a glassy substance. This phenomenon is called the freeze-concentration effect. The freeze-concentration effect affects the pH, ionic strength, osmotic fluid and viscosity of the buffer. These physicochemical changes may cause protein denaturation. In addition, proteins are also subject to pressure from the ice-water interface. During the freezing process, proteins interact with the ice surface, and their hydrophobic bonds are weakened, resulting in the destruction of the natural structure of the protein.

[0005] Since the freeze-thaw process will affect the quality of the protein, it is necessary to develop a freeze-thaw process during drug development to reduce the negative impact of freeze-thaw on product quality. In the development of freeze-thaw processes, the degree of freeze-concentration effect can be characterized by the freeze-concentration factor. The freeze-concentration factor represents the change in the concentration of a component in the solution, generally referring to the multiple of the concentration of the component after the freeze-thaw process, specifically the ratio of the concentration of the component to the initial concentration at this time. For example, for a protein molecule, its freeze-concentration factor can be expressed by the ratio of the concentration of the protein in the sample to the initial protein concentration. If the freeze-concentration factor of a protein molecule is less than 1 at a certain position, it means that the concentration of the protein at this position is less than the concentration of the protein at that position before freeze-thaw. Conversely, if the freeze-concentration factor of a protein at a certain position is greater than 1, it means that the concentration of the protein at this position is greater than the concentration of the protein at that position before freeze-thaw, that is, the protein is "concentrated" after the freeze-thaw process. For electrolytes, such as sodium chloride, its freeze-concentration factor at a certain position can be expressed by the ratio of the conductivity value of the component at this position to the original conductivity value. For non-electrolytes, such as sucrose, the freeze concentration factor at a certain location can be expressed as the ratio of the osmotic pressure value of the component at this location to the original osmotic pressure value. For crystallizable excipients, such as sodium chloride, when they are gradually concentrated to their solubility limit during the freeze concentration process, they will crystallize out of the solution to form eutectics, so their freeze concentration factors are relatively low. For excipients that are not easy to crystallize, such as sucrose, they will be concentrated to a higher concentration during the freeze concentration process, so they have a relatively higher freeze concentration factor.

[0006] The pressure from the ice-water interface can often be characterized by the ice-water interface time. The ice-water interface time refers to the total time that the ice phase and the water phase coexist during the freeze-thaw process, which can be specifically divided into the ice-water interface time during the freezing process and the ice-water interface time during the thawing process. In the process of freeze-thaw process development, the total ice-water interface time is generally concerned. In freeze-thaw process research, a thermocouple temperature detection probe can be used to detect the temperature change of the entire solution system. In order to more accurately obtain the time that the ice phase and the water phase coexist, the ice-water interface time can be defined as the time that the temperature value recorded by the temperature detection probe is within the range of +3°C to -3°C.

[0007] Due to the differences in heat exchange area and the resulting heat transfer efficiency between different scales, the freeze-thaw process of biological preparations becomes more complicated as the loading volume increases. Although the freeze-thaw process is closely related to the loading volume, in the early stages of biological preparation development, biological preparation materials are relatively insufficient and expensive, and it is difficult to obtain large volumes of biological preparation materials for large-scale freeze-thaw process development. Therefore, the current main response strategy is to use a programmable temperature controller to control the freezing and thawing rates by manually setting programs, establish a freeze-thaw reduction model, and thus simulate a large-volume freeze-thaw process. This idea of ​​establishing a freeze-thaw reduction model is "active" and requires reliance on a programmable temperature controller, so it has high requirements for instruments and equipment. At the same time, when running a programmable temperature controller, liquid nitrogen is often used to cool the sample, resulting in a high overall operating cost. However, if a programmable temperature controller is not used and only a quick freezer or ultra-low temperature refrigerator is used, it is difficult to directly use a small volume of stock solution to simulate the freezing and thawing conditions of a large volume of stock solution. For example, the freeze-concentration effect of a small volume of stock solution cannot reach the freeze-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 process is an important part of biological product research and development. The current design ideas and development strategies of shrinkage models are "active". They are based on programmable temperature controllers and are highly dependent on instruments and equipment, resulting in high development thresholds and costs. Therefore, a new freeze-thaw shrinkage model design method and development strategy is urgently needed that does not rely on programmable 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 of biological preparations and its application, wherein the scaled-down model realizes the simulation of large-volume freeze-thaw processes by small-volume freeze-thaw. The freeze-thaw process developed based on the scaled-down model has high reliability, can save the amount of biological preparations used, does not rely on programmed temperature control, and reduces the development cost of the freeze-thaw process. Therefore, it is easy to apply to the development of freeze-thaw processes.

[0010] In order to solve the above technical problems, the first aspect of the present invention provides a reduced model for freeze-thaw process development and characterization of biological preparations, wherein when the reduced model is placed vertically, the ratio of the cross-sectional area value of the maximum cross section in the horizontal direction to the height value in the vertical direction is 1-10:1;

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

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

[0013] Preferably, the reduced model is a cylinder or a sphere.

[0014] In the present invention, the volume of the reduced model is in the range of 100-1000mL, which is suitable for small-volume freeze-thaw experiments, and the reduced model of this volume can greatly save the use of the freeze-thaw solution (i.e., the biological preparation, including its simulated liquid, or the biological preparation itself, i.e., the stock solution), saving development costs; the reduced model of the present invention is generally a cylinder or a sphere, the cylinder is such as a prism, a cylinder, an elliptical cylinder, etc., the prism is such as a triangular prism, a cuboid, a cube, a pentagonal prism, etc., the sphere is such as a sphere, etc.; when the reduced model is a cylinder, its opening can be located at the end faces of both ends of the cylinder , or it can be located on its side, but it needs to meet the requirement that when the opening is placed upward (vertically placed), the ratio of the cross-sectional area value of the maximum cross-section in the horizontal direction to the height value in the vertical direction (referring to the height value of the accommodation cavity) is 1-10:1. When the freeze-thaw liquid is added thereto, the amount of the freeze-thaw liquid added must also meet the following requirements: it accounts for 20%-80% of the volume of the reduced model, and when the reduced model is placed with the opening upward (vertically placed), the ratio of the cross-sectional area value of the maximum cross-section in the horizontal direction to the liquid level height value is 1-15:1, so that the reduced model can meet the needs of simulating an actual large-volume freeze-thaw process.

[0015] It should be noted that the biological preparations in the present invention refer to products obtained by various biotechnologies or various biological materials, such as biological drugs, selected from cytokines, erythropoietin, plasminogen activator, plasma factors, growth hormone, growth factors, insulin, immunoglobulins, antibodies (especially monoclonal antibodies) or vaccines (such as peptides, polypeptides or protein vaccines); for example, the biological preparations contain immunoglobulins or are composed of immunoglobulins; the simulation liquid in the present invention refers to a liquid used to simulate the freezing and thawing conditions of biological preparations in actual production, and its formula can be the same as that of biological preparations, such as biological preparations, or it can be different. The difference can be a difference in components or a difference in component concentrations. For example, the simulation liquid can be a combination of various candidate formulas of biological preparations; for example, the simulation liquid may not contain one or more biological products contained in the biological preparations in actual production; for example, the simulation liquid is the same as the placebo in the clinical trial, that is, the difference from the biological preparations in actual production is only that it does not contain biological products (such as proteins and monoclonal antibodies) as active ingredients.

[0016] In the present invention, the reduced model can meet the needs of simulating the actual large-volume freeze-thaw process, which is mainly determined by the freeze concentration factor and the time required for the coexistence of the ice-water interface (including the freezing process and the thawing process). Generally, the "worst case analysis" is adopted, that is, the protein is exposed to various pressure conditions in the reduced model that are greater than those faced in the actual large-volume production. If the quality attributes of the biological preparation are not significantly changed in the reduced model, the product quality of the biological preparation will not be reduced in the actual production. In the present invention, when the freeze concentration factor of the reduced model is greater than or close to the freeze concentration factor of the large volume freeze-thaw process (for example, the maximum freeze concentration factor value range of the large volume freeze-thaw process is obtained, and the freeze concentration factor of the reduced model is within the range), the simulated liquid or the original solution has no effect on the product quality (protein) in the reduced model, and it will not be affected in the large volume freeze-thaw process; the coexistence time of the simulated liquid or the original solution at the ice-water interface of the reduced model can be simulated by 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 ice-water interface coexistence time of the simulated liquid or the original solution in the large volume freeze-thaw process is not longer than the total ice-water interface coexistence time in the reduced model, the impact on the product quality of the simulated liquid or the original solution in the large volume freeze-thaw process is no greater than that of several rounds of freeze-thaw processes in the reduced model. It should be noted that the freeze concentration factor in the present invention is the change in the concentration of a component in the solution, specifically the ratio of the concentration of the component at the sampling position to the initial concentration. The concentration of the component is tested during sampling, and the initial concentration is the overall concentration of the freeze-thaw solution before freezing and thawing; the freeze concentration factor also varies depending on the component. When the component is protein, the freeze concentration factor is the ratio of the concentration of the protein at the sampling position before and after the freeze-thaw operation; when the component is an electrolyte (such as sodium chloride), the freeze concentration factor is the ratio of the conductivity values ​​of the electrolyte at the sampling position before and after the freeze-thaw operation; when the component is a non-electrolyte (such as sucrose), the freeze concentration factor is the ratio of the osmotic pressure values ​​of the non-electrolyte at the sampling position before and after the freeze-thaw operation.

[0017] In order to solve the above technical problems, the second aspect of the present invention is to provide an application of a reduced model in the development and characterization of a freeze-thaw process of a biological agent. Understandably, since the reduced model provided by the present invention can meet the needs of simulating an actual large-volume freeze-thaw process, it can be applied to the development and characterization of a freeze-thaw process of a biological agent.

[0018] In order to solve the above technical problems, the third aspect of the present invention is to provide a method for developing and characterizing a freeze-thaw process of a biological preparation using a scaled-down model, characterized in that it comprises the following steps:

[0019] S1, prepare the freeze-thaw solution;

[0020] S2, loading the freeze-thaw liquid into the reduced model, the loading amount being 20%-80% of the volume of the reduced model, and after loading, the ratio of the cross-sectional area value of the radial maximum cross-section of the reduced model to the liquid level value is 1-15:1;

[0021] S3, placing the scaled-down model containing the solution to be frozen and thawed in a refrigerator to freeze, recording the time required for the ice-water interface to coexist, and after being completely frozen, placing it at room temperature to thaw until it is completely thawed, recording the time required for the ice-water interface to coexist, and the freezing and thawing steps are repeated for 1-10 cycles;

[0022] S4. After the freezing and thawing step cycle is completed, sampling and testing are performed to obtain the freezing concentration factor of the solution to be frozen and thawed, and the influence of the freezing and thawing process on the quality of the solution to be frozen and thawed is obtained.

[0023] In the present invention, the preparation of the to-be-frozen-thawed liquid is to prepare the to-be-frozen-thawed liquid consistent with the large-volume freeze-thaw, which is generally a simulated liquid or a stock liquid. Due to the use of a reduced model (such as 100-1000 mL), the amount used is much lower than the large-volume freeze-thaw, which greatly reduces the cost; wherein, the amount of the to-be-frozen-thawed liquid loaded into the reduced model is preferably 20%-80%, and after loading, the ratio of the cross-sectional area value of the radial maximum cross-section of the reduced model to the liquid level height value is 1-15:1, so as to ensure that the simulation of the large-volume freeze-thaw process can be achieved; the refrigerator used in the present invention is, for example, a quick freezer or an ultra-low temperature refrigerator, which is the same as the refrigerator used for large-volume freeze-thaw, and does not require the use of a temperature control device to reduce the cost of the simulation; the refrigerator is always at a set temperature, and the reduced model containing the to-be-frozen-thawed liquid is directly placed in the reduced model. The small model can be placed in the refrigerator with the opening facing upward for the freezing operation. Generally speaking, it is necessary to insert a thermocouple temperature detection probe into the liquid to be frozen and thawed of the reduced model to monitor the temperature change of the liquid to be frozen and thawed, especially to monitor the duration of the temperature coexisting at the ice-water interface. After the frozen and thawed liquid is completely frozen, it is taken out of the refrigerator and brought to room temperature for natural thawing. During this period, the thermocouple temperature detection probe is used to detect the temperature change of the liquid to be frozen and thawed, especially to monitor the duration of the temperature coexisting at the ice-water interface. After it is completely thawed, it can be placed in the refrigerator again as needed for the next round of freezing and thawing process, and the total duration of the coexistence of the ice-water interface is recorded. It should be noted that the quality of the frozen and thawed liquid can be evaluated by conventional evaluation methods, which will not be repeated here.

[0024] In a specific scheme, the refrigerator temperature is maintained at minus 100°C to minus 5°C, or minus 80°C to minus 20°C; complete freezing means that the temperature of the freeze-thaw liquid drops to minus 100°C to minus 5°C, or minus 80°C to minus 20°C; complete thawing means that the temperature of the freeze-thaw liquid rises to 5°C to room temperature; the coexistence of the ice-water interface is determined as the time required for the freeze-thaw liquid to be maintained at minus 3°C to 3°C. In the present invention, the temperature interval for the coexistence of the ice-water interface is defined as minus 3°C to 3°C, therefore, the duration of the coexistence of the ice-water interface is the duration that the temperature of the freeze-thaw liquid detected by the thermocouple temperature detection device is in the temperature range of minus 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 a biological preparation using a reduced model provided by the present invention can achieve the simulation of a large-volume (such as 5L) freeze-thaw process using a reduced model (such as 100-1000mL), and achieve the development and characterization of the freeze-thaw process using a small amount of simulated liquid or stock liquid, and without the use of a temperature control device; and by limiting the size of the reduced model and the sample loading amount, the freeze concentration factor of the freeze-thaw liquid to be frozen and thawed using the reduced model is greater than or similar to (for example, the maximum freeze concentration factor value range of the large-volume freeze-thaw process is obtained, and the freeze concentration factor of the reduced model is within the range) the freeze concentration factor of the large-volume freeze-thaw process, therefore, after multiple rounds of freeze-thaw cycles on a reduced scale (the total ice-water interface coexistence time is not less than the ice-water interface coexistence time of the large-volume freeze-thaw process), the quality evaluation of the freeze-thaw liquid to be frozen and thawed is equivalent to or stricter than that of the large-volume freeze-thaw process.

[0026] In order to solve the above technical problems, the fourth aspect of the present invention is to provide a method for preparing a reduced model for freeze-thaw process development and characterization of biological preparations, comprising the following steps:

[0027] S1. Obtaining the range of freeze concentration factors for comparison of the liquid to be frozen and thawed in the container used in actual production, and the range of the total ice-water interface time during the freezing and thawing process;

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

[0029] S3, using the reduced model designed in step S2 to perform freezing and thawing operations on the liquid to be frozen and thawed, after one cycle is completed, sampling and testing are performed to obtain the freeze concentration factor of the liquid to be frozen and thawed in the reduced models of different shapes, and the reduced models whose freeze concentration factor is within the range of the freeze concentration factor for comparison or greater than the maximum value of the freeze concentration factor for comparison are selected for standby use, and the reduced models whose freeze concentration factor is less than the minimum value of the freeze concentration factor for comparison are discarded;

[0030] S4. In the selected reduced model, the amount of the liquid to be frozen and thawed is changed, and freezing and thawing operations are performed on the liquid to be frozen and thawed. After one cycle is completed, sampling and testing are performed to obtain the freeze concentration factor of the liquid to be frozen and thawed in the reduced models of different shapes, and the amount of the liquid to be frozen and thawed whose freeze concentration factor is within the range of the freeze concentration factor for comparison or greater than the maximum value of the freeze concentration factor for comparison is selected.

[0031] The preparation method of the reduced model provided by the present invention first screens the designed reduced model based on the parameters of large-volume freeze-thaw, including the shape of the reduced model, the ratio between the cross-sectional area of ​​the largest cross section and the height (referring to the height value of the containing cavity), and the ratio between the cross-sectional area of ​​the largest cross section and the liquid level when the freeze-thawed liquid is in the reduced model. The reduced model finally selected has a freeze concentration factor greater than or similar to the freeze concentration factor of the large-volume freeze-thaw process after freeze-thawing in the refrigerator used for large-volume freeze-thaw, and after multiple rounds of freeze-thaw cycles, the total coexistence time of the ice-water interface is not less than the total coexistence time of the ice-water interface in the large-volume freeze-thaw process; the selected reduced model The model includes a cylinder and a sphere, and the volume of the reduced model is in the range of 100-1000mL. Too large a volume will result in excessive consumption of the freeze-thaw solution, and too small a volume will make it difficult to simulate large-volume freeze-thaw. When the selected reduced model is placed with its opening upward (vertically placed), the ratio of the cross-sectional area value of the maximum cross-section in the horizontal direction to the height value in the vertical direction (referring to the height value of the accommodation cavity) is 1-10:1. When the freeze-thaw solution is added thereto, the amount of the freeze-thaw solution added must simultaneously meet the following requirements: accounting for 20%-80% of the volume of the reduced model, and when the reduced model is placed with its opening upward (vertically placed), the ratio of the cross-sectional area value of the maximum cross-section in the horizontal direction to the liquid level height value is 1-15:1. It should be noted that the total coexistence time of the ice-water interface during the large-volume freeze-thaw process (including the freezing process and the thawing process) can be obtained through testing; but generally speaking, after the large-volume container is confirmed (mainly the capacity), the total coexistence time of the ice-water interface can be basically determined based on the amount of solution actually loaded in mass production (the amount of biological agents loaded). The different types of biological agents have little effect on the total time, so it can also be determined based on usage experience. At the same time, the equipment manufacturer can also provide it; because when the large-volume container used and the amount of biological agents loaded in actual production are determined, the total coexistence time of the ice-water interface is basically determined. Therefore, for a reduced model of a given volume, after a certain amount of biological agents is loaded, the total coexistence time of the ice-water interface during a round of freeze-thaw is tested, and the number of freezing and thawing cycles can be determined to ensure that after multiple cycles of the reduced model, the total coexistence time of the ice-water interface is not shorter than the total coexistence time of the ice-water interface of the large-volume container. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solution of the present invention, the following briefly introduces the drawings required for use in the present invention. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0033] Figure 1 It is the large volume container and sampling position for actual production in Example 1;

[0034] Figure 2 is a temperature change diagram of the freezing process of the liquid to be frozen and thawed in the large volume container used in actual production in Example 1, wherein the changes at different temperature test positions are shown;

[0035] Figure 3 is a temperature change diagram of the thawing process of the to-be-frozen-thawed liquid in the large-volume container used in actual production in Example 1, wherein the changes at different temperature test positions are shown;

[0036] Figure 4 It is a three-view drawing of container No. 1 in Example 1;

[0037] Figure 5 It is a three-view drawing of container No. 2 in Example 1;

[0038] Figure 6 It is a three-view drawing of container No. 3 in Example 1;

[0039] Figure 7 It is a three-view drawing of container No. 4 in Example 1;

[0040] Figure 8 It is a three-view drawing of container No. 5 in Example 1;

[0041] Fig. 9 is a temperature variation diagram of the freezing process of the freeze-thaw solution in five scaled-down models in Example 1;

[0042] Fig.10 is a temperature variation diagram of the thawing process of the freeze-thaw solution in five scaled-down models in Example 1;

[0043] Fig.11 is a temperature change diagram of the freezing process of different addition amounts of the freeze-thaw solution in container No. 2 in Example 1;

[0044] Fig.12 is a temperature change diagram of the freezing process of different addition amounts of the freeze-thaw solution in container No. 2 in Example 1;

[0045] Fig.13 is a schematic diagram of container No. 6 in Example 2;

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

[0047] Fig.15 This is a temperature change diagram of the thawing process of the freeze-thaw solution in container No. 2 in Example 2. DETAILED DESCRIPTION

[0048] The following will be combined with the accompanying drawings to clearly and completely describe the technical solutions in the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0049] In the embodiments of the present invention, the ultra-low temperature refrigerator was purchased from Eppendorf (model: F740hi) or Thermofisher scientific (model: FDE60086FV); the thermocouple temperature detection probe (model: 5SRTC series) and the temperature detector (model: RDXL12SD) were purchased from OMEGA. The osmotic pressure detector (model: Osmotech Pro) was purchased from Advanced Instruments, Inc.; the multi-parameter measuring instrument (model: S470-B) was purchased from Mettler Toledo. 5L polycarbonate bottle (model: 3405) was purchased from Thermo fisher scientific. Bovine serum albumin (BSA, item number: KY-01014) was purchased from Kangyuan Biological Company.

[0050] Example 1 Preparation of reduced model

[0051] Step S1, testing the range of freeze concentration factors for comparison of the liquid to be frozen and thawed in the container used in actual production, and the range of the total ice-water interface time during the freezing and thawing process.

[0052] The formula of the simulated liquid is 20mM histidine buffer, 4% (w / v) sucrose, 70mM sodium chloride, 0.04% (w / v) polysorbate 80, pH 6.0, of which 20mM histidine buffer (pH 6.0) is a commonly used buffer system in marketed biological preparations. Sucrose and sodium chloride are not only commonly used excipients in biological preparations, but also typical non-electrolytes and electrolytes, respectively. Therefore, the freeze concentration effect of non-electrolytes and electrolytes during freeze-thaw can be studied. Polysorbate 80 is also a commonly used surfactant in biological preparations. The theoretical osmotic pressure of the simulated liquid is about 287mOsm / kg, which is an isotonic solution. The measured osmotic pressure of the evenly mixed simulated liquid is 275mOsm / kg, and the conductivity is 7.49mS / cm.

[0053] Among them, the large-volume container used in actual production is a 5L polycarbonate bottle commonly used in reagent production.

[0054] Among them, in order to simulate the freeze-thaw process of large-volume biological preparations, 4L of simulated liquid was placed in a 5L polycarbonate bottle, and the simulated liquid was transferred from room temperature (18-24°C) to an ultra-low temperature refrigerator (-70±10°C, model: FDE60086FV). After it was completely frozen, the frozen 5L polycarbonate bottle containing simulated liquid was transferred from the -70±10°C refrigerator to room temperature for static thawing. After the simulated liquid returned to room temperature, 6 sampling locations were selected for sampling and osmotic pressure and conductivity testing.

[0055] Among them, 6 sampling positions are selected, namely the top center position, the bottom corner and the center position, see Figure 1 As shown, the freezing concentration factors at 6 sampling positions were tested respectively (see Table 1); the thermocouple probes were respectively used to detect the temperature change at position 1 (the center of the solution) and 1 cm below position 1 (1 cm below the liquid surface) of the solution to be frozen and thawed to determine the total coexistence time of the ice-water interface during the freezing and thawing process (including the freezing and thawing processes, the temperature change during the freezing process is shown in Figure 2 , the temperature change during the thawing process is shown in Figure 3 , with +3℃ to -3℃ as the ice-water interface coexistence temperature, the obtained ice-water interface coexistence time is shown in Table 2).

[0056] Table 1

[0057]

[0058] It can be seen from Table 1 that for the prepared simulation solution, the maximum freeze concentration factor is located at the bottom, the maximum freeze concentration factor of sucrose is in the range of 6-8, and the freeze concentration factor of sodium chloride is 2-3.

[0059] Table 2

[0060]

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

[0062] Step S2, determining the volume of the reduced model and designing reduced models of various shapes;

[0063] The volume of the reduced model is about 500 mL, and the shape of the designed reduced model is as follows: Figure 4-Figure 8 As shown (the wall thickness is ignored, approximating the shape of the accommodating cavity), there are 5 types in total;

[0064] The first scaled-down model (container No. 1) has a rectangular body with an opening at its end (the end is square and the side length of the cavity is 7.5 cm). When placed vertically, the cross-sectional area is 56.3 cm. 2 , the height of the accommodation cavity is 9.3 cm, and the ratio of the maximum cross-sectional area to the height of the accommodation cavity is 6.05 cm; when about 80% of the freeze-thaw solution is put 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 No. 2) has a cylindrical body with an opening at its end (the end is circular and the diameter of the cavity is 6.4 cm). When placed vertically, the cross-sectional area is 32.2 cm. 2 , the height of the accommodation cavity is 16.3 cm, and the ratio of the maximum cross-sectional area to the height of the accommodation cavity is 1.98 cm; when about 80% of the freeze-thaw solution is put 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 No. 3) has a cylindrical body with an opening at its end (the end is circular and the diameter of the cavity is 7.5 cm). When placed vertically, the cross-sectional area is 44.2 cm. 2 , the height of the accommodation cavity is 11.6 cm, and the ratio of the maximum cross-sectional area to the height of the accommodation cavity is 3.81 cm; when about 80% of the freeze-thaw solution is put 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] Among them, the bottle body of the fourth scaled-down model (Container No. 4) is approximately a flat cylinder (ref. Figure 7 The side surface when placed vertically is a circle with a diameter of 11.5 cm, but the top and bottom are cut into horizontal planes. The thickness of the receiving cavity (the distance between the front and rear end surfaces of the flat cylindrical receiving cavity) is 6 cm. When placed vertically, the cross-sectional area of ​​the largest cross section (rectangular) is 69 cm 2 , the height of the accommodation cavity is 10.6 cm, and the ratio of the maximum cross-sectional area to the height of the accommodation cavity is 6.51 cm; when about 80% of the freeze-thaw solution is put 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] Among them, the bottle body of the fifth scaled-down model (container No. 5) is approximately spherical (ref. Figure 8 The side surface when placed vertically is a circle with a diameter of 10 cm, but the top and bottom are cut into horizontal planes; the largest cross section in the accommodating cavity is a circle with a diameter of 10 cm, and its cross-sectional area is 78.5 cm 2, the height of the accommodation cavity is 9.4 cm; the ratio of the maximum cross-sectional area to the height of the accommodation cavity is 8.35; when about 80% of the freeze-thaw solution is put 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, using the reduced model designed in step S2 to perform freezing and thawing operations on the liquid to be frozen and thawed, after one cycle, sampling and testing to obtain the freeze concentration factor of the liquid to be frozen and thawed in the reduced models of different shapes, selecting the reduced models whose freeze concentration factor is within the range of the freeze concentration factor for comparison or greater than the maximum value of the freeze concentration factor for comparison for standby use, and discarding the reduced models whose freeze concentration factor is less than the minimum value of the freeze concentration factor for comparison;

[0070] Specifically, five scaled-down models filled with about 80% simulated liquid were transferred from room temperature (18-24°C) to an ultra-low temperature refrigerator (-70±10°C, model: FDE60086FV). After they were completely frozen, the frozen containers No. 1 to No. 5 filled with simulated liquid were transferred from the -70±10°C refrigerator to room temperature for static thawing. After the simulated liquid returned to room temperature, samples were taken and osmotic pressure and conductivity tests were performed. 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. Fig. 9 , the temperature change during the thawing process is shown in Fig.10 , with +3°C to -3°C as the ice-water interface coexistence temperature, the ice-water interface coexistence time for one round and four rounds is shown in Table 4 (the probe position is 1 cm below the liquid surface).

[0071] Table 3

[0072]

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

[0074] Table 4

[0075]

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

[0077] Step S4, in the selected reduced model, changing the amount of the solution to be frozen and thawed, performing freezing and thawing operations on the solution to be frozen and thawed, and after one cycle, taking samples for testing to obtain the freeze concentration factor of the solution to be frozen and thawed in the reduced models of different shapes, and selecting the amount of the solution to be frozen and thawed whose freeze concentration factor is within the range of the freeze concentration factor for comparison or greater than the maximum value of the freeze concentration factor for comparison;

[0078] Among them, the freeze-thaw operation was carried out in container No. 2, and 100mL, 200mL, 300mL, and 400mL of simulated liquid were respectively loaded into container No. 2. After loading, the liquid level heights were 3.2cm, 7cm, 10.4cm, and 13.7cm, respectively. The simulated liquid was transferred from room temperature (18-24°C) to an ultra-low temperature refrigerator (-70±10℃, model: FDE60086FV). After it was completely frozen, the two frozen containers containing simulated liquid were transferred from the refrigerator at -70±10℃ to room temperature for static thawing. After the solution recovered to that time, samples were taken, and the osmotic pressure and conductivity were tested. The test results are shown in Table 5; the temperature change during the freezing process is shown in Table 5. Fig.11 , the temperature change during the thawing process is shown in Fig.12 , with +3°C to -3°C 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] As can be seen from Table 5, the loading volume has a significant effect on the freeze-concentration effect. In container No. 2, the larger the loading volume of the simulated liquid, the larger the maximum freeze-concentration factor of sucrose and sodium chloride. When the loading volume reaches 300mL, the maximum freeze-concentration factors of sucrose and sodium chloride no longer increase significantly with the increase in loading volume. Among them, when the loading volume is 200mL, 300mL, and 400mL, respectively, the maximum freeze-concentration factors that can be achieved are all the same as the maximum freeze-concentration factors that can be achieved during the freeze-thaw process of large-volume solutions (the maximum freeze-concentration factor of sucrose is about 6-8; the maximum freeze-concentration factor of sodium chloride is about 2-3). Therefore, the loading volume is 20%-80% of the reduced model volume, and the ratio of the maximum cross-sectional area to the liquid level is between 2.4-10.1:1, which can be used to simulate the freeze-thaw process of large-volume solutions.

[0082] Table 6

[0083]

[0084]

[0085] As can be seen from Table 6, with the increase of the amount of simulated liquid, the total ice-water interface time after a round of freeze-thaw process also increases. Therefore, the larger the amount of simulated liquid, the number of freeze-thaw cycles required to achieve the total ice-water interface time in large-volume freeze-thaw (5L polycarbonate bottle) gradually decreases. When the amount is 100mL, the total ice-water interface time is 4.2 hours, and 7 freeze-thaw cycles are required to be comparable to large-volume freeze-thaw; when the amount is 400mL, the total ice-water interface time is 9.0 hours, and 4 freeze-thaw cycles are required to be comparable to large-volume freeze-thaw. Therefore, the ice-water interface pressure faced by biological agents in large-volume freeze-thaw can be simulated by changing the amount and the corresponding number of freeze-thaw cycles.

[0086] Example 2 Effect of reducing model capacity on freeze-thaw process

[0087] In this embodiment, the capacity of the reduced model is expanded to 1000 mL and is determined as container No. 6. Fig.13 As shown (when placed vertically, the transverse cross-section is a square). The simulated liquid of this embodiment is the same as that of Example 1, and the amount of simulated liquid added is 80%. The simulated liquid is transferred from room temperature (18-24°C) to an ultra-low temperature refrigerator (-70±10°C, model: FDE60086FV). After it is completely frozen, transfer the frozen container No. 6 containing the simulated liquid from the refrigerator at -70±10°C to room temperature for static thawing. After the simulated liquid returns to room temperature, take a sample and perform osmotic pressure and conductivity tests. Since the shape of container No. 6 is similar to that of container No. 1, the test data of the two are compared synchronously. The osmotic pressure and conductivity test data are shown in Table 7; the temperature change during the freezing process is shown in Fig.14 , the temperature change during the thawing process is shown in Fig.15 , with +3°C to -3°C 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 value of container No. 1 is derived from Example 1.

[0088] Table 7

[0089]

[0090] It can be seen from Table 7 that the larger the size of the container, the stronger the maximum freezing concentration effect that the simulated liquid can achieve, especially the maximum freezing concentration factor of sucrose increases. Since the maximum freezing concentration factor of sodium chloride is relatively small as a whole, it is more susceptible to the influence of experimental errors, so the overall freezing concentration effect intensity can be judged according to the maximum freezing concentration factor of more sensitive sucrose. The maximum freezing concentration factors that can be achieved by container No. 1 and container No. 6 investigated here can reach the maximum freezing concentration factor range that can be achieved by the freeze-thaw process of a large volume solution of the embodiment (the maximum freezing concentration factor of sucrose is about 6-8; The maximum freezing concentration factor of sodium chloride is about 2-3), so it can be used to simulate the freeze-thaw process of a large volume solution.

[0091] Table 8

[0092]

[0093] Table 8 shows that the total ice-water interface time of the simulated liquid in container No. 1 during one round of freezing and thawing is 9.6 hours; while the total ice-water interface time of the simulated liquid in container No. 6 after one round of freezing and thawing is 13.1 hours. After containers No. 1 and No. 6 have undergone four or three rounds of freezing and thawing, respectively, their total ice-water interface time can be longer than that of the large volume (5L polycarbonate bottle) during freezing and thawing. Therefore, the ice-water interface pressure faced by biological agents in large-volume freezing and thawing can be simulated by increasing the number of freezing and thawing cycles of the reduced model.

[0094] Example 3: Development and characterization of a freeze-thaw process for bovine serum albumin using a scaled-down model

[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] Reduced model: Container No. 2 in Example 1;

[0097] For comparison, this embodiment simultaneously uses a 5L polycarbonate bottle for actual production to perform freeze-thawing of the stock solution.

[0098] The stock solution was added to the scaled-down model and the 5L polycarbonate bottle, and the added amount was 80%, that is, the added amount of the scaled-down model was 400mL, and the added amount of the 5L polycarbonate bottle was 4L; the scaled-down model and the 5L polycarbonate bottle with the stock solution were transferred from room temperature (18-24°C) to an ultra-low temperature refrigerator (-70±10℃, model: FDE60086FV). After it was completely frozen, the frozen container No. 2 containing the simulated liquid was transferred from the refrigerator at -70±10℃ to room temperature for static thawing. After the solution recovered to that time, samples were taken and the osmotic pressure, conductivity and protein concentration were tested.

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

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

[0101] Table 9

[0102]

[0103] Therefore, in general, the freeze-thaw reduction model established by rationally designing the geometry, size and content of small-volume containers can simulate the large-volume freeze-thaw process during production without the need for a temperature control device, and can be used for the research and development 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 in the scope of protection of the present invention.

Claims

1. A scaled-down model for freeze-thaw process development and characterization of biological preparations, characterized in that: When the reduced model is placed vertically, the ratio of the cross-sectional area value of the maximum cross section in the horizontal direction to the length value in the vertical direction is 1-10:1; The volume of the reduced model is 100-1000 mL; The amount of biological agent contained in the reduced model is 20%-80% of its volume, and when the reduced model is placed vertically, the ratio of the cross-sectional area value of its maximum cross section in the horizontal direction to the liquid level height value is 1-15:

1.

2. The reduced model according to claim 1, characterized in that: The reduced model is a cylinder or a sphere.

3. Use of the scaled-down model as claimed in claim 1 or 2 in developing and characterizing freeze-thaw processes of biological preparations.

4. A method for developing and characterizing a freeze-thaw process of a biological agent using the scaled-down model according to claim 1 or 2, characterized in that: The steps include: S1, prepare the freeze-thaw solution; S2, loading the freeze-thaw liquid into the reduced model, the loading amount being 20%-80% of the volume of the reduced model, and after loading, the ratio of the cross-sectional area value of the radial maximum cross-section of the reduced model to the liquid level value is 1-15:1; S3, placing the scaled-down model containing the solution to be frozen and thawed in a refrigerator to freeze, recording the time required for the ice-water interface to coexist, and after being completely frozen, thawing at room temperature until completely thawed, recording the time required for the ice-water interface to coexist, and the freezing and thawing steps are repeated for 1-10 cycles; S4. After the freezing and thawing step cycle is completed, sampling and testing are performed to obtain the freezing concentration factor of the solution to be frozen and thawed, and the influence of the freezing and thawing process on the quality of the solution to be frozen and thawed is obtained.

5. The method for developing and characterizing a freeze-thaw process for a biological agent using a scaled-down model as claimed in claim 4, wherein: The solution to be frozen and thawed is selected from a simulated solution or a stock solution.

6. The method for developing and characterizing a freeze-thaw process for biological preparations using a scaled-down model as claimed in claim 4, wherein: The refrigerator temperature should be maintained between -100℃ and -5℃, or between -80℃ and -20℃; Complete freezing means that the temperature of the freeze-thaw solution drops to -100℃ to -5℃, or -80℃ to -20℃; Thawing is complete when the temperature of the freeze-thaw solution rises to 5°C to room temperature; The ice-water interface coexistence was determined as the time required for the freeze-thaw solution to be maintained at -3°C to 3°C.

7. A method for preparing a scaled-down model for freeze-thaw process development and characterization of biological preparations, characterized in that: The steps include: S1. Obtaining the range of freeze concentration factors for comparison of the liquid to be frozen and thawed in the container used in actual production, and the range of the total ice-water interface time during the freezing and thawing process; S2. Determine the volume of the reduced model and design reduced models of various shapes; S3, using the reduced model designed in step S2 to perform freezing and thawing operations on the liquid to be frozen and thawed, after one cycle is completed, sampling and testing are performed to obtain the freeze concentration factor of the liquid to be frozen and thawed in the reduced models of different shapes, and the reduced models whose freeze concentration factor is within the range of the freeze concentration factor for comparison or greater than the maximum value of the freeze concentration factor for comparison are selected for standby use, and the reduced models whose freeze concentration factor is less than the minimum value of the freeze concentration factor for comparison are discarded; S4. In the selected reduced model, the amount of the liquid to be frozen and thawed is changed, and freezing and thawing operations are performed on the liquid to be frozen and thawed. After one cycle is completed, sampling and testing are performed to obtain the freeze concentration factor of the liquid to be frozen and thawed in the reduced models of different shapes, and the amount of the liquid to be frozen and thawed whose freeze concentration factor is within the range of the freeze concentration factor for comparison or greater than the maximum value of the freeze concentration factor for comparison is selected.

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

9. The preparation method according to claim 7, characterized in that: Scaled-down models in a variety of shapes including cylinders and spheres.

Images