CN116351094A - Method for synchronously coagulating and nucleating induced material in short time - Google Patents
Method for synchronously coagulating and nucleating induced material in short time Download PDFInfo
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Images
Classifications
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- F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
- F26—DRYING
- F26B—DRYING SOLID MATERIALS OR OBJECTS BY REMOVING LIQUID THEREFROM
- F26B5/00—Drying solid materials or objects by processes not involving the application of heat
- F26B5/04—Drying solid materials or objects by processes not involving the application of heat by evaporation or sublimation of moisture under reduced pressure, e.g. in a vacuum
- F26B5/06—Drying solid materials or objects by processes not involving the application of heat by evaporation or sublimation of moisture under reduced pressure, e.g. in a vacuum the process involving freezing
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D9/00—Crystallisation
- B01D9/02—Crystallisation from solutions
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a method for inducing synchronous coagulation nucleation of a material in a short time, which comprises the steps of pre-charging gas into the material in advance and saturating the gas; then sub-packaging the materials, placing the materials in a freeze dryer, and synchronously performing cooling and pressure increasing processes to enable the materials to be in a metastable state below a crystallization point; finally, the pressure of the freeze dryer is reduced to below 5psig in 10 seconds, so that the gas in the material is volatilized rapidly to induce the material to coagulate and nucleate. Compared with the prior art, the invention has the following advantages: (1) The method can realize synchronous crystallization within a narrow specific temperature range in a short time; (2) The method can ensure that the uniformity of the material is coagulated and nucleated in the freeze-drying process, and does not need to be maintained for a long time at a certain temperature below the crystallization point; (3) The method can be applied to any material treatment including nucleation phase transitions, such as in antibody-coupled drug production.
Description
Technical Field
The invention belongs to the technical field of freeze-drying, and relates to a method for controlling homogeneous coagulation nucleation in a freeze-drying process, in particular to a method for synchronously coagulation nucleation of an induced material in a short time.
Background
In the current freeze-drying process, the generation of coagulation nucleation is a random event, and the occurrence of coagulation nuclei and the time for initiating coagulation are also random for different samples frozen in the same batch, so that the phenomenon of asynchronous coagulation of samples in the same batch can occur. Thus, there is a need for improvements or control of nucleation methods to allow freezing of the drug or reagent in the freeze dryer to occur at a narrower temperature and time range, resulting in a lyophilized product with greater uniformity from vial to vial. How to control the nucleation conditions and greatly accelerate the freeze-drying process is a constant challenge to those skilled in the art.
Us patent 6682524 discloses a method of vacuum induced surface freezing by loading bottles containing an aqueous solution on a temperature controlled shelf in a freeze dryer and maintaining the initial temperature below 10 ℃. The freeze drying chamber is then evacuated to a near vacuum, thereby causing the aqueous surface to freeze to a depth of a few millimeters. Subsequent release of the vacuum and lowering of the shelf temperature below the freezing point of the solution allows ice crystals to grow from the pre-frozen surface layer through the remainder of the solution. The main drawbacks of this method are: the solution is easily boiled or degassed vigorously during the evacuation to release the gas from the liquid.
Chinese patent CN101379356B discloses a method of inducing nucleation of a material to a point below the thermodynamic freezing point and metastable state in a freeze drying chamber at a pressure of 7 to 50 psig; and rapidly reducing the pressure in the freeze-drying chamber in the vicinity of the material to induce nucleation of the material in the material within 40 seconds or less. In particular, the method is to start the depressurization in the freeze-drying chamber when the material reaches the desired nucleation temperature or at a desired time after the material temperature is below the thermodynamic freezing point temperature. The specification paragraph [ 0045 ] discloses that the material is preferably placed in a freeze drying chamber and that the temperature, pressure and gas atmosphere of the freeze drying chamber are allowed to be controlled. While the gas atmosphere acts on the one hand to control the pressure in the chamber and on the other hand to heat transfer fluid as a temperature of the material in each bottle or container. The prior art has the advantage of avoiding severe boiling or degassing of the solution caused by vacuum pumping, so that the problem of controlling the randomness of nucleation in the freeze-drying process caused by severe boiling or degassing of the solution can be improved to a certain extent. However, the method cannot ensure that the gas is saturated in each bottle or container by introducing the gas into the freeze drying chamber, so that the uniformity of nucleation cannot be ensured; in addition, the cooling and then depressurizing process takes a long time, and as described in the specification [ 0255 ], the process needs to be maintained for 16 hours at-14 ℃ in the freeze dryer frame. The above defects all affect the nucleation rate.
Thus, although the prior art charges the solution material with a gas (such as nitrogen), the prior art charges the freeze-dryer or the drying chamber with a gas, and thus, it is not guaranteed that each bottle or container is saturated. The freeze dryer or the method of reducing temperature and then reducing pressure in the drying chamber needs to be maintained for a long time at a temperature below the crystallization point to realize instant synchronous crystallization caused in the gas volatilization process. The above technique has the disadvantage that the homogeneous coagulation nucleation in the freeze-drying process cannot be ensured and the time is long, thereby affecting the nucleation rate.
There are fewer cases in the prior art where the controlled nucleation technique is truly useful in actual production. Synchronous cooling and boosting are very high in equipment requirement, and mature equipment with the function is few at present, so that the problems focused on equipment development by the technicians in the field are mostly focused. In addition, in the prior art, the prior instruments are mostly utilized to explore the nucleation temperature and the depressurization rate range of the material, namely, the research aims at nucleating the material, but the problems of long time consumption, uncontrollable nucleation rate and the like in the actual operation process cannot be solved.
Disclosure of Invention
The technical problems to be solved are as follows: in order to overcome the defects of the prior art, a process which ensures the uniformity of the material to coagulate and nucleate in the freeze-drying process and takes short time is obtained, namely, the process of enabling the gas in the material to reach saturation in a short time under the specific temperature condition and then utilizing the pressure mutation to realize instantaneous synchronous crystallization is realized. In view of this, the present invention provides a method of inducing synchronized coagulation nucleation of a material in a short period of time.
The technical scheme is as follows: a method for inducing synchronized coagulation nucleation of a material in a short period of time, comprising the steps of:
pre-charging gas into the material and saturating the gas;
subpackaging the material, placing the material into a freeze dryer, synchronously performing cooling and boosting processes, controlling the temperature in the freeze dryer to be-2 to-8 ℃ and the pressure to be 25-28 psig, so that the material is in a metastable state below a crystallization point;
the lyophilizer pressure was reduced to 2-5 psig within 10 seconds to quickly volatilize the gas within the material to induce material nucleation by condensation.
The metastable state referred to above refers to a state of a material at a temperature below its phase transition temperature, where the metastable state is an unstable and transient state of a chemical or biological system but relatively extends its lifetime. The metastable material will eventually transition from its non-equilibrium state to its equilibrium state without any change in the material or its environment.
Preferably, the material may be a pure substance, a gas, a suspension, a gel, a liquid, a solution, a mixture or a component within a solution or mixture. Specifically, biopharmaceutical materials, chemicopharmaceutical materials, live or attenuated viruses, nucleic acids, monoclonal or polyclonal antibodies, proteins, peptides or non-peptide analogs are included. The method of the invention needs to dissolve the materials in water, and can pretreat the water solution with a filter membrane to remove particles interfering with nucleation, promote the formation of a uniform ice crystal structure and reduce the freeze-drying time.
Preferably, the material is subjected to microfiltration prior to pre-aeration.
Preferably, the pre-filled gas within the material comprises at least one of nitrogen, argon, helium, neon, xenon, krypton.
Preferably, the purity of the gas pre-filled in the material is more than 99.99 percent, and the flow rate of the gas is 0.5-1L/min.
Preferably, the temperature conditions at which the pre-charge in the material reaches saturation are between 18 and 25 ℃.
Preferably, the freeze dryer is pre-cooled to 2-5 ℃ before being placed into the split charging materials.
Preferably, after being placed into a freeze dryer, the temperature is reduced from the precooling temperature to-2 to-8 ℃ within 10-20 min, the pressure is increased from normal pressure to 25-28 psig, and the temperature is maintained for 10-30 min.
Preferably, the pressure boosting process is to introduce a gas including at least one of nitrogen, argon, helium, neon, xenon, and krypton into the freeze dryer.
Preferably, the temperature in the freeze dryer is maintained at-2 to-8 ℃ in the depressurization process. In the above temperature range, the rapid depressurization allows the original elevated pressure within the lyophilizer to be rapidly reduced by the exiting gaseous atmosphere and the rapid depressurization can induce nucleation. Specifically, rapid depressurization causes the solubility of the pre-saturated dissolved gas within the material to decrease rapidly, and the rapid release of the gas from the metastable solution triggers phase change nucleation.
In principle, the method of the invention can be applied to any material treatment comprising a nucleation phase transition, for example: liquid freezing, crystal ice of medicine water solution, protein crystallization, food freezing, freezing concentration, etc. The most urgent application for the applicant however lies in the improvement of the current lyophilization process of drugs. For example, in industrial freeze dryers at pharmaceutical manufacturing facilities, there are batches of penicillin bottles containing pharmaceutical products that need to be frozen and dried. It is common practice in production to cool the temperature in the lyophilizer to a very low level to ensure that all of the drug in the penicillin vials therein freezes, but the drug in each vial freezes randomly in a temperature range below the freezing point, as the nucleation process is uncontrolled. The synchronous coagulation nucleation is realized in a short time in a specific application range such as the production process of antibody coupled drugs.
In view of this, for the present invention, the principle is that: the nucleation temperature is proportional to the size of the ice core, the higher the nucleation temperature is, the larger the ice core is, the coarser the pore channels are left after the ice crystal sublimates, the smaller the sublimation resistance is, and the faster the sublimation rate is. The temperature in the freeze dryer is raised to-2 ℃, and gas in the material is saturated in a short time, and then instantaneous synchronous crystallization is realized by utilizing pressure mutation. The method ensures that the nucleation temperatures of the feed liquid in different containers are more uniform in the same freeze dryer, and the nucleation temperatures are both improved, thereby being beneficial to generating larger ice core size and improving the drying efficiency of the material.
The beneficial effects are that: (1) The method can realize synchronous crystallization within a narrow specific temperature range in a short time; (2) The method can ensure that the uniformity of the material is coagulated and nucleated in the freeze-drying process, and does not need to be maintained for a long time at a certain temperature below the crystallization point; (3) The method can improve the nucleation temperature of the material to-2 ℃, and is beneficial to generating larger ice nucleus size, thereby being beneficial to improving the drying rate of the material.
Drawings
FIG. 1 is a schematic view showing the placement of a sample (e.g., penicillin bottle) in a freeze dryer;
FIG. 2 is a plot of pressure versus temperature versus time for a process of the present invention using reduced pressure control for nucleation for 30 minutes, wherein 1 is the cavity pressure psig,2 is the slab temperature, 3 is the product temperature, the vertical axis is temperature, and the horizontal axis is time;
FIG. 3 is a graph of pressure versus temperature versus time after the nucleation maintenance was reduced to 10 minutes using reduced pressure control for the process of the present invention, wherein 1 is the cavity pressure psig,2 is the slab temperature, 3 is the product temperature, and the vertical axis is temperature and the horizontal axis is time.
Detailed Description
The following examples further illustrate the invention but are not to be construed as limiting the invention. Modifications and substitutions to the method, steps or conditions of the invention without departing from the spirit and nature of the invention are intended to be within the scope of the invention. The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated.
The lyophilization process described herein is performed in an SP Scientific lyophilizer (model SP Hull
4.0 Is performed in the following manner). A freeze dryer clapboard is arranged in the freeze dryer, and a penicillin bottle filled with liquid medicine is arranged on the clapboard; the back side of the machine is provided with a ventilation valve, the ventilation valve is connected with a pipeline, and the ventilation valve releases or lets in gas to raise pressure. The liquid medicine is prepared in a biosafety cabinet, and particularly, the cabinet is filled with gas and reaches a saturated state. The gas of the present embodimentTaking nitrogen as an example and taking 5% sucrose solution as an example, the specific process of the method is as follows:
EXAMPLE 1 pressure maintenance time 30min
S1, 500mL of 5% sucrose solution in a PC material bottle is filtered by a 0.22 mu m filter membrane and then divided into two bottles of 250mL each, the two bottles are placed in a biosafety cabinet, the temperature in the biosafety cabinet is kept between 18 ℃ and 25 ℃, one bottle of solution is taken to be connected with a glass nozzle of a nitrogen pipeline with the purity of more than 99.99% and is introduced into the bottom of the liquid.
S2, closing a pressure control valve on the nitrogen tank, opening a nitrogen tank total valve, slowly unscrewing the pressure control valve, controlling the flow rate of nitrogen to be between 0.5 and 1L/min, and ensuring that the nitrogen is continuously filled and the liquid medicine is not flushed out by air flow at the flow rate, wherein the air filling process is continuously carried out for 30min, so that the nitrogen dissolved in the liquid medicine is saturated.
S3, respectively subpackaging the solution dissolved with saturated nitrogen and the solution which is not inflated into 15 bottles of penicillin in a biosafety cabinet at the temperature of 18-25 ℃, placing the 5mL bottles of the solution into a separator of a freeze dryer which is precooled to the temperature of 5 ℃, inflating and non-inflating sample solutions symmetrically left and right, and placing the inflated sample solutions in a V shape, as shown in figure 1, and closing a cavity door of the freeze dryer.
S4, setting a program of the freeze dryer, reducing the temperature of the separator of the freeze dryer to minus 5 ℃ within 10min, maintaining for 30min, reducing the temperature of the liquid medicine to minus 4 ℃ completely, and introducing nitrogen through a vent valve while reducing the temperature, so that the pressure of a cavity where the separator of the freeze dryer is positioned is increased to 28psig. At this time, the chemical solution is in a metastable state not higher than the crystallization point, but does not reach the supercooling point temperature, so that crystallization does not occur.
S5, when the temperature of the partition plate is minus 5 ℃, opening a vent valve to enable the partition plate to be communicated with the atmosphere, reducing the pressure to 2psig in less than 10S, and when the pressure is instantaneously released, volatilizing nitrogen dissolved in the liquid medicine in the pressure releasing process to cause the movement of water molecules in the solution to be aggravated, thereby achieving the critical size of nucleation and crystallizing.
The nucleation results of the high pressure maintenance for 30min are shown in Table 1
TABLE 1 high pressure maintenance for 30min to control nucleation results
As can be seen from FIG. 2, the sample solution is firstly cooled to about-4 ℃ and is lower than the nucleation temperature, and is maintained for 30min under the high pressure of 28psig, and when the pressure is released, the temperature of the liquid medicine is simultaneously changed suddenly, and the temperature is raised to 0 ℃, so that the water is changed into ice, the successful nucleation is realized, and the nucleation process is completed within a few seconds. As shown in table 1, the nucleation rates of both the two liquid medicines were 100% because the high pressure maintenance time was longer for 30min, the liquid medicine temperature was reduced to-4 ℃ below its crystallization point temperature, and the 28psig maintenance time was 30min enough to allow the nitrogen in the liquid medicine in the pre-aerated and non-pre-aerated penicillin bottles to reach a saturated state, so that there was no significant difference in the nucleation rates of the two liquid medicines.
Example 2 reduction of the pressure maintenance time to 10min
S1, 500mL of 5% sucrose solution in a PC material bottle is filtered by a 0.22 mu m filter membrane and then divided into two bottles of 250mL each, the two bottles are placed in a biosafety cabinet, the temperature in the biosafety cabinet is kept between 18 ℃ and 25 ℃, one bottle of solution is taken to be connected with a glass nozzle of a nitrogen pipeline with the purity of more than 99.99% and is introduced into the bottom of the liquid.
S2, closing a pressure control valve on the nitrogen tank, opening a nitrogen tank total valve, slowly unscrewing the pressure control valve, controlling the flow rate of nitrogen to be between 0.5 and 1L/min, and ensuring that the nitrogen is continuously filled and the liquid medicine is not flushed out by air flow at the flow rate, wherein the air filling process is continuously carried out for 30min, so that the nitrogen dissolved in the liquid medicine is saturated.
S3, respectively subpackaging the solution dissolved with saturated nitrogen and the solution which is not inflated into 15 bottles of penicillin in a biosafety cabinet at the temperature of 18-25 ℃, placing the 5mL bottles of the solution into a separator of a freeze dryer which is precooled to the temperature of 5 ℃, inflating and non-inflating sample solutions symmetrically left and right, and placing the inflated sample solutions in a V shape, as shown in figure 1, and closing a cavity door of the freeze dryer.
S4, setting a program of the freeze dryer, reducing the temperature of the separator of the freeze dryer to minus 5 ℃ within 10min, maintaining for 10min, reducing the temperature of the liquid medicine to minus 2 ℃ during the period, and introducing nitrogen through a vent valve while reducing the temperature, so that the pressure of a cavity where the separator of the freeze dryer is positioned is increased to 28psig. At this time, the chemical solution is in a metastable state not higher than the crystallization point, but does not reach the supercooling point temperature, so that crystallization does not occur.
S5, at the temperature of the baffle plate of minus 5 ℃, opening a vent valve to enable the baffle plate to be communicated with the atmosphere, and reducing the pressure to 2psig in less than 10S. The nucleation results of the 10min high pressure maintenance control are shown in Table 2
TABLE 2 high pressure maintenance for 10min to control nucleation results
After the lamina was cooled to-5 c, it was maintained at 28psig high pressure for 10min during which time the sample was cooled to about-2 c, slightly below the nucleation temperature, as can be seen from figure 3, the temperature probe temperature was slightly changed at the same time as the pressure was released, rising to about 0 c, indicating successful nucleation. However, since the high pressure holding time is shortened to 10min, the uninflated sample liquid is not fully nucleated due to incomplete saturation within 10min, and the nucleated sample has incomplete nucleation. While the pre-aerated nitrogen solution was saturated with dissolved nitrogen prior to packaging, when all penicillin bottles were cooled to-2 ℃ in the freeze dryer to a crystallization point temperature, all pre-aerated solutions were nucleated in a short period of time after pressure release from 28psig to 2psig, and the results are shown in table 2.
In summary, the pre-aeration method can ensure that the material is uniformly coagulated and nucleated in the freeze-drying process, can realize synchronous crystallization in a short time within a narrower specific temperature range, and does not need to be maintained for a long time at a certain temperature below a crystallization point; the pre-aeration can also increase the nucleation temperature of the sample solution to-2 ℃, which is beneficial to generating larger ice nucleus size, thereby being beneficial to increasing the drying rate of the liquid medicine.
Claims (10)
1. A method for inducing simultaneous coagulation nucleation of a material in a short period of time, comprising the steps of:
pre-charging gas into the material and saturating the gas;
subpackaging the material, placing the material into a freeze dryer, synchronously performing cooling and boosting processes, controlling the temperature in the freeze dryer to be-2 to-8 ℃ and the pressure to be 25-28 psig, so that the material is in a metastable state below a crystallization point;
the lyophilizer pressure was reduced to 2-5 psig within 10 seconds to quickly volatilize the gas within the material to induce material nucleation by condensation.
2. The method of inducing simultaneous clotting nucleation of a material in a short time according to claim 1, wherein the material comprises a biopharmaceutical material, a chemopharmaceutical material, a live or attenuated virus, a nucleic acid, a monoclonal or polyclonal antibody, a protein, a peptide or a non-peptide analog.
3. The method of inducing simultaneous nucleation of material in a short period of time according to claim 1, wherein said material is subjected to microfiltration prior to pre-aeration.
4. The method of inducing simultaneous nucleation of a material for a short period of time according to claim 1, wherein the gas pre-filled in the material comprises at least one of nitrogen, argon, helium, neon, xenon, krypton.
5. The method for inducing simultaneous coagulation nucleation of a material in a short period of time according to claim 1, wherein the purity of the gas pre-filled in the material is 99.99% or more and the flow rate of the gas is 0.5 to 1L/min.
6. The method for inducing simultaneous nucleation of a material according to claim 1, wherein the temperature at which the pre-charge of the material reaches saturation is between 18 and 25 ℃.
7. The method for synchronously coagulating and nucleating of an induced material in a short time according to claim 1, wherein the freeze dryer is pre-cooled to 2-5 ℃ before the material is put into the sub-packaging.
8. The method for synchronously nucleating induced materials according to claim 1 or 7, wherein the temperature in the freeze dryer is reduced from the pre-cooling temperature to-2 to-8 ℃ and the pressure is increased from normal pressure to 25 to 28psig within 10 to 20min, and maintained for 10 to 30min.
9. The method for inducing simultaneous nucleation of material for short time according to claim 1, wherein the pressure-increasing process is to introduce a gas comprising at least one of nitrogen, argon, helium, neon, xenon, krypton into the freeze dryer.
10. The method for inducing simultaneous coagulation nucleation of a material in a short period of time according to claim 1, wherein the temperature in the freeze dryer is maintained at-2 to-8 ℃ during the depressurization.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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