阅读说明：本技术 一种鸡胚培养疫苗的冻干工艺 (Freeze-drying process of chick embryo culture vaccine ) 是由 马光明 梁熔婷 许祥雨 钟伟锋 于 2020-12-29 设计创作，主要内容包括：本发明属于疫苗制备技术领域,公开了一种鸡胚培养疫苗的冻干工艺。将疫苗生产种毒接种鸡胚,经培养后收获病毒液,加入蔗糖作为冻干保护剂,然后经冷冻干燥处理,得到疫苗成品；所述冷冻干燥处理的工艺条件如下：预冻干：-20℃,4h；冻干：-40℃,120h；第一次升温：-20℃,8h；第二次升温：0℃,8h；第三次升温：15℃,8h；第四次升温：25℃,8h。本发明冻干工艺无需加入冻干保护剂,将原有-40℃冻干步骤直接升温至0℃保温的过程中,增加了第一次升温至-20℃保温8h的过程,所得冻干疫苗的形态得到了明显的改善,且疫苗效价得到了明显的提高。(The invention belongs to the technical field of vaccine preparation, and discloses a freeze-drying process of a chicken embryo culture vaccine. Inoculating the vaccine production seed virus into a chicken embryo, culturing to obtain a virus liquid, adding sucrose as a freeze-drying protective agent, and then performing freeze-drying treatment to obtain a vaccine finished product; the process conditions of the freeze drying treatment are as follows: pre-freeze-drying: 4h at-20 ℃; freeze-drying: 120h at-40 ℃; first temperature rise: 8h at the temperature of minus 20 ℃; and (3) second temperature rise: 0 ℃ for 8 h; and (3) heating for the third time: 15 ℃ for 8 h; fourth temperature rise: 25 ℃ and 8 h. According to the freeze-drying process, a freeze-drying protective agent is not required to be added, the original freeze-drying step at the temperature of-40 ℃ is directly heated to the temperature of 0 ℃ for heat preservation, the process of heating to the temperature of-20 ℃ for heat preservation for 8 hours for the first time is added, the form of the obtained freeze-dried vaccine is obviously improved, and the titer of the vaccine is obviously improved.)

1. A freeze-drying process of a chicken embryo culture vaccine is characterized by comprising the following steps:

inoculating the vaccine production seed virus into a chicken embryo, culturing to obtain a virus liquid, adding sucrose as a freeze-drying protective agent, and then performing freeze-drying treatment to obtain a vaccine finished product; the process conditions of the freeze drying treatment are as follows:

pre-freeze-drying: 4h at-20 ℃;

freeze-drying: 120h at-40 ℃;

first temperature rise: 8h at the temperature of minus 20 ℃;

and (3) second temperature rise: 0 ℃ for 8 h;

and (3) heating for the third time: 15 ℃ for 8 h;

fourth temperature rise: 25 ℃ and 8 h.

2. The freeze-drying process of the chick embryo culture vaccine as claimed in claim 1, wherein the freeze-drying process comprises the following steps: the vaccine production seed virus refers to newcastle disease virus, avian influenza virus, infectious bursal disease virus or avian adenovirus.

3. The freeze-drying process of the chick embryo culture vaccine as claimed in claim 1, wherein the freeze-drying process comprises the following steps: the addition amount of the freeze-drying protective agent sucrose is 10% of the mass of the virus liquid.

4. The freeze-drying process of the chick embryo culture vaccine as claimed in claim 1, wherein the freeze-drying process comprises the following steps: the pre-freeze-drying step is to cool the virus liquid with the temperature of 4-15 ℃ to-20 ℃ at the speed of 0.5-1.5 ℃/min and preserve the temperature for 4 h.

5. The freeze-drying process of the chick embryo culture vaccine as claimed in claim 1, wherein the freeze-drying process comprises the following steps: the freeze-drying step is to cool the mixture to-40 ℃ at the speed of 0.5-1.5 ℃/min and preserve the temperature for 120 h.

6. The freeze-drying process of the chick embryo culture vaccine as claimed in claim 1, wherein the freeze-drying process comprises the following steps: the first heating refers to heating to-20 ℃ at a heating rate of 0.4-0.8 ℃/min and preserving heat for 8 hours.

7. The freeze-drying process of the chick embryo culture vaccine as claimed in claim 1, wherein the freeze-drying process comprises the following steps: the second heating refers to heating to 0 ℃ at a heating rate of 0.4-0.8 ℃/min and preserving heat for 8 hours.

8. The freeze-drying process of the chick embryo culture vaccine as claimed in claim 1, wherein the freeze-drying process comprises the following steps: and the third heating is to heat the mixture to 15 ℃ at a heating rate of 0.5-1.0 ℃/min and keep the temperature for 8 hours.

9. The freeze-drying process of the chick embryo culture vaccine as claimed in claim 1, wherein the freeze-drying process comprises the following steps: the fourth heating is to heat to 25 ℃ at a heating rate of 0.5-1.0 ℃/min and keep the temperature for 8 hours.

10. The freeze-drying process of the chick embryo culture vaccine as claimed in claim 1, wherein the freeze-drying process comprises the following steps: the process conditions of the freeze drying treatment are as follows:

pre-freeze-drying: cooling the virus liquid at 4 ℃ to-20 ℃ at the speed of 1 ℃/min and preserving heat for 4 h;

freeze-drying: cooling to-40 deg.C at a rate of 1 deg.C/min and maintaining for 120 h;

first temperature rise: heating to-20 ℃ at the heating rate of 0.5 ℃/min and preserving heat for 8 h;

and (3) second temperature rise: heating to 0 ℃ at the heating rate of 0.5 ℃/min and preserving the heat for 8 hours;

and (3) heating for the third time: heating to 15 ℃ at the heating rate of 0.8 ℃/min and preserving the heat for 8 hours;

fourth temperature rise: heating to 25 ℃ at the heating rate of 0.8 ℃/min and preserving the heat for 8 h.

Technical Field

The invention belongs to the technical field of vaccine preparation, and particularly relates to a freeze-drying process of a chicken embryo culture vaccine.

Background

The chick embryo culture method is a culture method for culturing some animal viruses sensitive to chick embryos, and can be used for separating and culturing various viruses, carrying out virulence titration, carrying out neutralization tests, preparing antigens and vaccines and the like. The chicken embryo culture technology is easier to succeed than tissue culture, easier than animal sources for inoculating animals, free of special requirements for feeding management, isolation and the like, generally free of recessive infection of viruses, wide in sensitivity range and adaptable to various viruses, and therefore the chicken embryo culture technology is a commonly used method for culturing animal viruses.

The freeze dried vaccine is prepared through passage or gene modification of pathogenic microbe to make the pathogenic microbe non-pathogenic, amplification of pathogenic microbe, culture liquid in freeze drier, low temperature separation of water in the culture liquid by means of increasing vacuum degree in the freeze drier, and final freeze drying.

The novel freeze-dried inactivated vaccine is freeze-dried into powder or blocks according to the immunizing dose of a single-variety vaccine, and when the vaccine is used, corresponding immunologic adjuvant is used for on-site preparation, so that a fixed preparation form is formed in a short time. According to the requirements of farmers, the vaccine is selectively used in a combined manner, so that the problem that the content of combined vaccine antigen is difficult to meet is solved, and the puzzles of excessive immunization times and excessive immunization dosage of the traditional inactivated vaccine are solved.

Patent CN 103735521A discloses a freeze-dried inactivated vaccine for animals and a preparation process thereof. The inactivated vaccine is obtained by inactivating vaccine antigen and then performing a vacuum freeze drying process, and the immune adjuvant or diluent has good emulsifying property. The vaccine antigen is a viral antigen of PCV-2, PRRS, epidemic diarrhea or influenza virus, or a bacterial antigen of haemophilus parasuis, streptococcus or escherichia coli, or a mycoplasma, coccidium or eperythrozoon antigen. Although the patent discloses a preparation method of freeze-dried inactivated vaccine, the relationship between the freeze-drying process and the vaccine titer is not studied.

Patent CN101327323A discloses a freeze-drying process for preparing a freeze-dried live attenuated hepatitis a vaccine. The freeze-dried live attenuated hepatitis A vaccine is prepared by the process steps of prefreezing the vaccine, sublimating and drying the vaccine, eliminating the shrinkage and sublimation at the bottom of the vaccine, sublimating the invisible ice surface of the vaccine, resolving and drying the vaccine and the like. Avoids the continuous temperature rise and temperature rise mutation phenomena of the vaccine caused by the freeze-drying process in the sublimation period, reduces and eliminates the problem of larger loss of the infectious titer of the vaccine, and ensures that the residual moisture of the vaccine reaches the standard and the appearance is fine and full. However, the patent has complex process and high control difficulty, aims at the hepatitis A attenuated live vaccine purchased in the market, and is not suitable for the freeze-drying treatment of the chick embryo culture vaccine.

The prior freeze-drying process for chicken embryo culture vaccines generally has the following problems: 1. the freeze-drying condition is not optimized enough, so that the titer of the vaccine is reduced; 2. the appearance quality of the freeze-dried product is poor; 3. complex lyoprotectants need to be added.

Disclosure of Invention

Aiming at the defects and shortcomings of the prior art, the invention aims to provide a freeze-drying process of a chicken embryo culture vaccine. The process can obviously improve the appearance of the product and obviously improve the titer of the vaccine by adjusting the freeze-drying temperature and the program without adding any freeze-drying protective agent.

The purpose of the invention is realized by the following technical scheme:

a freeze-drying process of a chicken embryo culture vaccine comprises the following steps:

inoculating the vaccine production seed virus into a chicken embryo, culturing to obtain a virus liquid, adding sucrose as a freeze-drying protective agent, and then performing freeze-drying treatment to obtain a vaccine finished product; the process conditions of the freeze drying treatment are as follows:

pre-freeze-drying: 4h at-20 ℃;

freeze-drying: 120h at-40 ℃;

first temperature rise: 8h at the temperature of minus 20 ℃;

and (3) second temperature rise: 0 ℃ for 8 h;

and (3) heating for the third time: 15 ℃ for 8 h;

fourth temperature rise: 25 ℃ and 8 h.

Further, the type of vaccine production seed virus is not so limited, and any strain that can be cultured by inoculating chicken embryo cells and harvesting virus fluid can be used, including but not limited to newcastle disease virus, avian influenza virus, infectious bursal disease virus, avian adenovirus, and the like.

Further, the addition amount of sucrose serving as the freeze-drying protective agent is 10% of the mass of the virus liquid.

Further, the step of pre-freeze drying is to cool the virus liquid with the temperature of 4-15 ℃ to-20 ℃ at the speed of 0.5-1.5 ℃/min and keep the temperature for 4 h.

Further, the freeze-drying step is to cool the mixture to-40 ℃ at the speed of 0.5-1.5 ℃/min and preserve the temperature for 120 h.

Further, the first heating refers to heating to-20 ℃ at a heating rate of 0.4-0.8 ℃/min and keeping the temperature for 8 hours.

Further, the second heating refers to heating to 0 ℃ at a heating rate of 0.4-0.8 ℃/min and keeping the temperature for 8 hours.

Further, the third heating is to heat up to 15 ℃ at a heating rate of 0.5-1.0 ℃/min and keep the temperature for 8 hours.

Furthermore, the fourth heating is to heat to 25 ℃ at a heating rate of 0.5-1.0 ℃/min and keep the temperature for 8 hours.

Further preferably, the freeze-drying process has the following process conditions:

pre-freeze-drying: cooling the virus liquid at 4 ℃ to-20 ℃ at the speed of 1 ℃/min and preserving heat for 4 h;

freeze-drying: cooling to-40 deg.C at a rate of 1 deg.C/min and maintaining for 120 h;

first temperature rise: heating to-20 ℃ at the heating rate of 0.5 ℃/min and preserving heat for 8 h;

and (3) second temperature rise: heating to 0 ℃ at the heating rate of 0.5 ℃/min and preserving the heat for 8 hours;

and (3) heating for the third time: heating to 15 ℃ at the heating rate of 0.8 ℃/min and preserving the heat for 8 hours;

fourth temperature rise: heating to 25 ℃ at the heating rate of 0.8 ℃/min and preserving the heat for 8 h.

Compared with the prior art, the invention has the beneficial effects that:

(1) the key of the freeze-drying process is that the original-40 ℃ freeze-drying step is directly heated to 0 ℃ for heat preservation, and a process of heating to-20 ℃ for heat preservation for 8 hours for the first time is added. The shape of the freeze-dried vaccine obtained after the first heating process is increased is obviously improved through verification, and the titer of the vaccine is obviously improved.

(2) The freeze-drying process of the invention adopts simple sucrose components as freeze-drying protective agents, and the virus liquid after the chick embryo culture is subjected to freeze-drying treatment to obtain the high-titer vaccine, and the process is simple, the cost is low, and the effect is good.

Drawings

FIG. 1 is a graph showing the comparison result between the appearance and the form of the freeze-dried vaccine obtained in example 1 (right) and that obtained in comparative example 1 (left).

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto.

Example 1

Inoculating the avian adenovirus into SPF chick embryo for propagation culture, harvesting chick embryo allantoic fluid, centrifuging, and collecting supernatant for storage at 4 ℃. And adding 10% of sucrose into the obtained supernatant as a freeze-drying protective agent, and then carrying out freeze drying under the following process conditions to obtain a vaccine finished product.

The freeze drying treatment process conditions are as follows:

pre-freeze-drying: cooling the virus liquid at 4 ℃ to-20 ℃ at the speed of 1 ℃/min and preserving heat for 4 h;

freeze-drying: cooling to-40 deg.C at a rate of 1 deg.C/min and maintaining for 120 h;

first temperature rise: heating to-20 ℃ at the heating rate of 0.5 ℃/min and preserving heat for 8 h;

and (3) second temperature rise: heating to 0 ℃ at the heating rate of 0.5 ℃/min and preserving the heat for 8 hours;

and (3) heating for the third time: heating to 15 ℃ at the heating rate of 0.8 ℃/min and preserving the heat for 8 hours;

fourth temperature rise: heating to 25 ℃ at the heating rate of 0.8 ℃/min and preserving the heat for 8 h.

Comparative example 1

The comparative example is a freeze-drying process before improvement, and compared with example 1, the freeze-drying treatment process conditions are as follows:

pre-freeze-drying: cooling the virus liquid at 4 ℃ to-20 ℃ at the speed of 1 ℃/min and preserving heat for 4 h;

freeze-drying: cooling to-40 deg.C at a rate of 1 deg.C/min and maintaining for 120 h;

first temperature rise: heating to 0 ℃ at the heating rate of 0.5 ℃/min and preserving the heat for 8 hours;

and (3) second temperature rise: heating to 15 ℃ at the heating rate of 0.8 ℃/min and preserving the heat for 8 hours;

and (3) heating for the third time: heating to 25 ℃ at the heating rate of 0.8 ℃/min and preserving the heat for 8 h.

1. The comparison result of the appearance and the morphology of the freeze-dried vaccine obtained in example 1 (right) and comparative example 1 (left) is shown in fig. 1. As can be seen from the results in FIG. 1, the vaccine morphology obtained after the lyophilization process of the present invention is improved is significantly improved.

2. The lyophilized vaccines obtained in the above examples and comparative examples were subjected to Hemagglutination (HA) test to detect vaccine titer and Hemagglutination Inhibition (HI) method to detect antibody titer. The comparative results are shown in table 1 below.

TABLE 1

As can be seen from the results in Table 1, the titer of the vaccine obtained by the improved lyophilization process of the present invention is significantly improved.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.