阅读说明：本技术 一种冷冻保护剂在铝佐剂中的应用 (Application of cryoprotectant in aluminum adjuvant ) 是由 孙冰冰 李嘉欢 薛长颖 梁智慧 李敏 杨雪程 于 2021-09-25 设计创作，主要内容包括：本发明公开了一种冷冻保护剂在铝佐剂中的应用,用于保护铝佐剂悬浮液在冷冻过程中不受冻害。本发明提供的冷冻保护方法避免了在疫苗中使用的含铝佐剂冷冻过程中的大量聚集、减少了冷冻过程对其结构的破坏。本发明可以提高铝佐剂在冷冻过程中的稳定性,大大降低在冷链运输过程中由于意外冷冻事件的发生导致的佐剂效用损失可能性,可以避免不必要的损失和严重后果。(The invention discloses an application of a cryoprotectant in an aluminum adjuvant, which is used for protecting an aluminum adjuvant suspension from being frozen in a freezing process. The cryoprotection method provided by the invention avoids a large amount of aggregation in the process of freezing the aluminum-containing adjuvant used in the vaccine and reduces the damage of the freezing process to the structure of the vaccine. The invention can improve the stability of the aluminum adjuvant in the freezing process, greatly reduce the possibility of adjuvant effect loss caused by accidental freezing events in the cold chain transportation process, and avoid unnecessary loss and serious consequences.)

1. The application of the cryoprotectant in the aluminum adjuvant is characterized in that the cryoprotectant is added into an aluminum adjuvant suspension to obtain the aluminum adjuvant containing the cryoprotectant;

the cryoprotectant is a nonionic surfactant having a hydroxyl structure of 4-7, and C₈-C₁₇Alkyl chain of (2).

2. The use according to claim 1, wherein the aluminum adjuvant with cryoprotectant has a freezing temperature in the range of-20 ℃ to-80 ℃ and a freezing time of 12h or more.

3. Use according to claim 1, wherein the cryoprotectant is selected from one or more of octyl glucoside, coco glucoside, sucrose palmitate, polyglycerol 6-distearate, sucrose distearate.

4. The use according to claim 1, wherein the mass concentration of the cryoprotectant is between 0.05% and 5%.

5. The use according to claim 1, wherein the aluminum adjuvant is an aluminum oxyhydroxide adjuvant.

6. The use according to claim 1, wherein the concentration of the aluminum adjuvant suspension is 1-10 mg/mL.

7. The use according to claim 1, wherein the aluminum adjuvant suspension is prepared by a method comprising: dissolving the aluminum adjuvant in water, and uniformly dispersing.

8. Use of the aluminum cryoprotectant-containing adjuvant of claim 1 in a vaccine formulation.

9. The use according to claim 8, wherein the vaccine is diphtheria, tetanus and acellular pertussis vaccines, Haemophilus influenzae type b vaccines, hepatitis B vaccines, hepatitis A pneumonia vaccines.

Technical Field

The invention belongs to the technical field of vaccine adjuvant production processes, and particularly relates to application of a cryoprotectant in an aluminum adjuvant.

Background

Vaccines are currently the most effective way to prevent infectious diseases, early vaccines, which generally consist of live, attenuated and completely inactivated pathogens with endogenous adjuvants, which can elicit strong protective immune responses, whereas the antigens in most modern vaccines today are toxoids, polysaccharides, proteins and virus-like particles, which, although safer, have the disadvantage of poor immunogenicity of the antigen, often requiring the addition of additional adjuvants to elicit a protective immune response.

Adjuvant-enhanced immune response is a very complex process, and according to the current research, adjuvants mainly play two roles, on one hand, after adsorbing antigens, the adjuvants can present the antigens to immune cells, for example, the most common storage effect, and after adsorbing the antigens, the adjuvants can be used as a reservoir of the antigens, and the antigens can be presented to the immune cells for a long time to induce immune response. The FDA currently approved 6 human vaccine adjuvants including aluminum salt, AS03, MF59, AS10B, AS04, CpG 1018, all of which play irreplaceable roles in human vaccines, wherein aluminum salt adjuvants are the first approved adjuvants for human vaccines and are the most diverse and most widely used adjuvants.

According to the recommendations of the world health organization, aluminum-adjuvanted vaccine formulations containing diphtheria, pertussis, tetanus, hepatitis B, haemophilus influenzae type B, IPV and combinations thereof should all be stored at between 2 and 8 degrees celsius and should not be refrigerated. However, during cold chain transportation and storage, occasional instances of system malfunction may occur, exposing the vaccine to freezing temperatures. In China, due to the fact that the breadth of the population is broad, the climate difference between the south and the north is large, and the interference of factors such as the external environment of equipment, road conditions, air temperature and electric power energy in the cold chain transportation process can cause the problem of cold chain 'chain breakage' in the transportation link, so that accidental freezing events can occur.

The influence of freezing on the aluminum adjuvant is obviously higher than the influence of the freezing on the antigen, and researches show that the aluminum adjuvant is agglomerated due to the extrusion of mechanical stress generated by the formation of ice crystals on the aluminum adjuvant in the freezing process, and the surface structure of the aluminum adjuvant is damaged by the extrusion, so that the physicochemical property of the aluminum adjuvant is influenced. This directly undermines the stability and effectiveness of aluminum-adjuvanted vaccines.

Therefore, it is an urgent problem to provide a method for preventing the loss caused by accidental freezing in cold chain transportation, improving the stability and safety of vaccines, and reducing the cost and risk.

Disclosure of Invention

The invention aims to provide an application of a cryoprotectant in an aluminum adjuvant, wherein the cryoprotectant can inhibit agglomeration of the aluminum adjuvant in a freezing process, protect the structure and surface physicochemical properties of the aluminum adjuvant from being influenced, protect the immunogenicity of the aluminum adjuvant, reduce the production cost and solve the accidental freezing problem of a vaccine in cold chain transportation.

The invention has the first aim of providing the application of the cryoprotectant in the aluminum adjuvant, wherein the cryoprotectant is added into the suspension of the aluminum adjuvant to prepare the aluminum adjuvant containing the cryoprotectant; the cryoprotectant is a polyhydroxy surfactant which is a nonionic surfactant and has a polyhydroxy structure (4-7 carbons) and a certain length of alkyl chain (8-17 carbons), and comprises one or more of octyl glucoside, coco glucoside, sucrose palmitate, polyglycerol 6-distearate and sucrose distearate.

The freezing temperature range of the aluminum adjuvant containing the cryoprotectant is-20 ℃ to-80 ℃, and the freezing time is more than 12 hours.

The mass concentration of the cryoprotectant is 0.05-5% (w/v), more preferably 0.05-1%, and even more preferably 0.05%.

The aluminum adjuvant is an aluminum hydroxide adjuvant.

As one of the preferable embodiments of the invention, octyl glucoside is selected as the polyhydroxy surfactant used in the cryoprotectant for the aluminum adjuvant. The structural formula is as follows:

as one of the preferable embodiments of the invention, the polyhydroxy surfactant used in the aluminum adjuvant cryoprotectant disclosed by the invention is coco glucoside. The structural formula is as follows:

as one of the preferable embodiments of the invention, the polyhydroxy surfactant used in the aluminum adjuvant cryoprotectant provided by the invention is sucrose palmitate. The structural formula is as follows:

as one of the preferable embodiments of the invention, the polyhydroxy surfactant used in the aluminum adjuvant cryoprotectant provided by the invention is selected from polyglycerol 6-distearate. The structural formula is as follows:

as one of the preferable embodiments of the invention, the polyhydroxy surfactant used in the aluminum adjuvant cryoprotectant provided by the invention is sucrose distearate. The structural formula is as follows:

the second purpose of the invention is to provide a preparation method of the aluminum adjuvant containing the cryoprotectant; comprises the following steps:

(1) dissolving the aluminum adjuvant in water, and uniformly dispersing;

(2) adding the selected cryoprotectant into the aluminum adjuvant suspension, and uniformly mixing.

As a preferred embodiment of the invention, the final volume of the aluminum adjuvant suspension is 1ml calculated as: wherein, the aluminum adjuvant is 1-10 mg.

Wherein the dispersion mode in the step (1) comprises stirring, vortex and ultrasound; preferably, the dispersion mode is ultrasonic, and the time for ultrasonic dispersion is fifteen minutes.

Wherein the amount of the cryoprotectant in the step (2) is 0.5-50 mg.

Wherein, the mixing mode in the step (2) is mechanical mixing, including stirring and ultrasound; preferably, the mixing mode is ultrasonic mixing, and the time for ultrasonic mixing is five minutes.

The third object of the present invention is to provide the aluminum adjuvant containing cryoprotectant or the aluminum adjuvant containing cryoprotectant prepared by the above method, which can be applied to various vaccine preparations, such as diphtheria, tetanus and acellular pertussis (DTaP) vaccines, haemophilus influenzae type b (HiB) vaccines, hepatitis b (HBsAg) vaccines, hepatitis a pneumonia vaccines, etc., and comprises: the production, transportation and storage of the preventive and therapeutic aluminum adjuvant vaccine.

The aluminum adjuvant containing the cryoprotectant is frozen, the hydroxy aluminum oxide is not obviously agglomerated, the particle structure and the surface physicochemical property are not influenced, and the vaccine prepared by combining the aluminum adjuvant with various antigens is not inactivated.

The invention selects HBsAg and BSA as model antigens, and in vivo and in vitro experiments prove that the aluminum adjuvant containing the cryoprotectant can effectively protect the properties and functions of the adjuvant, and the freeze-thawed adjuvant can still exert good immune effect when being combined with the antigens.

As one of the preferable embodiments of the invention, when the content of the aluminum adjuvant in the formula of the prepared cryoprotectant is 1mg/mL, and the content of the cryoprotectant is 0.5mg/mL, the HBV antigen is adsorbed for immunization after freeze thawing, compared with the effect of the aluminum adjuvant which is not frozen, the levels of the generated antibody IgG and IgG1 have no significant difference, the level of the IgG1 subtype antibody reflects the humoral immune response level, and the application can effectively avoid the freezing damage of the aluminum adjuvant under the condition of lower dosage of the cryoprotectant and does not influence the vaccine response effect after the aluminum adjuvant adsorbs the antigen.

Advantageous effects

The cryoprotection method provided by the invention avoids a large amount of aggregation in the process of freezing the aluminum-containing adjuvant used in the vaccine and reduces the damage of the freezing process to the structure of the vaccine. The invention can improve the stability of the aluminum adjuvant in the freezing process, greatly reduce the possibility of adjuvant effect loss caused by accidental freezing events in the cold chain transportation process, and avoid unnecessary loss and serious consequences.

The aluminum adjuvant containing the cryoprotectant has good repeatability and high stability, and the cryoprotectant has low content, so the cryoprotectant has low cost.

The aluminum adjuvant containing the cryoprotectant can get rid of cold chain transportation, reduce the cost and improve the popularization rate of vaccines in remote areas.

Compared with the prior art, the invention has the following advantages:

the cryoprotectant used for the aluminum adjuvant has the advantages of single component, low dosage and difficult occurrence of adverse reactions such as allergy and the like.

The invention can realize the stable transportation and storage of the aluminum adjuvant suspension liquid at extremely cold temperature, greatly improve the stability of the aluminum adjuvant vaccine, save resources and reduce cost.

The preparation method of the aluminum adjuvant containing the cryoprotectant is simple and rapid, low in finished product, mild in condition, easy for expanded production and wide in application prospect.

Drawings

FIG. 1 is a graph showing the particle size distribution of aluminum oxyhydroxide in example 1.

FIG. 2 is the results of the immune response of the aluminum oxyhydroxide adjuvant in example 1 after binding to an antigen.

Detailed Description

The following non-limiting examples will allow one of ordinary skill in the art to more fully understand the present invention, but are not intended to limit the invention in any way.

Example 1

5mg of aluminum oxyhydroxide (purchased from InvivoGen (San Diego, CA)) was accurately weighed and dissolved in 5mL of an ultrapure aqueous solution, shaken, vortexed, and ultrasonically dispersed for fifteen minutes, and 2.5mg of octyl glucoside was accurately weighed and dissolved in an aluminum oxyhydroxide suspension after the aluminum oxyhydroxide particles were uniformly dispersed in the solution, and ultrasonically dispersed for five minutes to be completely dissolved, thereby preparing the aluminum adjuvant containing the cryoprotectant with an aluminum adjuvant concentration of 1 mg/mL. The aluminum adjuvant material containing the cryoprotectant is frozen at minus 80 ℃ for 24h to obtain an aluminum adjuvant frozen at minus 80 ℃ in the presence of the cryoprotectant, the aluminum adjuvant is detected, meanwhile, a hydroxy alumina suspension with the concentration of 1mg/mL is used as a fresh aluminum adjuvant, and a hydroxy alumina suspension with the concentration of 1mg/mL is frozen at minus 80 ℃ for 24h to obtain the aluminum adjuvant frozen at minus 80 ℃: (1) the particle size distribution of the frozen aluminum adjuvant in the formulation containing the protective agent was found to not increase significantly, as measured by DLS, and was substantially consistent with the curve for fresh aluminum adjuvant (see fig. 1); (2) the surface hydroxyl content is measured by an acid-base titration neutralization method, and the surface hydroxyl content of the frozen aluminum adjuvant in a formula containing the protective agent is not reduced and is maintained at 0.45mmol/g (see table 1); (3) the aluminum adjuvant frozen at-80 ℃ in the presence of the protective agent is combined with the hepatitis B antigen to prepare the hepatitis B vaccine with the vaccine concentration of 1mg Al/mL and 40ug HBV/mL, the hepatitis B vaccine is injected into a mouse body at the dose of 50 muL, the levels of antibody IgG and IgG1 are generated after 42 days as shown in figure 2, compared with the fresh aluminum adjuvant, the level of two antibodies obtained by combining the antigen after the aluminum adjuvant without the protective agent is frozen is obviously reduced, and the level of the antibodies of the aluminum adjuvant group frozen in the formula containing the protective agent is recovered to be normal.

TABLE 1 titration results of surface hydroxyl group content of aluminum oxyhydroxide in example 1

It will be apparent to those skilled in the art from this disclosure that many changes and modifications can be made, or equivalents modified, in the embodiments of the invention without departing from the scope of the invention. Therefore, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention shall still fall within the protection scope of the technical solution of the present invention, unless the contents of the technical solution of the present invention are departed.