KR101595949B1 - Producing method of adjuvant composition and vaccine composition - Google Patents

Abstract

The present invention relates to a method for manufacturing an adjuvant composition and a vaccine composition comprising the step of drying based on a spray drying technique. The method comprises the step of forming a powder by mixing and drying a biopolymer and two or more immune supplements. According to the present invention, the composition can be stably dispersed in an aqueous solution and can be stored in a powder form for a long period of time.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a multi-batch composition and a vaccine composition,

The present invention relates to a process for the preparation of a multi-batch composition and a vaccine composition, comprising drying using spray-drying to form a powder.

Aluminum (Alum) or MF59, which is a commercialized and widely used adjuvant, is effective in inducing the improvement of the humoral immune response, but it is effective in the application field related to anticancer or antiviral prevention / treatment requiring a strong cellular immune response There is a fatal disadvantage that it is not effective. Also, monophosphoryl lipid A (MPLA) and imiquimod (R837), which are mainly used as an immunosuppressant, are insoluble in water and thus involve many difficulties in using the vaccine. In addition, QS21, which is an extract of saponin component, has the property of promoting the immune reaction by increasing the reaction of the antigen-specific antibody and the T cell, but due to the property of making the cell membrane unstable like the surfactant, It is necessary to combine with other substances to reduce the toxicity. In order to solve this problem, a method of re-suspending in water by ultrasonic degradation or a method of delivering a substance by using liposome has been used (British Patent No. 10-0365373).

However, these methods have obvious disadvantages in that they are cumbersome to use and less stable and uniform in terms of formulation. Therefore, there is a need for other ways to have a form suitable for use in the body.

The present application is directed to providing a process for preparing a multi-batch composition and a vaccine composition, which comprises drying using spray-drying to form a powder.

However, the problems to be solved by the present invention are not limited to the problems described above, and other problems not described can be clearly understood by those skilled in the art from the following description.

The first aspect of the present invention provides a method for preparing a multibranch composition comprising mixing a biopolymer and an adjuvant, followed by drying to form a powder.

A second aspect of the present invention provides a method for preparing a vaccine composition, comprising mixing a biopolymer and an adjuvant, forming a powder, and adding an antigen to the dried powder.

A third aspect of the present invention provides a multiband powder composition prepared by the method of manufacture of the first aspect of the present application and comprising a biopolymer and an adjuvant.

The present invention relates to a method of preparing a biocompatible polymer by combining an immunoadjuvant such as Imiquimod, MPLA, QS21, etc., which is an immunostimulatory substance but not dissolvable in water, with a biocompatible polymer, followed by various drying methods such as spray drying To provide a powdery avastant composition and a vaccine composition which can be stably dispersed in an aqueous solution and stored for a long period of time. The powdery avant-lot composition and vaccine composition prepared by the method of the present invention can maintain stability even when stored at room temperature for more than 7 months. In addition, the vaccine composition prepared by the manufacturing method according to one embodiment of the present invention has an effect of excellently enhancing both humoral immunity and cellular immunity in vivo by binding of an immunosuppressant and a protein antigen.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a conceptual view schematically showing a preparation method of a cancerous vaccine composition and an avant-born composition according to an embodiment of the present invention. FIG.
FIGS. 2A and 2B are SEM images of the multibun composition according to one embodiment of the present invention and images showing the dispersion stability for 48 hours in a PBS solution. FIG.
FIG. 3 is a graph showing the immune response to the OVA antigen of the cancer vaccine composition dissolved in the injection solution after storage at room temperature in a powder form according to one embodiment of the present invention (1: OVA alone (10 ㎍), 2: OVA HA / MPL / QS21, 3: OVA and HA / MPL / R837, NS are not significant).
FIGS. 4A to 4C are graphs showing the immunoenhancing effect of the cancer vaccine composition according to one embodiment of the present invention after storage at room temperature for 3 months, 3 months, 5 months, and 7 months, respectively.
FIG. 5 is a graph showing T cell activity measured by a cancer vaccine composition dissolved in an injection solution after stored at room temperature in a powder form according to an embodiment of the present invention (1: OVA alone (10 μg), 2: OVA (10 OVA alone (20 μg), 4: OVA (10 μg) and HA / MPL / R837, 5: PBS).

Hereinafter, embodiments and examples of the present invention will be described in detail with reference to the accompanying drawings, which will be readily apparent to those skilled in the art to which the present invention pertains.

It should be understood, however, that the present invention may be embodied in many different forms and is not limited to the embodiments and examples described herein. In order to clearly illustrate the present invention, parts not related to the description are omitted, and similar parts are denoted by like reference characters throughout the specification.

Throughout this specification, when a member is "on " another member, it includes not only when the member is in contact with the other member, but also when there is another member between the two members.

Throughout this specification, when an element is referred to as "including " an element, it is understood that the element may include other elements as well, without departing from the other elements unless specifically stated otherwise. The terms "about "," substantially ", etc. used to the extent that they are used throughout the specification are intended to be taken to mean the approximation of the manufacturing and material tolerances inherent in the stated sense, Accurate or absolute numbers are used to help prevent unauthorized exploitation by unauthorized intruders of the referenced disclosure. The word " step (or step) "or" step "used to the extent that it is used throughout the specification does not mean" step for.

Throughout this specification, the term "combination thereof" included in the expression of the machine form means one or more combinations or combinations selected from the group consisting of the constituents described in the expression of the machine form, And the like.

Throughout this specification, the description of "A and / or B" means "A or B, or A and B".

Throughout this specification, " spray drying " means a method of rapidly drying a liquid or slurry with a hot gas to produce a dry powder.

The first aspect of the present invention provides a method for preparing a multibranch composition comprising mixing a biopolymer and an adjuvant, followed by drying to form a powder.

In one embodiment of the invention, the drying can be done, for example, by spray drying.

In one embodiment of the invention, the immunoadjuvant may include, but is not limited to, selected from the group consisting of toll-like receptor agonists, saponins, aluminum salts, and combinations thereof .

In one embodiment, the toll-like receptor agonist is selected from the group consisting of MPLA (monophosphoryl lipid A), imiquimod, recyquimod, flagellin, CpG, poly (I : C)], and combinations thereof, but are not limited thereto.

In one embodiment of the invention, the saponin may be selected from the group consisting of QS21, Quil A, QS7, QS17, beta-eskin, digitonin, and combinations thereof, but is not limited thereto.

In one embodiment of the present invention, the immunoadjuvant forms a complex with the biopolymer to allow the insoluble adjuvant to be dissolved in the aqueous solution, and may also reduce the toxicity, but may not be limited thereto. The immunoadjuvants may include, but are not limited to, two or more immunoadjuvants, including, for example, immunoadjuvants that activate the humoral immune response and immunoadjuvants that activate the cellular immune response. Such adjuvants may include, but are not limited to, MPLA and QS21, or MPLA and imiquimod. By using more than one adjuvant, both the humoral and cellular immune responses are activated.

In one embodiment of the present invention, the biopolymer may be selected from the group consisting of hyaluronic acid, polygamma glutamic acid, alginic acid, carboxymethylcellulose (CMC), and combinations thereof, but may not be limited thereto. In one embodiment herein, the biopolymer and the adjuvant may be included in a weight ratio of from about 10 to about 50: about 1 to about 50, and may be included, for example, in a weight ratio of about 13: about 1 However, the present invention is not limited thereto. The biopolymer, the adjuvant and the antigen may be included as a weight ratio of about 10 to about 50: about 1 to about 5: about 1 to about 10, and may be included, for example, as a weight ratio of about 13: about 1: But may not be limited thereto.

In one embodiment, the adjuvant is selected from the group consisting of ethanol, methanol, ethyl acetate, isopropanol, hexane, acetone, acetonitrile, tetrahydrofuran (THF), chloroform, dichloromethane, And may be, but not limited to, dissolved in a solvent.

The second aspect of the present invention provides a method for producing a vaccine composition, which comprises mixing a biopolymer and an immunosuppressant, followed by drying to form a powder, and adding an antigen to the dried powder.

In one embodiment of the invention, the drying can be done, for example, by spray drying.

In one embodiment of the invention, the immunoadjuvant may include, but is not limited to, selected from the group consisting of toll-like receptor agonists, saponins, aluminum salts, and combinations thereof .

In one embodiment, the toll-like receptor agonist is selected from the group consisting of MPLA (moophosphoryl lipid A), imiquimod, recyquimod, flagellin, CpG, poly (I : C)], and combinations thereof, but are not limited thereto.

In one embodiment of the invention, the saponin may be selected from the group consisting of QS21, Quil A, QS7, QS17, beta-eskin, digitonin, and combinations thereof, but is not limited thereto.

In one embodiment of the invention, the biopolymer may be selected from the group consisting of hyaluronic acid, polygamargamic acid, alginic acid, carboxymethylcellulose (CMC), and combinations thereof, but may not be limited thereto.

In one embodiment of the present invention, the immunoadjuvant forms a complex with the biopolymer so that the insoluble adjuvant is dissolved in the aqueous solution, and the toxicity may be reduced, but the present invention is not limited thereto. The immunoadjuvants may include, but are not limited to, two or more immunoadjuvants, including, for example, immunoadjuvants that activate the humoral immune response and immunoadjuvants that activate the cellular immune response. Such adjuvants may include, but are not limited to, MPLA and QS21, or MPLA and imiquimod. By using more than one adjuvant, both the humoral and cellular immune responses are activated.

In one embodiment of the invention, the antigen may be selected from the group consisting of proteins, recombinant proteins, glycoproteins, peptides, polysaccharides, lipid polysaccharides, polynucleotides and combinations thereof of pathogens, but may not be limited thereto . Such proteins may include, but are not limited to, for example, over-albumin, recombinant proteins, subunits, split protein antigens. For example, the antigen promotes an immune response to an antigen in the body, thereby inhibiting the growth of cancer cells when cancer cells expressing the antigen are generated at a later time, thereby effectively preventing cancer.

In one embodiment herein, the biopolymer and the adjuvant may be included in a weight ratio of from about 10 to about 50: about 1 to about 50, and may be included, for example, in a weight ratio of about 13: about 1 However, the present invention is not limited thereto. The biopolymer, the adjuvant, and the antigen may be included as a weight ratio of about 10 to about 50: about 1 to about 5: about 1 to about 5, for example, as a weight ratio of about 13: about 1: 1 But may not be limited thereto.

In one embodiment of the invention, the vaccine composition may be in powder form, but is not limited thereto. The powdery vaccine composition can be stored for a long period of time and dissolved in a desired volume in a desired solvent for convenient use.

In one embodiment, the adjuvant is selected from the group consisting of ethanol, methanol, ethyl acetate, isopropanol, hexane, acetone, acetonitrile, tetrahydrofuran (THF), chloroform, dichloromethane, And may be, but not limited to, dissolved in a solvent.

FIG. 1 is a conceptual view schematically showing the preparation process of the avant-lot composition and the vaccine composition of the present invention, wherein MPLA and QS21 are mixed with hyaluronic acid (HA) as biopolymer, MPLA and imiquimode (R837) Drying by drying to produce a very stable composition, and a step of preparing a cancer vaccine composition by adding an antigen dissolved in PBS after spray drying.

A third aspect of the present invention provides a multiband powder composition prepared by the method of manufacture of the first aspect of the present application and comprising a biopolymer and an adjuvant.

In one embodiment of the invention, the immunoadjuvant may include, but is not limited to, selected from the group consisting of toll-like receptor agonists, saponins, aluminum salts, and combinations thereof .

In one embodiment, the toll-like receptor agonist is selected from the group consisting of MPLA (monophosphoryl lipid A), imiquimod, recyquimod, flagellin, CpG, poly (I : C)], and combinations thereof, but are not limited thereto.

In one embodiment of the invention, the saponin may be selected from the group consisting of QS21, Quil A, QS7, QS17, beta-eskin, digitonin, and combinations thereof, but is not limited thereto.

In one embodiment of the present invention, the immunoadjuvant forms a complex with the biopolymer so that the insoluble adjuvant is dissolved in the aqueous solution, and the toxicity may be reduced, but the present invention is not limited thereto. The immunoadjuvants may include, but are not limited to, two or more immunoadjuvants, including, for example, immunoadjuvants that activate the humoral immune response and immunoadjuvants that activate the cellular immune response. Such adjuvants may include, but are not limited to, MPLA and QS21, or MPLA and imiquimod. By using more than one adjuvant, both the humoral and cellular immune responses are activated.

In one embodiment of the invention, the biopolymer may be selected from the group consisting of hyaluronic acid, polygamargamic acid, alginic acid, carboxymethylcellulose (CMC), and combinations thereof, but may not be limited thereto. In one embodiment herein, the biopolymer and the adjuvant may be included in a weight ratio of from about 10 to about 50: about 1 to about 50, and may be included, for example, in a weight ratio of about 13: about 1 However, the present invention is not limited thereto. The biopolymer, the adjuvant and the antigen may be included as a weight ratio of about 10 to about 50: about 1 to about 5: about 1 to about 10, and may be included, for example, as a weight ratio of about 13: about 1: But may not be limited thereto.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the scope of the present invention is not limited by these Examples.

[ Example ]

Example  1: Spray DRYING  Used Very Bunt  Preparation of composition

A very bunted composition was prepared according to the process as shown in Fig. Hyaluronic acid was dissolved in distilled water at 1 mg / ml, and MPLA (monophosphoryl lipid A) and imiquimod (R837) were dissolved in DMSO and QS21 was dissolved in 20% THF aqueous solution at 5 mg / ml. 10 쨉 g of MPLA (monophosphoryl lipid A) was collected per 133 쨉 g of hyaluronic acid, stirred vigorously, and dialyzed against distilled water for 3 days. After the dialysis, 10 쨉 g of QS21 per 133 쨉 g of hyaluronic acid was vigorously stirred, reacted for 2 hours, and THF was evaporated for one day to prepare a HA / MPL / QS21 avant-lot composition. 10 쨉 g of imiquimod (R837) was mixed with 133 쨉 g of hyaluronic acid, followed by vigorous stirring, followed by reaction for 2 hours to prepare a HA / MPL / R837 enrichment composition. After thoroughly stirring the two solution mixtures, a powder formulation was prepared using a spray dryer (Mini Spray Dryer B-290, BUCHI Labortecnnik AG).

FIG. 2A is an electron micrograph (SEM) photograph of a zombant material of the powder form prepared as described above, and FIG. 2B shows dispersion stability when the zombie powder of the powder form prepared as described above is redispersed in PBS . And it was confirmed that they were stably dispersed in the aqueous solution for 48 hours. In the case of MPLA and QS21, it can be seen that the dispersion is well dispersed and has a relatively crystalline property. Even in the case of R837 having a low solubility in aqueous solution, the powder is stabilized in the aqueous solution without any precipitation or aggregation for 48 hours As shown in FIG.

Example  2: Evaluation of OVA-specific antibody production of cancer vaccine composition

In order to investigate whether the AMBA composition of the powdered form prepared as in Example 1 exhibited a specific immunoenhancing effect on the OVA antigen, it was found that, in the antibody-specific immune response, And the effect on immune response was investigated.

The avant-burnt composition powder prepared as in Example 1 was dissolved in 50 [mu] l PBS so as to contain 133 [mu] g of hyaluronic acid to prepare a solution of a lot composition. Ovalbumin (OVA), a model antigen, was dissolved in 10 mg / ml PBS, and then mixed with the above-mentioned adjuvant composition solution so as to have a ratio of 100 μg per 133 μg of hyaluronic acid. The cancer prevention vaccine used 133 ㎍ of hyaluronic acid per mouse and the total volume was 100 ㎕.

A cancer vaccine composition in which OVA (20 쨉 g) alone was administered as a control, and the avant-bleed compositions HA-MPL / QS21 and HA / MPL / R837 of powder form prepared as in Example 1 were added to C57BL / 6 mice -7 week old female) by intramuscular injection. Mouse serum was collected at 4 weeks after 2 doses every 2 weeks and the antibody titer against OVA was measured by ELISA (enzyme linked immunosorbent assay) method. As can be seen from Fig. 3, it was confirmed that the antibody titer (IgG) was higher in the group to which the cancer vaccine compositions (HA / MPL / QS21 and HA / MPL / R837) of the two powder formulations were added. These experimental results show that the prepared vaccine formulation, Zambant, is effective in enhancing humoral immunity.

Example  3: Powder formulation Very Bunt  The stability test of the composition at room temperature

In order to investigate the storage stability at room temperature of the vaccine Azbunt material of the powder formulations, four types of biopolymers (hyaluronic acid, polygamma glutamic acid, alginic acid and carboxymethylcellulose (CMC)) and two immunostimulatory complexes (MPLA / QS21, MPLA / R837) were mixed to produce eight samples (Table 1). The sample To 50 ml of the PBS solution was mixed 133 mg of hyaluronic acid, 10 mg of MPLA, 10 mg of QS21, and 10 mg of R837 in a weight ratio . Three samples per each sample were prepared and the average value of each sample activity was calculated. For the stability test at room temperature, OVA-specific antigen production was measured at 3, 5, and 7 months after storage for 7 months at room temperature. The activity value was calculated as a scale for viewing. Here, the activity was calculated as the ratio of the activity (Amonth) value of the sample after a certain number of months to the activity (AFresh) of the sample immediately after preparation (Table 1). Table 1 shows numerical results of these experiments, and FIG. 4 shows experimental results of fitting the data to the graph. As can be seen from the data, it was found that about 94-99% after 7 months without significant difference, the original immunostimulatory effect was maintained by the biomolecules used.

Example  4: Powder formulation Very Bunt  A cell-mediated specific T cell response confirmation experiment of the composition

In order to investigate the effect of enhancing the cellular immunity by the powder formulation AJB composition, the cellular immune response of mouse spleen T cells was examined. Three mice out of the mice inoculated in Example 2 were selected from among OVA alone and Ovata / OVA groups. After 2 weeks, spleens were removed from each mouse, the spleen tissues were transferred to a sterilized Petri dish, cell strainer) to separate the cells from the tissue capsule. All the contents in the Petri dish were transferred to a 50 ml tube, filled with RPMI medium, centrifuged at 1,500 rpm for 5 minutes, and then the red blood cell lysing buffer (Sigma Aldrich, Germany) was added to the pellet from which the supernatant was removed. And the mixture was allowed to stand in a water bath at 30 ° C for 5 to 10 minutes to hemolyze red blood cells. Cells contained in the tubes were washed with PBS and suspended in RPMI medium to isolate splenocytes. Separated splenocytes were plated in 96 wells at ⁵ x 10 cells / 100 μl on plates coated with IFN-r and MHC class I-restricted OVA peptides were treated at a concentration of 5 μg / ml for 48 hours. Then, IFN-y with horse raddish peroxidase was added. As the substrate, 100 μl of ACE (3-amino-9-ethyl-carbazole, BD biosciences, USA) was added and the reaction was developed and measured by ELSPOT (enzyme-linked immunospot) method. As a result, it was confirmed that IFN-y showed a remarkably high response in the control group (Fig. 5).

It will be understood by those of ordinary skill in the art that the foregoing description of the embodiments is for illustrative purposes and that those skilled in the art can easily modify the invention without departing from the spirit or essential characteristics thereof. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive. For example, each component described as a single entity may be distributed and implemented, and components described as being distributed may also be implemented in a combined form.

The scope of the present invention is defined by the appended claims rather than the detailed description, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be interpreted as being included in the scope of the present invention .

Claims (18)

Mixing the biopolymer and two or more immunoadjuvants, and then drying to form a powder,
Wherein the biopolymer is selected from the group consisting of hyaluronic acid, polygamargamic acid, alginic acid, carboxymethylcellulose (CMC), and combinations thereof,
Wherein the two or more immunoadjuvants comprise MPLA (monophosphoryl lipid A) and imiquimod, or MPLA and saponin,
Wherein said saponin is selected from the group consisting of QS21, Quil A, QS7, QS17, and combinations thereof,
Wherein the biopolymer and the immunosuppressant are capable of forming a complex to be dissolved in an aqueous solution.
≪ / RTI >
The method according to claim 1,
Wherein the drying is performed by spraying drying.
delete delete delete delete A biopolymer and two or more immunosuppressants are mixed and dried to form a powder,
Adding the antigen to the dried powder
/ RTI >
Wherein the biopolymer is selected from the group consisting of hyaluronic acid, polygamargamic acid, alginic acid, carboxymethylcellulose (CMC), and combinations thereof,
Wherein the two or more immunoadjuvants comprise MPLA (monophosphoryl lipid A) and imiquimod, or MPLA and saponin,
Wherein said saponin is selected from the group consisting of QS21, Quil A, QS7, QS17, and combinations thereof,
Wherein the biopolymer and the immunosuppressant are capable of forming a complex to be dissolved in an aqueous solution.
Lt; / RTI >
8. The method of claim 7,
Wherein the drying is performed by spray drying.
delete delete delete delete 8. The method of claim 7,
Wherein the antigen is selected from the group consisting of proteins, recombinant proteins, glycoproteins, peptides, polysaccharides, lipid polysaccharides, polynucleotides, and combinations thereof of the pathogen.
3. A composition according to claim 1 or 2,
Biopolymers selected from the group consisting of hyaluronic acid, polygamma glutamic acid, alginic acid, carboxymethylcellulose (CMC), and combinations thereof; And
Two or more immunoadjuvants including MPLA (monophosphoryl lipid A) and imiquimod or MPLA and saponin
/ RTI >
Wherein said saponin is selected from the group consisting of QS21, Quil A, QS7, QS17, and combinations thereof.
Very bunt powder composition. delete delete delete delete

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