WO2020242199A1 - Method for producing immunogenic conjugate of streptococcus pneumoniae serotype 23f - Google Patents

Abstract

The present invention provides a method for producing a conjugate of Streptococcus pneumoniae serotype 23F and a carrier protein, the method comprising: (i) a step for mixing a purified Streptococcus pneumoniae (S. pneumoniae) serotype 23F capsular polysaccharide with a sodium acetate (NaOAc) buffer solution to obtain a mixture; (ii) a step for reacting the mixture with an oxidizing agent to produce an activated serotype 23F polysaccharide; (iii) a step for filtering the activated serotype 23F polysaccharide with sodium acetate to select an activated serotype 23F polysaccharide having at least 300 kDa; (iv) a step for mixing the activated serotype 23F polysaccharide with a carrier protein; and (v) a step for adding a reducing agent to a mixture of the activated serotype 23F polysaccharide and the carrier protein and conjugating same to form a conjugate of the activated serotype 23F polysaccharide and the carrier protein. The present invention can provide a highly immunogenic conjugate in which the size of a Streptococcus pneumoniae (S. pneumoniae) serotype 23F capsular polysaccharide can be kept constant.

Description

Method for preparing immunogenic conjugate of Streptococcus pneumoniae serotype 23F

This application claims priority based on Korean Application No. 10-2019-0062757 filed on May 28, 2019, and all contents disclosed in the specification and drawings of the application are incorporated in this application. It relates to a method for preparing an immunogenic conjugate of Streptococcus pneumoniae serotype 23F, and more particularly, to a vaccine comprising an immunogenic conjugate of Streptococcus pneumoniae serotype 23F and a method for preparing the same.

Infection with the bacteria Streptococcus pneumoniae can cause several types of diseases, such as pneumonia (inflammation of the lungs), otitis media (inflammation of the middle ear), and meningitis (inflammation of the membranes surrounding the brain and spinal cord). Etc. Streptococcus pneumoniae is a major causative agent of pneumonia. In addition, it causes invasive diseases such as sepsis, bacteremia, and meningitis. According to Statistics Korea [2014 Death Cause Statistics], the mortality rate from pneumonia in 2014 was 23.7 per 100,000 people, an increase of 11% compared to 2013, and an increase of 2.8 times compared to 2015, and the mortality rate due to pneumonia continued to increase. Showed a trend. In addition, according to the WHO in 2012, 476,000 children under the age of 5 who were HIV-negative worldwide died from infections caused by Streptococcus pneumoniae in 2008, accounting for 5% of the causes of death for infants and young children under the age of 5.

The Streptococcus pneumoniae is encapsulated with a chemically linked polysaccharide that confers serotype specificity. This is called a capsule, a thick mucous layer that surrounds Streptococcus pneumoniae, and is used to defend itself or attach it to a specific surface. The capsular polysaccharide has been widely used in immunology for many years for the prevention of Streptococcus pneumoniae disease. There are approximately 90 known Streptococcus pneumoniae serotypes, and the capsular membrane not only protects the inner surface of the Streptococcus pneumoniae from complement, but is itself an incomplete immunogen, so the capsular membrane is in the Streptococcus pneumoniae. It is a major determinant of toxicity to Polysaccharides are T-independent antigens and cannot be processed or presented to MHC molecules to interact with T-cells. However, they can stimulate the immune system through alternative mechanisms including crosslinking of surface receptors on B cells.

Streptococcus pneumoniae serotype 23F is known to exhibit high distribution in pediatric and adult pneumococcal-invasive patients. However, there are still difficulties in producing vaccines that can effectively prevent infection by Streptococcus pneumoniae with serotype 23F. For example, a vaccine using a conjugate made by conjugating a polysaccharide and a protein carrier has problems such as reduction in the size of the polysaccharide due to oxidation in the process of conjugation of the polysaccharide, or a low binding rate between the polysaccharide and the protein carrier. have.

The present invention is to provide a vaccine of Streptococcus pneumoniae serotype 23F.

An object of the present invention is to provide a method in which the capsular polysaccharide of Streptococcus pneumoniae serotype 23F maintains the size of a conjugate conjugated to a carrier protein and induces immunity.

An object of the present invention is to provide a method for preparing a conjugate with a small reduction in size of the capsular polysaccharide of Streptococcus pneumoniae serotype 23F.

In addition, in another embodiment, the conjugate prepared by the method for preparing the conjugate is to be provided as an immunogenic composition.

The present invention is to provide a conjugate capable of preventing or treating diseases caused by Streptococcus pneumoniae serotype 23F, and to provide the conjugate for vaccine use.

The present invention relates to a method for preparing a Streptococcus pneumoniae serotype 23F conjugate. Preferably, the present invention provides a method for preparing a conjugate of a capsular polysaccharide of Streptococcus pneumoniae serotype 23F and a carrier protein.

In one embodiment of the present invention, when preparing a conjugate of S. pneumoniae, the activated S. pneumoniae serotype 23F capsular polysaccharide decreases in size and is effective for conjugation. It provides a method of preparing a conjugate of S. pneumoniae serotype 23F and a carrier protein capable of obtaining a polysaccharide of. An embodiment of the present invention is to provide an activated polysaccharide having a size of about 50 to 60% of the size of the natural 23F serotype polysaccharide. Preferably, the activated polysaccharide may have a constant size of about 300 kDa. One embodiment of the present invention can provide a method of maintaining the size of about 50 to 60% of the polysaccharide of the natural 23F serotype.

The term "activated polysaccharide" as used herein refers to "activated S. pneumoniae serotype 23F polysaccharide", and refers to a polysaccharide chemically modified to form a reactive group in the polysaccharide chain. Activated polysaccharides do not necessarily mean that all available activation sites have been chemically modified. Activated saccharides can be understood to refer to capsular polysaccharides subjected to oxidation treatment, for example, and can be understood to mean polysaccharides in a state before conjugation of capsular polysaccharides to carrier proteins. The capsular polysaccharide subjected to the oxidation treatment may be further subjected to a lyophilization step, and in this case, it may be understood to mean a polysaccharide in a state before conjugation with a protein after lyophilization.

In one embodiment of the present invention, the conjugate of S. pneumoniae may include the following steps.

(i) reacting the purified serotype 23F polysaccharide with an oxidizing agent to produce activated serotype 23F polysaccharide; (ii) lyophilizing the activated serotype 23F polysaccharide and the carrier protein; (iii) resuspending in lyophilized activated serotype 23F polysaccharide and carrier protein dimethyl sulfoxide (DMSO); (iv) mixing the activated serotype 23F polysaccharide resuspended in dimethyl sulfoxide (DMSO) and the carrier protein; And (v) reacting the mixed activated serotype 23F polysaccharide and carrier protein with a reducing agent to generate a conjugate of serotype 23F polysaccharide and carrier protein. The method may further comprise capping the unreacted aldehyde in the carrier protein conjugate with the serotype 23F polysaccharide.

Serotype 23F polysaccharides from which the conjugates of the present invention are prepared can be prepared using purification procedures known to those skilled in the art (see, eg, US Patent Application Publication No. 2008/0286838, etc.). It can also be produced using synthetic protocols. Typically capsular polysaccharides are prepared by growing each serotype 23F in a medium (eg, soy-based medium), and the polysaccharide can then be prepared from the bacterial culture. In one embodiment of the present invention, the capsular polysaccharide can be purified using purification techniques known in the art.

In one embodiment of the present invention, the purified serotype 23F polysaccharide may react with an oxidizing agent in a buffer solution. The buffer may be preferably deionized water or acetic acid/sodium acetate (NaOAc), and most preferably, acetic acid/sodium acetate (NaOAc) may be used. The inventors of the present invention confirmed that the yield of the conjugate can be improved and the size of the polysaccharide can be adjusted according to the characteristics of the buffer solution used in the process of activation of the polysaccharide, and the present invention was completed.

In one embodiment, when the acetic acid/sodium acetate (NaOAc) buffer is used, a phenomenon in which the size of the polysaccharide rapidly decreases in the oxidation process may be less observed, and the conjugation yield of the polysaccharide and the carrier protein is improved, thereby ultimately resulting in immunogenicity. Can be improved. This effect is also seen in high concentration buffers above 100 mM.

In one embodiment, the acetic acid/sodium acetate (NaOAc) buffer may have a concentration of 1 to 550 mM, a concentration of 5 to 540 mM, a concentration of 8 to 520 mM, or a concentration of 9 to 510 mM. It may preferably have a concentration of 10 to 500mM.

The pH in step (i) is pH 2.5-8, pH 3-6, or pH 4-5, or pH 4.5. Preferably, when the pH 4.5 acetic acid/sodium acetate (NaOAc) buffer has a concentration of 1 to 550 mM, it may be advantageous in achieving the object of the present invention.

In another embodiment, the inventors of the present invention confirmed that the yield of the conjugate can be improved and the size of the polysaccharide can be adjusted according to the molar equivalent of the oxidizing agent along with the properties of the buffer solution used in the process of activating the polysaccharide. Preferably, the oxidizing agent includes sodium periodate, and periodate and periodic acid may also be included as an oxidizing agent of the present invention. When the polysaccharide of the present invention reacts with periodate, periodate oxidizes adjacent hydroxyl groups to form carbonyl or aldehyde groups, causing decomposition of C-C bonds. For this purpose, the term'reacting an antigen with periodate' includes the oxidation of adjacent hydroxyl groups by periodate.

In order to prepare a conjugate for use as a serotype 23F immunogenic composition of S. pneumoniae, the inventors of the present invention are preferably 0.07 to 0.23, 0.01 to 0.2, 0.015 to 0.19, 0.02 to 0.18, 0.05 To 0.17, 0.1 to 0.165 or 0.16 molar equivalent of periodate may be reacted to activate it. When oxidized to the molar equivalent of the above range, the size of the polysaccharide can be obtained in an appropriate range. The above range is preferred for the purposes of the present invention.

In one embodiment of the present invention, the size change rate of activated S. pneumoniae serotype 23F is reduced, including the following steps, S. pneumoniae serotype 23F and a transporter. It provides a method of preparing a conjugate of a protein.

(i) mixing the purified S. pneumoniae serotype 23F capsular polysaccharide with sodium acetate (NaOAc) buffer to obtain a mixture;

(ii) reacting the mixture with sodium periodate to produce activated serotype 23F polysaccharide;

(iii) diafiltration of the activated serotype 23F polysaccharide with sodium acetate to select an activated serotype 23F polysaccharide having at least 300 kDa;

(iv) mixing the activated serotype 23F polysaccharide with the carrier protein; And

(v) adding a reducing agent to a mixture of activated serotype 23F polysaccharide and carrier protein for conjugation to form a conjugate of activated serotype 23F polysaccharide and carrier protein.

In another embodiment, deionized water may be used instead of the sodium acetate (NaOAc) buffer of step (i).

The capsular polysaccharide of S. pneumoniae serotype 23F activated through the steps (i) and (ii) was diafiltered with sodium acetate (pH4.5) using 100 kDa MWCO ultrafiltration to obtain the weight of the polysaccharide. -The average molecular weight may be 100kDa or more, 200kDa or more, 300kDa or more, 400kDa or more, 500kDa or more. Preferably, for the purposes of the present invention, it may have a size of 300 to 500 Da. The molecular weight or average molecular weight of the saccharide herein refers to the weight-average molecular weight (Mw) of the bacterial saccharide measured before conjugation, which is measured by MALLS. MALLS technology is well known in the art. MALLS analysis can be performed using an Ultrahydrogel analytical column and 100 mM sodium phosphate (pH 7.2)/0.05% sodium azide as an elution buffer at 0.5 ml/min using an RI/DAWN-EOS detector.

The term “carrier protein” includes both small peptides and large polypeptides (>10 kDa). The carrier protein can be any peptide or protein. It may include one or more T-helper epitopes. In one embodiment of the present invention, the carrier protein is composed of tetanus toxoid (TT), fragment C of tetanus toxoid, diphtheria toxoid (DT), CRM197, pneumolysin (Ply), protein D, PhtD, PhtDE and N19. It can be selected from the group. In a further embodiment, the carrier protein is CRM197. In one embodiment, CRM197 is used as the carrier protein. CRM197 protein is a non-toxic variant of diphtheria toxin, immunologically indistinguishable from diphtheria toxin. CRM197 is produced from a culture of Corynebacterium diphtheria strain C7 (β197) grown in media based on casamino acid and yeast extract. Typically, CRM197 is purified through a combination of ultrafiltration, ammonium sulfate precipitation and ion exchange chromatography. In another further embodiment, the carrier protein is tetanus toxoid (TT). The inventors of the present invention believe that the serotype 23F polysaccharide activated by reacting with a specific molar equivalent of periodate in sodium acetate buffer or activated in 1 to 550 mM sodium acetate buffer is particularly stable in conjugate formation with the carrier protein and free It was confirmed that the incidence of polysaccharides and free proteins can be reduced. In the case of free protein, less than 1% was hardly produced. In particular, such stable conjugate formation can be obtained by using CRM197 as a carrier protein.

The method of the present invention may include mixing the activated serotype 23F polysaccharide with the carrier protein after step (iii). The mixing step

(a) mixing any one sugar selected from sucrose, trehalose, raffinose, stachyose, mellegitose, dextran, mannitol, and lactitol with activated serotype 23F and a carrier protein;

(b) freeze-drying the activated serotype 23F and the carrier protein mixed with the sugar, respectively; And

(c) suspending the activated serotype 23F polysaccharide and carrier protein obtained in step (b) in a solvent.

Specifically, any one sugar selected from sucrose, trehalose, raffinose, stachyose, mellegitose, dextran, mannitol, and lactitol, and activated serotype 23F and carrier protein are first mixed, In the presence of the sugar, polysaccharides and carrier proteins may be lyophilized. Preferably, the sugar contains sucrose.

Then, it is suspended in the presence of a solvent, and the solvent may be a DMSO (dimethyl sulfoxide) solvent for the purposes of the present invention.

In step (v) of the method of the present invention, a mixture of activated serotype 23F polysaccharide and carrier protein may be mixed in a weight ratio of 1:0.1 to 5, and preferably, the weight ratio of activated serotype 23F polysaccharide: carrier protein is 1 May be :1.

After step (v), after the polysaccharide and the carrier form a conjugate, the remaining unreacted carbonyl groups may be present, which may be capped using a suitable capping agent. In one embodiment, such a capping agent may comprise sodium borohydride, for example, the product after step (v) is 15 minutes-15 hours, 15 minutes-45 minutes, 2-10 hours or 3- It can be reacted with sodium borohydride for 5 hours, about 30 minutes or about 4 hours. In a further embodiment, capping is achieved by mixing the product of step (v) with about 0.8 to 0.12 molar equivalents of sodium borohydride.

The present invention may also include an additional step of purifying the conjugate, and diafiltration of the unreacted serotype 23F polysaccharide and carrier protein with 100 kDa ultrafiltration.

In one embodiment, a method of preparing an immunogenic conjugate comprising a Streptococcus pneumoniae serotype 23F polysaccharide covalently bound to a carrier protein may have the following specific examples.

(1) The purified serotype 23F polysaccharide was diluted with 1mM ~ 550mM sodium acetate (NaOAc) (pH4.5);

(2) reacting serotype 23F polysaccharide with sodium periodate to produce activated serotype 23F polysaccharide;

(3) The activated serotype 23F polysaccharide was diafiltered with sodium acetate (pH 7.4) using 100 kDa MWCO ultrafiltration;

(4) mixing activated serotype 23F polysaccharide with sucrose;

(5) lyophilized activated serotype 23F and carrier protein, respectively;

(6) activated serotype 23F polysaccharide and carrier protein were resuspended in DMSO;

(7) Resuspended activated serotype 23F polysaccharide and carrier protein are mixed 1:1 and reacted with sodium cyanoborohydride to generate serotype 23F polysaccharide:carrier protein conjugate;

(8) unreacted aldehyde in the serotype 23F polysaccharide:carrier protein conjugate was capped with sodium borohydride;

(9) Unreacted serotype 23F polysaccharide and carrier protein were diafiltered through 100kDa ultrafiltration;

(10) It involves generating an immunogenic conjugate comprising a Streptococcus pneumoniae serotype 23F polysaccharide covalently linked to a carrier protein.

In another embodiment, it may be diluted with Deionized Water instead of sodium acetate of (1).

Another embodiment of the present invention provides a conjugated conjugate of Streptococcus pneumoniae serotype 23F and a carrier protein obtained by the method embodied by the present invention. It also provides an immunogenic composition comprising the conjugate.

Another embodiment of the present invention provides a conjugated conjugate of Streptococcus pneumoniae serotype 23F and a carrier protein obtained by the method embodied by the present invention. It also provides an immunogenic composition comprising the conjugate.

As used herein, an “immunogenic composition” contains an antigen, such as a bacterial capsular polysaccharide or polysaccharide-protein conjugate, that has the ability to elicit an immune response mediated from a host, such as a mammal to a body fluid or to a cell or both. Means composition. The immunogenic composition can protect the host from infection by bacteria, can reduce its severity, or can protect the host from death due to bacterial infection. Immunogenic compositions can also be used to generate antibodies to a subject.

In one embodiment, there is provided a conjugate of activated Streptococcus pneumoniae serotype 23F and CRM197 obtained by the above method, and an immunogenic composition comprising the same. In one embodiment, there is provided a conjugate of S. pneumoniae serotype 23F and CRM197 obtained by the above method of at least 300 kDa and an immunogenic composition comprising the same. In one embodiment, a conjugate of Streptococcus pneumoniae serotype 23F and CRM197 having a size of 250kDa to 400kDa obtained by the above method and an immunogenic composition comprising the same is provided.

The immunogenic composition of the present invention may contain a pharmaceutically acceptable additive and may contain a pharmaceutically acceptable excipient. Additives or excipients included in the immunogenic composition may include examples commonly used in the industry.

The present invention also provides for use as a vaccine or vaccine against Streptococcus pneumoniae serotype 23F comprising the immunogenic composition of the present invention. The vaccine may be administered through a vaccine administration route generally used for administration to a subject. It provides a use as a medicine for the treatment of Streptococcus pneumoniae serotype 23F infection comprising the immunogenic composition of the present invention.

The present invention can also prevent or treat pneumonia, meningitis, or bacteremia caused by Streptococcus pneumoniae serotype 23F by administering to an individual a conjugate in which Streptococcus pneumoniae serotype 23F is bound to a carrier protein.

Another embodiment of the present invention provides a method for activating a Streptococcus pneumoniae serotype 23F polysaccharide for the preparation of an immunogenic conjugate of a S. pneumoniae serotype 23F polysaccharide. The method is to dilute Streptococcus pneumoniae serotype 23F polysaccharide purified by a conventional method with sodium acetate (NaOAc) (pH4.5), preferably sodium acetate at a concentration of 1 mM to 550 mM, and react with sodium periodate. And activating it. Preferably, the sodium periodate may contain a molar equivalent of 0.07 to 0.23, preferably 0.16 molar equivalent.

When the inventors of the present invention obtained an activated polysaccharide that can be used in the conjugation process of the activated serotype 23F polysaccharide obtained using the method of the present invention, the degree of oxidation (Do), molecular weight, and yield were similar after activation, and as a result of conjugation, the polysaccharide/ It was confirmed that the properties of the conjugate were similar, such as protein ratio (PS/PR), free sugar (unconjugated polysaccharide, Free PS), size of the conjugate (MSD, MALLS), and yield.

In particular, the activated 23F polysaccharide obtained by diluting and oxidizing purified serotype 23F polysaccharide using KH ₂ PO ₄ (pH 7.4) is 100 mM water for injection (WFI) regardless of the amount of sodium periodate added. In phosphate, the activated serotype 23F polysaccharide molecular weight was 50 kDa or less, but in the case of the present invention, an activated 23F polysaccharide of at least 300 kDa or more could be obtained. In the case of the phosphate buffer, the effective concentration is limited to 10 mM, but sodium acetate (pH4.5) can obtain activated PS suitable for the purposes of the present invention in the range of 10 to 500 mM.

The present invention provides a vaccine of Streptococcus pneumoniae serotype 23F.

The conjugate of the present invention can induce immunity.

The present invention is to provide an optimal method for obtaining a size suitable for immunogenicity even if the polysaccharide size is reduced by treatment with an oxidizing agent.

The present invention provides a conjugate capable of preventing or treating diseases caused by Streptococcus pneumoniae serotype 23F.

Hereinafter, in order to describe the present invention in more detail, it will be described with reference to the following examples. However, the embodiments according to the present invention may be modified in various forms, and the scope of the present invention should not be construed as being limited to the embodiments described below. Embodiments of the present invention are provided by way of example to aid in a specific understanding of the present invention.

Example 1. Preparation of S. pneumoniae capsular polysaccharide

S. pneumoniae culture and purification of capsular polysaccharide were performed by methods known to those skilled in the art. S. pneumoniae serotype 23F can be obtained from various trustees and research institutes including ATCC (Strain: Spain 23F-1 [Sp264]). S. pneumoniae was identified as alpha hemolysis in capsular, non-motile, Gram-positive, lancet-shaped dicocci, and blood agar medium. The serotype was confirmed based on the Quellung test using a specific antisera (US Patent No. 5,847,112).

Preparation of cell bank

In order to increase the strain and remove components of animal origin, seed stocks were cultured for several generations (F1, F2 and F3 generations). Two additional generations of protozoa were cultured. Additional first generations were cultured from F3 vials, and subsequent generations were cultured from additional first generation vials. As a cryopreservative, the seed vial was stored frozen together with synthetic glycerol (-70° C. or less). For cell bank preparation, all cultures were grown in soy-based medium. Before freezing, the cells were concentrated by centrifugation, the used medium was removed, and the cell pellet was resuspended in a fresh medium containing a cryopreservative (eg, synthetic glycerol).

inoculation

A culture derived from the cell bank for production was used to inoculate a seed bottle containing a soybean-based medium. A seed bottle was used to inoculate a seed fermentor containing a soy-based medium.

Seed culture

Fermentation was carried out in a seed fermenter while controlling temperature and pH. After reaching the target absorbance, it was inoculated into a production fermentor containing a soy-based medium.

Production culture

Production culture is the final step in fermentation. The temperature, pH and stirring speed were adjusted.

Inactivation

After growth was stopped, the pH was allowed to decrease, and then an inactivating agent was added to terminate the fermentation. After inactivation, the contents of the fermentor were cooled.

refine

The culture broth was centrifuged and filtered to remove bacterial cell debris. Impurities were removed and the capsular polysaccharide was purified using several concentration/diafiltration operations, precipitation/elution and multi-layer filtration.

Example 2. Preparation of conjugate of S. pneumoniae capsular polysaccharide and CRM197

The serotype 23F polysaccharide was activated and conjugated to CRM197. The activation process involves reducing the size of the capsular polysaccharide to a target molecular weight, chemically activating it, and buffer exchange through ultrafiltration. Purified CRM197 is conjugated with activated polysaccharide, and the conjugate is purified using ultrafiltration and finally filtered through a 0.22 μm filter. Process parameters such as pH, temperature, concentration and time are as described below.

(1) activation process

Step 1

Each serotype polysaccharide was diluted with water for injection and sodium acetate so that the final concentration range was 1.0 to 2.0 mg/mL. The final concentration of the sodium acetate buffer was 0.5mM, 10mM, 120mM, 150mM, 180mM, 200mM, 300mM and 500mM.

Step 2: periodate reaction

Sodium periodate required for serotype 23F polysaccharide activation was 0.16 molar equivalent to the polysaccharide content. While thoroughly mixing, the oxidation reaction proceeded for 16 to 20 hours at room temperature (21-25°C).

Step 3: Ultrafiltration

The activated serotype 23F polysaccharide was concentrated with a 100 kDa MWCO ultrafiltration filter and diafiltered with 0.01M sodium acetate buffer (pH 4.5). The permeate was discarded and the residue was filtered through a 0.22 μm filter.

Activation result by serotype 23F polysaccharide buffer concentration in the activation process

Oxidation	DW	0.5mM NaOAc	10mM NaOAc	120mM NaOAc	150mM NaOAc	180mM NaOAc	200mM NaOAc	300mM NaOAc	500mM NaOAc
Do (degree of oxidation)	7.4	6.4	8.8	8.8	8.2	8.3	8.8	8.2	7.9
M.W. (kDa)	178	292	351	320	316	315	316	321	316
Yield (%)	67.3	53.7	49.3	51.5	56.5	53.9	56.2	60.1	60.0

As can be seen in Table 1, as a result of the activation using an Acetate buffer, a similar Do value of 7.5 or higher and a similar molecular weight of 300 kDa or higher were obtained using NaOAc at a concentration of 10 mM -500 mM. Through these results, the concentration of the NaOAc buffer solution capable of obtaining a polysaccharide of a serotype 23F having a uniform size using NaOAc at a concentration of 10 mM-500 mM was confirmed.

Step 4: freeze drying

To the activated serotype 23F polysaccharide was added a specific amount of sucrose calculated to reach a 5%±3% sucrose concentration. The concentrated saccharide and CRM197 carrier protein were each filled into a glass bottle and freeze-dried.

(2) bonding process

Step 1: dissolution

Lyophilized activated sugar serotype 23F and lyophilized CRM197 carrier protein were equilibrated at room temperature and resuspended in DMSO.

Step 2: conjugation reaction

Activated saccharide and CRM197 carrier protein were mixed in a ratio ranging from 0.8 to 1.25 g saccharide/g CRM197. The conjugation reaction was initiated by adding a sodium cyanoborohydride solution (100 mg/mL) in a ratio of 0.8 to 1.2 molar equivalents of sodium cyanoborohydride to 1 mole of activated saccharide. Water for injection at a target concentration of 1% (v/v) was added to the reaction mixture and the mixture was incubated at 23° C.±2° C. for 24 hours. 100 mg/mL of sodium borohydride solution (typically 1.8 to 2.2 molar equivalents of sodium borohydride per mole of activated sugar) and water for injection (target concentration 5% v/v) were added to the reaction and the mixture was added at 23°C Incubated at 2° C. for 4.5 hours. Through this process, any unreacted aldehyde present in the sugar was reduced. Then, the reaction mixture was diluted with 0.9% sodium chloride, and the diluted conjugate mixture was filtered through a 0.45 μm filter.

Step 3: Ultrafiltration

The diluted conjugation mixture was concentrated and diafiltered on a 100 kDa MWCO ultrafiltration filter using at least 20 volumes of 0.9% sodium chloride or buffer. The permeate was discarded.

Step 4: sterile filtration

The residual liquid after diafiltration of 100 kDa MWCO was filtered through a 0.22 μm filter. The filtered product 23F-CRM197 conjugate was subjected to control (sugar content, free protein, free sugar, residual cyanide and residual DMSO) during the manufacturing process. The filtered residue was subjected to in-process control to determine whether additional concentration, diafiltration and/or dilution were required. If necessary, the filtered conjugate was diluted with 0.9% sodium chloride to a final concentration of less than 0.55 g/L. At this stage, glass tests were conducted for sugar content, protein content and sugar:protein ratio. The conjugate was filtered (0.22 μm) and subjected to a free test (appearance, free protein, free sugar, endotoxin, molecular size determination, residual cyanide, residual DMSO, sugar identity and CRM197 identity). The final conjugate concentrate was stored refrigerated at 2 to 8°C.

Conjugation results of activated polysaccharides by activation buffer

Sodium acetate Conc. (mM)	Activated PS M.W. (kDa)	Scale (mg)	Ratio (PS/PR)	Free PS (%)	MSD (%)	MALLS (kDa)	Conjugation yield (%)
0 (DW)	178	196.0	0.75	11.7	32	721	56.6
0.5	292	192.4	0.85	13.8	40	753	51.7
10	351	162.1	0.72	1.6	47	953	50.7
120	320	170.3	0.72	0.2	44	900	49.5
150	316	183.6	0.74	0.7	46	907	53.1
180	315	178.6	0.71	1.5	45	897	52.4
200	316	185.9	0.71	0.6	42	825	47.4
300	321	211.6	0.76	3.0	51	1106	54.4
500	316	214.5	0.66	0.0	58	1490	37.0

As can be seen in Table 2, when using DW and when using 5 mM sodium acetate, activated polysaccharides of 300 kDa or less were obtained, and the immunogenicity of the conjugates prepared using them was also found to be poor (immunogenicity The result is Example 4).

Example 3. Formulation of pneumococcal conjugate vaccine

The required amount of the final stock solution was calculated based on the batch volume and the serotype 23F conjugate saccharide concentration. The required amount of 0.85% sodium chloride (physiological saline), polysorbate 80, and succinate buffer were added to the previously labeled formulation container, followed by the addition of the conjugate concentrate. Mix thoroughly and filter through 0.22 μm filter. After the addition of aluminum phosphate, the formulated final liquid was slowly mixed. The formulated product was stored at 2-8°C. The resulting vaccine composition contained 2 μg of saccharide, about 2.5 μg of CRM197 carrier protein in a total of 0.5 mL; 0.125 mg elemental aluminum (0.5 mg aluminum phosphate) adjuvant; About 4.25 mg sodium chloride; About 295 μg of succinate buffer; And about 100 μg of polysorbate 80.

Example 4. Evaluation of immunogenicity of 23F conjugate

Serum was analyzed by OPA assay to evaluate the function of the antibody. The same amount of serum was taken for each individual and diluted 10-fold. Using opsonization buffer, 10uL of S. pneumoniae diluted appropriately diluted with 20 uL of serial dilution (step dilution) serum was mixed and reacted at room temperature for 30 minutes. To the serum-S. pneumoniae reaction solution, a mixture of previously differentiated HL-60 cells and complement was added and reacted in a CO ₂ incubator (37° C.) for 45 minutes. The temperature was lowered to stop phagocytosis, and 10 μL of the reaction solution was plated on dried THY agar medium for 30 to 60 minutes in advance. After smearing, when the reaction solution was completely absorbed in the THY agar medium, the THY agar medium containing TTC was additionally overlaid. Incubated for 12 to 18 hours in a CO ₂ incubator (37° C.) and the number of clusters was counted. OPA titer was expressed as the dilution factor at which 50% killing was observed. The results are shown in Table 3 below.

2 weeks after the second inoculation, titer of 23F conjugate by concentration of buffer solution in activation process

Group	Buffer concentration in the activation process	Geometric mean titer (95% CI * )
One	DW	1002 (462.6-2171.9)
2	0.5mM NaOAc	340 (122.4-943.5)
3	10mM NaOAc	3013 (1596.6-5686.3) +
4	150mM NaOAc	2976 (857.9-10321.0) +

^* CI: confidence interval

Significance of groups 3 and 4 to groups 1 and 2: ⁺ ( P <0.05)

Conjugates (groups 3 and 4) prepared by activating polysaccharides of serotype 23F at a buffer concentration of 10 mM or 150 mM showed significantly higher MOPA titers than conjugates (groups 1 and 2) activated at a buffer concentration of DW or 0.5 mM. .

The present invention can be used as a pneumococcal vaccine. The present invention can be provided as a drug for preventing pneumococcal infection.

Claims (16)

1. (i) mixing the purified S. pneumoniae serotype 23F capsular polysaccharide with sodium acetate (NaOAc) buffer to obtain a mixture;

   (ii) reacting the mixture with an oxidizing agent to produce an activated serotype 23F polysaccharide;

   (iii) filtering the activated serotype 23F polysaccharide with sodium acetate to select an activated serotype 23F polysaccharide having at least 300 kDa;

   (iv) mixing the activated serotype 23F polysaccharide with the carrier protein; And

   (v) adding a reducing agent to a mixture of activated serotype 23F polysaccharide and carrier protein for conjugation to form a conjugate of activated serotype 23F polysaccharide and carrier protein,

   A method of preparing a conjugate of S. pneumoniae serotype 23F and a carrier protein.
2. The method of claim 1, wherein the sodium acetate (NaOAc) in the step (i) has a concentration of 1 to 550 mM at pH 4.5, a conjugate of S. pneumoniae serotype 23F and a carrier protein. How to manufacture.
3. The method of claim 2, wherein the sodium acetate (NaOAc) in step (i) has a concentration of 10 to 500 mM at pH 4.5, to prepare a conjugate of S. pneumoniae serotype 23F and a carrier protein. How to.
4. The method of claim 1, wherein the oxidizing agent in step (ii) is sodium periodide having 0.07 to 0.23 molar equivalents, Streptococcus pneumoniae serotype 23F and a conjugate of a carrier protein.
5. The method of claim 4, wherein the oxidizing agent in step (ii) is sodium periodate having a molar equivalent of 0.16, S. pneumoniae serotype 23F and a conjugate of a carrier protein.
6. The method of claim 1, wherein the filtration in step (iii) is performed by diafiltration with a sodium acetate buffer solution having a pH of 4.5 using 100kDa MWCO ultrafiltration. S. pneumoniae serotype 23F and A method of preparing a conjugate of a carrier protein.
7. According to claim 1, After step (iii), any one or more sugars selected from sucrose, trehalose, raffinose, stachyose, mellegitose, dextran, mannitol, and lactitol and activated serotypes A method of preparing a conjugate of a S. pneumoniae serotype 23F and a carrier protein, characterized in that after mixing 23F and a carrier protein and freeze-drying the same.
8. The method of claim 1, wherein (iv) mixing the activated serotype 23F polysaccharide with a carrier protein

   (a) mixing any one or more sugars selected from sucrose, trehalose, raffinose, stachyose, mellegitose, dextran, mannitol, and lactitol with activated serotype 23F and carrier protein;

   (b) freeze-drying the activated serotype 23F and the carrier protein mixed with the sugar, respectively; And

   (c) comprising the step of suspending the activated serotype 23F polysaccharide and carrier protein obtained in step (b) in a solvent,

   A method of preparing a conjugate of S. pneumoniae serotype 23F and a carrier protein.
9. The method of claim 8, wherein the sugar in the step (a) is sucrose. S. pneumoniae serotype 23F and a carrier protein conjugate.
10. The method of claim 8, wherein the solvent in step (c) is DMSO. The method of preparing a conjugate of S. pneumoniae serotype 23F and a carrier protein.
11. The method of claim 1, wherein in step (v), a mixture of activated serotype 23F polysaccharide and carrier protein is mixed in a weight ratio of 1:0.1 to 5, S. pneumoniae serotype. A method of preparing a conjugate of 23F and a carrier protein.
12. The method of claim 1, wherein the reducing agent in step (v) comprises sodium cyanoborohydride. S. pneumoniae serotype 23F and a conjugate of a carrier protein.
13. An immunogenic composition comprising a conjugate in which a Streptococcus pneumoniae serotype 23F polysaccharide is bound to a carrier protein obtained by the method of any one of claims 1 to 12.
14. 14. The immunogenic composition of claim 13, wherein the serotype 23F polysaccharide in the conjugate has a size of 250 kDa to 400 kDa.
15. Pneumonia caused by Streptococcus pneumoniae serotype 23F by administering to an individual a conjugate in which Streptococcus pneumoniae serotype 23F obtained by the method of any one of claims 1 to 12 is bound to a carrier protein. How to prevent or treat.
16. A conjugate in which Streptococcus pneumoniae serotype 23F obtained by the method of any one of claims 1 to 12 is bound to a carrier protein as a vaccine or therapeutic drug against Streptococcus pneumoniae serotype 23F Usage.