AU2026200210A1

AU2026200210A1 - Streptococcus pneumoniae capsular polysaccharides and immunogenic conjugate thereof

Info

Publication number: AU2026200210A1
Application number: AU2026200210A
Authority: AU
Inventors: Kyung-Jun An; Dong Soo Ham; Hun Kim; Sung-Hyun Kim; Jin-Hwan Shin
Original assignee: SK Bioscience Co Ltd
Current assignee: SK Bioscience Co Ltd
Priority date: 2018-04-18
Filing date: 2026-01-14
Publication date: 2026-01-29
Also published as: US11951162B2; KR20250006789A; EP3782642A4; AU2019256218B2; US12440548B2; US20240207384A1; TW202011985A; BR112020021296A2; AU2019256218A1; EP3782642A1; WO2019203599A1; NZ770014A; US20260007732A1; KR20190121713A; JP2021522213A; US20210154287A1; CN111989114A; CA3096358A1

Abstract

【ABSTRACT】 The present invention provides an immunogenic composition comprising a Streptococcus pneumoniae polysaccharide-protein conjugate, comprising a capsular polysaccharide derived from one or more selected from the group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F, derived from Streptococcus pneumoniae; and one or 2 or more of carrier proteins conjugated to the respective capsular polysaccharide, and method of preparation thereof. Through one example of the present invention, an immunogenic composition for preventing or treating pneumococcal infection can be provided. 20 26 20 02 10 14 J an 2 02 6 2 0 2 6 2 0 0 2 1 0 1 4 J a n 2 0 2 6

Description

【DESCRIPTION】【INVENTION TITLE】 STREPTOCOCCUS PNEUMONIAE CAPSULAR STREPTOCOCCUS PNEUMONIAE CAPSULARPOLYSACCHARIDE POLYSACCHARIDE AND AND

IMMUNOGENIC CONJUGATETHEREOF THEREOF 2026200210

IMMUNOGENIC CONJUGATE

5 5 【TECHNICAL FIELD】

The present application claims the benefit of priority based on Korean Patent

Application No. 10-2018-0045245 filed on April 18, 2018, Korean Patent Application No. 10-

2018-0045246 filed on April 18, 2018, Korean Patent Application No. 10-2018-0045247 filed

on April 18, 2018, and Korean Patent Application No. 10-2018-0045248 filed on April 18,

10 10 2018, and the entire contents disclosed in the description and drawings of the applications

are incorporated herein by reference.

The present invention relates to an immunogenic composition and a vaccine of

Streptococcus pneumoniae, and more specifically, the present invention relates to an

immunogenic composition and a vaccine which comprise a capsular polysaccharide-carrier

15 15 protein conjugate of Streptococcus pneumoniae.

【BACKRGROUND ART】

Streptococcus pneumoniae is a major causative bacterium of pneumonia. In

addition, it causes invasive diseases such as septicemia, bacteremia, meningitis and the like.

According to National Statistical Office [2014 causes of death statistics], the death rate by 2026200210

5 5 pneumonia in 2014 was 23.7 people per hundred thousand people, and it increased 2.8

times compared with 2015, and the death rate by pneumonia has been continuously

increasing. Furthermore, according to 2012 WHO, in 2008, 476,000 infants under the age

of 5 who were HIV-negative died from Streptococcus pneumoniae infection globally,

accounting for 5% of causes of death of infants under the age of 5. Pneumococcus is

10 classified into more than about 90 serotypes, depending on structural and immunological

characteristics of the capsular polysaccharide, which is a major virulence factor surrounding

its outside (cell membrane).

To prevent diseases caused by Streptococcus pneumoniae, a 14-valent

polysaccharide vaccine was developed by Dr. Robert Austrian in 1977, and after that, it was

15 developed into a 23-valent polysaccharide vaccine. It has been demonstrated that a

multivalent pneumococcus polysaccharide vaccine is useful for preventing Streptococcus

pneumoniae diseases in the elderly and high-risk patients. However, infants and children

do not have an immune response to most of Streptococcus pneumoniae polysaccharides,

because of the T-cell independent immune response. Thus, a conjugate vaccine of a 2026200210

5 5 Streptococcus pneumoniae capsular polysaccharide and a carrier protein, which can cause

a T-cell dependent response, has been developed.

The 7-valent Streptococcus pneumoniae conjugate vaccine (Prevnar®) comprises

capsular polysaccharides derived from 7 most frequent serotypes 4, 6B, 9V, 14, 18C, 19F

and 23F. It has been demonstrated that it is highly immunogenic and effective against

10 10 invasive pneumococcal diseases and otitis media in infants and children, since it was

primarily approved in America in 2000. After that, Prevnar 13®, the 13-valent conjugate

vaccine in which 6 serotypes 1, 3, 5, 6A, 7F, 19A were added, and Synflorix, the 10-valent

conjugate vaccine in which 3 serotypes 1, 5, 7F were added, have been developed in order,

and the number of invasive diseases caused by Streptococcus pneumoniae was further

15 15 reduced. However, as the serotype change due to introduction of Prevnar, Prevnar 13 and

3

Synflorix appeared and the number of diseases caused by serotypes comprised in a

vaccine was generally reduced, the importance of non-vaccine serotypes which were of

relatively low importance is rather emphasized.

In particular, the increase of incidence of invasive pneumococcal diseases caused 2026200210

5 5 by Streptococcus pneumoniae serotype 20 occurred in North America and Brazil (See, e.g.,

[Kendall B. et al., Vaccine. 34:474-478, 2016], [Yildirim I. et al., Pediatr Infect Dis J. 31(10):

1016-1021, 2012] or [Caierγo J. et al., PLoS ONE 9(10): e111129, 2014]). In addition, in the

CASPER research conducted in Canada, the increase of incidence of serotypes 8 and 12F

in addition to serotype 19A was observed (Sα-Leγo A. et al., J Clin Microbiol., 49(4): 1369-

10 10 75, 2011). A recently announced research has reported that diseases caused by non-vaccine

serotypes are increased after introduction of Prevnar 13 in Norway and Israel, and has

exemplified 23A, 23B, 12F, 15A/15B/15C, 31, 33F, 7C, and 8 as such non-vaccine serotypes

(Martin J., Pediatr Infect Dis J., 33(11):e286-90, 2014).

Despite the steady increase in incidence of pneumococcal diseases caused by

15 15 Streptococcus pneumoniae serotypes 2, 9N, 17F, and/or 20, there is a lack of research that

4

can effectively prevent or treat infection by serotypes.

Thus, there is an increasing need for an immunogenic conjugate and an

immunogenic composition against non-vaccine serotypes comprising serotypes which are

comprised in multivalent polysaccharide vaccines but are not comprised in conjugate 2026200210

5 5 vaccines to provide a broader protection range.

【DISCLOSURE】【TECHNICAL PROBLEM】

Accordingly, a problem to be solved by the present invention is to provide a

10 10 multivalent vaccine capable of providing a wide range of protection.

In addition, it is also intended to provide an immunogenic composition comprising

a new serotype that has not previously been comprised in a conjugate vaccine.

In addition, a problem to be solved by the present invention is to provide a

pneumococcal conjugate vaccine having excellent antibody titer.

15 15 【TECHNICAL SOLUTION】

5

One embodiment of the present invention provides an immunogenic composition

comprising a Streptococcus pneumoniae polysaccharide-protein conjugate, comprising a

capsular polysaccharide derived from one or more selected from serotypes 1, 2, 3, 4, 5, 6A,

6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F, derived 2026200210

5 from Streptococcus pneumoniae; and a carrier protein conjugated to the each capsular

polysaccharide.

One embodiment provides an immunogenic composition comprising a

Streptococcus pneumoniae polysaccharide-protein conjugate, in which when the

immunogenic composition comprises a polysaccharide derived from serotype 2, the

10 polysaccharide is activated and binds to the carrier protein at a molecular weight of 100 to

400 kDa to form a conjugate, or

when the immunogenic composition comprises a polysaccharide derived from

serotype 17F, the polysaccharide is activated and binds to the carrier protein at a molecular

weight of 400 to 900 kDa to form a conjugate, or

15 15 when the immunogenic composition comprises a polysaccharide derived from

6

serotype 20, the polysaccharide is activated and binds to the carrier protein at a molecular

weight of 400 to 800 kDa to form a conjugate.

One embodiment of the present invention provides an immunogenic composition

comprising a Streptococcus pneumoniae polysaccharide-protein conjugate, in which when 2026200210

5 5 the immunogenic composition comprises a polysaccharide derived from serotype 2, an

immunogenic conjugate comprising a polysaccharide derived from serotype 2 has a

molecular weight of 1,000 to 16,000 kDa, or

when the immunogenic composition comprises a polysaccharide derived from

serotype 17F, an immunogenic conjugate comprising a polysaccharide derived from

10 10 serotype 17F has a molecular weight of 300 to 4,500 kDa, or

when the immunogenic composition comprises a polysaccharide derived from

serotype 20, an immunogenic conjugate comprising a polysaccharide derived from

serotype 20 has a molecular weight of 1,000 to 4,000 kDa.

One embodiment of the present invention provides an immunogenic composition

15 comprising a Streptococcus pneumoniae polysaccharide-protein conjugate, characterized

7

by that the carrier protein is TT (Tetanus toxoid) or CRM197, and preferably, an

immunogenic composition comprising only one serotype may comprises CRM197 as the

carrier protein.

One embodiment of the present invention provides an immunogenic composition 2026200210

5 5 comprising a Streptococcus pneumoniae polysaccharide-protein conjugate, in which when

the immunogenic composition comprises a polysaccharide derived from serotype 2, the

ratio of the serotype 2 capsular polysaccharide to the carrier protein in the immunogenic

conjugate (polysaccharide/protein, W/W) is 0.5 to 2.0, or

when the immunogenic composition comprises a polysaccharide derived from

10 serotype 17F, the ratio of the serotype 17F capsular polysaccharide to the carrier protein in

the immunogenic conjugate (polysaccharide/protein, W/W) is 0.5 to 18, or

when the immunogenic composition comprises a polysaccharide derived from

serotype 20, the ratio of the serotype 20 capsular polysaccharide to the carrier protein in

the immunogenic conjugate (polysaccharide/protein, W/W) is 1 to 5.

15 15 One embodiment of the present invention provides an immunogenic composition

8

comprising a Streptococcus pneumoniae polysaccharide-protein conjugate, in which 20 to

60% of the total molecular weight is present within 0.3 Kd in a CL-4B column, in case of

the immunogenic conjugate comprising a polysaccharide derived from serotype 2, or

15 to 60% of the total molecular weight is present within 0.3 Kd in a CL-4B column, 2026200210

5 5 in case of the immunogenic conjugate comprising a polysaccharide derived from serotype

17F, or

70 to 90% of the total molecular weight is present within 0.3 Kd in a CL-4B column,

in case of the immunogenic conjugate comprising a polysaccharide derived from serotype

20. 20.

10 10 One embodiment of the present invention provides an immunogenic composition

comprising a Streptococcus pneumoniae polysaccharide-protein conjugate, in which

the degree of oxidation of the polysaccharide conjugated to the conjugate is 2 to

18, in case of the immunogenic conjugate comprising a polysaccharide derived from

serotype 2, or

15 the degree of oxidation of the polysaccharide conjugated to the conjugate is 1 to

9

22, in case of the immunogenic conjugate comprising a polysaccharide derived from

serotype 17F, or

the degree of oxidation of the polysaccharide conjugated to the conjugate is 4 to

16, in case of the immunogenic conjugate comprising a polysaccharide derived from 2026200210

5 5 serotype 20.

One embodiment of the present invention provides an immunogenic composition

comprising a Streptococcus pneumoniae polysaccharide-protein conjugate, in which the

immunogenic composition is that polysaccharides derived from 15 serotypes different each

other are conjugated to respective carrier proteins, and

10 the serotypes are 1, 2, 3, 4, 5, 6A, 6B, 7F, 9N, 9V, 14, 18C, 19A, 19F, and 23F, and

the serotypes are conjugated to CRM 197.

One embodiment of the present invention provides an immunogenic composition

immunogenic composition is that polysaccharides derived from 23 serotypes different

15 15 each other are conjugated to respective carrier proteins, and

10

the serotypes are 1, 2, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F,

20, 22F, 23F, and 33F, and

among the serotypes, capsular polysaccharides derived from serotypes 3 and 5 are

conjugated to carrier protein TT and capsular polysaccharides derived from serotypes 1, 2, 2026200210

5 5 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F are

conjugated to carrier protein CRM197.

One embodiment of the present invention provides an immunogenic composition

immunogenic composition is that polysaccharides derived from 24 serotypes different

10 10 each other are conjugated to respective carrier proteins, and

the serotypes are 2, 3, 4, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A,

19F, 20, 22F, 23F, and 33F, and

capsular polysaccharides derived from serotypes 1 and 5 are conjugated to carrier

protein TT and capsular polysaccharides derived from serotypes 2, 3, 4, 6A, 6B, 7F, 8, 9N,

15 15 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F are conjugated to carrier

11

protein CRM197.

One embodiment of the present invention provides an immunogenic composition

comprising a Streptococcus pneumoniae polysaccharide-protein conjugate, which

comprises a physiologically acceptable vehicle. 2026200210

5 5 One embodiment of the present invention provides an immunogenic composition

immunogenic composition is a vaccine.

One embodiment of the present invention provides a preparation method of an

immunogenic composition comprising a Streptococcus pneumoniae polysaccharide-

10 10 protein conjugate comprising

(a) a step of fermenting and dissolving a bacterial cell which produces a capsular

polysaccharide derived from one or more serotypes selected from the group consisting of

Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14,

15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F;

15 15 (b) a step of purifying a capsular polysaccharide derived from Streptococcus

pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A,

19F, 20, 22F, 23F, and 33F in the dissolved cell;

(c) a step of reacting the purified polysaccharide with an oxidizing agent to activate

it; and 2026200210

5 5 (d) a step of combining the activated polysaccharide with a carrier protein to form

a Streptococcus pneumoniae polysaccharide-protein conjugate bound to the carrier

protein.

In one embodiment of the present invention, the preparation method may further

comprise a step of hydrolyzing the purified Streptococcus pneumoniae capsular

10 10 polysaccharide to size it, before the (c) step, in case of the capsular polysaccharides derived

from serotypes 2 and 17F.

One embodiment of the present invention provides a preparation method of an

immunogenic composition comprising a Streptococcus pneumoniae polysaccharide-

protein conjugate, characterized by that the combined carrier protein of the (d) step forms

15 a conjugate with the polysaccharide activated by reacting with one or more reducing

13

agents selected from the group consisting of cyanoborohydride, borane-pyridine and

borohydride exchange resin.

One embodiment of the present invention provides a preparation method of an

immunogenic composition comprising a Streptococcus pneumoniae polysaccharide- 2026200210

5 5 protein conjugate, characterized by that the (c) step is reacting 0.01 ~ 0.22 ㎍ of periodate

per 1 ㎍ polysaccharide at a temperature of 20 to 25 ℃ for 15 to 20 hours.

One embodiment of the present invention provides a Streptococcus pneumoniae

polysaccharide-protein conjugate for preventing or treating Streptococcus pneumoniae

infection, obtained by the method.

10 10 One embodiment of the present invention provides a method for preventing or

treating infection of Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N,

9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and/or 33F in a subject,

by administering an effective dose of an immunogenic composition comprising a

Streptococcus pneumoniae polysaccharide-protein conjugate, comprising

15 15 a capsular polysaccharide derived from one or more selected from serotypes 1, 2,

3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F,

derived from Streptococcus pneumoniae; and

one or 2 or more of carrier proteins conjugated to the respective capsular

polysaccharide, into a subject. The and/or means ‘and’ or ‘or’. 2026200210

5 5 The method may prevent or treat infection against one or more serotypes selected

from the group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14,

15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F, or it may be one or more, and it may prevent

infection against 15 serotypes, or it may prevent infection against 23 serotypes, or it may

prevent infection against 24 serotypes.

10 10 One example of the present invention provides a use for prevention or treatment

of pneumococcal infection of a Streptococcus pneumoniae polysaccharide-protein

conjugate, comprising

a capsular polysaccharide derived from one or more selected from serotypes 1, 2,

15 15 derived from Streptococcus pneumoniae; and

15

one or 2 or more of carrier proteins conjugated to the respective capsular

polysaccharide.

One example of the present invention provides an immunogenic conjugate of

Streptococcus pneumoniae serotype 2, comprising a capsular polysaccharide of serotype 2026200210

5 5 2, derived from Streptococcus pneumoniae; and a carrier protein bound to the capsular

polysaccharide.

The polysaccharide of serotype 2 may be activated and bind to the carrier protein

at a molecular weight of 100 to 400 kDa to form a conjugate.

In one example of the present invention, the immunogenic conjugate may have a

10 molecular weight of 1,000 to 16,000 kDa, and for example, the carrier protein may be

CRM197.

In one example of the present invention, the ratio of serotype 2 capsular

polysaccharide to the carrier protein in the immunogenic conjugate (W/W) may be 0.5 to

2.0. 2.0.

15 15 20 to 60% of the immunogenic conjugate of serotype 2 may be present within 0.3

16

Kd in a CL-4B column. Another example provides an immunogenic conjugate of

Streptococcus pneumoniae serotype 2 having a degree of oxidation of 2 to 18.

When the polysaccharide of Streptococcus pneumoniae serotype 2 is oxidized by

adding 0.02 to 0.12 ㎍ of periodate per 1 ㎍ of the sugar content and is conjugated to a 2026200210

5 5 protein, the molecular weight of the conjugate may be 1,000 ~ 16,000 kDa, and the

distribution of the molecular weight may be 20 ~ 60% (0.3kd or less), and the ratio of

polysaccharide/protein may be 0.5 to 2.0.

One example of the present invention may provide an immunogenic composition

comprising the immunogenic conjugate and a physiologically acceptable vehicle.

10 10 Other example of the present inventio may provide a vaccine comprising the

immunogenic composition.

Other example of the present invention may provide a preparation method of an

immunogenic conjugate of Streptococcus pneumoniae serotype 2, comprising

(a) a step of fermenting and dissolving a bacterial cell producing a Streptococcus

15 15 pneumoniae serotype 2 capsular polysaccharide;

17

(b) a step of purifying the Streptococcus pneumoniae serotype 2 capsular

polysaccharide in the dissolved cell;

(c) a step of hydrolyzing the purified Streptococcus pneumoniae serotype 2

capsular polysaccharide to size the polysaccharide; 2026200210

5 5 (d) a step of reacting the sized polysaccharide of the (c) step to activate the

polysaccharide; and

(e) a step of combining the activated polysaccharide with a carrier protein to form

a conjugate of Streptococcus pneumoniae serotype 2 capsular polysaccharide bound to

the carrier protein.

10 The carrier protein combined of the (e) step may form a conjugate with the

activated polysaccharide by reacting it with a reducing agent.

The (d) step may comprise a process of reacting 0.02 ~ 0.12 ㎍ of periodate per 1

㎍ polysaccharide at a temperature of 20 to 25 ℃ for 15 to 20 hours.

The activated polysaccharide to be combined with the carrier protein of the (e)

15 15 step may have a molecular weight of 100 to 400 kDa.

18

A preparation method of an immunogenic conjugate of Streptococcus

pneumoniae serotype 2, in which the carrier protein is CRM197, may be provided.

The immunogenic conjugate may have a molecular weight of 1,000 to 16,000 kDa.

In one example, the initial input ratio of the activated serotype 2 capsular 2026200210

5 5 polysaccharide versus the carrier protein (carrier protein:polysaccharide) may be 0.5 to 2:1.

Other example may provide a preparation method of an immunogenic conjugate

of Streptococcus pneumoniae serotype 2, in which at least 20 to 60% of the immunogenic

conjugate is present within 0.3 Kd in a CL-4B column.

One example of the present invention may provide an immunogenic conjugate

10 10 obtained by the method.

One example of the present invention may provide an immunogenic composition

comprising an immunogenic conjugate obtained by the method and a physiologically

acceptable vehicle.

Other example may provide a vaccine comprising the immunogenic composition.

15 15 The present invention provides an immunogenic conjugate of Streptococcus

19

pneumoniae serotype 9N, comprising a capsular polysaccharide of serotype 9N, derived

from Streptococcus pneumoniae; and a carrier protein bound to the capsular

polysaccharide.

In one example of the present invention, the polysaccharide of serotype 9N may 2026200210

5 5 be activated and bind to the carrier protein at a molecular weight of 200 to 700 kDa to

form a conjugate.

The immunogenic conjugate may have a molecular weight of 500 to 4,000 kDa

and the carrier protein may be CRM197.

The ratio of the serotype 9N capsular polysaccharide to the carrier protein in the

10 10 immunogenic conjugate (W/W) is 0.1 to 5, and preferably, it may be 0.5 ~ 2.5.

In one example, 15 to 60% of the immunogenic conjugate may be present within

0.3 Kd in a CL-4B column.

In one example, the conjugate may have a degree of oxidation of 2 to 19.

In one example of the present invention, when the polysaccharide of Streptococcus

15 15 pneumoniae serotype 9N is oxidized by adding 0.02 ~ 0.19 ㎍ of periodate per 1 ㎍ of

20

the sugar content and is conjugated with a protein, the molecular weight of the conjugate

may be 500 ~ 4,000 kDa, and the distribution of the molecular weight may be 15 ~ 60%

(0.3kd or less), and the polysaccharide/protein ratio may be 0.5 ~ 2.5.

One example of the present invention may provide an immunogenic composition 2026200210

5 5 comprising an immunogenic conjugate and a physiologically acceptable vehicle.

Other example of the present invention may provide a vaccine comprising the

immunogenic composition.

Other example of the present invention may provide a preparation method of an

immunogenic conjugate of Streptococcus pneumoniae serotype 9N, comprising

10 10 (a) a step of fermenting and dissolving a bacterial cell producing a Streptococcus

pneumoniae serotype 9N capsular polysaccharide;

(b) a step of purifying the Streptococcus pneumoniae serotype 9N capsular

polysaccharide in the dissolved cell;

(c) a step of reacting the polysaccharide with an oxidizing agent to activate it; and

15 15 (d) a step of combining the activated polysaccharide with a carrier protein to form

21

a conjugate of Streptococcus pneumoniae serotype 9N capsular polysaccharide bound to

the carrier protein.

A preparation method of an immunogenic conjugate of Streptococcus

pneumoniae serotype 9N, characterized by that the combined carrier protein of the (d) 2026200210

5 5 step is reacted with a reducing agent to form a conjugate with the activated polysaccharide,

may be provided.

The (c) step may comprise a process of reacting 0.02 ~ 0.19㎍ of periodate per 1

㎍ of polysaccharide at a temperature of 20 to 25 ℃ for 15 to 20 hours.

The polysaccharide reacting with the oxidizing agent of the (c) step may have a

10 10 molecular weight of 400 to 900 kDa.

The activated polysaccharide to be combined with the carrier protein of the (d)

step may have a molecular weight of 200-700 kDa.

The immunogenic conjugate may have a molecular weight of 500 to 4,000 kDa.

The initial input ratio of the activated serotype 9N capsular polysaccharide versus

15 15 the carrier protein (carrier protein:polysaccharide) may be 0.5 to 2.5:1.

22

In one example, at least 15 to 60% of the immunogenic conjugate may be present

within 0.3 within 0.3 Kd Kd in in aa CL-4B column. CL-4B column.

One example of the present invention may provide an immunogenic conjugate

obtained by the method. One example of the present invention may provide an 2026200210

5 5 immunogenic composition comprising an immunogenic conjugate obtained by the

method and a physiologically acceptable vehicle. Other example may provide a vaccine

comprising the immunogenic composition.

One example of the present invention provides an immunogenic conjugate of

Streptococcus pneumoniae serotype 17F, comprising a capsular polysaccharide of

10 serotype 17F, derived from Streptococcus pneumoniae; and a carrier protein bound to the

capsular polysaccharide.

The polysaccharide of serotype 17F may be activated and bind to the carrier protein

at a molecular weight of 400 to 900 kDa to form a conjugate.

In one example of the present invention, the immunogenic conjugate may have a

15 15 molecular weight of 300 to 4,500 kDa, and for example, the carrier protein may be CRM197.

23

In one example of the present invention, the ratio of the serotype 17F capsular

18. 18.

15 to 60% of the immunogenic conjugate of serotype 17F may be present within 2026200210

5 5 0.3 Kd in a CL-4B column. Another example provides an immunogenic conjugate of

Streptococcus pneumoniae serotype 17F having a degree of oxidation of 1 to 22.

When the polysaccharide of Streptococcus pneumoniae serotype 17F is oxidized

by adding 0.01 to 0.22 ㎍ of periodate per 1 ㎍ of the sugar content and is conjugated

with a protein, the molecular weight of the conjugate may be 300 to 4,500 kDa, and the

10 10 distribution of the molecular weight may be 15 ~ 60% (0.3kd or less), and the

polysaccharide/protein ratio may be 0.5 to 18.

One example of the present invention may provide an immunogenic composition

comprising the immunogenic conjugate and a physiological acceptable vehicle.

Other example of the present invention may provide a vaccine comprising the

15 15 immunogenic composition.

24

Other example of the present invention may provide a preparation method of an

immunogenic conjugate of Streptococcus pneumoniae serotype 17F, comprising

pneumoniae serotype 17F capsular polysaccharide; 2026200210

5 5 (b) a step of purifying the Streptococcus pneumoniae serotype 17F capsular

polysaccharide in the dissolved cell;

(c) a step of hydrolyzing the purified Streptococcus pneumoniae serotype 17F

capsular polysaccharide to size the polysaccharide;

(d) a step of reacting the sized polysaccharide of the (c) step to activate the

10 10 polysaccharide; and

a conjugate of Streptococcus pneumoniae serotype 17F capsular polysaccharide bound to

the carrier protein.

A preparation method of an immunogenic conjugate of Streptococcus

15 15 pneumoniae serotype 17F, characterized by that the carrier protein combined of the (e)

25

step may form a conjugate with the activated polysaccharide by reacting it with a reducing

agent, may be provided.

The (d) step may comprise a process of reacting 0.01 ~ 0.22 ㎍ of periodate per 1

㎍ polysaccharide at a temperature of 20 to 25 ℃ for 15 to 20 hours. 2026200210

5 5 The activated polysaccharide to be combined with the carrier protein of the (e)

step may have a molecular weight of 400 to 900 kDa.

The immunogenic conjugate may have a molecular weight of 300 to 4,500 kDa.

The initial input ratio of the activated serotype 17F capsular polysaccharide versus

the carrier protein (carrier protein:polysaccharide) may be 1:1.

10 10 In one example, at least 15 to 60% of the immunogenic conjugate molecular weight

may be present within 0.3 Kd in a CL-4B column.

One example of the present invention may provide an immunogenic conjugate

obtained by the method. One example of the present invention may provide an

immunogenic composition comprising an immunogenic conjugate obtained by the

15 15 method and a physiologically acceptable vehicle. Other example may provide a vaccine

26

comprising the immunogenic composition.

One example of the present invention provides an immunogenic conjugate of

Streptococcus pneumoniae serotype 20, comprising a capsular polysaccharide of serotype

20, derived from of Streptococcus pneumoniae; and a carrier protein bound to the 2026200210

5 5 capsular polysaccharide.

The polysaccharide of serotype 20 may be activated and bind to the carrier protein

at a molecular weight of 400 to 800 kDa to form a conjugate.

In one example of the present invention, the immunogenic conjugate may have a

molecular weight of 1,000 to 4,000 kDa, and for example, the carrier protein may be CRM197.

10 10 In one example of the present invention, the ratio of the serotype 20 capsular

polysaccharide to the carrier protein in the immunogenic conjugate (W/W) may be 1 to 5.

70 to 90% of the immunogenic conjugate of serotype 20 may be present within

0.3 Kd in a CL-4B column. Another example provides an immunogenic conjugate of

Streptococcus pneumoniae serotype 20 having a degree of oxidation of 4 to 16.

15 15 When the polysaccharide of Streptococcus pneumoniae serotype 20 is oxidized by

27

adding 0.01 to 0.04 ㎍ of periodate per 1 ㎍ of the sugar content and is conjugated with

a protein, the molecular weight of the conjugate may be 1,000 to 4,000 kDa, and the

distribution of the molecular weight may be 70 ~ 90% (0.3kd or less), and the

polysaccharide/protein ratio may be 1 to 5. 2026200210

5 One example of the present invention may provide an immunogenic composition

comprising the immunogenic conjugate and a physiological acceptable vehicle.

Other example of the present invention may provide a vaccine comprising the

immunogenic composition.

Other example of the present invention may provide a preparation method of an

10 10 immunogenic conjugate of Streptococcus pneumoniae serotype 20, comprising

pneumoniae serotype 20 capsular polysaccharide;

(b) a step of purifying the Streptococcus pneumoniae serotype 20 capsular

polysaccharide in the dissolved cell;

15 15 (c) a step of reacting the polysaccharide with an oxidizing agent to activate it; and

28

(d) a step of combining the activated polysaccharide with a carrier protein to form

a conjugate of Streptococcus pneumoniae serotype 20 capsular polysaccharide bound to

the carrier protein.

The carrier protein combined of the (d) step may be reacted with a reducing agent 2026200210

5 5 to form a conjugate with the activated polysaccharide.

The (c) step may comprise a process of reacting 0.01 ~ 0.04 ㎍ of periodate per 1

㎍ polysaccharide at a temperature of 20 to 25 ℃ for 15 to 20 hours.

The activated polysaccharide to be combined with the carrier protein of the (d)

step may have a molecular weight of 400 to 800 kDa.

10 10 In one example of the present invention, the immunogenic conjugate of the

present invention by the method may have a molecular weight of 1,000 to 4,000 kDa.

The initial input ratio of the activated serotype 20 capsular polysaccharide versus

the carrier protein (carrier protein:polysaccharide) may be 1:1.

In one example, at least 70 to 90% of the immunogenic conjugate molecular

15 15 weight may be present within 0.3 Kd in a CL-4B column.

29

One example of the present invention may provide an immunogenic conjugate

obtained by the method.

One example may provide an immunogenic composition comprising an

immunogenic conjugate obtained by the method and a physiologically acceptable vehicle. 2026200210

5 5 Other example may provide a vaccine comprising the immunogenic composition.

One embodiment of the present invention provides an immunogenic composition

6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F, derived

10 from Streptococcus pneumoniae; and a carrier protein conjugated to the respective

capsular polysaccharide.

One embodiment provides an immunogenic composition comprising a

Streptococcus pneumoniae polysaccharide-protein conjugate, in which when the

immunogenic composition comprises a polysaccharide derived from serotype 2, the

15 15 polysaccharide is activated and binds to the carrier protein at a molecular weight of 100 to

30

400 kDa to form a conjugate, or

when the immunogenic composition comprises a polysaccharide derived from

weight of 400 to 900 kDa to form a conjugate, or 2026200210

5 5 when the immunogenic composition comprises a polysaccharide derived from

weight of 400 to 800 kDa to form a conjugate.

One embodiment of the present invention provides an immunogenic composition

comprising a Streptococcus pneumoniae polysaccharide-protein conjugate, in which when

10 10 the immunogenic composition comprises a polysaccharide derived from serotype 2, an

immunogenic conjugate comprising a polysaccharide derived from serotype 2 has a

molecular weight of 1,000 to 16,000 kDa, or

when the immunogenic composition comprises a polysaccharide derived from

serotype 17F, an immunogenic conjugate comprising a polysaccharide derived from

15 15 serotype 17F has a molecular weight of 300 to 4,500 kDa, or

31

when the immunogenic composition comprises a polysaccharide derived from

serotype 20, an immunogenic conjugate comprising a polysaccharide derived from

serotype 20 has a molecular weight of 1,000 to 4,000 kDa.

5 5 comprising a Streptococcus pneumoniae polysaccharide-protein conjugate, characterized

carrier protein.

One embodiment of the present invention provides an immunogenic composition

10 comprising a Streptococcus pneumoniae polysaccharide-protein conjugate, in which when

conjugate (polysaccharide/protein, W/W) is 0.5 to 2.0, or

when the immunogenic composition comprises a polysaccharide derived from

15 15 serotype 17F, the ratio of the serotype 17F capsular polysaccharide to the carrier protein in

the immunogenic conjugate (polysaccharide/protein, W/W) is 0.5 to 18, or

when the immunogenic composition comprises a polysaccharide derived from

the immunogenic conjugate (polysaccharide/protein, W/W) is 1 to 5. 2026200210

5 5 One embodiment of the present invention provides an immunogenic composition

15 to 60% of the total molecular weight is present within 0.3 Kd in a CL-4B column,

10 10 in case of the immunogenic conjugate comprising a polysaccharide derived from serotype

17F, or

20. 20.

33

serotype 2, or 2026200210

5 5 the degree of oxidation of the polysaccharide conjugated to the conjugate is 1 to

serotype 17F, or

16, in case of the immunogenic conjugate comprising a polysaccharide derived from

10 serotype 20.

One embodiment of the present invention provides an immunogenic composition

other are conjugated to the respective carrier proteins,

15 and the serotypes are 1, 2, 3, 4, 5, 6A, 6B, 7F, 9N, 9V, 14, 18C, 19A, 19F, and 23F, and

34

the serotypes are conjugated with CRM 197.

One embodiment of the present invention provides an immunogenic composition

immunogenic composition is that polysaccharides derived from 23 serotypes different 2026200210

5 5 each other are conjugated to the respective carrier proteins,

and the serotypes are 1, 2, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A,

19F, 20, 22F, 23F, and 33F,

and among the serotypes, the capsular polysaccharides derived from serotypes 3

and 5 are conjugated to the carrier protein TT, and the capsular polysaccharides derived

10 10 from serotypes 1, 2, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F,

23F, and 33F are conjugated to the carrier protein CRM197.

One embodiment of the present invention provides an immunogenic composition

15 15 each other are conjugated to the respective carrier proteins,

and the serotypes are 2, 3, 4, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C,

19A, 19F, 20, 22F, 23F, and 33F,

and among the serotypes, the capsular polysaccharides derived from serotypes 1

and 5 are conjugated to the carrier protein TT, and the capsular polysaccharides derived 2026200210

5 5 from serotypes 2, 3, 4, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20,

22F, 23F, and 33F are conjugated to the carrier protein CRM197.

One embodiment of the present invention provides an immunogenic composition

immunogenic composition comprises a physiologically acceptable vehicle.

10 One embodiment of the present invention provides an immunogenic composition

comprising a Streptococcus pneumoniae polysaccharide-protein conjugate, characterized

by that the immunogenic composition is a vaccine.

One embodiment of the present invention provides a preparation method of an

immunogenic composition comprising a Streptococcus pneumoniae polysaccharide-

15 15 protein conjugate, comprising

36

(a) a step of fermenting and dissolving a bacterial cell producing a capsular

15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F; 2026200210

5 5 (b) a step of purifying a capsular polysaccharide derived from Streptococcus

19F, 20, 22F, 23F, and 33F in the dissolved cell;

it; and

10 10 (d) a step of combining the activated polysaccharide with a carrier protein to form

protein.

In one embodiment of the present invention, the preparation method may further

comprise a step of hydrolyzing the purified capsular polysaccharide of Streptococcus

15 15 pneumoniae to size the polysaccharide, before the (c) step, in case of capsular

37

polysaccharides derived from serotypes 2 and 17F.

One embodiment of the present invention provides a preparation method of an

immunogenic composition comprising a Streptococcus pneumoniae polysaccharide-

protein conjugate, characterized by that the combined carrier protein of the (d) step forms 2026200210

5 5 a conjugate with the polysaccharide activated by reacting it with one or more reducing

agents selected from the group consisting of cyanoborohydride, borane-pyridine, and

borohydride exchange resin.

One embodiment of the present invention provides a preparation method of an

immunogenic composition comprising a Streptococcus pneumoniae polysaccharide-

10 10 protein conjugate, characterized by that the (c) step is reacting 0.01 ~ 0.22㎍ of periodate

per 1 ㎍ of polysaccharide at a temperature of 20 to 25 ℃ for 15 to 20 hours.

One embodiment of the present invention provides a Streptococcus pneumoniae

infection obtained by the method.

15 15 One embodiment may provide a method for preventing or treating infection of

38

15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and/or 33F in a subject,

by administering an effective dose of an immunogenic composition comprising a

Streptococcus pneumoniae polysaccharide-protein conjugate, comprising 2026200210

5 5 a capsular polysaccharide derived from one or more selected from serotypes 1, 2,

derived from Streptococcus pneumoniae; and

one or 2 or more of carrier proteins conjugated to the respective capsular

polysaccharide. The and/or means ‘and’ or ‘or’.

10 10 The method may prevent or treat infection against one or more serotypes selected

prevent infection against 24 serotypes.

15 15 One example of the present invention provides a use for prevention or treatment

39

of pneumococcal infection of a Streptococcus pneumoniae polysaccharide-protein

conjugate, comprising

3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F, 2026200210

5 derived from Streptococcus pneumoniae; and

one or 2 or more of carrier proteins conjugated to the respective capsular

polysaccharide.

One example of the present invention provides an immunogenic conjugate of

Streptococcus pneumoniae serotype 2, comprising a capsular polysaccharide of serotype

10 10 2, derived from Streptococcus pneumoniae; and a carrier protein bound to the capsular

polysaccharide.

at a molecular weight of 100 to 400 kDa to form a conjugate.

In one example of the present invention, the immunogenic conjugate may have a

15 15 molecular weight of 1,000 to 16,000 kDa, and for example, the carrier protein may be

40

CRM197.

In one example of the present invention, the ratio of the serotype 2 capsular

2.0. 2026200210

2.0.

5 20 to 60% of the immunogenic conjugate of serotype 2 may be present within 0.3

Kd in a CL-4B column. Another example provides an immunogenic conjugate of

Streptococcus pneumoniae serotype 2 having a degree of oxidation of 2 to 18.

When the polysaccharide of Streptococcus pneumoniae serotype 2 is oxidized by

adding 0.02 to 0.12 ㎍ of periodate per 1 ㎍ of the sugar content and is conjugate with

10 10 a protein, the molecular weight of the conjugate may be 1,000 ~ 16,000 kDa, and the

distribution of the molecular weight may be 20 ~ 60% (0.3kd or less), and the

polysaccharide/protein ratio may be 0.5 to 2.0.

One example of the present invention may provide an immunogenic composition

comprising the immunogenic conjugate and a physiologically acceptable vehicle.

15 15 Other example of the present invention may provide a vaccine comprising the

41

immunogenic composition.

Other example of the present inventio may provide a vaccine comprising the

immunogenic composition.

Other example of the present invention may provide a preparation method of an 2026200210

5 immunogenic conjugate of Streptococcus pneumoniae serotype 2, comprising

pneumoniae serotype 2 capsular polysaccharide;

(b) a step of purifying the Streptococcus pneumoniae serotype 2 capsular

polysaccharide in the dissolved cell;

10 10 (c) a step of hydrolyzing the purified Streptococcus pneumoniae serotype 2

capsular polysaccharide to size the polysaccharide;

(d) a step of reacting the sized polysaccharide of the (c) step to activate the

polysaccharide; and

15 15 a conjugate of Streptococcus pneumoniae serotype 2 capsular polysaccharide bound to

42

the carrier protein.

The carrier protein combined of the (e) step may form a conjugate with the

activated polysaccharide by reacting it with a reducing agent.

The (d) step may comprise a process of reacting 0.02 ~ 0.12 ㎍ of periodate per 1 2026200210

5 5 ㎍ polysaccharide at a temperature of 20 to 25 ℃ for 15 to 20 hours.

The activated polysaccharide combined to the carrier protein of the (e) step may

have a molecular weight of 100 to 400 kDa.

A preparation method of an immunogenic conjugate of Streptococcus

pneumoniae serotype 2, in which the carrier protein is CRM197, may be provided.

10 10 The immunogenic conjugate may have a molecular weight of 1,000 to 16,000 kDa.

In one example, the initial input ratio of the activated serotype 2 capsular

polysaccharide versus the carrier protein (carrier protein:polysaccharide) may be 0.5 to 2:1.

Other example may provide a preparation method of an immunogenic conjugate

15 15 conjugate is present within 0.3 Kd in a CL-4B column.

43

One example of the present invention may provide an immunogenic conjugate

obtained by the method.

One example of the present invention may provide an immunogenic composition

comprising an immunogenic conjugate obtained by the method and a physiologically 2026200210

5 5 acceptable vehicle.

Other example may provide a vaccine comprising the immunogenic composition.

The present invention provides an immunogenic conjugate of Streptococcus

from Streptococcus pneumoniae; and a carrier protein bound to the capsular

10 polysaccharide.

In one example of the present invention, the polysaccharide of serotype 9N may

be activated and bind to the carrier protein at a molecular weight of 200 to 700 kDa to

form a conjugate.

The immunogenic conjugate may have a molecular weight of 500 to 4,000 kDa

15 15 and the carrier protein may be CRM197.

44

immunogenic conjugate (W/W) may be 0.1 to 5, and preferably, it may be 0.5 ~ 2.5.

In one example, 15 to 60% of the immunogenic conjugate may be present within

0.3 0.3 Kd in aa CL-4B column. 2026200210

Kd in CL-4B column.

5 5 In one example, the conjugate may have a degree of oxidation of 2 to 19.

pneumoniae serotype 9N is oxidized by adding 0.02 ~ 0.19 ㎍ of periodate per 1 ㎍ of

10 10 (0.3kd or less), and the polysaccharide/protein ratio may be 0.5 ~ 2.5.

One example of the present invention may provide an immunogenic composition

comprising an immunogenic conjugate and a physiologically acceptable vehicle.

Other example of the present invention may provide a vaccine comprising the

immunogenic composition.

15 15 Other example of the present invention may provide a preparation method of an

immunogenic conjugate of Streptococcus pneumoniae serotype 9N, comprising

pneumoniae serotype 9N capsular polysaccharide;

(b) a step of purifying the Streptococcus pneumoniae serotype 9N capsular 2026200210

5 5 polysaccharide in the dissolved cell;

the carrier protein.

10 10 A preparation method of an immunogenic conjugate of Streptococcus

pneumoniae serotype 9N, characterized by that the combined carrier protein of the (d)

step is reacted with a reducing agent to form a conjugate with the activated polysaccharide,

may be provided.

15 15 ㎍ of polysaccharide at a temperature of 20 to 25 ℃ for 15 to 20 hours.

46

The polysaccharide reacting with the oxidizing agent of the (c) step may have a

molecular weight of 400 to 900 kDa.

The activated polysaccharide to be combined with the carrier protein of the (d)

step may have a molecular weight of 200-700kDa. 2026200210

5 5 The immunogenic conjugate may have a molecular weight of 500 to 4,000 kDa.

the carrier protein (carrier protein:polysaccharide) may be 0.5 to 2.5:1.

In one example, at least 15 to 60% of the immunogenic conjugate may be present

within 0.3 within 0.3 Kd Kd in in aa CL-4B column. CL-4B column.

10 10 One example of the present invention may provide an immunogenic conjugate

obtained by the method. One example of the present invention may provide an

immunogenic composition comprising an immunogenic conjugate obtained by the

comprising the immunogenic composition.

15 15 One example of the present invention provides an immunogenic conjugate of

47

Streptococcus pneumoniae serotype 17F, comprising a capsular polysaccharide of

serotype 17F, derived from Streptococcus pneumoniae; and a carrier protein bound to the

capsular polysaccharide.

The polysaccharide of serotype 17F may be activated and bind to the carrier protein 2026200210

5 5 at a molecular weight of 400 to 900 kDa to form a conjugate.

In one example of the present invention, the immunogenic conjugate may have a

molecular weight of 300 to 4,500 kDa, and for example, the carrier protein may be CRM197.

In one example of the present invention, the ratio of the serotype 17F capsular

10 10 18. 18.

15 to 60% of the immunogenic conjugate of serotype 17F may be present within

0.3 Kd in a CL-4B column. Another example provides an immunogenic conjugate of

Streptococcus pneumoniae serotype 17F having a degree of oxidation of 1 to 22.

When the polysaccharide of Streptococcus pneumoniae serotype 17F is oxidized

15 15 by adding 0.01 to 0.22 ㎍ of periodate per 1 ㎍ of the sugar content and is conjugated

48

distribution of the molecular weight may be 15 ~ 60% (0.3kd or less), and the

polysaccharide/protein ratio may be 0.5 to 18.

5 5 comprising the immunogenic conjugate and a physiological acceptable vehicle.

Other example of the present invention may provide a vaccine comprising the

immunogenic composition.

Other example of the present invention may provide a preparation method of an

immunogenic conjugate of Streptococcus pneumoniae serotype 17F, comprising

pneumoniae serotype 17F capsular polysaccharide;

(b) a step of purifying the Streptococcus pneumoniae serotype 17F capsular

polysaccharide in the dissolved cell;

(c) a step of hydrolyzing the purified Streptococcus pneumoniae serotype 17F

15 15 capsular polysaccharide to size the polysaccharide;

49

(d) a step of reacting the sized polysaccharide of the (c) step to activate the

polysaccharide; and

a conjugate of Streptococcus pneumoniae serotype 17F capsular polysaccharide bound to 2026200210

5 5 the carrier protein.

A preparation method of an immunogenic conjugate of Streptococcus

pneumoniae serotype 17F, characterized by that the carrier protein combined of the (e)

agent, may be provided.

10 10 The (d) step may comprise a process of reacting 0.01 ~ 0.22 ㎍ of periodate per 1

㎍ polysaccharide at a temperature of 20 to 25 ℃ for 15 to 20 hours.

The activated polysaccharide to be combined with the carrier protein of the (e)

step may have a molecular weight of 400 to 900 kDa.

The immunogenic conjugate may have a molecular weight of 300 to 4,500 kDa.

15 15 The initial input ratio of the activated serotype 17F capsular polysaccharide versus

50

the carrier protein (carrier protein:polysaccharide) may be 1:1.

In one example, at least 15 to 60% of the immunogenic conjugate molecular weight

may be present within 0.3 Kd in a CL-4B column.

One example of the present invention may provide an immunogenic conjugate 2026200210

5 5 obtained by the method. One example of the present invention may provide an

immunogenic composition comprising an immunogenic conjugate obtained by the

comprising the immunogenic composition.

One example of the present invention provides an immunogenic conjugate of

10 10 Streptococcus pneumoniae serotype 20, comprising a capsular polysaccharide of serotype

20, derived from of Streptococcus pneumoniae; and a carrier protein bound to the

capsular polysaccharide.

at a molecular weight of 400 to 800 kDa to form a conjugate.

15 15 In one example of the present invention, the immunogenic conjugate may have a

51

In one example of the present invention, the ratio of the serotype 20 capsular

70 to 90% of the immunogenic conjugate of serotype 20 may be present within 2026200210

5 0.3 Kd in a CL-4B column. Another example provides an immunogenic conjugate of

Streptococcus pneumoniae serotype 20 having a degree of oxidation of 4 to 16.

When the polysaccharide of Streptococcus pneumoniae serotype 20 is oxidized by

10 10 distribution of the molecular weight may be 70 ~ 90% (0.3kd or less), and the

polysaccharide/protein ratio may be 1 to 5.

One example of the present invention may provide an immunogenic composition

comprising the immunogenic conjugate and a physiological acceptable vehicle.

Other example of the present invention may provide a vaccine comprising the

15 15 immunogenic composition.

Other example of the present invention may provide a preparation method of an

immunogenic conjugate of Streptococcus pneumoniae serotype 20, comprising

pneumoniae serotype 20 capsular polysaccharide; 2026200210

5 5 (b) a step of purifying the Streptococcus pneumoniae serotype 20 capsular

polysaccharide in the dissolved cell;

10 10 the carrier protein.

The carrier protein combined of the (d) step may be reacted with a reducing agent

to form a conjugate with the activated polysaccharide.

㎍ polysaccharide at a temperature of 20 to 25 ℃ for 15 to 20 hours.

15 15 The activated polysaccharide to be combined with the carrier protein of the (d)

53

step may have a molecular weight of 400 to 800 kDa.

In one example of the present invention, the immunogenic conjugate of the

The initial input ratio of the activated serotype 20 capsular polysaccharide versus 2026200210

5 the carrier protein (carrier protein:polysaccharide) may be 1:1.

In one example, at least 70 to 90% of the immunogenic conjugate molecular

weight may be present within 0.3 Kd in a CL-4B column.

One example of the present invention may provide an immunogenic conjugate

obtained by the method.

10 10 One example may provide an immunogenic composition comprising an

immunogenic conjugate obtained by the method and a physiologically acceptable vehicle.

Other example may provide a vaccine comprising the immunogenic composition.

Characteristics of serotype 20 polysaccharide-carrier protein conjugate

15 15 In one embodiment, the conjugate may have a molecular weight of 1,300 to 4,300

54

kDa, or a molecular weight of 1,250 to 4,250 kDa, or a molecular weight of 1,200 to 4,200

kDa, or a molecular weight of 1,150 to 4,150 kDa, or a molecular weight of 1,100 to 4,100

kDa, or a molecular weight of 1,000 to 4,000 kDa. All integers within any of the above

ranges are considered as the embodiment. 2026200210

5 5 In the above molecular weight ranges, a conjugate having an excellent yield of the

conjugate may be formed stably. In addition, the ratio of free sugar may be reduced.

Furthermore, it may contribute to excellent immunogenicity in the above molecular weight

ranges.

The immunogenic composition of the present invention is formulated by

10 10 combining them after purifying respective polysaccharide-protein conjugates.

The polysaccharide-protein conjugate of serotypes of the present invention may

be characterized by the ratio of the saccharide to the carrier protein (amount of

polysaccharide/amount of protein) (weight/weight).

In some embodiments, the ratio of the saccharide to the carrier protein of each

15 15 serotype among the polysaccharide-protein conjugate (w/w) may be 0.1 to 7, 0.2 to 7.5,

55

0.3 to 7, 0.4 to 6.5, 0.5 to 6, 0.6 to 6.5, 0.7 to 6, 0.8 to 5.8, 0.9 to 5.6, 0.95 to 5.3, or 1 to 5.

For example, it may be about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about

1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4,

about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2, about 2026200210

5 5 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4, about 4.1,

about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about

5. 5.

In another embodiment, the ratio of the saccharide to the carrier protein (w/w)

may be 1 to 5, 1.2 to 4.5, or 1.3 to 4.

10 10 Preferably, the carrier protein may be CRM197.

When the ratio of the saccharide to the carrier protein is same as above, a

conjugate having an excellent yield of the conjugate may be formed stably. In addition,

the ratio of free sugar may be reduced. Moreover, in case of the above range, not only the

immunogenicity is excellent, but also the conjugate may be maintained stably without

15 15 interference of other serotypes.

56

The conjugate and immunogenic composition of the present invention may

comprise a free saccharide which is not conjugated to the carrier protein covalently but is

present in the polysaccharide-protein conjugate composition. The free saccharide may be

non-covalently associated with the polysaccharide-protein conjugate (that is, it may be 2026200210

5 5 non-covalently bound or adsorbed to the polysaccharide-protein conjugate, or be

encapsulated in the polysaccharide-protein conjugate or by the polysaccharide-protein

conjugate).

In a preferable embodiment, the polysaccharide-protein conjugate comprises a

polysaccharide of each free serotype less than about 70%, about 60%, about 50%, 45%,

10 10 40%, 35%, 30%, 25%, 20%, 15%, or 10% to the total amount of the polysaccharide of each

serotype.

The polysaccharide-protein conjugate of each serotype may be also characterized

by its molecular size distribution (Kd). Using a size exclusion chromatography medium (CL-

4B, Cross-linked Agarose beads, 4%), the relative molecular size distribution of the

15 15 conjugate may be measured. The molecular size distribution of the conjugate is profiled

57

using the size exclusion chromatography (SEC) in a gravity feed column. Big molecules

excluded from pores in the medium are eluted faster than small molecules. Using a fraction

collector, the column eluates are collected. The fractions are tested by colorimetry by

saccharide analysis. For measurement of Kd, the column is scaled to set fractions in which 2026200210

5 5 molecules are completely excluded (VO), (Kd = 0) and fractions showing the maximum

maintenance (Vi), (Kd = 1). The fractions in which the specified sample characteristic is

reached (Ve) is related to Kd by the equation Kd = (Ve - V0)/(Vi - V0).

In a preferable embodiment, at least 30% of the polysaccharide-protein conjugate

of each serotype may be present within 0.3 Kd in a CL-4B column.

10 10 In a preferable embodiment, at least 95% of the polysaccharide-protein conjugate

of each serotype may be present within 0.3 Kd in a CL-4B column. In a preferable

embodiment, at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the

polysaccharide-protein conjugate of each serotype may be present within 0.3 Kd in a CL-

4B column. In a preferable embodiment, at least 60% of the polysaccharide-protein

15 15 conjugate of each serotype may be present within 0.3 Kd in a CL-4B column. In a preferable

58

embodiment, 50 to 90% of the polysaccharide-protein conjugate of each serotype may be

present within 0.3 Kd in a CL-4B column. In a preferable embodiment, 65 to 90% of the

4B column. In a preferable embodiment, 70 to 90% of the polysaccharide-protein 2026200210

5 5 conjugate of each serotype may be present within 0.3 Kd in a CL-4B column.

1.4 Combination of capsular saccharide-carrier protein

In one embodiment, the immunogenic composition of the present invention

comprises a conjugate of one or more polysaccharide selected from the group consisting

10 of Streptococcus pneumoniae serotypes 2, 9N, 17F and 20 and a protein. In one

embodiment, any one of the immunogenic compositions may comprise a conjugate in

which a polysaccharide derived from one or more selected from the group consisting of

1,3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F is further

conjugated to the carrier protein.

15 15 In one embodiment, any one of the polysaccharide-protein conjugates among the

59

immunogenic composition is conjugated to CRM197 and/or TT, respectively. Preferably, a

23-valent or 24-valent immunogenic composition may comprise both CRM197 and TT as

the carrier protein, and in this case, preferably, serotype 5 comprises TT as the carrier

protein. In one example, in the 23-valent immunogenic composition, the polysaccharides 2026200210

5 derived from serotype 3 and 5 are conjugated to TT, and polysaccharides derived from

serotypes 1, 2, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F,

and 33F are conjugated to CRM197. In one example, in the 24-valent immunogenic

composition, the polysaccharides derived from serotypes 1 and 5 are conjugated to TT,

and serotypes 2, 3, 4, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F,

10 23F, and 33F are conjugated to CRM197.

In one embodiment, the immunogenic composition may comprise a

polysaccharide-protein conjugate derived from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,

16, 17, 18, 19, 20, 21, 22, 23, or 24 different serotypes.

15 15 2. Dose of immunogenic composition

60

The amount of the capsular saccharide-carrier protein conjugate(s) among each

dose is selected as the amount of inducing an immune protection response without

significant side effects among typical vaccines. The amount as above may change

depending on how a specific immunogen is used and how it is provided. 2026200210

5 5

2.1 Amount of capsular polysaccharide-carrier protein conjugate

The amount of the specific capsular polysaccharide-carrier protein in the

immunogenic composition may be calculated on the basis of the total polysaccharides to

the conjugate (conjugated and unconjugated). For example, the capsular polysaccharide-

10 10 carrier protein conjugate having 20% free polysaccharides means having about 80 ㎍

conjugated polysaccharides and about 20 ㎍ unconjugated polysaccharides in 100 ㎍

polysaccharide dose. The amount of the polysaccharide-protein conjugate may change

depending on the pneumococcal serotypes. The polysaccharide concentration may be

measured by anthrone or uronic acid analysis.

15 15 The “immunogenic amount” of the different polysaccharide components in the

61

immunogenic composition may vary, and it may comprise any specific polysaccharide

antigen of about 1 ㎍, about 2 ㎍, about 3 ㎍, about 4 ㎍, about 5 ㎍, about 6 ㎍, about

7 ㎍, about 8 ㎍, about 9 ㎍, about 10 ㎍, about 15 ㎍, about 20 ㎍, about 30 ㎍, about

40 ㎍, about 50 ㎍, about 60 ㎍, about 70 ㎍, about 80 ㎍, about 90 ㎍, or about 100 2026200210

5 ㎍, respectively.

In general, each dose may comprise polysaccharides of 0.1 ㎍ to 100 ㎍,

particularly, 0.5 ㎍ to 20 ㎍, more particularly, 1.0 ㎍ to 10 ㎍, and more particularly 2.0

㎍ to 5.0 ㎍ to the given serotype. All integers within any of the above ranges are

considered asthe considered as theembodiment. embodiment.

10 10 In one embodiment, each dose may comprise polysaccharides of about 1.0 ㎍,

about 1.2 ㎍, about 1.4 ㎍, about 1.6 ㎍, about 1.8 ㎍, about 2.0 ㎍, about 2.2 ㎍, about

2.4 ㎍, about 2.6 ㎍, about 2.8 ㎍, about 3.0 ㎍, about 3.2 ㎍, about 3.4㎍, about 3.6 ㎍,

about 3.8 ㎍, about 4.0 ㎍, about 4.2 ㎍, about 4.4 ㎍, about 4.6 ㎍, about 4.8 ㎍, about

5.0 ㎍, about 5.2 ㎍, about 5.4 ㎍, about 5.6 ㎍, about 5.8 ㎍ or about 6.0 ㎍ to each

15 15 specific capsular saccharide-carrier protein conjugate.

62

In one embodiment, each dose may comprise polysaccharides of 1 to 3㎍ to the

polysaccharide-protein derived from Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A,

7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and/or 33F. For example,

it may comprise polysaccharides of about 1.1 ㎍, about 1.2 ㎍, about 1.3 ㎍, about 1.4 ㎍, 2026200210

5 5 about 1.5 ㎍, about 1.6 ㎍, about 1.7 ㎍, about 1.8 ㎍, about 1.9 ㎍, about 2.0 ㎍, about

2.1 ㎍, about 2.2 ㎍, about 2.3 ㎍, about 2.4 ㎍, about 2.5 ㎍, about 2.6 ㎍, about 2.7

㎍, about 2.8 ㎍, about 2.9 ㎍, or about 3.0 ㎍.

In one embodiment, in case of further comprising a polysaccharide-protein

conjugate derived from serotype 6B, polysaccharides of 2 to 6㎍ may be comprised.

10 10

2.2 Amount of carrier protein

In one embodiment, the carrier protein may be CRM197 and/or TT.

In one embodiment, when the carrier protein is CRM197, each dose of the carrier

protein comprised in the immunogenic composition may comprise carrier proteins of 10

15 15 ㎍ to 150 ㎍, 20 ㎍ to 100 ㎍, 25 ㎍ to 95㎍. For example, the 24-valent immunogenic

63

composition according to one embodiment of the present invention may comprise carrier

proteins of 70 to 90 ㎍.

In one embodiment, when the carrier protein is TT, each dose of the carrier protein

comprised in the immunogenic composition may comprise carrier proteins of 5 ㎍ to 15 2026200210

5 5 ㎍, 8 ㎍ to 10 ㎍.

3. Adjuvant

In some embodiments, the immunogenic composition disclosed in the present

invention may further comprise one or more adjuvants. The term “adjuvant” refers to a

10 compound or mixture which increases an immune response against an antigen. The

adjuvant may enhance an immune response against an antigen exhibiting weak

immunogenicity and/or, may increase an antibody titer against an antigen and/or, may

reduce an effective dose of an antigen for achieving an immune response in a subject, in

case of single administration, as inducing no or weak antibody titer or cell mediated

15 15 immune response. Thus, the adjuvant mostly plays a role of increasing an immune

64

response, and this is known to those skilled in the art. The suitable adjuvant enhancing the

efficacy of a composition includes the followings, but it is not limited thereto:

In one example, the adjuvant may include aluminum salts (alum), for example,

aluminum hydroxide, aluminum phosphate, aluminum sulfate, and the like. 2026200210

5 5 In a specific embodiment, the adjuvant is an aluminum salt. The aluminum salt

adjuvant may be an alum-precipitated vaccine or an alum-adsorbed vaccine. The

aluminum salt adjuvant is known in the art. The aluminum salt includes hydrated alumina,

alumina hydrate, alumina trihydrate (ATH), aluminum hydrate, aluminum trihydrate,

Alhydrogel, Superfos, Amphogel, aluminum hydroxide (III), aluminum hydroxyphosphate

10 10 sulfate (aluminum phosphate adjuvant (APA)), amorphous alumina, trihydrated alumina,

or trihydroxyaluminum, but not limited thereto.

APA is an aqueous suspension of aluminum hydroxyphosphate. APA is prepared

by blending aluminum chloride and sodium phosphate at a volume ratio of 1:1 and

precipitating aluminum hydroxyphosphate. After the blending process, by reducing in a

15 15 size of a material using a high shear mixer, target aggregate particles having a size in a

65

range of 2-8 ㎛ are obtained. Subsequently, products are diafiltrated for saline solution

and aresteam and are steam sterilized. sterilized.

In a specific embodiment, a protein is adsorbed at a ratio of 50-200g protein/mg

aluminum hydroxide using a commercially available Al(OH)3 (for example, Alhydrogel or 2026200210

5 5 Superfos of Denmark/Accurate Chemical and Scientific Co. (U.S. New York Westbury). In

another embodiment, the adsorption of the protein differs depending on pI (isoelectric

pH) of the protein and pH of a medium. The protein having lower pI are adsorbed to

positively charged aluminum ion more strongly than the protein having higher pI. The

aluminum salt may establish Ag depot slowly released over 2-3 weeks, and/or may involve

10 in non-specific activation of macrophage and complement activation, and/or may

stimulate a congenital immune mechanism (possibly through uric acid stimulation).

In a preferable embodiment, the adjuvant is an aluminum-based adjuvant selected

from the group consisting of aluminum phosphate, aluminum sulfate and aluminum

hydroxide. In one embodiment, the immunogenic composition disclosed herein comprises

15 15 an aluminum phosphate adjuvant.

66

4. Formulation

The immunogenic composition of the present invention may be formulated in a

liquid form (that is, solution or suspension) or in a lyophilized form. Advantageously, the 2026200210

5 5 liquid formulation may be directly administered in its packaged form, and therefore the

formulation is ideal for injection without re-composition in an aqueous medium as

required for the lyophilized composition of the present invention.

The formulation of the immunogenic composition of the present invention may be

carried out using a method approved in the art. For example, the composition may be

10 10 prepared by formulating an individual pneumococcal conjugate with a physiologically

acceptable vehicle. The example of the vehicle as above unlimitedly includes water,

buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol)

and dextrosesolution. and dextrose solution.

The present invention provides an immunogenic composition comprising any one

15 of combinations of the polysaccharide-protein conjugates disclosed and a

67

pharmaceutically acceptable excipient, carrier or diluent.

In one embodiment, the immunogenic composition of the present invention is in

a liquid form, preferably in an aqueous liquid form.

The immunogenic composition of the present invention may comprise one or 2026200210

5 more kinds among buffer, salt, divalent cation, non-ionic detergent, cryoprotectant, e.g.

sugar, and anti-oxidant, e.g. free radical scavenger and chelating agent, and any of various

combinations thereof. combinations thereof.

In one embodiment, the immunogenic composition of the present invention

comprises a buffer. In one embodiment, the buffer has pKa of about 3.5 to about 7.5. In

10 10 some embodiments, the buffer is phosphate, succinate, histidine or citrate. In some

embodiments, the buffer is succinate at a final concentration of 1 mM to 10 mM. In one

specific embodiment, the final concentration of succinate is about 5 mM.

In one embodiment, the immunogenic composition of the present invention

comprises a salt. In some embodiments, the salt is selected from the group consisting of

15 15 magnesium chloride, potassium chloride, sodium chloride and combinations thereof. In a

68

preferable embodiment, the salt is sodium chloride. In one specific embodiment, the

immunogenic composition of the present invention comprises sodium chloride of 150 mM.

In one embodiment, the immunogenic composition of the present invention

comprises a surfactant. The surfactant is selected from the group consisting of 2026200210

5 5 polyoxyethylene sorbitan fatty acid ester, polysorbate-80 (Tween 80), polysorbate-60

(Tween 60), polysorbate-40 (Tween 40) and polysorbate-20 (Tween 20), polyoxyethylene

alkyl ether (including Brij 58, Brij 35, but not limited thereto), as well as other materials, for

example, one or more kinds of non-ionic surfactants which include Triton X-100; Triton X-

114, NP40, Span 85 and pluronic series of non-ionic surfactants (for example, pluronic 121),

10 10 but not limited thereto. In a preferable embodiment, the immunogenic composition

comprises polysorbate-80 or polysorbate-20, preferably polysorbate-20. In a preferable

embodiment, the immunogenic composition comprises polysorbate-20 at a concentration

of about 0.001% to about 2% (less than about 0.005% is preferable).

In one embodiment, the container of the present invention is prepared by glass,

15 15 metal (for example, steel, stainless steel, aluminum, etc.) and/or polymers (for example,

69

thermoplastic materials, elastomers, thermoplastic-elastomers). In one embodiment, the

container of the present invention is prepared by glass.

In one embodiment, the present invention provides an injection filled with any one

of the immunogenic compositions disclosed in the present invention. In one specific 2026200210

5 embodiment, the injection is treated with silicon and/or is prepared by glass.

5. 5. Use Use

In one embodiment, the immunogenic composition disclosed in the present

invention is to be used as a pharmaceutical. The amount of the conjugate in the

10 10 composition is selected as an amount of inducing an immune protective response without

significant side effects. Such an amount may vary depending on serotypes of

pneumococcus.

The immunogenic composition disclosed in the present invention may be used by

various therapeutic or prophylactic methods for prevention, treatment or improvement of

15 15 bacterial infection, diseases or conditions in a subject. In particular, the immunogenic

70

composition disclosed in the present invention may be used for prevention, treatment or

improvement of Streptococcus pneumoniae infection, diseases or conditions in a subject.

All references or patent applications cited in the present patent specification are

incorporated by reference herein. 2026200210

5 5 The present invention is illustrated by accompanying examples. The following

examples are conducted by using common standard techniques known to those skilled in

the art, except where otherwise specifically described. These examples are illustrative, but

does not limit the present invention.

The immunogenic composition disclosed in the present invention may be used by

10 10 various therapeutic or prophylactic methods for prevention, treatment or improvement of

bacterial infection, diseases or conditions in a subject. In particular, the immunogenic

In one embodiment, the present invention provides a method for preventing,

15 15 treating or improving Streptococcus pneumoniae infection, diseases or conditions in a

71

subject, comprising administering an immunologically effective dose of the immunogenic

composition of the present invention into the subject.

In some embodiments as above, the infection, diseases or conditions are selected

from the group consisting of pneumonia, sinusitis, otitis media, acute otitis media, 2026200210

5 5 cerebromeningitis, bacteriemia, septicemia, pyothorax, conjunctivitis, osteomyelitis, septic

arthritis, endocarditis, peritonitis, pericarditis, mastoiditis, cellulitis, soft tissue infection and

brain brain abscess. abscess.

In one embodiment, the present invention provides a method of inducing an

immune response against Streptococcus pneumoniae in a subject, comprising

10 administering an immunologically effective dose of immunogenic composition of the

present invention into the subject.

In one embodiment, the immunogenic composition disclosed in the present

invention is to be used as a vaccine. In an embodiment as above, the immunogenic

composition disclosed in the present invention may be used for preventing Streptococcus

15 15 pneumoniae infection. Thus, in one embodiment, the present invention provides a method

for preventing infection by Streptococcus pneumoniae in a subject, comprising

administering an immunologically effective dose of the immunogenic composition of the

present invention into the subject.

In some embodiments as above, the infection is selected from the group consisting 2026200210

5 5 of pneumonia, sinusitis, otitis media, acute otitis media, cerebromeningitis, bacteriemia,

septicemia, pyothorax, conjunctivitis, osteomyelitis, septic arthritis, endocarditis, peritonitis,

pericarditis, mastoiditis, cellulitis, soft tissue infection and brain abscess. In one

embodiment, the subject to be vaccinated is a mammal, for example, human, cat, sheep,

pig, horse, cow or dog.

10 10 In one embodiment, the immunogenic composition disclosed in the present

invention is to be used for a method for prevention, treatment or improvement of infection,

diseases or conditions related to Streptococcus pneumoniae in a subject. In some

embodiments as above, the infection, diseases or conditions are selected from the group

consisting of pneumonia, sinusitis, otitis media, acute otitis media, cerebromeningitis,

15 bacteriemia, septicemia, pyothorax, conjunctivitis, osteomyelitis, septic arthritis,

73

endocarditis, peritonitis, pericarditis, mastoiditis, cellulitis, soft tissue infection and brain

abscess.

In one embodiment, the immunogenic composition disclosed in the present

invention to be used as a vaccine. In an embodiment as above, the immunogenic 2026200210

5 5 composition disclosed in the present invention may be used for Streptococcus

pneumoniae preventing infection in a subject. Thus, in one embodiment, the immunogenic

composition disclosed in the present invention is to be used for a prophylactic method of

infection by Streptococcus pneumoniae in a subject. In some embodiments as above, the

infection is selected from the group consisting of pneumonia, sinusitis, otitis media, acute

10 otitis media, cerebromeningitis, bacteriemia, septicemia, pyothorax, conjunctivitis,

osteomyelitis, septic arthritis, endocarditis, peritonitis, pericarditis, mastoiditis, cellulitis, soft

tissue infection and brain abscess. In one embodiment, the subject to be vaccinated is a

mammal, for example, human, cat, sheep, pig, horse, cow or dog.

The immunogenic composition of the present invention may be used for

15 protecting or treating human sensitive to pneumococcal infection by administering the

immunogenic composition through a systemic or mucosal route. In one embodiment, the

immunogenic composition disclosed in the present invention is administered through an

intramuscular, intraperitoneal, intracutaneous or subcutaneous route. In one embodiment,

the immunogenic composition disclosed in the present invention is administered by 2026200210

5 5 intramuscular, intraperitoneal, intracutaneous or subcutaneous injection. In one

embodiment, the immunogenic composition disclosed in the present invention is

administered by intramuscular or subcutaneous injection.

In the ELISA (enzyme-linked immunosorbent assay) method, an antibody from

serum of the vaccinated subject is cultured with a polysaccharide adsorbed to a solid

10 10 support. The bound antibody is detected by using an enzyme-conjugated secondary

detection antibody.

The ELISA measures a type-specific IgG anti-Streptococcus pneumoniae capsular

polysaccharide (PS) antibody present in human serum. When the dilution of human serum

is added to a type-specific capsule PS-coated microtitration plate, the antibody specific to

15 15 the capsule PS binds to the microtitration plate. The antibody bound to the plate is

75

detected by using a goat anti-human IgG alkaline phosphatase-labeled antibody, followed

by a p-nitrophenyl phosphate substrate.

The optical density of the colored final product is proportional to the amount of

the anti-capsule PS antibody present in the serum. 2026200210

5 5 In one embodiment, the immunogenic composition comprising one or more of

polysaccharide-protein conjugates from Streptococcus pneumoniae serotypes 2, 9N, 17F

and 20 may induce an IgG antibody capable of binding to the Streptococcus pneumoniae

serotype 15B polysaccharide at a concentration of at least 0.05, 0.1, 0.2, 0.3, 0.35, 0.4 or 0.5

㎍/㎖, as measured by ELISA analysis in human.

10 10 In one embodiment, the immunogenic composition comprising one or more of

and 20 may induce formation of an antibody capable of phagocytosis of Streptococcus

pneumoniae of one or more serotype selected from Streptococcus pneumoniae serotypes

2, 9N, 17F and 20 in the phagocytosis analysis initiated by opsonin as disclosed in the

15 15 present invention.

In one embodiment, the immunogenic composition comprising one or more

and 20 has a bigger OPA titer than an OPA titer obtained for a natural Streptococcus

pneumoniae capsular polysaccharide which is not conjugated during the test in the OPA 2026200210

5 analysis.

The pneumococcus opsonin-initiated phagocytosis analysis (OPA) measuring

killing of Streptococcus pneumoniae cells by cells having a phagocytosis effect under the

present of a functional antibody and a complement is considered as an important

substitute in evaluating the efficacy of a pneumococcal vaccine.

10 10 The opsonin-initiated phagocytosis analysis (OPA) may be performed by culturing

the mixture of Streptococcus pneumoniae cells, heat-inactivated human serum to be

tested, differentiated HL-6 cells (phagocytes) and exogenous complement sources of

supply (for example, baby rabbit complement) together. The opsonin-initiated

phagocytosis is progressed during the culturing, and bacterial cells coated with an

15 antibody and a complement are killed during the opsonin-initiated phagocytosis. The

colony forming unit (cfu) of survived bacteria escaping from the opsonin-initiated

phagocytosis is measured by painting out the analysis mixture. The OPA titer is defined as

mutual dilution producing 50% reduction of the number of bacteria in a control well

without test serum. The OPA titer is interpolated from 2 diluents comprising the 50% killing 2026200210

5 cutoff.

The terminal titer of 1:8 or more is considered as the result of the amount in the

killing type OPA.

In one embodiment, the immunogenic composition comprising one or more

10 and 20 may induce at least 1:8 titer against one or more serotypes selected from

Streptococcus pneumoniae serotypes 2, 9N, 17F and 20, as measured by the opsonin-

initiated phagocytosis killing analysis (OPA). In one embodiment, the immunogenic

composition comprising one or more polysaccharide-protein conjugates from

Streptococcus pneumoniae serotypes 2, 9N, 17F and 20 may induce at least 1:8 titer against

15 15 Streptococcus pneumoniae serotypes 2, 9N, 17F and 20 in at least 60%, 70%, 80%, 90% or

78

at least 93% of the subject, as measured by the opsonin-initiated phagocytosis killing

analysis (OPA).

6. Subject to be treated by the immunogenic composition of the present invention 2026200210

5 5 As disclosed in the present invention, the immunogenic composition disclosed in

the present invention may be used for various therapeutic or prophylactic methods for

preventing, treating or improving bacterial infection, diseases or conditions in a subject.

In a preferable embodiment, the subject is human. In the most preferable

embodiment, the subject is newborn babies (that is, 3 months or less), infants (that is, 3

10 10 months to 1 year), or toddlers (that is, 1 year to 4 years).

In one embodiment, the immunogenic composition disclosed in the present

invention is to be used as a vaccine. In the embodiment as above, the subject to be

vaccinated may be less than 1 year. For example, the subject to be vaccinated may have

an age of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9,

15 15 about 10, about 11 or about 12 months. In one embodiment, the subject to be vaccinated

79

is about 2, about 4 or about 6 months. In another embodiment, the subject to be

vaccinated is less than 2 years. For example, the subject to be vaccinated has an age of

about 12 months to about 15 months. In some cases, one dose of the immunogenic

composition according to the present invention may be required, but in some 2026200210

5 circumstances, a second, third or fourth dose may be provided.

In an embodiment of the present invention, the subject to be vaccinated is an adult

over 50 years old, more preferably, an adult over 55 years old.

In one embodiment, the subject to be vaccinated is an adult over 65 years old, 70

years old, 75 years old, or 80 years old.

10 In one embodiment, the subject to be vaccinated is an immune-deficient individual,

in particular, human. The immune-deficient individual is generally defined as people with

reduced or decreased ability to initiate normal body fluids or cell defense against attacks

by infectious agents.

In one embodiment of the present invention, the immune-deficient subject to be

15 15 vaccinated suffers from diseases or conditions damaging the immune system and

80

produces an antibody response insufficient for protecting from pneumococcal disease or

treating the diseases.

In one embodiment, the diseases are primary immunodeficiency diseases.

Preferably, the primary immunodeficiency diseases are selected from the group consisting 2026200210

5 5 of complex T- and B- cells immunodeficiency, antibody deficiency, well defined syndrome,

immunodysregulation disease, phagocyte disease, congenital deficiency,

autoinflammatory disease and complement deficiency.

In a specific embodiment of the present invention, the immune-deficient subject

to be vaccinated may suffer from a disease selected from the group consisting of the

10 10 followings: HIV-infection, acquired immune deficiency syndrome (AIDS), cancer, chronic

heart or lung disease, congestive heart failure, diabetes, chronic liver disease, alcohol

abuse, cirrhosis, spinal fluid leak, cardiomyopathy, chronic bronchitis, emphysema, chronic

obstructive pulmonary disease (COPD), spleen dysfunction (for example, sickle cell disease),

spleen function deficiency (alienia), hematologic malignancy, leukemia, multiple myeloma,

15 15 Hodgkin disease, lymphoma, renal insufficiency, nephrotic syndrome and asthma.

81

In one embodiment of the present invention, the immune-deficient subject to be

vaccinated may suffer from malnutrition.

In a specific embodiment of the present invention, the immune-deficient subject

to be vaccinated may be on medication or treatment that reduces the body’s resistance to 2026200210

5 infection.

In a specific embodiment of the present invention, the immune-deficient subject

to be vaccinated may be a smoker.

In a specific embodiment of the present invention, the immune-deficient subject

to be vaccinated may have the number of white blood cells (leukocytes number) of 5 x 109

10 10 cell/liter or less, or 4 x 109 cell/liter or less, or 3 x 109 cell/liter or less, or 2 x 109 cell/liter or

less, or 1 x 109 cell/liter or less, or 0.5 x 109 cell/liter or less, or 0.3 x 109 cell/liter or less, or

0.1 x 109 cell/liter or less.

The number of white blood cells (leukocytes number): the number of white blood

cells (WBC) in blood. The WBC is measured as a part of CBC (complete blood cell number).

15 15 The white blood cells are infection-struggling cells and are different from red (oxygen-

82

delivery) blood cells as red blood cells.

There are different types of white blood cells, for example, neutrophils

(polymorphous nucleus white blood cell; PMN), stab cells (some immature neutrophils), T-

type lymphocytes (T-cell), B-type lymphocytes (B-cell), monocytes, eosinophils and 2026200210

5 5 basophils. All the above types of white blood cells are reflected to the number of white

blood cells. The normal range of the white blood cells is mostly 4,300 to 10,800 cell/blood

cubic milliliter. This is also referred as the number of white blood cells, and it may be

represented by the international unit as 4.3 to 10.8 x 109/liter.

In a specific embodiment of the present invention, the immune-deficient subject

10 10 to be vaccinated is suffering from neutropenia. In a specific embodiment of the present

invention, the immune-deficient subject to be vaccinated may have the number of

neutrophils of 2 x 109 cell/liter or less, or 1 x 109 cell/liter or less, or 0.5 x 109 cell/liter or less,

or 0.1 x 109 cell/liter or less, or 0.05 x 109 cell/liter or less.

The low number of white blood cells or “neutropenia” is a condition characterized

15 15 by abnormally low level of neutrophils in circulating blood. The neutrophil is a unique kind

of white blood cells, which helps prevent infection and struggle infection. The most

common reason why cancer patients suffer from neutropenia is as side effects of

chemotherapy. The chemotherapy-caused neutropenia increases infection riskiness of

patients and halts cancer treatment. 2026200210

5 5 In a specific embodiment of the present invention, the immune-deficient subject

to be vaccinated may have a CD4+ cell number of 500/㎣ or less, or a CD4+ cell number

of 300/㎣ or less, or a CD4+ cell number of 200/㎣ or less, or a CD4+ cell number of

100/㎣ or less, or a CD4+ cell number of 75/㎣ or less, or a CD4+ cell number of 50/㎣

or less. or less.

10 10 The CD4 cell test is commonly reported as the cell number of ㎣. The normal CD4

number is 500 to 1,600, and the CD8 number is 375 to 1,100. The CD4 number falls

significantly in people with HIV.

In one embodiment of the present invention, any subject of the immune-deficient

subject disclosed in the present invention may be a human male or human female.

15 15

84

7. Prescribed diet 7. Prescribed diet

In some cases, one dose of the immunogenic composition according to the present

invention may be required, but in some circumstances, for example, under the condition

of greater immune-deficiency, a second, third or fourth dose may be provided. Following 2026200210

5 the initial vaccination, the subject may receive one or more additional immunizations at

appropriate intervals.

In one embodiment, the vaccination schedule of the immunogenic composition

according to the present invention is a single dose. In a specific embodiment, the single

dose schedule is for a healthy human of at least 2 years.

10 10 In one embodiment, the vaccination schedule of the immunogenic composition

according to the present invention is a multiple dose schedule. In a specific embodiment,

the multiple dose schedule consists of a series of 2 doses separated by an interval of about

one month to about two months. In a specific embodiment, the multiple dose schedule

consists of a series of 2 doses separated by an interval of about 1 month, or a series of 2

15 15 doses separated by an interval of about 2 months.

85

In another embodiment, the multiple dose schedule consists of a series of 3 doses

separated by an interval of about 1 month to about 2 months. In other embodiment, the

multiple dose schedule consists of a series of 3 doses separated by an interval of about 1

month, or a series of 3 doses separated by an interval of about 2 months. 2026200210

5 In other embodiment, the multiple dose schedule consists of a series of 3 doses

separated by an interval of about 1 month to about 2 months and subsequent fourth dose

of about 10 months to about 13 months after the first dose. In other embodiment, the

month and subsequent fourth dose of about 10 months to about 13 months after the first

10 10 dose, or a series of 3 doses separated by an interval of about 2 months and subsequent

fourth dose of about 10 months to about 13 months after the first dose.

In one embodiment, the multiple dose schedule consists of at least 1 dose (for

example, 1, 2 or 3 doses) at 1 year and subsequent at least 1 dose of toddler dose.

In one embodiment, the multiple dose schedule consists of a series of 2 or 3 doses

15 15 separated by an interval of about 1 month to about 2 months (for example, 28 to 56 days

between doses) after starting at 2 months, and a subsequent toddler dose of 12 to 18

months. In one embodiment, the multiple dose schedule consists of a series of 3 doses

separated by an interval of about 1 month to about 2 months (for example, 28 to 56 days

between doses) after starting at 2 months, and a subsequent toddler dose of 12 to 15 2026200210

5 5 months. In other embodiment, the multiple dose schedule consists of a series of 2 doses

separated by an interval of about 2 months after starting at 2 months, and a subsequent

toddler dose toddler doseof of 12 12 to to 18 18 months. months.

In one embodiment, the multiple dose schedule consists of a series of 4 vaccine

doses at 2, 4, 6 and 12 to 15 months.

10 10 In one embodiment, the initial dose is provided at 0 day, and once or more of

additional doses are provided at an interval of about 2 to about 24 weeks, preferably, at

an administration interval of 4 to 8 weeks.

In one embodiment, the initial dose is provided at o day, the additional dose is

provided about 3 months later.

15 15

【ADVANTAGEOUS EFFECTS】

The present invention can provide an immunogenic conjugate for a new serotype

such as serotype 17F which has not provided a protection range previously.

The present invention can provide a multivalent pneumococcal vaccine capable of 2026200210

5 5 providing a wide range of protection by comprising a new serotype conjugate which has

not provided a protection range previously.

In addition, it is possible to form an antibody against various pneumococcal

serotypes without any special interference phenomenon, thereby providing a wide

spectrum of immunity.

10 10 The present invention can provide excellent antibody titer.

The multivalent pneumococcal vaccine of the present invention exhibits less side

effects.

The vaccine of the present invention can be inoculated to infants and toddlers and

can be inoculated to the elderly.

15 15

88

【MODE FOR INVENTION】

Hereinafter, the present invention will be described with reference to the following

examples and the like in order to describe it more specifically. However, the examples

according to the present invention may be modified into various other forms, and the 2026200210

5 5 scope of the present invention should not be construed as being limited to the examples

described below. The examples of the present invention are provided to illustrate the

present invention in order to facilitate a specific understanding of the present invention.

Example 1. Preparation of serotype 2, 9N, 17F or 20-derived polysaccharide-protein

10 10 conjugate vaccine

[1. Streptococcus pneumoniae serotype 2-derived polysaccharide-protein

conjugate]

Preparation of polysaccharide-protein conjugate from Streptococcus pneumoniae

15 15 serotype 2

89

Preparation of cell bank for master and preparation

Streptococcus pneumoniae serotype 2 was acquired from American Type Culture

Collection (ATCC) (strain ATCC 6302). In order to enhance the strain and remove

components of animal origin, a seed stock was cultured for several generations. The seed 2026200210

5 5 vial was frozen with synthetic glycerol as a cryopreservative (< -70℃). For preparation of

cell bank, all cultures were proliferated in a soybean-based medium. Before freezing, cells

were concentrated by centrifugation and the used medium was removed, and then a cell

pellet was resuspended in a new medium containing a cryopreservative (e.g.: synthetic

glycerol).

10 10 Fermentation Fermentation

The cultures derived from the cell bank for preparation was used and inoculated

into a seed bottle containing a soybean-based medium. Before satisfying the growth

requirements, it was cultured at a certain temperature without stirring. Using the seed

bottle, it was inoculated to a seed fermenter containing the soybean-based medium in

15 15 which the temperature, pH and stirring speed were controlled. After the growth was

90

stopped, or at the time of reaching the work capacity of the fermenter, fermentation was

terminated. After terminating the fermentation process by adding an inactivator, cell

residuals were removed using the combination of continuous flow centrifugation and

filtration. 2026200210

filtration.

5 5 Purification

The purification of the pneumococcal polysaccharide was composed of multiple

media filtration, several times of concentration/diafiltration work and precipitation/elution

steps.

Activation Activation

10 10 The final polysaccharide concentration was adjusted to be about 2.0 g/L in 0.01N

hydrochloric acid solution by adding a calculated amount of 0.1N hydrochloric acid

solution and WFI in order. For hydrolysis of the polysaccharide, the hydrolysis reaction was

carried out at 60℃ for 60 minutes. After lowering the temperature of the reaction solution

to the room temperature, the reaction pH was adjusted to approximately 6.0 by adding

15 15 0.1M sodium monohydrogen phosphate solution. After adjusting the pH, the temperature

91

was adjusted to 23℃. The oxidation was initiated by adding sodium periodic acid of

approximately 0.023~0.114 mg per 1 mg sugar. The oxidation reaction was carried out at

23℃ for 18 hours.

The concentration and diafiltration of the activated polysaccharide were performed 2026200210

5 5 using 100 kDa MWCO ultrafiltration membrane. Diafiltration was performed on WFI of 10-

fold diafiltration volume. Then, the purified activated polysaccharide was stored at 2 ~ 8 ℃.

The purified activated polysaccharide was characterized by in particular, (i) polysaccharide

concentration by colorimetric determination, (ii) aldehyde concentration by colorimetric

determination, (iii) degree of oxidation and (iv) molecular weight by SEC-MALLS.

10 10 SEC-MALLS is used for determining the molecular weights of the polysaccharide

and polysaccharide-protein conjugate. SEC is used for separating the polysaccharide by

fluid dynamical volume. The refractive index (RI) and multi-angle laser light scattering

detector are used for molecular weight determination. When light interacts with a material,

light is scattering and the amount of scattered light is related to concentration, square of

15 15 dn/dc (unique refractive index increase) and molar mass of a material. The molecular

92

weight measured value is calculated on the basis of the reading value from scattered light

signal from MALLS detector and the concentration signal from RI detector.

The degree of oxidation (DO) of the activated polysaccharide was determined by

‘mole of sugar repeating unit ÷ mole of aldehyde’. By various colorimetry methods, for 2026200210

5 5 example, using Anthrone method, the mole of sugar repeating unit was determined. In

addition, at the same time, using Park-Johnson colorimetry method, the mole of aldehyde

was determined. was determined.

Preferably, the activated Streptococcus pneumoniae serotype 2 capsular

polysaccharide obtained by the method has a degree of oxidation of 2 to 18 and a

10 10 molecular weight of about 100 kDa to 400 kDa.

Conjugation process

The activated polysaccharide was combined with sucrose at a ratio of sucrose of 2

to 8 g per the activated polysaccharide gram. Subsequently, the bottle of the combined

mixture was lyophilized. Following lyophilization, the bottle containing the lyophilized

15 15 activated polysaccharide was stored at -20 to -30 ℃. The calculated amount of CRM197

93

protein was separately lyophilized. The lyophilized CRM197 was stored at -20 to -30 ℃.

The lyophilized activated polysaccharide was recomposed in anhydrous dimethyl

sulfoxide solution (DMSO). When completing dissolution of the polysaccharide, for

recomposing anhydrous DMSO, it was added to lyophilized CRM197. The activated 2026200210

5 5 polysaccharide recomposed in the reaction container was combined with the recomposed

CRM197 (input ratio 0.5 to 2:1) and then it was mixed thoroughly. The conjugation reaction

was initiated by adding sodium cyanoborohydride (NaBH3CN) of 1.0 mole equivalent to

the reaction mixture. WFI was added to the reaction mixture at a target concentration of

1% (v/v) and it was reacted at 23℃ for 22 to 26 hours. The conjugation reaction was

10 10 terminated by adding sodium borohydride (NaBH4) of 2.0 mole equivalent and adding WFI

at a target concentration of 5%(v/v) to the reaction mixture, thereby capping unreacted

aldehyde. The capping reaction was conducted at 23℃ for 4.5 hours.

The conjugate solution was diluted with 0.9% sodium chloride solution during the

preparation for purification by concentration and diafiltration using 100kDa MWCO

15 15 membrane. The diluted conjugate solution was passed through a 0.8 ㎛ filter and

94

diafiltration was carried out using 0.9% sodium chloride at a 15-fold to 40-fold diafiltration

volume. After completing the diafiltration, the residual solution was filtrated through a

0.2㎛ filter. The conjugate solution was diluted with 0.9% sodium chloride solution so as

to be less than approximately 0.55 mg/mL concentration and was under sterile filtration 2026200210

5 5 and wasstored and was storedatat22to to 88 °C. ℃.

The purified serotype 2 conjugate was characterized by (i) polysaccharide

concentration by colorimetric determination, (ii) protein concentration by colorimetric

determination (Lowry), (iii) ratio of polysaccharides to protein, (iv) molecular size

distribution by size exclusion chromatography (CL-4B), (iv) content of free sugar and (v)

10 10 molecular weight by SEC-MALLS.

The characteristic change of the serotype 2 conjugate was observed by controlling

the degree of oxidation (DO) based on the preparation method. The result was

summarized summarized in in Table Table 1.1.

【Table 1】

Conjugate 1-1 1-2 1-3 1-4

95

number number

Activated 365 313 300 231

polysaccharide

molecular 2026200210

weight, kDa

DO DO 14.2 8.6 6.7 2.7

Input ratio (P:S) 1:1

% Conjugate 72 47 57 67

yield

Ratio of 1.2 1.1 1.1 1.2 1.2 1.0 1.0

saccharides to

protein

% Free 27 13 13 8 8 11

polysaccharide

% Molecular 93 92 88 64

96

weight

distribution distribution

Conjugate 4,918 3,845 3,485 1,988

molecular 2026200210

molecular

weight, kDa

the mixing ratio of the activated polysaccharides and CRM197 during the lyophilization on

the basis of the preparation method. The result was summarized in Table 2.

5 【Table 2】

Conjugate 1-5 1-6 1-6 1-7 1-7 1-8 1-8 1-9 1-10 1-10 1-11 1-11 1-12 1-12 1-13 1-13 1-14

number number

Activated Activated 260 260 147 147

polysaccharid

e e molecular molecular

97

weight, kDa

DO 9.4 3.0 3.0

Input ratio 2:1 2:1 1.5:1 1.5:1 1:1 1:1 0.7:1 0.7:1 0.5:1 0.5:1 2:1 2:1 1.5:1 1.5:1 1:1 1:1 0.7:1 0.7:1 0.5:1 0.5:1

(P:S) 2026200210

% Conjugate 47 53 57 58 58 64 70 70 73 68

yield

Ratio Ratio of 0.53 0.69 0.99 1.44 1.81 0.58 0.69 1.04 1.41 1.86

saccharides to

protein

% Free 14 15 18 18 26 26 27 27 11 0 11 9 14 14

polysaccharid

e

% Molecular 94 96 96 97 97 96 96 94 94 76 71 71 67 67 65 65 63 63

weight

distribution

98

Conjugate 15,81 11,38 5,53 3,54 2,62 8,25 4,07 2,18 1,22 1,05

molecular molecular 9 9 9 9 2 2 4 4 9 9 5 0 0 5 5 6 6 0 0

weight, kDa 2026200210

Research on immunogenicity of serotype 2 polysaccharide-protein conjugate

A monovalent conjugate composition comprising a polysaccharide-protein

conjugate from Streptococcus pneumoniae serotype 2 all individually conjugated to

5 CRM197 was formulated.

The immunogenicity of the monovalent immunogenic composition of the Table 1

and Table 2 was analyzed using ELISA in a rabbit, thereby measuring a serotype-specific

IgG concentration in serum.

5 female New Zealand white rabbit group of 2.5 kg to 3.5 kg was immunized via

10 10 intramuscular route at the 0th week with the proposed human clinical dose (conjugate 2.2

㎍; + aluminum 0.25 ㎎/㎖ as AlPO4). The rabbit was further immunized at the 2nd week

with the same dose of conjugate vaccine, and subsequently blood-gathering was carried

99

out at the 4th week. The serotype-specific ELISA was performed in the 0th and 4th serum

samples.

The analysis result was shown in Table 3. The rabbit immunized with the

monovalent conjugate composition (conjugate number 1-6) exhibited a significant 2026200210

5 increase of the total IgG titer against serotype 2. In the rabbit immunized with other

conjugate, a significant increase of the total IgG titer was shown.

The values of the following Table 3 are the result of showing the measured IgG

concentration after immunizing with the conjugate number 1-6 of the Table 1.

【Table 3】

IgG concentration (U/mL)

Serotype Pre-immunization Pre-immunization Post-immunization Post-immunization

2 2 130.0 62,164.7

10

[2. Streptococcus pneumoniae serotype 9N-derived polysaccharide-protein

conjugate]

100

Preparation of Streptococcus pneumoniae serotype 9N-derived polysaccharide-

protein conjugate

Preparation of cell band for master and preparation

Streptococcus pneumoniae serotype 9N was obtained from American Type Culture 2026200210

5 5 Collection (ATCC) (strain ATCC 6309). It was progressed in the same manner as serotype

2. 2.

Fermentation Fermentation

It was progressed in the same manner as serotype 2.

Purification Purification

10 10 It was progressed in the same manner as serotype 2.

Activation Activation

The final polysaccharide concentration of about 2.0 g/L was provided by adding a

calculated amount of WFI in order. If needed, the reaction pH was adjusted to

15 15 approximately 6.0. After adjusting the pH, the reaction temperature was adjusted to 23℃.

101

The oxidation was initiated by adding sodium periodic acid of 0.024~0.189 mg per

approximately 1 mg sugar. The oxidation reaction was carried out at 23℃ for 18 hours.

The concentration and diafiltration of the activated polysaccharide were performed

using 100 kDa MWCO ultrafiltration membrane. Diafiltration was performed on WFI of 10- 2026200210

5 fold diafiltration volume. Then, the purified activated polysaccharide was stored at 2 ~ 8 ℃.

SEC-MALLS is used for determining the molecular weights of the polysaccharide

10 10 and polysaccharide-protein conjugate. SEC is used for separating the polysaccharide by

dn/dc (unique refractive index increase) and molar mass of a material. The molecular

15 15 weight measured value is calculated on the basis of the reading value from scattered light

signal from MALLS detector and the concentration signal from RI detector.

The degree of oxidation (DO) of the activated polysaccharide was determined by

‘mole of sugar repeating unit ÷ mole of aldehyde’. By various colorimetry methods, for

example, using Anthrone method, the mole of sugar repeating unit was determined. In 2026200210

5 5 addition, at the same time, using Park-Johnson colorimetry method, the mole of aldehyde

was determined. was determined.

Preferably, the activated Streptococcus pneumoniae serotype 9N capsular

polysaccharide obtained by the method has a degree of oxidation of 2 to 19 and a

molecular weight of about 200 kDa to 700 kDa.

10 10 Conjugation process

The activated polysaccharide was combined with the carrier protein, CRM197 at a

ratio of CRM197 of 0.5 to 2 grams per the activated polysaccharide gram. Subsequently,

the combined mixture was lyophilized. Following lyophilization, the lyophilized mixture of

the activated polysaccharide and CRM197 was stored at -20℃.

15 15 The lyophilized mixture of the activated polysaccharide and CRM197 was

103

recomposed in 0.1M sodium phosphate solution and then was mixed sufficiently. The final

polysaccharide concentration in the reaction solution is about 10 to 20 g/L. The

conjugation was initiated by adding sodium cyanoborohydride (NaBH3CN) of 1.2 mole

equivalent to the mixture, and it was reacted at 37℃ for 48 hours. The conjugation 2026200210

5 5 reaction was terminated by adding 0.9% sodium chloride solution at the same volume as

the conjugation reaction solution and then adding sodium borohydride (NaBH4) of 2.0

mole equivalent, thereby capping unreacted aldehyde. The capping reaction was

conducted at 23℃ for 4.5 hours.

10 10 preparation for purification by concentration and diafiltration using 100kDa MWCO

membrane. The diluted conjugate solution was passed through a 0.45 ㎛ filter and

purification by concentration and diafiltration was carried out. Diafiltration using 100kDa

MWCO membrane was carried out using 0.9% sodium chloride solution at a 15-fold to 40-

fold diafiltration volume. After completing the diafiltration, the residual solution was

15 15 filtrated through a 0.2㎛ filter. The conjugate solution was diluted so as to be less than

104

approximately 0.55 mg/mL concentration and was under sterile filtration, and was stored

at at 2 to 88 °C. 2 to ℃.

The purified serotype 9N conjugate was particularly characterized by (i)

polysaccharide concentration by colorimetric determination, (ii) protein concentration by 2026200210

55 colorimetric determination (Lowry), (iii) ratio of polysaccharides to protein, (iv) molecular

size distribution by size exclusion chromatography (CL-4B), (iv) content of free sugar and

(v) molecular weight by SEC-MALLS.

The characteristic change of the serotype 9N conjugate was observed by

controlling the degree of oxidation (DO) based on the preparation method. The result was

10 10 summarized summarized in in Table Table 4.4.

【Table 4】

Conjugate number 2-1 2-2 2-3 2-4 2-5 2-6

Activated polysaccharide 582 619 459 563 490 427

molecular weight, kDa

DO 18.2 9.4 7.4 6.7 4.3 2.3

105

Input ratio (P:S) 0.8:1

20.0 Polysaccharide concentration 20.0

in conjugation reaction

solution, g/L 2026200210

% Conjugate yield 53 43 39 32 33 39

Ratio of saccharides to 2.1 1.5 1.3 1.1 1.0 0.78

protein

% Free polysaccharide 44 28 22 20 21 31

% Molecular weight 52 49 50 55 44 31

distribution

Conjugate molecular weight, 860 860 1,110 1,912 1,168 1,189 1,160

kDa

The characteristic change of the serotype 9N conjugate was observed by

controlling the mixing ratio of the activated polysaccharides and CRM197 during the

106

lyophilization on the basis of the preparation method. The result was summarized in Table

5.

【Table 5】

Conjugate number 2-7 2-8 2-9 2-10 2-11 2026200210

2-7 2-8 2-9 2-10 2-11

Activated polysaccharide 287 287

molecular weight, kDa

DO 5.6

Input ratio (P:S) 2:1 1.5:1 1:1 0.67:1 0.5:1

Polysaccharide 20.0 20.0

concentration concentration in in

conjugation reaction reaction

solution, g/L

% Conjugate yield 25 25 50 50 43 43 41 41 66 66

Ratio of saccharides Ratio of saccharidesto to 0.71 0.71 0.85 0.85 1.0 1.0 1.2 1.2 1.8 1.8

protein

107

% Free polysaccharide 5 6 15 27 27 62 62

% Molecular weight 52 58 50 40 22

distribution

Conjugate molecular 3,720 3,713 1,327 1,016 545 2026200210

weight, kDa

The characteristic change of the serotype 9N conjugate was observed by

controlling the polysaccharide concentration in the conjugation reaction solution on the

basis of the preparation method. The result was summarized in Table 6.

5 5 【Table 6】

Conjugate number 2-12 2-12 2-13 2-13 2-14 2-14 2-15 2-15 2-16 2-16

Activated polysaccharide 560 560

molecular weight, kDa

DO 6.1

Input ratio (P:S) 0.8:1

108

Polysaccharide 10.0 10.0 12.5 15.0 17.5 20.0 20.0

concentration concentration in in

conjugation reaction reaction

solution, g/L 2026200210

% Conjugate yield 20 20 31 31 28 28 40 42 42

Ratio of saccharides to 1.0 1.0 1.0 1.0 0.93 0.93 0.99 0.99 0.97 0.97

protein

% Free polysaccharide 32 30 22 21 18

% Molecular Molecular weight 17 27 40 47 54 %

distribution

Conjugate molecular 560 546 845 932 1,438

weight, kDa

Research on immunogenicity of serotype 9N polysaccharide-protein conjugate

A monovalent conjugate composition comprising a polysaccharide-protein

109

conjugate from Streptococcus pneumoniae serotype 9N all individually conjugated to

CRM197 was formulated.

The immunogenicity of the monovalent immunogenic composition of the Table 4

to Table 6 was analyzed using ELISA in a rabbit, thereby measuring a serotype-specific IgG 2026200210

5 5 concentration in serum. concentration in serum.

The female New Zealand white rabbit group was immunized via intramuscular

route in the same manner as serotype 2.

The analysis result was shown in Table 7. The rabbit immunized with the

monovalent conjugate composition (conjugate number 2-8) exhibited a significant

10 10 increase of the total IgG titer against serotype 9N. In the rabbit immunized with other

conjugate, a significant increase of the total IgG titer was shown.

The values of the following Table 7 are the result of showing the measured IgG

concentration after immunizing with the conjugate number 2-8 of the Table 5.

【Table 7】

IgG concentration (U/mL)

110

Serotype Pre-immunization Post-immunization

9N 130.0 656,345.3

[3. Streptococcus pneumoniae serotype 17F-derived polysaccharide-protein 2026200210

conjugate]

Preparation of Streptococcus pneumoniae serotype 17F-derived polysaccharide-

5 protein conjugate

Preparation of cell band for master and preparation

Streptococcus pneumoniae serotype 17F was obtained from American Type

Culture Collection (ATCC) (strain ATCC 6317). In order to enhance the strain and remove

components of animal origin, a seed stock was cultured for several generations. It was

10 10 progressed in the same manner as serotype 2.

Fermentation

It was progressed in the same manner as serotype 2.

111

Purification Purification

It was progressed in the same manner as serotype 2.

Activation 2026200210

Activation

5 5 The final polysaccharide concentration was adjusted so as to be about 2.0 g/L in

0.01N hydrochloric acid solution by adding a calculated amount of 0.1N hydrochloric acid

carried out at about 60 ℃ for 60 minutes. After lowering the temperature of the reaction

solution to a room temperature, the reaction pH was adjusted to approximately 6.0 by

10 10 adding 0.1M sodium monohydrogen phosphate solution. After adjusting the pH, the

temperature was adjusted to 23℃. The oxidation was initiated by adding sodium periodic

acid of approximately 0.008~0.219 mg per 1mg sugar. The oxidation reaction was carried

out at 23 out at ℃ for 23 °C for 18 18 hours. hours.

15 15 using 100 kDa MWCO ultrafiltration membrane. Diafiltration was performed on WFI of 10-

determination, (iii) degree of oxidation and (iv) molecular weight by SEC-MALLS. 2026200210

5 5 SEC-MALLS is used for determining the molecular weights of the polysaccharide

10 10 dn/dc (unique refractive index increase) and molar mass of a material. The molecular

signal from MALLS detector and the concentration signal from RI detector.

The degree of oxidation (DO) of the activated polysaccharide was determined by

15 15 example, using Anthrone method, the mole of sugar repeating unit was determined. In

113

was determined. was determined.

Preferably, the activated Streptococcus pneumoniae serotype 17F capsular

polysaccharide obtained by the method has a degree of oxidation of 1 to 22 and a 2026200210

5 5 molecular weight of about 400 kDa to 900 kDa.

Conjugation process

The activated polysaccharide was combined with the carrier protein, CRM197 at a

ratio of CRM197 of 1.0 gram per the activated polysaccharide gram. Subsequently, the

combined mixture was lyophilized. Following lyophilization, the lyophilized mixture of the

10 10 activated polysaccharide and CRM197 was stored at -20℃.

The lyophilized mixture of the activated polysaccharide and CRM197 was

recomposed in 0.1M sodium phosphate solution (pH 7.2±0.1). The final polysaccharide

concentration in the reaction solution is 15.0 to 25.0 g/L. The conjugation was initiated by

adding sodium cyanoborohydride (NaBH3CN) of 1.2 mole equivalent to the mixture, and it

15 15 was reacted at 37℃ for 48 hours. The conjugation reaction was terminated by adding 0.9%

sodium chloride solution at the same volume as the conjugation reaction solution and

then adding sodium borohydride (NaBH4) of 2.0 mole equivalent, thereby capping

unreacted aldehyde. The capping reaction was conducted at 23℃ for 4.5 hours.

The conjugate solution was diluted with 0.9% sodium chloride solution during the 2026200210

5 5 preparation for purification by concentration and diafiltration using 100kDa MWCO

fold diafiltration volume. After completing primary diafiltration, the residual solution was

10 10 filtrated through a 0.2㎛ filter and was stored at 2 to 8 ℃.

The purified serotype 17F conjugate was particularly characterized by (i)

polysaccharide concentration by colorimetric determination, (ii) protein concentration by

colorimetric determination (Lowry), (iii) ratio of polysaccharides to protein, (iv) molecular

15 15 (v) molecular weight by SEC-MALLS.

115

The characteristic change of the serotype 17F conjugate was observed by

controlling the degree of oxidation (DO) and the polysaccharide concentration in the

reaction solution based on the preparation method. The result was summarized in Table

8. 2026200210

8.

5 5 【Table 8】

Conjugate 3-1 3-2 3-3 3-4 3-5 3-6 3-7 3-8 3-9

number number

Activated 551 560 577 628 801

polysaccharide

molecular

weight, kDa

DO 21.3 9.4 7.5 3.8 1.3

Input ratio (P:S) 1:1

Polysaccharide 20.0 15.0 17.5 20.0 22.5 25.0

concentration in

conjugation

reaction

solution, g/L

% Conjugate 31 48 58 35 28 25 28 37 37 2026200210

31 48 58 35 28 25 28 37 37

yield

Ratio of 14.9 5.3 4.9 2.8 0.68 0.65 0.67 0.70 0.71

saccharides to

protein

% Free 84 84 82 82 78 78 60 60 8 8 4 4 4 4 6 6 4 4

polysaccharide

% Molecular -- 18 18 38 38 48 48 49 49 58 58

weight

distribution

Conjugate 372 706 706 456 456 1,064 1,346 2,115 2,531 3,150 4,423

molecular

117

weight, kDa

Research on immunogenicity of serotype 17F polysaccharide-protein conjugate

A monovalent conjugate composition comprising a polysaccharide-protein 2026200210

conjugate from Streptococcus pneumoniae serotype 17F all individually conjugated to

5 5 CRM197 wasformulated. CRM197 was formulated.

The immunogenicity of the monovalent immunogenic composition of the Table 8

to Table 6 was analyzed using ELISA in a rabbit, thereby measuring a serotype-specific IgG

concentration in serum.

The female New Zealand white rabbit group was immunized via intramuscular

10 10 route in the same manner as serotype 2.

The analysis result was shown in Table 9. The rabbit immunized with the

monovalent conjugate composition (conjugate number 3-8) exhibited a significant

increase of the total IgG titer against serotype 17F. In the rabbit immunized with other

conjugate, a significant increase of the total IgG titer was shown.

The values of the following Table 9 are the result of showing the measured IgG

concentration after immunizing with the conjugate number 3-8 of the Table 8.

【Table 9】

IgG Concentration (U/mL) 2026200210

Serotype Pre-immunization Post-immunization Post-immunization

17F 17F 130.0 130.0 227,590.3

5 [4. Streptococcus pneumoniae serotype 20-derived polysaccharide-protein

conjugate]

Preparation of Streptococcus pneumoniae serotype 20-derived polysaccharide-

protein conjugate

Preparation of cell band for master and preparation

10 10 Streptococcus pneumoniae serotype 20 was obtained from American Type Culture

Collection (ATCC) (strain ATCC 6320). It was progressed in the same manner as serotype

2.

Fermentation

It was progressed in the same manner as serotype 2. 2026200210

5 5 Purification Purification

It was progressed in the same manner as serotype 2.

Activation Activation

The final polysaccharide concentration was adjusted so as to be about 2.0 g/L in

10 10 0.01N hydrochloric acid solution by adding a calculated amount of 0.1N hydrochloric acid

solution and WFI in order. The oxidation was initiated by adding sodium periodic acid of

approximately 0.010~0.038 mg per 1 mg sugar. The oxidation reaction was carried out at

23 ℃for 23 °C for18 18 hours. hours.

120

signal from MALLS detector and the concentration signal from RI detector.

The degree of oxidation (DO) of the activated polysaccharide was determined by

121

was determined. was determined.

Preferably, the activated Streptococcus pneumoniae serotype 20 capsular

polysaccharide obtained by the method has a degree of oxidation of 4 to 16 and a 2026200210

5 5 molecular weight of about 400 kDa to 800 kDa.

Conjugation process

The activated polysaccharide was combined with the carrier protein, CRM197 at a

10 10 activated polysaccharide and CRM197 was stored at -20℃.

The lyophilized mixture of the activated polysaccharide and CRM197 was

concentration in the reaction solution is 15.0 g/L. The conjugation was initiated by adding

sodium cyanoborohydride (NaBH3CN) of 1.2 mole equivalent to the mixture, and it was

15 15 reacted at 37℃ for 48 hours. The conjugation reaction was terminated by adding 0.9%

then adding sodium borohydride (NaBH4) of 2.0 mole equivalent, thereby capping

unreacted aldehyde. The capping reaction was conducted at 23℃ for 4.5 hours.

membrane. The diluted conjugate solution was passed through a 0.45 ㎛ filter and 2-step

MWCO membrane was carried out using 0.9% sodium chloride solution at a 20-fold

diafiltration volume. After completing primary diafiltration, the residual solution was

10 10 filtrated through a 0.2㎛ filter and was stored at 2 to 8 ℃. The conjugate solution was

diluted so as to be less than approximately 0.55 mg/mL concentration and was under

sterile filtration, and was stored at 2 to 8 ℃.

The purified serotype 20 conjugate was particularly characterized by (i)

15 15 colorimetric determination (Lowry), (iii) ratio of polysaccharides to protein, (iv) molecular

123

(v) molecular weight by SEC-MALLS.

The characteristic change of the serotype 20 conjugate was observed by

controlling the degree of oxidation (DO) based on the preparation method. The result was 2026200210

5 5 summarized summarized in in Table Table 10. 10.

【Table 10】

Conjugate number 4-1 4-2 4-2 4-3 4-4 4-4 4-5 4-6 4-7

Activated 651 749 675 463 613 444 712

polysaccharide

molecular weight,

kDa

DO 15.7 15.4 8.9 7.3 7.3 6.7 6.7 4.8 4.8 4.6

Input ratio (P:S) 1:1

% Conjugate yield 65 57 56 49 45 24 20

Ratio of saccharides 3.7 2.9 2.5 2.7 2.2 2.1 1.5

124

to protein

% Free 29 28 16 15 16 11 8

polysaccharide

% Molecular weight 84 -- 79 78 82 78 -- 2026200210

distribution

Conjugate 1,968 1,271 3,349 2,458 3,645 2,123 2,563

molecular weight,

kDa

Research on immunogenicity of Streptococcus pneumoniae serotype 20

polysaccharide-protein conjugate

A monovalent conjugate composition comprising a polysaccharide-protein

5 5 conjugate from Streptococcus pneumoniae serotype 20 all individually conjugated to

CRM197 was formulated.

The immunogenicity of the monovalent immunogenic composition of the Table 10

125

was analyzed using ELISA in a rabbit, thereby measuring a serotype-specific IgG

concentration in serum. concentration in serum.

The female New Zealand white rabbit group was immunized via intramuscular

route in the same manner as serotype 2. 2026200210

5 5 The analysis result was shown in Table 11. The rabbit immunized with the

monovalent conjugate composition (conjugate number 4-7) exhibited a significant

increase of the total IgG titer against serotype 20. In the rabbit immunized with other

conjugate, a significant increase of the total IgG titer was shown.

The values of the following Table 11 is the result of showing the measured IgG

10 10 concentration after immunizing with the conjugate number 4-7 of the Table 10.

【Table 11】

IgG concentration (U/mL)

Serotype Pre-immunization Pre-immunization Post-immunization

20 20 166.2 166.2 277,210.1

126

Example 2. Preparation of multivalent Streptococcus pneumoniae polysaccharide-

protein conjugate

[5. Streptococcus pneumoniae 15-valent polysaccharide-protein conjugate] 2026200210

5 Preparation of Streptococcus pneumoniae 15-valent polysaccharide-protein

conjugate

A 15-valent conjugate composition comprising the polysaccharide-protein

conjugate derived from Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9N,

9V, 14, 18C, 19A, 19F, and 23F all individually conjugated to CRM197 (15vPnC) was

10 10 formulated. formulated.

For serotypes 2 and 9N, the conjugate was prepared by the afore-mentioned

method, and for other serotypes, the conjugate was prepared according to the method

disclosed in Korean Patent Application 2012-0065893.

The required volume of the final bulk concentrate was calculated based on batch

15 15 volume and bulk saccharide concentration. A required amount of 0.85% sodium chloride,

127

polysorbate 80 and succinate buffer were added to a pre-labeled formulation container,

and then the bulk concentrate was added. It was sufficiently mixed and was filtrated

through a 0.22㎛ filter. During and after addition of bulk aluminum phosphate, the

formulated bulk solution was slowly mixed. pH was checked and was adjusted if necessary. 2026200210

5 5 The formulated bulk product was stored at 2 to 8℃. The obtained vaccine composition

contained each saccharide of 2.2 ㎍, but 6B of 4.4 ㎍; CRM 197 carrier protein of about

32 ㎍; adjuvant of aluminum element of 0.125 mg (0.5 mg aluminum phosphate); sodium

chloride about 4.25 mg; succinate buffer about 295 ㎍; and polysorbate 80 about 100 ㎍

in in the total 0.5 the total 0.5 mL. mL.

10 10

Research on immunogenicity of Streptococcus pneumoniae 15-valent

polysaccharide-protein conjugate

IgG concentration measurement

The immunogenicity of the 15-valent immunogenic composition was analyzed in a

15 15 rabbit using ELISA, thereby measuring the serotype-specific IgG concentration in serum.

128

6 female New Zealand white rabbit group of 2.5 kg to 3.5 kg was immunized via

intramuscular route at the 0th week with the proposed human clinical dose (conjugate 2.2

㎍, except for serotype 6b determined as 4.4 ㎍; + aluminum 0.25 ㎎/㎖ as AlPO4). The

rabbit was further immunized at the 3rd week with the same dose of conjugate vaccine, 2026200210

5 5 and subsequently blood-gathering was carried out at an interval of 3 weeks. The serotype-

specific ELISA was performed in serum samples of each week.

The serotype specific immune response for the vaccine formulation according to

the present invention and the vaccine formulation of the comparative example was

evaluated by IgG ELISA. The analysis result was summarized in Table 12. It shows IgG

10 10 concentrations (U/mL) as time passes after inoculation. It was shown that the rabbit

immunized by the 15vPnC produced antibodies against serotypes 2 and 9N which could

not be obtained with Prevnar13, and particularly, it could induce an equivalent or excellent

serum IgG titer compared to Prevnar13, even though the valence number increased by

serotype addition.

15 15

129

【Table 12】

ELISA Prevnar13 SK-15

Type Day0 Day21 Day42 Day63 Day0 Day21 Day42 Day63 2026200210

11 0 0 8522 8522 14187 14187 10207 10207 0 0 2053 2053 17110 17110 7752 7752

2 2 -- -- -- -- 134 134 47864 47864 36482 36482 23790 23790

3 3 0 968 7229 5330 0 2084 11069 10947

4 4 0 0 3831 3831 23100 23100 16654 16654 0 0 2592 2592 15000 15000 10179 10179

5 97 97 5597 5597 13083 14819 14819 111 111 4866 19615 19615 16062 16062

6A 6A 0 0 15810 15810 28609 28609 20581 20581 0 0 3634 3634 20313 20313 10141 10141

6B 0 12920 43575 34932 0 4296 29763 14204

7F 7F 0 0 48129 48129 26694 26694 18014 18014 0 0 43979 43979 21590 10878 10878

9N 9N -- -- -- -- 0 0 9197 9197 17085 17085 13021 13021

130

9V 436 17281 35392 9442 373 15128 12143 12304

14 194 8365 10403 10403 10786 10786 416 7066 7066 13160 13160 16043 16043

18C 0 19101 23989 24862 0 19385 14192 14565 2026200210

19A 0 71160 136042 139273 0 24713 64138 59135

19F 0 40695 53165 56443 0 8382 38304 30852

23F 0 11171 62331 47522 0 7116 65352 67085

OPA analysis result

In order to confirm whether the 15-valent polysaccharide-protein conjugate

induced a functional antibody reaction, multiplexed opsonophagocytic killing assay

5 (MOPA) was carried out.

By collecting the same amount of serum by each subject, serum was pooled

between the same groups. Streptococcus pneumoniae was cultured in a THY medium by

each serum and was diluted to be 1000CFU/10uL. Opsonization buffer 200uL, diluted

131

serum 10uL, and diluted Streptococcus pneumoniae 10uL were mixed and it was reacted

at a room temperature for 1 hour. The mixed solution of a pre-differentiated HL-60 cell

and a complement was added and it was reacted in a CO2 incubator (37℃) for 1 hour. The

phagocytosis was stopped by lowering the temperature and the reaction solution 5uL was 2026200210

5 5 painted out in an agar medium dried for 30 to 60 minutes in advance. It was cultured in

the CO2 incubator (37℃) for 12 to 18 hours and the number of colonies was counted. The

OPA titer was represented by the dilution rate in which 50% death was observed. As a

comparative example, a 13-valent vaccine (Prevnar13, Pfizer) was used to evaluate in the

same manner, and the result was summarized in Table 13.

10 10 【Table 13】

OPA OPA Prevnar13 Prevnar13 SK-15 SK-15

Type Day21 Day42 Day63 Day21 Day42 Day63

11 16 16 64 64 64 64 4 4 64 64 64 64

2 2 -- -- -- 128 128 512 512 512 512

3 3 11 2 2 4 4 11 4 4 4 4

4 128 1024 1024 128 1024 1024

5 64 256 512 32 256 512

6A 512 2048 2048 256 2048 2048

6B 256 2048 2048 128 2048 2048 2026200210

7F 1024 2048 2048 1024 2048 2048

9N -- -- -- 512 2048 2048

9V 256 512 512 256 512 512

14 256 1024 1024 256 1024 1024

18C 1024 1024 2048 1024 512 2048 2048

19A 512 512 1024 1024 2048 2048 256 256 1024 1024 1024 1024

19F 256 1024 1024 128 512 512

23F 256 2048 2048 256 2048 2048

[6. Streptococcus pneumoniae 23-valent polysaccharide-protein conjugate]

Preparation of Streptococcus pneumoniae 23-valent polysaccharide-protein

133

conjugate

A 23-valent conjugate composition comprising the polysaccharide-protein

conjugate in which polysaccharides derived from Streptococcus pneumoniae serotypes 1,

2, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F were 2026200210

5 5 conjugated to CRM197 and the polysaccharide-protein conjugate in which Streptococcus

pneumoniae serotypes 3 and 5 were conjugated to TT (Tenus toxoid) (23vPnC) was

formulated. formulated.

For serotypes 2, 9N, 17F and 20, the conjugate was prepared by the afore-

mentioned method, and for other serotypes, the conjugate conjugated to CRM197 or TT

10 10 was prepared according to the methods disclosed in Korean Patent Application 2012-

0065893, U.S. Patent Applications 62/371,529, 62/371,553 and 62/626,482.

The required volume of the final bulk concentrate was calculated based on batch

volume and bulk saccharide concentration. A required amount of 0.85% sodium chloride,

15 15 and then the bulk concentrate was added. It was sufficiently mixed and was filtrated

134

formulated bulk solution was slowly mixed. pH was checked and was adjusted if necessary.

The formulated bulk product was stored at 2 to 8℃. The obtained vaccine composition

contained each saccharide of 2.2 ㎍, but 6B of 4.4 ㎍; CRM 197 carrier protein of about 2026200210

5 50 to 85 ㎍; an experimental amount of aluminum adjuvant (for example, in case of Group

4 in Table 14, aluminum element of 0.125 mg, that is, 0.5 mg aluminum phosphate); sodium

in in the total 0.5 the total 0.5 mL. mL.

10 10 Research on immunogenicity of Streptococcus pneumoniae 23-valent

polysaccharide-protein conjugate

IgG concentration measurement

The immunogenicity of the 23-valent immunogenic composition was analyzed in

a rabbit using ELISA, thereby measuring the serotype-specific IgG concentration in serum.

15 15 The ability of inducing a serotype-specific immune response of the 23vPnC vaccine

135

containing an adjuvant was investigated.

5 female New Zealand white rabbit group of 2.5 kg to 3.5 kg was immunized via

㎍, except for serotype 6b determined as 4.4 ㎍) in which aluminum of 0.0625 ㎎/㎖, 0.125 2026200210

5 5 ㎎/㎖, 0.25 ㎎/㎖, 0.5 ㎎/㎖ and 1 ㎎/㎖, respectively was comprised as AlPO4. The rabbit

was further immunized at the 2nd week with the same dose of conjugate vaccine, and

subsequently blood-gathering was carried out at the 4th week. The serotype-specific ELISA

was performed in the 0th and 4th serum samples.

As a comparative example, a 13-valent vaccine (Prevnar13, Pfizer) was used to

10 10 evaluate in the same manner, and the analysis result was summarized in Table 14. It shows

IgG concentrations (U/mL) as 4th week passes after inoculation.

The geometric mean titer (GMT) measured in the pooled serum sample after

administering the 23vPnV vaccine and Prevnar13 twice was proposed. These data

demonstrate that a higher level of IgG antibody is induced compared to the same vaccine

15 15 which does not contain an adjuvant, when an adjuvant is comprised in the 23vPnV

136

formulation. In particular, it was confirmed that a 23-valent immunogenic vaccine

comprising all serotypes 2, 9N, 17F and 20 could be obtained. As could be seen in the

following result, in particular, an antibody against serotype 2 and the like, which could not

be obtained by Prevnar13, could be produced. In addition, it was confirmed that it could 2026200210

5 induce an immune response against all the comprised serotype without greatly affecting

production of an antibody against an antigen of other serotypes, despite greatly increased

valence number. valence number.

【Table 14】

Group 1 : Group 2 : Group 3 : Group 4 : Group 5 : Group 6 : Group 7

PCV24 PCV24 I I PCV24 PCV24 I I PCV24 PCV24 I I PCV24 PCV24 I I PCV24 I PCV24 PCV24 I I (Prevnar®1

alum alum 00 alum 62.5 alum 62.5 alum 125 alum 250 alum 250 alum 500 alum 1000 3)

Control Reference Reference

group group

Type 1 4421 10951 11205 9595 12821.5 12038.5 9131.0

Type 4850.7 4850.7 7347 7347 7358.2 7358.2 6833.1 6833.1 9909.4 9909.4 7875.3 7875.3 --

2 2

137

Type 15552.6 15552.6 17362.7 17362.7 23574.8 23574.8 11718 11718 24425.5 24425.5 18313.2 18313.2 4265.9 4265.9

3 3

Type 5676.1 5676.1 11391.8 11391.8 10274.5 10274.5 10699.7 10699.7 13622.7 13622.7 15469 15469 5930.1 5930.1

5 5 2026200210

Type 15265.3 13034.2 30631.9 17203.6 21335.1 21011.1 5697.2

6A

Type 3566.3 3566.3 8567.3 8567.3 17964.5 17964.5 8101.7 8101.7 21985.6 21985.6 19993.7 19993.7 4136.6 4136.6

6B 6B

Type 10474.8 10474.8 32904.5 32904.5 36935.5 36935.5 28650.3 28650.3 45878.8 45878.8 48559 48559 31991.5 31991.5

7F 7F

Type 18684.5 18684.5 26486.5 26486.5 39449.2 39449.2 26369.3 26369.3 43372.9 43372.9 56812.6 56812.6 --

8 8

Type 29350.5 29350.5 52915.1 52915.1 61918.7 61918.7 25465.9 25465.9 73406.3 95465.2 95465.2 --

9N 9N

Type 9041 9041 19108 19108 24479.9 24479.9 24602.9 24602.9 30087.5 30087.5 36732.8 36732.8 23053.6 23053.6

9V 9V

Type 19833.3 19833.3 38644.7 38644.7 39959.5 39959.5 73948.6 73948.6 40524.8 40524.8 60464.4 60464.4 --

10A 10A

Type 893.6 893.6 3845.9 3845.9 4447.6 4447.6 2474.3 5683.2 5683.2 8358.5 --

11A 11A

Type 4785.2 10497.8 7381.7 7381.7 8444.8 8444.8 8113.6 8113.6 14211.8 14211.8 --

12F 12F 2026200210

Type 9177.7 9177.7 17574 17574 12811.4 12811.4 15834.6 15834.6 13543.4 13543.4 23876.5 23876.5 11178.8 11178.8

14 14

Type 4908.7 4908.7 17293.7 17293.7 18345.2 18345.2 4936.3 19436.1 19436.1 28797.9 28797.9 --

15B 15B

Type 7441.1 7441.1 9373.4 9373.4 14848.8 14848.8 10186.3 10186.3 14319.7 14319.7 32906.8 32906.8 --

17F 17F

Type 16747.2 16747.2 27864.6 27864.6 44605.6 44605.6 29416.3 29416.3 37658 37658 38860.5 38860.5 34163.2 34163.2

18C 18C

Type 781.9 781.9 4088.9 5846.5 4114.6 4114.6 8824.5 8824.5 12216.5 12216.5 14381.8 14381.8

19A 19A

Type 4501.9 4501.9 32543.6 32543.6 27502.3 27502.3 28629.2 28629.2 36290.5 36290.5 68218.5 68218.5 15315.5

19F 19F

Type 18252.8 18252.8 32553.5 32553.5 34663.5 34663.5 21760.3 21760.3 37978.2 37978.2 44630 44630 --

20 20

139

Type 6790.8 6790.8 24687.1 24687.1 24800.1 24800.1 19953 19953 42634.5 42634.5 55543.4 --

22F 22F

Type 823.6 823.6 3859.8 3859.8 7938 7938 6634 6634 9468.7 10348.8 9440.5

23F 23F 2026200210

Type 7261.6 7261.6 23864.8 23864.8 24843.6 20105.5 20105.5 22586.9 22586.9 28958 28958 --

33F 33F

[7. Streptococcus pneumoniae 24-valent polysaccharide-protein conjugate]

Preparation of Streptococcus pneumoniae 24-valent polysaccharide-protein

conjugate

5 5 A 24-valent conjugate composition comprising the polysaccharide-protein

conjugate in which polysaccharides derived from Streptococcus pneumoniae serotypes 2,

3, 4, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F

were conjugated to CRM197 and the polysaccharide-protein conjugate in which

Streptococcus pneumoniae serotypes 1 and 5 were conjugated to TT (Tenus toxoid)

10 10 (24vPnC) was formulated. For serotypes 2, 9N, 17F and 20, the conjugate was prepared by

the afore-mentioned method, and for other serotypes, the conjugate conjugated to

CRM197 or TT was prepared according to the methods disclosed in Korean Patent

Application 2012-0065893, U.S. Patent Applications 62/371,529, 62/371,553 and

62/626,482. 2026200210

5 5 The required volume of the final bulk concentrate was calculated based on batch

10 10 formulated bulk solution was slowly mixed. pH was checked and was adjusted if necessary.

50 to 90 ㎍; adjuvant of aluminum element of 0.125mg (0.5 mg aluminum phosphate);

sodium chloride about 4.25 mg; succinate buffer about 295 ㎍; and polysorbate 80 about

15 15 100 ㎍ in the total 0.5 mL.

141

Research on immunogenicity of Streptococcus pneumoniae 24-valent

polysaccharide-protein conjugate

OPA analysis result 2026200210

5 5 In order to confirm whether the 24-valent polysaccharide-protein conjugate

induced a functional antibody reaction, opsonophagocytic killing assay (OPA) was carried

out in three out in three rabbits rabbits in inthe thesame same manner manner asasthe the15-valent 15-valentvaccine. vaccine.

【Table 15】

Serotype PCV24 Prevnar 13

1 309 54

2 490 --

3 407 393

4 1290 2072

5 1516 306

6A 1817 1817 2355

6B 2888 2888 1614

7F 1277 952

8 279 -I

9N 653 52 2026200210

9N 653 52

9V A6 178 324

10A 658 -I

11A 675 -

12F 471 471 -I

14 959 539

15B 371 -

17F 348 -I

18C 1357 1996

19A 642 1870

19F 1521 1516

20 306 -

143

22F 22F 1703 --

23F 23F 1531 928

33F 33F 428 428 -- 2026200210

Through the result, it was confirmed that a 24-valent vaccine comprising all the

serotypes 2, 9N, 17F and 20 could be obtained. As could be seen in the result, in particular,

an antibody against serotypes 2, 17F and the like, which could not be obtained by

5 5 Prevnar13, could be produced. In addition, it was confirmed that the immune response

against all comprised serotypes could be induced without greatly affecting production of

an antibody against an antigen of other serotypes, although 11 serotypes were added to

Prevnar13.

10 10 【INDUSTRIAL APPLICABILITY】

The immunogenic composition of one example of the present invention may be

used used as as medicament. medicament.

144

The immunogenic composition of one example of the present invention may be

used as various therapeutic or prophylactic methods for prevention, treatment or

improvement of bacterial infection, diseases or conditions. In particular, the immunogenic

composition disclosed in the present invention may be used for prevention, treatment or 2026200210

5 5 improvement of Streptococcus pneumoniae infection, diseases or conditions in a subject.

In one embodiment, a method for inducing an immune response against

Streptococcus pneumoniae in a subject, comprising administering an effective dose of the

immunogenic composition of the present invention into the subject may be provided.

Claims

【CLAIMS】

【Claim 1】

An immunogenic composition comprising a Streptococcus pneumoniae

polysaccharide-protein conjugate, comprising 2026200210

a capsular polysaccharide derived from one or more selected from serotypes 1, 2,

3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F,

derived from Streptococcus pneumoniae; and

one or 2 or more of carrier proteins conjugated to the respective capsular

polysaccharide.

【Claim 2】

The immunogenic composition comprising a Streptococcus pneumoniae

polysaccharide-protein conjugate according to claim 1, wherein the polysaccharide is

activated and binds to the carrier protein at a molecular weight of 100 to 400 kDa to form

a conjugate, when the immunogenic composition comprises a polysaccharide derived

from serotype 2, or

146

the polysaccharide is activated and binds to the carrier protein at a molecular

weight of 400 to 900 kDa to form a conjugate, when the immunogenic composition

comprises a polysaccharide derived from serotype 17F, or

the polysaccharide is activated and binds to the carrier protein at a molecular 2026200210

weight of 400 to 800 kDa to form a conjugate, when the immunogenic composition

comprises a polysaccharide derived from serotype 20.

【Claim 3】

The immunogenic composition comprising a Streptococcus pneumoniae

polysaccharide-protein conjugate according to claim 1, wherein an immunogenic

conjugate comprising the polysaccharide derived from serotype 2 has a molecular weight

of 1,000 to 16,000 kDa, when the immunogenic composition comprises a polysaccharide

derived from serotype 2, or

an immunogenic conjugate comprising the polysaccharide derived from serotype

17F has a molecular weight of 300 to 4,500 kDa, when the immunogenic composition

comprises a polysaccharide derived from serotype 17F, or

147

an immunogenic conjugate comprising the polysaccharide derived from serotype

20 has a molecular weight of 1,000 to 4,000 kDa, when the immunogenic composition

comprises a polysaccharide derived from serotype 20.

【Claim 4】 2026200210

The immunogenic composition comprising a Streptococcus pneumoniae

polysaccharide-protein conjugate according to claim 1, wherein the carrier protein is TT

(Tetanus toxoid) or CRM197.

【Claim 5】

The immunogenic composition comprising a Streptococcus pneumoniae

polysaccharide-protein conjugate according to claim 1, wherein the ratio of the serotype

2 capsular polysaccharide to the carrier protein in the immunogenic conjugate

(polysaccharide/protein, W/W) when the immunogenic composition comprises a

polysaccharide derived from serotype 2 is 0.5 to 2.0, or

the ratio of the serotype 2 capsular polysaccharide to the carrier protein in the

immunogenic conjugate (polysaccharide/protein, W/W) when the immunogenic

148

composition comprises a polysaccharide derived from serotype 17F is 0.5 to 18, or

the ratio of the serotype 20 capsular polysaccharide to the carrier protein in the

immunogenic conjugate (polysaccharide/protein, W/W) when the immunogenic

composition comprises a polysaccharide derived from serotype 20 is 1 to 5. 2026200210

【Claim 6】

The immunogenic composition comprising a Streptococcus pneumoniae

polysaccharide-protein conjugate according to claim 1, wherein the immunogenic

composition is that

20 to 60% of the total molecular weight is present within 0.3 Kd in a CL-4B column,

in case of the immunogenic conjugate comprising a polysaccharide derived from serotype

2, or

15 to 60% of the total molecular weight is present within 0.3 Kd in a CL-4B column,

in case of the immunogenic conjugate comprising a polysaccharide derived from serotype

17F, or

70 to 90% of the total molecular weight is present within 0.3 Kd in a CL-4B column,

149

in case of the immunogenic conjugate comprising a polysaccharide derived from serotype

20. 20.

【Claim 7】

The immunogenic composition comprising a Streptococcus pneumoniae 2026200210

polysaccharide-protein conjugate according to claim 1, wherein the immunogenic

composition is that

the degree of oxidation of the polysaccharide conjugated to the conjugate is 2 to

18, in case of the immunogenic conjugate comprising a polysaccharide derived from

serotype 2, or

the degree of oxidation of the polysaccharide conjugated to the conjugate is 1 to

22, in case of the immunogenic conjugate comprising a polysaccharide derived from

serotype 17F, or

the degree of oxidation of the polysaccharide conjugated to the conjugate is 4 to

16, in case of the immunogenic conjugate comprising a polysaccharide derived from

serotype 20.

150

【Claim 8】

The immunogenic composition comprising a Streptococcus pneumoniae

polysaccharide-protein conjugate according to claim 1, wherein the immunogenic

composition is that polysaccharides derived from 15 serotypes different each other are 2026200210

conjugated to respective carrier proteins, and

the serotypes are 1, 2, 3, 4, 5, 6A, 6B, 7F, 9N, 9V, 14, 18C, 19A, 19F, and 23F, and

the serotypes are conjugated to CRM 197, respectively.

【Claim 9】

The immunogenic composition comprising a Streptococcus pneumoniae

polysaccharide-protein conjugate according to claim 1, wherein the immunogenic

composition is that polysaccharides derived from 23 serotypes different each other are

conjugated to respective carrier proteins, and

the serotypes are 1, 2, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F,

20, 22F, 23F, and 33F, and

among the serotypes, capsular polysaccharides derived from serotypes 3 and 5 are

151

conjugated to carrier protein TT and capsular polysaccharides derived from serotypes 1, 2,

6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F are

conjugated to carrier protein CRM197, respectively.

【Claim 10】 2026200210

The immunogenic composition comprising a Streptococcus pneumoniae

polysaccharide-protein conjugate according to claim 1, wherein the immunogenic

composition is that polysaccharides derived from 24 serotypes different each other are

conjugated to respective carrier proteins, and

the serotypes are 2, 3, 4, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A,

19F, 20, 22F, 23F, and 33F, and

capsular polysaccharides derived from serotypes 1 and 5 are conjugated to carrier

protein TT and capsular polysaccharides derived from serotypes 2, 3, 4, 6A, 6B, 7F, 8, 9N,

9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F are conjugated to carrier

protein CRM197, respectively.

【Claim 11】

The immunogenic composition comprising a Streptococcus pneumoniae

polysaccharide-protein conjugate according to claim 1, wherein the immunogenic

composition comprises a physiologically acceptable vehicle.

【Claim 12】 2026200210

The immunogenic composition comprising a Streptococcus pneumoniae

polysaccharide-protein conjugate according to claim 1, wherein the immunogenic

composition is a vaccine.

【Claim 13】

A preparation method of an immunogenic composition comprising a

Streptococcus pneumoniae polysaccharide-protein conjugate comprising

(a) a step of fermenting and dissolving a bacterial cell which produces a capsular

polysaccharide derived from one or more serotypes selected from the group consisting of

Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14,

15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F;

(b) a step of purifying a capsular polysaccharide derived from Streptococcus

153

pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A,

19F, 20, 22F, 23F, and 33F in the dissolved cell;

(c) a step of reacting the purified polysaccharide with an oxidizing agent to activate

it; and 2026200210

(d) a step of combining the activated polysaccharide with a carrier protein to form

a Streptococcus pneumoniae polysaccharide-protein conjugate bound to the carrier

protein.

【Claim 14】

The preparation method of an immunogenic composition comprising a

Streptococcus pneumoniae polysaccharide-protein conjugate according to claim 13,

wherein the preparation method further comprises a step of hydrolyzing the purified

Streptococcus pneumoniae capsular polysaccharide to size it, before the (c) step, in case

of the capsular polysaccharides derived from serotypes 2 and 17F.

【Claim 15】

The preparation method of an immunogenic composition comprising a

154

Streptococcus pneumoniae polysaccharide-protein conjugate according to claim 13,

wherein the combined carrier protein of the (d) step forms a conjugate with the

polysaccharide activated by reacting with one or more reducing agents selected from the

group consisting of cyanoborohydride, borane-pyridine and borohydride exchange resin. 2026200210

【Claim 16】

The preparation method of an immunogenic composition comprising a

Streptococcus pneumoniae polysaccharide-protein conjugate according to claim 13,

wherein the (c) step is reacting 0.01 ~ 0.22 ㎍ of periodate per 1 ㎍ polysaccharide at a

temperature of 20 to 25 ℃ for 15 to 20 hours.

【Claim 17】

[Claim 17]

A Streptococcus pneumoniae polysaccharide-protein conjugate for preventing or

treating Streptococcus pneumoniae infection, obtained by the preparation method

according to any one of claims 13 to 16.

【Claim 18】

A method for preventing or treating infection of Streptococcus pneumoniae

serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F,

23F, and/or 33F in a subject,

by administering an immunogenic composition comprising a Streptococcus

pneumoniae polysaccharide-protein conjugate, comprising 2026200210

a capsular polysaccharide derived from one or more selected from serotypes 1, 2,

3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F,

derived from Streptococcus pneumoniae; and

one or 2 or more of carrier proteins conjugated to the respective capsular

polysaccharide, into a subject.

【Claim 19】

A use for prevention or treatment of pneumococcal infection of a Streptococcus

pneumoniae polysaccharide-protein conjugate, comprising

a capsular polysaccharide derived from one or more selected from serotypes 1, 2,

3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F,

derived from Streptococcus pneumoniae; and

156

one or 2 or more of carrier proteins conjugated to the respective capsular

polysaccharide. 2026200210

157