阅读说明：本技术 新型多价多糖-蛋白轭合物疫苗组合物及其制剂 (Novel multivalent polysaccharide-protein conjugate vaccine compositions and formulations thereof ) 是由 达温德·吉尔 桑迪普·沙玛 于 2018-04-26 设计创作，主要内容包括：一种新型多价多糖-蛋白轭合物疫苗制剂。所述制剂是血清群A、C、Y、W和X脑膜炎奈瑟菌荚膜多糖(Men A、C、Y、W、X血清群)的液体或冻干或液体-冻干联合五价制剂,每种所述多糖分别与破伤风类毒素(TT)载体蛋白结合,获得Men A、C、Y、W、X-TT轭合物,具有一种或多种缓冲液,且具有或不具有佐剂及药学上可接受的成分/赋形剂。(A novel multivalent polysaccharide-protein conjugate vaccine formulation. The formulations are liquid or lyophilized or liquid-lyophilized combined pentavalent formulations of serogroup A, C, Y, W and X neisseria meningitidis capsular polysaccharides (Men a, C, Y, W, X serogroup), each of which is conjugated to Tetanus Toxoid (TT) carrier protein, respectively, to obtain Men a, C, Y, W, X-TT conjugates, with one or more buffers, with or without adjuvants and pharmaceutically acceptable ingredients/excipients.)

1. A novel multivalent polysaccharide-protein conjugate vaccine formulation, wherein said formulation is a liquid or lyophilized or liquid-lyophilized combined pentavalent formulation of serogroup A, C, Y, W and X neisseria meningitidis capsular polysaccharide (Men a, C, Y, W, X serogroup), each of said polysaccharides being separately conjugated to Tetanus Toxoid (TT) carrier protein, to obtain Men a, C, Y, W, X-TT conjugates, with one or more buffers, with or without adjuvants and pharmaceutically acceptable ingredients/excipients, wherein said formulation provides the required osmotic pressure, the required pH, high stability and the required immunogenicity.

2. The novel vaccine formulation of claim 1, wherein the liquid formulation comprises:

3. the novel vaccine formulation of claim 1, wherein the liquid formulation comprises:

4. the novel vaccine formulation of claim 1, wherein the liquid formulation comprises:

5. the novel vaccine formulation of claim 1, wherein the liquid formulation comprises:

6. the novel vaccine formulation of claim 1, wherein the lyophilized formulation comprises:

7. the novel vaccine formulation of claim 1, wherein the liquid-lyophilized cocktail formulation comprises:

8. the novel vaccine formulation according to claim 1, wherein the Men a, C, Y, W, X-TT conjugates are obtained by an optimized combination of conjugation chemistries.

9. The novel vaccine formulation according to claim 8, wherein the conjugation chemistry used to obtain the at least one conjugate is carbamate chemistry.

10. The novel vaccine formulation according to claim 9, wherein said at least one conjugate obtained with said carbamate chemistry is preferably a Men X-TT conjugate.

11. The novel vaccine formulation of claim 8, wherein the conjugation chemistry used to said obtain the at least one conjugate is cyanation chemistry.

12. The novel vaccine formulation according to claim 11, wherein the at least one conjugate obtained with the cyanation chemistry is preferably the Men a, C, Y, W-TT conjugate.

13. The novel vaccine formulation according to claim 1, wherein each of the capsular polysaccharides degrades within a range of 0.38 ± 0.1Kd, preferably within a range of 0.38 ± 0.06Kd, when molecular weight distribution is tested using HPLC PWXL4000 and 5000 tandem columns, resulting in a conjugate with high antigenicity, high immunogenicity and high stability.

14. The novel vaccine formulation according to any one of claims 1 to 8, wherein the conjugate produced has a linker arm between the polysaccharide and the carrier protein, wherein the linker arm is linked to the polysaccharide or to the carrier protein, or to both the polysaccharide and the carrier protein.

15. The novel vaccine formulation according to claim 14, wherein the linker arm is selected from adipic acid dihydrazide or hydrazine.

16. The novel vaccine formulation according to claim 1, wherein the Men X-TT conjugate preferably has a hydrazine linker arm and the Men a, Y, W-TT conjugate preferably has an adipic acid dihydrazide linker arm.

17. The novel vaccine formulation of claim 1, wherein the ratio of the carrier protein to polysaccharide of each of the conjugates is between 0.3-1.0.

18. The novel vaccine formulation according to claim 1, wherein the pharmaceutically acceptable excipient is selected from adjuvants, buffers, preservatives, stabilizers, surfactants, alone or in combination.

19. The novel vaccine formulation according to claim 6, wherein the excipient is selected from sucrose, maltose, arginine, lactose, sorbitol, histidine, glycine, alone or in any combination.

20. The novel vaccine formulation according to claim 6, wherein the diluent is selected from the group consisting of water, 5-20mM phosphate buffer, 500-⁺⁺⁺Aluminum phosphate per ml, alone or in any combination.

21. The novel vaccine formulation according to claim 7, wherein the lyophilized (lyo) fraction comprises Men a-TT conjugates, Men C-TT conjugates, alone or in any combination.

22. The novel vaccine formulation according to claim 7, wherein the excipient is selected from sucrose, maltose, arginine, lactose, sorbitol, histidine, glycine, alone or in any combination.

23. The novel vaccine formulation of claim 7, wherein the diluent is selected from the group consisting of water comprising Men C-TT, Men Y-TT, Men X-TT, Men W-TT, 520mM phosphate buffer, 500-⁺⁺⁺Aluminum phosphate per ml, alone or in any combination.

24. The novel vaccine formulation of claim 1, wherein the formulation is a liquid or lyophilized formulation or a mixed formulation thereof with a single or multiple dose regimen, with or without a preservative.

25. The novel vaccine formulation according to claim 1, wherein the optimal human dose range for serogroups A, C, Y, W and X is 2-10 μ g polysaccharide per dose per serogroup, preferably 5-10 μ g polysaccharide per serogroup A and X and 5 μ g polysaccharide per serogroup C, Y and W.

26. The novel vaccine formulation according to claim 1, wherein the desired osmolality of the formulation is 240-330 mOsmlo/Kg.

27. The novel vaccine formulation of claim 1, wherein the desired pH of the formulation is 6.5-7.5.

28. The novel vaccine formulation of claim 1, wherein the moisture content of the lyophilized portion of the lyophilized formulation or liquid-lyophilized formulation does not exceed 3%.

29. The novel vaccine formulation of claim 1, wherein the vaccine formulation is stable at elevated temperatures of 37 ± 2 ℃ for at least 21 days and at temperatures of 25 ± 2 ℃ for at least 3 months, with less than 40% free polysaccharide present.

30. The novel vaccine formulation of claim 1, wherein the vaccine formulation has high antigenicity and high immunogenicity.

31. The novel vaccine formulation of claim 1, wherein the vaccine formulation has the same immunogenicity even after 7 days of exposure at 37 ± 2 ℃ as compared to a stored formulation under real-time storage conditions.

32. The novel vaccine formulation of claim 1, wherein the formulation is preferably a liquid pentavalent meningococcal conjugate vaccine formulation with a single or multiple dose regimen, with or without a preservative.

33. The novel vaccine formulation of claim 1, wherein the formulation and the components can be used to produce a liquid or lyophilized or liquid-lyophilized pentavalent meningococcal ACYWX-TT combination vaccine.

Technical Field

The present invention relates to a novel polysaccharide-protein conjugate vaccine composition. More particularly, the invention relates to a pentavalent conjugate vaccine formulation of serogroup A, C, Y, W and X neisseria meningitidis capsular polysaccharide (Men a, C, Y, W, X serogroup) in combination with Tetanus Toxoid (TT) carrier protein and pharmaceutically acceptable ingredients/excipients. The pentavalent conjugate vaccine formulations of the present invention are in liquid or lyophilized form or a combination thereof.

Background

Vaccines are complex to produce and formulate, and require strict supervision to ensure personal safety and to maximise their efficacy and stability. All vaccines contain active ingredients that generate a protective immune response. The vaccine may also contain pharmaceutically acceptable additional components to improve the stability and/or immunogenicity of the formulation, thereby generating a strong protective immune response.

The world health organization recommends that countries with moderate or high incidence rates or frequent outbreaks of vaccine to prevent disease should be vaccinated regularly. For countries with lower risk of disease, it is recommended that high risk populations should be vaccinated.

Meningococcal disease is an acute, potentially serious disease caused by neisseria meningitidis bacteria. Neisseria meningitidis (meningococcus) is an aerobic gram-negative bacterium that has been serologically divided mainly into A, B, C, D, 29E, H, I, K, L, W135, X, Y and Z13 serogroups. The grouping system is based on capsular polysaccharides of organisms.

The WHO official website states that neisseria meningitidis is one of the most common causes of bacterial meningitis worldwide, being the only bacteria that can cause a large-scale epidemic of meningitis. The incidence of major epidemics is reported to be as high as 1000 per 10 ten thousand residents, especially in sub-saharan africa.

Neisseria meningitidis is transmitted by aerosol or direct contact with respiratory secretions of patients or healthy carriers. Endemic disease occurs primarily in the population of children and young adults, with infants at the highest incidence of 3-12 months, and older children and young adults more susceptible to epidemic disease. However, the rapid progression of meningococcal disease often leads to death within 1-2 days after the onset. Neisseria meningitidis infection may be prevented by vaccination.

Immunization is the only rational way to control meningitis disease. The most effective vaccine of neisseria meningitidis is a polysaccharide-protein conjugate vaccine formed by covalently binding activated protein to active polysaccharide. Since most natural polysaccharides are not capable of effective chemical attachment to a carrier protein without activation, chemical modification, also referred to as an "activation" process, of the polysaccharide is required prior to attachment to the carrier protein.

There are many conjugation reactions in which polysaccharides are covalently linked to proteins. Three more common methods include:

1) reductive amination wherein an aldehyde or ketone group on one component of the reaction reacts with an amino or hydrazine group on the other component, the C ═ N double bond thus formed being subsequently reduced to a C — N single bond by a reducing agent;

2) cyanation coupling, in which the polysaccharide is activated by cyanogen bromide (CNBr) or 1-cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP), the cyanate groups are introduced into the hydroxyl groups, which form covalent bonds with amino or hydrazine groups after addition of the protein component; and

3) carbodiimide reactions, in which carbodiimide activates a carboxyl group on one component of a conjugation reaction, the activated carboxyl group reacting with an amino or hydrazino group on the other component. These reactions are also often used to activate components of the conjugate prior to the conjugation reaction.

According to one publication, historically serogroup a (mena) was the most common cause of epidemic disease in regions of sub-saharan africa. In developed countries, serogroups B and C are the cause of most cases, with the remainder being due to serogroups W135 and Y. Serogroup C cases have recently been observed in regions of africa south of sahara, where historically this serogroup is not prevalent. Recently, serogroup X has also emerged in a few countries. Xie O et al (occurrence of African serogroup X meningococcal disease: vaccine required;. vaccine 2013Jun 12; 31 (27): 2852-6), serogroup X meningococcus was once considered to be a rare cause of sporadic meningitis, but serogroup X meningitis was developed in Nigerl, Uganda, Kenya, Mugo and Bukenna during 2006-2010, and at least 1300 serogroup X meningitis were found in 6732 reported cases of the latter.

According to another publication by NCBI, serogroup X meningococcus accounts for 16% of 702 cases of diagnosed bacterial meningitis in many cases during 2006-2009. In 3 months 2007, a outbreak of serogroup X meningococcus occurred in the claza region, with a seasonal cumulative incidence of 33/100000. During the year 2007-2010, the incidence of serogroup X meningococcus of bukinxol accounted for 7% of 778 cases of diagnosed bacterial meningitis, with an increase in the year 2009-2010 (4-35% of all cases diagnosed, respectively). In 2010, serogroup X meningococcal epidemics occurred in northern and middle regions of bukinawa; the highest regional cumulative incidence of serogroup X meningococcus during months 3-4 was estimated to be 130/100000.

Based on the above facts, the pentavalent meningococcal ACYWX polysaccharide-protein conjugate vaccine can provide a broader range of meningococcal disease coverage, except for serogroup B, because the MenB capsular polysaccharide is structurally similar to the molecule found in human neuronal cells, the conjugate vaccine is less likely to be prepared. Currently, various monovalent or multivalent vaccines, including serogroup A, C, Y and the W135 polysaccharide conjugate, have been approved for sale on the market. The prior art discloses vaccines against a variety of serogroups, including serogroup A, B, C, W and Y neisseria meningitidis, as disclosed in US patent application US2015/0044253, immunogenic components including saccharide fragments obtained from haemophilus influenzae type B (Hib), serogroup A, B, C, W, Y neisseria meningitidis and conjugated to a carrier protein. However, in US2015/0044253, a carbamate linker arm is directly attached to the amino group of the carrier protein, resulting in low conjugation efficiency.

Indian patent application 281/MUM/2012 discloses a lyophilized pentavalent neisseria meningitidis polysaccharide-protein conjugate component. However, the application is limited to a specific Men X strain and uses multiple carrier proteins.

PCT/EP2006/006188 discloses an immunogenic component comprising a meningococcal capsular polysaccharide from at least one of groups A, C, W and Y conjugated to a carrier protein, but does not disclose a MenX conjugate in the immunogenic component.

The existing multivalent meningococcal conjugate vaccines are relatively costly and complex in formulation. None of the prior art presents neisseria meningitidis polysaccharide-protein conjugate formulations that utilize an optimized combination of conjugation chemistries to present Men X conjugates in liquid or lyophilized form or combinations thereof.

There is an urgent need for a meningococcal conjugate vaccine that is highly homogeneous, immunogenic, and inexpensive, and is simple to prepare and administer.

Object of the Invention

In order to obviate the disadvantages of the prior art, it is a primary object of the present invention to provide a novel polysaccharide-protein conjugate vaccine composition.

It is another object of the present invention to provide a novel polysaccharide-protein conjugate vaccine composition wherein the conjugate is produced using an optimized combination of conjugation chemistries.

It is another object of the present invention to provide a novel polysaccharide-protein conjugate vaccine composition wherein the components can be used to produce a pentavalent meningococcal ACYWX-TT combination vaccine comprising serogroup ACYWX polysaccharides, each conjugated to tetanus toxoid as a carrier protein.

It is another object of the present invention to provide a novel vaccine formulation of the polysaccharide-protein conjugate vaccine composition comprising a polysaccharide-protein conjugate and pharmaceutically acceptable ingredients/excipients.

It is another object of the present invention to provide a novel vaccine formulation of the polysaccharide-protein conjugate vaccine composition comprising a pentavalent meningococcal ACYWX-TT polysaccharide-protein conjugate and a pharmaceutically acceptable ingredient/excipient.

It is another object of the present invention to provide a novel polysaccharide-protein conjugate vaccine formulation wherein the pentavalent meningococcal ACYWX-TT formulation is a liquid or lyophilized vaccine formulation or a combination of lyophilized and liquid portions.

It is another object of the present invention to provide a novel pentavalent meningococcal ACYWX polysaccharide-tetanus toxoid carrier protein conjugate vaccine composition and formulation, wherein the components have a novel conjugate combination prepared using optimized conjugation chemistry.

It is another object of the invention to provide preferred dosages of each conjugate in the vaccine compositions and formulations.

It is another object of the present invention to provide a pentavalent vaccine formulation that is stable at high temperatures and has high immunogenicity and antigenicity.

Disclosure of Invention

Accordingly, the present invention provides a novel polysaccharide-protein conjugate vaccine composition and formulations thereof. More specifically, the present invention relates to a conjugate vaccine formulation comprising a polysaccharide-protein conjugate produced using conjugation chemistry. The conjugation chemistry used includes, but is not limited to, one or more of carbamate chemistry and/or cyanation chemistry. The preparation can be used for producing a pentavalent meningococcal combined vaccine.

By optimizing the fermentation process, polysaccharides useful for preparing the conjugates of the invention are obtained.

Novel vaccine formulations of polysaccharide-protein conjugate vaccine compositions include a polysaccharide-protein conjugate and a pharmaceutically acceptable ingredient/excipient. All conjugates in the vaccine compositions and formulations have the same carrier protein. The invention provides a pentavalent vaccine preparation.

The novel polysaccharide-protein conjugate vaccine formulation of the present invention comprises five polysaccharide-protein conjugates. The polysaccharide is selected from gram-negative bacteria belonging to serogroup A, C, Y, W and Neisseria meningitidis pod polysaccharide X. Tetanus toxoid was used as the carrier protein in the preparation of all conjugates.

The capsular polysaccharide is degraded to a smaller size suitable for conjugation to a carrier protein to obtain a conjugate with high antigenicity and high immunogenicity. When molecular weight distribution is tested using HPLC PWXL4000 and 5000 tandem columns, the capsular polysaccharide degrades in the range of 0.38 + -0.1 Kd. The capsular polysaccharide preferably degrades within a range of 0.38 ± 0.06 Kd. Both the polysaccharide and the carrier protein are activated prior to the conjugation reaction. The conjugate was obtained with hydrazine or adipic dihydrazide as linker arm.

Tetanus toxoid carrier protein is conjugated to the size-specific degraded capsular polysaccharide (size-specific capsular polysaccharide). The formulations comprise conjugates prepared from optimized carbamate chemistry for one or more serogroups, preferably serogroup X polysaccharides, and conjugates prepared from cyanation chemistry for one or more serogroups, preferably serogroup A, C, Y and W polysaccharides.

The pharmaceutically acceptable excipient may be an adjuvant, a buffer, a preservative, a stabilizer, a surfactant, alone or in combination. The formulations of the present invention are liquid or lyophilized formulations or a combination of liquid and lyophilized formulations with a single or multiple dose regimen (liquid-lyophilized formulations) with or without a preservative.

The invention also provides preferred dosages of each conjugate in the vaccine compositions and formulations.

Drawings

FIGS. 1a-e depict real-time stability studies of liquid pentavalent meningococcal ACYWX-TT conjugate vaccines at 5 + -3 deg.C.

FIGS. 2a-e depict accelerated stability studies of liquid pentavalent meningococcal ACYWX-TT conjugate vaccines at 25 + -2 deg.C.

FIGS. 3 a-e: stress stability studies of liquid pentavalent meningococcal conjugate vaccines at 37 ± 2 ℃.

FIGS. 4 a-e: anti-meningococcal mouse IgG and SBA titers of adjuvanted and non-contained liquid pentavalent meningococcal ACYWX-TT conjugate vaccine are depicted compared to the registered ACYW conjugate vaccine and/or vector control after 2 doses.

FIGS. 5 a-e: anti-meningococcal rabbit IgG and SBA titers of adjuvanted and unadjuvanted liquid pentavalent meningococcal ACYWX-TT conjugate vaccines are depicted after 2 doses compared to the registered ACYW conjugate vaccine or vector control.

FIGS. 6 a-e: anti-meningococcal mouse IgG and SBA titers of the adjuvanted liquid pentavalent meningococcal ACYWX-TT conjugate vaccine are depicted after 3 doses compared to VVM7 adjuvanted liquid pentavalent vaccine and the registered ACYW conjugate vaccine or vector control.

Detailed Description

Accordingly, the present invention provides a novel polysaccharide-protein conjugate vaccine composition and formulations thereof. More specifically, the present invention relates to a conjugate vaccine composition comprising a polysaccharide-protein conjugate produced using conjugation chemistry. The conjugation chemistry used included a combination of optimized carbamate chemistry and cyanation chemistry. The components of the invention can be used for producing a pentavalent combination vaccine.

The polysaccharides used to produce the conjugates of the invention are obtained by an animal component-free fermentation process.

The novel vaccine formulations of the present invention comprise a polysaccharide-protein conjugate and a pharmaceutically acceptable ingredient/excipient. All of the conjugates in the vaccine compositions and formulations have the same carrier protein. The invention provides a pentavalent vaccine preparation.

The novel polysaccharide-protein conjugate vaccine compositions and formulations of the present invention comprise five separate polysaccharide-protein conjugates. The polysaccharide is selected from gram negative bacteria serogroup A, C, Y, W and Neisseria meningitidis pod polysaccharide X. The carrier protein used to prepare all of the conjugates was Tetanus Toxoid (TT).

The capsular polysaccharide is degraded to a smaller size suitable for conjugation to a carrier protein to obtain a conjugate with high antigenicity, high immunogenicity, and high stability. When molecular weight distribution is tested using HPLC PWXL4000 and 5000 tandem columns, the capsular polysaccharide degrades within a range of 0.38 + -0.1 Kd, preferably within a range of 0.38 + -0.06 Kd. Both the polysaccharide and the carrier protein are activated prior to conjugation. The conjugates produced have a linker arm between the polysaccharide and protein moieties. The linker arm is attached to the polysaccharide or the carrier protein, or both.

The novel multivalent polysaccharide-protein conjugate vaccine formulation of the present invention comprises serogroup A, C, Y, W, X meningococcal polysaccharides, each conjugated individually to tetanus toxoid (Men ACYWX-TT), wherein serogroup X polysaccharide is conjugated to tetanus toxoid via optimized carbamate chemistry and serogroup A, C, Y and W polysaccharide are conjugated via optimized cyanation chemistry. The ratio of protein to polysaccharide of each of said conjugates is between 0.3 and 1.0.

The novel multivalent polysaccharide-protein conjugate vaccine formulations of the present invention comprise serogroup A, C, Y, W, X meningococcal polysaccharides, each conjugated individually to tetanus toxoid (Men ACYWX-TT), mixed with one or more buffers and one or more pharmaceutically acceptable excipients, with or without adjuvants.

The pharmaceutically acceptable excipient may be an adjuvant, a buffer, a preservative, a stabilizer, a surfactant, alone or in combination. The formulations of the present invention are liquid or lyophilized formulations or a combination of liquid and lyophilized formulations with a single or multiple dose regimen, with or without a preservative. The vaccine formulations of the present invention are stable at high temperatures. With the maximum target of 40% free PS, the formulation is stable at elevated temperatures of 37 + -2 deg.C for at least 21 days, at temperatures of 25 + -2 deg.C for at least 3 months, and at 5 + -3 deg.C for at least 9 months.

In a preferred embodiment, the novel pentavalent liquid polysaccharide-protein conjugate vaccine formulation of the present invention comprises:

stirring at room temperature for 0.5-2 hr, mixing the components, and then filling the components into vials and storing at 2-8 deg.C.

In another embodiment, the novel pentavalent liquid polysaccharide-protein conjugate vaccine formulation of the invention comprises:

stirring at room temperature for 0.5-2 hr, mixing the components, and then filling the components into vials and storing at 2-8 deg.C.

A preferred embodiment of the formulation with an adjuvant, which provides the desired osmotic pressure, high stability and the desired immunogenicity, comprises:

stirring at room temperature for 0.5-2 hr, mixing the components, and then filling the components into vials and storing at 2-8 deg.C.

Another embodiment of the formulation without adjuvant, which provides the desired osmotic pressure, high stability and desired immunogenicity, comprises:

stirring at room temperature for 0.5-2 hr, mixing the components, and then filling the components into vials and storing at 2-8 deg.C.

The formulations of the invention, with or without adjuvant, have been tested for osmotic pressure, stability and immunogenicity. The formulations were exposed to a high temperature (VVM) of 37 + -2 deg.C for 21 days to check for an increase in free polysaccharide content. The free polysaccharide in the pentavalent vaccine formulation was isolated using Deoxycholate (DOC) precipitation filtration and its content was estimated using high performance anion exchange chromatography-pulsed amperometric detector (HPAEC-PAD) method.

The formulation is stable at high temperatures. With the maximum target of 40% free PS, the formulation is stable at elevated temperatures of 37 + -2 deg.C for at least 21 days, at temperatures of 25 + -2 deg.C for at least 3 months, and at 5 + -3 deg.C for at least 9 months.

The formulations of the present invention are in liquid or lyophilized form or a combination thereof. The invention also provides said optimal dose of each of said conjugates in said vaccine compositions and formulations. Said optimal dose of serogroup A and X polysaccharides is 5-10 μ g/human dose, and said optimal dose of serogroup C, Y and W polysaccharides is 5 μ g/human dose, either without adjuvant or with 500 μ gAl⁺⁺⁺Human dose of aluminum phosphate adjuvant.