Production of bacterial polysaccharides
阅读说明：本技术 细菌多糖的生产 (Production of bacterial polysaccharides ) 是由 桑迪普·沙玛 尼廷·库马尔 萨麦德·哈尼夫 马诺伊·库马尔·奇卡拉 达温德·吉尔 于 2018-04-26 设计创作，主要内容包括：本发明尤其涉及脑膜炎奈瑟氏菌(Neisseria meningitidis)多糖的营养培养基组分、补料组分和发酵条件。本发明描述了快速、可工业规模化、成本有效的生产脑膜炎奈瑟氏球菌的方法。本发明的脑膜炎奈瑟氏菌多糖可用于生产经济型的抗脑膜炎球菌感染的多糖蛋白结合疫苗。(The invention relates in particular to the nutrient medium components, feed components and fermentation conditions of the Neisseria meningitidis (Neisseria meningitidis) polysaccharide. The present invention describes a rapid, industrially scalable, cost-effective method for producing neisseria meningitidis. The neisseria meningitidis polysaccharides of the present invention are useful in the production of economical polysaccharide protein conjugate vaccines against meningococcal infection.)
1. A method of producing neisseria meningitidis polysaccharides from serogroups A, C, Y, W and X using a modified animal component-free nutrient medium, a modified animal component-free feed medium, and modified fermentation process parameters, resulting in rapid growth and increased production of neisseria meningitidis.
2. The method of claim 1, wherein the modified animal component-free nutrient medium comprises a combination of two or more components selected from the group consisting of:
-monosodium glutamate
Disodium hydrogen phosphate
-potassium chloride
-selective plant peptones
-TC-yeast powder
-glucose
-L-cystine
-magnesium chloride
-nicotinamide adenine dinucleotide
Wherein the combination results in rapid growth and increased production of Neisseria meningitidis polysaccharides from serogroups A, C and X.
3. The method of claim 2, wherein the nutrient medium comprises the following concentration ranges of ingredients:
4. the method of claim 1, wherein the modified animal component-free nutrient medium comprises a combination of two or more of the following components:
-monosodium glutamate
Disodium hydrogen phosphate
-potassium chloride
-selective plant peptones
-TC-yeast powder
-glucose
-L-cystine
-magnesium chloride
-nicotinamide adenine dinucleotide
-ammonium chloride
Wherein the combination results in rapid growth and increased production of N.meningitidis polysaccharides from serogroups Y and W.
5. The method of claim 4, wherein the modified animal component-free nutrient medium comprises the following concentration ranges of components:
6. the method of claim 1, wherein the modified feed medium comprises a combination of two or more of:
-L-glutamic acid
-glucose
-L-serine
-L-arginine
-Glycine
-L-tryptophan
-TC-yeast powder
Wherein the combination results in rapid growth and increased production of Neisseria meningitidis polysaccharides.
7. The method of claim 6, wherein the modified animal component-free feed medium comprises the following concentration ranges of components:
8. the method of claim 1, wherein the nutrient medium and feed medium result in the following enhanced yields of neisseria meningitidis polysaccharides from serogroups A, C, Y, W and X:
。
9. The method according to claim 1, wherein the fermentation process is carried out according to the following process parameter ranges:
。
10. The fermentation process of claim 9, wherein the fermentation process is completed rapidly within 11 ± 3 hours.
11. The method of producing neisseria meningitidis polysaccharides according to claim 1, wherein the method produces neisseria meningitidis polysaccharides that are useful in producing economical polysaccharide protein conjugate vaccines against meningitidis infection.
Technical Field
The present invention relates to an improved process for the production of bacterial polysaccharides. The invention relates in particular to a nutrient medium composition, feed composition, fermentation conditions and purification process for producing Neisseria meningitidis polysaccharides. The neisseria meningitidis polysaccharides of the present invention are useful in the production of economical polysaccharide protein conjugate vaccines against meningococcal infection.
Background
Neisseria meningitidis, commonly referred to as meningococcus, is a gram-negative bacterium that can cause meningitis and other forms of meningococcal disease, such as meningococcemia.
Based on the types of capsular polysaccharides present on neisseria meningitidis (Men), 13 serogroups have been identified, 6 (A, B, C, W135, X and Y) of the 13 identified neisseria meningitidis capsular types account for the majority of the global meningococcal disease cases. MenA is the most prevalent serogroup in africa and asia, but is rare/scarce in north america. In europe and the united states, serogroup b (menb) is the leading cause of disease and death, followed by serogroups MenC and MenW. Recently, outbreaks of MenX have begun to occur in sub-Saharan Africa. The diversity of serogroups has hampered the development of universal vaccines against meningococcal disease.
Due to the urgent need to combat this fatal disease, the production of the first meningitis polysaccharide vaccine was completed in 1978. Later, pure polysaccharide based vaccines were found to be less effective in children under two years of age. These observations lead to further studies that indicate that infants have an immature immune system and are unable to mount an immune response to pure polysaccharide.
The immune response can be characterized as a T cell dependent (TD) immune response and a T cell independent (TI) immune response. Proteins and peptides are known to induce TD antigens by stimulating helper T lymphocytes and producing memory cells. In contrast, polysaccharides are TI antigens, do not induce T cell activation and do not form any memory B cells, which is a major drawback in the treatment of infants because of their immaturity of the immune system.
Thus, there is a need to combine bacterial polysaccharides with protein carriers that induce T cell dependent immune responses, characterized by increased immunogenicity, prolonged protection time and reduced nasopharyngeal carriage by meningococci in infants. This need was met by an original study that resulted in polysaccharide-protein conjugate vaccines, the first meningococcal conjugate vaccine approved in the uk in 1999.
The polysaccharides, particularly antigenic polysaccharides, used for the preparation of the vaccine may be monovalent, bivalent and multivalent vaccines comprising one, two or more polysaccharides, respectively. These vaccines are readily available on the market for the prevention of certain diseases or infections caused by a variety of microorganisms. Such multivalent polysaccharide based vaccines have been used for many years and are of significant value in the prevention of diseases such as pneumococci, meningococci or haemophilus influenzae.
The production of purified neisseria meningitidis capsular polysaccharides is a primary requirement for efficient conjugation to carrier proteins and development into conjugate vaccines. Traditionally, most bacterial fermentation media use animal components to grow meningococci for polysaccharide production. There is a need for animal component-free media that provide advantages in regional preferences and avoid infectious agent-induced diseases, such as Transmissible Spongiform Encephalopathies (TSEs) and Bovine Spongiform Encephalopathies (BSEs). The cost of culturing neisseria meningitidis for producing capsular polysaccharides is often high and involves long working times due to the series of production and quality control steps involved. An optimized animal component-free medium can avoid these problems.
Improvements in the polysaccharide production steps would be advantageous to formulate effective and economically viable conjugate vaccines.
There are a number of patents and non-patent documents describing methods for the production and purification of polysaccharides. One such document is the patent of application No. US 12/041,745, which discloses a process for the production of a meningococcal meningitis vaccine, which process comprises culturing neisseria meningitidis to produce capsular polysaccharides from serogroups A, C, Y and W-135 in neisseria meningitidis refined medium (NMFM), separating the capsular polysaccharides from the culture, purifying the capsular polysaccharides of any residual cellular biomass; the capsular polysaccharide is then physically depolymerised. The cited prior art uses a longer time to produce purified capsular polysaccharides.
Another U.S. patent publication No. US 20150299750 a1 discloses improved culture, fermentation and purification conditions for the preparation of neisseria meningitidis polysaccharides. Another U.S. patent publication No. 20080318285 a1 discloses a neisseria meningitidis refined medium designed to maximize capsular polysaccharide production and produce minimal cellular biomass and endotoxin in a shorter fermentation time.
ACFM (animal component free medium) of the present invention is different from the medium used by Shankar Pisal in U.S. patent publication No. US 2015/0299750 and Jeerri reddy in U.S. patent publication No. US 2008/0318285, neither of which uses selective plant peptone (Select phytone) nor TC yeast powder. SelectphytoneTMIs a peptone of plant origin. The nitrogen component of the plant peptone, in combination with naturally occurring vitamins, contributes to the growth of bacteria. The phytone peptone has an endotoxin level of less than or equal to 500 EU/g. TC yeast powder is a mixture of peptides, amino acids, carbohydrates and vitamins. The TC yeast powder product is animal free and is a water soluble component of autolysed yeast. TC Yeast powder UF has been ultrafiltered at 10,000MWCO (molecular weight cut-off). The endotoxin value of TC yeast powder UF is less than 500 EU/g. It is a universal nutritional supplement and can promote bacterial growth. This is a new class of media components that replaces most of the individual components used to promote growth. The medium components of the present invention do not even include casamino acids that other inventors have used in the prior art. The greatest advantage of animal component-free media is that because of regional preference, the need for ACFM vaccines (millions of doses) is high in the middle east, GCC (gulf Council) countries and other countries, and in addition such vaccines do not contain BSE and TSE.
Thus, current methods for producing neisseria meningitidis serogroups use animal component media, and fermentation processes require relatively long periods of time (up to 20-24 hours or more) to culture the polysaccharide, thereby increasing production costs, and making the process commercially impractical due to its inability to scale-up in a cost-effective and rapid manner and having animal components.
It is an object of the present invention to provide improved nutrient and feed media (feed media) for better production of neisseria meningitidis polysaccharides by short and high yield fermentation. The improvements will lead to the production of polysaccharide protein conjugate vaccines at a lower price, which can then be provided to children in developing countries at a substantial price.
Object of the Invention
The main object of the present invention is to provide a method for producing bacterial polysaccharides.
It is another object of the invention to provide a process for producing capsular polysaccharides of different serogroups of neisseria meningitidis.
It is yet another object of the present invention to provide an optimized medium and feed medium composition that is free of animal components.
It is a further object of the invention to provide improved nutrient medium and feed medium components for the growth of Neisseria meningitidis serogroups A, C, W, X and Y.
It is yet another object of the present invention to provide a process for fermentation in a shorter time by a simple, efficient, improved and commercially scalable process that results in better polysaccharide yields in a very short time with lower impurities.
It is a further object of the present invention to purify neisseria meningitidis polysaccharides while eliminating impurities in a short time by a simple, efficient, improved and commercially viable process.
It is a further object of the present invention to produce high quality products with better yields that meet the relevant quality specifications.
Disclosure of Invention
The present invention describes a rapid, industrially scalable, cost-effective method for growing bacteria, preferably neisseria meningitidis, to produce bacterial polysaccharides. The method provides a purification process for purifying neisseria meningitidis polysaccharides in a significantly reduced time.
The invention describes a nutrient medium of Neisseria meningitidis, which comprises but is not limited to monosodium glutamate with the concentration of 1.00 +/-0.5 g/L, disodium hydrogen phosphate with the concentration of 3.25 +/-1.0 g/L, potassium chloride with the concentration of 0.09 +/-0.1 g/L, selective plant peptone with the concentration of 10.0 +/-2.0 g/L, yeast powder with the concentration of 4.0 +/-2.0 g/L, glucose with the concentration of 5.00 +/-2.0 g/L, L-cystine with the concentration of 0.03 +/-0.1 g/L, magnesium chloride with the concentration of 0.60 +/-0.5 g/L, nicotinamide adenine dinucleotide with the concentration of 0.25 +/-0.1 g/L and ammonium chloride with the concentration of 1.00 +/-0.2 g/L. The nutrient medium components described above provide optimal growth for the neisseria meningitidis serogroup.
The invention also describes a feed medium of Neisseria meningitidis, including but not limited to L-glutamic acid with concentration of 6.00 +/-2.0 g/L, glucose with concentration of 20 +/-2.0 g/L, L-serine with concentration of 0.50 +/-0.1 g/L, L-arginine with concentration of 0.20 +/-0.1 g/L, glycine with concentration of 0.20 +/-0.1 g/L, L-tryptophan with concentration of 0.20 +/-0.1 g/L, TC-yeast powder with concentration of 5 +/-2.0 g/L, and other components such as L-cystine, magnesium chloride, calcium chloride, ferrous sulfate and ammonium chloride according to requirements. The feed medium component provides optimal growth for the neisseria meningitidis serogroup when added to a fermentation broth during fermentation culture with the nutrient medium.
The present invention describes a fermentation process at predetermined temperature, pH, gas flow, dissolved oxygen and stirring rate such that the fermentation is completed within 11 ± 3 hours.
The present invention describes purification steps for producing high yields of neisseria meningitidis serogroup W and Y capsular polysaccharides. The crude polysaccharide in the fermentation broth was concentrated and diafiltered with MilliQ water (MQW) to form a concentrate with reduced impurity levels. The concentrate thus obtained is treated with a base, for example 1. + -. 0.2M NaOH, at a predetermined temperature for an optimum time. The resulting partially purified polysaccharide was diafiltered again with MQW, then carbon filtered and finally sterile filtered.
Drawings
FIG. 1 depicts the growth curves of Shake flask study 1(6 ACFM fractions)
FIG. 2 depicts the growth curves of Shake flask study 2(5 ACFM and 1 ACM fractions)
FIG. 3 depicts growth curves of the MenA group with ACFM
FIG. 4 depicts growth curves of MenC group with ACFM
FIG. 5 depicts growth curves of MenY group with ACFM
FIG. 6 depicts growth curves of MenW group with ACFM
FIG. 7 depicts growth curves of MenX group with ACFM
Detailed Description
The present invention discloses optimized nutrient and feed media free of animal components that can be used to grow difficult to breed Neisseria meningitidis in a shorter period of time.
The greatest advantage of animal component-free media is that due to regional preferences for ACFM vaccines, the demand for ACFM vaccines (millions of doses) is high in the middle east, in GCC (gulf Council) countries, and in other countries. Furthermore, such vaccines are not at risk for TSE and BSE.
Before the preferred embodiments of the present invention are described, it is to be understood that this invention is not limited to particular materials described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention in any way.
The invention describes a nutrient medium of Neisseria meningitidis, which comprises but is not limited to monosodium glutamate with the concentration of 1.00 +/-0.5 g/L, disodium hydrogen phosphate with the concentration of 3.25 +/-1.0 g/L, potassium chloride with the concentration of 0.09 +/-0.1 g/L, selective plant peptone with the concentration of 10.0 +/-2.0 g/L, yeast powder with the concentration of 4.0 +/-2.0 g/L, glucose with the concentration of 5.00 +/-2.0 g/L, L-cystine with the concentration of 0.03 +/-0.1 g/L, magnesium chloride with the concentration of 0.60 +/-0.5 g/L, nicotinamide adenine dinucleotide with the concentration of 0.25 +/-0.1 g/L and ammonium chloride with the concentration of 1.00 +/-0.2 g/L.
The nutrient medium components described above provide optimal growth for the neisseria meningitidis serogroup.
In a preferred embodiment, the invention describes a nutrient medium for Neisseria meningitidis, comprising monosodium glutamate at a concentration of 1.00g/L, disodium hydrogen phosphate at a concentration of 3.25g/L, potassium chloride at a concentration of 0.09g/L, selective phytone peptone at a concentration of 10.00g/L, TC-yeast powder at a concentration of 4g/L, glucose at a concentration of 5.00g/L, L-cystine at a concentration of 0.03g/L, magnesium chloride at a concentration of 0.60g/L, nicotinamide adenine dinucleotide at a concentration of 0.25g/L, and ammonium chloride at a concentration of 1.00 g/L.
All the above optimized concentrations are listed in table 4 of the specification. The nutrient medium components described above provide optimal growth for neisseria meningitidis serogroups MenA, MenC, MenY, MenW and MenX. Ammonium chloride was added only to serogroups W and X ACFM to optimize polysaccharide production.
The invention also describes a feed culture medium of the neisseria meningitidis, which comprises but is not limited to L-glutamic acid with the concentration of 6.00 +/-2.0 g/L, glucose with the concentration of 20 +/-2.0 g/L, L-serine with the concentration of 0.50 +/-0.1 g/L, L-arginine with the concentration of 0.20 +/-0.1 g/L, glycine with the concentration of 0.20 +/-0.1 g/L, L-tryptophan with the concentration of 0.20 +/-0.1 g/L and TC-yeast powder with the concentration of 5 +/-2.0 g/L. The feed medium composition described above provides optimal growth for the neisseria meningitidis serogroup.
In a preferred embodiment, the invention also describes a feed medium for Neisseria meningitidis comprising L-glutamic acid at a concentration of 6.00g/L, glucose at a concentration of 20.00g/L, L-serine at a concentration of 0.50g/L, L-arginine at a concentration of 0.20g/L, glycine at a concentration of 0.20g/L, L-tryptophan at a concentration of 0.20g/L, TC-yeast powder at a concentration of 5.00 g/L. The feed medium components described above provide optimal growth for neisseria meningitidis serogroups MenA, MenC, MenY, MenW and MenX. The optimized feed composition is listed in table 6 of the specification.
After culturing the bacteria in shake flasks with optimized nutrient medium, the bacteria were fermented as described in example 5 and example 6 of the specification. The fermentation conditions are optimized such that the resulting fermentation product (liquor) has a higher polysaccharide yield and low levels of impurities and the fermentation process is completed within 11 ± 3 hours, more preferably within 10 to 12 hours.
In a preferred embodiment, the fermentation is carried out at a temperature in the range of 36. + -. 1 ℃ and a rotation speed of 150 to 600rpm, the air flow rate of the fermenter being maintained during the entire fermentation in the range of 0.2 to 0.8l/m, the oxygen partial Pressure (PO)2) The pH was maintained at 20% and 7.2. + -. 0.1.
Thus, the present invention provides a method for producing Neisseria meningitidis serogroups MenA, MenC, MenY, MenW and MenX rapidly, industrially, economically and efficiently on a large scale using optimized nutrient and feed media, which provides for maximal growth of Neisseria meningitidis.
The various aspects of the invention described in detail above are now illustrated by way of non-limiting examples: