Improved process for high level production of CRM
阅读说明：本技术 用于高水平生产crm的改进方法 (Improved process for high level production of CRM ) 是由 巴拉穆拉利·马西拉马尼 拉扬·斯里拉曼 曼达尔·希里什·迪克西特 德维普拉萨纳·查卡 叙尔迪 于 2018-04-19 设计创作，主要内容包括：本发明提供了用于使用具有增加的CRM197基因的拷贝数的工程化白喉棒状杆菌菌株以高产率生产CRM197的改进方法,其中该方法包括使该菌株在具有一种或多种氨基酸的无动物来源组分的培养基中生长。(The present invention provides an improved process for the production of CRM197 in high yield using an engineered strain of corynebacterium diphtheria with increased copy number of the CRM197 gene, wherein the process comprises growing the strain in an animal derived component free medium having or more amino acids.)
1, methods for using CRM with additions197Engineered Corynebacterium diphtheriae (Corynebacterium diphtheria) strains with gene copy number to produce CRM in high yield197Wherein the method comprises growing the strain in a fermentation medium comprising more than 10 amino acids and no animal-derived components.
2. The method of claim 1, wherein the method comprises supplementing the medium with nutrients.
3. The method of claim 2, wherein the supplemental nutrients are based on a metabolic flow model.
4. The method of claim 1, wherein the amino acid is selected from the group consisting of alanine, arginine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, valine, and salts thereof.
5. The method of claim 4, wherein each amino acid is used in an amount of about 0.05 to2 g/L.
6. The method of claim 1, wherein the fermentation medium comprises a combination of phenylalanine, arginine, and other amino acids, wherein the amount of phenylalanine and arginine is less than about 1 g/L.
7. The process according to claim 2, wherein the nutrients are selected from vitamins such as niacin, thiamine, pantothenic acid, biotin, riboflavin, folic acid; pimelic acid; phosphate, nitrogen source and trace metals.
8. The method of any of the preceding claims, wherein the fermentation medium is non-deferipred and completely free of animal-derived components.
9. The method of , wherein the fermentation medium composition comprises a basal fermentation medium comprising yeast extract, vegetable peptone, potassium dihydrogen phosphate (KH)2PO4) Tryptophan, glucose, YC trace salt solution.
10. The method of any of the preceding claims, wherein the method includes supplementing the medium with glucose throughout the fermentation process.
11, used for producing CRM197Wherein the method comprises:
i) culturing CRM in a fermentation medium free of animal-derived components and comprising a basal medium and more than 10 amino acids selected from the group consisting of alanine, arginine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, valine, and salts thereof197Engineered Corynebacterium diphtheriae strain with copy number of gene, and
ii) supplementing the medium with glucose and nutrients based on a metabolic flow model.
12, kinds of CRM for producing CRM197Comprising CRM increased in a fermentation medium that is free of animal-derived components and comprises more than 10 amino acids197An engineered diphtheria strain with copy number of genes, and supplementing the medium with nutrients based on a metabolic flux model, wherein each amino acid is used in an amount of about 0.05 to2 g/L.
13. The process of any of the preceding claims, wherein fermentation is carried out at a temperature of 30 to 40 ℃ and a pH in the range of 7.0 to 8.0, preferably 7.4 to 7.6.
14. The method of any of the preceding claims, wherein the obtained CRM197The yield of (b) is more than 150mg/L, 200mg/L, 300mg/L, 500mg/L, 1g/L, 1.5g/L, 2g/L, 2.5g/L, 3g/L, 3.5g/L, 4g/L, 4.5g/L, 5 g/L.
15, A process for the manufacture of a conjugate vaccine, said process comprising combining a vaccine derived from Salmonella typhi , Streptococcus pneumoniae (Streptococcus pneumoniae)e) Polysaccharide of Haemophilus influenzae (Haemophilus fluuenzae) and CRM prepared according to any of the preceding claims 197And (4) combining.
Technical Field
The invention relates to using CRM with augmentation197Engineered Corynebacterium diphtheriae (Corynebacterium diphtheria) strains with gene copy number to produce CRM in high yield197The improved method of (1).
Background
CRM197Is a genetically detoxified form of diphtheria toxin. A single mutation at position 52 replaces the glycine with glutamic acid, resulting in loss of ADP-ribosyltransferase activity of the native toxin. CRM lacking toxicity197The structural basis of has been elucidated, and is broadly useful as a carrier protein for conjugate vaccines, like diphtheria toxin , CRM197Is a single polypeptide chain of 535 amino acids (58.4KD) consisting of two subunits connected by disulfide bridges.
CRM197Used as many approved conjugate vaccines, such as Haemophilus influenzae type b (Haemophilus influenza) conjugate, sold under the trade name Hibtiter (TM), under the trade name Haemophilus influenza)
Ruth m.drew et al, bacteriol.1951 Nov; 62, (5) 549-59; chemically defined media suitable for the production of high titre diphtheria toxin is disclosed and the amino acid requirements of the Toronto strain Corynebacterium diphtheriae of Park-Williams No.8 are summarized. The medium contains amino acids that effectively replace the animal-derived component, i.e., casein hydrolysate. Amino acids include glutamic acid, cystine, proline, tryptophan, leucine, valine, methionine and glycine.
Rappuoli et al; applied and Environmental Microbiology 1983 Vol.46(3):560-197It is up to three times higher than that of the original lysogen alone.
Et al, Applied Microbiology and Biotechnology, April 1995, Volume 43(1):83-88, discloses high density growth methods that produce mutant diphtheria toxin from two strains of corynebacterium diphtheria, C7(β) (tox-201, tox-9) and C7(β) (tox-107.
Parag P.Nagarkar et al, Journal of Applied Microbiology 2002,92, 215-220; the amino acid usage pattern during the growth of corynebacterium diphtheriae is disclosed and shows that only four of the nine amino acids tested, namely cystine, histidine, aspartic acid and methionine, are important for the growth and toxin production of corynebacterium diphtheriae.
European patent No. 1849860B 1 discloses the use of non-animal derived protein materials such as proteins from soybean, cottonseed, potato, etc., as a medium component for the culture of pathogenic bacteria.
U.S. Pat. No. 6,962,803B 2 discloses a method for purifying diphtheria toxin by fermenting a strain of microorganism capable of producing diphtheria toxin, which comprises adding glucose to the growing culture, whereby the addition of glucose maintains the growth of microorganisms effective to support production of diphtheria toxin it is disclosed in step that in addition to a carbon source there are other minimum nutritional requirements for growth, including trace metals, phosphates, nitrogen sources, typically casamino acids and yeast extract.
U.S. patent application publication No. 2011/0097359 a1 discloses a medium for culturing a strain of corynebacterium diphtheriae to produce levels of diphtheria toxin or analog thereof, wherein the medium is substantially free of animal-derived products and comprises water, a carbohydrate source, a nitrogen source, and a plurality of initial concentrations of free amino acids, wherein the initial concentration of each free amino acid is not limited to the production level of diphtheria toxin or analog thereof, further discloses that the carbohydrate source is free of glucose.
WO 2006/100108 a1 discloses fermentation processes comprising the steps of culturing a strain of corynebacterium diphtheriae in the medium of a fermentor under agitation conditions sufficient to maintain homogenous culture and limited aeration such that the pO2 in the culture is reduced to less than 4% for most of the fermentation steps, and further discloses maintaining the pH in the fermentor between 7.0 and 7.8 by the degree of aeration without the addition of acid or base.
CRM197The process is typically sensitive to small variations in process components as well as process parameters. Despite limited success in commercial implementation, CRM from Corynebacterium diphtheriae is used worldwide197And (4) production. None of the above references disclose the use of engineered Corynebacterium diphtheriae strains with high yields such as>150mg/L CRM production197The method of (1). The inventors of the present invention have developed the use of engineered Corynebacterium diphtheriae strains for CRM197A metabolic flux model of high yield production.
Objects of the invention
The main object of the present invention is to provide a CRM for high-level production197The improved method of (1).
Yet another objects of the invention are to provide for high level production of CRM197Is cost effective and can be used to produce conjugate vaccines.
Disclosure of Invention
The present invention provides uses with increased CRM197Engineered Corynebacterium diphtheriae strain with copy number of genes to produce CRM in high yield197Wherein the method comprises growing the strain in a fermentation medium comprising or more amino acids and no animal-derived components.
The invention provides CRM products were supplied197The method comprising culturing CRM with an increase in CRM in a fermentation medium that is free of animal-derived fractions and comprises greater than 10 amino acids197Engineered corynebacterium diphtheriae strain with copy number of the gene, and supplementing the medium with nutrients.
The invention also provides CRM products197The method comprising culturing CRM with an increase in CRM in a fermentation medium that is free of animal-derived components and comprises greater than 10 amino acids197Engineered diphtheria corynebacteria with copy number of genes, and supplementation of the medium with nutrients based on metabolic flux models.
Drawings
FIG. 1 construction of the plasmid named pBE33
FIG. 2-metabolic flow balance model flow chart.
Detailed Description
The invention provides for using CRM with an increase197Engineered Corynebacterium diphtheriae strain with copy number of genes to produce CRM in high yield197Wherein the process comprises growing the strain in a fermentation medium comprising more than 10 amino acids and no animal-derived components.
The engineered diphtheria corynebacteria strain (C7Ep) of the present invention is an episomal strain in which CRM, a host, is placed197CRM regulated by same mechanism of gene197Multiple copies of the gene and the genetic background of the strain was corynebacterium diphtheriae with a single lysogen.
The amino acid used in the present invention is selected from the group consisting of alanine, arginine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, valine and salts thereof, each of which is used in an amount of about 0.05 to2 g/L.
Amino acids, vitamins and process conditions were investigated for the production of CRM197The influence of (c). It was found that certain amino acids show a negative effect on the growth of the bacteria. However, it was found that if the concentration is optimizedMore than 10 amino acids are used, then CRM197The yield of (2) can be increased. Amino acid concentrations were optimized using experiments designed by Plackett Burma.
The Plackett Burman design is an experimental design that uses a limited number of experiments to study the dependence of certain measured quantities on a variety of independent variables (factors), in such a way as to minimize the estimated variance of these dependencies. The results show that amino acids such as aspartic acid, glutamine, glycine, isoleucine, leucine, valine are directed against CRM at temperatures of 35 to 36 ℃197The synthesis is positively influenced, whereas amino acids such as alanine, isoleucine, valine are responsible for CRM at temperatures of 35 to 36 ℃197Have negative effects. Several levels of experimental design were performed at different concentrations to achieve high yield of CRM197. Use of tyrosine and asparagine results in CRM197The overall yield of (a) is reduced and therefore these amino acids are not part of the invention.
In a preferred embodiment of the invention, the fermentation medium comprises a combination of phenylalanine, arginine and or more other amino acids, wherein the amount of phenylalanine and arginine used is less than about 1 g/L.
In a more preferred embodiment of the invention, the fermentation medium comprises a combination of phenylalanine, arginine and or more other amino acids, wherein the amount of phenylalanine is about 0.25 to 0.75g/L and arginine is about 0.1 to 0.5 g/L.
The invention provides for using CRM with an increase197Engineering of copy number of genes Corynebacterium diphtheriae strain to produce CRM197The improved process of (a), wherein the process comprises growing the strain in a fermentation medium comprising greater than 10 amino acids supplemented with a range of about 0.05 to 20mg/L of vitamins, wherein the medium is free of animal-derived components.
In another embodiments of the invention, amino acids, vitamins, trace elements, etc. are added to the fermentation medium as nutrients during the cultivation.
Various nutrients useful as supplements in the present invention include vitamins selected from the group consisting of niacin, thiamine, pantothenic acid, biotin, riboflavin, folic acid; pimelic acid; phosphates, nitrogen sources, trace metals, and the like, and each vitamin is used in an amount ranging from about 0.05 to 20 mg/L.
The fermentation medium used in the present invention is non-deferipred and completely free of animal derived components. In addition, the medium is free of traditional meat-based Loeffler medium and casamino acid-based de-ferrated low-iron YC medium. The components of the fermentation medium of the invention comprise yeast extract, vegetable peptone and potassium dihydrogen phosphate (KH)2PO4) Tryptophan, glucose, YC trace salt solution.
In another embodiments of the invention, the culture medium is for CRM with increased yield197Engineered Corynebacterium diphtheriae strain media compositions of copy number of genes include a basal fermentation medium comprising yeast extract, vegetable peptone, potassium dihydrogen phosphate (KH)2PO4) Tryptophan, glucose, YC trace salt solution, kinds or more of amino acids, vitamins, trace elements, etc.
Suitable trace metals include potassium, magnesium, calcium, chloride, choline, copper, manganese, sulfate, zinc, and the like.
The invention provides for using CRM with an increase197Engineering of copy number of genes Corynebacterium diphtheriae strain to produce CRM197Wherein the process comprises supplementing the fermentation medium with nutrients based on a metabolic flow model.
The metabolic flow model refers to the complete network of heat transfer, mass transfer, Oxygen Transfer Rate (OTR), Oxygen Uptake Rate (OUR), and ion transfer kinetics, where the OTR is maintained by stirring, backpressure, and pumping pure oxygen. The model is shown in fig. 2.
Development of metabolic flux models
The model is based on the accumulation of hydrogen ions as the main variable in the process, followed by dissolved oxygen, oxygen to CO2And the on-line interpretation of the heat released during the process. For example, the model is applicable to carbon source limitation conditions. The carbon source was pulsed and the response was evaluated during this procedure. Response-based generation of hydrogen ionsAnd (6) carrying out correction. The feed is then optimized to achieve a stable pH and Dissolved Oxygen (DO) as well as heat transfer rate (e.g., pH 7.4; residual DO ≧ 2 to 20; heat transfer rate HTR).
In an embodiment of the invention, the method comprises supplementing the medium with glucose during the entire fermentation process.
The present invention does not involve the use of maltose as a carbon source and a deferrization step.
CRM produced according to the invention197Can be used for preparing conjugate vaccine such as pneumococcal conjugate, typhoid conjugate, Hib conjugate, etc.
In yet another embodiments, the invention provides for the production of CRM197Comprising CRM increased in a fermentation medium that is free of animal-derived components and comprises more than 10 amino acids197Engineered corynebacterium diphtheriae strain with copy number of genes, and supplementing the medium with nutrients based on metabolic flux models, wherein the amino acids are selected from the group consisting of alanine, arginine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, valine, and salts thereof.
In yet another embodiments, the invention provides for the production of CRM197Comprising CRM increased in a fermentation medium that is free of animal-derived components and comprises more than 10 amino acids197A copy number of the gene in an engineered strain of corynebacterium diphtheriae, wherein each amino acid is used in an amount of about 0.05 to2 g/L.
In yet another embodiments, the invention provides monomers for the production of CRM197Comprising CRM increased in a fermentation medium that is free of animal-derived components and comprises more than 10 amino acids197An engineered strain of corynebacterium diphtheriae with a copy number of the gene, and supplementing the medium with nutrients based on a metabolic flux model, wherein each amino acid is used in an amount of about 0.05 to2 g/L.
In another embodimentsIn which the invention provides for the production of CRM197Comprising culturing CRM with an increase in CRM in a fermentation medium that is free of animal-derived components and comprises a basal medium, more than 10 amino acids197An engineered strain of corynebacterium diphtheriae with a copy number of the gene, and supplementing the medium with nutrients according to a metabolic flux model, wherein each amino acid is used in an amount of about 0.05 to2 g/L.
In yet another embodiments, the invention provides for the production of CRM197Comprising CRM increased in a fermentation medium that is free of animal-derived components and comprises more than 10 amino acids197An engineered corynebacterium diphtheriae strain with a copy number of a gene, and supplementing the medium with a nutrient based on a metabolic flux model, wherein the amino acids are selected from the group consisting of alanine, arginine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, valine, and salts thereof, wherein each amino acid is used in an amount of about 0.05 to2 g/L.
In yet another embodiments, the invention provides for the production of CRM197Comprising culturing CRM increased in a fermentation medium free of animal-derived components and comprising more than 10 amino acids197Engineered diphtheria corynebacteria in copy number of genes, wherein the amino acids are selected from the group consisting of alanine, arginine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, valine and salts thereof, wherein each amino acid is used in an amount of about 0.05 to2 g/L.
In a preferred embodiment, the present invention provides for the production of CRM197The method comprising
i) In a medium free of animal-derived components and comprising a basal medium and more than 10 of the substances selected from the group consisting of alanine, arginine, aspartic acid, cysteine, glutamic acid, glutamine, glycineCulture of CRM with increased CRM in fermentation media of amino acids with histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, valine and salts thereof197An engineered diphtheria corynebacteria strain of copy number of genes, and
ii) supplementing the medium with glucose and nutrients according to the metabolic flow model.
In yet another embodiments, the temperature is maintained in the range of 30 to 40 ℃ and the pH is maintained at 7.0 to 8.0, preferably 7.4 to 7.6, using 20% orthophosphoric acid, 12.5% ammonium hydroxide during the fermentation process, which is carried out for 15 to 24 hours, preferably 16 to 20 hours.
In embodiments, the method of the present invention does not contain the low iron YC medium for the deferrization of casamino acids, maltose as a carbon source, and a deferrization step.
The invention provides a method for producing CRM197The method comprising culturing a microorganism having increased CRM in a fermentation medium197Engineered corynebacterium diphtheriae strain with copy number of the gene, the fermentation medium being free of animal-derived components and containing a combination of phenylalanine, arginine, and or more additional amino acids in an amount of less than about 1 g/L.
In embodiments, the invention provides a method for producing a CRM197Comprising CRM increased in a fermentation medium that is free of animal-derived components and comprises more than 10 amino acids197Engineered Corynebacterium diphtheriae strain for copy number of genes, and supplementing the culture medium with nutrients according to metabolic flux model, wherein the obtained CRM197The yield of (a) is more than 150 mg/L.
In embodiments, the present invention enables the preparation of conjugate vaccines comprising combining polysaccharides from Salmonella typhi (Salmonella typhi), Streptococcus pneumoniae (Streptococcus pneumoniae), meningococcus (menigococcus), Haemophilus influenzae (Haemophilus influenzae) with polysaccharides made according to the present inventionPrepared CRM197And (4) combining.
The invention provides for using CRM with an increase197Engineered Corynebacterium diphtheriae strain with copy number of genes to produce CRM in high yield197Wherein the method comprises growing the strain in a medium that is free of animal-derived components and comprises more than 10 amino acids, and supplementing the medium with nutrients based on a metabolic flux model.
In embodiments, the invention provides methods for producing CRM in yields of, e.g., 150mg/L, 200mg/L, 300mg/L, 500mg/L, 1g/L, 1.5g/L, 2g/L, 2.5g/L, 3g/L, 3.5g/L, 4g/L, 4.5g/L, and 5g/L197The improved method of (1).
In embodiments, the present invention provides an engineered corynebacterium diphtheriae strain C7(β 197) wherein the pBE33 plasmid was transferred by electroporation.
CRM produced according to the invention197Quantitation was performed using an immunocapture enzyme-linked immunosorbent assay (IC-ELISA).
Development of CRM with increases on expression vector plasmids197Engineered corynebacterium diphtheriae strains with copy numbers of genes
Corynebacterium diphtheriae C7(β -197)
The present invention is more specifically illustrated with reference to the examples given below. It should be understood, however, that this invention is not limited in any way to the examples, but includes variations thereof within the parameters described herein, as would be known to one skilled in the art.
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