OA20586A

OA20586A - An improved method for high level production of CRM

Info

Publication number: OA20586A
Application number: OA1201900385
Authority: OA
Inventors: Narender Dev MANTENA; Mahima DATLA; Ramesh Venkat Matur; Balamurali MASILAMANI; Mandar Shirish Dixit; Deviprasanna CHAKKA; Sureddisatyam NAIDU; Rajan Sriraman
Original assignee: Biological E Limited
Priority date: 2017-04-22
Filing date: 2018-04-19
Publication date: 2022-11-29

Abstract

The present invention provides an improved method for the production of CRM197 with high yield using engineered Corynebacterium diphtheria strain having increased copy number of CRM197 gene, wherein the method comprises growing the strain in a media free of animal derived components with one or more amino acids.

Description

AN IMPROVED METHOD FOR HIGH LEVEL PRODUCTION OF CRM|₉₇Field of the Invention

The présent invention relates to an improved method for the production of CRM]9₇ with high yield using engineered Corynebacterium dîphtheria strain having increased copy number of CRM|9₇ gene.

Baekground ofthe invention

CRM μ;-? is a genetically detoxified form of dîphtheria toxin. A single mutation at position 52, substituting glutamic acid for glycine, causes the loss of ADP-ribosyltransferase activity of the native toxin. The structural basis of CRM197 for the lack of toxicity has been elucidated and is widely used as a carrier protein for conjugate vaccines. CRM 197, like dîphtheria toxin, is a single polypeptide chain of 535 amino acids (58.4 KD) consisting of two subunits (linked by disulfide bridges).

CRMi97 is used as a carrier protein in a number of approved conjugate vaccines such as Haemophilus influenza type b conjugate marketed under the trade name Hibtiter TM, 13-valenl pneumococcal polysaccharide conjugate marketed under the trade name PREVNAR 13® and the like.

Ruth M. Drew et al., BaclerioL 195! Nov; 62(5):549-59} disclosed a chemically defined media suitable for the production of high titer diphtherial toxin and summarized the amino acid requirements of Corynebacterium diphlheriae, Toronto strain of Park-Williams no. 8. The media contains amino acids which effectively replace the animal derived component i.e. casein hydrolysate. The amino acids include glutamic acid, cystine, proline, tryptophan. leucine. valine. méthionine, and glycine.

Rappuoli et al; Applied and Environmenlal Microbiology 1983Vol. 46(3):560-564 hâve disclosed that the nontandem double lysogens were stable and capable of giving high yields of CRM|9₇, up to threefold higher than monolysogens.

R Fass. et al., Applied Microbiology and Biotechnology: April 1995, Volume 43(1): 83-88} disclosed a high-density growth approach to produce mutated dîphtheria toxin from two strains of Corynebacterium dîphtheria·. C7 (β) (tox-201. lox-9) and C7 (β)(ΐοχ-107). The procedure involves the use of a modified, non-deferrated growth media that provided fast and high-density growth ofthe bacteria, and which, when associated wiih simnhaneous depletton of glucose and lion, enhanced the toxin production. Oxygen-enriched air was supplied to enable the bacteria to grow to a cell density giving an absorbance of 70 at 600 nm (15-20 g/L dry weight). The maximum toxin concentration in the culture supernatant was 150 mg/L

Parag P.Nagarkar et al.. Journal of Applied Microbiology 2002, 92, 215-220· disclosed the annno acid utdization pattern during growth of Corynebacierium diphtheria and showed that only four ofthe nine amino acids tested, namely cystine, histidine, aspartate and méthionine, were cntical for growth and toxin production by Corynebacierium diphtheria.

Européen Patent No. i 849 860 Bl disclosed the use of proteinaceous material of non animal ongin, such as proteins from soy beans, cotton seeds. potatocs. etc., as a media constituent for the cultivation of pathogenic bacteria.

US Patent No. 6,962,803 B2 disclosed a method of purifying diphtheria toxin by means of fermenting a microorganism strain capable of producing diphtheria toxin, said method comprising adding glucose to a growing culture whereby the addition of glucose maintins microorganism growth effective to support diphtheria toxin production. It further disclosed that in addition to a carbon source, there are other minimum nutritional requirements for growth 20 which include trace metals, phosphate, a nitrogen source, generally casamino acids and yeast extract.

US Patent Application Publication No. 2011/0097359 Al disclosed a media for culturing a strain of Corynebacierium diphtheriae to produce a level of diphtheria toxin or an analog thereof, 25 wherein the media is substantiality free of animal derived products and comprises: water; a carbohydrate source; a nitrogen source; and a number of free amino acids in an initial concentration wherein the initial concentration of each free amino acid is not limiting for the level of production of the diphtheria toxin or the analog thereof. Further discloses that the carbohydrate source is free of glucose.

WO 2006/100108 Al disclosed a fermentation process comprising a step of growing a strain of Corynebacierium diphtheria in a media within the fermenter under conditions of agitation suffirent to maintam a homogenous culture and limited aération such that pO2 within the culture falls to iess than 4% For the majority of the fermentation step. Further disclosed that the pH within the fermenter is held between 7.0 and 7.8 by the degree of aération without requiring addition of acid or base.

The CRM|₉₇ piocess is generally sensitive to small changes in process components as well as process parameters. The CRM₁₉₇ production from Corynebacterium diptheriae is exercised across the globe albeit with limited success for commercial realization. None of the above référencés disclosed method of producing CRM_!97 with hîgh yields such as >150 mg/1 using engineered Corynebacterium diphiheria strain. The inventors of the présent invention hâve developed a metabolic flux model for h i g h yield production of CRM ]₉₇ using engineered Corynebacterium diphiheria strain.

Objective of the Invention

It is the main objective ofthe présent invention to provide an improved process for the high level production ofCRM_i97.

Yet another objective of the présent invention is to provide an improved process for the high level production of CRM|₉₇ which is cost effective and can be used for manufacturing conjugale vaccines.

Summary ofthe Invention

The présent invention provides an improved method for the production of CRM 197 with high yield using engineered Corynebacterium diphiheria strain having increased copy number of CRM |₉₇ gene, wherein the method comprises growing the strain in a fermentation media comprising one or more amino acids and is free of animal derived components.

The présent invention provides an improved method for the production of CRM|₉₇ which method comprises culturing the engineered Corynebaclerium diphiheria strain having increased copy number of CRM,₉₇ gene in a fermentation media which is free of animal derived components and comprises more than 10 amino acids and supplementing the media with nutrients.

The présent invention also provides an improved method for the production of CRM₁₉₇ which method comprises culturing the engineered Corynebacterium diphiheria strain having increased copy number of CRM|₉₇ gene in a fermentation media which is free of animal derived components and comprises more than 10 amino acids and supplementing the media with nutrients based on metabolic flux model.

Brief Description of the Drawings

Figure 1- Construction of a plasmid designated as pBE33

Figure 2- The metabolic flux balance model flow chart.

Detailed Description ofthe Invention

The présent invention provides an improved method for the production of CRM₁₉₇ with high yield using engineered Corynebacterium diphtheria strain having increased copy number of CRM]g? gene, wherein the method comprises growing the strain in a fermentation media compiising moie than 10 amino acids and is free of animal derived components.

Engineeied Corynebacterium diphtheriae strain (C7Ep) of the présent invention refers to the C orynebacterium diphtheriae having episomally placed multiple copies of CRM|₉₇ gene legulated by the same mechanism that of hosl CRM|₉₇ gene and the said strain’s genetic background is single lysogen.

The amino acids used in the présent invention are selected from Alanine, Arginine, Aspartic acid, Cysteine, Glutamic acid. Glutamine, Glycine, Histidine, Isoleucine, Leucine, Lysine, Méthionine, Phenyl alanine, Proline. Serine, Thréonine, Tryptophan, Valine and its salis and each amino acid is used in an amount of about 0.05 to 2 g/1.

The effect of amino acids, vitamins and the process conditions for the production of CRM]₉₇ has been sludied. It has been found that certain amino acids hâve shown négative effect on the growth of bacteria. However, it is found that the production ofCRM|₉₇ is increased if more than 10 amino acids are used in an optimum concentration. The concentration of amino acids is optimized using Plackett Burman design of experiments.

Plackett Burman designs are experimental designs for invesligating the dependence of some measured quantity on a number of independent variables (factors), in such a way as to minimize the variance ol the estimâtes of these dependencies using a limited number of experiments. The outcome showed amino acid such as Aspartic acid, Glutamine, Glycine isoleucine leucine, valine and al a température of 35 to 36 °C hâve positive impact on the CRM|₉₇ synthesis and amino acids such as Alanine, Isoleucine, Valine and at température of 35 to 36 °C hâve négative effect m the synthesis of CRMi₉₇. Several levels of Design of experiments were carried out at different concentrations to achieve high level production of CRM₁₉₇ The use of tyrosine and asparagine resulted in decrease in the overall yield of CRM|₉₇ and hence these amino acids are not part of the invention.

In a preferred embodiment of the présent invention, the fermentation media contains combination of Phenyl alanine, Arginine and one or more other amino acids wherein the amount ot Phenyl alanine and Arginine used is about less than about Ig/L.

In a moie preferred embodiment of the présent invention, the fermentation media comprise combination of Phenyl alanine. Arginine and one or more other amino acids wherein amount of Phenyl alanine is about 0.25 to 0.75g/l and Arginine is about 0.1 to 0.5 g/1.

Ihe présent invention provides an improved method for the production of CRM|₉₇ using engineered Corynebacterium diphtheria strain having increased copy number of CRM₁₉₇ gene, wherein the method comprises growing the strain in a fermentation media comprising more than 10 amino acids, supplemenling the media with vitamins in the range of about 0.05 to 20 mg per liter, wherein the media is tree of animal derived components.

In another embodiment of the présent invention, amino acids, vitamins, trace éléments and the lîke are added as nutrients to the fermentation media during the cultivation.

Varions nutrients used in the présent invention as suppléments include vitamins selected from Nicotinic acid, Thiamine, Pantothenic acid, Biotin, Riboflavin, FolicAcid; Pimelic acid; phosphate, a nitrogen source and trace metals and the like and each vitamin is used in an amount in the range from about 0.05 to 20 mg per liter.

The fermentation media used in the présent invention is a non-defferated and completely free from animal derived components. Further, the media is also devoid of traditional méat based Loeffler media and casamino acid based defferated low iron YC media. The components of fermentation media of the présent invention comprises Yeast Extract, Veg.Peptone, Potassiumdihydrogen phosphate (KH₂PO₄), Tryptophan, Glucose, YC Trace sait solution.

In another embodiment of the présent invention, the media composition for the cultivation of engineeied Corynebacteriwn diphtheria strain having increased copy number of CRM 197 gene comprises base fermentation media comprising Yeast Extract, Veg.Peptone, Potassiumdihydrogen phosphate (KH₂PO₄), Tryptophan, Glucose, YC Trace sait solution; one or more amino acids, vitamins, trace éléments and the like.

Suitable trace metals include Potassium, Magnésium, Calcium, Chloride, Choline, Copper, Manganèse, Sulfate, Zinc and the like.

The présent invention provides an improved method for the production of CRM197 using engineeied Corynebaclerium diphtheria strain having increased copy number of CRM₁₉₇ gene, wherein the method comprises supplementing the fermentation media with nutrients based on metabolic flux model.

Metabolic flux model refers to the complété network of Heat Transfer, Mass Transfer, Oxygen Iran s fer rate (OTR), Oxygen uptake rate (OU R) and Ion Iran s fer kinetics wherein OTR i s being maintained by means of agitation, back pressure and pump in pure oxygen. This model is depicted in Figure 2.

Development of Metabolic flux model.

This model is created on the basis ol online data interprétation of hydrogen ions accumulations în the process as a primary variable which is followed by the dissolved oxygen, oxygen to CO₂conversion along with the heat liberated in the process. For example the model Works on the carbon source limitation conditions. The carbon source is pulsed and the response is evaluated in the process. The corrective action was taken on hydrogen ions génération based on the response. Then the leed was optimized to get a steady pH and dissolved oxygen (DO) and heat transfer rate (ForegpH of 7.4; residual DO of> 2 to 20; Heat transfer rate HTR).

In an embodiment of the présent invention, the method comprises supplementing the media with glucose during the entire fermentation process.

The piesent invention does not involve the use of maltose as a carbon source and deferration step.

CRM|₉₇ produced according to the présent invention can be used for manufacturing conjugale vaccines such as pneumococcal conjugale, typhoid conjugale, Hib conjugale and the like.

In yet another embodiment, the présent invention provides an improved method for the production of CRM,₉₇ which method comprises culturing the engineered Corynebacterium diphtheria strain having increased copy number of CRM_I97 gene in a fermentation media which is fiee of animal derived components and comprises more than 10 amino acids and supplementing the media with nutrients based on metabolic flux model, wherein the amino acids are selected from Alanine, Arginine, Aspartic acid, Cysteine, Glutamic acid. Glutamine, Glycine, Histidine, Isoleucine, Leucine, Lysine, Méthionine, Phenyl alanine, Proline, Serine, Threonine, Tryptophan. Valine and its salts.

In yet another embodiment, the présent invention provides an improved method for the production of CRM|₉₇ which method comprises culturing the engineered Corynebacterium diphtheria strain having increased copy number of CRM|₉₇ gene in a fermentation media which is free of animal derived components and comprises more than 10 amino acids, wherein each amino acid is used in an amount of about 0.05 to 2 g/L

In yet another embodiment. the présent invention provides an improved method for the production of CRMi₉₇ which method comprises culturing the engineered Corynebacterium diphtheria strain having increased copy number of CRM|₉₇ gene in a fermentation media which is free oi animai derived components and comprises more than 10 amino acids and supplementing the media with nutrients based on metabolic flux model, wherein each amino acid is used în an amount of about 0.05 to 2 g/J.

In yet another embodiment, the présent invention provides an improved method for the production of CRM_f97 which method comprises culturing the engineered Corynebacterium diphtheria strain having increased copy number of CRM_i97 gene in a fermentation media which is free of animal derived components and comprises base media, more than 10 amino acids and supplementing the media with nutrients based on metabolic flux model, wherein each amino acid is used in an amount of about 0.05 to 2 g/1.

In yet another embodiment, the présent invention provides an improved method for the production of CRM|₉₇ which method comprises culturing the engineered Corynebacterium diphthena strain having increased copy number of CRM_[97 gene in a fermentation media which is free oi animal derived components and comprises more than 10 amino acids and supplementing the media with nutrients based on metabolic flux mode!, wherein the amino acids are selected from Alanine, Arginine, Aspartic acid, Cysteine, Glutamic acid, Glutamine, Glycine, Histidine, Isoleucine, Leucine, Lysine, Méthionine, Phenyl alanine, Proiine, Serine, Threonine, Tiyptophan, Valine and its salts wherein each amino acid is used in an amount of about 0.05 to 2 g/i.

In yet another embodiment, the présent invention provides an improved method for the production of CRM|₉₇ which method comprises culturing the engineered Corynebacterium diphtheria strain having increased copy number of CRM_I97 gene in a fermentation media which is free of animal derived components and comprises more than 10 amino acids, wherein the amino acids are selected from Alanine, Arginine, Aspartic acid, Cysteine, Glutamic acid. Glutamine, Glycine, Histidine, Isoleucine, Leucine, Lysine, Méthionine, Phenyl alanine, Proiine, Serine, Threonine, Tryptophan, Valine and its salts wherein each amino acid is used in an amount of about 0.05 to 2 g/1.

In a piefeired embodiment, the présent invention provides an improved method for the production of CRM197 which method comprises

i) cultuiing the engineered Corynebaclerium diphtheria strain having increased copy number of CRM 197 gene in a fermentation media which is free of animal derived components and comprises base media and more than 10 amino acids selected from Alanine, Arginine, Aspartic acid, Cysteine, Glutamic acid, Glutamine, Glycine, Histidine, Isoleucine, Leucine, Lysine, Méthionine, Phenyl alanine, Proline, Serine, Threonine, Tryptophan, Valine and its salts and ii) supplementing the media with glucose and nutrients based on metabolic flux model.

In yet anothei embodiment, during the fermentation process température is maintained in the range from 30 to 40 °C and pH is maintained at 7.0 to 8.0, preferably 7.4 to 7.6 using 20% orthophosphoric acid. 12.5% ammonium hydroxide. The fermentation process is carried out for a period of 15 to 24 hours, preferably 1 6 to 20 hours.

In an embodiment, the présent invention provides fermentation composition and process which is devoid of tyrosine and asparagine. In an embodiment, the process of the présent invention is devoid of casamino ac.d based defferated low iron YC med.a, maltose as a carbon source and deferration step,

The présent invention provides an improved method for the production of CRM₁₉₇ which method comprises cultunng the engineered Corynebacterium diphtheria strain having increased copy number of CRM 197 gene in a fermentation media which is free of animai derived components and contains combination of Phenyl alanine, Arginine in an amount of about less than 1 g/L and one or more other amino acids.

In an embodiment, the présent invention provides an improved method for the production of CRM197 which method comprises culturing the engineered Corynebacterium diphtheria strain having increased copy number of CRM_iy7 gene in a fermentation media which is free of animal derived components and comprises more than 10 amino acids and supplementing the media with nutrients based on metabolic flux model, wherein the yield of CRM_W obtained is more than 150 mg/L.

In an embodiment, the présent invention enables making a conjugate vaccine which comprises conjugating polysaccharides from Salmonella typhi, Streplococcus pneumoniae, meningococcus, Haemophilus influenzae with CRM197 prepared according to the présent invention.

The présent invention provides an improved method for the production of CRM|₉₇ with high yield using engineered Corynebacterium diphtheria strain having increased copy number of CRM i97 gene, wherein the method comprises growing the strain in media free of animal derived components comprising more than 10 amino acids and supplementing the media with nutrients based on metabolic flux model.

in an embodiment, the présent invention provides an improved method for the production of CRM197 with yields such as 150mg/L, 200mg/L, 300mg/L. 500mg/L. Ig/L. 1.5g/L, 2 g/L, 2.5g/L, 3g/L, 3.5g/L, 4g/l, 4.5g/L, and 5g/L.

In an embodiment. the présent invention provides an engineered Corynebacterium diphtheria strain C7 (β 197) wherein pBE33 plasmid was transferred by electroporation.

The CRM,₉₇ produced according to the présent invention has been quantified with ImmunoCapture Enzyme Linked Immuno-Sorbent Assay (IC-ELISA).

Development of Engineered Corynebacterium diphtheria strain having încreased copy number of CRM₁₉₇ gene on an expression vector plasmid.

Corynebcaterium diphtheriae C7 (β-197) ATCC 53821, the gene encoding for CRM_W exists as single copy and mutant CRM₁₉₇ protein is secreted into the culture media under iron régulation. The yield of CRM₁₉₇ increases three-fbld in Corynebacterium. diphtheriae C7 strain that hâve two copies of the corynephage-beta (ATCC 39255). This suggests that gene copy number is linked to încreased productivity. In order to make CRM₍₉₇ production from Corynebacterium diphtheriae commercially viable it is desired to increase the expression level. Integrating more copy number into bacterial genome is technically challenging. The multi copy phage intégrants are genetically unstable and lose the additional copies of the CRM gene. The présent inventors intended to improve the expression levels ot the CRMj₉₇ by increasing the gene copies of CRM₁₉₇ delivered on plasmids having an antibiotic sélection marker, along with the native icgulatory éléments oi CRM_!97. Accordîngiy, the inventors hâve developed Corynebacterium diphtheria strain having încreased copy number of CRMi₉₇ gene, evaluated the strain’s stability. The modified strain had higher levels of CRM₁₉₇ expression compared to un modified Corynebacterium strains.

The présent invention is more specificaily illustrated with reference to the examples given below. However, ît should be understood that the présent invention is not limited by an example in any manner but includes variations thereof within the parameters described herein, as can be known to those well-versed in the art.

Example 1 Improvement of CRM_I97 expression by episomal copy of CRMI97:

A vector that can stably replicale in Corynebacterium diphtheria Cl (β 197) was prepared. Isolation of Native plasmid from Corynebacterium Cl diphtheriae has been carried ont using large plasmid isolation protocol as described in T.C Currier et al., Anal Biochem. 1976; 76(2): 431441. Using the native plasmid DNA préparation, 1.87Kb origin of réplication (priR) was amphfied. Simultaneously, 1.033Kb kanR sequence was amplified using pUC4-KIXX template DNA and blunt end ligated to oriR to generate pBE30. Further a 2.13 Kb gene sequence of CRM|₉₇ comprising pTox promoter région (Boyd et ah, 1988), cru and predicted terminator sequence was amplified from Corynebacterium diphtheria C7 (β 197) genomic DNA and cioned into the unique Spel restriction site designed in the pBE30, The GAG mutation in this amplicon was verified by allele spécifié PCR assay (Pushnova et al., Anaiytical Biochemistry. 1998; 260: 24 to 29) and DNA sequencing. The plasmid thus obtained was designated as pBE33 (Figure 1).

The pBEoj plasmîd was transferred by electropo ration folio wing the procedures optimîzed for Corynehacterium diphtheria C7 (β 197). The transformed Corynehacterium diphtheria C7 (β 197) (pBE33) colonies were confirmed by spécifie colony PCR and plasmid isolation and restriction digestion. Expression of CRM197 in colonies was analyzed by SDS-PAGE and Western blotting. The CRM197 in the media was quantified by EL1SA and HPLC and were presented as concentration of CRM|₉₇ expressed per liter of the culture media.

Exaniple 2

Production of CRM197 with engineered Corynebacterium diphtheria C7Ep strain on base media - free of animal derived components.

Corynebacterium diphtheria C7Ep strain was used for this process. A two-step cultivation was followed for inocuhim préparation in shake flask. Media for shake flask cultivation comprises of Yeast Extract (YE) 10 g/L, Veg. Peptone 15g/L, KH₂PO₄ 4.3g/L, Tryptophan 50mg/L, Glucose 4.0g/L, Nicotinic acid 0.8 mg/L, Pimelic acid 0.08 mg/L, CuSO₄. 5H₂O 25 mg/L, ZnSO₄. 5H₂O 12.5 mg/L, MnCl₂. 4H₂O 6.25 mg/L, Cystine 0.5 g/L, Kanamycin 25 mg/L, Glucose 4.0 g/L.

To a 20 I, fermenter media. 5% inoculum was added to start the process. Media for fermenter cultivation comprises of YE 15g/L, Veg.Peptone 30 g/L, KH₂PO₄ 4.3g/L, Tryptophan 50mg/L, Nicotinic acid 0.8 mg/L, Pimelic acid 0.08 mg/L, CuSO₄. 5H₂O 25 mg/L, ZnSO₄. 5H₂O 12.5 mg/L, MnCl₂. 4H₂O 6.25 mg/L, Cystine 0.5 g/L, Kanamycin 25mg/L, Glucose 4.0 g/L.

The culture was stirred al 300 RPM throughout the batch. Température was maintained al 35 °C and pH was maintained at 7.4 using 20% orlhophosphoric acid and 5 N NaOH. The culture was aerated using 1.0 wm air. Culture was enriched with oxygen to maintain Dissolved Oxygen (DO) level to 20%. After first few hours, DO levels keeps falling below 20% when limit of O₂enrichment is reached, The DO level for rest of the batch remains close to zéro with rise towards end hours. When residual glucose in the broth fells below 0.5 g/L, 40% glucose feed was given with 2 g/L/hr. Batch was harvested aller 14 hrs of cultivation when cell densities reached about units of OD 600nm. The foam in the reactor was controlled by 30% organic antifoam. The CRMi97 production was reached to a titer of 60 to 65 mg/L of fermentation broth.

A fermentation lun under similar conditions using unengineered Corynebacterium diphtheriae Cd CRM|97 stiaîn produced about 35 to 40 mg/L of CRM 197 which is less than engineered strain. This shows the influence of the extra gene copies towards increased production of the CRM197.

Example 3:

Production of CRM₍<j7 with engineered Corynebacterium diphtheria C7Ep strain on base media free of animal derived components by following the metabolic flux balance model.

Corynebacterium diphtheria C7Ep strain was used for this process. A two-step cultivation was foliowed for inoculum préparation in shake flask. Media for shake flask cultivation comprises of YE 10 g/L, Veg. Peptone 15g/L, KH₂PO₄ 4.3g/L, Tryptophan 50mg/L, Glucose 4.0g/L Nicotinic acid 0.8 mg/L. Pimelic acid 0.08 mg/L, CuSO₄. 51-bO 25 mg/L, ZnSO₄. 5H₂O 12.5 mg/L, MnCb. 4H2O 6.25 mg/L. Cystîne 0.5 g/L, Kanamycin 25mg/L, Glucose 4.0 g/L.

To a 20 L fermenter media, 5% inoculum was added to start the process. Media for fermenter cultivation comprises of YE 15 g/L, Veg.Peptone 30 g/L, KH₂PO₄ 4.3g/L, Tryptophan 50mg/L, Nicotinic acid 0.8 mg/L, Pimelic acid 0.08 mg/L, C11SO4. 5H₂O 25 mg/L, ZnSO₄. 5H2O 12.5 mg/L, MnCb. 4H₂O 6.25 mg/L, Cystine 0.5 g/L, Kanamycin 25mg/L, Glucose 4.0 g/L,

The culture was stirred at 300 RPM throughout the batch. Température was maîntained at 35 °C and pH was maîntained at 7.4 using 20% orthophosphoric acid and 12.5% ammonium hydroxîde. The culture was aerated using 1.0 vvm air. Culture was enriched with oxygen to maintaîn DO level to 20%. A model was introduced which follows the metabolic rate of conveision in the given point ol time, this model takes the input from the online parameter of residual DO in correspondence with the changes with respect to pH, CO₂, heat génération. In log phase. DO levels keeps falling below 20% despite O₂ enrichment limit has reached. The DO level for resl of the batch remains close to zéro with vise towards end hours. When residual glucose in the broth fall below 0.5 g/L, 40% glucose feed was fed in order to justify the metabolic flux model. Batch was harvested aller 14 hrs of cultivation when cell densities reached about 90 units of OD 600nm. The foam in the reactor was controlled by 30% organic antifoam. The CRM 197 production was reached to a titer of 150 mg/L of fermentation broth.

Example 4: Production of CRM₁₉₇ according to the présent invention

A thiee-step cultivation was followed for inoculum préparation. First two steps were done in the shake flasks. Media for shake flask cultivation comprises of YE 10 g/L, Veg.Peptone 15g/L, 5 KH₂PO₄ 4.3g/L, Tryptophan 50mg/L, Glucose 4.0g/L, YC Trace sait solution 2 ml/L, Cystine supplément 1 ml/L, Kanamycin 25mg/L, Glucose 4.0 g/L.

The base media composition for seed fermenter was YE 15g/L, Veg.Peptone 30 g/L_} KH2PO4

4.3g/L, Tryptophan 50mg/L, Nicotinic acid 0.8 mg/L, Pimelic acid 0.08 mg/L, CuSO₄. 5H₂O 25 10 mg/L, ZnSO₄. 5H₂O 12.5 mg/L, MnC^. 4H₂O 6.25 mg/L, Cystine 0.5 g/L, Kanamycin 25mg/L, Glucose 4.0 g/L

Given below is the fermenter media composition (Table 1) and the ingrédients which lias been designed following the Placket Burmann design.

Table 1

S. No	Media components	Quanti ty	Cuits	S. No	Media components	Quantity	Units
1	Alanine	0.1		18	Valine	1	g/L
2	Arginine	0.1	g/L	19	Potassium	2	g/L
3	Aspartic acid	0.5	g/L	20	Magnésium	1	g/L
4	Cysteine	0.5	g/L	21	Thiamine	0.1	mg/L
5	Gkitamic acid	1	g/L	22	Biotin	4	mg/L
6	Glutamine	0.1	g/L	23	Calcium	2	mg/L
7	Glycine	0.5	g/L	24	Chloride	0.5	mg/L
8	Histidine	1	g/L	25	Choline	Γ 0.1	mg/L
9	Isole ucine	1	g/L	26	Copper	10	mg/L
10	Leucine	0.5	g/L	27	Folie Acid	1,6	mg/L
11	Lysine	0.1	g/L	28	Manganèse	1	mg/L
12	Méthionine	1	g/L	29	Nicotinamîc Acid	100	mg/L
13	Phenyl alanine	0.5	g/L	30	Pantothenic Acid	50	mg/L
bd	Praline	0.1	g/L	31	Pimelic acid	20	mg/L

15	Serine	0.1	g/L	32	Riboflavin	50	mg/L
16	Threonine	0.1	g/L	33	Sulfate	20	mg/L
17	Tryptophan	0.1	g/L	34	Zinc	7	mg/L

To a 20 L fermenter with above components with the base media (YE 15 g/L, Veg.Peptone 30 g/L, KH₂PO₄ 4,3 g/L·, Tryptophan 50 mg/L, Nicotinic acid 0.8 mg/L, Pimelic acid 0.08 mg/L, CuSO₄. 5H₂O 25 mg/L, ZnSO₄. 5H₂O 12.5 mg/L, MnCl₂. 4II₂O 6.25 mg/L, Cystine 0.5 g/L, Kanamycin 25mg/L, Glucose 4.0 g/L·.), 5% inoculum from seed fermenter was added to start the process. Température was maintained at 35 °C and pH was maintained at 7.4 to 7.6 using 20% orthophosphoric acid. 12.5% ammonium hydroxide. and model based on metabolic flux (Figure 2) modulation of glucose feeding and OTR. The culture was aerated using 1.0 vvm air. Culture was enriched with oxygen to maintain DO Ievel to 20%. In log phase, DO levels keeps falling below 20% despite O₂ enrichment limit has reached. The DO Ievel for rest ofthe batch remains close to zéro with ri se towards end hours. When residual glucose in the broth falls below 0.5 g/L, feed with 40% glucose solution was given at 4.5 g/L/hr. Intermittent!y vitamins and trace éléments were supplemented at following levels ; Nicotinic acid 6.425 mg/L, Pimelic acid 0.465 mg/L, CuSO₄. 5H₂O 25 mg/L, ZnSO₄. 5H₂O 12.5 mg/L, MnCl₂. 4H₂O 6.25 mg/L, Thiamine 0.08 mg/L, Pantothenic acid 0.25 mg/L, Biotin 0.006 mg/L, ,Ribollavm 0.3 mg/L, Folie Acid 0.06 mg/L . The batch was harvested after 18 hrs of cultivation when cell densilies reached about 132 units of OD 600nm. The foam in the reactor was controlled by 30% organic antifoam. The CRMtç>7 production reached a titer of 450 to 500 mg/L of fermentation broth.

Example 5

Comparison of CRM197 yiclds using native Corynebacterium diphtheriae C7 and an engin eered s train of Corynebacterium diphtheriae C7Ep.

A fermentation was carried ont using basic animal free media, Pre model Process Improvements and Media and process as per the Présent invention using Native C7 Corynebacterium diphtheria and engîneered Corynebacterium diphtheria strain.

The single lysogénie Corynebacterium diphtheriae strain C7 and engineered strain with episomally placed extra copies of CRM 197 gene (C7Ep) were used for process optimization. In a typical non deferrated YC media having animal components, C7 gave 22 to 25 mg/L of CRM 197 whereas C7Ep gave 40 to 45 mg/L. When media was modified by replacing the animal components by veg peploues, the C7 strain resulted in 40 to 45 mg/L of CRM|₉₇ and C7Ep gave mg/L of CRM₁₉₇. Process conditions when modified by changing the parameters, C7Ep produced 120 to 150 mg/L of CRM_i97. A mode! was developed using inputs from the base process. The final outcome of the optimized method which resulted in 120 mg/1 of CRM_I97 from C7 and >500 mg/L of CRM₁₉₇from C7Ep, The yields are compared in Table II given below:

Table II - Yield comparison of Native C7 - Corynebacterium diphtheriae with that of episomally modified C7Ep al different process condition.

S. No	Organism ___	Yields of CRMi₉₇in mg/L by ELISA
Basic Animal free media	Pre model Process Improvements	Media and per the invention	process as Présent
1	Corynebacterium C7 ( Native)	20 -25	35 -40	110- 120
2	Corynebacterium C7 Ep ( Epîsome incorporated extra	60-65	120-150	> 500

copy of gene)

Claims

I. A method for the production of CRM₁₉₇ with a bigh yield using an engineered Corynebacterium diphtheria strain having an increased copy number of the CRM 197 gene, wherein the method comprises growing the strain in a non-defferated fermentation medium comprising more than 10 amino acids other than tyrosine and asparagine and free of animal derived components, wherein no maltose is used as a carbon source.
2. The method as claimed in claim 1, wherein the method comprises supplementing the medium with nutrients.
3. The method as claimed in claim 2, wherein the supplément with nutrients is based on a metabolic flux model.
4. The method as claimed in claim 1, wherein the amino acids are selected from Alanine, Arginine, Aspartic acid. Cysteine, Glutamic acid, Glutamine, Glycine, Histidine, Isoleucine, Leucine, Lysine, Méthionine, Phenyl alanine, Proline, Serine, Threonine. Tryptophan, Valine and their salts.
5. The method as claimed in claim 4, wherein each amino acid is used in an amount of about 0.05 to 2 g/L.
6. The method as claimed in claim 1, wherein the fermentation medium contains a combination of Phenyl alanine, Arginine and other amino acids wherein the amount of Phenyl alanine and Arginine is less than about l g/L.
7. The method as claimed in claim 2, wherein the nutrients are selected from vitamins such as Nicotinic acid, Thiamine, Pantothenic acid, Biotîn, Riboflavin, FolicAcid, pimelic acid, phosphate, a nitrogen source and trace metals.
8. The method as claimed in any of the preceding claims, wherein the fermentation medium is non-defferated and completely free from animal derived components.
9. The method as claimed in any of the preceding claims, wherein the fermentation medium comprises a base fermentation medium comprising Yeast Extract, Veg.Peptone, Potassium η

dihydrogen phosphate (KH₂PO₄), Tryptophan, Glucose, and YC Trace sait solution.
10. The method as claimed in any of the precedîng daims, wherein the method comprises supplementing the medium with glucose during the entire fermentation process.
11. A method for the production of CRM 197, wherein the method comprises:

i) culturing an engineered Corynebacterium diphtheria strain having an increased copy number of the CRM₁₉₇ gene in a non-defferated fermentation medium which is free of animal derived components and comprises a base medium and more than 10 amino acids selected from Alanine, Arginine, Aspartic acid, Cysteine, Glutamic acid. Glutamine, Glycine, Histidîne, Isoleucine, Leucine, Lysine, Méthionine, Phenyl alanine, Proline, Serine, Threonine, Tryptophan, Valine and theîr salis, and ii) supplementing the medium with glucose and nutrients based on a metabolic flux model, wherein no maltose is used as a carbon source.
12. A method for the production of CRM 197, which method comprises culturing an engineered Corynebacterium diphtheria strain having an increased copy number of the CRM 197 gene in a non-defferated fermentation medium which is free of animal derived components and comprises more than 10 amino acids other than tyrosine and asparagine and supplementing the medium with nutrients based on metabolic flux model, wherein each amino acid is used in an amount of about 0.05 to 2 g/L, wherein no maltose is used as a carbon source.
13. The method as claimed in any of the preceding daims, wherein the fermentation is camed out at a température between 30 to 40 °C and at a pH in the range of 7.0 to 8.0, preferably 7.4 to 7.6
14, The method as claimed in any of the preceding daims, wherein the yield of CRM197 obtained is more than I50mg/L, 200mg/L, 300mg/L, 500mg/L, Ig/L, L5g/L, 2 g/L, 2.5g/L, 3g/L, 3.5g/L, 4g/l, 4.5g/L, or 5g/L.
15. A method for manufacturing a conjugate vaccine which comprises conjugating polysaccharides from Salmonella typhi, Streptococcus pneumoniae, or Haemophilus infhienzae with CRM197 prepared according to any of the preceding daims.