MÉTODOS PARA A PRODUÇÃO DE CRM 197 E MÉTODO PARA A FABRICAÇÃO DE VACINA CONJUGADA

Abstract

The present invention relates to an improved method for the high-yield production of crm197 using a manipulated strain of Corynebacterium diphtheriae with an increased copy number of the crm197 gene, wherein the method comprises cultivating the strain in a medium free of animal-derived components containing one or more amino acids.

Description

1 / 19 “METHODS FOR THE PRODUCTION OF CRM197 AND METHOD FOR THE MANUFACTURE OF CONJUGATE VACCINE” FIELD OF TECHNIQUE

[001] The present invention relates to an improved method for producing CRM197 in high yield using a manipulated Corynebacterium diphtheriae strain having an increased number of copies of the CRM197 gene. FUNDAMENTALS OF THE INVENTION

[002] CRM197 is a genetically detoxified form of diphtheria toxin. A single mutation at position 52, replacing glutamic acid with glycine, causes the loss of ADP-ribosyltransferase activity of the native toxin. The structural basis of CRM197's lack of toxicity has been elucidated and it is widely used as a carrier protein for conjugate vaccines. CRM197, like diphtheria toxin, is a single polypeptide chain of 535 amino acids (58.4 kDa) consisting of two subunits (linked by disulfide bridges).

[003] CRM197 is used as a carrier protein in several approved conjugate vaccines, such as the Haemophilus influenzae type b conjugate marketed under the trade name Hibtiter™, the 13-valent pneumococcal polysaccharide conjugate marketed under the trade name PREVNAR 13®, and similar vaccines.

[004] Ruth M. Drew et al., Bacteriol. 1951 Nov; 62 (5): 549-59; described a chemically defined medium suitable for the production of high-titer diphtheria toxin and summarized the amino acid requirements of Corynebacterium diphtheriae, Park-Williams Toronto strain no. 8. The medium contains amino acids that effectively replace the animal-derived component, i.e., casein hydrolysate. The amino acids include glutamic acid, cystine, proline, tryptophan, leucine, valine, methionine, and glycine.

[005] Rappuoli and others; Applied and Environmental Microbiology 1983, vol. 46 Petition 870260056488, dated 10 / 06 / 2026, page 10 / 57 2 / 19 (3): 560 to 564 described that non-tandem double lysogens were stable and capable of providing high CRM197 yields, up to three times higher than monolysogens.

[006] R Fass. et al., Applied Microbiology and Biotechnology; April 1995, Volume 43 (1): 83 to 88; described a high-density growth approach to produce mutated diphtheria toxin from two strains of Corynebacterium diphtheriae: C7(β) (tox-201, tox-9) and C7(β) (tox-107). The procedure involved the use of a modified, iron-free (non-deferred) growth medium, which provided rapid, high-density growth of the bacteria and which, when associated with simultaneous depletion of glucose and iron, increased toxin production. Oxygen-enriched air was supplied to allow the bacteria to grow to a cell density, providing an absorbance of 70 to 600 nm (15 to 20 g / L dry weight). The maximum toxin concentration in the culture supernatant was 150 mg / L.

[007] Parag P. Nagarkar et al., Journal of Applied Microbiology 2002, 92, 215-220; described the pattern of amino acid utilization during the growth of Corynebacterium diphtheriae and showed that only four of the nine amino acids tested, namely cystine, histidine, aspartate, and methionine, were critical for the growth and production of toxins by Corynebacterium diphtheriae.

[008] European Patent No. 1 849 860 B 1 described the use of protein material of non-animal origin, such as proteins from soybeans, cottonseed, potatoes, etc., as a constituent medium for the cultivation of pathogenic bacteria.

[009] US Patent No. 6,962,803 B2 described a method for purifying diphtheria toxin by fermenting a strain of microorganisms capable of producing diphtheria toxin, said method comprising adding glucose to a growing culture, wherein the addition of glucose maintains the growth of microorganisms. Petition 870260056488, dated 10 / 06 / 2026, page 11 / 57 3 / 19 effective organisms to support diphtheria toxin production. It further described that, in addition to a carbon source, there are other minimum nutritional requirements for growth, which include trace metals, phosphate, a nitrogen source, usually casing amino acids, and yeast extract.

[010] Publication of US Patent Application No. 2011 / 0097359 A1 described a medium for culturing a strain of Corynebacterium diphtheriae and for producing a level of diphtheria toxin or an analogue thereof, wherein the medium is substantially free of animal-derived products and comprises: water; a carbohydrate source; a nitrogen source; and a number of free amino acids at an initial concentration wherein the initial concentration of each free amino acid is not limiting for the level of production of diphtheria toxin or its analogue. Furthermore, it describes that the carbohydrate source is glucose-free.

[011] Document WO 2006 / 100108 A1 described a fermentation process comprising a growth step of a Corynebacterium diphtheriae strain in a medium within the fermenter under conditions of sufficient agitation to maintain a homogeneous culture and limited aeration, such that pO2 within the culture falls to less than 4% for most of the fermentation step. It is also described that the pH within the fermenter is maintained between 7.0 and 7.8 by the degree of aeration without the need for the addition of acid or base.

[012] The CRM197 process is generally sensitive to small changes in process components as well as process parameters. The production of CRM197 from Corynebacterium diphtheria is carried out worldwide, although with limited success for commercial realization. None of the above references described the method of producing CRM197 with high yields, such as > 150 mg / L, using the manipulated Corynebacterium diphtheriae strain. The inventors of the present invention have developed a metabolic flux model for the production of high-yield CRM197 using the manipulated Corynebacterium strain. Petition 870260056488, dated 10 / 06 / 2026, page 12 / 57 4 / 19 diphtheriae. OBJECTIVE OF THE INVENTION

[013] The main objective of the present invention is to provide an improved process for the high-level production of CRM197.

[014] Yet another objective of the present invention is to provide an improved process for the high-level production of CRM197, which is cost-effective and can be used for the manufacture of conjugate vaccines. SUMMARY OF THE INVENTION

[015] The present invention provides an improved method for the high-yield production of CRM197 using the manipulated strain of Corynebacterium diphtheriae with an increased copy number of the CRM197 gene, wherein the method comprises cultivating the strain in a fermentation medium comprising one or more amino acids and being free of components of animal origin.

[016] The present invention provides an improved method for the production of CRM197, a method comprising cultivating the manipulated strain of Corynebacterium diphtheriae with an increased number of copies of the CRM197 gene in a fermentation medium free of components of animal origin and comprising more than 10 amino acids and supplementing the medium with nutrients.

[017] The present invention also provides an improved method for the production of CRM197, a method comprising cultivating the manipulated strain of Corynebacterium diphtheriae with an increased number of copies of the CRM197 gene in a fermentation medium free of components of animal origin and comprising more than 10 amino acids and supplementing the medium with nutrients based on the metabolic flux model. BRIEF DESCRIPTION OF THE DRAWINGS

[018] Figure 1 shows the construction of a plasmid designated as pBE33 Petition 870260056488, dated 10 / 06 / 2026, page 13 / 57 5 / 19

[019] Figure 2 presents a flowchart of the metabolic flow equilibrium model. DETAILED DESCRIPTION

[020] The present invention provides an improved method for the high-yield production of CRM197 using the manipulated strain of Corynebacterium diphtheriae with an increased copy number of the CRM197 gene, wherein the method comprises cultivating the strain in a fermentation medium comprising more than 10 amino acids and being free of components of animal origin.

[021] The manipulated strain of Corynebacterium diphtheriae e (C7Ep) of the present invention refers to Corynebacterium diphtheriae that has multiple episomally localized copies of the CRM197 gene regulated by the same mechanism as the host CRM197 gene and the genetic background of said strain is a single lysogen.

[022] The amino acids used in the present invention are selected from alanine, arginine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, valine and their salts, and each amino acid is used in an amount of about 0.05 to 2 g / L.

[023] The effect of amino acids, vitamins and process conditions on the production of CRM197 was studied. Certain amino acids were found to have a negative effect on bacterial growth. However, it was found that the production of CRM197 is increased if more than 10 amino acids are used at an optimal concentration. The amino acid concentration is optimized using the Plackett-Burman experimental model.

[024] Plackett-Burman models are experimental models for investigating the dependence of some measured quantity on a number of independent variables (factors), in order to minimize the variation of estimates of these dependencies using a limited number of experiments. The result showed that Petition 870260056488, dated 10 / 06 / 2026, p. 14 / 57 6 / 19 amino acids such as aspartic acid, glutamine, glycine, isoleucine, leucine, and valine, at a temperature of 35°C to 36°C, have a positive impact on the synthesis of CRM197, while amino acids such as alanine, isoleucine, and valine, at a temperature of 35°C to 36°C, have a negative effect on the synthesis of CRM197. Several model levels of experiments were performed at different concentrations to achieve high-level production of CRM197. The use of tyrosine and asparagine resulted in a decrease in the total yield of CRM197, and therefore these amino acids are not part of the invention.

[025] In a preferred embodiment of the present invention, the fermentation medium contains a combination of Phenylalanine, Arginine and one or more other amino acids wherein the amount of Phenylalanine and Arginine used is less than about 1 g / L.

[026] In a more preferred embodiment of the present invention, the fermentation medium comprises a combination of Phenylalanine, Arginine and one or more other amino acids wherein the amount of Phenylalanine is about 0.25 to 0.75 g / L and that of Arginine is about 0.1 to 0.5 g / L.

[027] The present invention provides an improved method for the production of CRM197 using the manipulated strain of Corynebacterium diphtheriae with an increased copy number of the CRM197 gene, wherein the method comprises cultivating the strain in a fermentation medium comprising more than 10 amino acids, supplementing the medium with vitamins in the range of about 0.05 to 20 mg per liter, wherein the medium is free of components of animal origin.

[028] In another embodiment of the present invention, amino acids, vitamins, trace elements and the like are added as nutrients to the fermentation medium during cultivation.

[029] Several nutrients used in the present invention as supplements include selected vitamins of nicotinic acid, thiamine, pantothenic acid, biotin, riboflavin, folic acid; pimelic acid; phosphate, a nitrogen source and Petition 870260056488, dated 10 / 06 / 2026, p. 15 / 57 7 / 19 trace metals and similar substances, and each vitamin is used in an amount ranging from about 0.05 to 20 mg per liter.

[030] The fermentation medium used in the present invention is a product without iron deficiency and completely free of components of animal origin. In addition, the medium is also devoid of the traditional meat-based Loeffler medium and the iron-deficient, low-iron casamino acid-based YC medium. The components of the fermentation medium of the present invention comprise yeast extract, vegetable peptone, potassium dihydrogen phosphate (KH2PO4), tryptophan, glucose, YC trace salt solution.

[031] In another embodiment of the present invention, the composition of the medium for cultivating the manipulated strain of Corynebacterium diphtheriae with an increased number of copies of the CRM197 gene comprises basic fermentation media comprising yeast extract, vegetable peptone, potassium dihydrogen phosphate (KH2PO4), tryptophan, glucose, YC trace salt solution; one or more amino acids, vitamins, trace elements and the like.

[032] Suitable trace metals include potassium, magnesium, calcium, chloride, choline, copper, manganese, sulfate, zinc and the like.

[033] The present invention provides an improved method for the production of CRM197 using the manipulated strain of Corynebacterium diphtheriae with an increased copy number of the CRM197 gene, wherein the method comprises supplementing the fermentation medium with nutrients based on the metabolic flux model.

[034] The metabolic flow model refers to the complete network of kinetics of heat transfer, mass transfer, oxygen transfer rate (OTR), oxygen uptake rate (OUR), and ion transfer, where the OTR is maintained by means of agitation, backpressure, and pumping in pure oxygen. This model is represented in Figure 2.

[035] Development of a Metabolic Flux Model. Petition 870260056488, dated 10 / 06 / 2026, page 16 / 57 8 / 19

[036] This model is created based on the interpretation of direct online data of hydrogen ion accumulations in the process as a primary variable, followed by dissolved oxygen, oxygen conversion to CO2, and heat released in the process. For example, the model works under limiting carbon source conditions. The carbon source is pulsed, and the response is evaluated in the process. Corrective action was taken on hydrogen ion generation based on the response. Then, the feed was optimized to obtain a constant pH and dissolved oxygen (DO) and heat transfer rate (e.g., pH of 7.4; residual DO > 2 to 20; heat transfer rate HTR).

[037] In one embodiment of the present invention, the method comprises supplementing the medium with glucose throughout the fermentation process.

[038] The present invention does not involve the use of maltose as a carbon source and the deferronization step.

[039] CRM197 produced according to the present invention can be used for the manufacture of conjugate vaccines, such as pneumococcal conjugate, typhoid conjugate, Hib conjugate and the like.

[040] In yet another embodiment, the present invention provides an improved method for the production of CRM197, which comprises cultivating the manipulated strain of Corynebacterium diphtheriae with an increased copy number of the CRM197 gene in a fermentation medium free of components of animal origin and comprising more than 10 amino acids and supplementing the medium with nutrients based on the metabolic flux model, wherein the amino acids are selected from alanine, arginine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, valine and salts thereof.

[041] In yet another embodiment, the present invention provides an improved method for the production of CRM197, a method comprising cultivating the strain of Petition 870260056488, dated 10 / 06 / 2026, p. 17 / 57 9 / 19 Corynebacterium diphtheriae manipulated with an increased number of copies of the CRM197 gene in a fermentation medium free of animal-derived components and comprises more than 10 amino acids, where each amino acid is used in an amount of approximately 0.05 to 2 g / L.

[042] In yet another embodiment, the present invention provides an improved method for the production of CRM197, a method comprising cultivating the manipulated strain of Corynebacterium diphtheriae with increased copy number of the CRM197 gene in a fermentation medium free of components of animal origin and comprising more than 10 amino acids and supplementing the medium with nutrients based on the metabolic flux model, wherein each amino acid is used in an amount of about 0.05 to 2 g / L.

[043] In yet another embodiment, the present invention provides an improved method for the production of CRM197, a method comprising cultivating the manipulated strain of Corynebacterium diphtheriae with increased copy number of the CRM197 gene in a fermentation medium free of components of animal origin and comprising base media with more than 10 amino acids and supplementing the medium with nutrients based on the metabolic flux model, wherein each amino acid is used in an amount of about 0.05 to 2 g / L.

[044] In yet another embodiment, the present invention provides an improved method for the production of CRM197, a method comprising cultivating the manipulated strain of Corynebacterium diphtheriae with an increased copy number of the CRM197 gene in a fermentation medium free of animal-derived components and comprising more than 10 amino acids and supplementing the medium with nutrients based on the metabolic flux model, wherein the amino acids are selected from 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 a Petition 870260056488, dated 10 / 06 / 2026, page 18 / 57 10 / 19 quantity of approximately 0.05 to 2 g / L.

[045] In yet another embodiment, the present invention provides an improved method for the production of CRM197, a method comprising cultivating the manipulated strain of Corynebacterium diphtheriae with increased copy number of the CRM197 gene in a fermentation medium free of components of animal origin and comprising 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, methionine, phenylalanine, proline, serine, threonine, tryptophan, valine and salts thereof wherein each amino acid is used in an amount of about 0.05 to 2 g / L.

[046] In a preferred embodiment, the present invention provides an improved method for the production of CRM197, a method comprising: i) cultivate the manipulated strain of Corynebacterium diphtheriae with an increased number of copies of the CRM197 gene in a fermentation medium free of components of animal origin and comprising base media and more than 10 amino acids selected from alanine, arginine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, valine and salts thereof, and ii) supplement the medium with glucose and nutrients based on the metabolic flux model.

[047] In yet another method, during the fermentation process, the temperature is maintained in the range of 30°C to 40°C 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. The fermentation process is carried out for a period of 15 to 24 hours, preferably 16 to 20 hours.

[048] In one embodiment, the present invention provides a composition and fermentation process that is devoid of tyrosine and asparagine. In a Petition 870260056488, dated 10 / 06 / 2026, p. 19 / 57 In embodiment 11 / 19, the process of the present invention is devoid of iron-deficient YC media with low iron content based on casamino acid, maltose as a carbon source and deferronization step.

[049] The present invention provides an improved method for the production of CRM197, a method comprising cultivating the manipulated strain of Corynebacterium diphtheriae with increased copy number of the CRM197 gene in a fermentation medium free of components of animal origin and containing the combination of phenylalanine and arginine in an amount of about less than 1 g / L and one or more other amino acids.

[050] In one embodiment, the present invention provides an improved method for the production of CRM197, a method comprising cultivating the manipulated strain of Corynebacterium diphtheriae with an increased number of copies of the CRM197 gene in a fermentation medium free of components of animal origin and comprising more than 10 amino acids and supplementing the medium with nutrients based on the metabolic flux model, wherein the yield of CRM197 obtained is greater than 150 mg / L.

[051] In one embodiment, the present invention allows the manufacture of a conjugate vaccine comprising conjugating polysaccharides from Salmonella typhi, Streptococcus pneumoniae, meningococcus, Haemophilus influenzae with CRM197 prepared in accordance with the present invention.

[052] The present invention provides an improved method for the high-yield production of CRM197 using the manipulated strain of Corynebacterium diphtheriae with an increased copy number of the CRM197 gene, wherein the method comprises cultivating the strain in media free of animal-derived components comprising more than 10 amino acids and supplementing the medium with nutrients based on the metabolic flux model.

[053] In one embodiment, the present invention provides a method Petition 870260056488, dated 10 / 06 / 2026, p. 20 / 57 12 / 19 improved for the production of CRM197 with yields such as 150 mg / L, 200 mg / L, 300 mg / L, 500 mg / L, 1 g / L, 1.5 g / L, 2 g / L, 2.5 g / L, 3 g / L, 3.5 g / L, 4 g / L, 4.5 g / L and 5 g / L.

[054] In one embodiment, the present invention provides a manipulated C7 strain of Corynebacterium diphtheriae (β 197) into which the pBE33 plasmid has been transferred by electroporation.

[055] CRM197 produced according to the present invention was quantified using the Enzyme-Linked Immunosorbent Assay - Immunocapture (IC-ELISA).

[056] Development of a Manipulated Corynebacterium diphtheriae Strain with Increased Copy Number of the CRM197 Gene in an Expression Vector Plasmid.

[057] Corynebacterium diphtheriae C7 (β-197) ATCC 53821, the gene encoding CRM197 exists as a single copy, and the mutant CRM197 protein is secreted into the culture medium under iron regulation. CRM197 yield increases threefold in the Corynebacterium diphtheriae C7 strain that has two copies of the beta corynephage (ATCC 39255). This suggests that gene copy number is linked to increased productivity. In order to make CRM197 production from Corynebacterium diphtheriae commercially viable, it is desirable to increase the expression level. Integrating more copy numbers into the bacterial genome is technically challenging. Members of multi-copy phages are genetically unstable and lose the additional copies of the CRM gene.The present inventors aimed to improve CRM197 expression levels by increasing the number of CRM197 gene copies delivered on plasmids carrying an antibiotic selection marker, along with native CRM197 regulatory elements. Therefore, the inventors developed a Corynebacterium diphtheriae strain with an increased number of CRM197 gene copies and evaluated the strain's stability. The modified strain showed higher levels of CRM197 expression. Petition 870260056488, dated 10 / 06 / 2026, p. 21 / 57 13 / 19 comparison with unmodified strains of Corynebacterium.

[058] The present invention is illustrated more specifically with reference to the examples given below. However, it should be understood that the present invention is not limited by any one example in any way, but includes variations thereof within the parameters described herein, as may be known to those skilled in the art. Example 1

[059] Enhancement of CRM197 expression by episomal copying of CRM197:

[060] A vector capable of stably replicating in Corynebacterium diphtheriae C7 (β 197) was prepared. Isolation of the native plasmid of Corynebacterium diphtheriae C7 was performed using a large plasmid isolation protocol as described by TC Currier et al., Anal Biochem. 1976; 76 (2): 431-441. Using the native plasmid DNA preparation, the 1.87Kb origin of replication (oriR) was amplified. Simultaneously, the 1.033Kb kanR sequence was amplified using the pUC4-KIXX template DNA and the blunt end attached to oriR to generate pBE30. Furthermore, a 2.13 Kb gene sequence of CRM197 comprising the pTox promoter region (Boyd et al., 1988), the predicted raw and terminator sequence was amplified from Corynebacterium diphtheriae C7 (β 197) genomic DNA and cloned into the unique Spel restriction site designed on pBE30.The GAG ​​mutation in this amplicon was verified by allele-specific PCR assay (Pushnova et al., Analytical Biochemistry. 1998; 260: 24-29) and DNA sequencing. The plasmid thus obtained was designated pBE33 (Figure 1).

[061] The pBE33 plasmid was transferred by electroporation following the optimized procedures for Corynebacterium diphtheriae C7 (β 197). The transformed colonies of Corynebacterium diphtheriae C7 (β 197) (pBE33) were Petition 870260056488, dated 10 / 06 / 2026, page 22 / 57 14 / 19 confirmed by colony-specific PCR and plasmid isolation and restriction digestion. CRM197 expression in colonies was analyzed by SDS-PAGE and Western blotting. CRM197 in the medium was quantified by ELISA and HPLC and presented as CRM197 concentration expressed per liter of culture medium. Example 2

[062] Production of CRM197 with the manipulated strain of Corynebacterium diphtheriae C7Ep in base media - free of components of animal origin.

[063] The C7Ep strain of Corynebacterium diphtheriae was used for this process. A two-step culture was followed for the preparation of the inoculum in the shake flask. The culture medium in shake flasks comprises Yeast Extract (YE) 10 g / L, Vegetable Peptone 15 g / L, KH2PO4 4.3 g / L, Tryptophan 50 mg / L, Glucose 4.0 g / 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.

[064] To 20 L of fermenting medium, 5% inoculum was added to start the process. The medium for cultivating fermenters comprises YE 15 g / L, Vegetable 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, MnCl2.4H2O 6.25 mg / L, Cystine 0.5 g / L, Kanamycin 25 mg / L, Glucose 4.0 g / L.

[065] The culture was agitated at 300 RPM throughout the batch. The temperature was maintained at 35°C and the pH was maintained at 7.4 using 20% ​​orthophosphoric acid and 5N NaOH. The culture was aerated using 1.0 vvm of air. The culture was enriched with oxygen to maintain the Dissolved Oxygen (DO) level at 20%. After the first few hours, DO levels continue to fall below 20% when the O2 enrichment limit is reached. The DO level for the remainder of the batch remains close to zero with an increase in the final hours. When the residual glucose in the broth falls below 0.5 g / L, 40% glucose is supplied at 2 g / L / h. The batch was harvested after 14 hours. Petition 870260056488, dated 10 / 06 / 2026, page 23 / 57 15 / 19 cultivation was carried out when cell densities reached approximately 90 OD units at 600 nm. Foaming in the reactor was controlled with a 30% organic antifoaming agent. CRM197 production was achieved at a titration of 60 to 65 mg / L of fermentation broth.

[066] A fermentation carried out under similar conditions using the unmanipulated strain of Corynebacterium diphtheriae C7 CRM197 produced approximately 35 to 40 mg / L of CRM197, which is less than the manipulated strain. This shows the influence of extra gene copies on increasing CRM197 production. Example 3:

[067] Production of CRM197 with the manipulated strain of Corynebacterium diphtheriae C7Ep in a base medium free of components of animal origin, following the metabolic flux equilibrium model.

[068] The C7Ep strain of Corynebacterium diphtheriae was used for this process. A two-step culture was followed for the preparation of the inoculum in the shake flask. The culture medium in shake flasks comprises YE 10 g / L, Vegetable Peptone 15 g / L, KH2PO4 4.3 g / L, Tryptophan 50 mg / L, Glucose 4.0 g / 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.

[069] To 20 L of fermentation medium, 5% inoculum was added to start the process. The medium for cultivating fermenters comprises YE 15 g / L, Vegetable 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, MnCl2.4H2O 6.25 mg / L, Cystine 0.5 g / L, Kanamycin 25 mg / L, Glucose 4.0 g / L.

[070] The culture was agitated at 300 RPM throughout the batch. The temperature was maintained at 35°C and the pH was maintained at 7.4 using 20% ​​orthophosphoric acid and 12.5% ​​ammonium hydroxide. The culture was aerated using 1.0 vvm of air. The culture was Petition 870260056488, dated 10 / 06 / 2026, page 24 / 57 16 / 19 enriched with oxygen to maintain DO level at 20%. A model was introduced that follows the metabolic conversion rate at a given point in time; this model receives input of the residual DO parameter corresponding to changes in pH, CO2, and heat generation. In the logarithmic phase, DO levels continue to fall below 20%, despite the O2 enrichment limit having been reached. The DO level for the remainder of the batch remains close to zero with an increase in the final hours. When the residual glucose in the broth falls below 0.5 g / L, 40% glucose is fed to justify the metabolic flux model. The batch was harvested after 14 hours of cultivation when cell densities reached approximately 90 DO units at 600 nm. Foaming in the reactor was controlled with an organic antifoaming agent at 30%. CRM197 production was achieved with a titration of 150 mg / L of fermentation broth. Example 4:

[071] Production of CRM197 according to the present invention

[072] A three-step culture was followed for the preparation of the inoculum. The first two steps were carried out in shake flasks. The medium for the shake flask culture comprised YE 10 g / L, Vegetable Peptone 15 g / L, KH2PO4 4.3 g / L, Tryptophan 50 mg / L, Glucose 4.0 g / L, YC trace salt solution 2 ml / L, cystine supplement 1 ml / L, kanamycin 25 mg / L, glucose 4.0 g / L.

[073] The composition of the base medium for the seed fermenter was YE 15 g / L, Vegetable 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, MnCl2.4H2O 6.25 mg / L, Cystine 0.5 g / L, Kanamycin 25 mg / L, Glucose 4.0 g / L.

[074] The composition of the fermentation medium (Table I) and the ingredients that were designed following the Placket Burmann model are presented below. Table I Petition 870260056488, dated 10 / 06 / 2026, page 25 / 57 17 / 19 s. No. Components of the Medium Quantity Units s. No. Medium Components Quantity Units 1 Alanine 0.1 g / L 18 Valine 1 mg / L 2 Arginine 0.1 g / L 19 Potassium 2 mg / L 3 Aspartic acid 0.5 g / L 20 Magnesium 1 mg / L 4 Cysteine ​​0.5 g / L 21 Thiamine 0.1 mg / L 5 Glutamic 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 0.1 mg / L 9 Isoleucine 1 g / L 26 Copper 10 mg / L 10 Leucine 0.5 g / L 27 Folic Acid 1.6 mg / L 11 Lysine 0.1 g / L 28 Manganese 1 mg / L 12 Methionine 1 g / L 29 Nicotinamic Acid 100 mg / L 13 Phenylalanine 0.5 g / L 30 Pantothenic Acid 50 mg / L 14 Proline 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

[075] A 20 L fermenter with the above components with the base medium (YE 15 g / L, Vegetable 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 Petition 870260056488, dated 10 / 06 / 2026, page 26 / 57 18 / 19 The following components were used: 12.5 mg / L, MnCl2.4H2O 6.25 mg / L, Cystine 0.5 g / L, Kanamycin 25 mg / L, Glucose 4.0 g / L), and 5% inoculum from the seed fermenter was added to start the process. The temperature was maintained at 35°C and the pH was maintained at 7.4 to 7.6 using 20% ​​orthophosphoric acid, 12.5% ​​ammonium hydroxide, and a model based on the modulation of metabolic flux (Figure 2) of glucose and OTR feed. The culture was aerated using 1.0 vvm of air. The culture was enriched with oxygen to maintain the DO level at 20%. In the logarithmic phase, DO levels continued to fall below 20%, despite the O2 enrichment limit having been reached. The DO level for the remainder of the batch remained close to zero with an increase in the final hours. When the residual glucose in the broth fell below 0.5 g / L, feeding with a 40% glucose solution was administered at 4.5 g / L / h.Intermittently, vitamins and trace elements were supplemented at the 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, Riboflavin 0.3 mg / L, Folic acid 0.06 mg / L. The batch was harvested after 18 hours of cultivation when cell densities reached approximately 132 OD units at 600 nm. Foaming in the reactor was controlled with a 30% organic antifoaming agent. CRM197 production reached a titer of 450 to 500 mg / L of fermentation broth. Example 5

[076] Comparison of CRM197 yields using native Corynebacterium diphtheriae C7 and a manipulated strain of Corynebacterium diphtheriae C7Ep.

[077] A fermentation was carried out using basic medium not derived from animal origin, Pre-model Process Enhancements and Medium and Process according to the present invention using native Corynebacterium diphtheriae C7 and modified strain of Corynebacterium diphtheriae.

[078] The only lysogenic strain of Corynebacterium diphtheriae C7 and the strain Petition 870260056488, dated 10 / 06 / 2026, page 27 / 57 A 19 / 19 strain manipulated with episomally localized extra copies of the CRM197 gene (C7Ep) was used for process optimization. In a typical iron-free YC medium containing animal-derived components, C7 yielded 22 to 25 mg / L of CRM197 while C7Ep yielded 40 to 45 mg / L. When the medium was modified by replacing the animal components with plant peptones, the C7 strain yielded 40 to 45 mg / L of CRM197 and C7Ep yielded 65 mg / L of CRM197. Under process conditions, when modified by altering parameters, C7Ep produced 120 to 150 mg / L of CRM197. A model was developed using inputs from the baseline process. The final result of the optimized method yielded 120 mg / L of CRM197 from C7 and > 500 mg / L of CRM197 from C7Ep. Yields are compared in Table II below: Table II - Comparison of the yield of native Corynebacterium diphtheriae C7 with the yield of episomally modified C7Ep under different process conditions. S. No Organism Yields of CRM197 in mg / L by ELISA Basic medium free of animal-derived derivatives Premodel Process Improvements Medium and process as per the present invention 1 Corynebacterium C7 (native) 20-25 35-40 110-120 2 Corynebacterium C7 Ep (episome incorporated in extra copy of the gene) 60-65 120-150 >500 Petition 870260056488, dated 10 / 06 / 2026, page 28 / 57

Claims

1 / 3 CLAIMS 1. Method for the production of CRM197 CHARACTERIZED in that the method comprises cultivating a strain of Corynebacterium diphtheriae in an iron-free fermentation medium free of animal-derived components and comprising 18 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 and valine, wherein each of the amino acids is present in an amount of 0.05 g / L, and wherein the iron-free fermentation medium is free of maltose.

2. Method according to claim 1, CHARACTERIZED in that it comprises supplementing the fermentation medium with nutrients.

3. Method, according to claim 2, CHARACTERIZED in that the supplementation of the fermentation medium with nutrients comprises using a metabolic flux model.

4. Method according to claim 1, CHARACTERIZED in that the iron-free fermentation medium comprises phenylalanine and arginine in an amount less than 1 g / L.

5. Method, according to claim 2, CHARACTERIZED in that the nutrients are selected from the group consisting of nicotinic acid, thiamine, pantothenic acid, biotin, riboflavin, folic acid, pimelic acid, phosphate, a nitrogen source and trace metals.

6. Method, according to any one of claims 1 to 5, CHARACTERIZED in that the composition of the iron-free fermentation medium comprises a basic fermentation medium, wherein the basic fermentation medium comprises yeast extract, vegetable peptone, potassium dihydrogen phosphate (KH2PO4), tryptophan and glucose. Petition 870260056488, dated 10 / 06 / 2026, p. 29 / 57 2 / 3 7. Method, according to any one of claims 1 to 6, CHARACTERIZED in that it further comprises supplementing the iron-free fermentation medium with glucose.

8. Method for the production of CRM197 CHARACTERIZED in that the method comprises: i) cultivating a strain of Corynebacterium diphtheriae having an increased number of copies of the CRM197 gene in an iron-free fermentation medium comprising a base medium and 18 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 and valine; and ii) supplementing the iron-free fermentation medium with glucose and nutrients using metabolic flux, wherein the iron-free fermentation medium is maltose-free.

9. Method for the production of CRM197 CHARACTERIZED in that the method comprises cultivating a strain of Corynebacterium diphtheriae having an increased number of copies of the CRM197 gene in an iron-free fermentation medium free of animal-derived components and comprising 18 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 and valine, and supplementing the iron-free fermentation medium with nutrients using metabolic flux, wherein each amino acid is present in the iron-free fermentation medium in an amount of 0.05 g / L to 2 g / L, wherein the amino acids are not tyrosine or asparagine, and wherein the iron-free fermentation medium is free of maltose.

10. Method, according to claim 8 or 9, CHARACTERIZED by Petition 870260056488, dated 10 / 06 / 2026, p. 30 / 57 3 / 3 fact that the method is carried out at a temperature between 30°C and 40°C and at a pH of 7.0 to 8.

0.

11. Method, according to claim 8 or 9, CHARACTERIZED in that the yield of the method is from 50 mg / L to 500 mg / L of CRM197.

12. Method for manufacturing a conjugate vaccine CHARACTERIZED in that it comprises: a. preparing CRM197 by the method as defined in any one of claims 1 to 11; and b. conjugating polysaccharides from Salmonella typhi, Streptococcus pneumoniae or Haemophilus influenzae with the prepared CRM197. Petition 870260056488, dated 10 / 06 / 2026, p. 31 / 57

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