Method for high level production of CRM197
Inventors
Masilamani, Balamurali • Sriraman, Rajan • DIXIT, Mandar Shirish • Chakka, Deviprasanna • Sureddi, Satyam Naidu • Matur, Ramesh Venkat • Mantena, Narender Dev • Datla, Mahima
Assignees
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Publication Number
US-11098089-B2
Publication Date
2021-08-24
Expiration Date
Abstract
The present invention provides an improved method for the production of CRM197 with high yield using engineered Corynebacterium diphtheria strain having an increased copy number of the CRM197 gene, wherein the method comprises growing the strain in media free of animal-derived components with one or more amino acids.
Core Innovation
The invention relates to improved high-yield production of CRM197. It describes growing an engineered Corynebacterium diphtheriae strain having an increased copy number of the CRM197 gene, including episomal copies, to increase CRM197 gene copy number and titer.
The production is carried out in a non-deferrated fermentation media that is free of animal-derived components and free of maltose. The non-deferrated fermentation media comprises 10 or more amino acids, where the amino acids are not tyrosine or asparagine, and further includes base media components and nutrients, and may be supplemented with glucose.
The document also describes metabolic-flux modeling using online parameters to support nutrient/glucose feed optimization, including pH, dissolved oxygen, CO2, and heat, together with glucose feeding and oxygen transfer related control variables. The document reports increased CRM197 titers under different approaches, including examples with and without metabolic-flux modeling, and higher titers associated with media optimization.
Claims Coverage
The document contains three independent claims that cover producing CRM197 in non-deferrated, animal-derived-component-free, maltose-free fermentation media containing 10 or more amino acids excluding tyrosine and asparagine. Two independent claims further require a Corynebacterium diphtheriae strain having an increased CRM197 gene copy number, and two claims additionally require supplementation with nutrients using metabolic flux, including glucose supplementation or specified amino-acid presence amounts.
Non-deferrated maltose-free animal-derived-component-free CRM197 fermentation
A method for producing CRM197 by growing a Corynebacterium diphtheriae strain in a non-deferrated fermentation media that is free of animal derived components, includes 10 or more amino acids, where the amino acids are not tyrosine or asparagine, and where the non-deferrated fermentation media is free of maltose.
Increased CRM197 gene copy number with metabolic-flux glucose/nutrients supplementation in maltose-free media
A method of producing CRM197 by culturing a Corynebacterium diphtheriae strain having an increased copy number of the CRM197 gene in a non-deferrated fermentation media comprising a base media and 10 or more amino acids selected from a defined group of standard amino acids and salts, and supplementing the non-deferrated fermentation media with glucose and nutrients using metabolic flux, wherein the non-deferrated fermentation media is free of maltose.
Metabolic-flux supplemented maltose-free amino-acid-level-defined CRM197 fermentation
A method of producing CRM197 by culturing a Corynebacterium diphtheriae strain having an increased copy number of the CRM197 gene in a non-deferrated fermentation media free of animal derived components and comprising 10 or more amino acids, and supplementing the non-deferrated fermentation media with nutrients using metabolic flux, wherein each of the 10 or more amino acids is present in the non-deferrated fermentation media in an amount of about 0.05 g/L to 2 g/L, wherein the amino acids are not tyrosine or asparagine, and wherein the non-deferrated fermentation media is free of maltose.
Coverage centers on producing CRM197 using a Corynebacterium diphtheriae strain with increased CRM197 gene copy number in non-deferrated, animal-derived-component-free, maltose-free media containing 10 or more amino acids excluding tyrosine and asparagine. The independent claims further recite metabolic-flux supplementation and either glucose/nutrient feed optimization or defined amino-acid presence amounts, while dependent claims narrow to conjugate vaccine manufacturing with polysaccharides from specified pathogens.
Stated Advantages
Improved high-yield production of CRM197.
Increased CRM197 titers, with examples showing higher titers under metabolic-flux modeling and/or media optimization.
Documented Applications
Manufacturing a conjugate vaccine by conjugating polysaccharides from Salmonella typhi, Streptococcus pneumoniae, and Haemophilus influenzae with CRM prepared according to the CRM197 production methods.
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