Mutant C. difficile toxins reduce cytotoxicity

The patent applies parameter changes by introducing specific point mutations at residues 285, 287, 700, 972, and 978 in toxin A, and residues 286, 288, 698, 970, and 976 in toxin B. These mutations alter the amino acid sequence and biochemical properties of the toxins, reducing their cytotoxic activity while preserving their immunogenicity. The mutations specifically affect the glucosyltransferase domain and cysteine protease domain, modifying the toxins' ability to glucosylate host cell proteins and cause cell death.

The patent applies local quality by making targeted mutations at specific residue positions while maintaining the overall structure and immunogenic epitopes of the toxins. The mutations are localized to specific regions (residues 285-287 in the glucosyltransferase domain and residues 700, 972-978 in other domains) that are less critical for immune recognition, thereby preserving the toxin's ability to elicit neutralizing antibodies while reducing cytotoxicity.

The patent applies parameter changes by modifying the composition of the culture medium, specifically using soy hydrolysate as the carbon source instead of traditional glucose or other simple sugars. This parameter change in the culture medium composition optimizes the metabolic pathway of C. difficile, enhancing toxin production yield and purity while maintaining reasonable manufacturing simplicity. The soy hydrolysate provides a more suitable carbon source that promotes efficient toxin synthesis.