Stainer and Scholte developed a simple, chemically defined medium for large-scale production of phase I Bordetella pertussis, consisting of sodium glutamate, proline, cystine, salts, and growth factors. The medium, designated '1G+1P', eliminated unnecessary components from previously published formulations, removing nucleic acid derivatives, most amino acids, liver co-enzymes, and additives like charcoal and resin. In shake flasks and fermentors, the medium yielded 30-40 × 10⁹ organisms/ml within 48-72 hours. Cultures were successfully detoxified using 0.14% formalin for 5 days, producing vaccines that were non-toxic to mice and guinea pigs while maintaining good protective antigen levels. Vaccines showed satisfactory antigenic stability when stored at elevated temperatures. The work confirmed that glutamic acid, proline, and cystine are essential amino acids for B. pertussis growth. When grown without glutamate or proline, the organism synthesized amino acids de novo, suggesting distinct metabolic pathways. The simplicity and excellent vaccine yields make this medium attractive for routine production compared to conventional casein hydrolysate methods.

Key findings

• A simplified chemically defined medium (1G+1P) containing only sodium glutamate, proline, cystine, salts, and growth factors produced 30-40 × 10⁹ B. pertussis organisms/ml in 48-72 hours
• Formalin detoxification at 0.14% for 5 days or 0.12% for 7 days yielded vaccines with low toxicity in mice and guinea pigs and good mouse-protective antigen potency
• The medium maintained antigenic stability for at least 44 weeks at 4°C and 8 weeks at 37°C, either as plain pertussis vaccine or combined with diphtheria and tetanus toxoids
• Different metabolic pathways exist for glutamate and proline utilization, as evidenced by distinct amino acid synthesis patterns when each amino acid was omitted from the medium
• Vaccines produced in the chemically defined medium demonstrated phase I characteristics with appreciable quantities of agglutinogens 1, 2, and 3