A high-density growth approach was utilized to produce mutated diphtheria toxin 
from two strains of Corynebacterium diphtheria: C7 (beta)(tox-201,tox-9) and C7 
(beta)(tox-107). The cross-reacting mutants (CRM) of the diphtheria toxin are 
CRM9 and CRM107; both of them carry the mutation in their binding site and, as a 
result, have 1/300 of the systemic toxicity of the wild-type diphtheria toxin. 
Since iron inhibits diphtheria toxin production, the traditional approach has 
been to grow the bacteria in a very low iron concentration. The procedure 
described here involved the use of a modified, non-deferrated, growth medium 
that provided fast and high-density growth of the bacteria, and which, when 
associated with simultaneous depletion of glucose and iron, 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. The CRM 
products, which remained stable following microfiltration and ultrafiltration, 
could be easily purified using a two-step chromatography procedure.