Optimization of the production process for the anticancer lead compound illudin M II. Process development in stirred tank bioreactors

Background: The fungal natural products illudin S and M have been investigated as precursors for the development of semisynthetic anticancer agents such as Irofulven (illudin S derivative) which is currently in phase II clinical trials. Recently, illudin M derivatives have shown in vitro improved selectivity towards cancer cells encouraging further investigations. This requires a stable supply of the precursor which is produced by Basidiomycota of the genus Omphalotus . We have recently developed a stable process for the production of gram quantities of illudin M from Omphalotus nidiformis in shake flasks, but transferring such findings into stirred tank bioreactors - which can be used in a commercial production set-up - is often not straightforward and is particularly challenging when the producer can undergo morphological changes. There are only few reports addressing bioprocess development for production of compounds from Basidiomycota since these organisms have been underexplored i.a because they have complex life cycles and many of them are difficult to cultivate under laboratory conditions. Results: : The recently developed shake flask process yielding stable titers of ~940 mg L -1 of illudin M was evaluated using off-gas analysis, and critical parameters were identified to facilitate the transfer from shaken into stirred tank bioreactors. The process was adapted in 2 L stirred tank bioreactors (1.5 L working volume) by controlling growth of biomass with a carefully timed pH-shift combined with an improved precursor-feeding strategy. This resulted in comparable titers as in shake flasks. A scale-up experiment in a 15 L bioreactor (10 L working volume), resembling the process at 1.5 L resulted in illudin M >500 mg L -1 and will be the starting point for optimization of the identified parameters at that scale. Conclusion: By identifying and controlling key process parameters, the production process for illudin M was transferred from shake flasks into stirred tank bioreactors reaching comparable titers (> 900 mg L -1 ) which is significantly higher than any previously reported yield. The established bioprocess at bench-scale (~500 mg L -1 ) paves the way towards further scale-up studies that will enable industrial supply of illudin M to support preclinical and clinical development programs.