Optimization of the production process for the anticancer lead compound illudin M: downstream processing

Abstract

Background: Secondary metabolites have played a key role as starting points for drug development programs due to their often unique features compared with synthetically derived molecules. However, limitations related to the discovery and supply of these molecules by biotechnological means led to the retraction of big pharmaceutical companies from this field. The reasons included problems associated with strain culturing, screening, re-discovery, purification and characterization of novel molecules from natural sources. Nevertheless, recent reports have described technical developments that tackle such issues. While many of these reports focus on the identification and characterization of such molecules to enable subsequent chemical synthesis, a biotechnological supply strategy is rarely reported. This may be because production processes usually fall under proprietary research and/or few processes may meet the requirements of a pharmaceutical development campaign. We aimed to bridge this gap for illudin M-a fungal sesquiterpene used for the development of anticancer agents-with the intention to show that biotechnology can be a vital alternative to synthetic processes dealing with small molecules.

Results: We used µL-scale models to develop an adsorption and extraction strategy for illudin M recovery from culture supernatant of Omphalotus nidiformis and these findings were successfully transferred into lab-scale. By adsorbing and eluting the product using a fixed resin-bed we reduced the working volume by ~ 90% and removed the aqueous phase from the process. After a washing step, a highly concentrated illudin M fraction was obtained by isocratic elution with 80% methanol. The fraction was dried and extracted using a water/heptane mixture, enriching illudin M in the heptane phase. From heptane illudin M could be instantly crystalized by concentrating the solution, achieving a final purity > 95%.

Conclusion: We have developed a robust, scalable and low-cost downstream process to obtain highly pure illudin M. By using solid phase extraction we reduced the production of solvent waste. Heptane from the final purification step could be recycled. The reduced amounts of solvents required, and the short purification time render this method a very economic and ecologic alternative to published processes.

Keywords: Anti-cancer agents; Basidiomycota; Crystallization; DSP; Fungal secondary metabolites; Liquid–liquid extraction; Natural products; Small-scale model; Solid phase extraction.

Fig. 1

Pressure build-up and flow rate during depth filtration of fermentation broth. Centrifuged culture broth was filtered with comercial depth filters to further reduce particle content. The blue scatter plots indicate the pressure during flitration. The red scatter plots indicate the LMH (Liter per m² per hour) calculated based on filtrate weight and filter area. a Pressure and LMH of the run with the 60SP02A filter capsule plotted over weight of the filtrate. The pressure reached 2 bar after about 1.6 L and the flow rate dropped by a factor of 4. b Pressure and LMH of the run with the 30SP02A filter capsule plotted over weight of the filtrate. The pressure reached 2 bar after about 9 L while the flow rate dropped by a factor of 3

Fig. 2

OD600 of different samples to compare efficiency of depth filtration and centrifugation. The bar diagram illustrates the absorbance at 600 nm of samples taken before and after depth filtration with two different filters (30SP02A, 60SP02A). To compare the efficiency of filtration and centrifugation a sample of the starting material was centrifuged for two hours (SM centrifuged). Supernatant filtered with the 60SP02A filter had the lowest absorbance among all samples while centrifugation and filtration with 30SP02A achieved a similar level of clearance. The Y axis does not start at 0.00 for better comparison

Fig. 3

Binding capacity of XAD16N in batch operation. The blue scatter plot illustrates the percentage of illudin M bound to increasing concentrations of XAD16N using 10 mL of cell-free supernatant containing 293 mg L⁻¹ of illudin M

Fig. 4

Concentration of Illudin M in extracts of two subsequent elutions from XAD16N incubated with culture supernatant. The gray bars illustrate the illudin M content in the fractions after a first extraction with incresing methanol concentrations. The blue bars illustrate the concentration of illudin M after a second extraction of the resin with 100% methanol. The observed drop at 90% methanol (grey bar) is probably a measurement error since it is not reflected with a higher concentration in the second elution step (blue bar)

Fig. 5

Illudin M concentration indicated in weight percent (wt%) in the dry mass of eluates from two subsequent extraction steps. Blue dots illustrate the wt% of illudin M in the extract from the first elution with increasing methanol concentrations and the respective regression curve. Red squares illustrate the wt% of illudin M in the extract after the second elution with 100% methanol and the respective regression curve. Outliers of the second extraction are illustrated as black squares and may result from measurement errors due to the low weight of the samples. Both data sets were generated with triplicate samples of each eluted fraction

Fig. 6

Illudin M concentration in the flow through after loading two XAD16N columns at different flow velocities with culture supernatant containing 200 mg L⁻¹ of illudin M. The scatter plots illustrate the illudin M titers in each fraction collected after loading with flow velocity ~ 7.5 cm min⁻¹ (blue) and ~ 30 cm min⁻¹ (red) with the respective linear fit

Fig. 7

Illudin M concentrations in fractions after stepwise elution of loaded XAD16N column. The red squares (illustrated as a curve to highlight the chromatographic elution) indicate the absolute illudin M concentration in each fraction after extraction from the resin with increasing methanol concentrations. Blue bars illustrate the wt% of illudin M in the total mass of each fraction. The gray area illustrates the percentage of methanol used in each elution step

Fig. 8

UV signal at 325 nm and illudin M concentration in the flow through during loading experiment. The black curve illustrates the UV signal at 325 nm recorded while loading illudin M on a XAD16N column. Blue squares illustrate the illudin M concentration in samples taken every 6 min from the flow through. The experiment was performed loading 3 L of clear supernatant containing ~ 529 mg L⁻¹ of illudin M

Fig. 9

UV signal at 325 nm and illudin M concentration during isocratic elution with different methanol mixtures. The black curve illustrates the UV signal at 325 nm while eluting illudin M from XAD16N with isocratic steps. The blue squares illustrate the illudin M concentrations in samples taken over the run. The colored blocks indicate the methanol concentration and the duration of the isocratic wash and elution steps

Fig. 10

UV signal and illudin M concentration measured while overloading a packed column. A XAD16N column was loaded with 10 L of clarified supernatant (~ 700 mg L⁻¹ illudin M). The scatter plot illustrates the concentration of illudin M in the flow through pointing out a breakthrough after 1.5 h (4.5 L). The two different trends of the linear fits are shown in red and blue. The black curve illustrates the UV signal obatined from the chromatography system

Fig. 11

Percentage of illudin M distribution during liquid–liquid extration of eluate with hepane. a liquid–liquid extraction of methanol-containing eluates diluted with increased amounts of water and extracted with heptane; the stacked bars illustrate the distribution of illudin M (%) between the methanol/water phase (gray) and the heptane phase (blue). b liquid–liquid extraction of dry eluate dissolved in increasing amounts of water and extracted with heptane; the stacked bars illustrate the distribution of illudin M (%) between the aqueous (gray) and the heptane phase (blue)

Fig. 12

Dried illudin M crystals after concentrating a heptane fraction. A purity > 95% was confirmed using NMR spectroscopy