Department of Microbial Drugs, Helmholtz Centre for Infection Research, Brunswick, Germany.
German Centre for Infection Research (DZIF), Partner Site Hannover-Braunschweig, Brunswick, Germany.
Microb Cell Fact. 2022 Aug 17;21(1):165. doi: 10.1186/s12934-022-01886-2.
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.
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%.
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.
与合成衍生分子相比，次生代谢物具有独特的特征，因此在药物开发计划中一直是重要的起点。然而，生物技术手段发现和供应这些分子的局限性导致大型制药公司退出了这一领域。原因包括与菌株培养、筛选、从天然来源重新发现、纯化和表征新分子相关的问题。然而，最近的报告描述了解决这些问题的技术发展。虽然许多这些报告都集中在识别和表征这些分子，以实现随后的化学合成，但很少有生物技术供应策略的报道。这可能是因为生产工艺通常属于专有研究，或者很少有工艺可能满足药物开发活动的要求。我们旨在弥合这一空白，以真菌倍半萜 illudin M 为例，用于开发抗癌药物，旨在表明生物技术可以作为处理小分子的合成工艺的重要替代方法。
我们使用 µL 规模的模型开发了一种从 Omphalotus nidiformis 培养上清液中回收 illudin M 的吸附和提取策略，并且这些发现成功地转移到了实验室规模。通过使用固定树脂床吸附和洗脱产物，我们将工作体积减少了约 90%，并从工艺中去除了水相。经过洗涤步骤后，通过等度洗脱用 80%甲醇获得高度浓缩的 illudin M 级分。将该级分干燥并用水/庚烷混合物提取，使 illudin M 在庚烷相中富集。从庚烷中，通过浓缩溶液可以立即结晶出 illudin M，最终纯度>95%。
我们已经开发了一种稳健、可扩展且低成本的下游工艺，以获得高纯度的 illudin M。通过使用固相萃取，我们减少了溶剂废物的产生。最后纯化步骤中的庚烷可以回收。所需溶剂的减少量和短的纯化时间使该方法成为与已发表工艺相比非常经济和环保的替代方法。
