Developing downstream processes for the purification of recombinant proteins and small molecules from Nicotiana benthamiana biomass

Summary Nicotiana benthamiana (Australian tobacco) has become a major host for plant‐based recombinant protein production, especially using transient expression. Once a candidate protein has been designed and produced in a suitable variety (e.g. facilitated humanized glycosylation) under reproducible conditions (e.g. in a vertical farm), the product must be extracted from the biomass and purified to meet the application requirements. For example, >95% purity is often expected for biopharmaceuticals. Here, we review options for and challenges of recombinant protein isolation from N. benthamiana biomass taking inspiration from related host systems when appropriate. Specifically, we first introduce typical properties of the biomass to be processed and then discuss options to harvest it and extract the product, for example, using homogenizers. This includes conditioning steps like heat treatment and means to ensure product integrity. Next, we highlight the advantages and limitations of different clarification operations given the high particle loads typically present in N. benthamiana extracts. Afterwards, we look at product purification using both membrane and chromatographic unit operations focusing on the specific aspects of plant molecular farming. We briefly discuss the potentials of modelling and artificial intelligence in downstream process development and conclude with a short outlook on future developments.