Living cellular systems such as bacteria, yeasts, plants, and mammalian cells have become the workhorse to produce a myriad of biologics and compounds. More recently, lyophilized cell extracts (LCE) prepared from living cells have enabled decentralized pharmaceutical production and room temperature storage, which offer opportunities for safe deployment of genetically encoded materials. However, current LCE systems have not been proven to be stable for several years without loss of activity at room temperature, and no evidence has shown that they are resistant to various harsh conditions to perform their functions in extreme environments. Furthermore, additional limitations for LCE include: 
1) unlike living hosts, which can replicate, a cell extract cannot replicate, and therefore is difficult to scale up
2) some LCE systems use the bacterium Escherichia coli, which necessitates stringent procedures to remove endotoxin contamination
3) mixtures of proteins and small molecules in cell lysates require extensive downstream product purification 
4) there exists batch-to-batch variability in cell-free protein expression systems
Technology Overview
The proposed invention describes engineering and installing of genetic components encoding desired biologics in Bacillus subtilis spores for long term storage with superior stability. Spores are highly resistant to desiccation and also to radiation at doses that exceed the threshold for damage to humans. Despite their dormancy, spores can rapidly return to a vegetative state of growth in one to two hours via germination and then proliferate in the presence of nutrients. 
Importantly, in this technology, researchers have utilized the secretory expression system in B. subtilis in the vegetative state to produce nanobodies (Nbs), which are a distinct class of proteins derived from single-domain antibody fragments found in Camelidae. Nbs feature small sizes (12-17 kDa), high solubility, and high thermal stability compared to conventional antibodies. In this work, however, it's been observed that high thermostability and relatively small size may render Nbs amenable to the secretory system in B. subtilis, and it demonstrated that a panel of four different Nbs specific for small molecules (caffeine and methotrexate) and cell surface-associated protein antigens (PD-L1 and CTLA-4) can be readily secreted by B. subtilis.
- Ease of high yield protein production and purification through direction secretion from host cells
- Superior stability of the microbial factory system against environmental extremes
- Low cost
- Portability
- Ultra‑long shelf life
- Production of nanobodies, animal vaccines and long peptides
- License
- Research collaboration
- Partnering
Patent Information:
For Information, Contact:
Colin Sullivan
Commercialization Consultant
Northeastern University
Jiahe Li
Antibody manufacturing
Protein production