Efficient bioprocess design drives enzyme development for pharma applications

Producing CRISPR nucleases for use as RNPs can be difficult - but we found a solution

Increasing the production yield while dealing with an enzyme difficult to purify due to strong DNA/RNA binding forces - this customer challenge has made our contract research for a pharmaceutical application particularly intriguing.

CRISPR nuclease genes can either be expressed in a host in vivo, or the host can be transformed by the purified enzyme in situ to function as a genome editing tool. CRISPR nucleases consist of two components, the enzyme and a so-called guide RNA. The complex is referred to as a ribonucleoprotein (RNP). Because RNP-mediated CRISPR genome editing can be more effective and avoid certain problems associated with the use of plasmids, we developed a bioprocess to produce RNPs.  

Leveraging our expertise in microbial expression technology, we strategically selected the optimal  Escherichia coli  strain, expression cassette design, and plasmid to establish an efficient production system. Once we achieved significant nuclease production at a small scale, we rapidly transitioned to bioreactors for further development. By systematically optimizing key process parameters - including temperature, pH, induction timing, and feed composition - across fermentation scales ranging from 2 L to 10 L, we successfully enhanced production yield by 100%.  

Addressing the specific binding characteristic of the enzymes during downstream processing

One of the key challenges in downstream processing was the inherent tendency of nuclease enzymes to bind to DNA and RNA molecules.

For our application, it was crucial to eliminate these unwanted interactions, ensuring the nuclease remained completely free of nucleic acids for precise loading in its intended use. Commercially available nucleases were not an option, as the final product needed to be entirely nuclease-free.

To overcome this, we implemented a data-driven optimization strategy. Using a design of experiments (DOE) approach, we enhanced DNA removal through flocculation and further refined flocculation agent removal via a second DOE-optimized precipitation process. Additionally, we systematically optimized buffer conditions to improve enzyme solubility, enabling high product concentration.  

In the end, we achieved a scalable and commercially viable multi-step purification process achieving > 95 % enzyme purity enabling our partner in the pharmaceutical sector to commercially use the nucleases as RNP. 

This successful project relied on interdisciplinary collaboration which is one of our strengths. Microbial strain and bioprocess development as well as analytics - driven by scientific exchange and a shared goal.

Dr. Christian Naumer - Director, Unit Head Bioprocess Development

Technologies & services

Our advanced bioprocess solutions ensure superior enzyme purity and functionality, making our technology a valuable asset for industrial applications. Learn more about the technologies & services we offer for industry.

Innovative enzyme solutions for life sciences & pharma

Committed to the pharmaceutical industry, we harness the strengths of BRAIN Biotech and Biocatalysts Ltd. to provide innovative enzyme solutions through our BRAINBiocatalysts Life Science Solutions tailored specifically for pharmaceutical applications.