Biologics manufacturing by high-density microbial fermentation can have the advantages of development speed, low cost and scalability. Though E. coli is a preferred host for expression of non-glycosylated proteins, KBI is also experienced with more novel yeast and Pseudomonas expression systems, designed for disulfide-bond formation, product folding and soluble accumulation. In other cases, insoluble protein accumulation in inclusion bodies (IBs) is an advantage, allowing high recovery and conversion to active product by solubilization and refolding procedures. KBI’s expertise in this area has allowed the use of microbial hosts for the expression of several known “difficult-to-refold” products.

KBI has been applying established microbial recombinant protein expression systems to provide rapid cell line generation services using:

E. coli expression systems

• Arabinose, rhamnose, phosphate depletion, and IPTG-inducible gene promoters
• Removable leader sequences for localizing proteins, increasing expression or editing the amino-terminus
• Soluble accumulation
• Insoluble (IB) accumulation
• Periplasmic accumulation

Expression strains support proof-of-concept (POC) material generation for preclinical programs, designed to meet the client’s timeline and budgetary objectives. Cell line development activities can be performed as stand-alone services, or are integrated into larger-scope process development programs. Selected cell lines are used to generate KBI internal research cell banks (RCBs).

For full scope process development and cGMP manufacturing programs, master cell bank (MCB) generation is outsourced to established third party vendors. KBI conducts quality audit of a vendor, transfers distribution vials of the selected RCB to the third party facility, manages and provides quality oversight of the pre-bank testing, the MCB production, testing and release under cGMP.

The key considerations for microbial cell line development programs include:

• Understanding of business and science driven objectives related to the expression platform choice, e.g. product quality, IP status and licensing terms
• Applying established, commercially proven expression platform systems in order to streamline subsequent process development activities
• Implementing ICH Q5D and ICH Q5B guidelines
• Gene and codon optimization of the expression plasmids,
• Clone selection based on product yield, quality, and cell culture performance,
• Verification of critical product quality attributes by integrating analytical support, e.g. mass spec, into the clone selection process.