E. coli is an extensively used bacterial host cell for biopharmaceutical manufacturing with many associated regulatory approvals. It is a popular candidate for initial biologic production for discovery work and/or pre-clinical testing as the methods of establishing the strains are tried, tested and fairly widely available. However, being a prokaryotic host, it has limited capabilities compared to the eukaryotic yeast and mammalian expression systems, especially for manufacturing therapeutic products based on human proteins. Regardless of these limitations, in the cases E. coli is suitable, it offers the advantages of rapid growth, contributing to lower CoGs due to a higher number of production batches generated per year. This means a greater yield on the initial capital expenditure required to construct a manufacturing facility and/or from the capacity rental of E. coli fermentation infrastructure within the CDMO sector.
Non protein secretion causes recovery losses
However, there are some well-known problems with E. coli as a host system for biologics manufacture. While it is generally preferable to secrete the recombinant protein into the culture media for simple recovery, E. coli naturally lacks an efficient secretion pathway for recombinant proteins. Most proteins accumulate intracellularly, and when secretion is required, the low efficiency of translocation across the inner and outer membranes can dramatically limit yields and jeopardise process viability. Many recombinant proteins expressed in E. coli tend to accumulate as insoluble aggregates known as inclusion bodies. These inclusion bodies can lead to difficulties in recovering the active protein.
Protein folding inability causes issues of quality, cost and efficiency
Refolding steps using high concentrations of hazardous denaturants such as urea or guanidine hydrochloride and toxic reducing agents such as β-mercaptoethanol or dithiothreitol are likely to be required, which are also labour-intensive and reduce overall yield. The in vitro refolding process will often not correctly fold proteins with complex disulphide bond assignments or will be unsuitable for large-scale manufacturing. In addition to E. coli being unable to fold many biopharmaceutical proteins and introduce disulphide bonds correctly, it can also not perform most other post-translational modifications typically required for the functional activity of eukaryotic proteins, such as glycosylation.
Host cell toxicity and endotoxin risks
In some cases, the biopharmaceutical product itself may be toxic to the E. coli host, which results in reduced cell growth and lower protein yield. E. coli will degrade many of these proteins, further reducing yields. E. coli also produces endotoxins – pyrogenic lipopolysaccharides from the outer membrane of Gram-negative bacteria, leading to the release of pro-inflammatory cytokines causing fever – which are harmful if present in therapeutic proteins for human use. These endotoxins must be removed during purification, adding extra complexity and cost to the manufacturing process.These issues all limit the suitability of E. coli for the manufacture and regulatory approval of many eukaryotic-based therapeutic proteins and present a range of scale-up problems for commercial production that can jeopardise the viability of the product.
