Team:UCL London/Bioprocess Flowsheet Development

Bioprocess Flowsheet

According to Makrides (1996) the periplasm of E.coli contains only about 4% of the total cell protein. The periplasmic expression allows expression of fragments to be effectively concentratd. Other reports (Humphreys, 2004) that the antibody expression of Fab fragments can take place in the periplasm of E.coli and purified with an aqueous periplasmic heat extraction, which eliminates most of the host cytoplasmic and membrane proteins, followed by ion exchange chromatography.

E. coli is an established production system of choice for antibody fragments used in therapeutic applications. One reason is that E. coli provides the means to progress from antibody selection to Good Manufacturing Practice (GMP) production of antibodies in a rapid manner. The other reason is the fact that high production levels of antibody fragments are attainable when using E. coli. As of 2004, there have been several Fabs or Fab’s in clinical trials run by Genentech and Celltech, where the fragments have been produced using E. coli (Anderson et al., 2004).   Fermentative Pathways Host: Escherichia Coli Advantages:

Provides a wide choice of cloning vectors Easily controlled gene expression Gives large yields Secretes good protein Provides fast growth rate

Disadvantages  Lacks post-translational modifications  Posses high levels of endotoxins  Forms inclusion bodies (i.e. protein aggregates) Escherichia coli produces antibody fragments rather than whole antibodies due to the fact that it lacks post-translational modifications and also since polymeric polypeptide assembly is not well supported (Johansson, 2007)