Development of a GMP Phase III purification process for VB4-845, an immunotoxin expressed in E. coli using high cell density fermentation

Process development of periplasmatically produced single chain fragment variable against epidermal growth factor receptor in Escherichia coli

“…In comparison to the similar processes like Premsukh et al (2011) published the production yield of soluble scFv was with 750 g/L nearly identically. In addition, the overall recovery of the five-step column purification procedure was with around 40% comparable.…”

“…Finally, 8 mM of sodium phosphate buffer pH 6.5 was added. At this phosphate and sodium chloride concentration the target protein remained in the flow-through whereas, the CHT resin only bound HCPs comparable to the work of Premsukh et al (2011).…”

“…Therefore, a HCP binding chromatography was adapted from the published method of Premsukh et al (2011). The used ceramic hydroxyapatite resin (CHT) works basically as mixed mode chromatography and is described as a combination between cation exchange and calcium based metal affinity chromatography (Gorbunoff, 1984).…”

“…Frequently it might be difficult to get an approval for tagged biopharmaceuticals. For example, the most commonly used polyHis-tag can be used for fast and easy purification by immobilized metal ion affinity chromatography (Franzreb et al, 2006;Premsukh et al, 2011). The inevitable (and subsequent) proteolytic cleavage of these tags might, however, be expensive or difficult to achieve (Gottschalk, 2009).…”

“…Using high cell density cultivation (HCDC), various pharmaceutical antibody fragments have been already produced (Premsukh et al, 2011;Casey et al, 1995). By employing a pelB leader sequence, the construct is secreted into the periplasmic space (Schlegel et al, 2013;Lei et al, 1987).…”