Fedbatch design for periplasmic product retention in Escherichia coli.

The feed profile of glucose during fedbatch cultivation could be used to influence the retention of the periplasmic product ZZ-cutinase. An increased feed rate led to a higher production rate but also to an increased specific leakage, which reduced the periplasmic retention. Three growth rates: 0.

Alternative antibody Fab' fragment PEGylation strategies: combination of strong reducing agents, disruption of the interchain disulphide bond and disulphide engineering.

Antigen-binding fragments (Fab') of antibodies can be site specifically PEGylated at thiols using cysteine reactive PEG-maleimide conjugates. For therapeutic Fab'-PEG, conjugation with 40 kDa of PEG at a single hinge cysteine has been found to confer appropriate pharmacokinetic properties to enable infrequent dosing. Previous methods have activated the hinge cysteine using mildly reducing conditions in order to retain an intact interchain disulphide.

Engineering of Escherichia coli to improve the purification of periplasmic Fab' fragments: changing the pI of the chromosomally encoded PhoS/PstS protein.

Escherichia coli is a widely used host for the heterologous expression of proteins of therapeutic and commercial interest. The scale and speed at which it can be cultured can result in the rapid generation of large quantities of product. However, to achieve low costs of production a simple and robust purification process is also required.

Comparison of techniques for monitoring antibody fragment production in E. coli fermentation cultures.

The use of an optical biosensor for monitoring antibody fragment accumulation following induction in a batch fermentation of recombinant E. coli is compared to the more traditional method of ELISA quantification. Using the biosensor, concentration data can be obtained within minutes of sample addition to the device, compared to an average assay time of 3-4 h for the ELISA.

A plasmid system for optimization of Fab' production in Escherichia coli: importance of balance of heavy chain and light chain synthesis.

We demonstrate the importance of optimizing the balance of light chain (LC) and heavy chain (HC) expression to achieve high level production of Fab' fragments in the Escherichia coli periplasm. The LC:HC balance has been controlled by varying the codon usage of the signal peptide (SP) and 5' mature domain coding regions. Different SP coding regions have been identified from a codon wobble-based library using alkaline phosphatase (AP) as a reporter gene.