High‐level accumulation of a recombinant antibody fragment in the periplasm of Escherichia coli requires a triple‐mutant (degP prc spr) host strain

Abstract

During production of a humanized antibody fragment secreted into the periplasm of Escherichia coli, proteolytic degradation of the light chain was observed. In order to determine which protease(s) were responsible for this degradation, we compared expression of the F(ab')(2) antibody fragment in several E. coli strains carrying mutations in genes encoding periplasmic proteases. Analysis of strains cultured in high cell density fermentations showed that the combination of mutations in degP prc spr was necessary for the cells to produce high levels of the desired recombinant antibody fragment. In order to eliminate the possible effects of mutations in other genes, we constructed E. coli strains with protease mutations in isogenic backgrounds and repeated the studies in high cell density fermentations. Extensive light chain proteolysis persisted in degP strains. However, light chain proteolysis was substantially decreased in prc and prc spr strains, and was further decreased with the introduction of a degP mutation in prc and prc spr mutant strains. These results show that the periplasmic protease Prc (Tsp) is primarily responsible for proteolytic degradation of the light chain during expression of a recombinant antibody fragment in E. coli, and that DegP (HtrA) makes a minor contribution to this degradation as well. The results also show that spr, a suppressor of growth defects in prc strains, is required for a prc mutant to survive throughout high cell density fermentations.

Proteomic Profiling of Recombinant Escherichia coli in High-Cell-Density Fermentations for Improved Production of an Antibody Fragment Biopharmaceutical

“…While many 2-D PAGE studies have been performed with cells cultured in shake flasks, there is also a need to study high-cell-density, fed-batch fermentations of Escherichia coli, which are widely used in industry to obtain high productivity (9,26). Results of 2-D PAGE analyses can identify targets for bioprocess improvement, such as genes to delete from the host cell chromosome (9) or to coexpress with a product (17,18). Ultimately, proteome analysis may improve the design and control of industrial fermentation processes.…”

“…Studies of fed-batch fermentations conducted in Genentech laboratories have focused on end-of-run analyses to assess multiproduct host cell protein immunoassays (7,8) or product proteolysis (9). The latter studies (9) involved production of the same antibody fragment, as discussed in this paper. The same fermentation process was utilized in both cases, and the host strains were similar.…”

“…Recombinant anti-CD18 was measured in two ways. First, total light chain and total heavy chain were measured by reversed-phase chromatography following denaturation with guanidine-HCl and dithiothreitol as previously described (9). Second, anti-CD18 was measured in its intact, soluble form by using affinity chromatography.…”

Degradation of C-terminal tag sequences on domain antibodies purified fromE. colisupernatant

“…21 It is well known that E. coli contains several proteases that can degrade recombinant protein expressed in the bacteria. 22,23 Tsp is a periplasmic serine protease that degrades protein from the C-terminal end based on the recognition of the C-terminal residues. A range of amino acids are substrates for Tsp when placed at the C-terminal, but there is a preference for substrates with non-polar carboxyl termini and preferably at least 2 alanines out of the 3 C-terminal positions.…”

Production of antibody fragment (Fab) throughout Escherichia coli fed-batch fermentation process: Changes in titre, location and form of product

“…Promoters and secretion signals from a few outer membrane proteins have been used to direct recombinant proteins into the periplasm (Moir and Mao, 1990;Carter et al 1992;Joly et al 1998;Chen et al 2004;Laird et al 2005;Yoon et al 2010). The oxidizing environment of the periplasm in cooperation with the periplasmic disulfide oxidoreductases/isomerases (Berkmen et al 2007) and periplasmic chaperones (Betton, 2007) assists the formation of correctly folded recombinant proteins.…”