When having an appropriate signal sequence, recombinantly produced polypeptides can be secreted into the periplasmic space of E. coli cells (Joly, J. C. and Laird, M. W., in The Periplasm ed. Ehrmann, M., ASM Press, Washington D.C., (2007) 345-360). In the chemically oxidizing environment the formation of disulfide bonds and thereby the functionally correct folding of polypeptides is favored. Selective isolation of these polypeptides from the periplasmic space is desired to avoid contamination with host cell proteins (HCPs) from E. coli. Thereby, the subsequent purification of the polypeptides is facilitated.
An exemplary isolation method is the osmotic shock as described e.g. by Ren, G. et al., J. of Bacteriology 189 (2007) 2777-2786. Using this method, EDTA dissolved in TRIS buffer (pH 7.2) destabilizes the outer membrane of prokaryotic cells, which enables the penetration of sucrose into the periplasmic space. The inner membrane is not permeable for sucrose. Subsequently, the TRIS-EDTA buffer is removed by centrifugation and the cells are quickly resuspended in ice-cold, distilled water. Thereby water is soaked into the sucrose-filled periplasmic space and the destabilized outer membrane is disintegrated via the increase of the periplasmic volume. Addition of MgCl2 re-stabilizes the outer membrane.
Additional methods can be found in Humphreys, D. P. (in The Periplasm (ed.) Ehrmann, M. pp. 361-388. Washington, D.C.: ASM Press (2007)) and Middelberg, A. P. (Biotechnol. Adv. 13 (1995) 491-551).
According to Joly and Laird (see supra) “common methods for isolating periplasmic fractions at small scale such as spheroplasting or osmotic shock treatment are not practically accomplished once cultures reach volumes at and above 10 liters”. The “recovery of the protein from the external medium or after a disruption of only the outer membrane and peptidoglycan is still a goal that has not been put into practice on large scale” (page 354).
In WO 2012/013930 purification of recombinant protein from sample or extract of Gram-negative bacterial host cell expressing recombinant protein and disulfide isomerase DsbC, involves adjusting pH of sample or extract to precipitate and separate DsbC is reported. New antibody specific for human tumor necrosis factor (TNF)-alpha, useful for treating TNF-alpha mediated diseases, e.g. congestive heart failure, septic or endotoxic shock, cachexia and adult respiratory distress syndrome is reported in WO 01/94585. In US 2005/048056 preparing a tumor necrosis factor-alpha antibody having a heavy and light chain comprises fermenting a cell mixture, forming a cell pellet and allowing the pellet to stand for a hold time is reported.
In WO 2007/106120 modulating tissue distribution of a peptide molecule comprises administering to the tissue a conjugate molecule comprising serum albumin binding peptide is reported. Peptide ligands with affinity for immunoglobulin (Ig) G, IgM and/or human serum albumin which may be conjugated and used to prolong the elimination halftime of active agents from the circulation are reported in WO 01/45746. In US 2004/001827 modulating tissue distribution of a peptide molecule, for enhancing therapeutic efficacy and reducing side effects, comprises administering to the tissue a conjugate molecule comprising a peptide ligand domain and an active domain is reported.