A critical assessment of the impact of mixing on dilution refolding

Abstract

Refolding often presents a bottleneck in the generation of recombinant protein expressed as inclusion bodies. Few studies have looked at the effect of physical factors on the yield from refolding steps. Refold reactors typically operate in fed-batch mode with a slow injection rate. This paper characterizes mixing in a novel reactor, and seeks to relate the conditions of mixing to native lysozyme yields after refolding. A novel twin-impeller system incorporating a mini-paddle impeller located in the vicinity of the injection point was used to increase the local levels of energy dissipation experienced by the injected material, and to improve refolding yields. Mixing only affected yields during and immediately after denatured protein addition. Analysis of lysozyme refolding yield, under a variety of conditions, revealed that dispersive mixing affected the yield. The beneficial effect of the mini-paddle impeller in providing a source of localized energy dissipation was limited to conditions where the bulk impeller intensity was low. The effects appeared to become more significant when injection times were longer, because of increased exposure of the injected material to the energy dissipation of the mini-impeller. The results suggest that for fed-batch protein refolding systems, where mixing has been shown to be a critical factor, the local energy dissipation experienced in the vicinity of the injection point is critical to the refolding yields.

Ultra scale‐down of protein refold screening in microwells: Challenges, solutions and application

“…Therefore, until the complete volume is added the liquid can undergo greater displacement relative to the microwells and experience more turbulence. The results suggest that inefficient mixing can limit the eventual refolding yields in microwells, which is consistent with previous observations at larger scales (Lee et al, 2002;Mannall et al, 2006). Reproducibility of refolds varied considerably, although the pipette and shaker III methods exhibited the least deviation.…”

“…Evidence suggests that low concentrations of chaotrope (0.3 M GdHCl) included in refold buffers can help prevent aggregation (Hevehan and De-Bernardez Clark, 1997;Lee et al, 2002;Mannall et al, 2006) and a similar effect may also prevent non-specific binding to the plate surface. As expected, the cell culture plates bound the most protein, particularly at 0 M GdHCl.…”

“…There are many factors, both chemical (De-Bernardez Clark et al, 1998;Hevehan and De-Bernardez Clark, 1997;Yasuda et al, 1998) and physical Lee et al, 2002;Mannall et al, 2006) which can affect refolding steps. While some proteins refold very easily, for the majority of proteins a large number of refold conditions will need to be tested in order to identify the conditions necessary to achieve a high yield in a practical time-frame.…”

“…Such an effect is undesirable when performing refolds and must be minimized. A third factor that has been shown to have a significant effect upon protein refolding is mixing Lee et al, 2002;Mannall et al, 2006). However, large-scale mixing effects are typically very difficult to mimic in microplates.…”

Increased yield of high purity recombinant human interferon-γ utilizing reversed phase column chromatography

“…In the food and animal industries, IFN-γ is effective against a variety of economically important animal viral diseases [16,17]. Hence, the commercial interests in efficiently optimizing the production yields and specific activities of IFN-γ preparations are tremendous [18][19][20][21][22].…”