Shear stress analysis of mammalian cell suspensions for prediction of industrial centrifugation and its verification

Abstract

This article describes the use of ultra scale-down studies requiring milliliter quantities of process material to study the clarification of mammalian cell culture broths using industrial-scale continuous centrifuges during the manufacture of a monoclonal antibody for therapeutic use. Samples were pretreated in a small high-speed rotating-disc device in order to mimic the effect on the cells of shear stresses in the feed zone of the industrial scale centrifuges. The use of this feed mimic was shown to predict a reduction of the clarification efficiency by significantly reducing the particle size distribution of the mammalian cells. The combined use of the rotating-disc device and a laboratory-scale test tube centrifuge successfully predicted the separation characteristics of industrial-scale, disc stack centrifuges operating with different feed zones. A 70% reduction in flow rate in the industrial-scale centrifuge was shown to arise from shear effects. A predicted 2.5-fold increase in throughput for the same clarification performance, achieved by the change to a centrifuge using a feed zone designed to give gentler acceleration of the bioprocess fluid, was also verified at large-scale.

Effects of antibody disulfide bond reduction on purification process performance and final drug substance stability

“…Hutchinson and co‐workers observed significant mAb fragmentation of an IgG4 molecule with increasing centrifugation shear conditions. They hypothesized that the mechanical forces in the centrifuge were responsible for reducing the molecule into half‐antibodies (Hutchinson et al, 2006). Trexler‐Schmidt and co‐workers further demonstrated that antibody disulfide reduction can be attributed to the cell lysis and the release of intra‐cellular reducing enzymes (primarily thio‐redoxin reductase/thioredoxin) as a result of harsh centrifugation conditions (Trexler‐Schmidt et al, 2010).…”

Study of robustness of filamentous bacteriophages for industrial applications

“…The use of a rotating disk shear device to mimic the shear conditions experienced during bioprocessing has been previously described (Hutchinson et al, 2006;Levy et al, 1999;Zhang et al, 2007). Here, shearing was performed in a device consisting of a single rotating stainless steel disk (diameter 40 mm, thickness 0.14 mm) mounted centrally in a 20-mL capacity stainless steel cylindrical chamber (diameter 50 mm).…”

“…The disk rotation speed within the device has been previously characterized using computational fluid dynamics (CFD) in terms of the resulting maximum energy dissipation rate (Boychyn et al, 2004), a general parameter for which estimates exist for many largescale equipment designs (Yim and Shamlou, 2000). In particular, verification studies using shear sensitive material have shown a good correlation between the shear effects of the feed-zone of large-scale centrifuge designs and the rotating disk mimic (Boychyn et al, 2004;Hutchinson et al, 2006). In this study, the disk device was operated at rotation speeds of 300, 400, 467, 500, and 533 revolutions per second (rps).…”

“…The benefit of this ultra scale-down (USD) approach is that predictions of the performance or effect of large-scale operations can be made more rapidly using considerably less material. In particular, a USD rotating disk shear device has been developed to predict the effects of turbulent fluid flow from large-scale pumps and continuous centrifuges (Boychyn et al, 2004;Hutchinson et al, 2006;Zhang et al, 2007). A solid base of small and large-scale verification experimentation exists, validating its use to determine patterns of shear-sensitive behavior for materials such as plasmid DNA, mammalian cell and antibody feeds (Biddlecombe et al, 2007;Hutchinson et al, 2006;Levy et al, 1999).…”

IMAC capture of recombinant protein from unclarified mammalian cell feed streams

“…These techniques can account for up to 25% COG/g when factors such as capital cost and energy consumption are included (Marichal‐Gallardo and Álvarez, 2012). Primary processing of high cell density feed streams requires particular consideration to avoid cell stresses and shearing that could result in protein aggregation and cell lysis (Hutchinson et al, 2006; Kiese et al, 2008). The latter affects the quantity and quality of product through the release of host cell proteins (HCP), proteases, and DNA (Sandberg et al, 2006).…”