Ultra scale‐down prediction using microwell technology of the industrial scale clarification characteristics by centrifugation of mammalian cell broths

Abstract

This article describes how a combination of an ultra scale-down (USD) shear device feeding a microwell centrifugation plate may be used to provide a prediction of how mammalian cell broth will clarify at scale. In particular a method is described that is inherently adaptable to a robotic platform and may be used to predict how the flow rate and capacity (equivalent settling area) of a centrifuge and the choice of feed zone configuration may affect the solids carry over in the supernatant. This is an important consideration as the extent of solids carry over will determine the required size and lifetime of a subsequent filtration stage or the passage of fine particulates and colloidal material affecting the performance and lifetime of chromatography stages. The extent of solids removal observed in individual wells of a microwell plate during centrifugation is shown to correlate with the vertical and horizontal location of the well on the plate. Geometric adjustments to the evaluation of the equivalent settling area of individual wells (Sigma(M)) results in an improved prediction of solids removal as a function of centrifuge capacity. The USD centrifuge settling characteristics need to be as for a range of equivalent flow rates as may be experienced at an industrial scale for a machine of different shear characteristics in the entry feed zone. This was shown to be achievable with two microwell-plate based measurements and the use of varying fill volumes in the microwells to allow the rapid study of a fivefold range of equivalent flow rates (i.e., at full scale for a particular industrial centrifuge) and the effect of a range of feed configurations. The microwell based USD method was used to examine the recovery of CHO-S cells, prepared in a 5 L reactor, at different points of growth and for different levels of exposure to shear post reactor. The combination of particle size distribution measurements of the cells before and after shear and the effect of shear on the solids remaining after centrifugation rate provide insight into the state of the cells throughout the fermentation and the ease with which they and accumulated debris may be removed by continuous centrifugation. Hence bioprocess data are more readily available to help better integrate cell culture and cell removal stages and resolve key bioprocess design issues such as choice of time of harvesting and the impact on product yield and contaminant carry over. Operation at microwell scale allows data acquisition and bioprocess understanding over a wide range of operating conditions that might not normally be achieved during bioprocess development.

Assessment of the manufacturability of Escherichia coli high cell density fermentations

“…The availability of scale-down tools allows this information to be gathered while the fermentation proceeds rather than in a retrospective fashion. Here, we use a microscale methodology reported by Tait et al 20 to predict the centrifugation performance of samples obtained at different time points over the postinduction phase. The centrifugation performance of the unsheared samples is included, which gives an indication of the shear sensitivity of the broth over time.…”

“…[16][17][18][19] The performance of a large scale centrifuge can be related to bench-top centrifugation using the correlation: where R 1 is the equivalent settling area for the pilot-scale centrifuge, Q is the volumetric flow rate, V is the volume of material used in the small scale centrifugation, t is the time of centrifugation at small scale, and c is the experimentally derived calibration factors to allow for flow conditions not considered in the theoretical derivation of R (c 1 ¼ 0.4) for disc-stack centrifuges 23 and c 2 ¼ 1 for bench-top centrifugation. R 2 is the equivalent settling area at the laboratory scale determined according to the relationship described by Maybury et al 17 Centrifugation studies were carried out using an Eppendorf 5180R bench-top centrifuge (Cambridge, UK) with an A-4-62 swing-out rotor with temperature controlled to 20 C. Microwell plate centrifugation was performed using a 96-deep square well (Abgene, Epsom, UK) microwell plate format. In the interest of minimizing wall effects on particle settling characteristics, the smallest size plate configuration was selected (8.25 mm wide).…”

“…11 This has been extended to microscale using microwell plates allowing for automation and high throughput. 20 Wenger et al 21 described the use of adaptive focus acoustics (AFA), a technique that delivers highly focused acoustic energy through the use of ultrasonication, as a microscale yeast cell disruption mimic.…”

“…Research has shown that the prediction of large-scale centrifugation through the use of scale-down centrifugation approaches is possible using a shear device and a bench-top centrifuge, [16][17][18][19] requiring milliliters of processing materials, both in mammalian 20 and bacterial systems. 11 This has been extended to microscale using microwell plates allowing for automation and high throughput.…”

Rapid whole monoclonal antibody analysis by mass spectrometry: An Ultra scale‐down study of the effect of harvesting by centrifugation on the post‐translational modification profile

“…This was undertaken in conjunction with the development of whole antibody analysis using LC/MSD-TOF in line with the process analytical technology (PAT) initiative to introduce fast, at-line in-depth analysis of product attributes. Whilst we have previously shown using a scale-down approach that disc-stack centrifugation of mammalian cells can have a significant impact upon the contaminating solids carry over (Hutchinson et al, 2006;Tait et al, 2009) the structural integrity of the product was not considered. Here we specifically describe how post-translational modifications change according to the time of harvest of a mammalian cell culture, and study how conditions in the subsequent centrifugation step affects these.…”