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.

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Short running title:High-throughput measurement of protein stability SummaryThe direct determination of protein stability at high throughput has applications in proteomics, directed evolution, and formulation. Each application places different requirements on the accuracy of stability or transition mid-point determination. The measurement of protein stability by chemical denaturation has been previously performed at medium throughput and high accuracy using autotitrating fluorometers, after removing proteins from the 96-well plate format in which they were expressed and purified. Here we present a higher-throughput method for measuring and indexing the stability of proteins maintained within the 96-well format using a fluorescence microplate reader. Protein unfolding transitions were monitored by tryptophan fluorescence at 340 nm, and assessed using bovine and equine cytochrome c (cyt c), as well as bovine serum albumin (BSA) stabilised with various amounts of palmitic acid. Two different approaches for generating unfolding curves in microtiter plates have been evaluated for their accuracy and applicability.Unfolding curves generated by the serial addition of denaturant into single wells, allowed high-throughput stability screens capable of identifying protein variants with unfolding mid-point differences of 0.15 M denaturant concentration or larger. Such a method would be suitable for screening large numbers of proteins, as typically generated for directed evolution. Unfolding curves generated using one well per denaturant concentration allowed for medium-throughput stability screening and generated more accurate and precise stability values (C 1/2 ±0.05 M, m G andfor cyt c that are similar to values reported in literature. This method is suitable for screening the smaller numbers of proteins generated in proteomic research programmes. By using BSA stabilised with various palmitate concentrations, and simple numerical indexing, it was shown that both experimental methods can successfully rank the order of protein stability.