Ultra scale-down approaches to study the centrifugal harvest for viral vaccine production

Large scale continuous cell‐line cultures promise greater reproducibility and efficacy for the production of influenza vaccines, and adenovirus for gene therapy. This paper seeks to use an existing validated ultra scale‐down tool, which is designed to mimic the commercial scale process environment using only milliliters of material, to provide some initial insight into the performance of the harvest step for these processes. The performance of industrial scale centrifugation and subsequent downstream process units is significantly affected by shear. The properties of these cells, in particular their shear sensitivity, may be changed considerably by production of a viral product, but literature on this is limited to date. In addition, the scale‐down tool used here has not previously been applied to the clarification of virus production processes. The results indicate that virus infected cells do not actually show any increase in sensitivity to shear, and may indeed become less shear sensitive, in a similar manner to that previously observed in old or dead cell cultures. Clarification may be most significantly dependent on the virus release mechanism, with the budding influenza virus producing a much greater decrease in clarification than the lytic, non‐enveloped adenovirus. A good match was also demonstrated to the industrial scale performance in terms of clarification, protein release, and impurity profile.UK Engineering and Physical Sciences Research Council, EPSRC. Grant Number: EP/G034656/1Published versio

Effects of centrifugal stress on cell disruption and glycerol leakage from Dunaliella salina

Dunaliella salina accumulates large amounts of intracellular glycerol in response to the increases in salt concentration, thus is a potential source for producing fuel grade glycerol as an alternative to biodiesel-derived crude glycerol. D. salina lacks a cell wall; therefore the mode of harvesting Dunaliella cells is critical to avoid cell disruption caused by extreme engineering conditions. This study explored cell disruption and glycerol leakage of D. salina under various centrifugal stresses during cell harvesting. Results show a centrifugal g-force lower than 5000 g caused little cell disruption, while a g-force higher than 9000 g led to ~40 % loss of the intact cells and glycerol yields from the recovered algal pellets. Theoretical calculations of the centrifugal stresses that could rupture Dunaliella cells were in agreement with the experimental results, indicating optimisation of centrifugation conditions is important for recovering intact cells of from D. salina enriched in glycerol

An ultra scale-down methodology to characterize aspects of the response of human cells to processing by membrane separation operations

Tools that allow cost-effective screening of the susceptibility of cell lines to operating conditions which may apply during full scale processing are central to the rapid development of robust processes for cell-based therapies. In this paper, an ultra scale-down (USD) device has been developed for the characterization of the response of a human cell line to membrane-based processing, using just a small quantity of cells that is often all that is available at the early discovery stage. The cell line used to develop the measurements was a clinically relevant human fibroblast cell line. The impact was evaluated by cell damage on completion of membrane processing as assessed by trypan blue exclusion and release of intracellular lactate dehydrogenase (LDH). Similar insight was gained from both methods and this allowed the extension of the use of the LDH measurements to examine cell damage as it occurs during processing by a combination of LDH appearance in the permeate and mass balancing of the overall operation. Transmission of LDH was investigated with time of operation and for the two disc speeds investigated (6,000 and 10,000 rpm or ϵmax  ≈ 1.9 and 13.5 W mL-1 , respectively). As expected, increased energy dissipation rate led to increased transmission as well as significant increases in rate and extent of cell damage. The method developed can be used to test the impact of varying operating conditions and cell lines on cell damage and morphological changes. Biotechnol. Bioeng. 2017;114: 1241-1251. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc

High throughput automated microbial bioreactor system used for clone selection and rapid scale-down process optimization

High throughput automated fermentation systems have become a useful tool in early bioprocess development. In this study, we investigated a 24 x 15 mL single use microbioreactor system, ambr 15f, designed for microbial culture. We compared the fed-batch growth and production capabilities of this system for two Escherichia coli strains, BL21 (DE3) and MC4100, and two industrially relevant molecules, hGH and scFv. In addition, different carbon sources were tested using bolus, linear or exponential feeding strategies, showing the capacity of the ambr 15f system to handle automated feeding. We used power per unit volume (P/V) as a scale criterion to compare the ambr 15f with 1 L stirred bioreactors which were previously scaled-up to 20 L with a different biological system, thus showing a potential 1,300 fold scale comparability in terms of both growth and product yield. By exposing the cells grown in the ambr 15f system to a level of shear expected in an industrial centrifuge, we determined that the cells are as robust as those from a bench scale bioreactor. These results provide evidence that the ambr 15f system is an efficient high throughput microbial system that can be used for strain and molecule selection as well as rapid scale-u

Scale-down principles for the accelerated design of protein purification processes.

With speed to market as a critical factor determining the economic success of a therapeutic product, bioprocess development must be carried out as efficiently as possible. This relies heavily on the ability to predict industrial-scale operation with only small (< 0.5 L) quantities of available feed; laboratory scale-down is superior to pilot- plant work in terms of speed, ease, and cost. Application of such a strategy is particularly relevant to early solid-liquid separation stages, where the performance of a centrifuge or filter may largely determine process yield and the quality of material to be delivered to subsequent guard filtration and chromatographic stages. Previous work focused on the modification of a disc stack centrifuge and laboratory prediction of clarification for shear-insensitive species such as cell debris. This thesis extends this work by developing laboratory-scale methods to mimic sediment dewatering and recovery of shear-sensitive protein precipitates in continuous-flow centrifuges. Initial experiments conducted with a multichamber-bowl, resulted in a great difference between predicted and pilot clarifications (50% lower) due to particle break-up occurring in the high-velocity entrance zone of the pilot centrifuge. The hydrodynamic forces in this region were analysed by computational fluid dynamics and reproduced in a small highspeed rotating disc device. Exposing suspension to the device prior to laboratory centrifugation permitted accurate prediction of pilot clarification. This technique was translated to other continuous centrifuges (disc stack, CARR, production multichamber-bowl). Importantly, the performance of the production centrifuge was more accurately predicted by the scale-down process than the pilot one. A simpler scaling tactic of constant tip velocity of the distributor in the centrifuge feed zone was also examined and found to be a good predictor of large-scale clarification. Conventional filtration (with precoat and body feed) was investigated as an alternative primary separation step. A production rotating vertical leaf filter was scaled down by transformation of a laboratory-batch, Nutsche pressure-filter; the continuous filter resulted in cakes of more uniform composition with lower specific resistances. Filtration gave significantly better clarification and sediment dryness than centrifugation. Finally, chromatographic performance was shown to depend moderately on the extent but principally on the type of solid-liquid separation, with centrifugation samples resulting in significantly greater column dynamic capacities than filtrates, which was not predicted by the response of a guard (cartridge) filter