Use of scale-down methods to rapidly apply natural yeast homogenisation models to a recombinant strain

Abstract

Models for high pressure homogenisation developed using natural bakers' yeast were applied to a recombinant yeast strain. The models were found to be generic in nature and generally adequate for initial process design (30%). More accurate descriptions (10%) of the release of protein engineered enzyme product, the release of total soluble protein and the change in debris size distribution required a small amount of extra information which was readily acquired using a scale-down unit. Utilisation of such scale-down and modelling techniques will enable design calculations, unit integration and identi®-cation of optimum operating conditions to be carried out for the high pressure homogenisation of recombinant yeast early in process development with a high degree of con®dence.List of symbols b constant in Eq. (5) C u soluble protein assay result (mg/ml) C y yeast concentration on a wet weight basis (g/l) d particle diameter (lm) d 50 Boltzman average diameter (lm) d 50N0 whole cell Boltzman average diameter (lm) d à 50 dimensionless form of d 50 de®ned as d 50N0 À d 50 ad 50N0 À D fraction of cells disrupted À F aqueous fraction À f D S stress distribution imposed on cells by homogeniser À f S S cell strength distribution À f d NYP cumulative (undersize) volume PSD for a given size range at given P and N i number of data points À k constant in Eq. (7) K constant in Eq. (1) m constant in Eq. (5) n constant in Eq. (5) N number of passes À Q¯owrate (m/s 3 ) P pressure (barg) P t threshold pressure (barg) r regression coef®cient À R soluble protein release (mg protein/ g wet weight yeast) R max maximum soluble protein release (mg protein/g wet weight yeast) S effective strength À S m mean effective strength À Greek symbols a exponent in Eq. (1) r standard deviation À x Boltzman parameter in Eq. (6) x N0 whole cell Boltzman parameter in Eq. (6) x à dimensionless form of x de®ned as (x N0 À xx N0 À v chi statistic R centrifuge sigma factor (m 2 ) IntroductionThe use of the yeast Saccharomyces cerevisiae as a host organism for the production of recombinant proteins often results in an intracellular product [9]. Ef®cient cell disruption is therefore required to achieve product release and enable subsequent recovery. High pressure homogenisation is usually the preferred unit operation for achieving this disruption at a large scale [20]. In determining process feasibility and for subsequent engineering design it is essential that the homogenisation stage can be described by relevant mathematical models. With increasing pressure on biotechnology companies to bring bioproducts to market in the shortest possible time and at low cost, there is also a need to produce unit and process models early in the process development cycle from only small amounts of sample material and with a minimum of effort. This provides the incentive for scaledown research in which accurate mimics of key unit operations are developed which require only limited quantities of feed material but can generate useful process information [13].

Micro biochemical engineering to accelerate the design of industrial‐scale downstream processes for biopharmaceutical proteins

“…The minimum quantity of test material for a small pilot high pressure homogenizer is 2 L but a laboratory device with a similar rupture valve can operate with 40 mL. Varga et al (1998) described the use of this device to predict large scale performance in disrupting a recombinant Saccharomyces cerevisiae to yield an intracellular protein. Models of rupture of microorganisms had been established in the 1970s (Hetherington et al, 1971) and more recently Siddiqi et al (1996) developed an empirical model describing the change in cell debris particle size distribution under various homogenizing conditions.…”

“…Miller et al (2002) applied a CFD model to a high pressure homogenizer to understand further the fluid dynamics. The ultra scale-down approach was used by Varga et al (1998) to collect the basic parameters for the model, which differed between the recombinant organism and natural yeast. The particle size distribution model was also refined.…”

UCL biochemical engineering

“…With this framework, it was then possible to examine how to acquire the same information at a much faster rate by ultra scaledown beyond the smallest industrial homogenizers (Siddiqi et al, 1997) and centrifuges (Maybury et al, 1998). By linking the ultra scale-down to a set of experimentally validated process models, it was subsequently possible to predict the performance of a process for a recombinant yeast with a protein-engineered alcohol dehydrogenase by acquiring a minimum of data on the new system (Varga et al, 1998). The alcohol dehydrogenase target provided the basis for whole bioprocess studies of plasmid genes (Ciccolini et al, 1998) and their complexes with delivery and cell targeting molecules such as liposomes and antibody fragments.…”

Visualizing integrated bioprocess designs through ?windows of operation?

“…Recent modeling studies at UCL have concentrated on the development of predictive models where the model outputs are tested, verified, by comparison with pilot-scale data Clarkson et al, 1996;Siddiqi et al, 1996;Varga et al, 1997). The verification process is seen as critical since it confirms the accuracy and limitations of the models and can be used to establish the generic nature of the modeling framework (Varga et al, 1998). This research has led to the generation of a suite of unit operation models capable of being linked together as a flowsheet for mass balancing and the estimation of process performance.…”