Prediction and verification of centrifugal dewatering of P. pastoris fermentation cultures using an ultra scale-down approach

Recent years have seen a dramatic rise in fermentation broth cell densities and a shift to extracellular product expression in microbial cells. As a result, dewatering characteristics during cell separation is of importance, as any liquor trapped in the sediment results in loss of product, and thus a decrease in product recovery. In this study, an ultra scale-down (USD) approach was developed to enable the rapid assessment of dewatering performance of pilot-scale centrifuges with intermittent solids discharge. The results were then verified at scale for two types of pilot-scale centrifuges: a tubular bowl equipment and a disk-stack centrifuge. Initial experiments showed that employing a laboratory-scale centrifugal mimic based on using a comparable feed concentration to that of the pilot-scale centrifuge, does not successfully predict the dewatering performance at scale (P-value <0.05). However, successful prediction of dewatering levels was achieved using the USD method (P-value ≥0.05), based on using a feed concentration at small-scale that mimicked the same height of solids as that in the pilot-scale centrifuge. Initial experiments used Baker's yeast feed suspensions followed by fresh Pichia pastoris fermentation cultures. This work presents a simple and novel USD approach to predict dewatering levels in two types of pilot-scale centrifuges using small quantities of feedstock (<50 mL). It is a useful tool to determine optimal conditions under which the pilot-scale centrifuge needs to be operated, reducing the need for repeated pilot-scale runs during early stages of process development.     

Ultra scale‐down device to predict dewatering levels of solids recovered in a continuous scroll decanter centrifuge

During centrifugation operation, the major challenge in the recovery of extracellular proteins is the removal of the maximum liquid entrapped within the spaces between the settled solids–dewatering level. The ability of the scroll decanter centrifuge (SDC) to process continuously large amounts of feed material with high concentration of solids without the need for resuspension of feeds, and also to achieve relatively high dewatering, could be of great benefit for future use in the biopharmaceutical industry. However, for reliable prediction of dewatering in such a centrifuge, tests using the same kind of equipment at pilot‐scale are required, which are time consuming and costly. To alleviate the need of pilot‐scale trials, a novel USD device, with reduced amounts of feed (2 mL) and to be used in the laboratory, was developed to predict the dewatering levels of a SDC. To verify USD device, dewatering levels achieved were plotted against equivalent compression (Gt_comp) and decanting (Gt_dec) times, obtained from scroll rates and feed flow rates operated at pilot‐scale, respectively. The USD device was able to successfully match dewatering trends of the pilot‐scale as a function of both Gt_comp and Gt_dec, particularly for high cell density feeds, hence accounting for all key variables that influenced dewatering in a SDC. In addition, it accurately mimicked the maximum dewatering performance of the pilot‐scale equipment. Therefore the USD device has the potential to be a useful tool at early stages of process development to gather performance data in the laboratory thus minimizing lengthy and costly runs with pilot‐scale SDC. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1494–1502, 2013     

The influence of precipitation reactor configuration on the centrifugal recovery of isoelectric soya protein precipitate

The strength and rheological characteristics of isoelectric soya protein precipitate, prepared in continuous tubular and batch-stirred tank reactors, were determined in relation to the separation efficiency and sludge discharge characteristics of intermittent disk and scroll discharge centrifuges. The batch tank precipitate showed greater resistance to aggregate shear breakup than the tubular reactor precipitate. This difference was of significance to the separation efficiency of the scroll centrifuge only. The tubular reactor precipitate sludge showed greater resistance to shear deformation and resulted in drier sludge for a given scroll differential speed. some compressive dewatering occurred for both types of precipitate sludge but shear-induced dewatering is proposed as the major mechanism. This study illustrates the need to integrate the design of protein precipitate formation and centrifugal recovery operations.     

An ultra scale-down approach to assess the impact of the choice of recombinant P. pastoris strain on dewatering performance in centrifuges

Pichia pastoris is becoming a desirable host in the biopharmaceutical industry for therapeutics production. It grows on methanol to high cell densities ≥100 g DCW/L and secretes foreign proteins at high titers. However, the culture conditions to reach high cell densities pose a challenge to the processability by primary recovery operations, in particular centrifugation, used for cell removal. This work aims to assess the impact of recombinant P. pastoris strain selection on centrifugal dewatering. Normally, the choice of P. pastoris recombinant strain is based on best target protein expression levels; however, it is unknown whether the choice of strain will have an impact on performance of centrifugation operation. To achieve this aim, a previously developed laboratory ultra‐scale down (USD) methodology that successfully predicted centrifugal dewatering of pilot‐scale disk‐type machines, was used in this work. Two recombinant P. pastoris strains, namely a X‐33 and a glycoengineered Pichia strain, were used to perform fermentations secreting different products. The resulting harvested fermentation culture properties were analyzed and the dewatering performances of a pilot‐ and a large‐scale disk‐type centrifuge were evaluated using the USD methodology. The choice of P. pastoris strain was found to have a considerable impact on dewatering performance, with P. pastoris X‐33 strain reaching better dewatering levels than the glycoengineered strain. The USD method proved to be a useful tool to determine optimal conditions under which the large scale centrifuge needed to be operated, reducing the need for repeated pilot‐scale runs during early stages of process development for therapeutic products. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 1029–1036, 2012     

Modeling industrial centrifugation of mammalian cell culture using a capillary based scale-down system

Continuous-flow centrifugation is widely utilized as the primary clarification step in the recovery of biopharmaceuticals from cell culture. However, it is a challenging operation to develop and characterize due to the lack of easy to use, small-scale, systems that can be used to model industrial processes. As a result, pilot-scale continuous centrifugation is typically employed to model large-scale systems requiring a significant amount of resources. In an effort to reduce resource requirements and create a system which is easy to construct and utilize, a capillary shear device, capable of producing energy dissipation rates equivalent to those present in the feed zones of industrial disk stack centrifuges, was developed and evaluated. When coupled to a bench-top, batch centrifuge, the capillary device reduced centrate turbidity prediction error from 37% to 4% compared to using a bench-top centrifuge alone. Laboratory-scale parameters that are analogous to those routinely varied during industrial-scale continuous centrifugation were identified and evaluated for their utility in emulating disk stack centrifuge performance. The resulting relationships enable bench-scale process modeling of continuous disk stack centrifuges using an easily constructed, scalable, capillary shear device coupled to a typical bench-top centrifuge.     

Performance prediction of industrial centrifuges using scale-down models

Computational fluid dynamics was used to model the high flow forces found in the feed zone of a multichamber-bowl centrifuge and reproduce these in a small, high-speed rotating disc device. Linking the device to scale-down centrifugation, permitted good estimation of the performance of various continuous-flow centrifuges (disc stack, multichamber bowl, CARR Powerfuge) for shear-sensitive protein precipitates. Critically, the ultra scale-down centrifugation process proved to be a much more accurate predictor of production multichamber-bowl performance than was the pilot centrifuge.     

Scale-down of continuous filtration for rapid bioprocess design: Recovery and dewatering of protein precipitate suspensions

The early specification of bioprocesses often has to be achieved with small (tens of millilitres) quantities of process material. If extensive process discovery is to be avoided at pilot or industrial scale, it is necessary that scale-down methods be created that not only examine the conditions of process stages but also allows production of realistic output streams (i.e., streams truly representative of the large scale). These output streams can then be used in the development of subsequent purification operations. The traditional approach to predicting filtration operations is via a bench-scale pressure filter using constant pressure tests to examine the effect of pressure on the filtrate flux rate and filter cake dewatering. Interpretation of the results into cake resistance at unit applied pressure (alpha) and compressibility (n) is used to predict the pressure profile required to maintain the filtrate flux rate at a constant predetermined value. This article reports on the operation of a continuous mode laboratory filter in such a way as to prepare filter cakes and filtrate similar to what may be achieved at the industrial scale. Analysis of the filtration rate profile indicated the filter cake to have changing properties (compressibility) with time. Using the insight gained from the new scale-down methodology gave predictions of the flux profile in a pilot-scale candle filter superior to those obtained from the traditional batch filter used for laboratory development.     

Ultra scale‐down approaches for clarification of mammalian cell culture broths in disc‐stack centrifuges

Ultra‐scale down (USD) methodology developed by University College London for cell broth clarification with industrial centrifuges was applied to two common cell lines (NS0 and GS‐CHO) expressing various therapeutic monoclonal antibodies. A number of centrifuges at various scales were used with shear devices operating either by high speed rotation or flow‐through narrow channels. The USD methodology was found effective in accounting for both gravitational and shear effects on clarification performance with three continuous centrifuges at pilot and manufacturing scales. Different shear responses were observed with the two different cell lines and even with the same cell line expressing different products. Separate particle size analysis of the treated broths seems consistent with the shear results. Filterability of the centrifuged solutions was also evaluated to assess the utility of the USD approach for this part of the clarification operation. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

MELISSA: a loop of interconnected bioreactors to develop life support in space

The development of a loop of interconnected continuous bioreactors, aimed to provide life support in space, is reported. The complete loop concept consists of four bioreactors and one higher plant compartment. For its realization the continuous and controlled operation of the bioreactors is characterized, up to the pilot scale level, first for each individual reactor, second for the interconnected reactor operation. The results obtained with the two more advanced bioreactors in the Micro Ecological Life Support System Alternative (MELISSA) loop are described more specifically. These reactors consist of a packed-bed reactor working with an immobilized co-culture of Nitrosomonas and Nitrobacter cells, and an external loop gas-lift photobioreactor for the culture of the cyanobacteria Spirulina platensis. Their individual operation for long duration runs has been achieved and characterized, and their interconnected operation at pilot scale is reported.     

Scale-down of microalgae cultivations in tubular photo-bioreactors--a conceptual approach

Rational design of large-scale bioreactors is still suffering from inadequate scale-up of technical parameters from lab to large scale and from missing kinetic information concerning the physiological reactions of the specific strain under cultivation. Therefore, simulations of processes expected in large-scale have to be carried out as far as possible and experiments have to be performed in small-scale reactors mimicking the situation in large scale. This procedure is referred to as scale-down. In this paper a concept to accomplish this task is proposed. Firstly, interactions between light transfer, fluid dynamics, and microbial metabolism are described. Secondly, a procedure is given to decompose the interactions by simulation on the one hand and by finding physiological parameters in model reactors on the other. Light transfer can be calculated by Monte Carlo methods, while fluid dynamics is handled by CFD. Ideally illuminated model photo-bioreactors and pilot reactors with enforced flow field are proposed to measure physiological parameters especially induced by light/dark cycles generated by interaction of turbulences and light attenuation.     

Scale up aspects of sparged insect-cell bioreactors

Conclusion In this chapter we have attempted to evaluate the most important parameters which can be useful for the pur-pose of design and scale up. Insect cells and animal cells in general can be grown well in large vessels. However, none of the theories and parameters discussed in this chapter have been validated on a larger scale than laboratory and small pilot reactors. Selection of the most suitable design and scale-up method there-fore needs in particular studies in larger vessels. The Kolmogorov theory and the killing-volume model are in this respect the most promising approaches for the optimal design of large-scale animal-cell bioreactors.     

Hydrodynamic performance of pilot-scale duckweed,algal-based,rock filter and attached-growth media reactors used for waste stabilisation pond research

The specific nature of fluid dynamics within waste stabilisation ponds can have a determining influence on their functional treatment performance. This paper presents the results of hydraulic tracer experiments undertaken to characterise the hydrodynamic behaviour of several pilot-scale advanced pond treatment systems (a duckweed-based pond, a conventional algal-based ‘open’ pond, a rock filter and a novel horizontal-flow attached-growth media reactor) investigated for their effectiveness at polishing a full-scale tertiary pond effluent. Duplicate tracer studies were undertaken for each of the four experimental reactors with the aid of the fluorescent dye rhodamine WT. Results from tracer studies showed flow distribution in all reactors to be highly dispersed with varying degrees of dead space volume and short-circuiting in all pilot reactors, indicating that a percentage of the total reactor volume across all treatment systems was inactive. Results from a number of calculated parameters of hydraulic performance showed that hydrodynamic efficiency was greatest in the novel horizontal-flow attached-growth media system, where a possible baffling of inflowing wastewater was thought to have promoted improved hydraulic operation. Outcomes from this research in general highlight the importance of undertaking detailed characterisations of the hydrodynamic operation of experimental pond systems and also emphasise the value of pre-validating the hydraulic design of experimental reactors used for stabilisation pond research.     

An automated laboratory‐scale methodology for the generation of sheared mammalian cell culture samples

Continuous disk‐stack centrifugation is typically used for the removal of cells and cellular debris from mammalian cell culture broths at manufacturing‐scale. The use of scale‐down methods to characterise disk‐stack centrifugation performance enables substantial reductions in material requirements and allows a much wider design space to be tested than is currently possible at pilot‐scale. The process of scaling down centrifugation has historically been challenging due to the difficulties in mimicking the Energy Dissipation Rates (EDRs) in typical machines. This paper describes an alternative and easy‐to‐assemble automated capillary‐based methodology to generate levels of EDRs consistent with those found in a continuous disk‐stack centrifuge. Variations in EDR were achieved through changes in capillary internal diameter and the flow rate of operation through the capillary. The EDRs found to match the levels of shear in the feed zone of a pilot‐scale centrifuge using the experimental method developed in this paper (2.4×10⁵ W/Kg) are consistent with those obtained through previously published computational fluid dynamic (CFD) studies (2.0×10⁵ W/Kg). Furthermore, this methodology can be incorporated into existing scale‐down methods to model the process performance of continuous disk‐stack centrifuges. This was demonstrated through the characterisation of culture hold time, culture temperature and EDRs on centrate quality.     

The use of laboratory centrifugation studies to predict performance of industrial machines: studies of shear-insensitive and shear-sensitive materials

A method for using a bench-top centrifuge is described in order to mimic the recovery performance of an industrial-scale centrifuge, in this case a continuous-flow disc stack separator. Recovery performance was determined for polyvinyl acetate particles and for biological process streams of yeast cell debris and protein precipitates. Recovery of polyvinyl acetate particles was found to be well predicted for these robust particles. The laboratory centrifugation scale-down technique again predicted the performance of the disc stack centrifuge for the recovery of yeast cell debris particles although there was some suggestion of over-prediction at high levels of debris recovery due to the nature of any cell debris aggregates present. The laboratory centrifuge scale-down technique also proved to be an important investigative probe into the extent of shear-induced breakup of shear-sensitive protein precipitate aggregates during recovery in continuous high speed centrifuges. Such breakup can lead to over 10-fold reduction in separator capacity.     

A methodology for centrifuge selection for the separation of high solids density cell broths by visualisation of performance using windows of operation

Expression systems capable of growing to high cell densities are now readily available and are popular due to the benefits of increased product concentration. However, such high solids density cultures pose a major challenge for bioprocess engineers as choosing the right separation equipment and operating it at optimal conditions is crucial for efficient recovery. This study proposes a methodology for the rapid determination of suitable operating conditions for the centrifugal recovery of high cell density fermentation broths. An ultra scale-down (USD) approach for the prediction of clarification and dewatering levels achieved in a range of typical high-speed centrifuges is presented. Together with a visualisation tool, a Window of Operation, this provides for the rapid analysis of separation performance and evaluation of the available operating conditions, as an aid in the selection of the centrifuge equipment most appropriate for a given process duty. A case study examining centrifuge selection for the processing of a high cell density Pichia pastoris culture demonstrates the method. The study examines semi-continuous disc-stack centrifuges and batch-operated machines such as multi-chamber bowls and Carr Powerfuges. Performance is assessed based on the variables of clarification, dewatering and product yield. Inclusion of limits imposed by the centrifuge type and design, and operation itself, serve to constrain the process and to define the Windows of Operation. The insight gained from the case study provides a useful indication of the utility of the methodology presented and illustrates the challenges of centrifuge selection for the demanding case of high solids concentration feed streams.     

Inoculation and tissue distribution in pilot-scale plant root culture bioreactors

Summary Inoculation of large-scale plant root culture reactors can be carried out by briefly homogenizing bulk root tissue, followed by aseptic transfer as a slurry to the reactor. Uniform root distribution can be achieved in bioreactors by entrapment of the growing root inoculum onto process packing elements (e.g. distillation packings), randomly distributed within the reactor, in a bubble column operation. These two techniques have been successfully used to inoculate a 14 L pilot-scale reactor which is subsequently operated as a trickle bed reactor.     

Assessing the feasibility of achieving biological nutrient removal from wastewater at an Irish food processing factory

In Ireland, wastewaters emanating from the food industry typically contain elevated levels of nitrogen and phosphorus before treatment. Two pilot scale studies were performed to determine the feasibility of achieving biological N and P removal on-site at a food ingredients plant. The wastewater treated by the pilot reactors was that which resulted from the day-to-day production in the full-scale food ingredients plant. Both reactors were of the anaerobic/anoxic/oxic (A/A/O) design, however the sizing of the zones was varied in this study. In the first pilot study, while treating a wastewater of the following strength: 1008 mg COD/l; 30.1 mg NH4-N/l and 26.7 mg P/l, removal efficiencies of 93%, 99% and 98% were obtained for COD, NH4-N and P, respectively. In the second study, while operating at reduced hydraulic retention times and lower recycle rates, the pilot plant treated a wastewater of the following strength: 1757 mg COD/l; 62 mg NH4-N/l and 57 mg P/l, with removal efficiencies of 94%, 97% and 75% obtained for COD, NH4-N and P, respectively. This work showed that biological nutrient removal could be successfully applied to treatment of food industry wastewaters.     

A CFD model for predicting protein aggregation in low-pH virial inactivation for mAb production

Significant amounts of soluble product aggregates were observed in the low-pH viral inactivation (VI) operation during an initial scale-up run for an immunoglobulin-G 4 (IgG4) monoclonal antibody (mAb IgG4-N1). Being earlier in development, a scale-down model did not exist, nor was it practical to use costly Protein A eluate (PAE) for testing the VI process at scale, thus, a computational fluid dynamics (CFD)-based high-molecular weight (HMW) prediction model was developed for troubleshooting and risk mitigation. It was previously reported that the IgG4-N1 molecules upon exposure to low pH tend to change into transient and partially unfolded monomers during VI acidification (i.e., VIA) and form aggregates after neutralization (i.e., VIN). Therefore, the CFD model reported here focuses on the VIA step. The model mimics the continuous addition of acid to PAE and tracks acid distribution during VIA. Based on the simulated low-pH zone (≤pH 3.3) profiles and PAE properties, the integrated low-pH zone (ILPZ) value was obtained to predict HMW level at the VI step. The simulations were performed to examine the operating parameters, such as agitation speed, acid addition rate, and protein concentration of PAE, of the pilot scale (50–200 L) runs. The conditions with predictions of no product aggregation risk were recommended to the real scale-up runs, resulted in 100% success rate of the consecutive 12 pilot-scale runs. This study demonstrated that the CFD-based HMW prediction model could be used as a tool to facilitate the scale up of the low-pH VI process directly from bench to pilot/production scale.     

Scale-up fermentation of recombinant <Emphasis Type="Italic">Candida rugosa</Emphasis> lipase expressed in <Emphasis Type="Italic">Pichia pastoris</Emphasis> using the GAP promoter

The high-cell-density fermentation of Candida rugosa lipase in the constitutive Pichia pastoris expression system was scaled up from 5 to 800 l in series by optimizing the fermentation conditions at both lab scale and pilot scale. The exponential feeding combined with pH-stat strategy succeeded in small scale studies, while a two-stage fermentation strategy, which shifted at 48 h by fine tuning the culture temperature and pH, was assessed effective in pilot-scale fermentation. The two-stage strategy made an excellent balance between the expression of heterogeneous protein and the growth of host cells, controlling the fermentation at a relatively low cell growth rate for the constitutive yeast expression system to accumulate high-level product. A stable lipase activity of approximately 14,000 IU ml⁻¹ and a cell wet weight of ca. 500 g l⁻¹ at the 800-l scale were obtained. The efficient and convenient techniques suggested in this study might facilitate further scale-up for industrial lipase production.