Centrifugal Separation in Biotechnology Plenary Presentation American Filtration & Separation Society Annual Conference Valley Forge, PA May 20-22 Wallace

Woon-Fong Leung Director, Research Institute of Innovative Products & Technologies, and Chair Professor, Department of Mechanical Engineering, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong Email: riwl@polyu.edu.hk Abstract: Disk-stack and tubular centrifuges have been used to separate soluble and insoluble proteins for biopharmaceutical processing. There are three common host-cell technology platforms, namely yeast, mammalian cells, and bacteria. The first two use the host cells to express extracellular protein, while the latter uses the host cell to express intracellular protein in form of submicron-sized inclusion bodies. For the yeast host-cell process, the first step is to separate yeast cells using centrifugation from dissolved protein, followed by clarification of protein solution product using centrifugation to meet the minimal turbidity requirement. The flow sheet of separating mammalian cells such as Chinese Hamster Ovaries (CHO) cells using centrifuge from the protein product is presented. The key concern is to reduce shear on the cells in the suspension (maintain high cell viability) through gentle feed acceleration to high-speed rotation to effect separation in a centrifuge. The subsequent step is to use a depth filter or microfiltration to clarify the separated liquid product removing any submicron fine solids. It is critical to ensure the centrifuge-filter work together as a system. The flow sheet of processing bacteria bearing protein is to concentrate using centrifugation the bacteria solution followed by lysing the bacteria to release the inclusion bodies. The resultant suspension is reslurried and washed, with the protein-bearing inclusion bodies separated from any impurities by centrifugation until the desired purity is reached suitable for downstream processing. Separation by centrifugation is used extensively in these three host-cell platforms, which is a major application in production of antibotics and intermediate drug substances for the biopharmaceutical market. Other important large-scale centrifugal separation in biotech processes has also been discussed. Finally, throughput and sizing of centrifuge and simulation tools are presented. Keywords: centrifugation, centrifuge, disk-stack, tubular, biopharmaceutical, biotechnology

The lighter liquid phase is displaced to the smaller diameter flowing toward the effluent exit near the axis of the machine. Note the bowl volume and separation surface are not as large as the disk stack centrifuge. However. 2.1. the machine is adaptable with appropriate adjustment. centripetal pumps or paring disks are used. Introduction Due to robustness and compactness. centrifugal acceleration which is over thousands of earth gravity is generated and heavier solids settle to the underside of the disk and slide down to the large diameter by centrifugal force. 1a shows an intermittent discharge disk-stack centrifuge. Separated heavy phase collected at the large diameter is discharged periodically through exit ports. The scientific basis has been presented recently based on experiments and theory in great details [1]. 1a – Intermittent-discharge disk stack centrifuge To avoid foaming. centrifugal separation has an unique application in biopharmaceutical production [1. Under high-speed rotation. Basically the rotating clarified liquid is channeled into the opening of convoluted passages from which the kinetic energy of the rotating liquid is gradually transformed into static pressure head with minimal head loss. the feed is stopped and the pool of liquid is drained before the sediment cake is discharged by various mechanisms. Feed is brought in. 1b shows a tubular centrifuge. Heavier solids settle to the bowl wall and are allowed to accumulate until the layer thickness become too thick interfering with the effluent which stays close to the pool surface. It has a long and slender bowl. accelerated to angular speed to acquire centrifugal acceleration before feeding into the annular pool. Disk-stack and tubular centrifuges have been used to separate soluble and insoluble proteins for biopharmaceutical production. Special provisions have been made [1] on disk stack centrifuges in feed acceleration zones to avoid generating high shear especially for processing delicate cells such as mammalian cells. At this point.3 mm – 2 mm depending on the process [1]. The former benefit is applicable to various situations and environment especially those for which change is norm than an exception. Most common designs are suspended and driven from the top. Clarified Overflow R1 D R2 Feed Suspension disks Concentrate Discharge Dropping Bottom Feed Feed acceleration θ Clarified Liquid Fig. Fig. Fig. separation of solids and clarification of liquid broth with small amount of dispersed solids/light phase are most effective. The machine has numerous conical disks each separated by a small distance from 0. Centrifuge In this section the basic machines are briefly introduced. It has been a workhorse in the biotech industry.2]. it finds application in . Because of the small distance and many disks with large surface area.

and bacteria. 2b – Animal cell. and a bacteria cell. see Fig. 3.various bioprocessing that does not require high feed rate and relatively inexpensive capital requirement. Fig 2a. This is illustrated in Fig. Fig. Yeast cells are typically 8-10 microns in size. while the latter uses the host cell to express intracellular protein in form of submicron-sized inclusion bodies. an animal cell that resembles closely a mammalian cell. Fig. Fig. namely yeast. 2a. 1b – Tubular centrifuge 3. followed by clarification of protein solution product using centrifugation to meet the maximal turbidity requirement. Fig. In short. The tubular bowl has a long history for separation of E coli bacteria in biotech process. the first step is to separate yeast cells using centrifugation from dissolved protein. 4a shows an example where a two-stage centrifugal separation is used to remove expressed dissolved protein at harvest with the first-stage centrifugation carrying out separation and the second-stage centrifugation carrying out clarification of the product liquid protein which may contain a small residual amount of yeast cells and debris. 2a – Yeast cell. an engineered gene segment is inserted in the DNA for which it is implanted in host cells for large manufacturing of an engineered protein. There are three common host-cell technology platforms. 2b and 2c shows respectively a schematic of a yeast cell. For the yeast host-cell process. mammalian cells. 2c – Bacteria cell. Both disk stack and tubular centrifuges can be used for carrying out separation. Biopharmaceutical Process Recombinant DNA process has found popular applications in protein manufacturing. D Overflow Dp Dh Annular Pool L Particle trajectories Hub Feed Fig. The first two use the host cells to express extracellular protein. The end result is that over 98% of feed yeast solids are .

A single-stage and double-stage washing are shown in Fig. The key success is to reduce shear on the cells in the suspension (maintain high cell viability) through gentle feed acceleration to high-speed rotation to effect separation in a centrifuge [1].4% =9. . The resultant suspension is reslurried and washed. The number of stages employed depends on the required product purity [1]. with the protein-bearing inclusion bodies separated from any impurities by centrifugation until the desired purity meets the requirement for downstream processing (such as purification). The flow sheet of separating mammalian cells such as Chinese Hamster Ovaries (CHO) cells using centrifuge from the protein product is similar for the first stage separation except the second stage clarification of the liquid product is usually carried out by depth filtration with filter aid or with microfiltration to remove particulates less than 1 micron.15% loss Fig. 4a – Yeast cell platform and subsequent separation The flow sheet of processing bacteria bearing protein is to concentrate using centrifugation the bacteria solution followed by lysing the bacteria to release the inclusion bodies.4% Product Clarification stage 98.56%x9. 4b.4%=0.4%x9.recovered. 3 – Host cell platform and subsequent separation processes 100% feed 90. It is critical to ensure the centrifuge-filter work together as a system.6% recovery 99. Fermentation & Bioreaction Mammalian Cell Extracellular Centrifuge Separation Solid Product Depth Filter Polishing Bacteria Intracellular Yeast Extracellular Centrifuge Clarification Liquid Product Centrifuge Lysate Clarification Centrifuge Polishing Lysing Centrifuge IBClassification IB Washing Centrifuge/Filter Polishing Downstream Processing Drug Substance & Monoclonal Antibodies Fig.85% total recovery Separation stage 9.25% 1.

4a Insulin Production Insulin is an important hormone controlling the glucose level of blood in human. A generic insulin production sheet is shown in Fig. The resulting product is frozen to maintain freshness prior to finish processing. 4. coli Thickening Concentrate Centrifuge Homogenizer Reslurrying Separation Centrifuge Reslurrying Separation Centrifuge Reslurrying Separation Centrifuge Inclusion Bodies protein Cell Debris Cell Debris Excess liquid 3-5 μm "Single Wash" "Double Wash" Cell Debris Fig. An enzyme trypsin is used for cleavage. 4b shows processing of E.1 μm E. . First the bacteria. such as E. are genetically modified to express a specific protein under fermentation. The protein is further concentrated by crystallization. Other Important Biotech Processes Two examples are used to illustrate the many important biotech processes that are being widely used today. The product solution is purified with chromatography column. The pre-insulin is treated with buffer liquid to activate to tertiary form. coli and inclusion bodies bearing protein. Separation by centrifugation is used extensively in these three host-cell platforms. coli bacteria. physiological conditions are closely monitored to ensure the process is well controlled and optimal protein is released in solution. The bacteria go through a fermentation process where temperature. the bacteria cells are lysed and the protein in solids is recovered by centrifugation followed by depth filtration. 5. which is a major application in production of antibiotics and intermediate drug substances for the biopharmaceutical market. washing and centrifugation. This process is repeated several times until the desired purity is reached. agitation rate. During harvesting.

The inclusion bodies free from contaminants are sent downstream for further processing. Micro-organism. Liquid Waste Substrate Micro-organism Air Cell Disruption Cellular-Liquid with Cell Debris (Waste) Fermenter Nozzle Disk Biomass Homogenizer Nozzle Disk Solids Liquid Waste Wash/Buffer Liquid Nozzle Disk Solids Wash/Buffer Liquid Nozzle Disk Inclusion Bodies Product for Downstream Processing Fig. 6 shows a generic process of separating and related processing of hormones. substrate and air are typically ingredients to the fermenter. 7. Working with the nervous system. . is classified by another nozzle disk in the centrate with moderate G-force. the broth is fed to a nozzle disk where the liquid phase is removed as waste stream. coli Fermentation Harvest . Separation by centrifugation has been widely used to process insulin for use by diabetic patients. 6.Innoculation with genetically modified E. regulates the blood glucose of the body. such as insulin. A common hormone. Fig. 6 . Subsequently the biomass after resuspended in wash/buffer liquid is sent to a homogenizer for cell disruption releasing the solid inclusion bodies. This is much lower from the process viewpoint and the specific feed rate depends on the requirements of a given process. As shown in Fig. and the separated biomass concentrate discharges continuously through the nozzles. Throughput and Sizing of Centrifuges The maximum and minimum feed rates of respective tubular and disk centrifuges are shown in Fig.Hormones processing flowsheet 5. The feed rate varies approximately as the third power of the bowl diameter from the hydraulic capacity consideration. After fermentation. 5 – Generic insulin flow sheet using centrifugation 4b Hormones Processing Hormones are chemicals in the bodies that regulate and control the metabolism and organ functions. they co-ordinate all essential functions of the body. the concentrate with solid inclusion bodies containing contaminants is reslurried and separated by centrifugation sequentially in two stages. The cellular liquid. with cell debris typically in the submicron sizes.Lyse cells and recover protein by centrifuge&filtration Refolding .treatment of preinsulin w/ buffer solution to active tertiary form Enzyme clevage with trypsin Finish Deep Freezing Centrifugation Reslurrying Crystallization Chromatography Washing & Separation Fig.

the projected area parallel to the axis of the machine Lp. Le depends on centrifuge design geometry. Le is directly correlated with the cut size – the maximum size in the supernatant. Despite one might argue that there is no cut size that provides a sharp demarcation of the minimum size of cake/concentrate sample and the maximum size of the centrate due to various complexities (entrainment of sediment etc. 7 – Disk and tubular size versus feed rate. Eq. 6. the slant length of disk L. 3) In the above. the number of disks n. tubular and decanter centrifuges as used for separation and clarification sizing. 1a is very similar to that of the decanter centrifuge except that R′ replaces the pool radius Rp for the case of a decanter. the inner disk radius R1. the approach presented herein provides a unified approach for sizing different types of centrifuges. Sizing for Disk-Stack Centrifuge The dimensionless Le number for a disk stack centrifuge incorporates the feed rate Q. disk. L/min 100 Max Rate 10 Min Rate 1 100 Tubular 1000 Diameter. The geometry is captured in Fig.). the liquid viscosity μ. The Le number has been developed for batch sedimentation (spin tube) and continuous-feed centrifuges (disk. and the characteristics particle size xo. R′ can be . and properties of suspended particles (size distribution and density) and liquid (density and viscosity). The Le number was used successfully [3] in many applications for fine particle sedimentation (0. The Le number for the disk centrifuge [1] is given by. 8. Le = Q μ L p Δρ ΩR' xoη 1/ 2 ⎡ R 2 + R2 R1 + R12 ⎤ R' = ⎢ 2 ⎥ 3 ⎣ ⎦ L p = nL cos θ ⎛ ⎞ ≈ R1 ⎜1 + 0. the density difference Δρ.1000 Disk Rate. Most importantly. 7.5536⎡ R2 − 1⎤ ⎟ ⎢ R1 ⎥ ⎠ ⎣ ⎦ ⎝ (1a. chamber bowl. with maximum rate limited by the hydraulic capacity. 2. or the minimum size in sediment. the outer disk radius R2. the angle of the disk stack with respect to the vertical θ. yet this provides a concept whereby one can quantitatively size or scale-up centrifuges. mm Fig. the effective radius R′ is related to R1 and R2 as expected. tubular and decanter centrifuges). Dimensionless Le Number A new dimensionless Leung number (hereafter abbreviated as “Le”) has been developed for spintube.1 – 100 μm).

000g (7270 rpm). The results are depicted in Fig. 8 .Disk Centrifuge Centrifuge Bowl Diameter D=400 mm Disk Outer Diameter 2R2=300 mm Disk Inner Diameter 2R1=177 mm Disk Angle θ=40o from vertical Density difference/density Δρ/ρ=0.0 Separated Cell Size. microns Fig. It can also be shown that Le can be expressed in a more compact form as follows: Le = ⎞ tan θ ⎛ 3Q ⎞⎛ μ ⎜ n ⎟⎜ [ρ − ρ ]⎟ 3 3 ⎠⎝ ⎝ s L ⎠ R2 − R1 ( ) Ωxoη (1b) It is to be noted that both Eq.1 1. 9 – Performance of a disk for a feed suspension with 5 cP.1 μ=5 cP n =100 disks 10 6000g 12000g 1 0.1 Viscosity μ=5 cP G = 12. 9 in which the feed rate depends on the size of particles that need to be separated. 1a and 1b yields identical result in calculation of the Le number. Axis Lcosθ θ L L R1 Total Projected Area Lp= n Lcosθ R2 G Fig. .approximated by a linear relationship. G=6000 (5140 rpm) n=100 disks Overall Efficiency = 70% 100 Feed Rate. L/min Δρ/ρ=0.Projected area of disk centrifuge Example .0 10.

Also it is important to characterize (such as cell size. pH. toxicity. 11. solids concentration in various appropriate streams etc.5% 97. %Cumulative Undersize 100% 80% 60% 40% 20% 0% 0 10 20 30 40 50 100.8. ionic strength.0% 98. Numerical simulations can be made to simulate respectively separation in spin tubes in lab screening stage. 12 shows simulation result on a large tubular bowl centrifuge for separating with mammalian cells with the cell size distribution as displayed in Fig. Also process optimization can be facilitated for the installed equipment. cell concentration and sizes.5% 99. Tools Testing and simulation are useful tools to evaluate and optimize the separation process.). Fig. viscosity etc. as well as disk stack and tubular centrifuges in pilot/demonstration and production stages. Fig. flow rates.0% 99. 10 – Simulation used in various stages from lab discovery to production. Fig. pressure. 11 – Simulation of separation of CHO cells using a disk-stack centrifuge. see the strategies outlined in Fig. This provides a more systematic way of monitoring the outcome of separation and making performance assessment on the machine and process. 11a. disk 6000g 12000g %Solids Recovery x. The test rig should be equipped with variable-speed drive (VFD) and appropriate measurement devices to quantify operating variables (e. in-line turbidity in centrate. Pilot testing is an important ingredient to determine if laboratory success in spintube can be realized under larger-scale and continuous-flow in practice.) and performance parameters (yield.0% 0 20 40 60 80 100 400-mm dia. this provides a fast and inexpensive way of predicting outcome before committing to the capital equipment. microns Q. 10.g. L/min Fig. In addition. concentration. b show respectively simulation result on a large disk stack centrifuge separating mammalian cells with 20 microns median in diameter.5% 98. viability) the biological sample for making separation.0% 97. (a) Feed particle size .

References [1] Wallace Woon-Fong Leung. 100.0% %Solid Recovery 99. UK.5% 98. Desai (ed).5% 97. McGraw-Hill. 12 – Simulation of separation of CHO cells using a tubular centrifuge with diameter 457 mm. gentle feed acceleration.distribution. Centrifugal Separation in Biotechnology. to classifying submicron sized cell debris. Acknowledgement The author wishes to acknowledge the Hong Kong Polytechnic University for their support. . Academic Press. Humana Press. The density difference between cells and liquid is 10% and viscosity is 5 cP. and increased separation surface area offers the flexibility from separating small delicate cells. L/min 30 40 Fig. (b) solids recovery by centrifugation under 2 G’s for a 400-mm disk bowl. 1998. Downstream Processing of Proteins.000g 20. NY. 9. 610 bowl length and 95% efficiency. Conclusion Centrifugal separation is a robust means for separating high-value biotech intermediate products and final products. 2007. Totowa.5% 99.0% 0 10 20 10. [2] M. concentrating valuable materials. [3] Wallace Woon-Fong Leung.000g Q. NJ 2000. clarifying broth. High-speed.0% 97.0% 98. Industrial Centrifugation Technology.

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