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

1. Introduction Due to robustness and compactness, centrifugal separation has an unique application in biopharmaceutical production [1,2]. The former benefit is applicable to various situations and environment especially those for which change is norm than an exception, the machine is adaptable with appropriate adjustment. 2. Centrifuge In this section the basic machines are briefly introduced. Disk-stack and tubular centrifuges have been used to separate soluble and insoluble proteins for biopharmaceutical production. Fig. 1a shows an intermittent discharge disk-stack centrifuge. It has been a workhorse in the biotech industry. The machine has numerous conical disks each separated by a small distance from 0.3 mm 2 mm depending on the process [1]. Under high-speed rotation, 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. The lighter liquid phase is displaced to the smaller diameter flowing toward the effluent exit near the axis of the machine. Because of the small distance and many disks with large surface area, separation of solids and clarification of liquid broth with small amount of dispersed solids/light phase are most effective. Separated heavy phase collected at the large diameter is discharged periodically through exit ports. 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.
Clarified Overflow
R1 D R2

Feed Suspension

disks Concentrate Discharge Dropping Bottom Feed Feed acceleration

Clarified Liquid

Fig. 1a Intermittent-discharge disk stack centrifuge

To avoid foaming, centripetal pumps or paring disks are used. 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. Fig. 1b shows a tubular centrifuge. Most common designs are suspended and driven from the top. It has a long and slender bowl. Feed is brought in, accelerated to angular speed to acquire centrifugal acceleration before feeding into the annular pool. 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. The scientific basis has been presented recently based on experiments and theory in great details [1]. At this point, the feed is stopped and the pool of liquid is drained before the sediment cake is discharged by various mechanisms. Note the bowl volume and separation surface are not as large as the disk stack centrifuge. However, it finds application in

various bioprocessing that does not require high feed rate and relatively inexpensive capital requirement. The tubular bowl has a long history for separation of E coli bacteria in biotech process.
D Overflow Dp Dh Annular Pool L Particle trajectories
Hub

Feed

Fig. 1b Tubular centrifuge

3. Biopharmaceutical Process Recombinant DNA process has found popular applications in protein manufacturing. In short, 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, namely yeast, mammalian cells, and bacteria. Fig 2a, 2b and 2c shows respectively a schematic of a yeast cell, an animal cell that resembles closely a mammalian cell, and a bacteria cell.

Fig. 2a Yeast cell.

Fig. 2b Animal cell.

Fig. 2c Bacteria cell.

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. This is illustrated in Fig. 3. 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 maximal turbidity requirement. Yeast cells are typically 8-10 microns in size, see Fig. 2a. Both disk stack and tubular centrifuges can be used for carrying out separation. Fig. 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. The end result is that over 98% of feed yeast solids are

recovered. 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. 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]. It is critical to ensure the centrifuge-filter work together as a system.
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. 3 Host cell platform and subsequent separation processes

100% feed 90.6% recovery 99.85% total recovery

Separation stage

9.4%
Product Clarification stage

98.4%x9.4% =9.25%

1.56%x9.4%=0.15% 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. The resultant suspension is reslurried and washed, 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). A single-stage and double-stage washing are shown in Fig. 4b. The number of stages employed depends on the required product purity [1].

1 m E. 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 m

"Single Wash"

"Double Wash"

Cell Debris

Fig. 4b shows processing of E. coli and inclusion bodies bearing protein.

Separation by centrifugation is used extensively in these three host-cell platforms, which is a major application in production of antibiotics and intermediate drug substances for the biopharmaceutical market. 4. Other Important Biotech Processes Two examples are used to illustrate the many important biotech processes that are being widely used today. 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. 5. First the bacteria, such as E. coli bacteria, are genetically modified to express a specific protein under fermentation. The bacteria go through a fermentation process where temperature, agitation rate, physiological conditions are closely monitored to ensure the process is well controlled and optimal protein is released in solution. During harvesting, the bacteria cells are lysed and the protein in solids is recovered by centrifugation followed by depth filtration. The pre-insulin is treated with buffer liquid to activate to tertiary form. An enzyme trypsin is used for cleavage. The product solution is purified with chromatography column. The protein is further concentrated by crystallization, washing and centrifugation. This process is repeated several times until the desired purity is reached. The resulting product is frozen to maintain freshness prior to finish processing.

Innoculation with genetically modified E. coli

Fermentation

Harvest - 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. 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. Working with the nervous system, they co-ordinate all essential functions of the body. A common hormone, such as insulin, regulates the blood glucose of the body. Separation by centrifugation has been widely used to process insulin for use by diabetic patients. Fig. 6 shows a generic process of separating and related processing of hormones. Micro-organism, substrate and air are typically ingredients to the fermenter. After fermentation, the broth is fed to a nozzle disk where the liquid phase is removed as waste stream, and the separated biomass concentrate discharges continuously through the nozzles. Subsequently the biomass after resuspended in wash/buffer liquid is sent to a homogenizer for cell disruption releasing the solid inclusion bodies. The cellular liquid, with cell debris typically in the submicron sizes, is classified by another nozzle disk in the centrate with moderate G-force. As shown in Fig. 6, the concentrate with solid inclusion bodies containing contaminants is reslurried and separated by centrifugation sequentially in two stages. The inclusion bodies free from contaminants are sent downstream for further processing.
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 - Hormones processing flowsheet

5. Throughput and Sizing of Centrifuges The maximum and minimum feed rates of respective tubular and disk centrifuges are shown in Fig. 7. The feed rate varies approximately as the third power of the bowl diameter from the hydraulic capacity consideration. This is much lower from the process viewpoint and the specific feed rate depends on the requirements of a given process.

1000

Disk

Rate, L/min

100

Max Rate
10

Min Rate

1 100

Tubular

1000

Diameter, mm

Fig. 7 Disk and tubular size versus feed rate, with maximum rate limited by the hydraulic capacity.

6. Dimensionless Le Number A new dimensionless Leung number (hereafter abbreviated as Le) has been developed for spintube, disk, tubular and decanter centrifuges as used for separation and clarification sizing. The Le number was used successfully [3] in many applications for fine particle sedimentation (0.1 100 m). Le is directly correlated with the cut size the maximum size in the supernatant, or the minimum size in sediment. 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.), yet this provides a concept whereby one can quantitatively size or scale-up centrifuges. Most importantly, the approach presented herein provides a unified approach for sizing different types of centrifuges. Le depends on centrifuge design geometry, and properties of suspended particles (size distribution and density) and liquid (density and viscosity). The Le number has been developed for batch sedimentation (spin tube) and continuous-feed centrifuges (disk, chamber bowl, tubular and decanter centrifuges). 7. Sizing for Disk-Stack Centrifuge The dimensionless Le number for a disk stack centrifuge incorporates the feed rate Q, the angle of the disk stack with respect to the vertical , the inner disk radius R1, the outer disk radius R2, the slant length of disk L, the projected area parallel to the axis of the machine Lp, the number of disks n, the liquid viscosity , the density difference , and the characteristics particle size xo. The geometry is captured in Fig. 8. The Le number for the disk centrifuge [1] is given by,

Le =

Q L p R' xo
1/ 2

R 2 + R2 R1 + R12 R' = 2 3 L p = nL cos

R1 1 + 0.5536 R2 1 R1

(1a, 2, 3)

In the above, the effective radius R is related to R1 and R2 as expected. Eq. 1a is very similar to that of the decanter centrifuge except that R replaces the pool radius Rp for the case of a decanter. R can be

approximated by a linear relationship. It can also be shown that Le can be expressed in a more compact form as follows:

Le =

tan 3Q n [ ] 3 3 s L R2 R1

xo

(1b)

It is to be noted that both Eq. 1a and 1b yields identical result in calculation of the Le number.
Axis

Lcos

L L

R1

Total Projected Area Lp= n Lcos

R2 G

Fig. 8 - Projected area of disk centrifuge

Example - Disk Centrifuge Centrifuge Bowl Diameter D=400 mm Disk Outer Diameter 2R2=300 mm Disk Inner Diameter 2R1=177 mm Disk Angle =40o from vertical Density difference/density /=0.1 Viscosity =5 cP G = 12,000g (7270 rpm); G=6000 (5140 rpm) n=100 disks Overall Efficiency = 70%

100

Feed Rate, L/min

/=0.1 =5 cP n =100 disks

10 6000g 12000g

1 0.1 1.0 10.0

Separated Cell Size, microns

Fig. 9 Performance of a disk for a feed suspension with 5 cP.

The results are depicted in Fig. 9 in which the feed rate depends on the size of particles that need to be separated.

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

Fig. 10 Simulation used in various stages from lab discovery to production.

%Cumulative Undersize 100% 80% 60% 40% 20% 0% 0 10 20 30 40 50
100.0% 99.5% 99.0% 98.5% 98.0% 97.5% 97.0% 0 20 40 60 80 100 400-mm dia. disk 6000g 12000g

%Solids Recovery

x, microns

Q, L/min

Fig. 11 Simulation of separation of CHO cells using a disk-stack centrifuge. (a) Feed particle size

distribution, (b) solids recovery by centrifugation under 2 Gs for a 400-mm disk bowl.
100.0%

%Solid Recovery

99.5% 99.0% 98.5% 98.0% 97.5% 97.0% 0 10 20

10,000g 20,000g

Q, L/min

30

40

Fig. 12 Simulation of separation of CHO cells using a tubular centrifuge with diameter 457 mm, 610 bowl length and 95% efficiency. The density difference between cells and liquid is 10% and viscosity is 5 cP.

9. Conclusion Centrifugal separation is a robust means for separating high-value biotech intermediate products and final products. High-speed, gentle feed acceleration, and increased separation surface area offers the flexibility from separating small delicate cells, clarifying broth, concentrating valuable materials, to classifying submicron sized cell debris. References [1] Wallace Woon-Fong Leung, Centrifugal Separation in Biotechnology, Academic Press, UK, 2007. [2] M. Desai (ed), Downstream Processing of Proteins, Humana Press, Totowa, NJ 2000. [3] Wallace Woon-Fong Leung, Industrial Centrifugation Technology, McGraw-Hill, NY, 1998.

Acknowledgement The author wishes to acknowledge the Hong Kong Polytechnic University for their support.