Centrifugation Application Notes

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INTRODUCTION beckmancoulter.com

Beckman Coulter has been at the forefront of centrifuge innovation since we introduced the first commercial ultracentrifuge in 1947. Beckman Coulter General Purpose, High Performance and Ultracentrifuges provide systematic excellence through their superior quality, reproducibility, and reliable performance. Beckman Coulter is a name laboratories around the world have grown to trust.

“Perhaps the greatest reward is the knowledge that Beckman products have contributed and are contributing to the progress of mankind.” Arnold O. Beckman, PhD


Proteins & Centrifugation Avanti JXN Series - Complete solution for protein purification workflow.


Automating a Linear Density Gradient for Purification of a Protein: Ligand Complex. p.4 Enhancing Vaccine Development and Production - Efficient Centrifuge Systems for Vaccine Development and Production. p.9

Exosomes, Microparticles and More Complete Solutions for Your Specific Exosom Workflow – from Preparation to Analysis. p.14 High-Performance Exosome Purification and Characterization via Density Gradient Ultracentrifugation and Dynamic Light Scattering. p.18

Viruses, Lentivirus, Virus Particles... Lentiviral Vector Preparation.


Lentiviral Vector Production.


Nanoparticles and Nano-Structures Density Gradient Separation of Gold Nanorods.

p.27 High-Performance Automation and Centrifugation Preparation of Carbon Nanotubes for Analytical Ultracentrifugation p.30 Particle Characterization and Centrifuge - Nanotoxicity Application Note p.37

Cell Separation by Centrifugation Optimizing Cell Separation with Beckman Coulter’s Centrifugal Elutriation System.


Developing Elutriation Protocols


Cell Separation Beckman Coulter Cell Culture Flask Adapters.


Centrifugation Techniques & Special Labware Principles of Continuous Flow Centrifugation


Automating a Linear Density Gradient for Purification of a Protein: Ligand Complex


The Avanti One-Liter Bottle - Your capacity will never be the same.


g-Max: Added Capabilities to Beckman Coulter’s Versatile Ultracentrifuge Line


Get Control in GMP Environments - Centrifugation that supports compliance and eliminates hassle. p.88 High-Performance Automation and Centrifugation Preparation of Carbon Nanotubes for Analytical Ultracentrifugation p.30 Optima XPN and Avanti JXN Series Centrifuges Designed to Handle the Multi-User Lab with Ease. p.90 Optimizing Centrifugal Separations Sample Loading p.93 Predicting Protein Separation in Rate Zonal Centrifugation Using the ESP™ Rate Zonal Run Simulation From the Optima Expert™ Software p.96 Using k-Factor to Compare Rotor Efficiency p.100 Biopharma Solutions Enhancing Vaccine Development and Production - Efficient Centrifuge Systems for Vaccine Development and Production. p.9 Principles of Continuous Flow Centrifugation. p.62 The Avanti One-Liter Bottle - Your capacity will never be the same. p.80 Get Control in GMP Environments - Centrifugation that supports compliance and eliminates hassle. p.88

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© 2016 Beckman Coulter, Inc.


Avanti JXN Series Complete solution for protein purification workflow.

Purified proteins are required for many proteomics applications such as X-ray crystallography, NMR, mass spec, and in vitro biochemical assays. Proteins can be isolated from tissue or, more often, by their overexpression in an organism, like bacteria, yeast, or mammalian cells in culture. Characteristics unique to each protein, like amino acid composition, size, shape, isoelectric point, and solubility are used to develop unique strategies for isolation of the protein of interest. The objective is to isolate the largest amount of functional protein of interest with the least amount of other contaminants present. Centrifugation is an important, and often the first step, in any protein purification protocol. The Avanti JXN Series has an array of rotors available to help with any stage of protein purification. The wide selection of rotors with different combinations of volume and g-force makes it a versatile instrument capable of meeting the needs of any proteomics lab. It is an excellent solution for various proteomics applications such as cell pelleting, protein precipitation, subcellular and membrane isolation, density gradients, and protein concentration.

Typical protein purification workflow for overexpressed protein:

Step 1 Harvest and Lyse

Step 2 Lysate Clarification

Step 3 Crude Purification/Precipitation

Step 4 Chromatography/Secondary Purification

Step 5 Density Gradients/Tertiary Purification

Step 6 Concentration/Buffer Exchange

Avanti JXN 26

1. Harvesting: Pelleting the sample from which the protein of interest has to be purified (for example bacterial cells, insect cells, mammalian cells or tissues, etc.) is the first step in protein purification. This step typically requires low-speed but high-volume rotors. The Avanti JXN Series has fixed- angle JLA-8.1000/JLA-9.1000/JLA-10.500 for volumes up to 6 liters which can help with this. The JCF-Z continuous flow rotor can be used for even higher volumes of pelleting required in bioprocessing setup. 2. Lysate clarification: After the cells have been pelleted and lysed, the second important step in protein purification is efficient separation of protein from non-protein components and cellular debris. High-speed clarification is used for this step. The rotor required for this step is a high-speed, low- volume rotor. The Avanti JXN Series has various rotors like JA-25.15, JA-25.50 and JA-30.50 Ti which can go above 100,000 x g needed for this step. 3. Crude purification: Precipitation steps using precipitants like ammonium sulfate, polyethylene glycol, etc., help in recovering the bulk protein from a crude extract and are used as primary purification methods. The separation of these precipitates requires medium speeds and low- to medium-volume rotors. The Avanti JXN Series has rotors like JA-17, JA-18, JA-20 and JLA-16.250 which provide the right combination of speed and capacity for this step.

JLA-10.500 Rotor

4. Chromatography columns: Secondary purification of target protein is usually based on various chromatographic principles such as affinity, ion- exchange, hydrophobic, size exclusion, etc. Microcentrifuge spin columns and 96-well kits help purify small quantities of recombinant proteins in a fast and simple manner. These columns are perfect for prescreening ideal chromatographic conditions as well as high-throughput proteomics. The JS-5.3 and JS-5.9 rotors can be used for 96-well format protein prep columns. 5. Density gradient: Density gradients are used often to isolate subcellular organelles like mitochondria, plasma membranes, etc., from which a specific protein needs to be isolated. Density gradients are also used as a tertiary purification step for protein prep. The JS-24.15 and JS-24.38 rotors can be used for both rate zonal, as well as isopycnic density gradients. 6. Buffer exchange and concentration: For any proteomics assay, the right buffer and concentration of the pure protein are a must. There are various filter-based columns available which are used for buffer exchange as well as protein concentration. The JS-4.0, JS-4.3 and JS-5.3 have multiple adapters available for these concentrators.



JLA-8.1000 (6 x 1L) JLA-9.1000 (4 x 1L)

JLA-10.500 (6 x 500mL)


JA-17 (14 x 50mL) JA-18 (10 x 100mL) JA-20 (8 x 50mL) JA-14.50 (16 x 50mL conical) JLA-16.250 (6 x 250mL) Crude Purification and Protein Precipitation



JA-30.50 (8 x 50mL) JA-25.50 (8 x 50mL) JA-25.15 (24 x 15mL) Lysate Clarification


Density Gradients

JS-5.3 JS-5.9 JA-18.1 (24 x 1.8mL) Microfuge Columns and 96-Well Kits

JS-24.38 (6 x 38.5mL) JS-24.15 (6 x 15mL)

Note: Maximum speeds and capacity might vary based on adapter, labware and instrument model. Please refer to applicable rotor/instrument manual for exact details.


42K x g

109K x g

6.6K x g

Relative Centrifugal Force

Beckman Coulter, Avanti, and the stylized logo are trademarks of Beckman Coulter, Inc. and are registered with the USPTO.

For Beckman Coulter’s worldwide office locations and phone numbers, please visit “Contact Us” at www.beckmancoulter.com CENT-IAPP03.14-A © 2014 Beckman Coulter, Inc. PRINTED IN U.S.A.

Automating a Linear Density Gradient for Purification of a Protein:Ligand Complex

Introduction Proteins have a variety of cellular functions, structures, and mechanisms of action. Routinely, proteins bind other biomolecules, or ligands, in order to complete a task. Researchers gain valuable knowledge on how proteins work in a cellular environment by purification of proteins bound to their appropriate substrate. Typically, protein:ligand complexes are further analyzed by cryo-EM, NMR, and/or x-ray crystallography for structural information. However, purification of protein:ligand complexes remains challenging due to the lack of robust, reproducible separation techniques. Linear (also known as continuous), rate-zonal density gradients are formed in several ways, but the process always starts with layering a discontinuous (also known as step) gradient first. In the most popular technique, an aliquot of a less dense solution is first pipetted into a centrifuge tube and successively denser solutions are introduced to the bottom of the tube by a long syringe as to not disturb the previous layer, leaving a sharp interface between the different density layers. Another approach layers decreasingly dense solutions gently on top of a more dense solution. In order to generate a continuous gradient from a discontinuous gradient, 3 main techniques are used: 1 ) incubating at 4˚ to 8˚C for 1 6 hours or overnight; 2) spinning in a centrifuge for a set speed and duration; or 3) using a commercial gradient maker that spins the tube at a specific angle for a set speed and duration. In all techniques, the solution diffuses such that a gradual increase in density is produced from the top to the bottom of the tube. Both layering techniques are tedious and time-consuming and are often not reproducible among researchers, requiring practice and a whole lot of patience to generate strong interfaces between densities.

Here, we present a simplified, fourth method for gradient preparations. To eliminate user variability, Beckman Coulter’s Biomek 4000 Workstation provides consistent and reproducible results in layering discontinuous density gradients (see Application Notes DS- 1 8638A, IB- 1 8433A, and CENT-447APP08. 1 4-A). The Biomek 4000Workstation offers ease of use and outstanding precision in liquid handling. Additionally, the Biomek 4000 Workstation is easily equipped with a cooling, static peltier Automated Labware Position (ALP) that provides an incubation platform on-deck for linear gradient formation overnight without the need for a refrigerator or cold room. After centrifugation, gradients are typically fractionated by puncturing a hole at the bottom of a tube and collecting a specific number of drops per aliquot, or by manually pipetting from the meniscus. There are commercially available systems that are capable of automatically fractionating gradients, but these systems are typically expensive and not compatible with all tube types. Since the Biomek 4000 Workstation offers solutions to accurate liquid handling, the instrument was tested to determine whether or not it was also suitable to fractionate a sucrose gradient. The ATPase of phi29 and DNA ligand Double stranded (ds) DNA viruses package their genomic dsDNA into a pre-formed protein shell, called procapsid, during maturation. 1 ,2 This entropically unfavorable process is accomplished by a nanomotor which uses ATP as an energy source. 3-6 Bacteriophage phi29 is an extensively

a gradient was made either manually by layering 5% solution on top of 20% solution with a pipette, or automatically using the Biomek 4000 Workstation. Solutions were incubated at 4˚C overnight in a refrigerator or on the pre-cooled peltier ALP of the Biomek 4000 Workstation. In the Biomek 4000 Workstation method, 1 5 mL conical tubes containing either 5% or 20% sucrose solution in a holding rack were placed on-deck along with a pre- chilled static peltier holding up to six, 1 3.2 mL Beckman Coulter polypropylene centrifuge tubes (P/N 33 1 372). The Biomek 4000 Workstation was first prompted to add 5.5 mL of 5% solution to the bottom of the centrifuge tube using a P 1 000SL tool in 9 1 6.6 µl aliquots. Next, the Biomek Workstation was asked to add 5.5 mL of 20% sucrose underneath the 5% solution at the bottom of the tube. Beckman Coulter Span-8 P 1 000 pre-sterile tips (P/N B0 11 24) are long and cylindrical, causing minimal damage to the sucrose interface during this step. The peltier ALP was then set to hold temperature overnight, allowing the sucrose to generate a linear gradient. Gp 1 6 was re-engineered in 2009 to contain a fluorescent arm, enhanced green fluorescent protein (eGFP), which did not affect the protein’s folding or activity 18 but provided an easily identifiable marker for in vitro and single molecule assays. Additionally, it has been shown that eGFP-gp 1 6 binds non-specifically to dsDNA, and fluorescent tags—such as cy3—are easily conjugable for further identification. Ultrapurified 40 bp cy3-conjugated dsDNA was purchased from Integrated DNA Technologies (IDT) and resuspended in DEPC-H 2 O. γ -S-ATP was purchased from Roche Diagnostics. Samples were prepared by mixing eGFP-gp 1 6, cy3-dsDNA, and γ -S-ATP in final concentrations of 1 µM, 250 nM, and 1 .25 mM, respectively. Samples were subsequently gently added to the top of the gradient as not to disrupt the formed gradient, balanced, and then placed in a Beckman Coulter Optima XPN Ultracentrifuge and spun for 7 hours at 40,000 rpm at 4˚C. Samples were then either fractionated directly from the top of the tube using a pipette set at 250 µl into an opaque microplate, or using the Biomek 4000 Workstation

investigated phage due to its simplistic design, comprised of an ATPase packaging enzyme—deemed gp 1 6— a connector portal protein (gp 1 0), and packaging RNA (pRNA). Guo et al. 7 first proposed that the mechanism by which dsDNA is packaged into the procapsid resembles the action of other AAA+ (ATPases Associated with Diverse Cellular Activities) proteins using ATP as energy. Recently, it has been determined that gp 1 6 utilizes a sequential action mechanism with dsDNA and ATP to accomplish packaging. 8 Furthermore, it was validated that gp 1 6 exists as a hexamer—similar to other AAA+ proteins—on the viral packaging motor 9 and that cooperativity exists among the ATPase and ATP, generating a high affinity state for dsDNA after binding a non- hydrolyzable ATP substrate, γ -S-ATP. 1 0 A revolution mechanism for DNA packaging was proposed 1 0 and subsequently substantiated. 11 - 1 3 This motor is of particular interest as it has been extensively shown to be utilized in several applications of nanotechnology. 1 4- 1 7 In the study of this motor, it was critical to research how cer tain components interact with others in cellular conditions to better understand the phage’s mechanism and biology. One such assay involved isolating the gp 1 6/dsDNA complex by rate-zonal centrifugation. In a previous experiment, published in Nucleic Acids Research , 8 complexes were purified in a 5–20% sucrose gradient in a Beckman Coulter SW-55 rotor at 35,000 rpm and subjected to further kinetic analysis to determine rate of ATP hydrolysis. Additional experiments were also performed on the purified complex, gaining valuable information that allowed researchers to elucidate the mechanism of DNA packaging in phi29 phage maturation. In the example to follow, purification of the gp 1 6/dsDNA complex will be assayed by mimicking the previous published experiment but in a larger rotor and using an automated layering and fractionating method with the Biomek 4000 Workstation. Methods Sucrose was diluted at 5% and 20% (w/v) using a dilution buffer (50 mM NaCl, 25 mM Tris pH 8.0, 2% glycerol, 0.0 1 % Tween-20, 2 mM MgCl2, 0. 1 5 mM γ -S-ATP) and

The same data was plotted in Figure 2 but compared the 2 methods in individual graphs for each signal. The overall plot shape is very similar, suggesting that the automation method is robust and can replace the storied manual method. Again, it appears the resolution is greater for the Biomek 4000 Workstation method (red line), which can be attributed to better pipetting techniques and less physical movement after layering. The standard deviation is also comparable between both techniques, signifying reproducibility.

to liquid level track the meniscus and automatically transfer fractions of the same volume to the microplate sitting on the deck. In the Biomek 4000 Workstation method, a rack holding the spun centrifuge tubes was placed on-deck along with a black-bottom microplate. P 1 000 tips were used to fractionate 250 µl from the very top of the meniscus and added directly to consecutive wells of the 96-well microplate. Based on user-defined parameters for the geometry of the tube, the Biomek 4000 Workstation is capable of precisely tracking the liquid level of the tube as fractions are removed. The fractions were subsequently analyzed by a Molecular Devices SpectraMax ® i3 Multi-Mode Detection Platform at both GFP and cy3 wavelengths (488 nm and 540 nm, respectively) and data was exported to a Microsoft ® Excel ® file for analysis. The data was then transferred into Origin Pro v9.0 for plotting. Results The data from the Molecular Devices SpectraMax ® i3 microplate reader was plotted and overlaid for both wavelengths and methods in Figure 1 . The direction of sedimentation is from left to right on the graphs as fractions were taken from the top of the tube. The black cy3 line represents cy3-DNA and the red GFP line denotes eGFP-gp 1 6. In Figure 1 a, the manual layering and manual fractionation technique was able to resolve free protein and DNA (fraction 2–6) from the protein-DNA complex (fractions 1 7–32). Using the Biomek 4000 Workstation for both layering and fractionation (Figure 1 b), again the free protein and free DNA (fractions 3–6) was separated from the gp 1 6/DNA complex (fractions 1 7–27). However, in the Biomek technique, two distinct peaks exist in the complex region, especially evident looking at the cy3 signal. Fractions 1 6–2 1 are clearly an independent peak from fractions 24–27, suggesting that 2 separate conformations or oligomeric state of the protein exist bound to DNA. In fact, this phenomenon has previously been discussed in a recent Virology paper 10 and is a profound finding that provides relevant information to the packaging mechanism. It is believed that gp 1 6 first binds to dsDNA as a dimer and then assembles into a hexamer to complete the packaging function. It is hypothesized that fractions 1 6–2 1 represent the dimeric state, whereas fractions 24–27 consist of the hexamer.

Manual Layering/Fractionation


Biomek 4000 Layering/Fractionation


Fig. 1a and 1b. Manual versus Biomek 4000 Workstation preparation of a 5–20% linear sucrose gradient.

advantages; however, often times, the method requires large dilution factors, protein incompatibilities, and low resolution. Density gradient centrifugation allows the user to quickly modify parameters, offering efficient separations, and is governed by the laws of thermodynamics. Here, we described an automated method for purifying protein:ligand complexes by rate-zonal centrifugation. It is important to note that this method is amenable to almost all proteins, after optimization of spin time, speed, and gradient conditions. The protocol offers significant advantages over manual preparations as the following outlines. Reproducibility Human error is common in an array of scientific experiments and compounded in difficult tasks, such as layering and fractionating a density gradient by different users in a lab group. Layering a gradient manually, using either the needle and syringe method or by pipette, requires a steady hand and patience. By automating the process, the Biomek 4000 Workstation provides a distinct interface and consistent fraction every time. No more worrying about adding samples to the same well twice or manually counting irregular drops out of the bottom of a centrifuge tube. Additionally, the automated approach for layering includes a chilled peltier step that reduces the jostling of tubes that occur when moving gradients to the cold room or refrigerator. This advantage is important as this movement often causes the interface to become turbid. Ease of Use It is diligent, tedious work to manually layer and fractionate a density gradient. Let the Biomek do the brunt of the work by just pressing a button. Walk-Away Approach The automated methods took just about the same amount of time to layer and fractionate a gradient than doing so manually. In fact, layering 2 gradients took less than 20 minutes and fractionating 2 tubes took less than 50 minutes. The difference is that a researcher can simply walk-away from the machine and perform other work during these methods. Lastly, the Biomek Automated Liquid Handling

GFP Signal


Cy3 Signal


Fig. 2a and 2b. Overlaid images of different preparation techniques for eGFP-gp16 (a) and cy3-dsDNA (b).

Discussion Purification of protein:ligand complexes are important to understanding biological processes. Often times, purified complexes are used in downstream analyses such as high-resolution imaging, sequencing, or crystallography for discovery of protein-based therapeutics. In the previous example, the purified gp 1 6/dsDNA complex revealed the mechanism of a unique biological function known as DNA packaging in a bacteriophage. Lack of a robust, reproducible method for protein:ligand purification has hindered research for many years. Chromatography has several

product line is capable of being integrated with several types of downstream analysis equipment, including microplate readers such as Molecular Devices SpectraMax ® i3 Multi-Mode Detection Platform. This allows researchers to focus on more important matters, such as data analysis and grant writing. Author The author would like to thank Dr. Peixuan Guo, Endowed Chair in Nanobiotechnology, and Zhengyi Zhao at the University of Kentucky College of Pharmacy for kindly providing material and direction. References 1. Guo P X and Lee T J. Viral nanomotors for packaging of dsDNA and dsRNA. Mol. Microbiol. 64; 886–903: (2007). 2. Rao V B and Feiss M. The bacteriophage DNA packaging motor. Annu. Rev. Genet. 42; 647–681: (2008). 3. Guo P, Peterson C and Anderson D. Prohead and DNA-gp3-dependent ATPase activity of the DNA packaging protein gp16 of bacteriophage f29. J. Mol. Biol. 197; 229–236: (1987). 4. Chemla Y R, Aathavan K, Michaelis J, Grimes S, Jardine P J, Anderson D L and Bustamante C. Mechanism of force generation of a viral DNA packaging motor. Cell. 122, 683–692: (2005). 5. Hwang Y, Catalano C E and Feiss M. Kinetic and mutational dissection of the two ATPase activities of terminase, the DNA packaging enzyme of bacteriophage lambda. Biochemistry. 35; 2796–2803: (1996). 6. Sabanayagam C R, Oram M, Lakowicz J R and Black L W. Viral DNA packaging studied by fluorescence correlation spectroscopy. Biophys. J. 93; L17–L19: (2007). Chad Schwartz, PhD, Application Scientist Beckman Coulter, Inc., Indianapolis, IN USA Acknowledgements

7. Guo P, Zhang C, Chen C, Trottier M and Garver K. Inter-RNA interaction of phage phi29 pRNA to form a hexameric complex for viral DNA transportation. Mol. Cell. 2; 149–155: (1998). 8. Schwartz C, Fang H, Huang L and Guo P. Sequential action of ATPase, ATP, ADP, Pi and dsDNA in procapsid-free system to enlighten the mechanism in viral dsDNA packaging. Nucl. Acids Res . 40(6); 2577–2586: (2012). 9. Schwartz C, De Donatis G M, Fang H and Guo P. The ATPase of the phi29 DNA packaging motor is a member of the hexameric AAA+ superfamily. Virology. 443; 20–27: (2013). 10. Schwartz C, De Donatis G M, Zhang H, Fang H and Guo P. Revolution rather than rotation of AAA+ hexameric phi29 nanomotor for viral dsDNA packaging without coiling. Virology. 443; 28–39: (2013). 11. Zhao Z, Khisamutdinov E, Schwartz C and Guo P. Mechanism of one-way traffic of hexameric phi29 DNA packaging motor with four electropositive relaying layers facilitating antiparallel revolution. ACS Nano. 7(5); 4082–4092: (2013). 12. De-Donatis G M, Zhao Z, Wang S, Huang L P, Schwartz C, Tsodikov O, Zhang H, Haque F and Guo P. Finding of widespread viral and bacterial revolution dsDNA translocation motors distinct from rotation motors by channel chirality and size. Cell & Bioscience. 4(30); eCollection 2014: (2014). 13. Guo P, Schwartz C, Haak J and Zhao Z. Discovery of a new motion mechanism of biomotors similar to the earth revolving around the sun without rotation. Virology. 446(0); 133–143: (2013). 14. Schwartz C and Guo P. Ultrastable pRNA hexameric ring gearing hexameric phi29 DNA-packaging motor by revolving without rotating and coiling. Curr Opin Biotechnol. 24(4); 581–590: (2013). 15. Wendell D, Jing P, Geng J, Subramaniam V, Lee T J, Montemagno C and Guo P. Translocation of double stranded DNA through membrane adapted phi29 motor protein nanopore. Nature Nanotechnology. 4(11); 765–72: (2009). 16. Shu D, Shu Y, Haque F, Abdelmawla S and Guo P. Thermodynamically stable RNA three-way junction for constructing multifunctional nanoparticles for delivery of therapeutics. Nature Nanotechnology. 6(10); 658–67: (2011). 17. Khisamutdinov E, Jasinski D and Guo P. RNA as a boiling-resistant anionic polymer material to build robust structures with defined shape and stoichiometry. ACS Nano . 8(5); 4771–81; (2014). 18. Lee T J, Zhang H, Chang C, Savran, C and Guo P. Engineering of the fuorescent-energy-conversion arm of phi29 DNA packaging motor for single- molecule studies. Small . 5(21); 2453–9: (2009).

© 2014 Beckman Coulter, Inc. All rights reserved. Beckman Coulter, the stylized logo, Optima, and Biomek are trademarks of Beckman Coulter, Inc. and are registered with the USPTO. All other trademarks are the property of their respective owners.

For Beckman Coulter’s worldwide office locations and phone numbers, please visit “Contact Us” at www.beckmancoulter.com CENT-686APP11.14-A

Enhancing Vaccine Development and Production Efficient Centrifuge Systems for Vaccine Development and Production

Avanti JXN-26

The increasing ability of vaccines to impact quality of life is fueling demand for ever-better vaccine products, from new therapeutics for cancer and seasonal influenza, to aver ting threats of bioterrorism and emerging diseases. Beckman Coulter, Inc. offers a full continuum of centrifuge-related products to enhance vaccine development and production. From initial cell clarification to inactivated viral purification or splitting, Beckman Coulter, Inc. has the perfect centrifuge for your laboratory. Drawing on more than 6 decades of expertise, our full line of centrifugation products delivers quality separations in a short amount of time. Whether you are focused on viral, bacterial, or cell-based vaccines, our centrifuge technology speeds your processes, improves your yield, and allows you to achieve demanding development and production goals. In addition to centrifuges, Beckman Coulter offers complementary solutions for the vaccine environment, including automated cell viability analyzers, flow cytometry solutions, and protein characterization systems. Consider us your partner in creating and delivering high-quality vaccine products from beginning to end.

Optima XPN 100

Performance Our instruments, rotors, tubes, bottles and adapters are designed to work together as a comprehensive system. A modular approach satisfies cross-contamination issues by providing lot-to-lot comparability, which enables faster time-to-market, reduces pharmaceutical development costs and ensures optimum dose efficacy, all with the reliability and long-term commitment you’ve come to expect from Beckman Coulter. Our instruments and rotors are designed with the consumer in mind as researchers want faster speeds, better controlled temperature and vacuum systems, larger capacities, and increased ease of use for quicker, more reliable, higher throughput, and reproducible separations.

Compliance The traceability and electronic run records of the Avanti JXN and Optima XPN software support 2 1 CFR Part 11 compliant and GLP/GMP environments. The embedded software tracks a long list of run parameters, usage histories, rotor cycles and more. Please see application note CENT-5 1 2APP09. 1 4-A for a more detailed look on how Avanti JXN/Optima XPN centrifuges support 2 1 CFR Part 11 compliance. Flexibility As a global leader in centrifugation, Beckman Coulter delivers innovative centrifuge solutions that enhance the productivity, efficiency and compliance of vaccine facilities worldwide. Beckman Coulter centrifuge solutions meet your facility’s specific needs throughout the vaccine process, from research and development to production and validation. Beckman’s product line offers an array of optimized centrifuge and rotor systems that allows researchers to select the best fit for their vaccine-related needs. An integrated library of centrifuge instruments, rotors, tubes and accessories enables a high degree of customization and versatility in the use of your Beckman Coulter systems, ensuring efficiency, productivity and safety at each step in the process.

Ease of Use Beckman Coulter’s intuitive, user-friendly instrument software makes it easy to program centrifuge runs from within the laboratory or even remotely. The Optima XPN/Avanti JXN software provides large digital displays, a comprehensive set of help options, user programmed workflows, and printable run reports, all in a touch- screen format. The software systems for both the Optima XPN and Avanti JXN are consistent with each other, making it easy for customers to go back and forth between instruments. Furthermore, our ergonomic system design enhances operator safety, comfort and productivity, keeping researchers happy and healthy. Clean Room Suitability Beckman Coulter’s BioSafe * centrifuge systems are equipped with a pharmaceutical-grade sterilizing filter system which alleviates concerns over unwanted particles escaping into the vaccine production environment. Our pharmaceutical-grade sterilizing filters are manufactured in a controlled environment and undergo extensive integrity testing. The filter components have met the specifications for biological tests listed in the current revision of the United States Pharmacopeia (USP) for Class VI- 1 2 1 ˚C plastics and bacterial retention in conformance with the applicable requirements of the FDA Guideline Sterile Drug Products Produced by Aseptic Processing—Current Good Manufacturing Practice. Furthermore, the filter system undergoes tests of cleanliness in accordance with Title 2 1 of the U.S. Code of Federal Regulations (CFR) parts 2 11 .72 and 2 1 0.3 (b) (6), oxidizable substances, pH, and pyrogens.

Workflow screen capture from Optima XPN.

JCF-Z reorienting gradient rotor for high-throughput virus and cell isolation.

Exemplary Service Our reputation for excellence, quality and customer service is widely recognized—and is the reason biological facilities around the world look to us for answers to their centrifugation challenges. Our centrifuge technology for vaccine development and production is designed to meet the exacting standards required of the biologics environment. Our technology is backed by Beckman Coulter’s ISO 900 1 Certified Field Service Organization. Comprised of factory trained and cer tified engineers, our service operation has a reputation that is unmatched in the industry. In addition, we offer Installation Qualification and Operational Qualification (IQ/OQ), and our knowledgeable customer service representatives handle your requests promptly to keep your facility running efficiently.

A Powerful Lineup Beckman Coulter manufactures and supports a long list of high-performance ultracentrifuges; Table 1 is not a comprehensive list, but highlights 4 of Beckman Coulter’s most popular centrifuges for vaccine development and accompanying commonly-used rotors in vaccine production. Many rotors are interchangeable among centrifuge series’. Additionally, Beckman Coulter offers HarvestLine single-use centrifuge bottle liners that provide a significant improvement in the centrifugation of biological material. HarvestLine System liners eliminate time-consuming manual scraping of harvested solids from labware and enhance operator biosafety. HarvestLine liners can be sterilized (gamma irradiation) and frozen to facilitate your production process.

Table 1. Beckman Coulter Centrifuges and Accompanying Rotors for Vaccine Production.

Centrifuge Series



Vaccine-Related Applications

Max. Speed: 32,000 rpm Capacity: 1,675 mL Max. Flow Rate: 50 mL/min Max. Speed: 32,000 rpm Capacity: 231 mL Max. Speed: 32,000 rpm Capacity: 430 mL Flow Rate: 150 mL/min Max. Speed: 45,000 rpm Capacity: 564 mL

Efficient isolation of subcellular particles and antigens

Ti-15 Zonal

SW-32 Ti

Rate-zonal separation of particles

Optima XPN

High-throughput isolation of large viruses and bacteria

CF-32 Ti Continuous Flow

Type 45 Ti Fixed Angle

Rate-zonal separation of particles

Large-volume processing of cells, viruses, and precipitates Large-volume processing of cells, viruses, and precipitates Processing of small or large viruses, bacteria, and/or whole cells High-throughput isolation and concentration of viruses and bacteria

JLA-8.1000 FIxed-Angle Max. Speed: 5,000 rpm Capacity: 6,000 mL

JLA-9.1000 Fixed-Angle Max. Speed: 9,000 rpm Capacity: 4,000 mL

Avanti JXN-26

JCF-Z Reorienting Gradient Max. Speed: 20,000 rpm Capacity: 1,750 mL

Max. Speed: 20,000 rpm Capacity: 1,900 mL Max. Speed: 5,000 rpm Capacity: 9,000 mL Max. Speed: 4,200 rpm Capacity: 6,000 mL

JCF-Z Continuous Flow Standard Pellet Core

Avanti J-HC JS-5.0 Swinging Bucket

Cell harvesting with very high throughput

Rapid pelleting of large quantities of cells, cell debris, and precipitates

JS-4.2 Swinging Bucket


JE-5.0 Elutriator

Max. Speed: 5,000 rpm Gentle, yet powerful technique for

harvesting large populations of viable cells



1st Inactivation (Formalin)

Embryonated Chick Eggs




Zonal Centrifugation


Detergent Fragmentation



Chilling Overnight

2nd Inactivation (Formalin)

UF Diafiltration

Harvest (Pool Allantoic Fluid)

Sterile Filtration


Final Concentrate Product

Fig. 1. Typical workflow for egg-based influenza vaccine manufacturing. 1

Beckman Coulter centrifuges are compatible with both egg-based and cell-based vaccine development laboratories. Many potential advantages exist with cell-based vaccine development including the lack of dependence on eggs for global production. Additionally, the procedure is more reproducible, standardized, and potentially faster. 2,3 The downstream process of cell-based vaccine development is similar to egg-based manufacturing, requiring rounds of centrifugation, inactivation, and filtration. Upstream, the process star ts by culturing frozen, preserved cells in an incubator at 37˚C in small volumes. The culture is scaled-up and after reaching a specific density of cells, seed virus is added to the cell-containing bioreactor, such that the virus infects the cell lines and multiplies. The virus is subsequently harvested and clarified by centrifugation. Vaccine production requires sophisticated instruments performed by cutting-edge researchers. Beckman Coulter is a world leader in innovative centrifuges and here to help your laboratory select the right equipment to generate the most efficient workflow. Author Chad Schwartz, PhD, Senior Application Scientist Beckman Coulter, Inc., Life Sciences, Indianapolis, IN USA References 1. Matthews J T. Egg-Based Production of Influenza Vaccine: 30 Years of Commercial Experience. The Bridge. 17–24: (Fall 2006). 2. International Federation of Pharmaceutical Manufacturers & Associations. Cell-Culture Based Vaccine. 2014. http://www.ifpma.org/resources/influenza- vaccines/influenza-vaccines/cell-culture-based-vaccine.html 3. Dormitzer P R et al. Synthetic Generation of Influenza Viruses for Rapid Response to Pandemics. Sci Transl Med. 5; 185ra68: (2013). doi: 10.1126/ scitranslmed.3006368

Vaccine Development Workflow and Beckman Coulter

There are two main types of vaccine development: egg-based and cell-based. Figure 1 demonstrates a typical workflow for egg-based influenza vaccine manufacturing 1 . The upstream process starts with acquiring embryonated eggs from biosecure flocks, which are subsequently inoculated and incubated for several days to allow the virus to sufficiently multiply. The eggs are then candled to ensure there are no cracks and then chilled overnight. The next day, the allantoic fluid is harvested and clarified by the appropriate means of centrifugation, depending on the required volumes for production. The Avanti JXN-26 is a perfect complement to your workflow in this step, providing ample volume and more than enough speed. The virus is then inactivated by chemical means, filtered, and concentrated. Zonal centrifugation is now performed to separate the virus from other contaminating particles by size and shape in a sucrose solution. Here, the Optima XPN can be utilized, capable of speeds fast enough to ensure proper separation. The virus is recovered and split by detergent to solubilize the viral membrane, and clarified again by centrifugation to remove large contaminants. Researchers should again take advantage of the Optima XPN in this step. The subunit hemagglutinin and neuraminidase proteins are isolated, inactivated by a second round of formalin, and ultrapurified and concentrated by filtration. Most egg-based vaccine development protocols call for 3 to 5 centrifugation steps at varying speeds and with different volumes depending on production demands. The protocol lasts around 7 days with further validation, formulation, quality control, and lot release following the purification procedure.

*BioSafe and BioSafety are terms intended to describe the enhanced biocontainment features of our products. © 2015 Beckman Coulter, Inc. All rights reserved. Beckman Coulter, the stylized logo, Avanti, and Optima are trademarks of Beckman Coulter, Inc. and are registered with the USPTO. HarvestLine is a trademark of Beckman Coulter, Inc. All other trademarks are the property of their respective owners. For Beckman Coulter’s worldwide office locations and phone numbers, please visit “Contact Us” at www.beckmancoulter.com CENT-751APP01.15-A

Complete Solutions for Your Specific Exosome Workflow – from Preparation to Analysis

Beckman Coulter provides solutions customized for each step in your specific exosome research workflow – from sample preparation to sample analysis – and can also help improve processes and assist with related challenges.

Sample Preparation



Jurkat cells grown in log phase density - 1 x 1 0 6 cells/mL



2 Centrifugation to remove cells and debris

Allegra X- 1 5R

3 Ultracentrifugation to remove smaller cellular debris

Optima XPN

This chart provides a sample workflow and the solution for each step.

4 Ultracentrifugation to pellet exosomes

Optima XPN

5 Automated method for density gradient set-up

Biomek 4000Workstation

6 Density gradient ultracentrifugation to isolate exosome from co-purified proteins and other membrane vesicles

Optima XPN

7 Ultracentrifugation to exchange solvent from Iodixanol and sucrose to phosphate buffered saline

Optima MAX-XP

Step Sample Analysis


8 Size determination and analysis

DelsaMax CORE

Contact your Beckman Coulter Life Sciences Sales Representative for options to improve your processes and to address your specific exosomes workflow:

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Solutions for Exosome Sample Preparation

High Performance / High Capacity Centrifugation

The Avanti JXN High Performance Series of centrifuges including the (4L) Avanti JXN-30 and the (6L) Avanti JXN-26 offer many advantages: • Perfect for shared labs and GMP environment. Password protection allows appropriate security levels. • Flexibility with remote monitoring and


control from your Apple® iOS and Android™ device with MobileFuge. • Intuitive interface with large, user- friendly LCD screen. • Detailed run history and rotor tracking by serial number. • Backward compatibility with existing Avanti rotor library (please refer to the centrifuge User Guide for a list of compatible rotors).

Floor Ultracentrifugation For over 65 years, Beckman Coulter has been the global leader in ultracentrifugation. The Optima XPN Series is the premier ultracentrifuge, available in three configurations. The versatile rotor library which includes swinging bucket (SW), fixed angle (FA), near vertical (NVT), vertical (VT), zonal and continuous flow rotors allow for various types of applications that require a high RCF – from pelleting to density gradient fractionation and more. Among many other advantages, this series offers an intelligent user interface, networking and remote control capabilities, and is energy efficient.

Tabletop Ultracentrifugation The Optima MAX-XP tabletop ultracentrifuge features all the functionality and efficiency of our entry-level model, the Optima MAX-TL, plus offers additional advantages such as: • Maximum RCF of 1,019,000 x g with the MLA 130 rotor. • User ID/password protection. • Rotor tracking by serial number. • Remote monitoring and control option. • Choose from a Library of 17 rotors,* among many other features.

General Purpose Benchtop Centrifugation

Laboratory Automation The Biomek 4000 Laboratory Automation Workstation is truly intelligent automation. From its enhanced work surface with interchangeable tools to its flexible, icon– driven software, the Biomek 4000 system helps overcome human variables and provide a consistent reproducible, high-throughput method for gradient setup. An elegant solution to scale -up hurdles so you can conduct your research with ease and move your discovery process forward. For more information, check out www.Biomek4k.com The (120V) Allegra X-14 Series Benchtop centrifuges, available as refrigerated and constant temperature models, and the (208V) Allegra X-15R available as a refrigerated model, offer the optional ARIES (SX4750A) swinging bucket rotor which automatically detects and corrects an imbalance of up to 50 grams opposing loads, allowing you to complete the run without interruption. The Allegra X-14R and Allegra X-15R have a powerful refrigeration system. The centrifuge pre-cools from room temperature to 4°C in less than 4 minutes preserving temperature sensitive samples.

Solutions for Exosome Sample Analysis

Cell Viability and Cell Concentration The Vi-CELL** XR Cell Viability Analyzer automates the process of determining cell concentration and viability, eliminating variabilities inherent in manual sample preparation and counting. • Automatic and cost effective means to perform the trypan blue dye exclusion method.

• Automation improves the accuracy and repeatability of cell concentration measurements resulting in a more efficient exosome isolation process.

Zeta Potential and Submicron Particle Size The DelsaMax Series† is an advanced system for measuring size and zeta potential of exosomes. Research laboratories worldwide have benefitted from the highly accurate, fast, and reproducible results using the DelsaMax Series. The unique parallel design enables deep insight into exosome formation and function. Research Flow Cytometry The ultra-sensitive CytoFLEX† Flow Cytometer empowers your laboratory with an additional tool for the study of biological nanoparticles. With the option of a 405nm laser for side scatter detection of particles as small as 200nm, in combination with a flexible number of fluorescent detector parameters, you’ll discover more about molecular mechanisms of cellular interaction. To learn more visit www.cytoflexflow.com

References: Beckman Coulter Application Note: High-Performance Exosome Purification and Characterization via Density Gradient Ultracentrifugation and Dynamic Light Scattering DS- 1 8638A Native-condition Characterization The Beckman Coulter XL-A and XL-I analytical ultracentrifuges allow for the characterization of proteins, oligomers, aggregates, particles, colloids and small structures in native conditions allowing you to determine the sample testing environment that best suits your application. Interference optics (XL-I system) delivers increased accuracy and the ability to examine a greater concentration range with a wider selection of samples.††

* For the Optima MAX-XP. ** Vi-CELL is For Laboratory Use Only. Not for use in diagnostic procedures. † DelsaMax and CytoFLEX are For Research Use Only. Not for use in diagnostic procedures. †† When compared to the XL-A system.

Beckman Coulter, the stylized logo, Allegra, Avanti, Optima, Biomek and Vi-CELL are trademarks of Beckman Coulter, Inc. and are registered in the USPTO. DelsaMax and MobileFuge are trademarks of Beckman Coulter, Inc. CytoFLEX is a trademark of Xitogen Technologies (Suzhou), Inc., a Beckman Coulter company. Apple is a registered trademark of Apple, Inc. IOS is a trademark or registered trademark of Cisco in the U.S. and other countries and is used under license. Android is a trademark of Google, Inc. For Beckman Coulter’s worldwide office locations and phone numbers, please visit www.beckmancoulter.com/contact

High-Performance Exosome Purification and Characterization via Density Gradient Ultracentrifugation and Dynamic Light Scattering

biomarkers. Additionally, exosomes have been shown to be part of intercellular communication functions, with implications toward both anti-tumor and pro-tumor activity. 6 Previous work has provided insight to the isolation of exosomes using density gradient ultracentrifugation, 2,7 although there is an effort to gain more concrete confirmation of the size and concentration after purification. Here we present a simple workflow using automation, centrifugation and dynamic light scattering (DLS), to purify and analyze exosome samples. The Biomek 4000 Laboratory Automation Workstation helps overcome the human variable and provides a consistent, reproducible, high-throughput method for density gradient setup— an elegant solution to scale-up hurdles. Preparative ultracentrifugation helps to maintain reliability between runs and high reproducibility. Importantly, preparative ultracentrifugation reaches the g -force necessary for timely separation of biological macromolecule samples to their isopycnic point in density gradients. The DelsaMax CORE DLS platform is used for size analysis of the fractions, because exosome particles can be: (1) analyzed in solution, (2) with statistical significance, (3) with less cost and time, compared to Electron Microscopy. Instead of taking several hours to analyze a few hundred particles, the DelsaMax CORE is able to analyze and size thousands of exosomes in one minute.

Faster, more accurate exosome analysis.

Abstract Exosomes purification and analyses comprise a fast evolving research area; more than 70% of published research on exosomes has been done within the last six years. Challenges to researchers working with exosomes include setting up density gradients by hand, because it is tedious, time-consuming and subject to user, lab, and method variability. There also is a need for greater accuracy in size and concentration analysis. At the same time, experts in the field have called for the establishment of standard protocols. 1 This paper focuses on solutions to those challenges through cost-effective, large-scale purification, and fast analysis of exosomes. Specifically, the Biomek 4000 Workstation helps overcome human variables and provides a consistent, reproducible, high-throughput method for gradient setup, representing a breakthrough solution to scale-up problems. Optima Ultracentrifugation Series helps researchers maintain reliability between runs, making outcomes highly reproducible. The DelsaMax CORE saves time and cost of TEM analysis for size and concentration. Introduction Although scientists have known about extracellular vesicles for decades, only recently have techniques been able to distinguish exosomes from microvesicles and apoptotic bodies. Classification of membrane vesicles— and the most appropriate, and effective protocols for their isolation—continue to be intense areas of investigation. When isolating vesicles, it is crucial to use systems that are able to separate them, to avoid cross-contamination. At the same time, there is a need for increased size and concentration accuracy, as well as enhanced workflow. Exosomes are membrane vesicles (~ 30–120 nm in diameter) released by almost all cell types. 2,3 They are freely available in plasma as well as other body fluids and contain proteins, mRNA and miRNA, representing the cells they are secreted from. 4,5 Exosomes have come into focus as diagnostic as well as therapeutic

Figure 1. Deck Layout of the Biomek 4000 Workstation Showing the Basic Tools Required for Gradient Prep. (1) One 24-position tube rack for placing nanotubes: the centrifuge tubes fit the existing 24-position tube rack, but new labware type had to be created to accommodate the height of the tubes; (2) one P1000 tip box for P1000 wide bore tips; (3) one Biomek 4000 Workstation P1000SL Single-Tip Pipette Tool for liquid transfer; (4) one Modular Reservoir for gradient reagents.

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