Calypso® IICalypso® IIRapid, automated, quantitative

characterization of macromolecular interactions

Wyatt technology corporation

The Calypso® IIIlluminate protein-protein association and aggregation with CG-MALS

Macromolecular interactions play key roles in a host of scientific and biotechno-

logical studies—from basic biomole-cular research to protein therapeutic R&D—and the Wyatt Calypso is a ver-satile, unique tool for characterizing a wide range of interactions, quickly and reliably.

Utilizing a novel twist on a common technique –static light scattering – sim-

ple and repeatable, automated measure-ments of macromo-lecular interactions without tagging, immobilization, or other sample modifi-

cations are now available to every analytical biochemist and biophysicist.

The Power of CG-MALS Composition Gradient Multi-Angle

static Light Scattering (CG-MALS) is one of the few analytical techniques that can characterize both specific and nonspecific solute-solute interac-tions over a wide range of interaction strengths. Depending on the molar masses and buffer conditions, concen-trations from less than 1 µg/mL to over 100 mg/mL can be probed with the Calypso system.

Calypso’s unique software provides the tools for analyzing•Self-andhetero-association;•Nonspecificinteractions,bothattractiveandrepulsive;

•Reversibleandirreversiblekineticsof aggregation and dissociation.A comprehensive variety of inter-

action parameters may be determined from CG-MALS signals, such as virial coefficients(A2 ), equilibrium dissocia-tion constants (Kd ), stoichiometry, and

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Figure 1: Calypso II syringe pump accessory connected in series to light scattering and concentration detectors

reaction rates. The absence of columns and surfaces plus the inherently large dynamic range of Wyatt MALS detec-tors means a large range of concentra-tion and, hence, of measurable interac-tion strengths, enabling biomolecular scientists to•Validate drug efficacy requiringstrong,specificinteractionsbetweenbiopharmaceuticalsandtargets;

•Optimizeweak repulsion betweenmacromolecules to ensure solubility andstabilityofaformulation;

• Study cooperative and competitiveallostery of compounds exhibiting multiplebindingsites;

•Fine-tunebufferconditionstomini-mize solution viscosity due to self-attractive forces.

CG-MALS and Calypso Designed with maximum automa-

tion, f lexibility, and ease-of-use in mind, the Calypso system provi-des hands-off operation for excellent repeatability and unattended runs. The end result is enhanced productivity, whether you are investigating complex homo/hetero-associations or require an

extensive series of A2 measurements u nder v a r y i ng buffer conditions.

The Calypso system consist s of hardware and software for car-

rying out CG–MALS measurements, working in conjunction with any Wyatt MALS instrument, such as the DAWN®HELEOS®orminiDAWN™ TREOS®, and an optional concen-trationdetector, suchas theOptilab® T-rEX™ Differential Refractometer, or a third-partyUV/Vis absorption

No sample modification means capturing true label-free, solution-phase interactions.

Fully synchronized sample processing and data collection for reliable, repeatable measurements

spectrometer. The Calypso II hardware generates

and delivers accurate sample compo-sition gradients to the detectors. The Calypso II comprises a set of three computer-controlled syringe pumps together with associated degassers, filters,mixers, and valves.DC-servosyringe pumps provide pulse-free deli-very and precise mixing ratios.

The Calypso software integrates all aspects of CG-MALS: controlling the pumps, synchronizing injections with data acquisition, storing and analyzing the data, and reporting the results.

Users will appreciate the built-in automation resulting in simple, robust measurements. Compared to manual batch measurements, sample prepara-tion times are significantly reduced since only one sample solution is pre-pared manually. The Calypso per-forms all further dilutions and mixing, minimizing human error in the pre-paration of composition series, resul-ting in improved reproducibility and consistency of experimental results. Optional externalmodules like theOrbit recycle valve further enhanceoperations with minimal user inter-vention.

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Sample and solvent reservoirs are easily installed to prevent contamination during an experiment.

Inline degassers and filter membranes deliver clean, debubbled sample to downstream detectors.

Precision syringe pumps compatible with syringe sizes 0.01-12.5 mL.

Solvent wash for unattended cleaning.

Back panel features connections for additional automation accessories.

A

B

C

D

E

Streamline MeasurementsNon-Specific Interactions:

•Self-virialcoefficientsA2, A3•Cross-virialcoefficients

Specific Interactions:•Reversibleself-andhetero-

association•Equilibriumdissociation

constant Kd (pM-mM)•Truesolution-phase

stoichiometry

Kinetics:•Aggregation,association,or

dissociation reaction times from seconds to hours

Molecular Sizes:•Weight-averagedmolarmass

Mw•Rootmeansquareradius rg

(a.k.a. radius of gyration)

Refractive Index:•Differentialrefractiveindex

increment, dn/dc

B

A

C

E

D

Macromolecular interactions are measured with unfrac-tionated samples, or “batch”

measurements, via the technique known as Composit ion Grad ient M A LS (CG-MALS). Historically, collecting these data has been labor-intensive and time-consuming, potentially leading to operator error and poor repeatability. With the advent of the Wyatt Calypso, quick, reliable batch MALS measure-ments are now feasible.

The CG-MALS technique entails automatically preparing and injecting into a light scattering detector a series of com-positions or concentrations of a macro-molecular solution. After each injection the flow stops to permit the reaction to reach equilibrium. The apparent weight-average molar mass, Mw,app, is determined for each step in the gradient by analy-zing light scattering and concentration data. Significant interactions between macromolecules manifest as changes in Mw,app vs. composition—decreasing with repulsive interactions and increasing with attractive interactions. Depending on the application, each composition gradient can comprise dilutions of a single species or different ratios of two or even three species mixed together.

A rich assortment of interact ion models is available for data analysis. The virial expansion and effective hard sphere approximation are best suited for nonspecific interactions.Self-associationmay be represented as equilibrium bet-ween monomers and one or more larger oligomers. Heteroassociation models include standard monovalent and mul-tivalent interactions, as well as more complex stoichiometries—combinations of self- and hetero-association and even metacomplex formation.

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Macromolecular InteractionsCG-MALS quantifies interaction strengths spanning orders of magnitude.

Figure 2: The light scattering signal from a single-species concentration gradient determines molecular weight and interactions:

– Virial coefficients (A2) for nonspecific attraction and repulsion

– Affinity (Kd) and stoichiometry of oligomerization

Figure 3: Macromolecular interactions are highly influenced by solvent conditions. The Calypso II fully automates repeat experiments to probe the effects of pH, ionicity, and concentrations of other excipients.

Figure 4: Identifying the magnitude and stoichiometry of interactions between two binding partners—entirely in solution—has never been easier, with a full suite of models to analyze multiple stoichiometries and high order complex formation!

Buffer Conditions

Hetero-associations

Self Interactions

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HowCG-MALSWorks

Self-association and Hetero-associationReversible binding phenomena—involving molecules of the same or different spe-

cies—are governed by thermodynamic equilibrium between free monomers and com-plexes, described in terms of equilibrium association constants Ka. Specific complexes form at different concentration ranges and, in the case of hetero-association, different ratios of species A and B. The presence of associated complexes manifests through changes in the solution’s weight-average molar mass, Mw, which is reflected in turn by the excess Rayleigh ratio R—the fraction of incident light scattered by the sample.

In the case of an ideal, dilute solution, the total excess light scattering measured is just the sum of intensity from each species present:

The concentration of each species ci is dependent on Ka,i and the monomer concen-trations cA and cB. The Calypso software fits MALS data from a series of compositions to association models in order to solve for both the affinity, in terms of association constants, and stoichiometry. CG-MALS clearly distinguishes between different stoichi-ometries with the same stoichiometric ratio, e.g. 1:1 vs. 2:2.

The High Concentration ChallengeRepulsive interactions, often termed “thermodynamic non-ideality,” can usually be

ignored when characterizing specific interactions under dilute conditions. As concentra-tions rise above a few mg/mL, repulsions must be factored into the analysis for an accu-rate representation of the data. The comprehensive mathematical equations describ-ing multi-component light scattering and accounting for thermodynamic nonideality become quite complex when more than two or three species are present. To overcome this challenge, the Calypso software implements a simplified approximation that works well to analyze solutions up to 100 mg/mL and beyond, distinguishing attractive versus repulsive components of the overall interaction as independent quantities.

Figure 5: Measuring specific interactions by light scattering allows for the calculation of both the interaction strength (Kd) and complex stoichiometry.

Nonspecific InteractionsIn the limit of low concentrations, the fundamental relationship linking the intensity of scattered light, the scattering angle, and the molecular properties of a single non-associating species is simply:

•R(θ) is the excess Rayleigh ratio, •MW is the weight-average molar

mass (g/mol), •cisthesampleconcentration

(g/L), •A2 is the second virial coefficient, •A3 is the third virial coefficient,•K* is an optical parameter that

depends on system constants such as the laser wavelength and solvent refractive index

•P(θ) describes the scattered light’s angular dependence. For most proteins and other compact macromolecules with diameters below 25 nm, P(θ)~1.

A plot of R/K*c vs. c yields a curve whose intercept gives MW and whose slope at low concentrations gives A2, an indicator of non-specific interactions between molecules, as mediated by the solvent. The figure below shows qualitatively the effect of attractive and repulsive interactions on the concentration dependence of light scattering. At higher concentrations the curve may be fit to yield A3 as well.

R(θ) =

ΜW P(θ) K*c 1 + 2A2Mc + 3A3Mc2 + ...

R = MAcA + MBcB + S

M i ciK*

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ApplicationsFrom high-affinity protein-protein binding to high-concentration formulationcharacterization,theWyattCalypsoIIdoesitall!

Antibody-antigen interactionsIn pharmaceutical development, desirable anti-

body-antigen interactions typically exhibit strong affinity andhigh specificity.Standard antibody-anti-gen complexes bind with a 1:1 or 1:2 stoichiometry, but other stoichiometries are possible when one or both molecules self-associate. Many interaction assays cannot identify conclusively the true antibody-antigen stoichiometries present in solution. In this example (showninFigure6&7) of a commercial drug product, CG-MALS—automated by Calypso—determined the affinityandstoichiometrywithout labeling or immobili-zation for an antibody-antigen pair.

The dramatic increase in light scattering signal observed during the hetero-association portion of the experiment, despite a nearly constant overall protein concentration, is a direct indicator of antibody-antigen binding.

Using concentration gradients of 5-50 µg/mL for each protein, the expected bivalent association was con-firmed;otherstoichiometricmodelsjustdonotfitthedata. A calculated equilibrium dissociation constant Kd of 2 nM per binding site matched previous analysis by an orthogonal technique, surface plasmon resonance (SPR).

Nonspecific Interactions vs. pH At intermediate concentrations above ~ 1 mg/

mL, all macromolecules will exhibit some form of nonspecific interaction. Understanding how these interactions vary with buffer conditions is critical to optimizingbuffer formulation, purification andpro-tein crystallization.

Concentration gradients of a protein in buffer solu-tion were automated to produce a series of pH states by programmatically titrating a neutral buffer and sample stock solution with a low-pH buffer. Around the iso-electric point (pI), the charge of the protein approaches neutral, and electrostatic repulsion is at a minimum. This is evidenced by a minimum in the repulsive sec-ond virial coefficient,A2 (Figure8 reproduced fromSome, D. et al. (2009) Am. Biotechnol. Lab.;27(2),16-20).Referencing these A2 values to the hard-core repulsion—the excluded-volume A2 is 9⋅10-5 mol⋅mL/g²—indicates net“soft”forcesareattractiveinthepHrange3-6.

Figure 6: Light scattering and concentration data for antibody-antigen interaction, utilizing the method of Figure 4 to identify possible self- and hetero-association.

Figure 7: Best fit of light scattering data for hetero-association gradient in Figure 6, showing the contribution of each species to the overall light scattering signal.

Figure 8: The second virial coefficient for BSA varies as a function of buffer pH, with a minimum at pI.

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Analysis of Protein Formulation at High Concentration

Macromolecules that appear relatively benign at low concentrations can behave unpredictably at high protein concentrations,whereweak attractive forces—insignifi-cant at low concentrations—lead to aggregation, poor stabi-lity and high viscosity. Especially challenging are therapeutic antibodies, often formulated at 00-200 mg/mL, especially for non-intravenous delivery. Such systems must be cha-racterized under true, not dilute, conditions in order to fullyelucidatethefinalproduct.Forwell-formulated proteins, repulsive interactions

dominate, manifesting as a highly sub-linear relationship betweenlightscatteringandconcentration.Figure9plotsthe light scattering and concentration data for a stable solu-tionofbovineserumalbumin(BSA)atpH7,upto100mg/mL. Even at the maximal concentration there is no evidence of aggregation.In contrast, specific self-association of chymotrypsin

evolveswith concentration, as shown in Figure 10.Atconcentrations below 10 mg/mL, monomer-dimer equili-brium dominates the interaction landscape. At higher con-centrations, pentamers form, and the dimer concentration decreases (C.Fernández&A.P.Minton,Biophys. J., 96, 1992-1998(2009)).

Time-Dependent MALS and Kinetics of Small Molecule Inhibition

Although small molecules cannot be detected reliably by light scattering, their effect on the association of larger macromolecules can be readily observed with CG-MALS. Chymotrypsin is known to self-associate reversibly at low pH.The smallmoleculeAEBSF (4-(2-aminoeythyl)ben-zenesulfonyl fluoride) covalently binds chymotrypsin at the dimerization site, irreversibly inhibiting protein binding.

In the experiment, an initial stock solution of chymo-trypsin contains monomers and dimers in equilibrium. UponmixingwithAEBSF, dimers dissociate, dynami-cally altering the light scattering signal which reflects the changing weight-average molar mass. Monitoring the dependence of the dissociation rate on inhibitor concentra-tion enabled calculation of the Michaelis constant and rate constant for the inhibitor (Some, D. & Hanlon, A. (2010) Am. Biotechnol. Lab. 28(1), 9-12).

*Reprinted from The Biophysical Journal,Vol.96,C.Fernández&A.P.Minton,“StaticLightScatteringFromConcentratedProteinSolutionsII: Experimental Test of Theory for Protein Mixtures and Weakly Self-AssociatingProteins,”1992-1998,Copyright(2009),withpermissionfrom Elsevier.

Figure 12: The characteristic reaction time, τ, is inversely proportional to the inhibitor concentration, cI .

Figure 11: Protein dissociation kinetics follows an inhibitor dose-dependent response. The final plateau is pure chymotrypsin in the absence of AEBSF.

Figure 10: CG-MALS data show that specific oligomers of chymotrypsin appear only at higher protein concentrations. Left: Raw light scattering data and best fit analysis for chymotrypsin self-association; reference lines for incomplete models, as indicated. Right: Concentrations of each species present in best-fit analysis.*

Figure 9: BSA, 10-100 mg/mL. A sub-linear relation between light scattering and concentration indicates strong repulsion between macromolecules.

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Comparing CG-MALS to Other Label-free Interaction MethodsOther prevalent biophysicalmethodsfor quantifying protein-protein and related macromolecular interactions include surface plasmon resonance (SPR), isothermal titration calorime-try (ITC), and analytical ultracen-trifugation sedimentation equilibri-um (AUC-SE). As shown in the table below, Calypso provides the broadest range of analyses with no sample modificationrequired:•No immobilization or labeling,

hence no questions about the effect on the interactions

•Range ofmacromolecular binding

affinitiesequaltoorgreaterthananyother technique: pM to mM

•Self-andhetero-associationanalysiswith rich possibilities for arbitrary association models, e.g., simulta-neous self- and hetero-association, aggregation of complexes, multiple binding sites, protein-lipid or prote-in-carbohydrate complexes, etc.

•Kinetics of association, dissociati-on, and aggregation: kon < 107 M-1s-1 (typical for 100 kDa molecule), koff < ~1 s-1

•Characterize non-specific interac-tions,aswellasbindingaffinityand

stoichiometryofspecificcomplexes• Allostericinhibitionorcooperativity•Accountforinactivesample• Self- and hetero-associat ion of

macromolecules at high concentra-tion

•Share detectorswith size exclusionchromatography or fieldflow frac-tionation for absolute determination of molar mass distributions

•Add in a WyattQELS™ module for simultaneous measurement of hydrodynamic radius

SPR ITC AUC-SE CalypsoSample modification Immobilized on chip surface In solution, label-free In solution, label-free In solution, label-free

Measurement time Minutes to hours 1-2 hours Hours to days 0.5- hour

Range of Kd pM - µM nM - mM nM - mM pM – mM100 kDa: ~30 pM1

10 kDa: ~300 pM1

Self-association No Yes Yes Yes

Stoichiometry 1:1;atbest1:n Stoichiometric ratio only, self+hetero

Any, self+hetero Any, self+hetero, metacomplexes

Non-specific interactions No No Yes •Self+crossvirialcoefficients•Reversibleassociationathigh concentration

Kinetics Yes, but not solution- phase;masstransportlimitations on true rates

Onlyveryslow No Yes—moderate to slow reaction rates

Enthalpy & Entropy Yes (from temperature dependence)

Yes (single run) Yes (from tempera-ture dependence)

Yes (from temperature dependence)

Indicates aggregation state

No No Yes Yes

Related separation technique

No No Yes (sedimentation velocity)

Yes(SEC-MALS,FFF-MALS)

Integrated complimentary technique

No No No Yes(optionalQELSforrh)

Additional information None Thermal capacitance, Cp Frictioncoefficient Radius of gyration (>10 nm)

Manual cell cleaning No Yes Yes No

Sample volume/run2 (2-3) mL× Kd (5-15) mL× Kd (1.5-3) mL× Kd (10-50) mL× Kd

Challenges •Masstransport•Immobilization chemistry•Molecularorientation•Regeneration

•Kinetics•Dialysis•Bufferselection

•Sampledegradation over course of long measurement•Kinetics•Smallmolecules

•Samplepurification(particle removal)•Molecules<1kDa

Comparison of Different Measurement Techniques*

*Shaded cells indicate the advantages of each technique.1 Assuming most sensitive Wyatt MALS detectors.2Forexample,measuringKd of 100 nM for 100 kDa proteins would optimally require 10-7 M × 105 g/mol × (2-20) mL = 20-200 µg across thedifferenttechniques;therequiredvolumeincreasessomewhatasKd decreases.

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Calypso SoftwareIntuitive interface for method design, data acquisition, and analysis

The Calypso software integrates smart method design, simple hardware manipulation and robust data acqui-sition for a smooth experimental run. Once themeasurement is complete,the intuitive graphical user interface steps the user through data proces-sing for rapid but powerful analysis of macromolecular interactions.

Smart method development & design

Calypso provides a number of user-friendly features to facilitate experi-ment design. Templates for typical composition gradients are preloaded with the software and can be used alone or as a jumping off point for more complex analyses. Additional com-mands for hardware accessories, such as theWyattOrbit solvent recycler,are programmable to enable full auto-

mation of post-experiment cleaning cycles. Unattended CG-MALS data collection and system maintenance are robust, reliable, and fully custo-mizable.

Moreover, the Calypso software provides vital simulation capabilities to aid in experiment design. Given a

priori knowledge or educated guesses, users may input parameters such as molecularweights, virial coefficients,association stoichiometries and Ka values applicable to their system, and the Simulation tool plots predicted light scattering data for up to 5 models. This information lets users determine appropriate solution concentrations for their experiment as well as the opti-mal composition gradient steps.

An initial sample analysis may indicate the need for additional mea-surements acquired under different conditions, for example as a means of discriminating between multiple associationmodelsthatfitthefirstdataset. The Simulation tool is invaluable in designing follow-up experiments servingtorefinetheanalysisandinter-pretation of complex interactions.

Data Analysis: Versatile interaction modeling

CG-MALS experiments address a wide variety of macromolecular interactions. The Calypso software is unique in its ability to interpret light scattering and concentration data in terms of different models of speci-fic equilibrium association aswell asnonspecificinteractions(viathevirialexpansion) and aggregation or dissoci-ation kinetics.

In the case of nonspecific inter-actions,aglobalfitof lightscatteringintensity to scattering angle and con-centration yields molecular weight, radius of gyration rg, and interac-tion parameters in the form of viri-al coefficients—both self- and cross-species. The “Effective Hard Sphere Approximation” is a model of non-specific repulsion particularly suitedfor dealing with high concentration protein solutions.For specific macromolecular asso-

ciation, the Calypso software incor-porates common interaction models, including•Homo-dimers•Progressiveself-association• 1:1hetero-association• 1:nhetero-associationwith equiva-

lent binding sites. More complicated binding schemes,

including simultaneous self- and hete-ro-association, cooperativity, and meta-complex formation, may also be modeled with some of the advanced features.Foranalysisofhighconcen-trations above ~1 g/L, thermodynamic non-ideality may also be taken into account.

With such a complete set of interac-tion parameters, the Calypso system is unparalleled in its ability to investigate a wide variety of systems, allowing users to unravel the true solution-phase behavior of their systems.

Figure 14: Reversible Associations analysis in Calypso software, with several data fitting models shown. Analysis outputs include molecular weight of each monomer species, interaction strength and stoichiometry, and goodness of fit as χ².

Figure 13: Lines in a typical Calypso method timeline. A single line creates an entire composition gradient. Calypso methods automate operations such as saving data files, post-experiment sample clean-up, programmatically pausing the run, and controlling the detector hardware.

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Additional Applications & Products

Multi-Angle Light Scattering (MALS)The MALS family of instruments includes theDAWN®HELEOS® andminiDAWN™TREOS®. These instruments measure the absolute molar mass, size, and shape of macro-molecules in solution. They may be used in batch mode (off-line) or con-nectedon-linetoanHPLC/FPLC/AFFF,etc.Eachinstrumentcontainsconnections and on-board digital signal processing hardware for four externaldevices.TheHELEOSIIhas18detectorsforunparalleledmul-ti-angle measurements (from a low angle of about 10° to a high angle of almost180°),a120mWGaAslinearlypolarized laser, and options for a vari-ety of thermostatic-control systems to go up to 210ºC or as low as -15ºC.

TheDAWNHELEOSistheultimateina research-oriented light scattering instrument. Its 18 angles of detection give it the widest angular range of any commercially-made light scattering detector, and its numerous options enable it to be customized for virtually any application.

Möbiuζ™

The Möbiuζ isthefinest,mostver-satile laser-based light scattering instrument for reliable, reproducible, and non-destructive electrophore-tic mobility measurements of nano-particles (including proteins and small macromolecules), extending the measurable molecular size range of zeta potential down to 1.0 nm. Simultaneous measurement of the hydrodynamic radius is available with theembeddedWyattQELSoption.

The Möbiuζ is the finest, most versatile light scattering instrument for reliable electrophoretic mobility measurements of proteins particles and macromolecules, extending the measurable molecular size range of zeta potential down to 1.0 nm.

Wyatt’sOptilabT-rEX™has256timesthe detection power and up to 50 times the dynamic range of any other RI detector.

Dynamic Light Scattering (DLS)When you want to have the best of both worlds (classical and dynamic light scattering), our MALS instru-ments can be ordered with the embe-ddedWyattQELS™, or they can inter-face with our DynaPro™NanoStar®. The combined instruments allow simultaneous stat ic and dynamic light scattering measurements from which both absolute molar masses and hydrodynamic radii can be obtained. Or, if high throughput DynamicLight Scattering is desired, the DynaPro™ Plate Reader™ system can automatically measure sizes of prote-insandnanoparticlesin96or384or1536wellplates.

The DynaPro Plate Reader is the only DLS instrument that can measure directly in standard plate formats of 96, 384 or 1536 wells. Temperature control and temperature ramping from 4°C to 70°C is available.

Optilab T-rEX™

TheOptilabT-rEX(refractometerwith EXtended range) is an RI detec-torthathas256timesthedetectionpower and up to 50 times the dynamic range of any RI detector in existence today.TheOptilabT-rEXmayalsobe

used to measure the absolute refrac-tive index increment, dn/dc, at the same wavelength as the light scatte-ringinstrument.Finally,theOptilabmay be used to measure the absolute refractive index of a solution.

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Service & SupportWorld-classserviceandsupportfromtheleading

developer of light scattering instrumentation.

The founders of Wyatt Technology commercialized the first light scattering instruments incorpora-

tinglasersmorethan40yearsago.It’snosurprise that today, we’re the world leader in thefield.No instrumentationdevelo-per has greater resources devoted to the manufacture, service, and support of light scattering instruments than we do. We have more post-graduate degreed scientists involved in the support of this type of equipmentthanalloftheotherfirmsinthebusiness combined. In fact, our company has more than 300 person-years of cumulative experience in light scattering.

We’re proud to offer our unique Light Scattering University® course that includes three days of inten-sive hands-on trai-ning in Santa Barbara, California. The cur-riculum covers light scattering theory, data collection and analysis, troubleshooting, and maintenance. During training, our customers interact with the peo-ple who develop our software and hardware. Customers learn not only about hardware and software operation but also about sample preparation and data interpretat ion. Ourinternationally-acclaimed immersion trai-ning also insures that operating the instru-ments will be second nature by the time the course is done.

When questions do arise, our superb staff answers telephone, fax, and e-mail inquiries quickly. By means of our interac-

tive customer support, our service perso-nnel are always just a phone call away, and a proprietary Internet link can bring us directly into your lab for “hands-on” help and guidance with our Remote Assistant support feature.

Since the company has so many bio-technology, pharmaceutical, and polymer customers, we have developed complete IQOQservicepackages,whichareavaila-blewithon-sitesupport.OurInternationalLight Scattering Colloquium is held once a year to stimulate the exchange of informa-tion among our customers and to promote greater understanding of the capabilities of MALS and other technologies develo-

ped by Wyatt. All of Wyatt Technology’s customers are eligible to attend and partici-pate in this colloqui-um that is often called the most interesting scientific conference around.

The company also maintains an online Support Center where customers can view their instrument ser-vice status, down-load current software updates, read our Frequent ly AskedQuestions, andorderspare parts and sup-plies from the on-line

store.OurWebsite provides frequentadditions to its extensive bibliogra-phy of thousands of refereed scien-tific papers that have utilizedWyattinstruments, training schedules, and ApplicationNotes.You canfind themall at www.wyatt.com.

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Absolute Macromolecular Characterization™

Compatible Detectors: MALS detectors:DAWNHELEOS®orminiDAWNTREOS® Concentration detectors:Optilab®differentialrefractometer,UV/Visabsorptionorsimilar

Syringe Pumps: 3, dc-servomotor;4-portdistributionvalveoneachpump

Syringe Volumes: 12.5 µL-12.5 mL (1 mL syringes supplied as standard)

Degasser Channels: 1 per pump, 100 µL internal volume

Pressure Limit: 1-mL syringes: 200 psi 0.5-mL syringes: 350 psi

Auxiliary Autoinject out, injector valve control, recycle valve control Inputs/Outputs: digitalandanalogI/Oforspecificapplications

Solvents: Aqueous

WettedMaterials: Borosilicateglass,PEEK,PTFE,polyethylene,alumina,tita-nium, stainless steel

Typical Sample 1.5-7mL,dependingondetectorconfiguration

Volume Per Analysis:

Repeatability of Mw: ±5% Measurement: A2: ±0.5x10-4 mol⋅mL/g² for sample molar mass of 50-100 kDa log(Kd): ±0.3

Range of Equilibrium Dissociation Constant, Kd: 100 pM-1 mM typical for Measurement: 100 kDa molecules (actual range varies with molecular weight and association stoichiometry) Association Stoichiometry Models: arbitrary self + hetero- assocation models, equivalent binding site for self- and hetero- association, aggregation of complexes, incompetent fractions

Power Requirements: 100-240VAC

Host PC Requirements: 2.8GHzorbetterprocessor,1GBRAM EthernetconnectionforHELEOS/TREOScommunications

Software Microsoft Windows®XP,VistaorWindows7 Compatibility:

Dimensions: 37cm(W)x35cm(H)x58cm(D)

*Specificationssubjecttochangewithoutnotice.

Warranty: All Calypso instruments are guaranteed against fabrication defects for twelve months. Should any unit become defective due to normal use within the warranty period, we will repair or replace it at no charge.