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Wyatt Technology

Calypso II

Calypso II

 

Rapid, quantitative characterization of protein-protein and other macromolecular interactions with composition-gradient multi-angle light scattering.

The Calypso II, in conjunction with a DAWN or miniDAWN MALS detector, measures binding affinities and absolute, molecular stoichiometries of complex biomolecular interactions.

In addition to equilibrium association constants KD and absolute stoichiometry of reversible self- and hetero-associations, it can also help determine reaction or aggregation rates, non-specific interaction parameters (virial coefficients) and even automate measurements of weight-average molar mass and dn/dc for polymers.

Included with each Calypso is a copy of CALYPSO software, the most versatile software package available for analysis of biomolecular interactions in solution by light scattering.

  • Calypso Experimental SetupCreating composition gradients...

    CG-MALS employs a Calypso II connected to a MALS and optional concentration detector. The Calypso II performs sample preparation and delivery, combining up to three different solution in precise mixing ratios and injecting into the detectors.

    Accurate compositions result from setting the Calypso's pumps to appropriate flow rate ratios, all controlled from the CALYPSO software through an intuitive GUI. Mixing is achieved by dispensing the solutions simultaneously from all three pumps via a static mixer.

    ...that work with MALS detectors

    Reliable light scattering measurement require the samples to be degassed and filtered. The Calypso II incorporates built-in, low-volume degassers and filter housings, connected via PEEK tubing and finger-tight fittings. Even the filter housing is finger-tight, making use of a proprietary design for maximum convenience.

    Calypso II features

    • Biocompatible wetted materials.
    • Flexibility to re-organize fluid paths and utilize different wetted materials as needed.
    • Convenient loading of sample and diluent solutions via conical tubes - 5, 15 or 50 mL.
    • Autoinject Out contact closure signal, to trigger data acquisition by ASTRA or DYNAMICS software for data acquisition and analyses not supported by the CALYPSO software (e.g., DLS acquisition).
    • Built-in Wash port, allowing the pumps to draw on reservoirs of wash solutions for post-experimental clean-up. Automation of up to two sequential wash solutions, e.g. buffer (to clean out proteins) followed by water (to clean out buffer salts) or detergent followed by water, is possible when a COMET solvent selector valve is added to the setup. These operations are appended to the Method for unattended operation.
  • Label-free, immobilization-free

    Composition-Gradient Multi-Angle Light Scattering (CG-MALS) employs a series of unfractionated samples of different composition or concentration in order to characterize a wide range of macromolecular interactions. No special modifications (e.g., sample tagging or immobilization procedures) are necessary: samples are unlabeled and entirely in solution.

    Addressing a host of biomolecular phenomena

    The primary analysis techniques supported by Calypso hardware and software are:

    • Dynamic equilibria: specific binding and complex assemblies of proteins, oligonucleotides and other biomolecules; KD (equilibrium dissociation constant) from picomolars to millimolars; absolute molecular stoichiometry of associating complexes; self and/or heteroassociations
    • Non-specific macromolecular interactions: self- and cross-virial coefficients
    • Stop flow kinetics: aggregation, dissociation and other time-dependent reactions: equilibration time from seconds to hours
    • Zimm plots: concentration gradients for determining solution-average molar mass MW, size rg, and second virial coefficients
    • Refractive index increment: dn/dc

    Calypso's automation enhances productivity while the CALYPSO software provides an unparalleled selection of interaction models. Relative to other techniques for characterizing protein interactions, such as surface plasmon resonance, sedimentation equilibrium, kinetic exclusion or isothermal titration calorimetry – as well as manual CG-MALS measurements – the Calypso II provides fast and accurate results.

    Applications

    Drug Discovery

    • Quantify binding affinity and stoichiometry of enzyme/inhibitor or antibody/antigen interactions, including complex multi-valent and multi-protein complex formation
    • Study the impact of small molecules on protein-protein interactions

    Process Improvement

    • Determine second virial coefficient and adjust buffer parameters to improve formulation stability and viscosity
    • Determine cross virial coefficients to optimize antibody purification and understand the effects of large excipients on formulations

    Self-assembly / Aggregation

    • Quantify impact of solvent ionic strength, pH, or excipients on polymerization or protein associations
    • Measure kinetics of self-assembly and aggregation via rate of change of molar mass and radius of gyration, and hydrodynamic radius (with a WyattQELS module or NanoStar and parallel analysis in ASTRA or DYNAMICS)

    Biotech R&D

    • Characterize macromolecular binding affinity and associated complex stoichiometry over a wide range of buffer compositions, time, and temperature scales
  • Deceptively simple

    Conceptually, the Calypso II is 'just' a box with some syringe pumps and tubing, right? Well, that's what we thought it would be - until coming up against the juxtaposition of challenging biomolecular interaction research and the demands of exquisitely sensitive light scattering instruments. In order to simultaneously achieve:

    • high precision
    • minimal sample consumption
    • compatibility from peptides to to high-concentration monoclonal antibodies to nanogels
    • affinities ranging from pM to mM

    while still providing exceptional ease-of-use, accuracy, robustness and reasonable cost in capital and consumables, we had to stretch the limits of available technology.

    We worked hard, so you don't have to

    We left no stone unturned looking for the rare components that met all our demands, such as one-of-a-kind syringe pumps and pressure transducers as well as hard-to-find, 30 nm pore size, filter membranes and low-volume, high-efficiency degassers. What we couldn't buy, we innovated and designed from scratch.

    Users have been particularly pleased with our unique, ultra-low-dead-volume, finger-tight filter housings, which help ensure sufficient solution cleanliness for high-affinity analyses yet retain very little sample. Special valves and syringes were co-developed with suppliers to push syringe pump performance and ease-of-use to the max.

    Recognizing that CG-MALS is unfamiliar territory for most users, we've also developed Tutorials, Application Guides, Method Templates, and additional support literature and training to help you get started and keep you on track. Make the most of this amazingly powerful technique - we're here to help at every turn.

    Some additional Calypso II features

    The layout and design were fine-tuned to reduce sample use while enhancing automation and maintaining compatibility with the DAWN HELEOS II and miniDAWN TREOS, the ultimate in MALS detectors. Special features of the Calypso II include:

    • Most tubing is 0.010" (0.25 mm) i.d. PEEK for biocompatibility and low volume. May be changed to PTFE or stainless steel for non-aqueous solutions, and larger i.d. for viscous solutions
    • A range of syringe sizes to meet specific needs
    • In-line degassing of samples and buffers
    • Easy access to all tubing and filters, for rapid maintenance
    • Pressure warnings indicate when a filter membrane is saturated or a capillary tube clogged
  • Protein-Ligand Binding

    Most protein-ligand and similar biomolecular interactions are best measured at or below 1 mg/mL. The CG-MALS configuration should include:

    • Calypso II
    • DAWN II H/C for temperature regulation of the interaction and highest sensitivity, though a miniDAWN (ambient only) may suffice
    • Third-party on-line UV/Vis absorption detector - please This email address is being protected from spambots. You need JavaScript enabled to view it. for recommended models

    Non-Specific Interactions

    Non-specific interactions are usually measured in the range of 1 - 25 mg/mL. The configuration should include:

    • Calypso II
    • DAWN II H/C when measuring temperature-sensitive samples or polymers with rg > 40 nm; otherwise miniDAWN
    • Optilab T-rEX for concentration measurements

    High-Concentration Protein-Protein Interactions

    These measurements may go to concentrations of hundreds of mg/mL protein.

  • MALS

    DAWN HELEOS II - The most sensitive MALS detector available, anywhere. Incorporates detectors at 18 angles to determine molar masses from 200 Da to 1 GDa and radii from 10 – 500 nm.

    • Heated/cooled (H/C) option -15°C to +150°C

    The HELEOS offers advanced options such as fluorescence or polarization filters and a 785 nm laser.


    miniDAWN TREOS - Second only to the DAWN in sensitivity. Incorporates detectors at 3 angles to determine molar masses from 200 Da to 20 MDa and radii from 10 – 50 nm. Ambient only.


    Options

    WyattQELS - A dynamic light scattering (DLS) module which integrates inside the DAWN or miniDAWN to provide simultaneous DLS measurements in the same scattering volume. The optical fiber replaces any one MALS detection angle.

    COMET - An ultrasonic transducer that cleans MALS flow cells automatically by agitating the fluid and loosening particles from the glass surfaces. Integrates inside the DAWN or miniDAWN.


    Accessories

    Orbit - The Orbit helps conserve mobile phase by programmatically directing the solvent to either a waste bottle or back to the solvent reservoir. In conjunction with the Calypso II, the Orbit is used to programmatically select from one of two Wash solutions in order to enhance automated post-experimental clean-up.

    Solaris - Prevents growth of bacteria, fungi or algae in solvents with high efficiency and protects your system from down-time due to contamination.


    dRI

    Optilab T-rEX - A unique on-line differential refractometer for measuring concentration of any macromolecule, regardless of chromophores. The high-concentration Optilab accommodates protein concentration up to 180 mg/mL.

  • CALYPSO softwareCALYPSO - Simulation and method design of CG-MALS experiments; Calypso II fluidics control & MALS/dRI data acquisition; data analysis for determination of equilibrium dissociation constants KD, absolute molecular stoichiometry and other parameters.

  • Syringe Pumps
    3 computer-controlled pumps; dc-servo motor, for minimum pulsation;
    3-port distribution valve on each pump for Input, Output and Wash
    Syringe Volumes
    12.5 µL – 2.5 mL (1 mL and 0.5 mL syringes supplied as standard)
    Degasser Channels
    1 per pump, 60 µL internal volume
    Pressure Limit
    1 mL syringes: 200 psi
    0.5 mL syringes: 400 psi
    Auxiliary Inputs/Outputs
    Autoinject out, injector valve control, recycle valve control, digital and analog I/O for specific applications
    Solvents
    Aqueous
    Wetted Materials
    Borosilicate glass, PEEK, PTFE, polyethylene, alumina, titanium, stainless steel
    Typical Sample Volume Per Analysis
    2.5 – 7 mL, depending on detector configuration and complexity of analysis
    Range of Measurement

    Equilibrium Dissociation Constant, KD: 100 pM – 1 mM typical for 100 kDa molecules (actual range varies with molecular weight and association stoichiometry)

    Repeatability of Measurement

    Mw: ± 5%
    A2: ± 0.5 x 10-4 mol⋅mL/g² for sample molar mass of 50 – 100 kDa
    log(KD): ± 0.3

    Analyses
     
       Specific Binding
    See CALYPSO Software for more details.
    • Self-association
    • Hetero-association
    • Multi-valent
    • Combined self- and hetero-association
    • incompetent (inactive) fraction
    • Cooperativity and competitive binding
    • Binding with thermodynamic non-ideality (high concentration)
    • Concatenate multiple data sets for global fit
       Non-Specific Interactions
    • Self-interaction: 2nd and 3rd virial coefficients
    • Hetero-interactions: cross virial coefficients with 2nd self-virial coefficients
       Kinetics

    1st-order analyzed in software; export data for higher-order kinetics

       Additional analyses
    dn/dc
    Host PC Requirements
    USB port for Calypso II
    Ethernet connection for HELEOS / TREOS communications
    Dimensions
    58 cm (L) x 37 cm (W) x 35 cm (H)

    Facilities requirements may be found in Wyatt Instrument Facilities Specifications.
    Host PC requirements may be found in Computer Requirements.

    Specifications subject to change without notice.

   

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Wyatt Technology is the recognized leader in light scattering instrumentation and software for determining the absolute molar mass, size, charge and interactions of macromolecules and nanoparticles in solution.

Wyatt's line of multi-angle static light scattering products couple to size exclusion chromatography (SEC-MALS), field-flow fractionation (FFF-MALS), and stop-flow composition-gradient systems (CG-MALS). Our dynamic light scattering (DLS) products operate in traditional cuvette as well as on-line and automated, high-throughput modes. We also offer unique instruments for electrophoretic light scattering (MP-PALS), differential refractometry, and differential viscosity.



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