OBSERVER is a compact application that does no more and no less than it needs to:

• Set up instrument control and parameters for real-time Mw and Rg measurement in inline or online mode.
• In online mode, an HPLC pump is controlled by OBSERVER to pull a constant flow from the main process stream or container, with optional dilution. Supported pumps are: Agilent 1100 series, 1200 series and 1200 Infinity and 1290 Infinity II quaternary pumps.
• Set up trigger conditions: permissible ranges or end point bands for molar mass or size. Band tolerances and required duration of trigger conditions may also be set.
• Click the ‘Play’ button to begin monitoring. Data are saved for later review in OBSERVER and may transferred via OPC to a central processor.
• End the process when end point is reached or deviation identified, via analog voltage output or OPC.
• Alarms are generated if the hardware diagnostics indicate loss of communication, a pump leak or other improper operation conditions.

Specific, Reversible Interactions – Protein Binding Studies

The CALYPSO software uses CG-MALS data to calculate the affinity and stoichiometry of specific, reversible complexes formed in solution, such as antibody-antigen interactions and protein dimerization. The software enables quick data fitting to simple models of dimer formation, 1:n stoichiometries, and infinite self-association. Alternatively, the highly versatile analysis tools allow you to choose and design more complex binding schemes, including simultaneous self- and hetero-association, cooperativity, and meta-complex formation.

Nonspecific Interactions –Virial Coefficients

The second virial coefficient (A2) is used to quantify nonspecific attractive and repulsive interactions for proteins, polymers, and colloids. Batch light scattering data collected with the CALYPSO software can be analyzed using a Zimm formalism, Debye formalism, or effective hard sphere approximation. Higher virial coefficients may be determined as well.

Time-Dependent MALS – Association and Dissociation Kinetics

Some protein association or dissociation reactions can take seconds or minutes to reach completion. The CALYPSO software gives you the power to set the time for allowing each composition to come to equilibrium—from a few seconds to several hours. In the example pictured below, different concentrations of a small molecule inhibitor were used to disrupt the dimerization of a protein; the light scattering decrease corresponds to the dissociation of dimers into monomers over time.

Light Scattering and Concentration Detection

Maximum versatility of experimentation is provided by the real time display of your light scattering and concentration data with a method timeline showing you exactly how far your experiment has run. Analyze data as it is being collected to monitor and evaluate progress of your experiment. Pause and modify your method at any time to optimize and change parameters on the fly, such as increasing measurement times to accommodate long kinetics.

Simultaneous collection with ASTRA

Light scattering using either the DAWN® or miniDAWN® and concentration data from a differential refractive index detector (such as the Optilab®) or a UV detector can be collected simultaneously with ASTRA®.

Flexible Concentration Detection

Use either UV or RI concentration detectors or nominal concentrations based on the calculated dilutions. These values are automatically computed and saved with your data. Concentration detection modes can be easily selected post-acquisition.

Calypso Pump Controls

The CALYPSO software allows you to customize every aspect of your CG-MALS experiment, giving you full control of your interacting system. Power on degassers, set maximum allowable flow rates and pressures, and perform priming and wash steps to prepare your hardware for an experiment. Flow and wash parameters can be programmed into an experiment method for complete, unattended experiments.

Useful Accessories

External devices may be readily integrated with the Calypso. The Calypso can control external valves to simplify system maintenance. Use an Orbit Solvent Recycler to keep clean solvent pumping through your system when not in use. Switch between two wash buffers using an external two-position valve for thorough washing after an experiment. Both functions can be automated within a method for quick and easy cleanup.

Method Design

Design your own composition gradients or use one of the software’s built-in methods to investigate different macromolecular interactions. Any number of composition gradients may be run in a single experiment, repeat runs can be saved to their own data files, and the system washed and ready for the next use in the morning all from a single method page. In addition to the provided example methods, user-designed methods can be saved for later use.

Simulations

Whether you’ve already performed an experiment and like to optimize parameters or whether you like to hit the ground running designing your first Calypso Experiment, the CALYPSO software provides vital simulation capabilities to aid in experiment design. Input parameters, such as molecular weights, virial coefficients, association stoichiometries, and equilibrium association constants (KA), and the Simulation tool plots predicted light scattering data for up to 5 models. This information lets you determine appropriate solution concentrations for your experiment as well as the optimal composition gradient steps.

Calculator

Calculators are included in the CALYPSO software to aid in experiment design and evaluation of results: Use the A2 Calculator to predict the excluded volume A2 and select an appropriate concentration range. The dn/dc Calculator helps you estimate the change in your molecule’s dn/dc in buffers of varying refractive index. The KD Calculator calculates the equilibrium dissociation constant (KD) for one or two binding sites and helps identify cooperativity.

VISION CSH

VISION Chromatography Software Hub serves as FFF Central Station on your lab PC. Open VISION to launch any and all of the primary Wyatt software packages for FFF-MALS:

• SCOUT DPS: Combines simplified, rationalized FFF method development with FFF data analysis to determine distributions of hydrodynamic radii based on first-principles physics.
• VOYAGER CDS: Complete, integrated solution for FFF experimental runs. Instrument control and data acquisition for Eclipse and Agilent HPLC components.
• ASTRA: Wyatt’s flagship software for characterization of macromolecules and nanoparticles by SEC/FFF-MALS. Acquires, analyzes and reports measurements from multi-angle light scattering, dynamic light scattering, UV/Vis, dRI and differential viscometry detectors. Determines molar mass, size, concentration, intrinsic viscosity, conformation, shape, particle concentration (number density) and more.

SCOUT DPS

The flexibility of FFF allows users to achieve excellent separations for most applications, but in some cases finding the optimal method for the separation run can require tedious trial-and-error.

SCOUT DPS is an indispensable tool for FFF method development which utilizes in-silico, “virtual” experiments to eliminate most of the time and effort required for FFF method development.

Typical method optimization in SCOUT combines the results of one or two real FFF runs with an unlimited number of virtual FFF runs. A proprietary simulation algorithm quickly calculates the effect of changing parameters such as:

• Channel flow rate
• Cross-flow rate gradients
• Spacer height
• Membrane porosity

From virtual to physical

SCOUT makes use of first-principles FFF physics to simulate the separation process. The results are shown immediately, including the effect of band broadening and dilution of the sample at the channel outlet.

The example to the right shows the process for optimizing a nanoparticle separation.

• The blue experiment is the result of a default method for separating a sample containing three nanoparticle sizes. The dashed blue line shows the cross-flow gradient used, and the solid blue line is the measured concentration signal.
• The red experiment is the result of a series of simulations tested to improve the resolution. With a modified cross-flow gradient, the peaks are expected to exhibit baseline resolution.

The final measurement shows that the optimized method performed well, with enhanced separation between the three peaks.

Added value: Rh

An additional benefit of SCOUT is the analysis of fractograms to determine size distributions according to basic FFF physical theory.

This graph shows the size distributions determined for the nanoparticles in terms of hydrodynamic radius.

SCOUT DPS and the VOYAGER CDS software for performing FFF experiments exchange methods and data seamlessly.

VOYAGER CDS

VOYAGER CDS is a complete chromatography data system that incorporates everything you need for maximum productivity in FFF-MALS with an Eclipse and Agilent HPLC components:

• Eclipse: Complete control over all flows plus readings of pressures and flow rates
• Agilent® HPLC: Control and digital data acquisition from Agilent HPLC components including pumps, degassers, autosamplers, fraction collectors and detectors
• ASTRA control: Digital synchronization of multi-sample sequences with ASTRA

Extra benefits

VOYAGER CDS offers additional flexibility beyond OpenLab or ChemStation, such as storing all data files (Eclipse method, Agilent data, ASTRA) in one project folder.

A primary benefit is the ability to exchange files with SCOUT DPS:

• Read in FFF methods designed in SCOUT DPS
• Export HPLC detector data to SCOUT DPS for method feedback and size distribution analysis

ECLIPSE plug-ins

An ECLIPSE plug-in module controls the Eclipse DualTec and Eclipse AF4 Field-Flow Fractionation (FFF) systems. Coupled with ASTRA for data collection and analysis, an ECLIPSE plug-in provides users with a basic turn-key solution for macromolecular characterization.

ECLIPSE modules are available for these chromatography software packages:

• Agilent
• Dionex
• Shimadzu

Plug-ins benefits:

• Familiarity: The Eclipse instrument and the associated HPLC components are all operated from a single, familiar interface.
• Simple configuration: The Eclipse instrument is configured using the standard configuration dialog.
• Simple setup: Setup of all flow modes and flow rates is controlled by standard software menus within the HPLC software.

ASTRA Features and Capabilities

Browse these tabs to learn about the many capabilities and features in ASTRA which make it is the most powerful and versatile software available for the characterization of macromolecules and nanoparticles via multi-angle and dynamic light scattering.

ASTRA 7 increases productivity for routine analyses

Running a SEC-MALS analyses has never been easier. ASTRA’s One-Click MW feature complete an entire method at the click of a button.

Click on Run Default and ASTRA will:

• Synchronize data collection with your HPLC
• Autoset parameters to determine MW and Rg
• Generate custom reports and graphs
• Prepare for the next run
• run-default-button

Method Builder

ASTRA’s Method Builder simplifies the specification of methods for the most common protein and polymer analyses, in three intuitive steps:

• Choose an analysis profile
• Specify the instruments
• Enter additional parameters

Then save the method, load the sample(s), click Run Default and return later to find out the MW results in the final report.

Freedom to Explore

With ASTRA, you are never locked in to default methods or standard procedures. You always* have access to each parameter and processing step to review, validate and improve.

*Except network accounts where access is limited under the 21CFR(11) compliant Security Pack.

Ahead of the Curve

As you build expertise and confidence, and move on to complex analyses, ASTRA allows you to refine the results of automated processing with fine adjustments to peak selection, baseline specification, detector filtering and other settings.

HPLC Control

Full digital synchronization between your HPLC system, Wyatt detectors and ASTRA 7:

• Minimizes user errors
• Runs SEC-MALS from a single, familiar environment
• Saves you time and money

ASTRA 7’s HPLC control modules works with standard Agilent hardware including pumps, autosamplers, column ovens and UV/Vis detectors.*

*Control of additional HPLC instruments coming soon

Integrated HPLC control

Let ASTRA take control of your HPLC stack for effortless synchronization between injections and data collection. ASTRA 7 eliminates the need for separate HPLC and light scattering software. HPLC modules are listed together with Wyatt detectors in a common experiment configuration, and key parameters such as vial position, injection volume and flow rate specified in ASTRA’s sequences.

HPLC Manager

The HPLC Manager app controls and monitors HPLC modules including pumps, column ovens, UV detectors and autosamplers, right from the desktop. Included free with the HPLC Control module.

High Throughput Processing

Built from the ground up to handle with ease large sets of data, ASTRA has the tools you need to process and report on sequences containing many samples, including replicates. “One-to-Many” processing interface lets you focus on your goals, and leave the drudgery to the computer.

EASI Table

Quickly compare multiple data runs with the EASI Table feature to assess compound stability and consistency. The table data can be exported in a variety of formats, or copied and pasted into your favorite word processing software.

Specify which data should be displayed in EASI Table, as well as the format and precision used to display the values. Choose from percent or numerical uncertainties, or omit them entirely.

Sequence Processing

Sequences are used to specify and control the automated collection of data as well as system functions. A sequence can be configured with the default method or a different method can be specified for each sample. Sequences also perform post-collection maintenance, such as running the COMET flow cell cleaner or switching the Orbit to solvent recycle mode.

In the Samples view, it is possible to modify sample parameters on-the-fly, adding samples, changing collection times, or skipping samples.

Let your computer do the work.

ASTRA allows you to process an experiment as you wish, then instruct the computer to apply the same settings and analysis steps on any number of additional data files. You can quickly and easily specify which elements of your ‘source’ experiment should be incorporated in the target experiments.

Once the processing is finished, your resulting data files are fully analyzed and ready to be reviewed.

Unmatched Analysis

Building on over 40 years of light scattering research and development, ASTRA 7 includes an unrivaled range of analysis features. ASTRA takes full advantage of modern multicore computers, processing data at blazing speeds and with superior accuracy.

Molar Mass Determination

Determine molar mass and size.

Molar Mass via Light Scattering

Light scattering stands alone in its ability to measure the molar mass of macromolecules in solution both accurately and quickly.

Molar Mass via Conventional and Universal Calibration

Build a bridge between legacy conventional and universal calibration results and modern, accurate light scattering analysis.

Protein Conjugate and Copolymer Analysis

If you have an RI detector, a UV detector, and a light scattering instrument, you can directly measure the molar mass, size, and relative fractions of copolymers and protein conjugates.

Particles and Number Density

It is possible to measure the size and number density of a sample with just a light scattering instrument. ASTRA supports several sophisticated models to determine the size of a macromolecule based solely on the angular variation in the light scattering intensity.

On-Line QELS

With the support of a QELS instrument module, you can extend your data analysis to determine the translational diffusion and corresponding hydrodynamic radius of your samples.

QELS batch Regularization and Cumulants

Determine translational diffusion and corresponding hydrodynamic radius of your unfractionated samples.

Viscometry

ASTRA provides several analysis features that take advantage of viscosity data. Use these features to further quantify your macromolecules to determine Mark-Houwink-Sakurada terms, hydrodynamic radius, and more. Learn more…

Molecular Conformation

The shape of molecules can be assessed from the molar mass and rms radius of the sample.

Distributions

Sample properties are determined for individual peaks in a chromatography run. ASTRA also supports visualizing these data in terms of the distribution of these measured quantities. This is extremely powerful when assessing how much of a sample falls with a particular range of molar mass or size.

Second Virial Coefficient (A2) Measurements

The second virial coefficient (A2) is a measure of macromolecular self-association, and is one of the few parameters that can be used to predict the crystallization properties of a sample. ASTRA allows you to measure this important characteristic.

dn/dc and UV extinction coefficient determination. Measure the dn/dc and UV extinction coefficients for your novel samples.

While dn/dc and UV extinction coefficients are documented for many existing compounds, they must be experimentally determined for novel samples. ASTRA provides analysis features to measure these critical sample characteristics.

Band Broadening Correction

While most analysis packages force you to live with the undesirable effects of passing a sample through a series of detectors. ASTRA’s revolutionary band broadening correction accounts for these effects to give you most accurate results possible.

Absorbance Correction

If a sample absorbs light at the MALS laser wavelength, molar mass calculations will be skewed due to less light reaching the scattering volume than expected. ASTRA uses the Forward Monitor detector signal available in all Wyatt MALS instruments to calculate the laser attenuation produced by sample absorption and correct the scattering signal levels. In this manner accurate molecular weights are ensured, even in the presence of absorbtion.

Blank Subtraction

Certain analysis techniques, such as FFF, cause large changes in refractive index and viscosity measurements as flow rates are modified during the run. This can make it difficult to properly analyze the file, as the RI and viscosity changes due to flow and pressure changes mask those caused by the sample. ASTRA provides a mechanism to subtract a data collection run without sample (a ‘blank’) from a sample run to reveal the sample-specific signal changes.

Error Analysis

Wyatt Technology software is written by scientists for scientists. ASTRA was designed from the ground up to properly compute and propagate uncertainties to ensure our results are publication-quality.

Cutting Edge Technology

Wyatt Technology is committed to pushing macromolecular characterization to new levels. Consequently, we have a very strong program to develop intellectual property. Many of the algorithms in ASTRA are on the cutting edge of scientific analysis, and are either patented or have patents pending. ASTRA is protected by:

• U.S. patent numbers: 5,528,366; 6,411,383; 6,651,009; 6,774,994; 7,386,427; 7,911,594;
• German patent Numbers: 694 33 615.7-08; 603 19 078.2-08;
• British patents based on European application numbers: EP 0 665 433; EP 1 510 807;
• Japanese patent Numbers: 3,580,380; 4,439,211; 4,381,914; and other patents pending.

State of the Art Reporting

Take control of your analysis reporting with ASTRA’s custom reporting system. With just a few mouse clicks, adjust which data to display and how it should be formatted. Add your own logo, incorporate some personal notes, or just pare the default reports to focus on what you care about.

Personalize your reports.

In addition to the numerical and content selections provided in the Report Designer, ASTRA allows you to specify a logo image, and where it should be displayed. You can specify the title as well as additional summary information or notes that will appear on the report.

Personalized Data Selection

Control the content and presentation of your reports with a few mouse clicks. Any bit of data generated during analysis is now available for you to incorporate into your report, with just a click of the mouse.

Built from the ground up to comply with 21 CFR Part 11, ASTRA 7 with Security Pack makes compliance complete and intuitive.

• Data Safety and Integrity
• User Security
• Audit Trails
• Electronic Signatures
• Enforcing Permitted Sequences
• Human Readable Records
• Validation

Full Numerical Formatting

Click on a result, and ASTRA presents a simple, intuitive interface for controlling the numerical precision and display format of the result. Quickly and easily change results from fixed point to scientific notation, increase or decrease the number of significant digits to display, and determine if you wish uncertainties to be displayed as numerical values or percentages.