Benefits of RT-MALS

• Accelerate process development and scale-up
• Increase yield, reduce overall time until lot release
• Maintain product quality with confidence

Where MALS and PAT meet

The ultraDAWN is a unique member of the DAWN family of MALS instruments. Instead of residing in the analytical lab like Wyatt’s other MALS instruments, it is adapted to pilot plants and production environments. OBSERVER software is used to calculate and report molar mass and size, 30 times per minute, as opposed to ASTRA’s post-separation, detailed off-line analysis and reporting.

PAT instrumentation is well-known and typically monitors process parameters such as pH, temperature, pressure and feedstock, which are tied indirectly to product properties and quality. UltraDAWN revolutionizes process development with its continuous, direct measurements of actual product properties.

With an intelligent user interface indicating molar mass and size alongside system health diagnostics, photodetectors at eighteen scattering angles plus one more to measure light transmitted through the flow cell, the ultraDAWN provides a solution for:

• Real-time monitoring of Mw from 103 to 109 g/mol and Rg from 10 to 250 nm. For molar mass analysis, the current version of OBSERVER software requires fixed analyte concentration and solvent composition
• Immediate detection of particulate contamination
• Triggering process endpoints or critical alarms

Process integration options

• In-line or on-line configuration
• Process or slipstream flow rates from 0.1 mL/min to 5 mL/min
• Integration with production control software via OPC

Real-Time Operation

In-line – For continuous processes that take place at flows below 5 mL/min, such as microfluidic drug encapsulation or chromatographic purification steps, in-line RT-MALS provides the shortest feedback loop while the ultraDAWN samples the entire process stream.

On-line

When processing takes place within a fixed volume such as a homogenizer, or when the process flow is greater than 5 mL/min, a bypass stream is diverted from the manufacturing process to the ultraDAWN. RT-MALS data are produced in near-real-time.

At-line / Off-line

For detailed analysis, products can be characterized by techniques such as SEC-MALS or FFF-MALS. These provide complete, high-resolution distributions of molar mass and size, though not in real time. A standard DAWN® MALS instrument is employed along with an SEC system or an Eclipse field-flow fractionation system.

RT-MALS for PAT

A PAT strategy that incorporates ultraDAWN for RT-MALS sees reduced development time and fewer samples sent to off-line analytical labs. With real-time CQA analysis, process margins can be optimized and attribute drifts detected immediately, reducing discarded product and increasing yield.

RT-MALS in Lab R&D

RT-MALS can be implemented early in process development – at pilot plant scales and even smaller, such as regular production of small batches of material for R&D use. Experience with lab-scale RT-MALS paves the way for effective implementation on the production floor.

RT-MALS in Scale-up from Lab to Manufacturing

Scaling up often means re-developing the process, requiring off-line analysis of numerous samples collected from intermediate and final steps. RT-MALS greatly accelerates scale-up: it provides real-time feedback, indicating the effect of method tweaks on CQAs, while simultaneously reducing effort and the number of samples analyzed off-line.

Unlike complex at-line PAT devices, ultraDAWN can simply be left in the process flow upon completion of scale up, for continuous or periodic monitoring of the final process.

RT-MALS on the Manufacturing Floor

ultraDAWN and OBSERVER software are simple, robust technologies that may be employed in different ways during serial production. During initial stages, technicians can visually monitor molar mass and size, intervening in the process as needed. With increasing confidence in the capabilities and reliability of RT-MALS, engineers may implement fully-automated process control using OBSERVER’s configurable triggers. In both cases, all data can be reviewed post-production to determine if any deviations were encountered.

Distinctly from the miniDAWN, the microDAWN is uniquely suitable for use in conjunction with UHPLC Size Exclusion Chromatography (SEC-MALS) thanks to its small interdetector dispersion. In some cases it may be used with ion-exchange or other types of chromatography. Regardless of the specifics, the microDAWN produces minimal band broadening in order to maintain the narrow peaks typical of UHPLC.

• Mw from 200 Da to 10 MDa
• Sensitivity: 1.0 µg/mL BSA (66.4 kDa) in PBS; 50 ng/mL 100 kDa polystyrene in THF.
• Rg from 10 to 50 nm
• Ultra-stable laser

The microDAWN and its companion microOptilab Refractive Index Detector interface to most standard UHPLC systems. As these detectors are based on the advanced technology of Wyatt’s miniDAWN and Optilab, respectively, users may be confident in the underlying capabilities cited in thousands of peer-reviewed publications.

Ultrasonic Cleaning Included

Each microDAWN includes a COMET ultrasonic transducer that cleans MALS flow cells automatically by agitating the fluid and loosening particles from the glass surfaces.

Online Operation

The microDAWN will work downstream of all types of UHPLC systems for analysis of molar mass and size distributions (see SEC-MALS), and derivatives such as conformational analysis. For those systems equipped with a UV, RI, fluorescence or other type of concentration detector, the microDAWN accepts up to 4 analog inputs so a 3rd-party concentration signal may be used in MALS calculations of molar mass. The microDAWN is set up downstream of a UV detector and upstream of a RI detector.

In most cases it is beneficial to connect an microOptilab as well as a UV detector. This permits universal concentration detection regardless of fluorophores or chromaphores. The combination of UV and RI signals is utilized in ASTRA’s Conjugation Analysis algorithm to characterize binary complexes such as glycoproteins, PEGylated proteins, membrane proteins enveloped in surfactant, or block co-polymers.

Molar Mass

The miniDAWN® determines absolute molar mass and size to characterize proteins, peptides, small polymers and nanoparticles in solution. Coupled to size exclusion chromatography (SEC or GPC), it eliminates the uncertainty and hassle of column calibration, for true confidence in macromolecular characterization.

Superb Sensitivity

The miniDAWN not only offers superb sensitivity—it further enhances productivity thanks to greatly enhanced field serviceability and easy data analysis with One-Click Mw.

Compatible with ASTRA

The miniDAWN is accompanied by ASTRA, the most versatile software package available for analysis of online multi-angle and dynamic light scattering. ASTRA and the miniDAWN incorporate advanced ease-of-use features that help guarantee excellent measurements with a single click.

Molar mass, absolutely

Wyatt’s miniDAWN is most often used in conjunction with size exclusion chromatography (SEC-MALS) or field-flow fractionation (FFF-MALS) to determine distributions of molecular weight, size and composition. No need to make assumptions about molecular conformation or ideal column interactions.

Characterize:

• Proteins, small peptides and oligonucleotides
• Synthetic and natural polymers
• Nanoparticles, virus-like particles and vesicles

Measurement Range:

• MW from 200 Da to 10 MDa
• rg from 10 to 50 nm, up to 150 nm with shape-specific models
• sensitivity down to 25 ng of 100 kDa polystyrene in THF, injected on a standard GPC column

Ultrasonic Cleaning Included

Each miniDAWN includes an embedded COMET ultrasonic transducer that cleans the flow cell automatically by agitating the fluid and loosening particles from the glass surfaces.

Modularity = Productivity

The miniDAWN incorporates a fully modular design. Each module can be tested independently by the software to verify that it is functioning correctly. This imparts nearly complete field serviceability, since malfunctioning modules can be replaced and validated on-site by service engineers with no further calibration or alignment.

The miniDAWN is also field-upgradeable: the embedded WyattQELS DLS Module for measuring hydrodynamic radii from 0.5 to 50 nm can be installed on-site as well as in new units shipped from the factory.

Not just SEC-MALS…

Wyatt’s miniDAWN may be used in batch (off-line) mode or in conjunction with a Calypso CG-MALS system for characterization of average solution properties as well as macromolecular interaction properties including protein-protein binding affinity and complex stoichiometry.

Simplicity personified

With miniDAWN and ASTRA, SEC-MALS became a whole lot easier. Sample-specific methods are set up in three short steps with ASTRA’s Method Wizard, and new capabilities for automatic data processing mean that it takes just one click from your sample to a full report of the molecular weight and size results.

The miniDAWN’s System Ready monitor will even let you know when the system is fully equilibrated with low baseline noise, ready to run.

Basic On-line Configuration

The miniDAWN may be positioned downstream of all types of standard GPC, HPLC or FPLC systems for analysis of molar mass and size distributions (see SEC-MALS page), and derivatives such as conformational analysis. For those systems equipped with a UV, RI, fluorescence or other type of concentration detector, the miniDAWN accepts 2 analog inputs so a 3rd-party concentration signal may be used in MALS calculations of molar mass. The miniDAWN instrument is generally set up downstream of a UV detector and upstream of a RI detector.

In most cases it is beneficial to connect an Optilab as well as a UV detector. This permits universal concentration detection regardless of fluorophores or chromaphores. The combination of UV and RI signals is utilized in ASTRA’s Conjugation Analysis algorithm to characterize binary complexes such as glycoproteins, PEGylated proteins, membrane proteins enveloped in surfactant, or block co-polymers.

Advanced On-line Configurations

Wyatt’s miniDAWN may also be connected to an Eclipse Field-Flow Fractionation system for characterization of macromolecules and nanoparticles from 1 nm to > 100 nm via FFF-MALS.

Both SEC-MALS and FFF-MALS will benefit from an additional WyattQELS module for measuring nanoparticle size via dynamic light scattering. The WyattQELS utilizes the same laser and flow cell volume as MALS detection. Its collection fiber, placed at 135° to the laser beam, does not displace any of the three detectors in the miniDAWN.

For analysis of natural and synthetic polymers, a ViscoStar online viscometer may also be added as well. Differential viscometry is an orthogonal means of determining molar mass and conformation.

MALS detectors may be operated in batch (unfractionated) mode when the analysis is targeted at:

Macromolecular interactions

• In order to characterize macromolecular or nanoparticle complexes which are disrupted by column shear or dilution
• In order to measure binding affinity or reaction kinetics of macromolecules such as protein-ligand interactions

Average solution properties

• If the sample is not amenable to separation by SEC or FFF, to measure weight-average molar mass, and size as z-average radius
• In order to validate SEC-MALS measurements:
  – to determine if large aggregates or other species are filtered out on the column
  – to determine if reversible aggregates dissociate subsequent to dilution on the column or FFF separation channel

Calypso

Automated sample preparation and delivery system for carrying out and analyzing composition-gradient MALS in a miniDAWN. Analyses include biomolecular interactions analyses high-concentration protein-protein interactions, Zimm plots and dn/dc as well as time-resolved MALS. Utilizes the CALYPSO software for interaction analysis.

Microbatch Kit

Used to inject samples into the flow cell of a MALS detector, and in determining dn/dc with an RI detector.

Premier MALS

The DAWN® is the premier Multi-Angle static Light Scattering (MALS) detector for absolute characterization of the molar mass and size of macromolecules and nanoparticles in solution. The DAWN offers the highest sensitivity, the widest range of molecular weight, size and concentration, and the largest selection of configurations and optional modules for enhanced capabilities.

Used with SEC-MALS

The DAWN is most often used in conjunction with Gel Permeation Chromatography (GPC-MALS) or Size Exclusion Chromatography (SEC-MALS) or Field-Flow Fractionation (FFF-MALS) to determine distributions of mass, size and composition independent of column calibration by reference standards.

Compatible with ASTRA

The DAWN is accompanied by ASTRA, the most versatile software package available for analysis of online multi-angle and dynamic light scattering.

Overview

SEC-MALS with a DAWN and Optilab characterizes conformation and branching of synthetic and natural polymers larger than 10 nm in rms radius Rg. The addition of a ViscoStar differential viscometer creates a GPC triple-detection system that can analyze molecular conformation and size below the rg limit of 10 nm and determine Mark-Houwink Sakurada coefficients.

The combination of a UV/Vis absorbance detector with a DAWN and Optilab refractive index detector creates a UV-MALS-RI triple-detection system, for analysis of copolymers and protein conjugates (glycoproteins, PEGylated proteins, surfactant-bound membrane proteins, etc.). With the addition of a WyattQELS DLS module for size determination, protein conformation may be analyzed.

• Mw from 200 Da to 1000 MDa
• Sensitivity: 10 ng/mL 100 kDa polystyrene in THF, 0.4 µg/mL BSA (66.4 kDa) in PBS
• Rg from 10 to 500 nm, up to 1000 nm with shape-specific models
• Ultra-stable, high-power laser

The DAWN may also be used in batch (off-line) mode to characterize the weight-average molar mass, z-average size and second virial coefficient of unfractionated samples.

In conjunction with a Calypso CG-MALS system, the DAWN characterizes macromolecular interactions, label-free and immobilization-free: affinity and absolute stoichiometry of self- and hetero-association of simple or complex molecular systems. The Calypso system also automates the determination of second virial coefficients, cross virial coefficients, average molar masses and sizes.

Ultrasonic Cleaning Included

Each DAWN includes an embedded COMET ultrasonic transducer that cleans the flow cell automatically by agitating the fluid and loosening particles from the glass surfaces.

Temperature-Controlled Models

• Ambient model
• Ultra-High-Temperature (UHT) model, from ambient up to +210°C
• Standard Peltier heated/cooled (H/C) model, temperature control from -15°C to +150°C

Online Operation

The DAWN may be positioned downstream of all types of standard GPC, HPLC or FPLC systems for analysis of molar mass and size distributions (see SEC-MALS), and derivatives such as conjugation and conformation analyses, all included in the ASTRA data collection and analysis software. For those systems equipped with a UV, RI, fluorescence or other concentration detector, the DAWN accepts up to 4 analog inputs so a 3rd-party concentration signal may be used in MALS calculations of molar mass. The DAWN is generally set up downstream of a UV detector and upstream of a RI detector.

In most cases it is beneficial to connect an Optilab RI detector to the eluent stream. This permits universal concentration detection regardless of fluorophores or chromaphores. The combination of UV and RI signals, acquired simultaneously, is utilized in ASTRA’s Conjugation Analysis algorithm to characterize binary complexes such as glycoproteins, PEGylated proteins, or block co-polymers.

The DAWN may also be connected to an Eclipse FFF system for characterization of macromolecules and nanoparticles from 1 nm to 1 µm via FFF-MALS.

Both SEC-MALS and FFF-MALS will benefit from an additional WyattQELS module for measuring nanoparticle size via dynamic light scattering (DLS). The WyattQELS utilizes the same laser and flow cell volume as MALS detection. The optical fiber may be located in one of the 4 angular positions.

Both SEC-MALS and FFF-MALS will benefit from an additional WyattQELS module for measuring nanoparticle size via dynamic light scattering (DLS). The WyattQELS utilizes the same laser and flow cell volume as MALS detection. The optical fiber may be located in one of the 4 angular positions.

For analysis of natural and synthetic polymers, the ViscoStar may also be added as well. Differential viscometry is an orthogonal means of determining molar mass and conformation.

Batch Operation

MALS detectors may be operated in batch mode when the analysis does not benefit from separation:

• If the sample is not amenable to separation by SEC or FFF (e.g., very large and diffuse polymers such as PMMA) to measure weight-average molar mass, and size as z-average radius
• In order to validate SEC-MALS measurements:
  – to determine if large aggregates or other species are filtered out on the column
  – to determine if reversible aggregates dissociate subsequent to dilution on the column or separation channel
• In order to characterize macromolecular or nanoparticle complexes which are disrupted by column shear or dilution
• In order to measure binding affinity or reaction kinetics of macromolecules such as protein-ligand interactions

Calypso

Automated sample preparation and delivery system for carrying out and analyzing composition-gradient MALS in a DAWN. Analyses include biomolecular interactions, high-concentration protein-protein interactions, Zimm plots and dn/dc as well as time-resolved MALS. Utilizes the CALYPSO software for interaction analysis.

microCuvette

Allows static and dynamic light scattering measurements to be made simultaneously in a MALS detector using a quartz cuvette, requiring only 10 µL.

NanoFilter Kit

Makes it easy to filter, recover and refilter precious sample. This is especially useful when your total sample volumes are below 300 µL.

Microbatch Kit

Used to inject samples into the flow cell of a MALS detector, and in determining dn/dc with an RI detector.