Real-time multi-angle light scattering = RT-MALS for PAT
Whereas MALS is traditionally used in the analytical laboratory, it can also be utilized to develop, monitor and control production processes for nanoparticles, biopharmaceuticals and polymers. RT-MALS, which determines molar mass (Mw), radius (R or Rg) and particle concentration in real time, is used in PAT environments to indicate a process endpoint, collect the appropriate fraction or flag deviations from acceptable product attribute values. This technique is implemented with an ultraDAWN™ MALS instrument and OBSERVER™ software.
Benefits of RT-MALS:
- Accelerate process development and scale-up
- Increase yield, reduce overall time until lot release
- Maintain product quality with confidence
What is RT-MALS?
RT-MALS adds multi-angle light scattering instrumentation and real-time software to process equipment such as preparative chromatography, tangential flow filtration, homogenizers, reactors or nanoparticle production systems. It is a powerful technique for monitoring attributes of macromolecules, nanoparticles and small gene vectors like AAV in terms of:
- Molar mass
- Particle concentration or titer
- Full:total capsid ratio
The process is sampled continuously. At lower flow rates the entire process stream may pass through the ultraDAWN’s flow cell, where it is probed by a laser beam, while for higher flow rates or static vessels like a reactor, a small fraction of the product is continuously drawn off and delivered to the ultraDAWN with optional dilution up to 10x.
The MALS signals, along with UV absorbance signals conveyed by an external UV detector for certain applications, are analyzed to quantify the analyte’s physical properties. Measurements may be made up to 5 times per second for rapid feedback and process control.
An ultraDAWN may be added as a PAT tool in-line or on-line (using an HPLC pump). OBSERVER software controls the ultraDAWN and pump, providing feedback to the process via analog or digital signals.
Measure the product, not the process
Traditional PAT monitors process parameters such as time, concentration, temperature, pH and feedstock. RT-MALS represents a breakthrough in process analytical technology for production of nanoparticles, biopharmaceuticals, gene vectors and polymers: critical quality attributes (CQAs) can be monitored directly, in-line or on-line, for rapid feedback on product attributes and process quality.
RT-MALS is appropriate for advanced products such as polysaccharide antigens, monoclonal antibodies, therapeutic proteins, drug-loaded liposomes, viral vectors or RNA-encapsulating lipid nanoparticles. It quantifies size, molecular weight and particle concentration, and generates triggers to the process when the values meet or deviate from specified ranges. In conjunction with OBSERVER RT-MALS software, ultraDAWN measures primary CQAs for enhanced yield and quality, when and where it counts most: on the manufacturing floor.
RT-MALS is absolute
For most unit operations, RT-MALS provides first-principles measurements that are independent of product-specific calibration curves.
- Basic physical equations connect molar mass with scattered light intensity (measured by MALS) and concentration (measured by UV or dRI).
- Similar equations are used to calculate particle size and concentration from the scattered intensity and angular dependence.
- Genomic payload of AAV and other small viral vectors is calculated from light scattering plus UV absorbance at 260 nm and 280 nm.
- The analysis does not depend on molecular shape/conformation or flow rate and is not impacted by multiple scattering.
RT-MALS determines molecular weight from 1000 g/mol to 1 billion g/mol. It can also determine molecular or nanoparticle radius from 10 nm to 250 nm. MALS detectors only require annual calibration.
Aggregates and impurities
The presence of aggregates and similar product-related impurities, as well as process-related impurities such as free DNA accompanying a gene vector, is inferred from differences in molar mass or size relative to the desired product.
Who uses RT-MALS?
RT-MALS is primarily suitable for monitoring downstream processing (DSP) of biologics as well as the production of synthetic nanoparticles like LNPs.
Bioprocess development scientists working on DSP who normally have to wait days or weeks for fractions or samples to be analyzed in analytical labs, in order to understand the outcome of a new process or modifications to an existing process, gain immediate information on the impact of process parameters, shortening development cycles and reducing time-to-market of a new product.
At the same time, users of RT-MALS gain invaluable process knowledge that can be related directly to scale-up and transferred to process engineers.
Bioprocess engineers who implement RT-MALS can ensure that products are within specifications for each process step, regardless of small changes in process parameters. They will benefit from process knowledge gained in early process development via RT-MALS.
PAT champions immediately recognize the QbD advantages imparted by RT-MALS: direct measurements of relevant product attributes, in real-time, in-line or on-line with unit operations, which will guarantee reliable scale-up, scale-out and site transfer.
Integrate with lab-scale FPLC – and much more
While integrating an ultraDAWN in-line with preparative FPLC is perhaps the most straightforward RT-MALS setup, in fact there are multiple ways to make use of RT-MALS, with a variety of process equipment types.
The ultraDAWN is suitable for flow rates from 0.3 to 150 mL/min and beyond, and so may be placed in-line immediately following the UV or conductivity detector. For larger scale FPLC systems an HPLC pump is used to draw a continuous slipstream at 0.1 to 10 mL/min. The ultraDAWN/OBSERVER system can exchange analog and digital signals with the FPLC to synchronize data collection, transfer analog data between the systems and optimize fraction collection.
RT-MALS operation with other unit operations typically follows one of these configurations: in-line or on-line.
For chromatographic operations at high flow rates, ultraDAWN is configured on-line using an HPLC pump to draw a slipstream. At lower flow rates the ultraDAWN is placed directly in-line with the FPLC.
How does MALS work?
The MALS detector incorporates 18 photodiodes positioned at different angles θ relative to the laser beam to measure the scattered light function R(θ), and one more that measures transmission through the flow cell. At each data point, typically twice or more per second:
- The plot of scattered intensity vs. angle, R(θ), is fit to determine R(0) (the y-intersect at angle θ = 0).
- For macromolecules:
- The weight-average molar mass, Mw, is calculated from the ratio of R(0) and the concentration. Concentration is either derived from UV/Vis absorption, conveyed via analog output to the ultraDAWN, or entered into OBSERVER by the user when concentration is known and constant over the process.
- The z-average radius of gyration, Rg, is calculated by fitting an appropriate equation to the angular dependence of R(θ).
- The z-average radius R is calculated from the angular dependence of R(θ).
- The particle concentration (e.g. viral particles/mL) is calculated from R(0) and the particle radius.
- R(0) is combined with UV absorbance at 260 and 280 nm to determine the molar masses of the proteinaceous capsid and the nucleic acid genome.
- These molar masses are analyzed to determine the full:total capsid ratio Vg/Cp. UV absorbances and molar masses are combined to calculate the total, full and empty capsid titers.
- The rms radius Rg is calculated from the angular dependence of R(θ).
Most of the constants used to determine Mw, radius, particle concentration, Vg/Cp, etc. are related to system optical properties such as laser wavelength and solvent (mobile phase) refractive index. In addition, the analyte’s refractive index (for particles) or dn/dc value, the specific refractive index increment (for macromolecules) in the mobile phase must be known or measured (this is easier than it sounds!) and when using a UV detector the extinction coefficients must be provided. For more details on the theory of MALS, please see our MALS Theory page.
Multi-angle light scattering analyzes the amount of light scattered by the analyte into detectors positioned at various angles relative to the illuminating beam. The extrapolation to zero angle gives molar mass and the angular variation provides the size.
When RT-MALS is insufficient
RT-MALS can only determine average molar masses, particle sizes, particle concentrations and Vg/Cp. Particle size distributions as well as concentrations can be measured at-line with a DynaPro® NanoStar® or DynaPro Plate Reader dynamic light scattering instrument. Another at-line approach is to combine a microDAWN® MALS detector for UHPLC with a sampling front end and UHP-SEC separation, providing detailed quantitation every 3 – 5 minutes.
In-process AAV quantitation using a DynaPro Plate Reader for dynamic light scattering analysis of size distributions and virion concentration.
What constitutes a complete RT-MALS system?
In-line with FPLC, non-GMP
For RT-MALS in-line with FPLC, all you need are a standard preparative FPLC system with an I/O box for analog input/output and digital signals; ultraDAWN; and OBSERVER (for real-time analysis with feedback to the FPLC) and/or ASTRA software (for post-process analysis). Typically this combination will function well for flows up to 150 mL/min.
When monitoring proteins, nucleic acids or AAVs, the FPLC UV detector signal(s) are conveyed via analog cables to the ultraDAWN. Measurements of proteins or nucleic acids require one UV wavelength, while AAVs require two (260 nm and 280 nm). UV is not needed for monitoring larger viral vectors, though the signals may be read into OBSERVER or ASTRA for comparison and synchronization purposes.
Data collection between the FPLC software and OBSERVER or ASTRA is synchronized via exchange of digital pulses. In addition, OBSERVER can output two types of analog signals to the FPLC:
- Trigger: A +2 V ‘trigger’ signal used to indicate that the eluting material meets the pooling criteria;
- Data: An analog voltage proportional to one of the measured properties (e.g. molar mass or Vg/Cp) which may be read into the FPLC software, displayed and stored with the native data and used for fraction collection logic.
ultraDAWN works with popular FPLC systems in protein purification labs.
In-line with other systems, non-GMP
ultraDAWN can be operated in-line with other systems providing relatively low flow, e.g. TFF or a microfluidic nanoparticle production setup. The limit for in-line operation is usually the backpressure on the process equipment: ultraDAWN produces 0.7 bar (9 psi) at 150 mL/min, so if the process can tolerate higher backpressures, higher flow rates can be achieved.
Most other systems with relatively low flow rates will not have the full-featured control software that FPLC offers, with the exception perhaps of TFF. Data collection etc. will usually be controlled manually via OBSERVER or ASTRA. However, if the user provides appropriate analog-to-digital capabilities, similar integration can be performed as with FPLC.
On-line with chromatography, non-GMP
For pilot- and process-scale chromatography, the flow rate is usually too high to go through the ultraDAWN, so the on-line configuration is employed. A quaternary HPLC pump, controlled by OBSERVER, draws off a small continuous slipstream from the main process flow, at 0.1 – 10 mL/min.
The pump can optionally be configured to perform continuous dilution of the slipstream sample. Dilution is important for these specific conditions:
- when the mobile phase has low ionic strength, the sample is diluted with moderate- or high-ionic-strength buffer in order to screen electrostatic interactions between molecules
- When the mobile phase has varying ionic strength and high concentration (> 2 mg/mL), the sample is diluted with moderate-ionic-strength buffer at a high dilution ratio in order to achieve a nearly constant, well-buffered condition. When the sample is extremely turbid, it is diluted in order to enable light transmission.
- When the sample is not diluted, it can be returned to the process, but — depending on the relative sampling volume and dilution ratio — this may be undesirable when dilution is applied.
In the on-line configuration, OBSERVER can exchange analog signals with the FPLC as explained in the in-line FPLC setup section, but cannot exchange digital signals for synchronizing data collection – that must be done manually.
On-line with other process equipment, non-GMP
Using the quaternary HPLC pump, ultraDAWN can also be connected to high-flow-rate TFF or fill-finish equipment, as well as stationary vessels like chemical reactors. Usually these do not offer analog-digital interfaces, but if they are available, ultraDAWN can take advantage of them, for example using the analog trigger output to control quenching a reaction or switching a valve between pool and waste. If UV measurements are needed they can be provided with a stand-alone UV detector placed between the pump and ultraDAWN.
- For post-process analysis with 21 CFR Part 11 compliance, ASTRA is available with the ASTRA Security Pack.
- For real-time analysis, OBSERVER is available with OPC-UA connectivity. Users may write an OPC-UA client that takes care of compliance requirements such as a secure database or audit trails. The OBSERVER UI is locked against user intervention when connected to an OPC-UA client.
- The ultraDAWN is not, at this time, suitable for spray-down sterilization or gamma irradiation/autoclaving of the flow cell.
- The flow path may be cleaned internally with 1 M NaOH or most other standard sterilization solutions, and cleaning is validated along with the entire skid.
- The instrument may be housed in a cabinet suitable for spray-down sterilization.
For chromatographic operations at high flow rates, ultraDAWN is configured on-line using an HPLC pump to draw a slipstream. At lower flow rates the ultraDAWN is placed directly in-line with the FPLC.
Quantify product attributes when and where it counts most
The key application areas of RT-MALS are biotherapeutics, vaccines, viral and non-viral gene vectors and nanoparticle drug delivery vehicles. Below you will find a select set of application. Additional links to application notes, webinars, our searchable bibliography and more are ravailable in the Resources tab. If you find something promising, please contact us by clicking on the Request Info button, and an expert Application Scientist will be in touch to help ascertain if SEC-MALS meets your specific characterization goals.
In order to collect virus-enriched fractions during ion-exchange chromatographic purification, RT-MALS monitors particle radius R. The trigger is set to turn on and activate a collection valve when R is within a suitable range for differentiating monomeric virus particles from smaller proteins, DNA, virus aggregates or cell lysate fragments. At the same time, it calculates viral particle concentration for immediate determination of titer in the pool.
Learn how MALS is used in rapid at-line monitoring of virus-like particle purification in Aguilar et al., “At-line multi-angle light scattering detector for faster process development in enveloped virus-like particle purification”, J. Sep. Sci. (2019)
RT-MALS optimizes chromatographic purification of viral vectors by discriminating viruses from impurities.
A tangential flow filtration step for concentrating viral vectors can be controlled by an ultraDAWN set up to monitor particle concentration. The ultraDAWN is connected to the TFF flow path in an on-line configuration by a pump and continuously receives sample (which is subsequently returned to the TFF system). In OBSERVER, the trigger is programmed to turn on when the concentration is above the specified end point, signaling to the control system to terminate the process.
Simultaneously, the viral size is monitored to immediately identify potential particle degradation.
View recent webinars on the use of RT-MALS for monitoring downstream viral vector production processes.
UF/DF of viral vectors is both controlled and monitored for product quality by ultraDAWN with OBSERVER real-time software.
In this process, hydrolysis of the polysaccharide must be quenched when it reaches the target molecular weight of 350 kDa. Using the on-line configuration, sample is continuously drawn from the reactor, diluted with buffer and delivered to the ultraDAWN. RT-MALS monitors molecular weight and sets the trigger when MW is below the specified end point.
Download the application note AN8005: RT-MALS end-point determination of a polysaccharide depolymerization process. MALS has also been demonstrated in-line with preparative size-exclusion chromatography for real-time monitoring of protein-polysaccharide conjugate purification in Rajendar et al., “Multi angle light scattering as a process analytical technology tool for real-time monitoring of molar mass of protein-polysaccharide conjugate fractions”, J. Chromat. Open (2022).
RT-MALS ensures product quality in this depolymerization step, regardless of variations in the starting material.
The weight-average molecular weight determined by RT-MALS can be used to estimate aggregate content in a polishing step using an ultraDAWN in-line with flow-through hydrophobic interaction chromatography. In this example with a 146.8 kDa mAb, OBSERVER’s trigger is programmed for a band between 145 and 152.5 kDa, the latter corresponding to 3.5% aggregate. The trigger turns on at 31 minutes and off at 91 minutes for overall aggregate content in the pool of 0.9%.
Read how RT-MALS was first demonstrated to provide highly accurate monitoring of aggregate load during flow-through mAb purification in Patel et al., “Multi-angle light scattering as a process analytical technology measuring real-time molecular weight for downstream process control”, mAbs 2018.
An example of in-line MALS for monitoring and optimizing the pooling of a monoclonal antibody Fab arm purified by bind-and-elute ion-exchange chromatography appears in AN8004: Coupling MAS with preparative ion-exchange (pIEX) for structural biology applications.
Real-time molecular weight monitoring identifies aggregate content in order to optimize yield while maintaining overall pool quality.
Ion-exchange chromatography (IEX) is a common method for separating empty and full adeno-associated viral vectors as well as removing residual impurities. RT-MALS may be implemented in prep-scale IEX systems in order to monitor the capsid particle and viral genome concentrations of every eluting fraction, and signal when Vg/Cp is above a specified value in order to control pooling. At the same time ultraDAWN and OBSERVER provide other key product attributes that can be related to aggregation and discrimination between AAVs and process impurities like free DNA.
Wagner et al., “Biophysical Characterization of Adeno-Associated Virus Vectors Using Ion-Exchange Chromatography Coupled to Light Scattering Detectors”, Intl. J. Mol. Sci (2022) presents the use of MALS in-line with IEX for comprehensive characterization of AAVs: capsid titer, full-to-total ratio, absolute molar mass of the capsid and nucleic acid, size and polydispersity. These CQAs and other attributes can now be determined in real time for prep-scale IEX with ultraDAWN and OBSERVER.
Both capsid particle and viral genome concentrations are monitored by RT-MALS, along with the capsid ratio Vg/Cp, capsid and genome molar masses and virus size. Together these indicate pooling for enrichment while eliminating aggregates and impurities.
Nanoparticle drug delivery
Incoming liposomes begin with a wide size range but are reduced to a uniform size via homogenization, emerging within a narrow size band. RT-MALS monitors product attributes such as radius and concentration; if they fall outside the specified range, OBSERVER signals the process control system to divert out-of-spec product to waste in order to avoid contaminating the collection vessel.
Download the application note AN8006: In-line monitoring of liposome size by RT-MALS.
ultraDAWN monitors nanoparticle production processes such as extrusion, homogenization of microfluidic precipitation in order to flag deviations from specified product attributes.
Shed new light on your process
No more long waits for offline sample analyses. No more wasted batches. With RT-MALS you can finally see what you need to know – true product attributes – in your PD lab or on the production floor, in real time, measured directly, no models.
How do I use RT-MALS?
Since RT-MALS is used with a variety of processing steps and equipment, the specifics of how it is used will depend on the application. A general outline is:
- Get the sample to the ultraDAWN.
- If the ultraDAWN is in-line with the process flow, this is trivial.
- If the on-line configuration is used, connect inlet line A of the quaternary HPLC pump to the sample source (reactor vessel or main process flow line). Inlet line B is placed in a bottle with the process buffer, line C may be placed in a bottle of diluent and line D may be placed in a bottle of wash solution.
- A reactor vessel might have a constant concentration
- Other systems will require a UV analog signal or other detection method, and the delay volume between the concentration detector and ultraDAWN must be established.
- AAV monitoring requires two UV wavelengths
- In-line configuration: run elution buffer or whatever solvent will be present with the product.
- On-line configuration: The process buffer/solvent is provided from inlet line B.
ultraDAWN is generally calibrated once or twice per year and does not require calibration curves for most products. If a new solvent is introduced that differs significantly in refractive index from the previous solvent, it may be necessary to perform ‘normalization’, a simple procedure that involves flowing through the ultraDAWN 40 KDa dextran or a similarly small polymer or protein dissolved in the new solvent.
When connected to OBSERVER, the ultraDAWN’s touch-screen display provides real-time readings of current and recent measured attributes.
Operation of OBSERVER is based on customizable workflows:
- Inline or Online Macromolecule – for monitoring and controlling processing of polysaccharides, proteins, etc.
- Inline or Online Nanoparticle – for monitoring and controlling processing of viruses, LNPs, liposomes, etc.
- Inline AAV – for monitoring processing of AAV, other small viral vectors or virus-like particles encapsidating nucleic acids, or small UV/Vis-active nanoparticles (up to ~ 50 nm in diameter) that encapsulate drug substances like peptide or small-molecule drugs.
- Performance Qualification
- Automated cleaning
In-line Macromolecule, Nanoparticle or AAV
OBSERVER’s in-line workflows:
- Synchronize with an FPLC or microfluidic system
- Report for the main process flow
- Macromolecules: weight-average molar mass Mw and z-average rms radius Rg
- Nanoparticles: z-average rms radius R and particle concentration PC
- AAV: total, full and empty particle concentration; total, capsid and payload molar mass; full-total ratio; z-average rms radius Rg
- Macromolecules: Mw and total mass
- Nanoparticles: PC and total number of particles
- AAV: total, full and empty particle concentration; total, capsid and payload molar mass; full-total ratio; z-average rms radius Rg, and total number of capsids, number of full capsids and number of empty capsids.
On-line Macromolecule or On-line Nanoparticle
OBSERVER’s on-line workflows:
- Control an auxiliary pump to:
- Draw a sample flow from the main process flow
- Dilute the sample up to 10:1
- Pass the sampled product to the ultraDAWN
- Macromolecules: Mw and Rg
- Nanoparticles: R and PC
Basic OBSERVER operation is controlled and data are reviewed from a Windows graphical user interface. For GMP operation, OBSERVER becomes an OPC-UA server. A user-supplied client program communicates with OBSERVER to perform control and data retrieval.
21 CFR Part 11 compliance for RT-MALS is achieved via OPC-UA control.
ASTRA software is used when real-time measurements are not needed and it is sufficient to acquire the data during the process, then analyze afterwards. ASTRA is built in many ways like typical chromatography software.
For those in GMP/GLP-regulated environments, ASTRA’s Security Pack add-on enables 21 CFR Part 11 compliance including full audit trails and electronic signatures.
Wyatt Technology provides several avenues for supporting novice and advanced users, including tutorials, training materials and technical notes in the online Wyatt Support Center; phone and email support; and Light Scattering University. Site visits may be arranged for IQ/OQ, service and preventive maintenance as well as group training.
With ultraDAWN, ASTRA provides comprehensive characterization methods for post-process analytics.
Based on Wyatt’s award-winning MALS technology
More than 45 years ago, Wyatt Technology's scientists invented the very first commercial light scattering instruments incorporating lasers as their light source. We've been defining and redefining the state-of-the-art for laser light scattering hardware, software, training and services to meet customer needs for over three and a half decades.
With RT-MALS, Wyatt brings that same commitment to PAT for process development scientists and production engineers.
Founded in 1982 by Dr. Philip Wyatt (center), Wyatt Technology still maintains its core values of quality and personal service.
ultraDAWN - The only MALS detector for process and real-time operation. Incorporates detectors at 18 angles to determine molar masses from 200 Da to 1 GDa and radii from 10 to 250 nm, as well as a transmission detector for obtaining accurate scattering measurements under turbid conditions.
- Standard option: ambient temperature; accommodates flow rates from 0.1 to 150 mL/min with tubing changes
- Heated/cooled option: -15° C to +150 °C; accommodates flow rates from 0.1 to 5 mL/min
Both ultraDAWN versions accept optical attenuators in order to measure turbid solutions. Learn more.
OBSERVER Real-time MALS measurements for process analytics and process control.
- Accepts MALS data and UV analog data (digitized by the ultraDAWN) to quantify product attributes of macromolecules, nanoparticles and AAVs or other sub-50 nm vehicles for gene or drug delivery.
- In-line: integrates with FPLC and similar systems via analog and digital signal exchange. Outputs analog trigger signal and analog data to FPLC system.
- On-line: controls a quaternary HPLC pump to draw a slipstream with optional dilution. Outputs analog trigger signal to process control.
- Macromolecules: determines molar mass and rms radius. Aggregate level can be estimated from changes in molar mass.
- Nanoparticles: determines average particle size and concentration. Particles are discriminated from free macromolecules by size.
- AAVs (and other small vectors): determines Vg/Cp, total and full concentrations, capsid and payload molar mass, rms radius. Aggregate levels can be estimated from changes in capsid molar mass.
- OPC-UA connectivity for commercial processes and GMP operation
ASTRA - Comprehensive post-process analysis of in-MALS measurements to determine molar mass, size, particle concentration and more. Also analyzes online DLS data.
- The Security Pack add-on enables 21CFR(11) compliance, including full audit trails and electronic signatures.
Delve deeper to learn how RT-MALS works and performs real-time quantification of product attributes for proteins, viral vectors, lipid nanoparticles and more. These resources are all available on our web site.
On-demand webinars presenting the theory and applications of RT-MALS are available for unrestricted viewing on the RT-MALS Webinars page. Listed below are some of the most popular:
Application notes and white papers
Application notes and white papers highlighting the use of RT-MALS are available for unrestricted viewing on the RT-MALS Application Notes page. Listed below are some of the most popular:
An extensive searchable bibliography of publications Wyatt SEC-MALS instruments is available at www.wyatt.com/Bibliography. Just open the Advanced Search and click the Multi-Angle Light Scattering box, then enter your search terms below.
Wyatt offers its customers comprehensive online support via the Wyatt Support Center, including many technical notes that can help make the most of RT-MALS experiments. If you are a customer and do not have access, please request an account on the Support Log-in page.
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