RT-MALS: Real-time product attributes, from lab to plant


Wyatt’s analytical instruments, based on multi-angle static light scattering (MALS) and dynamic light scattering (DLS), have played essential roles in the development of biotherapeutics for over three decades. Early applications specialized in determining absolute molar mass, size, charge, conformation, conjugation, aggregation and interactions of therapeutic proteins such as IgG(1); polysaccharides like hyaluronic acid(2); antigenic glycoproteins including viral surface motifs (3); and viruses or virus-like particles as vaccines(4). More recently the DAWN® MALS instrument and DynaPro® DLS detectors have been enhanced with the latest cutting-edge capabilities to enable characterization of the titer and genomic content of viral and non-viral gene vectors such as adeno-associated virus(5) and RNA-bearing lipid nanoparticles(6). Common to all of these applications of light scattering is their place in the scheme of biologic development and bioprocess: primarily in the analytical and formulation lab, with a few specific instances in quality control.

Given the importance of MALS and DLS instruments in product development, it makes sense to ask how they might benefit process development and as process analytical technologies (PAT). In fact, MALS and DLS are often used for off-line analytics during process development. Depending on the properties of the specific product, especially (but not always) size range, fractions collected during downstream processing (DSP) may be analyzed by SEC-MALS, FFF-MALS and/or batch DLS in automated plate format to assess size or molar mass, polydispersity, aggregate and impurity content, titer and payload. The value of such analyses notwithstanding, off-line measurements of product attributes cannot provide the level of immediate feedback that would constitute a game-changing productivity boost for biomanufacturers.

Click to enlarge. Advanced analytical applications of MALS include quantifying multiple key attributes of AAV (top graphic) or LNP-RNA (bottom graphic) in a single, short, reagent-free, automated run, replacing a multitude of individual methods that may require reagents as well as significant time and sample preparation efforts.

PAT and light scattering

PAT is commonly implemented to great effect in many chemical manufacturing environments, but the challenges of bioprocess, including regulatory aspects, have presented significant obstacles to its acceptance in biologics production. Those barriers are starting to come down: increasingly, the FDA is encouraging sponsors to improve yield and quality by adopting PAT, and at the same time there is growing recognition of the potential benefits of PAT on the part of the bioprocessing industry. These benefits include:

  • Monitoring and controlling the process in real time
  • Streamlining processing, improving yield
  • Stopping a bad process early to minimize wasted time, material and resources
  • Effectively delivering consistent, quality product
  • Advancing to the next stage with confidence
  • Transferring successfully across scales and sites

Recognizing the potential of light scattering-based PAT to make a substantial contribution to DSP productivity, Wyatt Technology released its first real-time MALS (RT-MALS) products in November of 2019. These consist of the ultraDAWN RT-MALS instrument accompanied by OBSERVER real-time software. Unlike standard PAT, which monitors process parameters and attributes, RT-MALS sets a new paradigm: it measures, in real or near-real time, actual product attributes. Currently, those attributes are weight-average molar mass, z-average size, and/or particle concentration (physical titer).

The ultraDAWN measures molar mass, size and particle concentration in real time in order to monitor product attributes inline or online with downstream processes.

Monitor the product, not the process

The prevalent paradigm in biologic manufacturing is ‘the process is the product’. This means that if you understand and have good control over all aspects of the process, you can assure consistent product quality. Hence, standard PAT tools monitor process conditions and indirect product attributes. For example, in a protein purification process, a process attribute (elution volume) or indirect product attribute (UV absorption) may be monitored to decide when to cut a pool in order to minimize aggregate content, even though these parameters are not direct indicators of aggregate content. This approach to PAT may rely on process modelling, entailing a major investment in acquiring the process knowledge needed to build up a reliable model. It also presents an impediment to process improvements which may result in different process parameters, even though the end product is identical or better.

RT-MALS, on the other hand, addresses relevant product attributes head-on. In the previous example, the weight-average molar mass of a protein solution will change in a very specific, predictable and well-understood manner when aggregates begin eluting. Unlike elution volume or UV, variations in the process feedstock or column aging will not cause errors in the pooling cutoff when it is tied to molar mass.

In another application, purification of a virus may require identifying impurities such as free proteins or nucleic acids. UV absorption does not differentiate well between an intact virus and free macromolecules. With RT-MALS, the large size of the virus is exploited to discriminate between it and impurities. In both cases, reliance on process parameters or indirect product attributes necessitates large safety margins resulting in low yield; directly monitoring relevant product attributes with RT-MALS enables smaller safety margins, correspondingly higher yield and enhanced process flexibility – the ability to improve the process without undue burden in re-validation.

Reducing time to market

RT-MALS bridges off-line analytics with real-time response. Whereas off-line measurements might take minutes to hours per fraction (not including sampling time, transit time and lab queues), in-line RT-MALS is essentially instantaneous, providing measurements up to five times per second. For some applications, RT-MALS may be configured on-line, automatically sampling the process vessel or flow path with a pump; in this case, depending on the specifics, it provides data at the same rate as in-line monitoring, but with delay times relative to the process from several seconds up to a few minutes.

In process development and scale-up, RT-MALS means rapid turn-around on the analytics. Instead of sending fractions off to the analytical lab and waiting days or weeks to learn how each tweak to process conditions impacted the production profile, the results are immediate. More conditions may be tested in less time, for faster optimization – and that means that a product can be commercialized and marketed in much less time than usual.

RT-MALS is not a complete substitute for detailed off-line characterization; the tradeoff is time versus detail. An off-line SEC-MALS run provides complete molar mass distributions, while RT-MALS only provides a weight average. On the other hand, SEC-MALS generally runs for 30 minutes or more and requires sampling, transfer to the analytical lab and at least some sample modification, while RT-MALS takes less than a second and is performed in situ. In the end, process development can be sped up greatly with RT-MALS, minimizing—but not completely eliminating—off-line analytics.

The ultraDAWN may be configured to sample and measure either in-line or on-line, depending on the type of process.


Click to enlarge image. OBSERVER may be programmed to provide a trigger upon passing pre-determined limits (green vertical line), and turn the trigger off upon exiting an acceptable attribute range (red vertical line).


New capabilities and applications

The first publication of RT-MALS for protein purification, by Patel et al. (7), described a prototype in-line RT-MALS system for a flow-through hydrophobic-interaction chromatography (HIC) process. Fortuitously, in flow-through mode the protein concentration and buffer composition are essentially constant during HIC elution, eliminating variability in MALS analysis that may arise from protein-protein interactions that depend on concentration and buffer. The authors monitored aggregate content via changes in molar mass, determined by combining MALS and UV data. The limit of detection was found to be less than 0.25% dimer, enabling precise, real-time pooling decisions that would potentially result in acceptable aggregate content with maximum yield, adding considerable value to each product lot.

OBSERVER 1.0: Online Macromolecule WF

The inital commercial release of ultraDAWN and OBSERVER 1.0 was directed to similar applications, where 1) product concentration and buffer conditions do not change, and 2) the desired product attributes involve molar mass (Mw) and/or size (Rg). Besides continuous-flow protein purification, these include polymerization, depolymerization (e.g. the depolymerization of polysaccharides for vaccines) or the conjugation of proteins and polysaccharides in a reaction vessel.

The polymer reactions inherently require on-line sampling in order to bring sample to the pump, so OBSERVER 1.0 only included an online workflow for molar mass and size reporting: the Online Macromolecule workflow. A quaternary pump, controlled by OBSERVER, is used to aspirate sample from the reactor and dilute with buffer in order to achieve optimal measurement conditions in the ultraDAWN. OBSERVER provides continuous analysis of Mw and Rg, and generates triggers upon entering or exiting a pre-determined range of one of these attributes. An example of RT-MALS application to polysaccharide depolymerization is provided in AN8005: RT-MALS end-point determination of a polysaccharide depolymerization process.

Click to enlarge image. RT-MALS provides immediate indication of increasing aggregate breakthrough in continuous-flow HIC purification. See Patel et al. (7)

OBSERVER’s Online Macromolecule workflow controls a pump to either sample a reactor vessel or a draw a slipstream from a flowing process, and determines average molar mass and size.

OBSERVER 1.1: Online Nanoparticle WF

In response to the COVID-19 pandemic and the emergence of novel vaccines based on viral vectors and nanoparticles, OBSERVER 1.1 implemented on-line nanoparticle monitoring. Unlike the Online Macromolecule workflow, OBSERVER 1.1’s Online Nanoparticle workflow calculates particle size and concentration. It is not restricted to constant-concentration scenarios, and because these measurements are not particularly susceptible to changes in solvent composition, it does not require a constant buffer. This makes the Online Nanoparticle workflow suitable for a variety of applications related to nanoparticles, such as:

  • viral-vector purification, ultrafiltration / diafiltration and fill-and-finish (especially larger viruses such as adenovirus or lentivirus)
  • lipid nanoparticle or liposome formulation development, production and homogenization.

OBSERVER’s Online Nanoparticle workflow determines average size and particle concentration of virions or lipid nanoparticles.

Integration concept of RT-MALS into a fill and finish process via the on-line configuration. Since the ultraDAWN may be sterilized with 1 m NaOH, a slipstream drawn from the main F/F line may be returned to the process following measurement of nanoparticle size and concentration.

Integration concept of RT-MALS into an ultrafiltration / diafiltration process via the on-line configuration to determine titer and size. Sampling may occur in the reservoir, retentate prior to DF, retentate following DF or permeate.

OBSERVER 1.3: Inline Nanoparticle & UV

OBSERVER 1.2 introduced many GUI and behavioral improvements to RT-MALS, but the next key feature appeared in OBSERVER 1.3, the Inline Nanoparticle workflow. This workflow supports applications such as lab-scale chromatographic purification of viral vectors or LNP-RNA formulation and production.

Whereas in online workflows, OBSERVER fully controls sampling and flow to the ultraDAWN, during inline workflows the flow is set by an external system such as an FPLC or a microfluidic LNP formulation device. In these scenarios, OBSERVER must synchronize with the external system. Synchronization is carried out through the simple exchange of digital pulses and analog signals. Example instructions for Cytiva’s UNICORN software to synchronize with OBSERVER are included in the OBSERVER User’s Guide.

In addition to synchronization with the external system, the Inline Nanoparticle workflow exports an analog voltage that is proportional to one of the calculated attributes—size or particle concentration. Hence the real-time data may be imported to the external system for recording, display and even pooling decisions made by its own software, e.g. UNICORN.

RT-MALS has been demonstrated for in-line monitoring of the elution of an adenovirus-based vaccine product from a lab-scale preparative anion-exchange column.: The study was presented by Clara Pérez Peinado, Janssen Infectious Diseases and Vaccines, at the 2021 International Symposium on the Separation of Proteins, Peptides and Polynucleotides, with example data shown here to the right. Particle radius was used as the criteria for pooling, and viral titer calculated for the total pool. As seen in the figure, instantaneous concentrations above 6 x 1012 virions/mL were measured. Size and final pool titer were in excellent agreement with expected values and off-line analytical methods.

Another new feature in OBSERVER 1.3 is the ability to couple ultraDAWN to a UV absorption detector, in order to determine the molar mass of UV-active molecules such as proteins or nucleic acids, without the constraint of constant concentration. The UV concentration signal is imported as an analog voltage. This feature is relevant to macromolecule workflows (currently only the Online Macromolecule workflow is available, but an Inline Macromolecule workflow is planned for release in early 2022).

Molar mass analysis using concentration from UV should be limited to conditions where intermolecular interactions are either negligible (concentration in the MALS flow cell is below roughly 1-2 mg/mL and ionic strength > 50 mM) or constant (buffer conditions do not change).

Post-processing for advanced applications

Wyatt’s analytical software for MALS, ASTRA®, supports a variety of advanced analyses that are not currently implemented in OBSERVER. A prominent example is viral vector analysis, which calculates the VgCp ratio (genomic fill factor) and capsid concentration of small viral gene vectors such as adeno-associated virus (AAV). While mostly used in the analytical lab, this analysis would also be of great value in process development labs engaged in DSP such as ion-exchange chromatography for purifying full AAVs from empty capsids.

An intermediate, near-real-time solution for determining VgCp and capsid concentration during IEX purification utilizes the ultraDAWN in ‘post-processing’ mode. In this scenario, the ultraDAWN is placed in-line with the FPLC system and ASTRA collects MALS and UV data during the elution. While there are no real-time results calculated, the data are analyzed immediately following elution, in situ in the PD lab, for timely information on the content of each fraction. The development scientist obtains pertinent results in a very short time, in contrast with the delay of days or weeks incurred when fractions must be sent off to the analytical lab. Post-processing analysis using ASTRA and an ultraDAWN in-line with FPLC affords early benefits, and a smooth transition to real-time PAT once the application is implemented in OBSERVER.

OBSERVER’s Inline Nanoparticle workflow synchronizes with an external system such as an AKTA avant with UNICORN chromatography software to optimize pooling based on size. OBSERVER also provides immediate determination of virion titer in the collected pool.

Real-time measurement of adenovirus elution from an anion-exchange purification column, showing particle size and concentration, and the region identified for pooling based on size. Data courtesy Clara Pérez Peinado, Janssen Infectious Diseases and Vaccines.


The current ultraDAWN offering fulfills a sizeable subset of DSP PAT applications in the realm of biologics and nanomedicines, primarily in-line for lab-scale process development, and on-line, for sampling of in-place processes (reactors, homogenizers) or high flow rate processes (pilot plant and full production scale). Recognizing that a central goal of process development scientists and engineers is to scale up their PAT tools from the lab to the plant, Wyatt is committed to further developing RT-MALS technology to support larger scales and higher flow rates, so that PAT knowledge obtained in the process development lab can be utilized during all phases of drug product commercialization.


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2Mendichi, R. and Schieroni, A.G. “Fractionation and characterization of ultra-high molar mass hyaluronan: 2. On-line size exclusion chromatography methods”, Polymer 43(23):6115-6121 (2002). https://doi.org/10.1016/S0032-3861(02)00586-4

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