Dynamic Light Scattering (DLS) for Monitoring Protein Aggregation

To ensure the stability and usability of injectable biopharmaceutical products, protein aggregation and active principle ingredients (API) are of the utmost concern. Protein aggregation is a process that can occur at any point during the lifetime of a therapeutic protein, whose stages include protein expression, refolding, purification, sterilization, shipping, storage and delivery.

While the exact mechanism by which protein aggregation occurs remains unclear, it has been established that certain manufacturing processes such as formulation composition, the presence of microbial or vial contaminants during cell culture, as well as the potential influence of storage conditions propagating the risk of chemical degradation can play a role in increasing the risk of physical degradation and the formation of protein aggregates.

In particular, the conditions of the storage containers, such as prefilled syringes that can leak silicone oil from the rubber stopper, play an important role in producing protein aggregates. In an effort to develop more stringent international health regulations regarding the control of biopharmaceutical products, researchers are interested in improving the in-situ monitoring of the potential denaturation and degradation of therapeutic proteins during production processes and subsequent storage.

Current Measurement Techniques and Limitations

There are various different analytical techniques that can be used to investigate the presence of protein aggregates within the micrometer (μm) range. Some examples of these methods include light obscuration (LO), dynamic imaging particle analysis (DIPA), micro-flow imaging (MFI) and Coulter counter (CC).

To detect and quantify the presence of protein aggregates at an early stage, other techniques including multi-angle static light scattering (MALS) coupled with separating techniques such as size-exclusion chromatography (SEC), analytical ultracentrifugation (AUC) and asymmetrical-flow field-flow fractionation (AF4) are also commonly used.

Additionally, the use of batch Dynamic Light Scattering (DLS) can be useful in the characterization of protein aggregates when the dilution or shear encountered in SEC or FFF causes disassociation of the aggregates, or when measuring protein aggregation under differing conditions and/or temperatures.

While each of these techniques are highly efficient in their range of use, they often require specific preparation and handling of the sample either prior to or during measurement, both of which have the potential to modify the sample aggregation state.

Therefore, the most preferred method of sample manipulation and handling involves directly measuring samples into the storage medium, such as through the use of a hermetically sealed vial or syringe. Unfortunately, none of the aforementioned techniques are capable of directly measuring protein aggregates in an injectable.

In situ Contactless Measurement: The VASCO Kin © Concept

Cordouan’s VASCO Kin, which is based on real time in-situ contactless remote DLS measurements, has recently emerged to address the limitations of traditional protein aggregation test methods. DLS, which is a prevalent technique that has been used in both colloidal sciences and proteins characterization studies, is a mature and very powerful technique that is based on the analysis of scattered light fluctuations that occur as a result of the Brownian motion of particles.

This phenomenon allows for the accurate measurement of particle sizes ranging from one nanometer to several microns in a minute. Different measurement configurations are available for commercial DLS systems, however each of these options require the user to withdraw the sample with a pipette or automatic pump and subsequently place and/or inject the sample into the instrument prior to the measurement. In doing so, this procedure has the potential to negatively affect the sample protein aggregation state.

To avoid this issue, the VASCO Kin is a fully agile DLS system that utilizes its unique Optical Fiber Remote Probe (OFRP), which is an optimized and highly robust optomechanical assembly that is designed to make direct and contactless measurements that do not require any sample batching process.

The OFRP, which is connected to an Optical Unit by a special optical fiber umbilical, injects a laser beam into the sample and collects scattered light from the sample in the backward direction at an angle 170°.

The Avalanche Photodiode Detector (APD) is a highly sensitive single photon that is connected to a dedicated fast acquisition electronic board to provide a real time monitoring of the intensity of the fluctuations of scattered light. These fluctuations are then converted into time-resolved particle size kinetic analysis through powerful mathematical algorithms.

Measurement in Vaccines Syringes

To demonstrate the capabilities of the VASCO kin in achieving contactless in situ measurements into a syringe, a series of particle size measurements on a commercial injectable flew vaccine were performed.

This vaccine is a complex medium that is comprised of a mixture of many different ingredients including both deactivated and fragmented Flew Viruses that are derived from three different stem cell lines, several excipients including ppi water, potassium chloride, sodium chloride, buffered saline solution, as well as traces of chicken eggs proteins, which is otherwise known as ovalbumin, formaldehyde, neomycin and octoxinol 9, to name a few.

During the measurement procedure, the vaccine syringe was removed from its plastic packaging and placed in a dedicated mount 6 cm in front of the probe.

To obtain adequate comparisons as well as evidence on the possible aging effects of the vaccine, one vaccine was stored at 7 °C, whereas a second vaccine was stored at room temperature for 8 months. The two vaccines were then measured simultaneously in the same conditions the same day and the particle size distribution was evaluated.

For the vaccine stored at 7 °C, the sample exhibited a relative complexity of the sample particle size distribution with three distinct peaks ranging from 30 nm up to 800 nm, all of which correspond to virus fragments and proteins. Additionally, several aggregates were also visualized beyond 10 μm.

The VAXIGRIP vaccine syringe out of its blister (left); Measurement setup (right) with the VASCO Kin remote head mounted on a dedicated translatable stage and placed in front of holder designed for the purpose of the experiment

Figure 1. The VAXIGRIP vaccine syringe out of its blister (left); Measurement setup (right) with the VASCO Kin remote head mounted on a dedicated translatable stage and placed in front of holder designed for the purpose of the experiment

In comparison, the vaccine stored at room temperature showed noticeable changes in the particle size distribution within a broad continuum of 10 nm to 10 μm and higher. As these preliminary studies were used for the in situ DLS measurement into injectable syringe, further investigation must be performed.

Particle size distribution measurement results (X axis: size in nm; Y axis Amplitude in arbitrary unit) of a vaccine stored in a fridge (top) and of a vaccine stored at room temperature for 8 months (bottom)

Figure 2. Particle size distribution measurement results (X axis: size in nm; Y axis Amplitude in arbitrary unit) of a vaccine stored in a fridge (top) and of a vaccine stored at room temperature for 8 months (bottom)


The first practical demonstration of a contactless in situ particle size measurement of a commercial injectable vaccine directly into a syringe was performed here. By eliminating sample batching steps, the VASCO Kin system utilizes an innovative optical fiber remote head that can be applied to a wide range of particle size measurement systems, such as for the in situ monitoring of protein aggregation within biopharmaceutical injectable products.

The VASCO Kin is also a useful tool for monitoring the real time nanoparticle synthesis kinetics of various types of reactor configuration, such as a double jacket glass reactor, high pressure and high temperature Super Critical CO2 autoclaves, microwave reactors, micro fluidic chips or for instrumental coupling.


1. E.Y. Chi, “Excipients and their Effects on the Quality of Biologics”, AAPS J. (2012), accessed January 2015.

2. M. Hasija, L. Li, and N. Rahman et al., Vaccine: Development and Therapy

3. R. Manning et al., “Review of Orthogonal Methods to SEC for Quantitation and Characterization of Protein Aggregates,” BioPharm International 27 (12)

4. Berne, B.J.; Pecora, R. Dynamic Light Scattering: Willey, New York, 2nd edition- 2000 (ISBN 0-486-41155-9)

5. VASCO Kin : https://www.cordouan-tech.com/products/vasco-kin/

6. A. Schwamberger & al, “Combining SAXS and DLS for simultaneous measurements and time-resolved monitoring of nanoparticle synthesis”, Nuclear Instruments and Methods in Physics Research B 343 (2015) 116–122

This information has been sourced, reviewed and adapted from materials provided by Cordouan Technologies.

For more information on this source, please visit Cordouan Technologies.

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