Monitoring Protein Aggregation with DLS


Florian Aubrit*, David Jacob, Sylvain Boj

* actually at Organic Polymers Chemistry Laboratory (LCPO), CNRS-University of Bordeaux, 33600 Pessac, France

Active principle ingredients (API) and protein aggregation are of the utmost importance when ensuring the usability and stability of injectable biopharmaceutical products.

Protein aggregation is a process that can materialize at any stage in the lifetime of a therapeutic protein. Its stages are as follows: protein expression, refolding, purification, sterilization, shipping, storage, and delivery.

The specific mechanism by which protein aggregation occurs is unknown. However, it has been found that particular manufacturing processes can play a part in increasing the risk of the formation of protein aggregates and physical degradation.

The presence of vial or microbial contaminants during cell culture, the formulation composition, along with the possible effects of storage conditions all maximize the risk of chemical degradation.

The storage container condition can play an important part in the production of protein aggregates in particular, for example, prefilled syringes that can exude silicone oil from the rubber stopper.

Researchers are focused on the improvement of in-situ monitoring of possible degradation and denaturation of therapeutic proteins during systems of production and subsequent storage to create stricter international health regulations concerning the control of biopharmaceutical products.

Current Measurement Techniques and Limitations

Many varied analytical methods can be utilized to evaluate the presence of protein aggregates within the micrometer (μm) range. Some examples of these techniques include Coulter counter (CC), micro-flow imaging (MFI), dynamic imaging particle analysis (DIPA),  and light obscuration (LO).

Methods such as multi-angle static light scattering (MALS) can be used to detect and measure the presence of protein aggregates at an early stage.These can be used in combination with commonly used separating methods, for example, asymmetrical-flow field-flow fractionation (AF4), analytical ultracentrifugation (AUC), and size-exclusion chromatography (SEC).

Moreover, batch Dynamic Light Scattering (DLS) can be beneficial for use in the characterization of protein aggregates when the shear or dilution encountered in FFF or SEC creates disassociation of the aggregates, or when evaluating protein aggregation under various temperatures and/or conditions.

Although each of these methods are very efficient in their range of applications, they frequently require particular handling and preparation of the sample, either before or during measurement, both of which can alter the state of the sample aggregation.

As such, the favored technique for the handling and manipulation of the sample entails the direct analysis of samples into the storage medium, for example using a hermetically sealed syringe or vial.

Regrettably, none of the methods described can directly measure protein aggregates in an injectable.

In Situ Contactless Measurement: The VASCO Kin © Concept

Cordouan’s VASCO Kin, which is grounded in live, in-situ, contactless, and remote DLS measurements, has been recently created to overcome the challenges of conventional protein aggregation test techniques.

DLS is an advanced and highly effective technique which is widely used in both proteins characterization studies and colloidal sciences.

It is founded on the evaluation of scattered light fluctuations that happen due to the Brownian motion of particles.

This event enables the precise measurement of particle sizes varying from one nanometer to several microns in a minute.

For commercial DLS systems, various measurement configurations can be selected. Although, each selection requires the user to withdraw the sample with an automatic pump or pipette and then place and/or inject the sample into the instrument before the measurement.

By doing this, this process may negatively influence the sample protein aggregation state.

To overcome this challenge, the VASCO Kin is a completely mobile DLS system that employs its novel Optical Fiber Remote Probe (OFRP).

The OFRP is a highly robust and optimized optomechanical assembly that is engineered to make contactless and direct measurements that lack the requirement for any sample batching procedure.

Connected to an Optical Unit by a unique optical fiber umbilical, the OFRP injects a laser beam into the sample and gathers scattered light from the sample in the backward direction at an angle of 170°.

The Avalanche Photodiode Detector (APD) is an incredibly sensitive single photon that is attached to an exclusive and fast acquisition electronic board to offer live monitoring of the fluctuations in the intensity of scattered light.

These fluctuations are subsequently translated into time-resolved particle size kinetic analysis through the use of powerful mathematical algorithms.

Measurement in Vaccines Syringes

A range of particle size measurements on a commercial injectable flu vaccine was carried out to verify the ability of the VASCO Kin to achieve contactless in situ measurements into a syringe.

This vaccine is a multifaceted medium containing a combination of several different ingredients, including both fragmented and deactivated flu viruses sourced from three different stem cell lines.

Many excipients are involved such as buffered saline solution, sodium chloride, potassium chloride, ppi water, along with traces of chicken egg proteins, also called ovalbumin, and neomycin, formaldehyde, and octoxinol 9, to name a few.

During the measurement process, the vaccine syringe was taken from its plastic packaging and placed in a special mount located 6 cm in front of the probe.

To acquire sufficient comparisons along with evidence of the potential aging influence of the vaccine, one vaccine was stored at 7 °C and a second vaccine was stored for eight months at room temperature.

Both vaccines were measured at the same time, date, and in equivalent conditions to evaluate the particle size distribution.

The sample of the vaccine stored at 7 °C showed a relative complexity of the sample particle size distribution with three distinct peaks varying from 30 nm up to 800 nm, all of which relate to virus proteins and fragments. Moreover, several aggregates were 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 positioned in front of the holder created for the aim 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 positioned in front of the holder created for the aim of the experiment

Comparatively, the vaccine stored at room temperature demonstrated obvious modifications in the particle size distribution within a wide continuum of 10 nm to 10 μm and greater. More investigations must be performed because these preliminary studies were utilized for the in situ DLS measurement into the injectable syringe.

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 eight 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 eight months (bottom)


This investigation was the first of its kind to demonstrate a contactless in situ particle size measurement of a commercial injectable vaccine directly into a syringe.

By removing sample batching procedures, the VASCO Kin system uses an original optical fiber remote head that can be applied to a broad range of particle size measurement systems, for example, the in situ monitoring of protein aggregation within biopharmaceutical injectable products.

The VASCO Kin is also beneficial in the observation of the real-time nanoparticle synthesis kinetics of different kinds of reactor configuration, for example a double jacket glass reactor, high temperature and high-pressure Supercritical CO2 autoclaves, microfluidic chips, microwave reactors, or instrumental coupling.

References and Further Reading

  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:
  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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