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Dynamic light scattering presents a good analytical technique for testing protein stability.
Dynamic light scattering (DLS) is a well-known technique for determining sample interactions, particle size, and aggregation of molecules dispersed or dissolved in solution. The technique can also be a reliable method for characterizing protein stability (1).
Among the most challenging aspects of conducting analytical stability studies on protein therapeutics is protein instability, which can be caused by many different mechanisms, says Hanna Jankevics-Jones, PhD, segment manager, pharmaceuticals, at Malvern Panalytical. A change in the local environment around the protein, such as change in pH, ionic strength, or temperature, or an external factor; for example, agitation, can cause stress that affects the non-covalent bonds in the protein’s structure. These stresses can reduce or increase the strength of these non-covalent bonds (depending on the magnitude and direction of change) and thereby affect the likelihood of the protein maintaining its structure and stability, she explains.
“If the stresses are significant enough to cause unfolding, this often manifests itself in secondary effects, such as protein aggregation,” she adds.
Hanna Jankevics-Jones also notes that many analytical techniques used in stability studies measure the secondary effects of instability, such as aggregation, rather than the primary cause of instability, such as reduced repulsion due to increased ionic concentration in the buffer. “A combination of analytical techniques is often needed to better understand or pinpoint the likely cause of instability and identify potential remedies,” she states.
Another challenge with protein therapeutics is that they are quite complex molecules, remarks John Bak, principal scientist, at PPD Laboratories GMP Lab. Proteins are more fragile than typical small-molecule therapeutics, and their function depends not only on their chemical identity (primary structure), but on the shape of the molecule (secondary and tertiary structures), and even multi-subunit proteins (quaternary structure). Proteins are, in addition, particularly sensitive to temperature changes, pH, and conductivity, he says. It is thus imperative that proteins be reconstituted and/or diluted with a diluent in an ionic environment in which the protein is stable. “Ensuring that the proteins are produced in the proper form, and that the form remains stable all the way from production to the patient, is required,” he states.
Because a stability study involves setting aside quantities of candidate formulations for long periods of time to evaluate shelf life, it becomes essential to minimize the number of candidates and pre-optimize the selection as much as possible, adds Dan Some, principal scientist at Wyatt. “This process reduces the risk of not finding a suitably stable formulation and consequently delaying commercialization. Effectively screening a wide range of formulations in order to select the best candidates for long-term stability studies is a major challenge.”
DLS, also known as photon correlation spectroscopy (PCS) or quasi-elastic light scattering (QELS), is just one analytical technique that can offer a solution to the challenges of studying protein therapeutics to determine their stability, says Bak. For ideal samples, DLS has sensitivity starting at a few nanometers. For instance, he points out, one can see sodium dodecyl sulfate (SDS) micelles at about 4 nm. The upper range of detection is about 1000 nm or more, depending on how quickly particles settle. In comparison, laser diffraction methods tend to lose sensitivity under 100 nm, and electron microscopy or X-ray methods tend to have more difficult sample preparation and cannot always be performed in solution. DLS, however, can provide measurements in a few minutes using under 100 µL of solution, Bak explains.
“A protein is far from an ideal scatterer,” remarks Eric Olson, senior research scientist at PPD Laboratories GMP Lab. “Proteins are not hard spheres, but instead deformable and porous, so many of the assumptions of DLS are broken. For instance, DLS assumes particles are moving by Brownian motion, but, since proteins are not hard spheres, the collisions with solvent molecules could be considered inelastic rather than elastic,” he states. Olson uses the example of punching a pillow instead of a basketball to illustrate the difference between protein and small-molecule detection using DLS. “Some of the energy is lost in the collision as it deforms the protein instead of sending it flying in a different direction,” he says.
Proteins may not be ideal scatterers, Bak chimes in, but DLS can still be used to measure them, although it will not have the same sensitivity as with more ideal particles. “Typically, a protein will need to have a minimum diameter of several tens of nanometers to be detected. The sensitivity of a protein will depend on several factors: the relative refractive index; optical density; and the relative degree of hydration inside the structure of the protein to name a few. A very rough estimate of the radius (not diameter) of a globular protein is the cube root of its molecular weight in kilodaltons,” he emphasizes.
Meanwhile, sample preparation is also a significant issue in that the structure of the protein cannot be allowed to change in response to preparing the test solution, Olson points out. “Ideally, the sample should be measured neat. If it needs to be diluted, the formulation buffer should be used to keep the environment the same. Temperature is also an issue, so it must be controlled as well, though most modern instruments will do that,” he says.
In stability studies, DLS plays two major roles, says Some. One is to detect large aggregates in the course of long-term stability studies—aggregates that are too large for size-exclusion chromatography (SEC). The second role is the most important, he notes, which is to screen a matrix of formulations to identify those that are most likely to succeed in the course of long-term stability and other stability stress tests.
Some points to several different measurements that can be made with DLS to quantify stability predictors and help optimize the candidate selection process: aggregation, colloidal stability, thermal stability, and conformational stability:
Size distributions measured by DLS—these measurements are useful in determining the average protein size and detecting the presence of small and large aggregates. A relatively large average size may be indicative of physical instability, and the presence of aggregates is a definitive sign of poor formulation.
Measurement of kD (diffusion interaction parameter)—this parameter is derived from the change in diffusion coefficient (determined directly by DLS) with increasing protein concentration and provides a measure of colloidal stability. Formulations with poor colloidal stability cannot be expected to do well over the long term.
Accelerated stability tests–these tests hold the formulation at an elevated temperature, typically 40 °C, while monitoring aggregation rate. DLS provides rapid feedback on aggregate content of the solution, and the measurement cell may be held at temperature for extended periods, making this method a core predictor of stability, one that is fully enabled by DLS.
Temperature ramp experiment—here, DLS monitors the change in size and molar mass of a protein solution as a function of temperature. The temperatures that experience the onset of unfolding (Tonset) and aggregation (Tagg) are also considered key predictors of stability.
Isothermal chemical denaturation—DLS can also be used to monitor conformational stability via isothermal chemical denaturation. In this technique, the size of the protein is measured as a function of the amount of a denaturant, such as urea or guanidine hydrochloric acid, added to the formulation, which causes the protein to unfold. A higher resistance to chemical denaturation is a sign of higher conformational stability.
Other DLS-based techniques—other techniques are being developed to provide more robust formulation ranking. A technique published in 2018 (2) monitors the protein size upon denaturation and subsequent dilution back to near-formulation conditions. According to the study, proteins that are more stable tend to refold more compactly as the degree of denaturation is reduced.
DLS’ sensitivity is based on the impact of particle size change on the amount of light that particle scatters, adds Jankevics-Jones. The light scattered by particles in the typical size range for proteins “scales as the sixth power of the particle diameter, meaning that even a small change, such as the formation of dimers from monomers, can be detected by an increase in light scattering,” she states.
DLS alone, however, is not sufficient as a standalone analytical technique to evaluate protein stability studies, Jankevics-Jones further states. “As previously mentioned, DLS reports on aggregation, which is really an indicator for many types of instability. It should therefore be applied as part of a portfolio of techniques whose data can be used collectively to identify degradation pathways or compare stability under different environmental conditions,” she says. An example would be the use of DLS in combination with differential scanning calorimetry (DSC) to cross-correlate any aggregation reported by DLS with changes in the higher-order structure and content of folded protein material, she adds.
The level of complexity in protein molecules requires the use of many other technologies in addition to those used in the development of small-molecule therapeutics, Olson says. Including other analytical detection technologies can help ensure that the different levels of protein structure are maintained for the highest therapeutic efficacy, safety, and quality. “One property of proteins in solution that can be monitored is the particle size.Particle size is difficult because proteins can be small. They range in size from a few nanometers to tens of nanometers for individual molecules and up to hundreds of nanometers for multi-subunit structures,” Olson explains.
“Full characterization of a protein therapeutic requires a large battery of tests,” Bak concurs. “Focusing only on particle size, an excellent way to improve the performance of DLS is to use it as a detector on a separation technique such as SEC or field flow fractionation,” he says. Bak explains that the limitations on the minimum size that can be detected will still be there and will be exacerbated with the sample going through a separation; the concentration will be lower, so there will be less signal generated for the DLS detector. “When it does work though, we can use the particle size as a rough check on the molecular weights reported by SEC,” he says.
In long-term storage studies, DLS is primarily useful for its ability to quickly detect large aggregates, adds Some. Some also agrees that DLS needs to be combined with other analytical techniques, including SEC coupled to multi-angle light scattering (SEC–MALS), which identifies small aggregates and fragments; field-flow fractionation coupled to multi-angle light scattering (FFF–MALS), which provides detailed information on both small and large aggregates; various types of high-performance liquid chromatography (HPLC) coupled to mass spectrometry to identify chemical degradation; and other techniques that can identify modifications such as charge variants.
“In the preliminary candidate selection process, DLS should be combined with SEC for fragment/aggregate quantification and techniques mentioned above that evaluate chemical degradation, especially with respect to the different stress tests. It may also be valuable to add dye-based extrinsic fluorescence assays that can detect changes in hydrophobicity and significantly small amounts of aggregates,” Some says. He further adds that another commonly used analytical technique during this stage is differential scanning calorimetry, which is considered the “gold standard” for measuring the protein melting onset temperature of each domain in a multi-domain protein.
An important area of improvement for DLS is that the technique can use multi-angle detectors. Olson says that most DLS instruments only have one angle of detection; however, by using multiple angles one can more accurately perform molecular weight measurements and measure the radius of gyration. These measurements would help account for particles that are not spherical, he states. In addition, using a green or violet laser will increase light scatter and improve sensitivity as well as resolution. “The one drawback of green and violet lasers is that any fluorescence caused by them would interfere with the DLS measurement,” Olson cautions.
“Other techniques that can access this size range also include analytical ultra-centrifugation and resonant mass measurement. X-ray (SAXS) and neutron scattering (SANS) are used for micelle work and can be applied to proteins. Each of these techniques has its strengths and weaknesses, but they can be considered for use,” says Olson.
Meanwhile, another technique that uses Brownian motion is nanoparticle tracking analysis, adds Bak. Rather than monitoring light scatter intensity, a camera monitors the locations where particles are emitting scattered light and analyzes their Brownian motion, allowing for the sizing of individual particles. “Unfortunately, it is much less sensitive than DLS, so it only will detect larger proteins, starting at about 75 nm to 100 nm. For larger aggregates in the micron size range, flow microscopy can be used,” he states.
“DLS can often be a bit of a black box in that it’s easy to get a quantity out, but much harder to confirm that the value is correct and not subject to experimental artifacts, so as to filter out any poor data,” states Some, who points out that one of the main challenges for DLS users sifting through high-throughput experiments is to receive robust indications from the software that the results are valid, when they have many measurements yet to review.
Furthermore, DLS is extremely sensitive to the presence of small amounts of aggregates forming in the sample because of the light scattering’s dependency on size, which means that even tiny changes are magnified in the millions, Jankevics-Jones says. This extreme sensitivity can, however, also be a drawback for DLS as a technique, as low levels of contamination can influence DLS measurements. “This has, up to recently, meant that sample handling has had to be meticulous in order to avoid any extraneous contamination,” she says.
“Recent developments have meant that the analysis algorithms applied to collect and analyze DLS data, such as adaptive correlation (3), have become ‘cleverer’, scanning the profile of a sample at all times and identifying those particle populations that can be detected only occasionally. This reduces the likelihood of results being skewed by extraneous contaminants. It also extends the application of the method to novel, challenging modalities, such as gene therapy vectors and next-generation vaccines,” Jankevics-Jones explains.
1. B. Lorbe, et al., Biochemistry and Molecular Biology Education 40 (6) 372–382 (2012).
2. H. Svilenov, et al., Journal of Pharmaceutical Sciences 107 (12) 3007–3013 (2018).
3. A.V Malm and J.C.W. Corbett, Sci Rep 9, 13519 (2019).
Feliza Mirasol is the science editor for BioPharm International.
Vol. 34, No. 4
Pages: 41–43, 46
When referring to this article, please cite it as F. Mirasol, “Stability Testing of Protein Therapeutics Using DLS,” BioPharm International 34 (4) 2021.