Many techniques are available to study protein aggregation, each with their own particular strengths and limitations.
One of the reasons for the relative abundance of analysis techniques is the sheer variety of proteins, their aggregates and their interactions. For example, proteins can be soluble or insoluble and the aggregation process can be reversible (noncovalent) or irreversible (covalent). In addition, the large size range between monomer or active forms of a protein and its aggregate add further complexity to their analysis. For these reasons, it is typically recommended that a variety of orthogonal tools are used to initially characterize protein aggregation. These studies can then enable the selection of a primary analysis technique which is suitable for routine analysis.
Key considerations when determining which analysis tool(s) to use to measure aggregation include protein size and state.
While the average molecular weight of a human protein is approximately 50 kDa, the range of molecular weights is vast, going from a few kDa all the way to 4 MDa for the largest protein titin (whose length is over 1 μm). It stands to reason that the molecular weight and size of protein aggregates can be significantly larger than their monomeric or active forms.
Few technologies are capable of measuring the complete spectrum of protein sizes, necessitating knowledge and use of multiple technologies. Figure 1 shows the size of proteins which can detected using a number of commonly used analysis tools; however, while such comparisons are useful, specific understanding of each technique is essential. As we will see later, the application of dynamic light scattering (DLS), for example, can provide inaccurate measurement of protein aggregation due to overrepresentation of larger particles.
Figure 1: Typical protein size detection ranges of commonly used analysis techniques. Detection limit data (with the exception of DS) obtained from den Engelsman et al (2011). Figure adapted from Siew (2015)
As previously discussed, some proteins aggregates are reversible and, as such, it is advantageous to minimize sample perturbation when measuring aggregation. Ideally, the technique chosen should utilize the sample directly without change to the buffer composition.
Furthermore, the sample analysis technique should not introduce additional sources of protein interaction, as can be the case with column or matrix-based techniques. Techniques that approach this ideal of not or minimally perturbing the aggregation state in a sample include, dynamic light scattering (DLS), analytical ultracentrifugation (AUC) and microfluidic diffusional sizing (MDS).
Limitations of common protein aggregation analysis techniques
In this section we will examine some of the more commonly used protein aggregation analysis techniques, highlighting some of the key considerations and limitations to be aware of. This review will focus on techniques which can be used to measure protein size; however, there are additional techniques that address other characteristics such as changes in structure and interactions. More information on these can be found in the “Further reading” section at the end of this article.
Visual and microscopic techniques
Some large protein aggregates (>50 µM) can be seen with the naked eye, while for others (>1 µM) standard light microscopy can be used. Such assessments are both cheap and easily accessible; however, they reveal little about the amount of isomerization in solution. Fluorescent molecules may also be added to help study the interactions between two molecules but there is some debate as to whether such molecules may change the aggregation properties of the protein. Microscopic techniques are also typically low throughput with just one sample analyzed at a time.
Detecting obscuration or blocking of light caused by a particle in solution is a commonly used technique for protein size analysis. The main limitation of this technique is that many proteins are translucent and so cannot be accurately detected. In addition, the technique requires large sample volumes, typically >1 mL.
Flow imaging microscopy utilizes a high magnification camera to capture a series of images as the solution of interest passes through an illuminated flow cell. Particles with a different refractive index than the solution decrease the light intensity compared to the background and can be detected and analyzed using the captured images. Analysis of these images allows the concentration, size and morphology of micron-sized particles (1–400 μm) to be determined. One of the limitations of this techniques is the inability to analyses particles smaller the 1 μm.
Dynamic light scattering (DLS)
DLS measures light scattered by particles in solution, which can be used to calculate their hydrodynamic radius. A commonly known challenge with DLS is the over representation of larger particles, which means that even a small amount of oligomerization or aggregation can have a large impact on size measurement. This also means that the presence of small protein molecules can be masked. A further limitation of DLS is that, in order to resolve two species in a mixture there needs to be approximately three-fold difference in diameter between the species. Find out more about what to look out for when performing DLS in our recent article here.
Analytical ultracentrifugation (AUC)
This technique creates a high gravity field that causes the protein in solution to sediment and settle with a clear boundary to the rest of the solution. As the sample is being spun, absorption and refractive index scans follow the movement of the particles at the boundary and yield a parameter called the sedimentation co-efficient, which is linked to the size of the molecule.
It has been reported that reproducibility of AUC can be poor. This may be caused by misalignment of the cell which holds the sample and the quality of the centerpiece which holds the cell. To ensure correct functioning, regular calibration and maintenance of the system and its accessories are required. A further consideration for AUC is that is both slow and expensive. In addition, highly trained staff are required to optimize the system and interpret the data.
Size exclusion chromatography (SEC)
SEC works by separating molecules based on their size by filtration through a gel. The gel comprises beads containing pores of specific size distributions. Small molecules diffuse into the pores, slowing their movement though the column containing the gel. Larger molecules flow more quickly though the gel. The fractionated eluate can then be analyzed using traditional protein analysis techniques such as UV-absorbance or light scattering.
A well-known limitation of SEC is the potential for interaction between the protein and the gel/matrix, which may affect aggregation and flow though the column. The requirement to use non-native sample conditions can also affect protein isomerization and the range of protein sizes that can be analyzed using this method is limited.
Nanoparticle tracking analysis (NTA)
NTA utilizes the properties of both light scattering and Brownian motion in order to obtain the nanoparticle size distribution of samples in liquid suspension. A laser beam is passed through the sample chamber and the particles in the path of the beam scatter light which is detected using a video camera, allowing for real-time monitoring of protein interactions.
One challenge of NTA is the low refractive index of protein, which means the lower limit of detection is approximately 30 nm diameter. As a result, monomers may not be measurable; however larger aggregates can be sized and concentration determined.
Microfluidic diffusional sizing (MDS)
MDS is a relatively new technique that utilizes rate of diffusion to determine protein size. This allows researchers to perform a quick but accurate analysis of proteins of interest to identify aggregation. A key advantage of this technique is that samples are analyzed in their native state under biologically relevant solution conditions. Furthermore, a wide range of protein sizes can be detected (0.5 to 20 nm, or 0.3 kDa to 8 MDa) and quantified. This offers a much lower limit of size detection than most orthogonal techniques, increasing the range of proteins that can be analyzed.
The Fluidity One uses MDS and enables analysis of protein samples in just 8 minutes using just 5 µL of sample.
It is clear that, as yet, no single technology can provide detailed insight into all proteins and their aggregates; however, new techniques such as MDS can deliver rapid, low-cost size and concentration analysis of a wide range proteins. Future developments in this area promise to expand the utility of this technique to enable analysis of protein interactions over time and increase sample throughput, further streamlining the analysis of protein aggregates.
For more information about analyzing protein aggregates, view our recent blog post.
den Engelsman, J. et al. (2011) Strategies for the assessment of protein aggregates in pharmaceutical biotech product development. Pharm Res. 28(4): 920–933.
Siew, A. (2015) Analyzing protein aggregation in biopharmaceuticals. BioPharm International. 28(1).
Berkowitz, S. et al (2015). Challenges in the determination of protein aggregates. LCGC North America 33(7): 478–489
Manning, M.C. et al (2014). Review of orthogonal methods to SEC for quantitation and characterization of protein aggregates. BioPharm International. 27(42)