Protein aggregation is when protein molecules stick together to form larger structures, ranging from small clusters to large complexes. It usually follows misfolding or partial unfolding, and aggregated protein commonly loses its function and may come out of solution altogether. It can happen naturally or under stress, and it affects both how a protein performs in an experiment and how safe a drug product is.

Why proteins aggregate

After synthesis, proteins typically fold into a particular three-dimensional conformation, the native state. That folding process can be impaired, creating misfolding or unfolding and leading to aggregate formation. In general, aggregation can be the result of various interactions, including hydrophobic, electrostatic or van der Waals forces. Proteins have hydrophobic regions that tend to interact with each other, leading to aggregation when they are exposed to an aqueous environment.

The conditions that trigger it are well known. Extremely high protein concentrations can exceed the saturation point. High temperatures can denature proteins and promote their aggregation. Changes in pH can disrupt the electrostatic interactions between proteins. Variations in salt concentration affect protein solubility. Mechanical stress, such as pipetting or vortexing, can disrupt protein structure. Freeze and thaw cycles, improper storage and prolonged storage or incubation all allow proteins to gradually lose stability. Contaminants or impurities in the sample can promote aggregation, mutations in the protein sequence can make a protein aggregation-prone, and post-translational or chemical modifications can alter a protein's properties and lead to aggregation.

Most of these are conditions under experimental control, which is why aggregation is normally studied as a function of one of them rather than measured once.

The two types, and why the distinction matters

Aggregates are not a single population. In relation to FIDA measurements there are two categories.

Large aggregates, also called insoluble aggregates, are particles that are not undergoing diffusion. They are therefore not observed as a regular gaussian shape, but rather as individual spikes along the Taylorgram. Generally, the amplitude of the spike is related to the size of the aggregate.

Source: course "What is an aggregate and how does it look?" on Fida Learning Hub, a Taylorgram showing large insoluble protein aggregates as sharp spikes, with the main diffusing protein population appearing as a gaussian peak.

Intermediate aggregates, also called soluble aggregates, are for example oligomers that are not diffusing sufficiently to satisfy Taylor's conditions. In this case the aggregates can be seen as a shoulder before the gaussian shape.

**Source: course "What is an aggregate and how does it look?" on Fida Learning Hub. Taylorgram showing soluble protein aggregates as a shoulder rising before the main gaussian peak.**

The distinction matters because the two classes appear differently in the data and are quantified differently: spikes are counted, while the soluble fraction is quantified from the fluorescent area of the larger species.

How aggregation is measured

Aggregation is rarely a single number. It is normally followed against another variable: time, temperature, buffer composition, or the number of freeze and thaw cycles. The quantity of interest is the trend, and the condition at which the trend changes.

Aggregation and disease

Aggregation is not only an experimental nuisance. It is central to several neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease and Huntington's disease, in which particular proteins misfold and accumulate as deposits that damage neurons. Much of the interest in measuring aggregation in solution, under native conditions, follows from this.

How aggregation is measured in FIDA

FIDA captures both classes of aggregate within a single in-solution measurement, because both alter the recorded signal in ways that can be separated.

Soluble aggregates contribute to the dispersion profile and can be quantified from the fluorescent area of the larger species when several populations are fitted, which gives an amount rather than a simple indication that aggregation is present. Insoluble aggregates appear as discrete spikes in the raw signal, and each spike represents at least one aggregate. The number of spikes is quantified automatically as the data is loaded, and with several files the counts can be tabulated and plotted to follow how aggregation develops.

The position of the spikes along the profile indicates which sample they came from. Spikes arriving in the later portion of the profile originate from the indicator sample, while spikes distributed throughout come from the analyte, or from both samples together. Where spikes are numerous and large enough, they can obscure the underlying peak entirely, in which case the size of the diffusing species cannot be recovered from that run.

Because nothing is immobilised and nothing is separated, the sample is observed as it is, including in serum, plasma, lysate and formulation buffers, and aggregation is recorded alongside size and binding in the same experiment. Nanolitre volumes per data point make series against temperature, time or freeze and thaw cycles practical on limited material. Further reading: Aggregation readout.