Understanding Peptide and Protein Aggregation: A Guide to Terminologies, Implications, Factors, and the Role of Liquid Chromatography

The aggregation or clumping of peptides and proteins is a crucial phenomenon in biology, influenced by various factors [1]. Understanding these factors, such as the quantity of peptide/protein, solution pH, impurities, metal particles, salts, ionic strength, temperature, stirring, and shear forces, is not only important but paramount [2]. This understanding significantly affects the physical stability and activity of the peptide and can lead to serious issues such as toxicity and immune responses.

Peptide or protein aggregation is a process where molecules clump together, sometimes leading to changes in their structure. A protein or peptide's primary structure is the amino acid sequence. Secondary structure is local interactions that arise from the hydrogen bonds formed between atoms of the polypeptide chain. These structures include α-helix and β-pleated sheet structures. Tertiary structure is their overall three-dimensional folding.

The terms "aggregation" and "self-assembly" are often used interchangeably, but they don't have precise definitions. "Aggregation" refers to a process that changes the structure of the peptide, often leading to the formation of various aggregates. On the other hand, "self-assembly" specifically refers to the formation of larger molecules without changing the peptide's structure. Understanding this difference is important to avoid confusion and ensure clarity.

Aggregation can happen in different ways in a liquid solution or on surfaces where particles stick. It can

Figure 1. Depiction of protein aggregation. (A) Individual protein structures. (B) Protein aggregate forms. Images adapted from reference 3.

be shapeless (amorphous) or have a specific structure. These forms can be temporary or permanent. One well-studied type of aggregation is when protein molecules join together to form structured amyloid fibrils, which is important because of its connection to brain disorders like Alzheimer’s disease. Peptide aggregation is also a common and troublesome process in the development of biological drugs [2].

Aggregation and liquid chromatography. When using analytical reverse phase chromatography (HPLC), we can identify protein and peptide aggregation by analyzing the chromatogram's shape and certain characteristics such as peak broadening, tailing, fronting, retention time, and sensitivity. However, it's challenging to detect and separate these aggregates during the purification process of proteins and peptides.

In analytical chromatography, a small sample, typically 1-2% or less of the column volume, is injected into the column, while in preparative chromatography, an overloaded column is used to increase purification capacity. Overloading the column can alter the chromatogram's characteristics. The effects of the purification method conditions, such as mobile phase pH, buffer, ionic strength, and organic additives, should also be considered early in the method development process, as they may influence aggregation.

Summary, ongoing, and future efforts. Lately, many research programs have been looking into how peptides and proteins aggregate. There's been a lot of progress in understanding what factors affect this process. Most of the research has focused on peptides and proteins related to diseases, especially ones that affect the brain. But what we've learned from studying how disease-related peptides and proteins aggregate can also be useful for understanding how peptide-based drugs might aggregate. Even though we've made a lot of progress, we still don't fully understand how peptides in drug development might aggregate [2].

At Millennial Scientific, we develop next-generation products for separation and purification of peptides and proteins. Get in touch with us to discuss how we can support your chromatography separation needs. For more information or to request samples, please email us at inquiry@millennialscientific.com, call us at 855 388 2800, or fill in our online form.

References

[1] M.F. Pignataro, M.G. Herrera, V.I. Dodero, Evaluation of Peptide/Protein Self-Assembly and Aggregation by Spectroscopic Methods, Molecules, 2020.

[2] K.L. Zapadka, F.J. Becher, A.L. Gomes dos Santos, S.E. Jackson, Factors affecting the physical stability (aggregation) of peptide therapeutics, Interface Focus 7(6) (2017) 20170030.

[3] P. Faller, C. Hureau, Reproducibility Problems of Amyloid-β Self-Assembly and How to Deal With Them, Frontiers in Chemistry, 2021.