Journal of Chromatography & Separation Techniques
Open Access

Protein Quality Control using Size-Exclusion Chromatography

Marcus Hao^{* *}Correspondence: Marcus Hao, Department of Analytical Chemistry, University of Paris-Saclay, Orsay, France, Email:

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Description

Size-Exclusion Chromatography (SEC), often known as gel filtering, is a useful analytical technique for isolating compounds based on their sizes. Often referred to as gel filtration chromatography, Size-Exclusion Chromatography (SEC) a useful analytical technique for isolating compounds based on their sizes. chromatography. This method is particularly valuable in the study of protein aggregation and stability, providing critical insights into the behavior of proteins in solution, their molecular weight distribution, and the formation of aggregates. By separating molecules according to their hydrodynamic size as they flow through a column filled with porous beads, sizeexclusion chromatography operates on this basic concept. As the sample is injected into the column, smaller molecules penetrate the pores of the beads and travel through a longer path, while larger molecules are excluded from the pores and move more quickly through the column. The separation occurs because larger molecules are less able to enter the pores and thus elute from the column earlier than smaller molecules, which have more opportunities to diffuse into the pores. This size-based separation results in a chromatogram where different protein species, including aggregates, appear as distinct peaks.

Proteins can aggregate into various forms, from dimers and trimmers to large multimers. SEC allows for the detection and quantification of these aggregates by separating them based on size. Aggregates are detected as additional peaks or shoulders on the chromatogram, which can be analyzed to determine their size and concentration. Protein stability is often assessed by monitoring changes in the aggregation profile over time. SEC can be used to track the formation of aggregates in protein samples under different conditions, such as varying potential of Hydrogen (pH), temperature, or the presence of stabilizing agents. By comparing chromatographic profiles before and after stress conditions, researchers can gain insights into the stability of the protein. In the pharmaceutical industry, SEC is employed to evaluate the quality of protein-based drugs and formulations. Aggregation can affect the efficacy and safety of therapeutic proteins, and SEC provides a means to ensure that products meet quality standards by monitoring aggregate levels. It helps ensure that the final product is of high purity and contains minimal aggregates, which is essential for the safety and efficacy of biopharmaceutical products. SEC can be combined with other techniques, such as Multi-Angle Light Scattering (MALS), to study protein-protein and protein-ligand interactions. These interactions can influence aggregation behavior, and understanding them is essential for developing stable protein formulations. SEC is used in stability studies to monitor the formation of aggregates under different storage conditions. By analyzing samples at various time points, researchers can identify conditions that promote aggregation and develop strategies to improve protein stability. SEC helps in the formulation development process by assessing how different excipients or formulation conditions affect protein aggregation. This information is used to optimize formulations to enhance protein stability and extend shelf life. In research settings, SEC is used to investigate the aggregation of proteins associated with various diseases, such as neurodegenerative disorders.

Conclusion

Its ability to separate proteins based on size makes it ideal for detecting and quantifying aggregates, assessing protein stability under different conditions, and characterizing protein formulations. By providing detailed information about the size distribution and aggregation state of proteins, SEC plays a important role in ensuring the quality and safety of proteinbased products in the pharmaceutical industry, as well as advancing our understanding of protein behavior in various research contexts. The technique's versatility and effectiveness in analyzing protein aggregation make it indispensable for both industrial and research applications. Continued advancements in SEC technology and methodologies will further enhance its utility, providing deeper insights into protein aggregation and stability, and supporting the development of more effective and stable therapeutic proteins.