The Analytical Characteristics of Size Exclusion Chromatography in Biopharmaceuticals

About the Study

In the ever-evolving landscape of biopharmaceuticals, the importance of rigorous characterization cannot be overstated. Among the myriad analytical techniques available, Size Exclusion Chromatography (SEC) stands out as a stalwart method for unravelling the complexities of these large, intricate molecules. In this article, we delve into the significance of Size Exclusion Chromatography in the characterization of biopharmaceuticals, shedding light on its principles, applications, and transformative role in ensuring the safety and efficacy of these therapeutic agents [1].

Biopharmaceuticals, including monoclonal antibodies, therapeutic proteins, and nucleic acid-based drugs, have redefined the landscape of modern medicine. Unlike small-molecule drugs, these large and structurally diverse molecules demand sophisticated analytical approaches to guarantee their quality, safety, and efficacy [2].

Size exclusion chromatography

At its core, Size Exclusion Chromatography is a liquid chromatography technique designed to separate molecules based on their size. Also known as gel filtration chromatography, it relies on a porous stationary phase through which molecules of different sizes can navigate. The larger molecules, unable to penetrate deep into the pores, traverse a shorter path and elute first, while smaller molecules venture deeper into the matrix, resulting in a more extended elution time [3].

The separation achieved by SEC is rooted in the steric hindrance principle. As the sample passes through the column, smaller molecules can access internal pores, delaying their elution, while larger molecules pass through the column more swiftly. This process generates a chromatogram, a visual representation of the distribution of different-sized species within the sample.

Applications across the biopharmaceutical spectrum

Monoclonal Antibodies (mAbs): SEC plays a pivotal role in assessing the aggregation state of mAbs, a critical parameter influencing their stability and efficacy. Size variants, such as monomers, dimers, and higher-order aggregates, are effectively separated and quantified.

Therapeutic proteins: For proteins with multiple isoforms and glycosylation patterns, SEC offers a means of discerning these variants and ensuring batch-to-batch consistency. Analysis of protein-protein interactions and the determination of protein conformation are also within the purview of SEC.

Nucleic acid-based drugs: SEC aids in characterizing the size distribution of nucleic acid-based drugs, ensuring their integrity and functionality. The detection of impurities, such as truncated sequences or contaminants, is achieved with high precision.

While SEC provides valuable information about the size distribution of biopharmaceuticals, it is often complemented by other analytical techniques to paint a comprehensive picture [4].

Multi-detector SEC: Combining SEC with detectors such as light scattering and refractive index enhances accuracy in determining absolute molecular weight and size. Information on molecular shape and conformation can be obtained by integrating additional detectors.

Hyphenated techniques: SEC coupled with Mass Spectrometry (SEC-MS) allows for the identification of individual species within a sample. Integrating SEC with Dynamic Light Scattering (DLS) provides insights into the hydrodynamic radius and aggregation state.

Ensuring quality and safety

Regulatory bodies, including the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA), emphasize the need for comprehensive biopharmaceutical characterization. SEC, with its ability to provide detailed information on size variants and aggregation states, aligns seamlessly with regulatory expectations. It is a crucial tool in ensuring the quality, safety, and efficacy of biopharmaceutical products.

High-resolution SEC and emerging innovations

While SEC is a robust technique, challenges persist, particularly in the analysis of large and complex biopharmaceuticals. High-Resolution SEC, with smaller particle sizes and longer columns, addresses these challenges by providing improved resolution and separation efficiency. Innovation continues to drive the evolution of SEC. The development of advanced column chemistries, novel stationary phases, and miniaturized SEC systems holds promise for overcoming existing limitations and pushing the boundaries of analytical capabilities.

As biopharmaceuticals continue to diversify and advance, the role of SEC is poised to expand. Tailoring SEC methodologies to accommodate emerging modalities, such as gene therapies and cell-based therapeutics, represents the next frontier. This involves adapting SEC to handle larger sizes, more intricate structures, and the evolving landscape of therapeutic agents [5,6].

In the biopharmaceutical development, size exclusion chromatography plays an important role. From elucidating the structural intricacies of monoclonal antibodies to ensuring the purity of therapeutic proteins, SEC stands as a steadfast ally in the pursuit of safe and effective biopharmaceuticals. As technologies advance and therapeutic modalities evolve, it continues with SEC harmonizing the molecular notes that define the future of medicine.

References

1. Borel JF, et al. Biological effects of cyclosporin A: A new antilymphocytic agent. Agents Actions. 1976;6:468-475.

   [Crossref] [Google Scholar] [PubMed]

2. Rydel TJ, et al. The structure of a complex of recombinant hirudin and human alpha-thrombin, Science. 1990; 249:277-280.

   [Crossref] [Google Scholar] [PubMed]

3. Chen Q, et al.  Amphibian-derived natural anticancer peptides and proteins: Mechanism of action, application strategies, and prospects. Int J Mol Sci. 2023;24:13985.

   [Crossref] [Google Scholar] [PubMed]

4. Li J, et al. Identification of peptides of cinobufacini by gel filter chromatography and peptidomics. J Sep Sci. 2022;45:2845-2854.

   [Crossref] [Google Scholar] [PubMed]

5. Kálmán A, et al. Isolation and identification of peptide conformers by reversed-phase high-performance liquid chromatography and NMR at low temperature. J Chromatogr A. 1996;729:155-171.

   [Crossref] [Google Scholar] [PubMed]

6. Guthals A, et al. Peptide identification by tandem mass spectrometry with alternate fragmentation modes. Mol Cell Proteomics. 2012;11:550-557.

   [Crossref] [Google Scholar] [PubMed]