Size-Exclusion Chromatography Faces New Challenges for Complex Therapeutics

Size-exclusion chromatography (SEC) differs from most types of analytical chromatography in that molecular size, conformation, and conjugation ratio, rather than affinity, determine elution order. Smaller species become trapped or slowed inside SEC media’s pores while larger species that can’t squeeze into the pores elute first.

We tend to think of SEC as separation by molecular weight, and analytical SEC columns are often specified in terms of molecular weight resolving capability. “But size in solution or hydrodynamic radius is what determines elution order,” explains Karen-Lorena Lopez, Pre-Sales Application Engineer at Agilent Technologies.

That is why molecules with the same molecular weights may have very different SEC elution times.

“Cylindrical molecules will elute earlier than predicted based on their molecular weight because they will have a larger hydrodynamic radius than more compact proteins,” Lopez says.

As is typical for liquid chromatography, decreasing column resin particle size improves resolving power by increasing the theoretical plate count, loading capacity, and separation efficiency. But as with other common chromatography modes, smaller particles mean higher backpressures whose resulting shear forces can damage proteins, especially very large, complex biologics.

SEC’s facile combination with standard chemical or spectroscopic detection methods makes it suited to many development-stage studies, particularly those related to aggregation and quality. For example SEC pairs with chemical/spectroscopic detectors such as ultraviolet, infrared, Raman, or fluorescence, with or without mass detection. Spectroscopy-based detection, however, requires an active chromaphore.

Analytes lacking chromophores may be derivatized, but this adds one or more steps to analytical workflows. Another approach is to detect eluents through physical methods like viscometry, sedimentation, refractive index, or light scattering.

Capabilities and considerations

Together, SEC’s elution and detection options open opportunities for orthogonal analyses involving both chemical and physical properties—an approach suited for many development-stage quality and characterization studies like aggregate type and count, degradation or forced degradation, feedback for process changes, antibody-drug conjugate characterization, and related regulatory submission to name a few.

SEC is particularly suited to studying aggregates arising from stresses occurring at any point in a products lifecycle, for example excursions from normal temperatures, concentrations, or environmental exposure. “Aggregates can lead to activity loss, decreased solubility, and enhanced immunogenicity,” Lopez tells Biocompare, “so monitoring them at every stage of development and production is essential.”

“The most common form of SEC, high-performance SEC (HP-SEC), is regularly used for forced degradation, comparability, and characterization, studies,” says Kevin Wons, Pharma/Biopharma Market Manager at Shimadzu Scientific Instruments. “SEC is frequently paired with multi-angle light scattering (MALS), but usually just once to characterize a new lot,” Wons adds, “while SEC fractionation is useful in several later stages of development.”

Despite its widespread adoption, ease of use, and compatibility with standard LC and detection systems, SEC requires care to execute successfully. Things to consider:

• Selecting a stationary phase whose pore size range is appropriate for the separation
• Sample preparation that includes the removal of interfering aggregates or contaminants
• Buffers and mobile phases compatible with both analytes and column
• Column care and equilibration, sample loading, and mobile phase flow optimization to minimize peak broadening while keeping elution times under control
• Multiple detection modalities, used individually or combined, based on UV absorbance, refractive index, and multi-angle light scattering for absolute mass calculations
• Molecular weight calibration standards
• Data analysis and quality practices

The challenges of advanced therapies

SEC separates monomers from dimers, trimers, and higher-order aggregates, and can even resolve native proteins or fragments from their misfolded counterparts. However, its resolving ability is limited, as very large aggregates may stick to the column even under strongly eluting conditions.

This becomes an issue for advanced biotherapeutics like antibody-drug conjugates, therapeutic viruses, or viral vectors. According to Lopez, particle sizes of up to around 50 nm are a practical limit for SEC. Beyond that, she says, “analysis becomes very difficult. Consider choosing an SEC pore size at least three times larger than the molecule of interest.”

A recent paper co-authored by scientists at Waters described an SEC-MALS combination method for analyzing adeno-associated viral vectors which, due to their size and composition, fall outside the capabilities of conventional SEC. Waters’ approach, involving 2.5 mm (as opposed to sub-2-micron) particles and operation at relatively low pressure, reduced run times, improved sensitivity, and generated more measurements with lower per-run sample consumption.

Since SEC is established and well-understood for conventional biologics, one can expect ongoing, incremental improvements for monoclonal antibodies and other first-generation drugs. Developers still grapple with SEC specifics, however, for advanced cell- and gene-based therapies (CGTs).

“Despite many advances, there are inherent challenges associated with these complex, intricate modalities, each with its unique manufacturing requirements” says Mandana Fasth, Global Principal Product Manager for Cell & Gene Therapy at Waters. “Their geometries are complex and intricate, necessitating the development of robust and precise methodologies to ensure standard quality for drug safety and effectiveness.”

These issues, including a lack of high-resolution particle columns with low adsorption, are limiting the growth and accessibility of global CGTs.

“Addressing these challenges will require rapid, reproducible, high-resolution, and low sample consumption analytical tools to assess heterogeneous and lower-purity samples to ensure the products’ stability and potency,” Fasth says. It also requires a broader understanding of how a process works.

Because of CGT diversity, developers of analytical systems require specific R&D domain knowledge as well as experience in regulatory, manufacturing, supply chain management, and commercialization.

“Conventional protein-based drugs may be characterized at the amino acid level, and their binding understood through chromatography and other methods. By contrast viral vectors may incorporate hundreds of individual proteins arranged three-dimensionally, and their structure and genomic variability create a large ensemble of structures that complicate upstream and downstream processing.”

Scaleup, moreover, becomes challenging as viral vectors are traditionally grown from adherent cells, not more easily scaled suspension cultures.

“Additionally, purifying and formulating the vectored therapy involves multiple steps, which increases the risk of contamination (and the need for monitoring) at each step,” Fasth adds. “New SEC technology for analytical assays can be applied to conserve drug substances for patients and to give insights to guide the development of higher yield manufacturing processes.”