New Study Compares Wide Pore Size Exclusion Chromatography Columns for Gene Therapy Product Characterization

To better characterize new-generation gene therapy products, researchers from the University of Geneva led efforts to compare new wide pore size-exclusion chromatography columns. Their findings were published in the Journal of Chromatography A (1). The research team included Mathias Buff and Davy Guilarme (an LCGC International Editorial Advisory Board member) of the University of Geneva, Alexandre Goyon and Kelly Zhang of Genentech, and Carsten Elger, Raphael Ruppert, and Markus Haindl of Roche Diagnostics.

The Human Genome Project, which was completed in 2003, was created to sequence approximately 92% of the total human genome (2). This has helped pharma companies target complex, previously thought undruggable, biological mechanisms. This has led to the development of novel classes of therapeutic modalities beyond traditional small molecules. Over the past two decades, there has been increasing interest in new drug modalities, with an emphasis on monoclonal antibodies (mAbs), and on cell and gene therapies (CGTs). Gene therapy typically involves the repair, replacement, or deactivation of dysfunctional genes in a patient body in order to restore normal functions. CGTs have allowed major progress to be made, largely due to their ability to cure inherited diseases, such as melanoma, pancreatic cancer, and spinal muscular atrophy, among others. CGT approaches typically involve two critical components: the drug modality and the delivery system (drug carrier).

Messenger RNAs (mRNAs) can encode the protein sequence of gene editing enzymes, and its versatility can make it well-adapted to quickly respond to urgent therapeutic needs. Additionally, platform manufacturing technologies enable large-scale productions that are needed for their global distribution. Viral vectors and lipid nanoparticles (LNPs) are the most widely used carriers to deliver the gene editing materials. Thorough analytical characterization of complex gene therapies is required by health agencies prior to patient administration to ensure product quality, safety, and efficacy. These therapies can undergo various changes during their preparation, formulation, and storage, necessitating the evaluation of various critical quality attributes (CQAs) with various analytical techniques.

In this research, a new generation of wide pore size-exclusion chromatography (SEC) columns were systemically evaluated for determining size variants for mRNAs ranging from 1000–5000 nucleotides and for multiple recombinant adeno-associated viruses’ (rAAVs) serotypes. Among the six SEC columns (450 to 700 Å) used for rAAC analysis, the DNACore AAV-SEC column showed the highest efficiency (11,000 plates), probably due to its monodisperse 3 µm silica particles. Meanwhile, the SRT SEC-500 column showed the lowest efficiency (< 1000 plates), likely due to its 5 µm particle packing.

Optimal rAAV selectivity was found with columns that had larger pore sizes (550–700 Å). However, the final resolution for different rAAV serotypes was highly sample-dependent, with no single column consistently providing the best separation of size variants. Further, the six columns deemed suitable to study rAAV degradation trends were subjected to accelerated stress conditions. Among the five SEC columns (700–1000 Å) evaluated for mRNA analysis, the Biozen dSEC-7 LC column (700 Å) column systematically achieved the highest efficiency and was particularly well suited for analyzing small mRNA (∼1000 nts). However, columns with larger pore sizes were more appropriate for larger mRNA (> 1000 nts).

Despite these findings, separating low molecular weight species (LMWS) and high molecular weight species (HMWS) of mRNA remained limited across all tested columns; this makes integration challenging and the quantification of LMWS and HMWS less accurate. Regardless, similar amounts of impurities eluting before and after the main peak were obtained across every tested column, with few exceptions.

References

(1) Buff, M.; Goyon, A.; Elger, C.; Ruppert, R.; et al. Systematic Comparison of Wide Pore Size Exclusion Chromatography Columns for the Characterization of Gene Therapy Products. J. Chromatogr. A 2025, 1752, 465972. DOI: 10.1016/j.chroma.2025.465972

(2) First Complete Sequence of a Human Genome. National Institutes of Health 2022. https://www.nih.gov/news-events/nih-research-matters/first-complete-sequence-human-genome (accessed 2025-7-29)