IP-RPLC Assesses Integrity of Recombinant Adeno-Associated Virus Genomes as Next-Generation Therapeutics


Recombinant adeno-associated viruses (rAAV) have been the subject of much scientific investigation as their use in gene therapy enters the mainstream.

A new submission to Analytical Chemistry proposes a first-of-its-kind ion pairing-reversed-phase liquid chromatography method (IP-RPLC) for determining quality control of recombinant adeno-associated viruses (rAAV), which are gaining attention for their promise as delivery vehicles for gene therapy, but for which analytical techniques have to this point been difficult to develop (1). rAAV are genetically modified versions of adeno-associated viruses. They are created by introducing specific genes of interest into the viral genome, allowing them to deliver and express these genes in target cells, making them useful for gene therapy and biomedical research.

Genetic engineering and gene manipulation concept. Hand is replacing part of a DNA molecule. | Image Credit: © vchalup - stock.adobe.com

Genetic engineering and gene manipulation concept. Hand is replacing part of a DNA molecule. | Image Credit: © vchalup - stock.adobe.com

IP-RPLC is a separation technique that combines both reversed-phase chromatography and ion pairing agents to enhance the separation of ionizable analytes. In this method, an ion pairing reagent is added to the mobile phase, forming complexes with charged analytes and enabling their retention on the hydrophobic stationary phase. The use of ion pairing agents can improve the resolution and selectivity of the separation, particularly for polar or charged compounds that may be difficult to separate using conventional reversed-phase chromatography alone.

This research, mainly based at the Center for Proteomics and Metabolomics at Leiden University Medical Center in Leiden, The Netherlands, obtained results while being supported by two orthogonal techniques, analytical ultracentrifugation (AUC) and capillary gel electrophoresis (CGE). AUC is able to analyze intact, unmodified viruses and can distinguish between rAAV capsids that contain single-stranded DNA (ssDNA) with large size differences, but requires high volumes of samples. CGE’s sample consumption is very low, with injection volumes in the nanoliter range, but that limits sensitivity especially when ultraviolet (UV) detection is employed. IP-RPLC was favored in this study for not only its robustness compared to these techniques, but also its user-friendliness.

IP-RPLC is based on a lipophilic ion-pair reagent such as triethylamine (TEA) that binds to the negatively charged DNA phosphate backing, resulting in increases in both hydrophobicity and DNA length. That eases the process of separation, particularly in differently-sized DNA species in the reversed-phase column. While IP-RPLC has previously been used in the analysis of RNA samples and short- to medium-sized DNA fragments of up to 100 bp, this research demonstrates for the first time a usage in rAAVs of transgene size 2.5 to 4.6 kbp. In addition, the approach can be performed above DNA melting temperatures, which helps avoid the detection of secondary isoforms, and does not require the use of dyes because of UV detection.

Recombinant adeno-associated viruses (rAAV) possess several attributes that make them attractive options for gene delivery – namely, favorable site-specific genome integration, lack of pathogenicity in humans, and dependency on helper viruses. As of 2022, the researchers said, as many as 136 clinical trials of rAAVs in different phases had been reported. Two rAAV therapies, for retinal dystrophy and spinal muscular atrophy, have been approved by the Food & Drug Administration (FDA). In this study, the research team sought to add IP-RPLC to the toolbox of analysis methods for rAAV genome assessment.

One of the primary results reported in the study was IP-RPLC’s ability to separate the genome of a rAAV2-S sample from a contaminant differing in 800 bp, with IP-RPLC requiring only approximately 10–20 μL per injection and CGE, comparatively, able to perform multiple injections out of the same amount. An AUC-based method was unable to resolve smaller differences in ssDNA under normal conditions. IP-RPLC also provided peak fractionation and further and more straightforward characterization, although the researchers conceded that the CGE method demonstrated higher peak efficiency. Still, the study concluded, a possible integration in 2D-LC with a mass spectrometry (MS)-compatible second dimension, or the use of volatile ion pairing reagents, could bring IP-RPLC to the potential of peak identification.


(1) Gstöttner, C.; Hutanu, A.; Boon, S.; et al. Reversed Phase-Liquid Chromatography for Recombinant AAV Genome Integrity Assessment. Anal. Chem. 2023, 95 (22), 8478-8486. DOI: 10.1021/acs.analchem.3c00222

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Toby Astill | Image Credit: © Thermo Fisher Scientific