by Lucia Geis Asteggiante, Senior Technical Specialist at Phenomenex
Nucleic acid-based drugs and gene therapies are innovative approaches that manipulate genes to treat, prevent or cure a medical condition or disease.1 Within these approaches, messenger RNAs (mRNAs) are a key class of drugs with demonstrate potential for the treatment of a broad array of diseases.
Exogenous mRNAs can be introduced into targets cells, where they use the host translation machinery to produce one or more therapeutic proteins. Depending on the therapeutic goal, these proteins could be antigens, monoclonal antibodies, immune modulators, proteins replacing a crucial missing or defective protein, or gene editing machinery, such as Cas 9 in CRISPR-based therapies.2 These drugs are manufactured by a process called in vitro transcription, which is a cell-free, versatile, scalable and cost-effective synthetic platform.
In vitro transcribed (IVT) mRNAs are produced upstream by a sequence of enzymatic processes and then purified downstream through liquid chromatography and ultrafiltration approaches. However, the production process does not end there. Since mRNAs cannot be administered by themselves, a delivery system needs to be set in place to provide both the protection of the mRNA from nucleases and the specificity needed to deliver the genetic material to the appropriate target tissue.
Several delivery systems have been developed. Among them, adeno-associated viruses (AAVs) and lipid nanoparticles (LNPs) are the most often used.3 These delivery systems further the complexity of the drug product, emphasizing the importance of developing reliable and reproducible analytical methods to ensure the quality, safety, and efficacy of these groundbreaking treatments. The USP Draft guidelines provide a framework on the type of methodologies to use to assess the critical quality attributes of both the drug substrate and drug product.4 Size exclusion chromatography (SEC) is one of the suggested approaches for drug purity assessment, providing information regarding the presence of impurities and quantitation of aggregates.
SEC is a chromatographic technique that separates analytes of interest based on their size, or more accurately, their hydrodynamic radius. As the sample is introduced into the column, no interactions (or minimal interactions) between the analytes and the packed media are expected. Separation should then proceed strictly based on the frequency with which each analyte partitions in and out of the pores of the media. The outcome—smaller analytes, which enter the pores more easily and have a longer path to elution, will elute last.
In the case of mRNA therapeutics, SEC can provide lab professionals with information regarding the IVT mRNA (drug substance) molecular mass, the quantity of possible high molecular weight species or aggregates, and the quantity of fragments that remain due to process-related impurities. SEC also characterizes the encapsulated mRNA (drug product), providing information regarding the particle size distribution, the presence of free mRNA, empty AAV or LNP, and an estimate of the empty vs. full particle ratios.
SEC media
Thorough research has been carried out to better explore which SEC media physicochemical properties drive separation and provide chromatographic success. The major findings from this work are that separation selectivity will depend more markedly on both the column’s pore and particle size distribution.
For example, AAVs containing mRNAs have a more compact hydrodynamic radius of ~ 20-25 nm, and achieve satisfactory separation with columns of pore sizes between 550 – 700 Å for most serotypes.5 In the case of mRNAs, column selection can be more challenging, as these molecules can vary significantly in length and have a very dynamic structure. In one specific study, mRNAs with length <4000 nucleotides (nt) were sufficiently separated from their aggregates using a 700 Å SEC column, with best results achieved for mRNAs <2000 nt.6 However, in general, larger mRNA often benefit from wider pore sizes (1000 Å) for optimal separation.5 LNP-encapsulated mRNAs havea much larger hydrodynamic radius than AAVs (e.g. COVID-19 vaccines ~ 93 nm), necessitating ultra-wide pore sizes.7 Note additionally that, even in situations where the evaluated columns had similar manufacturer’s reported specifications, differences in performance were still observed depending on the nature of the sample. Hence, the resulting column choice will strongly depend on the sample under study and may require a column screening process.
Another application of SEC on mRNA therapeutics uses smaller pore size columns (~200 Å) to shed light on the mRNA’s polyA tail average length.8 This approach utilizes the sample preparation of mRNA mapping, where the mRNA is first digested using an enzyme that will simultaneously free the intact polyA tail (100 - 150 nt) and cleave the rest of the mRNA sequence into smaller oligos (~ 30 nt) so the oligonucleotide mixture is separated on a ~200 Å SEC column. This provides significant separation between the shortmers and the polyA tail and allows researchers to quickly estimate average length. This is particularly useful for peptide mapping analyses carried out using Ion Pairing Reversed Phase (IP-RP)-LC-MS/MS, an approach that takes longer to analyze and requires advanced users capable of interpreting MS data.
SEC advancements
Three major technological advancements have allowed SEC technology to become suitable for mRNA therapeutic analyses. The first advancement is the ever-improving hardware inertness, which is crucial to achieve highly reliable analyses with high confidence in the recovery of higher and smaller molecular weight species and robust quantitation capabilities. The modifications done to the column hardware to achieve mitigated adsorption is different depending on the manufacturer. For example, biocompatibility has been achieved by infusing a layer of titanium on the column hardware9, using a hydrophilically modified hybrid surface technology (h-HST) 10 or using a metal free PEEK-lined hardware.11
The second advancement relates to media inertness, which translates into simpler and straightforward platform method development where analyte separation is not significantly affected by the mobile phase composition. As stated earlier, SEC media should, in theory, show no interactions with the analyte, but this is often a challenge to achieve. For example, a highly hydrophilic diol-based silica will do a great job reducing secondary hydrophobic interactions. However, as silanol groups will be negatively charged at pH above ~5, we notice that often higher salt contents are needed to reduce secondary electrostatic interactions. Conversely, a SEC media that better covers or renders unavailable the free silanol groups by using technologies like bridged ethylene polyethylene oxides will offer reduced secondary electrostatic interactions, with these columns performing well with mobile phases with lower salt contents. But, the mild hydrophobicity added by the surface modification may induce secondary hydrophobic interactions with the analyte, often requiring higher organic solvent concentrations. The disadvantage of increasing the organic content is the undesirable denaturing effects on a native approach.
The third advancement in the field is the manufacturing of more reliable and robust ultra-wide pore size SEC columns, which are key for larger biomolecules such as LNPs. Even with the above-mentioned achievements by SEC column manufacturers, there is still room for improvement.
The scientific community would benefit from ultra-wide pore materials that are more robust to pressure fluctuations to achieve longer column lifetimes. Addtionally, columns that provide higher efficiencies would allow for shorter run times, a bottleneck often seen in the fast-paced biopharmaceutical industry.
About the author
Lucia Geis Asteggiante is a Senior Technical Specialist at Phenomenex. She has extensive experience in analytical chemistry, specializing in LC-MS. Lucia has worked on projects from small molecules to large protein complexes, holds a Ph.D. from the University of Maryland, and conducted post-doctoral research at the University of Oxford. She has authored/co-authored 23 scientific publications and 2 book chapters.