The 21st century has been particularly productive for the biopharmaceutical industry, with the introduction of several classes of innovative therapeutics, such as monoclonal antibodies and related compounds, gene therapy products, and RNA-based modalities. All these new molecules are susceptible to aggregation and fragmentation, which necessitates a size variant analysis for their comprehensive characterization. Size exclusion chromatography (SEC) is one of the reference techniques that can be applied. The analytical techniques for mAbs are now well established and some of them are now emerging for the newer modalities. In this context, the objective of this review article is: i) to provide a short historical background on SEC, ii) to suggest some clear guidelines on the selection of packing material and mobile phase for successful method development in modern SEC; and iii) to highlight recent advances in SEC, such as the use of narrow-bore and micro-bore columns, ultra-wide pore columns, and low-adsorption column hardware. Some important innovations, such as recycling SEC, the coupling of SEC with mass spectrometry, and the use of alternative detectors such as charge detection mass spectrometry and mass photometry are also described. In addition, this review discusses the use of SEC in multidimensional setups and shows some of the most recent advances at the preparative scale. In the third part of the article, the possibility of SEC for the characterization of new modalities is also reviewed. The final objective of this review is to provide a clear summary of opportunities and limitations of SEC for the analysis of different biopharmaceutical products.

Stapled peptides with an enforced α-helical conformation have been shown to overcome major limitations in the development of short peptides targeting protein–protein interactions (PPIs). While the growing arsenal of methodologies to staple peptides facilitates their preparation, stapling methodologies are not broadly embraced in synthetic library screening. Herein, we report a strategy leveraged on hybridization of short PNA–peptide conjugates wherein nucleobase driven assembly facilitates ligation of peptide fragments and constrains the peptide’s conformation into an α-helix. Using native chemical ligation, we show that a mixture of peptide fragments can be combinatorially ligated and used directly in affinity selection against a target of interest. This approach was exemplified with a focused library targeting the p-53/MDM2 interaction. One hundred peptides were obtained in a one-pot ligation reaction, selected by affinity against MDM2 immobilized on beads, and the best binders were identified by mass spectrometry.

Biopharmaceutical products, in particular messenger ribonucleic acid (mRNA), have the potential to dramatically improve the quality of life for patients suffering from respiratory and infectious diseases, rare genetic disorders, and cancer. However, the quality and safety of such products are particularly critical for patients and require close scrutiny. Key product-related impurities, such as fragments and aggregates, among others, can significantly reduce the efficacy of mRNA therapies. In the present work, the possibilities offered by size exclusion chromatography (SEC) for the characterization of mRNA samples were explored using state-of-the-art ultra-wide pore columns with average pore diameters of 1000 and 2500 Å. Our investigation shows that a column with 1000 Å pores proved to be optimal for the analysis of mRNA products, whatever the size between 500 and 5000 nucleotides (nt). We also studied the influence of mobile phase composition and found that the addition of 10 mM magnesium chloride (MgCl₂) can be beneficial in improving the resolution and recovery of large size variants for some mRNA samples. We demonstrate that caution should be exercised when increasing column length or decreasing the flow rate. While these adjustments slightly improve resolution, they also lead to an apparent increase in the amount of low-molecular-weight species (LMWS) and monomer peak tailing, which can be attributed to the prolonged residence time inside the column. Finally, our optimal SEC method has been successfully applied to a wide range of mRNA products, ranging from 1000 to 4500 nt in length, as well as mRNA from different suppliers and stressed/unstressed samples.

Therapeutic drugs and multivalent vaccines based on the delivery of mRNA via lipid nanoparticle (LNP) technologies are expected to dominate the biopharmaceutical industry landscape in the coming years. Many of these innovative therapies include several nucleic acid components (e.g., nuclease mRNA and guide RNA) posing unique analytical challenges when monitoring the quantity and quality of each individual payload substance in the formulated LNP. Current methods were optimized for single payload analysis and often lack resolving power needed to investigate nucleic acid mixtures. Ion pairing reversed phase (IP-RP) and size exclusion chromatography (SEC) are increasingly being used to characterize nucleic acids. Here, we studied their application for payload quantification in formulated LNP drug-like products. Using a detergent to disrupt the LNPs, the liberated payloads can be separated on an octadecyl RP column using a fast gradient. Reproducible results were obtained as lipids, and surfactants were efficiently eluted using a high organic solvent wash protocol. Alternatively, we also established an online SEC disruption analysis of the mRNA/LNPs wherein an alcohol and detergent containing a mobile phase was applied. Such conditions universally deformulated all tested LNP samples, indicating that a 5 min-long SEC separation can be used as a high-throughput platform method. In both approaches, the measurements facilitate a multiattribute analysis. Apart from quantitation, the characterization of specific impurities is achieved: IP-RP reveals mRNA-lipid adducts, while SEC informs on size variants, which in turn reduces a laboratory's analytical workload. These easy-to-adopt LC-based assays are expected to fortify the analytical toolbox for emerging gene therapeutics.

More and more transformative gene therapies (GTx) are reaching commercialization stage and many of them use Adeno Associated Viruses (AAVs) as their vector. Being larger than therapeutic antibodies, their size variant analysis poses an analytical challenge that must be addressed to speed up the development processes. Size exclusion chromatography (SEC) can provide critical information on the quality and purity of the product, but its full potential is not yet utilized by currently applied columns that are (i) packed with relatively large particles, (ii) prepared exclusively in large formats and (iii) built using metal hardware that is prone to secondary interactions. In this paper, we investigate the use of state-of-the-art sub-3 µm particles to address existing limitations. A prototype 2.5 µm column was found to deliver superior kinetic efficiency, significant reduction in run times and increased resolution of separations. No evidence for shear or sample sieving effects were found during comparisons with conventional 5 µm columns. Moreover, use of low adsorption hardware enabled the application of a wide range of mobile phase conditions and a chance to apply a more robust platform method for several AAV serotypes. The resulting method was tested for its reproducibility as well as utility for critical quality attribute assays, including multiangle light scattering based (MALS) measurements of size and molar mass. Thus, a new tool for higher resolution, higher throughput size variant analysis of AAVs has been described.

Lipid nanoparticles (LNPs) are a key platform for delivering mRNA vaccines and therapeutics with numerous innovative drugs under development. However, characterizing these complex and unstable products remains challenging. Developing fast, reliable methods to assess critical quality attributes (CQAs) of the mRNA component is crucial for ensuring the safety and efficacy of these medicines. Currently, evaluating key CQAs, such as mRNA integrity and encapsulation efficiency, often involves a labor-intensive manual extraction protocol, which requires LNP disruption prior to analysis. However, these additional offline steps contribute to mRNA degradation and measurement uncertainties, highlighting the urgent need for rapid and effective methods capable of performing an online LNP disruption. Hydrophilic interaction chromatography (HILIC) might offer a promising solution to address this need. Due to the presence of high concentrations of organic solvent and the possibility to work at elevated temperatures, HILIC might enable on-column disruption of LNPs while preserving the full integrity of the mRNA payload, facilitating a streamlined characterization process. To evaluate this, we developed two proof of concept HILIC methods. The first one disrupts LNPs and retains the mRNA payload using a high percentage of organic solvent and elevated temperatures. The second one, relying on milder conditions, retains only the unencapsulated mRNA, which can be used to evaluate the encapsulation efficiency. Both methods were used on Comirnaty and Spikevax vaccines and on Sanofi's in-development mRNA product as model samples. Our preliminary findings suggest that HILIC holds potential for online LNP disruption, mRNA integrity assessment, and encapsulation efficiency analysis. They also highlight the limitations of small-pore-sized columns currently available on the market.