Size exclusion chromatography of biopharmaceutical products: From current practices for proteins to emerging trends for viral vectors, nucleic acids and lipid nanoparticles.

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.

Considering the selectivity of pore size gradient size exclusion chromatography columns.

One of the most significant performance determining variables of a size exclusion column is the pore size of its packing material. This is most definitely the case for assigning the suitability of a given column for differently sized analytes. As technologies for particle and column manufacturing continue to advance, it is worth contemplating the value of more finely controlled manipulation of this parameter. The change in a packing material's pores across the length of a size exclusion column was thus explored. A change in average pore diameter and pore size distribution was studied by means of theoretical modeling. These parameters were investigated for independent and combinatorial effects. From our predictions, versus tandem column chromatography, a gradient column apparatus does not yield sizable increases in monomer to dimer selectivity of any given critical pair. Instead, our modeling suggests it can yield more universally effective separations of multiple pairs of species at once, as is sometimes necessary when analyzing the high molecular weight components of highly aggregated drug substances.

Monitoring stability indicating impurities and aldehyde content in lipid nanoparticle raw material and formulated drugs.

Lipid nanoparticles (LNPs) are designed to protect and transport sensitive payloads or active pharmaceutical ingredients as part of new therapeutic modalities. As a multi-component particle, a high degree of quality control is necessary to ensure raw materials are free of critical impurities that could adversely impact the drug product. In this study, we demonstrate a reversed phase liquid chromatography method hyphenated with a single quadrupole mass spectrometer (RPLC-MS) as an alternative platform to methods that incorporate evaporative light scattering or charged aerosol detectors in the detection and quantitation of critical impurities associated with LNPs. The proposed RPLC-MS method offers an increase of up to 2 orders of magnitude in dynamic range and 3 orders of magnitude in sensitivity in the analysis of impurities associated with LNPs compared to conventional detectors. Access to complementary mass data enabled the detection and identification of stability indicating impurities as part of stress studies carried out on an ionizable lipid. In addition to confirmation of peak identity, complementary mass data was also used to assess residual aldehydes in raw material and formulated LNPs in accordance with regulatory guidance. Following derivatization using 2,4-dinitrophenylhydrazine, aldehyde content in the ionizable lipid raw material was determined to exceed the reporting threshold of 0.05% in 30% of the test cases. The experimental findings observed in this study demonstrate the utility of the proposed RPLC-MS method in the identification and monitoring of stability-indicating attributes associated with LNPs as part of current Good Manufacturing Practices for improved consumer safety in drug products.

Fast and efficient size exclusion chromatography of adeno associated viral vectors with 2.5 micrometer particle low adsorption columns.

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.

Separation of Plasmid DNA Topological Forms, Messenger RNA, and Lipid Nanoparticle Aggregates Using an Ultrawide Pore Size Exclusion Chromatography Column.

Health authorities have highlighted the need to determine oligonucleotide aggregates. However, existing technologies have limitations that have prevented the reliable analysis of size variants for large nucleic acids and lipid nanoparticles (LNPs). In this work, nucleic acid and LNP aggregation was examined using prototype, low adsorption ultrawide pore size exclusion chromatography (SEC) columns. A preliminary study was conducted to determine the column's physicochemical properties. A large difference in aggregate content (17.8 vs 59.7 %) was found for a model messenger RNA (mRNA) produced by different manufacturers. We further investigated the nature of the aggregates via a heat treatment. Interestingly, thermal stress irreversibly decreased the amount of aggregates from 59.7 to 4.1% and increased the main peak area 3.3-fold. To the best of our knowledge, for the first time, plasmid DNA topological forms and multimers were separated by analytical SEC. The degradation trends were compared to the data obtained with an anion exchange chromatography method. Finally, unconjugated and fragment antigen-binding (Fab)-guided LNPs were analyzed and their elution times were plotted against their sizes as measured by DLS. Multi-angle light scattering (MALS) was coupled to SEC in order to gain further insights on large species eluting before the LNPs, which were later identified as self-associating LNPs. This study demonstrated the utility of ultrawide pore SEC columns in characterizing the size variants of large nucleic acid therapeutics and LNPs.

High-Throughput Chromatographic Separation of Oligonucleotides: A Proof of Concept Using Ultra-Short Columns.

Ion-pairing reversed-phase liquid chromatography (IP-RPLC) is the reference separation technique for characterizing oligonucleotides (ONs) and their related impurities. The aim of this study was to better understand the retention mechanism of ONs, evaluate the applicability of the linear solvent strength (LSS) retention model, and explore the potential of ultra-short columns having a length of only 5 mm for the separation of model ONs. First, the validity of the LSS model was evaluated for ONs having sizes comprised between 3 and 30 kDa, and the accuracy of retention time predictions was assessed. It was found that ONs in IP-RPLC conditions follow an "on-off" elution behavior, despite a molecular weight lower than that of proteins. For most linear gradient separation conditions, a column length between 5 and 35 mm was found to be appropriate. Ultra-short columns of only 5 mm were therefore explored to speed up separations by considering the impact of the instrumentation on the efficiency. Interestingly, the impacts of injection volume and post-column connection tubing on peak capacity were found to be negligible. Finally, it was demonstrated that longer columns would not improve selectivity or separation efficiency, but baseline separation of three model ONs mixtures was enabled in as little as 30 s on the 5 mm column. This proof-of-concept work paves the way for future investigations using more complex therapeutic ONs and their related impurities.

Studying effective column lengths in liquid chromatography of large biomolecules.

Based on their nature, large molecules tend to exhibit on-off elution such that only a small segment of a column bed participates in their separation. We were intrigued to investigate empirical data on this behavior and to apply a simple method to estimate the length of column bed that is needed to produce an effective separation. Models were derived by rearranging the linear solvent strength (LSS) model equations, and data sets from almost 100 different separation conditions were treated to illustrate effects for various types of solutes as separated by reversed phase (RP), ion-pair reversed phase (IP-RP), ion-exchange (IEX), hydrophobic interaction (HIC) and hydrophilic interaction (HILIC) chromatography. By empirically measuring S parameters (S is a solute dependent model parameter, it describes how sensitive is the solute retention to mobile phase composition), and calculating for an exit retention factor of 0.5, we have determined that there is little to no benefit to separating moderately sized solutes (5 - 10 kDa) with a column bed that is longer than 3 cm, particularly when a less than 20 min gradient is desired. Moreover, even shorter columns would be predicted to be adequate for 100 - 150 kDa molecules. Interpretations of this sort have become possible because there is some correlation between a solute's molecular weight and its S parameter. That is, empirical observations on retention behavior are not needed to select appropriate column lengths; molecular weight provides a sufficient approximation. With these insights, we suggest reconsidering the routine use of 5 - 15 cm long columns for >10 kDa biomolecule separations and instead propose that a new focus be placed on 1-2 cm long columns.

Theoretical study on solute migration and band broadening occurring in pressure-enhanced liquid chromatography.

Upon recently studying the use of pressure gradients during liquid chromatography (LC), it was noted that pressure differentials across a column can have a significant impact on peak shape, not just retention as has been noted several times before. Theoretical models and thought experiments were performed here to more carefully study these effects. Two situations have been elucidated. The first is one that reflects a protein reversed phase separation wherein solute retention increases with pressure. In this condition, it has been found that a positive pressure gradient will result in band broadening while a negative pressure gradient will help yield sharper peaks. The second case that has come to be better appreciated is when solute retention decreases with pressure, which can occur in protein ion exchange (IEX) and hydrophobic interaction chromatography (HIC). In this situation, a positive pressure gradient will conversely result in peak sharpening, and a negative pressure gradient will introduce band broadening. These observations have facilitated making new fundamental understandings on pressurized separations which has in turn made it possible to begin envisioning new ways of and reasons for applying pressure enhanced LC methods.

Insights on further improving fast size exclusion chromatography separations of biopharmaceuticals using 2.1 millimetre column diameters.

Recent trends in the pharmaceutical and biotechnology industries call for the miniaturization of size exclusion chromatography. The thought of such a future has been tantalizing but there are many practical and theoretical considerations that have impeded progress. Here, the capabilities of a narrow bore 2.1 mm ID SEC column have been studied and compared to reference 150 × 4.6 mm SEC columns when using UV detection. While our study reconfirms the importance of having very low system dispersion for SEC separations, it goes on to show that a 150 × 2.1 mm 1.7 µm particle SEC column can offer a balanced compromise of performance. Despite the fact that the 150 × 2.1 mm ID 1.7 µm column's intrinsic efficiency was not fully utilized, it still performed with an apparent efficiency similar to that of a 150 × 4.6 mm ID 2.5 µm column. Beyond this, our study provides insights on what more will need to be achieved to robustly establish low flow SEC separations. If SEC chromatographers aim to miniaturize sizing separations to 1 mm diameters or below, there is more work to do on chromatographic instruments and flow paths. In order for an instrument to be optimized for 1 mm ID SEC it would need to exhibit a system variance of less than 0.5 µL2.

Capillary liquid chromatography coupled with mass spectrometry for analysis of nanogram protein quantities on a wide-pore superficially porous particle column in top-down proteomics.

In top-down proteomics experiments, intact protein ions are subjected to gas-phase fragmentation for MS analysis without prior digestion. This approach is used to characterize post-translational modifications and clipped forms of proteins, avoids several "inference" problems associated with bottom-up proteomics, and is well suited to the study of proteoforms. In the past decade, top-down proteomics has progressed rapidly, taking advantage of MS instrumentation improvements and the efforts of pioneering groups working to improve sample handling and data processing. The potential of this technology has been established through its successful use in a number of important biological studies. However, many challenges remain to be addressed like improving protein separation capabilities such that it might become possible to expand the dynamic range of whole proteome analysis, address co-elution and convoluted mass spectral data, and aid final data processing from peak identification to quantification. In this study, we investigated the use of a wide-pore silica-based superficially porous media with a high coverage phenyl bonding, commercially packed into customized capillary columns for the purpose of top-down proteomics. Protein samples of increasing complexity were tested, namely subunit digests of a monoclonal antibody, components of purified histones and proteins extracted from eukaryotic ribosomes. High quality mass spectra were obtained from only 100 ng of protein sample while using difluoroacetic acid as an ion pairing agent to improve peak shape and chromatographic resolution. A peak width at half height of about 15 s for a 45 min gradient time was observed on a complex mixture giving an estimated peak capacity close to 100. Most importantly, efficient separations were obtained for highly diverse proteins and there was no need to make method specific adjustments, suggesting this is a highly versatile and easy-to-use setup for top-down proteomics.

Challenges and emerging trends in liquid chromatography-based analyses of mRNA pharmaceuticals.

Lipid encapsulated messenger RNA (LNP mRNA) has garnered a significant amount of interest from the pharmaceutical industry and general public alike. This attention has been catalyzed by the clinical success of LNP mRNA for SARS-CoV-2 vaccination as well as future promises that might be fulfilled by the biotechnology pipeline, such as the in vivo delivery of a CRISPR/Cas9 complex that can edit patient cells to reduce levels of low-density lipoprotein. LNP mRNAs are comprised of various chemically diverse molecules brought together in a sophisticated intermolecular complex. This can make it challenging to achieve thorough analytical characterization. Nevertheless, liquid chromatography is becoming an increasingly relied upon technique for LNP mRNA analyses. Although there have been significant advances in all types of LNP mRNA analyses, this review focuses on recent developments and the possibilities of applying anion exchange (AEX) and ion pairing reversed phase (IP-RP) liquid chromatography for intact mRNAs as well as techniques for oligo mapping analysis, 5' endcap testing and lipid compositional assays.

Ultra-Fast Middle-Up Reversed Phase Liquid Chromatography Analysis of Complex Bispecific Antibodies Obtained in Less Than One Minute.

This work illustrates the benefits and limitations of using ultra-short reversed phase liquid chromatography (RPLC) columns for the characterization of various complex bispecific antibodies after prolonged thermal stress at the middle-up level of analysis. First, we have demonstrated that alternative organic modifiers, such as isopropanol, can be used in RPLC mode without generating excessive pressure, thanks to the prototype 10 × 2.1 mm, 2.7 µm particle column. However, compared to acetonitrile, the selectivity was not improved, at least for the selected biopharmaceutical products. Importantly, very fast separations (sub-1 min) of high quality were systematically obtained for the different samples when using a spectroscopic detector, but a severe loss of performance was observed with mass spectrometry (MS) detection due to dispersion effects. Based on these results, there is a clear need to improve the interfacing between LC and MS (shorter/thinner tubing) to mitigate band broadening.

Expediting the chromatographic analysis of COVID-19 antibody therapeutics with ultra-short columns, retention modeling and automated method development.

The COVID-19 pandemic necessitated the emergency use authorization (EUA) of several new therapeutics and vaccines. Several monoclonal antibodies (mAbs) were among those authorized for use, and they have served a purpose to provide passive immunity and to help minimize dangerous secondary effects in at-risk and hospitalized patients infected with SARS-CoV-2. With an EUA submission, scientific data on a drug candidate is often collected near simultaneously alongside drug development. In such a situation, there is little time to allow misguided method development nor time to wait on traditional turnaround times. We have taken this dilemma as a chance to propose new means to expediting the chromatographic characterization of protein therapeutics. To this end, we have combined the use of automated, systematic modeling and ultrashort LC columns to quickly optimize high throughput RP, IEX, HILIC and SEC separations for two COVID-19-related mAbs. The development and verification of these four complementary analytical methods required only 2 days of experimental work. In the end, one chromatographic analysis can be performed with a sub-2 min run time such that it is feasible to comprehensively characterize a COVID-19 mAb cocktail by 4 different profiling techniques within a 1-hour turnaround time.

Studying the possibilities of dual stationary phase gradients to explore alternative selectivities in liquid chromatography.

Previous studies on stationary phase gradients have inspired a new phase of theoretical work and an expansion of the concept to include multiple retention mechanisms. The theoretical work presented here corroborates previous reports that a stationary phase gradient can produce selectivity in a separation as effectively as careful adjustments to a mobile phase gradient program, especially when such column is operated in mobile phase gradient elution mode. In reality, no column is singularly based on one type of interaction mechanism, and many columns are nowadays designed to solicit multiple solute to stationary phase interactions. These mixed-mode columns inspired us to give more careful consideration to the idea of dual stationary phase gradients. With the theory applied here, it is suggested that the modulation of two unique interaction mechanisms across a chromatographic column has the potential to open up previously unseen selectivities. With the increasing prevalence of mixed-mode columns, we believe there will be ample opportunity to explore these new concepts in experimental work.

Investigating the secondary interactions of packing materials for size-exclusion chromatography of therapeutic proteins.

Size exclusion chromatography has become an essential tool for the protein therapeutics industry. Conceptually, it is a simple form of chromatography that is driven by entropy and sieving effects. An ideal size exclusion column would exhibit no adsorptive interactions between its internal surfaces and the solutes being analysed, but that is not easily achieved. To this end, we have studied the utility of three unique packing materials in pursuit of additional column chemistries that might be less prone to interacting with proteins. These packing materials were each prepared from bridged ethylene hybrid organic/inorganic particles but uniquely derivatized into either hydroxy terminated PEO bonded, methoxy terminated PEO bonded, or diol bonded packing materials. All three materials were packed into column hardware modified with hydrophilic hybrid surface technology (h-HST) so that packing material effects could be more clearly observed without any influence from the secondary interactions that can originate from metal hardware. Non-specific interactions were compared for various challenging protein samples in the presence of ammonium acetate (volatile) and phosphate buffered saline (non-volatile) buffers. It was reconfirmed that the h-HST column hardware mitigates a majority of non-desired secondary interactions. However, during studies on hydrophobic interactions, the new hydroxy terminated PEO packing material showed clear benefit to obtaining higher apparent recoveries to better ensure accurate aggregate quantitation. Further experiments were explored to show that a hydroxy terminated PEO column could be effectively paired with a mobile phase comprised of standard strength phosphate buffered saline to make a fast platform method capable of baseline resolving monoclonal antibody monomer and aggregate peaks within a 3 min analysis time.

The impact of low adsorption surfaces for the analysis of DNA and RNA oligonucleotides.

As interest in oligonucleotide (ON) therapeutics is increasing, there is a need to develop suitable analytical methods able to properly analyze those molecules. However, an issue exists in the adsorption of ONs on different parts of the instrumentation during their analysis. The goal of the present paper was to comprehensively evaluate various types of bioinert materials used in ion-pairing reversed-phase (IP-RPLC) and hydrophilic interaction chromatography (HILIC) to mitigate this issue for 15- to 100-mer DNA and RNA oligonucleotides. The whole sample flow path was considered under both conditions, including chromatographic columns, ultra-high-performance liquid chromatography (UHPLC) system, and ultraviolet (UV) flow cell. It was found that a negligible amount of non-specific adsorption might be attributable to the chromatographic instrumentation. However, the flow cell of a detector should be carefully subjected to sample-based conditioning, as the material used in the UV flow cell was found to significantly impact the peak shapes of the largest ONs (60- to 100-mer). Most importantly, we found that the choice of column hardware had the most significant impact on the extent of non-specific adsorption. Depending on the material used for the column walls and frits, adsorption can be more or less pronounced. It was proved that any type of bioinert RPLC/HILIC column hardware offered some clear benefits in terms of adsorption in comparison to their stainless-steel counterparts. Finally, the evaluation of a large set of ONs was performed, including a DNA duplex and DNA or RNA ONs having different base composition, furanose sugar, and modifications occurring at the phosphate linkage or at the sugar moiety. This work represents an important advance in understanding the overall ON adsorption, and it helps to define the best combination of materials when analyzing a wide range of unmodified and modified 20-mer DNA and RNA ONs.

Pressure-Enhanced Liquid Chromatography, a Proof of Concept: Tuning Selectivity with Pressure Changes and Gradients.

Many chromatographers have observed that the operating pressure can dramatically change the chromatographic retention of solutes. Small molecules show observables changes, yet even more sizable effects are encountered with large biomolecules. With this work, we have explored the use of pressure as a method development parameter to alter the reversed-phase selectivity of peptide and protein separations. An apparatus for the facile manipulation of column pressure was assembled through a two-pump system and postcolumn flow restriction. The primary pump provided an eluent flow through the column, while the secondary pump provided a pressure-modulating flow at a tee junction after the column but ahead of a flow restrictor. Using this setup, we were able to quickly program various constant pressure changes and even pressure gradients. It was reconfirmed that pressure changes impact the retention of large molecules to a much greater degree than small molecules, making it especially interesting to consider the use of pressure to selectively separate solutes of different sizes. The addition of pressure to bring the column operating pressure beyond 500 bar was enough to change the elution order of insulin (a peptide hormone) and cytochrome C (a small serum protein). Moreover, with the proposed setup, it was possible to combine eluent and pressure gradients in the same analytical run. This advanced technique was applied to improve the separation of insulin from one of its forced degradation impurities. We have referred to this method as pressure-enhanced liquid chromatography and believe that it can offer unseen selectivity, starting with peptide and protein reversed-phase separations.

Development of optimized drug-like small molecule inhibitors of the SARS-CoV-2 3CL protease for treatment of COVID-19.

The SARS-CoV-2 3CL protease is a critical drug target for small molecule COVID-19 therapy, given its likely druggability and essentiality in the viral maturation and replication cycle. Based on the conservation of 3CL protease substrate binding pockets across coronaviruses and using screening, we identified four structurally distinct lead compounds that inhibit SARS-CoV-2 3CL protease. After evaluation of their binding specificity, cellular antiviral potency, metabolic stability, and water solubility, we prioritized the GC376 scaffold as being optimal for optimization. We identified multiple drug-like compounds with <10 nM potency for inhibiting SARS-CoV-2 3CL and the ability to block SARS-CoV-2 replication in human cells, obtained co-crystal structures of the 3CL protease in complex with these compounds, and determined that they have pan-coronavirus activity. We selected one compound, termed coronastat, as an optimized lead and characterized it in pharmacokinetic and safety studies in vivo. Coronastat represents a new candidate for a small molecule protease inhibitor for the treatment of SARS-CoV-2 infection for eliminating pandemics involving coronaviruses.

Direct coupling of size exclusion chromatography and mass spectrometry for the characterization of complex monoclonal antibody products.

The present study describes the possibilities offered by an innovative bioinert size exclusion chromatography column for size variant characterization of complex monoclonal antibody products. This size exclusion chromatography column includes a novel column hardware surface. The column was prepared from metallic hardware components that were treated to have prototype hydrophilically modified hybrid organic-inorganic silica surfaces called hybrid surface technology. This provides a significant reduction in nondesired hydrophobic and electrostatic interactions that can occur between column and analyte when performing size exclusion chromatography analysis with volatile mobile phase. Compared to a reference stainless-steel column packed with the same batch of packing material, peak tailing, band broadening, and above all recovery of high molecular weight species were distinctly improved for all types of monoclonal antibody products. Based on our observations, we found that 50 mM ammonium acetate in water was a suitable mobile phase offering good compromise in terms of liquid chromatography performance and mass spectrometry sensitivity. In addition, method repeatability (intra- and interday relative standard deviations) on elution times and high molecular weight species peak areas were found to be excellent. By using this innovative size exclusion chromatography material, the low and high molecular weight species contained in various stressed and nonstressed monoclonal antibody products were successfully characterized with mass spectrometry detection.

Size Exclusion and Ion Exchange Chromatographic Hardware Modified with a Hydrophilic Hybrid Surface.

Certain biomolecules have proven to be difficult to analyze by liquid chromatography (LC), especially under certain chromatographic conditions. The separation of proteins in aqueous mobile phases is one such example because there is the potential for both hydrophobic and ionic secondary interactions to occur with chromatographic hardware to the detriment of peak recovery, peak shape, and the overall sensitivity of the LC analysis. To decrease non-specific adsorption and undesired secondary interactions between column hardware and biomolecules, we have developed and applied a new hydrophilically modified hybrid surface (h-HST) for size exclusion chromatography (SEC) and anion exchange (AEX) separations of proteins and nucleic acids. This surface incorporates additional oxygen and carbon atoms onto an ethylene bridge hybrid siloxane polymer. As a result, it exhibits reduced electrostatic properties and hydrophilicity that facilitates challenging aqueous separations. Flow injection tests with a phosphate buffer showed superior protein recovery from an h-HST frit when compared to unmodified ethylene-bridged hybrid HST, titanium, stainless steel, and PEEK frits. When applied to SEC of rituximab, ramucirumab, and trastuzumab emtansine with a 50 mM ammonium acetate buffer, this new hydrophilic chromatographic hardware yielded improved monomer and aggregate recovery, higher plate numbers, and more symmetrical peaks. AEX columns also benefited from h-HST hardware. An acidic mAb (eculizumab) showed improved recovery, more stable retention, and a sharper peak when eluted from an h-HST versus SS column. Moreover, AEX separations of intact mRNA samples (Cas9 and EPO mRNA) were improved, where it was seen that h-HST column hardware provided higher sensitivity and more repeatable peak areas from injection to injection. As such, there is significant potential in the use of h-HST chromatographic hardware to facilitate more robust and more sensitive analyses for a multitude of challenging separations and analytes.