Quantitative description of the quality of size exclusion chromatography separations.

Size exclusion chromatography (SEC) is unique among chromatographic methods as it allows separation of non-retained analytes. However, such mechanism often put an analytical scientist in front of relatively poorly resolved set of peaks that may have strikingly different abundance. The description of such chromatograms needs a particular approach to accurately capture the overall quality of separation. Consequently, use of a single parameter description may not be accurate enough and therefore we introduce a dimensionless separation quality factor, which is based on five SEC specific measures (peak-to-valley, elution window width, peak widths, peak-positioning and recovery). Combining several factors allowed detailed differentiation of various simulated separations, clearly correlating column characteristics with specific contributions to separation quality whether they concern a single peak pair or entire peak landscape. The method could be further elaborated by the addition of normalized priority weighting allowing for flexible quality quantification of a relevant portion of real-life nucleic acid separation on different columns. With growing complexity of biotherapeutics to be separated, such a term is predicted to be a useful response function for purposes of factorial method optimization.

Optimizing Messenger RNA Analysis Using Ultra-Wide Pore Size Exclusion Chromatography Columns.

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.

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.

Improved sample introduction approach in hydrophilic interaction liquid chromatography to avoid breakthrough of proteins.

When therapeutic proteins are analysed under hydrophilic interaction liquid chromatography (HILIC) conditions, there is an inherent mismatch between the sample diluent (proteins must be solubilised in aqueous media) and the mobile phase, which is mostly composed of aprotic solvent (acetonitrile). This difference in eluent strength between sample diluent and mobile phase is responsible for severe analyte breakthrough and peak distortion. As demonstrated with therapeutic proteins of different sizes (insulin of 6 kDa, anakinra of 17 kDa and rituximab subunits of 25 and 50 kDa), only very small volumes of 0.1-0.2 µL can be injected without breakthrough effects, when performing rapid analysis on short HILIC columns of 20-50 mm, leading to poor sensitivity. In order to avoid the undesired effect of the strong sample diluent, a special injection program should be preferred. This consists in the addition and automatic injection of a defined volume of weak solvent (acetonitrile) along with the sample to increase retention factors during sample loading. Various injection programs were tested, including the addition of a pre-injection or post-injection or both (bracketed injection) of acetonitrile plugs. Several weak to strong injection solvent ratios of 1:1, 1:2, 1:4 and 1:10 were tested. Our work proves that the addition of a pre-plug solvent with a weak vs. strong injection solvent ratio of 1:10 is a valuable strategy to inject relatively large volumes of proteins in HILIC, regardless of column dimensions, thus maximising sensitivity. No peak deformation or breakthrough was observed under these conditions. However, it is important to note that peak broadening (40 % larger peaks) was observed when the injection program increased the injection solvent ratio from 1:1 to 1:10. Finally, this strategy was applied to a wide range of therapeutic mAb products with different physico-chemical properties. In all cases, relatively large volumes can be successfully injected onto small volume HILIC columns using a purely aqueous sample diluent, as long as an appropriate (weak) solvent pre-injection is applied.

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.

[How could forensic psychiatry be made more attractive?] / Hogyan lehetne az igazságügyi pszichiátriát vonzóbbá tenni?

INTRODUCTION: Decrease in the number of forensic psychiatric experts recently reached a critical level. Shortage of forensic experts caused difficulties in the health care as well as in the justice system. OBJECTIVE: Surveying of how the field of forensic psychiatry and the forensic psychiatric expert work can be made more attractive. METHOD: We performed an online survey among forensic psychiatric experts and specialists in psychiatry. We complied a questionnaire that beside demographic data contained questions in 4 areas. The first 2 areas of the questionnaire - simplifying the forensic psychiatry training (10 items), decreasing the cost of the forensic psychiatry training (5 items) - consisted of simple tatements. Responders had to indicate on a 10-point Likert scale their level of agreement. In the last 2 areas, participants were asked to describe in unstructured format whether they see any circumstance that makes forensic psychiatry attractive or unattractive. The questionnaire was sent out in electronic form to the forensic psychiatric experts on the mailing list of the Hungarian Forensic Expert Chamber, and all specialists in psychiatry on the mailing list of the Hungarian Medical Chamber. RESULTS: Altogether 171 persons filled in the questionnaire with a mean age of 57.26 ± 11.57 years. There were 122 (71.3%) females among the participants. The following proposals received the highest ratings from the forensic psychiatric experts as well as from the specialists in psychiatry: increasing the number of the training institutes; decreasing the costs of the training; making the Hungarian Forensic Expert Chamber course free of charge; and introducing a stipend to cover the costs of the training. DISCUSSION: Results of the survey indicate that active forensic psychiatric experts and specialists in psychiatry representing a potential professional resource, concur that forensic psychiatry training can be made more attractive mainly with increasing the number of training institutes and decreasing the total costs of the training which is compiled from different constituents. CONCLUSION: Due to the critical lack of necessary professionals, urgent action is needed to make forensic psychiatry more popular and the forensic psychiatry qualification easier to obtain. Orv Hetil. 2023; 164(35): 1373-1380.

Practical study on the impact of injection conditions in gradient elution mode for the analysis of therapeutic proteins when using very short columns.

The impact of injected sample volume on apparent efficiency has been studied for very short columns in a systematic way. For large molecules such as therapeutic proteins, it was found that relatively large volumes can be injected onto ultra-short RPLC and IEX columns (i.e. L < 50 mm) without significantly affecting the quality of the separation. This favourable behavior is due to the on-off elution mechanism of large molecules and to the fact that therapeutic protein samples are formulated in aqueous-based media, which is the weakest solvent in RPLC and IEX. Therefore, their peak is strongly focused at the column inlet even when large volume is injected, and pre-column peak dispersion is compensated. However, ultra-short HILIC columns do not seem to be favorable, as they require for very low injection volume to avoid detrimental peak splitting and breakthrough effects. Such peak distortion is related to the inherent solvent mismatch between sample diluent (aqueous) and mobile phase strength (highly organic in HILIC). When studying mass load, the ranking of the elution modes was the same, and the largest relative mass could be injected onto IEX columns (as large as 10% sample to sorbent mass), without affecting the separation quality.

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.

Ultra-short columns for the chromatographic analysis of large molecules.

Today, reverse phase liquid chromatography (RPLC) analysis of proteins is almost exclusively performed on conventional columns (100-150 mm) in gradient elution mode. However, it was shown many years ago that large molecules present an on/off retention mechanism, and that only a very short inlet segment of the chromatographic column retains effectively the large molecules. Much shorter columns - like only a few centimetres or even a few millimetres - can therefore be used to efficiently analyse such macromolecules. The aim of this review is to summarise the historical and more recent works related to the use of very short columns for the analysis of model and therapeutic proteins. To this end, we have outlined the theoretical concepts behind the use of short columns, as well as the instrumental limitations and potential applications. Finally, we have shown that these very short columns were also possibly interesting for other chromatographic modes, such as ion exchange chromatography (IEX), hydrophilic interaction chromatography (HILIC) or hydrophobic interaction chromatography (HIC), as analyses in these chromatographic modes are performed in gradient elution mode.

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.

Investigation of several chromatographic approaches for untargeted profiling of central carbon metabolism.

Monitoring the central carbon metabolism (CCM) network using liquid chromatography/mass spectrometry (LC-MS) analysis is hampered by the diverse chemical nature of its analytes, which are extremely difficult to analyze using single chromatographic conditions. Furthermore, CCM-related compounds present non-specific adsorption on metal surfaces, causing detrimental chromatographic effects and sensitivity loss. In this study, polar reversed-phase, mixed-mode (MMC), and zwitterionic hydrophilic interaction chromatography (HILIC) featuring low-adsorption hardware were investigated towards untargeted analysis of biological samples with a focus on energy metabolism-related analytes. Best results were achieved with sulfoalkylbetaine HILIC with different supports, where polymeric option featured the highest coverage and inert hybrid silica facilitated best throughput and kinetic performance at a cost of less selectivity for small carboxylic acids. MMC demonstrated excellent performance for strongly anionic analytes such as multiresidue phosphates. The obtained experimental data also suggested that an additional hydrophilic modulation might be necessary to facilitate better resolution of carboxylic acids in zHILIC mode, as found during the application of the developed method to study the effect of two different mutations on the energy metabolism of S. aureus.

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.

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.

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.

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.