Conventional Size Exclusion Chromatography Research Articles

Purification of plasmid DNA using a novel two stage chromatography process

The chromatography process of large-scale plasmid purification with high efficiency and low cost has always been a major challenge. We established a two-step plasmid chromatography purification process combining multimodal and thiophilic chromatography with an overall chromatography yield of nearly 70%. Capto Core 700, a multimodal core–shell particle, was firstly used to remove the impurities from the crude lysate. The effects of different experimental conditions on chromatography recovery and impurity removal were screened. Compared to conventional size exclusion chromatography, the sample load and flow rate of this step were enhanced by 40-fold and 5-fold, respectively, while maintaining a 90% yield. For the thiophilic chromatography (Capto PlasmidSelect), the method of Design of Experiments (DoEs) was used to study the influence of parameters on the results. The effects of ammonium sulfate concentration, sodium chloride concentration and flowrate in the elution phase were studied and optimized with a central composite design model consisting of 17 experiments. The versatility of this process was demonstrated by successfully purifying three different lentiviral packaging plasmids (pLP1, pLP2 and pLP/VSVG) and the target plasmid containing green fluorescent protein (GFP). Purified plasmids consistently achieved a supercoiled purity of at least 90% with endotoxin levels below 5 EU/mg. Lentiviral vectors packaged using these plasmids exhibited high infectious titers of 1 × 107 TU/mL, thereby verifying the process applicability for diverse plasmid purification requirements.

Simple and fast one-step FRET assay of therapeutic mAb bevacizumab using anti-idiotype DNA aptamer for process analytical technology

We developed an aptamer-based fluorescence resonance energy transfer (FRET) assay capable of recognizing therapeutic monoclonal antibody bevacizumab and rapidly quantifying its concentration with just one mixing step. In this assay, two fluorescent dyes (fluorescein and tetramethylrhodamine) labeled aptamers bind to two Fab regions on bevacizumab, and FRET fluorescence is observed when both dyes come into close proximity. We optimized this assay in three different formats, catering to a wide range of analytical needs. When applied to hybridoma culture samples in practical settings, this assay exhibited a signal response that was concentration-dependent, falling within the range of 50–2000 μg/mL. The coefficients of determination (r2) ranged from 0.998 to 0.999, and bias and precision results were within ±24.0 % and 20.3 %, respectively. Additionally, during thermal and UV stress testing, this assay demonstrated the ability to detect denatured samples in a manner comparable to conventional Size Exclusion Chromatography. Notably, it offers the added advantage of detecting decreases in binding activity without changes in molecular weight. In contrast to many existing process analytical technology tools, this assay not only identifies bevacizumab but also directly measures the quality attributes related to mAb efficacy, such as the binding activity. As a result, this assay holds great potential as a valuable platform for providing highly reliable quality attribute information in real-time. We consider this will make a significant contribution to the worldwide distribution of high-quality therapeutic mAbs in various aspects of antibody manufacturing, including production monitoring, quality control, commercial lot release, and stability testing.

Size exclusion chromatography as green support for forced degradation study of adalimumab.

Size exclusion chromatography (SEC) has become a powerful tool for analysing size variants of biologic drugs in their native form. Modern SEC can be defined by the use of chromatographic columns packed with sub-3 µm particles, allowing an increase in method throughput compared to that of conventional SEC. We performed the forced degradation study of adalimumab, the first genetically engineered fully humanised immunoglobulin G1 monoclonal antibody, and evaluated tha possibilities of an advanced SEC column packed with sub-3 µm particles for elucidation of the degradation pathway. Our results revealed the main adalimumab degradation products to be antibody fragments. Acidic and basic conditions had the most intensive effect on the degradation of the adalimumab while the drug exhibits relative stability under thermal and photolytic stress conditions. The AGREE and AGREEprep calculators were used for the evaluation of the environmental performance of the forced degradation procedure. The results of the green score evaluation are presented as round pictograms with a circle in the centre that shows the overall score of 0.81 and 0.61, respectively. Both pictograms are highlighted in green, indicating the eco-friendly conditions.

Fractionation and Absolute Molecular Weight Determination of Organosolv Lignin and Its Fractions: Analysis by a Novel Acetone-Based SEC─MALS Method

Accurate and reliable structural characterization of technical lignins is still challenging and inhibits industrial utilization as only poor understanding of structure–property relationship is available. Especially, molar mass analysis of technical lignins is of paramount interest; however, the usage of conventional size exclusion chromatography (SEC) and synthetic polymer standards as a consequence of inaccessible lignin standards is predominantly found in academia. This leads to a huge discrepancy in molecular weight analyses of lignins between different laboratories and consequently to fragile comparability. In this study, organosolv (OS) lignin from beech wood was exemplarily used to develop and evaluate a new acetone–water-based SEC method with the ability to be coupled to an electrospray ionization-mass spectrometer (ESI-MS) or to a multi-angle light scattering detector (MALLS or short MALS). The eluent system used shows very good solubility for the lignins investigated and hence lowers the probability for molecular aggregation of lignin molecules in solution. Solvent fractionation in acetone–water mixtures was conducted to acquire molecular weight classes. The starting lignin (parent lignin) was initially dissolved in 80 wt % acetone–water. Various fractions of the parent lignin were produced by a stepwise reduction of the acetone content. Molecular weights based on narrow polyethylene glycol (PEG)/polyethylene oxide (PEO) standards and absolute molar masses by coupling SEC with MALS were obtained, and the drawback of using polymer standards is discussed in detail. At a lower acetone content, low-molecular-weight fractions were found. Additionally, the specific refractive index increments (dn/dc) were determined for the parent lignin and its fractions. The impact of dn/dc on the final molecular weight (MW) was evaluated considering the chemical composition obtained by nuclear magnetic resonance spectroscopy (NMR) analysis. Furthermore, light scattering revealed that the absorption behavior for this OS lignin is low and neglectable. This article proposes a new acetone-based analytical method for direct determination of absolute molar masses of OS lignin molecules.

A New Approach for Increasing Speed, Loading Capacity, Resolution, and Scalability of Preparative Size-Exclusion Chromatography of Proteins

Low speed, low capacity, and poor scalability make size-exclusion chromatography (SEC) unattractive for use in the preparative separation of proteins. We discuss a novel z2 cuboid SEC device that addresses these challenges. A z2 cuboid SEC device (~24 mL volume) was systematically compared with a conventional SEC column having the same volume and packed with the same resin. The primary objective of this study was to use the same volume of SEC medium in a much more efficient way by using the novel device. At any given flow rate, the pressure drop across the z2 cuboid SEC device was lower by a factor of 6 to 8 due to its shorter bed height and greater cross-sectional area. Under overloaded conditions, the peaks obtained during protein separation with the conventional column were poorly resolved and showed significant fronting, while those obtained with the z2 cuboid SEC device were much better resolved and showed no fronting. At any given flow rate, better resolution was obtained with the z2 cuboid SEC device, while for obtaining a comparable resolution, the flow rate that could be used with the z2 cuboid SEC device was higher by a factor of 2 to 3. Hence, productivity in SEC could easily be increased by 200 to 300% using the z2 cuboid SEC device. The scalability of the z2 cuboid SEC device was also demonstrated based on a device with a 200 mL bed volume.

Rapid Isolation of Extracellular Vesicles Using a Hydrophilic Porous Silica Gel-Based Size-Exclusion Chromatography Column.

Extracellular vesicles (EVs) are nanoscale lipid bilayer vesicles released by almost all cell types and can be found in biological fluids, such as blood and urine. EVs play an important role in various physiological and pathological processes via cell–cell communication, highlighting their potential applications as diagnostic markers for diseases and therapeutic drug delivery carriers. Although various methods have been developed for the isolation of EVs from biological fluids, most of them exhibit major limitations, including low purity, long processing times, and high cost. In this study, we developed a size-exclusion chromatography (SEC) column device using hydrophilic porous silica gel (PSG). Owing to the resistance to pressure of the device, a rapid system for EV isolation was developed by connecting it to a flash liquid chromatography system furnished with a UV detector and a fraction collector. This system can be used for the real-time monitoring of eluted EVs by UV absorption without further analysis and separation of high-purity EVs from urine samples with high durability, reusability, and reproducibility. In addition, there were no significant differences between the PSG column- and conventional SEC column-isolated EVs in the proteome profiles and cellular uptake activities, suggesting the good quality of the EVs isolated by the PSG column. These findings suggest that the PSG column device offers an effective and rapid method for the isolation of intact EVs from biological fluids.

Revisiting the Molar Mass and Conformation of Derivatized Fractionated Softwood Kraft Lignin.

The limited utilization of reliable tools and standards for determination of the softwood kraft lignin molar mass and the corresponding molecular conformation hampers elucidation of the structure-property relationships of lignin. At issue, conventional size exclusion chromatography (SEC) is unable to robustly measure the molar mass because of a lack of calibration standards with a similar structure to lignin. In the present work, the determination of the absolute molar mass of acetylated technical lignin was revisited utilizing SEC combined with multi-angle light scattering with a band pass filter to suppress the fluorescence. Fractionated lignin isolated using sequential techniques of solvent and membrane methods was used to enhance the clarity of light-scattering profiles by narrowing the molar mass distribution of lignin fractions. Further information on the molecular conformation of derivatized samples was studied utilizing a differential viscometer, and chemical structures were identified by NMR spectroscopy analysis. Through the help of fractionation, intrinsic viscosity values were determined for the different fractions as a function of molecular weight cut-off membranes. The derivatized acetone-soluble lignin was found to possess a lower molecular weight and an extremely compact structure relative to the derivatized acetone-insoluble fraction based on a significantly lower "α" value in the Mark-Houwink-Sakurada plot (0.15 acetone-soluble vs 0.33 acetone-insoluble). The differences in geometry were supported by the linkage analysis from NMR showing the acetone-soluble part containing fewer native linkages. In both of these examples, kraft lignin behaved like a solid sphere, limiting the ability to provide entanglements between molecular chains. From this standpoint, macroscopic properties of lignin are justified with this knowledge of a dense and extremely compact structure.

An optimized method for plasma extracellular vesicles isolation to exclude the copresence of biological drugs and plasma proteins which impairs their biological characterization.

Extracellular vesicles (EVs) are cell membrane-derived phospholipid bilayer nanostructures that contain bioactive proteins, enzymes, lipids and polymers of nucleotides. They play a role in intercellular communication and are present in body fluids. EVs can be isolated by methods like ultracentrifugation (UC), polyethylene-glycol-precipitation (PEG) or size exclusion chromatography (SEC). The co-presence of immunoglobulins (Ig) in EV samples isolated from plasma (pEVs) is often reported and this may influence the assessment of the biological function and phenotype of EVs in bio- and immunoassay. Here, we studied the presence of an Ig-based therapeutic (etanercept) in pEV samples isolated from rheumatoid arthritis (RA) patients and improved the isolation method to obtain purer pEVs. From plasma of etanercept (Tumor-necrosis-factor (TNF)-α antibodies)-treated RA patients pEVs were isolated by either UC, PEG or SEC. SEC isolated pEVs showed the highest particle-to-protein ratio. Strong TNF-α inhibition determined in a TNF-α sensitive reporter assay was observed by pEVs isolated by UC and PEG, and to a lesser extent by SEC, suggesting the presence of functional etanercept. SEC isolation of etanercept or labelled immunoglobulin G (IgG) showed co-isolation of these antibodies in the pEV fraction in the presence of plasma or a high protein (albumin) concentration. To minimize the presence of etanercept or immunoglobulins, we extended SEC (eSEC) column length from 56mm to 222mm (total stacking volume unchanged). No effect on the amount of isolated pEVs was observed while protein and IgG content were markedly reduced (90%). Next, from six etanercept- treated RA patients, pEVs were isolated on a eSEC or standard SEC column, in parallel. TNF-α inhibition was again observed in pEVs isolated by conventional SEC but not by eSEC. To confirm the purer pEVs isolated by eSEC the basal IL-8 promoter activation in human monocytes was determined and in 4 out of 5 SEC isolated pEVs activation was observed while eSEC isolated pEVs did not. This study shows that extended SEC columns yielded pEVs without detectable biologicals and with low protein and IgG levels. This isolation method will improve the characterization of pEVs as potential biomarkers and mediators of disease.

Comparative Study of Chemical Composition, Molecular and Rheological Properties of Silicone Oil Medical Devices.

PurposeWe evaluated chemical composition, and molecular and rheological properties in 10 commercially available silicone oils (SilOils), focusing on siloxane chains of low molecular weight (LMW components, LMWC) that are known to be “impurities” produced during the SilOil synthesis process.MethodsWe assessed the type of SilOil polymer and molecular weight distribution (MWD) by spectroscopy and conventional size exclusion chromatography, respectively. From the Cumulative MWD, we calculated the fractions of LMWC with molecular weight (M): ≤2000, ≤5000, and ≤10,000 g/mol. Due to the low MW, the content of LMWC with M ≤1000 g/mol was determined by gas chromatography-mass spectrometry. The dynamic viscosity (η) was assessed by rotational rheometry.ResultsFor all SilOils, the polymer was polydimethylsiloxane. The samples differed significantly in terms of MWD and relative LMWC fractions. Specifically, the relative fraction of all LMWC (M ≤10,000 g/mol) ranged from 2.31% to 9.40% and the content of LMWC with M ≤1000 g/mol also varied significantly (range, 51–1151 ppm). The η values were different between the SilOils, and, for many of them, from the declared viscosity.ConclusionsCommercially available SilOils differ significantly in molecular and rheologic features. These compounds contain a significant amount of LMWC, “impurities” generated during the synthesis process, acting as emulsifier, potentially inducing ocular inflammation and toxicity.Translational RelevanceThe amount of impurities in different SilOils may influence significantly their biocompatibility.

Raman spectroscopy as a quick tool to assess purity of extracellular vesicle preparations and predict their functionality

ABSTRACTExtracellular vesicles (EVs) from a variety of stem cell sources are believed to harbour regenerative capacity, which may be exploited for therapeutic purposes. Because of EV interaction with other soluble secreted factors, EV activity may depend on the employed purification method, which limits cross-study comparisons and therapeutic development. Raman spectroscopy (RS) is a quick and easy method to assess EV purity and composition, giving in-depth biochemical overview on EV preparation. Hereby, we show how this method can be used to characterise EVs isolated from human liver stem cells and bone marrow mesenchymal stem/stromal cells by means of conventional ultracentrifugation (UC) and size exclusion chromatography (SEC) protocols. The obtained EV preparations were demonstrated to be characterised by different degrees of purity and a specific Raman fingerprint that represents both the cell source and the isolation procedure used. Moreover, RS provided useful hints to explore the factors underlying the functional diversity of EV preparations from the same cell source, thus representing a valuable tool to assess EV quality prior to functional assays or therapeutic application.

Molar Mass Characterization of Crude Lignosulfonates by Asymmetric Flow Field-Flow Fractionation

Lignosulfonates (LSs) are the main components of the byproduct of sulfite pulping processes accumulated in large quantities of hundreds of kilotons per year. Furthermore, they are currently the most widely utilized form of lignin due to their chemical and physical properties. LS molar mass characteristics determine their functional properties; however, there is a lack of fast and precise methods for LS molar mass analysis. The complex LS structure after pulping as well as the presence of considerable amounts of impurities in the raw sulfite spent liquor require tedious and time-consuming sample purification steps if conventional size exclusion chromatography (SEC) is to be used for analysis. Asymmetric flow field-flow fractionation (AsFlFFF) represents a good alternative to the conventional SEC technique, as the absence of a stationary phase allows purification and molar mass analysis of nonpurified industrial black liquors in one step, resulting in a drastically simplified procedure. The method’s applica...

Getting Closer to Absolute Molar Masses of Technical Lignins.

Determination of molecular weight parameters of native and, in particular, technical lignins are based on size exclusion chromatography (SEC) approaches. However, no matter which approach is used, either conventional SEC with a refractive index detector and calibration with standards or multi‐angle light scattering (MALS) detection at 488 nm, 633 nm, 658 nm, or 690 nm, all variants can be severely erroneous. The lack of calibration standards with high structural similarity to lignin impairs the quality of the molar masses determined by conventional SEC, and the typical fluorescence of (technical) lignins renders the corresponding MALS data rather questionable. Application of MALS detection at 785 nm by using an infrared laser largely overcomes those problems and allows for a reliable and reproducible determination of the molar mass distributions of all types of lignins, which has been demonstrated in this study for various and structurally different analytes, such as kraft lignins, milled‐wood lignin, lignosulfonates, and biorefinery lignins. The topics of calibration, lignin fluorescence, and lignin UV absorption in connection with MALS detection are critically discussed in detail, and a reliable protocol is presented. Correction factors based on MALS measurements have been determined for commercially available calibration standards, such as pullulan and polystyrene sulfonate, so that now more reliable mass data can be obtained also if no MALS system is available and these conventional calibration standards have to be resorted to.

Size-exclusion chromatography using reverse-phase columns for protein separation

Reverse-phase (RP) liquid chromatography (RPLC) and size-exclusion chromatography (SEC) are methods commonly used for protein/peptide separation, and they are based on distinct principles. This study develops a method using RP columns for size-based separation of protein mixtures. Results show that high concentrations of acetonitrile with trifluoroacetic acid as an acid modifier successfully suppressed interactions between proteins and the stationary phase and allowed the RP column to act as a SEC column to separate proteins based on their molecular weight. The reduction of protein disulfide bonds resulted in an improved correlation between the retention time and molecular weight in the RP-based SEC, which indicates that conformation-dependent SEC retention is less important for disulfide-reduced proteins using a current mobile phase. Importantly, the employed salt-free mobile phase allowed the RP-based SEC system to be directly coupled with online mass spectrometry (MS) analysis. Furthermore, by reducing the flow rate and increasing the column length, the separation efficiency was further improved and no adverse effects due to the prolonged separation were observed, which indicates the potential of this strategy to serve as a first-dimensional separation method for constructing an online multi-dimensional LC system. The size-based separation phenomenon with RP columns was further evaluated using a complex protein mixture (a cell lysate), and compared to conventional SEC, more proteins of the cell lysate were observed following the SEC separation principle, which implies the better generality of usage of the RP-based SEC method for protein separation. In summary, results show that the RP-based SEC is highly efficient in achieving protein fractionation. In addition, the employed salt-free mobile phase provides excellent compatibility of the RP-based SEC with other separation strategies or online mass spectrometric analysis. We anticipate that laboratories using RP-HPLC for protein separation will easily be able to move to the size-based separation mode using the same RP-HPLC system.

Conversion of calibration curves for accurate estimation of molecular weight averages and distributions of polyether polyols by conventional size exclusion chromatography

Accurate measurement of molecular weight averages (M¯n,M¯w,M¯z) and molecular weight distributions (MWD) of polyether polyols by conventional SEC (size exclusion chromatography) is not as straightforward as it would appear. Conventional calibration with polystyrene (PS) standards can only provide PS apparent molecular weights which do not provide accurate estimates of polyol molecular weights. Using polyethylene oxide/polyethylene glycol (PEO/PEG) for molecular weight calibration could improve the accuracy, but the retention behavior of PEO/PEG is not stable in THF-based (tetrahydrofuran) SEC systems. In this work, two approaches for calibration curve conversion with narrow PS and polyol molecular weight standards were developed. Equations to convert PS-apparent molecular weight to polyol-apparent molecular weight were developed using both a rigorous mathematical analysis and graphical plot regression method. The conversion equations obtained by the two approaches were in good agreement. Factors influencing the conversion equation were investigated. It was concluded that the separation conditions such as column batch and operating temperature did not have significant impact on the conversion coefficients and a universal conversion equation could be obtained. With this conversion equation, more accurate estimates of molecular weight averages and MWDs for polyether polyols can be achieved from conventional PS-THF SEC calibration. Moreover, no additional experimentation is required to convert historical PS equivalent data to reasonably accurate molecular weight results.

Mapping the mAb Aggregation Propensity Using Self-Interaction Chromatography as a Screening Tool.

The osmotic second virial coefficient ( B2), which describes protein-protein molecular interactions in solution, was determined using self-interaction chromatography (SIC) for an IgG1-type mAb across a wide range of solution conditions. These data were compared with its time dependent aggregation behavior, as determined using size-exclusion chromatography (SEC), and its temperature dependent aggregation behavior using dynamic light scattering (DLS) over a four-week period (SEC) or overnight (DLS). DLS and SEC gave consistent data on aggregation behavior, which correlated well with experimental B2 trends across the wide pH (4-9) and NaCl concentration (0-1.0 M) ranges studied. The IgG aggregated at pH 4 for 0.5-1.0 M NaCl concentrations and for 0 M NaCl concentrations at pH 8. Best stability against aggregation was exhibited for the pH range from 5 to 8 at 0.8-1.0 M NaCl. SIC data were able to be classified within the one-day solution conditions for aggregation, which were not identified for 2-3 weeks in the accelerated SEC stability study. The ability of SIC to provide such data rapidly reflects the fundamentally thermodynamic nature of this parameter and of the aggregation process itself. Proteins with attractive protein-protein interactions and negative B2 coefficients in the range -3 to -6 clearly exhibit aggregation behavior, while B2 values in the range 0 to 2 showed good stability toward aggregation. SIC allows the rapid screening of solution conditions for which mAbs will exhibit stability to aggregation while requiring 90% less time and material compared with that required for a conventional SEC aggregation study.

Interlaced Size Exclusion Chromatography for faster protein analysis

In this research note, interlaced Size Exclusion Chromatography (SEC) is used to shorten the duration of protein analysis. The applied method has a threefold higher sample throughput than comparable ones using conventional SEC. Further important advantages are the identical area’s precision and the preservation of protein stability. Two different applications are presented where a short time interval of analysis is very important. One application is the determination of aggregates of monoclonal antibodies (mAbs). A high number of antibody samples have to be analyzed during the development process. The antibody sample was measured in a series of 60 injections within only 5.5 h instead of 16 h. The second application is the two-dimensional (2 D) separation of protein mixtures. The number of samples typically increases from the first to the second dimension. Thus, interlaced SEC is a reasonable option for the second dimension. This work confirms the functionality of interlaced SEC and widens the applicable range. Interlaced SEC can become a convenient option for method acceleration in the future.

Improving the accuracy of hyaluronic acid molecular weight estimation by conventional size exclusion chromatography.

There is an unreasonably high variation in the literature reports on molecular weight of hyaluronic acid (HA) estimated using conventional size exclusion chromatography (SEC). This variation is most likely due to errors in estimation. Working with commercially available HA molecular weight standards, this work examines the extent of error in molecular weight estimation due to two factors: use of non-HA based calibration and concentration of sample injected into the SEC column. We develop a multivariate regression correlation to correct for concentration effect. Our analysis showed that, SEC calibration based on non-HA standards like polyethylene oxide and pullulan led to approximately 2 and 10 times overestimation, respectively, when compared to HA-based calibration. Further, we found that injected sample concentration has an effect on molecular weight estimation. Even at 1g/l injected sample concentration, HA molecular weight standards of 0.7 and 1.64MDa showed appreciable underestimation of 11-24%. The multivariate correlation developed was found to reduce error in estimations at 1g/l to <4%. The correlation was also successfully applied to accurately estimate the molecular weight of HA produced by a recombinant Lactococcus lactis fermentation.

Consecutive determination of softwood kraft lignin structure and molar mass from NMR measurements

Abstract The general molecular properties and in particular, the molar mass of lignin are of central importance for industrial applications, as these data govern important thermal and mechanical characteristics. The focus of the present paper is pulsed field gradient-nuclear magnetic resonance (PFG-NMR), which is suitable for determination of lignins’ weight-average molar mass, based on diffusion constants. The method is calibrated by lignin fractions characterized by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). It could be demonstrated on a set of softwood kraft lignins that the PFG-NMR approach gives results in very good agreement with those obtained using conventional size exclusion chromatography (SEC).

Size-exclusion chromatography using core-shell particles

Size-exclusion chromatography (SEC) is an indispensable technique for the separation of high-molecular-weight analytes and for determining molar-mass distributions. The potential application of SEC as second-dimension separation in comprehensive two-dimensional liquid chromatography demands very short analysis times. Liquid chromatography benefits from the advent of highly efficient core-shell packing materials, but because of the reduced total pore volume these materials have so far not been explored in SEC.The feasibility of using core-shell particles in SEC has been investigated and contemporary core-shell materials were compared with conventional packing materials for SEC. Columns packed with very small core-shell particles showed excellent resolution in specific molar-mass ranges, depending on the pore size. The analysis times were about an order of magnitude shorter than what could be achieved using conventional SEC columns.

Ultra-high performance size-exclusion chromatography in polar solvents

Size-exclusion chromatography (SEC) is amongst the most widely used polymer characterization methods in both academic and industrial polymer research allowing the determination of molecular weight and distribution parameters, i.e. the dispersity (Ɖ), of unknown polymers. The many advantages, including accuracy, reproducibility and low sample consumption, have contributed to the worldwide success of this analytical technique. The current generation of SEC systems have a stationary phase mostly containing highly porous, styrene-divinylbenzene particles allowing for a size-based separation of various polymers in solution but limiting the flow rate and solvent compatibility. Recently, sub-2μm ethylene-bridged hybrid (BEH) packing materials have become available for SEC analysis. These packing materials can not only withstand much higher pressures up to 15000psi but also show high spatial stability towards different solvents. Combining these BEH columns with the ultra-high performance LC (UHPLC) technology opens up UHP-SEC analysis, showing strongly reduced runtimes and unprecedented solvent compatibility. In this work, this novel characterization technique was compared to conventional SEC using both highly viscous and highly polar solvents as eluent, namely N,N-dimethylacetamide (DMAc), N,N-dimethylformamide (DMF) and methanol, focusing on the suitability of the BEH-columns for analysis of highly functional polymers. The results show a high functional group compatibility comparable with conventional SEC with remarkably short runtimes and enhanced resolution in methanol.