High-performance Size-exclusion Chromatography Column Research Articles

High Performance Size Exclusion Chromatography and High-Throughput Dynamic Light Scattering as Orthogonal Methods to Screen for Aggregation and Stability of Monoclonal Antibody Drug Products

The presence of aggregates in monoclonal antibody (mAb) drug product (DP) formulations can present product quality challenges. Here we show that use of High Performance Size Exclusion Chromatography (HP-SEC), in conjunction with high-throughput dynamic light scattering (HT-DLS) analyses of mAb DPs can be a useful strategy to determine monomer content and the presence of aggregates under simulated stress conditions. This analytical approach was used to evaluate four commercially available mAb DPs under different conditions i.e.; original formulations, diluted, and thermo-mechanical stressed condition. Due to particle size limitations of HP-SEC columns, resulting in particles accumulating in the column frits prior to reaching the detector for analysis, there is a possibility that large mAb aggregates may not be detected. Both HP-SEC and HT-DLS were able to detect and resolve the mAb monomer (~10–12 nm) of the DPs in their recommended storage conditions. However, the ability to detect large aggregates (>40 nm) by both analytical methods differed, and HT-DLS was able to detect aggregates between 60 nm and 1400 nm under stress conditions. Our data indicates that HP-SEC, in conjunction with HT-DLS, may be beneficial to detect both mAb DP monomer content and multiple aggregate species (1–1000 nm) in the submicron size range.

Synthesis of Mesoporous Siliceous Materials in Choline Chloride Deep Eutectic Solvents and the Application of These Materials to High-Performance Size Exclusion Chromatography

Mesoporous materials were prepared in choline chloride deep eutectic solvents by triblock copolymer-templated hydrothermal synthesis. The mesoporous materials were characterized by thermogravimetric analysis, scanning electron microscopy and nitrogen sorption analysis. The mesoporous material-based choline chloride-ethylene glycol (1:2 mol ratio) deep eutectic solvent had a flower-type shape, but the material-based choline chloride-urea (1:2) and based choline chloride-acetic acid (1:2) had mesoporous spherical shapes. These mesoporous materials were applied to high-performance size exclusion chromatography column packing. The separation effect of the two dextrans was better from the choline chloride-acetic acid deep eutectic solvent-based mesoporous spheres packed in a high-performance size exclusion chromatography column. In addition, the three polysaccharides, dextran-1 (molecular weight: 670 KD), dextran-2 (molecular weight: 50 KD) and dextran-3 (molecular weight: 5 KD), were separated successfully using the choline chloride-acetic acid deep eutectic solvent-based mesoporous spheres packed in a high-performance size exclusion chromatography column. The mesoporous spheres-based deep eutectic solvents are a potential packing material in high-performance size exclusion chromatography. The separation conditions of more target compounds will be explored in future studies.

Exploration of Mesoporous Siliceous Particle-Based High-Performance Size Exclusion Chromatography for the Quantitation of Biomacromolecular Polysaccharides

Based on a modification of normal hydrothermal synthesis, uniform and spherical mesoporous siliceous particles (MSP) were prepared and applied to high-performance size exclusion chromatography (HPSEC). Sodium acetate buffer solution at pH = 4.0 was the optimal mobile phase for the separation of the three biomacromolecular polysaccharides (alginic acid, fucoidan, and laminarin) in the MSP-based HPSEC. Based on the optimal separation conditions, the calibration curves of the three polysaccharide standards showed good linearity (R2 > 0.999). In addition, the limits of detection of the three standards ranged from 0.02 to 0.13 mg mL−1, the limits of quantitation ranged from 0.07 to 0.43 mg mL−1, the related recoveries were 97.7–101.2%, and the relative standard deviations of the retention time were 0.2–0.5. All the quantitative parameters showed that the developed approach was suitable for quantifying biomacromolecular polysaccharides. Compared to a common commercial HPSEC column, the MSP-based HPSEC column was low cost and had a similar separation efficiency for the three polysaccharides. In particular, 2.8 mg g−1 of the alginic acid fractions, 4.4 mg g−1 of the fucoidan fractions, and 6.9 mg g−1 of the laminarin fractions were separated from the Thallus Laminariae extracts by MSP-based HPSEC. Similar results were obtained from the commercial HPSEC.

In vitro digestibility, structural and functional properties of starch from pigeon pea (Cajanus cajan) cultivars grown in India

Abstract Starches isolated from two pigeon pea cultivars (AL-15 and AL-201) were evaluated for their in vitro digestibility, structural and functional properties. Both the cultivars exhibited a characteristic C-type diffraction pattern with relative crystallinity values of 31.9% and 34.2%, for AL-15 and AL-201, respectively. The structural characterization obtained using high performance size exclusion column chromatography revealed that molecular weight of amylopectin and amylose was higher for AL-201 (396 and 3.92 × 106 g/mol, respectively) in comparison to AL-15 starch (354 and 3.31 × 106 g/mol, respectively). AL-201 exhibited higher values for transition temperatures, enthalpy of gelatinization, pasting temperature and percentage retrogradation in comparison to AL-15 starch. Slowly digestible starch, readily digestible starch and resistant starch contents of AL-15 and AL-201 cultivars were observed to be 31.0, 8.1 and 60.9% and 29.6, 5.2 and 65.2%, respectively. The hydrolysis index and RS values indicated that pigeon pea starches were highly resistant to digestion.

Structural characteristics and in vitro digestibility of Mango kernel starches ( Mangifera indica L.)

Structural characteristics and digestibility of starches isolated from the kernels of two mango cultivars (Chausa and Kuppi) were studied and compared with those of a commercial normal corn starch. Mango kernel starches showed an A-type X-ray diffraction pattern, with relative crystallinities of 35.4% and 38.3%, respectively for Kuppi and Chausa cultivars. The structural characterisation obtained, using high performance size exclusion column chromatography connected to multi-angle laser light scattering and refractive index detectors (HPSEC-MALLS-RI), revealed that the mango kernel starches had lower molecular weight ( M w) and radius of gyration ( R g) of amylopectin and amylose compared to those of corn starch. The M w of amylopectin for Chausa and Kuppi starches were 179 × 10 6 and 140 × 10 6 g/mol, respectively. The amounts of readily digestible starch (RDS) and slowly digestible starch (SDS) were lower for mango kernel starch than those of corn starch. Resistant starch (RS) contents in the mango kernel starches (75.6% and 80.0%, respectively) were substantially higher than those of corn starch (27.3%). The glycemic index (GI) values for mango kernel starches were 48.8 and 50.9 (for Chausa and Kuppi, respectively), whereas that of corn starch was 74.8, indicating that the mango kernel starch granules were highly resistant to digestion with significant contents of RS.

Retention Behaviors and Analytical Methods of High Performance Size-Exclusion Chromatography of Polysaccharides from Duanaliella salina

There are many polysaccharides with biological activities including anti-virus and anti-tumor in the residue produced by the extraction of beta-carotene from Duanaliella salina. In this paper, a method of high performance size-exclusion chromatography coupled with refractive index detection has been developed for the relative molecular mass analysis of the polysaccharides isolated from the residue. The effects of salt and pH values of mobile phase on retention behaviors of five polysaccharides fractions were investigated on two HPSEC columns (Waters Ultrahydragel Linear, 7.8 mm i. d. x 300 mm) connected in series. The results showed that 0. 1 mol/L NaAc buffer solution may be utilized as mobile phase during HPSEC under the conditions of column temperature of 45 degrees C and flow rate of 0.9 mL/min to minimize nonspecific interaction of sulfated polysaccharides fraction (PD4a), complex carbohydrate containing oligonucleic acid (PD4b) and acidic polysaccharide fraction (PD1) with the column matrix. In addition, under the conditions the association effect of the polysaccharide molecules was eliminated. Thereby, the polysaccharide molecules were eluted and separated following equilibrium exclusion mechanism mainly. The weight mean molecular masses (M(w)) of five polysaccharide fractions from D. salina determined under optimum chromatographic conditions were 1 548 000 for PD1, 33 000 for PD2, 67 000 for PD3, 424 000 for PD4a and 10 000 for PD4b. For sulfated polysaccharide fraction PD4a, the relative standard deviations were 1.7% and 0.88% for M(w) and peak area, respectively.

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Structural characteristics and digestibility of starches isolated from the kernels of two mango cultivars (Chausa and Kuppi) were studied and compared with those of a commercial normal corn starch. Mango kernel starches showed an A-type X-ray diffraction pattern, with relative crystallinities of 35.4% and 38.3%, respectively for Kuppi and Chausa cultivars. The structural characterisation obtained, using high performance size exclusion column chromatography connected to multi-angle laser light scattering and refractive index detectors (HPSEC-MALLS-RI), revealed that the mango kernel starches had lower molecular weight (Mw) and radius of gyration (Rg) of amylopectin and amylose compared to those of corn starch. The Mw of amylopectin for Chausa and Kuppi starches were 179 × 106 and 140 × 106 g/mol, respectively. The amounts of readily digestible starch (RDS) and slowly digestible starch (SDS) were lower for mango kernel starch than those of corn starch. Resistant starch (RS) contents in the mango kernel starches (75.6% and 80.0%, respectively) were substantially higher than those of corn starch (27.3%). The glycemic index (GI) values for mango kernel starches were 48.8 and 50.9 (for Chausa and Kuppi, respectively), whereas that of corn starch was 74.8, indicating that the mango kernel starch granules were highly resistant to digestion with significant contents of RS.

Separation and characterization of NOM by high-performance liquid chromatography and on-line three-dimensional excitation emission matrix fluorescence detection.

By using high-performance size exclusion chromatography (HPSEC) and reversed-phase high-performance liquid chromatography with on-line three-dimensional excitation emission matrix fluorescence detection, we measured fluorescence properties of natural organic matter (NOM) as a function of molecular size (MS) and polarity. The work was carried out with Suwannee River Fulvic Acid, Aldrich Humic Acid, and a naturally occurring river NOM sample. Significant differences in fluorescence maximum pattern were found as NOM was separated chromatographically based on MS and polarity. There existed a strong relationship between MS, fluorescence pattern, and polarity. Humic-, fulvic-, and protein-like fluorescence fractions had distinct hydrophilic/hydrophobic nature. The results suggest that HPSEC may be better for characterizing major fulvic-like fluorescence and smaller MS fractions but not those having humic- and protein-like fluorescence and larger MS, which may be strongly adsorbed onto the HPSEC column because of their hydrophobic nature. This study has significant implications for further understanding the nature of NOM and its complexation with trace metals.

Conductivity detection for molecular mass estimation of per- O-sulfonated glycosaminoglycans separated by high-performance size-exclusion chromatography

Chemically per- O-sulfonated polysaccharides, including glycosaminoglycans (GAGs) and hyaluronan oligosaccharides were analyzed using high-performance size-exclusion chromatography (HPSEC) with suppressed conductivity detection. The results were compared to those obtained by gel filtration HPLC using UV detection or fluorescence detection after the post-column reaction with 2-cyanoacetamide in strong alkaline solution. Analysis was performed on a TSKgel G3000SWXL HPSEC column in 5 m M boric acid (pH 7.0 adjusted by 10 m M NaOH). The use of conductivity detection, in the absence of any derivatization and under isocratic conditions gave a limit of detection in the picogram range. Preliminary studies suggest that this approach may be particularly useful in examining sulfonated polysaccharides and oligosaccharides having no UV chromophore, such as those prepared from O-sulfonated fucans and galactans isolated from algae.

Reduced heterogeneity of a lignite humic acid by preparative HPSEC following interaction with an organic acid. Characterization of size-separates by Pyr-GC-MS and 1H-NMR spectroscopy.

Preparative high performance size exclusion chromatography (HPSEC) was used to size-fractionate a humic acid (HA) solution with 0.05 M ionic strength before and after having made the humic solution 0.5 x 10(-3) M in acetic acid (AcOH). Size-fractions were characterized by pyrolysis-gas-chromatography/mass spectrometry (Pyr-GC-MS) and 1H NMR spectroscopy. Pyr-GC-MS showed that the AcOH treatment altered the distribution of humic molecular components in the size-fractions. The unsaturated alkyl chains were moved from size-fractions of larger molecular-size into those of lower molecular-sizes. Most of the aromatic moieties, which were found in larger molecular-size fractions for the untreated HA, were spread into fractions of lower molecular size after AcOH addition to HA. Carbohydrates, which were undetectable in anyfraction of the untreated HA, appeared instead in the pyrogram of the lowest molecular-size and most hydrophilic fraction after treatment with AcOH. Our results suggested that AcOH disrupted the weakly bound association of humic supramolecularstructures and HPSEC elution separated size-fractions of different composition without losing humic matter by adsorption on the HPSEC column. The fractions with the largest apparent molecular size were the richest in alkyl chains, thereby suggesting that humic molecules were stabilized into supramolecular associations by multiple weak interactions among apolar groups such as alkyl chains and aromatic moieties. 1H NMR spectra of size-fractions were greatly simplified and more resolved after AcOH treatment. This was attributed to a less complex molecular association in the size separates which provided a larger solubility in the NMR solvent and more favorable relaxation times. Combination of the procedure used here for size-fractionation with NMR and Pyr-GC-MS methods appears to be promising to advance knowledge on the molecular composition of humic substances.

Molar Masses and Radii of Humic Substances Measured by Light Scattering

A bulk aquatic natural organic material (NOM) was characterized by high-performance size exclusion chromatography (HPSEC) coupled with multi-angle laser light scattering (MALLS), UV-Vis, and refractive index detection. This technique was used to measure the molar mass and radius of the natural organic matter in solution. Results indicate that the NOM aggregates to a large extent at the concentrations used in this study (5...15 mg mL -1 ). The solution conditions of pH and ionic strength were also shown to affect the measured size of NOM. The number and type of HPSEC columns also influenced these measurements. The importance of using multiple detectors was illustrated by peaks that were not detected by all the detectors. While these data are preliminary and have large errors associated with them, it is nevertheless an indication that HPSEC-MALLS can be used to investigate the effects of solution conditions and aggregation on the size of humic substances in solution.

Evaluation of on-line coupling size exclusion chromatography electrothermal atomic absorption spectrometry for selenium speciation

Chromatographic effluents were on-line analyzed by Zeeman-ETAAS, using a flow-through cell placed in a graphite furnace autosampler as interface. To obtain high sampling rates, the use of fast graphite furnace programmes was studied. Conventional programmes of 96 s were reduced to 18 s by using hot injection (120 °C) and reducing the charring step to 2 s. The increase of the injection volume from 20 to 60 μl lengthened the programme to 46 s. Nickel had to be added to get a comparable response for both inorganic and organic selenium species (selenite and selenomethionine) and to reduce the interferent effect produced in presence of the chromatographic eluent (TRIS 0.01 mol l-1, NH4NO3 0.1 mol l-1, pH 7). The optimized conditions were applied to the speciation of selenium in human erythrocyte lysates by size exclusion LC-ETAAS. Using a high performance size exclusion column selenium could be assigned to proteins of 100 and 35 kDa. Detection limits in the range of 1 ng (2 μg l-1 for 500 μl injection volume) were obtained for the combined technique.

Macromolecular Complexation between Bovine Serum Albumin and the Self-Assembled Hydrogel Nanoparticle of Hydrophobized Polysaccharides

Macromolecular complexation between bovine serum albumin (BSA) and self-assembled hydrogel nanoparticle formed by the self-aggregation of cholesterol-bearing pullulan (CHP) was studied by high performance size exclusion column chromatography (HPSEC) and circular dichroism (CD). The CHP self-aggregates complexed with one BSA molecule to give colloidally stable nanoparticles (RG = 17 nm) at pH 7.0 and 25 °C. This was almost irrespective of the substitution degree of the cholesterol group of CHP. The helical content of BSA decreased upon complexation. Unfolding of BSA by either heating or a denaturant such as urea was largely suppressed upon complexation. BSA would be incorporated inside into the hydrogel matrix of the CHP nanoparticle. Kinetic analysis of the complexation suggested a two-step process: namely, the fast pre-equilibrium of looser binding of BSA to the CHP self-aggregate followed by the slower process of tighter inclusion into the hydrogel network. The substitution degree of the cholesterol group in CHP significantly affected the complexation kinetics.

Characterization of starches dissolved in water by microwave heating in a high pressure vessel

Starch granules derived from four corn varieties were dispersed in water and depending on variety about 49–71% of the granules dissolved by microwave heating in a high pressure vessel (MWHPV). The apparent ratios of amylopectin to amylose were 1:0, 3:1, 1:1 and 3:7. High performance size exclusion chromatography (HPSEC) was carried out using two μ Bondagel † and one Synchropak † HPSEC columns placed in series. These had size exclusion limits specified by their manufacturers as 400 nm, 100 nm, and 10 nm, respectively. The mobile phase was 0.05 m NaNO 3. For each starch composition, refractive index and viscosity chromatograms were obtained and fitted with the same six Gaussian components by nonlinear regression analysis. Calibration of the column set with pullulan and dextran standards in hydrodynamic volume and root mean square radius of gyration ( R g) enabled calculation of the intrinsic viscosity ( IV), molecular weight ( M), and R g for each component in addition to global values of these quantities for the entire distribution. Analysis of the data revealed that as starches eluted from the column set, there were large changes in M and R g and rather small changes in IV. Furthermore, MWHPV containing water as employed here produces starch of relatively large molecular weight and size but low intrinsic viscosity leading to the conclusion that dense starch granule fragments were solubilized.

High performance size exclusion chromatography in environmental method development. 1. The effect of mobile phase and temperature on the retention of dioxins, furans, and polychlorinated biphenyls

The thermodynamics of retention for PCBs, dioxins, and furans have been studied for a high performance size exclusion column containing a poly(styrene) stationary phase. The dioxins and furans exhibit a tendency to sorb to this relatively inert surface in the presence of chloroform and dichloromethane. The data indicate that both mobile phase and temperature can be adjusted to improve the separation of these analytes from unwanted matrix components in samples such as sediments, motor oil, and fat. The data also provide some insight into the sorption behavior of these analytes in non-polar solvents.

Determination of molecular size distributions of humic acids by high-performance size-exclusion chromatography

High-performance size-exclusion chromatography (HPSEC) was applied to the study of humic acids and a method for the rapid determination of reliable molecular size distributions of these substances was developed. Using commercial HPSEC columns, in conjunction with a neutral saline solution as eluent, molecular size distributions of four samples of humic acids of various origin and nominal average molecular weights were obtained. Measurements were independent of the operating conditions and based on differences in the molecular sizes of the humic acids examined.

High-performance size-exclusion chromatography of porcine colonic mucins : Comparison of Bio-Gel ® TSK 40XL and Sepharose ® 4B columns

A high-performance size-exclusion chromatography (HPSEC) method was developed for the separation of porcine colonic mucins using a Bio-Gel ® TSK 40XL HPSEC column (300 mn × 75 mm). In addition, porcine gastric and bovine submaxillary mucin preparations were used to describe more fully the separation characteristics of the HPSEC column. For comparison, the same preparations were also separated using a Sepharose ® 4B column (100 cm × 2.6 cm). The colonic and gastric mucins eluted in the void volume ( V 0) of both columns. Bovine submaxillary mucin was in the elution volume ( V e) of both columns. Analytical HPSEC of fractions ( V 0 and V e) of the various preparations obtained by Sepharose 4B chromatography exhibited retention times identical to those for fractions obtained by HPSEC. After separation by both methods, purified mucins were obtained by CsCl 2 density gradient ultracentrifugation; analytical HPSEC profiles, protein contents, and monosaccharide compositions of both gastric and colonic mucins from either column were similar. The HPSEC method, however, is ideally suited to separate microgram to milligram quantities of colonic mucin preparations quickly: 2 to 4 h, compared with 24 to 30 h for the Sepharose 4B method.

Molecular weight distribution of cellulose as its tricarbanilate by high performance size exclusion chromatography

AbstractCellulose tricarbanilates (CTCs) were prepared from a range of cellulose samples (cotton linters, wood pulps, Avicel, amorphous cellulose, and cellulose II) for molecular weight distribution (MWD) studies by high performance size exclusion chromatography (HPSEC). The HPSEC columns were calibrated using CTC standards with the aid of a microcomputer. CTCs were prepared by reaction of cellulose samples with phenylisocyanate in pyridine at 80°C. For some samples, e.g., cellulose II, activation with liquid ammonia and pyridine was necessary prior to reaction in pyridine. All samples tested were also derivatised in dimethylsulfoxide at 70°C, although for high molecular weight (MW) cellulose samples some MW reduction occurred in this solvent. Conditions were determined for optimum precipitation of CTCs in aqueous methanol without coprecipitation of low MW impurities.

Efficient high-performance liquid chromatographic system for protein purification.

An efficient high-performance liquid chromatographic system, consisting of an affinity column and a high-performance size-exclusion column, was developed and applied to the purification of growth hormone receptors from rabbit livers. When a 6-ml sample of Triton X-100 extracts containing 16 mg of protein was applied to the system, 1200-fold purified receptor with a 10% recovery of binding activity from homogenates was obtained within 3-4 h. The purified receptor exhibited one main band on sodium dodecyl sulphate polyacrylamide gel electrophoresis, and the affinity constant (Ka = 6.0.10(9) M-1) was found to be comparable with that of 1% Triton X-100 extract (4.4.10(9) M-1). The injection of 1 ml of 3 M urea solution prior to receptor elution with 10 ml of 6 M urea solution was effective in removing non-specific binding proteins.

Calibration of High Performance size Exclusion Chromatography for Small Epoxy Molecules

Abstract Many epoxy resin formulations contain small oligomers (e.g., less than 1000 molecular weight units) as left over starting materials, reaction by products, or as active parts of the formulation. Modern high performance size exclusion columns are available which enable molecular weights of these oligomers to be determined if proper calibration techniques are employed. Data are presented which demonstrate the differences in calibration curves for epoxy molecules and polystyrene standards, n-alkanes, and phthalate esters for molecular weights under 1000 g/mole. Low MW epoxies are used to calibrate directly for epoxies rather than using molecular size conversion factors calculated from other standards such as polystyrenes.