Size Exclusion High Performance Liquid Chromatography Method Research Articles

Extractability and chromatographic separation of proteins from potato (Solanum tuberosum L.) trimmings

By-products from the potato processing industry, like potato trimmings, are sustainable sources of proteins. Here, a size-exclusion high performance liquid chromatography (SE-HPLC) method was applied to simultaneously determine the extractability and aggregation state of proteins from three batches of potato trimmings of different cultivars. Obtained SE-HPLC profiles allowed distinguishing between the patatin and protease inhibitor fractions of potato proteins. Moreover, only 75% of the crude proteins could be extracted in phosphate buffer containing sodium dodecyl sulfate and a reducing agent, indicating the presence of physical extraction barriers. Ball milling for 5 min significantly increased protein extractability, but prolonged treatment resulted in aggregation of native patatin and a reduced protein extractability. Microwave-dried trimmings had a lower protein extractability than freeze-dried trimmings. In future research, the SE-HPLC method can be used to examine changes in potato protein (fractions) as a result of processing.

Genetic and environmental variation in protein composition of Belgian soy determined with a novel size-exclusion chromatography method

To allow the determination of soy protein composition on protein subunit level in a quantitative manner, a novel method combining size-exclusion high-performance liquid chromatography (SE-HPLC) and peak deconvolution was developed. Peaks corresponding to the different soy protein (subunits) were identified based on the analysis of (i) marker proteins, (ii) 7S and 11S globulin-enriched fractions, and (iii) cross-validation with SDS-PAGE analysis. Given the recent introduction of soybean cultivation in Belgium, the optimized SE-HPLC method was here applied to study the impact of genotype, growing location, harvest year and inoculation strategy on the protein composition of Belgian soy. Especially genotype and harvest year were key to determining protein composition, while all factors influenced the chemical composition of soy flour. The developed SE-HPLC method can be used to further understand the impact of soybean cultivation practices in Belgium and elsewhere on protein composition, which is highly relevant for novel applications of soy. In addition, this SE-HPLC method can be used to determine how various soy processing methods may affect protein composition.

Osborne extractability and chromatographic separation of protein from quinoa (Chenopodium quinoa Willd.) wholemeal

The composition of quinoa proteins has hardly been studied. We here applied an Osborne type extraction procedure. High protein yield and little protein denaturation was obtained with extraction (2x) with water followed by extraction (2x) with 0.4 mol/l sodium chloride. Albumins, globulins, prolamins and glutelins accounted for 41–50, 7–11, 4–6 and 14–16% of the total protein of four quinoa cultivars, respectively. Protein extracts were separated by size exclusion high performance liquid chromatography (SE-HPLC) with a sodium dodecyl sulfate containing elution medium. Electrophoretic separation followed by mass spectrometry allowed identifying the proteins in the Osborne type extracts. Aqueous extracts contained different chloroplastic and cytoplasmic enzymes as well as legumin-like and vicilin-like proteins. Dilute salt extracts mainly contained legumin-like proteins. Aqueous alcohol extracts contained small proteins while dilute base extracts proteins were similar to those present in the aqueous and dilute salt extracts. In one cultivar, only the monomer of the 11S globulin seed storage protein 2-like was observed while the other cultivars studied also contained its acidic and basic subunits separately. This SE-HPLC method allows analyzing food processing induced changes in quinoa protein extractability and size distribution.

Experimental Testing of a Size-Exclusion Chromatography Method Used for Evaluation of Molecular Parameters of Equine Anti-Ebola Immunoglobulin

Haemorrhagic fever caused by the Ebola virus is a highly hazardous infectious disease with a mortality rate of 50– 90 %. Heterologous immunoglobulins with a high virus-neutralizing titer are an important element of the WHO-endorsed set of measures for emergency prevention and treatment of the disease. Specific activity of these products is largely determined by their fractional composition, and, in particular, by molecular mass distribution (MMD). The size-exclusion-high-performance liquid chromatography (SEC-HPLC) has traditionally been used for determination of the MMD of the target protein in human immunoglobulin-based products. The use of this method for evaluation of molecular parameters of heterologous immunoglobulin requires confirmation of its specificity, accuracy and precision, and establishment of the chromatographic system suitability criteria in the context of a new test object.The aimof the study was to test the applicability of the SEC-HPLC method to the assessment of molecular parameters of anti-Ebola immunoglobulin derived from horse serum.Materials and methods: three batches of purified equine anti-Ebola immunoglobulin were used in the study. Normal equine and human immunoglobulins of the IgG isotype were used as reference standards. The HPLC test procedures described in the European Pharmacopoeia 9.6 and State Pharmacopoeia of the Russian Federation, 14th ed., were used for determination of monomers and other immunoglobulin fractions. An Agilent 1260 Infinity (Agilent, USA) HPLC system with a diode array detector and an Agilent Bio SEC-3 HPLC column were used for quality evaluation of the tested products.Results: the resolution factor between IgG monomer and dimer peaks (1.69 and 2.10), and the chromatographic column efficiency (>2000) make it possible to use the SEC-HPLC system for evaluation of molecular parameters of heterologous immunoglobulin. The study demonstrated reproducibility of the test procedure.Conclusions: the study confirmed the applicability of the SEC-HPLC procedure for evaluation of molecular parameters of anti-Ebola immunoglobulin derived from horse serum. It demonstrated the compliance of the purified immunoglobulin to the national and international quality requirements in terms of «Molecular parameters».

A new size-exclusion chromatography method for fast rapeseed albumin and globulin quantification

The method described in the article aims at the quantification of both main storage proteins, globulins and albumins, in aqueous extract from rapeseed, as an alternative to the current reference methods, Kjeldahl and SDS-PAGE electrophoresis. The new method lies on the analytical separation of extracted compounds by Size-Exclusion High Performance Liquid Chromatography (SE-HPLC) (Biosep-SEC-s2000, 5 µm). The elution of rapeseed extracts with water/acetonitrile/trifluoroacetic acid (45/55/0.1% v/v) during 30 min yields two distinct peaks for the main proteins of rapeseed. Based on the protein extinction coefficients, a calibrationless methodology was developed for their quantification on the basis of the UV signal. The SE-HPLC method was successfully compared to references: Kjeldahl and SDS-PAGE densitometry for the determination of the proportion of each protein. Then, it was successfully applied on two other oleoproteagineous plants, linseed and sunflower.

Size-exclusion HPLC provides a simple, rapid, and versatile alternative method for quality control of vaccines by characterizing the assembly of antigens

The assembly of antigen structure is often crucial to the potency of vaccines. Currently adopted methods like animal testing and ultracentrifugation take long time and are difficult to automate for multiple samples. Here we develop a size-exclusion high-performance liquid chromatography (SE-HPLC) method to characterize the assembly of antigen structure during both manufacturing process and storage. Three important vaccine antigens including inactivated foot and mouth disease virus (FMDV), which is a virus vaccine; and two virus-like particles (VLPs) vaccines involving hepatitis B core antigen (HBcAg) VLPs, and hepatitis B surface antigen (HBsAg) VLPs, were successfully analyzed using commercially available TSK gel columns with pore size above 45nm. Combined with other analytical methods including SDS–PAGE, dynamic light scattering, wavelength scan, and multi-angle laser light scattering, the SE-HPLC method was proven to be a simple, rapid, and reliable tool for antigen particles assembly analysis. Specifically, for FMDV whole virus particle, SE-HPLC was used to analyze 146S content in vaccine preparations and the thermal dissociation of the 146S. For HBcAg-VLPs that are expressed in recombinant Escherichia coli, its expression level during cell culture process was quantitatively monitored by SE-HPLC. The SE-HPLC also showed applicability for quality check of HBsAg vaccine preparations by monitoring the product consistency of different lot number and the product stability during storage. Results shown in this work clearly demonstrated that SE-HPLC method has potential as a versatile alternative technology for control of the final product by both manufacturers and the regulatory agencies.

Quantitative Determination for Nerve Growth Factor Concentration in Commercial Product by SEC‐HPLC

The commercial product, Mouse Nerve Growth Factor (NGF) for Injectioni¼Nobex®, is consisted of NGF and human albumin. These two components are both proteins and make it difficult to determine the concentration of NGF by conventional methods. In order to establish a new efficiency quality control process for NGF, this work choose Size Exclusion High Performance Liquid Chromatography (SEC-HPLC) to detect stock solutions and products under screened chromatographic conditions: using Shodex KW-802.5 column, at room temperature with the 0.05 M PBS pH=7 buffer as mobile phase, 0.8 mL/min flow rate and detected under 280 nm wavelength. The standard curves with correlation coefficient of 0.999 82 (for stock solutions) and 0.999 97 (for products) were obtained by external standard method. The NGF concentrations tested in HPLC were compared with which in BCA for the same batches, and the RSD values were less than 3%, which corresponded with China Pharmacopoeia (2010). The results show that SEC-HPLC method is sensitive, accurate and reliable for NGF on-line quality control and has the potential for the quantitative determination of other mixed protein preparations.

Relationship between SDS-Unextractable Glutenin Polymeric Protein and Mixograph Parameters

Improvement of end-use quality has become a major breeding objective in China. In order to further improve gluten quality, effective selection parameter in early generation is necessary in wheat quality breeding program. In total, 108 wheat (Triticum aestivum L.) varieties and advanced lines were used to test kernel hardness, protein content, sedimentation value, mixogragh parameters, glutenin polymeric protein content by biuretium protein fraction procedure, and the amount and ratio of gluten protein fractions by size-exclusion high-performance liquid chromatography method (SE-HPLC). The relationship between flour protein content, sedimentation volume, and mixograph parameters and gluten protein fractions in quantity were investigated. The results showed that broad variations for quality traits and quantitativedata for protein fractions were observed in this set of germplasm. The range of mixograph peak time was 1.12–7.19 min, and the range of SDS-unextractable polymeric protein was very broader than that of SDS-extractable polymeric protein. There were less correlation between grain hardness, flour protein content, whole meal sedimentation value, and mixograph peak time with r of 0.24–0.49. The amount of gliadin was not signifi- cantly correlated with mixograph parameters, while the ratio of gliadin to glutenin (Gli/Glu) was crucial in determining mixograph parameters [r = (–0.52)– (–0.61), P0.001], which was independent of flour protein content. The strong associations between percent SDS-unextractable fraction in total polymeric protein (%UPP) and mixograph parameters were observed with r of0.70–0.85 (P0.001). %UPP and Gli/Glu tested by SE-HPLC can be used as effective parameters for early generation selection to improve wheat gluten strength due to their automatic micro-analyzing method.

Development of physicochemical methods for analysis of pandemic influenza vaccine

The utility of a size exclusion high-performance liquid chromatography (SE-HPLC) method, in the identification and quantitation of haemagglutinin (HA) protein in monovalent, inactivated influenza vaccines for the purposes of potency assessment is reported. This method is sufficiently eveloped to provide a distinct peak separation of HA from other vaccine constituents, and a correlation with HA potency determined by single radial-immunodiffusion (SRID) assay. Sensitivity of the method is demonstrated, with each HA peak accurately titrated to the HA content of the protein load injected. Highly reproducible chromatographic profiles (on a G4000SWxl column) were achieved, demonstrating vaccine protein integrity and stability. Potency assessments, determined by SE-HPLC peak area analysis, provided very good correlation with the quantitative HA protein values, determined by SRID, reported by the manufacturer and by Health Canada's Centre for Biologics Evaluation, Pandemic Influenza Division (PID). Peak resolution was further enhanced by expanding to a tandem SE-HPLC system, utilizing a G4000SWxl SE column coupled to a G3000SWxl column. HA was detected in the 11-17 minute (min) elution, collected in 30 second (sec) fractions, with the smallest, most distinct peak width detected to date at the 15.5 min fraction. This SE-HPLC method has considerable potential for widespread use as a physicochemical method for HA identification and quantification, in quality control testing for seasonal and pandemic influenza vaccines, and as a practical alternative for potency measures by the reagent dependent SRID assay. Increased resolution and further method development will facilitate the collection of separated fractions for further analysis, to correlate immunoactivity to HA type and content.

Studying the formation of aggregates in recombinant human granulocyte-colony stimulating factor (rHuG-CSF), lenograstim, using size-exclusion chromatography and SDS-PAGE

The stability of proteins is a subject of intense current interest. Aggregation, as a dominant degradation pathway for therapeutic proteins, may cause multiple adverse effects, including loss of efficacy and immunogenicity. In the present study, the formation of aggregates in lenograstim under physiological conditions was monitored. For this purpose, a simple and selective size-exclusion high-performance liquid chromatography method for detection and separation of aggregates from intact protein was developed. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis was performed under reducing and non-reducing conditions to determine the nature of aggregate bond formation. Using both techniques, the presence of a low aggregate content attached via disulfide bonds was detected.

Development of a sensitive size exclusion HPLC method with fluorescence detection for the quantitation of recombinant human erythropoietin (r-HuEPO) aggregates

Human erythropoietin produced by recombinant DNA technology, is now marketed worldwide for the treatment of anemias associated with chronic renal failure and chemotherapy. No sensitive methods, which can determine r-HuEPO dimer or oligomer aggregate content in formulated products, have been published to date. This report describes the development and validation of a sensitive size exclusion high performance liquid chromatography (HPLC) method for the quantitation of r-HuEPO aggregates in formulations containing 0.03% polysorbate 80. A Waters Alliance 2690 HPLC system connected to a TosoHaas TSKgel G3000 SWxl (7.8 mm × 30 cm, 250 Å pore size, 5 μm particle size) column and a Waters 474 fluorescence detector was used. The mobile phase for the SEC-HPLC method consists of isopropyl alcohol–potassium phosphate (0.1 M)/potassium chloride buffer (pH 6.8 ± 0.1, 0.2 M) (25:75, v/v). The flow rate was 0.3 mL/min and the method run time was 60 min. The SEC-HPLC method presented here was shown to be specific for r-HuEPO total aggregates (dimer and oligomers) and allows for their quantitation at 80 ng/mL or 4 ngs/injection, in the presence of r-HuEPO monomer and the pharmaceutical excipients, glycine (5 mg/mL), sodium chloride (4.3 mg/mL), and 0.03% polysorbate 80. The finalized method is stability-indicating and is suitable for determining r-HuEPO aggregates between 0.2 and 0.5% levels in the formulated product of r-HuEPO. This method offers a robust way to measure total aggregates on a routine basis with a high sensitivity for use in product quality control.

Extractability and chromatographic separation of rice endosperm proteins

The molecular weight (MW) distribution of proteins extracted with different solvents from defatted rice endosperm was examined by size exclusion-high performance liquid chromatography (SE-HPLC) with 2.0% sodium dodecyl sulfate (SDS) (w/v) as mobile phase. The resulting protein peaks were further characterized by SDS-PAGE. Under the experimental conditions, 2.0% SDS extracted 64% of the proteins. Adding 6.0 M urea resulted in a 15% increase in extractability (up to 79%). With using 20–100 mM NaOH, 70–81% of the proteins were extractable. Maximum extractability was reached with 2.0% SDS, 6.0 M urea and 0.5–1.5% dithiothreitol (DTT). Apparent MW profiles of rice endosperm proteins allowed classification into six fractions of decreasing apparent MW. Fraction VI contained the low MW albumin, globulin, and prolamin protein material. Fractions IV and V originated from α and β glutelin subunits, respectively. The polypeptides of fraction III consisted of an α and a β subunit linked by an intermolecular disulfide bond. The polypeptides of fractions I and II were dimers, trimers or more highly polymerized forms of the (α–β) glutelin subunit dimer in fraction III. While the work confirmed that rice glutelin is composed of polymers of α and β subunits, remarkably, higher MW glutelin aggregates (fractions I–III) only partly dissociated on reduction. Low MW protein material (fraction VI) was entrapped in the aggregated protein network and was released on reduction. The rapid and reproducible SE-HPLC method developed for rice protein separation allows a more quantitative approach than SDS-PAGE.