Reversed-phase High-performance Liquid Chromatography Profiles Research Articles

Impact of Poloxamer 188 Material Attributes on Proteinaceous Visible Particle Formation in Liquid Monoclonal Antibody Formulations

Surfactants such as Poloxamer 188 (PX188) play an important role in controlling particle formation in biotherapeutic formulations due to interfacial stresses. This study demonstrates for the first time that hydrophobicity of PX188 is a potential critical material attribute (CMA) as far as control of visible particle (VP) formation is concerned. We have found that within PX188 lots satisfying pharmacopeial specifications, there is variability in material attributes such as hydrophobicity, as determined from their reversed-phase high-performance liquid chromatography profiles. However, it currently remains unknown how such variability in hydrophobicity of PX188 affects surfactant function and VP formation. Here, we compared the effect of seven PX188 lots in two monoclonal antibody drug product formulations under various stress conditions. Notably, proteinaceous VP formation was reduced while using a PX188 lot with higher hydrophobicity. Our findings emphasize the importance of monitoring lot-to-lot variability of PX188 and provide insight into potential CMA for improving and controlling material attributes of PX188 for use in liquid biotherapeutic formulations.

Dromedary Milk Protein Hydrolysates Show Enhanced Antioxidant and Functional Properties.

SUMMARYResearch backgroundMilk protein hydrolysates have received particular attention due to their health-promoting effects. Dromedary milk differs from the milk of other dairy animals in the composition and structure of its protein components, which give it unique properties. The bioactivity and functionality of whole dromedary milk proteins and their enzymatic hydrolysates have not received much attention, hence this study aims to investigate the effect of enzymatic hydrolysis of dromedary milk proteins on their antioxidant activities and functional properties.Experimental approachDromedary milk proteins were treated using four proteolytic enzymes (pepsin, trypsin, α-chymotrypsin and papain) and two mixtures of enzymes (pancreatin and pronase). The degree of hydrolysis was measured to verify the hydrolysis of the proteins. The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography served to determine the molecular mass distribution of the hydrolysates while reversed phase-high performance liquid chromatography (RP-HPLC) was conducted to explore their hydrophobicity. The antioxidant activities were evaluated using various in vitro tests, including 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical scavenging capacities, iron(III) reducing ability and chelating activity. Besides, functional properties such as solubility, foaming and emulsification were assessed.Results and conclusionsDromedary milk protein hydrolysates exhibited different degrees of hydrolysis ranging from 17.69 to 41.86%. Apart from that, the hydrolysates showed different electrophoretic patterns, molecular mass distribution and RP-HPLC profiles demonstrating the heterogeneity of the resulting peptides in terms of molecular mass and polarity. The hydrolysates displayed significantly higher antioxidant capacities than the undigested proteins at all the tested concentrations. Iron(II) chelating activity was the most improved assay after proteolysis and the hydrolysate generated with pancreatin had the highest chelating power. Dromedary milk protein hydrolysates possessed good solubility (>89%). Further, foaming and emulsifying properties of dromedary milk proteins were enhanced after their proteolysis. These interfacial properties were influenced by the enzymes employed during proteolysis.Novelty and scientific contributionEnzymatic hydrolysis of dromedary milk proteins is an effective tool to obtain protein hydrolysates with great antioxidant and functional properties. These results suggest that dromedary milk protein hydrolysates could be used as a natural source of antioxidant peptides to formulate functional foods and nutraceuticals.

Proteolysis and related enzymatic activities in ten Greek cheese varieties

The objective of this study was to assess indices of proteolysis and related enzymatic activities of various cheese varieties produced in Greece. Physicochemical composition, extent of proteolysis (nitrogen fraction and reversed-phase high-performance liquid chromatography (RP-HPLC) profiles of cheese soluble fraction) and residual chymosin and plasmin activities were analyzed in 57 commercial samples, grouped according to cheesemaking technologies. The mean values of soluble nitrogen fraction on total nitrogen and trichloroacetic acid-soluble nitrogen fraction on total nitrogen did not differ significantly between the different cheese varieties, with mean values of 16.03% and 9.97%, respectively. Brined cheeses had the highest mean residual chymosin activity 0.166 International Milk Clotting units (IMCU).g−1 of cheese dry matter and the lowest mean plasmin plus plasminogen-derived activity 3.88 U.g−1 of cheese. The respective values for Gruyere and hard type cheeses were 0.029 and 0.047 IMCU.g−1 of cheese dry matter for chymosin and 6.22 and 6.94 U.g−1 of cheese for plasmin. It was interesting that both enzymatic activities were high in pasta filata type cheeses, i.e., respective mean values of 0.086 IMCU.g−1 of cheese dry matter and 7.42 U.g−1 of cheese. Intermediate regions on the RP-HPLC profiles were positively correlated with residual chymosin activity and negatively correlated with plasmin activity. It was concluded that cooking at pH close to cheesemilk pH and pressing are the most important technological factors both for proteolysis and activity of major proteolytic enzymes in various cheese varieties.

STUDY OF CONFORMATIONAL CHANGES OF EWE'S HOLO (NATIVE) AND APO-α-LACTALBUMIN BY SPECTROSCOPY AND TRYPSINOLYSIS

Conformational changes of ewe's α-lactalbumin (ALA) upon removal of Ca 2+ were determined by surface hydrophobicity, calorimetry and circular dichroism. Native ewe's ALA resisted trypsinolysis, showing 4% maximum degradation after 20 h of hydrolysis. Removal of bound calcium by addition of either ethylenediaminetetraacetic acid or ethylene glycol bis β-aminoethyl ether-N,N,N,N-tetraacetic acid induced major protein conformational changes, enhancing its susceptibility to trypsinolysis, and leading to complete degradation of the protein. Reversed-phase high-performance liquid chromatography profiles of tryptic hydrolysate of Ca 2+ -free ALA were nearly the same through the whole enzymatic incubation period (24 h) showing the absence of sequential hydrolytic mechanism. They were characterized by the presence of five main peaks representing hydrophobic large-sized peptides. Cleaving the S-S bonds in the resulting hydrolysates with 2-mercaptoethanol gave rise to new peaks representing more hydrophilic and hydrophobic peptides.

Protein homogeneity testing by reversed-phase high-performance liquid chromatography of reduced and non-reduced samples

The concept of this study was based on the assumption that protein standards obtained using the same method may reveal heterogeneity explainable by changes in the disulphide bonding pattern. Two lots of bovine a-lactalbumin standard were subjected to reversed-phase high-performance liquid chromatography (RP-HPLC). One of them was homogenous. The other revealed the presence of two major fractions when non reduced. Neither RP-HPLC after protein reduction nor mass spectrometry showed differences between them. This indicates that one of the lots contained isomers with different locations of disulphide bonds. A comparison of RP-HPLC profiles obtained with and without reduction may be thus recommended as a tool for testing homogeneity of protein standards, especially those used in experiments involving denaturation.

Characterization of storage proteins in different soybean varieties and their relationship to tofu yield and texture

The contribution of the total soybean proteins, the storage proteins [glycinin (11S) and β-conglycinin (7S) fractions] and their respective subunits to tofu yield and texture was studied. Protein contents were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) coupled with densitometry and reversed phase-high performance liquid chromatography (RP-HPLC), for seven soybean varieties, and correlated to tofu yield and texture. Results from SDS-PAGE, coupled with densitometry, showed that the 11S fraction proteins ( r=0.863, P<0.01), the α′ polypeptide of 7S ( r=0.917, P<0.01) and the basic polypeptide of 11S ( r=0.775, P<0.01) appeared to each affect tofu yield; however, no relationship between storage protein fractions and tofu firmness was observed. On the other hand, the RP-HPLC profiles of the total soybean proteins and the 11S and 7S fractions indicated relationships between tofu firmness and the 11S fraction, the 7S fraction, and their ratio. The inverse correlation ( r=−0.832, P<0.01) between peak 7 of the 7S fraction and tofu firmness seems to point toward its important role in defining tofu quality.

Characterization of proteolysis during the ripening of semi-dry fermented sausages

The respective contribution of indigenous enzymes and enzymes from starter bacteria to proteolysis in fermented sausages were determined by comparing the proteolytic changes occurring in sausages resulting from the presence of a proteolytic strain of Staphylococcus carnosus, i.e. S. carnosus MC 1 to the proteolytic changes occurring in control sausages containing glucono-δ-lactone (GDL) and an antibiotic mixture. Proteolysis was quantified by assaying for non-protein nitrogen (NPN) and free amino acids. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and reversed phase high performance liquid chromatography (RP-HPLC) were used to qualitatively assess the proteolytic changes in the sarcoplasmic and myofibrillar proteins as ripening progressed. The concentration of NPN and free amino acids increased in both sausages initially, but subsequently decreased towards the end of ripening in sausages inoculated with the starter culture. SDS-PAGE showed a similar pattern of proteolysis of sarcoplasmic proteins in both sausages, while of the two sausage types; the S. carnosus MC 1 inoculated sausages exhibited the most intense degradation of myofibrillar proteins, especially myosin and actin. RP-HPLC profiles of 2% trichloroacetic acid (TCA)-soluble peptides for the two sausage types were similar, with the production of numerous hydrophilic peptides. N-Terminal amino acid sequence analysis and sequence homology with proteins of known primary structure showed that six of the TCA-soluble peptides were released from the sarcoplasmic (myoglobin and creatine kinase) and myofibrillar (troponin-I, troponin-T and myosin light chain-2) proteins. In addition, the initial degradation of sarcoplasmic proteins was due to the activity of indigenous proteinases, while both indigenous and bacterial enzymes contributed to the initial degradation of myofibrillar proteins. Furthermore, indigenous enzymes were responsible for the release of TCA-soluble peptides, which, were further hydrolysed by bacterial enzymes.

Characterization of beta-conglycinin and glycinin soy protein fractions from four selected soybean genotypes.

The beta-conglycinin and glycinin fractions of soy protein were isolated from Macon, Ohio FG1, Enrei, and IL2 genotypes that were grown under the same environmental conditions. The soy protein fractions were evaluated to determine whether chemical composition and gel-forming properties were related. Amino acid analyses suggested that the hydrophobic residues may be the primary cause of differences in soy protein gel characteristics as the storage moduli increased with higher percentages of hydrophobic residues. Reversed-phase high-performance liquid chromatography profiles revealed variations in the composition of each fraction that corresponded to differences observed among the storage moduli. The gel-forming properties may be related to more than just protein content, such as the amount and type of amino acid in the fraction.

Bitterness in processed cheese caused by an overdose of a specific emulsifying agent?

In Austria, a big manufacturer of processed cheese was faced with a mysterious problem of bitterness and a texture defect in processed cheese slices occurring in distinct batches every once in a while and unexpectedly. To clear up this phenomenon, reference samples of good quality as well as problem cheeses were analysed for chemical composition. Proteolytic changes were studied by urea-polyacrylamide gel electrophoresis (PAGE), SDS-PAGE and isoelectric focusing of caseins as well as of soluble nitrogen fractions. Peptides of the water-soluble nitrogen fractions were analysed by reversed phase-high performance liquid chromatography (RP-HPLC). Surprisingly, bitter cheese samples showed very weak or even no bands in the α s1 - and β -casein region, but only γ -caseins and mainly low molecular weight peptides, which was also confirmed by Kjeldahl analysis. In comparison with the respective reference samples, RP-HPLC profiles of bitter cheese samples showed extremely high concentrations of hydrophilic and hydrophobic peptides. Since significantly higher concentrations of ash and phosphorus were detected in bitter cheese samples, it was noticed that the respective batches of processed cheese slices had been produced apparently using an overdose of a specific emulsifying agent (of high phosphorus content).

Multiple prolactin-releasing activity in the bovine hypothalamic extract.

The prolactin (PRL)-releasing activity (PRA) in the bovine hypothalamic extract (BHE) was compared to that of known substances with PRA and further characterized by gel filtration and reversed-phase high performance liquid chromatography (RP-HPLC). Crude BHE produced marked dose-dependent stimulation of PRL secretion from the cultured rat adenohypophysial cells. Among the synthetic substances examined, vasoactive intestinal peptide (VIP), thyrotropin-releasing hormone (TRH) and beta-endorphin (END) showed significant PRA. However, the flatter dose-response slope for TRH compared with BHE or the small amounts of VIP and END in BHE suggested that these peptides could not account for the major active elements of BHE. Oxytocin and interleukin-1beta were also tested, but they exhibited no PRA in our assay system. Gel filtration of BHE on the Sephadex G-100 column yielded two peaks of PRA distinct from TRH, VIP and END. One eluted in the void and the other in more retarded fractions. The latter fractions were pooled and subjected to the two-step RP-HPLC. The PRA was separated into three peaks designated peaks I, II and III in the first RP-HPLC experiment. Furthermore, the second RP-HPLCs with finer resolution revealed that peak II as well as peak III consisted of three peaks, while peak I eluted as a single peak. Most of these seven PRA peaks exhibited different RP-HPLC profiles from those of the newly characterized PRL-releasing peptides. These findings again provide confirmatory evidence that BHE contained unique factors different from the above known substances.

Analysis of wheat storage proteins by exhaustive sequential extraction followed by RP-HPLC and nitrogen determination

Wheat storage proteins are responsible for the viscoelastic properties of dough. Their effect on dough rheology depends on the glutenin components and the proportion of gliadins, high and low molecular weight glutenin subunits (HMW-GS, LMW-GS). A prediction of dough behaviour is possible when the concentration of each prolamin group is known. A method of sequential extraction and quantification of wheat flour proteins was developed. Reversed-phase high-performance liquid chromatography (RP-HPLC) was used to quantify gliadins and HMW-GS and LMW-GS. Non-prolamin proteins and lipids were extracted with phosphate buffer and phosphate buffer containing Triton X114 respectively and 70% (v/v) ethanol was used to extract gliadins. Several solvent systems were tested to exhaustively extract glutenins and a buffer containing 0·05 M tetraborate pH 8·5, 2% (v/v) 2-mercaptoethanol, 1 g litre−1 glycine, and 8 M urea was selected. It facilitated the most complete extraction and was compatible with RP-HPLC analysis of extracts. The quantities of extracted prolamins were estimated from RP-HPLC profiles using peak area determination. The concentration of different classes of prolamins obtained by RP-HPLC was compared with their determination by nitrogen (N) content. The average yield of extractions performed on 45 cultivars was 0·99 (coefficient of variation (CV)=7·6%) for gliadins and 0·89 (CV=9·1%) for glutenins. Quantifications of proteins by N determination and RP-HPLC were strongly correlated: regression coefficients were 0·86 for total prolamins and gliadins, and 0·71 for glutenins with the gradient of regression of approximately 1. Therefore, RP-HPLC could be used to quantify prolamin groups without using N determination. © 1998 SCI.

Synthesis and structural characterization of charybdotoxin, a potent peptidyl inhibitor of the high conductance Ca2(+)-activated K+ channel.

Charybdotoxin (ChTX), a potent inhibitor of the high conductance Ca2(+)-activated K+ channel (PK,Ca) is a highly basic peptide isolated from venom of the scorpion Leiurus quinquestriatus hebraeus, whose primary structure has been determined (Gimenez-Gallego, G., Navia, M. A., Reuben, J. P., Katz, G. M., Kaczorowski, G. J., and Garcia, M. L. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 3329-3333). The synthesis of this peptide using continuous flow solid phase fluorenylmethyloxycarbonyl-pentafluorophenyl ester methodology has now been achieved. The 1-37-amino acid hexasulfhydryl peptide oxidizes readily to give the tricyclic disulfide structure in good yield. This folded synthetic material is identical to native toxin based on three criteria: co-migration with ChTX on reversed phase high performance liquid chromatography (HPLC); competitive inhibition of 125I-labeled monoiodotyrosine charybdotoxin binding to bovine aortic sarcolemmal membrane vesicles with a Ki (10 pM) identical to that of native toxin; blockade of PK,Ca activity in excised outside-out patches from bovine aortic smooth muscle with the potency and inhibitory properties characteristic of ChTX (i.e. appearance of silent periods interdispersed with normal bursts of channel activity in single channel recordings). Selective enzymatic digestion of native or synthetic ChTX by simultaneous exposure to chymotrypsin and trypsin yields identical reversed phase HPLC profiles. Analysis of the sequence and amino acid composition of the resulting fragments defines a disulfide bond arrangement (Cys7-Cys28, Cys13-Cys33, Cys17-Cys35) which differs from that previously suggested. This configuration predicts a highly folded tertiary structure for ChTX which, together with observations from electrophysiological and binding experiments, suggests a possible mechanism by which ChTX interacts with PK,Ca to block channel function.