Size-exclusion High-performance Liquid Chromatography Research Articles

(-)-Epicatechin-An Important Contributor to the Antioxidant Activity of Japanese Knotweed Rhizome Bark Extract as Determined by Antioxidant Activity-Guided Fractionation.

The antioxidant activities of Japanese knotweed rhizome bark extracts, prepared with eight different solvents or solvent mixtures (water, methanol, 80% methanol(aq), acetone, 70% acetone(aq), ethanol, 70% ethanol(aq), and 90% ethyl acetate(aq)), were determined using a 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical-scavenging assay. Low half maximal inhibitory concentration (IC50) values (2.632–3.720 µg mL−1) for all the extracts were in the range of the IC50 value of the known antioxidant ascorbic acid at t0 (3.115 µg mL−1). Due to the highest extraction yield (~44%), 70% ethanol(aq) was selected for the preparation of the extract for further investigations. The IC50 value calculated for its antioxidant activity remained stable for at least 14 days, while the IC50 of ascorbic acid increased over time. The stability study showed that the container material was of great importance for the light-protected storage of the ascorbic acid(aq) solution in a refrigerator. Size exclusion–high-performance liquid chromatography (SEC-HPLC)–UV and reversed phase (RP)-HPLC-UV coupled with multistage mass spectrometry (MSn) were developed for fractionation of the 70% ethanol(aq) extract and for further compound identification, respectively. In the most potent antioxidant SEC fraction, determined using an on-line post-column SEC-HPLC-DPPH assay, epicatechin, resveratrol malonyl hexoside, and its in-source fragments (resveratrol and resveratrol acetyl hexoside) were tentatively identified by RP-HPLC-MSn. Moreover, epicatechin was additionally confirmed by two orthogonal methods, SEC-HPLC-UV and high-performance thin-layer chromatography (HPTLC) coupled with densitometry. Finally, the latter technique enabled the identification of (−)-epicatechin. (−)-Epicatechin demonstrated potent and stable time-dependent antioxidant activity (IC50 value ~1.5 µg mL−1) for at least 14 days.

Isolation of Extracellular Vesicles (EVs) Using Size-Exclusion High-Performance Liquid Chromatography (SE-HPLC).

Extracellular vesicles (EVs), are membrane-bound nanoparticles of biological origin. These signature molecules of health and disease have raised remarkable attention of the biomedical arena due to its potential diagnostic and therapeutic applicability. Among the many different techniques available for EV isolation, size-exclusion chromatography (SEC) is widely accepted.In this chapter, we present a protocol of size-exclusion high-performance liquid chromatography (SE-HPLC) as a method of EV isolation. This method can be adapted as a low cost but a reliable and scalable method of EV isolation in those laboratories having access to the HPLC systems.

An Investigation to Examine the Effect of the Elastomeric Surface Treatment on Protein Stability.

Various kinds of treatments on the surface of the elastomeric components can have negative impacts on the quality of protein therapeutics. We compared the effects of bare (non-siliconized and nonlaminated), siliconized, and fluoropolymer-laminated elastomeric components on the stability of ß-lactoglobulin, human serum albumin, adalimumab, abatacept, and immunoglobulin antibodies. The study was conducted in two main parts. Part I was to evaluate the stability of proteins under agitation-induced stress. Protein aggregate formation, turbidity, and protein recovery were analyzed using dynamic FI, absorbance at 350 nm, and size-exclusion high-performance liquid chromatography, respectively. Proteins were found to be more stable with laminated stoppers as compared with bare or siliconized stoppers. Part II was to identify the chemical modifications when the proteins were stored in contact with the same three stoppers. Capillary isoelectric focusing analysis of the adalimumab samples showed formation of acidic variants in siliconized and bare stoppers. Reverse-phase high-performance liquid chromatography suggested chemical changes to the human serum albumin. Analysis of tryptic digest of human serum albumin by liquid chromatography/mass spectrometry/mass spectrometry indicated that the amino acids most susceptible to oxidation (cysteine, tryptophan, and methionine) were also the ones that were modified. Part III of this study investigated the barrier property of the fluoropolymer film with no drug product. Our results were consistent with the suggestion that the fluoropolymer lamination provides a barrier that prevents leachables from the elastomeric components into the protein therapeutics. Our work provided an in-depth understanding of the effects of elastomeric surface treatments on the biophysical and chemical stability of protein drugs.

Physicochemical stability of the bevacizumab biosimilar, ABP 215, after preparation and storage in intravenous bags

Study Objectives: To evaluate extended in-use stability of bevacizumab biosimilar, ABP 215, after dilution into intravenous bags, extended storage, and simulated infusion to enable advanced preparation and storage. Methods: Two lots of ABP 215 were diluted to high- (16.5 mg/mL) and low- (1.4 mg/mL) dose concentrations in two types of intravenous bag under ambient light conditions. Dosed intravenous bags were stored at 2°C–8°C for 35 days, followed by 30°C for 2 days, and each bag was infused on Day 37. Analysis of purity and physicochemical stability was performed using size-exclusion high-performance liquid chromatography (SE-HPLC), cation-exchange high-performance liquid chromatography (CEX-HPLC), reduced capillary electrophoresis-sodium dodecyl sulphate (rCE-SDS), subvisible particle detection assays, visual inspection, and by measuring protein concentration and potency. Results: No meaningful changes were seen in ABP 215 purity when analysed by SE-HPLC, CEX-HPLC and rCE-SDS following dilution, storage and infusion of two lots, bags, and doses. Protein concentration remained consistent throughout the study for all samples and no significant loss in potency was detected. No potentially proteinaceous particles or increases in subvisible particles were observed. Discussion: This study investigated the in-use stability of ABP 215 following dilution, extended storage, and infusion, that represent worst-case handling conditions. ABP 215 exhibited consistent product quality and activity, with no significant degradation observed under the conditions tested. Conclusion: ABP 215 retains physicochemical stability after dilution over the recommended dosing concentrations, extended storage, and simulated infusion. This supports the advance preparation and storage of ABP 215 in intravenous bags for infusion.

Autolysis of rainbow trout (Oncorhynchus mykiss) by-products: Enzymatic activities, lipid and protein oxidation, and antioxidant activity of protein hydrolysates

Autolysis (at fixed temperatures (40, 50, 60 °C) or gradually increasing temperatures (40-60 °C) with different times) was used to produce protein hydrolysates from rainbow trout processing by-products. Total proteolytic and trypsin activities with fixed temperatures was higher than gradually increasing temperatures, decreased with increasing autolysis time and temperature with fixed temperatures (P ≤ 0.05). TCA-soluble peptides decreased with increasing autolysis times from 1 to 3 h (P ≤ 0.05). Autolysis increased peroxide value and thiobarbituric acid-reactive substances in both lipids and hydrolysates, particularly with gradually increasing temperatures (P ≤ 0.05) and was associated with oxidation of polyunsaturated fatty acids. Sulfhydryl groups decreased, while protein carbonyls and solubility were increased with fixed temperatures at higher temperatures (50 and 60 °C) or with gradually increasing temperatures at longer autolysis time (P ≤ 0.05). Free radical scavenging activity and the metal chelating ability of hydrolysates were governed by autolysis times and temperatures. Results of size exclusion chromatography-high performance liquid chromatography indicated that autolysis for 1 h at lower temperature (40 °C) was sufficient to produce autolysates rich in small peptides from rainbow trout by-products. Autolytic hydrolysis might be useful for production of such protein hydrolysates.

Moment of Bentonite Addition, Co-Addition of Tannins, and Bentonite Type Affect the Differential Affinity of Pathogenesis-Related Grape Proteins towards Bentonite during Fermentation.

To test the effect of the moment of bentonite addition, co-addition of tannins, and bentonite type on the differential affinity of pathogenesis-related (PR) proteins towards bentonite during grape must fermentation, three separate experiments were set up. PR proteins in the obtained wines were analyzed by reverse phase and size exclusion high-performance liquid chromatography (HPLC). The most significant reduction of bentonite dose and PR protein concentration was achieved by applying bentonite in the last third of fermentation. Particular thaumatin-like proteins (TLP) and proteins with lower molecular mass in general were more affected than others, while TLPs were more affected than chitinases. Exogenous enological tannins interacted with particular PR proteins, mostly TLPs, and lowered the total bentonite dose required. The combined application of tannins and bentonite in fermentation removed more PR proteins than bentonite alone, but did not achieve a synergistic effect in reducing the bentonite dose. Various bentonite types, including two Na-activated bentonites, an activated Na bentonite with specifically adsorbed silica, and an active Na-Ca bentonite, exhibited differential affinity towards different PR proteins. The results obtained could be used in developing wine fining protocols which combine treatments with complementary affinity for adsorption and removal of PR proteins, and in this way achieve greater efficiency of bentonite fining by reducing its total dose, which is of significant interest to the wine industry.

ANAVIP® interaction with western pygmy rattlesnake venom: In vitro assesment of reactivity using SE-HPLC

Every year large numbers of venomous snake bites occur around the world, especially in tropical areas. The World Health Organization classifies venomous snake bites as one of their highest priority neglected tropical diseases, one of the reasons being the short supply of antivenom compared to the number of snake envenomations. The standard of care for snake envenomation is administration of antivenom. Many antivenoms are polyvalent, which are produced using venoms from multiple species of snakes. The polyvalent antivenoms can treat envenomation from snake venoms used in the production, but also show cross-reactivity against snake venoms with similar composition. Determining cross-reactivities of antivenoms could help improve the quality of treatment and provide a better understanding of venom-antivenom binding. One antivenom only has been available in the United States for treatment of North American Crotaline envenomation, with the recent introduction of an F(ab’)2 antivenom (ANAVIP®). Size-exclusion high performance liquid chromatography (SE-HPLC) was used to assess cross-reactivity of the western pygmy rattlesnake, Sistrurus miliarius streckeri (S. m. streckeri), against ANAVIP®. Estimates of venom-antivenom reactivity was measured in reaction mixtures based on the increase in elution profile area of higher molecular weight complexes (region 1) and on the decrease in elution profile area of reactants (region 2). Reaction mixtures contained ANAVIP® (1.0 mg/ml) and S. m. streckeri venom (0.125, 0.25, 0.5, or 1.0 mg/ml). Controls were ANAVIP® and S. m. streckeri (1.0 mg/ml). Mixtures were incubated at 37 °C for 30 min, then stored at 4 °C (5 min) prior to SE-HPLC. Relative binding, estimated from the increase in region 1 (immune complexes) and decrease in region 2 (reactants) region areas, suggested saturation of reactive antivenom binding sites at 0.125 (and above) mg venom/mg antivenom. SE-HPLC data indicate that binding of ANAVIP® to S. m. streckeri venom does occur, consistent with protective effects observed clinically. Further studies are needed to compare the binding of S. m. streckeri venom to other commercially available antivenoms, and the binding of ANAVIP® to other venoms of clinical significance.

New insights on the interaction mechanism of rhTNFα with its antagonists Adalimumab and Etanercept.

TNFα is a pro-inflammatory cytokine that is a therapeutic target for inflammatory autoimmune disorders. Thus, TNFα antagonists are successfully used for the treatment of these disorders. Here, new association patterns of rhTNFα and its antagonists Adalimumab and Etanercept are disclosed. Active rhTNFα was purified by IMAC from the soluble fraction of transformed Escherichia coli. Protein detection was assessed by SDS-PAGE and Western blot. The KD values for rhTNFα interactions with their antagonists were obtained by non-competitive ELISA and by microscale thermophoresis (MST). Molecular sizes of the complexes were evaluated by size-exclusion chromatography-high performance liquid chromatography (SEC-HPLC). Surprisingly, both antagonists recognized the monomeric form of rhTNFα under reducing and non-reducing conditions, indicating unexpected bindings of the antagonists to linear epitopes and to rhTNFα monomers. For the first time, the interactions of rhTNFα with Adalimumab and Etanercept were assessed by MST, which allows evaluating molecular interactions in solution with a wide range of concentrations. Biphasic binding curves with low and high KD values (<10-9 M and >10-8 M) were observed during thermophoresis experiments, suggesting the generation of complexes with different stoichiometry, which were confirmed by SEC-HPLC. Our results demonstrated the binding of TNFα-antagonists with rhTNFα monomers and linear epitopes. Also, complexes of high molecular mass were observed. This pioneer investigation constitutes valuable data for future approaches into the study of the interaction mechanism of TNFα and its antagonists.

Effects of catechins on the polymerisation behaviour, conformation and viscoelasticity of wheat gluten

SummaryThe effects of catechins on the structural properties of wheat gluten were investigated in terms of their polymerisation behaviour, conformation and viscoelasticity. Depolymerisation of glutenin macropolymers was analysed by size‐exclusion high‐performance liquid chromatography (SE‐HPLC) of gluten with catechins, revealing major variation in sulfhydryl (SH) content. Compared with controls, the SH content of catechin‐enhanced gluten was significantly increased due to breakage of disulphide (SS) bonds. Reversed‐phase HPLC revealed that catechins had minimal effect on subunit distribution of gliadin and glutenin. Fourier transform infrared (FTIR) spectroscopy showed that catechins induced the transformation of α‐helices to β‐sheets, β‐turns and antiparallel β‐sheets. Rearrangement of secondary structure when supplemented with catechins might be a consequence of altered non‐covalent interactions. Catechins increased tan δ, indicating enhanced gluten fluid properties. These results imply that catechins might weaken the network structure of gluten and change the rheological properties by inhibiting SS cross‐linkage formation and influencing non‐covalent bonds.

Analytical comparability study of anti-CD20 monoclonal antibodies rituximab and obinutuzumab using a stability-indicating orthogonal testing protocol: Effect of structural optimization and glycoengineering

Glycoengineering and biosimilarity are the key factors for growing, promising and progressive approaches in monoclonal antibodies development. In this study, the physicochemical stability of anti-CD20 rituximab (RTX); originator and biosimilar was compared to its glycoengineered humanized version; obinutuzumab (OBZ). An orthogonal stability-indicating protocol using a set of validated bioanalytical techniques; size exclusion high performance liquid chromatography (SE-HPLC), reversed phase liquid chromatography (RP-HPLC), quantitative gel electrophoresis by TapeStation, receptor binding assay and dynamic light scattering (DLS) was used to investigate the effect of different stress factors on the pattern and kinetics of degradation. SE-HPLC results supported with spectral purity showed similar degradation extent with a different pattern of degradation between RTX and OBZ. A lower tendency to form degraded fragments and a relatively higher favorability for degradation through aggregate formation has been revealed in case of OBZ. Results were in agreement with those of DLS and receptor binding assay which showed specificity to the intact antibodies in the presence of their degradation products. Furthermore, results were additionally confirmed through denaturing quantitative gel electrophoresis which suggested reducible covalent bonds as the mechanism for aggregates formation. RP-HPLC results showed two oxidized forms via excessive oxidation of RTX and OBZ with nearly the same degradation percent. Comparability data of RTX and OBZ using the applied methodologies showed that although glycoengineering; carried out to enhance the therapeutic and biological activity of OBZ altered the pattern of degradation but did not significantly affect the overall stability. Results showed also consistent stability profile between the biosimilar and its originator RTX products.

Impact of Simulated Gastrointestinal Digestion on the Biological Activity of an Alcalase Hydrolysate of Orange Seed (Siavaraze, Citrus sinensis) by-Products.

In this study, orange seed proteins were hydrolyzed by Alcalase enzyme at different enzyme concentrations 1–3% (v/w) and hydrolysis times (2–5 h), to obtain bioactive peptides showing antioxidant, Angiotensin-converting enzyme (ACE) -inhibitory, and hypoglycemic activities. The highest biological activities (p < 0.05) were achieved by using a hydrolysis time of 5 h and an enzyme concentration of 2%. Orange seed protein hydrolysate (OSPH) was prepared under these conditions, and peptides were isolated and purified by using size-exclusion chromatography and high-performance liquid chromatography, respectively. The fractions that showed the highest biological activities were analyzed by mass spectrometry in tandem, and a total of 63 peptide sequences were found. Moreover, the effect of simulated gastrointestinal digestion on the bioactivity of the fractions was studied, and the novel peptide sequences generated were also identified. Overall, despite there being some differences in the profile of peptide sequences obtained, the main results showed non-significant differences in the analyzed bioactivities after simulated gastrointestinal digestion.

Conjugation of Pea Protein Isolate via Maillard-Driven Chemistry with Saccharide of Diverse Molecular Mass: Molecular Interactions Leading to Aggregation or Glycation.

Diverse saccharides are effectively grafted to pea protein isolate (PPI) through Maillard-driven chemistry. The development of conjugates (glyco-PPI) was validated by ultraviolet-visible spectroscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and size exclusion chromatography-high performance liquid chromatography. The impact of covalent conjugation on color development, structural modification, solubility, thermal stability, and volatiles of glycoprotein was examined. The protein solubility was improved, while its thermal stability seemed to be negatively influenced. The principle proposed involves Maillard-driven generation of the conjugates, which enhanced the surface hydrophilicity and unfolding of protein architecture of glyco-PPI. Additionally, both molecular mass and the grafted number of saccharides played a vital role in determining the solubility and thermal stability of glyco-PPI. Protein tends to denature at reaction conditions of 80 °C and pH 10.0, and its cross-linkage occurred in the aqueous system. The two potential routes of molecular interactions between PPI and saccharides were denaturation and glycation or self-cross-linkage. Flavor profile alteration of glycoprotein before and after conjugation was depicted, and relevant off-odors were quantified via headspace solid-phase microextraction gas chromatography-mass spectrometry. These outcomes could furnish valuable in-depth information for dictating functionalities of plant-based protein for food application.

Evaluation of the Physico-Chemical and Biological Stability of SB8 (Aybintio), a Proposed Biosimilar to Bevacizumab, Under Ambient and In-Use Conditions.

No stability data of SB8 (proposed biosimilar to bevacizumab) are available for opened vials at 2-8°C or for unopened vials stored outside of this range. Furthermore, limited stability data are available for dilutions. Stability of unopened vials of SB8 at ambient temperature and in-use stability for opened vials as well as diluted SB8 in infusion bags were evaluated. SB8 stability was assessed in three scenarios in the absence of light: unopened 100-mg vials 36 months after manufacture stored at 30 ± 2°C with 65 ± 5% relative humidity for 1month, opened 100-mg and 400-mg vials stored at 5 ± 3°C for 72h and diluted (1.4mg/ml or 16.5mg/ml in 100ml 0.9% NaCl polyolefin bags) stored for 45days at 5 ± 3°C and then 3days at 30 ± 2°C; the United Kingdom's National Health Service protocol was used for the study design after dilution. Physicochemical stability (appearance, pH, protein concentration, size-exclusion high-performance liquid chromatography, non-reducing capillary electrophoresis-sodium dodecyl sulfate, imaged capillary isoelectric focusing), biological activity [vascular endothelial growth factor (VEGF) neutralization assay, VEGF binding assay] and potential safety impact properties (subvisible particulates, submicronic aggregation by dynamic light scattering) were determined. All stability-indicating criteria including those for biological activity were met for both unopened vials at ambient condition and for in-use conditions of opened vials as well as both dilutions. No noteworthy changes in terms of physicochemical stability, biological activity and properties with a potential safety impact occurred. Under the studied aspetic extreme conditions, SB8 was stable. Our data may support advanced SB8 preparation and may help prevent SB8 wastage because of exceptional temperature excursions or unused product. Sterility assurance is the responsibility of the user and is of utmost importance when opened or diluted SB8 is not immediately used.

The role of exogenous lipids in starch and protein mediated sponge cake structure setting during baking

While it is well established that using exogenous lipids (ELs) such as monoacylglycerols and polyglycerolesters of fatty acids improves gas cell incorporation and stability in sponge cake batter (SCB) and allows producing sponge cakes (SCs) with very high volume, fine grained crumb and soft texture, their impact on starch gelatinization and protein polymerization remained unknown. Here, differential scanning calorimetry and size-exclusion high performance liquid chromatography were performed on SC(B) samples prepared with or without ELs. Starch gelatinization and protein denaturation and polymerization started at temperatures exceeding 67 °C and mostly occurred up to a temperature of 96 °C. During further isothermal treatment at 96 °C the rigidity of the cake matrix (for which temperature-controlled time domain 1H NMR T2 relaxation times are a predictor) further increased mainly because of protein polymerization. While the temperature range of starch crystal melting was not affected by the use of ELs, protein polymerized more intensively in an 88 to 94 °C temperature range when SCB contained ELs. The more intense protein polymerization and the high water binding capacity of ELs presumably made the cake matrix more rigid at that point in time. The present results allow concluding that ELs not only impact air-liquid interface stability but also cake structure setting. Hence, both aspects most likely contribute to the superior quality of SCs containing ELs.

Identification and Quantification of Major Faba Bean Seed Proteins

Faba bean (Vicia faba L.) holds great importance for human and animal nutrition for its high protein content. However, better understanding of its seed protein composition is required in order to develop cultivars that meet market demands for plant proteins with specific quality attributes. In this study, we screened 35 diverse Vicia faba genotypes by employing the one-dimensional sodium dodecyl sulfate–polyacrylamide gel electrophoresis (1D SDS-PAGE) method, and 35 major protein bands obtained from three genotypes with contrasting seed protein profiles were further analyzed by mass spectrometry (MS). Twenty-five of these protein bands (MW range: ∼ 9–107 kDa) had significant (p ≤ 0.05) matches to polypeptides in protein databases. MS analysis showed that most of the analyzed protein bands contained more than one protein type and, in total, over 100 proteins were identified. These included major seed storage proteins such as legumin, vicilin, and convicilin, as well as other protein classes like lipoxygenase, heat shock proteins, sucrose-binding proteins, albumin, and defensin. Furthermore, seed protein extracts were separated by size-exclusion high-performance liquid chromatography (SE-HPLC), and percentages of the major protein classes were determined. On average, legumin and vicilin/convicilin accounted for 50 and 27% of the total protein extract, respectively. However, the proportions of these proteins varied considerably among genotypes, with the ratio of legumin:vicilin/convicilin ranging from 1:1 to 1:3. In addition, there was a significant (p < 0.01) negative correlation between the contents of these major fractions (r = −0.83). This study significantly extends the number of identified Vicia faba seed proteins and reveals new qualitative and quantitative variation in seed protein composition, filling a significant gap in the literature. Moreover, the germplasm and screening methods presented here are expected to contribute in selecting varieties with improved protein content and quality.

Evaluation on the analytical methods for tissue plasminogen activator (tPA) in pharmaceutical formulations

Tissue plasminogen activator, tPA, is a thrombolytic agent for treatment of the brain or myocardium infarction. Clot lysis assay is a functional determination method of tPA described in the pharmacopeia. However, the accuracy of clot lysis assay is insufficient because the lysis-time is not determined by the end-point detection. Therefore, an alternative analytical method for the post-marketing quality survey of tPA pharmaceutical formulations is needed. This study was aimed to establish a rapid, precise and cost-saving analytical method for tPA routine test. We have compared methods of capillary zone electrophoresis (CZE), size-exclusion high performance liquid chromatography (SE-HPLC), in vitro clot lysis, enzyme-linked immunosorbent assay (ELISA), and chromogenic substrate assay for the evaluation of recombinant tPA drug products. The results showed that S-2288™, as substrate of chromogenic assay for tPA, exhibited a good linear relationship at tPA concentration of 5-50 μg/mL (correlation coefficient: 0.9988). The intraday precision ranged between 0.5% - 4.3%. Results obtained from our comparative study, suggested that the chromogenic substrate assay is among the best for tPA assay. The features of precision, rapidity and inexpensiveness of this method are suitable for a routine test for tPA in pharmaceutical formulations.

Aggregate Removal Nanofiltration of Human Serum Albumin Solution Using Nanocellulose-Based Filter Paper.

This study is dedicated to the rapid removal of protein aggregates and viruses from plasma-derived human serum albumin (HSA) product to reduce the risk of viral contamination and increase biosafety. A two-step filtration approach was implemented to first remove HSA aggregates and then achieve high model virus clearance using a nanocellulose-based filter paper of different thicknesses, i.e., 11 μm (prefilter) and 22 μm (virus filter) at pH 7.4 and room temperature. The pore size distribution of these filters was characterized by nitrogen gas sorption analysis. Dynamic light scattering (DLS) and size-exclusion high performance liquid chromatography (SE-HPLC) were performed to analyze the presence of HSA aggregates in process intermediates. The virus filter showed high clearance of a small-size model virus, i.e., log10 reduction value (LRV) > 5, when operated at 3 and 5 bar, but a distinct decrease in LRV was detected at 1 bar, i.e., LRV 2.65–3.75. The throughput of HSA was also dependent on applied transmembrane pressure as was seen by Vmax values of 110 ± 2.5 L m−2 and 63.6 ± 5.8 L m−2 at 3 bar and 5 bar, respectively. Protein loss was low, i.e., recovery > 90%. A distribution of pore sizes between 40 nm and 60 nm, which was present in the prefilter and absent in the virus filter, played a crucial part in removing the HSA aggregates and minimizing the risk of virus filter fouling. The presented results enable the application of virus removal nanofiltration of HSA in bioprocessing as an alternative to virus inactivation methods based, e.g., on heat treatment.

Scalable High-Performance Production of Recombinant Horseradish Peroxidase from E. coli Inclusion Bodies.

Horseradish peroxidase (HRP), an enzyme omnipresent in biotechnology, is still produced from hairy root cultures, although this procedure is time-consuming and only gives low yields. In addition, the plant-derived enzyme preparation consists of a variable mixture of isoenzymes with high batch-to-batch variation preventing its use in therapeutic applications. In this study, we present a novel and scalable recombinant HRP production process in Escherichia coli that yields a highly pure, active and homogeneous single isoenzyme. We successfully developed a multi-step inclusion body process giving a final yield of 960 mg active HRP/L culture medium with a purity of ≥99% determined by size-exclusion high-performance liquid chromatography (SEC-HPLC). The Reinheitszahl, as well as the activity with 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and 3,3′,5,5′-tetramethylbenzidine (TMB) as reducing substrates, are comparable to commercially available plant HRP. Thus, our preparation of recombinant, unglycosylated HRP from E. coli is a viable alternative to the enzyme from plant and highly interesting for therapeutic applications.

Immunomodulatory effect of a purified peptide fraction isolated from Mesobuthus eupeus scorpion venom on astrocytoma cell line (1321N1)

Objective(s): Multiple Sclerosis is a central nervous system disease which belongs to the category of autoimmune diseases. The prevalence of this disease in Iran is approaching the European level. Astrocyte cells are nerve tissues that regulate the immune system activity by secreting various cytokines such as IL- 17. The aim of this study was partial purification of toxin from M.eupeus scorpion venom that has immunomodulatory effect on astrocyte cell line (1321N1) Materials and Methods: In the present study, purified crude venom of M.eupeus scorpion. Size exclusion and reverse-phase high-performance liquid chromatography was used for fractionation. The fractional molecular weight was determined by Using SDS and Tricine electrophoresis, Astrocyte cells (1321N1) were selected as functional cells in testing the immunomodulatory effect of venom. The viability of cells were determined by MTT and LDH assays. Astrocyte cells were activated by lipopolysaccharide and the release of interleukin-17 in activated cells was estimated using ELISA kit. Results: fraction 331 (F331) from RP-HPLC contain the purified peptide with molecular weight of about 4500 Dalton. When activated cells exposed to purified peptide the rate of interleukin-17 release was found to be 85 pg/ml which is almost similar to un-activated cells (78 pg/ml). However in activated cells by LPS without treatment with purified peptide the rate of IL-17 release was found to be 147 pg/ml which was significantly (p <0.05) higher than control group. Conclusion: The purified peptide (F331) from venom of Mesobouthus eupeus can inactivate the astrocyte 1321N1 cells activated by LPS as indicated by decreased secretion of IL-17 from the cells.

Comparison of intracellular trace element distributions in the liver and gills of the invasive freshwater fish species, Prussian carp (Carassius gibelio Bloch, 1782)

Prussian carp (Carassius gibelio) is an invasive freshwater fish known for its high tolerance to aquatic pollution. Our aim was to try to clarify its tolerance to increased exposure to metals/nonmetals, by determining their cytosolic distributions among peptides/proteins of different molecular masses (MM), which form a part of the fish protective mechanisms. The applied approach consisted of fractionation of gill and hepatic cytosols of Prussian carp from the Croatian river Ilova by size-exclusion high performance liquid chromatography, whereas Cd, Cu, Zn, Fe, Mo, and Se analyses were done by high resolution inductively coupled plasma mass spectrometry. The results indicated high detoxification of Cd by its binding to metallothioneins (MTs) in both fish organs. In addition, binding to MTs was observed for Cu in both organs and for Zn in the liver, whereas clear Zn binding to MTs in the gills was not recorded. Zinc in the gills was predominantly bound to proteins of higher MM (50–250 kDa) and to biomolecules of MM below 2 kDa. Predominant Fe binding to proteins of MM of ~400 kDa (presumably storage protein ferritin) was observed in the liver, whereas in the gills Fe was mainly associated to proteins of MM of ~15–65 kDa (presumably hemoglobin oligomers). Maximum Mo and Se elutions in the liver were noted at 235 kDa and 141 kDa, respectively, and in the gills below 10 kDa. The striking difference was observed between two organs of Prussian carp, with predominant metal/nonmetal binding to high MM proteins (e.g., enzymes, storage proteins) in the liver, and to very low MM biomolecules (<10 kDa) in the gills (e.g., antioxidants, metallochaperones, nonprotein cofactors). Such metal/nonmetal distributions within the gills, as the first site of defense, as well as association of several metals to MTs, indicated highly developed defense mechanisms in some organs of Prussian carp.