A key aspect that must be supervised during the development of recombinant therapeutic products is the potential presence of impurities.
Residual host cell proteins (HCPs) are a major class of ...process-related impurities derived from the host organism that even in trace amount have the potential to affect product quality, safety, and efficacy. Therefore, the product purification processes must be optimized to consistently remove as many HCPs as feasible, with the goal of making the product as pure as possible. The workhorse of HCP monitoring and quantitation during bioprocessing manufacturing is sandwich ELISA (enzyme‐linked immunosorbent assay), which employs polyclonal anti-HCP antibodies for both capture and detection. Commercial ELISA kits developed from Chinese Hamster Ovary (CHO) cell lines are widely applied in early drug development stages (preclinical, phase I, and phase II), but are not specifically designed for a given manufacturer's proprietary cell line, and users do not have control over reagent availability and lot-to-lot consistency. For later development stages, the upstream process-specific method is preferred to guarantee an improved sensitivity and coverage. In agreement with the USP General Chapter 〈1132〉, a platform assay can be used in place of the commercial one through all stages of product development, if already available when product development starts.
This proof-of-concept study was carried out to demonstrate the feasibility and the advantages of the development of a proprietary CHO HCPs platform ELISA. Different proprietary mock materials have been characterized and compared by orthogonal bidimensional electrophoresis techniques (SDS-PAGE coupled to SS/WB and 2D DIGE) with the scope of selecting the best antigen-antibody couple for setting up the in-house ELISA. A preliminary evaluation of the in-house method performance has been done in comparison with the commercial assay, demonstrating that the platform method is promising for an accurate and precise CHO HCPs quantification during the early phase product and process development.
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Biochar, a waste biomass-derived adsorbent, holds promise for decentralised wastewater treatment. However, limited research exists on its efficacy in adsorbing anionic surfactants in wastewater. To ...address this, the adsorption of sodium dodecyl sulphate (SDS), a common anionic surfactant, was studied using various biochar types: rice husk biochar (RH-550 and RH-700), wheat straw biochar (WS-550 and WS-700) produced at 550°C and 700°C, wood-based biochar (OB), and activated carbon (AC) as a control. The study investigated the impact of pH (3-9), adsorbent loading (1-10 g/L), adsorbent size (<0.5-2.5 mm), contact time (5-180 min), and initial concentration (50-200 mg/L) on SDS removal. Under optimised conditions (100 mg/L SDS, 4 g/L adsorbent, 1-2 mm particle size, pH 8.3, and 180 min contact time), maximum SDS removals were RH-550 (78%), RH-700 (82.4%), WS-550 (89.5%), WS-700 (90.4%), AC (97%), and OB (88.4%). Among the tested adsorbent materials, WS-550 exhibited the highest SDS adsorption capacity at 66.23 mg/g compared to AC (80.65 mg/g), followed by RH-550 (49.75 mg/g), OB (45.87 mg/g), RH-700 (43.67 mg/g), and WS-700 (42.74 mg/g). SDS adsorption followed a pseudo-second-order kinetic model, indicating chemisorption on the adsorbent surface. The Freundlich isotherm model exhibited a better fit for the experimental data on SDS adsorption using all tested adsorbents except for RH-550. This study showed that biochars produced from agricultural and forestry residues are effective adsorbents for SDS in aqueous solutions and can be a promising sustainable and low-cost material for the treatment of greywater containing anionic surfactants (e.g. handwashing, laundry, kitchen, and bathroom greywaters).
The glycine transporter GlyT2 plays an important role in glycine-inhibitory neurotransmission of the hindbrain and spinal cord. Its special feature is the extended N-terminus, which contains a large ...number of potentially phosphorylated serine and threonine residues. Due to its unstructured nature, it is difficult to address the changes introduced by potential phosphorylation. Here, we used relatively simple methods such as calpain sensitivity, Bradford instability, and SDS electrophoretic mobility shift to investigate the effect of multiple phosphomimetic mutations versus neutral mutations on GlyT2N properties. The replacement of several serines or threonines with neutral alanines did not have a significant effect on the studied GlyT2N properties. Replacement of the same residues with phosphomimetic aspartate resulted in significant alterations in calpain cleavage patterns, Bradford instability, and SDS gel protein mobility. Interestingly, a correlation between the relative intensity of the measured effects was observed, indicating that they all reflect similar structural changes introduced by potential phosphorylation in vivo. Results indicate that a potential single or multiple phosphorylation significantly alters the proteomic properties of the glycine transporter GlyT2 N-terminus. Assays can be helpful in the first screening of structurally significant and possibly phosphorylated residues in the N-terminus of GlyT2.
•Blockade of major GlyT2 N calpain cleavage sites reveals multiple calpain sensitivity.•GlyT2 N-terminal phosphomimetic mutations cause a SDS gel mobility shift.•GlyT2 N-terminal SDS gel mobility shift is correlated with its Bradford instability.
Phytopathogenic fungi devastate agricultural crops worldwide. The biological agents, such as Trichoderma spp., antagonize phytopathogenic fungi by secreting various cell wall‐degrading enzymes, for ...example, endochitinase and β‐1,3‐glucanase that target glycosidic linkages in β‐glucan and chitin polymers of fungal cell walls, thus inhibiting pathogen growth. In this study, two antifungal genes endochitinase and β‐1,3‐glucanase cloned from local Trichoderma spp. were ligated in pET28a+ expression vector individually to generate two recombinant vectors. The vectors were mobilized into Escherichia coli host strain Rosetta‐gami 2 for protein expression, and the 6xHis‐tagged recombinant proteins were purified through Ni‐NTA affinity chromatography. The purified proteins were individually confronted in vitro with pure cultures of Phytophthora parasitica (destructive pathogen affecting several hundred plant species worldwide) for analyzing their effect on pathogen growth. In vitro confrontation assay revealed P. parasitica growth inhibition by purified β‐1,3‐glucanase. The pathogen growth inhibition was due to hyphal morphological distortions, such as breakages, swelling, and holes evinced through electron micrography confirming direct role of β‐1,3‐glucanase in pathogen structural degradation.
The present study aimed to investigate the effect of N-Ethylmaleimide (NEM) on the evaluation of disulfide formation in the oxi dized myofibrillar proteins during the sample preparation of the ...non-reducing SDS-PAGE procedure. For this purpose, extracted myofibrillar proteins were oxidized firstly via a Fenton oxidation reaction, and non-oxidized proteins were used as a control. Before running SDS-PAGE, in the sample preparation, these oxidized and non-oxidized proteins were prepared according to the three dif ferent sample preparation methods with or without the presence of N-Ethylmaleimide or β-mercaptoethanol. Results showed that oxidized proteins treated with NEM regardless of sample preparation methods presented attenuated bands of myosin heavy chain monomer in the non-reducing SDS-PAGE gels, suggesting that the disulfide bonds formed as a result of protein oxidation could be preserved by NEM during sample preparation. Meanwhile, a possible mechanism for the effect of NEM was proposed.
Yellow field peas were subjected to soaking (SY), germination (SGY), fermentation (SFY), fermentation and pasteurization (SFPY), combined germination and fermentation (SGFY), as well as SGFY upon ...pasteurization (SGFPY). Soaking generally had a negligible impact on the proximate composition and nutritive quality of field peas except that a 23.4% reduction in trypsin inhibitor activity (TIA) was observed. Both germination and fermentation led to a significant reduction in starch content by 11.2–11.7%. Processed peas including SFY, SFPY, SGFY, and SGFPY exhibited higher in vitro protein digestibility (IVPD) values (P < 0.05) of 87.80–89.16%, which was further strengthened by the negative correlation between IVPD and antinutrients including tannin and TIA. The electrophoretic profile of their protein hydrolysates confirmed the IVPD results where the above four treatments resulted in an extensive degradation and breakdown of pea seed's storage proteins with the peptide subunits appeared at molecular weight <16 kDa. SFY, SFPY, SGFY, and SGFPY also showed higher levels of digestible starch (114.1–257.7 g/kg) which agreed with their DSC gelation temperatures and the most distinguishable alternations on their microstructure. The fermentation of germinated peas led to an enhanced nutritional property, the following pasteurization process further changed the starch crystalline organization and protein resembling structure of field peas.
•Germination is less effective than fermentation in affecting pea composition.•Combined germination and fermentation greatly enhanced nutritional profile of peas.•The fermentation of germinated peas upon pasteurization led to highest digestibility.•Variation in protein digestibility was verified by electrophoresis.•Peas with higher digestible starch exhibit distinguishable microscopical changes.
•An experimental method for determination of bubble sizes is thoroughly studied.•If conditions are not optimized, bubble coalescence leads to too big sizes.•The presence of a standing wave enhances ...coalescence.•Conditions for measurement of the ambient size of cavitation bubbles are determined.•An ambient radius of 2.9–3.0 µm is derived for Ar-saturated water at 362 kHz.
A powerful experimental approach to measure the size distribution of bubbles active in sonoluminescence and/or sonochemistry is a technique based on pulsed ultrasound and sonoluminescence emission. While it is an accepted technique, it is still lacking an understanding of the effect of various experimental parameters, including the duration of the pulse on-time, the nature of the dissolved gas, the presence of a gas flow rate, etc. The present work, focusing on Ar-saturated water sonicated at 362 kHz, shows that increasing the pulse on-time leads to the measurement of coalesced bubbles. Reducing the on-time to a minimum and/or adding sodium dodecyl sulfate to water allows to reducing coalescence so that natural active cavitation bubble sizes can be measured. A radius of 2.9–3.0 µm is obtained in Ar-saturated water at 362 kHz. The effects of acoustic power and possible formation of a standing-wave on coalescence and measured bubble sizes are discussed.
Dynamic rheological properties of dough and gluten were studied and related to the empirical rheology (Farinograph) and the protein fractions of different wheat varieties. The relationship between ...high molecular weight-glutenin subunits (HMW-GS) and dough strength was evaluated. Dynamic rheology of under-, optimum- and over-mixed dough was also related to the farinograph characteristics and the protein fractions. The moduli (G′ and G″) of over- and optimally-mixed dough were significantly correlated with Farinograph characteristics and SDS-unextractable polymeric protein (UPP) content. The temperature-induced rheology of glutens revealed that the differences in G′ and G″ during heating as well as cooling were related to the UPP content. Principal component analysis revealed wide range of differences in the dynamic rheology among the wheat varieties mainly attributed to UPP.
Viscoelastic behavior of gluten measured during heating (25–90 °C) and cooling (90–25 °C) from a wheat variety. Storage modulus (G′; ▪) and loss modulus (G″; ×). Display omitted
•Dynamic rheology of wheat dough & gluten was related to farinographic properties.•Rheological properties also related to the wheat protein fractions.•UPP content contributed to differences in dynamic & farinographic properties.
In this work, we observed the effect of grape juice (% concentrated juice/% concentrated camel milk: GJ20/80, GJ50/50) and pomegranate juice (PJ20/80, PJ40/60) fortification on camel milk (CM) ...protein solubility and digestibility. Proteins were dissolved in sodium phosphate buffer to 50 mg/ml and defatted prior Bradford assay of protein concentration, then analyzed by Size Exclusion-Ultra High-Performance Liquid chromatography (SE-UHPLC). The CM protein aggregation and their stability were further monitored at different pH 2.0, 4.0, and 7.5 via sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Freeze dried CM (FDCM) was the reference sample and our results showed that GJ50/50 and PJ40/60 with the highest fruit juice ratio had the lowest protein content in the supernatant, hence the decreased solubility. SE-UHPLC of supernatants showed a slight decrease in retention times of 11 kDa and 62 kDa proteins for GJ50/50 and PJ40/60 suggesting a possibility of adduct formation due to fortification leading to higher molecular weight. The simulated static in vitro gastrointestinal digestion of samples revealed that most soluble proteins were readily digested by pepsin, trypsin and chymotrypsin enzymes leading to small peptides. However, the SDS PAGE of pellets showed the partial resistance of casein and α-lactalbumin against peptic digestion.
•Fruit juice fortification affects CM protein aggregability and digestibility.•CM fortified with higher fruit juice ratio had the lowest soluble protein content.•SE-UHPLC of supernatants revealed protein aggregates in fortified CM.•Most of the soluble CM proteins were readily digested under simulated digestion.•Casein and α-LA in digesta pellets were less susceptible to peptic digestion.
•Microfluidisation induced molecular unfolding and rearrangement of almond proteins.•α helix reduced by 11 % & β sheets by 24 % at 80 MPa pressure compared to control.•80 MPa pressure significantly ...improved almond protein’s digestibility & PDCAAS.•Almond meal proteins functionality improved with increasing pressure up to 80 MPa.•High pressure >80 MPa reversed beneficial functional properties of almond proteins.
Almond protein isolate (API) obtained from almond meal was processed using dynamic high-pressure microfluidisation (0, 40, 80, 120, and 160 MPa pressure; single pass). Microfluidisation caused significant reductions in the particle size and increased absolute zeta potential. SDS-PAGE analysis indicated reduction in band intensity and the complete disappearance of bands beyond 80 MPa. Structural analysis (by circular dichroism, UV–Vis, and intrinsic-fluorescence spectra) of the API revealed disaggregation (up to 80 MPa) and then re-aggregation beyond 80 MPa. Significant increments in protein digestibility (1.16-fold) and the protein digestibility corrected amino acid score (PDCAAS; 1.15-fold) were observed for the API (80 MPa) than control. Furthermore, significant improvements (P < 0.05) in the functional properties were observed, viz., the antioxidant activity, protein solubility, and emulsifying properties. Overall, the results revealed that moderate microfluidisation treatment (80 MPa) is an effective and sustainable technique for enhancing physico-chemical and functional attributes of API, thus potentially enabling its functional food/nutraceuticals application.
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