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Analytical Chemistry Apr 2024The number of therapeutic monoclonal antibodies (mAbs) is growing rapidly due to their widespread use for treating various diseases and health conditions. Assessing the...
The number of therapeutic monoclonal antibodies (mAbs) is growing rapidly due to their widespread use for treating various diseases and health conditions. Assessing the glycosylation profile of mAbs during production is essential to ensuring their safety and efficacy. This research aims to rapidly isolate and digest mAbs for liquid chromatography-tandem mass spectrometry (LC-MS/MS) identification of glycans and monitoring of glycosylation patterns, potentially during manufacturing. Immobilization of an Fc region-specific ligand, oFc20, in a porous membrane enables the enrichment of mAbs from cell culture supernatant and efficient elution with an acidic solution. Subsequent digestion of the mAb eluate occurred in a pepsin-modified membrane within 5 min. The procedure does not require alkylation and desalting, greatly shortening the sample preparation time. Subsequent LC-MS/MS analysis identified 11 major mAb N-glycan proteoforms and assessed the relative peak areas of the glycosylated peptides. This approach is suitable for the glycosylation profiling of various human IgG mAbs, including biosimilars and different IgG subclasses. The total time required for this workflow is less than 2 h, whereas the conventional enzymatic release and labeling of glycans can take much longer. Thus, the integrated membranes are suitable for facilitating the analysis of mAb glycosylation patterns.
Topics: Glycosylation; Antibodies, Monoclonal; Humans; Tandem Mass Spectrometry; Polysaccharides; Chromatography, Liquid; Pepsin A; Immunoglobulin G; Animals; Membranes, Artificial
PubMed: 38607313
DOI: 10.1021/acs.analchem.4c00030 -
Small (Weinheim An Der Bergstrasse,... Apr 2024Helicobacter pylori (H. pylori) is the major etiological factor of a variety of gastric diseases. However, the treatment of H. pylori is challenged by the destruction of...
Helicobacter pylori (H. pylori) is the major etiological factor of a variety of gastric diseases. However, the treatment of H. pylori is challenged by the destruction of targeted drugs by gastric acid and pepsin. Herein, a dual-targeted cascade catalytic nanozyme PtCo@Graphene@Hemin-2(L-arginine) (PtCo@G@H2A) is designed for the treatment of H. pylori. The dual-targeting ability of PtCo@G@H2A is derived from directly targeting the receptor protein of H. pylori through hemin and responding to the acidic environment to cause charge reversal (protonation of L-arginine) to capture H. pylori, achieving efficient targeting effect. Compared with the single-targeting strategy relying on hemin, the dual-targeting strategy can greatly improve the targeting rate, achieving an increase of 850% targeting rate. At the concentration of NaHCO in intestinal fluid, the surface potential of PtCo@G@H2A can be quickly restored to avoid side effects. Meanwhile, PtCo@G@H2A has pH-responsive oxidase-like activity, which can generate nitric oxide (NO) through a cascade catalytic process that first generates reactive oxygen species (ROS) with oxygen, and further oxidizes L-arginine through ROS, realizing a superior acid-selective bactericidal effect. Overall, it proposes a promising strategy for the treatment of H. pylori that maintains high targeting and therapeutic effects in the environment of gastric acid and pepsin.
Topics: Helicobacter pylori; Pepsin A; Reactive Oxygen Species; Graphite; Hemin; Arginine
PubMed: 37991257
DOI: 10.1002/smll.202306155 -
Nutrition and Health Jun 2024Whey proteins and their peptide derivatives have attracted a great attention of researchers in the pharmaceutical and nutritional fields, due to their numerous...
Whey proteins and their peptide derivatives have attracted a great attention of researchers in the pharmaceutical and nutritional fields, due to their numerous bio-functionalities. In the present research study, enzymatic protein hydrolysates (CWPHs) from camel whey proteins (CWPs) were produced and investigated for their antioxidant and antimicrobial potentials. Herein, Pepsin (gastric), and Trypsin and Chymotrypsin (pancreatic) enzymes were used to produce CWPHs. The obtained hydrolysates were characterize to ascertain the level of protein degradation and studies on their antimicrobial and antioxidant potential were conducted. Among all CWPHs, a complete degradation of all different protein bands was perceived with Chymotrypsin-derived CWPHs, whereas, light bands of serum albumin and α-lactalbumin were observed with Trypsin and Pepsin-derived CWPHs. After enzymatic degradation, both CWPHs antioxidant and antimicrobial activities were improved. Chymotrypsin-derived CWPHs demonstrated higher DPPH and ABTS radical scavenging activities, anent the increase in proteolysis time. Compared to unhydrolyzed CWPs, higher metal chelating activities were displayed by Trypsin-derived CWPHs. No significant increase in the FRAP activities was noticed after CWPs hydrolysis using Trypsin and Chymotrypsin, while Pepsin-derived CWPHs showed higher reducing power. In terms of antimicrobial activity, significantly higher bacterial growth inhibition rates were exhibited by CWPHs compared to the unhydrolyzed CWP. Overall, CWPHs displayed enhanced antioxidative and antimicrobial properties.
Topics: Whey Proteins; Camelus; Antioxidants; Animals; Digestion; Protein Hydrolysates; Trypsin; Chymotrypsin; Anti-Infective Agents; Pepsin A; Proteolysis; Gastrointestinal Tract
PubMed: 36065597
DOI: 10.1177/02601060221122213 -
Journal of Nutritional Science and... 2024The Ussing chamber is a tool for analyzing drug absorption. We investigated whether the Ussing chamber can be used to analyze the process from digestion to absorption of...
The Ussing chamber is a tool for analyzing drug absorption. We investigated whether the Ussing chamber can be used to analyze the process from digestion to absorption of protein in the gastrointestinal tract. Mixtures containing infant formula, whole cow's milk, processed soy milk, enteral nutrition, or human breast milk, were placed in the apical membrane side equipped with Caco-2 cells. After the addition of first pepsin then pancreatin, samples from the apical and basal membranes were collected. Infant formula showed the highest digestibility and absorption rate. This may be attributed to the presence of whey protein, which is rapidly digested and absorbed. The digestion and absorption of human breast milk showed different results in each donor, suggesting that digestion and absorption may vary among individuals. We concluded that the Ussing chamber can continuously analyze the process from digestion to absorption of proteins in the gastrointestinal tract.
Topics: Digestion; Humans; Caco-2 Cells; Gastrointestinal Tract; Milk, Human; Infant Formula; Animals; Intestinal Absorption; Whey Proteins; Milk Proteins; Milk; Dietary Proteins; Enteral Nutrition; Soy Milk; Infant; Pepsin A
PubMed: 38684386
DOI: 10.3177/jnsv.70.158 -
The Laryngoscope Jul 2024This study aimed to evaluate the role of pepsin inhibitors in the inflammatory response and their effects on laryngeal mucosal integrity during gastroesophageal reflux...
OBJECTIVE
This study aimed to evaluate the role of pepsin inhibitors in the inflammatory response and their effects on laryngeal mucosal integrity during gastroesophageal reflux (GERD) under in vivo conditions.
METHODS
A surgical model of GERD was used, in which mice were treated with pepstatin (0.3 mg/kg) or darunavir (8.6 mg/kg) for 3 days. On the third day after the experimental protocol, the laryngeal samples were collected to assess the severity of inflammation (wet weight and myeloperoxidase activity) and mucosal integrity (transepithelial electrical resistance and paracellular epithelial permeability to fluorescein).
RESULTS
The surgical GERD model was reproduced. It showed features of inflammation and loss of barrier function in the laryngeal mucosa. Pepstatin and darunavir administration suppressed laryngeal inflammation and preserved laryngeal mucosal integrity.
CONCLUSION
Pepsin inhibition by the administration of pepstatin and darunavir improved inflammation and protected the laryngeal mucosa in a mouse experimental model of GERD.
LEVEL OF EVIDENCE
NA Laryngoscope, 134:3080-3085, 2024.
Topics: Animals; Mice; Gastroesophageal Reflux; Disease Models, Animal; Pepsin A; Pepstatins; Laryngeal Mucosa; Male; Inflammation
PubMed: 38214310
DOI: 10.1002/lary.31239 -
International Journal of Biological... Jan 2024This study develops hemp seed globulin (GLB)-alginate (ALG) nanoparticles (GANPs) for Cannabisin A (CA) stabilization under environmental stress and during pepsin...
This study develops hemp seed globulin (GLB)-alginate (ALG) nanoparticles (GANPs) for Cannabisin A (CA) stabilization under environmental stress and during pepsin digestion. The optimal GLB: ALG mass ratio of 1: 1.5 was determined for GANPs formation at pH 3.5, resulting in a high yield of 95.13 ± 0.91 %, a ζ-potential of -35.73 ± 1.04 mV, a hydrodynamic diameter of 470.67 ± 11.36 nm, and a PDI of 0.298 ± 0.016. GANPs were employed to encapsulate CA, achieving a high loading capacity of 13.48 ± 0.04 μg mg. FTIR analysis demonstrated that the formation of CA-GLB-ALG nanoparticles (CGANPs) involves electrostatic interactions, hydrogen bonding, and hydrophobic interactions. XRD and DSC analyses revealed that CA is amorphous within the CGANPs. CGANPs demonstrated remarkable dispersion stability as well as resistance to high ionic strength and high-temperature treatments, indicating their potential as efficient hydrophobic drug-delivery vehicles. When compared to free CA, CA coated within CGANPs displayed greater DPPH/ABTS scavenging activity. Furthermore, the ALG-shelled nanoparticles protected GLB from pepsin digestion and slowed the release of CA throughout the release process, extending their stay on the intestinal wall mucosa. These findings imply that CGANPs is an ideal delivery vehicle for CA as they may expand the application of CA in food items.
Topics: Antioxidants; Alginates; Cannabis; Pepsin A; Nanoparticles; Globulins
PubMed: 38000582
DOI: 10.1016/j.ijbiomac.2023.128380 -
Experimental Eye Research Sep 2023The corneal stroma is primarily composed of collagen fibrils, proteoglycans, and glycosaminoglycans (GAGs). It is known that corneal crosslinking (CXL) treatment...
The corneal stroma is primarily composed of collagen fibrils, proteoglycans, and glycosaminoglycans (GAGs). It is known that corneal crosslinking (CXL) treatment improves mechanical properties of the cornea. However, the influence of stromal composition on the strengthening effect of CXL procedure has not been thoroughly investigated. The primary objective of the present research was to characterize the effect of keratan sulfate (KS) GAGs on the efficacy of CXL therapy. To this end, the CXL method was used to crosslink porcine corneal samples from which KS GAGs were enzymatically removed by keratanase II enzyme. Alcian blue staining was done to confirm the successful digestion of GAGs and uniaxial tensile experiments were performed for characterizing corneal mechanical properties. The influence of GAG removal and CXL treatment on resistance of corneal samples against enzymatic pepsin degradation was also quantified. It was found that removal of KS GAGs significantly softened corneal tensile properties (P < 0.05). Moreover, the CXL therapy significantly increased the tensile stiffness of GAG-depleted strips (P < 0.05). GAG-depleted corneal buttons were dissolved in the pepsin digestion solution significantly faster than control samples (P < 0.05). The CXL treatment significantly increased the time needed for complete pepsin digestion of GAG-depleted disks (P < 0.05). Based on these observations, we concluded that KS GAGs play a significant role in defining tensile properties and structural integrity of porcine cornea. Furthermore, the stiffening influence of the CXL treatment does not significantly depend on the density of corneal KS GAGs. The findings of the present study provided new information on the relation between corneal composition and CXL procedure mechanical effects.
Topics: Swine; Animals; Glycosaminoglycans; Keratan Sulfate; Pepsin A; Collagen; Cornea; Corneal Stroma; Cross-Linking Reagents; Photosensitizing Agents; Riboflavin; Ultraviolet Rays; Keratoconus
PubMed: 37454921
DOI: 10.1016/j.exer.2023.109570 -
International Journal of Biological... Apr 2024Pepsin is one of the major enzymes with significant importance in the food industry, biomedicines, and pharmaceutical formulations. In this work, the main objective was...
Pepsin is one of the major enzymes with significant importance in the food industry, biomedicines, and pharmaceutical formulations. In this work, the main objective was to biochemically characterize a pepsin-like enzymatic extract obtained from Pygocentrus nattereri, a predatory freshwater fish, focusing on their potential industrial application. The obtained extract exhibited optimal activity at 45 °C and pH 1.0-2.0. These proteases remained stable after 2 h of incubation at temperatures ranging from 0° to 45 °C and within pH range of 1.0 to 7.0. Their activity was significantly affected in presence of pepstatin A and SDS, 10 μM and 3.46 mM respectively, while EDTA and PMSF showed partial inhibitory effects. Divalent cations (Ca2+ and Mg2+) did not inhibit the proteolytic activity of the extract; in fact, it improved at a 5 mM CaCl2 concentration. As the NaCl concentration increased, the enzyme activity decreased. However, after desalination, 90 % of the activity was recovered within the tested exposure time. Besides, this extract demonstrated exceptional versatility across diverse industrial applications, including collagen extraction augmentation, IgG hydrolysis facilitation, and silver and polyester recovery from X-ray films. Our results suggest that the obtained enzymatic extract has a wide range of potential applications.
Topics: Animals; Peptide Hydrolases; Pepsin A; Characidae; Stomach; Perciformes; Hydrogen-Ion Concentration
PubMed: 38431015
DOI: 10.1016/j.ijbiomac.2024.130548 -
World Journal of Gastroenterology May 2024Heartburn is a common symptom shared by both gastroesophageal reflux disease (GERD) and functional heartburn (FHB), which can make it challenging to differentiate...
Heartburn is a common symptom shared by both gastroesophageal reflux disease (GERD) and functional heartburn (FHB), which can make it challenging to differentiate between the two conditions. However, examining oral manifestations of GERD can be a cost-effective and readily available method to aid in this differentiation process. It may serve as a valuable tool in distinguishing GERD from FHB.
Topics: Humans; Gastroesophageal Reflux; Saliva; Heartburn; Pepsin A; Diagnosis, Differential; Biomarkers
PubMed: 38817654
DOI: 10.3748/wjg.v30.i19.2612 -
Spectrochimica Acta. Part A, Molecular... Oct 2024The effects of common migration substances in milk packaging on digestive protease were studied. We choose the common migrants found in eight types of multi-layer...
The effects of common migration substances in milk packaging on digestive protease were studied. We choose the common migrants found in eight types of multi-layer composite milk packaging. Enzyme activity experiments revealed that pepsin activity decreased by approximately 18 % at 500 μg/mL of stearic acid and stearamide treatment, while trypsin activity decreased by approximately 18 % only by stearic acid treatment (500 μg/mL). Subsequently, fluorescence spectroscopy, circular dichroism spectroscopy, and molecular docking technology were employed to investigate the inhibition mechanism of protease activity by migrating substances in three systems: stearic acid-trypsin, stearic acid-pepsin, and stearamide-pepsin. Results showed that the inhibitory effect of stearic acid on trypsin is a reversible mixed inhibition, whereas the inhibitory effects of stearic acid and stearamide on pepsin are non-competitive. In all three systems, ΔH < 0, ΔS < 0, and ΔG < 0, indicating the binding process between the migrant and the protease is a spontaneous exothermic process primarily driven by hydrogen bonding and van der Waals forces. In addition, their binding constants are all around 10 L/moL, indicating that there are moderate binding affinities exist between migrants and proteases. The binding process results in the quenching of the protease's endogenous fluorescence and induces alterations in the enzyme's secondary structure. Synchronized fluorescence spectroscopy showed that stearic acid enhanced the hydrophobicity near the Tyr residue of trypsin. The molecular docking results indicated that the binding affinity of stearic acid-trypsin, stearic acid-pepsin, and stearamide-pepsin was -22.51 kJ/mol, -12.35 kJ/mol, -19.28 kJ/mol respectively, which consistent with the trend in the enzyme activity results. This study can provide references for the selection of milk packaging materials and the use of processing additives, ensuring food health and safety.
Topics: Molecular Docking Simulation; Animals; Milk; Spectrometry, Fluorescence; Trypsin; Food Packaging; Stearic Acids; Pepsin A; Circular Dichroism; Peptide Hydrolases; Thermodynamics
PubMed: 38801790
DOI: 10.1016/j.saa.2024.124517