scholarly journals Development and validation of a method for the determination of the specific activity of recombinant monoclonal antibody eculizumab

2020 ◽  
Vol 15 (2) ◽  
pp. 77-85
Author(s):  
D. I. Zybin ◽  
A. S. Seregin ◽  
A. D. Askretkov ◽  
N. V. Orlova ◽  
Yu. A. Seregin ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Pharmaceutical Analysis ◽  
Comparative Evaluation ◽  
Specific Activity ◽  
In Vitro Method ◽  
Recombinant Monoclonal Antibody ◽  
Development And Validation ◽  
First Time

Objectives. Developing reliable and accurate analytical methods is necessary for comparative pharmaceutical analysis using physicochemical, biological (in vitro), preclinical, and clinical trials. The main objective of this study was to develop and validate an in vitro method for determining the specific activity of the recombinant monoclonal antibody eculizumab.Methods. The method of indirect enzyme immunoassay was used in the study.Results. A method for determining the specific activity of the humanized recombinant monoclonal antibody eculizumab was described and validated for the first time. A comparative evaluation of the specific activity of Soliris® (Alexion Pharmaceuticals Inc., USA), and its biosimilar PRK-001 (Pharmapark, Russia) was performed using the developed method.Conclusions. The similarity of PRK-001 and the original Soliris® in relation to their specific activity, that is, binding to the human complement system C5 protein, was proved. 

Carbon dots derived from Fusobacterium nucleatum for intracellular determination of Fe3+ and bioimaging both in vitro and in vivo

2021 ◽  
Author(s):  
Lijuan Liu ◽  
Shengting Zhang ◽  
Xiaodan Zheng ◽  
Hongmei Li ◽  
Qi Chen ◽  
...  
Keyword(s):  
Carbon Dots ◽  
Fusobacterium Nucleatum ◽  
Living Cells ◽  
First Time ◽  
Fluorescent Carbon Dots ◽  
Fluorescent Carbon

Fusobacterium nucleatum has been employed for the first time to synthesize fluorescent carbon dots which could be applied for the determination of Fe3+ ions in living cells and bioimaging in vitro and in vivo with excellent biocompatibility.

scholarly journals Conformation-Specific Inhibitory Anti-MMP-7 Monoclonal Antibody Sensitizes Pancreatic Ductal Adenocarcinoma Cells to Chemotherapeutic Cell Kill

Cancers ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1679
Author(s):  
Vishnu Mohan ◽  
Jean P. Gaffney ◽  
Inna Solomonov ◽  
Maxim Levin ◽  
Mordehay Klepfish ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Active Site ◽  
Drug Targets ◽  
Fas Ligand ◽  
Specific Activity ◽  
Matrix Metalloproteases ◽  
Ductal Adenocarcinoma ◽  
Enzyme Active Site ◽  
Induced Apoptosis

Matrix metalloproteases (MMPs) undergo post-translational modifications including pro-domain shedding. The activated forms of these enzymes are effective drug targets, but generating potent biological inhibitors against them remains challenging. We report the generation of anti-MMP-7 inhibitory monoclonal antibody (GSM-192), using an alternating immunization strategy with an active site mimicry antigen and the activated enzyme. Our protocol yielded highly selective anti-MMP-7 monoclonal antibody, which specifically inhibits MMP-7′s enzyme activity with high affinity (IC50 = 132 ± 10 nM). The atomic model of the MMP-7-GSM-192 Fab complex exhibited antibody binding to unique epitopes at the rim of the enzyme active site, sterically preventing entry of substrates into the catalytic cleft. In human PDAC biopsies, tissue staining with GSM-192 showed characteristic spatial distribution of activated MMP-7. Treatment with GSM-192 in vitro induced apoptosis via stabilization of cell surface Fas ligand and retarded cell migration. Co-treatment with GSM-192 and chemotherapeutics, gemcitabine and oxaliplatin elicited a synergistic effect. Our data illustrate the advantage of precisely targeting catalytic MMP-7 mediated disease specific activity.

scholarly journals An in vitro method for the determination of protein turnover in incubated proximal tubule segments

1993 ◽  
Vol 43 (5) ◽  
pp. 1156-1159 ◽  
Author(s):  
Robert May ◽  
Brian Logue ◽  
Byrad Edwards ◽  
Swati Patel
Keyword(s):  
Proximal Tubule ◽  
Protein Turnover ◽  
In Vitro Method ◽  
Proximal Tubule Segments

scholarly journals Development and Validation of an Eco-friendly HPLC-UV-DAD Method for the Determination of Allopurinol in Pharmaceuticals with Application to In vitro Dissolution Studies

2021 ◽  
Vol 11 (5) ◽  
pp. 13089-13101
Keyword(s):  
Factorial Design ◽  
Matrix Effects ◽  
Dissolution Kinetics ◽  
In Vitro Dissolution ◽  
Dissolution Test ◽  
Dissolution Studies ◽  
Dissolution Tests ◽  
Development And Validation

In this study, a sustainable HPLC-UV-DAD method was developed and validated for the determination of allopurinol in tablets and optimization of the dissolution test using factorial design. The separation of the analyte from the sample matrix was achieved in 3.01 minutes in a C8 column (4.6 mm X 150 mm X 5 μm), using mobile phase 0.1 mol L-1 HCl (25%) + ethanol (50%) + ultrapure water (25%) by UV detection at 249 nm. The method presented satisfactory analytical parameters of validation (specificity, selectivity, linearity, stability, precision, accuracy, and robustness), showing no matrix effects. The dissolution test was optimized by complete factorial design 23 and, the optimal conditions were: HCl 0.001 mol L-1, apparatus II (paddle) and 75 rpm. The analytical procedures and dissolution tests were applied to allopurinol tablets marketed in Bahia, Brazil, to evaluate the dissolution studies. The pharmaceuticals had similar dissolution profiles and first-order dissolution kinetics. This new and sustainable HPLC-UV-DAD method is friendly to the environment and can be used for the routine pharmaceutical analysis of allopurinol in fixed dosage forms.

scholarly journals Quantification and characterization of the 5’exonuclease activity of the lysosomal nuclease PLD3 by a novel cell-based assay

2020 ◽  
pp. jbc.RA120.015867
Author(s):  
Cedric Cappel ◽  
Adriana Carolina Gonzalez ◽  
Markus Damme
Keyword(s):  
Lupus Erythematosus ◽  
Specific Activity ◽  
Nuclease Activity ◽  
Hydrolytic Activity ◽  
Proteolytic Processing ◽  
Exonuclease Activity ◽  
Fluorescence Quencher

Phospholipase D3 (PLD3) and phospholipase D4 (PLD4), the most recently described lysosomal nucleases, are associated with Alzheimer`s disease, spinocerebellar ataxia, and systemic lupus erythematosus. They exhibit 5’ exonuclease activity on single-stranded DNA, hydrolyzing it at the acidic pH associated with the lysosome. However, their full cellular function is inadequately understood. To examine these enzymes, we developed a robust and automatable cell-based assay based on fluorophore- and fluorescence-quencher coupled oligonucleotides for the quantitative determination of acidic 5’ exonuclease activity. We validated the assay under knockout and PLD-overexpression conditions, and then applied it to characterize PLD3 and PLD4 biochemically. Our experiments revealed PLD3 as the principal acid 5’ exonuclease in HeLa cells, where it showed a markedly higher specific activity compared to PLD4. We further used our newly developed assay to determine the substrate specificity and inhibitory profile of PLD3, and found that proteolytic processing of PLD3 is dispensable for its hydrolytic activity. We followed the expression, proteolytic processing, and intracellular distribution of genetic PLD3 variants previously associated with Alzheimer’s disease and investigated each variant's effect on the 5’ nuclease activity of PLD3, finding that some variants lead to reduced activity, but others not. The development of a PLD3/4-specific biochemical assay will be instrumental in understanding better both nucleases and their incompletely unknown roles in vitro and in vivo.

scholarly journals DEVELOPMENT AND VALIDATION OF ULTRAVIOLET-SPECTROPHOTOMETRIC METHOD FOR DETERMINATION OF FEBUXOSTAT FOR CONDUCTING IN-VITRO QUALITY CONTROL TESTS IN BULK AND PHARMACEUTICAL DOSAGE FORMS

2018 ◽  
Vol 11 (9) ◽  
pp. 115
Author(s):  
Jaspreet Kaur ◽  
Daljit Kaur ◽  
Sukhmeet Singh
Keyword(s):  
Spectrophotometric Method ◽  
Limit Of Detection ◽  
Pharmaceutical Formulations ◽  
Dosage Forms ◽  
Pharmaceutical Dosage Forms ◽  
Relative Standard ◽  
Limit Of Quantitation ◽  
Development And Validation

Objective: A simple, accurate, and selective ultraviolet-spectrophotometric method has been developed for the estimation of febuxostat in the bulk and pharmaceutical dosage forms.Method: The method was developed and validated according to International Conference on Harmonization (ICH Q2 R1) guidelines. The developed method was validated statistically with respect to linearity, range, precision, accuracy, ruggedness, limit of detection (LOD), limit of quantitation (LOQ), and recovery. Specificity of the method was demonstrated by applying different stressed conditions to drug samples such as acid hydrolysis, alkaline hydrolysis, oxidative, photolytic, and thermal degradation.Results: The study was conducted using phosphate buffer pH 6.8 and λmax was found to be 312 nm. Standard plot having a concentration range of 1–10 μg/ml showed a good linear relationship with R2=0.999. The LOD and LOQ were found to be 0.118 μg/ml and 0.595 μg/ml, respectively. Recovery and percentage relative standard deviations were found to be 100.157±0.332% and <2%, respectively.Conclusion: Proposed method was successfully applicable to the pharmaceutical formulations containing febuxostat. Thus, the developed method is found to be simple, sensitive, accurate, precise, reproducible, and economical for the determination of febuxostat in pharmaceutical dosage forms.

Eine Methode zur qualitativen und quantitativen Bestimmung von drei Juvenilhormonen von Insekten

1974 ◽  
Vol 29 (3-4) ◽  
pp. 161-168 ◽  
Author(s):  
K. H. Trautmann ◽  
A. Schuler ◽  
M. Suchý ◽  
H.-K. Wipf
Keyword(s):  
Quantitative Determination ◽  
Specific Activity ◽  
High Specific Activity ◽  
Ether Extract ◽  
Qualitative And Quantitative ◽  
Work Up ◽  
First Time ◽  
Very High ◽  
Tenebrio Molitor Larvae

Abstract A method is presented permitting the qualitative and quantitative determination of all three presently known hormones (JH1-3). The determination is based on the method of radioactive isotope dilution, whereby a very small known amount of tritium-labelled JH-1 is added to the ether extract of the particular species. The addition of radioactive JH-1 permits the isolation of all three hormones, because of their similar behaviour during the chosen work up. The quantitative determination was carried out by gas chromatography and the identification was confirmed with the help of retention-times and GC-MS combination. The method was checked by using an extract of Hyalophora cecropia. For the first time methyl 10,11-epoxy-3,7,11-trimethyl-2-trans-6-trans-dodecadienoate (JH-3) could also be identified as the juvenile hormone of Melo­lontha melolontha. In Vanessa io larvae, Tenebrio molitor larvae and adults and in Musca domestica larvae none of the three known hormones could be detected. The preparation of JH-1 labelled with tritium in the methyl group of the ester was accomplished with very high specific activity (4.34 Ci/mmol) of the tritiated acid with diazomethane.

Control of Recombinant Monoclonal Antibody Effector Functions by Fc N-Glycan Remodeling in Vitro

2005 ◽  
Vol 21 (6) ◽  
pp. 1644-1652 ◽  
Author(s):  
J. Hodoniczky ◽  
Y.Z. Zheng ◽  
D.C. James
Keyword(s):  
Monoclonal Antibody ◽  
Effector Functions ◽  
Recombinant Monoclonal Antibody

An in vitro method for the quantitative determination of the antimicrobial efficacy of silver-containing wound dressings

2009 ◽  
Vol 366 (1-2) ◽  
pp. 111-116 ◽  
Author(s):  
Simon Gaisford ◽  
Anthony E. Beezer ◽  
Alistair H. Bishop ◽  
Michael Walker ◽  
David Parsons
Keyword(s):  
Quantitative Determination ◽  
Wound Dressings ◽  
Antimicrobial Efficacy ◽  
In Vitro Method

scholarly journals Development and Validation of HPTLC Method for Estimation of Carbamazepine in Formulations and Its In Vitro Release Study

2011 ◽  
Vol 2011 ◽  
pp. 1-8 ◽  
Author(s):  
Rashmin B. Patel ◽  
Mrunali R. Patel ◽  
Kashyap K. Bhatt ◽  
Bharat G. Patel
Keyword(s):  
High Performance ◽  
Linear Regression Analysis ◽  
In Vitro Release ◽  
Analysis Data ◽  
Limit Of Quantitation ◽  
Bulk Drug ◽  
Development And Validation ◽  
Vitro Release

A new, simple, and rapid high-performance thin-layer chromatographic method was developed and validated for quantitative determination of Carbamazepine. Carbamazepine was chromatographed on silica gel 60 F254 TLC plate using ethyl acetate-toluene-methanol (5.0 + 4.0 + 1.0 v/v/v) as mobile phase. Carbamazepine was quantified by densitometric analysis at 285 nm. The method was found to give compact spots for the drug (Rf=0.47 ± 0.01). The linear regression analysis data for the calibration plots showed good linear relationship with r2=.9995 in the concentration range 100–600 ng/spot. The method was validated for precision, recovery, repeatability, and robustness as per the International Conference on Harmonization guidelines. The minimum detectable amount was found to be 16.7 ng/spot, whereas the limit of quantitation was found to be 50.44 ng/spot. Statistical analysis of the data showed that the method is precise, accurate, reproducible, and selective for the analysis of Carbamazepine. The method was successfully employed for the estimation of equilibrium solubility, quantification of Carbamazepine as a bulk drug, in commercially available preparation, and in-house developed mucoadhesive microemulsion formulations and solution.

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