L-Asparaginase and HCP quantification by SWATH LC-MS/MS for new and improved purification step in Erwinia chrysanthemi L-asparaginase manufacture.

Author(s):  
Tapasvi Modi ◽  
Sergio Regufe Da Mota ◽  
David Gervais
2020 ◽  
Vol 7 (2) ◽  
pp. 121-133
Author(s):  
Ayesha Akhtar ◽  
Shivakumar Arumugam ◽  
Shoaib Alam

Background:: Protein A affinity chromatography is often employed as the most crucial purification step for monoclonal antibodies to achieve high yield with purity and throughput requirements. Introduction:: Protein A, also known as Staphylococcal protein A (SPA) is found in the cell wall of the bacteria staphylococcus aureus. It is one of the first discovered immunoglobulin binding molecules and has been extensively studied since the past few decades. The efficiency of Protein A affinity chromatography to purify a recombinant monoclonal antibody in a cell culture sample has been evaluated, which removes 99.0% of feed stream impurities. Materials and Method:: We have systematically evaluated the purification performance by using a battery of analytical methods SDS-PAGE (non-reduced and reduced sample), Cation Exchange Chromatography (CEX), Size-exclusion chromatography (SEC), and Reversed phased-Reduced Chromatography for a CHO-derived monoclonal antibody. Results and Discussion:: The analytical test was conducted to determine the impurity parameter, Host Cell Contaminating Proteins (HCP). It was evaluated to be 0.015ng/ml after the purification step; while initially, it was found to be 24.431ng/ml. Conclusion:: The tests showed a distinct decrease in the level of different impurities after the chromatography step. It can be concluded that Protein A chromatography is an efficient step in the purification of monoclonal antibodies.


1993 ◽  
Vol 48 (1-2) ◽  
pp. 10-15 ◽  
Author(s):  
Sabine Gubatz ◽  
Rolf Wiermann

The results of tracer experiments performed with anthers of Tulipa cv. Apeldoorn have already shown that a high level of incorporation into the sporopollenin fraction was achieved, when [14C]phenylalanine was applicated as a precursor. In order to investigate whether the substantial incorporation of [14C]phenylalanine is a unique phenomenon restricted to Tulipa, tracer experiments were carried out on anthers of Cucurbita maxima. The sporopollenin fraction was isolated and purified by a gentle method including extractions with various solvents, incubations with hydrolysing enzymes and fractionated saponifications. The remaining, as well as the released radioactivity, was determined after each purification step. After the application of [U-14C]phenylalanine, a substantial incorporation into the sporopollenin fraction was determined. The values were clearly higher than those obtained with [1-14C]glucose or those from corresponding experiments on Tulipa anthers. After potash fusion of sporopollenin fractions labeled via [U-14C]phenylalanine, p-hydroxybenzoic acid was shown to be the main com ponent among the ether soluble acids; moreover it showed the highest level of radioactivity. No radioactivity was detected in the degradation products oxalic acid, benzoic acid, phthalic acid and terephthalic acid or octanedioic acid- and decanedioic acid-dimethylester.


2019 ◽  
Vol 102 (1) ◽  
pp. 255-261 ◽  
Author(s):  
Ji-cai Fan ◽  
Quan Jin ◽  
Hua-li He ◽  
Ren Ren ◽  
Shu-ting Wang

Abstract Background: Phthalate esters (PAEs) are a group of chemical compounds widely used as plasticizers to increase the flexibility of plastics that are used in the manufacturing of kitchen utensils and food containers. Objective: In this study, a simple, rapid, and sensitive method for the determination of 20 PAEs in different kinds of food packaging materials has been developed. Methods: Samples injected with five internal standards were extracted with acetonitrile saturated with n-hexane and then detected by GC-MS/MS without a purification step. Results: The standard calibration curves were linear for all analytes over the concentration range of 5–500 μg/L, and the correlation coefficients ranged from 0.9913 to 0.9999. The LODs and LOQs were in the ranges of 1.7–62.5 and 5.5–208.3 μg/kg, respectively. The accuracy of this method was evaluated by measuring the recovery from spiked samples. The recoveries of all 20 phthalates from samples spiked at three different concentrations were measured, and the recovery was in the range of 82.1–110.8% and the relative standard deviation range of recovery result (n = 6) was 0.3–9.7%. Conclusions: The method presented here is simple, rapid, and sensitive and can be applied to large-scale detection of PAEs in plastic materials. Highlights: Instead of only one solvent, acetonitrile saturated with n-hexane was used as the extraction solvent. Samples were pretreated without a purification step. Five internal standards were used for quantitative determination.


Virology ◽  
1984 ◽  
Vol 137 (1) ◽  
pp. 102-112 ◽  
Author(s):  
A. Resibois ◽  
M. Colet ◽  
M. Faelen ◽  
E. Schoonejans ◽  
A. Toussaint
Keyword(s):  

2006 ◽  
Vol 72 (3) ◽  
pp. 1956-1965 ◽  
Author(s):  
Anne-Marie Grenier ◽  
Gabrielle Duport ◽  
Sylvie Pagès ◽  
Guy Condemine ◽  
Yvan Rahbé

ABSTRACT Dickeya dadantii (Erwinia chrysanthemi) is a phytopathogenic bacterium causing soft rot diseases on many crops. The sequencing of its genome identified four genes encoding homologues of the Cyt family of insecticidal toxins from Bacillus thuringiensis, which are not present in the close relative Pectobacterium carotovorum subsp. atrosepticum. The pathogenicity of D. dadantii was tested on the pea aphid Acyrthosiphon pisum, and the bacterium was shown to be highly virulent for this insect, either by septic injury or by oral infection. The lethal inoculum dose was calculated to be as low as 10 ingested bacterial cells. A D. dadantii mutant with the four cytotoxin genes deleted showed a reduced per os virulence for A. pisum, highlighting the potential role of at least one of these genes in pathogenicity. Since only one bacterial pathogen of aphids has been previously described (Erwinia aphidicola), other species from the same bacterial group were tested. The pathogenic trait for aphids was shown to be widespread, albeit variable, within the phytopathogens, with no link to phylogenetic positioning in the Enterobacteriaceae. Previously characterized gut symbionts from thrips (Erwinia/Pantoea group) were also highly pathogenic to the aphid, whereas the potent entomopathogen Photorhabdus luminescens was not. D. dadantii is not a generalist insect pathogen, since it has low pathogenicity for three other insect species (Drosophila melanogaster, Sitophilus oryzae, and Spodoptera littoralis). D. dadantii was one of the most virulent aphid pathogens in our screening, and it was active on most aphid instars, except for the first one, probably due to anatomical filtering. The observed difference in virulence toward apterous and winged aphids may have an ecological impact, and this deserves specific attention in future research.


Fuel ◽  
2013 ◽  
Vol 113 ◽  
pp. 50-58 ◽  
Author(s):  
Marta Serrano ◽  
Abderrahim Bouaid ◽  
Mercedes Martínez ◽  
José Aracil

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