High‐yield production of recombinant CRM197, a non‐toxic mutant of diphtheria toxin, in the periplasm of <i>Escherichia coli</i>

Goffin, Philippe; Dewerchin, Marianne; Rop, Philippe De; Blais, Normand; Dehottay, Philippe

doi:10.1002/biot.201700168

Cited by 19 publications

(22 citation statements)

References 31 publications

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“…Production of CRM 197 is hindered, however, by the strict growth requirements of the traditional expression system (C diphtheriae) and generally lower yields compared to other expression systems. 8 Given these challenges, the evaluation of recombinant CRM 197 expressed in multiple heterologous organisms (e.g., E coli, P fluorescens, Bacillus subtilis) is being pursued as an alternative source of carrier proteins. 9 Given the complex process of manufacturing conjugate vaccines, 10 delineating the physicochemical properties of recombinant CRM 197 , expressed in C diphtheriae or a heterologous system, is essential to mitigate risk during manufacturing, including subsequent polysaccharide conjugation, to ensure the immunogenicity of the final drug product.…”

Section: Introductionmentioning

confidence: 99%

Analytical Comparability Assessments of 5 Recombinant CRM 197 Proteins From Different Manufacturers and Expression Systems

Hickey

Toprani

Kaur

et al. 2018

Journal of Pharmaceutical Sciences

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Cross-reacting material 197 (CRM), a single amino acid mutant of diphtheria toxoid, is a commonly used carrier protein in commercial polysaccharide protein conjugate vaccines. In this study, CRM proteins from 3 different expression systems and 5 different manufacturers were obtained for an analytical comparability assessment using a wide variety of physicochemical and in vitro antigenic binding assays. A comprehensive analysis of the 5 CRM molecules demonstrate that recombinant CRM's expressed in heterologous systems (Escherichia coli and Pseudomonas fluorescens) are overall highly similar (if not better in some cases) to those expressed in the traditional system (Corynebacterium diphtheriae) in terms of primary sequence/post-translational modifications, higher order structural integrity, apparent solubility, physical stability profile (vs. pH and temperature), and in vitro antigenicity. These results are an encouraging step to demonstrate that recombinant CRM expressed in alternative sources have the potential to replace CRM expressed in C diphtheriae as a source of immunogenic carrier protein for lower cost polysaccharide conjugate vaccines. The physicochemical assays established in this work to monitor the key structural attributes of CRM should also prove useful as complementary characterization methods (to routine quality control assays) to support future process and formulation development of lower cost CRM carrier proteins for use in various conjugate vaccines.

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Section: Introductionmentioning

confidence: 99%

Analytical Comparability Assessments of 5 Recombinant CRM 197 Proteins From Different Manufacturers and Expression Systems

Hickey

Toprani

Kaur

et al. 2018

Journal of Pharmaceutical Sciences

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“…Recently, the notable result for periplasmic soluble expression of CRM197 in E . coli was reported [ 22 ]. It was reported to achieve production of 3 g/l of CRM197 by a high cell density fed-batch process.…”

Section: Resultsmentioning

confidence: 99%

“…More recently, successful periplasmic expression of soluble CRM197 in E . coli was reported [ 22 ]. The authors showed that the purity of the primary cell extract reached 36.5% of total protein after periplasmic extraction by osmotic shock, while not exceeding 8.3% of total protein by whole cell disruption.…”

Section: Discussionmentioning

confidence: 99%

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Efficient recovery of recombinant CRM197 expressed as inclusion bodies in E.coli

Park¹,

Jang²,

Kim³

et al. 2018

PLoS ONE

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CRM197, which retains the same inflammatory and immune-stimulant properties as diphtheria toxin but with reduced toxicity, has been used as a safe carrier in conjugated vaccines. Expression of recombinant CRM197 in E. coli is limited due to formation of inclusion bodies. Soluble expression attempts in Bacillus subtilis, P. fluorescens, Pichia pastoris, and E. coli were partially unsuccessful or did not generate yields sufficient for industrial scale production. Multiple approaches have been attempted to produce CRM197 in E. coli, which has attractive features such as high yield, simplicity, fast growth, etc., including expression of oxidative host, concurrent expression of chaperones, or periplasmic export. Recently, alternative methods for recovery of insoluble proteins expressed in E. coli were reported. Compared to traditional denaturation/refolding, these methods used the non-denaturing solubilization agent, N-lauroylsarkosine to obtain higher recovery yields of native proteins. Based on this work, here, we focused on solubilization of CRM197 from E. coli inclusion bodies. First, CRM197 was expressed as inclusion bodies by high-level expression of recombinant CRM197 in E. coli (126.8 mg/g dcw). Then bioactive CRM197 was isolated from these inclusion bodies with high yield (108.1 mg/g dcw) through solubilization with N-lauroylsarkosine including Triton X-100 and CHAPS, and purified by Ni-affinity chromatography and size-exclusion chromatography. In this study, we present a cost-effective alternative for the production of bioactive CRM197 and compare our recovery yield with yields in other production processes.

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“…This Special issue of the Biotechnology Journal , the official journal of the society, showcases some of the technical programe of the symposium and the advances and topics of interest in biochemical engineering, ranging from bioeconomy , sustainability and solvent free extractions , discovery and optimization of new biocatalysts , design of new biotherapeuticals , as well as new process control and design approaches based on modeling and predictive tools .…”

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confidence: 99%

Editorial: The European Symposium on Biochemical Engineering Sciences, Dublin 2016

Ferreira

Glassey

2017

Biotechnology Journal

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High‐yield production of recombinant CRM197, a non‐toxic mutant of diphtheria toxin, in the periplasm of Escherichia coli

Cited by 19 publications

References 31 publications

Analytical Comparability Assessments of 5 Recombinant CRM 197 Proteins From Different Manufacturers and Expression Systems

Analytical Comparability Assessments of 5 Recombinant CRM 197 Proteins From Different Manufacturers and Expression Systems

Efficient recovery of recombinant CRM197 expressed as inclusion bodies in E.coli

Editorial: The European Symposium on Biochemical Engineering Sciences, Dublin 2016

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