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About

CRM197

Consistency:  Pfenex CRM197 shows higher purity than expression in alternate platforms

Scalability:  Continuity from milligrams for research to kilograms for commercial production

Compliance:  Pfenex CRM197 is the only recombinant host with demonstrated cGMP availability since 2011

Pfenex CRM197 is a non-toxic mutant of diphtheria toxin having a single amino acid substitution of glutamic acid to glycine at position 52. CRM197 is a well-defined protein and functions as a carrier for polysaccharides and haptens, making them immunogenic. It is utilized as a carrier protein in a number of approved conjugate vaccines for diseases such as Streptococcus pneumoniaeHaemophilus influenzae b and Neisseria meningitidis.  Pfenex CRM197 is a recombinant, soluble form produced in Pseudomonas fluorescens using the Pfenex Expression Technology™ platform.  For more information, download our data sheet.

Formerly sold under the Reagent Proteins division of Pfenex, CRM197 is the ideal carrier protein or immunological adjuvant for conjugate vaccines.  CRM197 has been used commercially in prophylactic vaccines since 1990.  Current research and development areas include immunotherapies and anti-addiction vaccines.  Pfenex CRM197 is similar in both structure and function to the protein produced in C. diphtheriae, as shown in physicochemical and in vivo studies.

Regulatory

A Biologics Master File (BMF) is a confidential, detailed dossier submitted to the US FDA which contains the proprietary information on the manufacture and safety of a drug component. Pfenex will submit a letter authorizing FDA to review the CRM197 BMF in conjunction with their review of a customer-filed IND using Pfenex CRM197 as an active substance starting material in the manufacture of their product(s). Pfenex will also provide a copy of this authorization letter to each customer for inclusion in their IND. Since the BMF mechanism is not available in the EU or elsewhere in the world, Pfenex will supply the necessary information regarding CRM197 required to complete our customers’ regulatory filings. This information will be supplied in a format consistent with CTD Module 2 and Module 3, to be easily incorporated into non-US regulatory filings.

References

Please see below for a sample of references that discuss CRM197 from Pfenex:

The amino-acid sequence of two non-toxic mutants of diphtheria toxin: CRM45 and CRM197.
Nucleic Acids Research | By G Giannini, R Rappuoli, and G Ratti | May 25, 1984

Abstract: The amino-acid sequences of two diphtheria toxin-related, non-toxic proteins, CRM45 and CRM197 , were deduced from the complete sequence of their genes: tox 45 and tox 197. CRM45 lacks the last 149 C-terminal amino-acid residues, but is otherwise identical to diphtheria toxin: a single C—-T transition introduces an “ochre” (TAA) termination signal in tox 45, after the codon for threonine-386. A single G—-A transition was also found in tox 197, leading to the substitution of glycine-52, present in the wild-type toxin, with glutamic acid in CRM197 . This aminoacid change is responsible for the loss of the NAD:EF2 ADP-ribosyltransferase activity in CRM197 , due most probably to an alteration of the NAD+ binding site.

Identification of diphtheria toxin receptor and a nonproteinous diphtheria toxin-binding molecule in Vero cell membrane
Journal of Cell Biology | By: Mekada, E., and Uchida, T. J. | August 1, 1988

Abstract: Two substances possessing the ability to bind to diphtheria toxin (DT) were found to be present in a membrane fraction from DT-sensitive Vero cells. One of these substances was found on the basis of its ability to bind DT and inhibit its cytotoxic effect. This inhibitory substance competitively inhibited the binding of DT to Vero cells. However this inhibitor could not bind to CRM197, the product of a missense mutation in the DT gene, and did not inhibit the binding of CRM197 to Vero cells. Moreover, similar levels of the inhibitory activity were observed in membrane fractions from DT-insensitive mouse cells, suggesting the inhibitor is not the DT receptor which is specifically present in DT-sensitive cells. The second DT-binding substance was found in the same Vero cell membrane preparation by assaying the binding of 125I-labeled CRM197. Such DT-binding activity could not be observed in membrane preparation from mouse L cells. From competition studies using labeled DT and CRM proteins, we conclude that this binding activity is due to the surface receptor for DT. Treatment of these substances with several enzymes revealed that the inhibitor was sensitive to certain RNases but resistant to proteases, whereas the DT receptor was resistant to RNase but sensitive to proteases. The receptor was solubilized and partially purified by chromatography on CM- Sepharose column. Immunoprecipitation and Western blotting analysis of the partially purified receptor revealed that a 14.5-kD protein is the DT receptor, or at least a component of it.

Dedicated Facility Injects CRM197 Into Conjugate Vaccine Market
in-Pharma Technologist | By: Dan Stanton | January 21, 2013

Excerpt: Reagent Proteins says its ability to produce scalable amounts of protein CRM197 supports the current demand in production of conjugate vaccines.

Pfenex Division Begins Product Capacity Expansion
Genetic Engineering & Biotechnology News | January 16, 2013

Excerpt: Pfenex’s Reagent Proteins division’s manufacturing partner, Serum Institute of India, began the construction of a new large scale cGMP CRM197 production facility, which is expected to be completed next year. Annual capacity will be in excess of 50 kilograms of cGMP grade CRM197, according to Pfenex.

Recombinant Production of Carrier Proteins
Genetic Engineering & Biotechnology News | December 1, 2012

Excerpt: Within the rapidly growing vaccine space, conjugate vaccines—polysaccharide antigens covalently linked to carrier proteins—have been shown to be effective against several bacterial pathogens.

Publications