TMEcoCRM : Aﬀordable CRM197 Vaccine Carrier Protein#A. Lees* , N. Oganesyan*, I. Krauss**, D. Nguyen**#*Fina BiosoluBons LLC, **Brandeis University, University of Maryland

Aﬀordable carrier protein
CRM197 , a geneBcally detoxiﬁed diphtheria toxin, is widely used
as a carrier protein in conjugate vaccines. It was originallyexpressed as a secreted protein in Corynebacterium diphtheriae.
Recently recombinant CRM197 expressed in the periplasm ofPseudomonas ﬂuorescens has become available.
Fina BioSoluBons has developed a new CRM197, EcoCRMTM,
produced in E.coli. EcoCRMTM is expressed as soluble protein in
the cytoplasm of a widely-used E.coli expression strain. Highexpression of EcoCRMTM and a simple puriﬁcaBon method
allows low cost producBon and the promise of signiﬁcantly
reducing the cost of this component of conjugate vaccines.
The presented data show that EcoCRMTM as a carrier protein is
equivalent to CRM197 from established sources.

GlycopepBde-CRM conjugate
Thio-ether conjugaBon is frequently used for linking pepBdes,
oligosaccharides and other anBgens to a carrier protein. EcoCRMTM wasderivaBzed with an excess of the NHS maleimide reagent sulfo-EMCS.

The MW of labeled EcoCRMTM increased
from 58,413 to 62,525, a mass increaseof 4,100. Each maleimide linker adds
about 208 mass units, indica/ng about
20 of CRM’s 39 lysines have beenlabeled.

Overview!EcoCRMTM is a new aﬀordable CRM197 carrier protein!Highly expressed as a soluble single polypepBde in thecytoplasm of E.coli!Equivalent immunogenicity as a carrier protein to other rCRM197!Progress towards clinical development!Biophysical analysis completed by independent lab tobe presented at PEGS (Boston, May 1, 2017)!GMP master cell banking completed!CMO selected!GMP EcoCRMTM anBcipated by Q2 2018

11+DTT

2 2+DTTMW

12

129 kDa
80 kDa60 kDa40 kDa30 kDa20 kDaA

B

1- CRM197 (Pfenex) 2- EcoCRMTM

A: SDS-PAGE Reduced vs non
reduced. Coomassie Blue staining.
B:Western Blot analysis, detec/on
with polyclonal rabbit@CRM197(AIC Biotech)

Glycopep/de conjugates ofEcoCRMTM and Pfenex CRM wereprepared using EMCS-labeled CRM.
Rabbits were immunized twice.
Aeer the ﬁnal bleed, IgG an/glycopep/de /ters were measured
using glycopep/de-BSA as theELISA an/gen.

GlycopepBdes conjugates with EcoCRMTM and rCRM197 (Pfenex) inducecomparable anB-glycopepBde Bters.

Protein-CRM conjugate
AnBbodies to the malaria protein Pfs25 have been shown to prevent themalaria parasite from passing through the mosquito gut.
Poorlyimmunogenic by itself, Pfs25-protein conjugates induce anB-Pfs25
anBbodies in mice that can block malaria mosquito transmission.
AnB-Pfs25 anBbody Bter

TMSDS-PAGE and WB analysis of EcoCRM and CRM197 (Pfenex)

EcoCRMTM is expressed as soluble properly folded single polypepBdeand highly puriﬁed. Recombinant CRM197 from Reagent Proteins(Pfenex), made in Pseudomonas ﬂuorescens has generally been shown
to be equivalent to “naBve” CRM197 expressed by Corynebacterium
diphtheriae (Cassidy et al., Bioprocessing Int.,p.50 Nov. 2012).

Thiol-glycopep/de (MW ~15kDa) wasconjugated to EMCS-labeled CRM.
Mass spec of the conjugates indicatedan average of 7 glycopep/des/CRM.

Mice were immunized 3 /mes with
pfs25-CRM conjugates made with
EcoCRMTM or Pfenex CRM. Eachdose contained equal amounts of 4µg Pfs25 in soluble or conjugate
form, adsorbed to Alum.
The authors thank the Lab. Of Malaria &Vector Research at NIAID/NIH for the
ELISA and SMFA and the MVI for
providing Pfs25 expressed in baculovirus(Malar J 15:405, 2016).

Standard Membrane Feeding Assay

The ability of the an/body to block
Plasmodium falciparum malaria
parasite transmission in themosquito gut was evaluated using
the “Standard Membrane FeedingAssay”, SMFA; (Miura et al.
Vaccine 34:4145,2016). An/body
4B7 is a posi/ve control.

Conjugates with EcoCRMTM Pfenex CRM induced comparable ani-Pfs25
anBbody response in mice and were equally eﬀecBve at killing malaria
parasites in a membrane feeding assay (SMFA).
Acknowledgments: Pfs25 conjugaBon was funded by the PATH Malaria Vaccine

IniBaBve. The pfs25 assays were performed by the SMFA reference center at the NIAID/NIH.
For more informaBon: www.FinaBio.com

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