TM
EcoCRM -Economical Recombinant CRM

197

Significantly reducing the cost of conjugate vaccines
Natalia Oganesyan* , Sanjay Jain**, Andrew Lees**Fina Biosolutions LLC, Rockville, MD, USA, ** Novo Biosolutions Inc., Shrewsbury, MA, USA

AFFORDABLE CARRIER PROTEIN

EXPRESSIONSDS-PAGE and Western Blot

STABILITY

CRM197 , a genetically detoxified diphtheria toxin, iswidely used as a carrier protein in conjugate
vaccines. Highly effective conjugate vaccines against
Streptococcus pneumonaie, Haemophilus influenzaeb and Neisseria meningiditis have been made usingthis carrier protein. CRM197 has been expressed as asecreted protein in Corynebacterium diphtheriae but
effective CRM197 production requires precise controlof growth parameters and yields are typically low(<100 mg/L). Recently, CRM197 expressed in the
periplasm of Pseudomonas fluorescens has become
available, but it is not generally priced to be anaffordable alternative.
Fina BioSolutions has developed a highly efficient
Escherichia coli expression system for CRM197, alongwith a simple purification scheme. Previous methods
for CRM197 expression in E.coli have involved
refolding from insoluble inclusion bodies orsecretion into the periplasm. We have developed amethod to produce soluble CRM197 in the cytoplasmof E.coli and achieved expression yield of grams perliter in a fermenter. The production process
comprises of a simple highly efficient purification
method with minimal number of steps. EcoCRMTM ,economical CRM from E. coli, offers the promise ofsignificantly reducing the cost of conjugate vaccines.

EcoCRMTM is expressed in E. coli intracellularly as asoluble protein. High expression yield of correctly
folded* CRM197 is achieved by using an E. coli strain
developed by Fina BioSolutions, along with anoptimized coding sequence.

EcoCRMTM is expressed in E.coli as an intact single chainpolypeptide.

EcoCRMTM shows minimal “nicking” after storage at 4°C
for 60 days and no sign of degradation.

1 1+DTT

2 2+DTTMW

1

2

129 kDa
80 kDa60 kDa40 kDa20 kDa80 kDa60 kDaEcoCRMTM

40 kDaA

B

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

20 kDa10 kDa* Fina BioSolutions has developed an ELISA method toevaluate CRM197 functional integrity.

PURIFICATIONSOLUBILITY
EcoCRMTM can be concentrated to >30 mg/ml, in PBS,
without precipitation or oligomerization.

EcoCRMTM, expressed in E.coli, is purified using astraightforward and economical purification scheme tomore than 95% purity

3480 kDa

EcoCRMTM

60 kDa40 kDa30 kDa

1- CRM197 (Pseudomonas) 2- EcoCRMTM (E. coli)

30 kDa2129 kDa

30 kDa129 kDa

MW 1+DTT -DTT

20 kDaA: SDS-PAGE analysis
B:Western Blot analysis, detection withrabbit@CRM197 (AIC Biotech)
Additional stability studies are underway.

polyclonal

LONG TERM STORAGE
EcoCRMTM cancryoprotectant.
monomeric.

be stored at -80°C with a
The thawedprotein remains

5SUMMARY
129 kDa

EcoCRMTM is a new and affordable CRM197

A

80 kDa60 kDaEcoCRMTM

40 kDaSimple, low cost fermentation in wellaccepted E. coli strain
Straightforward purification process
High yields of soluble CRM197
Native sequenceNo nicking
Excellent solubility propertiesRESEARCH POSTER PRESENTATION DESIGN © 2015

www.PosterPresentations.com

B

A: SDS-PAGE analysis of concentrated EcoCRMTM
B: SEC-HPLC analysis of concentrated EcoCRMTM

30 kDa20 kDa10 kDaMASS SPEC ANALYSIS

SEC-HPLC analysis: overlay of EcoCRMTM samples before
and after freezing.
Lyophilization studies are underway.

ACKNOWLEDGMENTS
Frank Robb (U Maryland) for contributions to the1-pellet; 2-cleared lysate; 3-CRM197 after IEX1, 4EcoCRMTM after IEX2, 5µg, 5-EcoCRMTM after IEX2 +DTT

CRM197 native sequence: 58,412 DaEcoCRMTM:
58,413 Da

DNA work and helpful discussions.
*This work was supported by PATH.
CONTACT: Fina BioSolutions LLC
Andrew Lees alees@finabio.com
301 633-0464 www.FinaBio.com