TM
Characterization of EcoCRM , an E. coli Expressed CRM197

Conjugate Vaccine Carrier Protein*#*Natalia Oganesyan*, John Van Druff , Frank Robb , Morgane Ollivault-Shiflett ,#*#Sarah Kronheim*, Raphael Simon and Andrew Lees
*Fina Biosolutions LLC, Rockville, MD. #University of Maryland Baltimore, MD

INTRODUCTION
CRM197, a genetically detoxified diphtheria toxin, iswidely used as a carrier protein in conjugate vaccines.
Effective conjugate vaccines against Streptococcus
pneumoniae, Haemophilus influenzae b and Neisseria
meningiditis have been made using the strain specificcapsule polysaccharide linked to this carrier protein. The
cost of CRM 197 production represents a significant
contribution to the cost of goods for these vaccines
however. Fina BioSolutions has improved the production
process by development of a highly efficient Escherichia
coli expression system for CRM197, along with a simple
purification scheme, whereby we have achieved expression
yields of grams per liter.
Here, we show that EcoCRMTM maintained equivalency
to “native” (expressed in Corynebacterium diphtheriae) or
Pf CRM197 (expressed in Pseudomonas fluorescens) forcomposition and sequence (amino acid analysis and peptidemapping), stability (differential scanning calorimetry),molecular weight (multi-angle light scattering, and mass
spectrometry), structure (circular dichroism, intrinsic
fluorescence). EcoCRMTM also maintained solubility at highconcentrations and over a broad pH range.
We alsoconfirmed the carrier function ofEcoCRMTM
using
conjugation to Salmonella LPS derived O-polysaccharide asa model system.

22+DTTMW

1Size Exclusion ChromatographyMulti Angle Light Scattering (SEC MALS)

Chromatogram overlay of EcoCRMTM
and Pf CRM197

Chromatogram overlay of EcoCRMTM , “native” CRM197
and Pf CRM197

By SEC HPLC, both Pf CRM and EcoCRMTM show
comparable separation patterns and <1% dimer.

Chromatograms of EcoCRMTM, native CRM and Pf CRM all overlay. Whilenative CRM exhibited dimerization due to sample age, Pf CRM andEcoCRMTM had virtually no aggregation. The MW calculated by MALS waswithin experimental range of theoretical MW of 58,408. MW of native CRM
was not calculated due to dimerization.

Solubility at high concentrations

High concentrations after modification

Chromatogram overlay of EcoCRMTM
samples concentrated at different pH

EcoCRMTM was prepared in buffers from pH 6 to pH 9,
without additives, and concentrated to 28 mg/ml using anAmicon Ultra device. Recovery after concentration was >95%(BCA assay) and the monomeric state of the protein wasconfirmed by SEC HPLC.

EcoCRMTM was labeled with a 30 fold molar excess of the NHS
maleimide reagent (GMBS) and concentrated to 30 mg/ml at
pH 6.8 using an Amicon Ultra spin device. Protein recovery
was >95% (BCA assay). Monomeric state of the modified
protein was confirmed by SEC HPLC.

EcoCRMTM : Stability at 4°C

EcoCRMTM: SDS-PAGE and WB
1 1+DTT

Size Exclusion Chromatography

MW2+DTT -DTT

MW+DTT

EcoCRMTM : Secondary structure
Circular dichroism spectra

Intrinsic FluorescenceProtein Thermal Denaturation
Differential Scanning Calorimetry

129 kDa
129 kDa
80 kDa60 kDa60 kDa40 kDa(Degree cm2 dmol−1)

80 kDaEcoCRMTM

30 kDa20 kDa40 kDaEcoCRMTM (Green)
Pf CRM (Blue)

30 kDa20 kDaB1- Pf CRM197 (Pseudomonas)
2- EcoCRMTM (E. coli)
A: SDS-PAGE, reduced vs non reduced, Coomassie Blue staining
B:Western Blot, detection with polyclonal rabbit@CRM197 (AIC Biotech)

EcoCRMTM : LC MS Peptide Mapping

AB

EcoCRMTM shows minimal “nicking” and degradation
after 60 days at 4°C

EcoCRMTM : Nuclease Activity
DNAfragment+ EcoCRMTM+ Control

1 µg 10 µg

1 µg 10 µg

The similarity of the far UV CD spectra
indicates similar
percentages of alpha-helix and beta-sheet of EcoCRMTM and PfCRM.
Tryptophan fluorescence (excitation at 295 nm) is diagnostic of theconformational state of the protein as the emission peaks areinfluenced by the microenvironment. The overlay of the the spectraof EcoCRMTM and Pf CRM are indicative of similar conformations
for each.

Conjugates Immunogenicity

Acknowledgement
This work was partially funded by PATH
www.PATH.org

The nuclease activity of EcoCRM TM was assayedby 30 min incubation with linearized DNA andelectrophoresis on 1% agarose. Tetanus toxin
heavy chain fragment (TTHC) expressed andpurified from E.coli was used as a negativecontrol. EcoCRM TM but not TTHC caused
degradation of the DNA fragment.

SUMMARY
EcoCRMTM!Purified from E. coli as single chain
monomeric protein!Remains monomeric at pH 6-9 even athigh concentrations!Excellent immunogenicity and carrier
function

APeptide maps of EcoCRMTM and “native” CRM were compared.
Following trypsin, chymotrypsin or Glu-C digestion, thepeptides were analyzed by nano LC/MS/MS. Over 95% coverage
was achieved. The peptide maps were identical except for theN-terminal fragment.
EcoCRMTM was found to have approximately 50% of N formyl
methionine as the starting amino acid.

EcoCRMTM and “native” CRM197 have similar Tm and ΔH
values within experimental error, Indicating that they have
similar thermal stability. This confirms that EcoCRMTM has
its full complement of disulfides.

BS. typhimurium core outer polysaccharide (COPS) )was covalently linked to EcoCRMTM via its KDO
group (A).
Groups of 10 female outbred CD-1 mice were immunized 3 times
with 2.5 µg of conjugated COPS at 28 day intervals. Serum wastaken 21 days after the final dose and assessed for anti- S.
typhimurium COPS IgG titers by ELISA (B).

Equivalence with CRM197
from other sources:!Amino acid sequences match except
for the presence of f-Met!Possesses intrinsic nuclease activity!Biophysical similarity: DSC, CD,intrinsic fluorescence An economical alternative 
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A

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