scarabgenomics/CRM197/Purified Protein, 20 mg/CRM197-20

Cross-Reactive Material 197 (CRM197) is a genetically non-toxic form of diphtheria toxin (DT). A single base change (glutamic acid to glycine at position 52) in CRM197 disables the ADP-ribosylation activity of the A chain attenuating toxicity^{1, 2}. Although CRM197 is non-toxic, it is immunologically indistinguishable from diphtheria toxin. CRM197 functions as a carrier for polysaccharides and haptens making them immunogenic in a number of conjugate vaccines.

Scarab Genomics’ CRM197 is a recombinant form expressed in Clean Genome^® E. coli, our proprietary platform.

SOURCEClean Genome^® E. coli expressed recombinant CRM197

PRODUCT MOLECULAR MASS: 58.4 kDa

Figure 1: Scarab Genomics’ CRM197 is exceptionally pure and consistent from lot to lot. Five replicates of each lot of Scarab’s CRM197 were run on a Bis-Tris 4-12% gradient SDS polyacrylamide gel and stained with GelCode™ Blue, then purity assessed via gel scan. The average purity for each lot was calculated and assigned as follows: Panel A 98.3% pure, Panel B 95.5% pure.

Figure 2: Scarab Genomics’ CRM197 is higher purity than other commercially available sources. Two micrograms of CRM197 from each source were run on a Bis-Tris 4-12% gradient SDS polyacrylamide gel and stained with GelCode™ Blue. Breakdown products (indicated as bands A and B), are virtually absent in the Scarab product. Smeared background in some lanes is due to protein degradation and carry-over of host proteins, both are minimal in the Scarab product. Lane 1 Size Marker, Lane 2 empty Lane 3 Scarab Genomics CRM197,SG1 Lane 4 Scarab Genomics CRM197, SG2, Lane 5 vendor F, Lane 6 vendor M, and Lane 7 vendor L.

Figure 3: LC MS peptide mapping of Scarab’s CRM197 confirms it is biosimilar to native CRM197. Amino acid sequencing and mass spectrometry (MS) on were performed on Scarab’s highly pure CRM197. Extensive bioinformatics analysis of MS material revealed no post-translational modifications. Trypsin and chymotrypsin digestion followed by nano-liquid chromatography-MS/MS identified very high protein coverage (~97-98%). Electrospray ionization mass spectrometry (ESI-MS) on Scarab CRM197 confirmed the expected peptide sequence. Neither differences to the reported amino acid composition nor side chain modifications were observed.

Figure 4: Scarab’s CRM197 conjugates as effectively. Not all lysines in CRM197 are exposed on the protein surface for potential conjugation. Employing Solulink’s ChromaLink™ Biotin conjugation assays (San Diego, CA), which utilize a NHS ester reaction scheme to conjugate Biotin to primary amines, we confirmed at least 10 sites on Scarab’s CRM197 are accessible for conjugation. This optically quantitative (absorbance at 354 nm) functional test confirms the lysines on Scarab’s CRM197 are accessible for conjugation with hapten, like native CRM197.

Quality Control

Purity:	≥95% CRM197 by SDS-PAGE
	A and B chain content ≤5% of total CRM197
Concentration:	4 mg/mL in 150mM NaCl, 25mM HEPES, pH7.4 via absorbance at 280 nm using an E0.1%=1.07 for a 1 mg/mL solution
Endotoxin:	≤25 EU/mg of protein by the kinetic turbidimetric LAL method (maximum sensitivity = 0.01 EU/mL)
Dimer:	≤5%

Grade: CRM197 is for Research Use Only, Not for use in humans or as a diagnostic agent.

Storage Conditions:

• CRM197 is a 0.2 μm-filtered frozen solution of 4 mg/mL CRM197, 150mM NaCl, 25mM HEPES, pH 7.4. Store at -70°C upon receipt.
• To minimize aggregation, thaw CRM197 in 37°C water bath for 1 hour.
• Repeated freeze-thaw cycles can degrade material. To minimize freeze-thaw cycles, aliquot CRM197 to match desired use.
• Handle product gently, DO NOT VORTEX.

Product ManualsScarab Genomics CRM197 Liquid Data SheetPosters

1. Economic QBD Production of the Conjugate Vaccine Carrier Protein, CRM197 by a Continuous Manufacturing Process Using Scarab Genomics’ Clean Genome® E. coli

1. Scarab Genomics Proprietary Platform for Continuous Manufacturing of Pharmaceutical Biologics Applied to CRM197

1. Giannini, G., Rappuoli, R., and Ratti, G. (1984) Nucleic Acids Research, 12 (10):4063-4069.
2. Mekada, E., and Uchida, T. (1985) Journal of Biological Chemistry, 260:12148-12153.

Scarab is providing you with this Material subject to the non-transferable right to use the subject amount of the Material for your research at your academic institution. The Recipient agrees not to sell or otherwise transfer this Material, or anything derived or produced from the Material to a third party. NO RIGHTS ARE PROVIDED TO USE THE MATERIAL OR ANYTHING DERIVED OR PRODUCED FROM THE MATERIAL FOR COMMERCIAL PURPOSES. If the Recipient makes any changes to the chromosome of the Material that results in an invention in breach of this limited license, then Scarab will have a worldwide, exclusive, royalty-free license to such invention whether patentable or not. If the Recipient is not willing to accept the terms of this limited license, Scarab is willing to accept return of this product with a full refund, minus shipping and handling costs. For information on obtaining a license to this Material for purposes other than research, please contact Scarab’s Licensing Department. Scarab Genomics’ technology is covered by the following Patent Applications: US, 15/122,891, PCT/US2016/25588 and their related foreign applications.Clean Genome^® is a registered trademark of Scarab Genomics, LLC.