Analysis Note

SILuMAb K4 Heavy ChainEVQLVESGGGLVQPGGSLRLSCVASGFTLNNYDMHWVRQGIGKGLEWVSKIGTAGDRYYAGSVKGRFTISRENAKDSLYLQMNSLRVGDAAVYYCARGAGRWAPLGAFDIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGSILuMAb K4 Light ChainQSALTQPRSVSGSPGQSVTISCTGTSSDIGGYNFVSWYQQHPGKAPKLMIYDATKRPSGVPDRFSGSKSGNTASLTISGLQAEDEADYYCCSYAGDYTPGVVFGGGTKLTVLTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECTarget overlap areas are underlinedPackage size based on protein content determined by A₂₈₀ using an extinction coefficient (E^0.1%) of 1.4

QuantitativeMRM settings provided (xls)

Application

Multiplex Universal Peptide Quant Approach Using Microflow LC/MS/MS for Non-human Preclinical PK Study of Therapeutic Monoclonal Antibodies ( Webinar )Applying Best-in-Class HPLC Column Technologies to the Analysis of Proteins and Biotherapeutics Using SILuMab ( Webinar )

Features and Benefits

Universal Peptide Sequence LocationYGPPCPPCPAPEFLGGPSVFLFPPKPK Heavy Chain (IgG4 Stabilized)VVSVLTVLHQDWLNGK Heavy Chain (IgG1, IgG3, IgG4)GFYPSDIAVEWESNGQPENNYK Heavy Chain (IgG1, IgG4)TTPPVLDSDGSFFLYSR Heavy Chain (IgG4)SGTASVVCLLNNFYPR Light Chain (kappa)VDNALQSGNSQESVTEQDSK Light Chain (kappa)DSTYSLSSTLTLSK Light Chain (kappa)Underlined is a Serine-Proline substitution in the hinge region that is present in many human IgG4-based therapeutic monoclonal antibodiesSILuMab has been validated as an internal standard for quantitation of relevant biotherapeutics in a complex biological matrix by MRM-based LC-MS/MS. SILuMab yielded reproducible, linear curves from 0.1 µg/mL to 1000 µg/mL without enrichment or depletion. Good agreement was observed between multiple peptides derived from the same target. Label incorporation was determined to be >98% by mass spectrometry. Sequence coverage was confirmed by peptide mapping.

Legal Information

SILu is a trademark of Sigma-Aldrich Co. LLC

This product is licensed under U.S. Patent No. 7,396,688 and foreign counterparts from E. I. du Pont de Nemours and Company. The purchase of this product conveys to the buyer the nontransferable right to use the purchased amount of the product for research and development only, including services for a third party for consideration. The buyer cannot sell or otherwise transfer this product, its components or materials made using this product or its components to a third party. Information about licenses for excluded uses is available from: E. I. du Pont de Nemours and Company; Attn: Associate Director, Commercial Development; DuPont Experimental Station E268; 200 Powdermill Rd.; Wilmington, DE 19803; 1-877-881-9787 (voice), 1-302-695-1437 (fax), licensing@dupont.

Physical form

Supplied as a lyophilized powder containing phosphate buffered saline

Preparation Note

SILuMab design is optimized to be used as an internal standard for quantitation of monoclonal antibodies as well as Fc-fusion therapeutics. Because of overlap with the common sequences in the Fc region with candidate antibodies, SILuMab provides univer­sal utility, thus eliminating the need for production of candidate-specific internal standards.

Produced utilizing enriched media containing stable isotope labeled amino acids are ¹³C6, ¹⁵N4-labeled arginine and ¹³C6, ¹⁵N2-labeled lysine

Reconstitution

SILuMab recovery is maximized when 0.1% formic acid is used for reconstitution of the lyophilized product. Reconstitution with other solvents may reduce recovery. Do not freeze after reconstitution.Procedure Briefly centrifuge the vial at ~10,000 x g to collect the product at the bottom of the vial. Add 500 µL of purified water containing 0.1% formic acid to the vial. Mix the contents by gently inverting the vial a minimum of 5 times. Allow the vial to stand at room temperature for a minimum of 15 minutes and repeat mixing by inversion.