Analysis Note

SILuMAb K1 Heavy ChainEVQLVESGGGLVQPGGSLRLSCVASGFTLNNYDMHWVRQGIGKGLEWVSKIGTAGDRYYAGSVKGRFTISRENAKDSLYLQMNSLRVGDAAVYYCARGAGRWAPLGAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SILuMAb K1 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

SILu^™ MAB K1 Stable-Isotope Labeled Universal Monoclonal Antibody has been used to spike peptide samples to assess preparation reproducibility.

Features and Benefits

Universal Peptide Sequence LocationFNWYVDGVEVHNAKHeavy Chain (IgG1)VVSVLTVLHQDWLNGKHeavy Chain (IgG1, IgG3, IgG4)GFYPSDIAVEWESNGQPENNYKHeavy Chain (IgG1, IgG4)SGTASVVCLLNNFYPRLight Chain (kappa)VDNALQSGNSQESVTEQDSKLight Chain (kappa)DSTYSLSSTLTLSKLight Chain (kappa)SILu^™Mab has been validated as an internal standard for quantitation of relevant biotherapeutics in a complex biological matrix by MRM-based LC-MS/MS. •SILu^™Mab 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), [email protected].

Physical form

Supplied as a lyophilized powder containing phosphate buffered saline

Preparation Note

SILu^™Mab 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, SILu^™Mab 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 ¹³C﹤SUB>6﹤/SUB>, ¹⁵N﹤SUB>4﹤/SUB>-labeled Arginine and ¹³C﹤SUB>6﹤/SUB>, ¹⁵N﹤SUB>2﹤/SUB>-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.