Analysis Note

SILuMab Heavy ChainEVQLVESGGGLVQPGGSLRLSCVASGFTLNNYDMHWVRQGIGKGLEWVSKIGTAGDRYYAGSVKGRFTISRENAKDSLYLQMNSLRVGDAAVYYCARGAGRWAPLGAFDIWGQGTMVTVSS|ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SILuMab Light ChainQSALTQPRSVSGSPGQSVTISCTGTSSDIGGYNFVSWYQQHPGKAPKLMIYDATKRPSGVPDRFSGSKSGNTASLTISGLQAEDEADYYCCSYAGDYTPGVVFGGGTKLTVL|GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSTarget overlap areas are underlined

Package size based on protein content determined by A₂₈₀ using an extinction coefficient (E^0.1%) of 1.4

QualitativeIntact heavy and light chains (FASTA file)QuantitativeMRM settings provided (Skyline, xls)

Application

SILu^™MAB Stable-Isotope Labeled Universal Monoclonal Antibody Standard human has been used in a comparative study of automated antibody de novo sequencing.

Features and Benefits

Universal Peptide Sequence LocationDTLMISR Heavy Chain (IgG1, IgG2, IgG3, IgG4)FNWYVDGVEVHNAK Heavy Chain (IgG1)VVSVLTVLHQDWLNGK Heavy Chain (IgG1, IgG3, IgG4)NQVSLTCLVK Heavy Chain (IgG1, IgG2, IgG3, IgG4)GFYPSDIAVEWESNGQPENNYK Heavy Chain (IgG1, IgG4)AGVETTTPSK Light Chain (lambda)YAASSYLSLTPEQWK Light Chain (lambda)

SILuMab 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.

General description

SILu^™Mab is a recombinant, stable isotope-labeled, human monoclonal antibody which incorporates [¹³C₆, ¹⁵N₄]-Arginine and [¹³C₆, ¹⁵N₂]-Lysine. Expressed in CHO cells, SILuMab is designed to be used as a universal internal standard for bioanalysis of monoclonal antibodies. Because of overlap with common sequences in the Fc region with candidate antibodies, SILuMab provides universal utility and thus eliminates the need to produce candidate-specific internal standards. SILuMab is an IgG1 antibody with lambda light chain, but contains peptide sequences common to other IgG isotypes.

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

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.