MSQC3

Sigma-Aldrich

SILuMAB Stable-Isotope Labeled Universal Monoclonal Antibody Standard human

recombinant, expressed in CHO cells

NACRES:
NA.12

Quality Level

recombinant

expressed in CHO cells

assay

≥90% (SDS-PAGE)

packaging

vial of 100 μg (± 10% Lot-specific vial content given on certificate of analysis)

shipped in

wet ice

storage temp.

−20°C

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General description

SILuMab 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.

Application

SILuMAB 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 SequenceLocation
DTLMISRHeavy Chain (IgG1, IgG2, IgG3, IgG4)
FNWYVDGVEVHNAKHeavy Chain (IgG1)
VVSVLTVLHQDWLNGKHeavy Chain (IgG1, IgG3, IgG4)
NQVSLTCLVKHeavy Chain (IgG1, IgG2, IgG3, IgG4)
GFYPSDIAVEWESNGQPENNYKHeavy Chain (IgG1, IgG4)
AGVETTTPSKLight Chain (lambda)
YAASSYLSLTPEQWKLight 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.

Physical form

Supplied as a lyophilized powder containing phosphate buffered saline

Preparation Note

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

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.

Analysis Note

SILuMab Heavy Chain
EVQLVESGGGLVQPGGSLRLSCVASGFTLNNYDMHWVRQGIGKGLEWVSKI
GTAGDRYYAGSVKGRFTISRENAKDSLYLQMNSLRVGDAAVYYCARGAGRW
APLGAFDIWGQGTMVTVSS|ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF
PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
HKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR
TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV
VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL
YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

SILuMab Light Chain
QSALTQPRSVSGSPGQSVTISCTGTSSDIGGYNFVSWYQQHPGKAPKLMIY
DATKRPSGVPDRFSGSKSGNTASLTISGLQAEDEADYYCCSYAGDYTPGV
VFGGGTKLTVL|GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTV
AWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQ
VTHEGSTVEKTVAPTECS

Target overlap areas are underlined
Package size based on protein content determined by A280 using an extinction coefficient (E0.1%) of 1.4
Qualitative
Intact heavy and light chains (
FASTA file)

Quantitative
MRM settings provided (
Skyline, xls)

Legal Information

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.com.
SILu is a trademark of Sigma-Aldrich Co. LLC

RIDADR

NONH for all modes of transport

WGK Germany

WGK 3

Flash Point(F)

Not applicable

Flash Point(C)

Not applicable

Certificate of Analysis
Certificate of Origin
Qian Zhang et al.
Analytical chemistry, 86(17), 8776-8784 (2014-07-11)
Quantitation of therapeutic monoclonal antibodies (mAb) using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for pharmacokinetic (PK) studies is becoming an essential complement to traditional antibody-based ligand binding assays (LBA). Here we show an automated method to perform LC-MS/MS-based quantitation, with IgG1...
Sandor Schokker et al.
mAbs, 12(1), 1795492-1795492 (2020-08-04)
Given the increasing use of combination therapy with multiple monoclonal antibodies (mAbs), there is a clinical need for multiplexing assays. For the frequently co-administered anti-human epidermal growth factor receptor 2 (HER2) mAbs trastuzumab and pertuzumab, we developed a high-throughput and...
Automated Antibody De Novo Sequencing and Its Utility in Biopharmaceutical Discovery.
Sen K I, et al.
Journal of the American Society For Mass Spectrometry, 28(5), 803-810 (2017)
Carlo O Martins et al.
The Journal of molecular diagnostics : JMD, 22(7), 901-911 (2020-04-18)
Multiple myeloma is a systemic malignancy of monoclonal plasma cells that accounts for 10% of hematologic cancers. With development of highly effective therapies for multiple myeloma, minimal residual disease (MRD) assessment has emerged as an important end point for management...
Liyun Zhang et al.
Bioanalysis, 10(13), 1039-1054 (2018-06-29)
The requirements for developing antibody biotherapeutics benefit from understanding the nature and relevant aspects of the entire molecule. The method presented herein employs on-line multidimensional LC-quadrupole time-of-flight (QTOF)-MS for the quantitative determination of an antibody isolated from biological samples while...

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