Differentiation and quantification of endogenous and recombinant-methionyl human leptin in clinical plasma samples by immunocapture/mass spectrometry.
Therapeutic recombinant-methionyl human leptin (r-metHu-Leptin, Mr 16155) shares an identical amino acid sequence with endogenous leptin (endo-leptin, Mr 16024), with the addition of an N-terminal methionyl incorporated during recombinant expression in Escherichia coli. Current immunochemistry-based assays do not allow discrimination between the drug and endo-leptin because of cross reactivity. Using the immunoassay, the total plasma concentration measured in some clinical study subjects receiving r-metHu-Leptin can reach supra-physiological levels. To determine which leptin species contributes to the elevated concentrations detected in some subjects, a mass spectrometry-based method allowing discrimination and quantification of both leptin species was developed. Endo-leptin and r-metHu-Leptin were enriched from plasma matrix proteins by immuno capture, and subsequently injected onto a reversed phase analytical column coupled to an API 4000 Q-TRAP LC-MS/MS system. Multiple charge state ions and specific MRMs were monitored to provide unambiguous differentiation between endo-leptin and r-metHu-Leptin. A "top down" assay distinguishing the two forms of leptin was successfully developed and had a linear range from 15.63 to 1000 ng/ml, low limit of quantification of 15.63 ng/ml. The method was applied to selected clinical samples and revealed that the elevated leptin concentrations observed in some subjects reflected accumulation of r-metHu-Leptin. An LC-MS/MS method was developed for unambiguous differentiation of r-metHu-Leptin from endogenous leptin in human plasma. Using this method, specific quantitative information was obtained for pharmacokinetic studies in a clinical trial. The method should prove useful in quantifying this investigational drug against endo-leptin background in future clinical studies.
Amylin Pharmaceuticals, Inc., 9360 Towne Centre Drive, San Diego, CA 92121, USA. yan.wang@amylin.com
Yan
Y
Wang
Joseph S
JS
Heilig
D019440:Anti-Obesity Agents / Q000008:administration & dosage / Q000097:blood* / Q000493:pharmacokinetics; D002138:Calibration; D056148:Chromatography, Reverse-Phase; D004797:Enzyme-Linked Immunosorbent Assay / Q000592:standards; D006801:Humans; D020738:Leptin / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000097:blood / Q000493:pharmacokinetics; D057230:Limit of Detection; D016014:Linear Models; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000592:standards
D019440:Anti-Obesity Agents; D020738:Leptin; C122136:recombinant methionyl human leptin