A multiplexed immunocapture liquid chromatography tandem mass spectrometry assay for the simultaneous measurement of myostatin and GDF-11 in rat serum using an automated sample preparation platform.

Myostatin, also known as growth differentiation factor 8 (GDF-8), is a protein acting as a negative regulator in skeletal muscle growth. Inhibition of myostatin by therapeutic agents provides opportunities for current unmet medical needs. In order to better understand drug engagement to aid the drug development, we have developed a hybrid LC-MS/MS method which can differentially measure myostatin and another protein from the same GDF family, GDF-11. Although the two proteins share high homology, the LC-MS/MS assay provided the specificity based on monitoring of unique surrogate peptide generated from enzymatic digestion. An automated sample preparation platform, Agilent AssayMap Bravo, was used for automated immunocapture. Capture antibody that is non-competing with our investigational drug and has similar binding affinity to both myostatin and GDF-11 was used. Therefore, total myostatin and GDF-11 including both free form and drug-bound form were captured and measured. The enriched sample was digested after reduction and alkylation. Two surrogate peptides (IPAMVVDR for myostatin and IPGMVVDR for GDF-11) were monitored and the lower limit of quantitation (LLOQ) was established at 1.0 ng/mL for myostatin and 0.1 ng/mL for GDF-11. The accuracy was demonstrated with recovery for IPAMVVDR between 99.2% and 103.1% and for IPGMVVDR between 90.3% and 114.5%. The developed hybrid assay exhibits sufficient sensitivity, accuracy and specificity to differentiate between the highly structurally similar myostatin and GDF-11. This analytical approach was successfully applied to a rat toxicology study, and was demonstrated to be a powerful tool for biomarker measurement in the present of a therapeutic agent.

Quantification of in vivo site-specific Asp isomerization and Asn deamidation of mAbs in animal serum using IP-LC-MS.

BACKGROUND: Isomerization of aspartic acid and deamidation of asparagine are two common amino acid modifications that are of particular concern if located within the complementarity-determining region of therapeutic antibodies. Questions arise as to the extent of modification occurring in circulation due to potential exposure of the therapeutic antibody to different pH regimes. RESULTS: To enable evaluation of site-specific isomerization and deamidation of human mAbs in vivo, immunoprecipitation (IP) has been combined with LC-MS providing selective enrichment, separation and detection of naive and modified forms of tryptic peptides comprising complementarity-determining region sequences. CONCLUSION: IP-LC-MS can be applied to simultaneously quantify in vivo drug concentrations and measure the extent of isomerization or deamidation in PK studies conducted during the drug discovery stage.