Analytical and Bioanalytical Operations, Research & Development, Bristol-Myers Squibb Co., Route 206 and Province Line Road, Princeton, NJ 08543, United States.
Analytical and Bioanalytical Operations, Research & Development, Bristol-Myers Squibb Co., Route 206 and Province Line Road, Princeton, NJ 08543, United States.
Anal Chim Acta. 2017 Aug 1;979:36-44. doi: 10.1016/j.aca.2017.04.028. Epub 2017 Apr 26.
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.
肌肉生长抑制素，又称生长分化因子 8（GDF-8），是一种在骨骼肌生长中起负调控作用的蛋白质。通过治疗剂抑制肌肉生长抑制素为当前未满足的医疗需求提供了机会。为了更好地了解药物结合以帮助药物开发，我们开发了一种混合 LC-MS/MS 方法，该方法可以区分测量来自同一 GDF 家族的肌肉生长抑制素和另一种蛋白质，GDF-11。尽管这两种蛋白质具有高度同源性，但 LC-MS/MS 测定法基于监测来自酶消化产生的独特替代肽提供了特异性。自动化样品制备平台，安捷伦 AssayMap Bravo，用于自动化免疫捕获。使用非竞争性与我们的研究药物结合并且对肌肉生长抑制素和 GDF-11 具有相似结合亲和力的捕获抗体。因此，捕获并测量了包括游离形式和药物结合形式在内的总肌肉生长抑制素和 GDF-11。还原和烷基化后，对富集的样品进行消化。监测了两个替代肽（用于肌肉生长抑制素的 IPAMVVDR 和用于 GDF-11 的 IPGMVVDR），并将肌肉生长抑制素的定量下限（LLOQ）设定为 1.0ng/mL，GDF-11 的定量下限（LLOQ）设定为 0.1ng/mL。IPAMVVDR 的回收率为 99.2%至 103.1%，IPGMVVDR 的回收率为 90.3%至 114.5%，证明了准确性。开发的混合测定法具有足够的灵敏度，准确性和特异性，可以区分高度结构相似的肌肉生长抑制素和 GDF-11。该分析方法已成功应用于大鼠毒理学研究，并被证明是在治疗剂存在下进行生物标志物测量的有力工具。
