LC–MS/MS multiplexed assay for the quantitation of a therapeutic protein BMS-986089 and the target protein Myostatin
Background: Therapeutic protein discovery study highlights the need for the development of quantitative bioanalytical methods for determining the levels of both the therapeutic protein and the target protein, as well. Results: For the quantitation of BMS-986089, both accuracy (99–103%) and precision (2.4–12%) were obtained for the analysis of the surrogate peptide (ITYGGNSPVQEFTVPGR), in addition to the accuracy (100–108%) and precision (0.7–18%) that were obtained for the analysis of the surrogate peptide (VVSVLTVLHQDWLNGK). For Myostatin, accuracy (94–103%) and precision (2.4–14.9%) were obtained for the analysis of the surrogate peptide (IPAMVVDR). Conclusion: The developed method was applied to the analysis of samples following dosing of BMS-986089 to mice. This method highlights the potential of LC–MS/MS-based methods to eventually assess in vivo drug–target engagement.
Myostatin, also known as GDF-8, is a secreted TGF-β superfamily protein [1,2]. It is well known as a negative regulator of muscle development [3]. Myostatin is produced primarily in skeletal muscle, circulates in the blood and acts on muscle tissue as an autocrine and paracrine growth factor to regulate its size. Myostatin signals through a receptor complex consisting of Activin receptor type-2B (ActRIIb) and Activin receptor type-1B (ACVR1B or ALK4) [4,5]. Mouse Myostatin is a 376 amino acid (AA) preprotein that consists of a 24 AA signal peptide, a 243 AA propeptide and a 109 AA mature protein. In vivo proteolytic processing of the Myostatin precursor protein generates a Myostatin propeptide and mature protein. Dimerization of the approximately 12 kDa mature Myostatin protein creates the bioactive form of Myostatin with total molecular weight approximately 25 kDa. Myostatin is highly conserved across species. At the AA sequence level, mature human, mouse, rat and cow Myostatin are 100% identical.
It is thought that binding of Myostatin to the soluble Activin receptor prevents it from interacting with cell-surface receptors and inhibits signaling [6]. Further research on Myostatin and its genetic disposition has shown that blocking the activity of Myostatin may have therapeutic benefits in treating muscle wasting diseases such as cachexia, sarcopenia and Duchenne muscular dystrophy [7,8]. The strategy employed is to introduce a therapeutic agent that blocks Myostatin from binding. As an example, a monoclonal antibody specific to Myostatin has been shown to increase muscle mass in mice [9], and similar results in monkeys were also demonstrated [10].
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