Fig 2: Confirmation of luspatercept presence in serum samples. (a) Analysis by SDS‐PAGE and double‐blot (anti‐ActRIIB Sino Biological Inc. 10229‐T20). (b) Analysis by SAR‐PAGE and double‐blot (anti‐ActRIIB Sino Biological Inc. 10229‐T16). (c) Analysis by IEF‐PAGE (2‐10) and single‐blot (biotinylated anti‐ActRIIB R&D systems BAF339). A signal corresponding to the reference luspatercept was present in all samples evaluated post‐administration (D49 to D70) and absent before administration (C1). Positive QC (serum spiked with luspatercept) and negative QC (blank serum) were prepared with the samples and included in the analysis

Fig 3: In vitro study of BM ACE‐031 using human and rat liver microsomes. SDS‐PAGE and Western blot with biotinylated clone 60408 anti‐ACVR2B antibody and streptavidin‐POD.

Fig 4: Results of the inter‐laboratory myostatin inhibitors' study. Of the eight blind serum samples, the one containing black market ACE‐031 (Sample B) was correctly identified. IP with polyclonal ACVR2B antibody coated magnetic beads. Immunoblot after SDS‐PAGE with clone 60408 anti‐ACVR2B antibody. The red arrow indicates the monomer, and the green indicates the dimer.

Fig 5: (A) Amino acid sequence of human activin receptor type‐2B (1–18: signal peptide (green), 19–137/138–158/159–512: extracellular/transmembrane/cytoplasmatic domains; see numbered amino acids in the sequence). [23] Sequences identified by mass spectrometry are marked in red (underlined: spectra shown in Figure 8). (B) IdeS protease digest of several BM ACE‐031 products on SDS‐PAGE with Coomassie stain. Compared with the ACVR2B‐Fc fusion protein luspatercept (Reblozyl), no cleavage was observed for the BM products. ACE‐031 (red) was adjusted to match the amount of Reblozyl (0.5–1.0 μg; blue). Without/with IdeS protease: −/+.