Figure 1. The initial (a) and improved (b) workflow for MK-1454 biocatalysis and API isolation to remove residual proteins.
Figure 2. Standard curves plotted using the ELISA signal generated from kit standard (green curve) and an in-house lysate supernatant standard (red curve) against their respective standard concentrations. The A, B, C, D values of the curve are listed on the right.
Figure 3. The dilutional non-linearity of prep-lab batch MK-1454 and the differences in HCP (ng/mg) measurement by using different standards. a., the ng/mL protein concentration (y-axix) measured by using an in-house standard (red curve) and Cygnus kit standard (green curve) at different concentrations of MK-1454 API (mg/mL, x-axis); b., The 4-parameter fit binding curve of plotted using the mean absorbance at 450 nm (y-axis) against Cygnus kit standard (ng/mL, green curve), in-house lyste standard (ng/mL, red curve), and MK-1454 API (mg/mL, blue curve) at various concentrations on x-axis.; c., the relative protein concentration (ng/mg, y-axis) measured by using an in-house standard (red curve) and Cygnus kit standard (green curve) respectively at different concentrations (mg/mL) of MK-1454 API.
Figure 4. SDS-PAGE comparing the protein profile between Cygnus kit standard and in-house lysate supernatant standard and their immunoreactivity to the kit antibody. a, SDS-PAGE gel with different concentrations of each standard and Coomassie blue staining. b, Western blot of a membrane transferred from a duplicate SDS-PAGE gel showing the immunoreactivity of Cygnus kit antibody to the proteins in each standard. c, Immunoreactivity detected by Cygnus kit ELISA on enzymes A, B, and C, which were used in the early steps of MK-1454 biocatalysis prior to the last reaction step. d, Immunoreactivity of proteins detected in Prep. Lab, API to Cygnus kit antibody on Western blot.