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.