ELISA measurement of residual E. coli HCPs in Prep. Lab
batch MK-1454 API.
A commercial E. coli HCP kit from Cygnus Technologies was used to
estimate the total amount of residual E. coli HCPs in MK-1454 early
process API samples (Prep. Lab. batch). Given that the reaction mixture
contains not only E. coli HCPs but also evolved recombinant
enzyme along with chaperone proteins and antibiotics resistance genes
co-expressed with the enzyme and that process-specific antibodies
targeting those proteins are not available, an attempt was made to use
an in-house produced E. coli cell lysate supernatant expressing
cGAS to represent the proteins used in the synthesis with the assumption
that all proteins used in the process are non-specifically purged with a
similar purging factor through the purification process. A standard
curve was plotted using the absorbance signals
(A450-650, y-axis) generated from this in-house standard
at different concentrations against its concentrations (ng/mL, x-axis)
to calibrate the amount of proteins remaining in the API. As shown in
Figure 2, the binding affinity as indicated by the EC50value (or C value) of the 4-parameter non-linear logistic fitting curve
with the in-house standard (EC50 = 5199) is much lower
than that with the Cygnus kit E. coli HCP standard (EC50= 170.5). When using the Cygnus kit standard to calibrate the
immune-equivalent amount of proteins included in the in-house standard,
the standard series have a % recovery ranging from 0.83% to 11.49% of
its nominal value (Table 1). Differences in binding affinity to the
anti-HCP antibodies in ELISA between the two standard series suggest the
composition differences between the kit standard and in-house standard
and that a portion of proteins included in the in-house standard may not
be reactive to the antibodies used in the kit. Not surprisingly, the
amount of proteins in Prep. Lab. MK-1454 API samples at different
concentrations measured by using each standard curve differs
significantly, with the numbers obtained using in-house standard being
~16-78 times higher than those obtained using the kit
standard (Figure 3a). When plotting the dilution curves of API (mg/mL,
blue) along with the kit standard (ng/ml, green) and in-house standard
(ng/ml, red) using the absorbance values generated by ELISA against its
concentrations, we noticed the differences between API response curve
and the two standard curves, indicating protein composition differences
among all three of them (Figure 3b). As shown in Figure 3c, the HCP to
API mass ratio (ng/mg) increases along with the dilution of API samples
from 50 mg/mL to 1.28 mg/mL, regardless of using which standards to
back-calculate residual HCP concentrations, then stayed relatively
consistent between 1.28 mg/mL to 0.082 mg/mL and increased again along
with the dilution of API samples from 0.082 to 0.013 mg/ml. This
dilutional non-linearity often indicates the presence of matrix
interference and/or co-purifying HCPs that have antigen excess to the
antibodies used in the assay (Zhu-Shimoni et al., 2014). It also shows
the complexity of protein measurement by immunoassay, where matrix
interference, specificity, sensitivity, sample linearity, precision and
accuracy of measurement must be carefully assessed to qualify and
validate the assay for its intended purpose(FDA, 1997; Guideline, 1999).
Given that multiple enzymes have been over-expressed and added to the
biocatalytic route for MK-1454 synthesis(John A. McIntosh1*, 2022), the
commercial kit, with its antibody raised against generic E. coliHCPs, is not expected to accurately quantify the recombinant enzymes,
the associated chaperones and co-expressed antibiotic resistant
proteins. The inability of kit antibody to detect cGAS was confirmed by
Western blot, where purified cGAS was loaded onto the SDS-PAGE gel and
blotted with anti-E. coli HCP antibody obtained from the
commercial kit (Figure 4a and 4b). The immunoreactivity of other enzymes
added to the process prior to the last reaction step was also tested by
the commercial ELISA kit and shown as the baseline (Figure 4c). No
concentration-dependent responses were observed for each of these three
purified enzymes, enzyme A (AK, adenylate kinase), B (GK, guanylate
kinase), and C (AcK, acetate kinase)), indicating the lack of antibody
coverage to those evolved enzymes (Figure 4c). Therefore, before a
process specific ELISA can be developed, orthogonal methods are required
to monitor the clearance of total residual proteins including those
evolved enzymes.