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.