Conclusions
Residual HCP control and their risk assessment has been an industry-wide challenge for biotechnology companies. This is mostly due to the complexity and heterogeneity of HCP makeup in the upstream and downstream process and the relative low abundance of HCP in final drug substance. The latter makes analyzing residual HCPs like finding needles in a haystack and thus requires highly sensitive and specific detection methods that can detect and distinguish HCPs from a dominant matrix of therapeutic proteins. A sandwich ELISA utilizing polyclonal antibodies that can recognize and capture residual HCPs is often used as the workhorse for residual HCP measurement. However, since the ELISA relies on the antibodies to detect and quantify residual HCP amount in samples, the ability of such antibodies to detect potential HCPs that can reside in the process intermediates and final drug substance need to be demonstrated and the method validated for its precision, accuracy, linearity, range, LOQ and robustness (Gunawan et al., 2018). In bioprocess development, a generic or platform HCP ELISA that is commercially available or developed in-house is often used in the early phase development up to the stage of process validation with appropriate assay qualification to gain insight on the HCP clearance trend and batch consistency. From phase III and beyond, either a platform assay or upstream process specific assay is preferred to mitigate the risk of inadequate coverage of HCPs specific to the manufacturing process by a generic HCP ELISA (USP Monograph Chapter 1132). However, even with a well-validated process specific assay, chances are that not all residual proteins are quantified accurately given the difficulty to achieve 100% coverage and to find relevant standards to quantify residual HCPs in all process intermediates. More commonly, an upstream process mock HCP culture from a null cell line is used as the calibration standard for the ELISA assay. This often leads to quantitation error when certain HCPs are enriched during this process, especially when the amount of HCPs present is in excess of the antibodies available to capture and detect the HCPs (Zhu-Shimoni et al., 2014). As indicated in Figure 3, the use of different calibration standards can lead to significantly different measurement of protein values, despite being uncommon to see such a large extent of difference with well-qualified assay standard. Therefore, orthogonal methods are often needed to supplement the results obtained from ELISA testing to evaluate the overall risk of residual proteins while the ELISA method needs demonstrate its fitness for its intended purpose in early phase development and fully validated at late phase development to demonstrate its precision, accuracy, linearity, range, sensitivity, specificity, and robustness.
Unlike biologics, residual HCPs in small molecule APIs often have distinct biochemical properties and can be easily separated from API by SEC-HPLC or Tangential flow filtration (TFF). However, the use of TFF and column-based separation is not desired in small molecule process development. Process chemists tend to use phase cut and crystallization as the main means of isolation (Wells et al., 2012; Wells et al., 2016). In the case of MK-1454 discussed in this manuscript, an initial isolation of API using these traditional process chemistry techniques achieved high API yield and purity comparable to chemical synthesis. However, E. coli proteins along with the enzymes added to the process were not completely removed or polished, leaving a large pool of proteins present at trace levels in the Prep. Lab batch API. The amount of residual E. coli proteins were estimated using the commercialE. coli HCP kit while the enzymes used in the reaction are not reactive to the kit antibody (Figure 4b and 4c). Although efforts have been made and some success has been achieved to use the total input level of proteins (reactive or not) to estimate the residual amount of proteins in the API, quantification by this approach have the risk of over-estimating E. coli proteins if contamination occurs during the process. To overcome these challenges, 1D SDS-PAGE gel with silver stain and LC-MS was used to estimate the total protein amount in API and assess the risks associated with those proteins by their relative abundance level and in silico predicted immunogenicity. Although the proteins present in the Prep. Lab. batch API are not considered to pose significant immunogenicity risk, these materials haven’t been assessed for immunotoxicity in animal studies or clinical trials. Instead, the chemically synthesized API was used in early clinical study and the biocatalytic route is developed for commercial chemistry. To minimize the potential immunogenicity risk and allow a direct use of biocatalytic route synthesized API in clinical trials, further reduction of residual proteins is achieved by process optimization. The new workup process has barely any detectable level of proteins as analyzed by ELISA, 1D SDS-PAGE with silver stain and proteomic LC-MS/MS. This case study demonstrates the importance of a holistic analytical control strategy in HCP characterization for biocatalytic route synthesized API. This holistic analytical control strategy allows process chemistry to design new commercial manufacturing process to remove residual proteins (HCP and enzymes) to insignificant levels (<10 ng/mg) in three representative batches of API. With a robust process and holistic analytical characterization, a process-specific ELISA using antibody reagents developed for matching cell lysate used for MK-1454 biocatalysis may not be needed in late phase development weighing in the time and resources investment in developing such an assay, the API comparability and the low demand in MK-1454 quantity in commercial manufacturing. However, the holistic analytical characterization presented here, together with the API stability monitoring, will be essential to reduce patient safety and product quality risk associated with the presence of residual E. coli HCPs and enzymes.