While overall HCP levels are usually monitored by ELISA, MS-based approaches have been emerging as alternative providing qualitative and quantitative information. However, a major challenge for LC-MS-based methods is to deal with the wide dynamic range of DPs and the sensitivity required to detect trace-level HCPs. Reproducible MS-based analytical workflows coupling optimized and efficient sample preparations, the library-free data-independent acquisition (DIA) method, and stringent validation criteria will be discussed.  

A working stream consisting of 26 member companies initiated a collaboration among its members to align industry best practices and generated a generic risk assessment tool to manage HCP-related risks identified during biologics development from both an assay development and process development perspective. A sub team within the HCP working stream also compiled a list of “high-risk” HCPs from CHO-produced biologics through literature review and cross-company discussions. The presentation provides an update on those efforts within BPDG HCP Workstream.

HCPs are process related impurities of biopharmaceuticals and application of MS to identify and quantify them is becoming more routine. This presentation describes the analytical challenges that LC-MS based HCP analysis faces, the developments that have been made to cope with those challenges,  our efforts in development and optimization of HCP analysis, and its implementation in the bioprocess.

Historically, immunoassays have been relied upon to characterize residual host cell proteins (HCPs) in biopharmaceutical drug substances. However, in recent years, due to the biochemical diversity found in HCPs, mass spectrometry has gained increasing acceptance as a tool for characterizing HCP profiles. As a relatively time consuming technique, it is important to understand how mass spectrometry-based assays fit most effectively within the whole analytical strategy for bioprocess development.

Mass spectrometry (MS) has become an increasingly common approach for identification and quantitation of host cell proteins (HCPs) in biotherapeutic products. Based on feedback from industry stakeholders, USP has formed an expert panel comprised of subject matter experts to write a new general chapter on best practices for identification and quantitation of HCPs by MS. Here we present an update on the Expert Panel’s progress and the chapter contents.

CHO HCPs are processed-related impurities that are derived from the host cell expression system.  Throughout process development HCPs are evaluated using ELISA. ELISAs are labor intensive with multiple manual steps with a timeframe of 7 hours.  Ella is an automated “ELISA”-like technology that quantitates HCPs in 75 minutes.  Both the Ella versus ELISA were comparable in CHO HCP trends, which resulted in the development of a fully automated CHO HCP analysis laboratory.

Host cell proteins (HCPs) are process-related impurities that are monitored to ensure product purity, stability, efficacy, and safety. HCP levels were collected into a database from the analyses of hundreds of samples from the harvest and purification of biologics. Knowing the most abundant, problematic, and dynamic HCPs along with their physiochemical properties will help optimize upstream and downstream processes for a given protein product.

This study reports a powerful strategy to identify HCPs in antibody drug substance by applying ProteoMiner enrichment with optimized conditions followed by shotgun proteomic analysis. Using this strategy, we observed that the low abundance HCPs were enriched up to 1000-fold. When applying this methodology to the study of HCPs in NIST monoclonal antibody (NISTmAb), more than 500 HCPs were confidently identified.

A broadly reactive and well qualified HCP ELISA is critical for monitoring purification process consistency and final drug substance purity.  Here we present an overview of assay qualification steps and demonstrate how the orthogonal antibody affinity extraction and mass spectrometry methods assess ELISA Ab coverage to HCPs present in a given process and identify process-specific HCPs that may co-purify with a drug substance.

In exploring Takeda cell-therapy pipeline, the retro-viral vector DS is generated by collecting culture media supernatant producing cells derived from a specific mouse cell line. Since no appropriate ELISA HCP kit is available, LC/MS-based shotgun proteomics is applied for HCP identifications during early phase. This presentation describes the HCP profiling procedure in DS and DP.  It also includes a preliminary immunogenicity risk assessment based on in silico tool.

Host Cell Proteins (HCP) are process-related impurities that must be carefully considered during commercialization of biological products as they may impact safety and/or efficacy. Process changes in the upstream/downstream can potentially induce different HCP profiles. LC-MS based proteomics methods are an orthogonal approach to ELISA to identify/characterize individual HCP. LC-MS comparison of HCP process clearance and populations before and after process changes help ensure a robust commercial control strategy.

Host cell protein (HCP) impurities generated during production of therapeutic recombinant proteins are removed during downstream processing. Low levels of residual HCPs may reduce drug efficacy or pose safety risks to patients. Using a fast, robust 1D LC-MS/MS method for HCP identification, we can monitor HCPs present at low levels (0.4 - 50 ppm) in harvested cell culture fluid, in-process pools and representative drug substance. For specific HCPs identified, additional quantitation using high sensitivity (< 0.4 ppm) analytical methods support the implementation of end-to-end mitigations, reducing overall HCP burden and total HCPs present in purified pools.

Host cell protein and other process-related impurities levels are critical quality attributes in biotherapeutic manufacturing. Gyrolab® systems perform automated immunoassays within nanoliter-scale microfluidic structures in Compact Disk format, increasing throughput, the sample and reagent volumes dramatically as compared to plate-based ELISA. We present case studies showing titer and impurities analytics of mAb bioprocess samples, CHO cells, and adeno-associated viral vectors, purified from HEK 293 cells, using the 5-CD Gyrolab xPand system.

The control strategy to monitor residual HCP content in biotechnological products undergoes a steady evolution to ensure the best possible product quality and patient safety. With focus on HCPs derived from mammalian cells, this talk discusses different technologies for HCP detection considering the lifecycle status of the product (“doing the right thing”) and highlights relevant quality aspects for selected analytical methods (“doing the right thing right”).

PS80 degradation was observed in a monoclonal antibody (mAb) within 18 hours at 5 ºC with a low level of host cell proteins (HCPs) presented. This study focused on identification of the unique HCPs presented in this mAb that was responsible for the rapid degradation by applying a novel targeted HCP analysis method (Activity Based Protein Profiling (ABPP)). A twist of the ABPP method can facilitate the clearance of these HCPs.

Individual CHO host-cell protein (HCP) levels may persist in mAb process streams despite the apparent effectiveness of separation processes, especially Protein A chromatography, at removing them. Several potential mechanisms have been explored for such persistence, such as HCP-mAb binding. This presentation will present results of recent such investigations and discuss possible mitigation approaches.

A high-throughput and high sensitivity LC-MS based platform has been developed to monitor HCP clearance during process development. Two residual HCPs were identified as ‘difficult to remove’ during downstream purification process. Risk assessment was performed to understand the impact of the HCPs and to guide process development. An end-to-end approach from upstream to downstream was utilized to understand HCP removal capability to establish a robust process for biopharmaceutical manufacturing.