Host cell proteins (HCPs) are low-level process-related impurities that must be adequately cleared from recombinant biopharmaceuticals during the downstream process to ensure product quality, purity, and patient safety. It is well known that some HCPs with very little abundance have a strong impact on product quality. One example is the degradation of the formulation component polysorbate accompanied by increased product aggregation. The detection of those HCPs is a prerequisite for developing a suitable HCP depletion process. Here we assess our research on methods for detection of low-abundant HCPs by MS/MS and enrichment by hexapeptides or antibodies (IAC).

Host cell proteins are process-related impurities, some of which can be problematic. For those that are immunogenically, enzymatically, or biologically active, they are high-risk and often impact patient safety and product stability. In this study, a case study is presented to demonstrate that such a host cell protein was identified and confirmed as the cause of product protein cleavage in one of the process aimed at high yield, and an effective mitigation strategy was implanted based on our finding.   

The ELISA is still the gold standard to monitor HCP reduction during drug purification and for product release testing. The performance of a HCP ELISA strongly depends on the respective polyclonal antibodies, which needs to match the HCP composition in terms of absolute abundance and affinity for each individual HCP. A sophisticated ELISA development strategy supported by orthogonal methods for reagent characterization is essential for robust and reproducible ELISA-based HCP monitoring.

Highly active hydrolytic enzymes at sub-ppm levels can negatively impact the shelf life of drug products but are challenging to identify by LC-MS/MS due to the high dynamic range between HCPs and biotherapeutic proteins. We have employed strategies that increased the sensitivity for HCP identification by 10- to 100-fold over previous reports and shown the robustness as low as 0.1 ppm for identifying HCPs (34.5 to 66.2 kDa MW). The method capability was further confirmed by identifying the largest number (746) of mouse proteins from NIST antibody reported to date by a single analysis.

Host cell proteins (HCPs) are impurities in the biological process development. HCP ELISA has been widely used in process development for detecting and quantifying HCPs owing to its high sensitivity and throughput. This presentation describes the implementation of a mass spectrometry-based HCP workflow in the process development of antibody-based biologics and demonstrates the strength and weakness of HCP analysis by mass spectrometry-based methods.

Characterization and quantification of biologics, is essential to ensure that critical quality attributes are achieved. It is valuable to utilize strong analytical methods and instrumentation to help researchers monitor biologic production and accurately characterize samples. PerkinElmer’s LabChip GXII Touch provides high throughput characterization of proteins and nucleic acids in as little as 42 seconds, delivering data comparable to other methods of quantitation with as much as 70x increase in throughput. 

Data from LC-MS analyses of host cell proteins focusing on quantitation aspects and application to recombinant proteins and AAV-based gene therapy will be presented. Additionally, combination with ribosomal footprint profiling (Ribo-seq) to improve the depth and quality of the database used for MS data searching will also be discussed. Using this approach, we identified microprotein-based HCPs present in commercial drug products and investigated their behaviour during cell culture.

The implementation of new MS-compatible standards for robust and accurate quantification of Host Cell Proteins (HCP) will be presented. The potentialities of Data Independent Acquisition approaches for HCP profiling will be highlighted and benchmarked against commonly used targeted and Data Dependent Acquisition methods. Finally, benefits of the addition of an ion mobility separation in the LC-MS/MS workflow will be assessed on various instrumental platforms including High-Field Asymmetric-Waveform Ion-Mobility Spectrometry (FAIMS) and Trapped Ion Mobility Spectrometry (TIMS). Results will illustrate how the methods can support bioprocess understanding and development and ultimately be applied for final drug product purity assessment.

Custom reagents that are host strain and process specific can help ensure good characterization and clearance of HCP impurities. However, some non-immunogenic proteins will not be covered by the anti-HCP antibodies. Initial detection and identification of these HCPs requires the use of LC-MS, but protein-specific immuno-based methods are sometimes needed to support process development. A case study will be presented detailing the development and application of a single-protein immunoassay method.

Process-related impurities such as HCPs are considered critical quality attributes. Removal of HCPs to the lowest possible level is required by regulatory guidance documents. For patient safety, the development and adaption of suitable HCP control strategies throughout the product life cycle are a measure to guarantee high-quality medicines on the market. The choice of the most appropriate HCP assay and the life cycle management of the critical reagents are important decision points during product development and for continuous product monitoring. The case studies discussed will provide guidance for licensing applications for the European and the US markets.

For each platform-specific ELISA, it is beneficial to show that the ELISA antibodies recognize the most abundant HCPs in the purified protein drug as well as in upstream samples. The combination of affinity enrichment along with 2D PAGE and mass spectrometry is useful to demonstrate good coverage as well as individual abundant HCPs understanding. Practical tips and lessons learned in the development of these methods will also be discussed.

In recent years we’ve learned that Host Cell Proteins (HCP) can cause problems with respect to patient safety, Drug Substance (DS) stability, and DS efficacy. It’s more important than ever to fully understand the HCPs in your process. This talk will focus on using advanced methods immunoaffinity chromatography and mass spectrometry to fully characterize the Host Cell Protein ELISA as well as the individual HCPs in in-process and DS samples.

Having an appropriate assay to detect HCP is critical in biopharmaceutical development, including the development of gene therapy products. To ensure accurate quantitation of HCP from your bioproduction process, process- or platform-specific HCP assays are preferred over generic assays. We describe the development of custom reagents for process-specific HCP assays, as well as the development of custom HCP assays on two different immunoassay platforms for product release and in-process support.

We developed a new method for improving HCP detection in biotherapeutics, which relies on hydrophilic interaction chromatography for HCP enrichment followed by in situ concentration and digestion prior to LC-MS. Our results indicate that HILIC is able to separate most HCPs from the therapeutic proteins tested, and also improve sensitivity for a population of HCPs which are difficult to observe when using other LC-MS approaches, such as the native digestion method.

Host cell proteins (HCPs), one of the major process-related impurities of biologics production, can affect product quality and patient safety. Our study focuses on a holistic approach based on fractionation, LC-MS and dendritic cell assay to investigate the immunogenic potential of HCPs contained in a CHO culture harvest. A varying activation of dendritic cells was observed depending on the HCPs present in fractions, indicating an HCP-specific immune response.

Antibody titre improvement has led to process intensification, which has challenged DSP with high cell densities and associated process-related impurities. Breakthrough studies and a novel confocal imaging technique have been applied to Millistak+ HC (cellulose) and SP (synthetic) depth filters to show differences in performance and foulant distribution at different feed viability and loading. Protein A resin lifetime studies show higher fouling, particularly DNA, when SP filters are used.

Residual HCP titer is a critical quality attribute of biotherapeutics – from mAbs to viral vectors for gene therapy and vaccines. Specific HCPs, however, resist removal by chromatography and persist through the purification pipeline. Responding to this challenge, we developed LigaGuard, an adsorbent designed to remove HCPs in flow-through mode: CHO HCP removal in mAb manufacturing, HEK293 HCP removal in viral vector manufacturing, and VERO HCP removal in vaccine manufacturing.

Enzymatic activity from residual host cell enzymes such as lipases and esterases plays a major role in polysorbate degradation. Their high activity, often at very low concentration, constitutes a major analytical challenge in the biopharmaceutical industry. In this study, we evaluated and optimized the activity-based protein profiling (ABPP) approach to identify active enzymes responsible for polysorbate degradation, which enables more meaningful polysorbate degradation investigations for biotherapeutic development.

Host cell proteins (HCPs) are a class of process-related impurities during the manufacturing of biologics. HCPs must be removed in biologics manufacturing to ensure final product purity, manufacturing robustness, and safety. HCP analysis is often performed using an enzyme-linked-immunosorbent-assay (ELISA) due to method sensitivity and ease of use. When using an ELISA kit for HCP monitoring best practices involve determining kit sensitivity (quantification and coverage), product dilutional linearity, and robustness.