Host Cell Protein Analysis in Biologics Manufacturing

Impurity control is a fundamental step in biologics manufacturing, crucial for safeguarding the safety, efficacy and consistency of therapeutic products. A critical component of this process is the removal of impurities originating from the host cells. These impurities – such as host cell DNA, lipids, protein aggregates and host cell proteins (HCPs) – can hamper drug purity, disrupt manufacturing consistency and, most importantly, compromise patient safety.

HCPs, endogenous proteins expressed by the production cell line, are vital for cellular processes like gene transcription, protein synthesis, cell growth, proliferation and survival. However, during fermentation, cell death and lysis can cause these proteins to be released into the product stream. Even trace levels of HCPs can have significant consequences. They may degrade the therapeutic products or their excipients, provoke unwanted immune responses in patients and ultimately hamper the quality, stability and shelf life of the final biologic product. Because of these risks, regulatory authorities require that HCPs be rigorously identified, quantified and controlled throughout the biomanufacturing process.

Tracking and measuring HCPs is a major challenge

Accurately detecting and quantifying HCPs is critical for supporting process development and mitigating risks. This requires analytical methods that are not only highly sensitive but also offer rapid turnaround times.

However, identifying and measuring HCPs is a major challenge. HCPs are typically present at very low concentrations (1 to 100 parts per million) and exhibit wide variability in molecular mass, isoelectric point, hydrophobicity and structural characteristics. Some possess protease activity, which can alter the protein composition in the culture supernatant and interfere with downstream purification. Furthermore, post-translational modifications can complicate analysis, making quantification and characterization difficult.

In an ideal scenario, a HCP assay should detect the majority of HCPs early in the production process, particularly those likely to persist through purification, as well as trace levels of residual protein in the final product. The assay should also be sensitive enough to identify changes in the HCP profile resulting from process failures, such as a leaking chromatography column. Similarly, if manufacturing processes are modified, the assay must be capable of detecting any newly introduced or altered HCPs.

How testing methods have evolved

HCPs represent a heterogeneous group of impurities, and as such, a range of analytical technologies and regulatory expectations have emerged to support their detection and characterization. Traditionally, the industry has used a threshold of 100 ng/mg of therapeutic protein as an acceptable limit for residual HCPs. However, the goal is always to minimize HCP levels as much as possible, since the most significant risks are often linked to specific individual proteins rather than total levels.

Importantly, the HCP profile can be influenced by several upstream factors, including cell culture duration, feeding strategies, culture temperature and process scale-up during commercial manufacturing. This variability highlights the importance of continuous and precise monitoring of HCPs throughout all stages of the production process.

Enzyme-linked immunosorbent assays (ELISAs) are widely used for HCP analysis due to their sensitivity, scalability and high throughput. However, ELISAs have certain limitations. They only provide a total concentration of HCPs without identifying the individual proteins present, and their antibody-based detection does not offer complete coverage.

Since the potential for immunogenicity or degradation of monoclonal antibodies often results from specific HCPs, regardless of the overall level, there is a growing need for alternative methods. As a result, mass spectrometry (MS) has emerged as a promising tool for HCP monitoring, enabling the identification and quantification of individual HCPs for more accurate risk assessment through an unbiased, protein-specific analysis.

“HCP analysis has significantly evolved over the past few decades, primarily driven by the change in regulatory expectations on the topic,” Dr. Anurag Rathore, professor in the Department of Chemical Engineering at the Indian Institute of Technology, Delhi, said. “From the time when just showing the HCPs were < 10 ppm using a generic ELISA, to now where a more detailed analysis is expected to be able to elucidate the amounts and identity of the most copious HCPs. Given the enormous diversity in HCPs that are present, there is room for innovation both in analytical tools and approaches,” he continued.

 “The most common method for detecting HCPs in biopharmaceuticals today is ELISAs, which remains the industry standard due to its high throughput and established regulatory acceptance,” said Dr. Jared Auclair, Dean, College of Professional Studies and Director, Bioinnovation at Northeastern University. “That said, over the last several years, MS has emerged as a powerful alternative due to its superior sensitivity and ability to identify the specific (problematic) HCPs. One must consider the trade-offs in sensitivity, specificity, throughput and cost when determining the appropriate tool. For example, ELISA will be faster and cheaper at the expense of sensitivity and specificity,” he added.

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MS offers a significant advantage in its ability to simultaneously detect and identify multiple protein analytes within a single sample, enabling rapid and high-throughput analysis. However, achieving absolute quantification across a broad range of proteins remains a technical challenge. Despite this, MS allows researchers to move beyond total HCP quantification, providing detailed insight into the identity and relative abundance of individual proteins, including low-abundance proteins. As a result, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become a valuable tool for the in-depth and efficient monitoring of HCPs.

What do regulators expect?

In recent years, regulatory agencies have placed increasing emphasis on the detection and control of process-related impurities, particularly HCPs. Key guidance documents from the US Pharmacopeia  and the European Pharmacopeia outline recommended strategies for monitoring HCPs, alongside guidance from the International Council for Harmonisation and regulatory bodies such as the US Food and Drug Administration and the European Medicines Agency.

These guidelines do not establish a fixed numerical limit for acceptable HCP levels, as the risk associated with HCPs is highly contextual. Factors such as dosage, route of administration, frequency of exposure, disease indication, patient population and the specific nature of the HCP impurity all contribute to the risk.

Regulatory agencies stress the importance of establishing a robust HCP assay early in process development. If the assay lacks sufficient coverage or fails to detect the dominant HCPs in the final product, this can lead to significant delays in later development stages.

To enhance detection, regulators recommend using orthogonal analytical techniques, such as LC-MS, electrophoresis, high-performance liquid chromatography and western blotting. These methods help validate HCP-ELISA results and support the characterization of profiles throughout the manufacturing process.

Best practices for HCP testing in biopharmaceutical development

As regulatory expectations increase and analytical technologies advance, biopharmaceutical companies are reassessing their strategies for HCP testing. Experts in the field emphasize the importance of more tailored and comprehensive approaches.

“A multi-method approach that combines immunoassays (i.e., ELISA) with orthogonal techniques (i.e., MS) to overcome the limitations of any single method is recommended,” Auclair said. “As important as instrumentation is to invest in, developing process- and product-specific methods using the appropriate reference standards that better reflect the actual HCP profile should also be explored. Lastly, stay current with regulatory guidance, as expectations for HCP characterization continue to evolve toward more comprehensive identification and risk assessment of individual proteins, rather than just total HCP content.”

Measurement via generic ELISA may not be enough for getting regulatory approval for your product,” Rathore added. “A deeper understanding of the HCPs that are present in the final product – both identify and quantity – is expected from industry. While large companies may have the expertise and means to do this, smaller companies are likely to need support from analytical CMOs,” he concluded.

A clearer path to safer biologics

As biologics become more sophisticated, the demand for precise impurity control, especially for HCPs continues to rise. Advances in analytical technologies, such as MS, are reshaping how manufacturers understand and manage impurity profiles. Rather than relying solely on total HCP counts, developers can identify and quantify individual proteins, track how they co-purify and evaluate their potential risk to product quality and patient safety. This shift represents a critical step forward in biological drug development.