The Importance of HCP Monitoring

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In biologics production, biomanufacturers need to demonstrate clearance of impurities and contaminants during the manufacturing process. Host cell impurities (HCPs) are a major class of process-related impurities (1) that must be monitored, and their presence must be documented throughout the development and manufacturing process of a biologic.

“The presence of HCPs beyond a permissible regulatory limit can potentially result in adverse immune response, toxicity, possible biological activity, [and] hence, interfere with the potency and stability of the drug as well as cause disruption during the purification process,” states Anis H. Khimani, PhD, senior strategy leader, Life Sciences, Revvity, a Waltham, Mass.-based provider of health science solutions, technologies, expertise, and services. “It is therefore important to monitor and control the presence of HCPs in large-molecule drug formulations all along upstream through downstream processes.”

Olaf Stamm, PhD, technical business development director, Biopharmaceutical Testing Solutions, Charles River Laboratories, highlights several reasons why monitoring the presence of HCPs during biologic drug manufacture is critical:

• Safety: HCPs can cause adverse immune reactions in patients, even in small amounts. These proteins can trigger allergic responses or other immune-mediated effects, which can compromise patient safety.
• Efficacy: HCPs can interfere with the biological activity of the drug, such as by degrading the drug product, altering its function, or inhibiting its intended biological activity, thus reducing the biologic’s efficacy.
• Product stability: HCPs can affect the stability of the biologic by, for example, inducing aggregation, oxidation, or other forms of degradation. These effects can reduce product shelf life and affect product quality over time.
• Consistency and quality control: Consistent monitoring of HCPs ensures consistency from batch-to-batch in the manufacturing process. Variability in HCP levels can signal an issue in the production process, such as changes in cell-culture conditions.
• Regulatory compliance: Regulatory agencies such as FDA and the European Medicines Agency have stringent guidelines for HCPs in biologic products. Therefore, manufacturers must demonstrate that HCP levels are within acceptable limits in their product to gain approval for that product’s purification inefficiencies, which then needs to be addressed to maintain product quality.

“Overall, monitoring HCPs is essential to ensure that biologic drugs are safe, effective, and of high quality, meeting both regulatory standards and patient needs,” Stamm says.

Effective HCP clearance

Effective HCP clearance starts in the upstream processing stage, where optimization of cell-culture conditions with respect to optimal viability helps to reduce the load of HCP in the harvest material, notes Stamm. He explains that this approach works mainly for expression systems (e.g., Chinese hamster ovary [CHO] cells), which secrete their product. In the case of critical HCPs that might co-purify with the product protein, however, some production cell lines have been genetically engineered by knock out or silencing of such particular HCPs, he points out.

Khimani adds that, “Although most monoclonal antibodies and Fc-fusion proteins are expressed extracellularly, particularly from CHO cells, cell lysis and cell debris result in HCP contamination within the cellular milieu.” Therefore, various factors during cell growth and protein expression influence the level of HCPs, such as media composition, culture feeding, bioreactor controls, length of incubation, and even cell viability, all of which impact the profiles of HCPs as well as their detection and clearance.

In the downstream process, several tools can be implemented to manage the elimination or minimization of HCPs, Khimani says, while Stamm points out that the power horses for HCP removal are capture/affinity chromatography steps such as Protein A/G, which typically result in >95% of HCP removal; although, this clearance level is applicable for antibodies only.

“Additional [downstream processing] steps, such as hydrophobic interaction chromatography, ion exchange chromatography, size exclusion and mixed-mode chromatography, aid in the removal of HCPs,” Stamm adds.

Khimani emphasizes that, “optimization of each step is critical to achieve a robust workflow resulting in efficient removal of HCPs. To monitor the levels of contaminating proteins, it is critical to utilize analytical tools that offer sensitive and reproducible detection.”

Analytical detection

HCP identification and profiling is a designated critical quality attribute (CQA), Khimani points out. Therefore, covering this CQA is key to the development and production of biological drugs. Khimani explains that HCP detection has evolved from traditional techniques to next-generation technologies that offer greater sensitivity, reproducibility, and higher throughput.

“Immunoassays have been considered the gold-standard method for monitoring HCPs along upstream and downstream workflows of large-molecule drug production. ELISA [enzyme-linked immunosorbent assay] has been one of the most commonly used assays to identify and measure total HCP in large-molecule drug preparations; however, it is unable to provide a uniform comparison across various types of drugs where the HCP content from each cell type may differ and their related immune specificity may be undefined. Therefore, ELISA is limited by providing a coverage analysis of HCPs,” says Khimani.

On the other hand, detection using two-dimensional gels resolves proteins by size and charge followed by transfer onto membranes and subsequent Western blot detection. However, this method is challenged by the gel size, protein population complexity, and lower throughput, Khimani states.

Stamm says that the advantages of ELISA are high sensitivity, ability to handle multiple samples simultaneously, quantitative results, unexpensive equipment, ease of use, and good manufacturing practice (GMP) compliance. However, he also points out its disadvantages, the main one being that the performance of ELISA rests entirely on the quality of the antibodies generated for this assay. “The challenge is to generate polyclonal antibodies against several hundred to thousands of proteins which vary in size, immunogenicity, and physio-chemical properties. As these are critical reagents, those antibodies need to be thoroughly characterized before they can be used in the ELISA development. The entire process from the antigen generation to the immunization and qualification need to get thoroughly documented as such information is reviewed by the authorities and might be required a couple of years later to replenish antibodies of equal quality,” Stamm explains.

Khimani, meanwhile, points to mass spectrometry (MS) as an alternate or orthogonal technique that is more powerful in profiling HCPs because it has greater sensitivity and specificity. However, MS is limited by throughput due to complexity, requirements, and higher cost, he adds. Ultimately, the choice of methods to detect HCPs depends upon the complexity of the host cell lines’ protein profiles, regulatory requirements to monitor HCP levels as a CQA, and risk management strategies during large-molecule drug development, he states.

“Each technique varies in its sensitivity, specificity, and suitability for different stages of the purification process,” Stamm adds. He also states that the advantages of techniques such as liquid chromatography with tandem mass spectrometry (LC–MS/MS) is a high specificity and ability to identify individual HCPs in complex mixtures, such as bulk harvest, as well as in drug substance. The disadvantages of LC–MS/MS lay in the fact that it requires sophisticated equipment along with deep biochemical and bioinformatics expertise.

Improving HCP monitoring

Improving the monitoring of HCPs during the manufacturing process involves a combination of immunoassays and MS, says Stamm. Combining these techniques also enables HCP clearance in downstream processing. During early process development, generic ELISA kits are typically used for the quantitation of the HCP load in the cell harvest, process intermediates, and drug substance; however, instead of trusting such ELISA results blindly, Stamm emphasizes, the use of MS (e.g., shotgun LC–MS/MS approach) on the same samples will provide unbiased quantitative results as well as help identify potential critical HCPs that may be co-purifying (i.e., hitchhiking) with the product protein of interest.

Meanwhile, Khimani states that “a well-designed set of best practices that align with the type of cell line in use, the type of drug product in development with defined CQAs, the expected or previously defined profile of host proteins that may impact the drug preparation, and defined parameters for an optimal process should be implemented for greater efficacy and safety.”

Reference

1. Pilely, K.; Johansen, M.R.; Lund, R.R.; et al. Monitoring Process-Related Impurities in Biologics—Host Cell Protein Analysis. Anal Bioanal Chem. 2022, 414 (2), 747–758. DOI: 10.1007/s00216-021-03648-2

About the author

Feliza Mirasol is the science editor for Pharmaceutical Technology®.

Article details

Pharmaceutical Technology®
Vol. 48, No. 7
July 2024
Pages: 32–33

Citation

When referring to this article, please cite it has Mirasol, F. The Importance of HCP Monitoring. Pharmaceutical Technology 2024, 48 (7), 32–33.