Minimizing Risk & Maximizing Precision Using HCP Detection Advances

Regulatory standards in biopharmaceutical manufacturing require rigorous host cell protein (HCP) detection to ensure product safety and efficacy. Customization, optimization, and balance are essential to meet these standards while minimizing risk. Since each biologic drug presents unique challenges, advanced detection technologies, real-time monitoring, and computational tools help maintain production efficiency and quality while reducing the risk of undetected impurities.

Improvements in mass spec for more comprehensive HCP profiling

A broad spectrum of HCPs can pose risks to the efficacy and overall quality of the final drug substance (DS). Cygnus Technologies’ Vice President of Technical Marketing, Alla Zilberman, explained that “many HCPs are benign, but some are immunogenic, may interact with the drug substance, can cause drug degradation, or reduce effective product dosage through direct action on the drug or its stability by interfering with the formulation buffer.”

To distinguish which HCPs may be problematic, a comprehensive characterization of proteins present at various points within the process development is necessary. Liquid chromatography-mass spectrometry (LC-MS) is often used in the initial assessment of HCPs, as it detects low-abundance proteins and provides an in-depth view of HCPs. However, there are limitations of using LC-MS that cause the assay to miss proteins, especially those in low-abundance.

Zilberman shared that “a major challenge for LC-MS-based methods for identification of HCPs in drug substances is that there can be a more than five orders of magnitude difference in the concentration between HCPs and the DS. To overcome this challenge, efforts have been made to optimize the sample preparation to improve the dynamic range, such as online or offline fractionation, removal of mAbs by affinity depletion or molecular weight cut-off, and HCP enrichment by native digestion method.”

Cygnus Technologies has pioneered the use of Antibody Affinity Extraction (AAE) for sample prep, which is highly effective at enriching HCPs and depleting the DS. The technique uses custom polyclonal antibodies to enrich HCPs and deplete the DS before LC-MS. This preparation dramatically increases the resolution of the HCP profile and allows for detection and quantification of very low-abundance proteins for an in-depth assessment of the HCP profile and a more comprehensive risk assessment.

In addition to sample preparation for improved MS profiling, the use of data-independent acquisition (DIA) is now widely available with several companies, including SCIEX, Bruker, and Thermo Fisher Scientific, offering advanced instrumentation and data analysis software. DIA detects more HCPs than data-dependent acquisition (DDA) because it captures data from all proteins in a sample, while DDA only selects the most abundant ones. The significant increase in data acquisition also results in challenges to data analysis, driving the need for specialized software and workflows that are constantly in development to improve on sensitivity and throughput.

Customizing ELISAs for deeper coverage of HCPs

Still the gold standard for routine HCP quantification is the enzyme-linked immunosorbent assay (ELISA), which quantifies total HCPs without identifying individual proteins. While there are many off-the-shelf kits to perform ELISA for an array of standard cell lines, Hartmut Pohl, Application Scientist from Enzo Life Sciences, explained that when testing for HCPs, “broad coverage is important. Many standard kits will only have a coverage of around 50% of input proteins. Our kits operate in the range of 75–80%, which is good, but that doesn’t mean it covers 80% of the proteins in any given sample. We work closely with our customers to analyze total protein coverage of their samples.”

Many standard processes are able to use off-the-shelf kits for HCP analysis throughout development, however, genetically modifying cell lines, changing process conditions, or using unestablished processes will require development of custom ELISA assays. Customization can involve tailored polyclonal antibody production, specially formulated reagents, high-quality plate coating, and tailored assay protocols.

Customized ELISAs for HCP detection are often developed in partnership with outside service providers. Many companies offer custom ELISA development, including Cygnus Technologies, GTP Bioways, Enzo Life Sciences, BioGenes, Rockland, and Charles River Laboratories. Choosing the right partner is important not only for the efficacy of the ELISA, but also for achieving regulatory compliance.

Providing proof of validation of the custom ELISA is an important aspect of the process due to regulatory oversight. Pohl shared that “we've seen a growing demand for specific certifications and deeper analyses of factors that were not previously required. Customers are also requesting more frequent audits of production sites, making the regulatory workflow and exchanges between manufacturers and customers more intensive than ever.”

Novel high-throughput and miniaturized HCP detection technologies

Process optimization in biopharmaceutical manufacturing requires testing many conditions—from cell lines and media formulations to purification steps—resulting in large numbers of samples that must be analyzed efficiently. Advances in automation, microfluidics, and real-time analytics now enable faster, scalable HCP analysis, reducing bottlenecks and accelerating decision-making in bioprocess development.

Moving away from ELISAs entirely, the Sartorius Octet platform offers a fluidics-free system, in a 96-well format, using biolayer interferometry (BLI) for analyte detection. The system’s biosensors are coated with anti-HCP antibodies that detect HCPs using the interference of light waves. Unlike ELISAs, the platform requires no incubation steps or reliance on fluorescent probes and is able to process 96 samples in as little as an hour. Additionally, the platform can be used with an anti-CHO HCP kit, which utilizes the Cygnus 3G Anti-CHO HCP antibody for a streamlined plug-and-play solution when CHO cells are used for bioprocessing.

Similar to ELISAs, but on a nano scale, the Gyrolab platform, from Gyros Protein Technologies, uses an innovative compact disc (CD) design, which runs miniaturized and automated immunoassays. The system’s CD design allows for the use of centrifugal force to move liquids, at nanoliter scale, in and out of microfluidic channels. Within the 96 chambers, immobilized beads are coated with anti-HCP antibodies and samples are added, incubated, and washed. Like ELISAs, the platform then uses a fluorescent detection agent to quantify total HCPs, yielding results in about an hour.

For in-depth analysis on a large scale, advances in LC-MS using the Evosep One platform have dramatically reduced run times from 1–2 hours, to just a few minutes. This advanced liquid chromatography system uses disposable trap columns, called Evotips, that allow for preformation of solvent gradients before entering the mass spectrometer. The disposable trap column eliminates carryover contamination, time-consuming real-time gradient formation, and system wear due to high-pressures. The novel technology is revolutionizing the high-throughput capabilities of LC-MS and even enhancing coverage of detected proteins.

The Evosep One platform can accelerate LC-MS runs 10x, but this means terabytes of data will need to be collected, stored, and analyzed. To solve the problem of MS data analysis throughput, Genedata has developed an automated workflow, named Expressionist, that handles all operations, from raw data management to generation of customized reports. The workflow, combined with the Evosep One and Bruker timsTOF instrumentation has enabled companies such as Novozymes to process 200 samples per day with data analysis reports generated within 24 hours.

Additional companies, such as SCIEX and Thermo Fisher Scientific, have also developed solutions for enhancing LC-MS data processing, making in-depth high-throughput HCP profiling possible for bioprocess optimization.

Final thoughts

As regulatory expectations become more stringent, manufacturers must integrate advanced detection technologies to ensure compliance and product safety. In addition, Paula Orens, Senior Global Market Development Manager, Biopharma at SCIEX, emphasized that “the challenge persists beyond detecting and identifying HCPs. Because mass spec detection capabilities continuously improve, process development must adapt to optimize purification steps while suiting the needs of stringent regulatory expectations.”