Detecting Host Cell Proteins in Biotherapeutics

When using cells to make a biotherapeutic, some of the cells’ proteins end up in the product. These host cell proteins (HCPs) must be removed. HCPs can be detected with various analytical methods, including ELISAs and liquid chromatography-mass spectrometry (LC-MS). Despite the selection of potential methods for detecting HCPs, it’s not easy.

“Depending on the analytical techniques employed, the challenges in detecting HCPs might be related to very low HCP concentration, poor HCP response, sample complexity, and adequate sample throughput,” says Catalin Doneanu, Principal Scientist, Scientific Operations, Biopharmaceutical Applications, Waters. “Any analytical method used for measuring HCPs faces a significant challenge due to the wide dynamic range of the protein concentration—five to six orders of magnitude—typically found in highly purified biopharmaceutical preparations.” He adds that “analyte interference can be challenging in the case of specific assays targeting a subset of HCPs.”

It’s important to remove HCPs from a bioproduct because they “are known to pose a safety risk in their potential to illicit an immunogenic response, and impact product efficacy and quality by causing degradation, or undesired alterations, to the product,” note a trio of experts at CPI—Vicky Smith, Principal Scientist, Higher Order Analytics; Nic Farrands, Scientist, Analytical; and Clare Trippet, Chief Technologist, Biologics.

HCPs in a biotherapeutic must be less than 100 nanograms per milligram. The methods for detecting and analyzing HCPs need to be effective and fit the overall flow of bioprocessing. “Given the heavy workloads in analytical labs, HCP assays must, despite their complexity, be efficient in addition to being highly sensitive, accurate, and reproducible, because HCPs must not only be identified, but also quantified,” says Lei Xiong, Biopharma Application Manager, Americas, at SCIEX.

Selecting a method

HCP ELISA has been the gold-standard method for process monitoring and release testing for HCPs. “ELISA is the one simple method that can measure nanogram per milliliter levels of hundreds of HCPs in the presence of milligram per milliliter levels of product protein,” says Alla Zilberman, Vice President, Technical Marketing, Cygnus Technologies. “Typically, a generic broadly reactive ELISA is used in early process development and clinical manufacturing; however, as a biotherapeutic candidate moves from early development phases to late clinical and commercial manufacturing, a process-specific ELISA method might need to be developed.” She adds, “This should be a data-driven decision.”

As noted, the best approach to HCPs depends on the stage of a therapeutic’s development. “In the early stage of process development, the HCP population to detect keeps changing,” says Joe Hirano, Program Manager, Imaging at Cytiva. “This requires an assay to detect a variety of HCP populations.” This could be a high-throughput ELISA kit.

When a process is fixed in late-stage development, the analysis is “more about the identity of HCPs,” Hirano says, and a bioprocessor needs to decide if it will “prepare a process-specific ELISA antibody or continue to use a generic kit or platform HCP assay.”

No matter what approach is taken with HCPs, more than one technique is needed. To detect all of the HCPs in a sample, bioprocessors use orthogonal methods, such as an ELISA and MS. Nonetheless, Hirano says, it’s “not easy to correlate those two approaches to have an entire view of HCPs.”

Other orthogonal methods include 2D Western blots and immunoaffinity chromatography, says Zilberman. Nonetheless, she points out that 2D Western blots “significantly underestimate the true antibody coverage to upstream HCPs” and do “not predict how that antibody will quantitatively react to the most important HCPs, which are those that co-purify with the drug substance.” An immunoaffinity chromatography method called Antibody Affinity Extraction can overcome those limitations, especially when combined with MS. Such a combination, Zilberman says, “can be extremely helpful in investigations of problematic HCPs, especially those HCPs that are present in the drug substance at concentrations below the LC-MS limit of detection.”

Areas being improved

The broad range of contaminants that can show up in biotherapeutics and the changes in these therapies requires ongoing improvements in detecting and analyzing HCPs. “There is always a need for greater sensitivity and more streamlined quantification, and such improvements are being introduced from a number of different angles,” says Xiong.

One of those angles in terms of sensitivity comes from advances in HCP-enrichment technologies, Xiong says. “One example is the improvement of protein digestion, in which a majority of the drug substance remains intact followed by selective digestion of the HCPs, which can then be separated.”

As Zilberman points out “using Antibody Affinity Extraction as a sample-preparation step to enrich HCPs and eliminate most of the drug substance significantly improves LC-MS sensitivity.” She adds that “combining AAE with MS helps identify HCPs that are quantified in the drug substance by the corresponding HCP ELISA.”

When only limited quantities of a biotherapeutic are available for analysis, such as in early development or personalized therapies, Xiong says, “micro-LC has advanced to the point where it now offers the dramatically improved sensitivity needed to detect very low-level HCPs even in small sample loading amount.” She adds, “It also offers a user-friendly workflow.”

When asked about the most interesting recent advances in HCP detection and analysis, the CPI experts replied that LC-MS and capillary electrophoresis-MS “can measure thousands of proteins simultaneously and quantify and identify these proteins.” These techniques, the CPI trio added, “can be applied to a wide range of gene-therapy products and biologics and work over a large dynamic range (10–100,000 ng/mg).”

Doneanu also points out the dynamic-range challenge. One way to address that involves a recent advance that “relies on using different sample-preparation protocols focused on removing a significant amount of the biopharmaceutical prior to the LC-MS assay,” he says.

In addition to new methods, better regulatory guidelines also exist. “The U.S., EU, and Japan have published HCP regulatory guidelines and more are coming,” Hirano says. “This helps analytical scientists to set up an HCP analysis strategy.”

An analytical strategy for HCPs is required throughout a biotherapeutic’s life cycle. Some analytical methods can be applied at many stages. For example, Zilberman says, “MS can play an important role in HCP analytics from investigational new drug through post-marketing, when evaluating the impacts of a process change, risk assessment, and characterizing reagent changes.”

In general, changes in the biopharmaceutical industry drive modifications in detecting, analyzing, and minimizing HCPs in products. Developers and manufacturers continually seek faster and more sensitive ways to analyze biotherapeutics for purity.