The success of biotherapeutics such as the current blockbuster drug Humira is encouraging for companies with biotherapeutics in development. One of the biggest challenges when developing a new biotherapeutic is to remove the unwanted proteins expressed by the host cell in which the biotherapeutic was originally expressed, known as host cell proteins (HCPs). “The biotherapeutic might hold on to a host cell protein, and it goes along for the ride in the purification process,” says John Gebler, director of biopharma business development at Waters. The presence of unwanted HCPs in biotherapeutics is dangerous because they can trigger strong immune responses in patients, or compromise the stability of the biotherapeutic.
Researchers mainly use two techniques to detect HCPs in a biotherapeutic sample—enzyme-linked immunosorbent assays (ELISAs), and mass spectrometry (MS). Here’s a look at the advantages of these technologies and examples of tools available today.
ELISAs for HCP detection
Compared to LC-MS, ELISAs are more affordable and don’t require technical expertise to run. ELISAs are quick, easy to run, and give a clear answer about the presence of HCPs. Cygnus Technologies, part of Maravai LifeSciences, has offered ELISA tools for HCP analysis since the late 1990s. Today they offer 26 HCP ELISA kits that comply with regulations for detecting HCP impurities in all stages of biotherapeutic production. “Regulatory agencies around the world have put measures in place to ensure the HCP ELISA is fit for monitoring purification process consistency as well as final drug substance purity,” says Alla Zilberman, director of technical marketing at Cygnus Technologies. “[Companies] must use orthogonal methods to demonstrate antibody coverage to individual HCPs and further qualify that HCP ELISA to support it is fit-for-purpose.”
Cygnus offers custom services for orthogonal methods, such as antibody coverage analysis by Antibody Affinity Extraction™ (AAE) followed by 2D PAGE or 2D-DIGE, AAE followed by mass spectrometry, and HCP identification in in-process samples and final drug substances by AAE-LC-MS. Companies often use Cygnus’s HCP ELISA kits in preclinical process development and phases 1 and 2 of clinical manufacturing, says Zilberman. Beginning in phase 3, companies need to decide whether to validate that kit, or develop an HCP ELISA specific for their process. Cygnus provides process-specific HCP antibody and assay development for those choosing the latter.
Cygnus’s customers develop various types of therapeutics. Some use CHO cell HCP ELISA kits to develop therapeutic monoclonal antibodies. Others use HCP ELISA kits for therapeutic recombinant proteins. Cygnus’s Vero HCP ELISA, as an example, is used by researchers working on cancer-fighting next-generation vaccines. “There is tremendous growth in efforts to develop novel cell and gene therapies that require clinical manufacturing of mostly AAV- and lentivirus-based viral vectors,” says Zilberman. “These manufacturing processes require rigorous analytics, including testing for HCP impurities.” Cygnus offers HCP ELISAs for cell lines commonly used for viral vectors, such as HEK293 and Sf9 cells.
A high-throughput twist on ELISAs
Chinese hamster ovary (CHO) cells are a common mammalian host cell type for producing recombinant protein therapeutics, but these too can leave behind HCPs. ForteBio’s Anti-CHO HCP Detection Kit for quantifying HCPs was designed to run on their Octet Platform, which conducts real-time, label-free, high-throughput protein analysis. The kit was developed with Cygnus Technologies and incorporates Cygnus’s 3G anti-CHO HCP antibody. “Customers mainly use our Anti-CHO HCP Detection kit for generic HCP analysis of bioprocess samples during downstream process development and manufacturing of biopharmaceuticals,” says Ivan Krylov, product manager for consumables at ForteBio.
Krylov notes that while LC-MS is appropriate for completely identifying and quantifying each HCP in a sample, it might be too low throughput, depending on particular situations. ForteBio’s Octet system cranks through antibody-based HCP identification with greater speed than manual ELISA methods. “If the objective is to screen a large number of samples and to quantify the total amount of HCPs, we recommend using ForteBio’s CHO kit with the Octet HTX system,” he says. “This platform allows for a completely hands-off, walk-away HCP analysis of 96 samples in one hour, which is three times faster than the manual, labor-intensive ELISA platform.”
Mass spectrometry for HCP detection
Despite the many advantages of ELISAs, one disadvantage is the time required to design one, including the creation of quality antibodies, which can take over a year. In addition, ELISAs can only test for the presence of known proteins. Mass spec, on the other hand, identifies any proteins that are present. Indeed, a benefit of MS is its ability to both identify and quantify proteins on a time scale that allows interaction with biotherapeutic process development. Because of this, biopharmaceutical companies can use MS to monitor which HCPs are persisting through a purification process, which allows them to adjust protocols to further reduce HCPs.
SCIEX offers high-performing LC-MS systems that include their SWATH Acquisition technology for unbiased detection of HCP peptides, and assays for HCP quantification on their QTRAP 6500+ System. The ability to identify HCPs quickly makes LC-MS particularly valuable in optimizing process development. In addition, “since different HCPs can have radically different physical properties, knowledge of their specific identity and relative abundance allows scientists to quickly and effectively optimize their preparative chromatography systems or other process steps to deal with these specific HCPs,” says Sean McCarthy, global technical marketing manager, biopharmaceuticals at SCIEX.
According to McCarthy, SCIEX’s customers use two main LC-MS workflows for HCP detection. One is the SWATH-based workflow on the TripleTOF 6600+ System for identification and quantification of host cell proteins, both in biotherapeutic process development, and viral vector-based gene therapies. Another is multiple reaction monitoring (MRM)-based quantitation workflows for the QTRAP 6500+ System for validated quantitation of specific HCPs. “Customers are investigating this as an orthogonal assay to their existing ELISAs,” says McCarthy. “Existing ELISAs would provide an overall picture of HCP burden while the MRM-based LC-MS assay would provide them highly accurate quantification on specific HCPs.”
Waters also offers MS tools for HCP identification, including the “high-end QTOF instruments with LC systems, such as our ACQUITY® UPLC® I-Class System on the front end, and our Xevo or SYNAPT QTOF mass spectrometers as a detector, along with the appropriate informatics tools that allow these systems to do protein identification,” according to Gebler. Waters recently purchased the robotics company Andrew Alliance with an aim to make MS more accessible for researchers without MS expertise. “This allows a scientist to take their current methods and program the system by simply recording pipetting steps,” says Gebler. “This allows scientists to take methods they already have and automate them without needing a programmer.”
Gebler notes that the actual operation of MS systems isn’t overly complicated. In adding the automation platform, Waters is trying to make it easier for researchers to do their own MS experiments, which in the past was viewed as too technically sophisticated for most scientists. “Five years ago, these experiments were not for the faint of heart, but that’s changing now,” says Gebler.
Beyond monoclonal antibodies, newer biotherapeutics are emerging on the market. “We’re seeing more fusion proteins, and we’re also beginning to see AAV complex proteins in gene therapy as a really hot area,” says Gebler. “These are much larger, as they're made up of recombinant proteins in a specific ratio.” Producing increasingly complex biotherapeutics will require researchers to take advantage of the best HCP detection tools available.