Whenever cells are used to produce biotherapeutics such as antibodies, process impurities known as host cell proteins (HCPs) find their way into downstream product flows. Some HCPs are benign. Others are harmful. But which are which? There’s one way to know for certain: detect and assess all HCPs. Once the harmful HCPs are identified, they can be removed by introducing the appropriate purification measures. Typically, these measures are called upon to remove the vast majority of HCPs.
Historically, most HCP monitoring served to affirm that bioprocesses remained stable, run after run. According to Eric Bishop, vice president of research and development, Cygnus Technologies, HCP clearance results just had to be consistent to be reassuring. Then, in the early 2000s, attitudes began to change. “We realized that some of the adverse events we were seeing in the clinic were actually due to HCPs,” Bishop recalls. “Regulatory agencies became much stricter about monitoring HCPs and making sure you had the right assay.”
Today, HCPs are considered a critical quality attribute (CQA). “HCPs need to be minimized and closely monitored,” notes Parul Angrish, PhD, director, biopharma/pharma market, Agilent Technologies. “HCP identification and quantification are needed for regulatory submissions.”
Building on the gold standard
“The HCP ELISA,” Bishop relates, “is the gold standard method for monitoring HCP clearance throughout your purification process and also as your lot release test.”
“The plate-based ELISA for HCP analysis can be used throughout the entire process. However, there are other promising future alternatives as well, such as real-time and label-free biosensor technologies,” says Fredrik Sundberg, global director of strategic customer relations, Cytiva. “For example, surface plasmon resonance detection holds a great promise as process analytical tool for rapid in-line monitoring of both drug substance and impurities.”
The polyclonal HCP ELISA is not an assay for measuring a single analyte. Instead, a single polyclonal assay is used to measure potentially thousands of HCPs. It’s really important that the selected assay be appropriate for monitoring the process, Bishop observes. In other words, the polyclonal antibodies must be broadly reactive to the HCPs that are found in the process and in the drug substance. Ensuring that this is the case can be very challenging because some HCPs are very immunogenic and elicit a strong immune response very easily, whereas other HCPs, such as housekeeping proteins and other highly conserved antigens, are weakly immunogenic. Once generated, the reagents are typically affinity purified to get rid of any nonspecific immunoglobulin G antibodies.
The next step is to demonstrate that the antibodies used in the ELISA are broadly reactive to the HCPs. It is necessary, Bishop points out, to ask, “How much of the total HCP that is in your starting material do your antibodies react to?” Answers to this question are found through “coverage analysis.”
A traditional coverage analysis technique involves running the harvest material—without the product—on a large-format 2D western blot and on a large-format 2D silver-stained gel, and comparing the spots on the two. The silver stain should contain all the HCPs that can be there, while the western blot should contain the subset of immunoreactive proteins. Differential in-blot gel electrophoresis (DIBE) is an alternative to the classical western blot.
Bishop adds, however, that the large-format 2D western blot presents “a lot of limitations,” including transfer inefficiencies and the need to denature the sample. The latter limitation occasions a concomitant loss of conformational epitopes.
In 2013, Cygnus devised an alternative technology—antibody affinity extraction (AAE). It is an immunoaffinity method in which HCP antibodies are immobilized on a chromatography support. “We pass the harvest material over that column so that all the antibody-antigen reactions are happening in a more natural environment,” Bishop explains. At the end, there are two fractions: the starting HCP from the clarified harvest, and the antibody elution fraction. Bishop indicates that the first fraction “represents all the HCPs that are in the process and could potentially find their way into your product,” and the second fraction represents “all of the HCPs that the antibodies specifically removed from the sample.”
Orthogonal analyses
As powerful as the traditional HCP ELISA and its elaborations are, “ELISA lacks the specificity and coverage to identify and quantify individual HCPs,” Angrish notes. To get around these limitations, it is necessary to use alternative HCP analysis technologies. One of them is liquid chromatography–mass spectrometry (LC-MS).
The main challenge for LC-MS analysis is the coelution of low-abundance HCP peptides along with high-abundance peptide drug products. Angrish says that detecting these “hitchhiker proteins” requires better separation of peptides and an LC-MS system that has a broad dynamic range.
“The benefit of physical methods, as opposed to immunological methods, is that you don’t need a specific antibody,” Sundberg observes. MS, for example, may be used to create impurity profiles that can inform purification efforts. However, MS may have issues of sensitivity and also interference from the matrix and the drug product itself.
LC-MS and other physical measures should serve as orthogonal assays, identifying and quantifying HCPs in a process step or even the final product, but they should not be used as replacements for ELISA. “The classical immunoassay is still the gold standard for HCPs,” Sundberg declares. “And it’s actually expected by the regulatory agencies.” He adds that ELISA has the sensitivity to quantify levels of protein in parts per million.
“With ELISA, we could see past the drug substance and get a reasonable idea of the HCP quantities, but we really had no idea of the HCP identities,” Bishop says. “But now with our AAE method, the chromatography column serves as an affinity column, selectively enriching HCP and depleting drug substance,” as well as setting the stage for MS analysis.
Unless AAE or some other sample preparation technique is used, MS can be pretty useless in the HCP analysis of drug substance samples. A typical sample might contain a high concentration of normal monoclonal antibody (100 mg/mL) and low concentrations of HCPs (ng/mL). So, it would be next to impossible to “see” those HCPs due to the drug substance saturating the detector.
Now that you know
Bishop asserts that it has become possible to determine the “wholesale proteins” that a drug substance contains. “We’re working toward making it so that companies can do realistic risk assessments based on that information,” he continues, “but right now, it’s still kind of challenging because not a lot of companies are sharing data.” At present, it’s usually unclear which HCPs or what HCP levels in a formulation may lead to different kinds of problems—problems such as toxicity, immunogenicity, and interference.
“If you could aggregate all of your information, you could use it to create a much better picture of which HCPs can cause problems and how they can cause problems,” Bishop suggests. “You could provide more meaningful risk assessments. We’re not there yet, but that’s kind of where we’re going.
“The work is to prove to yourself and to your regulators that the assay you’ve selected is doing the best job that you can. If you’re reporting low levels of HCP to them, they want you to make them feel comfortable that it’s because there are low levels of HCP and that you have an amazing purification process, and not just because you’re just using a poor detection method.”
Off-the-shelf and custom kits
Companies can develop their own HCP ELISA assays or contract out to have assays run for them. But firms using the most common expression platforms can purchase generic off-the-shelf kits.
Among these are assays that use the Ella automated platform to query HCPs from products manufactured in HEK and CHO lines. “We partnered with Cygnus, which uses its industry-leading gold-standard antibodies,” says Julia Hatler, PhD, senior director, Immunoassay Business Unit, Bio-Techne. “We’ve shown correlation data to the Cygnus plate-based ELISA.”
“A lot of regulatory agencies are totally fine with companies using generic kits early in clinical development—pre-IND through Phase II,” Bishop remarks. “But they really like to see a custom assay going into Phase III through post-marketing.”
Many companies in later stages develop their own process-specific kits by culturing the host cell line without it expressing the product, under the same conditions that would be used for product manufacture. This, then, is used to immunize animals to generate anti-HCP sera for use in the custom ELISA. The process often lasts a year or more and requires some expertise, so many companies avoid it as long as they can.
HCPs are not the only contaminants that biomanufacturers need to be concerned with. Host cell DNA is a critical testing parameter as well, says Mark White, PhD, associate director of biopharma product marketing, Bio-Rad Laboratories. He notes that Bio-Rad offers “multiple droplet digital PCR kits to quantify the amount of host cell DNA from common production cell lines.”
If HCPs are found
After identifying an HCP, a manufacturer may decide that the best course of action would be to minimize the amount of the HCP in the process or product, or to remove the HCP altogether. One mitigation strategy is to address processes such as lysis that damage cells and cause HCPs to accumulate.
“Find conditions in the process that keep the cells happy and intact,” advises Andreas Castan, PhD, strategic technology and business development leader, Cytiva. Once these conditions are defined, they can be maintained by controlling simple parameters such as temperature and pH, and by choosing the best harvest time. Castan adds that it may also be possible to make helpful downstream adjustments, provided one “knows the load that can be put on the chromatography step and how to collect the pool to get rid of HCPs.”
Ion-exchange chromatography resins can be used to remove HCPs during purification and polishing of biologics, notes Jean Luo, vice president and general manager, Purification and Pharma Analytics, Thermo Fisher Scientific, and different resins can be designed for different conditions—high capacity, high resolution, or salt tolerance, for example. Some HCPs can be removed at upstream and midstream steps by filtration and centrifugation as well, depending on their sizes. And once the purification and polishing processes have been carried out, it’s important to verify that the offending HCPs have been mitigated.
“A company should fully understand the HCPs that are in its products,” Luo insists. “It is better to identify and remove a problematic HCP from the drug substance before it goes into patients than it is to find out in the middle of clinical trials that you’re going to have adverse events.” She adds that companies may have opportunities to exceed regulatory expectations. Indeed, going above and beyond may be in companies’ best interests.