Modern medicine relies on our ability to genetically engineer cells and turn them into miniature protein production plants. The trouble is that, in addition to expressing the material that is desired by drug industry scientists, the cells also make their own polypeptides—host cell proteins (HCPs)—as they grow and multiply in culture.
Considerable efforts have been made to identify and quantify HCPs and
exclude them from finished biopharmaceuticals. And with good reason,
according to Sanjay Gupta, PhD, scientist, impurity analytics, Roche Diagnostics. He says that HCPs can cause a range of serious problems: “HCPs are inherent process-related impurities in biopharmaceuticals arising from the host organism in which they are produced. HCPs in biopharmaceuticals require close monitoring, as they can be biologically active—enzymatically active in deleterious ways or immunogenic—and can therefore have an impact on product quality and in turn on patient safety.”
Gupta cites phospholipase B-like 2 (PLBL2) as an example of an immunogenic HCP. “PLBL2 is co-purified with lebrikizumab,” he elaborates. “In clinical studies evaluating the immunogenicity of lebrikizumab, a measurable immune response to PLBL2 was observed in about 90% of participants.”
Other HCPs are a problem because of the impact they have on the biopharmaceutical formulation in which they are present. “Enzymatically active HCPs can affect the integrity of either product molecules or formulation excipients,” Gupta points out.
Excipients vulnerable to enzymatically active HCPs include polysorbates, which are commonly used surfactants in protein formulations. “Polysorbates can act as stabilizers and prevent protein aggregation, denaturation, and interfacial stresses,” Gupta continues. “But polysorbate hydrolysis can lead to undesired particle formation in parenteral formulations.” Unfortunately, HCPs that break down polysorbate can be harmful in low concentrations and very hard to detect with current methods.
“In general, there are two approaches to assessing HCP-related impurities,” Gupta relates. “The first approach relies on detecting the trace levels of HCPs in drug products via enzyme-linked immunosorbent assay (ELISA) technologies or mass spectrometry (MS). The second approach involves assays that measure the hydrolytic activities in samples and offer a more direct readout based on functional activity of the enzymes.
“However, these methods lack sufficient sensitivity to detect either trace HCPs or are inappropriate for high-throughput testing. These methods are often material and labor intensive, limiting their suitability for bioprocess development and optimization.”
Substrate substitution
Gupta and his colleagues at Roche have developed what they claim is a highly sensitive, fast, and high-throughput method to detect the presence of hydrolytic activity in process samples. The method combines a surrogate substrate and a well-established detection platform, one that enables electro-chemiluminescence-based visual detection. According to Gupta, the surrogate substrate was designed in-house at Roche. He adds that the detection platform was the Roche Elecsys cobas e 411, an instrument that can enable “fast and highly sensitive readout compatible with high-throughput testing.”
“Our electrochemiluminescence-based polysorbase activity (EPA) assay measures hydrolytic activity in biotherapeutics throughout the drug substance manufacturing process,” Gupta asserts. “The EPA substrate is made up of different building blocks that mimic the polysorbate substrate and act as a target for the enzymes. The substrate also incorporates various tags from the diagnostics toolbox to facilitate the highly sensitive and specific detection on the Elecsys cobas e 411 analyzer.
“Our newly developed EPA assay can detect enzymatic activity in biopharmaceuticals at different stages of biopharmaceutical purification and do so with high sensitivity, fast turnaround time, and less sample manipulation. Our recent work, which was published in the European Journal of Pharmaceutics and Biopharmaceutics,1 showed that our assay was suitable for measuring the enzymatic activity of various HCPs that were identified in antibody products and found to increase poylsorbate hydrolysis. High sensitivity and wide applicability of the EPA assay were confirmed by performing a head-to-head comparison between this method and an established liquid chromatography–mass spectrometry (LC-MS)–based assay for the quantification of free fatty acids.”
Gupta maintains that the EPA approach offers several additional benefits. For example, the EPA approach can eliminate the need to pretreat samples, provide a four- to five-hour turnaround time, and measure hydrolytic activity throughout the entire purification process. Gupta notes that Roche has patented the EPA method2 but currently has no plans to commercialize it. He confides, however, that Roche has been “experiencing a considerable, industry-wide interest in utilizing it.”
LC-MS vs. ELISAs
Alphalyse, a contract research organization, specializes in performing customized analyses for the advanced characterization of biopharmaceuticals. With respect to HCP analysis, the company favors LC-MS-based analyses. Indeed, the company indicates that it has used LC-MS on over 400 client projects. The company also reports that it performed the world’s first GMP-certified MS-based HCP analysis for product release testing for a Phase III trial. Finally, the company points out that its LC-MS methods are generic and can be applied immediately to new biologics.
According to Ejvind Mørtz, PhD, Alphalyse’s co-founder and COO, understanding HCPs is a critical part of process development, particularly in chemistry, manufacturing, and controls (CMC) management: “For CMC purposes, it is vital to develop an efficient and consistent purification process to remove problematic or abundant HCPs. Such a process is key if you aim to make comparably pure products when the manufacturing process is scaled up or moved to a new contract manufacturing organization. Also, such a process can help you continuously monitor HCPs in GMP batches of marketed biopharmaceuticals.”
Mørtz observes that in HCP analysis, more detail is always better. On this point, he adds that ELISAs may suffer from comparison with LC-MS-based techniques.
“An HCP ELISA measures a total sum of immunologically weighted impurities,” he explains. “The antibodies used in the ELISA detect the most immunogenic HCPs, potentially overlooking HCPs that are non-immunogenic. Less immunogenic HCPs will give a very low or no response in the ELISA at all. ELISA does not provide any information about individual HCPs.
“In contrast, LC-MS can identify and quantify each individual HCP, as well as the total sum of HCPs. This more detailed information about each protein impurity enables the process developers to design better upstream process parameters and a better purification process to remove specific unwanted impurities.”
Mørtz stresses that detailed HCP information is used by regulators to determine whether an “efficient and robust purification process is in place” and whether “purity and impurity profiles are comparable between batches or even across products.” He adds that if potentially problematic HCPs are present in the product, the “risks for the patient population can be evaluated and monitored.”
Detail-rich LC-MS-based analyses have already enhanced clinical development. Besides identifying individual HCPs that can cause adverse effects in patients, LC-MS-based analyses can quantify individual HCPs and enable risk assessments for patients.
Regulatory support for LC-MS-based analyses was underlined last May when the U.S. Pharmacopoeia proposed a new general chapter, specifically, General Chapter <1132.1> (Residual Host Cell Protein Measurement in Biopharmaceuticals by Mass Spectrometry).3 At the time, the U.S. Pharmacopoeia announced that its aim was to provide guidance for the industry to develop and use LC-MS methods for HCP characterization.
Mørtz stresses that LC-MS-based analyses offer practical advantages for biopharmaceutical companies. “The development time for a new ELISA is typically two years, with an inherent risk that the new assay does not measure enough impurities, or that abundant and potentially problematic HCPs are not being measured,” he details. “This uncertainty puts a significant risk to the timelines of a project if clinical GMP batches cannot be released. To reduce this risk, most companies today are using LC-MS (for process and product HCP characterization) in combination with ELISAs (for batch release testing).
“The actual laboratory costs depend on availability of already established analysis methods. It is expensive to develop a new ELISA, and it is much more expensive to set up an MS laboratory. If a commercial ELISA kit can be validated as fit for purpose, it reduces the development costs and risks.”
Combination approach
Ultimately, the question is not whether a developer should stick with traditional ELISAs, use LC-MC-based methods, or even take an EPA assay–based approach to HCP analysis. What is important is understanding that each method has plusses and minuses. Ultimately, as Mørtz suggests, the best approach may be to combine different methods: “For many projects, it makes very good sense to use a combination of both ELISA and LC-MS, as they together provide a more detailed understanding of the impurities and the manufacturing process to ensure a pure and safe product.”
“For some projects, it is not possible to develop an ELISA,” Mørtz explains. “Such projects include the development of complex vaccines and viral vector–based therapies, products that contain protein impurities from multiple sources and different organisms. Also, projects that seek accelerated regulatory approval may not have enough time to develop a new ELISA, as we’ve seen with the development of COVID-19 vaccines. Wherever ELISA development is problematic, LC-MS will become important as a GMP release testing method for HCPs.”
References
1. Gupta SK, Graf T, Edelmann FT, et al. A fast and sensitive high-throughput assay to assess polysorbate-degrading hydrolytic activity in biopharmaceuticals. Eur J Pharm Biopharm. 2023; 187: 120–129. DOI: 10.1016/j.ejpb.2023.04.021.
2. Edelmann F, Falkenstein R, Graf Tobias, et al., inventors; Hoffmann-La Roche, assignee. Method for determination of hydrolytic activity. WIPO patent WO/2023/117325. June 29, 2023.
3. U.S. Pharmacopeia and National Formulary. 〈1132〉 Residual Host Cell Protein Measurement in Biopharmaceuticals. General Chapter. 2023. USP-NF. Rockville, MD.
