Article 
The Risk of Detection Failure
Failure to detect and remove these contaminants reliably and predictably can compromise efficacy and stability of the therapeutic protein and jeopardize patient safety. Additionally, the presence of contaminants can be costly, forcing the shutting down of processes, delaying of timelines, and the discarding of batches. The full breadth of ramifications can be even more wide-ranging. For example, even if no contaminated lots are released, the presence of impurities can lead to drug shortages, multi-million-dollar investigations, cleanup, corrective actions, lost sales, manufacturing downtime, and damaged corporate reputations (Liu et al. 2000). The risk of contamination is exemplified by the recent formation of a BioPhorum Development Group HCP Workstream (Jones et al. 2021). The industry alignment around high-risk HCPs which are immunogenic, biologically active, or enzymatically active, and have the potential to degrade the target protein or excipients used in the formulation.
This article provides an overview of detection methods used to identify some of the most common impurities that may be present in the mAb bioprocessing workflow.
| Containment | Regulatory Guidance |
| HCPs | HCPs (host cell proteins) in the final product must be reduced to consistently low levels as detected by a sensitive analytical method (U.S. Food and Drug Administration [FDA] 1997). The most likely range of HCPs in biologic products reviewed by the US FDA is 1–100 ppm (Champion et al. 2005). |
| DNA | Residual DNA should be less than 10 ng/dose and the DNA size to below approximately 200 base pairs (U.S. FDA 2010). |
| Viruses | There is no minimal level of virus clearance defined in regulatory guidelines (International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use [ICH] 1999). |
| Endotoxin | The amount of endotoxin administered should not exceed the pyrogenic threshold (for example, 5 EU/kg/hr for intravenous administration or 0.2 EU/kg/hour for intrathecal administration) (McCullough 2018). |
| Mycoplasma | The amount of endotoxin administered should not exceed the pyrogenic threshold (for examTherapeutics must be free of Mycoplasma contamination (U.S. FDA 1997). |
Table 1. Common contaminants in bioprocessing workflows and limits defined by regulatory authorities.
See more...Host Cell Proteins
Cell lines such as the Chinese hamster ovary (CHO) and human embryonic kidney (HEK) cell lines are frequently used as the expression systems for production of recombinant proteins, including mAbs. Proteins from these cell lines can contaminate the bioprocessing workflow, and if not effectively detected and removed from the final product, can lead to decreased drug efficacy and adverse events in patients due to immunogenicity. As shown in Table 1, regulatory guidelines require the reduction of these proteins to the lowest possible levels. Enzyme-linked immunosorbent assays (ELISA) are typically used to identify HCPs in the bioprocess workflow and can help optimize the chromatographic methods used for their removal. These assays are also used during lot release testing and quality control. The antibodies used in cell line–specific ELISAs are generated against a lysate of the respective cells and typically provide at least 80–90% coverage of expected HCPs. However, if significant genetic manipulation of a cell line occurs, these ELISA antibodies must be requalified to ensure sufficient coverage for adequate sensitivity and detection.
DNA
Residual DNA from the expression system must be less than 10 ng/dose with remaining fragment sizes smaller than 200 bp. In addition to the potential for immunogenicity, there is a risk, albeit rare, that oncogenes used to immortalize expression system cell lines could insert into the cells of the patient following administration of a drug in which DNA was not reduced to the regulated level.
Several methods are available to detect host cell DNA in the bioprocessing workflow. Fluorescent nucleic acid stain and ethidium bromide are available for quantitating double-stranded DNA.
Quantitative PCR (qPCR) offers rapid and sensitive quantification of nucleic acid. qPCR measures the accumulation of DNA during a PCR reaction with the increase in quantity of DNA at each cycle measured by the change in intensity of a fluorescent signal. Comparison to a reference sample determines the number of original copies of template DNA in the reaction. With Droplet Digital™ PCR (ddPCR™), each sample is partitioned into thousands of individual reactions. Each partition is analyzed after endpoint PCR cycling for the presence or absence of a fluorescent signal, and the absolute number of molecules present in the sample is calculated. Advantages of using ddPCR technology is that the method does not require sample preparation nor development of a standard curve for quantification, saving time and increasing precision.
Viruses
Mycoplasma are a type of bacteria that pose a safety hazard to patients if not removed by downstream purification. They are gram-negative bacteria that lack a cell wall and pose a particular challenge in the bioprocessing workflow because they are very small (2–3 μm) and cannot be detected by light microscopy. Mycoplasma are characterized by slow growth and because they do not cause death of infected cells, there are no obvious signs if a cell culture is contaminated.
Given the risk presented by Mycoplasma, time is of the essence when testing for the presence of these organisms. Unfortunately, traditional detection tests are culture-based and require more than 28 days to complete; the bioproduction workflow continues during this time. If a positive result is returned four weeks later, a significant amount of time, labor, and materials have already been invested in a batch that must be discarded.
Several alternative methods are available, each with advantages and disadvantages:
- Fluorescent staining is easy, but data interpretation can be difficult and labor intensive
- ELISA is moderately sensitive and rapid, but only a narrow range of Mycoplasma species can be detected
- qPCR offers a short time to result but can produce non-specific signals (i.e., false-positive results) and has poor resolution with low DNA input
Given the shortcomings of these methods, Droplet Digital PCR is gaining favor as a fast, precise, and reproducible molecular detection method (Wu M et al. 2020). Compared to other techniques, ddPCR technology provides higher sensitivity and a quantitative readout that reports genome copies per reaction and colony forming units (CFU) per ml. ddPCR technology also provides an advantage in terms of minimizing the risk of false positives. The most widely used qPCR Mycoplasma assay uses SYBR® chemistry which can generate non-specific signals and poor resolution at very low input DNA range (<10 copies). The time needed to confirm whether a positive signal does in fact indicate an actual contamination adds further time and cost to the production workflow.
Conclusion
A diverse range of process- and product-derived impurities may be present during the mAb production workflow and can have a deleterious effect on efficacy and stability of the target protein, compromise patient safety, and result in drug shortages. A contamination event can result in lost time and material, and a substantial investment in time and effort to identify and resolve the issue. Given the potential impact, the sooner an impurity can be detected the better, as the resulting investment to address the situation will be reduced.
Given these risks, a robust, rigorous, and proactive approach to impurity detection is essential. Many strategies are available and should be selected based on the specificity and selectivity needed to ensure confidence in the detection process.
Tips from our Experts
Our team has extensive experience in the detection of impurities during the mAb processing workflow. Here are some tips for optimizing your process.
- Keep in mind that contamination can happen at any stage of the bioprocessing workflow. Be sure to design the right processes and checkpoints using tests that are specific and sensitive
- Understand the point at which your process is at risk of having more impurities and contaminants and potentially a reduction in yield
- Fully characterize the purification process to thoroughly monitor each run and proactively identify the presence of possible impurities
- Monitor liquid media and buffers that will come into contact with cells during upstream processing for possible contamination
- Provide regular training to staff to reinforce strict adherence to Good Laboratory Practices (GLP)
References
Champion K et al. (2005). Defining your product profile and maintaining control over it. BioProcess International 3, 52–57.
ICH (1999). International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use. Q5A(R1). Viral Safety Evaluation of Biotechnology Products Derived From Cell Lines of Human or Animal Origin. https://database.ich.org/sites/default/files/Q5A%28R1%29%20Guideline_0.pdf, accessed March 21, 2023.
Jones M et al. (2021). "High-risk" host cell proteins (HCPs): A multi-company collaborative view. Biotechnol Bioeng 118, 2,870–2,885.
Liu S et al. (2000). Development and qualification of a novel virus removal filter for cell culture applications. Biotechnol Prog 16, 425–34.
Morris C et al. (2021). Adventitious agent detection methods in bio-pharmaceutical applications with a focus on viruses, bacteria, and mycoplasma. Curr Opin Biotechnol 71, 105–114.
U.S. Food & Drug Administration (1997). Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use. https://www.fda.gov/regulatory-information/search-fda-guidance-documents/points-consider-manufacture-and-testing-monoclonal-antibody-products-human-use, accessed March 21, 2023.
U.S. Food & Drug Administration (2010). Guidance for Industry: Characterization and Qualification of Cell Substrates and Other Biological Materials Used in the Production of Viral Vaccines for Infectious Disease Indications. https://www.fda.gov/regulatory-information/search-fda-guidance-documents/characterization-and-qualification-cell-substrates-and-other-biological-materials-used-production, accessed March 21, 2023.
Wu M et al. (2020). Transitioning from quantitative PCR to Droplet Digital PCR for mycoplasma detection. Bio-Rad Bulletin 7427.
McCullough KZ (2018). Calculating endotoxin limits for drug products. https://www.americanpharmaceuticalreview.com/Featured-Articles/353977-Calculating-Endotoxin-Limits-for-Drug-Products/, accessed March 21, 2023.
BIO-RAD, DDPCR, and DROPLET DIGITAL are trademarks of Bio-Rad Laboratories, Inc. in certain jurisdictions. SYBR is a trademark of Thermo Fisher Scientific Inc. All trademarks used herein are the property of their respective owner. © Bio-Rad laboratories, Inc.
