Literature
Ubiquitin in mAb Products: Detection, Risks, and Why ELISA May Miss It
March 30 2021, by Ejvind Mørtz, PhD
Why investigate ubiquitin in monoclonal antibody (mAb) products?
Ubiquitin is increasingly detected as a low-level host cell protein (HCP) in final mAb drug substances - despite rigorous purification processes. Understanding why this protein escapes typical purification methods and how it may affect drug quality or patient safety is critical for CMC managers and quality assurance teams working in biopharma.
At Alphalyse, we regularly encounter ubiquitin-related impurities across mAb characterization projects. This article answers key questions:
- Why does Protein A purification not remove ubiquitin effectively?
- Why might process-specific ELISA tests fail to detect it?
- Is the presence in the final product a cause for concern?
Ubiquitin in biologics – Harmless or risk factor?
Ubiquitin is a small protein (8 kDa) that is highly conserved in all eukaryotic cells. It consists of 76 amino acids and plays a central role in protein degradation through the ubiquitin-proteasome system (UPS). When attached to target proteins via ubiquitination, it can alter their function, location, or stability.
While ubiquitin has essential intracellular roles, its presence in biologic drug products - especially monoclonal antibodies - raises questions about potential risks:
- Could it trigger immunogenic responses?
- Might it interfere with drug function or stability?
Current data suggest that ubiquitin, in the low ppm range, has not been linked to patient harm or loss of efficacy. However, consistent detection and monitoring remain crucial for regulatory documentation and ensuring batch comparability.
Why ubiquitin may escape Protein A purification
Protein A chromatography is the gold standard for purifying monoclonal antibodies, yet ubiquitin often co-elutes with the mAb. Why?
- Tight Binding: Ubiquitin may associate with the mAb non-covalently or covalently, acting like a “hitchhiker” HCP.
- Physicochemical Properties: Despite being much smaller than the mAb, ubiquitin’s charge, hydrophobicity, or interaction motifs may allow it to mimic the antibody’s behavior during chromatography.
- Post-translational Modifications (PTMs): In some cases, ubiquitin may be bound as a low-level PTM - not visible through ELISA alone, but picked up by LC-MS.
In multiple Alphalyse projects, we’ve observed these patterns, confirming that even validated purification protocols may not effectively remove all ubiquitin-related impurities.
Why ELISA may miss it
In our client's case, as shown in the table below, mass spectrometry detected 1-25 ppm of ubiquitin, well within the detection range of the applied HCP ELISA. So why didn’t the ELISA pick it up?
There are several reasons:
- Low molecular weight: ELISAs often fail to detect small proteins, such as ubiquitin, because they do not elicit strong immune responses (poor immunogenicity).
- Sequence homology: Ubiquitin is highly conserved across species. This can make it non-immunogenic in animals used for antibody production (e.g., rabbits or goats), resulting in inadequate antibody coverage.
- Antibody specificity: The polyclonal antibodies in each ELISA originate from individual animals. If the animal did not produce antibodies against ubiquitin, it will simply not be detected.
This illustrates a key point: You cannot rely on ELISA alone for full HCP surveillance. ELISAs measure immune-equivalents, not individual proteins.
Ubiquitin quantification using mass spectrometry
Mass spectrometry (MS) offers a powerful orthogonal method for detecting and quantifying individual HCPs, including low-ppm and low-Mw proteins like ubiquitin. At Alphalyse, we use:
- Native digest protocols to retain PTMs and reduce detection limits
- Data-independent acquisition (DIA) methods like SWATH-MS for reproducibility and accuracy
- Custom-built databases specific to each expression system and process
This allows us to track ubiquitin and other problematic HCPs across batches, purification steps, or manufacturing changes - essential for regulatory submissions like INDs and BLAs.
How to act on ubiquitin detection
If you detect ubiquitin in your product, consider the following actions:
- Perform orthogonal confirmation using LC-MS to validate ELISA results
- Analyze its binding behavior - covalent or non-covalent attachment?
- Optimize purification protocols if it consistently co-purifies
- Document its clearance and variability across batches for regulatory readiness
Ensuring control over ubiquitin in biologic drug development
Although ubiquitin has not been linked to patient harm in approved mAb therapies, its consistent appearance in downstream processes - combined with its biological activity - demands careful scrutiny.
Biopharmaceutical developers must demonstrate that potential process-related impurities, such as ubiquitin, are well-characterized and consistently removed. Relying solely on ELISA may leave blind spots. That’s why regulatory agencies now expect orthogonal analytical methods - such as mass spectrometry - to strengthen impurity assessments.
At Alphalyse, we specifically designed our LC-MS-based HCP workflows to meet these demands. With our approach, you can:
- Identify and quantify ubiquitin down to low ppm levels
- Monitor batch-to-batch variability and purification efficiency
- Confirm ELISA limitations and fill detection gaps
- Provide precise documentation for regulatory submissions
With extensive experience across hundreds of mAb products, we help you make confident, data-driven decisions about product purity and patient safety.
Need support with impurity profiling or regulatory documentation?
Contact Alphalyse to discuss your project with our mass spectrometry experts.
References
[1] Goldstein et al.: “Isolation of a polypeptide that has lymphocyte-differentiating properties and is probably represented universally in living cells,” Proceedings of the National Academy of Sciences of the United States of America, 1975
[2] Wilkinson, KD: “The discovery of ubiquitin-dependent proteolysis,” Proceedings of the National Academy of Sciences of the United States of America, 2005
[3] Kimura et al.: “Regulatory mechanisms involved in the control of ubiquitin homeostasis,” Journal of Biochemistry, 2010
[4] Glickman et al.: “The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction,” Physiological Reviews, 2002
[5] Lin et al.: “Exploitation of the host cell ubiquitin machinery by microbial effector proteins,” Journal of Cell Science, 2017
[6] Bracewell et al.: “The Future of Host Cell Protein (HCP) Identification During Process Development and Manufacturing Linked to a Risk-Based Management for Their Control,” Biotechnology and Bioengineering, 2015
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