Post-translational modifications (PTMs) govern protein structure, function, and stability, influencing nearly every biological process. In biopharmaceuticals (biologics), PTMs such as glycosylation and phosphorylation are critical quality attributes that affect efficacy, stability, and safety, with variability linked to altered activity and immunogenicity.1 Beyond bio-therapeutics, PTMs can serve as translational biomarkers that connect disease biology, as well as pharmacology to clinical outcomes. Comprehensive and systematic characterisation of these modified proteoforms remains challenging due to analytical limitations in resolution, sensitivity, and specificity. This article explores the essentiality of PTM measurements at scale across the drug development continuum and highlights emerging separation and mass spectrometry technologies that close the analytical gap, thereby improving specificity, sensitivity and overall fidelity to elucidate biological processes and associated pharmacology.
The Biological and Commercial Imperative for PTM Characterisation
In the high-stakes, high-cost environment of drug discovery and development, where capitalised costs exceed $2.5 billion and clinical attrition rates remain high,2,3 a comprehensive understanding of PTMs is not merely an academic exercise but is a core risk mitigation strategy (Figure 1). Incomplete molecular characterisation of the biopharmaceutical as well as its underlying mechanism of action introduces significant risk, with the potential to trigger late-stage failures that may compromise patient safety and eventually impact financial investment.
PTMs as Critical Quality Attributes (CQAs)
In the context of biopharmaceutical manufacturing, a Critical Quality Attribute (CQA) is a physical, chemical, or biological property that must be controlled within an appropriate limit to ensure the desired product quality.4,5 PTMs are among the most important CQAs for protein-based therapeutics. A prime example is the glycosylation profile on monoclonal antibodies. The specific Carbohydrate structures (glycans) attached to the antibody’s constant region directly modulate its effector functions, i.e. the ability to engage with the immune system to eliminate target cells.5,6 Furthermore, the cell lines used for production, such as Chinese Hamster Ovary (CHO) or murine myeloma (NS0) cells, can introduce glycan structures not found in humans, such as galactose-α-1,3-galactose or N-glycolylneuraminic acid. These non-human glycans can be immunogenic, triggering an anti-drug antibody response that neutralises the therapeutic and can cause adverse events.6
Other common PTMs routinely monitored as CQAs for therapeutic proteins include:
• Oxidation: Particularly of methionine residues, which can affect protein stability and function.7
• Deamidation: The conversion of asparagine or glutamine residues, which introduces charge heterogeneity and can impact biological activity.6
• N-terminal Pyroglutamate Formation: The intramolecular cyclisation of an N-terminal glutamine residue, which removes a charge and introduces heterogeneity. It is often monitored as a CQA to ensure manufacturing process consistency.6
