Stability Studies for Biopharmaceutical Formulations and the Role of Methionine Oxidation
Why Stability Studies Matter
Stability studies in monoclonal antibody (mAb) formulations are critical for ensuring product efficacy, safety, and regulatory compliance. These studies assess how a mAb formulation degrades over time under different stress conditions, such as:
1. Temperature fluctuations (accelerated and long-term stability)
2. pH shifts
3. Oxidative stress
4. Light exposure
5. Agitation and freeze-thaw cycles
One of the major degradation pathways in mAbs is methionine oxidation, which can significantly impact the structure, function, and immunogenicity of the therapeutic protein.
Methionine Oxidation and Its Impact
Methionine (Met) is a sulfur-containing amino acid that is highly susceptible to oxidation, particularly when exposed to oxygen, light, metal ions, and peroxides.
The oxidation process occurs in two steps:
...
1. Methionine → Methionine Sulfoxide (MetO)(mild oxidation)
2. Methionine Sulfoxide → Methionine Sulfone (MetO₂) (severe oxidation, irreversible)
In mAbs, oxidation typically occurs at solvent-exposed methionine residues—often in the Fc region, complementarity-determining regions (CDRs), or hinge regions. This can lead to:
- Reduced binding affinity to Fc receptors (affecting antibody-dependent cellular cytotoxicity, ADCC)
- Loss of potency due to structural alterations in antigen-binding regions
- Aggregation and increased immunogenicity risks
- Altered pharmacokinetics and reduced in vivo stability
How to Control and Monitor Methionine Oxidation in Stability Studies
With the MIRA Analyzer and CLADE™ QA Scan, we leverage ultra-sensitive FTIR spectroscopy to precisely monitor methionine oxidation kinetics. The data below demonstrate that FTIR fingerprints for Methionine (Met), Methionine Sulfoxide (MetO), and Methionine Sulfone (MetO₂) are distinctly unique, allowing real-time tracking of oxidation progression.
Key Findings from Accelerated Stability Studies:
- No visible spectral changes at first glance across timepoints.
- Zooming in at 1285 cm⁻¹ reveals a subtle yet significant spectral shift, correlating precisely with oxidation progression.
- Highly accurate quantification of MetO and MetO₂, providing detailed insights into degradation kinetics.
- Comprehensive analysis in just 4–6 minutes, with near-instant data analytics.
Why It Matters:
This breakthrough enables rapid, high-throughput stability testing, capturing excipient stability, API concentration, and degradation characterization in a single measurement. One instrument, vast amounts of critical data in minutes—empowering biopharma companies with unparalleled formulation insights.
See the difference. Control oxidation early.
Ensure stability. Contact us to learn how we can support your stability studies.
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| Attribute | T1 | T2 | T3 | T4 |
|---|---|---|---|---|
| Protein Concentration | 45.0 mg/ml | 44.8 mg/ml | 44.7 mg/ml | 44.7 mg/ml |
| Sucrose | 36.87 mg/ml | 37.00 mg/ml | 36.75 mg/ml | 36.80 mg/ml |
| Histidine | 3.20 mg/ml | 3.20 mg/ml | 3.22 mg/ml | 3.19 mg/ml |
| Methionine | 1.40 mg/ml | 1.08 mg/ml | 0.88 mg/ml | 0.71 mg/ml |
| Methionine Sulfon | 0 mg/ml | 0.10 mg/ml | 0.11 mg/ml | 0.15 mg/ml |
| Methionine Sulfoxide | 0 mg/ml | 0.23 mg/ml | 0.38 mg/ml | 0.59 mg/ml |
| Polysorbate 20 | 0.31 mg/ml | 0.31 mg/ml | 0.29 mg/ml | 0.27 mg/ml |
| Protein secondary structure - Alpha Helix | 4.31 % | 4.29 % | 4.10 % | 3.90 % |
| Protein secondary structure - Beta sheet | 46.18 % | 46.10 % | 42.10 % | 40.50 % |
| Protein ID similarity | 0.998 | 0.998 | 0.991 | 0.980 |
| pH | 6.8 | 6.8 | 6.8 | 6.8 |