Freeze-Thaw Stability of mRNA and RNA-Based Therapeutics: Strategies for Preservation and Regulatory Compliance

Messenger RNA (mRNA) and other RNA-based therapeutics have revolutionized modern medicine, particularly with the success of mRNA vaccines in global pandemic responses. However, RNA molecules are inherently fragile, susceptible to hydrolysis, enzymatic degradation, and structural instability. Repeated freeze-thaw cycles—common in manufacturing, transport, and clinical settings—pose a significant risk to their integrity and efficacy. This article explores the unique stability challenges of RNA-based drugs under freeze-thaw stress, offering detailed guidance for designing robust stability studies and aligning with regulatory expectations for global product deployment.

1. Why Freeze-Thaw Stability Is Critical for RNA-Based Therapeutics

Unique Vulnerabilities of RNA Molecules:

• Prone to hydrolysis due to 2’-hydroxyl group on ribose
• Susceptible to RNase contamination, even in trace amounts
• Dependent on structural folding for biological function
• Often formulated in lipid nanoparticles (LNPs), which are themselves sensitive to thermal stress

Common Exposure Points:

• Manufacturing interruptions during storage or batching
• Transport via air freight or extended customs delays
• Thawing during clinical use or pharmacy reconstitution

2. Regulatory Expectations for Freeze-Thaw Testing of RNA Drugs

FDA Guidance:

• Freeze-thaw studies are essential for mRNA vaccines and RNA-based gene therapies
• Validation of formulation stability under real-use conditions is mandatory
• Stability data must justify cold chain handling and excursion limits

ICH Q1A(R2) and WHO PQ:

• Stress testing of new drug substances must include temperature extremes
• Thermal excursion data supports labeling, storage, and packaging claims

EMA Position:

• RNA-based products require freeze-thaw resilience for central authorization
• Label statements must be data-backed and risk-assessed

3. Designing Freeze-Thaw Studies for mRNA and RNA-Based Drugs

Protocol Considerations:

Sample Considerations:

• Final LNP formulation in filled vials or prefilled syringes
• Include RNA-only controls and placebo formulations
• Evaluate both bulk and final packaged form if applicable

Monitoring Tools:

• Calibrated data loggers for temperature validation
• Environmental monitoring of freezers and thawing stations

4. Common Freeze-Thaw Degradation Pathways in RNA Products

1. Hydrolysis of Phosphodiester Bonds:

• Mechanism: Water-mediated cleavage under thawing or humidity
• Detection: Capillary electrophoresis, RT-qPCR, UV absorbance

2. RNase Contamination and Enzymatic Breakdown:

• Mechanism: Introduction of trace RNases during handling
• Detection: Fluorescent RNA integrity assays, Bioanalyzer profiles

3. Lipid Nanoparticle Disruption:

• Mechanism: Phase transition or fusion of LNPs during freeze/thaw
• Detection: DLS, zeta potential, particle size distribution

4. Aggregation or Precipitation:

• Mechanism: Ice crystal-induced destabilization of encapsulated RNA
• Detection: SEC, turbidity, TEM imaging

5. Analytical Methods for Assessing Freeze-Thaw Stability

RNA Quality & Integrity:

• Bioanalyzer RNA Integrity Number (RIN)
• RT-qPCR for gene expression capacity
• UV absorbance (A260/A280 ratios)

LNP Formulation Analysis:

• Dynamic Light Scattering (DLS)
• Zeta potential measurement
• Encapsulation efficiency by fluorescence-based assays

Potency and Functional Testing:

• Cell transfection assays
• In vitro translation efficacy (luciferase or reporter protein)
• mRNA degradation kinetics modeling

6. Case Studies from mRNA and RNA Therapeutic Programs

Case 1: mRNA Vaccine Stored at –80°C

Subjected to 3 freeze-thaw cycles between –80°C and 25°C. RIN values remained above 8.0, and LNP integrity preserved. Functional in vitro translation confirmed consistent expression. Supported regulatory labeling as “Stable up to 3 thaw cycles for single-use.”

Case 2: RNAi Drug in Prefilled Syringe

Exposed to –20°C 2–8°C cycles. DLS analysis showed particle aggregation beyond acceptable limits after 5 cycles. Reformulation using cryoprotectants like trehalose mitigated the issue in subsequent batches.

Case 3: Nasal Spray RNA Formulation

Exhibited pH drift and encapsulation drop-off after 4 thermal cycles. Stabilizers and surfactants were optimized to support field-use excursions during pandemic distribution campaigns.

7. Labeling and Regulatory Filing Considerations

Include in CTD Modules:

• 3.2.P.2.4: Justification for stress condition and formulation robustness
• 3.2.P.5.6: Validation of analytical methods for RNA integrity and LNP analysis
• 3.2.P.8.3: Study results, freeze-thaw data summaries, labeling impact

Label Claim Examples:

• “Do Not Freeze. Product loses activity after freeze-thaw exposure.”
• “Stable for up to 3 freeze-thaw cycles when stored at –20°C and thawed at 5°C.”
• “Use immediately after thawing. Do not refreeze.”

8. SOPs and Tools for Freeze-Thaw Testing of RNA-Based Drugs

Available from Pharma SOP:

• mRNA Freeze-Thaw Stability Protocol Template
• RNA Integrity and RIN Evaluation SOP
• Lipid Nanoparticle Stress Testing Template
• CTD Summary Sheet for Freeze-Thaw Study (RNA Products)

Further guidance available at Stability Studies.

Conclusion

The stability of mRNA and RNA-based therapeutics under freeze-thaw stress is a cornerstone of their development and global deployment. These fragile molecules demand precise handling, scientifically validated storage conditions, and robust testing strategies. By understanding the degradation mechanisms, employing targeted analytical techniques, and integrating findings into regulatory filings, pharmaceutical teams can ensure their RNA products maintain efficacy and safety across all stages of the supply chain.