Advanced Impurity Profiling & Characterization

We conducted a comprehensive comparative impurity study against the Reference Listed Drug (RLD) to ensure the highest purity standards. Our rigorous characterization included:

• Sequence Integrity: Full-Length Product (FLP), Shortmers (N-1), and Longmers (N+1).
• Modification Variants: Detailed analysis of 2'-MOE and 2'-O-EOE substitutions, including base-specific variants (mocG, mocA, moe-meU, moe-meC).
• Process-Related Species: Characterization of CNET, ADP/IDP artifacts, and comprehensive monitoring of front-eluting and post-eluting impurities.

Definitive Sameness Studies

• Mass Spectrometry (MS/MS):
• Determines the exact molecular weight of nusinersen to verify its chemical composition.
• Tandem MS (MS/MS) fragments the oligonucleotide to confirm its sequence integrity and identify any impurities or degradation products.
• Nuclear Magnetic Resonance (NMR) Spectroscopy:
• Validates the three-dimensional structure of nusinersen, ensuring correct base pairing and backbone conformation.
• Detects potential structural deviations or modifications.
• Liquid Chromatography (LC):
• Separates nusinersen from impurities, excipients, or process-related byproducts.
• Quantifies purity and monitors stability under storage conditions.
• Duplex Melting Temperature (Tm):
• Measures the thermal stability of the duplex formed between nusinersen and its complementary RNA strand.
• Reflects binding strength and hybridization efficiency, critical for its mechanism of action.
• Circular Dichroism (CD) Spectroscopy:
• Analyzes the secondary structure (e.g., A-form helix) of nusinersen.
• Ensures proper folding and conformational stability.
• Differential Scanning Calorimetry (DSC):
• Assesses thermal stability by measuring heat absorption during structural transitions (e.g., denaturation).
• Evaluates batch-to-batch consistency and formulation robustness.
• Size Exclusion Chromatography (SEC):
• Determines the oligonucleotide size distribution and detects aggregates or fragments.
• Ensures nusinersen remains monomeric and within specified size limits.
• Sedimentation Velocity Analytical Ultracentrifugation (SV-AUC):
• Provides high-resolution analysis of molecular weight, shape, and aggregation state.
• Validates homogeneity and confirms the absence of higher-order aggregates.