Determination of Molecular Weight Distribution for Glatiramer Acetate by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering and RI Detector (SEC-MALS-RI)

Glatiramer acetate (GA) is a heterogeneous synthetic polypeptide used as a disease-modifying therapy for relapsing forms of multiple sclerosis, and its molecular weight distribution (MWD) is a key attribute for demonstrating lot consistency and comparability across reference and follow-on products. In this work, GA MWD was characterized by size-exclusion chromatography coupled online to multi-angle light scattering and refractive index detection (SEC-MALS-RI), enabling calibration-independent (absolute) molar-mass determination across the elution profile. GA exhibited a broad, asymmetric SEC envelope consistent with inherent compositional and chain-length heterogeneity. Using RI-derived concentration (refractive index increment (dn/dc) established by a linear RI response–concentration relationship with R² = 1.0000; dn/dc ≈ 0.1834 mL/g, fit error ~ 0.38%), absolute molar-mass moments were obtained across defined peak regions: number-average molecular weight, Mn≈6.34–7.56 kDa; weight-average molecular weight, Mw≈8.90–9.56 kDa; with polydispersity index, Mw/Mn≈1.26–1.41; and average molecular weight, Mz≈12.34–14.49 kDa, indicating a measurable contribution from higher-mass species. Repeatability and intermediate precision were strong, with day-wise %RSD values < 2% for Mn and Mw (n = 6/day across three days). Robustness testing showed that small dn/dc perturbations (± 0.005 mL/g around nominal) produced < 2% change in Mw under stable integration and detector alignment. A BSA performance check yielded Mw/Mn = 1.005, supporting detector normalization and inter-detector synchronization. Overall, SEC-MALS-RI provides a reliable, absolute approach to quantify GA MWD and supports scientifically defensible comparability assessments for complex generic development and lifecycle quality control.