A multi-platform characterization of aggregation in morpholino oligonucleotide drug products: From soluble aggregates to subvisible particles.

Antisense oligonucleotides (ASOs) represent a promising class of therapeutic agents; however, their efficacy and quality can be compromised by aggregation, a critical quality attribute that remains inadequately understood and analytically challenging. To address these methodological limitations, we evaluated the feasibility of both conventional and emerging analytical techniques for characterizing phosphorodiamidate morpholino oligomer (PMO) aggregates across their complete size distribution, from nanometer-scale soluble oligomers to micrometer-scale subvisible particles (SVP). Four FDA-approved PMO drug products were analyzed using five complementary methods selected to cover distinct size ranges: flow imaging (FI) microscopy for SVP (3-100 μm), microfluidic resistive pulse sensing (MRPS) for submicron particles (250-2000 nm), size-exclusion chromatography with multi-angle light scattering (SEC-MALS) and asymmetric flow field-flow fractionation (AF4) for soluble aggregates, and intact mass spectrometry for rapid qualitative assessment of noncovalent assemblies. Method performance, recovery, and orthogonality were systematically evaluated. Additionally, we assessed the effectiveness of filtration (0.2 μm and 0.02 μm) as a risk mitigation strategy and examined the impact of product age on aggregation extent. Aggregates were detected across all size ranges in all four marketed PMO products, with substantial product-specific variability in both quantity and size distribution. SEC-MALS achieved >80% recovery and detected oligomeric species ranging from dimers to tetramers, with soluble aggregate levels ranging from 0.27% to 6.75% for the products tested. MRPS quantified submicron particles at concentrations exceeding 10⁵ particles/mL, while FI detected SVP concentrations ranging from 129 to 3,544 particles/mL. These PMO aggregates exhibited moderate transparency (transparency < 0.6), suggesting potential undercounting by traditional light obscuration methods. AF4 and intact mass spectrometry-applied here for the first time to PMOs-confirmed the presence of high molecular weight species, although AF4 separation of monomers and dimers was incomplete for some products. Intact mass spectrometry enabled direct detection of noncovalent aggregates without chromatographic separation. Assessment of product stability revealed that expired products exhibited up to a 1,000-fold increase in particle burden compared to products within shelf life. Evaluation of filtration as a control strategy demonstrated that 0.2 μm filtration reduced SVP by >95% while leaving soluble aggregates unaffected. This multi-platform analytical approach enables comprehensive characterization of PMO aggregation spanning from nanometer to micrometer scales. The current study provides analytical scientists with enhanced understanding of analytical technologies for subvisible particles measurements which may be selected as part of a control strategy and used to monitor this critical quality attribute in oligonucleotide therapeutic during development and manufacturing.