From Narrow to Ultrawide Pores: Characterization of mAbs, AAVs, mRNA, and pDNA

Significance of the topic

Size heterogeneity is a central quality attribute for modern biopharmaceuticals. Modalities from monoclonal antibodies (mAbs) to viral vectors (AAV), mRNA and plasmid DNA (pDNA) differ by orders of magnitude in molecular weight and hydrodynamic size; aggregates, fragments and topology variants affect potency, immunogenicity and stability. Size‑exclusion chromatography (SEC) under native conditions remains the preferred analytic approach to quantify and characterize these size variants, and advances in column particle technology (sub‑3 µm particles, expanded pore sizes up to 1000 Å, improved stationary phases and higher pressure tolerance) extend SEC applicability across this broad size range.

Objectives and overview of the study

This application note evaluated the Agilent AdvanceBio SEC column family (2.7 µm particles; pore sizes 130 Å, 300 Å, 500 Å, 1000 Å) for characterization of representative biopharmaceuticals: trastuzumab (mAb), adeno‑associated virus serotype 5 (AAV5) with and without genome, several mRNA constructs and 7.5 kbp pDNA. The intent was to demonstrate how pore size selection and detector combinations (UV, fluorescence, serial column coupling) optimize resolution of monomers, aggregates, fragments, dsRNA impurities and topological forms across modalities spanning ~66 kDa to multi‑MDa and tens to hundreds of nm in hydrodynamic diameter.

Methodology and experimental workflow

• Common mobile phase: 50 mM phosphate, 400 mM NaCl, pH 7.2 (used as a general go‑to buffer across sample types).
• Columns tested: AdvanceBio SEC 2.7 µm, column formats 4.6×300 mm and 4.6×150 mm with pore sizes 130 Å, 300 Å, 500 Å and 1000 Å. Serial coupling of 1000 Å + 300 Å columns was evaluated to expand separation space.
• Flow rate: typical analytical method 0.35 mL/min for 4.6 mm columns (run time 24 min); higher flow rates explored for pDNA to probe hydrodynamic/slalom behavior and column backpressure.
• Detection: diode array UV (260 nm for nucleic acids, 280 nm for proteins) and fluorescence detection (Ex 280 nm / Em 348 nm) to increase sensitivity for capsid/aggregate species.
• Samples and treatments: temperature stress for trastuzumab; RNase T1 and RNase 4 digests, thermal melting, and concentration for mRNA; restriction enzyme linearization for pDNA; intact and genome‑filled/empty AAV5 samples.

Used instrumentation

• Agilent 1260 Infinity II Bio‑Inert Pump (G5654A)
• Agilent 1260 Infinity II Bio‑Inert Multisampler (G5668A)
• Agilent 1260 Infinity II Multicolumn Thermostat (G7116A)
• Agilent 1260 Infinity II Diode Array Detector (G7115A) with bio‑inert 13 µL, 10 mm UV flow cell
• Agilent 1260 Infinity II Fluorescence Detector (G7121B) with standard 8 µL FLD flow cell
• Data system: Agilent OpenLab CDS v2.7

Key results and discussion

• Pore size selection principle: optimal pore size places the main analyte peak centrally in the column separation window (between exclusion and permeation limits) to maximize resolution of aggregates and fragments.
• Proteins and mAbs: 300 Å columns provided best performance for proteins such as BSA and trastuzumab, resolving monomer, noncovalent dimer and low‑molecular‑weight fragments (e.g., Fab, mAb‑Fab). A smaller particle/pore column (1.9 µm, 200 Å) can be advantageous when finer resolution of ~100 kDa fragments from the monomer is required.
• AAVs: 500 Å pore columns combined with fluorescence detection sensitively separated AAV monomer and aggregate species and allowed estimation of genome packaging via A260/A280 ratios (~1.2 for genome‑filled capsids, ~0.7 for empty capsids in the examples). Differences in aggregate profiles between empty and genome‑filled samples were observed.
• mRNA: 1000 Å pore columns are required to resolve large ssRNA (0.5–9 kb) and to separate dsRNA impurities from main mRNA peaks. Serial coupling of 1000 Å + 300 Å columns improved separation of RNase digestion products and co‑formulated constructs (e.g., Cas9 mRNA + sgRNA), revealing shorter oligonucleotide fragments and the resistant poly(A) tail.
• pDNA and large nucleic acids: very large constructs (7.5 kbp pDNA, >100 nm) are excluded from pores and primarily separate by hydrodynamic chromatography/slalom effects rather than classic SEC. Flow rate strongly influences elution behavior: higher flow promotes shear stretching and slalom retention, with linearized pDNA showing larger relative shifts in elution volume than supercoiled forms—enabling topology profiling by exploiting differential elastic relaxation under shear.
• Operational note: serial column configurations raise backpressure (measured up to ~280 bar for 1000 Å + 300 Å tandem) but remained within the column mechanical limit (≥400 bar).

Benefits and practical applications

• Single‑platform SEC strategy: by selecting appropriate pore size(s), the same SEC chemistry and buffer can be used to assess proteins, viral vectors, mRNA and even large pDNA, simplifying method development and QC workflows.
• Sensitivity and orthogonality: combining UV and fluorescence detectors improves sensitivity for capsid aggregates and allows rapid estimation of genome content via absorbance ratios; serial‑column setups expand resolving power for complex digests and co‑formulations.
• Topology and aggregation profiling: SEC/HDC/slalom phenomena provide practical handles to distinguish topological forms of pDNA and to resolve distinct aggregate populations in AAV and protein samples relevant to release testing and formulation development.

Future trends and potential applications

• Further expansion of pore sizes and particle engineering will continue to push SEC applicability toward larger nucleic acids and complex nanoparticle formulations (LNPs) while maintaining high efficiency and pressure stability.
• Integration with orthogonal detectors (MALS, light scattering, mass photometry, MS) and automated multi‑column schemes will enhance absolute sizing, molar mass determination and impurity identification in a single analytical workflow.
• Exploiting controlled slalom/HDC behavior and high‑pressure operation may become a standardized approach for topology profiling of large DNA/RNA in gene therapy analytics.
• Regulatory and QC adoption will likely grow as column chemistries that minimize nonspecific interactions and bleed become more robust, enabling SEC to serve as a routine release and stability assay across diverse biologics.

Conclusion

Advances in SEC column technology (sub‑3 µm particle sizes, expanded pore sizes to 1000 Å and improved mechanical stability) allow a single chromatographic platform to characterize a wide dynamic range of biopharmaceutical modalities. Appropriate pore selection (300 Å for mAbs; 500 Å for AAVs; 1000 Å or 1000+300 Å tandem for mRNA and large nucleic acids) and detector choices (UV, FLD) yield sensitive, informative separations of monomers, aggregates, fragments and topological variants. Additionally, hydrodynamic/slalom separation phenomena can be intentionally leveraged to profile very large constructs such as pDNA. The workflow described provides practical guidance for QC and R&D labs working with contemporary biologics and genetic medicines.

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