Enhance Biopharma Insights with Advanced Size Exclusion Chromatography

Advanced Size Exclusion Chromatography for Biopharma: Overview of Agilent SEC Solutions

Significance of the topic

Size exclusion chromatography (SEC) is a cornerstone analytical technique for biopharmaceutical development and quality control. It provides critical information on aggregation, molecular weight, and size—key quality attributes for monoclonal antibodies, viral vectors, lipid nanoparticles (LNPs), mRNA therapeutics, and other large biomolecules. Robust SEC workflows that combine reliable separation with absolute detection are essential to ensure product stability, efficacy, and regulatory compliance.

Objectives and study overview

This material presents an integrated SEC workflow packaged by Agilent for advanced biopharma characterization. The goals are to: provide absolute molecular weight and size metrics using light scattering; offer user-friendly, compliance-ready software to analyze complex datasets; and cover complete analytical solutions including columns, standards, LC hardware, and detectors. Several application examples demonstrate performance for monoclonal antibodies, mRNA, and LNPs.

Methodology and approach

The promoted workflow couples conventional SEC separation with orthogonal light scattering detectors: static light scattering (MALS) for absolute molecular weight and radius of gyration (Rg), and dynamic light scattering (DLS) for hydrodynamic radius (Rh) and aggregation behavior. Low-volume detector flow cells are used to minimize band broadening. Data processing and quality control are handled within Agilent OpenLab CDS using the Advanced GPC/SEC software module, enabling overlay, multi-angle analysis, and tabulated light-scattering results for reproducible reporting.

Used instrumentation

Key instrumentation and system components described include:

• Agilent InfinityLab and Infinity III / Infinity II Bio LC platforms — biocompatible, bio-inert LC systems with robust pump and sampling options suited for bioanalysis.
• Agilent 1260 Infinity II Multi-Angle Light Scattering (MALS) Detector — high-precision MALS with up to 20 angles for accurate absolute molecular weight and Rg determination.
• Agilent 1260 Infinity II Bio-SEC Multidetector System — low-volume flow cell design with dual-angle and DLS detection to preserve chromatographic resolution while providing Rh and aggregation data.
• Agilent 1290 Infinity II Bio LC System — used in conjunction with detectors for high-performance SEC-MALS workflows.
• Advanced GPC/SEC Software for OpenLab CDS — integrated analysis environment for conventional and advanced SEC, with LS plotting, compound-detail displays, result tables, and compliance features.
• Wide SEC column portfolio (AdvanceBio SEC series, Bio SEC-5, PROTEEMA Lux) and dedicated SEC standards for system suitability and calibration.

Key results and discussion

Representative application highlights presented:

• Monoclonal antibody (Rituximab biosimilar): SEC-MALS analysis determined the monomer molecular weight at ~148 kDa. Precision across seven consecutive injections was excellent, with 0.253% relative standard deviation (RSD) for molecular weight determination, demonstrating high reproducibility.
• mRNA (IVT mRNA) characterization: SEC-MALS provided molecular weight distribution across chromatographic peaks and reported Rg values, allowing assessment of biophysical and structural attributes relevant to production, formulation, and storage effects.
• Lipid nanoparticles (LNPs): Online SEC combined with DLS and MALS yielded precise Rh, Rg, and shape factor (ρ = Rg/Rh) values for both loaded and empty particles. Example results (n = 5) include: LNP A (mRNA) Rh ≈ 38 nm (RSD 5.0%), Rg ≈ 22.9 nm (RSD 8.6%), ρ ≈ 0.608; LNP B (mRNA) Rh ≈ 58 nm (RSD 2.2%), Rg ≈ 37.4 nm (RSD 12.6%), ρ ≈ 0.646. Shape factors were used to infer internal structure (e.g., dense-core spheres expected ρ ≤ 0.77).

Columns and separation ranges (summary)

Column pore size and particle size are emphasized as primary determinants of SEC applicability. Agilent offers multiple chemistries and pore sizes to cover analyte ranges from small oligonucleotides to very large assemblies (up to tens of MDa):

• AdvanceBio SEC (various formats): particle sizes 1.9–2.7 µm; pore sizes 120–1000 Å; useful ranges extend from ~1 kDa up to several MDa depending on phase.
• Bio SEC-5: 5 µm, 2000 Å for very large oligonucleotides and VLPs (range up to ~10 MDa).
• PROTEEMA Lux: polymer-protein applications covering up to several MDa.

Benefits and practical applications

Main advantages of the integrated SEC + LS/DLS workflow:

• Absolute molecular weight and size measurement independent of column calibration, reducing uncertainty when analyzing non-globular or heterogeneous biomolecules.
• High precision and reproducibility enabled by low-volume flow cells and multi-angle detection.
• Simultaneous structural metrics (Rg, Rh, and shape factor) to inform on particle morphology and aggregation state.
• Compatibility with regulatory and QA/QC environments via OpenLab CDS and compliance-ready reporting.
• Broad applicability across biopharma modalities: mAbs, oligonucleotides, AAVs/VLPs, mRNA, and LNPs for formulation development, stability studies, and release testing.

Future trends and opportunities for use

Emerging directions that will further enhance SEC-based biopharma analytics include:

• Tighter integration of orthogonal detectors — combining MALS/DLS with intrinsic fluorescence, refractive index, and mass spectrometry to deliver richer physicochemical profiles.
• Higher-resolution column chemistries and sub-2 µm stationary phases tailored to large biomolecules to increase peak capacity while maintaining biocompatibility.
• Automation and online fraction collection to link SEC separations to downstream assays (e.g., potency, structural MS, or bioassays).
• Improved software workflows with AI-assisted peak deconvolution and automated QC reporting to accelerate data interpretation and regulatory submissions.
• Miniaturized and low-flow SEC formats for material-limited samples and enhanced sensitivity with coupling to advanced detectors.

Conclusions

Combining robust, biocompatible LC hardware with multi-angle static light scattering and dynamic light scattering detectors, supported by integrated compliance-capable software, provides a powerful platform for modern SEC characterization in biopharma. The approach delivers absolute molecular weight and size information with high precision, enabling informed decisions in development and quality control across diverse modalities including mAbs, mRNA, LNPs, and viral-like particles. Selecting appropriate columns and standards remains essential to maximize the analytical value of SEC-MALS/DLS workflows.

References

The summary is based on Agilent Biopharma SEC Solutions product literature and application examples (Agilent Technologies technical brochure, DE-013426, published March 23, 2026).