Aggregate Analysis of a Recombinant L-Asparaginase from E. coli

Význam tématu

Aggregation of biotherapeutic proteins like L-asparaginase is critical for product safety and efficacy in oncology therapies. Changes in pH, temperature, shear stress, and concentration can induce formation of aggregates, which may compromise potency, stability, and immunogenic profile of the final drug product.

Cíle a přehled studie

This application note evaluates two SEC columns—a silica-diol stationary phase (Agilent ProSEC 300S) and a hydrophilic polymer-coated phase (Agilent AdvanceBio SEC)—for quantifying aggregates of recombinant L-asparaginase. The study also examines method robustness through prolonged testing and lot-to-lot consistency.

Použitá instrumentace

• Agilent 1260 Infinity II Bio-inert LC system with quaternary pump, multisampler, column thermostat, and multi-wavelength detector
• Agilent 1260 Bio-SEC multidetector system for light scattering
• Agilent OpenLab CDS 2.6 and GPC/SEC software for data acquisition and analysis

Hlavní výsledky a diskuse

• AdvanceBio SEC column displayed approximately 30 % larger pore volume and higher efficiency, yielding superior resolution (e.g., BSA dimer–monomer Rs 2.64 vs 2.16) compared with ProSEC 300S.
• Calibration curves demonstrated consistent molecular weight discrimination; larger proteins eluted earlier on AdvanceBio SEC.
• Over 300 injections (6 days), AdvanceBio SEC maintained stable retention times and resolution, confirming robustness and lot-to-lot reproducibility.
• L-asparaginase exhibited atypical retention behavior; coupling UV detection with light scattering enabled accurate determination of the main tetramer peak at ~139 kDa.

Přínosy a praktické využití metody

The hydrophilic polymer-coated SEC method offers improved sensitivity and reliability for aggregate quantification in quality control environments, facilitating biologics development, batch release testing, and formulation screening with enhanced precision and extended column lifetime.

Budoucí trendy a možnosti využití

• Integration of multi-angle light scattering and refractive index detectors for comprehensive aggregate characterization.
• Application to other aggregation-prone biotherapeutics, including antibodies and enzyme formulations.
• Adaptation to high-throughput and automated workflows for process analytical technology (PAT) in continuous manufacturing.

Závěr

The Agilent AdvanceBio SEC column outperforms ProSEC 300S for L-asparaginase aggregate analysis, delivering higher resolution, consistent long-term performance, and compatibility with light scattering detection, thus supporting more accurate and robust biotherapeutic QC assays.

Reference

1. Kidd J.G. Regression of Transplanted Lymphomas Induced In Vivo by Means of Normal Guinea Pig Serum, Horse Serum, or Rabbit Serum. J. Exp. Med. 1953, 98(6):565–582.
2. Broome J.D. Evidence That the L-asparaginase of Guinea Pig Serum Is Responsible for Its Antilymphoma Effects. I. Properties of the L-Asparaginase of Guinea Pig Serum in Relation to Those of the Antilymphoma Substance. J. Exp. Med. 1963, 118(1):99–120.
3. Gervais D. et al. Robust Quantitation of Basic-Protein Higher-Order Aggregates Using Size-Exclusion Chromatography. J. Pharm. Biomed. Anal. 2017, 139:215–220.