High-Resolution SEC-HPLC Aggregation Analysis of a Pembrolizumab Biosimilar Using Welch Xtimate™ SEC Columns
Abstract
Pembrolizumab is a humanized IgG4/kappa monoclonal antibody widely used in immuno-oncology. SEC-HPLC is a critical analytical tool for evaluating aggregation and fragmentation in monoclonal antibodies. This application note describes a robust SEC-HPLC method using Welch Xtimate SEC-300 column, demonstrating efficient separation of monomer, HMW aggregates, and LMW fragments.
Introduction
Structural integrity of monoclonal antibodies is directly linked to safety and efficacy. Pembrolizumab biosimilar development requires sensitive techniques to monitor aggregation. SEC-HPLC separates molecules based on hydrodynamic volume under non-denaturing conditions, making it the preferred method for size heterogeneity analysis.
| Structural Integrity | Biosimilar Development | SEC-HPLC Method |
| Direct link to safety and efficacy of monoclonal antibodies | Requires sensitive techniques to monitor aggregation | Preferred method for size heterogeneity analysis under non-denaturing conditions |
Sample Prepration
Pembrolizumab biosimilar drug substance samples were prepared by diluting the stock solution with the SEC mobile phase to obtain a final concentration of 2.0 mg/mL. Samples were gently mixed by inversion to avoid shear-induced aggregation. Prepared samples were transferred into HPLC vials and maintained at 10 ± 2 °C in the autosampler prior to analysis. Filtration was avoided to prevent potential loss of high molecular weight aggregates.
System Suitability Standard: Bovine Serum Albumin (BSA) was used as a system suitability standard. A 1.0 mg/mL BSA solution was prepared in Milli-Q water and injected to verify column performance, resolution, and reproducibility.
Reagents and Chemicals
| Reagent / Material | Grade |
| Sodium dihydrogen phosphate monohydrate | Analytical grade |
| Disodium hydrogen phosphate (anhydrous) | Analytical grade |
| Sodium chloride | Analytical grade |
| Acetonitrile (ACN) | HPLC grade |
| Milli-Q water | HPLC grade |
| Bovine Serum Albumin (BSA) | Reference standard |
| HPLC vials and caps | Certified |
Chromatographic Conditions
| Parameter | Condition |
| Column | Welch Xtimate SEC-300 (4.6 × 300 mm, 3 µm) |
| Mobile Phase | 100 mM phosphate buffer + 300 mM NaCl : ACN (80:20) |
| Flow Rate | 0.2 mL/min |
| Column Temperature | 30 ± 2 °C |
| Injection Volume | 25 µL |
| Detection | 214 nm (monitoring at 280 nm) |
| Run Time | 45 min |
Results and Discussion
The Welch Xtimate SEC-300 column provided excellent resolution between Pembrolizumab monomer and higher molecular weight aggregates. The monomer peak (~146–147 kDa) was sharp and symmetrical, indicating minimal secondary interactions.
Key Observations:
- Predominantly monomeric species at expected retention time
- Distinct HMW species at earlier elution times
- LMW species at later retention times
- Heterogeneous mixture in process intermediate samples
Note: The process intermediate samples contain a heterogeneous mixture of monomeric Pembrolizumab along with HMW aggregates and LMW fragments.
Conclusion
The developed SEC-HPLC method employing the Welch Xtimate™ SEC-300 column demonstrated robust performance and reliable resolution of monomer, high-molecular-weight aggregates, and low-molecular-weight species. The method is therefore well-suited for routine quality control testing and for biosimilar comparability assessment of Pembrolizumab.