Sub Topic | Secondary Topic: Analytical Methodology | HPLC (Biopharmaceutical Molecule)
Authors: Jingyi Li, Pfizer Inc. (Main Author, Presenting Author)
Presenting Author: Jingyi Li
Purpose: Size-Exclusion Chromatography (SEC) has emerged as the most widely used analytical technique to determine invisible and semi-visible aggregates in peptide and protein biopharmaceuticals. Common protein standards such as Bovine serum albumin (BSA, 66 kDa), Ribonuclease A (13kDa) and Aprotinin (6.5kDa) are often used as system suitability standards to verify routine performance of SEC system for analysis of aggregates in peptide products. Although peptides, like proteins, have amino acids in the primary structures, they may not have secondary and tertiary structures as proteins do because peptides usually contain less than 50 amino acids. Therefore, the chromatographic merits of peptides may not vary with the chromatographic conditions in the same way as protein standards in SEC. It is necessary to evaluate the suitability of protein standards as system suitability standards and choose the proper protein standards to verify the routine performance of SEC for quality control of peptide products. This poster compares the chromatographic merits of small and medium size peptides to those of the protein standards when the concentrations and types of salts, % organic solvents in mobile phases and column temperature are varied in SEC. Also, the effect of % organic solvent and sample dilution on the amount of aggregates in a peptide product is determined by SEC-PDA. The presence of aggregates in the peptide product is also confirmed by SEC-refractive index detector (RI), SEC-multi-angle light scattering detector (MALD) and analytical ultracentrifugation analysis (AUC).
Methods: The effects of salt types and concentrations, % organic solvent in mobile phases and column temperatures on the retention times and tailing factors are compared for the peptides and protein standards in SEC-PDA. Additionally, the impact of sample dilution and % organic solvent in mobile phases on the aggregates and peptide are investigated to accurately determine the aggregates in a peptide product. SEC with multi-angle light scattering (MALD) and analytical ultracentrifugation (AUC) are used to confirm the presence of aggregates in the peptide product.
Results: It is found that the tailing factors and retention times of the larger protein standards do not change as much as the small and medium size peptides do when the %organic solvent, salt types and concentrations and column temperature are varied. However, the smaller protein standard Aprotinin behaves chromatographically similar to peptides. Organic solvent of 10% or higher in the mobile phases can degrade both the peptide product and aggregates for diluted and undiluted samples. Other orthogonal analytical techniques such as AUC, SEC-RI and SEC-MALD result in similar amount of aggregates as SEC-PDA does for the peptide product.
Conclusion: The study indicated that the tailing factors and retentions times of peptides and proteins varied differently with the change of chromatographic conditions in SEC. Therefore, it is necessary to evaluate the suitability of a protein standard used in a system suitability test to ensure a protein standard can properly verify the performance of SEC for routine analysis of peptide products. Also, effect of % organic solvent the in mobile phases should be investigated to accurately determine aggregates and peptide product for diluted and undiluted samples
See attached abstract pdf for images.