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Introduction
The latest publication from the CuriRx team illustrates an improved HPLC method to ensure better analyses and better separation of peaks regarding theoretical plate, peak width, height, symmetry, and mitigating impurities’ interaction with the column.
Link:
https://link.springer.com/article/10.1007/s11095-018-2446-x
Abstract
Purpose
Accurate quantification of the intact proteins, antibodies, or peptides and their impurities without interaction with silanols of the HPLC column.
Methods
Hydroxypropyl ß Cyclodextrin (HPCD) is added in the mobile phase at different concentrations. Commercial SEC-HPLC columns and biologics with molecular weights ranging from 5.8 kDa to 150 kDa were assessed with and without cyclodextrin.
Results
The addition of non-ionic sugars such as Hydroxypropyl ß Cyclodextrin in the mobile phase resulted in improved peak performance such as theoretical plates, peak resolution, peak width, peak height, and improved quantification of aggregates in biologics such as antibodies Humira and Actemra, and peptides such as insulin. There is an increase in peak height, reduced retention time, increased plate, and reduced peak width with increasing concentration of cyclodextrin studied.
Discussion
High ionic strength, basic amino acids such as arginine, organic solvents (with a concentration low enough not to precipitate protein), sodium perchlorate, and ion pairing agents in the mobile phase used for the separation of peptides, proteins, and antibodies to prevent silanol interaction. These commonly used solutions are not always successful, as they not only interact with the biological but are sometimes not compatible. The non-ionic cyclodextrin itself does not cause protein aggregation. Still, it prevents the nonspecific binding or interaction of the protein itself, allowing for improved resolution and accurate quantification of aggregates in antibodies and peptides. The data on the separation in the presence of cyclodextrin in the mobile phase showed higher peak resolution, improved peak shape, accurate apparent molecular weight, improved efficiency, and less peak tailing for biological products.
Conclusion
Hydroxypropyl ß Cyclodextrin in the mobile phase resulted in improved SEC-HPLC resolution and quantitation of aggregates in biologics by preventing the interaction of biologics to silanol of the commercial SEC-HPLC columns.
