Simultaneous determination of protein aggregation, degradation, and absolute molecular weight by size exclusion chromatography?multiangle laser light scattering - Analytical Methods & Tools for Lyophilization

Simultaneous determination of protein aggregation, degradation, and absolute molecular weight by size exclusion chromatography?multiangle laser light scattering
Received 10 March 2006. Available online 9 June 2006.
Hongping Ye
Analytical Biochemistry
Volume 356, Issue 1 , 1 September 2006
ScienceDirect
aDivision of Pharmaceutical Analysis, Food and Drug Administration, 1114 Market Street, Room 1002, St. Louis, MO 63101, USA
Abstract
The feasibility of size exclusion chromotography (SEC)?multiangle laser-light scattering as a technique to investigate aggregation and degradation of glycosylated and nonglycosylated proteins, and antibodies under various conditions such as addition of detergent, changes in pH, and variation of protein concentration and heat stress temperature was examined. Separation of proteins and their aggregates was performed using SEC?high-performance liquid chromatography. Detection of analytes was carried out with on-line UV, refractive index, and multiangle laser light-scattering detectors. Quantification and molecular weight determination were performed using commercial software. Aggregation and degradation were examined under various conditions and quantitative results are presented for bovine serum albumin, choriogonadotropin, glyceraldehyde-3-phosphate dehydrogenase, Herceptin, and ReoPro. This method can simultaneously determine both the quantities and the molecular weights of macromolecules from a single injection. The determination of molecular weight is absolute which avoids misleading results caused by molecular shape or interactions with the column matrix. This technique is valuable not only for assessing the extent of aggregation but also for effectively monitoring molecule degradation as evidenced by molecular weight reduction and change in monomer amount.
Keywords: SEC-MALLS; Protein aggregation; Absolute molecular weight; Protein degradation; Pharmaceutical proteins; Glycoproteins

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