Low pH induced aggregation of equine immunoglobulin G
Abstract
Protein aggregation during pharmaceutical processing results in loss of biological potency, particulate matter in formulations and even anaphylactic reactions in the patient. Equine IgG, purified from horse sera for use in preventing organ transplant rejections, readily produces soluble and insoluble aggregates under acidic conditions, such as those required for protein G affinity chromatography. In the present work, it has been found that the kinetics of equine IgG aggregation in the pH 3.4-4.3 range and ionic strength between 0.02-0.5 M are explained by either diffusion limited, reaction limited, or transitional kinetics depending upon solution pH and ionic strength. Aggregation is hindered by electrostatic repulsion between the proteins in the pH 4.0-4.5 range. Below pH 4.0, a greater degree of attractive force is present and electrostatic repulsion is no longer the rate limiting barrier for aggregation. Analysis of the aggregation kinetics also revealed that the aggregate growth rate increased exponentially with decreasing pH in the above stated range, and that increasing ionic strength produced aggregates of larger mean size. The aggregation is attributed to unfolding of the protein and exposure of previously buried hydrophobic surface. It was also discovered that equine polyclonal IgG molecules with different susceptibilities to aggregation could be separated by ion exchange chromatography. The increased aggregation found in later eluting fractions from the ion exchange column was most likely the result of a relatively greater amount of the IgGb subclass. The subclass is more likely to aggregate because of a lower net surface charge and a greater degree of hydrophobic surface exposure at reduced pH. Finally, aggregation during protein G affinity chromatography was inhibited by the presence of 0.5 M sucrose, a preferentially excluded solute which stabilizes native protein structure by inhibiting unfolding.
Degree
Ph.D.
Advisors
Nail, Purdue University.
Subject Area
Biochemistry|Pharmaceuticals
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