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Rapid, automated, two-dimensional high-performance liquid chromatographic analysis of immunoglobulin G and its
T K Nadler1, S K Paliwal, F E Regnier
1Chemistry Department, Purdue University, West Lafayette, IN 47907.
It is important to determine the amount of IgG multimers in immunoglobulin-containing pharmaceuticals because these aggregates can cause adverse reactions in patients. Previous methods for determining aggregates either suffered from interference of other proteins or required fraction collection and sample purification. A new, automated two-dimensional approach has been developed in which size-exclusion chromatography is performed in the first dimension followed by protein A affinity chromatography in the second dimension. This method is robust in that the aggregates are not disturbed by a preliminary purification step. Further, the presence of contaminating proteins has no effect on the analysis since affinity chromatography is used to determine the presence of IgG in the second dimension. The entire automated two-dimensional analysis can be performed in ca. 1 h.
It is important to determine the amount of IgG multimers in immunoglobulin-containing pharmaceuticals because these aggregates can cause adverse reactions in patients. Previous methods for determining aggregates either suffered from interference of other proteins or required fraction collection and sample purification. A new, automated two-dimensional approach has been developed in which size-exclusion chromatography is performed in the first dimension followed by protein A affinity chromatography in the second dimension. This method is robust in that the aggregates are not disturbed by a preliminary purification step. Further, the presence of contaminating proteins has no effect on the analysis since affinity chromatography is used to determine the presence of IgG in the second dimension. The entire automated two-dimensional analysis can be performed in ca. 1 h.