ABSTRACT
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.
Subject(s)
Immunoglobulins, Intravenous/analysis , Autoanalysis , Blood Proteins/analysis , Chromatography, Affinity , Chromatography, Gel , Chromatography, High Pressure Liquid , Humans , Staphylococcal Protein A/chemistryABSTRACT
The production of recombinant gamma-interferon was monitored using high-performance liquid chromatographic methods. These methods were able to distinguish between glycosylated and non-glycosylated forms of gamma-interferon by complexing the carbohydrate with borate. Sufficient quantities of standard glycosylated gamma-interferon were not available for peak identification so immunological techniques were used to identify gamma-interferon variants. These techniques were validated with the non-glycosylated form. The non-glycosylated form was then shown to be retained only on a cation-exchange column, while the glycosylated form, complexed with borate, was retained only on an anion-exchange column. Samples were drawn at 2-h intervals over a 60-h production cycle and analyzed by both anion- and cation-exchange chromatography. Results indicated that the production of each form was coincidental and that the glycosylated form of gamma-interferon is produced in greater abundance than non-glycosylated.
Subject(s)
CHO Cells/metabolism , Interferon-gamma/metabolism , Animals , Chromatography, High Pressure Liquid/methods , Chromatography, Ion Exchange/methods , Cricetinae , Recombinant Proteins , Time FactorsABSTRACT
Antifibronectin, monoclonal antibody was monitored through 52 h of production. Samples were automatically drawn from a bioreactor into the injection valve of an HPLC system without prior sample preparation. The hybridoma cell line was nonadherent, so whole cells were injected directly onto the perfusable protein A affinity column. There was only a modest column back pressure (ca. 1700 psi at a linear flow rate of 1.5 cm/s) after over 75 injections over the 52-h experiment. These experiments demonstrate the utility of high-speed chromatography for rapid process monitoring.
Subject(s)
Antibodies, Monoclonal/biosynthesis , Fibronectins/immunology , Immunoglobulin G/biosynthesis , Animals , Chromatography, High Pressure Liquid , Hybridomas , Hydrogen-Ion Concentration , Spectrophotometry, UltravioletABSTRACT
Detection of protein variants in the production of recombinant DNA products is an important and complex task. Rapid acquisition of this information permits feedback control of the production process and continuous validation of the product. Much of the technology required for rapid process monitoring is currently available or under development.
