Analytical immunology.
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Alkaline Phosphatase↗
Biomedical subjects
S K Paliwal
Publications and source records attributed to S K Paliwal.
Rapid separations of proteins by liquid chromatography.
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Buffers↗
Rapid, automated, two-dimensional high-performance liquid chromatographic analysis of immunoglobulin G and its multimers.
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.
Autoanalysis↗
Ensemble averaging and digital filtering in chromatography and electrophoresis.
Ensemble averaging and digital filtering were implemented for signal-to-noise ratio improvement in the separation techniques of size-exclusion chromatography, immunoaffinity chromatography, capillary zone electrophoresis and capillary ion analysis. Results of ensemble averaging were always greater than statistically predicted. Techniques included five to nine replicate separations and yielded signal-to-noise improvement factors of 2.5 to 9.3. Running-average and time constant (RC)-convolution digital filters yielded increases in the signal-to-noise ratio ranging from zero to twelve. This paper will discuss and illustrate the usage of ensemble averaging and digital filtering in liquid-phase separation techniques.
Algorithms↗
Process monitoring of the production of gamma-interferon in recombinant Chinese hamster ovary cells.
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.
Animals↗
Automated process monitoring of monoclonal antibody production.
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.
Animals↗
Liquid chromatography based enzyme-amplified immunological assays in fused-silica capillaries at the zeptomole level.
Through the use of fused-silica capillaries it was shown that reducing the liquid volume of an enzyme-amplified immunological assay increases the rate of amplification and sensitivity of the assay by several orders of magnitude. Human immunoglobulin G (hIgG) captured on protein G-coated 100-microns-i.d. columns was saturated with F(ab) anti-hIgG conjugated to alkaline phosphatase (ALP). Conjugated enzyme captured by the antigen was subsequently assayed in a stop-flow incubation with p-nitrophenyl phosphate. p-Nitrophenol produced in the stop-flow incubation was then swept to the detector and quantitated at 405 nm. The detection limit in the stop-flow mode was approximately 3 fmol. Three problems were identified in this flow-through, capillary assay format. The first was that the rate of immunological complex formation within the capillary was too slow. Preforming the immunological complex before application to the column increased the sensitivity by 2 orders of magnitude. Another problem was that the rate of mass transfer within the capillary limited capture of the preformed immunological complex. This problem was solved by stop-flow incubation of the complex in the column. The combination of preformation of the immunological complex and stop-flow binding within the column reduced the detection limit to approximately 3 amol. Finally, reducing the amount of F(ab)-ALP used in the assay minimized nonspecific binding of the conjugated enzyme and reduced the detection limit further to 333 zmol.
Alkaline Phosphatase↗
Rapid process monitoring in biotechnology.
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.
Biotechnology↗