PubMed · 14186680
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G C MARTIN, M W WILLIAMS. 1964. QUANTITATIVE PHLORIDZIN ANALYSIS.. https://doi.org/10.1016/0003-2697(64)90053-3
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The chromatographic purification of biological macromolecules requires a novel approach to overcome some of the pore size limitations of commercially available resins. Membrane adsorbers offer the potential for better resolution as well as productivity. Sharp peaks are gained by the rapid exchange rate with the adsorbing membranes associated with the convective flow path, in contrast to the pore diffusion requirement for resin exchange. The resolution advantage is preserved even when the very short bed heights of membranes are exploited for the purpose of exceptionally high flow rates and productivity. Breakthrough experiments were used to assess the membrane dynamic loading capacities of flexible macromolecules using supercoiled (SC) DNA as a model system. In contrast to reports for smaller biomolecules such as proteins and antibodies, the dynamic capacity for DNA was found to be highly dependent on flow rates and concentrations. Increasing flow rates induced DNA elongation, which increased the surface coverage and, in turn, lowered the capacity. Increasing concentrations beyond C*, the overlap concentration, led to exclusion-volume interactions, which reduced the size of DNA and increased the membrane adsorber capacity. In the chromatographic mode, membranes with a strongly positive charge were able to resolve various isoforms of DNA, surpassing the capabilities of analogous chromatographic resins. In this study, we found that the convective-flow-induced-structural behavior of DNA is responsible for the resolution in separation.
In accordance with the US Code of Federal Regulations 21CFR 211.84 (6)(d)(1), a specific identity test must be performed for the release of chromatography media (stationary phase) before use in production of human pharmaceuticals. Due to the complexity of the physical and chemical properties of these media, i.e., variable particle morphology, insolubility, and chemical inertness, the development of specific identity tests presents a challenge. In this paper we report a new strategy for media identification that uses a combination of three relatively simple techniques: Fourier transform infrared (FT-IR) and near infrared (NIR) spectroscopy in conjunction with search libraries, and particle size distribution analysis. The methods are well established and suitable for routine application in a quality control laboratory. A hierarchical selection procedure utilizing these methods permits assignment of a unique identity for each of the chromatography media in use at a given facility, and form the basis of release tests for the media. Although this strategy was developed using specific media, the generic nature of the technology and the selection strategy proposed would permit its application to other chromatography media as well.
Protein analysis techniques are developing fast due to the growing number of proteins obtained by recombinant DNA techniques. In the present paper we compare selected techniques, which are used for protein sizing, quantitation and molecular weight determination: sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), lab-on-a-chip or microfluidics technology (LoaC), size exclusion chromatography (SEC) and mass spectrometry (MS). We compare advantages and limitations of each technique in respect to different application areas, analysis time, protein sizing and quantitation performance.