The Development of Gel Media and Columns for Large-Scale Chromatography of Proteins, a Historical Review and Visions for the Future

摘要

The history of modern protein purification begins around 1950. Before then the separation and purification of enzymes and other proteins was primarily performed by precipitation and adsorption methods using a variety of inorganic as well as organic materials. The first attempts to separate proteins by elution chromatography were based on the weakly acidic resin cation exchanger Amberlite IRC-50 (a co-polymer of methacrylic acid and divinylbenzene) and on calcium phosphate. The early resin ion exchangers suffered primarily from low porosity and too high hydrophobicity, restricting their application area to low molecular weight basic proteins. The calcium phosphate crystallites were both chemically and physically unstable. These pioneering efforts were followed by two major breakthroughs: the introduction of cellulose ion exchangers and of hydroxyapatite The hydrophilic nature of these materials made them especially suited for purification of high molecular weight proteins with a wide variety of iso-electric points and polarities. The possibility of separating water soluble molecules according to their size by molecular sieving in neutral gel materials were discussed already in the late 1940s. Some years later, preliminary reports on starch were followed by the development of more suitable materials such as cross-linked dextran, cross-linked polyacrylamide and agarose. An account of the history of the development of cross-linked dextran (Sephadex~R) has been published. The cross-linked dextran gels were also derivatized to ion exchangers which were rapidly adopted also to industrial applications. In the late 1960s the CNBr-method for activation and coupling of water soluble, primary amino group-containing molecules to agarose gels was developed. By the utilization of this technology for the synthesis of biospecific adsorbents for the purification of enzymes and other proteins the technique of affinity chromatography was introduced. Its rapid acceptance was facilitated by the commercial availability of CNBr preactivated 4% agarose by Pharmacia in 1971. The polysaccharide matrix of agarose gels is held together primarily by hydrogen bonds. Such gels are thermoreversible and, furthermore, substitution with e.g. ionizable groups would lead to gel instability and eventually leakage. To overcome these weaknesses, cross-linked bead shaped agarose was introduced by Porath et al. in 1971. Later work showed that it is possible to accomplish considerably higher rigidity using improved cross-linking chemistries for agarose enabeling the synthesis of 10μm particles for HPLC of proteins and also the manufacturing of media suitable for industrial applications. Other important chromatographic materials developed during the 70:es and 80:es include various porous silica bonded phases and several synthetic organic polymer based materials. Particular interest has been focused on monosized polystyrene-divinylbenzene particles. So, the first dedicated protein chromatography media were introduced during the fifties and sixties. As mentioned above, there was an early awareness of the possibility of using these for production applications within the biopharmaceutical industry, especially the human plasma protein fractionation industry. However, the crucial limitation was the fact that those media that were most compatible with proteins lent themselves less favourably to scaling-up. The problems were primarily physical. Thus the fibrous cellulose media showed bed cracking tendencies and the bead shaped polyacrylamide, dextran, and agarose gel media, then available, were too soft to stand the hydrodynamic forces acting in large columns, leading to bed compaction and increased pressure drop.