Protein Standardization I: Protein Purification. Procedure for the Purification of Human Prealbumin, Orosomucoid and Transferrin as Primary Protein Preparations

A procedure for the purification of human prealbumin, orosomucoid and transferrin as primary protein preparations has been developed. The procedure describes in detail the chemicals, the fractionation equipment and the purification of the three proteins from the same starting material (a 90-donor plasma pool). The fractionation steps involve methods like salting out, various anion and cation exchange chromatographies, preparative electrophoresis, and finally, size chromatography. Only mild and highly reproducible fractionation methods are used in order to obtain high recoveries. All of these provide the best guarantee that no serious subfractionation has taken place. At the same time, high recoveries are a necessity for the pure protein to be representative of the same protein in vivo. The following recoveries were obtained: prealbumin 55%, orosomucoid 70% and transferrin 85%. The pure proteins are produced as liquid calibrators (primary protein preparations) dissolved in one electrolyte (0.1 mol/l KCl) and stored in sealed glass ampoules at -80 degrees C. These three pure proteins were used as primary reference preparations in the certification of the international reference preparation CRM 470.