Characterising of chromatography gels for purification of erythropoietin

Abstract: Erythropoietin is a human natural hormone which task is to regulate the amount of red blood cells in the body. At Centro de Inmunología Molecular, situated in Havana, erythropoietin is produced by recombinant DNA-technique. The protein is purified through several chromatography steps. Among other things, Centro de Inmunología Molecular uses affinity chromatography and ion exchange chromatography. To both of these chromatographic methods, gel is used as stationary phase. The aim of this study was to investigate and determine parameters for characterising of two gels, this because Centro de Inmunología Molecular have to exchange the gels. The reason for the gel exchange is that the currently used gels will not be manufactured any more. The gel used in the affinity chromatography is Chelating Sepharose Fast Flow and the gel used in the ion exchange chromatography is Q Sepharose Fast Flow. For both of this gels kinetic parameters and isotherm parameters were determined by experiments. The isotherm parameters qmax and Kd were calculated from an adsorption isotherm. To be able to calculate qmax and Kd for both Q Sepharose Fast Flow gel and Chelating Sepharose Fast Flow gel different experiments were made. A kinetic adsorption and an isotherm adsorption were made on each gel. The kinetic adsorption was made in due to find out how long the two different processes were supposed to run and to understand which part of the mass transfer that is controlling the rate. There is no use to let the process to be in progress any longer than until the adsorption ceases. For the Q Sepharose Fast Flow gel this was after 200 seconds. The adsorption with the Chelating Sepharose Fast Flow gel never ceased completely, but after 1000 seconds the adsorption was so slow that it would be no use to continue the process. If the processes continue after the calculated times only money, hours and recourses will be wasted. The data that were achieved was plotted in two different isotherm adsorption models both the Freundlich- and the Langmuir model, this to determine which model that had the best fit. One could see that the Q Sepharose Fast Flow gel was following the model of Langmuir better and because of this the Langmuir equation was used to calculate qmax and Kd. The qmax for the Q Sepharose Fast Flow gel agreed a lot with the value that Centro de Inmunología Molecular had assumed. When it came to the Chelating Sepharose Fast Flow gel, the same kind of plotting was made. But one could see that this time the data was following the model of Freundlich much better. Therefore a calculation of the desired qmax was impossible. Only the value of Kd was calculated. Because the company Centro de Inmunología Molecular still needed the value of qmax an assumption that the gel was following the model of Langmuir was made. qmax was calculated but without any satisfied results. The programs Excel, Statgraphic and Matlab have been used in all calculations.