Scale‐up of free‐flow electrophoresis: II. Purification of alcohol dehydrogenase from a crude yeast extract by field step electrophoresis and combined field step‐zone electrophoresis

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Abstract
Results of the purification of alcohol dehydrogenase (ADH) by field step electrophoresis and combined field step‐zone electrophoresis are presented. In field step electrophoresis, optimization of voltage, residence time and pH of the sample solution led to a maximal purification factor of 2.8 and a yield of 89% ADH. The limit of loading capacity was reached at a protein concentration of the sample solution of approximately 4 g/L, allowing a maximal throughput of 1.14 g/h with a yield of 86% and a 2.8‐fold purification in the Elphor VaP 22 apparatus. With a production scale apparatus a throughput of 2.07 g/h without any loss of separation quality could be achieved. By introducing the sample solution into the separation chamber through 3 inlets, simultaneously, the throughput was increased to 3.2 g/h with a purification factor of 2.7 and a yield of 82% ADH. For the combined field step‐zone electrophoresis method a maximum purification factor of 3.6 and a yield of 80% ADH were achieved. The loading capacity was limited to a 4.13 g/L protein concentration of the sample solution, resulting in a throughput of 440 mg/h. Injecting the sample solution simultaneously into 3 inlets resulted in a maximum throughput of 1.92 g/h with 3.1‐fold purification and a yield of 80% ADH. Zone electrophoresis, field step electrophoresis and a combination of both are compared with respect to resolution, throughput and the application potential in a protein purification scheme. A scale‐up to 3 g/h is possible in zone electrophoresis and field step electrophoresis. The highest yields and the best purification were achieved in zone electrophoresis with a purification factor of 4.7 while on field step electrophoresis this factor was only 2.8. The combination of both technique was less efficacious with respect to throughput, yield and purification factor. Due to the different sample concentrations used, zone electrophoresis and field step electrophoresis are complementary in a purification scheme.