11.25 Diafiltration of recombinant protein A glycosylated recombinant protein is
ID: 1047046 • Letter: 1
Question
11.25 Diafiltration of recombinant protein A glycosylated recombinant protein is produced using mink lung epithelial cells. In the first step of protein recovery from the culture liquor, the protein is precipitated by addition of (NH)2S04 and the precipitate harvested by centrifugation. When redissolved in buffer, the precipitate yields 150 litres of solution containing 18 mg1 of recombinant protein and 50 g1 of (NH)2SO4. Constant-volume diafiltration is used to desalt the protein and replace the suspending buffer with a tris(hydroxymethyl)aminomethane (Tris) solution prior to additional protein purification by chromatography. Diafiltration is carried out using a 2.5-m2 membrane that fully retains the protein while allowing (NH)2SO4 and Tris to pass through freely. The membrane filter is operated under gel polarisation conditions with a mass transfer coefficient of 1.4x 10 5m s1. The gelation concentration of the recombinant protein is 120 mg1-1. The (NH2SO4 concentration in the final protein solution must be no greater than 0.1 g 1-1. Fouling effects can be neglected (a) What volume of Tris solution is needed? (b) Determine the time required for diafiltration -1Explanation / Answer
ANSWER :
Approximately 800 ml of Tris solution will be needed for diafiltration of recombinant protein.
Approximately 90 minutes would be required for the diafiltration.
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