Purification of antibody and antibody-fragment from E. coli homogenate using 6,9-diamino-2-ethoxyacridine lactate as precipitation agent.
To obtain a more efficient purification process for antibody fragments from an Escherichia coli homogenate, the precipitant, Ethodin (6,9-diamino-2-ethoxyacridine lactate) was introduced to the homogenate. By adding the precipitant a drastic reduction of host cell protein was obtained. The majority of the proteins were recovered in a precipitate with the cell debris, while the antibody or antibody-fragment was recovered in the clarified supernatant. In addition, DNA was also efficiently precipitated when using Ethodin as a precipitation agent. The improved purity of the clarified extract obtained by using the precipitant allows for the use of smaller chromatography columns and may reduce the number of chromatographic steps required in the recovery process. The effect of Ethodin concentration, pH, temperature, and conductivity were investigated. The investigation was performed on two different antibody-fragments, e.g., F(ab')(2) molecules and a full-length antibody produced in E. coli. The two F(ab')(2) proteins were F(ab')(2)A and F(ab')(2)B, which have a similar molecular mass (100 kDa) but different isoelectric points (pIs), i.e., 8.9 and 7.5, respectively. The full-length antibody, Ab (the full IgG form of F(ab')(2)B) has a pI of 7.8 and molecular mass of 150 kDa. The investigation showed that the highest purification factors were obtained at neutral pH, low conductivity, and Ethodin concentrations of 0.6%.
AstraZeneca, 151 85 Södertälje, Sweden.
Josefine
J
Persson
Philip
P
Lester
D000906:Antibodies / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D002457:Cell Extracts / Q000737:chemistry* / Q000276:immunology*; D005591:Chemical Fractionation / Q000379:methods*; D004926:Escherichia coli / Q000276:immunology*; D004975:Ethacridine / Q000737:chemistry*; D005590:Fractional Precipitation; D006863:Hydrogen-Ion Concentration; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013696:Temperature
D000906:Antibodies; D002457:Cell Extracts; D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins; D004975:Ethacridine