Purification of antibody and antibody‐fragment from E. coli homogenate using 6,9‐diamino‐2‐ethoxyacridine lactate as precipitation agent

Abstract

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(abV) 2 molecules and a full-length antibody produced in E. coli. The two F(abV) 2 proteins were F(abV) 2 A and F(abV) 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(abV) 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%. B

Improved procedure for separation and purification of Arthronema africanum phycobiliproteins

“…Similar effect of lowering the concentration of NaCl on the purification of recombinant antibodies during rivanol treatment has been observed (Persson and Lester 2004).…”

ALLOPHYCOCYANIN FROM A LOCAL ISOLATEGEITLERINEMASP. A28DM (CYANOBACTERIA): A SIMPLE AND EFFICIENT PURIFICATION PROCESS

“…Hence, we used ethodin as an alternative precipitating agent. The ethodin molecule is positively charged over a wider pH range (Persson and Lester 2004). However, changing the pH will change the charge on the proteins.…”

“…As ethodin precipitates partly due to the charged characteristics of the molecule, it seems probable that the conductivity of the sample might have an effect on the purity. Hence, addition of a low concentration of NaCl (150 mM) in the buffer considerably improved the effective interaction of ethodin (Persson and Lester 2004) with the crude extract components, leading to a significant purification of C‐APC.…”

“…The concentration of ethodin plays a major role in protein purification (Persson and Lester 2004). We exploited this property in C‐APC purification.…”

A Role of the 9-Aminoacridines and their Conjugates in a Life Science

“…The 6, 9-diamino-2-ethoxyacridine lactate was used for precipitation of cell homogenate in order to purify the synthesized anti-bodies. The advantage of such precipitant consists in its ability to remove cellular protein and also cellular DNA from medium [171].…”