Comparison the efficacy of two sources of butyl media in downstream processing of monoclonal antibody and fusion protein
,1 Fatemeh ashouri
,2 Mansoureh askari
,3 Neda abadian
2. Biopharmaceutical Research Center, AryoGen Pharmed Inc., Alborz University of Medical Sciences, Karaj, Iran
3. Biopharmaceutical Research Center, AryoGen Pharmed Inc., Alborz University of Medical Sciences, Karaj, Iran
4. Biopharmaceutical Research Center, AryoGen Pharmed Inc., Alborz University of Medical Sciences, Karaj, Iran
Hydrophobic interaction chromatography (hic) separates proteins according to differences in their surface hydrophobicity by utilizing a reversible interaction between proteins and the hydrophobic surface of a hic media. generally, hic media with optimized particle and pore size have significantly improved binding capacity which can increase hic purification efficiency . toyopearl butyl-650 media from tosoh company is available in three particle sizes, 35 µm (s-grade), 65 µm (m-grade), and 100 µm (c-grade). on the other hand, butyl sepharose 4 fast flow from ge healthcare company is part of the sepharose fast flow hic platform with cross-linked 4 % agarose and ~90 μm particle size. in this study, the efficacy of toyopearl butyl-650m with 65 µm particle size was compared to butyl sepharose 4 fast flow in purification process.
In this study, two different hic media, toyopearl butyl-650m (butyl-tosoh) and butyl sepharose 4 fast flow (butyl-ge), were selected and the efficacy of them were evaluated in purification of a monoclonal antibody and a fusion protein. to follow this purpose, 6 ml of each media was packed in xk 16/20 column separately. in purification of the monoclonal antibody, hics chromatography were applied in binding mode in which the columns were equilibrated with 3 cv of equilibration buffer containing ammonium sulfate, ph 7.1 and protein solutions were loaded onto both columns separately. target protein was eluted using 20 mm tris, ph 7.1 with gradient elution procedure. to purify fusion protein, hics chromatography were used in flow-through mode and after the column equilibration with 3 cv containing nacl, ph 7.1, protein solutions were loaded onto both equilibrated columns separately.
The results of the monoclonal antibody purification showed that suitable binding capacity was gained by use of butyl-tosoh media and equilibration buffer containing 1 m ammonium sulfate. higher concentration of ammonium sulfate (1.2 m) was applied to bind the target protein to butyl-ge media. in addition, the results of butyl-tosoh cleared that appropriate resolution was obtained in the elution peaks which the main peak was clearly separated from the undesired isoforms with 0-100 % gradient in 6 cv. on the other hand, slight resolution was observed by butyl-ge media in spite of applying 0-100 % gradient in 20 cv. in the following, the results of fusion protein purification presented that the target protein was excluded in flow through mode in both media. in addition, like the results observed in antibody, butyl-tosoh media was able to separate impurities from fusion protein by buffer containing 1 m nacl which was about 2 fold less than the concentration of nacl in butyl-ge media.
The results of purification of the monoclonal antibody demonstrated that butyl-tosoh media in compare to butyl-ge not only has the better resolution in elution peak but also higher binding capacity is obtained with the similar concentration of salt in equilibration buffer. furthermore, the efficacy of these two media in flow-through mode in purification of the fusion protein showed the similar results. the difference between these media can be attributed to their particle sizes which influences the efficacy of them in purification of the monoclonal antibody and fusion protein.
Butyl sepharose 4 fast flow, hic, media, toyopearl butyl-650m