MEP HyperCel chromatography II: binding, washing and elution.
Binding, washing and elution conditions for MEP HyperCel chromatography were examined using two conditioned media (CM) containing monoclonal antibodies (humanized IgG1) and bovine serum albumin (BSA). Monoclonal antibodies derived from mammalian expression system bound to the column without pretreatment, although a majority of contaminating proteins present in the CM also showed binding. Inorganic salts, ethanol and glycerol were ineffective in eluting proteins under the conditions examined, suggesting that electrostatic or hydrophobic interactions are not a major factor for antibody binding to the MEP resin. Ethylene glycol, 2-propanol, urea and arginine were effective, to varying degrees, in elution of the bound proteins. The bound contaminating proteins and BSA were effectively eluted with ethylene glycol and the bound antibodies were finally eluted with aqueous arginine solutions at neutral pH. MEP showed selectivity toward BSA and hence utility for removing BSA from the samples. Interestingly, Fc-fusion proteins derived from silkworm larvae showed no detectable binding. Serum proteins present in silkworm larvae strongly competed with the Fc-fusion proteins and monoclonal antibody for binding to MEP resin, while the same Fc-fusion proteins can be readily purified in one-step by Protein-A resin, again confirming weak selectivity of the MEP resin.
Arakawa
Tsutomu
T
Alliance Protein Laboratories, Thousand Oaks, CA 91360, USA. tarakawa2@aol.com
Futatsumori-Sugai
Mutsumi
M
Tsumoto
Kouhei
K
Kita
Yoshiko
Y
Sato
Haruna
H
Ejima
Daisuke
D
D019840:2-Propanol / Q000378:metabolism; D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism*; D001120:Arginine / Q000737:chemistry / Q000378:metabolism; D012831:Bombyx / Q000378:metabolism; D002417:Cattle; D002845:Chromatography / Q000379:methods*; D057927:Hydrophobic and Hydrophilic Interactions; D007136:Immunoglobulins / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000378:metabolism*; D012996:Solutions / Q000378:metabolism; D013205:Staphylococcal Protein A / Q000737:chemistry / Q000378:metabolism; D014508:Urea / Q000737:chemistry / Q000378:metabolism; D014867:Water / Q000378:metabolism
D000906:Antibodies; D000911:Antibodies, Monoclonal; D007136:Immunoglobulins; D011506:Proteins; D011994:Recombinant Proteins; D012996:Solutions; D013205:Staphylococcal Protein A; D014867:Water; D012710:Serum Albumin, Bovine; D014508:Urea; D001120:Arginine; D019840:2-Propanol