Membrane chromatography for rapid purification of recombinant antithrombin III and monoclonal antibodies from cell culture supernatant.
The task of purifying monoclonal antibodies (MAbs) and human recombinant antithrombin III (rATIII) from cell culture supernatant was carried out using two different approaches, both based on the use of membraneous matrices. The first approach employed a strongly acidic and a strongly basic membrane ion exchanger, which were evaluated for their ability to purify monoclonal antibodies and the human active recombinant antithrombin III from cell culture supernatant. Within minutes gram amounts of product could be purified in a high-flux system, specially developed for this purpose, achieving purities of 80% for MAbs and 75% for rATIII, respectively. The capacity of the acidic membrane ion exchanger for MAbs was found to be 1 mg/cm2 with recoveries up to 96% and that of the basic membrane ion exchanger for rATIII was 0.15 mg/cm2 with recoveries up to 91%. The second approach consisted of using heparin, a mucopolysaccharide with a strong affinity towards ATIII, coupled to amine-modified or epoxy-activated membranes by reductive amination, for the purification of rATIII. The ATIII binding capacities of the membranes were found to be 91 micrograms/cm2 for the amine-modified and 39 micrograms/cm2 for the epoxy-activated membrane, achieving purities of 75%. The coupling proved to be fairly stable over a period of 5 months and the membranes remained operable even after steam sterilization and treatment with sodium dodecyl sulphate. Final purification in both instances was carried out by gel filtration.
Lütkemeyer
D
D
Institute for Cell Culture Technology, University of Bielefeld, Germany.
Bretschneider
M
M
Büntemeyer
H
H
Lehmann
J
J
D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D000990:Antithrombin III / Q000302:isolation & purification*; D016466:CHO Cells; D002852:Chromatography, Ion Exchange / Q000379:methods*; D006224:Cricetinae; D016895:Culture Media, Serum-Free / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006493:Heparin / Q000737:chemistry; D006801:Humans; D006825:Hybridomas; D051379:Mice; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D016895:Culture Media, Serum-Free; D011994:Recombinant Proteins; D000990:Antithrombin III; D006493:Heparin