Rapid, two-step purification process for the preparation of pyrogen-free murine immunoglobulin G1 monoclonal antibodies.
A cost-efficient process was specifically designed for the preparation of gram amounts of highly pure murine immunoglobulin (Ig) G1 monoclonal antibodies (mAbs). This rapid, simple and scalable purification process employs a unique binding and elution protocol for IgG1 mAbs on a silica-based, mixed-mode ion-exchange resin followed by conventional anion-exchange chromatography. mAbs are bound to BakerBond ABx medium at pH 5.6 directly from serum-supplemented hybridoma culture supernatants. Contaminating proteins and nucleic acids are removed by an intermediate wash at pH'6.5, followed by the specific elution of IgG1 mAbs with 100 mM Tris-HCl (pH 8.5). The mAb eluate is then loaded directly on to QAE-Sepharose Fast Flow medium and eluted with 10 mM sodium phosphate buffer (pH 7.4), containing 150 mM sodium chloride. The resulting IgG1 mAbs are greater than 98% pure, free from measurable endotoxin, formulated in a physiological buffer and suitable for in vivo applications.
Neidhardt
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EA
PROCEPT, Inc., Cambridge, MA 02139.
Luther
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MA
Recny
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MA
D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D006825:Hybridomas; D007074:Immunoglobulin G / Q000302:isolation & purification*; D007525:Isoelectric Focusing; D051379:Mice; D011749:Pyrogens
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D011749:Pyrogens