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Journal of Biochemistry Advance Access originally published online on October 3, 2008
Journal of Biochemistry 2008 144(6):733-739; doi:10.1093/jb/mvn128
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© The Authors 2008. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved

Single-step Purification of Pepsin-derived Monoclonal Antibody Fragments from Crude Murine Ascitic Fluids by Ceramic Hydroxyapatite High-performance Liquid Chromatography

Ayaka Moro1, Tomohiko Yoshitake2, Tetsuro Ogawa2,* and Tohru Ichimura1,{dagger}

1Department of Applied Chemistry, National Defense Academy, Yokosuka, Kanagawa 239-8686; and 2R&D Department of New Ceramic Division, Life Care Business Division, PENTAX Corporation, Itabashi-ku, Tokyo 174-8639, Japan

{dagger}To whom correspondence should be addressed. Tel: +81-468-41-3810 (ext. 3563), Fax: +81-468-41-5901, E-mail: ichimura{at}nda.ac.jp

Received July 9, 2008; Accepted September 15, 2008


  Abstract

Ceramic hydroxyapatite (CHT) high-performance liquid chromatography (HPLC) is used to purify a variety of classes of monoclonal antibodies (mAbs) from crude murine ascites fluids. We report here that this method is also applicable for simple and efficient purification of many mAb fragments that are generated by pepsin treatment of crude ascites. F(ab')2 fragments were quantitatively generated from IgG1 mAbs in ascitic fluids by incubation with pepsin for 6 h at pH 3.9–4.1. Under the same conditions, pepsin also cleaved unwanted ascites components, such as albumin and transferrin to very low molecular weight polypeptides. The F(ab')2 fragments, but not the low molecular weight products, selectively bound to and were eluted from the CHT column using a linear gradient of phosphate ion concentration over 15 min. The recovery of the F(ab')2 fragments by CHT-HPLC was >90%. This method also allowed single-step purification of mAb fragments from distinct IgG subclasses (IgG2a and IgG2b) and IgM directly from crude digested ascitic samples. This CHT-HPLC method combined with direct pepsinolysis of murine ascites is a useful strategy for rapid purification and characterization of many types of mAb fragments.

Key Words: antibody fragment, ceramic hydroxyapatite, high-performance liquid chromatography, monoclonal antibody, purification

Abbreviations: CHT, ceramic hydroxyapatite; HPLC, high-performance liquid chromatography; mAb, monoclonal antibody

*Present address: Laboratory Skin Care Inc., Taboe City, CA 96145-7469, USA.


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