title: Recombinant IgA production: single step affinity purification using camelid ligands and product characterization.
abstract: Immunoglobulins of isotype A (IgA) mediate a key role in mucosal immunity and are promising new immunotherapeutic candidates, but difficulties in obtaining enough material often hamper their in vivo exploration. We established recombinant Chinese hamster ovary (CHO) cells which stably expressed two IgA1 antibodies under serum-free conditions. The two cell lines achieved significantly different specific productivities of 16 pg per cell and day and 100 times less, a common phenomenon in recombinant antibody expression which challenges the production and purification process. Polymeric IgA in crude culture supernatants was assembled with J chain and showed expected specificity. We employed an immobilized camelid anti-human alpha-chain VHH ligand and isolated both recombinant IgAs at high purity and yield in a single chromatographic step. The described method was irrespective of the light chain and specificity and may be used as a generic capture step for the isolation of any IgA. Results were compared with a multistep purification process consisting of an affinity step based on immobilized jacalin followed by anion exchange and hydrophobic interaction chromatography.
authors.affiliation: Department of Biotechnology, University of Natural Resources and Life Sciences, Muthgasse 11, 1190 Vienna, Austria. David.reinhart@boku.ac.at
authors.forename: David
authors.initials: D
authors.lastname: Reinhart
authors.forename: Robert
authors.initials: R
authors.lastname: Weik
authors.forename: Renate
authors.initials: R
authors.lastname: Kunert
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000302:isolation & purification; D016466:CHO Cells; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods; D006224:Cricetinae; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007070:Immunoglobulin A / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D007147:Immunoglobulin Light Chains / Q000276:immunology / Q000302:isolation & purification; D007137:Immunoglobulin alpha-Chains / Q000276:immunology / Q000302:isolation & purification; D008024:Ligands; D037121:Plant Lectins / Q000276:immunology; D010957:Plasmids / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
chemical_list: D000906:Antibodies; D007070:Immunoglobulin A; D007147:Immunoglobulin Light Chains; D007137:Immunoglobulin alpha-Chains; D008024:Ligands; D037121:Plant Lectins; D011994:Recombinant Proteins; C043696:jacalin; C034639:polymeric IgA