title: Generation of stable cell clones expressing mixtures of human antibodies.
abstract: Therapeutic monoclonal antibodies, a highly successful class of biological drugs, are conventionally manufactured in mammalian cell lines. A recent approach to increase the therapeutic effectiveness of monoclonal antibodies has been to combine two or more of them; however this increases the complexity of development and manufacture. To address this issue a method to efficiently express multiple monoclonal antibodies from a single cell has been developed and we describe here the generation of stable cell clones that express high levels of a human monoclonal antibody mixture. PER.C6 cells were transfected with a combination of plasmids containing genes encoding three different antibodies. Clones that express the three corresponding antibody specificities were identified, subcloned, and passaged in the absence of antibiotic selection pressure. At several time points, batch production runs were analyzed for stable growth and IgG production characteristics. The majority (11/12) of subclones analyzed expressed all three antibody specificities in constant ratios with total IgG productivity ranging between 15 and 20 pg/cell/day under suboptimal culture conditions after up to 67 population doublings. The growth and IgG production characteristics of the stable clones reported here resemble those of single monoclonal antibody cell lines from conventional clone generation programs. We conclude that the methodology described here is applicable to the generation of stable PER.C6(R) clones for industrial scale production of mixtures of antibodies.
authors.affiliation: Merus Biopharmaceuticals, Postvak 133, Padualaan 8, 3584 CH Utrecht, The Netherlands. j.dekruif@merus.nl
authors.forename: John
authors.initials: J
authors.lastname: de Kruif
authors.forename: Arjen
authors.initials: A
authors.lastname: Kramer
authors.forename: Roy
authors.initials: R
authors.lastname: Nijhuis
authors.forename: Vanessa
authors.initials: V
authors.lastname: van der Zande
authors.forename: Renate
authors.initials: R
authors.lastname: den Blanken
authors.forename: Carina
authors.initials: C
authors.lastname: Clements
authors.forename: Therèse
authors.initials: T
authors.lastname: Visser
authors.forename: Rob
authors.initials: R
authors.lastname: Keehnen
authors.forename: Marcel
authors.initials: M
authors.lastname: den Hartog
authors.forename: Mark
authors.initials: M
authors.lastname: Throsby
authors.forename: Ton
authors.initials: T
authors.lastname: Logtenberg
mesh_terms: D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000235:genetics; D001709:Biotechnology / Q000379:methods*; D018929:Cell Culture Techniques; D002460:Cell Line; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins