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.

4.2 Manufacturing of Complex Biotherapeutic Protein Products: Medical Need and Rational for Monoclonal Antibody Mixtures, Multispecific Formats, and Fc-fusion Proteins

“…This strategy leads to generation of a mixture of three different mAbs of the types AA, AB, and BB when two heavy chains are transfected, and AA, AB, AC, BB, BC, and CC when three heavy chains are transfected each with a unique specificity (Figure 4.2.2(3)). Published data on clone stability studies suggest that the individual clones are stable over extended generations with respect to growth, production, and ratio of the different molecules [9]. The single-batch manufacturing approach (Figure 4.2.2(4)) also offers the possibility of simultaneous manufacturing of several constituent antibodies, however, with few limitations to the desired composition as compared to the Oligoclonic TM approach.…”

Monoclonal antibodies for the prevention of rabies: theory and clinical practice

“…96 Homologous products (recombinant) -fully human intact igG Important parameters that drive selection of the most optimal systems for expression of antibodies include basic structure, protein folding and glycosylation, 97 productivity, ease of purification, 98 product quality and quantity, safety issues, time to clinic and market, and economic considerations such as cost of goods and regulatory issues. 99 In general, eukaryotic systems such as mammalian cells, 99,100 insect cells, 97 yeast, 101 plants, 74,102 green algae, 103 and transgenic animals 104 hold the greatest promise for expressing recombinant human MAbs. However, mammalian cells are predominantly used because they can correctly fold, assemble, glycosylate, and secrete antibodies.…”

Promises and pitfalls for recombinant oligoclonal antibodies-based therapeutics in cancer and infectious disease

“…Several single pot technology platforms have been developed to overcome the challenges of reproducible cell growth and constant antibody ratios. Merus has developed a platform that utilizes PER.C6 cells stably expressing one common light chain and three heavy chains that is limited to the production of oligoclonal antibodies that do not include antigen specificity via the light chains [111]. A second approach was developed by Symphogen and is based on the site specific integration of the antibody expression cassette in order to favor consistent growth and expression (e.g.…”