In recent years, targeted gene integration (TI) has been introduced as a strategy for the generation of recombinant mammalian cell lines for the production of biotherapeutics. Besides reducing the immense heterogeneity within a pool of recombinant transfectants, TI also aims at shortening the duration of the current cell line development process. Here we describe the generation of a host cell line carrying Matrix‐Attachment Region (MAR)‐rich landing pads (LPs), which allow for the simultaneous and site‐specific integration of multiple genes of interest (GOIs). We show that several copies of each chicken lysozyme 5'MAR‐based LP containing either BxB1 wild type or mutated recombination sites, integrated at one random chromosomal locus of the host cell genome. We further demonstrate that these LP‐containing host cell lines can be used for the site‐specific integration of several GOIs and thus, generation of transgene‐expressing stable recombinant clones. Transgene expression was shown by site‐specific integration of heavy and light chain genes coding for a monospecific antibody (msAb) as well as for a bi‐specific antibody (bsAb). The genetic stability of the herein described LP‐based recombinant clones expressing msAb or bsAb was demonstrated by cultivating the recombinant clones and measuring antibody titers over 85 generations. We conclude that the host cell containing multiple copies of MAR‐rich landing pads can be successfully used for stable expression of one or several GOIs.

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