Engineering mammalian cell lines that stably express many transgenes requires the precise insertion of large amounts of heterologous DNA into well-characterized genomic loci, but current methods are limited. To facilitate reliable large-scale engineering of CHO cells, we identified 21 novel genomic sites that supported stable long-term expression of transgenes, and then constructed cell lines containing one, two or three ‘landing pad’ recombination sites at selected loci. By using a highly efficient BxB1 recombinase along with different selection markers at each site, we directed recombinase-mediated insertion of heterologous DNA to selected sites, including targeting all three with a single transfection. We used this method to controllably integrate up to nine copies of a monoclonal antibody, representing about 100 kb of heterologous DNA in 21 transcriptional units. Because the integration was targeted to pre-validated loci, recombinant protein expression remained stable for weeks and additional copies of the antibody cassette in the integrated payload resulted in a linear increase in antibody expression. Overall, this multi-copy site-specific integration platform allows for controllable and reproducible insertion of large amounts of DNA into stable genomic sites, which has broad applications for mammalian synthetic biology, recombinant protein production and biomanufacturing.

As CHO cell line development for biotherapeutic production becomes more sophisticated through the availability of the CHO genome sequence, the ability to accurately and reproducibly engineer the host cell genome has become increasingly important. Multiple well characterized systems for site-specific integration will enable more complex cell line engineering to generate cell lines with desirable attributes. We built and characterized a novel recombinase mediated cassette exchange (RMCE) system using Bxb1 integrase and compared it to the commonly used Flp/FRT RMCE system. We first integrated a DNA construct flanked by either Bxb1 attachment sites or FRT sequences (referred to as a landing pad) into the Fer1L4 genomic locus of CHO-S cells using CRISPR/Cas9 mediated homologous recombination. We characterized the resulting clones harboring either the Bxb1 or Flp/FRT landing pad using whole genome resequencing to compare their genomes with the parental host cell line. We determined that each landing pad was specifically integrated into the Fer1L4 locus in the selected clones and observed no major structural changes in the genome or variations in copy number as a result of CRISPR/Cas9 modification. We subsequently tested the ability of the Bxb1 and Flp/FRT landing pad clones to perform proper RMCE with donor vectors containing identical mAb expression cassettes flanked by either Bxb1 attachment sites or FRT sites. We demonstrated that both RMCE systems were able to generate stable pools in a similar time frame with comparable mAb expression. Through genetic characterization of up to 24 clones derived from either system, we determined that the BxB1 RMCE system yielded higher fidelity RMCE events than the Flp/FRT system as evidenced by a higher percentage of clones with expected integration of the mAb cassette into the landing pad in the respective cell lines. We conclude that Bxb1 RMCE is an excellent alternative to Flp/FRT RMCE and valuable addition to our toolbox enabling the engineering of more sophisticated cell lines for biotherapeutic production. Biotechnol. Bioeng. 2017;114: 1837-1846. © 2017 Wiley Periodicals, Inc.

Phage-derived integrases can catalyze irreversible, site-specific integration of transgenic payloads into a chromosomal locus, resulting in mammalian cells that stably express transgenes or circuits of interest. Previous studies have demonstrated high-efficiency integration by the Bxb1 integrase in mammalian cells. Here, we show that a point mutation (Bxb1-GA) in Bxb1 target sites significantly increases Bxb1-mediated integration efficiency at the Rosa26 locus in Chinese hamster ovary cells, resulting in the highest integration efficiency reported with a site-specific integrase in mammalian cells. Bxb1-GA point mutant sites do not crossreact with Bxb1 wild-type sites, enabling their use in applications that require orthogonal pairs of target sites. In comparison, we test the efficiency and orthogonality of ϕC31 and Wβ integrases, and show that Wβ has an integration efficiency between those of Bxb1-GA and wild-type Bxb1. Our data present a toolbox of integrases for inserting payloads such as gene circuits or therapeutic transgenes into mammalian cell lines.

Photocleavage of chicken hen egg lysozyme by three Co(III)ammine complexes, hexamminecobalt(III) chloride ([Co(NH3)6]+3), pentamminechloro cobalt(III)chloride ([Co(NH3)5Cl]+2), and tetramminecarbonato cobalt(III) nitrate ([Co(NH3)4CO3]+), is reported here. Photocleavage resulted in two fragments of molecular masses of approximately 10.5 kDa and approximately 3.5 kDa which add-up to that of the parent molar mass. Detailed studies on the influence of irradiation time, excitation wavelength, the type of ligand coordinated to Co(III), concentration of the metal complex, the addition of competing metal ions, and quenchers on the protein photocleavage are reported. The Co(III) complexes also photocleaved apotransferrin, bovine serum albumin, and yeast enolase. Near-equimolar concentrations of Ni(II), Co(II) or Gd(III) inhibited the photocleavage, and therefore, binding of Co(III) metal complexes to Ni(II)/Co(II)/Gd(III) binding sites on lysozyme is necessary for the observed photocleavage. Since these ions are known to bind to Asp52 on lysozyme, we suspect that the above Co(III) complexes bind at this site, and initiate the protein cleavage. The Co(III) complexes have appropriate photochemical reactivities to cleave the peptide backbone, and they may be useful in the design of novel photochemical approaches to cleave the protein backbone.

Chinese Hamster Ovary (CHO) cells are used for the production of therapeutic proteins. This work examines improving passaging growth rate of two CHO clones. Growth rates were significantly improved for both clones with supplementation of the nucleosides cytidine, hypoxanthine, uridine, and thymidine to the culturing media at the optimal concentration of 100 lM of each nucleoside. We investigated supplementing the same combination of nucleosides to seed bioreactors and production fed batch bioreactors. In the seed bioreactors, growth rate and harvest density were improved. However, in the production fed batch bioreactors, no improvements in growth rate or peak viable cell density were observed. Cell cycle analysis of the passaging cells provides evidence that nucleosides can affect the cell cycle. It is not clear from our work how the nucleosides impact the cell cycle regulatory pathways. Overall, nucleoside supplementation in cell culture media is an effective approach for improving growth rate in passaging and seed bioreactors of certain CHO cells.