Genome-scale functional genomics screening highlights genes impacting protein fucosylation in Chinese hamster ovary cells.

N-linked glycosylation is a common post-translational modification that has various effects on multiple types of proteins. The extent to which an N-linked glycoprotein is modified and the identity of glycans species involved is of great interest to the biopharmaceutical industry, since glycosylation can impact the efficacy and safety of therapeutic monoclonal antibodies (mAbs). mAbs lacking core fucose, for example, display enhanced clinical efficacy through increased antibody-dependent cellular cytotoxicity. We performed a genome-wide CRISPR knockout screen in Chinese hamster ovary (CHO) cells, the workhorse cell culture system for industrial production of mAbs, aimed at identifying novel regulators of protein fucosylation. Using a lectin binding assay, we identified 224 gene perturbations that significantly alter protein fucosylation, including well-known glycosylation genes. This functional genomics framework could readily be extended and applied to study the genetic pathways involved in regulation of other glycoforms. We hope this resource will provide useful guidance toward the development of next generation CHO cell lines and mAb therapeutics.

Improving the efficiency of CHO cell line generation using glutamine synthetase gene knockout cells.

Although Chinese hamster ovary (CHO) cells, with their unique characteristics, have become a major workhorse for the manufacture of therapeutic recombinant proteins, one of the major challenges in CHO cell line generation (CLG) is how to efficiently identify those rare, high-producing clones among a large population of low- and non-productive clones. It is not unusual that several hundred individual clones need to be screened for the identification of a commercial clonal cell line with acceptable productivity and growth profile making the cell line appropriate for commercial application. This inefficiency makes the process of CLG both time consuming and laborious. Currently, there are two main CHO expression systems, dihydrofolate reductase (DHFR)-based methotrexate (MTX) selection and glutamine synthetase (GS)-based methionine sulfoximine (MSX) selection, that have been in wide industrial use. Since selection of recombinant cell lines in the GS-CHO system is based on the balance between the expression of the GS gene introduced by the expression plasmid and the addition of the GS inhibitor, L-MSX, the expression of GS from the endogenous GS gene in parental CHOK1SV cells will likely interfere with the selection process. To study endogenous GS expression's potential impact on selection efficiency, GS-knockout CHOK1SV cell lines were generated using the zinc finger nuclease (ZFN) technology designed to specifically target the endogenous CHO GS gene. The high efficiency (∼2%) of bi-allelic modification on the CHO GS gene supports the unique advantages of the ZFN technology, especially in CHO cells. GS enzyme function disruption was confirmed by the observation of glutamine-dependent growth of all GS-knockout cell lines. Full evaluation of the GS-knockout cell lines in a standard industrial cell culture process was performed. Bulk culture productivity improved two- to three-fold through the use of GS-knockout cells as parent cells. The selection stringency was significantly increased, as indicated by the large reduction of non-producing and low-producing cells after 25 µM L-MSX selection, and resulted in a six-fold efficiency improvement in identifying similar numbers of high-productive cell lines for a given recombinant monoclonal antibody. The potential impact of GS-knockout cells on recombinant protein quality is also discussed.

Zebrafish primordial germ cell cultures derived from vasa::RFP transgenic embryos.

Although embryonic germ (EG) cell-mediated gene transfer has been successful in the mouse for more than a decade, this approach is limited in other species due to the difficulty of isolating the small numbers of progenitors of germ cell lineage (PGCs) from early-stage embryos and the lack of information on the in vitro culture requirements of the cells. In this study, methods were established for the culture of PGCs obtained from zebrafish embryos. Transgenic embryos that express the red fluorescent protein (RFP) under the control of the PGC-specific vasa promoter were used, making it possible to isolate pure populations of PGCs by fluorescence-activated cell sorting (FACS) and to optimize the culture conditions by counting the number of fluorescent PGC colonies produced in different media. Cultures initiated from 26-somite-stage embryos contained the highest percentage of PGCs that proliferated in vitro to generate colonies. The effect of growth factors, including Kit ligand a and b (Kitlga and Kitlgb) and stromal cell-derived factor 1a and 1b (Sdf-1a and Sdf-1b), on PGC proliferation was studied. Optimal in vitro growth and survival of the zebrafish PGCs was achieved when recombinant Kitlga and Sdf-1b were added to the culture medium through transfected feeder cells, resulting in a doubling of the number of PGC colonies. Results from RT-PCR and in situ hybridization analysis demonstrated that PGCs maintained in culture expressed the kita receptor, even though receptor expression was not detected in PGCs isolated by FACS directly from dissociated embryos. In optimal growth conditions, the PGCs continued to proliferate for at least 4 months in culture. The capacity to establish long-term PGC cultures from zebrafish will make it possible to conduct in vitro studies of germ cell differentiation and EG cell pluripotency in this model species and may be valuable for the development of a cell-mediated gene transfer approach.

Comparative study of therapeutic antibody candidates derived from mini-pool and clonal cell lines.

The long journey of developing a drug from initial discovery target identification to regulatory approval often leaves many patients with missed window of opportunities. Both regulatory agencies and biopharmaceutical industry continue to develop creative approaches to shorten the time of new drug development in order to deliver life-saving medicine to patients. Generally, drug substance materials to support the toxicology and early phase clinical study can only be manufactured after creating the final Master Cell Bank (MCB) of the clonally derived cell line, which normally takes 1-2 years. With recent advances in cell line development, cell culture process and analytical technologies, generating more homogeneous bulk/mini-pool population with higher productivity and acceptable quality attributes has become a norm, thereby making it possible to shorten the timeline to initiate First in Human (FIH) trial by using bulk/mini-pool generated materials to support toxicology and FIH studies. In this study, two monoclonal antibodies of different subclasses (IgG1 and IgG4) were expressed from the mini-pool cells as well as clonally derived cell lines generated from the same mini-pool. Cell growth, productivity, and product quality were compared between the materials generated from the mini-pool and clonally derived cell line. The results demonstrate the similarity of the antibody products generated from mini-pool cells and clonally derived cell lines from the same mini-pool, and strongly support the concept and feasibility of using antibody materials produced from mini-pool cultures for toxicology and FIH studies. The strategy to potentially shorten the FIH timeline is discussed. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1456-1462, 2017.

Zebrafish embryonic stem cells.

Methods are presented for the derivation of zebrafish embryonic stem (ES) cell cultures that are initiated from blastula and gastrula stage embryos. To maintain pluripotency, the ES cells are cocultured with rainbow trout spleen cells from the RTS34st cell line. ES cells maintained for multiple passages on a feeder layer of growth-arrested RTS34st exhibit in vitro characteristics of pluripotency and produce viable germ cells following transplantation into a host embryo. The ES cells are able to undergo targeted plasmid insertion by homologous recombination, and methods are described for the introduction of a targeting vector by electroporation. Two strategies are described for the efficient isolation of homologous recombinants using a visual marker screen and positive-negative selection.

Development of cell cultures with competency for contributing to the zebrafish germ line.

The zebrafish is an established model for the genetic analysis of vertebrate development. Forward-genetic screens have generated thousands of mutations, and antisense-based methods have been used to transiently knockdown gene expression during embryogenesis. Although these methods have made the zebrafish a valuable system for the identification and functional characterization of developmentally important genes, one deficiency of the zebrafish model is the absence of methods to introduce targeted mutations to generate knockout lines of fish. Application of gene-targeting methods has been limited in nonmurine species due to the absence of germ-line competent embryonic stem (ES) cell lines. Recently, progress was made in addressing this problem by the derivation of zebrafish embryo cell lines that remain pluripotent and germ-line competent for multiple passages in culture. Zebrafish germ-line chimeras were generated using cultures derived from embryos at two different developmental stages, and targeted insertion of vector DNA by homologous recombination was demonstrated in both cultures. Several strategies are being used to optimize the production and identification of germ-line chimeras. The zebrafish embryo cell culture system should provide the basis of a gene-targeting approach that will complement other genetic strategies and improve the utility of the zebrafish model for studies of development and disease.

Development of a highly-efficient CHO cell line generation system with engineered SV40E promoter.

Chinese hamster ovary (CHO) cells have been one of the most widely used host cells for the manufacture of therapeutic recombinant proteins. An effective and efficient clinical cell line development process, which could quickly identify those rare, high-producing cell lines among a large population of low and non-productive cells, is of considerable interest to speed up biological drug development. In the glutamine synthetase (GS)-CHO expression system, selection of top-producing cell lines is based on controlling the balance between the expression level of GS and the concentration of its specific inhibitor, l-methionine sulfoximine (MSX). The combined amount of GS expressed from plasmids that have been introduced through transfection and the endogenous CHO GS gene determine the stringency and efficiency of selection. Previous studies have shown significant improvement in selection stringency by using GS-knockout CHO cells, which eliminate background GS expression from the endogenous GS gene in CHOK1SV cells. To further improve selection stringency, a series of weakened SV40E promoters have been generated and used to modulate plasmid-based GS expression with the intent of manipulating GS-CHO selection, finely adjusting the balance between GS expression and GS inhibitor (MSX) levels. The reduction of SV40E promoter activities have been confirmed by TaqMan RT-PCR and GFP expression profiling. Significant productivity improvements in both bulk culture and individual clonal cell line have been achieved with the combined use of GS-knockout CHOK1SV cells and weakened SV40E promoters driving GS expression in the current cell line generation process. The selection stringency was significantly increased, as indicated by the shift towards higher distribution of producing-cell populations, even with no MSX added into cell culture medium. The potential applications of weakened SV40E promoter and GS-knockout cells in development of targeted integration and transient CHO expression systems are also discussed.

Initiation of a zebrafish blastula cell line on rainbow trout stromal cells and subsequent development under feeder-free conditions into a cell line, ZEB2J.

A continuous cell line, ZEB2, was developed from zebrafish blastula-stage embryos expressing enhanced green fluorescent protein (GFP). Originally the rainbow trout spleen cell line, RTS34st, was used as feeders to initiate and maintain the cells through several passages. ZEB2 was then grown for 2 years without feeders in L-15 with 15% fetal bovine serum (FBS) for 120 population doublings. This new cell line, ZEB2J, was heteroploid, had detectable telomerase activity, and was adherent. After growing into monolayers, some cells continued to grow into mounds. Cultures expressed Pou-2 mRNA and contained many alkaline phosphatase and a few stage-specific embryonic antigen-1-positive cells. In dishes coated with a phospholipid polymer (2-methacryloxyloxyethyl phosphorylcholine, MPC), ZEB2J formed spherical aggregates. Aggregates attached to conventional culture plastic, and most cells that emerged from aggregates had typical epithelial-like shapes of ZEB2J, which suggests that ZEB2J had limited differentiation potential, despite expressing some stem cell properties. The fluorescence of ZEB2J allowed relationships with feeder cells to be studied. In MPC dishes, ZEB2J formed mixed spheroids with RTS34st. In adherent cocultures, RTS34st and other fish cell lines strongly stimulated the ZEB2J growth, which could be quantified specifically because ZEB2J expressed GFP. ZEB2J should be useful for optimizing culture conditions for zebrafish embryonic stem cells.

Homologous recombination in zebrafish ES cells.

Targeted insertion of a plasmid by homologous recombination was demonstrated in zebrafish ES cell cultures. Two selection strategies were used to isolate ES cell colonies that contained targeted plasmid insertions in either the no tail or myostatin I gene. One selection strategy involved the manual isolation of targeted cell colonies that were identified by the loss of fluorescent protein gene expression. A second strategy used the diphtheria toxin A-chain gene in a positive-negative selection approach. Homologous recombination was confirmed by PCR, sequence and Southern blot analysis and colonies isolated using both selection methods were expanded and maintained for multiple passages. The results demonstrate that zebrafish ES cells have potential for use in a cell-mediated gene targeting approach.

Progress towards cell-mediated gene transfer in zebrafish.

Although the zebrafish possesses several favourable characteristics that make it an ideal model for genetic studies of vertebrate development, one disadvantage of this model system is the absence of methods for the production of gene knockouts. The authors' laboratory, and others, are working to develop zebrafish pluripotent embryonic stem (ES) and primordial germ cell (PGC) cultures that can be used for cell-mediated gene transfer and the production of knockout mutant lines of fish. Progress has been made in developing short-term cell cultures that possess the ability to contribute to multiple tissues, including the germ line of a host embryo, and transgenic lines of zebrafish have been established using the embryo cell cultures. Work is in progress to extend the length of time that the embryo cells can be maintained in culture without losing their ability to generate germ-line chimeras.

Zebrafish embryo cells remain pluripotent and germ-line competent for multiple passages in culture.

Mouse embryonic stem (ES) cell lines are routinely used to introduce targeted mutations into the genome, providing an efficient method to study gene function. Application of similar gene knockout techniques to other organisms has been unsuccessful due to the lack of germ-line competent ES cell lines from non-murine species. Previously, we reported the production of zebrafish germ-line chimeras using short-term primary embryo cell cultures. Here we demonstrate that zebrafish embryo cells, maintained for several weeks and multiple passages in culture, remain pluripotent and germ-line competent. Zebrafish germ-line chimeras were generated from passage 5 and 6 cultures initiated from blastula- and gastrula-stage embryos. In addition to the germ line, the cultured cells contributed to multiple tissues of the host embryo, including muscle, liver, gut, and fin. To facilitate the identification of germ-line chimeras, ES cells expressing the green fluorescent protein (GFP) were introduced into host embryos, and germ-line contribution was detected by the presence of GFP+ cells in the region of the gonad. The germ-line competent embryo cell cultures will be useful for the development of a gene targeting strategy that will increase the utility of the zebrafish model for studies of gene function.