Development of a targeted integration Chinese hamster ovary host directly targeting either one or two vectors simultaneously to a single locus using the Cre/Lox recombinase-mediated cassette exchange system.

Cell line development (CLD) by random integration (RI) can be labor intensive, inconsistent, and unpredictable due to uncontrolled gene integration after transfection. Unlike RI, targeted integration (TI) based CLD introduces the antibody-expressing cassette to a predetermined site by recombinase-mediated cassette exchange (RMCE). The key to success for the development of a TI host for therapeutic antibody production is to identify a transcriptionally active hotspot that enables highly efficient RMCE and antibody expression with good stability.

Concurrent transfection of randomized transgene configurations into targeted integration CHO host is an advantageous and cost-effective method for expression of complex molecules.

Complex recombinant proteins are increasingly desired as potential therapeutic options for many disease indications and are commonly expressed in the mammalian Chinese hamster ovary (CHO) cells. Generally, stoichiometric expression and proper folding of all subunits of a complex recombinant protein are required to achieve the desired titers and product qualities for a complex molecule. Targeted integration (TI) cell line development (CLD), which entails the insertion of the desired transgene(s) into a predefined landing-pad in the CHO genome, enables the generation of a homogeneous pool of cells from which clonally stable and high titer clones can be isolated with minimal screening efforts.

Comparison of central corneal thickness measurement by scanning slit topography, infrared, and ultrasound pachymetry in normal and post-LASIK eyes.

Objectives: To compare central corneal thickness (CCT) measurements by scanning slit topography (SST), infrared pachymetry (IRP), and ultrasound pachymetry (USP), and their agreement in normal and post-laser in situ keratomileusis (LASIK) eyes.

Methods: Sixty normal and 35 post-LASIK subjects were recruited. Only one eye from each subject was analyzed.

Maximizing antibody production in a targeted integration host by optimization of subunit gene dosage and position.

Historically, therapeutic protein production in Chinese hamster ovary (CHO) cells has been accomplished by random integration (RI) of expression plasmids into the host cell genome. More recently, the development of targeted integration (TI) host cells has allowed for recombination of plasmid DNA into a predetermined genomic locus, eliminating one contributor to clone-to-clone variability. In this study, a TI host capable of simultaneously integrating two plasmids at the same genomic site was used to assess the effect of antibody heavy chain and light chain gene dosage on antibody productivity.

Endothelial intercellular cell adhesion molecule 1 contributes to cell aggregate formation in CHO cells cultured in serum-free media.

Chinese hamster ovary (CHO) cells have been adapted to grow in serum-free media and in suspension culture to facilitate manufacturing needs. Some CHO cell lines, however, tend to form cell aggregates while being cultured in suspension. This can result in reduced viability and capacity for single cell cloning (SCC) via limiting dilution, and process steps to mitigate cell aggregate formation, for example, addition of anti-cell-aggregation agents.

Achieving greater efficiency and higher confidence in single-cell cloning by combining cell printing and plate imaging technologies.

In recent years, health authorities have increased emphasis on demonstrating that a cell line, which is used for the generation of biologics, is clonally derived. Within the past few years, single-cell manipulation technologies, especially microfluidic drop-on-demand dispensing, have gained increased attention in the biopharmaceutical industry. This work discusses the development and characterization of a single-cell printing workflow followed by plate imaging.

Probing the importance of clonality: Single cell subcloning of clonally derived CHO cell lines yields widely diverse clones differing in growth, productivity, and product quality.

In the past few decades, a large variety of therapeutic antibodies and proteins have been expressed in Chinese hamster ovary (CHO) cells. This mammalian expression system is robust, scalable, relatively inexpensive, and importantly allows for post-translational modifications that are important for some therapeutic proteins. Historically, CHO cell lines were derived from colonies of cells grown in semi-solid or liquid plates using either serum-containing or serum-free media.

Development and characterization of an automated imaging workflow to generate clonally-derived cell lines for therapeutic proteins.

In the development of biopharmaceutical products, the expectation of regulatory agencies is that the recombinant proteins are produced from a cell line derived from a single progenitor cell. A single limiting dilution step followed by direct imaging, as supplemental information, provides direct evidence that a cell line originated from a single progenitor cell. To obtain this evidence, a high-throughput automated imaging system was developed and characterized to consistently ensure that cell lines used for therapeutic protein production are clonally-derived.

Beating the odds: The poisson distribution of all input cells during limiting dilution grossly underestimates whether a cell line is clonally-derived or not.

Establishing that a cell line was derived from a single cell progenitor and defined as clonally-derived for the production of clinical and commercial therapeutic protein drugs has been the subject of increased emphasis in cell line development (CLD). Several regulatory agencies have expressed that the prospective probability of clonality for CHO cell lines is assumed to follow the Poisson distribution based on the input cell count. The probability of obtaining monoclonal progenitors based on the Poisson distribution of all cells suggests that one round of limiting dilution may not be sufficient to assure the resulting cell lines are clonally-derived.

Supplementation of Nucleosides During Selection can Reduce Sequence Variant Levels in CHO Cells Using GS/MSX Selection System.

In the process of generating stable monoclonal antibody (mAb) producing cell lines, reagents such as methotrexate (MTX) or methionine sulfoximine (MSX) are often used. However, using such selection reagent(s) increases the possibility of having higher occurrence of sequence variants in the expressed antibody molecules due to the effects of MTX or MSX on de novo nucleotide synthesis. Since MSX inhibits glutamine synthase (GS) and results in both amino acid and nucleoside starvation, it is questioned whether supplementing nucleosides into the media could lower sequence variant levels without affecting titer.

Evaluation of heavy chain C-terminal deletions on productivity and product quality of monoclonal antibodies in Chinese hamster ovary (CHO) cells.

Monoclonal antibodies (mAbs) have been well established as potent therapeutic agents and are used to treat many different diseases. During cell culture production, antibody charge variants can be generated by cleavage of heavy chain (HC) C-terminal lysine and proline amidation. Differences in levels of charge variants during manufacturing process changes make it challenging to demonstrate process comparability.

Taming hyperactive hDNase I: Stable inducible expression of a hyperactive salt- and actin-resistant variant of human deoxyribonuclease I in CHO cells.

While the most common causes of clonal instability are DNA copy number loss and silencing, toxicity of the expressed protein(s) may also induce clonal instability. Human DNase I (hDNase I) is used therapeutically for the treatment of cystic fibrosis (CF) and may have potential benefit for use in systemic lupus erythematosus (SLE). hDNase I is an endonuclease that catalyzes degradation of extracellular DNA and is inhibited by both salt and G-actin.

FX knockout CHO hosts can express desired ratios of fucosylated or afucosylated antibodies with high titers and comparable product quality.

During antibody dependent cell cytotoxicity (ADCC) the target cells are killed by monocytes and natural killer cells. ADCC is enhanced when the antibody heavy chain's core N-linked glycan lacks the fucose molecule(s). Several strategies have been utilized to generate fully afucosylated antibodies.

Noninvasive Tracking of Encapsulated Insulin Producing Cells Labelled with Magnetic Microspheres by Magnetic Resonance Imaging.

Microencapsulated islets are usually injected free-floating into the peritoneal cavity, so the position of the grafts remains elusive after transplantation. This study aims to assess magnetic resonance imaging (MRI) as a noninvasive means to track microencapsulated insulin producing cells following transplantation. Encapsulated insulin producing cells (MIN6 and human islets) were labelled with magnetic microspheres (MM), assessed for viability and insulin secretion, and imaged in vitro using a clinical grade 3 T MRI and in vivo using both clinical grade 3 T and research grade 11.