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.

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.

A strategy to accelerate protein production from a pool of clones in Chinese hamster ovary cells for toxicology studies.

In the biopharmaceutical industry, a clonally derived cell line is typically used to generate material for investigational new drug (IND)-enabling toxicology studies. The same cell line is then used to generate material for clinical studies. If a pool of clones can be used to produce material for IND-enabling toxicology studies (Pool for Tox (PFT) strategy) during the time a lead clone is being selected for clinical material production, the toxicology studies can be accelerated significantly (approximately 4 months at Genentech), leading to a potential acceleration of 4 months for the IND submission.

Carboxypeptidase D is the only enzyme responsible for antibody C-terminal lysine cleavage in Chinese hamster ovary (CHO) cells.

Heterogeneity of C-terminal lysine levels often observed in therapeutic monoclonal antibodies is believed to result from the proteolysis by endogenous carboxypeptidase(s) during cell culture production. Identifying the responsible carboxypeptidase(s) for C-terminal lysine cleavage in CHO cells would provide valuable insights for antibody production cell culture processes development and optimization. In this study, five carboxypeptidases, CpD, CpM, CpN, CpB, and CpE, were studied for message RNA (mRNA) expression by qRT-PCR analysis in two most commonly used blank hosts (DUXB-11 derived DHFR-deficient DP12 host and DHFR-positive CHOK1 host), used for therapeutic antibody production, as well an antibody-expressing cell line derived from each host.

Industry view on the relative importance of "clonality" of biopharmaceutical-producing cell lines.

Recently, several health authorities have requested substantial detail from sponsor firms regarding the practices employed to generate the production cell line for recombinant DNA-(rDNA) derived biopharmaceuticals. Two possible inferences from these regulatory agency questions are that (1) assurance of "clonality" of the production cell line is of major importance to assessing the safety and efficacy of the product and (2), without adequate proof of "clonality", additional studies of the cell line and product are often required to further ensure the product's purity and homogeneity. Here we address the topic of "clonality" in the broader context of product quality assurance by current technologies and practices, as well as discuss some of the relevant science and historical perspective.

Effects of copper on CHO cells: cellular requirements and product quality considerations.

Recent reports highlight the impact of copper on lactate metabolism: CHO cell cultures with higher initial copper levels shift to net lactate consumption and yield lower final lactate and higher titers. These studies investigated the effects of copper on metabolite and transcript profiles, but did not measure in detail the dependences of cell culture performance and product quality on copper concentrations. To more thoroughly map these dependences, we explored the effects of various copper treatments on four recombinant CHO cell lines.

Complete knockout of the lactate dehydrogenase A gene is lethal in pyruvate dehydrogenase kinase 1, 2, 3 down-regulated CHO cells.

Accumulation of high level of lactate can negatively impact cell growth during fed-batch culture process. In this study, we attempted to knockout the lactate dehydrogenase A (LDHA) gene in CHO cells in order to attenuate the lactate level. To prevent the potential deleterious effect of pyruvate accumulation, consequent to LDHA knockout, on cell culture, we chose a pyruvate dehydrogenase kinase 1, 2, and 3 (PDHK1, 2, and 3) knockdown cell line in which to knock out LDHA alleles.

Fast identification of reliable hosts for targeted cell line development from a limited-genome screening using combined φC31 integrase and CRE-Lox technologies.

The use of targeted integration (TI) in cell line development (CLD) usually introduces one copy of a recombinant gene into a predetermined transcriptionally active locus. This reduces the heterogeneity typically associated with traditional random integration (RI) CLD with regards to varied productivity and instability, resulting from diverse chromosomal influences, varied copy numbers, and repeat-induced rearrangement. As such, TI CLD offers the hope of a predictable and consistent CLD process for establishing stable clones.

Chinese hamster ovary K1 host cell enables stable cell line development for antibody molecules which are difficult to express in DUXB11-derived dihydrofolate reductase deficient host cell.

Therapeutic monoclonal antibodies (mAb) are often produced in Chinese hamster ovary (CHO) cells. Three commonly used CHO host cells for generating stable cell lines to produce therapeutic proteins are dihydrofolate reductase (DHFR) positive CHOK1, DHFR-deficient DG44, and DUXB11-based DHFR deficient CHO. Current Genentech commercial full-length antibody products have all been produced in the DUXB11-derived DHFR-deficient CHO host.

Controlling glycation of recombinant antibody in fed-batch cell cultures.

Protein glycation is a non-enzymatic glycosylation that can occur to proteins in the human body, and it is implicated in the pathogenesis of multiple chronic diseases. Glycation can also occur to recombinant antibodies during cell culture, which generates structural heterogeneity in the product. In a previous study, we discovered unusually high levels of glycation (>50%) in a recombinant monoclonal antibody (rhuMAb) produced by CHO cells.

Decreasing lactate level and increasing antibody production in Chinese Hamster Ovary cells (CHO) by reducing the expression of lactate dehydrogenase and pyruvate dehydrogenase kinases.

Large-scale fed-batch cell culture processes of CHO cells are the standard platform for the clinical and commercial production of monoclonal antibodies. Lactate is one of the major by-products of CHO fed-batch culture. In pH-controlled bioreactors, accumulation of high levels of lactate is accompanied by high osmolality due to the addition of base to control pH of the cell culture medium, potentially leading to lower cell growth and lower therapeutic protein production during manufacturing.

Mechanisms of unintended amino acid sequence changes in recombinant monoclonal antibodies expressed in Chinese Hamster Ovary (CHO) cells.

An amino acid sequence variant is defined as an unintended amino acid sequence change and contributes to product heterogeneity. Recombinant monoclonal antibodies (MAbs) are primarily expressed from Chinese Hamster Ovary (CHO) cells using stably transfected production cell lines. Selections and amplifications with reagents such as methotrexate (MTX) are often required to achieve high producing stable cell lines.

Proteomic profiling of recombinant Escherichia coli in high-cell-density fermentations for improved production of an antibody fragment biopharmaceutical.

By using two-dimensional polyacrylamide gel electrophoresis, a proteomic analysis over time was conducted with high-cell-density, industrial, phosphate-limited Escherichia coli fermentations at the 10-liter scale. During production, a recombinant, humanized antibody fragment was secreted and assembled in a soluble form in the periplasm. E.

High-level accumulation of a recombinant antibody fragment in the periplasm of Escherichia coli requires a triple-mutant (degP prc spr) host strain.

During production of a humanized antibody fragment secreted into the periplasm of Escherichia coli, proteolytic degradation of the light chain was observed. In order to determine which protease(s) were responsible for this degradation, we compared expression of the F(ab')(2) antibody fragment in several E. coli strains carrying mutations in genes encoding periplasmic proteases.

The cryptic ushA gene (ushA(c)) in natural isolates of Salmonella enterica (serotype Typhimurium) has been inactivated by a single missense mutation.

Two mutational mechanisms, both supported by experimental studies, have been proposed for the evolution of new or improved enzyme specificities in bacteria. One mechanism involves point mutation(s) in a gene conferring novel substrate specificity with partial or complete loss of the original (wild-type) activity of the encoded product. The second mechanism involves gene duplication followed by silencing (inactivation) of one of these duplicates.