Residue-Specific Impact of EDTA and Methionine on Protein Oxidation in Biotherapeutics Formulations Using an Integrated Biotherapeutics Drug Product Development Workflow.

The rational design and selection of formulation composition to meet molecule-specific and product-specific needs are critical for biotherapeutics development to ensure physical and chemical stability. This work, based on three antibody-based (mAb) proteins (mAbA, mAbB, and mAbC), evaluates residue-specific impact of EDTA and methionine on protein oxidation, using an integrated biotherapeutics drug product development workflow. This workflow includes statistical experimental design, high-throughput experimental automation and execution, structure-based in silico modeling, inferential statistical analysis, and enhanced interactive data visualization of large datasets.

Mammalian cell-produced therapeutic proteins: heterogeneity derived from protein degradation.

Therapeutic glycoproteins, for example, antibodies (Abs) and Fc fusion proteins when produced in mammalian cells, such as Chinese hamster ovary (CHO) cells generally exhibit heterogeneity. Both the oligosaccharide moiety and the protein moiety contribute to this phenomenon. Non-enzymatic and enzymatic pathways of protein fragmentation generate heterogeneity in the polypeptide backbone.

Early prediction of instability of Chinese hamster ovary cell lines expressing recombinant antibodies and antibody-fusion proteins.

One of the most important criteria for the successful manufacture of a therapeutic protein (e.g., an antibody) is to develop a mammalian cell line that maintains stability of production.

Development of mammalian production cell lines expressing CNTO736, a glucagon like peptide-1-MIMETIBODY: factors that influence productivity and product quality.

In an attempt to develop a high producing mammalian cell line expressing CNTO736, a Glucagon like peptide-1-antibody fusion protein (also known as a Glucagon like peptide-1 MIMETIBODY), we have noted that the N-terminal GLP-1 portion of the MIMETIBODY was susceptible to proteolytic degradation during cell culture, which resulted in an inactive product. Therefore, a number of parameters that had an effect on productivity as well as product quality were examined. Results suggest that the choice of the host cell line had a significant effect on the overall product quality.

Correlation of heavy and light chain mRNA copy numbers to antibody productivity in mouse myeloma production cell lines.

Manufacturing cell line development at Centocor involves transfection of antibody genes into host cell lines and isolating primary transfectomas that upon subcloning yield high expressing cell lines for the desired antibody. In an attempt to increase productivity of these cell lines, we set out to identify the rate-limiting step in the process of antibody expression and secretion. For this purpose, 30 antibody expressing cell lines with variable antibody expression levels were analyzed for heavy-chain and light-chain mRNA expression levels.

Comparative studies of active site-ligand interactions among various recombinant constructs of human beta-amyloid precursor protein cleaving enzyme.

Amyloid precursor protein (APP) cleaving enzyme (BACE) is the enzyme responsible for beta-site cleavage of APP, leading to the formation of the amyloid-beta peptide that is thought to be pathogenic in Alzheimer's disease (AD). Hence, BACE is an attractive pharmacological target, and numerous research groups have begun searching for potent and selective inhibitors of this enzyme as a potential mechanism for therapeutic intervention in AD. The mature enzyme is composed of a globular catalytic domain that is N-linked glycosylated in mammalian cells, a single transmembrane helix that anchors the enzyme to an intracellular membrane, and a short C-terminal domain that extends outside the phospholipid bilayer of the membrane.