Cell function profiling to assess clone stability.

In cell line development the identification of stable Chinese hamster ovary cells for production is a critical but onerous task. The stability trial focus upon high-level attributes can mask profound underlying cellular changes, leading to unstable clones mistakenly being chosen for production. The challenge is to assay underlying cell pathways and subsystems without pushing up cell line development costs. ChemStress® cell function profiling is a simple, multiwell plate-based assay that uses a panel of active chemicals to mimic known bioprocess stresses and challenge key pathways. After 3 days of static culture on the plate, functional responses are assayed, for example, titer and growth. Here this approach is used to monitor 131 clones as they change over real stability trials. A novel stability metric is defined over the data to identify stable clones that remain unperturbed across many components of cell function. This allows stability trials to look beneath the titer to identify clones that are internally more stable.

High-throughput quantitation of Fc-containing recombinant proteins in cell culture supernatant by fluorescence polarization spectroscopy.

Measurement of recombinant protein product titer critically underpins all biopharmaceutical manufacturing process development, as well as diverse research and discovery activity. Here, we describe a simple rapid (<2 min per 96 samples) 96-well microplate-based assay that enables high-throughput quantitation of recombinant immunoglobulin G and Fc-containing IgG derivatives in mammalian cell culture supernatant over a wide dynamic range of 2.5-80 mg/L, using microplate fluorescence polarization (FP) spectroscopy. The solution-phase FP assay is based on the detection of immunoglobulin Fc domain containing analyte binding to FITC-conjugated recombinant Protein G ligand to measure analyte concentration dependent changes in emitted FP. For ease of use and maximal shelf life, we showed that air-dried assay microplates containing pre-formulated ligand that is re-solubilized on addition of analyte containing solution did not affect assay performance, typically yielding an across plate coefficient of variation of <1%, and a between-plate standard deviation below 1%. Comparative assays of the same samples by FP and other commonly used IgG assay formats operating over a similar dynamic range (Protein A HPLC and bio-interferometry) yielded a coefficient of determination >0.99 in each case.

Tardive dyskinesia and essential fatty acids.

Tardive dyskinesia (TD) is a movement disorder described in individuals who have been treated with anti-dopaminergic agents. The pathophysiology of this condition remains to be fully elucidated. Several mechanisms like dopaminergic supersensitivity, dysfunction of striatonigral, GABAergic neurons and disturbed balance between dopaminergic and cholinergic systems have been described. Essential fatty acids (EFAs) are important components of neuronal membrane and the EFA content of these membranes can significantly influence neuronal functioning. Lower levels of EFAs have been reported in red blood cells (RBC) and plasma of individuals with moderate to severe TD. Supplementation with EFAs (omega-3 and omega-6 and ethyl-EPA) have been tried to alleviate TD in open and double-blind clinical trials and in some animal models of TD. In addition, antioxidants (Vitamin E) and melatonin have been tried. However, smaller numbers of patients and shortened length of clinical studies make it difficult to draw any definitive conclusions. Large multi-centre studies with sound methodology of both EFAs and antioxidants are needed.