Rohini Deshpande | PubMed Health

PubMed2006

Serum-free suspension large-scale transient transfection of CHO cells in WAVE bioreactors.

Here, we report the development of a large-scale transient expression platform utilizing Chinese hamster ovary (CHO) cells. The majority of recombinant proteins and antibodies that are produced for preclinical models and clinical trials are expressed in stably transfected CHO cells. A protocol for transient transfection of CHO cells that is rapid, reproducible, and cost-effective would therefore streamline the process from research to development and help avoid any potential host species induced variation in the molecule of interest. CHO cells were adapted to grow in serum-free suspension conditions in spinner flask cultures in a proprietary in-house developed growth medium. In developing this transient transfection protocol, the parameters optimized included the transfection reagent of choice, the cell density at the time of transfection, the plasmid DNA concentration, and the transfection reagent concentration. Using this optimized protocol, we have expressed recombinant proteins, including antibodies, at an expression level of up to 9.4 mg/L. We also report transient transfections from 500 mL working volume (w.v.) up to 20 L w.v. in a WAVE bioreactor. Using this optimized protocol, it is possible to rapidly (within 10 d) produce up to 100 mg of recombinant protein for further study.

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PubMed2005

Quantitative methods for developing Fc mutants with extended half-lives.

Fc mutants with increased binding affinity for the neonatal receptor, FcRn, exhibit increased half-lives in vivo, and represent an attractive means for extending the half-lives of therapeutic antibodies. The half-lives of other therapeutic molecules (e.g., proteins) may also be extended by conjugating them to Fc fragments, thus decreasing the frequency of patient injections and allowing the administration of low and potentially nontoxic concentrations of the therapeutics. To investigate the possibility for further increasing the half-life of Fc, a pair of quantitative methods is presented to complement combinatorial screening and in vivo testing. Specifically, a simple molecular modeling procedure was developed to predict relative Gibbs free energies of binding values (DeltaDeltaGbind) between Fc and FcRn across different mutants and species. This procedure was found to reasonably reproduce experimental DeltaDeltaGbind values from our experiments and the literature, and may be used as an initial screen to explore Fc sequence space more fully prior to experimental testing. In addition, a mathematical model of Fc trafficking was formulated and combined with a cell-level pulse-chase assay to obtain a quantitative recycling parameter in human T84 cells. This Fc recycling parameter was found to be correlated with binding affinity, but captures the pH dependent nature of the interaction between Fc and FcRn and may serve as an additional screen following combinatorial experiments.

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