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In the biopharmaceutical industry, therapeutic monoclonal antibodies are primarily produced in mammalian cell culture systems. During the scale-up of a monoclonal antibody production process, we observed excessive mechanical cell shear as well as significant reduction of the antibody's interchain disulfide bonds during harvest operations. This antibody reduction event was catastrophic as the product failed to meet the drug substance specifications and the bulk product was lost. Subsequent laboratory studies have demonstrated that cells subjected to mechanical shear release cellular enzymes that contribute to this antibody reduction phenomenon (manuscript submitted; Kao et al., 2009). Several methods to prevent this antibody reduction event were developed using a lab-scale model to reproduce the lysis and reduction events. These methods included modifications to the cell culture media with chemicals (e.g., cupric sulfate (CuSO(4))), pre- and post-harvest chemical additions to the cell culture fluid (CCF) (e.g., CuSO(4), EDTA, L-cystine), as well as lowering the pH and air sparging of the harvested CCF (HCCF). These methods were evaluated for their effectiveness in preventing disulfide bond reduction and their impact to product quality. Effective prevention methods, which yielded acceptable product quality were evaluated for their potential to be implemented at manufacturing-scale. The work described here identifies numerous effective reduction prevention measures from lab-scale studies; several of these methods were then successfully translated into manufacturing processes.

␤-Adrenergic receptor stimulation regulates the activity of several different cardiac ion channels through an adenylate cyclase/cAMP/protein kinase A-dependent mechanism. Previous work has suggested that basal tyrosine kinase activity attenuates the ␤-adrenergic responsiveness of these cardiac ion channels, supporting the idea that tyrosine phosphorylation exerts an inhibitory effect at some point in the common signaling pathway. To determine which element in the ␤-adrenergic pathway is regulated by tyrosine kinase activity, we studied the effects of various protein tyrosine phosphatase (PTP) inhibitors on the cAMP-dependent regulation of the L-type Ca 2ϩ current in guinea pig ventricular myocytes. Three such compounds, sodium orthovanadate, peroxovanadate, and bpV(phen), had no effect on the basal Ca 2ϩ current, yet each caused a pronounced inhibition of the Ca 2ϩ current stimulated by the ␤-adrenergic receptor agonist isoproterenol. These observations are consistent with the idea that basal tyrosine kinase activity is capable of inhibiting ␤-adrenergic responses. However, these PTP inhibitors had no effect on cAMP-dependent stimulation of the Ca 2ϩ current via activation of adenylate cyclase with forskolin or activation of H 2 -histaminergic receptors with histamine. These results are consistent with the idea that inhibition of PTP activity produces an inhibitory effect involving a tyrosine kinase-dependent mechanism acting selectively at the level of the ␤-adrenergic receptor. This signaling mechanism does not seem to be linked to tyrosine kinase activity associated with insulin and insulin-like growth factor receptors, because acute exposure to agonists of these receptors did not inhibit isoproterenol regulation of the Ca 2ϩ current.

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