There has been a rapid increase in the number and demand for approved biopharmaceuticals produced from animal cell culture processes over the last few years. In part, this has been due to the efficacy of several humanized monoclonal antibodies that are required at large doses for therapeutic use. There have also been several identifiable advances in animal cell technology that has enabled efficient biomanufacture of these products. Gene vector systems allow high specific protein expression and some minimize the undesirable process of gene silencing that may occur in prolonged culture. Characterization of cellular metabolism and physiology has enabled the design of fed-batch and perfusion bioreactor processes that has allowed a significant improvement in product yield, some of which are now approaching 5 g/L. Many of these processes are now being designed in serum-free and animal-component-free media to ensure that products are not contaminated with the adventitious agents found in bovine serum. There are several areas that can be identified that could lead to further improvement in cell culture systems. This includes the down-regulation of apoptosis to enable prolonged cell survival under potentially adverse conditions. The characterization of the critical parameters of glycosylation should enable process control to reduce the heterogeneity of glycoforms so that production processes are consistent. Further improvement may also be made by the identification of glycoforms with enhanced biological activity to enhance clinical efficacy. The ability to produce the ever-increasing number of biopharmaceuticals by animal cell culture is dependent on sufficient bioreactor capacity in the industry. A recent shortfall in available worldwide culture capacity has encouraged commercial activity in contract manufacturing operations. However, some analysts indicate that this still may not be enough and that future manufacturing demand may exceed production capacity as the number of approved biotherapeutics increases.

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The effect of ammonium chloride was determined on a culture of CHO cells transfected with the human erythropoietin (EPO) gene. Cell growth was inhibited above a culture concentration of 5 mM NH4Cl with an IC‐50 determined to be 33 mM. The specific production of EPO increased with the addition of NH4Cl above 5 mM. At 10 mM NH4Cl, the final cell density after 4 days in culture was significantly lower but the final yield of EPO was significantly higher. This appeared to be due to continued protein production after cell growth had ceased. The metabolic effects of added NH4Cl included higher specific consumption rates of glucose and glutamine and an increased rate of production of alanine, glycine, and glutamate. The EPO analyzed from control cultures had a molecular weight range of 33–39 kDa and an isoelectric point range of 4.06–4.67. Seven distinct isoforms of the molecule were identified by two‐dimensional electrophoresis. This molecular heterogeneity was ascribed to variable glycosylation. Complete enzymatic de‐glycosylation resulted in a single molecular form with a molecular mass of 18 kDa. Addition of NH4Cl to the cultures caused a significant increase in the heterogeneity of the glycoforms as shown by an increased molecular weight and pI range. Enzymatic de‐sialylation of the EPO from the ammonia‐treated and control cultures resulted in identical electrophoretic patterns. This indicated that the effect of ammonia was in the reduction of terminal sialylation of the glycan structures which accounted for the increased pI. Selective removal of the N‐glycan structures by PNGase F resulted in two bands identified as the O‐glycan linked structure (19 kDa) and the completely de‐glycosylated structure (18 kDa). The proportion of the O‐linked glycan structure was reduced, and its pI increased in cultures to which ammonia was added. Thus, the glycosylation pattern altered by the presence of ammonia included a reduction in terminal sialylation of all the glycans and a reduction in the content of the O‐linked glycan. The addition of a sialidase inhibitor to the cultures had no effect on the ammonia‐induced increase in EPO heterogeneity. Also, the effect of ammonia on glycosylation could not be mimicked using the weak base chloroquine in our system. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 68: 370–380, 2000.

Clients who had completed psychotherapy were interviewed about the significant experiences and moments they recalled within their sessions. These interviews were analyzed using grounded theory, creating a hierarchy of categories that represent what clients find important in therapy. From the hermeneutic analysis of the content of these categories, a list of principles was constructed to guide the moment-to-moment process of psychotherapy practice. The authors respond to the call for qualitative outcome studies and demonstrate how qualitative psychotherapy research can lead to empirically derived principles that then can become the foundation of future research and psychotherapy integration efforts.