Biotherapeutic proteins represent a mainstay of treatment for a multitude of conditions, for example, autoimmune disorders, hematologic disorders, hormonal dysregulation, cancers, infectious diseases and genetic disorders. The technologies behind their production have changed substantially since biotherapeutic proteins were first approved in the 1980s. Although most biotherapeutic proteins developed to date have been produced using the mammalian Chinese hamster ovary and murine myeloma (NS0, Sp2/0) cell lines, there has been a recent shift toward the use of human cell lines. One of the most important advantages of using human cell lines for protein production is the greater likelihood that the resulting recombinant protein will bear post-translational modifications (PTMs) that are consistent with those seen on endogenous human proteins. Although other mammalian cell lines can produce PTMs similar to human cells, they also produce non-human PTMs, such as galactose-α1,3-galactose and N-glycolylneuraminic acid, which are potentially immunogenic. In addition, human cell lines are grown easily in a serum-free suspension culture, reproduce rapidly and have efficient protein production. A possible disadvantage of using human cell lines is the potential for human-specific viral contamination, although this risk can be mitigated with multiple viral inactivation or clearance steps. In addition, while human cell lines are currently widely used for biopharmaceutical research, vaccine production and production of some licensed protein therapeutics, there is a relative paucity of clinical experience with human cell lines because they have only recently begun to be used for the manufacture of proteins (compared with other types of cell lines). With additional research investment, human cell lines may be further optimized for routine commercial production of a broader range of biotherapeutic proteins.

E2F directs the cell cycle-dependent expression of genes that induce or regulate the cell division process. In mammalian cells, this transcriptional activity arises from the combined properties of multiple E2F-DP heterodimers. In this study, we show that the transcriptional potential of individual E2F species is dependent upon their nuclear localization. This is a constitutive property of E2F-1, -2, and -3, whereas the nuclear localization of E2F-4 is dependent upon its association with other nuclear factors. We previously showed that E2F-4 accounts for the majority of endogenous E2F species. We now show that the subcellular localization of E2F-4 is regulated in a cell cycle-dependent manner that results in the differential compartmentalization of the various E2F complexes. Consequently, in cycling cells, the majority of the p107-E2F, p130-E2F, and free E2F complexes remain in the cytoplasm. In contrast, almost all of the nuclear E2F activity is generated by pRB-E2F. This complex is present at high levels during G 1 but disappears once the cells have passed the restriction point. Surprisingly, dissociation of this complex causes little increase in the levels of nuclear free E2F activity. This observation suggests that the repressive properties of the pRB-E2F complex play a critical role in establishing the temporal regulation of E2F-responsive genes. How the differential subcellular localization of pRB, p107, and p130 contributes to their different biological properties is also discussed.E2F is a transcriptional regulator that plays a pivotal role in the regulation of cellular proliferation (reviewed in reference 59; 8). Many E2F-responsive genes have been identified, and their products are components of either cell cycle control (e.g., cyclin E, cyclin A, and cdc2) or DNA synthesis (e.g., dihydrofolate reductase, thymidine kinase, or DNA polymerase ␣) machinery. In each case, E2F is thought to restrict the expression of these genes to the point of the cell cycle at which their products act (38).E2F is regulated by the retinoblastoma protein (pRB) (3, 5, 12), a tumor suppressor that is functionally inactivated in a large proportion of all human tumors (reviewed in reference 67). Consistent with its antiproliferative role, pRB blocks the ability of E2F to activate transcription (32, 33). In addition, overexpression studies have indicated that the resultant pRB-E2F complex can act as a transcriptional repressor, in which E2F provides the sequence-specific DNA binding activity and pRB inhibits transcription by sequestering adjacent transcription factors (2,10,62,69,70). This idea suggests that E2F participates in both the activation and the inhibition of cellular proliferation. Consistent with this hypothesis, homozygous deletion of the murine E2F-1 gene causes atrophy in some tissues and tumors in others (26, 73).The growth-inhibitory properties of pRB are regulated by its cell cycle-dependent phosphorylation (reviewed in reference 7). Phosphorylation is catalyzed by one or more of the cell cycle-dependent kinases...

Glycosylation in the Fc region of antibodies has been shown to play an important role in antibody function. In the current study, glycosylation of human monoclonal antibodies was metabolically modulated using a potent alpha-mannosidase I inhibitor, kifunensine, resulting in the production of antibodies with oligomannose-type N-glycans. Growing Chinese hamster ovary cells for 11 days in batch culture with a single treatment of kifunensine was sufficient to elicit this effect without any significant impact on cell viability or antibody production. Antibodies expressed in the presence of kifunensine at a concentration as low as 60 ng/mL contained mainly oligomannose-type glycans and demonstrated increased ADCC activity and affinity for FcgammaRIIIA, but reduced C1q binding. Although the kifunensine-mediated shift to oligomannose-type glycans could, in theory, result in rapid clearance of the antibody through increased mannose receptor binding, the serum levels of antibody in mice were not significantly altered up to 168 h following injection. The use of kifunensine provides a simple and rapid method for the production of antibodies with increased ADCC without the time-consuming need to re-engineer either the antibody molecule or the host cell line.