Human cell lines for biopharmaceutical manufacturing: history,status, and future perspectives

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.     

Effect of Temperature, Light, Nutrients and Dehardening on Abscisic Acid Induced Cold Hardiness in Bromus inermis Leyss Suspension Cultured Cells

The effects of culture conditions on abscisic acid (ABA)-inducedfreezing tolerance were determined in smooth bromegrass Bromusinermis Leyss cv. Manchar) cell suspension cultures. Bromegrasscultures initiated with 2 g fr wt of cells achieved maximumfreezing tolerances (greater than –32?C) at 25 to 30?Cin the presence of 75 to 100 µM ABA. High levels of freezingtolerance induced by ABA were correlated with high growth ratesat 25 and 30?C. In control cells, incubation at 10?C inducedoptimum levels of hardiness with minimal growth. Prolonged exposure(6 weeks) of cells to 3?C, with or without ABA, increased freezingtolerance only by several degrees. Exogenous ABA concentrationsgreater than 100 µM were not inhibitory to growth. Repeatedexposure to ABA, however, retarded growth and made the cellstolerant to temperatures below –40?C. Removal of ABA fromthe medium resulted in dehardening of the cells both at 25 and3?C. Nitrogen had a marginal effect on ABA-induced hardeningat 25?C, but inhibited age-dependent hardening of control cellcultures. Light had no effect on the freezing tolerance of culturedcells. Addition of 10% sucrose, 30 min prior to freezing, tobromegrass cells treated with ABA for 4 days increased freezingtolerance more than 15?C. These observations are discussed inrelation to the contrasting behaviour of the low temperatureand photoperiod dependent cold acclimation of plants (Received July 14, 1989; Accepted October 23, 1989)     

Mutation of Threonine 34 in Mouse Podoplanin-Fc Reduces CLEC-2 Binding and Toxicity in Vivo While Retaining Anti-lymphangiogenic Activity

The lymphatic system plays an important role in cancer metastasis and inhibition of lymphangiogenesis could be valuable in fighting cancer dissemination. Podoplanin (Pdpn) is a small, transmembrane glycoprotein expressed on the surface of lymphatic endothelial cells (LEC). During mouse development, binding of Pdpn to the C-type lectin-like receptor 2 (CLEC-2) on platelets is critical for the separation of the lymphatic and blood vascular systems. Competitive inhibition of Pdpn functions with a soluble form of the protein, Pdpn-Fc, leads to reduced lymphangiogenesis in vitro and in vivo. However, the transgenic overexpression of human Pdpn-Fc in mouse skin causes disseminated intravascular coagulation due to platelet activation via CLEC-2. In the present study, we produced and characterized a mutant form of mouse Pdpn-Fc, in which threonine 34, which is considered essential for CLEC-2 binding, was mutated to alanine (PdpnT34A-Fc). Indeed, PdpnT34A-Fc displayed a 30-fold reduced binding affinity for CLEC-2 compared with Pdpn-Fc. This also translated into fewer side effects due to platelet activation in vivo. Mice showed less prolonged bleeding time and fewer embolized vessels in the liver, when PdpnT34A-Fc was injected intravenously. However, PdpnT34A-Fc was still as active as wild-type Pdpn-Fc in inhibiting lymphangiogenesis in vitro and also inhibited lymphangiogenesis in vivo. These data suggest that the function of Pdpn in lymphangiogenesis does not depend on threonine 34 in the CLEC-2 binding domain and that PdpnT34A-Fc might be an improved inhibitor of lymphangiogenesis with fewer toxic side effects.     

c-erbB-2 expression in primary breast cancer.

c-erbB-2 is an oncoprotein which is overexpressed in up to 40% of primary breast cancers. c-erbB-2 overexpression is a bad prognostic factor in patients with lymph node-positive disease. Unfortunately, there has been no agreement to date on whether c-erbB-2 overexpression is of prognostic significance in patients with lymph node-negative disease. c-erbB-2 overexpression is correlated with the absence of estrogen receptor expression in a number of publications. Correlation between c-erbB-2 overexpression and hormone sensitivity in the clinical setting is less well established and is the focus of ongoing studies. Both preclinical and clinical studies support an association between c-erbB-2 receptor overexpression and resistance to alkylating agents. In contrast, the data for c-erbB-2 and anthracyclines should be viewed in a slightly different manner. Anthracyclines appear to have a greater therapeutic effect in c-erbB-2-positive disease which may be dose sensitive. In c-erbB-2-negative disease not only is the therapeutic effect reduced but there does not appear to be any improved response to higher doses of anthracyclines. The data for c-erbB-2 and the taxanes is still not clear enough to provide any definite conclusions. If there is a correlation it would at present appear to be between paclitaxel and response rates, but this needs to be confirmed in larger studies. Few studies have looked at changes in c-erbB-2 on therapy. Those that have seem to show no significant change on either tamoxifen or chemotherapy.     

Does Height Off the Ground Affect Bird Visitation and Fruit Set in the Pollen‐Limited Mistletoe Peraxilla tetrapetala (Loranthaceae)?

For pollination studies of forest species it is sometimes only possible to work on those flowers nearest to the ground. We test whether using low flowers introduces bias, by measuring height effects on bird visitation and fruit set in one mistletoe species pollinated by bellbirds in New Zealand. At this site, previous studies have shown fruit set near the ground to be pollen limited. We measured fruit set on 32 mistletoes at different heights in 11 host trees. Mistletoe fruit set varied significantly among host trees but did not vary with height. Although bellbirds generally forage preferentially in the upper part of the forest, mistletoe flowers appear to be attractive enough to ensure adequate visitation and fruit set at all heights.     

Determination of high-affinity oestrogen-receptor sites in uterine supernatant preparations

An assay method was developed for the determination of high-affinity oestradiol receptors in uterine supernatant preparations. When only high-affinity sites are present in such preparations, or when they predominate, the analysis of the equilibrium between oestradiol and receptor sites based on the Scatchard (1949) plot may be used to determine the dissociation constant of the equilibrium and the molar concentration of the high-affinity sites. When both high-affinity and low-affinity sites are present the Scatchard plot is no longer linear and cannot be used directly to determine high-affinity sites. Determination of the reverse velocity constants of the reaction between high-affinity (k(-1)) and low-affinity (k(-2)) receptor sites and [(3)H]oestradiol has shown that these constants differ by at least one order of magnitude. Advantage has been taken of this difference to introduce an additional step into the assay procedure that eliminates oestradiol bound to low-affinity sites and permits the determination of high-affinity sites in different species and under a variety of physiological conditions.