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.     

Distinctive roles of receptor‐interacting protein kinases 1 and 3 in caspase‐independent cell death of L929

Upon tumour necrosis factor alpha (TNFα) stimulation, cells respond actively by way of cell survival, apoptosis or programmed necrosis. The receptor‐interacting proteins 1 (RIP1) and 3 (RIP3) are responsible for TNFα‐mediated programmed necrosis. To delineate the differential contributions of RIP3 and RIP1 to programmed necrosis, L929 cells were stimulated with TNFα, carbobenzoxy‐valyl‐alanyl‐aspartyl‐[O‐methyl]‐fluoromethylketone (zVAD) or zVAD along with TNFα following RNA interference against RIP1 and RIP3, respectively. RIP1 silencing did not protect cells from TNFα‐mediated cell death, while RIP3 down‐regulation made them refractory to TNFα. The heat shock protein 90 inhibitor geldanamycin (GA) down‐regulated both RIP1 and RIP3 expression, which rendered cells resistant to zVAD/TNFα‐mediated cell death but not to TNFα‐mediated cell death alone. Therefore, the protective effect of GA on zVAD/TNFα‐stimulated necrosis might be attributed to RIP3, not RIP1, down‐regulation. Pretreatment of L929 cells with rapamycin mitigated zVAD‐mediated cell death, while the autophagy inhibitor chloroquine did not affect necrotic cell death. Meanwhile, necrotic cell death by zVAD and TNFα was caused by reactive oxygen species generation and effectively diminished by lipid‐soluble butylated hydroxyanisole. Taken together, the results indicate that RIP1 and RIP3 can independently mediate death signals being transduced by two different death stimuli, zVAD and TNFα. Copyright © 2013 John Wiley & Sons, Ltd.     

Intraband optical activity of semiconductor nanocrystals

Here we review our three recently developed analytical models describing the intraband optical activity of semiconductor nanocrystals, which is induced by screw dislocations, ionic impurities, or irregularities of the nanocrystal surface. The models predict that semiconductor nanocrystals can exhibit strong optical activity upon intraband transitions and have large dissymmetry of magnetic‐dipole absorption. The developed models can be used to interpret experimental circular dichroism spectra of nanocrystals and to advance the existing techniques of enantioseparation, biosensing, and chiral chemistry.     

Genome‐wide analysis of TNF‐alpha response in pigs challenged with porcine circovirus 2b

Tumor necrosis factor alpha (TNF‐α) is a pro‐inflammatory cytokine with a role in activating adaptive immunity to viral infections. By inhibiting the capacity of plasmacytoid dendritic cells to produce interferon‐α and TNF‐α, porcine circovirus 2 (PCV2) limits the maturation of myeloid dendritic cells and impairs their ability to recognize viral and bacterial antigens. Previously, we reported QTL for viremia and immune response in PCV2‐infected pigs. In this study, we analyzed phenotypic and genetic relationships between TNF‐α protein levels, a potential indicator of predisposition to PCV2 co‐infection, and PCV2 susceptibility. Following experimental challenge with PCV2b, TNF‐α reached the peak at 21 days post‐infection (dpi), at which time a difference was observed between pigs that expressed extreme variation in viremia and growth (P < 0.10). A genome‐wide association study (n = 297) revealed that genotypes of 56 433 SNPs explained 73.9% of the variation in TNF‐α at 21 dpi. Major SNPs were identified on SSC8, SSC10 and SSC14. Haplotypes based on SNPs from a SSC8 (9 Mb) 1‐Mb window were associated with variation in TNF‐α (P < 0.02), IgG (P = 0.05) and IgM (P < 0.13) levels at 21 dpi. Potential overlap of regulatory mechanisms was supported by the correlations between genomic prediction values of TNF‐α and PCV2 antibodies (21 dpi, r > 0.22), viremia (14–21 dpi, P > 0.29) and viral load (r = 0.31, P < 0.0001). Characterization of the QTL regions uncovered genes that could influence variation in TNF‐α levels as well as T‐ and B‐cell development, which can affect disease susceptibility.     

SOCS2 Balances Metabolic and Restorative Requirements during Liver Regeneration

After significant injury, the liver must maintain homeostasis during the regenerative process. We hypothesized the existence of mechanisms to limit hepatocyte proliferation after injury to maintain metabolic and synthetic function. A screen for candidates revealed suppressor of cytokine signaling 2 (SOCS2), an inhibitor of growth hormone (GH) signaling, was strongly induced after partial hepatectomy. Using genetic deletion and administration of various factors we investigated the role of SOCS2 during liver regeneration. SOCS2 preserves liver function by restraining the first round of hepatocyte proliferation after partial hepatectomy by preventing increases in growth hormone receptor (GHR) via ubiquitination, suppressing GH pathway activity. At later times, SOCS2 enhances hepatocyte proliferation by modulating a decrease in serum insulin-like growth factor 1 (IGF-1) that allows GH release from the pituitary. SOCS2, therefore, plays a dual role in modulating the rate of hepatocyte proliferation. In particular, this is the first demonstration of an endogenous mechanism to limit hepatocyte proliferation after injury.     

Speciation of Phlebotomus sandflies of the subgenus Larroussius coincided with the late Miocene-Pliocene aridification of the Mediterranean subregion

The phylogcny and mode of speciation of Mediterranean Phlebotomus of the subgenus Larroussius were inferred by comparative sequence analyses of a fragment of mitochondrial DNA (Cytochrome b) and of a nuclear gene (Elongation factor alpha). The molecular phytogenies were congruent basally, where their clades matched the species complexes defined by a few genitalic characters of each sex. Reticulate evolution was suggested for the most derived species complex [Phlebotomus perniciosus): the molecular phytogenies were incongruent, and mitochondrial-marker distribution was consistent with introgressive hybridizations not between sister species but between species whose ranges now overlap or abut. By considering the molecular phytogenies, the mitochondrial molecular clock and the ecological niches of the species, as well as the historical biogeography and palaeoecology of the Mediterranean subregion, we propose that the derived lineages arose from a sequential series of speciation events associated with habitat shifts promoted by progressive aridification. This 'taxon pulse'-like speciation occurred in the Pliocene, later than previously proposed in a vicariance hypothesis that invoked only tectonic events, but too early for Pleistocene Ice-age refugia to have played any role other than the isolation of geographical races. Speciation occurred before the proposed divergence of members of the Leishmania donovani complex and this helped to rule out any vector-parasite co-speciation or co-cladogenesis.     

Differential susceptibility to hydrogen sulfide-induced apoptosis between PHLDA1-overexpressing oral cancer cell lines and oral keratinocytes: Role of PHLDA1 as an apoptosis suppressor

Hydrogen sulfide (H₂S) is a novel gasotransmitter that plays multiple biological roles in various body systems. In addition to its endogenous production, H₂S is produced by bacteria colonizing digestive organs, including the oral cavity. H₂S was previously shown to enhance pro-apoptotic effects in cancer cell lines, although the mechanisms involved remain unclear. To properly assess the anti-cancer effects of H₂S, however, investigations of apoptotic effects in normal cells are also necessary. The aims of this study were (1) to compare the susceptibility to H₂S-induced apoptosis between the oral cancer cell line Ca9-22 and oral keratinocytes that were derived from healthy gingiva, and (2) to identify candidate genes involved in the induction of apoptosis by H₂S. The susceptibility to H₂S-induced apoptosis in Ca9-22 cells was significantly higher than that in keratinocytes. H₂S exposure in Ca9-22 cells, but not keratinocytes, enhanced the expression of pleckstrin homology-like domain, family A, member 1 (PHLDA1), which was identified through a differential display method. In addition, PHLDA1 expression increased during actinomycin D-induced apoptosis in Ca9-22 cells. Knockdown of PHLDA1 expression by small interfering RNA in Ca9-22 cells led to expression of active caspase 3, thus indicating apoptosis induction. The tongue cancer cell line SCC-25, which expresses PHLDA1 at a high level, showed similar effects. Our data indicate that H₂S is an anti-cancer compound that may contribute to the low incidence of oral cancer. Furthermore, we demonstrated the role of PHLDA1 as an apoptosis suppressor.     

Phenotypic and genetic relationships between growth and feed intake curves and feed efficiency and amino acid requirements in the growing pig

Improvement of feed efficiency in pigs has been achieved essentially by increasing lean growth rate, which resulted in lower feed intake (FI). The objective was to evaluate the impact of strategies for improving feed efficiency on the dynamics of FI and growth in growing pigs to revisit nutrient recommendations and strategies for feed efficiency improvement. In 2010, three BWs, at 35±2, 63±9 and 107±7 kg, and daily FI during this period were recorded in three French test stations on 379 Large White and 327 French Landrace from maternal pig populations and 215 Large White from a sire population. Individual growth and FI model parameters were obtained with the InraPorc^R software and individual nutrient requirements were computed. The model parameters were explored according to feed efficiency as measured by residual feed intake (RFI) or feed conversion ratio (FCR). Animals were separated in groups of better feed efficiency (RFI⁻ or FCR⁻), medium feed efficiency and poor feed efficiency. Second, genetic relationships between feed efficiency and model parameters were estimated. Despite similar average daily gains (ADG) during the test for all RFI groups, RFI⁻ pigs had a lower initial growth rate and a higher final growth rate compared with other pigs. The same initial growth rate was found for all FCR groups, but FCR⁻ pigs had significantly higher final growth rates than other pigs, resulting in significantly different ADG. Dynamic of FI also differed between RFI or FCR groups. The calculated digestible lysine requirements, expressed in g/MJ net energy (NE), showed the same trends for RFI or FCR groups: the average requirements for the 25% most efficient animals were 13% higher than that of the 25% least efficient animals during the whole test, reaching 0.90 to 0.95 g/MJ NE at the beginning of the test, which is slightly greater than usual feed recommendations for growing pigs. Model parameters were moderately heritable (0.30±0.13 to 0.56±0.13), except for the precocity of growth (0.06±0.08). The parameter representing the quantity of feed at 50 kg BW showed a relatively high genetic correlation with RFI (0.49±0.14), and average protein deposition between 35 and 110 kg had the highest correlation with FCR (−0.76±0.08). Thus, growth and FI dynamics may be envisaged as breeding tools to improve feed efficiency. Furthermore, improvement of feed efficiency should be envisaged jointly with new feeding strategies.