No abstract

The continued need to improve therapeutic recombinant protein productivity has led to ongoing assessment of appropriate strategies in the biopharmaceutical industry to establish robust processes with optimized critical variables, that is, viable cell density (VCD) and specific productivity (product per cell, qP). Even though high VCD is a positive factor for titer, uncontrolled proliferation beyond a certain cell mass is also undesirable. To enable efficient process development to achieve consistent and predictable growth arrest while maintaining VCD, as well as improving qP, without negative impacts on product quality from clone to clone, we identified an approach that directly targets the cell cycle G1-checkpoint by selectively inhibiting the function of cyclin dependent kinases (CDK) 4/6 with a small molecule compound. Results from studies on multiple recombinant Chinese hamster ovary (CHO) cell lines demonstrate that the selective inhibitor can mediate a complete and sustained G0/G1 arrest without impacting G2/M phase. Cell proliferation is consistently and rapidly controlled in all recombinant cell lines at one concentration of this inhibitor throughout the production processes with specific productivities increased up to 110 pg/cell/day. Additionally, the product quality attributes of the mAb, with regard to high molecular weight (HMW) and glycan profile, are not negatively impacted. In fact, high mannose is decreased after treatment, which is in contrast to other established growth control methods such as reducing culture temperature. Microarray analysis showed major differences in expression of regulatory genes of the glycosylation and cell cycle signaling pathways between these different growth control methods. Overall, our observations showed that cell cycle arrest by directly targeting CDK4/6 using selective inhibitor compound can be utilized consistently and rapidly to optimize process parameters, such as cell growth, qP, and glycosylation profile in recombinant antibody production cultures.

The addition of the non-ionic surfactant, Pluronic F-68, to serum-free CHO cultures causes multi-functional effects that enhance cell yield in agitated cultures and reduce cell adhesion in stationary cultures. Three independent CHO cell lines were subjected to high liquid shear in assay systems that either included or excluded a liquid-gas interface. In the absence of Pluronic, there was a loss in cell viability in either assay system, although there was an intrinsic variability in sensitivity of the cell lines to shear damage. Supplementation with Pluronic prevented loss of cell viability, indicating protection in either a gas sparged or bubble-free environment. However, we found no evidence of long-term protection of cells once Pluronic was removed. Pluronic was capable of repairing trypsin-damaged cells as evidenced by enhanced growth, reduced membrane porosity, and improved robustness under liquid shear. The proportion of adherent cells was reduced to a minimal level by the presence of Pluronic although its effect was rapidly reversible with a high proportion (70%) of adherent cells observed within a few culture passages of its removal. The observed effects of Pluronic on these cultures are compatible with a mechanism in which the polymer forms a protective layer on the cell membrane, which has a significantly lower hydrophobicity.

Mucus within the cervical canal represents a hormonally regulated barrier that reconciles the need to exclude the vaginal microflora from the uterus during progesterone dominance, while permitting sperm transport at estrus. Its characteristics change during the estrous cycle to facilitate these competing functional requirements. Hydrated mucin glycoproteins synthesized by the endocervical epithelium form the molecular scaffold of this mucus. This study uses the bovine cervix as a model to examine functional groups of genes related to mucin biosynthesis and mucus production over the periestrous period when functional changes in cervical barrier function are most prominent. Cervical tissue samples were collected from 30 estrus synchronized beef heifers. Animals were slaughtered in groups starting 12 h after the withdrawal of intravaginal progesterone releasing devices (controlled internal drug releases) until 7 days postonset of estrus (luteal phase). Subsequent groupings represented proestrus, early estrus, late estrus, metestrus, and finally the early luteal phase. Tissues were submitted to next generation RNA-seq transcriptome analysis. We identified 114 genes associated with biosynthesis and intracellular transport of mucins, and postsecretory modifications of cervical; 53 of these genes showed at least a twofold change in one or more experimental group in relation to onset of estrus, and the differences between groups were significant (P < 0.05). The majority of these genes showed the greatest alteration in their expression in the 48 h postestrus and luteal phase groups.

Glycosylation is a critical characteristic of biotherapeutics because of its central role in in vivo efficacy. Multiple factors including medium composition and process conditions impact protein glycosylation and characterizing cellular response to these changes is essential to understand the underlying relationships. Current practice typically involves glycosylation characterization at the end of a fed-batch culture, which in addition to being an aggregate of the process, reflects a bias towards the end of the culture where a majority of the product is made. In an attempt to rigorously characterize the entire time-course of a fed-batch culture, a real-time glycosylation monitoring (RT-GM) framework was developed. It involves using the micro sequential injection (μSI) system as a sample preparation platform coupled with an ultra-performance liquid chromatography (UPLC) system for real-time monitoring of the antibody glycan profile. Automated sampling and sample preparations were performed using the μSI system and this framework was used to study manganese (Mn)-induced glycosylation changes over the course of a fed-batch culture. As expected, Mn-supplemented cultures exhibited higher galactosylation levels compared to control while the fucosylation and mannosylation were consistent for both supplemented and control cultures. Overall, the approach presented in the study allows real time monitoring of glycosylation changes and this information can be rapidly translated into process control and/or process optimization decisions to accelerate process development.