Evaluating the impact of high Pluronic® F68 concentrations on antibody producing CHO cell lines.

Pluronic® F68 (P-F68) is an important component of chemically-defined cell culture medium because it protects cells from hydrodynamic and bubble-induced shear in the bioreactor. While P-F68 is typically used in cell culture medium at a concentration of 1 g/L (0.1%), higher concentrations can offer additional shear protection and have also been shown to be beneficial during cryopreservation. Recent industry experience with variability in P-F68-associated shear-protection has opened up the possibility of elevated P-F68 concentrations in cell culture media, a topic that has not been previously explored in the context of industrial cell culture processes. Recognizing this gap, we first evaluated the effect of 1-5 g/L P-F68 concentrations in shake flask cultures over ten 3-day passages for cell lines A and B. Increase in terminal cell density and cell size was seen over time at higher P-F68 concentrations but protein productivity was not impacted. Results from this preliminary screening study suggested no adverse impact of high P-F68 concentrations. Subsequently fed-batch bioreactor experiments were conducted at 1 and 5 g/L P-F68 concentrations with both cell lines where cell growth, viability, metabolism, and product quality were examined under process conditions reflective of a commercial process. Results from these bioreactor experiments confirmed findings from the preliminary screen and also indicated no impact of elevated P-F68 concentration on product quality. If additional shear protection is desired, either due to raw material variability, cell line sensitivity, or a high-shear cell culture process, our results suggest this can be accomplished by elevating the P-F68 concentration in the cell culture medium without impacting cell culture performance and product quality.

A framework for real-time glycosylation monitoring (RT-GM) in mammalian cell culture.

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.

Use of a small molecule cell cycle inhibitor to control cell growth and improve specific productivity and product quality of recombinant proteins in CHO cell cultures.

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.

Glycans as cancer biomarkers.

BACKGROUND: Non-invasive biomarkers, such as those from serum, are ideal for disease prognosis, staging and monitoring. In the past decade, our understanding of the importance of glycosylation changes with disease has evolved. SCOPE OF REVIEW: We describe potential biomarkers derived from serum glycoproteins for liver, pancreatic, prostate, ovarian, breast, lung and stomach cancers. Methods for glycan analysis have progressed and newly developed high-throughput platform technologies have enabled the analysis of large cohorts of samples in an efficient manner. We also describe this evolution and trends to follow in the future. MAJOR CONCLUSIONS: Many convincing examples of aberrant glycans associated with cancer have come about from glycosylation analyses. Most studies have been carried out to identify changes in serum glycan profiles or through the isolation and identification of glycoproteins that contain these irregular glycan structures. In a majority of cancers the fucosylation and sialylation expression are found to be significantly modified. Therefore, these aberrations in glycan structures can be utilized as targets to improve existing cancer biomarkers. GENERAL SIGNIFICANCE: The ability to distinguish differences in the glycosylation of proteins between cancer and control patients emphasizes glycobiology as a promising field for potential biomarker identification. Furthermore, the high-throughput and reproducible nature of the chromatography platform have highlighted extensive applications in biomarker discovery and allowed integration of glycomics with other -omics fields, such as proteomics and genomics, making systems glycobiology a reality. This article is part of a Special Issue entitled Glycoproteomics.

Strategies for the profiling, characterisation and detailed structural analysis of N-linked oligosaccharides.

Many post-translational modifications, including glycosylation, are pivotal for the structural integrity, location and functional activity of glycoproteins. Sub-populations of proteins that are relocated or functionally changed by such modifications can change resting proteins into active ones, mediating specific effector functions, as in the case of monoclonal antibodies. To ensure safe and efficacious drugs it is essential to employ appropriate robust, quantitative analytical strategies that can (i) perform detailed glycan structural analysis, (ii) characterise specific subsets of glycans to assess known critical features of therapeutic activities (iii) rapidly profile glycan pools for at-line monitoring or high level batch to batch screening. Here we focus on these aspects of glycan analysis, showing how state-of-the-art technologies are required at all stages during the production of recombinant glycotherapeutics. These data can provide insights into processing pathways and suggest markers for intervention at critical control points in bioprocessing and also critical decision points in disease and drug monitoring in patients. Importantly, these tools are now enabling the first glycome/genome studies in large populations, allowing the integration of glycomics into other 'omics platforms in a systems biology context.

A HPLC-based glycoanalytical protocol allows the use of natural O-glycans derived from glycoproteins as substrates for glycosidase discovery from microbial culture.

Many disorders are characterised by changes in O-glycosylation, but analysis of O-glycosylation has been limited by the availability of specific endo- and exo-glycosidases. As a result chemical methods are employed. However, these may give rise to glycan degradation, so therefore novel O-glycosidases are needed. Artificial substrates do not always identify every glycosidase activity present in an extract. To overcome this, an HPLC-based protocol for glycosidase identification from microbial culture was developed using natural O-glycans and O-glycosylated glycoproteins (porcine stomach mucin and fetuin) as substrates. O-glycans were released by ammonia-based ß-elimination for use as substrates, and the bacterial culture supernatants were subjected to ultrafiltration to separate the proteins from glycans and low molecular size molecules. Two bacterial cultures, the psychrotroph Arthrobacter C1-1 and a Corynebacterium isolate, were examined as potential sources of novel glycosidases. Arthrobacter C1-1 culture contained a ß-galactosidase and N-acetyl-ß-glucosaminidase when assayed using 4-methylumbelliferyl substrates, but when defucosylated O-glycans from porcine stomach mucin were used as substrate, the extract did not cleave ß-linked galactose or N-acetylglucosamine. Sialidase activity was identified in Corynebacterium culture supernatant, which hydrolysed sialic acid from fetuin glycans. When both culture supernatants were assayed using the glycoproteins as substrate, neither contained endoglycosidase activity. This method may be applied to investigate a microbial or other extract for glycosidase activity, and has potential for scale-up on high-throughput platforms.

Molecular aspects of mucin biosynthesis and mucus formation in the bovine cervix during the periestrous period.

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.

Polyclonal hyper immunoglobulin: A proven treatment and prophylaxis platform for passive immunization to address existing and emerging diseases.

Passive immunization with polyclonal hyper immunoglobulin (HIG) therapy represents a proven strategy by transferring immunoglobulins to patients to confer immediate protection against a range of pathogens including infectious agents and toxins. Distinct from active immunization, the protection is passive and the immunoglobulins will clear from the system; therefore, administration of an effective dose must be maintained for prophylaxis or treatment until a natural adaptive immune response is mounted or the pathogen/agent is cleared. The current review provides an overview of this technology, key considerations to address different pathogens, and suggested improvements. The review will reflect on key learnings from development of HIGs in the response to public health threats due to Zika, influenza, and severe acute respiratory syndrome coronavirus 2.

Discovering new clinical markers in the field of glycomics.

Glycosylation modifications have been reported in a number of disease states and, as a result, there is significant focus on the discovery and development of glycan-based biomarkers. Glyco-biomarkers have the potential to enhance the efficacy and efficiency of the diagnostic procedures for these diseases.

Characterization of phenotypic and genotypic diversity in subclones derived from a clonal cell line.

Regulatory guidelines require the sponsors to provide assurance of clonality of the production cell line, and when such evidence is not available, additional studies are typically required to further ensure consistent long-term manufacturing of the product. One potential approach to provide such assurance of clonal derivation of a production cell line is to characterize subclones generated from the original cell line and assess their phenotypic and genotypic similarity with the hypothesis that cell lines derived from a clonal bank will share performance, productivity and product quality characteristics. In this study, a production cell line that was cloned by a validated FACS approach coupled with day 0 imaging for verification of single-cell deposition was subcloned using validated FACS and imaging methods. A total of 46 subclones were analyzed for growth, productivity, product quality, copy number, and integration site analysis. Significant diversity in cell growth, protein productivity, product quality attributes, and copy number was observed between the subclones, despite stability of the parent clone over time. The diversity in protein productivity and quality of the subclones were reproduced across time and production scales, suggesting that the resulting population post sub-cloning originating from a single cell is stable but with unique properties. Overall, this work demonstrates that the characteristics of isolated subclones are not predictive of a clonally derived parental clone. Consequently, the analysis of subclones may not be an effective approach to demonstrate clonal origin of a cell bank. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:613-623, 2018.

In vitro generation of polysialylated cervical mucins by bacterial polysialyltransferases to counteract cytotoxicity of extracellular histones.

Neutrophil extracellular traps (NET) are formed against pathogens. However, various diseases are directly linked to this meshwork of DNA. The cytotoxic properties of extracellular histones especially seem to be an important trigger during these diseases. Furthermore, NET accumulation on implants is discussed to result in an impaired efficiency or failure, depending on the category of implant. Interestingly, mucins have been investigated as surface coatings potentially capable of reducing neutrophil adhesion. Similarly, polysialic acid was shown to inactivate the cytotoxic properties of extracellular histones. We wanted to combine the probability to decrease the adhesion of neutrophils using mucins with the capability of sialic acid polymers to counteract histone-mediated cytotoxicity. To this end, we elongate cervical mucins using bacterial polysialyltransferases. Subsequent cell-based experiments demonstrated the activity of elongated mucins against histone-mediated cytotoxicity. Thus, polysialylated mucins may represent a novel component to coat implants or to combat diseases with exaggerated NET formation.

Platform technology to identify potential disease markers and establish heritability and environmental determinants of the human serum N-glycome.

Glycomics has been proven to be challenging when compared to genomics, transcriptomics, or proteomics. Understanding glycans is difficult as they have a non-template driven biosynthesis and their structural characterization requires highly efficient techniques. This review will describe the robotic platform developed in our laboratory for high-throughput analysis of N-glycans, and some of the advances obtained in the Glyco-biomarker field obtained in the last three years, including the first Genome-Wide Association Study showing a direct link between DNA polymorphisms and glycosylation.

High yields of monomeric recombinant beta-interferon from macroporous microcarrier cultures under hypothermic conditions.

Macroporous microcarriers such as Cytopore entrap mammalian cells in a mesh network allowing growth to high cell concentrations in a protected environment within a stirred culture. Chinese hamster ovary (CHO) cells producing recombinant human beta-interferon (IFN-beta) and grown in Cytopore microcarriers showed a 2.6- to 2.8-fold increase in the volumetric product titer compared with cells grown in an equivalent suspension culture. In an attempt to maximize production of IFN-beta, microcarrier cultures were subjected to a low temperature regime. Low temperature culture conditions (32 degrees C) have been shown previously to enhance cell specific productivity in suspension cultures although at reduced cell growth rates. These conditions can be optimized by a timely shift from physiological to hypothermic conditions during the culture run to maximize volumetric protein production. In the case of IFN-beta production the lower temperature has the added advantage of stabilizing the product and reducing intramolecular aggregation. Using a biphasic temperature-shift regime from 37 to 32 degrees C the volumetric production of IFN-beta was enhanced to 4.2-fold compared with a single temperature suspension culture in a controlled bench-top bioreactor. Furthermore, the degree of intramolecular aggregation of IFN-beta was reduced significantly (59%) compared with control cultures, largely due to the lower temperature but also partially due to the presence of microcarriers. These results indicate that the hypothermic conditions in a Cytopore culture had a combined and possibly synergistic effect of increasing volumetric production of the recombinant protein.

CHO cells adapted to hypothermic growth produce high yields of recombinant beta-interferon.

Mild hypothermic conditions (30-33 degrees C) have previously been shown to increase cell-specific productivity (Q(p)) of recombinant proteins from mammalian cells. However, this is often associated with a lower growth rate which off-sets any potential advantage of higher product titers. We report the isolation of a population of Chinese Hamster Ovary (CHO) cells that have been adapted to low-temperature growth by continuous subculture at low temperature for up to 300 days. This adapted cell population achieved a growth rate twofold greater than nonadapted cells under low-temperature conditions (32 degrees C) while maintaining an elevated level of cell-specific expression of recombinant beta-interferon. The volumetric titer of beta-interferon was enhanced by 70% in stationary cultures and by more than twofold by application of a temperature-shift strategy involving a growth to production phase. However, the low-temperature-adapted cells were fragile and demonstrated an increased sensitivity to hydrodynamic stress in agitated cultures. This problem was resolved by the use of macroporous microcarriers which protected the cells and allowed growth of high-density cultures under hypothermic conditions. This eventually resulted in a threefold enhancement of volumetric titer of monomeric beta-interferon compared to the original control culture at 37 degrees C.