An integrated modelling/experimental framework for protein-producing cell cutures

Imperial Users onl

A theoretical estimate for nucleotide sugar demand towards Chinese Hamster Ovary cellular glycosylation

Glycosylation greatly influences the safety and efficacy of many of the highest-selling recombinant therapeutic proteins (rTPs). In order to define optimal cell culture feeding strategies that control rTP glycosylation, it is necessary to know how nucleotide sugars (NSs) are consumed towards host cell and rTP glycosylation. Here, we present a theoretical framework that integrates the reported glycoproteome of CHO cells, the number of N-linked and O-GalNAc glycosylation sites on individual host cell proteins (HCPs), and the carbohydrate content of CHO glycosphingolipids to estimate the demand of NSs towards CHO cell glycosylation. We have identified the most abundant N-linked and O-GalNAc CHO glycoproteins, obtained the weighted frequency of N-linked and O-GalNAc glycosites across the CHO cell proteome, and have derived stoichiometric coefficients for NS consumption towards CHO cell glycosylation. By combining the obtained stoichiometric coefficients with previously reported data for specific growth and productivity of CHO cells, we observe that the demand of NSs towards glycosylation is significant and, thus, is required to better understand the burden of glycosylation on cellular metabolism. The estimated demand of NSs towards CHO cell glycosylation can be used to rationally design feeding strategies that ensure optimal and consistent rTP glycosylation

Population balance modelling captures host cell protein dynamics in CHO cell cultures

Monoclonal antibodies (mAbs) have been extensively studied for their wide therapeutic and research applications. Increases in mAb titre has been achieved mainly by cell culture media/feed improvement and cell line engineering to increase cell density and specific mAb productivity. However, this improvement has shifted the bottleneck to downstream purification steps. The higher accumulation of the main cell-derived impurities, host cell proteins (HCPs), in the supernatant can negatively affect product integrity and immunogenicity in addition to increasing the cost of capture and polishing steps. Mathematical modelling of bioprocess dynamics is a valuable tool to improve industrial production at fast rate and low cost. Herein, a single stage volume-based population balance model (PBM) has been built to capture Chinese hamster ovary (CHO) cell behaviour in fed-batch bioreactors. Using cell volume as the internal variable, the model captures the dynamics of mAb and HCP accumulation extracellularly under physiological and mild hypothermic culture conditions. Model-based analysis and orthogonal measurements of lactate dehydrogenase activity and double-stranded DNA concentration in the supernatant show that a significant proportion of HCPs found in the extracellular matrix is secreted by viable cells. The PBM then served as a platform for generating operating strategies that optimise antibody titre and increase cost-efficiency while minimising impurity levels

Hybrid dynamic model of monoclonal antibody production using CHO cells

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Whole-cell Escherichia coli lactate biosensor for monitoring mammalian cell cultures during biopharmaceutical production

Many high-value added recombinant proteins, such as therapeutic glycoproteins, are produced using mammalian cell cultures. In order to optimise the productivity of these cultures it is important to monitor cellular metabolism, for example the utilisation of nutrients and the accumulation of metabolic waste products. One metabolic waste product of interest is lactic acid (lactate), overaccumulation of which can decrease cellular growth and protein production. Current methods for the detection of lactate are limited in terms of cost, sensitivity, and robustness. Therefore, we developed a whole-cell Escherichia coli lactate biosensor based on the lldPRD operon and successfully used it to monitor lactate concentration in mammalian cell cultures. Using real samples and analytical validation we demonstrate that our biosensor can be used for absolute quantification of metabolites in complex samples with high accuracy, sensitivity and robustness. Importantly, our whole-cell biosensor was able to detect lactate at concentrations more than two orders of magnitude lower than the industry standard method, making it useful for monitoring lactate concentrations in early phase culture. Given the importance of lactate in a variety of both industrial and clinical contexts we anticipate that our whole-cell biosensor can be used to address a range of interesting biological questions. It also serves as a blueprint for how to capitalise on the wealth of genetic operons for metabolite sensing available in Nature for the development of other whole-cell biosensors

Immunoglobulin G N-glycan biomarkers for autoimmune diseases: Current state and a glycoinformatics perspective

The effective treatment of autoimmune disorders can greatly benefit from disease-specific biomarkers that are functionally involved in immune system regulation and can be collected through minimally invasive procedures. In this regard, human serum IgG N-glycans are promising for uncovering disease predisposition and monitoring progression, and for the identification of specific molecular targets for advanced therapies. In particular, the IgG N-glycome in diseased tissues is considered to be disease-dependent; thus, specific glycan structures may be involved in the pathophysiology of autoimmune diseases. This study provides a critical overview of the literature on human IgG N-glycomics, with a focus on the identification of disease-specific glycan alterations. In order to expedite the establishment of clinically-relevant N-glycan biomarkers, the employment of advanced computational tools for the interpretation of clinical data and their relationship with the underlying molecular mechanisms may be critical. Glycoinformatics tools, including artificial intelligence and systems glycobiology approaches, are reviewed for their potential to provide insight into patient stratification and disease etiology. Challenges in the integration of such glycoinformatics approaches in N-glycan biomarker research are critically discussed

Host cell protein removal from biopharmaceutical preparations: toward the implementation of quality by design

Downstream processing of protein products of mammalian cell culture currently accounts for the largest fraction of the total production cost. A major challenge is the removal of host cell proteins, which are cell-derived impurities. Host cell proteins are potentially immunogenic and can compromise product integrity during processing and hold-up steps. There is an increasing body of evidence that the type of host cell proteins present in recombinant protein preparations is a function of cell culture conditions and handling of the harvest cell culture fluid. This, in turn, can affect the performance of downstream purification steps as certain species are difficult to remove and may require bespoke process solutions. Herein, we review recent research on the interplay between upstream process conditions, host cell protein composition and their downstream removal in antibody production processes, identifying opportunities for increasing process understanding and control. We further highlight advances in analytical and computational techniques that can enable the application of quality by design

CHO cell cultures in shake flasks and bioreactors present different host cell protein profiles in the supernatant

Several studies on the impact of cell culture parameters on the profile of host cell protein (HCP) impurities have been carried out in shake flasks. Herein, we explore how transferable the findings and conclusions of such investigations are to lab-scale bioreactors. Experiments were performed in both systems in fed-batch mode under physiological temperature and with a shift to mild hypothermia and the impact on key upstream performance indicators was quantified. Under both temperatures, bioreactors produced a richer HCP pool despite the overall concentration being similar at both scales and temperatures. The number of different HCPs detected in bioreactor supernatants was four times higher than that in flasks under physiological temperature and more than eight times higher under mild hypothermia. The origin of HCPs was also altered from mostly naturally secreted proteins in flasks to mainly intracellular proteins in bioreactors at the lower temperature. Although the number of species correlated with apoptotic cell density in bioreactors, this was not the case in flasks. Even though the level of HCP impurities and mAb/HCP concentration ratio were similar under all four conditions with an average of approximately 330 μg HCP/mL culture and 0.3 mg HCP/mg IgG4, respectively, the fact that culture method significantly affects the number of species present in the supernatant can have implications for downstream processing steps

Osmolality effects on CHO cell growth, cell volume and antibody productivity and glycosylation

The addition of nutrients and accumulation of metabolites in a fed-batch culture of Chinese hamster ovary (CHO) cells leads to an increase in extracellular osmolality in late stage culture. Herein, we explore the effect of osmolality on CHO cell growth, specific monoclonal antibody (mAb) productivity and glycosylation achieved with the addition of NaCl or the supplementation of a commercial feed. Although both methods lead to an increase in specific antibody productivity, they have different effects on cell growth and antibody production. Osmolality modulation using NaCl up to 470 mOsm kg−1 had a consistently positive effect on specific antibody productivity and titre. The addition of the commercial feed achieved variable results: specific mAb productivity was increased, yet cell growth rate was significantly compromised at high osmolality values. As a result, Feed C addition to 410 mOsm kg−1 was the only condition that achieved a significantly higher mAb titre compared to the control. Additionally, Feed C supplementation resulted in a significant reduction in galactosylated antibody structures. Cell volume was found to be positively correlated to osmolality; however, osmolality alone could not account for observed changes in average cell diameter without considering cell cycle variations. These results help delineate the overall effect of osmolality on titre and highlight the potentially negative effect of overfeeding on cell growth