Integration Approaches to Model Bioreactor Hydrodynamics and Cellular Kinetics for Advancing Bioprocess Optimisation.

Large-scale bioprocesses are increasing globally to cater to the larger market demands for biological products. As fermenter volumes increase, the efficiency of mixing decreases, and environmental gradients become more pronounced compared to smaller scales. Consequently, the cells experience gradients in process parameters, which in turn affects the efficiency and profitability of the process.

CHOmpact: A reduced metabolic model of Chinese hamster ovary cells with enhanced interpretability.

Metabolic modeling has emerged as a key tool for the characterization of biopharmaceutical cell culture processes. Metabolic models have also been instrumental in identifying genetic engineering targets and developing feeding strategies that optimize the growth and productivity of Chinese hamster ovary (CHO) cells. Despite their success, metabolic models of CHO cells still present considerable challenges.

CHOGlycoNET: Comprehensive glycosylation reaction network for CHO cells.

Chinese hamster ovary (CHO) cells are extensively used for the production of glycoprotein therapeutics proteins, for which N-linked glycans are a critical quality attribute due to their influence on activity and immunogenicity. Manipulation of protein glycosylation is commonly achieved through cell or process engineering, which are often guided by mathematical models. However, each study considers a unique glycosylation reaction network that is tailored around the cell line and product at hand.

Rapid Antibody Glycoengineering in Chinese Hamster Ovary Cells.

Recombinant monoclonal antibodies bind specific molecular targets and, subsequently, induce an immune response or inhibit the binding of other ligands. However, monoclonal antibody functionality and half-life may be reduced by the type and distribution of host-specific glycosylation. Attempts to produce superior antibodies have inspired the development of genetically modified producer cells that synthesize glyco-optimized antibodies.

Rapid Antibody Glycoengineering in CHO Cells Via RNA Interference and CGE-LIF N-Glycomics.

The impact of the glycan distribution on the in vivo function and half-life of monoclonal antibodies has long motivated the genetic engineering of producer cells to achieve structures that enhance efficacy, safety and stability. To facilitate glycoengineering of IgG-producing Chinese hamster ovary cells, we present a rapid protocol that involves the use of RNA interference for the knockdown of genes of interest coupled with capillary gel electrophoresis and laser-induced fluorescence detection (CGE-LIF) for fast, high-throughput glycan analysis. We apply this methodology to the Fut8 gene, responsible for the addition of core fucose, which is a typical target for increasing antibody-dependent cellular cytotoxicity.

An integrated modular framework for modeling the effect of ammonium on the sialylation process of monoclonal antibodies produced by CHO cells.

Background: Monoclonal antibodies (mABs) have emerged as one of the most important therapeutic recombinant proteins in the pharmaceutical industry. Their immunogenicity and therapeutic efficacy are influenced by post-translational modifications, specifically the glycosylation process. Bioprocess conditions can influence the intracellular process of glycosylation.

Model-based optimization of antibody galactosylation in CHO cell culture.

Exerting control over the glycan moieties of antibody therapeutics is highly desirable from a product safety and batch-to-batch consistency perspective. Strategies to improve antibody productivity may compromise quality, while interventions for improving glycoform distribution can adversely affect cell growth and productivity. Process design therefore needs to consider the trade-off between preserving cellular health and productivity while enhancing antibody quality.

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.

Dynamics of immature mAb glycoform secretion during CHO cell culture: An integrated modelling framework.

Ensuring consistent glycosylation-associated quality of therapeutic monoclonal antibodies (mAbs) has become a priority in pharmaceutical bioprocessing given that the distribution and composition of the carbohydrates (glycans) bound to these molecules determines their therapeutic efficacy and immunogenicity. However, the interaction between bioprocess conditions, cellular metabolism and the intracellular process of glycosylation remains to be fully understood. To gain further insight into these interactions, we present a novel integrated modelling platform that links dynamic variations in mAb glycosylation with cellular secretory capacity.

A multi-pronged investigation into the effect of glucose starvation and culture duration on fed-batch CHO cell culture.

In this study, omics-based analysis tools were used to explore the effect of glucose starvation and culture duration on monoclonal antibody (mAb) production in fed-batch CHO cell culture to gain better insight into how these parameters can be controlled to ensure optimal mAb productivity and quality. Titer and N-glycosylation of mAbs, as well as proteomic signature and metabolic status of the production cells in the culture were assessed. We found that the impact of glucose starvation on the titer and N-glycosylation of mAbs was dependent on the degree of starvation during early stationary phase of the fed-batch culture.

Amino acid and glucose metabolism in fed-batch CHO cell culture affects antibody production and glycosylation.

Fed-batch Chinese hamster ovary (CHO) cell culture is the most commonly used process for IgG production in the biopharmaceutical industry. Amino acid and glucose consumption, cell growth, metabolism, antibody titer, and N-glycosylation patterns are always the major concerns during upstream process optimization, especially media optimization. Gaining knowledge on their interrelations could provide insight for obtaining higher immunoglobulin G (IgG) titer and better controlling glycosylation-related product quality.

Towards controlling the glycoform: a model framework linking extracellular metabolites to antibody glycosylation.

Glycoproteins represent the largest group of the growing number of biologically-derived medicines. The associated glycan structures and their distribution are known to have a large impact on pharmacokinetics. A modelling framework was developed to provide a link from the extracellular environment and its effect on intracellular metabolites to the distribution of glycans on the constant region of an antibody product.

An optimized method for extraction and quantification of nucleotides and nucleotide sugars from mammalian cells.

Glycosylation is a critical attribute of therapeutic proteins given its impact on the clinical safety and efficacy of these molecules. The biochemical process of glycosylation is inextricably dependent on metabolism and ensuing availability of nucleotides and nucleotide sugars (NSs) during cell culture. Herein, we present a comprehensive methodology to extract and quantify these metabolites from cultured cells.

Integration of models and experimentation to optimise the production of potential biotherapeutics.

Despite decades of clinical and commercial success, the current paradigm for drug discovery and development is still empirical and costly. The many hundreds of therapeutic proteins (TPs) in the development pipeline and the FDA-led quality-by-design initiative represent opportunities to address this issue. Advances in our understanding of cellular mechanisms as well as the physicochemical and biological characteristics of TPs have enabled researchers to develop computational models that analyse or even predict molecular and cellular behaviour under different conditions.

Metabolic network reconstruction: advances in in silico interpretation of analytical information.

Mathematical modelling is a powerful tool for the organisation and analysis of biological data. Both stoichiometric and kinetic models have been applied to the investigation of cellular metabolism in a variety of bacterial, yeast and mammalian hosts to elucidate metabolic network structure, optimise fermentation conditions and improve genetic engineering strategies among others. The current challenge is to interrelate different levels of information, from the genome to the transcriptome, the proteome and the metabolome, and experimental data from widely used high-throughput techniques to recreate a given phenotype and ultimately to make predictions about network and cellular behaviour.

A dynamic mathematical model for monoclonal antibody N-linked glycosylation and nucleotide sugar donor transport within a maturing Golgi apparatus.

Monoclonal antibodies (mAbs) are one of the most important products of the biopharmaceutical industry. Their therapeutic efficacy depends on the post-translational process of glycosylation, which is influenced by manufacturing process conditions. Herein, we present a dynamic mathematical model for mAb glycosylation that considers cisternal maturation by approximating the Golgi apparatus to a plug flow reactor and by including recycling of Golgi-resident proteins (glycosylation enzymes and transport proteins [TPs]).

Towards the implementation of quality by design to the production of therapeutic monoclonal antibodies with desired glycosylation patterns.

Quality by design (QbD) is a scheme for the development, manufacture, and approval of pharmaceutical products. The end goal of QbD is to ensure product quality by building it into the manufacturing process. The main regulatory bodies are encouraging its implementation to the manufacture of all new pharmaceuticals including biological products.