Ioscani Jimenez del Val

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. Monoclonal antibodies (mAbs) are currently the leading products of the biopharmaceutical industry. It has been widely reported that glycosylation directly influences the therapeutic mechanisms by which mAbs function in vivo. In addition, glycosylation has been identified as one of the main sources of monoclonal antibody heterogeneity, and thus, a critical parameter to follow during mAb manufacture. This article reviews the research on glycosylation of mAbs over the past 2 decades under the QbD scope. The categories presented under this scope are: (a) definition of the desired clinical effects of mAbs, (b) definition of the glycosylation‐associated critical quality attributes (glycCQAs) of mAbs, (c) assessment of process parameters that pose a risk for mAb glycCQAs, and (d) methods for accurately quantifying glycCQAs of mAbs. The information available in all four areas leads us to conclude that implementation of QbD to the manufacture of mAbs with specific glycosylation patterns will be a reality in the near future. We also foresee that the implementation of QbD will lead to the development of more robust and efficient manufacturing processes and to a new generation of mAbs with increased clinical efficacy.

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. In this work, different fed‐batch processes with two chemically defined proprietary media and feeds were studied using two IgG‐producing cell lines. Our results indicate that the balance of glucose and amino acid concentration in the culture is important for cell growth, IgG titer and N‐glycosylation. Accordingly, the ideal fate of glucose and amino acids in the culture could be mainly towards energy and recombinant product, respectively. Accumulation of by‐products such as NH4+ and lactate as a consequence of unbalanced nutrient supply to cell activities inhibits cell growth. The levels of Leu and Arg in the culture, which relate to cell growth and IgG productivity, need to be well controlled. Amino acids with the highest consumption rates correlate with the most abundant amino acids present in the produced IgG, and thus require sufficient availability during culture. Case‐by‐case analysis is necessary for understanding the effect of media and process optimization on glycosylation. We found that in certain cases the presence of Man5 glycan can be linked to limitation of UDP‐GlcNAc biosynthesis as a result of insufficient extracellular Gln. However, under different culture conditions, high Man5 levels can also result from low α‐1,3‐mannosyl‐glycoprotein 2‐β‐N‐acetylglucosaminyltransferase (GnTI) and UDP‐GlcNAc transporter activities, which may be attributed to high level of NH4+ in the cell culture. Furthermore, galactosylation of the mAb Fc glycans was found to be limited by UDP‐Gal biosynthesis, which was observed to be both cell line and cultivation condition‐dependent. Extracellular glucose and glutamine concentrations and uptake rates were positively correlated with intracellular UDP‐Gal availability. All these findings are important for optimization of fed‐batch culture for improving IgG production and directing glycosylation quality. Biotechnol. Bioeng. 2015;112: 521–535.

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]). The glycosylation reaction rate expressions were derived based on the reported kinetic mechanisms for each enzyme, and transport of nucleotide sugar donors [NSDs] from the cytosol to the Golgi lumen was modeled to serve as a link between glycosylation and cellular metabolism. Optimization‐based methodologies were developed for estimating unknown enzyme and TP concentration profile parameters. The resulting model is capable of reproducing glycosylation profiles of commercial mAbs. It can further reproduce the effect gene silencing of the FucT glycosylation enzyme and cytosolic NSD depletion have on the mAb oligosaccharide profile. All novel elements of our model are based on biological evidence and generate more accurate results than previous reports. We therefore believe that the improvements contribute to a more detailed representation of the N‐linked glycosylation process. The overall results show the potential of our model toward evaluating cell engineering strategies that yield desired glycosylation profiles. Additionally, when coupled to cellular metabolism, this model could be used to assess the effect of process conditions on glycosylation and aid in the design, control, and optimization of biopharmaceutical manufacturing processes.

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. The main focus of this work is the mechanistic in silico reconstruction of the nucleotide sugar donor (NSD) metabolic network by means of 34 species mass balances and the saturation kinetics rates of the 60 metabolic reactions involved. NSDs are the co-substrates of the glycosylation process in the Golgi apparatus and their simulated dynamic intracellular concentration profiles were linked to an existing model describing the distribution of N-linked glycan structures of the antibody constant region. The modelling framework also describes the growth dynamics of the cell population by means of modified Monod kinetics. Simulation results match well to experimental data from a murine hybridoma cell line. The result is a modelling platform which is able to describe the product glycoform based on extracellular conditions. It represents a first step towards the in silico prediction of the glycoform of a biotherapeutic and provides a platform for the optimisation of bioprocess conditions with respect to product quality.

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.

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. To establish the full protocol, two methods for the extraction of these compounds were evaluated for efficiency, and the requirement for quenching and washing the sample was assessed. A chromatographic method based on anion exchange has been optimized to separate and quantify eight nucleotides and nine NSs in less than 30 min. Degradation of nucleotides and NSs under extraction conditions was evaluated to aid in selection of the most efficient extraction protocol. We conclude that the optimized chromatographic method is quick, robust, and sensitive for quantifying nucleotides and NSs. Furthermore, our results show that samples taken from cell culture should be treated with 50% v/v acetonitrile and do not require quenching or washing for reliable extraction of nucleotides and NSs. This comprehensive protocol should prove useful in determining the impact of nucleotide and NS metabolism on protein glycosylation.

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. Higher degree of glucose starvation reduced intracellular concentrations of UDP‐GlcNAc and UDP‐GalNAc, but increased the levels of UDP‐Glc and UDP‐Gal. Increased GlcNAc and Gal occupancy correlated well with increased degree of glucose starvation, which can be attributed to the interplay between the dilution effect associated with change in specific productivity of mAbs and the changed nucleotide sugar metabolism. Herein, we also show and discuss that increased cell culture duration negatively affect the maturation of glycans. In addition, comparative proteomics analysis of cells was conducted to observe differences in protein abundance between early growth and early stationary phases. Generally higher expression of proteins involved in regulating cellular metabolism, extracellular matrix, apoptosis, protein secretion and glycosylation was found in early stationary phase. These analyses offered a systematic view of the intrinsic properties of these cells and allowed us to explore the root causes correlating culture duration with variations in the productivity and glycosylation quality of monoclonal antibodies produced with CHO cells. Biotechnol. Bioeng. 2015;112: 2172–2184.

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. Two alternative mechanistic representations of how mAb specific productivity (qp ) influences glycosylation are compared. In the first, mAb glycosylation is modulated by the linear velocity with which secretory cargo traverses the Golgi apparatus. In the second, glycosylation is influenced by variations in Golgi volume. Within our modelling framework, both mechanisms accurately reproduce experimentally-observed dynamic changes in mAb glycosylation. In addition, an optimisation-based strategy has been developed to estimate the concentration of glycosylation enzymes required to minimise mAb glycoform variability. Our results suggest that the availability of glycosylation machinery relative to cellular secretory capacity may play a crucial role in mAb glycosylation. In the future, the modelling framework presented here may aid in selecting and engineering cell lines that ensure consistent mAb glycosylatio.

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. Coupled with new analytical tools, these models are increasingly used to systemise and expedite the design and optimisation of protein production processes throughout the discovery and development stages.

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.