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Process development is typically associated with lengthy wet-lab experiments for the identification of good candidate setups and operating conditions. In this paper, we present the key features of a model-based approach for the identification and assessment of process design space (DSp), integrating the analysis of process performance and flexibility. The presented approach comprises three main steps: (1) model development & problem formulation, (2) DSp identification, and (3) DSp analysis. We demonstrate how such an approach can be used for the identification of acceptable operating spaces that enable the assessment of different operating points and quantification of process flexibility. The above steps are demonstrated on Protein A chromatographic purification of antibody-based therapeutics used in biopharmaceutical manufacturing., Pre-print paper under review

Transient gene expression (TGE) is a methodology employed in bioprocessing for the fast provision of recombinant protein material. Mild hypothermia is often introduced to overcome the low yield typically achieved with TGE and improve specific protein productivity. It is therefore of interest to examine the impact of mild hypothermic temperatures on both the yield and quality of transiently expressed proteins and the relationship to changes in cellular processes and metabolism. In this study, we focus on the ability of a Chinese hamster ovary cell line to galactosylate a recombinant monoclonal antibody (mAb) product. Through experimentation and flux balance analysis, our results show that TGE in mild hypothermic conditions led to a 76% increase in qP compared to TGE at 36.5°C in our system. This increase is accompanied by increased consumption of nutrients and amino acids, together with increased production of intracellular nucleotide sugar species, and higher rates of mAb galactosylation, despite a reduced rate of cell growth. The reduction in biomass accumulation allowed cells to redistribute their energy and resources toward mAb synthesis and Fc‐glycosylation. Interestingly, the higher capacity of cells to galactosylate the recombinant product in TGE at 32°C appears not to have been assisted by the upregulation of galactosyltransferases (GalTs), but by the increased expression of N‐acetylglucosaminyltransferase II (GnTII) in this cell line, which facilitated the production of bi‐antennary glycan structures for further processing.

Over 8 million tonnes of sugar beet are grown annually in the UK. Sugar beet pulp (SBP) is the main by-product of sugar beet processing which is currently dried and sold as a low value animal feed. SBP is a rich source of carbohydrates, mainly in the form of cellulose and pectin, including D-glucose (Glu), L-arabinose (Ara) and D-galacturonic acid (GalAc). This work describes the technical feasibility of an integrated biorefinery concept for the fractionation of SBP and conversion of these monosaccharides into value-added products. SBP fractionation is initially carried out by steam explosion under mild conditions to yield soluble pectin and insoluble cellulose fractions. The cellulose is readily hydrolysed by cellulases to release Glu that can then be fermented by a commercial yeast strain to produce bioethanol at a high yield. The pectin fraction can be either fully hydrolysed, using physico-chemical methods, or selectively hydrolysed, using cloned arabinases and galacturonases, to yield Ara-rich and GalAc-rich streams. These monomers can be separated using either Centrifugal Partition Chromatography (CPC) or ultrafiltration into streams suitable for subsequent enzymatic upgrading. Building on our previous experience with transketolase (TK) and transaminase (TAm) enzymes, the conversion of Ara and GalAc into higher value products was explored. In particular the conversion of Ara into L-gluco-heptulose (GluHep), that has potential therapeutic applications in hypoglycaemia and cancer, using a mutant TK is described. Preliminary studies with TAm also suggest GluHep can be selectively aminated to the corresponding chiral aminopolyol. The current work is addressing the upgrading of the remaining SBP monomer, GalAc, and the modelling of the biorefinery concept to enable economic and Life Cycle Analysis (LCA).

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.

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Recombinant proteins have been extensively studied for their wide therapeutic and research applications. The main therapeutic product category is that of monoclonal antibodies (mAbs), which have been widely approved to treat a variety of chronic and life-threatening diseases. Increasing mAb titre has been achieved mainly by cell culture medium improvement and genetic engineering to increase cell density and productivity. However, this improvement has caused many technical issues in both upstream (USP) and downstream (DSP) processes. The higher accumulation of the main cell-derived impurities, host cell proteins (HCPs), in the supernatant has proved to negatively affect product integrity and immunogenicity in addition to increasing the subsequent cost of capture and polishing steps. It has severely affected the performance of antibody drug candidates in at least two cases in which clinical trials have been put on hold as a result of HCP-related problems. Certain HCPs are naturally secreted, while others are inevitably released because of cell death and lysis. Exploring the relationship between critical process parameters (CPPs) and critical quality attributes (CQAs) in the context of HCP dynamics at a minimum cost is a highly important factor from an industrial point of view. Mathematical modelling of bioprocess dynamics is a valuable tool to improve industrial production at fast rate and low cost. A single stage volume-based population balance model (PBM) has been built to capture Chinese hamster ovary (CHO) cell behaviour in fed-batch bioreactors. The model includes two operating modes; the first at physiological temperature and the second, which represents a common industrial practice, with a shift to mild hypothermic conditions (32 ℃) in mid-exponential growth phase. The model considers the dynamic profile of substrates and metabolites, product titre and HCPs. Culture osmolality is also considered as a determining factor for cell growth rate and cell volume increase. The model was then used to optimise titre by controlling CPPs such as feed volume and frequency, the time point of temperature downshift as well as the harvesting time. The optimisation is subject to constraints such as maintaining culture viability above 80% and no feeding in the first 48 hours interval in all model optimisation runs. Four specific optimisation scenarios have been explored based on optimising titre and the titre/HCP ratio. This has been done on both operating modes; physiological temperature and initial physiological temperature with the possibility of temperature downshift after the second culture day. Total nutrients volume can be efficiently minimised by changing feeding volume and time point to satisfy the cellular metabolic need. This approach yields higher purity and more economical operating conditions. In general, higher product titres, up to 30%, and prolonged culture viability can be attained at the expense of higher feeding pulses. However, when a constraint on HCP concentration is also applied model-based optimisation results in shorter culture duration and, in turn, overall lower antibody titre. This thesis shows the usefulness of mathematical modelling for exploring trade-offs in bioprocess performance. Integrating this model with a downstream purification model to evaluate the cost of removing these fractions of impurities, can help determine what concentration of HCPs can be economically tolerated in the cell culture supernatant and aid whole bioprocess design. Open Access

N-linked glycosylation, is the covalent linkage of an oligosaccharide to an amido-group of an asparagine in a consensus sequence motif. The biosynthesis and transfer of a carbohydrate chain onto a nascent protein occurs within the ER and these initial steps are highly conserved throughout eukaryotes. Further processing of the carbohydrate chain occurs in the Golgi apparatus, where differences in processing between species and cell-types arise, resulting in different saccharide composition of the end-product. These differences are important in nature as N-linked glycosylation can influence a protein’s function and interaction. However, this trait can be problematic when manufacturing a biopharmaceutical, due to the potential pharmacokinetic influence of the glycan structure. Consequently, it is important to ensure a suitable N-linked glycan is produced. The removal of the N-linked glycosylation processing from the constraints of a cell will allow for greater control of the end saccharide composition of the glycan chain. A cell-free protein expression system will reduce heterogeneity allowing for the production of a homogenesis glycan chain. With this in mind, a project was conceived to create a cell free protein synthesis system for the expression of glycoproteins. The project commenced with implementation of a glycoengineering approach on P. pastoris to generate pentamannosyl oligosaccharide. This oligosaccharide is seen as substrate for the synthesis of a complex-type glycoprofile. To process the pentamannosyl oligosaccharide the expression of a glycosidase and two glycotransferases was attempted. The feasibility of producing a cell-free protein synthesis (CFPS) system from yeast cell lysate was also tested. This project resulted in a knockout P. pastoris strain which prevented the hypermannosylation glycoprofile. This strain can viably be used to generate the pentamannosyl oligosaccharide. A recombinant human GlcNAcT-I was produced which demonstrated the in vitro modification of an oligosaccharide chain. Finally, the project developed an active CFPS with a S. cerevisiae cell lysate but not with P. pastoris, suggesting that not all cell lysates can be utilised to generate an active CFPS. Together, these results highlight the difficulties in utilising a non-conventional yeast for genetic and metabolic engineering. This research has suggested targets for future work which could yet see the generation the first cell free glycoprotein synthesis with a complex-type glycoprofile. Open Access

One of the reasons for the rejection of new drugs during clinical trials is the presence of host cell proteins (HCPs) in the drug formulation. HCPs are immunogenic impurities that can compromise patient safety. Moreover, proteolytic and binding HCPs compromise the integrity, and, hence, the stability and efficacy of a recombinant protein. Therefore, HCPs should be removed from the bioprocess train as soon as possible. Current downstream purification platforms are challenged by HCP-mAb co-elution. A Quality by Design strategy to overcome this problem is to reduce the number of HCPs in the downstream feedstock by tracing their source back to upstream culture and eliminating it. Previous studies have shown that upstream cell culture parameters, e.g. harvest time and culture temperature, significantly affect HCP profiles. However, little is known about how host cells coordinate and regulate their molecular machinery under different cell culture environment that results in different HCP profiles. This study presents experimental results linking cell culture temperature and key process indicators of CHO cell cultures and post-Protein A purification (mAb titre, HCP level and HCP species) by considering the cellular behaviour in terms of cell growth, cell cycle distribution and cell health). This study involved the application of single-use fed-batch bioreactors to culture IgG4 producing GS-CHO cell line, cell health and cell cycle analysis with NucleoCounter, Protein A purification and proteomic analysis with HCP ELISA kits and LC-MS/MS. Cells were more robust under mild hypothermia: over 90% of cells were maintained in a healthy state until the decline phase, and the onset of apoptosis was less evident compared to the results for physiological temperature. IgG4 titre and HCP level at harvest were comparable between the two cases. However, mild hypothermia reduced the HCP variety in HCCF by 36%, including 44% and 27% lower proteases and chaperones, respectively. The differences in HCP variety at harvest resulted in a significantly different HCP profile post-Protein A purification between the two cases. Half of the critically immunogenic HCPs species as determined by the CHOPPI tool were different between the two cases. This study shows that cell culture conditions significantly affect the HCP profile at harvest and that of purified samples. Open Access

Glycoproteins make up the bulk of biologically-derived medicines, and are taking up an ever increasing share of the prescription pharmaceuticals market. As opposed to small molecule drugs, glycoproteins are large complex molecules with heterogeneity arising from a multitude of glycan moieties. Glycans are complex post-translation modifications, which result from a number of enzymatic reactions in the ER and Golgi collectively known as glycosylation and play an important role in pharmacokinetics such as drug safety, efficacy and half-life. It is known that the availability of the nucleotide sugar donors (NSDs), which are the co-substrates to the enzymatic glycosylation reactions of the Golgi, can be affected by a number of process conditions such as nutrient availability, as well as the addition of precursor molecules to the culture medium. Consequently, feeding strategies of nucleotide and nucleotide sugar precursors have been explored to exert control over the glycoform. In this work, a mathematical model platform is presented to quantify the impact of nutrient availability and feeding strategies on the glycosylation process with the aim to enable the design of feeding strategies to optimise the product glycoform. A modelling platform was developed and trained to link the extracellular environment, through the availability of intracellular metabolites in the cytoplasm and the Golgi apparatus, to the glycosylation of the conserved glycan site of the IgG heavy chain. The model platform comprises four parts, which are interlinked through dynamic fluxes and metabolite concentrations: A modified cell growth model based on Monod kinetics capturing cell culture dynamics and the impact of various hexose and nucleotide precursor additions to the cell culture media; A semi-structured purine and pyrimidine synthesis network describing the intracellular concentrations of nucleotide triphosphates, which are the co-substrates of NSD synthesis; A structured and mechanistic representation of the NSD synthesis pathway; The del Val et al. model describing the N-linked glycosylation process of the conserved glycan structure of the IgG antibody heavy chain. An initial proof-of-concept model framework was able to reproduce hybridoma batch cell growth dynamics, extracellular nutrient availability, dynamic intracellular NSD and nucleotide concentrations, product titer and the antibody product glycoform. Refinement of the original model framework and further training was achieved through a two-step CHO cell-based experimental process. In a first set of experiments the cells’ dynamic response to mannose and guanosine feeding was observed. The data allowed a model extension to fed-batch operation as well as inclusion of additional control and inhibitory mechanisms in the in silico representation of the nucleotide and NSD de novo synthesis networks. A second set of experiments probed the dynamic impact of galactose and uridine feeding strategies on antibody product galactosylation. The effects were dynamic perturbations with respect to cell culture dynamics, nucleotide and NSD synthesis as well as the antibody product glycoform. This formed as a basis for further model refinement and to provided a calibrated operating space with respect to galactose and uridine additions to cell culture media. The calibrated model platform was able to produce an optimal dynamic feeding strategy in silico with a predicted increase in galactosylation of 46.9% through pulse feeding of galactose and uridine on even days of culture. This framework is a first step towards a platform for the in silico optimisation of bioprocess conditions with respect to product quality and safety in line with the Quality by Design (QbD) paradigm. Open Access

With positive outcomes from medical treatments, monoclonal antibodies (mAbs) are to date the best-selling biologics in the pharmaceutical market. The fact that a lot of blockbuster drugs are facing the period of patent cliffs and patents of many of them are due to expire in the next 5 years, places an urgency for better, cheaper and more efficient bioproduction processes, as well as the development of novel drugs and biosimilars. To address to this issue, application of the Quality by Design paradigm that was introduced by the Food and Drug Administration (FDA) is of paramount importance. Medical values and safety of monoclonal antibodies have been reported to rely on the carbohydrate structures that are attached to the mAb N-linked glycosylation site on each constant region. Fc-N-linked glycosylation is considered as a critical quality attribute (CQA) of these therapeutic proteins under the scope of Quality by Design. It was also reported that different bioprocess conditions during recombinant mAb production directly impact glycan compositions and their distribution on the molecules, although the mechanism behind this change is not fully understood. This lack of understanding limits process design and optimisation. To address this issue we examined the effect of mild hypothermia (32oC) and the different recombinant expression systems on mAb N-linked glycosylation, using experiments, flux balance analysis (FBA) and mechanistic modelling to identify resulting differences in cell metabolism. A defined mathematical model that mechanistically and quantitatively describes CHO cell behaviour and metabolism, mAb synthesis and its N-linked glycosylation profiles before and after the induction of mild hypothermia in SGE and TGE expression systems was also constructed, which we believe is the first quantitative model that relates mild hypothermia and TGE system to the four elements mentioned above. Not only does the model aid understanding of the way bioprocess conditions affect product quality, it also provides a platform for bioprocess design, control and optimisation in industry and helps the implementation of the Quality by Design principles. Results obtained from our computational studies suggested glycosyltransferases to be the key players for changes observed among different bioprocess conditions, based on results obtained from this thesis we then manipulated the expression of galactosyltransferase in particular, through a proof-of-concept experiment using miRNAs. Open Access

Alzheimer’s disease (AD) is the most common form of dementia disorders and, yet, there is no preventative or curative treatment. It is associated with the progressive loss of memory and cognition and clinically divided into sporadic and familial forms. Familial Alzheimer’s disease (FAD) has predominantly a genetic predisposition with inherited mutations in the amyloid-β precursor protein (APP) and presinilin genes, which promote APP processing through the amyloidogenic pathway. This results in the release of the Aβ peptide, a major neurotoxic agent in AD progression. Accumulation of unfolded and misfolded disease-specific proteins (including Aβ and tau proteins) in neuronal cells perturbs endoplasmic reticulum (ER) homeostasis, leading to the onset of a cellular stress cascade called unfolded protein response (UPR), markers of which are upregulated in AD brain specimens. This suggests a possible role for ER stress in activation and the pathogenesis of AD. The research aimed to investigate the dynamic response of the UPR in an experimental model system of the disease combined with a computational model. For this purpose human neuroblastoma cell lines overexpressing the wild-type (APPWT) and two mutant forms of APP (APPMUT) associated with FAD were generated. Gene expression analysis of UPR markers revealed that overexpression of APP induces preconditioning of ER stress in all cell lines but with a stronger response in FAD-associated mutants. The progression sequence of UPR in APPWT and APPMUT was investigated in a time-course manner following the application of chemical stress. The results revealed that APPMUT exhibited the highest global response to chemically induced stress with a similar pattern. A computational model of the mammalian UPR was then generated and used to understand the dynamics of UPR. The model was able to reproduce our experimental data, which included pre-existing genetic factors (mutations in APP-associated with FAD) and a mimic of environmental triggers (induction of stress) consequently triggering the stress response. It suggested a different protein load and magnitude of transcriptional activation upon stress among the three cell lines. This was followed by in silico case studies exploring the effect of drugs targeting different branches of the UPR. This study proposes a novel multidisciplinary platform that could be further used for the development of therapeutics for AD. As the familial and sporadic form of the disease have similar neuropathological characteristics, drugs efficacious for FAD will also be beneficial for the most common form of AD. Open Access

It has long been a goal of the bioprocessing field to be able to produce proteins in a cost-effective and efficient manner. The current method of choosing high-producing mammalian cells is labour-intensive and time-consuming, representing an opportunity to employ new analytical methodologies in an effort to expedite progress. It would be advantageous to measure the intracellular concentration of key metabolites as the cells are growing. This would allow the user to have more information regarding the wellbeing and growth potential of cells. In this thesis the construction, optimisation and use of FRET biosensors for the in vivo measurement of glucose and glutamine in Chinese Hamster Ovary cells will be discussed. Experiments have been conducted in batch and fed-batch cultures as well as small-scale investigations using the BioLector™. The work presented here suggests the use of genetically encoded FRET biosensors allows for quantification of intracellular metabolite concentrations via FRET ratios during cell growth. These FRET biosensors also show that it may be possible to predict intracellular metabolite concentrations based solely on the FRET ratio of these cells. It has also been suggested that in a smaller volume micro fermentation system (BioLector™) these biosensor transfected cell lines can be used to detect changes in intracellular metabolite levels. These biosensor cell lines were then used for cell line selection, results indicate that the FRET ratio may also be used as a tool to discard various cell lines based on time of progression. Using data in the final study, it may be concluded that the early progressed cell lines may not necessarily be the highest protein producers and with, respect to scFv production, no product specific differences exist. The results indicate the usefulness of these FRET biosensors as tools in aiding the process of information gathering at an early stage during cell line selection. Open Access

The present thesis focuses on amino acids (a.a.) and their metabolism by Chinese hamster ovary cells, the workhorse of the multibillion dollar biopharmaceutical industry. The aim of the research was to explore a.a. transport and metabolism and define optimal operating conditions during fed-batch culture, which is the most common process mode used industrially. A fast and reliable way to calculate a.a. concentration ranges in media and feeds is of vital importance, as a.a. are the monomers of proteins, which account for 70% of dry cell weight. The desired recombinant product of bioprocesses is typically also a protein. The transport of a.a. into the cells was studied at the mRNA level of a.a. transporters for the first time in a bioprocessing context. The presented results demonstrate that a.a. transport is not the limiting step for recombinant protein formation. Also, the study allowed for a staged feeding strategy to be designed, where a.a. were not fed altogether. Following linear projection of an integral of viable cell concentration target and using the specific a.a. consumption rates during batch culture, six feeds were formulated containing a.a. and glucose. Three designs were based on the results of the a.a. transport study; however, they underperformed in comparison to the other feeds. In the latter, all nutrients were fed at the same time, resulting in cell culture performance comparable to that obtained with a commercial feed that was tested in parallel. This renders the presented method the first to define a traceable quantitative way to calculate amount of nutrients in the feeds. Flux balance analysis, a powerful technique that allows for investigation of intracellular dynamics, was used to analyse the metabolic data. An enhanced intracellular network was created by coupling two pre-existing in the literature that also for the first time included the glycosylation of the host proteins in the biomass equation. Finally, a novel methodology was developed and coded in R to calculate specific rates of consumption/production of various metabolites in cell culture. The methodology couples mass balances for fed-batch culture operation with constructed vectors of the sampling and feeding schemes. This can be further developed to a bioprocess relevant software platform for analysing cell culture data. Open Access

Pichia pastoris is a commonly used recombinant protein expression host, predominantly due to ease of genetic manipulation and its capacity for high cell densities in cheap culture media. While considerable yields can be achieved in this way, the specific productivity is relatively low. Consequently, the full impact of this host on industrial biotechnology has not yet been realised. This research aimed to develop a strategy to optimise production of single chain antibody fragments (scFvs), an industrially relevant protein, using an integrated modelling and experimental approach. Initially, a dynamic model was constructed from literature sources to reproduce the scFv production pathway in P. pastoris. It incorporated aspects of transcription, translation, folding and misfolding in the endoplasmic reticulum (ER). Moreover, the unfolded protein response (UPR) and ER-associated degradation (ERAD) were added as these two stress pathways are crucial to productivity. Simulations qualitatively reproduced phenomena including secretion saturation and the negative influence of high gene copy numbers on yield. The model was used to target evaluation of the experimental system: P. pastoris strains expressing the scFvs BC1 and MFE23. RT Q-PCR and LC-MS/MS results revealed some surprising correlations between certain factors, such as the concentration of Kar2 and PDI, and yield. Moreover, it showed that there was more than one route to high productivity. Finally, it suggested that there may be an internal regulation of Kar2 that would be crucial to strategies aiming to increase yield through overexpression of that chaperone. Together, the results revealed a more complex picture of productivity than previously understood. In order to develop a strategy for optimal scFv production in P. pastoris, a greater understanding of the underlying biology and biochemistry is required. This research has suggested targets for future work which should generate insight into the network of factors responsible. Open Access

The pharmaceutical industry is going through a rather turbulent period. Many blockbuster drugs have fallen off patent over the past two years and many more are expected to do so in the near future. In response, pharmaceutical companies have continued searching for products that will replace those that have lost patent protection. However, drug development and approval is extremely time-consuming and costly. So that this critical issue is addressed, industry experts and regulatory agencies have jointly proposed the implementation of Quality by Design (QbD) principles in the development and manufacture of all new drugs. Adoption of QbD is expected to reduce drug development cost and approval time. It is also expected to encourage innovation by developing drugs, and the processes used to manufacture them, around the mechanisms that relate process inputs with end product quality. Within this context, monoclonal antibodies (mAbs) are currently the highest-selling products of the biopharmaceutical industry and are projected to account for nearly half of the world’s top-selling drugs by 2018. All currently commercialized mAbs contain N-linked glycans (complex carbohydrates) bound to their protein backbone. These carbohydrates, in turn, have been widely reported to impact the safety and efficacy of mAbs. Furthermore, it has widely been reported that bioprocess conditions heavily impact the composition and distribution of these glycans. For these reasons, mAb glycosylation is considered a critical quality attribute (CQA) of these therapeutic proteins under the QbD scope. Based on QbD principles, the objective of this thesis was to generate a mathematical model that mechanistically relates the effect of nutrient availability throughout cell culture with the glycan profile of a mAb. The model was constructed from three individual ones. The first model describes the N-linked glycosylation process which occurs in the Golgi apparatus. The second model is unstructured and describes cell culture dynamics. The third and final model describes the biosynthetic pathway for nucleotide sugars. All three models were developed independently, but were adapted with features so that they could be interconnected. The glycosylation model approximates the Golgi apparatus to a single plug flow reactor where resident proteins (glycosylation enzymes and transport proteins) are recycled from distal portions of the Golgi space to proximal ones. Optimisation-based methods were developed to estimate unknown parameters of the model. The cell culture dynamics model was developed to represent cell growth, nutrient consumption and mAb synthesis. It was originally based on Monod kinetics, but was adapted to include experimentally-encountered complexity. The model for nucleotide metabolism was heuristically reduced from 35 constituting reactions to 7. Additional mechanistic features were adapted or included to ensure model fidelity. Experimentally, batch cultures were performed with hybridoma (CRL-1606 from ATCC). Data for viable cell density, glucose, glutamine, lactate, ammonia and mAb titre were collected. Intracellular samples were produced by perchloric acid extraction. These samples were then analysed for nucleotide sugar content using a high performance anion exchange chromatographic method which was optimized to quantify eight nucleotide sugars and four nucleotides in 30min. mAb bound glycans were analysed by MALDI mass spectrometry. The experimental data was used to estimate the unknown parameters of the models. The models – along with their associated parameters – were then combined to produce a coupled model that mechanistically relates nutrient availability with mAb glycosylation-associated quality. With further validation, such a model could be used for bioprocess design, control and optimization.