CHO Cell Culture Research Articles

Protocol for bevacizumab purification using Ac-PHQGQHIGVSK-agarose

Bevacizumab is a monoclonal antibody, produced in CHO cells, used for the treatment of many human cancers. It is an anti-vascular endothelial growth factor (antsi-VEGF) that blocks the growth of tumor blood vessels. Nowadays its purification is achieved by affinity chromatography (AC) using protein A which is a very expensive ligand. On the other hand, the peptide Ac-PHQGQHIGVSK contained in the VEGF fragment binds bevacizumab with high affinity. This short peptide ligand has higher stability and lower cost than protein A and it can be prepared very easily by solid phase peptide synthesis. The present protocol describes the synthesis of Ac-PHQGQHIGVSK-agarose and its use for affinity chromatography purification of bevacizumab from a clarified CHO cell culture.•Ac-PHQGQHIGVSK-agarose capacity and selectivity are equivalent to those of protein A matrices.•The peptide ligand shows a greater stability and lower cost. The lack of Trp, Met or Cys in the peptide ligand prevents its oxidation and extends the useful life of the chromatographic matrix.•Mild conditions used during chromatography preserved the integrity of bevacizumab.

Novel peptoid-based adsorbents for purifying IgM and IgG from polyclonal and recombinant sources

Polyclonal immunoglobulin therapeutics comprising dosed IgG and IgM combinations are powerful tools in fighting cancer and severe infections. The inability of protein ligands to produce polyclonal IgG- and IgM-enriched formulations and recover monoclonal IgM calls for novel ligands with superior biorecognition activity. In this study, a peptoid ligand discovered by our group, and integrated into affinity adsorbents LigaTrap Technologies’ “Human IgG” and “Human IgM”, were utilized to purify IgG and IgM from complex fluids. IgG purification from human serum using LigaTrap IgG afforded 94.6% purity and 62.9% yield, on par with Protein A/G resins. When challenged with CHO and HEK cell culture harvests with low IgG titer (<1 mg/mL), LigaTrap IgG returned values of yield and purity well above 60% and 90%. LigaTrap IgM was evaluated for purifying IgM in comparison with commercial adsorbents, and afforded a product purity of 93% from a CHO harvest (IgM titer of 1 mg/mL) and 75.1% yield from a HEK harvest (0.5 mg/mL). LigaTrap-M provided IgM enrichment up to 11-fold higher than HiTrap resin. The peptoid adsorbents separated IgG-depleted human serum into IgM- and IgA-enriched fractions. These results demonstrate the potential of the peptoid ligand for manufacturing polyclonal Ig formulations and monoclonal IgM therapeutics.

Support Vector and Locally Weighted regressions to monitor monoclonal antibody glycosylation during CHO cell culture processes, an enhanced alternative to Partial Least Squares regression

Since monoclonal antibodies (mAb) are sensitive to the manufacturing process, several mAb variants can be the result of a single batch production. The most critical source of heterogeneity is glycosylation which has a profound impact on safety and efficacy of the final product. Implementation of monitoring and control of the process using the Quality by Design (QbD) approach may help to ensure mAb specifications, although its implementation is limited by the availability of real-time specific measurements. All current approaches to elucidate mAb glycoforms require sampling and labour-intensive efforts. Thus, glycosylation analysis is often performed with the objective of detecting quality defects at the end of the culture process. In this work, the capability of Near Infrared spectroscopy and chemometric treatment to accurately monitor mAb glycosylation during CHO cells cultures using in situ probes is shown for the first time. Real-time monitoring of glycosylation, in terms of high mannose isoforms, fucosylated, sialylated and galactosylated isoforms as well as non-glycosylated mAb, has been successfully performed by the novel use of Locally Weighted Regression (LWR) and Support Vector Regression (SVR). These encouraging results open the way for the implementation of control systems on the impact of cell culture operating parameters on mAb heterogeneity, particularly glycosylation, during CHO cell culture processes through the QbD approach.

In silico model-based characterization of metabolic response to harsh sparging stress in fed-batch CHO cell cultures

Mammalian cell culture platform has been successfully implemented for industrial biopharmaceutical production through the advancements in early stage process development including cell-line engineering, media design and process optimization. However, late stage developments such as scale-up, scale-down and large-scale cell cultivation still face many industrial challenges to acquire comparable process performance between different culture scales. One of them is the sparging strategy which significantly affects productivity, quality and comparability. Currently, it is mainly relying on the empirical records due to the lack of theoretical framework and scarcity of available literatures to elucidate intracellular metabolic features. Therefore, it is highly required to characterize the underlying mechanism of physiological changes and metabolic states upon the aeration stress. To this end, initially we cultivated antibody producing CHO cells under mild and harsh sparging conditions and observed that sparging stress leads to the decreased cell growth rate, viability and productivity. Subsequent in silicomodel-driven flux analysis suggested that sparging stress rewires amino acid metabolism towards the enriched H2O2 turnover rate by up-regulated fluxes of amino acid oxidases. Interestingly, many of these H2O2-generating reactions were closely connected with the production of NADH, NADPH and GSH which are typical reducing equivalents. Thus, we can hypothesize that increased amino acid uptake caused by sparging stress contributes to restore redox homeostasis against oxidative stress. The current model-driven systematic data analysis allows us to quickly define distinct metabolic feature under stress condition by using basic cell cultivation datasets.

Interest of locally weighted regression to overcome nonlinear effects during in situ NIR monitoring of CHO cell culture parameters and antibody glycosylation.

Animal cell culture processes have become the standard platform to produce therapeutic proteins such as recombinant monoclonal antibodies (mAb). Since the mAb quality could be subject to significant changes depending on manufacturing process conditions, real time monitoring and control systems are required to ensure mAb specifications mainly glycosylation and patient safety. Up to now, real time monitoring glycosylation of proteins has received scarce attention. In this article, the use of near infrared (NIR) to monitor mAb glycosylation has been reported for the first time. Whereas monitoring models are mainly constructed using linear partial least squares regressions (PLSR), evidences presented in this study indicate nonlinearity relationship between in situ captured spectra and compound concentrations, compromising the PLSR performances. A novel and simple approach was proposed to fit nonlinearity using the locally weighted regression (LWR). The LWR models were found to be more appropriate for handling information contained in spectra so that real time monitoring of cultures were accurately performed. Moreover, for the first time, the LWR calibration models allowed mAb glycosylation to be monitored, in a real time manner, by using in situ NIR spectroscopy. These results represent a further step toward developing active-control feedback of animal cell processes, particularly for ensuring properties of biologics.

Multivariate data analysis of capacitance frequency scanning for online monitoring of viable cell concentrations in small-scale bioreactors

Viable cell concentration (VCC) is one of the most important process attributes during mammalian cell cultivations. Current state-of-the-art measurements of VCC comprise offline methods which do not allow for continuous process data. According to the FDA’s process analytical technology initiative, process monitoring and control should be applied to gain process understanding and to ensure high product quality. In this work, the use of an inline capacitance probe to monitor online VCCs of a mammalian CHO cell culture process in small-scale bioreactors (250 mL) was investigated. Capacitance sensors using single frequency are increasingly common for biomass monitoring. However, the single-frequency signal corresponds to the cell polarization that represents the viable cell volume. Therefore single-frequency measurements are dependent on cell diameter changes. Measuring the capacitance across various frequencies (frequency scanning) can provide information about the VCC and cope with changing cell diameter. Applying multivariate data analysis on the frequency scanning data successfully enabled direct online monitoring of VCCs in this study. The multivariate model was trained with data from 5 standard cultivations. The model provided a prediction of VCCs with relative errors from 5.5 to 11%, which is a good agreement with the acceptance criterion based on the offline reference method accuracy (approximately 10% relative error) and strongly improved compared with single-frequency results (16 to 23% relative error). Furthermore, robustness trials were conducted to demonstrate the model’s predictive ability under challenging conditions. The process deviations in regard to dilution steps and feed variations were detected immediately in the online prediction of the VCC with relative errors between 6.7 and 13.2%. Thus in summary, the presented method on capacitance frequency scanning demonstrates its suitability for process monitoring and control that can save batches, time, and cost.Graphical abstract

Monitoring online biomass with a capacitance sensor during scale-up of industrially relevant CHO cell culture fed-batch processes in single-use bioreactors

In 2004, the FDA published a guideline to implement process analytical technologies (PAT) in biopharmaceutical processes for process monitoring to gain process understanding and for the control of important process parameters. Viable cell concentration (VCC) is one of the most important key performance indicator (KPI) during mammalian cell cultivation processes. Commonly, this is measured offline. In this work, we demonstrated the comparability and scalability of linear regression models derived from online capacitance measurements. The linear regressions were used to predict the VCC and other familiar offline biomass indicators, like the viable cell volume (VCV) and the wet cell weight (WCW), in two different industrially relevant CHO cell culture processes (Process A and Process B). Therefore, different single-use bioreactor scales (50–2000 L) were used to prove feasibility and scalability of the in-line sensor integration. Coefficient of determinations of 0.79 for Process A and 0.99 for Process B for the WCW were achieved. The VCV was described with high coefficients of determination of 0.96 (Process A) and 0.98 (Process B), respectively. In agreement with other work from the literature, the VCC was only described within the exponential growth phase, but resulting in excellent coefficients of determination of 0.99 (Process A) and 0.96 (Process B), respectively. Monitoring these KPIs online using linear regression models appeared to be scale-independent, enabled deeper process understanding (e.g. here demonstrated in monitoring, the feeding profile) and showed the potential of this method for process control.

Evaluating the impact of suramin additive on CHO cells producing Fc-fusion protein.

To examine the effects of suramin in CHO cell cultures in terms of the cell culture performance and quality of the Fc-fusion protein. Suramin had positive effects on the CHO cell cultures. The addition of suramin caused an increase in the viable cell density, cell viability, and titer of the Fc-fusion protein. Moreover, suramin had no impact on protein aggregation and enhanced the sialic acid contents of Fc-fusion protein by 1.18-fold. The enhanced sialylation was not caused by the increased nucleotide sugar level but by the inhibition of sialidase activity. The results showed that suramin inhibited apoptosis and had positive impacts on the productivity and quality of Fc-fusion protein. The addition of suramin increased the production of Fc-fusion protein and enhanced sialylation when added as a supplement to the media component in CHO cell cultures. This study suggested that suramin could be a beneficial additive during the biological production in terms of the productivity and quality of Fc-fusion protein.

Concomitant consumption of glucose and lactate: A novel batch production process for CHO cells

The rapid consumption of large quantities of glucose in conventional CHO batch processes yields large quantities of lactate, leading to the inhibition of cell growth. We have observed that under certain culture conditions, CHO cells are able to co-metabolize glucose and lactate, even during the growth phase.Taking advantage of this metabolic behavior, a new batch process has been defined where there is a concomitant consumption of glucose and lactate from the beginning of the culture. This batch process requires the addition of lactate to the culture media as well as keeping the pH around 6.8. As a result, an exponential growth phase without lactate production together with a substantially diminished glucose consumption is achieved.Flux Balance Analysis (FBA) confirmed that in glucose consumption/lactate generation, most of the pyruvate generated through glycolysis is converted to lactate to fulfill the NADH regeneration requirements into the cytoplasm, whereas only a 31% of pyruvate enters the TCA through acetyl-CoA. In contrast, in glucose-lactate concomitant consumption, glucose uptake is reduced up to 3-fold, balancing glycolysis and TCA cycle fluxes and thus yielding to a more efficient substrate consumption. Such metabolic behavior in which glucose and lactate are simultaneously consumed from the beginning of the culture has never been reported in CHO cell cultures, and it is promising for the design of new culturing strategies in batch, fed-batch or perfusion processes.

A new tetrapeptide biomimetic chromatographic resin for antibody separation with high adsorption capacity and selectivity

Biomimetic affinity chromatography with short peptide ligands is a developing technology, which has great potential for antibody separation and purification. In this study, a tetrapeptide library with critical residues of natural ligands to hIgG was constructed and a novel tetrapeptide ligand (Ac-FYHE) with high LibDock scores was selected by molecular docking. Then, Ac-FYHE ligand was linked to agarose bead to prepare a new chromatography resin. The properties of antibody adsorption were measured and evaluated by static/dynamic adsorption. It was found that the resin with ligand Ac-FYHE has high binding capacity and selectivity for hIgG. The results showed the Qm-hIgG of Ac-FYHE-4FF resin was 87.9 mg/g resin while the Qm-BSA of this resin was only 16.5 mg/g resin at pH 7.0. Moreover, at pH 7.0, Q10% of Ac-FYHE-4FF resin was 24.1 mg/mL for hIgG but just 2.1 mg/mL for BSA, which presented high selectivity of the screened resin at pH 7.0. Subsequently, the adsorption and separation properties of the Ac-FYHE-4FF resin were further investigated. As a result, with the addition of 0.5 M NaCl, Qm decreased by less than 20% but Qm decreased by 70% with the addition of 50% (v/v) ethylene glycol, which indicated that hydrophobic interaction would be the driving force for the binding between resin and hIgG. Besides, pH 7.5 and pH 4.5 could be the optimal loading and elution condition for hIgG, respectively. Finally, the Ac-FYHE-4FF resin was applied to separate mAb or/and hIgG from BSA containing feedstock, CHO cell culture supernatant and human serum, and the purity and recovery were both more than 90% with only one-step separation. The results indicate that the Ac-FYHE-4FF resin developed in this work would be promising for antibody separation and purification.

Perfusion CHO cell culture applied to lower aggregation and increase volumetric productivity for a bispecific recombinant protein

Secreted recombinant proteins can aggregate during cell culture. We studied a poorly-behaved bispecific scaffold that increasingly aggregated (up to 62% high molecular weight species, HMW) as a function of culture time in a fed-batch and intensified cell culture processes. We identified that protein aggregates increased with accumulated protein concentration inside the bioreactor. Furthermore, results indicated that a maximum product concentration was reached beyond which no additional soluble protein accumulated in culture even when doubling the integrated viable cell density with the intensified process, suggesting additional secreted protein was precipitating. To overcome this limitation and maintain the cell-specific productivity (qp) in culture, we explored a perfusion process where recombinant protein was continuously removed from the bioreactor to maintain low product concentration and consequently, minimize protein aggregation. We studied different viable cell densities (VCDs) inside the bioreactor (one to five-fold) and found a corresponding two-fold modulation of monomer levels. In all VCD conditions, qp was maintained. On the contrary, the previous intensified process showed an “apparent” 2.5-fold decrease in qp at the end of culture because of the presumed limited protein solubility at higher concentrations. The combination of lower aggregate levels and constant qp resulted in up to four to five-fold increase in recoverable product (i.e., monomer) with the improved perfusion process.

Transcriptome and proteome analysis of steady-state in a perfusion CHO cell culture process.

Long-term continuous protein production can be reached by perfusion operation. Through the continuous removal of waste metabolites and supply of nutrients, steady-state (SS) conditions are achieved after a certain transient period, where the conditions inside the reactor are not only uniform in space but also constant in time. Such stable conditions may have beneficial influences on the reduction of product heterogeneities. In this study, we investigated the impact of perfusion cultivation on the intracellular physiological state of a CHO cell line producing a monoclonal antibody (mAb) by global transcriptomics and proteomics. Despite stable viable cell density was maintained right from the beginning of the cultivation time, productivity decrease, and a transition phase for metabolites and product quality was observed before reaching SS conditions. These were traced back to three sources of transient behaviors being hydrodynamic flow rates, intracellular dynamics of gene expression as well as metabolism and cell line instability, superimposing each other. However, 99.4% of all transcripts and proteins reached SS during the first week or were at SS from the beginning. These results demonstrate that the stable extracellular conditions of perfusion lead to SS also of the cellular level.

Impact of depth filtration on disulfide bond reduction during downstream processing of monoclonal antibodies from CHO cell cultures.

Monoclonal antibody interchain disulfide bond reduction was observed in a Chinese Hamster Ovary manufacturing process that used single-use technologies. A similar reduction has been reported for processes that involved high mechanical shear recovery unit operations, such as continuous flow centrifugation and when the clarified harvest was stored under low dissolved oxygen (DO) conditions (Trexler-Schmidt et al., 2010. Biotechnology and Bioengineering, 106(3), 452-461). The work described here identifies disposable depth filtration used during cell culture harvest operations as a shear-inducing unit operation causing cell lysis. As a result, reduction of antibody interchain disulfide bonds was observed through the same mechanisms described for continuous flow centrifugation. Small-scale depth-filtration models were developed, and the differential pressure (Δ P) of the primary depth filter was identified as the key factor contributing to cell lysis. Strong correlations of Δ P and cell lysis were generated by measuring the levels of lactate dehydrogenase and thiol in the filtered harvest material. A simple risk mitigation strategy was implemented during manufacturing by providing an air overlay to the headspace of a single-use storage bag to maintain sufficient DO in the clarified harvest. In addition, enzymatic characterization studies determined that thioredoxin reductase and glucose-6-phosphate dehydrogenase are critical enzymes involved in antibody reduction in a nicotinamide adenine dinucleotide phosphate (NADP + )/NADPH-dependent manner.

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. In this work, we present a modeling platform that quantifies the impact of glycosylation precursor feeding - specifically that of galactose and uridine - on cellular growth, metabolism as well as antibody productivity and glycoform distribution. The platform has been parameterized using an initial training data set yielding an accuracy of ±5% with respect to glycoform distribution. It was then used to design an optimized feeding strategy that enhances the final concentration of galactosylated antibody in the supernatant by over 90% compared with the control without compromising the integral of viable cell density or final antibody titer. This work supports the implementation of Quality by Design towards higher-performing bioprocesses.

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.

High-throughput Carbonyl Content Method of Therapeutic mAb using size-exclusion chromatography with ultraviolet and fluorescence detection

Monoclonal antibody (mAb), one of the major types of therapeutic proteins in the pharmaceutical industry, is predominantly manufactured using mammalian cell culture [1]. Oxidative stress, potentially present during cell culture process, may increase the protein carbonyl content in the mAb product, which was reported to positively correlate with aggregate burst rate during storage [2]. In order to monitor carbonyl content during mAb process development, we developed a high-throughput screening method for therapeutic mAbs using size-exclusion chromatography followed by ultraviolet and fluorescence detection (SEC-UV/FL), optimized from a fluorescein thiosemicarbazide (FTC) semi-microplate method. The method demonstrated a good correlation with conventional ELISA assay and FTC-based fluorometric semi-microplate method with improved throughput and precision. The method was successfully applied in three case studies to improve our understanding of mAb carbonylation, including the impact of metal-catalyzed oxidation on an IgG4 mAb, comparison of carbonyl content between several mAbs expressed by CHO cell culture with human serum antibody pool, as well as the surface charge property of carbonylated mAb assessed by ion-exchange chromatography.

Dissecting N-Glycosylation Dynamics in Chinese Hamster Ovary Cells Fed-batch Cultures using Time Course Omics Analyses

SummaryN-linked glycosylation affects the potency, safety, immunogenicity, and pharmacokinetic clearance of several therapeutic proteins including monoclonal antibodies. A robust control strategy is needed to dial in appropriate glycosylation profile during the course of cell culture processes accurately. However, N-glycosylation dynamics remains insufficiently understood owing to the lack of integrative analyses of factors that influence the dynamics, including sugar nucleotide donors, glycosyltransferases, and glycosidases. Here, an integrative approach involving multi-dimensional omics analyses was employed to dissect the temporal dynamics of glycoforms produced during fed-batch cultures of CHO cells. Several pathways including glycolysis, tricarboxylic citric acid cycle, and nucleotide biosynthesis exhibited temporal dynamics over the cell culture period. The steps involving galactose and sialic acid addition were determined as temporal bottlenecks. Our results show that galactose, and not manganese, is able to mitigate the temporal bottleneck, despite both being known effectors of galactosylation. Furthermore, sialylation is limited by the galactosylated precursors and autoregulation of cytidine monophosphate-sialic acid biosynthesis.

Принцип оперативного выбора лидерных клонов-продуцентов моноклональных антител при создании стабильных клеточных линий на основе CHO

Growth and productive characteristics of monoclonal cell lines based on CHO cells and producing a therapeutic protein have been monitored using the robot Ambr Tap Biosystems, which permitted to identify the leading line. Twenty four clones producing a recombinant monoclonal antibody were studied under the close to industrial conditions in a fed-batch culturing mode. The ambr®15 cell culture workstation controls 24 disposable mini bioreactors, and offers parallel processing and evaluation of multiple (24) experiments in an automated bench-top system. The volumetric productivity of 24 clones determined by ELISA was 120-450 mg /L. A protocol was shown to select a leader among producing clones for further research. producing clones, mini bioreactor; Ambr Tap Biosystems, fed-batch, monoclonal antibodies, CHO cell culture

Novel column generation-based optimization approach for poly-pathway kinetic model applied to CHO cell culture

Mathematical modelling can provide precious tools for bioprocess simulation, prediction, control and optimization of mammalian cell-based cultures. In this paper we present a novel method to generate kinetic models of such cultures, rendering complex metabolic networks in a poly-pathway kinetic model. The model is based on subsets of elementary flux modes (EFMs) to generate macro-reactions. Thanks to our column generation-based optimization algorithm, the experimental data are used to identify the EFMs, which are relevant to the data. Here the systematic enumeration of all the EFMs is eliminated and a network including a large number of reactions can be considered. In particular, the poly-pathway model can simulate multiple metabolic behaviors in response to changes in the culture conditions.We apply the method to a network of 126 metabolic reactions describing cultures of antibody-producing Chinese hamster ovary cells, and generate a poly-pathway model that simulates multiple experimental conditions obtained in response to variations in amino acid availability. A good fit between simulated and experimental data is obtained, rendering the variations in the growth, product, and metabolite uptake/secretion rates. The intracellular reaction fluxes simulated by the model are explored, linking variations in metabolic behavior to adaptations of the intracellular metabolism.

Multi-Omics Study on the Impact of Cysteine Feed Level on Cell Viability and mAb Production in a CHO Bioprocess.

There is continual demand to maximize CHO cell culture productivity of a biotherapeutic while maintaining product quality. In this study, a comprehensive multi-omics analysis is performed to investigate the cellular response to the level of dosing of the amino acid cysteine (Cys) in the production of a monoclonal antibody (mAb). When Cys feed levels are insufficient, there is a significant decrease in protein titer. Multi-omics (metabolomics and proteomics, with support from RNAseq) is performed over the time course of the CHO bioprocess producing an IgG1 mAb in 5 L bioreactors. Pathway analysis reveals that insufficient levels of Cys in the feed lead to Cys depletion in the cell. This depletion negatively impacts antioxidant molecules, such as glutathione (GSH) and taurine, leading to oxidative stress with multiple deleterious cellular effects. In this paper, the resultant ER stress and subsequent apoptosis that affects cell viability and viable cell density has been considered. Key metabolic enzymes and metabolites are identified that can be potentially monitored as the process progresses and/or increased in the cell either by nutrient feeding or genetic engineering. This work reinforces the centrality of redox balance to cellular health and success of the bioprocess as well as the power of multi-omics to provide an in-depth understanding of the CHO cell biology during biopharmaceutical production.