Elucidating uptake and metabolic fate of dipeptides in CHO cell cultures using 13C labeling experiments and kinetic modeling.

The rapidly growing market of biologics including monoclonal antibodies has stimulated the need to improve biomanufacturing processes including mammalian host systems such as Chinese Hamster Ovary (CHO) cells. Cell culture media formulations continue to be enhanced to enable intensified cell culture processes and optimize cell culture performance. Amino acids, major components of cell culture media, are consumed in large amounts by CHO cells. Due to their low solubility and poor stability, certain amino acids including tyrosine, leucine, and phenylalanine can pose major challenges leading to suboptimal bioprocess performance. Dipeptides have the potential to replace amino acids in culture media. However, very little is known about the cleavage, uptake, and utilization kinetics of dipeptides in CHO cell cultures. In this study, replacing amino acids, including leucine and tyrosine by their respective dipeptides including but not limited to Ala-Leu and Gly-Tyr, supported similar cell growth, antibody production, and lactate profiles. Using 13C labeling techniques and spent media studies, dipeptides were shown to undergo both intracellular and extracellular cleavage in cultures. Extracellular cleavage increased with the culture duration, indicating cleavage by host cell proteins that are likely secreted and accumulate in cell culture over time. A kinetic model was built and for the first time, integrated with 13C labeling experiments to estimate dipeptide utilization rates, in CHO cell cultures. Dipeptides with alanine at the N-terminus had a higher utilization rate than dipeptides with alanine at the C-terminus and dipeptides with glycine instead of alanine at N-terminus. Simultaneous supplementation of more than one dipeptide in culture led to reduction in individual dipeptide utilization rates indicating that dipeptides compete for the same cleavage enzymes, transporters, or both. Dipeptide utilization rates in culture and cleavage rates in cell-free experiments appeared to follow Michaelis-Menten kinetics, reaching a maximum at higher dipeptide concentrations. Dipeptide utilization behavior was found to be similar in cell-free and cell culture environments, paving the way for future testing approaches for dipeptides in cell-free environments prior to use in large-scale bioreactors. Thus, this study provides a deeper understanding of the fate of dipeptides in CHO cell cultures through an integration of cell culture, 13C labeling, and kinetic modeling approaches providing insights in how to best use dipeptides in media formulations for robust and optimal mammalian cell culture performance.

Completely noninvasive multi-analyte monitoring system for cell culture processes.

Although online monitoring of dissolved O2, pH, and dissolved CO2 is critical in bioprocesses, nearly all existing technologies require some level of direct contact with the cell culture environment, posing risks of contamination. This study addresses the need for an accurate, and completely noninvasive technique for simultaneous measurement of these analytes. A "non-contact" technique for simultaneous monitoring of dissolved O2, pH, and dissolved CO2 was developed. Instead of direct contact with the culture media, the measurements were made through permeable membranes via either a sampling port in the culture vessel wall or a flow cell. The efficacy of the "non-contact" technique was validated in Escherichia coli (E.coli), Chinese hamster ovary (CHO) culture processes, and dynamic environments created by sparging gases in cell culture medium. The measurements obtained through the developed techniques were comparable to those obtained through control methods. The noninvasive monitoring system can offer accurate, and contamination-minimized monitoring of critical process parameters including dissolved O2, pH, and dissolved CO2. These advancements will enhance the control and optimization of cell culture processes, promising improved cell culture performance.

A genome-scale dynamic constraint-based modelling (gDCBM) framework predicts growth dynamics, medium composition and intracellular flux distributions in CHO clonal variations.

Optimizing mammalian cell growth and bioproduction is a tedious task. However, due to the inherent complexity of eukaryotic cells, heuristic experimental approaches such as, metabolic engineering and bioprocess design, are frequently integrated with mathematical models of cell culture to improve biological process efficiency and find paths for improvement. Constraint-based metabolic models have evolved over the last two decades to be used for dynamic modelling in addition to providing a linear description of steady-state metabolic systems. Formulation and implementation of the underlying optimization problems require special attention to the model's performance and feasibility, lack of defects in the definition of system components, and consideration of optimal alternate solutions, in addition to processing power limitations. Here, the time-resolved dynamics of a genome-scale metabolic network of Chinese hamster ovary (CHO) cell metabolism are shown using a genome-scale dynamic constraint-based modelling framework (gDCBM). The metabolic network was adapted from a reference model of CHO genome-scale metabolic model (GSMM), iCHO_DG44_v1, and dynamic restrictions were imposed to its exchange fluxes based on experimental results. We used this framework for predicting physiological changes in CHO clonal variants. Because of the methodical creation of the components for the flux balance analysis optimization problem and the integration of a switch time, this model can generate sequential predictions of intracellular fluxes during growth and non-growth phases (per hour of culture time) and transparently reveal the shortcomings in such practice. As a result of the differences exploited by various clones, we can understand the relevance of changes in intracellular flux distribution and exometabolomics. The integration of various omics data into the given gDCBM framework, as well as the reductionist analysis of the model, can further help bioprocess optimization.

Leveraging mathematical models for optimizing filter utility at manufacturing scale.

In the production of biopharmaceuticals depth filters followed by sterile filters are often employed to remove residual cell debris present in the feed stream. In the back drop of a global pandemic, supply chains associated with the production of biopharmaceuticals have been constrained. These constraints have limited the available amount of depth filters for the manufacture of biologics. This has placed manufacturing facilities in a difficult position having to choose between running processes with reduced number of depth filters and risking a failed batch or the prospect of plants going into temporary shutdown until the depth filter resources are replenished. This communication describes a modeling based method that leverages manufacturing scale filtration data to predict the depth filter performance with a reduced number of filters and an increased operational flux. This method can be used to quantify the acceptable level of area reduction before which the filtration process performance is affected. This enables facilities to manage their filter inventory avoiding potential plant shutdowns and reduces the risks of negative depth filter performance.

Rapid at-line early cell death quantification using capacitance spectroscopy.

Cell death is one of the failure modes of mammalian cell culture. Apoptosis is a regulated cell death process mainly observed in cell culture. Timely detection of apoptosis onset allows opportunities for preventive controls that ensure high productivity and consistent product quality. Capacitance spectroscopy captures the apoptosis-related cellular properties changes and thus quantifies the percentage of dying cells. This study demonstrated a quantification model that measures the percentage of apoptotic cells using a capacitance spectrometer in an at-line setup. When predicting the independent test set collected from bench-scale bioreactors, the root-mean-squared error of prediction was 8.8% (equivalent to 9.9% of the prediction range). The predicted culture evolution trajectory aligned with measured values from the flow cytometer. Furthermore, this method alarms cell death onset earlier than the traditional viability test, that is, the trypan blue exclusion test. Compared to flow cytometry (the traditional early cell death detection method), this method is rapid, simple, and less labor-intensive. In addition, this at-line setup can be easily transferred between scales (e.g., lab-scale for development to manufacturing scale), which benefits process transfers between facilities, scale-up, and other process transitions.

Host Cell Binding Mediated by Leptospira interrogans Adhesins.

Leptospirosis is a neglected infectious disease with global impact on both humans and animals. The increase in urban development without sanitation planning is one of the main reasons for the disease spreading. The symptoms are similar to those of flu-like diseases, such as dengue, yellow fever, and malaria, which can result in a misleading clinical diagnosis. The characterization of host-pathogen interactions is important in the development of new vaccines, treatments, and diagnostics. However, the pathogenesis of leptospirosis is not well understood, and many gaps remain to be addressed. Here, we aimed to determine if Leptospira strains, virulent, culture-attenuated, and saprophytic, and the major outer membrane proteins OmpL37, OmpL1, LipL21, LipL41, and LipL46 are able to adhere to different endothelial, epithelial and fibroblast cell lines in vitro. We showed that virulent leptospires robustly bind to all cells compared to the culture-attenuated and saprophytic lines. The recombinant proteins exhibited certain adhesion, but only OmpL1 and LipL41 were able to bind to several cell lines, either in monolayer or in cell suspension. Blocking OmpL1 with polyclonal antibodies caused a decrease in bacterial binding to cells, contrasting with an increase observed when anti-LipL41 antibodies were used. The adhesion of OmpL1 to HMEC-1 and EA.hy926 was inhibited when cells were pre-incubated with collagen IV, suggesting that both compete for the same cell receptor. We present here for the first time the interaction of five leptospiral outer membrane proteins with several cell lines, and we conclude that LipL41 and OmpL1 may have an impact on leptospiral adhesion to mammalian cells and may mediate the colonization process in leptospiral pathogenesis.

Magnetoelastic sensors for real-time tracking of cell growth.

Magnetoelastic (ME) sensors, which can be remotely activated via magnetic fields, are an excellent choice for wireless monitoring of biological parameters due to their ability to be scaled into different sizes and have their surface functionalized for chemical or biological sensing. In this study, we present the application of a commercially available ME material (Metglas 2826 MB) to develop a sensor system that can monitor the attachment of anchorage-dependent mammalian cells in two-dimensional in vitro cell cultures. Results obtained with the developed sensors and detection system correlated with microscopic image analysis of cell quantification, which showed a linear relationship between the sensor response and attached fibroblast cells on the sensor surface. It was also revealed that the developed ME sensor system is capable of providing temporal profiles of cell growth corresponding to different stages of cell attachment and proliferation in real-time.

Tailoring translational strength using Kozak sequence variants improves bispecific antibody assembly and reduces product-related impurities in CHO cells.

Optimal production of bispecific antibodies (bsAb) requires efficient and tailored co-expression and assembly of two distinct heavy and two distinct light chains. Here, we describe a novel technology to modulate the translational strength of antibody chains via Kozak sequence variants to produce bsAb in a single cell line. In this study, we designed and screened a large Kozak sequence library to identify 10 independent variants that can modulate protein expression levels from approximately 0.2 to 1.3-fold compared with the wild-type sequence in transient transfection. We used a combination of several of these variants, covering a wide range of translational strength, to develop stable single cell Chinese hamster ovary bispecific cell lines and compared the results with those obtained from the wild-type sequence. A significant increase in bispecific antibody assembly with a concomitant reduction in the level of product-related impurities was observed. Our findings suggest that for production of bsAb it can be advantageous to modify translational strength for selected protein chains to improve overall yield and product quality. By extension, tuning of translational strength can also be applied to improving the production of a wide variety of heterologous proteins.

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.

Expression vector cassette engineering for recombinant therapeutic production in mammalian cell systems.

Human tissue plasminogen activator was the first recombinant therapy protein that successfully produced in Chinese hamster ovary cells in 1986 and approved for clinical use. Since then, more and more therapeutic proteins are being manufactured in mammalian cells, and the technologies for recombinant protein production in this expression system have developed rapidly, with the optimization of both upstream and downstream processes. One of the most promising strategies is expression vector cassette optimization based on the expression vector cassette. In this review paper, these approaches and developments are summarized, and the future strategy on the utilizing of expression cassettes for the production of recombinant therapeutic proteins in mammalian cells is discussed.

Long-Term Culture of Giardia lamblia in Cell Culture Medium Requires Intimate Association with Viable Mammalian Cells.

Giardia lamblia is usually cultured axenically in TYI-S-33, a complex medium which does not permit survival and growth of mammalian cells. Likewise, medium commonly used to maintain and grow mammalian cells does not support healthy trophozoite survival for more than a few hours. The inability to coculture trophozoites and epithelial cells under optimal conditions limits studies of their interactions as well as interpretation of results. Trophozoites of the WB isolate but not the GS isolate were repeatedly adapted to grow stably in long-term cocultures with Caco2, Cos7, and mouse tumor rectal (RIT) cell lines using hybridoma-screened Dulbecco's modified Eagle's medium and 10% fetal calf serum. Giardia did not grow in spent cell culture medium or when separated by a permeable membrane using transwell methodology. Giardia chronically cocultured with specific cell lines became adapted (conditioned). These Giardia cocultures grew better than nonconditioned trophozoites, and the cell lines differed in their ability to support trophozoite growth in the order of RIT > Cos7 > Caco2. Trophozoites conditioned on one cell line and then grown in the presence of a heterologous cell line changed their growth rate to that seen in conditioned Giardia from the heterologous cell line. Trophozoite survival required intimate contact with cells, suggesting that trophozoites obtain an essential nutrient or growth factor from mammalian cells. This may explain why Giardia trophozoites adhere to the small intestinal epithelium during human and animal infections. This coculture system will be useful to understand the complex interactions between the host cells and parasite.

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.

New developments in online OUR monitoring and its application to animal cell cultures.

The increasing demand for biopharmaceuticals produced in mammalian cells has driven the industry to enhance the productivity of bioprocesses through intensification of culture process. Fed-batch and perfusion culturing strategies are considered the most attractive choices, but the application of these processes requires the availability of reliable online measuring systems for the estimation of cell density and metabolic activity. This manuscript reviews the methods (and the devices used) for monitoring of the oxygen consumption, also known as oxygen uptake rate (OUR), since it is a straightforward parameter to estimate viable cell density and the physiological state of cells. Furthermore, as oxygen plays an important role in the cell metabolism, OUR has also been very useful to estimate nutrient consumption, especially the carbon (glucose and glutamine) and nitrogen (glutamine) sources. Three different methods for the measurement of OUR have been developed up to date, being the dynamic method the golden standard, even though DO and pH perturbations generated in the culture during each measurement. For this, many efforts have been focused in developing non-invasive methods, such as global mass balance or stationary liquid mass balance. The low oxygen consumption rates by the cells and the high accuracy required for oxygen concentration measurement in the gas streams (inlet and outlet) have limited the applicability of the global mass balance methodology in mammalian cell cultures. In contrast, stationary liquid mass balance has successfully been implemented showing very similar OUR profiles compared with those obtained with the dynamic method. The huge amount of studies published in the last years evidence that OUR have become a reliable alternative for the monitoring and control of high cell density culturing strategies with very high productivities.

Impact of mammalian cell culture conditions on monoclonal antibody charge heterogeneity: an accessory monitoring tool for process development.

Recombinant monoclonal antibodies are predominantly produced in mammalian cell culture bioprocesses. Post-translational modifications affect the micro-heterogeneity of the product and thereby influence important quality attributes, such as stability, solubility, pharmacodynamics and pharmacokinetics. The analysis of the surface charge distribution of monoclonal antibodies provides aggregated information about these modifications. In this work, we established a direct injection pH gradient cation exchange chromatography method, which determines charge heterogeneity from cell culture supernatant without any purification steps. This tool was further applied to monitor processes that were performed under certain process conditions. Concretely, we were able to provide insights into charge variant formation during a fed-batch process of a Chinese hamster ovary cell culture, in turn producing a monoclonal antibody under varying temperatures and glucose feed strategies. Glucose concentration impacted the total emergence of acidic variants, whereas the variation of basic species was mainly dependent on process temperature. The formation rates of acidic species were described with a second-order reaction, where a temperature increase favored the conversion. This platform method will aid as a sophisticated optimization tool for mammalian cell culture processes. It provides a quality fingerprint for the produced mAb, which can be tested, compared to the desired target and confirmed early in the process chain.

Development of a shake tube-based scale-down model for perfusion cultures.

The use of benchtop bioreactors (BRs) for the development of mammalian cell perfusion cultures is expensive and time consuming, given its complexity in equipment and operation. Scale-down models, going from liter to milliliter scale, are needed to support the rapid determination of suitable operating conditions in terms of viable cell density (VCD), perfusion rate, and medium composition. In this study, we compare the performance of steady-state perfusion cultures in orbitally shaken tube and BR systems for a given Chinese hamster ovary cell line. The developed scale-down model relied on a daily workflow designed to keep the VCD constant at specific target values. This includes: cell count, removal of excessive cells (bleeding), spin down of remaining cells, harvest of cell-free supernatant, and resuspension in fresh medium. Steady-state cultures at different VCD values, medium exchange rates and working volumes were evaluated. Shake-tube perfusion cultures allowed the prediction of cell-specific growth, glucose consumption, ammonia, and monoclonal antibody production rates for much larger BRs, but not lactate (LAC) production rates. Although charge variant profiles remained comparable, different glycosylation patterns were obtained. The differences in LAC production and glycosylation probably resulted from the discontinuous medium exchange, the poor carbon dioxide removal, and the deficient pH control. Therefore, if requested by the specific process to be developed, product quality has to be fine-tuned directly in the BR system. Altogether, the developed strategy provides a useful scale-down model for the design and optimization of perfusion cultures with strong savings in time and media consumption.

A comparative study of the bispecific monoclonal antibody, blinatumomab expression in CHO cells and E. coli.

The "bispecifics" market improved over the past decade due to the development of many technological platforms including bispecific T cell engagers (BiTEs). The approval of blinatumomab, the most advanced bispecific T-cell engager (BiTE) in clinical trials, can be a significant milestone in the development of bispecific antibodies. Both Chinese hamster ovary (CHO) cells and E. coli strain are considered as the most widely used hosts for the large-scale production of therapeutic monoclonal antibodies. Since both of the economic and qualitative aspects of protein production are important in industry, selection of a suitable protein expression system is very critical. The BsAb gene was cloned into the expression vectors FC550A-1, pcDNA3.1 (+), and PET22b and 6 × His-tagged BsAb then purified on a Ni-NTA chromatography column. Both SDS-PAGE and Western blotting analysis of the purified protein demonstrated that blinatumomab was successfully expressed as a 55 kDa in both expression systems. The antigen-binding properties of blinatumomab were compared in the mammalian system versus Escherichia coli. The results showed that the purified antibody from a mammalian expression system has better binding activity than the one from E. coli host.

Evaluation of different vector design strategies for the expression of recombinant monoclonal antibody in CHO cells.

Monoclonal antibodies (mAbs) have emerged as the most promising category of recombinant proteins due to their high efficiency for the treatment of a wide range of human diseases. The complex nature of mAbs creates a great deal of challenges in both upstream and downstream manufacturing processes. Proportional expression and correct folding and assembly of the light chain and heavy chain are required for efficient production of the mAbs. In this regard, expression vector design has proven to have profound effects on the antibody expression level as well as its stability and quality. Here, we have explored the efficiency of different vector design strategies for the expression of a recombinant IgG1 antibody in Chinese hamster ovary (CHO) cells. The antibody expression level was analyzed in transient expression and stable cell pools followed by expression analysis on single-cell clones. While detectable amounts of antibody were observed in all three systems, dual-promoter single-vector system showed the highest expression level in transient and stable expression as well as the highest productivity among clonal cells. Our results here show the importance of vector design for successful production of whole mAbs in CHO cells.

13 C-metabolic flux analysis of human adenovirus infection: Implications for viral vector production.

Adenoviruses are human pathogens increasingly used as gene therapy and vaccination vectors. However, their impact on cell metabolism is poorly characterized. We performed carbon labeling experiments with [1,2-13 C]glucose or [U-13 C]glutamine to evaluate metabolic alterations in the amniocyte-derived, E1-transformed 1G3 cell line during production of a human adenovirus type 5 vector (AdV5). Nonstationary 13 C-metabolic flux analysis revealed increased fluxes of glycolysis (17%) and markedly PPP (over fourfold) and cytosolic AcCoA formation (nearly twofold) following infection of growing cells. Interestingly, infection of growth-arrested cells increased overall carbon flow even more, including glutamine anaplerosis and TCA cycle activity (both over 1.5-fold), but was unable to stimulate the PPP and was associated with a steep drop in AdV5 replication (almost 80%). Our results underscore the importance of nucleic and fatty acid biosynthesis for adenovirus replication. Overall, we portray a metabolic blueprint of human adenovirus infection, highlighting similarities with other viruses and cancer, and suggest strategies to improve AdV5 production. Biotechnol. Bioeng. 2017;114: 195-207. © 2016 Wiley Periodicals, Inc.

Segmented linear modeling of CHO fed-batch culture and its application to large scale production.

We describe a systematic approach to model CHO metabolism during biopharmaceutical production across a wide range of cell culture conditions. To this end, we applied the metabolic steady state concept. We analyzed and modeled the production rates of metabolites as a function of the specific growth rate. First, the total number of metabolic steady state phases and the location of the breakpoints were determined by recursive partitioning. For this, the smoothed derivative of the metabolic rates with respect to the growth rate were used followed by hierarchical clustering of the obtained partition. We then applied a piecewise regression to the metabolic rates with the previously determined number of phases. This allowed identifying the growth rates at which the cells underwent a metabolic shift. The resulting model with piecewise linear relationships between metabolic rates and the growth rate did well describe cellular metabolism in the fed-batch cultures. Using the model structure and parameter values from a small-scale cell culture (2 L) training dataset, it was possible to predict metabolic rates of new fed-batch cultures just using the experimental specific growth rates. Such prediction was successful both at the laboratory scale with 2 L bioreactors but also at the production scale of 2000 L. This type of modeling provides a flexible framework to set a solid foundation for metabolic flux analysis and mechanistic type of modeling. Biotechnol. Bioeng. 2017;114: 785-797. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

Cell-controlled hybrid perfusion fed-batch CHO cell process provides significant productivity improvement over conventional fed-batch cultures.

A simple method originally designed to control lactate accumulation in fed-batch cultures of Chinese Hamster Ovary (CHO) cells has been modified and extended to allow cells in culture to control their own rate of perfusion to precisely deliver nutritional requirements. The method allows for very fast expansion of cells to high density while using a minimal volume of concentrated perfusion medium. When the short-duration cell-controlled perfusion is performed in the production bioreactor and is immediately followed by a conventional fed-batch culture using highly concentrated feeds, the overall productivity of the culture is approximately doubled when compared with a highly optimized state-of-the-art fed-batch process. The technology was applied with near uniform success to five CHO cell processes producing five different humanized monoclonal antibodies. The increases in productivity were due to the increases in sustained viable cell densities. Biotechnol. Bioeng. 2017;114: 1438-1447. © 2017 Wiley Periodicals, Inc.