ABSTRACT
Biomarkers are valuable tools in clinical research where they allow to predict susceptibility to diseases, or response to specific treatments. Likewise, biomarkers can be extremely useful in the biomanufacturing of therapeutic proteins. Indeed, constraints such as short timelines and the need to find hyper-productive cells could benefit from a data-driven approach during cell line and process development. Many companies still rely on large screening capacities to develop productive cell lines, but as they reach a limit of production, there is a need to go from empirical to rationale procedures. Similarly, during bioprocessing runs, substrate consumption and metabolism wastes are commonly monitored. None of them possess the ability to predict the culture behavior in the bioreactor. Big data driven approaches are being adapted to the study of industrial mammalian cell lines, enabled by the publication of Chinese hamster and CHO genome assemblies which allowed the use of next-generation sequencing with these cells, as well as continuous proteome and metabolome annotation. However, if these different -omics technologies contributed to the characterization of CHO cells, there is a significant effort remaining to apply this knowledge to biomanufacturing methods. The correlation of a complex phenotype such as high productivity or rapid growth to the presence or expression level of a specific biomarker could save time and effort in the screening of manufacturing cell lines or culture conditions. In this review we will first discuss the different biological molecules that can be identified and quantified in cells, their detection techniques, and associated challenges. We will then review how these markers are used during the different steps of cell line and bioprocess development, and the inherent limitations of this strategy.
Subject(s)
Biomarkers , Bioreactors , Cricetulus , Animals , CHO Cells , Biomarkers/metabolism , Cricetinae , Cell Culture Techniques/methodsABSTRACT
Improving current cell line development workflows can either focus on increasing the specific productivity of the cell lines or shortening timelines to reach the clinic as fast as possible. In this work, using the Beacon platform, we have combined two distinct protocols - early cloning with low-viability pools, and IgG membrane staining-, to concomitantly reach both objectives, and generate highly productive CHO clones in shorter timelines. Fast-sorting approaches using low-viability pools in combination with the Beacon platform have recently been reported to shorten CLD timelines. However, the low recovery led to a drastic reduction in the clone number obtained postcloning. Here, we report a combined approach of fast-sorting and fluorescent membrane staining. With this new protocol, the cells reach a correct recovery, allowing to fully exploit the Beacon screening capacities. In addition, by using a fluorescent staining recognizing the secreted IgG, we were able to enrich the fraction of highly secreting cells prior to cloning and we obtained significant increases in the cell's specific productivity. The combination of these two protocols has a synergistic effect, and as they help discarding the dead and nonproducing populations prior to cloning, they increase the throughput power of the Beacon platform and the detection of super productive clones.
Subject(s)
Cricetulus , CHO Cells , Animals , Immunoglobulin G/genetics , Lab-On-A-Chip Devices , Flow CytometryABSTRACT
Controlling high-mannose (HM) content of therapeutic proteins during process intensification, reformulation for subcutaneous delivery, antibody-drug conjugate or biosimilar manufacturing represents an ongoing challenge. Even though a range of glycosylation levers to increase HM content exist, modulators specially increasing M5 glycans are still scarce. Several compounds of the polyether ionophore family were screened for their ability to selectively increase M5 glycans of mAb products and compared to the well-known α-mannosidase I inhibitor kifunensine known to increase mainly M8-M9 glycans. Maduramycin, amongst other promising polyether ionophores, showed the desired effect on different cell lines. For fed-batch processes, a double bolus addition modulator feed strategy was developed maximizing the effect on glycosylation by minimizing impact on culture performance. Further, a continuous feeding strategy for steady-state perfusion processes was successfully developed, enabling consistent product quality at elevated HM glycan levels. With kifunensine and maduramycin showing inverse effects on the relative HM distribution, a combined usage of these modulators was further evaluated to fine-tune a desired HM glycan pattern. The discovered HM modulators expand the current HM modulating toolbox for biotherapeutics. Their application not only for fed-batch processes, but also steady-state perfusion processes, make them a universal tool with regards to fully continuous manufacturing processes.
Subject(s)
Lactones , Mammals , Animals , Glycosylation , Perfusion , Mannose , Polyether Polyketides , PolysaccharidesABSTRACT
BACKGROUND: Despite technological advances ensuring stable cell culture perfusion operation over prolonged time, reaching a cellular steady-state metabolism remains a challenge for certain manufacturing cell lines. This study investigated the stabilization of a steady-state perfusion process producing a bispecific antibody with drifting product quality attributes, caused by shifting metabolic activity in the cell culture. MAIN METHODS: A novel on-demand pyruvate feeding strategy was developed, leveraging lactate as an indicator for tricarboxylic acid (TCA) cycle saturation. Real-time lactate monitoring was achieved through in-line Raman spectroscopy, enabling accurate control at predefined target setpoints. MAJOR RESULTS: The implemented feedback control strategy resulted in a three-fold reduction of ammonium accumulation and stabilized product quality profiles. Stable and flat glycosylation profiles were achieved with standard deviations below 0.2% for high mannose and fucosylation. Whereas galactosylation and sialylation were stabilized in a similar manner, varying lactate setpoints might allow for fine-tuning of these glycan forms. IMPLICATION: The Raman-controlled pyruvate feeding strategy represents a valuable tool for continuous manufacturing, stabilizing metabolic activity, and preventing product quality drifting in perfusion cell cultures. Additionally, this approach effectively reduced high mannose, helping to mitigate increases associated with process intensification, such as extended culture durations or elevated culture densities.
Subject(s)
Antibodies, Monoclonal , Pyruvic Acid , Cricetinae , Animals , Pyruvic Acid/metabolism , Antibodies, Monoclonal/chemistry , Bioreactors , Cricetulus , Mannose , Lactic Acid/metabolism , CHO CellsABSTRACT
Hollow fiber-based membrane filtration has emerged as the dominant technology for cell retention in perfusion processes yet significant challenges in alleviating filter fouling remain unsolved. In this work, the benefits of co-current filtrate flow applied to a tangential flow filtration (TFF) module to reduce or even completely remove Starling recirculation caused by the axial pressure drop within the module was studied by pressure characterization experiments and perfusion cell culture runs. Additionally, a novel concept to achieve alternating Starling flow within unidirectional TFF was investigated. Pressure profiles demonstrated that precise flow control can be achieved with both lab-scale and manufacturing-scale filters. TFF systems with co-current flow showed up to 40% higher product sieving compared to standard TFF. The decoupling of transmembrane pressure from crossflow velocity and filter characteristics in co-current TFF alleviates common challenges for hollow fiber-based systems such as limited crossflow rates and relatively short filter module lengths, both of which are currently used to avoid extensive pressure drop along the filtration module. Therefore, co-current filtrate flow in unidirectional TFF systems represents an interesting and scalable alternative to standard TFF or alternating TFF operation with additional possibilities to control Starling recirculation flow.
Subject(s)
Bioreactors , Filtration , Cell Culture Techniques , PerfusionABSTRACT
The COVID-19 pandemic highlighted the urgent need for life-saving treatments, including vaccines, drugs, and therapeutic antibodies, delivered at unprecedented speed. During this period, recombinant antibody research and development cycle times were substantially shortened without compromising quality and safety, thanks to prior knowledge of Chemistry, Manufacturing and Controls (CMC) and integration of new acceleration concepts discussed below. Early product knowledge, selection of a parental cell line with appropriate characteristics, and the application of efficient approaches for generating manufacturing cell lines and manufacturing drug substance from non-clonal cells for preclinical and first-in-human studies are key elements for success. Prioritization of established manufacturing and analytical platforms, implementation of advanced analytical methods, consideration of new approaches for adventitious agent testing and viral clearance studies, and establishing stability claim with less real-time data are additional components that enable an accelerated successful gene to clinical-grade material development strategy.
Subject(s)
COVID-19 , Pandemics , Humans , Recombinant Proteins/therapeutic useABSTRACT
Implementation of continuous in lieu of batch upstream processing (USP) and downstream process (DSP) for the production of recombinant therapeutic protein is a significant paradigm change. The present report describes how the first kilograms of monoclonal antibody were produced with equipment originally designed for batch operations while using continuous manufacturing processes and principles. Project timelines for the delivery of clinical material have driven this ambition and helped the transition. Nevertheless, because of equipment availability, a tradeoff between the envisaged continuous downstream process (cDSP) operations and the ones described in this article had to be taken. A total of 2.1 kg of monoclonal antibody were produced in two GMP runs for clinical trials. For USP, a 200-L single-use pilot scale bioreactor was upgraded to enable perfusion operation. DSP steps were designed to be easily transferable to cDSP for later clinical or commercial productions. An in-line conditioning buffer preparation strategy was tested in a discontinuous way to prove its efficiency and the purification cascade was structured in parallel to the continuous collection of antibody-containing cell culture supernatant. This strategy will avoid any process change when later moving to the continuous equipment that is currently under qualification. Alignment between small-scale references runs and the GMP runs in terms of productivity and quality confirmed that the presented approach was valid. Thus, we demonstrate that existing fed-batch infrastructure can be adapted to continuous manufacturing without significant additional investments. Such approach is useful to evaluate next-generation manufacturing processes before making large investments.
Subject(s)
Antibodies, Monoclonal , Bioreactors , Cell Culture TechniquesABSTRACT
Media preparation for perfusion cell culture processes contributes significantly to operational costs and the footprint of continuous operations for therapeutic protein manufacturing. In this study, definitions are given for the use of a perfusion equivalent nutrient feed stream which, when used in combination with basal perfusion medium, supplements the culture with targeted compounds and increases the medium depth. Definitions to compare medium and feed depth are given in this article. Using a concentrated nutrient feed, a 1.8-fold medium consumption (MC) decrease and a 1.67-fold increase in volumetric productivity (PR) were achieved compared to the initial condition. Later, this strategy was used to push cell densities above 100 × 106 cells/ml while using a perfusion rate below 2 RV/day. In this example, MC was also decreased 1.8-fold compared to the initial condition, but due to the higher cell density, PR was increased 3.1-fold and to an average PR value of 1.36 g L-1 day-1 during a short stable phase, and versus 0.46 g L-1 day-1 in the initial condition. Overall, the performance improvements were aligned with the given definitions. This multiple feeding strategy can be applied to gain some flexibility during process development and also in a manufacturing set-up to enable better control on nutrient addition.
Subject(s)
Antibodies, Monoclonal/metabolism , Cell Culture Techniques/methods , Culture Media , Recombinant Proteins/metabolism , Animals , Bioreactors , CHO Cells , Cell Count , Cricetinae , Cricetulus , Culture Media/analysis , Culture Media/chemistry , Culture Media/metabolismABSTRACT
Multivariate latent variable methods have become a popular and versatile toolset to analyze bioprocess data in industry and academia. This work spans such applications from the evaluation of the role of the standard process variables and metabolites to the metabolomics level, that is, to the extensive number metabolic compounds detectable in the extracellular and intracellular domains. Given the substantial effort currently required for the measurement of the latter groups, a tailored methodology is presented that is capable of providing valuable process insights as well as predicting the glycosylation profile based on only four experiments measured over 12 cell culture days. An important result of the work is the possibility to accurately predict many of the glycan variables based on the information of three experiments. An additional finding is that such predictive models can be generated from the more accessible process and extracellular information only, that is, without including the more experimentally cumbersome intracellular data. With regards to the incorporation of omics data in the standard process analytics framework in the future, this works provides a comprehensive data analysis pathway which can efficiently support numerous bioprocessing tasks.
Subject(s)
Bioreactors , Cell Culture Techniques/methods , Metabolomics/methods , Models, Biological , Multivariate Analysis , Animals , CHO Cells , Cricetinae , Cricetulus , Glycosylation , Least-Squares Analysis , Recombinant Proteins/metabolismABSTRACT
Integrated continuous manufacturing is entering the biopharmaceutical industry. The main drivers range from improved economics, manufacturing flexibility, and more consistent product quality. However, studies on fully integrated production platforms have been limited due to the higher degree of system complexity, limited process information, disturbance, and drift sensitivity, as well as difficulties in digital process integration. In this study, we present an automated end-to-end integrated process consisting of a perfusion bioreactor, CaptureSMB, virus inactivation (VI), and two polishing steps to produce an antibody from an instable cell line. A supervisory control and data acquisition (SCADA) system was developed, which digitally integrates unit operations and analyzers, collects and centrally stores all process data, and allows process-wide monitoring and control. The integrated system consisting of bioreactor and capture step was operated initially for 4 days, after which the full end-to-end integrated run with no interruption lasted for 10 days. In response to decreasing cell-specific productivity, the supervisory control adjusted the loading duration of the capture step to obtain high capacity utilization without yield loss and constant antibody quantity for subsequent operations. Moreover, the SCADA system coordinated VI neutralization and discharge to enable constant loading conditions on the polishing unit. Lastly, the polishing was sufficiently robust to cope with significantly increased aggregate levels induced on purpose during virus inactivation. It is demonstrated that despite significant process disturbances and drifts, a robust process design and the supervisory control enabled constant (optimum) process performance and consistent product quality.
Subject(s)
Antibodies , Automation/methods , Bioreactors , Cell Culture Techniques/methods , Perfusion/methods , Animals , Antibodies/analysis , Antibodies/isolation & purification , Antibodies/metabolism , CHO Cells , Cricetinae , Cricetulus , Recombinant Proteins/metabolism , Virus InactivationABSTRACT
Monoclonal antibodies (mAbs) and related recombinant proteins continue to gain importance in the treatment of a great variety of diseases. Despite significant advances, their manufacturing can still present challenges owing to their molecular complexity and stringent regulations with respect to product purity, stability, safety, and so forth. In this context, protein aggregates are of particular concern due to their immunogenic potential. During manufacturing, mAbs routinely undergo acidic treatment to inactivate viral contamination, which can lead to their aggregation and thereby to product loss. To better understand the underlying mechanism so as to propose strategies to mitigate the issue, we systematically investigated the denaturation and aggregation of two mAbs at low pH as well as after neutralization. We observed that at low pH and low ionic strength, mAb surface hydrophobicity increased whereas molecular size remained constant. After neutralization of acidic mAb solutions, the fraction of monomeric mAb started to decrease accompanied by an increase on average mAb size. This indicates that electrostatic repulsion prevents denatured mAb molecules from aggregation under acidic pH and low ionic strength, whereas neutralization reduces this repulsion and coagulation initiates. Limiting denaturation at low pH by d-sorbitol addition or temperature reduction effectively improved monomer recovery after neutralization. Our findings might be used to develop innovative viral inactivation procedures during mAb manufacturing that result in higher product yields.
Subject(s)
Antibodies, Monoclonal , Biotechnology/methods , Virus Inactivation , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/isolation & purification , Antibodies, Monoclonal/metabolism , Bioreactors , CHO Cells , Cricetinae , Cricetulus , Hydrogen-Ion Concentration , Hydrophobic and Hydrophilic Interactions , Osmolar Concentration , Protein Aggregates , Protein Unfolding , Spectrometry, FluorescenceABSTRACT
N-linked glycosylation plays a fundamental role in determining the thermodynamic stability of proteins and is involved in multiple key biological processes. The mechanistic understanding of the intracellular machinery responsible for the stepwise biosynthesis of N-glycans is still incomplete due to limited understanding of in vivo kinetics of N-glycan processing along the secretory pathway. We present a glycoproteomics approach to monitor the processing of site-specific N-glycans in CHO cells. On the basis of a model-based analysis of structure-specific turnover rates, we provide a kinetic description of intracellular N-glycan processing along the entire secretory pathway. This approach refines and further extends the current knowledge on N-glycans biosynthesis and provides a basis to quantify alterations in the glycoprotein processing machinery.
Subject(s)
Carbohydrate Metabolism , Glycoproteins/metabolism , Polysaccharides/metabolism , Animals , CHO Cells , Cricetulus , GlycosylationABSTRACT
Biologically manufactured monoclonal antibodies (mAb) can strongly vary in their efficacy and affinity. Therefore, engineering and production of the mAb is highly regulated and requires product monitoring, especially in terms of N-glycosylation patterns. In this work, we present a high-throughput matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) method based on a microarray technology to monitor N-glycopeptides of IgG1 produced in a perfusion cell culture. A bottom-up approach combined with zwitterionic-hydrophilic interaction liquid chromatography for sample purification was used to determine the day-by-day variation of the terminal galactose within two major N-glycoforms. Our results show that microarrays for mass spectrometry (MAMS) are a robust platform for the rapid determination of the carbohydrate distribution. The spectral repeatability is characterized by a low coefficient of variations (1.7% and 7.1% for the FA2 and FA2G1 structures, respectively) and allows to detect the N-glycosylation variability resulting from operating conditions during the bioreactor process. The observed trend of released N-glycans was confirmed using capillary gel electrophoresis with laser-induced fluorescence detection. Therefore, the microarray technology is a promising analytical tool for glycosylation control during the production process of recombinant proteins.
Subject(s)
Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Bioreactors , GlycosylationABSTRACT
Perfusion cell culture technologies for the production of therapeuthic recombinant proteins are currently on the rise for diverse applications with the aim of process intensification (Bielser et al., 2018; Chen et al., 2018; Fisher et al., 2018; Jordan et al., 2018). This study reports a unique comparison of low (LS) and high (HS) seeding fed-batch bioreactors, corresponding to traditional and intensified operation using perfusion at the N-1 stage, respectively, with perfusion (PF) bioreactors, using a bispecific conjugated fusion protein as a model. It is found that the gain in daily volumetric productivity compared to the traditional LS fed-batch, increases by a factor 3 with HS and 7 with PF. Critical quality attributes (CQAs) also benefited from the perfusion operation. In particular, levels of clipping, that is the fragmentation of the fusion protein, are significantly reduced compared to both fed-batch operations. In PF the clipping varied between 0.6 and 1.5% while in the LS and HS it reached up to 8.7 and 4.9%, respectively. Aggregate levels were also decreased using PF, while the charge variant distribution was more homogeneous and the glycosylation pattern was also significantly affected. The comparison of LS, HS and PF for the manufacturing of a bispecific conjugated fusion protein reported here highlight some productivity and quality benefits inherent to the nature of continuous processing.
Subject(s)
Batch Cell Culture Techniques/methods , Bioreactors/microbiology , Recombinant Fusion Proteins/metabolism , Recombinant Proteins/metabolismABSTRACT
The development of mammalian cell perfusion cultures is still laborious and complex to perform due to the limited availability of scale-down models and limited knowledge of time- and cost-effective procedures. The maximum achievable viable cell density (VCDmax ), minimum cell-specific perfusion rate (CSPRmin ), cellular growth characteristics, and resulting bleed rate at steady-state operation are key variables for the effective development of perfusion cultures. In this study, we developed a stepwise procedure to use shake tubes (ST) in combination with benchtop (BR) bioreactors for the design of a mammalian cell perfusion culture at high productivity (23 pg·cell-1 ·day-1 ) and low product loss in the bleed (around 10%) for a given expression system. In a first experiment, we investigated peak VCDs in STs by the daily discontinuous medium exchange of 1 reactor volume (RV) without additional bleeding. Based on this knowledge, we performed steady-state cultures in the ST system using a working volume of 10 ml. The evaluation of the steady-state cultures allowed performing a perfusion bioreactor run at 20 × 106 cells/ml at a perfusion rate of 1 RV/day. Constant cellular environment and metabolism resulted in stable product quality patterns. This study presents a promising strategy for the effective design and development of perfusion cultures for a given expression system and underlines the potential of the ST system as a valuable scale-down tool for perfusion cultures.
Subject(s)
Bioreactors , Cell Culture Techniques/instrumentation , Perfusion/instrumentation , Animals , CHO Cells , Cell Count , Cell Survival , Cricetulus , Equipment DesignABSTRACT
On-line monitoring tools for downstream chromatographic processing (DSP) of biotherapeutics can enable fast actions to correct for disturbances in the upstream, gain process understanding, and eventually lead to process optimization. While UV/Vis spectroscopy is mostly assessing the protein's amino acid composition and the application of Fourier transform infrared spectroscopy is limited due to strong water interactions, Raman spectroscopy is able to assess the secondary and tertiary protein structure without significant water interactions. The aim of this work is to implement the Raman technology in DSP, by designing an in-line flow cell with a reduced dead volume of 80 µL and a reflector to increase the signal intensity as well as developing a chemometric modeling path. In this context, measurement settings were adjusted and spectra were taken from different chromatographic breakthrough curves of IgG1 in harvest. The resulting models show a small average RMSEP of 0.12 mg/mL, on a broad calibration range from 0 to 2.82 mg/mL IgG1. This work highlights the benefits of model assisted Raman spectroscopy in chromatography with complex backgrounds, lays the fundamentals for in-line monitoring of IgG1, and enables advanced control strategies. Moreover, the approach might be extended to further critical quality attributes like aggregates or could be transferred to other process steps.
Subject(s)
Chromatography/methods , Recombinant Proteins , Spectrum Analysis, Raman/methods , Animals , CHO Cells , Cricetinae , Cricetulus , Equipment Design , Protein Conformation , Recombinant Proteins/analysis , Recombinant Proteins/chemistry , Recombinant Proteins/metabolismABSTRACT
The semicontinuous twin-column multicolumn countercurrent solvent gradient purification (MCSGP) process improves the trade-off between purity and yield encountered in traditional batch chromatography, while its complexity, in terms of hardware requirements and process design, is reduced in comparison to process variants using more columns. In this study, the MCSGP process is experimentally characterized, specifically with respect to its unique degrees of freedom, i.e., the four switching times, which alternate the columns between interconnected and batch states. By means of isolation of the main charge isoform of an antibody, it is shown that purity is determined by the selection of the product collection window with negligible influence from the recycle phases. In addition, the amount of weak and strong impurities can be specifically attributed to the start and end of the collection, respectively. Due to higher abundance of weakly adsorbing impurities, the start of product collection influences productivity and yield more than the other switching times. Furthermore, most of the encountered tendencies scale between different loadings. The found trends can be rationalized from the corresponding batch chromatogram and therefore used during process design to obtain desirable process performances without extensive trial-and-error experimentation or complete model development and calibration.
Subject(s)
Countercurrent Distribution/methods , Recombinant Proteins/isolation & purification , Animals , Antibodies, Monoclonal/isolation & purification , Batch Cell Culture Techniques , Biotechnology , CHO Cells , Cricetinae , Cricetulus , Protein IsoformsABSTRACT
Perfusion cell culture, confined traditionally to the production of fragile molecules, is currently gaining broader attention in the biomanufacturing of therapeutic proteins. The development of these processes is made difficult by the limited availability of appropriate scale-down models. This is due to the continuous operation that requires complex control and cell retention capacity. For example, the determination of an optimal perfusion and bleed rate for continuous cell culture is often performed in scale-down bioreactors and requires a substantial amount of time and effort. To increase the experimental throughput and decrease the required workload, a semi-continuous procedure, referred to as the VCDmax (viable cell density) approach, has been developed on the basis of shake tubes (ST) and deepwell plates (96-DWP). Its effectiveness has been demonstrated for 12 different CHO-K1-SV cell lines expressing an IgG1. Further, its reliability has been investigated through proper comparisons with perfusion runs in lab-scale bioreactors. It was found that the volumetric productivity and the CSPRmin (cell specific perfusion rate) determined using the ST and 96-DWP models were successfully (mostly within the experimental error) confirmed in lab-scale bioreactors, which then covered a significant scale-up from the half milliliter to the liter scale. These scale-down models are very useful to design and scale-up optimal bioreactor operating conditions as well as screening for different media and cell lines.
Subject(s)
Batch Cell Culture Techniques/instrumentation , Bioreactors , Animals , Batch Cell Culture Techniques/methods , CHO Cells/chemistry , CHO Cells/cytology , Cell Count , Cell Survival , Cricetinae , Cricetulus , Kinetics , PerfusionABSTRACT
Glycosylation, a critical product quality attribute, may affect the efficacy and safety of therapeutic proteins in vivo. Chinese hamster ovary fed-batch cell culture batches yielded consistent glycoprofiles of a Fc-fusion antibody comprizing three different N-glycosylation sites. By adding media supplements at specific concentrations in cell culture and applying enzymatic glycoengineering, a diverse N-glycan variant population was generated, including high mannose, afucosylated, fucosylated, agalactosylated, galactosylated, asialylated, and sialylated forms. Site-specific glycosylation profiles were elucidated by glycopeptide mapping and the effect of the glycosylation variants on the FcγRIIIa receptor binding affinity and the biological activity (cell-based and surface plasmon resonance) was assessed. The two fusion body glycosylation sites were characterized by a high degree of sialic acid, more complex N-glycan structures, a higher degree of antennarity, and a site-specific behavior in the presence of a media supplement. On the other hand, the media supplements affected the Fc-site glycosylation heterogeneity similarly to the various studies described in the literature with classical monoclonal antibodies. Enzymatic glycoengineering solely managed to generate high levels of galactosylation at the fusion body sites. Variants with low core fucosylation, and to a lower extent, high mannose glycans exhibited increased FcγRIIIa receptor binding affinity. All N-glycan variants exhibited weak effects on the biological activity of the fusion body. Both media supplementation and enzymatic glycoengineering are suitable to generate sufficient diversity to assess the effect of glycostructures on the biological activity.
Subject(s)
Antibodies, Monoclonal/biosynthesis , Immunoglobulin Fc Fragments/biosynthesis , Mannose/metabolism , Polysaccharides/metabolism , Receptors, IgG/metabolism , Animals , Antibodies, Monoclonal/genetics , CHO Cells , Cricetulus , Glycosylation , Immunoglobulin Fc Fragments/genetics , Mannose/genetics , Polysaccharides/genetics , Receptors, IgG/geneticsABSTRACT
Mammalian cell perfusion cultures represent a promising alternative to the current fed-batch technology for the production of various biopharmaceuticals. Long-term operation at a fixed viable cell density (VCD) requires a viable culture and a constant removal of excessive cells. Product loss in the cell removing bleed stream deteriorates the process yield. In this study, the authors investigate the use of chemical and environmental growth inhibition on culture performance by either adding valeric acid (VA) to the production media or by reducing the culture temperature (33.0 °C) with respect to control conditions (36.5 °C, no VA). Low temperature significantly reduces cellular growth, thus, resulting in lower bleed rates accompanied by a reduced product loss of 11% compared to 26% under control conditions. Additionally, the cell specific productivity of the target protein improves and maintained stable leading to media savings per mass of product. VA shows initially an inhibitory effect on cellular growth. However, cells seemed to adapt to the presence of the inhibitor resulting in a recovery of the cellular growth. Cell cycle and Western blot analyses support the observed results. This work underlines the role of temperature as a key operating variable for the optimization of perfusion cultures.