There is a trend across the pharmaceutical sector toward process intensification and continuous manufacturing to produce small-molecule drugs or biotechnology products. For biotechnology products, ...advancing the manufacturing technology behind upstream and downstream processes has the potential to reduce product shortages and variability, allow for production flexibility, simplify scale-up procedures, improve product quality, reduce facility footprints, increase productivity, and reduce production costs. On the upstream side of biotechnology manufacturing, continuous perfusion cell cultures are fairly well established. However, truly integrated continuous biomanufacturing requires the uninterrupted connection of continuous unit operations (upstream and downstream) with no isolated intermediate or hold steps occurring between them. This work examines the current scientific and regulatory landscape surrounding the implementation of integrated continuous biomanufacturing.
There is a trend across the pharmaceutical sector toward process intensification and continuous manufacturing to produce small-molecule drugs or biotechnology products.
For biotechnology products, advancing the manufacturing technology behind upstream and downstream processes has the potential to reduce product shortages and variability, allow for production flexibility, simplify scale-up procedures, improve product quality, reduce facility footprints, increase productivity, and reduce production costs.
Nevertheless, some scientific and regulatory challenges still exist in implementing integrated continuous biomanufacturing.
N-Linked glycosylation is a cellular process transferring sugars from glycosyl donors to proteins or lipids. Biopharmaceutical products widely produced by culturing mammalian cells such as Chinese ...hamster ovary (CHO) cells are typically glycosylated during biosynthesis. For some biologics, the N-linked glycan is a critical quality attribute of the drugs. Nucleotide sugars are the glycan donors and impact the intracellular glycosylation process. In current analytical methods, robust separation of nucleotide sugar isomers such as UDP glucose and UDP galactose remains a challenge because of their structural similarity. In this study, we developed a strategy to resolve the separation of major nucleotide sugars including challenging isomers based on the use of ion-pair reverse phase (IP-RP) chromatography. The strategy applies core-shell columns and connects multiple columns in tandem to increase separation power and ultimately enables high-resolution detection of nucleotide sugars from cell extracts. The key parameters in the IP-RP method, including temperature, mobile phase, and flow rates, have been systematically evaluated in this work and the theoretical mechanisms of the chromatographic behavior were proposed.
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N-linked glycosylation is a post-translational modification that occurs on many proteins during biosynthesis. The profile of different glycans on the protein is a critical quality attribute of some ...recombinant biopharmaceutical proteins including monoclonal antibodies (mAbs). Methods for profiling glycan should be robust, fast, and sensitive. Isolating glycans from proteins and tagging a label on glycans is the most commonly used technique for glycan profiling. Currently, existing protocols for sample preparation can be complicated, time-consuming, and expensive, which can limit the wide adaptation of glycan profiling methods. As a further barrier to use, an expensive ultra-high-pressure liquid chromatography (UHPLC) system is frequently required for the profile. In this article, a low cost and easily-used workflow of sample preparation is coupled with a standard high-performance liquid chromatography (HPLC) system to achieve comparable results to UHPLC. The number of steps required in the protocol and the time, as well as the cost associated with the sample preparation, is significantly reduced, while maintaining robust analytical performance. We describe the creation and validation of a human serum IgG glycan library to be used as the calibration standard, and successful profiling of glycoforms from a variety of mAbs.
•A HPLC-based method is developed for high-resolution separation of mAb glycoforms.•A low-cost, simple and robust process is developed for glycan sample preparation.•Analytical results are robust in the optimized method.
In this work, a three-stage population balance model (TSPBM) was developed for a pulsed top-spray fluidized bed granulation (FBG) process. The batch top-spray FBG process was divided into three ...stages based on the analysis of granule properties evolution and different granulation mechanisms were considered in each stage of the TSPBM. In each stage, population balance model (PBM) describes the evolution of granule size distribution (GSD), and partial least square (PLS) regressions describes the relationship between the operating variables and kernel parameters in PBM. By fitting coefficients of PLS regressions using experimental data, the developed TSPBM establishes a predictive relationship between the manipulated binder spray parameters of pulsed frequency, binder flow rate and atomization pressure and granule critical quality attributes (CQAs). A model-based multi-stage optimization strategy was proposed to improve the granule quality of the pulsed-spray FBG. The optimization strategy reduced the process error caused by mismatch between developed model and actual system. In the optimization strategy, volume average granule diameter is only measured online at the end of each stage. Based on the online measurement, the optimization strategy adjusts process operating variables to remedy any drift between measured and predicted GSD. Validation experiments and simulation tests were carried out to validate the effectiveness of the proposed TSPBM and optimization strategy. The developed TSPBM is shown to accurately predict experimental GSD and shows high accuracy comparing to the one-stage PBM. The proposed optimization strategy can improve the prediction capability by >50% compared to an offline optimization.
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•A developed TSPBM was well validated for a pulsed top-spray fluidized bed granulation process.•Customized granulation mechanism on each stage to accurately simulate the GSD evolution•Online optimization to reduce influence of mismatch between model and actual system•Abnormal operating mistake could be remedied by the online optimization strategy.
The fluidization velocity and mean particle size were selected to be numerically investigated pertaining to their effects on the gas-particle circulation pattern within a fluidized bed granulator by ...three-dimensional computational fluid dynamics (CFD) simulation applying an Eulerian-Eulerian two-fluid model. The CFD simulations were designed by full factorial design method and the developed CFD model was experimentally validated. The fluidization process was proved to reach a quasi-steady state. The gas-particle circulation pattern and particle concentration distribution were analyzed based on fluidization velocity and mean particle size. A mathematical model was developed to provide guidance on how to change fluidization level during one experiment.
Celotno besedilo
Dostopno za:
BFBNIB, DOBA, GIS, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
The variability of trace metals in cell culture media is a potential manufacturing concern because it may significantly affect the production and quality of therapeutic proteins. Variability in trace ...metals in CHO cell culture has been shown to impact critical production metrics such as cell growth, viability, nutrient consumption, and production of recombinant proteins. To better understand the influence of excess supplementation, zinc and copper were initially supplemented with 50-μM concentrations to determine the impact on the production and quality of β-glucuronidase, a lysosomal enzyme, in a parallel bioreactor system. Ethylenediaminetetraacetic acid (EDTA), a metal chelator, was included as another treatment to induce a depletion of trace metal bioavailability to examine deficiency. Samples were drawn daily to monitor cell growth and viability, nutrient levels, β-glucuronidase activity, and trace zinc flux. Cell cycle analysis revealed the inhibition of sub-G0/G1 species in zinc supplemented cultures, maintaining higher viability compared to the control, EDTA-, and copper-supplemented cultures. Enzyme activity analysis in the harvests revealed higher specific activity of β-glucuronidase in reactors supplemented with zinc. A confirmation run was conducted with supplementations of zinc at concentrations of 50, 100, and 150 μM. Further cell cycle analysis and caspase-3 analysis demonstrated the role of zinc as an apoptosis suppressor responsible for the enhanced harvest purity of β-glucuronidase from zinc-supplemented bioreactors.
•The challenges and bottlenecks arising due to batch chromatography are identified.•Merits of continuous multi-column chromatography are provided.•The modelings and technologies currently available ...for the continuous chromatography are summarized.•The control strategy and gaps for implementation of continuous chromatography are described.
The potential of continuous bioprocessing is hindered by the bottlenecks of chromatography processing, which continues to be executed in batch mode. Highlighting the critical drawbacks of batch chromatography, this review underscores the transition that the industry has made by implementing continuous upstream process without devising a working model for downstream chromatography operations. Even though multitude of process development initiatives have commenced, the review emphasizes the first principle models of chromatography on which these initiatives are built. Various models of continuous chromatography, which are essential, but not limited to multi-column systems, employed to congeal a unified process are reviewed. Advancements made by several mechanistic models and simulations to maximize productivity and performance are described, in an attempt to provide the integral tools. The modeling tools can be used for development of a strong model based control strategy and can be embedded into the continuous chromatography framework. The review addresses the limitations and challenges of the current modeling methods for development of robust mechanistic modeling and efficient unit operation platform in continuous chromatography.
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In Chinese Hamster Ovary (CHO) cell lines, the establishment of the ideal fed-batch regimen promotes metabolic conditions advantageous for the bioproduction of therapeutic molecules. A tailored, cell ...line-specific feeding scheme is typically defined during process development (PD) activities, through the incorporation of Design of Experiment (DOE) and late stage cell culture approaches. The feeding during early stage cell line development (CLD) was a simplified “one-fits-all” design, inherited from PD lab, that didn’t account for CLD needs of throughput and streamlined workflow. The “one-fits-all” efficiency was not routinely verified when novel technologies were incorporated in CLD and sub-optimal feeding carried the risk of not selecting the most desirable cell lines amenable to late stage PD. In our work we developed the DOE-feed method; a streamlined, three-stages framework for identifying efficient feeding schemes as the CLD technologies evolved. We combined early stage cell culture input data with late-stage techniques, such as statistical modelling, principal component analysis (PCA), DOE and Prediction Profiler. Novel in our DOE-feed work, we deliberately anticipated the application of statistics and approached the method development as an early-stage, continuously updated process, by building iterative datasets and statistically interpreting their responses. We capitalized on the statistical models defined by the DOE-feed methodology to study the influence of feeds on daily productivity and growth and to extrapolate feeding-schemes that improved the cell line screening. The DOE-feed became a methodology suited for CLD needs at AbbVie, and optimized the early stage screening, reduced the operational hands-on time and improved the overall workstream efficiency.
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•n-layer BET isotherm model explains the binding characteristics of a rProtein A ligand.•Binding capacity decreases with exposure to caustic, except at time zero for new resin.•Adding ...salt to sanitization regime improves the binding capacity for a limited time.•Static and dynamic adsorption confirms the improved binding with caustic for new resin.•Fundamental representation of a multimeric Protein A ligand is the novelty of this paper.
Langmuir and other single-layer adsorption isotherms show the binding behavior of natural Protein A ligands immobilized on a column. However, no models have been shown in literature to explain the adsorption phenomena on the recombinant high binding capacity Protein A resins. This study has characterized the Protein A binding domain distribution across the ligand with multi-layer adsorption isotherms for a recombinant Protein A resin. The adsorption data was analyzed using the Langmuir, Freundlich, Brunauer–Emmett–Teller (BET) and various other mathematical equations. The best fit of experimental data was obtained with n-layer BET model wherein the isotherms of Protein A exhibited Type IV behavior according to BET classification. Furthermore, the binding capacity was studied throughout the shelf life using the multi-layer adsorption isotherm model. Antibody adsorption isotherms of Protein A resin were obtained at preset duration of caustic incubation. The experiments were carried out for two conditions of sanitization agent, namely, caustic and caustic with salt. Static and dynamic isotherm analysis showed that a new resin had a lower binding capacity and the initial sanitization improved the binding capacity, probably by making the binding domains more accessible. The binding capacity at equilibrium, dynamic breakthrough and batch were also evaluated and reported in this paper. The study modeled the multimeric Protein A ligand and established the requirement of optimization for cleaning regime. This study provides a fundamental understanding of the binding patterns in the recombinant Protein A ligands through a working mathematical equation and improves the current knowledge of Protein A resin lifetimes.
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•Transcriptomics analysis was used to study metabolism change over the batch culture.•Metabolic pathway alternations were analyzed between high and low producers.•Key pathways related ...to productivity increase and high productivity were elucidated.•Combined transcriptomics and flux analysis allow to identify metabolic bottlenecks.
In this study, we presented an integrated systems biology approach to elucidate the key characteristics of cellular metabolism in Chinese hamster ovary (CHO) cells producing monoclonal antibodies (mAb). The cellular metabolism in high and low producers under batch conditions was interrogated dynamically both within and among cells. First, transcriptomics analysis was used to study the time-course change in the metabolic pathway within cells that was correlated with mAb productivity increase. Second, differentially regulated pathways between high and low producers were sought at each growth phase. Several up-regulated pathways were identified in the high producer at the late growth phase, including citrate cycle, oxidative phosphorylation, and pentose phosphate pathway. These activities were further analyzed by intracellular flux distributions estimated through a genome-scale CHO model. Our results revealed that these key pathways are identified to be characteristics of high mAb production, not only for the high-producing cell line but also a dynamic phenomenon in mAb-producing cell cultures. This study showed that the approach of integrating transcriptomics and flux analysis leads to a better understanding of cellular metabolism related to mAb productivity. In turn, this allows for the identification of metabolic bottlenecks and potential engineering targets for cell line development and process optimization.
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