9 research outputs found
Establishment of a Perfusion Process with Antibody-Producing CHO Cells Using a 3D-Printed Microfluidic Spiral Separator with Web-Based Flow Control
Monoclonal antibodies are increasingly dominating the market for human therapeutic and diagnostic agents. For this reason, continuous methodsâsuch as perfusion processesâare being explored and optimized in an ongoing effort to increase product yields. Unfortunately, many established cell retention devicesâsuch as tangential flow filtrationârely on membranes that are prone to clogging, fouling, and undesirable product retention at high cell densities. To circumvent these problems, in this work, we have developed a 3D-printed microfluidic spiral separator for cell retention, which can readily be adapted and replaced according to process conditions (i.e., a plug-and-play system) due to the fast and flexible 3D printing technique. In addition, this system was also expanded to include automatic flushing, web-based control, and notification via a cellphone application. This set-up constitutes a proof of concept that was successful at inducing a stable process operation at a viable cell concentration of 10â17 Ă 106 cells/mL in a hybrid mode (with alternating cell retention and cell bleed phases) while significantly reducing both shear stress and channel blockage. In addition to increasing efficiency to nearly 100%, this microfluidic device also improved production conditions by successfully separating dead cells and cell debris and increasing cell viability within the bioreactor
Implementation of QbD strategies in the inoculum expansion of a mAb production process
The quality by design approach was introduced to the biopharmaceutical industry over 15 years ago. This principle is widely implemented in the characterization of monoclonal antibody production processes. Anyway, the early process phase, namely the inoculum expansion, was not yet investigated and characterized for most processes. In order to increase the understanding of early process parameter interactions and their influence on the later production process, a risk assessment followed by a design of experiments approach was conducted. The DoE included the critical parameters methotrexate (MTX) concentration, initial passage viable cell density and passage duration. Multivariate data analysis led to mathematical regression models and the establishment of a designated design space for the studied parameters. It was found that the passage duration as well as the initial viable cell density for each passage during the inoculum expansion have severe effects on the growth rate and viability of the early process phase. Furthermore, the variations during the inoculum expansion directly influenced the production process responses. This carry-over of factor effects highlights the crucial impact of early process failures and the importance of process analysis and control during the first part of mAb production processes. © 2020 The Authors. Engineering in Life Sciences published by Wiley-VCH Gmb
Stress-induced increase of monoclonal antibody production in CHO cells
Monoclonal antibodies (mAbs) are of great interest to the biopharmaceutical industry due to their widely used application as human therapeutic and diagnostic agents. As such, mAb require to exhibit human-like glycolization patterns. Therefore, recombinant Chinese hamster ovary (CHO) cells are the favored production organisms; many relevant biopharmaceuticals are already produced by this cell type. To optimize the mAb yield in CHO DG44 cells a corelation between stress-induced cell size expansion and increased specific productivity was investigated. CO2 and macronutrient supply of the cells during a 12-day fed-batch cultivation process were tested as stress factors. Shake flasks (500 mL) and a small-scale bioreactor system (15 mL) were used for the cultivation experiments and compared in terms of their effect on cell diameter, integral viable cell concentration (IVCC), and cell-specific productivity. The achieved stress-induced increase in cell-specific productivity of up to 94.94.9%â134.4% correlates to a cell diameter shift of up to 7.34 ÎŒm. The highest final product titer of 4 g/L was reached by glucose oversupply during the batch phase of the process
Stressâinduced increase of monoclonal antibody production in CHO cells
Monoclonal antibodies (mAbs) are of great interest to the biopharmaceutical industry due to their widely used application as human therapeutic and diagnostic agents. As such, mAb require to exhibit humanâlike glycolization patterns. Therefore, recombinant Chinese hamster ovary (CHO) cells are the favored production organisms; many relevant biopharmaceuticals are already produced by this cell type. To optimize the mAb yield in CHO DG44 cells a corelation between stressâinduced cell size expansion and increased specific productivity was investigated. CO(2) and macronutrient supply of the cells during a 12âday fedâbatch cultivation process were tested as stress factors. Shake flasks (500Â mL) and a smallâscale bioreactor system (15Â mL) were used for the cultivation experiments and compared in terms of their effect on cell diameter, integral viable cell concentration (IVCC), and cellâspecific productivity. The achieved stressâinduced increase in cellâspecific productivity of up to 94.94.9%â134.4% correlates to a cell diameter shift of up to 7.34Â ÎŒm. The highest final product titer of 4Â g/L was reached by glucose oversupply during the batch phase of the process
Optimization of a mAb production process with regard to robustness and product quality using quality by design principles
Quality by Design principles are well described and widely used in biopharmaceutical industry. The characterization of a monoclonal antibody (mAb) production process is crucial for novel process development and control. Yet, the application throughout the entire upstream process was rarely demonstrated. Following previously published research, this study marks the second step toward a complete process characterization and is focused on the effect of critical process parameters on the antibody production efficiency and quality of the process. In order to conduct the complex Design of Experiments approach with optimal control and comparability, the ambrÂź15 micro bioreactor platform was used. Investigated parameters included the pH and dissolved oxygen set points, the initial viable cell density (iVCD) as well as the N-1 duration. Various quality attributes (e.g., growth rate, viability, mAb titer, and peak proportion) were monitored and analyzed using multivariate data analysis to evaluate the parameter effects. The pH set point and the initial VCD were identified as key process parameters with strong influence on the cell growth as well as the mAb production and its proportion to the total protein concentration. For optimization and improvement in robustness of these quality attributes the pH must be increased to 7.2, while the iVCD must be lowered to 0.2 Ă 106 cells/mL. Based on the defined design space, additional experiments verified the results and confirmed the intact bioactivity of the antibody. Thereby, process control strategies could be tuned toward high cell maintenance and mAb production, which enable optimal downstream processing. © 2022 The Authors. Engineering in Life Sciences published by Wiley-VCH GmbH
Monitoring cell productivity for the production of recombinant proteins by flow cytometry : An effective application using the cold capture assay
Due to the increasing economic and social relevance of biotherapeutics, their production processes are continually being reconsidered and reoptimized in an effort to secure higher product concentrations and qualities. Monitoring the productivity of cultured cells is therefore a critically important part of the cultivation process. Traditionally, this is achieved by determining the overall product titer by high performance liquid chromatography (HPLC), and then calculating the specific cell productivity based on this titer and an associated viable cell density. Unfortunately, this process is typically timeâconsuming and laborious. In this study, the productivity of Chinese Hamster Ovary (CHO) cells expressing a monoclonal antibody was analyzed over the course of the cultivation process. In addition to calculating the specific cell productivity based on the traditional product titer determined by HPLC analysis, culture productivity of single cells was also analyzed via flow cytometry using a cold capture assay. The cold capture assay is a cell surface labelling technique described by Brezinsky et al., which allows for the visualization of a product on the surface of the producing cell. The cell productivity results obtained via HPLC and the results of cold capture assay remained in great accordance over the whole cultivation process. Accordingly, our study demonstrates that the cold capture assay offers an interesting, comparatively timeâeffective, and potentially cheaper alternative for monitoring the productivity of a cell culture
Establishment of a Perfusion Process with Antibody-Producing CHO Cells Using a 3D-Printed Microfluidic Spiral Separator with Web-Based Flow Control
Monoclonal antibodies are increasingly dominating the market for human therapeutic and diagnostic agents. For this reason, continuous methodsâsuch as perfusion processesâare being explored and optimized in an ongoing effort to increase product yields. Unfortunately, many established cell retention devicesâsuch as tangential flow filtrationârely on membranes that are prone to clogging, fouling, and undesirable product retention at high cell densities. To circumvent these problems, in this work, we have developed a 3D-printed microfluidic spiral separator for cell retention, which can readily be adapted and replaced according to process conditions (i.e., a plug-and-play system) due to the fast and flexible 3D printing technique. In addition, this system was also expanded to include automatic flushing, web-based control, and notification via a cellphone application. This set-up constitutes a proof of concept that was successful at inducing a stable process operation at a viable cell concentration of 10â17 Ă 106 cells/mL in a hybrid mode (with alternating cell retention and cell bleed phases) while significantly reducing both shear stress and channel blockage. In addition to increasing efficiency to nearly 100%, this microfluidic device also improved production conditions by successfully separating dead cells and cell debris and increasing cell viability within the bioreactor
Optimization of a mAb production process with regard to robustness and product quality using quality by design principles
Abstract Quality by Design principles are well described and widely used in biopharmaceutical industry. The characterization of a monoclonal antibody (mAb) production process is crucial for novel process development and control. Yet, the application throughout the entire upstream process was rarely demonstrated. Following previously published research, this study marks the second step toward a complete process characterization and is focused on the effect of critical process parameters on the antibody production efficiency and quality of the process. In order to conduct the complex Design of Experiments approach with optimal control and comparability, the ambrÂź15 micro bioreactor platform was used. Investigated parameters included the pH and dissolved oxygen set points, the initial viable cell density (iVCD) as well as the Nâ1 duration. Various quality attributes (e.g., growth rate, viability, mAb titer, and peak proportion) were monitored and analyzed using multivariate data analysis to evaluate the parameter effects. The pH set point and the initial VCD were identified as key process parameters with strong influence on the cell growth as well as the mAb production and its proportion to the total protein concentration. For optimization and improvement in robustness of these quality attributes the pH must be increased to 7.2, while the iVCD must be lowered to 0.2Â ĂÂ 106Â cells/mL. Based on the defined design space, additional experiments verified the results and confirmed the intact bioactivity of the antibody. Thereby, process control strategies could be tuned toward high cell maintenance and mAb production, which enable optimal downstream processing