scholarly journals Cell-culture growth conditions resulting in the oxidation of a recombinant antigen-binding fragment

2019 ◽  
Vol 6 (1) ◽  
Author(s):  
Khandaker Siddiquee ◽  
Cheng Zhao ◽  
Michelle A. Stemler ◽  
Bill Zeck ◽  
Jeffrey R. Fishpaugh ◽  
...  
Keyword(s):  
Cell Culture ◽  
Feeding Strategy ◽  
Scale Up ◽  
Chinese Hamster ◽  
Recombinant Antigen ◽  
Small Scale ◽  
Cell Culture Process ◽  
Antigen Binding ◽  
Scale Production ◽  
Target Molecule

Abstract Use of Quality-by-Design (QbD) tools is becoming an important part of the bioprocessing industry when developing a process for manufacturing operations to ensure the robustness and reproducibility of the biologic product. In the present study, a QbD tool, Design of Experiments (DOE), was utilized to optimize a bioprocess for the production of a CHO recombinant antigen-binding fragment (rFab) in small-scale bioreactors. DOE studies evaluated percent dissolved oxygen, temperature, and feeding strategy specific to this Chinese Hamster Ovary (CHO) clone. It was determined that these factors influenced cell viability, yield of the recombinant protein, and metabolic byproduct formation. To ensure the quality of the target molecule in the cell-culture process, small-scale purifications and analytical evaluation of the target molecule were completed prior to cell-culture scale-up to ensure that oxidation of the rFab, presence of free light chain, and truncation of thiol group were not observed. Analysis of the purified rFab by mass spectrometry indicated that rFab oxidation occurred under poor cell-culture conditions. PCR profile array results also revealed increased transcription of the oxidative genes Superoxide Dismutase 3, Myeloperoxidase, Dual Oxidase Like 2, Nuclear Receptor Coactivator 7, NADPH Oxidase Organizer 1, Mitochondria Uncouple Protein 3, Eosinophil Peroxidase, Lactoperoxidase Like, Serum Albumin Like, and Glutathione S-Transferase Pi 1 in this CHO strain. The present study suggests a mechanism and pathway for the oxidation of an rFab molecule during cell-culture bioprocess optimization. The present study also demonstrated the importance of utilizing the QbD tool of DOE to optimize the cell-culture bioprocess prior to scaling up into the large-scale production bioreactor.

Miniature Bioreactors for Rapid Bioprocess Development of Mammalian Cell Culture

2012 ◽  
Vol 59 (1) ◽  
Author(s):  
Mohd Helmi Sani ◽  
Frank Baganz
Keyword(s):  
Cell Culture ◽  
Mammalian Cell ◽  
Process Development ◽  
Mammalian Cell Culture ◽  
Small Scale ◽  
Cell Culture Process ◽  
Deep Wells ◽  
Working Volume ◽  
Culture Process ◽  
And Control

At present, there are a number of commercial small scale shaken systems available on the market with instrumented controllable microbioreactors such as Micro–24 Microreactor System (Pall Corporation, Port Washington, NY) and M2P Biolector, (M2P Labs GmbH, Aachen, Germany). The Micro–24 system is basically an orbital shaken 24–well plate that operates at working volume 3 – 7 mL with 24 independent reactors (deep wells, shaken and sparged) running simultaneously. Each reactor is designed as single use reactor that has the ability to continuously monitor and control the pH, DO and temperature. The reactor aeration is supplied by sparging air from gas feeds that can be controlled individually. Furthermore, pH can be controlled by gas sparging using either dilute ammonia or carbon dioxide directly into the culture medium through a membrane at the bottom of each reactor. Chen et al., (2009) evaluated the Micro–24 system for the mammalian cell culture process development and found the Micro–24 system is suitable as scaledown tool for cell culture application. The result showed that intra-well reproducibility, cell growth, metabolites profiles and protein titres were scalable with 2 L bioreactors.

Scale-up analysis for a CHO cell culture process in large-scale bioreactors

2009 ◽  
Vol 103 (4) ◽  
pp. 733-746 ◽  
Author(s):  
Zizhuo Xing ◽  
Brian M. Kenty ◽  
Zheng Jian Li ◽  
Steven S. Lee
Keyword(s):  
Cell Culture ◽  
Large Scale ◽  
Scale Up ◽  
Cell Culture Process ◽  
Cho Cell ◽  
Culture Process ◽  
Cho Cell Culture

scholarly journals Upstream process optimization and micro- and macrocarrier screening for large-scale production of the oncolytic H-1 protoparvovirus

2021 ◽  
Author(s):  
Daniel Wohlfarth ◽  
Veronika Frehtman ◽  
Marcus Müller ◽  
Martin Vogel ◽  
Linh Minh Phuc Phan ◽  
...  
Keyword(s):  
Cell Culture ◽  
Production Process ◽  
Large Scale ◽  
Culture Medium ◽  
Feeding Strategy ◽  
Virus Yield ◽  
Seeding Density ◽  
Scale Production ◽  
Cell Culture Medium ◽  
Transfer Capability

Abstract The oncolytic virus H-1PV is a promising candidate for various cancer treatments. Therefore, production process needs to be optimized and scaled up for future market release. Currently, the virus is produced with minimum essential medium in 10-layer CellSTACK® chambers with limited scalability, requiring a minimum seeding density of 7.9E3 cells/cm2. Production also requires a 5% fetal bovine serum (FBS) supplementation and has a virus yield up to 3.1E7 plaque-forming units (PFU)/cm2. Using the animal-free cell culture medium VP-SFM™ and a new feeding strategy, we demonstrate a yield boost by a mean of 0.3 log while reducing seeding density to 5.0E3 cells/cm2 and cutting FBS supplementation by up to 40% during the production process. Additionally, FBS is completely removed at the time of harvest. Eleven commercial micro- and macrocarriers were screened regarding cell growth, bead-to-bead transfer capability, and virus yield. We present a proof-of-concept study for producing H-1PV on a large scale with the microcarrier Cytodex® 1 in suspension and a macrocarrier for a fixed-bed iCELLis® bioreactor. A carrier-based H-1PV production process combined with an optimized cell culture medium and feeding strategy can facilitate future upscaling to industrial-scale production. Key points • Virus yield increase and FBS-free harvest after switching to cell culture medium VP-SFM™. • We screened carriers for cell growth, bead-to-bead transfer capability, and H-1PV yield. • High virus yield is achieved with Cytodex® 1 and macrocarrier for iCellis® in Erlenmeyer flasks.

scholarly journals Effect of Sialic Acid on Mammalian Cell Culture and Protein Expression: A Potential Productivity Enhancer for Biopharmaceutical Cell Culture Processes

Processes ◽  
2020 ◽  
Vol 8 (11) ◽  
pp. 1449
Author(s):  
Xiangsong Chen ◽  
Shang Xiao ◽  
Jinyong Wu ◽  
Jianming Yao
Keyword(s):  
Cell Culture ◽  
Sialic Acid ◽  
Cell Lines ◽  
Hek293 Cell ◽  
Chinese Hamster ◽  
Lactate Production ◽  
Production Costs ◽  
Cell Culture Process ◽  
Cho Cell ◽  
Potential Productivity

Improved productivity of the two most commonly used cell lines in the biopharmaceutical industry, such as human embryonic kidney 293 (HEK293) and Chinese hamster ovary (CHO), could reduce production costs and increase manufacturing capacity. One method for increasing protein productivity is the addition of antioxidants during the cell culture process. In this study, we examined the effect of sialic acid (SA) on one HEK293 cell line and two CHO cell lines. The addition of SA to HEK293 cell led to a higher viable cell density (VCD), viability (Via), and a lower lactate content in the later stage of cultures. Further results showed that SA reduced the reactive oxygen species (ROS), improved cell viability, reduced lactate production, and increased antibody expression by more than 20% in the later stage of the two CHO cell lines cultures. Besides, an optimized dose of SA had no significant effect on acidic variants level aggregation level, N-linked glycosylation pattern, and SA content on antibodies. These results suggest that the addition of SA can improve the productivity of biopharmaceutical cell culture processes.

Ultrasonic-Impregnation for Fiber-Reinforced Thermoplastic Prepreg Production

2017 ◽  
Vol 742 ◽  
pp. 17-24
Author(s):  
Steve Sockol ◽  
Christoph Doerffel ◽  
Juliane Mehnert ◽  
Gerd Zwinzscher ◽  
Steffen Rein ◽  
...  
Keyword(s):  
Large Scale ◽  
Matrix Material ◽  
Scale Up ◽  
Small Scale ◽  
Hydraulic Pressure ◽  
Scale Production ◽  
Fiber Reinforced ◽  
Technological Parameters ◽  
Ultrasonic Technology ◽  
High Processing

Fiber-reinforced thermoplastics have a high potential for big scale light weight process applications due to low processing times and recyclability. Further advantages are the low emissions during the manufacturing process and beneficial handling and storing properties of the semi finished materials. Thermoplastic composites are made of reinforcement fibers and a thermoplastic polymer matrix by applying two essential sub processes: (1) melting of the matrix-material and (2) impregnating the textile component with molten matrix-material. At present state of art both sub-processes are applied by using double-belt-presses, characterized by high processing temperatures and high processing forces. Therefore, a large amount of energy is needed to create the necessarily high compaction forces and temperatures with hydraulic cylinders and electric heating. Convection, infrared-radiation and the cooling (dynamic) of tempered machine parts leads to a significant dissipation of energy. Especially the process for generating the hydraulic pressure has a low level of efficiency. Therefore, in respect to economic and ecologic reasons, novel energy-efficient impregnation processes need to be investigated and developed. The represented novel impregnation process is based on ultrasonic technology. A stack of polymer film (outer layers) and a textile ply (inner layer) is formed and the energy is applied with an ultrasonic sonotrode. The efficient, fast and strongly concentrated energy application into the thermoplastic films allows the development of novel and highly flexible machine concepts. These can be used for development of small scale up to large scale production processes. The ultrasonic-technology allows a continuous impregnation of the textile component with molten matrix-material. A custom-designed prototype was developed. First material samples were produced and the technological parameters studied. A characterization of the experimental results, material samples, prototype machine and process is the focus of this paper.

scholarly journals Designing a Strategy for pH Control to Improve CHO Cell Productivity in Bioreactor

2021 ◽  
Author(s):  
Zohreh Ahleboot ◽  
Mahdi Khorshidtalab ◽  
Paria Motahari ◽  
Rasoul Mahboudi ◽  
Razieh Arjmand ◽  
...  
Keyword(s):  
Cell Culture ◽  
Large Scale ◽  
Chinese Hamster ◽  
Ph Control ◽  
Agitation Speed ◽  
Cell Culture Process ◽  
Co2 Removal ◽  
Bioreactor Culture ◽  
Product Titer ◽  
Culture Ph

Background: Drastic pH drop is a common consequence of scaling up a mammalian cell culture process, where it may affect the final performance of cell culture. Although CO2 sparging and base addition are used as common approaches for pH control, these strategies are not necessarily successful in large scale bioreactors due to their effect on osmolality and cell viability. Accordingly, a series of experiments were conducted using an IgG1 producing Chinese Hamster Ovary (CHO-S) cell culture in 30 L bioreactor to assess the efficiency of an alternative strategy in controlling culture pH.   Methods: Factors inducing partial pressure of CO2 and lactate accumulation (as the main factors altering culture pH) were assessed by Plackett-Burman design to identify the significant ones. As culture pH directly influences process productivity, protein titer was measured as the response variable. Subsequently, Central Composite Design (CCD) was employed to obtain a model for product titer prediction as a function of individual and interaction effects of significant variables.   Results: The results indicated that the major factor affecting pH is non-efficient CO2 removal. CO2 accumulation was found to be affected by an interaction between agitation speed and overlay air flow rate. Accordingly, after increasing the agitation speed and headspace aeration, the culture pH was successfully maintained in the range of 6.95-7.1, resulting in 51% increase in final product titer. Similar results were obtained during 250 L scale bioreactor culture, indicating the scalability of the approach.   Conclusion: The obtained results showed that pH fluctuations could be effectively controlled by optimizing CO2 stripping.

Comparative metabolite analysis to understand lactate metabolism shift in Chinese hamster ovary cell culture process

2011 ◽  
Vol 109 (1) ◽  
pp. 146-156 ◽  
Author(s):  
Jun Luo ◽  
Natarajan Vijayasankaran ◽  
Jennifer Autsen ◽  
Rodell Santuray ◽  
Terry Hudson ◽  
...  
Keyword(s):  
Cell Culture ◽  
Chinese Hamster Ovary Cell ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Cell Culture Process ◽  
Lactate Metabolism ◽  
Metabolite Analysis ◽  
Culture Process

scholarly journals High shear resistance of insect cells: the basis for substantial improvements in cell culture process design

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Florian Strobl ◽  
Mark Duerkop ◽  
Dieter Palmberger ◽  
Gerald Striedner
Keyword(s):  
Cell Culture ◽  
Gas Flow ◽  
Insect Cells ◽  
Negative Impact ◽  
Chinese Hamster ◽  
Shear Resistance ◽  
Pure Oxygen ◽  
Cell Culture Process ◽  
Foam Formation ◽  
Shear Rates

AbstractMulticellular organisms cultivated in continuous stirred tank reactors (CSTRs) are more sensitive to environmental conditions in the suspension culture than microbial cells. The hypothesis, that stirring induced shear stress is the main problem, persists, although it has been shown that these cells are not so sensitive to shear. As these results are largely based on Chinese Hamster Ovary (CHO) cell experiments the question remains if similar behavior is valid for insect cells with a higher specific oxygen demand. The requirement of higher oxygen transfer rates is associated with higher shear forces in the process. Consequently, we focused on the shear resistance of insect cells, using CHO cells as reference system. We applied a microfluidic device that allowed defined variations in shear rates. Both cell lines displayed high resistance to shear rates up to 8.73 × 105 s−1. Based on these results we used microbial CSTRs, operated at high revolution speeds and low aeration rates and found no negative impact on cell viability. Further, this cultivation approach led to substantially reduced gas flow rates, gas bubble and foam formation, while addition of pure oxygen was no longer necessary. Therefore, this study contributes to the development of more robust insect cell culture processes.

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