Therapeutic Fc fusion protein misfolding: A three-phasic cultivation experimental design

Autor: Sepideh Samavat, Farnoush Jafari, Shayan Maleknia, Paria Motahari, Rasoul Mahboudi, Atefeh Ghorbani Aghdam, Saeed Moradhaseli
Jazyk: angličtina
Rok vydání: 2019
Předmět:
0106 biological sciences
0301 basic medicine
Protein Folding
Protein Expression
01 natural sciences
Biochemistry
Cell Fusion
Mathematical and Statistical Techniques
Bioreactors
Protein biosynthesis
Macromolecular Structure Analysis
Process optimization
Multidisciplinary
Chemistry
Chinese hamster ovary cell
Statistics
Temperature
Recombinant Proteins
Protein Misfolding
Physical Sciences
Cell lines
Medicine
Protein folding
Target protein
Biological cultures
Research Article
Protein Structure
Cell Physiology
Recombinant Fusion Proteins
Science
CHO Cells
Research and Analysis Methods
03 medical and health sciences
Cricetulus
010608 biotechnology
Bioreactor
Gene Expression and Vector Techniques
Animals
Response surface methodology
Statistical Methods
Molecular Biology Techniques
Molecular Biology
Molecular Biology Assays and Analysis Techniques
Analysis of Variance
Biology and Life Sciences
Proteins
Cell Biology
Cell Cultures
Models
Theoretical
Immunoglobulin Fc Fragments
030104 developmental biology
Cell culture
Biophysics
Mathematics
Zdroj: PLoS ONE, Vol 14, Iss 1, p e0210712 (2019)
PLoS ONE
ISSN: 1932-6203
Popis: Cell culture process optimization is a critical solution to most of the challenges faced by the pharmaceutical manufacturing. One of the major problems encountered in large-scale production of therapeutic proteins is misfolded protein production. The accumulation of misfolded therapeutic proteins is an immunogenic signal and a risk factor for immunogenicity of the final product. The aim of this study was the statistical optimization of three-phasic temperature shift and timing for enhanced production of correctly folded Fc-fusion protein. The effect of culture temperatures were investigated using the biphasic culture system. Box–Behnken design was then used to compute temperature and time of shifting optimum. Response surface methodology revealed that maximum production with low level of misfolded protein was achieved at two-step temperature shift from 37°C to 30°C during the late logarithmic phase and 30°C to 28°C in the mid-stationary phase. The optimized condition gave the best results of 1860 mg L−1 protein titer with 24.5% misfolding level. The validation experiments were carried out under optimal conditions with three replicates and the protein misfolding level was decreased by two times while productivity increased by ~ 1.3-fold. Large-scale production in 250 L bioreactor under the optimum conditions was also verified the effectiveness and the accuracy of the model. The results showed that by utilizing two-step temperature shift, productivity and the quality of target protein have been improved simultaneously. This model could be successfully applied to other products.
Databáze: OpenAIRE
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