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

Abstract

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.

Sodium chloride impacts glycosylation and N‐ and O‐glycan site occupancy of an Fc‐fusion protein

“…In the literature, Fc‐fusion size heterogeneity is known to be caused by proteinogenic fragments, resulting from the activity of host cell‐related proteases such as cysteine endopeptidase (Mitsuo et al, 1990) or matrix metalloproteases (Sandberg et al, 2006). Several mitigation strategies have been developed including changes in bioprocess conditions (culture temperature and time, partial pressure of CO 2 [Chakrabarti et al, 2016; Ghorbani Aghdam et al, 2019; Menthe et al, 2022]) or change in cell culture media composition (addition of ferric citrate, or protease inhibitor cocktail [Chakrabarti et al, 2016; Hou et al, 2019]). Other recent studies on complex N‐ and C‐ terminal Fc‐fusion proteins (Datola et al, 2023) showed that LMW proteoforms may be caused by reshuffled disulfide bridges and can lead to protein misfolding responsible for aggregation and precipitation (Zhang et al, 2011), further affecting the biological activity and safety of the therapeutic molecule.…”

“…Another extensive process development study was performed to obtain correctly folded erythropoietin (EPO) Fc‐fusion protein (Ghorbani Aghdam et al, 2019). The final process included a two‐step temperature shift, which led to an upregulation of genes encoding unfolded protein response specific transcriptional activators and ER‐resident proteins associated with processing and folding in the secretory compartments (Torres et al, 2021).…”

Impact of SARS-CoV-2 RBD Mutations on the Production of a Recombinant RBD Fusion Protein in Mammalian Cells

“…Some of these production issues can be influenced by the amino acid sequence of the produced protein [ 20 , 21 , 22 ], which is of particular concern for the efficient production of mutated SARS-CoV-2 RBD recombinant proteins. To overcome these challenges, engineering and optimisation approaches have been proposed [ 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 ]. Additionally, mammalian cells have become a favoured system to produce recombinant proteins for clinical and diagnostic applications because they allow proper post-translational modifications and protein folding [ 35 , 36 ].…”

A Computational Approach to Modeling an Antagonistic Angiogenic VEGFR1-IL2 Fusion Protein for Cancer Therapy

“…In designing the recombinant multi-domain proteins, the fusion proteins are more prone to misfolding or assembling in an incorrect 3D shape when compared with a single-domain protein, as different peptide domains interact with each other in different ways. 29 To overcome this limitation, in silico analyses are performed to generate the recombinant fusion proteins followed by the protein modeling and Molecular Dynamic (MD) simulations. In the current report, molecular modeling of an antagonistic angiogenic fusion protein—ie, soluble ECD of human VEGFR-1 (sVEGFR-1) and human IL-2—uses threading assembly refinement to predict the 3D structure and energy minimization.…”