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Antibody-drug conjugate (ADC) is a class of targeted cancer therapies that combine the advantages of monoclonal antibody (mAb)’s specific targeting and chemotherapy’s potent cytotoxicity. The therapeutic effect of ADC is significantly affected by its bioproduction process. This study aims to develop an effective ADC production process using anti-HER2 mAb-drug as a model therapeutic. First, a high titer (>2 g/L) of mAb was produced by Chinese hamster ovary cells from fed-batch cell culture. Both live-cell confocal microscopy imaging and flow cytometry analysis demonstrated that the produced mAb and ADC had strong and specific binding to HER2+ cell line BT474. Second, various conjugation conditions of mAb and drug, including linker selection, ratio of drug and mAb, and conjugation approaches, were investigated to improve the production yield and product quality. Finally, the ADC structure and biological quality were evaluated by SDS-PAGE and anti-breast cancer toxicity study, respectively. The ADC with integral molecular structure and high cytotoxicity (IC50 of 1.95 nM) was produced using the optimized production process. The robust bioproduction process could guide the development of ADC-based biopharmaceuticals.

The adoptive transfer of human T cells or genetically-engineered T cells with cancer-targeting receptors has shown tremendous promise for eradicating tumors in clinical trials. The objective of this study was to develop a novel T cell biomanufacturing platform using stirred-tank bioreactor for large-scale and high-quality cellular production. First, various factors, such as bioreactor parameters, media, supplements, stimulation, seed age, and donors, were investigated. A serum-free fed-batch bioproduction process was developed to achieve 1000-fold expansion within 8 days after first stimulation and another 500-fold expansion with second stimulation. Second, this biomanufacturing process was successfully scaled up in bioreactor with dilution factor of 10, and the robustness and reproducibility of the process was confirmed by the inclusion of different donors' T cells of various qualities. Finally, T cell quality was monitored using 12 surface markers and 3 intracellular cytokines as the critical quality assessment criteria in early, middle and late stages of cell production. In this study, a new biomanufacturing platform was created to produce reliable, reproducible, high-quality, and large-quantity (i.e. > 5 billion) human T cells in stirred-tank bioreactor. This platform is compatible with the production systems of monoclonal antibodies, vaccines, and other therapeutic cells, which provides not only the proof-of-concept but also the ready-to-use new approach of T cell expansion for clinical immune therapy.

Chinese hamster ovary (CHO)1 cells have been widely used to express heterologous genes and produce therapeutic proteins in biopharmaceutical industry. Different CHO host cells have distinct cell growth rates and protein expression characteristics. In this study, the expression of about 1,307 host proteins in three sublines, i.e. CHO K1, CHO S and CHO/dihydrofolate reductase (dhfr)−, were investigated and compared using proteomic analysis. The proteins involved in cell growth, glycolysis, tricarboxylic acid cycle, transcription, translation and glycosylation were quantitated using Liquid chromatography tandem-mass spectrometry (LC-MS/MS). The key host cell proteins that regulate the kinetics of cell growth and the magnitude of protein expression levels were identified. Furthermore, several rational cell engineering strategies on how to combine the desired features of fast cell growth and efficient production of therapeutic proteins into one new super CHO host cell have been proposed.

Human induced-pluripotent stem cells (hiPSCs) can be efficiently differentiated into cardiomyocytes (hiPSC-CMs) via the GiWi method, which uses small-molecule inhibitors of glycogen synthase kinase (GSK) and tankyrase to first activate and then suppress Wnt signaling. However, this method is typically conducted in 6-well culture plates with two-dimensional (2D) cell sheets, and consequently, cannot be easily scaled to produce the large numbers of hiPSC-CMs needed for clinical applications. Cell suspensions are more suitable than 2D systems for commercial biomanufacturing, and suspended hiPSCs form free-floating aggregates (i.e., spheroids) that can also be differentiated into hiPSC-CMs. Here, we introduce a protocol for differentiating suspensions of hiPSC spheroids into cardiomyocytes that is based on the GiWi method. After optimization based on cardiac troponin T staining, the purity of hiPSC-CMs differentiated via our novel protocol exceeded 98% with yields of about 1.5 million hiPSC-CMs/mL and less between-batch purity variability than hiPSC-CMs produced in 2D cultures; furthermore, the culture volume could be increased ∼10-fold to 30 mL with no need for re-optimization, which suggests that this method can serve as a framework for large-scale hiPSC-CM production.

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Biobutanol is a sustainable green biofuel that can substitute for gasoline. Carbon flux has been redistributed in Clostridium tyrobutyricum via metabolic cell engineering to produce biobutanol. However, the lack of reducing power hampered the further improvement of butanol production. The objective of this study was to improve butanol production by rebalancing redox. Firstly, a metabolically-engineered mutant CTC-fdh-adhE2 was constructed by introducing heterologous formate dehydrogenase (fdh) and bifunctional aldehyde/alcohol dehydrogenase (adhE2) simultaneously into wild-type C. tyrobutyricum. The mutant evaluation indicated that the fdh-catalyzed NADH-producing pathway improved butanol titer by 2.15-fold in the serum bottle and 2.72-fold in the bioreactor. Secondly, the medium supplements that could shift metabolic flux to improve the production of butyrate or butanol were identified, including vanadate, acetamide, sodium formate, vitamin B12 and methyl viologen hydrate. Finally, the free-cell fermentation produced 12.34 g/L of butanol from glucose using the mutant CTC-fdh-adhE2, which was 3.88-fold higher than that produced by the control mutant CTC-adhE2. This study demonstrated that the redox engineering in C. tyrobutyricum could greatly increase butanol production.

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