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As of early 2022, the coronavirus disease 2019 (COVID-19) pandemic remains a substantial global health concern. Different treatments for COVID-19, such as anti-COVID-19 neutralizing monoclonal antibodies (mAbs), have been developed under tight timelines. Not only mAb product and clinical development but also chemistry, manufacturing, and controls (CMC) process development at pandemic speed are required to address this highly unmet patient need. CMC development consists of early- and late-stage process development to ensure sufficient mAb manufacturing yield and consistent product quality for patient safety and efficacy. Here, we report a case study of late-stage cell culture process development at pandemic speed for mAb1 and mAb2 production as a combination therapy for a highly unmet patient treatment. We completed late-stage cell culture process characterization (PC) within approximately 4 months from the cell culture process definition to the initiation of the manufacturing process performance qualification (PPQ) campaign for mAb1 and mAb2, in comparison to a standard one-year PC timeline. Different strategies were presented in detail at different PC steps, i.e., pre-PC risk assessment, scale-down model development and qualification, formal PC experiments, and in-process control strategy development for a successful PPQ campaign that did not sacrifice quality. The strategies we present may be applied to accelerate late-stage process development for other biologics to reduce timelines.

Abstract 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.

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.

Meningiomas are primary tumors of the central nervous system with high recurrence. It has been reported that somatostatin receptor 2 (SSTR2) is highly expressed in most meningiomas, but there is no effective targeted therapy approved to control meningiomas. This study aimed to develop and evaluate an anti-SSTR2 antibody–drug conjugate (ADC) to target and treat meningiomas. The meningioma targeting, circulation stability, toxicity, and anti-tumor efficacy of SSTR2 ADC were evaluated using cell lines and/or an intracranial xenograft mouse model. The flow cytometry analysis showed that the anti-SSTR2 mAb had a high binding rate of >98% to meningioma CH157-MN cells but a low binding rate of 50 value of

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.

OBJECTIVES/SPECIFIC AIMS: (1) To delineate the function of the heavy-chain receptor binding domain (HCR), a portion of botulinum neurotoxin type A (BoNT/A) and synaptic vesicle 2 (SV2) signaling pathway, which provide a novel multipurpose biologic with potential clinical applications in tumor detection/imaging, inhibition of tumor progression, and reduction of bioactive hormone secretion in metastatic neuroendocrine (NE) cancers. (2) To evaluate the expression pattern of SV2 receptors in NE cancer patient-derived tissues for prediction of patient response for recombinant HCR (rHCR) treatment. (3) To assess the in vivo efficacy and toxicity of rHCR in a NE cancer liver metastasis mouse models and in the NE patient-derived 3D MicroTumor system. (4) To collect preclinical data to design and conduct a clinical trial with NE cancer patients, a major goal toward translating our discoveries into much needed therapies. METHODS/STUDY POPULATION: Recombinant botulinum heavy chain (rHCR) was produced using an IPTG-inducible expression vector in E. coli BL21. The rHCR was His-Tag purified and stored in PBS buffer before usage. Cytotoxicity: H727, TT, and MZ cells were plated at a density of 5000 cells/well in 96-well plates and incubated under standard conditions overnight. The next day, cells were treated with 10, 100, or 500 nmol/L of rHCR and incubated for 72 hours. Following incubation, cell viability was assessed by ATP quantification using the CellTiter-Glo (Promega) assay. Fresh NE tumors were dissociated and injected into polydimethylsiloxane bioreactors in a matrigel and collagen suspension for 3D culture experiments. The viability of 3D cultures incubated with various doses of rHCR was assessed by measuring the uptake of the near-infrared dye IR-783 using an IVIS imaging system. Western blot: H727, TT, and MZ cells were seeded in 6-well plates at a density of 3×105 cells/well for 24 hours followed by treatment with 100 nmol/L for 72 hours. Total cellular proteins were isolated and analyzed to assess the level of SV2A expression and the effect of rHCR on the expression levels of NET marker proteins. Immunohistochemistry: Deparaffinized tissue culture slides were incubated with SV2A primary antibody in 1% BSA and incubated overnight at 4°C. Slides were rinsed twice with TBS containing 0.025% Triton, followed by 0.3% H2O2 for 15 minutes. Slides were then incubated with HRP-conjugated secondary antibody for 1 hour at room temperature. Detection of protein-protein interaction: Precleared cell lysate was incubated with glutathione-agarose beads in the presence of 10 μg of GST-tagged rHCR for 2 h at 4°C with end-over end mixing. Samples were then centrifuged at 10,000 g for 2 minutes and the supernatant was analyzed by SDS-PAGE. Preclinical models: To allow rHCR testing on NET patient derived cells in a very novel 3D surrogates, sterile collected fresh NET tissues will be obtained from the UAB Tissue Procurement, dissociated into a single cell suspension and injected into a polydimethylsiloxane bioreactors containing extracellular matrix composed of bovine collagen and matrigel. Such 3D cell culture will be maintained in bioreactors with constant supply of media through the microchannels and treated with rHCR at concentrations ranging from 10 nM to 1 mM. Following histologic confirmation of growth and morphology of NET patient-derived 3D surrogates we will test the anticancer activity of rHCR in this system using the standard cytotoxicity assays as well as we will validate the NET hormone expression using immunohistochemistry assay. To create an animal model of NE cancer progression, we will perform intrasplenic injection of NET cell lines. In approximately 4 weeks, the animals should develop NE liver metastases based upon our previous experience. rHCR-iFPs accumulation in the tumor mass: rHCR-iFPs will be injected to the tumor bearing mice after 4 weeks of cells implantation in 1 week interval for total of 4 treatments at the concentrations of 0.125, 1.25, and 12.5 mg/kg. RESULTS/ANTICIPATED RESULTS: Based on the preliminary data, we expect to detect rHCR-iFPs in NE cancer xenografts and in patient derived 3D explants. Our preliminary data revealed that treating NETcells with rHCR significantly reduced NE peptide expression in 3 days. Thus, we expect to see the decrease of NE tumor markers even if the fluorescent detection method is not sensitive enough to monitor the signal. The reduction of the NET markers can be used as an indicator of the rHCR-iFPs uptake by the tumor mass. If HCR exhibit high binding affinity to SV2 receptors in NET models but moderate anticancer efficacy, we plan to use rHCR peptide to conjugate with the nanocarrier for targeted drug delivery. In this case rHCR peptide can be used as a ligand that specifically binds to NE cancer cells and delivers anticancer drug. In 3D NET patient derived explants we expect significant reduction of NET markers and hormones (serotonin and calcitonin) in NE cancer cells upon long-term rHCR-iFPs treatment. In addition, we will perform multiplex protein quantification assay using Luminex to assess the various hormones, cytokines, and growth factors to be repurposed into a diagnostic and detection reagent, or a drug delivery ligand for targeted therapies. DISCUSSION/SIGNIFICANCE OF IMPACT: NE cancers are highly metastatic: NE cancers such as carcinoid, islet cell tumors, and medullary thyroid cancer frequently metastasize to the liver. They are the second most prevalent GI malignancy. Ninety percent of patients with pancreatic carcinoid tumors and 50% of patients with islet cell tumors develop isolated hepatic metastases. Patients with untreated, isolated NE liver metastases have

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.