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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.

A consolidated bioprocess for cellulosic n-butanol production has been developed by engineering Clostridium cellulovorans to overexpress a bifunctional aldehyde/alcohol dehydrogenase. Rational metabolic engineering is important to further improve butanol production. This study aimed to investigate intracellular metabolism and identify the key regulators of cellulosic butanol formation in C. cellulovorans via integrated Omics and fermentation kinetics data analysis. First, comparative proteomics and metabolomics analyses of wild type and n-butanol producing mutant strain were conducted, which quantified 624 host cell proteins and 474 primary and secondary metabolites. Compared to wild type, most cellulases in cellulolysis were up-regulated, but three glycolysis enzymes and three enzymes in central pathway were down-regulated in the n-butanol producing strain. Second, a dynamic model integrating Omics and fermentation data was developed to identify key regulators in butanol biosynthesis, which were ranked by further metabolic control analysis. Finally, rational metabolic engineering was performed in C. cellulovorans by overexpressing two genes (thl and hbd) identified as important factors limiting butanol biosynthesis, which improved butanol yield and C4/C2 ratio. This study demonstrated a research approach to integrate multi-Omics and fermentation data of C. cellulovorans and guide its rational metabolic engineering, which can also be applied to other microorganisms.

Introduction: Improvements in stem cell viability, differentiation efficiency, and functional activity opened the door for advanced research applications in cardiac regeneration. However, the use of cell-based cardiac therapies, including implantable patches and cell products as well as in-vitro models for mechanistic studies and drug development requires large numbers of high-quality cardiomyocytes (CMs). Typically, differentiation is conducted in 6-well culture plates via the GiWi method, which uses small-molecule inhibitors of glycogen synthase kinase (GSK) and tankyrase to first activate and then suppress Wnt signaling. This monolayer method requires significant manual labor and cannot be easily scaled. Suspension culture and differentiation of free-floating hiPSC aggregates following similar biochemical cues offers advantages for commercial biomanufacturing purposes and provides a more physiological environment for cell development. Hypothesis: We assessed the hypothesis that when hiPSCs are cultured and differentiated in suspension they will produce CMs with improved yield, purity, and quality. Methods: hiPSCs were expanded as aggregates in culture flask using a fed-batch approach then differentiated over a 12 day period with Wnt activation on day 0 and inhibition on day 3. Optimization studies examined purity on day 9 and complete characterization via morphological assessments and CM-specific genes expression was done on day 12 following metabolic purification. All studies were conducted with a minimum of 4 replicates. Results: Optimal conditions for both purity and yield based on cardiac troponin T staining were achieved with an initial cell density of 1.6x10 6 cells/mL, 6 μM CHIR99021, and 55 rpm shaking. After scaling to 30mL culture volume the purity of hiPSC-CMs differentiated via our novel protocol was 98.2±0.8% with yields of 1.47±0.18 million cells/mL and less between-batch purity variability than hiPSC-CMs produced in 2D cultures. Conclusions: Suspension differentiation of hiPSCs into CMs in a widely available format results in improved biomanufacturing endpoints. Therefore, this method functions as a groundwork for future bioreactor systems to produce the large number of cells needed for clinical applications.

The optimization of cell culture modes and basal media is very important to improve the production of therapeutic monoclonal antibodies (mAbs) by Chinese hamster ovary (CHO) cells, but the underlying cellular mechanism has not been well investigated. The objective of this study was to elucidate the interaction between the intracellular proteome and key process parameters by employing comparative proteomics. Three cell culture operations and four basal media were characterized in the production of anti-HER2 mAb using CHO DG44. The representative host cell proteins that demonstrated significant responses to various culture modes and media, including the enzymes involved in carbohydrate metabolism, transcription, translation, and post translational modification, were described. The fed-batch culture in a 2-L bioreactor produced mAb with a titer of 2411 mg/L using Dynamis medium. The purified mAb showed strong and specific targeting to HER2+ MDA-MB-361 cell and similar anti-cancer toxicity to the FDA approved Trastuzumab. Moreover, this proteomics study also indicated several key considerations for future rationally engineering a mAb production process, which could benefit biopharmaceutical manufacturing.

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 &gt, 98% to meningioma CH157-MN cells but a low binding rate of &lt, 5% to the normal arachnoidal AC07 cells. The In Vivo Imaging System (IVIS) imaging demonstrated that the Cy5.5-labeled ADC targeted and accumulated in meningioma xenograft but not in normal organs. The pharmacokinetics study and histological analysis confirmed the stability and minimal toxicity. In vitro anti-cancer cytotoxicity indicated a high potency of ADC with an IC50 value of &lt, 10 nM. In vivo anti-tumor efficacy showed that the anti-SSTR2 ADC with doses of 8 and 16 mg/kg body weight effectively inhibited tumor growth. This study demonstrated that the anti-SSTR2 ADC can target meningioma and reduce the tumor growth.

Neuroendocrine (NE) tumors include a diverse spectrum of hormone-secreting neoplasms that arise from the endocrine and nervous systems. Current chemo- and radio-therapies have marginal curative benefits. The goal of this study was to develop an innovative antibody-drug conjugate (ADC) to effectively treat NE tumors (NETs). First, we confirmed that somatostatin receptor 2 (SSTR2) is an ideal cancer cell surface target by analyzing 38 patient-derived NET tissues, 33 normal organs, and three NET cell lines. Then, we developed a new monoclonal antibody (mAb, IgG1, and kappa) to target two extracellular domains of SSTR2, which showed strong and specific surface binding to NETs. The ADC was constructed by conjugating the anti-SSTR2 mAb and antimitotic monomethyl auristatin E. In vitro evaluations indicated that the ADC can effectively bind, internalize, release payload, and kill NET cells. Finally, the ADC was evaluated in vivo using a NET xenograft mouse model to assess cancer-specific targeting, tolerated dosage, pharmacokinetics, and antitumor efficacy. The anti-SSTR2 ADC exclusively targeted and killed NET cells with minimal toxicity and high stability in vivo. This study demonstrates that the anti-SSTR2 ADC has a high-therapeutic potential for NET therapy.

The cellulolytic Clostridium cellulovorans has been engineered to produce n -butanol from low-value lignocellulosic biomass by consolidated bioprocessing (CBP). The objective of this study was to establish a robust cellulosic biobutanol production process using a metabolically engineered C. cellulovorans . First, various methods for the pretreatment of four different corn-based residues, including corn cob, corn husk, corn fiber, and corn bran, were investigated. The results showed that better cell growth and a higher concentration of n -butanol were produced from corn cob that was pretreated with sodium hydroxide. Second, the effects of different carbon sources (glucose, cellulose and corn cob), basal media and culture pH values on butanol production were evaluated in the fermentations performed in 2-L bioreactors to identify the optimal CBP conditions. Finally, the engineered C. cellulovorans produced butanol with final concentration >3 g/L, yield >0.14 g/g, and selectivity >3 g/g from pretreated corn cob at pH 6.5 in CBP. This study showed that the fermentation process engineering of C. cellulovorans enabled a high butanol production directly from agricultural residues.

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.

Neuroendocrine (NE) cancers include a diverse spectrum of hormone-secreting neoplasms that arise from the endocrine and nervous systems. Current chemo- and radio- therapies have marginal curative benefits. This study aimed to develop an innovative antibody-drug conjugate (ADC) to effectively treat NE tumors (NETs). We first confirmed that somatostatin receptor 2 (SSTR2) is an ideal surface target by analyzing 38 patient-derived NET tissues, 33 normal organs, and 3 NET cell lines. We then developed a new monoclonal antibody (mAb, IgG1 and kappa) to target two extracellular domains of SSTR2, which showed strong and specific surface binding to NETs. The ADC was constructed by conjugating the anti-SSTR2 mAb and antimitotic monomethyl auristatin E. In vitro evaluations indicated that the ADC can effectively bind, internalize, release payload, and kill NET cells effectively. Finally, the ADC was evaluated in vivo using a NET xenografted mouse model to determine cancer targeting, maximal tolerated dosage, pharmacokinetics, and anti-cancer efficacy. The anti-SSTR2 ADC was able to exclusively target and kill NETs with minimal toxicity and high stability in vivo. This study demonstrates that the anti-SSTR2 mAb-based ADC has high therapeutic values for NET therapy.

Background As patient-derived xenografts and other preclinical models of neuroendocrine tumors for testing personalized therapeutics are lacking, we have developed a perfused, 3D bioreactor model to culture tumor surrogates from patient-derived neuroendocrine tumors. This work evaluates the duration of surrogate culture and surrogate response to a novel antibody-drug conjugate. Methods Twenty-seven patient-derived neuroendocrine tumors were cultured. Histologic sections of a pancreatic neuroendocrine tumor xenograft (BON-1) tumor were assessed for SSTR2 expression before tumor implantation into 2 bioreactors. One surrogate was treated with an antibody-drug conjugate composed of an anti-mitotic Monomethyl auristatin-E linked to a somatostatin receptor 2 antibody. Viability and therapeutic response were assessed by pre-imaging incubation with IR-783 and the RealTime-Glo AnnexinV Apoptosis and Necrosis Assay (Promega Corporation, Madison, WI) over 6 days. A primary human pancreatic neuroendocrine tumor was evaluated similarly. Results Mean surrogate growth duration was 34.8 days. Treated BON-1 surrogates exhibited less proliferation (1.2 vs 1.9-fold) and greater apoptosis (1.5 vs 1.1-fold) than controls, whereas treated patient-derived neuroendocrine tumor bioreactors exhibited greater degrees of apoptosis (13- vs 9-fold) and necrosis (2.5- vs 1.6-fold). Conclusion Patient-derived neuroendocrine tumor surrogates can be cultured reliably within the bioreactor. This model can be used to evaluate the efficacy of antibody-guided chemotherapy ex vivo and may be useful for predicting clinical responses.

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 Chinese Hamster Ovary (CHO K1) cell was used to express a targeted anti-cancer monoclonal antibody by optimizing the platform of the construction of production cell line in this study. The adherent CHO K1 was first adapted to suspension culture in chemical defined medium. Then the glutamine synthetase (GS) vector was applied to construct a single plasmid to overexpress a monoclonal antibody IgG1. Post transfection, the production of cell pool was optimized by glutamine-free selection and amplification using various concentrations of methionine sulfoximine. The best cell pool of CHO K1/IgG1 was used to screen the top single clone using the limiting dilution cloning. Finally, a high IgG1 production of 780 mg/L was obtained from a batch culture. This study demonstrated that the construction of high producing cell line, from gene to clone, could be completed within six month and the gene amplification improved protein production greatly.

The objective of this study was to improve the production of butyric acid by process optimization using the metabolically engineered mutant of Clostridium tyrobutyricum (PAK-Em). First, the free-cell fermentation at pH 6.0 produced butyric acid with concentration of 38.44 g/L and yield of 0.42 g/g. Second, the immobilizedcell fermentations using fibrous-bed bioreactor (FBB) were run at pHs of 5.0, 5.5, 6.0, 6.5 and 7.0 to optimize fermentation process and improve the butyric acid production. It was found that the highest titer of butyric acid, 63.02 g/L, was achieved at pH 6.5. Finally, the metabolic flux balance analysis was performed to investigate the carbon rebalance in C. tyrobutyricum. The results show both gene manipulation and fermentation pH change redistribute carbon between biomass, acetic acid and butyric acid. This study demonstrated that high butyric acid production could be obtained by integrating metabolic engineering and fermentation process optimization. Open image in new window

Butanol is a promising biofuel with high energy intensity and can be used as gasoline substitute. It can be produced as a sustainable energy by microorganisms (such as Clostridia) from low-value biomass. However, the low productivity, yield and selectivity in butanol fermentation are still big challenges due to the lack of an efficient butanol-producing host strain. In this article, we systematically review the host cell engineering of Clostridia, focusing on (1) various strategies to rebalance metabolic flux to achieve a high butanol production by regulating the metabolism of carbon, redox or energy, (2) the challenges in pathway manipulation, and (3) the application of proteomics technology to understand the intracellular metabolism. In addition, the process engineering is also briefly described. The objective of this review is to summarize the previous research achievements in the metabolic engineering of Clostridium and provide guidance for future novel strain construction to effectively produce butanol. Open image in new window

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.

Aims Currently, there is no direct method for detecting Clostridium thermocellum in the insoluble medium. In this study, a quantitative real-time PCR assay was developed for the direct growth detection of C. thermocellum at the single-cell level in lignocellulosic biomasses. Methods and Results The assay targeted the cipA gene and was able to distinguish C. thermocellum from other species with good reproducibility which quantitative detection limit was 10 cell equivalents (CE) per reaction. OD600-based counting and qPCR quantification of C. thermocellum cultured in soluble medium were compared and an excellent consistency was revealed, indicating the appropriateness of the developed qPCR method. Analysis based on yellow affinity substrate and fermentation products may incorrectly estimate its population. Conclusions The developed assay can serve as a specific, sensitive and reproducible method for the detection of C. thermocellum in lignocellulosic biomass at the single-cell level. Significance and Impact of the Study With the ability to rapidly detect C. thermocellum, this method will contribute substantially to the understanding of the lignocellulosic biomass degradation mechanism. Moreover, it can also be applied to detect C. thermocellum growth in certain co-culture system for the understanding of the metabolic interactions.