Review
Biotechnology & Applied Microbiology
Nicolas Marx, Peter Eisenhut, Marcus Weinguny, Gerald Klanert, Nicole Borth
Summary: Recent advances have greatly improved our understanding of Chinese hamster ovary (CHO) cells as hosts for producing biopharmaceuticals. Despite progress in characterizing CHO cells on different omics levels, our understanding of its epigenetic regulation is still incomplete. This review provides an overview of epigenetic regulation in CHO cells and discusses its significance in shaping cell phenotype. The review also presents current state-of-the-art technologies for manipulating epigenetic networks and controlling gene expression.
BIOTECHNOLOGY ADVANCES
(2022)
Article
Biochemical Research Methods
Madolyn L. MacDonald, Kelvin H. Lee
Summary: EvalDNA is a tool that utilizes supervised machine learning to score the quality of genome assemblies without the need for a reference genome. It has been successfully applied in various studies for evaluating human and bacterial assemblies, providing scientists with an easy way to identify the best genome assembly for their organism of interest.
BMC BIOINFORMATICS
(2021)
Article
Plant Sciences
Luca Marcolungo, Leonardo Vincenzi, Matteo Ballottari, Michela Cecchin, Emanuela Cosentino, Thomas Mignani, Antonina Limongi, Irene Ferraris, Matteo Orlandi, Marzia Rossato, Massimo Delledonne
Summary: High-throughput chromosome conformation capture (Hi-C) is commonly used in de novo assembly for its ability to produce highly contiguous genomes. However, its statistical approach can introduce connection errors. We used optical mapping to evaluate the quality of Hi-C reconstructed scaffolds and found numerous inconsistencies. Manual inspection confirmed that some of these conflicts were caused by Hi-C joining errors. We conclude that integrating optical mapping data after Hi-C-based scaffolding improves the assembly quality by identifying and investigating misjoins.
Article
Biochemistry & Molecular Biology
Raffaele Altamura, Jiten Doshi, Yaakov Benenson
Summary: Cell line development is crucial in biopharmaceutical manufacturing, but current protocols rely on random transgene integration. A novel strategy has been proposed to reduce screening effort, improve stability, and predict product titers through engineered multi-copy gene constructs.
NUCLEIC ACIDS RESEARCH
(2022)
Article
Engineering, Chemical
Beichen Zhao, Xueliang Li, Wanqiang Sun, Juntao Qian, Jin Liu, Minjie Gao, Xin Guan, Zhenwu Ma, Jianghua Li
Summary: The field of industrial biotechnology has shown a growing interest in using digital twins to improve process productivity and management efficiency. However, digital-twin-based biomanufacturing has not been fully implemented yet. As a preliminary step, an open-source digital twin framework for cell culture was developed, along with a web-based, cloud-native HMI application. A microbioreactor digital twin system was implemented using this framework, and its practical applicability was demonstrated through a case study involving the construction of real-time glucose and lactic acid soft sensors.
Article
Genetics & Heredity
Gangcai Xie, Xu Zhang, Feng Lv, Mengmeng Sang, Hairong Hu, Jinqiu Wang, Dong Liu
Summary: In this study, a high-quality reference genome for Trachidermus fasciatus was successfully assembled, containing 19,147 protein-coding genes and 35,093 mRNA transcripts. Additionally, a novel gene-finding strategy was introduced, leading to the identification of 51 new genes.
Article
Biochemistry & Molecular Biology
Stephanie H. Chen, Maurizio Rossetto, Marlien van der Merwe, Patricia Lu-Irving, Jia-Yee S. Yap, Herve Sauquet, Greg Bourke, Timothy G. Amos, Jason G. Bragg, Richard J. Edwards
Summary: This study presents the first chromosome-level genome of Telopea speciosissima, providing valuable insights into the speciation, introgression, and adaptation processes. The genome assembly is of high quality and the annotation revealed important genetic information, such as the presence of CYCLOIDEA genes and potential gene duplications. The T. speciosissima reference genome will contribute to the conservation efforts of Proteaceae plants in Australia and beyond.
MOLECULAR ECOLOGY RESOURCES
(2022)
Article
Oncology
Bo Ren, Jinshou Yang, Chengcheng Wang, Gang Yang, Huanyu Wang, Yuan Chen, Ruiyuan Xu, Xuning Fan, Lei You, Taiping Zhang, Yupei Zhao
Summary: The study found that in metastatic pancreatic cancer cells, A/B compartments, contact domains, and chromatin loops changed significantly, associated with epigenetic state alterations. Upregulated genes in metastatic pancreatic cancer cells were related to cancer metastasis and poor prognosis. Transcription factors in specific enhancer-promoter loop formation were also associated with metastasis.
JOURNAL OF HEMATOLOGY & ONCOLOGY
(2021)
Article
Biochemistry & Molecular Biology
Bingyan Li, Zhenyong Du, Li Tian, Limeng Zhang, Zhihua Huang, Shujun Wei, Fan Song, Wanzhi Cai, Yanbi Yu, Hailin Yang, Hu Li
Summary: The study constructed a high-quality reference genome of Aphidius gifuensis, revealing significantly expanded gene families involved in metabolic processes, cell signal communication, and other functions, along with contracted olfactory receptors. This provides a solid foundation for future research on the molecular mechanisms of population decline and host-parasitoid relationship for parasitoid wasps.
MOLECULAR ECOLOGY RESOURCES
(2021)
Article
Biochemistry & Molecular Biology
Grace Yao, Kathryn Aron, Michael Borys, Zhengjian Li, Girish Pendse, Kyongbum Lee
Summary: The research identified potential high productivity indicators in CHO cell clones, such as citrate, which improved qP by up to 490% when used as a medium additive. This demonstrates the potential of metabolomics in discovering novel metabolite additives for higher volumetric productivity in biologics production processes.
Article
Biotechnology & Applied Microbiology
Stephen Bevan, Stefan Schoenfelder, Robert J. Young, Lin Zhang, Simon Andrews, Peter Fraser, Peter M. O'Callaghan
Summary: Chinese hamster ovary (CHO) cell lines are crucial for the biopharmaceutical industry, but their unstable transgene expression poses challenges. This study presents a 3D genome map of CHOK1SV (R) 10E9 cell line and an improved genome assembly, enhancing understanding of the CHO genome organization. Our findings reveal higher order chromatin structures and assist in identifying regulatory elements, ultimately improving transgene integration and cell line development.
BIOTECHNOLOGY AND BIOENGINEERING
(2021)
Article
Biotechnology & Applied Microbiology
Thu M. Cao, Dayue Chen, Gavin C. Barnard, Amy Shen
Summary: This study explores the potential of using CHO cells for rAAV production and identifies cap protein expression as a limiting factor. By increasing cap protein expression, rAAV titers in CHO cells were comparable to those in HEK-derived rAAV, providing a foundation for potential rAAV production in CHO cells.
BIOTECHNOLOGY AND BIOENGINEERING
(2023)
Article
Microbiology
Ignat V. Sonets, Nikita V. Dovidchenko, Sergey V. Ulianov, Maria S. Yarina, Stanislav I. Koshechkin, Sergey V. Razin, Larissa M. Krasnopolskaya, Alexander V. Tyakht
Summary: The complete genome sequence of G. lucidum strain 5.1 was obtained using high-throughput shotgun sequencing. The improved assembly process resulted in 12 chromosome-level scaffolds. Comprehensive annotation of the genome identified genes likely involved in polysaccharide biosynthesis. This study is important for elucidating the medicinal potential of Ganoderma species, discovering novel pharmaceutically valuable compounds, and understanding fungal genomic organization.
Article
Biotechnology & Applied Microbiology
Benjamin Strain, James Morrissey, Athanasios Antonakoudis, Cleo Kontoravdi
Summary: This study evaluates the reliability of Chinese hamster ovary (CHO) cell metabolic models and introduces a new model that outperforms the existing ones in predicting extracellular phenotypes and intracellular fluxes. However, there is still room for improvement in predicting intracellular metabolic states. This work provides an updated CHO cell model and lays the foundation for further research in flux analysis techniques.
BIOTECHNOLOGY AND BIOENGINEERING
(2023)
Article
Biotechnology & Applied Microbiology
Eleftheria Skrika-Alexopoulos, C. Mark Smales
Summary: Exosomes were isolated from CHO cells using a novel method and characterized to determine their protein and RNA content. These findings contribute to the future research and improvement of exosome manufacturing from CHO cells.
BIOTECHNOLOGY LETTERS
(2023)
Article
Biotechnology & Applied Microbiology
Philipp N. Spahn, Xiaolin Zhang, Qing Hu, Huiming Lu, Nathaniel K. Hamaker, Hooman Hefzi, Shangzhong Li, Chih-Chung Kuo, Yingxiang Huang, Jamie C. Lee, Anthony J. Davis, Peter Ly, Kelvin H. Lee, Nathan E. Lewis
Summary: This study identified deleterious single-nucleotide variants in DNA repair genes in CHO cells, which contribute to productivity loss and genome instability. Correcting these DNA repair genes improves genome stability and protein expression in CHO cells.
BIOTECHNOLOGY AND BIOENGINEERING
(2022)
Article
Biotechnology & Applied Microbiology
Sumit K. Singh, Kelvin H. Lee
Summary: Glycosylation is a critical quality attribute of monoclonal antibody therapeutics. Hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) is a valuable technology for glycosylation characterization. Traditional glycan analysis based on glucose units (GU) and accurate mass (AM) is not suitable for complex samples or high-throughput applications. This study presents a two-step strategy for targeted glycan analysis and its application in different scenarios.
FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY
(2022)
Article
Biotechnology & Applied Microbiology
Nathaniel K. Hamaker, Lie Min, Kelvin H. Lee
Summary: The biomanufacturing industry is moving towards continuous processes, which may pose challenges due to fluctuations in host cell protein (HCP) levels. A study on Chinese hamster ovary (CHO) cells showed that HCP expression changes over time, but not as dramatically as originally expected.
BIOTECHNOLOGY AND BIOENGINEERING
(2022)
Review
Chemistry, Applied
Alana C. Szkodny, Kelvin H. Lee
Summary: This review highlights the key milestones in the production of biopharmaceuticals and emphasizes the maturity of the technologies used and the importance of understanding the interaction between product quality and processing conditions.
ANNUAL REVIEW OF CHEMICAL AND BIOMOLECULAR ENGINEERING
(2022)
Article
Biotechnology & Applied Microbiology
Sandeep B. Kedia, Jeffrey C. Baker, Ruben G. Carbonell, Kelvin H. Lee, Christopher J. Roberts, John Erickson, John E. Schiel, Kelley Rogers, Gene Schaefer, Stefanie Pluschkell
Summary: The Manufacturing Readiness Levels (MRLs) are well-established tools for describing technology maturity, and the Biomanufacturing Readiness Levels (BRLs) provide a specific viewpoint for the biomanufacturing industry.
BIOTECHNOLOGY AND BIOENGINEERING
(2022)
Article
Biotechnology & Applied Microbiology
Young Hoon Oh, Matthew L. Becker, Kerri M. Mendola, Leila H. Choe, Lie Min, Kelvin H. H. Lee, Yinges Yigzaw, Alexander Seay, Jerome Bill, Xuanwen Li, David J. Roush, Steven M. Cramer, Stefano Menegatti, Abraham M. Lenhoff
Summary: In the production of biopharmaceuticals, the presence of residual host-cell proteins (HCPs) is a critical quality concern. Measurements on seven monoclonal antibody (mAb) processes revealed that HCP aggregates, not necessarily derived from chromatin, are important factors in the persistence of HCPs. These aggregates can also interfere with accurate detection of HCPs using proteomics methods. The study also highlights the difficulty in removing certain HCPs due to their functional complementarity to the biopharmaceutical product.
BIOTECHNOLOGY AND BIOENGINEERING
(2023)
Article
Biotechnology & Applied Microbiology
Lauren T. Cordova, Hussain Dahodwala, Kathryn S. Elliott, Jongyoun Baik, Daniel C. Odenewelder, Douglas Nmagu, Bradley A. Skelton, Lisa Uy, Stephanie R. Klaubert, Benjamin F. Synoground, Dylan G. Chitwood, Venkata Gayatri Dhara, Harnish Mukesh Naik, Caitlin S. Morris, Seongkyu Yoon, Michael Betenbaugh, Jon Coffman, Frank Swartzwelder, Michael Gilmeister, Sarah W. Harcum, Kelvin H. Lee
Summary: Due to the limitations in cell, media, and feed diversity, as well as the limited public availability of industrially relevant CHO cells, the knowledge translation and research progress in the field have been hindered. To address this issue, AMBIC has acquired two CHO reference cell lines from different lineages and developed detailed technology transfer protocols and a chemically defined media formulation, aiming to provide a universal and industrially relevant CHO culture platform.
BIOTECHNOLOGY AND BIOENGINEERING
(2023)
Article
Biotechnology & Applied Microbiology
Nathaniel. K. K. Hamaker, Kelvin. H. H. Lee
Summary: Efforts to improve CRISPR/Cas9 efficiency in targeted genomic modifications in mammalian cells led to the development of an all-in-one reporter system, including a new superfolder orange fluorescent protein (sfOrange), to quantify gene disruption, site-specific integration (SSI), and random integration (RI). Different donor plasmid formats and Cas9 nuclease variants were evaluated for accuracy and efficiency in Chinese hamster ovary cells. Double-cut and double-nick donor formats significantly improved targeting accuracy, and Cas9-mediated donor linearization increased RI events while donor nicking minimized RI without sacrificing SSI efficiency.
BIOTECHNOLOGY AND BIOENGINEERING
(2023)
Review
Biotechnology & Applied Microbiology
Gene Schaefer, John Balchunas, Timothy Charlebois, John Erickson, Roger Hart, Sandeep B. Kedia, Kelvin H. Lee
Summary: The challenge of introducing new technologies into established industries is not unique to biopharmaceuticals, but it is crucial for the long-term competitiveness and delivery of therapies, particularly for new treatment modalities such as cell and gene therapies. This review identifies barriers to technology adoption and proposes enablers and suggestions for synergy and connection between innovation in product discovery and manufacturing, and across supplier, discovery, manufacturing, and regulatory arms.
BIOTECHNOLOGY AND BIOENGINEERING
(2023)
Article
Biotechnology & Applied Microbiology
William Hilliard, Kelvin H. Lee
Summary: The use of targeted integration for industrial CHO cell line development requires significant upfront effort to identify genomic loci capable of supporting high therapeutic protein production. To address this barrier, the Transgene Integrated in Parallel screening method was used to characterize transgene expression from thousands of stable hotspots in the CHO genome. Cell lines with integrations at retargeted hotspot candidates consistently exhibited higher transgene mRNA expression than a commercially viable hotspot. These findings provide valuable resources for targeted integration platform development in the CHO community.
BIOTECHNOLOGY AND BIOENGINEERING
(2023)
Article
Biotechnology & Applied Microbiology
Chase E. Herman, Lie Min, Leila H. Choe, Ronald W. Maurer, Xuankuo Xu, Sanchayita Ghose, Kelvin H. H. Lee, Abraham M. Lenhoff
Summary: Recent research suggests that host-cell proteins (HCPs) and high molecular weight (HMW) species in monoclonal antibody downstream processing may be linked. Proteomic analysis reveals substantial overlap of HCP profiles between harvest cell culture fluid (HCCF) and protein A eluate, with HCPs conserved in aggregates that persist through the protein A capture step. These findings support previous hypotheses and highlight the importance of this persistence mechanism.
BIOTECHNOLOGY PROGRESS
(2023)
Article
Biotechnology & Applied Microbiology
Young Hoon Oh, Kerri M. Mendola, Leila H. Choe, Lie Min, Ashton R. Lavoie, Sobhana A. Sripada, Taufika Islam Williams, Kelvin H. Lee, Yinges Yigzaw, Alexander Seay, Jerome Bill, Xuanwen Li, David J. Roush, Steven M. Cramer, Stefano Menegatti, Abraham M. Lenhoff
Summary: This study investigates the characterization and persistence of host-cell proteins (HCPs) in downstream processing steps for monoclonal antibody (mAb) manufacturing. The results show variability in HCP identities during processing steps, but consistency in the most abundant HCPs across different processes. The abundance of HCPs is likely related to the reproducibility of quantification measurements, and certain groups of HCPs may hinder characterization. The study suggests that factors such as HCP abundance and mAb-HCP interactions contribute to the persistence of individual HCPs and the identification of common, persistent HCPs in mAb manufacturing.
BIOTECHNOLOGY AND BIOENGINEERING
(2023)
Article
Biotechnology & Applied Microbiology
Qiang Fu, Yong Suk Lee, Erica A. Green, Yongdan Wang, So Young Park, Ashli Polanco, Kelvin H. Lee, Michael Betenbaugh, David McNally, Seongkyu Yoon
Summary: This study investigated the influence of four process parameters on the quality attributes of rAAV products. Regression models and a design space were established, and the predictive capability of the models was confirmed.
BIOTECHNOLOGY AND BIOENGINEERING
(2023)
Article
Biochemical Research Methods
Chase E. Herman, Lie Min, Leila H. Choe, Ronald W. Maurer, Xuankuo Xu, Sanchayita Ghose, Kelvin H. Lee, Abraham M. Lenhoff
Summary: Recent research has revealed that aggregates in mAb solutions may contain not only mAb oligomers but also hundreds of host-cell proteins (HCPs), which suggests a relationship between aggregate persistence and HCP clearance during downstream purification operations. This study found that the phenomenon is relevant to depth filtration, protein A chromatography, and flow-through anion-exchange (AEX) polishing. Confocal laser scanning microscopy showed that aggregates compete with mAb during protein A chromatography, and column chromatography confirmed the presence of high aggregate concentrations in the protein A elution tail. Similar observations were made in flow-through AEX chromatography, with aggregate retention depending on resin surface chemistry. The quantification of aggregate mass fraction may serve as a surrogate for informing early process development decisions regarding HCP clearance strategies.
JOURNAL OF CHROMATOGRAPHY A
(2023)
Article
Biochemical Research Methods
Lauren T. Cordova, Hussain Dahodwala, Rebecca Cooley, Kelvin H. Lee
Summary: In this study, researchers evaluated the expression of five DNA repair genes in over 40 CHO cell lines and found a correlation between two genes and cell line stability. Additionally, they found a negative correlation between the expression levels of DNA repair genes and cell age.
BIOTECHNOLOGY JOURNAL
(2023)
