About

Professor Michael Betenbaugh leads the Betenbaugh Group at Johns Hopkins University, focusing on research in Sustainable Biomanufacturing and Synthetic Biology. His laboratory specializes in a diverse range of areas including Mammalian Biomanufacturing, AAV optimization, Genetic Stability of CHO cells, Machine Learning and Modeling, CRISPR technologies, Vaccine Analytics, Microbial Engineering, Ammonia Biomanufacturing, Living Wall Bio-leaching, Waste Recovery and Circular Economy, as well as Cellular Agriculture. The group integrates computational modeling approaches, such as those applied to recombinant AAV production, to advance their research objectives. Located at Maryland Hall 25 in Baltimore, Maryland, the Betenbaugh Lab is engaged in training postdoctoral researchers, master’s students, and undergraduates, contributing to the development of expertise in biomanufacturing and synthetic biology fields.

Research topics

• Biology
• Virology
• Medicine
• Immunology
• Pathology
• Computational biology
• Engineering
• Cell biology
• Internal medicine
• Chemistry
• Biochemical engineering
• Genetics
• Biotechnology
• Biochemistry

Selected publications

• A continuous viral vaccine biomanufacturing platform utilizing multiple bioreactor configurations

  Journal of Biological Engineering · 2026-04-24

  articleOpen accessSenior authorCorresponding

  Scalable, continuous biomanufacturing processes have grown in importance to meet demand for smaller bioreactor sizes, lowered production costs, and improved quality attribute consistency. The Sf9/recombinant baculovirus (rBV) expression system demonstrates promise for virus-like particle (VLP) vaccine and gene therapy production. Here, we present a continuous rBV platform integrating an infection plug flow reactor (PFR) between stirred tank growth (gCSTR) and production (pCSTR) bioreactors. Cell expansion in the gCSTR included a ramp-up stage followed by continuous growth, reaching a steady state of 5$$\times$$106 cells/mL and >90% viability. Péclet number-fit tracer studies confirmed near-ideal plug flow in the PFR, yielding a 10 h residence time and progressive infection as measured by gp64 signaling. Finally, a pCSTR with a residence time of 48 h exhibited sustained recombinant protein production. An integrated pilot cascade incorporating all reactors ran continuously for 5 days, maintaining stable CSTR cell densities and a measurable increase in infected cell diameter from 14.5 μm to 16.1 μm. Western blotting and EM of ~ 100 nm VLPs in pCSTR effluent demonstrated platform success. Digital twin mechanistic models across four distinct stages of bioreactor operation and Hill-type relationships for rBV infection kinetics predicted cell growth and death for a 7-day run, demonstrating promise for designing continuous systems in silico and building a quantitative framework for scale-up and optimization. Our multi-stage reactor configuration represents a cell host- and product-agnostic production scheme, particularly for processes prone to product heterogeneity, and paves the way towards a true end-to-end continuous platform for myriad modalities in the future.

  PublisherDOI

• Continuous purification of a parvovirus using two aqueous two‐phase extraction steps

  Biotechnology Progress · 2025-04-15 · 3 citations

  articleOpen access

  Aqueous two-phase systems (ATPS) are a liquid-liquid extraction method that offers low-cost, continuous-adaptable virus purification. A two-step ATPS using polyethylene glycol (PEG) and sodium citrate that recovered 66% of infectious porcine parvovirus with 2.0 logs of protein removal and 1.0 logs of DNA removal in batch has now been run continuously. The continuous system output of <10 ng/mL DNA regardless of starting DNA titer agreed with batch studies. However, the continuous system had a five-fold higher contaminating protein titer than batch studies, likely because of incomplete mixing or settling. Turbidity was tested as a measure of mixing and settling efficiency. Monitoring in-line absorbance at 880 nm directly after mixing and before collection in the settling reservoir could track both mixing and settling during operation. Settling time was reduced by changing the settling line material from PVC to PTFE, which is more hydrophobic. A flow-through AEX filter tested to make impurity removal more robust recovered 90% of PPV and removed an additional 87% of host cell DNA. The filter did not add any additional protein removal. In the future, in-line absorbance sensors could be implemented along with conductivity sensors to measure salt concentration, refractive index sensors to track the PEG-citrate interface, and scales to track mixer and reservoir volumes to enable automated, continuous ATPS. Our vision is to integrate continuous ATPS into a fully continuous end-to-end production for viral vectors.

  PublisherOA PDFDOI

• Correction: Polyhydroxyalkanoate production by Paramecium caudatum isolated from industrial wastewater: a micro eukaryotic host for bioplastic accumulation

  Biotechnology Letters · 2025-09-22

  erratumOpen access
  PublisherOA PDFDOI

• Unraveling Cytotoxicity in HEK293 Cells During Recombinant AAV Production for Gene Therapy Applications

  Biotechnology Journal · 2025-03-01 · 5 citations

  articleSenior authorCorresponding

  Transient transfection of HEK293 cells represents the dominant technique for the production of recombinant adeno-associated virus (AAV) vectors. However, recombinant AAV (rAAV) production is cytotoxic, potentially impacting process performance, product yields, and quality, complicating downstream processing. This study characterizes cell death response for rAAV producing HEK293 cells and explores the potential to control cytotoxicity. Initial analysis of triple transfected cells revealed caspase-mediated apoptosis as a likely mechanism of cellular death. Next, the causes of this cytotoxicity were investigated by dissecting transfection steps. Exposing cells to polyethyleneimine (PEI) alone or complexed with a blank plasmid at typical concentrations had a limited impact on cell growth. However, the inclusion of plasmid constructs containing genes to produce rAAVs triggered significant cell death, with the helper plasmid being the most toxic both independently and in combination with packaging and transgene plasmids. Additionally, apoptosis in transfected cultures could be inhibited using the pan-caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD.fmk), leading to a 65% increase in peak viable cell density (VCD). Although the rAAV genome titer remained relatively unaltered, capsid levels declined upon cell death inhibition. Consequently, the ratio of full to empty capsids, an important product quality attribute (PQA) for rAAVs increased following caspase inhibition. This study provides insights into apoptosis activation in rAAVs and uncovers avenues for its modulation to alter PQAs.

  PublisherDOI

• Polyhydroxyalkanoate production by Paramecium caudatum isolated from industrial wastewater: a micro eukaryotic host for bioplastic accumulation

  Biotechnology Letters · 2025-08-01 · 4 citations

  article
  PublisherDOI

• Deep Learning-Powered Colloidal Digital SERS for Precise Monitoring of Cell Culture Media

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-02-04 · 1 citations

  preprintOpen access

  Maintaining consistent quality in biopharmaceutical manufacturing is essential for producing high-quality complex biologics. Yet, current process analytical technologies (PAT) struggle to achieve rapid and highly accurate monitoring of small molecule critical process parameters and critical quality attributes. While Raman spectroscopy holds great promise as a highly sensitive and specific bioanalytical tool for PAT applications, its conventional implementation, surface-enhanced Raman spectroscopy (SERS), is constrained by considerable temporal and spatial intensity fluctuations, limiting the achievable reproducibility and reliability. Herein, we introduce a deep learning-powered colloidal digital SERS platform to address these limitations. Rather than addressing the intensity fluctuations, the approach leverages their very stochastic nature, arising from highly dynamic analyte-nanoparticle interactions. By converting the temporally fluctuating SERS intensities into digital binary "ON/OFF" signals using a predefined intensity threshold by analyzing the characteristic SERS peak, this approach enables digital visualization of single-molecule events and significantly reduces false positives and background interferences. By further integrating colloidal digital SERS with deep learning, the applicability of this platform is significantly expanded and enables detection of a broad range of analytes, unlimited by the lack of characteristic SERS peaks for certain analytes. We further implement this approach for studying AMBIC 1.1, a chemically-defined, serum-free complete media for mammalian cell culture. The obtained highly accurate and reproducible results demonstrate the unique capabilities of this platform for rapid and precise cell culture media monitoring, paving the way for its widespread adoption and scaling up as a new PAT tool in biopharmaceutical manufacturing and biomedical diagnostics.

  PublisherDOI

• A community-consensus reconstruction of Chinese Hamster metabolism enables structural systems biology analyses to decipher metabolic rewiring in lactate-free CHO cells

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-04-17 · 3 citations

  preprint

  Abstract Genome-scale metabolic models (GEMs) are indispensable for studying and engineering cellular metabolism. Here, we present i CHO3K, a community-consensus, manually-curated reconstruction of the Chinese Hamster metabolic network. In addition to accounting for 11004 reactions associated with 3597 genes, i CHO3K includes 3489 protein structures and structural descriptors for &gt;70% of its 7377 metabolites, enabling deeper exploration of the link between molecular structure and cellular metabolism. We used i CHO3K to contextualize transcriptomics and metabolomics data from a CHO cell line in which lactate secretion is abolished. We found the reduced glycolytic flux and enhanced TCA cycle flux were accompanied by an elevated NADH and PEP levels in these cells, consistent with experimental measurements. Leveraging i CHO3K’s structural annotations, we identified candidate binding interactions of NADH and PEP with glycolytic enzymes showing model-predicted differential flux, suggesting novel allosteric regulation associated with the observed decrease in glucose uptake and glycolysis. Overall, i CHO3K offers a valuable framework for systematic integration of omics data, improved flux predictions, and structure-guided insights, thus advancing CHO cell engineering and enhancing biomanufacturing efficiency.

  PublisherDOI

• Comparative omics profiling reveals differences in biomass, energy production, and vesicle transport between CHO and fast-growing CHL-YN cells

  Scientific Reports · 2025-11-13

  articleOpen access

  Chinese hamster lung (CHL)-YN cells are promising novel hosts for producing therapeutic antibodies with the potential to shorten the research, development, and manufacturing timelines in biopharmaceutical production. CHL-YN cells grow twice as fast as Chinese hamster ovary (CHO) cells, with a doubling time of 8.1 h. These cells possess strong glutamine synthetase activity, allowing them to be cultured in glutamine-free media. In this study, we conducted comparative transcriptomics and proteomics among CHL-YN cells, CHO cells, and lung tissue from Chinese hamster to better understand the global characteristics of CHL-YN cells and determine whether these features are tissue-derived or unique to the cell line. Omics profiling revealed that CHL-YN cells, in contrast to CHO cells, exhibit highly activated processes and pathways related to biomass and energy production, such as translation and biosynthesis of amino acids, but less activated vesicle transport processes, such as Golgi-related vesicle transport. This study highlights distinct characteristics of CHL-YN cells, contributing to streamlined operations, shortened development timelines, and expanded options for selectable cell lines. The findings here could contribute to identifying potential biomarkers and targets for cell engineering toward improving antibody productivity and growth rate in both traditional CHO cells and next-generation host cells, CHL-YN.

  PublisherDOI

• Investigating subpopulation dynamics in clonal CHO-K1 cells with single-cell RNA sequencing

  Journal of Biotechnology · 2025-01-15 · 3 citations

  article
  PublisherDOI

• Integration of Bayesian optimization and solution thermodynamics to optimize media design for mammalian biomanufacturing

  iScience · 2025-07-03 · 2 citations

  articleOpen accessSenior author

  Rapid, cost-effective biomanufacturing of products like therapeutics, materials, and lab-grown foods depends on optimizing cell culture media, a complex and expensive task due to the combination of components and processing variables. This is especially important for therapeutic production using mammalian systems like Chinese Hamster Ovary (CHO) cells, where long development timelines contribute to high drug costs. Using Bayesian optimization (BO), adapted for bioprocess applications, our method supports multiple parallel experiments and incorporates thermodynamics-based constraints on media solubility to ensure feasible medium formulations. The approach is validated both in-silico and in experimental bioreactor settings, showing improved product titers compared to classical design of experiments (DOE) methods. This work bridges machine learning and physical modeling to create a more data-efficient process optimization strategy. The integration of this method into biomanufacturing pipelines together with robotics-assisted bioreactors paves the way for automated bioprocess optimization and more rapidly available and affordable biotherapeutics.

  PublisherDOI

Recent grants

• EFRI-CBE: An Integrated Computational and Experimental Model for Biochemical and Electrical Interactions in Ion Channels and the Impact of Sialic Acid on Neuronal Function

  NSF · $2.0M · 2007–2012

• Evolving membrane proteins for high-level expression in modified mammalian cells

  NIH · $1.2M · 2010–2015

• IUCRC Phase II+: Johns Hopkins University: Advanced Mammalian Biomanufacturing Innovation Center (AMBIC)

  NSF · $1.6M · 2021–2026

• Collaborative Research: GOALI: Exploiting metabolism-apoptosis interactions to enhance mammalian cell culture

  NSF · $101k · 2011–2014

• Collaborative Research: Synthetic Lichen Co-Cultures for Sustainable Generation of Biotechnology Products

  NSF · $315k · 2018–2025

Frequent coauthors

• Joseph Shiloach

  48 shared

• Julian N. Rosenberg

  Johns Hopkins University

  33 shared

• Nathan E. Lewis

  University of California, San Diego

  29 shared

• George A. Oyler

  28 shared

• Aliaksandr Druz

  National Institute of Allergy and Infectious Diseases

  24 shared

• Andrew Pekosz

  Johns Hopkins University

  22 shared

• Chia Chu

  Pfizer (United States)

  22 shared

• Pavlo Bohutskyi

  21 shared

Education

• Ph.D., Chemical Engineering

  University of Delaware

  1988

• B.S., Chemical Engineering

  University of Virginia

  1981

Awards & honors

• D.I.C. Wang Award for Excellence in Biochemical Engineering…
• Marvin J. Johnson Award from the American Chemical Society’s…
• James Van Lanen Award from the American Chemical Society’s D…
• Cell Culture Engineering Award (2010)
• Fellow of the American Institute for Medical and Biological…