Evidence map›Paper›PMID 40197832›Full record

ArticleBiotechnology and bioengineering2025

Development of an HEK293 Suspension Cell Culture Medium, Transient Transfection Optimization Workflow, and Analytics for Batch rAAV Manufacturing.

Erica A Green, Qiang Fu, Nelson Ndahiro, Thomas M Leibiger, Yongdan Wang, Yongsuk Lee, Kelvin H Lee, Michael Betenbaugh, Seongkyu Yoon, David J McNally

Abstract read
PubMed Full text at PMC Publisher
In one paragraph

Article in Biotechnology and bioengineering, 2025. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 5 papers.

0numbers the graph read from it
0cells of the map it votes in
5citing papers in PubMed
–field-weighted citation impact
1 · What the graph read from it

What it found

Each row is one number read from the abstract, on the scale the paper reported it, with its interval. Left of the dashed line favours the treatment, right favours the comparator. Under each row is the sentence it came from. New to these charts? A ten-minute tutorial.

The abstract states no effect estimate the extractor could read, or names no intervention and outcome on the map, so this paper lights no cell and moves no belief. It is still indexed, cited and linked below.

2 · The registry

The trial behind it

Trials whose registry record cites this paper, or whose number appears in the abstract. A trial that started after this paper was published is citing it as background, not reporting it.

Neither the registry nor the abstract names a trial number. If this is a trial report, that itself is worth knowing.

3 · Its place in the literature

Who cites it

5 citing papers in PubMed.

  1. Article
  2. Review
  3. Article
  4. Article
  5. Article
4 · The record

Corrections and comments

PubMed lists nothing against this paper. Absence here is not a guarantee, only a check that was made.

5 · Who and what money

Authors and funding

10 authors.

Erica A GreenDepartment of Chemical and Biomolecular Engineering, University of Delaware, Newark, Delaware, USA.
Qiang FuDepartment of Biomedical Engineering and Biotechnology, University of Massachusetts Lowell, Lowell, Massachusetts, USA.
Nelson NdahiroDepartment of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland, USA.ORCID http://orcid.org/0000-0003-1937-1920
Thomas M LeibigerDepartment of Chemical and Biomolecular Engineering, University of Delaware, Newark, Delaware, USA.
Yongdan WangDepartment of Chemical Engineering, University of Massachusetts Lowell, Lowell, Massachusetts, USA.
Yongsuk LeeDepartment of Pharmaceutical Sciences, University of Massachusetts Lowell, Lowell, Massachusetts, USA.
Kelvin H LeeDepartment of Chemical and Biomolecular Engineering, University of Delaware, Newark, Delaware, USA.ORCID http://orcid.org/0000-0003-0908-2981
Michael BetenbaughDepartment of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland, USA.ORCID http://orcid.org/0000-0002-6336-4659
Seongkyu YoonDepartment of Chemical Engineering, University of Massachusetts Lowell, Lowell, Massachusetts, USA.ORCID http://orcid.org/0000-0002-5330-8784
David J McNallyDepartment of Chemical Engineering, University of Massachusetts Lowell, Lowell, Massachusetts, USA.ORCID http://orcid.org/0000-0002-6993-4368

Funding

This study was funded and supported by the Advanced Mammalian Biomanufacturing Innovation Center (AMBIC) through the Industry - University Cooperative Research Center Program of the U.S. National Science Foundation (grant numbers 1624684, 1624698, 2100075, 2100502). We would like to express our gratitude to all AMBIC member companies for their mentorship and financial support, with particular gratitude to Kim Schrag for advice on media development This study was partially funded by The National Institute for Innovation in Manufacturing Biopharmaceuticals (NIIMBL, grant/award 70NANB17H002). The authors also appreciate Sartorius Data Analytics for providing MODDE and SIMCA licenses for the process optimization portion of this study.
6 · The paper itself

Abstract

Recombinant adeno associated virus (rAAV) vectors have become popular delivery vehicles for in vivo gene therapies, but demand for rAAVs continues to outpace supply. Platform processes for rAAV production are being developed by many manufacturers, and transient chemical transfection of human embryonic kidney 293 (HEK293) cells is currently the most popular approach. However, the cutting edge nature of rAAV process development encourages manufacturers to keep cell culture media formulations, plasmid sequences, and other details proprietary, which creates hurdles for small companies and academic labs seeking to innovate in this space. To address this problem, we leveraged the resources of an academic-industry consortium (Advanced Mammalian Biomanufacturing Innovation Center, AMBIC) to develop an rAAV production system based on transient transfection of suspension HEK293 cells adapted to an in-house, chemically defined medium. We found that balancing iron and calcium levels in the medium were crucial for maintaining transfection efficiency and minimizing cell aggregation, respectively. A design of experiments approach was used to optimize the transient transfection process for batch rAAV production, and PEI:DNA ratio and cell density at transfection were the parameters with the strongest effects on vector genome (VG) titer. When the optimized transient process was transferred between two university sites, VG titers were within a twofold range. Analytical characterization showed that purified rAAV from the AMBIC process had comparable viral protein molecular weights versus vector derived from commercial processes, but differences in transducing unit (TU) titer were observed between vector preps. The developed media formulation, transient transfection process, and analytics for VG titer, capsid identity, and TU titer constitute a set of workflows that can be adopted by others to study fundamental problems that could improve product yield and quality in the nascent field of rAAV manufacturing.

Indexed as

Batch Cell Culture TechniquesCell Culture TechniquesCulture MediaDependovirusGenetic VectorsTransfectionVirus CultivationHEK293 CellsHumansCulture Mediaanalytical method developmentdesign of experiments (DOE)media formulationrecombinant adeno‐associated virus (rAAV) productionsuspension HEK293 cell culturetransient transfection optimization

Identifiers

PMID40197832
PMCPMC12152525

What Socratic holds

Textmetadata
LicenceCC BY-NC
Read underepoch 390

Registered trials

None linked

Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the Socratic graph.