Augmenting recombinant antibody production in HEK293E cells: Optimizing transfection and culture parameters

Heng, Zealyn Shi Lin, Yeo, Joshua Yi, Koh, Darius Wen Shuo, Gan, Samuel Ken-En, and Ling, Wei Li (2022) Augmenting recombinant antibody production in HEK293E cells: Optimizing transfection and culture parameters. Antibody Therapeutics, 5 (1). pp. 30-41.

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Background: Optimizing recombinant antibody production is important for cost-effective therapeutics and diagnostics. With impact on commercialization, higher productivity beyond laboratory scales is highly sought, where efficient production can also accelerate antibody characterizations and investigations.

Methods: Investigating HEK293E cells for mammalian antibody production, various transfection and culture parameters were systematically analyzed for antibody light chain production before evaluating them for whole antibody production. Transfection parameters investigated include seeding cell density, the concentration of the transfection reagent and DNA, complexation time, temperature, and volume, as well as culture parameters such as medium replacement, serum deprivation, use of cell maintenance antibiotic, incubation temperature, medium volume, post-transfection harvest day, and common nutrient supplements.

Results: Using 2 mL adherent HEK293E cell culture transfections with 25 kDa linear polyethylenimine in the most optimized parameters, we demonstrated a ~2-fold production increase for light chain alone and for whole antibody production reaching 536 and 49 μg, respectively, in a cost-effective manner. With the addition of peptone, κ light chain increased by ~4-fold to 1032 μg, whereas whole antibody increased to a lesser extent by ~2.5-fold to 51 μg, with benefits potentially for antibodies limited by their light chains in production.

Conclusions: Our optimized findings show promise for a more efficient and convenient antibody production method through transfection and culture optimizations that can be incorporated to scale-up processes and with potential transferability to other mammalian-based recombinant protein production using HEK293E.

Item ID: 78242
Item Type: Article (Research - C1)
ISSN: 2516-4236
Keywords: antibody therapeutics manufacturing, HEK293E, recombinant antibody production, transfection parameters, transient transfection
Copyright Information: © The Author(s) 2022. Published by Oxford University Press on behalf of Antibody Therapeutics. All rights reserved. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (, which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact
Date Deposited: 09 May 2023 04:00
FoR Codes: 31 BIOLOGICAL SCIENCES > 3101 Biochemistry and cell biology > 310103 Cell metabolism @ 100%
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