Human-derived protein is crucial for initial characterization studies, and depending on the protein, may be required to ensure activity as a therapeutic or functionality in a medical device. Of these mammalian cell lines, HEK293 provides the additional benefit of conferring fully human PTMs that are less immunogenic. Cell lines such as Chinese hamster ovary (CHO), murine myeloma (NS0) and human embryonic kidney 293 (HEK293) are advantageous host platforms versus bacteria and yeast due to their ability to correctly assemble complex proteins, secrete them at high levels, and add human-like post-translational modifications (PTMs). Mammalian cell lines are commonly used for viral vector and recombinant protein production at research and commercial scales. While a bio-layer interferometry (BLI) assay was crucial for efficiently screening DWP batch titers, an enzyme-linked immunosorbent assay (ELISA) was used to compare titers from the flask-scale batches due to varying matrix effects from different cell culture media compositions. Flask-scale transient transfection and stable fed-batch processes were then established that produced rRBD up to 100 mg/L and 140 mg/L, respectively. In parallel, four clonal cell lines with titers higher than that of the selected stable pool were identified in a batch screen. Use of the cytomegalovirus (CMV) promoter to drive expression at 32 ☌ led to the highest transient protein titers, but inclusion of episomal expression elements did not augment titer. Nine DNA vectors that drove rRBD production under different promoters and optionally contained Epstein-Barr virus (EBV) elements to promote episomal expression were screened for transient rRBD production at 37 ☌ or 32 ☌. Initial process development was done at 24 deep well plate (DWP) -scale to screen transient processes and stable clonal cell lines for recombinant SARS-CoV-2 receptor binding domain (rRBD) titer. The Creative Commons Public Domain Dedication waiver ( ) applies to the data made available in this article, unless otherwise stated in a credit line to the data. ![]() If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.
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