A rapid procedure to generate stably transfected HEK293 suspension cells for recombinant protein manufacturing: Yield improvements, bioreactor production and downstream processing
Bibliographic record
Abstract
The human cell line HEK293 is one of the preferred choices for manufacturing therapeutic proteins and viral vectors for human applications. Despite its increased use, it is still considered in disadvantage in production aspects compared to cell lines such as the CHO cell line. We provide here a simple workflow for the rapid generation of stably transfected HEK293 cells expressing an engineered variant of the SARS-CoV-2 Receptor Binding Domain (RBD) carrying a coupling domain for linkage to VLPs through a bacterial transpeptidase-sortase (SrtA). To generate stable suspension cells expressing the RBD-SrtA, a single two plasmids transfection was performed, with hygromycin selection. The suspension HEK293 were grown in adherent conditions, with 20% FBS supplementation. These transfection conditions increased cell survival, allowing the selection of stable cell pools, which was otherwise not possible with standard procedures in suspension. Six pools were isolated, expanded and successfully re-adapted to suspension with a gradual increase of serum-free media and agitation. The complete process lasted four weeks. Stable expression with viability over 98% was verified for over two months in culture, with cell passages every 4-5 days. With process intensification, RBD-SrtA yields reached 6.4 μg/mL and 13.4 μg/mL in fed-batch and perfusion-like cultures, respectively. RBD-SrtA was further produced in fed-batch stirred tank 1L-bioreactors, reaching 10-fold higher yields than perfusion flasks. The trimeric antigen displayed the conformational structure and functionality expected. This work provides a series of steps for stable cell pool development using suspension HEK293 cells aimed at the scalable production of recombinant proteins.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".