A rapid procedure to generate stably transfected HEK293 suspension cells for recombinant protein manufacturing: Yield improvements, bioreactor production and downstream processing DOI Creative Commons
Shantoshini Dash, Omar Farnós, Zeyu Yang

и другие.

Protein Expression and Purification, Год журнала: 2023, Номер 210, С. 106295 - 106295

Опубликована: Май 16, 2023

The human cell line HEK293 is one of the preferred choices for manufacturing therapeutic proteins and viral vectors applications. Despite its increased use, it still considered in disadvantage production aspects compared to lines such as CHO line. We provide here a simple workflow rapid generation stably transfected cells expressing an engineered variant SARS-CoV-2 Receptor Binding Domain (RBD) carrying coupling domain linkage VLPs through bacterial transpeptidase-sortase (SrtA). To generate stable suspension RBD-SrtA, single two plasmids transfection was performed, with hygromycin selection. were grown adherent conditions, 20% FBS supplementation. These conditions survival, allowing selection pools, which otherwise not possible standard procedures suspension. Six pools isolated, expanded successfully re-adapted gradual increase serum-free media agitation. complete process lasted four weeks. Stable expression viability over 98% verified months culture, passages every 4-5 days. With intensification, RBD-SrtA yields reached 6.4 μg/mL 13.4 fed-batch perfusion-like cultures, respectively. further produced stirred tank 1L-bioreactors, reaching 10-fold higher than perfusion flasks. trimeric antigen displayed conformational structure functionality expected. This work provides series steps pool development using aimed at scalable recombinant proteins.

Язык: Английский

A rapid procedure to generate stably transfected HEK293 suspension cells for recombinant protein manufacturing: Yield improvements, bioreactor production and downstream processing DOI Creative Commons
Shantoshini Dash, Omar Farnós, Zeyu Yang

и другие.

Protein Expression and Purification, Год журнала: 2023, Номер 210, С. 106295 - 106295

Опубликована: Май 16, 2023

The human cell line HEK293 is one of the preferred choices for manufacturing therapeutic proteins and viral vectors applications. Despite its increased use, it still considered in disadvantage production aspects compared to lines such as CHO line. We provide here a simple workflow rapid generation stably transfected cells expressing an engineered variant SARS-CoV-2 Receptor Binding Domain (RBD) carrying coupling domain linkage VLPs through bacterial transpeptidase-sortase (SrtA). To generate stable suspension RBD-SrtA, single two plasmids transfection was performed, with hygromycin selection. were grown adherent conditions, 20% FBS supplementation. These conditions survival, allowing selection pools, which otherwise not possible standard procedures suspension. Six pools isolated, expanded successfully re-adapted gradual increase serum-free media agitation. complete process lasted four weeks. Stable expression viability over 98% verified months culture, passages every 4-5 days. With intensification, RBD-SrtA yields reached 6.4 μg/mL 13.4 fed-batch perfusion-like cultures, respectively. further produced stirred tank 1L-bioreactors, reaching 10-fold higher than perfusion flasks. trimeric antigen displayed conformational structure functionality expected. This work provides series steps pool development using aimed at scalable recombinant proteins.

Язык: Английский

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