Comprehensive Comparison of Baculoviral and Plasmid Gene Delivery in Mammalian Cells.

(1) Recombinant protein production in mammalian cells is either based on transient transfection processes, often inefficient and underlying high batch-to-batch variability, or on laborious generation of stable cell lines. Alternatively, BacMam, a transduction process using the baculovirus, can be employed. (2) Six transfecting agents were compared to baculovirus transduction in terms of transient and stable protein expression characteristics of the model protein ACE2-eGFP using HEK293-6E, CHO-K1, and Vero cell lines. Furthermore, process optimization such as expression enhancement using sodium butyrate and TSA or baculovirus purification was assessed. (3) Baculovirus transduction efficiency was superior to all transfection agents for all cell lines. Transduced protein expression was moderate, but an 18-fold expression increase was achieved using the enhancer sodium butyrate. Ultracentrifugation of baculovirus from a 3.5 L bioreactor significantly improved the transduction efficiency and protein expression. Stable cell lines were obtained with each baculovirus transduction, yet stable cell line generation after transfection was highly unreliable. (4) This study demonstrated the superiority of the BacMam platform to standard transfections. The baculovirus efficiently transduced an array of cell lines both transiently and stably and achieved the highest efficiency for all tested cell lines. The feasibility of the scale-up of baculovirus production was demonstrated and the possibility of baculovirus purification was successfully explored.

Media on-demand: Continuous reconstitution of a chemically defined media directly from solids.

Chemically defined media are reconstituted batchwise and stored in hold tanks until use. To avoid large hold tanks and batchwise production of media, we developed continuous on-demand reconstitutions directly from solids consisting of a hopper and a screw conveyor capable of feeding dry powdered media with the required precision ±5% at low dosing rates of 0.171 g min-1 . A commercially available dry powdered cell culture medium was continuously fed over a duration of 12 h into a mixer which was connected to a UV-cell for monitoring and the media were compared to a batchwise production. A comparable amino acid, carbohydrate, and osmolality profile to a batchwise reconstitution could be obtained. Cell cultivation showed comparable performance of batch and continuous reconstitution for two CHO cell lines producing the antibodies adalimumab and trastuzumab on a small and benchtop scale. In-depth analysis of the produced antibodies showed the same glycosylation pattern, other posttranslational profiles such as methionine oxidation and deamidation compared to batchwise reconstitution. Therefore, we conclude a continuous reconstitution of the medium results in the same quality of the product. A continuous on-demand media reconstitution will impact the supply chain and significantly reduce the floor space necessary for preparation and storage.

Continuous capture of recombinant antibodies by ZnCl 2 precipitation without polyethylene glycol.

The capture of recombinant antibodies from cell culture broth is the first critical step of downstream processing. We were able to develop a precipitation-based method for the capture and purification of monoclonal antibodies based on divalent cations, namely ZnCl2. Traditional precipitation processes have to deal with high dilution factors especially for resolubilization and higher viscosity due to the use of PEG as precipitation or co-precipitation agent. By the use of the crosslinking nature of divalent cations without the use of PEG, we kept viscosity from the supernatant and resolubilization dilution factors very low. This is especially beneficial for the solid-liquid separation for the harvest and wash of the precipitate in continuous mode. For this harvest and wash, we used tangential flow filtration that benefits a lot from low viscosity solutions, which minimizes the membrane fouling. With this precipitation based on ZnCl2, we were able to implement a very lean and efficient process. We demonstrated precipitation studies with three different antibodies, Adalimumab, Trastuzumab, and Denosumab, and a continuous capture case study using tangential flow filtration for precipitate recovery. In this study, we achieved yields of 70%.