Experiments on the preparation of recombinant proteins by transient gene transfer in mammalian cells

Summary

Reports show that in the past decade, a variety of highly efficient transient transfection methods have been developed that meet or exceed these requirements. Currently, transient expression of protein is primarily performed in HECD-derived cell lines, while researchers are working to develop methods with comparable efficiencies for CHO cells. This facilitates the maintenance of the same host cell background, which allows for consistency of product properties throughout the development of therapeutic proteins. A detailed summary of current T G E can be found in a number of recently published reviews.

Authors: Burgess et al, Translator: Chen Wei, This experiment is from "Protein Purification Guide".

Operation method

Experiments on the preparation of recombinant proteins by transient gene transfer in mammalian cells

Move

I. Transfection methods

For liposome transfection (Upofection), there are many formulations commercially available, all of which have a single patented component, and most of which are undoubtedly very effective at transfecting plasmid DNA into cells. The disadvantage is the high price - when using these reagents, transient transfection on scales exceeding hundreds of milliliters is not economically feasible. Large-scale transient transfection methods require that the transfection reagents be readily available in large quantities, with little batch-to-batch variation to ensure consistent transfection. Although some alternatives exist, such as the use of chitosan and its derivatives and 14D E A 2 as transfection reagents (D ag and L e o n g , 2006; Jiang and Sharfstein, 2008; K u s u m o t oet a L , 2006), calcium phosphate-mediated transfection as well as polymer-based transfection are not economically feasible. Calcium phosphate-mediated transfection and polyethylenimine (PEI)-based plasmidcomplexation and cellular uptake are the only two commonly used techniques to fulfill these requirements.

1. Large-scale transient transfection using polyethylenimine in the Wave™ bioreactor

In recent years, one-off Wave™ bioreactors have gained widespread acceptance because of the ease of maintenance and operation compared to classic stirred kettle reactors. Although 100 L working volume reactors are commercially available and prototypes with 300 L working volume are being developed, the most commonly used reactors have 10 L and 20 L working volumes. Whether or not to use the Wave ™ Bioreactor for transfections below 10 L depends on economic feasibility - we prefer to use the Wave ™ Bioreactor for transfections below 10 L.
We prefer to use shake flasks for small-scale production ( F e m b a c h flasks, Corning). Other reactors or shaking devices have been reported to be successfully used for transient cultures (Muller et al., 2005; Pham et al., 2006; Stettler et al., 2007).

The following describes a method for 10 L scale antibody production using M 11V 3 serum-free medium (Novartis proprietary) with HEC 293 T cells as host. Both chains of the antibody were cloned into the same expression vector.

(1) A 20 L W a v e ™ bag (Sartorius Stedim Biotech, G 6ttingen, G e r m a n y ) was mounted onto the W a v e ™ workbench (W a v e - Bioreactor S P S 5O ) and connected to a D A S G I P gas mixing module (D A S G I P , Juelich, G e r m a n y ). r m a n y ). The bags were then inoculated with 4 L H E K 293T cell cultures at a cell density of approximately I.8 X ^IO6 cells/m L .

(2) The following process parameters and conditions were used: gas flow rate of 200 L/h; gas mixture of 21% to 25% 〇2, 0% CO2; temperature of 37°C; p H 6. 8 to 7, 4; shaking rate of 10 r Z million; shaking angle of 7°.

(3) Mix 10 m g of plasmid DNA (1 m g /m L ) with M 11V 3 medium to a final volume of 500 m L and incubate for 10 m i n at room temperature. Then, a 0.22 p m G P E X P R E S S P L U S filter (Millipore, Billerica, USA) was used to remove the plasmid from the culture medium.
The diluted DNA solution was then filtered and sterilized using a 0.22 p m G P E X P R E S S P L U S filter (Millipore, Billerica, M A ).

(4) Mix 300 m L of P E I solution (I m g /m L ) with 500 m L of M l l V 3 medium and incubate for 10 m i n at room temperature. The diluted P E I solution was then filtered through a 0.22um G P E X P R E S S P L U S filter membrane to remove bacteria.

Note: The PEI stock solution should be sterile, filtered before use, dispensed and frozen at 1 80°C until use.

(5) Next, add the P E I solution to the DNA solution and incubate at room temperature for 15 m i n to form the polycomplex.

(6) The DNA-PEI-M 11V 3 mixture was added to the cell culture in the Wave ™ bag under sterile conditions to a final volume of 5 L. The final volume was then incubated for 5 to 6 h with the following parameters: gas flow rate, gas flow rate, gas flow rate, gas flow rate and gas flow rate. Incubation was continued for 5 to 6 h with the following parameters: gas flow rate M L /h; gas mixture consisting of 25 % O2, 0 % _CO2; temperature 37° 0; mouth 1^6.8 to 7.4; shaking rate 1 0 r/min; shaking angle 7°.

(7) Then, 100 m L of R X l combination supplement (containing amino acids, glucose, and glutamine; customized by Irvine Scientific, Santa Ana, CA) was added to 5 L of M 11V 3 medium, and added to the cells of the Wave ™ bags under sterile conditions. During the production period, the following parameters were used: gas flow rate 30 to 40 L/h; gas mixture consisting of 30 to 40 % O2, 0 % CO2; temperature 37°C; p H 6.8 to 7.4 (adjusted with a bicarbonate solution with p H 9.2, Sigmunda Aldrich, Buches, Switzerland); gas saturation 60 % to 100 %; shaking rate 14~18 r/mi.n ; Swing angle 7°.

(8) Transfected cells were cultured in a W a v e ™ bioreactor for 10 days for antibody production.
Note: For the production of recombinant proteins, the production period is usually 5 to 7 days, but this time can be shortened if the target protein is sensitive to proteolysis. The production of antibody molecules can be extended to 10 days.

(9) Samples are taken daily and cell density and viability are measured using a V1-Cell Counter (Beckman Coulter). Nutritional status, p H
and gas saturation. I g G concentration was determined by high-pressure liquid chromatography using protein A (Fig. 15. 1).

(10) After 10 days, cells are collected under sterile conditions and removed by cross-flow filtration (Fresenius Filter Plasmafilter, 0.2 fim). The cell-free supernatant was then concentrated 10-fold by hollow fiber filtration (Hemoflow F10HPS, Fresenius, Stans, Switzerland, 10 kD acutoff).

(11) Purification of the concentrate by Protein A affinity chromatography and molecular sieve chromatography. Various modifications of these standard methods have been tested and published, some of which are briefly listed below.

In the production process using the CHO cell line, the expression rate was increased by switching the temperature to 30-32°C (Backliwal et al., 2008a; Galbraith et al., 2006).

Cells were treated with the microtubule depolymerizing agent nocodazole to arrest the cell cycle at the G2/M stage (Tait et a L, 2004).

(iii) Treatment with inhibitors such as sodium butyrate and valproic acid to inhibit histone deacetylase and D N A methyl transferase in C HO and H E K 293 cells (Backliwal et al., 2004). Backliwal et al., 2008c).

(iv) Genetic modification of cell lines to prevent apoptosis or to overcome the inhibitory effects of the unfolded protein response (U P R) (Backliwal et al., 2008c; Majors et al. Fussenegger, 2006) "

Conclusion

As reflected in the large amount of data that has been published in the field, the preparation of recombinant proteins by transient transfection has reached an impressive state of affairs over the past few years in terms of technological development and yield. However, although it has been reported that the production of proteins in bioreactors using large-scale transient transfection techniques has enabled the achievement of fairly high antibody titers-greater than I g / W B a c k l i w a l et al. , 2008a)-it is too early to think of replacing the process-intensive constructed-cell-line method for the production of biotherapeutic drugs. Our observation of more than 100 transient transfection expression experiments shows that the expression amount varies greatly for different genes: the expression amount of general proteins ranges from I m g / L to more than 170 m g /L; the average expression amount of antibodies is 20 to 40 m g /L, but is often distributed at the two extremes. Undoubtedly, further technical improvements are needed in the design of expression vectors, optimization of culture conditions, and possible modification of cell lines, but the overall impressive success of this method proves that such efforts are worthwhile.

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