TITLE: IgG expression and purification
AUTHORS: Bei Wang, Wen-Hsin Sandy  Lee, Helen Huang, Patricia Ng, Eve Ngoh, Chia Yin Lee, Hwee Ching Tan, Rabiatul Adawiyah, Mun Kuen Soh, Frannie Teo, Yvonne Yeap, Yuanyu Hu, Cheng-I Wang

[STEPS]
?. [IgG Expression]
Day 0: Transfection of ExpiCHO-S cells
?. [IgG Expression]
Sterile the plasmids by filtering through 0.22 µm spin-X column.
?. [IgG Expression]
According to the transfection manual of ExpiFectamine™ CHO Transfection Kit, 0.8 µg of plasmid per ml of ExpiCHO-S cells (20 µg of heavy chain plasmid and 20 µg of light chain) were diluted in cold OptiPRO™ SFM in a 50 ml tube.Mix by swirling the tubes.
?. [IgG Expression]
Gently invert the ExpiFectamine™ CHO Reagent bottle 4-5 times to mix.
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Add respective volume of ExpiFectamine™ CHO Reagent to the tube of diluted DNA. Mix reaction by swirling the tube.
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Incubate reaction at room temperature for at least 1 minute but no more than 5 minutes.
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Take out the flask of ExpiCHO-S suspension cells that was prepared in step 3 out from the incubator andadd the transfection mix dropwise into the cells while swirling the flask gently during addition.
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Place cells into back into incubator at 37°C with humidified atmosphere of 8% CO2 on an orbital shaker.
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Day 1: Add ExpiFectamine™ CHO Enhancer and ExpiCHO Feed.
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After 18-22 hours of transfection, add the required amount stated in of ExpiFectamine™ CHO Enhancer and ExpiCHO Feed.
?. [IgG Expression]
Gently swirl the flask during addition.
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Place cells into back into incubator at 37°C with humidified atmosphere of 8% CO2 on an orbital shaker for another 7 days before harvesting.
?. [Harvest the cell supernatant containing IgGs.]
Day 8 : Harvest ExpiCHO-S cell culture supernatant
?. [Harvest the cell supernatant containing IgGs.]
Collect transfected ExpiCHO-S suspension cells in 50 ml Falcon tubes.
?. [Harvest the cell supernatant containing IgGs.]
Centrifuge the ExpiCHO-S suspension cells at 2000 rpm for 10 minutes.
?. [Harvest the cell supernatant containing IgGs.]
Filter the supernatant through a 0.20 µm syringe filter to remove the cells and debris.
?. [Harvest the cell supernatant containing IgGs.]
Add 10x PBS to neutralize pH of supernatant.
?. [Purification of antibodies]
Day 9: Add Protein G agarose to the harvested ExpiCHO-S cell supernatant.
?. [Purification of antibodies]
Wash 1ml of Protein G agarose (2 ml slurry) 3X with 5 ml of cell culture grade PBS in a 50 ml tube to remove storage buffer from Protein G agarose and add it into the supernatant.
?. [Purification of antibodies]
Allow Protein G agarose to bind to the antibodies in supernatant overnight in 4°C or for 2 hours in room temperature with rotation before purification.
?. [Purification of antibodies]
Day 10: Wash Protein G agarose and elute IgG antibodies
?. [Purification of antibodies]
Centrifuge tube with supernatant and agarose at 150 g for 5 minutes.
?. [Purification of antibodies]
Remove supernatant without disturbing the protein G agarose and collect in a new tube.
?. [Purification of antibodies]
Wash 1 ml polypropylene column with cell culture grade water three times and with cell culture grade PBS for one time.
?. [Purification of antibodies]
Transfer protein G agarose into column.
?. [Purification of antibodies]
Wash protein G agarose with 3 column volume of cell culture grade PBS.
?. [Purification of antibodies]
After washing protein G agarose with PBS, elute antibodies with 0.1 M of Glycine solution, pH 3.5 into 1.5 ml tubes with 200 µl of 1.0 M Tris, pH 8.0 to neutralise reaction.
?. [Purification of antibodies]
Test 10 µl of various fractions of eluted antibody in 200 µl of 1X Bio-Rad Protein Assay Dye Reagent already prepared in a 96 well plate.
?. [Purification of antibodies]
Pool the fractions of elute that turn blue in 1X Bio-Rad Protein Assay Dye Reagent together.
?. [Purification of antibodies]
Day 10: Dialysis of antibodies
?. [Purification of antibodies]
Transfer pooled fractions of elute into 10K MWCO Slide-A-Lyzer™ G2 Dialysis Cassettes and allow it to dialyse 3X, each time with 4 liters of 1x PBS prepared from 10X PBS. All buffer exchange should be done at 4°C with at least one of the dialysis done overnight.
?. [IgG Expression]
Seed 50 ml of 6 x 106 cells/ml of ExpiCHO-S suspension cells in a 250 ml flask. Ensure percentage of viability is >95% before proceeding. Gently pipette cells into the medium and swirl flask to mix the cells. Put flask into incubator and prepare transfection mix.
?. [IgG Expression]
Pre-warm ExpiCHO™ Expression Medium to 37°C in water bath.
?. [Purification of antibodies]
Day 11: Collection of dialysed IgG antibodies
?. [Purification of antibodies]
Collect dialysed antibodies from cassettes and concentrate to desired concentration using VIVASPIN 6 30,000 MWCO if necessary.