PG9 chimeric light chain IgG expression

PG9 chimeric light chain IgG expression

1.     Under sterile conditions, prepare a 200 ml culture of FreeStyle 293-F cells (ThermoFisher, R79007) in 293 Freestyle media (ThermoFisher, 12338-018) to 1.0 x 10⁶ cells/ml with >90% viability in a 500ml baffled shake flask for each antibody you are expressing.

2.     For each transfection, prepare 2 tubes each containing 5 ml Transfectagro (MediaTech, 40-300-CV).

3.     To one tube add 200 ml PEImax 40K solution (Polysciences, 24765-2).

4.     To the other tube, add 25 mg each purified light chain and heavy chain expression plasmid DNA maxipreps (Qiagen), sterile filter through 0.22 mm syringe filter (MilliporeSigma, SLGV033RB).

5.     Add the filtered Transfectagro/plasmid solution to the Transfectagro/PEI solution, mix gently then incubate at room temperature 25-30minutes.

6.     Add the Transfectagro solution dropwise to the culture, gently swirling as you add.

7.     Place the transfected culture in a shaker running at 135 rpm with 8% CO₂ and 80% humidity for 5 days.

8.     On the 5^th day, centrifuge the culture at 3000 x g for 20 minutes then vacuum filter using a 0.22 mm Stericup (MilliporeSigma).

9.     Prepare a 1:1 Protein A/G Sepharose slurry to produce a 2 ml column bed by mixing 1.3 ml nProtein A Sepharose (Cytiva formally GE Healthcare, 7528002) and 1.3 ml Protein G Sepharose (Cytiva, 17061805).

10.  Exchange the slurry buffer to PBS by bringing up the volume to 10 ml with PBS, centrifuge 3000 x g for 5 minutes then carefully pour off as much PBS as possible without losing Sepharose. Repeat 2 more times. 

11.  Bring the slurry up in 2-3 ml PBS then add entire volume to the filtered culture supernatant and place at 4°C on a rocker to gently mix overnight.

12.  Set up a column (Bio-Rad, 7321010) with a stockcock (Bio-Rad, 7328102), then add the supernatant/slurry mix, allowing it to flow through at full speed and form a bed.

13.  Wash column with 250 ml PBS, letting flow at full-speed.

14.  Once the wash has flowed through but before the bed runs dry, stop the flow.

15.   Set up a collection tube under the column containing 2 ml Neutralization Buffer (1M Tris pH 9.0)

16.  Apply 12 ml Elution Buffer (50 mM citric acid buffer pH 2.2) to the column with the stopcock set to slow drip (1 drop/5 seconds).

17.  Immediately, concentrate and PBS buffer exchange the eluted sample to a volume of 500 ml using a 50 KDa Vivaspin concentrator (Sigma-Aldrich).

18.  (Recommended for chimeric IgG) Using an AKTA FPLC (Cytiva), purify by size exclusion chromatography (SEC) on a S200 10/30 column (Cytiva) in PBS buffer and then pool the peak fractions, concentrate to 500 ml as described above.

19.  Measure the IgG concentrations using a Nanodrop (ThermoFisher)

20.  Sterile filter using a Spin-X 0.22 mm filter device (Corning, 8160). 

21.  As chimeric IgG do not store as well as normal IgG at 4°C, assays and tests should be completed within 1 week of purification or they should be frozen and stored at -20°C.

Solutions:

PEImax 40K Solution

Dissolve 0.1g PEIMax 40K (Polysciences, 24765-2) in100ml Milli-Q water

Neutralization Buffer (1M Tris pH 9.0)

Dissolve 121.1g Trizma Base in 1L Milli-Q water, adjust pH to 9.0, sterile filter through 0.22 mm pore.

Elution Buffer (50 mM Citric Acid pH 2.2)

Dissolve 9.6g Citric Acid in 1L Milli-Q water, adjust pH to 2.2, sterile filter through 0.22 mm pore.

Category