Construction and small-scale production of hIgG1Fc-VHH chimeric antibodies

Cells: HEK 293T (adherent) are grown in DMEM/ 10%FBS/NEAA to 90% confluence in 10 cm dish and transfected with the linearized expression plasmid with lipofectamine 2000 agent according to the manufacturer instructions. At the time of transfection, there are no antibiotics present in the medium. At least 5 hours later, medium is removed, cells are re-suspended in medium with Strep/Pen plated 1:100 and 1:10 on 10 cm dishes and remaining cells plated on a 15 cm dish. 1:10 and 1:100 dilutions can be used for picking individual clones under microscope after selection. Usually for small scale production, the amount of antibody purified from pools of clones will be satisfactory for majority of assays.

The selection with hygromycin starts next day when medium is replaced with expansion medium. Cells not containing inserted plasmid will start dying. Usually, after 4-6 days the only growing cells are those with integrated construct. If deciding to pick individual clones, they can be grown in 96 or 48 well plate and tested in an antigen specific Elisa for the expression of antibodies, so that clones with best expression are chosen for further expansion.

I you decide for small scale expression from pools of clones, you can expand to two to four 15 cm dishes and when they are full, proceed with the production, i.e. changing to the production medium. 

EXPANSION MEDIUM

500 ml DMEM (BioWhittaker/Lonza , catN BE12-604F/41)

-10%FBS (Foetal calf serum, also from Whittaker) 

-100X NEAA (Non-essential amino acids) Biowhittaker Lonza Cat BE13-114E)

-PEN/STREP (P0781-100ml Sigma 070M0765)

-Hygromycin ( HYGROMYCIN B, Roche Diagnostics GmbH , 20ml 50mg/ml REF10843555001) final concentration 200micrograms/ml of medium

Expansion medium is used for growing cells until ready for production purposes and for freezing cells in liquid N2 with addition of 10% DMSO.

Cells are attached but do not need trypsin for de-attaching and making single cell suspension. By repeating pipetting up and down cells will de-attach. Usually they need splitting 1 in 3 every 3-4 days.

PRODUCTION MEDIUM

500ml Optimem +GlutaMax^TK-I (Gibco, 500ml, 51985)

5ml of 100X NEAA

PEN/STREP

Hygromicin

Normally HEK cells grow as attached and for full 15cm tissue culture dish we use 20ml of medium. After removing the expansion medium and for the first time applying the serum free medium, carefully wash cells with pre –wormed PBS or serum free medium or do not use first collection for antibody production. This is to prevent having traces of FBS antibodies in your antibody preparations. The production medium is collected and replaced with fresh medium every 3 to 4 days. Usually from the same 15 cm dish you can collect medium for at least 3 weeks. If you are not planning to purify the antibody immediately you can go trough steps 1, 2 and 3 of purification and freeze supernatants before attempting final purification.

Purification of Antibodies:

1. Collect supernatant from HEK cells grown on the production medium

2. Spin supernatants in 50 ml tube (such as Greiner Bio one Cellstar tubes Cat No 227261) for 5 min at 1000 rpmi

3. Transfer supernatant into a new tube (leaving behind possible cells and cell debris)

5. Add prot A beads to supernatant (Protein A-Agarose Fast Flow, SIGMA P3476-5ml ) 100 microliters per 50 ml

6. Rotate at 4°C over night (cold room)

7. spin down at 4°C, 5 min at 1300 rpmi

8. Remove the medium safely 

9. Add PBS to the beads and

10. Load the beads on a home made column  

Home made column could be made from 1ml syringe. Remove the plunger and push the piece of cotton wool to the very end/ opening. Optionally, column could be made from sterile plastic 5ml pipette, by pushing the cotton wool at the end of pipette to the tip (by attaching it to the high air pressure).

Wash the column with PBS and load with the prot A beads. You can load on 1ml column protein A beads from 100ml of collected medium. Wash the column 3X with 1ml of PBS/tween and 1X PBS and elute with 400 microliters of 3M CKNS (Potassium Thiocyanate, Merck).

Perform the dialysis to remove toxic 3M CKNS. Dialysis could be done against PBS, but also with another physiological solution that you might use for injection of antibody into animal.  

We do 3 rounds of dialysis in the cold room (at 4°C), each time with the 1000 X volume of the sample.  We use dialysis tubing high grade regenerated cellulose tubular membrane with the cut off of 8000 KD. Cellu SepH1 Part 0810-24 , 10m when doing a big batch since flat width is 24mm or  part 0810-12 width 12mm when doing smaller volumes. (Membrane Filtration Products.Inc, USA).   

As an alternative to dialysis you can use Amicon Ultra 4 centrifugal filters (cut of 30 KD for VH fusion with Fc) for concentrating and buffer exchange according to the instruction provided by supplier. 

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