Ig Depletion from Foetal Calf Serum (FCS)

1. Introduction
This protocol describes the use of Amersham-Pharmacia 1ml HP protein G columns for the purification of monoclonal antibodies (mAb). Scale-up to the use of 5ml columns is simple (5x flow rate etc.). There are some important considerations ...

A. In general a single column is used for the purification of only one mAb, to eliminate possible cross-contamination. If this is not possible for reasons of expense, the optional step of cleaning the column with guanidine hydrochloride should be used between purifications of different antibodies.

B. The bovine IgG in culture media containing FCS will be co-purifiued along with the mAb. Therefore it is essential to use either serum-free media, or media containing FCS that has been depleted of protein-A-binding Ig. (See separate protocol for pA/pG depletion of FCS).

2. Related documentation
See Amasham protocol that comes with the columns, and on which this protocol is based.

3. Materials
3.1 Biorad peristaltic ECONO pump and appropriate tubing (1.6 mm diameter pump tube).
3.2 Filter unit (Nalgene, MF75 series, polystyrene housing, 0.2um PES membrane: 250ml capacity Cat no 168-0020, 500ml capacity Cat no 166-0020) (can use 0.45um filter and alternatives).
3.3 Hi Trap Protein G HP column, 1ml (Amersham, Cat no 17-04040-01).
3.4 PBSa sterile (IAH, microbiological services).
3.5 0.22uM syringe filter (Millipore, Cat no SLG033RS).
3.6 0.45uM syringe filter (Millipore, Cat no SLHA025OS).
3.7 500ml sterile conical flasks.
3.8 200-400ml beakers for waste collection, lL cylinder for dialysis.
3.9 10ml syringe to pre-soak syringe filters.
3.10 Pipetteman and filter tips.
3.11 Retort stand and clamps.
3.12 1.5ml eppendorf tubes for collection of elution fluid.
3.13 0.1M Glycine pH 2.7.
3.14 1M Tris-HCl pH 9.
3.15 70% ethanol.
3.16 20% ethanol (made with sterile water).
3.17 pH paper.
3.18 Vacuum pump.
3.19 Stopwatch.
3.20 Sticky tape.
3.21 Float-A-Lyser (Spectrum laboratories, 5ml sample volume, Cat no 235054).
3.22 Acrodisc Syringe Filter 0.2µm Low Protein Binding (PALL Gelman Laboratory, product no 4454).



4. Responsibilities

4.1 Personnel using this method / protocol are responsible for ensuring that they have read and understood the contents, and the procedure is followed. They must be suitably trained and competent, as documented in their training records. It is the duty of the Head of Department / Group (or nominee) to ensure that staff are aware of this responsibility.

4.2 Health and Safety: This method / protocol should be used in accordance with the current Health and Safety Policy, and other relevant instructions as issued by the Institute for Animal Health. Due consideration must be given to National standards and regulations.

5. Procedure
VITAL INSTRUCTIONS

Work throughout at +4oC

Although this procedure is done on the bench try to keep it as sterile as possible by cleaning the bench with 70% Ethanol and using good sterile technique.

Always avoid any air getting into the column.

The bottle or container used to fill the column should never empty, it should be set at an angle and tubing should be taped in place to hold secure.

5.1 Stretch the 1.6mm brown/yellow tube around the pump. The tube should be stretched, and the end sections should be right round the back of the pump. The ends will fit into the appropriate slots.

5.2 Turn the pump on (switch underneath pump). Ensure that the direction of pumping is correct.




5.3 Check the ‘diameter of tubing’ setting on the front of the pump is set to 1.6mm.

5.4 Connect a piece of the clear tubing, so that liquid can be fed into the pump. This tubing must ALWAYS be held in place in the bottle or flask using sticky tape. Place this tubing into a 400ml bottle of sterile PBSa and tape in place.

5.5 Ensure the pump is set to run at 1ml/min. Turn the pump on, and run until there is a good sized drop at the end of the brown tubing.

5.6 Connect a 0.45uM and a 0.22uM syringe filter together. Pre-wet the filters using a syringe filled with PBSa. Hold the syringe vertically with the filters on top to ensure the air will be pushed out upwards.

5.7 Connect the filters to the right hand end of the brown tubing (outlet), pushing the filters firmly in place. Ensure the fluid will flow through the 0.45uM filter first then the 0.22uM.

5.8 Attach the appropriate piece of clear tubing to this filter to allow connection of the pump to the column. Pump to fill tubing with PBSa.

5.9 Take the Protein G column from storage and support in a clamp stand.

5.10 Unscrew the black cap from the top of the column and set aside ensuring it is kept as clean as possible. Use a 200ul pipetteman with filter tips to fill the top of the column with PBSa. Push the tip down as far as possible without damaging the column, and ensure you don’t introduce any bubbles.

5.11 Connect the clear tubing to the top of the column. First turn the pump on to allow a drop of PBSa to form at the tip of the tubing, this will ensure no air is taken into the column. TURN THE COLUMN TO ATTACH IT TO THE TUBING. Make sure the seal is good and not leaking.

5.12 Remove the snap-off end of the column outlet, or remove screw cap if re-using a column. Place a waste beaker under column.

5.13 Turn on the pump until PBSa drips from the bottom of the protein G column.

5.14 Switch off the pump and attach a piece of tubing from the bottom of the Protein G column to a waste beaker. The whole of the tubing and column have a volume of approximately 4ml.

5.15 Check all joints in the pump-column train for leaks and tighten/reassemble as needed. Allow 10 column volumes or so of PBSa to run through the column. Collect some of the flow through.

5.16 (Mock-elution to clean column. Not necessary if the column is new or has been stored for only a few days). Switch off the pump and transfer the inlet tubing to a universal containing 2-3ml 0.1M Glycine pH2.7. Switch on the pump. The first 3-4ml of fluid will be PBSa. Collect some of the flow through when the pH of the flow through drops to pH 3.

5.17 Transfer the inlet tube to a universal of PBSa to wash the tubing then back to the bottle of PBSa. Check pH of flow through. Pump until the pH returns to 7. Now pump at least 10ml PBSa to wash column.

5.18 Calibrate the speed of pumping through the column. Attach a 1ml syringe onto the end of the tubing going to waste. Turn on the pump and run until the liquid reaches the 0ml point on the syringe. Now time how long it takes the pump to reach a certain marker in the syringe. If necessary adjust the speed set on the front of the pump.

5.19 Filter the medium through a 0.22uM syringe filter or Nalgene filtration unit (500ml, or appropriate volume) using a vacuum pump to draw the medium though the filter:

• Switch on the vacuum pump and then connect the pump tubing to the filter unit.
• When all the medium has passed through the filter remove the tubing from the filter unit and THEN switch off the pump.
• You never want to turn off the vacuum pump while it is attached to the filter unit as you could get suck back, where dirty air/liquid from pump is sucked into your sterile filtered medium.

5.20 Transfer the inlet tube to the bottle of filtered medium. Pump through the column at 1ml/min. Watch until medium is passing out the outlet tube then transfer the outlet tube so that the flow through collects into a sterile conical flask. Make sure you do not leave the pump unattended towards the end of the pumping period. Otherwise you risk ruining the column by pumping air through it.

5.21 Transfer the inlet tube back to the bottle of PBSa, and wash column collecting to waste. Collect an aliquot of the flow through.

5.22 Before eluting mix 500µl of 0.1M Glycine pH2.7 and 60-200µl 1M Tris-HCL pH 9. Using pH paper ensure this mix is neutralised at pH7. Prepare 10-12 1.5ml eppendorf collection tubes containing an appropriate volume of 1M Tris-HCL pH 9 to neutralise 500µl of 0.1M Glycine pH2.7.

5.23 Switch off the pump and transfer the inlet tubing to a universal containing 3-4ml 0.1M Glycine pH2.7. Switch on the pump. Pump through the column for 90 sec. Place the first elution collection tube under the column, continue to pump whilst collecting 0.5ml of elute every 30 sec. When the Glycine levels get low replace with PBSa.

5.24 Transfer the inlet tube back to the bottle of PBSa and wash the column collecting to waste.

5.25 Pump at least 20 mls of 20% Ethanol through the column. (You can check when Ethanol is emerging to waste by looking for refractive index changes and floating of the eluting fluid).

5.26 Stop the pump and remove the tubing from the bottom of the column. Switch on the pump and hold the bottom seal of the protein G column under the column to allow it to fill with ethonal before attaching loosely while the pump is still running. This will ensure all air is expelled. Turn of the pump and tighten seal. Loosen the top fitting of the protein column (by turning the column), then start the pump and run while removing the top fitting completely. Stop the pump and replace the top seal of the protein G column. Tighten both top and bottom seals as tight as possible by hand before returning the column to its box and to storage at 4oC. WRITE THE DATE, INITIALS AND USAGE ON THE BOX.

5.27 Analyse protein levels in the collected fractions by reading A280. The protein tends to collect in three aliquots.

5.28 Take the peak fractions and transfer to a Float-A-Lyser Dialysis Cassette (or equivalent) to dialise. If using a Float-A-Lyser remove from packaging by holding it firmly at the collar of the floatation disk. Detach the packaging tube and discard the 0.1% sodium azide preservative. Remove the cap and discard the 0.1% sodium azide from inside the membrane. Dispense PBSa inside the membrane to wash and discard. Using a pipette carefully load sample and snap cap back into place. Float in a 1L cylinder of PBSa on a magnetic stirrer. Work at 4oC. Change the PBSa three times over a 24 hour period. After dialysis open the cap and recover sample using a pastette.

5.29 Analyse protein levels by reading A280. Adjust to 1mg/ml with PBSa. If necessary concentrate. Pass through a 0.2µm low protein binding small volume syringe filter to sterilise. Store main aliquot at 40C and open only under sterile conditions. Take a small aliquot and add 10% sodium azide, diluting 1 in 100 to give 0.1% final concentration. This working aliquot can be opened on the bench. Store at 40C.







6. Results

A2801=0.7mg/ml
7. Maintenance
Rinse all pump and associated tubing with 70% Ethanol, drain, and store in the bags provided to avoid contamination.


8. Troubleshooting
Air bubbles before column. You can pump the system backwards for a VERY short time, before the drop at the outlet of the last column reaches the packed medium. This can be used to flush small bubbles from end of the inlet tube. In other case, you will have to dismantle the tubing arrangements and re-prime them. Care must be taken to avoid gravity driven flows by keeping disconnected tubing at appropriate levels. The in-line filters will provide some protection against pumping air onto the columns should the tubing accidentally come out of the source vessel, or should the source vessel become emptied. High surface tension will provide back-pressure preventing flow when the filters are full of air. However, this may not work for high-protein solutions, such as serum, with reduced surface tension, and may lead to explosive disconnection of the tubing under the back-pressure. Therefore this should NEVER be used as a substitute for appropriate surveillance of the column. DO NOT RUN COLUMN OVERNIGHT.