Purification of Endotoxin Free mAbs

The following method is recommended for purification of monoclonal antibodies produced by tissue culture (hollow fibre cartridges or roller cultures) for laboratory use.

General notes:

1) Endotoxin - you must assume that all standard laboratory glassware will be coated with loads of endotoxin. Wherever possible use sterile, specifically endotoxin-free (clinical grade) disposable plastics. If you need to use glassware or other materials that are not guaranteed endotoxin free you should soak them in 0.5M NaOH and then rinse thoroughly with endotoxin free water. Likewise, assume any source of water or buffers are contaminated unless you have checked them using the standard LAL assay or similar (all starting reagents, including medium and buffers should be less than 1EU per ml). Generally speaking, once you have endotoxin in any of your reagents or samples it is alomost impossible to remove it (even though you will find adverts that claim various columns can remove endotoxin, we have not found those we have tried to be very efficient).

2) Concentration - if convenient volumes (eg. less than 1 litre) are being processed you can proceed straight to the ammonium sulphate precipitation step. If you have larger volumes, or your antibody is present at low concentrations/poor yields, we find it useful to concentrate the supernatants by circulating them through the same type of hollow fibre dialysis cartridges we use for cell growth, allowing the slight pressure of circulation to force water out through the membrane, and rapidly concentrating the antibody/proteins up to 100 fold. The advantage of this method is very large volumes can be processed using sterile and endotoxin free components.

Ammonium Sulphate Precipitation

Prepare saturated (NH4)2SO4, using Analar Grade reagent, 1kg per litre endotoxin free water, and autoclave in NaOH treated glass bottles. Store at 4 deg C. Mix supernatant above excess crystals before use to ensure even saturation.

Add equal volume of saturated (NH4)2SO4 to your (concentrated) antibody sample. Mix well and leave to stand at 4 deg C (overnight if not previously concentrated). Spin out antibody precipitate (eg 30 mins 3500rpm using 50ml Falcon tubes in bench centrifuge). Discard supernatant, and redissolve pellet in endotoxin free water. Repeat saturated (NH4)2SO4 precipitation.

Redissolve Ig fraction pellet in endotoxin free water and dialyse against PBS using at least 3 changes of 100x volume. (Dialysis can be performed either in hollow fibre cartridges, with antibody circulating through the fibres and buffer circulating in the opposite direction outside the fibres, for very large volumes, or using standard dialysis tubing that has been boiled in endotoxin free water containing 10mM EDTA and subsequently stored sterile in endotoxin free water containing 0.1% sodium azide).

Finally, centrifuge (at least 3500rpm for 30 min in bench centrifuge) and 0.2 micron sterile filter to remove any grossly aggregated material, and store frozen at -20 deg C.

If your supernant was produced in a hollow fibre cartridge with good yields (ie more than 1mg/ml with less than 5% FCS) then you should expect that more than 90% of the protein visible on a native gel electrophoresis will be the monoclonal antibody. In this case we normally estimate antibody concentration simply by the OD280/1.4 value. This material is suitable for most purposes, including in vitro conjugation to FITC or biotin, or for experimental use in vivo. Levels of endotoxin at this point should be less than 20EU per ml (or less than 10EU per mg of protein) using the LAL assay.

Ion Exchange Purification

This step is necessary if your starting yield was lower than 1mg/ml or if higher purity is required. Ion exchange, rather than affinity purification (eg protein A or G) is recommended, because ion exchange materials are high capacity, cheap enough to be disposable, and can easily be sanitized with 0.5M NaOH. The following method is applicable to most rat and mouse IgG antibodies (not IgM), although individual monoclonal antibodies may require modification of either the pH or ionic strength for optimal purification. Cation exchange using Fast Flow S-Sepharose (Sigma S 1264) at pH5.4 has the advantage that most antibodies will bind, while albumin and degraded antibody will not. This allows reasonable yields in a simple, step-wise absorption, wash, elution method performed entirely in tubes with centrifugation, minimizing the risks of introducing endotoxin by the use of columns etc. However, for maximal purity, and elimination of all residual bovine Ig and transferrin, elution and fractionation with a slowly increasing NaCl concentration would be advantageous.

Dialyse your (NH4)2SO4 precipitated antibody preparation into 1x Malonate buffer (buffer A). At this point you will probably get quite a large precipitate - this is mostly degraded proteins - cellular components, FCS and denatured antibody (NB. you get very little precipitate from ascitic fluid preparations).

        To make a 10x stock of buffer A:

        Malonic Acid         104g
        NaOH                  60g
        Betaine               20g
        Make to 2 litres with endotoxin free water.
        (NB. This gets quite hot) 
        should be pH 5.2 - pH 5.4 at 25 deg C 

(pH very temperature sensitive - always use at 20-25 deg C) Sterile filter to 0.2micron and store at 4 deg C (bugs will grow in this very well if not kept sterile!)

        To make Buffer B (1x):

Make 1x buffer A, but containing 0.5M NaCl Filter to 0.2micron if not used immediately

Spin out precipitate and discard. If you dialysed at 4 deg C allow to warm to room temperature. Ensure all steps from here are with room temperature buffers etc.

Measure OD280 and adjust to between 1-20 mg/ml in buffer A. You will need to use the minimum amount of Fast Flow S-Sepharose to maximise yield, so estimate the total amount of Ig in your preparation (ie. approx. purity on gel x OD280/1.4) and use 1ml of packed gel for every 20mg of antibody. If you use excess the eluted antibody will be more dilute.

Prepare Fast Flow S-Sepharose: if this is from a fresh bottle it is usually sufficient just to wash it three times (by centrifugation for 2 mins at 2000 rpm in bench centrifuge) in buffer A. Otherwise you should first wash once in 0.5M NaOH, followed by buffer B, and then three washes with buffer A.

Incubate the antibody previously dialysed into buffer A with correct volume of S-Sepharose for 1 hour at room temperature, with gentle rotation.

Remove supernatant (keep and check that antibody has been adsorbed by running analytical gel at end), and wash three times with buffer A (each wash at least 5 gel volumes).

Elute antibody by adding 1 gel volume of buffer B, rotating at room temperature for 5 mins, centrifuging, and collecting supernatant. Repeat with another column volume and pool eluates.

It is a good idea to make certain that no Sepharose remains that could re-adsorb antibody in the eluate, either by spinning again or passing the antibody through a sterile 0.2 micron filter.

Dialyse against two changes of PBS, check final OD280 and purity on SDS reducing PAGE and native PAGE. Generally, approx. 95% of the protein should be antibody, appearing as a single band (or cluster of close bands) on native PAGE. With optimal conditions, yields should exceed 70% and the final Ig concentration should be from 5-15 mg/ml.

Steve Cobbold 31/7/96