Enzymic fragmentation of monoclonal antibodies

2.12 Enzymic fragmentation of monoclonal antibodies

Removal of the Fc region of monoclonal antibodies, to leave a divalent fragment which is no longer able to bind to Fc receptors or to activate the complement pathway, is often desirable but, in practice, is often difficult or impossible to achieve. Amongst the IgG sub-

classes there is a differential sensitivity to proteolysis; with pepsin, for example, IgG 2b is most sensitive followed by IgG 3 > IgG 2a > IgG 1 , while for papain, IgG 1 is most sensitive followed by IgG 2a > IgG 3 > IgG 2b .

There are individual sensitivities unique to each monoclonal antibody, so it is essential to per- form a range of trial digests on each antibody preparation to optimize conditions. Each batch of the same monoclonal antibody will be structurally identical; once the digestion conditions are determined they will not need to be varied.

In the experiment described below, a batch of purified antibody is mixed with the enzyme, under digestion conditions and samples taken for fragment analysis by SDS-PAGE to determine the optimal time of incubation, i.e. that which gives maximum yield of the desired fragment with minimum subfragmentation to smaller peptides.

2.12ENZYMIC FRAGMENTATION OF MONOCLONAL ANTIBODIES

Note: Only small amounts of monoclonal IgG will be available; slightly lower concentrations are used than for rabbit or human IgG.

2.12.1 Pepsin digestion

MATERIALS AND EQUIPMENT

0.1 M citrate buffer, pH 3.5 IgG, monoclonal antibody Pepsin (1 mg/ml) in 0.1 M citrate buffer, pH 3.5 SDS-PAGE (see Appendix B.2.1)

METHOD

1 Dissolve or dialyse the IgG in the citrate buffer and adjust concentration to 1 mg protein/ml.

2 Place 300 µl in a small tube and add 6 µl of a 1 mg/ml solution of pepsin in the same buffer and incubate at 37°C.

3 Immediately (time 0) and then at 5, 10, 15 and 30 min, 1, 2, 4, 8, 16 and 24 h, remove a

20 µl aliquot.

4 The pH of each aliquot should be adjusted to just above 7.0 to stop the reaction.

5 At the end of the time course, analyse the fractions by SDS-PAGE under reducing and non-reducing conditions (see Appendix B.2.1).

TECHNICAL NOTE From an examination of these gels, for example Fig. 2.2, it should be possible to select a suitable set of conditions to apply to a bulk preparation of the monoclonal antibody. The fragments may then

be separated by a combination of gel filtration and protein A chromatography.

2.12.2 Papain digestion

Papain needs activation by cysteine accomplished by either: (a) incorporating cysteine in the reaction mixture or (b) pre-activating the papain prior to addition to the digestion mixture.

Using pre-activated papain with mouse IgG sometimes gives a F(ab′) 2 fragment similar to the pepsin F(ab′) 2 . If cysteine is present during the enzyme reaction the fragmentation proceeds to give Fab and Fc.

MATERIALS AND EQUIPMENT Papain Phosphate-buffered saline (PBS) IgG monoclonal antibody (2 mg/ml in PBS) 0.002 M ethylene diamine tetra-acetic acid (EDTA) in PBS

0.5 M cysteine in PBS

0.1 M iodoacetamide Sephadex G-25 Chromatography column, e.g. 10-ml disposable syringe plugged with glass wool (Caution: Wear

gloves when handling glass wool )

64 C H A P T E R 2: Monoclonal antibodies: production, purification and enzymatic fragmentation

Fig. 2.2 Time course of papain digestion of monoclonal IgG. To determine the optimum conditions for the papain digestion of a monoclonal antibody a time course experiment was performed. Samples were taken, at intervals, and run on an SDS-PAGE system in non-reducing conditions.

0 Sample prior to digestion. 0 Reaction stopped immediately on addition of papain. 5 Reaction stopped 5 min after addition of papain.

10 Reaction stopped 10 min after addition of papain. 15 Reaction stopped 15 min after addition of papain. 30 Reaction stopped 30 min after addition of papain.

1 Reaction stopped 1 h after addition of papain. 2 Reaction stopped 2 h after addition of papain. 4 Reaction stopped 4 h after addition of papain.

Easily detectable digestion products are visible at first sampling at 5 min. The reaction proceeds through a number of intermediates, such as one Fab linked to Fc, before arriving at the 50 kDa products. With prolonged incubation small molecular weight degradation products begin to appear. Choosing an optimum time is a balance between efficient usage of starting material and not too much degradation.

METHOD Pre-activation of papain

1 Place 100 µg papain in a small tube containing 50 µl 0.002 M EDTA in PBS and 10 µl 0.5 M cysteine in PBS.

2 Incubate at 37°C for 30 min.

3 Prepare a small Sephadex G-25 column. A disposable 10-ml disposable syringe plugged with glass wool at the bottom would be suitable. The column should be filled with Sephadex G-25 and equilibrated with PBS containing 0.002 M EDTA.

4 Apply the activated papain to the column and elute with PBS-EDTA. Collect 0.25 ml fractions and determine their protein content to locate the papain peak. Use the peak tube for the digestion; this should contain about half the total absorbance units of enzyme.

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2.12ENZYMIC FRAGMENTATION OF MONOCLONAL ANTIBODIES

Digestion of IgG

5 Add the papain to 1 mg IgG in 0.5 ml PBS containing 0.002 M EDTA.

6 Incubate at 37°C.

7 Immediately remove 20 µg IgG and mix with 0.1 M iodoacetamide to a final concentration of 0.025 M to stop the reaction.

8 Remove further 20 µg samples at 5, 10, 15 and 30 min, 1, 2, 4 and 8 h and each time stop the reaction with iodoacetamide.

9 Analyse the fractions by SDS-PAGE in both non-reducing and reducing conditions. As the samples contain iodoacetamide, add 2-mercaptoethanol to a concentration of 0.02 M before proceeding to the normal sample preparation conditions for the reducing gel (see Appendix B.2.1).

Having established the optimal conditions for digestion, proceed with the bulk preparation and isolate the resultant fragments by gel filtration and protein A affinity chromatography.