Separation of Fab and Fc Fragments from Monoclonal Antibody Papain
Digest on Ceramic Hydroxyapatite and Ceramic Fluoroapatite
Cheryl S. Aberin* and Mark Snyder, Bio-Rad Laboratories Inc., Research and Development Department, Process Chromatography Division

Abstract

Chromatographic Conditions:

Antibody fragments have a wide variety of uses in analytical and diagnostic
applications. They are also becoming a major biotherapeutic agent fortreatment of human diseases. Protein A and Protein G affinity separations, ion
exchange, and gel filtration chromatography are techniques commonly used toseparate Fab and Fc antibody fragments.
This study describes an alternative technique for separating Fab and Fc
antibody fragments using CHT™ ceramic hydroxyapatite Type I and Type IIand CFT™ ceramic fluoroapatite Type II.
Materials and Methods
Purified monoclonal antibody (Mab-G) was digested with immobilized
papain for 6 and 17 hr. The digestion mixtures were analyzed using aBio-Sil® SEC 250-5 size based separation column. A small quantity of
undigested antibody remained.
Separations of Fab and Fc fragments were evaluated on CHT ceramichydroxyapatite and CFT ceramic fluoroapatite using a sodium phosphate
gradient at two different pH values, 6.5 and 7.0. Crude digested antibody wasloaded onto the column with 0.005 M sodium phosphate as the loading bufferat 311 cm/hr (2 ml/min) using a BioLogic DuoFlow™ chromatography system.
Fab and Fc fragments were eluted off the column from 0–0.25 M sodium
phosphate buffer for 40 column volumes. Fractions collected were analyzed bySDS-PAGE under nonreducing conditions with Precision Plus Protein™
molecular weight standards and Fab standard. Fab standard was obtained bypurifying the crude digested material in CHT support equilibrated with 25 mM
HEPES plus 2.5 mM CaCl2, pH 7.0 (Gagnon et al. 2009). The results ofseparations of Fab into Fc and residual antibody (Ab) fragments on both apatite
media were analyzed by nonreducing SDS-PAGE. Equal volumes of pooled
fractions were loaded into the gel except the original material (undigested) anddigested monoclonal antibody where differing volumes were used.

Sample:Columns:
Bed volume:
Buffer A:
Buffer B:
Buffer B2:
Flow rate:
Gradient profile:All chromatographic data were analyzed by nonreducing SDS-PAGE.
Electropherograms showed the same results on all chromatography
experiments (data not shown).

Crude digested antibody (digested for 6 hr)
CHT Type I, 20 and 40 µm
CFT Type II, 40 µm
1 ml0.005 M sodium phosphate, pH 6.5 and 7.00.250 M sodium phosphate, pH 6.5 and 7.00.500 M sodium phosphate, pH 6.5 and 7.02 ml/min (311 cm/hr)
100% A for 7 CV; 0–100% B1 for 40 CV;
100% B1 for 5 CV; 100% B2 for 5 CV

Figure 2A represents the CFT Type II, 40 µm, chromatogram fromFigure 1. Figure 2B is representative of pooled fractions analyzed bySDS-PAGE on all experimental runs performed on CHT and CFT media.

Chromatograms presented in Figure 1 have the same general elution profile.
CHT Type I, 40 µm, at pH 6.5 and 7.0 has broader peaks than CHT Type I, 20µm, at pH 6.5 and 7.0 because of its larger particle size. The antibody fragments
and intact IgG eluted earlier on CFT support than on CHT support, whichindicates lower affinity to the media.

Fab and Fc fragments were converted into smaller fragments after 6 hrof digestion. Digestion yielded low molecular weight fragments of Fcand Fab shown in Figure 2B, lane 3, which were identified by LC-MS(liquid chromatography and mass spectrometry) analysis (courtesy ofDr Tim Wehr).
The conditions used for separations of Fab to Fc and intact IgG after
17 hr digestion, evaluated on CHT and CFT media were as follows:

Separations of antibody fragments were performed also using low-phosphatebuffer with a high concentration of sodium chloride. There were no separations ofFab to Fc and intact IgG observed; hence all components eluted as one peak(data not shown).

A.

Sample:Columns:
Bed volume:
Buffer A:
Buffer B1:
Buffer B2:
Flow rate:
Gradient profile:

Crude digested antibody (digested for 17 hr)
CHT Type I, 20 and 40 µm
CHT Type II, 40 µm
CFT Type II, 40 µm
1 ml
0.005 M sodium phosphate, pH 7.00.250 M sodium phosphate, pH 7.00.500 M sodium phosphate, pH 7.02 ml/min (311 cm/hr)
100% A for 7 CV; 0–100% B1 for 40 CV;
100% B1 for 5 CV; 100% B2 for 5 CV

The results obtained for these experiments were shown in Figure 3.
Chromatographic profiles of all the experimental runs were the same
except for the retention time. Antibody fragments were retained less on
CHT Type II than in the rest of the experimental runs, which exhibited
weak binding to the media.

Lane 8CHT Type II 40 µm at pH 7.0

Lane 5The separations of Fab from Fc and intact IgG from the mixture that was digested 6 hr wereevaluated on CHT Type I, 20 and 40 µm particle sizes, and CFT Type II, 40 µm particle size,with the following results (Figure 1):

Lane 9

CFT Type II 40 µm at pH 7.0

FabFc

CHT Type I 20 µm at pH 7.0

IgG

B.

CFT Type II 40 µm at pH 6.5
CHT Type I 40 µm at pH 6.5
CHT Type I 20 µm at pH 6.5

123456789150
100

4567891011100
7550FcFab37252015Figure 4. Nonreduced SDS-PAGE of Fab to Fc separations on CFT TypeII, 40 µm, at pH 7.0. Lanes 1, 5, and 11, Precision Plus Protein molecularweight standards; lane 2, Fab standard; lane 3, undigested Ab; lane 4,digested Ab; lane 6, Fc fragments; lane 7, Fc and Fab fragments; lane 8,Fab standard; lane 9, Fab fragments; lane 10, intact IgG.
Increased amounts of low molecular weight Fc and Fab antibody fragments
were seen throughout the gel in Figure 4. This might be due to overdigestionof the monoclonal antibody sample. It was apparent from the SDS-PAGE that
prolonged digestion time resulted in further cleavage of the antibody
fragments into smaller fragments.

Conclusions
Separations of Fab to Fc and intact IgG can be performed on CHT and CFT
media using a sodium phosphate gradient system alone. This technique isvery simple and can be implemented in a large-scale manufacturing process.
Overdigestion or prolonged digestion time of monoclonal antibody in papain
generated increased amounts of low molecular weight fragments of Fc andFab antibody fragments.

Acronyms- Fragment having the antigen binding site
- Fragment that crystallizes- Size exclusion chromatography- Immunoglobulin G- Column volume- Sodium dodecyl sulfate polyacrylamide gelelectrophoresis

Reference755037Fc
Fab2520Gagnon et al. 2009, Reverse calcium affinity purification of Fab with calcium
derivatized hydroxyapatite, J. Immnunol. Methods 342, 115-118.

10Figure 3. Separations of Fab to Fc and intact IgG on CHT TypeI and II, 20 and 40 µm, and CFT Type II, 40 µm, at pH
7.0.

Figure 2. A) Fab, Fc, and intact IgG chromatographic elutions on CFTType II 40 µm; B) Nonreduced SDS-PAGE of Fab, Fc, and intact IgG;
lanes 1 and 4, Precision Plus Protein molecular weight standards; lane
2, undigested Ab; lane 3, digested Ab; lane 5, Fc fragment; lane 6, Fcand Fab fragments; lane 7, Fab standard; lane 8, Fab fragment; lane 9,intact IgG.

The first major peak on CHT Type I, 20 µm, was an artifact peak due to thedifference in buffer composition. This was determined when the digested
monoclonal antibody sample was buffer exchanged to low sodium phosphatebuffer before loading to the column (see Figure 3 for the experimental runs).
With or without buffer exchange of the digested antibody sample, selectivity
was not affected on CHT and CFT media.

15Figure 1. Separations of Fab to Fc and intact IgG on CHT and CFTmedia on a sodium phosphate gradient at pH 6.5 and 7.0.

3150

FabFc
SECIgGCVSDS-PAGE

250

CHT Type I 20 µm at pH 7.0

2

CHT Type I 40 µm at pH 7.0

Lane 6CHT Type I 40 µm at pH 7.0

1250

10

The first major peak on Figure 1 was caused by the difference in buffer
composition; this was determined later in this study. The second major peak onFigure 1 represents the Fc fragment; the third peak represents the Fab fragment
and intact IgG was eluted at the end. Eluted peaks at pH 6.5 were more strongly
retained than those at pH 7.0. This exhibits strong affinity at lower pH value.

Results

CFT Type II 40 µm at pH 7.0

More of the intact IgG was converted to Fab and Fc fragments after 17 hr ofdigestion than was converted after 6 hr of digestion. Residual amounts ofintact IgG were still present (see Figure 4, lane 4).

Acknowledgement
Thanks to Dr Tim Wehr for performing LC-MS identification of Fab and Fc
fragments on CHT and CFT fractions.

Presented at: 5th Conference on Hydroxyapatite and Related

Products, October 11-14, 2009, Rottach-Egern (Munich), Germany.
*Corresponding author: Cheryl_Aberin@Bio-Rad.com
Bulletin 5913 Rev A