chromatographytech note 2849

Protein A Removal From IgG on
CHT™ Ceramic Hydroxyapatite Support
Samuel G Franklin, PhD, Bio-Rad Laboratories, Inc., 2000 Alfred Nobel Drive,Hercules, CA 94547 USA

Introduction
Affinity chromatography with immobilized protein A from
Staphylococcus aureus or from recombinant sources has
been used for the purification of immunoglobulins since the1970s (Gagnon 1996). Much of its popularity is due to thesimplicity of the method itself, the high product purity andrecovery, and protein A’s selectivity to IgG class and species.
Good clearance of DNA, virus, and endotoxin is often
obtained, plus the immobilized protein A is relatively resistant
to chemical regeneration and sanitization procedures.
Despite these advantages, conditioned media and cellular
lysates frequently contain varying amounts of proteases,
which can release fragments of protein A from immobilizing
supports (Bloom et al. 1989). This is of great concern to thediagnostics and biopharmaceutical industries due to the wide
range of immunomodulatory effects associated with protein
A and its fragments (Gagnon 1996).
A number of chromatographic methods have been used
to separate protein A from IgG. Cation exchange
chromatography is attractive because conditions can bedevised in which protein A does not bind to the support while
IgG is retained. However, process development is often
required and chaotropic agents may be needed to disrupt theprotein A-IgG complex. Anion exchange chromatography
has been used, but suffers from the disadvantage that thestrength of the protein A-IgG association is enhanced byelevated pH. Since both IgG and protein A are retained,coelution of protein A fragments with IgG can occur (Bloomet al. 1989). Use of hydrophobic interaction chromatographyfor protein A-IgG separation has also been reported. Processdevelopment is again required since high salt concentrations
increase the affinity of protein A for IgG. Coelution of protein
A and IgG is common, reducing contaminant clearance andproduct yield.

Mariani et al. (1989) developed a simple method for thepurification of IgG1 in the presence of protein A onhydroxyapatite (Bio-Rad HPHT). They started with an ascites
fluid ammonium sulfate fraction. In recent years, HPHT
has been replaced by the more robust CHT ceramichydroxyapatite, which was designed for process-scale
separations. In this report, we show that CHT can resolve
IgG1 from an IgG1-protein A complex in unfractionatedconditioned media. We have also optimized conditions for theseparation of protein A and an IgG4 by cation exchange
chromatography on UNOsphere™ S support.
Results and Discussion
Initial studies in our laboratory indicated that recombinant
protein A and IgGs from various sources exhibited virtually
identical retention times on CHT under several elution
conditions. We noted, however, that when small amounts ofrecombinant protein A were mixed with mouse ascites fluid
containing IgG1, an additional peak of higher retention timethan any other component in the system was generated.
This finding inspired more detailed studies.
We obtained a conditioned medium containing about
0.24 mg/ml of mouse IgG1. When this medium orrecombinant protein A was applied directly to a CHT column
at pH 7.0 and eluted with a sodium phosphate gradient,peaks with essentially identical retention times were obtained(Figure 1, pink and green traces). However, upon mixing
increasing amounts of recombinant protein A with the IgG1containing medium, an additional peak was seen with ahigher retention time than any other in the chromatogram.
In concert, the IgG1 peak appeared to diminish in intensity
until, at the highest recombinant protein A concentration,
most of the IgG1 peak was depleted. This suggested that astable complex between IgG1 and protein A was being
formed in these experiments and that this complex had ahigher retention time than the other species present.

A0.2A280

BC0.1

0.0
02550Run time, minFig. 1. CHT chromatography of conditioned medium containing murine IgG1and spiked with increasing amounts of recombinant protein A. Pink line, murine
IgG1, 6.0 mg; green, recombinant protein A, 4.2 mg; dark blue, 0.48 mg
IgG1 + 0.05 mg protein A; light blue, 0.48 mg IgG1 + 0.1 mg protein A; purple,0.48 mg IgG1 + 0.2 mg protein A. Proteins were mixed in a total volume of
6.0 ml buffer A (5 mM sodium phosphate buffer, pH 7.0). The column was a7 x 50 mm Bio-Scale™ column packed with CHT Type I, 20 µm (catalog#158-2000) equilibrated with buffer A. Elution was with a 20 column volume
gradient to 200 mM sodium phosphate buffer, pH 7.0. at a flow rate of
1.5 ml/min. Detection was by absorption at 280 nm on a BioLogic™ HRsystem. The fractions at the apex of peaks A, B, and C were analyzed by
SDS-PAGE (Figure 2). Scale offset for comparison.

Mariani et al. (1989) did not attempt to demonstrate that theadditional peak they had observed on HPHT upon mixing
partially purified IgG1 and protein A was actually a complex ofthe two proteins. To show this, we picked a ratio of IgG1 torecombinant protein A of about 0.48 to 0.15 mg, which we
had calculated would produce IgG1 and IgG1-protein Acomplex peaks of approximately equal intensity on elution
from the CHT column. The peaks were well resolved and ofnearly equal height.
Fractions were collected and fractions from each of the threemain peaks (A, B, and C) were examined with SDS-PAGE
under reducing and nonreducing conditions. The gel was
stained with an experimental fluorescent stain that is about
50 times more sensitive than Coomassie Blue (Figure 2).
Lanes 2 and 3 of this gel show a commercially purified
recombinant protein A standard, which appears to containnumerous trace impurities or degradation products. Lanes 4and 5 contain the conditioned medium and show that IgG1 is
the main component. The unbound peak A (lanes 6 and 7)

appears to contain little or no protein and is probably
indicator dye and other medium additives. Lanes 8 and 9 arefrom peak B and show this fraction to be the purified IgG1 asexpected. However, lanes 10 and 11 are from peak C, theputative IgG1-protein A complex, which show two bands inaddition to the IgG1 components. One of these bands
comigrates with the main protein A band. The other band has
a slightly higher mobility and is probably a proteolytic fragmentof the recombinant protein A.
12

3

4

56

78

9101112– 200 kD
– 116
– 97
– 66
– 45
– 31
– 22
– 14– 7Fig. 2. SDS-PAGE analysis of CHT column fractions. Lanes 1 and 12, broadrange molecular weight standards (catalog # 161-0317); lanes 2 and 3,protein A standard, reduced and unreduced; lanes 4 and 5, IgG1 in
conditioned medium, reduced and unreduced; lanes 6 and 7, peak A,reduced and unreduced; lanes 8 and 9, peak B, reduced and unreduced;
lanes 10 and 11, peak C, reduced and unreduced. The gel was a 4–20%
Ready Gel® precast gel.

In biopharmaceutical production processes, additional
polishing steps are often included to ensure a pure product.
We evaluated cation exchange chromatography onUNOsphere S as a potential additional polishing step.
Protein A has an isoelectric point of 5.1, and an examinationof its calculated titration curve revealed that below pH 5.1,
the negative charge on protein A increases rapidly, while
above this pH, the change is much more gradual. Thus,
protein A and a human IgG4 both bind to UNOsphere S
below pH 5.1. At pH 6.0, which does not favor interaction
between protein A and IgG4, the two proteins could beseparated (Figure 3). The proteins were run individually andmixed at various ratios, but retention times and peak shape
did not vary significantly. This suggests that little or no protein
A-IgG4 complex was formed under these conditions(unpublished data).

0.850References

45Bloom JW et al., Detection and reduction of protein A contamination in
immobilized protein A purified monoclonal antibody preparations, J ImmunolMethods 117, 83–89 (1989)

403530

A280

0.6

250.4

20
150.2

Conductivity, mS/cm

1.01050.00010203040Run time, minGagnon P, Purification Tools for Monoclonal Antibodies, Chapter 9, Validated
Biosystems, Inc., Tucson, AZ (1996)
Lai H and Franklin SG, Purification of transgenic antibody from corn seedusing UNOsphere S and CHT ceramic hydroxyapatite supports, Bio-Radbulletin 2774
Lai H and Franklin SG, Purification of murine IgG using UNOsphere S andCHT ceramic hydroxyapatite chromatography, Bio-Rad bulletin 2780
Mariani M et al., Purification of monoclonal antibodies for in vivo use: a twostep HPLC shows equivalent performance to immunoaffinity chromatography,
Biochromatography 4, 149–155 (1989)
Coomassie is a trademark of Imperial Chemical Industries PLC.

Fig. 3. Separation of recombinant protein A and human IgG4 on UNOsphere Ssupport. The protein A and IgG4 (30 and 4.8 mg, respectively) were mixed in a
total volume of 6.0 ml of buffer containing 50 mM MES, 1.0 mM NaH2PO4, pH
6.0. Proteins were applied to a 7 x 50 mm Bio-Scale column containing
UNOsphere S (catalog # 156-0111). Elution was by a 20 column volume
gradient from 0 to 0.5 M NaCl. Flow rate was 2.0 ml/min. Light blue line, A280;
pink line, conductivity. A BioLogic HR chromatography system was used.

Recovery of IgG from the protein A spiking experiment onCHT (see table) was about 64%. Almost 30% of the applied
IgG was recovered in the protein A-IgG complex. About
85% of the IgG and 98% of the protein A applied to theUNOsphere S column was recovered.
We have shown that both supports offer high recovery andare effective in removing protein A from IgG from conditioned
medium. These and other studies (Bio-Rad bulletins 2774
and 2780) also show high purity of the IgG recovered from acomplex mixture using CHT and UNOsphere S. CHT andUNOsphere S chromatography are effective for protein Aremoval and as polishing steps in biopharmaceuticalpurification processes.
Table. Protein recovery from CHT and UNOsphere S columns.
Protein Recovery (%)*
SupportIgG

IgG-Protein A Complex

CHT**

63.8

28.9‡

ND

Protein A

UNOsphere S†

84.8

ND

98.6

* Recovery calculated by A280 of all pooled fractions.
** The CHT column load was conditioned culture medium containing 0.48 mg
IgG1 and 0.1 mg recombinant protein A. Conditions were as in Figure 1.
†The UNOsphere S column load was 5.0 mg purified human IgG4 and 30 mg
recombinant protein A. Conditions were as in Figure 3.

‡Expressed as %IgG1 recovered. This value was reduced by 10% to account forthe protein A contribution to absorbance (the extinction coefficient of protein A
is 10% that of IgG).

Information in this tech note is current as of the date of writing (2002) and notnecessarily the date of this revision or printing.

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