Removal of Aggregate from an IgG4 ProductUsing CHT™ Ceramic Hydroxyapatite Resin
TechNote

Samuel G Franklin, PhD
Bio-Rad Laboratories, Inc., 2000 Alfred Nobel Drive, Hercules, CA 94547

Process Separations

CHT Ceramic Hydroxyapatite exhibits electrostatic, repulsive,and coordinate covalent bond formation in interacting withprotein species. We have explored its potential in removing
aggregate from a purified IgG4 biopharmaceutical known tocontain aggregate, and report here on a simple and effective
process to do so.
Results
Size exclusion chromatography analysis of the IgG4 sample(derived from chromatogram a, Figure 1) indicated that it
contained 86.6% monomer of 141.9 kD and 10.1% aggregate,
which consisted of nearly equal amounts of 485.3 kD and>1.1 MD species. Possible degradation products withapparent molecular weights below 3,000 were also present(about 1.0% of total protein).

Fig. 1. Size exclusion chromatography
analysis of IgG4 samples.
Chromatogram a (—), starting material;
chromatogram b (—), main peak(Figure 2, peak a) from the CHT Column(scales are offset for comparison).
Column, Bio-Sil® SEC 250-5,7.8 x 300 mm (catalog #125-0062);
buffer, 0.1 M sodium phosphate, pH
6.8, containing 0.15 M NaCl; flow rate,
1.0 ml/min. Molecular weights werecalculated by calibration with standards(IgG, ovalbumin, myoglobin, vitamin B12,
catalog #151-1901)

0.7A280

0.6
0.5
0.4
0.3
0.2

a0.1

b0.0
05

101520Run time, minFractions

2 3 4 5 6 7 8 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55 57

1.50abc45

1.25

40
35

1.00

30

0.75

25
20

0.50

15
100.250.00

50

Conductivity, mS/cm

Most chromatographic techniques would be expected to
be able to remove aggregated or multimeric species of
immunoglobulin from the monomer. However, techniques suchas ion exchange and hydrophobic interaction chromatography
may also induce the formation of additional aggregate or
multimer due to increased protein concentration or changes
in salt concentration, pH, or both required for elution. Sizeexclusion chromatography does not suffer from thesedisadvantages, but does result in significant dilution of theproduct. If large quantities of product are required, large
size exclusion columns, concentration steps, and support
equipment will add considerably to production costs. In somecases, additional product loss due to denaturation may occur
during dilution.

0.8

A280

Introduction
The presence of even trace amounts of aggregate inimmunoglobulin biopharmaceuticals is of significant concern
during all phases of drug development, from process design
to quality control. The formation of aggregates can adversely
impact process economics by causing decreased productyield, peak broadening (requiring the addition of polishingsteps), and loss of activity (Gagnon 1996). Aggregate formation
can also adversely affect product safety because it can cause
complement activation or even anaphylaxis upon injection(Ritchie and Navolotskaia 1996, WHO Report 1995).

Bulletin 2940

50102030Run time, min

40500Fig. 2. CHT chromatography of IgG4 sample containing aggregate.
Chromatography was performed on a BioLogic DuoFlow™ 10 System(catalog #760-0037). Sample (12 mg) was loaded onto a 7 x 52 mm Bio-Scale™
MT Column (catalog #751-0081) containing CHT, 20 µm (catalog #158-2000)
in 20 mM sodium phosphate, pH 7.0; elution was from 20 to 160 mM sodiumphosphate in 20 column volumes. The flow rate was 1.5 ml/min, and thefraction size was 1.5 ml. Peaks were pooled as indicated in the figure:a, fractions 31–35; b, fractions 37–40; c, fractions 41–47. A 280 (—);
conductivity (—).

Removal of Aggregate From an IgG4 Product Using CHT™ Ceramic Hydroxyapatite Resin

The IgG4 sample was applied to a CHT Column and eluted
with a linear phosphate gradient. A single major peak, flanked
by two or three broader minor peaks, was obtained (Figure 2).
The major peak from the CHT Column (peak a) was analyzed
on a size exclusion column and was found to consist of only
monomeric IgG4 (Figure 1, chromatogram b). Spectrophotometric analysis at 280 nm indicated that it represented about
72.4% of the protein applied to the CHT Column. The major
CHT column pools were also analyzed by nonreducing
5% SDS-PAGE (Figure 3).
A major band comigrating with the 150 kD standard waspresumed to be IgG4 monomer. Some nine or ten components
with molecular weights greater than that of the IgG4 monomer
were apparent in the CHT column load (Figure 3, lane 2) andwere presumed to be aggregates. The major CHT columnpeak (lane 3) appeared to be free of these aggregates and12

345– 250 kD– 150– 100– 75LFig. 3. Nonreducing SDS-PAGE analysis of CHT column pools. Lane 1, MWstandards; lane 2, IgG4 starting material; lane 3, main CHT column peak (peaka); lane 4, CHT peak b; lane 5, CHT peak c; lane 6, human IgG (Sigma). Thegel was a 5% Ready Gel® Precast Gel (catalog #161-1213), and the stain was
an experimental stain about 50-fold more sensitive than Coomassie Blue.
Positions of the IgG4 monomer (M) and light chain (L) are indicated by arrows.
123Fig. 4. Reducing SDS-PAGE analysis ofCHT column pools. Lane 1, IgG starting

4– 250 kD
material; lane 2, main CHT column pool– 150(peak a); lane 3, MW standards. The gel– 100– 75was a 4–20% Ready Gel Precast Gel

H– 50– 37L– 25(catalog #161-1159); the stain was thesame as in Figure 3. Positions of theIgG4 heavy (H) and light (L) chains areindicated by arrows.

– 15– 10

A reducing 4–20% SDS gel was also run (Figure 4), whichshows that some aggregated species were present underthese conditions as well and may have arisen from covalentcrosslinking. These components were also removed by CHT
chromatography. The heavy and light chains migrated as
expected in this gel.
Discussion
The results reported here indicate that CHT column
chromatography is capable of removing essentially all of theaggregation and degradation products found in a human IgG4
biopharmaceutical in a single step.

6M

had a trace of a single lower molecular weight species, which
may be the F(ab’)2 fragment. Lanes 4 and 5, which contained
the later eluting minor peaks (b and c, respectively) fromthe CHT Column, contained an array of aggregates andsome lower molecular weight species, which may be IgG4
degradation products. A major band, presumed to be the IgG4light chain, ran slightly ahead of the 75 kD standard underthese conditions. Some of the components observed in theIgG4 fractions were also found in a commercially available
human IgG preparation (lane 6).

The aggregates found in this preparation did not appear tobe simple multimers of the 141.9 kD IgG4 monomer. The
absence of simple multimers may have been due to theformation of various complexes with the monomer, F(ab’)2
fragment, and other degradation products. Both disulfide
and nonreducible crosslinks (possibly enzyme-mediated)appeared to exist in this preparation.
The CHT Column performed well at a relatively high sample
load (6 mg/ml) and with a relatively short bed height (52 mm).
No reaggregation was evident in these experiments.
The procedure is simple and should be easily scaled up.
This suggests CHT chromatography may be useful for
immunoglobulin polishing in numerous circumstances.
References
Gagnon P (1996). Hydroxyapatite Chromatography. In Purification Tools for
Monoclonal Antibodies (Arizona: Validated Biosystems, Inc.), p. 95.
Ritchie RF and Navolotskaia O (1996). Serum Proteins in Clinical Medicine, Vol 1(Maine: Foundation for Blood Research).
Veterinary Public Health Unit (1995). Report of a WHO Consultation on
Intradermal Application of Human Rabies Vaccines (Geneva, Switzerland: WorldHealth Organization).
Information in this tech note was current as of the date it was written (2002),
not necessarily the date this version (Rev B, 2015) was printed.

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Bulletin 2940 Rev B

US/EG15-0853

0415

Sig 1214