chromatographytech note 5735

Purification of a Monoclonal Antibody Using a Combination ofUNOsphere™ Rapid S and UNOsphere Q Media Ion Exchange
Chromatography After Protein A Capture
Introduction
Antibody-based drugs represent a growing segment of thepharmaceutical industry and one of the most promisingclasses of therapeutic drugs. The demand for protein-based
drugs is expected to grow steadily for the foreseeable
future. However, production of monoclonal antibody(MAb)–based drugs remains very costly, and solutions tolower production costs are needed. Downstream purification
steps are becoming a target for cost reduction. The use ofion exchange chromatography to directly capture MAbs fromcell culture streams or as a polishing step after an affinity
separation may be a way to reduce costs.
Ion exchange chromatography is an established step inthe purification of MAbs following protein A capture. In thisapplication note, we demonstrate the utility of the combinationof a cation exchange step and an anion exchange stepusing UNOsphere Rapid S media and UNOsphere Q
media, respectively. The mirrored binding characteristics
of UNOsphere Rapid S and UNOsphere Q media provide
complementary purification steps for the removal of host cell
proteins (HCPs), fragmented or aggregated MAbs, leachedprotein A ligands, DNA, and virus particles (Figure 1).
In the separation method described in this note, a protein A
support was used to isolate a MAb from a cell culture feed.
A large proportion of HCPs and other contaminants did not
CAPTURE
Affinity chromatography

INTERMEDIATE PURIFICATION
UNOsphere Rapid S media

POLISHING
UNOsphere Q media

Fig. 1. Complementary ion exchangers used after protein A to removecontaminants.

bind to the protein A support. However, along with the intact
MAb, variant forms were retained by the protein A support andfound in the eluted fractions. We used UNOsphere Rapid S
media to separate a deamidated degradation product from theintact MAb and used UNOsphere Q media to further reduce
the remaining contaminants to acceptable levels.
Materials and Methods
MAb was captured from a cell culture filtrate by affinity
chromatography using immobilized protein A (0.5 x 5 cmcolumn), equilibrated with 50 mM sodium phosphate, 100 mM
sodium chloride, pH 7.0. Bound MAb was eluted with 100 mM
arginine, 50 mM sodium acetate, pH 3.8. The MAb pool fromprotein A capture was adjusted to pH 4.5 prior to cationexchange chromatography.
For cation exchange chromatography, a column (0.5 x 5 cm)
was packed with UNOsphere Rapid S media and equilibrated
in 10 mM sodium acetate, pH 4.5 (buffer 1). An aliquot of theprotein A eluate was diluted 1:4 with buffer 1 and applied tothe column at a linear velocity of 300 cm/hr. The column waswashed with buffer 1 and then with buffer 2 (10 mM sodiumcitrate, 10 mM sodium phosphate, pH 5). Elution was performed
with a 15-column-volume pH gradient (pH 5–7.4) formed
between buffer 2 and buffer 3 (buffer 2 adjusted to pH 7.4).
The main peak of MAb eluted from UNOsphere Rapid S media(8.0 ml) was adjusted to pH 7.0 by addition of 0.8 ml of 500
mM sodium phosphate, pH 7.0, and applied at a linear velocityof 300 cm/hr to a 0.5 x 5 cm column packed with UNOsphere
Q media. In this flow-through step, the column was equilibrated
with buffer 4 (50 mM sodium phosphate, pH 7.0). Following
sample application, the column was washed with 30 ml ofbuffer 5 (75 mM sodium phosphate, pH 7.0) and then waswashed with buffer 6 (500 mM sodium phosphate, pH 7.0) tostrip the column of remaining bound components.
Protein fractions were analyzed by SDS-PAGE using a Criterion™
Tris-HCl 4–20% gradient gel (Bio-Rad). Mass spectrometry
analysis of gel-purified proteins was carried out with an Autoflex IIMALDI-TOF mass spectrometer (Bruker Corporation).

but also in the leading shoulder. Identification of the leading
shoulder of the peak as a modified or fragmented MAb bygel electrophoresis was not conclusive. Protein bands fromUNOsphere Rapid S media elution fractions 1 and 2 (shoulder),and 3–6 (main peak) were extracted from the gel and analyzed
by mass spectrometry (Figure 4). Comparison of the measured
mass of the shoulder fraction proteins and main peak proteins is
consistent with three deamidations on a single MAb peptide.

UV1_280 nm
CondpH

8.0
7.5
7.0

pH6.56.05.5300

5.02138.646

shoulder4.0Fig. 2. UNOsphere Rapid S media purification of MAb. pH-induced elution
profile from UNOsphere Rapid S media. The distinct shoulder on the front side ofthe main peak is a modified MAb fragment.

Intensity, au

200

4.5

10002136.142

1501005002125

Results
The UNOsphere Rapid S media cation exchange chromatogramis shown in Figure 2. The bulk of the MAb eluted as a sharp
peak during pH gradient elution. Recovery of MAb in the main
peak, estimated by A280 nm, was >95%. Mass spectral data
demonstrated a peak at 2138.6 amu (atomic mass unit) for theeluate pool and 2136.1 amu for the leading shoulder. This isconsistent with deamidation of three aspargine or glutamine
residues of the MAb. Such a deamidation event, which converts
neutral asparagine, glutamine, or both to their respective acidicforms, would be expected to result in a more acidic protein thatwould elute earlier than the unmodified MAb in a pH gradient.
With a shallower pH gradient than the one employed here,baseline separation of such species is possible.
Aliquots from different fractions collected throughout the entire
procedure were analyzed by SDS-PAGE (Figure 3). IgG heavy
and light chains were present not only in the main elution peak,
12 3 4567 82130

2135

2140

2145
m/Z

2150

2155

2160

2165

Fig. 4. Mass analysis of MAb. Analysis of gel-purified proteins was carried out
with an Autoflex II MALDI-TOF mass spectrometer. Mass spectrometer analysisof the shouldered peak (top spectrum) and the main peak (lower spectrum)
show a mass shift of about 2.5 amu between the two peaks, which is consistent
with three deamidations on a single peptide.

The reduction of HCPs is shown in Table 1. HCP reduction wasdetermined using ELISA quantification. UNOsphere Rapid S
media chromatography achieved a tenfold reduction in HCP,
and the UNOSphere Q media step further reduced the HCPs tobelow the limit of detection of the assay.
Table 1. Purification process. HCP content after each step of the MAb purification procedure was determined using standard ELISA techniques.
Chromatographic Step

HCP (ng/ml)
A 280
HCP (ng/ml)

Protein AcaptureUNOsphere
Rapid S media

UNOsphere
Q media

30.4
3.4
8.94

2.14
2.42
0.88

<0.7
0.865
<0.8

9 10 11 12 13 14 15 16 17 18Conclusions

25015010075503725
20
15
10Fig. 3. SDS-PAGE analysis of chromatographic fractions. 1, Precision Plus
Protein™ standard; 2, MAb cell culture harvest; 3, protein A capture eluate. Lanes
4–16 show UNOsphere Rapid S media chromatography: 4–5, flowthrough 1 and2; 6, wash; 7–14, elutions 1–8; 15, pooled elution fractions 3–6; 16, strip. Lanes
17 and 18 show UNOsphere Q media chromatography: 17, flowthrough; 18, strip.
The bands circled in red correspond to the bands used for mass spectrometry
analysis in Figure 4.

UNOsphere Rapid S media chromatography with anappropriate pH gradient elution scheme can be used to remove
unwanted degradation products from a process stream withnearly quantitative recovery of IgG. In addition to providing a
powerful separation technique, pH-based elution is well-suited
to a process flow. Use of pH rather than ionic strength forelution means that the eluate pool will have low conductivity;
the pool can thus be applied directly to a subsequent anion
exchange step, if desired, with minimal feed stream dilution.
Autoflex II is a trademark of Bruker Corporation.
Information in this tech note was current as of the date of writing (2008) and notnecessarily the date this version (rev A, 2008) was published.

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Life ScienceGroup

Bulletin 5735 Rev A

US/EG08-0340

0508

Sig 0308