Practical Guide: Selecting the Optimal Resinsfor Immunoglobulin G (IgG) Process Purification
Payal Khandelwal, PhD
Bio-Rad Laboratories, Inc., 6000 James Watson Drive, Hercules, CA 94547

Purification Solutions

Bulletin 6793

Comprehensive Solutions for IgG Purification

Immunoglobulins (Ig), or antibodies (Ab), are the heavy globular plasma proteins produced in response to foreign
bodies or antigens. They exist in five isoforms: IgA, IgD, IgE, IgG, and IgM. IgG provides the majority of antibodybased immunity. The ability of animal immune systems to produce antibodies against an injected antigen caneasily be exploited to generate application-specific antibodies for both basic research and diagnostic applications.
Additionally, the proliferation of recombinant DNA and protein technology has opened up previously uncharted
territories in antibody construction. Substantial refinements in the upstream expression and production ofantibodies have created a need for advanced downstream purification processes to remove contaminants such as
albumin, transferrin, and a2-macroglobulin from native serum and host cell proteins (HCPs) from the recombinantsource.
A number of factors will affect which chromatography resin you choose to purify the target antibody, including thedesired purity for the final application and the required physicochemical characteristics of the purified antibody.
Since each resin has unique properties, purification strategies are often broken down into steps such as capture,intermediate, and polish to maximize antibody purity. The resins used at each step have to be selected based onwhether they can deliver the desired process-related costs, time lines, and purity, among other factors.
Bio-Rad has provided a progressive selection of chromatography resins for process-scale purification of antibodies
for more than 50 years. This guide provides a brief snapshot of the different resins that can be used for theprocess-scale purification of polyclonal IgGs and the specific features that make them useful for such purifications.
For more information about resins appropriate for the purification of mAbs specifically, refer to bulletin 6875 —Selecting the Optimal Resins for Monoclonal Antibody Process Purification.

Affinity Purification
UNOsphere SUPrA™ Resin

Protein A affinity chromatography is widely used for IgGpurification since Protein A–based resins bind with high affinity
to the Fc regions of most immunoglobulins, including IgG.
UNOsphere SUPrA Resin was developed with recombinantProtein A (includes only the IgG binding site to ensure specificpull down of IgG from serum) conjugated to UNOsphere™
beads for robust and scalable IgG purification. This resin
provides multiple advantages over other Protein A resins,
including predictable performance over a wide range ofantibody concentrations and stability at high pH (<11). It
has a binding capacity of 25–30 mg/ml of IgG (bulletin 5729)

and typically provides >95% recovery of target antibodies
at ~90% purity (Figure 1 and Table 1) (bulletin 6053). Therefore,UNOsphere SUPrA is an excellent choice for process-scale
purification of IgGs.

LSUPrA
FT W E

Affi-Prep®
FT W E

HiTrap HP
FT W E

50 kD heavy chain
25 kD light chain

Fig. 1. Purity of IgG obtained with UNOsphere SUPrA Resin relative
to two other Protein A resins. L, load; FT, flowthrough; W, wash; E, eluate.

Practical Guide: Selecting the Optimal Resins for Immunoglobulin G (IgG) Process Purification
Table 1. Quantitation of remnant HCP and DNA after mAb purification
using UNOsphere SUPrA and MabSelect Resins.
Resin

HCP, ng/mg

DNA, ng/mg

rProtein A, ppm

MabSelect
SUPrA

39.2
33.2

26.0
21.6

6.4
20.5

2468

101618202224262830
76pHA 280

40.5
32
1
0.0

00

10Ion Exchange Chromatography

2030

Run time, min

UNOsphere™ S Resin

UNOsphere S is a cation exchange (CEX) resin that delivers
high productivity through high binding capacity and lowbackpressure. In general, it has been shown to bind 40–60 mg
IgG/ml resin in the linear velocity range of 150–600 cm/hr(bulletin 2669). More specifically, it has been used successfullyin the capture purification of a recombinant antibody fromcorn seeds with 97% recovery and 80% purity (bulletin 2774)(Table 2) and the high efficiency capture of murine IgG1 witha demonstrated recovery of 97% (Figure 2). It possesses aunique ability to self-generate a pH gradient during certain
elution schemes, which gives it a resolving power unattainable
by other CEX resins. Additionally, harsh clean-in-place andsanitization cycles do not affect its long-term stability, making
it very amenable to regeneration and reuse. This resin has
also been shown to successfully clear Protein A contamination(bulletin 2849).
Table 2. Summary of transgenic IgG purification by UNOsphere Sand CHT™ Ceramic Hydroxyapatite.
Purification
Step

Total IgG,
mgTotal Protein, Recovery,mg%

Purity,%Crude corn
extract + IgG

1.0

6.1

100

22UNOsphere S 1.07

1.47

9780

CHT0.918295© 2016 Bio-Rad Laboratories, Inc.

145In summary, UNOsphere SUPrA is ideal as a standalone
for antibodies that do not have to be exceptionally pure. Inaddition, it can be used for capture purification followed byother purification steps, which would help in the removal ofleached Protein A.

0.88

121.0Fig. 2. Purification of murine IgG1 on a UNOsphere S Column. A280 (—);
buffer pH (—).

Macro-Prep® DEAE Resin

Macro-Prep DEAE is a weak anion exchange (AEX) resin withexcellent flow properties and the ability to maintain its resolving
power at flow rates ≥1,000 cm/hr. Most IgGs have a basic
isoelectric point and thus do not bind to AEX resins. Therefore,
this AEX resin serves well in binding contaminants such as
negatively charged HCPs and host cell DNA, ensuring thata purer fraction of unbound basic IgG can be obtained fromthe flowthrough. Macro-Prep DEAE Resin is stable in a wide
variety of organic solvents, making it possible to thoroughly
sanitize and regenerate the material. It operates well at low andmedium pressures and exhibits minimal volume changes in
response to variations in pH or ionic strength. It has been used
as a capture purification resin for horse IgGT (bulletin 2524).
Mixed-Mode Chromatography
CHT Ceramic Hydroxyapatite MediaCHT offers unique selectivities, enabling the resolution ofmixtures that appear homogenous with other media. It is an
excellent choice for intermediate or final polishing. Its robust
properties improve IgG purification quality, efficiency, yield,
and financial value through its large capacity for high-titer
upstream feedstocks. CHT Media is available in two formats,Type I and Type II. In addition to increasing purity, CHT has
also been shown to decrease the amount of IgG4 aggregatesfrom a biopharmaceutical product (Figure 3) (bulletin 2940).
Furthermore, CHT can also be utilized for the removal ofProtein A contaminants (bulletin 2849), which is advantageous
due to the wide range of immunomodulatory effects
associated with Protein A and its fragments. It provides easy
process-scale separation of Protein A from unfractionatedconditioned media without the need for additional steps oragents. CHT can also be used for endotoxin removal from feedusing a 0–1 M phosphate gradient (bulletin RP0033).

Bulletin 6793

Practical Guide: Selecting the Optimal Resins for Immunoglobulin G (IgG) Process Purification

123– 250 kD
– 150
– 100
– 75

H– 50
– 37L– 25– 15– 10Fig. 3. Reducing SDS-PAGE analysis of samples after CHT Columnpurification. Lane 1, IgG4 starting material; lane 2, CHT-purified sample;
lane 3, MW standards; H, IgG4 heavy chain; L, IgG4 light chain.

CFT™ Ceramic Fluoroapatite Media

CFT is an apatite-based media that is a composite offluoroapatite and hydroxyapatite. One of its unique properties
is that it can be used in low pH (~5.0) purifications. Its tensile
strength, chemical durability, and density provide excellent
throughput and consistent performance for IgG separations.
It can be used for the polish step in IgG purification after
the target molecules have been captured and eluted from aProtein A column (bulletin 5853).
For the purification of monoclonal IgG, other resins such as
Nuvia™ Q, Nuvia™ S, Nuvia™ HR-S, and Nuvia™ cPrime™ can beused as well. See bulletin 6875 for details about these resins in
the purification of monoclonal IgG.
The details provided here can help you design a process
purification strategy for your polyclonal IgG. For technical/product support or to request a quote, email your regional
Bio-Rad representative at process@bio-rad.com or contact
our customer service at 1-800-4-BIORAD (1-800-424-6723).
Explore our extensive selection of process-scalechromatography resins and their performance characteristics
and applications (bulletin 6713). For process optimization ofyour IgG purification, request a sample.

© 2016 Bio-Rad Laboratories, Inc.

Bulletin 6793

Practical Guide: Selecting the Optimal Resins for Immunoglobulin G (IgG) Process Purification

HiTrap and MabSelect are trademarks of GE Healthcare.

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