Process Separations

Monoclonal Antibody Purification:
Capture Purification Resins■■■■

Bulk impurities
such as HCPs
and DNA removed
from initial feed
Target mAb
concentration
increased bydecreasing impurities
and feed volume

Affinity and Ion Exchange Resins for
Monoclonal Antibody Capture Purification
Monoclonal antibody (mAb) purification processes
typically involve a multistep workflow consisting of
two or three steps for capture, intermediate, andpolish purification. The resins selected for each ofthese steps must be compatible with the specificpurification challenges that exist at that particularphase of purification.
Capture Purification Objectives■■ Remove the bulk of the impurities, including
host cell proteins (HCPs), endotoxins, and DNA,
from the initial feed■■

Increase target mAb concentration by
decreasing the impurities and feed volume

CAPTURE

Ideal Features for Capture Purification
Resins■■ Able to withstand high flow rates in order tominimize– the potential of mAb inactivation/misfolding– target antibody’s exposure to proteases 				 and nucleases present in the cell culture 				feedstream■■

High binding capacity to get the most efficient
separation of impurities from the mAb

Bio-Rad’s Resins for mAb Capture
Purification■■ UNOsphere SUPrA™ Affinity Resin■■Nuvia™ S Cation Exchange Resin

INTERMEDIATEPOLISH

UNOsphere SUPrA

UNOsphere™ Q

CHT ™ Ceramic Hydroxyapatite

UNOsphere SUPrA

Nuvia™ Q

Nuvia™ cPrime™

Nuvia S

Nuvia HR-S

Nuvia cPrime™

UNOsphere SUPrA Affinity Resin
UNOsphere SUPrA Affinity Resin combines UNOsphere beadtechnology with a recombinant Protein A ligand, making it an ideal
candidate for the production of mAbs. Its outstanding pressure/flow performance allows for its use in large-scale processapplications.
Bead Properties
Property

Description

LigandRecombinant Protein A

Particle size

53–61 µm

Total ligand density

10 mg/ml
30 ± 3 mg/ml

Dynamic binding capacity

10% BT capacity determined with
1.0 mg/ml polyclonal hIgG in
1.1 x 10 cm column

Recommended linear flow rate

100–600 cm/hr

A. pH 3.7mAb G
100
9080

Under 2 bar at flow rate of 300 cm/hrin DI water(20 x 20 cm packed bed,
1.15 compression factor)

pH stability

3–11

Shipping solution

50% slurry in 20% ethanol

Regeneration

1–2 M NaCl

CIP/sanitization

6 M guanidine-HCl, 10 mMhydrochloric acid, 0.1 M sodiumhydroxide, 1 M acetic acid/20%
ethanol

Storage conditions

2–8°C

Chemical stability
No significant change in chromatographic
performance after storage at RT for 24 hr

10 mM HCI, 6 M guanidine-HCl, 0.1 M
arginine (pH 2.8), 0.1 M citrate (pH 2.8),
0.1 M glycine (pH 2.8)

Shelf life

5 years (4–8°C)

70
Recovery, %Pressure vs. flow performance

Percent recovery of two mAbs at different pH by UNOsphere
SUPrA and two other Protein A resins. mAb G and mAb R
were screened on three Protein A resins. Each mAb (5.5 mg) wasloaded onto columns with 1 ml of each resin, run at 300 cm/hr,
and eluted with 0.1 M glycine at pH 3.7. More than 80% of eachmAb was recovered from UNOsphere SUPrA, whereas recovery
was much less from the two other resins (Figure 1A). Since a lower
pH is known to improve the performance of the other two resins,the experiments were repeated at pH 3.5. Total percent recoveryfor both mAb G and mAb R was more consistent with UNOsphere
SUPrA than with the other resins (Figure 1B). Greater than 80%
recovery can be achieved in 3–5 CV only with UNOsphere SUPrA.

60504030
20100UNOsphere SUPrA

Resin RResin SResin R

Resin S

mAb R
100
90

BT, breakthrough; hIgG, human immunoglobulin G.

80

■■

■■

■■

■■

■■

High dynamic binding capacity (DBC) at high bed heights —increased bed height leads to increased residence time, whichleads to increased mAb binding to the resin

70
Recovery, %Performance Advantages■■ Excellent flow properties — fast flow without proportionalpressure spikes

6050403020Narrow elution profile — reduced requirement for elution buffer

10Rapid mass transfer — minimized aggregation or precipitationof low pH–sensitive mAbs

0

Predictable performance — over a wide range of mAbconcentrations
High recovery — typically >95% target mAb recovery

Competitive Data
The low pH requirement for elution from Protein A–based resins
can result in higher aggregate content and precipitation and lower
monomer concentration in the recovery pool. The rapid masstransfer characteristics of UNOsphere SUPrA help overcome thischallenge.

UNOsphere SUPrA

Fig. 1A. Comparison of percent recovery using UNOsphere SUPrA and twoother Protein A resins at pH 3.7. Percent recovery was evaluated at pH 3.7. 3 CV (n);5 CV (n); 7 CV (n).

Nuvia S Cation Exchange Resin

B. pH 3.5
mAb G

Nuvia S Resin is built on the industry proven UNOsphere basematrix technology, designed for robust and scalable processapplications. It is a next-generation resin that provides flexible
design for both capture and intermediate purifications.

1009080

Recovery, %7060

Bead Properties

50

Property

Description

40

Type of ion exchanger

Strong cation

30

Functional group–SO3–Particle size

85 ± 15 μm

Total ionic capacity

90–150 μeq/ml

2010>110 mg/ml at 300 cm/hr

0UNOsphere SUPrA

Resin R

Resin SmAb R
100

80

Recovery, %50–300 cm/hr
Under 2.5 bar at a flow rate of600 cm/hr in DI water(20 x 20 cm packed bed,1.17 compression factor)

60
50

pH stability

40

Shipping solution

20% ethanol + 0.1 M NaCl

30

Regeneration

1–2 M NaCl

20Sanitization

0.5–1.0 N NaOHStorage conditions

20% ethanol or 0.1 N NaOH

10UNOsphere SUPrA

Resin RResin S

Fig. 1B. Comparison of percent recovery using UNOsphere SUPrA and twoother Protein A resins at pH 3.5. Percent recovery was evaluated at pH 3.5. 3 CV (n);5 CV (n); 7 CV (n).

Other Resources■■ Instruction manual, bulletin 10014430
Product information sheet, bulletin 5729

Short term: 2–14
Long term: 4–13

Application of UNOsphere SUPrA Resin in industrial antibodypurification, bulletin 6053

Description

Bulk Resin
1560218 	UNOsphere SUPrA Affinity Chromatography Media, 25 ml
1560219	UNOsphere SUPrA Affinity Chromatography Media, 100 ml
156-0220	UNOsphere SUPrA Affinity Chromatography Media, 500 ml

Up to 1 week

0.1 N NaOH (20°C)

Up to 5 years
5 years

Performance Advantages■■ Best-in-class DBC — superior DBC over a broad range of pH,
conductivity, and flow rates due to unique design■■■■

Ordering Information
Prepacked Screening Tools
732-4729		
Foresight™ UNOsphere SUPrA Column, 1 ml
732-4749		 Foresight UNOsphere SUPrA Column, 5 ml
732-4834		Foresight UNOsphere SUPrA RoboColumn Unit, 200 µl
732-4835		Foresight UNOsphere SUPrA RoboColumn Unit, 600 µl

1.0 N NaOH (20°C)
Shelf life

Monoclonal antibody purification using UNOsphere SUPrA
Resin, bulletin 5728

Catalog #1.15–1.20

Chemical stability

0■■

Recommended linear flow rate

Compression factor(settled bed volume/packed bed volume)

70■■

10% BT capacity determined with
4.5 mg/ml hIgG in 40 mM Na acetate +30 mM NaCl, pH 5.0.

Pressure vs. flow performance

90■■

Dynamic binding capacity■■■■

Replacement of affinity capture in a three-step process— efficient HCP and DNA clearance to effectively replace anaffinity-based capture in a three step process
Base stability and consistent performance — no decrease
in DBC or recovery when exposed to 840 hr of 1.0 M NaOH
with typically used cleaning cycles; delivers value and process
stability by maintaining peak performance over an extended life
Economical — in terms of cost, buffer consumption, and spacerequirements
Superior productivity — with decreased cycle timeCompetitive Data
DBC at high flow rates on Nuvia S and other commerciallyavailable CEX resins. Binding of human immunoglobulin G (hIgG)
was tested on Nuvia S and three other commercially available
CEX resins (Figure 2). Nuvia S exhibited the most efficient bindingof hIgG in the linear velocity range 150–600 cm/hr, significantly
outperforming the other CEX resins. The binding capacity of hIgG
on Nuvia S was found to be 114 mg/ml even at a high flow rateof 600 cm/hr. When tested against six other commercial CEX
resins, Nuvia S showed the highest DBC under similar conditions(Figure 3). Such performance allows Nuvia S to deliver the speed
and throughput needed in the process manufacturing of mAbs.

10% breakthrough (hIgG), mg/ml

140

Other Resources■■ Instruction manual, bulletin 10018215■■■■

Product information sheet, bulletin 5987
High-capacity CEX resin for the process purification ofmonoclonal antibodies, bulletin 5984

Ordering Information
Catalog #Bulk Resin
15603111560313156-0315
156-0317

130120110

Description

Prepacked Screening Tools
732-4701 		
Foresight Nuvia S Plates, 2 x 96-well, 20 μl
732-4801		
Foresight Nuvia S RoboColumn Unit, 200 μl
732-4802 		Foresight Nuvia S RoboColumn Unit, 600 μl
732-4720		
Foresight Nuvia S Column, 1 x 1 ml
732-4740 		
Foresight Nuvia S Column, 1 x 5 ml
Nuvia S Media, 25 ml
Nuvia S Media, 100 ml
Nuvia S Media, 500 ml
Nuvia S Media, 10 L

100908070300

350

400

450
500
Linear velocity, cm/hr

550

600

Fig. 2. Binding of hIgG by Nuvia S Resin and three other resins at various linear
velocities. Column size, 1.1 x 9.5 ± 0.3 cm. Nuvia S (—); CEX 1 (—); CEX 2 (—);
CEX 3 (—).

All our resins come with full regulatory support backed by
Bio-Rad’s global application and development team. Contact
your regional Bio-Rad process chromatography specialistat process@bio-rad.com or call customer service at
1-800-4-BIORAD (1-800-424-6723) for more information.
Test drive our resins for your mAb purification.
Visit bio-rad.com/web/ResinSample to order your sample.
RoboColumn is a trademark of Atoll GmbH.

DBC polyclonal hIgG, 10% BT, mg/ml

140120100806040200Nuvia S
Resin

Resin
1Resin2Resin3Resin4Resin5Resin6Fig. 3. Comparison of Nuvia S and other commercially available CEX resins.
Column size, 0.7 x 5.5 cm. Sample was loaded onto the column, in 40 mM sodiumacetate, pH 5.0 and 30 mM sodium chloride, washed, and eluted with
40 mM sodium acetate, pH 5.0 and 1 M sodium chloride. BT, breakthrough.

Bio-RadLaboratories, Inc.
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Life ScienceGroup

Bulletin 6861 Ver A

US/EG

16-09531216

Sig 1215