•Broadutility—POROS®A,G,andCaptureSelect®affinityligandsaddressmostbiologicaldrugclasses

•Rapid—POROS®resinsofferfastquantitationorpurificationofimmunoglobulinsandfusionproteins

•Reducedhands-ontime—HPLCmethodcanbeautomatedforstreamlinedoperation

•Robust—deliverhigh-performancepurificationoverhundredsofchromatographycycles

Analyticalgroupssupportingbiologicaldrugdevelopmenteffortsrequirerapid,sensitive,andprecisemethodsforquantifyingproteinsfromcellcultures,fermentationharvests,orpurificationintermediates.Itiscriticalthatbiopharmaceuticalmanufacturersbeabletomonitorbioreactorproducttiterstodeterminewhentoharvestorabortareactorcycle.

POROS®AffinityChromatographyColumnsincorporatetheadvantagesofhigh-speed,high-performanceanalytical

POROS®AffinityChromatographyColumnsHigh-speed quantitation or purification

PRODUCTBULLETINPOROS®AffinityChromatographyColumns

Protein A or G

CaptureSelect®

LC-Kappa or LC-Lambda

Protein L

CaptureSelect®

IgG Fc

CH2

CH1 CL

CH3

VH VL

Figure 1. POROS® A, G, and CaptureSelect® ligands target multiple IgG epitopes. This graphic shows the binding sites for affinity ligands Protein A, Protein G, and Protein L. Novel POROS® CaptureSelect® ligands provide alternative specificities that enable rapid binding for the quantitation or purification of engineered immunoglobulins (POROS® Affinity Chromatography Columns represented in orange).

20µmPOROS®chromatographyresinswiththeuniqueselectivityofProteinA,ProteinG,andCaptureSelect®ligands(Figure1).Thecombinationallowsfor3-minutequantitationofimmunoglobulins,fusionproteins,Fabfragments,andbispecificantibodies.Alternativeassayslackthecombinedadvantagesofspeed,selectivity,methodautomation,andhighprecision.Inaddition,theaffinity-purifiedproteinissuitableforsubsequentanalysissuchasmassspectrometry.

High speed with high specificityThe combination of Protein A, Protein G, and CaptureSelect® affinity ligands combined with POROS® 20 µm resin provides a high–flow rate, high-performance chromatography solution for the rapid quantitation and small-scale purification of:

• IgG—all subclasses, specific for human and many other species (Table 1)

• IgM—specific for human, mouse, or rat

• Kappa light chain—specific for human, all subclasses

• Lambda light chain—specific for human, all subclasses

• Human serum albumin• Fusion proteins, Fc, or human

serum albumin

POROS® perfusion chromatography allows the quantitation of product from clarified harvest or partially purified intermediates—typically in under 3 minutes. Moreover, only minimal sample preparation (0.2 µm filtration) is required, and the eluted product is highly purified and suitable for further analysis. Figure 2 illustrates the chromatographic performance of POROS® CaptureSelect® LC-Kappa with five unique kappa Fab fragments. The associated standard curves, Figure 3, exhibit excellent linearity. Additionally, Figure 4 displays the specificity of the CaptureSelect® HSA ligand.

TheBACandLifeTechnologiescollaborationcreatesarangeofready-to-useHPLCcolumnsprepackedwithCaptureSelect®affinityligandsimmobilizedonPOROS®chromatographymedia.Designedforthehighflowratesnecessarytoprovidethereal-timeresultsdemandedforbiopharmaceuticaldevelopmentandqualityassuranceneeds,theuniqueselectivityofthesecolumnswillsupportProcessAnalyticalTechnology(PAT)formostbiopharmaceuticalmanufacturingprocess.

Figure 3. Standard curves for POROS® CaptureSelect® LC-Kappa exhibit good linear range (8x) and linear correlation coefficients (R2 range: 0.9991 to 1).

Column: POROS® CaptureSelect® LC-Kappa column (Cat. No. 4469149, 2.1 x 30 mm, PEEK™ polymer). System: Agilent 1200, flow rate 2 mL/min. Sample injection: Specified volumes of purified kappa light chain, 0.5 mg/mL. Equilibration buffer: phosphate buffer, 150 mM NaCl, pH 7.5. Elution buffer: 12 mM HCl, 150 mM NaCl, pH 1.9. UV detection: 280 nm.

Figure 2. POROS® CaptureSelect® resins exhibit high-speed and efficient chromatography, regardless of the type of kappa light chain. This overlay of 5 kappa light chain runs on a POROS® CaptureSelect® LC-Kappa column illustrates the speed and efficiency of the analysis.

Column: POROS® CaptureSelect® LC-Kappa column (Cat. No. 4469149, 2.1 x 30 mm, PEEK™ polymer). System: Agilent 1200, flow rate 2 mL/min. Sample injection: 50 µL purified kappa light chain, 0.5 mg/mL. Equilibration buffer: phosphate buffer, 150 mM NaCl, pH 7.5. Elution buffer: 12 mM HCl, 150 mM NaCl, pH 1.9. UV detection: 280 nm.

Ab

sorb

ance

(mA

U)

Time (min)

0 0.5 1.0 1.5 2.0

163150

125

100

75

50

25

0

-20

Ab

sorb

ance

(mA

U•m

in)

Injection volume (μL)

0 10 20 30 40 50 60 70 80 90

70

60

50

40

30

20

10

0

MaterialsComponent Material of construction

Column PEEK™ polymer or stainless steel

Frit 2 µm, titanium

POROS® resin Crosslinked poly (styrenedivinylbenzene)

CaptureSelect® ligandSingle-domain monospecific antibody fragment produced in Saccharomyces cerevisiae

Protein A, Protein G Recombinant Protein A and Protein G

SpecificationsProduct Protein-binding capacity (mg/mL) Species specificity

POROS® CaptureSelect® IgG-FC 8.9 Human

POROS® CaptureSelect® LC-Kappa 14.5 Human

POROS® CaptureSelect® LC-Lambda 10.0 Human

POROS® CaptureSelect® HSA 2.6 Human

POROS® CaptureSelect® IgM 4.0 Human, mouse, rat

POROS® Protein A 19.0 Human, mouse, and 5 other species

POROS® Protein G 9.0 Human, mouse, rat, and 8 other species

Automation POROS® Affinity Chromatography Columns are suitable for use with HPLC systems, which allow for the automation of chromatography methods, data collection, and data analysis. In addition, the immobilized affinity ligands enable one method and buffer system to be used across a class of molecules, streamlining analytical approaches.

Multiple cyclesProper sample preparation, adherence to column-operating instructions, and the use of an optimized column-cleaning regime result in strong reuse performance. POROS® Affinity Chromatography Columns can be cycled hundreds of times for cost-effective and reproducible quantitation or purification.

Table 1. Selection guide.

POROS® Protein A, G resins POROS® CaptureSelect® resins

Bimolecular type Protein A Protein G IgG-Fc LC-Kappa LC-Lambda HSA IgM

IgG1, IgG2, IgG4 • • • • •

IgG3 • • • •

IgG, CH2 domain deleted • • •

IgM • • •

IgA, IgD, IgE • •

Fab, Fab´2 • •

Human serum albumin •

Albumin fusion protein •

IgG Fc fusion protein • • •

Ab

sorb

ance

(mA

U)

Time (min)

0 1 3 52 5 6 7

120

100

80

60

40

20

0

Ab

sorb

ance

(mA

U)

Time (min)

0 1 2 43 5

400

300

200

100

0

Ab

sorb

ance

(mA

U)

Time (min)

0 1 2 43 5

400

500

300

200

100

0

B

C

Figure 4. POROS® CaptureSelect® HSA resin exhibits high specificity for the target molecule. The specificity of a POROS® CaptureSelect® HSA column was demonstrated by loading (A) 1 mg/mL bovine serum albumin (BSA) and (B) 1 mg/mL monoclonal antibody samples. The chromatograms show both BSA and antibody eluting in the void volume, whereas HSA fusion protein (C) elutes at approximately 2.9 min.

Column: POROS® CaptureSelect® HSA column (Cat. No. 4469151, 2.1 x 30 mm, PEEK™ polymer). System: Agilent 1200, flow rate 1 mL/min. Sample injection: 100 µL purified HSA fusion protein, 0.2 mg/mL. Equilibration buffer: phosphate buffer, pH 7.0. Elution buffer: 0.5 M acetic acid, pH 2.5. UV detection: 280 nm.

A

Ordering informationProduct Column dimension

(diameter (mm) x length (mm))Volume (mL) Cat. No.

POROS® Protein A

20 μm (stainless steel column) 2.1 x 30 0.1 1-5024-12

20 μm (PEEK™ polymer column) 2.1 x 30 0.1 2-1001-00

20 μm (PEEK™ polymer column) 4.6 x 50 0.8 1-5022-24

20 μm (PEEK™ polymer column) 4.6 x 100 1.7 1-5022-26

20 μm (PEEK™ polymer column) 10 x 100 7.9 1-5022-46

POROS® Protein G

20 μm (PEEK™ polymer column) 2.1 x 30 0.1 2-1002-00

20 μm (PEEK™ polymer column) 4.6 x 50 0.8 1-5122-24

20 μm (PEEK™ polymer column) 4.6 x 100 1.7 1-5122-26

20 μm (PEEK™ polymer column) 10 x 100 7.9 1-5122-46

POROS® CaptureSelect® HSA

20 μm (PEEK™ polymer column) 2.1 x 30 0.1 4469151

20 μm (PEEK™ polymer column) 4.6 x 50 0.8 4469165

20 μm (PEEK™ polymer column) 4.6 x 100 1.7 4469170

20 μm (PEEK™ polymer column) 10 x 100 7.9 4469175

POROS® CaptureSelect® IgG-Fc

20 μm (PEEK™ polymer column) 2.1 x 30 0.1 4469148

20 μm (PEEK™ polymer column) 4.6 x 50 0.8 4469153

20 μm (PEEK™ polymer column) 4.6 x 100 1.7 4469166

20 μm (PEEK™ polymer column) 10 x 100 7.9 4469171

POROS® CaptureSelect® IgM

20 μm (PEEK™ polymer column) 2.1 x 30 0.1 4469152

20 μm (PEEK™ polymer column) 4.6 x 50 0.8 4469164

20 μm (PEEK™ polymer column) 4.6 x 100 1.7 4469169

20 μm (PEEK™ polymer column) 10 x 100 7.9 4469174

POROS® CaptureSelect® LC-Kappa

20 μm (PEEK™ polymer column) 2.1 x 30 0.1 4469149

20 μm (PEEK™ polymer column) 4.6 x 50 0.8 4469162

20 μm (PEEK™ polymer column) 4.6 x 100 1.7 4469167

20 μm (PEEK™ polymer column) 10 x 100 7.9 4469172

POROS® CaptureSelect® LC-Lambda

20 μm (PEEK™ polymer column) 2.1 x 30 0.1 4469150

20 μm (PEEK™ polymer column) 4.6 x 50 0.8 4469163

20 μm (PEEK™ polymer column) 4.6 x 100 1.7 4469168

20 μm (PEEK™ polymer column) 10 x 100 7.9 4469173

Speak to a technical specialist about how these columns can improve your current process, at bp@lifetech.com, or go to www.appliedbiosystems.com/poros/captureselect.

lifetechnologies.com

For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.© 2012 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners. CaptureSelect is a registered trademark of BAC BV. PEEK is a trademark of Victrex PLC. Printed in the USA. CO24355 0112