AGILENT ASSAYMAP SOLUTION FOR ANTIBODY PURIFICATION High throughput analytical scale purification and quantitative titering using Protein A and Protein G

BackgroundHistorically, the preparation of biological samples for analysis has been both a source of significant error as well as a difficult process to automate. Typically, the purification step is followed by quantitation and analysis. With increased demands to rapidly purify small amounts of antibodies and Fc-fusion proteins from multiple samples in parallel, there is a need for high throughput, quantitative purification output to support both the direct measurement of the antibody concentration in the starting sample and for high precision analytical methods.

Introducing the AssayMAP Solution Agilent Technologies introduces an intelligently automated workflow for the purification and quantification of antibodies for analysis. The results are rapid, quantitative, robust, and reproducible. The AssayMAP Antibody Purification solution is also flexible, and can be used alone or in combination with other AssayMAP workflows, such as N-glycan sample preparation.

The AssayMAP Solution for Antibody Purification offers two options: Protein A for human biotherapeutics and Protein G for reagents such as mouse antibodies. The workflow is shown in Figure 1.

Highlights

• Bestinclassliquidtransferwiththepowerofprecisionflowcontrol

• Simple,interactiveuserinterfaceforpushbuttonoperationofabind,washandeluteworkflow

• Industrystandardprotein-AHPLCadaptedtoprocess96samplesinparallelwiththesamereagents

pH 2 1.2

0.00 3000Concentration

(µg/mL)

A 280

Figure 1. Antibody purification using Protein A or Protein G.

Quantitative capture and elution The standard curve in Figure 2 was generated using Protein A cartridges and shows a linear relationship between IgG loaded and IgG eluted up to ~100 µg. This demonstrates the dynamic capacity of the cartridge with no breakthrough sample loss. For quantitative binding of the target to the immobilized Protein A, sufficient sample exposure (residence time) to the resin bed is required. The flow rate (and resultant residence time) is easily optimized using the Bravo Platform. A saturation capacity of about 150 µg is possible when maximum quantities of pure sample are desired, but will result in some loss of total sample.

Intra-assay variability Intra-assay variability was demonstrated using 96, AssayMAP Protein A wide pore cartridges which were loaded with 100 µg hIgG and eluted with 25 µL of elution solution at a flow rate of 5 µL/min. The absorbance data for the eluates are shown in the graph in Figure 3 (% CV = 1.26).

Keybenefits

• Processes96,50µLsamplesin1hour

• Fullwalkawayautomationwithnoexpertiserequired

• Excellentanalyticalprecision,with2%CVs

• Quantitativemeasurementandrecoveryofpurifiedproduct

• EasyintegrationwithotherAssayMAPworkflows

Product description Part number

Bravo platform for AssayMAP (Includes Agilent Bravo Platform with AssayMAP head, tip, and cartridge seating station, wash station and pump module)

G5542A

AssayMAP Protein A, wide pore cartridges G5496-60000

AssayMAP Protein G, wide pore cartridges G5496-60008

AssayMAP Streptavidin, wide pore cartridges (for user-defined affinity purification) G5496-60010

Figure 2. Demonstration of quantitative capture and elution; micrograms of human IgG eluted versus amount loaded.

Figure 3. Demonstration of intra-assay variability with sample number versus absorbance (% CV = 1.26).

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www.agilent.com/lifesciences/AssayMAP This item is intended for Research Use Only. Not for usein diagnostic procedures. This information is subject to change without notice.

© Agilent Technologies, Inc. 2012Published in the USA, September 19, 20125991-1178EN

Ordering information

Contact your Agilent Automation Solutions product specialist to find out how AssayMAP technology can work for you.

50 100 150 200

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0 250

hIgG

elu

ted

(µg)

hIgG loaded (µg)

PAW Identity

0

.2

.4

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A 280

Sample number

% CV = 1.26