IEX ChromatographyPresented by:

Nikki ApostolakisHelen SoTiffany Yu

CHEE450: Engineering Biology

Chromatography Introduction

The term “chromatography” applies to a wide range of separation techniques that are based on the differential interaction of molecules between a moving phase and a stationary phase.

Different column chromatography methods exploit different properties of the protein; Ion exchange chromatography separates molecules based on charge.

Column chromatography is a commonly used technique for protein purification.

Anion Exchanger

CationExchangervs.

Ion Exchange Principles

There are four main steps to IEX Chromatography:

o Equilibrationo Load and Washo Elutiono Regeneration

Resins are the medium for separation and represent the stationary phase. The surface of these particles provide sites for selective binding. Separation is based on reversible adsorption of ions onto resin

Ion Exchange Principles

EquilibrationStationary Load Wash Elution Regeneration

Ion Exchange Purpose

Intermediate separation and purification of MABs from bulk of impurities

Inlet stream Composition:o Acetic Acid

o Impurities

o MAB

o Polysorbate 80

o WFI

Design Considerations

COLUMN DESIGN• Flow rates • Aspect ratio (H, d)• Column material• Space constraints

COLUMN INTERNALS• Packing material• Buffer pH’s • Buffer composition

Chosen Design

Chromaflow ColumnsWhy the Chromaflow line?

• Easily validated

• Cost-effective, safe packing/unpacking

• Appropriate for most chromatography

techniques

• Suitable for large scale GMP production

Chosen Design

Specifications

Resin Type: Sepharose High PerformanceResin Volume: 207.88 LColumn Type: GE ChromaflowColumn Dimensions:

Inner diameter: 1800 mmHeight: 100-300mm

Pressure Rating: 3 bar

Chromaflow Columns

IEX Column Costing

BPG Family(Model #)

ID(mm)

Bed Height(cm)

Packing Volume(L)

Purchase Cost($)

BPG 100/500 100 0-26 0-2 $5,678.40

BPG 100/950 100 45-54 3.5-4.2 $6,171.90

BPG 200/500 200 0-26 0-8.2 $7,677.60

BPG200/950 200 45-54 14.1-16.9 $8,922.90

BPG 300/500 296 0-26 0-17.9 $15,682.80

BPG 300/950 296 45-54 31-37.2 $18,244.80

BPG 450/500 446 3-23 4.7-35.9 $35,631.75

BPG 450/1000 446 47-58 73.4-90.6 $43,548.75

BPG BioProcess Columns

Alternatives

Can make batch process continuous by running columns in parallel

Change resin to have impurities bind and allow MAB to pass

CONFIGURATION

SEPARATION

Use a different basis for separation (e.g. molecular size)

Downstream Processing

THE END

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