Основы белковой хроматографии

Hydrophobic Interaction Chromatography

Life Science Group I Chromatography

Mixed-Mode Chromatography

−Separates molecules according to size, isoelectric point and charge ...

−The best known media is Ceramic Hydroxyapatite - (Ca5(PO4)3OH)2

−It has a calcium (Ca+) phosphate (PO4-) backbone. Biomolecules interact with both.

−Monoclonal & polyclonal antibody purification

−Viruses, DNA and endotoxins removal

−DNA purification

Life Science Group I Chromatography

Mixed-Mode Chromatography

Media for Mixed-mode chromatography

–Ceramic Hydroxyapatite

–Ceramic Fluoroapatite

–New – Mixed hydrophobic and weak cation exchange

–Combines hydrophobic spacer with cation exchange functional group based on macroporous hydrophilic polymer

Life Science Group I Chromatography

Purification Strategy – Putting the Techniques Together!

Typical purification experiments will require several purification steps

As examples:

Life Science Group I Chromatography

Using a 3-Step Purification Strategy

 Initial separation or clarification

Removal of insoluble material, with cell disruption

Filtration and extraction

Step 1: Capture (of target)

Concentrates product from clarified lysate; emphasis is on speed and recovery

Adsorption (Affinity, IEX)

Step 2: Intermediate

Separates the components of the now concentrated, partially purified sample; emphasis is on maximum resolution

Other Chromatography (IEX, HIC)

Step 3: Polishing

Achieves the final purity; a high resolution step that remove all other impurities

 Other chromatography (CHT, Gel filtration)

Life Science Group I Chromatography

Example of a Purification Strategy in Industry

Life Science Group I Chromatography

Goals For Chromatography Purification Process

Define your objectives

–Purity (>90% vs. ≥ 99.9%, type of contaminant removal needed)

–Activity (active vs. inactive)

–Quantity (ng or mg levels)

–Log reductions of key contaminants

What is known about your protein

–pH, temp stability

–Effects of salt, detergents, organic solvents, metal ions

–Post-translational modifications that must be present

Selecting the type of chromatography

–Denaturing/non-denaturing

–Resolution

–Yield

–Speed

Life Science Group I Chromatography

Typical Reasons why Proteins are Purified

•Identify the function of the protein (eg enzymes)

•Establish basic biochemical parameters eg Km

•Establish effects of activators and inhibitors on a protein function

•Crystallization to identify the structure of the protein

•Use the protein to generate a desired product eg antibodies, brewing, baking

•Produce a commercial product such as a therapeutic or diagnostic

Life Science Group I Chromatography

Applications that require biochromatography, a few examples

Purifying Proteins for Downstream assays (Functional Biology)

–Requires a few mg of proteins

–Requires the protein to be active

–Sometimes can use Tags, but the tag might affect the activity of the protein

Life Science Group I Chromatography

Applications that requires biochromatography, a few examples

Structural Biology

–Requires 10-100 mg of proteins

–Sometimes can use Tags, but the tag might affect the conformation of the protein

Usually require a tag clivage step

Life Science Group I Chromatography