Box 41 Schematic Presentation Of Common Chromatography Techniques Used For Purification Of Target Proteins From Host Proteins

Taking advantage of the unique properties of each protein, protein products can be purified from host proteins by:

1. Charge low high

Time

Column Matrix Proteins Mixture

2. Size (molecular weight, shape of molecules, etc.) excluded included in set matrix

Protein separation principle

High vs. low charge density

Large vs. small molecular diameter

Time

3. Affinity to bind to specific ligand or receptor leluted free ligand

Time

Ligand-receptor specificity and affinity

■TABLE 4.5. Some analytical techniques use in protein characterization and stability assessment

Techniques

Analysis

Protein Examples

Analytical centrifugation

Aggregation

Rop proteins [45]

Capillary electrophoresis

Degradation

Hirudin [46]

(CE)

Determination of Tm

RNase [47]

Circular dichroism (CD)

Estimation of secondary structures

a-Spectrin [48]

Determination of Tm

muscle acylphosphatase [49]

Conformation and interactions

IL-4 [50]

Determination of multimers

a-Lactablumin [51]

Differential scanning

Determination of Tg

hGH [52]

calorimetry (DSC)

Determination of Tm

a-Fibroblast growth factor [53]

Electron paramagnetic

Ligand-protein interactions

rhGH, rhlFN-g [54]

resonance (EPR)

Fluorescence

Protein unfolding interaction

a/ß MerP protein [55]

Conformation

a-Antitrypsin [56]

Degradation and aggregation

hGH [57], ßFGF [58]

HPLC-ion exchange

Degradation and aggregation

hGH [52]

HPLC-reversed phase

Estimation of contamination

rh-Parathyroid hormone [59]

HPLC-size exclusion

Degradation and aggregation

hGH [52]

Estimation of secondary structures

Chymotrysinogen [60]

Infrared spectroscopy

Determination of Tm

h-phenylalanine hydroxylase [61]

Confirmation

IL-1a [62]

Karl Fischer

Water determination

Insulin [63]

Light scattering

Aggregation

h-Relaxin [64]

Mass spectrometry

Determination of molecular

ßFGF [58]

weight, degradation products,

and contaminants

Protein glycosylation

Epoietin [65]

NMR spectroscopy

Determination of 3D and

IL-6 [66]

secondary structures

Protein relaxation and softening

g-globulin [67]

Protein glycosyaltion

h-ß-Galacto-sulfotransferase [68]

Raman spectroscopy

Determination of secondary

Insulin [69]

structures

Refractometry

Ligand-protein interactions

rhGH, rhlFN-g [54]

Two-dimensional gel

Protein glycosylation

Epoietin [70]

electrophoresis

Protein degradation

Product contaminant

determination

UV visible spectroscopy

Protein aggregation

aFGF [53]

Estimation of contamination

hGH [71]

Probing protein conformation

IL-2, insulin [72,73]

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