Основы белковой хроматографии
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IEX Advantages and Disadvantages
Advantages
Concentrating step High capacity Fast
Inexpensive Easy to scale up
Equilibration &Binding: Low Salt (20mM)
Elute with Linear gradient/stepwise with salt
Disadvantages
Sample often elutes in high salt
Low selectivity
Life Science Group I Chromatography
Ion Exchange Chromatography
–Strong ion exchangers
–Q (Quaternary amine, -N(CH3)3+) for Anion Excahge
–S (Sulfate, -SO3-) for Cation Exchange
–Weak ion exhangers:
–DEAE (DiEthylAminoEthyl, C2H4-N(C2H5)2) for AEX
–CM (CarboxyMethyl, -CH2-COO-) for CEX
–Various media show different selectivity => need for media screening to optimize purification
Life Science Group I Chromatography
Size Exclusion Chromatography (Gel Filtration)
–Separation based on the size and conformation
–The smaller the protein, the longest time it takes to elute
–Diluting technique
–Can be used for desalting (or buffer exchange)
Life Science Group I Chromatography
Size Exclusion Summary
Advantages |
Disadvantages |
–Simple, inexpensive
–Separation unaffected by buffer conditions
Desalting/buffer exchange
Solvent removal
–Non-adsorptive technique
Mild conditions
Good recoveries
–Separates monomers from aggregates
–Moderate resolution
–Slow
Low flow rates
Low capacity
–Diluting
Non-adsorptive technique - dilutes product, BUT quicker than dialysis!
Fractionation requires large columns relative to sample size
Life Science Group I Chromatography
Size Exclusion Chromatography (Gel Filtration)
Egg Ovalbumin, 44,7 kDa
Myoglobin, 17 kDa
Bovine γ-globulin, 150 kDa
Thyroglobulin, 660-690 kDa |
Vitamin B12, 1,25 kDa |
Life Science Group I Chromatography
Affinity Chromatography
– Separation based on the reversible affinity between the target protein and a ligand
Technique |
Tag |
Ligand |
Elution buffer (Typical) |
IMAC |
6-Histidine |
Ni+ |
500mM imidazole |
GST |
Glutathion S-Transferase (GST) |
Glutathione |
10mM reduced glutathione |
Mabs purification |
None (Antibody heavy chain) |
Protein A/G |
0,1 M glycine-HCl pH 2,7 |
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– Concentrating Technique
– Do not require high pressure
– Better results when Reverse Flow Chromatography
Life Science Group I Chromatography
Affinity Chromatography
Single step purification!
–High specificity (referred to as “lock and key”)
–High capacity
–Concentrating
Adsorption technique that exploits affinity between a target molecule and a ligand covalently bound to a stationary phase
–Binding interactions are specific
–Binding is reversible
Highly specific and effective purification method
In the BioProcessing industry, Protein A affinity chromatography is used as the primary capture step
Life Science Group I Chromatography
Affinity Chromatography Summary
Advantages
Excellent capture step
–Generally used as 1st step for production of drug therapeutics
Very specific – high purity achieved in single step!
Purification of MAb on Protein A media very easy!
Concentrates sample
Disadvantages
Protein columns are very Expensive
Ligand leaching
–Contamination issues
–Toxicity issues (i.e. Protein A)
Coupling of ligands to activated matrix – not always straightforward
Requires cloning of target protein sequence to a tag sequence
Life Science Group I Chromatography
Hydrophobic Interaction Chromatography
–Separation based on hydrophobicity of part of proteins
–Works better in high salt, thus good after Ion Exchange
–A little more tricky, because need to work in harsher conditions
Life Science Group I Chromatography
HIC Applications
Anything with moderately hydrophobic groups will bind
Often used upstream after ammonium sulfate precipitation
After ion exchange step since the sample will be in high salt which enhances binding
Life Science Group I Chromatography