4. → Purpose
→ Principle of HIC
→ Advantages of
using HIC
→ What are the
factors affecting
HIC
→ Conclusion
Source of protein
Extraction
Separation
Purity &
characterization
5. ■ Purpose-
• Downstream purification
• Separation of biomolecuoles
• Exploits differences in hydrophobicity.
→ Number of hydrophobic aminoacids.
→ Distribution of these aminoacids.
6. Principle
• Separation of substances is based
on their varying strength of
interaction with hydrophobic
groups attached to an uncharged
gel matrix
• Hydrophobic groups on proteins are
sufficiently exposed to bind to the
hydrophobic groups on the matrix.
• How is this achieved?
Source of
protein
Extraction
Separation
Purity &
characterizati
on
9. ■ Choice of column → XK colums for
HIC.
• Column dimensions → Short bed
height (5-15 cm) suitable for HIC
■ Packing of Column: a modern,
highly crosslinked agarose-based gel
such as Sepharose Fast Flow is
however easier than packing a gel
filtration column since the bed
height required is much smaller.
■ Sample preparation
• Sample composition
• Sample volume
• Sample viscosity
■ Sample application
■ Batch Separation
14. Large volume of sample can be
loaded
Samples with high ionic strength
can be used
Well suited to use before gel
filtration, ion-exchange and
affinity chromatography
Sample eluted with low salt
Purification steps that
generate large sample volume
can be coupled with this
method
Good for samples after
ammonium sulfate
fractionation.
These techniques may require
pretreatment of samples (e.g.
reducing ionic strength)
Sample can be used in ion
exchange chromatography
step
15. Type and
concentration of ligand
Type of base matrix.
Type and
concentration of salt
pH
Temperature
Additives
Effects of ligand density-
Degree of substitution
‰ Binding capacity of protein to
HIC increases with increased alkyl
chain length (A) and increased
degree of substitution of
immobilized ligand (B)
‰Caution: protein can bind via
multipoint attachment, thus difficult
to elute
16. O CH2 CH CH2 O ( CH2) 3 CH3
OH
O CH2 CH CH2 O ( CH2) 7 CH3
OH
O CH2 CH CH2 O
OH
Butyl
Octyl
Phenyl
HIC Ligands
17. ‰Important to take note that selectivity will not be exactly
the same even with the same type of ligand if the base matrix
is different
‰Two widely used supports are cross-linked agarose and
synthetic copolymer materials
‰May be necessary to modify adsorption and elution
conditions
18. Type of salt
▪ salt effect follow the
Hofmeister series.
▪ Hydrophobic interaction
increases at increased salt
concentration
Increasing salting out effect
Anions: PO43- >SO42- >Cl-
>Br- >NO3- >ClO4- >I- >SCN-
Cations: NH4+ > K+ >Na+
>Li+ >Mg2+
Decreasing surface tension
Increasing chaotropic effect
Effect of pH on HIC is not
straight forward.
‰In general an increase in pH
weakens hydrophobic
interactions. It could be due to
increased titration of charged
groups leading to an increase in
hydrophilicity of the proteins
‰Decrease in pH leads to an
apparently increase in
hydrophobic interaction
‰Implication: Important factor
to consider for optimization of
HIC interaction. It is observed
that proteins which do not bind
to HIC adsorbent at neutral pH,
bind at acidic pH.
19. Visser and Strating (1975): that
role of temperature is a
complex issue and differ from
observation of Hierten.
‰ Binding of proteins to HIC
adsorbents is entropy driven
(Hjerten, 1976), i.e. interaction
increases with increase in
temperature
‰ Discrepancy in views could
be due to differential effects
by temperature on the
conformational state of
different
proteins and solubility in
solution
‰ Practical terms: To be aware
that procedure developed
at room temperature may be
different if used in the cold
room
Salts that cause “salting-
in” will weakenprotein-
ligand interactions.
‰Alcohols and
detergents (non-polar
parts) can compete with
protein for HIC absorbent
sites and may displace
proteins.
20. 1.HIC in combination with ammonium sulphate precipitation
Crude purification of human autotaxin
HIC was used for initial purification of autotaxin, a human 125K protein which
stimulates tumour cell motility (49).
2. HIC in combination with ion exchange chromatography
Purification of recombinant HIV reverse transcriptase
Purification of mammalian transcription factors
Micropurification of a GTPase activating protein
3. HIC in combination with gel filtration
A major advantage with adsorption chromatography is the possibility to achieve
a
decrease in sample volume concomitant with an increase in purity. In a
purification
scheme, HIC and other adsorption chromatography techniques are therefore
frequently used prior to gel filtration, in which sample volume is limited.
4. HIC as a ‘‘single step’’ purification technique
21. 5.Other HIC application areas in the research laboratory
HIC using Phenyl Sepharose CL-4B has been used for exchange of protein-bound detergent
(54).
Octyl Sepharose CL-4B has been used for the separation of different forms of dermatan
sulphate proteoglycans (55). HIC of nucleic acids, viruses and cells has also been described.
6. Preparative, large scale applications
Purification of a monoclonal antibody for clinical studies of passive immunotherapy of HIV-
1.
Purification of recombinant human Epidermal Growth Factor (h-EGF) from yeast.
Purification of a monoclonal antibody for in vitro diagnostic use.
Purification of a recombinant Pseudomonas aeruginosa exotoxin produced in E. coli.
22. M.M. Diogoa, J.A. Queirozb, D.M.F.
Prazeres (2003, March) Assessment of
purity and quantification of plasmid DNA in
process solutions using high-performance
hydrophobic interaction chromatography
Journal: The Journal of Chromatography A
23. The purpose of the study is to demonstrate the usefulness of HIC for
monitoring the performance of a plasmid DNA isolation process .
Many genetic engineering techniques require a highly pure plasmid DNA
sample , hence it is important to develop a reliable analytical method for
quantification of plasmid DNA and assessing its purity.
Quantification of total plasmid DNA in pure solutions is easily
accomplished , by spectrophotometry at 260 nm . However a
spectrometer cannot be used for the quantification of impure DNA
samples
Techniques like Agarose gel electrophoresis , capillary electrophoresis
and HPLC can be used to measure the purity of the sample , but all these
methods have considerable disadvantages
Agarose gel electrophoresis is not reproducible ,CE and HPLC are
expensive and time consuming .
24. The researchers used a a column filled with Sepharose CL-6B gel
derivatized with 1,4-butanediol diglycidyl ether.The HIC column was
fitted with a HPLC system , to qualitatively and quantitatively analyze
the plasmid sample.
The column was equilibriated with Tris-Cl buffer (ph 8.0)
The technique takes advantage of the more hydrophobic character of
nucleic acid impurities (RNA, proteins etc )compared to ds DNA
A E coli lysate is injected into the column in two stages :
In the first stage . The sample was eluted with no ammonium sulphate
added .
In the second stage , the sample was eluted with 450 mM and 1050 mM
of ammonium sulphate added to the sample .
The chromatograms of both the stages were obtained from the HPLC
system , the chromatograms were then compared
25.
26. The above slide shows four chromatograms each representing the
resolved components of the E coli lysate.
The chromatogram A was obtained during the first stage of the
experiment , where the ammonium sulphate concentration was 0 . The
components are not properly resolved , and only a single peak is
observed .
The chromatogram B , C , D were obtained when the ammonium
sulphate concentration was 450 mM , 1050 mM and 1.5 M respectively
The above mentioned chromatograms show distinct peaks.The first one
representing the eluted plasmid DNA ,the remaining representing the
impurities eluted out after the ammonium sulphate concentration has
been increased
Chromatogram D shows the resolved components of the impurities.
27. The results obtained showed that that HIC can be used as for
quantification and purity assessment of a plasmid DNA
sample.
The method used involved a simple and a rapid (7 Minute
long ) procedure for detecting the purity of a Plasmid
sample.
Results obtained from repeating the experiment several
times, showed a standard deviation value of less than
10%,which shows that the results are reproducible.
The technique had the ability to handle highly contaminated
samples ( <5% of plasmid DNA ) without any pre-treatment
such as digestion of high molecular mass RNA
28. Very useful technique for mAb purification.
Mainly used in the third step as a complementary
technique to protein A and IEC (in-vivo).
HIC can be used in both binding and removal mode.
Can be a useful alternative to SEC for aggregate
removal.
HIC is also very useful for purification of antibodies
in 2-step techniques (non-protein A) for in-vitro
applications.
Equilibrate the column and the sample to binding conditions. essak "General" starting point: equilibration buffer 50 mM sodium phosphate with 1-1.5 M ammonium sulphate Buffer choice will be discussed in more detail later 5 to 10 column volumes is normal for equilibration
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Salts that promote hydrophobic interactions are known as kosmo or lyotropes and are found to the left in the Hofmeister series. The chemical basis for the Hofmeister ranking reflects a balance between the effects of a given salt´s component ions on water structure and their interaction with protein surfaces. Salting-out ions increase order in water structure, making solubilization water less available. This effect is equated with increased surface tension and favors entropic association of protein hydrophobic residues with immobilized hydrophobic ligands. The most commonly used salt in HIC is (NH4 )2SO4 but other salts like Na2SO4 (sodium sulfate) and NaCl are often used when modifications in binding strength are desirable. Ammonium sulfate is the binding salt most frequently used and has an exceptional solubility (not sodium sulfate). Its major limitation is that above pH 7.5, strongly basic free ammonia can cause denaturation of some proteins and can also cause instability of buffer pH. At elevated pH, use potassium phosphate. The use of NaCl is limited to strongly hydrophobic proteins, on strongly hydrophobic ligands and even then only at very high salt concentrations. By varying both the type of salt and the concentration, good separation may be achieved over a wide range. The concentration of salt is very important. By choosing the right concentration the product may be bound to the matrix while most of the contaminants pass through the column.
