Hydrophobic interaction chromatography

■ Alternatives• Gel filtration chromatography• Ion exchange chromatography• Reverse phase chromatographyWhy HIC? • Different basis of separation• Weaker interactions→ Less structural damage→ Maintain high activity

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→ Purpose→ Principle of HIC→ Advantages of

using HIC

→ What are the factors affecting HIC

→ Conclusion

Source of protein

Extraction

Separation

Purity & characterization

■ Purpose-• Downstream purification• Separation of biomolecuoles• Exploits differences in

hydrophobicity.→ Number of hydrophobic aminoacids.→ Distribution of these aminoacids.

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 &

characterization

dta4n/biochem503/Hydrophobic Interaction.html

■ 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

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macromol.sbcs.qmul.ac.uk

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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

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

O CH2 CH CH2 O ( CH2) 3 CH3

OH

O CH2 CH CH2 O ( CH2) 7 CH3

OH

O CH2 CH CH2 OOH

Butyl

Octyl

Phenyl

HIC Ligands

‰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

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.

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.

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 protein3. 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

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.

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

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 .

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

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.

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

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.

1. Teachline.Is.huji.ac.il2.http://

www.separations.eu.tosohbioscience.com3. www. http://en.wikibooks.org www.biotech.kth.se/courses/gru/

courselist/.../ChromMethods.pdf people.virginia.edu/~dta4n/.../

Hydrophobic%20Interaction.html www.google.com/patents/

US20070037966 www.med.unc.edu/.../... - United States

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