Successfully reported this slideshow.
We use your LinkedIn profile and activity data to personalize ads and to show you more relevant ads. You can change your ad preferences anytime.
Hydrophobic interaction
chromatography
■ Alternatives
• Gel filtration chromatography
• Ion exchange chromatography
• Reverse phase chromatography
Why HIC?
• Dif...
tainano.com
→ Purpose
→ Principle of HIC
→ Advantages of
using HIC
→ What are the
factors affecting
HIC
→ Conclusion
Source of protein...
■ Purpose-
• Downstream purification
• Separation of biomolecuoles
• Exploits differences in hydrophobicity.
→ Number of h...
Principle
• Separation of substances is based
on their varying strength of
interaction with hydrophobic
groups attached to...
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 Col...
onubiol217.blogspot.com
jkirkbrown.com
macromol.sbcs.qmul.ac.uk
gelifesciences.com
 Large volume of sample can be
loaded
 Samples with high ionic strength
can be used
 Well suited to use before gel
filt...
 Type and
concentration of ligand
 Type of base matrix.
 Type and
concentration of salt
 pH
 Temperature
 Additives
...
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
 ‰Important to take note that selectivity will not be exactly
the same even with the same type of ligand if the base matr...
 Type of salt
▪ salt effect follow the
Hofmeister series.
▪ Hydrophobic interaction
increases at increased salt
concentra...
 Visser and Strating (1975): that
role of temperature is a
complex issue and differ from
observation of Hierten.
 ‰ Bind...
1.HIC in combination with ammonium sulphate precipitation
 Crude purification of human autotaxin
 HIC was used for initi...
5.Other HIC application areas in the research laboratory
 HIC using Phenyl Sepharose CL-4B has been used for exchange of ...
M.M. Diogoa, J.A. Queirozb, D.M.F.
Prazeres (2003, March) Assessment of
purity and quantification of plasmid DNA in
proces...
 The purpose of the study is to demonstrate the usefulness of HIC for
monitoring the performance of a plasmid DNA isolati...
 The researchers used a a column filled with Sepharose CL-6B gel
derivatized with 1,4-butanediol diglycidyl ether.The HIC...
 The above slide shows four chromatograms each representing the
resolved components of the E coli lysate.
 The chromatog...
 The results obtained showed that that HIC can be used as for
quantification and purity assessment of a plasmid DNA
sampl...
 Very useful technique for mAb purification.
 Mainly used in the third step as a complementary
technique to protein A an...
1. Teachline.Is.huji.ac.il
2.http://www.separations.eu.tosohbioscience.c
om
3. www. http://en.wikibooks.org
 www.biotech....
THANK
YOU
Hydrophobic interaction chromatography (1)
Hydrophobic interaction chromatography (1)
Upcoming SlideShare
Loading in …5
×

Hydrophobic interaction chromatography (1)

7,466 views

Published on

Published in: Education, Technology
  • Be the first to comment

Hydrophobic interaction chromatography (1)

  1. 1. Hydrophobic interaction chromatography
  2. 2. ■ Alternatives • Gel filtration chromatography • Ion exchange chromatography • Reverse phase chromatography Why HIC? • Different basis of separation • Weaker interactions → Less structural damage → Maintain high activity
  3. 3. tainano.com
  4. 4. → Purpose → Principle of HIC → Advantages of using HIC → What are the factors affecting HIC → Conclusion Source of protein Extraction Separation Purity & characterization
  5. 5. ■ Purpose- • Downstream purification • Separation of biomolecuoles • Exploits differences in hydrophobicity. → Number of hydrophobic aminoacids. → Distribution of these aminoacids.
  6. 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
  7. 7. dta4n/biochem503/Hydrophobic Interaction.html
  8. 8. ■ 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
  9. 9. onubiol217.blogspot.com
  10. 10. jkirkbrown.com
  11. 11. macromol.sbcs.qmul.ac.uk
  12. 12. gelifesciences.com
  13. 13.  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
  14. 14.  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
  15. 15. 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
  16. 16.  ‰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
  17. 17.  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.
  18. 18.  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.
  19. 19. 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
  20. 20. 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.
  21. 21. 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
  22. 22.  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 .
  23. 23.  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
  24. 24.  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.
  25. 25.  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
  26. 26.  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.
  27. 27. 1. Teachline.Is.huji.ac.il 2.http://www.separations.eu.tosohbioscience.c om 3. 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
  28. 28. THANK YOU

×