1. Umair Saleem Methods in protein chemistry Hydrophobic interaction chromatography

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

3. Contents → Purpose → Principle of HIC → Advantages of using HIC → What are the factors affecting HIC → Conclusion Source of proteinExtraction Separation Purity & characterization

4. ■ Purpose Downstream purification Downstream purification Separation of biomolecuolesSeparation of biomolecuoles Exploits differences in hydrophobicity. Exploits differences in hydrophobicity. → Number of hydrophobic aminoacids. → Distribution of these aminoacids.

5. Principle Separation of substances is based on their varying strength of interaction with hydrophobic groups attached to an uncharged gel matrix 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. Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix. How is this achieved?How is this achieved? Source of proteinExtraction Separation Purity & characterization

6. General Concept

7. Hydrophobic Interaction Chromatography

8. Experimental Technique ■ Choice of column → XK colums for HIC. Column dimensions → Short bed height (5-15 cm) suitable for HIC Column dimensions → Short bed height (5-15 cm) suitable for HIC ■Packing of Column: ■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.

9. ■ Sample preparation Sample composition Sample composition Sample volume Sample volume Sample viscosity Sample viscosity ■ Sample application ■ Batch Separation

10. Advantages of HIC Large volume of sample can be loaded Large volume of sample can be loaded Samples with high ionic strength can be used Samples with high ionic strength can be used Well suited to use before gel filtration, ion-exchange and affinity chromatography Well suited to use before gel filtration, ion-exchange and affinity chromatography Sample eluted with low salt Sample eluted with low salt Purification steps that generate large sample volume can be coupled with this method Purification steps that generate large sample volume can be coupled with this method Good for samples after ammonium sulfate fractionation. Good for samples after ammonium sulfate fractionation. These techniques may require pretreatment of samples (e.g. reducing ionic strength) These techniques may require pretreatment of samples (e.g. reducing ionic strength) Sample can be used in ion exchange chromatography step Sample can be used in ion exchange chromatography step

11. Factors affecting HIC  Type and concentration of ligand  Type of base matrix.  Type and concentration of salt  pH  Temperature  Additives

12. HIC Ligands OCH 2 CHCH 2 O(CH 2 ) 3 CH 3 OH OCH 2 CHCH 2 O(CH 2 ) 7 CH 3 OH OCH 2 CHCH 2 O OH Butyl Octyl Phenyl

13. 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)‰ 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‰Caution: protein can bind via multipoint attachment, thus difficult to elute

14. Type of base matrix ‰ 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 ‰ 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 ‰Two widely used supports are cross-linked agarose and synthetic copolymer materials ‰May be necessary to modify adsorption and elution conditions ‰May be necessary to modify adsorption and elution conditions

15. Influence of salts Type of salt Type of salt salt effect follow the Hofmeister series. salt effect follow the Hofmeister series. Hydrophobic interaction increases at increased salt concentration Hydrophobic interaction increases at increased salt concentration Increasing salting out effect Increasing salting out effect Anions: PO43- >SO42- >Cl- >Br- >NO3- >ClO4- >I- >SCN- Anions: PO43- >SO42- >Cl- >Br- >NO3- >ClO4- >I- >SCN- Cations: NH4+ > K+ >Na+ >Li+ >Mg2+ Cations: NH4+ > K+ >Na+ >Li+ >Mg2+ Decreasing surface tension Decreasing surface tension Increasing chaotropic effect Increasing chaotropic effect

16. PH Effect of pH on HIC is not straight forward. 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 ‰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 ‰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. ‰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.

17. Temperature Visser and Strating (1975): that role of temperature is a 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 ‰ 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 ‰ 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 ‰ Practical terms: To be aware that procedure developed at room temperature may be different if used in the cold room room

18. Additives Salts that cause “salting-in” will weaken protein-ligand interactions. Salts that cause “salting-in” will weaken protein-ligand interactions. ‰Alcohols and detergents (non-polar parts) can compete with protein for HIC absorbent sites and may displace proteins. ‰Alcohols and detergents (non-polar parts) can compete with protein for HIC absorbent sites and may displace proteins.

19. Hydrophobic Interaction Chromatography - Overall Conclusions Very useful technique for mAb purification Very useful technique for mAb purification Mainly used in the third step as a complementary technique to protein A and IEC (in-vivo) 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 HIC can be used in both binding and removal mode Can be a useful alternative to SEC for aggregate removal 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. HIC is also very useful for purification of antibodies in 2-step techniques (non-protein A) for in-vitro applications.

20. THANKS