Hydrophobic Interaction Chromatography

Abstract

Hydrophobic interaction chromatography (HIC) – today a key method in the purification of monoclonal antibodies – involves the separation of protein molecules in their native and biologically active state owing to a differential interaction of these molecules with hydrophobic sites on the surface of a solid support. Therefore, an understanding of the principles involved in this method is crucial. In the separation process, hydrophobic patches on the protein surface interact with hydrophobic molecules (e.g. alkyl residues) immobilised on the hydrophilic solid‐phase surface (e.g. agarose). In the article, a historical overview is followed by a consideration of the biochemical and biophysical mechanisms involved in HIC and a discussion of the critical hydrophobicity method and other approaches to hydrophobic interaction chromatographic methods. A detailed protocol for the individual synthesis and screening of gel libraries for specific needs has been published elsewhere. The treatise is concluded with a discussion on the predictability of chromatographic results, new developments and an outlook into the future.

Key Concepts

  • Hydrophobic interaction chromatography is a biological recognition process corresponding to a two‐dimensional lock‐and‐key model, that is, the dynamic pairing of a 2D key (protein) with its complementary 2D ‘lock’ (alkyl lattice).
  • Proteins are adsorbed to hydrophobic agaroses as metastable states of adsorption–desorption hysteresis, indicating energetically that a local energy minimum exists (i.e. nonequilibrium state) in distinction to the global energy minimum, that is, true equilibrium.
  • Critical hydrophobicity is the threshold hydrophobicity under low salt concentrations (e.g. 50 mM Tris, 150 mM NaCl and pH 7.0) and characterises a gel for reversible HIC binding properties.
  • A homologous gel library consists of gels carrying covalently immobilised hydrocarbons (e.g. alkyl residues) comprising a homologous series.
  • A quantised hydrophobicity gradient is a hydrophobicity gradient separated into a library of distinct gels of varying hydrophobicity for determining the critical hydrophobicity.
  • The lattice‐site binding function is a protein adsorption isotherm (often showing positive cooperativity) as a function of the surface concentration of immobilised hydrocarbons (e.g. alkyl residues).
  • The bulk ligand‐binding function is a protein adsorption isotherm on immobilised hydrocarbons (often showing negative cooperativity) as a function of the bulk protein concentration.
  • Predicting chromatographic behaviour from sorption isotherms and protein structure are the ultimate aim in understanding and utilising HIC.
  • The critical hydrophobic patch area hypothesis is a method of predicting the chromatographic behaviour of proteins in HIC from protein structure, utilising the area of hydrophobic patches on the surface of proteins.

Keywords: agarose/Sepharose 4B ; adsorption hysteresis; cooperativity; critical hydrophobicity; fibrinogen; Hofmeister series; irreversibility; multivalent interactions; phosphorylase; salting‐in; salting‐out

Figure 1. The lattice‐site concept and parameters of HIC. (a) Simplified scheme of a planar two‐dimensional binding site lattice on a butyl‐Sepharose 4B disregarding variations in the orientation of butyl‐residues. The spacing between residues of c. 1.5–2.0 nm is in the range of a surface concentration of 30 µmol mL−1 packed gel; the length of a butyl residue is c. 0.5 nm. Phosphorylase b (6.3 × 5.5 × 10.9 nm) will cover an area containing more than 10 residues (Jennissen, ). (b) Variation of the chain length parameter in an uncharged homologous series of alkyl agaroses. The surface concentration of the immobilised residues should be constant, no charges should be introduced and leakage should be low. The CDI method is optimal for coupling of alkyl amines (Jennissen, ). (c) Variation of the surface concentration parameter of immobilised alkyl residues of a fixed chain length. The surface concentration shown here as a one‐dimensional array of residues increases from (i) to (iii). The number of alkyl residues capable of interacting with the protein (i.e. the valence) and thus the affinity of binding increases as the surface concentration of alkyl residues and concomitantly the hydrophobicity is enhanced.
Figure 2. Model curves of the interrelationship between the chain length parameter and the surface concentration parameter for the binding of a single protein to a series of alkyl agaroses. The sigmoidal curves shown here for an idealised system correspond to adsorption isotherms of the lattice‐site binding function type (Jennissen, , ). This form of the curves is due to the cooperative interaction of multiple alkyl residues with the protein (multivalence). For simplicity, the curves were calculated with the same increment, although there are indications that the increment can vary (Jennissen and Heilmeyer, ). An increase in the chain length of the immobilised alkyl residue shifts the curves from the right to the left. CH indicates the threshold region of critical hydrophobicity, which corresponds to the three different points of origin for the sigmoidal curves on the x‐axis. n, number of carbon atoms (n > 3).
Figure 3. Inverse salt dependence of the chromatography of purified phosphorylase b (phos. b) on Seph‐C1 (30 µmol mL−1 of packed gel). The equilibration buffer contained 10 mmol L−1 of sodium β‐glycerophosphate, 20 mmol L−1 of mercaptoethanol, 2 mmol L−1 of EDTA, 20% sucrose, 0.5 µmol L−1 of phenylmethylsulfonyl fluoride and pH 7.0 (buffer A), to which either 1.1 mol L−1 of ammonium sulfate or NaCl was added. Phosphorylase b (6 mg 3 mL−1) was added to 20 mL Seph‐C1 in a 2.0 cm i.d. × 17 cm column at 5 °C. Fractions of 6.5 mL were collected. The gel was prepared by the CNBr procedure. (a) Application of enzyme to a column equilibrated with buffer without ammonium sulfate (NH4)2SO4 or NaCl. (1) Application of phosphorylase b in buffer A. (2) Elution with buffer A + 1.0 mol L−1 of NaCl. (b) Application of enzyme to a column equilibrated with buffer of (NH4)2SO4. (1) Application of phosphorylase b in buffer A + 1.1 mol L−1 of (NH4)2SO4. (2) Elution with buffer A. (3) Elution with buffer A + NaCl. For further details, see the text and Jennissen .
Figure 4. Determination of the critical surface concentration (critical hydrophobicity) of Seph‐C4‐6 for the adsorption of purified fibrinogen. The uncharged alkyl‐agaroses were synthesised by the CDI method. Purified human fibrinogen (1.0 mg) was applied in 1.0 mL to a column (0.9 cm i.d. × 12 cm) containing 2.0 mL of packed gel in 50 mmol L−1 of Tris HCl, 150 mmol L−1 of NaCl, 1.0 mmol L−1 of EGTA and pH 7.4. Fractions of 1.5 mL were collected at room temperature. The column was washed with 15 mL of buffer followed by elution either with 7.5 mol L−1 of urea or, at high hydrophobicity of the gel, with 1% sodium dodecyl sulfate for the determination of the amount of protein bound. 100% (= saturation value) equals 1.0 mg of fibrinogen adsorbed to 2.0 mL of packed gel. Half‐saturation values correspond to 48, 22 and 8 µmol mL−1 for Seph‐C4, ‐C5 and ‐C6, respectively. The total amount of adsorbed fibrinogen, not corrected for the amount adsorbed to unsubstituted control Sepharose 4B, is shown in %. The solid lines were traced through the data points by the program Prism (Graph Pad Software Inc., La Jolla, CA) according to a variable slope sigmoidal equation. The arrows denote the critical hydrophobicities (c. 5% saturation value) at 31, 14 and 4.5 µmol mL−1 for Seph‐C4, ‐C5 and ‐C6, respectively. The ratio of critical hydrophobicity to half‐saturation values is ca. 0.6. For further details, see the text and Jennissen ; Jennissen and Demiroglou .
Figure 5. One‐step purification of fibrinogen from human blood plasma by HIC at the critical hydrophobicity point of Seph‐C5. (a) 16 mL (53.8 mg) of fresh unclotted human blood plasma to which NaCl (1.35 g) had been added was applied at fraction zero to 20 mL of packed Seph‐C5 (13.6 µmol mL−1 of packed gel in a column 1.4 cm i.d. × 11.6 cm) equilibrated with 50 mmol L−1 of Tris HCl and 1.5 mol L−1 of NaCl (pH 7.4), at a flow rate of 52 mL h−1 and a fraction volume of 8.6 mL at room temperature. The nonadsorbed protein was washed out with 200 mL equilibration buffer. Elution was facilitated at fraction 24 by elution buffer containing a 10‐fold lower salt concentration of 150 mmol L−1 NaCl. (b) Fractions 29–33 were pooled, giving a clottability of 96% with a yield of 61%. The total yield of fractions 27–34 was 82%. For further details, see the text, legend to Figure and Jennissen ; Jennissen and Demiroglou .
Figure 6. Comparison of equilibrium adsorption/desorption isotherms of phosphorylase b with zonal chromatography on the same low‐affinity butyl‐Sepharose 4B (5 µmol mL−1 packed gel). The experiments were performed in 10 mM Tris/maleate, 5 mM dithioerythritol, 1.1 M ammonium sulphate, 20% sucrose and pH 7.0 at 5 °C. (a) The adsorption and desorption isotherms are described by the Freundlich equations (a = α c1/n), a = 1.4 (c0.82) (mg mL−1 packed gel) and a =1.2 (c0.39) (mg mL−1 packed gel), respectively (a: amount adsorbed, c: equilibrium concentration, α: Freundlich constant and 1/n: Freundlich exponent). For further details on the isotherms, apparent binding constants and saturation and thermodynamic data, see Table. Data from Jennissen and Botzet ; Jennissen . (b) Isocratic chromatography of phosphorylase b: (○) control run with unsubstituted Sepharose 4B (sample: phosphorylase b, 0.39 mg mL−1); (□), butyl Sepharose 4B, (sample: 0.39 mg mL−1); (▪) butyl Sepharose 4B, (sample: 3.6 mg mL−1). Column: 0.62 cm i.d. × 16.5 cm bed height; flow rate: 5 mL h−1; fraction volume: 2.9 mL; sample volume 0.2 mL. Vt = 5.0 mL, Vo/Vt = 0.4 and the dead volume Vd = 3.2 mL. In the chromatogram no corrections for Vd were made. The protein yields were between 77% and 100%. Vm and Ve were determined according to the one‐half peak width at one‐third maximum height method (Crone, ) to Vm = 8.41 mL, = 17.98 mL and = 24.9 mL (corrected for Vd). The peak width itself (in mL) at one‐third maximum height was determined to (○) 5.5 mL, (□), 13.1 mL and (▪) 18.9 mL (data from Jennissen, ). For further details, equations and corrected elution volumes, see Table with legend.
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Further Reading

Nienhaus GU (2005) Protein–Ligand Interactions: Methods and Applications, pp. 1–580. Totowa, New Jersey: Humana Press.

Vijayalakshmi MA (2002) Biochromatography Theory and Practice, pp. 1–544. London, New York: Taylor and Francis.

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Jennissen, Herbert P(Sep 2016) Hydrophobic Interaction Chromatography. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0002678.pub4]