Purification Techniques: Influencing Factors and Applications of Hydrophobic Interaction Chromatography Resin

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As we all know, hydrophobic interaction chromatography resin separates substances through reversible interaction between protein and hydrophobic surface of chromatography resin according to the distribution of hydrophobic ligands on protein surface. Due to its excellent removal performance of high polymer and oligomer in separation and purification, hydrophobic resin play an important role in the purification of fusion proteins, antibodies, ADCs, etc. How to better use hydrophobic resin is inseparable from an in-depth understanding of hydrophobic resin.

Influencing Factors of Hydrophobic Interaction Chromatography Resin

1. Hydrophobic Properties of Resin

Hydrophobicity is an important reference data in resin selection, which is mainly determined by ligand type (Phenyl > Octyl > Butyl > Butyl-S), ligand density and matrix, and hydrophobicity also directly affects the recovery rate of target protein. BioLink also ranked the hydrophobicity of existing mainstream hydrophobic resins.

purification-techniques-influencing-factors-and-applications-of-hydrophobic-interaction-chromatography-resin1.jpg

Figure 1: The hydrophobicity of BioLink hydrophobic resins is sorted from weak to strong

2. Selection of Salt Solution

According to the principle of hydrophobic chromatography, different salt solution and its concentration can directly change the number of hydrophobic ligands on the molecular surface, thus affecting the interaction between the target protein and hydrophobic resin. Therefore, the selection of salt solutions is generally screened and optimized at the initial stage.

Salt Solution (Elution Capacity):

NaSO4＞KH2PO4＞Na2HPO4＞（NH4)2SO4＞K2CO3＞Na2CO3＞NaCl＞NaNO3

3. Effect of Temperature

Generally, the temperature increases, the hydrophobic interaction increases, and the binding capacity increases; The temperature decreases, the hydrophobic interaction decreases, and the binding capacity decreases. During process development, it should be kept consistent with the ambient temperature of scale-up production to prevent excessive temperature difference and offset of process scale-up.

4. Selection of Additives

In the process of hydrophobic interaction chromatography, it is quiet common for the amount of target protein in the eluate to be small or even uneluted. Based on this, adding additives in the elution process is a commonly used way to improve the solubility of protein and promote protein elution. Common additives include organic solvents (sopropanol, ethylene glycol, etc.) or surfactants and amino acids (arginine), etc.

Application of Hydrophobic Interaction Chromatography Resin

Application 1

Resin Name : MaXtar® Phenyl HR

Column Volume: 4.7 mL

Target Protein: BsAb (130 mS/cm)

Binding Capacity: 30 mg/mL

Equilibration Buffer: 20 mM Tris-HCl, 1M (NH4) 2SO4, pH 7.4

Elution Buffer: 20 mM Tris-HCl, pH 7.4

purification-techniques-influencing-factors-and-applications-of-hydrophobic-interaction-chromatography-resin2.jpg

Figure 2: MaXtar® Phenyl HR Chromatogram

Sample Name	SEC Purity
Sample Name	HMW(%)	Monomer(%)	LMW(%)
Load	4.59	95.41	0.0
Elution	0.59	99.4	0.0

Table 1: MaXtar® Phenyl HR Before and after purification

SEC _ HPLC (%) Results

Application 2

Resin Name : Phenyl Chromstar® HP

Column volume: H = 15 cm, D = 26 mm

Target Protein: Herpes zoster vaccine (VZV) (90-105 mS/cm)

Equilibration Buffer: PBS, 0.6 M (NH4) 2SO4

Elution Buffer: purified water

purification-techniques-influencing-factors-and-applications-of-hydrophobic-interaction-chromatography-resin3.jpg

Figure 3: Phenyl Chromstar® HP chromatogram

Sample Name	SEC Purity
Sample Name	HMW(%)	Monomer(%)	LMW(%)
Elution	2.0	98.0	0.0

Table 2: Phenyl Chromstar® Before and after HP purification

SEC _ HPLC (%) Results

Summary:

Application 1: After purifying bispecific antibody by MaXtar® Phenyl HR, SEC _ HPLC (%) increased from 95.41% to 99.4%, and the high polymer decreased by 4%, which showed a remarkable high polymer removal effect.

Application 2: After purifying herpes zoster vaccine by Phenyl Chromstar® HP, SEC _ HPLC (%) reached 98%, and the purification effect was remarkable.

Application Tips:

1. Selection of Hydrophobic Resins

The hydrophobic ligand can be selected according to the properties of the target protein according to the different hydrophobicity. If you don't know the properties of the target protein, you can give priority to the Phenyl ligand for testing. Based on the specific situation, consider whether to replace the resin.

2. Selection of Loading Buffer

Commonly used salt solutions in hydrophobic buffers are (NH4)2SO4 and NaCl. The general concentration range of (NH4)2SO4 is 0.5-1.5 M (NH4)2SO4, while the general concentration range of NaCl is within 3 M NaCl. The salt solution can be selected according to specific conditions.

3. Elution Process Optimization

The hydrophobic resin is eluted linearly, and the process is adjusted according to the elution situation. If the eluted target protein is well separated from the heteroprotein, one-step elution can be tried; If the resolution is poor, the linear range can be reduced and the slope can be reduced to improve the resolution; If the eluted protein is less or difficult to elute, it can be optimized by additives or changing the salt solution.

References

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13 Feb 2023