Purification Techniques | Is the Effect of Hydrophobic Chromatography Related to Temperature?

In the beginning of winter, although the weather is getting colder, the experimental staff's enthusiasm for their work is undiminished. Recently, we often hear various complaints from the experimental staff: why are the results of the hydrophobic chromatography not as good as before? Why can't we reproduce the previous results? Today's hydrophobic classroom can help you out.

In the early stage of purification, we mentioned the effective method of removing antibody variants - hydrophobic chromatography resin. The separation of samples and impurities by hydrophobic chromatography resin is mainly based on the hydrophobicity of the chromatography resin, the properties and composition of the separated substances, the distribution of exposed hydrophobic groups on the surface, and the type and concentration of salts in the binding buffer.

The main factors affecting the purification effect of hydrophobic chromatography resin include the properties of the chromatography resin (base frame type, structure, ligand density, etc.), the composition of the buffer solution (types, concentrations, pH values, temperature of the equilibrium and elution buffers), the properties of the feed solution, and other components of the sample.

We thought that among these influencing factors, did the experimenters overlook any of them when repeating the hydrophobic chromatography? Considering the recent cooling, the first thing we thought of was temperature.

Generally, the effect of temperature on chromatography is complex. The binding of proteins to hydrophobic chromatography resin is driven by entropy. Therefore, when the temperature decreases, the interaction between proteins and chromatography resin decreases, as does the van der Waals force.

BioLink can provide hydrophobic chromatography resin with different particle sizes, including the MaXtar substrate and Chromstar substrate. For different experimental needs, there are also different hydrophobic ligands such as butyl, octyl, phenyl, and other combinations.

Purification Techniques | Is the Effect of Hydrophobic Chromatography Related to Temperature?

Given that temperature can affect hydrophobic interactions, the consistency of sample temperature should be fully considered in the development of downstream purification processes and subsequent scale-up production: during the R&D stage, when the sample size is relatively small, the sample can be taken out of the frozen state and returned to room temperature in a very short period of time; when it comes to large-scale production, the sample takes longer to rewarm.

Application

Case of purification of double antibodies using hydrophobic chromatography resin

Resin: Butyl Chromstar HP

Chromatography column: Chrom-Screen 4.7ml; 10cm

Sample: double antibody sample (adjust the conductivity to 130mS/cm), sample temperature 25℃

Equilibrium buffer: 20 mM Tris, 1 M (NH₄)2SO₄, pH 7.4

Elution buffer: 20 mM Tris, pH 7.4

Retention time: 5min

Purification Techniques | Is the Effect of Hydrophobic Chromatography Related to Temperature?

Figure 1 Chromatogram

Purification Techniques | Is the Effect of Hydrophobic Chromatography Related to Temperature?

Figure 2 SEC-HPLC detection spectra before and after purification (left: before purification; right: after purification)

Summary: From the chromatogram and SEC detection spectra, it can be seen that Butyl Chromstar HP can increase the purity of the sample from 95.41% to 98.8%, while decreasing the aggregate content from 4.59% to 1.20%.

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