Analysis of Protein Aggregates Charge Properties by Hydrophobic Interaction Chromatography (HIC) (CAT#: STEM-B-0308-CJ)

Introduction

The generic term 'aggregates' refers to species characterized by a wide size range, diverse morphologies and structures. Protein aggregates may start in the low nanometer size range but then can grow into the micrometer and even visible size range.

Most protein therapeutics and many other biopharmaceutical compounds are inherently unstable and can undergo aggregation through various pathways. Aggregates of various kinds can be formed, such as reversible and non-reversible, soluble, and non-soluble etc. In addition,Aggregation maybe occur because of exposure to air-liquid or liquid-solid interfaces, e.g., during mixing, during filling and shipping, during reconstitution of lyophilized products, or through contact with chromatography columns, pumps, pipes, vessels, filters, etc. Aggregation can directly influence the efficacy of the therapy by reducing the number of functional molecules, but also indirectly influence efficacy as well as safety of a therapy by inducing side-effects, such as unwanted immunogenicity.

Hydrophobicity is basically a measure of the degree of affinity between water and the side chain of an amino acid. Amino acids with non-polar side chains have higher hydrophobicities compared to amino acids with polar or charged side chains.

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Principle Applications Procedure Materials Notes Related Products

Principle

The principle for protein adsorption to HIC media is complementary to ion exchange and size exclusion chromatography. Sample molecules containing hydrophobic and hydrophilic regions are applied to an HIC column in a high-salt buffer. The salt in the buffer reduces the solvation of sample solutes. As solvation decreases, hydrophobic regions that become exposed are adsorbed by the media. The more hydrophobic the molecule, the less salt is needed to promote binding. Usually a decreasing salt gradient is used to elute samples from the column in order of increasing hydrophobicity. Sample elution may also be assisted by the addition of mild organic modifiers or detergents to the elution buffer.

Applications

Biopharmaceutica

Procedure

1. The proteins are bound to the hydrophobic ligand on the HIC resin in a binding buffer with a high salt concentration. 2. When the ionic strength of the buffer is reduced, the interaction is reversed. The protein with the lowest degree of hydrophobicity is eluted first. The most hydrophobic protein elutes last, requiring a greater reduction in salt concentration to reverse the interaction.

Materials

• Sample: Proteins
• Equipment: Hydrophobic Interaction Chromatography (HIC)

Notes

• The formation of aggregates in your biopharmaceutical product can have a negative effect on safety, efficacy and function. Regulatory authorities expect that orthogonal characterization techniques are used to fully understand the aggregation profile of any molecule.
• Measurable range: 5–50 nm

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