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Analysis of Protein Aggregates Hydrodynamic Diameter by Ultra-high- Performance Size-exclusion Chromatography (UP-SEC) (CAT#: STEM-B-0303-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.<br /><br />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.<br /><br />Molecular weight, also called molecular mass, mass of a molecule of a substance, based on 12 as the atomic weight of carbon-16. It is calculated in practice by summing the atomic weights of the atoms making up the substance's molecular formula.




Principle

In principle, an UP-SEC system works just like a HP-SEC instrument, however, under much increased system pressures of up to 1,000 bar. This allows for reduced analysis times, requiring lower sample and mobile phase volumes and delivering better resolution.

Applications

Biopharmaceutica

Procedure

1. Sample preparation.
2. Size exclusion chromatography (SEC) separates molecules based on their size by filtration through a gel.
3. Separation occurs when molecules of different sizes are included or excluded from the pores within the matrix.
4. Collect and analyse data.

Materials

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

Notes

If the increased sample throughput or better resolution is not required, HP-SEC is preferred as it is a more established technology.