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Analysis of Quaternary Structure by High Performance Size-Exclusion Chromatography (HP-SEC) (CAT#: STEM-B-0383-CJ)

Introduction

Structure and conformation of a biological molecule is key for its function. The higher order structure of a biopharmaceutical molecule is, thereby, often directly connected to the quality, stability, safety, and efficacy of a therapy. The higher order structure is considered a critical quality attribute and, thus, a detailed understanding of the higher order structure of a biopharmaceutical compound is critical in every research and development phase. Characterizing the secondary, tertiary and, if present, quaternary structure of a biopharmaceutical compound requires multiple analytical techniques.<br /><br />Many proteins are made up of more than one polypeptide chain to perform their function. The complete structure of such a protein is designated its quaternary structure, and each polypeptide chain is referred to as a subunit. The quaternary structure is stabilized by the same bonds as for the tertiary structure, including different noncovalent bonds and disulfide bonds. These bonds hold the subunits together and arrange themselves to form a larger protein complex.




Principle

In HP-SEC, the sample is injected onto a column that is constantly flushed with mobile phase. The chromatography column itself contains fine and porous beads, which are composed of dextran, agarose, silica or polyacrylamide (the stationary phase). The beads allow small species to migrate into their pores, increasing their retention inside the column, while larger species migrate with the mobile phase without entering the bead pores. Thus, larger species reach the column end faster than smaller species. After separation, the various species are detected using Ultraviolet (UV),absorption , Fluorescence, Refractive index (RI), Multi-angle laser light scattering(MALLS), Charged aerosol detection(CAD) detectors.

Applications

Biopharmaceutica

Procedure

1. Sample preparation.
2. Samples were injected onto a column that was continuously flushed with mobile phase and species were separated.
3. Various species were detected using ultraviolet (UV), absorption, fluorescence, refractive index (RI), multi-angle laser scattering (MALLS), and charged aerosol detection (CAD) detectors.
4. Analyse data.

Materials

• Sample: Protein monomers, Fragments and Aggregates
• Equipment: High performance size-exclusion chromatography (HP-SEC)

Notes

• HP-SEC allows the sizing, quantification and molecular weight determination of fragments, monomers and aggregates.
• The size range of HP-SEC is defined by the pore size of the column and the method set-up (e.g., mobile phase, flow settings and column dimensions).
• HP-SEC is commonly employed in all stages of research and development of biopharmaceuticals. It is highly valuable during formulation development, stability studies and forced degradation studies, but is most frequently used for batch release testing.