Purity analysis SEC and RPLC

Overview

In recent years, the use and number of biotherapeutics has increased significantly. For these protein-based therapies, the quantitation of aggregates is of particular concern, given their potential effect on efficacy and immunogenicity. This need has renewed interest in size-exclusion chromatography (SEC). SEC is a chromatographic method, which acts as a sieve and separates molecules in solution by their size, and in some cases molecular weight. Molecular sieves are materials containing precise and single tiny holes, which can be used to adsorb gases or liquids. SEC has long residence time for small molecular weight compounds into gel pores, whereas large molecular compounds are washed away earlier. The advantage of molecular sieve chromatography is that it can efficiently process at least 1nmol of target protein. The separation time is also very short, generally within 3 hours, and the chromatographic separation peak obtained at the same time is also smaller.

SEC analysis of biopharmaceuticals

Advantages of SEC in Protein Purification

1. The buffer solution used for SEC can be exchanged and desalted.
2. Similar varieties (such as protein fragments and oligomers) can be separated.
3. Compatible with a variety of solvents.
4. Independent on any particular form of protein for preservation and elution.

The liquid chromatography is divided into positive and reverse phase chromatography according to the relative polarity of the mobile phase and the stationary phase. Reversed-phase liquid chromatography (RPLC) refers to chromatographic method that uses a hydrophobic stationary phase. RPLC has the characteristics of high resolution and high repeatability, therefore it has been widely used in protein and polypeptide analysis, esp. for the separation of small molecular weight proteins and protein fragments with molecular weight <20000 Da. MtoZ Biolabs has established a platform with both SEC and RPLC technologies, to meet the requirements of analyzing various biopharmaceutical samples.

RPLC analysis of biopharmaceuticals

Sample Requirement

Total conc. of protein >0.5 ug/ L
Volume of protein sample >50 uL
Note: Please specify the composition of the buffer, if desalt or concentration of protein sample is needed.

Reports

• Experiment procedures
• Parameters of liquid chromatography
• Protein purity results
• Bioinformatics analysis

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