Authors: Alexis Oliva; Matias Llabres; Jose B. Farina

Source: Current Drug Discovery Technologies, Volume 1, Number 3, October 2004 , pp. 229-242(14)

Publisher: Bentham Science Publishers

Abstract:

The proliferation of new peptides and proteins requiring characterisation is a direct result of recent advances in genomics and proteomics, but protein aggregation is particular problem in the biotechnology industry, where aggregation is encountered throughout the lifetime of a therapeutic protein, including during refolding, purification, sterilization, shipping, and storage process. To ensure that it meets quality standards, the size, molecular weight and / or molecular weight distribution, and aggregate state must be accurately determined. Traditional analytical methods for determining molecular weight include size-exclusion chromatography (SEC), gel electrophoresis, analytical ultracentrifugation and time-of-flight mass spectrometry. These technologies are time-consuming (some take days), provide data based on relative standards, or cannot characterise very high molecular weight aggregates.

Laser light-scattering (LS) detection coupled with SEC system have been used for over a decade to determine the size and molecular weight of bio-molecules such as proteins, peptides, polysaccharides, oligonucleotides, and antibodies, the method of choice being for molar mass determinations and the study of self-association and heterogeneous interaction under native, equilibrium conditions in solution.

The purpose of the current review is to describe and discuss the capability of the SEC / LS system to determine absolute molecular weight of proteins and their complexes and the association state of the conjugate, either with itself or with protein receptor / ligands. For this, the “two or three detector” methods, each with its advantages and limitations, can be used to calculate the molecular weight of a simple protein or glycoprotein, and the stoichiometry of their complexes.

Also, some alternative techniques for determining the molecular weight are discussed in this review. Applications of all these methodologies are described.

Keywords: light-scattering; protein; peptide; size-exclusion chromatography; molecular weight; stoichiometry

Document Type: Review article

DOI: http://dx.doi.org/10.2174/1570163043334938

Affiliations: 1: Dpto. Ingenieria Quimica y Tecnología Farmacéutica, Facultad de Farmacia, Universidad de La Laguna, 38200, La Laguna, Tenerife, Spain.

Publication date: 2004-10-01

More about this publication?

• Due to the plethora of new approaches being used in modern drug discovery by the pharmaceutical industry, Current Drug Discovery Technologies has been established to provide comprehensive overviews of all the major modern techniques and technologies used in drug design and discovery. The journal is the forum for publishing both original research papers and reviews describing novel approaches and cutting edge technologies used in all stages of drug discovery. The journal addresses the multidimensional challenges of drug discovery science including integration issues of the drug discovery process.
  
  Current Drug Discovery Technologies is an essential journal for all scientists and research managers involved in drug discovery who wish to keep abreast of all the modern techniques and technologies used in drug discovery.

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