Properties, in theory and practice, of novel gel filtration media for standard liquid chromatography

Hagel, Lars; Lundström, Håkan; Andersson, Torvald; Lindblom, Hans

doi:10.1016/s0021-9673(01)93880-4

Cited by 36 publications

(13 citation statements)

References 23 publications

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“…Evaluation of these smaller particles has shown the advantages in resolution as compared to larger particles. [106,110–112] Liu et al [94] demonstrated these effects for the analysis of a reduced antibody: improved resolution and higher efficiencies were observed for the light and heavy chain using columns with smaller particle sizes (Table 1). [94] …”

Section: Methods Of Optimizationmentioning

confidence: 99%

A Review Size-Exclusion Chromatography for the Analysis of Protein Biotherapeutics and Their Aggregates

Hong

Koza

Bouvier

2012

Journal of Liquid Chromatography & Related Technologies

459

291

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In recent years, the use and number of biotherapeutics has increased significantly. For these largely 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). In the following review we will outline the history and background of SEC for the analysis of proteins. We will also discuss the instrumentation for these analyses, including the use of different types of detectors. Method development for protein analysis by SEC will also be outlined, including the effect of mobile phase and column parameters (column length, pore size). We will also review some of the applications of this mode of separation that are of particular importance to protein biopharmaceutical development and highlight some considerations in their implementation.

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Section: Methods Of Optimizationmentioning

confidence: 99%

A Review Size-Exclusion Chromatography for the Analysis of Protein Biotherapeutics and Their Aggregates

Hong

Koza

Bouvier

2012

Journal of Liquid Chromatography & Related Technologies

459

291

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“…The optimal experimental conditions will depend on the size differences of the solutes and on the processing rate. Using established theory, it is possible to obtain guidelines for fractionation by gel filtration (Hagel et al, 1989).…”

Section: Applicationsmentioning

confidence: 99%

Gel Filtration

Hagel¹

2001

Encyclopedia of Life Sciences

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Gel filtration is a chromatographic technique that separates solutes according to decreasing size. Typical applications are desalting (buffer exchange), fractionation (purification) and determination of molecular mass (solute size). The theory of gel filtration is well established and simulations of experiments may readily be performed to aid in selection and optimization of experimental conditions.

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“…zone broadening due to non-equilibrium effects at high solvent velocity) and the number of cycles (i.e. zone broadening due to large sample volumes) in order to process a given volume within a given time (22). The effect of increasing the bed diameter to allow for application of larger sample volumes without sacrificing the resolution is also illustrated in Figure 5.…”

Section: Molecular Sizementioning

confidence: 99%

Characteristics of Modern Media for Aqueous Size Exclusion Chromatography

Hagel

1996

ACS Symposium Series

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Important characteristics of media for size exclusion chromatography include particle size, pore volume, sorptive properties and matrix rigidity. However, the single most decisive parameter is the optimization of the selectivity to cope with the demands of high resolution separations. Whereas traditionally media are manufactured to encompass a broad separation range we now see a focus to a few key applications. These include a renewed interest in separation of smaller macromolecules, e.g. peptides and protein fragments and in dedicated separations such as monoclonal antibodies from growth media additives. The increase in selectivity may be obtained by selective grafting of polymers to reduce accessibility of pores to a small size range of solutes and in principle mimic the functionality of a single pore size support. The properties of such a medium designed for aqueous size exclusion chromatography, Superdex, is reviewed to elucidate the effect of important characteristics.By definition, aqueous size exclusion chromatography is carried out using a porous support. The molecules are separated due to the varying degrees of steric exclusion from the pore volume as caused by different molecular sizes of the solutes. This will result in the molecules being eluted from the column in order of decreasing size. Furthermore, the separation will only take place over an elution volume corresponding to the pore volume. Thus, the effective separating volume in aqueous size exclusion is small as compared to adsorptive techniques (i.e. solutes will ideally only be eluted within one column volume). In preparative purifications this limitation is generally of little importance since the desired separation is achieved through employing a ge)l medium with a suitable selectivity. Also for analytical applications where one component is of major interest it is often possible to find a gel of matching selectivity. However, the analysis of broad molar mass distributions may demand a wide separation range to encompass the whole distribution and this may be obtained by

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Properties, in theory and practice, of novel gel filtration media for standard liquid chromatography

Cited by 36 publications

References 23 publications

A Review Size-Exclusion Chromatography for the Analysis of Protein Biotherapeutics and Their Aggregates

A Review Size-Exclusion Chromatography for the Analysis of Protein Biotherapeutics and Their Aggregates

Gel Filtration

Characteristics of Modern Media for Aqueous Size Exclusion Chromatography

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