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

Abstract

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.

Surface Properties of Heat‐Induced Soluble Soy Protein Aggregates of Different Molecular Masses

“…The size (hydrodynamic molecular mass, m ) of heat‐induced soluble SPI aggregates and the m distribution were characterized with size exclusion HPLC. The method yielded reproducible results in agreement with literature reports (Hong and others ). Attempts were made to also use light scattering techniques, including multiangle laser light scattering (MALLS) and dynamic light scattering (Zetasizer), to measure the particle sizes.…”

Surface Properties of Heat‐Induced Soluble Soy Protein Aggregates of Different Molecular Masses

“…The size (hydrodynamic molecular mass, m ) of heat‐induced soluble SPI aggregates and the m distribution were characterized with size exclusion HPLC. The method yielded reproducible results in agreement with literature reports (Hong and others ). Attempts were made to also use light scattering techniques, including multiangle laser light scattering (MALLS) and dynamic light scattering (Zetasizer), to measure the particle sizes.…”

Cholinium-Based Ionic Liquids as Efficient Media for Improving the Structural and Thermal Stability of Immunoglobulin G Antibodies

“…Moreover, as verified by the SE-HPLC chromatograms, a band with high molecular weight is observed, indicating the presence of IgG agglomerates in the control and IL solutions. However, the intensity of this band is not observed to increase, thus corroborating the SE-HPLC results, that is, no new IgG aggregates other than originally present are formed. − In the previous studies, the structural stability of IgG was evaluated in the presence of imidazolium-, ammonium-, and phosphonium-based ILs by SDS-PAGE, after its extraction from rabbit serum, no degradation of IgG was detected, except in the presence of tetrabutylphosphonium bromide. In this previous work, Ferreira et al concluded that the high hydrophobicity of phosphonium-based ILs was the main reason behind its stronger negative effect on the integrity of IgG.…”

Heat-Induced Soluble Protein Aggregates from Mixed Pea Globulins and β-Lactoglobulin

“…It seems that better separation and M w distribution of the protein aggregates and fractions were obtained in relation to Mession et al 23 This could be due to the properties of the broader range separation column selected and the UV wavelength (280 vs 215 nm) retained, with which the response is more sensitive for aromatic amino acids and which is commonly used for quantifying proteins with greater linearity. 50 Moreover, the M w of the Glob aggregates in G′ 1 were close to the results obtained by Li et al 17 for heat-induced soy protein aggregates, as well as the results obtained by Fang et al 25 The latter had considered that the M w distribution of the soy globulin aggregates was (4−8) × 10 3 kDa at a protein concentration between 0.5 and 5%. 22 At the same protein concentration for Glob and βlg (2 wt %), the G′ 1 signal intensity was low in relation to the expected quantity of high-M w aggregates formed.…”

Surface Properties of Heat‐Induced Soluble Soy Protein Aggregates of Different Molecular Masses

“…The size (hydrodynamic molecular mass, m ) of heat‐induced soluble SPI aggregates and the m distribution were characterized with size exclusion HPLC. The method yielded reproducible results in agreement with literature reports (Hong and others ). Attempts were made to also use light scattering techniques, including multiangle laser light scattering (MALLS) and dynamic light scattering (Zetasizer), to measure the particle sizes.…”

Cholinium-Based Ionic Liquids as Efficient Media for Improving the Structural and Thermal Stability of Immunoglobulin G Antibodies

“…Moreover, as verified by the SE-HPLC chromatograms, a band with high molecular weight is observed, indicating the presence of IgG agglomerates in the control and IL solutions. However, the intensity of this band is not observed to increase, thus corroborating the SE-HPLC results, that is, no new IgG aggregates other than originally present are formed. − In the previous studies, the structural stability of IgG was evaluated in the presence of imidazolium-, ammonium-, and phosphonium-based ILs by SDS-PAGE, after its extraction from rabbit serum, no degradation of IgG was detected, except in the presence of tetrabutylphosphonium bromide. In this previous work, Ferreira et al concluded that the high hydrophobicity of phosphonium-based ILs was the main reason behind its stronger negative effect on the integrity of IgG.…”

Heat-Induced Soluble Protein Aggregates from Mixed Pea Globulins and β-Lactoglobulin

“…It seems that better separation and M w distribution of the protein aggregates and fractions were obtained in relation to Mession et al 23 This could be due to the properties of the broader range separation column selected and the UV wavelength (280 vs 215 nm) retained, with which the response is more sensitive for aromatic amino acids and which is commonly used for quantifying proteins with greater linearity. 50 Moreover, the M w of the Glob aggregates in G′ 1 were close to the results obtained by Li et al 17 for heat-induced soy protein aggregates, as well as the results obtained by Fang et al 25 The latter had considered that the M w distribution of the soy globulin aggregates was (4−8) × 10 3 kDa at a protein concentration between 0.5 and 5%. 22 At the same protein concentration for Glob and βlg (2 wt %), the G′ 1 signal intensity was low in relation to the expected quantity of high-M w aggregates formed.…”

Surface Properties of Heat‐Induced Soluble Soy Protein Aggregates of Different Molecular Masses

“…The size (hydrodynamic molecular mass, m ) of heat‐induced soluble SPI aggregates and the m distribution were characterized with size exclusion HPLC. The method yielded reproducible results in agreement with literature reports (Hong and others ). Attempts were made to also use light scattering techniques, including multiangle laser light scattering (MALLS) and dynamic light scattering (Zetasizer), to measure the particle sizes.…”

Cholinium-Based Ionic Liquids as Efficient Media for Improving the Structural and Thermal Stability of Immunoglobulin G Antibodies

“…Moreover, as verified by the SE-HPLC chromatograms, a band with high molecular weight is observed, indicating the presence of IgG agglomerates in the control and IL solutions. However, the intensity of this band is not observed to increase, thus corroborating the SE-HPLC results, that is, no new IgG aggregates other than originally present are formed. − In the previous studies, the structural stability of IgG was evaluated in the presence of imidazolium-, ammonium-, and phosphonium-based ILs by SDS-PAGE, after its extraction from rabbit serum, no degradation of IgG was detected, except in the presence of tetrabutylphosphonium bromide. In this previous work, Ferreira et al concluded that the high hydrophobicity of phosphonium-based ILs was the main reason behind its stronger negative effect on the integrity of IgG.…”

Heat-Induced Soluble Protein Aggregates from Mixed Pea Globulins and β-Lactoglobulin

“…It seems that better separation and M w distribution of the protein aggregates and fractions were obtained in relation to Mession et al 23 This could be due to the properties of the broader range separation column selected and the UV wavelength (280 vs 215 nm) retained, with which the response is more sensitive for aromatic amino acids and which is commonly used for quantifying proteins with greater linearity. 50 Moreover, the M w of the Glob aggregates in G′ 1 were close to the results obtained by Li et al 17 for heat-induced soy protein aggregates, as well as the results obtained by Fang et al 25 The latter had considered that the M w distribution of the soy globulin aggregates was (4−8) × 10 3 kDa at a protein concentration between 0.5 and 5%. 22 At the same protein concentration for Glob and βlg (2 wt %), the G′ 1 signal intensity was low in relation to the expected quantity of high-M w aggregates formed.…”