Presentation Information
[P04-531]Separation of Antibody-Modified Liposomes with Protein A and Protein L Affinity Chromatography
Ayano Tsutsumi1, Sakura Takeshima1, ○Noriko Yoshimoto1 (1. Yamaguchi University (Japan))
Keywords:
IgG-modified liposomes,protein A affinity chromatography,protein L affinity chromatography,cation-exchange chromatography
[Purpose]
Antibody modification of liposomes often produces heterogeneous IgG-liposome populations that differ in antibody density and binding orientation, making the purification and characterization of modified liposomes an important analytical challenge. In this study, we demonstrate the feasibility of using antibody affinity chromatography to distinguish and separate liposomes according to their antibody modification states, providing a new analytical approach for characterizing immunoliposomes and other antibody-functionalized nanoparticles.
[Method]
Human polyclonal immunoglobulin G (IgG), pre-purified by protein A and protein L chromatography and showing affinity for both columns, was used for liposome modification. Liposomes composed of POPC (15 mol%) and NG-POPE (85 mol%) containing vitamin B12 (VB12) were prepared by a lipid film hydration/extrusion method. The glutaryl groups on the liposome surface were activated with sulfo-NHS and EDC, followed by dialysis and mixing with IgG. The reaction mixture was separated using a Sepharose 4B gel filtration column to obtain IgG-modified liposomes and remove unreacted IgG. The resulting IgG-liposomes were further separated by affinity chromatography using protein A and protein L columns (MabSelect SuRe LX and KANEKA KanCap L), as well as by cation-exchange chromatography using a self-packed SP Sepharose HP column.
[Results]
The diameter of the IgG-liposomes fractionated by GFC increased from 110 nm for unmodified liposomes to 126 nm, while the ζ-potential shifted from -41.8 mV to -8.98 mV. The increase in diameter was smaller than the approximately 30 nm expected if IgG molecules (approximately 15 nm in length) were fully extended on the liposome surface, suggesting heterogeneous binding orientations. The fractionated IgG-liposomes were loaded onto a protein A column connected in series to a protein L column at pH 8.0 (Step I). IgG-liposomes that bound to the protein A column, including those also capable of binding to protein L, were eluted with a pH 3.0 buffer (Type A). IgG-liposomes that passed through the protein A column but bound to the protein L column were subsequently eluted with a pH 3.0 buffer (Type L). The Type A fraction eluted from the protein A column was mixed with binding buffer (pH 8.0) and reloaded onto a protein L column. Liposomes exhibiting affinity for both protein A and protein L were then eluted with pH 3.0 buffer (Type A-L). IgG-liposomes that showed no affinity for either protein A or protein L were collected during the initial loading step as the non-binding fraction (Type N).
[Consideration]
The IgG-liposomes were successfully fractionated using the affinity columns, although a substantial fraction was classified as Type N. These IgG-liposomes did not bind to the cation-exchange column and eluted immediately after sample loading, as indicated by VB12 absorbance at 360 nm. In contrast, unconjugated IgG was retained on the column. These results indicate that the positive surface charge of the IgG-liposomes was insufficient for retention by cation-exchange chromatography, which is consistent with the ζ-potential measurements.
[Conclusion]
IgG-modified liposomes exhibiting affinity for protein A and protein L were successfully separated by chromatographic methods. However, several issues remain, particularly the low recovery yields from the columns.
Antibody modification of liposomes often produces heterogeneous IgG-liposome populations that differ in antibody density and binding orientation, making the purification and characterization of modified liposomes an important analytical challenge. In this study, we demonstrate the feasibility of using antibody affinity chromatography to distinguish and separate liposomes according to their antibody modification states, providing a new analytical approach for characterizing immunoliposomes and other antibody-functionalized nanoparticles.
[Method]
Human polyclonal immunoglobulin G (IgG), pre-purified by protein A and protein L chromatography and showing affinity for both columns, was used for liposome modification. Liposomes composed of POPC (15 mol%) and NG-POPE (85 mol%) containing vitamin B12 (VB12) were prepared by a lipid film hydration/extrusion method. The glutaryl groups on the liposome surface were activated with sulfo-NHS and EDC, followed by dialysis and mixing with IgG. The reaction mixture was separated using a Sepharose 4B gel filtration column to obtain IgG-modified liposomes and remove unreacted IgG. The resulting IgG-liposomes were further separated by affinity chromatography using protein A and protein L columns (MabSelect SuRe LX and KANEKA KanCap L), as well as by cation-exchange chromatography using a self-packed SP Sepharose HP column.
[Results]
The diameter of the IgG-liposomes fractionated by GFC increased from 110 nm for unmodified liposomes to 126 nm, while the ζ-potential shifted from -41.8 mV to -8.98 mV. The increase in diameter was smaller than the approximately 30 nm expected if IgG molecules (approximately 15 nm in length) were fully extended on the liposome surface, suggesting heterogeneous binding orientations. The fractionated IgG-liposomes were loaded onto a protein A column connected in series to a protein L column at pH 8.0 (Step I). IgG-liposomes that bound to the protein A column, including those also capable of binding to protein L, were eluted with a pH 3.0 buffer (Type A). IgG-liposomes that passed through the protein A column but bound to the protein L column were subsequently eluted with a pH 3.0 buffer (Type L). The Type A fraction eluted from the protein A column was mixed with binding buffer (pH 8.0) and reloaded onto a protein L column. Liposomes exhibiting affinity for both protein A and protein L were then eluted with pH 3.0 buffer (Type A-L). IgG-liposomes that showed no affinity for either protein A or protein L were collected during the initial loading step as the non-binding fraction (Type N).
[Consideration]
The IgG-liposomes were successfully fractionated using the affinity columns, although a substantial fraction was classified as Type N. These IgG-liposomes did not bind to the cation-exchange column and eluted immediately after sample loading, as indicated by VB12 absorbance at 360 nm. In contrast, unconjugated IgG was retained on the column. These results indicate that the positive surface charge of the IgG-liposomes was insufficient for retention by cation-exchange chromatography, which is consistent with the ζ-potential measurements.
[Conclusion]
IgG-modified liposomes exhibiting affinity for protein A and protein L were successfully separated by chromatographic methods. However, several issues remain, particularly the low recovery yields from the columns.
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