Evaluation of Phenomena that Determine the Performance of Immunoaffinity, Peptide-Based and Ion Exchange Affinity Sorbents
Insufficient supply and pathogen safety concerns regarding plasma-derived therapeutic proteins, such as fibrinogen and immunoglobulins, have been the impetus for the development of genetic engineering techniques and separations methods for the economical and safe production of these proteins. This s...
Main Author: | |
---|---|
Other Authors: | |
Format: | Others |
Published: |
Virginia Tech
2014
|
Subjects: | |
Online Access: | http://hdl.handle.net/10919/29841 http://scholar.lib.vt.edu/theses/available/etd-12012000-111836/ |
id |
ndltd-VTETD-oai-vtechworks.lib.vt.edu-10919-29841 |
---|---|
record_format |
oai_dc |
collection |
NDLTD |
format |
Others
|
sources |
NDLTD |
topic |
affinity chromatography protein purification |
spellingShingle |
affinity chromatography protein purification Sines, Brian James Evaluation of Phenomena that Determine the Performance of Immunoaffinity, Peptide-Based and Ion Exchange Affinity Sorbents |
description |
Insufficient supply and pathogen safety concerns regarding plasma-derived therapeutic proteins, such as fibrinogen and immunoglobulins, have been the impetus for the development of genetic engineering techniques and separations methods for the economical and safe production of these proteins. This study is concerned with the isolation of these important therapeutics from complex media. Immunoaffinity chromatography has been an important method in the isolation of these products, typically being implemented as the final cleanup step yielding an extremely pure, homogenous final product. However, the use of immunoaffinity chromatography in large-scale purification processes have been precluded due to high capital costs and the inherent lability of immunosorbents. Peptide-based affinity sorbents are being developed in order to surmount the inherent limitations posed by monoclonal antibodies that are used as ligands in immunosorbents.
The objective of this research is to quantitatively assess the impact of affinity ligand orientation, local density and transport phenomena on peptide-based affnity sorbent performance. The peptides under study herein can form high-affinity complexes with their protein targets, thus these ligands are one of the newest technologies arising from combinatorial chemistry with applications to the difficult problem of purifying high-molecular weight proteins from complex mixtures. Two types of structural motifs which are common to small peptide affinity ligands derived from combinatorial chemistry are studied here: a linear peptide which is comprised of the affinity recognition sequence in its entirety and a chain structure which displays multiple branches of the recognition sequence emanating from a central lysinic core structure. Two recognition sequences are studied here which bind plasma proteins. One peptide recognition sequence forms a high affinity complex with fibrinogen. Another peptide recognition sequence binds the Fc region of immunoglobulins. Immunglobulins are plasma proteins which range in molecular weight from 155 to 900-kDa and are valuable for therapeutic uses for imparting passive immunity.
This study seeks to identify factors analogous to those manifested in immunosorbent performance that may also be important in the optimal design of peptide-based affinity sorbents. In general, previous research with the design of immunosorbents have found that immunosorbent performance, i.e., target-binding efficiency or activity, is substantially dependent upon several factors which include effects associated with ligand orientation, and local density as related to steric incumbrance of target binding sites, and transport phenomena as related to under utilization of intra matrix volume. In summary, this study asks the questions: (1) What factors regarding ligand orientation, local ligand density, and intraparticle transport phenomena, are important in the optimal design of peptide affinity sorbents?; and (2) Are these effects analogous to those manifested in immunosorbent performance?
This study seeks to investigate the use of techniques used to mitigate the effects associated with these negative factors upon immunosorbent performance in order to elucidate the nature of these same effects upon peptide-based affinity sorbents. For example, oriented ligand immobilization can be facilitated through selective coupling chemistries and the premasking of ligand binding domains prior to immobilization. In addition, the manipulation of local ligand density using novel spatially controlled matrix activation and ligand immobilization methods can be assessed and implemented for the optimization of the performance and design of peptide-based affinity sorbents. This study has found that enhanced transport phenomena into the matrix interior volume can be achieved by using low solids content cellulose matrices having a low extent of crosslinking. This study demonstrates the effective use of these large-particle diameter, low-solids content cellulose hydrogel matrices in immunoaffinity, peptide-based affinity and ion exchange chromatography in the separation of high-molecular weight therapeutic proteins. === Ph. D. === This report presents an evaluation of the design of low-solids content, large-particle diameter beaded cellulose supports for column-mode protein purification. The study presented here optimizes the molecular accessibility of the cellulose support to high molecular weight proteins relative to the mechanical stability of the support at high operating linear velocities by the manipulation of bead particle diameter and solids content. A novel epoxidegradient activation method (epoxy-GAM) is developed for creating a gradient of support activation for support crosslinking and affinity ligand installation in the preparation of DEAE hydrogel matrices. These cellulose hydrogel supports were evaluated with regards to structure, dynamic and static binding capacity, pressure-flow stability, chemical stability and intraparticle transport phenomena. The utility of the low-solids content, large-particle diameter DEAE hydrogel matrices was demonstrated in a column-mode protein purification using albumin and fibrinogen mixtures. |
author2 |
Chemical Engineering |
author_facet |
Chemical Engineering Sines, Brian James |
author |
Sines, Brian James |
author_sort |
Sines, Brian James |
title |
Evaluation of Phenomena that Determine the Performance of Immunoaffinity, Peptide-Based and Ion Exchange Affinity Sorbents |
title_short |
Evaluation of Phenomena that Determine the Performance of Immunoaffinity, Peptide-Based and Ion Exchange Affinity Sorbents |
title_full |
Evaluation of Phenomena that Determine the Performance of Immunoaffinity, Peptide-Based and Ion Exchange Affinity Sorbents |
title_fullStr |
Evaluation of Phenomena that Determine the Performance of Immunoaffinity, Peptide-Based and Ion Exchange Affinity Sorbents |
title_full_unstemmed |
Evaluation of Phenomena that Determine the Performance of Immunoaffinity, Peptide-Based and Ion Exchange Affinity Sorbents |
title_sort |
evaluation of phenomena that determine the performance of immunoaffinity, peptide-based and ion exchange affinity sorbents |
publisher |
Virginia Tech |
publishDate |
2014 |
url |
http://hdl.handle.net/10919/29841 http://scholar.lib.vt.edu/theses/available/etd-12012000-111836/ |
work_keys_str_mv |
AT sinesbrianjames evaluationofphenomenathatdeterminetheperformanceofimmunoaffinitypeptidebasedandionexchangeaffinitysorbents |
_version_ |
1719341003711709184 |
spelling |
ndltd-VTETD-oai-vtechworks.lib.vt.edu-10919-298412020-09-26T05:31:27Z Evaluation of Phenomena that Determine the Performance of Immunoaffinity, Peptide-Based and Ion Exchange Affinity Sorbents Sines, Brian James Chemical Engineering Velander, William H. Sullivan, Joseph T. Williams, Kimberly Forsten Tyson, John J. Conger, William L. Davis, Richey M. affinity chromatography protein purification Insufficient supply and pathogen safety concerns regarding plasma-derived therapeutic proteins, such as fibrinogen and immunoglobulins, have been the impetus for the development of genetic engineering techniques and separations methods for the economical and safe production of these proteins. This study is concerned with the isolation of these important therapeutics from complex media. Immunoaffinity chromatography has been an important method in the isolation of these products, typically being implemented as the final cleanup step yielding an extremely pure, homogenous final product. However, the use of immunoaffinity chromatography in large-scale purification processes have been precluded due to high capital costs and the inherent lability of immunosorbents. Peptide-based affinity sorbents are being developed in order to surmount the inherent limitations posed by monoclonal antibodies that are used as ligands in immunosorbents. The objective of this research is to quantitatively assess the impact of affinity ligand orientation, local density and transport phenomena on peptide-based affnity sorbent performance. The peptides under study herein can form high-affinity complexes with their protein targets, thus these ligands are one of the newest technologies arising from combinatorial chemistry with applications to the difficult problem of purifying high-molecular weight proteins from complex mixtures. Two types of structural motifs which are common to small peptide affinity ligands derived from combinatorial chemistry are studied here: a linear peptide which is comprised of the affinity recognition sequence in its entirety and a chain structure which displays multiple branches of the recognition sequence emanating from a central lysinic core structure. Two recognition sequences are studied here which bind plasma proteins. One peptide recognition sequence forms a high affinity complex with fibrinogen. Another peptide recognition sequence binds the Fc region of immunoglobulins. Immunglobulins are plasma proteins which range in molecular weight from 155 to 900-kDa and are valuable for therapeutic uses for imparting passive immunity. This study seeks to identify factors analogous to those manifested in immunosorbent performance that may also be important in the optimal design of peptide-based affinity sorbents. In general, previous research with the design of immunosorbents have found that immunosorbent performance, i.e., target-binding efficiency or activity, is substantially dependent upon several factors which include effects associated with ligand orientation, and local density as related to steric incumbrance of target binding sites, and transport phenomena as related to under utilization of intra matrix volume. In summary, this study asks the questions: (1) What factors regarding ligand orientation, local ligand density, and intraparticle transport phenomena, are important in the optimal design of peptide affinity sorbents?; and (2) Are these effects analogous to those manifested in immunosorbent performance? This study seeks to investigate the use of techniques used to mitigate the effects associated with these negative factors upon immunosorbent performance in order to elucidate the nature of these same effects upon peptide-based affinity sorbents. For example, oriented ligand immobilization can be facilitated through selective coupling chemistries and the premasking of ligand binding domains prior to immobilization. In addition, the manipulation of local ligand density using novel spatially controlled matrix activation and ligand immobilization methods can be assessed and implemented for the optimization of the performance and design of peptide-based affinity sorbents. This study has found that enhanced transport phenomena into the matrix interior volume can be achieved by using low solids content cellulose matrices having a low extent of crosslinking. This study demonstrates the effective use of these large-particle diameter, low-solids content cellulose hydrogel matrices in immunoaffinity, peptide-based affinity and ion exchange chromatography in the separation of high-molecular weight therapeutic proteins. Ph. D. This report presents an evaluation of the design of low-solids content, large-particle diameter beaded cellulose supports for column-mode protein purification. The study presented here optimizes the molecular accessibility of the cellulose support to high molecular weight proteins relative to the mechanical stability of the support at high operating linear velocities by the manipulation of bead particle diameter and solids content. A novel epoxidegradient activation method (epoxy-GAM) is developed for creating a gradient of support activation for support crosslinking and affinity ligand installation in the preparation of DEAE hydrogel matrices. These cellulose hydrogel supports were evaluated with regards to structure, dynamic and static binding capacity, pressure-flow stability, chemical stability and intraparticle transport phenomena. The utility of the low-solids content, large-particle diameter DEAE hydrogel matrices was demonstrated in a column-mode protein purification using albumin and fibrinogen mixtures. 2014-03-14T20:19:30Z 2014-03-14T20:19:30Z 2000-11-03 2000-12-01 2002-01-04 2001-01-04 Dissertation etd-12012000-111836 http://hdl.handle.net/10919/29841 http://scholar.lib.vt.edu/theses/available/etd-12012000-111836/ ChapterThreeFigures11to13.pdf ChapterThree.pdf ChapterFiveFigures11to20.pdf ChapterFourFigures.pdf ChapterFourFigure9.pdf ChapterThreeFigure18.pdf ChapterFiveFigures26to31.pdf ChapterThreeFigures9&10.pdf ListofFigures.pdf ChapterThreeFigures6to8.pdf ChapterFourTables.pdf ChapterSix.pdf ListofTables.pdf ChapterThreeFigures41to52.pdf ChapterThreeFigures26to28.pdf ChapterFive.pdf ChapterThreeFigures1to5.pdf ChapterFourFigure8.pdf DissertationAbstract.pdf ChapterThreeFigures19&20.pdf ChapterFiveFigures21to25.pdf ChapterTwo.pdf TableofContents.pdf ChapterOne.pdf ChapterFiveTables.pdf ChapterThreeFigures16&17.pdf ChapterThreeFigures29&30.pdf Vita.pdf Appendix.pdf ChapterFour.pdf ChapterFourFigure7.pdf ChapterFourFigure10.pdf ChapterThreeFigures14&15.pdf ChapterThreeFigures21to25.pdf ChapterThreeFigures31to40.pdf ChapterFiveFigures1to10.pdf ChapterThreeTables.pdf DissertationTitlePage.pdf In Copyright http://rightsstatements.org/vocab/InC/1.0/ application/pdf Virginia Tech |