Polymer Surface-Modification Studies for Ion-Exchange and Affinity Bioseparations

181 Pages·2016·5.06 MB·English

Clemson University
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8-2014
High-Productivity Membrane Adsorbers: Polymer
Surface-Modification Studies for Ion-Exchange and
Affinity Bioseparations
Heather Chenette
Clemson University, [email protected]
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Bioseparations" (2014).All Dissertations. 1319.
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HIGH-PRODUCTIVITY MEMBRANE ADSORBERS: POLYMER SURFACE-
MODIFICATION STUDIES FOR ION-EXCHANGE AND AFFINITY
BIOSEPARATIONS

A Dissertation
Presented to
the Graduate School of
Clemson University

In Partial Fulfillment
of the Requirements for the Degree
Doctor of Philosophy
Chemical Engineering

by
Heather C. S. Chenette
August 2014

Accepted by:
Dr. Scott M. Husson, Committee Chair
Dr. Charles Gooding
Dr. Douglas E. Hirt
Dr. Igor Luzinov
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ABSTRACT

This dissertation centers on the surface-modification of macroporous membranes
to make them selective adsorbers for different proteins, and the analysis of the
performance of these membranes relative to existing technology. Traditional
chromatographic separations for the isolation and purification of proteins implement a
column packed with resin beads or gel media that contain specific binding ligands on
their exposed surface area. The productivity of this process is balanced by the effective
use of the binding sites within the column and the speed at which the separation can take
place, in addition to the need to maintain sufficient protein purity and bioactivity.
Because of the nature of the densely packed columns and, in the case of resin columns,
the limited access to the binding sites internally located within the resin, the operating
speed of this separation process may be constrained by mass-transfer and pressure
limitations. Other constraints include the time-intensive measures taken to properly pack
the columns, and the challenges associated with scaling chromatography columns to
industrial-sized processes. Because of the excellent selectivity, chromatography processes
are the workhorse for biopharmaceuticals drugs, and other plant- and animal- based
protein products. Thus, there are many markets that could benefit by improvements to
this technology.
My strategy focused on modifying porous membranes with surface-initiated atom
transfer radical polymerization (ATRP) to grow polymer chains containing functional
groups that target three different protein-ligand interactions for three different types of
chromatography: cation-exchange, carbohydrate affinity, and Arginine-specific affinity
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chromatography. Although each of these types of separation has different challenges and
different possibilities for impact among their unique applications, they all have the
common need for a stationary phase platform with the potential for fast separations and
specific interactions.
The common approach used in these studies, which is using membrane
technology for chromatographic applications and using ATRP as a surface modification
technique, will be introduced and supported by a brief review in Chapter 1. The specific
approaches to address the unique challenges and motivations of each study system are
given in the introduction sections of the respective dissertation chapters.
Chapter 2 describes my work to develop cation-exchange membranes. I discuss
the polymer growth kinetics and characterization of the membrane surface. I also present
an analysis of productivity, which measures the mass of protein that can bind to the
stationary phase per volume of stationary phase adsorbing material per time. Surprisingly
and despite its importance, this performance measure was not described in previous
literature. Because of the significantly shorter residence time necessary for binding to
occur, the productivity of these cation-exchange membrane adsorbers (300 mg/mL/min)
is nearly two orders of magnitude higher than the productivity of a commercial resin
product (4 mg/mL/min).
My work studying membrane adsorbers for affinity separations was built on the
productivity potential of this approach, as articulated in the conclusion of Chapter 2.
Chapter 3 focuses on the chemical formulation work to incorporate glycoligands into the
backbone of polymer tentacles grown from the surface of the same membrane stationary
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phase. Emphasis is given to characterizing and testing the working formulation for ligand
incorporation, and details about how I arrived at this formulation are given in Appendix
B. The plant protein, or lectin, Concanavalin A (conA) was used as the target protein.
The carbohydrate affinity membrane adsorbers were found to have a static binding
capacity for con A (6.0 mg/mL) that is nearly the same as the typical dextran-based
separation media used in practice. Binding under dynamic conditions was tested using
flow rates of 0.1−1.0 mL/min. No bound lectin was observed for the higher flow rate.
The first Damkohler number was used to assess whether adsorption kinetics or mass
transport contributed the limitation to conA binding. Analyses indicate that this system is
not limited by the accessibility of the binding sites, but by the inherently low rate of
adsorption of conA onto the glycopolymer.
The research described in Chapter 4 focuses on reaction chemistry experiments to
incorporate a phosphonate-based polymer in the membrane platform to develop a new
class of affinity adsorbers that function based on their affinity for Arginine (Arg) amino
acid residues. The hypothesis was that benzyl phosphonate-containing functional
polymers would form strong complexes with Arg-rich proteins as a result of multivalent
binding. Introducing a new class of affinity membranes for purification of Arg-rich and
Arg-tagged proteins may have an impact similar to the introduction of immobilized metal
ion affinity chromatography (IMAC), which would be a significant achievement. Using
Arg-tags would overcome some of the associated drawbacks of using metal ions in
IMAC. Additionally, some cell penetrating peptides are said to be Arg-rich, and this
would be a convenient feature to exploit for their isolation and purification. Lysozyme
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was used as a model Arg-rich protein. The affinity membranes show a static binding
capacity of 3 mg/mL.
This dissertation is a demonstration of the potential and challenges associated
with development of membrane materials for cation-exchange chromatography for
protein capture, affinity purification of conA and other lectins, and affinity separations
targeting Arg-rich regions of proteins and peptides.
v
DEDICATION

I dedicate this dissertation to my loving parents, Mark and Marilyn Schalliol.
v i
ACKNOWLEDGMENTS

I am extremely indebted to my advisor, Dr. Husson, who has assisted me in my
studies not only as an expert in the field of membrane science and surface modification,
but also as a mentor and a teacher. Dr. Husson has taught me many lessons by example,
and by acting with respect, integrity, and honesty. For his encouragement when I wanted
to explore teaching, and for his support when I had the opportunity to work at BMS for
the summer, I am grateful.
My studies would not be as complete, nor would my days in the lab be as
enjoyable without my current and former lab colleagues: Bharat, Daniel, Jinxiang,
Milagro, Juan, Nikki, Christine, Joe, Steven, Julie, and Sid. I am especially appreciative
of Bharat for introducing me first-hand to membranes for bioseparations, and for inviting
me to join him for a special summer research project. I also need to express my thanks to
Milagro for her cherished advice and guidance.
I would like to thank my committee members Dr. Charles Gooding, Dr. Douglas
Hirt, and Dr. Igor Luzinov for sharing their valuable time and for giving me helpful
feedback and suggestions to complete this work.
For the many times I needed assistance and access to special equipment, I thank
Dr. Terri Bruce, Dr. David Bruce, Dr. Igor Luzinov, Dr. Christopher Kitchens, and their
research students. I would like to thank Kim Ivey for her assistance with analytical
equipment, trouble-shooting, and most importantly for sharing her valuable time,
patience, and cheerfulness with me.
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I would not be where I am today if it were not for my parents, who first
recognized my love for science and math, who helped me seize the opportunity to study
it, and who continue to believe in me. For their unconditional love, support, and
encouragement, I am truly grateful. My most steady source of support has been my
husband, Nate. My most recent source of joy has been my daughter. I cannot express how
important they have been on this journey.
Lastly, I would like to thank the National Science Foundation and the National
Institutes of Health for providing me with the funding that enabled me to pursue my
doctoral studies.
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TABLE OF CONTENTS

Page

TITLE PAGE .................................................................................................................... i

ABSTRACT ..................................................................................................................... ii

DEDICATION ................................................................................................................ vi

ACKNOWLEDGMENTS ............................................................................................. vii

LIST OF TABLES .......................................................................................................... xi

LIST OF FIGURES ....................................................................................................... xii

CHAPTER

1. INTRODUCTION ............................................................................................... 1

1.1. Membrane chromatography for protein purification .................................. 1
1.2. Purification challenges and developing strategic
solutions ..................................................................................................... 3
1.3. Grafting polymer tentacles via surface-initiated
polymerization to enhance protein binding within
macroporous membranes ............................................................................ 8
1.4. Outline of the dissertation .......................................................................... 9
1.5. References ................................................................................................ 12

2. DEVELOPMENT OF HIGH-PRODUCTIVITY CATION-
EXCHANGE MEMBRANES FOR PROTEIN CAPTURE
BY GRAFT POLYMERIZATION .................................................................... 18

2.1. Introduction .............................................................................................. 18
2.2. Experimental method ............................................................................... 23
2.3. Results and discussion .............................................................................. 32
2.4. Conclusions .............................................................................................. 54
2.5. References ................................................................................................ 55

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