Peptide Affinity Adsorbents for Purification of High-Value Biotherapeutics

275 Pages·2016·5.64 MB·English

by  William S. Kish

ABSTRACT
KISH, WILLIAM STANLEY. Peptide Affinity Adsorbents for Purification of High-Value
Biotherapeutics. (Under the direction of Dr. Ruben G. Carbonell.)

Over the last 30 years, biopharmaceuticals have dramatically impacted society by
providing life-changing treatments for devastating diseases. Monoclonal antibodies (mAbs)
and erythropoietin (EPO) are two biotherapeutics used to help millions of patients worldwide
each year. Notably, mAbs are employed in the fight against cancer, multiple sclerosis and
other diseases, while EPO is used to treat anemia in patients suffering from cancer and
chronic kidney disease. Manufacturing high volumes of these biologics at a low cost
requires a comprehensive re-thinking of downstream processes.
This dissertation describes the design, development and assessment of novel affinity
technologies for downstream processing of mAbs and EPO. In past studies, HWRGWV
adsorbents have shown promise as mAb purification tools, but were hindered by a low
dynamic binding capacity (~20 mg IgG/mL resin). In order to increase the binding capacity
of these adsorbents, two synthetic peptide coupling strategies were studied. The best
resulting adsorbent, HWRGWVC-WorkBeads displayed a static binding capacity of 48 mg
IgG/mL. In addition, this resin exhibited a dynamic binding capacity (DBC) with 5 min
residence time of 42 mg IgG/mL. HWRGWVC-WorkBeads was tested for 20 DBC cycles,
which included cleaning in place with 0.1 M NaOH. During these cycles, the resin
maintained 90% of its initial capacity. HWRGWVC-WorkBeads was used to purify IgG1
from CHO cell culture fluid (CCF), resulting in yield and purity of 93% and 96%,
respectively.
In order to identify ligands targeting EPO, a cyclic depsipeptide library of the form
cyclo[(N -Ac)-Dap(A)-X -X -A-E]) on a solid phase was designed to mimic interactions of
α 1 6
EPO with the EPO receptor. Screening of the library resulted in identification of 29 lead
candidates that were synthesized on chromatographic resin and characterized through binding
studies against rHuEPO to select a pool of candidate ligands. Sequences with higher
hydrophobicity consistently showed stronger binding to rHuEPO, with the exception of
FSLLSH, which was noted for its lower hydrophobicity and high rHuEPO binding. To gain
insight on binding mechanisms, top performing ligands were studied by mutagenesis and in
silico docking experiments. First, the crystal structures of the cyclic peptides were docked
against EPO and the resulting complexes were utilized to estimate the binding affinity in
solution. Mutagenesis studies were performed on top candidate FSLLSH with natural and
non-natural amino acid substituents. These studies led to the identification of critical
rHuEPO-binding determinants and the discovery of new peptide ligands. Isotherm adsorption
studies were performed on linear and cyclic versions of FSLLSH to determine the
dissociation constant (K ) of the ligands on solid phase. Notably, the cyclic peptide bound
D
rHuEPO with K = 0.46 μM, displaying an affinity three times higher than its corresponding
D
linear form (K = 1.44 μM). Further, histidine-scanning mutagenesis was performed on three
D
lead sequences, resulting in discovery of variants, whose rHuEPO-binding is more pH-
sensitive, and therefore offer improved rHuEPO recovery.
Cyclic peptide resins were employed to purify rHuEPO from albumin, myoglobin and
hIgG. During studies of FSLLHH, inclusion of CHAPS detergent in wash and elution buffers
improved the yield and purity of recovered rHuEPO by 22% and 23%, respectively. FSLLSH
and VVFFVH afforded the most successful purifications of rHuEPO, with yield and purity of
85% and 95-97%, respectively. The cyclic peptide resins were used to purify spiked rHuEPO
from CHO CCF. FSLLSH provided the most successful separations from CHO, with
rHuEPO yield, purity and HCP log reduction value (LRV) of 91%, 90% and 0.98,
respectively. These results compare well with the yields and purities obtained using wheat
germ agglutinin agarose. Finally, FSLLSH was used to purify rHuEPO from Pichia pastoris
CCF, resulting in recovery of rHuEPO with yield, purity and DNA LRV of 96%, 84%, and
1.3, respectively.
© Copyright 2016 William Stanley Kish
All Rights Reserved
Peptide Affinity Adsorbents for Purification of High-Value Biotherapeutics

by
William Stanley Kish

A dissertation submitted to the Graduate Faculty of
North Carolina State University
in partial fulfillment of the
requirements for the degree of
Doctor of Philosophy

Chemical Engineering

Raleigh, North Carolina
2016

APPROVED BY:
_______________________________ _______________________________
Dr. Ruben G. Carbonell Dr. Stefano Menegatti
Committee Chair

_______________________________ _______________________________
Dr. Patrick V. Gurgel Dr. Michael B. Goshe

_______________________________ _______________________________
Dr. Michael C. Flickinger Dr. Nathaniel G. Hentz
DEDICATION
I dedicate this dissertation to the love of my life, Amy Elizabeth Kish, who continually
encouraged me during my PhD studies with Biblical truths like these:

“Let us not become weary in doing good, for at the proper time we will reap a harvest if we
do not give up.” – Galatians 6:9
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BIOGRAPHY
William Stanley Kish was born on October 12, 1986, in Corning, NY, USA, to
William John Kish, an electrical engineer, and Linda Lukasiak Kish, a saleswoman. His
family moved to Wilmington, NC, in 1990, where he lived for 19 years. While in
Wilmington, he developed a deep love for athletics as he competed in roller hockey, baseball,
basketball, and cross country. Notably, his father and various athletic coaches taught him that
success depends heavily on one’s work ethic. William attended E.A. Laney High School,
where he earned his diploma and developed a strong curiosity for mathematics, physics,
chemistry and biology. In 2009, he earned his BS degree in Chemistry with a concentration
in Biochemistry from the University of North Carolina at Wilmington. William graduated
with departmental honors and completed an honors thesis under Dr. Christopher Halkides. In
addition to academic endeavors, he also competed on UNCW’s cross country and track teams
and won two CAA titles with the latter. In the fall of 2010, he matriculated into the graduate
program in the Department of Chemical and Biomolecular Engineering at North Carolina
State University and joined the Carbonell Bioseparations group. In 2012, he earned his M.
Sc. in Chemical and Biomolecular Engineering with a minor in Biomanufacturing and then
began a Ph.D. under Dr. Ruben Carbonell’s tutelage. William married the love of his life,
Amy Kish, on May 30, 2015. In March 2016, William won the graduate student poster
competition at the ISPE CaSa conference and will compete in the national competition. Upon
defense of his PhD dissertation, he will join Pfizer-Bamboo Therapeutics as a Sr. Scientist,
where he will develop downstream processes for recombinant adeno associated viral vectors
in the fight against neurological and neuromuscular diseases.
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ACKNOWLEDGMENTS
The PhD degree is awarded to one individual, but is only possible through the
support, mentorship, and encouragement from various people. I would like to first
acknowledge my advisor, Dr. Ruben G. Cabonell for his tenacity, guidance, and belief in me
as a scientist. His commitment to innovative research is evidenced by the profound impact he
has made on me, and the 100+ former students that he has advised. Thanks Dr. Carbonell!
Next, I would like to thank Dr. Stefano “El Profo” Menegatti for his patience and
insight. Thank you for teaching me a wide range of techniques from peptide synthesis to
electrophoresis, and from scientific writing and metaphysics to barbeque. I will be keeping
an eye on NCSU’s next rising star. Shine bright, my friend.
I would like to thank Dr. Amith D. Naik for his practical advice on downstream
processing. You have always been kind, caring, and supportive. I would like to thank Dr.
Patrick V. Gurgel for his advice on science, religion, and of course, politics. Thank you for
organizing group activities and for challenging me to consider options outside my comfort
zone. I would like to thank Dr. Michael B. Goshe and Kevin Blackburn for access to their
mass spectrometer laboratory and helpful discussions and training. I would like to thank Dr.
Michael Flickinger for teaching me the principles of upstream processes in BTEC classes. I
would like to thank Dr. Nathaniel Hentz for his instruction and discussions on various
analytical techniques. I would like to thank Dr. Gary G. Gilleskie for teaching my all-time
favorite class: Introduction to Downstream Process Development. Dr. Benjamin G. Bobay is
thanked for his work on in silico docking studies presented in Chapter 4. I would like to
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thank Dr. Chris Halkides, of the UNCW Chemistry department, for teaching me important
fundamental research principles during my time in his lab.
I would like to offer my heartfelt appreciation to Hiroyuki Sachi of the Fuji Silysia
Chemical company, who synthesized many of the cyclic peptides found in this dissertation.
Thank you for being a great friend, hard worker, and for introducing me to authentic
Japanese food. Domo arigato, sinainaru tomodachi! I would like to thank Matthew K. Roach
for his relentless work ethic, excellent attitude and high attention to detail. I have no doubt
that you will be an outstanding contributor to the biopharma company that will be lucky
enough to hire you. I would like to thank Justin G. Thomas for his work ethic, smile, and
light-hearted attitude.
I would like to thank Dr. Michael L. Heller for his friendship throughout our tenure in
the Carbonell group. I have finished strong, my friend (c.f. Mike’s dissertation)! Mike, I hope
that your companies will succeed in the emerging sector of cannabis extraction. I would like
to thank Dr. Tuhidul Islam for helpful discussions and comedic relief. I would like to thank
Ashton Lavoie, Hannah Reese, John Schneible, Ria Domier, Dr. Yong Yow, Dr. Qian Li,
Jacopo Lembo, Giuilia Pierini, Brendan Turner, Sam Sarakbi and Sanketh Sukesh for being
excellent labmates and wonderful friends. To the rest of the Biopep group: thanks! I would
like to thank Carbonell group members Dr. Zhuo Liu, Dr. Haiyan Liu, and Dr. Nafisa Islam
for their patience, encouragement and help in my job search. I would like to thank Fuji
Silysia employees Takaaki Terasaka, Masato Horiguchi, Yasuhiro Hashimoto and Yuki
Ohara for teaching me Japanese and for laughing at my corny jokes.
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I would like to thank Dr. Edwin J. Walker for helping me through our first year of
graduate school and for being a strong brother in Christ. Thank you for your constant
support, prayers, encouragement and wisdom. Intel got a good one! I would like to thank Dr.
Patrick J. Fahey for his sarcasm, encouragement, and comradery. I would like to thank Dr.
Andreas Hoffie and Joe Miller, DPT, for prayers, encouragement and good times at
Providence Baptist. I would like to thank soon to be Dr. Weston Straka and Brett Bornhoft
for prayers and for being excellent accountability partners.
I would like to thank my parents, Bill and Linda Kish, for raising me in a Christian
home and for teaching me to value hard work. Mom, thank you for reading to me when I was
younger and for faithfully loving me and praying for me daily. Dad, thank you for the many
sacrifices you made for us and for displaying the true character of a Godly father. David and
Danella, thank you for your love, support and prayers. I love you both very much! I would
like to thank the Wilson family, especially Wendy, Chris, Melissa, Ben and Hailey Marie for
their undying support, love, and cuteness (Hailey). I would like to thank Dr. Sam Wilson for
encouraging conversations and for many Clif Bars! I would like to thank Nathan Boozell,
Corey Green, and Marcus Faison for their love, friendship, tolerance of my quirkiness, and
for the good times we have shared.
I would like to thank Amy Elizabeth Kish for her undying love, continual support,
and faithful encouragement. Thank you for every single late night in lab, for every
rescheduled date night, and most importantly, for constantly pointing me back to Jesus.
Lastly, I would like to thank the Alpha and Omega for bringing me to, and for bringing me
through this PhD.
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