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Optimizing Detoxi-Gel™ resin based affinity chromatography to prepare recombinant ELP therapies for in vivo evaluation

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Optimizing Detoxi-Gel™ resin based affinity chromatography to prepare recombinant ELP therapies for in vivo evaluation

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Content Optimizing Detoxi-Gel™ Resin Based Affinity
Chromatography To Prepare Recombinant ELP Therapies
For In Vivo Evaluation
By
Quratulain Bhatti
A Thesis Presented to the
FACULTY OF THE USC ALFRED E. MANN SCHOOL OF PHARMACY AND
PHARMACEUTICAL SCIENCE
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(PHARMACEUTICAL SCIENCES)
AUGUST 2024

ii
Acknowledgements
J. Andrew Mackay, Shin-Jae Lee, Sara Attia, Alvin Phan, Dr. Dillon P. Cogan, and Dr.
Martine Culty.
I would like to express my deepest gratitude to Dr. J. Andrew MacKay who made this
work possible. His kind guidance and advice carried me through the journey of writing my
project. I would also like to thank my committee members, Dr. Dillon P Cogan, and Dr.
Martine Culty for their valuable comments and suggestions.
I would also like to give special thanks to Shin-Jae Lee for mentoring me and providing
valuable instructions throughout the whole project.
Additionally, I would like to acknowledge Alvin Phan for cloning anti-IL6-A192 gene,
Changrim Lee and Jugal Dhandhukia for cloning the anti-HLADR10-A192 (Lym1-A192)
gene and Shin-Jae Lee for cloning VSI triblock gene.
Finally, I would like to thank lab members, Sara Attia, and Alvin Phan, for giving support
and advice.

iii

iv
List of Tables
Table 1. Standard dilutions preparation guide indicating
the volume of stock solution needed for serial dilutions
and corresponding wells on 96 well plate…………………………………………………... 22
Table 2. Guide to prepare serial dilutions of
sample to be tested indicating the volumes needed
for dilutions and corresponding wells on the dilution plate ……………………………….. 23
Table.3. ELP therapies from which LPS was effectively
removed by using this method ………………………………………………………………. 54

v
List of Figure
Fig.1 Structure of LPS present in cell wall of E.coli…………………………………………………..8
Fig. 2 Binding of LPS to TLR4 and MD-2 ……………………………………………………………..10
Fig. 3. The comparison between the lipopolysaccharide (LPS)
structure of a typical E. coli cell and the modified Lipid IVA
found in ClearColi cells…………………………………………………………………………12
Fig. 4 The dilution plate showing the rows
containing sample and standard dilutions…………………………………………………….24
Fig. 5 10ml gravity flow column parts
Provided by Fischer Scientific…………………………………………………………………33
Fig. 6 Assembled and resin packed
10 ml gravity flow column sample on top……………………………………………………33
Fig.7 Empty preassembled 2 ml Talon Column……………………………………………………...34
Fig. 8 Resin packed 2 ml column………………………………………………………………………34
Fig.9 Column being regenerated after being packed………………………………………………..36
Fig. 10 Rhodamine labeled sample being eluted
after 1 hr incubation……………………………………………………………………………….36
Fig.11 Coacervation observed after Pooled elutions of rhodamine
labeled sample are put in water bath at 37°C for 15 minutes……………………………….37
Fig. 12 Molecular weight and purity of rhodamine labeled Cry V96
before and after being passed through the column
was confirmed by SDS PAGE stained by coomassie blue…………………………………40
Fig. 13 A well of test plate showing 50000 HEK Blue cells per well……………………………….43

vi
Fig. 14 HEK Blue Assay detection plate after incubation with Quanti
blue showing different shades of purple , blue and pink corresponding
to the endotoxin concentration in the standard, sample, spike and blank………………...43
Fig.15 A192 (Clear Solution) after three spins compared to
Lym1 A192 (Cloudy solution) after 7 warm and cold spins…………………………………..44
Fig.16 HEK Blue Detection plate showing dark blue shade for higher
concentration of standard and undiluted concentration of
HAP A96 before and after being passed through the Detoxi gel column…………………...45
Fig. 17 Multiple passes through the Detoxi gel chromatography do
not promote endotoxin removal from HAP-A96……………………………………………...46
Fig. 18 Incubation in contact with Detoxi gel resulted in significant
reduction in endotoxin concentration of VSI that was expressed in Clear Coli……………47
Fig.19 Incubation in contact with Detoxi gel resulted in significant reduction in endotoxin
concentration of VSI that was expressed in Clear Coli………………………………………48
Fig. 20 1 hr Incubation in contact with Detoxi gel resulted in the most
reduction in endotoxin concentration of VSI that was expressed in Clear Coli…………...49
Fig. 21 Repeating the same method with Cry V96 gave the same result…………………………..50
Fig. 22 Incubating aIL6-A192 Nanoworm for 1 hr in contact with
Detoxi gel did not remove endotoxins from the sample…………………………………….51
Fig. 23 Incubating Lym1- A192 Nanoworm premixed with denaturing salt
(7M urea) overnight in contact with Detoxigel resulted in more
than ninety five percent reduction in endotoxin concentration……………………………52

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Abbreviations
LPS Lipopolysaccharide
ELP Elastin like Polypeptides
Tt The transition temperature
ITC Inverse Transition Cycling
ECM Extracellular Matrix
PRe-RDL Recursive Directional Ligation By Plasmid Reconstruction
PCR Polymerase Chain Reaction
TLR4 Toll-like receptor 4
MD-2 Myeloid Differentiation Factor 2
SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis
SEAP Secreted Embryonic Alkaline Phosphatase
QB QUANTI-Blue™
PBS Phosphate Buffer Saline
EU/kg Endotoxin Units per kilogram
IPTG Isopropyl β-D-1 thiogalactopyranoside
OD Optical density

viii
Abstract
Elastin-like polypeptides (ELPs), which are repetitive, high molecular weight peptides
derived from human tropoelastin, hold promise for various biomedical applications,
including drug delivery and tissue engineering. However, their production in E.coli often
leads to contamination with lipopolysaccharide (LPS), a potent endotoxin that can
confound in vitro and in vivo preclinical studies of biologic drug candidates. To address
this issue, this study investigated various chromatographic resins and columns to more
effectively remove LPS from ELPs while preserving protein yield. Among the methods
tested, polymyxin B resin packed in a gravity flow column demonstrated superior LPS
removal efficiency, achieving greater than 99 percent reduction in endotoxin levels after
a 1-hour incubation, as confirmed by a TLR 4 activation based colorimetric assay (HEK
Blue). While this method proved successful for purifying ELPs, it required the presence
of denaturing salt to remove LPS from ELP fusion proteins having single chain
antibodies attached to them. This highlights the need for further research to address this
challenge in the context of different ELP fusion peptides. The development of efficient
endotoxin removal strategies is crucial for advancing the translational potential of ELPbased biomaterials.

1
Chapter 1: Introduction
ELPs are biopolymers composed of repeating pentapeptide units with the sequence
Valine-Proline-Glycine-X-Glycine. The guest residue, represented by 'Xaa' can be any
amino acid and plays a crucial role in determining the ELP's properties. These polymers
exhibit a self-assembly pattern similar to tropoelastin, where hydrophobic domains
interact with each other. This self-assembly leads to coacervation, a process where the
ELPs separate from the solution into a dense, polymer-rich phase, at a specific
temperature known as the transition temperature. Below this temperature, the
thermosensitive ELPs remain soluble. Importantly, this phase separation is reversible,
and ELPs can be redissolved by lowering the temperature.[1]
The transition temperature(Tt) of an ELP is influenced by both the guest residue (Xaa)
and the number of pentameric repeats (n). A hydrophobic amino acid like Valine at the
guest residue position will lower the transition temperature, while a more hydrophilic
amino acid like Serine will raise it. Similarly, a higher number of repeats will lower the
transition temperature, while a lower number will increase it. This unique
thermoresponsive behavior makes ELPs attractive for various applications, including
drug delivery, tissue engineering, and biosensing. [1]
They are expressed in E.coli bacterial cell lines and their cell wall contains
Lipopolysaccharide (LPS). During expression and purification this LPS gets suspended
in the ELP solution and gets attached to the ELPs.[12] This presence of LPS poses risk
of septic shock once these ELPs are tested in vivo.[6][7] One way to avoid this from
happening is by using the modified E.coli system that has truncated Lipid A to express

2
the ELPs.[9] Unfortunately, in practice using the Clear Coli system does not completely
get rid of endotoxin and the risk of septic shock remains.
There are many methods which are used to remove the endotoxin from proteins
expressed in E.coli including two phase partitioning , ultrafiltration,chromatography,
electrophoresis , detergents etc. All these methods have their advantages and
disadvantages. One method that shows promise of effectively removing endotoxin from
ELPs is using polymyxin B based affinity chromatography and it has been proved to do
so for many ELP therapies.[17]

3
1.1 Elastin-Like Polypeptides
Elastin is a polymeric extracellular matrix (ECM) protein, found in tissues as varied as
the skin, lungs, blood vessels, and cartilage, which underpins the protractible nature of
vertebrate tissues.[2] Though only one gene encodes the ~60 kDa soluble precursor,
tropoelastin, it exists as polymorphs containing repetitive hydrophobic motifs, largely
valine and alanine, denoting elastomeric domains. Other amino acids present at
significant levels include glycine and proline, which disrupts alpha helix and beta-sheet
formation. These elastomeric domains occur between distinct peptides that are involved
in crosslinking other tropoelastin monomers through the action of lysyl oxidase on lysine
residues. As biosynthesis and extrusion into the ECM proceed, the final products
generated are insoluble elastin fibrils. Pioneering studies first revealed the temperaturesensitive nature of hydrolyzed α-elastin. The protein remained soluble below 25°C, but
when heated to 37°C, elastin phase-separated into a secondary amorphous phase
known as a coacervate. Interestingly, the investigators further noted that this process
was completely reversible. Those findings eventually facilitated the first chemical
synthesis of an elastin-like polypeptide prior to the emergence of molecular biology as a
discipline.[3]
Elastin-like polypeptides (ELPs) are an artificial, biomimetic class of protein polymers
inspired by the recurring hydrophobic motifs of tropoelastin. Due to their broad range of
applications, including in drug delivery and tissue engineering, ELPs have attracted
attention. The canonical ELP unit consists of a hydrophobic, five amino acid motif (ValPro-Gly-Xaa-Gly)n where the guest residue, Xaa, specifies any amino acid and n
determines the number of pentapeptide repeats. Proline is usually avoided at the fourth

4
residue since its presence can interfere with coacervation. It should be noted, however,
that inclusion of amino acid side chains capable of enhancing functionality does not
necessarily interfere with ELP phase behavior. The addition of tyrosine to facilitate
spectrophotometric analysis or lysine for crosslinking are two examples. Furthermore,
ELPs with other repeat motifs beyond the one described have similar properties.
Examples may range from other pentapeptides (e.g. IPGVG) to heptapeptide (e.g.
LGAGGAG) and nonapeptide (e.g. LGAGGAGVL) repeat sequences.[3]
A primary aspect of ELP biomaterials involves their ability to reversibly form
coacervates following temperature changes. This feature is known as the critical
transition temperature (Tt) and can be explained thermodynamically in terms of the
Gibbs free energy (ΔG = ΔH – TΔS). If ΔG during a temperature transition (ΔGt) is zero,
then ΔHt = TtΔSt which can be rearranged to Tt = ΔHt/ΔSt. The increase in order of
ELPs at Tt might appear to contradict the second law of thermodynamics— namely that
the order of a system has an inverse relationship with temperature— but the complete
system consisting of protein polymer and water must be considered. In the absence of a
stimulus, ELPs remain soluble in aqueous solutions as random coils and have been
described as intrinsically disordered proteins. The hydrophobic side chains of VPGXG
remain surrounded by ordered water molecules existing in a low entropy state. Once the
temperature rises above Tt, water molecules clustered around the hydrophobic amino
acids are expelled into the bulk water phase. This favors a gain in solvent entropy and
allows non-polar side chains to form intra- and intermolecular interactions with
neighboring ELP molecules. Hydrophobic interactions, meanwhile, facilitate folding and
dynamic assembly into more ordered secondary structures as type II β-turn spirals.[1]

5
1.1 (a) ELPs Genetic Engineering, Expression And Purification
ELPs can be linked to various molecules like drugs and antibodies. One method for
creating these linked ELPs is called PRe-RDL (recursive directional ligation by plasmid
reconstruction). This technique uses special enzymes to cut and paste DNA segments,
allowing scientists to precisely join an ELP gene with genes for other molecules.
Here's how PRe-RDL works:
1. Cutting the DNA: Type II restriction enzymes cut the DNA at specific locations
near the ELP gene, creating "sticky ends."
2. Joining the Pieces: The cut DNA segments, each containing a part of the ELP
gene, are then joined together.
3. Reconstructing the Plasmid: This process reconstructs the complete plasmid
(a circular DNA molecule) with the combined genes. [4]
PRe-RDL offers several advantages:
● Controlled ELP Size: It allows researchers to create ELPs of specific lengths by
repeatedly joining gene segments.
● Block Copolymer Formation: Different genes can be combined to create ELP
block copolymers with diverse functions.
● No Extra Amino Acids: The final protein product is precise, without any
unwanted amino acids. [4]
Beyond PRe-RDL, other techniques like PCR, seamless cloning, and OERCA are also
used to synthesize ELPs but PRe- RDL is most widely used in our lab.

6
Once a plasmid of interest is engineered, it is inserted into the Top 10 bacterial cell line.
Once this transformation is successful, the plasmid is harvested and inserted into E.coli
bacteria for ELP expression. The expressed ELP or ELP fusion proteins are then
purified by using a repeated hot and cold centrifugation process called inverse transition
cycling (ITC).[4]

7
1.2 Endotoxin (LPS) Structure and Function
Lipopolysaccharide molecule has a split nature—part of it is hydrophilic, and part of it
avoids water and is hydrophobic. This makes it an amphipathic molecule. It is like a tiny
pushpin:
● The Pin: The hydrophobic fatty acid chains act like the sharp pin, anchoring LPS
into the bacterial outer membrane.
● The Head: The hydrophilic portion, made of charged sugar molecules, sticks out
into the surrounding environment. Fig. 1. [5]
These two distinct regions are arranged as follows.
1. Lipid A: This is the "pin" part, made of a glucosamine sugar with fatty acid
chains attached. It's responsible for LPS's toxic effects. Lipid A has a consistent
structure across different Gram-negative bacteria, consisting of a glucosamine
sugar backbone with attached fatty acid chains.
2. Polysaccharide: This is the "head" part, made of a chain of sugars.This
hydrophilic region extends outward from the bacterial surface and determines the
bacteria's immune identity (immunogenicity). It has two sections:
○ Core Oligosaccharide: Attached directly to Lipid A.
○ O-polysaccharide (O antigen) : Attached to the core oligosaccharide,
this part varies between different bacteria and is involved in immune
response. It consists of one to eight glycosyl units. [5]

8
How LPS contributes to bacterial virulence:
● Lipid A: Acts as a potent immune stimulator, triggering a strong inflammatory
response in the host that can be harmful in large amounts.
● O-antigen: Helps bacteria evade the host's immune system by:
○ Aiding in adherence to surfaces.
○ Providing resistance to engulfment by immune cells (phagocytes).
○ Exhibiting variability to evade antibody recognition.
In essence, LPS is a key molecule that allows Gram-negative bacteria to colonize hosts,
evade immune defenses, and cause disease. [6]
Fig.1 Structure of LPS present in the cell wall of E.coli. Image has been
reproduced[6].

9
1.3 Endotoxin’s Ability To Activate TLR4 Receptors To Cause Septic Shock
The lipopolysaccharide of Gram-negative bacteria is a well-known activator of the innate
immune response. Toll-like receptor 4 (TLR4) and myeloid differentiation factor 2 (MD2) form a heterodimer that recognizes a common structural pattern in diverse
lipopolysaccharide (LPS) molecules. To elucidate the ligand specificity and receptor
activation mechanism of the TLR4-MD-2-LPS complex, its crystal structure was
determined. LPS binding induced the formation of an M-shaped receptor multimer
composed of two symmetrically arranged TLR4-MD-2-LPS complexes. LPS interacts
with a large hydrophobic pocket in MD-2 and directly links the two components of the
multimer.(Fig.2) Five of the six lipid chains of LPS are deeply buried within the pocket,
while the remaining chain is exposed on the surface of MD-2, forming a hydrophobic
interaction with conserved phenylalanine residues of TLR4. The F126 loop of MD-2
undergoes a localized structural change and supports the core hydrophobic interface by
making hydrophilic interactions with TLR4. Comparison with structures of tetra-acylated
antagonists bound to MD-2 indicates that two additional lipid chains in LPS displace the
phosphorylated glucosamine backbone by approximately 5 Å towards the solvent area.
This structural shift allows the phosphate groups of LPS to contribute to receptor
multimerization by forming ionic interactions with a cluster of positively charged residues
in TLR4 and MD-2. The TLR4-MD-2-LPS structure illustrates the remarkable versatility
of the ligand recognition mechanisms employed by the TLR family, which is essential for
defense against diverse microbial infections.[7] [8]

10
Fig. 2 Binding of LPS to TLR4 and MD-2. a, Chemical structure of the Ra
chemotype of E.coli LPS. The lipid chains are labeled. The carbons of the
glucosamines and lipid chains of Lipid A are numbered. Hydrogen bonds are shown by
broken blue lines. b, The molecular surface of the MD2 pocket is drawn in mesh. c,
Hydrogen bonds between Lipid A and TLR4-MD2. d,Ionic and hydrogen bond
interactions of the two phosphate groups of lipid A. Interaction distances in angstroms
are written.Inner core carbohydrates and carbon chains of the lipids are omitted for
clarity. [9]

11
1.4 Clear Coli® system designed to avoid activation of a Pro-Inflammatory
pathway
Genetically modified lipopolysaccharide (LPS) derived from a novel strain of E.coli
facilitates the production of recombinant proteins and plasmids that are functionally free
from endotoxic effects. ClearColi® BL21(DE3) cells represent the first commercially
available competent cells with an altered LPS structure (Lipid IVA - see Fig.3 ), which
does not induce an endotoxic response in mammalian cells. ClearColi® cells lack outer
membrane components that activate hTLR4/MD-2 signaling, resulting in a significantly
lower activation compared to wild-type E. coli cells. Heterologous proteins derived from
ClearColi are virtually devoid of endotoxic activity, making them suitable for various
applications with minimal purification steps from ClearColi cells.

12
Fig 3. The comparison between the lipopolysaccharide (LPS) structure of a
typical E. coli cell and the modified Lipid IVA found in ClearColi cells. In ClearColi,
the LPS has undergone genetic modifications: the oligosaccharide chain has been
eliminated, and two of the six acyl chains have been removed. These changes are
intended to disrupt the endotoxin signaling pathway.[10]
The genotype of ClearColi cells has been modified through the deletion of seven
specific genes, ensuring irreversibility to wild-type status and the production of typical
LPS. These genetic alterations include the removal of the oligosaccharide chain from
LPS, facilitating the easier extraction of resulting lipid IVA during downstream
processing. Notably, two of the six acyl chains in the LPS structure have also been
deleted. These acyl chains typically serve as triggers recognized by Toll-like receptor 4
(TLR4) in conjunction with myeloid differentiation factor 2 (MD-2), leading to NF-ƙB

13
activation and the production of inflammatory cytokines. Lipid IVA, containing only four
acyl chains, is supposed to lack recognition by TLR4 and thus not induce an endotoxic
response. However, in our lab despite using clear coli cell line to express ELP therapies
the purified product wasn’t endotoxin free. [11] [12]
How LPS attaches itself to ELPs:
Richard Pfeiffer, in 1892, first defined endotoxin as a heat-stable, toxic substance that
was released upon disruption of microbial envelopes . [13] During sonication LPS gets
separated from bacterial cells and stays suspended in the solution. This LPS stays in
the ELP solution despite repeated hot centrifugations because it is thermostable and
contaminate ELPs and their fusion proteins. [17]

14
1.5 FDA’s limit
The effects of endotoxin are related to the amount of endotoxin in the product dose
administered to a patient. Because the dose varies from product to product, the
endotoxin limit is expressed as K/M. K is 5.0 EU/kilogram (kg.), which represents the
approximate threshold pyrogenic dose for humans and rabbits. That is the level at which
a product is considered pyrogenic or non-pyrogenic. M represents the rabbit pyrogen
test dose or the maximum human dose per kilogram that would be administered in a
single one hour period, whichever is larger.[14]
LPS causes a wide spectrum of nonspecific pathophysiological reactions, such as fever,
changes in white blood cell counts, disseminated intravascular coagulation,
hypotension, shock and death. The injection of minimum doses of endotoxin results in
death in most mammals. The sequence of events follows a regular pattern:
(1) Latent period
(2) Physiological distress (diarrhea, prostration, shock)
(3) Death
How soon death occurs varies on the dose of the endotoxin, the route of administration,
and the species of animal. Animals vary in their susceptibility to endotoxin. [5]

15
1.6 Endotoxin removal methods
In biotechnological processes, Escherichia coli cells pose a risk due to their high
content of lipopolysaccharide (LPS), with a single cell containing about 2 million LPS
molecules. These molecules consist of a hydrophobic lipid A component, a complex
arrangement of sugar residues, and negatively charged phosphate groups. Importantly,
LPS, known as endotoxins, exhibit heat stability, rendering them resistant to
conventional sterilization methods. Consequently, separate tests are required to assess
both viable cell counts (bioburden) and endotoxin levels. [15]
Gram-negative bacteria, such as E. coli, are extensively utilized in biotechnology for
producing recombinant DNA products like peptides and proteins. However, these
products are invariably contaminated with endotoxins, necessitating specific procedures
for their removal from the final product [16]. There are two primary challenges
associated with endotoxin removal: first, the process must not alter the product itself
during clearance; second, despite the typically low concentrations of endotoxin relative
to the product, removing bound endotoxin can be particularly challenging. Binding
interactions between endotoxins and proteins can strengthen during concentration steps
in the production process.
Many techniques for endotoxin removal exploit the structural characteristics of
endotoxin complexes. Endotoxin molecules exhibit hydrophobic, hydrophilic, and
charged regions, which facilitate diverse interactions with other molecules. Additionally,
the mass of endotoxin complexes can be utilized as a secondary method for
removal.[17]

16
Two-Phase partitioning
The adoption of aqueous two-phase systems is gaining popularity in specific
applications as an alternative to organic-water solvent extraction methods. This
preference is partly due to the ability of aqueous two-phase systems to create gentler
conditions that minimize the risk of damaging or altering unstable or labile biomolecules.
In the context of endotoxin removal, the hydrophobic nature of endotoxins makes twophase partitioning a particularly effective method.
In this approach, optimization of conditions is crucial to achieve effective separation: the
target protein partitions into one phase while the endotoxin partitions into another,
thereby removing it from the product.[18] Manipulating two-phase systems involves
adjusting variables such as polymer mass, pH, ionic strength, and concentration of
phase components. Alternatively, the introduction of affinity ligands can enhance the
selectivity and efficiency of endotoxin removal within these systems.
Ultrafiltration
Endotoxin molecules typically aggregate into micelles or vesicles when suspended in
aqueous solutions, making them challenging to remove by conventional means.
Ultrafiltration offers a solution by leveraging molecular weight exclusion, utilizing ultrafine filters that selectively block molecules with a weight of 10,000 Daltons or higher.
[19]
In practice, ultrafiltration is often complemented with a 0.1 µm filter to control bioburden,
ensuring a high degree of purity in the final product. However, while ultrafiltration is

17
highly efficient for decontaminating water, its effectiveness diminishes significantly when
applied to protein solutions containing endotoxins. This limitation arises because
endotoxins, despite their aggregation state, can evade removal due to their interaction
with proteins or the lack of adequate size exclusion. Therefore, while useful in certain
contexts, ultrafiltration alone may not suffice for achieving stringent endotoxin levels in
protein-based solutions.ch as ultrafiltration, have little effect on endotoxin levels in
protein solutions.[20]
Chromatography
In many applications, negatively charged chromatographic techniques are favored for
effectively clearing endotoxins.[21] Affinity chromatography methods, employing ligands
like DEAE sepharose, poly-L-lysine, and polymyxin-B, selectively bind endotoxins
based on their affinity for specific ligands. This approach has been successfully
implemented in laboratory settings, where various column types such as spin and
gravity flow have been tested using poly-L-lysine and polymyxin-B resins.
In contrast, ion-exchange chromatography operates by attracting negatively charged
endotoxins to positively charged resins, facilitating their subsequent elution. Both affinity
and ion-exchange chromatography methods are influenced by factors such as pH,
temperature, flow rate, and electrolyte concentrations in the solution.[22] Recent
advancements include hydrogel-based methods using large beads, including
innovations like inside-out ligand attachment techniques.[24]
Size-exclusion chromatography offers another approach, depending on the size of the
proteins involved. However, its efficacy can be hindered by the tendency of endotoxins

18
to form micellar structures, which behave similarly to larger biological molecules. This
complexity poses challenges for both affinity and ion-exchange chromatography in
separating endotoxins from proteins, particularly when dealing with proteins that have
similar charge properties.[26]
Moreover, anion-exchange chromatography may inadvertently co-purify endotoxins due
to their negative charge interactions with the resin, potentially resulting in product
contamination. Furthermore, negatively charged proteins present additional
complications, including the risk of product loss during purification processes.
Specialized adsorber matrices designed for endotoxin removal through dissociation
from proteins offer potential solutions, though they must contend with the structural
characteristics and aggregation tendencies of endotoxins. These factors underscore the
intricate balance required in chromatographic methods to achieve effective endotoxin
clearance while maintaining product integrity in biotechnological applications.
Electrophoresis
While not commonly employed for removing endotoxins, certain researchers have
documented successful outcomes using slab-polyacrylamide or sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). These methods have shown promise
for separating lipopolysaccharides (LPS) from proteins.
Slab-polyacrylamide and SDS-PAGE gel electrophoresis are typically used to separate
proteins based on their molecular weight and charge properties. In the context of
endotoxin removal, these techniques can be adapted to effectively separate LPS

19
molecules, which are amphipathic and have distinctive hydrophobic lipid A regions, from
proteins. This separation is crucial in biotechnological processes where purified proteins
free of endotoxin contamination are required.
Despite their potential, these electrophoretic methods are not widely adopted for
endotoxin removal due to their specificity and the challenges involved in maintaining
protein stability and activity throughout the process. However, they remain valuable
tools in research settings for their ability to isolate and analyze LPS and protein
fractions separately.
To optimize their application for endotoxin removal, researchers continue to explore
modifications and refinements to these electrophoretic techniques, aiming to enhance
their efficacy while minimizing potential drawbacks related to protein denaturation or
loss during purification steps. [25]
Detergents
To dissociate endotoxins from protein solutions, non-ionic surfactants are sometimes
employed in a washing procedure. This method facilitates the separation of endotoxins
by disrupting their interactions with proteins.[26] However, a significant drawback is the
necessity to remove the detergent in subsequent purification steps, which can
complicate the process and potentially reduce the overall product yield.
The use of non-ionic surfactants represents an additional step in the purification
workflow aimed at enhancing the purity of proteins by eliminating endotoxin
contaminants. Despite its effectiveness, the presence of residual detergent after

20
dissociation necessitates thorough washing and purification procedures to ensure the
final product meets stringent quality standards.
In addition to surfactant-based methods, there are less commonly utilized techniques for
endotoxin removal. Ultracentrifugation, involving devices capable of achieving
accelerations as high as 1,000,000 x g, and enzymatic digestion of proteins are
examples. [27] These methods, while powerful in specific contexts, are generally not
suitable for many biotechnological processes due to their complexity, potential for
protein degradation, and limitations in scalability.
Researchers continue to explore and refine purification strategies to address the
challenges associated with endotoxin removal, aiming to develop robust and efficient
methods that preserve protein integrity and maximize yield in biopharmaceutical and
research applications.

21
1.7 HEK Blue Assay
To detect the removal of endotoxin after passing it through polymyxin B resin,
colorimetric TLR 4 activation cell-based assay for the detection of biologically active
endotoxin was used.
HEK blue 4 Cells are modified HEK 239 cells which are human embryonic Kidney cells.
HEK-Blue™ TLR cells stably co-express a human or murine TLR gene and an NF-κBinducible SEAP (secreted embryonic alkaline phosphatase) reporter gene. SEAP levels
produced upon TLR stimulation can be readily determined by performing the assay in
HEK-Blue™ Detection, a cell culture medium that allows for real-time detection of
SEAP. Alternatively, SEAP activity may be monitored using QUANTI-Blue™(QB), a
SEAP detection reagent. [28]
LPS in sample activates TLR4 -> NF kB formed -> SEAP activated -> cleaves QUANTIBlue™ and shows color.
Sensitivity : 0.01 EU/ml.
Sample Preparation
Material needed
1. 1 EU/ml HEK blue Endotoxin standard Solution* :
*Endotoxin Standard

22
1. Add 1 ml of Endotoxin free water to the contents of the tube to obtain initial stock
of 50 EU/ml.

Table 1. Standard dilutions preparation guide indicating the volume of
stock solution needed for serial dilutions and corresponding wells on 96
well plate. Table is reproduced from HEK-Blue LPS Detection kit 2 package
insert. (HEK-Blue™ LPS Detection Kit (InvivoGen®) )
Certificate of analysis / Vial will have X EU written on it. E.g 55 EU. When
reconstituted with 1 ml of endotoxin free water, we get 55 EU/ml.
2. Mix by vortexing as contents stick to walls.
3. Prepare 1 EU/ml by diluting 1/ X .
4. Store at 2-8°C. Stays stable for 1 week.
Prepare two fold dilutions.

23
Dilution Plate (Fig. 4)
Three fold serial dilutions of the sample by using endotoxin free water are prepared.
Table 2. Guide to prepare serial dilutions of sample to be tested indicating the
volumes needed for dilutions and corresponding wells on the dilution plate. Table
is reproduced from HEK-Blue LPS Detection kit 2 package insert. (HEK-Blue™ LPS
Detection Kit (InvivoGen®) )
Inhibition/ Activation Assay
1. Prepare a spike solution of 0.1 EU/ml and spike the sample dilutions with 20 ul of
spike. (to take into account involvement of sample with inhibition /activation of HEK
Blue 4 cells)

24
2. Only Spiked sample dilutions showing 50-200% absorbance in comparison to
unspiked sample dilutions are considered non-interfering and are used to
determine endotoxin concentration.
Fig. 4 The dilution plate showing the rows containing sample and standard
dilutions. Figure is reproduced from HEK-Blue LPS Detection kit 2 package insert.
(HEK-Blue™ LPS Detection Kit (InvivoGen®) )
HEK – Blue Cells Suspension Preparation
1. Use cells that are
● Passaged less than 30 times and within 48 hrs prior to testing
● Flat, Adherent with 60-80% confluency
2. Discard damaged or floating cells.
3. Aspirate media

25
4. Wash with 10 ml PBS ( pre-warmed at 37°C) , aspirate.
5. Add 7 ml PBS.
● Incubate for 2 minutes at 37 ̊C in CO2.
● Pipette up and down with 1 ml pipette.
6. Count, dilute up to 144k cells / ml with test medium. Pipette up and down.
Day 1
1. Add 20 ul of endotoxin free water to each well in dilution plate
2. Triplicate for Endotoxin standard dilutions
● Add 20 ul of Endotoxin standard dilutions per well from dilution plate to
96 well test plate.
3. Duplicate for samples
● 40 ul of each dilution transferred to the test plate.
4. Spike
● 20 ul of sample and 20 ul of spike solution per well in test plate.
5. Blank
● 20 ul of Endotoxin free water should be added to three or more wells
as blank.
If using Quanti Blue,
QUANTI-Blue™ is a colorimetric enzyme assay designed to measure alkaline
phosphatase (AP) activity in biological samples. When the solution encounters
secreted embryonic alkaline phosphatase (SEAP), it changes color from pink to

26
purple-blue. This change indicates the presence and activity level of AP in the
sample.(Fig.14)
Add 160 ul of cell suspension (50,000 Cells/well) to all wells.
Incubate at 37°C in a CO2 incubator for 18-24 hrs.
Day 2
Quanti Blue Preparation
1. 1ml QB reagent + 1 ml QB buffer + 98 ml of sterile water in sterile glass bottle
or flask =100 ml QUANTI-blue.
2. Vortex for mixing.
3. Incubate at room temp. 10 min. before use.
● Use immediately or store at 2-8°C for 2 weeks or -20°C for 2 months.
Avoid repeated freeze thaw.
● If using aliquot, warm at 37°C for 30 mins. Before use.
Detection Plate
1. Add 180 ul QB in each well.
2. Add 20 ul of supernatant from the test plate to corresponding wells.
3. Incubate 1-6 hrs at 37° C.
4. Read absorbance at 620-655 nm.

27
If using HEK – blue detection
Hek blue detection preparation
1. In Sterile vial/bottle: Contents of 1 pouch + 50 ml endotoxin free water.
2. Swirl gently to mix.
3. Warm contents at 37°C for 30 mins. - 1 hr.
4. Filter through 0.2 um membrane in sterile bottle.
Protect From Light
KEEP at 37°C before use
STORAGE: 2-8 °C for 2 weeks
Detection Plate
1. Mix cell suspension and HEK blue to get (140000) cells/ ml conc. Or 25k
cells/ well.
2. Add 180 ul of this mixture to each well ( Total needed 10,800 ul or 12 ml).
3. Add 20 ul of test plate contents in corresponding cells, pipette up and down.
4. Incubate at 37°C , 5% CO2 overnight.
5. Read optical density at 620-655 nm.
Reading And Interpretation
Validation : After 1-6 hrs incubation with QB , assay is validated if
1. Endotoxin Standard columns show blue purple pink gradient (top to bottom)

28
2. Blanks remain pink or light blue.
Calculation of Endotoxin concentration
1. Subtract mean blank absorbance from mean standard dilutions and sample
dilutions to get mean absorbance.
2. Plot this mean absorbance on Y axis and Conc. In EU/ml on the X axis.
3. Fit a linear trendline and get an equation for Y and R2 on the graph.
4. Assay is reliable if R2
is > or = 0.98.
5. Choose sample dilution y values in linear range of standard curve and
calculate x ( Conc.) using the equation on the graph.
6. Determine endotoxin value of these y ( sample conc.) by multiplying obtained
x values with respective dilution factor.

29
Chapter 2 : Materials and Methods
2.1 ELPs sequencing , expression and purification
The Spike-A192 vector design involves the modification and use of the pET-25(+) vector
for ELP (Elastin-Like Polypeptide) fusion cloning. Here’s a detailed breakdown of the
procedure:
1. Vector and Enzyme Preparation:
○ The pET-25(+) vector (Novagen #69753) is modified for ELP fusion
cloning.
○ The ELP A192 encoding plasmid is engineered.
2. Spike RBD Gene Sequence:
○ The spike RBD (Receptor Binding Domain) gene sequence is designed
with recognition sites for the restriction enzymes NdeI at the 5′ end and
BamHI at the 3′ end.
○ A BseRI recognition site is included for recursive directional ligation
plasmid reconstruction (PRe-RDL).
3. Plasmid Purification and Digestion:
○ Plasmids containing the spike RBD and A192 genes are purified from One
Shot TOP10 Chemically Competent cells (Thermo Fisher Scientific
#C404003) using a QIAprep Spin Miniprep Kit.
○ Double digestion of these plasmids is performed using BssHII and BseRI
enzymes (New England Biolabs).

30
4. Fragment Purification and Ligation:
○ Digested fragments containing the spike RBD and ELP are purified with a
QIAquick Gel Extraction Kit.
○ These purified fragments are then ligated together.
5. Transformation and Clone Selection:
○ The ligated plasmids are transformed into TOP10 cells and extracted
using a Miniprep Kit.
○ Successful clones are confirmed by diagnostic DNA digestion using NdeI
and BamHI restriction enzymes.
6. Validation:
○ The digestion results are visualized using electrophoresis on a 1%
agarose gel.
○ Sanger sequencing is performed with primers specific for the T7 promoter
(TAATACGACTCACTATAGG) and T7 terminator
(GCTAGTTATTGCTCAGCGG) to confirm the insertion into the pET-25(+)
vector.
This meticulous process ensures the correct insertion of the spike RBD gene into the
pET-25(+) vector, facilitating further experiments and applications involving ELP fusion
proteins.[29]

31
Expression and Purification of ELPs
ClearColi BL21(DE3) Electrocompetent cells (#60810, Lucigen, Middleton, WI) were
transformed with the ELP or spike-ELP plasmid via electroporation. This cell line
produces an incomplete lipopolysaccharide(lipidIVA) that lacks endotoxic activity in
human immune cells. Lipopolysaccharide is an antagonist of the humanToll-like receptor
(TLR4)/ myeloid differentiation factor 2 (MD-2)-mediated signaling. The transformed
colonies were cultured in 50mL of autoclaved TB containing 100 μg/mL of carbenicillin at
37°C for 16−18h . 25mL of ClearColi BL21 culture was transferred to an autoclaved 1L of
TB with 100 μg/mL carbenicillin to culture the bacteria at 37°C until the optical density at
600 nm (OD600) reached between 0.5 and 0.8. Each 1 L flask was then treated with 500
μLof 1M isopropyl β-D-1 thiogalactopyranoside (IPTG)to bring the final concentration to
500 μM IPTG. The IPTG-induced cultures were incubated overnight at room temperature.
The following day ,the bacteria were sonicated and the ELPs were purified as described
previously. This purification process uses the ELPs’ phase transition,which is sequentially
induced through at least three rounds of hot and cold centrifugation steps as necessary
to achieve the desired purity. [29]

32
2.2 Columns And Resin Used
Thermo Scientific™ Detoxi-Gel™ Endotoxin Removing Gel was degassed for 15-20
minutes in following steps.
● Loosen the cap of the resin bottle.
● Place it in the vacuum chamber.
● Attach it to the vacuum pump.
● Degass it for 15-20 minutes
● After degassing, turn off the pump , turn the valve slowly to allow the
vacuum to be released.
Thermo Scientific™ Pierce™ Disposable gravity flow propylene column, 10 mL Catalog
number: 29924 (Fig.5) was assembled by pushing the porous polyethylene disc to the
bottom of the column by using sterile spatula. Polypropylene caps were placed on both
top and bottom. Discs and caps are provided with the columns by Thermo Fisher.(Fig.6)
Total resin bed volume capacity of the column was calculated by measuring the height
of the column and internal diameter with a regular ruler.
Height of column = 7 cm
Internal diameter = 1.5 cm

33
Internal radius = 0.75 cm
Volume of resin it can hold = π r2 h
= 3.14 X 0.752 x 7
=12.3 cm3
0r 12.3 ml

This volume is consistent with the package insert provided by thermo fisher.
Fig. 5. 10ml gravity flow column parts Fig. 6 Assembled and resin packed
provided by Fischer Scientific. 10 ml gravity flow column
sample on top.
For Talon®, Catalog # 635606 2-ml disposable gravity flow preassembled columns,
total volume was measured by measuring the diameter of the disk and the height of the
smooth parallel lines structure. (Fig.7,8)

34
Fig.7. Empty preassembled 2 ml
Talon Column
Fig. 8 Resin packed 2 ml column
Endotoxin removal procedure
1. Slowly pipette up and down and swirl around the resin to form a homogeneous
solution without introducing bubbles.
2. Take 1 ml of resin at a time and slowly pipette it out in the column closer to the
disc.
3. Void volume is approx. 52%. So pack the resin volume more than twice the
volume of sample to be passed through the column e.g for 1.5 ml sample,
approx. 3 ml resin bed volume is needed so the height of the resin bed has to be
3/(3.14 X 0.752
) =1.699 0r 2 cm.
4. Let it settle for 30 minutes-1 hr.

35
5. Measure the height of the resin bed by using a ruler to ensure enough resin bed
volume.
6. Open the bottom cap and let the solvent of slurry (25% ethyl alcohol) flow
through.
7. Regenerate the resin by adding 3-5 Resin bed volume* 1% (w/v) sodium
deoxycholate followed by 4-6 Resin bed volume of endotoxin free water and 4-6
resin bed volume of PBS.(Fig.9).
*For 3ml resin bed volume , add 9-15 ml of 1% Na Deoxycholate.
● For lym1 A192 nanoworm, the sample was mixed 1:1 with 7M Urea before
passing it through the column.
8. Add your sample slowly and let it reach the middle of the resin bed, cap the
bottom and top. (Cover with aluminum foil if the sample is rhodamine labeled).
9. Let it sit in contact with resin for 1 hr.
10.Add the same volume of PBS as that of the sample to elute it out in 4
consecutive cycles to make sure that all the sample has been eluted out of the
column. (That is add 1.5 ml PBS once, collect the elution , add another 1.5 ml ,
collect the elution and so on). (Fig.10)
11.Pool the 4 elutions and use 1 warm spin cycle to reconcentrate the sample in
desired volume (Add Endotoxin free salt if needed. Endotoxin free NaCl is NaCl
baked at 150℃ for 48 hours). (Fig.11)
12.For Nanoworms, pooled elutions are to be dialyzed in endotoxin free PBS with 4
changes to remove Urea and then reconcentrated.

36

37
Fig.9 Column being regenerated after
elutions have been collected
Fig.10 A rhodamine labeled sample’s
elutions being collected

A) B)
C)

38
D)
Fig.11 Coacervation observed after pooled elutions of rhodamine labeled sample
were put in a water bath at 37°C for 15 minutes. A) Cloudy solution right after being
placed in a water bath. B) More pronounced cloudiness after 5 minutes in the water
bath.
C) Rhodamine chunks visible after 10 minutes in water bath D) prominent Coacervation
chunks sticking to walls of the tube after 15 minute incubation in water bath

39
2.2 a) Void volume estimation
The resin inserts claim a void volume range of 0.3-0.5 ml for 1 ml resin. In order to avoid
error in packing resin beds, the exact void volume was calculated. To get the exact void
volume, a rhodamine labeled ELP sample was added to the resin after regeneration.
The eluted PBS was collected until the pink color started to appear in the elutions.
Process was repeated by using different volumes of rhodamine labeled samples to
ascertain the consistency of the results which showed 50-52% void volume.

40
Stability and purity of processed samples was checked by using SDS PAGE stained by
coomassie blue. (Fig.12)
Fig. 12 Molecular weight and purity of rhodamine labeled Cry V96 before and
after being passed through the column was confirmed by SDS PAGE stained by
coomassie blue. It shows a single band at the expected Molecular weight position of
42 KDa indicating no impurities or difference in stability after endotoxin removal.
Ladder 1st
Elution
2nd
Elution
3rd
Elution
Original
Sample
Re-Concentrated
CryV96
Ladder
KDa
75
Cry
V96
25

41
2.2 b) Percentage Loss of Protein estimation
To optimize the method, loss of protein during the endotoxin removal was estimated by
a two step method. The rhodamine concentration of the sample before passing it
through the column was measured. To do so, 2 ul of sample was mixed with 6 ul of 6 M
guanidine HCL.Same measurements and method was used by using 2 ul of PBS to
prepare blank. The pedestal of Thermo Scientific™ NanoDrop™ 2000/2000c
Spectrophotometer was wiped with 70% alcohol to clean it and UV-Vis was set at 280
nm, 350 nm and 550 nm with 740 nm as baseline to measure Optical density (OD). To
blank the nanodrop 2 ul of blank was put on the pedestal and a blank button was
clicked. The blanking was repeated, if needed, to get the flat line on the wavelength vs
absorbance graph display. Then the pedestal was wiped with alcohol again and 2 ul of
the sample was run triplicate in the similar way the blank was run.The absorbance at
550 nm was recorded and averaged. Concentration of rhodamine was calculated by
using the Bradford formula which is multiplying the path length of 0.1mm with
Absorbance/ Molar extinction coefficient of rhodamine (6000) x dilution factor (6).
After pooling and re-concentrating the elutions , concentration was measured again by
using the same method. Both before and after concentrations were compared to assess
the loss of protein.

42
2.3 Hek Blue Assay
Endotoxin concentration before and after passing through Detoxi gel was measured by
using HEK Blue Assay. Frozen Hek Blue 4 cells (In vivo gen) were thawed and added
to prewarmed 20ml Hek Blue growth media and spun at 300 x g RCF for 5 minutes.
Pellet was resuspended in 5 ml prewarmed growth media and the content of the vial
was transferred to 25 cm2
tissue culture flask. It was incubated at 37°C and 5% CO2 till
80% confluency was reached. The cells were then trypsinized by 0.025% trypsin and
transferred to 75 cm2
tissue culture flask in 10 ml prewarmed Hek Blue Selection Media
and incubated. The selection media was changed periodically till 80% confluence was
reached.
To detect the endotoxin level of the sample, serial dilutions of Endotoxin standard(In
vivo gen stock conc. 50 EU/ml) were made in 96 well plate having concentration of 50
EU/ml, 5 EU/ml, 0.5 EU/ml, 0.05 EU/ml and 0.005 EU/ml . Sample was also diluted 10
fold in similar fashion .
To prepare a 96 well test plate, media from Hek Blue 4 cells were aspirated. After a 10
ml pre-warmed(room temperature) PBS wash, 7ml PBS was added to detach the cells.
The 75cm3
flask was left in the incubator at 37°C and 5% CO2 for 10 minutes. The flask
was observed under the microscope to confirm the detachment of cells. Contents of the
flask were pipetted up and down with 1000 ul pipette tip and 10 ul sample was taken to
estimate the cell count using a haemocytometer. Volume of cell suspension needed to
meet the criteria of 50k cells per well (Fig.13) for Hek blue test plate was calculated and
Prewarmed (37°C in water bath) HEK Blue test media was added to the cell suspension
to make up the final volume sufficient to fill the wells of 96 well plate.

43
160 ul of this cell suspension was added to each of the wells and 40 ul of standard
dilutions in descending order were added to wells B2 to F2. Sample's 40ul original
sample and dilutions were added from B3 to F3 in descending order.Rest of the
columns were also filled in similar fashion with processed sample. 40 ul PBS was added
to row G as a blank. The surrounding wells were filled with 200 ul PBS to minimize the
effect of heat on cells. The test plate was incubated for 24 hrs at 37°C and 5% CO2.
Next day, Quanti Blue (QB) reagent was warmed in a water bath and 180ul of QB was
added to the wells of the detection plate ( 96 well plate). Both endotoxin standard and
sample were run in duplicate. By using a multipipettor, 20 ul supernatant from endotoxin
standard wells ( B2 to G2) was added to B2 to G2 and B3 to G3 wells of the Detection
plate . Similarly, 20ul supernatant was taken from an undiluted sample and its dilutions
from B3 to G3 were added to B4 to G4 and B5 to G5 and so on. Surrounding wells
were filled with 200 ul PBS to avoid the effect of heat disrupting the results. The
detection plate was incubated at 37°C and 5% CO2 for one hour . Absorbance was
measured at 640 nm using BioTek Synergy H1 multi-mode microplate reader.
The data obtained was exported and absorbances of Endotoxin Standard and sample
were calculated by subtracting the mean blank absorbance from the absorbances of
Endotoxin standard and sample.
To obtain the standard curve, a semi-log plot of absorbance vs concentration was
created and absorbance of the sample was used to estimate the endotoxin
concentration in the sample.

44
Fig. 13 A well of test plate showing 50000 HEK Blue cells per well
Fig.14 HEK Blue Assay detection plate after incubation with Quanti blue showing
different shades of purple , blue and pink corresponding to the endotoxin
concentration in the standard, sample, spike and blank.

45
Chapter 3 : Results
3.1 ELPs sequencing , expression and purification
Different ELPs were sequenced , expressed and purified by vector design and inverse
transition cycling (ITC). These included HAP A96, VSI tri block, Cry V96, and aIL6-
A192. aIL6-A192 is a nanoworm.
All the ELPs except the nanoworms were purified into a glassy palette at the end of the
third warm spin and provided clear solution upon resuspension in ice cold PBS. On the
other hand nanoworms formed a loose palette and required at least 7 hot and cold
cycles to form a pure ELP . Fig. 15.
Fig.15 A192 (Clear Solution) after three spins compared to Lym1 A192 (Cloudy
solution) after 7 warm and cold spins.

46
3.2 Columns and Resin
Different methods were tried and tested to remove the maximum amount of endotoxin
from ELPs.
First method was passing a sample through a column multiple times. The endotoxin
units of the sample before and after each run was calculated by using HEK Blue Assay.
There was no significant difference in the endotoxin units after multiple runs.Fig.16,
Fig.17.
Fig.16 HEK Blue Detection plate showing dark blue shade for higher
concentration of standard and undiluted concentration of HAP A96 before and
after being passed through the Detoxi gel column. On the plate Column 2 and 3
showing standard dilutions, column 4 and 5 showing dilutions of HAP A96 not passed
through resin, columns 6 and 7 showing dilutions of HAP A96 passed once through the

47
resin , Columns 8 and 9 showing dilutions of HAP A96 passed twice through the column
and Columns 10 and 11 showing dilutions of HAP A96 passed thrice through the
column.
Figure 17. Multiple passes through the Detoxi gel chromatography do not
promote endotoxin removal from HAP-A96. Samples were passed directly through
the column and collected. Samples were quantified using a calibrated HEK-Blue assay
for human TLR4. Samples were compared using ANOVA followed by the Tukey Post
Hoc test with p<0.05 considered significant. *p = 0.03

48
For the second method, the sample(VSI CC) was incubated in contact with resin for 10
and 20 minutes .Significant difference between the sample not passed through resin,
and sample incubated for 10 minutes and sample incubated for 20 minutes was
observed. However, No significant difference between 10 min. And 20 min. Incubation
was observed.Fig.18.
Fig. 18 Incubation in contact with Detoxi gel resulted in significant reduction in
endotoxin concentration of VSI that was expressed in Clear Coli. Sample was
added directly to the column and the bottom cap was put in place to stop the flow for 10
and 20 minutes once it reached the resin bed. Then elutions were collected by adding
PBS. Samples were quantified using a calibrated HEK-Blue assay for human TLR4.
Samples were compared using ANOVA followed by the Tukey Post Hoc test with
p<0.05 considered significant. *p = 0.05

49
For the third method, a combination of multiple passes and incubation times was used.
A sample (VSI) was passed through the column thrice and the collected pooled elutions
were split into two parts.One was incubated for five minutes in contact with resin while
the other was incubated for thirty minutes. Difference in endotoxin concentration was
observed between samples passed thrice through the column and sample incubated in
contact with resin. However incubation time made no significant difference in reducing
endotoxin concentration. Fig.19
Fig.19 Incubation in contact with Detoxi gel resulted in significant reduction in
endotoxin concentration of VSI that was expressed in Clear Coli. Sample was
added directly to the column and was passed through it thrice.It was split into two parts
and one part was incubated for 5 minutes and the other, for 30 minutes. Then elutions
were collected by adding PBS. Samples were quantified using a calibrated HEK-Blue
assay for human TLR4. Samples were compared using ANOVA followed by the Tukey
Post Hoc test with p<0.05 considered significant. No difference between 3x+5 and 3X
+30 minutes.

50
The fourth method was incubating ELP in contact with the resin for 1 hr and it resulted
in up to ninety nine percent reduction in endotoxin concentration. Fig.20, Fig.21.
Fig.20 1 hr Incubation in contact with Detoxi gel resulted in the most reduction in
endotoxin concentration of VSI that was expressed in Clear Coli. Sample was
added directly to the column and the bottom cap was put in place to stop the flow once
it reached the resin bed.Sample was allowed to stay in contact with resin for an hour.
Then elutions were collected by adding PBS. Samples were quantified using a
calibrated HEK-Blue assay for human TLR4. Samples were compared using ANOVA
followed by the Tukey Post Hoc test with p<0.05 considered significant.

51
Fig. 21 Repeating the same method with Cry V96 gave the same result. No
observed significance difference between elutions, but ninety nine percent reduction in
endotoxin concentration of sample after being treated with Detoxi gel 1 hour incubation
was observed.

52
Using the same method for nanoworms (aIL6-A192) didn’t result in significant reduction
of endotoxins. Fig.22.
Fig. 22. Incubating aIL6-A192 Nanoworm for 1 hr in contact with Detoxi gel did not
remove endotoxins from the sample.

53
Adding 7M Urea (denaturing salt) 1:1 to Lym1 A192 before adding it to the column
followed by overnight incubation and dialysis followed by re-concentration of pooled
elutions with endotoxin free PBS resulted in more than ninety five percent reduction in
endotoxin concentration. Spike was 0.4 EU /ml added to different concentrations of
processed sample which seemed to not work. Fig.23.
Fig.23 Incubating Lym1- A192 Nanoworm premixed with denaturing salt (7M urea)
overnight in contact with Detoxigel resulted in more than ninety five percent
reduction in endotoxin concentration. Sample was added directly to the column and
the bottom cap was put in place to stop the flow once it reached the resin bed. Sample
was allowed to stay in contact with resin overnight . Then elutions were collected by
adding PBS, pooled and dialyzed with 1:10 of sample volume to endotoxin free PBS over
the course of one week with 3 PBS changes. After dialysis, pooled elutions were reconcentrated by using warm spin centrifugation. Before and after samples were quantified

54
using a calibrated HEK-Blue assay for human TLR4. Samples were compared using
ANOVA followed by the Tukey Post Hoc test with p<0.05 considered significant.
Following table summarizes the endotoxin removal success results obtained from after
testing different ELPs.

55
ELP
Construct
Name
Amino Acid Sequence Molecular
Weight
Da
Endotoxin
Removal
Success
αB-Crystallin
(VPGVG)96
or Cry V96
[30]
GDRFSVNLDVKHFSPEELKVK (VPGVG)96 Y 42,035 Yes
VSI G(VPGVG)48(VPGSG)48(VPGIG)48 Y 59,299 Yes
HAP A96
[31]
MIHVTIPADLWDWINKG(VPGAG)96 Y 38,790 Yes
aAIL6-A192
[32]
MSTVILSAAAPLSGVYAAMERGSHHHHHHGSGS
GSGIEGRPYNGTGSACELGTQVQLKESGPGLVPS
QSLSITCTVSDFSLTNYGVHWVRQSPGKGLEWLG
VIWSGGSTDYNAAFISRLSISKDNSKSQVFFEMNS
LQADDTAIYYCARNGNRYYGYALDYWGQGTSVT
VSSGGGGSGGGGSGGGGSDVVMTQTPLSLPVS
LGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQS
PKLLIYTVSNRLSGVPDRFSGSGSGTDFTLKISRV
EAEDLGVYYCFQGSHGPYTFGGGTKLEIKLQTCG
RKLSLNQN G(VPGAG)192 Y
106,330 No

56
Table.3 ELP therapies from which LPS was effectively removed by using Polymyxin
B resin.
Lym 1 A192
[33] [34]
MQVQLKESGPGLVAPSQSLSITCTISGFSLTSYGV
HWVRQPPGKGLEWLVVIWSDGSTTYNSALKSRL
SISKDNSKSQVFLKMNSLQTDDTAIYYCASHYGST
LAFASWGHGTLVTVSAGGGGSGGGGSGGGGSD
IQMTQSPASLSASVGETVTIICRASVNIYSYLAWYQ
QKQGKSPQLLVYNAKILAEGVPSRFSGSGSGTQF
SLKINSLQPEDFGSYYCQHHYGTFTFGSGTKLEIK
G(VPGAG)192Y
98,830 Yes (After
modification
of method)
Commented [1]: Also cite the source of the
Genes/peptide sequences
Lym1-A192 is in Changrim Lee/Mackay Biomaterials
paper 2020
Biomaterials. Author manuscript; available in PMC
2021 Dec 1.
Published in final edited form as:
Biomaterials. 2020 Dec; 262: 120338.
Published online 2020 Aug 31. doi:
10.1016/j.biomaterials.2020.120338
PMCID: PMC8386582
NIHMSID: NIHMS1732889
PMID: 32916604
Adaptable antibody Nanoworms designed for nonHodgkin lymphoma
Changrim Lee,a Santosh Peddi,a Caleb Anderson,c
Hao Su,c Honggang Cui,c Alan L. Epstein,b and J.
Andrew MacKaya,d,e,*
and this reference:
[22] Hu P, Glasky MS, Yun A, Alauddin MM, Hornick
JL, Khawli LA, Epstein AL, A human-mouse chimeric
Lym-1 monoclonal antibody with specificity for human
lymphomas expressed in a baculovirus system, Human
antibodies and hybridomas 6(2), 1995, pp. 57–67.
[PubMed] [Google Scholar]
IL6-A192 is derived from an antibody found:
(28) Shigemori, S.; Ihara, M.; Sato, T.; Yamamoto, Y.;
Nigar, S.; Ogita, T.; Shimosato, T. Secretion of an
immunoreactive single-chain variable fragment
antibody against mouse interleukin 6 by Lactococcus
lactis. Appl Microbiol Biotechnol 2017, 101 (1), 341-
349. DOI: 10.1007/s00253-016-7907-8 From NLM.
HAP peptide was derived from this paper:
Inhibiting complex IL-17A and IL-17RA interactions with
a linear peptide
Shenping Liu, Joel Desharnais, Parag V.
Sahasrabudhe, Ping Jin, Wei Li, Bryan D. Oates,
Suman Shanker, Mary Ellen Banker, Boris A. Chrunyk,
Xi Song, Xidong Feng, Matt Griffor, Judith Jimenez,
Gang Chen, David Tumelty, Abhijit Bhat, Curt W.
Bradshaw, Gary Woodnutt, Rodney W. Lappe, Atli
Thorarensen, Xiayang Qiu, Jane M. Withka & Lauren
D. Wood
Scientific Reports volume 6, Article number: 26071
(2016) Cite this article
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crystallin-V96 paper was Explained in Sara Attia's
...

57
Chapter 4 : Discussion
ELPs are expressed in E.coli. LPS is an integral part of the E.coli cell wall. During the
process of expression and purification, these LPS molecules become suspended in the
solution containing elastin-like polypeptides (ELPs) and can adhere to the ELPs. This
presence of LPS poses a significant risk of causing septic shock when these ELPs are
used in vivo experiments.
To mitigate this risk, one approach is to use modified E. coli systems where the Lipid A
portion of LPS is truncated. One such system, known as Clear Coli®, aims to reduce
endotoxin levels; however, complete elimination of endotoxin remains challenging with
this method, and the risk of septic shock persists.
Various methods are employed to remove endotoxins from proteins expressed in E. coli,
including two-phase partitioning, ultrafiltration, chromatography, electrophoresis, and the
use of detergents. Each of these methods has its strengths and limitations.
One promising method for effectively removing endotoxin from ELPs is based on
polymyxin B affinity chromatography. This technique has demonstrated efficacy in
removing endotoxins from various ELP-based therapies, thereby reducing the risk
associated with their in vivo applications.
While traditional methods for endotoxin removal have drawbacks, polymyxin B affinity
chromatography stands out as a reliable strategy to ensure the safety of ELPs intended
for biomedical use, by effectively eliminating endotoxin contamination.

58
Detoxi Gel™ polymyxin B resin was packed in gravity flow columns having different resin
bed volumes to form an affinity chromatography system. The endotoxin concentration
was measured by using Hek Blue colorimetric assay by first plotting a standard curve and
then using the equation on the graph to find the unknown concentration of the sample
corresponding to absorbance.
Different ELP therapies like HAP A96, VSI, CRY V96 , aIL6-A192 and LYM 1 A-192 were
passed through it. Different methods like multiple passes through the column, incubation
of samples in contact with resin and combination of multiple passes and incubation were
tested. Incubating simple chain ELPs for 1 hr in contact with resin gave most effective
results with more than ninety nine percent reduction in endotoxin concentration. However,
when the same method was used for a more complex structured ELP like nanoworm, this
method didn’t work without the addition of denaturing salt i.e 7M urea to the sample before
adding it to the column.
There is a need to optimize this method to make it efficient for nanoworms as well. It can
be done by creating different conditions of the salt environment by using various
concentrations of Urea. Different incubation times like one hour should be tested to speed
up the process. Furthermore, other nanoworms should be tested by using the same
method to ensure the robustness of this method.
HEK Blue assay can also be further optimized as no inhibition and activation was
considered while setting up the test plate. Furthermore, filtering the pooled elutions before
reconcentration can also prevent the effects that any material leaching from the column
can have on the Hek Blue results.

59
In terms of column and resin combination, there is room for considering height of the
column vis a vis width of the resin bed’s effect on results. There might have been a
difference in the effectiveness of the same method in different sized columns which needs
more streamlining.
More ELPs need to be treated in a similar way to find the consistency in the effectiveness
of this method. It can also help rule out the possibility of complexity of structure as a
hindrance to the success of this method.

60
Chapter 5 : Conclusion
ELPs are thermosensitive biopolymers which can be genetically engineered to be
attached to any cargo which can be protein in nature. They can also be modified to
coacervate at a certain temperature. They are expressed in E.coli strains which contain
LPS in their cell wall which gets attached to the purified ELPs and once injected ,poses a
risk of septic shock in animals used for in-vivo studies. Polymyxin B based affinity
chromatography was explored to remove endotoxins from ELP therapies to meet FDA’s
limit of 5 EU/kg. Incubating ELPS for 1 hr in contact with polymyxin B was effective in
removing more that ninety nine percent of endotoxin for chain peptide ELPs but this
method wasn’t successful for removing endotoxins from nanoworms unless the
nanoworms were incubated with a denaturing salt.

61
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Abstract (if available)

Abstract Elastin-like polypeptides (ELPs), which are repetitive, high molecular weight peptides derived from human tropoelastin, hold promise for various biomedical applications, including drug delivery and tissue engineering. However, their production in E.coli often leads to contamination with lipopolysaccharide (LPS), a potent endotoxin that can confound in vitro and in vivo preclinical studies of biologic drug candidates. To address this issue, this study investigated various chromatographic resins and columns to more effectively remove LPS from ELPs while preserving protein yield. Among the methods tested, polymyxin B resin packed in a gravity flow column demonstrated superior LPS removal efficiency, achieving greater than 99 percent reduction in endotoxin levels after a 1-hour incubation, as confirmed by a TLR 4 activation based colorimetric assay (HEK Blue). While this method proved successful for purifying ELPs, it required the presence of denaturing salt to remove LPS from ELP fusion proteins having single chain antibodies attached to them. This highlights the need for further research to address this challenge in the context of different ELP fusion peptides. The development of efficient endotoxin removal strategies is crucial for advancing the translational potential of ELP-based biomaterials.

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Asset Metadata

Creator Bhatti, Quratulain (author)

Core Title Optimizing Detoxi-Gel™ resin based affinity chromatography to prepare recombinant ELP therapies for in vivo evaluation

School School of Pharmacy

Degree Master of Science

Degree Program Pharmaceutical Sciences

Degree Conferral Date 2024-08

Publication Date 09/11/2024

Defense Date 07/23/2024

Publisher Los Angeles, California (original), University of Southern California (original), University of Southern California. Libraries (digital)

Tag affinity chromatography,elastin like polypeptides fusion proteins,ELPs,endotoxin,LPS.,OAI-PMH Harvest

Format theses (aat)

Language English

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Advisor Mackay, Andrew J . (committee chair), Cogan, Dillon P. (committee member), Culty, Martine (committee member)

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