Senior Design Project
Dr. Xuejun Wen
G-CSF Releasing Device:
Cost effective alternative to current G-CSF Delivery Methods for Treatment of
Neutropenia in Cancer Patients
Group Members:
Cabell Lamie
Aaron Rowane
Carolyn Song
Adil Suleman
Allison Yaguchi
Date of Submission: October 28, 2013
Table of Contents
1. Title Page ........................................................................................................................... 1
2. Table of Contents ............................................................................................................... 2
3. Executive Summary ........................................................................................................... 3
4. Introduction ........................................................................................................................ 4
5. Project Proposal ................................................................................................................. 6
6. Project Timing ................................................................................................................. 10
7. Project Considerations ..................................................................................................... 13
8. References ........................................................................................................................ 18
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Executive Summary
Granulocyte colony stimulating factor, or G-CSF, is used in the treatment of
neutropenia, which is a low level of white blood cells. Neutropenia is a common side effect
caused by chemotherapy. Current G-CSF treatments are very costly because the price of the
drug is high and treatment often requires many injections, which makes clinic and
administrative costs high as well. This project will focus on producing a more cost effective
way to deliver G-CSF to the patient. There were two methods of delivery considered:
transdermal drug delivery and cell encapsulation/hollow fiber tube implantation drug
delivery. Upon assessing the two methods, the hollow fiber tube method was determined to
be the most appropriate approach. The proposed product involves a hollow fiber tube lined
with irradiated cancer cells transfected to secrete G-CSF. This hollow fiber tube will be
subcutaneously implanted so that the G-CSF can be slowly secreted into the bloodstream.
The hollow tube will be made of poly(acrylonitrile/vinyl chloride) (PAN/PVC) or a
PAN/polysulfone polymer and will be lined with genetically modified HeLa cells irradiated
to halt cell growth.
This project aims to find the optimal permeability of the tube by evaluating the
material, thickness, and diameter of the tube so that G-CSF can best be delivered to the
bloodstream. The tube must be permeable enough to allow the G-CSF and cell waste to be
secreted out while also allowing nutrients and oxygen into the tube for the cells. Production
of the G-CSF utilizing a cell line dramatically decreases manufacturing costs. The method of
making the hollow tubes, phase inversion, is a highly effective, fast, and cheap way to create
the tubes. Overall, the production will be much cheaper while maintaining product efficacy.
Several factors must be considered during the development of this product such as health and
safety and process sustainability, which are discussed later in this proposal.
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Introduction
Neutropenia is a disorder in which patients have an abnormally low level of
neutrophils. Neutrophils, which make up the majority of white blood cells, are derived from
the bone marrow and are one of the first cells to respond in the event of an infection. Because
many chemotherapy treatments target fast growing cells, healthy cells in the bone marrow are
affected, leading to high incidences of neutropenia in cancer patients. After most
chemotherapy regimens, the patient often experiences one week of neutropenia. Fever is
often associated with neutropenia leading to febrile neutropenia. According to data from the
National Cancer Institute, more than 60,000 cancer patients are admitted for febrile
neutropenia annually3. Patients with neutropenia or febrile neutropenia are very susceptible
to serious, even fatal, infections, which can be costly to treat. To prevent neutropenic related
fevers and infections, the American Society of Clinical Oncology (ASCO) recommends that
hematopoetic colony-stimulating factors (CSFs) are used when the risk of febrile neutropenia
from chemotherapy is more than 20% and another treatment is not available, and when
patients receiving chemotherapy with less than 20% risk of febrile neutropenia become more
at risk due to factors such as age, medical history, or other reasons related to cancer19. There
are several types of CSFs, but the most commonly used is granulocyte colony stimulating
factor (G-CSF).
Granulocyte colony stimulating factor is a regulatory glycoprotein for the production
and functional activities of neutrophils10 and is essential for maintaining neutrophil levels. GCSF is a protein composed of 174 amino acids and is 18.7 kDa in size16. G-CSF works on all
stages of neutrophil development, particularly on increasing the proliferation and
differentiation of neutrophils. G-CSF also improves survival and function of mature
neutrophils. Currently, G-CSF is used clinically to offset the side effects associated with
myeloablative side effects, of chemotherapy. Additionally, G-CSF is used to increase
neutrophil count so the duration between chemotherapy treatments is decreased and
surviving cancer cells are prevented from proliferating, increasing cancer cell death. G-CSF
is generally not used on patients treated with radiation and chemotherapy3.
Currently, there are two forms of G-CSF analogs administered. Filgrastim, sold
commercial as Neupogen, has a shorter half-life (4 hours) and is given by subcutaneous
injection or through an IV for hospitalized patients. It is a small molecule so it is easily
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removed by the kidneys2. It is produced by E. coli that has been transfected by the G-CSF
gene. It is typically not administered to non-hospitalized patients except for special
circumstances, according to Dr. Kevin Brigle, a nurse practitioner at Virginia
Commonwealth University Health System’s Massey Cancer Center’s oncology unit4.
Neupogen is dosed at 5 mcg/kg/day and comes in vial sizes of 300 mcg and 480 mcg. The
does is always “rounded up” according to weight and is injected daily until neutrophil count
reaches 1,000.
Another G-CSF analog, called pegfilgrastim, sold commercially as Neulasta, has a
longer half-life (15-80 hours). It is the same chemical make-up of Neupogen, but has a
polyethylene glycol (PEG) molecule added to the N-terminus of the filgrastim protein. This
makes it too large for the kidney to eliminate Neulasta from the body so it has a much longer
half-life than Neupogen2. Neulasta is cleared by the neutrophils themselves as the number of
neutrophils increases. This form of G-CSF is administered only once subcutaneously
following chemotherapy due to the fact that it stays in the body for so long. It is injected at 6
mcg regardless of weight. It is given only in the outpatient setting as the injection is very
expensive. For both forms, the cheapest option for the patient is to administer the drug to
themselves at home4.
There are several problems with the current options. Every injection is expensive and
treatment of neutropenia often requires multiple injections. One 6-mg vial of pegfilgrastim is
$2,838 and 300-μg and 480-μg vials of filgrastim are $268 and $427, respectively3. Selfadministration of the drug can be unreliable because of noncompliance or inexperience with
administering injections. This senior design project intends to develop a low cost (<$200)
biodevice to deliver G-CSF using cell encapsulation technology to patients through a
minimally invasive subcutaneous injection. Two different methods of delivery methods were
considered, a hollow fiber tube and a transdermal patch. After analyzing the two different
methods, it was decided that the most practical method of delivery is through subcutaneous
injection. Unlike current methods however, this project proposes to develop a device that is
injected under the skin that will remain there over a specific range of time. During this time
frame, G-CSF will be slowly released into the bloodstream, therefore reducing the need for
multiple injections per treatment cycle and increasing the likelihood that the treatment will
effectively bring the neutrophil count up.
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Project Proposal
The intent of this project is to create a low cost method of administering G-CSF to
chemotherapy patients to treat neutropenia. Cancer cells will be transfected to express G-CSF.
The cells will then be placed inside of a hollow tube made by phase inversion. The hollow
tube device, equipped with the transfected cells, will then be injected subcutaneously into the
patient by needle.
Methods:
Transfection of cells for G-CSF production
Human cancer cells, likely HeLa cells, will be mutated by both phage integration
(chromosomal) and traditional DNA recombination (plasmid) to express human G-CSF.
Each cell will produce a certain amount of G-CSF. This amount will be determined through
an ELISA assay which will then determine how many cells are required for each hollow tube
in order to have proper dosage. The specified amount G-CSF to be delivered to the body is 5
mcg/kg/day. The cancer cells will be treated with irradiation in order to keep the cells from
growing out of the tube and into the rest of the body, eliminating the potential of forming a
tumor. The cells will be alive and producing G-CSF, but will not be actively proliferating.
The cells will slowly stop producing G-CSF as the cells die and then the tube may either
remain in the body or later be removed.
Phase inversion
Phase inversion, also known as wet spinning, involves a polymer being dissolved into
one solvent and then rapidly precipitated out to instantly form a solid. In this case, our
polymer of choice (either PAN/PVC or PAN/polysulfone) will be dissolved into an organic
solvent to create a polymer solution. DMF and DMSO are likely solvent choices. The
polymer solution needs to be slightly viscous because it will then be pressed through an
extruder. The extruder, called a spinerette, was developed by Dr. Wen. As the solution leaves
the extruder, it will contact a “non-solvent.” The non-solvent is a solvent that the
DMF/DMSO can mix freely with, but is highly repellent to the polymer. This causes the
polymer to instantly precipitate, resulting in a solid polymer. DMF/DMSO mixes freely with
water, so water will likely be our non-solvent. This is a very fast and simple process,
therefore optimal for commercial uses.
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Implantation of device into the body
Inject the tube using a needle of a larger diameter than the tube diameter. Over time, since
the cells are not proliferating, the cells will die. This will occur after the neutrophil count has
recovered. The tube can either be left in the body or later removed.
Design specifications:
● Length: 150 um to 5mm
● Diameter: 800 um to 1 mm
○ Ideal = 700 - 800 um
● Thickness is to be experimentally determined
● Device should be $200 or less
Variables to be analyzed:
● Polymer type (PAN/PVC vs. PAN/Polysulfone)
○ Depending on which combination is used, a .jmp files for each material
totaling two .jmp files.
● Cells
○ Cell life span
○ Secretion per cell
● Permeability Factors
○ Concentrations of PAN/PVC or PAN/Polysulfone
○ Density of material being secreted (fixed)
○ Solubility and diffusivity of material being secreted (fixed)
○ Rate of secretion
○ Solubility of tube material
○ Density of tube material
○ Tube thickness and length
○ Size of molecules secreted (fixed)
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● Solvent systems
○ Organic solvent: DMF or DMSO
○ Non-solvent: water
● We can use .jmp files to determine the optimal conditions for these variables to be
tested under
Figure 1: Schematic of the proposed device
An alternative proposal to the hollow fiber method is the use of transdermal patches.
Transdermal patches generally involve the use of an adhesive patch that would attach to the
skin to deliver a drug to the bloodstream. If such a product could be made, this is a low cost
alternative requiring little technical knowledge to apply. Additionally, a transdermal patch
allows for a controlled release of a drug over a period of time in a noninvasive manner. It
would be very optimal for those who are not hospitalized, as it is not necessary for someone
else to learn how to properly give an injection. However, the skin is an effective barrier, so
generally smaller molecules are able to penetrate. For example, nicotine, a common molecule
delivered by transdermal patches, has a molecular weight of 162 g/mol, whereas G-CSF has a
molecular weight of 19,600 g/mol. A good transdermal patch delivers a drug efficiently and
safely through the skin to the bloodstream.
Recently, there has been development on improving skin permeability via the use of
chemical enhancers and physical enhancers (such as ultrasound or microneedles)17. Such
technology is beyond the scope of this project; therefore patch method may not be the
appropriate approach. The need for the patch is not very high since G-CSF is already
subcutaneously injected. Because G-CSF is such a large molecule, it would likely be
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hindered by the skin, which would mean more G-CSF will be required to achieve the same
dosage. The cell-based injection this project proposes dramatically decreases the cost of the
injection, while a patch will actually increase the cost. Furthermore, there are similar hollow
fiber tube devices already being designed for other applications such as treatment of
Parkinson’s disease and diabetes that have been FDA-approved, setting a precedent for
approving this type of technology.
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Project Timing
Figure 2: Gantt chart detailing project timing
Note: Future deadlines are yet to be added
Project Schedule will be managed by Aaron Rowane using MS Project
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Scheduling
Team members have a set meeting time every Friday at 3pm. The team will meet on
the first floor of the IEM building to touch base with each other and discuss any results
and/or roadblocks. The team keeps up a high level of correspondence via email so meeting
often may not necessary. However, the team will put in the appropriate amount of work
(minimum of 6 hours) required for the project. Because the team has such different
scheduling due to classes and other research involvements, the team will primarily work in
shifts. For example, two people will come in on Monday, another couple on Tuesday, and so
on. This will ensure that the project experimentation is being completed on a daily basis and
lets the teamwork more efficiently. This will also allow all team members to put in the
required amount of hours.
As shown in Figure 2, a Gantt chart, developed in MS Project, will be utilized to track
project progress. It will also designate who is project leader during each phase of the project.
Aaron Rowane will be primarily responsible for keeping up the Gantt chart, as he has the
appropriate programming software on his computer. He will update the team with the
progress of the project every Friday during our 3pm meeting time.
General Timeline
The team has learned how to make hollow fiber tubes, so the experimental phase of
the project has just begun. The initial project designing will be fairly quick as Dr. Wen
already has a rather streamlined procedure already outlined for the project. The first task of
the project is to calculate permeability and potentially draft a model to calculate permeability
for ease of design later on. Experimentation of permeability should not take more than a few
months. The transfection of cells is a straightforward, traditional procedure that should take
no more than a couple of weeks. After these first few months of experimentation, the team
hopes to begin animal studies using mice. This phase will probably take to the end of the
spring semester in order to measure statistically relevant data.
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Technical Skills
The project will involve a significant amount of hollow tube synthesis, as a new set of
tubes will need to be synthesized as the permeability is adjusted. The team has just learned
how to set this up from Dr. Wen. It is a fairly easy process and each team member can come
in a take responsibility for making the tubes as needed. The design of experiment will be
discussed among all team members and proposed to Dr. Wen. As each experiment is
conducted, a new set of experiments will be derived as we learn more about how different
parameters impact the permeability.
The transfection of cells will be done by homologous recombination and through
phage insertion. Allison and Carolyn both have extensive experience with this and will
probably take the lead on this portion of the project. Once the cells are transformed to
produce G-CSF, the cells will only have to be kept alive and uncontaminated. ELISA assays
are a very standard protocol and while Allison and Carolyn have some experience with the
assay, other team members will learn and perform the protocol as well. Allison and Carolyn
can help Dr. Wen teach this protocol to other team members.
Thus, the team leader will vary as the level of technical skills varies. For example,
Carolyn will be team leader when we conduct mice studies.
● Mice studies: Carolyn
● Cell transfection: Allison and Carolyn
● Hollow tube synthesis: The team has completed this stage of the project
● ELISA assays: Allison and Carolyn
● Design of Experiment (DOE): All team members
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Project Considerations
Economic Issues
One of the most obvious issues with the current G-CSF treatment options is the price.
As mentioned above, one 6 mcg vial of pegfilgrastim is $2,838 and 300-μg and 480-μg vials
of filgrastim are $268 and $4273, respectively. On top of the raw cost of the drug, there are
administrative costs associated with the injections if someone responsible for the patients (i.e.
a family member, etc.) does not learn to inject it. If the patient were to have neutropenic
complications, the hospital expenses are also extremely expensive, as shown in the table 1
below.
Table 1: Cost per hospitalizations for neutropenia
Tumor Type
Cost per hospitalization
Leukemia
$28,200 ± $35,600
Lung and bronchus
$8,500 ± $9,700
Colon and rectum
$8,000 ± $9,700
Breast
$7,100 ± $10,200
Ovary
$7,600 ± $8,000
Stomach
$10,900 ± $12,200
Pancreas
$8,400 ± $8,800
Cervix
$8,600 ± $10,000
Testis
$9,200 ± $9,300
These values are the total cost of one hospitalization caused by neutropenia taking into account the length of stay. 6
The main cost for the project will be the PAN/PVC, which will only come out to
about a dollar per tube. An initial batch of the cells will be obtained and they will be
transfected and then grown as needed. This means that essentially the only other cost is the
upkeep of the cells. This will dramatically decrease the costs of the medicine and can allow
more people access to the drug.
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Manufacturability
Initial experimentation for hollow tube manufacturing has been performed with DMF
as the solvent and water as the non-solvent. The polymer component used PAN-PVC, but
PVC/polysulfone may be considered. The regulation of drug delivery of G-CSF will be
dependent on many factors of production. Following the transfection of stem cells to
produce G-CSF, the permeability of the hollow fiber membrane will be the determining
factor for the rate of secretion of G-CSF into the patient. It is intuitive that the efficacy of the
device is directly correlated to quality control.
There are many considerations that need to be taken into account when manufacturing
the device. There are many aspects of the hollow fiber membrane that influence the flux of
G-CSF across the hollow fiber membrane, which can primarily be attributed to its
morphology. Morphology includes the number of pore, thickness, smoothness/roughness,
and the patterns of the inner wall. All of these factors play a key role in diffusion in and out
of the device. The cells, which will be infused within the core of the membrane, require the
back diffusion of nutrients through the membrane. Most importantly, G-CSF must
successfully be able to cross the membrane for drug delivery to the patient. This requires
that the membrane attributes include a specific affinity for the molecules crossing the
membrane in either direction.
Hollow fiber membranes are created due to the coagulation of the polymer solution.
As the fiber is injected into the spinneret, the pressure is increased and the polymer
coagulates to a solid state. At the outflow of the spinneret the coagulated polymer is mixed
with a non-solvent to maintain its shape. Following this stage of the process, the polymer is
then washed in the non-solvent bath and the hollow fiber membrane is finished. The
structure of the spinneret is also of importance as it designates the width and thickness of the
tube15. The distance between the outflow of the polymer and when the solid tube contacts the
wash bath can affect how the layers of the tube form. For example, a shorter distance
between the bath and outflow will result in a single layer, which is desired in this project. A
longer distance will result in a double layer, which can be useful for other applications
requiring very fine diffusion of molecules requiring a high purity.
The key process parameters that need to be taken into account that influence the
quality control of the membrane are the temperature, ratio of input process streams (non-
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solvent/dissolved polymer), and the polymer concentration. As mentioned previously the
morphology of the membrane plays a key role in the diffusion of molecules across the
membrane. Once a final prototype of the device is developed, it is necessary that all of these
process parameters are held constant during commercialization for the device to operate as
specified.
Key aspects of design that cannot be neglected are the cells located in the interior of
the hollow fiber membrane. If the secretion of G-CSF is not properly regulated in
accordance to the membranes abilities it is needless to say it will not be effective. If not
enough G-CSF is produced the concentration gradient will not be sufficient enough for
delivery. On the other hand if too much is produced for too long, the patient will not be able
to follow their chemotherapy schedule, as chemotherapy cannot be completed when G-CSF
is being administered. Thus the cells need to be monitored frequently in their ability/inability
to produce G-CSF. Essentially the cells play a key role in the driving force of diffusion.
From Fick’s law of diffusion, the concentration gradient and permeability are ultimately what
influences mass flux. The ability for the G-CSF to diffuse is primarily attributed to the
diffusion coefficient, an experimentally determined constant that indicates a molecules
ability to diffuse through a medium13. With this implication, it is imperative that the number
of cells within the device is also regulated dependent on their ability to produce G-CSF,
which is determined by an ELISA assay.
Health and Safety Considerations
The subcutaneous implantation of a hollow fiber membrane tube introduces a foreign
object into a patient and may be a source of infection. In a patient that is already
immunocompromised due to chemotherapy, this may be dangerous. Chemotherapies are
often delivered via a central venous catheter, which also introduces a potential source of
infection. We believe the hollow fiber membrane tube will be less invasive and less costly
and the cell encapsulation method of delivering G-CSF ought to be explored. The device will
be implanted subcutaneously, so in the event of a serious infection, the device can be
removed and the patient can be treated with antibiotics.
Implantation of a device also brings a biomaterials issue. When evaluating a
biomaterial, several factors much be considered. A biomaterial must be biocompatible with
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the host without eliciting a negative response while performing its intended function. To
determine biocompatibility of a biomaterial, it is generally tested in in vitro and in vivo. The
polymers being used to make the hollow fiber tubes are well-researched biomaterials, so
biocompatibility should not be an issue.
Because G-CSF functions within the immune system, the potential for
immunodeficiency and autoimmunity should be considered. The proposed device is supposed
to treat neutropenia, a disorder characterized by abnormally low neutrophils, so
immunodeficiency is the problem we are attempting to treat. Developing autoimmunity after
treatment of G-CSF is unlikely because lymphopoiesis and lymphocyte receptor development
should not be affected. Additionally, it is very unlikely that a patient suffering from
neutropenia would produce too many neutrophils after G-CSF treatment.
Another and perhaps the most important health and safety consideration when
treating a cancer patient with G-CSF is the potential for the growth factor to facilitate cancer
growth. Some growth factors have been shown to promote cancer proliferation1,9,7. Results
from a clinical trial of same day treatment of G-CSF and chemotherapy have not
conclusively shown to have a negative effect on a patient11,5. One clinical study
recommended treating prostate cancer patients with G-CSF at the start of chemotherapy
treatment17. More recent mice studies have shown that G-CSF used after a chemotherapy
cycle may reduce antitumor activities of chemotherapy and promote angiogenesis, which
may promote tumor growth16. However, the same study concludes that there no studies
showing that simultaneous treatment of G-CSF and chemotherapy affects survival
outcomes18.
Process Sustainability
The goal of this work is to design cell encapsulation process via hollow fiber
membrane to sustain irradiated cancer cells that are mutated to produce human G-CSF for a
specified amount of time. Extended lifespan of the cells is preferred since clinical
applications for such devices are estimated to be expensive and inconvenient. Just like any
process there are some constraints to consider in order for the process to be sustainable. The
constraints are: biocompatibility (in vivo biocompatibility of encapsulated cells),
immunoprotection (protection from host’s immune system) and diffusion permeability.
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Cytocompatibility should be considered in the proposed design. The cells should
remain viable long enough to diffuse the necessary amounts of G-CSF to the host system.
Therefore extended lifespan is necessary to keep the costs of any clinical treatment
sufficiently low.
The design specification of hollow fiber membrane will be designed to allow for a
controlled release of G-CSF from irradiated cancer cells. The permeability of hollow fiber
membrane should be such that it readily allows G-CSF to readily diffuse out of the
membrane in order to ensure adequate G-CSF regulation.
The hollow fiber membrane should provide adequate protection from antibodies and
macrophages. This can achieved by having a molecular weight cutoff of hollow fiber
membrane at about 70-75 kDa, which will block the entry of antibodies and macrophages
and allow bidirectional diffusion of molecules such as the influx of oxygen and nutrients etc.
essential for cell metabolism and the outward diffusion of waste products and therapeutic
proteins, which in this case is G-CSF.
Validity of proposed design can only be achieved if the encapsulation method is
repeatable with desired results attained every time. Therefore the size and permeability of the
hollow fibers membrane must be predictable within certain ranges so as to ensure adequate
release of GCSF.
Ethical Considerations
In this project, the proposed device will be injected subcutaneously, a minimally
invasive procedure, in mice. After testing, the mice will be euthanized in a humane manner.
Because we plan to test on mice, there are ethical considerations. The argument against
animal studies is that animals suffer during experimentation. Some feel as though animals
used in testing are subjected to unnecessary torture. While it is unfortunate animals are used
in testing and may experience suffering, many developments in medicines and medical
technologies are due to successful animals studies. Furthermore, before FDA-approval of a
biomaterial, the material must undergo in vivo testing to assess toxicity and biocompatibility.
After animal studies, controlled clinical trials must be conducted before approval. Animal
testing is a crucial step in the biomedical research process.
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