Simulation of a Human Stomach as a Bioreactor
BSE-4126 Comprehensive Design Project
May 13, 2009
Purpose: The purpose of this report is to present the final paper, an updated executive
summary, project design, and conclusion on the bioreactor design.
Team: Proteinnovate
Group Members:
Marianita Avenida
Megha Maheshwari
Jennifer Kim
Kristen Pevarski
Advisors:
Dr. Zhang
Dr. Grisso
Executive Summary: Simulation of a Human Stomach as a Bioreactor
Due to rising costs of pharmaceutical testing and interest in the alternative delivery of
protein drugs, development of a device to test these proteins is essential. The harsh
environment of the stomach hinders the effectiveness of oral protein drugs by breaking
the polypeptides down into peptides. As a result, 98% of the protein is digested in this
process, making the subcutaneous route the most effective option. Injection has less
public appeal and increased chances of infections due to contaminated needles.
Furthermore, a lack of facilities and administrators to distribute these medications add
challenges for patients in developing countries. If pharmaceutical companies could
develop protein medications that could be taken orally, it would eliminate many issues
listed above.
Research and experiments are crucial in developing oral delivery protein drugs.
The bioreactor design by Proteinnovate will help resolve the issues involved with the
subcutaneous route of drug delivery by enabling drug developers to test what percent of
their potential oral protein drug is broken down during digestion. The bioreactor
includes: probes monitoring the pH, temperature, flow rate, and volume level, baffles,
stirrers, a stirrer motor, a cooling jacket, a conical bottom, and many other components.
The proposed design will monitor important parameters such as temperature, pH, flow
rate, agitation, and fluid level. Also, it will control them by changing the flow rate of
cooling water, adding hydrochloric acid, and changing the flow rate of the feed and
product. At the end of the bioreactor cycle, a SDS PAGE will detect the amount of
peptides. When an unprotected protein is inserted in the reactor, it will result in a 98%
digestion of the protein. Potential clients will be able to test their oral protein drug by
inserting it into the feed and measuring the unbroken protein. If clients believe the
amount of unbroken protein is sufficient enough to be taken by patients, they can start
producing them for the public.
The 2 1-liter bioreactors were calculated to cost approximately $100,000. This
figure can be compared to the cost of a previously constructed bioreactor made by British
scientists which was approximately $1 M. While the bioreactor by Proteinnovate has
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fewer functions than its British counterpart, it is significantly cheaper to construct and
maintain.
Bioreactors that can be used as an artificial organ can be beneficial as shown
below. Animal testing of a chemical can cost from $0.5 M to $1.5 M. By using a
bioreactor, companies can significantly reduce the amount of money spent on testing.
They can aid in changing the way medicine is administered in a more public friendly
way. This will ultimately make medicine more accessible to patients in developing
countries.
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Table of Content: Simulation of a Human Stomach as a Bioreactor
Page
Problem Statement……………………………………………………………..
6
Connection to Contemporary Issues…………………………………………..
6
Scope of Work…………………………………………………………………..
6
Deliverables…………………………………………………………………..
7
Introduction…………………………………………………………………….
7
Design Criteria and Constraints……………………………………………….
8
Literature Review………………………………………………………………
8
Stomach
Physical…………………………………………………………………….
8
Chemical Environment…………………………………………………...
9
Digestion
Digestion Process………………………………………………………….
11
Mechanisms………………………………………………………………..
11
Drug Delivery
Oral…………………………………………………………………………
13
Bioreactors
Overview……..…………………………………………………………….
13
History……………………………………………………………………...
14
Types of Bioreactors….……………………………………………………
15
Safety and Standards………………………………………………………...
17
Preliminary and Alternative Designs
Continuous Bioreactor……………………………………………………….
20
Batch Bioreactor……………………………………………………………...
21
Project Design…………………………………………………………………..
22
Project Evaluation………………………………………………………………
26
Conclusion / Summary…………………………………………………………
28
Work Plan……………………………………………………………………….
28
Project Reflections……………………………………………………………… 30
4
References……………………………………………………………………….. 34
Appendix A
Bioreactor Design……………………………………………………………...
38
Appendix B
Figure 1. Stomach’s Structural Components………………………………... 39
Table 1: pH for Optimum Activity for Each Stomach Enzyme…………….
39
Figure 2: pH Ranges Through the Human Digestive Tract………………...
40
Figure 3. Flowchart of the Digestive Process………………………………...
40
Figure 4. Artificial Stomach…………………………………………………..
41
Figure 5. Continuous Bioreactor Operation Modes (with time)……………
41
Figure 6. Continuous Bioreactor Operation Modes…………………………
41
Figure 7. Turbulent Impellers………………………………………………..
42
Figure 8. A Dual-Membrane Hollow-Fiber Reactor………………………..
42
Figure 9: Serial CSTRs with Step by Step Feed A…………………………
43
Figure 10: PFR with Lateral Feed B…………………………………………. 43
Figure 11: Flow Pattern in a Batch Bioreactor with the Help of Baffles…..
43
Table 2. Decision Matrix………………………………………………………
44
Figure 12: Bioreactor Stages………………………………………………….
44
Figure 13: Gantt Chart Fall Semester………………………………………..
45
Figure 14: Gantt Chart Spring Semester…………………………………….
45
Table 3. Work Plan Fall Semester……………………………………………
46
5
Title: Simulation of a Human Stomach as a Bioreactor
Problem Statement: Due to rising costs involved with pharmaceutical testing, interest
in the development of artificial organs has increased. The team’s focus will be on the
simulation of an artificial stomach as a bioreactor. The design of the artificial stomach
allows for a better understanding of how protein drug molecules are absorbed in the
human stomach.
Connection to Contemporary Issues: Drug testing on animals is a controversial issue.
Animals are often abused and most are euthanized at the end of testing. Also, the test
subjects and the potential drug users have inconsistencies in the components of the
stomach which could lead to unexpected and adverse side effects while the drug is still in
the testing phase. Currently, proteins, such as vaccines, are taken subcutaneously and are
unable to be taken orally without the protein being degraded in the stomach. {More of the
problem than CI}
Scope of Work: The goal is to create a bioreactor design that mimics the stomach’s
processes. The bioreactor design should be able to be used by pharmaceutical companies
for testing of new oral protein drugs, which will increase the drugs’ public appeal,
efficiency in distribution, and reduce the amount of money spent on testing. Some
deliverables will include a flow diagram outlining the drug’s progression through the
digestion process, a bioreactor process flowchart, an economic analysis, sensors to
monitor the bioreactor’s simulated stomach environment, and a final bioreactor design.
The key measurable outcome will be a measurement of the percent of the original protein
broken down during digestion, which will measure the success and subsequent accuracy
of the bioreactor. The deliverables, listed above, will present the client with a bioreactor
that mimics the stomach’s digestive process. Clients will be able to use the bioreactor to
test new oral protein drugs, negating the use for animal and human testing. The client
will also receive a process flowchart of both the human and bioreactor’s digestive system
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to better help them understand the process and an economic analysis to show why the
bioreactor is economically more feasible than human and animal testing.
Deliverables:
1. Flow diagram of the stomach’s processes
2. Bioreactor functioning as an artificial stomach {How would this be tested?}
3. Sensors to monitor the stomach’s environment (pH, temperature, etc)
4. Mass balance
5. Economic analysis
Introduction: Currently all protein drugs are taken administered subcutaneously.
However, there are issues associated with contaminated needles, the invasiveness of
needles, the difficulty distributing vaccinations in underdeveloped countries, and the
costs associated with the administration of shots. Currently, there is research for new oral
protein drugs to replace the need for drug injection. The current problem is when
proteins go through the digestive system, the majority is denatured and broken down in
the stomach as a result of the stomach’s harsh environment, the physical breakdown by
muscles, and the enzymatic activity targeting proteins. Team Proteinnovate is creating a
bioreactor design to simulate the stomach’s digestion of proteins that can ultimately be
built and tested using oral protein drugs. During digestion, 98% of proteins are broken
down in the stomach. The design challenge is to design a bioreactor that will yield the
same percent of protein breakdown after the simulated digestion. A bioreactor intended
for this purpose does not currently exist; the bioreactor most similar in scope of work to
our design is a bioreactor created by British scientists that mimics the stomach’s digestive
system. While their design successfully simulates the whole digestion process, our
design focuses mainly on protein digestion, making it specifically for the testing of oral
protein drugs.
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Design Criteria and Constraints: Some of the performance criteria include accuracy,
sensitivity, maintenance, and user-friendliness. Accuracy is defined by how well the
bioreactor follows the stomach’s process, i.e. 98% protein digestion. Our bioreactor
cannot produce too much physical breakdown as opposed to chemical breakdown; which
is shown as the bioreactor’s sensitivity towards the protein drugs. Due to the possibilities
of clients not having background knowledge in bioreactors, our design needs to be easy
to maintain and user-friendly. Other criteria include HCl, enzymes, and sensors to
monitor the pH and temperature of the bioreactor, smooth muscle simulation, and a
separation system. Design constraints include specific pH values during certain stages of
the digestion simulation and a temperature of 37 ºC. A successful design will need to
have a 98% protein digestion so that when clients input an oral protein drug, the stomach
will accurately simulate protein digestion. In order to be cost effective, the design will
need to cost less than $1 M. This number is in comparison to the $1 M that the British
stomach bioreactor cost and the $0.5 M to $1.5 M spent on animal testing per chemical.
Besides the above criteria, as yet, there are no additional design specifications.
Literature Review:
Stomach
Physical Environment
The stomach is only a part of the gastrointestinal system, which breaks down
particles of ingested food. Gastric juice is mainly composed of hydrochloric acid in the
stomach, which results in an environment with a pH that falls between 1 and 3 (Carter,
2004). The average temperature of the human stomach is approximately the same as the
body temperature of 37 ºC (Harvard Medical School, 2008). The stomach and its
different parts are depicted in Appendix B under Figure 1.
Waves of smooth muscle contractions along the stomach wall, known as
peristalsis, break food down into smaller pieces, mix it with the gastric juices produced
within the stomach lining, and move it through the stomach (Bowen, 2002). There are
three major regions in the stomach: fundus, corpus, and antrum. The fundus maintains
relatively constant intraluminal pressures with variable volumes and regulates the
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emptying of liquids, while the antrum mixes liquids and grinds solids. The antrum is
more important in the emptying of ingested solids than of liquids (Smout et. al., 1980).
Epithelial tissues line the gastrointestinal tract (Louvard et. al., 1986). Spatial
asymmetry creates a concentration of certain cell-receptors at one pole or at the side of a
cell, creating polarization on the surface (Louvard et. al., 1986). This polarization and
differentiation among the different areas on the surface of the cells is associated with a
specific set of enzymes and antigens (Louvard et. al., 1986). The enzymes and domains
of the epithelial cell are then associated with a specific cell function (Louvard et.
al.,1986). Two types of junctions are found within the epithelial cells: the gap junction
and the tight junction (Louvard et al., 1986). Gap junctions are essential in that they
allow the passage of small molecules through them (Louvard et al., 1986). Three
structures are found within the gap junction that helps to create a chemical composition
that assist in preventing the diffusion of various proteins (Louvard et al., 1986). The
other junction associated with the epithelial cells is that of the desmosome which serves
somewhat as a staple that keeps the cells joined (Louvard et al., 1986).
Chemical Environment
The stomach environment is occupied by several different enzymes. Gelatinase is
a gastric enzyme which helps in the digestion of meat. Gastric amylase contributes to the
breakdown of starch, although it is of minor significance. Gastric lipase breaks down
tributyrin (almost exclusively), which is a butter fat enzyme. Rennin digests milk protein
into peptides (SAPN, 2006).
Protein-digesting enzymes break the protein down into peptide fragments within
the stomach. In order to function properly, the contents of the stomach need to be at a pH
around 2 (Champbell, 1987). Most proteins have a fairly stable tertiary structure, making
them difficult to be broken down by proteases (Foltmann, 1986). To prevent pepsin
from digesting the cells that make it, the cells needs to be synthesized into their inactive
pepsinogen form. Low stomach pH and hydrochloric acid levels are required to convert
pepsinogen into pepsin. Pepsin can only break proteins into shorter chains called
polypeptides. It cannot break the proteins completely down into amino acids. Trypsin and
chymotrypsin are produced in the pancreas for the purpose of further breaking the
polypeptides down in the small intestine. Each enzyme has a specificity towards certain
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amino acids. Pepsin will only cleave to the N-terminal NH group that contains aromatic
amino acids (phenylalanine and tyrosine) and the C-terminal Co group of the dipeptidyl
unit (Tang, 1977). It will not cleave to bonds containing valine, alanine, or glycine.
Mucus, as one of the components that makes up gastric juice, is a “slimy”
material that protects the epithelial surfaces against acids and shear stresses and that is
produced by the mucous cells (Bowen, 1998). According to Forstner and Forstner (1986),
mucus functions as a “trap for the immobilization of enzymes, electrolytes,
microorganisms, products of digestion, and secretions”. Composed primarily of
glycopeptides, large mucin molecules bind together to form this layer of protection
(Forstner and Forstner, 1986). Mucus has several roles in its helping of the digestive
process. One is lubrication, mucus is able to lubricate nearby surfaces making them
easier to shear at the point of contact (Forstner and Forstner, 1986). Another function is
as a permeability barrier. Mucus has gaps in which certain nutrients and electrolytes are
able to pass through (Forstner and Forstner, 1986). In the intestinal tract, it protects the
outer linings for the high acid and is even able to stimulate gastrin mucin secretion
(Forstner and Forstner, 1986).
Hydrochloric acid (HCl), another component of gastric juice, is a strong acid that
makes sure the stomach maintains its acidic condition for proper digestion. HCl converts
pepsinogen to pepsin to break down proteins. The acidity also acts as an important barrier
to avoid infection caused by microorganisms (SAPN, 2006).
There are many important concentrations and rates that must be known in order
for the bioreactor to best be able to simulate the digestive process of the stomach. The
stomach secretes both H+ and Cl- ions against a concentration gradient. So, a great
amount of energy is required for the secretions. The energy to drive these two processes
is obtained from ATP hydrolysis. The concentration of hydrogen ions in the lumen of the
stomach can go up to 1.5*10-1 M. The concentration of chloride ions in the lumen of the
stomach can reach 170 mM (Smith & Morton, 2001). Gastric juice can reach up to 160
mM in the stomach. The gastric emptying rate has a large impact on the rate of drug
metabolism and the absorption of the drugs.
Stomach Concentrations and pH ranges
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Enzymes are only operable within their optimum pH ranges. Table 1, found in
Appendix B, depicts the pH optimums of various enzymes. The hydrogen ion
concentrations can be found utilizing the following formula: pH = -log (H+)
Figure 2 depicts the various pH ranges and the time allotted to each organ of the
digestive tract `The upper stomach has a pH that ranges from 4.0 to 6.5 and the lower
stomach has a pH that ranges from 1.5 to 4.0.
Digestion
Process
The digestive process begins in the mouth with the mastication or chewing of
food while the salivary glands secrete saliva to break down the carbohydrates and
starches. As shown in Figure 2, the nutrients then enter into the stomach where
simultaneously the stomach muscle physically breaks apart the proteins, the pH and
temperature denature the proteins, and specific enzymes target proteins for digestion.
Once leaving the stomach, 98% of the proteins are broken down into peptides to enter
into the small intestine.
Each secretion supplies enzymes, whose function is to permit the hydrolysis or
breakdown of certain dietary proteins, carbohydrates, or lipids (Corring, 1983). The
digestion of proteins begins in the gastric lumen by pepsins and concludes in the small
intestine by pancreatic proteolytic enzymes (Corring, 1983). Carbohydrate digestion is
initiated by salivary alpha-amylase. Lipids are digested in the intestinal lumen by
pancreatic bile salts-lipase complex.
Hydrolysis of protein begins with the action of pepsins secreted in the gastric
juices in the inactive form of pepsinogens. These are activated by the HCl in the stomach
causing the pH to quickly rise from 2 to 4. The proteins become denatured due to the
harsh pH environment of the stomach leading to the formation of polypeptides (Silk,
1983). Intraluminal digestion is due to inactive proteolytic enzymes (trypsinogen,
chymotrypsinogen, procarbodypeptidases A and B, and proelastases). Trypsinogen
becomes active by the enterokinase forming trypsin which then activates the
chymotrypsinogen, procarboxypeptidases A and B, and the proelastases (Corring, 1983).
These cleave the peptide bonds around the L-amino acids. The chrymotrypsin plays a
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significant role in the digestion of protein. After being broken down into small peptides,
the intestinal peptidases are further broken down. The majority of the absorption in
proteins takes place in the proximal jejunum while only small amounts are able to reach
the ileum for absorption (Silk, 1983).
Mechanisms
According to Silk and Keohane (1983), the following are two mechanisms
responsible for protein digestion: “transport of liberated free amino acids by group
specific active amino acid transport systems and the uptake of unhydrolysed peptides by
mechanisms independent of the specific amino acid entry mechanisms”.
Another important player in the digestive process is that of the salivary glands.
Secreting proteins, such as amylase, and mucin, along with water and electrolytes, saliva
has several important functions (Van Lennep et al., 1986). Along with wetting dry
nutrient, protecting mucus against dehydration, peroxidasing, and secreting lysozyme,
saliva is also important in the digestion of enzymes such as alpha-amylase and lingual
lipase (Van Lennep et al., 1986). According to Van Lennep et al. (1986), “the synthesis
of secretory proteins…is stimulated by beta-adrenoceptor activation but activation of
alpha-receptors inhibits synthesis.” Also able to inhibit the synthesis of certain proteins
is cycloheximide; however only when not stimulated with adrenaline or dibutyryl cyclicAMP (Van Lennep et al., 1986). Insulin, on-the-other-hand, is able to stimulate protein
synthesis (Van Lennep et al., 1986).
Starch is broken down by salivary alpha-amylase. The digestion of carbohydrates
also essentially begins in the lumen. The starch is broken down into maltose and other
forms. Starch digestion has its optimum capacity in a pH of 6.9, resulting in the stomach
not having as great of an effect. Some argue about whether or not starch is even broken
down in the stomach (Corring, 1983). Starch digestion mainly occurs in the small
intestine (Corring, 1983). In carbohydrate hydrolysis, brush border hydrolysis and the
use of the monosaccharide transport system is the primary method for digestion in
humans (Silk, 1983).
Lipids are also broken down in the lumen section of the stomach. The pH is also
relatively high for the optimum hydrolysis of lipids, resulting in a majority of the lipid
not being digested in the stomach (Corring, 1983). Chyme in the gastrointestinal tract
12
mixes, resulting in the dissolving of fat-soluble vitamins (Weber, 1983). These fat
droplets are then further broken down in the lumen into mixed micelles (Weber, 1983).
Lipase is essential in the step for the splitting of the dietary triglycerides into long-chain
fatty acids (Weber, 1983). Carboxylesterase and phospholipase are also important in the
hydrolysis of cholesterol ester and phospholipids (Weber, 1983).
Drug Digestion
Oral Delivery
Protein drugs’ efficiency is not maximized when taken orally, because of
protein’s high molecular weight, high hydrophilicity, and the likelihood of enzymatic
hydrolysis (Frokjaer and Hovgaard, 2000). However, an oral delivery of drugs has
caught the attention of some scientists. Due to invasiveness and possibilities of infections
while consuming insulin subcutaneously, some scientists have shown great interest in
oral delivery of insulin. Most of the insulin does not go into the bloodstream because it
gets broken down in the digestive system (Lin et al., 2008). In the study conducted by
Lin and other scientists in Taiwan, chitosan (CS) and poly-γ-glutamic acid (γ-PGA) was
developed to aid insulin to be taken orally. From the gastrointestinal tract to the small
intestines, the pH changes from acidic to alkaline. The authors used sodium
tripolyphosphate (TPP) and magnesium sulfate to make nanoparticles that can survive in
broader pH ranges (Lin et al., 2008).
Frokjaer and Hovgaard (2002) suggest that “the use of protease inhibitors,
adsorption enhancers, chemical modification, and special pharmaceutical formulations
can increase the bioavailability by bypassing enzymatic and absorption barriers”. The
authors indicate that aprotinin, amastatin, bestatin, boroleucine, and puromycin are
inhibitors that can be administered with the protein drugs. The major downfall of
protease inhibitors is that it has an effect on the absorption of other peptides and proteins
that are usually degraded in the system (Frokjaer and Hovgaard, 2000).
Bioreactors
Overview
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Bioreactors are generally characterized as closed containers where biological and
chemical reactions take place. There are a variety of different structures and components
of a bioreactor for organs. Usually, the bioreactors consist of a human cell culture, a
support structure, a port for the inflow of the fluid which is going to go through
processing, a port for the outflow for the processed fluid, a chamber to gather the fluid
which will be processed, a second chamber to collect the processed fluid, two sets of
hollow capillary fiber bundles, one for the fluid coming in to be processed and one for the
processed fluid coming out (Galvotti, 2008).
According to the New England Anti-Vivisection Society (NEAVS, 2008), a
bioreactor is an apparatus for growing organisms such as bacteria, viruses, or yeast.
These are used in the production of pharmaceuticals, antibodies, or vaccines, or for the
bioconversion of organic wastes. The design of a bioreactor utilizes the basic principles
of conservation of mass (stoichiometry) and energy (thermodynamics), and relies on
knowledge concerning the rate at which the process is expected to take place (kinetics). A
typical bioreactor has a control system that monitors the conditions inside the vessel, such
as: mass flow rate, pH, temperature, dissolved oxygen level, gases present (i.e., air,
oxygen, carbon dioxide, nitrogen) and the agitation speed, which is controlled in order to
keep the contents uniformly mixed, and to allow for oxygen transfer (NEAVS, 2008). A
heat exchanger or a cooling jacket is also needed to keep the bioprocess operating at a
constant temperature.
History
In 1886, Dr. Mackenzie simulated the digestion of milk in the human body by
means of a catheter, a vessel which acts as the artificial cavity, and a milk strainer. The
primary purpose of the simulation of this process was to feed sickly patients through their
rectums. Dr. Mackenzie saw that the fluid undergoes digestion in the artificial cavity and
absorption in the rectum. He inserted a celluloid catheter two inches into the anus and
saw that the sphincter closed on the catheter (Mackenzie, 1886). The celluloid turned
soft, after encountering the body heat and could hardly be felt by the patient (Mackenzie,
1886). The catheter is put through a thick piece of India-rubber, before it is inserted into
the patient (Mackenzie, 1886). The India-rubber was made up of attached tapes, which
14
connected around the loins and which were tied fairly close to the anus (Mackenzie,
1886).
Dr. Mackenzie warmed up some milk and allowed it to sit for thirty minutes
before passing it through the strainer into the vessel. He observed that there was no curd
left on the strainer and that after straining, the milk flowed easily through the tubes
(Mackenzie, 1886). The milk in the vessel is elevated by about two feet above the bed of
the patient. It is observed that the milk passes through the rectum in about three hours,
which is an accurate period of digestion for milk (Mackenzie, 1886). The understanding
and construction of this early artificial stomach allows for the basis and inventions of
modern bioreactors.
A group of British scientists were actually successful in designing and
constructing the first artificial stomach in 2006. The chief designer of the artificial
stomach is man named Dr. Martin Wickham (The Assoc. Press, 2006). The artificial
stomach is larger than the size of a desktop computer and cost about $1.8 M to build (The
Assoc. Press, 2006). The two parts that make up the artificial stomach are a funnel and a
metal tube enclosed within a box. The mixture of digestive enzymes, food, and stomach
acids take place in the funnel section of the artificial stomach. The food then moves
down into the silver metal tube section of the stomach, where it is grinded into minute
particles (The Assoc. Press, 2006). Figure 3 depicts the two parts of the stomach.
There is software which controls the parameters of the artificial stomach. Examples of
these parameters include different hormone responses and the time it takes food to remain
in a specific part of the stomach (The Assoc. Press, 2006). The artificial stomach is
particularly unique in that the stomach utilizes specific digestion movements, such as the
contractions of the stomach which serve to break food down (The Assoc. Press, 2006).
This innovation is an important tool that will allow for many breakthroughs in various
researches dealing with the stomach, such as studying the process of nutrient absorption
in the stomach.
Types of Bioreactors
In a continuous bioreactor, the feed rate and product discharge rate or both are
held constant. Chemostat, an alternative name for a single Continuously Stirred Tank
Reactor (CSTR), is the simplest and the most widely applied design. The changes in
15
volume of the culture broth relative to elapsed time for continuous bioreactor can be seen
in figure 4.
When using the Chemostat design, an assumption that the growth rate is
determined by the supply rate of the growth-limiting substrate is made. This single
essential nutrient could be nitrogen, carbon, trace elements, or vitamins (Asenjo, 1995).
Cells are removed at a rate equal to that of their growth rate, and the growth rate of cells
is equal to the dilution rate (Shuler, 2001). According to Center of Applied Catalysts
(2002), a CSTR is “an adaptation of a batch reactor in which the substrate is added
continuously to the reactor while the reaction mixture is removed at the same rate.”
Since the substrate concentration throughout the whole reactor stays the same, the
product yield is usually lower than in a batch process. A batch process is a one step
system. It is the most widely used method in the biopharmaceutical industry because of
its versatility. Changes in volume of the culture broth relative to elapsed time for a batch
bioreactor can be seen in Figure 5.
A typical batch reactor consists of an agitator and a heating/cooling system.
Impellers commonly used in batch reactors are shown below. In a batch reactor, there are
four distinct phases: lag, exponential growth, harvesting, and new batch preparation
(Shuler, 2001). The basic elements that determine a reactor’s productivity, and thus the
size of the bioreactor, are the rate equation (usually includes yield values), cell
concentration, and the reactor’s flow characteristics (Asenjo, 1995).
According to Asenjo (1995), a membrane reactor is a flow reactor within which
membranes are used to separate cells or enzymes from the feed or product streams. In
membrane reactors, feed streams are delivered continuously, while products are removed
continuously, although in some applications they are collected intermittently or at the end
of the run. The most common membranes used in membrane reactors are ultra-filtration
and polymeric microfiltration. Microfiltration membranes have pore sizes between 0.1
and 5 µm, and they are used to confine cells within a reactor without restricting the
passage of soluble nutrients and products (Asenjo, 1995). Ultra-filtration membranes’
pore sizes typically range from 2 to 100 nm and can be used to exclude macromolecules
with molecular weights from 103 to 106 (Asenjo, 1995).
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The most commonly used geometry for membrane reactors can be seen in the
hollow fiber design which is represented by Figure 7. This type of reactor was modeled
after the vertebrate circulatory system, wherein tissues are maintained by nutrients
provided through selectively permeable capillaries. A bundle of hollow fibers is sealed
into a cylindrical shell with epoxy or polyurethane resin. The thousands of hollow fibers
within the reactor provide a large surface area for mass transfer and cell adhesion
(Asenjo, 1995). The downside to using hollow fiber reactors is that cell observation and
harvesting can be problematic (Shuler, 2001).
One of the advantages of the membrane reactor is its ability to accomplish the
initial task of the membranes in a separation process within the body of the reactor.
Selective membranes can be used to facilitate both the removal of inhibitory metabolites
and the recovery of unstable products before degradation (Asenjo, 1995). Undesired
shear effects on protein are not significant except when gas-liquid interfaces are present,
assuming a Reynold’s number of less than 2300 and laminar flow of the fluid (Harrison,
2003). This denaturizing of protein at air-liquid interfaces is commonly encountered in
agitated vessels. This problem can be eliminated in a membrane reactor because the
enzymes are sequestered in a relatively quiescent region where they are protected from
mechanical damage and generally not in contact with air (Asenjo, 1995). The major
disadvantage of this system is the cost of the membranes and the need for their periodical
replacement (Chaplin, 2004).
Safety and Standards
The design project’s products will be a bioreactor and a testable protein drug.
When considering the testing process for this protein and the use of the bioreactor, certain
regulations must be observed. The following paragraphs review some of the pertinent
safety standards for the individuals handling the drugs that must be observed:
Drugs not in final form have additional protocol that must be followed during
the testing process. The Hazard Communication standard will be utilized throughout the
design. This standard is meant to enforce that all chemical hazards are looked at
carefully and the information about the risks of the hazards is sent to the employees and
employers who will be working with the chemicals. The standard also ensures the proper
17
labeling of all chemicals and that all employers make material safety data sheets available
to their employees. This standard will ensure the safety of the people working directly
with the protein drugs.
The Hazardous Waste Operations and Emergency Response standard will also be
followed throughout the design. This standard ensures the proper clean up of hazardous
chemicals, ensuring that the chemicals are removed or contained. If the proper waste
disposal procedure is not followed correctly, the laboratory will be at risk and might have
to shut down. In this case, refer to the TT OSHA Act of 2004, which discusses the
Industry Health and Safety. All chemical waste bottles must include Hazard Form
stickers with the start dates and the bottle contents. Since HCl is a strong acid, the bottle
must be labeled as Hazardous Waste and stored separately so that it may be carried out to
a proper hazardous waste management site. If a chemical is released and is not able to be
controlled quickly, an emergency response occurs, in which the fire department or some
other aid group comes to assist the employees (ACH, 2000).
The Access to Employee Exposure and Medical Records standard will give
employees, as well as the Assistant Secretary Representatives the right to look at medical
records. Access is needed by all of these people to ensure better detection, treatment, and
the prevention of diseases while working with these oral protein drugs.
In addition, the International Society for Pharmacoepidemiology has a set of Engineering
Pharmaceutical Innovation Standards and Practices and Guides that are used by
pharmaceutical and biotechnology professionals. These standards involve guidelines, as
to the testing and creation the drugs which would be pertinent to our design. The
environmental impact of the tested drugs would also need to be considered. The disposal
of the chemicals used in the creation of the drug and the composition of the bioreactor
would need to follow regulations set by the Environmental Protection Agency. The EPA
has numerous guidelines on the safe disposal of various chemicals and compounds.
The Federal Drug Administration also has many regulations for standard drug
quality. Some of the regulations include the Pharmacology/Toxicology standards, as well
as the Food and Drug Administration Amendment Acts of 2007 (CDER, 2003). The
Food and Drug Administration Act gives the FDA authority to regulate drugs, etc. The
team will have to follow the restrictions set forth by the FDA when obtaining drugs for
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the bioreactor. In order to regulate new drugs, there are several trials that the FDA needs
to conduct. The trials consist of three different phases. Phase I consists of determining
the side effects and the testing safety of the drug. Phase II includes the credibility and
dosage range. Phase III consists of a comparison of the drug with a placebo and other
treatments, as well as the side effects on the patient (CDER, 2003). Other sources of
guidelines for drug testing include the U.S. Pharmacopeia, the EP Pharmaceutical
Standards, and other Pharmaceutical professional organizations. Standard laboratory
safety procedures would need to be observed, such as those written by the American
Chemical Society. The handling of hydrochloric acid and other chemical compounds can
potentially be hazardous and safety regulations must be observed.
In addition to required and necessary guidelines that should be followed in the
creation and testing of the pharmaceutical proteins and the bioreactor, certain ethical
issues must be addressed. Since the pharmaceutical’s aim is to eliminate the need for
human testing, that issue commonly addressed is obsolete. The common ethical issue
that is always addressed when introducing a pharmaceutical to the population is that it is
important to consider the side effects and whether the outcome is more important than the
negative side effects the drugs may induce.
Preliminary/Alternative Designs:
In order to design a bioreactor that can simulate the human stomach, the basic
principles that account for the main components of every bioreactor have to be
considered, after which the idea can be expanded to make the design more specific. Since
the environment of the stomach has to be kept constant, the design needs to include a pH
and temperature probe for monitoring, as well as a pH controller system and heat
exchanger or cooling jacket for pH and temperature control respectively. A method of
providing the reactor with gastric juices is also necessary. Gastric juices enable the
chemical breakdown of protein in the stomach, and are composed mainly of hydrochloric
acid (HCl), mucus, pepsin, and rennin. In order to place any proteins being examined into
the reactor and collect the product at the end of the process, there needs to be influent and
effluent openings. The stomach also needs to provide physical breakdown by shear stress
produced between reactor walls. Using a type of biomaterial that can act as a stomach
19
wall was considered, as well as using baffles and a stirrer to produce the same effect.
After the protein has been broken down by gastric juices, there needs to be a way to
measure how much of the protein has survived the harsh conditions. A separation system
will separate the broken peptides from the “intact” protein.
Three alternative designs have been considered for a reactor that could mimic the
human stomach. Each reactor has its own advantages and disadvantages which will help
to decide on which system will work the best for this particular project. The alternative
designs are:
Continuous Bioreactor
In a continuous bioreactor, the feed is put in continuously at the beginning and the
product is then collected at the end of the process. The changes in the volume of culture
broth with elapsed time for a continuous bioreactor can be seen in Figure 9.
The group chose two types of systems for the continuous bioreactor. One of the
systems incorporates a type of mixing device and one does not.
i) Continuously Stirred Tank Reactor (CSTR)
A CSTR is a tank to which reactants are continuously fed and products are
constantly withdrawn. In a CSTR, the tank is continuously agitated to reach a
specific output. To make the system even more efficient, the group considered
designing a CSTR in a series of steps, as shown in the figure below. The feed and
the product flow rate will be kept constant throughout the system, which will
create a simple general mass balance. The concentration of the effluent will be the
same from the beginning to the end. The stirrers in the serial CSTR will mimic the
natural mixing movements found in the stomach. Another important step is to find
the right paddles to use, so that the protein is not broken and biodegraded. The
protein breakdown mostly occurs by chemical means, not physical stress. The
continuous bioreactor is easy to load and unload. The feed goes in and the
product is collected at the end of the line. Since the feed is continuously fed to the
bioreactor, the product yield is a lot higher. However, if one part of the series has
contamination, the whole system will need to be turned off, which in turns creates
a great amount of down time for the industry.
ii) Plug Flow Reactor (PFR)
20
In a PFR, the feed is fed into a continuously straight tube or pipe. Just like
in a CSTR, the feed and product flow rate are assumed to be constant. However,
the reaction rate is inversely proportional to the distance traveled along the tube.
The reaction rate will be substantially higher on the upstream and decrease over
time as it reaches downstream. The flow in PFR only flows in the axial direction
(parallel to the tube), which does not ensure a proper mixing within the reactor.
To overcome this, the feed is put in the direction perpendicular of the stream, as
the figure below indicates.
The maintenance cost of PFR is slightly higher than the maintenance cost
of a CSTR. This can be overcome by placing the reagent at different locations in
the reactor. PFR can run for a long period of time without maintenance, and it
also has a higher efficiency than CSTR. The higher efficiency in a PFR indicates
that the reactor will have a larger percentage of completion in a PFR, than if
conducted in a CSTR. Another modification of PFR is the placement of a
membrane separation system within the tube. As the protein is being degraded by
the enzyme, the membrane can diffuse and separate the peptides with the “intact”
protein.
Batch Bioreactor
A batch bioreactor is a one step system. It is the most widely used method in the
biopharmaceutical industries, due to its versatility. Pharmaceutical companies can
choose a specific protein to test on without drastically altering the principles of the
reactor. This type of process also reduces contamination within the system. Unlike the
CSTR, if something wrong occurs, the bioreactor just needs to be shut down, sterilized,
and the whole process can be repeated. The changes in volume of a culture broth with
elapsed time for a batch bioreactor can be seen in figure below.
A batch bioreactor also contains a stirrer and baffles, which aid in proper mixing.
The baffles are usually placed along the side wall of the bioreactor and are utilized to
ensure uniform mixing. The mixture being stirred can only move in one radial direction.
The purpose of the baffles is to break the flow pattern, as shown in the figure below.
21
The major disadvantage of a batch bioreactor is the need for its’ periodic shutdown and start-up which makes for a loss of production time. Waste can be easily
accumulated within the reactor because the mixture just stays in one tank.
Semi-Continuous Bioreactor
After consideration of both reactors, a combination of the batch and continuous
bioreactors was desired. The human stomach closely resembles a semi-continuous
system where the initial feed is the batch portion and the enzymes and HCl are
continuously fed into the bioreactor making the continuous system essential.
When determining which design to pick, the team used the Decision Matrix found
under Table 2. As mentioned previously, the accuracy is the most important criteria for
our design. It is vital that the design simulates the human stomach as accurately as
possible; this means the stomach bioreactor must have a 98% protein digestion and have
the same environment as the stomach. In the digestive process, a small portion of the
protein digestion is a result of the physical breakdown of the drug. The design cannot be
too aggressive and make the physical breakdown a bigger portion of the digestive process
than the chemical reaction. The design team found the semi-continuous bioreactor the
best option to meet these criteria.
Project Design: The final design of a membrane bioreactor consists of two cylindrical
drums chambers, the first containing enough HCl to produce a pH range of 6.5 to 4, and
the second containing enough HCl for a pH of 1-3, a cooling jacket to ensure a constant
temperature of 37 °C, protein digesting enzymes such as protease and pepsinase, two
agitators for continuous stirring, baffles for even mixing, pH and temperature probes with
controllers, and a detection system that outputs the amount of peptides present after
digestion in comparison to the number of undigested proteins present. The design can be
found under Appendix A: Bioreactor Design. Since the digestive fluids will be
approximately 0.8 liters and the liquid needs to be 80% of the total bioreactor volume, the
bioreactor has a total volume of 1.00 liters. As seen on the bioreactor design in Appendix
A, the sensors will be attached to a computer monitoring the bioreactor and prepared to
counteract any change in the environment by changing the input into the system. The
sensors relaying information can be seen by the dashed lines connecting the sensors to the
computer.
22
The design incorporates a conical bottom to enable easier draining. The content
in the reactor will be recycled through the use of a peristaltic pump by collecting the
liquid at the bottom of the bioreactor and pumping it back to the top. This ensures that no
protein is accumulated on the bottom of the bioreactor. A second agitator is essential to
the design since in a typical 1-liter bioreactor there would be inconsistencies in the
concentrations and temperature of the solution. A second agitator placed slightly lower
than the first agitator will ensure the solution in the conical area will be of the same
concentration as the upper region of the bioreactor. To ensure the pH of the bioreactor
will also be as consistent as possible, there will be two inlets for the HCl. This is more
accurate since in the human stomach there are multiple areas for the digestive acid to
enter.
After the construction of the bioreactor, the client will input the oral protein drug
into the influent opening of the bioreactor. During the very first stage of the reaction, the
drugs will go through a pretreatment process. This process is meant to mimic the
physical breakdown by saliva as the drugs enter the human mouth. The pretreatment will
be very quick at approximately 12 seconds. A block diagram of the whole reaction
process can be seen in Figure 12. The drug will remain in the bioreactor for one hour, or
the average amount of time spent by proteins in the stomach. After digestion, the
membrane separator in the bioreactor will allow the gastric juices and other materials to
flow through while retaining the proteins. The proteins will then be put into a separation
system where the number of peptides versus undigested proteins will be calculated. This
process will take place in a pharmaceutical laboratory under standard laboratory safety
procedures. One assumption will be that the bioreactor will continue having 98% protein
digestion after construction. Another assumption is the amount of protein input into the
bioreactor is in small enough amounts to not interact with the HCl to produce a
significant change in pH or polarity in the bioreactor’s environment.
Different enzymes in the stomach operate under specific pH ranges. The
bioreactor will consist of a series of two stages that operate at different pH values. The
first stage will consist of the lipase, trypsin, and invertase enzymes operating at a pH
range of 4-6.5. After 60 minutes, the reactor will enter stage two by the addition of HCl,
23
creating a pH range of 1.5-4. Figure 14 depicts the two stages with their respective
enzymes.
As mentioned before, HCl will be used. The amount of HCl entering will be
determined by the pH meter. A diluted HCl solution of concentration 0.1 M will enter in
by drops. Since the reaction of the acid with the water is immediate, the pH will rapidly
change. The pH meter is connected to a computer that is also connected to the input of
HCl. Once the pH becomes in the range of 4.5 – 5.5, the computer will shut off the input
of HCl. Once the second stage of the process is begun, the computer will restart the
addition of the HCl until the pH becomes in the range of 2.5-3.5. This is in the middle of
the target range for the second process. A very small diameter of 0.005 m lab hose will be
used to deliver the HCl into the reactor. Since it is necessary to put in the acid in the form
of drops, a small valve will be put in the middle of the hose to control the flow. The valve
is connected to the sensor that sends a signal to the computer which will physically open
and close the valve. As the valve is closed, there will not be any acid flowing into the
reactor. When more acid is needed, the valve will open to allow some acid to go through
and lower the pH of the solution. To ensure the HCl does not react with the air in the
bioreactor, the air will need to be replaced with an inert gas. Since argon is one of the
most cost effective inert gases available, it was chosen for the bioreactor. While the
human stomach does not have argon, the argon will make no difference in the interaction
between the enzymes, pH, and temperature in breaking down the protein. In fact, it will
ensure that no unnecessary reactions will occur. Since the design will use argon, an air
sparger will be placed between the two agitators. The following is the procedure done by
the pharmaceutical company personnel to run the bioreactor:
Start-Up Procedure
Preparation of Media (must be done one lab period before-hand):
For the “Saliva” pretreatment:
In 10mL flask, mix:
-7 mmol/L Sodium Chloride
-10 mmol/L Potassium
24
-1.2 mmol/L Calcium
-2.5 mmol/L Bicarbonate
-1.4 mmol/L Phosphate
-9.5 mL DI water
-Equal part of Mucopolysaccharide, Glycoprotein, Hydrogen Peroxide,
α- Amylase, Lysozyme
Running the bioreactor:
1. Only authorized personnel with proper protective equipments such as rubber
gloves, lab coat, and safety goggles is allowed to operate the bioreactor.
2. Remove the lid and attached hosing from the bioreactor and dump the contents (a
mild water and bleach solution) down the drain. Spray the inside of the reactor as
well as the stirrer, sparger, sample port, etc. with bleach and rinse thoroughly with
DI water. Rinse for as much time as necessary until you no longer smell any
bleach in the bioreactor.
3. Reattach the bioreactor to the mounting base and connect the water jacket hosing.
4. Turn on the water supply to the bioreactor by opening the appropriate valves.
Also check if the cooling water is flowing properly. Connect the 10% HCl tube
into the reactor, make sure that the tube is not clogged and the flow meter is
working properly.
5. There are 2 bioreactors being used. On the first bioreactor, insert the pretreatment
content along with 2 mg of the chosen protein and note the starting time. Replace
the lid, and attach the condenser water in and out of line and attach the
temperature probe and the pH probe.
6. Set the temperature to 37 ºC at the temperature controller and enter pH range 7.5 6.5 at the pH controller. Run the reaction at the lowest speed setting for 12
seconds.
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7. After 12 seconds, open the HCl valve and set the pH range to 6.4 – 4 and run the
reaction for 60 minutes.
8. After 60 minutes, continuously run the solution into the second bioreactor with
the same 37 ºC temperature and set the pH range to 4 - 1.5. Keep the 10% HCl
solution running constantly for 3 hours until the pH enters the set pH range.
9.
To collect samples, first connect a rubber bulb to the sampling port. Press a
sample tube tightly against the rubber seal on the collection port. Close the
sampling valve and squeeze the bulb. Slowly open the valve until a suitable
amount of sample has entered the tube.
Shutdown Procedure
1.
Stop the bioreactor by setting the control field of the agitator, temperature, etc. to
off. Switch off the main power supply.
2. Remove motor, all hosing, and attached probes from the bioreactor. Bring the
reactor to the sink.
3. Discard the contents to the designated drain area. Spray the inside of the
bioreactor including the sample port and all tubes with bleach solution twice and
rinse with water. Once everything has been thoroughly rinsed, fill the bioreactor
with enough water to cover the baffles and add approximately 150 mL of bleach.
4. Return the reactor to its base.
5. Make sure the area is clean for the next protein experiment.
After the digestive process is complete, the fluid inside the bioreactor will be run
through the SDS PAGE which will separate out the proteins by molecular weight. The
larger the protein the further it will travel towards the positive end of the gel following
electrophoresis which will enable the pharmaceutical companies to know what
percentage of the protein is still the original length and what percentage has been broken
down.
Project Evaluation: In promising the clients a dependable, accurate bioreactor that
emulates the human stomach, certain standards have to be met. After the construction of
26
the bioreactor, the physical environment has to consist of a temperature of 37 ºC, a pH
ranging from 6.5 to 1.5 over the course of four hours after the addition of HCl, and a size
of approximately 0.8L. After the input of a protein, the output on the SDS PAGE should
show approximately 98% breakdown of that protein.The economic analysis proves the
bioreactor’s competitiveness in the pharmaceutical market. The bioreactor’s digestive
process is not labor intensive due to a network of monitoring probes relaying data to a
computer which will change input conditions in order to maintain the preset design
criteria. The only labor associated with the process is in the initial construction, the input
of the materials, the cleaning of the bioreactor after completion of digestion, and reading
of the SDS PAGE to determine protein breakdown. While the two bioreactors together
cost approximately $60 K, the monitoring probes combined will cost under $2 K, the
specific computer software will cost minimum of $30 K, and the maintenance and
running costs will be well under $8 K, the total project is estimated to cost approximately
$100 K. This number is in comparison to the only other bioreactor designed to simulate
the human stomach which cost approximately $1 M after construction, fabrication, and
implementation. While the competitor bioreactor has a wider range of functions such as
producing vomit and monitoring all nutrients entering the bioreactor’s “stomach”,
Proteinnovate’s bioreactor design was created for a clientele solely interested in oral
protein drugs for a considerably less amount of money.
Certain areas of the design were speculated due to a lack of knowledge and
published material on oral protein drugs and an inability to construct the bioreactor to do
performance testing. One area of uncertainty was the size of the protein drug
administered. For the mass balance, a mass of 2 mg was used for the input protein since
oral protein drugs are still under development and there is currently no standard mass of
protein typically administered. The assumed 2 mg of protein, if substantially incorrect,
could result in insufficient amounts of enzymes put into the system. Another assumption
made was the amount of protein input into the system would not have a substantial
impact on the polarity or pH of the bioreactor’s chemical environment. If the amount of
protein typically administered were found to be a higher value than what was assumed,
this could result in a change in the stomach’s environment producing errors in the actual
pH of the system.
27
The bioreactor does not come with an air sparger because it is assumed that the
oxygen in the 20% reactor headspace and the dissolved oxygen contained in the water
would be enough to supply oxygen needed for the whole reaction. The group also
assumed that 100% of the feed protein is utilized and there no waste accumulation. With
this assumption, the following mass balance was obtained:
100% protein = 98% peptides + 2% protein
Conclusion/Summary: In order to create a bioreactor that would simulate the human
digestive system, three designs were considered. The CSTR, the semi-continuous, and
the batch bioreactor were all examined as possible reactor designs. The semi-continuous
bioreactor was considered to be the best design after much consideration and
comparisons with the other two reactors. The semi-continuous reactor was found to be
the best representation of the actual human stomach.
The semi-continuous bioreactor was designed with a conical bottom so that it
would be able to mimic digestion. Baffles, a cooling jacket, HCl, and enzymes were
added to simulate the physical and chemical environment of the stomach. The process
occurs in two stages after which the output is put through a SDS PAGE for the separation
of proteins by molecular weight to determine the protein digestion. The economic
analysis shows that the total cost of the bioreactor is around $100 K in comparison to the
only other bioreactor, costing approximately $1 M.
The deliverables that were promised at the beginning of the project included a
flow diagram of the stomach’s processes, a bioreactor functioning as an artificial
stomach, sensors to monitor the stomach’s environment, the mass and energy balance, as
well as the economic analysis. All of the deliverables, except for the energy balance,
were completed. The energy balance was not completed because the design is
theoretical, the group will not be able to physically test the accuracy of the bioreactor, so
it will be hard to determine the amount of energy needed to increase/decrease
temperature, etc. If the project were to be implemented, a possible environmental effect
would be that the waste would need to be distributed to the proper places, since strong
28
acid would be utilized. Team Proteinnovate’s bioreactor will hopefully be built
sometime in the future so that pharmaceutical companies will be able to reduce the cost
of testing as well as to reduce the invasiveness involved with needles.
Work Plan – Timeline – Design ScheduleSept. 25 - Finish and submit scope of work
Sept. 30 - Team meeting to possibly set a focus
Oct. 9 - Revision of cover page and scope of work
Submit project notebook
Oct. 23 - Submit Cover Page, Scope of Work, and References.
Nov. 6 - Turn in Project Notebook 2
Nov. 12 – Group Meeting: Revise Literature Review and Safety Standards
Nov. 13 - Submit Cover Page, Scope of Work, Resources, Safety, Regulatory, and
Environmental Considerations and Work Plan
Nov. 18 - Team meeting to discuss alternative designs
Nov. 20 - Turn in 3-Project Notebook, Finish and Submit Draft of Final report, Group
Meeting for outline update
Dec. 2 - Work on oral presentation and discussion.
Dec. 4 - Group Oral Presentation, Project notebook due
Dec. 16 - Revise final report
WINTER BREAK
Jan. 20 – Group meets to discuss new timeline and add necessary changes
Jan. 25 – Group discussion on gastric juice and research protein drugs
Jan. 26 – First class meeting of spring semester
Jan. 27 – Division of workload (chemical composition, protein drugs, paper editing)
Jan. 29 – Group meeting
Jan. 31 – Discussion of groups finding from previous research
Feb. 2 - Project notebook for spring semester due
Feb. 5 – Research on sensors
Feb. 7 – Research protein recovering method
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Feb. 9 – Find exact concentration of stomach’s chemical composition
Feb. 13 – Discussion on bioreactor choice (multiple reactor in series or one reactor)
Feb. 14 – Completing midterm project report
Feb. 16 – Midterm project report due/Jenn’s presentation to team on sensor choices/
decide on sensors
Feb. 19 – Begin mass balances/energy equations
Feb. 23 - 2-Project notebook due/Finish mass balances/energy equations – individual
research to begin on separation systems
Feb. 26 – Discussion and decision matrix on different types of separation systems found/
decide on system
Mar. 2 – Work on final design report/conclusion and final design
Mar. 5 – Meet with advisor to discuss progress
Mar. 7 to Mar. 15 – SPRING BREAK
Mar. 16 – Cost/Economic analysis to be completed
Mar. 19 – Work on midterm oral presentations
Mar. 23 - 3:30-6:30 midterm presentation
Mar. 30 - 3-Project notebook due
April 1 – Group meeting
April 20 Draft of final report (not graded) due
April 27 - May 1 Poster Presentation of final report
May 4 Final Project notebook due.
May 7-12 - Individual oral exam
May 13 - Final report approved, graded, and signed by advisors.
The summary of the above dates is depicted below in our team’s Gantt chart found under
Figure 15. The team added more items to the timeline compared to the fall timeline. As
the project progressed, there were many unforeseen objectives that had to be completed.
These objectives include instrumentation research, bioreactor design using CAD, and
economic analysis. Researching inputs took longer than expected due to team’s
uncertainties regarding the reaction that may take during the bioreactor operation. After
consulting with multiple professors, Proteinnovate’s project came to a conclusion on
30
March 16 which is a month and a half later than the fall timeline in Table 3. The other
objectives were completed on time. The fall semester timeline and Gantt chart can be
found in Table 3 and Figure 15 respectively.
Project Reflections:
Kristen Pevarski: Overall, the project was a success. One of the things our group and I
learned early on was the need for time management. We had team meetings but there
were many times we got together as a team and got very little accomplished. I didn’t
realize at first that even for a meeting you have to do some preliminary work to make
sure the meeting runs smoothly and the work gets completed. I found that when we all
split up and did our individual parts then met again twice a week to go over what
everyone had done, things went much smoother. If I had to do this over again, I would
also ensure that we had better communication with our advisor. We were behind
schedule many times and got our reports or drafts into our advisor late, giving him very
little time to offer us constructive feedback. Other groups I talked with mentioned they
had met with their advisor at least once a week and I feel like that would have been a
good idea for us to make sure that we were on track. This is more in relation to what
happened the first semester when there was miscommunication and it turned out our
project focus was entirely off. I also feel like it would have been more efficient with
fewer people. Overall, I feel like it was a very positive experience and I have gained
invaluable insight into how I work and communicate with others and vice versa.
Megha Maheshwari: I greatly enjoyed this class for it allowed me to better understand
the design process and to utilize what I have learned over the past four years to create a
suitable design. I learned a great deal about the different components of the design
process, such as coming up with a suitable problem statement, actually constructing the
design and coming up with alternative solutions, while keeping the cost in consideration.
This design was a fairly new concept for the team because none of the members were
very familiar with proteins and their breakdown. A great deal of research had to be
conducted on protein drugs and the digestive system, in order to figure out how to best
simulate the stomach within a bioreactor, so that protein breakdown could be tested. So,
I also had the opportunity to learn a great deal about the different protein drugs and about
31
the components of a bioreactor. The team has amazing dynamics. All of the team
members were committed to the design and we worked well together. We kept up with
all of the tasks that were assigned to us periodically throughout the year. The design
helped to improve my time management skills because it required a great deal of work
that had to be completed on certain deadlines. If I were to do this project again, I would
love to actually build it because I think the building would allow us to better test the
design. Right now, the design is just theoretical. Some project management items that I
would do differently would maybe be to do less research and actually concentrate more
heavily on the design, because I feel the whole first semester was just dedicated to
research. In closing, I greatly enjoyed this class and my team dynamics. I know it will
help me in the future.
Veni Avenida: We have been doing this Stomach Bioreactor project for two semesters
and I learned so much about working with a group of people throughout the process. We
had to learn about each other’s learning styles and we had to learn to work with different
schedules and opinions. This project was fairly new and there were not a lot of references
to use, so we had to brainstorm quite often and develop new ideas. I think we did a very
good job with the project considering the limited resources that we had. The project itself
taught me how to think critically and it gave me chances to apply some of the technical
knowledge that I have gained from the BSE department. If there are things that I could
change however, I would love to actually build an actual bioreactor so that we can get the
laboratory experience. Also, I feel like some of the classes that are being taught during
this Spring semester should have been taught a little earlier to prepare us better for the
project. It would have been nice if we could actually contact a person from an actually
pharmaceutical industry as a co-advisor so that we would know if our design is feasible
for the industry or not. If I could go back and change our management item, it would be
to finish all the goals in our timeline. We did not get a chance to do an energy balance or
the design layout using a SuperPro. However, our design is not being jeopardized by not
completing these goals. Overall, the project was a success. I learned a lot in term of
working with other team members; managing my time better, and most importantly, now
I know how to apply my BSE knowledge to solve a real problem.
32
Jennifer Kim: Senior design offered a different experience compared to all the other
group projects. Since the design project lasted a whole year, we were able to fix most of
the mistakes we made during the first semester. During the first semester, I only met
with the team members once a week. Every time the team had to turn a project item in, I
felt rushed. Since there are four members, it was very difficult to schedule a meeting due
to time conflicts. We managed to overcome this by splitting into two smaller groups
during the spring semester. The team operated more efficiently this way. Furthermore,
to resolve the issue of feeling overwhelmed, we met at least twice a week. Our frequent
meetings helped me accomplish more and get more feedback from other members. I
think better communications between team members and advisors would have helped
during the design process. In the beginning of the semester, our team’s focus was
completely different than what our advisors expected of us. Also, there were a few times
when team members got confused as to where to meet. In the future, I will make sure
that there is a clear understanding of expectations between supervisors and coworkers.
I wish our plant design took place during the fall semester. It provided a great
amount of information regarding instrumentation, designing a plant, and cost analysis. If
we had learned about it sooner, we would have been able to provide a more detail and
accurate cost analysis. Nevertheless, we were able to add information as the plant design
class went on. We were also able to get feedback from Dr. Agblevor on our
instrumentation.
If I had to redo this process, I would choose a team with fewer members or try to
find a more efficient way to work with many people. Also, I would have liked to have a
project that we could actually build. During in-class presentations and poster
presentations, I envied the fact that there were groups that were trying to solve problems
by visiting sites and growing bacteria to see if their project was feasible. During the first
semester, we took a wrong path. I ended up doing a lot of research on different drug
delivery methods. Although it was irrelevant to our final project, I learned so much from
research. Overall, I enjoyed the experience. Our group overcame our time constraints. I
got a feel for what working with others would be in the real world. I also learned a lot of
chemistry, biology, and CAD design from other members.
33
References:
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37
Appendix A
Bioreactor Design:
1. Control computer
2. Temperature transmitter
3. Temperature probe
4. pH probe
5. pH Indicator controller
6. Stirrer motor
7. Stirrers
8. Baffles
9. Level sensor
10. Cooling jacket
11. Cooling jacket
12. Level transmitter
13. Peristaltic pump
14. Flow transmitter
15. Valves
16. HCl
17. Pressure transmitter
18. Cooling water
19. Air filter
20. Air sparger
38
Appendix B
1. Body of stomach
2. Fundus
3. Anterior wall
4. Greater curvature
5. Lesser curvature
6. Cardia
9. Pyloric sphincter
10. Pyloric antrum
11. Pyloric canal
12. Angular notch
13. Gastric Canal
14. Rugal folds
Figure 1. Stomach’s Structural Components
Table 1: pH for Optimum Activity for Each Stomach Enzyme
Enzyme
Lipase (stomach)
Pepsin
Trypsin
Urease
Invertase
Maltase
Catalase
pH Optimum
4.0-5.0
1.5-1.6
7.8-8.7
7.0
4.5
6.1-6.8
7.0
39
Figure 2: pH Ranges Through the Human Digestive Tract
Input
Food
Mastication/Saliva breakdown of starches
Stomach
Muscle/
physical
breakdown of
food
pH and
temperature
denature
proteins
Enzymatic
Activity/
breakdown of
proteins/
carbohydrates
Output
Nutrients
Figure 3. Flowchart of the Digestive Process
40
Figure 4. Artificial Stomach
Figure 5: Continuous Bioreactor Operation Modes
Figure 6: Continuous Bioreactor Operation Modes
41
Figure 7: Turbulent Impellers
Figure 8: A Dual-Membrane Hollow-Fiber Reactor
42
Figure 9: Serial CSTRs with Step by Step Feed A
Figure 10: PFR with Lateral Feed B
Figure 11: Flow Pattern in a Batch Bioreactor with the Help of Baffles
43
Table 2: Decision Matrix to Determine a Preliminary Design
Design
Weight (%)
Continuous
Semi-
(CSTR)
continuous
Batch reactor
Accuracy
30
15
30
20
Versatility
25
15
25
25
Cost
20
5
5
15
Maintenance
15
5
10
10
User-
10
10
10
7
100
50
80
77
Friendliness
Total
44
Figure 12: Bioreactor Stages
8/28/2008
10/17/2008
12/6/2008
1/25/2009
3/16/2009
Research
Problem Statement
Literature Review
Alternative Designs
Safety and Environmental Standards
Research Inputs
Find Separation Apparatus
Test Alternative Designs
Find Solution
Final Report
Deliverables
Figure 13. Gantt Chart Fall Semester
45
Figure 14. Gantt Chart Spring Semester
46
Table 3: Work Plan (Fall Semester)
Sept. 25 - Finish and submit scope of work
Sept. 30
Oct. 9 - Revision of cover page and scope of work
Submit project notebook
Oct. 23 - Submit Cover Page, Scope of Work, and References.
Nov. 6 - Turn in Project Notebook 2
Nov. 12 – Group Meeting: Revise Literature Review and Safety Standards
Nov. 13 - Submit Cover Page, Scope of Work, Resources, Safety, Regulatory, and
Environmental Considerations and Work Plan
Nov. 18
Nov. 20 - Turn in 3-Project Notebook, Finish and Submit Draft of Final report,
Group
Meeting for outline update
Dec. 2 - Work on oral presentation and discussion.
Dec. 4 - Group Oral Presentation, Project notebook due
Dec. 16 - Revise final report
WINTER BREAK
Jan. 20 – Group meets to review the project status update.
Jan. 26 – First class meeting of Spring semester
Jan. 29 – Group meeting
Feb. 2 1 - Project notebook for Spring semester due
Feb. 9 - Group meeting
Feb. 10 - Work on midterm project report
Feb. 16 - Finish midterm project report; revised and signed final report
Feb. 19 – Group meeting
Feb. 23 - 2-Project notebook due
Feb. 26 – Work on project work plan
Mar. 2 - Revise work plan
Mar. 7 to Mar. 15 – SPRING BREAK
Mar. 16 – Group meeting
Mar. 19 – Work on midterm oral presentations
Mar. 23 - 3:30-6:30 midterm presentation
Mar. 30 - 3-Project notebook due
April 1 – Group meeting
April 20 Draft of final report (not graded) due
April 27 - May 1 Poster Presentation of final report
May 4 Final Project notebook due.
May 7-12 - Individual oral exam
May 13 - Final report approved, graded, and signed by advisors.
47