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Penicillin Production

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Kenzie Dupont, Gracie Kennard, Tim King, Fischer Robinson, Trey Schexnayder
April 11, 2019

Abstract:
Penicillin, the active ingredient produced by many fungi of the genus Penicillium, is an
antibiotic that uses an enzyme-inhibiting mode of action to prevent the growth of the
target cell wall. Discovered accidentally by a Scottish biological researcher, Alexander
Fleming, penicillin soon became the most widely used antibiotic in the world. The
production of penicillin occurs in a multi-stage, batch-fed process that occurs over
several days. The process begins by going through a growth and fermentation phase, in
which media is inoculated, the culture is grown, and penicillin is eventually produced.
The penicillin must then be isolated, extracted, and polished through a series of
downstream procedures. Each step is discussed in greater detail. By the end of the
process, a crystalline-powder of concentrated penicillin is formed.
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Table of Contents:

Simple Flow Chart 2

Objective: 3

Introduction 3

Unit Procedures 4
Penicillium chrysogenum Culturing/Growth Phase 4
Fermentation 4
Purification 8
High Performance Liquid Chromatography 8
Solvent Sublation 9
Adsorption 9
GAC Adsorption 9
Ion Exchange 10
Affinity Adsorption 10
Precipitation 10
Polishing 11
Crystallization 11
Drying 12

Chosen Methods 12
Culturing/Growth Phase 12
Fermentation 13
Removal of Insolubles 14
Extraction 14
Purification 15
Adsorption 15
Precipitation 15
Washing 16
Crystallization 16
Drying 17

Cost Analysis 18

Economic Evaluation Report 19

Conclusion 20

SuperPro Flowchart 22

References 23

Contributions 26
2

Simple Flow Chart


P. chrysogenum
culture
7 days @ 24 °C

P. chrysogenum
growth phase
(aeration)

1-2 days

Fermentation
pH = 6.5, 25-26 °C
Calcium Chloride
Polyelectrolyte Filtration Fungal Biomass

pH 2-3, 0 < T < 3 °C

Amyl/Butyl Acetate Extraction Solvent

Water
Acetone Precipitation
Potassium/Sodium

Isopropanol or Butanol Washing Solvent

Drying/Crystallization

Penicillin G
-Potassium/Sodium
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Objective:
The objective of this project is to create a process that will produce penicillin at a
concentration of at least 50 grams per liter. This process will occur over a period of eight
days.

Introduction
Penicillin was discovered 91 years ago as the first naturally occurring antibiotic
(Kalvaitis, 2008). Alexander Fleming, a Scottish biological research scientist and
pharmacologist, was the man deemed responsible for the discovery of the new, life-
saving drug (Alexander Fleming, n.d.). Fleming noticed a halo around a fungus that had
fallen onto a plate of bacteria, where the bacteria did not grow. He later identified the
mold to be part of the genus Penicillium, and named its active ingredient penicillin
(Gaynes, 2017). Due to lack of resources, Fleming could not fully develop and produce
the penicillin antibiotic. The first successful treatment of the new antibiotic occurred in
1942. When news that penicillin was effective, it became mass-produced and used
during WWII, decreasing the death rate from 18% to less than 1% (Bradford, 2017).
Penicillin has since become the most widely used antibiotic in the world
(Kalvaitis, 2008). It has been recorded to prevent the growth of Gram-positive
pathogens by inhibiting bacterial enzymes from constructing cell wall growth (Bradford,
2017). Penicillin cured diseases such as syphilis, gonorrhea, tuberculosis, gangrene,
pneumonia, diphtheria, and scarlet fever, saving at least 200 million lives upon its
discovery (Alexander Fleming, n.d.)
Though penicillin has worked in curing many diseases, many groups of bacteria
have now become non-susceptible to this antibiotic over the years. Streptococcus
pneumoniae remains the most commonly identified cause of community-acquired
pneumonia, even in comparison to the recent emergence of pathogens identified today
(Thomas et al., 2006). Historically, medical professionals prescribed penicillin to treat
Streptococcus pneumoniae without being concerned with how susceptible this
bacterium would be to the penicillin. But in the early 1990’s, there was a sharp increase
in the prevalence of resistant strains of S. pneumoniae to penicillin. Studies showed that
between the years of 1990-1991, prevalence among penicillin-nonsusceptible S.
pneumoniae was ~18% and was almost 34% by 2003 (Thomas et al., 2006.). Despite
many strains of bacteria becoming less susceptible to penicillin, it does not necessarily
mean that the bacteria is penicillin resistant. In order to further decrease the penicillin-
susceptible organisms, medical professionals should be mindful of the antibiotic
selections prescribed to patients. This can be best achieved by using low-resistance
potential antibiotics oral/intravenous mono-therapy at the full recommended dose
(Cunha, 2002).
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The main purpose of this study is to focus on the bioprocess through which
penicillin is manufactured. In order to promote product formation of penicillin over
biomass production, most industrial production processes operate in the manner of
multi-stage fed-batch modes (Posch et al., 2014). Batch processes are widely used in
order to produce low volumes of high value-added products (products that involve a
change of form to increase its value), such as antibiotics (Luo et al., 2016). All penicillin
processes are operated as batch fermentations, in which a volume of sterile medium in
a vessel is inoculated. The broth is fermented for a set parameter; the tank is then
emptied and the products are separated to obtain the final product, penicillin G
(Najafpour, 2015). With the production of penicillin G comes a host of other subsidiary
components, which must be separated through the process of filtration. Since beta-
lactamase-producing microorganisms have the ability to destroy or seriously damage
the penicillin, this process must be carried out without the contamination of the filtrate by
enzymes (Najafpour, 2015). In order to isolate the penicillin, the process of solvent
extraction, also known as liquid-liquid extraction, is typically used. Typically, the process
would then go through a purification step in which penicillin is further refined to separate
it from potential contaminants. Due to the effectiveness of the filtration step, no specific
step for purification will be needed. The penicillin is then combined with water and
ammonium salts to allow for precipitation in the form of ammonium penicillin. Finally, the
penicillin is washed, dried, and crystallized, yielding a product of extremely high purity.

Unit Procedures

Penicillium chrysogenum Culturing/Growth Phase


Penicillium cultures are typically grown using submerged culturing. In submerged
culturing, the fungi are grown below the surface of the media. Through consistent agitation
and aeration, the Penicillium chrysogenum will begin to grow homogeneously as small,
suspended colonies (Foster et al., 1946). Under these conditions, the fungi should have
optimum conditions that will accelerate the rate of growth of penicillin (Foster et al., 1946).
Conversely, improper agitation can negatively affect penicillin growth. Improper agitation
speeds can lead to the formation of pellets in the media and can potentially damage the
mycelium.
Additionally, the selection of a Penicillium strain will alter the final yield of
penicillin. Penicillium notatum was initially cultured to produce penicillin, but resulted in
an extremely low yield of 1 mg/L. Through the incorporation of a different species,
Penicillium chrysogenum, penicillin yields are typically around 50 g/L. Researchers have
also isolated individual mutant strains of Penicillium chrysogenum that contain physical
properties that lead to higher yield of Penicillin, such as the “selection of strains that
contain additional copies of the biosynthesis cluster that encodes the three key enzymes
for penicillin production: Using δ-(l-α-aminoadipyl)- L-cysteinyl-D-valine synthetase,
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isopenicillin N synthase, and isopenicillin N acyltransferase” (Weber et al., 2012). At the end
of the growth phase, the culture is ready for fermentation.

Fermentation

The fermentation process for penicillin production is typically executed as a


batch-fed, aerobic process that occurs in a batch fermenter. Penicillin is a secondary
metabolite, which means that it is produced at the highest rate when the growth phase
stops and enters the stationary phase (Meštrović, 2018). The composition of the media
is important in order to produce penicillin at a high yield. A key component of the
composition is a source of carbohydrates. Several “carbon sources have been adopted
for [fermentation] – including glucose, sucrose and other crude sugars” (Meštrović,
2018). Carbohydrates are used primarily for cell maintenance and eventual penicillin
production. Sugars can also be used to regulate the pH during the active penicillin-
production phase (Meštrović, 2018). Small variations in the percent composition of the
media can greatly affect the final yield of penicillin.

Figure 1. Graph of penicillin production.

Another factor to consider when deciding on the fermentation process is the state
of the media. Solid state fermentation is a process in which fermentation occurs on a
solid media and the metabolites are excreted into the surrounding media. In contrast,
liquid state fermentation involves the culture suspended in a liquid or broth. During the
production of penicillin, solid state fermentation “permits improved control of culture
conditions” which can improve yield and reduce production time (Barrios-Gonzalez et
al., n.d). Solid state fermentation has also shown an increase in efficiency of
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carbohydrate utilization and an increase in volume productivity (Barrios-Gonzalez et al.,


n.d.).

Removal of Insolubles
Filtration is a process of separation in which solid, undissolved, suspended
particles are removed from liquids by passage through a porous medium. As the feed is
being put through the process of filtration, the solid particles are collected on the filter
medium, and the deposit increases in depth, forming an accumulation of solute called a
filter cake. Filtration usually occurs in the early stages of purification of bioproducts
(Harrison et al., 2003).
Ultrafiltration is a beneficial form of crossflow filtration. An experiment performed
by Orr et al. used ultrafiltration in combination with anion-exchange membrane
chromatography to determine if the time and cost of the combination of processes
would be reduced. Their findings determined that the crossflow filtration process
removed oligopeptides and gave a 25-fold reduction when using a 3,000 Da membrane.
Nitrogen was used to pressurize the device to 25 psi. The low pressure decreased the
cost of production. However, the concentrate was highly polarized upon production.
Also, membrane fouling was a problem in the production process, but could be fixed by
either reversing the direction of the flow or increasing the flow rate (Orr et al., 2012).
An experiment run by Tessier et al. tested the separation capacity of ultrafiltration
and nanofiltration combined. The efficiency of three different ultrafiltration membranes
(5,000, 30,000, and 100,000 Da) as well as a nanofiltration membrane (300 Da) was
tested. Ultrafiltration removes emulsions, while nanofiltration reduces the ionic charge in
the broth. The ultrafiltration 30,000 Da and 100,000 Da membranes had an extraction
efficiency of around 90%, however, emulsions formed. The 5,000 Da ultrafiltration
membrane only yielded an 81% extraction, but was free of emulsions, giving it a higher
solvent extraction yield of 94.6%. The nanofiltration 30,000 Da membrane in
combination with the ultrafiltration 100,000 Da membrane lead to a recovery yield of
98%, but emulsions were again present, reducing the yield efficiency to about 81.3%.
The nanofiltration step after the 5,000 Da membrane ultrafiltration lead to no emulsions
and a 92.4% extraction yield. Tessier et al.’s method of filtration reduced the operating
pressure from an average of 145 psig to 25-80 psig, which reduced energy costs. The
lower pressure also lessened the occurrence of the fouling phenomenon, which was a
problem that was encountered throughout the experiment. The membrane in
nanofiltration is more porous than that of the membrane used in reverse osmosis, which
reduced the operating time (Tessier et al., 2005).
Diafiltration is a filtration method that is becoming more popular in experiments.
Diafiltration is a pressure-driven membrane filtration process where the solvent is added
to the process liqueur to aid in the separation of undissolved particles of various sizes
(Kovács, 1970). Nabais and Cardoso used ultrafiltration with diafiltration to filter a
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penicillin broth. They discovered that this method effectively filtered broths by removing
proteins, impurities, and colored substances, which allowed for a high yield of permeate.
They found that four volumes of discontinuous diafiltration followed by concentration of
30% PDFB lead to the recovery of 94.3% penicillin (Nabais et al., 1999). Cyganowski,
another scientist, agrees that diafiltration is very effective. However, Cyganowski
continues to explain in his article that although diafiltration can have as much as 98%
yield, it can also cause a product dilution anywhere from 15-30%, which could cause for
major error in production (Cyganowski, 2015).

Extraction
Extractions can be completed in differing methods depending on the target.
Solid-liquid, liquid-liquid combinations, or newer alternatives for extractions can be used
to select for particular antibiotics. The success rate of the extraction process can be
directly attributed to the compounds involved that can affect the molecular structure of
the product. Obtaining a high yield of penicillin can be difficult due to the instability and
high solubility of the molecule itself with an organic solvent that can make it difficult to
recover in solutions (Hossain et al., 2008). Four types of organic solvents that have
been implemented include: amphiprotic (i.e. 1-hexanol), hydrogen-bond acceptor
solvents (n-butyl acetate), hydrogen-bond donor solvents (i.e. chloroform), and apolar
aprotic inert solvents (i.e. cyclohexane) (Lee, 2008). The physical extraction of penicillin
was long-used but proven to be inefficient and not a very marketable means of
obtaining penicillin.
Over the years, penicillin was originally extracted physically using butyl acetate.
Using this method can lead to a yield of 85% max which is still quite inefficient. Physical
extractions of penicillin require it to be in an undissociated state, and for this to happen
the pH must be around 2.0 (Hossain et al., 2008). Such a low pH can result in an
unstable penicillin that is even more difficult to recover from the solvent. To improve
yield, liquid-liquid combinations can be used since they allow for the reactions to take
place with greater stability and selectivity for the desired product. For this reaction
combination, a carrier molecule that’s soluble in an organic phase is introduced to
penicillin G that is soluble in the aqueous phase. The complex that forms is soluble in
the organic phase. Ideal carrier molecules include secondary and tertiary amines that
can be chosen based on preference as each has its drawbacks and reactionary
benefits. These amines allow for higher pH’s to increase penicillin’s stability; however,
secondary amines, such as Amberlite LA-2, are prone to react with the solvent even
though they produce the highest yield. The distribution coefficient of a reaction is an
indicator for how much of the compound was converted from one phase to another. This
coefficient for tertiary amines is notably smaller than that of secondary amines (Hossain
et al., 2008). The latest extractions from Hossain and Dean’s experiment revealed a
combination of an amine carrier with a solvent, such as Shellsol TK or tributyl
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phosphate, can greatly increase efficiency of the extraction. This method, in


combination with a recycle mode where both feed and organic solutions are recirculated
to further increase this extraction percentage. Since penicillin is a weak acid, it can be
reacted with water readily to dissociate. The reaction with secondary amines uses a 2:2
ratio of penicillin G to carrier molecule to produce the complex used in extraction
(Hossain et al., 2008). Low pH’s induce a greater H⁺ concentration that allow for
penicillin to be in the undissociated phase needed to form the penicillin-amine complex.
The direct use of ionic liquid media in aqueous two-phase systems is becoming
an increasingly accepted means for extractions. The hydrophobic compound
[Bmim]PF₆ was tested and identified to have the highest efficiency for extraction in Liu
et al.’s experiment. The success of extractions can be directly attributed to parameters
for the aqueous solution that include: pH, initial concentration of antibiotic, and phase
ratio of the solution with the hydrophobic liquid. Extraction efficiency (EE) for the
aqueous solution was significantly deemed to range from 1.5-3 with extractions
performing best at a pH of 2 with an extraction efficiency of 91.5%. An initial
concentration ranging from 4.1-6.1 x 10⁴ units/mL and a backward extraction agent of
potassium bicarbonate, and phase ratio of 1.5-2.0 can be modified to maximize yield for
this method. [Bmim]PF₆ works so well since it has stronger intermolecular interactions
with penicillin G than water with penicillin G. Extraction efficiency can be improved by
using multiple stage extraction that can be seen as a more logical option since the
proteins remaining from the fermentation broth are not left denatured (Liu et al., 2015).

Purification
The purification process can be utilized for two principal reasons: to isolate the
desired product from any possible contaminants, and to refine the product if proven to
be too difficult to initially crystallize. Normally, the purification step is performed during
the final stages of downstream processing since it requires highly sophisticated
equipment which can prove to be extremely expensive. Depending on the effectiveness
of the filtration step, it is possible for the process of purification to converge with the
crystallization process, surrendering the use for the particular, expensive equipment that
would typically be necessary. In regards to beta-lactam antibiotics such as penicillin,
there are various methods in which the purification process can be carried out.

High Performance Liquid Chromatography


Utilized beginning in the 1970s, high performance liquid chromatography (HPLC)
has proven to play a key role in ascertaining the purity of beta-lactam compounds (Miller
et al., 1983). High performance liquid chromatography typically involves a series of
pumps that pass a pressurized solvent (referred to as the “mobile phase” of the column)
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through a column holding a solid adsorbent material (referred to as the “active phase” of
the column). The temperature and composition of each material and the means in which
they interact are important in the purification process, with these interactions mainly
consisting of hydrophilic bonds, ionic bonds, and/or dipole-dipole bonds. Penicillin can
be separated on a 5 μm Inertsil C8 analytical column, 250×4 mm2, at room temperature,
with a mobile phase of 0.1% TFA/ACN 50:50 v/v delivered isocratically at a flow rate of
1.1 mL/min (Samanidou et al, 2007).

Solvent Sublation
If in a fermentation broth, the penicillin can be separated and purified through the
method of solvent sublation. Solvent sublation is a process that is utilized to remove
complex compounds, hydrophobic compounds, and any other active components in
natural products to produce the highest yield of pure product (Bi et al., 2009). There are
various methods in which this process can be carried out. In one method, a 200-mL
aliquot of fermentation broth (consisting of glucose, inorganic salt, penicillin G, and P.
chrysogenum) was adjusted to pH 3.5 with HCl solution in a 300 mL beaker, and then
transferred to a flotation cell. Through the process of bubbling nitrogen gas at 40
mL/min from the bottom of the flotation cell, the penicillin was able to float at the top.
This sample was then extracted into an n-octanol solution containing 50% BA (v/v) on
the surface of the sample solution. This entire process is performed at room
temperature (Bi et al., 2009).

Adsorption
The adsorption process involves the adhesion of ions and/or molecules from a
liquid or gas to the surface of a solid, known as an adsorbent. The main goal of
adsorption is to secure the solute onto the surface of the adsorbent and discard the
excess material, leaving the resulting product to advance to the next step in the
downstream process.

Granulated Activated Carbon Adsorption


Granulated Activated Carbon adsorption utilizes activated carbon as its
adsorbent since the low-volume pore design allows for a larger surface area to aid in
the adsorption process. In order for this process to reach its maximum yield, the
adsorption process is typically performed at approximately 27 °C. This specific process
is mainly used in water treatment due to its success in adsorbing soluble and organic
compounds, which makes it highly valuable in the pharmaceutical industry as a means
of cleansing certain antibiotics and vitamins for maximum yield.

Ion Exchange
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Ion exchange adsorption is the process by which the ions of one particular
substance are replaced by similarly charged particles from a different substance. This
involves a column made of charged beads that form a gel matrix. In order to carry out
the separation, the charge of the column must be the opposite of the molecules that are
to be separated from the sample. As the sample passes through the column, the
charged molecules in the sample bind to the gel matrix and allows for the product to
flow through. Ion exchange is mainly used in the food industry to separate proteins from
foods to test the effects of individual macronutrients on health (Loughnane, 2007).
Although this process can prove to be successful when it is utilized, it was not the best
fit for the development of penicillin as it does not play a primary role in the
pharmaceutical industry.

Affinity Adsorption
Affinity adsorption involves highly-specific bio-molecular interactions in order to
separate a singular compound or molecule from a tested sample. This process involves
the interaction between a specific solute and ligand, a molecule that binds to a central
metal and forms a coordinate complex, to a product that then binds to the ligand (Odde,
1997). Similar to the ion exchange, affinity adsorption usually involves a bead-filled
column which the sample flows through and allows for the adsorbent material to latch
onto the column while the rest of the product flows through to the end of the column.
However, due to the limited availability of affinity adsorbents and their excessively high
price, this was not the best method for this process (Wang, Palanki, and Hyatt, 1989).

Precipitation
Penicillins are typically a class of antibiotics that are highly soluble in water.
Small amounts of water with the presence of an organic solvent can typically be used to
crystallize penicillin salts. Salivar et al. found that using a ratio of soluble ammonium
salts with tolerable concentrations of penicillin salts will yield a significant amount of
penicillin in return that will be in the form of ammonium penicillin. Since ammonium
penicillin can be removed easily from a mixture, it has been deemed the most logical
choice of salt to use. Ammonium salt concentration can influence the solubility of
penicillin contingent on the species used. Other precipitants can be used but ammonium
sulfate is likely the only feasible option as it can be vacuum dried and still produce a
large amount of ammonium penicillin since it is still able to be removed easily (Salivar et
al., 1948). The precipitate, once dry, can be added to a dioxane mixture as a solvent
that contains about 10% water.
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Polishing
Polishing is the last step in the idealized design process, ending with a final
product that is stable and can easily be packaged in a convenient form. It also refers to
the process needed in order to put the bioproduct, in this case penicillin, into its final
form which will be intended for use. There are two main procedures which can be
classified within the inclusive step of polishing itself: crystallization or drying. Both can
be used in the process of polishing for beta-lactam antibiotics.

Crystallization
During the method of crystallization, crystals are produced from the
homogeneous phase, which is always in a solution. The usefulness of crystallization is
to produce bioproducts with a very high purity level ~ 99.9%. For some bioproducts,
such as antibiotics, the final form of the bioproduct must be crystalline, and sometimes it
is necessary that a specific crystal from be obtained (Harrison et al., 2003). Because
crystallization is capable of producing a high purity level, it can be considered part of the
purification process, which in turn can replace a purification step such as
chromatography.
The method of crystallization begins with an already purified substance that is
aimed at establishing the most stable formula of the product in crystalline form. The
commercial benefit of this method would be to decrease the volume of the bulk
substance and also to reach a degree of stability that enables the storage of the product
(Wp., 2009). The main goal of crystallization is to achieve a small number of crystals
with sizes ranging from 0.2-0.9 mm and internal quality (Harrison et al, 2003).
Because penicillin polishing is the main focus, batch crystallizers will provide the
smallest impact financially. Batch crystallizers have the advantages of being flexible to
operate requiring less capital investment than continuous crystallizers (Harrison et al.,
2003). Batch crystallization is different from continuous crystallization in that the
withdrawal of crystal product for the batch system is made only once at the end of the
batch run and it is most widely used in the production of pharmaceuticals, like beta-
lactam antibiotics or penicillin (Wey et al., 2002). Generally, crystallization of
pharmaceutical drugs from a solution is conducted in a batch or semi-batch crystallizer
because it reduces the solubility of the solute in the solution and creates
supersaturation of the product. The solubility, which is the thermodynamic equilibrium
concentration of the solute, can be reduced by lowering the temperature, removing the
solvent (through evaporation), by adding antisolvent (drowning out), by reactive
crystallization (precipitation), or by adjusting the solution pH (isoelectric precipitation)
(Rohani, 2010). In the pharmaceutical drug industry, cooling crystallization provides the
most economical means but it is only applicable if there is a large enough temperature
sensitivity of the solubility. Though cooling crystallization is economically effective, it
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does not produce a high enough yield and it must be combined with other crystallization
techniques.
Another commonly used crystallization method in the pharmaceutical industry is
the method of micro-crystallization. Micro-crystallization is used when the final product
is needed in pulmonary applications. This is because this method produces crystals with
a size of 1 to 5 microns and can be applied to inhaler drugs. Crystallization from the
solution is used extensively during the synthesis and purification of active
pharmaceutical ingredients and its main goal is to produce tightly controlled crystal
properties such as crystal size distribution, crystal purity, polymorphic outcome, and
morphology (Rohani, 2010). Producing a high yield of penicillin is of paramount
importance which is why the use of a crystallization method is needed in this process.

Drying
Drying is the last procedure in the purification step of the idealized process. This
method thermally removes volatile substances in order to yield a solid. A common
reason for using drying in the procedure is to increase convenience of the final product.
In the production of antibiotics, this is useful because it allows for the final form of drugs
to be in a tablet form. The main dryers that generally have the greatest use in biological
processes are vacuum-shelf dryers, batch vacuum rotary dryers, freeze dryers, and
spray dryers.

Chosen Methods

Culturing/Growth Phase
Penicillium chrysogenum will be used as the inoculum for the production of
penicillin during this process because it is shown to have higher penicillin yield than
Penicillium notatum. A slant tube containing a wheat bran nutrient solution will be
inoculated with the P. chrysogenum culture and allowed to incubate at 24 °C for one
week. The culture is then transferred to inoculum tanks where the culture is grown for 1-
2 days in the presence of oxygen with consistent agitation. This step is included in the
process because it aids in producing elevated levels of mycelial growth, the vegetative
part of the fungus that produces mycotoxins.

Raw Materials:
● Culture of Penicillium chrysogenum
● Slant tube
● Wheat bran solution
● Oxygen
● Agitator
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Fermentation
The fermentation process for penicillin production will use a batch-fed, aerobic process
that occurs in a batch fermenter. In order to produce a penicillin concentration of the
highest yield, the media will contain the following:
● Corn steep liquor (Carbon source)
● Yeast extract (Nitrogen source)
● Minerals
● Phenylacetic acid (Precursor for Penicillin G)
The fermentation will be carried out using cultures that are submerged in the media
under a sterile air supply. A sterile air supply prevents potentially-contaminating
microorganisms from entering the fermentation tank. The temperature should be
maintained between 25-26 °C and the pH should be adjusted to around 6.5 by adding
phosphoric acid, a pH buffer. This buffer is added to the media because it helps
maintain the pH level around 6.5, an optimal level for penicillin growth. To keep the
process in a high-yield state, 20-40% of the culture will be removed and replaced with
new, sterile media (Meštrović, 2018). It has shown that repetition of this step can
actually increase the final yield of penicillin. As the carbohydrate source dwindles, the
culture growth slows, and ammonia is released, the pH level of the medium will begin to
climb until it becomes basic (around pH 8) and penicillin production will stop (SumiGK,
2017). The penicillin is excreted into the medium below the surface and must be
extracted via filtration.

Mass Balance Equation:


100 g/mol Corn Liquor + 18.02 g/mol P. chrysogenum + 136.15 g/mol Phenylacetic
acid + 180.16 g/mol Yeast extract → 17.031 g/mol Ammonia + 417.3 g/mol Penicillin

Table 1: SuperPro parameters for the fermentation of penicillin.

Cycle Duration Design Pressure Temperature

7.83 hours 1.52 Bar 25 ℃

Raw Materials:
● Corn steep liquor
● Yeast extract
● Minerals
● Phenylacetic Acid
● Phosphoric Acid
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Removal of Insolubles
A combination of ultrafiltration and nanofiltration will be used for the separation
and purification of penicillin. Together, the two processes have proved to be beneficial
in reducing cost as well as operation time, two very important factors in the production
process (Tessier et al., 2005). For both filtration methods, the parameters were
assumed based off of the findings during research. For ultrafiltration, the feed is a
combination of the fermented penicillin and calcium chloride, which formed larger
particles that could more easily be captured in the filter membrane. The fungal biomass
waste was discarded. The nanofiltration process’ feed was the filtrate from the
ultrafiltration step. The waste products were the remaining impurities from the fermented
penicillin. The parameters of the removal of insolubles can be seen in Table 2.

Table 2: SuperPro parameters for the removal of insolubles in penicillin.


Membrane Area Pore Size Cycle Duration Critical Temperature

Ultrafiltration 16.76m 2 .45 microns 11.25 hours 27∘C

Nanofiltration 13.4m 2 .045 microns 8 hours 26∘C

Raw Materials:
● Calcium Chloride

Extraction
The extraction of penicillin will use a membrane contactor. The membrane
contactor enables the differing phases to remain in contact while being separate at the
same time. For this, the contacting substances will be the “feed” solution, that contains
the penicillin, with an organic amine compound that’s dissolved in a solvent (Hossain et
al., 2008). The “feed” solution is penicillin that has dissolved in water. The pH will be
between 3.0-5.5. The organic amine-solvent solution used will be Amberlite LA-2 with
tributyl phosphate. There should be a 2:2 ratio of penicillin to the amine carrier to react
and form the complex necessary (Hossain et al., 2008). A hollow fiber module has a
region where penicillin molecules can be transported and allowed to react with the
organic solution and diffuse across pores. Allow for 1 hour to ensure it reaches steady
state then recirculate the solutions to flow near 4 L/s to allow for a greater extraction
yield (Hossain et al., 2008). This membrane contactor method was not available in
SuperPro; however, the “Centrifugal Extraction” method produced a similar output. The
centrifugal extractor works by using a rotor to mix the solvent solution of amyl acetate
with the feed solution and the mixture will react similar to the membrane contactor
method, but the liquids are separated according to density. Amyl acetate’s properties as
15

an organic solvent allow it to be substituted for the organic amine-solvent solution listed
above. This method was input into SuperPro and the cycle took 6 hours to complete
while yielding a rated throughput of 312 L/hr. Other operating parameters for this
method can be seen in Table 3 below.

Table 3: SuperPro parameters for extraction of penicillin.


Cycle Duration Rated Throughput Operating Pressure Critical Temperature

6 hours 312 L/hrs 1.01 Bar 26.4 ℃

Raw Materials:
● Amyl Acetate

Purification
Since the processes of ultrafiltration and nanofiltration prove to be especially
effective, no specific step for purification will be needed. This will ultimately save both
time and money in the downstream process of penicillin.

Adsorption
The addition of activated carbon into the adsorption process allows for a larger
internal surface in order to provide a shorter cycle duration per batch. Activated carbon
in itself, specifically granulated activated carbon, which was utilized in this procedure,
has the ability to adsorb many soluble substances, such as yeasts, poly-aromatic
hydrocarbons, and various fermentation products. The GAC adsorption in this process
was able to separate our penicillin from contaminants such as excess carbon and yeast
extract in order to prepare our product for the precipitation and polishing phase.

Table 4: SuperPro parameters for the adsorption of penicillin.

Cycle Duration Particle Density Particle Diameter Critical Temperature

0.38 hours 1030 g/L 5 mm 27 ℃

Raw Materials:
● Activated carbon

Precipitation
Obtaining a precipitate of penicillin from the solution could best be completed
with the use of ammonium sulfate as the salt. This salt should be used with ammonium
hydroxide and water to make a solution. Once the penicillin is added to the solution,
16

stirring for approximately 15 minutes is all that is needed to form the precipitated
penicillin (Salivar et al., 1948).

Table 5: SuperPro parameters for precipitation of penicillin.


Cycle Duration Critical Temperature Mass Flow Rate

13.5 hours 25℃ 600 kg/hour

Raw Materials:
● Ammonium Sulfate

Washing
After the process consideration of precipitation, the intermediate step of washing
is required. Washing is done to remove any undesirable ions that could still be present
post-precipitation. Liquids used must not interfere with the content of the precipitate.
The washing of the product with either isopropanol or butanol produces a waste product
(solvent) and leads to the crystallization and drying of penicillin to produce bioproduct in
its final form. Isopropanol is effective for the washing as it takes up particles that are
more soluble in the solution than the precipitate. Even though washing is recognized as
an intermediate step, the method used to for washing was the bulk flow method. Bulk
flow is the process of fluids down a temperature or pressure gradient. The parameters
for the bulk flow washer are represented in Table 6 below.

Table 6: SuperPro parameters for intermediate washing step.


Cycle Duration Critical Temperature Volumetric Flow Rate

½ hour 25 ℃ 66.7 liter/hr

Raw Materials:
● Isopropanol

Polishing

Crystallization
The desire to achieve the following is what led to choosing the method of
crystallization for processing penicillin: (1) sufficient product purity to meet established
quality standards, (2) isolation of the chose crystal form, which is the most
thermodynamically stable form, (3) a high yield, (4) good volume productivity with final
slurry concentrations typically targeted for 10 ± 5 wt %, and (5) reasonable batch cycle
time for crystallization (Variankaval, 2008). Typically, factors (1), (2), and (3) are
required to ensure the safety of the patients who will have direct contact with the drug.
17

Crystallization of penicillin represents a convenient and scalable method to polish the


drug substance. Because of its very high purity percentage (~99.9%), it can also be
accounted for in the purification process. This allows for a conversion of both the
purification and polishing step, which saves process timing and money in whole. In
order to promote the growth of crystals, the optimum pH for the solution was to be at
around 5. Table 7 below identifies the remaining parameters required in order to
promote crystallization. Based on the review of different literatures found that included
details of the process of producing penicillin, assumptions were made for the
parameters with best judgement.

Table 7: SuperPro parameters for crystallization of penicillin.

Cycle Duration Pressure Evaporation Crystallization


Temperature Temperature

6.08 hours 12.06 bar 100 ℃ 25 ℃

Drying
For the use of penicillin, the freeze drying process was the drying procedure
chosen to ensure the final product was at the intended quality. The freeze dryer requires
both the temperature and pressure be controlled during the entire drying process. The
product being dried can be dried using vials or trays, which is why this drying process
works for penicillin production. Pharmaceutical freeze dryers can dry a liquid product
contained in vials. When the vials are placed on trays, the stoppers are closed only
partially so that water vapor has the opportunity to escape (Harrison et al., 2003). There
is a hydraulic piston attached which allows for the stoppers to completely push into the
vials at the end of the drying process and then a heat transfer fluid is circulated through
the trays to provide temperature control of the vials (Harrison et al., 2003). Freeze
drying provides the advantage for delicate, unstable or heat-sensitive drugs to be dried
at low temperatures without damaging their physical structure. Figure 2 below
represents a pharmaceutical freezer dryer which can be used during the process of
producing penicillin. It also allows the freeze-dried products to be reconstituted quickly
and easily, which is valuable in the case of emergencies for vaccines and antibiotics,
like penicillin, that need to be administered as quickly as possible (Cuddon, 2015).
Choosing the method of freeze drying lead to the advantages of removing 95%-99.5%
of water from our penicillin G which helps increase preservation time and provided
minimal changes in the properties of the product. The process itself took 15 hours to
complete due to the fact that water must slowly be removed in order to prevent any
damage to the product. The remaining process parameters for the method of freeze
drying are represent in Table 8 below. With the use of these parameters, the process
successfully produced the desired product of penicillin G.
18

Figure 2. Pharmaceutical Freeze Dryer

Table 8: SuperPro parameters for freeze drying of penicillin.

Cycle Duration Tray Area Final Temperature

15 hours .64 square meters 12 ℃

Cost Analysis
Per the Economic Evaluation Report, the total capital investment for penicillin production
was $24,940,000 with an obtained revenue of $1,726,000 per year. Gross margin is the net
sales revenue minus the cost of goods sold. The gross margin here is -198.78%. This indicates
a negative amount of revenue retained for every dollar sold. This could be combated in future
evaluation reports by using more cost efficient goods that would be able to produce a greater
output or by minimizing the amount spent on good investments.
19

Economic Evaluation Report


20

Conclusion
Using the four main steps of the idealized engineering process, a concentration
of 50 g/L of penicillin was desired to be produced within an eight-day period through a
batch process. A batch process was ideal for the production of penicillin, as it yields low
volume, high-value products. The fungus Penicillium chrysogenum was the main raw
material for this process which had to be cultured and fermented over a nine-day time
frame before beginning the main batch process. For the removing of the insolubles,
calcium chloride was added through an ultrafiltration process before the filtrate was sent
to the nanofiltration system. Both filters were used for their cost efficiency and
decreasing operating time. With this filtration, fungal biomass and impurities was
produced as waste and the remaining reagents lead into the extraction procedure. In
order to correctly extract the reagent, amyl/butyl acetate was added through a
membrane contactor extraction method because it provides the highest yield. The next
step of the process was purification, but because purification of penicillin would include
the use of chromatography, an expensive process, crystallization replaced it. The
crystallization step produced penicillin at a purity level of ~ 99.9%, confirming that the
purification step could be omitted. The last steps focused on precipitation, where water
acetone and potassium was added to flow into the washing phase. Though washing is
not part of the main steps of the idealized process, it was an intermediate step which
called for the washing of the penicillin product with isopropanol or butanol. Freeze-
drying was the very last step of penicillin production. The freeze-drying method was
used because it allowed for penicillin to be placed at very low temperature without
damaging the physical structure. It also allows for the penicillin to be easily accessible in
case of emergencies. Using the parameters mentioned throughout this paper, the
21

process was successfully mapped and executed in SuperPro Designer. It was found
that the initial objective was exceeded, producing 83.46 grams per liter in approximately
3 days (68.54 hours).
22

SuperPro Flow Chart


23

References
Alexander Fleming. (n.d.). Retrieved from
http://www.newworldencyclopedia.org/entry/Alexander_Fleming

Barrios-González, J., Tomasini, A., Viniegra-González, G., & López, L. (n.d.). Penicillin
production by solid state fermentation. Retrieved from
https://link.springer.com/article/10.1007/BF01027575

Bi, Pengyu, et al. (2009) “Separation and Purification of Penicillin G from Fermentation Broth
by Solvent Sublation.” Separation and Purification Technology, 2nd ed., vol. 65, Elsevier
Science B.V., 2009, pp. 228–231.

Bradford, A. (2017). Penicillin: Discovery, Benefits and Resistance. Retrieved from


https://www.livescience.com/58038-bacteria-facts.html

Cuddon Freeze Drying. (2015). Retrieved from


https://www.cuddonfreezedry.com/pharmaceutical-freeze-dryng/

Cunha, B. A. (2002). The Clinical Relevance of Penicillin-Resistant Streptococcus


Pneumoniae. Retrieved February 19, 2019, from https://academic-oup-
com.libezp.lib.lsu.edu/cid/article/42/6/798/286393

Cyganowski, J. (2015). Ultrafiltration for Bioprocessing (H. Lutz, Ed.). Ultrafiltration in


Bioprocessing, 244.

Foster, J. W., Woodruff, H. B., & McDaniel, L. E. (1946). Microbiological Aspects of Penicillin:
IV. Production of Penicillin in Submerged Cultures of Penicillium Notatum. Journal of
bacteriology, 51(4), 465-78.

Gaynes, R. (2017). The discovery of penicillin—new insights after more than 75 years of
clinical use. Emerging Infectious Diseases.

Harrison, Roger G. Todd, Paul Rudge, Scott R. Petrides, Demetri P. (2003). Bioseparations
Science and Engineering. Oxford University Press. Retrieved from
https://app.knovel.com/hotlink/toc/id:kpBSE00003/bioseparations-
science/bioseparations-science

Hossain, M. M., & Dean, J. (2008). Extraction of penicillin G from aqueous solutions: Analysis
of reaction equilibrium and mass transfer. Separation and Purification Technology,
62(2), 437-443. doi:10.1016/j.seppur.2008.02.012

Kalvaitis, K. (2008). Penicillin: An accidental discovery changed the course of medicine.


Endocrine Today.

Kovács, Z. (1970). Diafiltration. Retrieved from


https://link.springer.com/referenceworkentry/10.1007/978-3-642-40872-4_171-5
24

Lee, S. C. (2009). Extraction equilibria of penicillin G in four different types of organic solvent
systems. Journal of Industrial and Engineering Chemistry, 15(3), 403-409.
doi:10.1016/j.jiec.2008.12.009

Liu, Q., Li, Y., Li, W., Liang, X., Zhang, C., & Liu, H. (2015). Efficient Recovery of Penicillin G
by a Hydrophobic Ionic Liquid. ACS Sustainable Chemistry & Engineering, 4(2), 609-
615. doi:10.1021/acssuschemeng.5b00975

Loughnane, Sara. “Ion Exchange Chromatography.” Science Learning Hub, 16 Nov. 2007,
www.sciencelearn.org.nz/resources/2046-ion-exchange-chromatography.

Luo, L., Bao, S., Mao, J., & Tang, D. (2016). Quality prediction and quality-relevant monitoring
with multilinear PLS for batch processes. Chemometrics and Intelligent Laboratory
Systems.

Meštrović, T. (2018). Penicillin Production. Retrieved from https://www.news-


medical.net/health/Penicillin-Production.aspx

Miller R.D., Neuss N. (1983) High-Performance Liquid Chromatography of β-Lactam


Antibiotics. In: Demain A.L., Solomon N.A. (eds) Antibiotics. Handbook of Experimental
Pharmacology (Continuation of Handbuch der experimentellen Pharmakologie), vol 67 /
1. Springer, Berlin, Heidelberg

Nabais, A. M., & Cardoso, J. P. (1999). Purification of benzylpenicillin filtered broths by


ultrafiltration and effect on solvent extraction. Bioprocess Engineering, 21 (2), 157.

Najafpour, Ghasem D. (2015). Biochemical Engineering and Biotechnology (2nd Edition) - 11.
Production of Antibiotics. Elsevier.

Odde, D. J. “Affinity Adsorption.” SpringerLink, Springer, Dordrecht, 1 Jan. 1997,


link.springer.com/chapter/10.1007/978-94-009-1563-3_4.

Orr, V., Scharer, J., Moo-Young, M., Honeyman, C. H., Fenner, D., Crossley, L., Chou, C. P.
(2012). Simultaneous clarification of Escherichia coli culture and purification of
extracellularly produced penicillin G acylase using tangential flow filtration and anion-
exchange membrane chromatography (TFF-AEMC). Journal of Chromatography B,900,
71-78.

Posch, A. E., & Herwig, C. (2014). Physiological description of multivariate interdependencies


between process parameters, morphology and physiology during fed-batch penicillin
production. Biotechnology Progress.

Rohani, S. (2010). Applications of the crystallization process in the pharmaceutical industry.


Frontiers of Chemical Engineering in China, 4(1), 2. Retrieved from https://search-
ebscohost-com.libezp.lib.lsu.edu/login.aspx?
direct=true&db=edb&AN=50120640&site=eds-live&scope=site
25

Salivar, C. J., Grenfell, T. C., & Brown, E. V. (1948). Studies on the naturally occurring
penicillins. II. Precipitation of crystalline ammonium penicillins. JOUR BIOL CHEM,
176((2)), 977–981. Retrieved from http://libezp.lib.lsu.edu/login?
url=https://search.ebscohost.com/login.aspx?
direct=true&db=boh&AN=BACD19492300014260&site=eds-
live&scope=site&profile=eds-main

Samanidou, V. F., Nisyriou, S. A. and Papadoyannis, I. N. (2007), Development and validation


of an HPLC method for the determination of penicillin antibiotics residues in bovine
muscle according to the European Union Decision 2002/657/EC. J. Sep. Science, 30:
3193-3201

SumiGK. (2017). Fermentation of Penicillin Antibiotic. Retrieved from


https://www.generalmicroscience.com/industrial-microbiology/fermentation-penicillin-
antibiotic/

Tessier, L., Bouchard, P., & Rahni, M. (2005). Separation and purification of benzylpenicillin
produced by fermentation using coupled ultrafiltration and nanofiltration technologies.
Journal of Biotechnology.

Thomas M., Tan, et al. “Clinical Relevance of Penicillin-Resistant Streptococcus Pneumoniae:


A New Perspective.” OUP Academic, Oxford University Press, 15 Mar. 2006,
academic.oup.com/cid/article/42/6/798/286393.

Variankaval, N., Cote, A. S., & Doherty, M. F. (2008). From form to function: Crystallization of
active pharmaceutical ingredients. AIChE Journal, 54(7), 1682-1688.

Wang, Henry Y., et al. “Application of Affinity Adsorption in Thienamycin Fermentation.”


SpringerLink, Springer-Verlag, 1989, link.springer.com/article/10.1007/BF00263996.

Weber, S. S., Polli, F., Boer, R., Bovenberg, R. A., & Driessen, A. J. (2012). Increased
Penicillin Production in Penicillium chrysogenum Production Strains via Balanced
Overexpression of Isopenicillin N Acyltransferase. Retrieved from
https://aem.asm.org/content/78/19/7107

Wey, J., & Karpinski, P. (2002). Batch Crystallization. Retrieved February 19, 2019, from
https://www.researchgate.net/publication/279436168_Batch_crystallization

Wp. (2009). Healthcare industry. Retrieved from https://www.gesundheitsindustrie-


bw.de/en/article/news/protein-crystallisation-an-attractive-alternative-to-protein-
purification/
26

Contributions
 Kenzie Dupont wrote the history of penicillin and process considerations of
filtration and chosen methods of removal of insolubles, as well as edited the
paper post-midterm.

 Gracie Kennard wrote about the relevance of penicillin in the introduction,


washing, the process considerations for polishing including crystallization and
drying, and the chosen methods for crystallization/drying and the conclusion.

 Fischer Robinson wrote about the culturing, growth, and fermentation of


Penicillium chrysogenum. He also wrote the abstract for the paper and created
the simple flow chart for the process.

 Tim King wrote about and compared the methods of extraction that would
produce the highest yield, wrote about the chosen method of precipitation used,
worked on the washing
step. He also wrote about
the cost analysis from the
economic evaluation report.

 Trey Schexnayder wrote


about how extraction can be
used to improve penicillin
recovery in the introduction,
frequently used methods of
purification and its
importance in the
downstream process, and
the chosen method for
purification. He also worked
on the SuperPro flowchart.
27

References
Alexander Fleming. (n.d.). Retrieved from
http://www.newworldencyclopedia.org/entry/Alexander_Fleming
Barrios-González, J., Tomasini, A., Viniegra-González, G., & López, L. (n.d.). Penicillin
production by solid state fermentation. Retrieved from
https://link.springer.com/article/10.1007/BF01027575
Bi, Pengyu, et al. (2009) “Separation and Purification of Penicillin G from Fermentation
Broth by Solvent Sublation.” Separation and Purification Technology, 2nd ed.,
vol. 65, Elsevier Science B.V., 2009, pp. 228–231.
Bradford, A. (2017). Penicillin: Discovery, Benefits and Resistance. Retrieved from
https://www.livescience.com/58038-bacteria-facts.html
Cuddon Freeze Drying. (2015). Retrieved from
https://www.cuddonfreezedry.com/pharmaceutical-freeze-dryng/
Cunha, B. A. (2002). The Clinical Relevance of Penicillin-Resistant Streptococcus
Pneumoniae. Retrieved February 19, 2019, from https://academic-oup-
com.libezp.lib.lsu.edu/cid/article/42/6/798/286393
Cyganowski, J. (2015). Ultrafiltration for Bioprocessing (H. Lutz, Ed.). Ultrafiltration in
Bioprocessing, 244.
Foster, J. W., Woodruff, H. B., & McDaniel, L. E. (1946). Microbiological Aspects of
Penicillin: IV. Production of Penicillin in Submerged Cultures of Penicillium
Notatum. Journal of bacteriology, 51(4), 465-78.
Gaynes, R. (2017). The discovery of penicillin—new insights after more than 75 years of
clinical use. Emerging Infectious Diseases.
Harrison, Roger G. Todd, Paul Rudge, Scott R. Petrides, Demetri P. (2003).
Bioseparations Science and Engineering. Oxford University Press. Retrieved
from https://app.knovel.com/hotlink/toc/id:kpBSE00003/bioseparations-
science/bioseparations-science
Hossain, M. M., & Dean, J. (2008). Extraction of penicillin G from aqueous solutions:
Analysis of reaction equilibrium and mass transfer. Separation and Purification
Technology, 62(2), 437-443. doi:10.1016/j.seppur.2008.02.012
Kalvaitis, K. (2008). Penicillin: An accidental discovery changed the course of medicine.
Endocrine Today.
Kovács, Z. (1970). Diafiltration. Retrieved from
https://link.springer.com/referenceworkentry/10.1007/978-3-642-40872-4_171-5
28

Lee, S. C. (2009). Extraction equilibria of penicillin G in four different types of organic


solvent systems. Journal of Industrial and Engineering Chemistry, 15(3), 403-
409. doi:10.1016/j.jiec.2008.12.009
Liu, Q., Li, Y., Li, W., Liang, X., Zhang, C., & Liu, H. (2015). Efficient Recovery of
Penicillin G by a Hydrophobic Ionic Liquid. ACS Sustainable Chemistry &
Engineering, 4(2), 609-615. doi:10.1021/acssuschemeng.5b00975

Loughnane, Sara. “Ion Exchange Chromatography.” Science Learning Hub, 16 Nov.


2007, www.sciencelearn.org.nz/resources/2046-ion-exchange-chromatography.
Luo, L., Bao, S., Mao, J., & Tang, D. (2016). Quality prediction and quality-relevant
monitoring with multilinear PLS for batch processes. Chemometrics and
Intelligent Laboratory Systems.
Meštrović, T. (2018). Penicillin Production. Retrieved from https://www.news-
medical.net/health/Penicillin-Production.aspx
Miller R.D., Neuss N. (1983) High-Performance Liquid Chromatography of β-Lactam
Antibiotics. In: Demain A.L., Solomon N.A. (eds) Antibiotics. Handbook of
Experimental Pharmacology (Continuation of Handbuch der experimentellen
Pharmakologie), vol 67 / 1. Springer, Berlin, Heidelberg
Nabais, A. M., & Cardoso, J. P. (1999). Purification of benzylpenicillin filtered broths by
ultrafiltration and effect on solvent extraction. Bioprocess Engineering, 21 (2),
157.
Najafpour, Ghasem D. (2015). Biochemical Engineering and Biotechnology (2nd
Edition) - 11. Production of Antibiotics. Elsevier.
Odde, D. J. “Affinity Adsorption.” SpringerLink, Springer, Dordrecht, 1 Jan. 1997,
link.springer.com/chapter/10.1007/978-94-009-1563-3_4.
Orr, V., Scharer, J., Moo-Young, M., Honeyman, C. H., Fenner, D., Crossley, L., Chou,
C. P. (2012). Simultaneous clarification of Escherichia coli culture and purification
of extracellularly produced penicillin G acylase using tangential flow filtration and
anion-exchange membrane chromatography (TFF-AEMC). Journal of
Chromatography B,900, 71-78.
Posch, A. E., & Herwig, C. (2014). Physiological description of multivariate
interdependencies between process parameters, morphology and physiology
during fed-batch penicillin production. Biotechnology Progress.
Rohani, S. (2010). Applications of the crystallization process in the pharmaceutical
industry. Frontiers of Chemical Engineering in China, 4(1), 2. Retrieved from
https://search-ebscohost-com.libezp.lib.lsu.edu/login.aspx?
direct=true&db=edb&AN=50120640&site=eds-live&scope=site
29

Salivar, C. J., Grenfell, T. C., & Brown, E. V. (1948). Studies on the naturally occurring
penicillins. II. Precipitation of crystalline ammonium penicillins. JOUR BIOL
CHEM, 176((2)), 977–981. Retrieved from http://libezp.lib.lsu.edu/login?
url=https://search.ebscohost.com/login.aspx?
direct=true&db=boh&AN=BACD19492300014260&site=eds-
live&scope=site&profile=eds-main
Samanidou, V. F., Nisyriou, S. A. and Papadoyannis, I. N. (2007), Development and
validation of an HPLC method for the determination of penicillin antibiotics
residues in bovine muscle according to the European Union Decision
2002/657/EC. J. Sep. Science, 30: 3193-3201
SumiGK. (2017). Fermentation of Penicillin Antibiotic. Retrieved from
https://www.generalmicroscience.com/industrial-microbiology/fermentation-
penicillin-antibiotic/
Tessier, L., Bouchard, P., & Rahni, M. (2005). Separation and purification of
benzylpenicillin produced by fermentation using coupled ultrafiltration and
nanofiltration technologies. Journal of Biotechnology.
Thomas M., Tan, et al. “Clinical Relevance of Penicillin-Resistant Streptococcus
Pneumoniae: A New Perspective.” OUP Academic, Oxford University Press, 15
Mar. 2006, academic.oup.com/cid/article/42/6/798/286393.
Variankaval, N., Cote, A. S., & Doherty, M. F. (2008). From form to function:
Crystallization of active pharmaceutical ingredients. AIChE Journal, 54(7), 1682-
1688.
Wang, Henry Y., et al. “Application of Affinity Adsorption in Thienamycin Fermentation.”
SpringerLink, Springer-Verlag, 1989,
link.springer.com/article/10.1007/BF00263996.
Weber, S. S., Polli, F., Boer, R., Bovenberg, R. A., & Driessen, A. J. (2012). Increased
Penicillin Production in Penicillium chrysogenum Production Strains via Balanced
Overexpression of Isopenicillin N Acyltransferase. Retrieved from
https://aem.asm.org/content/78/19/7107
Wey, J., & Karpinski, P. (2002). Batch Crystallization. Retrieved February 19, 2019,
from https://www.researchgate.net/publication/279436168_Batch_crystallization
Wp. (2009). Healthcare industry. Retrieved from https://www.gesundheitsindustrie-
bw.de/en/article/news/protein-crystallisation-an-attractive-alternative-to-protein-
purification/
30

Contributions
- Kenzie Dupont wrote the history of penicillin and process considerations of
filtration and chosen methods of removal of insolubles, as well as edited the
paper post-midterm.

- Gracie Kennard wrote about the relevance of penicillin in the introduction,


washing, the process considerations for polishing including crystallization and
drying, and the chosen methods for crystallization/drying and the conclusion.

- Fischer Robinson wrote about the culturing, growth, and fermentation of


Penicillium chrysogenum. He also wrote the abstract for the paper and created
the simple flow chart for the process.

- Tim King wrote about and compared the methods of extraction that would
produce the highest yield, wrote about the chosen method of precipitation used,
worked on the washing step. He also wrote about the cost analysis from the
economic evaluation report.

- Trey Schexnayder wrote about how extraction can be used to improve penicillin
recovery in the introduction, frequently used methods of purification and its
importance in the downstream process, and the chosen method for purification.
He also worked on the SuperPro flowchart.

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