Amoxicillin Production: Designing a Process

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Amoxicillin Production: Designing a

Process

Written by Ethan Byrne, Derek Bordelon, Taylor Boudreaux,

Daniel Davison, and Sam Politz

BE 3340 Section 2 Dr. Sabliov 29 February 2016

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Amoxicillin is a commonly used antibiotic and is a derivative of penicillin. Most companies that produce amoxicillin use the same chemical process; however, recent studies have shown the benefits of a one-pot batch enzymatic process. The purpose of this paper is to outline a process for the production of 5 kg of amoxicillin in one day. Multiple methods for unit operations are outlined, and the best fit for this process was chosen. The process for the production of amoxicillin begins with two compounds, penicillin G potassium salt, and d-hydroxyphenylglycine methyl ester hydrochloride, in a medium consisting of an enzyme (penicillin G acylase), water, 2-butanol to increase the yield, and a potassium phosphate buffer maintain pH. After the reaction, the solution is filtered using a plate and frame filter. The slope of the filtration graph for a thirty-minute filtration was found to be 2162.9 s/m2. Once filtration takes place, the plate and frame filter must be washed to remove the filter cake. The filter cake is then placed in water to undergo high-pressure liquid chromatography in order to obtain the desired amount of amoxicillin of excellent purity. The final step in the process is freeze-drying, which will dry the amoxicillin in less than eight hours. A comprehensive flowchart outlining the steps of the chosen process is included. The total cost, including material and equipment expenses, was estimated to be $1,967,664. The amoxicillin would be sold at $5.40/g. At a production rate of 5 kg per day, the gross income would be $27,000 a day and $9,855,000 a year. Mass balances and energy usage for the process were calculated as well.

II.Objective

The objective of this paper is to design a process to produce 5 kilogram of purified amoxicillin per day.

III. Introduction History of antibiotics

The discovery of the first antibiotic, the β-lactam antibiotic penicillin, by Alexander Fleming in 1928 led to a revolution in the treatment of infectious disease. This important discovery, like many great discoveries throughout history, happened by pure chance. One of Fleming’s cultures containing a species of Staphylococcus bacteria, which is known to cause boils, sore throats, and abscesses, was accidentally contaminated with a rare strain of Penicillium fungus1,

Penicilliumchrysogenum2. Fleming noticed that the Staphylococcus was not growing around the fungus. Further investigation proved that the secretions from the fungus killed other harmful bacteria. The difficult nature of isolating pure penicillin led Fleming to only briefly mention its potential therapeutic benefits when he published his findings in the British Journal of Experimental Pathology in June 19291.

The revolution in the processing of penicillin began in 1938 with Howard Florey and Ernst Chain at the Sir William Dunn School of Pathology at Oxford University. They decided to study the biochemical and biological properties of selected antibacterial substances produced by certain microorganisms, including penicillin. Their work on penicillin earned them and Fleming a Nobel Prize in 1945 in physiology and medicine. Florey and Chain were instrumental in developing a successful technique for purifying and concentrating penicillin by controlling pH, reducing temperatures, and evaporating the product repeatedly, essentially freeze drying it3. Initially 500

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liters a week of mold filtrate had to be processed for Flory and Chain to carry out animal experiments and clinical trials. To keep up with demand, they used a strange array of culture vessels, including baths, milk churns, bedpans, and food tins. Eventually, a customized fermentation vessel was designed for easier production and harvesting of the fermentation products. Purification of the product became easier with the discovery of alumina column chromatography and led to an increase in production1_.

While Florey and Chain were having success refining and amplifying the process of penicillin production, World War II made industrial production of penicillin in Britain difficult since its chemical industry was fully absorbed in the war effort1. At that time, the United States was still a noncombatant, which caused Florey to seek assistance from the U.S. in furthering penicillin production. In July 1941, Florey and one of his associates, a biochemist named Norman Heatley, flew to the U.S. for a meeting with the Department of Agriculture’s Northern Regional Research Laboratory (NRRL) in Peoria, Illinois, which led to large-scale fermentation processes to be studied3. This study led to major increases in penicillin yield by substituting lactose for sucrose and by adding corn-steep liquor and penicillin precursors, such as phenylacetic acid, to the fermentation medium. Further advances in penicillin production were made when a more productive strain of Penicillium was found on a moldy cantaloupe from a Peoria fruit market. This strain was mutated twice, once with the use of X-rays and once with the use of ultraviolet radiation, producing an even more productive strain1.

A huge surge in penicillin production came when the U.S. entered WWII, but it was not without its challenges. Leading the charge in production were pharmaceutical companies Merck, Squibb, Pfizer, and Lederle. During the scale-up phase of production, Pfizer’s John L. Smith remarked, “the mold is as temperamental as an opera singer, the yields are low, the isolation is difficult, the extraction is murder, the purification invites disaster, and the assay is unsatisfactory.” Despite early difficulties, several advances in the fermentation, recovery, purification, and packaging led to Pfizer opening the first commercial plant for large-scale penicillin production by submerged culture in 1944 in Brooklyn, New York. The proposed D-Day invasion of Europe caused the War Production Board (WPB) to take responsibility of penicillin production in 1943. The WPB selected 21 companies to participate in the penicillin production program with the goal of having an adequate supply of penicillin available for the D-Day invasion. Production of penicillin in the U.S. jumped from 21 billion units in 1943 to 6.8 trillion units in 1945. The cost of penicillin dropped from $20 per 100,000 units in 1943 to less than $0.10 in 1949. In 1949, Florey recognized the great joint-effort of American chemists, chemical engineers, microbiologists, mycologists, government agencies, chemical manufacturers, and pharmaceutical manufacturers that worked on the penicillin production problem by saying, “too high a tribute cannot be paid to the enterprise and energy with which the American manufacturing firms tackled the large-scale production of the drug, had it not been for their efforts there would certainly not have been sufficient penicillin on D-Day in Normandy in 1944 to treat all severe casualties, both British and American.”1

Beta-lactam antibiotics

While manufacturers were improving the production process of penicillin, the advent of X-ray crystallography allowed Dorothy Hodgkin at Oxford University to accurately describe its structure. Its structure consists of a four-membered highly labile β-lactam ring fused to a thiazolidine ring1. Lactam rings can be either β (four member ring), γ (five member ring), or δ (six

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member ring). The strained nature of the four-member β-lactam ring makes it highly reactive; nucleophilic reagents easily open the β-lactam ring4_{. Accurately describing the structure of the} penicillin was instrumental in developing penicillin-like antibiotics that could be administered more conveniently, be more effective, and have fewer side effects3.

The unique mode of action of β-lactam antibiotics make them incredibly useful in the treatment of infectious disease in humans and in other animals. There are thousands of known antibiotics; however, less than one percent of them are clinically useful due to their toxicity to the host or the lack of the host cell being able to uptake the antibiotic. Neither of these problems is associated with β-lactam antibiotics. The β-lactam antibiotics work by inhibiting cell wall synthesis in bacteria, particularly in gram-positive bacteria, by impeding transpeptidation. Transpeptidation is the reaction that results in crosslinking of two glycan-linked peptide chains during the formation of the cell wall. Without adequate cell walls, the bacterial cells lyse. Since humans and other animals have no cell walls, the unique mode of action of β-lactam antibiotics makes them nontoxic to host cells2.

The efficacy of penicillin and the knowledge of its structure allowed researchers to begin to develop semisynthetic β-lactam antibiotics by altering the side chain of the molecule. One method to accomplish this was to provide side chain precursors in the fermentation broth. However, the range and diversity of compounds produced in this method were limited. Another method by scientists at Beecham Research Laboratories was to produce p-aminobenzylpenicillin through broth fermentation with the intentions of modifying its side chain later. In this method, the absence of sufficient side chain precursors produced the molecule 6-aminopenicillinic acid (6-APA), which lacked a side chain altogether. The lack of a side chain made 6-APA a suitable platform upon which to create synthetic β-lactam antibiotics, such as methicillin, ampicillin, and amoxicillin5.

Amoxicillin

Amoxicillin is a broad-spectrum antibiotic that was first synthesized from the penicillin derivative 6-APA in 1970. It is very closely related to ampicillin, which was first synthesized in 19615. The main structural difference between the two is that amoxicillin has a hydroxyl on the benzene ring6. They both have the same spectrum of activity and potency, however amoxicillin is much better absorbed when given orally, achieving blood concentrations approximately twice those obtained with ampicillin5. Some common uses of amoxicillin are the treatment of sexually transmitted diseases and infections, such as ear, nose, throat, genitourinary tract, skin and skin structure, lower respiratory tract, helicobacter pylori infections6.

Beta-lactamase

Despite a high level of clinical success with β-lactam antibiotics, by 1965 a serious mechanism of resistance in bacteria had emerged. That mechanism was the production of the enzyme β-lactamase that could render β-lactam antibiotics inactive. This enzyme was identified as the basis of staphylococcal resistance to penicillin. It was found that the genetic code for this resistance could be passed between bacteria, even bacteria of different species and genera, via plasmids. This horizontal and vertical gene transfer allowed the resistance to be spread rapidly. Large-scale screening of microorganisms for the production of β-lactamase inhibitors began in 1967. It was not until 1972 that a potent inhibitor of β-lactamase was identified. The inhibitor was produced by Streptococcus clavuligerus and named clavulanic acid. The β-lactam ring of

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clavulanic acid binds irreversibly to bacterial β-lactamase preventing it from inactivating β-lactam antibiotics. Clavulanic acid was found to have low antibacterial activity and thus needed to be paired with an antibiotic. Amoxicillin was chosen to be paired with clavulanic acid and administered in tablet form because of its good oral absorption and broad-spectrum antimicrobial activity. The tablet was named Augmentin and launched in the United Kingdom in 1981. Even today, it plays an important and unique role in the treatment of a range of community-acquired infections, especially respiratory infections5.

IV.Process considerations Synthesis methods of amoxicillin

There are several ways that amoxicillin can be synthesized. The conventional route adopted for the production of amoxicillin is by chemical synthesis. The problem with this method is that it is a complex process requiring several reaction steps at low temperature. It also requires anhydrous conditions and the use of highly toxic solvents that generate considerable amounts of non-biodegradable wastes7. One way that these problems can be minimized or eliminated is through enzymatic synthesis, which is a process that is becoming increasingly more interesting as an industrial process. Enzymatic synthesis reduces the number of reaction steps and decreases the amount and toxicity of waste products per kilogram of antibiotic. The use of enzymes such as penicillin G acylase (PGA) and 6-APA in the synthesis of amoxicillin could be of great interest due to their high selectivity, speciﬁcity, and activity in mild reaction conditions (aqueous media, neutral pH, and moderate temperatures)8_{. Another environmentally friendly process for the} synthesis of amoxicillin is using microorganisms to catalyze its enzymatic production. One particular method involves using β-lactamase-deficient mutants of Pseudomonas melanogenum

KY 3987. Lactamase-deficient mutants were isolated from amoxicillin-sensitive mutants derived from this strain. These mutants became sensitive to other penicillins and cephalosporin as well as amoxicillin and did not produce 3-lactamase even in the presence of an inducer, such as penicillin V. Amoxicillin was effectively synthesized from D-2-p-hydroxyphenylglycine methyl ester and 6aminopenicillanic acid (6-APA) with these mutants as the enzyme sources9.

Reactor

The industrial production of amoxicillin in the modern era is plagued with flaws. First, many of the organic solvents used in the pharmaceutical industry are detrimental to environmental health10. Even if one chooses not to consider the fact that these solvents, such as methylene chloride and silylation reagents, are not biodegradable, they are widely considered toxic. To make this even more concerning, these toxic solvents cannot be reused. For every 1 kg of amoxicillin that is made, 30-40 kg of non-recyclable toxic waste is produced, most of which stems from activation and deprotection steps in the Dane anhydride method that is so often used11. Activation is the process of exciting a compound to undergo a reaction12 and deprotection is the process of removing a protecting group to prevent oxidation13_{. Both of these steps involve toxic byproducts that cannot} be reused. In addition to being unsafe for the environment, the chemical methods used in industrial production can be tedious. These methods involve many reaction steps that take place in temperatures reaching -30°C. Because of these many problems, enzymatic synthesis is gaining more and more interest as environmental regulations become increasingly strict. By using enzymes as catalysts in the production of amoxicillin, not only is the amount of toxic waste reduced, but the number of the reaction steps from the chemical process is also reduced to a single session in a batch reactor11. In addition, the enzyme used to catalyze the reaction can be reused for the next

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reaction14. It is safe to say that the future of amoxicillin production will be based more on the enzymatic means of synthesis as opposed to the current chemical route used in industries.

Nearly every form of synthetic penicillin begins with the reagent (2S, 5R, 6R)-6-amino-3, 3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid, more commonly referred to as 6-aminopenicillanic acid, or 6-APA. Technical information for 6-APA and all other compounds can be found in Table 1. 6-APA forms the main component in the amoxicillin structure; in fact, all types of penicillin follow the structure of 6-APA, only differing in functional groups that are attached. 6-APA is extracted by the fermentation of a fungus containing penicillin,

Penicillium chrysogenum being the most common one that is used15. However, 6-APA can also be formed through the hydrolysis of penicillin G potassium salt, or PGK, by penicillin G acylase, or PGA. It should also be noted that 6-aminopenicillanic acid is not known to be harmful to humans and is a perfectly safe organic compound to work with11.

One method of enzymatic amoxicillin production employs the use of 6-APA along with p-hydroxyphenyl glycine (HPG). Both are used in high purity (greater than 98%). The reaction takes place in a low concentration solution containing a potassium phosphate buffer, which helps to keep the solution slightly acidic (pH 5-6). A stock enzyme solution is added in order to catalyze the reaction. The thermos-jacketed reactor in which the reaction takes place should be kept at approximately 25°C, which is much more feasible than the current method at -30°C. The reactions are carried out for approximately 30 minutes. This process has been proven in the lab to give high yields16_.

Another method for the enzymatic synthesis of amoxicillin involves many of the same compounds as the previous method. However, it eliminates the need for a supply of 6-APA. The synthesis of 6-APA and amoxicillin both take place in the same batch, thus reducing the costs of production. This “one-pot” process utilizes PGK along with D-HPGM, which is a derivative of HPG, as the

substrates of the reaction. Both substrates are typically used in low concentrations (less than 1 mol/L for each of them). A synthesis diagram for the one-pot enzymatic synthesis versus a two-step enzymatic synthesis can be found in Figure 1. The reaction takes place in small vials that are placed in a shaker kept at 25°C. The vials are shaken at approximately 200 rpm. The buffer used in the reaction is similar to the previous process described, except that sodium phosphate was used as opposed to potassium phosphate. The reaction media contains the aforementioned sodium phosphate buffer and ethylene glycol. The alcohol ethylene glycol is used to help produce better yields. Additionally, an aromatic carboxylic acid and methanol will be produced as byproducts11.

Figure 1. The one-pot enzymatic structure is shown at the top,

as opposed to the two-step “traditional” enzymatic process shown on the bottom11_.

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Immobilized penicillin G acylase is used as an enzyme to catalyze the reaction. The immobilized penicillin G acylase can be extracted from the mixture through filtering and used repeatedly for future syntheses. Immobilized penicillin G acylase is a very powerful enzyme to be used in industrial processes. Because the PGA is immobilized, it is still active over a vast range of conditions, including different temperatures and pHs. The immobilization also means that PGA is highly resistant to many solvents. Therefore, it will not dissolve into the medium and it can be easily separated from the other byproducts for reuse. Because enzymes are very expensive to manufacture or buy, the recovery of the immobilized PGA is crucial. This enzymatic process can also result in a high yield17.

Many other minor studies have been done in the hopes for a more efficient and safe way to produce amoxicillin. For example, in one study, researchers found that when using 6-APA to produce amoxicillin, the addition of certain alcohols can greatly improve the yield. When no alcohol was added to the reaction mixture, the conversion of 6-APA to amoxicillin was approximately 50%. However, when ethanol was added, 65% of the 6-APA was converted into amoxicillin. In fact, with the addition of 2-butanol, 91% of the 6-APA can be converted into amoxicillin, a large increase in comparison to the original 50%. The alcohol helps to repress the hydrolysis of HPG (and its derivatives), thus allowing more 6-APA undergo the reaction1718.

Table 1. The table below is a list of all compounds used in the production of amoxicillin along

with their abbreviation, molecular formula, and molecular weight. Molecular weights were rounded to six significant figures19, 20

Compound Abbreviation Molecular Formula Molecular Weight (g/mol) Amoxicillin AMOX C16H19N3O5S 365.404 6-Aminopenicillanic acid 6-APA C8H12N2O3S 216.257 Penicillin G potassium salt PGK C16H17KN2O4S 372.480 P-hydroxyphenyl glycine HPG C8H9NO3 167.162

Penicillin G-acylase PGA - 89.500

Potassium phosphate PPB H2KO4P 136.085

Sodium phosphate - H2NaO4P 119.977

Ethylene glycol - C2H6O2 62.0678

Ethanol - C2H6O 46.0684

2-Butanol - C4H10O 74.1216

Hydroxyphenylglycine methyl ester hydrochloride

HPGMEHCl C9H12ClNO3 217.650

Hydroxyphenylglycine methyl ester

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Although not much information can be found on the outdated method of production used by many companies today, it is clear that there are exciting new methods being developed. With increasing environmental regulations and the ever-growing concern for public health, it is evident that the enzymatic production of amoxicillin is the future of the anti-biotic industry.

Chemical Selection

After consideration of the different methods in the production of amoxicillin, the one-pot enzymatic synthesis method will be the method used in production due to the environmental considerations and efficiency. The reagents used in this method will be PGK, HPGMEHCL, PGA, PPB, 2-butanol, and water. PGK was selected because of its use in the one-pot method. HPGMEHCL was not discussed previously, but was selected because an assumption is being made that the attached hydrogen chloride will dissociate in water lowering the pH. Lowering the pH to below 5 is advantageous because amoxicillin has very low solubility at low pH compared to the rest of the reagents21. The use of HPGMEHCL should prevent or limit having to add more reagents to lower the pH. The pH will be tested during the reaction and adjusted accordingly with the addition of HCl and NaOH. PGA was chosen because it is the enzyme necessary for the synthesis reaction. PPB is a buffer, 2-butanol increases product yield, and water is the solvent.

Separation

Unlike the options available for the reaction methods, there are many methods for the separation of amoxicillin from pharmaceutical solution. Aminopenicillins break down in solution to their penicilloic acids. Therefore, the method used for separation should be conducted as quickly as is feasible, but the purity of the product must not be sacrificed. Amoxicillin is more stable once dried into a crystalline form. The main molecules of interest are amoxicillin penicilloic acids, p-hydroxyphenyl-glycine (p-HPG), and the enzyme 6-aminopenicillanic acid (6-APA). Various methods for separation were studied to find the most efficient process for removing impurities in the solution22.

First, an article in Separation Purification Technology studying side flow filtration using nanofiltration membranes. This type of filtration is mainly used to treat pharmaceutical wastewater, and is not widely used in the production of amoxicillin. However, the relationship of pore size of the membrane and pH of the buffer solution is examined very closely in the publication. During the research, UV photopolymerization of a polysulfone ultrafiltration membrane combined with polyethylene glycol (PEG) of varying molecular weights was used to create a polysulfone nanofiltration membrane. The results of this test illustrated two important factors to take into consideration when choosing pore size. First, the filter that was created using 400 Da PEG showed unusually high amoxicillin rejection. The study concludes that the dominant mechanism of separation of this pore is molecular sieving. Then, the membrane containing PEG with a molecular weight of 4000 Da also had high amoxicillin rejection. This is attributed to molecular sieving and charge repulsion of the negatively charged amoxicillin molecule. The results that detail this are shown in Figure 2. A pH of 10 decreased the fouling of amoxicillin on the membrane23_{. This illustrates how impurities with varying size and surface charge can be separated} by carefully choosing pore size and buffer pH.

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Figure 2: Amoxicillin rejection of membranes produced with varying polyethylene glycol

molecular weights23_.

Ultrafiltration is another mechanical filtration technique used to remove solutes with high molecular weights or suspended solids. The sample is pumped through a closed inline filter that consists of a membrane. The membrane has a particular pore diameter, and molecules that are larger than the pores are not passed through. This method has the added advantage of being very simple. However, fouling of the membranes can decrease the flux across the filter24.

Capillary zone electrophoresis (CE) is another separation method studied in the separation of amoxicillin. This method separates molecules by how well they are propelled through a specially designed capillary that is immersed in an electromagnetic field. The molecular size and surface charge both affect the rate at which the molecules move. This method produced very consistent results, and recovered a very high percentage of the amoxicillin present in the solution25. However, the analysis time and reagent loss is higher than other techniques26.

Chromatography also requires the movement of molecules in order to separate them. It is one of the most varied separation methods for amoxicillin. All chromatography consists of a mobile phase and a stationary phase. The mobile phase that dissolves the sample, and moves through the system. The stationary phase is used to take advantage of the different properties of the molecules in the sample, and change the speed that the molecules travel through the stationary phase. After the stationary phase, the various molecules are identified by a detector. This detector is used to determine the presence of a molecular compound in the mobile phase as it passes through the system, and the molecule of interest can be isolated and removed27_{. The methods focused on} are high performance liquid chromatography (HPLC), gel filtration chromatography (GFC), reverse phase liquid chromatography (RPLC), and sequential injection chromatography (SIC).

0 10 20 30 40 50 60 70 400 1500 3000 4000 MW of PEG t=0 min t=60 min t=120 min t= 180 min High MW PEG: Molecular Sieving +Charge Repulsion Low MW PEG:

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During HPLC, the mobile phase is pumped through the system at high pressures, and the stationary phase consists of a sorbent filled tube called a column. The particle size of HPLC is much smaller (3 to 5 microns) than that of traditional liquid chromatography techniques. This increases the resolution of the chromatography. The mobile phase then passes through a detector that registers the presence of molecules in the tube. The type of detector used will vary with the product and impurities to be determined. Therefore, the product can be removed from the impurities. The time it takes for HPLC separation is 10 to 30 minutes28.

The next method is GFC. It is a separation method that, due to the high molecular weight of the gel used, high-density molecules can be separated from the sample. It also has advantages in the simplicity of the setup necessary for separation. The size of the particles is the main factor that affects transit time through the gel. Studies at The National Institute for the Control of Pharmaceutical and Biological Products in Beijing have conducted studies that show that Superdex peptide and TSK G2000SW columns can be used to separate amoxicillin. There are also no organic solvents left over from the mobile phase during this method29.

Unlike GFC, RPLC is a separation method that uses a polar mobile phase and a nonpolar stationary phase. The stationary phase consists of non-polar silica C18 molecule. These molecules slow down the non-polar molecules in the stationary phase. RPLC is a more complex separation method than gel chromatography. This method uses liquid as the mobile phase and generates substantial waste from the separation process29.

SIC is a relatively new chromatographic method compared to the others that can be used for the separation of amoxicillin. It uses a computer program to control the flow of the solution through the chromatography system. This method produces much less reagent waste than other methods. In a recent study to separate 875 mg of amoxicillin, high performance liquid chromatography produced 500 ml of reagent waste, capillary electrophoresis produced 50 ml, and the sequential injection chromatography produced 3.26 ml. Table 2 displays these results26. This efficiency results in reduced cost of reagents purchased and maintains the high precision of other chromatography methods.

All of these methods have been successful in separating amoxicillin from impurities. However, Chromatography has the added advantage of separating all molecules, both known and unknown, in suspension because the choice of column can be varied. This makes it possible to extract amoxicillin only when an optical detector indicates that it is present. Additionally, the sequential injection chromatography seems to be much faster, less expensive to operate, and the limited reagent waste makes it ecological as well30.

Another method of separating solids from liquids is the plate-and-frame filter press. This method can be used for batch processes by compressing the feed suspension between one or more plates. The dewatered cake is discharged by self-weight when the plates are opened. The plates are typically made of stainless steel, cast iron, rubber, or polypropylene. Plates typically have areas ranging from 150 mm2 to 2 m2, and they are covered with a polymer filter cloth. The plates are mounted vertically, and they form chambers when pressed together. These chambers are where the filtrate collects forming cakes when the feed stream is pumped through the filter press. The unwanted liquid exits the filter press, leaving the cakes with as little as 20% moisture content. Plate-and-frame filter presses can be fully automated making the production process run more efficiently31.

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Table 2: Results from various separation techniques26_.

Analytical feature SIC HPLC [13] CE [16]

Sample matrix Tablets Tablets Injection

Separation column C18 (4.6×25 mm) C18 (3.9×300 mm) (75 μm×60 cm) Mobile phase composition 25 mmol l−1 PHSa:MeOHb (85:15, v/v) at pH 3.0 0.1 mol l−1 PHS:MeOH at pH 4.0 PHS:boratec (pH 8.7) MP/Elec Vd _(ml) ₂ ₅₀₀ ₅₀ UV detection (nm) 228 230 214 Resolution 1.6 10.3 14.5

Analysis time (min) 1.8 240 150

Waste production (ml) 3.24 500 50

System stabilize time (s) 612 612 7200

Sample frequency (samples h−1)

33.0 0.3 0.4

AMX CLV AMX CLV AMX CLV

Retention time (min) 0.3 0.5 10.1 5.6 7.1 8.6

Peak symmetry 1.15 1.19 1.07 1.25 1.80 2.25 Theoretical plates 3868 2743 2741 2686 16,266 42,428 Linear range (μg ml−1₎ _5–100 _5–100 _– _– _50–3030 _20– 2020 Linearity (correlation coefficient) 0.9997 0.9996 – – 0.9998 0.9998 Recovery (%) 97.5 100.5 – – – – Intra-day precision of recovery (RSD%) 0.29 1.27 0.99 0.62 1.52 1.30 LOD (μg ml−1₎ _0.37 _0.33 _– _– _0.4 _0.3 LOQ (μg ml−1₎ _1.2 _1.1 _– _– _0.8 _0.5

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Filtration Calculations

Calculations for filtration can be performed using the following equation:

𝐴𝑡 𝑉 = 𝛼𝜌˳µ 2𝛥𝑃 ( 𝑉 𝐴) + µ𝑅𝑀 𝛥𝑃

where A = area, t = time, V = volume, µ = viscosity, P = pressure, RM = media

resistance, and 𝜌˳ = mass of

solid cake/volume of filtrate

(assume RM is negligible). Figure 3 is a graph of the filtration of amoxicillin based on available information and assumptions made by the investigators of this paper. The slope of the graph is 𝛼𝜌˳µ

2𝛥𝑃 =

2162.9 s/m2_{when the time of} filtration was calculated at 30 minutes. Testing of this model can be validated or rejected through testing using the lab-scale pathway illustrated in the flowchart, Figure 4.

Separation Selection

After careful consideration of the product stream, several different separation techniques were chosen. Plate and frame filtration was selected for primary filtration directly following the batch reaction process. It was selected for the capability to filter large amount of material that fits with the production output of 5 kilograms per day. A 20-µm wire filter mesh is chosen for durability to minimize replacement costs. The filter press will operate under a pressure differential of 15 bars (225 psi) 32 and the other filtration constants (αρ̥μ) will be calculated using the lab scale process shown in Figure3. For the lab scale, ultrafiltration will be utilized.

Before the precipitate and solution are filtered, a sample will be subjected to ultrafiltration with a pore size of 0.001 µm. The filter cake obtained will be analyzed by the same chromatography method as our large-scale process. The small pore size of the ultrafiltration will aide in determining the amount of amoxicillin that has precipitated out of solution, and the amount of amoxicillin that has undergone hydrolysis. This analysis allows the batch to be optimized before large-scale filtration.

Once the batch has been optimized and filtered, the filter cake will be washed and the filtrate will undergo further separation. The separation technique chosen was gel chromatography. This technique was chosen because it separates substances from the waste stream based on their molecular weights. Methanol, which is our smallest molecule in the waste stream leaving filtration, will be removed. The gel column used will be a Sphadex G-25. This medium has been used

Figure 3. Graph of amoxicillin filtration assuming an

incompressible cake is formed. The equation is in y = mx + b form

where b = 0. Variables usedA = 0.0279 m2_{, t = 30 minutes, V =}

25.45 L, 𝜌˳=0.2725 kg/L. y = 2162.9x 0 500 1000 1500 2000 2500 0 0.2 0.4 0.6 0.8 1

A

t/

V

(

se

c/

m

et

er

s)

V/A (meters)

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successfully to separate amoxicillin and the byproducts of synthesis29. The water in the mother liquor will be the mobile phase in this step. Once the methanol is removed from the waste stream, the stream can be used again for another precipitation.

After the waste stream is addressed, the amoxicillin can be purified. Initially, sequential injection chromatography was selected for the purification step in the process. However, this method has not been adapted to large-scale production, and it would not produce the amount of product needed for this process. The chromatography method chosen was HPLC. It has the same benefits as the SIC method but is much faster. A continuous multi-column HPLC system, Prochem Varicol, was chosen. This method includes a solvent storage and recycling system that the company claims can reduce the use of solvents by 99.7%. The multi-column design and automation allows for 24-hour operation. This equipment allows for greater production rates than a system that requires an operator. Due to the hydrophobic nature of amoxicillin, a reverse phase liquid chromatography method was selected33_{. A hydrophobic 5 mm C18 column was chosen as} the stationary phase. Water was chosen as the mobile phase. In addition, a pH buffer consisting of 0.5 mole/liter dipotassium phosphate at a ratio of 1/100, 0.5 mole/liter monopotassium phosphate at a ratio of 9/100, and distilled water at a ratio of 90/100 will be constructed. These chemicals will maintain the pH at 9.5 during chromatography. The spectrophotometer for detection of amoxicillin will be set at 220 nm. The effluent not recycled by the chromatography system should be treated with 1% KOH before disposal30.

Precipitation

After chromatography, the purified solution will be sent to a mixing vessel where it will be precipitated again. This second precipitation will be carried out by the addition of HCl to lower the pH below 5. As HCl dissociates into ions, the solution becomes more acidic causing a significant reduction in the solubility of the amoxicillin. The excess ions in the solution force the amoxicillin out of the solution yielding a precipitate.

Drying

In the production of most biological materials, such as amoxicillin, a point is reached where the final product is a precipitate or powder-like state. Small amounts of moisture (usually water) are left within the amoxicillin powder that tarnishes the purity of the product. To reduce impurities, the final step in production is usually drying and/or polishing. Drying methods remove all excess moisture in the last step of production so that only the final solid remains. Polishing methods improve purity by removing all excess chemicals or substances from the product. The separation step of amoxicillin production usually yields a product of high purity requiring no polishing, only drying34.

Air Drying Methodsare simplistic in design and are widely used in many commonplace production fields such as on farm grain-drying operations. Models for air-drying methods utilize the simple principle of circulating air to dry the product. Normally, the product is secured while a fan propels and circulates the air through the area in which the product is, in order to remove excess moisture from the product. Two examples of air dryers are the cabinet dryers and tunnel dryers. Cabinet dryers are sold in small scale to households, and tunnel dryers are simply a large-scale modification of the cabinet dryer concept. In both cases, the undried products are loaded into the drying system and strapped down in some fashion while air is slightly heated (with coils or burners), and blown through the system with fans. The only real difference is that in the tunnel

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dryer multiple trays of product are able to be dried at one time. This method of drying provides several benefits. For one, with the simple application of recirculation energy savings could be boosted by up to 50%. Another benefit is that much research and experimentation has been done using this method. Finally, this process is one of the cheaper drying methods due to its simple design and set up. On the other hand, this method does very little to prevent contamination. Because it is so commonly used in agricultural or other larger product drying, small contaminants can be overlooked, but with a product of much higher quality than grain or some other agricultural product, problems may be encountered when it comes time to make sure the product is as pure as possible. The simplicity and efficiency of this method make it favorable, but the time that is required for air-drying is another drawback to this method34.

Freeze Drying Method is a more complex method than air-drying. It changes the surrounding pressure of a frozen material in order to rid it of the frozen water within it through the process of sublimation. This method is widely used by many food and bioprocessing companies to improve the long-term storage stability of the products. This method allows for much longer shelf life, longer and safer storage options, and greater shipping options. However, this method gives many companies trouble in scaling up production, as it is a very complicated process. Every step within the process presents a unique product development and scale up challenge. These challenges means that for the process to be sized up from the small-scale laboratory to large-scale manufacturing requires a lot of time, effort, and calculations to make sure that the products do not crystallize during the freeze drying process and all of the thermal-based reactions are not affected35.

Unlike freeze-drying, the spray drying method uses hot gas to rapidly dry a liquid into a dry powder. This drying is achieved by a nozzle dispersing the liquid in which the desired product is in through a controlled filtered spray size. This spraying forces the liquid into a mist state. This mist is then mixed with hot gas to further cause vaporization. Finally, the product and hot gases normally reach a fork in the system in which the product collects on the bottom in a collection vessel and the now cooling gas is recycled back up to the beginning of the system. Because this process causes large-scale evaporation, which is a cooling process, this method is suitable for heat-sensitive materials, despite the high temperatures of the drying gas. When compared to the freeze-drying method, the spray method is a faster and more economical single-step freeze-drying method. This method also provides the unique opportunity to control the particle size and the morphology of the dried powder by carrying the parameters and the formulation factors. However, this process does have its drawbacks. A large sample volume is required, 50ml minimum, to yield a maximum yield of 70% using a minimum 2µM particle size. A new advanced method called Nano Spray drying is available that allows a sample volume to be as small as 2mL. It yields products closer to 90% with a decreased particle size of up to 300 nm with narrow size distribution and eventually fast drying process (up to 150ml/h). This method gives more control over the conditions in a process36_.

Because of the inquisitive nature of this literature review, it would be remiss to not search out for the newest and, in some cases, not very commonplace drying methods in order to properly provide options for production, in addition to the more common methods already discussed. As such, the following paragraphs are dedicated to the newest drying methods that seem most applicable to bioprocessing, which include heat-pump-assisted drying, superheated steam drying, variable pressure drop drying, and molecular sieves.

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Heat-pump-assisted drying is one of these newer drying methods. The dryer consists of a heat pump system, which can be mechanical, chemical, or other types, and a convection dryer that are coupled. What makes the heat-pump-assisted drying method unique is that it uses energy efficient, low-temperature, dehumidified air as the convective drying medium. This drying is done by recovering the condensing moisture from the drying air and blowing that condensed air back, which then heats as it dehumidifies. This increases the overall energy efficiency of the dryer by capitalizing on the recovered heat, which would otherwise be lost to the atmosphere in conventional dryers. The drawbacks of this type of dryer are higher capital cost, more maintenance, and negative environmental impacts compared to regular conventional dryers. However, from a monetary standpoint the heat pump assisted dryer would be worth consideration over other drying methods37.

Another method that uses heat is superheated steam drying. This method is to be considered for cases in which higher drying rates and safer drying systems are needed. This drying system functions by replacing drying air, as common in convection dryers, with superheated steam. Superheated steam then functions to remove the moisture from the wet solids. By removing it, the superheated steam eliminates the risk of fire and explosion hazards because of an absence of oxygen. This method gives a very short drying time, but it can affect product quality with the added variable of the fast moving steam disturbing the finished product. In addition, it is a method very seldom used by most scientist, which should be taken into consideration. However, the overall safety and quick drying rates that it offers make it appealing37_.

Unlike the methods that use heat, variable pressure drop drying is a series of methods, which all revolve around the idea of changing the pressure surrounding the product to induce vaporization of moisture within the product. This pressure drop is usually achieved by creating a vacuum around the product. The next step is slightly varied throughout different designs. Some methods shoot in steam to allow for a sudden reduction of pressure due to the addition of the fast moving fluid (the steam). Other methods simply control the vacuum pressure in which the product is in and vent out pressure until vaporization occurs. In all cases, these methods allow for a shorter drying time than most conventional drying methods, and results in highly porous dried products with no shrinkage. This method has been tested successfully in many food-drying productions as well. Utilizing the variable pressure drop method affords a unique opportunity. It allows manufacturers to safely control the drying of the product without the addition of any contaminants or reactants to tarnish the purity of the final product37.

Molecular sieves do not require pressure of heat to dry products. Instead, theyare a series of absorbents introduced to organic compounds (especially solvents) in order to dry out trace amounts of moisture left within them. This drying is achieved by the sieves having pores about the same dimensions as small moisture-type molecules such as water. When they are heated, the water from the sieves evaporates. This process then allows the sieves to take up the moisture from the surroundings effectively drying them. These drying agents usually come in a bead-like form, and they are commonly seen in the form of silica. Silica packs can be found in packages that are being shipped to keep the contents dry. There are four main types of sieve used in laboratories. Crystalline potassium aluminosilicate has the smallest pore size at about 3 Angstroms. It is used to dry liquids such as acetone, acetonitrile, methanol, ethanol, and 2-propanol. Crystalline sodium aluminosilicate has the next biggest pore size at 4 Angstroms. It is used to dry many variations of ethane, chloroform, ether, ethyl, and benzene; however, it cannot absorb water. Crystalline calcium aluminosilicate has the next largest pore size at 5 Angstroms. It can absorb a larger class of

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alcohol-like molecules such as butane, hexane, butane, higher olefins, butyl alcohol, and higher n-alcohols. Crystalline sodium aluminosilicate has the largest pore size, at 10 Angstroms. Because of its large size it absorbs many molecules removed by other sieves, and enables drying of such large molecules as branched-chain and cyclic compounds38.

Because molecular sieves are drying agents, the price of drying a product could be as low as the price of the specific sieve that works best with it. This could greatly improve the efficiency of an experiment, and is a positive quality of molecular sieves. There would be no need for expensive ovens and maintenance thereof, but sieves are also limited. At best, sieves only absorb 18% by weight of water. This result is incredibly low and would require a large amount of the sieve in which to properly dry a product38. Furthermore, with all the different sizes of molecular sieves it would almost certainly require time and effort in order to experiment to find the specific sieve best for drying the amoxicillin product.

Drying Selection

Amoxicillin is a highly unstable particulate, and is very sensitive to changes in humidity and temperature19. Due to these characteristics, any drying technique requiring increased temperature will not be used for the final product. Originally, the drying method selected was crystalline sodium aluminosilicate molecular sieve drying because ethyl acetate, an organic solvent, was used to wash the amoxicillin. Molecular sieves target the solvent. However, after consideration of the HPLC's parameters, it was determined that the process needed to include re-dissolving the amoxicillin in H2O and re-precipitating it. Consideration of these parameters led to the use of water as the primary solvent, meaning the drying technique must be selected to target water and retain a low temperature. After reviewing the literature, freeze-drying was the optimal drying technique for the process parameters. Freeze-drying is an extremely complex procedure consisting of three stages: freezing, primary drying, and secondary drying. Freezing is the first stage of the freeze-drying process, and is the stage that the majority (>86%) of the water content will be removed from the product. This step separates the solution in to multiple phases, creating ice to drug interactions. Freezing generally takes between 2-4 hours to complete, and it is followed by primary drying. Primary drying is the most complex step. The unbound water/ice undergoes sublimation, and it is removed from the final product. Primary drying is also one of the longest steps in the entire process, and is heavily dependent on several factors. One factor that directly affects the time of primary drying is margin of safety, which is the temperature difference between the product temperature and the critical temperature. Increasing the margin of safety temperature by 1°C deceases the primary drying time by 13%. In order to accommodate for the 5kg/day production a margin of safety of 7°C will be used to ensure that primary drying will be completed in less than 8 hours. Primary drying is governed by the rate at which ice undergoes sublimation given by the equation:

𝑑𝑚

𝑑𝑡 =

𝑃_𝑖𝑐𝑒− 𝑃_𝑐 𝑅𝑝+ 𝑅𝑠

𝑤ℎ𝑒𝑟𝑒 𝑑𝑚_𝑑𝑡 = 𝑡ℎ𝑒 𝑟𝑎𝑡𝑒 𝑎𝑡 𝑤ℎ𝑖𝑐ℎ 𝑖𝑐𝑒 𝑢𝑛𝑑𝑒𝑟𝑔𝑜𝑒𝑠 𝑠𝑢𝑏𝑙𝑖𝑚𝑎𝑡𝑖𝑜𝑛, Pice = vapor pressure of ice,

Pc = Chamber pressure, Rp= Resistance of dry layer, Rs = Resistnace of stopper.

Primary drying is followed by secondary drying. In this stage the trace amount of water that did not freeze and undergo sublimation is removed by desorption. After this stage, the final product

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should contain <1% moisture content on a dried-solid basis. Overloading the freeze dryer is the last constraint that affects the time it will take for the product to be completely purified. The flow of material cannot exceed 1kg∙m2_{/h for the typical manufacturing freeze dryer}39_.

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V. Flowchart

Figure 4.The above figure shows a combination of the research conducted and the possible process design. The upper pathway of

the process is for lab-scale testing and the lower pathway is for large-scale production.

 HPGMEHCL: D-Hydroxyphenylglycine

methyl ester Hydrochloride

 PGA: Penicillin G acylase

 PGK: Penicillin G Potassium salt

 PPB: Potassium Phosphate Buffer

 HPLC: High Pressure Liquid Chromatography

 K2HPO4: Dipotassium Phosphate

 Mother Liquor o PPB o PGK o HPGMEHCL o 2-Butanol o Water

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The flowchart depicted in Figure 4 represents a lab-scale pathway when testing is required, and a pathway for large-scale production. It starts with the introduction of the selected reagents into the reactor. The optimal temperature and pH for the reaction are 25°C and 6.5 respectively. As the HPGMEHCL dissociates, the pH will drop, and the amoxicillin will precipitate. The lab-scale portion of the production pathway, shown at the top of the flowchart, allows samples to be taken for testing without disrupting the production pathway. When samples are needed for testing, a small stream can be diverted into the lab-scale pathway. Since the size of the stream is insignificant in comparison to the production stream, it can be quickly processed ahead of the rest of the stream. When the streams leave the reactor, they will both be carrying the precipitated amoxicillin product, some unreacted reagents that are still in solution, and a methanol byproduct. The enzymes will be immobilized in gel, and remain in the batch reactor so they will not be in the streams.

The stream for the production pathway will leave the reactor, then enter the plate and frame filtration. The amoxicillin will be compressed into cakes and the waste stream will be sent to gel filtration where the methanol, which is detrimental to the production process, will be removed from the stream and disposed of properly. The stream will be stored and reintroduced into the process later after analysis. The amoxicillin filter cakes will be removed from filtration, and the filters will be washed with water to remove remaining product. The filter cakes will then be dissolved in water and combined with the wash from filtration, mixed with the appropriate buffer, and sent to HPLC. Since the reagents used in the reaction are of high purity, the HPLC should yield a product that is also of high purity. After HPLC, the solution will enter a blending/storage vessel where it will be precipitated by dropping the pH using HCl. The final product will be freeze-dried, which will leave it ready for packaging. The final amoxicillin produced will be an off-white powder of high purity (approximately 99.9%).

VI.Mass Balance

With the overall process described, the mass balance of the process will now be addressed in Tables 3-7. The best ratio of reagents to maximize yield, and minimize the unwanted hydrolysis reaction is 2.5:1 of HPGMEHCl to 6-APA respectively40. However, PGK is used in this process instead of 6-APA. Since 6-APA is a derivative of PGK, it is assumed that the ratios will hold true in this production method. Using the 2.5:1 ratio, 20% extra reagents will be added to account for a less than 100% conversion of reagents to products. The mass balances were calculated assuming no losses at any of the unit ops.

Table 3. Reaction: All reagents and masses are listed that go into and exit the reactor. PGK and

HPGMEHCL are the primary reagents used to produce amoxicillin. PGA is immobilized in gel, and it stays in the reactor. PPB, water, and 2-butanol are necessary components in the reaction media. In (kg) Out (kg) PPB 0.1 0.1 PGK 1.9 0.29 PGA 0.05 N/A HPGMEHCL 4.58 0.71 Water 20 20 2-Butanol 4 4 Amoxicillin N/A 5.00

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Table 4. Plate/Frame Filtration: A list of all the chemicals and their masses that go into and exit

the Plate/Frame filter. 86% of the liquid is removed in the plate/frame filter and transported through a waste stream and into storage. The assumption is that there would be no loss of the mass of the amoxicillin.

In (kg) Filter Cake: Out (kg) Waste Stream Out (kg)

PPB 0.1 0.014 0.086

PGK 0.29 0.0406 0.2494

PGA N/A N/A N/A

HPGMEHCL 0.71 0.0994 0.6106

Water 20 2.8 17.2

2-Butanol 4 0.56 3.44

Amoxicillin 5.00 5.00 N/A

Table 5. Washing: A list of all chemicals and their masses as it pertains to washing the filter. 5

kg of water is used to wash the plate/frame press to remove the filter cake. Again, an assumption that no losses was made when considering the washing step.

In (kg) Out (kg)

PPB 0.014 0.014

PGK 0.0406 0.0406

PGA N/A N/A

HPGMEHCL 0.0994 0.0994

Water 7.8 7.8

2-Butanol 0.56 0.56

Amoxicillin 5.00 5.00

Table 6. HPLC: A list of all chemicals and their masses as it pertains to the chromatography step.

12.2 kg of water is added to the solution to dissolve the cake. The rest of the chemicals remain in negligible amounts, and they can be considered absent for all practical purposes.

In (kg) Out (kg)

PPB 0.014 Negligible (0)

PGK 0.0406 Negligible (0)

PGA N/A N/A

HPGMEHCL 0.0994 Negligible (0)

Water 20 18.5

2-Butanol 0.56 Negligible (0)

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Table 7. Freeze Drying: A freeze-drying method is used to remove the remaining water. All that

remains after drying is 5 kg of amoxicillin in high purity.

In (kg) Out (kg)

PPB N/A N/A

PGK N/A N/A

PGA N/A N/A

HPGMEHCL N/A N/A

Water 14 N/A

2-Butanol N/A N/A

Amoxicillin 5 5

VII. Energy Usage

The energy that each piece of equipment uses per day was calculated using the power consumption of similar processes and it is represented in Tables 8 and 9. The cost was then calculated using the current price for electricity.

Table 8. Equipment energy usage per day.

Equipment Power Column2 Energy KWh Cost

Bioreactor 1883.73 kW 30 min 3.4 MJ 941.865 $113.02

Ultrafiltration insignificant 0

washing insignificant 0

blending 1883.73kW 30 min 3.4MJ 941.865 $113.02 freeze drying 230 kW 11 hour 9.1MJ 2530 $303.6 plate and frame filter 4kW 30 min 7200kJ 2 $0.24 gel chromatography 600kW 60 min 2.16MJ 600 $72

HPLC 800W 30 1.44MJ 400 $48

Table 9. Total cost of energy used per day

Current Price of Electricity Total Cost per day total energy

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VIII. Cost Analysis

Table 10. Budget Analysis: The objective for this process was to produce 5kg or amoxicillin per

day using the aforementioned process and equipment. The table below shows the cost of equipment, chemicals, and final product required to meet this objective.

IX.Conclusion

The production of β-lactam antibiotics has been successfully conducted utilizing many different methods. This production has become much more efficient through many experiments and technological advancements. The effectiveness of amoxicillin to as a broad-spectrum antibiotic has driven research forward. This research has generated several methods of producing a cheaper and higher quality product. The one pot enzymatic production process was researched to analyze the cost and feasibility. The results of the cost analysis in Table 10 illustrate that sales of amoxicillin at the retail price of $5.4/g, and a production rate of 5kg/day could generate a net profit of $25,000 per run and $9,125,000 per year. From the research done in this paper, the runtime of production would be 13.5 hours and it would yield $1852 of amoxicillin per hour. After 80 runs of the process, the debt from the equipment cost would be cleared, resulting in pure profit. This

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research and its findings indicate that this design for the production of amoxicillin is both feasible and profitable.

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Amoxicillin Production: Designing a Process