Production of antibiotics

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Production of antibiotics is a naturally occurring event, that thanks to advances in science can now be replicated and improved upon in laboratory settings. Due to the discovery of penicillin by Alexander Fleming, and the efforts of Florey and Chain in 1938, large-scale, pharmaceutical production of antibiotics has been made possible. As with the initial discovery of penicillin, most antibiotics have been discovered as a result of happenstance. Antibiotic production can be grouped into three methods: natural fermentation, semi-synthetic, and synthetic. As more and more bacteria continue to develop resistance to currently produced antibiotics, research and development of new antibiotics continues to be important. In addition to research and development into the production of new antibiotics, repackaging delivery systems is important to improving efficacy of the antibiotics that are currently produced. Improvements to this field have seen the ability to add antibiotics directly into implanted devices, aerosolization of antibiotics for direct delivery, and combination of antibiotics with non antibiotics to improve outcomes. The increase of antibiotic resistant strains of pathogenic bacteria has led to an increased urgency for the funding of research and development of antibiotics and a desire for production of new and better acting antibiotics.

Contents

Identifying useful antibiotics

An agar plate streaked with microorganisms Klebsiella pneumoniae 01.png
An agar plate streaked with microorganisms

Despite the wide variety of known antibiotics, less than 1% of antimicrobial agents have medical or commercial value. For example, whereas penicillin has a high therapeutic index as it does not generally affect human cells, this is not so for many antibiotics. Other antibiotics simply lack advantage over those already in use, or have no other practical applications.

Useful antibiotics are often discovered using a screening process. To conduct such a screen, isolates of many different microorganisms are cultured and then tested for production of diffusible products that inhibit the growth of test organisms. Most antibiotics identified in such a screen are already known and must therefore be disregarded. The remainder must be tested for their selective toxicities and therapeutic activities, and the best candidates can be examined and possibly modified.

A more modern version of this approach is a rational design program. This involves screening directed towards finding new natural products that inhibit a specific target, such as an enzyme only found in the target pathogen, rather than tests to show general inhibition of a culture.

Research into antibiotic identification has shown the opportunity exists to move away from lawn spotting methodology, a methodology which increases the chances of cross contamination. This new methodology involves using Lactobacillus species and shows a clear zone of inhibition as well as allowing for a determination of minimum inhibitory concentration. [1]

Industrial production techniques

Fermentation

Industrial microbiology can be used to produce antibiotics via the process of fermentation, where the source microorganism is grown in large containers (100,000–150,000 liters or more) containing a liquid growth medium. Oxygen concentration, temperature, pH and nutrient are closely controlled. As antibiotics are secondary metabolites, the population size must be controlled very carefully to ensure that maximum yield is obtained before the cells die. Once the process is complete, the antibiotic must be extracted and purified to a crystalline product. This is easier to achieve if the antibiotic is soluble in organic solvent. Otherwise it must first be removed by ion exchange, adsorption or chemical precipitation.

Semi-synthetic

A common form of antibiotic production in modern times is semi-synthetic. Semi-synthetic production of antibiotics is a combination of natural fermentation and laboratory work to maximize the antibiotic. Maximization can occur through efficacy of the drug itself, amount of antibiotics produced, and potency of the antibiotic being produced. Depending on the drug being produced and the ultimate usage of said antibiotic determines what one is attempting to produce.

An example of semi-synthetic production involves the drug ampicillin. A beta lactam antibiotic just like penicillin, ampicillin was developed by adding an addition amino group (NH2) to the R group of penicillin. [2] This additional amino group gives ampicillin a broader spectrum of use than penicillin. Methicillin is another derivative of penicillin and was discovered in the late 1950s, [3] the key difference between penicillin and methicillin being the addition of two methoxy groups to the phenyl group. [4] These methoxy groups allow methicillin to be used against penicillinase producing bacteria that would otherwise be resistant to penicillin.

Synthetic

Not all antibiotics are produced by bacteria; some are made completely synthetically in the lab. These include the quinolone class, of which nalidixic acid is often credited as the first to be discovered. [5] Like other antibiotics before it the discovery of nalidixic acid has been chalked up to an accident, discovered when George Lesher was attempting to synthesize chloroquine. However a recent investigation into the origin of quinolones have discovered that a description for quinolones happened in 1949 and that patents were filed concerning quinolones some 5 years before Lesher's discovery. [6]

Strains used for the production

In the earliest years of antibiotic discovery the antibiotics being discovered were naturally produced antibiotics and were either produced by fungi, such as the antibiotic penicillin, or by soil bacteria, which can produce antibiotics including streptomycin and tetracycline. [7]

Microorganisms used in fermentation are rarely identical to the wild type. This is because species are often genetically modified to yield the maximum amounts of antibiotics. Mutation is often used, and is encouraged by introducing mutagens such as ultraviolet radiation, x-rays or certain chemicals. Selection and further reproduction of the higher yielding strains over many generations can raise yields by 20-fold or more. Another technique used to increase yields is gene amplification, where copies of genes coding for enzymes involved in the antibiotic production can be inserted back into a cell, via vectors such as plasmids. This process must be closely linked with retesting of antibiotic production.

Some antibiotics are produced naturally by fungi. These include the cephalosporin producing Acremonium chrysogenum. [8]

Geldanamycin is produced by Streptomyces hygroscopicus. [9]

Erythromycin is produced by what was called Streptomyces erythreus and is now known as Saccharopolyspora erythraea. [10]

Streptomycin is produced by Streptomyces griseus . [11]

Tetracycline is produced by Streptomyces aureofaciens [12]

Vancomycin is produced by Streptomyces orientalis, now known as Amycolatopsis orientalis. [13]

Advancements

Penicillin was the first of the antibiotics to be discovered. After the discovery there was the issue of taking the raw naturally produced penicillin and developing a method so that wide-scale production of a clinically significant antibiotic could occur. Over the course of many years a team led by Florey and Chain and based in Oxford was able to successfully purify, concentrate, and produce the antibiotic.

Advances in scientific technology have not always led to better conditions for the production of antibiotics. Since 1987 there have been no new classes of antibiotics discovered for industrial production and widespread usage. However new developments in genomic sequencing and technology have led to improvements and discovery in the field of antibiotic production. Genomic engineering of antibiotic gene clusters has already been shown to lead to an increase in production of different antibiotics. [14]

Antibiotic production and delivery method

Antibiotics do not render themselves fully functional and deliverable simply by being produced. Often modifications must be made to the antibiotics so that maximum efficiency is attained. Post-production modifications include making antibiotics aerosolized so as to bypass doing unnecessary damage to bacteria located in other parts of the body and instead going directly to the lungs. Nosocomial infections can lead to serious complications during and in the recovery following surgery or a hospital stay in general. By merging surgical implants with antibiotics, healthcare providers are able to strike at a specific high risk area of infection without having to use a body wide size dosage of antibiotics.

Meropenem is an antibiotic that is delivered into the body via injection. When produced meropenem is a crystalline antibiotic, so it must be mixed in with solution before injection can occur. During this process meropenem is mixed with sodium carbonate, then diluted in water after which it can be injected. [15]

Aerosolization of antibiotics is necessary because infections of the lung are especially troublesome, which is why direct targeting of the infection is needed. Broad spectrum antibiotics can have detrimental side effects when their action is also taken against necessary non-pathogenic bacteria residing in the human microbiome. Aerosolization is effective in bypassing the microbiome that exists in the gastrointestinal tract by directing the antibiotic directly to the lungs. This process is undertaken after the production of the antibiotic itself.

The rise of antibiotic-resistant bacteria has affected implantation of medical devices. In some cases it is no longer enough for devices to be sterile when they are implanted into an individual, now they must be proactive in fighting off bacterial infection. As such antibiotics are now being added into the surface of implanted devices as an added layer of defense against the threat of infection. One such infection is Osteomyelitis which can offer a unique challenges in treatment efforts, one novel approach has been the creation of antibiotic cement nails which can be inserted into the infected bone. First described by Paley and Herzenberg [16] antibiotic cement nails have dual purpose, both of stabilization of the bone being treated, and prevention against post-procedure infection. Antibiotic cement nails are inserted during surgery, and are produced around the time of procedure using materials available in the operating room setting. Antibiotics are mixed in with cement filling then molded around a support anchor, often chest tubes are used to ensure proper molding. Chest tubes have the advantage of being cheap and ubiquitous and have been shown to have uniformity in the production of antibiotic cement nails. The antibiotics fill the voids within the cement matrix, and upon drying and setting can be inserted into the bone. The antibiotic has a direct contact with the area of infection and retains its properties in acting upon the infection. In addition to cement nails, antibiotic cement spacers have been used to treat and prevent osteomyelitis, and for a longer period of time. In producing the antibiotic cement material it is necessary to choose antibiotics that will be effective in this hybrid form, it has been found that antibiotics in powder form that are broad spectrum are of best use. There are recommendations for the amount of antibiotic that is used when mixing in with the cement, but industry wide guidelines have not been established.

Challenges

Development of antibiotics is difficult, whereas many drug discoveries have been a result of concerted effort and intensive research and development, antibiotics have seemingly been discovered by chance. Since 1987 there have been no discoveries or development of a new class of antibiotics. This is partly due to the finicky nature of antibiotics. As most are produced biosynthetically they require an organism to produce. Historically this has meant that different species are grown and observed for any antimicrobial activity. Not only does this require a culturable species to start off with, but the conditions the species are grown in must be adequate for production of antibiotics as well as having the number of antibiotics produced reach a density threshold so that their function can be observed.

Another reason behind the lack of new antibiotic production is the diminishing amount of return on investment for antibiotics and thus the lack resources put into research and development by private pharmaceutical companies. [17] The World Health Organization has recognized the danger of antibiotic resistance bacteria and has created a list of "priority pathogens" that are of the utmost concern. [18] In doing so the hope is to stimulate R&D that can create a new generation of antibiotics. In the United States, the Biomedical Advanced Research and Development Authority (BARDA) aims to support the work of the industry to produce new antibiotics. [19]

The buildup of inorganic phosphate can limit the biosynthetic production of certain antibiotics, researchers found that by using an inorganic phosphate trapping agent, the phosphate would be sequestered away and antibiotic production would return to normal levels, [20] thus allowing production to continue.

Meropenem is mixed with sodium carbonate post-production before being injected into the body, subsequent analysis of this mixture using Nuclear Magnetic Resonance has shown that a second form of Meropenem is produced. This second form has an additional carbon dioxide on it, and exists alongside the pure form. [21] In order to ensure that Meropenem stays in the correct form a four-step process was developed wherein the crude form is mixed together with a base in water, a proper pH is established, the product is treated with alkanols, and then the pure form is isolated. [22]

Related Research Articles

<span class="mw-page-title-main">Antibiotic</span> Antimicrobial substance active against bacteria

An antibiotic is a type of antimicrobial substance active against bacteria. It is the most important type of antibacterial agent for fighting bacterial infections, and antibiotic medications are widely used in the treatment and prevention of such infections. They may either kill or inhibit the growth of bacteria. A limited number of antibiotics also possess antiprotozoal activity. Antibiotics are not effective against viruses such as the ones which cause the common cold or influenza; drugs which inhibit growth of viruses are termed antiviral drugs or antivirals rather than antibiotics. They are also not effective against fungi; drugs which inhibit growth of fungi are called antifungal drugs.

<span class="mw-page-title-main">Ampicillin</span> Antibiotic

Ampicillin is an antibiotic belonging to the aminopenicillin class of the penicillin family. The drug is used to prevent and treat a number of bacterial infections, such as respiratory tract infections, urinary tract infections, meningitis, salmonellosis, and endocarditis. It may also be used to prevent group B streptococcal infection in newborns. It is used by mouth, by injection into a muscle, or intravenously.

<span class="mw-page-title-main">Beta-lactamase</span> Class of enzymes

Beta-lactamases (β-lactamases) are enzymes produced by bacteria that provide multi-resistance to beta-lactam antibiotics such as penicillins, cephalosporins, cephamycins, monobactams and carbapenems (ertapenem), although carbapenems are relatively resistant to beta-lactamase. Beta-lactamase provides antibiotic resistance by breaking the antibiotics' structure. These antibiotics all have a common element in their molecular structure: a four-atom ring known as a beta-lactam (β-lactam) ring. Through hydrolysis, the enzyme lactamase breaks the β-lactam ring open, deactivating the molecule's antibacterial properties.

<span class="mw-page-title-main">Penicillin</span> Group of antibiotics derived from Penicillium fungi

Penicillins are a group of β-lactam antibiotics originally obtained from Penicillium moulds, principally P. chrysogenum and P. rubens. Most penicillins in clinical use are synthesised by P. chrysogenum using deep tank fermentation and then purified. A number of natural penicillins have been discovered, but only two purified compounds are in clinical use: penicillin G and penicillin V. Penicillins were among the first medications to be effective against many bacterial infections caused by staphylococci and streptococci. They are still widely used today for different bacterial infections, though many types of bacteria have developed resistance following extensive use.

This is the timeline of modern antimicrobial (anti-infective) therapy. The years show when a given drug was released onto the pharmaceutical market. This is not a timeline of the development of the antibiotics themselves.

<span class="mw-page-title-main">Ertapenem</span> Antibiotic medication

Ertapenem, sold under the brand name Invanz, is a carbapenem antibiotic medication used for the treatment of infections of the abdomen, the lungs, the upper part of the female reproductive system, and the diabetic foot.

<span class="mw-page-title-main">Methicillin</span> Antibiotic medication

Methicillin (USAN), also known as meticillin (INN), is a narrow-spectrum β-lactam antibiotic of the penicillin class.

<span class="mw-page-title-main">Broad-spectrum antibiotic</span> Treatment for a wide range of bacteria

A broad-spectrum antibiotic is an antibiotic that acts on the two major bacterial groups, Gram-positive and Gram-negative, or any antibiotic that acts against a wide range of disease-causing bacteria. These medications are used when a bacterial infection is suspected but the group of bacteria is unknown or when infection with multiple groups of bacteria is suspected. This is in contrast to a narrow-spectrum antibiotic, which is effective against only a specific group of bacteria. Although powerful, broad-spectrum antibiotics pose specific risks, particularly the disruption of native, normal bacteria and the development of antimicrobial resistance. An example of a commonly used broad-spectrum antibiotic is ampicillin.

<span class="mw-page-title-main">Meropenem</span> Broad-spectrum antibiotic

Meropenem, sold under the brand name Merrem among others, is an intravenous β-lactam antibiotic used to treat a variety of bacterial infections. Some of these include meningitis, intra-abdominal infection, pneumonia, sepsis, and anthrax.

<span class="mw-page-title-main">Clavulanic acid</span> Molecule used to overcome antibiotic resistance in bacteria

Clavulanic acid is a β-lactam drug that functions as a mechanism-based β-lactamase inhibitor. While not effective by itself as an antibiotic, when combined with penicillin-group antibiotics, it can overcome antibiotic resistance in bacteria that secrete β-lactamase, which otherwise inactivates most penicillins.

<span class="mw-page-title-main">Carbapenem</span> Class of highly effective antibiotic agents

Carbapenems are a class of very effective antibiotic agents most commonly used for treatment of severe bacterial infections. This class of antibiotics is usually reserved for known or suspected multidrug-resistant (MDR) bacterial infections. Similar to penicillins and cephalosporins, carbapenems are members of the beta-lactam antibiotics drug class, which kill bacteria by binding to penicillin-binding proteins, thus inhibiting bacterial cell wall synthesis. However, these agents individually exhibit a broader spectrum of activity compared to most cephalosporins and penicillins. Furthermore, carbapenems are typically unaffected by emerging antibiotic resistance, even to other beta-lactams.

Ampicillin/sulbactam is a fixed-dose combination medication of the common penicillin-derived antibiotic ampicillin and sulbactam, an inhibitor of bacterial beta-lactamase. Two different forms of the drug exist. The first, developed in 1987 and marketed in the United States under the brand name Unasyn, generic only outside the United States, is an intravenous antibiotic. The second, an oral form called sultamicillin, is marketed under the brand name Ampictam outside the United States, and generic only in the United States. Ampicillin/sulbactam is used to treat infections caused by bacteria resistant to beta-lactam antibiotics. Sulbactam blocks the enzyme which breaks down ampicillin and thereby allows ampicillin to attack and kill the bacteria.

<span class="mw-page-title-main">Flucloxacillin</span> Penicillin

Flucloxacillin, also known as floxacillin, is an antibiotic used to treat skin infections, external ear infections, infections of leg ulcers, diabetic foot infections, and infection of bone. It may be used together with other medications to treat pneumonia, and endocarditis. It may also be used prior to surgery to prevent Staphylococcus infections. It is not effective against methicillin-resistant Staphylococcus aureus (MRSA). It is taken by mouth or given by injection into a vein or muscle.

<span class="mw-page-title-main">Lincosamides</span> Group of antibiotics

Lincosamides are a class of antibiotics, which include lincomycin, clindamycin, and pirlimycin.

<span class="mw-page-title-main">Dicloxacillin</span> Chemical compound

Dicloxacillin is a narrow-spectrum β-lactam antibiotic of the penicillin class. It is used to treat infections caused by susceptible (non-resistant) Gram-positive bacteria. It is active against beta-lactamase-producing organisms such as Staphylococcus aureus, which would otherwise be resistant to most penicillins. Dicloxacillin is available under a variety of trade names including Diclocil (BMS).

<span class="mw-page-title-main">Cefoxitin</span> Chemical compound

Cefoxitin is a second-generation cephamycin antibiotic developed by Merck & Co., Inc. from Cephamycin C in the year following its discovery, 1972. It was synthesized in order to create an antibiotic with a broader spectrum. It is often grouped with the second-generation cephalosporins. Cefoxitin requires a prescription and as of 2010 is sold under the brand name Mefoxin by Bioniche Pharma, LLC. The generic version of cefoxitin is known as cefoxitin sodium.

<span class="mw-page-title-main">Thienamycin</span> Chemical compound

Thienamycin is one of the most potent naturally produced antibiotics known thus far, discovered in Streptomyces cattleya in 1976. Thienamycin has excellent activity against both Gram-positive and Gram-negative bacteria and is resistant to bacterial β-lactamase enzymes. Thienamycin is a zwitterion at pH 7.

β-Lactamase inhibitor Family of enzymes

Beta-lactamases are a family of enzymes involved in bacterial resistance to beta-lactam antibiotics. In bacterial resistance to beta-lactam antibiotics, the bacteria have beta-lactamase which degrade the beta-lactam rings, rendering the antibiotic ineffective. However, with beta-lactamase inhibitors, these enzymes on the bacteria are inhibited, thus allowing the antibiotic to take effect. Strategies for combating this form of resistance have included the development of new beta-lactam antibiotics that are more resistant to cleavage and the development of the class of enzyme inhibitors called beta-lactamase inhibitors. Although β-lactamase inhibitors have little antibiotic activity of their own, they prevent bacterial degradation of beta-lactam antibiotics and thus extend the range of bacteria the drugs are effective against.

References

  1. ↑ Dubourg, Grégory; Elsawi, Ziena; Raoult, Didier (November 2015). "Assessment of the in vitro antimicrobial activity of Lactobacillus species for identifying new potential antibiotics". International Journal of Antimicrobial Agents. 46 (5): 590–593. doi:10.1016/j.ijantimicag.2015.05.011. PMID   26163158.
  2. ↑ Raynor, B.Denise (1997). "Penicillin and ampicillin". Primary Care Update for OB/GYNS. 4 (4): 147–152. doi:10.1016/s1068-607x(97)00012-7.
  3. ↑ Waness, Abdelkarim (2010-01-01). "Revisiting methicillin-resistant Staphylococcus aureus infections". Journal of Global Infectious Diseases. 2 (1): 49–56. doi: 10.4103/0974-777x.59251 . PMC   2840971 . PMID   20300418.
  4. ↑ STAPLETON, PAUL D.; TAYLOR, PETER W. (2002-01-01). "Methicillin resistance in Staphylococcus aureus". Science Progress. 85 (Pt 1): 57–72. doi:10.3184/003685002783238870. ISSN   0036-8504. PMC   2065735 . PMID   11969119.
  5. ↑ Emmerson, A. M.; Jones, A. M. (2003-05-01). "The quinolones: decades of development and use". Journal of Antimicrobial Chemotherapy. 51 (suppl_1): 13–20. doi: 10.1093/jac/dkg208 . ISSN   0305-7453. PMID   12702699.
  6. ↑ Bisacchi, Gregory S. (2015-06-25). "Origins of the Quinolone Class of Antibacterials: An Expanded "Discovery Story"". Journal of Medicinal Chemistry. 58 (12): 4874–4882. doi: 10.1021/jm501881c . ISSN   0022-2623. PMID   25738967.
  7. ↑ Clardy, Jon; Fischbach, Michael; Currie, Cameron (2009-06-09). "The natural history of antibiotics". Current Biology. 19 (11): R437–R441. doi:10.1016/j.cub.2009.04.001. ISSN   0960-9822. PMC   2731226 . PMID   19515346.
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  9. ↑ "Geldanamycin and process for producing same". 1969-03-26.{{cite journal}}: Cite journal requires |journal= (help)
  10. ↑ Wu, Jiequn; Zhang, Qinglin; Deng, Wei; Qian, Jiangchao; Zhang, Siliang; Liu, Wen (2011-11-01). "Toward Improvement of Erythromycin A Production in an Industrial Saccharopolyspora erythraea Strain via Facilitation of Genetic Manipulation with an Artificial attB Site for Specific Recombination". Applied and Environmental Microbiology. 77 (21): 7508–7516. Bibcode:2011ApEnM..77.7508W. doi:10.1128/AEM.06034-11. ISSN   0099-2240. PMC   3209160 . PMID   21841022.
  11. ↑ de Lima Procópio, Rudi Emerson; Silva, Ingrid Reis da; Martins, Mayra Kassawara; Azevedo, João Lúcio de; Araújo, Janete Magali de (2012). "Antibiotics produced by Streptomyces". The Brazilian Journal of Infectious Diseases. 16 (5): 466–471. doi: 10.1016/j.bjid.2012.08.014 . PMID   22975171.
  12. ↑ Darken, Marjorie A.; Berenson, Herman; Shirk, Richard J.; Sjolander, Newell O. (1960-01-01). "Production of Tetracycline by Streptomyces aureofaciens in Synthetic Media". Applied Microbiology. 8 (1): 46–51. doi:10.1128/AEM.8.1.46-51.1960. ISSN   0003-6919. PMC   1057549 . PMID   13814119.
  13. ↑ Martin, Elizabeth (2015). Vancomycin - Oxford Reference. doi:10.1093/acref/9780199687817.001.0001. ISBN   9780199687817.
  14. ↑ Du, Deyao; Wang, Lu; Tian, Yuqing; Liu, Hao; Tan, Huarong; Niu, Guoqing (2015-03-04). "Genome engineering and direct cloning of antibiotic gene clusters via phage ϕBT1 integrase-mediated site-specific recombination in Streptomyces". Scientific Reports. 5: 8740. Bibcode:2015NatSR...5E8740D. doi:10.1038/srep08740. ISSN   2045-2322. PMC   4349145 . PMID   25737113.
  15. ↑ "Meropenem For Injection, USPFOR INTRAVENOUS USE ONLY". dailymed.nlm.nih.gov. Retrieved 2017-04-10.
  16. ↑ Paley, Dror; Herzenberg, John E. (2002-11-01). "Intramedullary infections treated with antibiotic cement rods: preliminary results in nine cases". Journal of Orthopaedic Trauma. 16 (10): 723–729. doi:10.1097/00005131-200211000-00007. ISSN   0890-5339. PMID   12439196. S2CID   46632207.
  17. ↑ Nathan, Carl; Goldberg, Frederick M. (2005). "Outlook: The profit problem in antibiotic R&D". Nature Reviews Drug Discovery. 4 (11): 887–891. doi: 10.1038/nrd1878 . PMID   16247440. S2CID   37126421.
  18. ↑ "WHO publishes list of bacteria for which new antibiotics are urgently needed". World Health Organization. Retrieved 18 April 2017.
  19. ↑ "BARDA Broad Spectrum Antimicrobials (BSA) program - ASPR Blog". www.phe.gov. Retrieved 18 April 2017.
  20. ↑ MASUMA, ROKURO; TANAKA, YOSHITAKE; TANAKA, HAJIME; OMURA, SATOSHI (1 January 1986). "Production of nanaomycin and other antibiotics by phosphate-depressed fermentation using phosphate-trapping agents". The Journal of Antibiotics. 39 (11): 1557–1564. doi: 10.7164/antibiotics.39.1557 . ISSN   0021-8820. PMID   3793625.
  21. ↑ Almarsson, Örn; Kaufman, Michael J.; Stong, John D.; Wu, Yunhui; Mayr, Suzanne M.; Petrich, Mark A.; Williams, J. Michael (1998-05-01). "Meropenem Exists in Equilibrium with a Carbon Dioxide Adduct in Bicarbonate Solution". Journal of Pharmaceutical Sciences. 87 (5): 663–666. doi:10.1021/js970370u. ISSN   0022-3549. PMID   9572920.
  22. ↑ EP 2303225,George, Vinod; Vashishta, Bhupendra& Prasad, Mohan,"Compositions of Carbon Dioxide Adduct of Ertapenem and Polymorphic Forms of Ertapenem Monosodium Salt",published 2013-11-20

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