Introduction

Marketing & pharmaceutical marketing

Pharmaceutical marketing, sometimes called medico-marketing or pharma marketing in some countries, is the business of advertising or otherwise promoting the sale of pharmaceuticals or drugs. There is some evidence that marketing practices can negatively affect both patients and the health care profession. Many countries have measures in place to limit advertising by pharmaceutical companies. Pharmaceutical company spending on marketing far exceeds that spent on research. In Canada, $1.7 billion was spent in 2004 to market drugs to physicians; in the United States, $21 billion was spent in 2002. In 2005 money spent on pharmaceutical marketing in the US was estimated at $29.9 billion with one estimate as high as $57 billion. When the US numbers are broken down, 56% was free samples, 25% was detailing of physicians, 12.5% was direct to user advertising, 4% on hospital detailing, and 2% on journal ads. The marketing of medication has a long history. The sale of miracle cures, many with little real potency, has always been common. Marketing of legitimate nonprescription medications, such as pain relievers or allergy medicine, has also long been practiced, although, until recently, mass marketing of prescription medications has been rare. It was long believed that since doctors made the selection of drugs, mass marketing was a waste of resources; specific ads targeting the medical profession were thought to be cheaper and just as effective. This would involve ads in professional journals and visits by sales staff to doctors offices and hospitals. An important part of these efforts was

marketing to medical students

To health care providers

Marketing to health care providers takes four main forms: gifting, detailing, drug samples, and sponsoring continuing medical education (CME).Of the 237,000 medical sites representing 680,000 physicians surveyed in SK&A's 2010 Physician Access survey, half said they prefer or require an appointment to see a rep (up from 38.5% preferring or requiring an appointment in 2008), while 23% won't see reps at all, according to the survey data. Practices owned by hospitals or health systems are tougher to get into than private practices, since appointments have to go through headquarters, the survey found. 13.3% of offices with just one or two doctors won't see reps, compared with a no-see rate of 42% at offices with 10 or more docs The most accessible physicians for promotional purposes are allergists/immunologists only 4.2% won't see reps at all followed by orthopedic specialists (5.1%) and diabetes specialists (7.6%). Diagnostic radiologists are the most rigid about allowing details 92.1% won't see reps followed by pathologists and neuroradiologists, at 92.1% and 91.8%, respectively. Edetailing is widely used to reach "no see physicians"; approximately 23% of primary care physicians and 28% of specialists prefer computer-based edetailing, according to survey findings reported in the April 25, 2011, edition of American Medical News (AMNews), published by the American Medical Association (AMA).

New pharma code & guidelines

The Pharmaceutical Research and Manufacturers of America (PhRMA) released updates to its voluntary Code on Interactions with Healthcare Professionals on July 10. The new guidelines take effect January 2009." In addition to prohibiting small gifts and reminder items such as pens, notepads, staplers, clipboards, pill boxes, etc., the revised Code: 1. Prohibits company sales representatives from providing restaurant meals to healthcare professionals, but allows them to provide occasional meals in healthcare professionals offices in conjunction with informational presentations" 2. Includes new provisions requiring companies to ensure their representatives are sufficiently trained about applicable laws, regulations, and industry codes of practice and ethics. 3. Provides that each company will state its intentions to abide by the Code and that company CEOs and compliance officers will certify each year that they have processes in place to comply 4. Includes more detailed standards regarding the independence of continuing medical education. 5. Provides additional guidance and restrictions for speaking and consulting arrangements with healthcare professionals. However, the Good Works Health government-approved platform offers physicians and other health care professionals the opportunity to direct donations to charities of their choice in exchange for participation in pharmaceutical promotional/educational programs.

Free samples
Free samples have been shown to affect physician prescribing behaviour. Physicians with access to free samples are more likely to prescribe brand name medication over equivalent generic medications. Other studies found that free samples decreased the likelihood that physicians would follow standard of care practices. Receiving pharmaceutical samples does not reduce prescription costs. Even after receiving samples, sample recipients remain disproportionately burdened by prescription costs. It is argued that a benefit to free samples is the try it before you buy it approach. Free Samples give immediate access to the medication and the patient can begin treatment right away. Also, it saves time from going to a pharmacy to get it filled before treatment begins. Since not all medications work for everyone, and many do not work the same way for each person, free samples allow patients to find which dose and brand of medication works best before having to spend money on a filled prescription at a pharmacy.

Sale and techniques of sales

To become a successful sales representative you need to have a solid understanding of selling concepts. These concepts give you the foundation to understand why customers act the way they do and provide a guide as to how to use your selling skills to overcome obstacles and increase sales.

Successful Selling Steps If we consider the steps most successful sales representatives go through to increase their effectiveness on sales calls, we will see there are three general areas for consideration:

1. 2. 3.

Business Planning The Sales Call (pre-call, sales call, post-call) Follow-Up

Business planning is a critical step in ensuring your selling skills are most effective. Sales representatives in the healthcare industry manage their territories as if they were running their own business. To be successful in your sales territory you need to fully understand it before you attempt to call on customers. This includes knowing which customers drive your business, what their buying style is so you can match your selling style to it, managing an investment budget, managing your daily and weekly schedule, and working with your team to implement the territory business plans. You can view the business planning process as creating a blueprint for success. Most healthcare companies will give you a territory, a list of target customers, the sales tools needed to influence your target customers and a budget. It is up to you and your territory team to come up with a plan that will most effectively make use of these resources to increase sales. Following the business planning process, there are three main steps to consider to improve your sales call effectiveness: 1. 2. 3. Pre-call planning (before you meet the customer) The sales call (face to face with the customer) Post-call analysis and follow-up (after your sales call)

Pre-call planning done prior to meeting your customer involves several steps such as reviewing past customer call notes, setting call objectives and preparing sales tools to be most effective in the short time with your customer. This will be covered in more detail shortly.

During the actual sales call itself, there are several steps to be aware of: opening, presenting features and benefits, probing, objection handling, etc. Making an effective sales call involves excellent skills within each of these areas and we will cover them more in-depth in the coming chapters.

After leaving each and every sales call, post-call analysis needs to be completed, and any follow-up required accurately recorded and completed. These notes which you make post-call are what you will be reviewing prior to your next appointment with your customers. Without having these notes, your effectiveness on the next call will not be as high.

One major goal of a healthcare sales representative is to be seen as a valued consultant by customers. Once you are viewed by your target customers as an excellent and credible resource (and not just another sales representative) you will have increased access and time with them and increased face-to-face selling time is a key ingredient for success in healthcare sales. Ensuring timely follow-up to customer requests in a professional manner will go a long way in ensuring you become that value added consultant in their practices.

The Sales Cycle A sales cycle is the stages that a typical customer goes through before agreeing to purchase or use your product. Although the ultimate goal is a sale, there are several stages which you must take the customer through before you can ask for the business. In the first stage, called Introduction and Awareness, the customer is not yet aware that you or your products exist. They may not even know about any problems they are currently facing because they have not taken the time to consider them. Your goal with a customer at this stage is to introduce your products and services and try to identify any problem areas which your product can solve for them, their patients or the healthcare system. Moving a customer through this stage is sometimes not easy and requires certain sales techniques. Techniques used to move a customer from this stage to the next one are rapport building, probing, objection handling, and gaining feedback. Throughout this book you will learn how to improve each of these skills to be successful in a healthcare sales role.

The second stage is Identification. At this stage you need to convince the customer that a problem does indeed exist. This is sometimes the hardest stage to get through as many customers will tell you that they are happy with their current product. To move the customer through this stage, sales skills such as presenting features and benefits, probing, gaining feedback, and the ability to handle objections will be needed. In the third stage, the Knowledge stage, you must educate the customer on what your company and products offer using the features and benefits of your products and or services. Without the proper knowledge of what you and your company can offer (to the

customer, the patient or the healthcare system), the customer will not be able to evaluate his options properly. Excellent use of features and benefits, probing, and objection handling will help you to move the customer through this stage to the evaluation stage.

The Evaluation stage is where the customer takes the time to compare your product to that which he is currently using. It is sometimes extremely difficult to change the habits of a customer, so persuasion and persistence is needed. Realizing where the customer is at within this sales cycle will help you to maintain perspective and use the appropriate sales skills to move him along the sales cycle. This is where your needs analysis is critical, as you want to ensure you are reinforcing, on a frequent basis, the benefits your product or service offers versus your competition in addressing THAT particular customers needs. To move the customer through this stage you need to present strong features and benefits, use excellent competitive selling techniques in addition to probing and strong skills.

In the final stage, the customer makes a Decision to use your product or service or your competitors. If he has chosen your product, you can feel good about taking your customer through the sales cycle and convincing him of the benefits of your product versus the competition. If the customer does not choose your product or service, then your job will be to take him back to the evaluation stage so he can re-evaluate the options and reconsider your product. Sometimes this is very difficult to do, as they may be very happy with what they are currently using. You may have to take them all the way back to the identification stage to find new opportunities or problems which your product can address and your competitor cannot. This is a very dynamic model, and each customer you deal with will be at a different stage in this cycle. It is your job to identify where that

customer is and try to get him to the next stage using effective selling techniques.

Think of the sales cycle as a series of steps that you are climbing. Before you reach the top step (the sale), you have to walk the customer up each of the lower steps first. It is very rare that you will meet a customer for the first time and walk away with a sale. You need to complete the steps in the cycle and earn the right to ask for the business.

Another concept to keep in mind is that the time needed to move a customer through the sales cycle varies based on several factors:

1.

Type of product Healthcare sales products which are more expensive (such as

capital equipment for use in hospitals) often requires consensus from a committee before the contract is signed. As a result, to get all members of the committee to agree takes quite a bit longer than a sale where only one customer needs to say yes.

2.

Frequency of sales calls The more frequently you are able to get face to face

selling time with a customer, the quicker you will be able to move them through the sales cycleIF you have developed excellent selling skills.

3.

Representative selling skills Without effective selling skills, you will not be able

to move customers through the sales cycle. To move them more quickly through the sales cycle, you must continually develop and improve on all of the sales skills listed in this book.

Of the three listed here, representative selling skills is by far the most important one which affects the time needed to move a customer through the sales cycle.

The Customer Environment

Before we discuss each stage and the selling skills needed, we will consider the customer environment. The healthcare customer environment and selling into it requires a significant amount of planning, preparation, persistence and versatility to be effective. As you will learn, time is a very valuable commodity in the healthcare office or clinic, and making effective use of your time is critical to move that customer along the sales cycle to a decision which is favorable for your product or service. The key to success in this environment is ensuring you are calling on the right customer, the right number of times using the right message!

Why is it getting more difficult to get time with our customers?

Over the past several years, it has been getting more and more difficult to access customers in the healthcare industry, and those that can be accessed are giving less and less time to sales representatives. We will now have a look at some of the main reasons this is occurring:

1.

Time Pressures Todays professionals try to squeeze more work into a day.

Some physicians see between 30 and 60 patients per day. In addition to seeing patients, physicians need to finish patient charts, fill out insurance and lawyers reports, return phone calls and attend meetings. This is in addition to trying to have a life outside the office and spending quality time with their families. Five minutes with a representative is five minutes they could have spent completing other tasks.

2.

Lack of Monetary Gain Physicians do not get paid to see representatives but

they do get paid to see patients. If they could see a patient in the time it takes to talk to a representative then they are losing money every time they talk to a representative. This reiterates the fact that you must provide value to your target customers on each and every call if you want to continue to get face time with them.

3.

Number of Representatives In many offices that see representatives, it is not

uncommon for six or more representatives to visit on any given day. The sheer number representatives can be overwhelming, not only to the physician, but to the medical office assistants who have to deal with the patients in the waiting room.

4.

Lack of Value Seen This is the most important factor to keep in mind as you

work on your territory. If a physician truly believes that seeing a particular sales representative will add value to his practice, he will take the time away from his patients to hear what that representative has to say. However, if he feels that a representative will bring no value, he will likely not spend any time with him or her. There can be various reasons for this, but the number one reason is that only the best representatives take the time to identify true needs of each customer and sell to that particular need. There are a

lot of representatives who do not take the time to identify needs, involve the customer in the sales call, or prepare for each call to ensure value is seen every time. Without providing value to your key customers you will not be able to get adequate selling time to be effective.

Key Terms

To be most successful as a healthcare sales representative, you must understand two key terms: targeting and frequency.

Targeting: The Right Customer

When considering who your target customers are, there are two key questions to ask: how important is this customer to your business and how accessible is the customer to you?

How important is this customer to your business?

When considering this factor you need to look at not only how busy their practice is, but also what their patient demographics are. Yes, you must call on customers who see many patients per day, but if they are not the right types of patients for your products, then you are wasting your time. For example, if you are selling a medication for high blood pressure and you are calling on a busy physician who sees mainly younger women and

children, this is not likely a good use of your time. It is crucial that you look at both how busy your customer is and what types of patients they most commonly treat. Generally speaking, the more patients a customer sees the better, as long as these are the types of patients your product will help.

How does a representative find out what types of patients a customer may see?

There are many ways including viewing the waiting room to profile the patients waiting to see the physician, looking to see what types of product samples are on the shelves, and asking the medical office assistant or local pharmacist what types of patients the area services. If all else fails, ask your physician! They will usually give you a straight up answer as to the types of patients they look after. Just remember, its not just the busy physicians who are important to you they may be busy, but may not be seeing your targeted patient population. If this is the case, they may not be a true target physician for you.

How accessible is your customer?

If you have a customer who is extremely busy and services the types of patients that would be a great fit for your product, it makes sense that they should be a target for you. However, what if this physician is not willing to see you as a sales representative even though you have tried many ways to see him or her? In this situation, it may be wiser to take them off your target list and replace them with another customer that you have

access to. However, it is important to remember that just because you have tried a couple of times to see this customer and have not had success, it does not mean they are not accessible. You need to try every means possible (using all of the activities you read about in the Introduction to the Pharmaceutical Sales Industry from PharmaCareer) to ensure that there is not some way that you can access this customer. There are many times a customer seems inaccessible but somehow, someway, you manage to make contact with them. These can end up being your best supporters as much of your competition may have given up calling on them, giving you the opportunity to sell to them in a less competitive environment. It is important to remember that on average you will need to make contact with your targeted customers six to twelve times a year to make an impact. Seeing a customer once every two years is not going to make much of an impact on your sales!

Gentamicin is an aminoglycoside antibiotic, used to treat many types of bacterial infections, particularly those caused by Gram-negative organisms.[1] However, gentamicin is not used for Neisseria gonorrhoeae, Neisseria meningitidis or Legionella pneumophila. Gentamicin is also ototoxic and nephrotoxic, with this toxicity remaining a major problem in clinical use.[1] It is synthesized by Micromonospora, a genus of Gram-positive bacteria widely present in the environment (water and soil). To highlight their specific biological origins, gentamicin and other related antibiotics produced by this genus (verdamicin, mutamicin, sisomicin, netilmicin, retymicin) generally have their spellings ending in ~micin and not in ~mycin. Gentamicin is a bactericidal antibiotic that works by binding the 30S subunit of the bacterial ribosome, interrupting protein synthesis.

Like all aminoglycosides, when gentamicin is given orally, it is not systemically active. This is because it is not absorbed to any appreciable extent from the small intestine. It is administered intravenously, intramuscularly or topically to treat infections. It appears to be completely eliminated unchanged in the urine. Urine must be collected for many days to recover all of a given dose because the drug binds avidly to certain tissues. E. coli has shown some resistance to gentamicin, despite being Gram-negative. Reluctance to use gentamicin for empirical therapy has led to increased use of alternative broad-spectrum antibiotics, which some experts suggest has led to the prevalence of antibiotic-resistant bacterial infections by MRSA and other so-called "superbugs".[1] Gentamicin is one of the few heat-stable antibiotics that remain active even after autoclaving, which makes it particularly useful in the preparation of some microbiological growth media. It is used during orthopaedic surgery when high temperatures are required for the setting of cements (e.g. hip replacements).[2] Spectrum of activity Active against a wide range of human bacterial infections, mostly Gram-negative bacteria including Pseudomonas, Proteus, Serratia, and the Gram-positive Staphylococcus.[3] Gentamicin is not used for Neisseria gonorrhoeae, Neisseria meningitidis or Legionella pneumophila bacterial infections (because of the risk of the patient going into shock from lipid A endotoxin found in certain Gram-negative organisms). Gentamicin is also useful against Yersinia pestis, its relatives, and Francisella tularensis (the organism responsible for Tularemia seen often in hunters and/or trappers).[4] Some Enterobacteriaceae, Pseudomonas spp., enterococci, Staphylococcus aureus and other staphylococci are resistant to Gentamicin Sulfate, USP to varying degrees.

GENTAMICIN is the fourth generation antibiotic belongs to a group of aminoglycosides. Definitions: Gentamicin is an aminoglycoside antibiotic used to treat infections caused by a wide range of bacteria. It can be administered by injection or applied in a cream to the skin or in drops to the ears and eyes. Kidney and ear damage may occur at high doses. Trade names: Garamycin, Genticin, Cidomycin. Gentamicin is an antibiotic derived from the fungi of genes micromonospora. Gentamicin is an antibiotic complex consisting of the closely related components, Gentamicin C1, C2, C1a produced by fermentation of micromonospora pupurea or M. echinospora and their variants.

Some historical information suggests manufacturer and year of introduction of the same antibiotic in the respective country.

Tell your doctor if your skin infection persists or if it worsens.

What conditions does gentamicin Top treat? Next: Side Effects >

Related to gentamicin top

Is Your Child Contagious? Anatomy of an Ear Infection Urinary Tract Infections Living Healthy Guide Mouth and Tooth Infections Antibiotic Q&As

Conditions & Treatments Related to gentamicin top

Presence of Boils On the Skin Medications Inflammation and Infection of Nail Cuticle Medications Skin Infection due to a Bacteria Medications Inflammation of a Hair Follicle Medications Skin Ulcer Medications

Side effects
These aminoglycosides are toxic to the sensory cells of the ear, but they vary greatly in their relative effects on hearing versus balance. Gentamicin is a vestibulotoxin, and can cause permanent loss of equilibrioception, caused by damage to the vestibular apparatus of the inner ear, usually if taken at high doses or for prolonged periods of time, but there are well documented cases in which gentamicin completely destroyed the vestibular apparatus after three to five days.[citation needed] A small number of affected individuals have a normally harmless mutation in their mitochondrial RNA (m1555 A>G), that allows the gentamicin to affect their cells. The cells of the ear are particularly sensitive to this, sometimes causing complete hearing loss.[6] However, gentamicin is sometimes used intentionally for this purpose in severe Mnire's disease, to disable the vestibular apparatus. These side effects are most common when the drug is administered via drops directly to the eye. Gentamicin can also be highly nephrotoxic, particularly if multiple doses accumulate over a course of treatment. For this reason gentamicin is usually dosed by body weight. Various formulae exist for calculating gentamicin dosage. Also trough and peak serum levels of gentamicin are monitored during treatment, generally before and after the third dose is infused. Gentamicin, like other aminoglycosides, causes nephrotoxicity by inhibiting protein synthesis in renal cells. This mechanism specifically causes necrosis of cells in the proximal tubule, resulting in acute tubular necrosis which can lead to acute renal failure.[7]

Side effects of gentamicin toxicity vary from patient to patient. Side effects may become apparent shortly after or up to months after gentamicin is administered. Symptoms of gentamicin toxicity include:

Balance difficulty Bouncing, unsteady vision Ringing in the ears (tinnitus) Difficulty multi-tasking, particularly when standing

Psychiatric symptoms related to gentamicin can occur. These include anorexia, confusion, depression, disorientation and visual hallucinations.[8] Immediate professional help should be sought if any of these symptoms or others appear after administration of aminoglycosides. General medical practitioners should refer patients with such symptoms to an otolaryngologist, commonly known as an 'ear, nose, and throat doctor', for comprehensive tests. A number of factors and determinants should be taken into account when using gentamicin, including differentiation between empirical and directed therapy which will affect dosage and treatment period.[1] Many medical practitioners freely administer gentamicin as an antibiotic without advising patients of the severe and permanent potential ramifications of its use. Gentamicin is well known to be a cheap, low-cost yet old medicine when compared with modern alternatives, and is typically US$36 per dosage less than modern alternatives. Many people recover from gentamicin toxicity naturally over time if the drug is discontinued, but they recover slowly and usually incompletely.[citation needed] Sometimes the toxicity of gentamicin can still increase over months after the last dose. Upon cessation of

gentamicin therapy symptoms such as tinnitus and imbalance may become less pronounced. Sensori-neural hearing loss caused by gentamicin toxicity is permanent however.

CLINICAL PHARMACOLOGY
After intramuscular administration of gentamicin sulfate, peak serum concentrations usually occur between 30 to 60 minutes and serum levels are measurable for 6 to 8 hours. When gentamicin is administered by intravenous infusion over a two-hour period, the serum concentrations are similar to those obtained by intramuscular administration. In patients with normal renal function, peak serum concentrations of gentamicin (mcg/mL) are usually up to four times the single intramuscular dose (mg/kg); for example, a 1 mg/kg injection in adults may be expected to result in a peak serum concentration up to 4 mcg/mL; a 1.5 mg/kg dose may produce levels up to 6 mcg/mL. While some variation is to be expected due to a number of variables such as age, body temperature, surface area and physiologic differences, the individual patient given the same dose tends to have similar levels in repeated determinations. Gentamicin administered at 1 mg/kg every eight hours for the usual 7- to 10-day treatment period to patients with normal renal function does not accumulate in the serum. Gentamicin, like all aminoglycosides, may accumulate in the serum and tissues of patients treated with higher doses and for prolonged periods, particularly in the presence of impaired renal function. In adult patients, treatment with gentamicin dosages of 4 mg/kg/day or higher for seven to ten days may result in a slight, progressive rise in both peak and trough concentrations. In patients with impaired renal function, gentamicin is cleared from the body more slowly than in patients with normal renal function. The more severe the impairment, the slower the clearance.

Dosage must be adjusted. Since gentamicin is distributed in extracellular fluid, peak serum concentrations may be lower than usual in adult patients who have a large volume of this fluid. Serum concentrations of gentamicin in febrile patients may be lower than those in afebrile patients given the same dose. When body temperature returns to normal, serum concentrations of the drug may rise. Febrile and anemic states may be associated with a shorter than usual serum half-life. (Dosage adjustment is usually not necessary.) In severely burned patients, the half-life may be significantly decreased and resulting serum concentrations may be lower than anticipated from the mg/kg dose. Protein binding studies have indicated that the degree of gentamicin binding is low. Depending upon the methods used for testing, this may be between 0 and 30%. After initial administration to patients with normal renal function, generally 70% or more of the gentamicin dose is recoverable in the urine in 24 hours; concentrations in urine above 100 mcg/mL may be achieved. Little, if any, metabolic transformation occurs; the drug is excreted principally by glomerular filtration. After several days of treatment, the amount of gentamicin excreted in the urine approaches the daily dose administered. As with other aminoglycosides, a small amount of the gentamicin dose may be retained in the tissues, especially in the kidneys. Minute quantities of aminoglycosides have been detected in the urine weeks after drug administration was discontinued. Renal clearance of gentamicin is similar to that of endogenous creatinine. In patients with marked impairment of renal function, there is a decrease in the concentration of aminoglycosides in urine and in their penetration into defective renal parenchyma. This decreased drug excretion, together with the potential nephrotoxicity of

aminoglycosides, should be considered when treating such patients who have urinary tract infections. Probenecid does not affect renal tubular transport of gentamicin. The endogenous creatinine clearance rate and the serum creatinine level have a high correlation with the half-life of gentamicin in serum. Results of these tests may serve as guides for adjusting dosage in patients with renal impairment (see DOSAGE AND ADMINISTRATION). Following parenteral administration, gentamicin can be detected in serum, lymph, tissues, sputum, and in pleural, synovial, and peritoneal fluids. Concentrations in renal cortex sometimes may be eight times higher than the usual serum levels. Concentrations in bile, in general, have been low and have suggested minimal biliary excretion. Gentamicin crosses the peritoneal as well as the placental membranes. Since aminoglycosides diffuse poorly into the subarachnoid space after parenteral administration, concentrations of gentamicin in cerebrospinal fluid are often low and dependent upon dose, rate of penetration and degree of meningeal inflammation. There is minimal penetration of gentamicin into ocular tissues following intramuscular or intravenous administration. Microbiology In vitro tests have demonstrated that gentamicin is a bactericidal antibiotic which acts by inhibiting normal protein synthesis in susceptible microorganisms. It is active against a wide variety of pathogenic bacteria including Escherichia coli, Proteus species (indolepositive and indole-negative); Pseudomonas aeruginosa, species of KlebsiellaEnterobacter-Serratia group; Citrobacter species, and Staphylococcus species (including

penicillin and methicillin-resistant strains). Gentamicin is also active in vitro against species of Salmonella and Shigella. The following bacteria are usually resistant to aminoglycosides: Streptococcus pneumoniae, most species of streptococci, particularly group D and anaerobic organisms, such as Bacteroides species or Clostridium species. In vitro studies have shown that an aminoglycoside combined with an antibiotic that interferes with cell wall synthesis may act synergistically against some group D streptococcal strains. The combination of gentamicin and penicillin G has a synergistic bactericidal effect against virtually all strains of Streptococcus faecalis and its varieties (S. faecalis var. liquifaciens, S. faecalis var. zymogenes), S. faecium and S. durans. An enhanced killing effect against many of these strains has also been shown in vitro with combinations of gentamicin and ampicillin, carbenicillin, nafcillin or oxacillin. The combined effect of gentamicin and carbenicillin is synergistic for many strains of Pseudomonas aeruginosa. In vitro synergism against other gram-negative organisms has been shown with combinations of gentamicin and cephalosporins. Gentamicin may be active against clinical isolates of bacteria resistant to other aminoglycosides. Bacteria resistant to one aminoglycoside may be resistant to one or more other aminoglycosides. Bacterial resistance to gentamicin is generally developed slowly.

Trade name manufacturer country Year introduced Garamycin Schering U.S. 1966 Refobasin Merck W.Germany 1967 Garramycin Kirby-Warrick U.K. 1966 Gentalyn Essex Italy 1967 Gentalline Unicet France 1968 Genoptc Allergan U.S. 1979 U-gencin Upjohn U.S. 1980 Bristagen Bristol U.S. 1980 Apogen Beecham U.S. 1980 Jenamicin Hauck U.S. 1982 Gentafair Pharmafair U.S. 1983 Biogen Cusi Spain Biomargen Biologia Marina Spain Cidomycin Roussel U.K. Duramycin Durachemie W. Germany Espectrosina Centrum Spain Gensumycin Roussel Genta I.E.Kimya Evi Turkey Genta-gobens Normon Spain Gentabac Infan Mexico Getacin Schering-Shionogi Japan Gentadavur Davur Spain Gentamedical Medical Spain Gentamicin-pos Ursapharm W.Germany Gentamin Medix Spain Gentamina Essex Argentina Gentamival Valles Mestre Spain Gentamorgens Morgens Spain Gentamytrex Mann W.Germany Gentaroger Roger Spain Antibiotics are secondary metabolism products which inhibits growth of other microbial species even at low levels. They are used as antimicrobials for treating human diseases. There are 6000 antibiotics known in the world, 100 of which are produced commercially via fermentation process. They are having 100000 tons world annual production and 4billion dollars sale.

Antibiotics are classified on the basis of: 1) breadth of antimicrobial action ( narrow or broad)

2) basis of activity (mechanism) 3) source (production strain) 4) biosynthetic pathway 5) molecular structure

Classification of antibiotics according to their chemical structure: (An example of each is given in parentheses) 1. Carbohydrate-containing antibiotics Pure sugars Aminoglycosides Orthosomycins N-Glycosides C-Glycosides Glycolipids 2. Macrocyclic lactones Macrolide antibiotics Polyene antibiotics Ansamycins Macrotetrolides 3. Quinones and related antibiotics Tetracyclines Anthracyclines Naphthoquinones Benzoquinones 4. Amino acid and peptide antibiotics (Tetracycline) (Adriamycin) (Actonorhodin) (Mitomycin) (Erythromycin) (Candicidin) (Rifamycin) (Tetranactin) (Nojirimycin) (Streptomycin) (Everninomicin) (Streptothricin) (Vancomyciun) (Moenomycin)

Amino acid derivatives -Lactam antibiotics Peptide antibiotics Chromopeptides Depsipeptides Chelate-forming peptides 5.

(Cycloserine) (Penicillin) (Bacitacin) (Actinomycines) (Valinomycin) (Bleomycins)

Heterocyclic antibiotics containing nitrogen Nucleoside antibiotics (Polyoxins)

6.

Heterocyclic antibiotics containing oxygen Polyether antibiotics (Monensin)

7.

Alicyclic derivatives Cycloalkane derivatives Steroid antibiotics (Cycloheximide) (Fusidic acid)

8.

Aromatic antibiotics Benzene derivatives Condensed aromatic antibiotics Aromatic ether (Chloramphenicol) (Griseofulvin) (Novobiocin)

9.

Aliphatic antibiotics Compounds containing phosphorus (Fosfomycins)

There is a group of antibiotics which contain carbohydrates and they are classified as aminoglycosides. Aminoglycosides which are aminoglycosidic are bactericidal inhibitors of protein synthesis. Although relatively toxic compared to other classes of antibiotics, they remain useful primarily in the treatment of infections caused by aerobic gram negative bacteria.

They are originally obtained from various species of streptomyces and sharing chemical, antimicrobial, pharmacologic and toxic characteristic. The group includes streptomycin, neomycin, kanamycin, amikacin, Gentamicin, tobramycin, sisomycin, netilmicin and others. They are most widely used against gram-negative bacteria, especially in bacteremia and sepsis, in combination with vancomycin or penicillin for endocarditic and for treatment of tuberculosis. Streptomicin is the oldest and best studied of the all. Gentamicin, tobramycin and amikacin are the most widely employed aminoglycosides till late 1990s. Neomycin and kanamycin are now largely limited to topical or oral use. Aminoglycosides contain amino sugars linked to an amino cyclitol ring by glycosidic bonds. They are poly cations and their polarity is in part responsible for pharmacokinetic properties shared by all members of the group. Mutations affecting proteins in the bacterial ribosome, the target for these drugs, can confer marked resistance to their action. Resistance most commonly results from the acquisition of plasmids or transposonencoding genes for aminoglycoside-metabolizing enzymes or from impaired transport of drug into the cell. There can be cross resistance between some members of the class. Although they are widely used and important agents, serious toxicity is a major limitation to the usefulness of the aminoglycosides. The same spectrum of toxicity is shared by all members of the group. Most notable are nephrotoxicity and ototoxicity.

Chemistry
The aminoglycosides consist of two or more amino sugars joined in glycosidic linkage to a hexose nucleus, which is usually in a central position. This hexose, or aminocyclitol, is either streptidine (found in streptomycin) or 2-deoxystrcptamine (all other available aminoglycosides). These compounds are thus aminoglycosidic aminocyclitols, although the simpler term aminoglycoside is commonly used to describe them. The aminoglycoside families are distinguished by the amino sugars attached to the aminocyclitol. In the neomycin family, an aminoglycoside used orally for the treatment of intestinal parastic infections, three amino sugars attached to the central 2-deoxystreptatine. The kanamycin and Gentamicin families have only two such amino sugars. The gentamicin family, which includes gentamicin C1, C1a and C2, sisomicin, and netilmicin (the 1-N-ethyl derivative of sisomicin contains a different 3-amino sugar (garosamine). Different components of gentamicin are resulted from variation in methylation of the other amino sugar. (Figure). These modifications appear to have little effect on biological activity.

Mechanism of Action: The aminoglycoside antibiotics are rapidly bactericidal.

Bacterial killing is concentration-dependent: the higher the concentration, the greater the rate at which bacteria are killed. A post antibiotic effect, that is, residual bactericidal activity persisting after the serum concentration has fallen below the minimum inhibitory concentration, also is characteristic of aminoglycoside antibiotics, and the duration of this effect is concentration-dependent. These properties probably account for the efficacy of once-daily dosing regimens of aminoglycosides. Although much is known about their capacity to inhibit protein synthesis and decrease the fidelity of translation of mRNA at the ribosome, the precise effect is unknown. Aminoglycosides diffuse through aqueous

channels formed by porin proteins in the outer membrane of gram-negative bacteria to enter the periplasmic space (Nakae and Nakae, 1982). Transport of aminoglycosides across the cytoplasmic (inner) membrane depends on electron transport, in part because of a requirement for a membrane electrical potential (interior negative) to drive permeation of these antibiotics (Bryan and Kwan, 1983). This phase of transport has been termed energy-dependent phase I, It is rate-limiting and can be blocked or inhibited by di-valent cations (e.g., Ca2+ and Mg2+), reduction in pH, and anaerobiasis. The antimicrobial activity of aminoglycosides is reduced markedly in the anaerobic environment of an abscess, in hyperosmolar acidic urine. Once inside the cell, aminoglycosides bind to polysomes and interfere with protein synthesis by causing misreading and premature termination of translation of mRNA. The aberrant proteins produced may be inserted into the cell membrane, leading to altered permeability and further stimulation of aminoglycoside transport. This phase of aminoglycoside transport is termed as an energy-dependent phase II (EDP2). Progression of the leakage of small ions, followed by larger molecules and, eventually, by proteins from the bacterial cell prior to aminoglycoside-induced death. This progressive disruption of the cell envelope, as well as other vital cell processes, may help to explain the lethal action of aminoglycosides (Bryan, 1989).

Microbial Resistance to the Aminoglycosides: Bacteria may be resistant to the

antimicrobial activity of aminoglycosides because of failure of permeation of the antibiotic, low affinity of the drug for the bacterial ribosome, or inactivation of the drug by microbial enzyme. Penetration of drug through the pores in the outer membrane of gram-negative microorganisms into the periplasmic space may be retarded; resistance of this type is unimportant clinically. Once the aminoglycoside does reach the periplasmic

space, it may be altered by microbial enzymes that phosphorylate, adenylate, or acetvlate specific hydroxyl or amino groups. The genes for these enzymes are acquired primarily by conjugation and the transfer of DNA as plasmids and resistance transfer factors (Davies, 1994). These plasmids have become widespread in nature (especially in hospital environment), and code for a large number of enzymes (more than 20) that have markedly reduced the clinical usefulness of aminoglysides. Acquisition of aminoglysides inactivating enzymes by enterococci has become a source of concern. In several centers, a significant percentage of clinical isolates of these organisms are highly resistant to all aminoglysides. Because different enzymes are responsible for inactivation of gentamicin and streptomycin, a small proportion of gentamicin-resistant strains of enterococci will be susceptible to streptomycin. Resistance to gentamicin indicates resistance to tobramycin, amikacin, kanamycin, and netilmicin, because the inactivating enzyme is bi-functional and modifies all of these aminoglycosides (Murray, 1991). Another common form of natural resistance to aminoglycosides is caused by failure of the drug to penetrate the cytoplasmic (inner) membrane. As mentioned above, transport of aminoglycosides across the cytoplasmic is an oxygen-dependent, active process. Strictly anaerobic bacteria are thus resistant to these drugs. There is no synergistic effect of penicillin and streptomycin against these strains demonstrable in vitro. Because ribosomal resistance often is specific for streptomycin, these strains of enterococci generally are sensitive to combination of penicillin and gentamicin in vitro.

Antibacterial Activity of the Aminoglycosides: The antibacterial activity of

gentamicin, tobramycin, kanamycin, netilmicin, and amikacin is primarily directed against aerobic, gram-negative bacilli. As noted above, these antibiotics have little activity against anaerobic microorganisms or facultative bacteria under anaerobic conditions. Their action

against most gram-positive bacteria is limited. Although not active when used alone, either streptomycin or gentamicin in combination with a cell wall-active agent, such as penicillin or vancomycin is active against "sensitive" strains of enterococci and streptococci at concentrations that can be achieved clinically. Such combinations result in a more rapid bactericidal effect than is produced by either drug alone (they are synergistic). Both gentamicin and tobramycin are active in vitro against more than 90% of strains of Staphylococcus aureus and 75% strains of staphylococcus epidermidis. Sensitive microorganisms are defined as those inhibited by concentrations that can be achieved Clinically in plasma without a high incidence of toxicity: when given at 8 or 12 hour intervals, these therapeutic peak values range from 4 to 12 g/ml for gentamicin, tobramycin. Many gram-negative bacilli that are resistant to gentamicin because of plasmid-mediated inactivating enzymes also will inactivate tobramycin Nosocomial-flora have shown gradual increase, in resistance to gentamicin and tobramycin over the last 20 to 30 years.

Absorption: The aminoglycosides are highly polar cations and therefore are very poorly
absorbed from the gastrointestinal tract. Less than 1 % of a dose is absorbed following either oral or rectal administration. Long-term oral or rectal administration may result in accumulation of aminoglycosides to toxic concentrations in patients with renal impairment. Absorption of gentamicin from the gastrointestinal tract may be increased by gastrointestinal disease. Instillation of these drugs into body cavities with serosal surfaces may result in rapid absorption and unexpected toxicity

i.e., neuromuscular blockade. Similarly, intoxication may occur when aminoglycosides are applied topically for long periods to large wounds, burns, or particularly if there is renal insufficiency. All of the aminoglycosides are absorbed rapidly from intramuscular

sites of injection. In critically ill patients, especially those in shock, absorption of drug may be reduced from intramuscular sites because of poor perfusion.

Distribution: Because of their polar nature, the aminoglycosides largely are excluded
from most cells, from the central nervous system, and from the eye. Concentrations of aminoglycosides in secretion and tissues are low. High concentrations are found only in the renal cortex and in the endolymph and prilymph of the inner ear; this may contribute to the nephrotoxicity and ototoxicity caused by the drugs. Intrathecal or intraventricular administration of aminoglycosides has been used to achieve therapeutic levels, but the availability of third generation cephalosporins has now made this unnecessary in most cases. Administration of aminoglycosides to women late in pregnancy may result in accumulation of drug in fetal plasma and amniotic fluid. Gentamicin can cause hearing loss to children born to women who receive the drug during pregnancy. All aminoglycosides have the potential to produce reversible and irreversible vestibular, cochelar, and renal toxicity. These side effects complicate the use of these compounds and make their proper administration difficult. Ototoxicity is largely irreversible and results from progressive destruction of vestibular or cochelar sensory cells, which are highly sensitive to damage by aminoglycosides. Nephrotoxicity effects are usually reversible. Approximately 8% to 12% of patients who receive aminoglycosides for more than several days will develop mid renal impairment that is almost always from accumulation and retention of aminoglycoside in the proximate tubular cells. The initial manifestation of damages at this site is excretion of enzymes of the renal tubular brush border.

Gentamicin is an aminoglycoside isolated from micromonospora purpurea. It is an

important agent for the treatment of many serious gram-negative bacillary infections. It is

the aminoglycoside of first choice because of its low cost and its reliable activity against all but the most resistant gram-negative aerobes. However, emergence of resistant microorganisms in some hospitals has become a serious problem and may limit the future use of this agent.

Structure:

Antimicrobial activity: Gentamicin sulfate, 2-10 g/ml inhibits in vitro many strains of staphylococci and coliforms and other gram-negative bacteria. It is active alone, but also as synergistic companion with beta-lactam antibiotics, against pseudomonas, proteus. enterobaeter. klebsiella, serratia, stenotrophomonas and other gram-negative rods/that may be resistant to multiple other antibiotics. Like all aminoglycoside, it has no activity against anaerobes.

Pharmacology: All of the aminoglycosides have similar pharmacokinetic properties. They are poorly absorbed orally and must be used parenterally for systemic infection. As the aminoglycosides are toxic and are absorbed from areas of denuded skin, such as burns, it is important to be cautious even in their topical use. They are absorbed well from

the peritoneum, pleural cavity, and joints and should never be instilled in these cavities. Penetration of aminoglycosides into the eye and respiratory secretions is poor even with inflammation. Aminoglycosides are inactivated in vitro by antipseudomonal penicillins (eg, car-bcnieillin) and the 2 agents should not-be mixed together in vitro. In vivo inactivation of the aminoglycoside can occur in patients with renal failure receiving both a penicillin and aminoglycoside with long intervals between doses of aminoglycoside.

Indication: Gentamicin, tobramycin, amikacin, and netilmicin should be used only in the treatment of serious gram-negative bacillary infection. Gentamicin is also used as a companion drug with a penicillin or vancomycin in the treatment of enterococci or S. aureus endocarditic or in prophylaxis of enterococcal endocarditis. Gentamicin and tobramycin are very similar in antimicrobial activity against gram-negative bacilli with only two differences: Tobramycin is more active against aeruginosa and gentamicin is more active against S.marcescens. Resistance of gram-negative bacilli to gentamicin and tobramycin has occurred in some hospitals, and such infections in these institutions cannot be treated with these agents. The resistance is most commonly due to a plasmid-mediated enzymatic alteration of the aminoglycoside.

Resistance: Streptococci and enterococci are relatively resistant to gentamicin owing to failure of the drug to penetrate into the cell. However, gentamicin in combination with vancomycin or a penicillin produces a potent bactericidal effect, which in part is due to enhanced uptake of drug that occurs with inhibition of ceil wall synthesis. Among gramnegative bacteria, resistance is most commonly due to plasmid-encoded aminoglycoside

modifying enzymes. Gram-negative bacteria that are gentamicin resistant usually will be susceptible to amikacin, which is much more resistant to modifying enzyme activity. The enterococcal enzyme that modifies gentamicin is a bi-functional enzyme that also modifies amikacin, netilmicin, and tobramycin, but not streptomycin; the latter is modified by a different enzyme. This is why some gentamicin-resistant enterococci arc susceptible to streptomycin.

Therapeutic uses of Gentamicin with other Aminogly cosides: Gentamicin, tobramycin, amikacin, and netilmicin can be used interchangeably for the treatment of most of the following infections and are therefore discussed together. For most indications, gentamicin is the preferred agent because of long experience with its use and its relatively low cost. A large variety of infections have been treated success-fully with these aminoglycosides; however, due lo their toxicities, prolonged use should be restricted to the therapy of life-threatening infections and those for which a less toxic agent is contraindicated or less effective. These antibiotics frequently are used (often in combination with a penicillin or a cephalosporin) for the therapy of proven or suspected serious gram-negative microbial infectionsespecially those due to P. aeruginosa, Enterobactei; Klebsiella, Serratia, and other species resistant to less toxic antibiotics urinary tract infections, bacteremia, infected bones, osteomyelitis, pneumonia, peritonitis, and otitis. Penicillin and aminoglycosides must never be mixed in the same bottle because the penicillin inactivates the aminoglycosides to a significant degree. Similar incompatibilities exist in vitro to different degrees between gentamicin and heparin, amphoteriein B and the various cephalosporins.

Gentamicin has applications alone or in combination with penicillin for urinary tract infections, pneumonia, meningitis, peritonitis, gram-positive infections & sepsis.

Topical applications: gentamicin is very slowly absorbed when applied in an ointment but absorption may be more rapid when cream is used topically. When the antibiotic is applied to large areas included body surfaces as may be the case in the burned patients, plasma concentration can reach 4g/ml and 25 to 5% of the drug used may appear in the urine. Untoward effect: The most important and serious effects of gentamicin are nephrotoxicity and irreversible ototoxicity. Intrathecal or intraventricular administration may cause local inflammation and can result in radiculitis and other complication and therefore is rarely used.

Adverse Reactions: Nephrotoxicity is reversible and usually mild. It occurs in 5-25% of patients receiving the drug for longer than 3-5 days. Nephrotoxicity requires, at the very least, adjustment of the dosing regimen and should prompt reconsideration of the necessity of using the drug, particularly if there is a less toxic alternative agent. Measurement of gentamicin serum levels is essential. Ototoxicity, which tends to be irreversible, manifests itself mainly its vestibular dysfunction, perhaps due to destruction of hair cells by prolonged elevated drug levels. Loss of hearing can also occur. The incidence of ototoxicity is 1-5% for patients receiving the drug for more than 5 days. Hypersensitivity reactions to gentamicin are uncommon. Aminoglycosides can cause paresthesias and peripheral neuropathy. Gentamicin may be more nephrotoxic than tobramycin, amikacin and netilmicin. Nephrotoxicity is reversible and is more likely to occur with large doses, high blood levels, or long duration of therapy, and in elderly

patients, those with preexisting renal disease, those who are dehydrated, and those receiving furosemide or cephalothin. Streptomycin and gentamicin are more likely to produce vestibular damage than hearing loss.

To minimize the possibility of adverse reactions, smaller doses can be given at the customary intervals or normal doses can be given at increased intervals. It is probably best to give decreased doses at normal or somewhat increased intervals to avoid ling time periods with subtherapeutic blood levels. Nomograms are available to calculate dosages based on serum creatinine clearance values, but they are inaccurate.

Precautions: Ototoxic antibiotics should be avoided in pregnancy. Elderly persons and those with a preexisting hearing loss should not be treated with ototoxic drugs if other effective drugs are available, if possible, before treatment is began with an ototoxic drug (especially an ototoxic antibiotic), the hearing should be measured in order to document a preexisting hearing loss. Hearing should be monitored audiometrically as often as daily while treatment is continued. The highest frequencies are usually affected first, highpitched tinnitus or vertigo may develop though they are not reliable warning symptoms. If renal function is impaired, the dosage of renally eliminated ototoxic drugs should be adjusted so that the blood levels do not exceed those required therapeutically, Serum levels of the agent should be monitored to insure that adequate therapeutic levels have been achieved but not exceeded.

CHEMICAL AND PHYSICAL PROPERTIES OF GENTAMICIN

Gentamicin, as produced, is a white amorphous powder having the following physical and chemical characteristics:

I. Melting point: (Koeffler block) softening at 102 C and completely melts at 108C. II. Analysis: (a) Elementary C=50.20% H=9.52% N= 13.47% 0=27.81% (by difference) No other elements are present. (b) Qualitative/quantitative 1. 2. 3. No methoxy groups N-mefhyl groups=2.75% C-methyl groups=2.97%

III.

Molecular weight543 (C21H43N5O7)

IV.

Calculated molecular weight = 545 (based on assumption of one N-

methyl group per molecule).

V. Rotation [a]D25= + 146 (1% in water).

VI.

CAS No: 1403-66-3

VII.

Solubility: Extremely soluble in water. It is soluble in polar media such as

pyridine and dimethylformamide. It is Soluble in acidic media with salt formation. Gentamicin is moderately soluble in methanol, ethanol and acetone. Insoluble in ether, benzene, halogenated hydrocarbons.

VIII.

Ultraviolet spectrum: Completely transparent in the range 220-400 mp.

IX. Packaging & storage: preserve in collapsible tubes or in other tight containers and avoid exposure to excess heat. X. Salt formation: Gentamicin, being a moderately strong base forms salts with any strong organic or inorganic acids. The salts of strong inorganic acids are extremely water soluble. A. Hydrochloride Melting point: 194-209 C. (dec.) Rotation [a] D25 +113 (1% in water) Elemental analysis: C=35.47%

H=7.27% N=9.69% 0=22.46% Cl=24.90% Antibiotic activity = 820 units/mg. Solubilityvery soluble in water and methanol, slightly soluble in ethanol, insoluble in other common organic solvents

B. Sulfate Melting point: 218-237 C. (dec.) Elemental analysis: C=31.22% H=6.57% N=8.46% 0=41.63% SO4=31.22% Rotation: [a]D25= + 102 (1% in water)

Antibiotic activity=800 units/mg.

Miscellaneous properties:

*Antibiotic activity of gentamicin completely destroyed by hydrolysis with 6N hydrochloric acid at 140 C. in 15 minutes. With 2 N hydrochloric acid at 100 C, 7 hours reaction time necessary to completely destroy activity.

*StabilityThe activity of gentamicin is not significantly altered when an aqueous solution of the antibiotic is subjected to a temperature of 100 C. for 30 minutes throughout the pH range of 2 through 12.

*Other derivativesGentamicin like other antibiotics having primary amino groups, is transformed into N-methylenesulfonic acid derivatives by the action of sodium formaldehyde bisulfite (preferably upon gentamicin sulfate). The extent of conversion of the primary amino groups of gentamicin to the N-methylenesulfonic acid derivative is governed by the amount of formaldehyde-sodium bisulfite addition compound used in the reaction. The pH of the medium from which the product is isolated determines whether the product is a methane sulfonic acid or a salt thereof. Other analogous sulfonate derivatives are prepared by the action upon gentamicin of the addition compound of an alkyl or aryl ketone or aldehyde with bisulfite. These derivatives exhibit altered therapeutic indices as compared with the parent antibiotic.

*Antibiotic activity: The antibiotic activity of gentamicin is 1120 units/mg. A unit being that amount of substance required to produce an 18 mm zone of inhibition.

BIOLOGICAL PROPERTIES OF GENTAMICIN Gentamicin possesses a broad anti-bacterial and anti-rickettsial spectrum. Gentamicin is a useful anti-infective agent capable of effectively inhibiting certain disease manifestations caused by Staphylococcus aureus, Klebsiella pneumoniae and other pathogenic organisms. It is also useful in treating mastitis in cattle. It is particularly effective in combatting urinary tract infections caused by gram-negative organisms such as species of Proteus and Psedomonas. It is known that the number of hospital infections due to gramnegative organisms is increasing. Gentamicin appears particularly useful in treating such chronic gram-negative infections of the kidney and bladder even in refractory patients. The high antibiotic activity of Gentamicin against Proteus and Pseudomonas infections easily permits clinical utilization. As a particular example of such effect, Gentamicin has been successfully employed in treating a chronic Pseudomonas kidney infection at a dose of 1 mg/kg administered three times daily. This infection which did not respond well to other therapy was completely eliminated from the urine after one day of treatment and

did not recur even after 10 days. Similar responses have been elicited against several Proteus infections. It thus appears that Gentamicin possesses an advantageous combination of unique types of activity in conjunction with low toxicity permitting its use to other antibiotics.

PROCESS SELECTION

Gentamicin is formed be cultivation under controlled conditions of hitherto undescribed species of the genus micromonospora of the order actinomycelates.

MICROORGANISM SELECTION The microorganisms useful for the preparation of gentamicin, and its co-produced antibiotics, are species of Micromonospora. Cultures of the living organisms have been deposited and made a part of the stock culture collection of the United States Department of Agriculture, Northern Utilization Research and Development Division, Peoria, Illinois, from which they are available under their respective NRRL identifications. One of the species, designated as Micromonospora purpurea n. sp., hereinafter called M. purpurea, has been assigned the number NRRL 2953 and was originally isolated from a sample of park loam in Syracuse, New York. The other novel species of this invention has been designated as Micromonospora echinospora n. sp., hereinafter called M. echinospora. This latter species was isolated from a mud sample in Jamesville, New York, and has been assigned the number NRRL 2985.

M. purpurea is characterized by a fine mycelium averaging 0.5m in diameter; does not produce a true aerial mycelium; produces single spores which average l m in diameter at the ends of simple sporophores; is non-acid-fast; gram positive; digests certain types of protein and starch; is aerobic, grows well between 22-37 C. but not at 46 C. or above. The colony form and orange color on most rich organic agar media are typical. On other agar media low in nitrogen, the color of the colony is reddish purpose to purple. While

this reddish purple pigment is characteristic of the fresh soil isolate, it may be temporarily or permanently lost in strains obtained by repeated isolation and transfer. There are numbers of micromonospora species available which can be used for antibiotic production but for our selection, we will consider comparative taxonomy of M. purpurea and M. echinospora. The colony observations made after 14 days of incubation at 2426C in the designated media. Medium: 3% NZ Amine type A, 1% dextrose, 1.5% agar

Organism M. pupurea NRRL 2953

Observations Macroscopic No aerial

Microscopic mycelium, Mycelium long, branched,

colony raised, convolute regular, nonseptate, 0.5m abundant growth, waxy, no in diameter, sporophores diffusible pigment single, terminally, spores borne spores

spherical to ellipsoidal, 0.1 M.echinospora NRRL 2985 No aerial m in diameter. mycellium, Mycellium long, branched, crenate- regular, non-septet, 0.5m

colony

raised,

convolute good growth, in diameter., no spores. waxy, slight amber

diffusible pigment. For isolating the microorganisms, a portion of the soil sample is shaken in sterile distilled water and after making suitable dilutions, the suspension is plated on screen agar medium. A useable medium contains 1% glycerol, 0.2% sodium nitrate, 0.1% dipotassium hydrogen phosphate, 0.05% magnesium sulfate, 0.05% potassium chloride,

0.001% ferrous sulfate, 1.5% agar, all in distilled water. A preferred medium contains 0.5% tryptose, 2.0% soluble starch, 0.3% calcium propionate and 2.0% agar all in distilled water. The cultures are tested for antibiotic activity by first growing in the following medium for up to 60 days at 26 C: 0.3% beef extract, 0.5% tryptose, 0.1% dextrose, 2.4% soluble starch, 0.5% yeast extract, 1.5% agar, all in tap water. The whole aqueous agar is then extracted with butanol and the butanol-water extract is concentrated. Although the antibiotic is only minimally soluble in butanol, sufficient antibiotic is extracted by the butanol-water mixture to provide a concentrate, which, by disc test, inhibits the growth of Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa Klebsiella pneumoniae and Mycobacterium smegmatis. Antibiotic activity against the same organisms is observed after growth in submerged fermentation for 96 hours in an aqueous medium such as one containing 1 % yeast extract, 1% cerelose and 0.1% calcium carbonate.

The two species are being compared at laboratory scale for growth on various media and results are as shown in the table.

Medium Bennetts agar Emersons agar Tomato paste oatmeal agar Glucose asparagines agar Glucose yeast extract agar

M. pupurea NRRL 2953 Good growth Good growth Good growth Fair growth Good growth

M.echinospora NRRL 2985 Good Growth Good growth Fair growth Pair growth Good growth

M. purpurea and M. echinospora are capable of utilizing various carbon and nitrogen sources. In Table carbon utilization is compared with other Micromonospora species by

a visual estimate of growth on agar plates in a medium consisting

of 0.5% yeast extract,

Difco (Difco Laboratories Inc., Detroit, Michigan), 1% carbohydrate and 1.5% agar all in distilled water. In Table nitrogen utilization is compared with other Micromonospora

species by visual estimate of growth on agar plates in a medium consisting of 1% glucose, 1.5% agar, nitrogen source concentration as indicated, all in distilled water.

Carbon & Nitrogen utilization:

Carbohydrate Arbinose Cellulose Glucose Lactose Sucrose Strach Nitrogen 1% Aspargin 1% Glutamic acid 1% sodium nitrate

M. pupurea NRRL 2953 Good Poor Good Poor Good Good Fair Growth Fair Growth Poor Growth to

M. chalcea ATCC 12452 Good Poor Good Fair Good Good Fair Growth Fair Growth no No Growth

M. fusca NRRL B-943 Good Poor Good Poor Good Good Poor Growth Poor Growth No Growth

M. sp. ATCC10026 Good Poor Good Poor Good Good Poor Growth Poor Growth No Growth

1% Ammonium nitrate

Poor Growth

to

no No Growth

No Growth

No Growth

Therefore Research & results discussed until now suggests M. pupurea Gives good result than any other micromonospora species. So we here select same organism for Gentamicin production for our scope of project.

GROWTH MEDIUM SELECTION M. Pupurea can be adequately cultivated as described above in the presence of a source of assimilable carbon such as starch, dextrose, sugars and the like coupled with a source of assimilable nitrogen such as soybean meal, peptones and the like. In the absence of cobalt supplement, profuse growth may be obtained. However it has been found that adding a cobalt supplement to the growth medium will significantly increase the gentamicin production over that obtainable in the absence of cobalt. It has been determined to get optimum enhancement of gentamicin, at least 0.01 micrograms of cobalt as COCl2.2H2O must be present per ml of medium. This is equivalent to 2.510-9 gram of cobalt per ml. Cobalt can be added in the form of cobaltous chloride (or hexahydrate), cobaltous nitrate and the like. But it has been found that cobaltic ion also enhances antibiotic production but apparently not to the same degree as cobaltous ion. It would appear that any ionizable water soluble salt of cobalt will affect the increase in antibiotic production such as the sulfate, lower slanoate, halide and the like. There are limit on the addition of the amount of the cobalt. The upper limit appears dependent upon the medium; whether it is purely synthetic or derived from natural sources. Thus a toxic level of cobalt is reached at a much lower level than with a medium

derived from natural sources. As such the ability of the enzyme to act is enhanced by the presence of cobalt: however cobalt exerts a toxic effect on the enzyme and gentamicin production falls off rapidly.

Effect of varying concentration of cobalt on gentamicin production

(A) In a synthetic medium: Component gm/lit

Dextrin.............................30 NaNO3...............................2 K2HPO4.............................1 MgSO40.5 KCI.0.5 FeSO4....0.01

Co (gm/ml) as CoCl2.6H2O 0.0 5.0 10 20 40

Zone of inhibition (mm) 16 18.6 Trace Trace No antibiotic produced

(B) Medium derived from natural sources

Component

gm/lit

Soybean meal30 Cerelose.40 Calcium carbonate...1 Tap water...1000

Co (gm/ml) as CoCl2.6H2O 0.0 0.2 0.4 0.6 0.8 1.0 3.0 6.0 8.0 10.0 12.0 15.0 20.0 40.0 60.0

Yield of gentamicin (units/ml) 82.5 181.3 187.5 273.0 287.5 268.8 246.0 256.0 206.0 194.0 188.0 180.0 158.0 41.0 33.0

So the details described here in suggest utilizing Co concentration of about 0.1 gm per ml which more than adequately effects enhancement.

PROCEDURE TO BE FOLLOWED:

M. purpurea and M. echinospora and variants thereof, by the fermentation methods described herein, produce a mixture of antibiotics. In which Gentamicin represents the major component. The other components, as determined by paper chromatographic studies, comprise essentially two antibiotic substances. These co-produced substances are sometimes referred to as BA-3 (fraction A) and BA-3 (fraction B). In order to form gentamicin, M. purpurea is grown at a suitable temperature of about 25 C to 40 C, preferably 30 C, under submerged aerobic conditions in an aqueous nutrient medium containing an assimilable carbon and nitrogen source. Suitable carbon sources include carbohydrates such as starch, dextrin, sugars, etc. Suitable nitrogen sources include both organic and inorganic nitrogen, preferably the former, such as soybean meal, peptones and the like. The fermentation is carried out for about 24 to 48 hours at a pH in the range of about 6 to 8. At about the end of this period, peak antibiotic production has been attained. Since most of the activity resides in the mycelium, the fermentation broth is filtered and the filtrate is discarded. Gentamicin is separated from the mycelium by acid extraction with mineral acid at a pH of about 2. The acid extracts are neutralized to a pH of about 6 and may be either passed through an ion exchange resin, preferably of the IRC-50 Amberlite type; or precipitated by formation of an insoluble salt. When the resin procedure is employed, the aforementioned solution of the antibiotic at pH 6 is passed through the ion

exchange resin and the antibiotic is eluted from the resin with dilute sulfuric acid. The eluate is made alkaline to a pH of about 10 with sodium hydroxide. Upon the addition of acetone, inorganic salts are precipitated. The supernatant liquor is adjusted to pH 4.5 and concentrated in vacuo to give a concentrated solution of gentamicin sulfate. Methanol is added to this solution and crude gentamicin sulfate is precipitated. By this methodology we get 56 gm of gentamicin sulfate with 500units/mg. When the insoluble salt method is utilized, the neutralized mycellium extract is treated with Santomerse-S and the precipitated salts removed by filtration. The precipitate is washed with water and dissolved in methanol. The methanolic solution is filtered and then passed through an anionic exchange resin, for example, of the Amberlite IRA-401 S type. The eluate is concentrated, adjusted to pH 4.5 with sulfuric acid resulting in a concentrated solution of Gentamicin sulfate. The sulfate is obtained by adding methanol to the solution and separating the precipitated salt by filtration. Gentamicin is a moderately strong base which readily forms salts with strong organic and inorganic acids. In general, the mineral acid salts, such as that formed with hydrochloric acid, are completely water soluble. The salts are obtained by concentration of their aqueous solution and precipitation with a water miscible organic solvent, preferably a lower aliphatic alcohol or ketone. Gentamicin, as isolated by the resin technique, is further separated from its co-produced antibiotics preferably by fractional precipitation of its Santomerse S (sodium dodecylbenzene sulfonate) salt. By this optional procedure of insoluble salt method we get gentamicin yield of 20-30 gm in the form of sulfate salt and 675 units/mg of activity of antibiotic which is preferable option than previous one.

CO-PRODUCED ANTIBIOTICS SEPERATION

The co-produced antibiotic substances comprise essentially two 15 fractions identified as BA-3 (fraction A) and BA-3 (fraction B). Advantage is taken of the greater solubility of gentamicin in acetone-methanol mixture as compared with BA-3 (fraction A) and BA-3 (fraction B). Upon the addition of acetone to a concentrated methanolic solution of gentamicin plus the two co-produced fractions, precipitation of the latter occurs. After washing with acetone, the precipitate is dried and consists essentially of a mixture of the two fractions A and B. An alternate method for isolating these A and B fractions is one which takes advantage of the greater solubility of their dodecylbenzene sulfonate salts as compared with gentamicin. The dodecylbenzene sulfonate (Santomerse-S) salt of gentamicin is precipitated by the addition of a solution of Santomerse-S to a solution of crude gentamicin sulfate. After filtering and washing the filter cake the filtrates are combined and represent a solution of the dodecylbenzene sulfonate salts of BA-3 (fraction A) and BA-3 (fraction B).

The mixture of fractions A and B as obtained above exhibit the following properties: (1) Sulfate: white amorphous powder, soluble in water, insoluble in ethanol, methanol, acetone and other common organic solvents. (2) Solubility: similar to that of gentamicin. (3) Elemental content: C, H, N, O; no other elements present.

CHAPTER

PROCEDURE & MATERIAL BALANCE

Detailed procedure with material balance

Detailed procedure from US patent is described over here. Gentamicin production is carried out in main three steps: 1) germination 2) fermentation 3) downstream processing How gentamicin production is carried out? We will understand germination by taking case of laboratory scale.

Germination stage: Add a lyophilized culture of M. purpurea to a 300ml. shake flask containing 100ml. of the following sterile medium:

Bacto-beef extract Tryptose Dextrose Starch (soluble) Yeast extract Tap water

gm gm gm gm gm gm

3 5 1 24 5 1000

Incubate the flask and its contents for 5 days at 37 C. on a rotary shaker (280 r.p.m., 2 inch. stroke)

Inoculum preparation stage: Transfer a 25ml. inoculum (from the germination stage) to each of four 2-liter flasks, each containing 500ml. of the sterile medium utilized for germination. Incubate the flasks and contents for 5 days at 28 C. on a rotary shaker (280 r.p.m., 2 inch. stroke). Pool the contents of the flasks. Add a 25ml inoculum (taken from the pool) to each of twenty 2liter flasks, each containing 500ml. of the following sterile medium:

Soybean meal Dextrose (cerelose) Calcium carbonate Tap water

gm gm gm gm

30 40 1 1000

Incubate the flasks and their contents for 3- 5 days at 28 C. on a rotary shaker (280 r.p.m., 2 stroke). Pool and aseptically transfer the broth into a sterile inoculum flask having a side arm (total volume about 10 liters).

Production-batch time calculation: Aim: 250 kg of gentamicin per annum.

1year = 365 days. We will assume 300 working days.

1 batch takes 30 hours production time and if we assume 6 hours of cleaning time then total batch time = 30+6 = 36 hours. We will run simultaneously 4 batches and second batch will start after 10 hours from staring of first batch.

66 hours time is necessary for 4 batches. Therefore, 7200 hours (300 days) time is necessary for 430 batches.

437 batches produce 250 kg of gentamicin. So, 1 batch produces 0.5720 kg of gentamicin.

Now we will consider material balance following detail procedure as shown in the flow diagram:

Fermentation stage: As described above, inoculum is prepared at laboratory scale. We required this inoculum of 365.85 lit. Aseptically transfer the 365.85 liters of inoculum to a 1780.47 gallon fermenter containing following sterile medium:

Bacto-beef extract Bacto-tryptose Dextrose (cerelose) Starch (soluble) Yeast extract

Anti-foamer Tap water, q.s.

Adjust pH to 6.9-7.0 before sterilization. Aerobically ferment for 20-30 hours (until the packed cell volume is about 10-15%) under the following conditions:

Temperature Pressure Agitation

37 C 7 psi 180 r.p.m

Aseptically transfer the contents of the fermenter to a 16463.25 gallon fermentation tank containing 10975.5 gallons of sterile medium having the following composition:

Yeast extract Dextrose Calcium carbonate Anti-foam (GE-60) Water Ferment at 35C, while agitating at 120 r.p.m. and introducing air at 7 psi and 365.85 ft3/min for 24-31 hours. At the end of this period, the potency of the produced antibiotic reaches a peak which remains substantially constant. During the fermentation the pH remains substantially in the range of 6.6 to 7.4. The packed, cell volume reaches a constant value of 2-2.5 ml. The potency of the produced antibiotic is approximately 25units/ml.

O2 supplied 365.85 ft3/min of air is supplied. 21% of which is oxygen. 0.21 365.85 = 76.828 ft3/min of O2 is supplied. In 1 min In 30 hours (30 60 min) = 76.828 ft3/min of O2 is supplied = 138290.4 ft3 of O2 is supplied per batch = 3915.94 m3 of O2 is supplied per batch = 3915.94 m3 1.087 kg /m3 = 4256.62 kg / 32 kg/kmol = 133.019 kmol = 133019 gmol of O2 is supplied/batch

N2 supplied 365.85 ft3/min of air is supplied. 78% of which is oxygen. 0.78 365.85 = 285.36 ft3/min of N2 is supplied. In 1 min 285.36 ft3/min of N2 is supplied In 30 hours (30 60 min) = 513653.4 ft3 of N2 is supplied per batch = 14545.04 m3 of N2 is supplied per batch = 14545.04 m3 1.087 kg /m3 = 15810.45 kg / 32 kg/kmol = 494.076 kmol = 494076 gmol of N2 is supplied/batch

Dextrose consumed 402.74 kg of dextrose is supplied = 402.74 kg /198 kg/kmol = 2.0340 kmol = 2034 gmol

Gentamicin sulfate 1365.84 gm of gentamicin sulfate is produced. Molecular weight of gentamicin sulfate = 638 gm/gmol. 1365.84/638 = 2.14 gmol of gentamicin sulfate is produced.

C6H12O6 + 6O2 6CO2 + 6H2O

Composition of dextrose 402.74 kg of dextrose is supplied C = 40% H = 6.67% O = 53.33% 0.40 (402.74) = 161.096 kg 0.0667 (402.74) = 26.862 kg 0.5333 (402.74) = 222.71 kg

Gentamicin sulfate composition 1.365 kg of Gentamicin sulfate is produced C = 43.26% H = 8.15% O = 32.60% N = 10.97% S = 5.01% 1.365 (0.4326) = 0.5065 kg 1.365 (0.0815) = 0.0954 kg 1.365 (0.3260) = 0.3817 kg 1.365 (0.1097) = 0.1497 kg 1.365 (0.0501) = 0.0683 kg

Material balance:

Material balance of Carbon: Carbon in: Carbon supplied = 161.096 kg

Carbon out:

C6H12O6 + 6O2 6CO2 + 6H2O 180 32 44 18

180 kg of dextrose produces 264 kg of CO2 (402.74 /2) =201.37 kg of dextrose produces 295.38 kg of CO2

44 kg of CO2 contains 12 kg of Carbon 295.38 kg of CO2 contains = 80.558 kg of Carbon

Carbon librated by combustion = 80.558 kg of Carbon

Unconverted Carbon + Carbon librated from respiration = 80.031 kg

Carbon used in Gentamicin sulfate formation = 0.5904 kg Carbon consumed in other metabolic activity = 160.58 kg Total Carbon out = 161.096 kg

Material balance of hydrogen: Hydrogen in: Hydrogen supplied = 26.862 kg

Hydrogen out: C6H12O6 + 6O2 6CO2 + 6H2O 180 32 44 18

180 kg of dextrose produces 108 kg of H2O (402.74 /2) =201.37 kg of dextrose produces 120.82 kg of H2O

18 kg of H2O contains 2 kg of Hydrogen 120.82 kg of H2O contains = 13.42 kg of Hydrogen

Hydrogen used in Gentamicin sulfate formation Hydrogen out in form of water Hydrogen consumed in other metabolic activity Total Hydrogen out = 26.862 kg

= 0.5065 kg = 13.42 kg = 12.93 kg

Material balance of oxygen Oxygen in: Oxygen in dextrose = 222.71 kg Oxygen in form of air Now, total Oxygen in = 4256.62 kg =222.71 kg + 4256.62 kg = 4479.33 kg Oxygen out: 50% of Carbon source supplied will be used as energy source for maintenance and growth of microorganism.

C6H12O6 + 6O2 6CO2 + 6H2O 180 32 44 18

O2 required for combustion of (402.74 /2) =201.37 kg of dextrose

50% will be used as energy source and it will get converted into H2O Oxygen consumed in combustion + remaining used for respiration = 214.74 kg

180 kg of dextrose takes 192 kg of O2 201.37 kg of dextrose takes 214.74 kg of O2

44 kg of CO2 contains 32 kg of Oxygen 49.22 kg of CO2 contains = 35.79 kg of Oxygen

180 kg of dextrose produces 108 kg of H2O (402.74 /2) =201.37 kg of dextrose produces 120.82 kg of H2O

18 kg of H2O contains 16 kg of Oxygen 120.82 kg of H2O contains = 107.39 kg of Oxygen

Oxygen consumed Oxygen in form of CO2 Oxygen in gentamicin sulfate

= 214.74 kg = 35.79 kg = 0.4449 kg

Unconsumed oxygen + oxygen in unconverted biomass = 4121.02 kg Oxygen in form of water Total oxygen out = 107.39 kg = 4479.33 kg

Material balance of Nitrogen Nitrogen will be supplied in the form of soybean meal that will get converted into biomass and a faction of which will be used in gentamicin formation. We will require 0.0061 kg of Nitrogen for gentamicin sulfate production.

Material balance on individual equipment:

Seed fermentor Capacity = 1780.47 gallons = 6739.07lit Volume filled up = 80% (1780.47)

= 1424.37 (gallon) = 5391.26lit Material in from shake flask fermentation = 365.85 lit

Material in as sterile medium: Component Bactobeef extract Bacto tryptose Dextrose (cerelose) Starch (soluble) Yeast extract Antifoamer Water Volume Liter 14.92 39.43 8.15 177.47 24.90 2.36 4731.66 Density gm/cc 0.95 0.60 0.58 0.64 0.95 0.10 Mass Kg 14.18 23.66 4.73 113.58 23.66 4731.66 Mass Gm 14180 23660 4730 113580 23660 4731660

Total material in = 5365.8 lit Total material out = 5365.8 lit Assume 5195.07 lit of material from seed fermentor are transferred to process fermentor.

Process fermentor Material in = innoculum = 5195.07 lit = 213 10-3 m3 1050 kg / m3 = 5454.82 kg

Material in Yeast Dextrose CaCO3 Anti foamer CoCl2.6H2O Water

Volume (lit) 421.70 694.38 57.31 9.477 40358.13

Density (gm/cc) 0.95 0.58 0.70 0.10

Mass (kg) 400.61 402.74 40.12 9477.95 (ml) 4878 (mg) 40358.13

Total biomass = 5454.82 + 400.61 + 402.74 = 6257.98 Kg 6257.98 / 2 = 3128.99 Kg, is used as the energy source for production of

gentamicin sulfate. 3128.99 kg is consumed for other metabolic activities. 48% of 3128.99 kg is converted into CO2 and H2O. Therefore 1073.16 kg of CO2

and 439.02 kg of H2O are produced. 1512.18 kg (CO2 + H2O) + 1616.81 kg (unconverted biomass + antibiotic) = 3128.99 kg 2146.32 kg (CO2) + 975.6 kg (H2O) = 3128.99 kg

Material out: Component CO2 H2O Unconverted biomass Mass (kg) 1073.16 + 2146.32 = 3219.48 40358.13 + 439.02 + 975.6 = 41780.07 1616.81

Mixture of antibiotics Anti foamer CaCO3

4739.22 (gm) 9477.95 (ml) 4878

Assumption: (1) 100% conversion in fermentor (2) Total CO2 produced, will be removed from the vent provided at the top of fermentor.

Rotary vaccum filter: Assumption: Alcohol solubility of gentamicin is 10-3 mg/lit. It is almost negligible. For ease of separation, we will assume 15% solubility of gentamicin sulfate in the methanol. Gentamicin sulfate is totally soluble in water but it is insoluble in cold water. Material in: Biomass Antibiotic mixture Water Filter out: Antibiotic mixture Water Cake discarded: Biomass = 1616.81 kg = 4739.22 gm = 41780.07 kg = 1616.81 kg = 4739.22 gm = 41780.07 kg

H2SO4 tank: H2SO4 required: Normality = gm / lit (1 / Equivalent weight)

Mass of H2SO4

= Normality total volume Molecular weight / valency = 6 [(41780.07/1000) + (4.739/ 1050)] 1000 98 / 2 = 12284511.3 gm/ lit

Material in: Cold water Antibiotic mixture Water H2SO4 = 8536.5 lit = 8536.5 kg = 4739.22 gm = 41780.07 kg = 12284.51 kg

H2SO4 is used to maintain the pH = 2 Material out: Therefore total material in = total material out

Plate & frame filter press (1) Material in: Cold water Antibiotic mixture Water H2SO4 = 8536.5 lit = 8536.5 kg = 4739.22 gm = 41780.07 kg = 12284.51 kg

Filterate discarded: Cold water Water H2SO4 Antibiotic mixture (soluble) = 8536.5 lit = 8536.5 kg = 41780.07 kg = 12284.51 kg = 710.72 gm

Cake out: Antibiotic mixture (insoluble) = 4028.49 gm

NaOH tank: NaOH required: Normality Mass of NaOH = gm / lit (1 / Equivalent weight) = Normality total volume Molecular weight / valency = 4 1243.89 40 / 1 = 199022.4 gm/ lit Material in: Cold water Antibiotic mixture NaOH NaOH is used to maintain the pH Therefore total material in Material out: Total mass of all the component added =11370.35 kg = 1219.5 lit = 1219.5 kg = 710.72 gm = 10150.14 kg = 6.5 = total material out

Membrane filter: Material in: Cold water Antibiotic mixture NaOH = 1219.5 lit = 1219.5 kg = 710.72 gm = 10150.14 kg

Filterate discarded: Cold water NaOH Antibiotic mixture (soluble) = 1219.5 lit = 1219.5 kg = 10150.14 kg = 604.14 gm

Cake out: Antibiotic mixture (insoluble) = 3424.35 gm

Air dryer: Material in: Antibiotic mixture (insoluble) 5% of moisture will be removed Material out: Antibiotic mixture = 3253.13 gm = 3424.35 gm = 5% (3424.35) = 171.21 gm

Methanol tank: Material in: Methanol added = 975.6 lit 779.17 gm/lit = 760158.25 gm Antibiotic mixture = 3253.13 gm

Therefore total material in

= total material out

Material out: Total mass of all the component added =763.41 kg

Membrane filter: Material in: Methanol Antibiotic mixture = 760158.25 gm = 3253.13 gm

Filterate discarded: Methanol Antibiotic mixture (soluble) = 760158.25 gm = 487.8 gm

Cake out: Antibiotic mixture (insoluble) = 2765.2 gm

Anion exchange column:

Material in: Methanol = 24390 lit 779.17 gm/lit= 19003956.3 gm This 24390 lit of methanol contains 121.95 lit of Amberlite 40-S anion exchange resin. Antibiotic mixture Material out: Methanol = 19003956.3 gm = 2765.2 gm

Gentamicin sulfate Impurities removed

= 2033.54 gm = 731.7 gm

Vaccum evaporator Material in: Methanol Gentamicin sulfate Material out: Methanol Gentamicin sulfate = 13302.76 kg = 1984.76 gm = 19003956.3 gm = 2033.54 gm

Assume 48.78 gm of gentamicin sulfate is lost during evaporation.

Membrane filter: Material in: Methanol Gentamicin sulfate Filterate discarded: Methanol Gentamicin sulfate (soluble) Cake out: Gentamicin sulfate (insoluble) = 1687.05 gm = 13302.76 kg =297.70 = 13302.76 kg = 1984.76 gm

H2SO4 tank: H2SO4 required: Normality = gm / lit (1 / Equivalent weight) Mass of H2SO4 = Normality total volume Molecular weight / valency = 2 9756 10-3 98 / 2 = 956.08 gm/ lit

Material in: Gentamicin sulfate H2SO4 H2SO4 is used to maintain the pH Therefore total material in = 1687.05 gm = 956.08 gm = 4.5 = total material out

Material out: Total mass of all the component added = 2643.13 gm

Methanol tank: Material in: Methanol added = 243.9 lit 779.17 gm/lit = 193095.63 gm Gentamicin sulfate H2SO4 = 1687.05 gm = 956.08 gm

Therefore total material in Material out:

= total material out

Total mass of all the component added = 195738.43 gm

Membrane filter: Material in: Methanol Gentamicin sulfate H2SO4 Filterate discarded: Methanol Gentamicin sulfate (soluble) H2SO4 Cake out: Gentamicin sulfate (insoluble) = 1434.13 gm = 193095.63 gm = 252.92 gm = 956.08 gm = 193095.63 gm = 1687.05 gm = 956.08 gm

Vaccum dryer: Material in: Antibiotic mixture (insoluble) 5% of moisture will be removed Material out: Gentamicin sulfate = 1365.84 gm = 1434.13 gm = 5% (1434.13) = 68.29 gm

Thus produced gentamicin sulfate is further purified by following same series of equipments as considered above. This is well described and self explanatory as shown flow diagram-2. After purification of Gentamicin sulfate = 1365.84 gm, yield is Gentamicin sulfate = 902.43 gm. This gentamicin sulfate is used for producing gentamicin which is having antibiotic activity of 1120 units/ml.

Anion exchange adsorption column:

Material in: Cold Water = 36585 ml 10-6 m3 1000 kg/m3 = 36.585 kg This 36.585 lit of water contains 2.439 lit of IRA-400 (-OH form) anion exchange resin. Gentamicin sulfate = 902.43 gm.

Material out: Cold Water Gentamicin Impurities removed = 36.5 kg = 731.7 gm = 170.73 gm

Vaccum evaporator Material in:

Cold Water Gentamicin sulfate Material out: Cold Water Gentamicin

= 36.5 kg = 731.7 gm

= 36.5 kg = 600.48 gm

Assume 131.21 gm of gentamicin sulfate is lost during evaporation.

Vaccum dryer: Material in: Antibiotic mixture (insoluble) 5% of moisture will be removed Material out: Gentamicin = 571.94 gm = 600.48 gm = 5% (600.48) = 30.02 gm

CHAPTER DESIGNING OF EQUIPMENTS

FERMENTOR DESIGN

For fermentation broth: Density Viscosity Dia. of agitator Da Agitation speed n = 1050-1090 Kg/m (M. Verrall, P.No.11) = 0.10-1.5 Pa-S = 1m (A Scragg, P.No. 278) (Bailey & Ollis, P.No. 624)

= 300 rpm (assumed as generally taken between 150-500 rpm)

NRe = ( Da n ) / = ( 1 300/60 1050 ) / 0.10 = 5.25 104

From Ludwig, graph of Npo vs NRe: Npo = 2 As aeration is also taking place simultaneously, effective density of aeration will decrease resulting in lower power consumption.

Npog / Npo Npog

= 0.7 = 0.7 2 = 1.4

Npog Power P

= P / ( n (Da)5 ) = 1.4 1050 (300/60) (1)5 = 183750 W

= 183.750 KW

Calculation of oxygen transfer rate: The maximum possible mass transfer rate is simply that found by putting concentration = 0 All oxygen entering the bulk solution is assumed to be rapidly consumed. The maximum possible oxygen rate = X max / YO2

X = cell density YO2 = moles of cells formed per mole of oxygen consumed Combine X max = net rate of cell mass growth = cell mass / (culture volume time) (Bailey & Ollis, P.No. 386) X max = 14 gm/l = (14/ 30) gm/l hr = 0.467 gm/l hr

Taking Mass balance at steady state condition, K1a ( C1 C ) = X max / YO2

YO2 = 1.35 mg/l hr (filamentous fungi) (Bailey & Ollis, P.No. 284) C1 = bulk liquid concentration of oxygen in water = 10 mg/l It is kept higher than the critical oxygen value.

Critical oxygen value of filamentous fungi at 35 C = 0.009 mmol/l = 0.009 32 mg/l = 0.288 mg/l K1a ( C1 C ) K1a ( 10 0.288 ) K1a = X max / YO2 = 0.467 10 3 / 1.35 = 35.6 hr-1

(P/V) Ugs

= power required/ m = 5927.41 W/ m = superficial gas velocity

K1a 35.6 / 3600 Ugs

= 2.6 10-2 (P/V)0.4 (Ugs)0.5 sec-1 = 2.6 10-2 5927.410.4 (Ugs)0.5 sec-1 = 1.38 10-4 m/sec

Ugs C.S.A

= gas feed vol. flow rate / C.S.A = / 4 D = / 4 3.16 = 7.8426 m

Gas feed vol. flow rate = (0.467 103/1.35) (1/3600) = 0.096 10-3 Kg/ m sec

Oxygen transfer rate = 0.096 10-3 100 62 3600 = 2142 Kg/hr

Cooling water requirements Rate of oxygen transfer = 0.096 10-3 /32 Kmol 3600hr / 1000 l = 1.08 10-5 Kmol/ l hr (Bailey & Ollis, P.No. 293) Corresponding heat produced = 2 Kcal/l hr Total volume Heat produced = 62 m 1000 lit = 62 1000 lit 2 1000 cal = 1.24 108 cal/hr

Q Cp

= m Cp T = 0.45 cal/gm c (From Perrys handbook)

Assume water is supplied at 25 c and outlet temp of water is 40 c. T Q 1.24 108 m = 15 c = m Cp T = m 0.45 15 = 1.83 107 gm/hr = 1.83 104 Kg/hr

Cooling water supply = 18.3 m3/hr

FERMENTER MECHANICAL DESIGN:

From the standards Capacity Diameter Height Shell material Allowable stress Operating pressure = 62 m = 3.16m = 7.9 m = SS 316 = 80 MN/m = 1.5 atm

Pd = design pressure (5% excess of operating pressure) Pd = 5% (1.5) + (1.5) = 1.575 atm = 1.575 1.013 105 N/m = 1.59 105 N/m = 0.159 MN/ m

F = Allowable stress of MOC at given design temperature T = design temperature = 35C J = weld efficiency = 0.85 Di = internal diameter of the vessel = 3.16m

Shell thickness ts = (Pd Di) / [(2F J) Pd] Shell thickness ts = 1.59 105 3.16 / [ ( 2 80 106 0.85 ) ( 1.59 105 ) ] = 3.71 10-3 m = 3.71 mm So, we will provide 4mm thickness.

Do = outer diameter of the vessel = 3.16 + 0.004 = 3.164 m

Head design We will consider torispherical head Knuckle radius (ri) = 0.06 Do = 0.06 3.164 = 0.2528 m = 252.8 mm

Crown radius (ro) Where W

= Do = 3.164 m = 0.25 ( 3 + (ro/ri )0.5 ) = 0.25 ( 3 + (3.164/0.2528)0.5 ) = 0.9735

Corrosion allowance = 0

Thickness of head is given by Th Th = ( P ro w ) / [ ( 2 F J ) + c ] = ( 0.159 3.164 0.9735 ) / [ ( 2 80 0.85 ) + 0 ] = 0.00360 m = 3.6 mm So, we will consider 4 mm thickness for both top and bottom head because they are subjected to same external pressure.

Jacket design We will use half coil jacket. Internal design pressure for jacket Pd = 10% (1.0) + (1.1) = 1.1 1.013 105 N/m = 1.11 105 N/m = 1.1 Kg/cm Assume internal diameter of half coil = 100 mm = 1.1 atm

Thickness of jacket = ((Pd Di) / [(2F J) Pd] = (1.11 105 0.1 ) / [2 ( 80 106 0.85 ) ( 1.11 105 )] = 8.16 * 10-5 m = 0.0816 mm Minimum thickness of jacket should be 3 mm For half coil jacket,

Circumferential stress occurring at the junction with shell, fpc tc = ( P di ) / ( 2 tc ) = (1.11 105 0.1) / ( 2 80 106 ) = 6.93 * 10-5 m = 0.0693 mm Use Minimum thickness of coil to be 3 mm

Longitudinal stress is given by fac = ( P * di ) / [ ( 4 * tc ) + ( 2.5 * ts )

fac

= ( 1.11 105 * 0.1 ) / [ ( 4 * 0.003 ) + ( 2.5 * 3.71 * 10-3) ] = 5.40 * 105 N/m2

Circumferential stress in the shell, fp = fac + fpc = 5.40 * 105 + 80 106 = 80.54 106 N/m2 These both the stresses are less than allowable stress value = 96.13 106 N/m2

Agitator design Diameter of tank Dt Diameter of agitator Da Diameter of impeller Di = 3.16 m = 1.00 m = 0.333 Dt = 1.05 m L/Di = 0.25 length of agitator L = 0.2625 m

W/Di = 0.20 width of agitator W = 0.21 m Wb/Dt = 0.1 width of baffle Wb Da/W = 6 = 0.316 m

width of blade W = 0.16 m

Speed of agitator = 300 rpm M.O.C = SS 316

We have already calculated power required = 183.750 KW

= 246.3 HP 10% gland losses 15% transmission losses We will take motor of = 270.93 HP = 286.9 HP = 300 HP

Permissible shear stress Elastic limit in tension Modulus of elasticity

= 55 N/mm =275 N/mm = 1.83 105 N/mm

Torque on shaft is given by Tc = (HP 746 60) / (2 n) = (300 746 60) / (2 300) = 7123.77 Nm

Maximum torque resistible by shaft is given by Tm = 1.5 Tc = 1.5 7123.77 = 10685.65 Nm

Modulus of shear section Zp = Tm / f permissible = 10685.65 / 55 = 194.28 103 mm Zp = /16 d3

Diameter of shaft (d) = 99.64 mm

Diameter of shaft based on bending moment: Fm = Tm / ( 0.75 Rb ) = 10685.65 / ( 0.75 850) = 16761.8 N

Max. Bending moment ( M )

= Fm L

L is the overhang between agitator and bearing = 2 m ( M ) = 16761.8 2 = 50285.4 Nm

Equivalent bending moment is given by Me = [ M + ( M + Tm )0.5 ] = [ 50285.4 + (50285.4 + 10685.65 )0.5 ] = 50846.80 Nm

Permissible stress in the elastic limit is given by F = Me / ( / 32 d3 )

275 = 50846.80 1000 / ( / 32 d3 ) d = 200 mm = 20 cm

Diameter of shaft based on critical speed: Moment of inertia I = / 64 d4 = / 64 2004 = 7.85 107 Deflection of shaft is given by Deflection Deflection = W(=Fm) L3 / 3 E I = ( 16761.8 20003 ) /( 3 183 103 7.85 107 ) = 0.3111 mm = 3.111 cm

Critical speed Nc

= ( 60 4.987 ) / 0.5 = (60 4.987 ) / 3.1110.5 = 536.46

Operating speed should be 45-65% of Nc. Condition is fulfilled here. So, we are selecting shaft diameter = 20 cm

Jacket design If we select half coil, Selecting tube of 7/8 inch, 14 BWG Outside diameter Wall thickness Inside diameter Pitch of coil = 22.58 mm = 2.11 mm = 18 mm = 2.5 D = 2.5 3.16

= 7.9 m If coil is provided to one diameter vessel then no. of turns in the coil is 850 So here, no. of turns = 850 / 7.9 = 107.5

Length of tube

= 108 0.62 = 210 m

Outside area of coil for heat transfer = 0.02258 210 = 14.9 m

Bracket design Diameter of vessel Height of vessel No. of brackets = 3.168 m = 7.9 m =6 =1m =1m =3m

Diameter of anchor bolt circle Height of vessel from bottom Height of bracket from foundation

Permissible stresses for structural steel: Tension Bending = 140 N/mm = 157.5 N/mm = 3.5 N/mm

Permissible bearing pressure of concrete

Weight of reactor

Dblank = ( Do ) + ( Do/42 ) + ( 2/3 ri ) + ( 2 Sf ) = ( 3.168 ) + ( 3.168 / 42 ) + ( 2/3 0.2528 ) + ( 2 4 ) = 3.483 m

= [ ( D L ) + ( 2 ( /4) Dblank ) ] t = [ ( 3.168 7.9 ) + ( 2 (/4) 3.483 ) ] 0.004 7800 = 3045.3 Kg

Maximum weight of content = 47000 Kg Maximum weight of content = 200 Kg Total weight = 50545.3 Kg = 495.8 103 N

Maximum compressive load P =W/6 = 495.8 103 / 6 = 82263.34 N

Base plate for bracket Suitable base plate size A = 140 mm & B = 150 mm Average pressure on plate PAVG = P / ( A B ) = 82263.34 / ( 140 150 ) = 3.91 N/ mm = 140 150 mm

Maximum bending stress I a rectangular plate subjected to pressure P AVG and fixed at the end is f bending = 0.7 PAVG ( B / T12 ) [ A4 / A4 + b4] 157.5 = 0.7 3.9 ( 150 / T12 ) [ 1404 / 1404 + 1504] T12 T1 = 168.65 = 12.98 mm = 14 mm (for ease of weld ability)

Web plate design Bending stress in the we plate, C = Eccentricity = (1.65 1.5) / 2 = .075 m Angle with horizontal of web plate cut = = 45

f bending = ( 3 P C ) / ( T2 h2 COS ) 157.5 T2 = ( 3 82263.34 75 ) / ( T2 1402 0.707 ) = 8.46 mm

We will consider thickness of plate as 10 mm

Column support for bracket

It is proposed to use channel section as column. The size chosen is ISMC 150 Size 150 75 Area of cross section Modulus of section Radius of gyration = 20.88 cm2 = 19.4 cm3 = 2.21 cm

Weight Height from foundation

= 16.4 Kg/m =3m

Equivalent length for fixed ends le = l / 2 = 3 / 2 = 1.5 m Slenderness ratio = le / r = 1.5 100 / 2.21 = 68.18

Maximum combined bending and direct stress is given by, f bending = ( P / A ) + ( P e / Z ) f bending = (82263.34 / 2088 ) + (61975 75 / 19400 ) = 359.4 N/mm2

Direct stress, f = [ ( P / A ) ( 1 + a (le / r ) 2 ) ] + ( P e / Z ) = [ (82263.34/2088) (1 + { (1/75000) (68.18) 2}) ] + (61975 75/19400) = 359.76 N/mm2

As both stresses exceed permissible limit, we should use base plate of larger cross section area than that is suggested over here.

Base plate for column The size of the column is 200 100 Base plate extends 20 mm & 30 mm on respective sides.

Side B = 0.8 100 + 2 20 = 120 mm Side C = 0.95 200 + 2 30 = 250 mm

Bending moment, Pb =P(1/(bc)) = 82263.34 ( 1 / ( 120 250 ) ) = 2.72 N/mm2 This is less than the permissible stress for concrete = 3.5 N/mm2

Maximum bending moment in the plate, M = Pb L2 / 2 = 2.72 (20+30) 2 / 2 = 850 N

Maximum stress in the plate f = 6 M / t2

157.5 = 6 850 / t2 t2 t = 32.38 = 5.69 mm

So we should use plate of thickness 6 mm

FILTER DESIGN: PLATE AND FRAME FILTER:

Plate and frame filters are commonly used for batch processes in fermentation techniques. The slurry is fed to the filter press through the continuous channel formed by the holes in the corner of the plates and frames. The filtrate runs through down grooves in the filter plates and is then discharged through outlet into a pipe for maintaining aseptic conditions. Advantages of Plate & Frame filter press for this process: On industrial scale it is one of the cheapest filters per unit of filtering space for batch process It is most suitable for separating low solid content from fermentation broth and low resistance to filteration.

DESIGINING OPERATING DATA: Filterate discarded: Cold water Water H2SO4 Antibiotic mixture (soluble) Cake out: Antibiotic mixture (insoluble) = 4028.49 gm = 8536.5 lit = 8536.5 kg = 41780.07 kg = 12284.51 kg = 710.72 gm

Total filterate out

= 62601.7 Kg.

Average density of filterate

(f) = [ (50316.57 / 62601.7 1000 ) + (12284.51 / 62601.7 1830 ) + ( 0.7107 / 62601.7 1050 ) ] = 1162.8 Kg/m Volume of filterate (Vf) = total filterate out / Avg. density of filterate = 62601.7 Kg / 1162.8 Kg/m = 53.83 m

Total amount of cake Density of solids (s)

= 4.082 Kg = 1050 Kg/m

Total liquid out with cake

= 0 Kg

Cake moisture

(m)

= Wet cake mass / dry cake mass = 4.082 / 4.082 =1

Mass fraction of solid in slurry (S) = mass of cake / (mass of cake + filterate) = 4.082 / ( 4.082 + 62601.7 ) = 6.52 10-5 Filter pressure drop Specific resistance Medium resistance (P) () (Rm) = 10 KN/m = 5 108 m/Kg = 4 109 m1

Filteration time

(t f)

= 20 min

FILTER AREA REQUIREMENTS:

Solid concentration is given by (C) = ( f S ) / ( 1 ( m S ) ) = ( 1162.8 6.52 10-5 ) / ( 1 ( 1 6.52 10-5 ) = 0.075 Kg/m

Viscosity of solution () Filteration time is given by

= 0.1 Pa-sec (N-sec/m)

(t f) = [ ( C Vf ) / ( 2 A P ) ] + [ ( Rm Vf ) / (A P) ]

15 60 =

[(0.10 0.075 5 108 53.83 ) / ( 2 A 107 ) ] + [ ( 0.10 4 10 9 53.83 ) / (A 107 ) ]

Area of filter (A) = 2.62 m

TOTAL CYCLE TIME:

Let us take down line time (td) = 5 min

Down line time is time required for filling the filter and discharging filter cake.

K1 and K2 are filter parameter. K1 = ( C ) / ( 2 A ) = (5 108 0.10 0.075 ) / ( 2 2.62 ) = 2.73 105

K2 K2

= ( Rm) / A = ( 0.10 4 109) / 2.62 = 1.52 108

Rate of wash = 0.25 P / [( 2 K1 Vf ) + K2 ] = ( 0.25 10 106 ) / [( 2 2.73 105 53.83 ) + 1.52 108]

= 0.0137 m/sec

Volume of wash

= 0.1 Vf = 0.1 (53.83 ) = 5.383 m

Calculation of wash time (tw) (tw) = volume of wash / wash rate = 5.383 m / 0.0137 m/sec = 392.9/60 = 6.54 min 7min

We will consider wash time of 10 min for ease of operation.

Total cycle time

= t f + td + tw = 15 + 5 + 10 min = 35 min

CAKE THICKNESS Average porosity of solid can be calculated from its density and moisture content by E = ( s ( m -1 ) ) / ( + s ( m -1 ) ) = ( 1050 ( 1 -1 ) ) / (1162.8 + 1050 ( 1 -1 ) ) =0 Porosity is taken as zero because we have assumed that no liquid is going out with cake. Optimum cake thickness l* = w* / ( A ( 1 E ) s ) = ( C Vf ) / ( A ( 1 E ) s ) = (0.075 53.83) / (2.62 ( 1 0 ) 1050) = 0.0014 m

NO OF FRAMES: Take total area 25% excess that from calculated Actual Area A* = 1.25 A = 1.25 2.62 = 3.275 m Frame area A = A* / 2

= 3.275 / 2 = 1.63 m

If we take 600 600 mm frame size,

Area of one frame

= 0.36 m

No. of frames

= A / A = 1.63 / 0.36 = 4.52

Here solid content of slurry is so less that number of frames calculated is approximately 5. We can use 6 no. of frames for ease of operation. Better alternative will be simpler membrane filtration techniques.

CHAPTER INSTRUMENTAION & PROCESS CONTROL

Instrumentation:

Instruments are provided to monitor the key process variables during plant operation. They may also be part of an automatic computer data logging system, instruments monitoring critical process variables will best fitted with automatic alarms to alert the operators to critical and hazardous situations. Instrumentation and control objective: The primary objectives of the designer when specifying instrumentation and control are: 1) Safe plant operation. - To keep the process variables within known safe operating limits. - To detect dangerous situations and to provide alarms and automatic shut down systems. - To provide interlocks and alarms to prevent dangerous operating procedures. 2) Production rates: - To achieve the desired product output. 3) To maintain product composition within specified quality-standards. 4) Cost The typical control system consists of the following parts: (A) The process (B) measuring element (C) controller (D) final control element Control system used within the plant Volume measurement: There are tow systems which are generally used for on line volume determinations: - Liquid level sensors and - Weight or mass measurement devices. At the plant level the pressure difference measurement method is usually used, where a

flush mounted diaphragm pressure cell is located in the base of the vessel for liquid height, and hence volume measurement. Weight/mass measurement: Weight or mass of the liquid can be determined by scales of various types. In this method the vessel is suspended on a scale and the confined with of the vessel and liquid is electronically measured, usually by strain type gauges. Several methods for electronically or mechanically eliminating the tare weight of the system can be used. Liquid metering: Liquid metering is an important physical process variable since it serves as the basis for material balance analysis when fresh nutrient feed is added during the culture. Liquid flow measurement devices like flow monitors (meters) and metering pumps (steam utilizablein place) can be used for better liquid metering purpose. Gas flow measurement: Introduction of air into aerobic biological processes serves essentially two purposes. It supplies the necessary oxygen and assures the removal of carbon dioxide and other volatile gaseous products of metabolism. On line monitoring of gas flow can be performed by mean of a simple variable area meter such as rotameter. Gas flow meters with electrical transducers, are also available. These include devices such as - Magnetic flow rate sensing elements - Thermal mass flow rate sensing elements - Laminar flow rate sensing devices. Temperature measurement:

Heat energy is considered to be the only environmental variable which freely penetrates the cell wall and directly influences intracellular metabolism. Therefore temperature measurement and control is considered to be the most important direct process variable. Typical sensor devices which can be used for fermentation application are described below: - Platinum resistance sensor - Thermistors - Thermocouples - Filled bulbs

Pressure measurement Static head pressure inside the bioreactor influences the partial pressure conditions and hence solubility of gases and volatile compounds. A positive head pressure can also contribute to the maintenance of sterility inside bioreactors operating under aseptic conditions. Also, it is important to measure internal vessel pressure conditions during sterilization as well as the pressure condition of the main supply services (steam water and air). Depending on the mode of application, there are two pressure measuring systems generally accepted for biochemical reactors: - pressure devices without electrical signal outputs and - pressure transducers In their applications there are several points to note: At non-sterile points, generally bourdon gauges are used. For steam pressure measurement a gauge protector is usually employed. For determination of vessel pressure under aseptic conditions a diaphragm is placed between the sterile environment and the gauge. For fermenter head pressure measurement, which also provides an electronic signal useful for

on line analysis as ell as for automatic control of a given head pressure when gas flow rates are changed, pressure transducers are required. Agitation speed measurement: The measurement of agitation speed can be by either direct or indirect monitoring devices. For low speed rpm ad in our plant direct gear or belt driven tachometers are used for monitoring of agitator speed. For higher speed monitoring indirect magnetic devices are preferred. The diameter of the impeller will determine the absolute rotation speed of the tip of the impeller, which in turn defines the liquid shear rate and liquid turnover rate in the culture vessel. Power input to the fluid: Power measurement of the energy imparted the fermentation fluid is the use of an in-line torque measuring device treated external to the vessel. The losses due to bearings and seals will be induced in this type of measurement but by suitable no load testing it can be effectively eliminated from the reading and a reasonable approximation of power imparted to fluid can be made. A Hall Effect wattmeter measures at the drive motor armature the total energy consumed by the agitator. A torsion dynamometer may also be used to measure shaft power input. A disadvantage of both the instruments is inclusion of frictional losses in the shaft bearings and seals. Newtonian and non-Newtonian broths have been observed to respond differently during such a brief agitation transient.

PH measurement: The only steam sterilizable truly direct reading sensor with high selectivity is the pH probe, therefore requires only the penetration in the fermenter.

Medium chemical sensors

Electrodes which can be repeatedly sterilized in place are now available of redox potential and dissolved oxygen and CO2 partial pressure. Measurement of medium redox potential is possible using a combined platinum and reference electrode. While the influence of pH biochemical kinetic is clearly established and the physical significance of a pH measurement is straightforward, interpretation of redox potential measurement and understanding the relationship between redox potential and cell activity can be difficult. The various types of dissolved oxygen probes now available are of galvanic or poarographic (amperometric or clark) types. These electrodes measure the partial pressure (or activity) of the dissolved oxygen and not the dissolved concentration. The fig below shows the schematic summary of biochemical instrumentation. A striking feature of this illustration is the predominance of measurement of medium and gas chemical & physical properties while shortage of measurement of cell properties.

Process control:
During operation, a chemical plant must satisfy several requirements imposed by its designed and the general technical, economic and social conditions. Among such the requirements are the following. (A) Safety (B) production specifications (C) environmental regulations (D) operational constraints (E) economics There are three general classes of needs that a control system is called on to satisfy. - Suppressing the influence of external disturbances, - Ensuring the stability of a chemical process. - Optimizing the performance of a chemical process.

Control system for fermenter The objectives of applying instrumentation to fermenter are: (A) To analyze the process status and (B) To establish an optimum set of environmental conditions in order to maximize specific cellular or enzymatic activities. Analysis of conditions in biological process is performed by two general methods. (A) Off-line operating instruments (B) On-line operating instruments

Off-line operating instruments: Off-line instruments are not connected directly to the fermenter consequence, sampling (sample preparation or treatment) and sample analysis are performed and the analysis results provide information on the process status. Current examples of off-line operating instruments include spectrophotometers, mass spectrometers, gas chromatography, enzyme activity analyzers and electron microscopes.

On-line operating instruments On-line operating instruments are directly connected to the fermenter and have the capability to sense a specific variable which directly or indirectly reflects a process condition and /or the performance characteristics of a controller. The predominant types of on-line operating instruments indirectly reflect a process condition in the whole broth culture.

Measurement and control of environmental variables: The environmental variables include temperature hydrogen ion concentration (pH) partial pressure of dissolved oxygen (DO) and concentration and proportion of certain nutrients.

The fermentor temperature is controlled by measuring the temperature via an electronic signal producing thermometer, by comparing the deviation between the actual and desired temperature, and by manipulating a control valve, the position of which control the flow rate of cooling water to a cooling surface. In a complex situation more than one manipulated variable performs the control of a process variable. In the case of dissolved oxygen measurement, the controller based on DO level measurement and comparison with a set point, governs both the inlet air (O2) flow rate control valve and the impeller motor speed controller in an interactive way to maintain a dissolved oxygen level under dynamic conditions where the oxygen consumption and oxygen input rates must be in balance. The air (O2- gas) flow rate and the agitation speed interactively influence the oxygen mass transfer coefficient (KLa) resulting in the dissolution of gaseous O2 into the culture broth.

Cascade control of metabolism The ultimate objective of any cellular bioreactor control scheme is to provide an environment or an environmental history which drive the metabolic controls in the organisms to maximize the production of the desired compounds. Accordingly, instead of thinking pH or temperature at some particular value, it may be more useful; to consider controlling of the so that the culture growth rate or respiratory quotient is kept at a desired value. This is feasible since, we estimated some metabolic properties of culture based on the available measurements. Thus estimated properties are compared with set point value. Error over here determines the metabolic control action using one of the feed back controller algorithms. The output of the metabolic controller may be used directly to alter a process input such as a pumping rate. Alternatively, the metabolic controller output may be used to change the set point of an environmental controller say for pH or DO. The pH or DO controller which in a good will change the metabolic variable to reduce its deviation from the metabolic variable set point. When the environmental variables are controlled by local single loop controllers and the environmental controller set points come from a digital computer the scheme is called supervisory control or digital set point control (DSC). Of course, everything may be done by digital computer/controllers.

CHAPTER PLANT LAYOT & LOCATION

Plant location
Plant location strictly means to discover an exact place where an industrial enterprise can be started more profitably and plant is place where man, materials, money and machinery are brought together for manufacturing products. Thus problem of plant location is concerned with selection of a profitable geographical region and then a favorable site

within that region. If the plant location is not carefully selected then it wipes out the advantages of the process and careful engineering research and development work. The final selection of plant location has a strong influence on the success of industrial venture.

Factors to be considered for plant location selection 1) Primary factors

Raw material supply The source of raw materials is one of the most important factors which influence the selection of the plant. The raw material should be easily and cheaply available at the plant site. It is necessary that raw material is available at the plant site when required without any delay. The major raw material used for manufacturing gentamicin is micro organisms, which can be easily available from soil. Some protein, minerals and nitrogen sources are also required to feed these micro organisms which includes soybean flour, starch etc.

Market The location of plant affects the cost of product. The proximity to large market can be beneficial in three ways; cost of transporting finished goods to the market is brought down. The delay in supplying goods to market is avoided. The market can be studied properly and easily. Gentamicin is mainly used as base for preparing antibiotic injections and ointments.

Energy availability Power and fuel can be considered as major factors in choice of plant. The local cost of power can help to determine whether it should be purchased or self generated.

Water supply The chemical process industries use large quantities of water for cooling, heating, washing steam generation, sanitary purposes etc and in erythromycin plant fermentation process requires a large quantity of water. Hence plant site should be nearer to the source of water.

Climate Weather has seasonal effect on the economic operation of the plant. The temperature and the humidity should be favorable. Also the wind velocity, hurricanes, earthquakes, floods etc. factors should be considered.

2) Secondary factors

Transportations A plant should have easy access to transport facilities. The transport facilities available to the plant must not only easily accessible but they must be enough, quick and available at reasonable rates also. Water, rail road and highways are common means of transportation. These facilities are necessary for transfer of raw materials and products.

Labor supply Availability of skilled labors with constant supply and reasonable wages should be considered in the selection of plant site. Labor problems should be minimized.

Waste disposal

The site should be such that it should have the best and adequate facilities for waste disposal without polluting the environment. In choosing plant site the permissible tolerance levels for various methods of waste disposal should be considered carefully.

Taxes and legal phases The state and local tax rates on property, income, transportation, and facilities have a major influence on the site selection.

Site characteristics The characteristics of the land at the proposed plant site should be examined carefully. The topography of the tract of the land and the soil structure must be considered since either or both have pronounced effect on the construction costs. Future expansion of the plant should be envisaged.

Fire fighting The site should be such that it should have best possible fire protection facilities during emergency.

Advanced library and training To develop the plant property and efficiently trained staff is very much essential and for further research, advanced library facilities covering the subjects in details are necessary.

Community attitude Success of the industry depends on attitude of local people and their mutuality.

Plant layout
Once the location of plant is decided and the process flow diagrams are completed, the immediate step is to have specific plant layout. Plant layout means the allocation of space, arrangement of equipment and machinery in such a way that maximum utilization of man power, machinery and materials is achieved and minimum material handling is required. Therefore the manufacturing costs of the unit are the least. The following factors should be considered while selecting plant layout.

New site developments or addition to previously developed site. a) Type and quantity of product to be produced. b) Possible future expansions. c) Economic distribution of utilities and services. d) Type of building and building code requirements. e) Health and safety considerations. f) Waster disposal problems. g) Sensible use of floor and elevations space. h) Operational convenience and accessibility. i) Proper usage of space available.

Maximum advantage of gravity flow is taken to reduce operational costs. An optimum layout is one which provides maximum satisfaction to all concerned i.e. employees, management etc. The basic principles of plant layout are-

1. Principle of minimum distance moved 2. Principle of flow 3. Principle of flexibility 4. Principle of satisfaction and safety.

Equipment layout A primary route for equipment layout is that an operator should have at least two different routes to escape form any point in the unit. Also equipment should be located in such a way that flow of material is stream lined. Since a clean well maintained area is often a safe area.

Plant expansion Plant expansion is an important aspect and area must be provided before hand. Generally plant costs are bound to increase with passage of time, hence purchasing more than required is always a better idea.

Building Plant offices, mechanical shops and laboratories should be located as far as possible from the operating units. Ware housing and loading facilities should be located at the plant properties line so they are easily accessible from the public roads and as far as possible from the area of possible danger.

Utilities The generation and distribution of utilities in a process plant is such an important function that these facilities should be located as remote as practicable from the operating units.

Storage layout

Storage facilities for raw materials and finished product should be located in an isolated area. Arranging storage of materials so as to facilitate or simply handling is also an aspect to be considered.

Gravity flow The higher area of the site may be used advantageously for storage of raw materials and products so that gravity loading of takers is possible. Also storage area must be nearer to the existing highway and on the plant road lines. Adequate likes must be built around there tanks to prevent rapid spread of fire. Existing highway and rail and road facilities Offices and ware house must be readily accessible from the main highway and rail roads.

Arrangement of process units If the product from one process unit is fed directly to another, the unit should be adjacent to reduce piping and pumping costs. Operation, maintenance and utility distribution are simplified by location similar units in one section of the plant.

Plant services The power generations, shops, ware houses, cafeteria and change house should be located not only for maximum efficiency and convenience but also for minimum interference with the process operation.

Safety

Combustible and inflammable materials must be stored away from equipments and units processes generation heat or flame. The plant area should be declared as " non smoking zone'' and a separate zone or chambers must be provided for smoking also medical and fire fighting facilities must be situated in such a way that they are accessible to the workers during the time of need.

CHAPTER SAFETY AND POLLUTION CONTROL

SAFETY:

The safety of working personnel in a chemical plant is of prime most consideration. The principal ways in which chemicals may enter our body are: (1) Ingestion (2) Skin absorption (3) Inhalation For safety of every individual working in the plant one should not ignore the standard procedures for operating the equipment. One should never take risks to save time and avoid work and use shortcuts or approximate readings. Safety of the workers as well as the personnel safety depends upon the sincerity and accuracy of the operator. Safety should never be compromised by anything .The loss of individual life can never be compromised by cash profit. Hence during operation of a plant, safety should be of prime importance.

HAZARDS COMMONLY ENCOUNTERED IN CHEMICAL INDUSTRY:

TOXICITY Most of the materials used in manufacture of chemicals are poisonous to some extent. The potential of hazard depend upon the toxicity of the material. There are two types of effects of chemicals: (A) Short term effects or acute effects (B) Long term effects or chronic effects. Inherent toxicity of the materials is determined by tests on animals. It is usually expressed as lethal dose LD50 at which 50% of test animals are killed. It is expressed as mg of toxic substance /kg body weight of test animal. LD only gives idea of acute effects.

For long term effects a quantity called threshold limit value is defined. TLV is defined as concentration to which it is believed the average worker could be exposed to day by day for 8hrs a day, 5days a week without suffering harm. It is expressed as ppm for vapors.

Control of toxic materials: ~ Containment: Safe design, leak proof joints. ~ Disposal: Provision of venting, scrubbers ~ Ventilation ~ Emergency equipment ~ Regular medical Check-up ~ Monitoring of environment ~ Good general hygiene.

FLAMMABILITY Hazards caused by flammable materials depend upon following factors: (a) Flammability limits - they are the lowest and highest concentration of material in air at normal pressure and temperature. (b) Auto ignition temperature - it is the temperature at which the liquid will ignite spontaneously in air without any external source of ignition. (c) Flash point of material - it is measure of ease of ignition of liquid, lower the flash point more flammable the liquid is.

EXPLOSIONS

Explosion is a sudden, catastrophic, release of energy, causing pressure wave. Explosion can occur without fire, such as failure through over pressure of steam boiler or an air receiver. Explosion can occur by release of flammable gases, vapors or dust.

SOURCE OF IGNITION Following are different sources of ignitionElectrical equipment - sparking in electrical equipment may cause ignition, thus flame proof material is required. The equipment should be intrinsically safe without any faults and risk of ignition. Static Electricity - movement of any no conducting material, powder, liquid or gas can generate static electricity producing sparks. Precautions should be taken that all equipment is properly earthed.

Process flames- open flames from process furnace are source of ignition and must be away from flammable materials in plant.

IONIZING RADIATION The radiation emitted by radioactive materials is harmful to living matter; radioactive materials should be handled carefully.

PRESSURE Over pressure in the system can cause lot of hazards. It can associate with failure of vessel or pipelines causing explosion. Hence different pressure relief devices are used to avoid hazard.

TEMPERATURE Temperature, higher than the operating temperature may cause failure of the equipment and loss of control of reactors and heaters. Following protection against high temperature should be ensured: High temperature alarms, internodes, shut down of reactor feeds, heating system, emergency cooling systems, and structural design of equipment to withstand high temperature.

NOISE Excessive noise is a hazard to health and safety. Long exposures to noise can cause permanent damage to ear or fatigue. The unit of sound level is decibel.

RESPIRATORY PERSONNEL EQUIPMENT

PROTECTION - Hazardous gases should be vented outside the building away from work area. - Provide exhaust facilities like providing exhaust fan. - Protection equipment should be easily available.

There are three types of equipments uses: (1) Industrial Canister Air from contaminated area is allowed to pass through the canister containing suitable absorbing agent which will absorb the harmful gas. The clean air which will come out will be going to mouth piece which is worm by the person.

(2) Airline respirator It consists of a half or full mask face piece or a loose fitting hood or helmet to which air is supplied through a small diameter hose.

(3) Self contained Breathing Apparatus (SCBA) It is a self sufficient breathing apparatus which permits freedom of movement as the wearer carries the supply of breathable air. This is the apparatus in which user obtained air or oxygen from compressed air or oxygen cylinders.

GOOD MANUFACTURE TECHNIQUES TO PREVENT ACCIDENTS

Filling burette - Keep burette below eye level.

Filling drum - Keep hose pipe little inside the drum rather than on the hole.

Using fuming chamber - In laboratory while working with hazardous chemicals like H2S, N02 use fuming chamber.

Reduce heat of reaction -Add sulfuric acid to bucket full of water and not water to bucket full of sulfuric acid.

Opening flanges - While opening a flange on pipeline containing corrosive liquid, chances of liquid coming out with a spray are there. To avoid accident due to such spray or acid or alkali use plastic sheet while opening valve. So that it will not contact with body.

Location of gauge glass - Gauge glass for reading level in the tank should be located away from path where many people may be working.

Location of safety valve/ vent line - The vent pipe should not be located in a closed area.

Location of flammable material - Storage should be away from any source of flame.

Smoking - Do not smoke in unauthorized area where flammable materials are likely to be present.

Purging with inert atmosphere - Before entering a reactor or a distillation column containing hazardous vapors, the equipment must be purged with air/inert gas for sufficiently long time.

Machinery guards - Install guards on moving machinery parts.

Incompatible chemicals - Do not mix incompatible materials together.

Earthling of equipment - When two phase mixture is being separated into different tanks, the tank should be earthed to avoid spark due to accumulation of static electricity. Explosion due to dust - In the operation of fine grinding, solid temperature increases which can lead to dust explosion initiated by hot metal. It can be prevented by cooling grinder with water or inert gas purging.

Drying and ignition of flammable liquids - Keep air flow rate high so that air vapor mixture is not near flammable limit.

Mixing - It should be effective to take care of exothermic heat of reaction.

Good house keeping - Do not store waste flammable materials near flame source.

Labeling of chemicals - Label the chemicals so that they do not get mixed up with incompatible chemicals.

Pipetting - Do not suck with mouth, use rubber bulb.

Free excess energy exit - Do not store anything in passage way destructing free movement in emergency.

 Common safety measures for various chemicals handled in the gentamicin plant:

GENTAMICIN

Hazard identification Emergency overview: Danger! Irritant. Toxic if swallowed. May cause organ failure or death.

Toxic if absorbed. Toxic by inhalation. May cause sensitization. Possible reproductive system hazard based on animal data.

Potential health effects:

Eye: Can cause moderate irritation, tearing and reddening, but not likely to permanently injure eye tissue.

Skin: It can cause moderate skin irritation, defatting, and dermatitis. Not likely to cause permanent damage. It may cause sensitization. Upon prolonged or repeated exposure, toxic if absorbed through the skin. It is likely to cause systemic damage.

Inhalation: It can cause moderate respiratory irritation, dizziness, weakness, fatigue, nausea and headache. It may cause allergic respiratory reaction. Toxic! It can cause systemic damage. Respiratory failure is possible at high doses.

Ingestion: Irritating to mouth, throat and stomach. Can cause abdominal discomfort, nausea, vomiting and diarrhea.toxic if swallowed. May cause target organ failure and/or death.

Chronic: No data on cancer. Contains a substance(s) that is a possible reproductive system hazard based on high dose tests with laboratory animals.

Fire fighting measures: Extinguishing media: Not combustible. Use extinguishing media appropriate for surrounding fire.

Firefighting techniques/equipment: Do not enter fire area without proper protection including self-contained breathing apparatus and full protective equipment. Fight fire from a safe distance and a protected location due to the potential of hazardous vapors and decomposition products.

Hazardous combustion products: Includes carbon dioxide, carbon monoxide, dense smoke.

Handling and storage: Storage of some materials is regulated by federal, state, and/or local laws. Storage pressure: Ambient

Handling procedures: Harmful or irritating material. Avoid contacting and avoid breathing the material. Use only in a well ventilated area. Keep closed or covered when not in use. Storage procedures: Store in a cool dry ventilated location. Isolate from incompatible materials and conditions. Keep container(s) closed. Suitable for most general chemical storage areas.

Personal protection: Eye: Safety glasses should be the minimum eye protection. Wear chemical goggles.

Skin: Prevent contact with this product. Wear protective gloves, apron, face and further skin protection depending upon condition of use. Inspect gloves for chemical break through and replace at regular intervals. Clean protective equipment thoroughly after each use. Do not wear surgical style latex gloves. Wash hands and other exposed areas with mild soap and water before eating, drinking, and when leaving work. Gloves should be used as minimum hand protection.

Respiratory: A respiratory protection program that meets OSHA's 29 CFR 1910.134 and ANSI Z88.2 requirements must be followed whenever workplace conditions warrant a respirator's use.

Stability and reactivity: Stability: Stable under normal conditions. Conditions to avoid: Strong oxidizing agents. Hazardous decomposition products: Carbon monoxide. Carbon dioxide. Sulfur containing gases. Nitrogen oxides. Hazardous polymerization: Hazardous polymerization will not occur.

Soybean oil

* characteristics : pale yellow to brownish-yellow liquid * composition : glcerides of fatty acids * melting point : 22.2 C * flash point : 540C * auto ignition temp : 833C * density : 0.925 gm/cc

Hazard analysis: a substance which migrates to food from packaging materials Fire hazard: slight, when exposed to heat or flame. Spontaneous heating: moderate Fire extinguishers: foam, carbon dioxide dry chemical or carbon tetrachloride.

Caustic soda

* formula: NaOH * molecular weight: 40.01 * boiling point: 1390C * melting point: 318.4C * vapor pressure: 1 mm at 739C * hazard analysis:

Acute effects: irritant, ingestion, inhalation

Toxicity: It has a markedly corrosive action upon a body tissue, which causes burns and frequently deep ulceration. Prolonged contact with dilute solutions has a destruction effect on tissue. Effects of inhalation may vary from mild irritation of the mucous membranes to a severe pneumonitis. Disaster hazard: It is dangerous. It will react with water or steam to produce heat and will attack living tissue.

Treatment: Speed in removing this caustic from contact with the skin of one who has come in contact with it is important to avoid injury. Remove all contaminated clothing at once and if possible give patient a shower. If the eyes are involved, they should be irrigated at once with plenty of warm water for 15 minutes. Persons so injured should be referred to a physician.

YEAST EXTRACT

Product description: Tastone 154 is a primary grown strain of Saccharomyces cerevisiae. It is a water soluble yeast extract produced by the natural autolytic action of yeast proteases. Tastone 154 is a light brown spray dried powder. Usage and storage: 0.5 2 lb. per 1000 gallons. Tastone 154 is used as a nutrient in all fermentations. It has a high concentration of natural protein and free amino nitrogen. It also supplies peptides, vitamins, minerals, and trace elements. The product

is hygroscopic, and storage under cool, dry conditions is recommended. Opened packages should be tightly resealed after evacuating the air.

Composition: Protein Moisture AN/TN Ratio Ash Salt (NaCl) pH (5% solution) - (%N x 6.25) 67% - 3% - 34% - 15% - <1% - 5.6

Heavy metals (as lead) <10 ppm Arsenic <3 ppm

Microbiological composition: Total bacterial count -15,000/g Mold Coliform - 50/g - 10/g

Solubility in water: Completely soluble Appearance and odor: A light tan spray-dried powder with an overall mild, slightly oat-like aroma. Unusual fire and explosion hazards: Fine powder, may present dust explosion hazard. Fire will generate CO, CO2 and smoke. Stability: unstable: its stable under normal conditions of use and storage.

Health hazard data Health hazards (Acute and Chronic): Excessive dust exposure should be avoided by individuals with respiratory conditions.

Signs and symptoms of exposure: Skin irritation, allergic types symptoms.

Emergency and first aid procedures: Remove to fresh air.

Precautions for safe handling and use Steps to be taken in case material is released or spilled: Scoop or vacuum so as not to generate excessive dust.

Waste disposal method: Dispose of in accordance with local regulations. Material is water soluble. Precautions to Be Taken in Handling and Storing: Keep containers tightly closed. Store it in a cool, dry area. Control Measures Protective Gloves: Yes Eye Protection: Goggles Other Protective Clothing or Equipment: Use in a well ventilated area.

DEXTROSE

Name Dextrose monohydrate

CAS # 5996-10-1

% by Weight 100

Hazards identification Potential acute health effects: Slightly hazardous in case of eye contact (irritant), of ingestion, of inhalation.

First aid measures Eye contact: Immediately flush eyes with running water for at least 15 minutes, keeping eyelids open. Cold water may be used. Inhalation: Allow the victim to rest in a well ventilated area. Seek immediate medical attention. Ingestion: Do not induce vomiting. Loosen tight clothing such as a collar, tie, belt or waistband. If the victim is not breathing, perform mouth-to-mouth resuscitation. Seek immediate medical attention.

Fire and explosion data

Flammability of the product: May be combustible at high temperature. Products of combustion: These products are carbon oxides (CO, CO2). Fire fighting media and instructions: Small fire: Use DRY chemical powder. Large fire: Use water spray, fog or foam. Do not use water jet.

Handling and Storage

Precautions: Keep away from heat. Keep away from sources of ignition. Empty containers pose a fire risk, evaporate the residue under a fume hood. Ground all equipment containing material. Do not ingest. Do not breathe dust. If ingested, seek medical advice immediately and show the container or the label.

Storage: Keep container dry. Keep in a cool place. Ground all equipment containing material. Keep container tightly closed. Keep in a cool, well-ventilated place. Combustible materials should be stored away from extreme heat and away from strong oxidizing agents.

Exposure controls/personal protection

Engineering controls: Use process enclosures, local exhaust ventilation, or other engineering controls to keep airborne levels below recommended exposure limits. If user operations generate dust, fume or mist, use ventilation to keep exposure to airborne contaminants below the exposure limit.

Personal protection: Safety glasses. Lab coat. Personal protection in case of a large Sspill: Splash goggles. Full suit. Boots. Gloves. Suggested protective clothing might not be sufficient; consult a specialist before handling this product.

Physical and Chemical Properties

Physical state and appearance: Solid. Molecular weight: 198.17 g/mole Boiling point: Decomposes. Melting point: 86C (186.8F) Specific gravity: 1.54 (Water = 1) Dispersion properties: See solubility in water. Solubility: Easily soluble in cold water. Stability and reactivity data Stability: The product is stable. Corrosivity: Non-corrosive in presence of glass.

Toxicological information Toxicity to animals: Acute oral toxicity (LD50): 25800 mg/kg [Rat]. Other toxic effects on humans: Slightly hazardous in case of ingestion, of inhalation. Special remarks on chronic effects on humans: Passes through the placental barrier in human.

Ecological information Products of biodegradation:

Possibly hazardous short term degradation products are not likely. However, long term degradation products may arise. Toxicity of the products of biodegradation: The products of degradation are more toxic.

METHANOL

Chemical Product Identification Product Name: Methanol Trade name: Methanol Chemical name: Methanol Chemical formula: CH3OH Chemical family: alcohol Synonyms: methyl alcohol, carbinol, wood spirit, wood alcohol, pyroxylic

Hazards identification Emergency Overview Flammable liquid, poison. May form explosive mixtures with air. May be fatal or cause blindness if swallowed. Harmful or fatal if inhaled or absorbed through the skin. May cause dizziness and drowsiness. Self-contained breathing apparatus may be required by rescue workers.

Routes of vapour exposure

Inhalation. Swallowing. Skin absorption. Skin contact. Eye contact. Inhalation: Dizziness, drowsiness and disturbances of vision, and tingling, numbness, and shooting pains in the hands and forearms

Effects of single(acute) exposures: Skin contact: Prolonged contact with the skin may cause reddening and defatting of the skin. Prolonged or widespread skin contact with the liquid may result in the absorption of harmful amounts of material.

Skin absorption: Swallowing: Nausea, abdominal pain, vomiting, headache, dizziness, shortness of breath, weakness, fatigue, leg cramps, restlessness, confusion, drunken behavior, visual disturbances, drowsiness, coma and death. There may be a delay of several hours between ingestion of methanol and the onset of signs and symptoms. The effects observed are in part due to acidosis and partially to cerebral edema. Visual effects include blurred vision, diplopia, changes in colour perception, restrictions of visual fields, complete blindness. Ingestion of moderate quantities of methanol also produces metabolic acidosis. Onset of symptoms may be delayed up to 48 hours. 60 200 ml is a fatal dose for most adults. Ingestion of as little as 10 ml has caused blindness. With massive overdoses, liver, kidney and heart muscle injury has been described. Eye contact: Liquid may cause mild redness and swelling of the conjunctiva with transient superficial injury of the cornea.

Medical conditions aggravated by over exposure: Due to its defatting properties, methanol may aggravate an existing skin condition, e.g., eczema. Due to its liver and kidney-injuring potential, may exacerbate existing liver and/or kidney diseases.

First Aid Measures Eye contact: Check for and remove any contact lenses. In case of contact, immediately flush eyes with plenty of water for at least 15 minutes. Cold water may be used. Get medical attention. Skin contact: Remove contaminated clothing and flush skin thoroughly with water. Inhalation: If inhaled, remove to fresh air. If not breathing, give artificial respiration. If breathing is difficult, give oxygen. Get medical attention immediately. Swallowing: If swallowed, do not induce vomiting unless directed to do so by medical personnel. Never give anything by mouth to an unconscious person. Loosen tight clothing such as a collar, tie, belt or waistband. Get medical attention immediately.

Fire Fighting Measures Flammable: Yes. (Forms explosive mixtures with air and oxidizing agents. May ignite in presence of flames or sparks.) Flash point (test method) closed cup: 11.1C (52F). (Tag.) Auto ignition temperature: 385C (725F) Flammable limits in air, % by volume: Lower: 6 Upper: 36.5

Extinguishing media: CO2, dry chemical, foam. Water may be ineffective. Use water spray or fog to reduce flammable vapours.

Fire fighting procedure: DANGER! Evacuate all personnel from danger area. Immediately cool cylinders with water spray from maximum distance taking care not to extinguish flames. Remove ignition source if without risk. If flames are accidentally extinguished. Explosive reignition may occur; therefore, appropriate measures should be taken; e.g., total evacuation. Reapproach with extreme caution. Use self-contained breathing apparatus. Stop flow of gas if without risk while continuing cooling water spray. Remove all containers from area if without risk. Allow fire to burn out.

Unusual fire and explosion hazard: Flammable liquid. Forms explosive mixtures with air and oxidizing agents. Container may rupture due to heat of fire. Do not extinguish flames due to possibility of explosive re-ignition. Vapours form from this product and may travel or be moved by air currents an ignited by pilot lights, other flames, smoking, sparks, heaters, electrical equipment, static discharges, or other ignition sources at locations distant from product handling point. Explosive atmospheres may linger. Before entering area, especially confined areas, check atmosphere with approved device. No part of a container should be subjected to temperature higher than 52 C. Burns with an almost invisible flame.

Hazardous combustion product: These products are carbon oxides (CO, CO2) and water. Sensitivity to impact Avoid impact against container.

Sensitivity to discharge: Possible.

Waste disposal method: Prevent waste from contaminating the surrounding environment. Keep personnel away. Discard any product, residue, disposable container, or liner in an environmentally acceptable manner, in full compliance with federal, provincial, and local regulations. If necessary, call your local supplier for assistance.

Handling and storage Precautions to be taken in storage: DANGER: Flammable volatile liquid. May form explosive mixtures with air. Ground all equipment. Only use spark-proof tool and explosion-proof equipment. Keep away from head, sparks, and open flame. Store and use with adequate ventilation at all times. Safety shower and eyewash fountain should be immediately available. May be fatal or cause blindness if swallowed. Do not leave container open. Furnace gas atmospheres produce by the use of methanol and nitrogen are similar to endothermic gas atmospheres in that they contain substantial quantities of carbon monoxide, hydrogen and nitrogen. Carbon monoxide is toxic having a TLV-TWA of 50 parts-per million (ACGIH) in air. Flammable carbon monoxide and highly flammable hydrogen form explosive mixtures with air. Since the atmospheres contain no oxygen they will

not support life. It is therefore, imperative that he spent atmospheres be burned and vented to a save location.

Precautions to be taken in handling: Furnace gas atmospheres produce by the use of methanol and nitrogen are similar to endothermic gas atmospheres in that they contain substantial quantities of carbon monoxide, hydrogen and nitrogen. Carbon monoxide is toxic having a TLV-TWA of 50 parts-per-million (ACGIH) in air. Flammable carbon monoxide and highly flammable hydrogen form explosive mixtures with air. Since the atmospheres contain no oxygen they will not support life. It is therefore, imperative that he spent atmospheres be burned and vented to a save location.

Other hazardous conditions of handling, storage and use: Extremely flammable, volatile liquid. Do not get liquid or vapours in eyes, on skin, or clothing. Safety showers and eyewash fountains should be immediately available. Use only in a closed system. Use only spark-proof tools and explosion-proof equipment. Keep away from heat, sparks, and open flame. Forms explosive mixtures with air. Ground all equipment. Store and use with adequate ventilation at all times. Close valve after each use; keep closed even when empty. Prevent reverse flow. Reverse flow into container may cause rupture. Use a check valve or other protective device in any line or piping from the container. When returning container to supplier, be sure valve is closed, then install valve outlet plug tightly. Never work on

a pressurized system. If there is a leak, close the cylinder valve. Vent the system down in a safe and environmentally sound manner in compliance with all federal, provincial, and local laws; then repair the leak. Never place a container where it may become part of an electrical circuit.

Exposure controls/personal protection Engineering controls: Personal protection Local exhaust: An explosion-proof local exhaust system is acceptable. Special: Use only in a closed system. Use local exhaust ventilation to maintain exposure below the applicable limits. Respiratory protection: Use respirable fume respirator or air supplied respirator when working in confined space or where local exhaust or ventilation does not keep exposure below TLV. Select in accordance with the provincial regulations or guidelines. Selection should also be based on the current CSA standards Z94.4, "Selection, care and use of respirators". Respirators should be approved by NIOSH and MSHA. Skin protection: Wear work gloves when handling cylinders. Nitrile gloves. Eye protection: Wear safety glasses when handling cylinders. Select in accordance with the current CSA standard Z94.3, "Industrial Eye and Face Protection", and any provincial regulations, local bylaws or guidelines. Protective equipments: Metatarsal shoes for cylinder handling. Protective clothing where needed. Cuffless trousers should be worn outside the shoes.

Physical and Chemical Properties Physical state: Liquid. Boilinf point: 64.66C (148.4F) Freezing point -97.7C (-143.9F) Appearance and odor: Colourless. Odour: Alcohol-like. Molecular weight: 32.04 g/mole Specific gravity: LIQUID ( Water = 1) 0.79 Specific gravity: VAPOUR (air = 1) 1.11 Vapour pressure: 17.1 kPa (@ 20C) Solubility in water: completely soluble

Stability and Reactivity Stability: The product is stable. Incompatibility (materials to avoid): Acids, alkali metals, halogens, halogen compounds, oxidizing agents, lead and its alloys, magnesium and fluoroelastomer Hazardous decomposition products: Thermal decomposition or burning may produce carbon monoxide/carbon dioxide. Hazardous polymerization: Will not occur.

Pollution control:

Nowadays the government of India has become very strict in matter of pollution control. Many industries have been given notice to close their plant as they were not following the rules set by the laws. The problems such as global warming have made all this restrictions to be put strictly. The method used for production of erythromycin is not so much a pouting one. It does not give any hazardous gases or polluting chemicals. But than also some precaution have to be made so that all the rules set up by the government are followed. The various materials produced by industrial plants, may be terrified in general as solid, gaseous or liquid.

Water pollution: Gentamicin manufacturing plant consumes millions of liters of water for different purposes. This water is contaminated with different chemicals, chlorobenzene, methyl chloride, sodium chloride etc. most of these substances are non biodegradable and their removal before discharging the water to river, ponds lakes etc is essential. Water pollution in the plant can be considerably reduced if the raw materials are properly recovered and reutilize various chemical, mechanical and biological methods can be used to treat waste water.

Air pollution: This plant no toxic gases or flames are produced and hence it causes very less pollution

Environmental auditing: The company should go for a systematic examination of how a business operation affects the environment. It will include all emissions to air, land and water cover the

legal constraints the effects on the community the landscape and the ecology. Environmental impact assessment can be followed at the design stage of the project. Following are some of the objectives of the environmental audit: 1. To identify environmental problems associated with the manufacturing process and the use of the products before they become liabilities. 2. 3. 4. 5. To develop standards for good working practices To ensure compliances with environmental legislation To satisfy requirements of insurers To be seen to be concerned with environmental questions: important for public relation 6. To minimize the production of waste: an economic factor

The ISO-14000 guide lines may be followed for better performance and environmental friendly functioning of the unit.