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CHAPTER 1

HISTORICAL PROFILE

1.1. INTRODUCTION

The cephalosporins, a subgroup of-lactam antibiotics, consist of a 4-membered lactam ring fused through the nitrogenand the adjacent tetrahedral carbon atom to a second heterocycle forming a 6-membered dihydrothiazine ring. Other

structural features common to all the cephalosporins are a carboxyl group on the dihydrothiazine ring on the carbonnext to the ring nitrogen and a functionalized amino group on C-7, the carbon of the -lactam ring opposite the nitrogen.These features are evidenced in 7-aminocephalosporanic acid [957-68-6] (7-ACA), C10H12N2O5S. Cephalosporins, likeall -lactam antibiotics, exert their antibacterial effect by interfering with the synthesis of the bacterial cell wall. Theseantibiotics tend to be irreversible inhibitors of cell wall biosynthesis and they are usually bactericidal atconcentrations close to their bacteriostatic levels. Cephalosporins are widely used for treating bacterial infections. Theyare highly effective antibiotics and have low toxicity.[1]

1.2. NATURAL OCCURENCES

The first cephalosporin was extracted from sewage-contaminated water, and other naturally occurring ones have been

isolated from moulds taken from soil samples.

1.3. BIOSYNTHESIS

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1.4. HISTORY

The first chemical compounds of the cephalosporin group were isolated from Cephalosporium acremonium, acephalosporin-producing fungus first discovered by Brotzu in 1948 from a sewage outfall off the Sardinian coast.In1953, during studies on the chemical nature of cephalosporin, Guy Newton and Edward Abraham found a secondhydrophilic antibiotic in the culture broths of Acremonium that was named cephalosporin C (Newton and Abraham,1954). The new antibiotic was easily obtained in its sodium salt form and showed antibiotic activity againstStaphylococcus aureus, Salmonella typhi, and Escherichia coli.

1.5. CLASSIFICATION

Cephalosporin antibiotics are divided in four subgroups called generations. The individual drugs are arranged intogenerations according their spectrum of antibacterial activity (including the susceptibility/resistance to beta-lactamases)not according to their date of synthesis or introducing to the market.[1]

1.5.1. FIRST GENERATIONFirst-generation cephalosporins are characterized by good gram-positive activity and modest to weak gram-negative activity. Their spectrum further includes corynabacteria, meningococci, and some community-acquired stems of gram-negative rods like Escherichia coli or Proteus mirabilis. The drugs are active againstanaerobes in the extent similar to penicillin.

Fig.1.5.1.Examples of 1st generation cephalosporins : A) Cephadrine B) Cefadroxil C) Cefalexin

1.5.2.SECOND GENERATIONThe second-generation cephalosporins have a broader spectrum and generally are more active against aerobicgram-negative organisms than the first-generation cephalosporins.Most second-generation cephalosporinshave some activity against indole-positive Proteus, Enterobacter (except E. cloacae), and Haemophilusinfluenza.

Fig.1.5.2. Examples of 2nd generation cephalosporins : A) Loracarbef B) Cefuroxime C) Cefotiam

1.5.3.THIRD GENERATION

Third-generation cephalosporins have an expanded gram-negative spectrum and are the most active againstenteric gram-negative bacilli, including penicillinase-producing strains, as well as Serratia and Citrobacter.They are also highly active against Haemophilus influenzae and Neisseria gonorrhoeae, Streptococcus

pneumoniae and pyogenes, moderately active against Pseudomonas aeruginosa and anaerobes, but somewhatless active against staphylococci than the first-generation cephalosporins.

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Fig.1.5.3. Examples of 3rd generation cephalosporins : A) Cefdinir B) Cefixime C) Ceftibuten

1.5.4.FOURTH GENERATION

Antibiotics of this group have a broad spectrum summarizing the 1st

, 2nd

and 3rd

generation. They can resistsome potent beta-lactamases. Nevertheless, their activity against staphylococci is not better than withcephalotin and activity against P.aeruginosa is not better than with ceftazidim.

Fig.1.5.4. Examples of 4th generation cephalosporins : A) Cefzopran B) Cefclidine C) Cefepime

1.5.5.FIFTH GENERATIONCurrently there are only two drugs in this category, Ceftobiprole and Ceftaroline. These new drugs are alsothe only -lactam antibiotics that are effective against methicillin-resistant-Staphylococcus-aureus (MRSA).[16]

Fig.1.5.5. 5th generation cephalosporins : A) Ceftaroline phosfamil (prodrug) and B) Ceftobiprole medocaril(prodrug)

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CHAPTER-22. APPLICATIONS AND GRADES

2.1. APPLICATIONS

The cephalosporins are used for treating infectious diseases of bacterial origin in both humans and animals. First-

generation cephalosporins such as cephalothin and cephalexin are the most active against taphylococci and

nonenterococcal streptococci and are effective alternatives to the penicillins in patients with endocarditis, osteomyelitis,

septic arthritis, and cellulitis. They are especially useful for treating patients who are allergic to the pencillins or who

have mixed infections from gram-positive and gramnegative bacteria. The first-generation cephalosporins have been

widely used for prophylaxis in cardiovascular, orthopedic, biliary, pelvic, and intra-abdominal surgery. Cefazolin,

which has a longer half-life than the other first-generation compounds, is the first-generation agent of choice for

surgical prophylaxis.

Cefuroximeis effective against community-acquired pneumonia in which ampicillin-resistant Haemophilus influenzaeis the probable etiologic agent. Cefoxitinis used to treat mixed aerobicanaerobic infections including pelvic infections,

intra-abdominal infections, and nosocomial aspiration pneumonia. Cefonicid, because of its long half-life has been used

in a once-a-day regimen to treat a variety of mild to moderate infections including community-acquired pneumonias,

urinary tract infections, and infections of the skin and soft tissue.

The third-generation cephalosporins are effective in the treatment of bacteremias, pneumonias, urinary tract infections,

intra-abdominal infections, and skin and soft tissue infections. Because of its long half-life, cetriaxonehas been used for

outpatient treatment of skin and soft tissue infections. Single dose ceftriaxone has also been effectively used in the

treatment of urinary tract infections.

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CHAPTER-33. ECONOMIC SCENARIO

3.1.WORLD SCENARIOCephalosporins first entered the marketplace in 1964, when cephalothin and cephaloridine, which are both injectable,

were launched. By the late 1970s, the injectable cephalosporins had become important therapeutic agents in the

hospitals. Also in 1964 the first oral cephalosporin, cephaloglycin [3577-01-3], C18H19N3O6S, was launched only to be

displaced by the end of the year by cephalexin. For years cephalexin was the leading oral cephalosporin on the market.

It has since been displaced by cefaclor. With the advent of the more -lactamase stable cephalosporins such as cefoxitin

and cefuroxime , and the more potent agents such as cefotaxime and other third-generation compounds, cephalosporins

now dominate the antibiotic market worldwide. On a worldwide basis, total pharmaceutical sales for 1983 were $71

billion, antibiotic sales totaled $9.2 billion, and cephalosporins accounted for $1.2 billion, or 13% of the an tibacterial

market. By 1986 the worldwide antibiotic market was valued at around $11 billion and cephalosporins represented

approximately 41% of the total. Marketing projections for antibiotics from 1986 to 1991 show an average annual

growth rate of 3.7% to $13,144 million. Cephalosporin sales are projected to reach 40% or $5,234 million. Worldwide,

in 1988, the top selling cephalosporin was the oral cephalosporin Ceclor which had 1988 sales of $605 million and

projected 1989 sales of $696 million. The second best selling, and top parenteral, cephalosporin was Rocephin which

had 1988 sales of $475.8 million and projected sales of $597 million for 1989. The third cephalosporin on the list was

Claforan which had 1988 sales of $376.2 million and a projected decline in sales for 1989 to $353 million as it is

displaced by the newer cephalosporins. Cephalosporins are continuing to gain market share, and their final product cost

still justifies continued development and manufacture in Europe, Japan and the United States. New and effective

cephalosporins continue to be introduced and there is still a major need to find more effective cephalosporins for

methicillin-resistant (MRSA) and other resistant pathogens.[15]

S.no Antibiotic World market($ million)

1. Cephalosporins 11,000

2. Pencillin G and V 4,400

3. Erythromycin 3,500

4. Tetracyclines 1,400

5. Vancomycin & Teichoplanin 1,000

6. Semisynthetic cephalosporin 1,000

Table.3.2.Table showing World Antibiotic market 2006

3.2. INDIAN SCENARIOIndian pharmaceutical industry looks set for a solid long term growth, currently it ranks 14th by value and 2nd in

volume globally. It is estimated to be amongst top 10 markets by 2020. Indian Cephalosporin formulation market is

valued at US$ 1.1 billion out of which 1st& 2nd generation Cephalosporin derived from Pen-G which contributes about

US $ 400million. However this segment of market is too commoditized and thus is volume driven with relatively low

realization both the top & bottom line. As against this, 3rd & 4th generation Cephalosporin are derived from 7ACA &

GCLE which are value added non commodised derivatives which accounts about US $ 700million and is mainly

restricted to only 3 highly integrated players in markets. This segment is more lucrative and not so price sensitive as 1st

and 2nd generation.[15]

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FIG.3.2(a). Chart showing market share of various segment of pharmaceutical sectors in India(2003).

Market segment Market size Rs

billion(2003)

Market share Market growth

rate

Top most selling

category

Alimentary and

metabolism

58.07 25%

Anti-infectives 44.9 19% 0.5% Cephalosporin,Quinoline

Respiratory 23.6 10% 6.1% Cough

preparations

Cardiovascular 22.1 10% 14.2% Calcium channel

blockers

Musculoskeletal 18.1 8% 3.5% Anti-rheumatics

Others 64.7 28% 6% ---Table.3.2. Information related to the above chart.

19%

10%

10%

8%

28%

25%

Sales

Anti-infectives Respiratory Cardiovascular

Musculosketal others Alimentary and metabolism

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INDIAN COMPANIES MANUFACTURING CEPHALOSPORIN

S.no Company Location

1. Nectar Lifesciences Limited (NecLife) Chandigarh

2. Orchid Chemicals & Pharmaceuticals Ltd Alathur(Chennai),Auranga

bad Irungattukottai,

3. Parabolic Drugs Punjab

4. Aurobindo pharma Hyderabad,Andhra Pradesh

5. Lupin Pharmaceuticals Mumbai, Maharashtra

6. Apex Pharma Mumbai

7. Wockhardt Limited Bharuch, Gujarat

8. Zota Pharmaceuticals Pvt. Ltd. Chennai

9. Nootan Pharmaceuticals Baddi,Himachal pradesh

10. Phlox Pharmaceuticals Limited Vadodra,Gujarat

Table. 3.2: Projected growth by 2013.

S.no Projected Growth Countries Additional in global sales by 2013

1. China US$40 billion

2. Brazil,Russia,India US$5-15 billion each

3. 13 fast followers* US$1-5 billion each

4. Total US$90 billion

*13 fast followers comprise Venezuela,Poland,Argentina,Turkey,Mexico,Vietnam,South

Africa,Thailand,Indonesia,Romania,Egypt,Pakistan and Ukraine.

http://www.indianindustry.com/nootan-pharmaceuticals/http://www.indianindustry.com/nootan-pharmaceuticals/

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Fig.3.2(b): Chart depicting Table.3.2

0

5

10

15

20

25

30

35

40

45

China India 13 fast followers

Projected Sales by 2013

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CHAPTER-44. PROPERTIES, HANDLING AND STORAGE

4.1. PROPERTIES

4.1.1.PHYSICAL PROPERTIES

Most cephalosporin antibiotics are white, off-white, tan, or pale yellow solids that are usually amorphous, but cansometimes be obtained crystalline. The cephalosporins do not usually have sharp melting points, but rather decomposeupon heating at elevated temperatures. One of the distinguishing physical characteristics of the cephalosporins is theinfrared stretching frequency of the -lactam carbonyl. This absorption occurs at higher frequencies (1770 1815cm1) than those of either normal secondary amides (1504 1695 cm1) or ester carbonyl groups (1720 1780cm1).

4.1.2.CHEMICAL PROPERTIES

The cephalosporins are more resistant to ring opening than the penicillins. For example, although alcohols readily attackthe penicillin -lactam, cephalosporins are sufficiently stable to permit the use of methanol as a recrystallizationsolvent.

Fig.4.1.2 Cephalosporin -lactam reactivity where Nu is a nucleophile and X is a leaving group; ( a) path followed fornucleophiles; (b) when Nu is the serine OH of an enzyme (ie, Nu=Enz SerOH) deacylation may precede expulsion ofthe leaving group and both pathways (a) and (b) may be observed.

Cephalosporins are attacked by nucleophiles such as alkoxide or hydroxylamine, but because of the nature of thedihydrothiazine ring, products equivalent to the penicilloic or penilloic acids derived from the penicillins under similarconditions, are not obtained. Rather, studies indicate involvement of the 3_-substituent as a leaving group (66). Thus 3-deacetoxycephalosporins are found to be more stable than cephalosporins having 3-substituents that can be readily lost.

4.1.3. BIOLOGICAL PROPERTIES

The clinical effectiveness of Cephalosporins depends on the antibacterial and pharmacokinetic properties.Pharmacokinetic properties include the efficiency and rate of adsorption, the rate of metabolism and excretion, tissue

distribution, serum binding, and serum and tissue inactivation. New compounds are also tested for the ability to resistinactivation by -lactamases (penicillinases, cephalosporinases) produced by bacteria.

4.2.MATERIAL SAFETY DATA SHEET (MSDS)HAZARDS IDENTIFICATION

APPEARANCE AND ODOUR

White, off-white, tan, or pale yellow solids

SAFETY INFORMATION:

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HEALTH EFFECTS

Exposure might occur via skin; eyes; ingestion; inhalation. May cause sensitisation by inhalation or skin contact.

FIRST AID MEASURES

INGESTION

Never attempt to induce vomiting. Do not attempt to give any solid or liquid by mouth if the exposed subject isunconscious or semi-conscious. Wash out the mouth with water. Obtain medical attention.

INHALATIONPhysical form suggests that risk of inhalation exposure is negligible.

SKIN CONTACT

Using appropriate personal protective equipment, remove contaminated clothing and flush exposed area with largeamounts of water. Obtain medical attention if skin reaction occurs.

EYE CONTACT

Wash immediately with clean and gently flowing water. Continue for atleast 15 minutes. Obtain medical attention Ifrequired.

HEALTH HAZARD ACUTE AND CHRONIC

This item may cause hypersensitivity (Rash, Hives) or anaphylaxis. May cause blood, liver or kidney dysfunction.

MEDICAL TREATMENTTreat according to locally accepted protocols. For additional guidance, refer to the current prescribing information or tothe local poison control information center. Protect the patients airway and support ventilation and perfusion.Meticulously monitor and maintain, within acceptable limits, the patients vital signs, blood gases, serum electrolyte s,etc. Absorption of drugs from the gastrointestinal tract may be decreased by giving activated charcoal, which, in manycases, is more effective than emesis or lavage; consider charcoal instead of or in addition to gastric emptying. Repeateddoses of charcoal over time may hasten elimination of some drugs that have been absorbed. Safeguard the patientsairway when employing gastric emptying or charcoal. Forced diuresis, peritoneal dialysis, hemodialysis, or charcoalhemoperfusion have not been established as beneficial for an overdose of cephalexin; however, it would be extremelyunlikely that one of these procedures would be indicated. In allergic individuals, exposure to this material may requiretreatment for initial or delayed allergic symptoms and signs. This may include immediate and/or delayed treatment ofanaphylactic reactions.

FIRE FIGHTING MEASURES

Fire and Explosion Hazards

Assume that this product is capable of sustaining combustion.

Extinguishing MediaWater, dry chemical, carbon dioxide, foam.

Special Firefighting Procedures

Use a self contained breathing apparatus and full protective equipment.

Hazardous Combustion Products

Hazardous combustion or decomposition products are expected when the product is exposed to fire.

ACCIDENTAL RELEASE MEASURES

PERSONAL PROTECTION

Wear protective clothing and equipment consistent with the degree of hazard.

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ENVIRONMENTAL PROTECTION

For large spills, take precautions to prevent entry into waterways, sewers, or surface drainage systems.

METHODS FOR CLEANING UP RECOVERY

Pump into an inert labelled emergency container. Clean up puddle of Product immediately. Dilute the puddles with

water & recover it.

HANDLING AND STORAGE

SAFE HANDLING ADVICE

No special control measures required for the normal handling of this product. Normal room ventilation is expected tobe adequate for routine handling of this product.

STORAGE

Prior to mixing store at 20 to 25 C (68 to 77F) [See USP Controlled Room Temperature]. After mixing, store in a

refrigerator. Keep tightly closed.

EXPOSURE CONTROL / PERSONAL PROTECTION

Wear appropriate clothing to avoid skin contact. Wash hands and arms thoroughly after handling .

STABILITY AND REACTIVITY

Stable under recommended storage conditions.

TOXICOLOGICAL INFORMATION

Oral Toxicity

Not expected to be toxic following ingestion.Inhalation Toxicity

Can produce respiratory irritation. Adverse effects might occur following Inhalation.

Skin Effects

Irritation might occur following direct contact.

Eye Effects

Irritation might occur following direct contact with eyes.Gastrointestinal Reactions

Symptoms of pseudomembranous colitis may appear either during or after antibiotic treatment. Nausea and vomitinghave been reported rarely. The most frequent side effect has been diarrhea. It was very rarely severe enough to warrantcessation of therapy. Dyspepsia, gastritis, and abdominal pain have also occurred. As with some penicillins and someother cephalosporins, transient hepatitis and chloestatic jaundice have been reported rarely.

Hypersensitivity Reactions

Allergic reaction in the form of rash, urticara, angioedema, and rarely, erythema multiforme, Stevens-Johnsonsyndrome, or toxic epidermal necrolysis have been observed. These reactions usually subsided upon discontinuation ofthe drug. In some of these reactions, supportive therapy may be necessary. Anaphylaxis has also been reported.

Genetic Toxicity

Not expected to be genotoxic based on animal studies.

CarcinogenicityNot expected to be carcinogenic based on animal studies.

Reproductive Effects

Not expected to produce adverse effects on fertility or development based on animal studies.No adequate and well-controlled studies in pregnant women.No studies during labor and delivery.Should be used during pregnancy only if clearly needed.

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Pharmacological Effects

This material is an antibiotic; a cephalosporin. It is an agent intended for the treatment of bacterial infections.

Fig.4.2. Pharmacokinetic and adverse effects of Cephalosporins

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CHAPTER-55. MANUFACTURING PROCESS

5.1.INTRODUCTION TO MANUFACTURING STRATEGIESCephalosporins are economically and therapeutically as important as the pencillins. These drugs are broad-spectrum

antibiotics,active against respiratory,ear,skin and bone infections caused y G+ and G- bacteria and they have low

toxicity. In a maner similar to the penicillins, they act by inhibiting bacterial cell wall(peptidoglycan) synthesis and they

kill growing cell .Natural cephalosporin C is the main fermentation product but is not very therapeutically active and is

converted to caphalexin or other active forms which are.Cephalosporin C is produced by the same fermentation process

used for penicillns but utilizes diffewrnt growth media and producer organisms. However final fermentation broth

concentrations are much lower and include many similar compounds resulting in a more difficult separation and

purification process.

Fig.5.1(a): Production Flowchart

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Fig.5.1(b). Block flow diagram for a general antibiotic production process.[2]

5.2.GENERAL PRODUCTION TECHNIQUES:Antibiotic manufacturing plants contain two main processing segments :

A highly specialized fermentation section

A large energy intensive separation and purification section often termed as Downstream Processing

While the fermentation section generally occupies about 50% of the space and acoounts for about 50% of the equipment

cost ,it accounts for a relatively small portion of the processing steps required for antibiotic production. Highly

specialized fermentation equipment is necessary including fermenters,airand fluid filters,agitators,heat

exchangers,pumps etc. whereas separation and purification include centrifuges and filters for separating microbes from

broth and solvent extractors or membrane separators to separate antibiotic from fermentation broth .Distillation columns

are used for solvent recovery and purification.[6]

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Each production process is generally divided into the following key stages as shown in figure:

Fig.5.2. A generalized,schematic representation of fermentation process.[]

5.2.1 RAW MATERIALS AND UTILITIESThe yield and final fermentation broths are strongly dependent on the particular organism used. The antibiotic

precursors are also important in order to produce the desired type of antibiotic.the growth medim is quite important and

specific to the particular organism.

Utility requirements consist of sterilized air, purified water, cooling and chilled water ,electricity and steam. Large

amounts of cooling and chilled water are required to control the large amount of heat involved during aerobic

fermtnation .Consumption of electricity is hifgh due to the large amount of agitation required for Oxygen transfer in the

aerobic fermentations.[9]

5.2.2. FERMENTATIONOperating an antibiotic fermentation unit requires special consideration be given to culture preservation ,growth media,

air and media sterilization ,oxygen transfer and temperature control in the fermenter. Long term preservation of an

efficient culture is vital to antibiotic manufacturing. Three methods for culture preservation are subcultivation ,low

temperature storage and freeze drying. To prevent contamination a sterile environment must be maintained.

Fermentations require large volume of air which must be sterilized to remove bacteria which have particle size down to

0.5.Antibiotic fermentations are aerobic with optimum operating temperatures in the 20 to 30 C range. Oxygen transfer

in fermentation broth can be controlling variable with vigorous agitation required which often produces foaming

problems so we use a standard antifoaming agent.

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5.2.3. DOWNSTREAM PROCESSINGDownstream processing is an essential part of bioprocess technology in that the desired product needs to be isolated,

purified and formulated for different en uses. the products are manufactured using variety of equipment. Besides

fermenters other special reactors such as airlift, membrane and immobilized cell reactors are also used. the products

formed are usually in low concentrations necessitating the handling of large volume and in some cases the broths are

highly viscous. The downstream processing steps are based on steady as well as unsteady state techniques. The unit

operations involved are filtration, centrifugation ,sedimentation, adsorption and liquid-liquid extraction .The choice of

the separation methodology depends to a large extent on the nature of the product ,its quantity and the extent of purity

required.[7]

5.2.4. QUALITY CONTROLChromatography and fluorescence detection are standard methods of assaying antibitotics .High performance liquid

chromatography(HPLC) uses regular and reverse phase and ion exchange packing with ultraviolet light as the detector

source, to analyze for penicillins and cephalosporins.

HPLC assays are particularly important for the B-lactum antibiotics in order to detect impurities.

5.2.5. PRODUCTION OF CEPHALOSPORIN ANTIBIOTICSHigh yielding strains of Acremonium chrysogenum are used in large scale, fed-batch fermentations.

Additives :

Production scale fermentations are fed-batch with carbon supplied as simple or complex carbohydrate feeds

during the growth phase.

As the fermentation progresses ,sugar feeds are reduced are usually replaced by higher energy oils such as

soyabean or peanut oil. Oil addition leads to multicellular arthropores stage which leads to greater oxygen

availability to the organism increasing cephalosporin production. DL-Methionine addition which results in the onset of arthrospore formation is often added during the early

growth phase of the fermentation.

Organic nitrogen is often supplied as a combination of soybean and cottonseed meals supplemented with

ammonium sulfate, ammonia used to help control the pH throughout fermentation. The pH of the fermentation

is maintained between 6.2 and 7.0 and the temperature range is controlled between 24 and 28 C.

5.2.6. CEPHALOPORIN C RECOVERY AND PURIFICATION:The purification and recovery of harvest cephalosporin C broth begins with the rapid chilling of t he active broth to 3-5 c

followed by removal of mycelia solids either by filtation or by centrifugation. The active broth contains not only the

desired cephalosporin component but also small quantities of the biosynthetic precursors, penicillin N-

deacetylcephalosporin C and degraded cephalosporin C.[11]

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Thus two major strategies can be used for the recovery and purification of cephalosporin C

STRATEGY ONE:

This involves the use of activated carbon or the use of a non ionic resin .Because of the high selectivity of the resin

,cephalosporin C is preferentially adsorbed over penicillin N or the contaminating biosynthetic precursor molecules

.Most of the penicillin is removed in the pH 2.0 acidification step. An additional anion and cation exchange step

usually results in high quality of cephalosporin C.

STRATEGY TWO:

This strategy involves the substitution of the amine moiety on the -aminoadipyl side chain at C-7.Two substituted

derivatives ,N-2,4-dichlorobenzoyl cephalosporin C and tetrabromocarboxybenzoyl cephalosporin C can be crystallized

from the acidic aqueous solution N-Substituted cephalosporin C salts containing small amounts of contaminant can be

easily converted to 7-ACA.About two third of the commercial cephalosporins are derive from 7-ACA by either using

chemical or enzymatic deacylation. Enymatic processes are now used by the major producers of cephalosporis C.Recent bulk market costs for 7-ACA ranges from $115 to $130/kg

S.no Component Medium A% Medium B% Medium C% Medium D%

1. Peanut meal 4.0 4.0 4.0 4.0

2. Soybean meal 2.0 2.0 2.0 2.0

3. Beet molasses 3.45 3.45 3.45 3.45

4. Methyl oleate 0.575 0.575 0.575 0.575

5. Lard oil 6.0 6.0 6.0 6.0

6. Sodium sulfate 0.4 0.3 --- ---7. Ammonium sulfate --- 0.1 --- ---

8. Methionine --- --- 0.4 ---

9. Calcium carbonate 0.2 0.2 0.2 ---

10. Calcim sulfate --- --- --- 0.4

Yield:g

Cephalosporin /ml

3240 3560 4190 3920

Table5.2.6. Table depicting growth medium for fermentation process

5.2.7. PRODUCT RECOVERY:Cephalsporins are extracellular fermentation product, allowing a bulk filtration to be used to separate the solid matter

from broth. Recovery from the filtrate is difficult due to low concentration of product and the need to remove high

molecular weight biological compounds which can lead to allergic and toxic manifestations when administerd.Thus

many separation schemes are employed but most appropriate is to

Filter the broth at acidic pH(5.0) followed by adsorption of filtrate on activated carbon .

Removal of the adsorbed antibiotic by contacting the carbon with a mixture of water and polar organic

solvent.

Contacting the eluate with an anion exchange resin and eluting the resin with a salt solution at a pH of 5.5-10.

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CHAPTER-66. PROCESS SELECTION

6.2. CAPACITY SELECTIONMATERIAL BALANCE FOR CAPACITY SELECTION:

Material balance for a capacity of= 50 tonnes/annum

Amount of feed in : 50 tonnes/annum

Outlet concentration of the fermentation broth : 25 g\l

No of working days : 300 days

Total time taken for the fermentation of the broth :120 hours.

Total feed in (kg/hr ): =6.94 kg/hr

Concentration out in (kg/hr l):2.083e-4 kg/hr l

Therefore volume required will be: total feed in / concentration out=33,333.33l or 33 Kilo Litre

RESULT:After calculations we derived for a 50tonnes per annum plant capacity volume of the bioreactor required is 33kiloliters.

6.3. PROCESS SELECTIONThe selected process strategy for the manufacture Cephalosporins C is Strategy One.

6.4. BLOCK FLOW DIAGRAM

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FiG.6.3. Figure depicting the block flow diagram for the process.

Fermentation MF 0-

2m

10000

MWCO

RO

Concentrate

cell harvest cell

washing

(CELL MASS)

Filtrate

Concentrate

protein ,Pigment

removal

Permeate

Adsorption

Permeate (Low

dissolved solid

Further

downstream

processing

concentrate

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REFERENCES1. Antibiotics (-Lactams) in ECT 3rd ed., Vol. 2, pp. 871919, by J. R. E. Hoover and C. H. Nash, Smith

Kline and French Laboratories; Antibiotics, -Lactams (Cephalosporins) in ECT 3rd ed., Supplement, pp.6483, by C.OCallaghan and J. Elks, Glaxo Pharmaceuticals Ltd.

2. Chemical Technology Division EXPEDIENT ANTIBIOTICS PRODUCTION P. R. Bienkowski, C. H.Byers,D. D. Lee Date Published - May 1988

3. P. W. Muggleton, "The Preservation of Cultures," Progress in Industrial Microbiology, 4 191 (1962).4. C. F. Mascone, "Separations are key to Biotech Scale-up," Chem.Engr. January 19, 1987.5. D. W. Blackburn, "Cephalosporin Purification Process,'' US Patent #4 028 355 July 18, 1975.6. G. T. Stewart, "Purified Cephalosporins and Their Production,"British Patent #1 232 656 May 23, 1967.7. H. Coenen, "Isolation and Purification of Antibiotics," German Patent #33 18 194 May 19, 1983.8. M. Matsumura, T. Imanaka, T. Yoshida and H. Taguchi, "Effect of Glucose and Methionine Consumption

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