Gentamicin

MICROBIOLOGY II TO:

Assistant Professor HADAYAT RASOOL

COLLEGE OF PHARMACY

Government College University Faisalabad Pharm. D 4th semester (evening) SESSION 2007-2012

CONTENTS GENTAMICIN  Introduction  Fermentation

EXTRACTION LYOPHILIZATION PACKAGING AND LABELLING

BROAD SPECTRUM ANTIBIOTIC COMPLEX OF THE AMINOGLYCOSIDE GROUP

GENTAMICIN

HISTORY Isolated from Micromonospora purpurea in 1963 BY Weinstein and colleagues

Chemistry

1. A mixture of closely related antimicrobials known as getamicin c1, c2, and C1a 4. Precipitates heparin in syringe

SPECTRUM  More active than Streptomycin  Active against       

Entrobacter aerogenes Escherichia coli Klebsiella pneumoniae Proteus Pseudomonas aeruginosa Non pigmented Serratia Salmonella and Shigella

PHARNACODYNAMICS

 Inhibits

production of bacterial protein, causing bacterial cell death.

PHARMACOKINETICS 1. Higher tissue levels versus plasma levels when compared to other aminoglycosides 4. Therapeutic blood levels for about 12 hours

CLINICAL USE Systemic 4. Administer 1. Intramuscular (IM) 2. Subcutaneous (SC)

5. Approved for use in respiratory and urinary tract infections in dog and cats 6. Also used for GI tract, skin, and soft tissue infections 7. Approved for use in swine

CLINICAL USE Topical 3. Topical preparation approved for use in canine ear infections 5. Topical conjunctival administration for ocular infections of Pseudomonas aeruginosa

PRODUCTION PRODUCTION INDUSTRIAL SCALE

PRODUCTION STEPS: 3. Fermentation 4. Extraction 5. Lyophilization 6. Packaging and Labelling

FERMENTATION FERMENTATION by Micromonospora purpurea

Micromonospora purpurea Important To Learn:  Isolation  Characteristics  Colonial characters  Microscopic characters Without staining With staining

ISOLATION SAMPLE Micromonospora pupurea Most common in 1. Soil 2. Lake mud 3. River sediments

CHARACTERISTICS  Colonial:  no diffusible pigment  no aerial mycelium

 Microscopic:  long, branched mycelia  0.5 microns in diameter  Smooth-walled spore-like structures  sporophores are not observed.

SHAKE FLASK CULTURE SHAKE FLASK CULTURE METHOD METHOD METHOD OF FERMENTATION

FERMENTATION DEFINITION Drug producing microbe is grown and cultivated in appropriate culture vessels or in large fermentors on a suitable medium in which respective drug is accumulated.

FERMENTATION STEPS 2. Isolation of microbe 3. Characterization of microbe 4. Purification of culture 5. Preparation of inoculum 6. Quality control 7. Lab scale fermentation 8. Quality control 9. Seed cultivation 10.Quality control

Isolation Yeast Extract Nutrient Agar 3.Beef extract..……………………………3 g 4.Tryptone….……………………………5 g 5.Dextrose, ..………………………….......1 g 6.Potato starch, …………………………24 g 7.Yeast extract,…………………………… 5

ISOLATION Medium

Sampl e  Conditions  280C  3-7 days  pH - 7

Autoclave at 1200C for 15 minutes

FERMENTATION STEPS Isolation of microbe q Characterization of microbe q Purification of culture q Preparation of inoculum q Quality control q Lab scale fermentation q Quality control q Seed cultivation q Quality control

CHARACTERISTICS  Colonial:  no diffusible pigment  no aerial mycelium

 Microscopic:  long, branched mycelia  0.5 microns in diameter  Smooth-walled spore-like structures  sporophores are not observed.

FERMENTATION STEPS Isolation of microbe Characterization of microbe q Purification of culture q Preparation of inoculum q Quality control q Lab scale fermentation q Quality control q Seed cultivation q Quality control

PURIFICATION Colloidal chitin agar Colloidal chitin ….…………………….…..….. 2g K2HPO4 ………..………………….….……. 0.7g KH2PO4 ………..……………………..……. 0.3g MgSO4.5H2O . ………………………….… 0.5g FeSO4.7H2O ……..……….……….…… 0.01g ZnSO4 ….………………………..……

PURIFICATION Medium

Autoclave at 1200C for 15 minutes

 Conditions  280C  3-7 days  pH - 7

Sampl

MacCorten y bottle

FERMENTATION STEPS Isolation of microbe Characterization of microbe Purification of culture q Preparation of inoculum q Quality control q Lab scale fermentation q Quality control q Seed cultivation q Quality control

CHARACTERISTICS  Colonial:  no diffusible pigment  no aerial mycelium

 Microscopic:  long, branched mycelia  0.5 microns in diameter  Smooth-walled spore-like structures  sporophores are not observed.

PREPARATION OF INOCULUM 5ml water + glass beeds Pour in MacCorteny Bottle Shake Pour out Inoculum

FERMENTATION STEPS Isolation of microbe Characterization of microbe Purification of culture Preparation of inoculum q Quality control q Lab scale fermentation q Quality control q Seed cultivation q Quality control

QUALITY CONTROL Check No. of spores in inoculum

FERMENTATION STEPS Isolation of microbe Characterization of microbe Purification of culture Preparation of inoculum Quality control q Lab scale fermentation q Quality control q Seed cultivation q Quality control

SHAKE FLASK CULTIVATION "SEED STAGE" MEDIUM (per flask) Potato dextrin……………………………50 g Dextrose……………….………………….5 g Soybean meal….…….…………………35 g Calcium carbonate ....………………….. 7 g Cobalt chloride…….……..……….10-6

SHAKE FLASK CULTIVATION 

2. 3. 4. 5.

Conditions: 28° C 300 rpm pH 7 48 hours

FERMENTATION STEPS Isolation of microbe Characterization of microbe Purification of culture Preparation of inoculum Quality control Lab scale fermentation q Quality control q Seed cultivation q Quality control

Quality control Sampling After every 8 hours 2. Contents examination  Morphological properties of biomass  Average no of spores  Occurrence of pellet

3. pH 4. Nutrient concentration

PRODUCTION ANALYSIS Microbiological disk test gentamicin samples up to 30 micro litre with water applied to filter paper disks

PRODUCTION ANALYSIS The filters dried at 37°C placed in Petri dish on a soft agar top layer seeded with Bacillus subtilis

PRODUCTION ANALYSIS The soft agar contained 2. Yeast extract………………….…0.5g 3. Tryptone………………………...….1g 4. NaCl ………………………….……0.5g 5. Bactoagar ……………….........0.75g 6. Water …………………………..100ml The Petri dishes were incubated overnight at 30°C.

PRODUCTION ANALYSIS

The diameter of the inhibition zones formed around the filters determined area of inhibition calculated

FERMENTATION STEPS Isolation of microbe Characterization of microbe Purification of culture Preparation of inoculum Quality control Lab scale fermentation Quality control q Seed cultivation q Quality control

Seed cultivation  In

huge fermentors of capacity 10L to 1000L

FERMENTATION STEPS Isolation of microbe Characterization of microbe Purification of culture Preparation of inoculum Quality control Lab scale fermentation Quality control Seed cultivation q Quality control

Quality control Sampling After every 8 hours 2. Contents examination  Morphological properties of biomass  Average no of spores  Occurrence of pellet

3. pH 4. Nutrient concentration

PRODUCTION ANALYSIS Microbiological disk test gentamicin samples up to 30 micro litre with water applied to filter paper disks

PRODUCTION ANALYSIS The filters dried at 37°C placed in Petri dish on a soft agar top layer seeded with Bacillus subtilis

PRODUCTION ANALYSIS The soft agar contained 2. Yeast extract………………….…0.5g 3. Tryptone………………………...….1g 4. NaCl ………………………….……0.5g 5. Bactoagar ……………….........0.75g 6. Water …………………………..100ml The Petri dishes were incubated overnight at 30°C.

PRODUCTION ANALYSIS

The diameter of the inhibition zones formed around the filters determined area of inhibition calculated

EXTRACTION EXTRACTION CEPHALOSPORIN

EXTRACTION Step 1 damp-dry mycelium extracted with ice-cold 70% (v/v) ethanol 3ml/g for 5min.

EXTRACTION Step 2 The mycelium filtered and extracted with icecold water 3ml/g for 5min filtered again and washed on the filter with water 1 ml/g

EXTRACTION Step 3 The ethanol and water extracts were combined water had been added to reduce the ethanol content of the extracts to 10% (v/v). freeze-dried

EXTRACTION EXTRACTION GENTAMICIN

EXTRACTION STEP 1

pH of the broth Acidic ( 2 ) using H2SO4 Stirrer Filtration by gravity

EXTRACTION  STEP

2 pH of the broth neutral (7)

By using concentrated ammonium hydroxide

EXTRACTION Step 3 the neutralized filtrate passed through a column of IRC-50 resin (NH4+ + form) The resin was washed with water eluted with 2N NH4OH the elute concentrated and air dried

EXTRACTION Step 4 The resulting dark brown crude passed through a column of IRS-401S resin (OH- form) eluted with water

EXTRACTION Step 5 Elutes collected between ph 8 to 12 combined and air dried. The decolorized crude was absorbed on a column packed with Dowex 1 × 2 resin (OH- form) the gentamicin complex eluted with water.

LYOPHILIZATION LYOPHILIZATION 4 STEPS

LYOPHILIZATION Introduction The purpose of freeze-drying is to remove a solvent (usually water) from dissolved or dispersed solids. Use Freeze-drying is method for preserving materials, which are unstable in solution.

LYOPHILIZATION Step 1 Freezing The product is frozen. This provides a necessary condition for low temperature drying.

LYOPHILIZATION Step 2 Vacuum After freezing, the product is placed under vacuum. This enables the frozen solvent in the product to vaporize without passing through the liquid phase, a process known as sublimation.

LYOPHILIZATION Step 3 Heat Heat is applied to the frozen product to accelerate sublimation.

LYOPHILIZATION Step 4 Condensation Low-temperature condenser plates remove the vaporized solvent from the vacuum chamber by converting it back to a solid. This completes the seperation process.

PACKAGING AND PACKAGING AND LABELING LABELING RULES AND REGULATIONS

PACKGING AND LABELING As given by good manufacturing practices With respect to different dosage forms

PACKGING AND LABELING Labeling should be as given by official data Examples Generic name Name of manufacturer Manufacturing date Expiry date Batch no Dosage precautions

THANK S QUESTIONS ?