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PHAR457 Asst. Prof. Dr. Mehmet İLKTAÇ Lecture 3: 30/10/2017 - FALL
PHAR457
Asst. Prof. Dr. Mehmet İLKTAÇ
Lecture 3: 30/10/2017 - FALL
Raw material, ingredient and solvent in manufacturing pharmaceuticals
Washing and cooling processes
WATER
The most Significant source of microbial contamination of pharmaceuticals.
Over 50% of non-sterile product recalls were as a result of contamination of water-borne bacteria (1991-2001)
Pseudomonas Burkholderia Ralstonia
WATER Three grades of water in pharmaceutical industry
1. POTABLE WATER
a) PURIFIED WATER
b) WATER FOR INJECTION
2. WATER FOR PHARMACEUTICALS
WATERBORNE OUTBREAKS-2011-2012-USA
WATER MONITORING IN PHARMACEUTICAL INDUSTRY
Potable water is used as raw material (source water) in the preparation of water for pharmaceutical purposes
Source water is among the most common causes of contamination of water for pharmaceutical purposes
Contamination of water for pharmaceuticals is responsible for the contamination of paranteral and non-paranteral drugs.
HEALTH THREAT TO CONSUMER
SPOILAGE OF PRODUCT
1. Product recalls 2. Production shutdown 3. Losses in labor and manufacturing 4. Financial loss
WATER MONITORING Continuous process of:
Measuring Detecting Recording
CHANGES IN MICROBIAL POPULATION
Change in number and composition in relation to
previous monitoring
MORE IMPORTANT THAN DETERMINING ABSOLUTE NUMBER AND COMPLETE TYPES OF MICROORGANISMS IN WATER
RECORD THE RESULTS IN ORDER TO BE AWARE OF
CHANGES!!!!!
POTABLE WATER MONITORING
POTABLE WATER Municipal water is chlorinated at 0,5 ppm free chlorine residual
Chlorination is effective against many waterborne pathogen bacteria/viruses.
Salmonella typhi Vibrio cholerae Shigella dysanteriae E. coli O:157 H:7 Legionella pneumophila
Entamoeba histolytica Giardia intestinalis Cryptosporidium Microsporidium
Hepatitis A Poliovirus Norovirus
Waterborne bacteria Waterborne protozoons
Waterborne viruses Ascaris Echinococcus Taenia Trichuris
Waterborne helminthes
POTABLE WATER Explosive epidemics = Common source epidemic = Point source epidemic
Waterborne infections
1. Heterotrophic Plate Count
2. Total Coliform Count
3. Fecal Coliform Count
PARAMETERS FOR MONITORING POTABLE
WATER
PARAMETERS FOR MONITORING POTABLE WATER
1. Heterotrophic Plate Count (HPC)
Total number of viable heterotrophic, aerobic bacterial population is determined.
Heterotrophic: Microorganisms living in high nutrient environment.
The lower the HPC, the better maintained the water is.
HPC of potable water should be lower than 500 CFU per ml.
Oligotrophic: Microorganisms living in environment low in nutrient at lower temperature .
PARAMETERS FOR MONITORING POTABLE WATER
1. Heterotrophic Plate Count (HPC) Methods
a) Pour Plate Method
Volumes of sample or diluted sample ranging from 0.1 to 2.0 mL is added to petri dish.
Molten non-selective non-differentiating medium at 45 °C is poured into petri dish and mixed with sample. Solidify at room temperature.
Incubate at 35 C for 48 hours and Count.
a) Pour Plate Method
Pour Plate Method – Serial Dilution
PARAMETERS FOR MONITORING POTABLE WATER
1. Heterotrophic Plate Count (HPC) Methods b) Spread Plate Method
Small volume of sample or diluted sample ranging 0.1 to 0.5 mL is added to the surface of medium and spread all over the surface.
Pour 15 ml non-selective non-differentiating medium into petri dish and solidify at room temperature.
Incubate at 35 C for 48 hours and Count.
b) Spread Plate Method
PARAMETERS FOR MONITORING POTABLE WATER
1. Heterotrophic Plate Count (HPC) Methods
c) Membrane Filtration Method
Large volumes of sample or diluted sample ranging upto 100 ml can be used.
Sample or diluted sample is filtered through 0.45 µm diametered filters.
Filter is transferred onto non-selective non-differentiating medium and incubated for 48 hours at 35 °C.
PARAMETERS FOR MONITORING POTABLE WATER
2. Total Coliform Count
Intestinal tract and feces of warm blooded animals
May also be found in the environment (soil, plant.....)
E. coli Klebsiella Enterobacter Citrobacter
Coliform bacteria: Rod shaped Gram negative Non-spore forming Ferment lactose with the production of acid and gas
when incubated at 35–37°C for 24 hours.
PARAMETERS FOR MONITORING POTABLE WATER
2. Total Coliform Count
As HPC increases, the probablity of coliform presence increases
During coliform testing, water borne pathogens are not investigated.
I. Coliform testing are used as a screen for the fecal contamination of water (INDICATOR of water pollution).
II. Coliform count provides the best assessment of water treatment effectiveness and the sanitary quality of source water.
2. Total Coliform Count A. Most probable number method
METHODS FOR TOTAL COLIFORM COUNT
a) Presumptive Test
Inoculate 5 tubes containing 10 ml lactose containing selective broth (lauryl tryptose broth) with 10 ml water, 5 tubes with 1 ml and 5 tubes with 0.1 ml water.
Incubate 35 °C for 24-48 hours.
The presence of gas and turbidity is a positive presumptive test.
Determine the MPN using positive tubes for each volume.
Most probable number method- Presumptive test
Lauryl tryptose broth
GROWTH ON LAURYL TRYPTOSE BROTH
DETECTION OF GAS IN DURHAM TUBES
Gas negative
Gas positive
2. Total Coliform Count
Most probable number method
PARAMETERS FOR MONITORING POTABLE WATER
b) Confirmed Test
Add a small quantity from positive presumptive test tube into lactose containing selective broth (brillant green lactose bile broth) and incubate 35 °C for 48 hours.
The formation of gas and turbidity is positive confirmed test.
GROWTH ON BRILLANT GREEN LACTOSE BILE BROTH
2. Total Coliform Count Most probable number method
PARAMETERS FOR MONITORING POTABLE WATER
c) Completed Test
From positive confirmed test tube, transfer a small volume to Endo agar. Incubate at 35 C for 24 hours.
Selective and differential media for coliforms
Metallic sheen colony is positive.
Transfer to lauryl tryptose broth and confirm gas once more time.
Perform Gram staining. Gram negative rods.
Most probable number method- Confirmed and Completed
2. Total Coliform Count
PARAMETERS FOR MONITORING POTABLE WATER
Filtration Method
More rapid (2-3 d) than MPN method (5 d)
The standard volume to be tested is 100 ml (large).
After filtration, transfer the filter onto selective and differentiating medium (eg: Endo agar)
Incubate under 35 °C for 48 hours. Typical colonies of coliform according to indicator media used are counted.
Verify the colonies by subculturing them in lactose containing broth medium (lauryl tryptose broth, brillant green lactose broth).
COLIFORM BACTERIA ON ENDO AGAR
2. Total Coliform Count
PARAMETERS FOR MONITORING POTABLE WATER
Regulatory requirement 1. Total coliform count should be 0 per 100 ml water.
Every sample positive for total coliforms must be analysed for fecal coliforms
2. Not more than 5% of monthly samples being positive for coliform bacteria.
3. If one of two consecutive total coliform positive sample is positive for fecal coliform, it is a maximum contaminant level violation
3. Fecal Coliform Count
PARAMETERS FOR MONITORING POTABLE WATER
Fecal Coliform bacteria: Rod shaped Gram negative
Non-spore forming Ferment lactose with the production of acid and gas
when incubated at 44.5°C for 24 hours.
BETTER INDICATOR FOR FECAL
CONTAMINATION
Since not found in the environment and only in intestinal tract of warm blooded animal.
BETTER INDICATOR FOR FECAL
CONTAMINATION
3. Fecal Coliform Count PARAMETERS FOR MONITORING POTABLE WATER
Most Probable Number
2 methods
A.Take the all total coliform presumptive test positive samples into lactose containing selective broth (EC medium). Incubate under 44.5 °C for 24 h. Calculate MPN.
B. Inoculate 10 ml, 1 ml and 0.1 ml of water each of five tubes of A1 broth (lactose containing medium). İncubate at 35 C for 3 hours and then for 44.5 C for 21 hours. Check for gas formation (positive result). Calculate MPN.
GROWTH ON EC BROTH
3. Fecal Coliform Count
PARAMETERS FOR MONITORING POTABLE WATER
After filtration, transfer the filter onto lactose containing selective and differentiating solid medium (eg: mFC medium (agar)).
Incubate under 44.5 °C for 24 hours. Typical colonies of coliform according to indicator media used are counted.
Verify the colonies by considering biochemical characteristics (lactose fermentation, indole production, urease test etc....).
Filtration Method
SAMPLE COLLECTION
Sterile plastic or glass bottles.
Add a reducing agent like sodium thiosulphate to the bottle to neutralize chlorine or other halogens.
The volume of sample should be min. 100 mL
Hold samples at 5 °C for 24 h till analysis. Transport on ice if possible.
PHARMACEUTICAL WATER MONITORING
EXOGENOUS
SOURCES OF CONTAMINATION OF PHARMACEUTICAL WATER
ENDOGENOUS
SOURCE WATER
Unprotected vents
Backflow from contaminated outlets
Unsanitized distribution system
openings
Biofilms in unit operation.
.Carbon bed .Deionizer resins
.Filters.... Biofilms in distibution system
.Pipe surfaces .Dead legs
.Valves
Source water DRINKING WATER
2. Total Aerobic Microbial Count (TAMC) should be lower than 500 cfu/ml.
1. No coliform bacteria
REDUCING CONTAMINATION OF PHARMACEUTICAL WATER
Biofilm formation reducing
2. Absence of dead legs.
1. High turbulence
3. Periodic sanitization by hot water, steam or ozone.
4. Providing smooth surfaces during design
MICROBIOLOGICAL ANALYSIS OF PHARMACEUTICAL WATER - USP
Method (Media)
Minimum sample size
Incubation conditions
Recommended limits
Potable Water Pour plate
count (Plate count agar)
1 ml 48-72 h at 30-35 C 500 cfu/ml
Purified Water
Pour plate or membrane filtration
(Plate count agar, R2A
agar)
1 ml 48-72 h at 30-35 C 100 cfu/ml
Water for Injection
Membrane filtration
(Plate count agar)
100 ml 48-72 h at 30-35 C 10 cfu/100 ml
MICROBIOLOGICAL ANALYSIS OF PHARMACEUTICAL WATER - EP
Method (Media)
Minimum sample size
Incubation conditions
Recommended limits
Potable Water Pour plate
count (Plate count agar)
1 ml 5 days at 30-35 C 500 cfu/ml
Purified Water Membrane
filtration (R2A agar)
1 ml 5 days at 30-35 C 100 cfu/ml
Water for Injection
Membrane filtration
(R2A agar)
200 ml 5 days at 30-35 C 10 cfu/100 ml
HIGH NUTRIENT MEDIA OR LOW NUTRIENT MEDIA ??????
High nutrient media
Low nutrient media
Heterotrophic bacteria
Oligotrophic bacteria
Plate count agar
mHPC
R2A
RWRI
Gives higher counts
USP
EP
LONGER OR SHORTER INCUBATION vs
HIGHER OR LOWER INCUBATION TEMPERATURE ???
High nutrient media 30-35 C for 48-72 h.
Low nutrient media 20-25 C for 1-2 week.
Longer incubation time increases the recovered count by recovering injured or slow growing microorganisms.
The advantages gained by incubating for longer times should be balanced against the need to have a timely information and to take corrective action
Highest count: Low nutrient, 28 C, 2 weeks.
LONGER OR SHORTER INCUBATION vs
HIGHER OR LOWER INCUBATION TEMPERATURE ???
Whether or not a particular system needs to be monitored using high- or low-nutrient media with higher or lower incubation temperatures or shorter or longer incubation times should be determined during or prior to water system validation.
NEED TO MONITOR THERMOPHILIC BACTERIA IN WATER FOR INJECTION ?
Generally photosynthetic, chemolithotrophic or anaerobic
Unlikely to be present in water for injection
Even if present they donot pose health threat humans.
NO NEED TO INVESTIGATE THERMOPHILIC BACTERIA
