Pham aceutical

M icrobiology
Subtitle
Growth Promotion Test

▪ The purpose of the Growth promotion test is to assure the nutritive
properties of medium by challenging it with a small number of
microorganisms (not more than 100 CFU).
▪ The GPT should be performed on each batch of purchased ready-prepared
medium, each batch of dehydrated medium or medium prepared from
components in the laboratory.
▪ EZ-CFU One step are standardized, lyophilized microorganism
preparation designed to deliver an inoculum of less than 100 CFU per 0.1
ml.

2
Growth Promotion Test

▪ If testing agar, the number of colonies on the new batch of medium must be
within a factor of two of the number of colonies on the previously approved
medium. If testing broth, the new and previously approved medium must be
comparable in appearance.
▪ For example, if the average number of colonies on the previously approved
agar is 40, then the acceptable number of colonies on the new agar should be
between 20 and 80.
▪ In addition to growth promotion test, inhibition test is also performed.
Inhibition test is used for selective media which can support the growth of
particular microorganism and inhibit the growth of other type of
microorganisms.
3
Microbial Enumeration Tests

▪ The tests are designed primarily to determine whether a substance or
preparation complies with an established specification for microbiological
quality and
▪ Should be carried out the determination under conditions designed to
avoid extrinsic microbial contamination of the product to be examined.
▪ If the product to be examined has antimicrobial activity, this is, in so far
as possible, removed or neutralized.

4
Bacterial Endotoxin Test

▪ The gel-clot LAL test method is a simple, reproducible test to detector
measure endotoxin that is conducted by mixing ENDOSAFE LAL
reagent and test specimen and promptly incubating the mixture
undisturbed for 60 minutes at 37 C.
▪ A positive result is defined as the formation of a firm gel capable of
maintaining its integrity when the test tube is inverted 180 and a negative
test is characterized by the absence of gel when the tube is inverted.
▪ Test results are only valid when the positive control and product positive
control are positive at 2λ endotoxin concentration and the negative
controls are negative.

5
Bacterial Endotoxin Test

▪ Frederick Bang observed that bacteria caused intravascular coagulation in
the American horseshoe crab, Limulus polyphemus.
▪ In collaboration, Levin and Bang, found that that agent responsible for the
clotting phenomena resided in the crab's amoebocyte or circulating blood
cells and that pyrogen (bacterial endotoxin) produced a gelation reaction
of amoebocyte lysate by an enzymatic process.
▪ Serine proteases zymogens found in amoebocyte lysate are activated by
endotoxin in the presence of divalent cations to initiate an enzymatic
coagulation cascade that alters an abundant protein called coagulogen to
produce a proteinaceous gel.

6
Bacterial Endotoxin Test

▪ The
  LAL test is generally limited to aqueous solutions or extracts of test
specimen.
▪ The reaction requires a neutral pH (6.0-8.0) and is time and concentration
dependent. If the specimen, contains interfering substances, dilute or
modify the specimen to an extent that eliminates interference.
▪ The maximum valid dilution is the maximum allowable dilution of a
specimen at which the endotoxin limit can be determined.
MVD

7
Bacterial Endotoxins Test

▪ The minimum concentration of endotoxin required to cause the lysate to
clot under standard conditions is the labeled sensitivity of the lysate
reagent.
▪ If the sample under test does not comply with the test at a dilution less
than the MVD, repeat the test using a greater dilution, not exceeding the
MVD.

8
Bacterial Endotoxins Test

Reagents
 Lyophilized LAL Reagent
 LAL Reagent Water
 Control Standard Endotoxin (CSE)

Test Procedure
 Add 0.2 mL of test specimen to assay tubes.
 Mix the contents gently until the contents are dissolved.
 Immediately place the reaction tubes in a 37 C water bath for 60
minutes (±2 minutes)

9
Bacterial Endotoxins Test

▪ Assay
  Controls
▪ Negative Control
▪ LAL Reagent plus LRW
▪ Verifies suitability of LAL reagent water
▪ Positive Control
▪ LAL Reagent plus 2 CSE
▪ Product Positive Control
▪ LAL Reagent plus 2 CSE & product
▪ P

10
Water For Pharmaceutical Purposes

▪ Water is widely used as a raw material, ingredient and solvent in the
processing, formulation and manufacture of pharmaceutical products,
active pharmaceutical ingredients (APIs) and analytical reagents.
▪ Water should be monitored at a frequency that is sufficient to ensure that
the system is in control and continues to produce water of acceptable
quality.
▪ The sampling plan should take into consideration the desired attributes of
the water being sampled. There is no single method that is capable of
detecting all of the potential microbial contaminants of a water system.

11
Water For Pharmaceutical Purposes

▪ The number of recoverable bacteria in a sample can change positively or
negatively over time after sample collection, it is best to test the samples
as soon as possible after being collected.
▪ If it is not possible to test the sample within about 2 h of collection, the
sample should be held at refrigerated temperatures (2°–8°) for a
maximum of about 12 h to maintain the microbial attributes until analysis.

12
Validation Of Aseptic Manufacturing Process

▪ The process simulation (also known as a media fill) is the performance of
an aseptic manufacturing procedure using a sterile microbiological growth
medium in place of the drug solution.
▪ To test whether the aseptic procedures are adequate to prevent contamination
during actual drug production.
▪ The media fill should evaluate
– the aseptic assembly and operation of the critical (sterile) equipment,
– qualify the operators and assess their technique, and
– demonstrate that the environmental controls are adequate to meet the basic
requirements necessary to produce a sterile drug by aseptic processing.

13
Validation Of Aseptic Manufacturing Process

14
Validation Of Aseptic Manufacturing Process

▪ To initially validate an aseptic process at a specific facility, 3 media fills
should be conducted on three separate days at that facility using the
specific production process that is being validated. For validated process,
1 media fill should be conducted to revalidate the aseptic process.
▪ In addition, media fills should be conducted whenever significant changes
are made to the aseptic process e.g., changes in personnel, components or
equipment.
▪ Key elements to be taken into account include number and frequency of
runs, number of units filled, container (vial) size, fill volume, filling
speed, duration of fill, operators shifts, monitoring activities,
Interventions etc.
15
Validation Of Aseptic Manufacturing Process

16