Biological Methods in Quality

Control

1
 Learning Objectives

At the end of the chapter the students will be able to:

 Describe the significance of conducting microbial
quality control

 Elaborate the importance and methods of

evaluation of various preservatives

 Discuss on the issues of microbial limits,

standards and quality assurance of sterile
products
 Conduct biological assay for pharmaceutical
products, endotoxins, and related substances

2
Pharmaceutical Microbiology
 Pharmaceutical Microbiology deals with
microbiological safety of pharmaceuticals, medical
devices, and related issues.

 The topic gives an overview of microbial quality

assurance methods for isolation, identification, and
enumeration of microorganisms for pharmaceutical
products :
 Tests for Microbial Limits for Non-sterile Pharmaceutical
and Biological Products
 Microbiological Assay of Antibiotic Drugs
 Bacterial Endotoxins (LAL) Test for Pyrogen
 Sterility Test of Sterilized Pharmaceutical and Biological
Products
 Test for Effectiveness of Antimicrobial Preservatives
3
 Microbial Quality Control

 Contamination of pharmaceutical products by

objectionable microorganisms is a major risk within the
pharmaceutical industry as
 it may impact product integrity and patient safety
 To prevent contamination events from occurring, licensed
pharmaceutical manufacturing companies worldwide are
required to adhere to:
 strict regulations and
 robust quality control procedures issued by their
respective government agencies.
 These regulatory processes and procedures comprise
various quality control methods, such as those
described in the USP, EP, and JP.
 When properly followed, these procedures can help
identify microbial contamination prior to product
release, thus avoiding the pitfalls of product recalls.
 Microbiological quality control is an essential part of
the pharmaceutical manufacturing process.
 Pharmaceutical companies must safeguard the quality
and safety of their products by thoroughly testing:
 raw materials,
 equipment,
 environmental surfaces, and
 final preparations for microbial contaminants
 Microbial Quality Control…..
 Microbial QC is applied to
 Pharmaceuticals
 Medical devices

WHY We Need to Conduct Microbial Quality Control?

Health Significance (infection, disease....)

Economic significance (deterioration of products...)

7
Microbial Limit Tests

 They are applied to all raw materials and

preparations not required to be sterile.
 They cover the total microbial viable aerobic count
(TAC) and the absence of specified pathogens.
 Microbial limit tests are carried out according to
the method mentioned in different Pharmacopoeias.

 Non-sterile medicaments (products)

 May not be totally free of microbes
 Bio-burden
 Limited permissible level of microbes.
Microbial limit requirements for pharmaceutical dosage
forms
Microbial Limit Tests...

Table 1: Criteria for microbiological quality of

nonsterile dosage forms
 Entries for total aerobic microbial counts
(TAMC)
 Entries for total combined yeasts/molds count
(TYMC)
 Entries for specified microorganisms
Microbial Limit Tests...

 Tests for Microbial Limits are designed for the

determination of the level of bioburden and the absence
of specific pathogens in pharmaceutical raw materials
and finished products, which are not compulsorily
sterile.

 Species of concern:
 According to USP, the four major species of concerns
are:
a. Escherichia coli
b. Staphylococcus aureus
c. Pseudomonas aeruginosa
d. Salmonella species

11
Microbial Limit Tests...

E. coli
 Its presence is an index of faecal pollution of water.

Staph. aureus
 Workers with poor sanitary quality could be the
source of product contamination

Pseu. aerugenosa
 Universally distributed microbe and highly resistant
to antibiotics

Salmonella
 The common enteric micro-flora of human and other
animals
 Associated with Low hygienic quality
12
Microbial Limit Tests...

 when testing for the presence of microbes,

CONSIDER THE FOLLOWING:

◦ Culture media preparation → should be as per the

USP specification
 Fresh, specificity, selectivity, and sensitivity
◦ Media sterilization → always by autoclaving

◦ If agar is employed, it should not normally contain

> 15% agar
→ this helps to avoid over-solidification of media.

13
Microbial Limit Tests...

◦ pH → be adjusted at 25 oC; @6-8

◦ Incubation Temp. →between 30-35 oC.

◦ Duration of Incubation: 24-48hrs.

 18-24 h→ is too early for injured bacteria

Injured bacteria require Longer lag phase
→ Could be extended to 72h.
 Microbial Limit Tests...

1. Total Viable Aerobic Count

This test is conducted to determine mesophilic
bacteria and fungi which grow under aerobic
conditions.

 There are four methods for this test (TAC):

1. membrane filtration method
2. pour plate method
3. spread plate method and
4. serial dilution method (Most Probable Number
method).

An appropriate method should be taken from among these

four, depending on purposes.
15
Microbial Limit Tests...

2. Specific Tests for the designated microbes

Test for absence of pathogens

 Escherichia coli- IMViC tests

 Salmonella species-Brilliant Green Agar
 Staphylococcus aureus-Coagulase Test
 Pseudomonas aeruginosa-Oxidase Test
 Microbiological Assay for
Pharmaceuticals
Biological assay
 It is a practical procedure where by the potency of a material
of unknown potency is estimated by comparison of its effect
in a biological system with that of the reference standard of
known or defined potency.
Microbiological assay
 The biological system is a culture of MOs
Potency: measurement of power to kill or inhibit the
growth of certain microorganism
17
 Antibiotic -Microbiological Assay…
 Why antibiotic should be analysed ?
 The use of antimicrobial agent are increasing rapidly
 Resistance of many pathogenic microbes
 To confirm the effectivity of available antimicrobial
agent against new strains

18
 Microbiological Assay for
Pharmaceuticals………
 Objective of the test
 As a standard to confirm the potency of
antibiotic to kill or inhibit the growth of certain
microorganism .
 Microbial Assay for Antibiotics
 It requires the fulfilment of 3 factors:
i. The test compound → Antibiotic
ii. Reference standard
iii. Sensitive reference strain
19
 Microbial Assay for Antibiotics

 The basis of microbial assay of antibiotic is the

quantitative comparison of the effect of the test
compound and the reference standard on the
growth of a suitable or susceptible organism in a
nutrient medium.
 Expected effect:
 Inhibition of growth of the sensitive test
strain.
 The test micro-organism should:
 be sensitive to the antibiotic, and show graded
response.  successive dilution should
show ↓ or ↑ in effect based on the
conc.
20
Reference standard to be used for Microbiological Assay:

 Is required to be PURE.
– Since the basis of any Microbiological Assay (MBA)
is comparison of the test substance with reference
standard, the success of MBA is ensured only when
the reference standard itself is pure.
 Ideally, a reference standard should be:
a. Available in sufficient quantity
b. Completely homogenous
c. Highly stable
d. Qualitatively be identical with the test cpd that is
being assayed
e. Be preferably a single substance.
21
Media and Diluents

 Choice of media is as per USP/other compendial

guidelines.

 Media must provide

 adequate nutrition/physical conditions
 facilitate antibiotic diffusion.
 While being sterilized-not excessively heated-
why?

 Before use all media should be checked for

sterility and growth promotion.
 Media and Diluents…

 Media always be stored under defined

conditions.
 Refrigerator
 Room Temperature

 It requires taking extreme care specially

during the preparation of stock solutions and
dilutions.
 Reference Standard Preparations:……

The essential requirement is that:

 The antibiotic and reference material be completely
soluble in the solvent/diluent,
 If the antibiotic is to be extracted from an
ointment/cream base, then the extraction process be
validated.

24
MBA of Antibiotics could be conducted in 2 ways:

1. Plate or Cylinder plate /Diffusion Assay

2. Tube Assay (Turbidimetric Assay)

25
A. Plate Assay
Principles:
 It depends upon diffusion of the antibiotic through a
solidified agar layer in a Petri dish or plate to an extent
such that growth of the added MO is prevented.
 Then zone of inhibition is measured and compared with
that of standard.
 The Zone of inhibition is a circular area around the spot of the
antibiotic in which the bacteria colonies do not grow.
 The zone of inhibition can be used to measure the susceptibility of
the bacteria to wards the antibiotic.
Turbidimetric Method

Principles:
 The antibiotic will inhibit the growth of
microbes in liquid media in test tubes.
 Turbidity intensity of the culture will express
the population of microbes
 Turbidimetric method is suitable for antibiotic
which is not soluble in water

27
Tube/Turbidimeteric Assay of Antibiotics
 Activities:
1. A series of concentrations of Ref/samples/ are prepared,
2. 1ml of each solution is added to a separate tube,
3. About 9ml of Nutrient broth is added to the above tubes,
followed by inoculation of the test microbe,
4. The tubes are incubated for 4 hrs,
5. Growth is stopped by heating at 80 0C.
6. The inhibition of growth is estimated by measuring Turbidity
using Spectrophotometer .
7.  the lesser the Turbidity, the higher the efficacy of the
Test sample (antibiotic) 28
Preservatives Efficacy Testing

 Why preservatives?
 To protect the user against possible infections by any
pathogens which the product may contain

 To improve the safety of non-sterile medicaments

 preservatives are added mostly to prevent spoilage during
transportation and storage. Ex. Food, phar. products

 Preservatives serve as either antimicrobials or antioxidants

or both.
o As antimicrobials, → they prevent the growth of molds, yeasts
and bacteria.
o As antioxidants, → they keep the product from becoming rancid,
or browning.

29
Preservatives and their properties
 should not be irritant or toxic to tissues to which
they will be applied
 must be effective in preventing growth of
microorganisms likely to contaminate them and
 must have sufficient solubility and stability to
remain active.
 Commonly used preservative agents include
1. Organic acids such as benzoic acid and salts,
2. The parabens, (alkyl esters of p-
hydroxybenzoic acid),
3. Sorbic acid and salts,
4. Phenolic compounds,
5. Quaternary ammonium compounds, Alcohols…
30
Test organisms -To be used for Efficacy test
 Standard species/strain should be employed.
Include:
1. Bacteria
a. Staphylococcus aureus
b. Pseudomonas aeroginosa
c. Escherichia coli
2. Yeasts – Candida albicans
3. Molds - Aspergillus niger

31
Test organisms…
Why the above microbes as Test strains?

They are representative of typical

contaminants during manufacturing and
use/treatment:

They have undemanding nutritional

requirements and are commonly associated
with preservative resistance

They have pathogenic potential

32
Basic Test Protocol/ Conditions

1. In those cases where the product containers

can be entered aseptically (with needle),
conduct the test in 5 original containers
2. If the container can not be entered, then
transfer 20 ml sample or the entire amount (if
< 20ml) to the 5 sterile capped tubes

33
 Basic Test Protocol/ Conditions...

3. Inoculate each tube or container with standardized

inoculum at a ration of 0.1 ml inoculum to 20ml of
the product and mix.
4. Incubate the containers at 20-25 ˚C. →Examine at
7, 14, 21, 28 days:
5. Determine the total count at those intervals and
express the count as a percentage change in the no
of microbes.
Interpretation of Result
 The preservative is considered effective if:

a. The number of viable bacteria is reduced by at

least 0.1% of the initial count by the 14th day

b. The no of yeasts and molds remain at or below

the initial number during the first 14 days

c. The no of each test organism remains at or

below the designated levels during the
remainder of the 28 days of the test period
35
Sterility Test
 Required for all articles or substances to be
introduced into raw tissue (injections and
ophthalmics).

 Sterility testing attempts to reveal the presence

or absence of viable micro-organisms in a sample
number of containers taken from batch of
product.

 Based on results obtained from testing the

sample a decision is made as to the sterility of
the batch.
 The test shows that samples tested were free
from living bacteria and fungi but not viruses.
Sterility Test…

☻ It is not possible to claim that a batch is sterile

unless
a. The entire contents of every container is
tested and
b. The test provides optimum conditions for
growth of all possible microbes.
BUT, Neither of the above 2 conditions can be
fulfilled.

►Thus, sterility test can only show that “ microbes

capable of growing in the tested media employed
under selected conditions are absent from a
fraction of a batch tested.”
37
Sterility Test…
Two generalization could be made:

1. To obtain reliable results, it is necessary

to take sufficient samples and use
sensitive enough media

2. Sterility testing should not be used as a

sole means of controlling sterile
processing.
General Methodology – for sterility Test

 Tests for sterility are carried out by the method of:

 Membrane Filtration, or
 Direct inoculation.
► The method of Membrane Filtration should be
used as the method of choice wherever feasible.

 Media:
◦ Fluid Thioglycollate Medium (Medium 1) - usually
used to detect anaerobic organisms

◦ Soybean-Casein Digest Medium (Medium 2) - used to

detect aerobic MOs

 Containers of Medium 1 are incubated at 30 -

35°C and Medium 2 at 20 - 25°C. 39
General Methodology – for sterility Test…

Where the preparation to be tested has

antimicrobial effects, these effects may
be reduced or neutralized by 3
approaches:

1. the sample is added to a volume of

medium sufficient to dilute
2. treat the sample with a neutralizing
agent
3. use membrane filtration
41
 General Methodology…

Stages of sampling:
1. For terminally sterilized products – the
final end product stage is where samples
are collected,
2. For aseptically processed products – two
sampling stages are required:
i. The bulk from which final container
are filled
ii. The final container after they are
sealed.
42
General Methodology…
Selection of samples
 The selected sample must be
representative of the entire lot of final
container,
 For bulk product, the entire
liquid/material be well mixed.
 For terminally sterilized lot, select
at random
General Methodology…

Sample Size

 USP generally insists on a minimum of:

 10 units – for each lot of
steam/terminal sterilized
products
 20 units – for each lot of continuously
sterilized products (radiation/gas
sterilization)
 30 units – from each filling operation

44
General Methodology …
Culture media
- Ve control:
◦ Sensitive
- growth is not expected
Control tests - growth may occur if:
+ Ve control
-Ve control i. improperly sterilized
medium
+ Ve control: ii. Contamination during
- growth is expected testing
- growth may not occur if:
Purpose of the –ve control if no
i. inadequate medium growth is seen:
ii.overheating of medium
during sterilization  it confirms that medium is
sterile
iii.inhibition by active
ingredient or excipient  serves as a standard with
which test can be compared
 General Methodology …

Test procedure
 Opening the container:
◦ Cleanse external surface with a suitable
disinfecting agent & gain access inside aseptically.

How to inoculate:
 If it is a liquid, remove sample with a sterile
pipette, syringe.
◦ After adding to medium gently mix.
 Incubate as directed for not less than 14 days
◦ Bact- 30-35 oC
◦ Fungi-20-25 oC
 Test procedure…
 Immersion method or direct inoculation method
 Prepare the appropriate media
 Directly inoculate the product or test solution
 Incubate the inoculums for14 days (check the + /- of
Mos in certain interval of time)
Advantage
 It require small number of samples
 used for non liquid article – cream, ointment, etc
Limitation
 Not used for article having antimicrobial activity
 It require pretreatment step

47
Test procedure…

 Membrane filtration method

 Test article is made to pass through a filter
 Organisms of interest are retained in the
filtering material but Test article pass through
the filter
 Incubate the filtered microbe for 14 days
 Check if growth is present in certain interval
of time
Advantage; it used for
 Large volume of sterile product
 Articles having antimicrobial activity
Limitation:
 Used only for liquid samples
 Require large volume of sample 48
 Recording and interpretation of results

 Examine the media for growth at least as often as

on 3/4/5/7/8/14th day
 Record- the nature, size, composition, of
item/product, the method, nature/ Vol. of medium.

1st stage
 At various times- no evidence of growth
 product passes

 If growth is seen, but a review of procedure,

materials suggests a possible contamination and also
the –ve control shows growth.
 the entire test is declared invalid and repeated.
Recording and interpretation of results…

2nd stage
 In this repeat testing- the minimum number of
samples to be tested is doubled.
 If no growth is observed
 product passes

But if growth occurs again-it conclusively proves that

product is non-sterile

[to be repeated if –ve control also shows growth]

Pyrogens testing

 Pyrogens are defined as substances that when injected

in sufficient amounts gives rise to a variety of
responses and symptoms.
 The most prominent response/symptom is raise in body
temperature.

 Pyrogen is “a fever producing agent”.

 All microbes appear to be capable of producing
pyrogens but the most potent are associated with
Gram-negative bacteria.
51
 Gram-negative bacteria vs Pyrogen????
Bacterial Endotoxins

 Bacterial endotoxins, found in the outer membrane of

gram-negative bacteria are members of a class of
phospholipids called lipopolysaccharides (LPS).
 Human exposure to endotoxins triggers a strong
inflammatory reaction, which can be life-threatening.

 To avoid this, the FDA requires that all

pharmaceuticals and medical devices be tested for
endotoxin levels.

52
Bacterial Endotoxins …..

 The LPS is pyrogenic and responsible for some of

the symptoms that accompany infections caused by
gram-negative bacteria.
 LPS are not exogenous products of gram negative
bacteria. The release of LPS from bacteria takes
place after death and lysis of the cell.
 Good examples of pyrogen producing gram-
negative bacteria are Escherichia coli, Proteus,
Pseudomonas, Enterobacter and Klebsiella.
Properties of Endotoxins

Chemical property:
 Pyrogens are polysaccharides attached to a lipid,
thus called Lipopolysaccharides.
Physical property:
 Highly stable at higher temperature.
 Autroclaving can not destroy pyrogens.
 Long drastic heating of more than 180 oC is
necessary.
 Dry heating is the method of choice for
depyrogenation of glass wares, other equipment. 54
Biological Properties of Endotxins:
 Induce Fever, chilly sensation
 Intravascular coagulation,
 Bone marrow necrosis,
 Renal/liver damage,
 Decrease respiration
 Increase in arterial BP
 Pain in the back & leg
 Diarrhea
55
Pyrogen Testing...

 In vivo – Rabbit Pyrogen Test ( biological)

 In vitro – Limulus Amebocyte Lysate (LAL)

Test (chemical)

56
 Pyrogen Testing...
1. Rabbit Pyrogen Testing
WHY rabbit?
i. In expensive, Easy to handle
ii. Rabbits show some response to pyrogens as
humans on per kg body weight basis
iii. Rabbits have labile thermoregulatory
mechanisms
BUT, rabbits frequently produce false positive
reactions. 57
Pyrogen Testing...

Basic principles: after administration of the

product;

 If ↑ in body temp. of rabbit above the limit

→ the product is pyrogenic;
 If the body temp. of the rabbit below the
limit →the product is free from pyrogen
Test Procedure:
1. Three healthy rabbits are selected ,
2. Accurate temperature (rectal) is initially taken by a
thermometer. (To be taken for not more than 30 min prior to
the administration of the product), (control To)
3. Test solution is warmed to 37 oC ±2 oC and
administered, (≈ 10ml/kg)
4. Rectal temperatures are detected every 30 min for 1-3
hrs,
5. Conclusion: The product passes if no rabbit shows a
temperature raise of 0.5 oC or more,
6. If any rabbit shows > 0.5 oC raise, the test is
repeated on 5 more rabbits
7. Now, if not more than 3 of the eight rabbits show an
individual To raise 0.5 oC and if the sum of the eight
individual rabbits To increase is not greater than 3.3,
then the product passes. 59
 Pyrogen Testing …
Advantages
 Not specific to any one class of pyrogens, but
can detect all types of endotoxins.
 Best replicate & domenstrate the production of
fever in human
 Disadvantage
 Time taking, laborious
 Chance of biological variation ( extremly
sensitive to Env’t)
 Development of tolerance by the rabbits
 Less sensitive
 A pass/fail test, rather than an assay

60
2. In-vitro pyrogen Testing
Limulus Amoebocyte Lysate (LAL)Test

 The Limulus amebocyte lysate (LAL) test was commercially

introduced in the 1970s.
 LAL is derived from the blood cells, or amebocytes, of the
horseshoe crab, Limulus polyphemus.
 Frederick Bang and Jack Levin observed that blood cells from
horseshoe crabs were found to clot in the presence of endotoxin.
 The blood of horseshoe crabs contains an enzyme that promotes
clotting upon exposure to endotoxin.
 This provides a reliable method for detecting and quantifying
endotoxin levels.
 It is a test for the determination of G-ve bacterial endotoxin
61
Limulus Amoebocyte Lysate (LAL)Test…………

 It employs a lysate protein obtained by lysing the blood

cells (amoebocytes) of the horseshoe crab (Limulus
polyphemous).
 LAL test is an aqueous extract of blood cells
(amoebocytes) which obtain from the horseshoe crab
 The lysate protein is the most sensitive substance known
for endotoxins.
 Principle: If the lysate protein (diagnostic reagent) is
added to the test material with endotoxin, there will be
formation of a ‘firm gel’ in < 60 min at 37 ˚C.
 LAL reagent is used to test the presence of
pyrogen in: end product, raw material, medical
supplies

 Three ways of conducting LAL Test

A. Gel-clot End Point Test
B. Turbidimeteric Test
C. Chromogenic Substrate Test
64
 1. Gel-clot Test

 Original method
 The specimen is incubated with LAL of a known
senstivity.
 Formation of a gel clot is positive for endotoxin.

65
 Gel-clot Test …
Steps:
 Equal volumes of test solution and LAL reagent (usually 0.1ml
each) are mixed in depyrogenated test tubes of 10x75 mm size
 Spin gently and keep for incubation at 370C ± 1 0C for 60 min ±
2 min and then carefully remove to observe
 A positive reaction is characterized by the formation of
firm gel that will not collapse on inverting the tube.
 A negative rxn is characterized by absence of a gel OR
formation of a gel that does not maintain its integrity
 Compare with +ve control (Reference Standard Endotoxin, RSE)
and –ve control ///
66
Gel-clot Test …

 Advantages:
 Simple
 Economical

 Disadvantages:
 Subjective nature of its end point
 Semi quantitative
 2. Turbidimetric Method

 During the LAL– endotoxin reaction, the

solution mixture becomes increasingly turbid.
 The turbidity is proportional to the amount
of endotoxin present.
 The endotoxin concentration of a sample can
be estimated by comparing its turbidity to that
of an endotoxin standard curve.

68
 2. Turbidimetric Method…

 Usually more sensitive than gel-clot end point tests

because turbidity occurs prior to gelation, and it is
thus possible to detect endotoxin concentrations
that are lower than needed to form a solid gel clot.
 Turbidity is read by a spectrophotometer.
 Quantitative

Disadvantage: additional cost is incurred for the

spectrophotometer.
 3. Chromogenic Substrate Test

 Endotoxin catalyzes the activation of a

proenzyme in LAL which will cleave a colorless
substrate to produce a colored end product
which can be measured spectrophotometrically
and compared to a standard curve.

70
Chromogenic Substrate Test …
Synthetic substrate is added to the reaction after the pro
enzyme has been activated.
Endotoxin + LAL reagent

Activation of proenzyme
- substrate (as attached to a chromophore)

Chromophore is released

Color change

71
 Intensity of the color produced is
proportional to the amount of endotoxin
present in the test sample.

 Measured by spectrophotometer.

 Advantage & disadvantage – similar to

that of the turbidimetric LAL test.
 Advantages and Disadvantages of LAL Test

 Advantages
 More sensitive
 Less variable
 Quantification possible
 Easier, Not time consuming
 Disadvantages
 Specific to Gram -ve endotoxins
 Interference problems from chemicals
 It can not measure the fever producing potential of
pyrogen in human
73
Thank you
Quiz

1. What is the role of conducting microbial

QC?
2. What activities are performed while doing
microbila limit test and mention the
methods used for this purpose
3. What are the mechanisms applied for
decreasing antimicrobial effect of
components in sterility testing
4. Describe chromogenic substrate testing