SECTION OF BIOENGINEERING

TECHNOLOGY
UNIKL MALAYSIAN INSTITUTE OF
CHEMICAL AND BIOENGINEERING
TECHNOLOGY
ALOR GAJAH, MELAKA

CSB 30603

QA & QC IN BIOPRODUCT

LABORATORY MANUAL

GUIDELINE:

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A. BASIC GOOD LABORATORY PRACTICE

1. Prepare for each laboratory period by reading each exercise and becoming
familiar with the principles and methods involved. By being familiar with the
exercise you decrease the chances of an accident. Also, advance preparation
allows you to use your time efficiently in the laboratory to complete the
experiment.
2. No eating, drinking, or smoking is permitted in the laboratory.
3. Laboratory coats or aprons must be worn at all times in the laboratory. This is to
ensure that culture material is not accidentally deposited on your clothes or skin,
and as a safeguard to protect your clothes and yourself from chemical spills and
stains.
4. Only those materials pertinent to your laboratory work, such as laboratory
manuals, laboratory notebooks, and other laboratory materials, should be brought
to your laboratory work space. All other items, such as coats, books, and bags,
should be stored away from your work area.
5. Begin each laboratory session by disinfecting your work area. Saturate the area
with a disinfectant, spread the disinfectant with a paper towel, and allow the area
to dry. Repeat this procedure after you have finished your work to ensure that any
material you have deposited on the work surface is properly disinfected.
6. All culture material and chemicals should be properly labeled with your name,
class, date, and experiment. Labeling is critical to avoid improper use or disposal
of material.
7. Be very careful with Bunsen burners. To avoid injuries, burners should be turned
off when not in use. When reaching for objects, be careful not to place your hands
into the flame.
8. All contaminated material must be disinfected before disposal or reuse. All
material to be autoclaved should be placed in a proper receptacle for collection.
Used pipets should be placed in disinfectant.
9. After the laboratory session, observe good hygiene by washing your hands before
leaving the laboratory.
10. In the event of any accident or injury, report immediately to the laboratory
instructor so that prompt and proper action can be taken.

EXPERIMENT 1

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 IDENTITY TEST FOR ASCORBIC ACID TABLETS (A BASIC TEST)

INTRODUCTION
The basic tests represent one of the many elements of quality assurance in
pharmaceutical supply system. They have been devised with the following objectives:
a. to provide a simple and readily applicable method for verifying the identity of
a drug substance, using a limited range of easily available reagents, when the
labeling and physical attributes give rise to doubt
b. to provide a practicable means of confirming the identity of a drug, when a
fully equipped laboratory is not available.

OBJECTIVES
a. To perform identity test for different types of ascorbic acid tablet available in
market.

EXPERIMENTAL PROCEDURE
Description. Each tablet usually contains 50 mg of ascorbic acid.
Preparation of the sample
1. Weigh 1 tablet and calculate the amount equivalent to 0.3 0 g of ascorbic acid.
2. Grind the tablets, weigh out the above-calculated equivalent amount as
powdered material and use directly as the test substance, dividing it into 6
equal parts.
3. Shake 4 parts of the test substance with 20 ml of water, filter and use the
fíltrate as the test solution.
4. Suspend 1 part of the test substance in 10 ml of ethanol (~750 g/1) TS. Place 3
strips of filter-paper into the suspension and allow the solution to ascend for
about 4 cm. Take out the strips, cut away the lower dipped portion as well as
the part that has not been wetted by the solution and dry the remaining part of
the strips in air at room temperature (test papers).
Identity tests
a. Heating behaviour.
Heat a small quantity of the test substance in a test-tube; it melts, acquires a
brown colour and has an odour resembling caramel. Ignite the melt; it swells and
burns.

b. Colour and other reactions

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 1. To 2.0 ml of the test solution add l.0 g of sodium hydrogen carbonate R and
20 mg of ferrous sulfate R; a violet colour is produced. Add 2.0 ml of
hydrochloric acid (~70 g/l) TS; the colour disappears.
Alternative test by filter-paper technique:
On a test paper place 1 small drop of sodium hydrogen carbonate (40 g/l) TS,
followed by 1 drop of ferrous sulfate (15 g/l) TS; a violet spot is produced.
Then apply, at the same place on the test paper, 1 drop of hydrochloric acid
(~70 g/1) TS; the spot disappears.
2. To 2.0 ml of the test solution add 0.5 ml of potassium permanganate (10 g/1)
TS; the initial violet colour is immediately discharged but a slight brown
precipitate may appear.
Alternative test by filter-paper technique:
On a test paper place 1 drop of potassium permanganate (10 g/1) TS; the
violet colour is discharged but a brown spot appears.
3. To 2.0 ml of the test solution add 2 – 3 drops of silver nitrate (40 g/1) TS; a
dark grey precipitate is produced.
Alternative test by filter-paper technique:
On a test paper place 1 drop of silver nitrate (40 g/1) TS; a dark grey spot is
produced.

EXPERIMENT 2

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 MICROBIAL QUALITY TESTING OF HERBAL PRODUCTS

INTRODUCTION
In the manufacture, packaging, storage and distribution of pharmaceutical
preparations, suitable means must be taken to ensure their microbiological quality.
The pharmaceutical preparations should comply with the criteria given below:
 Herbal medicinal products consisting solely of one or more herbal drugs (whole,
reduced or powdered).
 a. Herbal medicinal products to which boiling water is added before use.
Total viable aerobic count. Not more than 107 bacteria and not more than 105
fungi per gram or per millilitre.
Not more than 102 Escherichia coli per gram or per millilitre, using suitable
dilutions).
 b. Herbal medicinal products to which boiling water is not added before use.
   Total viable aerobic count. Not more than 105 bacteria and not more than 104
fungi per gram or per millilitre.
Not more than 103 enterobacteria and certain other gram-negative bacteria per
gram or per millilitre .
Absence of Escherichia coli (1 g or 1 ml).
Absence of Salmonella (10 g or 10 ml).

OBJECTIVES
a) To determine total viable aerobic count in herbal products.
b) To determine the presence of E. coli in herbal products.

EXPERIMENTAL PROCEDURE

a. Preparation of sample

Dissolve or dilute 1 g of the product to be examined in buffered sodium chloride-

peptone solution pH 7.0 or in another suitable liquid. In general a one in ten dilution
is prepared. However, the characteristics of the product or the required sensitivity
may necessitate the use of other ratios. If the product is known to have antimicrobial
activity, an inactivating agent may be added to the diluent. If necessary adjust the pH
to about pH 7 and prepare further serial tenfold dilution using the same diluent.

Preparation of Buffered sodium chloride-peptone solution pH 7.0

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Potassium dihydrogen phosphate 3.6 g
Disodium hydrogen phosphate dihydrate 7.2 g, equivalent to 0.067 M phosphate
Sodium chloride 4.3 g
Peptone (meat or casein) 1.0 g
Purified water 1000 ml
To this solution surface-active agents or inactivators of antimicrobial agents may be
added, such as:
Polysorbate 80 or Tween 80 1 g/l to 10 g/l
Sterilise by heating in an autoclave at 121 °C for 15 min.

b. Total viable aerobic count

Pour-plate method  

Using Petri dishes 9 cm in diameter, add to each dish 1 ml of the sample prepared as
described in the section (a) Preparation of the sample and 15 ml to 20 ml of a
liquefied agar medium suitable for the cultivation of bacteria (Nutrient Agar), or 15
ml to 20 ml of a liquefied agar medium suitable for the cultivation of fungi (Potato
Dextrose Agar) at not more than 45 °C. Incubate the plates at 30 °C to 35 °C (20 °C
to 25 °C for fungi) for five days, unless a reliable count is obtained in a shorter time.
Select the plates corresponding to one dilution and showing the highest number of
colonies less than 300 (100 colonies for fungi). Take the arithmetic average of the
counts and calculate the number of colony-forming units per gram or millilitre.

Surface-spread method  

Using Petri dishes 9 cm in diameter, add 15 ml to 20 ml of a liquefied agar medium

suitable for the cultivation of bacteria (Nutrient Agar) or a liquefied agar medium
suitable for the cultivation of fungi (Potato Dextrosa Agar) at about 45 °C to each
Petri dish and allow to solidify. Dry the plates, for example in a LAF bench or in an
incubator. Spread a measured volume of not less than 0.1 ml of the sample prepared
as described in the section Preparation of the sample over the surface of the medium.
Use at least two Petri dishes for each medium and each level of dilution. For
incubation and calculation of the number of colony-forming units proceed as
described for the pour-plate method.

c. Test for specified microorganisms

Escherichia coli

Prepare the product to be examined as described in the (a) Preparation of the sample
and use 10 ml or the quantity corresponding to 1 g or 1 ml to inoculate 100 ml of
Nutrient broth, homogenise and incubate at 35-37 °C for 18-48 h. Shake the
container, transfer 1 ml to 100 ml of MacConkey broth and incubate at 43-45 °C for
18-24 h. Subculture on plates of MacConkey agar at 35-37 °C for 18-72 h. Growth
of red, non- mucoid colonies of gram-negative rods indicates the possible presence of
E. coli. This is confirmed by suitable biochemical tests, such as indole production.

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The product passes the test if such colonies are not seen or if the confirmatory
biochemical tests are negative.

Indole test: Add 5 drops of Kovac's reagent to the culture broth. A positive result is
shown by the presence of a red or red-violet color in the surface alcohol layer of the
broth. A negative result appears yellow.

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 II. HEAVY METAL DETECTION IN HERBAL PRODUCTS

INTRODUCTION

The use of herbal medicines has been increasing rapidly in the last decades world
wide and therefore, a critical evaluation of their safety is extremely important.
Dramatic uses in herbal medicines have prompted Drug Control Authority (DCA),
Malaysia, to implement the phase three registration of traditional medicines on 1
January, 1992. The registration criteria for traditional medicines include limits for
heavy metals (Poison Act 1952, Revised 1989), limits of microbial contamination and
the absence of steroids and other adulterants (Poison Act 1952, Revised 1989), limits
of disintegration time (Pharmacopoeial Standards), claimed indications (Medicines
Act, Advertisement and Sale, 1956, Revised 1983), prohibition of herbs with known
adverse effects, prohibition of endangered animal species (Wildlife Protection Act,
1972) and compliance to good manufacturing practice (GMP) and good storage
practice (GSP). the latest specification for Quality Control of Traditional Medicine
Products for heavy metals are as follows:
1.1 Lead : = 10.0 mg/kg or mg/litre (= 10.0ppm)
1.2 Arsenic : = 5.0 mg/kg or mg/litre (= 5.0ppm)
1.3 Mercury : = 0.5 mg/kg or mg/litre (= 0.5ppm)
1.4 Cadmium: 0.3mg/kg or mg/litre (= 0.3 ppm)

OBJECTIVES

a. To determine the quality of herbal products

b. To quantify the heavy metals contamination in selected herbal products

Methods

Treatment of glasswares
All glasswares are soaked with aqua regia (HCl:HNO 3 = 3:2) (Sastre et al., 2002) for
2 h and then washed with deionized water.

Solution
Acetate buffer (pH=3.5), thioacetamide TS, thioacetamide-basic glycerin TS, and
glycerin basic are prepared according to USP.

Standard lead solution

Lead nitrate (or plumbum nitrate) (160 mg) is dissolved in double distilled water
(ddW) and nitric acid (6N, 1 ml) and is made to volume (100 ml). One ml of this
solution is diluted to 100 ml with ddW to contain 10 μg/ml of Pb.

General screening method

The 3rd method of USP was applied for monitoring the total heavy metal content in
herbal medicines under study. In this method, three different solutions should be
prepared, i.e., a standard, a monitor and herbal sample solutions.

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Standard solution
A mixture of sulfuric acid (8 ml) and nitric acid (10 ml) is transferred to a Kjeldal
flask (100 ml). The mixture is heated until the appearance of white vapors. Double
distilled water (10 ml) is added cautiously and boiled until a white vapor appeared.
After cooling, ddW (5 ml) is added to reach a volume of 2-3 ml, mixed and boiled.
After cooling, ddW (5 ml) and lead standard solution (2 ml, 20 μg Pb2+) are added
and mixed. For color comparison the mixture was transferred to a 50 ml test tube
(TT), the flask is washed, transferred to the TT and the volume is made to 25 ml.

Preparation of the herbal product under study for heavy metal limit test
Herbal sample (2.0 g) is transferred to a dry Kjeldal flask, moistened by a mixture of
sulfuric and nitric acids (8:10 v/v) and heated gently until the reaction started. This
process is repeated until 18 ml of the acid mixture is consumed. The solution is boiled
gently until the appearance of a deep color, and then cooled. Nitric acid (2 ml) was
added, and re-heated until the solution became deeply colored. Heating and adding
nitric acid are repeated until the solution is no more deeply colored. After cooling,
ddW (5 ml) is added and boiled until the appearance of white vapors. The volume is
reduced to 2-3 ml and then cooled. If the solution is colored yellow, it should have
been decolorized using hydrogen peroxide. The solution is transferred to a 3rd TT, the
flask is rinsed by ddW and transferred to the same TT and the volume was made to 25
ml.

Monitor solution
The solution is prepared and processed similar to the preparation method of the
herbal product under study and is then cooled and diluted with ddW. Then, the
standard lead solution (2 ml, 20 μg) is added and mixed. The mixture is transferred to
the 2nd comparison TT, the flask is washed and transferred to the same TT, and made
to 25 ml by ddW.

Heavy metal levels of the herbal products under study

The pH values of the monitor, standard and herbal solutions are adjusted to 3-4 using
dilute ammonia solution. The volumes are made to 40 ml by ddW, acetate buffer (2
ml, pH=3.5) and thioacetamide glycerin base TS solution (2 ml) are added, to each
TT, the volumes are made to 50 ml by ddW, and left for 2 min. By watching of
comparing the solutions against a white surface, the test solution color intensity
should not exceed that of the standard and monitor solutions.

Reagents preparation
1. Acetate Buffer pH 3.5
Dissolve 25 g of ammonium acetate in 25 ml of water and add 38 ml of 7M
hydrochloric acid. Adjust the pH to 3.5 with either 2M hydrochloric acid or 6M
ammonia and dilute to 100 ml with water.
2. Thioacetamide TS
Dissolve 4 g of thioacetamide in 100 ml of water
3. Glycerin base TS
To 200 g of glycerin add water to bring the total weight to 2.3g. add 140 ml of 1N
NaOH and 50 ml of water.
4. Thioacetamide-glycerin base TS
Mix 0.2 ml of thioacetamide TS and 1 ml of glycerin base TS and heat in a
boiling water for 20 s. use the mixture immediately.

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Reference
A Quantitative investigation on some toxic and non-toxic metals in popular medicinal
herbs in Iranian market
Fazel Shamsa, Shamsali Reza Zadeh, Hashim Shamsa and Khosro Abdi
Iranian Journal of Pharmaceutical Research (2009), 8 (2): 95-99

Lead contamination in Eugenia dyeriana herbal preparations from different

commercial sources in Malaysia
H.H. Ang
Food and Chemical Toxicology 46 (2008) 1969–1975

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 GMP PRACTICE

INTRODUCTION

“Good manufacturing practice” or “GMP” is part of a quality system covering the

manufacture and testing of active pharmaceutical ingredients, diagnostics, foods,
pharmaceutical products, and medical devices.
Good Manufacturing Practice is concerned with both production and quality control.
The basic requirements of GMP are that:
1. all manufacturing processes are clearly defined, systematically reviewed in the
light of experience and shown to be capable of consistently manufacturing
medicinal products of the required quality and complying with their
specifications;
2. critical steps of manufacturing processes and significant changes to the process
are validated;
3. all necessary facilities for GMP are provided including:
1 a. appropriately qualified and trained personnel;
2 b. adequate premises and space;
3 c. suitable equipment and services;
4 d. correct materials, containers and labels;
5 e. approved procedures and instructions;

4. instructions and procedures are written in an instructional form in clear and

unambiguous language, specifically applicable to the facilities provided;
5. operators are trained to carry out procedures correctly;
6. records are made, manually and/or by recording instruments, during
manufacture which demonstrate that all the steps required by the defined
procedures and instructions were in fact taken and that the quantity and quality
of the product was as expected. Any significant deviations are fully recorded
and investigated;
7. records of manufacture including distribution which enable the complete history
of a batch to be traced, are retained in a comprehensible and accessible form;
8. the distribution (wholesaling) of the products minimises any risk to their
quality;
9. a system is available to recall any batch of product, from sale or supply;
10. complaints about marketed products are examined, the causes of quality defects
investigated and appropriate measures taken in respect of the defective products
and to prevent re-occurrence.

Therefore this exercise is design with the aim to expose students with the basic
activity involve in GMP production.

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GOAL: Make a batch of hard gelatin capsule

OBJECTIVE:
 Understand the complexity of a GMP process.
 Gain appreciation of teamwork and cooperation of all departments.

HOW IT MIRRORS INDUSTRY:

 You will be frustrated at times
 You will be rushed
 You will feel a sense of accomplishment once you made a batch.
 The actual time for the chemistry/ fermentation etc is very small compared to
the time takes to get all GMP documentation in place.

TEAMS:
Material control: 1 people
QC: 1 people that like to test material
QA: 2 people that have an eye for written details
Production: 2 people that like to work

SUPPLIES:
Department tasks and deliverables
Approved labels
Quarantine labels
Box for approved, quarantine and finished products
Capsule filler
Empty gelatin capsules
Flour

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 HARD GELATINE CAPSULE

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 HARD GELATINE CAPSULE

Capsule filler

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 HARD GELATINE CAPSULE

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 HARD GELATINE CAPSULE

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 HARD GELATINE CAPSULE

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 HARD GELATINE CAPSULE

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 EMPTY HARD GELATINE
CAPSULE

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 WHEAT FLOUR

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 POWDER FILLED HARD GELATINE CAPSULE

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 PROCEDURE OF THE PRODUCT
CHANGEOVER, CLEANING OF
CAPSULE FILLER

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 Capsule Filler

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