INDUSTRIAL

MICROBIOLOGY

PRESENTERS
1. MTWEVE, Luka v
2. JAFFU, Dyness R
3. DARAJANI, Shaidu
OUTLINE

This presentation will cover the following

1. Good Manufacturing Practice
2. Aseptic technique – design and function
3. Pharmaceutical products from microorganisms
4. Recombinant DNA pharmaceutical production
Introduction
• Quality in pharmaceutical products is taken to
mean “fitness for purpose”
• Have desired therapeutic properties
• Safe for administration by route intended
• A Quality product is therefore free from
“Manufacturing faults”
• Manufacture-complete cycle of production of
the medicinal product:
• Raw material  processing  packaging, 
distribution
• Quality assurance-The total sum of the
procedures needed to ensure the fitness for a
pharmaceutical product for its intended use.
• QA is a wide-ranging concept covering all matters
that individually or collectively influence the quality
of a product.
• It is the totality of the arrangements made with
the object of ensuring that pharmaceutical
products are of the quality required for their
intended use.
• It incorporates GMP and other factors
• GMP is part of QA aimed at ensuring the
product is consistently manufactured to
the quality appropriate for intended use
and meets regulatory requirements.
• Requires that:
• The manufacturing process fully defined
before it starts
• The necessary facilities are provided.

GMP monitors the fulfillment of

predetermined specifications
Exp: Normal Saline Specifications as per BP
• GMP is aimed primarily at diminishing the
risks inherent in any pharmaceutical
production.
• Such risks are essentially of two types:
• Cross-contamination (in particular of unexpected
contaminants) and
• Mix ups (confusion) caused by, for example,false
labels being put on containers.

(WHO GMP guideline)

• Quality control QC is that part of GMP
concerned with sampling, specifications and
testing, as well as the organization,
documentation and release procedures which
ensure that the necessary and relevant tests
are carried out, and that materials are not
released for use, nor products released for
sale or supply, until their quality has been
judged satisfactory.
• For sterile include sterility and pyrogen testing
• QC must be independent of production
• In process control-Any test on product,
environment or the equipment that is used
during the manufacturing process.

• Validation-A documented programme that

provides high assurance that specific process,
method or system will consistently produce
results meeting predetermined acceptance
criteria.
 GMP
• There should be a comprehensive system to
provide assurance that products will consistently be
of a quality appropriate to their intended use.
• GMP ensures that quality work is built in to all
stages of the manufacturing process, including:
• Selection of ingredients,
• Cleaning of equipment,
• Suitability of plant construction,
• Process validation, etc.
Why GMP?

• Selective testing of products after

manufacture is not considered to be a reliable
method can only test sample, not everything
• Poor chance that patient will detect if
anything wrong
• Potential danger to patients using product
• High assurance of overall product quality
comes best from a detailed specification, and
control and monitoring of all stages that
contribute to the manufacturing process
before manufacturing starts.
GMP Requirements
• Qualified staff do the work after appropriate
training.
• The premises are suitable for the intended
purpose and maintained accordingly.
• Suitable equipment and services are provided
(eg. easy cleaning / sterilisation).
• Quality starting materials are provided with low
bioburden (low bacterial content).
GMP Requirements

• Validated procedures for all production steps.

• Environmental monitoring and in-process
testing procedures are in place.
• Detailed equipment and process records are
maintained .

NOTE:
GMP (If well implemented) gets things right all
along the line!!
Sources of Contamination.
• Contamination means the introduction of a
microorganism either during:
• manufacture (from raw materials, personnel
or process) or
• after opening the product.
• In pharmaceutical manufacturing,
microbiological contamination comes from a
number of factors in the environment.
 Sources of Contamination.
1. Atmosphere.
• Suspended spores, moulds and yeasts contaminate
most air supplies. Dusty environments are more
heavily contaminated.
• Clean rooms and surfaces have lower levels of
atmospheric contamination.
• Personnel movements cause air currents and also
liberate contaminated particles from hair, skin,
breathing, coughing, sneezing, etc. Clean body
coverings will reduce contamination results.
Sources of Contamination.

Addressing:

Air contamination can be controlled by:

• Dry air filtration through polytetrafluorethylene
(PTFE) or ultimately,
• High efficiency air filters (HEPA).
• Routinely checking for integrity of Clarifying
filters
 Sources of Contamination.
2. Water.
• Water can be used in several ways in manufacturing
processes; as a constituent, for washing or as a
coolant.
• It is always surprising how many microbes exist in
even clean water!
• Levels of contamination must be in line with the
desired outcomes.
• Raw or supply water is generally used only for
washing.
• Distilled water is close to sterile out of the still and
should be used promptly before contamination levels
accumulate.
 Sources of Contamination.
Addressing
• Reverse Osmosis: water is prepared by forcing water
through a semi-permeable membrane at pressure.
• Distillation
• Outcome: water with low contamination levels
• Note: Use as soon as possible after
preparation
• Keeping water for longer periods at a temperature
above 65 ° C (typically 80 ° C) to prevent bacterial
growth with consequent pyrogen production.
• Vessels and pipes for storage / transport of water
should have smooth surfaces for easy cleaning /
decontamination. “Dead legs” should be avoided.
• Membrane (0.22 µm) filtration after prior clarification.
 Sources of Contamination.
Chemical decontamination (of water) may be
done as follows:
• Hypochlorite at 0.5 -1.5 ppm at controlled pH
around neutral. Higher concentration up to 5
ppm used in some industrial processes.
• 1% HCHO (formaldehyde) is a general
disinfectant for water.
• UV light at 254 nm is satisfactory for good
optical grade water over very short distances.
UV tubes need to be checked regularly for
calibration.
 Sources of Contamination.
3. Operators.
• Staph. aureus and other common skin pathogens can
cause serious contamination of products.
• Microorganisms from wounds, nasal passages or faecal
sources must be reduced to an absolute minimum.

Addressing:
• Staff training is the most appropriate solution.
• Foot-operated taps on wash basins, antiseptic soaps
and hot air driers assist control.
• Complete coverage with sterilised clothing coupled with
gloves after hand washing is recommended practice for
controlled areas.
 Sources of Contamination.
4. Raw materials.
• Sometimes raw materials from
• Animals e.g. gelatine, pancreas.
• Plant e.g. Gum acacia, tragacanth, agar, starch
• Type of contamination varies with the source and
include bacteria and fungi
• Appropriate treatments must be designed to
address these as needed.
• Synthetic raw materials are usually free from
microbial contamination
 Sources of Contamination.
5. Packaging.
• The package has to: contain the product, exclude
contaminants as well as permit identification via
label.
• Microflora of packaging depend on type:
Packaging Material Type of contaminant
Cardboards Mold
Glass Glass spicules
Plastic Mold
Cork, Rubber Mold
 Sources of Contamination.
Addressing.
• A sterilisation step after sealing will complete
some products but not eye and ear preparations.
• Vials, liners and seals may need to be sterilised
separately.
• Containers with narrow openings or single dose
containers reduce contamination on the
consumer’s hands.
 Sources of Contamination.
6.Walls and Floors.
• Poor ventilation and soft paints on walls leads to
poor results.
• Smooth, easy-to-clean surfaces and shiny gloss
paint helps reduce contamination.
• Joints in surfaces must be impervious and smooth.
• Floor-to-wall junctions must be coved.
• No ledges, horizontal surfaces or overhead pipes
should be present in manufacturing areas.
• Lagged pipes must be avoided.
 Sources of Contamination.
7. Equipment.
• Should be easy to clean, smooth surfaces and easy
to dismantle for cleaning / decontamination.
• The requirement for clean, decontaminated
equipment and its contribution to quality products
cannot be over-emphasised.
 ASEPTIC
MANUFACTURING
Introduction
• Many products for human use must be
provided in a sterile condition.
• Terminally sterilised products. The product is
sealed in its final container and then sterilised,
usually by heat but irradiation may be used
(Terminally sterilized).
• This method reduces the subsequent sterility
risks
• It is validated with good sterility assurance level
• However it is not suitable for heat sensitive
material, thus necessitating the aseptic
production
Introduction
• Aseptic technique refers to a set of specific practices
and procedures performed under carefully controlled
conditions with the goal of minimizing contamination.
• Performed in medical, laboratory and in certain
pharmaceutical productions.
• In pharmaceutical production, the components must
be sterilised prior to being placed in sterile containers
under strict aseptic conditions.
• E.g. Vaccines...Inactivated (killed) /Live attenuated
vaccines.
• This is achieved using adequate condition and
facilities designed to prevent microbial
contamination
Terminal sterilization Aseptic Manufacturing
 Design of aseptic areas
• This specialised work must be done in a purpose-
built unit, separated from other manufacturing
areas and staff thoroughfares.
• The aim is to ensure that the entry of
microorganisms / contaminated materials is
minimised.
• The facility design should separate each stage of
production while allowing a safe and organised
workflow.
• Completed products must be held in a separate
quarantine area within the facility while awaiting
the results of sterility testing.
• Walls and ceilings must have smooth,
impervious surfaces to minimise dust
accumulation, allow easy cleaning and
decontamination.
• Junctions between walls, ceilings and floors
must be coved for the same reasons.
• Floor coverings should be welded sheets of
polyvinyl chloride without cracks.
• Windows must not be openable.
• Cupboards and other essential furniture must
not interfere with the intended flow of
filtered air.
• Plumbing and other fittings -Stainless steel or
laminated plastic.
• Trolleys- smooth surfaces and easily cleaned
and disinfected. Stainless steel.
• Lighting must be adequate and fittings
recessed in false ceilings.
• Electrical switches and sockets must be flush with the
wall.
• Pipes that bring liquids or gases into the units must be
sealed through the walls.
• Drains should be avoided as these are specialised areas
where spills are expected to be rare. Sinks should be
avoided.
• Air pressure differentials must be at least 15 Pa
between adjacent areas. Twenty air changes / hour are
usual in aseptic clean rooms.
• Dispensing areas are usually equipped with laminar
flow cabinets through which HEPA filtered air is passed
over the product and past the operator. Airflow rates of
0.45 m / sec are usual.
• SEQUENCING OF ENTRY AND FLOW OF MATERIALS
 SEQUENCE OF ENTRY PROCEDURES /
WORKFLOW IN CLEAN AND ASEPTIC UNITS.

ZONE PRESSURE DESCRIPTION

BLACK ROOM +15-25 Pa Removal of domestic clothing.
GREY ROOM +25-40 Pa • Washing hands & arms
• Putting on sterile head wear.
• Rewash hands and arms and
dry.
WHITE ROOM +35-40 Pa Suiting room for putting on suit,
gloves, over boots, etc.
ASEPTIC +50 Pa Lowest contamination level;
FILLING staff ready for work
• Design and workflow in
typical sterile products
manufacturing facility.
• The changing area using the
black (A), grey(B), white (C)
principle serves two units:
• Clean areas, 4i-4iv, for
terminally sterilised products.
Staff proceed via A and B.
Some products are finished
and exit the unit (to the left).
Others pass through a double-
ended autoclave (steam
steriliser) to the aseptic unit
(box 5). Alternatively,
ingredients may be sterilised
by filtration before entering
the aseptic unit.
• Aseptic area, 3, for
sterile dispensing. Staff
proceed via A, B and C
(see table below). Pre-
sterilised articles may be
passed through the
double-doored
hatchway, area 6.
• For HEPA filtered air
supply, air enters the
most critical zone, area
3, and flows through
areas 6, 4, B2 and A2.
 Personnel attire and movement
• Clothing worn in clean / aseptic areas must be
made of non-shedding fibres (terylene) and be
close fitting at the neck, wrists and ankles.
• In clean areas, clothing should be clean each
day, but fresh gloves, headwear and overshoes
each time they are removed.
• The hair is a major source of particles and
microbial contamination so must be covered at
all times.
Personnel attire and movement
• In aseptic areas, complete, fresh sterile
clothing should be provided each time a
person enters.
• Staff entry to clean and aseptic areas are
through interlocking doors to prevent the
influx of non-sterile air.
• Regular cleaning and disinfection procedures
must be implemented.
Personnel attire and movement
• Containers made from paper, cardboard or other
fibrous materials should not enter the clean or
aseptic areas.
• Any articles entering the aseptic area must be pre-
sterilised.
• Speed, accuracy and economy of movement are
essential elements of good aseptic technique.
• Thus workers must be well trained, motivated and
have a good understanding of the tasks to be done.
• Double-doored autoclaves, hatches and change
rooms must be designed so that only one door may
be opened at a time.
Personnel attire and
movement
• Speed, accuracy and economy of movement
are essential elements of good aseptic
technique.
• Thus workers must be well trained,
motivated and have a good understanding of
the tasks to be done.
• Double-doored autoclaves, hatches and
change rooms must be designed so that only
one door may be opened at a time.
 INDUSTRIAL
PRODUCTS FROM
MICROORGANISMS

46
• Industrial microbiology uses microorganisms grown in
large scale to produce valuable commercial products
or carry out important chemical transformations.
• The major organism used are fungi (yeast and molds)
• Certain prokaryotes in genus Streptomyces
• For high yields genetic manipulation e.g.
mutation/recombination have been used e.g. P
chrystogenum from 60mg/ml to 85,000mg/ml.

47
• Important exploitation of micro-organisms
include:
• Food products eg beers and wines; food additives
• Pharmaceuticals products production
• Recombinant DNA industry Technology
• The products include both cells (e.g. yeast)
and substances made by the cells (e.g.
enzymes, alcohol)

48
• Pharmaceuticals products include:
1. Antibiotics
2. Vaccines
3. Hormones
4. Vitamins and amino acids
5. Enzymes - table
6. Chemicals e.g. Butanol, gluconic acid and citric
acid
7. Polymers e.g. Dextrans-table 26.1 H&R

49
Dextrans
• Dextrans are high molecular weight polysaccharides
produced by Leuconostoc species. ( Gram-positive
bacteria of family Lactobacillaceae)
• Used as plasma substitutes by intravenous infusion to
maintain blood volume.
• Dextrans are long chain glucose polymers 15,000 to
20,000,000 daltons.

50
Vitamins and amino acids
• Vitamin B12 (cyanocobalamin) is produced by yeast and
many fungi / moulds. After extraction, it is a
component of many vitamin supplements. (Lack of
vitamin B12 causes pernicious anaemia).
• Vit B2 (riboflavin) by mold and bacteria.
• Biotin is a B-group vitamin once extracted from yeasts
but now often chemically synthesised.
• Essential amino acids produced from large scale
fermentations include glutamic acid, aspartic acid,
phenylalanine and lysine. Used e.g. for parenteral
nutrition.

52
Hormones
• Steroids an be produced by complete chemical
synthesis but this complicated and expensive.
• Certain steps carried out more efficiently by
microorganisms! Therefore production of steroid
involve a microbial step.
• Steroid produced in process called biotransformation
i.e. the sterol precursor is bio-transformed to the sterol.
• Rhizopus nigricans used in the production of
hydrocortisone and cortisone.

54
Iron chelating agents

• Many microorganisms need Fe for growth. The human

body has a capacity to recycle Fe so strongly that many
Fe-requiring bacteria are unable to grow in the body.

• In iron-deficient environments, the organisms secrete

iron-chelating agents called SIDEROPHORES.

56
• Desferrioxamine is from a Streptomyces species. It
has a very strong binding constant for Fe+++, in excess
of 1030 .
• It is used for treating accidental cases of Fe poisoning.
• Also used for removing excess Fe from the
blood in cases of haemolytic anaemia such as
thalassaemia.
• Some cancers also require iron, so it is possible
in the future that products like desferrioxamine
may have a role in anti-cancer therapy.
58
Enzymes
• Organisms can produce enzymes released in the
medium called exoenzymes which are capable of
digesting polymers such as cellulose, protein and starch.

• Produced by both fungi and bacteria.

• Include proteases, amylases, lipases, reductases etc
(used in laundry detergents).

• In health, Streptococcus produce an enzyme,

streptokinase, that can convert plasminogen (a normal
component of blood) to plasmin a proteolytic enzyme
that can dissolve mammalian blood clots.

59
Enzymes
• Streptokinase is used to treat patients suffering from
blood clot disease such as arterial occlusions,
myocardial infarction, deep vein thrombosis, etc.

• Streptodornase is produced similarly and acts to

dissolve pus in chest cavities, etc.

• Both enzymes may be dispensed together for a

combined effect.

• Neuraminidase- From V. cholerae strip cells of sialic

acid residues. Can be used to increase immunogenicity
of tumor cells.
60
Other enzymes
• L-asparaginase. Asparagine is an essential amino acid
requirement for some tumour cells but much less for
normal cells.
• The enzyme can therefore be used to deplete some
leukaemia cells of asparagine and so slow tumour
growth.
• β-lactamase causes a problem for antibiotic
manufacture but can be useful in other circumstances
e.g.:
• E.g. Aid in isolation of the infecting organism from a
patient who is being treated with penicillin or
cephalosporin for a urinary tract infection.
• Sterility testing of certain antibiotics
• Treating patients showing severe allergy to 61penicillin
Recombinant DNA pharmaceutical
products

63
In tro d u c tio n

• Genetic engineering involves altering DNA molecule

outside an organism them making the resultant
DNA work in living cells
• Such cells can produce substances of medical importance
• Pharmaceutical biotechnology involve use of living
microorganisms to create new pharmaceutical
products, or safer or more effective version of what
is available
• Include-new recombinant drugs
• Development of subunit vaccines, attenuated
vaccines, DNA vaccines
• Potential gene therapy- i.e. modify genetic material
of living cells to prevent, control or cure diseases
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DNA Manipulating techniques.

1. Cutting and joining DNA molecules.

• DNA isolated from any type of cell can be
fragmented using restriction endonuclease. The
enzyme act at specific point called Restriction
point. Two types of end can be formed after
fragmentation i.e. blunt end and cohesive/sticky
end
• DNA fragments obtained by restriction enzyme
digestion can be covalently joined together using
the enzyme DNA ligase.
2. Cloning vectors.
• After ligation the genes present in DNA fragments
can be maintained and expressed by inserting
them into vectors. These vectors have the ability
to self-replicate in the host cell.
• Types of cloning vectors.
• Plasmid
• Bacteriophage
• Cosmid
• Bacterial artificial chromosome (BAC)
Biotech in Pharm. Industry
• Development of subunit vaccines, attenuated
vaccines, DNA vaccines
• Potential gene therapy- i.e. modify genetic material
of living cells to prevent, control or cure diseases
• Gene coding genetic engineering of important
proteins allows commercial application of this
technology in pharmacy.
• Bacteria are cheap and can be grown large scale in
fermentors and synthesize large quantities of
protein.
• Examples of recombinant drugs:
68
 Egs of clinical important proteins
S/N Protein Source Application
1 Human insulin E.coli Treat diabetes mellitus
2 Human E.coli HGH deficiency in
somatotropin children
3 Interferon α2a E.coli Various cancers and viral
and α2b diseases
4 Interferon γ1b E.coli Chronic granulomatous
disease
5 Tissue E.coli, yeast, Acute myocardial infarct
plasminogen animal cells and pulmonary embolism
activator
6 Interleuckin-2 E.coli, kidney carcinoma and
animal cells metastatic melanoma
69
S/N Protein Sourse Aplication
7 Human serum Yeast In hypovolemic shock and
albumin adjunct in haemodialysis
8 Factor VIII Mammalian Treatment of
cells haemophilia
9 Factor IX Mammalian Treatment of
cells haemophilia B
10 Erythropoietin Mammalian Treatment of anaemia
cells associated with dialysis
and AIDS
11 Hep B surface Yeast, Vaccination
Ag mammalian
cells
12 Influenza virus Insect cells Vaccination
hemaglutinin

70
Carcinogen and mutagen testing
• Carcinogens and mutagens cause genetic changes in
DNA and so are potentially harmful.
• They may cause point mutations in DNA, or major
changes at the transcription or chromosome levels.
• As well as ‘forward’ mutations that change a wild type
organism to a new genotype, there are ‘backward’ or
reverse mutations that can return a mutant organism to
its wild genotype.

71
• In the modern world, a vast number of food additives,
drugs, and other products must be tested prior to
registration to ensure that are not carcinogenic.
• This reverse mutation principle is used in the widely
accepted Ames Test.
• This test is used to test a wide variety of compounds.
• Known carcinogens such as cigarette smoke, aflatoxin,
mitomycin C, vinyl chloride, adriamycin, etc. all yield a
positive Ames test result.

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Ames Test
• In simple form, several carefully studied mutants
derived from Salmonella typhimurium (or Escherichia
coli) are used that contain mutations in the cluster of
genes that synthesise the essential amino acid
histidine.
• A large number of cells from these mutant strains are
treated with the suspect carcinogen.
• Growth in medium without histidine allow revertant
colonies to be detected on agar plates.

73
Ames Test
• Colonies grown from individual cells that have
reverted (changed back) to the wild type property /
genotype and can make their own histidine.
• The frequency or rate at which revertants appear gives
an index of the carcinogenic properties of the test
compound.
• It is not a perfect test, but combined with other
information, provides a cheap, quick indicator for
further investigations.

74
 Phenylketonuria (PKU) testing
• PKU is an inborn error of metabolism; phenylalanine
cannot be converted to tyrosine and consequently, phenyl
ketones accumulate in the urine.
• Unless addressed, babies with PKU will become mentally
retarded. Diagnosis of PKU at birth is important.
• This can be done microbiologically; Bacillus subtilis
growth is inhibited by β-2-thienylalanine but this can be
reversed in the presence of phenylalanine or the phenyl
ketone.
• Using a sample of urine or blood on a filter disk, the test
can be quantified by measuring the diameter of bacterial
growth on an agar plate.
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Diagnostics using recombinant DNA technology

1. Diagnosis of infectious diseases

• This uses two major techniques
• DNA hybridization technique
• involves the formation of double stranded DNA
molecules from single stranded DNA or RNA
sequence through base pairing (Dx: TB HBV, HCV)
• Polymerase chain reaction (PCR) – involve
amplification of specific DNA sequences
• Infectious diseases identified by PCR are like
malaria, chagas disease, AIDs, gastroenteritis
e.t.c.
2. Diagnosis of genetic disorders (hereditary
disease)
• The major technique used in diagnosis of genetic
disorder is PCR
• Hereditary diseases identified by PCR are like
hemophilia A and B, familial
hypercholesterolemia e.t.c.
Reference

• Hugo and Russell’s (2011): Pharmaceutical Microbiology, 8th Edition

• Page (381 – 459)