10 microbiological control.ppt

Microbiological control of medicines in pharmaceutical

manufacturing and pharmaceutical companies.

Fundamentals of biotechnology and genetic engineering.

Microbiological control of medicines in pharmaceutical manufacturing and

pharmaceutical companies.

Microbial Control Considerations

Product Development Routine Monitoring Water systems and Usage Active Ingredients Equipment Design and Use Conditions Personnel Manufacturing Environment

Guidance and Recommendations for performing a microbiological assessment a microbiological assessment considering a total program of facility, material and personnel management

recommend a program of control for the manufacturing environment rather than control by direct environmental monitoring of the manufacturing area.

•Injectable products (sterile)•Ophthalmic products (sterile)•Inhalation solutions (sterile)•Metered-dose dose and dry powder inhalants and dry powder inhalants•Nasal sprays•Otics•Vaginal suppositories•Topicals•Oral liquids (aqueous)•Oral liquids (non-aqueous)•Rectal suppositories•Liquid-filled capsules•Oral tablets and powder-filled capsules

The order of risk of pharmaceutical products based on the invasiveness of the route of administration:

Microbiological Samplings Methods

Air Sampling: Active Passive Surface Sampling: Contact Plates Swabs Rinse Sampling

Manufacturing facility Appropriate design and layout of the facility : Crucial to

the production of safe and effective medicines Commonly contains : - Specific production of a target drug - Quality control, Storage areas, etc

cf) Injectable bio-drugs : Require unique facility design and operation safety of product

- Clean room technology - Generation of ultra pure water (WFI : water for injection)

- Proper design and maintenance of non-critical areas : storage, labeling, and packing areas

Clean rooms Environmentally controlled areas for injectable/sterile

biopharmaceutricals : specifically designed to protect the product from contamination (microorganisms and particulate matters etc.)

Designed in a way that allows tight control of entry of all substances and personnel (e.g., equipment, in-process product, air etc..)

A basic feature of design : Installation of high efficiency particulate air (HEPA) filters in the ceilings :

- Layers of high-density glass fiber : Depth filter

- Flow pattern of HEPA-filtered air : - Air is pumped into the room via the filters, generating a

constant downward sweeping motion Clean rooms with various levels of cleanliness : - Classified based on the number of airborne particles and viable microorganisms in the room - Maximum permitted number of particles or microorganisms

per m3 of clean room air

Europe : 5 μm particle dia viable microorganisms Grade A : 0 < 1 B : 0 5 C : 2,000 100 D : 20,000 500

USA : class 100 (grade A/B), class 10,000(grade C), class 100,000 (grade D)

Factors affecting the clean room condition Use of HEPA filters with high particulate-removing

efficiency Generation of a unidirectional downward air distribution

pattern (i.e. laminar flow)

Additional elements critical to maintaining intended clean room conditions

- All exposed surfaces : a smooth, sealed impervious finish in order to minimize accumulation of dirt/microbial particles to facilitate effective cleaning procedures

- Floors, walls, and ceilings : coated with durable, chemical-resistance

materials like epoxy resins, polyester, PVC coatings

- Fixtures (work benches, chairs, equipments etc..) : designed and fabricated to facilitate cleaning processes

- Air-lock systems : buffer zone - prevention of contamination - entry of all substances/personnel into a clean room must occur via air-lock systems- An interlocking system : doors are never simultaneously

open, precluding formation of a direct corridor between the uncontrolled area and clean area Generalized clean room design: - Separated entries and exits for personnel, raw materials, and products

- Personnel represent a major potential source of process contaminants: required to wear specialized protective clothing when working in clean area

- Operators enter the clean area via a separated air-lock

- High standard of personnel hygiene

- Only the minimum number of personnel required should be present in the clean area at any given time

Cleaning, decontamination, and sanitation (CDS) CDS regime : essential to the production of a safe and effective

biopharmaceuticals

- Cleaning : removal of “dirt” (organic/inorganic materials) - Decontamination : inactivation and removal of undesirable substances, which generally exhibit some specific biological activity ex) endotoxins, viruses, prions - Sanitation : destruction and removal of viable microorganisms

Effective CDS procedures are routinely applied to : - Surfaces are not direct contact with the product (e.g. clean room walls and floors) - Surfaces coming into direct contact with the product (e.g. manufacturing vessels, product filters, columns)

CDS of process equipment - surfaces/equipment in direct contact with the product : special CDS requirement - no trace of the CDS reagents product contamination Final stage of CDS procedures involves exhaustive rinsing with highly pure water (water for injections (WFI))

CDS of processing and holding vessels as well as equipment that is easily detachable/dismantled (e.g., homogenizer, centrifuge rotors etc.,) straightforward

Cleaning in place(CIP) : large equipment/process fixtures due to the impracticality/undesirability of their dismantling

ex) internal surfaces of fermentation equipment, fixed piping, large processing/storage tanks, process-scale chromatographic column

- General procedure: A detergent solution in WFI, passage of sterilizing live steam generated from WFI

CDS of process-scale chromatography systems : challenging

ex) Processing of product derived from microbial sources : contamination with lipid, endotoxins, nucleic acids, proteins

Water for pharmaceutical processing Water : One of the most important raw materials : used as a basic ingredient - Cell culture media, buffers, solvent in extraction and purification, solvent in preparation of liquid form and freeze-dried products

- used for ancillary processes : cleaning - ~ 30,000 liters of water : production of 1 kg of a recombinant biopharmaceutical produced in a microbial system Generation of water of suitable purity : central to successful operation of facility

Two levels of water quality : purified water and WFI - Outlined in international pharmacopoeias

Use of purified water: - Solvent in the manufacture of aqueous-based oral products (e.g., cough

mixtures, ) - Primary cleaning of some process equipment/clean room floors in class D

or C area, - Generation of steam in the facilities, autoclaves - Cell culture media

Water for injection (WFI) - Highest purity - Extensive use in biopharmaceutical manufacturing

Generation of purified water and WFI Generated from potable water Potential impurities in potable water : Multi-step purification steps for purified water and WFI: Monitoring of each step : continuous measurement of the

resistivity of the water ex) Deionization : anion/cation exchangers Increased resistivity with purity up to 1- 10 MΩ

Filters to remove microorganisms: 0.22 µm, 0,45 µm

Reverse osmosis (RO) membrane : Semi-permeable membrane (permeable to the solvent, water, but impermeable to solute, i.e., contaminants)

General procedure for WFI

Potable water depth filtration organic trap (resin) activated charcoal Anion exchanger Cation exchanger Deionization step : monitored by measuring the water resistivity Filtration with membrane to remove microorganisms - “purified water” Distillation (or reverse osmosis) Water for injection(WFI)

Sterility Testing Sterility test is a quality control test used as part of

product release for product required to be sterile Has significant statistical limitations - will really only

detect gross contamination Sampling

No of containers and volume to be tested defined in Pharmacopoeia

Samples from aseptically manufactured product should be taken from beginning, middle and end of batch fill and also after interventions and stoppages

Samples from terminally sterilized product should be taken from previously identified cool spots within load

Sampling should be sufficient to allow for retests if needed

Sterility Testing Facilities

Sterility testing should be carried out under the same conditions as aseptic manufacture

In a Grade A laminar air flow cabinet in a Grade B background (may also be carried out in an isolator)

Air supply through HEPA filters, pressures should be monitored and alarmed

Access to area should be through airlocks Operators should be appropriately gowned is sterile

garments Operators should be appropriately trained and validated Appropriate cleaning, sanitisation and disinfection

procedures should be in place Environmental monitoring should be conducted

Sterility Testing Methods are defined in Pharmacopoeia

membrane filtration is the preferred method if product is filterable direction innoculation is alternative

Media types Soybean Casein Digest medium (SCD), (also knows as Trypticase

Soy Broth(TSB)) and Fluid Thioglycollate medium (FTM) is usually used (to detect aerobic and anaerobic organisms)

validation studies should demonstrate that the media are capable of supporting growth of a range of low numbers of organisms in the presence of product. May need to incorporate inactivators

growth should be evident after 3 days (bacteria), 5 days (moulds)

media may be purchased or made in-house using validated sterilization procedures

Sterility Testing Media

should be tested for growth promoting qualities prior to use (low number of organisms)

should have batch number and shelf life assigned

Incubation Period At least 14 days incubation 20-25°C for SCD/TSB, 30-35°C for FTM Test containers should be inspected at intervals temperatures should be monitored and temperature monitoring

devices should be calibrated if product produces suspension, flocculation or deposit in media,

suitable portions (2-5%) should be transferred to fresh media, after 14 days, and incubated for a futher 7 days

Sterility Testing Negative Contols

media should be incubated for 14 days prior to use, either a portion or 100% of batch (may be done concurrently with test)

negative product controls - items similar in type and packaging to actual product under test should be included in each test session

facilitate interpretation of test results negative control contamination rate should be calculated

and recorded

Sterility Testing Positive Test Controls

bactiostasis/fungistasis test should demonstrate that media are capable of supporting

growth of a range of low numbers of organisms in the presence of product. May need to incorporate inactivators

growth should be evident after 3 days (bacteria), 5 days (moulds)

Sterility Testing Positive Controls

should be performed on all new products and when any changes are made.

Should be repeated annually Stasis test recommended particularly for product with

antibiotics or preservatives or slow release tested by direct innoculation

demonstrates that media can support growth at the end of the incubation period and has not been affected by product

Results Any growth should be identified (genotypic) Automated/Semi-automated systems used for

identification should be periodically verified using reference strains

Sterility Testing

Interpretation and Repeat TestsNo contaminated units should be foundA test may only be repeated when it can be

demonstrated that the test was invalid for causes unrelated to the product being examined

Sterility Testing Interpretation and Repeat Tests

No contaminated units should be found A test may only be repeated when it can be demonstrated that

the test was invalid for causes unrelated to the product being examined

European Pharmacopoeia criteria (a) the data of the micro monitoring of the sterility test facility

show a fault (b) a review of the testing procedure used during the test in

question reveals a fault (c) microbial growth is found in negative controls (d) after determination of the identity of the microorganisms

isolated from the test, the growth of this species or these species may be ascribed unequivocally to faults with respect to the material and/or technique used in conducting the sterility test procedure

Sterility Testing Interpretation and Repeat Testing

When conditions (a), (b) or (c) apply the test should be aborted If a stasis test performed at the end of the test shows no

growth of challenge organisms, this also invalidates the test For conditions (d) to apply must demonstrate that the

orgamisms isolated from the sterility test is identical to an isolate from materials (e.g. media) and/or the environment

must use genotypic identification methods

Repeat test is carried out with same number of samples as first test

Any contamination detected in repeat test, product does not comply

Other Microbiological Laboratory Issues

Testing of Biological Indicators if tested in-house the method should include a

heat-shock step (this verifies that the indicators do actually contain spores and not vegetative organisms)

BIs should occasionally be tested in house to verify the suppliers count


Endotoxin Testing Parenteral products should be free from

endotoxin Endotoxin is a lipopolysaccharide present in the

cell wall of gram negative bacteria which can cause fever if introduced into the body

Raw materials, WFI used in manufacture and some finished product must be tested for endotoxin

Other Microbiological Laboratory IssuesEndotoxin Testing (2) LAL (Limulus Amebocyte Lysate) test is used for

detecting endotoxin (previously a rabbit test) based on clotting reaction of horseshoe crab blood to

endotoxin Types of LAL test

Gel Clot Turbidimetric Colorimetric

Equipment used in test must be endotoxin free

Validation of accuracy and reliability of the method for each product is essential


Gel Clot Method Original method

The official “referee test”

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

Endotoxin Testing (3)


Turbidimetric Method

A kinetic method


The specimen is incubated with LAL and either the rate of increase in turbidity or the time taken to reach a particular turbidity is measured spectrophotometrically and compared to a standard curve.


Colorimetric Method Endotoxin catalyzes the

activation of a proenzyme in LAL which will cleave a colorless substrate to produce a colored endproduct which can be measured spectrophotmetrically and compared to a standard curve.

Can be kinetic or endpoint



* (Sensitivities vary by reagent manufacturer, instrumentation and testing conditions)

Sensitive down to .001 EU/ml *

Sensitive down to .005 EU/ml

Sensitive down to 0.1 EU/ml

Sensitive down to 0.03 EU/ml

Can be automated,allows electronicdata storage



Manually read and recorded

Requiresincubating plate or tube reader

Requiresincubating plate or tube reader

Requiresspectrophotometeror plate reader

Simple Least expensive,Requires 37 degree bath

QuantitativeQuantitativeQuantitativeSemi-quantitative

TurbidimetricChromogenicKinetic

ChromogenicEndpoint

Gel Clot


Fundamentals of biotechnology and genetic

engineering.

What is Biotechnology?

“The use of microbial, animal or plant cells or enzymes to synthesize, break down or transform materials”.

It mainly depends upon the expertise of biological systems in recognition and catalysis.

The Biotechnology Tree

Biotechnology and Genetic Engineering

•Genes are the fundamental basis of all life.•They determine the properties of all living forms. •Genes are defined segments of DNA. •DNA structure and composition in all living forms is essentially the same.• Any technology that can isolate, change or reproduce a gene is likely to have an impact on almost every aspect of society.•Genetic recombination, as occurs during normal sexual reproduction, consists of the breakage and rejoining of the DNA molecules of the chromosomes, and is of fundamental importance to living organisms for their assortment of genetic material.

The flow of genetic materialRNA and DNA

Bacterial chromosome and plasmid

Bacteriophage

Historical Development of Biotechnology•Sumarians and Babylonians were drinking beer by 6000 BC, they were the first to apply direct fermentation to product development.

• Egyptians were baking leavened bread by 4000 BC; wine was known in the Near East by the time of the book of Genesis.

•Microorganisms were first seen in the seventeenth century by Anton van Leeuwenhoek who developed the simple microscope;

•The fermentative ability of microorganisms was demonstrated between 1857 and 1876 by Pasteur – the father of Microbiology/Biotechnology

•Cheese production has ancient origins, as does mushroom cultivation.•Biotechnological processes initially developed under non-sterile conditions

•Ethanol, acetic acid, butanol and acetone were produced by the end of the nineteenth century by open microbial fermentation processes.

Historical Development of Biotechnology•Waste-water treatment and municipal composting of solid wastes represents the largest fermentation capacity practiced throughout the world.

•Introduction of sterility to biotechnological processes In the l940s complicated engineering techniques were introduced to the mass production of microorganisms to exclude contaminating microorganisms. Examples include the production of antibiotics, amino acids, organic acids, enzymes, steroids, polysaccharides, vaccines and monoclonal antibodies.

• Applied genetics and recombinant DNA technology:Traditional strain improvement of important industrial organisms has long been practiced; recombinant DNA techniques together with protoplast fusion allow new programming of the biological properties of organisms.

Recent developments in BiotechnologyCategory Examples

1 -Medicine

2- Agriculture

3- Chemicals

4- Environment

5- Food

6- Industry

-Production of antibiotics, steroids, monoclonal antibodies, vaccines, gene therapy, recombinant DNA technology drugs and improving diagnosis by enzymes and enzyme sensors.Plant tissue culture, protoplast fusion, introduction of foreign genes into plants and nitrogen fixation.

-Organic acids (citric, gluconic), mineral extraction.

-Improvement of waste treatment, replacement of chemical insecticides by biological ones and biodegradation of xenobiotics.

-Single cell protein (SCP), use of enzymes in food processing and food preservation.

- Use of enzymes in detergent industry, textile and energy production

Genetic engineering The formation of new combinations of heritable material by the insertion of nucleic acid molecules, produced by whatever means outside the cell, into any virus, bacterial plasmid or other vector system so as to allow their incorporation into a host organism in which they do not naturally occur.Princple: DNA can be isolated from cells of plants, animals or microorganisms (the donors) and can be fragmented into groups of one or more genes.Passenger DNA fragments can then be coupled to another piece of DNA (the vector ) and then passed into the host or recipient cell. The host cell can then be propagated in mass to form novel genetic properties and chemical abilities that were unattainable by conventional ways of selective breeding or mutation.

1. DNA is isolated from the cells and purified.

2. Restriction enzymes are used to cut the DNA for cloning.

3. Ligases are the used to join the DNA fragments together.

4. The new cloned plasmid is the transformed into competent cells (Cells treated chemically to allow passage of foreign DNA).

Steps:

Overview of a Biotechnological Process

1- Therapeutic proteins and peptides

A. A- Insulin productionInsulin = protein = 2 polypeptide chains

A chain = 30 amino acids B chain = 21 amino acids

Synthesize A-chain gene and insert into a plasmid

Synthesize B-chain gene and insert into a plasmid

Cloned plasmids are inserted separately into E. coli

Lyse cells and purify the proteins

A chain B chain

A chain B chain Mix and connect

Insulin

Applications in Genetic Engineering

B- Interferons:•Interferons are proteins produced by eukaryotic cells in response to viral infection. They prevent replication of the infecting virus in adjacent cells.

•There are several kinds of interferons each made by a different cell type: •α-Interferon is produced by leukocytes.• -interferon is produced by fibroblasts. • γ-interferon is produced by sensitized T cells.

Interferon can be produced (commercially) by two methods:

1- Cultures of human diploid fibroblasts attached too a solid support.

2- Bacteria in which the interferon gene is cloned and expressed , the interferon is then purified.

•Used for treatment of Hepatitis B and C and many other Cancer and autoimmune diseases.

•PEGylated interferons are interferons conjugated with PEG to allow for slow release inside the body, injected once a week.

C- Human-growth hormone:

•Human growth hormone is another pharmaceutical product made more efficiently by a genetically engineered bacterium.

•Previously the hormone was obtained only in extremely small quantities by extracting it from the pituitary glands of the animals.

•The genetically engineered product is being used to treat children pituitary dwarfism and other conditions related to growth hormone deficiency.

• Production of certain vaccines such as hepatitis B, has been difficult because the virus was unable to grow in cell cultures and the extreme hazards of working with large quantities of the virus which can be obtained from the blood of humans and experimentally infected chimpanzees.

• Using DNA from HBV, it was possible to clone the gene for hepatitis B surface antigen (HBs antigen) into cells of the yeast Saccharomyces cerevisiae.

• The yeast expressed the gene and made HBs antigen particles that could be extracted after the cells were broken.

• Since yeast cells are easy to propagate, it was possible to obtain-unlimited quantities of HBs antigen particles.

• This was the first vaccine against a human disease produced with genetic engineering methods.

D- Hepatitis B vaccine:

• The dye indigo is a plant product but was manufactured chemically to reduce the cost.

• However, it was possible to clone naphthalene oxidase gene from Pseudomonas sp. into E. coli.

• The modified E. coli produced indigo, as the naphthalene oxidase enzyme oxidized indole of E. coli to 2-3 dihydrodiol which spontaneously oxidize and dimerize to indigo resulting in blue E. coli.

• It is the “blue” of blue genes that is why the commercial importance.

2- Chemicals: Indigo dye

•An interesting ecological relationship between bacteria and plants involves the role of Pseudomonas syringae which produce a surface protein initiating ice crystals formation, which results in frost damage to the plant.

•These bacteria are conditional plant- pathogens, causing death due to frost damage only at temperatures that can initiate the freezing process.

• A genetically engineered ice-minus strain (with the surface protein deleted) is sprayed to replace the indigenous population and protect the crop.

•The release of genetically engineered raised environment questions.

3- Construction of new microbesIce-minus Pseudomonas syringae:

The key control gene for an important product can be identified and manipulated to be insensitive to repression.

The manipulated gene is cloned and reintroduced at a high copy number.

Ex: The genes of antibiotic-producing organisms.

4- Improvement of performance and productivity:

5- Protein engineering:Knowledge of the tertiary structure of an enzyme with knowledge of its DNA sequence can enable the rational modification of the molecule to produce the desired change such as substrate specificity and temperature stability.

Substitution of certain amino acid at a specific position can be achieved by site-directed mutation in the cloned gene.

• Transgenic animals: Transgenesis is the use of gene manipulation to permanently modifying germ cells of animals.

• For example the production of super mice was a result of the over-production of human growth hormone.

• Over-expression of growth hormone has also been tried in order to increase the rate of growth of livestock, poultry and fish.

• Production of foreign proteins in transgenic farm animals find a more significant progress.

• For example α1-antitrypsin, a protein used as replacement therapy for genetically-deficient individuals at risk from emphysema, have been produced in transgenic sheep. The compound is excreted in their milk.

6 -Modification of macroscopic animals:

7- Plant biotechnologyIntroduction of genes into plant that enables the plant to degrade or detoxify the herbicide

Herbicide tolerant crops:To allow the use of non-selective herbicides to remove all “weeds” in a single and quick application.Advantages: Less spraying, less traffic on the field, and lower operating costs.

Genetically Modified Products

Genetically engineered Tomatoes with reduced polygalacturonase enzyme. This enzyme is involved in softening and over ripening of tomatoes.

Advantages:Faster growth, better yield ,quality and longer shelf life)

Gene Therapy

Any treatment strategy that involves the introduction of genes or genetic material into human cells to alleviate or eliminate disease.

The aim of gene therapy is to replace or repress defective genes with sequences of DNA that encode a specific genetic message.

Within the cells, the DNA molecules may provide new genetic instructions to correct the host phenotype.

Ex Vivo gene therapy:

What factors have kept gene therapy from becoming an effective treatment for genetic disease?

1- Short-lived nature of gene therapy Problems with integrating therapeutic DNA into the genome and the rapidly dividing nature of many cells prevent gene therapy from achieving any long-term benefits. Patients will have to undergo multiple rounds of gene therapy.

2- Immune response Anytime a foreign object is introduced into human tissues, the immune system is designed to attack the invader. The risk of stimulating the immune system in a way that reduces gene therapy effectiveness is always a potential risk.

3- Problems with viral vectors Viruses, while the carrier of choice in most gene therapy studies, present a variety of potential problems to the patient --toxicity, immune and inflammatory responses, and gene control and targeting issues. In addition, there is always the fear that the viral vector, once inside the patient, may recover its ability to cause disease.

4- Multigene disorders Conditions or disorders that arise from mutations in a single gene are the best candidates for gene therapy.

Unfortunately, some the most commonly occurring disorders, such as heart disease, high blood pressure, Alzheimer's disease, arthritis, and diabetes, are caused by the combined effects of variations in many genes.

Multigene or multifactorial disorders such as these would be especially difficult to treat effectively using gene therapy

10 microbiological control.ppt

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