principles of aseptic processing
TRANSCRIPT
PRINCIPLES OF ASEPTIC PROCESSING
Jim HardyGHGBioSciences, [email protected]@gmail.com
Agenda Background Microbiology Basics Contamination Control
Sources of Contamination
Cleaning & Disinfection 4 Pillars of Aseptic
Techniques
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What are Aseptic Practices?Definitions
Aseptic = without microorganismsA methodology that prevents the introduction
of unwanted organisms into an environment.A practice that removes or kills
microorganisms from hands and objects.
Sterile = the complete destruction of all forms of microbial life, including bacterial spores.
The meaning of this word is absolute; there is no such thing as "partially sterile“
Something is either sterile or non-sterile
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Aseptic TechniquesHistory & Milestones
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1600 1700 1800 1850 1900 2000
Microscope
Iodine
Carbolic Acid
HEPA
19th Century SurgeryIgnaz Semmelweis
Useful:-E.coli for production of rDNA/insulin
-Aspergillus for production of penicillin
-Production of foods, for example:
-Dairy industry: Streptococcus thermophilus to make cheese
-Yeast in beer / wine making
-Bakers yeast
Harmful:-contamination
-food decay
-contamination of medication
-germs
When are Aseptic Practices Used?
Sampling Raw Materials for Qualification Release Sampling of Utilities (WFI, pure steam,
compressed air, and specialty gases) Bioburden & Sterility Testing Environmental Monitoring Manufacturing in classified areas (i.e. cleanrooms) Compounding/Formulation Fill/Finish Operations
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Good Aseptic PracticesRegulatory Aspects
21 CFR 211 211.28 (b) Personnel shall practice good sanitation and health habits. 211.113 (b) Appropriate written procedures, designed to prevent microbiological
contamination of drug products purporting to be sterile shall be established and followed.
211.84 (c) Sterile equipment and aseptic sampling techniques shall be used when necessary.
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To ensure our drug products are safe, pure, and effective
Eudralex Vol. 4 Part 1, Ch. 2 Personnel working in areas where contamination is a hazard, e.g. clean areas or areas
where highly active, toxic, infectious or sensitizing materials are handled, should be given specific training. (2.10)
EC Guide to Good Manufacturing Practice Revision to Annex 1: Manufacture of Sterile Medicinal Products The manufacture of sterile products should be carried out in clean areas entry to which
should be through airlocks for personnel and/or equipment and materials. Clean areas should be maintained to an appropriate cleanliness standard and supplied with air which is passed through filters of an appropriate efficiency.
Microbiology and Microorganisms
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Why is Microbiology Important?
Defined as “the study and science of microorganisms” Most microbial contaminants are not pathogens;
however, their presence in drug product and raw materials can affect the safety, purity, and efficacy by: Causing turbidity Causing product degradation Shifting the pH Introducing endotoxins and other toxins
Microbial testing (bioburden) is performed to ensure that an adulterated product does not reach the patient
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Types of Microorganisms Bacteria Fungi
YeastMold
Mycoplasma Microscopic algae Viruses
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T4 VirusMycoplasma Bacteria
Saprolegnia Mold E Coli Bacteria
Bacteria Prokaryotes: Unicellular microorganisms with no
nucleus & rarely have membrane-bound organelles Ubiquitous to every habitat on Earth 0.5 -1.0 microns in length : 3 million can fit
into an area the size of a pin head Wide range of shapes, spheres, rods, spiral Approximately 10X as many bacterial cells as human
cells in the human bodyLarge populations on the skin and in digestive tract
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Bacterial Reproduction
Binary FissionOne cell divides into
two identical cellsA single bacterial cell can
multiply to 9 x1030 in a 24-hour period in ideal conditions
Refrigeration slows bacterial growth, but does not stop it completely
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Bacterial Binary Fission
E.coli can double their number every 20 minutes!!
Fungi
Eukaryotic organismA cell that has a complex structure
enclosed within a membraneContains a nucleus Ubiquitous to every habitat on Earth
TypesYeastMold
Can be multi-cellular (mold) or single-celled (yeast)
Reproduction is sexual or asexualCommonly via spores
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Mold
Yeast
Yeast Unicellular (single-celled) fungi Average size = 3 - 4 µm in diameter Both aerobic and anaerobic respiration Ideal growth conditions:
Neutral pHTemperature 10˚ – 37˚ C
Part of normal flora of human body Reproduce asexually by budding or
sexually by spores like other fungi Converts sugar into alcohol = alcohol
tolerant
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Budding Yeast Cells
Yeast Cells with Spores
Molds Multi-cellular filaments called “hyphae”
A colony of hyphae is called a mycelium Functions as a decomposer Ability to survive extreme temperature and
pH Visible as a downy or furry coating on food
or surfaces Reproduce through small spores
Spores can be asexual (mitosis) or sexual (meiosis)
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Aspergillus niger
Penicillium
Microbes and the Human Body
Where are microbes found on humans?SkinEyesNoseMouthUpper throatIntestines
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Hands10,000 -100,000 cm2
Groin1-20 million / cm2
Feet1 million / cm2
Scalp~ 1 million / cm2Forehead
100 -1,000 / cm
Saliva~ 10 million / gm
Nasal Fluid~ 10 million / gm
Armpit1-10 million / cm2
Feces>100 million / gm
Identification of Microorganisms Why?
Identify sources of contamination Identify trends in the environment Monitor disinfectant effectiveness
How?1. Appearance or morphology2. Gram staining to identify Gram-negative or Gram-positive bacteria3. Secondary tests to identify families4. Biochemical tests to further speciate (Vitek and API)5. Genetic sequencing analysis (MicroSeq)
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Fewer than 1% of the world’s microbes have been identified!!
Morphology: Size, ShapeTexture, Color
Bacterial Identification Gram Positive Microorganisms
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Gram Positive Cocci Staphylococcus aureus
Gram Positive Rods Bacillus sp.
Bacterial Identification Gram Negative Microorganisms
A source of endotoxin
A single E Coli can contribute 2 million Endotoxin Molecules!
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Gram Negative Rods Escherichia coli
Gram Negative Cocci Neisseria gonorrhoeae
Endotoxin
Cell Wall
Contamination Control
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What is Contamination?The introduction of undesirable impurities
into an environment Viable (living) Contamination:
Examples: bacteria, yeasts, and molds
Non-Viable Contamination: Examples: lint, dust particles, skin flakes, hair, pollen, smoke, chemical
substances, etc.
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ContaminantsCommon Particulates
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Fungi
Water Vapor Smoke
Bacteria
Skin Flakes
Influenza Virus
Contamination Control
Contamination – any effect or action that has a negative impact on a product's integrity making it unfit for useChemical composition pH Sterility & Pyrogenicity Biological or Therapeutic Potency Physical appearance Particulate Matter (e.g. dust, glass or precipitation)
Importance of Aseptic TechniqueParenteral (needle injection) administration bypasses the skin
and gastrointestinal tract, the body’s natural barriers to infectionGiving a patient a contaminated product can cause serious
adverse events including DEATH
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ContaminationSources
Personnel (most common)○ Touch Contamination○ Skin / Hair / Mucous
Membranes○ Clothing
Equipment Improper Cleaning
/Contaminated SuppliesAir
○ HVAC / HEPA Failure○ Infiltration○ Internal Generation
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Particle GenerationPersonnel
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NOTE: Men shed more particles than women
Activity Particles Generated
Standing or Sitting 100,000
Slight head/hand movement
500,000
Body, arm movement w/ toe tapping
1 million
Changing from sitting to standing
2.5 million
Slow walk 5 million
Running 30 million
Contamination ControlPreventative Practices
Gowning Labcoats in the labs Scrubs/Cleanroom suits for manufacturing areas Face masks, beard covers, hair nets, etc.
Good hygiene practices Personnel/material/waste flow in critical areas HEPA filters Sanitization of equipment/materials Slow, deliberate movements in controlled areas Cleaning techniques
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The HEPA Filter
High Efficiency Particulate Attenuation
Unidirectional Air FlowAir Flow Patterns•Air moving devices displace the air in a unidirectional (one direction) pattern to reduce turbulence of the air and displace contaminants.
•Air is directed using walls, curtains, or panels.
•Smoke studies are done to allow the airflow to be visualized.
•Machinery and large objects can affect airflow by creating local zones of air turbulence
Room Air Changes
Air is circulated through the Cleanroom and carries contaminants as air passes through in unidirectional flow.
Recirculated air passes through HEPA filters.
To maintain ISO 5 (Grade A, Class 100) conditions requires the air volume to be replaced a minimum of 25 air changes per hour (ACH) ; air velocity should at least be 0.45 m/s.
20 ACH are typically required for ISO 7 areas (Grade C areas; Class 10,000) and ISO 8 (Grades D areas; Class 100,000.
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AHU1:Recirculated Air
AHU2: Outdoor Air
HEPA Filters
Types of Cleaners/Disinfectants
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Spor Klenz Active Ingredient 22% Acetic Acid 4.5% Peracetic Acid 10% Hydrogen Peroxide
70% IPA
LpHsePhenolic Disinfectant Acidic, (pH 2.6 – 3.0)
Vesphene IIse Alkaline pH 10.4-10.6
Bleach Sodium Hypochlorite
Disinfectant Action
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UV Light
SurfaceProteins
Protein Denaturation by Base
Disruption of Cell Membrane
Protein Hydrolysis by Acid
Microbial Killing Efficiency Antimicrobial Agents Compared
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Contamination Control Personal Resposibility
Personal Hygiene Regular Bathing/showering (includes washing
hair) Practice good oral hygiene Clean clothes and shoes
Illness Notify your management of illness or open
wounds Restrict access to controlled areas
When working in Controlled Areas No make-up, jewelry, or cologne No food, drinks, or gum/candy Avoid getting sunburned (flaking skin)
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Contamination Control Personal Responsibility
Frequent hand washing If you smoke, drink water
after to reduce the introduction of smoke particles into controlled areas
Maintain a clean, organized workspace
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4 Pillars of Aseptic Techniques
Personnel training & monitoringEnvironmental monitoringFacilities design & HVAC validationProcess simulation (media fills)
Let’s Review! What does “aseptic” mean? Name 3 scenarios that require aseptic technique Name 3 types of microorganisms List the different techniques that QC employs for microbial
identification What are the two categories of particulate contamination? Name 3 sources of contamination List 3 ways to control contamination Describe YOUR personal responsibilities toward contamination
control
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Q & A
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