Principles of Aseptic Processing

PRINCIPLES OF ASEPTIC PROCESSING Jim Hardy GHGBioSciences, Inc. f [email protected] [email protected]

Transcript of Principles of Aseptic Processing

Page 1: Principles of Aseptic Processing


Jim HardyGHGBioSciences, [email protected]

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Agenda Background Microbiology Basics Contamination Control

Sources of Contamination

Cleaning & Disinfection 4 Pillars of Aseptic



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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


1600 1700 1800 1850 1900 2000



Carbolic Acid


19th Century SurgeryIgnaz Semmelweis

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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


-food decay

-contamination of medication


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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.


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.

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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


Mycoplasma Microscopic algae Viruses


T4 VirusMycoplasma Bacteria

Saprolegnia Mold E Coli Bacteria

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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


Bacterial Binary Fission

E.coli can double their number every 20 minutes!!

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Eukaryotic organismA cell that has a complex structure

enclosed within a membraneContains a nucleus Ubiquitous to every habitat on Earth


Can be multi-cellular (mold) or single-celled (yeast)

Reproduction is sexual or asexualCommonly via spores




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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



Budding Yeast Cells

Yeast Cells with Spores

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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)


Aspergillus niger


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Microbes and the Human Body

Where are microbes found on humans?SkinEyesNoseMouthUpper throatIntestines


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

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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)


Fewer than 1% of the world’s microbes have been identified!!

Morphology: Size, ShapeTexture, Color

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Bacterial Identification Gram Positive Microorganisms


Gram Positive Cocci Staphylococcus aureus

Gram Positive Rods Bacillus sp.

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Bacterial Identification Gram Negative Microorganisms

A source of endotoxin

A single E Coli can contribute 2 million Endotoxin Molecules!


Gram Negative Rods Escherichia coli

Gram Negative Cocci Neisseria gonorrhoeae


Cell Wall

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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



Water Vapor Smoke


Skin Flakes

Influenza Virus

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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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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


NOTE: Men shed more particles than women

Activity Particles Generated

Standing or Sitting 100,000

Slight head/hand movement


Body, arm movement w/ toe tapping

1 million

Changing from sitting to standing

2.5 million

Slow walk 5 million

Running 30 million

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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

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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

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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.


AHU1:Recirculated Air

AHU2: Outdoor Air

HEPA Filters

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Types of Cleaners/Disinfectants


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

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Disinfectant Action


UV Light


Protein Denaturation by Base

Disruption of Cell Membrane

Protein Hydrolysis by Acid

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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)

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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



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Q & A