Principles of Aseptic Processing

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

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Principles of Aseptic Processing

Principles of Aseptic Processing

Jim HardyGHGBioSciences, [email protected]

1Introduce yourself.

Welcome to todays training.

AgendaBackgroundMicrobiology Basics Contamination ControlSources of ContaminationCleaning & Disinfection4 Pillars of Aseptic Techniques2

2Today were going to cover the following topics: read slide

What are Aseptic Practices?DefinitionsAseptic = 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 sterileSomething is either sterile or non-sterile


3Sepsis: Comes from Greek for decay or putrid. As in Septic Tank. Indicates bacterial contamination.Asepsis: Absence of significant contamination.Aseptic techniques are used to prevent contamination of surgical instruments, medical personnel, and the patient during surgery. Aseptic techniques are also used to prevent bacterial contamination in food industry.

Aseptic TechniquesHistory & Milestones4

160017001800 1850 1900 2000

MicroscopeIodineCarbolic Acid

HEPA19th Century SurgeryIgnaz Semmelweis

4Ancient records show that antiseptics date far back into history; the ancient Chinese, Persians, and Egyptians had methods for water sanitation and antisepsis for wounds. The ancient Greeks and Romans used silver vessels to store fresh liquids and wine. Middle Ages silver and copper coins added to water to inhibit the growth of microbes. It was observed that water stayed fresher for longer. Mercuric chloride was used to prevent sepsis in wounds by Arabian physicians in the Middle Ages. Early civilizations practiced salting, smoking, pickling, drying, and exposure of food and clothing to sunlight to control microbial growth.Use of spices in cooking was to mask taste of spoiled food. Some spices prevented spoilage. First microscope designed and built by Anton van Leeuwenhoek in 1675Hypochlorite and iodine were introduced as a treatment for open wounds in 1825 and 1839. In 1861 Louis Pasteur proved that microorganisms caused spoilage and could be transported via the air. He placed broth in flasks with long S-shaped necks, then boiled the broth and observed that no microorganisms grew in the flasks. These experiments were the basis for the development of aseptic techniques. Pasteur showed that heat could kill microorganisms; this process was later named pasteurization. Using the knowledge gained from Louis Pasteur, a scientist named Dr. Ignaz Semmelweis reduced the number of postpartum infections (puerperal sepsis) in the wards of Viennas lying-in hospitals by urging doctors to wash their hands between patients.By the mid-nineteenth century, post-operative sepsis infection accounted for the death of almost half the patients who underwent major surgery. Later in the 1860s, an English surgeon named Joseph Lister heard about Pasteurs work. He began soaking his surgical dressings in carbolic acid (phenol) because he had heard the previous year that carbolic acid had been used to treat sewage in a nearby town. The fields that had been treated were now free of parasite-causing disease. This led to a dramatic decrease in the number of post-operational infections. Before the discovery of antisepsis by Lister, about 80% of surgical patients contracted gangrene. In mid 1800s Semmelweiss and Lister helped developed aseptic techniques to prevent contamination of surgical wounds. Before then:Nosocomial infections caused death in 10% of surgeries.Up to 25% mothers delivering in hospitals died due to infection

In 1870, Listers antiseptic methods were used by Germany during the Franco-Prussian war, where they saved the lives of many Prussian soldiers. Although Germany and several other countries followed Listers procedure of sterilization, England and America were still in opposition to his germ theory.

The turning point for Lister came on October 26, 1877, when he had the opportunity to perform a simple knee operation (wiring a fractured kneecap, which entailed deliberate conversion of a simple fracture into a compound fracture), which often resulted in generalized infection and death. News of this operation was widely publicized; its success forced people to accept that his methods greatly added to the safety of operative surgery. The culmination of his emphasis on the principle of preventative medicine was the opening of the Institute of Preventative Medicine in 1891. This is why Joseph Lister is often referred to as the father of the antiseptic.Paul Ehrlich, a German scientist, later advanced the idea of using chemicals to kill microorganisms by testing many more compounds. He eventually found a chemical that was successful against syphilis. Another scientist that had a significant impact on the field of sterilization was Ernst von Bergmann. He is credited with introducing steam sterilization under pressure for treating instruments and all other medical equipment used for a surgical patient. A famous surgeon from John Hopkins, William Stewart Halsted, introduced sterile rubber gloves to the field of medicine when his fiances hands became irritated from constant washings and antiseptics.

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

Micro-organisms are both useful and harmful. For example, we all have micro-organisms that live in our intestines that help in the digestion and absorption of food. Without our "gut bacteria" we would not be able to survive.BEFORE revealing the slide ASK the learners of they can think of types of good micro-organismsADVANCE slide to reveal answers and then REPEAT with Bad micro-organismsbacteria "don't have legs or wings," so they are transported from place to place by contact, movement and air currents. (Bacteria are hitchhikers)


When are Aseptic Practices Used?Sampling Raw Materials for Qualification ReleaseSampling of Utilities (WFI, pure steam, compressed air, and specialty gases)Bioburden & Sterility TestingEnvironmental MonitoringManufacturing in classified areas (i.e. cleanrooms)Compounding/FormulationFill/Finish Operations6

6Aseptic technique is used in the following areas

Operating rooms and hospitalsFood industryElectronics

Good Aseptic PracticesRegulatory Aspects21 CFR 211211.28 (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.

7To ensure our drug products are safe, pure, and effectiveEudralex Vol. 4 Part 1, Ch. 2Personnel 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 ProductsThe 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.

7The regulations tells us that we need to employ aseptic technique to avoid compromising our products. Therefore, if we do not practice aseptic technique we can face punishment/consequences from the regulatory agencies, loss of public confidence in our products/company, increased product complaints, lost batches, etc.

Microbiology and Microorganisms8

8"Before we get into the Techniques, let's review the reason behind using Aseptic Processing Techniques in the first place: Microorganisms"

Why is Microbiology Important?Defined as the study and science of microorganismsMost 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 degradationShifting the pHIntroducing endotoxins and other toxinsMicrobial testing (bioburden) is performed to ensure that an adulterated product does not reach the patient9


Types of MicroorganismsBacteriaFungiYeastMoldMycoplasmaMicroscopic algaeViruses


T4 VirusMycoplasma Bacteria

Saprolegnia MoldE Coli Bacteria

10BacteriaFungiYeastMoldMycoplasmaMicroscopic algaeViruses

BacteriaProkaryotes: Unicellular microorganisms with no nucleus & rarely have membrane-bound organellesUbiquitous to every habitat on Earth0.5 -1.0 microns in length : 3 million can fit into an area the size of a pin headWide 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 tract11

11Last bullet: There is more bacteria on and in the human body than human cells.

Bacterial ReproductionBinary FissionOne cell divides into two identical cellsA single bacterial cell can multiply to 9 x1030 in a 24-hour period in ideal conditionsRefrigeration slows bacterial growth, but does not stop it completely12

Bacterial Binary FissionE.coli can double their number every 20 minutes!!


FungiEukaryotic organismA cell that has a complex structure enclosed within a membraneContains a nucleus Ubiquitous to every habitat on EarthTypesYeastMoldCan be multi-cellular (mold) or single-celled (yeast)Reproduction is sexual or asexualCommonly via spores13




YeastUnicellular (single-celled) fungiAverage size = 3 - 4 m in diameterBoth aerobic and anaerobic respirationIdeal growth conditions: Neutral pHTemperature 10 37 CPart of normal flora of human bodyReproduce asexually by budding or sexually by spores like other fungiConverts sugar into alcohol = alcohol tolerant14

Budding Yeast Cells Yeast Cells with Spores

14Unicellular (single-celled) in the Fungi familyAverage size = 3 - 4 m in diameter, but can be as large as 40 m Both aerobic and anaerobic respirationUnderpinning Knowledge: this means they can live with or without oxygenIdeal growth conditions: Neutral pHTemperature 10 37 CPart of normal flora of human bodyReproduce asexually by buddingConverts sugar into alcohol = alcohol tolerant

MoldsMulti-cellular filaments called hyphaeA colony of hyphae is called a myceliumFunctions as a decomposerAbility to survive extreme temperature and pHVisible as a downy or furry coating on food or surfacesReproduce through small sporesSpores can be asexual (mitosis) or sexual (meiosis)15

Aspergillus niger


15Mold contamination can be difficult to get rid of because of their ability to survive in extreme conditions. They can even survive in Outer Space!

Microbes and the Human BodyWhere are microbes found on humans?SkinEyesNoseMouthUpper throatIntestines16

Hands10,000 -100,000 cm2

Groin1-20 million / cm2Feet1 million / cm2

Scalp~ 1 million / cm2Forehead100 -1,000 / cmSaliva~ 10 million / gmNasal Fluid~ 10 million / gmArmpit1-10 million / cm2Feces>100 million / gm

16Bacteria is part of the body. They are more prevalent on warm, moist areas, like the armpits and nasal cavity. Without bacteria in our gut, we would not be able to break down food into essential nutrients and we would die.When bacteria grows out of control or foreign bacteria enters our bodies, we develop a fever to fight off the infection.

Identification of MicroorganismsWhy?Identify sources of contaminationIdentify trends in the environmentMonitor disinfectant effectiveness

How?Appearance or morphologyGram staining to identify Gram-negative or Gram-positive bacteriaSecondary tests to identify familiesBiochemical tests to further speciate (Vitek and API)Genetic sequencing analysis (MicroSeq)17Fewer than 1% of the worlds microbes have been identified!!

Morphology: Size, ShapeTexture, Color

17Because this is a regulated environment, we are also required to identify microorganisms to ensure the safety, efficacy, and purity of our product.

Bacterial Identification Gram Positive Microorganisms18

Gram Positive Cocci Staphylococcus aureusGram Positive Rods Bacillus sp.

18Part of the identification process is gram staining. These are examples of Gram + organisms after gram staining.

Bacterial Identification Gram Negative MicroorganismsA source of endotoxin

A single E Coli can contribute 2 million Endotoxin Molecules!19

Gram Negative Rods Escherichia coli

Gram Negative Cocci Neisseria gonorrhoeae


Cell Wall

19This slide shows Gram negative organisms after gram staining. They stain RED and are a source of Endotoxin. Endotoxin will persist after the bacteria are removed and can cause a serious medical condition, sepsis, if injected.

Contamination Control20

20Introduce to the participants Contamination ControlREMIND: Our goal here at this facility it to successfully manufacture drugs for human consumption, so lets discuss one of the most critical elements of the production process: Controlling Contamination

What is Contamination?The introduction of undesirable impurities into an environmentViable (living) Contamination:Examples: bacteria, yeasts, and molds

Non-Viable Contamination: Examples: lint, dust particles, skin flakes, hair, pollen, smoke, chemical substances, etc.


21DISCUSS: Have audience define - What is contamination?Facilitate the participants to the correct answer.What is Contamination Definition: An undesirable material in or on the material of interest such as Examples: Dust particles, skin flakes, hair, cleaning solvents, paper flakes, cigarette smoke, pollen, molds, bacteria, micro organismsASK "What did you do to leave particles of contamination on you today?" Facilitate the participants to the correct answer.ANSWERS Smoke, pet the cat, combed hair, walked outside, went to the gym. Just about anything you do causes a potential source of contamination.

ContaminantsCommon Particulates 22

FungiWater VaporSmokeBacteriaSkin FlakesInfluenza Virus

22ASK: How many different contamination types are there?Facilitate discussion amongst the participants. Write the responses on a dry erase board or flip chart. Explain: There are two contamination types: #1 Non Viable Contamination Not Alive Examples: solvent residue, dust, hair, paper flakes ASK "What are some other types of non-viable contamination?" Facilitate the participants to the correct answer.ANSWERS lint, metal shavings from tools, another product, condensate water from the air, grease

You can engage your audience by asking them what type of contamination, viable or non-viable, is each example.

Contamination ControlContamination 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 bodys natural barriers to infectionGiving a patient a contaminated product can cause serious adverse events including DEATH23


ContaminationSourcesPersonnel (most common)Touch ContaminationSkin / Hair / Mucous MembranesClothing Equipment Improper Cleaning /Contaminated SuppliesAirHVAC / HEPA FailureInfiltrationInternal Generation24

24InfiltrationParticles from adjacent spaces (e.g. anteroom)Internal generation Walls, floors, ceilings, packaging, equipment

Particle GenerationPersonnel25 NOTE: Men shed more particles than womenActivityParticles GeneratedStanding or Sitting100,000Slight head/hand movement500,000Body, arm movement w/ toe tapping1 millionChanging from sitting to standing2.5 millionSlow walk5 millionRunning30 million

25Emphasize that in classified areas and when using aseptic technique, movements should be slow and deliberate to reduce excess particle generation.

Contamination ControlPreventative PracticesGowningLabcoats in the labsScrubs/Cleanroom suits for manufacturing areasFace masks, beard covers, hair nets, etc.Good hygiene practicesPersonnel/material/waste flow in critical areasHEPA filtersSanitization of equipment/materialsSlow, deliberate movements in controlled areas Cleaning techniques26

26What are some of the ways that we can reduce contamination?Discuss each item above. Ask the audience how each contributes to contamination control.

The HEPA Filter

High Efficiency Particulate Attenuation

27HEPA filtration and laminar flow serve as contamination control devices. HEPA (High Efficiency Particulate Attenuation) filters are used to remove particulates and microorganisms from the air supply to the manufacturing/filling rooms, laminar flow hoods, biosafety cabinets, etc.

Most HEPA filters consist of a rigid frame into which a filter pack, constructed by folding a continuous sheet of media into closely spaced pleats, is sealed. Conventional HEPA media is an all-glass paper which is composed of an extremely large number of randomly oriented micro-fibers which utilizes some fairly complex mechanical principles to achieve its effectiveness on sub-micron particles

HEPA Fact: HEPA filters are made of boron silicate microfibers formed into a flat sheet by a process similar to papermaking. The flat sheets are pleated to increase the overall surface area. A HEPA filter is able to trap 99.99% of particles of a diameter greater than or equal to 0.3 microns.

There are many HEPA filter specifications and standards. Some have been developed by individual companies for their own use, some are for specific applications or industries. It is also common to find some specifications or standards misapplied to a HEPA filter or HEPA filtration, in general. HEPA Filters are placed in Terminal locations for Class 10K and above

Unidirectional Air Flow

Air 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

Unidirectional AirflowAir Flow PatternsAir 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

TIP: Use the image as a guide and point to the picture as you explain unidirectional airflowPoint out the HVAC on the top (explain this is just a model and not exactly what our facility looks like), HEPA filters in the ceiling, people and objects in the room and vents along the floorPOINT OUT the black specs are representative of dirt or particles


Room Air ChangesAir 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 100,000.29

AHU1:Recirculated AirAHU2: Outdoor Air

HEPA Filters

Room Air ChangesAir 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.

ACH means totally replacing the volume of air in a given space with new, filtered air a part of which is fresh and a portion is recirculated.Need to have some fresh, outside air to replace the oxygen humans are consuming through respiration. one AHU is using fresh, make-up air and one is using recirculated airSHOW one AHU is much larger than the other, because it has to move more airthe size of the AHU depends on the size of the room and how many ACH are required29

Types of Cleaners/Disinfectants30

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

30DISCUSS How cleaning is an important piece to controlling contamination. There have been many studies within the industry to test various cleaning methods and cleaners/disinfectants.UNDERPINNING KNOWLEDGEIt is important to cycle cleaning reagents to target different types of microorganisms and avoid creating strains of potential contaminants that are resistant to cleaning solutions.

Disinfectant Action


UV LightSurfaceProteins

Protein Denaturation by Base

Disruption of Cell MembraneProtein Hydrolysis by Acid

This graphic shows HOW disinfectants work.UNDERLYING SCIENCEUV light penetrates the cell and damages DNA, killing the cellSome disinfectants break the cell open and kill it through disruption of the cell membraneIn some cases, surface proteins are hydrolyzed by acid or denatured by base. This is how LpHse and Vesphene work.31

Microbial Killing Efficiency Antimicrobial Agents Compared32

It is not as important to read this whole graph.DISCUSS how after 120 seconds (2 minutes) using soap and water 90% of the bugs are still alive, versus only less than 10% with 70% Ethanol32

Contamination Control Personal ResposibilityPersonal HygieneRegular Bathing/showering (includes washing hair)Practice good oral hygieneClean clothes and shoesIllnessNotify your management of illness or open woundsRestrict access to controlled areasWhen working in Controlled AreasNo make-up, jewelry, or cologneNo food, drinks, or gum/candyAvoid getting sunburned (flaking skin)33

33What can YOU do to help control contamination? Follow the established rules. Be aware of how YOU can affect the environment.READ THROUGH this slide and advance to the next

Contamination Control Personal Responsibility

Frequent hand washingIf you smoke, drink water after to reduce the introduction of smoke particles into controlled areasMaintain a clean, organized workspace34


4 Pillars of Aseptic TechniquesPersonnel training & monitoringEnvironmental monitoringFacilities design & HVAC validationProcess simulation (media fills)


Lets Review!What does aseptic mean?Name 3 scenarios that require aseptic techniqueName 3 types of microorganismsList the different techniques that QC employs for microbial identificationWhat are the two categories of particulate contamination?Name 3 sources of contaminationList 3 ways to control contaminationDescribe YOUR personal responsibilities toward contamination control36??

36What does aseptic mean? Without organismsName 3 scenarios when aseptic technique is required. Isolation of a microbial cultureWorking within a Biosafety cabinet or Laminar Air FlowBioburden TestingSterility TestingEnvironmental MonitoringSampling of Utilities (WFI, steam, air, and gases)Manufacturing in classified areas (i.e. cleanrooms)Fill/Finish OperationsName 3 types of microorganisms.BacteriaFungiYeastMoldMicroscopic algaeVirusesList the different techniques that QC employs for microbial identificationAppearance or morphologyGram staining to identify gram-negative or gram-positive bacteriaSecondary tests to identify familiesBiochemical tests to speciateWhat are the two types of contamination?Viable Contamination: living contamination Examples: bacteria, yeast, and moldsNon-Viable Contamination: non-living contaminationExamples: lint, dust particles, skin flakes, hair, pollen, smoke, chemical substances, etc.Name 5 sources of contamination.PEOPLE (Skin, Hair, Mucous membranes, Clothes & shoes)Equipment and MaterialsAirWaterSpills Improper cleaning techniques List 3 ways to control contamination.Clothing regulationsLabcoats in the labsScrubs/Cleanroom suits for manufacturing areasFace masks, beard cover, hair nets, etc.Personnel/material flow in critical areasHEPA filtersSanitization of equipment/materialsGood hygiene practicesSlow, deliberate movements in controlled areas (i.e. BSC or class 100 curtain)Cleaning techniques (rotating cleaners)Describe YOUR personal responsibilities toward contamination control.Personal HygieneReporting illnessNo make-up or jewelry in controlled areasNo food, gum, or candy in controlled areasFrequent handwashingIf you smokeMaintain a clean, organized workspaceName 5 tips for working in a BSC.Reference slides 45, 46, and 47List 3 things to remember when performing EM.Do not touch the surface of an open plateAvoid reaching your hand or arm over an open plateDo not cough or sneeze around or over an open plateDiscard the plate if you suspect that you contaminated it and resampleRoutinely check gloves for damageDisinfect gloves frequentlyKeep movements slow and methodical to reduce airflow disturbance and shedding of particles

Q & A37