akers 2009 steriles

Sterile Product Processing

The ABCs of Sterile Manufacturing

Michael Akers and Matt ErvinBaxter BioPharma Solutions

AAPS Webinar June 3, 2009

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Outline of Topics• Microbiology 101• Process Flow• Manufacturing Facility Design• Sterilization and Depyrogenation

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• Sterilization and Depyrogenation• Environmental Control and Monitoring• Personnel Training• Aseptic Process Control and Validation• In-Process and End-Product Testing


Microbiology 101

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Bacteria• Bacterial cell envelop most important physical feature.

Disinfectants and antibiotics work again cell envelope–– Gram +:Gram +: peptidoglycan, teichoic acid

» Staph, Bacillus, Strep, Clostridia–– Gram Gram -- : peptidoglycan, lipoprotein, lipopolysaccharide layers

sources of LPS endotoxinLPS endotoxin» Pseudomonas, E coli, Salmonella, Klebsiella, Serrati

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» Pseudomonas, E coli, Salmonella, Klebsiella, Serrati• Various classifications

– Gram positive or gram negative– Anaerobic or aerobic or facultative– Pathogenic or non-pathogenic or opportunisitic– Vegetative or spore (Bacillus and Clostridium)

• Spores--form by vegetative cells in response to environmental shock such as nutrient depletion; hundreds more resistant than vegetative forms; selected as biological indicators to measure effectiveness of sterilization methods


Fungi (yeasts and molds) and VirusesFungi (yeasts and molds) and VirusesFungiFungiFungiFungi VirusesVirusesVirusesViruses

• Cellular forms more like human cells which makes these organisms harder to kill without killing human cells

• ~ 100 of 1,000 known

• Intracellular parasites; do not need food

• Very small, thus can pass through even 0.1 micron filters

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• ~ 100 of 1,000 known fungi are pathogenic

• Candida spp. and some dermatophytes are only known fungi transmitted from person to person

• Mold spores exist, but much less resistant than bacterial spores

• Readily inactivated by heat > 60C

• Very susceptible to surface disinfectants

• Environmental detection very costly

• Sterile facility conditions too harsh for viruses to survive


Kinetics of Microbial Destruction• Microoganisms grow and die logarithmically.

• Geometric progression: One cell dividing every 20 minutes will in 10 hours produce >10 billion cells!

• Knowing a term called the “D-Value” for the biological indicator used to validate a sterilization (heat, gas, radiation) process is the basis for sterilization validation.

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sterilization validation.• D stands for decimal reduction time that is the time

required for a one log reduction in the microbial population

• Various mathematical approaches for calculating the D-value

• D-value is the basis for calculating required Z and F values


Common Terms Used in Microbial Death Kinetics

• Bioburden– No = Initial population of surviving microorganisms per

defined unit or surface. Typical limit: <10 CFU/100 mL• Survival Rate

– D Value = Time or dose required for a one log reduction in the microbial population. Depends on many conditions

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– D Value = Time or dose required for a one log reduction in the microbial population. Depends on many conditions

• Resistance – Z Value = Number of degrees or dosage (Mrad) units

required for a one log reduction in the D value• Sterilization Process Equivalent Time

– F or Fo Value = The equivalent time at a given temperature (Fo refers to 121ºC and Z = 10ºC) that a lethal amount of sterilization is delivered to a unit of product


Sources of Microbial Contamination• The atmosphere

– Air is not a natural environment for microbial growth (too dry, absent of nutrients), but organisms such as Bacillus, Clostridium, Staph, Strep, Penicillin, Aspergillus can survive

– Degree of contamination depends on particle level• Buildings

– Potential mold contamination; nutrients come from plaster, worry about cracks, inadequate sealings

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– Potential mold contamination; nutrients come from plaster, worry about cracks, inadequate sealings

• Water– Always concerned about Pseudomonas

• Raw Materials– Less concerned these days because little/no natural sources

• Packaging– Mold spores, especially if any paper sources

• People (!)


Greatest Source of Microbial Contamination: People!!

• People generate millions of particles every hour—breathing, talking, hair, skin, body movements, clothing, etc. �� > 1.2 million aerobic bacteria per m> 1.2 million aerobic bacteria per m22 in head and neck region in head and neck region �� 0.9 0.9 –– 3 million per m3 million per m22 on hands and armson hands and arms�� Much higher numbers of viable anaerobes (Much higher numbers of viable anaerobes (Proprionibact. acnesProprionibact. acnes) )

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�� Much higher numbers of viable anaerobes (Much higher numbers of viable anaerobes (Proprionibact. acnesProprionibact. acnes) ) �� Fully gowned person sitting in cleanroom releases ~ 15,000 particles Fully gowned person sitting in cleanroom releases ~ 15,000 particles

per minuteper minute�� Walking person releases ~ 157,000 particles/minWalking person releases ~ 157,000 particles/min�� Ratio of total particles > 0.5µm and viable aerobic organisms = 600Ratio of total particles > 0.5µm and viable aerobic organisms = 600--

7000 to 1.7000 to 1.�� People release 600People release 600--1300 total particles per hour in > 0.5µm size range 1300 total particles per hour in > 0.5µm size range

with ~ 40 CFU viable aerobic organisms among thesewith ~ 40 CFU viable aerobic organisms among these�� Properly gowned cleanroom worker will contribute 10Properly gowned cleanroom worker will contribute 10--100 CFU of 100 CFU of

viable aerobic organisms to the environment per hourviable aerobic organisms to the environment per hour

-- Berit Reinmueller papers, presentations-- Berit Reinmueller papers, presentations


Process Flow

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Table 1 of FDA Aseptic Processing Guidelines—Air ClassificationsClass (0.5µ parts/ft3)

ISO Desig-nation

≥ 0.5µ par-ticles/m3)

Micro Air Action Levels (cfu/ m3)

Micro Settle Plate Action Levels (cfu/4h)

100 5 3,520 1 1

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100 5 3,520 1 (expect 0)

1 (expect 0)

1,000 6 35,200 7 3

10,000 7 352,000 10 5

100,000 8 3,520,000 100 50


Flow of Manufacturing

Warehouse (unclassified)

Preparation (US: ISO 8 or better; EU: ISO 7 or better)

Formulation (US: ISO 8 or better; EU: ISO 7 or better)

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Filling (ISO 5)

Capping/Sterilization (ISO 5)

Sampling (unclassified)

Finishing (unclassified)

Dispense raw materials(active and excipients)

Prepare solution in appropriatemixing tank (add ingredients to Water For Injection)

Wash and sterilizeprimary containers and closures

Thaw and poolactive biopharmaceutical

Add active to solution,pH adjustment, final QSThis is formulation bulk solution

Schematic Overview of Processing Solution and Freeze-Dried Biopharmaceutical Dosage Forms

ISO 8, Grade D, Class 100,000

The EMEA requires estab. of “buffer area”where intermediate air

Sterile filter formulatedbulk solution

Aseptically fill formulated bulk solutioninto primary package and stopper(partial stopper if product is to be freeze-dried

Transfer to Freeze-dryersand lyophilize

Fully insert stopper, remove from freeze dryer Apply aluminum overseal

100% inspectionStore finished dosageforms at appropriate temperature (usually 2-8ºC)

Label, sec package, storage, distribution

ISO 5, Grade AClass 100

where intermediate airquality separates GradeA and D areas


What Can Go Wrong During Solution Manufacturing?

§ Foreign particles

§ Time limits exceeded

§ Sterility assurance issues, e.g.§ Personnel mistakes§ Filter integrity failures

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§ Filter integrity failures§ Environmental monitoring action levels

exceeded

§ Mechanical problems, e.g.§ Filling machines§ Rubber closure hoppers§ Freeze-dryers

§ Many other possibilities


Flow Diagram for Sterile Suspension Manufacturing

Sterilized and Milled Drug

Vehicle and Excipients

Sterile Filtration

Sterile RecrystallizationSpray DryingFreeze DryingEtO Powder

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Sterile ReceivingVessel

Aseptic Combination

Aseptic Mixing and Milling

Filling and Sealing

EtO PowderDry Heat PowderGamma Irradiation Powder


What Can Go Wrong During Suspension Manufacturing?

§ Sterility assurance§ Contamination produced from gasket materials, metal

parts from pumps, homogenizers, metal surfaces§ Tubing ruptures, e.g. at recirculating pump

§ Particle size failure§ Outlier particles and / or agglomerates

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§ Outlier particles and / or agglomerates

§ Lack of homogeneity§ Problems with dispersion of certain components§ Foaming due to “dumping” of recirculated suspension

in the tank§ Production stops and starts

§ Inadequate flushing of filling tubing§ Particle settling / separation


Manufacturing Facility Design

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Facilities—GMP Requirements• Section 211.42: Must be separate or defined areas of operation

to prevent contamination, and that for aseptic processing there be, as appropriate, an air supply filtered through HEPA filters under positive pressure, and systems for monitoring the environment and maintaining equipment used to control aseptic conditions.

• Section 211.46: Equipment for adequate control over air pressure, microorganisms, dust, humidity, and temperature be provided where appropriate and that air filtration systems,

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provided where appropriate and that air filtration systems, including prefilters and particulate matter air filters, be used when appropriate on air supplies to production areas.

• Section 212.42 (proposed GMP for LVP)– Walls, floors, ceilings, fixtures, and partitions in controlled

environment areas shall– Have a smooth, cleanable finish that is impervious to water

and to cleaning and sanitizing solutions– Be constructed of materials that resist chipping, flaking,

oxidizing, or other deterioration.


Facility Layout

• “Sterile Block” design– Control ∆P among various

areas– Proper directional flow of

air, materials and people movement

– Avoidance of “clean to dirty” crossovers

Mechanical AreaGeneral Area

Clean AreaAseptic Adjacent

andAseptic Area

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dirty” crossovers– Minimize critical area work

force• “Cleanability” and

sanitization of all surfaces • “Technique” as important as

chemical actions• Trend toward modular

construction

• Floors: Epoxy terrazo, urethane, Mipolam (solid vinyl) on concrete

• Walls: Mipolam, cement plaster, Kydex (acrylic/PVC) shields

• Ceilings: Mipolam, lighting and fixtures recessed

• Curtains: Vinyl or lexan


• Basic materials of construction much like a normal building (Concrete, Steel, Gypsum)

• Each module built separately, then entire set of modules put together (Sweden)

• Entire setup tested to ensure that everything works properly with customer approval

Modular Constructione.g. www.pharmadule.comModular Constructione.g. www.pharmadule.com

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that everything works properly with customer approval (Sweden)

• Modules disassembled (equipment, utilities, etc remain within each module), shipped to customer site, reassembled

• Everything re-qualified• 12-18 months average from

start of design to commissioning and qualification


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“Air Handling Systems for Cleanroom Control”, Sterile Pharmaceutical Products, Avis, ed., 1996, p. 57.


Examples of Facility 483 Observations• Separate defined areas not provided to assure prevention of

contamination or product mix-up• Capping area not under auspices of controlled environment; same for

loading vials into F-D• Evidence of cracks, deterioration of walls, ceilings, floors; debris in

clean rooms• Lack of proper certification of HEPA filters• Lack of smoke tests during operations

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• Lack of smoke tests during operations• Lack of EM data during operations• Inadequate sanitization validation• Sampling of water system does not reflect actual use of water in

production– Water collected after 3 minute flush, yet no flush prior to water

used in actual production• Lack of documentation regarding facility maintenance

– Cleaning, DP checks, gas filter integrity, improper storage of equipment


Sterilization and Depyrogenation

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Methods of SterilizationMethods of Sterilization

• Thermal– Moist heat (saturated steam under pressure)– Dry heat

• Chemical

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• Chemical– Gaseous– Radiation– Bright Light

• Filtration


Thermal SterilizationThermal Sterilization• Lethality depends on

– Degree of heat– Duration of exposure– Humidity

• Lethal mechanism is coagulation of protein in the cell• Moist heat is more effective than dry heat

– e.g. Clostridium botulinum destruction @ 121°C

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– e.g. Clostridium botulinum destruction @ 121°C» Moist heat: 10 minutes» Dry heat: 120 minutes

• Basic principle: Raising the boiling point of water from 100°C at atmospheric pressure to 121°C at 15psig above atm.At 121°C, saturated steam, when hitting a cooler surface, will condense, releasing up to 500+ cals/g degree. By comparison, dry heat at the same temperature will only release 1 calorie/g degree


Steam SterilizationSteam Sterilization• Primary applications—sterilizes

– Equipment—tanks, filling nozzles, aseptic attachments, lyo chambers

– Process tubing– Filters and apparatuses– Rubber closures

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– Rubber closures– Final products, if possible

• Basic cycle– Preconditioning--must remove air, then heat– Sterilization– Drying--removal of air and release of pressure


Steam SterilizationSteam Sterilization• Key Factors

– Steam must reach innermost recess of material being sterilized

» Biggest challenge—removal of all sources of air– For solutions in containers, wall of container must be

heated to raise temperature of solution inside the container to generate steam

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container to generate steam– There MUST be a source of water for steam to be

generated under temperature/pressure» Not suitable for anhydrous oils, powders or any system

that is enclosed and dry inside– Lag time and cycle based on nature of materials to be

sterilized and configuration of load• At least 7 different designs of steam sterilizers,

differentiated by the post-sterilization phase.


Dry Heat SterilizationDry Heat Sterilization• Death by dry heat is primarily the result of an

oxidation process• Temperatures and times:

– Sterilization: 160°C for 2-3h; 180°C for 1h– Depyrogenation: 230°C for 75 min; 250°C for 45 min– Industry actually uses higher temperatures

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– Industry actually uses higher temperatures

• Use either regular batch ovens or tunnel sterilizers• Most effective method to depyrogenate• Not used for drug product sterilization, but for

sterilization of processing items—glass, stainless steel


Radiation SterilizationRadiation Sterilization

• Damages nucleoproteins of microbes• Few variables: dose and time• Types

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• Types– Gamma radiation

» Cobalt 60 high energy photons– Accelerated electrons

» Beta particles (electron beam)» Ionizing radiations» Less penetrative than gamma

– Ultraviolet light


Radiation SterilizationRadiation Sterilization

• Big concern: formation of radiolytic by-products (e.g. *OH) which in turn can cause damage to ingredients and surfaces

• Typical dose: 25 kGray (2.5 mRad)• Sterilizes plastic devices, gowns, potentially drug products• Significant growth—nearly 50% of sterilization market• 12 D overkill approach always used• Bacillus pumulis is B.I.

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• Bacillus pumulis is B.I.– Typical D values = 1.7-2.0 kGray– Typical dose = 25 kGray– Can use as low as 2 to 8 kGray

• Dosimetric evaluation of load is critical– BI’s used with each cycle

• Gamma sterilization still method of choice; E beam sterilization being re-evaluated carefully, especially for finished products


Gaseous SterilizationGaseous Sterilization

Sterilization gases:– Formaldehyde– Ethylene oxide– Propylene oxide– Beta-propiolactone

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– Beta-propiolactone– Ozone– Peracetic ccid– Vapor hydrogen peroxide– Chlorine dioxide


Ethylene OxideEthylene Oxide

• Very potent and highly penetrating gas• Alkylating agent used for years and for

which standardized efficacy measurements exist

• Carcinogenic agent• Standard mixture: 12 EtO:88 Freon

– Profound environmental issues!– Alternative diluent: CO

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– Alternative diluent: CO2• Main issue: Residuals--ethylene glycol

and ethylene chlorhydrin• Must have specifications on residue

levels– Typically 1 mcg/mL or g for EtO– 50 mcg/mL or g for EtCh

• OSHA and EPA have tried to curb use because of residues


Vapor Phase Hydrogen Peroxide§ Sterilant of choice for isolators

§ Very effective sporicidal§ Relatively safe; environmentally friendly

§ Concentration depends on size of enclosure§ Liquid peroxide (30%)->Vapor peroxide (1-2mg/L)

§ Advantages§ Sterilization occurs at room temperature (non-corrosive)

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§ Sterilization occurs at room temperature (non-corrosive)§ In fact, increasing exposure temperature results in decreased

sterilization efficacy§ Very fast, efficient, effective against spores§ Environmentally friendly

§ Disadvantages§ Generators do not give consistent concentration§ Concern over B. stearo. spores developing resistance§ Absorption into plastics and other materials


Filtration• Objectives

– Clarification– Sterilization (majority of SVPs sterilized by filtration)

• Filter types– Different polymers and porosities

• Aseptic filtration validation

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• Aseptic filtration validation– Microbial retention– Extractables– Compatibility with drug product

• Filter integrity– Bubble point– Diffusion test

• Controversial issues– Non sterile filtrates through 0.2µm filters!


Pyrogens/Endotoxins• Pyrogen = “Pyro” (Greek = Fire) + “gen” (Greek = beginning).

• Fever producing, metabolic by-pdts of microbial growth and death.

• Bacterial pyrogens are called “Endotoxins”. Gram negative bacteria produce more potent endotoxins than gram + bacteria and fungi.

• Endotoxins are heat stable lipopolysaccharides (LPS) present in

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• Endotoxins are heat stable lipopolysaccharides (LPS) present in bacterial cell walls, not present in cell-free bacterial filtrates

• Stable to at least 175ºC; steam sterilization ineffective

• Water soluble; monomer unit of LPS can be 10,000 Daltons (1.8 nm)so endotoxins can easily pass through 0.22µm filters

• Sources: Water (main), raw mat’ls, equipment, process environment, people, and protein expression systems if using gram negative bac.


Depyrogenation MethodsDepyrogenation Methods• Endotoxin is removed or destroyed, not “killed”• Removal by rinsing (dilution), distillation, RO,

ultrafiltration (10,000 nmwl filters), electrostatic or hydrophobic attraction, activated carbon, affinity chromatography

• Destroyed/inactivated by

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• Destroyed/inactivated by– Acid and base hydrolysis– Oxidation--Use of hydrogen peroxide– Alkylation--Use of ethylene oxide (very questionable)– Dry heat– Moist heat--At least 3 hours exposure– Ionizing radiation--Must use fairly high doses


Environmental Control and Monitoring

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Environmental Monitoring and Control

• Controlling the environment is not the same thing as monitoring the environment. Control exists at all times; monitoring is only a snapshot.

• The main aspects of excellent environmental control include– Good training

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– Good training– Employee discipline to perform good aseptic practices– Expert management oversight

• Can monitor too much where monitoring can be a source of contamination (the units themselves, more people in the clean room, disruption of laminar flow)

JE Akers, PDA J Pharm Sci Tech., 51, 36-47, 1997


Environmental MonitoringEnvironmental Monitoring

• Room—surfaces and air• People• Utilities

– Water– Compressed gases– Clean steam

• Air Monitoring– Nutrient agar plates– Slit samplers– Electronic counters

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– Clean steam• HEPA filters• Filling nozzles after media

fill• Performance qualification

• Surface Monitoring– Rodac plates– Swab rinse


• Shift-by-shift monitoring of air and all surfaces • Written procedures to include

– List of locations to be sampled and when– Duration of sampling– Surface area and air volume sizes– Established frequencies– Limits

Environmental MonitoringEnvironmental Monitoring

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– Limits• Critical surface sampling may be performed at conclusion of

process• Air and surface samples should be taken at actual working

level/surface• Daily surface samples of each aseptic operator’s gown and finger

pads, at random intervals• Personnel monitoring program considered a separate procedure

from air and surface EM to accommodate different types of follow-up actions (e.g. increased scrutiny, retraining, re-qualification)


Environmental MonitoringEnvironmental Monitoring• Low level contamination not always detected. Because of

existence of false negatives, consecutive growth results should not be considered the only type of adverse trend.

– Look for increased evidence of contamination over a given period in comparison to that normally detected

• Establishing limits and trends– Limits established based on relationship of location to

operation– Both alert and action limits

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operation– Both alert and action limits

» Individual results exceeding alert limits should focus on trend data and deviation records and actions

» Individual results exceeding action limits will prompt more thorough documented inquiry

• Must have SOPs describing review, ID, and response to trends by QC unit and regular updating responsible mgmt.

• Routinely generate trend reports as function of location, shift, lot, room, operator, or other search parameters

• Investigate atypical microbes found


Personnel Training

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Personnel Training on Good Aseptic PracticesPersonnel Training on Good Aseptic Practices

� Characteristics of sterile dosage forms� Freedom from microbial, pyrogenic, and particulate

contamination� The problem of contamination

� Sources of contamination� Basic microbiology

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� Basic microbiology� Implications and consequences of a contaminated

product� Testing

� Objective� Hands-on—gowning, media fill, broth test� Remedial

� What FDA looks for when auditing training


FDA Aseptic Processing Guideline Emphasis on Personnel Training and Qualification

(211.22; 192; .25; .28)

• “Vigilant adherence to fundamental principles of aseptic techniques”

• Training should include– Proper aseptic technique– Microbiology

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– Microbiology– Hygiene– Gowning– Specific written procedures– Responsive and ongoing training

• Between media fills, regular training updates supplemented by routine evaluations by supervisory personnel of each operator’s conformance to written procedures and basic aseptic techniques during actual operations


• When objects or people interrupt the LAF pattern, it is re-established downstream within a distance equal to ~3 times the diameter of the object

• Beware of a false sense of security when working in LAF areas• Many rules involved in clean room procedures• Personal hygiene procedures• Always be aware of hands and fingers with respect to source

of HEPA filtered air

Some Basic Principles of Aseptic Techniques

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• Always be aware of hands and fingers with respect to source of HEPA filtered air

• Never touch the floor• Minimize talking• Minimize body movement• Understand rules of gowning• Avoidance of any particle shedding object--pencils, paper,

exposed hair and skin• When to re-sanitize or change gloves• Proper sanitization rules


What the FDA Evaluates in Aseptic Operator Training� Mechanisms used to evaluate training

� Media fills and/or broth tests� Sampling gowning surfaces� Documentation of observations of operators in actual

practice� Written tests

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� Written tests� Frequency of monitoring and evaluation

� Appearance and actions of operator during processing� Observation of hand washing and sanitation� Retraining procedures (failed plate counts, improper actions)� Degree of supervision


FDA Audit Findings on Aseptic Practices� Inappropriate techniques were observed within aseptic areas

� Different degrees of proper aseptic gowning were widely observed

� Not all personnel observed in the aseptic areas were wearing goggles as required

� Operator observed leaning over the accumulator for no apparent reason

� Exaggerated movements (dancing) was observed

� Plexi-panels were open on both sides of critical area; operators could talk to one another

� Too may people located within aseptic areas

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� One operator noted to run up to the filling line, arms waving

� A group of five operators congregated inside the Class 100 critical area

� Too much leaning over exposed vials observed

� Operator appeared to be touching sterile tweezers while hand stoppering

� Operator went into critical area three times without sanitizing their hands

� Operator not correctly using tweezers to remove overturned bottles on accumulator

� Cleaning/Sanitizing of aseptic areas not unidirectional

� Head covers did not always cover the face

� Beard covers did not always cover beard


Aseptic Process Control and Validation

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Control and Validation


Gloveboxes

ConventionalCleanrooms

Closed Isolators

Aseptic Processing Family Tree(Courtesy of James Agalloco)

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ConventionalCleanrooms

Barrier Systems

BFS/FFSRapid Assess

Barriers

Open Isolators

Closed Isolators


Some facts about aseptic processing

• About 80% of all small volume injectable products manufactured in the world are manufactured by aseptic processing.

• About 70% of all aseptically filled products are single dose products without an antimicrobial preservative.

• Aseptic process validation involves a large number of

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• Aseptic process validation involves a large number of considerations involving facilities, equipment, utilities, sterilization processes, personnel, monitoring, and constant evaluations.

• Lack of sterility assurance has been a top reason for sterile product recalls over the years.

• All drug products recalled due to non-sterility were produced by aseptic processing.


2004 Revised FDA Aseptic Processing Guidelines—Some Highlights

• Air particle count measured NMT one foot from the work site• Air velocity 90 to 100 feet/minute +/- 20%• Area immediately adjacent to aseptic processing line should meet

Class 10,000 standards (preferably better) under dynamic conditions. • Established specifications for acceptance /rejection of each

component lot for bioburden and endotoxin• In-process tests for delivery device functionality defects (e.g.

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• In-process tests for delivery device functionality defects (e.g. syringeability, delivery volume)

• Endotoxin control for all product contact surfaces prior to and after sterile filtration

• Includes maximum hold time for – Filtration processes– Unsterilized bulk solutions– Sterilized bulk solutions– Sterilized equipment– Sterilized containers and closures

• Daily surface samples of each aseptic operator’s gown and finger pads, at random intervals


When to conduct media fillsWhen to conduct media fills• Initial qualification

– Filling line– New product container configuration– Three repetitive successful media fills– Bracket all products filled on the line

» Size» Fill volume» Container opening» Line speed

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» Line speed» Manipulations

– Bracket worst case configurations» Largest opening» Slowest line speed» Most troublesome closure

• Periodic re-qualification– Assure validity of initial qualification– Typically done semi-annually

• Repeat of initial qualification– When significant changes occur—facility, filling line, personnel


Repeat of Initial QualificationRepeat of Initial Qualification

• Necessary under following situations– EM action levels exceeded on repetitive basis with no

assignable cause– Inoperative line for 1 year or more – Initiation of additional production shift– Significant change (defined by change control SOP) to the

room or equipment that influences air flow

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– Significant change (defined by change control SOP) to the room or equipment that influences air flow


Media FillsMedia Fills——What Must Be Considered……What Must Be Considered……• Duration of longest run• Worst case environmental conditions• Number and type of interventions, stoppages, adjustments, transfers• Aseptic assembly of equipment• Number and activities of personnel• Number of aseptic additions• Shift breaks, changes, multiple gownings

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• Shift breaks, changes, multiple gownings• Number/type of aseptic equipment disconnections and connections• Aseptic samples• Line speed/configuration• Manual weight checks• Operator fatigue• Container/Closure types run on the line• Temp/Relative humidity extremes• Conditions permitted before line clearance• C/C surfaces which contact formulation during aseptic process


Media Fill SpecificsMedia Fill Specifics• At least 3 consecutive, separate, successful runs, but this is a

minimal number• Routine semi-annual runs for each filling/closing line• Activities & interventions representative of each shift• All personnel must take part in a media fill at least once per

year• Any change should be evaluated for its level of significance (i.e.

need for new media fill validation) via change control system

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need for new media fill validation) via change control system• Media fill failures must be thoroughly investigated and followed

by multiple repeat runs.• Most accurate simulation model: the full batch size. • The number of units filled must be sufficient to reflect the

effects of worst case filling rates; eg. operator fatigue and maximum number of interventions and stoppages.

• Worst case line speed is generally considered to be slower than or equal to the slowest speed permitted by master production records


Media Fill DurationMedia Fill Duration——Regulatory Guidelines Regulatory Guidelines

• ISO: “sufficient duration to cover most manipulations”

• EU: “sufficient to enable a valid evaluation”• PIC: “Over the whole of the standard filling period”• FDA: “Duration of commercial aseptic process best

and preferred for larger simulations”

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• FDA: “Duration of commercial aseptic process best and preferred for larger simulations”

• Agalloco: “Sufficient to include all required manipulations, therefore, should be at least 3-4 hours long. Large batches will require longer fills, up to 16 or more hours long”


Acceptance Criteria (FDA Guidelines)Acceptance Criteria (FDA Guidelines)

• <5,000 units: no contamination– 1 failed unit considered cause for re-validation

• 5,000-10,000 units– 1 failed unit requires investigation, should

consider repeating media fill

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consider repeating media fill– 2 failed units considered cause for re-validation

• >10,000 units– 1 failed unit should result in an investigation– 2 failed units considered cause for re-validation


FDA 483s Related to Aseptic Processing• Inadequate investigation of media fill failure• Inadequate training of employees after media fill failure• Media fills did not follow SOP• Media fill aborted due to high particulate counts, but

inadequate investigation into reasons for high counts• Media fill did not start at point after product had been

sterilized

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sterilized• Defective vials discarded prior to incubation and not counted as

failures• Number of units filled too small• Media fills did not simulate what was documented in batch

records• Certain environmental data not collected during fill


In-Process and End-Product Testing

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• In-Process– Potency

» Preferably by UV, sometimes need HPLC– pH– Optical density (for suspensions)– Density– Osmolality (occasionally)

• End-Product– Appearance, other possible physical tests

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– Appearance, other possible physical tests – Assay– pH– Sterility– Endotoxin– Particulate matter– Lyo products—recon time, residual moisture– Dispersed systems—particle size, dispersibility, etc– Syringes/cartridges—functionality tests


100% Visual Inspection

• GMP required by USP (<1>) yet method not directly specified • Variety of opinions about what USP means by “essentially free”• EP provides method and describes apparatus• Performed by either/or

– Human visible inspection with the aided or unaided eye

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– Human visible inspection with the aided or unaided eye– Validated mechanical inspection

• Manual inspection highly variable– (e.g. particle motion, visual acuity, container volume, type and intensity of

lighting, background particle contrast, time of inspection, magnification, inspector attitude and concentration)

• Automated inspection equipment—Seidenader and Eisai– Containers ≥100 mL are generally not evaluated using automated

inspection– Vials spun rapidly and stopped just prior to evaluation

» Causes particles to rise to top and then settle» Too short of time between spinning and evaluation will not allow

enough time for air bubbles to escape


Sterile Manufacturing References--selected� Guidelines for Processing Aseptic Drug Products, FDA, 2004� Current Practices in the Validation of Aseptic Processing--2001,

Technical Report No. 36, Parenteral Drug Association� Validation of Aseptic Pharmaceutical Processes, FJ Carleton and JP

Agalloco, eds, Informa USA, Inc., 2007� RJ Harwood, JB Portnoff, and EW Sunbery, “The Processing of Small

Volume Parenterals and Related Sterile Products”, in Pharmaceutical Dosage Forms: Parenteral Medications, Vol 2, 2nd ed, KE Avis, HA

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Volume Parenterals and Related Sterile Products”, in Pharmaceutical Dosage Forms: Parenteral Medications, Vol 2, 2nd ed, KE Avis, HA Lieberman and L Lachman, eds, Marcel Dekker, NY, 1992.

� MJ Groves and R Murty, eds., Aseptic Manufacturing II, Interpharm Press, Buffalo Grove, IL, 1995.

� MJ Groves, WP Olson, and MH Anisfeld, eds., Sterile Pharmceutical Manufacturing, Vols 1&2, Interpharm Press, Buffalo Grove, IL, 1991.

� Development and Manufacture of Protein Pharmaceuticals, SL Nail and MJ Akers, eds., Kluwers-Wolters, New York, 2002.

� Articles in PDA Newsletter, PDA J PST, BioPharm, trade magazines


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