prueba de esterilidad consideraciones generales€¦ · retrospectiva de prueba de esterilidad...
TRANSCRIPT
Agenda
2
Introducción e historia
Conideraciones generales
1
3 Especificación y muestreo
4 Control de calidad
Esterilidad: Ausencia de microorganismos viables
Prueba de esterilidad: Prueba de control de calidad aplicada al lote de un
producto para verificar el cumplimiento con la regulación de (e.g.
<USP>; Eu.Ph.)
Droga farmacéutica:
Contenida en producto terminado, tableta, cápsula o solución que
contiene pocos aditivos farmacéuticos (CFR 21 314.3)
Parenterales:
Preparaciones estériles conteniendo una o más ingredientes activos, para su
administración mediante inyección, infusión o implantación en el
organsimo.
Empacados individualmente para su uso inmediato
(WHO http://apps.who.int/phint/en/p/docf/)
Propósito de la prueba de esterilidad
Detección de contaminación en una muestra
Prueba de presencia/ ausencia basada en el crecimiento de los microorganismos bacterias y hongos
¿Qué implica la prueba de esterilidad?
Presencia de microorganismos (<USP> 1211 – Sterilization and Sterility Assurance of Compendial Articles)
...significa la verificación de los materiales a analizar cumplimiento de los lineamientos European Pharmacopeia (Eu.Ph.5.1.9 –Guidelines for using the Test for Sterility)
¿Qué implica la prueba de esterilidad?
Exigida por CFR 21 610.12
Descrita <USP>, Eu.Ph. 2.6.1, WHO, GMP-Guidelines
Mitigación de riesgosImplica esfuerzos y costos...no es comercializableFactible de emplear riesgos de especímenes (USP <1211>)
¿Qué implica la prueba de esterilidad?
Pruebas no limitadas, implican implementación de otras técnicas o bien reflejan condiciones de mayor relevancia de GMP
Monitoreo ambiental
Validación de procesos asépticos
Validación de procesos de esterilización
Control del personal
Control de materias primas
e.g.
Sterility Testing
Muestras representativas de lotes, tomadas en el pasado su manejo adecuado mitiga risgos de contaminación
Relevancia muestreo Antes, durante y después del proceso En líneas de llenado y producto terminado
Ejemplos de Paraenterales & Productos que requieren prueba de estrilidad:
Vacunas
Antibióticos
Insulina
Suturas quirúrgicas
Ungüentos y cremas
Oftálmicos
e.g.
– Cell Cultivation & Purification – Injection/Infusion
Large Molecules Since1982
Biopharmaceuticals
Prueba de estrilidad requiere condiciones asépticas (USP <71>; Eu.Ph. 2.6.1)
Realizarse en campana de flujo laminar, cuarto limpio a bien en un aislador con monitoreo ambiental controlado(PIC/S Recomendaciones en prueba de esterilidad)
...material y equipos para moitoreo no debe interferir con flujo laminar(PIC/S Recommondations on sterility testing)
Retrospectiva de prueba de esterilidad
Invención de membrana en 1916
Prueba microbiológica 1932 (British Pharmacopeia; 632 – 633)
Comercialización de membranas para prueba de esterilidad
Richard Zsigmondy Membrane as a filtermaterial
Commercialization of the “membrane filtermethod“
Retrospectiva de prueba de esterilidad
Introducción en USP en 1935 (p. 469)
Re-evaluación de medios de cultivo – creación de estandares en USP 13
(1947 FTM)
Florenz Sartorius Sartorius instalaciones en 1930
Retrospectiva de prueba de esterilidad
Especificaciones: 1970 USP 18
14 días de incubación
Método de filtración de membrana (poro nominal inferior a 0.45
µm)
2009: Armonización de Farmacopeas relevantes
Eu.PH., USP & JP
Retrospectiva de la prueba de esterilidad
Sistemas:
1. Sistema abierto
2. Sistema cerrado, manualmente ensamblado
3. Pre-esterilizado, pre-ensamblado
Retrospectiva de la prueba de esterilidad
1. Sistema abierto: Filtración mediante vacío porta membranas de acero inoxidable o vidrio
glass
Procedimiento
Abrir el sistema
Colocar membranas
Cortar membranas una vez filtrado y
trnasferir en medios de cultivo
Restrospectiva de prueba de esetrilidad[Reusable Sartorius system (Schiller system)]
2. Sistema cerrado, manualmente ensamblado
Ventajas
Amplia variedad de membranas de 47 mm
comercialmente disponible
Incubación de los portamembranas
Sistema cerrado
Económico
Desventajas:
Tiempo consumido en ensamblar y
preparar el material
(limpiar, ensamblar, esterilizar)
Riesgo de daños
Riesgo de deterioro
Dificultad de evaluar / calificar
Restrospectiva de la prueba de esterilidad[Reusable Sartorius system (Schiller system)]
2. Sistema cerrado manualmente ensamblado
3. Sistema actual pre-esterilizado, pre-ensamblado, sistema cerrado
Ventajas
Sistema completamente cerrado –
elimina riesgo de contaminación o
resultados falsos positivos
elimina procedimiento de manipulación
de filtros
Pre-esterilizado Apto para uso Inmediato
no requiere validación de limpieza por
parte del usuario
Basado en la prueba de filtración de
membrana
Guía de validación disponible
Ventajas
3. Actual – pre-esterilizado, pre-ensamblado, sistema cerrado
Determinadas membranas disponibles
Inversión inicial mayor
Desventajas
3. Actual – previamente esterilizado, pre-ensamblado, sistema cerrado
Agenda
1 Introduction & History
Practical Approach & Considerations2
3 Specification & Sampling
4 Quality Control
Procedure of the sterility test in dependence of the aggregate state of the product
USP <71>
Eu.Ph. 2.6.1
Therapeutic Goods Administration (TGA)
Japanese Pharmacopeia 4.06
21 CFR 610.12 – General provisions
PIC/S
World Health Organization (WHO); 3.2 Test for sterility
Membrane Filtration
„The technique of membrane filtration is used whenever the nature of the product permits,...” (USP; Eu.PH)
Direct Inoculation
- for difficult to filter substances e.g. oils and emulsions
- for catgut and other surgical sutures for veterinary use: strands
Preparation / Process planning
Documentation of the samples to be analyzede.g. paperless by scanning -> reducing risk of false data
Verification of the correct quantity of samples
Compilation of materials
Nutrient media
Filtration units
Rinsing Buffer
Product samples
+ backup materials
Transfer of materials
Proper cleaning of work surfaces & materials
Using disinfected gloves (single use)
Transfer of materials
Prevention instead of decontamination
by using clean room dedicated materials and equipment
- pre-sterilized materials
(gamma irradiated or ETO sterilized)
- easy to clean
- VHP resistant packaging for direct use in Isolators
- e.g. double/triple packaged for easy transfer
Disinfection with proper agents
Avoiding additional contamination with particles and microorganism
Isopropyl alcohol Peroxides Quaternary ammoniume.g.
Peristaltic Sterisart®Universal pump in laminar air flow
Due to disinfection of material & devices, proper sealings needto be available
Aqueous Solutions
1. Pre-wetting of the membrane with sterile buffer Fluid A: Casein peptone solution (1g/l) pH 7,1 0,2
(PIC/S recommendation)
3. Add nutrient media FTM & TSB
4. Incubation2. immediate filtration of the sample;
Water soluble solids
2. Pre-wetting of the membrane with sterile buffer Fluid A: Casein peptone solution (1g/l) pH 7,1 0,2
(PIC/S recommendation)
4. Add nutrient media FTM & TSB
5. Incubation3. immediate filtration of the sample;
1. Dilution /dissolve Product with Fluid A
Ointments and Oils
1. Viscous oils may be diluted with isopropyl myristate, passage first by its own weight
6. Incubation5. Add nutrient media
3. Gradual pressure/ vacuum
increase during the
filtration of the sample
through the dry membrane
filter
4. Min. 3 times washing with Fluid A containingPolysorbate 80 at 10 g/L (Fluid K)(emulsifier)
2. Heating up to 40°Ceventually shortly up to 44°C (for
Ointments & Creams only)
Interpretation of Results
Periodically examination of the media for macroscopic evidence of microbiological growth at intervals during the incubation and at its conclusion (14 days)
Not turbid → complies with the test forsterility
Turbid → does not comply withthe test for sterility
High Identification to strain level
Low Identification to family level
Invalidation of sterility test
Isolates from critical aseptic processing areas
Environmental Isolates from Class A & B
Monitoring of WFI Isolates
Release testing of starting materials
Environmental Isolates from Class C & D
Rinsing and Diluting Fluids
Fluid A (Peptone water) for aqueous solutions and soluble solidsif necessary add ß-lactamase
Fluid K for oils and oily solutions (emulsifier)contains 10 g/l Polysorbate 80
Fluid D for sterile aerosol products
viability of microorganisms shouldnot be influenced
Nutrient Media
Bacteria: Fluid Thioglycollate medium (FTM)anaerobe/aerobe Incubation: 30 - 35°C, min. 14 days
Bacteria/Fungi: Soybean casein digest medium (TSB)aerobe Incubation: 20 - 25°C, min. 14 days
Other Media Equivalent commercial media may be used provided that they comply with the growth promotion test.
Antimicrobial Substances
Washing step min. 3 times max. 5 times 100 ml per filter USP <71>; Eu.Ph. 2.6.1
Modify the conditions in order to eliminate the antimicrobial activity and repeat the method suitability test (EP 2.6.1)
USP <1227> Validation of microbial recovery
Issues while performing the sterility test / validation
generation of foam reduce pump speed
low rate of filtration / optimize rinsing steps, or
blockage of membrane reduce quantity of product
difficult to filter products e.g. using direct method
(oils & emulsions)
Also check quality of materials, as rinsig Fluid A e.g., peptoneknown to interact with some products
Antimicrobial Agent / Product
Neutralizing Agent
Benzalkonium Chloride 0,01%
0,5% Lecithin & 3% Polysorbate 80
Chlorohexine Lecithine & Polysorbate 80
Parabens 5% Polysobate 80 or 0,07% Lecithin & 0,5% Polysorbate 80
Mercurial Compounds Thioglycollate (incubation at 20°C-25°C); Sodium Thiosulfate; Thioglycollate with Cystein
Azide Azolectin
Sorbic Acid Dilution & Polysorbate 80
Collagen Implant 3%Polysorbate 80
Source: Russell et al, 1978; Microbiological Update May 1999
Antimicrobial Agent / Product
Neutralizing Agent
Organic Acids Polysorbate 80
Penicillin / Cephalosporines Penicillinase (ß-Lactamase)
Chloramphenicol Chloramphenicol Acetyltransferase
Sulphonamide P-Aminobenzoic Acid
Glutaraldehyde Bisulfate
Aldehyde Glycin or Thiosulfate
Quaternary Ammonium Lecithin or Polysorbate 80
Source: Russell et al, 1978; Microbiological Update May 1999
Antimicrobial Agent / Product
Neutralizing Agent
EDTA Mg2+ or Ca2+ - Ions
Phenole Dilution
Alcohole Dilution
Bis Biguanides Lecithin
Iodine Polysorbate 80
Halogenes Thiosulfate
Source: Russell et al, 1978; Microbiological Update May 1999
Minimum Quantities to be Used for Each Medium acc. to Eu.Ph & USPQuantity per container Minimum quantity to be used for each medium unless
otherwise justified and authorised
Liquids
– less than 1 ml
– 1-40 ml
– greater than 40 ml and not greater than 100 ml
– greater than 100 ml
Antibiotic liquids
The whole contents of each container
Half the contents of each container but not less than 1ml
20 ml
10% of the contents of the container but not less than 20 ml
1 ml
Insoluble preparations, creams and ointments to be suspended or emulsified
The whole contents of each container to provide not less than 200 mg
Solids
– less than 50 mg
– 50 mg or more but less than 300 mg
– 300 mg to 5 g
– greater than 5 g
The whole contents of each container
Half the contents of each container but not less than 50 mg
150 mg
500 mg
Catgut and other surgical sutures for veterinary use 3 sections of a strand (each 30 cm long)
Minimum Number of Items to be Testedacc to Eu.Ph & USP
Number of items in the batch* Minimum number of items to be tested for each medium unless otherwise justified and authorised* *
Parenteral preparations- Not more than 100 containers
– More than 100 but not more than 500 containers
– More than 500 containers
10% or 4 containers, whichever is the greater
10 containers
2% or 20 containers, (10 containers for large-volume parenterals) whichever is less
Ophtalmic and other non-injectable preparations- Not more than 200 containers
- More than 200 containers
– If the product is presented in the form of single-dose containers, apply scheme shown for parenteral administration
5% or 2 containers, whichever is the greater
10 containers
Catgut and other surgical sutures for veterinary use 2% or 5 packages whichever is the greater, up to a maximum total of 20 packages
Bulk solid products- Up to 4 containers
– More than 4 containers but not more than 50 containers
– More than 50 containers
Each container
20% or 4 containers, whichever is the greater
2% or 10 containers, whichever is the greater
* If the batch size is not known, use the maximum number of items prescribed. * * If the contents of one container are enough to inoculate the 2 media, this column gives the number of containers needed for both the media together
Quality of materials & Sampling according to USP/Eu.Ph.
Each batch of media must be shown to be sterile by incubation at the indicated temperatures for 14 days.
In addition, each batch of medium must be shown to promote growth of test organisms — the Growth Promotion Test.
Studies showing extended shelf may be necessary
Agenda
1 Introduction & History
Practical Approach & Considerations
3
2
Specification & Sampling
4 Quality Control
Quality of materials & Sampling according to USP/Eu.Ph.
Use Membrane filters having a nominal pore size not greater than 0.45 µm
If the material being tested renders the medium turbid(...) 14 days after the beginning of incubation transfer portions (each not less than 1 ml) of the medium to fresh vessel of the same medium and then incubate the original and transfer vessels not less than 4 days.
?
?
Quality of materials & Sampling according to USP/Eu.Ph.
Nominal pore size: Any name of the pore size given by a manufacturer, without following(inter)national industrial standards e.g. because of absence of standards
not to confuse with retention rate!
Retention rate: Ability of a (membrane) filter to retain a certain quantity ofmicroorganismen / physical particles per square centimeter of activefiltration area.
What does thatmean?
*If validated as a sterilizing grade filter ≥10 Mio. microorganism per square centimeter must be retained under standard conditions
Poresize[µm]
In-House-Spec. International Spec.(ASTM)
Retention per cm2
Filtration area
0,1 A. laidlawii N.A.
0,2* B. diminuta B. diminuta ≥10 Mio
0,45 S. marcescens N.A. ≥10 Mio
0,65 L. lindneri N.A.
0,8
1,2
(Nominal) pore size defined by testing with a specific microorganism
Poresize [µm] In-House-Specification forBubble Point [bar]
International Specification
0,1 4,5 N.A.
0,2* 3,5 (3,2) N.A.
0,45 2,0 N.A.
0,65 1,8 N.A.
0,8 1,5 N.A.
1,2 1,0 N.A.
Nominal pore size in correlation to physical properties
*If validated as a sterilizing grade filter 10 Mio. microorgansim per squarecentimeter must be retained under standard conditions
Quality of materials & Sampling according to USP/Eu.Ph.
Physical test criteria for (membrane) filters:
(Water/Air) Flow rate
Integrity Test (Bubble Point , Diffusion Test, Pressure Hold Test)
ph
Thermal resistence
Thickness
Chemical compatibility
Weight
Extractables
e.g.
Quality of materials & Sampling according to USP/Eu.Ph.
Test criteria for (membrane) filters: with microbes:
Retention rate
Recovery rate
e.g.
Retention of microbes directly correlates to physical criteriassuch as Bubble Point (BP), Diffusion Test, Water Intrusion Test (WIT) e.g.
3. Integritätstestung von Membranfiltern
Safety margin
non sterile
sterile
Correlation of IT Data with Bacterial Challenge Tests Results
Quality of materials & Sampling according to USP/Eu.Ph.
...without compromising the integrity of the original sample
If the material being tested renders the medium turbid(...) 14 days after the beginning of incubation transfer portions (each not less than 1 ml) of the medium to fresh vessel of the same medium and then incubate the original and transfer vessels not less than 4 days.
Quality of materials & Sampling according to USP/Eu.Ph.
Current Situation for sampling / transfer:
Detaching of tubings
Sampling with a syringe by punching a hole into the tubing
Cutting the tubing and sampling with a syringe or pipette
All mentioned ways represent a high risk for getting a secondary contamination through environment / operator
...no Isolator form an absolute seal (cGMP-sterile drug products)
Quality of materials & Sampling according to USP/Eu.Ph.
What are possible technical solutions?
It needs to be a closed system for preventing secondary contamination to the original sample
It needs to have an accessible sampling port which
Quality of materials & Sampling according to USP/Eu.Ph.
What are the possible technical solutions?
Both solutions are already being used in medical industry
Needle less transfer Septum
Quality of materials & Sampling according to USP/Eu.Ph.
What are the possible technical solutions?
Needle less transfer
Positive:
No needle required
Negative:
Liquid can not be taken from different
positions of the Sterility Test Unit
expensive
Quality of materials & Sampling according to USP/Eu.Ph.
What are the possible technical solutions?
Positive:
Liquid can be taken from different positions
of the Sterility Test Unit
Long history in the market
Economical reasonable
Septum
Components of Sterisart®NF
Septum
SAN Housing
0,45 µm (nominal) membrane filterSartochem®
PVC TubingColour codedclamps Individual number & batch
number
Red caps tethered to thevent filter for protectionagainst overpressure & for filtration
0,2 µmHydrophobicsterilizing grade filter
Septum can be used for:
Dilution
Bacteriostasis/Fungiostasis Test
Sampling & Injection (deactivation of
antibiotics , e.g. by adding ß-lactamase)
Septum Version: Riskless Sampling & Injection
Septum can be used for:
Growth Promotion test of nutrient media
Stasis Test
Identification of positives
Rapid Microbiology
Agenda
1 Introduction & History
Practical Approach & Considerations
4
2
Specification & Sampling3
Quality Control
Quality Control of Sterisart®NF
Sartochem® Membrane:
BCT with Serratia marcescens
Bubble Point
Flow Rate
Thickness etc.
0,2 µm PTFE-Membrane
BCT with Brevundimonas diminuta
Bubble Point with IPA
Flow Rate with IPA
Flow Rate with air
Quality Control of Sterisart®NF
Tubing:
Measurement of dimension
Mesurement of inner diameter
Shore hardness
Leak Test
Equal conveyance of Liquid
Quality Control of Sterisart®NF