prueba de esterilidad consideraciones generales€¦ · retrospectiva de prueba de esterilidad...

1

Prueba de esterilidadConsideraciones Generales

Dominicana 2017

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)


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>)


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“


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


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.

Material loading of the Isolator

(Isolator)

Avoiding gas inaccessible areas

Peristaltic Sterisart®Universal pump integrated in an Isolator of SKAN AG

Peristaltic Sterisart®Universal pump integrated in an Isolator of Getinge La Calhene

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


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



Neutralizing Agent

EDTA Mg2+ or Ca2+ - Ions

Phenole Dilution

Alcohole Dilution

Bis Biguanides Lecithin

Iodine Polysorbate 80

Halogenes Thiosulfate


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


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



* 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


Practical Approach & Considerations

3

2

Specification & Sampling

4 Quality Control


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.

?

?


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


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.


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


...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.


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)


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


What are the possible technical solutions?

Both solutions are already being used in medical industry

Needle less transfer Septum



Needle less transfer

Positive:

No needle required

Negative:

Liquid can not be taken from different

positions of the Sterility Test Unit

expensive



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

+ one or two different adapters

+ plugs for closure

Components of Sterisart®NF

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


Practical Approach & Considerations

4

2

Specification & Sampling3

Quality Control

69

Sartorius contributes to health

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


Plastic Parts:

Visual Inspection

Burst Pressure of Container


Tubing:

Measurement of dimension

Mesurement of inner diameter

Shore hardness

Leak Test

Equal conveyance of Liquid


Growth Promotion Test

Firm connection of needles

Pressure-hold-test


We sincerely invite you

to our facilities for a

visit

technical meeting

audit

77

Thanks for your attention!

prueba de esterilidad consideraciones generales€¦ · retrospectiva de prueba de esterilidad...

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