EXCELLENCE IN MICROBIOLOGY

Microbiological Control
for Non-Sterile Pharmaceuticals

Pharmig Monograph No. 2

Pharmaceutical Quality Group Monograph No. 12
 Pharmig

Pharmaceutical Quality Group of The Chartered Quality Institute

Pharmig Monograph No. 2

PQG Monograph No. 12

MICROBIOLOGICAL CONTROL
FOR NON-STERILE PHARMACEUTICALS
November 2008

EXCELLENCE IN MICROBIOLOGY
 Contents

Contents
Page
General Introduction ii
Foreword v
From the Chairmen vi
Acknowledgements vii

Chapter 1 Introduction 1
Chapter 2 Roles and Responsibilities 5
2.1. Introduction
2.2. Microbial Governance
Chapter 3 Personnel 7
3.1. Introduction
3.2. Training
3.3. Hygiene
3.4. Dress Code/Changing
Chapter 4 Facilities 11
4.1. Introduction
4.2. Area Classification
4.3. Access to Areas
4.4. Building Requirements
4.5. Facility Qualification
Chapter 5 Cleaning and Disinfection 23
5.1. Introduction
5.2. Facilities
5.3. Equipment
5.4. Cleaning Agents
5.5. Validation of Cleaning
Chapter 6 Microbiology Laboratories 31
6.1. Introduction
6.2. Relationship to the Quality Unit
6.3. Microbiology Laboratory Facility Design
6.4. Autoclaves
6.5. Disinfectants
6.6. Media
6.7. Micro-organisms
6.8. Identification
6.9. Microbiological Testing of Product, Starting Materials and Intermediates
Chapter 7 Risk Assessment and Management 37
© 2008 Pharmig and The Chartered Quality Institute 7.1. Introduction
7.2. Risk Assessment Associated with Non-Sterile Products
All rights reserved. No part of this publication may be reproduced, stored 7.3. Water Activity
in a retrieval system, or transmitted in any form or by any means, without Chapter 8 Microbiological Monitoring 43
the written permission of Pharmig and The Chartered Quality Institute. 8.1. Introduction
8.2. Environmental Monitoring Policy
8.3. Designing the Microbiological Monitoring Programme
Published by Pharmig and The Chartered Quality Institute
Chapter 9 Reporting and Trending of Microbiological Data 61
ISBN 978-0-9560804-0-0 9.1. Introduction
9.2. Batch-Specific Data
9.3. Periodic Summary Reports
This monograph is available from: 9.4. Out of Specification/Out of Trend Handling
Pharmig, T5 The Maltings, Roydon Road, Stanstead Abbotts,
Hertfordshire SG12 8HG, United Kingdom. Appendix Mind Maps 65
www.pharmig.org.uk Glossary 69
Bibliography 73
The Chartered Quality Institute, 12 Grosvenor Crescent,
London SW1X 7EE, United Kingdom.
www.thecqi.org

Microbiological Control for Non-Sterile Pharmaceuticals Microbiological Control for Non-Sterile Pharmaceuticals i
 General Introduction

General Introduction

The Pharmaceutical Quality Group Pharmig EXCELLENCE IN MICROBIOLOGY

The Pharmaceutical Quality Group in the United Kingdom developed from a small group of
pharmaceutical quality executives who met initially in 1977 to assist in the preparation of
a supplier questionnaire for discussion at the 1978 seminar of the European Organisation
for Quality Control. The group has expanded since that time and is now incorporated with
the Chartered Quality Institute.
The objectives of the group are:
1. The open exchange of information and experience concerning pharmaceutical quality
matters.
2. The development of a unified approach to pharmaceutical quality assurance and good
manufacturing practices.
3. To promote and represent the status of pharmaceutical quality assurance professionals.
4. To promote education and training in the achievement of pharmaceutical quality.
The Pharmaceutical Quality Group initiated a project in 1983 with the intention of writing
a series of monographs, the prime objective of which was to augment the published,
official Codes of Good Manufacturing Practice by providing practical, comprehensive
and non-mandatory guidelines. The target audience was identified as management and
supervisory personnel engaged in the manufacture and supply of medicines, with particular
emphasis on their future training and education.
The Pharmaceutical Microbiology Interest Group (Pharmig) is a non-profit making
professional organisation, established in 1991, that represents the interests of individuals
who work in, have responsibility for, or work alongside microbiology.
Pharmig is the influential forum for microbiology in the pharmaceutical, healthcare and
allied industries. It provides a focus for continuing professional development and serves as
a unique network for the exchange of microbiological information through training courses,
conferences, publications and its website forum.
Initial meetings were held at members’ places of work and included site tours. The first Annual
Conference was held in 1992. This has now become a well-established annual diary event.
The Group has grown significantly since 1991 expanding the portfolio of products it now offers
to the Membership whilst remaining true to the initial needs of microbiologists which include:
Organising meetings, training courses and conferences that provide topical information
and views on microbiologically related topics
Advancing the science of microbiology and its practical application
Influencing the development of regulations and guidelines surrounding microbiology
Acting as a confidential forum for the dissemination of information concerning all
aspects of microbiology

The series of monographs currently contains the following titles: Publications

1. Pharmaceutical Premises and Environment Pharmig launched its first publication – ‘A Guide to Disinfectants and their use in the
Pharmaceutical Industry’ in 2006 (Monograph No.1).
2. Pharmaceutical Manufacturing (Processing and Packaging)
3. Elements and Philosophy of Pharmaceutical Quality Assurance A series of Limulus Amoebocyte Lysate (LAL) Fact Sheets on pyrogen/endotoxin testing
have also been produced by the LAL Action Group and range from ‘What is LAL/BET?’
4. Pharmaceutical Distribution
through to ‘Product Validations – Quantitative Methods’.
5. Pharmaceutical Auditing
Pharmig produces a quarterly technical newsletter for its members and has conducted,
6. Bulk Pharmaceutical Chemicals – (withdrawn)
in the past, various surveys that include Non-sterile manufacturing, Aseptic Manufacturing,
7. Pharmaceutical Contract Manufacture and Analysis Environmental Monitoring, Water Systems and Disinfectants.
8. Pharmaceutical Documentation
This monograph on ‘Microbiological Control for Non-Sterile Pharmaceuticals’ is Pharmig’s
9. Pharmaceutical Packaging Validation (produced jointly with The Packaging Society) first publication in collaboration with PQG.
10. Cleaning Validation
11. Good Quality Control Laboratory Practice

ii Microbiological Control for Non-Sterile Pharmaceuticals Microbiological Control for Non-Sterile Pharmaceuticals iii

Pharmaceutical Microbiology Advanced Training (PMAT):
Following a collaborative effort by Pharmig and the University of Manchester, a Master’s
Degree in Pharmaceutical Microbiology Advanced Training (PMAT) was launched in
September 2007. This is a university accredited qualification in pharmaceutical
microbiology for the medical and healthcare industry. It is a modular, distance-learning
programme designed for scientists and managers in the field of Pharmaceutical
Microbiology, the first of its kind in Europe.
iv Microbiological Control for Non-Sterile Pharmaceuticals
Foreword
Foreword
“Dead flies cause the ointment of the apothecary to send forth a stinking savour” was the
ancient observation of King Solomon as recorded in the Bible (Ecclesiastes Chapter 10,
verse 1). This early reference to a defective medicine was clearly the result of microbial
spoilage of a non-sterile pharmaceutical arising from inadequate control. Whilst this
historical reference might be expected through lack of scientific understanding,
pharmaceutical manufacturers today have no such excuse!
The focus of microbiological control of pharmaceuticals has been largely, and rightly,
directed to sterile products. However, there is a clear expectation from the MHRA and
other Regulators that the microbiological contamination risks associated with non-sterile
products are fully understood and that appropriate measures are taken to avoid
contamination during manufacture.
I welcome this monograph from Pharmig and PQG as the existing guidance on the
microbiological control of non-sterile pharmaceuticals is limited. This is a comprehensive
guide on all aspects of microbiological control from facility design to staff training which
taps into a great deal of useful experience from the industry. Whilst this guidance may
need to be interpreted for particular applications, the principles should be applicable
across the whole range of non-sterile manufacture.
It is good to see the emphasis on risk analysis to justify levels of microbiological control.
The reporting and investigation of microbiological excursions and the need for results
trending are also helpfully discussed.
I am delighted to commend this monograph which I feel sure will further strengthen the
endeavours of both Industry and Regulators to protect the public through the provision of
safe and effective medicines which are free from undesirable micro-organisms.
Gerald W Heddell
Director
Inspection, Enforcement and Standards Division
Medicines and Healthcare products Regulatory Agency
Microbiological Control for Non-Sterile Pharmaceuticals v

From the Chairmen
This monograph is the result of a collaborative effort between members of the PQG and
Pharmig. It represents the recommendations and opinions of a number of individuals
experienced in different aspects of microbiological control for non-sterile pharmaceuticals,
which have been integrated by our joint editors.
The PQG and Pharmig both strive to provide guidance on subjects where there is minimal
regulatory guidance or legislation available, as is the case with this subject. In the absence
of guidance, it is easy to either not do enough, leading to a risk of patient harm and/or
regulatory censure, or to do too much, thus adding costs without benefit. This is especially
the case with non-sterile pharmaceuticals due to the wide range of products that fall
under this banner and the consequent wide range of risk profiles. The information provided
is intended to be applicable to all non-sterile dosage forms, including liquids, creams,
ointments, tablets, capsules, powder inhalers, aerosols etc. It is applicable to herbal and
over-the-counter products as well as prescription pharmaceuticals.
Any responsible manufacturer of non-sterile dosage forms should ensure that:
A thorough assessment is conducted of the risks that may be posed to patients by
their particular product portfolio. This should include the raw materials used, the
process and the premises in which manufacture takes place
Based on this risk assessment document, actions are implemented to control the risks
to an acceptable level and appropriate monitoring is carried out to demonstrate this
control
All personnel involved in the manufacture are aware of the potential risks, their
sources and what steps they need to take to ensure that control is maintained
Using these principles and the detailed information that follows, manufacturers should be
able to select and apply the appropriate control and monitoring measures to protect the
well-being of their patient customers without excessive costs and use of resources.
Consequently, this monograph should contribute to the success of the organisation.
Ashley McCraight Stewart Green
Chairman, Pharmaceutical Quality Group Chairman, Pharmig
vi Microbiological Control for Non-Sterile Pharmaceuticals
Acknowledgements
Acknowledgements
The concept of a monograph for Microbiological Control for Non-Sterile Pharmaceuticals
was first discussed at the joint PQG/Pharmig meeting in 2001 and raised by PQG’s then
chairman Peter Gough. The monograph was initiated by Kerry Hawitt and overseen by a
steering team comprising Philip Butson, Stewart Green, Kerry Hawitt, Bob Johnson and
Ashley McCraight.
The monograph was edited by Philip Butson and Kerry Hawitt.
Original chapter drafts were prepared by Richard Burt, Derek Davey, Rob De Raaij, Kerry
Hawitt, Gail Henry, Julia Mottishaw and Trevor Munton with additional authoring by Philip
Butson and Kerry Hawitt.
Thanks are due to the following additional individuals for their contributions as reviewers:
Ian Anderson, Kathy Armstrong, David Begg, Elaine Doyle, Wanda Jay, Bob Johnson,
David Keen, Ashley McCraight, Colin Newbould, Wendy Pimblett, Tim Sandle, Kirit Sanghani.
Additional thanks are due to the following who provided feedback, either individually or on
behalf of their representative organisations, during the consultative review process:
Mike Murray – Association of the British Pharmaceutical Industry
Penny Viner – Herbal Forum
Mr R S Iyer – Indian Pharmacopoeia
Richard Funnell – UK Medicines and Healthcare products Regulatory Agency (MHRA)
John Harwood – UK National Health Service (NHS) Pharmaceutical Production Committee
Liz Allanson
Tony Cundell
Peter Gough
William Miele
Erica Notman
Norman Randall
Donald Singer
Scott Sutton
Not surprisingly given the lack of clear regulatory expectations and the range of individuals
consulted, a number of different opinions were expressed by the various reviewers. The final
decisions regarding content were made by the editors and the naming of an individual above
should not be taken as their agreement with the whole content of the final monograph.
Microbiological Control for Non-Sterile Pharmaceuticals vii
 Chapter 1 – Introduction

Chapter 1

Introduction
1.1. Microbiological Control for Non-Sterile Pharmaceuticals

The pharmaceutical industry is highly regulated by the application of the principles of Good
Manufacturing Practice (GMP). In most countries, government agencies provide guidance
to pharmaceutical manufacturers, which is intended to facilitate the manufacture of safe,
unadulterated and efficacious drug products.

The sterile pharmaceutical sector has a well defined set of expectations and regulations
which provide clear statements relating to microbiological controls and monitoring. In
contrast, the expectations for non-sterile pharmaceuticals are poorly defined, with few
specifics written in either legislation or guidance publications.

However the expectations are implied in various places within the GMP guidance, for example:
EU GMP 1.4 ‘Quality Control’ requires “procedures [to be] available... where
appropriate for monitoring environmental conditions for GMP purposes”
EU GMP Section 5 ‘Production’ states that “at every stage of processing, products and
materials should be protected from microbial and other contamination” (5.10); “cross-
contamination should be avoided by appropriate technical or organisational measures”
(5.19) and “measures to prevent cross-contamination and their effectiveness should
be checked periodically” (5.20)
EU GMP Annex 7 ‘Manufacture of Herbal Medicinal Products’ states that “effective
measures should be taken to prevent the spread of... micro-organisms brought in with
the crude plant and to prevent cross-contamination”
EU GMP Annex 9 ‘Manufacture of Liquids, Creams and Ointments‘ notes that “Liquids,
creams and ointments may be particularly susceptible to microbial and other
contamination during manufacture. Therefore special measures must be taken to
prevent any contamination”
CFR Part 211.113 ‘Control of microbiological contamination’ states that “appropriate
written procedures, designed to prevent objectionable micro-organisms in drug product
not required to be sterile, shall be established and followed”
USP 31-NF26 <1111> ‘Microbiological Attributes of Non-sterile Pharmaceutical
Products’ includes the following:
“Strict adherence to effective environmental control and sanitation, equipment
cleaning practices, and good personal hygiene practices in pharmaceutical
manufacture is vital in minimising both the type and the number of micro-organisms”
“Monitoring, in the form of regular surveillance, should include an examination of
the microbiological attributes of Pharmacopeial articles”

viii Microbiological Control for Non-Sterile Pharmaceuticals Microbiological Control for Non-Sterile Pharmaceuticals 1

“It is essential that ingredients and components be stored under conditions
designed to deter microbial proliferation”
The Therapeutic Goods Association of Australia (TGA) states that “it is not a
requirement that every batch of product be tested for microbial content at release.
Instead, for each product manufacturers should aim to develop confidence that the
manufacturing procedures do not permit contamination by excessive numbers of
microbes or by pathogenic organisms”. The TGA also require absence of all
pseudomonads not just Pseudomonas aeruginosa.
The regulatory agencies therefore expect the industry to take a risk based approach to
microbiological control and apply appropriately justified monitoring in the manufacture of
non-sterile pharmaceuticals.
Generally, companies understand and embrace the need for delivering microbiological
control. However, there is a broad spectrum of interpretation on how to deliver
appropriate control across industry.
For some companies, the establishment of initiatives to deliver microbiological control and
monitoring has required extensive efforts from Microbiology and QA staff to define and
implement procedures from basic principles. This effort is being repeated many-fold across
the industry; each company ‘reinventing the wheel’ in different forms and to different
extents owing to the lack of defined published criteria.
The original Parenteral Drug Association (PDA) Technical Guideline 13 indicated that the
approach used for the monitoring of sterile manufacturing environments could be applied
to non-steriles. If a company interprets this as applying the full remit of environmental
monitoring for sterile product manufacture to non-sterile manufacturing, then the result
will be costly and in excess of what is warranted by a risk based approach. Such an
approach is not recommended in this monograph.
The Pharmaceutical Research and Manufacturers of America (PhRMA) 1997 industry
review stated that routine monitoring of non-sterile manufacturing facilities should not be
mandatory, but dictated by the circumstances and required microbial quality of the drug
product manufactured. This is the forerunner of the application of risk based assessment
to determine and justify appropriate monitoring and controls, which is recommended by
this monograph.
Cundell (2005) summarises the results of three other surveys in the US dating from
1994, 1998 and 2002 in which most of industry was shown to conduct some form of
viable monitoring, albeit at varying frequencies and using various techniques.
In 2002, Pharmig conducted a survey of its membership and found that 81% of
respondent companies had a programme in place for their non-sterile manufacturing
facilities. However, there was considerable variation in the scope and execution of such
programmes. The survey was repeated in 2007 and showed an increase in the
percentage of companies surveyed who had adopted a programme, reflecting the evolution
2 Microbiological Control for Non-Sterile Pharmaceuticals
Chapter 1 – Introduction
of this expectation in the industry; all responders had a programme in place. There was
still significant variation in the scope and execution of monitoring programmes, and whilst
this could be partly due to variation in the facilities and operations, it was clear that it was
not always driven by a risk assessment.
This monograph has been created with the input of experts from across the industry
based in the UK, Europe, US and India. The principal objective is to provide the
pharmaceutical and associated industries with an international bench mark and guidance
on technical best practices for microbiological control and monitoring in manufacturing
facilities for non-sterile pharmaceuticals.
The monograph emphasises a risk based approach rather than providing a ‘one size fits
all’ solution and seeks to encourage organisations to understand their premises, products
and processes prior to deciding on their approach. In this way, manufacturers should be
able to select and apply appropriate control and monitoring measures to protect the well-
being of their patient customers without excessive costs and use of resources. In the
majority of cases, the microbiological risk associated with non-sterile products is low and
extensive monitoring should not be required.
Through the review process of this monograph, it was identified that there are currently
significant differences in approaches, largely driven by differing regulatory pressures,
between the US and EU. This is an important consideration for facilities that are part of a
global supply strategy and therefore need to comply with the minimum expectations of all
countries. However, the effective use of a risk based approach should result in decisions
regarding control and monitoring which have global application.
The considerations in this monograph may be applied to handling any non-sterile material
including starting materials, radio-pharmaceuticals, Investigational medical products
(IMPs), drug substances and all formulations of non-sterile drug products, in both the
private and public sectors.
The fundamental requirements for good microbiological control in non-sterile manufacturing
and quality assurance are established through cross functional management oversight,
qualified and trained personnel, and well designed and maintained facilities, supported by a
suitable microbiological laboratory function. This infrastructure is addressed in Chapters 2
to 6 respectively.
It should be recognised that microbiological monitoring is a mechanism for understanding
microbiological control and the potential risk to the product and patient, not a control
measure in itself. It is important that manufacturers understand and control the microbial
hazards within their facility. There should be an understanding of the ongoing performance
of the facility. The risk based assessment may be used to minimise and target any
monitoring activity to check periodically the effectiveness of the measures to prevent
cross-contamination required by EU GMP 5.20.
Microbiological Control for Non-Sterile Pharmaceuticals 3

Once the infrastructure has been evaluated a documented risk assessment can be carried
out to develop an appropriate monitoring programme which should be subject to periodic
review, this is considered in Chapters 7 and 8.
Data derived from such programmes should be analysed and interpreted to provide a
basis for the on-going risk assessment process. The reporting and trending of data is
considered in Chapter 9.
Some mind maps are included in an Appendix. These may be useful as memory joggers
or for audit purposes.
4 Microbiological Control for Non-Sterile Pharmaceuticals
Chapter 2 – Roles and Responsibilities
Chapter 2
Roles and Responsibilities
2.1. Introduction
Historically, the need for microbiological monitoring and control in non-sterile manufacture has
been challenged. Recent surveys suggest that monitoring is now generally practiced, but this
does not necessarily mean that the importance of comprehensive microbiological control has
been recognised. Just as quality is embedded as part of everybody’s responsibility,
safeguarding against microbial contamination should also be seen as everybody’s responsibility.
Quality Assurance’s role is to set standards, policy and provide guidance and oversight with the
site Microbiologist leading programmes of good microbiological control, in cooperation with the
Qualified Person (in Europe).
This is equally applicable and adaptable to manufacturing facilities of all sizes and types,
e.g. classic pharmaceutical manufacturers or specialist hospital units.
2.2. Microbial Governance
Top level microbial oversight ‘governance’ should be driven by senior level site management
and not limited to the senior Microbiologist or QA manager. Clear direction needs to be
given to the site emphasising that microbiological contamination control is a key factor in
Good Manufacturing Practice.
Multi-functional involvement of representatives from manufacturing (technical and
operations), engineering, QA management and QC Microbiology should have a collective
responsibility to ensure the appropriate quality systems are in place to ensure
microbiological control (Figure 2.1).
Within Europe the Qualified Person (QP) should also be included since The Code of Practice
for Qualified Persons (QP COP) includes requirements for QPs to consider all relevant
factors “including any not specifically associated with the output batch directly under review
(e.g. calibration and maintenance records, environmental monitoring)” (QP COP 7.9).
The Code of Practice for Qualified Persons also emphasises the need for the QP to consult
with other relevant experts on matters such as “potential environmental or microbiological
risks” (QP COP 7.11). The QP is dependant upon the knowledge and expertise of his/her
colleagues to enable them to fulfil their legal obligations (QP COP 7.14).
Microbiological Control for Non-Sterile Pharmaceuticals 5

There should be governance of every aspect of site functionality that could impact
microbial contamination and control. Direction, prioritisation and appropriate review and
action are required relating to:
Change control
Facility and engineering projects
New product introductions
Risk assessments
Monitoring data
Environmental monitoring performance, as determined by the risk assessment
Product, intermediate, starting material and water microbiological testing
Deviation handling
In addition to the normal roles and responsibilities in the quality management system,
specific microbiological expertise should be used in the following:
Risk assessment (Chapter 7)
Determination of appropriate monitoring as per the risk assessment (Chapter 8)
Setting of appropriate action and alert levels (Chapter 8)
Trending and Interpretation of testing and environmental monitoring data (Chapter 9)
In situ support to operational areas
Design and delivery of training (section 3.2)
Review of/consultation on equipment, system and facility designs to ensure they are
hygienic and thus minimise future contamination risks
Ensure appropriate product/starting material specifications are set from a
microbiological perspective (Ph Eur 5.1.4)
Investigation of Microbiological Deviations, Out of Specification (OOS)/Out of Trend
(OOT) data (section 9.4)
A site should have access to someone with appropriate training and background to make,
communicate and influence good assessments and decisions from a microbiological
perspective. This will typically, but not necessarily, be a graduate microbiologist but must
be someone with documented experience and appropriate qualifications. It may be
necessary to contract this resource from a reputable third party supplier/consultant.
6 Microbiological Control for Non-Sterile Pharmaceuticals
Chapter 3 – Personnel
Chapter 3
Personnel
3.1. Introduction
A potential source of microbiological contamination for pharmaceutical products comes
from the personnel. This includes not only the production operations staff but also
personnel from other disciplines including engineers, QA, QC sampling, management,
security personnel, cleaning staff and contractors; in short, anyone who may enter the
production areas.
All individuals who enter the production areas at any time therefore need training on
matters relating to microbiological control, in particular hygiene. Access to production
areas must be controlled to ensure entry is restricted to trained personnel and
accompanied visitors.
Production personnel are in a position to directly impact the microbiological quality of
products and should be given a basic microbiological awareness to underpin procedural
requirements for hygiene and good working practices.
3.2. Training
There is a general requirement for manufacturers to provide training in accordance with
GMP for all personnel whose activities may affect the quality of the product.
Pharmaceutical manufacturing sites have a broad spectrum of personnel in terms of
education and experience. Typically only a minority of staff will have microbiological
background knowledge. It is therefore important to provide appropriate levels of training to
all relevant functions as part of the overall microbiological control arrangements. The
training of staff, including any contract staff, should emphasise the importance of their
particular role in safeguarding the products made in the facility from contamination.
In relation to the manufacture of non-sterile pharmaceutical products it is important to
cover the following from a microbiological perspective:
All staff:
Introduction to micro-organisms and microbiological contamination control
Entry and exit of production facilities (including gowning)
Personal hygiene training
Microbiological Control for Non-Sterile Pharmaceuticals 7

More specialised training, dependant on role:
Environmental sampling, monitoring and control
Microbiological risks associated with specific production tasks
Risk assessment and design of microbiological control features
Classified area/Clean room practice (if required by facility/formulation)
Statistical/data analysis
Training must be documented and regularly reviewed. It is important that training is
effective and competency should be assessed to demonstrate this (EU GMP 2.9).
Training should be tailored on a facility by facility basis with an appreciation of the specific
risks associated with the product formulations and facility design.
Trainers should be appropriately communicative, knowledgeable and experienced in the
subject to provide adequate training and answers to technical queries.
Records/documents e.g curriculum vitae, training records etc, demonstrating the
trainer’s competency should be reviewed/archived.
3.3. Hygiene
There is a general requirement for manufacturers to ensure hygiene programmes are
established, understood and implemented. The programmes should be adapted to the
different needs of the facility and cover the following:
Health
Hygiene
Clothing of personnel (section 3.4)
Typically, for non-sterile manufacture the hygiene programme will include:
Medical examination upon recruitment
A procedure for the notification of health conditions that may affect product quality
and subsequent actions to prevent contact with starting materials, primary packaging
materials and manufactured products by those personnel with:
Infectious disease
Open lesions on any exposed part of body
Shedding skin conditions, such as eczema or psoriasis (skin scales may harbour
objectionable micro-organisms that may impact pharmaceutical products and
patients)
Gastric upsets
Steps to be taken to prevent direct contact between the operator’s skin and any
pharmaceutical product/product contact equipment
8 Microbiological Control for Non-Sterile Pharmaceuticals
Chapter 3 – Personnel
The prohibition of eating, drinking, chewing or smoking, or the storage of food, drinks,
smoking materials or personal medication in the production or storage areas
A requirement for personnel to wash their hands before entering the manufacturing
facility
3.4. Dress Code/Changing
Every person entering the manufacturing area should wear protective garments
appropriate to the operations to be carried out (EU GMP 2.16). Non dedicated clothing
should not be allowed in the production areas.
In general, a non-sterile manufacturing environment will require the wearing of suitable
protective over-garments, hair cover (e.g. mob cap), beard or moustache cover (where
applicable) and overshoes. Dedicated shoes are recommended for personnel working in
production areas for long periods.
Although it is only an explicit GMP requirement to remove jewellery and make up for sterile
manufacture (EU GMP Annex 1), consideration should be given to requiring the removal of
jewellery, in particular rings with stones and earrings, and make up by staff involved in
non-sterile manufacture.
One piece over-garments are often used as they provide a better level of protection to
both the operator and the product.
Visitors not entering production rooms may only require a laboratory coat, rather than a
one piece over-garment.
Over-garments may be disposable or washable. Procedures should be in place to manage
dirty garments and safeguard against cross-contamination. Additional procedural controls
should be in place to provide assurance over any laundry process. Consideration should be
given to:
The need for qualification of the laundry process
Repair and retirement/replacement of garments
Third party (i.e. outsourced laundry operations)
Quality assessment
Technical agreement
Gloves must be worn when performing production activities (including the handling of
components) to ensure there is no contact between operators’ hands and exposed
products or any part of equipment that comes into contact with product (EU GMP 2.18).
Frequently, the wearing of gloves is required as part of company procedures for entry into
production areas to provide a greater level of contamination control.
Microbiological Control for Non-Sterile Pharmaceuticals 9
 Chapter 4 – Facilities

Typically, the requirement for wearing of masks is driven by personnel protection Chapter 4
considerations. However, masks may be required for product protection when manufacturing
certain higher risk products such as aqueous inhaled products and those formulations with a
higher availability of water (Chapter 7). Facilities
4.1. Introduction

The design and operation of pharmaceutical facilities are critical to microbiological control.
Whereas there are well defined criteria for the design and operation of sterile manufacturing
facilities, there are few specific regulatory requirements provided for the manufacture of non-
sterile products. Whilst this provides for greater flexibility in interpretation, it also leads to a
greater need to assess risk and justify facility design and working practices.

There are a number of potential sources of microbial contamination within a

pharmaceutical facility, including:
Personnel
Water
Air/compressed gases
Product (active, starting materials, packaging components)
Equipment
Consumables and tools
Pests and vermin
Facility design and layout
Process design

Personnel and material flows should be considered when planning the facility and
processes. For example, when working with herbal products the risk of microbial
contamination from the starting materials may be significant and additional care should be
taken with staff movement and production scheduling.

Facility and equipment design cannot eliminate microbial contamination, but good hygienic
design can mitigate several of these risks. For example, ergonomic design can help people to
work more hygienically; well designed water and HVAC systems minimise microbial ingress
and aid monitoring; equipment designs with smooth surfaces of appropriate materials can
facilitate effective cleaning; points of access for pests and vermin should be minimised.

10 Microbiological Control for Non-Sterile Pharmaceuticals Microbiological Control for Non-Sterile Pharmaceuticals 11
 Chapter 4 – Facilities

4.2. Area Classification capability of the facility design but does not require the application of a formal area
classification. See Chapter 7 for further consideration of risk assessment.
This is a complex subject and this chapter can only summarise the overall position.
Typically for non-sterile manufacture, formal area classifications are not required. The The design of an appropriate facility will consider not only the air supply quality, but also
practical achievement of appropriate microbiological control should be a focus in addition aspects such as air change rates and pressure differentials which should reflect the
to chemical cross-contamination control and environmental health and safety concerns. potential risks.
However, facilities may be designed to achieve a given specification which will be assessed
during the commissioning. For example, a tablet manufacturing facility may be designed to There are no regulatory specifications for pressure differential or air change rates for non-
achieve an equivalent of EU GMP Grade D (at rest), if tested, but would not be formally sterile products. Current industry practice is to have pressure differentials of 10–20 Pa
classified as such. between areas of different standards. For lower risk activities, air change rates are
typically 10–15 per hour. Higher risk activities have higher air change rates, typically
The engineering standards for area classifications are based on the number and size of greater than 20 per hour.
particles in the air. Within the pharmaceutical industry it is the viable particles that are of
greater concern. This is recognised by the EU GMP Guide (Annex 1), for example, which The facility design also has a bearing on appropriate operational practices. For example,
incorporates recommended microbial limits within the area grades. The setting and where a processing room is at negative pressure to a corridor for the containment of
monitoring of appropriate microbial action and alert levels for non-sterile manufacturing is active ingredients, there is an increased risk of microbial ingress from the corridor.
discussed in Chapter 8. Consequently, an effective cleaning regime for the corridor is required to ensure that
microbial levels are kept low.
A number of different standards and nomenclature have been used across the
pharmaceutical industry. Table 4.1 summarises these and provides approximate Many pharmaceutical companies and engineering design houses have evolved their own
equivalencies. It should be noted that the US Federal Standard 209E has been withdrawn guidelines and engineering specifications based on experience. These are of value, but the
but it is given for reference because its terminology is still commonly used. importance of specifying and designing a facility based on an assessment of risk to the
product and patient is emphasised. The use of specific risk assessments as the basis for
the standards applied will result in different facilities applying different standards to the
EU GMP ISO 14644 US Federal 0.5µm 5.0µm manufacture of non-sterile products.
Annex 1 2008 1999 Pt 1 Standard 209E particles/m3 particles/m3
‘At Rest’ (withdrawn) Where higher levels of product protection are required, e.g., for inhaled products, a
graduated control standard with additional garment changes may be built into the design
A-B 5 100 3,520 Grade A =20
and incorporated within operational procedures.
Grade B = 29
C 7 10,000 352,000 2,900 Older facilities may have inherent design issues which are sub-optimal. For example some
D 8 100,000 3,520,000 29,000 manufacturing facilities may have been constructed from materials no longer considered
appropriate to the manufacturing environment, such as high and irregular ceilings or
unprotected light fittings. Where older facilities have such deficiencies compared to
Table 4.1: Different Standards for Air Classification current standards, these should be captured within a risk assessment and plans created
to address them through refurbishment, or justifications written to accept them in
association with appropriate operational procedures.

The EU GMP Guide Annex 1 also provides ‘in operation’ particle standards for each grade
(except class D), which are relevant for sterile manufacture, but typically not required for 4.3. Access to Areas
non-sterile production areas.
It does not matter how well a facility is designed unless it is operated appropriately and well
In the absence of specific requirements for non-sterile products, the design and monitoring maintained, and an important consideration is access to the area by personnel and materials.
requirements of the facility should reflect the risk profile of the products manufactured.
So, for example, the level of control applied might increase moving from tablets to
creams/liquids on the basis of available water and the level of control applied to inhaled
products enhanced due to route of administration. This may require an increase in the

12 Microbiological Control for Non-Sterile Pharmaceuticals Microbiological Control for Non-Sterile Pharmaceuticals 13

4.3.1. Personnel Access
Access to the facility must be restricted to personnel that are fully trained and assessed
as competent to work in that area, who are appropriately attired (see Sections 3.2 – 3.4).
All visitors should be accompanied at all times by fully trained personnel. Access is
normally restricted by the use of an electronic access control system, or similar.
Personnel access must only be made via changing rooms. The changing room design
contributes to the assurance of appropriate personnel access and microbial contamination
control.
Hand washing facilities should be included in the design of changing rooms and used prior
to access to the production areas (EU GMP 2.19). It is useful to have a ’step over’ line or
bench to define the boundaries of clothing requirements. An interlock may be appropriate
in some facilities.
The requirements for area entry must be written into procedures and it is usual to have
pictures of correctly clothed personnel and mirrors to enable staff to check them before
entry. A copy of the gowning procedure should be present in the changing room.
The changing room should be provided with filtered air. Intermediate (bag) filters will
typically be suitable for this purpose, though High Efficiency Particulate Air (HEPA) filtration
may be used. The air pressure should be negative with regards to the manufacturing area
corridor, but positive relative to external adjacent areas.
4.3.2. Materials Access
Materials include active pharmaceutical ingredients, starting materials, in process
materials, packaging components, consumables and tools, which may be brought into the
facility. These are all potential sources of microbial contamination and it is essential that
suppliers and delivery process of these items are assured to ensure that quality is
maintained. This is especially the case for materials of biological origin.
Warehouses should be well ventilated, cool and low in humidity; materials should be
checked regularly and used as quickly as possible especially in the case of herbal materials.
Dispensing operations and traffic within a facility should minimise dust carryover and hence
risk of cross-contamination of the production areas.
Materials access must be via specified routes – generally via air locks, although certain
items when justified may be introduced via the personnel changing rooms, e.g. equipment
for environmental monitoring.
14 Microbiological Control for Non-Sterile Pharmaceuticals
Chapter 4 – Facilities
Wooden pallets and cardboard cartons should not be taken into manufacturing or
packaging areas. Whenever possible, carts, pallet jacks, trolleys, etc. should be dedicated
to the production area.
Active pharmaceutical ingredients, starting materials and packaging components will have
been dispensed for a specific job and should be provided in appropriate clean, sealed
impermeable (e.g. plastic/stainless steel) containers.
In-process materials and consumables should also be supplied in sealed, clean plastic bags
and, if these are delivered in cardboard boxes, the plastic bags should be removed from
the boxes before being taken into the area.
All containers taken into processing areas should be clean and dry.
Tools (Chapter 5.3.1) have the potential to contaminate the equipment and product
contact parts and their introduction to the facility needs to be appropriately controlled with
consideration given to cleaning and sanitisation requirements. Similarly, the introduction of
new equipment and tooling into a production environment should be assessed and
appropriate actions taken to ensure that contamination is not brought into the facility.
4.4. Building Requirements
The design of the building and level of product protection provided by the facility should be
determined by the nature of the operations intended to be carried out.
4.4.1. Surfaces
Materials of construction, surface finish and general design also need to be reflective of
the different operating and cleaning regimes.
Non-sterile manufacturing areas need to have smooth surfaces that will not harbour
micro-organisms and can withstand the routine cleaning regimes required.
Floors should be constructed of durable cleanable materials with minimal joins and will
typically be epoxy resin* or sheet vinyl, not vinyl tiles. Terrazzo is used less in new facilities,
but may still be used for areas with potentially very high traffic and heavy moving loads.
*Note: Epoxy resin may have grit included to improve traction. However there can be
microbiological and general GMP implications arising from this – over time, this can
wear-out of the resin leading to potential for contamination and poorly sealed floors.
At points of entry to the facility or to rooms, “sticky” mats or polymeric flooring areas can be
beneficial in the prevention of cross-contamination by removing particulate material from the
soles of shoes or wheels. However, they should be frequently maintained (e.g. daily) so that a
fresh surface is presented and they do not themselves become a source of contamination.
Microbiological Control for Non-Sterile Pharmaceuticals 15

In older facilities, walls may be made of plaster and simply emulsioned. Typically,
elastomeric or flexible paints are now being used instead of emulsion. The latter may be
suitable for low risk applications only. Increasingly, areas are being fitted out with walls
made from proprietary pre-finished coated panels. These are more expensive but have the
advantages of quick construction, greater flexibility if room layouts need to be changed and
greater hygienic control.
Windows/viewing panels should be non-opening, encased, flush with the wall panels and
appropriately sealed to prevent collection of dust and microbial matter.
Ceilings can be a simple silicone sealed lay-in grid, but these are difficult to clean properly
and cannot withstand more than a minimal overpressure. It is therefore conventional to
have a solid surface in a similar finish to the walls.
Lights should be encased, flush with the ceiling or wall panels and appropriately sealed
with silicone to prevent collection of dust and microbial matter.
Coving or moulding should be used at the junctions between floor and wall and between
ceiling and wall to eliminate right-angle joints and aid cleaning. The joints should be sealed
with silicone and the coving maintained in a good condition to prevent collection of dust
and microbial matter.
Doors for a new facility should not be made of wood. In an older facility where wooden
doors still exist, they should be sealed and well maintained. A risk assessment should be
conducted regarding their potential replacement. The detailing around doors should be
sealed with silicone and well maintained to prevent collection of dust and microbial matter.
Sinks within the production areas should be made of stainless steel and designed to be
self-draining to minimise any pooling or trapping of water and to maximise cleanability.
Benches and trolleys should be made of cleanable, impermeable materials and have
smooth surfaces. The use of mobile benches can facilitate cleaning. However, where
mobile units are used, consideration must be given to maintaining cleanliness of the
wheels, e.g., through the use of sticky mats between rooms.
4.4.2. Drainage
Any waste water from sinks, wash-in-place systems or wash bays must be disposed of
and, if not correctly designed and maintained, there is the potential issue of back
contamination from the ‘foul’ waste pipework.
As drains are a potential source of contamination, they should be minimised. Where used,
they should be hygienically designed to reduce the risk of aerosols, standing water and
splash back. Drain covers, surfaces and joints should be smooth and appropriate
containment features should be incorporated within the design.
16 Microbiological Control for Non-Sterile Pharmaceuticals
Chapter 4 – Facilities
Water from any process system must have an ‘air break’ into a tundish before it is piped
away – conventionally to a floor drain. The floor drain will be trapped but there is still
potential for the water in the drain to be contaminated. The floor drain should be
connected via a break tank (as minimum) to the foul drain system. Although it does allow
for the containment of contaminated spillage (preventing it reaching the local foul system),
the primary purpose of the break tank is to prevent any backflow of contaminated waste
from the foul drain into the process drainage system.
Because drains are the primary link between the room and the external drainage system,
where present they require disinfection on a regular basis based upon a known
understanding of the microbial contamination risk presented.
Wash bays, which tend to remain wet for long periods, are major potential sources of
microbial contamination and particular attention needs to be paid to their design to ensure
‘puddling’ and stagnant water does not occur. It is important that air changes, robust
construction, excellent floor seals and good working practices are in place to ensure that the
wash bay does not encourage a residual microbial population or affect the surrounding facility.
Sinks and safety showers should be minimised to meet functional and safety requirements
and appropriately located. They present a potential risk as they are sources of water, often
potable water, and should be assessed and monitored appropriately.
4.4.3. Heating, Ventilation and Air Conditioning (HVAC) System
The HVAC system is crucial as it supplies air at the correct cleanliness and in the correct
volumes to help keep particles entrained within the airflow so they are passed out of the
room. The HVAC is also key to the correct maintenance of pressure differentials,
temperature and in most cases humidity throughout the facility including changing rooms.
All of these aspects play a part in microbial contamination control.
Humidity control is key for some storage areas if materials are hygroscopic, increased
moisture levels may present a risk to microbial proliferation.
Pressure differentials and air change rates are covered in Section 4.2.
Air ‘quality’ is improved by filtering the air through progressively more efficient filter media.
Absolute filtration, using High Efficiency Particulate Air (HEPA) filters removing 99.97% of
particles sized greater than 0.3µm, is commonly used throughout the industry and is
recommended for higher risk activities. However, less efficient filters, e.g. bag filters
removing 90% of particles sized greater than 0.5µm, with turbulent air flow are sufficient
for most lower risk activities, e.g. tablet manufacture.
Other factors to be considered in the design and maintenance of air systems is where the
filters are positioned, whether extract filters are required and how much ‘fresh air make
up’ is used. This chapter will only consider filter positioning as the other two areas tend to
be driven by the safety aspects of the product rather than microbial aspects.
Microbiological Control for Non-Sterile Pharmaceuticals 17

It is cheaper to install and easier to maintain filters housed centrally, with the air handling
plant, where the air is then distributed to all the rooms. From a microbial aspect it can
however be a totally false economy as the ductwork then becomes part of the controlled
environment and it can be virtually impossible to seal, clean and monitor the ductwork.
In addition, any changes of components or instrumentation, within the ductwork, are in
contact with ‘product contact air’ and need to be subjected to much more rigorous change
control. Depending on design it can also be much more difficult to spot problems in a
centrally filtered system and any problems will affect all areas served from that filter bank.
Terminal filters, which are actually situated above the production rooms, are more
expensive and are slightly more difficult to change but return the additional investment in
simplicity of operation and greater levels of quality assurance.
Deflectors are often incorporated between the filter/air duct and the room and are
important for air distribution and flow. However, they also create opportunities for the
collection of dust and micro-organisms. Consideration should be given to the positioning of
the air blades, to ensure that the flow of air is not directly onto product and contact
surfaces, and to cleaning, so that they do not become a source of contamination in
themselves.
4.4.4. Compressed Gases
Compressed gases and their distribution systems will have a microbiological impact if not
specified, installed and maintained to the appropriate standards.
See section 8.3.3.8 for further consideration.
4.4.5. Water
Water and its distribution systems will have a microbiological impact if not specified,
installed and maintained to the appropriate standards.
There are various qualities of water used within non-sterile manufacturing facilities, some of
which are detailed in pharmacopoeia and all of which need to be suitable for their intended
use. Regulatory requirements are specifically mentioned in the EMEA CPMP ‘Note for
guidance on quality of water for pharmaceutical use’ and the EU GMP Guide, e.g. Annex 9
relating to the manufacture of liquids, creams and ointments; Chapter 3 regarding
sanitisation of pipe work; Chapter 1 regarding utility qualification; Part II, 4.3 regarding
water for API manufacture.
Incoming water should be sampled at point of entry on a routine basis (section 8.3.3.6).
Where process water is treated by the manufacturer to create the appropriate quality, the
treatment process should be validated and routinely monitored.
18 Microbiological Control for Non-Sterile Pharmaceuticals
Chapter 4 – Facilities
4.4.5.1. Purified Water
Purified water is typically used as process water for non-sterile pharmaceutical products
and for the final wash/rinse while cleaning product contact surfaces.
The specifications for purified water are given in the pharmacopoeias and are not covered
in this monograph. Piped purified water systems are typically continually monitored on-line
for conductivity and Total Organic Carbon (TOC) and routinely monitored for chemical and
microbial quality (section 8.3.3.6).
Purified water is generated from potable water, e.g. by reverse osmosis or distillation,
though the latter is typically only used for the generation of Water for Injections.
The problems with purified water systems usually involve bacteria (Hutcheson 2008). Most
microbial problems arise from the storage and distribution of the water rather than its
generation – the development of biofilms can be extremely difficult to resolve. However,
improperly maintained generation components, e.g. carbon beds, softeners, reverse
osmosis membranes, can also contribute to subsequent contamination downstream within
the distribution system.
The design and installation of the components of the storage and distribution system
should enable validated cleaning and sanitisation.
Appropriate placement of outlets should be built into the design to assist operational
logistics, to ensure representative sampling and the minimisation of dead legs.
Various design features may be utilised to prevent the development of biofilms and control
contamination. The design of the system should include continual circulation with adequate
flow rate to aid the prevention of biofilm formation (typically 1–3 m/s). Other common
design features are the capacity to heat the water to elevated temperatures (typically
65–85°C) for sanitisation purposes and the inclusion of high intensity UV lamps. The
inclusion of UV lamps downstream of potential microbial reservoirs, e.g. carbon beds,
softeners, has the added advantage of enabling ozone to be used for sanitisation purposes,
if required. Where UV lamps are used they should be regularly checked and maintained to
ensure they are clean and provide the correct wavelength and energy output. The inclusion
of filters within the distribution loop is difficult to justify and is not advisable.
Older systems, without such design features may require regular disinfection using an
oxidising agent to control biofilm (for example, ozone, hydrogen peroxide, hypochlorite).
Each of these methods has disadvantages, e.g. additional design considerations (UV light
to destroy ozone) or Health and Safety risks, and none is fully effective. Extensive flushing
is required to remove chemical residues if hypochlorite is used, which is expensive and
disruptive.
Typically, stainless steel pipework will be used, though systems with plastic piping have also
been used. Sanitary design for valves is an expectation. The design must minimise
opportunities for stagnation, e.g. dead legs, or sites for residue accumulation, which can
Microbiological Control for Non-Sterile Pharmaceuticals 19
 Chapter 4 – Facilities

promote microbial proliferation. Good control over welding processes is required to ensure
that these do not introduce surfaces which provide opportunities for biofilm formation.
The system should also be suitably sized in relation to the anticipated consumption of the
water; general guidance is for a total water replacement every 24 hours. Where purified
water usage volumes are low, bottled purified water may be considered as an option.
Procedures should be in place to cover the maintenance of the water system, including
details of shut down and start up mechanisms.
4.5. Facility Qualification
The commissioning, qualification and validation of a facility is very important. There is a
GMP requirement to identify the validation required to prove control over critical
operations and a risk-based approach, with a multi-functional team, should be used to
determine the scope and extent of this work. Microbiological considerations should be
included within this risk assessment. This topic is not covered in detail in this monograph;
the reader is advised to refer to more specialised guides, such as those produced by ISPE,
2001, for further information on this topic.

Operating procedures should require outlets to be flushed before usage to ensure use of
the circulating water and to remove possible stagnant water or contamination from the
surface of the outlet. The flushing of outlets prior to sampling for monitoring purposes
should be equivalent to that applied in operational use.

The use of hoses and temporary piping is a major source of contamination to product in
non-sterile facilities and therefore their use should be minimised. Where used they should
be subject to appropriate controls to minimise the risk of contamination from this source.
For example, they should not be left on the outlets; they should be dried after use, hung
vertically in appropriate locations to ensure free drainage, monitored and regularly
cleaned, sanitised and replaced.

4.4.5.2. Hot and Cold Potable Water

Potable water may be used for hand washing and initial cleaning or wash-in-place systems.

These are very similar to domestic supplies (except that hot water is often in a re-
circulating loop) and do not conventionally cause many microbial issues. It is wise to
ensure the hot water does not get ‘too hot’ or staff will avoid using it for critical hand
washing activities.

In some cases potable water may be softened before use, which adds complexity to the
water system.

4.4.6. Steam

Clean steam is used for cleaning and sanitisation of production tools and equipment,
supply for autoclaves and humidification of air where required. The microbial risk from the
steam itself is low due to the physical characteristics of steam and its production from
purified water using a heat exchange. Care should be taken with respect to condensation
generated upon cooling of the steam on surfaces, which may be sources of contamination
that may then be spread by the condensate (Section 8.3.3.7).

20 Microbiological Control for Non-Sterile Pharmaceuticals Microbiological Control for Non-Sterile Pharmaceuticals 21
 Chapter 5 – Cleaning and Disinfection

Chapter 5

Cleaning and Disinfection

5.1. Introduction

Cleaning and disinfection are vital parts of the maintenance of facilities and equipment in
order to ensure that contamination risk is minimised.

Facilities and equipment need to be designed so that they are fit for the intended purpose
and cleanability is an important factor to consider in the design/technical specification.
The design should aid the cleaning and disinfection process rather than cleaning and
disinfection procedures being used to compensate for poor facility or equipment design.

The assurance of appropriate cleaning and selection of appropriate disinfection agents

through validation are also important.

For a more detailed consideration of cleaning and cleaning validation, including chemical
considerations, refer to the PQG monograph on ‘Cleaning Validation’, 1999.

For a more detailed consideration of disinfectants, refer to Pharmig’s ‘A Guide to

Disinfectants and their use in the Pharmaceutical Industry’, 2006.

5.2. Facilities

Areas must be regularly cleaned and, where necessary, disinfected to a written procedure
and programme. A cleaning log is required which records the areas cleaned, agents used,
type of cleaning (as appropriate) and the identity initials of the operator.

Sufficient time and resource should be allocated for cleaning activities.

Frequently, some or all GMP facility cleaning activities are carried out by contract staff.
Where contract GMP cleaners are used, a technical agreement should be in place with
the contract agency. Only named staff should have access to GMP areas and be involved
in GMP cleaning activities. A permanent member of staff should have oversight of the
contract cleaning operations to ensure that they are being conducted satisfactorily.

Whether contract or in-house staff are used for cleaning activities, they must have
documented training and competency (Chapter 3) and the responsibility for cleaning needs
to be clearly defined and traceable to accountable management.

Standard Operating Procedures (SOPs) are required and need to be part of the document
control system. SOPs need to state the areas to be cleaned and their frequency, together
with materials, equipment and methods.

22 Microbiological Control for Non-Sterile Pharmaceuticals Microbiological Control for Non-Sterile Pharmaceuticals 23

Risk assessment and hazard analysis tools should be used to formulate the cleaning
programme (Chapter 7).
The frequency of different cleaning and disinfectant activities will vary depending on the risk
assessment (which takes account of room usage and formulation). For example, floors,
fittings and benches may have a routine daily clean, while higher level walls and ceilings
may be cleaned less frequently, e.g. weekly or monthly.
The SOP needs to contain sufficient detail to allow the correct application of the cleaning
and disinfecting agents. Steps for preparing the correct concentrations are particularly
relevant. The manufacturer’s recommendations for preparation and use should be followed.
The method and sequences of cleaning is important; contact time, application
temperature, mechanical action and the chemistry of the cleaning agents should all be
considered during the design of the cleaning process.
Loose dirt and spillages of material require removal first to ensure that they are not
spread around the facility. This is typically done using a vacuum cleaner of appropriate
design, i.e. one that has a filtered exhaust to ensure that it does not present a source of
recontamination.
Fittings and fixtures including door handles, light switches and telephone receivers should
be included in the cleaning regime.
Hot water or hot water with detergent followed by a hot water rinse may be all that is
necessary in some areas, whereas other facilities will require detergent clean followed by
a disinfectant. This should be determined and documented by the risk assessment. Where
used, all residual cleaning and sanitising agents must be removed from product contact
surfaces to avoid product contamination.
Disinfectant rotation is a contentious issue attracting considerable debate within industry
and regulatory agencies. Annex 1 of EU GMP states that “where disinfectants are used,
more than one type should be employed”. However, this is specific to sterile manufacture
and not necessarily applicable or justifiable to non-sterile manufacture. Whilst this
requirement has lead to widespread application in non-steriles, there are arguments
against applying it. The principle scientific argument for rotation is to ensure activity
against the full spectrum of flora within the facility; one category of agent may not work
against all types of organisms (e.g. sporicides for use against spore forming organisms).
However, the counter argument is that the periodic use of an alternative and potentially
less effective disinfectant may have an adverse effect on the efficacy of the disinfection
procedure (Pharmig, 2006). One approach could be to use routinely a single disinfectant
only introducing a different disinfectant or sporicide in the event of an adverse trend in
environmental monitoring data. Another approach is to use a different disinfectant or
sporicide at a defined extended interval.
24 Microbiological Control for Non-Sterile Pharmaceuticals
Chapter 5 – Cleaning and Disinfection
Disinfectants should only be applied to relatively clean surfaces since they can be
inactivated by residues of dirt and other materials, e.g. product. Chemical disinfectants
need sufficient contact time and this can be aided by using repeated applications rather
than a brief wipe or mop application of the solution. Knowledge of the properties of the
disinfectant used is essential to ensure its optimal usage. It is important to use detergent
and disinfectants which are compatible with each other. This also applies to the
compatibility of disinfectants that are rotated with each other.
Disinfection programmes should be supported by laboratory studies with representative
micro-organisms (USP <1072> Disinfectants and Antiseptics).
Floor and sink drains, when present, are a potential source of microbial contamination.
Their design needs to include traps and screens where appropriate (see 4.4.2) and
consideration should be given to the risks of aerosols and splash back as part of the risk
assessment. A treatment with alkali may be used to eliminate blockages and a compatible
disinfectant should be used to reduce the microbial load. The effectiveness of the
disinfection should be determined and documented. The frequency of the disinfection
should be identified through risk assessment.
Utensils used for all aspects of cleaning must be managed so they do not present a
source of contamination. They should be selected to reflect the requirements of the area
and may be dedicated to specific areas of the facility. Utensils used in the preparation of
cleaning and disinfectant agents must be dedicated for this purpose. Whilst mop and
buckets remain a widely used method, consideration should be given to disposable mop
heads and wipes. Buckets and mop heads need to be of a design which allows them to be
stored clean and dry. It is important that mop heads are stored dry, frequently sanitised
and replaced on a regular basis. Water should never be left to stand in the buckets but
used and immediately discarded to ensure that a potentially significant source of
microbiological contamination is removed as soon as possible.
A bucket system which allows the separation of ‘used’ solutions of detergent/disinfectant
from fresh solutions is an aid to effective cleaning. The mop is wrung out in a separate
bucket from that used to contain the fresh cleaning agent. This reduces the rate at which
the disinfectant solution becomes dirty and diluted during use, and prevents the reapplication
of dirt to the cleaned surface, thus reducing the risk of recontamination and inactivation.
Mechanical cleaning equipment may be necessary to provide efficient, thorough cleaning.
Such items include cloths and brushes to enhance manual processes and scrubbing
machines and vacuum cleaners, wet or dry.
Machines used for mechanical cleaning need to be carefully maintained and operated in
accordance with SOPs and cleaned themselves on a routine basis, including any internal
filters or surfaces where they can be accessed. This cleaning and maintenance needs to
be documented.
Microbiological Control for Non-Sterile Pharmaceuticals 25
 Chapter 5 – Cleaning and Disinfection

All items, including machines used for mechanical cleaning, need to be stored clean and Biofilm development is a risk with some items of equipment and must be considered as
dry. Residues of liquid and moisture encourage microbial growth and the development of part of risk assessment both in relation to design and cleaning processes. Where biofilms
resistant strains. There should be a defined drying method/area and storage area, room do occur, remediation can be difficult and contingency plans for dealing with them should
or cupboard for dried cleaning utensils and materials. be considered.

Status labels should indicate the clean/dirty or quarantine/released status of the equipment.
5.3. Equipment

Methods for equipment cleaning vary from manual cleaning to automated systems, which
may be integral to the equipment. Automated systems, such as integral Clean-in-Place
(CIP) systems and stand alone automated washers, when fully validated, are preferable
due to their demonstrated reproducibility.
Equipment components (e.g. tooling) may require dismantling to enable effective Cleaning-
out-of-Place (COP). Items which prove difficult to clean (e.g. valves) may benefit from
additional steps such as sonication.
5.3.1. Tools and Tooling
Tools and tooling should not be forgotten:
Tools – items used to maintain or adjust equipment
Tooling – change parts for equipment (often product contact)
Tooling should be dealt with as part of the equipment as detailed in section 5.2.

Hosing and tubing that is not single use must be appropriately cleaned, dried and stored Tools generally fall into two categories:
to prevent pooling of water, i.e. hung with no loops or kinks to allow free flow drainage of Tools that stay within the facility
internal water by gravity and replaced at defined intervals. Specialist tools that are brought into the area, often by external service personnel

It is important that all cleaning regimes (automated and manual) are fully validated or
Tools that stay with the equipment should be cleaned on a regular basis and following use.
subject to verification (i.e. demonstrated efficacy) to ensure that the equipment used for
pharmaceutical production is free from residues of product or cleaning material and
Specialist tools should be checked for appropriate cleanliness before entry to the area and
microbial contamination.
appropriately cleaned before (as required) and after use.
Cleaning process hold times must be defined and validated in accordance with various
global GMP guidance and requirements (FDA guide to inspections validation of cleaning
processes; EU Annex 15 (38); PIC/S guidance 7.7.2; Health Canada – Cleaning Validation
5.4. Cleaning Agents
guidelines (part of 3.2)).
Cleaning provides an important mechanism for improving microbiological control by
removing physical contamination from surfaces which would otherwise reduce the ability of
The dirty equipment hold time (DEHT) is the time between the end of manufacture and
disinfectants to remove micro-organisms. When selecting a cleaning process it is
cleaning. The DEHT should be kept to a minimum because over time product residues may
important to consider both the chemical and microbiological cleaning capabilities of the
become harder to remove and the risk of microbial cross-contamination increases.
process to be adopted.
Maximum times should be defined and supported by validation.
Cleaning efficiency is derived from a number of parameters; time, temperature,
The clean equipment hold time (CEHT) is the time from cleaning to next use and is
mechanical action and cleaning chemistry. The relative efficiencies of different
important to assure equipment does not become re-contaminated during storage either by
combinations of these parameters are illustrated by Figure 5.1.
microbial contamination or dust. The CEHT should be a defined and documented period up
to which the cleanliness of the equipment has been demonstrated. This may be achieved
It is preferable not to use chemical cleaning agents, thus avoiding the need for additional
through a hold time study, i.e. validated.
assurance of the removal of chemical residue. However, some kinds of chemical cleaning
agent may be required to ensure a consistent and effective clean.
Equipment design and material of construction can significantly impact the ability to
achieve an effective clean. The Microbiologist should be involved in review of equipment
design to facilitate hygienic design and the inclusion and positioning of appropriate and
accessible sampling points where required.

26 Microbiological Control for Non-Sterile Pharmaceuticals Microbiological Control for Non-Sterile Pharmaceuticals 27
 Chapter 5 – Cleaning and Disinfection

Cleaning agents must be prepared in accordance with manufacturer’s instructions (unless

Water, chemical, mechanical and thermal action
otherwise justified and documented) to ensure appropriate concentrations are always
Water and thermal action used, otherwise this may negate the validation.
Water and chemical action

Water and 5.5. Validation of Cleaning

mechanical action
Trials should be carried out to provide evidence that the process will remove any residue
Cleanliness

adhering to the surface of the facility or equipment and leave the surface free from
Water only residues and microbial contamination. Validation should demonstrate the consistent
removal of cleaning agents and meeting of pre-defined limits for microbial and chemical
contamination.

Ongoing assurance of cleaning practices should be gained from the routine monitoring
programme.

For further details relating to cleaning validation and disinfectant validation refer to PQG
(1999) and Pharmig (2006).
Time

Figure 5.1: Relative Cleaning Efficiencies

(After Wilkinson, 2008)

If a cleaning agent is required, the agent should be selected based upon the following
considerations:
Maximise Active Pharmaceutical Ingredient (API), starting material, intermediate and
product removal
Compatibility with equipment surfaces
Supplier assurance
Environmental, toxicological, health and safety impact
Cost effectiveness
Rinsability

It is important to understand the chemistry of your cleaning agents and the mechanism of
residue removal.

Detergents are primarily used for the removal of soil/dirt residues whilst disinfectants are
primarily targeted at microbiological control. For further details relating to disinfectants
refer to Pharmig (2006).

All cleaning agents should be freshly prepared with water of a suitable quality (typically
purified water, although potable water may be acceptable). Cleaning agents and prepared
solutions should state the dates of preparation and expiry clearly on the container.

28 Microbiological Control for Non-Sterile Pharmaceuticals Microbiological Control for Non-Sterile Pharmaceuticals 29
 Chapter 6 – Microbiology Laboratories

Chapter 6

Microbiology Laboratories
6.1. Introduction

The microbiology laboratory and the microbiologists who support the site are the focal point
of the site’s mechanism for understanding microbiological control. An important objective of
the quality control microbiology laboratory is to provide information in the form of interpreted
microbiological data to the site. These data need to be generated and managed in
accordance with GMP. The following information provides a standard that is considered
appropriate to support the activities in a non-sterile facility. Further details on microbiological
best laboratory practices may be found in the USP general chapter <1117>.

6.2. Relationship to the Quality Unit

The microbiology unit is most commonly part of the quality control function owing to the
nature of the work being performed. However, the microbiology laboratory capability may
be managed through a third party contract arrangement overseen by the quality
assurance organisation.

Good quality control laboratory practice within the microbiology laboratory is essential to
the production of reliable and reproducible microbiological data.

It should be remembered that non-sterile environmental monitoring data will typically

provide an overall picture of the microbiological control within the facility, rather than data
relating to specific product batches.

6.3. Microbiology Laboratory Facility Design

The microbiology laboratory facility design should be hygienic, ergonomic and fit for purpose.
Its function requires a design that considers the product, people and process flows so as to
minimise the risk of cross-contamination. Thought needs to be given to the receipt, storage
and handling of the different types of material with respect to the level of contamination.

Statutory requirements to operate at Containment level 2 should be adhered to for health

and safety reasons and to facilitate good quality control laboratory practice (Advisory
Committee on Dangerous Pathogens, HSE Books, 2001).

30 Microbiological Control for Non-Sterile Pharmaceuticals Microbiological Control for Non-Sterile Pharmaceuticals 31

6.4. Autoclaves
Autoclaves should be validated and subject to revalidation depending upon the use. GMP
critical usage such as the sterilisation of in-house prepared media should be revalidated
annually. Non-GMP critical tasks, such as decontamination of lab waste prior to disposal,
may only require revalidation on a two yearly basis. The validation should cover the
different cycles and load patterns that may be required.
6.5. Disinfectants
Disinfectants qualified for use in the production area should be suitable for use in the
laboratory.
Validation includes assessing activity against pharmacopoeial organisms and local isolates
and checks to ensure that all detergents and disinfectants are mutually compatible.
Application of disinfectants as part of a cleaning/sanitisation programme in the
microbiology laboratory should be followed by a purified water rinse to ensure residue is
removed effectively.
6.6. Media
The quality of the work generated by a microbiology laboratory depends on the quality of
the culture media employed and so safeguarding the quality of this media is critical. Media
preparation, storage and quality control all need to be defined to assure the quality. It is
important to ensure that the manufacturer’s instructions are followed for the preparation
of media and that activities are recorded.
Special care should be taken to ensure that media used for environmental monitoring do
not introduce contamination into the production environment. In preference, commercially
manufactured irradiated and double bagged plates confer a greater level of assurance.
6.6.1. Vendor Assurance of Media Suppliers
Suppliers of media should be approved prior to use following an assessment of their
Quality Management System (QMS). Data supporting media shelf lives and storage
conditions should be available and each batch of media should be supplied with an
appropriate Certificate of Analysis (CoA).
Ready to use media supplied by a vendor with a good supply history may undergo a pre-
defined validation exercise leading to routine acceptance on the basis of the vendor’s CoA
and only periodic testing (e.g., every 10 lots or 6 months). Hence, the vendor’s data can
be used for growth promotion, selectivity and sterility.
32 Microbiological Control for Non-Sterile Pharmaceuticals
Chapter 6 – Microbiology Laboratories
6.6.2. Incoming Inspection
Received media should undergo a pre-defined incoming approval process before it is
released for use by the laboratory.
Media receipt from vendors should include a check that transport conditions have been
appropriate.
Each lot of ready made media should undergo a defined set of growth promotion, sterility
and chemical testing or be covered by validated skip lot testing.
6.7. Micro-organisms
6.7.1. Local Isolates
Each laboratory should maintain a collection of organisms representative of the local flora
typically isolated across the site for use in testing and validation activities. Since changes
in local flora may occur, it is recommended to review and, if appropriate, replace the
strains in the collection with fresh isolates regularly, e.g. annually.
6.7.2. Objectionable Organisms
Regulations require industry to prevent release of finished product that contain
objectionable organisms (21 CFR211.165(b), Ph Eur. 5.1.4). Some objectionable
organisms are specified in the pharmacopoeias but these are not exclusive and other
organisms may be objectionable depending on the nature of the product, route of
administration and intended patient population. There is an expectation that the
significance of other micro-organisms is evaluated (e.g. Ph Eur. 5.1.4, USP <1111>).
A risk based assessment should be conducted, including personnel with specialised
training in microbiology and data interpretation. In addition to dealing with isolates as they
arise, it is advisable that an assessment is done proactively to generate a documented list
of objectionable micro-organisms, which should be incorporated into procedures and
internal specifications as appropriate.
In certain production environments, such as for the production of herbal medicines,
by-products of objectionable organisms, for example, aflatoxins, should be considered.
Certain materials may need to be sampled if toxins are known potential contaminants
(Ph.Eur. monograph 1433).
Microbiological Control for Non-Sterile Pharmaceuticals 33
 Chapter 6 – Microbiology Laboratories

6.8. Identification
For non-sterile manufacturing an assessment should be made and procedures written
defining the extent of the identification required. A simple morphological description and
Gram stain may be justified for samples taken from low risk environmental isolates. A
more detailed identification, to genus or species level, may be required for higher risk
isolates such as those from starting materials or product, or for investigational purposes.
It is less likely that a level of identification beyond phenotypic identification will be required.
genotypic identification may be helpful when investigating adverse trends or product failure
but the benefit may not justify the additional cost.
Whichever method is chosen, the laboratory must show its ability to use the system and
identify known organisms, including local isolates, to ensure that any identifications
performed in routine analyses are valid.
6.9. Microbiological Testing of Product, Starting Materials
and Intermediates
6.9.1. Starting Materials
Where a starting material or packaging component is liable to microbiological
contamination testing will be required to ensure the absence of objectionable organisms
(21 CFR211.84(d)(6)).
Maximum hold times of aqueous solutions should typically not exceed 24 hours. For dried
intermediates longer hold times may be established. In order to provide a good level of
confidence, it is recommended that the validation period exceeds the defined maximum
hold time.
6.9.3. Product
There are general pharmacopoeial chapters giving expectations of microbiological purity of
non-sterile products (Ph Eur 5.1.4) and if tested the product should meet these minimum
requirements whether there is a registered specification or not. The registered
specification for non-sterile products will typically be based on these pharmacopoeial
requirements. Depending on development data, it may be possible to justify not performing
end product testing or to perform skip lot testing. For low risk non-sterile products, annual
or 6 monthly testing of finished product may be acceptable. Any such reduced testing
must be reflected in the Marketing Authorisation and specification for the product.
Where product is held in bulk form, hold times should be established and validated as for
intermediates.
All testing must be documented to controlled procedures and recorded
contemporaneously (in real time) following the verified method.

Consideration should also be given to the need for neutralisation agents in the media to
remove any inherent antimicrobial properties in the starting materials.

Microbiological testing of starting materials may be conducted on a skip lot basis as

determined and justified by documented risk assessment.

6.9.2. Intermediates

Intermediates include, for example, granulation solutions, coating solutions and

suspensions for spray drying. A risk assessment should be made to determine the risk of
microbial contamination; typically aqueous intermediates should have validated hold times
and be subject to periodic microbiological monitoring.

Where it is decided that there will not be any microbiological testing of intermediates the
justification of the risk assessment should be documented.

An appropriate in-house specification should be established for intermediates with the input
of a microbiologist.

34 Microbiological Control for Non-Sterile Pharmaceuticals Microbiological Control for Non-Sterile Pharmaceuticals 35
 Chapter 7 – Risk Assessment and Management

Chapter 7
Risk Assessment and Management
7.1. Introduction

A full consideration of pharmaceutical quality risk assessment and management is beyond

the scope of this monograph and the reader is encouraged to consult ICH Q9
(now Annex 20 of the EU GMP Guide) and more specialist publications to gain a greater
understanding of this important topic.

Key definitions that need to be understood are:

Risk: “The combination of the probability of occurrence of harm and the severity of
that harm” (ICH Q9; ISO/IEC Guide 51)
Harm: “Damage to health, including the damage that can occur from loss of product
quality or availability” (ICH Q9)
Hazard: “The potential source of harm” (ICH Q9; ISO/IEC Guide 51)

Risk management is fundamentally about understanding what is most important for the
control of product quality and then focussing resources on managing and controlling these
things to ensure that risks are reduced and contained. Before risks can be managed, or
controlled, they need to be assessed.

Two important points to remember for any risk assessment are:

1. There is no such thing as ‘zero risk’ and therefore a decision is required as to what is
‘acceptable risk’.
2. Risk assessment is not an exact science – different people will have a different
perspective on the same hazard.

Consequently, whichever tool is used, it is important that a team approach is taken

utilising an agreed set of definitions/scores.

Whichever tool is used, risk assessment involves:

Identifying hazards
Analysing the risk associated with each hazard
Evaluating how significant the risks are

36 Microbiological Control for Non-Sterile Pharmaceuticals Microbiological Control for Non-Sterile Pharmaceuticals 37

The outcome of the assessment can then be used to determine appropriate control
strategies to reduce the risk to a level that is deemed acceptable. These strategies are
typically focussed on reducing the probability of a risk occurring and/or increasing the
probability of it being detected.
Risk assessments and the actions arising from them need to be documented and subject
to periodic review to ensure that the assessment reflects the actions taken and the most
recent data.
7.2. Risk Assessment Associated with Non-Sterile Products
A science-based microbiological risk assessment should be carried out for non-sterile
manufacturing activities to establish what microbial risks are involved with the facility,
equipment and processes used. In many cases a generic risk assessment can cover a
number of products, but product-specific considerations should not be forgotten. The
outcome of the risk assessment will help determine appropriate controls associated with
the running of the facility or specific processes and an appropriate monitoring programme.
A description of the full manufacturing process is a useful starting point in assessing the
risks involved. This may be simply done as a process map or flow diagram, with activities,
equipment used and process parameters added on.
Important factors to consider in the manufacturing process include:
Solvents used
Water-based processes provide a more favourable environment for micro-
organisms; the use of other solvents might decrease the risk of microbial growth
pH
Values above 10 or below 2 generally being detrimental to micro-organisms
Osmolarity of solutions
High osmolarity typically being detrimental to micro-organisms
Temperatures used
25–35°C promoting microbial growth; significantly higher or lower temperatures
being detrimental to micro-organisms
Drying
If the water activity of the product is reduced below 0.6, then microbial growth will
be suppressed (section 7.3)
Hold times and overall campaign length
Longer processing times may increase the opportunity for microbial proliferation
unless the conditions are detrimental to microbial growth
38 Microbiological Control for Non-Sterile Pharmaceuticals
Chapter 7 – Risk Assessment and Management
Processes requiring heavy physical labour or increased numbers of personnel might
increase the risk of contamination from personnel through enhanced sweating or
shedding of skin particles
Whether the processing step has the potential to increase or reduce bioburden
Fundamental characteristics of the formulation, such as hygroscopicity, should also be
considered as part of the risk assessment.
Where possible, data should be used to support the risk assessment. Microbiological
process validation data can be a valuable aid in estimating the risk of microbial
contamination or proliferation in each stage of the process.
The growth of micro-organisms on manufacturing equipment and tools will be influenced by
the cleaning and sanitisation routine used and their design. Microbiological cleaning
validation data can be useful in estimating this risk.
Knowledge of the starting materials and their potential contribution to bioburden is essential.
Water is a frequently used material and there needs to be assurance of the quality of
water used. Typically, pharmacopoeial grade purified water will be used for non-sterile
manufacture. Modern purified water systems generate water of very high microbiological
quality and data should be available to demonstrate this.
Product-contact utilities, such as compressed air and Nitrogen, must not be forgotten.
These will typically be filtered close to the point of use, but reliance should not be placed
on filtration alone. Monitoring data should be available.
The manufacturing environment itself, including the design and classification (when applied)
of the facilities, contributes to the risk of microbiological contamination. Environmental
monitoring data should be obtained and used as part of the risk assessment.
GMP and personal hygiene routines are also important considerations as part of the risk
assessment.
Having gathered the information required, various tools may be used to conduct the risk
assessment. Methods commonly used for microbiological risk assessments include HACCP
(Hazard Analysis and Critical Control Points) and FMEA (Failure Mode and Effects Analysis).
Some tools deliver a numerical assessment of the risks from different sources. The risks
can then either be accepted or action taken to mitigate them. Where high risks of
microbiological contamination remain after mitigation, controls must be introduced at
these points with an appropriate level of monitoring. The Microbiologist and QP should be
involved in the risk assessment, the decisions on which risks to mitigate and the
appropriate mitigation actions.
Microbiological Control for Non-Sterile Pharmaceuticals 39
 Chapter 7 – Risk Assessment and Management

7.3. Water Activity Dosage Form aw

Nasal inhalant (aqueous based) 0.99
A fundamental component of assessing the risk for microbiological control in non-sterile
manufacturing is an understanding of whether the product or intermediates during the Antacid 0.99
production process are able to support growth or sustain viability of micro-organisms. Rectal cream 0.97
Oral liquid 0.90
Water activity (aw), is a measure of the free water in a material and is therefore a useful
measure to aid the determination of microbiological risk. The details on how to determine Oral suspension 0.87
water activity are outside the scope of this monograph. Topical ointment 0.55

Water activity ranges from 0.0 (completely dry) to 1.0 (pure water). It is a more accurate Liquid filled capsule 0.45
index for microbial growth than water content as micro-organisms have a limiting aw below Tablets 0.36
which they cannot grow (typically ~ aw 0.6) (Pharmig Water Activity – Technical Guidance
Suppositories 0.30
Document 1, 2008).

Typically, products with water activities less than 0.6 are not susceptible to microbial Table 7.1: Water Activity of Pharmaceutical Dosage Forms
growth of organisms found with compendial microbiological culture media. Friedel and Cundell, 1998

USP 31-NF26 <1112> ‘Application of Water Activity Determination to Non-Sterile

Pharmaceutical Products’ provides examples of the application of water activity
determination to non-sterile pharmaceutical products which can facilitate a risk based
approach and may be used to:
Develop product formulations
Set microbiological release specifications
Establish microbial testing programmes
Determine potential shelf life stability from microbial growth

It should be noted that water activity is temperature dependent and a lack of temperature
control or temperature stability may lead to variable water activity in your product.

Whilst the water activity provides a useful indicator of the ability of the formulation to
support growth it should be remembered that some organisms present may remain viable
and be pathogenic at low levels (e.g. Salmonella spp), therefore, good control during
manufacturing is still essential.

Table 7.1 shows the typical Water Activity of representative non-sterile pharmaceutical
drug products.

40 Microbiological Control for Non-Sterile Pharmaceuticals Microbiological Control for Non-Sterile Pharmaceuticals 41
 Chapter 8 – Microbiological Monitoring

Table 7.2 shows the water activity required by certain organisms for growth Chapter 8
(or the threshold at which they can no longer grow).

Organism aw Microbiological Monitoring

Most Bacteria >0.91
Pseudomonas spp 8.1. Introduction
Listeria monocytogenes
Mucor plumbeus 0.97 This chapter covers microbiological monitoring in non-sterile manufacturing. Other forms of
environmental monitoring, as listed below, are relevant to non-sterile manufacturing, but
Escherichia coli 0.95
are not detailed in this monograph:
Serratia marcesens Particulates
Shigella spp
Temperature
Bacillus subtilis 0.94
Humidity
Many yeasts 0.88
Differential pressures
Staphylococcus aureus
(reduced oxygen conditions) 0.86 Room air changes
Penicillium chrysogenum 0.83 Air flow patterns
Many moulds 0.80
Microbiological monitoring should deliver a monitoring programme that provides value
Halobacterium halobium
added information enabling successful microbiological control. However, the methods are
(halophilic bacterium) 0.75
very insensitive; increasing the number of samples taken increases the risk of diverse
Zygosachharomyces rouxii results and there is no direct correlation between number of organisms found and product
(osmophilic yeast) 0.60 contamination risk.
Xeromyces bisporus
(xerophilic fungi) 0.65 In general, the monitoring programme should demonstrate the effectiveness of production
and maintenance activities, operator discipline and housekeeping activities and compliance
No Microbial Proliferation <0.60 to defined standards.

Table 7.2: Minimum Water Activity required for Growth Monitoring should be targeted to gain detailed information which can help build a picture
Kabara and Orth, 1997 of the facility and enable appropriate risk assessment, thus helping trouble-shooting and
the identification of any ‘hot spots’.

It should be remembered that monitoring does not represent microbiological control in

itself but is a mechanism to understand the effectiveness of the various controls in place
and identify areas for improvement.

8.2. Environmental Monitoring Policy

It is recommended that an environmental monitoring policy be drafted, which once effected

should result in a state of control within the facility. All sampling techniques, sample
locations, and sample frequencies should be based on this policy. Sampling that does not
add to the understanding of the facility's state of control is a waste of resources and
should not be performed.

42 Microbiological Control for Non-Sterile Pharmaceuticals Microbiological Control for Non-Sterile Pharmaceuticals 43
 Chapter 8 – Microbiological Monitoring

The degree of environmental monitoring applied to non-sterile manufacturing activities Specific instructions regarding interpretation of results (e.g. in between read-outs to
should be aligned with the risks associated with the product, process and patient, which prevent problems with overgrowth, how to handle too-numerous-to-count results,
should be documented and defined in an environmental monitoring policy and standard whether scoring per colony type is performed)
operating procedures (section 8.2.1) following a robust risk analysis. How alert and action limits are set and revised

Surveys conducted over the last 14 years (PhRMA 1994, Pharmig 2001, Pharmig 2007) How a trend is defined
all indicate that there is no clear consensus on microbiological monitoring programmes. How to handle excursions of the alert or action limits, or when a trend is observed
However, regulatory guidelines and regulatory inspectors do expect microbiological (standardised investigation procedure)
monitoring in non-sterile manufacturing to be in place. EU GMP 5.20 requires periodic
assessment of measures to prevent cross-contamination and USP <1111> describes
Records of the microbiological monitoring programme data should be generated. The
monitoring in the form of regular surveillance. However, the nature of such a programme
records must allow full traceability allowing for information retrieval and data manipulation,
should be driven by circumstances, supported by risk assessment (Chapter 7) rather than
detailing:
a mandated set protocol as defined for sterile manufacturing.
How, when, where and by whom a sample was taken and further processed
If an area is determined as low risk, such as the manufacturing of tablets, the policy Which media and sampling methods were used:
should focus on the bioburden control measures and on targeted monitoring, for example, Type and lot numbers
the monitoring of relatively higher risk aqueous coating solutions. Routine monitoring can
be performed at a lower frequency in such cases. There is little value in generating large Equipment identification (where applicable)
amounts of data when the resource could be more effectively applied to preventive Room/Equipment status:
measures. For the highest risk activities, monitoring on a batch by batch basis may be
Clean status
deemed appropriate in addition to bioburden control measures.
Which activities were ongoing in the area at the moment of sampling:
Routine monitoring is recommended to provide qualitative information with regards to the flora Dynamic
present on site but should be supported through the risk assessment. A periodic species level
identification should be applied to routine monitoring to build up a general knowledge of the site At rest
flora to species level and enable identification of any changes in the facility.

8.3. Designing the Microbiological Monitoring Programme

8.2.1. Procedures and Documentation
In order to design an appropriate microbiological monitoring programme several aspects
The microbiological monitoring programme should be documented in standard operating should be considered which include but are not limited to:
procedures which should include the following topics: Location
For each sample (where applicable):
Frequency
Type of sample, media and disposables used
Technique type
Moment of sampling (at rest or in operation, but also related to operations,
Time of monitoring (in operation/at rest)
e.g. at the end of operations, prior to cleaning)
Clean status
Pre-treatment of the sampling location (disinfection at point of use, flushing of
media) should mimic actual use
Each aspect contributes different information regarding the efficiency of the control measures
Frequency and locations of sampling
and should be tailored to suit an individual site to provide a value added ‘complete’ picture.
Length of time of monitoring
Sample treatment, incubation and interpretation of the measurement
Post-treatment of the sampling location, e.g. removal of residual media from the
sampling event

44 Microbiological Control for Non-Sterile Pharmaceuticals Microbiological Control for Non-Sterile Pharmaceuticals 45
 Chapter 8 – Microbiological Monitoring

8.3.1. What to Monitor
The risk assessment process should highlight worst case areas and activities in the
facility; these may include areas and activities with proximity to product and product
contact, high human activity and/or traffic (e.g. door handles), areas with poor air flow,
where water is in use and where drains are present.
Suitable locations for monitoring should be identified, and if selected as part of the final
monitoring programme, mapped and diagrammatically or photographically represented in
procedures to help accurate identification of sampling points.
8.3.2. When to Monitor: Frequency

Special consideration should be given where contamination may be transferred and include: If the areas within the manufacturing facility have been formally classified, then regulatory
Splash back inspectors will expect the frequency and approach to monitoring defined by EU GMP Annex
1 to be followed.
Generation of aerosols
Pooling of water For manufacturing facilities and areas which have not been formally classified, the
Exposure to incoming contamination sources (e.g. starting materials, air, people etc.) frequency of monitoring should be based upon the risk assessment.

Cleaning materials and equipment Again, there is no clear consensus approach across the non-sterile pharmaceutical
The act of cleaning and storage industry and frequencies range from weekly to annual monitoring. Typically, a monitoring
programme will set different frequencies depending on the particular aspect being
Transfer by equipment wheels or by foot
monitored. A non-specific example illustrating the approach may assign a weekly monitor
Access to voids to final fill and wash bays, a monthly monitor to dispensary, unclassified production rooms,
Tools microbiology laboratory, preparation rooms and equipment and a quarterly monitor for
change areas, corridors and equipment storage rooms.

The areas covered by the environmental monitoring programme should be tailored based Once the frequency has been set and implemented, data review and trending should be
on risk. Areas that should be considered for monitoring include the following: used to assess the ongoing appropriateness of the frequencies chosen, which may be
Dispensary increased or decreased depending on data. Data should be presented in a form that will
Final fill (primary packaging) enable interpretation and identification of trends, e.g. graphical representation. In
response to an Out of Trend or Out of Specification result, the planned action may involve
Unclassified production areas
special cause monitoring or an increased frequency for a period of time; thus providing
Equipment more information to aid the resolution of the root cause and for the assessment of
Equipment storage product quality. The routine frequency may be reinstated once results have returned to
normal levels.
Wash bays
Packaging
8.3.3. How to Monitor: Techniques
Change areas
Corridors 8.3.3.1. Choice of Media
Preparation rooms
Pharmacopoeias stipulate media required for certain monitoring activities and techniques,
Storage vessels for example, water monitoring or absence of pathogens testing using selection and
Microbiology laboratory enrichment media. These media must be used unless supported by regulatory
commitments and validation to demonstrate equivalence to the stipulated media.
The baseline flora within the microbiology laboratory should be understood to provide
For bioburden determinations, it is an expectation to ensure monitoring can capture
information in the event of an investigation.
bacteria, yeasts and moulds. Common media and incubation conditions for monitoring are
given in table 8.1.

46 Microbiological Control for Non-Sterile Pharmaceuticals Microbiological Control for Non-Sterile Pharmaceuticals 47
 Chapter 8 – Microbiological Monitoring

Each particulate may be associated with thousands of micro-organisms and the final
Media Temperature Duration Comments dispersion of the particulates and therefore the resulting number of colony forming units
Tryptone Soy Agar 30–35°C up to 3 days predominantly for (CFUs) is influenced by the sampling method. The number of CFUs in a sample equals the
(TSA) (soybean-casein (48 hours USP the detection number of CFUs sampled from the environment. However, a limitation of these types of
digest agar) <1116>) of bacteria microbiological testing is that number of CFUs is not equivalent to the exact number of
micro-organisms that are present. For these purposes, the correlation is sufficient to
provide an indication of microbiological control.
Sabouraud 20–25°C up to 5 days predominantly for
dextrose agar (SDA) (72 hours USP the detection There are two principle mechanisms for monitoring air, active and passive capture.
<1116>) of yeasts and moulds
As discussed throughout this monograph, there are no stipulated requirements for
microbiological monitoring of unclassified areas and classification is only required in certain
Table 8.1: Common Media and Conditions for Monitoring specified conditions for non-sterile manufacturing. Monitoring during operation should be
considered as part of the risk assessment.

If separate selective media are used for bacteria and yeast/mould, then each sample
location requires sampling with each media to ensure representative data is obtained. 8.3.3.2.1. Passive Air Monitoring

Alternatively, if validation supports, bacteria, yeasts and moulds can all be monitored using
a single medium (e.g. TSA) with dual incubation (e.g., 20–25°C first, then 30–35°C). If
this approach is taken, recovery of normal flora needs to be demonstrated in the validation
after the prolonged incubation period.
Scientifically, the use of low nutrient media with longer incubation times may be justified
because environmental isolates are, by nature of the environment they are isolated from,
damaged. Nutrient rich media can shock damaged micro-organisms sufficiently to hinder
or prevent growth on that medium, rendering some isolates viable, non-culturable and
hence resulting in an unrepresentative low recovery rate. Validation data should always
demonstrate improved or equivalent recovery capability to more established media.
Neutralising agents added to media should be considered where sampling locations may
expose the samples to antimicrobial activity, for example, disinfectant residues on surfaces.
The passive air monitoring captures micro-organisms, which are typically attached to a
substrate particle, settling from the air by gravitational sedimentation onto ‘settle plates’
containing growth medium. Settle plates are typically Petri-dishes containing a nutrient
medium which are set out in predetermined locations for periods up to 4 hours. Longer
manufacturing periods may be monitored by sequential settle plates. Data obtained should
be corrected for the actual exposure time.
The advantages of passive air sampling include the ability to sample throughout the
production period with minimal intervention, hence minimal turbulence. As such, settle
plates are the method of choice for measuring air borne contamination in laminar air flows.
The disadvantages of passive air sampling include poor sensitivity which is unable to detect
rapid changes, no determination of the volume of air sampled and is semi-quantitative at
best. Settle plates may, however, be used to assess concentration of micro-organisms in
the environment and hence, as a tool for calculating contamination risk.

8.3.3.2. Air A quantitative value based upon the settling rate at which contaminants sediment from the
air could be applied. However, since the sedimentation rate is dependent on the size of
Airborne contamination includes bacteria (typically Gram positives), yeasts and moulds and aggregates and airflow patterns and is sensitive to disturbances in the airflow, this
may arise from several sources in the manufacturing environment, which may include: technique should be used as a qualitative or semi-quantitative measure of the environment.
Shedding skin cells from operators
Aerosols from contaminated liquids An exposure time of four hours is considered to be the maximum due to the potential for
agar media to lose moisture and hence alter the properties of the plates to support
Dust generated from building or engineering works growth. The maximum exposure time should be validated to demonstrate suitable recovery
Dust generated from the manufacturing process, for example, starting materials or of representative micro-organisms.
dry blends
Contaminated air

48 Microbiological Control for Non-Sterile Pharmaceuticals Microbiological Control for Non-Sterile Pharmaceuticals 49

8.3.3.2.2. Active Air Sampling
Active air sampling devices are designed to actively draw in air and capture the micro-
organisms contained within the sample. A defined volume of air is drawn into the device;
and the micro-organisms are captured either by impaction on the surface of media,
trapped in liquid or onto a filter which may then be transferred to growth media.
Factors that may influence the rate of capture include the velocity of air sucked in and
deflection of the flow; where strong air flows are present, attention must be given to the
exact placement of the equipment in the flow. The act of sampling using active methods
can cause disruption to laminar air flows. The equipment used should be covered by a
preventive maintenance and calibration programme.
8.3.3.3. Surfaces
Surfaces in the manufacturing environment are typically contaminated by micro-organisms
settling from the air, by direct touch of an operator, product or starting materials, tools or
ineffective cleaning.
The preferred method for sampling surfaces is determined by the properties of the surface.
Regular, flat and smooth surfaces with low levels of soil, moisture and contamination may
be sampled using contact plates. Irregular surfaces, liquids, or surfaces with higher levels
of soil or contamination may be sampled more effectively by swabs.
Surface monitoring forms a main mechanism for the microbiological assessment of
cleaning, by cleaning validation or monitoring and determination of clean equipment hold
times and dirty equipment hold times (section 5.3).
Surface sampling should be considered as part of the risk based assessment; the location
of sampling sites and frequency of sampling should be defined accordingly. Consideration
should be given to the benefits and limitations of the various techniques available when
choosing the most appropriate for each location.
8.3.3.3.1. Contact Plates
Contact plates, also called RODAC (Replicate Organism Detection and Counting), are
typically 55mm diameter plates where the media is slightly over filled and the surface, a
raised pad of agar, allows for direct application to a surface for sampling. In theory, micro-
organisms present on the surface in the area in contact with the agar, transfer to the
agar surface and develop in to CFUs when incubated. It is known, however, that the
pressure of application can impact recovery and that micro-organisms adhere to surfaces
differentially depending on multiple factors which include the surface type, organism type
and length of time it has resided on the surface. Although a better recovery rate and
efficacy is expected from RODAC plates, in relation to the recovery from swabs, the
transfer is not complete (in the region of 10–50%) and recovery capability of
50 Microbiological Control for Non-Sterile Pharmaceuticals
Chapter 8 – Microbiological Monitoring
representative micro-organisms from representative surfaces should be incorporated into
the validation of this method.
It is advisable to consider addition of neutralising agents to the media of RODAC plates to
counteract any residual antimicrobial activity of product, disinfectant or cleaning agents left
on the surface to be sampled.
RODAC plates are suitable for sampling flat and smooth surfaces only.
Application of the RODAC media to a surface leaves a residue of media on that surface
and sampling by this method must incorporate a subsequent cleaning of the surface to
remove traces of the residual media.
8.3.3.3.2. Swabs
Swabs are more suitable for sampling surfaces that are uneven or difficult to access.
Swabs should be moistened before sampling. Typically swabs are moistened in a sterile
isotonic buffered salt solution to help maintain viability of micro-organisms.
Several types of swabs are available for use, for example, alginate or cotton buds. Swabs
are wiped over a standardised surface area in a defined manner (templates may be
advantageous) and unlike RODAC sampling, may require further manipulation in the
laboratory to transfer the micro-organisms from the swab into the growth environment.
The viability data obtained from swabs may be impacted (proliferation or reduction) by the
lag time between sampling and processing in the laboratory or by the transportation
process to the laboratory, so both should be defined procedurally and incorporated into
method validation.
Neutralisation of the growth media should be considered and the sampled surface should
be cleaned following sampling to ensure no contaminating residues are left.
Swabs have poor transfer efficiency and recovery rates of micro-organisms from the swab
material are highly variable and depend on the technique used by the operator. Although it
is possible to validate swabs as semi-quantitative, swab sampling may be used more
effectively as a qualitative measure.
Note that the correlation between contact plates and swab samples can be very poor. In
addition to the variations in sampling efficacy, the mixing of swab solution can disrupt clumps
of bacteria leading to an increase in the number of colonies observed (section 8.3.7).
Microbiological Control for Non-Sterile Pharmaceuticals 51

8.3.3.4. Personnel
A site should consider monitoring of personnel in the risk assessment. Monitoring of
personnel is not mandatory for non-sterile manufacturing and is unlikely to be performed
routinely, but may be useful in investigations and training programmes.
If assessed as a risk, gloved fingers are typically sampled by applying finger dabs (including
the thumb print) to nutrient agar plates, for example, Tryptone Soy Agar and incubating for
3 to 5 days at 30–35°C.
Sampling of un-gloved hands can provide information on whether an individual carries a
particular micro-organism on the skin which may be of use for investigational purposes if
required.
8.3.3.5. Drains
Drains present the potential for back-contamination within the manufacturing environment
with respect to splash back and aerosols and are a source of standing contaminated
water (section 4.4.2). Hygienic design mitigates associated risks, but an effective
sanitisation programme should be incorporated as part of microbiological control. The
effectiveness of the microbiological control should be monitored.
The efficacy of disinfectants, method of application and contact time should be determined
and documented in a study to support microbiological control practices.
A qualitative monitor is typically achieved using swab sampling of the drain surfaces and or
liquid from the drain. Swabs provide easier access to difficult to reach locations and are
the method of choice for wet areas. Depending on the outcome of the risk assessment
with regards to the positioning of the drain in relation to manufacturing operations, areas
around the drain may also be sampled, for example, splash back areas.
Acceptance criteria should be based on objectionable organisms for the site (section
6.7.2) and new isolates should be identified to species level. If organisms defined as
objectionable to the product are identified there should be an assessment of the potential
risk to product and a decision made with regards to appropriate actions, e.g. additional
disinfection of the drain.
The frequency of monitoring drains should be justified through the risk assessment but
typically a quarterly monitor is advised once a baseline flora is understood.
8.3.3.6. Water
Micro-organisms in water systems are usually present in two forms: those attached to the
surfaces of the system in biofilms and micro-organisms circulating in the water which are
generally derived from organisms shed from the source biofilms. Water samples will
52 Microbiological Control for Non-Sterile Pharmaceuticals
Chapter 8 – Microbiological Monitoring
provide information on the organisms that are circulating in the water system but may
also be readily contaminated through the sampling process.
A possible source of microbial contamination in the system is the feed water. Feed water, in
most cases from the municipal supply, should be included in the monitoring programme. Action
limits may be derived from the national regulations for potable water and alert limits from
historical performance; the suggested minimal frequency for sampling and testing is monthly.
The method for testing of water and recommended action limits are provided in the
applicable monographs of the European Pharmacopoeia, however, it is expected to have
internally derived alert levels based upon the historical performance of the system.
The performance qualification of the water system requires frequent monitoring and
typically takes place over one year; in practice the performance qualification continues over
the life of the system through the ongoing monitoring programme.
The performance qualification may be divided into 3 phases:
Phase 1: for 2–4 weeks to:
Establish the operating ranges
Develop operating practices
Support cleaning and maintenance procedures
Demonstrate production and delivery of water of the required quality
Phase 2: for 2–4 weeks to demonstrate consistent operations producing
and delivering water
Phase 3: for one year to demonstrate extended performance over seasonal fluctuations
Sampling should be representative of point of use and all individual points-of-use should be
included in the monitoring programme. Points-of-use may be sampled according to a
rotating scheme, whereby each point-of-use is, for example, tested at least monthly with
representative samples obtained on a weekly basis.
Typical sampling frequencies are:
Potable feed Monthly/Quarterly
Product use Weekly
Washing Weekly
Sample points will depend on the design specification of the system and should ensure
coverage of the:
Start and end of the distribution system and generation plant
Feed water into the system
Intermediate points within the generation plant may also be considered to monitor the
performance of the different components.
Microbiological Control for Non-Sterile Pharmaceuticals 53

8.3.3.7. Steam
Refer to section 4.4.6.
The microbiological risk of steam usage should be considered in the risk assessment and
included in the monitoring programme if appropriate and at a suitable frequency
determined by the risk.
Extreme care should be taken if sampling steam and a condensate of the steam should be
generated for sampling. The sample should then be subjected to testing similar to that
used for water.
Limits should be established by historical evaluation and re-evaluated on a periodic basis.
8.3.3.8. Compressed Gases
Compressed gases which come into direct contact with product are a potential source of
contamination and should be considered in the risk assessment. However, they typically
represent a low risk due to the pressurised environment, low water availability and in line
filtration. Consequently, frequent monitoring is unlikely to be required and it may be possible to
demonstrate satisfactory control with a documented study as part of the initial qualification.
Microbiological sampling apparatus may be bespoke or commercially obtained and should
include a flow meter and mechanism for obtaining the sample securely; this may require a
flow rate controller to ensure integrity of the sample.
For anaerobic gases due attention should be paid to ensure maintenance of anaerobic
conditions at all stages including sampling, transportation to the laboratory and incubation.
A controlled amount of gas is directed through the equipment, on to the sampling surface;
the surface may be a filter membrane which will require transfer to suitable medium for
growth or impaction on to growth media.
Microbiological monitoring of compressed gases should complement physical monitoring of
particulates, moisture and hydrocarbons.
The frequency of microbiological monitoring should be determined by the risk assessment
and take into account filter performance etc. An approach for tabletting manufacture may
include microbiological sampling of the source each month with annual sampling of outlets.
Consideration should be given to the accessibility of sample outlets that are integral to
manufacturing equipment.
Limits should be established by historical evaluation and re-evaluated on a periodic basis.
54 Microbiological Control for Non-Sterile Pharmaceuticals
Chapter 8 – Microbiological Monitoring
8.3.4. Acceptance Criteria
Data generated by monitoring should be assessed against predetermined acceptance
criteria in order to understand if the area remains under microbiological control.
8.3.4.1. Setting Limits
For contamination levels, only upper limits are set. Typically, two limits are set for each
aspect tested; these are normally termed as “alert” for the tightest limit and “action” for
the widest limit. Unlike the situation for sterile product manufacture, limits for non-sterile
product manufacture are not typically set by regulations; it is for the manufacturer to set
appropriate limits. However, there is a regulatory expectation that limits will be based not
only on risk assessment but also on historical data, such that deviations from the norm
are readily detectable.
Excursions above the action limit will indicate a decline in the effectiveness of one or more
of the control measures; the excursion must initiate an investigation to determine the
cause and impact.
Excursion of the alert limit acts as an early warning signal for this decline, and may initiate
an investigation or an analysis of earlier data to detect a trend.
The limits set need to be a balance between taking unnecessary action or insufficient
action when needed. A brief outline is provided below, but it is recommended that a
statistician is consulted to ensure an appropriate data analysis is performed. The decision
regarding the approach to setting the appropriate limits should be captured in a written
justification document.
When very low counts are found, with a majority of the measurements revealing 0 colony
forming units, the results approach a Poisson distribution (but with tailing due to random
effects); this is normally the case for areas classified as ISO 6 or lower and Laminar Air
Flow cabinets.
When higher counts are found, the results may approach a normal distribution; this may be
the case for ISO classes 7 and up where more counts are typical. The data should be plotted
graphically to ensure the correct distribution is chosen for modelling. If the distribution is
unknown, then the use of cumulative probability will allow limits to be calculated.
For Poisson-distributed data, the alert limit may be set at the 95th percentile of the data-
set, and the action limit at the 99th percentile.
For normally distributed data, the limits may be set at the average plus 2x standard
deviation (equivalent to 95% probability) and 3x standard deviation (equivalent to 99.7%
probability) respectively.
Microbiological Control for Non-Sterile Pharmaceuticals 55

The figures calculated from the distributions should be critically assessed to ensure they
are commensurate with risk. For example, action limits significantly tighter than EU grade
C are unlikely to be required for most non-sterile products.
The data set used for determination of limits must include all seasonal variations; ideally a
period of 12 months or a multiple thereof. It is advisable to remove outliers from the data
set, since these may lead to limits being set which are too high. The method for removal
of outliers depends on the distribution, but must be described in the procedure for
establishment of limits. Individual values that are removed, e.g. due to assignable but
irrelevant contamination (special cause variation), must be justified.
In the beginning of execution of the monitoring plan, or after modifications to the
environment, equipment or processes, the data-set may be limited. It is recommended
that a period of intensive monitoring is performed at this stage. The number of data points
collected should allow for the detection of trends (to ensure a baseline is reached) and to
determine the correct statistical distribution. As guidance a minimum of 10 data points is
recommended for each aspect.
The limits should be reviewed and, if necessary, adapted periodically; typical review periods
are annually or bi-annually.
Adjustment to lower (tighter) limits is normally easy to justify; adjustment to higher limits
may be necessary due to, e.g. increased activity in the area, and can be acceptable if
investigations have revealed that the environment and processes are under control.
8.3.5. Performing Monitoring
Before monitoring commences, the following should be defined and in place:
An approved protocol or procedure
Approved and validated methods
Approved and validated equipment
Floor plan identifying sample points
Trained personnel to execute sampling (this may include production personnel)
Trained personnel to execute testing
Defined sample transit criteria to ensure timely and careful transfer to the laboratory
56 Microbiological Control for Non-Sterile Pharmaceuticals
Chapter 8 – Microbiological Monitoring
8.3.6. Investigations
In an established facility the majority of data collated from environmental monitoring should
confirm microbiological control. When excursions occur (see section 9.4), investigations
are typically led by microbiologically trained personnel. However, consideration should be
given to assigning ownership and accountability of the investigation to personnel from the
area where the excursion occurred, with support from microbiology staff, as this often
helps achieve a greater commitment to resolution.
For microbiological environmental monitoring the subsequent investigation typically includes:
Identification of the micro-organisms and comparison with the defined objectionable
organisms list for that site
Review of activities in the area, functioning and suitability of equipment and interview of
personnel
Review of cleaning records and review of data and trends of related environmental
monitoring samples
If it is necessary to gain a better understanding of the extent of a problem, the
monitoring programme can be increased with more sample locations and/or a higher
sampling frequency
Typical corrective actions upon excursion may include:
Communication with the area management from where the excursion occurred
Increase in monitoring
More frequent or more rigorous cleaning and/or disinfection
Retraining or communication with personnel
Modification of procedures for operations or cleaning
Maintenance or modification of the air handling system
The type of organism isolated may provide an indication of the contaminating source and
thus aid the determination of appropriate investigations and actions. For example:
isolation of airborne organisms (e.g. Moulds or Bacillus spp.)
Investigation of
The air supply
Maintenance of pressure differentials and air flow patterns
Containment of building work
Remedial actions may include use of a sporocide or disinfectant
Microbiological Control for Non-Sterile Pharmaceuticals 57
 Chapter 8 – Microbiological Monitoring

Isolation of water borne organisms (e.g. Pseudomonas spp.) Specificity

Investigation of Detection level/limit
Water data Quantification (limit, linearity and range – where appropriate)
Leaks, spills, opportunities for aerosol production and pooling water in the area Ruggedness
Drains Robustness
Humidity (e.g. washroom areas and equipment)
Methods used to recover micro-organisms from the environment are difficult to validate
Damaged surfaces or unsealed surfaces where water may accumulate and
reproducibly and accurately and are fraught with technical issues, for example:
harbour micro-organisms
Contaminants may not be completely removed during sampling
Remedial action may include
Contaminants may not be completely transferred from the sampling tool to the growth
Sanitisation of the water system medium
Disinfection of surfaces or drains Transit time from sampling to the laboratory for processing may proliferate or
Repair to damaged surfaces or seals deteriorate viability

Dehumidification Organisms may be sufficiently damaged by the environment to render them non-
culturable
The growth media may shock the organisms, hindering their growth
Isolation of personnel borne organisms (e.g. Staphylococcus spp.) may include:
Organisms are frequently isolated in clumps, which may or may not be broken up by
Investigation of
the analytical method, hence, a single clump of cells may be a colony forming unit but
Dressing and hygiene practices if agitated sufficiently during analysis multiple colony forming units may result in much
Personnel monitoring higher perceived recovery

Remedial action may include

Consequently, these methods provide an indication of the microbiological status of the area
Training in basic microbiology and hygiene sampled and validation should provide sufficient confidence of reproducibility to enable
Personnel counselling trends to be established, but an absolute value of the inherent population of micro-
organisms is unlikely to be obtained.

The validation may include in vitro style testing using spiked environments to provide a
8.3.7. Validation of Monitoring Techniques
measure of the method’s recovery capability. For example, coupons of various surface
types found in the manufacturing environment, may be spiked with known numbers of
Analytical methods should be validated (EU GMP Chapter 6). The variables and how the
viable cells and the method used to recover those cells from the surface. However, the
variables are controlled should be understood and accounted for in the design of the
micro-organisms’ differential ability to adhere to different surfaces, impacted by age of the
validation. This applies equally to both traditional and rapid microbiological methods.
culture, surface condition and type of organism, can all add further complexity to the
validation effort. A risk based assessment should be used to determine the approach to
Critical considerations for validation are:
method validation, ensuring validity of data generated.
Is the method appropriate?
Is the method reproducible? It is recommended that challenge organisms used during validation of monitoring methods
Is the method comparable (if the method replaces a current method)? includes a range of local isolates to demonstrate the ability to recover such isolates. The
validation should include a rationale for the approach taken including the choice of
representative local isolates. Consideration should also be given to how long local isolates
Recommended validation parameters include: are retained before there is a need to select new isolates from the local environment.
Accuracy
Precision

58 Microbiological Control for Non-Sterile Pharmaceuticals Microbiological Control for Non-Sterile Pharmaceuticals 59
 Chapter 9 – Reporting and Trending of Microbiological Data

When a method, medium or equipment is changed, a comparative analysis between the Chapter 9
methods must be made. Independent validation of both methods is insufficient. Preferably
both methods are used simultaneously for a certain period. Based on the obtained results,
new action and alert levels may be set, and a new baseline for trend evaluation may be Reporting and Trending of
determined.
Microbiological Data
9.1. Introduction

Raw monitoring data by itself is of little value and it needs to be appropriately analysed and
presented in order to provide trends and appropriate focus for microbiological control.

The reporting and trending of data provides an opportunity for the effectiveness of
microbiological control and the appropriateness of the monitoring programme to be reviewed
and modified. Where good control is demonstrated, there may be opportunities to reduce
the level of monitoring, thus reducing cost without compromise to product or patient.

9.2. Batch-Specific Data

In contrast to the situation with sterile product manufacture, environmental monitoring data
is typically not produced specifically to support batch disposition in non-sterile manufacturing.

Where batch-specific environmental monitoring is established as necessary to support the

quality assurance of the product this forms part of the batch disposition assessments.

In addition, if general monitoring action limits are exceeded they must be reported to the
Quality Unit and a boundary for investigation should be drawn taking into consideration the
impact on products manufactured in the facility since the previous satisfactory monitoring.

It is important that Qualified Persons are aware of any potential impact on the products
they are responsible for releasing.

9.3. Periodic Summary Reports

Data should be assessed and trended as it is generated. Data outside the set specification
or limits must be reported to the Quality Unit (9.2). If the facility is under good control, the
majority of data will be reported via periodic summary reports.

Periodic summary reports should be generated and reviewed by the applicable cross
functional team (Chapter 2) including functional management and the Quality Unit.

The frequency of generating a formal periodic summary report will depend on a number of
factors such as the sampling frequency, the facility history and the products manufactured.

60 Microbiological Control for Non-Sterile Pharmaceuticals Microbiological Control for Non-Sterile Pharmaceuticals 61
 Chapter 9 – Reporting and Trending of Microbiological Data

The summary report should be used to determine appropriate actions relating to the Laboratory environmental data
facility and its operations and reassess the testing frequencies and limits (Chapter 8). Data entry/calculations

Consideration should be given to the most appropriate means of presenting the summary
data to other site functions. A combination of graphical and tabulated formats providing a
visual representation with clear supporting summary text should be the aim. Data should be
presented covering an extended period of time, for example, over a rolling year, to
determine whether trends are present. It may also be useful to compare data with previous
rolling 12 months to establish understanding of successive seasonal variations. OOS and
OOT data should be specifically highlighted and addressed within the summary reports
(section 9.4).
Often microbiological data will contain many zero data points which can present issues in
statistical analysis of the data. It is important to select appropriate analysis tools which do
not lead to the masking of significant events or trends (Chapter 8).
Numerical methods (e.g. CUSUM calculations) or trend analysis software may be used for the
detection of trends; this ensures objectivity and speed in trend detection. Any software tool
used to generate graphical representations or analyse data must be qualified appropriately.
9.4. Out of Specification/Out of Trend Handling
Out of specification (OOS) results include all suspect results that fall outside the
specifications or acceptance criteria established in new drug applications, official
compendia, or by the manufacturer within their internal procedures, e.g. Action levels for
environmental monitoring data.
Out of Trend (OOT), with respect to environmental monitoring data, may be defined as the
exceeding of an alert level or a defined statistical signal.
If no attributable laboratory error is determined, then a wider investigation, involving a
cross-functional team will be required to determine the root cause.
Upon excursion of the alert limit (OOT) a limited investigation may follow; as a minimum an
analysis for trends of the applicable sample and related samples is expected.
For excursions outside of the specification or action limits, or when an adverse trend is
confirmed there should be a more extensive investigation. It is recommended to standardise
the investigation procedure which will be specific to the type of OOS or action limit breached.
If an OOS result is deemed to be valid, then further investigation is required to establish the
root cause(s) and appropriate corrective and preventive actions need to be determined.
Predetermined corrective action steps may be detailed in procedures. For example, in the
situation where an environmental monitoring sample shows an OOS (action level) or OOT
(alert level), particular steps to clean and retest may be taken and if the resulting retests
are satisfactory, no further corrective actions may need to be taken.
Even when procedure-driven corrective actions are taken, consideration should be given to
whether any actions are required to prevent recurrence of the root cause.
The information from the OOS and OOT investigations should be analysed and shared with
the quality unit and other relevant business functions to aid determination of root cause,
impact on product disposition and agreement of any further corrective and preventative
actions that may be appropriate. OOS and OOT data should also be specifically highlighted
and addressed within periodic summary reports.

The management of microbiological OOS and OOT results follows the same principles
applicable to chemical OOS and OOT investigations (see PQG, 2005) but requires additional
considerations, for example, microbiological contamination may not be homogeneous, and
may include specific requirements detailed in the compendial tests. These requirements
should be documented in procedures.

For OOS investigations, it is critical to perform an initial investigation to determine whether

the OOS result is due to laboratory error. A checklist may be useful to aid such
investigations and might include:
Sample handling/storage
Media/components
Equipment
Methods
Analyst technique

62 Microbiological Control for Non-Sterile Pharmaceuticals Microbiological Control for Non-Sterile Pharmaceuticals 63
 Appendix – Mind Maps

Appendix – Mind Maps

Figure A.1: Overall Microbiological Control Mind Map

Figure A.2: Personnel Microbiological Control Mind Map

64 Microbiological Control for Non-Sterile Pharmaceuticals Microbiological Control for Non-Sterile Pharmaceuticals 65
 Appendix – Mind Maps

Figure A.3: Facilities Microbiological Control Mind Map

Figure A.5: Laboratory Operations Microbiological Control Mind Map

Figure A.4: Cleaning Microbiological Control Mind Map

Figure A.6: Formulation Microbiological Control Mind Map

66 Microbiological Control for Non-Sterile Pharmaceuticals Microbiological Control for Non-Sterile Pharmaceuticals 67
 Glossary

Glossary
Aflatoxin
Naturally occurring mycotoxins that are produced by many species of Aspergillus. May be
associated with herbal starting materials.

At rest
A term for the static state with all services operating, but without personnel present.

Baseline
Observations or data used for comparison or as a control.

Bioburden
Figure A.7: Monitoring Microbiological Control Mind Map The total microbial load with which an object or sample is contaminated.

Biofilm
An organised microbial system consisting of layers of microbial cells associated with
surfaces, often with complex structural and functional characteristics. Biofilms have
physical/chemical gradients that influence microbial metabolic processes. They can form
on inanimate devices and also cause fouling (Prescott, Harley & Klein, 2005).

CFR (Code of Federal Regulations)

US regulations. Parts 210 and 211 relate to pharmaceutical Good Manufacturing Practice.

CFU (Colony Forming Unit)

Viable micro-organisms (bacteria, yeasts and mould) capable of growth under the
prescribed conditions (medium, atmosphere, time and temperature) develop into visible
colonies (colony forming units) which are counted. The term colony forming unit (CFU) is
used because a colony may result from a single micro-organism or from a clump/cluster
of micro-organisms. It is normally expressed as CFU per g or mL.

Cleaning
The removal of soil from a surface.

Clean room
A room in which the concentration of airborne particles is controlled to a defined
standard. This is achieved by controlling the introduction, formation and retention of
particles.

Contact time
The total time an organism is exposed to the antimicrobial action of a disinfectant.

68 Microbiological Control for Non-Sterile Pharmaceuticals Microbiological Control for Non-Sterile Pharmaceuticals 69
 Glossary

Containment level 2 Intermediate/in-process material

The facility requirements for the containment of Hazard Group 2 biological agents as A partly manufactured material which requires further processing before it becomes a
defined by the Advisory Committee on Dangerous Pathogens (Advisory Committee on bulk or filled product.
Dangerous Pathogens, 2001).
Local isolate
Contaminant An organism isolated from the local environment, e.g. production, laboratory.
A foreign agent not introduced as part of processing, such as airborne particulates or
adventitious micro-organisms. Medium (plural, media)
Any liquid or solid material prepared for the growth, maintenance, or storage of micro-
Dead leg organsims.
Any length of pipework that does not allow effective circulation of its contents, resulting in
the potential for contamination. Microbiological Control
The sum total of activities undertaken to manage and mitigate risks from microbiological
Disinfectant sources.
An agent that reduces the level of micro-organisms to one that is safe for the relevant
purpose. Usually a chemical agent but sometimes may be a physical one such as X-rays Microbiological Monitoring
or ultraviolet light (Pharmig 2006). Sampling, testing, trending and reporting activities undertaken to assess and understand
the state of microbiological control.
Dynamic state
Refers to environmental or particle monitoring when a room is occupied by personnel. Objectionable organism
An organism that is risk assessed to be objectionable with respect to its potential impact
FMEA (Failure Mode and Effects Analysis) to patients. Some objectionable organisms are specified in the pharmacopoeias but these
A risk assessment and risk mitigation tool. are not exclusive and other organisms may be objectionable depending on the nature of
the product, route of administration and intended patient population.
Genotype
The precise genetic constitution of an organism. Outlets
User points in a water system.
HACCP (Hazard Analysis and Critical Control Points)
A formalised process by which a manufacturer ensures that all steps critical to product safety Out-of-Specification (OOS)
are assessed and that adequate safety procedures are identified, implemented and reviewed. An OOS result is one which falls outside the specifications or acceptance criteria
established in new drug applications, official compendia, or by the manufacturer within
HEPA (High Efficiency Particulate Air) their internal procedures, e.g. Action levels for environmental monitoring data.
Describes the system for filtering (diluting) air into clean-rooms. Standard HEPA filters
remove 99.97% of 0.3µm particles. Out-of-Trend (OOT)
With respect to environmental monitoring data, the exceeding of an alert level or a defined
Herbal drugs statistical signal.
Herbal drugs are mainly whole, fragmented, or cut plants, parts of plants, algae, fungi or
lichen, in an unprocessed state, usually in dried form but sometimes fresh. Certain exudates Phenotype
that have not been subjected to a specific treatment are also considered to be herbal drugs. The observable properties of an organism.
Herbal drugs are precisely defined by the botanical scientific name according to the binominal
system (genus, species, variety and author). (European Pharmacopoeia, 6th edition, 2008). Potable water
Water which complies with local drinking water standards. Typically this is WHO (World
Incubation Health Organisation) drinking water quality.
The process of maintaining appropriate conditions (temperature, humidity, time) to allow
microbial cells to replicate. RODAC (Replicate Organism Detection And Counting)
The international acronym for a contact plate (that is an agar plate, with a raised surface,
applied to a surface for the enumeration of micro-organsims).

70 Microbiological Control for Non-Sterile Pharmaceuticals Microbiological Control for Non-Sterile Pharmaceuticals 71

Risk assessment
A systematic process of organising information to support a risk decision to be made
within a risk management process. It consists of the identification of hazard and the
analysis and evaluation of risks associated with the exposure to those hazards (ICH Q9).
Settle plate
An agar plate used for passive air-sampling. The plate is exposed for a fixed duration, after
which it is incubated, and the number of micro-organisms which have settled on it and
have produced colonies are counted.
Sporicide
An agent that destroys microbial spores, especially a chemical substance that kills
bacterial spores (Pharmig 2006).
Starting material
Any substance used in the production of a medicinal product but excluding packaging
materials (EU GMP Glossary).
Tundish
A receptacle within a drain forming a reservoir for water thus acting as a buffer,
smoothing out flow, between processing areas and the drainage system.
Water activity (aw)
A measurement of the energy status of the water in a system. It is defined as the vapour
pressure of water above a sample divided by that of pure water at the same temperature;
therefore, pure distilled water has a water activity of exactly one.
72 Microbiological Control for Non-Sterile Pharmaceuticals
Bibliography
Bibliography
ADVISORY COMMITTEE ON DANGEROUS PATHOGENS ‘The management, design and operation of
microbiological containment laboratories’
ISBN 0 7176 2034 4 (HSE Books, Her Majesty’s Stationery Office, 2001)
BAIRD, R.M.& BLOOMFIELD, S.F. , Microbial Quality Assurance in Cosmetics, Toiletries and Non-Sterile
Pharmaceuticals. 2nd Ed. Taylor & Francis. Bristol, PA. 1996. p 51 & 115
CFR Part 211.113 Control of microbiological contamination
CUNDELL, A. M. Environmental Monitoring in Non-Sterile Product Manufacturing. In Environment
Monitoring. Edited J. Moldenhauser Davis Horwood/PDA 2005
CUNDELL, A. M. ‘Managing the Microbiological Quality of Pharmaceutical Excipients’, PDA Journal of
Pharmaceutical Science and Technology, Vol 59, No 6, November – December 2005, p 381–395
EMEA CPMP ‘Note for guidance on quality of water for pharmaceutical use’ CPMP/QWP/158/01,
May 2002
EUROPEAN COMMISSION ‘The Rules Governing Medicinal Products in the European Union’ Volume 4
‘Good manufacturing practice (GMP) Guidelines’
http://ec.europa.eu/enterprise/pharmaceuticals/eudralex/vol4_en.htm
EUROPEAN PHARMACOPOEIA
FRIEDEL, RR & CUNDELL, AM; Pharmacopoeial forum vol 24 number 2 March/April 1998
p 6087-6090
HUTCHESON, J. ‘Validation of Purified Water Systems’ presentation at Pharmig conference on non-
sterile monitoring (2008)
INTERNATIONAL CONFERENCE ON HARMONISATION (ICH) ‘Q9 – Quality Risk Management’ (2005)
www.ich.org (also now incorporated as Annex 20 of European Commission GMP Guide and
available via Eudralex)
INTERNATIONAL CONFERENCE ON HARMONISATION (ICH) ‘Q4B ANNEX 4A
Evaluation and Recommendation of Pharmacopoeial Texts for Use in the ICH Regions on
Microbiological Examination of Non-Sterile Products: Microbial Enumeration Tests General Chapter’
INTERNATIONAL CONFERENCE ON HARMONISATION (ICH) ‘Q4B ANNEX 4B
Evaluation and Recommendation of Pharmacopoeial Texts for Use in the ICH Regions on
Microbiological Examination of Non-Sterile Products: Tests for Specified Micro-organisms General
Chapter’
INTERNATIONAL CONFERENCE ON HARMONISATION (ICH) ‘Q4B ANNEX 4C
Evaluation and Recommendation of Pharmacopoeial Texts for Use in the ICH Regions on
Microbiological Examination of Non-Sterile Products: Acceptance Criteria for Pharmaceutical
Preparations and Substances for Pharmaceutical Use General Chapter’
Microbiological Control for Non-Sterile Pharmaceuticals 73
 Bibliography

ISO/IEC Guide 51:1999 ‘Safety Aspects – Guideline for their inclusion in standards’ WILKINSON, S: ‘Cleaning Validation in Non-Sterile Manufacturing’
Presentation to Pharmig non-steriles conference, February 2008
ISPE Baseline pharmaceutical engineering guides for new and renovated facilities: Volume 2 Oral solid
dosage. 1998

ISPE Baseline pharmaceutical engineering guides for new and renovated facilities: Volume 5
Commissioning and Qualification. 2001

KABARA, JJ AND ORTH, DS (ed.) Preservative-free and self preserving cosmetics and Drugs:
Principles and Practise, Marcel Dekker, p 1–14 (1997)

MEDICINES AND HEALTHCARE PRODUCTS REGULATORY AGENCY (MHRA) Inspection and Standards
Division ‘Rules and Guidance for Pharmaceutical Manufacturers and Distributors’ Pharmaceutical
Press (2007) (Also referred to as ‘The Orange Guide’)

MONTGOMERY, E. & TAWIAH, W. M., Microbiological Considerations When Selecting Excipients During
Product Development, American Pharmaceutical Review. Vol. 7, Issue No. 4. July, 2004.

PDA TECHNICAL REPORT 13 ‘Fundamentals of an Environmental Monitoring Program’, 2001.

PHARMACEUTICAL QUALITY GROUP (PQG) ‘Monograph 10 – Cleaning Validation’ ISBN 0 906810 63 9

(Institute of Quality Assurance, 1999) Available from www.thecqi.org

PHARMACEUTICAL QUALITY GROUP (PQG) ‘Monograph 11 ‘Good Quality Control Laboratory Practice’
ISBN 0 906810 79 5 (Institute of Quality Assurance, 2005)
Available from www.thecqi.org

PHARMIG ‘A Guide to Disinfectants and their use in the Pharmaceutical Industry’ ISBN 0 9551030 0 3
(The Pharmaceutical Microbiology Interest Group, 2006)
Available from www.pharmig.org.uk

PHARMIG ‘Technical Guidance Document 1 – Water Activity’ (2008)

PHRMA ENVIRONMENTAL MONITORING WORK GROUP ‘Microbiological monitoring of environmental

conditions for Non-Sterile Pharmaceutical Manufacturing’. Pharmaceutical Technology March 1997;

PRESCOTT, HARLEY & KLEIN: ‘Microbiology’, 6th edition (2005)

THE CODE OF PRACTICE FOR QUALIFIED PERSONS: Institute of Biology, Royal Pharmaceutical Society of
Great Britain, Royal Society of Chemistry (Also included in ‘The Orange Guide’)

RANDALL, C. ‘Environmental monitoring of Non-Sterile Manufacturing’ presentation at Pharmig

Microbiological monitoring and control of Non-Sterile manufacturing meeting (2005)

UNITED STATES PHARMACOPOEIA

USP <1072> Disinfectants and Antiseptics

USP <1112> Microbiological Attributes of Non-Sterile Pharmaceutical Products – Application of Water

Activity Determinations

74 Microbiological Control for Non-Sterile Pharmaceuticals Microbiological Control for Non-Sterile Pharmaceuticals 75
 Notes

76 Microbiological Control for Non-Sterile Pharmaceuticals Microbiological Control for Non-Sterile Pharmaceuticals 77
 EXCELLENCE IN MICROBIOLOGY

Pharmig
T5 The Maltings, Roydon Road, Stanstead Abbotts
Hertfordshire, SG12 8HG, United Kingdom
T: + 44 (0)1920 871 999 F: + 44 (0)1920 871 156
E: info@pharmig.org.uk www.pharmig.org.uk

The Chartered Quality Institute

12 Grosvenor Crescent, London, SW1X 7EE
T: +44 (0)20 7245 6722 F: +44 (0)20 7245 6788
E: info@thecqi.org www.thecqi.org
9 780956 080400