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12075 : 1997
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Biotechnology Ð |
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Large scale process and |
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production Ð |
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Procedures for fermentation |
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and downstream processes |
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The European Standard EN 12075 : 1997 has the status of a |
British Standard |
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ICS 07.080 |
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NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAW
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BS EN 12075 : 1997

Committees responsible for this

British Standard
The preparation of this British Standard was entrusted to Technical Committee
CII/58, Biotechnology, upon which the following bodies were represented:

Association of Consultants to the Bioscience Industries (ACBI)

BLWA Ltd. (The Association of the Laboratory Supply Industry)
Bioindustry Association
Brewing Research Foundation International
British Agrochemicals Association Ltd.
Chemical Industries Association
Confederation of British Industry
Department of Health
Department of the Environment (Air Climate and Toxic Directorate)
Health and Safety Executive
Institution of Chemical Engineers
International Society for Pharmaceutical Engineering
Ministry of Agriculture, Fisheries and Food
National Engineering Laboratory
Public Health Laboratory Service
Society for Applied Bacteriology
Society for General Microbiology

This British Standard, having

been prepared under the
direction of the Sector Board for
Materials and Chemicals, was
published under the authority of
the Standards Board and comes
into effect on
15 August 1997

 BSI 1997
Amendments issued since publication
Amd. No. Date Text affected

The following BSI references

relate to the work on this
standard:
Committee reference CII/58
Draft for comment 95/124066 DC

ISBN 0 580 27949 9

 BS EN 12075 : 1997

Contents

Page
Committees responsible Inside front cover
National foreword ii
Foreword 2
Text of EN 12075 3

 BSI 1997 i
BS EN 12075 : 1997

National foreword

This British Standard has been prepared by Technical Committee CII/58, and is the
English language version of EN 12075 : 1997, Biotechnology Ð Large-scale process and
production Ð Procedures for fermentation and downstream processes, published by
the European Committee for Standardization (CEN).
Compliance with a British Standard does not of itself confer immunity
from legal obligations.

Summary of pages
This document comprises a front cover, an inside front cover, pages i and ii, the
EN title page, pages 2 to 8, an inside back cover and a back cover.

ii  BSI 1997
EUROPEAN STANDARD EN 12075
NORME EUROPEÂENNE
EUROPAÈISCHE NORM February 1997

ICS 07.080

Descriptors: Biotechnology, culture (biology), micro-organisms, management, fermentation, definitions, description, generalities, hazards,
inspection, contamination, accident prevention, environmental protection

English version

Biotechnology Ð Large-scale process and production Ð

Procedures for fermentation and downstream processes

Biotechnologie Ð ProceÂde aÁ grande eÂchelle et Biotechnik Ð Verfahren im Groûmaûstab und

production Ð ProceÂdures pour les proceÂdeÂs de Produktion Ð Vorgehensweise fuÈr die Bereiche
fermentation et traitement aval Fermentation und Aufarbeitung

This European Standard was approved by CEN on 1996-12-29. CEN members are
bound to comply with the CEN/CENELEC Internal Regulations which stipulate the
conditions for giving this European Standard the status of a national standard
without any alteration.
Up-to-date lists and bibliographical references concerning such national standards
may be obtained on application to the Central Secretariat or to any CEN member.
This European Standard exists in three official versions (English, French, German).
A version in any other language made by translation under the responsibility of a
CEN member into its own language and notified to the Central Secretariat has the
same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Denmark,
Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands,
Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom.

CEN
European Committee for Standardization
Comite EuropeÂen de Normalisation
EuropaÈisches Komitee fuÈr Normung

Central Secretariat: rue de Stassart 36, B-1050 Brussels

 1997 Copyright reserved to CEN members

Ref. No. EN 12075 : 1997 E
Page 2
EN 12075 : 1997

Foreword Contents
This European Standard has been prepared by Page
Technical Committee CEN/TC 233, Biotechnology, the
Secretariat of which is held by AFNOR. Foreword 2
This European Standard shall be given the status of a Introduction 3
national standard, either by publication of an identical 1 Scope 3
text or by endorsement, at the latest by August 1997, 2 Normative references 3
and conflicting national standards shall be withdrawn
at the latest by August 1997. 3 Definitions 3
According to the CEN/CENELEC Internal Regulations, 4 Process description 4
the national standards organizations of the following 5 Risk management 5
countries are bound to implement this European
Annex A (informative) Bibliography 8
Standard: Austria, Belgium, Denmark, Finland, France,
Germany, Greece, Iceland, Ireland, Italy, Luxembourg,
Netherlands, Norway, Portugal, Spain, Sweden,
Switzerland and the United Kingdom.

 BSI 1997

Introduction
This European Standard supports industrial activities
in the area of biotechnology covering operations with
both non-genetically modified micro-organisms and
genetically modified micro-organisms (GMMs), with
both non-pathogenic and pathogenic micro-organisms
(see annex A [1], [2]).
Fermentation processes vary widely in their nature and
design. Generally prokaryotic or eukaryotic
micro-organisms, plant cells, mammalian cells or insect
cells are cultivated and processed in such a way as to
produce a desired end-product such as biomass,
pharmaceuticals, additives, metabolites and foodstuffs.
1 Scope
This European Standard specifies the principles for the
assessment and selection of fermentation and
downstream operations so that they are carried out in
a manner which ensures the safety of personnel, the
environment and product and contributes to product
quality.
This European Standard is intended for use by those
designing and/or operating processes and by other
interested parties.
Unit operations are not described in detail and
individual production processes can require specific
equipment or unit operations which are not described
here.
2 Normative references
This European Standard incorporates by dated or
undated reference, provisions from other publications.
These normative references are cited at the
appropriate places in the text and the publications are
listed hereafter. For dated references, subsequent
amendments to or revisions of any of these
publications apply to this European Standard only
when incorporated in it by amendment or revision. For
undated references the latest edition of the publication
referred to applies.
prEN 12460 Biotechnology Ð Large-scale process
and production Ð Equipment
implementation according to the
degree of hazard
prEN 12461 Biotechnology Ð Large scale process
and production Ð Guidance for the
handling, inactivating and testing of
waste
 BSI 1997
Page 3
EN 12075 : 1997
3 Definitions
For the purposes of this standard, the following
definitions apply:
3.1 bioaerosol
Colloid dispersed solid or liquid particles in a gaseous
environment presenting negligible gravitational settling,
containing micro-organisms.
3.2 biocontamination
Presence of undesired micro-organisms.
3.3 closed system
System where a barrier separates
micro-organisms/organisms from the
environment. [EN 1620]
3.4 controlled area
Area constructed and/or operated in such a manner as
to limit contamination of the other areas by
micro-organisms/organisms from within the controlled
area [EN 1620].
3.5 downstream process
Sequence of operations following the fermentation.
3.6 fermentation
Biotechnical process where the target product is
formed while cultivating the process
micro-organism(s) [EN 1620].
3.7 fermenter
Closed or open vessel where a culture of
micro-organisms is grown under controlled conditions.
3.8 hazard
Intrinsic potential property or ability of something
(e.g. any agent, equipment, material or process) to
cause harm [EN 1620].
NOTE. Harm is an injury or damage to health of people and/or to
the environment.
3.9 inactivation
Destruction of micro-organisms.
3.10 master cell bank (MCB)
Stock of cells from which all subsequent cell banks are
derived [EN 1619].
NOTE 1. MCB stock is not normally intended for use directly in
production.
NOTE 2. The term MCB covers all type of cells i.e.
micro-organisms as defined in 3.11.
Page 4
EN 12075 : 1997
3.11 micro-organism
Microbiological entity, cellular or non-cellular, capable
of replication or of transferring genetic
material [EN 1619].
The term micro-organism covers the term of biological
agent, according to the Directive 90/679/EEC :
micro-organisms, including those which have been
genetically modified, cell cultures and human
endoparasites which may be able to provoke any
infection, allergy or toxicity.
3.12 pathogen
Micro-organism causing disease [EN 1620].
3.13 physical containment
System for confining a micro-organism/organism or
other entity within a defined space [EN 1620].
3.14 process micro-organism
Micro-organism used for production purposes in a
biotechnological process or constituting (part of) the
product itself.
3.15 risk
Probability of occurrence of a hazard causing harm
and the degree of severity of the harm.
3.16 sterilization
Validated process used to reach a state free from viable
micro- organisms.
NOTE. In a sterilization process, the nature of microbiological
death or reduction is described by an exponential function.
Therefore, the number of micro-organisms that survive a
sterilization process can be expressed in terms of probability.
While the probability can be reduced to a very low number, it can
never be reduced to zero.
Figure 1. Schematic diagram of a biotechnological manufacturing process
3.17 unit operation
Operation to perform a single chemical, physical or
mechanical activity.
NOTE 1. Examples of unit operations are heat transfer, mixing,
separating including filtration and centrifugation, and sterilization.
NOTE 2. Combinations of unit operations constitute a process
step. For example, downstream process step could consist of
separation, extraction, concentration and drying.
3.18 waste
By-product arising from a process or unwanted
substance or article derived from any activity.
NOTE. Examples of waste are scrap material, effluent, unwanted
residue or surplus arising from any process or activity or any
substance or article which is discarded or to be disposed of as
being broken, contaminated, spoiled, or worn out.
3.19 working cell bank (WCB)
Stocks of cells derived from the master cell bank
(MCB), which are used for inoculation [EN 1619].
NOTE. The term working cell bank covers all type of cells i.e.
micro-organisms as defined in 3.11.
4 Process description
4.1 General
Fermentation and downstream processes consist of a
number of unit operations linked together to produce a
product. There is a wide range of unit operations and a
general schematic diagram is shown in figure 1. The
unit operations can be categorized into process steps
which are described in 4.2 to 4.5.
 BSI 1997

4.2 Raw materials
Select, transport, store and treat raw materials so that
required characteristics are retained and contamination
with unwanted materials or micro-organisms is
prevented.
Carry out medium preparation according to
documented procedures for treatment, addition and
mixing of the raw materials.
NOTE. Guidance on control procedures for raw materials are
given in EN 1826.
4.3 Fermentation
Ensure that the fermenter and associated equipment
are cleaned and sterilized as appropriate to the process
and risk assessment (see clause 5).
Prepare the inoculum culture from the working cell
bank. Typically, this process consists of one or more
stages of increasing volume, such that the amount
transferred to the production stage in most cases is
between 2 % and 10 % of the production volume.
Checks on culture against specified requirements can
be carried out at each stage, especially for aseptic
operation.
Carry out transfers or terminations of each inoculum
stage as appropriate in accordance with documented
procedures for transfer, including contamination
criteria.
Run the main fermentation in an appropriate mode
such as batch, fed batch, continuous, semi-continuous
or multistep process or any combination of these. The
process micro-organism can be contained in the
fermenter in a variety of modes such as free
suspension, immobilized in a matrix or retained on a
membrane.
Terminate the fermentation and continue the
downstream process.
4.4 Downstream process
Use appropriate downstream processes for the
isolation and/or further purification, concentration and
stabilization of the product. These processes can occur
either during or after the fermentation process and, in
some cases, can occur in the fermenter itself.
Unit operations used in downstream processes can
include filtration, centrifugation, precipitation,
distillation, lyophilisation, crystallisation, ion exchange,
chromatography, electrodialysis, direct and tangential
filtration, liquid-liquid extraction, flotation, evaporation.
4.5 Output
Package, label, store and transport products with due
regard to safety and quality according to appropriate
documented procedures.
NOTE 1. Attention is drawn to relevant European and national
regulations.
 BSI 1997
Page 5
EN 12075 : 1997
Establish waste management procedures for the waste
products from the process including provisions for
contaminated or aborted process material. Waste from
biotechnological processes shall be handled and, if
necessary, inactivated in accordance with prEN 12461.
NOTE 2. Attention is drawn to European and national regulations
for the control of waste.
5 Risk management
5.1 Risk assessment
A documented risk assessment shall be made for the
micro-organism and process with regard to the general
hazards identified. This will typically be done at the
stages of process design, process implementation,
significant process change.
A relevant method of risk assessment shall be used
with regard to personnel, environment and product.
The risk group of the micro-organism shall be
considered in preparing the assessment
(see annex A [1], [2]).
NOTE 1. This assessment can be based on methods such as:
± HACCP (Hazard Analysis Critical Control Points)
(see annex A [3] [4])
± HAZOP (Hazard and Operability)
(see annex A [5] [6])
± IEC 812:1985 (see annex A [7])
± EN 1050 (see annex A [8])
NOTE 2. In some cases of traditional or empirically processes, the
risk assessment can take a simplified form based on a history of
safe use.
5.2 Description of relevant risks
The risk assessment shall take into account the
hazards from the potential exposure, such as:
a) release of bioaerosol;
b) biocontamination of raw materials or any part of
the process which can result in the production of a
pathogen or harmful substance;
c) deliberate or unintentional variation in the
composition of raw materials, or any variation of
process parameters which could allow physiological
variation, leading to the presence of harmful
substances or pathogens;
d) direct contact with micro-organisms;
e) failure of containment.
Page 6
EN 12075 : 1997
5.3 Control of risks
5.3.1 Process design
Based on the results of the risk assessment, the risks
to personnel, products and environment shall be
minimized by the choice of appropriate unit
operations, procedures and equipment in accordance
with prEN 12460. At the same safety level, this
selection will also take into account the type of
product and the economics of the process.
NOTE. Performance criteria which determine the suitability of
equipment for the intended operation of the plant are given in the
appropriate European Standards for biotechnological equipment.
5.3.2 Procedural reviews
Procedures to monitor the effectiveness of the
designated controls shall be established and reviews
held at regular intervals.
5.3.3 Biocontamination
The nature and extent of any biocontamination of the
process shall be monitored and the potential risks and
hazards arising shall be assessed taking into account
the experiences with the process. Levels of acceptable
biocontamination shall be determined. Procedures shall
be established to inactivate biocontaminated materials,
equipment and controlled areas in cases which present
unacceptable risks to personnel and/or the
environment.
NOTE. Occasional contamination of biotechnological processes
occurs, although the frequency of such occurrences can be
extremely low. It is recognized that in some traditional industries
(for example production of wines) natural introduction of
micro-organisms from the environment is a routine or essential
feature of the process.
5.3.4 Documentation
To ensure control of raw material and process, and to
contribute to product quality, documentation shall be
established so that the documented risk control
procedures are applied. The operating conditions and
methods of monitoring and control shall be defined
and results recorded such that control of the process
can be demonstrated. In the case of unexpected
events, training and procedures shall be established to
enable operating staff to regain control of or stop the
process in a safe manner.
NOTE. A system of quality management, such as
EN ISO 9000 series (see annex A [9]) should be used.
5.3.5 Preparation, cleaning and sterilization of
equipment
The methods of preparation of the equipment shall be
defined. Techniques for cleaning and sterilizing, such as
the use of chemicals, steam or heat, shall be verified.
NOTE. Guidance on testing procedures for cleanability,
sterilizability and leaktightness are given in prEN 12296
(see annex A [10]), prEN 12297 (see annex A [11]) and
prEN 12298 (see annex A [12]).
5.3.6 Procedural controls based on the risk
assessment
Depending on the risk group of the process
micro-organism (see 5.1) and on the specific
circumstances of the process in which it is present,
different parts of the process can require different
levels of physical containment as given in table 1.
 BSI 1997
 BSI 1997

Table 1. Physical containment levels based on the risk assessment

Requirement1) Physical containment level
1 2 3 4
Areas to production areas restricted to nominated workers only No2) Optional3) Yes4), recommended via Yes, via airlock
airlock
Viable micro-organisms containment in a closed system No Yes Yes Yes
Closed system to be located within a controlled area No Optional Yes Yes
Sample collection, addition of materials to a closed system and No Minimize release Prevent release Prevent release
transfer of viable micro-organisms to another closed system,
performed so as to:
Exhaust gases from the closed system treated so as to: No Minimize release Prevent release Prevent release
Bulk culture fluids not removed from the closed system unless the Optional Inactivated by Inactivated by validated Inactivated by validated
viable micro-organisms validated means chemical or physical chemical or physical
means means
1) When using this table, attention is drawn to existing national regulations concerning the requirements within a biotechnical area. See also annex A [1].
2) No : No special requirements for safety (but could be put in place for other reason for instance, quality).
3) Optional : Should be decided case by case basis subject to risk assessment, the extent to which these measures are to be applied.
4) Yes : Requirement.

EN 12075 : 1997
Page 7
Page 8
EN 12075 : 1997

Annex A (informative)
Bibliography

[1] Council Directive 90/219/EEC of 23 April 1990 on the contained use of genetically modified
micro-organisms. OJEC 08.05.1990, no L 117 p 1.
[2] Council Directive 90/679/EEC of 26 November 1990 on the protection of workers from risks related to
exposure to biological agents at work (seventh individual Directive within the meaning of
Article 16 paragraph 1 of Directive 89/391/EEC). OJEC 31.12.1990, no L 374 p 1.
[3] Hazard Analysis Critical Control Point : What the System is and What it is not. Frank L. Bryan, Ph. D.,
M.P.H. Journal of Environmental Health July/august 1988.
[4] Micro-organisms in Foods 4. Application of the Hazard Analysis Critical Control Point (HACCP).
System to ensure microbiological safety and quality. International Commission on Microbiological
Specifications for Food (ICMSF) 1988.
[5] Mayes T. & Klisby D. (1989). The use of HAZOP hazard analysis to identify critical control points for the
microbiological safety of food. Food Quality and Preference 1 : 53-57.
[6] Kletz T. (1992) HAZOP and HAZAN Identifying and Assessing Process Industry Hazards, 3rd edition,
London Institute of Chemical Engineers, ISBN 0 85295 285 6.
[7] IEC 812:1985 Analysis techniques for system reliability Ð Procedure for failure mode and effects
analysis (FMEA)
[8] EN 1050 Safety of machinery Ð Principles for risk assessment
[9] EN ISO 9000-1 Quality management and quality assurance standards Ð Part 1 : Guidelines for
selection and use (ISO 9000-1 : 1994)
EN ISO 9001 Quality systems Ð Model for quality assurance in design, development, production,
installation and servicing (ISO 9001 : 1994)
EN ISO 9002 Quality systems Ð Model for quality assurance in production, installation and
servicing (ISO 9002 : 1994)
EN ISO 9003 Quality systems Ð Model for quality assurance in final inspection and test
(ISO 9003 : 1994)
EN ISO 9004-1 Quality management and quality system elements Ð Part 1 : Guidelines
(ISO 9004-1 : 1994)
[10] prEN 12296 Biotechnology Ð Equipment Ð Guidance on testing procedures for cleanability
[11] prEN 12297 Biotechnology Ð Equipment Ð Guidance on testing procedures for sterilizability
[12] prEN 12298 Biotechnology Ð Equipment Ð Guidance on testing procedures for leaktightness
[13] EN 1826 Biotechnology Ð Large-scale process and production Ð Control procedures for raw
materials
[14] EN 1619 Biotechnology Ð Large-scale process and production Ð General requirements for
management and organization for strain conservation procedures
[15] EN 1620 Biotechnology Ð Large-scale process and production Ð Plant building according to the
degree of hazard

 BSI 1997
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