How water systems can go wrong and

what to do about it
Wednesday 24th May 2023
 Introductions

Annette Russell
Sterile & Non-Sterile Annette.russell@rssl.com
RSSL Microbiology Lead
+44(0)118 918 4075
www.rssl.com

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 RSSL – Who are we?

Reading based
40 mins from London with
excellent airport and rail links

Best CRO 2022 & MHRA and FDA Over 30 years of Committed CRO,
Employer of the approved experience and Flexible, Agile,
Year 2022 ISO 17025 expertise, from a Quality across the
accredited team of 250 whole
scientists in 12 organisation.
specialisms

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 Annex 1 regulations support
Contamination Control Quality Risk Management New Technologies

Environmental Extractables and Cleaning and

monitoring and leachables of SUP and disinfectant
consultancy packaging validation

Raw material testing

Microbial
including bioburden
identification
and endotoxins

Vial and stopper

Particulate control Sterility assurance testing, container
integrity

Training and consultancy

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Today’s speaker – Dr Tim Sandle

• 25 years experience in the field of pharmaceutical microbiology

• Member of several editorial boards
• Extensive publication record
• Works in the pharmaceutical industry
• Tutor at University of Manchester and UCL

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 Introduction

● Microbial contamination in water – risks and objectives

● Design and qualification
● Validation
● Effective user controls and practices
● Biofilm problems and remediation
● Trending

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 Contaminated water systems

• All water systems contain levels of microorganisms.

‒ What is of concern are the numbers of
microorganisms and, for non-sterile processing, the
presence of ‘objectionables’ like Burkholderia
cepacia and Pseudomonas aeruginosa.
• Contaminated water presents concern:
‒ Water is a growth source for certain
microorganisms, allowing them to survive and to
multiply.
‒ In process areas: water is an efficient vector for
spreading contamination (aerosols).

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 Microorganisms in water
• Dual problem:
‒ Microorganisms grow in water;
‒ Water spreads microorganisms easily.
• Most microorganisms have two ‘lifestyles’:
‒ Planktonic: freely floating in water
‒ Sessile: attached to a surface
‒ Main type: Gram-negative bacteria
• Microorganisms attach to surfaces by secreting a sticky polymer.
‒ This can lead to biofilms forming.

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 Objectives

Microbiome of water systems mainly Gram negative pseudomonads

Concern increases as biofilms

Water system biofilms release bacteria that contain endotoxin

Water distribution systems must prevent biofilm

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 Burkholderia cepacia complex

• Burkholderia cepacia poses a risk to water-based drugs.

• FDA Warnings.
‒ The agency has warned drug manufacturers about
Burkholderia cepacia complex after product recalls
due to contamination.
‒ BCC is a water-borne pathogen that can be found in
pharmaceutical water systems.
‒ Manufacturers need to establish procedures (e.g.,
sanitary design, equipment cleaning, environmental
monitoring) to prevent contamination of non-sterile
drug products.

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 Design and qualification

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 Basic design

A break tank

An activated carbon bed

A water softening column

De-ionisation columns or
devices

A reverse osmosis system

Distillation equipment

A distribution system and

storage system

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 Designing and maintaining good water systems #1

• Smooth internal surfaces in tanks and in pipe-

work.
‒ This is necessary because microorganisms
adhere less well to smooth surfaces than to
rough surfaces.
• Continuous movement of the water in tanks
and rapid flow in pipework is ideal.
‒ Shear forces.
• Avoidance of areas where water can remain
stagnant.
‒ Pockets of stagnant water can pose a
biofilm risk.
‒ These areas include so-called “dead legs”.

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Designing and maintaining good water systems #2

• Water storage tanks are normally

constructed from stainless steel.
‒ Regular water turnover helps prevent
contamination.
‒ Slow turnover presents a greater
potential contamination risk.
‒ Storage tanks should be vented to
manage water level fluctuations, with
vents fitted with a hydrophobic air
filter.

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Designing and maintaining good water systems #3

• Ring mains should be sloped (have “drop”)

from point of origin to the point of return
to ensure that systems are completely
drainable.
• Avoidance of leakage.
‒ Water leaks can cause bridging of water
to the external environment through
which bacteria may enter the system.

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 Designing and maintaining good water systems #4
• The requirements for well-designed water distribution pipes are:
‒ Smooth internal surfaces in tanks and in pipe-work.
• Microorganisms adhere less well.
‒ Continuous movement of the water.
• Shear forces mean microorganisms adhere poorly to surfaces.
‒ Avoidance of areas where water can remain stagnant.
• These include “dead legs” – water may stagnate in branch pipes branch.
• Water can also remain stagnant in valves, particularly at user points and even
more particularly at user points which are not in frequent and regular use.
‒ Avoidance of leakage.
‒ High temperature storage and distribution (for WFI).

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 Designing and maintaining good water systems #5

• Water points in production areas involve the transfer of water from the circulating water
loop to the point of use via transfer tubing.
‒ This should be made of a suitable nontoxic material, such as polyvinyl chloride (PVC),
chlorinated PVC, polypropylene, or PVDF.
‒ The transfer piping should be drained after use and changed regularly (such as every 24
hours).
‒ Care must be taken to avoid splash-back from sinks or recontamination from aerosols.
‒ New tubing should be sanitized before fitting.
‒ The tubing and outlet to be flushed prior to use (for a defined time or given volume of
water).

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 Poor design

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 Poor design and assessment

• Examples from FDA warning letters:

‒ Poorly designed cold system, not continuously circulating
‒ Inadequate monitoring
‒ Poor manufacturing controls
‒ Poor sampling plan
‒ Inadequate test methods
‒ Test methods not validated
‒ Limits not appropriate
‒ No defined list of objectionable organisms / screening methods insufficient

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 Design summary

• The design of a system is influential on

the size of the microbial populations
and the ability of the user to remove
them.
• Dead-legs, long pipework runs to taps,
un-drainable pipes and U-bends all
create microbiological problems once
installed.

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To address this, a rationale is required

Contamination control
• What are the risks?
• What is the sanitisation procedure and frequency?
Microbiological testing
• When?
• Sample size?
• Test method
Identification strategy

Defining and screening for objectionable organisms

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Validation of water systems

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Validation exercise

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 Phase 1 Testing

Sample after each step in treatment process

Sample at each point of use

Sample incoming feed water

Daily sampling

Chemistry testing specific to unit process

Microbiological testing for each unit process

At completion SOPs for operation, maintenance and trouble shooting finalised

Phase 1 may have to be repeated.

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 Phase 2

Typical duration 2-4 weeks

Demonstrate consistent operation within established ranges

Demonstrate consistent production and delivery of water of the

required quality when system operated to SOPs

Sampling scheme typically the same as Phase 1

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 Phase 3

Typical - 12 months/ 1 year

Captures the seasonality (especially of potable water supply), ensures that

the treatment systems can effectively treat the water to the required quality

Demonstrate extended performance

Confirms sample locations, frequencies and tests

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 Post Qualification

• Any changes to be managed per the Change

Control system
• Trend the laboratory data
• Investigate any change in trend data
• The water system is a process not a batch
and is not revalidated if there are no
significant changes

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 Sampling Frequency

Purified Water system outlets

• Typical outlet sampling frequencies vary from daily to
monthly.
• Vary the day sampled.
WFI:
• Daily beginning and end of the Loop
• All outlets sampled weekly

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Effective user controls and
practices

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 What not to do

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 Conduct audits

Use drawings
• Look for inconsistencies with drawings and what you see

Look at point of use tubing and sampling valves

Assess ergonomic issues – accessibility

Consider flush water limitations – nearby drain, bucket size

Assess environment

Hose management

Heat exchangers

Flow controllers/volume totalizers

Sampling procedure simulating use?

Additional devices used for sampling?

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 Operational issues

Softeners (prior filter, Carbon Beds (backwashing

Potentially problematic
backwashing/regeneration frequency, sanitization
purification units and
frequency, bed replacement frequency/how, bed
features
frequency?) replacement frequency?)

RO (cleaning and DI beds

Bisulphate Injection (fixed
sanitization frequency, last (regeneration/replacement
or variable feed)
membrane replacement) frequency)

EDI/CEDI (sanitization In-Line cartridge filters Vent filters (orientation,

frequency) (sanitization/service period) hydrophobic, heat traced/)

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 Sanitisation

• Distribution system sanitization approach

• Frequency/trigger
• Chemical or heat used and conditions
(concentration, temperature, time)
• Unsanitized areas bypassed?
• Efficacy assessment

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 Biofilms

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 Biofilms

• A biofilm is defined as bacterial populations adherent

to each other and/or to surfaces or interfaces -
opposed to planktonic cells, which are free motile
cells.
• Biofilm bacterial cell colonies secretes a slimy tacky
polysaccharide coating.
• The Polysaccharide coating:
‒ encourages the attachment of other organisms
‒ Is a trapping device for nutrients
‒ protects the biofilm
• Biofilm bacteria are not just simply planktonic cells
attached to a surface.

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 Biofilm Formation

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 Why is biofilm so attractive to Microbes?

• A survival strategy in low nutrient

environments.
• Nutrients tend to accumulate on
surfaces
• Protection: against drying, against
extremes of temperature, against
biocides, against UV, osmotic shock
etc.
• Cells change – size, population rate,
metabolic rate, adherence factors

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 Biofilm Defense Strategies
Extracellular Polymeric substances
(EPS)
Enzymatic protection
Constructed by the biofilm bacteria, provides
protection from desiccation, antimicrobials and
toxins
Catalase, beta lactamase can neutralise biocides

Altered microenvironments By-products produce slow growth and diversify the

ecology of the biofilm
Plastic phenotype Up to 50% of membrane proteins are different
from their planktonic counterparts

Heterogeneity When combined with slower growth heterogeneity

results in variation in the predominant species

Quorum sensing Bacterial pheromones

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 Biofilm Remediation

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 How do you control a biofilm?

• A well designed water system

‒ Correct materials of construction, flow rates, temperature, chemical dosing,
sampling,
‒ A good system on its own will not prevent biofilm formation, but it will help
control it
‒ The microbes that are found in a water system can potentially form a biofilm
(or already have)

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 System design pitfalls

• Typical requirements are:

‒ Hygienic design
‒ Sanitary instrumentation
‒ Minimise dead-legs
‒ Draining and pipe falls
‒ Sample location(s)
‒ Drain control
‒ Supply water quality and testing

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 Prevention

Biofilm will always form unless the environment is lethal to bacteria

Two options:
• Maintain a temperature continuously above approx. 65 °C
• Maintain an ozone environment continuously above 10 – 20 ppb

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 Control

Periodic sanitisation
System design
cycles:
• Raising the temperature • Water velocity
above 65 °C • Hygienic construction
• Circulating ozonated • Surface finish
water above 10 – 20 ppb
• Circulating other
sanitising chemicals

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 What happens when biofilm takes over

Temperature treatment

• Heat is the most effective

Chemical treatment

• The usual, plus ‘pickle and passivate’

Physical removal

• What about welds?

• Will this release high levels of planktonic bacteria?

The safest method? Throw away and start again.

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 Assessment

Biofilm can be
persistent and Bacteria
hard-to-kill definitively killed
Heat is very and cannot grow at
effective, chemicals high temps
are not (crevices!)

Hot WFI system

Heat, stills, and
problems can
heated systems are
emerge in cool sub-
very expensive
loops

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 Trend chart

• Sandle, T. An Approach for the Reporting of Microbiological Results from Water Systems, PDA
Journal of Pharmaceutical Science and Technology, Vol. 58, No.4, July-August 2004, pp231 – 237

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 Causes of contamination
Special causes are local,
sporadic problems such as the • Localised
poor management of a • Exceptions to the system
particular water outlet in a • Considered abnormalities
process area.:

• A certain process
Specific to a: • A certain outlet
• A certain method of sanitisation, etc.

Common causes are problems • The nature of the system

inherent in the system because • The way the system is managed
of: • The way the process is organised and operated

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 Summary

• Microbial contamination in water – risks

and objectives
• Design and qualification
• Validation
• Effective user controls and practices
• Biofilm problems and remediation
• Trending

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 Q&A session

Dr Tim Sandle

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How we can support you with key areas of Annex 1
Webinars - last in the Annex 1 series is ‘Design and qualification of isolators’ (21st June) Register now via our
website

Analytical services to support the manufacture of your sterile medicinal product

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Environmental monitoring Bacterial Endotoxins Microbial identification (MALDI-ToF)
Cleaning validation Raw materials testing Water testing (inc WFI)

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27 July Cleaning validation
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now via our website
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 Thank you for your time

Annette Russell
Sterile & Non-Sterile Annette.russell@rssl.com
RSSL Microbiology Lead
+44(0)118 918 4075
www.rssl.com

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