1. Pharmaceutical
Microbiology: Current and
Future Challenges
Dr. Tim Sandle
Pharmaceutical Microbiologist
http://www.pharmamicroresources.com/
Delivered to PDA Microbiology Europe, 15th
October 2018

2. Introduction
• The changing environment for pharmaceutical microbiology
• Limitations of methods
• Need for new (rapid) methods
• Separating people form processes
• Single-use technologies
• Environmental monitoring programme
• Best practices
• Rapid methods
• Contamination control strategy
• Objectionable organisms
• Burkholderia cepacia complex

3. The changing environment
for pharmaceutical
microbiology

4. Origins of pharmaceutical microbiology
• 1960s and 1970s – Pharmacy
schools, focus on sterilisation and
medicinal formulation.
• 1990 – The Pharmaceutical
Microbiology Forum in the U.S.
• 1991 - Pharmaceutical
Microbiology Interest Group
(Pharmig).
• 2000s - Pharmaceutical
microbiology courses
• 2006 – First Pharmaceutical
microbiology Master’s degree

5. Changing environment
• Range of activities associated with
pharmaceutical microbiology has
extended from the laboratory and
into the production environment.
• Also:
• Microbiological audits
• Rapid microbiological methods
• Conducting risk assessments
• Both proactive in terms of minimizing
contamination and reactive, in terms
of addressing microbial data
deviations;
• Ensuring that processes meet ‘quality
by design’ principles.

6. New understanding
• Knowledge
• Today’s pharmaceutical
microbiologist needs to have an
understanding of engineering,
regulation, the R&D process, and
production workflows.
• The microbiologist is expected to
understand industrial processes,
cleanrooms, and how to
effectively evaluate microbial risks
to products from people and
processes.

7. Limitations of monitoring methods
• Recognising the uncertainty with
environmental monitoring
methods
• Sample size
• Monitoring times
• Accuracy of methods
• ABNC phenomenon
• Some of these issues are greater
in ‘cleaner’ environments e.g.
Grade A / ISO class 5

8. Method limitations #1
• The classic techniques:
• Active air-sampling: volumetric
air-sampler
• Passive air-sampling: settle plates
• Surface samples: contact (RODAC)
plates and swabs
• Personnel samples: Finger plates
and gown plates
• New generation of ‘real time’
viable air samplers.

9. Method limitations #2
• Settle plates
• Position in relation to air currents
• Desiccation
• Meaningfulness of one CFU
• Attempt to quantify by Whyte’s
deposition rate
• Active air samplers
• Particle size cut-off (D50 value)
• Recovery ~50%
• Different instruments

10. Method limitations #3
• Contact plates
• Tidswell’s work on organism
recovery (~50%)
• Disinfectant residues on surfaces
and neutraliser selection
• Disinfectant rotation and
neutralisers
• Swabs
• Plain swabs ~10-20% recovery
• Flocked swabs 40-60% recovery

11. Ways to separate people from product
• Quality by design
• Control sterilisation
• Removing people from Grade A
environments
• Strengthening barriers between
people and product
• Such as: RABS, isolators or closed
systems for aseptic processing.

12. Single-use technologies #1
• Traditional technologies:
• Time-consuming,
• Expensive,
• Energy hungry,
• Require a large footprint,
• Prone to occasional control
breakdowns,
• Sterility assurance concerns.
• Single-use technologies:
• Reduce concerns around time,
• Can reduce costs,
• More security in avoiding non-
sterility

13. Single-use technologies #2
• Examples: tubing, capsule filters, ion exchange membrane
chromatography devices, mixers, bioreactors, product holding sterile
bags in place of stainless steel vessels (sterile fluid containment bags),
connection devices and sampling receptacles
• Need to assess:
• Leachables
• Extractables
• Method of sterilisation
• Robustness in relation to contamination e.g. aseptic connector
• Inhibition of microbial growth e.g. biocontainer bag

14. Environmental
monitoring programme

15. Environmental monitoring programme
• Forms part of the overall contamination control strategy.
• Purpose:
• To assess cleanrooms and controlled environments using viable and
particulate counting methods.
• To verify environmental control (how well is the cleanroom working?)
• To assess impact of staff behaviours
• To help evaluate risk in relation to excursions (location and event dependent)
• To assess event specific incidents e.g. HVAC losing power or maintenance
works
• Needs to be a written programme, with justifications.

16. Environmental monitoring programme
• Where monitoring takes place?
• Types of rooms and sample locations
• How often monitoring is performed?
• Pre-set sampling frequencies
• Types of samples required
• Methods describing how samples are taken and
how they are handled
• Who takes the samples?
• Data analysis and trending
• Level of microbial identification
• Investigating out of limits events
• CAPA for problems e.g. Cleaning and disinfection
• Types of culture media and incubation strategies

17. Importance of trending
• A good environmental monitoring programme will seek to show
consistent, high quality environmental conditions at any given time.
• The programme will seek to detect changes in the contamination
recovery rate that may be indicative of changes in the state-of-
control within the environment.
• This is achieved though a well-designed programme and an emphasis
upon trending.
• Trends are important:
• As counts
• As frequency of incidents
• As microflora

18. Rapid microbiological methods
• Rapid microbiological method technologies aim to provide more
sensitive, accurate, precise, and reproducible test results when
compared with conventional, growth-based methods.
• They normally involve some form of automation.
• They normal capture data electronically.
• Can support the contamination control strategy

19. Rapid microbiological methods
• Time
• To prepare the test
• Conduct the test
• Sample throughout
• Time to result
• Reduction in the time taken to conduct complimentary tests
• Less time for data analysis
• Simpler results reporting

20. Rapid microbiological methods
• Accuracy
• Reduction in human error
• Reduction in subjectivity
• To detect more accurately in comparison to a conventional method e.g. a
cultural method?
• To detect what a cultural method cannot?

21. Rapid microbiological methods
• Other areas:
• Real time data capture
• Electronic capture of data
• Automation
• Connecting apparatus
• Example - Real-time continuous
monitoring systems, based on
optical spectroscopy.

22. Contamination control
strategy

23. Holistic contamination control strategy
• Each facility - steriles and non-
steriles - should have a detailed,
facility-specific contamination
control strategy.
• To be effective, this needs to be an
approach that can assess
seemingly isolated contamination
events holistically.
• The process must be capable of
putting appropriate corrective and
preventive actions.
• The proposed revision to EU GMP
Annex 1 provides a working model.

24. Key points for a contamination control
strategy
• Understanding the design of both the plant and process.
• The quality of the design is important for controlling contamination.
• Detail of equipment and facilities,
• What is the equipment used?
• How does it work?
• How is it repaired?
• When is it calibrated?
• Are the best available technologies used?

25. Key points for a contamination control
strategy
• Training and control of personnel
• How well do personnel behave, especially in cleanrooms?
• Are staff trained in hygiene, cleanroom practices, contamination control,
aseptic techniques, loss of product sterility and in the basic elements of
microbiology?
• Control of utilities
• Utilities need to be controlled and operated within their design parameters.
• Compressed gasses, HVAC and water.
• Raw Materials Control
• Control of raw materials is not only a factor of testing and with controls in
place when containers are opened e.g. what safeguards are in place to
ensure containers are stored in dry environments?

26. Key points for a contamination control
strategy
• In-process controls
• Appropriate points in the production process need to be selected for
monitoring and appropriate limits set.
• Product containers and closures
• Aseptic processing requires a review of Grade A containment of vials; setting
maximum exposure time of sterilised containers and closures prior to closure
and ensuring crimping is conducted expediently; and establishing a robust
container closure integrity qualification.
• Vendor approval
• Understanding where materials come from, whether they have been
prepared and processed properly, and whether they remain the same as
those components used in previous validation.

27. Key points for a contamination control
strategy
• Outsourced services
• Cannot simply outsource services and accept certification when items are
delivered, especially with items that have undergone a sterilisation step.
Requires audit and expert review of the sterilisation process.
• Process risk assessment
• A formal risk assessment should be in place for each process step, beginning
with the start of the manufacturing process, capturing all processing steps
through to final packaging.
• Extend to good Distribution Practice.

28. Key points for a contamination control
strategy
• Process validation
• Process validation is an ongoing process and it should be frequently reviewed
and adapted as manufacturing feedback is gathered.
• An essential part of this is knowledge of microbial contamination rates,
particularly in relation to in-process bioburden and to the supporting
manufacturing environment.
• Preventative maintenance
• Control of the regular maintenance of equipment and premises, as either
planned and unplanned maintenance, is of importance to ensure product
quality and for a consistent process.

29. Key points for a contamination control
strategy
• Cleaning and disinfection
• Cleaning and disinfection are important steps for maintaining control. There
should be a rationale in place justifying the use of each disinfectant agent,
together with supporting data to show efficacy in terms of microcidal kill on
surfaces of a similar type used within the manufacturing area.
• Monitoring systems
• To assess whether cleanrooms and utilities are functioning as designed,
regular checks are required, such as checks of airflows or pressure
differentials. To be effective, the monitoring methods need to be continuous
so that data can be trended, and equipped with audible alarms.

30. Key points for a contamination control
strategy
• Prevention
• A contamination control strategy should have in place a good system for
addressing CAPAs. To arrive at an effective CAPA, there needs to be a system
in place for trending, conducting investigations, arriving at root causes, and
then for suggesting and implementing appropriate CAPAs.
• Continuous improvement
• No contamination control strategy should stand still and it should be regularly
reviewed and updated.

31. Objectionable
microorganisms and
BCC

32. Thinking about objectionable
microorganisms
• Need to move thinking beyond the organisms listed in USP and Ph.
Eur. Under ‘Microbial Limits Test’
• Undertake risk assessments to assess whether an organism is
objectionable
• Need to assess materials for:
• Need for testing
• Frequency of testing
• Appropriate limit
• Design test method

33. How to assess if an organism is
objectionable?
• The foremost factor is whether the organism is a pathogen for if the organism is known
to be a pathogen, and the route of infection is the same as the route of administration
for the product, the organism is most likely objectionable. This approach:
• Ranks administration routes in order of microbiological risk to patient,
• Discusses water activity & self-preservation/micro growth,
• Discusses preservative system for multi-use products.
• The ways by which objectionable microorganisms trigger a risk to the product or have
potential to cause patient harm include:
• Affecting product stability.
• Affecting the security of the container/ closure system
• Affecting the active ingredient.
• Producing off odors, flavors or undesirable metabolites.
• Having the potential to grow and exceed the total aerobic count specification.
• Possessing high virulence and a low infective dose.
• Resistance to antimicrobial therapy.

34. Burkholderia cepacia complex
• Burkholderia cepacia complex refers
to a group of Gram-negative, non-
spore forming rod-shaped.
• B. cepacia emerged as a human
respiratory opportunistic pathogen in
individuals with weakened immune
systems or chronic lung disease,
especially cystic fibrosis patients.
• BCC bacteria exist throughout the
environment, especially in soil and
water environments.
• In 2017, FDA sent out an alert
notification.

35. Burkholderia cepacia complex
• Burkholderia cepacia and related species
are human opportunistic pathogens
• They can cause pneumonia in
immunocompromised individuals.
• Other risks to susceptible patients
include:
• Endocarditis,
• Wound infections,
• Intravenous bacteremia,
• Foot infection,
• Respiratory infections.
• Some patient groups are at a greater risk
than others: elderly people, young
children, cancer patients, pregnant
women, people with chronic illnesses

36. Burkholderia cepacia complex
• As it is challenging to detect BCC, the FDA called for manufacturers of
non-sterile, water-based drug products to put in place risk
assessments and special tests for this group of organisms.
• 2019, there will be a new USP chapter.
• Standard bioburden tests are unlikely to be sufficient.
• The test for bile-tolerant Gram-negative bacteria described in USP<62> may
detect some types of BCC. But there are weaknesses.

37. Burkholderia cepacia complex
• 〈60 Microbiological Examination〉
of Nonsterile Products—Tests
for Burkholderia cepacia Complex
• BCSA contains peptones and sugars
that supply nutrients for the growth
of Burkholderia cepacia, and other
microorganisms.
• Crystal violet is added to inhibit
growth of other organisms.
• Anti-microbials are incorporated to
inhibit other organisms.
• Burkholderia cepacia colonies are
typically translucent and rough.
• On BCSA, the growth of B. cepacia
will cause a color change in the media
from red-orange to yellow.

38. Burkholderia cepacia complex
• Culture-independent rapid
microbiological methods which are
suitable, such as epiflourescence tests.
• Another possibility for testing is with the
use of polymerase chain reaction (PCR),
which amplifies specific segments of DNA.
• Researchers have developed a Ready-To-Go
PCR beads and specific DNA primers for B.
cepacia, which demonstrated a 100%
correlation between standard methods and
the PCR assay, with results obtained within
27 hours.
• A variant PCR method is with amplification
of the 16S rRNA gene, followed by
restriction enzyme-mediated fragmentation
of the amplicon.

39. Summary
• The changing environment for pharmaceutical microbiology
• Limitations of methods
• Need for new (rapid) methods
• Separating people form processes
• Single-use technologies
• Environmental monitoring programme
• Best practices
• Rapid methods
• Contamination control strategy
• Objectionable organisms
• Burkholderia cepacia complex

40. Thank you
Dr. Tim Sandle
Pharmaceutical Microbiologist
http://www.pharmamicroresources.com/