This document discusses updates being made to ISO 11737-1, the international standard for bioburden testing. It notes that microbiologists are less commonly employed directly by medical device companies now. This can lead to less expertise in critical areas like sterilization validation. The document advocates including knowledgeable microbiologists in product design and process validation decisions. It also provides guidance on challenges like low bioburden testing, sample pooling, atypical bioburden results, and updates being made to the standard to address these issues.

1. UPDATES TO THE ISO
BIOBURDEN STANDARD
P.S. WHAT HAPPENED TO THE MICROBIOLOGISTS?
Martell Winters
Senior Scientist
Nelson Laboratories
mwinters@nelsonlabs.com

2. ADDITIONS TO ISO 11737-1
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• ISO WG2 (Radiation) provided a list of
guidance to add to 11737-1
• Primarily information to help those involved in
sterilization
• Include main concepts here

3. P.S. WHAT HAPPENED TO THE
MICROBIOLOGISTS?
• Obvious trend to not have in-house
microbiologist/sterilization person
• Handed to QA, RA or manufacturing:
– “While you’re at it, do sterilization too…”
• Result: Relying on contract labs and
sterilization facilities for decisions
• NOT always the right decisions
• Often rationales are not documented properly
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4. • Have you ever heard this:
– “We can’t find the justification for…”
• How often do you ask RA to also be the
product engineers?
• …the finance person to also be the QA
manager?
• Is sterilization any less important than
engineering or QA?
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P.S. WHAT HAPPENED TO THE
MICROBIOLOGISTS?

5. • Having fewer manufacturing/sterilization
microbiologists in companies is reducing
knowledge base in the industry
• Standards bodies considering writing more
guidance
– Result is more information to be misunderstood
• Quandary…
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P.S. WHAT HAPPENED TO THE
MICROBIOLOGISTS?

6. WHAT AM I SAYING?
• Include knowledgeable people in your
company decisions
– Product design
– Manufacturing process design
– Sterilization process validation
– Routine monitoring of product and process
• If not hired people, contracted people
– As your people interact with true experts, they
learn and your company is better off
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7. • Attend seminars (plural) – they are a good
starting point
• Common discussion in standards meetings
– How much guidance can we give?
– Why do we have to provide this guidance? This
should be obvious!
• Don’t wait until you have a problem to involve
experts
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WHAT AM I SAYING?

8. LOW BIOBURDEN
• Seeing a lot of this with newer products
– Tissue/biologics
– Combination products with strict manufacturing
processes
– Automated processes
– Short cycles/low doses of sterilization are needed
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9. LOW BIOBURDEN
• Is this a problem?
– Often requires different bioburden test methods
• Pooling of products for testing
• Most probable number (MPN) testing
• Increased sensitivity in test (lower limit of detection)
– Often requires different approach to evaluation of
bioburden data
– Larger swings in bioburden counts can be seen
with low-bioburden products
• Larger implications for sterilized products
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10. • Sample pooling
– Facilitates low limit of detection
– Can usually resolve down to 0.1 CFU per sample
– Will mask individual product bioburden spikes
– Example:
• 5 samples pooled
• Result (5 pooled): 4 CFU
• Result (per sample): 4 / 5 = 0.8 CFU
• Not always a good option – be careful!
SAMPLE POOLING

11. POOLING IS A VIABLE OPTION
SAMPLE AEROBIC
BACTERIA
FUNGI TOTAL
BIOBURDEN
1 <4 <4 <8
2 4 <4 <8
3 <4 <4 <8
4 <4 4 <8
5 8 <4 <12
6 <4 <4 <8
7 4 <4 <8
8 <4 4 <8
9 <4 4 <8
10 <4 <4 <8
AVERAGE <4.4 <4.0 <8.4

12. CONSIDER TESTING SEPARATELY
SAMPLE AEROBIC
BACTERIA
FUNGI TOTAL
BIOBURDEN
1 <4 <4 <8
2 4 <4 <8
3 24 16 40
4 <4 4 <8
5 56 8 64
6 <4 <4 <8
7 4 <4 <8
8 <4 4 <8
9 120 32 152
10 <4 <4 <8
AVERAGE <22.8 <8.4 <31.2

13. INCREASED SENSITIVITY
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• Results in more accurate bioburden count
– Reduces the “less than” values (e.g. <4 CFU)
• Volume factor – change <4 to something less
• <1 – Filter all and re-incubate plates
• <2 – Filter ½ for bacteria and ½ for fungi
– If using an SIP, test larger portion of product
– Pool samples together
• Other options depending on product/situation

14. LOW BIOBURDEN
• How low is low?
– Varies per sterilization method
– Likely less of an issue with traditional overkill
methods (EO, steam, dry heat)
– Radiation (possibly in new bioburden standard)
• Low: Less than 10 CFU
• Ultra-low: Less than 1 CFU
– Repercussions for radiation
• Test more often - initially
• Test more samples – initially
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15. BIOBURDEN TEST METHODS
• Most common:
– Immersion in peptone tween solution
– Extraction using mechanical shaking
– Membrane filtration – 0.45µ cellulose filter
– TSA (SCDA) and SDA/PDA
– 30-35°C and 20-25°C
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16. BIOBURDEN TEST METHODS
• Is this always the right decision?
– No!
– How often is bioburden testing repeated due to
issues the first time around?
– Are all your microorganisms accounted for?
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17. BIOBURDEN TEST METHODS
• Keep in mind:
– Potential microorganisms on materials/components
• Understand their manufacturing process and origin
• Where are your microorganisms?
– Potential microorganisms in assembly
• Personnel/process involved
• Water?
• Material/component storage?
• Cleaning processes
– Which microorganisms are of greatest concern for
sterilization process?
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18. EVALUATING BIOBURDEN DATA
• Guidance will be provided on:
– Dealing with TNTC results
– Bioburden spikes
• Options for counting very high CFUs
• What can cause them
• Definition?
– Spreading microorganisms
– Zero CFU results on plates (less-than numbers)
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19. BIOBURDEN SPIKE
SAMPLE
AEROBIC
BACTERIA
FUNGI TOTAL SAMPLE
AEROBIC
BACTERIA
FUNGI TOTAL
1 21 3 24 1 68 3 71
2 33 3 36 2 95 6 101
3 39 3 42 3 50 3 53
4 694 3 697 4 56 3 59
5 15 6 21 5 55 3 58
6 82 3 85 6 17 3 20
7 33 3 36 7 27 3 30
8 27 3 30 8 26 3 29
9 12 3 15 9 95 3 98
10 36 3 39 10 151 3 154
Mean 99.2 3.3 102.5 Mean 64 3.3 67.3
Bioburden estimate 174.6 Bioburden estimate 114.7
Standard deviation 209.8 Standard deviation 40.9
Recovery efficiency 0.587 Recovery efficiency 0.587
What do you do with a spike?
Throw it out (outlier)?

20. BIOBURDEN SPIKE
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• Definition?
– Single value more than twice the average
• Similar option used in radiation validations
– Single value which is over 2 SD above the mean
• Mean + 2 SD….any value above that could be a spike
– Based on internal alert and action levels
• Perhaps best option – individualized for your product

21. BIOBURDEN SPREADER
Throw it out?
Assign a value?
Additional testing?
Identify?
Investigate?

22. TOO NUMEROUS TO COUNT
TNTC
Throw it out?
Assign a value?
Additional testing?
Identify?
Investigate?

23. THANK YOU
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