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May 2006

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From:
Daniel Prince <[log in to unmask]>
Reply To:
The Pharmaceutical Microbiology Forum Email List <[log in to unmask]>
Date:
Tue, 30 May 2006 17:57:48 -0400
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Scott and others,
I slightly disagree if the meaning here is to report all counts as less than
an accepted minimum countable range [e.g., 25] times 1 times the dilution
factor even when counts are detected.

This is because there is a dramatic difference in accurate disclosure if
detectable counts are ignored and the result is reported simply as < some
number, N [in this thread <250.]

<250 can mean all counts from 0-249cfus with any one of the possible 250
count outcomes as probable as another. Clearly, if you have two plates each
with 23 or 24 organisms you get a much different feel about the data if you
report about 230 cfu/gram versus <250 with no mention of what you so growing
on the plates.

How would you report the following hypothetical result? Assume there are no
lab errors or inhibitory issues at play. Would you conclude <250 or <2,500
cfu or average all of the detectable counts to obtain an estimate of 147
cfu/mL?
Dilution #cfu, duplicate counts
1:10 24, 10
1:100 1, 0

Furthermore, when all plates from a dilution range are 0 the proper
convention is to report the count as <10 not <250. This is as per FDA BAM.

In closing, the most valuable and powerful attribute that the classical
methods have is that they speak to real, living, viable, replicating
organisms. This must not be diminished or marginalized. Conversely, rapid
methods with greater "accuracy" do not measure living, viable, replicating
organisms. Is it possible that what they do measure is in fact not readily
interpretable in connection to our classical viability techniques? If so,
there will be some who accept this fact and consider the results obtained by
the new technique as valid while others will not, citing instead a
preference for a culture technique.


Sincerely,
Daniel L. Prince, Ph.D.
President
Gibraltar Laboratories, Inc.
122 Fairfield Road
Fairfield, NJ 07004
973-227-6882 ext. 519
973-227 0812 FAX
www.gibraltarlabsinc.com



 

-----Original Message-----
From: The Pharmaceutical Microbiology Forum Email List
[mailto:[log in to unmask]] On Behalf Of Scott Sutton
Sent: Tuesday, May 30, 2006 11:28 AM
To: [log in to unmask]
Subject: Re: [PMFLIST] Bioburden

Holly,

I agree that many of the specifications are not achievable by the plate
count method and that the USP/EP have contributed to the problem. However,
the plate count method is not the only one available. Matilde Soubes in an
earlier Email suggested the MPN method - that is a good alternative.

I am under no illusions about getting a more scientifically based regulatory
environment in the near future. However, after several validation protocols
for new (rapid) methods, reviewing the unworkable EP Chapter 5.1.6 on
validation of methods (did anyone on that committee ever try to get an RSD
of 10-15% for plate counts?), and several discussions on environmental
monitoring alert and action levels with clients and regulators I have to
admit to becoming increasingly agitated about the need for us to admit and
adhere to the limitations of the methods we employ.

Microbiologists have got to get to a point where we can measure the
uncertainty in our methods.

Scott

=========
Scott Sutton, Ph.D.
Pharma Consultant, Microbiology
Vectech Pharmaceutical Consultants
585-594-8273
[log in to unmask]
This Message is privileged, confidential and protected by law from
disclosure. If you received this message in error, then forward it to
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-----Original Message-----
From: The Pharmaceutical Microbiology Forum Email List
[mailto:[log in to unmask]] On Behalf Of Holly A Settles
Sent: Tuesday, May 30, 2006 9:20 AM
To: [log in to unmask]
Subject: Re: [PMFLIST] Bioburden

Scott,

While I may agree with your argument for reporting the LOQ as
scientifically supportable, I have to ask how we are to stop perpetuating
the accuracy charade.

The USP 2nd Supplement with the revised chapters for <61> Microbiological
Examination of Nonsterile Products: Microbial Enumeration Tests and <1111>
Microbiological Examination of Nonsterile Products: Acceptance Criteria
for Pharmaceutical Preparations and Substances for Pharmaceutical Use has
recently arrived on my desk.

The recommended acceptance criteria in <1111> for total aerobic microbial
count and total combined yeast/mold count for many of the nonsterile
dosage forms and substances for pharmaceutical use are less than 20 or 200
cfu/g or ml.

Many of these materials are not suitable for membrane filtration, and must
be diluted 1/10 to be able to be plated. Consequently, by using the
compendial method, and the scientifically justifiable LOQ of <250, it does
not seem possible to me to comply with the current guidance.

While new technologies and alternative methods may be the answer to
increasing the accuracy and sensitivity of the total count tests, it
should be expected that the compendial tests will continued to be used in
the industry. I would also suspect that the reporting counts of <10 cfu/g
or ml will continue to be convention.

Holly Settles
Microbiologist
Eli Lilly & Co.




Scott Sutton <[log in to unmask]>
Sent by: The Pharmaceutical Microbiology Forum Email List
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05/30/2006 07:42 AM
Please respond to
The Pharmaceutical Microbiology Forum Email List
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Subject
Re: [PMFLIST] Bioburden






Linda,

The question, as I see it, isn't so much whether or not it sounds
reasonable. "Reasonableness" is frequently based on our desires and
preconceptions. The question is rather is it scientifically supportable.

Let's look at the question fresh - we can agree that in normal situations
the valid number of bacterial colonies to count on a plate is 25-250 (my
apologies, I was a little sloppy with this range on my previous Email). We
can agree that if there are more than 250 colonies on a plate, we report
the
data out as TNTC (Too Numerous To Count). So we agree on a countable
range
and we agree on how to report excess at the high end.

How do we report excess at the low end of the countable range? Do we
ignore
it? I suggest we change "accepted practice" and opt for good science in
this regard. As we introduce more and more alternate, rapid
microbiological
methods, we are going to be more often faced with the questions of
accuracy
and precision in our test results.

ISO 17025 "General Requirements for the Competence of Testing and
Calibration Laboratories" is an interesting read in this regard. In
section
5.4.5.2 of "Validation of Methods" you can find the statement "The
techniques used for the determination of the performance of a method
should
be one of, or a combination of, the following: . . . assessment of the
uncertainty of the results based on scientific understanding of the
theoretical principles of the method and practical experience." This
statement is only one of several dealing with validation (accuracy,
precision, range, etc - see USP <1223> or Pharm Eur 5.1.6) and uncertainty
of measurements to be found in that ISO standard.

A question for testing labs certified under ISO 17025 - How do you meet
the
requirements for understanding and reporting uncertainty in the data?

Back to the question at hand. How do we report out data from a plate
that
has too few colonies to be in the countable range? Common practice is to
ignore the situation and report it out as if it were an accurate count. I
have even seen situations were a lab will take duplicate plates with
counts
of 0 and 1 and report out 0.5 CFU at a 1/10 dilution = 5 CFU/mL. This
practice is not scientifically supportable.

Merely because it would be convenient to report out CFU down to 1
CFU/plate
is not a justification for doing so. These numbers are not even remotely
accurate and we are only digging deeper holes for ourselves by
participating
in the charade - holes that will come to present major obstacles when we
attempt to validate an alternate method and are FORCED to apply rigorous
measures under USP or Pharm Eur guidance.

Yes, in answer to your question, the only defensible act in this situation
(in my opinion) is to acknowledge that the plate count method has a Limit
of
Quantification, and the LOQ is 25 CFU/plate. Pursuing this argument, if
you
have counts that fall below the countable range of 25-250 CFU/plate on the
1/10 dilution you would report the data out as <250 CFU/mL. Even if the
duplicate plate counts are 24 and 23 - they fall below the LOQ of the
plate
count method.

Scott

=========
Scott Sutton, Ph.D.
Pharma Consultant, Microbiology
Vectech Pharmaceutical Consultants
585-594-8273
[log in to unmask]
This Message is privileged, confidential and protected by law from
disclosure. If you received this message in error, then forward it to
[log in to unmask] and delete it from your system.

-----Original Message-----
From: The Pharmaceutical Microbiology Forum Email List
[mailto:[log in to unmask]] On Behalf Of Linda K. Skowronsky
Sent: Friday, May 26, 2006 5:57 PM
To: [log in to unmask]
Subject: Re: [PMFLIST] Bioburden

In reply to Scott's response....I'm confused. Based on what you said, then

if we have no growth on 2 plates from a 1:10 dilution and we can only
report cfu in the countable range of 20-200, does this mean the minimum
detection limit would be <200? Does this sound reasonable??? Or perhaps I
misunderstood...

Best Regards,
Linda

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the scientific community.

Please take a second to visit our sponsors' web sites and say thank you for
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ATS Laboratories - http://www.ats-labs.com

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Biomerieux - http://industry.biomerieux-usa.com

Dupont Qualicon - http://www.qualicon.com

EMD Chemicals - http://www.emdchemicals.com

EMSL Analytical, Inc. - http://www.emsl.com

Genomic Profiling Systems, Inc. - http://www.genprosys.com

MIDI, Inc. - http://www.midi-inc.com

Millipore - http://www.millipore.com

Pall - http://www.pall.com

NovaTek International - http://www.ntint.com

Raven Biological Labs - http://www.ravenlabs.com

Veltek Associates, Inc - http://www.sterile.com

=================================
The nature of this service is to provide a medium for communication. The
specific statements and endorsements of individuals participating in the
discussions are not necessarily those of The Microbiology Network, Inc., the
PMF, or the sponsors of the list.


------------------
The PMFList (http://microbiol.org/PMFList_info.htm) is operated from
The Microbiology Network (http://microbiol.org) and supported by
our sponsors (http://microbiol.org/sponsor.htm) as a service to
the scientific community.

Please take a second to visit our sponsors' web sites and say thank you for
their support of this service.

Accugenix - http://www.accugenix.com

American Type Culture Collection - http://www.atcc.org

ATS Laboratories - http://www.ats-labs.com

Biolog - http://www.biolog.com

Biomerieux - http://industry.biomerieux-usa.com

Dupont Qualicon - http://www.qualicon.com

EMD Chemicals - http://www.emdchemicals.com

EMSL Analytical, Inc. - http://www.emsl.com

Genomic Profiling Systems, Inc. - http://www.genprosys.com

MIDI, Inc. - http://www.midi-inc.com

Millipore - http://www.millipore.com

Pall - http://www.pall.com

NovaTek International - http://www.ntint.com

Raven Biological Labs - http://www.ravenlabs.com

Veltek Associates, Inc - http://www.sterile.com

=================================
The nature of this service is to provide a medium for communication. The
specific statements and endorsements of individuals participating in the
discussions are not necessarily those of The Microbiology Network, Inc., the
PMF, or the sponsors of the list.

------------------
The PMFList (http://microbiol.org/PMFList_info.htm) is operated from
The Microbiology Network (http://microbiol.org) and supported by
our sponsors (http://microbiol.org/sponsor.htm) as a service to
the scientific community.

Please take a second to visit our sponsors' web sites and say thank you for their support of this service.

Accugenix - http://www.accugenix.com

American Type Culture Collection - http://www.atcc.org

ATS Laboratories - http://www.ats-labs.com

Biolog - http://www.biolog.com

Biomerieux - http://industry.biomerieux-usa.com

Dupont Qualicon - http://www.qualicon.com

EMD Chemicals - http://www.emdchemicals.com

EMSL Analytical, Inc. - http://www.emsl.com

Genomic Profiling Systems, Inc. - http://www.genprosys.com

MIDI, Inc. - http://www.midi-inc.com

Millipore - http://www.millipore.com

Pall - http://www.pall.com

NovaTek International - http://www.ntint.com

Raven Biological Labs - http://www.ravenlabs.com

Veltek Associates, Inc - http://www.sterile.com

=================================
The nature of this service is to provide a medium for communication. The specific statements and endorsements of individuals participating in the discussions are not necessarily those of The Microbiology Network, Inc., the PMF, or the sponsors of the list.

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