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May 2006

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From:
Victor Mencarelli <[log in to unmask]>
Reply To:
The Pharmaceutical Microbiology Forum Email List <[log in to unmask]>
Date:
Tue, 30 May 2006 12:13:36 -0400
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Scott,

Therein lies the problem.  Simply put, until the regulators accept the 
inherent variability in the methods employed in microbiology, the 
microbiologist cannot "accept" the limitations.  Until the microbiologist 
accepts the limitations imposed by the method, the regulatory agencies 
will not even look at the issue.  Therefore, we are looking at a major 
impass based simply on the fact that 1) if the microbiologist simply 
accepts the limitations they are considered negligent in their duties by 
the regulators; 2) if the regulators simply accepts the limitation they 
appear to be "caving to industry"; and 3) until everyone sits down in a 
room with equal numbers and baseball bats, I really don't foresee much of 
a change in this issue.

While I realize my #3 above will garner either chuckles or jeers from the 
rest of list, it is simple.  Until everyone gets on the same page (and 
that includes the regulatory bodies hiring EXPERIENCED INDUSTRIAL 
MICROBIOLOGISTS) with this issue, we are going to have this charade 
continuing day after day after day.  Part of the problem is industry (not 
being willing to invite a "discussion" on the relative positions) (and by 
industry I am not referring to the consultants that are retained to 
present the industry views to FDA, I mean the actual COMPANIES 
themselves).  Part of the problem is the bureaucracy inherent in 
governmental regulations and regulators.  And truthfully, I believe that 
much of the issue is also lack of understanding of the issues by both 
groups.

These are just my opinions and in no way represent those of anyone else 
including my employer.  (Sorry, I had to put that disclaimer in for 
practical purposes here).

Vic Mencarelli
Regulatory Affairs Specialist
Engelhard Corp.
50 Health Sciences Dr.
Stony Brook, NY 11790
P: 631-380-2482
F: 631-689-6880
E-mail: [log in to unmask]

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Scott Sutton <[log in to unmask]> 
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05/30/2006 11:27 AM
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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
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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]> 
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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
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-----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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Genomic Profiling Systems, Inc. - http://www.genprosys.com

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Millipore - http://www.millipore.com

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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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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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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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