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May 2011

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Fri, 20 May 2011 14:26:11 -0400
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Dear Yatin,

Thank you for interesting information.

If the ultra filtration unit contains cellulosic material, there is a
possibility that beta-glucan is released from the filter.
If you use regular LAL which reacts with endotoxin and beta-glucan, the
released beta-glucan will cause false positives in the LAL test.
In this case, you can measure endotoxin in the sample with LAL
reconstituted with a beta-glucan blocker buffer (endotoxin-specific
buffer).
You can also measure beta-glucan with beta-glucan specific LAL product,
such as PTS Glucan cartridge.

Best regards, 

Masakazu Tsuchiya, Ph. D.
Senior Research Scientist
Endotoxin and Microbial Detection
Charles River
TEL: 843-766-7575
FAX: 843-766-7576
E-mail: [log in to unmask]
Experience  www.criver.com 
 
Accelerating Drug Development. Exactly.
 
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-----Original Message-----
From: The Pharmaceutical Microbiology Forum Email List
[mailto:[log in to unmask]] On Behalf Of Ajgaonkar, Yatin
Sent: 19 May 2011 1:40 AM
To: [log in to unmask]
Subject: Re: [PMFLIST] Interference in the LAL test

Dear Anders and Masakazu

In this regard even I would like to share my experience:

We have a KTA and it is often run with standard curve of 1-0.01.

Our water system has ozone in the storage tank that is why we get
<0.01EU/ml in all the water loop points but the generation point (Ultra
Filtration unit) tends to give a value E.g. 0.03EU/ml.

The recovery of loop points is always 90 to 120 but the generation point
which already has some endotoxin gives recovery of 180 to 220. 
Similarly LRW gives good (within idea range) recovery and water sample
with inherent endotoxin gives more recovery.

As if the presence of natural endotoxin in the sample triggers more
spike recovery.

I wonder if anyone else in the forum, is having similar experience.

Regards
Yatin 

-----Original Message-----
From: The Pharmaceutical Microbiology Forum Email List
[mailto:[log in to unmask]] On Behalf Of Tsuchiya, Masakazu
Sent: Tuesday, May 17, 2011 8:40 PM
To: [log in to unmask]
Subject: Re: [PMFLIST] Interference in the LAL test

Hello Anders,

There are several possibilities to obtain the recovery over 100% in the
Bacterial Endotoxins Test (BET).
Assuming you used a kinetic LAL method and the spiked endotoxin was
prepared from an endotoxin dilution used for the standard curve, there
are three possibilities.

(1) Bias from the shape of the standard curve
     A standard curve with a kinetic BET assay is usually bent.  Most of
KTA and KCA with microplate readers usually give concave standard
curves. Since the regular kinetic BET techniques use the linear
regression, there comes a bias from the shape of the standard curves.
Assuming the standard curve used was concave, a calculated endotoxin in
the middle of the standard range is larger than the actual endotoxin
concentration.  Spiked endotoxin concentrations are usually in the
middle of the standard range. Therefore, the recovery of the spiked
endotoxin is usually higher than 100% with a concave standard curve, if
there is no interference. You can see this bias if you calculate
endotoxin concentrations from the onset times of the standard dilution
used. I published some of our data relating this issue on the BioProcess
International in 2010. (See the website
http://www.bioprocessintl.com/journal/2010/July_August/Biases-in-the-Bac
terial-Endotoxin-Test-302020 )

(2) Endotoxin activity change by the sample
     Endotoxin activity can be changed by the sample. We see more
inhibition but some of them are enhancement. For example, detergents and
proteins can change the micelle size of endotoxin in the solutions to
change the activity.

(3) Enhancement of the LAL reaction by the sample
     If the pH of the reaction mixture is changed, the sensitivity of
the LAL reagent will be changed.  The  magnesium concentration in the
reaction mixture is also critical for the sensitivity of LAL reagent.
Denatured protein may increase the degree of turbidity change, resulting
in enhancement by providing shorter onset times in KTA.

I recommend you checking the bias from the shape of the standard curve
first because this bias is often observed and the degree of the bias is
usually beyond expectation.  If the bias is not so large, then the
interference is enhancement.  It is hard to know which caused the
enhancement, endotoxin or the LAL reaction.  However, more dilution
should provide a recovery close to 100%.  It is a good idea to test a
positive water control prepared by the same manner for the positive
product control.

The acceptance criteria of the endotoxin recovery in the sample is
between 50% and 200%, according to the BET. I guess this was decided
because of the agreement of the acceptance range for the gel-clot
method.  I personally like to see the recovery at least 75%-150% (maybe
80%-130%) for my samples with a kinetic method, although there is no
theoretical evidence.

I hope this information is helpful for you.

Thank you and best regards,

Masakazu Tsuchiya, Ph. D.
Senior Research Scientist
Endotoxin and Microbial Detection
Charles River
TEL: 843-766-7575
FAX: 843-766-7576
E-mail: [log in to unmask]
Experience  www.criver.com 
 
Accelerating Drug Development. Exactly.
 
Notice - This email and any files transmitted with it are confidential
and may contain privileged and/or proprietary information. You must not
disclose this message to another party without Charles River's express
written consent. If you are not the intended recipient you must not
copy, distribute or use this email or the information contained in it
for any purpose other than to notify us. If you have received this
message in error, please notify Charles River immediately, and delete it
from your system.
-----Original Message-----
From: The Pharmaceutical Microbiology Forum Email List
[mailto:[log in to unmask]] On Behalf Of ADET (Anders Thorn)
Sent: Monday, May 16, 2011 5:10 AM
To: [log in to unmask]
Subject: [PMFLIST] Interference in the LAL test

Dear Forum,

When you make a screening of different dilutions of a sample for LAL
test that cause inhibition of the assay the recovery of the positive
product (%PPC recovery) increase with the dilution. Often you will see
that the curve (% PPC as function of the dilution) level off when the
%PPC recovery is above 100 % (for instance at around 130 % which
corresponds to the %PPC recovery of LAL reagent water or WFI tested in
your assay) and not when the %PPC recovery is exactly 100 %.

When is a solution considered free for interference in the LAL test? Is
it when you dilute your sample so you get a %PPC recovery at around 100
% or is it when you dilute your sample to a degree where the curve (%
PPC as function of the dilution) level off (for instance at around 130
%)?

Thanks and best regards,
Anders

_______________________

Anders Thorn

Novo Nordisk A/S

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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

BD Diagnostic Systems - http://www.bd.com/ds/

Biolog - http://www.biolog.com

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

Biotest - http://www.BiotestUSA.com/micro

MicroBioLogics, Inc. - http://www.microbiologics.com

MODA Technology Partners: now a LONZA company http://lonza.com/moda

Pall - http://www.pall.com

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

Rapid Micro Biosystems - http://www.rapidmicrobio.com  (formerly GPS)

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

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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

BD Diagnostic Systems - http://www.bd.com/ds/

Biolog - http://www.biolog.com

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

Biotest - http://www.BiotestUSA.com/micro

MicroBioLogics, Inc. - http://www.microbiologics.com

MODA Technology Partners: now a LONZA company http://lonza.com/moda

Pall - http://www.pall.com

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

Rapid Micro Biosystems - http://www.rapidmicrobio.com  (formerly GPS)

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

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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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