BET Newsletter Jan2011 - · PDF fileBET Newsletter : A Charles River India Private Limited Communication Volume 2, Issue 1 : January 2011 Page 2 of 7 Don’t Print. Save Trees! Ex:

Volume 2, Issue 1 : January 2011A Charles River India Private Limited Communication

Don’t Print. Save Trees!

BET Newsletter

Dear Reader,

Happy New Year 2011!

I infer from my interactions with our customers, that the

BET Newsletters published during the year 2010 have

been useful. I also understand the need to continue our

endeavor of addressing pertinent issues through this medium.

As you are aware, LAL testing in India has come a long

way since 1996, whence it was introduced in the Indian

Pharmacopoeia. It has been noticed that the Indian Pharma

companies are now exploring the possibilities of establishing

the Kinetic Methods of LAL testing at their facilities which

is in line with the global trends. The global trends too

indicate a migration from the Gel Clot [qualitative] methods

to the Kinetic [quantitative] methods over a period of time as

more complex products with stringent limits are being

manufactured.

I, therefore, have devoted this current issue to the same topic

of migration from the Gel Clot method to the Kinetic method

of LAL testing. The process requires a frame shift in

understanding of a few vital aspects of the testing

methodology as well as the parameters that come into play.

An attempt has been made to address these aspects in

sufficient detail so as to bring out the relevance in the

process.

You are always welcome to send your suggestions and

comments to our email id: [email protected].

Sincerely,

Dr. P. K. Chitnis

Director

www.criverindia.com

Migration from Gel Clot to Kinetic LAL testingShibu Chakraborty and Dr. P. K. Chitnis

Introduction

The First Step: Getting your laboratory ready

The harmonized chapter of Bacterial Endotoxin Test includes

the photometric methods viz. the endpoint and kinetic LAL

assays. The endpoint methods have fallen in to disfavor

because the standard curve is limited to a one log range. Kinetic

BET methods dominate LAL testing because they provide the

most informative way to collect and manage LAL data. These

methods are quantitative, rapid, simple, precise and facilitate

good documentation and trend analysis.

The Kinetic LAL assays can are of two types viz. :

1. Kinetic Chromogenic Assay (KCA)

2. Kinetic Turbidimetric Assay (KTA)

The purpose of presenting this guide is to provide guidance to

the industry professionals who desire to migrate from the

existing gel clot method to the more advanced kinetic method.

Requirements for kinetic Assays:

1. Reagents

a. Licensed LAL with Certificate of Analysis for CSE for

Kinetic LAL

b.Control Standard Endotoxin (CSE)

c. LAL Reagent Water (LRW)

2. Testing Accessories & Disposables

a. Vortex Mixer

b.Calibrated mechanical pipettor (10-100uL) and fixed

volume 10 uL pipettor

c. Repeat Pipettor with disposable tip is also desirable

d.96 well microtiter plate

e. Endotoxin free pipette tips

3. Instrumentation & Software

a. Microplate Reader

i. Biotek ELX 808IU which has been qualified for

endotoxin analysis by Charles River Endosafe

b.Software

i. Endoscan V, a 21 CFR part 11 compliant software

developed for Endotoxin detection and

management

Volume 2, Issue 1 : January 2011BET Newsletter : A Charles River India Private Limited Communication

Don’t Print. Save Trees!Page 2 of 7

Ex: Testing of Gentamycin sulphate at a test concentration of

0.25 mg/mL on a Standard Curve of 5 - 0.05 EU/mL

Product Specific Sensitivity (PSS): 0.05EU/ml / 0.25mg/mL = 0.2 EU/mg

The above settings will enable you to quantify endotoxin values

as low as 0.2 EU/mg.

Quantifying endotoxin values at very low level requires greater 2sensitivity. With the availability of the Endosafe KTA , the

linearity can be extended for a more sensitive standard curve

range 50-0.005EU/mL.

For laboratories who are working with gel clot and kinetic

methods simultaneously, the Endosafe LAL 0.015EU/mL is an

ideal choice as it comes with a certificate of analysis for both the

gel clot and kinetic methods.

It is the EU/mL concentration range between the highest and

lowest points on the standard curve. Beginners are advised to

start with a 3 point 2 log curve like the 5.0 -0.05EU/mL. The

choice of the endotoxin standard is primarily determined by the

different kind of samples that a laboratory expects to receive for

analysis. For instance, the range of 1.0 – 0.01 EU/mL is best

suited for routine water testing. In case of product testing, the

most compatible concentration and endotoxin limit would help

determine the choice of the standard curve range.

A 4 point 3 log curve can also be employed as it covers a wide

range of standards like 10-1.0-0.1-0.01 EU/mL. There is merit in

selecting one standard curve and simplifying protocols in the

lab to enable the testing of different types of samples.

However, the end user must be cautious and judicious in

choosing the standard curve as a wide standard curve is less

forgiving and may lead to invalid assays.

The time required for a kinetic system to detect the onset times

for the lowest points on the standard curve is an indicator of the

speediness of a test.

Optical Density (OD) for a given wavelength is an expression of

the transmittance of an optical element. This time is termed as

onset time and the OD at that time is referred to as the onset OD.

Lambda ( ) and onset OD values influence the speed of the test.

For KTA, it takes about half an hour for an assay to get over

when working on a 5 - 0.05 EU/mL range as against 1.0 – 0.01

EU/mL which runs for about 40 minutes. It is recommended,

based on empirical data, to set the onset OD to 0.05 for KTA and

0.1 for KCA for obtaining the best results.

2. Standard Curve range

3. Speed

l

Endoscan V – Hardware and Software Requirements

Starting a Kinetic LAL system

1. Sensitivity

ProcessorIBM Compatible PC with Pentium4 or equivalent processorAtleast 1.4 GHz speed

Memory (RAM)128 MB minimum with Windows 2000 Pro and NT4256 MB minimum with Windows XP Pro512 MB recommended

DrivesHard disk with 100 MB of spaceCD-ROM or DVD-ROM

Operating System Windows 2003 Server Standard/Enterprise EditionWindows 2000 Advanced/ServerWindows XP Professional (Service Pack 1, 2*, or 3*)Windows 2000 Professional (Service Pack 4) orWindows NT4 Workstation/Server (Service Pack 6)**

OthersInternet Explorer 5.0 or laterAdobe Acrobat Reader 5 or later (recommended)

This discussion identifies and advises on critical choices that

must be made before the standard operating procedures are

finalized. When informed choices are made at the outset, there

is greater assurance that a kinetic system will perform as

required and pave a way for smooth transition.

In kinetic assays, the lowest point on the standard curve is the

sensitivity of the assay e.g. in a standard curve of 5.0 – 0.5 –

0.05 EU/ml, the sensitivity of the assay ( ) would be 0.05 EU/mL

The analyst has the liberty to decide the sensitivity to be used

for the assay based on a combination of factors viz. the

endotoxin limit of the product and the validated Non Interfering

Concentration (NIC). Further to this the analyst can estimate the

endotoxin content in his sample by using the following relation :

Product Specific Sensitivity (PSS):

The sensitivity of the test conditions in the profile must be

calculated and compared to the endotoxin limit to determine if

there is adequate sensitivity. The Product Specific Sensitivity

(PSS) is found by dividing the lambda by the test concentration

of a small volume parenteral, or by multiplying lambda by the

dilution factor for extracts.

l

Test Concentration = Lowest point on a standard curve (EU/mL)

Endotoxin Limit

Product Specific Sensitivity (PSS) = Lowest point on a standard curve (EU/mL)

Test Concentration (mg/mL)



4. Reagent

Code Description Sensitivity

Analyst Qualification

Results and Analysis

R Value

The reagent can be selected based on the above parameters

and objective of the test. If a wide range standard curve (5 point

curve) or greater sensitivity (0.005EU/mL) is a high priority for

kinetic assays, the KCA method would be favored because of its

speed and robustness. For the sole benefit of robustness, KCA

is again the reagent of choice. For a medium range standard

curve (5-0.05 or 1-0.01EU/mL), KTA is ideal as it yields equal

performance.

Endosafe kinetic LAL reagents at a glance:

R15003 50 test vial 0.03 EU/mL

R15015 50 test vial 0.015 EU/mL2R19000 KTA 100 - 0.001 EU/mL

R1708K Endochrome K 50 - 0.005 EU/mL

A qualified analyst is defined as the one who has demonstrated

the ability to achieve valid results while performing a standard

curve, as documented in training records.

A valid standard curve is the one that satisfies the criteria for

linearity, precision of replicates, number of points, slope and

acceptable resolution i.e. a significant separation between the

negative control of the assay and the lowest point on the

standard curve.

It is worthwhile to note that though the pharmacopoeia is silent on this

aspect, Dr. J. F. Cooper, the pioneer and a world acclaimed authority on

LAL testing, recommends at least a 300 second difference in reaction

times of the negative control of the assay and lowest point of the standard

curve in order for that assay to yield meaningful results. E.g. If the

Standard Curve of 5.0-0.05EU/mL has taken about 1800 seconds, the

negative water control must take atleast 2100 seconds to reach for it to be

valid.

As per the regulatory guidelines the assay is valid if the following

acceptance criteria are met. Acceptance criteria in a summary:

R value ≥ 0.980

CV % ≤10% Spike Recovery 50-200%

Linear Regression: A method wherein a straight line is fitted to a

set of data points to measure the effect of a single independent

variable. The slope of the line is the measured impact of that

variable.

When the endotoxin standards are prepared accurately, we

obtain a better linearity that meets the minimum criteria of

>0.980.

Polynomial Regression, on the other hand, fits a non-linear

relationship between the two variables viz: endotoxin

concentrations on the x axis and reaction times on the y axis.

There are two types of regression analysis available:

1. Linear Regression

2. Non-linear (polynomial) Regression

We at Charles River recommend the use of linear regression

over polynomial regression as the linear regression is more

restrictive than the polynomial regression, thereby making it

more accurate.

Spike recovery is performed in the positive control of the LAL

Kinetic assay wherein a known amount of endotoxin is added to the

sample and is recovered during the analysis. The measure of

recovery serves as a pointer of Interference Encountered while

conducting the assay.

In the Gel clot method, this is done by adding a 2 amount of the

control standard endotoxin to the positive control and recovering a

gelation of the positive control. E.g. for an assay employing a

sensitivity [ ] of 0.125 EU/mL, the spiking would be done at 2 i.e.

0.25 EU/mL concentration.

However, in the kinetic method, the spiking is carried out with an

amount of Control Standard Endotoxin equivalent to the mid point of

the standard curve. E.g. for a 5 – 0.5 – 0.05 EU/mL standard curve,

the spiking would be done at 0.5 EU/mL concentration.

The Endoscan software is designed to carry out calculation of the

mean recovery of the added endotoxin by subtracting the mean

endotoxin concentration in the solution (if any) from that containing

the added endotoxin. and report automatically as the spike

recovery. The regulatory guidelines specify that the acceptable

spike recovery is within 50 – 200% of the spiked value.

Slope -0.1 to -1.0 [USFDA guidelines]

The slope of the standard curve must be less than -0.1 and greater

than -1.0 for it to be valid. US FDA guidelines on the BET.

The slope signifies the speed of the assay and recognizes the

accuracy in the preparation of the standard solutions. When the

slope is too weak or strong, the endotoxin values from an unknown

sample can get under or over estimated.

Extrapolation is the process of constructing new data points

outside a discrete set of known data points whereas Interpolation

is the process which constructs new points between known points.

However, the results of extrapolations are often less meaningful,

and are subject to greater uncertainty.

Choice of Regression Analysis for Kinetic LAL Methods

Spike Recovery

Determination of Endotoxin Value

l

l l







In the above example, 2 samples have been tested and

analysed by the kinetic method while employing a standard

curve of 5.0-0.05EU/mL. Both samples were appropriately

diluted (1:40) and tested.

From the summary of the results, it can be observed that SPL1

is having an endotoxin value of < 2.0EU/mL as against SPL2

which has an endotoxin value of 5.64EU/mL.

Since the SPL1 value fell outside the range of the curve and

extrapolation is not allowed it could be interpreted as < 2EU/mL

which is obtained by multiplying dilution factor 40 to the bottom

point of the standard 0.05EU/mL.

On the other hand the SPL2 value fell within the range of the

standard curve and hence the exact value could be calculated

by interpolation on the standard curve.

While migrating from Gel Clot to Kinetic method, it is essential

for the lab personnel to gain sufficient confidence in handling

the kinetic assays. Thorough understanding of the ideal

analysis and instrument settings for kinetic methods will help

the analyst to perform the kinetic assays with consistency and

develop better confidence.

In the initial phase, only fewer products / samples must be

analysed at a time so that the risk of invalid results due to

analytical artifacts are minimized. Gradually, number of

samples per assay should be increased to optimize the

economy of the test.

Prior to product validation, it is important to set the calculations

right and conduct a thorough preliminary screening of the

product. The product testing and validation issues will be

addressed in a separate newsletter.

Comparison of Gel Clot versus Kinetic methods:

Phase of Transition

Cost implications of migrating a from Gel Clot assay

to a Kinetic Assay

Conclusion

Besides the capital investment of Kinetic Microplate Reader

and Software, other cost for reagents and accessories remain

more or less same for the Gel Clot Method and the Kinetic

Method. The most important advantage of kinetic assay is that it

is quantitative whereas the gel clot assay can at most be semi-

quantitative. In order to arrive at an estimate using the Gel Clot

assay it requires preparation of several dilutions to estimate an

endotoxin range of test sample, which is normally 2-fold.

The cost of actual test in terms of lysate remain same for Gel

Clot and Kinetic assay because the reagent volume used for

both is same i.e. 100uL. The cost of accessories viz. tips,

pipettes and dilution tubes remain the same, the only difference

being that the Kinetic assay is performed in 96 well microplate

instead of test tubes. It has been observed that the plate is

cheaper that the depyrogenated glass tubes.

For easy transition form Gel Clot to KTA, Endosafe – Charles

River Laboratories is the only supplier for reagents ( =

0.015EU/mL; 0.03EU/mL) which could be used for both Gel

Clot and KTA. In other words, the left over lysate after KTA can

be used for Gel Clot assay at the same time or later, preventing

wastage of lysate.

It is imperative for a laboratory to judiciously adopt a step wise

systematic approach to migrate successfully from the gel clot

technique to the advanced kinetic methods and provided better

quality control management for the test of bacterial endotoxins.

The pointers discussed in this newsletter should be taken as

guidance principles in achieving a successful and smooth

transition from gel clot testing to kinetic testing.

The rapidity, flexibility and changes in concept to read the

results must be correctly understood in the right perspective for

the analyst and laboratory manager. This will help them justify

the purpose and also meet the perquisites for which the kinetic

LAL system is introduced in the laboratory.

It is important to appreciate that even though there is capital

cost involved towards buying the reader and software, the

running cost is only slightly higher than the conventional Gel

Clot test while offering several advantages like rapidity,

quantification, audit trail and a valuable tool for corrective action

/preventive action in quality control and in-process

management.

In the current scenario when the global trend is looking towards

advanced high end technology in laboratory analysis, the

kinetic assay is a perfect choice to add value to the product and

earn better revenue for a quality product.

l

Gel Clot Kinetic Assay

Qualititative Analysis/Semi-Quantitative

Quantitative Analysis

60 minutes of run time required before observation and results can be drawn

Audit trail not possible

Employs the Gel clot reagents with sensitivity available up to 0.015 EU/mL.

Capital expenditure items include only heating block and vortex mixer.

An economical and convenient way to start BET when newly introduced in a laboratory.

Usually takes about 30 minutes depending on sensitivity of the assay

Audit trail possible, fully 21 CFR part 11 compliant

Turbidimetric Assay can be done with gel clot 0.015 EU/mL lysate

2and with KTA . Chromogenic Assay employs the KCA reagents.

Capital expenditure items include Biotek reader, Endoscan software, vortex mixer.

The most advanced tool that offers flexibility, better management due to quantification and compliance issues due to audit trail features.



The authors wish to thank Mr. Rajesh Puthiya and Dr. K. Nagarajan for their

valuable comments in writing up of this script.

1. USP 33 Chapter on <85> Bacterial Endotoxin Test

2. IP 2010, BET Monograph

3. LAL Times, Charles River Endosafe, Vol.7,No.3, September 2000.

4. LAL Times, Charles River Endosafe, Vol.7,No.1, March 2000.

5. LAL Times, Charles River Endosafe, Vol. 4, No. January 1997

Acknowledgements

References

Charles River Laboratories India Private Limited907, Barton Centre, 84, M.G. Road, Bangalore 560 001Tel: 080-25588175 - 177. Fax: 080-41659281. Email: [email protected]

Mumbai Tel: 022-25905203. Fax: 022-25911464. Email: [email protected]

Ahmedabad Tel: 079-26407713. Fax: 079-26406992. Email: [email protected]

Hyderabad Telefax: 040-27177710. Email: [email protected]

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BET Newsletter Jan2011 - · PDF fileBET Newsletter : A Charles River India Private Limited Communication Volume 2, Issue 1 : January 2011 Page 2 of 7 Don’t Print. Save Trees! Ex: