Volume 1. Issue 1 : January 2010A Charles River India Private Limited Communication

Dear Reader,

Happy New Year 2010 !

It gives me great pleasure to begin the year 2010 by bringing to

you the first 'BET Newsletter ' from Charles River Laboratories

India Pvt. Ltd. in “electronic format” which is in l ine with our

Eco-friendly policy.

Charles River Laboratories makes a conscious effort to share its

expertise, knowledge and advancements in the fie ld of Bacterial

E n d o t o x i n s Te s t i n g w i th i t s u s e r s t h r o u g h v a r i o u s

communications.

The present volume, the first in a series of newsletters, is

a concise review of the different methods employed for Bacterial

Endotoxins Testing in the pharmaceutical industry. It tries to

convey to the reader the pros and cons of using a qualitative

method versus a quantitative method while highlighting the

benefits of employing a kinetic method routinely in endotoxin

management.

It can be observed that the western countries have migrated

from the conventional Gel Clot method, a predominantly

qualitative method to the photometric or kinetic methods which

are quantitative. This transition has enabled them to get the

benefits of automation associated with these methods while

providing highly reproducible and valuable quantitative data.

The numbers that are generated by these methods can be

effectively employed for trend analysis and help move towards

assured quality manufacturing rather than assessing quality

after manufacturing.

The newslet ters to fo l low wi l l h ighl ight Charles River 's

advances in endotoxin testing and their immense potential in

bettering endotoxin management.

You are always welcome to send your suggestions and comments

to our email : india.customercare@crl.com

Sincerely,

Dr. P. K. Chitnis

Director

LAL Methods for Endotoxin Testing: A Review

Introduction The bacterial endotoxin test is used to detect or quantify endotoxins of gram negative bacterial origin using the lysate derived from amebocytes of the American Horse Shoe crab (Limulus polyphemus). There are two techniques for this test viz.:

1. Gel Clot method - which is based on gel formation 2. Photometric method:

a. Turbidimetric method - based on the development of turbidity after cleavage of the endogenous substrate;

b. Chromogenic method - based on the development

of color after cleavage of the synthetic peptide-chromogen complex.

The lysate derived from amebocytes of Limulus polyphemus is a complex mixture of serine proteases. Bacterial Endotoxin triggers the cascade mechanism of gel formation.

l l

Rajesh Puthiya, Shibu Chakraborty & Dr. P. K. Chitnis

Biochemical Pathway

Gel Clot Method

Endotoxin activates the clotting enzyme which then cleaves a soluble substrate. The resulting insoluble clotting protein stacks to form a gel. The gel thus formed is fragile and irreversible.

Fig 1. Mechanism of LAL gelation

The Gel Clot method is the simplest and described in detail by most of the regulatory guidelines. The reagent used for Gel Clot test is labeled with sensitivity ( ). LAL sensitivity ( ) is defined as the lowest concentration of purified endotoxin that will produce a firm gel.

The lysate for the Gel Clot method is available in a range of sensitivities viz. 0.25 EU/mL to 0.015 EU/mL. The sensitivity of the lysate to be used is typically decided considering the endotoxin limit of the test sample and its interference to the test.

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BET Newsletter

Interference to the test can be of two types viz. : Inhibition and Enhancement. Inhibition could be encountered due to extremes of pH, inappropriate divalent cation concentration, endotoxin depletion or the like while Enhancement could result from the presence of LAL Reactive Material [LAL-RM] or Glucans. Inhibition can be generally overcome by diluting the sample to a higher degree which can be facilitated by the use of a higher sensitivity of lysate which allows a higher dilution while Enhancement can be resolved by the use of an ES buffer.

The Gel Clot test result can either be a firm gel that remains 0in place upon inversion through 180 which is recorded as

positive (+) or everything flows down the tube which is recorded as negative (-). The test can be set up as a limits test or an endpoint assay. A limit test is to pass or fail a product at a particular endotoxin concentration whereas an endpoint assay includes testing of a series of two fold dilutions. The endpoint is the greatest dilution to give a positive test. The result of a typical Gel Clot endpoint assay are presented in Table 1.

The endotoxin concentration of the original sample is calculated by multiplying the reagent sensitivity by the dilution factor at the endpoint. In Table 1, therefore, the endotoxin concentration at the endpoint is 16 x 0.125EU/mL = 2EU/mL. Hence the endotoxin in the sample is between 2EU/mL and 4EU/mL.

Kinetic LAL assays can be performed by two methods:

1. Kinetic Turbidimetric Assay (KTA)2. Kinetic Chromogenic Assay (KCA)

On the backdrop of the Gel Clot test which is a qualitative or at the most a semi-quantitative test a need was felt to have a method which could quantify endotoxins. Quantification of endotoxins opens new vistas in Management of Endotoxins as it provides an excellent tool for trending of the data to assess the impact of various factors on endotoxin levels necessary for designing and implementing appropriate Corrective and Preventive Actions. Additionally, owing to the instrumentation and automation associated with the quantitative methods, a large number of samples can be analyzed efficiently while providing a very secure milieu owing to the use of 21CFR Part 11 compliant software.

Volume 1. Issue1 : January 2010BET Newsletter : A Charles River India Private Limited Communication

Kinetic Turbidimetric Method

Kinetic Chromogenic Method

Results Calculation of Endotoxin values

The turbidimetric assays measure the increase in turbidity as a function of endotoxin concentration. Endotoxin concentration in unknown is determined by comparing the resultant turbidity to a standard curve. Since turbidity is a precursor to gelation, these assays are more sensitive to endotoxin concentration. The absorbance is measured at 340 nm.

The chromogenic assays utilize the initial portions of the enzymatic cascade, but substitute a synthetic chromogenic peptide as a substrate for the activated clotting enzyme in place of the clotting protein. The chromogenic substrates like Boc-Leu-Gly-Arg-p-nitroanilide (pNA) are hydrolyzed by the clotting enzyme releasing the terminal chromogenic moiety and generating a yellow colour. The absorbance of the released pNA is measured at 405 nm.

The requirements of a harmonized kinetic BET include:a. three or more standard pointsb. curve linearity : r > -0.980 or betterc. recovery of 50% to 200% for positive controlsd. an incubation temperature suggested by the

manufacturer.

Kinetic analyses performed by either KTA or KCA are conducted using a incubating microplate reader that has a detection system to measure optical density over a period of time, a microprocessor and suitable software to analyze data by regression analysis. The Biotek ELx808IU with Endoscan software is an example of an incubating microplate reader with software designed to collect, process and store data using protocols for endotoxin measurement using LAL.

Each assay should include samples or dilutions of samples or products, positive product controls, a series of dilutions covering the desired standard curve and a negative water control. The assay has to be carried out in duplicate.

Routine analysis is most efficiently done with a two-log standard curve; each assay should contain a standard curve with at least 3 points, tested in duplicate, such as 5.0; 0.5 and 0.05 EU/mL. The positive product control (PPC) should contain endotoxin equal to or near the mid point of the standard curve. For example, when using a 5-0.05EU/mL standard curve, the spike value should be at 0.5EU/mL.

Throughout the assay, the microplate reader monitors the increase in absorbance. The reader records the time required for the absorbance to increase significantly over background, usually 0.05 OD units for KTA and 0.1 OD units for KCA.

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 OD at that time is termed as onset OD. The software then automatically produces a log/log correlation of the Onset time of each standard with its corresponding endotoxin concentration. Standard Curve features are then displayed and evaluated to determine the validity of the analysis.

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LAL Sensitivity ( ) = 0.125EU/mL

( - ) = No Gel

( + ) = Firm Gel

NPC = Negative Product Control

PPC = Positive Product Control

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Summary

References

The prime advantage of the Kinetic methods over the conventional Gel Clot method is the ability to Quantify the Endotoxins in the sample. The result of the Kinetic assay is objective whereas the results from the Gel Clot may be subjective especially in borderline cases where the gel formed may be viscous/semi-solid.

The Kinetic method is important as it is 21CFR Part 11 compliant and helps in maintaining audit trail, provides better documentation, helps in trend analysis and provides alerts to alarming rise of endotoxins in samples while in routine use.

While using advanced instrumentation, powerful software with latest computing abilities and endotoxin-free accessories the kinetic test has been shown to provide highly accurate quantification of endotoxin on a routine basis, thus, empowering the user the best possible means for endotoxin management.

· Chapter on “Bacterial Endotoxin Test” by Dr.James Cooper in the book

Microbiology in Pharmaceutical Manufacturing. Pages 546-549, December 2001.

· Endosafe newsletters Volume:Vol7, No3, September 2000.

· USP 27 Chapter on <85> Bacterial Endotoxin Test.

Performance Characterisitics

An example of kinetic assay

Linearity is the co-relation co-efficient (r) which describes how closely the points on a scatter-plot cluster lie in a straight line. Linearity is a function of the quality and speed of the LAL reagent formulation and the potency of the endotoxin standard solutions. The absolute value of the correlation co-efficient (r) must be greater than or equal to -0.980.

Recovery is the proportion of the positive product control (PPC) that is detected in a sample. A 50-200% recovery is allowed by the Interim Guidance of the LAL Guideline. (USP 28 NF23 Supplement 2 <85> BACTERIAL ENDOTOXIN TEST)

The recovery is dependent on the compatibility of the sample with the LAL reagent, linearity of the standard curve and temperature uniformity.

Calculation: % recovery = [Spike (well) Sample (well)] / Spike (conc.) * 100.

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Volume 1. Issue1 : January 2010BET Newsletter : A Charles River India Private Limited Communication

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