Pyrogen Testing

PYROGEN TESTING

Why Pyrogen Tests ?

A pyrogen is a protein that can induce a fever in a patient by triggering a series of immune reactions

Pyrogens are fever-inducing substances derived primarily from gram-negative and gram-positive bacteria. The guaranteed absence of pyrogens is a critical safety precaution for all drugs administered parenterally, since these contaminants can pose a life-threatening risk of shock to the patient.
Pyrogen testing defines a process used by drug manufacturers to determine if bacterial toxins are present in vaccines and drugs that might cause fever when used on humans. It determines if microbes or their metabolites are present in intravenous solutions during the manufacturing process.
The most common, reliable and oldest form of pyrogen testing consists of injecting drugs into rabbits to determine if a fever develops.

Why Rabbits are being used for testing ?

The Rabbit Pyrogen Test in an in vivo test to detect pyrogens qualitatively. Rabbits have a similar pyrogen tolerance to humans, so by observing a change in body temperature in rabbits it is possible to make a determination of the presence of pyrogens. This method can detect non-bacterial endotoxin pyrogens as well as bacterial endotoxins.

The rabbit pyrogen testing method surfaced in the 1940s after some patients became ill from intravenous drugs. Hypodermic devices at the time proved useful for administering drugs directly into the bloodstream for patients who were unable to tolerate oral medications. Even though hypodermics devices were sterile, the drugs were not always safe.

Patients sometimes developed high fevers, chills, and body aches, and some people suffered shock. Doctors didn’t know why this occurred, frequently calling the condition injection fever, saline fever, or distilled water fever. Researchers later discovered some drugs and vaccines were contaminated in production labs with pyrogen endotoxins, potent bacteria that withstand sterilization.

Endotoxins are compounds found in the cell walls of Gram negative bacteria. These compounds help to form a semi-permeable membrane which is designed to protect bacteria from threats. Once the bacteria die, the endotoxins are released, and many of these toxins cause health problems in people, animals, and other organisms, hence the “toxin” in their name.
The human body fights exposure to bacterial toxins in the environment through the skin. When contaminated drugs are injected into the bloodstream, toxins bypass normal defense mechanisms. White blood cells begin releasing another form of pyrogen that causes high fever, which might lead to shock and death.

The process of pyrogen testing on animals typically involves injecting several rabbits at a time within a 10-minute time frame. The dosage for each rabbit depends on body weight, age, and gender. The same group of rabbits might be tested repeatedly every few days until they develop a tolerance to the drugs.

What are the other Pyrogen Tests alternative to Rabbit Test ?

1. LAL Test – Limulus Amoebocyte Lysate Test

This test measures the coagulation of the amoebocytes of the horseshoe crab, initiated by cell wall components ( LPS) of gram-negative bacteria with a molecular weight of >8000 daltons.

The amebocytes, or circulating blood cells, of the horseshoe crab ( Limulus Polyphemus) contain a protein that clots in the presence of bacterial pyrogens. A test sample is incubated with amebocytes lysate from the horseshoe crab for 60 minutes. Pyrogenic substance will give a positive LAL-test characterized by the formation of a solid gel that remains intact in the bottom of the tube on inversion.

The drawback of this method is that this test cannot detect smaller LPS nor the LPS of gram-positive bacteria. Furthermore, the test cannot distinguish between the different types of endotoxins from gram-negative bacteria, which can vary in their fever-inducing potential in the mammal by a factor of 10,000.

LAL Test is based on GEL Clot method which is only qualitative- to indicate whether pyrogens are present or not. This test cannot be perfomed for many vaccines, because of inconsistency of the tests. Some of the vaccines may react with the standards / chemicals used for LAL test and may end up showing false test results. So many repeat tests are needed to judge the presence of pyrogens if the user select LAL test.

2. WBT – Human Whole Blood Test

This test, using an ELISA, was developed in 1996 by Hartung and Wendel. It measures cytokine production.

The main drawback of this method is that, the response to pyrogens in the WTB can be affected by the donor of the blood sample and may not be fully reliable.

Rabbit Tests are most reliable and accurate test method for Pyrogens.

What kind of drugs / vaccines should be checked using Pyrogen Temperature Testing :-

Most of the vaccines, especially Hepatitis B , Rabies Vaccine etc should be checked with Temperature of Rabbits. Because the chemicals like Adjuvant – Aluminum Phosphate, Aluminum Chloride cross reacts with KTA ( Kinetic Turbidometric Assay) reagents resulting false results.

What are the acceptable Accuracy of the Pyrogn monitoring System ?

The maximum tolerance acceptable during Pyrogen measurement is 0.6 to 0.7 deg C. So the measuring system should have an accuracy of at least +/- 0.1 deg C. If the sensor has an accuracy of only +/-0.5 deg C, the maximum tolerance will be 1 deg C which is beyond the required limits for performing Pyrogen Testing and analysis.

What are the allowed temperature limits of Pyrogen Testing ?

Normal Temperature of rabbits are 38 to 39.8 C and maximum allowed temp 39.8. So the allowed tolerance should be 0.6 to 0,7 deg C .

The rectal temperature of the rabbits is recorded at 1, 2 and 3 hours subsequent to injection.
The test passes if no rabbits shows an individual rise in temperature of 0.6 deg C or the sum of the temperature rise of the three rabbits does not exceed 1.4 deg C.

If the temperature rise exceeds these limits, continue he test using another 5 rabbits. The test will be passed if no temperature rise of more than 0.6 deg C for each individual rabbit or the sum of the temperature rise does not exceed 3.7 deg C for the whole 8 rabbits.

What are the basic requirements of a Good Pyrogen Testing Equipment / System :-

The measuring accuracy of the pyrogen testing system should be better than or equal to +/-0.1 deg C
And the equipment should be able to calibrate within the required accuracy as per the FDA norms.

The system should indicate alarms if the probe slips off from the rabbits during measurement. It should start measurement automatically after the probe is placed back properly.

The measuring probes should not irritate the rabbits and should have facilities to make sure that the measuring tip reaches the required depth for the precise measurements.

The system should fully complaint to FDA 21 CFR Part 11 and other international regulatory agencies

The system should be able to group the rabbits based on the Sham tests and make it ready for the real tests based on the results of Sham tests.

If Sham tests fails initially, the system should automatically try for the repeat tests.

The system should provide identification numbers to the rabbits and should automatically select for tests, if it passes Sham Tests

The test results should be documented properly both in tabular as well as graphical format and the test data should be tamper proof and not to be manipulated or adjusted

The system should have proper password protection to make sure that only authorized persons can change the parameters or operate the system

The system should have facilities to make tests and reports based on different international pharmacopeias and it should be able to make the test based on user defined pharmacopeias
The user should be able to check the final test results based on different pharmacopeias and make it sure that it passed all the required criteria.

. The system should have the facility to perform different pharmacopeia tests simultaneously

ELLAB PYROMON – Fully Automatic Pyrogen Testing System
Special Features and Benefits

 

 

ELLAB Pyrogen testing program has been developed in order to comply with every Pharmacopoeia that exists in the world now. Further on, the system gives the possibility to program user defined Pharmacopoeia to suit special requirements.

It has been developed in a way that allows maximum flexibility for the users without violating any form of security or limits the Pharmacopoeia states.

The main purpose for this system is to deliver tamper free documented evidence of the Products, Rabbits and Results. The special features in this system makes manual record taking obsolete and makes life so much easier for the operators and management i.e. Audit trail, Audit event trail,
Electronic Signature, List’s of new and sham animals, Daily Results etc.

Further on, it is possible to run different Pharmacopoeia (also Sham Test) at the same time without
fearing for injecting the wrong rabbits. The local monitors (UI 128) are equipped with background lights that help reading the information for each test (Rabbit No / Channel No. – Volume to inject for ea. Rabbit etc.)

For example, the system can perform the following pharmacopeias

IP – Indian
JP – Japanese
KP – Korean
BP – British
EP – European
USP – United States

Apart from the above, the system has the option to perform user defined Pharmacopoeias where in the user can define the parameters as per their SOP.

The user has the option to select the different pharmacopeias and on selection, the system make ready as per the international norms for that specific pharmacopeias. Or else, the user can program the rabbits body temperature, tolerance limits, time for stabilization, time or sham test, quarantine time, injection time, real test time etc as per own SOP.

The below picture shows how the user can pre-define the tests as per own Pharmacopeias.

Selection of Methods – here the user can select the required pharmacopeias or can program it as per their requirement.

The Phases can be programmed as follows. The time for stabilization, sham test, injection time etc can be selected or programmed here.

Here the user can select Rabbits required body temperature as per the Pharmacopeias or user defined. .

The criteria can be set as follows.

The number of animals to test per batch, pass criteria, fail criteria, over shoot temperature, tolerance limits, different levels etc can be set here.

Temperature Sensor

Special grade temperature sensors with fittings are provided to perfectly insert the measuring tip to the required depth inside the rectum of the rabbit without irritating it. It make sure that the probes would not slip off in case of normal jerking and shaking of rabbits during measurements.

PyroMon™ running on Windows XP, windows 98 or NT platform enables the user to use the computer efficiently to plan new tests. There are a lot of printing facilities, possibilities to program new Pharmacopoeia with limits that fits your products, simulate a previously performed test into other Pharmacopoeia, without violating traceability and security, all at the same time as the
program is capturing data from the animals.

The system controls that rabbits under quarantine cannot be used in analysis. The encrypted database has a phenomenal strength and keeps you safe and clear with every step you take.

 

 

The PyroMon software, in conjunction with Ellab equipment, offers you a complete system for Pyrogens test according to all the international pharmacopias demand.

Directives
IP 54 (International Protection Rating)
Ellab hardware has been tested to meet the demand to IP54 according to standard no. 60529.

EMC Directive
Ellab’s equipment has been tested to meet the European Electromagnetic Compatibility Directive (EMC Directive, 89/336/EEC). The Declaration of Conformity for your instrument lists the specific standards to which the unit was tested.

Low Voltage Directive (Safety)
In order to comply with the European Low Voltage Directive (73/23/EEC), Ellab equipment has been designed to meet the EN 50081-1, 50082-2 and the EN 50082-1 standards.

Number of Channels

The temperature module has a capacity of simultaneously monitor 16 temperatures ( rabbits). It is
manufactured of stainless steel to withstand a rugged industrial environment. The PyroMon™ system has a capacity of handling up to 5 TM 9616 temperature modules at the same time giving a total of 80 temperature channels ( rabbits). The user can group the animals ( 8 groups) and perform the tests separately as per different criteria.

You can mount the modules at different animal rooms and keep the central monitoring system at the main room to monitor and test upto 80 rabbits simultaneously including quarantine, sham test and injection tests.

Slipping Probe Alarm

If during test a probe slips out of an animal then the system will detect the error and respond by sending an error message to the Monitor, Alarm and Computer. The Monitor display will flash with slipping probe alarm information of channel and animal number. If action is not taken the rabbit will be will be aborted from the test after 30 minutes.

This guarantee that all the tests are performed as per the required parameters and conditions and no false results.

 

Sham Animals List

The Sham is training method for the rabbit; normally you need to trainings the rabbit before real
used. Working with the European pharmacopoeia it is needed to Re-Sham the rabbits that have been more than 14 days without performing tests. For this use the PyroMon system can generate a list of these specific animals so they can be trained without loss of time if they were forgotten.

Monitor Module :-

The monitor module can handle all the information of temperatures, dose, channel, test no., pin code, etc. in the network. It operates as an interactive interface to the system and serves the purpose of securing the system from illegal entry, starts and stops tests, deleting rabbits & tests, temperature monitor and alarm monitor.

The PyroMon™ system has a capacity of handling several UI128 modules at the same time. It is
manufactured of stainless steel to withstand a rugged industrial environment.

Connection Module

The picture shows the connection of 16 sensors to the TM 9616 module.

 

Graphic Presentation 

 

 

 

 

 

Master login

The PyroMon system have two login types. Master and Normal User. This feature allows the main user to protect the file from modifying or theft by others. The master user can provide limited access to the other users.

Audit Trail

Ellab defines an audit trail as “a record showing who has accessed a computer system and what
operations he or she has performed of critical change during a given period of time.”

In the PyroMon system we have two Audit trail. One Event audit trail will tell you about the
communication between boxes: Login user, Slipping probe, Record, and Injection start / stop
etc. We also have audit trail there mark information we due in database, users handling, animals
handling, methods handling, runs sheet handling etc.

The PyroMon software meets the guidelines for electronic signature as specified in FDA Regulation 21 CFR part 11, Electronic Records i.e. Electronic Signature.

Pharmacopoeias

This PyroMon™ system is designed to handle every possible pharmacopoeia existing today. Further on the system gives the possibility to program user defined Pharmacopoeia to suit special requirements. Since there are many possible combinations in the Method Definitions we recommend the users to start “learning” from the Pharmacopoeia already in the system, and from they’re on own programming.

The Pharmacopoeias already available in the system are EP-IV, JP-XIV, KP-VII, MRBP and USP-29NF24 etc. The system is capable of operating on any given Pharmacopoeia platform.

.Animal Database

Before starting to run test with the system needs basic information to the PyroMon Database. Among these information’s are Tag Number (Animal ID), Room Number (Cage), Sex and Weight. If a animal is used for other tests this might be indicated by adding a number to there “Room Number”. When a
rabbit dies or for any reason is wanted out it is possible to inactivate the animal. All inactivated animals can be printed out in a special list. When animals are created or shamed it is possibly to print an animal sham and new animal lists.

The database remember weight date, weight (last and current), weight change, arrival date, sham expire date, quarantine expire date, marker(s) (exclude), start date, stop date, last test date and last verdict.

Multi User
It is possible for more than one user to use each Monitor. Example: First test is selected and started by operator no.1. Operator no.2 would like to inject next preparation group. Operator no.2 presses Menu button on the Monitor. The content of the display changes to test selection. Operator no.2 can now select the wanted test on normal terms. Operator no.1 can anytime go back to the first test by follow the same procedure as operator no.2 and finish the injection.

Repeat Preparations / failed result (high temperature)

This procedure has been developed for enabling the user to perform a repeat test according to the defined demand in the test. The repeat results will be shown on the standard result formula. However the previous test will be included as well. This gives the operator a very easy and safe documentation. The validation and interpretation of the result will be given automatically

Simulate Pharmacopeias

The European Pharmacopeias has the most stringent norms. So if the user fails in EU, then he could use the same data to check the results of other Pharmacopeias without repeating the tests. This saves tremendous amount of time for performing repeat test of the same drug for different buyers as per their demand of meeting different pharmacopeias.

PyroMon Software and Reports

• Complies with all basic requirement in the Pharmacopoeias
• In the software, it is possible to change important requirements to accommodate local regulations
• The software covers these areas

1. Calibration
2. Methods
3. Preparations
4. Test run (Pre-set / injection / main test )
5. Simulation of methods
6. Database handling animals ( new / inactive)

Calibration Data – tabular form

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

The above data shows how the 16 sensors are calibrated as per international norms. The probes are kept in the high accurate water bath along with the master sensor and calibrated at time interval for stability, time for sham test, actual test etc.

Methods Overview – the parameters set for Sham test and real test as per the SOP.

PyroMon Special Features

• 80 channels capacity

• Online graphic and data

• Keep full database on rabbits and test results

• Capacity of doing several Pharmacopoeias simultaneously

• Electronic Signature

• Automatic control of rabbits used for protein

• User configurable Pharmacopoeia

• User configurable print out interval for temperature data

• Possibility of simulate runs under other Pharmacopoeia. Eg. EUR=USP

• Keep track of animals the need to be re-tested

Conclusion :

The PyroMon software, in conjunction with Ellab equipment, offers you a complete system for Pyrogens test according to all the international pharmacopias demand.

ELLAB offers the most accurate and reliable Pyrogen Testing System which is fully complaint to FDA 21 CFR Part 11 and are designed to meet the safe pyrogen testing analysis taking care of all the important critical parameters.

We offer the onsite installation and commissioning of the system with operational training as well as process training The customers can also avail the factory training at Ellab Denmark if necessary. We have application specialists on Automatic Pyrogen Testing Analysis who offer you onlne support through internet as well as onsite application support.

Please feel free to contact us for any further assistance on the mentioned subject. You can contact me on my mobile number +91 9447007056 or through email : josekthomas@hotmail.com.

Kindly visit Ellab web site www;ellab.com for more details about the mentioned system.