Pyrogen Testing, NIRAV

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Pyrogen Testing Presented By : MODI NIRAV M.Pharm-I(Q.A.) 1

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Introduction Classification of Endotoxins Properties of Bacterial Endotoxins Biological Properties Physical Properties Pyrogen Testing Rabbit test (sham test) LaL Test (Bacterial Endotoxin Test) Depyrogenation Techniques Physically removal of Pyrogens Inactivation of Pyrogens Content: 2


Introduction A Pyrogen is defined as “a fever producing agent” Pyrogens are products of metabolism of micro organisms. Most bacteria ,viruses & many moulds have been reported as pyrogen producing. Pyrogen are substances that initiate febrile reactions when sufficient numbers enter the circulatory system, therefore, the history of intravenous therapy and the history of pyrogen research are closely related. 3

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Endotoxins are part of the outer membrane of the cell wall of Gram-negative bacteria. Endotoxin is invariably associated with Gram-negative bacteria whether the organisms are pathogenic or not. Although the term "endotoxin" is occasionally used to refer to any cell-associated bacterial toxin, 4

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The scientific literature divides Pyrogen in two broad classes. Exogenous Pyrogen ( Exotoxins) Endogenous Pyrogen (Endotoxins) 5

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Non microbial Pyrogens Microbial Pyrogens Host origin Non host origin Exogenous Pyrogen ( Exotoxins) Most of non microbial pyrogen forms produce fever by inducing the synthesis and release of endogenous pyrogen from host phagocytic cells. Non microbial exogenous pyrogens of nonhost origin include pharmacologic agent like bleomycin, colchicin and polynucleotide poly- 1: C, Non microbial exogenous pyrogens of host origin include Antigen-Antibody complexes, products of lymphocytes and metabolic Products of androgenic steroids Bacteria (killed or live), Fungi (killed or live), plasmodia (malarial parasites),and viruses(live) can all act as pyrogens, additionally bacterial products, including streptococcal endotoxin, staphylococcal endotoxins. Microbial exogenous pyrogens are much more of a problem for pharmaceutical manufacturers; the lipopolysaccharide in the endotoxin is toxic in small Quantities. 6

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Exogenous pyrogen don't act directly on the thermoregulatory center of the hypothalamus of the brain to cause fever, Instead exogenous pyrogens stimulates the production of intermediate heat-labile, Protienacious, mediator substances,Since this mediator substance was first isolated from granulocytes, It was initially called granulocytic pyrogen or sometimes leukocytic pyrogen,After the mediator substance was found in the circulatory serum of febrile rabbits it was called endogenous pyrogen Endogenous pyrogen are produced by blood monocytes, Eiosinophils,alveolar macrophages and fixed- tissue macrophages such as splenic sinusoidal cells and hepatic kupffer cells, When injected in to animals, It also induced hepatic acute-phase protein synthesis,caused decrease in plasma iron and zinc levels produced neutrophilia, stimulated serum amyloid, a protein synthesis and augmented T cells response to mitogen and antigens. Endogenous Pyrogens 7

Chemical nature of Pyrogen: 

Chemical nature of Pyrogen Endotoxin has a polysaccharide structure that is covalently bound to a lipid component, called lipid A The polysaccharide component is composed of two distinct parts, the core oligosaccharide that is connected to lipid A and a longer oligosaccharide O-specific chain that is attached to the core and is the outer most part of the LPS Complex. The O-specific chain is the most variable component of the complex. 8

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It consist of 20-40 repeating units that include up to the eight sugars, and it is responsible for the specific immune reaction that each type of gram- negative bacteria is able to evoke in a host , The core oligosaccharide is much less variable than O-specific chain and its influence on the host is less profound, 9

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The lipid A is the least variable components of LPS. It consists of a disaccharides of glucosamine which is the highly substituted with amide- linked and structure-linked long chain fatty acids, Lipid A is the most interesting portion of LPS because it is responsible for the vast majority of the harmful and the beneficial biological activities that have been attributed to endotoxins 10


BIOLOGICAL PROPERTIES OF ENDOTOXINS : Endotoxins are potent substances which elicit a broad spectrum of the harmful physiologic responses, Profound alteration in organ function, such as hypotension and disseminated intravascular coagulation, which can lead to severe morbidity or death But on the other hand endotoxins are also active stimulators of the mammalian defense system, This capacity can enhance the body’s capacity to cope with both microbial infections and malignant tumors. 11

Physical properties of endotoxins : 

Physical properties of endotoxins It is important to be aware of the various physical properties of endotoxin in order to understand why they change their behavior when placed into different environments, An understanding of these physical properties is also essentials for designing effective process for depyrogenation . Heat stability Size Molecular weight Electrostatic properties 12



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The development of the large volume parental drug industry during the decade prior to world war II prompted the need for the Pyrogen testing. Pyrogen contamination is greater Problem for manufacturer of small volume parental because the initiation of patient fever by parental solution is dose depedent rather than concentration dependent. The onset & extent of injection fever depends on the total amount of pyrogen delivered to a patient 7 not on the concentration of pyrogen per milliliter of drug . 14

Pyrogen Testing : 

Pyrogen Testing Rabbit test (sham test) LAL Test (Bacterial Endotoxin Test) 15

Rabbit test (Sham test) : 

Rabbit test (Sham test) Selection of animal: Animal’s Quarters Equipment Preliminary test: Record the temperatures of the animals, beginning at least 90 minutes before injection and continuing for 3 hours after injection of the solution being examined. Any animal showing a temperature variation of 0.6 o C or more must not be used in the main test. 16

Main Test: 

Main Test Record the temperature of each animal at intervals of not more than 30 minutes, beginning at least 90 minutes before the injection of the solution being examined and continuing for 3 hours after the injection. Not more than 40 minutes immediately preceding the injection of the test dose, Record the "initial temperature " of each rabbit, which is the mean of two temperatures recorded for that rabbit at an interval of 30 minutes in the 40 minute period. Rabbits showing a temperature variation greater than 0.2 o C between two successive readings in the determination of "initial temperature " should not be used for the test. 17

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In any one group of test animals, use only those animals whose " initial temperatures" do not vary by more than 1 0 from each other ,and do not use any rabbit having a temperature higher than 39.8 o C and lower than 38 o C . Inject the solution being examined slowly into the marginal vein of the ear of each rabbit over a period not exceeding 4 minutes, unless otherwise prescribed in the monograph. The amount of sample to be injected varies according to the preparation being examined and is prescribed in the individual monograph. 18

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The volume of injection is not less than 0.5 ml per kg and not more than 10 ml per kg of body weight. Records the temperature of each animal at half-hourly intervals for 3 hours after the injection. The difference between the "initial temperature" and the "maximum temperature" which is the highest temperature recorded for a rabbit is taken to be its response. When this difference is negative, the results is counted as a zero response. 19

Interpretation of results: 

Interpretation of results If the responses of the group of three rabbits in which individual response is less than 0.6 o C , the preparation being examined passes the test. If the response of any rabbit is 0.6 o C or more, or if the sum of the response of the three rabbits exceeds 1.4 o C continue the test using five other rabbits. If not more than three of the eight rabbits show individual responses of 0.6 o C or more, and if the sum of responses of the group of eight rabbits does not exceed 3.7 o C , the preparation being examined passes the test. 20


LAL TEST( BACTERIAL ENDOTOXINS TEST ) LAL test = Limulus Amebocyte Lysate Test Test also referred by USP, IP, BP as BET test : Bacterial Endotoxin Test 21


Principle The addition of a solution containing endotoxins to a solution of the lysate produces turbidity, precipitation or gelation of the mixture. The rate of reaction depends on the concentration of endotoxin, the pH and the temperature. The reaction requires the presence of certain bivalent cations, a proclotting enzyme system and clottable protein all of which are provided by the lysate. 22


Methods The following six methods are described in the I.P. 2007 and B.P. 2007 Method A: Gel clot method: Limit test Method B: Gel clot method: Semi quantitative test. Method C: Turbidimetric Kinetic method Method D: Chromogenic kinetic method Method E: Chromogenic end- point method Method F: Turbidimetric end point method. Proceed by any of the six methods for the test. In the event of doubt or Dispute, the final decision is made based upon Method A , Unless otherwise indicated in the monograph: 23

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The Endotoxin limit for a given test material of preparation is calculated from the expression = K/M K is the maximum number of Units of endotoxin which the patient may receive without suffering toxic reactions M is the maximum dose administered to a patient per kg per hour. The value of K is 5.0 EU/kg for injectable preparations except for those administered intrathecally and is 0.2 EU/kg for intrathecal preparations. 24

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Before carrying out the test for endotoxins on the preparation being examined, it is necessary to verify (a) The sensitivity of the lysate; (b) The absence of interfering factors in the sample preparation which inhibit or enhance the reaction of otherwise interfere with the test; (c) The adequacy of the containers to resist absorbance of endotoxins. 25

Sensitivity of the lysate : 

Sensitivity of the lysate Confirm the labeled sensitivity using at least one vial of each batch of lysate. Prepare a series of two-fold dilutions of the CSE to give concentrations of 2 λ , λ , 0.5 λ and 0.25 λ , where λ is the labeled sensitivity of the lysate in EU per ml. Perform the test as given under Procedure on these four standard concentrations in duplicate or greater and include negative control consisting of water BET. 26

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Calculate the average of the logarithms of the lowest concentration of endotoxin in each series of dilutions for which a positive result is found. The antilogarithm of this average gives the estimated lysate sensitivity, which must be greater than or equal to 0.5 λ and less than or equal to 2.0 λ . Confirm the labeled sensitivity of each new batch of lysate prior to use in the test. 27

Test for interfering factors : 

Test for interfering factors For validation of the test results it must be demonstrated that the sample preparation does not inhibit or enhance the reaction or otherwise interfere with the test. The validation must be repeated if the lysate vendor or the method of manufacture or formulation of the sample is changed. Most preparation interferes with the bacterial endotoxin test. Dilution of the preparation with water BET is the easiest method for overcoming inhibition. 28

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The allowable dilution level or Maximum Valid Dilution (MVD) is dependent on the potency (concentration) of the product, the endotoxin limit for the product and the lysate sensitivity. Perform the test as described under Sensitivity of the lysate on aliquots of the sample preparation at the non-interfering dilution, i.e. the dilution that neither inhibits nor enhances the bacterial endotoxins test or at a dilution not exceeding the MVD which is calculated from the following expressions. 29

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When the endotoxin limit in the substance or preparation being examined is specified in terms of weight or units of active drug. 30

Preparation of test solutions (Preparatory testing) : 

Preparation of test solutions (Preparatory testing) Prepare the sample solution at any dilution between the non-interfering dilution and the MVD after treating, if necessary, to eliminate interfering factors (negative product control). Use as negative control water BET (blank) and two positive controls. One of the positive controls consists of the CSE at a concentration of 2 λ (positive water control) and the other consists of the sample solution at the same dilution as the negative product to which CSE has been added to give a concentration of 2 λ (positive product control ). 31

Procedure : 

Procedure Carry out the test of negative control, standard endotoxin concentrations or positive water control, test solution and positive product control. Add to each receptacle an equal volume of the appropriately constituted lysate unless single test vials are used. Mix the sample/lysate mixture gently and place in an incubating device such as a water-bath or a heating block, accurately recording the times at which the receptacles are so placed. Incubate each receptacles at 37 o C , undistributed and avoiding loss of water by evaporation, for 60 minutes. 32

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A positive reaction is characterised by the formation of a firm gel that retains its integrity when inverted through 180 o . Record this result as positive (+). A negative result is characterised by the absence of such a gel or by the formation of a viscous gel that does not maintain its integrity. Record such a result as negative (-). 33

Interpretation of results : 

Interpretation of results The substance or preparation being examined complies with bacterial endotoxins test if the positive product control is positive and the negative controls are negative. The test is invalid if the positive product control is negative or if any negative control is positive or the endotoxin standard does not show the end-point concentration. 34

Gel – Clot technique (Methods A and B) : 

Gel – Clot technique (Methods A and B) The gel-clot techniques allows detection or quantification of endotoxins and is based on clotting of the lysate in the presence to clot under the standard conditions is the labeled lysate sensitivity. To ensure both the precision and validity of the test confirm the labeled lysate sensitivity and perform the test for interfering factors as described at above. 35

Limit test (method-1): 

Limit test (method-1) Prepare solutions A,B,C, and D as show in following table, and perform the test on these solutions following the procedure described in (i) Preparatory study (ii) Confirmation of the labeled lysate sensitivity Prepare solution A and Solution B (Positive Product control) using a dilution not greater than the MVD and treatments as 1.Preparatory test 2. Test for interfering Factors. 36

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Solution B and C (positive control)contain the standard endotoxin at a concentration corresponding to twice the labeled lysate sensitivity. Solution D (negative control) consists of water for BET (water LAL). 37

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Solution Endotoxin conc./solution to which endotoxin is added Diluent Dilution factor Initial endotoxin concentration No. of replicant A None/Test solution - - - 4 B 2λ / Test solution Test sol. 1 2 λ 4 2 1 λ 4 4 0.5 λ 4 8 0.25 λ 4 C 2 λ/water for BET Water for BET 1 2 λ 2 2 1 λ 2 4 0.5 λ 2 8 0.25 λ 2 D None/Water for BET - - - 2 38

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39 Solution Endotoxin conc./solution to which endotoxin is added No. of Replicates A None/Test solution 2 B 2λ / Test solution 2 C 2 λ/water for BET 2 D None/Water for BET 2


Interpretation The test is not valid unless both replicates of the two positive control solution B and C are positive and those of the negative control solution D are negative. The preparation being examined complies with the test when a negative results is found for replicates of solution A When a positive results is found for both replicates of solution A 40

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If the preparation being examined is diluted to the MVD, it does not comply with the test If the preparation being examined is diluted to a dilution less than the MVD, the test is repeated at a dilution not greater than MVD Repeat the test if a positive results is found for one replicates of the solution A and a negative results is found for the other. The preparation being examined complies with the test if negative results is found for both replicates of solution A in the repeat test. 41

Semi- quantitative test (Method B) : 

Semi- quantitative test (Method B) The test quantifies bacterial endotoxins in the test solution by titration to an end- point, prepare solutions A,B,C and D as shown in following table 3, 42

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43 Solution Endotoxin conc./solution to which endotoxin is added Diluent Dilution factor Initial endotoxin conc. No. of replicant A None/Test solution Water for BET 1 2 λ 2 2 1λ 2 4 0.5 λ 2 8 0.25 λ 2 B 2λ / Test solution - 1 2 λ 2 C 2 λ/water for BET Water for BET 1 2 λ 2 2 1 λ 2 4 0.5 λ 2 8 0.25 λ 2 D None/Water for BET - - - 2

Calculation and Interpretation: 

Calculation and Interpretation The test is not valid unless the following three conditions are met: Both replicates of solution D(negative control) are negative. Both replicates of solution B(positive control) are positive The geometric mean end-point concentration of solution C is in the range of 0.5 λ to 2 λ. To determine the endotoxin concentration of solution A. calculate the end-point concentration for each replicates series of dilutions by multiplying each end-point dilution factor by λ 44

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The endotoxin concentration in the test solution is the geometric mean end point concentration of the replicates, if the test is conducted with a diluted test solution calculate the concentration of endotoxin in the original solution by multiplying the results by dilution factor. If none of the dilution of the test solution is positive in a valid test, record endotoxin concentration as less than λ (or if a diluted sample was tested, as less than λ× the lowest dilution factor of the sample) 45

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If all dilutions are positive, the endotoxin concentration is recorded as equal to or greater than the greatest dilution factor multiplied by λ (e.g. in table , the initial dilution factor ×8× λ. The preparation meets the requirements of the test if the endotoxin concentration is less than that specified in the individual monograph. 46

  Photometric techniques (Methods C, D, E and F) : 

Photometric techniques (Methods C, D, E and F) 1. Turbidimetric technique (Method C and F) This technique is a photometric test to measure the increase in turbidity, Based on the test principle employed, this technique is classified as being the end –point turnidimetric test or the kinetic- turbidimetric test. 47


CHROMOGENIC TECHNIQUES (METHODS D &E) This techniques is used to measure the chromophore released from a suitable chromogenic peptide by the reaction of endotoxins with the lysate.Depending on the principle employed , this technique is classified as being the end- point chromogenic test or the kinetic- chromogenic test. 48

The FDA LAL test validation guideline : 

The FDA LAL test validation guideline As the use of LAL test became more & more prevalent, the FDA desided that single standardize document was needed to govern all FDA regulated products that were subject to LAL testing, An FDA task force was formed by representatives from Center for drug evolution & Research. Center for biological evolution & Research. Center for devices & Radiologic health. Center for veterinary medicine. 49

Validating the LAL test : 

Validating the LAL test Three basic requirements The LAL reagent used in cell validation in process and end product LAL tests must be licensed by CBER The product manufacturer must perform an initial Qualification of their LAL test laboratory PERSONNEL Inhibition & enhancement tests must be performed on test products do not interfere with the enzymatic LAL endotoxin reaction. 50

LAL test inhibition and enhancement : 

LAL test inhibition and enhancement Inhibition Occurs when the test recovers less endotoxin than is spiked into solution Causes: Chemical nature of the drug product or excipients Factors that negatively effect serine protease enzyme reaction such as High or low pH Oxidants or antioxidants Proteolytic agents High heat Chelating agents Inadequately dispersed purified endotoxin Adsorption to the side of containers 51

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Enhancement: Occurs if more Endotoxins recovered than expected Causes: Chemical nature of the product Endotoxin contamination present in product Surfactants by increasing surface area of endotoxin. 52

Application of LAL test : 

Application of LAL test Pharmaceuticals: In Parenteral dosage form Large volume Parenterals Small volume Parenterals Biologicals In blood products & plasma fractions Vaccines Medical Device Nebulizers used in Respiratory therapy Diagnosis of disease caused by Gram –negative bacteria In food & drinking water Others: For validation of dry heat sterilization 53

Depyrogenation : 

Depyrogenation Removal of Pyrogens by physical Methods Dilution Ultra-filtration Reverse osmosis Distillation Adsorption on Charcoal Column Chromatography Charge Modified Media & Electrostatic Attraction Hydrophobic attraction to hydrophobic medium 54

(B) Inactivation of Pyrogens : 

(B) Inactivation of Pyrogens Dry heat sterilization Moist heat sterilization Use of dilute acids & Bases Oxidation Alkylation 55


Conclusion This Seminar conclude that Pyrogen testing from the rabbit test has some advantages like It detects all kinds of Injectable Pyrogen unlike LAL test Demonstrate the production of fever in human. But it is time consuming ,expensive procedure that require large capital investment, It can not be used to test of certain drugs that depress the fever. 56

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While LAL test is more advantageous than rabbits pyrogen test, it is more sensitive, less variable, easier to perform, can give quantitative results, less time consuming , less expensive In vitro test. 57

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But it has also some limitation like It is specific for gram negative pyrogens only, Clotting enzyme is heat labile, pH sensitive and possible interference problems. 58

Reference: : 

Reference: Encyclopedia of Pharmaceutical Technology, Volume:13,By James Swarbick & James Boylan. Indian Pharmacopoeia 2007,Vol.-1,Appendice-, Page no. British Pharmacopoeia 2007, United state pharmacopoeia Remington( The science and practice of pharmacy), Volume-I , Page no.562,832. The text book of pharmaceutical Technology, By Leon Lachman,Page no. 59

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Thank you 60