Gibraltar Labs Inc. is an ISO 17025 certified testing laboratory to perform Limulus Amebocyte Lysate (LAL) Test.  LAL, also known as Bacterial Endotoxin test, is a highly sensitive test used to detect or quantify bacterial endotoxins. Bacterial endotoxins, also known as pyrogens, are fever causing byproducts of gram negative bacteria. It is performed as an end-product endotoxin test for human and animal injectable drugs, biological products, and medical devices.

History: Rabbit pyrogen test was the first test used to detect endotoxins. LAL test officially replaced the rabbit pyrogen test due to its greater sensitivity because in rabbit pyrogen test, endotoxin amount wasn’t quantifiable. Amebocyte lysate from the horseshoe crab (Limulus poylphemus) was discovered to be the agent responsible for the blood clot in the amebocytes of the crab when came in contact with endotoxins. Amebocyte extract is now commercially prepared to be used as the major reagent used to detect and measure endotoxins. LAL test is a valuable test because it prevents the administration and or use of products and drugs which may cause fever, shock and death if highly endotoxic.

There are two methods of LAL endotoxin testing performed daily by Gibraltar’s highly experienced technicians. Gel-Clot method is based on the formation of clotting of the lysate in the presence of bacterial endotoxins in the test material. Kinetic Chromogenic method utilizes a microplate reader to automatically detect the amount of endotoxin concentration present in the sample when tested against a known amount of standards. Sensitivity of each test is based on the manufacturer’s label claim of the Lysate. Each new lot of reagents, a confirmation of label claim is performed by our certified technicians before a test can be considered valid and be performed on a routine basis. Gel-Clot has 0.03 EU/mL labeled lysate sensitivity and Kinetic Chromogenic has a sensitivity of 0.005 EU/mL.

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 and oldest form of pyrogen testing consists of injecting drugs into rabbits to determine if a fever develops. A newer test uses blood from the horseshoe crab to test for toxins.

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.

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.

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Pyrogen testing in drug labs involves heating equipment used to ensure sterilization. The drug is injected into ear veins of rabbits to see if a fever develops. Rectal temperatures of test animals are analyzed after 30 minutes and again one to three hours later. If the animals remain fever-free, the solution is free from toxins.

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.

A newer pyrogen testing technique is called the limulus amoebocyte lysate (LAL) test. Blood from horseshoe crabs contains high levels of toxins naturally found in marine life. Scientists discovered a way to use the blood to test for bacterial toxin in drugs and the raw materials used to manufacture medicine. The LAL pyrogen testing procedure might be 100 times more sensitive than the rabbit testing methods. Medical devices that are implanted into humans go through LAL testing, along with radioactive drugs and anesthesia.

Rabbit Pyrogen / General Safety Test A pyrogen is a foreign substance that causes a fever (temperature elevation) in an animal’s body. Typically, pyrogenic substances include endotoxin and other bacterial byproducts. Vaccines and other injectable drugs must be confirmed to be pyrogen free according to regulatory requirements of 21CFR, USP, and EP.

The typical assay for endotoxin contamination detection is the LAL test. In some cases, the LAL test may not be feasible. These cases might include:

Interference by the test article The presence of a compound capable of neutralizing endotoxins The presence of pyrogenic substances other than endotoxin, Certain regulatory requirements

BioReliance offers the rabbit pyrogen test, in addition to the LAL test, as an alternative assay for the detection of endotoxin and other pyrogens. The rabbit pyrogen test requires the injection of a small amount of batched test material into a rabbit’s blood stream, and monitoring for temperature increases.

Contact our experienced scientific staff to learn about more about rabbit pyrogen testing.

BioReliance also offers a general safety test to detect extraneous toxic contaminants.

Contact Us Phone Toll Free 1.800.756.5658 US - Local 1.301.738.1000 Email info@bioreliance.com Request Information General Inquiries

Analyst qualification: Each one of our technicians who are certified to perform endotoxin testing also perform a proficiency test yearly to show competence in LAL Gel-Clot and/or Kinetic Chormogenic Test.

There are many other  qualifications, calibrations and proficiencies that we routinely perform and document so as to be certified to offer this critical service to you our customers.

All methods used in Gibraltar Laboratories follow Current USP <85> and <161>, EP, JP, ANSI/AAMI ST72 and FDA guidelines and are performed based on Standard Operating Procedures under GMP/GLP environment.

Endotoxin Testing (LAL) Endotoxin testing (LAL test) ensures injectable therapeutics are safe for human use.

Bacterial endotoxins are lipopolysaccharides (LPS), components of Gram-negative bacterial cell walls known to cause fevers and disease when injected into the bloodstream. Bacterial endotoxins are heat stable and toxicity is not dependent on the presence of the bacterial cell.

Since many therapeutics are made in bacteria, endotoxin testing is employed to ensure a therapeutic product is endotoxin-free. BioReliance tests for endotoxin in several areas of drug manufacturing and production including:

Bulk Lot Release Testing Final Product Release Testing Raw Materials Testing

BioReliance research professionals have performed thousands of endotoxin testing assays for clients in Biopharma. We can provide a fast turnaround time and the highest quality testing to produce therapeutics that are bacterial endotoxin-free and safe for use in humans.

To see our endotoxin testing assays, use the search tool above.

How is endotoxin testing performed (the LAL test)?

The most common approach to endotoxin testing is the limulous amoebocyte lysate test (LAL test). This assay is based in the biology of the horseshoe crab (Limulous). These animals produce LAL enzymes in blood cells (amoebocytes) to bind and inactivate endotoxin from invading bacteria.

LAL serves as a primitive immune system. Inactivation of endotoxin also forms a clot, which can further protect the horseshoe crab from infection. The LAL test

exploits the action of this enzyme, by adding LAL reagent to the tested product, and assaying for clot formation.

BioReliance offers LAL - endotoxin tests with a variety of assay options including:

Quantitative and qualitative testing Gel-clot method (LAL test) Chromogenic methods (USP Chromogenic)

The LAL test was accepted by the FDA for endotoxin testing of pharmaceuticals and medical devices in 1983. USP requires pooled testing of a production lot for the presence of bacterial endotoxin.

To find the right endotoxin testing assay, use the search tool to the right or click here.

Endotoxin testing - the gel clot method vs. the chromogenic assay.

Both the gel clot method and the chromogenic method of endotoxin testing are appropriate for preclinical, Phase 1, Phase 2, Phase 3, and Licensed Product phases of therapeutic production and distribution.

Either test is approved for endotoxin testing of multiple therapeutic types including cell therapy, recombinant protein, monoclonal antibody, vaccines and gene therapy. The choice comes down to whether you need quantitative results or simply to detect endotoxin in your drug.

The gel clot assay with the LAL test is simplest method for the detection of bacterial endotoxins and the GMP format is typically used for lot release of final product intended for injection in humans.

To perform the assay, equal volumes of LAL reagents are mixed with undiluted or diluted test article and observed for clot formation. The dilutions are selected to cover the potential range of endotoxin in the sample and to reduce interference by the test material making the gel clot LAL test semi-quantitative. The levels of endotoxin allowed are strictly regulated and depend on the route and frequency of administration. The sensitivity of this assay is 0.06 EU/ml.

The USP chromogenic method is based on the activation of a serine protease (coagulase) by the endotoxin, which is the rate-limiting step of the clotting cascade. The assay measures the activation of the serine protease as opposed to the end result of this activation, which is clotting. The natural substrate, coagulogen, is replaced by a chromogenic substrate. On cleavage of this substrate a chromophore is released from the chromogenic peptide and is measured by spectrophotometry.

The USP chromogenic method is quantitative and can provide a greater sensitivity over a wider range. The sensitivity of this assay is 0.10 EU/ml.

BioReliance CRO is an expert in both types of endotoxin tests and can provide you with advice on the best assay for your drug study or lot release needs.

Pyrogenic/pro-inflammatory activities of medicines and vaccinesPyrogens are fever-causing agents which contaminate medicines and vaccines as a consequence of the manufacturing process.

Pyrogen-contaminated parenteral preparations can cause mild to severe clinical reactions including fever, rash, headache, myalgia, nausea, vomiting and hypotension. It is therefore vital that the level of pyrogenic contamination of parenteral preparations is carefully controlled to ensure their safety.

The most common pyrogen is endotoxin which is shed from the cell wall of Gram-negative bacteria. Medicines and vaccines produced by a process involving such bacteria are therefore at high risk of being contaminated with endotoxin.

The amount of endotoxin in a product can be quantified using one of the various methods of the bacterial endotoxins test (BET). In line with the 3Rs principle – the replacement, reduction and refinement of animal testing, the BET was a replacement for the rabbit pyrogen test (RPT). However, unlike the RPT, the BET does not detect non-endotoxin pyrogens or other pro-inflammatory contaminants of parenteral preparations which can stimulate unwanted clinical reactions.

For this reason a third test – the monocyte activation test (MAT) – was developed as a replacement for the RPT for quantifying pro-inflammatory/non-endotoxin pyrogenic contaminants of medicines and vaccines.

All three tests are described in the British and European Pharmacopoeias.

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

PowerPoint Presentation: 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: 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

PowerPoint Presentation: 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

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

PowerPoint Presentation: 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

PowerPoint Presentation: 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

PowerPoint Presentation: 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

PowerPoint Presentation: 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:: 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

NEED OF PYROGEN TESTING ????: NEED OF PYROGEN TESTING ???? 13

PowerPoint Presentation: 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

PowerPoint Presentation: 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

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

Principle: 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: 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

PowerPoint Presentation: 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

PowerPoint Presentation: 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

PowerPoint Presentation: 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

PowerPoint Presentation: 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

PowerPoint Presentation: 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

PowerPoint Presentation: 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

PowerPoint Presentation: 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

PowerPoint Presentation: 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

PowerPoint Presentation: 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: 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

PowerPoint Presentation: 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

PowerPoint Presentation: 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

PowerPoint Presentation: 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

PowerPoint Presentation: 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): 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

PowerPoint Presentation: 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: 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

PowerPoint Presentation: 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

PowerPoint Presentation: 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

PYROGEN & PYROGEN TESTING: PYROGEN & PYROGEN TESTING Subject: Biological evaluation & clinical research Prepared by: Swati Chauhan M.pharm-1(QA) ccprvbt

PYROGENS: PYROGENS Pyrogens are the agents which are responsible for increase in body temperature when injected into the body. Most common pyrogens which are of

significant in pharmaceutical industry are Gram-ve bacterial endotoxin. BACTERIAL ENDOTOXIN PYROGEN : Endotoxin are part of the outer membrane of the cell wall of gram –ve bacteria. These bacteria constantly shed endotoxin into environment as they grow & multiply ,as well as when they die & disintegrate. Bact. Endotoxin can either exist in a cell-associated state or in a free state. Heat stable

Chemical nature of endotoxin: Chemical nature of endotoxin The biologial activity of endotoxin is associated with the lipopolysaccharide (LPS) LIPOPOLYSACCHARIDE LIPID A POLYSACCHARIDE O-SPECIFIC CHAIN CORE OLIGOSACCHARIDE

Cont..: Cont.. LPS are complex amphiphilic molecule. Toxicity is associated with the lipid component (lipid A) Immunogenicity is associated with polysaccharide polysaccharide O-specific chain Outer core Inner core Lipid A

Biological properties of endotoxin: Biological properties of endotoxin Pyrogen elevate the circulatory levels of inflammatory cytokines which may be followed by fever, blood coagulation, hypotension Low doses of pyrogen: asymptomatic inflammation reaction Moderate doses: fever & changes in plasma composition High doses: cardiovascular dysfunction, vasodilation, vasoconstriction, endothelium dysfunction, multiple organ failure & finally death.

Sources of pyrogen: Sources of pyrogen Equipment Containers Solvent Solute glass plastic metal

Elimination of pyrogens: Elimination of pyrogens Dry heat sterilization : For glass wares, metal equipments, powders, waxes, oils, heat stable drugs 650 o c temp-1 min 250 o c temp-30 min 180 o c temp-240 min Ultra filtration Reverse osmosis : RO membrane is composed of cellulose acetate phthalate/ polyamide Distillation Adsorption method

PowerPoint Presentation: PYROGEN TEST

PYROGEN TEST: PYROGEN TEST Rabbits are used to perform this test because their body temp increases when pyrogen are introduced into their bodies by parenteral route 3 healthy adult rabbits of either sex, each weighing NLT 1.5 kg are selected Do not use any rabbit having a temp higher than 39.8 o c Showing temp variation >0.2 o c between two successive reading in the determination of initial temp Sham test is performed within 7 days of actual test Animal showing temp increase over 0.6 o c should be removed from pyrogen testing

Cont..: Cont.. Method : Dissolve the subs being examined in, or dilute it with a pyrogen free saline solution Warm the liquid being examined to approx. 38.5 o c temp before injection The volume of injection is NLT 0.5ml/kg & NMT 10ml/kg of body weight Withhold water during test Clinical thermometer is inserted into the rectum of rabbit to record body temp 2 normal reading of rectal temp are should be taken prior to the test injection at an interval of half an hr & its mean is calculated- initial temp

Cont..: Cont.. The solution under test is injected through an ear vein Record the temp of each rabbit in an interval of 30 min for 3 hrs The difference between initial temp & maximum temp is recorded- taken as response Interpretation of results if the sum of response of the group of 3 rabbits does not exceed 1.4 o c & if the response of individual rabbit is less than 0.6 o c, the prep being examined passes the test If the response of any rabbit is 0.6 o c or more / if the sum of response of 3 rabbits exceed 1.4 o c, continue the test using 5 other rabbits If NMT 3 rabbits show individual response of 0.6 o c or more, & if the sum of response of 8 rabbits does not exceed 3.7 o c, the prep being examined passes the test

BACTERIAL ENDOTOXIN TEST- LAL TEST: BACTERIAL ENDOTOXIN TEST- LAL TEST LAL-Limulus amoebocyte lysate It depends on gelling property of lysate of amoebocyte of limulus (horseshoe crab) In the presence of endotoxin , a firm gel is formed within 60 min, when incubated at 37 o c Procedure : The lysate is mixed with an equal volume of test solution in a depyrogenated container The tube is then incubated at 37 o c for 60 min The +ve reaction is indicated by formation of gel/clot LAL test needs +ve & -ve control For +ve control, a kn conc of endotoxin is added to lysate For –ve control, water free of endotoxin is added to lysate

Cont..: Cont.. B.P describes 6 separate methods for the test of endotoxin: Gel clot limit test Gel clot semi quantitative test Chromogenic kinetic method Chromogenic end point method Turbidometric kinetic method Turbidometric end point method Gel clot test : most commonly used method End point is determined by inversion of tubes. if a solid clot remains intact, the product is considered to contain endotoxin Chromogenic method : utilizes colourimetry. The colour produced is proportional to the amount of endotoxin

Cont..: Cont.. Turbidometric method : It is based on the fact that increase in endotoxin conc. Will cause increase in turbidity. The optical density is read spectrophotometrically either at fixed time or constantly for kinetic assay as turbidity develops

PYROGENS: PYROGENS PRESENTED BY YAJNESH PAI 1 st YR M.PHARM QA DEPT. 1

What are PYROGENS: What are PYROGENS Pyrogens are fever producing substances, which are metabolic products of microorganisms. Most potent pyrogens are the endotoxins produced from the cell walls of the Gram- negative bacteria. Pyrogens have a high molecular weight, often, more than 1,000,000. 2

History of PYROGENS: History of PYROGENS In 1894 Centanni showed that pyrogens were not proteinous and that they were not susceptible to heat. In 1912 Hort and Penfold started the Rabbit Assay and standardized it for fever. By correlating the fever effect in rabbits with the extent of pyrogens, and by characterizing the bacteria by staining as Gram positive and Gram negative, Hort and Penfold could prove that the pyrogenic bacteria were predominantly Gram negative whereas the Gram positive were mostly apyrogenic. Then they could correlate between the bacterial count in the injected distilled water and the extent of fever caused and also prove that dead bacteria also caused fever . 3

Nature of PYROGENS: Nature of PYROGENS A pyrogen is a fever producing substance. The most important pyrogen is the endotoxin produced by the Gram negative bacteria. Some pyrogens are not of microbial origin. They are in fact pharmacological agents, such as bleomycin and colchicines . Many organisms including live and dead bacteria, fungi, viruses, malarial parasites, and the bacterial products such as streptococcal exotoxins, staphylococcal enterotoxin, bacterial endotoxin lipopolysaccharide, and fungal products can act as pyrogens. It is the Gram negative bacteria produced endotoxin that is the most significant pyrogen for the manufacturers of injectables. This endotoxin is the most significant because its lipopolysaccharide component is very toxic in very small quantities. A quantity as small as 1ng/kg of the USP Reference endotoxin can cause fever. 4

Nature of PYROGENS: Nature of PYROGENS This endotoxin is present almost everywhere and is very difficult to remove. Its nature and omnipresence is as following : This is a high molecular weight complex molecule. This comes from the outer cell wall membrane of gram negative bacteria. These Gram –ve Bacteria keep shedding the endotoxin into the environment in which they live. The endotoxin is shed by the bacteria as they live, as they multiply and also after they die and disintegrate. So endotoxin may be present in a system in a form associated with live organisms or unassociated, in a free manner. 5

Nature of PYROGENS: Nature of PYROGENS 6 . Endotoxin associated with microorganisms may be removed by microporous sterilizing filters. 7. But unassociated endotoxin passes through these filters also. It cannot be destroyed by autoclaving as it is heat stable. 6

Nature of PYROGENS: Nature of PYROGENS Chemical Nature of endotoxin : Bacterial endotoxins contain lipid, protein and carbohydrate. 2. The primary chemical structure is a polysaccharide structure which is covalently bound to a lipid, called lipid A . 3. Because of this structure, the bacterial endotoxin is called as lipopolysaccharide or LPS . 4. The lipid A part is in the bacterial cell wall. 7

Nature of PYROGENS: Nature of PYROGENS The polysaccharide projects into the environment. The polysaccharide consists of two parts : a core oligosaccharide which is connected to Lipid A and a longer oligosaccharide O- specific chain which is attached to the core . This second oligosaccharide O- specific chain is the variable part of the molecular structure .

The minimum variable component is the lipid A. 9. Lipid A consists of a disaccharide of glucosamine. The glucosamine is highly substituted with ester linked and amide linked long chain fatty acids. 8

Nature of PYROGENS: Nature of PYROGENS 10. Each fatty acid has a backbone of 14 carbon atoms. 11. Lipid A is responsible for the harmful and useful activities of endotoxin. 9

METHODS: METHODS The following 6 methods for BACTERIAL ENDOTOXIN TESTING are described in I.P. 2010; B.P. 2013 and U.S.P 30-NF 25 : 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 (not mentioned in IP) The FINAL DECISION is made based upon Method A, unless otherwise indicated in the individual monograph. 10

METHODS: METHODS APPARATUS Depyrogenate all Glassware. Time and Temperature required is 60 minutes and 250°C. Plastic Apparatus which is FREE from DETECTABLE ENDOTOXINS. REAGENTS Water BET 0.1M Hydrochloric acid BET 0.1M Sodium hydroxide BET Tris-chloride buffer pH 7.4 BET – Dissolve 0.6057g of tris –(hydroxymethy l ) methylamine in 30ml of water BET, add 0.33ml of Hydrochloric acid, dilute to 100ml with water BET and mix. It gives a NEGATIVE result under the conditions of the test. 11

METHODS: METHODS LYSATE : A lysate of amoebocytes from either of the species of the horseshoe crab, Limulus polyphemus , Tachypleus gigas , Tachypleu s tridentatus or Carcinoscropius rotundicauda reconstituted as stated on the label. The species from which the lysate is obtained is stated on the label . Endotoxin Reference Standard 7. Standard Endotoxin Stock Solution Endotoxin is expressed in International Units (IU). 1 IU = 1 EU (ENDOTOXIN UNIT). 8. Control Standard Endotoxin 12

METHODS: METHODS 9. Preparation of Test Solutions Preparation of Test solution by dissolving or diluting active substance Adjust the pH of test solution (about 6 to 8) pH adjust with the use of acid,base or buffer Buffer must be validated to be free from Detectable Endotoxin 13

METHODS: METHODS ENDOTOXIN LIMIT Endotoxin Limit = K / M K = Threshold pyrogenic dose of Endotoxin per kg of body mass in single hour period. M = Maximum dose of product per kg of body mass in a single hour period. 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. 14

METHODS: METHODS DETERMINATION OF THE MAXIMUM VALID DILUTION (MVD) The Maximum Valid Dilution (MVD) is the maximum allowable dilution of a sample at which the endotoxin limit can be determined. MVD = Endotoxin limit x conc. of test solution λ λ = labeled lysate sensitivity in the gel clot, turbidimetric or chromogenic technique 15

METHODS: METHODS Gel Clot Technique (Methods A and B) The G el-Clot technique allows detection or quantification of Endotoxins and is based on clotting of the LYSATE in the presence of Endotoxins. The concentration of Endotoxins required to cause the Lysate to clot under standard conditions is the labelled lysate sensitivity. To ensure both the precision and validity of the test, confirm the labelled Lysate sensitivity and perform the test for interfering factors as described under Preparatory testing. 16

METHODS: METHODS PREPARATORY TESTING Sensitivity of the Lysate Confirm in 4 replicates the labelled lysate sensitivity ( λ ) Prepare Standard solutions of atleast 4 concentrations equivalent to 2 λ , λ , 0.5 λ and 0.25 λ from stock by diluting with water for BET. Mix lysate solution with Standard in each tube (1:1) Incubate the reaction mixture at 37 ± 1 °C for 60 ± 2 min (avoiding vibration) Invert it through approx. 180° in one smooth motion. 17

METHODS: METHODS If a firm gel has formed that remains in place upon inversion, record the result as positive. A result is negative if an intact gel is not formed. The end-point is the last positive result in the series of decreasing concentration of Endotoxin. Calculate Geometric Mean end-point concentration = ∑ e f Where ∑ e = Sum of the log end point concentrations. f = number of replicates. The geometric mean end point concentration is the measured sensitivity of the lysate solution (IU/ml). If this is not less than 0.5 λ and nmt 2 λ , the labelled sensitivity is confirmed. 18

METHODS: METHODS TEST FOR INTERFERING FACTORS Solution Endotoxin conc/sol to which Endotoxin is added DILUENTS DILUTION FACTOR INITIAL ENDOTOXIN CONC. NUMBER OF REPLICATES A (Sol. Of Prepn) None/Test solution - - - 4 B (Test for Interference) 2 λ / Test solution Test solution 1 2 4 8 2 λ 1 λ 0.5 λ 0.25 λ 4 4 4 4 C (+ve control) 2λ / Water for BET Water for BET 1 2 4 8 2 λ 1 λ 0.5 λ 0.25 λ 2 2 2 2 D (-ve control) None / Water for BET - - - 2 19

METHODS: METHODS Prepare solutions A,B,C and D as shown in the Table and use the test solutions at a dilution less than the MVD, not containing any detectable Endotoxins, operating as described under Confirmation of the Labelled Lysate Sensitivity. The geometric mean end-point concentrations of solutions B and C are determined. The test is not valid unless all replicates of solutions A and D show no reaction and the result of solution C confirms the Labelled Lysate Sensitivity. 20

METHODS: METHODS If the sensitivity of the Lysate determined with solution B is not less than 0.5 λ and not greater than 2 λ , the test solution does not contain interfering factors under the experimental conditions used. Otherwise, the solution interferes with the Test. If the preparation being examined interferes with the test at a dilution less than the MVD, repeat the test for interfering factors using a greater dilution, not exceeding the MVD. Interference may be overcome by suitable treatment, such as filtration, neutralization, dialysis or heat treatment. 21

METHODS: METHODS Limit test (Method-A ) Prepare solutions A,B,C and D as shown in the Table and perform the test on these solutions by following the procedure described in Confirmation of the Labelled Lysate Sensitivity. Solution Endotoxin conc/solution to which Endotoxin is added No. of replicates A None / Test solution 2 B 2 λ / Test solution 2 C 2 λ / Water for BET 2 D None / Water for BET 2 22

METHODS: METHODS Prepare Solution A and Solution B (Positive Product Control) using a dilution not greater than the MVD and Treatments. INTERPRETATION : If a positive result is found for one of the test duplicates and a negative result for the other, the test may be repeated as described above. The results of the retest should be interpreted as for the initial test. The substance or preparation being examined meets the requirements of the

test if the concentration of Endotoxin is less than the Endotoxin limit stated in the monograph. 23

METHODS: METHODS SEMI-QUANTITATIVE TEST (METHOD-B) Solution Endotoxin conc /solution to which endotoxin is added Diluents Dilution Factor Initial Endotoxin Conc. No. of Replicates A None / Test solution Water for BET 1 2 4 8 - - - - 2 2 2 2 B (+ve control ) 2 λ / Test solution - 1 2 λ 2 C 2 λ / Water for BET Water for BET 1 2 4 8 2 λ 1 λ 0.5 λ 0.25 λ 2 2 2 2 D (-ve control) None / Water for BET - - - 2 24

METHODS: METHODS CALCULATION AND INTERPRETATION The test is not valid unless the following 3 conditions are met : Both replicates of solution D (- v e control) are negative. Both replicates of solution B (+ve 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 . 25

METHODS: METHODS 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 . If all dilutions are positive, the Endotoxin concentration is recorded as equal to or greater than the greatest dilution factor multiplied by λ . 26

METHODS: METHODS The preparation meets the requirement of the test if the Endotoxin concentration is less than that specified in the individual monograph. 27

METHODS: METHODS PHOTOMETRIC METHODS (METHODS C,D,E,F) Turbidimetric technique (Method C) This technique is a photometric test to measure the increase in turbidity. Based on the test principle employed classified as, Kinetic Turbidimetric Method (C) Turbidimetric end-point Method (F) The kinetic turbidimetric method is a photometric assay measuring the increase in turbidity caused by the reaction of the endotoxin with the lysate. The kinetic turbidimetric assay is a method measuring either the time (onset

time) needed to reach a predetermined absorbance of the reaction mixture or the rate of turbidity development. 28

METHODS: METHODS The End-Point Turbidimetric Method (F) is based on the quantitative relationship between the Endotoxin concentration and the turbidity (absorbance or transmission) of the reaction mixture at the end of the incubation mixture. The test is carried out at the incubation temperature recommended by the Lysate manufacturer. (usually 37 ± 1°C) 29

METHODS: METHODS 2. CHROMOGENIC TECHNIQUES (METHODS D AND E) This technique 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, Kinetic chromogenic test ( D) End-point chromogenic test (E) 30

METHODS: METHODS Kinetic Chromogenic Test (D) Preparation of test solutions Unless otherwise prescribed, prepare the solutions to be employed in the test using water BET . If necessary, adjust the pH of the solution under examination to 6.0 to 8.0 using sterile 0.1 M hydrochloric acid BET , 0.1 M sodium hydroxide BET or a suitable buffer prepared with water BET . Prepare the test solution at a suitable dilution. Use not less than three CSE concentrations to prepare a linear standard curve . Use water BET as negative control and one positive control . The positive control consists of the test solution spiked with CSE to give an endotoxin concentration at the middle or below the middle point of the standard curve (PPC ). 31

METHODS: METHODS Method : Carry out the procedure described under Test for interfering factors . Interpretation of Results : The assay is valid only if (a) the standard curve is linear for the range of CSE concentrations used (b) the co-efficient of correlation, r, is not greater than - 0.980 (c) the mean percentage recovery of the added endotoxin in the positive product control is between 50 per cent and 150 per cent. 32

METHODS: METHODS The product under examination meets the requirements of the test if the mean endotoxin content of the replicates, after correction for dilution and concentration, is less than the endotoxin limit stated in the individual monograph. 33

METHODS: METHODS End-Point Chromogenic Test (E) Preparation of test solutions . Unless otherwise prescribed, prepare the solutions to be employed in the test using water BET . If necessary, adjust the pH of the solution under examination to 6.0 to 8.0 using sterile 0.1M hydrochloric acid BET , 0.1M sodium hydroxide BET or a suitable buffer prepared with water BET . Prepare the test solution at a suitable dilution. Prepare a reagent blank and not less than three dilutions of CSE in water BET to prepare a linear standard curve. Use water BET as negative control and one positive control. The positive control consists of the test solution spiked with CSE to give an endotoxin concentration at the middle or below the middle point of the standard curve (PPC). 34

METHODS: METHODS Method : Carry out the procedure described under Test for interfering factors. The chromogenic substrate and lysate are added to the solution and incubated for the recommended time . Stop the reaction and measure the absorbance at the wavelength specified by the lysate manufacturer . Perform the linear regression analysis of the absorbance on the endotoxin concentration using standard statistical methods (method of least squares is usually suitable). Do not average the absorbance values of the replicates of each standard before performing the linear correlation regression analysis . Determine the endotoxin concentration of the test solution from the standard curve. 35

METHODS: METHODS Interpretation of results : The assay is valid only if (a) the standard curve is linear for the range of CSE concentrations used (b) the co-efficient of correlation, r, is not less than 0.980; (c) the mean percentage recovery of the added endotoxin in positive product control is between 50 per cent and150 per cent. The product under examination meets the requirements of the test if the mean endotoxin content of the replicates, after correction for dilution and concentration, is less than the endotoxin limit stated in the individual monograph. 36

REFERENCES: REFERENCES 1. INDIAN PHARMACOPOEIA 2010, VOL I, PAGE NO. 28 – 33. 2. BRITISH PHARMACOPOEIA 2013, APPENDIX XIV C. 3. UNITED STATES PHARMACOPOEIA 30, NF 11,TOPIC NO. 85 37

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Packaging of sterile pharmaceutical

Comments Presentation Transcript Packaging Of Sterile Pharmaceuticals: Packaging Of Sterile Pharmaceuticals Monika V. Pawar M.Pharm – II Guided By : A.A.Joshi

Parenterals: Parenterals Parenteral preparations are sterile preparations containing one or more active ingredients intended for administration by injection, infusion or implantation into the body. Injections are sterile, pyrogen -free solutions or dispersions (emulsions or suspensions) of one or more active ingredients in a suitable vehicle.

Characteristics of Packaging Material: Characteristics of Packaging Material They should not interact with any of the ingredients of parenteral preparations. Container should protect the product from light,and microbial contamination. Glass container should be light amber colour to protect the parenterals from light. All glass containers for liquid preparations and for powders for parenteral use permit the visual inspection of the contents.

Containers: Containers Parenteral preparations are usually supplied in glass ampoules, bottles or vials, in plastic bottles or bags, or in prefilled syringes. In case of light-sensitive substances the container should protect the contents (for example, by the use of coloured glass). Containers are made, as far as possible, from material that is sufficiently transparent to permit the visual inspection of the contents. They do not adversely affect the quality of the preparation, allow diffusion of any kind into or across the material of the container or yield foreign substances into the preparation.

Glass Containers: Glass Containers Types of Glass : Type I : Commonly known as neutral glass. It has a high resistance to hydrolysis and withstands autoclaving,weathering.It is suitable for parenteral use. Type II ( Sulphated glass) : Container may be treated with moist sulphur dioxide at high temperature to create a neutral surface film with high hydrolytic resistance.Lower resistance to autoclaving than for type I glass. Glass containers are suitable for most acidic and neutral, aqueous preparations. Type III (Soda glass) : This offer very little resistance to hydrolysis and should only be used for powder for reconstitution prior to injection and for non-aqueous preparation.

Types of Container: Types of Container Single dose container Multiple dose container

Single Dose Containers: Single Dose Containers A single dose container for parenterals are containing single dose. When they open once they cannot be resealed with assurance of sterility of

product. Ampoules : A small, hermetically sealed glass or plastic container,eg.one containing medication for parenteral administration. An ampoule is a small sealed vial which is used to contain and preserve a sample, usually a solid or liquid. Ampoules are commonly made of glass, although plastic ampoules do exist.

Ampoule codes : Ampoule codes Ampoules often have colored rings of paint or enamel around their necks. A machine paints colored rings on the ampoule shortly after it's been sealed. These color codes identify the substance inside the ampoule so that it does not need to be tested to verify the contents. The machine-readable color codes allow for accurate handling of the substance for the purposes of storage, labeling, and secondary packaging. The dot above the neck identifies the location of a small cut in the glass to help breaking/opening the ampoule.

Filling and Sealing of Glass Ampoules: Filling and Sealing of Glass Ampoules Ampoules must first be rinsed out using WFI to remove any dust,particulate matter and/or glass fragments. Using a syringe gently draw the volume required to be filled plus an excess volume. Invert the syringe to allow the air to rise towards the needle and push up the plunger to remove all air. Invert the ampoule over the needle and expel the required volume of liquid into ampoule. Using a twin-jet burner, adjust the platform height and flame intensity. Position the ampoule between the flames.Grip the end of the neck with a blunt nosed forceps and when the glass is soft enough,pull of the top of the ampoule vertically and gently. A good seal will not deteriorate during the lifetime of the product.

Pre-filled syringe: Pre-filled syringe A prefilled syringe is a single-dose packet of parental drug to which a needle has been fixed by the manufacturer. Prefillable syringes have become an increasingly important option in the packaging and delivery of injectable drug products. Prefilled syringes are ready to use disposable syringes contain premeasured dosage, reduce dosing errors and increase patients compliance.

Slide 12: The prefilled syringe provides two major purposes to the administration of a drug. The first is primary packaging . The purpose of the primary package is to assure that there is no adverse effect on the identity, strength, quality,purity or potency of the drug over its shelf life. Second, a prefillable syringe is a delivery system designed to administer the approriate amount of medicament to the patients. There are 2 major types of prefilled systems available : Glass- based systems Plastic – based systems

Glass Based Systems: Glass Based Systems Traditionally, a prefilled syringe barrel has been made from glass tubing. These glass tubes are transformed by heat into the barrel that is hold the drug product. Benefits : 1.Deliver a more accurate dose of the drug to the patient. 2.Lass pain production at injection site. Drawbacks : 1.Breakage 2.Particulate contamination 3.Potential surface reactivity

Plastic Based systems: Plastic Based systems The most widely used prefilled plastic syringe systems are manufactured from cycloolefin polymer (COP) resins. Benefits : 1.Break resistance 2.Design flexibility

Filling Process: Filling Process Syringe filling involves vaccum filling coupled with online vaccum stoppering,known as bubble free filling. It eliminates the air bubble inside the syringe,& improves the stability of oxygen sensitive compounds. Sterilization is mainly done by autoclaving or by ionizing radiation. Frequently used Prefilled syringes : 1.Autojet syringes 2.Insulin prefilled pens

Cartridges: Cartridges Cartridges are similar to syringes with respect to having a product filled into a glass tube closed on either side by a rubber plunger and a rubber disk seal. Cartridge/pen delivery systems are used primarily for multiple dose proteins such as insulin and growth hormone, and, historically have been used for dental anesthesia and epinephrine emergency uses. Advantages : 1.Dose accuracy 2.Patient convenience. Disadvantage : 1.Slightly increased costs.

Multiple Dose Container: Multiple Dose Container Multiple dose container for parenterals is a hermetic container,which permits withdrawal of successive proportion of the contents without changing the strength,quality or purity of the remaining portion. VIALS : The most common packaging for liquid and freeze-dried injectables is the glass vial. The broad variety of vials from 1ml to 100 ml with different neck finish design. Industries also offer inside-coated vials for improved chemical stability,inertness and lyophilisation process. Vials are sealed using rubber closure which are held in place by aluminium sealing.A hand crimper is available in the laboratory to seal the rubber closures and aluminium sealing onto the vials.

Slide 18: PLASTIC VIALS : Due to excellent barrier properties, high chemical resistance and breakage resistance,this vial is an excellent alternative to glass.

Slide 19: BOTTLES : Bottles typically refer to containers larger than 100 ml, thus large volume injectable solutions or emulsions are contained in bottles rather than vials. Bottles are manufactured by the blow-molded process. Bottles can be glass or plastic, both are commonly used in hospital pharmacy practice.

Slide 20: INFUSION BAGS : Bags used for IV fluids include pre-filled or empty containers that range in size from 25 mL to greater than 1 L. Sizes that are 1 L or greater are often used in hospital settings for delivery of total parenteral nutrition. Bags of all sizes are used for ease of delivery and ease of transport. Printing on plastic bags is a challenge because of the flexibility of the bag material and labels adhered to the bags can become difficult to read. This was mostly resolved by the introduction of bar coding that allows traceability of bags from filling to patient use.

Slide 21: Plastic bags are manufactured by form-fill-finish processes where strips of plastic polymer are sealed on three sides, solution is filled into the ‘pouch’, then the bag is sealed with the fourth side that contains the spike and needle outlets.

Reconstitution packaging systems: Reconstitution packaging systems Lyophilized drugs were available in vials where the diluent used to reconstitute the freeze-dried powder was provided with the vial package (combination package of vial and syringe) The classic method of reconstituting freeze-dried powders is One is the use of vial adapters and pre-assembled systems facilitate the combination and transfer of diluents into the freeze-dried product vial. The other is the design of dual-chambered vials or syringes where the freeze-dried powder and the diluent are contained in the same packaging system separated by a rubber septum. At the time of reconstitution, the rubber septum is moved toward the powder compartment and the diluent combines with the powder via a bypass design.

Slide 23: A dual-chamber syringe offers safety during patient application, while minimizing product loss and packaging waste. The Lyo-Ject allows lyophilization of the drug to take place directly in the syringe, packaged with diluent for fast reconstitution and injection. Dual-chambered vials such as Solu-Medrol Mix-O-Vial™ are marketed.

Closure: Closure Closure for parenteral preparation containers should be equipped with a firm seal to prevent entry of microorganism and other contaminants while permitting the withdrawal of a part or the whole of the content without removal of the closure. They should not be made of components that react with the content ,nor should they allow foreign substance to diffuse into the preparation. Plastic materials of which the closure is composed should be sufficiently firm and elastic to allow the passage of a needle with least possible shedding of particles.

Slide 25: Closures for multidose container should be sufficiently elastic to allow the puncture to reseal when the needle is withdrawn and protect the content from contamination. A temper evident container is fitted with a device that reveals clearly whether it has ever been opened. It has compression recovery,coring resistance,solvent resistance,heat resistant,radiation resistant with very low water absorption and permeability properties.

Ophthalmics: Ophthalmics Ophthalmic preparation are sterile,liquid,semi -solid or solid preparation intended for administration upon the eyeball and/or to the conjuctiva or to be Inserted in the conjuctival sac. Characteristics of Ophthalmic Containers : 1. They should preserve the sterility of the content. 2. Not extract out anything to the container. 3. Not absorb anything from the contents. 4. It should be easy to sterilized. 5. They should be easy to handle and storage.

Packaging Of Ophthalmic Preparation: Packaging Of Ophthalmic Preparation EYE DROPS : Ophthalmic drops (eye drops) are sterile aqueous or oily solutions, suspensions, or emulsions intended for instillation into the conjunctival sac. Traditionally eye drops were stored in vertically fluted amber coloured glass bottles fitted with a bakelite cap carrying a dropper. Recently eye drops are packed in plastic container with applicators.

Slide 28: Eyedrops have been packed almost entirely in plastic dropper bottles. Advantages : Convenience of use by the patient. Decreased contamination potential Lower weight Lower cost The plastic bottle and dispensing top is made of low-density polyethylene(LDPE) resin which provide flexibility and inertness. The cap is made of harder resin than the bottle.Caps generally made from Polypropylene and basically seal the container to prevent contamination or leakage of the product.

Ophthalmic Cap Colour Coding: Ophthalmic Cap Colour Coding Colour Pharmaceutical Class Yellow or Blue Beta- Blocker Grey Non-steroids Pink Steroids Brown Anti- infectives Orange Carbonic anhydrase inhibitors Red mydriatics Green miotics

Slide 30: EYE OINTMENT : Eye ointment should be supplied in small sterilized collapsible tubes made of metal or in a suitable plastic. The tube is provided with nozzle of a suitable shape to prevent microbial contamination. The content of such a container is limited to not more than 5 g of the preparation.

Blow-fill-seal technology : Blow-fill-seal technology The blow-fill-seal process is a robust, advanced aseptic processing technology. Blow-fill-seal systems offer a unique combination of flexibility in packaging design, low operating cost and a high degree of sterility assurance. BFS is used for the filling of vials for parenteral preparations and infusions, eye drops, and inhalation products. The process is multi-stepped: First, pharmaceutical-grade plastic resin is vertically heat extruded through a circular throat to form a hanging tube called the parison . This extruded tube is then enclosed within a two-part mould, and the tube is cut above the mould.

Slide 32: The mould is transferred to the filling zone, or sterile filling space, where filling needles (mandrels) are lowered and used to expand the plastic to form the container within the mould. Following the formation of the container, the mandrel is used to fill the container with liquid. Following filling the mandrels are retracted and a secondary top mould seals the container.

Slide 33: SINGLE DOSE BLOW / FILL SEAL VIALS Unit dose blow/fill/seal vials for one-time ophthalmic use. The development of formulations that no longer contain preservatives,minimizing the risk of allergic reactions for patients. EYE WASH BLOW/FILL/SEAL PACKAGING : Emergency eye wash stations utilize a blow/fill/seal container, with a twist-off or snap-off top for quick dispensing of sterile water.

Conclusion: Conclusion Packages can eliminate waste, ensure correct dosages, and protect the patient . Packaging must combine ease of use with the ability to keep pharmaceuticals sterile. Concern for the safety of healthcare professionals and their patients has led to the creation of innovative parenteral systems. Safety and ease of use are the drivers for the new parenteral systems available to pharmaceutical packagers.

References: References Gregory A. Sacha , Wendy Saffell-Clemmer , Practical fundamentals of glass, rubber, and plastic sterile packaging systems, Pharmaceutical Development and Technology, 2010; 15(1): pp :6–34 Atul Kaushik,Bhaskar Chaurasia,Virendra Dhakar , Textbook of Pharmaceutical Packaging Technology,pp :331-363. C.F.Ross , Packaging Of Pharmaceuticals, pp : 24-30 H.Lockhart , F.A.Paine , Packaging Of Pharmaceuticals & Healthcare Product, pp : 38-40 Remington,The Science and Practice of Pharmacy,21 st edition, Vol-I,pp :523-530 http://www.healthcarepackaging.com/blowfillseal-offers-alternative-aseptic-processing-parenterals

Overview of gmp of sterile products: Overview of gmp of sterile products Guided by : Dr. Amol Tagalpallewar Presented by : Mr. Akash Patil (M. Pharm ) DEPARTMENT OF PHARMACEUTICS, SINHGAD INSTITUTE OF Pharmacy, Narhe, Pune-41. 1

Overview of presentation : Overview of presentation GMP – Good manufacturing practices Objective General consideration Premises Equipments Sanitation 5-Aug-11 2 OVERVIEW OF GMP

Slide 3: Continued… Personnel Processing Sterilization Packaging & labeling Finishing of products Holding & distribution 5-Aug-11 3 OVERVIEW OF GMP

Slide 4: Prerequisite programs which will provide the basic environmental and operating conditions that are necessary for the production of safe products. 5-Aug-11 4 OVERVIEW OF GMP

Objectives : Objectives To review basic GMP requirements in the manufacture of sterile pharmaceutical products. Air classifications for activities related to the manufacture of sterile products. Different types of sterilization methods. Quality assurance aspects in the manufacture and control of sterile products. 5-Aug-11 5 OVERVIEW OF GMP

General Considerations: General Considerations Production in clean areas Appropriate standard of cleanliness Filtered air supplied Airlocks for entry - equipment - materials Separate areas for operations - component preparation (containers and closures) - product preparation - filling, sterilization, etc. 5-Aug-11 6 OVERVIEW OF GMP

Premises: Premises Provide specifically defined area for certain operation. Including designed construction features. Lighting, ventilation, air filtration, air heating & cooling, plumbing, sewage disposal, washing & toilet facilities. 5-Aug-11 7 OVERVIEW OF GMP

Premises(continued): Premises(continued) - Avoid unnecessary entry of supervisors and control personnel Operations observed from outside In clean areas, all exposed surfaces - Smooth, unbroken - Minimize shedding and accumulation of particles, microorganisms - Permit cleaning and disinfection 5-Aug-11 8 OVERVIEW OF GMP

Premises (continued): Premises (continued) Changing rooms - Effective flushing with filtered air - Separate rooms for entry and exit desirable Hand washing facilities Proper installation of pipes & ducts - Visual and/or audible warning system 5-Aug-11 9 OVERVIEW OF GMP

Equipment : Equipment Use of automatic mechanical or electronic equipment Conveyer belts Effective sterilization of equipment Maintenance and repairs from outside the clean area

- If taken apart, re-sterilized before use - Use clean instruments and tools 5-Aug-11 10 OVERVIEW OF GMP

Equipment (continued): Equipment (continued) Planned maintenance, validation and monitoring (equipment, air filtration systems, sterilizers, water treatment systems) 5-Aug-11 11 OVERVIEW OF GMP

Environmental Monitoring - I: Environmental Monitoring - I Microbiological Air samples Surface swabs Personnel swabs Ref: www.copaninnovation.com 5-Aug-11 12 OVERVIEW OF GMP

Environmental Monitoring – II : Environmental Monitoring – II Physical Particulate matter Air changes, airflow patterns Filter integrity Temperature and relative humidity Airflow velocity Differential pressure 5-Aug-11 13 OVERVIEW OF GMP

Sanitation : Sanitation Frequent, thorough cleaning of areas necessary Written programme Regular monitoring to detect resistant strains of microorganisms Chemical disinfection 5-Aug-11 14 OVERVIEW OF GMP

Sanitation (continued) : Sanitation (continued) Monitoring of clean areas Monitoring of personnel and surfaces after critical operations Frequent monitoring in areas where aseptic operations are carried out - volumetric air samples, surface sampling (swabs and contact plates) - sampling methods should not contaminate the area 5-Aug-11 15 OVERVIEW OF GMP

Personnel : Personnel Training of persons : - Quality & sterile product - Types of contamination - Sources of contamination - Environmental - Water - Raw material - Container & closures - Control of contamination - Training on sterilization 5-Aug-11 16 OVERVIEW OF GMP

Personnel (continued) : Personnel (continued) Gowning & dress management - Non fiber shedding - Washing & sterilization - Head gear/ hood, face mask, one piece boiler suit, rubber/ plastic shoe, shoe cover, beard mask, safety goggles etc. 5-Aug-11 17 OVERVIEW OF GMP

Personnel (continued) : Personnel (continued) Some specific requirements - Restrict number of people in aseptic area - Only trained person is allowed - Medical check up is more frequent - Person with psychological problem in working area should not allowed 5-Aug-11 18 OVERVIEW OF GMP

Personnel (continued) : Personnel (continued) Minimum number of personnel in clean areas - especially during aseptic processing Inspections and controls from outside Training to all including cleaning and maintenance staff manufacturing, hygiene, microbiology 5-Aug-11 19 OVERVIEW OF GMP

Personnel (continued) : Personnel (continued) High standards of hygiene and cleanliness Periodic health checks No shedding of particles No introduction of microbiological hazards Changing and washing procedure No watches, jewellery and cosmetics 5-Aug-11 20 OVERVIEW OF GMP

Possible Issues : Possible Issues Poor design of the building Poor design of the systems, e.g. water, HVAC Flow of personnel Flow of material Old facilities not complying with current requirements Particulate levels/microorganism Temperature/humidity HEPA filters not checked regularly 5-Aug-11 21 OVERVIEW OF GMP

Two categories of manufacturing operations : Two categories of manufacturing operations Terminally sterilized - prepared, filled and sterilized Aseptic preparation - some or all stages 5-Aug-11 22 OVERVIEW OF GMP

Manufacture of sterile preparations : Manufacture of sterile preparations Classification of clean areas Manufacturing operation in an appropriate environment cleanliness level Minimize risks – particulate and microbiological contamination – product and material 5-Aug-11 23 OVERVIEW OF GMP

Manufacture of sterile preparations-II : Manufacture of sterile preparations-II For sterile pharmaceutical preparations: Grade A - local zone, high risk operations, e.g. filling, aseptic connections Grade B - background environment to Grade A (in case of aseptic preparation and filling) Grade C and Grade D - Clean areas for less critical operations 5-Aug-11 24 OVERVIEW OF GMP

Processing : Processing Minimize contamination - all stages including before sterilization and during processing No unsuitable materials, e.g. live micro organisms Minimize activities - staff movement - avoid shedding of particles Temperature and humidity comfortable Containers and materials in the area 5-Aug-11 25 OVERVIEW OF GMP

Processing (continued): Processing (continued) Control of components : Storage, handling, sampling, testing & approved/ rejection of components & drug products Prohibit the use rejected material Each lot is identified as to its status & that material is stored separately Rejected material is handled separately 5-Aug-11 26 OVERVIEW OF GMP

Processing (continued) : Processing (continued) Components, bulk product containers and equipment - fibre generation - no recontamination after final cleaning - sterilized when used in aseptic areas 5-Aug-11 27 OVERVIEW OF GMP

Processing (continued) : Processing (continued) Time intervals: components, bulk container, equipment Washing , drying and sterilization - as short as possible - time limit validated Time intervals: product Start of preparation of solution and sterilization (filtration) - as short as possible - maximum time set for each product 5-Aug-11 28 OVERVIEW OF GMP

Sterilization : Sterilization Methods of sterilization - moist or dry heat - irradiation (ionizing radiation) - sterilizing gaseous agents (e.g. ethylene oxide) - filtration with subsequent aseptic filling Whenever possible: terminal sterilization by heat in their final container - method of choice 5-Aug-11 29 OVERVIEW OF GMP

Sterilization (continued): Sterilization (continued) Differentiation between sterilized and not-yet-sterilized products Each basket/tray or other carrier, properly labelled - name of material - batch number - sterilization status Sterilization records for each run – approved as part of the batch release procedure 5-Aug-11 30 OVERVIEW OF GMP

Terminal Sterilization: Terminal Sterilization Sterilization by moist heat Sterilization by dry heat Sterilization by radiation Sterilization by gases 5-Aug-11 31 OVERVIEW OF GMP

Slide 32: Sterilization by moist heat (heating in an autoclave ) Water - wettable materials only, and aqueous formulations Temperature, time and pressure monitored Independent temperature indicator Regular leak test Material allows for penetration of steam Quality of the steam – no contamination 5-Aug-11 32 OVERVIEW OF GMP

Slide 33: Sterilization by dry heat For non-aqueous liquids, dry powders Air circulation in the chamber Positive pressure in chamber to prevent entry of non-sterile air HEPA filtered air supplied 5-Aug-11 33 OVERVIEW OF GMP

Slide 34: Sterilization by radiation Suitable for heat-sensitive materials and products High radioactive isotope ( Cobalt – 60 γ - rays ) Mechanical acceleration of electrons ( cathode rays, β - rays ) 5-Aug-11 34 OVERVIEW OF GMP

Sterilization by Filtration : Sterilization by Filtration Through a sterile filter of 0.22 µm or less, into previously sterilized containers - remove particles - remove bacteria and moulds Consider complementing with some degree of heat treatment Filter integrity testing immediately after use - also before use if possible 5-Aug-11 35 OVERVIEW OF GMP

Sterilization by Filtration - II: Sterilization by Filtration - II Same filter not used for more than one working day, unless validated No filter interaction with product, e.g. - removal of ingredients - releasing substances into product 5-Aug-11 36 OVERVIEW OF GMP

Sterilization by gases: Sterilization by gases Ethylene oxide ( EtO ): - Gas at room temperature - Highly flammable - Acts by alkylation of essential metabolites affecting particularly the reproductive process. 5-Aug-11 37 OVERVIEW OF GMP

Packaging & Labeling : Packaging & Labeling Labeling error or product mix–ups are avoided Use of appropriate electronic equipment Visual inspection to conduct 100 % examination of labeling All the inspection is done by single person independently & verified by second person 5-Aug-11 38 OVERVIEW OF GMP

Finishing of products : Finishing of products Containers closed by means of validated methods Samples checked for integrity Parenteral products inspected individually Visual inspection under suitable and controlled conditions 5-Aug-11 39 OVERVIEW OF GMP

Holding & Distribution: Holding & Distribution Covers ware housing & distribution Covers the Q.C. unit Storage of drug products at appropriate condition System for documenting the distribution of each lot 5-Aug-11 40 OVERVIEW OF GMP

References: References Liberman H. A., Rieger M. M., Banker G., Pharmaceutical Dosages Forms Disperse System, Vol. 3 rd , Ed. 2 nd , (2005),Page no. 425-426. Potdar M. A., Pharmaceutical Quality Assurance, Nirali Prakashan,(2007), Page no. 13.1 to 13.60. Nigel H., Microbiological contamination Control In Pharmaceutical Clean Rooms, Sue Horwood Publishing,(2004) , Page no. 1-23. 5-Aug-11 41 OVERVIEW OF GMP

Slide 42: 4) Linna A. et al., Developing a tool for the preparation of GMP audit of pharmaceutical contract manufacturer, European Journal of Pharmaceutics and Biopharmaceutics 69 (2008) 786–792. 5) Peyton T. H., Abdou O. A. Environmental Control Concepts for Industrial Clean-Room Facilities. Journal of Architectural Engineering. Vol. 1, No.1,(1995) 53-63. 5-Aug-11 42 OVERVIEW OF GMP

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