Introduction to Quality Management and Quality Control Presented by: M. Talha AfzalQuality:- "the totality of features and characteristics of a product or service that bears its ability to satisfy stated or implied needs.” Quality Management:- “The act of overseeing all activities and tasks needed to maintain a desired level of excellence. This includes the determination of a quality policy, creating and implementing quality planning and assurance, and quality control and quality improyement.”Quality Management System:- A quality management system (QMS) is a formalized system that documents pro s, procedures, and responsibilities for achieving quality policies and objectives. A QMS helps coordinate and direct an organization’s activities to meet customer and regulatory requirements and improve its effectiveness and efficiency on a continuous basis. Quality management systems serve many purposes, including: . Improving processes . Reducing waste . Lowering costs = Facilitating and identifying training opportunities . Engaging staff . Setting organization-wide directionQuality Assurance:- Quality assurance is the totality of the arrangements made with the object of ensuring that pharmaceutical products are of the quality required for their intended use. It incorperates GMP and other factors, including those outside the scope of this guide such as product design and development. Quality Control:- Quality control (QC) is a procedure or set of procedures intended to ensure that a manufactured product or performed service adheres to a defined set of quality criteria or meets the requirements of the client or customer. QC is similar to, but not identical with quality assurance.Difference b/w Quality Assurance and Quality Control >» The difference is that QA is process oriented and QC is product oriented. » Quality Assurance makes sure you are doing the right things, the right way. » Quality Control makes sure the results of what you've done are what you expected. » Quality Assurance is focuses on defect prevention, while quality control is focuses on defect identification.> Quality assurance(QA) in pharmaceutical industry is used to ensure that product or service will satisfy requirement for “Quality” and covers all matters related to factors influencing quality of a product. ® Quality control(QC) is concemed with sampling, specifications, testings' , organization, documentation and release procedures which ensures that necessary and relevant tests are actually carried out-and that materials are not released for sale, supply until their quality has been judged to be satisfactory.Comparison chart Definition QA isa set of activities for ensuring QC is a set of activities for ensuring Focus on Goal quality in the processes by which products are developed. to prevent defects with a focus on the process used to make the product. [t is a proactive quality process. is to improve development and test processes so that defects do not arise when the product is being developed. quality in products. The aetivities focus on identifying defects in the actual products produced. to identify (and correct) defects in the finished product. Quality control, therefore, is a reactive process. is to identify defects after a product is developed and before it’s released.bytes iirinecctt Creneenin) Responsibility Asa tool ‘Orientation Example Everyone on the team involved in developing the product is responsible for quality assurance. QA is a managerial tool QA is process oriented Verification is an example of QA Quality control is usually the responsibility of a specific team that tests the product for defects. QC is a corrective tool QC is product oriented Validation/Software Testing is an example of QCQuality Control Test of Tablets And CapsulesTABLETS + Tablets may be characterized or described by number of specifications. * These include: shape, diameter, thickness, weight variation, hardness, friability, disintegration time and dissolution characteristics. General Appearance: Size, shape, and thickness Organoleptic properties: Color and odor of the tablets.OFFICIAL AND UNOFFICIAL TESTS: »>Non-Official Tests: Hardness, Friability > Official Tests: Weight variation, Disintegration, Dissolution, Content uniformityGENERAL APPEARANCE > Diameter and Shape: + The diameter and shape depend on the die and punches selected for the compression of the tablet. + Generally, tablets are discoid in shape, although they may be oval, oblong, round, cylindrical, or triangular. + Their upper and lower surfaces may be flat, round, concave or convex to various degrees. + The top or lower surface may be embossed or engraved with a symbol or letters which serve as an additional mean of identifying the source of the tablets.Tablet Thickness: * The thickness of the tablet is the only dimensional variable related to the compression process. Tablet thickness is consistent batch to batch if the tablet granulation or powder blend is adequately consistent in particle size and size distribution. = The thickness of individual tablets may be measured with a micrometer. * Thickness should be controlled within + 5% variation of a standard value. = Thickness must be controlled for consumer acceptance of the product, and to facilitate packaging.ORGANOLEPTIC PROPERTIES: * Colour: Many pharmaceutical tablets use color as a vital means of rapid identification and consumer acceptance, >The color of a product must be uniform within a single tablet »Non-uniformity of coloring net only lacks esthetic appeal but could be associated with poor quality of the product. * Odour: » The presence of odor in a batch of tablets could indicate stability problems, such as the odor of acetic acid in degrading aspirin tablets. >» However, the presence of an odor could be characteristic for the drug (vitamins), added ingredients (flavoring agents) or the dosage form (film-coated tablets),NON-OFFICIAL TESTS * HARDNESS Tablet hardness can be defined as the force required to break a tablet in a diametric compression test. OR It is the load required to crush the tablet when placed on its edge. + Why do we measure hardness? »To determine the need for pressure adjustments on the tableting machine. > Hardness can affect the disintegration. So if the tablet is too hard, it may not disintegrate in the required period of time. And if the tablet is too soft, it will not withstand the handling during subsequent processing such as coating and packaging.+ Factors Affecting the Hardness: > Compression of the tablet and compressive force. > Amount of binder. > Method of granulation in preparing the tablet ¢ Hardness Test Apparatus >» Monsanto tester, Pfizer tester, strong cobb hardness tester are manually used & Heberlien schleuniger, Eweka, Casburt hardness tester are motor driven testers.* PROCEDURE: *MONSANTO TESTER: The apparatus consists of 2 jaws facing each other, one of which move towards the other. Measurements is carried out on 10 tablets, taking care to remove all the fragments of the broken tablets before each determination & then take the average hardness. * LIMITS: Normal tablet hardness ranges from 4 — 8 Kg (1 Kg = 10 Newton)° FRIABILITY: >It is expressed as a percentage loss in weight OR It is the tendency of tablets to powder, chip, or fragment and this can affect the elegance appearance, consumer acceptance of the tablet. >The measurement of friability is made by Roche friabilator. A number of tablets are weighed and placed in the tumbling apparatus where they are exposed to rolling and operated shocks resulting from free falls within the apparatus. After a given number of rotations, the tablets are weighed and the loss in weight indicates the ability of the tablets to withstand this type of wear.* Method: >Select 20 tablets randomly, dedust and weigh (W). Place the tablets in the Roche friabilator drum, switch on the apparatus adjusting the timer at 4 min and the speed at 25 rpm. >At the end of this operation, remove the tablets from the friabilator, dedust and reweigh (W). (Any tablet that breaks up should be rejected before reweighing). }friability is measured by using formula as: + Limit: >If the value of friability (% loss) is less than or equal to 1%, the batch is accepted,OFFICIAL TESTS * DISINTEGRATION: >It is the time required for the tablet to break into particles, >the disintegration test is a measure of the time required under a given set of conditions for a group of tablets to disintegrate into particles.* The Disintegration Apparatus: »This apparatus consists of a basket rack containing 6-open-ended glass tubes held in a vertical position. A number 10-mesh stainless steel wire sereen is attached to the bottom. One tablet is placed in each tube and the basket rack is positioned in a |- L beaker of water, simulated gastric fluid (pH 1.2), or simulated intestinal fluid (pH 7.4), at 37 °C. A standard motor driven device is used to move the basket assembly containing the tablets up and down through a distance of 5 to 6 cm at a frequency of 23 to 32 cycles per minute, perforated discs may also be used in the test. These are placed on the top of the tablets. These discs are useful for tablets that float. ®»To be in compliance with standards, the tablets must disintegrate, and all of the particles must pass through the 10-mesh screen in the time specified.* Limits: - Uncoated tablets should disintegrate within 30 minutes: Enteric coated tablets are to show no evidence of disintegration after 1 hour in simulated gastric fluid. These tablets are then tested in simulated intestinal fluid and are to disintegrate in 2 hours plus the time specified in the monograph. >If one tablet fails to disintegrate, the disintegration test is repeated on 12 additional tablets. Not less than 16 out oh the total 18 tablets tested disintegrate completely within 30 minutes,* Uniformity of Active Ingredients: It is measured to ensure a constant dose of drug between individual drugs > Traditionally, dose variation between tablet is tested in two separate tests namely >» A) Weight variation >B) Content uniformity* WEIGHT VARIATION: >Uniformity of weight is an in-process test parameter which ensures consistency of dosage units during compression. The weight of the tablets being made should be routinely measured to help ensure that the tablet contains the proper amount of drug.* Procedure: >Weigh 20 tablet selected at random, each one individually X1, X2, X3... X20 > Determine the average weight. X= (X1+X2 +X3+...+ X20 )/20 Not more than 2 of the individual weights deviate from the average weight (x) by more than the % deviation given below & none deviates by more than twice that %. + Limits: > Weight of tablet 80 mg or less, then % deviation = +10% > Weight of tablet >80 - <250 mg, then % deviation = +7.5 > Weight of tablet 250 mg or more, then % deviation = +5%+ CONTENT UNIFORMITY: >Uniformity of Content is a pharmaceutical analysis parameter for the quality control of tablets. Multiple tablets are selected at random and a suitable analytical method is applied to assay the individual content of the active ingredient in each tablet. * Method: ® Randomly select 30 tablets. 10 of these assayed individually. »The Tablet pass the test if 9 of the 10 tablets must contain not less than 85% and not more than 115% of the labeled drug content and the 10th tablet may not contain less than 75% and more than 125% of the labeled content. ¥If these conditions ate not met, remaining 20 tablet assayed individually and none may fall out side af the 85 to 115% range.* DISSOLUTION TEST: » The release of drug from the tablet into solution per unit time under standardize condition is called dissolution test. » Media used in dissolution testing may be purified water, simulated gastric fluid, simulated intestinal fluid or others. * Dissolution Apparatus 1. Rotating basket method 2. Paddle method 3. Reciprocating cylinder method 4. Flow through cell method 5. Paddle over disk method 6. Cylinder method 7. Reciprocating disk method* PROCEDURE: APPARATUS-1 (BASKET TYPE): ® A single tablet is placed in a small wire mesh basket attached to the bottom of the shaft connected to a variable speed motor. »The basket is immersed in a dissolution medium (as specified in monograph) contained in a 1000 ml flask. >The flask is cylindrical with a hemispherical bottom. The flask is maintained at 37+0.5 0 C by a constant temperature bath. The motor is adjusted to turn at the specified speed and sample of the fluid are withdrawn at intervals to determine the amount of drug in solutions.APPARATUS-2 (PADDLE TYPE): + It is same as apparatus-1, except the basket is replaced by a paddle. * The tablet is allowed to sink to the bottom of the flask before stirring. * For dissolution test U.S.P. specifies the dissolution test medium and volume, type of apparatus to be used, rpm of the shaft, time limit of the test and assay procedure. + The test tolerance is expressed as a % of the labeled amount of drug dissolved in the time limitQUALITY CONTROL TEST for Capsules Presented by: M. Talha AfzalCAPSULES * QUALITY CONTROL TEST for Capsules includes: > Weight variation > Content uniformity > Disintegration > Dissolution* CONTENT UNIFORMITY TEST: > This test is performed when specified by individual monographs. >In this case, 30 capsules are selected, 10 of which are assayed by the specified procedure. The requirements are met if 9 of the 10 are within the specified potency range of 85 to 115%, and the tenth is not outside 75 to 125 %. If more than 1, but less than 3, of the first 10 capsules fall out side the 85 fo 115 % limits, the remaining 20 are assayed The requirements are met if all 30 capsules are with in 75 to 125 % of the specified potency range, and not less than 27 of the 30 are within the 85 to 115% range.+ DISINTEGRATION: » The disintegration of capsules is different from those of tablets because the determination of end point is difficult owing to the adhesive nature of shell. The shell pieces after disintegration may agglomerate forming large mass of gelatin taking more time to dissolve and may adhere to the aries thus, blocking the holes. > According to USP, pes one dosage unit in each of the tubes of the basket with water or any other specified medium (depends on individual monograph) maintained at 37 + 2C. » Observe the capsules for a time limit (specified in individual monograph), at the end of prescribed time, all of the capsules must have been disintegrated excluding the fragments from the capsule shell. + The capsules pass the test ift >No residue remains on the screen of the apparatus, Ifa residue remains, it consists of fragments of shell.* WEIGHT VARIATION; >For hard capsules: Accurately weigh 10 capsules. By suitable means the contents of each capsule should be removed. The weights of emptied shells should be recorded individually. The difference of both the weights will yield the net weight of the contents. Then calculate acceptance value. ® For soft capsules: pre weigh 10 capsules. Cut the capsules by suitable means (either scissors or any open blade) remove the contents by washing with a suitable solvent and let the solvent evaporate by placing them at room temperature for about 30 mins. Weigh the individual shells. Calculate the acceptance value + Limit: Not more than two of the individual weights deviate from the average weight by more than the percentage deviation shown in the the table, and none deviates by more than twice that percentage.* DISSOLUTION TEST: » The dissolution test is carried out using the dissolution apparatus >The capsule is placed in a basket , and the basket is immersed in the dissolution medium and caused to rotate at a specified speed >The dissolution medium is held in a covered 1000mI glass vessel and maintained at 37¢ +-0.5 by means of a constant temperature suitable water bath. >The stirrer speed and type of dissolution medium are specified in the individual monographQuality Control tests for Syrups And ElixirsSyrup:- Syrup:- “Syrups are concentrated aqueous preparations of a sugar or sugar substance with or without flavoring agents and medicinal substances.” ‘Components of Syrup: (1) Sugar or its substitute (2) Medicinal agent (3) Flavourent (4) Colourent ete. Elixirs:- “A clear, sweetened, hydro-alcoholic liquid intended for oral use; elixirs contain flavoring substances and are used either as vehicles or for the therapeutic effect of the active medicinal agents.”Viscosity Measurement Viscosity is a property of liquids that is directly related to the resistance to flow. viscosity measurement is very important quality control test in case of syrups and elixirs. viscosity and consistency directly relates with stability of solutions. T viscosity Jomnee of stability.Methods used for Measurement of ViscosityMethod 1 (U tube viscometer) Apparatus:- The apparatus consists of a glass U tube viscometer made of clear borosilicate glass and constructed in accordance with the dimensions given in official books. The monograph states the size of viscometer to be used.Method * Fill the viscometer with the liquid being examined through tube L to slightly above the mark G, using a long pipette to minimize wetting the tube above the mark. * Place the tube vertically in a water bath and when it has attained the specified temperature, adjust the volume of the liquid so that the bottom of the meniscus settles at the mark G. + Adjust the liquid to a point about 5 mm above the mark E. + After releasing pressure or suction, measure the time taken for the bottom of the meniscus to fall from the top edge of mark E to the top edge of mark F.Method II (Capillary viscometer method) (Ph. Eur. method 2.2.9) + The determination of viscosity using a suitable capillary viscometer is carried out at a temperature of 20 £ 0.1 °C, unless otherwise prescribed. + The time required for the level of the liquid to drop from one mark to the other is measured with a stop-watch to the nearest one-fifth of a second. * The result is valid only if two consecutive readings do not differ by more than | per cent. + The average of not fewer than three readings gives the flow time of the liquid to be examined.Calculate the dynamic viscosity 4 in mili-pascal seconds using the formula: q=kpt ‘= constant of the viscometer p= density of the liquid to be examined expressed in milligrams per cubic millimeter, @ flow time, in seconds, of the liquid to be examined. The constant & is determined using a suitable viscometer calibration liquid.Method III (Rotating viscometer method) (Ph. Eur. method 2.2.10) The principle of the method is to measure the force acting on a rotor (torque) when it rotates at a constant angular velocity (rotational speed) in a liquid. * Rotating viscometers are used for measuring the viscosity of Newtonian (shear-independent viscosity) or non-Newtonian liquids (shear dependent viscosity or apparent viscosity), + Rotating viscometers can be divided in 2 groups, namely absolute and relative viscometers. + In absolute viscometers the flow in the measuring geometry is well defined. The measurements result in absolute viscosity values, which can be compared with any other absolute values.In relative viscometers the flow in the measuring geometry is not defined. The measurements result in relative viscosity values, which cannot be compared with absolute values or other relative values if not determined by the same relative viscometer method. Different measuring systems are available for given viscosity ranges as well as several rotational speeds.Weight/mL * Important for tinctures and extracts and defined as: “The weight per milliliter of a liquid is the weight expressed in grams of 1 milliliter of a liquid when weighed in air at the specific temperature”. * Almost equal to density of the liquid. Expressed in g/ml Procedure: For this, — Weigh a clean and dry pycnometer accurately — Fill it with liquid, remove any access and weigh — Determine the weight of liquid by subtraction Determine wt./ml by dividing the weight of liquid that filled the pycnometer by the capacity of pycnometerSUPPOSITORIES “Suppositories are solid dosage forms intended for insertion into body orifices where they melt, soften, or dissolve and exert localized or systemic effects’. It comes under semi solid preperation because it is prepared by melting all ingredient. ae1. APPEARANCE o It includes = Odour = Colour = Surface condition = Shape.WEIGHT UNIFORMITY © Weigh 20 suppositories individually. wl, w2, w3....w20 © Weigh all the suppositories together © Calculate the average weight = W/20. © Limit: Not more than 2 suppositories differ from the average weight by more than 5%, and no BaD DUSIEI differs from the average weight by more than 10%,MELTING RANGE TEST « 1-The suppository is completely immersed in the constant temperature water bath, andthe time for the entire suppository to melt or disperse in the surrounding water is measured.DISINTEGRATION TEST... © The suppository is considered disintegrated when: = A- It is completely dissolved or =B- Dispersed into its component part. =C- Become soft “change in shape” with formation of * core which is not resistant to pressure with glass rod.Method: © Place a suppository on the lower perforated disc of metal device and then insert the device into the cylinder and attach thisto the sleeve. Repeat the same operation with further two suppositories, metal device and sleeve. Place each piece of apparatus ina ve: containing at least five liters of water at 37C and fitted with a slow stirrer and by means of holding the top of apparatus 90 mm below the surface of water. After every 10 minutes invert each apparatus without removing it from water.Disintegration is complete when molded suppositories are: « Completely dissolved. Dispersed into its components. Have become soft. 1 Disintegration occurs in not more than 30 minutes for fat based suppositories and for water-soluble suppositories disintegration occurs in not more than 60 minutes.LIQUEFACATION TIME OR SOFTENING TIME TEST o In this test a U tube is partially immersed in a constant temperature bath and is maintained at a temperature between 35 to 37°C. There is a constriction in the tube in which the suppository is kept and above the suppository, a glass rod is kept. The time taken for the glass rod to go through the suppository and reach the constriction is known as the liquefaction time or softening time Another apparatus is there for finding “softening time” which mimics in vivo conditions. It uses a cellophane tube, and the temperature is smainiaines by water circulation. Time taken for the suppository to melt is noted.DISSOLUTION TEST © By using different types of apparatus such as wire mesh basket, or dialysis tubing is used to test for in vitro release from suppositories© Two types of apparatus are avaliable for testing the dissolution rate. They are, » SUPPOSITORY DIALYSIS CELL - lipophilic suppositories are tested using suppository dialysis cell. = STATIONARY BASKET — rotating paddle apparatus (USP dissolution test apparatus). Hydrophilic suppositories are tested using stationary basket.STABILITY TESTING © Cocoa butter suppositories on storage, “bloom”; i.e., they form a white powdery deposit on the surface. This can be avoided by storing the suppositories at uniform cool temperatures and by wrapping them in foils. Fat based suppository harden on storage. © There is an upward shift in melting range due to slow crystallization to the more stable polymeric forms of the base. © The softening time test and differential scanning calorimetry can be used as stability indicating test methods. if we store the suppositories at an elevated temperature, just below its melting range, immediately after manufacture, the aging process is speeded. oQUALITY CONTROL TESTS FOR PARENTRALS Presented by: M. Talha AfzalContents * Uniformity of content + Test for volume of liquid * Test for pyrogen * Test for sterility * Clarity of solution + Test for bacterial endotoxin + Leakage testPARENTERALS Term derived from Greek words “Para” outside & “Enteron” intestine. * Parenterals are sterile solution/suspension of drug in aqueous or oily vehicle. * Parenteral drugs are administered directly into the veins, muscles or under the skin, or more specialized tissues such as the spinal cord. + Term parenteral used for any drug/fluid whose delivery does not utilize the alimentary canal for entering into the body tissues. * Parenteral preparations are supplied in glass , plastic container and prefilled syringes with closures are made up of plastic or elastomer.‘Categories of parenteral preparations: Q Injections or infusions Q Concentrates for injections or infusions Q Powders for injection or infusions Q Gels for injections Q Implants Unique Characteristics of Parenterals: Sterile Particulate-free Pyrogen free Stable for intended use pH — not vary significantly Osmotic pressure similar to bloodQUALITY CONTROL TESTINGGeneral areas of Quality control Three General Are: * 1. Incoming Stock: Routine work testing * 2, Manufacturing: Include numerable tests, reading and observations through out the manufacturing process * 3, Finished Products: Sterility test, Pyrogen test, Clarity test, Leaker test.In process Quality control tests * Conduetivity measurement Volume filled + Temperature for heat sterilized product Environmental control tests Visual inspectionTESTS FOR PARENTERALS Finished product Quality control tests: There are mainly five Quality control test for the parenterals are performed. 1) LEAKER TEST 2) CLARITY TEST 3) PYROGEN TEST 4) STERILITY TEST 5) CONTENT UNIFORMITY TESTUNIFORMITY OF CONTENT + 30 sterile units are selected from each batch. © The weight of 10 individual sterile units is noted and the content is removed from them and empty individual sterile unit is weighed accurately again. + Then net weight is calculated by subtracting empty sterile unit weight from gross weight. + The dose uniformity is met if the amount of active ingredient is within the range of 85-115% of label claim.* Relative standard deviation is equal to or less than 6.0%. * If one unit is outside the range of 85-115.0%, and none of the sterile unit is outside the range of 75-125.0% or if the relative standard deviation of the resultant is greater than 6.0% ,or if both condition prevail, an additional 20 sterile unit should be tested. * The sterile units meet the requirements if not more than one unit is out side the range of 85-115%, no unit is outside the range of 75-125.0% and the calculated relative standard deviation is 7.8%.TEST FOR VOLUME OF LIQUID Test applies to liquid supplied in single dose , only part of the content is used. Empty the contents of one container & determine the volume of contents. For Emulsions & suspensions, shake the container before the determination The volume should not be less than the amount stated on the label.NOME rt Not more than 100 containers More than 100 but not more than 500 containers More than 500 containers For large volume parenterals 10% or 4 container 10 containers 2% or 20 containers whichever is less 2% or 20 containers whichever is less1) LEAKER TEST Q Leakage occurs when a discontinuity exists in the wall of a package that can allow the passage of gas under the action of a pressure or concentration differential existing across the wall. O Presence of capillary pores or tiny cracks can cause microbes or other dangerous contaminants to enter the ampoules or package or may lead to the leakage of contents to outside. This may cause contamination of the sterile contents and also spoilage of appearance of the package. OChanges in temperature during storage can cause expansion and contraction of the ampoule or package and thereby causing interchange of its contents if an opening exists,Leaker test is employed to detect incompletely sealed ampoule so that they may be discarded, * To test the package integrity. + Package integrity reflects its ability to keep the product in and to keep potential contamination out. Leaker tests are 4 types: a) visual inspeetion b) bubble test c) dye test d) vacuum ionizationa) Visual inspection + Visual inspection is the easiest leaker test method to perform. * The method is used for the evaluation of large volume parenterals. + To increase the sensitivity of the method the visual inspection of the sample container may be coupled with the application of vacuum to make leakage more readily observable. + This method is simple and inexpensive. + Disadvantage: less sensitive + Sensitivity is increased by applying pressure/vacuum.b)Bubble test = The test package is submerged in liquids. = A differential pressure is applied on the container. = The container is observed for bubbles. = Sometimes, surfactant added liquid is used for immersion of test package. = Any leakage is evident after the application of differential pressure as the generation of foaming in immersion liquid. = The method is simple and inexpensive. * The location of the leaks can be observed in this method.Generation of a differential positive pressure of 3 psi inside the vial and observation of any leakage using magnifying glass within a maximum test time of 15 minutes, However, it is relatively insensitive and the findings are operator dependent and are qualitative. The optimized conditions can be achieved using a surfactant immersion fluid along with the dark background and High intensity lighting.C)Dye test The test container is immersed in a dye bath. Vacuum and pressure is applied for sometime. The container is removed from the dye bath and washed. The container is then inspected for the presence of dye either visually or by means of UV spectroscopy. The dye used is usually 0.5% to 1% methylene blue. The dye test can be optimized by use of a surfactant and or a low viscosity fluid.The dye test is widely accepted in industry and is approved in drug use, The test is inexpensive and is requires no special equipment required for visual dye detection. However, the test is qualitative, destructive and slow. The test is used for ampoules.D)Vacuum ionization test > Vacuum ionization test is useful for testing leakage in the vials or bottled sealed under vacuum. » This test is used for testing of the lyophilized products. » High voltage, high frequency field is applied to vials which to cause residual gas, if present to glow. » Glow intensity is the function of headspace vacuum level. > The blue glow is the indicative of yacuum while the purple glow indicative of no vacuum.vv wv The sensitivity of the method is not documented. This test is rapid and is non destructive test. However, the proteins present in the test sample may be decomposed. This method is used for the lyophilized vials of biopharmaceuticals2) CLARITY TEST Definition: Clarity is a relative term, its mean a clear solution having a high polish conveys to the observer that the product is of exceptional quality and purity. Clarity test is carried out to check the particulate matter in the sample. It is practically impossible that every unit of lot is perfectly free from visible particulate matter ,that is, from particles that are 30 to 40 micrometer and large in size.PARTICULATE MATTER TEST * Particulate matter refers to the extraneous, mobile, undissolved particles, other than gas bubbles, unintentionally present in the solutions. 2 methods are used: (i)Light obstruction Particle Count Test (ii) Microscopic particle count testLIGHT OBSTRACTION PARTICLE COUNT TEST * Use a suitable apparatus based on the principle of light blockage which allows an automatic determination of the size of particles and the number of particles according to size. T of Particl © Deincted Particle $$ Lit Trap 90 Degree eet Detector l) —_ | Reflector J Flow Nozzle 90 Degree Optics <——$ Laser Beam ———————£§_ Ligh Source Laser Diode Or Hote LaserLimits: Ve taeCa Cece EOC particles. 10 Average in the units LVP= 100 ml 25 tested 25 per ml 3 per ml SVP — 100 ml and 10 6000 per container less than 100 ml 25 600 per containerMICROSCOPIC PARTICLE COUNT TEST + Wet the inside of the filter holder fitted with the membrane filter with several milliliter of particle-free water . + Transfer the total volume of a solution pool or of a single unit to the filtration funnel, and apply vacuum. Place the filter in a Petri dish and allow the filter to air-dry. + After the filter has been dried, place the Petri dish on the stage of the microscope, scan the entire membrane filter under the reflected light from the illuminating device, and count the number of particles.LVP > 100 ml SVP — 100 ml and less than 100 ml 10 25 Average in the units tested 12 per ml 2 per ml 3000 per container 300 per container3. PYROGENS TEST PYROGENS: * Pyrogens are fever producing substances. * Pyro means ‘pyrexia’, Gen means “producing”. * Pyrogens are the by-products of microorganisms mainly of bacteria, molds and viruses. + During the processing these pyrogens may come from water, active constituent or the excipient or from the equipments. * Chemically these pyrogens are lipid substances associated with carrier usually polysaccharides or may be proteins.Types of Pyrogen test For Detection and quantification of Pyrogens: Basically there are 2 tests performed to detect the presence of pyrogens in sterile parenteral products. they are: 1) In Vivo pyrogen test (Rabbit Test) 2) In Vitro pyrogen test (Limulus Ameabocyte Lysate Test)1) In Vivo Pyrogen test (Rabbit Test) PRINCIPLE: This test consists of measuring the rise in body temperature evoked in rabbits by the injection of a sterile solution of the substance being examined, Procedure: The test involves measurement of the rise in body temperature of rabbits following the IV injection of a sterile solution into ear vein of rabbit. * Dose not exceeding 10 ml per kg injected intravenously within a period of not more than 10 min.+ Test animals: Use healthy, adult rabbits of either sex, preferably of the same variety. + Recording of temperature: Clinical thermometer PRELIMINARY TEST (SHAM TEST) If animals are used for the first time in a pyrogen test or have not been used during the 2 previous weeks condition them | to 3 days before testing the substance by injecting IV 10m] per'kg pyrogen free saline solution warmed to about 38.5°C. + Record the temperature of the animals beginning at least 90 min before injection and continuing for 3 hours after injection. + Any animal showing a temperature variation of 0.6° or more must not be used in main test,MAIN TEST + Carry out the test using a group of 3 rabbits. Preparation of the sample: Dissolve the substance in or dilute with pyrogen free saline solution . Warm the liquid to approximately 38.5° before injection. iPROCEDURE + Inject the solution under examination slowly into the marginal veins of the ear of each rabbit over a period not exceeding 4 min. * Record the temperature of each animal at halfhourly intervals for 3 hours after 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.INTERPRETATION OF RESULT NUMBER OF Ole a RABBITS DOES N | 3 RABBITS 115°C 2,65°C 6 RABBITS 2.80°C 430°C 9 RABBITS 4.45°C 5.95°C 12 RABBITS. 6.10°C 7.60°CIn Vitro Pyrogen test Limulus Ameabocyte Lysate(LAL Test) Measures the concentration of bacterial endotoxin. Test is using lysate derived from hemolymph cells or amoebocytes of horse shoe crab. Endotoxin limit calculated by K/M K- maximum no.of endotoxin which receive the patient without suffering toxic reaction. M- maximum dose administered to a patient/kg/hrMechanism of LAL Test enzymes located within the crab's amoebocyte blood cells + endotoxins initiation of an i coagulation cascade proteinaceous gelLAL Reagent Bleeding adult crabs blood into an anticlotting solution Washing and centrifuging to collect the amaebocytes. Lysing in 3% NaCl. Lysate is washed and lyophilized for Storage. Procedure: Test: Equal volume of LAL reagent and test solution (usually 0.1 ml of each) are mixed in a depyrogenated test-tube Incubation at 37°C, for 1 hour Remove the tube — invert in one smooth motion (180°) Observe the resultDifferent Techniques: Three different techniques: The gel-clot technique — gel formation The turbidimetric technique — the development of turbidity after cleavage of an endogenous substrate The chromogenic technique — the development of color after cleavage of a synthetic peptide — chromogen complexGel Clot Technique A solid gel is formed in the presence of endotoxins This technique requires positive and negative controls Positive controls — a known concentration of endotoxin added to the lysate solution. Negative controls — water, free from endotoxins, added to the lysate solution. Turbidimetric Technique The test is based on the measurement of opacity change due to the formation of insoluble coagulin. * Opacity is directly proportional to the endotoxin concentration * This technique is used for water systems and simple pharmaceutical products.Chromogenic Technique * This is based on the measurement of color change which is caused by the release of the chromogenic chemical * p-nitroanilide * The quantity of the p-nitroanilide produced is directly proportional to the endotoxin concentrationTEST FOR STERILITY Sterility is defines as freedom from the presence of viable Microorganism. Vis TCO Seng reerMedia to be used in the sterility test Two types of media are used. ital Sierras) rabtorg A (Cahir) Thioglycolate AtenMEMBRANE FILTRATION METHOD:- + Amembrane has a nominal pore size not greater than 0.451 and diameter of approximately 50mm. « This method basically involves filtration of Sample through membrane filters. + The filtration is assisted under strict aseptic condition, after filtration complete the membrane is cut into 2 halves and one halve is placed in suitable volume of ( 100 ml usually) FIM, SCDM medium. * Incubate the media for not less than 14 days.DIRECT INOCULATION METHOD:- The DI methed is the more traditional sterility test method. Basically, the DI method involves three steps: |. Aseptically opening each sample container from a recently sterilized batch of product. 2. Using a sterile syringe and needle to withdraw the required volume of sample for both media from the container. 3. Injecting one-half of the required volume sample into a test tube containing the required volume of FTM and the other half volume of sample into a second test tube containing the required volume of SCD.MINIMUM QUANTITY TO BE USED FOR EACH MEDIUM Minimum quantity used is.. PC aT Cy Liquids 1. less than 1 ml The whole contents of each container 2. 1-40 ml Half the contents of each container but not less than | mi 3,Greater than 40 ml and not 20 ml greater than 100 ml 4. Greater than 100 ml 10 per cent of the contents of the container but not less than 20 ml Antibiotic liquids ImlINTERPRETATION OF RESULTS + If the material being tested renders the medium turbid so that the presence or absence of microbial growth cannot be easily determined by visual inspection,14 days after the beginning of incubation , transfer portion (< | ml) of the medium to fresh vessels of the same medium and then incubate original and transfer vessel for not less than 4 days. «If No evidence of microbial growth is found- complies with test for sterility. * If evidence of microbial growth is found- does not complies with test for sterility.