Parenterals Sterile products are those that are used to be administered by injection, infusion, or implantation into the body. (or) Sterile dosage forms intended for administration by injection under or through one or more layers of the skin; exert their action by directly entering into the systemic blood which are prepared with a higher degree of care and skills higher than utilized in preparing conventional oral or topical products. The finished parenteral product must be sterile, non-pyrogenic & free from extraneous insoluble materials. This is will be heavy responsibility on pharmaceutical industry & pharmacist to practice cGMPs & to practice good aseptic practices (GAPs).
QUALITY CONTROL OF PARENTERALSThere are 5 types of test for parenterals and they are: Content uniformity, Leaker Test/ Package Integrity Test, Clarity Test/ Particulate Matter Testing, Pyrogen Testing, Sterility Test. 1. Content uniformity The sterile solids used in parenteral preparation contents should be according to specifications.
For this purpose 10 sterile units are taken. The content of active ingredient in each sterile unit is calculated by performing assay which is according to individual monographs. Limits are 85 – 115% > for all 10 units.If 9 unit = 85 – 115% &1 unit = 75 – 125%;Then – test carried out on 20 more units. So for all 30 units – limits are 29 units = 85-115% &2.
unit = 75-125% 2. Leaker Test/ Package Integrity Test This test is employed for the package integrity. Package integrity means its ability to keep the product in & potential contamination out. Leakage occurs problem exists in wall of package which allow passage of gas under action of pressure or concentration differential existing across wall. Leaker Test MethodsA) Visual Inspection: The Package Visual inspection is the easiest leak test method to perform but is least sensitive.
This test is used for the evaluation of vials, ampoules & large volume parenterals. To increase sensitivity of the method may be coupled with application of vacuum to make leakage more readily observable. The advantage if this method is that it is inexpensive & disadvantage is that it is only qualitative and less sensitive. B) DYE BATH TEST: The test is used to detect leaks in parenterals. The presence of any cracks can cause the entrance of microbes or other dangerous to ampoules. Leaks in ampoule can also lead to contents outside.
This leads to contamination of sterile contents & also the appearance of the package. The procedure is by detection in a visible manner, the ampoules are placed in vacuum chamber and completely dipped in 0.5-1% methylene blue solution and negative pressure is applied, which cause penetration in opening. The negative pressure is applied for 30 minutes and about 27 inches Hg. C) Bubble test: This test is used for vials & bottles in which sufficient gaseous is allowed.
The formation of detectable bubbles or foaming of applied surfactant shows leakage in the package. There are 2 two methods, in first one the container are submerged in liquid and differential pressure is applied and inspect the bubbles. In 2nd method a surfactant is applied to package and pressurized and D) VACUUM IONIZATION TEST (Residual gas ionization test/ the spark coil test): This test is useful for testing leakage in vials or bottles sealed under vacuum. A high-frequency field, high-voltage is applied to bottles or vials which cause the ionization of residual headspace gas. As the ions and electrons recombine a visible light is observed. It will observe manually by locating a conducting plate near the bottle wall which measures the level of ionization current. The current or visible light indicates the loss of vacuum within the bottles which indicates the leakage.
E) WEIGHT CHANGE TEST: For this test filled & sealed containers are monitored for weight loss (for liquid-filled packages) or weight gain (for dry-filled packages) over time. The test conditions may include container storage at extremes of relative humidity and/or temperature. 3. Clarity Test/ Particulate Matter Testing: This test is carried out to check particulate matter in sample. Particulate matter is “Matter of biological or non-biological origin & with observable length, width, and thickness” e.g: bacteria, fungi, dirt, lint, fibres, plastic, dust, rubber, etc. It may me mixed accidentally during manufacturing in parenteral product. It is universally accepted that the particles size of 50 ?m is detected visually by naked eye.
Requirement for injectable products by USP: Each final container should be subjected individually to physical inspection. Every container whose contents show contamination with visible foreign material will be rejected. Inflammatory, Antigenic response or Occlusion of blood vessels is occurs due to these particulate matters.Sources of these particles may be add with excipients or in processes of packing materials including glass, rebber, plastics etc or personnel hairs or lint etc.
Methods of monitoring particulate matter contamination:
A) VISUAL METHOD: The filled containers are examined against illuminated screen by holding neck & rotating it slowly or inverted it to check out the foreign matter.
B) COULTER COUNTERS METHOD: The method Based on electrical resistance and is used for detection of particles less than 0.1 um in diameter. Sample is evaluated between two electrodes, If particle found resistance of electrodes is increased.
C) LIGHT BLOCKAGE HIAC COUNTER METHOD: This method is based on light scattering or light-blockage principle which give quantitative results of particles in parenteral solution.
D) FILTRATION METHOD: This method is used for counting particles in hydraulic fluids. Sample are passed through filter, the materials retain o filter are evaluated under microscope.
B.P LIMIT TEST FOR PARTICULATE MATTERFor Small volume parenterals (SVP) (Volume ?100ml) B.P has not yet proposed any numerical standards.
Container should be clear & practically free from visible particles. For Large volume parenterals (LVP) (Volume >100ml) If only one container is available for examination so the number of particles (? 2?m) = 2000/ml and for particles (? 5?m) = 200/mlUSP LIMIT TEST FOR PARTICULATE MATTERFor Small volume parenterals (SVP) (Volume ?100ml): USP recommends particle sizes of 10?m & 25?m via examining with light blockage HIAC counter method.For Large volume parenterals (LVP) (Volume >100ml): USP recommends particle sizes of 10?m & 25?m. It is Determined by using microscopic counting after membrane filtration.
NOTE: USP requires that all the parenterals (SVP or LVP) will be essentially free from visible particulate materials. 4. Pyrogen Testing All pharmaceutical parenterals must be free from pyrogens. Pyrogen are heat or fever generating substances, in sufficient amount it cause fever, chill, joint pain, headache, rise in arterial blood pressure, decrease in respiration etc within 30-120 min which may subside within 10-12 hours. The most common response is increase in body temperature.
Pyrogens are classified into exogenous: endotoxins i.e lipopolysacchrides(LPS) and endogenous pyrogens. Pyrogens are heat stable which can withstand with normal sterilization temperatures.Detection and quantification of Pyrogens: There are two tests for pyrogens recognized by pharmacopeia. (a) In-vivo pyrogen test/ RPT (Rabbit Pyrogen Test)and (b) In-vitro pyrogen test/ LAL (Limulus Amebocyte Lysate) Test A) In-vivo pyrogen test / Rabbit test: This test became an official QC test in USP, 12th edition for parenterals in 1942. It will detects LPS & other pyrogens, by measuring elevation in body temperature in rabbits by intravenous injection of a sterile solution of substance under test. The principle is based on the fact that the human & rabbits are equally responsive to pyrogen injected intravenously on a dose per weight basis.
Test conditions / Requirements: Some healthy adult rabbits having weight not less than 1500 gm (1.5kg) which is properly maintained in terms of environment & diet prior to performance of test. Their temperature should not differ more than 1°C from each other.
Rabbit having temperature more than 39.8°C or less than 38.0°C are excluded from the test. To hold the rabbits, boxes are required.
The boxes hold rabbits so that temperature can be noted easily during test. Before using a rabbit for the first time in a pyrogen test condition it not more than 7 days before use by a sham test. Sham test (Preliminary test): An I/V injection of pyrogen-free saline solution are administered 10ml/Kg of body weight and record temperature of the animals.
For 3 hrs after injection being examined. Any rabbit showing a temperature variation of 0.6 °C or more should not be used in the main test.
All the apparatuses, glassware, syringes, needles, containers, diluents (used in test) must be pyrogen free. Thermometer such type is used in test are automatic and record temperature during pyrogen testing. Procedure for the main test: The baseline temperature of three rabbits is determined. The sample (after dissolving or dilution & warming at 37±2°C) are injected into ear vein of 3 rabbits which are held in retaining boxes. Injected Dose (10ml/kg of body weight) is used or unless specified in individual monograph and injection are completely injected within 10 minutes. After injection of sample temperature of each rabbit is determined at 1, 2, & 3 hrs. The difference between temperatures of each rabbit is noted.
Any increase in temperature is taken to be the response of sample injected which Interpret of the results. The material under examination should meets USP requirements for apyrogenicity, if no rabbit shows an individual rise of 0.6°C, or more above its respective control temperature. The sum of 3 individual maximum temperature do not exceed 1.4°C.
If anyone of the rabbit doesn’t meet the above conditions then test is repeated by using additional 5 rabbits & result is considered for 8 rabbits. Requirements are met if not more than 3 out of 8 rabbits shows an individual rise in temperature of 0.6 °C or more, & the sum of 8 individual maximum rises in temperature does not exceed 3.7 °C. If differ- ence is negative, result of rabbit test is counted as zero response & sample is considered apyrogenic.
B) In-vitro pyrogen test / LAL Test: LAL (Limulus Amebocyte lysate) officially it is termed as bacterial endotoxin test (BET). The terms Amebocyte means mobile cells which can be moving pseudopodially like an amoeba in invertebrates body or blood. Term Lysate is an enzyme system present in amebocytes of horse shoe crab (Limulus Polyphemus). Horseshoe crab is arthropod that lives primarily in shallow ocean waters on soft sandy or muddy bottoms. Limulus Amebocyte lysate (LAL) is the aqueous extract of blood cells from horseshoe crab (Limulus Polyphemus). In this test LAL reagent reacts with bacterial endotoxin which detect and quantify bacterial endotoxins.
In contrast to rabbit test which detects LPS as well as other pyrogens LAL test detects only LPS. This test became official USP test in 1985. The principle of the test is based on clotting of lysate of Amebocyte in the presence of pyrogens (LPS). The extract from blood cells of horse shoe crab when reacts with pyrogens so increases in viscosity & opacity of mixture until an opaque gel is formed. This reaction completes in 15-60 minutes depending on concentration of pyrogens after mixing. If more pyrogens are there, the gel will be more turbid and thick.
Preparation of LAL: This enzyme is prepared by removing blood from horseshoe crab’s pericardium by puncturing his heart and then the crabs are returned to the water. The blood cells (Amebocytes) are separated from serum by centrifugation & then placed in distilled water, which causes them to swell up and burst due to osmotic effect. Enzyme Lysate is released from inside of cell which is then purified & freeze-dried. Lyophilization of enzyme is necessary for stability purposes.
LAL is extremely sensitive to heat and must be stored in the freezer. On the time of reconstitution must check date of production because LAL has a shelf life of 1 month storage at freezing conditions. On performing of LAL test lysates are assayed with purified endotoxins & accepted if it detects 0.001ug/ml or less concentration of purified endotoxins. Also any other interfering substances present in sample must also be removed before the test.
Procedure: The pH of test sample is adjusted. Then the test solution & standardized LAL are mixed in equal parts (0.05-0.2ml) performed in sterilized glass test tubes (10 x 75mm). The mixture is incubated immediately at 37±1°C for 1 hour in glass test tube and must be remained undisturbed completely because agitation may destroy the gel which leading to a false negative result. The tube is observed after specified time.
Formation of a firm gel is representative of positive test, while formation of a viscous gel indicates a negative reaction. Following criteria for LAL test result. Advantages of LAL test: In-vitro test & does not require animal handling, thus is more convenient to pyrogen test and require less laboratory facilities and minimum equipments. It is 10 times more sensitive than that of in-vivo rabbit test. It Consumes less time; i.e., 1 vs 3 hours required by rabbits test. It require less test volume; about 0.
1 ml of test solution5. Sterility TestSterility tests are attempts to detect the presence or absence of viable micro-organisms in a sample number of containers taken from batch of product. Sterility is uncompromising requirement of an injectable dosage form. Unlike internal administration, parenteral administration of drugs avoids many of the natural protective defences of the body. If Injection is contaminated product, it will cause multiple complications to a potentially immune-compromised patient. Sterility means the freedom from all viable micro-organisms and parenteral products must meet requirements of sterility test; when all culture media vessels incubated with product sample reveal no evidence of microbial growth (turbidity). If any type of microbial growth is detected sterility test is failed. Retesting is only allowed if clear proof of accidental contamination due to operator mistake.
Test when repeated in second stage, double number of samples will be tested. The evidence for microbial growth is determined by visual evaluated and inoculates the sample in culture medium. A noxious odour, cloudy or turbid appearance of solution of culture medium indicates microbial growth.. Culture media: USP & EP describe two primary types of culture media to be used in parenteral products for sterility testing.
(a)Fluid Thioglycollate Medium (FTM), (b) Trypticase Soy Broth (TSB) medium Soybean-Casein Digest (SCD).? Table: Ingredients of Fluid Thioglycollate medium and Their Purpose Table: Ingredients of Trypticase Soy Broth medium and Their Purpose STERILITY TEST METHODS: Their are two basic methods for performing sterility tests specified by USP and EP. Direct Transfer (DT) or direct inoculation method. Membrane Filtration(MF) method.
I. Direct Transfer Method: It is the most traditional sterility test method which involves the following steps: 1. Aseptically open each sample container from a recently sterilized batch. 2. Use a sterile needle & syringe to withdraw required volume of sample for both media from container. 3.
Inject half of required volume sample into a test tube having required volume of sterile FTM; & other half into a second test tube having required volume of sterile TSB. 4. Incubate media for some time at a temperature as specified in monographs. 5. After incubation period, media are observed for turbidity. 6. If microbial growth is found the product fails sterility test.
II. Membrane Filtration Method: This method became official in 1970 in 18th edition of USP. It is most widely used method over DT method, their steps are:1. Filter unit is properly assembled & sterilized prior to use. 2. The Contents of specified number of units are transferred to filter assembly under strict aseptic conditions. 3.
Contents are filtered with the addition of a vacuum. 4. Membrane (filter) is removed aseptically & cut in half.
5. One-half of membrane is placed in suitable volume (usually 100 ml) of sterilized FTM, & other membrane half is placed in an equal volume of sterilized TSB. 6. Media are incubated for specified period of time at specified temperature. 7.
After incubation period media are observed for turbidity. 8. Product will fail sterility test if microbial growth is found.
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