Presented ByNishath Fathima

M.Pharm., I year II SemUnder the guidance of

Dr. T. Mamatha, M.Pharm., Ph.D.

Department of Quality Assurance

DefinitionIn process quality control is a process of monitoring critical variables of manufacturing process to ensure a quality of the final product. In-process manufacturing controls are established and documented by quality control and production personnel to ensure that quality of the product is within the acceptable standard range.

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Definition

In the U.S., a biological product is defined as “a virus, therapeutic serum, toxin, antitoxin, vaccine, blood, blood component or derivative, allergenic product, or analogous product, or arsphenanaine, or derivative of arsphenamine (or any other trivalent organic arsenic compound), applicable to the prevention, treatment or cure of a disease or condition of human beings” (Public Health Services Act 42 U.S.C. § 262(i)).

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Biological products includeVaccineImmune sera

Antitoxin Antivenom

ToxoidsBlood and blood componentsAllergenic productsIn-vivo diagnostics

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Sterility test

1. Immersion (Direct Inoculation)

For bacteria - Using petri dishes 10 cm in diameter, add to each dish about 15 ml of a liquefied Soyabean casein digest medium, at not more than 45 °C. Alternatively, spread the preparation on the surface of the solidified medium of a petri dish. Prepare at least two such petri dishes using the same dilution and incubate 30 °C to 35 °C for 14 days. Count the number colonies that are formed.

For fungi - proceed as described in the test for bacteria but use Fluid thioglycollate medium and incubate the plates at 20 °C to 25 °C for 14 days.

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2. Membrane filtrationUse membrane filters which are 50 mm in diameter and having nominal pore size not greater than 0.45 µm. Transfer 10 ml or a quantity of each dilution containing 1 g of the preparation under examination to each of two membrane filters and filter immediately. The filter is then rinsed and then the membrane is transferred into the appropriate medium. (Soyabean Casein digest medium for bacteria and Fluid thioglycollate medium for fungi).Then the membrane is incubated for 14 days in the test medium.

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Interpretation of result: If there is no evidence of microbial growth then the preparation under examination passes the test. Test is repeated if(a) The data of the microbial monitoring of the sterility test facility show a fault(b) A review of the testing procedure used during the test in question reveals a fault(c) Microbial growth is found in negative controls(d) After determination of the identity of the microorganisms isolated from the test, the growth of this species or these species may be ascribed unequivocally to faults with respect to the material and/or technique used in conducting the sterility test procedure

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The test involves measurement of the rise in body temperature of rabbits following the intravenous injection of a sterile solution of the substance under examination.

Test animals: Healthy, adult rabbits of either sex, each weighing not less than 1.5 kg fed on a balanced diet are used for the test.

Dose: Not less than 0.5 ml/kg and not more than 10 ml/kg of the body weight. Method: Insert a clinical thermometer into the rectum of each rabbit and

normal readings of body temperature are taken prior to the injection of test solution. Two such readings are taken at an interval of 30 minutes and the mean is calculated. This reading is taken as initial temperature of the rabbit. The test solution is injected into the ear vein of each rabbit. Record the temperature of each rabbit at an interval of 30 minutes for 3 hours after the injection. The difference between the maximum temperature and initial temperature is taken as response.

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Interpretation

If the first test fails then repeat the test on additional 5 rabbits

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Vaccines are microbial preparations of killed or modified microorganisms that can stimulate an immune response in the body to prevent future infection with similar microorganisms.

Quality control tests for vaccines include1. Staining test2. Sterility test3. Inactivation test4. Pyrogen test5. Freedom from abnormal toxicity

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Approximately 10 mL of the test sample is centrifuged in a pointed centrifuge tube at approximately 2,000 × g for 30 minutes. The sediment or the bottom portion is spread on a slide glass, dried and heat-fixed over a flame. The smear is then stained by the Gram’s method and, unless otherwise specified, examined microscopically at an approximately 1,000-fold magnification.

Criterion for judgment

No bacteria shall be observed other

than those defined in the individual monographs.

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Each purified bulk material shall be tested in mice for effective inactivation of the virus before the addition of preservative and other substances.

The test should be performed with undiluted purified bulk material injected intra-cerebrally into at least 20 mice, each weighing between 15 and 20 g. these mice shall be observed for 14 days. Any symptoms caused by the virus shall be confirmed by immuno-florescence assay. At the end of the observation period, no cytopathetic effects should be observed.

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Test in mice Test in guinea pigs

Take 5 healthy mice weighing 17-22g.

Inject one human dose NMT 1 ml Intra-peritoneally

Observe the mice for 7 days

If more than one animal dies preparation fails the test

If one animal dies repeat the test

Preparation passes the test if no animal dies in the second group

Take 2 healthy guinea pigs (250-350 g)

Inject one human dose NMT 5 ml Intra-peritoneally

Observe guinea pigs for 7 days

If more than one animal dies/shows ill health preparation fails the test

If one animal dies/ shows ill health repeat the test

Preparation passes the test if no animal dies/ shows ill health in the second group

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It contain antibodies to specific bacteria or viruses It is of 2 types• Antitoxin• Antivenom Antitoxin: It contains an antibody capable of destroying

microorganisms including viruses and bacteria. Antivenom: it contains an antibody that is active against

the venom of a snake, spider, or other venomous animal or insect.

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Quality control test includeTest for immunoglobulin content Test for freedom from residual proteolytic enzymeSterility testPyrogen testTest for antitoxin/antivenom content

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1. Immunoglobulin testThe cellulose acetate membrane electrophoretic test is used to analyze the protein constituents in a test sample by differences in mobility of the protein solutions in electric fields. Dilute the test sample with diethylbarbiturate buffer solution (pH 8.6) to render the protein solution approximately 5%.Electrophoresed the solution. After electrophoresis, the membrane is stained with Ponceau 3R. Protein constituents and relative concentrations are analyzed by densitometry.Acceptance criterianot less than 95% of the total proteins shall be immunoglobulin.

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2.Test for residual proteolytic enzymes

When measured by a suitable method for the detection of proteolytic enzyme activity, the test material shall be practically free from residual proteolytic enzymes.

3.Test for antitoxin/ antivenom content.

In order to determine the antitoxin/antivenom content of the preparation under examination its potency is determined with respect to the standard antitoxin preparation of the test preparation by carrying out the assay.

The antitoxin/ antivenom content of each test sample shall be determined by statistical analysis of assay results. The final product shall contain antitoxin/ antivenom at no less than the value stated on the label.

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DefinitionA toxin that has been treated, as with chemicals or heat, so as to eliminate the toxic qualities while retaining the antigenic properties

Quality Control of Toxoids

1.Purity test2.Sterility test3.Detoxification test

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1. Purity TestEach bulk material shall be tested for protein nitrogen content and for toxoid content.

Protein nitrogen content

The protein nitrogen content test is a method used to determine protein content by measuring nitrogen in heated trichloroacetic acid-precipitable protein in the test sample by the micro-Kjeldahl method. The criterion for judgment shall be given in the individual monographs.

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Dilute the sample if necessary with water and transfer to centrifuge tube

Add 1/10th volume of 50% w/v trichloroacetic acid solution to render trichloroacetic acid concentration 4.5% w/v or higher.

Heat the mixture at 100 o C for 15 minutes and then cool it to room temperature.

Centrifuge the mixture at greater than 1400 x g for 10 minutes. Add appropriate amount of 5% w/v of trichloroacetic acid solution to

the precipitate, shake and centrifuge it again. Measure the nitrogen content in the precipitate using an appropriate

method such as the micro-Kjeldahl method. Calculation of protein content: The protein content is calculated from the nitrogen content by the

formula: 1 mg protein nitrogen (N) = 6.25 mg protein

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Toxoid content

The test shall be conducted by the flocculation test. The bulk material shall contain no less than 1,500 Lf toxoid of per mg protein nitrogen.

2. Detoxification testThe test shall be conducted on two kinds of the sample: the one shall be prepared by diluting the test sample with 0.017 mol/L phosphate-buffered sodium chloride solution (pH 7.0) to the concentration of x Lf/ml, and the other to a concentration higher than that of the final bulk not exceeding y Lf/ml. The latter sample shall be preserved at 37 °C for 20 days prior to the test. Following test shall be conducted on the samples with and without preservation at 37 °C for 20 days. Concentration of the diluted samples depend upon on the type of the toxoid and is given in the monograph of that particular toxoid.

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Procedure

Each sample shall be given by subcutaneous injection at a dose of 5 mL into at least 4 guinea pigs weighing 300−400 g. The inoculated animals shall be observed for at least 21 days. No animal shall die due to intoxication, or show specific symptoms of intoxication, or other abnormal signs during the observation period.

3. Sterility test

The test given in General Tests shall apply

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Whole human blood Concentrated red blood cells Platelet concentrate Human plasma protein fraction Human albumin Freeze dried human fibrinogen Dried human serum Human normal immunoglobulin

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Quality control testsIdentification testSterilityPyrogen testSolubilityAssay

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Identification testPrecipitation tests with specific antisera are used to show that only human serum proteins are present.

The characteristic mobilities of blood proteins in an electrophoretic field are a sensitive means of identifying fibrinogen, immunoglobulin, and the plasma protein fraction.

Proteins can also be identified by their sedimentation rate in an ultra-centrifuge., this method is suitable for identifying and quantifying the different types of gamma globulin

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2. Sterility And Pyrogen test All blood products must comply with the official tests for sterility, and those preparations (i.e., immunoglobulin and the plasma protein fractions) that are exposed to special risk of contamination with pyrogens due to lengthy processing must also pass the pyrogen test.

3. Solubility Complete solubility in an appropriate volume of the usual solvent, sometimes in a specified time, is required for all solid preparations except fibrin foam. This indicates that the protein constituents have not deteriorated.

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4. Assay

For whole blood and concentrated red cells the assay is a determination of the hemoglobin value. For the remaining products, except fibrin foam and thrombin, the protein content is determined chemically.

The hemoglobin value is a measurement of concentration and is the amount of hemoglobin present in a fixed volume of the patient’s blood. It is normally expressed as grams per deciliter (g/dl) or grams per liter (g/L).

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Quality control tests:Sterility TestThe test given in General Tests shall apply.Test for Hemoglobin contentMethodPipette 0.02 ml of the substance under examination into a stoppered tube, add 4.0 ml of ferricyanide cyanide reagent and mix well. Allow to stand for 10 minutes and measure the absorbance of the resulting solution at about 540 nm, using as blank the ferricyanide-cyanide reagent. Calculate the content of haemoglobin from the absorbance obtained by carrying out the determination simultaneously using a suitable volume of cyanmethaemoglobin RS and from the declared content of haemoglobin in cyanmethaemoglobin RS.

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1. InspectionFresh-frozen Human Plasma, upon visual inspection, shall be free from marked hemolysis, change in color or other abnormal findings

2. Coagulation test To 0.1 mL of the test material, taken in a test tube kept in a water-bath at 37 °C, 0.1 mL of thromboplastin solution and 0.1 mL of 0.025 M calcium chloride solution shall be added, and the time until the formation of a fibrin clot shall be recorded, and shall be within 20 seconds.

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1.Weight When weighed by a suitable method, not less than 63 g of red cells shall be recovered from 200 mL of source material.

2. Test for hemoglobin content When the test for Hemoglobin Content is applied, 1mL of the final product shall contain not less than 0.24 g of hemoglobin

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1. Inspection Platelet, upon visual inspection, shall be free from marked hemolysis, change in color or other abnormal findings.

2. Platelet count test The final product shall have a platelet count of not less than 0.2×1011 of Platelet count

3. Red blood cell count and white blood cell count tests The final product shall contain normal counts of red blood cells and white blood cells.

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Allergen products are used to diagnose and treat allergic diseases. Allergen extracts are biological products that are administered to humans to diagnose, prevent and treat allergic diseases.

Quality control tests Measurements of the total allergenic activity of individual

batches of an allergen extract should be undertaken preferably by IgE inhibition or by direct IgE-binding or other immunoassay.

The estimated potency derived from the assay of total allergenic activity should be not less than 50% and not more than 200% of the stated potency.

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In-vivo diagnostics are medical drugs produced using biotechnology. They include proteins, nucleic acids and living microorganisms like virus and bacteria where the virulence of viruses and bacteria is reduced by the process of attenuation; they can be used for therapeutic or in vivo diagnostic purposes.

The most widely used of these are the tuberculin PPD employed of infection to detect sensitization by mycobacterial proteins and hence the possible presence.

Apart from standardization of potency, which also serves as an identity test, the material must be checked for sterility and for the absence of viable mycobacteria.

The product is also checked for absence of reactogenicity in unsensitized guinea pigs and if required by the regulatory authority, for abnormal toxicity .

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