In process quality control tests for biological products

In-process Quality Control Tests For Biological Products: 

In-process Quality Control Tests For Biological Products Presented By Nishath Fathima M.Pharm., I year II Sem Under the guidance of Dr. T. Mamatha, M.Pharm., Ph.D. Department of Quality Assurance

Quality Control: 

Definition It includes operational techniques and individual activities that focus on controlling or regulating processes and materials to fulfill requirements for quality. The focus is on preventing defective products or services from being passed on. 2 Quality Control

BIOLOGICAL PRODUCT: 

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)). 3 BIOLOGICAL PRODUCT

Biological Products (Continued): 

Biological products include Vaccine Immune sera Antitoxin Antivenom Toxoids Blood and blood components Allergenic products In-vivo diagnostics 4 Biological Products (Continued)

General tests: 

Sterility test 1. Immersion (Direct Inoculation ) For bacteria - Using petri dishes 9 to 10 cm in diameter, add to each dish a mixture of 1 ml of the pretreated preparation and about 15 ml of a liquefied S oyabean 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 . 5 General tests

Sterility test: 

2. Membrane filtration Use 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. 6 Sterility test

Sterility test (Continued): 

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 7 Sterility test (Continued)

Pyrogen test: 

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. 8 Pyrogen test

Pyrogen test (Continued): 

Interpretation If the first test fails then repeat the test on additional 5 rabbits 9 Pyrogen test ( Continued)

Vaccines: 

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 include Staining test Sterility test Inactivation test Pyrogen test Freedom from abnormal toxicity 10 Vaccines

Staining test: 

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 bacterial shall be observed other than those defined in the individual monographs. 11 Staining test

Inactivation test: 

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. 12 Inactivation test

Freedom from abnormal toxicity: 

Freedom from abnormal toxicity 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 13

Immune sera: 

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. 14 Immune sera

Immune sera (Continued): 

Quality control test include Test for immunoglobulin content Test for freedom from residual proteolytic enzyme Sterility test Pyrogen test Test for antitoxin/ antivenom content 15 Immune sera (Continued)

Immune sera (Continued): 

1 . Immunoglobulin test The 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 criteria not less than 95% of the total proteins shall be immunoglobulin. 16 Immune sera ( Continued)

Immune sera (Continued): 

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 enzyme activity . 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. 17 I mmune sera ( Continued)

3. Toxoids: 

Definition A 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 Purity test Sterility test Detoxification test 18 3. Toxoids

Toxoids (Continued): 

1. Purity Test Each 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. 19 Toxoids ( Continued)

Protein nitrogen content: 

Dilute the sample if necessary with water and transfer to centrifuge tube Add 1/10 th 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 20 Protein nitrogen content

Toxoid (Continued): 

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 test The 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. 21 Toxoid ( Continued)

Toxoids (Continued): 

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 22 Toxoids ( Continued)

Blood products: 

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 23 Blood products

Blood products (Continued): 

Quality control tests Identification test Sterility Pyrogen test Solubility Assay 24 Blood products ( Continued)

Blood products (Continued): 

Identification test Precipitation 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 25 Blood products ( Continued)

Blood products (Continued): 

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. 26 Blood products ( Continued)

Blood products (Continued): 

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). 27 Blood products ( Continued)

Human plasma: 

1. Inspection Fresh-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. 28 Human plasma

Frozen thawed human blood cells : 

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 29 Frozen thawed human blood cells

Platelet concentrate: 

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 0.2×10 11 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. 30 Platelet concentrate

Allergenic products: 

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. 31 Allergenic products

In-vivo diagnostics: 

The most widely used of these are the tuberculin 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 . 32 In-vivo diagnostics