The test is not valid if the proportion of the protein in the principal band is not within the limits stated

complete exclusion of globulin proteins of molecular weight between 400.000 and 500.000 (Sephadex G 150 is suitable) at 20* to 35* with a saline-phosphate solution prepared by mixing 2 volumes of saline solution and 1 volume of mixed phosphate buffer pH 7 0 with azide. Apply to the column 2.5 ml of normal human serum, previously clarified by centrifuging, and elute with the saline-phosphate solution at a rate of 20 ml per hour Prepare a chromatogram by recording the aosorbance, Appendix 5.5 of the eluate at about 280 fun in relation to its volume The chromatogram exhibits three welt-defined peaks Determine the volume, V. of the eluate from the entry of the sample into the column to the apex of the first peak Dilute the substance being examined with the saline-phosphate solution to contain about 5% w/v of protein, apply ml to the column and elute under the above conditions Collecting the eluate m 5-ml portions. Three, peaks may appear in similar positions to those in the chromatogram obtained from the normal human serum but the relative peak heights may be different. To the fraction eluted between vo fume 0.85 V and 1.15 V, add for each 10 ml, 0.4 ml of a 7.5% w/v solution of sodium moiyfcbdate and 0.4 ml of a mixture of I part of nitrogen free sulphuric acid and 30 parts of water, shake, centrifuge for 5 minutes and complete the Assay described under Human Plasma beginning at the words 'decant the supernatant . The weight of protein in the fraction of the eluate is net more than 3. 5% of the weight of protein in the volume of the substance being examined applied to the column. Heat the substance being examined for 50 hours at 56. 5* to 57 5° and repeat the chromatographic separation and the determination of the weight of protein in the fraction eluted between 0 .85 V and 1. 15 V. When expressed as a percentage of the weight of protein in me volume of the substance being examined applied to the column, it exceeds the percentage obtained before heating by not more than 1. 5% w/v.Protein composition Not less than 95.0% w/V of the total protein as albumin, when determined by the following method Carry out Method II for cellulose acetate electrophoresis. Appendix 4. 1. using one strip of cellulose for each solution. For solution (1) dilute the substance being examined with saline solution to obtain a solution containing 2% w/v of total protein For solution (2) dilute human albumin for electrophoresis RS with saline solution to obtain a solution containing 2% w/v of total protein. Not more than 5% of total protein is contained in bands other than the principal band in the strip obtained with solution (1). The test is not valid if the proportion of the protein in the principal band is not within the limits stated in the leaflet supplied with human albumin for electrophoresis RS Stability .The contents of the final container remain unchanged, as determined by visual inspection, after heating at 57* for 50 hours, when compared to its control consisting of a sample from the same lot which has not undergone this heating Pyragens Complies with the lest for pyrogens. Appendix 2.6. using 3 ml per kg of the rabbits weight, irrespective of the protein content, in rabbits that have not previously received blood products Stenlity. Complies wilfi the tests for sterility. Appendix 9 .5 Abnormal toxicity Complies with the test for abnormal toxicity. Appendix 2 .2 using Method B and 0. 5 ml of the solution for each mouse and 5 ml of for each guinea-pig irrespective of the protein content Assay: For protein - Dilute to about 0.75H w/v of total protein with saline solution. Take 2 ml of this solution in a round-bottomed centrifuge, add 2 ml of a 7.5% w/v solution of sodium molybdate and 2 ml of a mixture of 30 volumes of water and 1 volume of nitrogen free sulphuric acid. Shake, centrifuge for 5 minutes, decant the supernatant liquid and let the inverted tube stand on a filter paper to drain the fluid. Carry out Method E for determination of nitrogen, Appendix 3. 20, on the residue thus obtained and multiply the result by 6. 25 to obtain the protein content For sodium - Dilute to 0.01 % w/V of protein with water and determine by Method A for atomic absorption spectrophotometry. Appendix 5. 2. or by Method B for flame photometry. Appendix 5.1. measuring at about 589 rim and using sodium solution FP suitabty diluted with water as the standard solution.For potassium -Dilute to 0.25% w/v of protein with water and determine by Method A for atomic absorpoon spectrophotometry. Appends 5. 2, or by Method B for flame photometry. Appendix 5.1, measuring at about 767 nm and using potassium solution FP suitably diluted with water as the standard solution. Human Albumin intended for administration to patients undergoing dialysis or to premature infants complies with the following additional requirement Aluminium; Not more than 200 ug of A1 per litre .Carry out the method for atomic absorption