Stability of the 4th International Standard for Insulin

Diabetologia, Dec 1975

Summary The stability of the (W.H.O.) 4th International Standard for Insulin, has been assessed by accelerated thermal degradation studies. This is a crystalline preparation of insulin, freed from proteolytic enzymes, sealed in ampoules containing air and with a moisture content of 5–6%. Of the original biological activity 95.8 (92.8–98.9; P=0.95)% was retained after storage for 12 years in the dark at 20°C and 65.7 (63.4–68.1; P=0.95)% after 14 years at 37°C. Degradation rate constants were calculated from these data for the Standard at 20°C and 37°C, assuming first order kinetics. The degradation constant at 37°C did not differ significantly from those found in earlier degradation studies at 37°C over shorter periods, thereby supporting the assumption that the degradation of crystalline insulin, at least at 37° C, is a first order reaction. Extrapolation of these data suggests that the Standard stored at −20° C for 20 years would have retained at least 99.93% (P=0.95) of its original activity and so for practical purposes can be considered to be stable.

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Stability of the 4th International Standard for Insulin

Stability of the 4th International Standard for Insulin P. L. Storring 0 P. L. Greaves 0 M. V. Mussett 0 D. R. Bangham 0 0 National Institute for Biological Standards and Control , London, England, WHO International Laboratory for Biological Standards Summary. The stability of the (W.H.O.) 4th International Standard for Insulin, has been assessed by accelerated thermal degradation studies. This is a crystalline preparation of insulin, freed from proteolytic enzymes, sealed in ampoules containing air and with a moisture content of 5-6%. Of the original biological activity 95.8 (92.8-98.9; P = 0.95)% was retained after storage for 12 years in the dark at 20 ~ C and 65.7 (63.4-68.1; P = 0.95)% after 14 years at 37~C. Degradation rate constants were calculated from these data for the Standard at 20~C and 37~C, assuming first order kinetics. The degradation constant at 37~ did not differ significantly from those found in earlier degradation studies at 37~ over shorter periods, thereby supporting the assumption that the degradation of crystalline insulin, at least at 37~C, is a first order reaction. Extrapolation of these data suggests that the Standard stored at -20~ for 20 years would have retained at least 99.93% (P = 0.95) of its original activity and so for practical purposes can be considered to be stable. Insulin standard; insulin stability; insulin accelerated thermal degradation; insulin biological activity - Two laboratories did not provide sufficient information for detailed statistical analysis of individual assays to be carried out. It was therefore decided to use relative potencies, calculated by each laboratory from their own data, as the starting point for the Materials and Methods analysis described below. Where necessary, relative potencies were adjusted so that activity of a degrada Four samples were included in the study and tion sample (A or D) was expressed as apercentage of coded: (A) 4th I. S. Insulin stored at 37 ~ C for 14 the Standard, (B or C). Laboratory 3 did not compare years; (B and C) two samples of 4th I. S. Insulin, preparations in the order requested, but assayed each stored at - 20 ~ C since 1966, and previously at - 10~ of the four samples against their own supply of the 4th C; and (D) 4th I. S. Insulin stored at 20 ~C for 12 years. I. S. Insulin. Potencies thus obtained for B and C did P. L. Storring et al.: 4th InternationalInsulinStandard Potency combined for all laboratories and both methods is 65.7, with fiducial limits (P = 0.95) of 63.4 and 68.1, and an assay weight of 16,094 Potency combined for all laboratories and both methods is 95.8, with fiducial limits (P = 0.95) of 92.8 and 98.9, and an assay weight of 20,709 not differ significantly from 100%; therefore ,the using results from all three degradation studies. In potencies estimated by this laboratory for A and D each of two earlier studies [ 9 ] in 1961 and 1963, were included with those obtained by the other two respectively, coded solutions were prepared, by the laboratories in direct comparison with the Standard. same person on the same day and in an identical Laboratory 2 provided three independent potency es- manner, from the 4th I. S. Insulin (then stored at timates for B and C by use of the mouse convulsion - 10~ and from degradation samples kept at 37~ method. Each of these potencies had been derived for 25 and 49 months, respectively. These samples from 10-20 individual assays as described in the Brit- were assayed by the mouse convulsion method, by ish Pharmacopoeia [ 10 ]. three laboratories in the first study and four Each log potency was weighted by the reciprocal laboratories in the second. The data obtained in the of its variance and by use of 22 it was shown that first study [ 9 ] gave a potency estimate combined for all estimates obtained within each laboratory and each laboratories, and for the second study combined poassay method formed a homogeneous set. Log poten- tency estimates for each laboratory (see Table 4). cies were therefore combined within these sub-sets These combined potency estimates were used to caland weighted means, together with fiducial limits, are culate the log K values shown in Table 4. A regression given in Tables 1 and 2. The assay method in rabbits line, and its 95% confidence limits were then calcuappeared to give higher estimates of potency for the lated from the log K values of all three studies. The 37~ sample than the method using mice and the additional data from the two earlier studies influenced reverse was true for the 20~ sample. However, log only the calculation of the degradation constant at potencies obtained by both methods were shown to be 37~ as a result the slope of the regression line was homogeneous for each sample (Table 3) and further little different, but the 95 % confidence limits were combination of results was considered justified, giving narrowed by inclusion of the extra information. By final estimates of 65.7% activity for the 37~ sample extrapolation, log K at - 2 0 ~ was calculated to be and 95.8% activity for the 20~ sample. - 1 2 . 8 9 , with 95% confidence limits of - 1 3 . 8 7 and The logarithm of the degradation constant (log K) - 1 1 . 9 1 . The calculations suggest that the potency of was calculated [ 12 ] for 20~ and 37~ from the com- the 4th I. S. Insulin, when stored at - 2 0 ~ for 20 bined potency estimates for each method and labora- years, would have retained 99.98% of its original tory (Table 4, Study 3). The linear regression of log K activity, with 95% confidence limits of 99.93% to on 1/T (the reciprocal of absolute temperature) was approximately 100%. then computed from these values, together with the 95 % confidence limits of the slope. By extrapolation of this regression line and the lines obtained from the D i s c u s s i o n and C o n c l u s i o n s confidence limits the value of log K at - 2 0 ~ was estimated to be - 1 2 . 9 1 with limits of - 1 4 . 3 4 to The present accelerated degradation studies on - 11.49. the 4th I. S. Insulin have shown that 95.8%, with Another estimate of log K at - 20~ was made by fiducial limits (P = 0.95) of 92.8% and 98.9%, of the a Mouse convulsion method (MC) and rabbit blood sugar method (RBS) original biological activity is retained after 12 years storage in the dark at 20~ and 65.7%, with fiducial limits (P = 0.95)of 63.4% and 68.1%, after 14 years in the dark at 37~ These data have been used to calculate the degradation rate constants, assuming first order kinetics, for the Standard at 20~ and 37~ The degradation rate constant at 37~ computed from these data, does not differ significantly from those found in earlier degradation studies at 37~ over shorter periods. Since the constants computed for different time intervals show such good agreement, the assumption that the degradation of crystalline insulin is a monomolecular reaction, at least at 37~ receives confirmation. First order kinetics for insulin degradation in solution have earlier been described [ 1, 7 ]. Extrapolation of these data gives an estimate of the degradation rate constants for the Standard stored at - 2 0 ~ This suggests that the Standard stored at - 2 0 ~ for 20 years would have retained at least 99.93% of its original activity (P = 0.95), and for practical purposes can therefore be considered to be stable. The great stability of this crystalline insulin may in part be due to the particular attention given during its preparation to the removal of contaminating proteolytic enzymes [ 8 ]. 1. Krogh , A. , Hemmingsen , A. M. : CLIII. The destructive action of heat on insulin solutions . Biochem. J . 22 , 1231 - 1238 ( 1928 ) 2. Sahyun , M. , Goodell , M. , Nixon , A. : Factors influencing the stability of insulin . J. biol. Chem . 117 , 685 - 691 ( 1937 ) 3. Sahyun , M. , Nixon , A. , Goodell , M. : Influence Ofcertainmetals on the stabilityof insulin . J. Pharmacol. exp. Ther . 65 , 143 - 149 ( 1939 ) 4. Lens , J.: The inactivation of insulin solutions . J. biol. Chem . 169 , 313 - 322 ( 1947 ) 5. Stephenson , N. R. , Romans , R. G.: Thermal stabilityof insulin made from zinc insulin crystals . J. Pharm. Pharmacol . 12 , 372 - 376 ( 1960 ) 6. Storvick , W. O. , Henry , H. J. : Effect of storage temperature on stability of commercial insulin preparations . Diabetes 17 , 499 - 502 ( 1968 ) 7. Pingel , M. , Volund , Aa.: Stability of insulin preparations . Diabetes 21 , 805 - 813 ( 1972 ) 8. Bangham , D. R. , Mussett , M. V. : The Fourth International Standardfor Insulin . Bull. WldHlth Org . 20 , 1209 - 1220 ( 1959 ) 9. Unpublished working document. W. H. O./BS/631 ( 1963 ) 10. British Pharmacopoeia , p. 1339 ( 1968 ) 11. The United States Pharmacopoeia 18 , p. 883 ( 1970 ) 12. Jerne , N. K. , Perry , W. L. M.: The stabilityof biologicalstandards . Bull. Wld Hlth Org . 14 , 167 - 182 ( 1956 ) Dr. P.L. Storring National Institute for Biological Standards and Controls, Holly Hill London NW3 6RB , England Dr . W. O. Storvick Eli Lilly and Company Indianapolis, Indiana U.S.A. Dr. G.A. Stewart The Wellcome Foundation Limited Dartford , Kent England


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P. L. Starring, P. L. Greaves, M. V. Mussett, D. R. Bangham. Stability of the 4th International Standard for Insulin, Diabetologia, 1975, 581-584, DOI: 10.1007/BF01222110