Insulin Human
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Insulin (human) [11061-68-0].
»Insulin Human is a protein corresponding to the active principle elaborated in the human pancreas that affects the metabolism of carbohydrate (particularly glucose),fat,and protein.It is derived by enzymatic modification of insulin from pork pancreas in order to change its amino acid sequence appropriately,or produced by microbial synthesis via a recombinant DNAprocess.Its potency,calculated on the dried basis,is not less than 27.5USP Insulin Human Units in each mg.The proinsulin content of Insulin Human derived from pork,determined by a validated method,is not more than 10ppm.The host cell derived proteins content of Insulin Human derived from a recombinant DNAprocess,determined by an appropriate and validated method,is not more than 10ppm.The host cell or vector derived DNAcontent and limit of Insulin Human derived from a recombinant DNAprocess that utilizes eukaryotic host cells are determined by a validated method.
NOTE—One USP Insulin Human Unit is equivalent to 0.0347mg of pure Insulin Human.
Packaging and storage—
Preserve in tight containers.Store in a freezer,and protect from light.
Labeling—
Label it to indicate that it has been prepared by microbial synthesis or that it is derived by enzymatic modification of insulin from pork pancreas.
USP Reference standards á11ñ—
USP Endotoxin RS.USP Insulin Human RS.USP Insulin (Pork)RS.USP Proinsulin (Pork)RS.
Identification—
A:
The retention time of the major peak in the chromatogram of the Assay preparationcorresponds to that in the chromatogram of the Standard preparation,as obtained in the Assay.
B:
Determine the peptide fragments,using the following peptide mapping procedure.
Sulfate buffer—
Mix equal volumes of 2.0Mammonium sulfate and 0.5Msulfuric acid,and filter.
Enzyme solution—
Prepare a solution of Staphylococcus aureusV-8protease in water having an activity of 500units per mL.
HEPESbuffer—
Dissolve 2.38g of HEPES(N-2-hydroxyethylpiperazine-N¢-2-ethanesulfonic acid)in about 90mLof water in a 100-mLvolumetric flask.Adjust with 5Msodium hydroxide to a pHof 7.5,dilute with water to volume,and mix.
Solution A—
Prepare a filtered and degassed mixture of 100mLof acetonitrile,700mLof water,and 200mLof Sulfate buffer.
Solution B—
Prepare a filtered and degassed mixture of 400mLof acetonitrile,400mLof water,and 200mLof Sulfate buffer.
Mobile phase—
Use variable mixtures of Solution Aand Solution Bas directed for Chromatographic system.Make adjustments,if necessary (see System Suitabilityunder Chromatography á621ñ).
Standard digest solution—
Dissolve about 6mg of USP Human Insulin RSin 3mLof 0.01Nhydrochloric acid,and transfer 500µLof the resulting solution to a clean vial.Add 2.0mLof HEPESbufferand 400µLof Enzyme solution,and incubate at 25
for 6hours.Quench the digestion by adding 2.9mLof Sulfate buffer.
for 6hours.Quench the digestion by adding 2.9mLof Sulfate buffer.
Test digest solution—
To 1mg of Insulin Human add 500µLof 0.01Nhydrochloric acid,and mix to dissolve.Proceed as directed for Standard digest solution,beginning with “Add 2.0mLof HEPESbuffer”.
Chromatographic system (see Chromatography á621ñ)—
Aliquid chromatograph is equipped with a 214-nm detector and a 4.6-mm ×10-cm column that contains packing L1.The flow rate is about 1mLper minute.The column temperature is maintained at 40.The chromatograph is programmed as follows.
Chromatograph the Standard digest solution,and record the peak responses as directed for Procedure:the chromatogram of the Standard digest solutioncorresponds to that of the standard chromatogram provided with USP Insulin Human RS.For the chromatogram of the Standard digest the tailing factor is not greater than 1.5;and the resolution,R,is not less than 3.4for digest fragments IIand III.
[NOTE*—Fragment Ielutes at the same time in insulin derived from pork and Insulin Human;Fragment IIelutes at the same time in all insulins;and Fragment IIIelutes at the same time in insulin derived from beef and pork.]
.The chromatograph is programmed as follows.
| Time (minutes) |
Solution A
% |
Solution B
% |
Elution |
| 0 | 90 | 10 | equilibration |
| 0–60 | 90®30 | 10®70 | linear gradient |
| 60–65 | 30®0 | 70®100 | linear gradient |
| 65–70 | 0 | 100 | isocratic |
| 70–71 | 0®90 | 100®10 | linear gradient |
| 71–86 | 90 | 10 | re-equilibration |
Procedure—
Using the gradient program,run a blank.Inject equal volumes of the Standard digest solutionand the Test digest solutioninto the chromatograph,and record the chromatograms.The chromatographic profile of the Test digest solutioncorresponds to that of the Standard digest solution.
Microbial limits á61ñ—
The total bacterial count does not exceed 300cfu per g,the test being performed on a portion of about 0.2g,accurately weighed.
Bacterial endotoxins á85ñ—
It contains not more than 10USP Endotoxin Units in each mg.
Loss on drying á731ñ—
Dry about 200mg,accurately weighed,at 105for 16hours:it loses not more than 10.0%of its weight.
for 16hours:it loses not more than 10.0%of its weight.
Related compounds—
Proceed as directed for the Related compoundstest under Insulinexcept to use the following gradient elution program.The program initially calls for isocratic elution for about 36minutes with a Mobile phaseconsisting of a mixture of 78%Solution Aand 22%Solution B.Following the gradient elution phase,the system is returned to the initial conditions of 78%Solution Aand 22%Solution B.Adjust the composition of the Mobile phaseso that the retention time of the main insulin human peak is between 15and 25minutes.The content of A-21desamido insulin and of other insulin related compounds is not more than 2.0%each of the total amount of insulin and total related compounds.
Limit of high molecular weight proteins—
Proceed as directed in the test for Limit of high molecular weight proteinsunder Insulin.Not more than 1.0%is found.
Assay—
Mobile phase,Standard preparation,Assay preparation,Resolution solution,Chromatographic system,and Procedure—
Proceed as directed in the Assayunder Insulinexcept to use USP Insulin Human RSand otherwise substitute Insulin Human for Insulin throughout.
*
Fragment Iconsists of amino acids A5to A17and B1to B13.Fragment IIconsists of amino acids A18to A21and B14to B21.Fragment IIIconsists of amino acids B22to B30.Fragment IVconsists of amino acids A1to A4.Arefers to the A-chain of Insulin Human,and Brefers to the B-chain of Insulin Human.
Auxiliary Information—
Staff Liaison:Larry N.Callahan,Ph.D.,Scientist
Expert Committee:(BNT)Biotechnology and Natural Therapeutics/Diagnostics
USP28–NF23Page 1022
Pharmacopeial Forum:Volume No.29(6)Page 1906
Phone Number:1-301-816-8385