Insulin Aspart Injection

Insulin Aspart Injection
Definition
Insulin Aspart injection is a sterile, neutral, aqueous solution of Insulin Aspart.
The injection complies with the requirements stated under Insulin Preparations with the
modifications described below.
Content of Insulin Aspart
Not less than 90% and not more than 110 % of the stated amount on the label.
Description
A colorless liquid, free from turbidity and foreign matter; during storage traces of a very fine
sediment may be deposited.
Identification
In the Assay, principal peak in the chromatogram of the test solution corresponds to that in the
chromatogram of the reference solution.
Tests
pH (2.4.24)
Between 6.9 to 7.8
Related proteins
Determine by Liquid Chromatography 2.4.14 as described under assay: use the normalization
procedure.
Limits:
• B28isoAsp insulin aspart: not more than 2.5% is found
• Total of B3Asp insulin aspart, A21Asp insulin aspart and B3isoAsp insulin aspart: not more
than 5.0%
• Total of other impurities: not more than 3.5%
Total Zinc
Between 10 and 40 µg per 100 units of Insulin Aspart
Atomic Absorption Spectrometry
Test solution: Shake the preparation gently and dilute a volume containing 100 units of insulin
aspart to 25.0 ml with 0.01M hydrochloric acid. Dilute, if necessary, to a suitable concentration
of zinc (for example 0.1 μg to 1.0 μg of Zn per milliliter) with 0.01M hydrochloric acid.
Reference solutions: Use solutions containing a suitable range of concentrations, for example
0.20 μg, 0.40 μg, 0.60 μg, 0.80 μg and 1.00 μg of Zn per milliliter, freshly prepared by diluting
zinc standard solution (5 mg/ml Zn) with 0.01M hydrochloric acid.
Measure the absorbance at 213.9 nm using a zinc hollow-cathode lamp as source of radiation and
an air-acetylene flame of suitable composition (for example 11 liters of air and 2 liters of
acetylene per minute).
Submission from NIB 1

Bacterial endotoxins (2.2.3)
It contains less than 80 Units per 100 units of Insulin Aspart.
Impurities with molecular masses greater than that of Insulin Aspart
Determine by size exclusion chromatography 2.4.16: use the normalization procedure.
Test solution: Add 4 µl of 6 M hydrochloric acid per ml of the preparation under examination,
whether a suspension or a solution, to obtain a clear acid insulin solution. When sampling a
suspension, agitate the material prior to sampling in order to obtain a homogenous sample. If a
suspension does not turn clear within 5 min. of the initial addition of hydrochloric acid, add
small aliquots of the acid (less than 4 µl/ ml) until a solution is obtained.
Mobile phase, Resolution solution and chromatographic system are as directed in the Impurities
with molecular masses greater than that of Insulin Aspart.
Limits: the sum of the areas of the peaks with a retention time less than that of the principal peak
is not more than 3.0% (protamine containing preparations) or 2.0% (non- protamine containing
preparations) of the total areas of the peaks. Disregard any peak with a retention time greater
than that of the peak due to insulin aspart monomer.
Assay
Determine by Liquid Chromatography (2.4.14)
Test solution: acidify each ml of the preparation being examined with 4 µl of 6 M hydrochloric
acid. Dilute, if necessary, a portion of the acidified solution with 0.01 M hydrochloric acid to
obtain a solution containing about 100 insulin aspart units per ml. Maintain the solution at 2-8°C.
Reference solution: Dissolve the contents of a vial of insulin aspart RS in 0.01 M hydrochloric
acid to obtain a known concentration of 100 insulin aspart units per ml. Add 4 µl of 6 M
hydrochloric acid per ml and mix. Maintain the solution at 2-8°C.
Resolution solution: Use an appropriate solution with a content of B3Asp insulin aspart and
A21Asp insulin aspart of not less than 1 per cent. This may be achieved by storing reference
solution at room temperature for about 1-3 days.
Column: Stainless steel column, 25 cm x 4.0mm that contains octadecyl silyl silica gel for
chromatography, 5 µm
Mobile phase A: dissolve 70.0 g of anhydrous sodium sulphate in approximately 4500 ml of
water; add 6.5 ml of phosphoric acid and adjust to pH 3.4 with sodium hydroxide. Dilute to 5000
ml with water. Mix 900 ml of this solution with 100 ml of acetonitrile.
Mobile phase B: mix equal volumes of water and acetonitrile.
The chromatogram is programmed as follows:
Time (min) Mobile phase A (%) Mobile phase B (%) Comments
0 - 35 58 42 Isocratic
35 - 40 58 20 42 80 Linear gradient
40 - 45 20 80 Isocratic
45 - 46 20 58 80 42 Linear gradient
46 - 60 58 42 Re-equilibration
Submission from NIB 2

Adjust the mobile phase composition and the duration of the isocratic elution to obtain a
retention time of about 22 min for insulin aspart and to ensure that B3isoAsp insulin aspart is
eluted before the gradient starts.
Column temperature: 35°C
Flow rate: 1 ml/min.
Detection wavelength: 214 nm.
Injection volume: 10 µl.
When the chromatograms are recorded under the prescribed conditions the relative retention
times with reference to insulin aspart (retention time, 20 to 26 min): B28isoAsp insulin aspart =
approximately 0.9; B3Asp insulin aspart and A21Asp insulin aspart (generally coeluted) =
approximately 1.3; B3isoAsp insulin aspart = approximately 1.5.
Procedure: separately inject the reference solution, the test solution and the resolution solution
into the chromatograph, record the chromatograms and measure the peak areas.
System suitability:
The test is not valid unless, in the chromatogram obtained with Resolution Solution, the
resolution between the peak due insulin aspart and the peak due to B3Asp insulin aspart and
A21Asp insulin aspart is greater than 1.6.
The test is not valid unless, the symmetry factor of the principal peak in the chromatogram
obtained with Reference Solution is not more than 1.8
The relative standard deviation of the insulin aspart peak area in the chromatograms obtained
with five replicate injections of the Reference Solution is not more than 1.4%.
Calculate the content of insulin aspart together with B28isoAsp insulin aspart, A21Asp insulin
aspart, B3Asp insulin aspart and B3isoAsp insulin aspart using the areas of the corresponding
peaks in the chromatograms obtained with Test Solution and Reference Solution and the
declared content of insulin aspart together with B28isoAsp insulin aspart, A21Asp insulin aspart,
B3Asp insulin aspart and B3isoAsp insulin aspart in insulin aspart RS
Storage
Preserve in the unopened, multi-dose container provided by the manufacturer. Store in a
refrigerator (5+3˚C), protect from sunlight and avoid freezing.
Labelling
The label states that (1) the strength in terms of insulin aspart units per ml; (2) the material is
produced by microbial synthesis via recombinant DNA technology; (3) the storage conditions.
Submission from NIB 3

Biphasic Insulin Aspart injection
Biphasic Insulin Aspart Injection complies with the monograph of Insulin Aspart Injection
monograph with the amendments prescribed below:
Description
A White suspension
Test for % Insulin aspart in solution in Biphasic Insulin aspart (30/70) samples:
Biphasic insulin aspart products are mixtures of insulin aspart in solution and crystalline
protamine insulin aspart. The content of insulin aspart in solution (after addition of Tris/EDTAbuffer)
relatively to the total content of insulin aspart is determined by GPC- HPLC.
Column:
- size l=0.3 m, Ø= 7.8 mm,
- stationary phase: hydrophilic silica gel for chromatography R (5-10 μm) with a pore size of
12-12.5 nm,
Method a)
Mobile phase: 2.5 M acetic acid.
Column temperature: ambient
Flow rate: 2.0 ml/min.
Detection wavelength: 280 nm.
Injection: 50 μl.
Run time: About 10 min
Method b)
Mobile phase: Mix 15 volumes of glacial acetic acid R, 20 volumes of acetonitrile for
chromatography R and 65 volumes of a 1.0 g/l solution of arginine R; filter and degas.
Column temperature: ambient
Flow rate: 0.5 ml/min.
Detection wavelength: 276 nm.
Injection: 100μl.
Run time: About 35 min
Equilibration: At least 3 injections of the resolution solution; the column is equilibrated when
repeatable results are obtained from 2 subsequent injections.
Resolution solution: Use a solution of insulin (about 4 mg/ml), containing more than 0.4 per cent
of high molecular mass proteins. An injectable insulin preparation, whether a solution or a
suspension, that has been clarified with a sufficient amount of 6M hydrochloric acid R,
containing the indicated percentage of high molecular mass proteins, or a solution prepared from
insulin, dissolved in 0.01M hydrochloric acid may be used. Insulin containing the indicated
percentage of high molecular mass proteins may be prepared by allowing insulin powder to stand
at room temperature for about 10 days. Maintain the solution at 2-8°C and use within 7 days.
Sample Preparation
Tris/EDTA buffer (approx. pH 8.6): dissolve 6.05g of Tris and 4.50g of EDTA in approx. 80ml
of water in a 100ml volumetric flask. Add 1.80ml 4M HCl and add water to volume. Do not
adjust pH.
Submission from NIB 4

Insulin aspart in solution (ins.asp.sol.): The whole content of a sample is transferred to a
centrifuge tube. Add 25µl Tris/EDTA-buffer per 1.5ml of sample and mix for 5 seconds by
means of a tube shaker.
The tube (plugged) is placed at 25°C for 1 hour. Centrifuge for 10min at 3000 rpm at 21- 25°C.
Decant the clear supernatant. Centrifuge this supernatant for 10min at 3000 rpm at 21- 25°C.
Decant the clear supernatant.
Add 4µl of 6M HCl per ml of the clear supernatant and mix thoroughly.
Total insulin aspart (ins.asp.tot.): Add 4µl of 6N HCl per ml of sample and mix thoroughly.
Determination of Insulin aspart in solution (ins.asp.sol.) as well as of Total insulin aspart
(ins.asp.tot.): the acidified sample solutions are subject to GPC-HPLC.
System suitability:
Method a)
The number of theoretical plates, N (5 sigma) calculated from the insulin aspart monomer peak
in the chromatogram obtained with the total insulin aspart solution is not less than 1000.
t
R
N = 25 ( )
W
t R is the retention time (approx. 5 minutes)
W is the peak width at 4.4 % of peak height
Method b)
Resolution solution: peak-to-valley ratio: minimum 2.0, where H p = height above the baseline of
the peak due to the dimmer and H v = height above the baseline of the lowest point of the curve
separating this peak from the peak due to the monomer.
Calculation for potency in solution
The relative content of insulin aspart in solution is calculated by means of the insulin aspart
monomer peak areas in the chromatograms obtained with the solution ‘insulin aspart in solution’,
A ins.asp.sol. , and with the solution ‘total insulin aspart’, A ins.asp.tot :
% insulin aspart in solution= A ins.asp.sol . 1525 . 100%
A ins.asp.tot . 1500
The concentration of Insulin aspart in solution is in the range of 24.2-36.0% for 30/70 Biphasic
Insulin Aspart Injection
Submission from NIB 5