Quality control of insulin formulations: API , protamine and aggregates follow-up

The prevalence of diabetes is increasing every year and insulin preparations are mostly prescribed for
treatment of both type 1 and type 2 diabetes. Since these biopharmaceutical formulations are expensive and
require a prescription, they are an important target for counterfeiting in developing countries. Therefore,
there is a need for fast and efficient methods for quality control of biopharmaceutical products such as insulin
formulations.
A fully validated micellar electrokinetic chromatography (MEKC) method was developed for
quantification of human insulin and NPH insulin (insulin combined with protamine) in formulations. The BGE
used was made of 50 mM ammonium acetate (pH 9.0), 20 mM Sodium dodecyl sulfate and 13 % (v/v)
acetonitrile and samples were introduced at the short capillary end allowing a separation of insulin and two
major excipients (phenol and m-cresol) within 3 minutes. This method was compared with HPLC method using
a mobile phase composed of water with 0.1% formic acid / acetonitrile with 0.1 % formic acid in gradient
mode.
Secondly a MEKC method was also optimized to analyze protamine peptides in insulin formulations.
The major protamine peptides could be separated using a BGE made of 100 mM phosphate buffer (pH 2) with
50 mM Thesit®. This method was used to check conformity of protamine in commercially available
formulations.
Finally, different approaches were investigated in order to follow insulin aggregation. Indeed, insulin
is prone to unfold when submitted to denaturating factors as temperature, ionic strength, agitation and pH. An
accumulation of unfolds protein in bloodstream results in a high tendency to aggregate and form amyloid
fibrils. A deposit of those fibrils in the subcutaneous tissue leads to a complication called “insulin-derived
amyloidosis”. On the other hand, during its production, insulin is often subjected to extreme conditions
making aggregation, as well as protein stability, important parameters to be controlled during its quality
control.
In this study, insulin aggregates were generated after optimization of incubation conditions (pH,
temperature, agitation…). Those aggregates were then analyzed by size-exclusion chromatography (SEC) and
capillary electrophoresis (CE). We showed that capillary gel electrophoresis (CGE) is a promising technique to
analyze covalent aggregates of insulin due to the fact that it separates the aggregates according to their size
and not to their size/charge ratio. The use of a laser-induced fluorescence detector was also found attractive
to enhance the sensitivity of the method.