Kromasil packing media are today the dominating materials used for large-scale production of human insulin and variants, among many other applications. Multi-ton volumes of Kromasil per year are used globally to produce these APIs, with high purity demands.
This article will show an example of the development work that has been performed to facilitate cost-effective processes for the final purification of human insulin, and to service our customers in order to lower their costs.
For human insulin (HI), two of the most demanding impurities to remove are A21 desamido HI, and B30 des-threonine HI. The A21 desamido HI is an impurity where the A21 asparagine is hydrolyzed to aspartic acid, and the B30 des-threonine HI is an impurity where the B30 threonine is missing.
Figure 1 shows the structure of insulin schematically, with the A and B chain being coupled together via disulfide bonds.
Figure 2 shows the analytical chromatogram of a crude insulin sample, where purity is 90%, and the two most demanding impurities are indicated.
When using RP chromatography to purify polypeptides or small proteins such as insulin, typically a shallow gradient is used. The chromatogram in figure 3 shows a case where some standard conditions are applied for the purification of human insulin containing a relatively high amount of des-threonine HI. It can be seen that the human insulin and the main impurity more or less co-elute, giving no possibility to collect any significant yield of pure insulin.
However, if the standard conditions as seen in figure 3 are replaced by a mobile phase where an ion-pairing agent has been added to the mobile phase, a significant improvement of the chromatography can be obtained. The des-threonine HI is pushed out due to a strong positive displacement effect, and > 98% pure insulin can be collected, with about 80% yield.
The above example shows the extreme importance of performing an extensive method development work before deciding the chromatographic conditions for the large-scale production. If the purification is to be run for a long time, even small improvements in yield and purity can result in huge savings in separation cost.
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