Application note

Detecting insulin oligomerization using microfluidic diffusional sizing

Insulin is a peptide hormone that plays an integral role in the regulation of protein, carbohydrate and fat metabolism by promoting absorption of glucose from the blood.  

Insulin is used as a biotherapeutic for the treatment of Diabetes Mellitus and is considered an essential medicine for a basic health system by the World Health Organisation. Insulin is commonly found in two oligomeric forms, a stable but inactive hexamer and an active monomer capable of diffusing rapidly through the capillary barrier and binding receptors. Insulin monomers associate to form dimers which further associate to form hexamers. Changes in the pH of an insulin solution can trigger changes in oligomeric state as can various additives such as Zn2+.

The diffusion of insulin in differing oligomeric states
Figure 1: Diffusion of insulin into the bloodstream in differing oligomeric states.

Identifying the oligomeric state of insulin is important for predicting the stability and activity of insulin, especially in the context of its role as an essential biotherapeutic (see Figure 1).

Method

An insulin solution was prepared in PBS, and separate portions were prepared to change the pH and add different additives as described in Figure 1 legend. The changing average hydrodynamic radius was monitored by Microfluidic Diffusional Sizing (MDS) to assess oligomerization.

For each analysis 5 µL of sample was tested on a Fluidity One instrument, with results obtained in around 8 minutes. The mode of the MDS analysis (3) means that the size was measured of the native species in solution, without any denaturation or labelling. It is only after the diffusional sizing step that label is added, so this does not affect the results obtained.

Comparison of insulin oligomerisation under different buffer conditions
Figure 2: Comparison of insulin oligomerisation under different buffer conditions. 
An 880 µM human insulin solution in 10 mM HCl was diluted down to 57 µM in PBS (final pH of 6), and in 10 mM HCl (final pH of 2). The size of insulin species was determined for each pH condition in triplicates using a Fluidity One instrument. Zinc chloride was then added to the pH 6 insulin at a final concentration of 25 µM and the sample sized before EDTA was added in excess (final concentration of 1 mM) to the sample, causing zinc chelation and consequently insulin dissociation into monomers. Finally, another fresh sample of human insulin solution at pH 6 was prepared as described above and reducing agent (BME) was added at a final concentration of 1 mM. The sample was incubated at 40 °C for 15 minutes, and aggregation was detected using MDS.

Results

We first measured the oligomerization of human insulin using the Fluidity One at pH 2 and pH 6. Under acidic conditions we observe primarily insulin monomers, whereas at neutral pH we observed a mixture of insulin monomers, dimers and hexamers, as reported previously (4).

We then added zinc ions to the human insulin solution at pH 6, which induced hexamer formation and an average size increase of the sample (Figure 2). The addition of excess EDTA to this solution chelated the zinc ions, causing dissociation of the sample into monomers (5), as detected by MDS.

Separately, an excess of reducing agent (BME) was added to a solution of human insulin at pH 6 and was incubated for 15 min at 40 °C. MDS revealed the formation of insulin aggregates larger than the hexamer after incubation.

Conclusion

Microfluidic Diffusional Sizing (MDS) was used to directly detect the oligomeric state of insulin in solution by measuring the average hydrodynamic radius of an insulin solution.

Monomers and hexamers can be readily distinguished, and intermediate sized mixtures of monomer, dimer and hexamer identified. Furthermore insulin aggregation can also be detected when the average hydrodynamic radius exceeds the size of hexameric insulin.

MDS on the Fluidity One allows the oligomerization state of human insulin to be detected under differing buffer conditions. This is achieved in a rapid test (8 minutes) with very low sample consumption (5 µL). Furthermore the instrument interface does not offer multiple adjustable test parameters, so results are readily and easily comparable.

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