Using Analytical Ultracentrifugation for Insulin Characterization

Patients suffering from Type 1 diabetes do not produce enough insulin and require daily insulin therapy as their primary treatment. Usually, patients with Type I diabetes need several insulin injections at mealtime daily.

However, the natural time action profile of insulin is not replicated by the insulin injections, even after proper administration. To improve efficacy, analogs have been created and are currently under development.

Much of the research on the action profile of insulin depends on the stoichiometry of the molecule in formulation and dilution conditions after administration of the injection.

Biopharmaceutical companies use a number of routine methods for particle size and heterogeneity measurements in a solution state.

Analytical ultracentrifugation (AUC) functions by defining particle size, heterogeneity and frictional ratio through manipulation of the Lamm equation, which describes particles as they sediment by a liquid under force.

The use of AUC to measure the effect of the formulation on the sedimentation coefficient of insulin has previously been documented, although never empirically to evaluate the effect of zinc and a chelating agent on higher order structure.

Materials

Fisher Scientific supplied zinc chloride, dibasic sodium phosphate, glycerol, EDTA and U.S. Pharmacopeia (USP) Human Insulin reference standard. Fluka and BDH Chemicals provided sodium hydroxide and hydrochloric acid, respectively.

Beckman Coulter supplied ProteomeLab XL I, quartz windows, two-sector sedimentation velocity analytical cells, An 50 Ti rotor, An 60 Ti rotor and torque stand.

Methods

Non formulated Insulin Concentration Titration

Insulin was resuspended to 50mg/mL in 0.04N HCl and diluted to respective concentrations. A reference buffer was created that matched the sample solvent, void of Insulin.

420μl of reference and sample were loaded into a two sector analytical cell aligned with An 60 Ti rotor and equilibrated at 20˚C for more than 1h. Samples were then spun at 60,000rpm, 20˚C, 4h scanning at Abs280nm in continuous mode.

Formulated Insulin Concentration Titration

Insulin was resuspended to 50mg/mL in 0.04N HCl, diluted to the respective concentrations and formulated with 150μM ZnCl2, 16mg/mL glycerol, 1.9mg/mL dibasic sodium phosphate, and 2.2mM NaOH.

Also, a reference buffer that matched the sample solvent, void of Insulin, was made. 420μl of reference and sample were loaded into a two sector analytical cell aligned with An 60 Ti rotor and equilibrated at 20˚C for more than 1h.

Samples were then spun at 60,000rpm, 20˚C, 4h scanning at Abs280nm in continuous mode.

Zinc Concentration Titration

Insulin was resuspended to 50mg/mL in 0.04N HCl, diluted to 4mg/mL formulated with different concentrations of ZnCl2, 1.9mg/mL dibasic sodium phosphate, 16mg/mL glycerol, and 2.2mM NaOH.

Also, a reference buffer that matched the sample solvent, void of Insulin, was made. 420μl of reference and sample were loaded into a two sector analytical cell, aligned with An 60 Ti rotor and equilibrated at 20˚C for more than 1h.

Samples were then centrifuged at 60,000rpm at 20˚C, 4h scanning at Abs280nm in continuous mode. The rotor was then stopped but left under vacuum at 20˚C for 15 days. Then, the cells were taken out of the rotor, strongly shaken, and re-run under the same conditions.

EDTA Concentration Titration

After Insulin was resuspended to 50mg/mL in 0.04N HCl, it was diluted to 2mg/mL formulated with 150μM ZnCl2, 1.9mg/mL dibasic sodium phosphate, 16mg/mL glycerol, and 2.2mM NaOH. Different concentrations of EDTA were added to the formulated product and incubated at room temperature for 20min.

Also, a reference buffer that matched the sample solvent, void of Insulin, was made. 420μl of reference and sample were loaded into a two sector analytical cell aligned with An 50 Ti rotor and equilibrated at 20˚C for over 1h. Samples were then spun at 50,000rpm, 20˚C, 5h scanning at Abs280nm in continuous mode.

Data Analysis

After extracting the data from the AUC controller, it was imported into SEDFIT 14.7g. A maximum entropy regularization confidence interval of 0.68 was used to analyze absorbance data in terms of a continuous c(s) distribution of sedimenting species.

All cases showed excellent fits with root mean square deviations ranging from 0.0016 to 0.0093 absorbance units. The partial specific volume of insulin was calculated based on the amino acid composition in SEDNTERP.

For each formulation buffer, the density (ρ) and viscosity (η) were also calculated in SEDNTERP. A c(s) model was used in which the frictional ratio f/fo is floated and the partial specific volume refined.

After extracting data into GUSSI, they were plotted for c(s) using an s value minimum constraint at 0.3S.

Results and Discussion

USP Human Insulin was diluted to three different concentrations in 0.04N HCl and loaded into two sector sedimentation velocity AUC cells. Analysis showed that the three concentrations were centered on 1.2 S through c(s) analysis by SEDFIT (Figure 1).

The c(s) values were left non normalized to show the change in signal with decreasing concentration. The value of 1.2 S obtained is most probably representative of monomeric insulin, which agrees with Pohl, et.al., and is consistent with monomer behavior.

Sedimentation velocity c(s) of Insulin in 0.04N HCl only.

Figure 1. Sedimentation velocity c(s) of Insulin in 0.04N HCl only. Image credt: Beckman Coulter

Insulin was sedimented again under typical formulation conditions and analyzed for sedimentation coefficient. In this case, the s20,w of insulin was between 2.95 – 3.1 S, but at 4.0mg/mL, a minor species at 2.12 S appeared at a signal weight value of 8.3% of the overall sedimenting material(Figure 2). The new peak most probably represents dimeric insulin.

Interestingly, it was at the highest concentration, that protein dissociated, implying that the assembly is not concentration dependent and that the dissociation is more likely from non-saturating conditions of one of the excipients.

Sedimentation velocity normalized c(s) of concentration titration of formulated Insulin. 0.25mg/mL (black), 1.0mg/mL (blue), and 4.0mg/mL (green) were analyzed for sedimentation coefficient following formulation.

Figure 2. Sedimentation velocity normalized c(s) of concentration titration of formulated Insulin. 0.25mg/mL (black), 1.0mg/mL (blue), and 4.0mg/mL (green) were analyzed for sedimentation coefficient following formulation. Image credt: Beckman Coulter

The effect was explored by probing the zinc concentration in the formulation buffer and the effect on sedimentation coefficient. When zinc was added at a fixed insulin concentration, complex formation was pushed toward the hexamer around 3.0 S (Figure 3A).

Therefore, zinc is crucial for proper hexamer formulation and the quantity of zinc is no longer at saturated levels at high insulin concentration, leading to a smaller species that is most probably representative of insulin dimer.

To verify whether this effect was time dependent, the same formulations were left under vacuum in the analytical cells at 20˚C for 15 days and sedimented for the second time.

The outcome was the same for both spins, indicating that insulin hexamer maintains stability for at least 15 days after formulation (Figure 3B).

Sedimentation velocity normalized

Figure 3. Sedimentation velocity normalized c(s) of concentration titration of formulated Insulin. 0.25mg/mL (black), 1.0mg/mL (blue), and 4.0mg/mL (green) were analyzed for sedimentation coefficient following formulation. Image credt: Beckman Coulter

The last step was titrating EDTA into the formulation to serve as a chelating agent in order to confirm the role of zinc in insulin hexamer formation. Here, the concentration of both zinc and insulin was fixed to hexamer stable conditions and EDTA was added and incubated for 20min.

The dissociation of hexameric insulin into monomer and dimer species occurred at increasing EDTA concentrations (Figure 4). Approximately 11% of the hexameric complex was dissociated into monomer at 75μM EDTA (Table 1).

An additional 15% was dissociated up to a total monomeric weight signal at 26.4%, at 150μM. The hexameric species was completely dissolved into approximately 60% monomer and 40% dimer at 300μM. Finally, insulin existed at roughly 68% monomer and 32% dimer, at 600μM.

Sedimentation velocity c(s) of EDTA titration with fixed Insulin concentration.

Figure 4. Sedimentation velocity c(s) of EDTA titration with fixed Insulin concentration. Image credt: Beckman Coulter

Table 1. Weight signal average of insulin oligomeric species following EDTA treatment

Zinc Titration

Hexamer

Dimer

Monomer

0μM EDTA

100% (3.201 S)

N/A

N/A

75μM EDTA

88.88% (3.015 S)

N/A

11.11% (1.373 S)

150μM EDTA

73.59% (2.949 S)

N/A

26.41% (1.400 S)

300μM EDTA

N/A

40.63% (2.258 S)

59.37% (1.52 S)

600μM EDTA

N/A

31.99% (2.262 S)

68.01% (1.551 S)

Conclusion

Insulin is a well characterized drug produced by various manufacturers across the world and has been used as a diabetes treatment for over eight decades.

Currently, biopharmaceutical companies are making great efforts to create insulin derivatives and novel formulations to enhance efficacy, time action profiles, and administration procedures.

In future, when developing insulin analogs, the impact of excipients such as zinc will need to be considered on the assembly condition of the drug. A number of analytical methods can be used to probe these effects.

The Human Insulin monograph SEC method has traditionally been used to control the insulin aggregation levels, measuring insulin covalent aggregates such as the covalent dimers as a result of the condensation reaction of free amines at the N-terminus and/or at free lysines.

Disruption and reformation of disulfide links can result in addition covalent aggregation. However, the relative levels of non-covalent aggregates such as insulin hexamer to monomer levels cannot be measured using this method.

These non-covalent aggregates are disrupted during SEC sample preparation and/or analyses procedure. This article has demonstrated the applicability of AUC for assessing the level of non-covalent aggregates such as the insulin hexamer.

This article has demonstrated a straight forward approach to characterizing the higher-order structure of a common biopharmaceutical with minimal development.

For most higher-order techniques such as SEC, the sample needs to be adulterated through large dilutions or column-matrix interactions that can further change the aggregation state.

The preferred method for measuring covalent aggregates is currently SEC, but the molar mass range of AUC is approximately from a few hundred to 109 daltons, a range that exceeds the capability of analytical SEC.

Moreover, AUC is capable of generating quantitative data around protein properties, such as shape, molecular weight, post-translational modifications, solvation properties, and conformational fluctuations at concentrations approaching 40-50mg/mL.

This makes the tool an independent or orthogonal method to the conventionally-used biopharmaceutical technique of SEC to study formulation, development, aggregation, lot-to-lot comparability and biosimilarity.

Acknowledgments

Produced from content authored by Chad Schwartz, Ph.D., Dean Clodfelter, M.S. | Beckman Coulter, Inc., Indianapolis, IN 46268.

References

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Last updated: May 16, 2020 at 12:36 PM

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