Shining a light on insulin

As a protein insulin is well known and it has now proved to be an ideal model to examine the oligomeric states of therapeutic proteins using light scattering techniques   Insulin plays a vital role in the regulation of blood glucose levels, cell growth, and fat metabolism – to name only a few of its manifold functions. Under physiological conditions, human insulin self-associates in the presence of zinc ions to form a hexameric complex with two zinc ions bound per hexamer. In pharmaceutical preparations, zinc is added to stabilise the hexamer and thereby produces a more stable formulation. Hence, it is of interest to investigate the oligomerisation from both a formulation and a pharmacological perspective.  The combination of size exclusion chromatography (SEC) with multi-angle light scattering (MALS), dynamic light scattering (DLS), and refractive index (RI) detection can be applied as a powerful tool to identify monomers, dimers, hexamers and higher aggregates of insulin. Using this approach, each preparation can be comprehensively characterised to determine optimal formulation, storage, and administration conditions for the patient’s benefit. Insulin, one of the most important mammalian hormones, regulates a multitude of metabolic functions, including the control of the blood glucose level in the body. Under healthy conditions, insulin is produced and stored in the islet tissues of the pancreas and released depending on the metabolic situation. In patients suffering from diabetes, insulin cannot be sufficiently produced by the body: It has to be administered via bolus injection, insulin pump, or other pathways. [caption id="attachment_38121" align="alignright" width="200"] Figure 1: Molar mass vs. elution time for insulin formulated with and without zinc, determined by SEC-MALS. Sample 1 (no zinc, red) shows a stable monomer with a small fraction of hexamer, while Sample 2 (0.1 mM zinc, blue) shows monomer-dimer equilibrium in conjunction with a stable hexamer. The UV signal at 280 nm is plotted as an overlay. The molar mass was determined by MALS using the concentration measured by RI.[/caption] The production and formulation of recombinant human insulin and its analogues have been evolving over the last 30 years, and understanding the oligomeric form of each formulation is essential. Under physiological conditions, insulin forms hexamers in the presence of zinc ions and is stored as zinc complexes in the pancreas. In pharmaceutical preparations insulin is normally formulated at concentrations where the self-association is pronounced and hence zinc is added to stabilise the hexamer and thereby a more stable formulation. Upon dilution after delivery the hexamer dissociates and ends up as monomers in the blood stream. Hence it is of interest to investigate the oligomerisation both from a formulation as well as pharmacological perspective. The first step in assessing the oligomeric state of a protein or peptide is usually the separation of the molecular species and their characterisation. Size exclusion chromatography (SEC) is often the method of choice for separating monomers, oligomers, and higher order aggregates for further characterisation. SEC separates the molecules according to their hydrodynamic volume. Although hydrodynamic volume is often proportional to molar mass, analytical SEC does not automatically provide for the exact mass determination. The coupling of SEC with light scattering overcomes this limitation and allows for absolute characterisation of the oligomeric state of the eluting macromolecules. Multi-angle light scattering (MALS) is a first principles method for quantifying the molar mass of macromolecules in solution. This technique makes no assumptions about the molecule’s structure, and no molar mass standards are required. Simultaneous dynamic light scattering (DLS) measurements can be made within the same detector to obtain the hydrodynamic radius of the molecules. [caption id="attachment_38122" align="alignleft" width="200"] Figure 2: Molar mass vs. elution time for Samples 1 (red) and 3 (green). The UV signal at 280 nm is plotted as an overlay. The molar mass was calculated by MALS and RI.[/caption] The reversible self-association of insulin and its analogues into dimers and higher-order oligomers is a well-known phenomenon¹. Previous studies have also shown the separation of insulin oligomers by SEC and detected by light scattering techniques^2,3. Light scattering measurements of insulin self-assembly are not limited to chromatography. Composition-gradient MALS (CG-MALS) measurements with unfractionated samples have also been applied to quantify the equilibrium stoichiometry and association constants for insulin^4,5. Here, we use SEC-MALS to compare a zinc-free insulin analogue formulation to human insulin formulated under two different zinc concentrations. Materials and methods Size exclusion chromatography was performed with an Agilent 1260 HPLC system, which included an auto sampler, isocratic pump, and degasser. The separation column was a Superose 12 300/10 from GE Healthcare. The eluent from the column flowed through a variable wavelength detector set to 280 nm (Agilent), a DAWN HELEOS multi-angle light scattering detector equipped with a WyattQELS for online DLS detection, and an Optilab rEX refractive index (RI) detector. The LS, DLS and RI detectors were from Wyatt Technology. SEC-MALS data were analysed using ASTRA software. The mobile phase consisted of 10 mM Tris, 140 mM NaCl, 2 mM phenol, and 200 ppm NaN₃ at pH 7.7. Three different insulin sample preparations all containing 0.6 mM insulin were analysed: Sample 1: insulin analogue without added zinc Sample 2: human insulin with 0.1 mM zinc Sample 3: human insulin with 0.3 mM zinc Results and discussion The presence and absence of zinc has a clear effect on the oligomerisation of insulin.  Figure 1 shows the measured molar mass by MALS overlaid on the UV chromatogram for Samples 1 and 2. The major fraction of Sample 1 (no zinc) has a measured molar mass of 5.8 kDa and corresponds to the insulin monomer (Figure 1, red). This species is well separated from the minor fraction corresponding to hexamer with molar mass ~35 kDa.  Moreover, the light scattering signal indicates that the measured molar mass for the secondary peak includes a range of oligomers from monomer to hexamer – a conclusion that could not be drawn from the UV signal alone. It should be emphasised that, because light scattering is extremely sensitive to high molecular-weight species, MALS identified the hexamer easily even though very little UV signal is present. In contrast, the majority of Sample 2 (0.1 mM zinc) exists as a hexamer (Figure 1, blue), and a significant fraction appears to be undergoing monomer-dimer equilibrium. This lower molecular weight peak elutes slightly faster than the monomer peak for Sample 1, and the measured-molar mass is heterogeneous across the peak – spanning the masses of the monomer and dimer (~6-12 kDa). [caption id="attachment_38123" align="alignright" width="200"] Figure 3: The change in molar mass as a function of eluting concentration (injection volume) for Sample 2 gives insight into the monomer-dimer equilibrium. The molar mass measured by SEC-MALS increases as more protein is loaded on the column, resulting in different monomer-dimer fractions. The molar mass was calculated by MALS and RI, and the UV signal at 280 nm is plotted as an overlay.[/caption] Further addition of zinc (to 0.3 mM) completely shifts the equilibrium towards the hexamer as seen in Sample 3 (Figure 2, green). The monomer-dimer peak is almost undetectable by UV, and the majority of the sample has a molar mass consistent with that of a hexamer. With this higher concentration of zinc, a small high-molecular weight fraction is also detected in Sample 3. Based on the measured molar mass (70 kDa), this fraction is identified as the insulin dodecamer. Although present only in a small amount, this peak generates a distinct signal clearly observed by the MALS detector. SEC-MALS allows the analysis of the strength of a reversible self-association. Increasing the volume injected on the column increases in the overall protein concentration as it passes through the detectors, thus shifting the equilibrium of the eluent toward the oligomeric complexes. For Samples 2 and 3, changes in equilibrium association were investigated by injecting either 50 µL or 200 ?L of the same sample solution onto the SEC column. The increased injection volume clearly affects the equilibrium of Sample 2 (Figure 3). As a higher insulin concentration is achieved, the monomer-dimer equilibrium is shifted towards dimer and higher order oligomers. The lowest measured molar mass observed for the 50 ?L injection is ~6 kDa, corresponding to the monomer. However, the smallest measured molar mass for the 200 µL injection has a molar mass consistent with a dimer. The hexamer fraction is unaffected by the increase in eluting concentration and shows no tendency to form larger oligomers. [caption id="attachment_38124" align="alignleft" width="200"] Figure 4: The molar mass profile for Sample 3 does not change as a function of concentration, indicating is “locked” in a stable hexameric conformation. The molar mass was calculated by MALS and RI. The RI signal is plotted as an overlay; the UV signal was saturated due the high sample load.[/caption] In the case of Sample 3, there is basically no effect of varying the injection volume (Figure 4). In presence of 0.3 mM zinc, the system is “locked” in its hexamer form, and identical molar masses are detected at both concentrations (50 µL and 200 ?L injections). There is also no increase in the mass fraction of dodecamer. Obviously, the self-association behaviour of the hexamer is significantly different from that of the monomer. Conclusions Multi-angle light scattering proves to be the method of choice for the examination of association phenomena not only for large proteins, such as antibodies, but also for peptide molecules with lower molecular masses. Since the use of proteins and peptides for therapeutic purposes is continually increasing, the application of light scattering as a powerful method of molecular characterisation will also become more and more important. References 1. D. Brett Ludwig, Jonathan N. Webb, Cristina Fernandez, John F. Carpenter, Theodore W. Randolph., (2011). Quaternary Conformational Stability: The Effect of Reversible Self-Association on the Fibrillation of Two Insulin Analogs. Biotechnology and Bioengineering, Vol. 108, 2359-2370. 2. M. H. Jensen, P.-O. Wahlund, J. K. Jacobsen, B. Vestergaard, M. van de Weert, S. Havelund., (2011). Self-association of long-acting insulin analogues studied by size exclusion chromatography coupled to multi-angle light scattering. Journal of Chromatography B, 879(28), 2945-2951. 3. I. Jonassen, S. Havelund, T. Hoeg-Jensen, D. B. Steensgaard, P.-O. Wahlund, U. Ribel., (2012). Design of the Novel Protraction Mechanism of Insulin Degludec, an Ultra-long-Acting Basal Insulin. Pharmaceutical Research, 29(8), 2104-2114. 4. Arun K. Attri, Cristina Fernández, Allen P. Minton., (2010). pH-dependent self-association of zinc-free insulin characterized by concentration-gradient static light scattering. Biophys. Chem., Vol. 148, 28-33. 5. Arun K. Attri, Cristina Fernández, Allen P. Minton., (2010) Self-association of Zn-insulin at neutral pH: Investigation by concentration gradient-static and dynamic light scattering. Biophysical J., Vol. 148, 23-27. Authors P. Wahlund, Novo Nordisk A/S, D. Roessner and T. Jocks, Wyatt Technology Contact www.wyatt.com info@wyatt.com