Characterization of seed nuclei in glucagon aggregation using light scattering methods and field-flow fractionation
© Hoppe et al; licensee BioMed Central Ltd. 2008
Received: 22 January 2008
Accepted: 09 July 2008
Published: 09 July 2008
Glucagon is a peptide hormone with many uses as a therapeutic agent, including the emergency treatment of hypoglycemia. Physical instability of glucagon in solution leads to problems with the manufacture, formulation, and delivery of this pharmaceutical product. Glucagon has been shown to aggregate and form fibrils and gels in vitro. Small oligomeric precursors serve to initiate and nucleate the aggregation process. In this study, these initial aggregates, or seed nuclei, are characterized in bulk solution using light scattering methods and field-flow fractionation.
High molecular weight aggregates of glucagon were detected in otherwise monomeric solutions using light scattering techniques. These aggregates were detected upon initial mixing of glucagon powder in dilute HCl and NaOH. In the pharmaceutically relevant case of acidic glucagon, the removal of aggregates by filtration significantly slowed the aggregation process. Field-flow fractionation was used to separate aggregates from monomeric glucagon and determine relative mass. The molar mass of the large aggregates was shown to grow appreciably over time as the glucagon solutions gelled.
The results of this study indicate that initial glucagon solutions are predominantly monomeric, but contain small quantities of large aggregates. These results suggest that the initial aggregates are seed nuclei, or intermediates which catalyze the aggregation process, even at low concentrations.
Glucagon is a 29-residue peptide hormone involved in the regulation of blood glucose. Glucagon has several uses as a therapeutic agent, including the emergency treatment of hypoglycemia . Pharmaceutical preparations of glucagon are formulated in the amorphous solid state, and must be solubilized immediately prior to administration. Once in solution, glucagon is physically unstable and must be discarded after 24 hours due to gel formation. In dilute acid, the medium in which lyophilized glucagon is normally solubilized, this gel formation has been shown to result from the growth of fibrillar aggregates . Protein aggregation is a problem in the manufacture, formulation, and delivery of biopharmaceutical products like glucagon. The presence of aggregates can result in reduced biological activity, and other complications with parenteral delivery, including increased immunogenicity . Understanding the aggregation process is important not only for the pharmaceutical production and therapeutic use of glucagon, but also for elucidating a general mechanism of fibril formation.
Much remains to be learned about the mechanism by which proteins associate into amyloid fibrils, the characteristic feature of over 20 degenerative conditions . The toxic species in such diseases has been identified as the intermediates in the aggregation pathway rather than the insoluble, mature fibrils and plaques [5, 6], although the exact molecular mechanism of pathogenesis is controversial . Protein aggregation has been described as a nucleation-dependent process, specifically that the aggregation rate can be seeded by the addition of intermediate aggregates . In general, determining the mechanism for fibril formation at the molecular level may be the key to understanding the basis for amyloid toxicity and disease prevention.
Glucagon has been presented as an ideal model for characterizing fibril formation, since the aggregation process can be studied at pharmaceutically and clinically relevant conditions . Recently, the structure of glucagon fibrils has been probed extensively by atomic force microscopy (AFM) [9–12] as well as electrophoretic and spectroscopic techniques. Various solution conditions have been shown to result in at least two different types of mature fibrils. However, the small oligomeric precursors which serve to initiate and nucleate the process remain relatively uncharacterized. In this study, these initial aggregates, or seed nuclei, are characterized in bulk solution using light scattering methods. The advantages of light scattering techniques over other methods for studying fibril formation are that light scattering experiments are non-invasive to the sample, and can provide absolute determination of hydrodynamic size and molar mass. Unlike AFM, protein aggregates are examined by light scattering in the bulk liquid phase rather than as deposited or adsorbed species on a solid substrate, where the growth and morphology of aggregates may be quite different. Static and dynamic light scattering have been used to monitor the aggregation process of fibril-forming proteins such as β-amyloid [14, 15], α-synuclein , and huntingtin .
Traditionally, polydisperse protein solutions have been separated by size-exclusion chromatography (SEC) for downstream light scattering analysis. In a recent study, the size of glucagon aggregates was evaluated by SEC . However, viscous gel-like protein aggregates have been known to cause plugging and fouling problems in chromatography columns, often leading to irreproducible results. For this reason, the separation method of field-flow fractionation (FFF) has been employed in this study. This technology is unique in that it can be used to separate materials over a much broader range of particle sizes than traditional analytical methods, from 1 nm up to 100 μm. Separation in FFF takes place in an open flow channel, greatly reducing shear forces due to the absence of stationary phase. Asymmetric flow FFF can be coupled with multi-angle laser light scattering for molar mass and size determination independent of molecular weight standards. This combination of analytical methods has recently been used to characterize polysaccharides , water-soluble polymers , and nanosized drug carrier systems [21, 22]. However, it has not been widely used protein applications. In this study, FFF is shown to be an effective method for separating intermediate aggregates from monomeric glucagon.
Static light scattering theory
c is the concentration of the scattering species in solution, R θ is the Rayleigh ratio or the excess intensity of scattered light at θ scattering angle, N A is Avogadro's number, λ is the wavelength of the laser light source, n0 is the refractive index of the solvent, and B22 is the second osmotic virial coefficient of the scattering species.
The M w is determined by plotting values of Kc/R θ versus particle concentration at a given scattering angle. This graphical representation is referred to as a Debye plot. The y-intercept determined through linear regression gives the reciprocal weight-averaged molar mass (1/M w ) of particles in solution.
Dynamic light scattering theory
where G2(∞) is the baseline of the experimentally measured intensity autocorrelation function.
For non-interacting, monodisperse particles that are small compared with the wavelength of light, the scattered electric field correlation function g1(t) decays exponentially,g1(t) = exp(-t/τ)
with scattering vector, q, given byq = (4πn0/λ) sin (θ/2)
where k is the Boltzmann constant and η is the solvent viscosity at absolute temperature, T. This simplified analysis ignores concentrative effects due to hydrodynamic and thermodynamic interactions which have been described elsewhere.
Information about relaxation time and apparent hydrodynamic radius distributions can be extracted from the data by regressing appropriate theoretical models to the autocorrelation function. For example, if a bimodal distribution is expected, a two-exponential fit is performed estimate the two relaxation times independently.
g1(t) = a1 exp(-t/τ1) + a2 exp(-t/τ2) (8)
Parameters a1 and a2 are expansion coefficients known as light intensity weighted amplitudes and τ1 and τ2 are relaxation times.
Generalizing from this approach to systems having multiple particle sizes or broad size distributions is straightforward in principle, but requires specialized computational approaches in practice. Each scattering species can be represented by a corresponding relaxation time, resulting in a sum of exponentials
g1(t) = a1 exp(-t/τ1) + a2 exp(-t/τ2)+... (9)
where a n values represent the relative contributions (amplitudes) of each particle size. The regularized inverse Laplace transform program CONTIN is routinely utilized to perform such multi-exponential analyses of relaxation-time distributions . Under the assumption that the scattering particles behave as hard spheres in dilute solution, the relaxation time distribution obtained can be converted into an apparent hydrodynamic size distribution using the Stokes-Einstein relationship.
The theory and principles of asymmetric flow FFF have been extensively reviewed elsewhere [24–26]. In summary, this type of FFF is performed inside a thin, ribbon-like channel approximately 30 cm in length, 2 cm wide, and ranging in thickness up to 500 μm. Carrier fluid is pumped through the channel from the inlet end exhibiting a laminar flow profile. A cross-flow is induced perpendicular to the channel flow, which exits the channel through the bottom wall fitted with an ultrafiltration membrane. The cross-flow forces the sample components toward this "accumulation wall" of the channel, where a concentration gradient is established. Small particles with higher diffusion coefficients achieve equilibrium positions at higher levels in the channel than larger particles, and are thus transported through the channel more rapidly due to the parabolic profile of the channel flow. Consequently, small particles elute first, opposite the order of elution in SEC.
In the manner described above, FFF accomplishes the separation of different sized particles in polydisperse solutions for analysis by downstream detectors. Molar masses of the fractionated species are determined using a multi-angle light scattering (MALS) detector which collects on-line SLS measurements at multiple scattering angles. The linear Zimm method  is used to obtain molar mass by setting B22 to zero and extrapolating to zero scattering angle.
Preparation of Protein Standards for FFF/MALS Method Validation
The following protein standards were obtained in lyophilized powder form from Sigma Aldrich (St. Louis, MO): bovine serum albumin (BSA), alcohol dehydrogenase, β-amylase, apoferritin, and thyroglobulin. All protein standards were dissolved in Dulbecco's phosphate buffer solution (PBS), pH 6.5, also obtained in powder form from Sigma and dissolved in chromatography grade Optima water from Fisher Scientific (Fair Lawn, NJ). PBS was filtered with 0.1 μm filters before use.
Preparation of Glucagon Solutions
SLS and DLS measurements
SLS and DLS experiments were conducted with an ALV-GmbH (Langen, Germany) SP-125 Compact DLS/SLS Goniometer. The majority of experiments presented here utilized a vertically polarized 400 mW diode-pumped, solid-state Coherent DPSS532-400 laser (Coherent Inc., Santa Clara, CA) operating at 532 nm wavelength as the light source. Glucagon solutions were prepared as previously described, placed in the sample compartment, fixed to restrict movement, and equilibrated at 30°C by a thermostatted bath. In DLS experiments, the apparent hydrodynamic size and PSD of glucagon samples were determined by obtaining autocorrelation functions of the scattered light intensity, G2(t), using an ALV-5000/E multiple tau digital correlator. Time correlation functions were analyzed using CONTIN software to determine R h and PSD. The specific refractive index (dn/dc) of glucagon was measured using a Bellingham & Stanley (Kent, UK) 60/ED Abbe refractometer with the laser light source described. The values obtained for dn/dc at 532 nm were 0.175 in HCl and 0.189 in NaOH.
Asymmetric flow FFF experiments were carried out with an Eclipse F separation system from Wyatt Technology Corporation (WTC, Santa Barbara, CA). For protein standard analysis, 250 μm channel thickness and 10 kDa MWCO membranes were used, also obtained from WTC. Protein standards were prepared in PBS as previously described, and this buffer was used as the mobile phase. For glucagon experiments, 450 μm channel thickness and 1 kDa MWCO ultrafiltration membranes were used, obtained from WTC. Glucagon solutions were prepared in certified 0.01 N HCl as previously described, and this solvent was used as the mobile phase. In all experiments, samples were injected into the FFF channel using a manual Rheodyne (Rohnert Park, CA) injection port. Channel flow was set at 1 mL/min, and cross-flow rates were controlled using the Eclipse2 software, version 2.3 (WTC).
FFF experiments were coupled with MALS detection for molar mass determination. The MALS detector used was an 18-angle DAWN EOS (WTC), which employs a 685 nm wavelength 30 mW linearly polarized Ga-As laser light source. Molar mass calculations were made using ASTRA software (WTC). On-line concentration measurements were made with a Shimadzu (Columbia, MD) SPD-10AV UV spectrophotometer set at a wavelength of 278 nm to monitor the absorbance of light by glucagon.
Results and Discussion
Validation of FFF/MALS method for protein characterization
Protein standards expected MW compared with M w inferred from FFF/MALS experiments
M w inferred from
Presence of high molecular weight glucagon aggregates
Removal of aggregates in acidic glucagon solutions slows gelation rate
DLS results indicate that acidic and alkaline glucagon solutions exhibit distinctly different behavior following filtration. Bimodal distributions were still present following the filtration of alkaline glucagon solutions (Fig. 6c), whereas the higher molecular weight aggregates were eliminated by filtration of acidic glucagon solutions (Fig. 6d). The bimodal distribution that remained after filtration of alkaline glucagon solutions showed the larger subpopulation peak hydrodynamic radius value shifting to a slightly smaller R h of 61 nm from its original size of 100 nm.
These DLS results indicate the presence of higher molecular weight aggregates that can be removed by filtration from acidic but not alkaline solutions. The filter retention may be related to the relative charge of the filter membrane or to the relative rigidity of the aggregates. Since the filters used were designed to have low affinity for protein, the aggregate rigidity seems to be the more plausible explanation for the observed effects.
Characterization of glucagon aggregates by FFF/MALS
To further quantify the two subpopulations within the unfiltered samples, SEC experiments were attempted. SEC proved ineffective in monitoring the molecular weight distribution of aggregating glucagon systems (data not shown). Aggregates present in glucagon solutions injected into a column using 0.01 N HCl eluent caused fouling and backpressure problems in the SEC system, and tended to dissociate inside the column, emerging in the monomeric state. Thus, this method was abandoned as a means to monitor the aggregation process.
where nM represents n glucagon monomers, and M n represents an oligomeric species of number n. It was demonstrated that filtration can remove the large aggregates in some situations.
These findings describe an important aspect of the aggregation behavior of glucagon, which is considered a model amyloidgenic protein. This work contributes to the field of biological engineering because of its relevance to drug formulation and delivery, as well as clinical applications.
We would like to gratefully thank Eli Lilly and Company for providing the glucagon used in these studies, and the NSF for fellowship funding.
- Kim DY, Shin NK, Chang SG, Shin HC: Production of recombinant human glucagon in Escherichia coli by a novel fusion protein approach. Biotechnology Techniques 1996,10(9):669-672. 10.1007/BF00168477View ArticleGoogle Scholar
- Beaven GH, Gratzer WB, Davies HG: Formation and structure of gels and fibrils from glucagon. Eur J Biochem 1969, 11: 37-42. 10.1111/j.1432-1033.1969.tb00735.xView ArticleGoogle Scholar
- Costantino HR, Langer R, Klibanov AM: Solid-phase aggregation of proteins under pharmaceutically relevant conditions. J Pharm Sci 1994,83(12):1662-1669. 10.1002/jps.2600831205View ArticleGoogle Scholar
- Stefani M: Protein misfolding and aggregation: new examples in medicine and biology of the dark side of the protein world. Biochim Biophys Acta 2004,1739(1):5-25.View ArticleMathSciNetGoogle Scholar
- Bucciantini M, Giannoni E, Chiti F, Baroni F, Formigli L, Zurdo JS, Taddei N, Ramponi G, Dobson CM, Stefani M: Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases. Nature 2002,416(6880):507-511. 10.1038/416507aView ArticleGoogle Scholar
- Kirkitadze MD, Bitan G, Teplow DB: Paradigm shifts in Alzheimer's disease and other neuro degenerative disorders: The emerging role of oligomeric assemblies. J Neurosci Res 2002,69(5):567-577. 10.1002/jnr.10328View ArticleGoogle Scholar
- Stefani M, Dobson CM: Protein aggregation and aggregate toxicity: new insights into protein folding, misfolding diseases and biological evolution. J Mol Med 2003,81(11):678-699. 10.1007/s00109-003-0464-5View ArticleGoogle Scholar
- Koo EH, Lansbury PT, Kelly JW: Amyloid diseases: Abnormal protein aggregation in neurodegeneration. Proc Natl Acad Sci USA 1999,96(18):9989-9990. 10.1073/pnas.96.18.9989View ArticleGoogle Scholar
- De Jong KL, Incledon B, Yip CM, DeFelippis MR: Amyloid fibrils of glucagon characterized by high-resolution atomic force microscopy. Biophys J 2006,91(5):1905-1914. 10.1529/biophysj.105.077438View ArticleGoogle Scholar
- Dong MD, Hovgaard MB, Xu SL, Otzen DE, Besenbacher F: AFM study of glucagon fibrillation via oligomeric structures resulting in interwoven fibrils. Nanotechnology 2006,17(16):4003-4009. 10.1088/0957-4484/17/16/001View ArticleGoogle Scholar
- Pedersen JS, Dikov D, Flink JL, Hjuler HA, Christiansen G, Otzen DE: The changing face of glucagon fibrillation: Structural polymorphism and conformational imprinting. J Mol Biol 2006,355(3):501-523. 10.1016/j.jmb.2005.09.100View ArticleGoogle Scholar
- Pedersen JS, Flink JM, Dikov D, Otzen DE: Sulfates dramatically stabilize a salt-dependent type of glucagon fibrils. Biophys J 2006,90(11):4181-4194. 10.1529/biophysj.105.070912View ArticleGoogle Scholar
- Onoue S, Iwasa S, Kojima T, Katoh F, Debari K, Koh K, Matsuda Y, Yajima T: Structural transition of glucagon in the concentrated solution observed by electrophoretic and spectroscopic techniques. J Chromatogr A 2006,1109(2):167-173. 10.1016/j.chroma.2005.11.130View ArticleGoogle Scholar
- Pallitto MM, Murphy RM: A mathematical model of the kinetics of beta-amyloid fibril growth from the denatured state. Biophys J 2001,81(3):1805-1822.View ArticleGoogle Scholar
- Lomakin A, Teplow DB, Kirschner DA, Benedek GB: Kinetic theory of fibrillogenesis of amyloid beta-protein. Proc Natl Acad Sci USA 1997,94(15):7942-7947. 10.1073/pnas.94.15.7942View ArticleGoogle Scholar
- Li J, Uversky VN, Fink AL: Conformational behavior of human alpha-synuclein is modulated by familial Parkinson's disease point mutations A30P and A53T. Neurotoxicology 2002,23(4-5):553-567. 10.1016/S0161-813X(02)00066-9View ArticleGoogle Scholar
- Georgalis Y, Starikov EB, Hollenbach B, Lurz R, Scherzinger E, Saenger W, Lehrach H, Wanker EE: Huntingtin aggregation monitored by dynamic light scattering. Proc Natl Acad Sci USA 1998,95(11):6118-6121. 10.1073/pnas.95.11.6118View ArticleGoogle Scholar
- Onoue S, Ohshima K, Debari K, Koh K, Shioda S, Iwasa S, Kashimoto K, Yajima T: Mishandling of the therapeutic peptide glucagon generates cytotoxic amyloidogenic fibrils. Pharm Res 2004,21(7):1274-1283. 10.1023/B:PHAM.0000033016.36825.2cView ArticleGoogle Scholar
- Wittgren B, Borgstrom J, Piculell L, Wahlund KG: Conformational change and aggregation of kappa-carrageenan studied by flow field-flow fractionation and multiangle light scattering. Biopolymers 1998,45(1):85-96. PublisherFullText 10.1002/(SICI)1097-0282(199801)45:1<85::AID-BIP7>3.0.CO;2-VView ArticleGoogle Scholar
- Viebke C, Williams PA: The influence of temperature on the characterization of water-soluble polymers using asymmetric flow field-flow-fractionation coupled to multiangle laser light scattering. Anal Chem 2000,72(16):3896-3901. 10.1021/ac991205xView ArticleGoogle Scholar
- Fraunhofer W, Winter G, Coester C: Asymmetrical flow field-flow fractionation and multiangle light scattering for analysis of gelatin nanoparticle drug carrier systems. Anal Chem 2004,76(7):1909-1920. 10.1021/ac0353031View ArticleGoogle Scholar
- Arifin DR, Palmer AF: Determination of size distribution and encapsulation efficiency of liposome-encapsulated hemoglobin blood substitutes using asymmetric flow field-flow fractionation coupled with multi-angle static light scattering. Biotechnol Prog 2003,19(6):1798-1811. 10.1021/bp034120xView ArticleGoogle Scholar
- Provencher SW: Inverse problems in polymer characterization - direct analysis of polydispersity with photon correlation spectroscopy. Makromol Chem 1979,180(1):201-209. 10.1002/macp.1979.021800119View ArticleGoogle Scholar
- Giddings JC: Nonequilibrium theory of field-flow fractionation. J Chem Phys 1968,49(1):81-85. 10.1063/1.1669863View ArticleGoogle Scholar
- Wahlund KG, Giddings JC: Properties of an asymmetrical flow field-flow fractionation channel having one permeable wall. Anal Chem 1987,59(9):1332-1339. 10.1021/ac00136a016View ArticleGoogle Scholar
- Fraunhofer W, Winter G: The use of asymmetrical flow field-flow fractionation in pharmaceutics and biopharmaceutics. Eur J Pharm Biopharm 2004,58(2):369-383. 10.1016/j.ejpb.2004.03.034View ArticleGoogle Scholar
- Wyatt PJ: Light scattering and the absolute characterization of macromolecules. Anal Chim Acta 1993, 272: 1-40. 10.1016/0003-2670(93)80373-SView ArticleGoogle Scholar
- Creighton TE: The general properties of protein structures. In Proteins: Structures and Molecular Properties. New York , W. H. Freeman and Company; 1993:229.Google Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.