Liquid-Liquid Phase Separation in Oligomeric Peptide Solutions.

Oligomeric peptides exist widely in living organisms and play a role in a broad range of biological functions. We report the first observation of liquid-liquid phase separation (LLPS) in peptide solutions, in particular, solutions of peptides consisting of noncovalent oligomers. We determined the binary phase boundary of the oligomeric peptide solution and compared the result to the well-established phase diagram of globular proteins. We also provide simple theoretical interpretations of the similarities and differences between the phase diagrams of peptides and proteins. Finally, by tuning inter-oligomer interactions using a crowding agent, we demonstrated that LLPS is a universal phenomenon that can be observed under different solution conditions for a variety of peptides.

The molecular basis for the prolonged blood circulation of lipidated incretin peptides: Peptide oligomerization or binding to serum albumin?

Hybrid incretin peptides are a new generation of drugs for the treatment of diabetes and obesity. Despite their biological potency, the effectiveness of these peptides as drugs is limited by their short circulation time in blood (typically within minutes). In this work, we show that lipid conjugated forms of a GLP-1/GIP/glucagon hybrid peptides stay in circulation for hours. We studied the oligomerization and albumin-binding of the unconjugated hybrid peptide as well as its lipidated variants. These lipidated peptides differ in the N-terminal mutation, the position of lipidation and the linkage to lipid. We found that these lipidated peptides form stable oligomers at concentrations above 1mg/mL. This concentration range is relevant to formulation and storage of the peptides. We observed no binding between the peptide oligomers and human serum albumin. However, at the expected therapeutic concentration range (~10-100ng/mL), the oligomers dissociate into monomers. The monomers of lipidated peptides bind to albumin. We have determined the dissociation constants of binding between the lipidated peptides and serum albumin. The dissociation constants of albumin-binding of our lipidated peptides are all very close and similar to that of the fatty acid binding of albumin. Our findings suggest that the monomeric lipidated peptides bind to HSA mainly by the fatty acid chain. Therefore, albumin binding is likely to be a universal mechanism of the prolonged circulating duration of lipidated pharmaceutical peptides.

Transformation of oligomers of lipidated peptide induced by change in pH.

Oligomerization of lipidated peptides is of general scientific interest and is important in biomedical and pharmaceutical applications. We investigated the solution properties of a lipidated peptide, Liraglutide, which is one of the glucagon-like peptide-1 (GLP-1) agonists used for the treatment of type II diabetes. Liraglutide can serve as a model system for studying biophysical and biochemical properties of micelle-like self-assemblies of the lipidated peptides. Here, we report a transformation induced in Liraglutide oligomers by changing pH in the vicinity of pH 7. This fully reversible transformation is characterized by changes in the size and aggregation number of the oligomer and an associated change in the secondary structure of the constituent peptides. This transformation has quite slow kinetics: the equilibrium is reached in a course of several days. Interestingly, while the transformation is induced by changing pH, its kinetics is essentially independent of the final pH. We interpreted these findings using a model in which desorption of the monomer from the oligomer is the rate-limiting step in the transformation, and we determined the rate constant of the monomer desorption.

Assessment and significance of protein-protein interactions during development of protein biopharmaceuticals.

Early development of protein biotherapeutics using recombinant DNA technology involved progress in the areas of cloning, screening, expression and recovery/purification. As the biotechnology industry matured, resulting in marketed products, a greater emphasis was placed on development of formulations and delivery systems requiring a better understanding of the chemical and physical properties of newly developed protein drugs. Biophysical techniques such as analytical ultracentrifugation, dynamic and static light scattering, and circular dichroism were used to study protein-protein interactions during various stages of development of protein therapeutics. These studies included investigation of protein self-association in many of the early development projects including analysis of highly glycosylated proteins expressed in mammalian CHO cell cultures. Assessment of protein-protein interactions during development of an IgG1 monoclonal antibody that binds to IgE were important in understanding the pharmacokinetics and dosing for this important biotherapeutic used to treat severe allergic IgE-mediated asthma. These studies were extended to the investigation of monoclonal antibody-antigen interactions in human serum using the fluorescent detection system of the analytical ultracentrifuge. Analysis by sedimentation velocity analytical ultracentrifugation was also used to investigate competitive binding to monoclonal antibody targets. Recent development of high concentration protein formulations for subcutaneous administration of therapeutics posed challenges, which resulted in the use of dynamic and static light scattering, and preparative analytical ultracentrifugation to understand the self-association and rheological properties of concentrated monoclonal antibody solutions.

Establishing a link between amino acid sequences and self-associating and viscoelastic behavior of two closely related monoclonal antibodies.

PURPOSE: To investigate the underlying cause for the observed differences in self-associating and viscoelastic behavior between two monoclonal antibodies, MAb1, and MAb2. METHODS: Several mutants were designed by swapping charged residues in MAb1 with those present in MAb2 at their respective positions and vice versa. Rheological analysis was done at low and high shear rates. Dynamic light scattering quantified intermolecular interactions in dilute solutions; sedimentation equilibrium analysis determined the corrected weight average molecular weight (M (wc)) to assess the self-associating behavior in high concentration. The molecular charge was estimated from electrophoretic mobility measurements. RESULTS: Replacing the charged residues in the CDR of MAb1 resulted in a lower M (wc) and solution viscosity. The corresponding changes in either just the variable light (VL) or variable heavy (VH) chain showed only a partial decrease in viscosity, whereas changes in both VL and VH chains resulted in a dramatic reduction in viscosity. The converse case where the VL and VH chains of MAb2 were made to look like MAb1 did not self-associate or show increased viscosity. CONCLUSIONS: Exposed charged residues in the CDR of MAb1 are critical in determining the self-associating and highly viscous behavior observed at high concentrations.

Increasing IgG concentration modulates the conformational heterogeneity and bonding network that influence solution properties.

Multiple molecular driving forces mediate protein stability, association, and recognition in concentrated solutions. Here we investigate the interactions that modulate the nonideal solution behavior of two immunoglobulins (IgG1s) in highly concentrated solutions using two-dimensional vibrational correlation spectroscopy (2D-COS) and principal components analysis (PCA). A specific sequence of changes is observed in the concentration-dependent vibrational spectra of the highly viscous IgG solution that deviates from ideality, whereas that sequence is reversed for all other conditions examined. The asynchronous spectra reveal variation in beta-sheet and turn regions occur before intensity variations in disordered and alpha-helical regions as the concentration is increased for the highly viscous regime. This is in contrast to the sequence observed for all other conditions studied and to the idea that beta-sheet regions are resistant to concentration-dependent affects. Finally, we show that increased hydrogen bonding and electrostatics primarily modulate the intermolecular association and nonideal behavior. Specifically, 2D-COS and PCA analysis of the amide II region suggests that Glu and Asp residues trigger the change resulting in increased viscosity and association of one IgG.

Carboxylate-dependent gelation of a monoclonal antibody.

PURPOSE: This paper shows the first ever assembly of monoclonal antibody using multivalent carboxylate ions into highly ordered structures that feature viscoelastic properties reminiscent of other filamentous proteins. METHODS: A monoclonal antibody was assembled into filamentous networks by adding multivalent carboxylates to the protein solution. Gelation and characterization of these networks were monitored using mechanical rheometry, electron microscopy, Fourier transform infra-red and Raman spectroscopy. RESULTS: Electron microscopy and mechanical rheometry suggest the formation of rigid filament bundles that feature strong interfilament interactions. Filament network elasticity increased with multivalent carboxylate and protein concentrations, hinting at the importance of multivalent carboxylates in the mechanism of assembly. CONCLUSION: Assembly is not triggered by high ionic strength but with multivalent carboxylates. A high protein concentration is required for filament formation and the elasticity of the networks are weakly dependent on concentration. The exact mechanism of assembly is still elusive, although we speculate that carboxylates could act as a bridge to crosslink antibody monomers. These monoclonal antibody monomers could be linked either through Fab-Fab or Fc-Fab regions, although previous reports have shown evidence of reversible self-association mediated through the Fab regions.

Reversible self-association of a concentrated monoclonal antibody solution mediated by Fab-Fab interaction that impacts solution viscosity.

Reversible self-association of a monoclonal antibody (MAb) in a high concentration formulation results in a solution with a high viscosity. The nature of the self-association of full-length as well as antibody fragments has been studied by rheometry. Chaotropic anions reduced solution viscosity more than kosmotropic anions, a result that can be explained by the Hofmeister series and the net charge of the MAb. The effect of strong chaotropes, such as urea and guanidine HCl at concentration below 300 mM on solution viscosity was also investigated. While the secondary and tertiary structure of the MAb was not altered, as determined by circular dichroism measurements, guanidine HCl reduced viscosity much more effectively than urea. Since urea is uncharged and guanidine HCl is monovalent, this study indicated that a charge effect may be a more important factor than the chaotropic nature of excipients in reducing solution viscosity by breaking network self-association of a MAb. To further understand which part of a MAb participates in this network self-association, a series of titration studies using the full-length MAb, F(ab')(2), and Fab fragments was conducted. From this study, the Fab was found to be the primary site of the network self-association.

Phase separation kinetics of polyelectrolyte solutions.

The kinetics of phase separation of aqueous solutions of sodium-poly(styrene sulfonate) (NaPSS) containing barium chloride (BaCl(2)) is studied by static and dynamic light scattering. We report a novel mechanism of phase separation, where an enrichment of polymer aggregates of well-defined size occurs in the very early stage of nucleation, which is then followed by a growth process in the formation of the new phase. In the latter stage, the polymer aggregates formed in the early stage act as the templating nuclei. Even in the homogeneous phase at higher temperatures above the upper critical phase boundary, polymer aggregates are present in agreement with previously reported results. Upon rapidly cooling the system below the phase boundary, the number concentration of the aggregates increases first by maintaining their size to be relatively monodisperse, before the growth process takes over at later times. The size and fractal dimension of aggregates in the homogeneous phase and the early nucleation stage of phase separation and the dependence of nucleation time and growth rate on quench depth and salt concentration are determined. The hydrodynamic radius (R(H)) of the unaggregated chains is of the order of 1-10 nm depending on the molecular weight of NaPSS, while R(H) of aggregates is of the order of 100 nm independent of the molecular weight of NaPSS. Unaggregated chains follow good solution behavior with a fractal dimension of 5/3 while the fractal dimension of aggregates is larger than 3.5 suggesting the branched nature of aggregates. Nucleation time is sensitive to quench depth and salt concentration. Increasing a quench depth or increasing BaCl(2) concentration shortens the nucleation time. After the nucleation time, during the growth period, the size of aggregates grows linearly with time, with growth rate being higher for deeper quench depths and higher BaCl(2) concentrations. The mechanism of phase separation of aqueous solutions of NaPSS and BaCl(2) is seen to proceed by utilizing the already-existing aggregates to nucleate the new phase, in marked contrast to hitherto known results on phase separation in uncharged polymer systems.

Extended, relaxed, and condensed conformations of hyaluronan observed by atomic force microscopy.

The conformation of the polysaccharide hyaluronan (HA) has been investigated by tapping mode atomic force microscopy in air. HA deposited on a prehydrated mica surface favored an extended conformation, attributed to molecular combing and inhibition of subsequent chain recoil by adhesion to the structured water layer covering the surface. HA deposited on freshly cleaved mica served as a defect in a partially structured water layer, and favored relaxed, weakly helical, coiled conformations. Intramolecularly condensed forms of HA were also observed, ranging from pearl necklace forms to thick rods. The condensation is attributed to weak adhesion to the mica surface, counterion-mediated attractive electrostatic interactions between polyelectrolytes, and hydration effects. Intermolecular association of both extended and condensed forms of HA was observed to result in the formation of networks and twisted fibers, in which the chain direction is not necessarily parallel to the fiber direction. Whereas the relaxed coil and partially condensed conformations of HA are relevant to the native structure of liquid connective tissues, fully condensed rods may be more relevant for HA tethered to a cell surface or intracellular HA, and fibrous forms may be relevant for HA subjected to shear flow in tight intercellular spaces or in protein-HA complexes.