Noncovalent PEGylation: different effects of dansyl-, L-tryptophan-, phenylbutylamino-, benzyl- and cholesteryl-PEGs on the aggregation of salmon calcitonin and lysozyme.

Protein aggregation is a major instability that can occur during all stages of protein drug production and development. Protein aggregates may compromise the safety and efficacy of the final protein formulation. In this paper, various new excipients [phenylbutylamino-, benzyl-, and cholesteryl-polyethylene glycols (PEGs)] and their use for the reduction of aggregation of salmon calcitonin (sCT) and hen egg-white lysozyme (HEWL) by noncovalent PEGylation are presented. The ability to suppress aggregation of sCT in various buffer systems at a 1:1 molar ratio was assessed by following changes in protein conformation and aggregation state over time. The results are compared with that of dansyl- and L-tryptophan (Trp)-PEGs described in earlier publications. Furthermore, the influence of the different PEG-based excipients on the aggregation of HEWL was measured. HEWL aggregation was completely suppressed in the presence of cholesteryl-PEGs (2 and 5 kDa), whereas deterioration was observed using benzyl-methoxy polyethylene glycols (mPEGs; 2 and 5 kDa). Phenylbutylamino- and Trp-mPEG (2 kDa), as well as dansyl-PEGs of different molecular weight prolonged the lag phase of aggregation and reduced the aggregation velocity of HEWL.

Molecular mass dependence of adsorbed amount and hydrodynamic thickness of polyelectrolyte layers.

Highly charged polyelectrolytes adsorbed on oppositely charged colloidal particles are investigated by electrophoresis and dynamic light scattering. The dependence of the adsorbed amount and of the hydrodynamic layer thickness on the molecular mass and the salt level is analyzed. The adsorbed amount increases with increasing salt level and decreases with increasing molecular mass. The hydrodynamic layer thickness is independent of the molecular mass at low salt levels, but increases with the molecular mass as a power law with an exponent 0.10 ± 0.01 at high salt. The same behavior was observed for different polyelectrolytes and substrates and therefore is suspected to be generic. Due to semi-quantitative agreement with computer simulations carried out by Kong and Muthukumar in 1998, the observed behavior is interpreted with conformational changes of single adsorbed polyelectrolyte chains.

Tryptophan-mPEGs: novel excipients that stabilize salmon calcitonin against aggregation by non-covalent PEGylation.

Protein aggregation, which is triggered by various factors, is still one of the most prevalent problems encountered during all stages of protein formulation development. In this publication, we present novel excipients, tryptophan-mPEGs (Trp-mPEGs) of 2 and 5 kDa molecular weight and suggest their use in protein formulation. The synthesis and physico-chemical characterization of the excipients are described. Possible cytotoxic and hemolytic activities of the Trp-mPEGs were examined. Turbidity, 90° static light scatter, intrinsic fluorescence, fluorescence after staining the samples with Nile Red and fluorescence microscopy were used to study the inhibitory effect of the Trp-mPEGs on the aggregation of salmon calcitonin (sCT) in different buffer systems and at various molar ratios. Aggregation of sCT was reduced significantly with increasing concentrations of Trp-mPEG 2 kDa. A 10-fold molar excess of Trp-mPEG 2 kDa suppressed almost completely the aggregation of sCT in 10mM sodium citrate buffer (pH 6) for up to 70 h. Trp-mPEG 5 kDa also reduced the aggregation of sCT, though less pronounced than Trp-mPEG 2 kDa. Low aggregation of sCT was measured after approximately 10 days in 10mM sodium citrate buffer, pH 5, with a 10-fold molar excess of Trp-mPEG 2 kDa. This paper shows that Trp-mPEGs are potent excipients in reducing the aggregation of sCT. Trp-mPEGs are superior to dansyl-PEGs concerning the stabilization of sCT in a harsh environment, wherein sCT is prone to aggregation. Trp-mPEGs might therefore also be used for stabilization of other biopharmaceuticals prone to aggregation.

Influence of the degree of ionization and molecular mass of weak polyelectrolytes on charging and stability behavior of oppositely charged colloidal particles.

Positively charged amidine latex particles are studied in the presence of poly(acrylic acid) (PAA) with different molecular masses under neutral and acidic conditions by electrophoresis and time-resolved dynamic light scattering. Under neutral conditions, where PAA is highly charged, the system is governed by the charge reversal induced by the quantitatively adsorbing polyelectrolyte and attractive patch-charge interactions. Under acidic conditions, where PAA is more weakly charged, the following two effects come into play. First, the lateral structure of the adsorbed layers becomes more homogeneous, which weakens the attractive patch-charge interactions. Second, polyelectrolyte adsorption is no longer quantitative and partitioning into the solution phase is observed, especially for PAA of low molecular mass.

Noncovalent pegylation by dansyl-poly(ethylene glycol)s as a new means against aggregation of salmon calcitonin.

During all stages of protein drug development, aggregation is one of the most often encountered problems. Covalent conjugation of poly(ethylene glycol) (PEG), also called PEGylation, to proteins has been shown to reduce aggregation of proteins. In this paper, new excipients based on PEG are presented that are able to reduce aggregation of salmon calcitonin (sCT). Several PEG polymers consisting of a hydrophobic dansyl-headgroup attached to PEGs of different molecular weights have been synthesized and characterized physicochemically. After addition of dansyl-methoxypoly(ethylene glycol) (mPEG) 2 kDa to a 40 times molar excess of sCT resulted in an increase in dansyl-fluorescence and a decrease in 90° light scatter suggesting possible interactions. The aggregation of sCT in different buffer systems in presence or absence of the different dansyl-PEGs was measured by changes in Nile red fluorescence and turbidity. Dansyl-mPEG 2 kDa in a 1:1 molar ratio to sCT strongly reduced aggregation. Reduction of sCT aggregation was also measured for the bivalent dansyl-PEG 3 kDa in a 1:1 molar ratio. Dansyl-mPEG 5 kDa deteriorated sCT aggregation. Potential cytotoxicity and hemolysis were investigated. This paper shows that dansyl-PEGs are efficacious in reducing aggregation of sCT.

Glycosaminoglycans as polyelectrolytes.

One of the barriers to understanding structure-property relations for glycosaminoglycans has been the lack of constructive interplay between the principles and methodologies of the life sciences (molecular biology, biochemistry and cell biology) and the physical sciences, particularly in the field of polyelectrolytes. To address this, we first review the similarities and differences between the physicochemical properties of GAGs and other statistical chain polyelectrolytes of both natural and abioitic origin. Since the biofunctionality and regulation of the structures of GAGs is intimately connected with interactions with their cognate proteins, we particularly compare and contrast aspects of protein binding, i.e. effects of both GAGs and other polyelectrolytes on protein stability, protein aggregation and phase behavior. The protein binding affinities and their dependences on pH and ionic strength for the two groups are discussed not only in terms of observable differences, but also with regard to contrasting descriptions of the bound state and the role of electrostatics. We conclude that early studies of the heparin-Antithromin system, proceeding to a large extent through the methods and models of protein chemistry and drug discovery, established not only many enabling precedents but also constraining paradigms. Current studies on heparan sulfate and chondroitin sulfate seem to reflect a more ecumenical view likely to be more compatible with concepts from physical and polymer chemistry.

Suppression of insulin aggregation by heparin.

The aggregation of insulin near its isoelectric point and at low ionic strength was suppressed in the presence of heparin. To understand this effect, we used turbidimetry and stopped-flow to study the pH- and ionic strength ( I)-dependence of the aggregation of heparin-free insulin. The results supported the role of interprotein electrostatic interactions, contrary to the commonly held view that such forces are minimized at pH = pI. Electrostatic modeling of insulin (DelPhi) revealed that attractive interactions arise from the marked charge anisotropy of insulin near pI. We show how screening of the interprotein attractions by added salt lead to maximum aggregation near I = 0.01 M, corresponding to a Debye length nearly equal to the diameter of the insulin dimer, consistent with a dipole-like protein charge distribution. This analysis is also consistent with suppression of aggregation by heparin, a strong polyanion that by binding to the positive domain of one protein, inhibits its interaction with the negative domain of another.

Prediction of binding sites in receptor-ligand complexes with the Gaussian Network Model.

Residues at the binding sites of the ligand and receptor of several enzyme-inhibitor and antibody-antigen complexes are predicted from the slowest (for the ligand) and fastest (for the receptor) modes of motion by the Gaussian Network Model applied to unbound molecules.

Nonspecific electrostatic binding characteristics of the heparin-antithrombin interaction.

We evaluated the role of nonspecific electrostatic binding in the interaction of antithrombin (AT) with heparin (Hp), a paradigmatic protein-glycosaminoglycan (GAG) system. To do so, we obtained the ionic-strength dependence of the binding constant, since a common feature in protein-polyelectrolyte systems is a maximum in affinity in the ionic strength range 10 mM

Electrostatically driven protein aggregation: beta-lactoglobulin at low ionic strength.

The aggregation of beta-lactoglobulin (BLG) at ambient temperature was studied using turbidimetry and dynamic light scattering in the range 3.8

Frontal analysis continuous capillary electrophoresis for protein-polyelectrolyte binding studies.

A novel technique, frontal analysis continuous capillary electrophoresis (FACCE), has been described as an effective way to study protein-polyelectrolyte binding. FACCE involves continuous sampling, integrating sample injection and separation into one process that provides advantages over conventional frontal chromatography. The method provides rapid and precise determination of binding isotherms, and allows for quantitative binding analysis in terms of binding constant and the binding-site size by considering the protein as the ligand and allowing the polyelectrolyte to bind to a number of proteins with variable levels of cooperativity. FACCE is particularly suitable for binding systems involving rapid binding kinetics because it allows for the determination of the concentrations of free or bound ligands under conditions that avoid perturbation of the binding equilibrium. This chapter focuses on studies of the binding of bovine serum albumin (BSA) to heparin using FACCE. These investigations are demonstrated within the context of this chapter as representative of a model protein-polyelectrolyte system from which extensions to other systems can be made.

Ionic strength dependence of protein-polyelectrolyte interactions.

The effect of univalent electrolyte concentration on protein-polyelectrolyte complex formation has been measured by frontal analysis continuous capillary electrophoresis (FACCE) and turbidimetry for the interaction of bovine serum albumin (BSA) with a synthetic hydrophobically modified polyacid, for BSA with (porcine mucosal) heparin (Hp), a highly charged polyanion, and for Hp and insulin. All three highly diverse systems display maxima or plateaus in complex formation in the range of ionic strength 5 < I < 30 mM, confirmed in the case of BSA-Hp by multiple techniques. Similar maxima are reported in the literature, but with little discussion, for BSA-poly(dimethyldiallylammonium chloride), lysozyme-hyaluronic acid, and lysozyme-chondroitin sulfate, always in the I range 5-30 mM. While inversion of salt effect has been discussed specifically for the interaction of gelatin and sodium polystyrenesulfonate with gelatin(28) and with beta-lactoglobulin,(10) the general nature of this phenomenon, regardless of polyelectrolyte origin, molecular weight, and charge sign has not been recognized. The position of the maxima and their occurrence when protein and polyelectrolyte have the same net charge imply that they arise when Debye lengths extend, at low I, beyond half the protein diameter so that addition of salt screens repulsions, as well as attractions. This appears to be a general effect caused by electrostatic repulsions that can coexist simultaneously with hydrophobic interactions. Modeling of protein electrostatics via Delphi is used to visualize this effect for BSA, lysozyme, insulin, and beta-lactoglobulin.