Hydrodynamic behavior and thermal stability of a PEGylated protein: Studies with hen egg lysozyme

We studied the effect of covalent attachment of polyethylene glycol (PEGylation) on the hydrodynamic behavior and thermal stability of a model protein---Hen Egg Lysozyme (HEL). HEL was modified with a linear, 20-kD, PEG to produce mono (PEG1-HEL), di (PEG2-HEL), and triPEGylated (PEG3-HEL) species. The hydrodynamic properties of HEL were altered upon PEGylation. A decrease in sedimentation (s) and diffusion (D) coefficients was observed for all three PEG-HEL molecules in comparison to HEL (1.81 s). Despite differences in molecular weights of the PEG-HEL molecules (∼34, 55 and 80 kD), their s values were very close (1.0--1.1 s). Significant hydrodynamic non-ideality was observed for the PEG-HEL molecules, however, their Stokes radii (Rh) calculated from Do1 values were in agreement with dynamic light scattering (DLS) measurements. The Rh of HEL increased dramatically from 20 A to ∼50 A upon modification with a single 20-kD PEG chain. PEG2-HEL and PEG3-HEL had even larger radii of ∼68 A and 74 A. DLS studies with various PEGS (MW 5000--40,000) indicated that PEG is a random coil in solution. The Rh of PEG1-HEL and PEG2-HEL were measured to be only ∼10% larger than the 20-kD (43 A) and 40-kD (60 A) PEG chains. These data suggest that the covalently tethered PEG(s) predominantly govern the solution conformation of the PEG-HEL molecules. The thermal stability of PEGylated HEL was evaluated by employing the Eyring-Lumry model ( N↔TmD →kaA )2 for protein aggregation. A decrease in the melting temperature (Tm) of HEL unfolding was observed with increasing degree of PEGylation, which is indicative of thermodynamic instability. A Tm drop of up to 2.5°C (DSC) and 4.0°C (Difference Spectrum method) was observed for the PEG-HEL molecules. In contrast, turbidimetric studies showed that the kinetic aggregation rate (ka) of the PEG-HEL molecules was dramatically lower in comparison to the native HEL. Size exclusion HPLC indicated that the extent of aggregation decreased with increasing degree of PEGylation; only 34% of the HEL monomer remained after incubation at 75°C for 30 minutes, while 68% and 79% of the PEG1-HEL and PEG2-HEL monomers were present. These data suggest that the thermal stability of PEGylated HEL is kinetically controlled. The Tm may not be a true indicator of the stability of a PEG-protein with respect to aggregation. 1Do---Diffusion coefficient value extrapolated to infinite dilution. 2N---native state, D---denatured state, A---aggregate

Particle Shape Enhances Specificity of Antibody-Displaying Nanoparticles

Monoclonal antibodies are used in numerous therapeutic and diagnostic applications; however, their efficacy is contingent on specificity and avidity. Here, we show that presentation of antibodies on the surface of nonspherical particles enhances antibody specificity as well as avidity toward their targets. Using spherical, rod-, and disk-shaped polystyrene nano- and microparticles and trastuzumab as the targeting antibody, we studied specific and nonspecific uptake in three breast cancer cell lines: BT-474, SK-BR-3, and MDA-MB-231. Rods exhibited higher specific uptake and lower nonspecific uptake in all cells compared with spheres. This surprising interplay between particle shape and antibodies originates from the unique role of shape in determining binding and unbinding of particles to cell surface. In addition to exhibiting higher binding and internalization, trastuzumab-coated rods also exhibited greater inhibition of BT-474 breast cancer cell growth in vitro to a level that could not be attained by soluble forms of the antibody. The effect of trastuzumab-coated rods on cells was enhanced further by replacing polystyrene particles with pure chemotherapeutic drug nanoparticles of comparable dimensions made from camptothecin. Trastuzumab-coated camptothecin nanoparticles inhibited cell growth at a dose 1,000-fold lower than that required for comparable inhibition of growth using soluble trastuzumab and 10-fold lower than that using BSA-coated camptothecin. These results open unique opportunities for particulate forms of antibodies in therapeutics and diagnostics

Effective charge measurements reveal selective and preferential accumulation of anions, but not cations, at the protein surface

Specific‐ion effects are ubiquitous in nature; however, their underlying mechanisms remain elusive. Although Hofmeister‐ion effects on proteins are observed at higher (\u3e0.3M) salt concentrations, in dilute (\u3c0.1M) salt solutions nonspecific electrostatic screening is considered to be dominant. Here, using effective charge (Q*) measurements of hen‐egg white lysozyme (HEWL) as a direct and differential measure of ion‐association, we experimentally show that anions selectively and preferentially accumulate at the protein surface even at low (\u3c100 mM) salt concentrations. At a given ion normality (50 mN), the HEWL Q* was dependent on anion, but not cation (Li+, Na+, K+, Rb+, Cs+, GdnH+, and Ca2+), identity. The Q* decreased in the order F− \u3e Cl− \u3e Br− \u3e NOurn:x-wiley:09618368:media:PRO591:tex2gif-stack-1 ∼ I− \u3e SCN− \u3e ClOurn:x-wiley:09618368:media:PRO591:tex2gif-stack-2 ≫ SOurn:x-wiley:09618368:media:PRO591:tex2gif-stack-3, demonstrating progressively greater binding of the monovalent anions to HEWL and also show that the SOurn:x-wiley:09618368:media:PRO591:tex2gif-stack-4 anion, despite being strongly hydrated, interacts directly with the HEWL surface. Under our experimental conditions, we observe a remarkable asymmetry between anions and cations in their interactions with the HEWL surface

Effect of PEGylation on protein hydrodynamics

We studied the effect of PEGylation on protein hydrodynamic behavior using hen egg-white lysozyme (HEWL) as a model protein. HEWL was PEGylated with a linear, 20 kDa PEG using reductive amination to produce PEG1-, PEG2-, and PEG3-HEWL. Near- and far-UV–CD spectroscopy revealed no significant effect of PEGylation on HEWL higher order structure. SDS–PAGE, mass spectrometry, online static light scattering (SLS) and sedimentation velocity analytical ultracentrifugation (SV-AUC) were employed to characterize the heterogeneity and molecular weights of the purified PEG-HEWL molecules, the results of which underscored the importance of using first-principle based methods for such analyses along with the underlying complexities of characterizing PEG–protein conjugates. Hydrodynamic characterization of various linear and branched PEGs (5–40 kDa) and PEG-HEWL molecules was performed using dynamic light scattering (DLS) and SV-AUC. The PEG polymer exhibited a random-coil conformation in solution with the Mw ∝ Rhn scaling relationship yielding a scaling exponent (n) = 2.07. Singly branched PEGs were also observed to exhibit random-coil behavior with Stokes radii identical to those of their linear counterparts. SV-AUC studies of PEG-HEWL showed PEG has a “parachute” like effect on HEWL, and dramatically increases the frictional drag; PEG-HEWL also exhibited random-coil-like characteristics in solution (n = 1.8). The sedimentation coefficient (s) of PEG-HEWL remained invariant with increasing degree of PEGylation, indicating that the increase in molecular mass from PEG was compensated by an almost equivalent increase in frictional drag. Our studies draw caution to using SV-AUC for the characterization of size heterogeneity of PEG–protein mixtures