Anders D. Nielsen

Thermochemistry of the specific binding of C12 surfactants to bovine serum albumin

The specific binding to bovine serum albumin (BSA) of anionic and non-ionic surfactants with C12 acyl chains has been studied by high sensitivity isothermal titration calorimetry. This method proved particularly effective in resolving the binding of anionic surfactants into separate classes of sites with different affinity. For sodium dodecylsulfate (SDS) the measured binding curves could be rationalized as association to two classes (high affinity/low affinity) of sites comprising, respectively, three and six similar (i.e. thermodynamically equivalent), independent sites. Changes in the thermodynamic functions enthalpy, standard free energy, standard entropy and heat capacity could be discerned for each class of binding site, as well as for micelle formation. These data suggest that binding to low affinity sites (in analogy with micelle formation) exhibits energetic parameters; in particular, a large negative change in heat capacity, which is characteristic of hydrophobic interactions. The thermodynamics of high affinity binding, on the other hand, is indicative of other dominant forces; most likely electrostatic interactions. Other anionic ligands investigated (laurate and dodecyl benzylsulfonate) showed a behavior similar to SDS, the most significant difference being the high affinity binding of the alkylbenzyl sulfonate. For this ligand, the thermodynamic data is indicative of a more loosely associated complex than for SDS and laurate. BSA was found to bind one or two of the non-ionic surfactants (NIS) hepta- or penta(ethylene glycol) monododecyl ether (C12EO7 and C12EO5) with binding constants about three orders of magnitude lower than for SDS. Hence, the free energy of the surfactant in the weakly bound BSA-NIS complex is only slightly favored over the micellar state. The binding process is characterized by very large exothermic enthalpy changes (larger than for the charged surfactants) and a large, positive increment in heat capacity. These observations cannot be reconciled with a molecular picture based on simple hydrophobic condensation onto non-polar patches on the protein surface.

Effects of PEG size on structure, function and stability of PEGylated BSA

The effects of PEGylation on the structural, thermal and functional stability of bovine serum albumin (BSA) were investigated using BSA and 6 linear mono-PEGylated BSA compounds. The secondary and tertiary structure of BSA measured by circular dichroism (CD) was independent of PEGylation. In contrast, the thermal stability of BSA was affected by PEGylation. The apparent unfolding temperature T(max) measured by differential scanning calorimetry (DSC) decreased with PEGylation, whereas the temperature of aggregation, T(agg), measured by dynamic light scattering (DLS) increased with PEGylation. The unfolding temperature and the temperature of aggregation were both independent of the molecular weight of the PEG chain. Possible functional changes of BSA after PEGylation were measured by Isothermal Titration Calorimetry (ITC), where the binding of sodium dodecyl sulphate (SDS) to BSA and PEGylated BSA was analysed. At 25°C, two distinct classes of binding sites (high affinity and low affinity) for BSA and one class of binding site (low affinity) for PEGylated BSA were identified. The binding isotherm was modelled assuming independence and thermodynamic equivalence of the sites within each class. From the present biophysical characterisation, it is concluded that after PEGylation BSA appears to be unaffected structurally (secondary and tertiary structure), slightly destabilised thermally (unfolding temperature), stabilised kinetically (temperature of aggregation) and has an altered functionality (binding profile). These biophysical characteristics are all independent of the molecular weight of the attached polymer chain.

Effect of calcium ions on the irreversible denaturation of a recombinant Bacillus halmapalus alpha-amylase: a calorimetric investigation.

The effect of temperature and calcium ions on the denaturation of a recombinant alpha-amylase from Bacillus halmapalus alpha-amylase (BHA) has been studied using calorimetry. It was found that thermal inactivation of BHA is irreversible and that calcium ions have a significant effect on stability. Thus an apparent denaturation temperature ( T (d)) of 83 degrees C in the presence of excess calcium ions was observed, whereas T (d) decreased to 48 degrees C when calcium was removed. The difference in thermal stability with and without calcium ions has been used to develop an isothermal titration calorimetric (ITC) procedure that allows simultaneous determination of kinetic parameters and enthalpy changes of the denaturation of calcium-depleted BHA. An activation energy E (A) of 101 kJ/mol was found for the denaturation of calcium-depleted BHA. The results support a kinetic denaturation mechanism where the calcium-depleted amylase denatures irreversibly at low temperature and if calcium ions are in excess, the amylase denatures irreversibly at high temperatures. The two denaturation reactions are coupled with the calcium-binding equilibrium between calcium-bound and -depleted amylase. A combination of the kinetic denaturation results and calcium-binding constants, determined by isothermal titration calorimetry, has been used to estimate kinetic stability, expressed in terms of the half-life of BHA as a function of temperature and free-calcium-ion concentration. Thus it is estimated that the apparent E (A) can be increased to approx. 123 kJ/mol by increasing the free-calcium concentration.

Stability of Monoclonal Antibodies at High-Concentration: Head-to-Head Comparison of the IgG1 and IgG4 Subclass

Few studies have so far directly compared the impact of antibody subclass on protein stability. This case study investigates two mAbs (one IgG1 and one IgG4 ) with identical variable region. Investigations of mAbs that recognize similar epitopes are necessary to identify possible differences between the IgG subclasses. Both physical and chemical stability were evaluated by applying a range of methods to measure formation of protein aggregates [size-exclusion chromatography (SEC)-HPLC and UV340 nm], structural integrity (circular dichroism and FTIR), thermodynamic stability (differential scanning calorimetry), colloidal interactions (dynamic light scattering), and fragmentation and deamidation (SEC-HPLC and capillary isoelectric focusing). The impact of pH (4-9) and ionic strength (10 and 150 mM) was investigated using highly-concentrated (150 mg/mL) mAb formulations. Lower conformational stability was identified for the IgG4 resulting in increased levels of soluble aggregates. The IgG1 was chemically less stable as compared with the IgG4 , presumably because of the higher flexibility in the IgG1 hinge region. The thermodynamic stability of individual mAb domains was also addressed in detail. The stability of our mAb molecules is clearly affected by the IgG framework, and this study suggests that subclass switching may alter aggregation propensity and aggregation pathway and thus potentially improve the overall formulation stability while retaining antigen specificity.

Biophysical characterisation of GlycoPEGylated recombinant human factor VIIa.

The effects of GlycoPEGylation on the structural, kinetic and thermal stability of recombinant human FVIIa were investigated using rFVIIa and linear 10 kDa and branched 40 kDa GlycoPEGylated(®) recombinant human FVIIa derivatives. The secondary and tertiary structure of rFVIIa measured by circular dichroism (CD) was maintained upon PEGylation. In contrast, the thermal and kinetic stability of rFVIIa was affected by GlycoPEGylation, as the apparent unfolding temperature T(m) measured by differential scanning calorimetry (DSC) and the temperature of aggregation, T(agg), measured by light scattering (LS) both increased with GlycoPEGylation. Both T(m) and T(agg) were independent of the molecular weight and the shape of the PEG chain. From the present biophysical characterisation it is concluded that after GlycoPEGylation, rFVIIa appears to be unaffected structurally (secondary and tertiary structure), slightly stabilised thermally (unfolding temperature) and stabilised kinetically (temperature of aggregation).

The molar hydrodynamic volume changes of factor VIIa due to GlycoPEGylation

The effects of GlycoPEGylation on the molar hydrodynamic volume of recombinant human rFVIIa were investigated using rFVIIa and two GlycoPEGylated recombinant human FVIIa derivatives, a linear 10kDa PEG and a branched 40kDa PEG, respectively. Molar hydrodynamic volumes were determined by capillary viscometry and mass spectrometry. The intrinsic viscosities of rFVIIa, its two GlycoPEGylated compounds, and of linear 8kDa, 10kDa, 20kDa and branched 40kDa PEG polymers were determined. The measured intrinsic viscosity of rFVIIa is 6.0mL/g, while the intrinsic viscosities of 10kDa PEG-rFVIIa and 40kDa PEG-rFVIIa are 29.5mL/g and 79.0mL/g, respectively. The intrinsic viscosities of the linear PEG polymers are 20, 22.6 and 41.4mL/g for 8, 10, and 20kDa, respectively, and 61.1mL/g for the branched 40kDa PEG. From the results of the intrinsic viscosity and MALDI-TOF measurements it is evident, that the molar hydrodynamic volume of the conjugated protein is not just an addition of the molar hydrodynamic volume of the PEG and the protein. The molar hydrodynamic volume of the GlycoPEGylated protein is larger than the volume of its composites. These results suggest that both the linear and the branched PEG are not wrapped around the surface of rFVIIa but are chains that are significantly stretched out when attached to the protein.

The effect of GlycoPEGylation on the physical stability of human rFVIIa with increasing calcium chloride concentration

The effects of calcium chloride on the structural, kinetic and thermal stability of recombinant human factor VIIa (rFVIIa) were investigated using rFVIIa and two GlycoPEGylated recombinant human FVIIa derivatives, a linear 10 kDa PEG and a branched 40 kDa PEG, respectively. Three different CaCl(2) concentrations were used: 10mM, 35 mM and 100mM. The secondary structure and tertiary structure of rFVIIa at 25°C, measured by circular dichroism (CD), were maintained upon GlycoPEGylation as well as CaCl(2) content. In contrast, the thermal stability of the three rFVIIa compounds, measured by differential scanning calorimetry (DSC) and circular dichroism (CD), and aggregation behaviour, measured by light scattering (LS), were affected by the increasing calcium concentration. Increasing the CaCl(2) concentration from 10mM to 35 mM resulted in a decrease in the apparent unfolding temperature, T(m), of rFVIIa, whereas the concentration of CaCl(2) has to be raised to 100mM in order to see the same effect on the GlycoPEGylated rFVIIa compounds. The temperature of aggregation of rFVIIa, T(agg), increased as the CaCl(2) concentration increased from 35 mM to 100 mM, while T(agg) for the GlycoPEGylated rFVIIa compounds was practically independent of the CaCl(2) concentration. From the obtained results, it is concluded that GlycoPEGylation postpones the calcium induced thermal destabilisation of rFVIIa, and a much higher calcium concentration also postpones the thermally induced aggregation of rFVIIa. The thermally induced aggregation of the GlycoPEGylated rFVIIa compounds is unaffected by an increasing calcium chloride concentration.