Nonnative aggregation of an IgG1 antibody in acidic conditions, part 2: nucleation and growth kinetics with competing growth mechanisms.

Aggregation mechanisms as a function of pH were assessed for the IgG1 antibody described in Part 1 (Brummitt RK, Nesta DP, Chang L, Chase SF, Laue TM, Roberts CJ. Non-native aggregation of an IGG1 antibody in acidic conditions: 1. Unfolding, colloidal interactions, and high molecular weight aggregate formation. J Pharm Sci. In press). Aggregation kinetics along with static light scattering and size-exclusion chromatography indicated that the aggregate nucleus was a dimer for all conditions tested, and this was semiquantitatively consistent with scaling of the characteristic time scale for nucleation (τ(n)) versus protein concentration at pH 4.5 and pH 5.5. Changing pH significantly altered the mechanism of aggregate growth, as well as the size and solubility of aggregates that were formed. Aggregates at pH 3.5 grew primarily by monomer addition and remained small and soluble. Aggregates at pH 4.5 grew first by chain polymerization, followed by condensation polymerization, leading ultimately to large insoluble particles. At pH 5.5, monomer loss resulted primarily in insoluble aggregate formation, with only low levels of soluble aggregate intermediates detected at early times. The influence of pH on aggregate solubility and the reversibility of aggregate phase separation were confirmed via cloud point titrations. Qualitatively, the global aggregation behavior was consistent with reduction of charge-charge repulsions as a primary factor in promoting larger aggregates and aggregate phase separation.

Nonnative aggregation of an IgG1 antibody in acidic conditions: part 1. Unfolding, colloidal interactions, and formation of high-molecular-weight aggregates.

Monomeric and aggregated states of an IgG1 antibody were characterized under acidic conditions as a function of solution pH (3.5-5.5). A combination of intrinsic/extrinsic fluorescence (FL), circular dichroism, calorimetry, chromatography, capillary electrophoresis, and laser light scattering were used to characterize unfolding, refolding, native colloidal interactions, aggregate structure and morphology, and aggregate dissociation. Lower pH led to larger net repulsive colloidal interactions, decreased thermal stability of Fc and Fab regions, and increased solubility of thermally accelerated aggregates. Unfolding of the Fab domains, and possibly the CH3 domain, was inferred as a key step in the formation of aggregation-prone monomers. High-molecular-weight soluble aggregates displayed nonnative secondary structure, had a semi-rigid chain morphology, and bound thioflavin T (ThT), consistent with at least a portion of the monomer forming amyloid-like structures. Soluble aggregates also formed during monomer refolding under conditions moving from high to low denaturant concentrations. Both thermally and chemically induced aggregates showed similar ThT binding and secondary structural changes, and were noncovalent based on dissociation in concentrated guanidine hydrochloride solutions. Changes in intrinsic FL during chemical versus thermal unfolding suggest a greater degree of structural change during chemical unfolding, although aggregation proceeded through partially unfolded monomers in both cases.

Mechanisms of protein stabilization in the solid state.

The solid state is preferred for many proteins due to their marginal stability in solution. However, even in the solid state, chemical and physical degradation can occur on the time scale of the drying process, distribution and use. This review summarizes the major degradation pathways of proteins in the solid state and the corresponding stabilization strategies. Specially, this review discusses the mechanisms of protein stabilization in the solid state. Understanding the mechanisms of protein stabilization is critical to efficient protein formulation development in the pharmaceutical industry.

The glass transition and sub-T(g)-relaxation in pharmaceutical powders and dried proteins by thermally stimulated current.

The main goal of the study was to evaluate the applicability of thermally stimulated current (TSC) as a measure of molecular mobility in dried globular proteins. Three proteins, porcine somatotropin, bovine serum albumin, and immunoglobulin, as well as materials with a strong calorimetric glass transition (T(g)), that is, indomethacin and poly(vinypyrrolidone) (PVP), were studied by both TSC and differential scanning calorimetry (DSC). Protein/sugar colyophilized mixtures were also studied by DSC, to estimate calorimetric T(g) for proteins using extrapolation procedure. In the majority of cases, TSC detected relaxation events that were not observed by DSC. For example, a sub-T(g) TSC event (beta-relaxation) was observed for PVP at approximately 120 degrees C, which was not detected by the DSC. Similarly, DSC did not detect events in any of the three proteins below the thermal denaturation temperature whereas a dipole relaxation was detected by TSC in the range of 90-140 degrees C depending on the protein studied. The TSC signal in proteins was tentatively assigned as localized mobility of protein segments, which is different from a large-scale cooperative motions usually associated with calorimetric T(g). TSC is a promising method to study the molecular mobility in proteins and other materials with weak calorimetric T(g).

Effect of sorbitol and residual moisture on the stability of lyophilized antibodies: Implications for the mechanism of protein stabilization in the solid state.

PURPOSE: To investigate the effect of plasticizers on the stability of protein formulations in the solid state and to apply these results to a study of mechanisms of protein stabilization by sugars in the solid sate. METHODS: The IgG1 antibody was formulated with either sucrose or trehalose alone or a mixture of sorbitol with sucrose or trehalose. After lyophilization, the pure protein and sucrose formulations were equilibrated at different relative humidities giving residual moistures from less than 1% to 5% for sucrose systems and up to 17% for pure protein systems. All the samples were stored at 50 degrees C for up to1 month and at 40 and 25 degrees C for up to 6 months. Aggregation and chemical degradation were monitored by size exclusion chromatography (SEC) and ion exchange chromatography (IEX), respectively. The secondary structure was characterized by FTIR using second derivative analysis of Amide I region. Structural relaxation times, tau, an indication of molecular mobility in the glassy matrix, were characterized using the thermal activity monitor (TAM). The tau values of the recombinant human monoclonal antibody (rhuMab) formulation with various water contents were also measured in this study and compared with stability data taken from the literature (Breen ED, Curley JG, Overcashier DE, Hsu CC, Shire SJ, 2001, Pharm Res 18:1345-1353). RESULTS: The structural relaxation time, tau, decreased sharply with increasing water content. However, the stability data suggest a minimum in degradation rate at 2%-3% water content. Addition of a small amount of sorbitol to a sucrose-based formulation resulted in greater retention of native structure, smaller relaxation time, but improved stability. However, with the trehalose-based formulations, addition of sorbitol had no effect on protein structure (FTIR), but the decrease in relaxation time and the improvement in stability were qualitatively similar to the corresponding data obtained with the sucrose-based formulation. CONCLUSION: Glass dynamics as measured by tau could not explain the stability results. Stability correlated best with the preservation of native structure for sucrose-based formulations, but with the trehalose-based formulation, neither structural relaxation time nor extent of native structure was predictive of stability. However, it is possible that the beta-relaxations rather than the alpha-relaxation (i.e., the tau we measured) is critical to the stability. Plasticizers like glycerol may decrease tau for "alpha-motion" but increase tau for "beta-motion" and stabilize proteins (Cicerone MT, Tellington A, Trost L, Sokolov A, 2003, BioProcess Inter 1:1-9).

Mechanism of protein stabilization by sugars during freeze-drying and storage: native structure preservation, specific interaction, and/or immobilization in a glassy matrix?

The purpose of this study is to investigate the mechanism of protein stabilization by sugars in the solid state. That is, explore whether the stabilization is controlled by "glass dynamics" or by native structure preservation through "specific interaction" between sugars and protein. The IgG1 antibody (150 kD) and recombinant human serum albumin (rHSA) (65 kD) were formulated with sorbitol, trehalose, and sucrose. Degradation of lyophilized formulations was quantified using size exclusion (SEC) and ion-exchange chromatography (IEX). The secondary structure of the protein in these formulations was characterized using Fourier Transform Infrared (FTIR) spectroscopy. The molecular mobility, as measured by the stretched relaxation time (tau(beta)) was obtained by fitting the modified stretched exponential (MSE) equation to the calorimetric data from the Thermal Activity Monitor (TAM). Compared with sucrose and trehalose, sorbitol could only slightly protect the protein against aggregation and had no effect on chemical degradation. The chemical degradation and aggregation rates of the protein decreased when the weight ratio of sucrose to protein increased from 0 to 2:1. Storage stability of the proteins showed a reasonably good correlation with the degree of retention of native structure of protein during drying as measured by the spectral correlation coefficient for FTIR spectra. The plots of tau(beta) as a function of fraction of sucrose passed through a maximum at 1:1 weight ratio of sucrose to protein. That is, the molecular mobility did not correlate with the stability of protein at high levels of sucrose content. Although the glass transition appears to be an important parameter for stability, protein stabilization by sugars in the solid state cannot be wholly explained by the glass dynamics mechanism, at least as measured by tau(beta). However, it is possible that the beta-relaxations rather than the alpha-relaxations (i.e., the tau we measured) are critical to stability. The data show that storage stability correlates best with "structure" as determined by FTIR spectroscopy. However, while a specific interaction between stabilizer and protein might be responsible for the preservation of native structure, the evidence supporting this position is not compelling.

Evaluation of glassy-state dynamics from the width of the glass transition: results from theoretical simulation of differential scanning calorimetry and comparisons with experiment.

The purpose of this study is to investigate the quantitative relationship between the width of the glass transition, DeltaTg, and glass fragility or activation energy for structural relaxation. The ultimate objective is the estimation of structural relaxation time as a function of temperature from the width of the glass transition region, allowing characterization of glass dynamics by a single simple measurement. The Moynihan correlation indicates that activation energy for structural relaxation should be inversely proportional to the width of the glass transition, but recent experimental studies suggest this relationship is a poor approximation for glasses of pharmaceutical interest. The present study is an effort to better understand the validity of the Moynihan correlation by selected experimental studies and a theoretical analysis of those factors that impact the glass transition width. Experimental data for glass transition widths for (poly)vinylpyrrolidone, sucrose, and trehalose are obtained using a variety of procedures, and relaxation time data are obtained using the thermal activity monitor. The theoretical analysis begins by simulating the temperature dependence of the heat capacity by breaking the cooling and heating scans into a large number of temperature steps followed by isothermal holds, during which relaxation of the material is calculated. Here, the modified VTF equation is used for relaxation time and the generalized Kohlraush-Williams-Watts stretched exponential function is used to describe the relaxation kinetics. Simulations are performed for materials of varying fragility and varying "stretched exponential" constants, beta, and the width of the glass transition region, DeltaTg, is evaluated from the simulated heat capacity versus temperature curves as one would do with experimental data. Agreement between the theoretical simulations and experimental DeltaTg data is excellent. The simulations demonstrate that although the Moynihan correlation is not valid for variable beta, a modification of the Moynihan correlation which includes variation in beta is a good approximation. Thus, an estimate of fragility may be obtained from glass transition width data provided an estimate of beta is available. Furthermore, it is shown that a first approximation for beta may be obtained from the magnitude (i.e., height) of the differential scanning calorimetry thermal overshoot. We also find that using the modified VTF equation to evaluate the temperature dependence of the structural relaxation time at the glass transition, and integrating this expression to lower temperatures, it is possible to obtain an evaluation of the relaxation time constant, tau(beta), in the glass at any temperature, using only the DeltaTg and beta values obtained from a single differential scanning calorimetry scan. These estimated time constants correlate very well with the values directly measured with the thermal activity monitor.

Thermophysical properties of pharmaceutically compatible buffers at sub-zero temperatures: implications for freeze-drying.

PURPOSE: To evaluate crystallization behavior and collapse temperature (Tg') of buffers in the frozen state, in view of its importance in the development of lyophilized formulations. METHODS: Sodium tartrate, sodium malate, potassium citrate, and sodium citrate buffers were prepared with a pH range within their individual buffering capacities. Crystallization and the Tg' were detected during heating of the frozen solutions using standard DSC and modulated DSC. RESULTS: Citrate and malate did not exhibit crystallization, while succinate and tartrate crystallized during heating of the frozen solutions. The citrate buffer had a higher Tg' than malate and tartrate buffers at the same pH. Tg' vs. pH graphs for citrate and malate buffers studied had a similar shape, with a maximum in Tg' at pH ranging from 3 to 4. The Tg' maximum was explained as a result of a competition between two opposing trends: an increase in the viscosity of the amorphous phase because of an increase in electrostatic interaction, and a decrease in the Tg' because of an increase in a water concentration of the freeze-concentrated solution. CONCLUSION: Citrate buffer was identified as the preferred buffer for lyophilized pharmaceuticals because of its higher Tg' and a lower crystallization tendency.