Conformational analysis of protein secondary structure during spray-drying of antibody/mannitol formulations.

Inhalation of spray-dried particles is a promising delivery route for proteins as an alternative to injection. Changes in the protein structure and aggregation have to be avoided. The effect of mannitol, a stabilizing agent typically used in both liquid and lyophilized protein formulations, on an antibody (IgG1) in a spray-dried powder was studied using different biophysical and chromatographic techniques. At first, different solutions composed of antibody (IgG1) and mannitol at a ratio between 20/80 and 100/0 IgG1/mannitol (100 mg/ml total solid) were investigated for their stability. Protein solutions containing the IgG1 showed mannitol-dependent aggregation. High amounts of mannitol (50-80%) exerted a destabilizing effect on the antibody and the aggregate 9 level increased to 2.6-4.2%. In contrast, solutions with only 20-40% mannitol showed the same amount of aggregates as the pure antibody solution. The antibody mannitol solutions were investigated by circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR) to evaluate whether changes in the protein secondary structure can be correlated with aggregation. Considering the sensitivity of the used methods and data evaluation, FTIR spectra did not reveal structural changes in the IgG1/mannitol solutions compared to the pure antibody, despite varying aggregate levels. Thermal stress was reflected in perturbations of the secondary structure, but mannitol-dependent aggregation could not be correlated to detectable alterations in the FTIR spectra. Analyzing the CD spectra revealed no distinctive change in the shape of the CD curve, indicating that the protein secondary structure is mainly retained. This is in agreement with the infrared data. Subsequently, the IgG1/mannitol solutions were spray-dried at Tin/Tout of 90/50 degrees C. Using ATR-FTIR for the investigation of the protein amide I band in the spray-dried powder revealed changes in the sub-components of the amide I band. This indicates that the peptide groups (CO and NH) of the protein are found in a different environment in the solid state, compared to the liquid protein formulation. After redissolution of the powders, the native structure of the pure antibody solution was found identical to the protein secondary structure before spray-drying, indicating that the protein secondary structure is not strongly altered in the dry state, and not affected by the spray-drying process. Thus, from the presented study it can be concluded that the formation of antibody aggregates in mannitol formulations cannot be correlated with significant perturbations of the protein secondary structure elements.     

Noninvasive determination of protein conformation in the solid state using near infrared (NIR) spectroscopy

Fourier transform infrared (FTIR) spectroscopy is a powerful tool for monitoring structural changes in lyophilized protein formulations. However, direct measurement of IR spectra requires significant handling time and effort. The possibility of using near infrared (NIR) spectroscopy as a rapid and noninvasive alternative to FTIR is explored in this study. NIR and conventional FTIR spectra were collected for two model proteins, alpha-chymotrypsinogen A and cytochrome c, under conditions of varying stability and structural perturbation. NIR was then compared to FTIR and whereby calibration model was generated by partial least square (PLS) regression to correlate NIR data with FTIR spectra. There is a strong correlation of certain NIR bands with the amide I region of FTIR spectra. It appears that NIR can distinguish damage caused by elevated temperatures and freeze-drying stresses. The ability of sucrose to stabilize the structure of these two proteins can be detected by both methods. It appears that NIR spectroscopy has the potential to provide detailed information on the secondary structure of proteins in the solid state. However, many more examples will be needed to demonstrate fully the ability of NIR to replace FTIR as the standard tool for characterizing lyophilized protein formulations.     

Optimizing storage stability of lyophilized recombinant human interleukin-11 with disaccharide/hydroxyethyl starch mixtures

Optimal storage stability of a protein in a dry formulation depends on the storage temperature relative to the glass transition temperature (T(g)) of the dried formulation and the structure of the dried protein. We tested the hypothesis that optimizing both protein structure and T(g)--by freeze-drying recombinant human interleukin-11 (rhIL-11) with mixtures of disaccharides and hydroxyethyl starch (HES)--would result in increased storage stability compared with the protein lyophilized with either disaccharide or hydroxyethyl starch alone. The secondary structure of the protein in the dried solid was analyzed immediately after lyophilization and after storage at elevated temperatures by infrared spectroscopy. After rehydration, aggregation was monitored by size exclusion chromatography. Oxidation levels and cleavage products were quantified by reversed-phase chromatography. For the formulation with HES alone, which has a relatively high T(g), storage stability of rhIL-11 was poor, because HES failed to inhibit lyophilization-induced unfolding. The sugar formulations inhibited unfolding, and had intermediate T(g) values and storage stabilities. Addition of hydroxyethyl starch to sucrose or trehalose increased T(g) without affecting the capacity of the sugar to inhibit protein unfolding during lyophilization. Optimal storage stability of lyophilized rhIL-11 was achieved by using a mixture of disaccharide and polymeric carbohydrates.     

The effects of Tween 20 and sucrose on the stability of anti-L-selectin during lyophilization and reconstitution.

We have chosen an anti-L-selectin antibody as a model protein to investigate the effects of sucrose and/or Tween 20 on protein stability during lyophilization and reconstitution. Native anti-L-selectin secondary structure is substantially retained during lyophilization in the presence of sucrose (1 or 0.125%). However, aggregation of the protein during reconstitution of lyophilized protein powders prepared without sucrose is not reduced by the presence of sucrose in the reconstitution medium. Aggregate formation upon reconstitution is completely inhibited by freeze drying the protein with sucrose and reconstituting with a 0.1% Tween 20 solution. Tween 20 (0.1%) also partially inhibits loss of native anti-L-selectin secondary structure during lyophilization. However, upon reconstitution the formulations lyophilized with Tween 20 contain the highest levels of aggregates. The presence of Tween in only the reconstitution solution appears to inhibit the transition from dimers to higher order oligomers. Potential mechanism(s) for the Tween 20 effects were investigated. However, no evidence of thermodynamic stabilization of anti-L-selectin conformation (e.g., by Tween 20 binding) could be detected.     

Characterization of Aerosols of Human Recombinant Deoxyribonuclease I (rhDNase) Generated by Jet Nebulizers

Recombinant human deoxyribonuclease I (rhDNase) is a new therapeutic agent developed to improve clearance of purulent sputum from the human airways. It is delivered by inhalation. Four jet nebulizers, T Up-Draft II (Hudson), Customized Respirgard II (Marquest), Acorn II (Marquest), and Airlife Misty (Baxter), were evaluated in vitro for their ability to deliver aerosols of rhDNase. The aerosols were generated from 2.5-mL aqueous solutions of rhDNase, at concentrations of either 1 or 4 mg/mL. In all experiments, the Pulmo-Aide Compressor (De Vilbiss) was used to supply the air to the nebulizers. Between 20 and 28% of the rhDNase dose initially placed in the nebulizers was delivered to the mouthpiece in the respirable range (1-6 µm). Evaluation of the rhDNase following nebulization in all four devices indicated that there was no loss in enzymatic activity and no increase in aggregation. Circular dichroism spectrophotometry indicated there was no change in either the secondary or the tertiary structure in rhDNase following nebulization. These results show that all four nebulizers are essentially equivalent in their ability to deliver respirable doses of rhDNase in an intact, fully active form. Changing the concentration of the solution in the nebulizer from 4 to 1 mg/mL rhDNase leads to a proportional reduction in the respirable dose delivered to the mouthpiece.     

The relation between moisture‐induced aggregation and structural changes in lyophilized insulin

Objectives Long‐term stability is a critical factor in the successful development of protein pharmaceuticals. Due to the relative instability of proteins in aqueous solutions, they are formulated frequently and stored as lyophilized powders. Exposure of such powders to moisture constitutes a substantial storage problem leading to aggregation and inactivation. We have investigated the structural consequences of moisture sorption by lyophilized insulin under controlled humidity conditions by employing Fourier transform‐infrared (FT‐IR) microscopy. Methods Lyophilized insulin samples were stored in humidity chambers under controlled conditions at 50°C. Protein aggregation studies were carried out by redissolving the insulin samples and measuring the amount of both soluble protein and insoluble aggregates. Near‐UV circular dichroism spectra were collected to assess the tertiary structure. FT‐IR microscopy studies were carried out to investigate secondary structural changes in solid‐state insulin after incubation at different relative humidities. Key findings It was found that sorption of moisture was accompanied by small structural changes in lyophilized insulin at low levels of relative humidity (i.e. 11%). At higher relative humidity levels, structural changes were becoming more pronounced and were characterized by a loss in the α‐helix and increase in β‐sheet content. The magnitude of the structural changes in tendency paralleled the solid‐state instability data (i.e. formation of buffer‐insoluble aggregates and loss in tertiary structure upon reconstitution). Conclusions The results support the hypothesis that water sorption by lyophilized proteins enables structural transitions which can lead to protein aggregation and other deleterious phenomena.     

Impact of sucrose level on storage stability of proteins in freeze-dried solids: II. Correlation of aggregation rate with protein structure and molecular mobility

The purpose of this study is to investigate the impact of sucrose level on storage stability of dried proteins and thus better understand the mechanism of protein stabilization by disaccharides in lyophilized protein products. Five proteins were freeze dried with different amounts of sucrose, and protein aggregation was quantified using Size Exclusion Chromatography. Protein secondary structure was monitored by FTIR. The global mobility was studied using Thermal Activity Monitor (TAM), and fast local dynamics with a timescale of nanoseconds was characterized by neutron backscattering. The density of the protein formulations was measured with a gas pycnometer. The physical stability of the proteins increased monotonically with an increasing content of sucrose over the entire range of compositions studied. Both FTIR structure and structural relaxation time from TAM achieved maxima at about 1:1 mass ratio for most proteins studied. Therefore, protein stabilization by sugar cannot be completely explained by global dynamics and FTIR structure throughout the whole range of compositions. On the other hand, both the fast local mobility and free volume obtained from density decreased monotonically with an increased level of sucrose in the formulations, and thus the local dynamics and free volume correlate well with protein storage stability. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3145–3166, 2009     

Prediction of the bitterness of single,binary- and multiple-component amino acid solutions using a taste sensor

Upon freeze-drying in the absence of lyoprotectants, Fourier transform infrared (FTIR) spectroscopy has detected changes in the secondary structures of proteins. Such FTIR studies have been typically conducted using protein/KBr pellets, where additional protein degradation could potentially occur due to pressure effects and partial dissolution of the chaotropic KBr. Diffuse reflectance FTIR spectroscopy, in which no sample preparation is necessary, was evaluated as an alternative spectroscopic method to examine protein structure upon freeze-drying. The therapeutic proteins recombinant human deoxyribonuclease I (rh-DNase) and recombinant human insulin like growth factor I (rh-IGF-I) were freeze-dried with mannitol, sucrose, trehalose, and two molecular weight dextrans (69 and 503 kDa) separately, at concentrations ranging from 0 to 100% (w/w). Upon freeze-drying, rh-DNase and rh-IGF-I underwent significant changes in their secondary structure. For both proteins, the presence of intermolecular beta-sheets due to aggregation was detected and the alpha-helix content decreased significantly. The addition of carbohydrates to the formulations inhibited the protein secondary structure rearrangement in a concentration-dependent manner. Sucrose and trehalose appeared to be the most efficient excipients in preventing secondary structure changes. The conformational changes observed for both proteins appeared to be reversible upon rehydration.     

Influence of Histidine on the Stability and Physical Properties of a Fully Human Antibody in Aqueous and Solid Forms

PURPOSE: The aim of the study was to investigate the effect of histidine on the stability and physical properties of a fully human anti-IL8 monoclonal antibody (ABX-IL8) in aqueous and solid forms. METHODS: Using a fractional factorial design, we tested many excipients, including histidine, sucrose, and other commonly used excipients, on the stability and physical properties of the antibody in both liquid and lyophilized forms. Antibody stability and physical properties were evaluated using size-exclusion high-performance liquid chromatography (SEC-HPLC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and a viscometer. Residual moisture content was determined by coulometric Karl Fischer titrator. Differential scanning calorimetry (DSC) was used to detect the glass transition temperatures (Tg) of the solid cakes and melting temperatures (Tm) of the antibody in liquid formulations. Fourier-transform infrared (FTIR) spectroscopy was used to examine the overall secondary structure. RESULTS: Increasing the histidine concentration in the bulk solution inhibited the increases of high-molecular-weight (HMW) species and aggregates upon lyophilization and storage. In addition, histidine bulk enhanced solution stability of the antibody under freezing and thermal stress conditions, as evidenced by the lower levels of aggregates. Furthermore, histidine reduced viscosity of the antibody solution, which is desirable for the manufacture of the dosage form. However, high concentrations of histidine in liquid formulations led to coloration of the solution and high levels of aggregates on storage at elevated temperature (40 degrees C) after the formulations were exposed to stainless steel containers during bulk freezing-thawing. CONCLUSIONS: Histidine enhanced the stability of ABX-IL8 in both aqueous and lyophilized forms. Histidine also improved the physical properties such as reducing the solution viscosity. Liquid formulations containing high concentrations of histidine should not be stored in stainless steel tanks at elevated temperatures.     

Effects of sugar additives on protein stability of recombinant human serum albumin during lyophilization and storage

Few researches on the protein stabilization of recombinant human serum albumin (rHSA) have been done. In the present study, we assessed the impact of sugar lyoprotectants on the protein stability of lyophilized rHSA (65 KDa) in the solid state. For the assessment, rHSA was formulated with sucrose and trehalose, respectively, alone or in combination with mannitol, which were lyophilized and stored at 35 degrees C. Degradation and aggregation of the resulting lyophilized formulations was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Induction of amorphous state by the lyophilactants with rHSA was determined by differential scanning calorimetry (DSC). The protein secondary structure of the rHSA in the formulations was analyzed by Fourier transform infrared spectroscopy (FT-IR). Results from SDS-PAGE analysis displayed that mannitol formulation caused aggregation resulting in a few bands that were greater than 65 KDa, whereas sucrose and trehalose formulations revealed no such aggregation. However, the aggregation of the protein decreased when mannitol was combined with sucrose or trehalose. DSC measurement supported the electrophoresis data showing that sucrose and trehalose formed complete amorphous state, but mannitol induced a partial amorphous state. These data indicate during lyophilization the most effective protein protection against aggregation was provided by sucrose and trehalose. The protection lasted during 4 months storage at 35 degrees C. FT-IR analysis displayed that the sucrose formulation inhibited deamidation. In conclusion, our data suggest that sucrose and trehalose as additives seems to be sufficient to protect from lyophilization of rHSA protein and also maintain its stability in the solid state during storage.     

Aggregation of rhDNase Occurred During the Compression of KBr Pellets Used for FTIR Spectroscopy

Purpose. To determine if a protein changes when it is compressed into a KBr pellet for FTIR spectroscopy measurement in the solid state, using recombinant human deoxyribonuclease I (rhDNase) as an example. Methods. Lyophilized rhDNase with KBr compressed at different pressures were analyzed by FTIR spectroscopy, size exclusion HPLC and enzymatic activity assay. Different protein/KBr weight ratios and residual water contents were studied for their possible effects on aggregation. Results. Depending on the pressure, a loss of enzymatic activity accompanied by an increase in soluble high molecular weight aggregates of the protein (up to ~15%) was demonstrated. Aggregation was reduced to less than 5% by a suitable dilution of the protein in KBr (1 in 1000). In contrast, water content variability (1–11 wt. %) did not affect aggregation. Conclusions. The findings emphasize the importance to examine for protein integrity when using the KBr method for FTIR sample preparation. Protein aggregation may be minimized by optimizing the sample preparation condition such as changing the protein/KBr weight ratio.     

Effects of sucrose on rFVIIa aggregation and methionine oxidation

The aim of this study was to characterize the effects of sucrose on the stability of recombinant factor VIIa (rFVIIa), with special emphasis on aggregation and methionine oxidation, as well as to investigate the impact of various environmental conditions on the rFVIIa conformation. The stability of rFVIIa was studied at pH 5. Aggregation was monitored using size exclusion high-performance liquid chromatography (SE-HPLC), whereas formation of methionine oxidation products was measured by reversed-phase high-performance liquid chromatography (RP-HPLC). Fourier transform infrared (FTIR) spectroscopy and circular dichroism (CD) spectroscopy were used to study protein conformation. Stability studies showed that increasing sucrose concentrations reduced the loss of monomeric rFVIIa, and decreased formation of dimeric/oligomeric and polymeric rFVIIa. Preferential exclusion of the sugar from the protein’s surface, which shifts the protein molecular population away from expanded aggregation competent species and toward the compact native state, is thought to account for these observations. rFVIIa is sensitive to methionine oxidation; two mono-oxidized and one di-oxidized product were formed upon incubation. Unlike aggregation, methionine oxidation was found to increase in the presence of sucrose. The two methionine residues susceptible to oxidation are presumably located at the protein surface, and the chemical potential increase in the presence of sucrose may account for the increase in oxidation rate. While FTIR spectroscopy suggested that sucrose induces small conformational changes in the rFVIIa structure, CD spectroscopy did not support this finding. The secondary structure of precipitated rFVIIa was changed when compared to the native solution secondary structure. Appearance of bands characteristic of intermolecular β-sheet structure were found coincident with a decrease in -helix and intramolecular β-sheet structure.     

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.     

Solid state chemistry of proteins: II. The correlation of storage stability of freeze-dried human growth hormone (hGH) with structure and dynamics in the glassy solid

This research presents storage stability of human growth hormone, hGH, in lyophilized di-saccharide formulations. Stability via HPLC assay was assessed at 40 and 50 degrees C. Structure of the protein in the solids was assessed by infrared spectroscopy. Molecular mobility was characterized by structural relaxation times estimated from DSC data and by measurement of atomic motion on a nanosecond time scale by neutron scattering. Very large stability differences were observed among the various formulations, with both chemical and aggregation stability showing the same qualitative trends with formulation. Near the T(g), T(g) appeared to be a relevant stability parameter, but for storage well below T(g), stability seems unrelated to T(g). Stability (chemical and aggregation) was weakly correlated with secondary structure of the protein, and there was a partial quantitative correlation between degradation rate and the structural relaxation time. However, at equivalent levels of disaccharide relative to protein, sucrose systems were about a factor of two more stable than trehalose formulations, but yet had greater mobility as measured by structural relaxation time. Secondary structure was equivalent in both formulations. Neutron scattering results documented greater suppression of fast dynamics by sucrose than by trehalose, suggesting that well below T(g), fast dynamics are important to stability.     

Effects of Additives on Heat Denaturation of rhDNase in Solutions

Purpose. To study the thermal stability of recombinant human deoxyribonuclease I (rhDNase) in aqueous solutions. Methods. Differential scanning calorimetry (DSC) was used to measure the denaturation or melting temperature (T_m) and enthalpy (H_m) of rhDNase. The effects of denaturants (guanidine HC1 and urea) and additives (mainly divalent cations and disaccharides) were investigated at pH 6–7. Results. The T_m and H_m of rhDNase in pure water were measured as 67.4 °C and 18.0 J/g respectively, values typical of globular proteins. The melting peak disappeared on re-running the sample after cooling to room temperature, indicating that the thermal denaturation was irreversible. The latter was due to the occurrence of aggregation accompanying the unfolding process of rhDNase. Size exclusion chromatography indicated that during heat denaturation, rhDNase formed soluble high molecular weight aggregates with a molecular size >300kD estimated by the void volume. Of particular interest are the divalent cations: Ca²⁺ stabilizes rhDNase against thermal denaturation and elevates T_m and H_m while Mg²⁺, Mn²⁺ and Zn²⁺ destabilize it. Sugars also stabilize rhDNase. As expected, denaturants destabilize the protein and lower the T_m and H_m. All destabilization of rhDNase can be prevented by adding Ca²⁺ to the solutions. Conclusions. CaCl₂ and sugars were found to stabilize rhDNase against thermal denaturation while divalent cations, urea and guanidine HC1 destabilize the protein. The effects could be explained by a mixture of mechanisms. For Ca²⁺ the protective effect is believed to be due to an ordering of the rhDNase structure in its native state, and by prevention of breaking of a disulfide bridge, thus making it less susceptible to unfold under thermal stress.     

The Impact of Thermal Treatment on the Stability of Freeze-Dried Amorphous Pharmaceuticals: II. Aggregation in an IgG1 Fusion Protein

The objective of this research was to investigate the impact of thermal treatment on storage stability of an IgG1 fusion protein. IgG1 protein formulations were prepared by freeze-drying the protein with sucrose. Some samples were used as controls, and others were subjected to a further heat treatment (annealing). The protein structure was investigated with Fourier transform infrared spectroscopy (FTIR), and protein aggregation was monitored with size exclusion HPLC. Enthalpy recovery was studied using DSC, and global mobility represented by the structural relaxation time constant (τ^β) was characterized by a thermal activity monitor (TAM). The local mobility of the protein system was monitored by both ¹³C solid-state NMR and neutron backscattering. Annealing increased the storage stability of the protein, as shown by the smaller aggregation rate and less total aggregation at the end of a storage period. The structural relaxation time constant of an annealed sample was significantly higher than the unannealed control sample, suggesting a decrease in global mobility of the protein system upon annealing. However, annealing does not significantly impact the protein secondary structure or the local mobility. Given the similar protein native structure and specific surface area, the improved stability upon annealing is mainly a result of reduced global molecular mobility. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:683–700, 2010     

Structure, stability, and mobility of a lyophilized IgG1 monoclonal antibody as determined using second-derivative infrared spectroscopy

There are many aspects of stabilization of lyophilized proteins. Of these various factors, retention of native structure, having sufficient amount of stabilizer to embed the protein within an amorphous matrix, and dampening β-relaxations have been shown to be critical in optimizing protein stability during storage. In this study, an IgG1 was lyophilized with varying amounts of sucrose. In some formulations, a small amount of sorbitol was added as a plasticizer. The structure of the protein in dried state was monitored using infrared (IR) spectroscopy. The IR spectra indicated increasing retention of the native structure, which correlated with stability as indicated by size-exclusion chromatography as well as micro-flow imaging. Maximal stability was achieved with a 2:1 mass ratio of sucrose to protein, which is more than that would be expected based on earlier studies. Analysis of both high and low frequency bands associated with intramolecular β-sheet structure provides additional information on the structure of antibodies in the solid state. Finally, there is a correlation between the bandwidth of the β-sheet bands and the enthalpy of relaxation, suggesting that amide I bands can provide some indication of the degree of coupling to the sugar matrix, as well as structural heterogeneity of the protein.     

Raman spectroscopic characterization of drying-induced structural changes in a therapeutic antibody: correlating structural changes with long-term stability

We characterized the secondary structure of a therapeutic recombinant humanized monoclonal antibody (rhuMAb), formulated with different concentrations of sucrose, trehalose, and histidine and in solution, lyophilized, and spray-dried states. Quantitative secondary structure estimates were obtained using amide I band Raman spectroscopy and a previously developed spectral deconvolution procedure. On lyophilization or spray drying in the absence of sugar, the antibody underwent significant structural perturbation. The beta-sheet content decreased with corresponding gain in the turn and unordered content. With increasing amount of sucrose or trehalose, the extent of structural perturbation decreased. Eventually, at sugar-to-protein molar ratios of > or =360, almost complete structural preservation was observed. Histidine also protected the antibody against lyophilization-induced structural changes. The extent of structural perturbation immediately after lyophilization or spray drying exhibited good correlation with the rate of aggregation for the antibody during long-term storage under accelerated conditions. The results demonstrate that amide I band Raman spectroscopy could be a quick and reliable way to screen excipients and their concentrations during lyophilized or spray dried formulation development.     

Heterogeneous nucleation-controlled particulate formation of recombinant human platelet-activating factor acetylhydrolase in pharmaceutical formulation

Clinical lots of recombinant human platelet-activating factor acetylhydrolase (rhPAF-AH) were prepared in a lyophilized formulation. After reconstitution with sterile water for injection to form an aqueous solution (10 mM sodium citrate, 7.5 w/v% sucrose, and 0.1 w/v% Pluronic-F68, pH 6.5), a few visible, slowly growing particles formed consistently within hours at room temperature. To investigate the mechanism of this phenomenon, immediately after reconstitution, all protein aggregates and exogenous particles were removed by filtration. During 20 days incubation at room temperature, no visible aggregates formed in these filtered samples. In contrast, when nano-sized hydrophilic silica particles were added, they seeded rapid and extensive aggregation of rhPAF-AH. This effect was exacerbated in solutions containing a lower Pluronic-F68 concentration at 0.01%. Aggregation occurred even under conditions where rhPAF-AH adsorption was reversible, and induced no detectable changes to protein secondary and tertiary structures. Decreasing the extent (e.g., adding Pluronic-F68) or affinity (e.g., increasing solution pH) of rhPAF-AH adsorption on nano-sized silica particles was found to be effective at reducing aggregation. Accelerated aggregation was not observed when rhPAF-AH formulation was seeded with aggregated rhPAF-AH. These results show that rhPAF-AH aggregation proceeds through a heterogeneous nucleation-controlled mechanism, where exogenous particles present in solution serve as seeds on which rhPAF-AH adsorb, nucleate, and grow into large aggregates.     

Effect of pH and Excipients on Structure, Dynamics, and Long-Term Stability of a Model IgG1 Monoclonal Antibody upon Freeze-Drying

Purpose

To investigate the mechanism of IgG1 mAb stabilization after freeze-drying and the interdependence of protein structural preservation in the solid state, glassy state dynamics and long-term storage stability under different formulation conditions.

Methods

IgG1 mAb was formulated with mannitol at pH 3.0, 5.0, and 7.0 in the presence and absence of sucrose and stability was monitored over 1 year at different temperatures. Physical and covalent degradation of lyophilized formulation was monitored using SEC, CEX, and light obscuration technique. Secondary and tertiary structure of the protein in the solid state was characterized using FTIR and fluorescence spectroscopy respectively. Raman spectroscopy was also used to monitor changes in secondary and tertiary structure, while SS-NMR ¹H relaxation was used to monitor glassy state dynamics.

Results

IgG1 mAb underwent significant secondary structural perturbations at pH 3.0 and conditions without sucrose, while pH 5.0 condition with sucrose showed the least structural change over time. The structural changes correlated with long-term stability with respect to protein aggregate formation and SbVP counts. SS-NMR data showed reduced relaxation time at conditions that were more stable.

Conclusions

Native state protein structural preservation and optimal solid-state dynamics correlate with improved long-term stability of the mAb in the different lyophilized formulations.