A specific molar ratio of stabilizer to protein is required for storage stability of a lyophilized monoclonal antibody

The selection of the appropriate excipient and the amount of excipient required to achieve a 2-year shelf-life is often done by using iso-osmotic concentrations of excipients such as sugars (e.g., 275 mM sucrose or trehalose) and salts. Excipients used for freeze-dried protein formulations are selected for their ability to prevent protein denaturation during the freeze-drying process as well as during storage. Using a model recombinant humanized monoclonal antibody (rhuMAb HER2), we assessed the impact of lyoprotectants, sucrose, and trehalose, alone or in combination with mannitol, on the storage stability at 40 degrees C. Molar ratios of sugar to protein were used, and the stability of the resulting lyophilized formulations was determined by measuring aggregation, deamidation, and oxidation of the reconstituted protein and by infrared (IR) spectroscopy (secondary structure) of the dried protein. A 360:1 molar ratio of lyoprotectant to protein was required for storage stability of the protein, and the sugar concentration was 3-4-fold below the iso-osmotic concentration typically used in formulations. Formulations with combinations of sucrose (20 mM) or trehalose (20 mM) and mannitol (40 mM) had comparable stability to those with sucrose or trehalose alone at 60 mM concentration. A formulation with 60 mM mannitol alone provided slightly less protection during storage than 60 mM sucrose or trehalose. The disaccharide/mannitol formulations also inhibited deamidation during storage to a greater extent than the lyoprotectant formulations alone. The reduction in aggregation and deamidation during storage correlated directly with inhibition of unfolding during lyophilization, as assessed by IR spectroscopy. Thus, it appears that the protein must be retained in its native-like state during freeze-drying to assure storage stability in the dried solid. Long-term studies (23-54 months) performed at 40 degrees C revealed that the appropriate molar ratio of sugar to protein stabilized against aggregation and deamidation for up to 33 months. Therefore, long-term storage at room temperature or above may be achieved by proper selection of the molar ratio and sugar mixture. Overall, a specific sugar/protein molar ratio was sufficient to provide storage stability of rhuMAb HER2.     

A practical approach to the use of nanoparticles for vaccine delivery

The objective of this work was to obtain a nanoparticle formulation that could be sterile filtered, lyophilized, and resuspended to the initial size with excipients appropriate for use as a vaccine formulation. Poly(lactide-co-glycolide) (PLG) polymers were used to create nanoparticles ranging in size from 110 to 230 nm. Protein antigens were adsorbed to the particles; the protein-nanoparticles were then lyophilized with the excipients. Vaccine compatible excipient combinations of sugars alone, surfactants alone, and sugars and surfactants were tested to find conditions where initial particle size was recovered. Sterile filtration of smaller nanoparticles led to minimal PLG losses and allowed the particle preparation to be a nonaseptic process. We found that the smaller nanoparticles of size approximately 120 nm required higher surfactant concentration to resuspend postlyophilization than slightly larger ( approximately 220 nm) particles. To resuspend 120 nm nanoparticles formulations of poly(vinyl alcohol) (PVA) with sucrose/mannitol or dioctyl sodium sulfosuccinate (DSS) with trehalose/mannitol were sufficient. The protein-nanoparticles resuspension with the same excipients was dependent on the protein and protein loading level. The nanoparticle formulations in vivo were either similar or had enhanced immunogenicity compared to aluminum hydroxide formulations. A lyophilized nanoparticle formulation with adsorbed protein antigen and minimal excipients is an effective vaccine delivery system.     

Comparative effects of some hydrophilic excipients on the rate of gabapentin and baclofen lactamization in lyophilized formulations

The aim of this study was to gain insights into the role played by some excipients on the stability of gabapentin 1 and baclofen 2 which can undergo degradation giving rise to the corresponding lactams 2-azaspiro[4.5]decan-3-one 3 and 4-(4-chlorophenyl)-2-pyrrolidone 4, respectively. A screening study was carried out on drug and drug-excipient freeze-dried mixtures at 50 degrees C and under three different humidity values by using a number of commonly available excipients. These include hydroxypropyl-beta-(HP-beta-CD), sulfobutyl-beta-cyclodextrin (SBE-beta-CD), lactose, raffinose, trehalose, PVP-K30 and mannitol. For most cases, it was found that the lactam formation can be satisfactory described by an apparent zero-order equation. Excipients shown to negatively impact gabapentin stability are HP-beta-CD, SBE-beta-CD, lactose and PVP K30 while only this last excipient had a significant effect on the degradation of baclofen. The results can be rationalized in terms of conformational factors favouring the intramolecular dehydration reaction. A positive effect of moisture on the lactamization process was observed under some circumstances. Water may provide a favourable environment for degradation. These findings, taken together, should be considered during the selection of excipients for a possible formulation of gabapentin and baclofen.     

Lyophilized Formulations of Recombinant Tumor Necrosis Factor

Recombinant tumor necrosis factor-alpha (TNF), an investigational biological response modifier, is a protein and is susceptible to particulate generation during handling in dilute aqueous solutions. TNF is prone to formation of nonreducible dimers and oligomers during formulation, lyophilization, and storage. The effect of various parameters, such as the pH, protein concentration, and nature of excipients present during lyophilization, on the formation of nonreducible dimers and oligomers was investigated. The results of these studies indicate that these parameters can significantly alter the rate of this reaction. Inclusion of an amorphous buffer and an appropriate amount of a crystallizing sugar (mannitol) combined with a suitable quantity of an amorphous protectant (dextran, sucrose, trehalose, or 2-hydroxypropyl--cyclodextrin) was shown to reduce the formation of these dimeric and oligomeric species during lyophilization. Representative lyophilized formulations of TNF based on selected amorphous excipients were found to be fully bioactive and stable over 9 months.     

Investigation of protein/carbohydrate interactions in the dried state. 1. Calorimetric studies.

Isoperibol calorimetry was used to evaluate protein/carbohydrate interactions after freeze drying. rh-DNase, rh-GH, rh-MetGH, and rh-IGF-I were freeze dried with either mannitol, sucrose, trehalose, or dextran at concentrations ranging from 0% to 100% (w/w). Enthalpies of solution for both freeze-dried and physical mixtures were measured in water at 25 degrees C. Differential scanning calorimetry was used to monitor changes in the melting or crystallization temperatures of the lyoprotectants. Linear relationships between enthalpies of solution and the percentage of protein in the formulations were observed for all physical mixtures. In contrast, nonlinear relationships between the enthalpies of solution and protein content were observed for the freeze-dried mixtures. Mannitol-containing mixtures were characterized by negative deviation from linearity, while positive deviations were detected for mixtures containing sucrose or trehalose. Using DSC, sucrose was found to be amorphous at low and not detected at high protein content in the freeze-dried mixtures. Melting of mannitol was observed through almost all of the protein concentration range examined. Two melting endotherms, however, were observed for mannitol at most protein/mannitol ratios, indicating the presence of protein/mannitol interactions. This work suggests that direct interactions occur between proteins and carbohydrates in lyophilized mixtures.     

Investigation of the stabilisation of freeze-dried lysozyme and the physical properties of the formulations.

The long-term stability of a protein formulation requires that the glass transition temperature (Tg) of the formulation should be maximised and the perturbation of the protein native structure in the dried form after processing minimised. In the present study, the stabilisation of lysozyme structure conferred by excipients was monitored using second derivative Fourier transform infrared spectroscopy and the physical properties of protein formulations were investigated using differential scanning calorimetry. The results showed that the preservation of protein native structure during freeze-drying and the Tg of freeze-dried formulations were excipient- and excipient to enzyme mass ratio-dependent. The freeze-dried lysozyme appeared to be less effectively stabilised compared with the spray-dried enzyme when the excipients and the excipient to enzyme mass ratios were the same. In terms of the preservation of the secondary structure of lysozyme, glycerol and sucrose seemed to be more efficient than trehalose, although the Tg of trehalose-containing formulations were found to be higher than the Tg of the equivalent sucrose-based ones. With adding either trehalose or dextran to sucrose-containing formulations, the stabilisation of lysozyme native structure could be as effective as with sucrose alone, whilst the Tg could be enhanced. The results in this study suggested that lysozyme, processed by freeze-drying, is stabilised primarily by the water substitution mechanism.     

Dextran or hydroxyethyl starch in spray-freeze-dried trehalose/mannitol microparticles intended as ballistic particulate carriers for proteins

The goal of this study was to clarify the effects of dextran 10 kDa on the properties of spray-freeze-dried microparticles for use with ballistic injectors. A novel carrier of trehalose, mannitol, and the polymer is known to maximize particle density. Measurements of T'(g) showed that the dextran anti-plasticizes the trehalose/mannitol, but also undergoes phase separation. The product temperature exceeded T'(g) during primary drying. The collapsed particles can therefore be explained by plastic flow of the freeze concentrate. DSC of the powder showed T(g) at 45 degrees C and, in the first scan, a wide endothermic melting peak caused by mannitol recrystallization. Catalase showed 35% activity loss on rehydration of its spray freeze-drying (SFD) powder, which was improved in the TM/D (3:3:4) formulation, but not up to that level seen with either trehalose or mannitol alone. The dextran 10 kDa, which is vital to maximize particle density, was therefore detrimental to protein integrity during SFD, as also found with a 65-72 kDa dextran. Hydroxyethyl starch (HES) 200 kDa gave similar, limited stabilizing effects on the protein. The proportion of polymer in the formulation should be low to minimize protein damage, whilst high enough to give required particle morphology and density.     

Physical characteristics and aerosolization performance of insulin dry powders for inhalation prepared by a spray drying method

The objective of this study was to investigate the influence of formulation excipients on the physical characteristics and aerosolization performance of insulin dry powders for inhalation. Insulin dry powders were prepared by a spray drying technique using excipients such as sugars (trehalose, lactose and dextran), mannitol and amino acids (L-leucine, glycine and threonine). High performance liquid chromatography and the mouse blood glucose method were used for determination of the insulin content. The powder properties were determined and compared by scanning electron microscopy, thermo-gravimetric analysis and size distribution analysis by a time-of-flight technique. The in-vitro aerosolization behaviour of the powders was assessed with an Aerolizer inhaler using a twin-stage impinger. Powder yield and moisture absorption were also determined. Results showed that there was no noticeable change in insulin content in any of the formulations by both assay methods. All powders were highly wrinkled, with median aerodynamic diameters of 2-4 microm, and consequently suitable for pulmonary administration. The tapped density was reduced dramatically when glycine was added. The powders containing mannitol, with or without L-leucine, were less sensitive to moisture. The highest respirable fraction of 67.3 +/- 1.3% was obtained with the formulation containing L-leucine, in contrast to formulations containing glycine and threonine, which had a respirable fraction of 11.2 +/- 3.9% and 23.5 +/- 2.5%, respectively. In addition, powders with good physical properties were achieved by the combination of insulin and trehalose. This study suggests that L-leucine could be used to enhance the aerosolization behaviour of the insulin dry powders for inhalation, and trehalose could potentially be used as an excipient in the formulations.     

Significance of Local Mobility in Aggregation of β-Galactosidase Lyophilized with Trehalose,Sucrose or Stachyose

Purpose The purpose of this study is to compare the effects of global mobility, as reflected by glass transition temperature (T_g) and local mobility, as reflected by rotating-frame spin-lattice relaxation time (T_1ρ) on aggregation during storage of lyophilized β-galactosidase (β-GA). Materials and Methods The storage stability of β-GA lyophilized with sucrose, trehalose or stachyose was investigated at 12% relative humidity and various temperatures (40–90°C). β-GA aggregation was monitored by size exclusion chromatography (SEC). Furthermore, the T_1ρ of the β-GA carbonyl carbon was measured by ¹³C solid-state NMR, and T_g was measured by modulated temperature differential scanning calorimetry. Changes in protein structure during freeze drying were measured by solid-state FT-IR. Results The aggregation rate of β-GA in lyophilized formulations exhibited a change in slope at around T_g, indicating the effect of molecular mobility on the aggregation rate. Although the T_g rank order of β-GA formulations was sucrose < trehalose < stachyose, the rank order of β-GA aggregation rate at temperatures below and above T_g was also sucrose < trehalose < stachyose, thus suggesting that β-GA aggregation rate is not related to (T-T_g). The local mobility of β-GA, as determined by the T_1ρ of the β-GA carbonyl carbon, was more markedly decreased by the addition of sucrose than by the addition of stachyose. The effect of trehalose on T_1ρ was intermediate when compared to those for sucrose and stachyose. These findings suggest that β-GA aggregation rate is primarily related to local mobility. Significant differences in the second derivative FT-IR spectra were not observed between the excipients, and the differences in β-GA aggregation rate observed between the excipients could not be attributed to differences in protein secondary structure. Conclusions The aggregation rate of β-GA in lyophilized formulations unexpectedly correlated with the local mobility of β-GA, as indicated by T_1ρ, rather than with (T-T_g). Sucrose exhibited the most intense stabilizing effect due to the most intense ability to inhibit local protein mobility during storage.     

β-Relaxation of Insulin Molecule in Lyophilized Formulations Containing Trehalose or Dextran as a Determinant of Chemical Reactivity

Purpose The purpose of this study was to elucidate whether the degradation rate of insulin in lyophilized formulations is determined by matrix mobility, as reflected in glass transition temperature (T_g), or by β-relaxation, as reflected in rotating-frame spin-lattice relaxation time . Methods The storage stability of insulin lyophilized with dextran was investigated at various relative humidities (RH; 12–60%) and temperatures (40–90°C) and was compared with previously reported data for insulin lyophilized with trehalose. Insulin degradation was monitored by reverse-phase high-performance liquid chromatography. Furthermore, the of the insulin carbonyl carbon in the lyophilized insulin–dextran and insulin–trehalose systems was measured at 25°C by ¹³C solid-state NMR, and the effect of trehalose and dextran on was compared at various humidities. Results The degradation rate of insulin lyophilized with dextran was not significantly affected by the T_g of the matrix, even at low humidity (12% RH), in contrast to that of insulin lyophilized with trehalose. The insulin–dextran system exhibited a substantially greater degradation rate than the insulin–trehalose system at a given temperature below the T_g. The difference in degradation rate between the insulin–dextran and insulin–trehalose systems observed at 12% RH was eliminated at 43% RH. In addition, the of the insulin carbonyl carbon at low humidity (12% RH) was prolonged by the addition of trehalose, but not by the addition of dextran. This difference was eliminated at 23% RH, at which point the solid remained in the glassy state. These findings suggest that the β-relaxation of insulin is inhibited by trehalose at low humidity, presumably as a result of insulin–trehalose interaction, and thus becomes a rate determinant. In contrast, dextran, whose ability to interact with insulin is thought to be less than that of trehalose, did not inhibit the β-relaxation of insulin, and thus, the chemical activational barrier (activation energy) rather than β-relaxation becomes the major rate determinant. Conclusions β-Relaxation rather than matrix mobility seems to be more important in determining the stability of insulin in the glassy state in lyophilized formulations containing trehalose and dextran.     

Stable Formulations of Recombinant Human Growth Hormone and Interferon-γ for Microencapsulation in Biodegradable Mircospheres

Purpose. The successful development of controlled release formulations for proteins requires that the protein not be denatured during the manufacturing process. The major objective was to develop formulations that stabilize two recombinant human proteins, human growth hormone (rhGH) and interferon- (rhIFN-), at high protein concentrations (>100 mg/mL) in organic solvents commonly used for microencapsulation, methylene chloride and ethyl acetate. Methods. Several excipients were screened to obtain the maximum solubility of each protein. These formulations (aqueous, lyophilized, milled, spray dried, or isoelectric precipitate) were then rapidly screened by emulsification in the organic solvent followed by recovery into excess buffer. Additional screening was performed with solid protein that was suspended in the organic solvent and then recovered with excess buffer. The recovery of native protein was determined by native size exclusion chromatography (SEC-HPLC) and circular dichroism (CD). The selected formulations were encapsulated in poly-lactic-coglycolic acid (PLGA) microspheres by either water-in-oil-in-water (W/O/W) or solid-in-oil-in-water (S/O/W) methods. The initial protein released from the microspheres incubated at physiological conditions was analyzed by SEC-HPLC, CD, and biological assays. Results. The stability of a given formulation in the rapid screening method correlated well with stability during encapsulation in PLGA microspheres. Formulations of rhGH containing Tween 20 or 80 resulted in lower recovery of native protein, while trehalose and mannitol formulations (phosphate buffer, pH 8.0) yielded complete recovery of native rhGH. Other additives such as carboxymethyl cellulose, gelatin, and dextran 70 were not effective stabilizers, and polyethylene glycol provided some stabilization of rhGH. Trehalose/rhGH (1:4 mass ratio) and mannitol/rhGH (1:2 mass ratio) formulations (potassium phosphate buffer, pH 8.0) were lyophilized, reconstituted to 200 and 400 mg/mL rhGH, respectively, and then encapsulated in PLGA micro-spheres. The protein was released from these microspheres in its native state. Lyophilized formulations of rhGH yielded analogous results indicating the ability of trehalose and mannitol to stabilize the protein. Small solid particles of rhGH generated by spray drying (both air and freeze-drying) formulations containing Tween 20 or PEG were stable in ethyl acetate, but not methylene chloride. Similar results were also obtained with rhIFN- (137 mg/mL in succinate buffer, pH 5.0), where both mannitol and trehalose were observed to stabilize the protein during exposure to the organic solvents resulting in the release of native rhIFN- from PLGA microspheres. Conclusions. The rapid screening method allowed the development of stable concentrated protein solutions or solid protein formulations that could be successfully encapsulated in PLGA microspheres. The excipients observed to stabilize these proteins function by preferential hydration of the protein, and in the dry state (e.g., trehalose) may stabilize the protein via water substitution yielding a protective coating around the protein surface. Studies of other proteins should provide further insight into this mechanism of protein stabilization during encapsulation.     

Do co-spray dried excipients offer better lysozyme stabilisation than single excipients?

The inherent instability of proteins when isolated from their native conditions creates the necessity of suitable stabilisation techniques. Because of the instability of proteins in solution it is often necessary to produce them as solid formulations. A method of producing relatively stable, solid protein pharmaceuticals is to incorporate them with a suitable excipient into an amorphous matrix by dehydration. The use of spray dried multiple excipient/single protein blends was compared to single excipient/protein systems using lysozyme as a model protein to establish the stabilising ability of such systems. Unprocessed controls and spray dried samples were characterised structurally by X-ray powder diffraction and Fourier transform Raman spectroscopy and also thermally by differential scanning calorimetry and thermogravimetric analysis. Retained lysozyme activity was assayed enzymatically. To assess long-term stability, samples were subjected to conditions of elevated temperature and relative humidity (RH) 40 degrees C/75% RH. Structural and thermal analysis of samples revealed that mannitol/trehalose spray dried excipient/lysozyme blends were completely amorphous upon production but partially recrystallised upon storage at elevated temperature and RH. Biological activity assays revealed that samples containing trehalose retained the highest percentage activity. Under the conditions employed mannitol/trehalose systems stabilise lysozyme more effectively than single excipient systems due to their ability to form amorphous products.     

Effect of Sucrose/Raffinose Mass Ratios on the Stability of Co-Lyophilized Protein During Storage Above the Tg

Purpose. To examine the potential of raffinose as an excipient in stabilizing protein and to study the effect of sucrose/raffinose mass ratios on the stability of co-lyophilized protein and amorphous solids during storage at an elevated temperature. Methods. Glucose-6-phosphate dehydrogenase (G6PDH) was co-lyophilized with sucrose and raffinose mixed at different mass ratios. The activity of dried G6PDH was monitored during storage at 44°C. Thermal properties of sucrose/raffinose matrices were determined by differential scanning calorimetry (DSC). Results. Mass ratios of sucrose to raffinose did not affect the recovery of G6PDH activity after freeze-drying, but significantly affected the stability of freeze-dried G6PDH during storage. The sucrose-alone formulation offered the best enzyme stabilization during storage. With increasing fraction of raffinose, the G6PDH stability decreased, sugar crystallization inhibited, and crystal-melting temperature increased. Conclusions. Despite the higher T_g of the formulations with higher fraction of raffinose, they provided less protection for G6PDH than did sucrose alone during storage. Our data do not support the prediction from recent thermophysical studies that raffinose should be superior to sucrose and trehalose as a potential excipient or stabilizer.     

Effect of pH on stability of recombinant botulinum serotype A vaccine in aqueous solution and during storage of freeze-dried formulations

The purpose of this study was to evaluate the importance of prelyophilization solution pH on the stability of botulinum neurotoxin, serotype A (rBoNTA(H(c))). This understanding is of significant importance for proteins such as rBoNTA(H(c)), a potential constituent of a multivalent vaccine product. For multivalent vaccines it may be difficult to identify a liquid formulation satisfying the stability requirements for all constituent protein antigens. Consequently, a lyophilized multivalent vaccine formulation may be a more viable alternative. Therefore evaluating the effect of prelyophilization pH (may be suboptimal) on the stability of antigens such as rBoNTA(H(c)) during lyophilization/storage becomes important. We hypothesize that when rBoNTA(H(c)) is lyophilized from a suboptimal pH, using the appropriate stabilizers can provide adequate physicochemical stability during lyophilization and long-term storage. We identified pH 5 and 8 in which the protein was stable and unstable against aggregation. Excipients were identified that could stabilize rBoNTA(H(c)) during lyophilization and storage in a stable solution of pH 5. These excipients were 7.5% (w/v) trehalose and 2.5% (w/v) trehalose with 2.5% (w/v) HES, with and without 0.01% (w/v) polysorbate 20. In support of our hypothesis, these excipients were found to provide adequate physicochemical stability to rBoNTA(H(c)) during lyophilization/storage, when freeze-dried from a prelyophilized solution of pH 8.     

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.     

Moisture sorption behavior of selected bulking agents used in lyophilized products

To develop a rational approach for the formulation of lyophilized products, six bulking agents commonly used in freeze-dried formulations were lyophilized under identical conditions, and their moisture sorption behavior, before and after lyophilization, were determined as a function of relative humidity at 25 degrees C. The bulking agents evaluated were mannitol, anhydrous lactose, sucrose, D(+)-trehalose, dextran 40 and povidone (PVP K24). The materials were also characterized for their crystal and thermal properties by powder X-ray diffraction, DSC and TG after exposure to various relative humidity conditions. Mannitol was crystalline and non-hygroscopic both before and after lyophilization with total moisture contents of 0.1 to 0.3% w/w between 10 and 60% RH. Anhydrous lactose, sucrose and trehalose were crystalline prior to lyophilization with moisture contents of 0.86, 0.15 and 9.2%, respectively, and the crystalline materials were relatively non-hygroscopic. Upon lyophilization, they converted to the amorphous form and had moisture contents of 1.6, 2.5 and 1.2%, respectively. The amorphous materials sorbed moisture rapidly upon exposure to increasing relative humidity conditions. The amorphous lactose converted to its crystalline hydrate form at 55% RH after sorption of an additional 10% moisture. This conversion to the crystalline hydrate form was accompanied by desorption of practically all the moisture sorbed by the amorphous form. Similarly, lyophilized sucrose converted to its crystalline form after the sorption of additional 4.5% moisture at 50% RH, and the lyophilized trehalose sorbed additional 10% moisture prior to its conversion to a crystalline hydrate form at 50% RH. Dextran and povidone were amorphous and hygroscopic both before and after lyophilization and they sorbed as much as 10-20% moisture at 50% RH. It is well established that different drugs, especially proteins, need different levels of moisture for optimal stability. The results of the present study show that moisture contents of lyophilized cakes may be varied and optimized by the selection of suitable excipients.     

Lyoprotection of aviscumine with low molecular weight dextrans

The aim of this research was to ascertain whether dextrans with low molecular weight will stabilize aviscumine. During freeze-drying increasing concentrations of dextran T1 (MW 1000) stabilized aviscumine. Eight percent of dextran resulted in a nearly 100% recovery of the activity and in addition a complete amorphous structure of the solid phase was obtained. By decreasing the molecular weight of the dextran from 75 to 1 kDa, the protein activity was increased by 20% in the lyophilisate. Combinations of dextran with either trehalose or mannitol showed no additional effects on stability. The improved stabilization of aviscumine using low molecular weight dextrans is explained by an increased interaction between the protein and the dextran molecules (like hydrogen bonds), whereas they are sterically hindered if larger dextran molecules are used. When the protein concentration was increased from 10 to 100 microg/ml (in formulas with 8% dextran T1), no influence on the protein activity could be found. With regard to the carbohydrate-binding activity of the protein, it was shown that the optimal content of residual water in the lyophilisate should be about 2%. Above and below this percentage a destabilization of the protein was observed. The often discussed failure of dextran as a stabilizing excipient in the freeze-drying of proteins seems to be a question of the selection of the correct molecular weight.     

Protective Mechanism of Stabilizing Excipients Against Dehydration in the Freeze-Drying of Proteins

Purpose. To investigate the influence of type and amount of excipient on the preservation of the native structure and the biologic activity of freeze-dried lysozyme and catalase. Methods. The secondary structure of protein in the dried form and in aqueous solution was obtained using second derivative infrared spectroscopy and circular dichroism spectra respectively whilst the activity was determined using bioassay. Results. Small molecular excipients (glycerol, sorbitol, 1,6-anhydroglucose, sucrose, and trehalose) were found to stabilize the activity and/or the native structure of freeze-dried lysozyme and catalase, despite the processing temperatures being above Tg of excipient-protein mixtures. The preservation of catalase activity required excipient to be present at a lower excipient to enzyme mass ratio than that necessary to preserve native structure in the dried form. Combining dextran with sucrose synergistically protected the native structure of catalase but preserved the activity in an additive manner. Conclusion. The results indicate that the stabilization of catalase and lysozyme by excipients during dehydration was mainly due to water substitution rather than the formation of glass; the latter appearing not to be a prerequisite during freeze-drying.     

Environmental risk assessment for excipients from galenical pharmaceutical production in wastewater and receiving water

Many different excipients are used in galenical pharmaceutical production, in addition to the active pharmaceutical ingredients. Excipients are little investigated regarding their environmental fate and impact, even though some of them are used in appreciable quantities. For 35 excipients used in galenical production at Roche Basle and Roche Kaiseraugst, both in Switzerland, in the years 2013 and 2014, the environmentally relevant properties were collated. A predicted environmental concentration (PEC) was calculated for the wastewater treatment plants (WWTPs) and the receiving water, the River Rhine for both sites, based on maximum daily losses of the single excipients to wastewater, derived by mass balance, and the site-specific dilution factor. Predicted no effect concentrations (PNECs) were derived for the WWTPs and the River Rhine. PECs and PNECs were compared for the WWTPs and the receiving water, in an environmental risk assessment. Additionally, to simulate a worst case scenario, certain galenical productions where given excipients are used in the highest amounts were assumed to take place in parallel on the same day, resulting in theoretical maximum excipient losses to wastewater. All PEC/PNEC risk characterisation ratios for the excipients currently used by Roche in Switzerland are well below 1 throughout. Together with the fact that based on biodegradability data many excipients will be removed in the WWTP, this indicates that the excipients currently used do not present a risk to the environment.     

The influence of various excipients on the conversion kinetics of carbamazepine polymorphs in aqueous suspension

The influence of various excipients on the conversion of carbamazepine polymorphs to the dihydrate in aqueous suspension has been investigated. Ten excipients having functional groups which were potentially able to form hydrogen bonds with carbamazepine (group 1: methylcellulose, hypromellose (hydroxypropyl methylcellulose), hydroxypropylcellulose (HPC), 2-hydroxyethylcellulose (HEC), carmellose sodium (sodium carboxymethylcellulose), cellobiose; group 2: povidone (polyvinylpyrrolidone), povidone-vinyl acetate copolymer (povidone/VA) and N-methyl-2-pyrrolidone; group 3: macrogol (polyethylene glycol) and polyethylene oxide-polypropylene oxide copolymer (PEO/PPO)) were selected. Carbamazepine polymorphic forms III and I were dispersed separately into each aqueous excipient solution (0.1%, w/v) for 30 min at room temperature. The inhibition effect of each excipient was quantified using Raman spectroscopy combined with multivariate analyses. The solubility parameter of each excipient was calculated and used for categorizing excipients. Excipients in groups 1 and 2, which had both low solubility parameters (< 27.0 MPa(1/2)) and strong hydrogen bonding groups, inhibited the conversion completely. With increasing solubility parameter, the inhibition effect decreased for group 1 excipients, especially for carbamazepine form I, which had a higher specific surface area. Also, the excipients of group 3, lacking strong hydrogen bonding groups, showed poor inhibition although they had low solubility parameters (< 21.0 MPa(1/2)). This study indicated the importance of both hydrogen bonding interaction and a suitable hydrophobicity (expressed by the solubility parameter) in the inhibition of the conversion of carbamazepine to the dihydrate.