Effect of peptide concentration and temperature on leuprolide stability in dimethyl sulfoxide

The effects of temperature and concentration on leuprolide degradation in dimethyl sulfoxide (DMSO) were explored. Leuprolide degradation products were analyzed by reverse phase high-performance liquid chromatography (RP-HPLC), size exclusion chromatography (SEC) and structurally characterized by mass spectrometry. Leuprolide solution stability in DMSO was characterized at 50, 100, 200, 400 mg/ml at 37-80 degrees C for 2 months to 3 years. Leuprolide degradation products were identified by mass spectrometry and could generally be attributed to isomerization, hydrolysis, oxidation, or aggregation. The hydrolytic degradation products consisted primarily of backbone cleavage C-terminal to Trp(3), Ser(4), Tyr(5), Leu(6) and Leu(7), and oxidation of Trp(3) and beta-elimination of Ser(4) were identified. Leuprolide degradation at 50 degrees C, 65 degrees C and 80 degrees C proceeded in an exponential fashion (E(a)=22. 6+/-1.2 kcal/mol); however, leuprolide degradation plateau'd after approximately 6 months at 37 degrees C. Upon closer examination, degradation product peak areas were seen to vary with temperature. For example, aggregation products did not increase with time at 37 degrees C, but aggregation peak intensities increased sharply with time at 80 degrees C. Increasing the temperature also increased the proportion of leuprolide degrading via isomerization/hydrolytic pathways, and decreased the proportion degrading via oxidation. These variations suggested that solvent dielectric, free H(+) in an aprotic solvent, oxygen solubility, impurities and residual moisture may play a role. Leuprolide solubilized in DMSO yields adequate stabililty for a 1 year implantable osmotic delivery system, where use of a dry aprotic solvent results in conditions similar to solid state stability.     

Dimerization of a PACAP peptide analogue in DMSO via asparagine and aspartic acid residues

To optimize the stability of a peptide development candidate for the treatment of type II diabetes, formulation studies were initiated in organic solvents and compared to results obtained in aqueous solutions. Stability was assessed by reversed phase liquid chromatography (RPLC) and electrospray ionization mass spectrometry (ESI-MS). Previous studies had shown deamidation and hydrolysis to be the primary mechanisms of degradation in aqueous formulations. Surprisingly, the use of an organic solvent did not decrease the rate of degradation and, as presented here, produced degradation products including dimers. We propose here that deamidation can readily occur in polar anhydrous organic solvents such as DMSO and that the dimer forms through intermolecular nucleophilic attack of an amino acid side chain on a stabilized cyclic imide intermediate.     

Effect of Gelation on the Chemical Stability and Conformation of Leuprolide

Purpose. The purpose of this study was to characterize the conformation, aggregation, and stability of leuprolide on gelation. Methods. Infrared spectra (FTIR) of leuprolide solutions and gels were collected in water, propylene glycol (PG), dimethyl sulfoxide (DMSO), and trifluoroethanol (TFE). Leuprolide solution and gel stability data were obtained by SEC and RP-HPLC. Results. Leuprolide was induced to gel with increasing peptide concentration, introduction of salts, and gentle agitation. Leuprolide dissolved in water (400 mg/ml) demonstrated FTIR spectra consisting of two major bands of equal intensity at 1615 cm^–1 and 1630 cm^–1, similar to inter- and intra-molecular -sheet structure in proteins. When samples were gently agitated for 24 hours at 25°C, the formulation was observed to change from a viscous liquid to an opaque gel with a concomitant shift in infrared spectra from the equal intensity bands to mostly 1630 cm^–1, indicating a shift to a preferred -sheet structure. Incubation of leuprolide with 20–200 mM salts at 25°C and 37°C also produced gels ranging from clear to cloudy and stringy white precipitates. The gel and precipitate were marked by a shift of the predominant p-sheet band to 1630 cm^–1 and 1615 cm^–1, respectively. Leuprolide was also observed to gel and/or precipitate in mixtures of water, PG or TFE, but not in DMSO. Conclusions. Birefringence was noted in many of the firmer gels. Both solutions and gels demonstrated minimal dimer or trimer formation, with no larger order aggregates detected. The chemical stability profile of gelled leuprolide was similar to that of the non-gelled water formulation by RP-HPLC.     

Glyceryl Monooleate Cubic Phase Gel as Chemical Stability Enhancer of Cefazolin and Cefuroxime

ABSTRACT

The primary objective of this study was to determine the ability of the glyceryl monooleate (GMO) cubic phase gel to protect drugs from chemical instability reactions such as hydrolysis and oxidation. Stability was assessed on cefazolin incorporated in cubic phase gel and in solution at two different concentrations (200 and 50 μg/g), at 22, 37, and 50°C. Degradation profiles, plotting percent cefazolin remaining on a logarithmic scale versus time, were constructed and the degradation rate constants calculated from the slopes. At both concentrations, degradation of cefazolin was found to be slower in the cubic phase gel than in solution at 22 and 37°C, but not at 50°C. The degradation rate constants were 3-to 18-fold lower in the gel than in solution at low concentration of cefazolin. At 22 and 37°C, the kinetics of degradation at high concentration of cefazolin was not first-order but showed a lag phase followed by an exponential loss of cefazolin, typical of oxidation. The potential oxidation of the thioether moiety of cefazolin was confirmed by its 18-fold higher stability in the presence of ethylenediaminetetraacetic acid (EDTA) and nitrogen in solution. Cefuroxime, a cephalosporin which degrades solely via β-lactam hydrolysis, degraded twice as fast in solution as it did in the gel. The enhanced stability of cefazolin and cefuroxime in the GMO cubic phase gel shows its potential as a chemical stability enhancer and this is the first report to demonstrate oxidation, in addition to β-lactam hydrolysis, as a mechanism for degradation of cefazolin.     

Solution stability of salmon calcitonin at high concentration for delivery in an implantable system

Abstract: Salmon calcitonin solutions (50 mg/mL and 100 mg/mL) were placed on stability at 37°C for 1 year in a variety of solvent systems including water, ethanol, glycerol, propylene glycol (PG) and dimethyl sulfoxide (DMSO). Calcitonin degradation was monitored by RP‐HPLC and size‐exclusion chromatography. DMSO and pH 3.3 solutions provided optimum stability. Conformational stability was also monitored by FTIR over the 1 year time course and compared with chemical and physical stability. After 12 months at 37°C, four major conformations were observed: a β‐sheet conformation (pH 3.3, pH 5.0, 70% DMSO and 70% glycerol), an aggregate conformation (pH 7.0 water), a strong α‐helical conformation (70% EtOH, 70% PG) and a weak α‐helical conformation (100% DMSO). No correlation between structure and chemical stability was observed in which both the β‐sheet structure (pH 3.3, water) and a loose α‐helical structure (100% DMSO) demonstrated good stability. However, some correlation was observed between structure and physical stability, where co‐solvents inducing an α‐helical structure resulted in a decrease in gelation. These two structural states associated with improved stability and minimal gelation, indicated that gelation can be reduced or eliminated by the use of pharmaceutically acceptable co‐solvents. Finally, salmon calcitonin (50 mg/mL) was formulated in 100% DMSO and delivered from a DUROS® implant over 4 months. Delivery at a target dose of 18 µg/day calcitonin at 37°C was confirmed.     

Stability of ricobendazole in aqueous solutions

The chemical stability of ricobendazole (RBZ) was investigated using a stability-indicating high performance liquid chromatographic (HPLC) assay with ultraviolet detection. The degradation kinetics of RBZ in aqueous solution was evaluated as a function of pH, buffer strength and temperature. The oxidation reaction in hydrogen peroxide solution was also studied. Degradation products were analyzed by mass spectroscopy and degradation pathways are proposed. Degradation of RBZ followed pseudo first-order kinetics and Arrhenius behavior over the temperature range 24–55 °C. A V-shaped pH-rate profile over the pH range 2–12 was observed with maximum stability at pH 4.8. The shape of the pH-rate profile was rationalized by catalytic effects of various components in the solution on each RBZ species. At pH 11 the activation energy for hydrolysis was 79.5 kJ/mol, and phosphate catalysis was not observed. Oxidation occurred in hydrogen peroxide solutions and was catalyzed by the presence of copper (Cu²⁺) ions. Ricobendazole amine and albendazole sulfone were identified by MS assay to be the degradation products of hydrolysis and oxidation respectively.     

Enzymatic Degradation of Leuprolide in Rat Intestinal Mucosal Homogenates

The purpose of this study was to evaluate in vitro enzymatic degradation and protection of leuprolide acetate in the mucosal homogenates of rat small intestine. When leuprolide was incubated at 37°C with the homogenates, it was degraded quickly. The apparent Michaelis–Menten constant, K_m, and the maximal reaction velocity, V_max, for leuprolide were 898 mM and 3.4 nmol/min/mg protein, respectively. At least four metabolites of leuprolide were observed in HPLC chromatograms, which were related to cleavages by some serine proteases. In the presence of protease inhibitors in the incubation medium, degradation of leuprolide was significantly suppressed by antipain and 3,4-dichloroisocoumarin (DCI), whereas bestatin and p-hydroxymercuribenzoic acid (PCMB) showed weaker protection than antipain and DCI, and α₂-macroglobulin (MG) exhibited no protection. When a w/o/w emulsion formulation was used, rapid degradation of the drug in intestinal homogenates was also inhibited. Therefore, the present study with representative protease inhibitors and a w/o/w formulation revealed that the enzymatic degradation of leuprolide is preventable in the rat intestinal mucosal homogenates.     

Coformulation of Broadly Neutralizing Antibodies 3BNC117 and PGT121: Analytical Challenges During Preformulation Characterization and Storage Stability Studies

In this study, we investigated analytical challenges associated with the formulation of 2 anti-HIV broadly neutralizing antibodies (bnAbs), 3BNC117 and PGT121, both separately at 100 mg/mL and together at 50 mg/mL each. The bnAb formulations were characterized for relative solubility and conformational stability followed by accelerated and real-time stability studies. Although the bnAbs were stable during 4°C storage, incubation at 40°C differentiated their stability profiles. Specific concentration-dependent aggregation rates at 30°C and 40°C were measured by size exclusion chromatography for the individual bnAbs with the mixture showing intermediate behavior. Interestingly, although the relative ratio of the 2 bnAbs remained constant at 4°C, the ratio of 3BNC117 to PGT121 increased in the dimer that formed during storage at 40°C. A mass spectrometry-based multiattribute method, identified and quantified differences in modifications of the Fab regions for each bnAb within the mixture including clipping, oxidation, deamidation, and isomerization sites. Each bnAb showed slight differences in the levels and sites of lysine residue glycations. Together, these data demonstrate the ability to differentiate degradation products from individual antibodies within the bnAb mixture, and that degradation rates are influenced not only by the individual bnAb concentrations but also by the mixture concentration.     

Oxidation and Deamidation of Monoclonal Antibody Products: Potential Impact on Stability,Biological Activity,and Efficacy

The role in human health of therapeutic proteins in general, and monoclonal antibodies (mAbs) in particular, has been significant and is continuously evolving. A considerable amount of time and resources are invested first in mAb product development and then in clinical examination of the product. Physical and chemical degradation can occur during manufacturing, processing, storage, handling, and administration. Therapeutic proteins may undergo various chemical degradation processes, including oxidation, deamidation, isomerization, hydrolysis, deglycosylation, racemization, disulfide bond breakage and formation, Maillard reaction, and β-elimination. Oxidation and deamidation are the most common chemical degradation processes of mAbs, which may result in changes in physical properties, such as hydrophobicity, charge, secondary or/and tertiary structure, and may lower the thermodynamic or kinetic barrier to unfold. This may predispose the product to aggregation and other chemical modifications, which can alter the binding affinity, half-life, and efficacy of the product. This review summarizes major findings from the past decade on the impact of oxidation and deamidation on the stability, biological activity, and efficacy of mAb products. Mechanisms of action, influencing factors, characterization tools, clinical impact, and risk mitigation strategies have been addressed.     

Dosage Form Development, <Emphasis Type="BoldItalic">in Vitro</Emphasis> Release Kinetics,and <Emphasis Type="BoldItalic">in Vitro–in Vivo</Emphasis> Correlation for Leuprolide Released from an Implantable Multi-reservoir Array

Purpose Implanted multi-reservoir arrays improve dosing control relative to osmotic pumps or polymer depots. The limited reservoir volume requires concentrated formulations. This report describes the development of a stable solid phase formulation of leuprolide acetate for chronic in vivo delivery from a multi-reservoir microchip and examines the correlation between in vitro release kinetics and serum pharmacokinetics. Materials and Methods Concentrated formulations (>10% w/v) were prepared using small volume processing methods. Drug yield, release kinetics, and formulation stability were evaluated in vitro by HPLC. The correlation between in vitro and in vivo kinetic data was determined for a solid formulation by direct comparison of data sets and using absorption kinetics calculated from the Wagner–Nelson equation. Results High yield and the control of release kinetics by altering peptide formulation or reservoir geometry were demonstrated. Lyophilized leuprolide in a soluble solid matrix exhibited reproducible release kinetics and was stable (>95% leuprolide monomer) after 6 months at 37°C. A strong correlation was found between in vitro release kinetics and in vivo absorption by direct comparison of data sets and using the Wagner–Nelson absorption (slopes of 1.01 and 0.91; R² 0.99). Conclusions Reproducible releases of a stable solid leuprolide formulation from a multi-reservoir microchip were achieved in vitro. Chronic pulsatile release was subsequently performed in vivo. Comparison of in vitro and in vivo data reveals that pharmacokinetics were controlled by the rate of release from the device.     

The effect of sucrose hydrolysis on the stability of protein therapeutics during accelerated formulation studies

Stability studies of protein therapeutics are often accelerated by storing potential formulations at elevated temperatures where the rates of various chemical and physical degradation pathways are increased. An often overlooked caveat of using these studies is the potential degradation of the formulation components themselves. In this report, we show that the monoclonal antibody MAB001 aggregated at a faster rate when formulated with sucrose compared to samples that contained sorbitol or no excipient during accelerated stability studies following an initial lag phase where the rates of aggregate formation were similar in all formulations. The duration of the lag phase was both pH and temperature dependent and a significant increase of protein glycation was noticed during this time. These observations indicate that the enhanced rate of antibody aggregation in sucrose containing formulations is likely due to protein glycation following sucrose hydrolysis under accelerated conditions. This hypothesis was confirmed by demonstrating that antibody directly glycated with glucose aggregated at a faster rate than nonglycated antibody stored in the identical formulation. These findings question the utility of using accelerated stability data for predicting protein stability in sucrose containing formulations stored at 2–8°C, where no glycation or change in aggregation rate was observed. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:4501–4510, 2009     

Enzymatic degradation of leuprolide in rat intestinal mucosal homogenates

The purpose of this study was to evaluate in vitro enzymatic degradation and protection of leuprolide acetate in the mucosal homogenates of rat small intestine. When leuprolide was incubated at 37 degrees C with the homogenates, it was degraded quickly. The apparent Michaelis-Menten constant, K(m), and the maximal reaction velocity, Vmax, for leuprolide were 898 mM and 3.4 nmol/min/mg protein, respectively. At least four metabolites of leuprolide were observed in HPLC chromatograms, which were related to cleavages by some serine proteases. In the presence of protease inhibitors in the incubation medium, degradation of leuprolide was significantly suppressed by antipain and 3,4-dichloroisocoumarin (DCI), whereas bestatin and p-hydroxymercuribenzoic acid (PCMB) showed weaker protection than antipain and DCI, and alpha 2-macroglobulin (MG) exhibited no protection. When a w/o/w emulsion formulation was used, rapid degradation of the drug in intestinal homogenates was also inhibited. Therefore, the present study with representative protease inhibitors and a w/o/w formulation revealed that the enzymatic degradation of leuprolide is preventable in the rat intestinal mucosal homogenates.     

Aqueous stability of leuprolide acetate: effect of temperature,dissolved oxygen,pH and complexation with β-cyclodextrin

In the present research, the aqueous stability of leuprolide acetate (LA) in phosphate buffered saline (PBS) medium was studied (pH?=?2.0–7.4). For this purpose, the effect of temperature, dissolved oxygen and pH on the stability of LA during 35 days was investigated. Results showed that the aqueous stability of LA was higher at low temperatures. Degassing of the PBS medium partially increased the stability of LA at 4?°C, while did not change at 37?°C. The degradation of LA was accelerated at lower pH values. In addition, complexes of LA with different portions of β-cyclodextrin (β-CD) were prepared through freeze-drying procedure and characterized by Fourier transform infrared (FTIR) and differential scanning calorimetry (DSC) analyses. Studying their aqueous stability at various pH values (2.0–7.4) showed LA/β-CD complexes exhibited higher stability when compared with LA at all pH values. The stability of complexes was also improved by increasing the portion of LA/β-CD up to 1/10.     

Stability of Benzoyl Peroxide in Aromatic Ester-Containing Topical Formulations

The chemical stability of benzoyl peroxide (BPO) was studied in solutions and gels. The solutions (1% w/v) were prepared in single solvents (alcohol USP, isopropyl alcohol USP, ethyl benzoate, C_12–15 alkyl benzoate, dimethyl isosorbide, propylene carbonate, and acetone) and in binary and tertiary combinations of these solvents, with and without the addition of antioxidant(s) (BHT, BHA, eugenol, tert-butyl hydroquinone, Tenox-2?, vitamin E, and vitamin C). The solutions were stored at 37°C for 5 weeks, and each week were analyzed for remaining BPO. Using first-order kinetics, the stability of BPO in solution was found to decrease in the order: ternary >binary >single solvent systems. Regardless of the number of solvents present, the highest stability of BPO (t_1/2 >7.5 weeks) was attained in the presence of ethyl benzoate and C_12–15 alkyl benzoate. The stability of BPO in solution did not change significantly with the addition of most antioxidants. The solutions in which BPO remained most stable were one in alcohol USP-ethyl benzoate-C_12–15 alkyl benzoate (60:20:20; t_1/2 = 18.15 weeks) and another in alcohol USP-C_12–15 alkyl benzoate-isopropanol plus 0.1% BHT (65:20:15; t_1/2 = 12.44 weeks). In turn, these two solutions were converted to homogeneous gels by the addition of Cab-O-Sil?. The chemical stability of BPO in these gels was evaluated at 37°, 45°, 50°, and 55°C for 5 weeks. Parallel experiments were conducted with two commercial BPO products, a 2.5% tinted gel and 5% vanishing lotion. BPO was less stable in commercial products (t_1/2 ≤ 13 weeks) than in the extemporaneously prepared gels (mean t_1/2 ～23 weeks). The present results suggest that aromatic esters can enhance the chemical stability of BPO in solutions and gel formulations to a significant extent.     

Kinetics and Mechanism of Degradation of Zileuton,a Potent 5-Lipoxygenase Inhibitor

Zileuton (N-(1-benzo[b]thien-2-ylethyl)N-hydroxyurea) is a powerful 5-lipoxygenase inhibitor. The chemical degradation of Zileuton and related hydroxyurea derivatives was studied in aqueous solutions as a function of pH and temperature. The pH profile for the degradation of Zileuton shows an acid-catalyzed region at pH values below 2, water hydrolysis of the protonated form at pH values from 3 to 8, and water hydrolysis of the unprotonated form at pH values greater than 9. Hydrolysis of the hydroxyurea moiety to give the hydroxylamine derivative represents the main degradation pathway for Zileuton. This product, however, is not stable and is present at low concentrations at pH values below 6 and not observed at pH values greater than 7. Further decomposition of the hydroxylamine derivative leads to the observed degradation products. Air oxidation to the isomeric oximes accounts for the observed products at pH values greater than 7. Hydrolysis of the oximes to the ketone derivative accounts for the observed products at pH values 2 to 6. Parallel decomposition pathways to the alcohol derivative were noted under strongly acidic conditions, pH 0 to 2.     

Capillary electrophoresis analysis of the degradation of the aspartyl tripeptide Phe-Asp-GlyOH at pH 2.0 and 7.4 under forced conditions

The degradation of the tripeptide l-Phe-α-l-Asp-GlyOH was studied at 80 °C and pH 2.0 and 7.4 by capillary electrophoresis. Separation of most known as well as unknown degradation products was achieved in a 50 mM sodium phosphate buffer, pH 3.0. The diastereomers l-Phe-α-l-Asp-GlyOH/l-Phe-α-d-Asp-GlyOH could only be separated upon addition of 16 mg/ml carboxymethyl-β-cyclodextrin and 5% acetonitrile to the background electrolyte. Compound identification was performed by capillary electrophoresis-electrospray ionization-mass spectrometry. In addition to Asp isomerization and epimerization products as well as hydrolysis products four diketopiperazine derivatives were identified. Moreover, two degradation products were observed containing the amino acids Asp, Gly and Phe but the unequivocal assignment could not be accomplished based on the mass spectra. Following validation with regard to linearity, range, limit of detection, limit of quantitation and precision the assay was applied to the analysis of the incubation solutions. While peptide backbone hydrolysis dominated at pH 2.0, isomerization and enantiomerization yielding β-Asp and d-Asp peptides as well as cyclization to diketopiperazine derivatives were observed at pH 7.4. The diketopiperazines were the dominant reaction products amounting to about 85% of the compounds detected after the maximal incubation time of 240 h. A kinetic model was used to fit the concentration versus time data.     

Chemical kinetics and aqueous degradation pathways of a new class of synthetic ozonide antimalarials

Chemical stability of a new class of ozonide (1,2,4 trioxolanes) antimalarial compounds was investigated. The effects of pH, ionic strength, dielectric constant and cyclodextrin-complexation on the chemical stability and degradation product formation of selected compounds were examined. The mechanism of degradation in aqueous solution was probed using (18)O-labelled water and kinetic solvent isotope effect studies. The effect of stereochemistry was investigated using selected pairs of stereoisomers. The degradation of the ozonides in aqueous solution followed apparent first-order kinetics, with no effect of ionic strength and no indication of any direct involvement of water in the degradation mechanism. All major degradation products were identified and mass balance was confirmed. Stereochemistry had a significant effect on degradation rate; trans isomers degrading approximately four-fold faster than the corresponding cis isomers. The degradation rates were essentially independent of pH above pH 2; however, an additional specific acid catalysed pathway was dominant below pH 2. Solvent dielectric constant had a significant effect on the degradation rate. It is proposed that the degradation observed in aqueous solution occurred through a concerted heterolytic scission of the central ozonide ring, with chemical substituents on the cyclohexyl ring having only a minor influence on degradation rate.     

Chemical Pathways of Peptide Degradation. IV. Pathways,Kinetics, and Mechanism of Degradation of an Aspartyl Residue in a Model Hexapeptide

In this study the hexapeptide Val-Tyr-Pro-Asp-Gly-Ala (Asp-hexapeptide) was used as a model to investigate the kinetics of aspartate degradation in aqueous solution. The apparent rate of degradation of the Asp-hexapeptide was determined as a function of pH, buffer concentration, and temperature. At very acidic pH levels (0.3, 1.1, 1.5, 2.0, and 3.0), the apparent rate of degradation followed pseudo-first-order kinetics. In this pH region, the Asp-hexapeptide predominantly underwent specific acid-catalyzed hydrolysis of the Asp-Gly amide bond (Asp-X hydrolysis) to form a tetrapeptide (Val-Tyr-Pro-Asp) and a dipeptide (Gly-Ala). In addition, parallel formation of a cyclic imide intermediate could be observed, although no isoAsp-hexapeptide was detected. At pH 4.0 and 5.0, the Asp-hexapeptide simultaneously isomerized via the cyclic imide to form the iso-Asp-hexapeptide and underwent Asp-X hydrolysis to produce the cleavage products. The pH-rate profiles (pH 0.3–5.0) for the Asp-X hydrolysis and the formation of cyclic imide revealed that the degree of ionization of the carboxylic acid side chain of Asp residue significantly altered the rate of reaction, with the ionized form being more reactive than the unionized form. Little or no buffer catalysis was observed for either pathway. Solvent isotope experiments were used to probe the mechanism of the Asp-X hydrolysis reaction. At pH values above 6.0, the apparent rate of degradation of the Asp-hexapeptide followed pseudo-first-order reversible kinetics, with the isoAsp-hexapeptide being the only observed product (isomerization). Above pH 8.0, the isomerization kinetics were found to be independent of pH and buffer concentration. The kinetics of degradation of Asp-hexapeptide (Val-Tyr-Pro-Asp-Gly-Ala) and Asn-hexapeptide (Val-Tyr-Pro-Asn-Gly-Ala) were compared to determine the relative instability of the Asp and Asn residues and to understand the mechanism of formation of cyclic imide at near neutral to basic pH.     

Effect of Moisture on the Stability of a Lyophilized Humanized Monoclonal Antibody Formulation

Purpose. To determine the effect of moisture and the role of the glass transition temperature (T_g) on the stability of a high concentration, lyophilized, monoclonal antibody. Methods. A humanized monoclonal antibody was lyophilized in a sucrose/histidine/polysorbate 20 formulation. Residual moistures were from 1 to 8%. T_g values were measured by modulated DSC. Vials were stored at temperatures from 5 to 50°C for 6 or 12 months. Aggregation was monitored by size exclusion chromatography and Asp isomerization by hydrophobic interaction chromatography. Changes in secondary structure were monitored by Fourier transform infrared (FTIR). Results. T_g values varied from 80°C at 1% moisture to 25°C at 8% moisture. There was no cake collapse and were no differences in the secondary structure by FTIR. All formulations were stable at 5°C. High moisture cakes had higher aggregation rates than drier samples if stored above their T_g values. Intermediate moisture vials were more stable to aggregation than dry vials. High moisture samples had increased rates of Asp isomerization at elevated temperatures both above and below their T_g values. Chemical and physical degradation pathways followed Arrhenius kinetics during storage in the glassy state. Only Asp isomerization followed the Arrhenius model above the T_g value. Both chemical and physical stability at T T_g were fitted to Williams-Landel-Ferry (WLF) kinetics. The WLF constants were dependent on the nature of the degradation system and were not characteristic of the solid system. Conclusion. High moisture levels decreased chemical stability of the formulation regardless of whether the protein was in a glassy or rubbery state. In contrast, physical stability was not compromised, and may even be enhanced, by increasing residual moisture if storage is below the T_g value.     

Parenteral formulation and thermal degradation pathways of a potent rebeccamycin based indolocarbazole topoisomerase I inhibitor

The development of a practical and pharmaceutically acceptable parenteral dosage form of 1 is described. A cosolvent formulation strategy was selected to achieve the necessary human dose of 1 for administration via intravenous infusion. The final market formulation of 1 chosen for commercial development and Phase II clinical supplies was the topoisomerase inhibitor dissolved in a 50% aqueous propylene glycol solution vehicle with 50 mM citrate buffered to pH 4. The thermal degradation pathways of 1 in this aqueous propylene glycol vehicle in the pH range of 3–5 were determined by relative kinetics and degradation product identification using LC/MS, LC/MS/MS, and NMR analysis. The primary mode of degradation of 1 in this aqueous cosolvent formulation is hydrolysis affording the anhydride 2 (in equilibrium with the dicarboxylic acid 3) and release of the hydrazine diol side chain 11. Subsequent oxidative degradation of 11 occurs in several chemical steps which yield a complicated mixture of secondary reaction products that have been structurally identified.