Comparison of Protein Particle Formation in IgG1 mAbs Formulated with PS20 Vs. PS80 When Subjected to Interfacial Dilatational Stress.

Polysorbates (PS) are nonionic surfactants that are commonly included in protein formulations to mitigate the formation of interfacial stress-induced protein particles and thus increase their long-term storage stability. Nonetheless, factors that dictate the efficiency of different polysorbates in mitigating protein particle formation, especially during the application of interfacial stresses, are often ill defined. Here, we used a Langmuir trough to determine the surface activity of two IgG1 monoclonal antibodies formulated with two different polysorbates (PS20 and PS80) when subjected to interfacial dilatational stress. Interfacial properties of these formulations were then correlated with characterization of subvisible protein particles measured by micro-flow imaging (MFI). Both mAbs, when formulated in PS20, demonstrate faster adsorption kinetics and higher surface activity compared to PS80 or surfactant-free formulations. Compression/expansion results suggest that when exposed to interfacial dilatational stresses, both mAb/PS20 formulations display interfacial properties of PS20 alone. In contrast, interfacial properties of both mAb/PS80 formulations suggest mAbs and PS80 are co-adsorbed to the air-water interface. Further, MFI analysis of the interface and the bulk solution confirms that PS20 is more effective than PS80 at mitigating the formation of larger particles in the bulk solution in both mAbs. Concomitantly, the efficiency of PS to prevent interface-induced protein particle formation also depended on the protein's inherent tendency to aggregate at a surfactant-free interface. Together, the studies presented here highlight the importance of determining the interfacial properties of mAbs, surfactants, and their combinations to make informed formulation decisions about the choice of surfactant.

Degradation of BMS-753493, a novel epothilone folate conjugate anticancer agent.

BMS-753493 is a folate-targeted candidate being developed for the treatment of cancer. As part of preformulation efforts, our aim was twofold - to understand the major degradation pathways and, study its kinetics of degradation to aid drug product development. Given the complexity of degradation, BMS-748285, the epothilone moiety of BMS-753493 was used as model compound to evaluate the major degradation pathway viz; macrolactone versus aziridine ring hydrolysis. Hydrolysis of BMS-753493 was studied in the pH range of 1.5-9.4 in 0.05 M buffers at 0.5 ionic strength and 5-40°C. Three major pathways were identified; carbonate ester hydrolysis and hydrolysis of aziridine and macrolactone rings resulting in addition products with identical masses (m/z = 794) in the pH range of 5-7.5. Similarly, two addition products, D1 and D2 (m/z = 555) were also formed on hydrolysis of BMS-748285 under neutral pH conditions. The reaction products from BMS-748285 were isolated and characterized using LC-MS and LC-SPE-NMR (1-D ¹H and 2-D HMBC, heteronuclear single quantum coherence) analyses. LC-NMR analysis indicated an intact aziridine ring and opened macrolactone ring, resulting in D1 and D2, an isomeric hydroxy acid pair resulting from an alkyl oxygen cleavage. By analogy to BMS-748285, BMS-753493 was also postulated to undergo alkyl cleavage of the macrolactone, forming two epimeric hydroxy acids under neutral pH. The pH-stability data were also consistent with these findings. Additionally, the degradation kinetics for BMS-753493, indicated a U-shaped pH-stability profile with maximum stability at pH 7. Based on the stability and solubility considerations, the pH range of 6-7 was optimal for an injectible drug product development.

Impact of Polysorbate 80 Grade on the Interfacial Properties and Interfacial Stress Induced Subvisible Particle Formation in Monoclonal Antibodies.

Polysorbate 80 is a nonionic surfactant that is added to therapeutic protein formulations to mitigate protein particle formation when subjected to various mechanical stresses. Variations in the PS80 grade has recently sparked questions surrounding the effect of oleic acid content (OAC) on surfactant's ability to mitigate interface-induced protein particle formation when stressed. In this work, a Langmuir trough was used to apply interfacial dilatational stress to two IgG molecules (mAb1 and mAb2) in formulations containing Chinese pharmacopeia (CP) and multicompendial (MC) grades of PS80. The interfacial properties of these mAb formulations, with and without interfacial dilatational stresses, were correlated with subvisible particle count and particle size/morphology distributions as measured by Micro-flow imaging (MFI). Overall, differences in interfacial properties correlated well with protein particle formation for both molecules in the two PS80 formulations. Further, the impact of grade of PS80 on the interfacial properties and interfacial stress-induced protein particle formation depends on the adsorption kinetics of the IgG molecules as well as the concentration of the surfactant used. This study demonstrates that measuring the interfacial properties of mAb formulations can be a useful tool to predict interfacial stress induced protein particle formation in the presence of different excipients of varying quality.

Glycosylation of aromatic amines I: Characterization of reaction products and kinetic scheme.

The reactions of aliphatic and aromatic amines with reducing sugars are important in both drug stability and synthesis. The formation of glycosylamines in solution, the first step in the Maillard reaction, does not typically cause browning but results in decreased potency and is hence significant from the aspect of drug instability. The purpose of this research was to present (1) unreported ionic equilibria of model reactant (kynurenine), (2) the analytical methods used to characterize and measure reaction products, (3) the kinetic scheme used to measure reaction rates and (4) relevant properties of various reducing sugars that impact the reaction rate in solution. The methods used to identify the reversible formation of two products from the reaction of kynurenine and monosaccharides included LC mass spectrometry, UV spectroscopy, and 1-D and 2-D (1)H-(1)H COSY NMR spectroscopy. Kinetics was studied using a stability-indicating HPLC method. The results indicated the formation of alpha and beta glycosylamines by a pseudo first-order reversible reaction scheme in the pH range of 1-6. The forward reaction was a function of initial glucose concentration but not the reverse reaction. It was concluded that the reaction kinetics and equilibrium concentrations of the glycosylamines were pH-dependent and also a function of the acyclic content of the reacting glucose isomer.

Dehydration and Stabilization of a Reactive Tertiary Hydroxyl Group in Solid Oral Dosage Forms of BMS-779788.

BMS-779788 contains a reactive tertiary hydroxyl attached to a weakly basic imidazole ring. Propensity of the carbinol toward dehydration to yield the corresponding alkene, BMS-779788-ALK, was evaluated. Elevated levels of BMS-779788-ALK were observed in excipient compatibility samples. Stability studies revealed that BMS-779788 degrades to BMS-779788-ALK in capsules and tablets prepared by both dry and wet granulation processes. An acid-catalyzed dehydration mechanism, in which the heterocyclic core contributes resonance stability to the cationic intermediate via charge transfer to the imidazole ring, was proposed. Therefore, neutralization via a buffered (pH 7.0) granulating solution was used to mitigate dehydration. Solution studies revealed degradation of BMS-779788 to BMS-779788-ALK over the pH range of 1-7.5. Reversibility was confirmed by initiating reactions with BMS-779788-ALK over the same pH range. Accordingly, a simple reversible scheme can be used to describe reactions initiated with either BMS-779788 or BMS-779788-ALK. To eliminate potential for charge delocalization across the heterocycle and probe the degradation mechanism, the imidazole ring of BMS-779788 was methylated (BMS-779788-Me). The propensity for acid-catalyzed dehydration was then evaluated. The acid stability of BMS-779788-Me confirmed that the heterocyclic core contributes to reactivity liability of the tertiary hydroxyl.

Quality by design development of brivanib alaninate tablets: degradant and moisture control strategy.

A quality by design approach was applied to the development of brivanib alaninate tablets. Brivanib alaninate, an ester pro-drug, undergoes hydrolysis to its parent compound, BMS-540215. The shelf-life of the tablets is determined by the rate of the hydrolysis reaction. Hydrolysis kinetics in the tablets was studied to understand its dependence on temperature and humidity. The BMS-540215 amount versus time profile was simulated using a kinetic model for the formation of BMS-540215 as function of relative humidity in the environment and a sorption-desorptiom moisture transfer model for the relative humidity inside the package. The combined model was used to study the effect of initial tablet water content on the rate of degradation and to identify a limit for initial tablet water content that results in acceptable level of the degradant at the end of shelf-life. A strategy was established for the moisture and degradant control in the tablet based on the understanding of its stability behavior and mathematical models. The control strategy includes a specification limit on the tablet water content and manufacturing process controls that achieve this limit at the time of tablet release testing.

Glycosylation of aromatic amines II: Kinetics and mechanisms of the hydrolytic reaction between kynurenine and glucose.

The kinetics of the weakly basic aromatic amine, kynurenine, with glucose were studied as model reactants aimed at mechanistic understanding of pharmaceutically relevant amine-aldehyde reactions. The reaction kinetics of the forward and reverse processes (glycosylamine formation and hydrolysis) were studied under first-order conditions in aqueous solutions at 40 degrees C in the pH range 1-6.5 in the presence of various buffers. The alpha-and beta-glycosylamines were reversibly formed via an acyclic imine that was not present in detectable quantities. Rate-limiting formation of the imine was complex and involved the addition of the amine and aldehyde to form the carbinolamine followed by the acid-catalyzed dehydration to the imine. The pH-rate profile was characterized by three kinetically distinguishable processes. At lower pH values, the profile was consistent with specific acid-catalyzed rate-determining addition of amine and aldehyde. In the pH range of 4-6 a downward bend was attributable to the change in rate determining step from addition to dehydration. In the pH region of 2-3 the rate law was described by specific acid catalysis and solvolysis of the zwitterionic form of kynurenine. Nonlinear buffer effects and Brönsted plots were shown to be consistent with this interpretation of the pH-rate profile.

Glycosylation of aromatic amines III: Mechanistic implications of the pH-dependent glycosylation of various aromatic amines (kynurenine, 2'-aminoacetophenone, daptomycin, and sulfamethoxzaole).

Glycosylation reaction kinetics of a series of aromatic amines (kynurenine, 2'-aminoacetophenone, daptomycin, and sulfamethoxazole) was compared to propose a unifying reaction mechanism. Kinetic studies were conducted in aqueous solutions containing glucose in the pH range 1-6.5 with 2'-aminoacetophenone and daptomycin. The resultant pH-rate profiles were compared to previously reported profiles for the reactions of glucose and kynurenine or sulfamethoxazole. Glycosylation of weakly basic aromatic amines involved the addition of the unprotonated amine to the aldehydic sugar leading to carbinolamine formation followed by specific acid catalyzed dehydration. All of the pH-rate profiles displayed characteristic downward bend at pH 4-5 due to a change from rate-determining addition to dehydration. In the pH-rate profile for kynurenine, a second downward bend was observed in the pH region 2-4. This feature was absent for the other substrates and was attributed to differences in reactivity of the two ionization states of the alpha carboxylic acid in kynurenine. This stabilization was not possible for the other amines studied.