Identification of leachables observed in the size exclusion chromatograms of a low concentration product stored in prefilled syringes.

Requisite leachables testing of pharmaceutical products is commonly conducted with pre-defined analytical methods on a subset of materials intended to be representative of the marketed product. Throughout product development, leachables may occasionally be detected in other methods not specifically intended for monitoring such impurities. We have identified two leachables, ethyl 4-ethoxybenzoate (E4E) and 2,6-di(t-butyl)-4-hydroxy-4-methyl-2,5-cyclohexadien-1-one (BHT-OH) in a low concentration product stored in prefilled syringes (PFS). The leachables were initially detected by size exclusion chromatography (SEC) as late-eluting impurity peaks. Syringe component extraction studies indicated that the impurities were related to the syringe stoppers. Positive identification of E4E was accomplished by reversed phase liquid chromatography- tandem mass spectrometry (RPLC-MS/MS). Positive identification of BHT-OH required RPLC-solid phase extraction-cryoflow NMR (RPLC-SPE-NMR), as initial RPLC-MS/MS investigations were unsuccessful in elucidating the structure. We focus specifically on the efforts required to identify the leachables, and the fortuitous mixed mode separation mechanism and low concentration nature of the product, which were the main factors contributing to the unlikely detection of the leachables by SEC. We note that our investigations were conducted independently of formal leachables and extractables (L&E) studies and we discuss challenges with designing and conducting such studies in a manner that captures the comprehensive L&E profile of a product.

Structural characterization of low level degradants in aztreonam injection and an innovative approach to aid HPLC method validation.

Three new degradants have been identified from drug product and active pharmaceutical ingredient stability samples of aztreonam, a marketed synthetic monocyclic beta-lactam antibiotic. The degradants were detected following the implementation of a new, more selective HPLC method for the determination of impurities and degradants. The new method was developed in response to changes in the regulatory requirement for mature products. Two of the new unknown Degradants (I and II) were observed in chromatograms from stability samples of aztreonam injection. The third new Degradant (III) was observed during a stability study of the aztreonam active pharmaceutical ingredient. These degradants were structurally characterized. A small amount (ca. 1-3mg) of each degradant was isolated via preparative HPLC for structure elucidation using accurate MS, one and two-dimensional NMR spectroscopy. The small amount of each NMR sample was then reused as a standard for HPLC purity/impurity method validation. Their exact concentrations were determined using quantitative NMR which enabled the execution of the quantitative elements of the HPLC method validation. This innovative approach eliminated the need to isolate or synthesize larger quantities of markers for HPLC/UV method validation, thus saving significant time and reducing costs.

Effect of pepsin on maintaining the supersaturation of the HCl salt of a weakly basic drug: a case study.

The impact of pepsin on the maintenance of supersaturated solution of the HCl salt of a weakly basic drug was evaluated in simulated gastric fluid by monitoring the drug solubility in the absence and presence of pepsin. In the presence of pepsin, the HCl salt maintained its apparent solubility through 24 h, whereas, no such solubility advantage was seen in the absence of pepsin. Consequently, a minimum inhibitory concentration of pepsin is required for maintenance of supersaturation. In addition, NMR study seems to indicate a molecular level interaction between pepsin and HCl salt leading to a weak binding between the two. Therefore, for the HCl salts of weak bases having disproportionation potential, it is preferred that preformulation solubility studies are conducted in the presence of pepsin to reflect their in vivo behavior in maintaining supersaturation solubility.

Synthesis of 2-(14)C-iminothiolane and 2-(13)C,(15)N-iminothiolane (Traut's reagent).

2-Iminothiolane has found utility in the growing area of antibody-drug conjugates by serving as a lysine-thiolating agent and the junction between the antibody and the cytotoxic payload during random conjugation of a monoclonal antibody. 2-(14)C-Iminothiolane was prepared from commercially available [(14)C]KCN using a four-step sequence in an overall 10% radiochemical yield. Stable-labeled 2-(13)C,(15)N-iminothiolane was also prepared from [(13)C(15)N]KCN in a similar manner. The ˙ labeled Traut's reagent produced by this sequence showed comparable reactivity as the commercially available unlabeled reagent with a representative monoclonal antibody and could serve as highly informative analytical tools to investigate antibody-drug conjugate formation via the random conjugation process.

Role of Self-Association and Supersaturation in Oral Absorption of a Poorly Soluble Weakly Basic Drug.

PURPOSE: Precipitation of weakly basic drugs in intestinal fluids can affect oral drug absorption. In this study, the implications of self-association of brivanib alaninate in acidic aqueous solution, leading to supersaturation at basic pH condition, on its solubility and oral absorption were investigated. METHODS: Self-association of brivanib alaninate was investigated by proton NMR spectroscopy, surface tension measurement, dynamic light scattering, isothermal titration calorimetry, and molecular modeling. Drug solubility was determined in various pH media, and its tendency to supersaturate upon pH shift was investigated in buffered and biorelevant aqueous solutions. Pharmacokinetic modeling of human oral drug absorption was utilized for parameter sensitivity analyses of input variables. RESULTS: Brivanib alaninate exhibited continuous, and pH- and concentration-dependent self-association. This phenomenon resulted in positive deviation of drug solubility at acidic pH and the formation of a stable supersaturated drug solution in pH-shift assays. Consistent with the supersaturation phenomenon observed in vitro, oral absorption simulations necessitated invoking long precipitation time in the intestine to successfully predict in vivo data. CONCLUSIONS: Self-association of a weakly basic drug in acidic aqueous solution can increase its oral absorption by supersaturation and precipitation resistance at the intestinal pH. This consideration is important to the selection of parameters for oral absorption simulation.

Semi-preparative LC-SPE-cryoflow NMR for impurity identifications: use of mother liquor as a better source of impurities.

Unambiguous structural elucidation of active pharmaceutical ingredients (API) impurities is a particularly challenging necessity of pharmaceutical development, particularly if the impurities are low level (0.1% level). In many cases, this requires acquiring high-quality NMR data on a pure sample of each impurity. High-quality, high signal-to-noise (S/N) one- and two-dimensional NMR data can be obtained using liquid chromatography-solid phase extraction-cryoflow NMR (LC-SPE-cryoflow NMR) with a combination of semi-preparative column for separation and mother liquor as a source of concentrated impurities. These NMR data, in conjunction with mass spectrometry data, allowed for quick and unambiguous structural elucidations of four impurities found at low level in the crystallized API but found at appreciable levels in the mother liquor that was used as the source for these impurities. These data show that semi-preparative columns can be used at lower than ideal flow rates to facilitate trapping of HPLC components for LC-SPE-cryoflow NMR analysis without compromising chromatographic resolution. Also, despite the complex chromatography encountered with the use of mother liquor as a source of impurities, acceptably pure analytes were obtained for acquiring NMR data for unambiguous structure elucidations.

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.