Analytical characterization of an orally-delivered peptide pharmaceutical product.

The characterization of orally-delivered peptide pharmaceuticals presents several challenges to analytical methods in comparison to characterization of conventional small-molecule drugs. These challenges include the analysis and characterization of difficult-to-separate impurities, secondary structure, the amorphous solid-state form, and the integrity of enteric-coated drug delivery systems. This work presents the multidisciplinary analytical characterization of a parathyroid hormone (PTH) peptide active pharmaceutical ingredient (API) and an oral formulation of this API within enteric-coated sucrose spheres. The analysis of impurities and degradation products in API and formulated drug product was facilitated by the development of an ultrahigh-performance liquid chromatography (UHPLC) method for analysis by high-resolution mass spectrometry (MS). The use of UHPLC allowed for additional resolution needed to detect impurities and degradation products of interest. The secondary structure was probed using a combination of solution-state NMR, infrared, and circular dichroism spectroscopic methods. Solid-state NMR is used to detect amorphous API in a nondestructive manner directly within the coated sucrose sphere formulation. Fluorescence and Raman microscopy were used in conjunction with Raman mapping to show enteric coating integrity and observe the distribution of API beneath the enteric-coating on the sucrose spheres. The methods are combined in a multidisciplinary approach to characterize the quality of the enteric-coated peptide product.

Development of quality-by-design analytical methods.

Quality-by-design (QbD) is a systematic approach to drug development, which begins with predefined objectives, and uses science and risk management approaches to gain product and process understanding and ultimately process control. The concept of QbD can be extended to analytical methods. QbD mandates the definition of a goal for the method, and emphasizes thorough evaluation and scouting of alternative methods in a systematic way to obtain optimal method performance. Candidate methods are then carefully assessed in a structured manner for risks, and are challenged to determine if robustness and ruggedness criteria are satisfied. As a result of these studies, the method performance can be understood and improved if necessary, and a control strategy can be defined to manage risk and ensure the method performs as desired when validated and deployed. In this review, the current state of analytical QbD in the industry is detailed with examples of the application of analytical QbD principles to a range of analytical methods, including high-performance liquid chromatography, Karl Fischer titration for moisture content, vibrational spectroscopy for chemical identification, quantitative color measurement, and trace analysis for genotoxic impurities.

LC-MS/MS and density functional theory study of copper(II) and nickel(II) chelating complexes of elesclomol (a novel anticancer agent).

Elesclomol (N-malonyl-bis(N'-methyl-N'-thiobenzoylhydrazide)), which is a novel anticancer agent, can form chelating complexes with Fe(II), Co(II), Ni(II), Cu(II), and Zn(II) in the gas phase during electrospray ionization (ESI) mass spectrometry. In the solution phase with acidic medium during chromatographic separation, however, only Cu(II) and Ni(II) to a lesser degree favor the formation of chelating complexes with elesclomol. The Cu(II)-chelating complex [Cu(II)+elesclomol-H]+· exhibits more complicated MS/MS fragmentation pathways than the Ni(II)-chelating complex [Ni(II)+elesclomol-H]+. One significant difference is the ready occurrence of the electron transfer upon collision-induced dissociation (CID) of [Cu(II)+elesclomol-H]+·. This leads to the reduction of Cu(II) to Cu(I). However, such phenomenon was not observed upon CID of [Ni(II)+elesclomol-H]+. On the basis of the density functional theory (DFT) calculations at the B3LYP/6-31+G(d)/LANL2DZ level, the Cu(II)- and Ni(II)-chelating complexes of elesclomol exist in the keto-form with tetra-coordinated trapezoid geometry in the gas phase but at different levels of distortion. As compared to the Ni(II)-elesclomol complex, the Cu(II)-elesclomol complex is more stable (by -55.25 kcal/mol). This relative stability of the chelating complexes of elesclomol is consistent with the Irving-Williams series of bindings to ligands.

Gas-phase derivatization via the Meerwein reaction for selective and sensitive LC-MS analysis of epoxides in active pharmaceutical ingredients.

A gas-phase derivatization strategy is reported by using the gas-phase Meerwein reaction for rapid and direct LC-MS analysis of epoxides, which are potential genotoxic impurities (GTIs) in active pharmaceutical ingredients (APIs). This class-selective ion/molecule reaction occurs between epoxides and the ethylnitrilium ion (CH(3)-C≡NH↔CH(3)-C=NH) that is generated by atmospheric pressure ionizations (when acetonitrile is used as the mobile phase). Density functional theory (DFT) calculations at the B3LYP/6-311+G(d,p) level show that the gas-phase Meerwein reaction is thermodynamically favorable. Commonly used atmospheric pressure ionization techniques including ESI, APCI and APPI were evaluated for optimal formation of the Meerwein reaction products. APCI appears to be the method of choice since it offers better sensitivity and more robust detection under typical LC-MS instrumentation conditions. Quantitative analysis of epoxides can be achieved by either single ion monitoring (SIM) or multiple reaction monitoring (MRM) of the Meerwein reaction products. We demonstrate herein quantitative analysis of two potential GTIs of SB797313 and SB719133 in APIs. The validated methods afford excellent linearity (r(2)≥0.999), sensitivity (LOD≤1 ppm by w/w in 10 mg/mL APIs) and recovery (ranging from 92% to 102%), as well as accuracy (≤2.8% difference) and precision (≤2.2% RSD) based on injections of six prepared standards. This novel strategy is particularly useful when a target analyte is difficult to be directly analyzed by LC-MS (e.g. due to poor ionization) or unstable in the course of solution-phase derivatization.

Gas-phase meerwein reaction of epoxides with protonated acetonitrile generated by atmospheric pressure ionizations.

Ethylnitrilium ion can be generated by protonation of acetonitrile (when used as the LC-MS mobile phase) under the conditions of atmospheric pressure ionizations, including electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) as well as atmospheric pressure photoionization (APPI). Ethylnitrilium ion (CH(3)-C≡N+H and its canonical form CH(3)-C+=NH) is shown to efficiently undergo the gas-phase Meerwein reaction with epoxides. This reaction proceeds by the initial formation of an oxonium ion followed by three-to-five-membered ring expansion via an intramolecular nucleophilic attack to yield the Meerwein reaction products. The density functional theory (DFT) calculations at the B3LYP/6-311+G(d,p) level show that the gas-phase Meerwein reaction is thermodynamically favorable. Collision-induced dissociation (CID) of the Meerwein reaction products yields the net oxygen-by-nitrogen replacement of epoxides with a characteristic mass shift of 1 Da, providing evidence for the cyclic nature of the gas-phase Meerwein reaction products. The gas-phase Meerwein reaction offers a novel and fast LC-MS approach for the direct analysis of epoxides that might be of genotoxic concern during drug development. Understanding and utilizing this unique gas-phase ion/molecule reaction, the sensitivity and selectivity for quantitation of epoxides can be enhanced.

Recent advances in trace analysis of pharmaceutical genotoxic impurities.

Genotoxic impurities (GTIs) in pharmaceuticals at trace levels are of increasing concerns to both pharmaceutical industries and regulatory agencies due to their potentials for human carcinogenesis. Determination of these impurities at ppm levels requires highly sensitive analytical methodologies, which poses tremendous challenges on analytical communities in pharmaceutical R&D. Practical guidance with respect to the analytical determination of diverse classes of GTIs is currently lacking in the literature. This article provides an industrial perspective with regard to the analysis of various structural classes of GTIs that are commonly encountered during chemical development. The recent literatures will be reviewed, and several practical approaches for enhancing analyte detectability developed in recent years will be highlighted. As such, this article is organized into the following main sections: (1) trace analysis toolbox including sample introduction, separation, and detection techniques, as well as several 'general' approaches for enhancing detectability; (2) method development: chemical structure and property-based approaches; (3) method validation considerations; and (4) testing and control strategies in process chemistry. The general approaches for enhancing detection sensitivity to be discussed include chemical derivatization, 'matrix deactivation', and 'coordination ion spray-mass spectrometry'. Leveraging the use of these general approaches in method development greatly facilitates the analysis of poorly detectable or unstable/reactive GTIs. It is the authors' intent to provide a contemporary perspective on method development and validation that can guide analytical scientists in the pharmaceutical industries.

Enhancing the detection sensitivity of trace analysis of pharmaceutical genotoxic impurities by chemical derivatization and coordination ion spray-mass spectrometry.

Many pharmaceutical genotoxic impurities are neutral molecules. Trace level analysis of these neutral analytes is hampered by their poor ionization efficiency in mass spectrometry (MS). Two analytical approaches including chemical derivatization and coordination ion spray-MS were developed to enhance neutral analyte detection sensitivity. The chemical derivatization approach converts analytes into highly ionizable or permanently charged derivatives, which become readily detectable by MS. The coordination ion spray-MS method, on the other hand, improves ionization by forming neutral-ion adducts with metal ions such as Na(+), K(+), or NH(4)(+) which are introduced into the electrospray ionization source. Both approaches have been proven to be able to enhance the detection sensitivity of neutral pharmaceuticals dramatically. This article demonstrates the successful applications of the two approaches in the analysis of four pharmaceutical genotoxic impurities identified in a single drug development program, of which two are non-volatile alkyl chlorides and the other two are epoxides.

Matrix deactivation: A general approach to improve stability of unstable and reactive pharmaceutical genotoxic impurities for trace analysis.

Trace analysis of unstable and reactive pharmaceutical genotoxic impurities (GTIs) is a challenging task in pharmaceutical analysis. Many method issues such as insufficient sensitivity, poor precision, and unusual (too high/low) spiking recovery are often directly related to analytes' instability. We report herein a matrix deactivation approach that chemically stabilizes these analytes for analytical method development. In contrast to the conventional chemical derivatization approach where the analytes are transformed into stable detectable species, the matrix deactivation approach chemically deactivates the hypothetical reactive species in the sample matrix. The matrix deactivation approach was developed on the premise that the instability of certain analytes at trace level is caused by reactions between the analytes and low level reactive species in the sample matrix. Thus, quenching the reactivity of the reactive species would be a key to stabilizing the unstable and reactive analytes. For example, electrophilic alkylators could be destabilized by nucleophiles or bases through either nucleophilic substitution or elimination reactions. One way to mask those reactive species is via protonation by adding acids to the diluent. Alternatively, one can use nucleophile scavengers to deplete reactive unknown species in the sample matrix completely, in analogy to the use of antioxidants and metal chelators to prevent oxidation in the analysis of compounds liable to oxidation. This paper reports the application of the matrix deactivation to the analyses of unstable and reactive pharmaceutical genotoxic impurities. Some of the methods have been used to support development of manufacturing processes for drug substances and a recent regulatory filing.

Analytical control of process impurities in Pazopanib hydrochloride by impurity fate mapping.

Understanding the origin and fate of organic impurities within the manufacturing process along with a good control strategy is an integral part of the quality control of drug substance. Following the underlying principles of quality by design (QbD), a systematic approach to analytical control of process impurities by impurity fate mapping (IFM) has been developed and applied to the investigation and control of impurities in the manufacturing process of Pazopanib hydrochloride, an anticancer drug approved recently by the U.S. FDA. This approach requires an aggressive chemical and analytical search for potential impurities in the starting materials, intermediates and drug substance, and experimental studies to track their fate through the manufacturing process in order to understand the process capability for rejecting such impurities. Comprehensive IFM can provide elements of control strategies for impurities. This paper highlights the critical roles that analytical sciences play in the IFM process and impurity control. The application of various analytical techniques (HPLC, LC-MS, NMR, etc.) and development of sensitive and selective methods for impurity detection, identification, separation and quantification are highlighted with illustrative examples. As an essential part of the entire control strategy for Pazopanib hydrochloride, analytical control of impurities with 'meaningful' specifications and the 'right' analytical methods is addressed. In particular, IFM provides scientific justification that can allow for control of process impurities up-stream at the starting materials or intermediates whenever possible.

Analytical control of genotoxic impurities in the pazopanib hydrochloride manufacturing process.

Pharmaceutical regulatory agencies are increasingly concerned with trace-level genotoxic impurities in drug substances, requiring manufacturers to deliver innovative approaches for their analysis and control. The need to control most genotoxic impurities in the low ppm level relative to the active pharmaceutical ingredient (API), combined with the often reactive and labile nature of genotoxic impurities, poses significant analytical challenges. Therefore, sophisticated analytical methodologies are often developed to test and control genotoxic impurities in drug substances. From a quality-by-design perspective, product quality (genotoxic impurity levels in this case) should be built into the manufacturing process. This necessitates a practical analysis and control strategy derived on the premise of in-depth process understanding. General guidance on how to develop strategies for the analysis and control of genotoxic impurities is currently lacking in the pharmaceutical industry. In this work, we demonstrate practical examples for the analytical control of five genotoxic impurities in the manufacturing process of pazopanib hydrochloride, an anticancer drug currently in Phase III clinical development, which may serve as a model for the other products in development. Through detailed process understanding, we implemented an analysis and control strategy that enables the control of the five genotoxic impurities upstream in the manufacturing process at the starting materials or intermediates rather than at the final API. This allows the control limits to be set at percent levels rather than ppm levels, thereby simplifying the analytical testing and the analytical toolkits to be used in quality control laboratories.