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Antibody-drug conjugates (ADCs) are an emerging class of biopharmaceuticals. As such, there are no specific guidelines addressing impurity limits and qualification requirements. The current ICH guidelines on impurities, Q3A (Impurities in New Drug Substances), Q3B (Impurities in New Drug Products), and Q6B (Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products) do not adequately address how to assess small molecule impurities in ADCs. The International Consortium for Innovation and Quality in Pharmaceutical Development (IQ) formed an impurities working group (IWG) to discuss this issue. This white paper presents a strategy for evaluating the impact of small molecule impurities in ADCs. This strategy suggests a science-based approach that can be applied to the design of control systems for ADC therapeutics. The key principles that form the basis for this strategy include the significant difference in molecular weights between small molecule impurities and the ADC, the conjugation potential of the small molecule impurities, and the typical dosing concentrations and dosing schedule. The result is that exposure to small impurities in ADCs is so low as to often pose little or no significant safety risk.

IPI-504 (retaspimycin hydrochloride) is an Hsp90 inhibitor that is the subject of multiple clinical trials for the treatment of cancer. IPI-504 is an aqueous soluble (>200 mg/ml) hydroquinone hydrochloride salt of 17-(allylamino)-17-demethoxygeldanamycin (17-AAG), a quinone derivative also undergoing clinical evaluation, albeit with suboptimal formulations that address its inferior aqueous solubility (∼50 µg/ml). IPI-504 interconverts with 17-AAG in vivo through oxidation-reduction reactions that result in a dynamic redox equilibrium. The development challenges associated with redox active molecules are significant due to the pH, oxygen, and temperature sensitivities associated with such chemotypes. The API and sterile drug product manufacturing processes thus warrant the monitoring and control of these key variables. Furthermore, the pharmaceutical development challenges associated with cancer agents that are often fast-tracked due to unmet medical needs mandate a rapid development cycle with associated regulatory hurdles. Drug Dev Res 71: 429–438, 2010. © 2010 Wiley-Liss, Inc.

With increasing frequency, new drug candidates being introduced into pharmaceutical drug pipelines are chiral. Often only one enantiomer exhibits the desired biological activity and the other enantiomer may exhibit undesired side effects, thereby making chiral purity an important parameter. The introduction of chiral analysis adds additional complications in drug development. The pharmaceutical industry is constantly striving to streamline processes and improve efficiencies in an effort to move molecules to market quickly. In order to simplify the process of chiral method development, chiral screening can be set up, however a successful chiral screen depends on optimizing two factors: the column and the detector. The following work investigated the second factor and evaluated two types of commercially available chiral detectors for their possible use in chiral method development and screening: polarimeters and circular dichroism (CD) detectors. Linearity, precision, and the limit of detection (LD) of six compounds (trans-stilbene oxide, ethyl chrysanthemate, propranolol, 1-methyl-2-tetralone, naproxen, methyl methionine) on four commercial detectors (three polarimeters and one CD detector) were determined experimentally and the limit of quantitation (LQ) calculated from the experimental LD. Trans-stilbene oxide worked well across all the detectors, showing good linearity, precision and low detection limits. However, the other five compounds proved to be more discriminating and showed that the circular dichroism detector performed better as a detector for chiral screens, over the polarimeters.

Modern liquid chromatographic (LC) analyses of targets in complex matrices, even with mass spectroscopic (MS) detection, more commonly are also requiring an orthogonal method to ensure peak purity and resolution of all sample impurities. This is especially true when stereoisomer identity and separation are critical criteria. To help address this gap in orthogonality, a novel coated cellulose carbamate stationary phase was developed using a reoptimized coating density, a type B 500-angstrom silica as its base entity, and a secondary amine to facilitate the coating to the base silica. The chiral phase can be used in reverse phase chromatography but is designed to work with mixed polar eluents in both polar organic and normal phase chromatography. It is also selective for achiral applications as well. A critical difference is the stability of this cellulose carbamate stationary phase at higher alcohol concentrations and the prediction capability of the elution order of analytes. This stationary phase showed greater chromatographic selectivity by requiring less alcohol modifier for elution of chiral compounds, when compared to other cellulose carbamate columns and by the use of acetonitrile (ACN) as a modifier. The Cogent EE phase is also rugged and can easily switch between different pH's without loss of resolution or memory effects. Finally this phase was used to separate optical isomers of gossypol and tramadol, and worked well in a case study where diastereomers were isolated from a previously unknown impurity.

Prior to the introduction of thalidomide, approved drugs could be racemic, but experience from that drug has now set new standards for purity assays. Not only must the pharmaceutical analytical chemist be able to separate the active pharmaceutical ingredient (API) from its process impurities and degradation products, but also they must demonstrate enantiomeric purity. This chapter focuses on chiral method development in the pharmaceutical industry. Starting with the historical and regulatory implications of chiral analysis, this chapter proceeds to demonstrate the types of analyses that are currently in use, as well as the columns and phases that are commercially available. Current method development strategies and the hardware that can aid in method development are discussed. The uses and conditions of three different chromatographic techniques, HPLC, SFC, and GC, are discussed.

The structural domains of the Escherichia coli CheA protein resemble 'beads on a string', since the N-terminal phosphate-accepting (P) domain is joined to the CheY/CheB-binding (B) domain through a flexible linker, and the B domain is in turn joined to the C-terminal dimerization/catalytic/regulatory domains by a second intervening linker. Dimerization occurs primarily via interactions between two dimerization domains, which is required for CheA trans-autophosphorylation. In this study, sedimentation equilibrium was used to demonstrate significant subunit interactions at secondary sites in the two naturally occurring (full-length and short) forms of CheA (CheA(1-654) or CheA(L), and CheA(98-654) or CheA(S)) by contrasting the dimerization of CheA(L) and CheA(S) to CheA(T), an engineered form that lacked the P domain entirely. The estimated dimer dissociation constant (K(1,2)) for CheA(T) (3.1 microM) was weaker than K(1,2) for CheA(L) (0.49 microM), which was attributed to the P domain-catalytic domain interactions that were present in CheA(L) but not CheA(T). In contrast, CheA(S) dimerization was unexpectedly stronger (K(1,2) approximately 20 nM), which arose through interactions between two P domain remnants in the CheA(S) dimer. This conclusion was supported by the results of sedimentation equilibrium experiments conducted with P domains and P domain remnants expressed in the absence of the dimerization/catalytic/regulatory domains. The P domain remnant had a measurable tendency to self-associate; the full-length P domain did not. Hydrophobic forces probably drive this interaction, since hydrophobic amino acids buried in the intact P domain are solvent-exposed in CheA(S). Also, the nascent N-terminus of CheA(S) bound to the phosphatase (CheZ) more effectively, a conclusion based on the demonstrably greater ability of the P domain remnant to co-sediment CheZ, compared to the intact P domain.