Automated High-Throughput Matrix Assisted Laser Desorption Ionization Mass Spectrometry Methodology for Formulation Assessment of Polyethylene-Glycol-Conjugated Cytokine Proteins.

Biological therapeutics are major contributors to the pharmaceutical pipeline and continue to grow in sales and scope. Additionally, the field's understanding of cancer biology has advanced such that biopharmaceuticals can harness the power of the immune system for oncology treatments. Several of these novel therapeutics are engineered versions of naturally occurring proteins designed to improve therapeutic properties including potency, target engagement and half-life extension. Cytokines, such as interferons and interleukins, are a broad class of signaling proteins which modulate the body's immune response; engineered cytokines have entered the clinic as promising new immuno-oncology therapies. While these therapies hold great promise, their additional structural complexity introduces analytical challenges, and traditional analytical platforms may be ill-suited to effectively assess product development risks. Further, the pharmaceutical industry relies on streamlining approaches for high-throughput experimentation to achieve speed and efficiency for the discovery and development of new modalities. These demands necessitate the use of state-of-the-art techniques to rapidly characterize these new modalities and guide process development and optimization. Matrix Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) is a rapid, sensitive and automatable technique amenable for high-throughput analysis of proteins. In this work, we have developed an automated MALDI-MS platform to prepare, acquire and analyze molecular degradation in engineered PEGylated cytokines formulation samples. This orthogonal technique integrated seamlessly with current developability risk assessment workflows, ultimately enabling selection of a final formulation strategy for clinical development.

The Absolute Bioavailability and Absorption, Metabolism, and Excretion of Ipatasertib, a Potent and Highly Selective Protein Kinase B (Akt) Inhibitor.

Ipatasertib (GDC-0068) is a potent, highly selective, small-molecule inhibitor of protein kinase B (Akt) being developed by Genentech/Roche as a single agent and in combination with other therapies for the treatment of cancers. To fully understand the absorption, metabolism, and excretion of ipatasertib in humans, an open-label study using 14C-radiolabeled ipatasertib was completed to characterize the absolute bioavailability (period 1) and mass balance and metabolite profiling (period 2). In period 1, subjects were administered a 200 mg oral dose of ipatasertib followed by an 80 µg (800 nCi) intravenous dose of [14C]-ipatasertib. In period 2, subjects received a single oral dose containing approximately 200 mg (100 µCi) [14C]-ipatasertib. In an integrated analytical strategy, accelerator mass spectrometry was applied to measure the 14C microtracer intravenous pharmacokinetics in period 1 and fully profile plasma radioactivity in period 2. The systemic plasma clearance and steady-state volume of distribution were 98.8 L/h and 2530 L, respectively. The terminal half-lives after oral and intravenous administrations were similar (26.7 and 27.4 hours, respectively) and absolute bioavailability of ipatasertib was 34.0%. After a single oral dose of [14C]-ipatasertib, 88.3% of the administered radioactivity was recovered with approximately 69.0% and 19.3% in feces and urine, respectively. Radioactivity in feces and urine was predominantly metabolites with 24.4% and 8.26% of dose as unchanged parent, respectively; indicating that ipatasertib had been extensively absorbed and hepatic metabolism was the major route of clearance. The major metabolic pathway was N-dealkylation mediated by CYP3A, and minor pathways were oxidative by cytochromes P450 and aldehyde oxidase. SIGNIFICANCE STATEMENT: The study provided definitive information regarding the absolute bioavailability and the absorption, metabolism, and excretion pathways of ipatasertib, a potent, novel, and highly selective small-molecule inhibitor of protein kinase B (Akt). An ultrasensitive radioactive counting method, accelerator mass spectrometry was successfully applied for 14C-microtracer absolute bioavailability determination and plasma metabolite profiling.

Phosphoramidates as Steering Elements for Highly Selective Access to Complementary Imidazo[1,2-a]pyrimidine Isomers.

We report that selective N-phosphorylation of aminoimidazoles results in a key steering element that controls isomeric selectivity in the condensation of ß-ethoxy acrylamides and aminoimidazoles to furnish imidazo[1,2-a]pyrimidines. We identified conditions that provide highly selective (99:1) phosphorylation at the endo- or exocyclic nitrogen. Either the 2-amino or 4-amino isomer of the (benzo)imidazo[1,2-a]pyrimidine products could be isolated in 64-95% yield. Mass spectrometric analysis and computational studies give insight into the mechanism of this exceptionally selective transformation.

Using Supercritical Fluid Technology as a Green Alternative During the Preparation of Drug Delivery Systems.

Micro- and nano-carrier formulations have been developed as drug delivery systems for active pharmaceutical ingredients (APIs) that suffer from poor physico-chemical, pharmacokinetic, and pharmacodynamic properties. Encapsulating the APIs in such systems can help improve their stability by protecting them from harsh conditions such as light, oxygen, temperature, pH, enzymes, and others. Consequently, the API's dissolution rate and bioavailability are tremendously improved. Conventional techniques used in the production of these drug carrier formulations have several drawbacks, including thermal and chemical stability of the APIs, excessive use of organic solvents, high residual solvent levels, difficult particle size control and distributions, drug loading-related challenges, and time and energy consumption. This review illustrates how supercritical fluid (SCF) technologies can be superior in controlling the morphology of API particles and in the production of drug carriers due to SCF's non-toxic, inert, economical, and environmentally friendly properties. The SCF's advantages, benefits, and various preparation methods are discussed. Drug carrier formulations discussed in this review include microparticles, nanoparticles, polymeric membranes, aerogels, microporous foams, solid lipid nanoparticles, and liposomes.

Identification of an acetonitrile addition impurity formed during peptide disulfide bond reduction using dithiothreitol and Tris(2-carboxyethyl)phosphine.

Identification and localization of modifications in peptides containing multiple disulfide bonds is challenging due to inefficient fragmentation in mass spectrometry (MS) analysis. In cases where MS fragmentation techniques such as electron capture dissociation (ECD), electron transfer dissociation (ETD), and ultraviolet photodissociation (UVPD) fail to achieve efficient fragmentation, off-line disulfide bond reduction techniques are typically employed prior to MS analysis. Some commonly used reducing agents include dithiothreitol (DTT) and tris(2-carboxyethyl)phosphine (TCEP). In this work, we describe the detection and identification of an unexpected impurity that formed during the reduction of Peptide A, containing multiple disulfide bonds, while using DTT or TCEP as reducing agents and acetonitrile as a co-solvent. The DTT reduced products were found to be a mixture of the expected linear Peptide A (fully reduced) and an unknown product (>50%) with a mass corresponding to linear Peptide A plus 41 Da ([reduced-M + 41]). A series of experiments were subsequently performed to investigate the identity and origin of this impurity. Disulfide bond reduction with DTT was performed in aqueous mixtures containing acetonitrile, methanol, and deuterated acetonitrile; and with TCEP in aqueous mixtures containing acetonitrile. Additionally, glycine amino acid was used as a surrogate to investigate the mechanism. The liquid chromatography-mass spectrometry (LCMSMS) results demonstrated that the [reduced-M + 41] impurity was an acetonitrile addition on the peptide's N-terminal glycine. The corresponding impurity [M + 41] was also found in the native Peptide A (non-reduced), suggesting that small amounts of this impurity may also be generated during the synthesis in the upstream process steps. By understanding the formation of this process-related impurity [M + 41], one could potentially reduce or eliminate its presence in Peptide A through chemical controls. Finally, this observation provides caution against using acetonitrile as a co-solvent during DTT- or TCEP-promoted reduction of peptides with an uncapped N-terminus primary amine.

Optimization of a two-dimensional liquid chromatography-supercritical fluid chromatography-mass spectrometry (2D-LC-SFS-MS) system to assess "in-vivo" inter-conversion of chiral drug molecules.

Enantioselective analysis is an essential requirement during the pharmaceutical development of chiral drug molecules. In pre-clinical and clinical studies, the Food and Drug Administration (FDA) mandates the assessment of "in vivo" inter-conversion of chiral drugs to determine their physiological effects. In-vivo analysis of the active pharmaceutical ingredient (API) and its potential metabolites could be quite challenging due to their low abundance (ng/mL levels) and matrix interferences. Therefore, highly selective and sensitive analytical techniques are required to separate the API and its metabolites from the matrix components and one another. Additionally, for chiral APIs, further analytical separation is required to resolve the API and its potential metabolites from their corresponding enantiomers. In this work, we demonstrate the optimization of our previously designed two-dimensional liquid chromatography-supercritical fluid chromatography-mass spectrometry (2D-LC-SFC -MS) system to achieve 10 ng/mL detection limit [1]. The first LC dimension, used as a desalting step, could efficiently separate the API from its potential metabolites and matrix components. The API and its metabolites were then trapped/focused on small trapping columns and transferred onto the second SFC dimension for chiral separation. Detection can be achieved by ultra-violet (UV) or MS detection. Different system parameters such as column dimensions, transfer volumes, trapping column stationary phase, system tubing internal diameter (i.d.), and detection techniques, were optimized to enhance the sensitivity of the 2D-LC-SFC-MS system. The limit of detection was determined to be 10 ng/mL. An application is described where a mouse hepatocyte treated sample was analyzed using the optimized 2D-LC-SFC-MS system with successful assessment of the ratio of API to its metabolite (1D-LC), as well as the corresponding enantiomeric excess values (% e.e.) of each (2D-SFC).

High throughput screening of active pharmaceutical ingredients by UPLC.

Ultra performance LC (UPLC) was evaluated as an efficient screening approach to facilitate method development for drug candidates. Three stationary phases were screened: C-18, phenyl, and Shield RP 18 with column dimensions of 150 mm x 2.1 mm, 1.7 microm, which should theoretically generate 35,000 plates or 175% of the typical column plate count of a conventional 250 mm x 4.6 mm, 5 microm particle column. Thirteen different active pharmaceutical ingredients (APIs) were screened using this column set with a standardized mobile-phase gradient. The UPLC method selectivity results were compared to those obtained for these compounds via methods developed through laborious trial and error screening experiments using numerous conventional HPLC mobile and stationary phases. Peak capacity was compared for columns packed with 5 microm particles and columns packed with 1.7 microm particles. The impurities screened by UPLC were confirmed by LC/MS. The results demonstrate that simple, high efficiency UPLC gradients are a feasible and productive alternative to more conventional multiparametric chromatographic screening approaches for many compounds in the early stages of drug development.

Peptide separations by slab gel electrophoresis in pluronic F127 polymer liquid crystals.

Proteomics and peptidomics could benefit from simple methods for high-resolution separation of oligopeptides analogous to slab gel electrophoresis of proteins. Gels of Pluronic F127 copolymer surfactant were investigated as media for slab gel electrophoresis of oligopeptides using a trypsin digest of myoglobin. Concentrated solutions of Pluronic F127 are fluid at low temperatures (