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Technology transfer of a small volume continuous (SVC) process and Current Good Manufacturing Practices (cGMP) manufacturing of merestinib are described. A hybrid batch-SVC campaign was completed at a contract manufacturing organization under cGMP. The decision process by which unit operations were selected for implementation in flow for the cGMP campaign is discussed. The hybrid process comprised a Suzuki–Miyaura cross-coupling reaction, a nitro-group hydrogenolysis, a continuous amide bond formation, and a continuous deprotection. A continuous crystallization using two mixed suspension, mixed product removal (MSMPR) crystallizers and a filtration with in situ dissolution were employed for purification between the two SVC steps. Impurity levels were monitored using both online process analytical technology (PAT) and offline measurements. The continuous processing steps operated uninterrupted for 18 days to yield the drug substance in solution at a throughput of 12.5 kg/day. Crystallization in batch mode ...

Development of a small volume continuous process that used a combination of batch and flow unit operations to manufacture the small molecule oncolytic candidate merestinib is described. Continuous processing was enabled following the identification and development of suitable chemical transformations and unit operations. Aspects of the nascent process control strategy were evaluated in the context of a 20 kg laboratory demonstration campaign, executed in walk-in fume hoods at a throughput of 5–10 kg of active pharmaceutical ingredient per day. The process comprised an automated Suzuki–Miyaura cross-coupling reaction, a nitro-group hydrogenolysis, a continuous amide bond formation, and a continuous deprotection. Three of the four steps were purified using mixed-suspension, mixed-product removal crystallizations. Process analytical technology enabled real-time or nearly real-time process diagnostics. Findings from the demonstration campaign informed a second process development cycle as well as decision mak...

Continued emphasis in the pharmaceutical industry toward development of hybrid and continuous processes has led to a resurgence in process analytical technologies. The need to consistently and rapidly monitor the quality of material being made from these processes is a significant challenge that requires integrated, online analytics. Often the most challenging aspect of implementing real-time monitoring is ensuring that the analytical instrumentation is receiving a representative sample from the process. The Small Molecule Design and Development organization at Eli Lilly and Company has developed a highly adaptable process-to-analytics interface that has been broadly implemented across the development portfolio. The automated sample carts use only gravity and low-pressure nitrogen to obtain representative process samples, typically on the order of 0.3–6 mL. Subsequent sample preparation operations including quenching, derivatization, and most commonly dilution (10–250× is customary, but not restrictive) i...

A new synthesis of a key indazole-containing building block for the MET kinase inhibitor merestinib was designed and demonstrated. Crucial to the successful construction of the challenging indazole is an SNAr reaction, which forges the heterocyclic ring. Continuous processing was applied to two of the five steps: nitration of a benzaldehyde and high-temperature hydrolysis of an aniline to phenol. Compared to a highly developed historical route, the new route shows clear benefits in terms of product quality and potentially manufacturability and robustness.

An NH4Cl-catalyzed ethoxy ethyl deprotection was developed for the synthesis of merestinib, a MET inhibitor. Alternative reactor technologies using temperatures above the solvent boiling point are combined with this mild catalyst to promote the deprotection reaction. The reaction is optimized for flow and has been used to synthesize over 100 kg of the target compound. The generality of the reaction conditions is also demonstrated with other compounds and protecting groups.

The IQ Consortium reports on the current state of process analytical technology (PAT) for active pharmaceutical ingredient (API) manufacturing in branded pharmaceutical companies. The article describes the application of PAT in manufacturing and provides representative examples in four common pharmaceutical unit operations: reaction and workup, crystallization, drying, and milling.

Serotonin norepinephrine reuptake inhibitor (SNRI) pyrrolidinyl ether 2 was synthesized by employing a dynamic kinetic resolution (DKR) with enantio- and diastereoselective hydogenation on β-keto-γ-lactam 8 to afford β-hydroxy-γ-lactam 9 with 96% ee and 94% de. Reduction of 9 and purification via the dibenzoyl-(L)-tartaric acid diastereomeric salt 16 enriched the ee and de to 100%. While screening hydrogenation reaction systems with ruthenium-BINAP catalysts to prepare 9, it was found that adding catalytic HCl and LiCl enabled higher yields. In addition, the rate and equilibrium of the DKR-hydrogenation of 8 to give 9 was studied by online NMR and chiral HPLC, which indicated that one of the enantiomers of 8 was reducing faster to 9 than the equilibration of the stereocenter of 8.

Fused-Core particles have recently been introduced as an alternative to using sub-2-microm particles in chromatographic separations. Fused-Core particles are composed of a 1.7 microm solid core surrounded by a 0.5 microm porous silica layer (d(p) = 2.7 microm) to reduce mass transfer and increase peak efficiency. The performance of two commercially available Fused-Core particles (Advanced Materials Technology Halo C18 and Supelco Ascentis Express C18) was compared with sub-2-microm particles from Waters, Agilent, and Thermo Scientific. Although the peak efficiencies were only approximately 80% of those obtained by the Waters Acquity particles, the 50% lower backpressure allowed columns to be coupled in series to increase peak efficiency to 92,750 plates. The low backpressure and high efficiencies of the Fused-Core particles offer a viable alternative to using sub-2-microm particles and very-high-pressure LC instrumentation.

The evolution of chromatography has led to the reduction in the size of the packing materials used to fabricate HPLC columns. The increase in the backpressure required has led to this technique being referred to as ultrahigh-pressure liquid chromatography (UHPLC) when the column backpressure exceeds 10000 psi (approximately 700 bar). Until recently, columns packed with sub-2-microm materials have generally fitted into two classes; either short (less than 5 cm) columns designed for use on traditional HPLC systems at pressures less than 5000 psi (350 bar), or capillary columns (inner diameters less than 100 microm). By using packing materials with diameters

In the increasingly scrutinized pharmaceutical industry, regulatory agencies are demanding validation of any and all analytical instrumentation, including documentation associated with its implemen...

Four different column packing techniques were used to pack capillary columns for comparison in capillary electrochromatography (CEC); the techniques were slurry pressure packing, using supercritical CO2, electrokinetic packing, and packing with centripetal forces. Capillary columns of 50 μm ID were packed with each technique and evaluated under electrochromatographic conditions. An initial evaluation suggested that among the packing techniques, supercritical CO2 packing and packing with centripetal forces produced the most efficient packed capillaries. However, examining the various protocols in detail revealed that experience of the practitioner and familiarity with packing equipment are critical factors in column fabrication. Upon further investigation of the packing techniques and optimization of the column fabrication protocol, columns packed by the four packing techniques showed similar performance when operated under CEC conditions. Efficiencies of 270,000–300,000 plates/m were observed for 50 μm ID capillary columns packed with 3 μm packing material.

We have prepared a series of bovine serum albumins (BSA) that have been site-selectively labeled at cysteine-34 with one of four different sulfhydryl-selective boron dipyrromethene difluoride (BODIPY) fluorescent probes (BODIPY FL IA, BODIPY FL C1 IA, BODIPY 530/550 IA, and BODIPY 493/503 MB). We determine how the choice of extrinsic probe structure dictates the recovered BSA-BODIPY dynamics under thermal (10–80°C) and chemical (0–5M guanidine hydrochloride) denaturation conditions. The results of these experiments show that the global protein dynamics are sensed equally by each fluorescent probe; however, the probe itself influences the local probe dynamics within the cybotactic region that surrounds cysteine-34. Thus, it seems inappropriate to think of these extrinsic fluorescent probes as passive, nonparticipatory viewers of local protein dynamics. © 2001 John Wiley & Sons, Inc. Biopolymers 59: 502–511, 2001

Capillary electrochromatography (CEC) continues to captivate many separation scientists. A remarkable activity is apparent from the numerous publications in the literature using CEC. A review of the most recent progress in CEC is presented herein, covering an extensive fraction of the literature on CEC published from the year 1997 until the beginning of 2000. Most of the recent developments have concentrated on column technology.

Considering the current interest in capillary electrochromatography (CEC), performed in packed columns, we present the different methods used to pack capillary columns for use in CEC. General considerations on column packing are given and the column fabrication process is discussed in sufficient detail to allow instruction to those who are not experienced in the field. Five different packing methods are discussed to deliver packing material into the capillary column from a practical view point: slurry pressure packing, packing with supercritical CO2, electrokinetic packing, using centripetal forces, and packing by gravity. Entrapment of particulate material by sintering and sol-gel technology is also mentioned. Although slurry pressure packing procedures are most common, higher separation efficiencies are obtained using other packing approaches. Electrokinetic packing seems to be the simplest technique to deliver the packing material into the capillary columns. Nevertheless, as with the other packing techniques, skill and experience are required to complete all the steps involved in the fabrication of packed columns for CEC.

Capillary columns have been packed for capillary electrochromatography (CEC) using centripetal forces. The packed columns were maintained under wet conditions or they were dried with nitrogen gas prior to forming the retaining frits. Upon fabrication of the retaining frits, the dried columns were resolvated with the mobile phase. The performance of the columns was evaluated to determine the effect of the drying step during the packing procedure. The columns submitted to the drying step showed improved separation efficiencies and stronger retention characteristics than those kept under wet conditions. The drying step allows the silica-based packing material to be better accommodated inside the capillary column. Upon solvation, the packing material "swells," resulting in a greater packing density, which allows for a stronger retention and improved separation efficiencies. The drying step led to a 13% increase in retention on columns packed with isopropanol. An increase of 15-20% in theoretical plates for the most retained compounds was also observed in such columns.

A strategy for developing chromatographic methods designed to determine impurities and degradation products in active pharmaceutical ingredients and drug products is presented. Selectivity is achieved by evaluating a chromatographic space comprised of 12 stationary/mobile phase combinations. Stationary phases predicted to be orthogonal based on their hydrophobic subtraction model parameters used. The particle sizes, column dimensions, and gradient times chosen provide high peak capacities and allow operation at backpressures that can be achieved with standard instrumentation. The mobile phases utilized are compatible with MS detection and cover a wide range of pH, solvent strength, and solvent selectivity. Analyte detection is accomplished using a combination of diode array and mass spectroscopic detectors which allow mixtures of project compounds to be injected and selectively detected. Automation of data acquisition and processing is accomplished using AutoChrom software from ACD\Labs. The strategy is illustrated with detailed data from two case studies and summary data from nineteen pharmaceutical projects.

Chromatography using sub-2 microm particles is becoming increasingly popular due to the potential for increased speed, resolution, sensitivity, and peak capacity. To meet the demand, various vendors have re-engineered traditional LC systems to operate at pressures of up to 15000 psi to accommodate the elevated backpressures associated with using sub-2 microm particles. This report investigates and compares the performance of three very high pressure LC (VHPLC) systems: Waters Acquity, Agilent 1200 SL, and Thermo Accela. Specifications for the pump, autosampler, column compartment, detector, and software for each instrument are presented. To assess the chromatographic performance of the three instruments, method development and validation were performed for three pharmaceutical compounds and the results are compared and discussed. The material presented herein serves to highlight the different features of the VHPLC instruments, and assess their suitability for the analysis of pharmaceutical compounds.

The increased interest in HPLC at elevated pressures, beyond the conventional 6000 psi (400 bar), has created a demand for injection systems capable of withstanding pressures beyond the 20,000 psi (1380 bar). To achieve high-resolution separations, an appropriate length of columns packed with sub 2-microm packing materials, a 30,000-40,000 psi (2070-2760 bar) pressure range is desirable. A new air-actuated needle valve injection system rated to withstand pressures of up to 40,000 psi (2760 bar) has been evaluated. Under isocratic chromatographic conditions, injecting 200 nL and operated at approximately 20,000 psi (1380 bar), the system showed a peak area reproducibility of approximately 2.5% RSD, contrasting the 5% RSD of a pressured-balanced injection system operated under similar conditions. Programmed for partial loop injections using injection times of 300-700 ms (injection volumes in the range of 1-2.5 microL) and operated at pressures close to 30,000 psi (2070 bar), the reproducibility in peak area for the amounts injected was approximately 1.5% RSD or lower, while an injection time of 100 ms resulted in a reproducibility of 3-4% RSD. The new injection system did not show any significant carryover, and after thousands of injections, the system has not shown sign of wear, loss of pressure during injection, or loss in chromatographic performance.

A key step towards a highly-selective antagonist of ionotropic glutamate receptors entails the diastereoselective arene hydrogenation of an enantiopure tetrahydroisoquinoline. An extensive screen using parallel reactors was conducted and led to the discovery of several Pd/C catalysts giving high yield and improved diastereoselectivity from 75 : 25 to 95 : 5. A detailed kinetic study of the best system was performed and supports the reduction occuring in two-steps.