Paul Schmitt

A low-cost second harmonic generation (SHG) microscope was constructed, and, for the first time, SHG microscopy was used for imaging agrochemical materials directly on the surface of common commercial crop leaves. The microscope uses a chromatically fixed (1560 nm) femtosecond fiber laser, a commercial 2D galvanometer mirror system, and a PCIe digital oscilloscope card, which together kept total instrument costs under $40 000 (USD), a significant decrease in cost and complexity from common systems (commercial and home-built) using tunable lasers and faster beam-scanning architectures. The figures of merit of the low-cost system still enabled a variety of measurements of agrochemical materials. Following confirmation of largely background-free SHG imaging of common crop leaves (soybean, maize, wheatgrass), SHG microscopy was used to image active ingredient crystallization after solution-phase deposition directly on the leaf surface, including at industrially relevant active ingredient concentrations (<0.05% w/w). Crystallization was also followed in real-time, with differences in crystallization time observed for different application procedures (spraying vs single droplet deposition). A strong dependency of active ingredient crystallization on the substrate was found, with an increased crystallization tendency observed on leaves vs on glass slides. Different crystal habits for the same active ingredient were also observed on different plant species. Finally, a model extended-release formulation was prepared, with a decrease in active ingredient crystallinity observed vs solution-phase deposition. These collective results demonstrate the need for making diagnostic measurements directly on the leaf surface and could help inform the next generation of pesticide products that ensure optimized agricultural output for a growing world population.

A statistical model enables auto-calibration of second harmonic generation (SHG) images for quantifying trace crystallinity within amorphous solid dispersions (ASDs) over a wide dynamic range of crystallinity. In this paper, we demonstrate particle-counting approaches for quantifying trace crystallinity, combined with analytical expressions correcting for particle overlap bias in higher crystallinity regimes to extend the continuous dynamic range of standard particle-counting algorithms through to the signal averaging regime. The reliability of the values recovered by these expressions was demonstrated with simulated data as well as experimental data obtained for an amorphous solid dispersion formulation containing evacetrapib, an Eli Lilly and Company compound. Since particle counting independently recovers the crystalline volume and the SHG intensity, the average SHG intensity per unit volume can be used as an internal calibrant for quantifying crystallinity at higher volume fractions, for which particle counting is no longer applicable.

•Second harmonic generation (SHG) microscopy was applied for sensitive detection of crystallinity in spray dried particles.•SHG detected crystallinity at an earlier time point than X-ray powder diffraction for two spray dried sytems.•The SHG technique presented herein can be potentially used to rapidly evaluate the stability of different spray dried formulations to crystallization. Various techniques have been used to detect crystallization in amorphous solid dispersions (ASD). However, most of these techniques do not enable the detection of very low levels of crystallinity (<1%). The aim of the current study was to compare the sensitivity of second harmonic generation (SHG) microscopy with powder X-ray diffraction (XRPD) in detecting the presence of crystals in low drug loading amorphous solid dispersions. Amorphous solid dispersions of the poorly water soluble compounds, flutamide (FTM, 15wt.% drug loading) and ezetimibe (EZT, 30wt.% drug loading) with hydroxypropyl methylcellulose acetate succinate (HPMCAS) were prepared by spray drying. To induce crystallization, samples were subsequently stored at 75% or 82% relative humidity (RH) and 40°C. Crystallization was monitored by XRPD and by SHG microscopy. Solid state nuclear magnetic resonance spectroscopy (ssNMR) was used to further investigate crystallinity in selected samples. For flutamide, crystals were detected by SHG microscopy after 8days of storage at 40°C/82% RH, whereas no evidence of crystallinity could be observed by XRPD until 26days. Correspondingly, for FTM samples stored at 40°C/75% RH, crystals were detected after 11days by SHG microscopy and after 53days by XRPD. The evolution of crystals, that is an increase in the number and size of crystalline regions, with time could be readily monitored from the SHG images, and revealed the formation of needle-shaped crystals. Further investigation with scanning electron microscopy indicated an unexpected mechanism of crystallization, whereby flutamide crystals grew as needle-shaped projections from the surface of the spray dried particles. Similarly, EZT crystals could be detected at earlier time points (15days) with SHG microscopy relative to with XRPD (60days). Thus, SHG microscopy was found to be a highly sensitive method for detecting and monitoring the evolution of crystals formed from spray dried particles, providing much earlier detection of crystallinity than XRPD under comparable run times.

The past decade has seen an increase in the use of nonlinear optical (NLO) techniques such as second harmonic generation, coherent antistokes Raman scattering, stimulated Raman scattering, and two-photon fluorescence for the solid-state characterization of pharmaceutical materials. These combined techniques offer several advantages (e.g., speed, selectivity, quantitation) of potential interest to the pharmaceutical community, as decreased characterization times in formulation development and testing could help decrease the time required to bring new, higher quality drugs to market. The large body of literature recently published in this field merits a review. Literature will be discussed in order of drug development, starting with applications in initial therapeutic molecule crystallization and polymorphic analysis, followed by final dosage form characterization, and ending with drug product performance testing.

The use of nonlinear optical Stokes ellipsometric (NOSE) microscopy for rapid discrimination of two polymorphic forms of the small molecule D-mannitol is presented. Fast (8 MHz) polarization modulated beam-scanning microscopy and a recently developed iterative, nonlinear least-squares fitting algorithm were combined to allow discrimination of orthorhombic and monoclinic crystal structures of D-mannitol with data acquisition times of <7 s per field of view with a signal-to-noise ratio (SNR) of similar to 300. Discrimination between polymorphic forms within the 99.99% confidence interval was achieved by standard statistical tests of the recovered probability density functions for the measured observables following two class linear discriminant analysis. These measurements target bottlenecks in small-volume, high-throughput solid form screening experiments for polymorph discovery in the development of emerging active pharmaceutical ingredients.

Second harmonic generation (SHG) microscopy was used to rapidly identify regions of interest for localized confocal Raman spectroscopy measurements in order to quantify crystallinity within lyophilized Abraxane powder (protein bound paclitaxel for injectable suspension). Water insoluble noncentrosymmetric crystalline particles ranging from ∼1 to 120 μm were identified by SHG, with wide variability in crystal size and frequency observed between several batches of Abraxane. By targeting the Raman analysis to these localized regions identified by SHG, the required measurement time was decreased over 2 orders of magnitude, from 8 h to 2 s. Experimental Raman spectra of SHG active domains in Abraxane were in good agreement with experimental spectra of pure crystalline paclitaxel. These collective results are consistent with up to 30% of the active ingredient being present as poorly soluble crystalline particulates in some batches of Abraxane.

A model for predicting the anticipated second harmonic generation (SHG) activity of crystalline active pharmaceutical ingredients (APIs) was developed based on combining time-dependent Hartree-Fock calculations of the molecular building block with analytical theory for the predicted intensity from the lattice. Predicted trends agreed well overall with experimental measurements for 18 representative APIs. Inspection of the SHG images suggests that outliers from the predicted trends likely arose from the presence of trace SHG-active metastable crystal forms not considered in the predictions. The success of this relatively simple computational approach suggests a route for reliably predicting the anticipated SHG activities of API target molecules, which in turn can serve to indicate their potential compatibility with analysis by SHG.

Fast 8 MHz polarization modulation coupled with analytical modeling, fast beam-scanning, and synchronous digitization (SD) have enabled simultaneous nonlinear optical Stokes ellipsometry (NOSE) and polarized laser transmittance imaging with image acquisition rates up to video rate. In contrast to polarimetry, in which the polarization state of the exiting beam is recorded, NOSE enables recovery of the complex-valued Jones tensor of the sample that describes all polarization-dependent observables of the measurement. Every video-rate scan produces a set of 30 images (10 for each detector with three detectors operating in parallel), each of which corresponds to a different polarization-dependent result. Linear fitting of this image set contracts it down to a set of five parameters for each detector in second harmonic generation (SHG) and three parameters for the transmittance of the incident beam. These parameters can in turn be used to recover the Jones tensor elements of the sample. Following validation of the approach using z-cut quartz, NOSE microscopy was performed for microcrystals of both naproxen and glucose isomerase. When weighted by the measurement time, NOSE microscopy was found to provide a substantial (>7 decades) improvement in the signal-to-noise ratio relative to our previous measurements based on the rotation of optical elements and a 3-fold improvement relative to previous single-point NOSE approaches.