Disparities of Single-Particle Growth Rates in Buried Versus Exposed Ritonavir Crystals within Amorphous Solid Dispersions.

Seeded growth rates of ritonavir in copovidone at 75% relative humidity (RH) and 50 °C were evaluated by single-particle tracking second harmonic generation (SHG) microscopy and found to be ∼3-fold slower for crystallites at the surface compared to the bulk. The shelf lives of final dosage forms containing amorphous solid dispersions (ASDs) are often dictated by the rates of active pharmaceutical ingredient crystallization. Upon exposure to elevated RH, the higher anticipated water content near the surfaces of ASDs has the potential to substantially impact nucleation and growth kinetics relative to the bulk. However, quantitative assessment of these differences in growth rates is complicated by challenges associated with discrimination of the two contributions (supersaturation and molecular mobility) in ensemble-averaged measurements. In the present study, "sandwich" materials were prepared, in which sparse populations of ritonavir single-crystalline seeds were pressed between two similar ASD films to assess bulk crystallization rates. These sandwich materials were compared and contrasted with analogously prepared "open-faced" samples, without the capping film, to assess the surface crystallization rates. Single-particle analysis by SHG microscopy time-series during in situ crystallization produced average growth rates of 3.8 µm/h for bulk columnar crystals with a particle-to-particle standard deviation of 0.9 µm/h. In addition, columnar crystal growth rates for surface particles were measured to be 1.3 µm/h and radiating crystal growth rates for surface particles were measured to be 1.0 µm/h, both with a particle-to-particle deviation of 0.4 µm/h. The observed appearance of radiating crystals upon surface seeding is attributed to reduced ritonavir solubility upon water adsorption at the interface, leading to higher defect densities in crystal growth. Despite substantial differences in crystal habit, correction of the surface growth rates by a factor of 4 from geometric effects resulted in relatively minor but statistically significant differences in the growth kinetics for the two local environments. These results are consistent, with viscosity being a relatively weak function of water absorption coupled with primarily diffusion-limited growth kinetics.

In Situ Crystal Growth Rate Distributions of Active Pharmaceutical Ingredients.

Single-particle tracking of crystal growth performed in situ enables substantial improvements in the signal-to-noise ratio (SNR) for recovered crystal nucleation and growth rates by nonlinear optical microscopy. Second harmonic generation (SHG) is exquisitely sensitive to noncentrosymmetric crystals, including those produced by many homochiral active pharmaceutical ingredients (APIs). Accelerated stability testing at elevated temperatures and relative humidity informs design of pharmaceutical formulations. In the present work, we demonstrate reduction in the Poisson noise associated with the finite number of particles present in a given field of view through continuous monitoring during stability testing. Single-particle tracking enables recovery of crystal growth rates of individual crystallites and enables unambiguous direct detection of nucleation events. Collectively, these capabilities provide significant improvements in the signal-to-noise for nucleation and crystal growth measurements, corresponding to approximately an order of magnitude reduction in anticipated measurement time for recovery of kinetics parameters.

Kinetic Modeling of Accelerated Stability Testing Enabled by Second Harmonic Generation Microscopy.

The low limits of detection afforded by second harmonic generation (SHG) microscopy coupled with image analysis algorithms enabled quantitative modeling of the temperature-dependent crystallization of active pharmaceutical ingredients (APIs) within amorphous solid dispersions (ASDs). ASDs, in which an API is maintained in an amorphous state within a polymer matrix, are finding increasing use to address solubility limitations of small-molecule APIs. Extensive stability testing is typically performed for ASD characterization, the time frame for which is often dictated by the earliest detectable onset of crystal formation. Here a study of accelerated stability testing on ritonavir, a human immunodeficiency virus (HIV) protease inhibitor, has been conducted. Under the condition for accelerated stability testing at 50 °C/75%RH and 40 °C/75%RH, ritonavir crystallization kinetics from amorphous solid dispersions were monitored by SHG microscopy. SHG microscopy coupled by image analysis yielded limits of detection for ritonavir crystals as low as 10 ppm, which is about 2 orders of magnitude lower than other methods currently available for crystallinity detection in ASDs. The four decade dynamic range of SHG microscopy enabled quantitative modeling with an established (JMAK) kinetic model. From the SHG images, nucleation and crystal growth rates were independently determined.

Solid phase assembly of defined protein conjugates.

We have developed a solid-phase procedure for protein-protein conjugation that gives greater control over product size and composition than previous methods. Conjugates are assembled by sequential addition of activated proteins to the support under conditions suitable for maintaining the activity of the proteins. The total number of conjugate units to be prepared is fixed in the first step by the quantity of the first protein absorbed by the support. In each following step, the added protein links only to previously bound protein. The final conjugate is released to solution by cleaving the linker holding the first protein to the support. This stepwise assembly provides uniformly sized conjugates of the desired size and composition with placement of components at the desired positions within the structure. Using this approach, we have prepared a series of conjugates containing R-phycoerythrin as the central protein, with varying quantities of alkaline phosphatase and IgG with expected molecular masses ranging from 1.6 to 11.5 MDa. Size-exclusion chromatography and atomic force microscopy demonstrate homogeneity and control of the conjugate size. In an immunoassay for human thyroid stimulating hormone, the conjugates show signals consistent with their compositions.