Publications

@article{noKey,

title = {Small-Angle X‑ray Scattering as a Powerful Tool for Phase and Crystallinity Assessment of Monoclonal Antibody Crystallites in Support of Batch Crystallization},

author = {Larpent, Patrick},

url = {https://pubs.acs.org/doi/abs/10.1021/acs.molpharmaceut.4c00418},

doi = {https://doi.org/10.1021/acs.molpharmaceut.4c00418},

year  = {2024},

date = {2024-07-03},

abstract = {Crystalline suspensions of monoclonal antibodies (mAbs) have great potential to improve drug substance isolation

and purification on a large scale and to be used for drug delivery via high-concentration formulations. Crystalline mAb suspensions

are expected to have enhanced chemical and physical properties relative to mAb solutions delivered intravenously, making them

attractive candidates for subcutaneous delivery. In contrast to small molecules, the development of protein crystalline suspensions is

not a widely used approach in the pharmaceutical industry. This is mainly due to the challenges in finding crystalline hits and the

suboptimal physical properties of the resulting crystallites when hits are found. Modern advances in instrumentation and increased

knowledge of mAb crystallization have, however, resulted in higher probabilities of discovering crystal forms and improving their

particle properties and characterization. In this regard, physical, analytical characterization plays a central role in the initial steps of

understanding and later optimizing the crystallization of mAbs and requires careful selection of the appropriate tools. This

contribution describes a novel crystal structure of the antibody pembrolizumab and demonstrates the usefulness of small-angle X-ray

scattering (SAXS) for characterizing its crystalline suspensions. It illustrates the advantages of SAXS when used to (i) confirm

crystallinity and crystal phase of crystallites produced in batch mode; (ii) confirm crystallinity under various conditions and detect

variations in crystal phases, enabling fine-tuning of the crystallizations for phase control across multiple batches; (iii) monitor the

physical response and stability of the crystallites in suspension with regard to filtration and washing; and (iv) monitor the physical

stability of the crystallites upon drying. Overall, this work highlights how SAXS is an essential tool for mAb crystallization

characterization.},

keywords = {SONICC},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Insights into Early Phases of Phycocyanin Crystal Formation via SONICC Spectroscopy},

author = {Pechkova, Eugenia},

url = {https://www.mdpi.com/2073-4352/14/5/395},

doi = {https://doi.org/10.3390/cryst14050395},

year  = {2024},

date = {2024-04-25},

abstract = {This research delves into the early nucleation stages of phycocyanin, a protein pivotal for its fluorescent properties and crystalline stability and holding considerable potential for biotechnological applications. The paper contrasts traditional crystallization methods with the innovative Langmuir–Blodgett nanotemplate approach, aiming to enhance molecular assembly and nucleation processes. The study employs Langmuir–Blodgett nanotemplates alongside second-order nonlinear imaging of chiral crystal (SONICC) spectroscopy. This combination is designed to orderly organize phycocyanin molecules and provide a sensitive visualization of early-stage crystal formation, capturing the intricate dynamics of protein crystallization. The experiments were conducted under controlled conditions, where surface pressure was maintained at 26 mN/m and barrier speed at 70 cm/min to optimize the monolayer formation at the air–water interface. The Langmuir–Blodgett method, compared to traditional vapor diffusion techniques, shows improvements in the uniformity and efficiency of nucleation. The sensitivity of SONICC spectroscopy significantly enhances the visualization of the nucleation process, revealing a more structured and uniform crystalline assembly in the early stages of formation. This method demonstrates a substantial improvement in nucleation dynamics, leading to a more orderly growth process and potentially larger, well-ordered crystals. Integrating Langmuir–Blodgett nanotemplates with SONICC spectroscopy offers a significant step in understanding protein crystallization processes with insights into the nucleation and growth of protein crystals and broad implications for refining crystallography methodologies of protein-based biomaterials, contributing to the advancement of structural biology and materials science.},

keywords = {SONICC},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Orthogonal Analytical Methods to Elucidate How Low Levels of Residual Crystallinity in Posaconazole Amorphous Solid Dispersions Impact Phase Behavior during Release},

author = {Shah, Dishan D., Takanti, Nita},

url = {https://pubs.acs.org/doi/abs/10.1021/acs.cgd.3c01147},

doi = {https://doi.org/10.1021/acs.cgd.3c01147},

year  = {2023},

date = {2023-12-01},

abstract = {For optimal bioperformance, the drug in an amorphous solid dispersion (ASD) should ideally not undergo crystallization in the solid dosage form during storage or from the supersaturated solution generated upon dissolution. Incomplete processing during hot melt extrusion (HME) can lead to residual crystallinity. Commonly, residual crystallinity is evaluated using techniques such as powder X-ray diffraction (pXRD). However, residual crystallinity at levels below the detection limit of pXRD can be detrimental to the ASD performance. The goal of this study was to evaluate the impact of different levels of residual crystallinity in an ASD containing the fast-crystallizing drug posaconazole (PCZ) and hydroxypropyl methylcellulose acetate succinate (HPMCAS) on dissolution and additional crystallization. ASDs with and without residual crystallinity at 10, 25, and 50 wt % drug loadings were prepared using HME, processing at temperatures below and above the critical temperature, which was calculated using the Flory–Huggins theory. Some of the ASDs contained levels of residual crystallinity that were below the quantification limit of pXRD, requiring the use of second harmonic generation (SHG) imaging. The impact of residual crystallinity on dissolution was studied by using two-stage dissolution. Additional characterization in support of dissolution measurements included SHG imaging and particle size evolution with focused beam reflectance measurement (FBRM) using pH-shift experiments. The 10 wt % ASD processed below the critical solution temperature contained residual crystallinity of 0.3%, which promoted rapid crystallization when the ASD was in a solution environment. Real-time monitoring of both the solid and solution phases revealed that PCZ in ASDs containing residual crystals underwent crystallization both in the matrix and from solution. The study supports the need to select a sufficiently sensitive crystallinity estimation technique, a suitable discriminatory dissolution technique, and appropriate HME processing conditions in order to optimize and achieve successful performance of ASDs of fast-crystallizing drugs.},

keywords = {SONICC},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Incoherent Nonreciprocal Absorbance Circular Dichroism of Uniaxial Assemblies},

author = {Turner, Gwendylan A, Hwang , Yechan},

url = {https://pubmed.ncbi.nlm.nih.gov/37722139/},

doi = {10.1021/acs.jpcb.3c03104},

year  = {2023},

date = {2023-09-28},

abstract = {Analytical theory is proposed predicting remarkably large and fully electric-dipole-allowed circular dichroism (CD) in electronic ultraviolet-visible (UV-vis) absorbance spectroscopy of uniaxial surface assemblies. Partial depolarization of the transmitted beam provides a pathway for surface-specific and chiral-specific dissymmetry parameters that are orders of magnitude greater than those from analogous measurements of isotropic systems. Predictions of the model generated using ab initio quantum chemical calculations with no adjustable parameters agreed with UV-vis absorbance CD measurements of naproxen microcrystals prepared on hydrophilic substrates. Notably, these calculations correctly predicted (i) the key spectroscopic features, (ii) the relative magnitudes of chiral-specific peaks in the CD spectrum, (iii) the absolute CD sign, and (iv) the reciprocal CD sign inversion arising from sample reorientation in the instrument. These results connect the molecular structure and orientation to large CD observable in oriented thin-film assemblies, with the potential for further extension to broad classes of chiral-specific spectral analyses.},

keywords = {SONICC},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Ultrafast structural changes direct the first molecular events of vision},

author = {Gruhl, Thomas, Weinert, Tobias},

url = {https://www.nature.com/articles/s41586-023-05863-6},

doi = {https://doi.org/10.1038/s41586-023-05863-6},

year  = {2023},

date = {2023-03-22},

abstract = {Vision is initiated by the rhodopsin family of light-sensitive G protein-coupled receptors (GPCRs)1. A photon is absorbed by the 11-cis retinal chromophore of rhodopsin, which isomerizes within 200 femtoseconds to the all-trans conformation2, thereby initiating the cellular signal transduction processes that ultimately lead to vision. However, the intramolecular mechanism by which the photoactivated retinal induces the activation events inside rhodopsin remains experimentally unclear. Here we use ultrafast time-resolved crystallography at room temperature3 to determine how an isomerized twisted all-trans retinal stores the photon energy that is required to initiate the protein conformational changes associated with the formation of the G protein-binding signalling state. The distorted retinal at a 1-ps time delay after photoactivation has pulled away from half of its numerous interactions with its binding pocket, and the excess of the photon energy is released through an anisotropic protein breathing motion in the direction of the extracellular space. Notably, the very early structural motions in the protein side chains of rhodopsin appear in regions that are involved in later stages of the conserved class A GPCR activation mechanism. Our study sheds light on the earliest stages of vision in vertebrates and points to fundamental aspects of the molecular mechanisms of agonist-mediated GPCR activation.},

keywords = {SONICC},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Crystalline Antibody-Laden Alginate Particles: A Platform for Enabling High Concentration Subcutaneous Delivery of Antibodies},

author = {Erfani, Amir, Schieferstein, Jeremy M.},

url = {https://onlinelibrary.wiley.com/doi/full/10.1002/adhm.202202370},

doi = {https://doi.org/10.1002/adhm.202202370},

year  = {2023},

date = {2023-02-06},

abstract = {Subcutaneous (SC) administration is a desired route for monoclonal antibodies (mAbs). However, formulating mAbs for small injection volumes at high concentrations with suitable stability and injectability is a significant challenge. Here, this work presents a platform technology that combines the stability of crystalline antibodies with injectability and tunability of soft hydrogel particles. Composite alginate hydrogel particles are generated via a gentle centrifugal encapsulation process which avoids use of chemical reactions or an external organic phase. Crystalline suspension of anti-programmed cell death protein 1 (PD-1) antibody (pembrolizumab) is utilized as a model therapeutic antibody. Crystalline forms of the mAb encapsuled in the hydrogel particles lead to stable, high concentration, and injectable formulations. Formulation concentrations as high as 315 mg mL−1 antibody are achieved with encapsulation efficiencies in the range of 89–97%, with no perceivable increase in the number of antibody aggregates. Bioanalytical studies confirm superior maintained quality of the antibody in comparison with formulation approaches involving organic phases and chemical reactions. This work illustrates tuning the alginate particles’ disintegration by using partially oxide alginates. Crystalline mAb-laden particles are evaluated for their biocompatibility using cell-based in vitro assays. Furthermore, the pharmacokinetics (PK) of the subcutaneously delivered human anti-PD-1 mAb in crystalline antibody-laden alginate hydrogel particles in Wistar rats is evaluated.},

keywords = {SONICC},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {DETECTING NANO- AND MICRO-SIZED PROTEIN CRYSTALS VIA NONLINEAR OPTICAL IMAGING METHODS},

author = {Cheng, Qingdi},

url = {https://ediss.sub.uni-hamburg.de/handle/ediss/9038},

doi = {Thesis},

year  = {2021},

date = {2021-05-21},

abstract = {Biological macromolecules, such as proteins and nucleic acids, are composed of linked monomers and play an important role in biological functions based on their three-dimensional (3D) structures. Proteins are composed of one or more polypeptide chains of different amino acid residues. These polypeptide chains fold into a 3D structure to constitute a functional protein. The 3D structure information of proteins can be applied to analyze protein-ligand processes and interactions. Furthermore, the 3D structure information of proteins can serve as the basis for structure-based target selection for drug discovery research. As it is not possible for protein 3D structures to be seen even under the most advanced light microscope, other methods are employed to determine their 3D structures. Since proteins can form crystals, X-ray crystallography can be used to solve the 3D structures of these proteins. In the deposited protein data bank (PDB), nearly 90% of protein structures are solved through X-ray crystallography. As a result, X-ray crystallography is the fundamental method for characterizing the atomic structure of proteins.

Notably, the primary and oldest method of X-ray crystallography is single-crystal X-ray diffraction. The major challenge of using this method is obtaining well-ordered crystals with a suitable size for crystallographic data collection. The demand for larger and well-ordered protein crystals has introduced difficulties for those proteins which cannot grow to larger dimensions.

With the development of synchrotron radiation, the brilliant beams achieved through synchrotron radiation have decreased the necessary protein crystal size for conventional X-ray diffraction crystallography. A free-electron laser (FEL) uses a much brighter beam, which decreases the dimensions of protein crystals that are required for diffraction data collection. Consequently, today micro-sized and nano-sized protein crystals are preferred. This preference for small crystals creates a strong demand to develop and establish new methods and instrumentation to identify, detect and analyze protein nano- and micro-crystals.

Current methods to detect micro-sized and nano-sized protein crystals mainly include bright-field imaging, ultraviolet fluorescence (UV) imaging, second harmonic generation (SHG) imaging and X-ray powder diffraction. However, each of these imaging methods has its own limitations. Because of this, a reliable and advanced imaging method is required.

The present work describes an in-house developed multi-modalities multiphoton instrument that is composed of three imaging methods, which are third-harmonic generation (THG), second-harmonic generation (SHG) and three-photon excited ultraviolet fluorescence (3PEUVF). To analyze the feasibility and detection sensitivity of the multimodal MPM system, different protein crystals and salt crystals were prepared with different symmetries. The combined effect of THG, SHG and 3PEUVF imaging is precise, as the system is able to identify nano- or micro-sized protein crystals and can distinguish between protein crystals, salt crystals and amorphous aggregates.

During the testing process, a detailed study of the angular-dependent SHG polarization response was conducted. The results demonstrated that the SHG polarization response of the crystal is highly sensitive to the lattice orientation of crystals. As a result, SHG polarization can extend its potential for protein crystal detection and characterization.

To better compare the differences between commercial imaging instruments and MPM system instruments, the in vitro nanocrystal samples were simultaneously tested with dynamic light scattering (DLS), depolarized dynamic light scattering (DDLS), transmission electron microscopy (TEM) and X-ray powder diffraction. For second-order nonlinear optical imaging of chiral crystals (SONICC) and MPM imaging instrument, the experimental results illustrate that the MPM imaging instrument processes a non-invasive detection method and high detection sensitivity to detect in vitro and in vivo protein nanocrystals. Notably, the nano-sized or sub-micro-sized protein crystals can be detected efficiently through the MPM system. For in vitro protein crystals, the MPM system reduces the risk of obtaining false-negative and false-positive results in crystal detection through providing a higher signal sensitivity. Moreover, the MPM imaging system offers the possibility for in vivo crystals to be detected. Furthermore, weak SHG signals from centrosymmetric crystals are also observed with the MPM system.},

keywords = {SONICC},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Remote Opener: Breaking Barriers to Crystallization Using Remote Crystal Growth Screening and Imaging},

author = {Bowman, Sarah},

url = {https://scripts.iucr.org/cgi-bin/paper?S0108767320097962},

doi = {Abstract},

year  = {2020},

date = {2020-08-01},

abstract = {The vast majority of biomolecular structural information is derived from macromolecular X-ray crystallography

methods, which serve as a foundation for structural biology and account for nearly 90% of the more than 165,000

biomolecular structures available in the PDB. Crystallography requires high-quality, well-diffracting crystals;

coaxing biomolecules into crystalline form is a rate-limiting step in structure determination. Searching for

conditions in which a biomolecule will crystallize often entails screening multiple different constructs against

thousands of crystallization conditions, requiring large sample amounts and many person-hours in a typical

laboratory set-up. In recent circumstances due to the COVID-19 pandemic, being physically in the laboratory for

setting up crystallization screening has become even more difficult. The Crystallization Center at HWI has been in

continuous operation as a crystallization resource for 20 years providing mail-in crystallization and remote access

to crystal growth monitoring. These services have become even more critical in the face of restrictions due to

COVID-19. The Crystallization Center is a high-throughput facility that provides expertise and access to state-ofthe-

art instrumentation to facilitate efficient and cost-effective crystallization. We have extensive robotics for

automated sample handling with very small sample volumes integrated with advanced imaging and a Formulatrix

Rock Imager with SONICC for rapid detection of crystal growth. The current pipeline in the Crystallization Center

screens for 1,536 conditions in one experimental plate and employs a robust imaging schedule, all of which is then

accessible remotely. Here, we will present details about the current capacity for high-throughput crystal growth

screening. We will also discuss innovations we are developing and opportunities for enhanced crystallization

services that will further facilitate crystallization for biomolecular structure determination, including scale up and

optimization, in situ diffraction experiments and enhanced imaging for crystal detection.},

keywords = {SONICC},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Enhanced X-ray diffraction of in vivo-grown lNS crystals by viscous jets at XFELs},

author = {Nagaratnam, N., Tang, Y.},

url = {http://scripts.iucr.org/cgi-bin/paper?S2053230X20006172},

doi = {https://doi.org/10.1107/S2053230X20006172},

year  = {2020},

date = {2020-06-01},

abstract = {μNS is a 70 kDa major nonstructural protein of avian reoviruses, which cause significant economic losses in the poultry industry. They replicate inside viral factories in host cells, and the �NS protein has been suggested to be the minimal viral factor required for factory formation. Thus, determining the structure of �NS is of great importance for understanding its role in viral infection. In the study presented here, a fragment consisting of residues 448-605 of �NS was expressed as an EGFP fusion protein in Sf9 insect cells. EGFP-�NS(448-605) crystallization in Sf9 cells was monitored and verified by several imaging techniques. Cells infected with the EGFP-�NS(448-605) baculovirus formed rod-shaped microcrystals (5-15 �m in length) which were reconstituted in high-viscosity media (LCP and agarose) and investigated by serial femtosecond X-ray diffraction using viscous jets at an X-ray free-electron laser (XFEL). The crystals diffracted to 4.5 � resolution. A total of 4227 diffraction snapshots were successfully indexed into a hexagonal lattice with unit-cell parameters a = 109.29, b = 110.29, c = 324.97 �. The final data set was merged and refined to 7.0 � resolution. Preliminary electron-density maps were obtained. While more diffraction data are required to solve the structure of �NS(448-605), the current experimental strategy, which couples high-viscosity crystal delivery at an XFEL with in cellulo crystallization, paves the way towards structure determination of the �NS protein.},

keywords = {SONICC},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Solid-state analysis of amorphous solid dispersions: Why DSC and XRPD may not be regarded as stand-alone techniques},

author = {Dedrooga, Sien, Pas, Timothy},

url = {https://doi.org/10.1016/j.jpba.2019.112937},

doi = {https://doi.org/10.1016/j.jpba.2019.112937},

year  = {2020},

date = {2020-01-30},

abstract = {Amorphous solid dispersions (ASDs) are single-phase amorphous systems, where drug molecules are molecularly dispersed (dissolved) in a polymer matrix. The molecular dispersion of the drug molecules is responsible for their improved dissolution properties. Unambiguously establishing the phase behavior of the ASDs is of utmost importance. In this paper, we focused on the complementary nature of (modulated) differential scanning calorimetry ((m)DSC) and X-ray powder diffraction (XRPD) to elucidate the phase behavior of ASDs as demonstrated by a critical discussion of practical real-life examples observed in our research group. The ASDs were manufactured by either applying a solvent-based technique (spray drying), a heat-based technique (hot melt extrusion) or mechanochemical activation (cryo-milling). The encountered limiting factors of XRPD were the lack of sensitivity for small traces of crystallinity, the impossibility to differentiate between distinct amorphous phases and its impossibility to detect nanocrystals in a polymer matrix. In addition, the limiting factors of (m)DSC were defined as the well-described heat-induced sample alteration upon heating, the interfering of residual solvent evaporation with other thermal events and the coinciding of enthalpy recovery with melting events. In all of these cases, the application of a single analytical technique would have led to erroneous conclusions, whilst the combination of (m)DSC and XRPD elucidated the true phases of the ASD.},

keywords = {SONICC},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Pembrolizumab microgravity crystallization experimentation},

author = {Reichert, Paul, Prosise, Winifred},

url = {https://www.nature.com/articles/s41526-019-0090-3},

doi = {https://doi.org/10.1038/s41526-019-0090-3},

year  = {2019},

date = {2019-12-02},

abstract = {Crystallization processes have been widely used in the pharmaceutical industry for the manufacture, storage, and delivery of small-molecule and small protein therapeutics. However, the identification of crystallization processes for biologics, particularly monoclonal antibodies, has been prohibitive due to the size and the flexibility of their overall structure. There remains a challenge and an opportunity to utilize the benefits of crystallization of biologics. The research laboratories of Merck Sharp & Dome Corp. (MSD) in collaboration with the International Space Station (ISS) National Laboratory performed crystallization experiments with pembrolizumab (Keytruda�) on the SpaceX-Commercial Resupply Services-10 mission to the ISS. By leveraging microgravity effects such as reduced sedimentation and minimal convection currents, conditions producing crystalline suspensions of homogeneous monomodal particle size distribution (39 �m) in high yield were identified. In contrast, the control ground experiments produced crystalline suspensions with a heterogeneous bimodal distribution of 13 and 102 �m particles. In addition, the flight crystalline suspensions were less viscous and sedimented more uniformly than the comparable ground-based crystalline suspensions. These results have been applied to the production of crystalline suspensions on earth, using rotational mixers to reduce sedimentation and temperature gradients to induce and control crystallization. Using these techniques, we have been able to produce uniform crystalline suspensions (1�5 �m) with acceptable viscosity (<12 cP), rheological, and syringeability properties suitable for the preparation of an injectable formulation. The results of these studies may help widen the drug delivery options to improve the safety, adherence, and quality of life for patients and caregivers.},

keywords = {SONICC},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Characterization of Phase Transformations for Amorphous Solid Dispersions of a Weakly Basic Drug upon Dissolution in Biorelevant Media},

author = {Elkhabaz, Ahmed, Sarkar, Sreya},

url = {https://link.springer.com/article/10.1007%2Fs11095-019-2718-0},

doi = {https://doi.org/10.1007/s11095-019-2718-0},

year  = {2019},

date = {2019-10-30},

abstract = {Purpose 

The overall goal of this study was to investigate the dissolution performance and crystallization kinetics of amorphous solid dispersions (ASDs) of a weakly basic compound, posaconazole, dispersed in a pH-sensitive polymeric matrix consisting of hydroxypropyl methylcellulose acetate succinate (HPMC-AS), using fasted-state simulated media. 



Methods 

ASDs with three different drug loadings, 10, 25 and 50 wt.%, and the commercially available tablets were exposed to acidic media (pH 1.6), followed by transfer to, and dissolution in, intestinal media (pH 6.5). Parallel single stage dissolution experiments in only simulated intestinal media were also performed to better understand the impact of the gastric stage. Different analytical methods, including nanoparticle tracking analysis, powder x-ray diffraction, second harmonic generation and two-photon excitation ultraviolet fluorescence microscopy, were used to characterize the phase behavior of these systems at different stages of dissolution. 



Results 

Results revealed that all ASDs exhibited some degree of drug release upon suspension in acidic media, and were also vulnerable to matrix crystallization. Upon transfer to intestinal media conditions, supersaturation was observed. This was short-lived for some dispersions due to the release of the crystals formed in the acid immersion stage which acted as seeds for crystal growth. Lower drug loading ASDs also exhibited transient formation of amorphous nanodroplets prior to crystallization. 



Conclusions 

This work emphasizes the significance of assessing the impact of pH change on dissolution and provides a fundamental basis of understanding the phase behavior kinetics of ASDs of weakly basic drugs when formulated with pH sensitive polymers.},

keywords = {SONICC},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Second harmonic generation microscopy and raman microscopy of pharmaceutical materials},

author = {Song, Zhengtian},

url = {https://hammer.purdue.edu/articles/thesis/Second_Harmonic_Generation_Microscopy_and_Raman_Microscopy_of_Pharmaceutical_Materials/8986256},

doi = {Thesis},

year  = {2019},

date = {2019-08-31},

abstract = {Second harmonic generation (SHG) microscopy and Raman microscopy were used for

qualitative and quantitative analysis of pharmaceutical materials. Prototype instruments and

algorithms for sampling strategies and data analyses were developed to achieve pharmaceutical

materials analysis with low limits of detection and short measurement times.

Manufacturing an amorphous solid dispersion (ASD), in which an amorphous active

pharmaceutical ingredient (API) within polymer matrix, is an effective approach to improve the

solubility and bioavailability of a drug. However, since ASDs are generally metastable materials,

they can often transform to produce crystalline API with higher thermodynamic stability.

Analytical methods with low limits of detection for crystalline APIs were used to assess the

stability of ASDs. With high selectivity to noncentrosymmetric crystals, SHG microscopy was

demonstrated as an analytical tool, which exhibited a limit of detection of 10 ppm for ritonavir

Form II crystals. SHG microscopy was employed for accelerated stability testing of ASDs, which

provided a four-decade dynamic range of crystallinity for kinetic modeling. An established model

was validated by investigating nucleation and crystal growth based on SHG images. To achieve in

situ accelerated stability testing, controlled environment for in situ stability testing (CEiST) was

designed and built to provide elevated temperature and humidity, which is compatible with a

commercial SHG microscope based on our research prototype. The combination of CEiST and

SHG microscopy enabled assessment of individual crystal growth rates by single-particle tracking

and nucleation rates for individual fields of view with low Poisson noise. In addition, SHG

microscopy coupled with CEiST enabled the study of heterogeneity of crystallization kinetics

within pharmaceutical materials.

Polymorphism of APIs plays an important role in drug formulation development. Different

polymorphs of identical APIs may exhibit different physiochemical properties, e.g., solubility,

stability, and bioavailability, due to their crystal structures. Moreover, polymorph transitions may take place during the manufacturing process and storage. Therefore, analytical methods with high

speed for polymorph characterization, which can provide real-time feedback for the polymorphic

transition, have broad applications in pharmaceutical materials characterization. Raman

spectroscopy is able to determine the API polymorphism, but is hampered by the long

measurement times. In this study, two analytical methods with high speed were developed to

characterize API polymorphs. One is SHG microscopy-guided Raman spectroscopy, which

achieved the speed of 10 ms/particle for clopidogrel bisulfate. Initial classification of two different

polymorphs was based on SHG images, followed acquisition of Raman spectroscopy at the

selected positions to determine the API crystal form. Another approach is implementing of

dynamic sampling into confocal Raman microscopy to accelerate Raman image acquisition for 6-

folds. Instead of raster scanning, dynamic sampling algorithm enabled acquiring Raman spectra at

the most informative locations. The reconstructed Raman image of pharmaceutical materials has

<0.5% loss of image quality with 15.8% sampling rate.},

keywords = {SONICC},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Small Is Beautiful: Growth and Detection of Nanocrystals},

author = {Coe, Jesse, Ros, Alexandra},

url = {https://link.springer.com/chapter/10.1007/978-3-030-00551-1_3},

doi = {https://doi.org/10.1007/978-3-030-00551-1_3},

year  = {2018},

date = {2018-12-28},

abstract = {With the advent of X-Ray free electron lasers (FELs), the field of serial femtosecond crystallography (SFX) was borne, allowing a stream of nanocrystals to be measured individually and diffraction data to be collected and merged to form a complete crystallographic data set. This allows submicron to micron crystals to be utilized in an experiment when they were once, at best, only an intermediate result towards larger, usable crystals. SFX and its variants have opened new possibilities in structural biology, including studies with increased temporal resolution, extending to systems with irreversible reactions, and minimizing artifacts related to local radiation damage. Perhaps the most profound aspect of this newly established field is that �molecular movies,� in which the dynamics and kinetics of biomolecules are studied as a function of time, are now an attainable commodity for a broad variety of systems, as discussed in Chaps. 11 and 12. However, one of the historic challenges in crystallography has always been crystallogenesis and this is no exception when preparing samples for serial crystallography methods. In the following chapter, we focus on some of the specific characteristics and considerations inherent in preparing a suitable sample for successful serial crystallographic approaches.},

keywords = {SONICC},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Calibration-Free Second Harmonic Generation (SHG) Image Analysis for Quantification of Trace Crystallinity Within Final Dosage Forms of Amorphous Solid Dispersions},

author = {Smith, Casey J., Dinh, Janny},

url = {https://journals.sagepub.com/doi/10.1177/0003702818786506},

doi = {https://doi.org/10.1177%2F0003702818786506},

year  = {2018},

date = {2018-10-03},

abstract = {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.},

keywords = {SONICC},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Rapid sample delivery for megahertz serial crystallography at X-ray FELs},

author = {Wiedorn, Max O., Awel, Salah},

url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6126653/},

doi = {https://doi.org/10.1107/S2052252518008369},

year  = {2018},

date = {2018-07-27},

abstract = {Liquid microjets are a common means of delivering protein crystals to the focus of X-ray free-electron lasers (FELs) for serial femtosecond crystallography measurements. The high X-ray intensity in the focus initiates an explosion of the microjet and sample. With the advent of X-ray FELs with megahertz rates, the typical velocities of these jets must be increased significantly in order to replenish the damaged material in time for the subsequent measurement with the next X-ray pulse. This work reports the results of a megahertz serial diffraction experiment at the FLASH FEL facility using 4.3 nm radiation. The operation of gas-dynamic nozzles that produce liquid microjets with velocities greater than 80 m s-1 was demonstrated. Furthermore, this article provides optical images of X-ray-induced explosions together with Bragg diffraction from protein microcrystals exposed to trains of X-ray pulses repeating at rates of up to 4.5 MHz. The results indicate the feasibility for megahertz serial crystallography measurements with hard X-rays and give guidance for the design of such experiments.},

keywords = {SONICC},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Kinetic modeling of accelerated stability testing enabled by second harmonic generation microscopy},

author = {Song, Zhengtian, Sarkar, Sreya},

url = {https://pubs.acs.org/doi/10.1021/acs.analchem.7b04260},

doi = {https://doi.org/10.1021/acs.analchem.7b04260},

year  = {2018},

date = {2018-03-05},

abstract = {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.},

keywords = {SONICC},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Impact of Eudragit EPO and hydroxypropyl methylcellulose on drug release rate, supersaturation, precipitation outcome and redissolution rate of indomethacin amorphous solid dispersions},

author = {Xie, Tian, Gao. et. al., Wei},

url = {https://www.sciencedirect.com/science/article/abs/pii/S0378517317308190?via%3Dihub},

doi = {https://doi.org/10.1016/j.ijpharm.2017.08.099},

year  = {2017},

date = {2017-10-05},

abstract = {The purpose of this work was to evaluate the impact of polymer(s) on the dissolution rate, supersaturation and 

precipitation of indomethacin amorphous solid dispersions (ASD), and to understand the link between precipitate characteristics and redissolution kinetics. The crystalline and amorphous solubilities of indomethacin 

were determined in the absence and presence of hydroxypropylmethyl cellulose (HPMC) and/or Eudragit � EPO 

to establish relevant phase boundaries. At acidic pH, HPMC could maintain supersaturation of the drug by 

effectively inhibiting solution crystallization while EPO increased both the crystalline and amorphous solubility 

of the drug, but did not inhibit crystallization. The HPMC dispersion dissolved relatively slowly without undergoing crystallization while the supersaturation generated by rapid dissolution of the EPO ASD was short-lived 

due to crystallization. The crystals thus generated underwent rapid redissolution upon pH increase, dissolving 

faster than the reference crystalline material, and at a comparable rate to the amorphous HPMC dispersion. A 

ternary dispersion containing both EPO and HPMC dissolved rapidly, generating an apparent drug concentration 

that exceeded the amorphous solubility of indomethacin, leading to the formation of a new nanosized droplet 

phase. These nanodroplets dissolved virtually immediately when the pH was increased. In conclusion, the 

concentration-time profiles achieved from indomethacin ASD dissolution are a complex interplay of drug release 

rate, precipitation kinetics and outcome, and precipitate redissolution rate, whereby each of these processes is 

highly dependent on the polymer(s) employed in the formulation.},

keywords = {SONICC},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Second harmonic generation microscopy as a tool for the early detection of crystallization in spray dried dispersions},

author = {Soto, Clara Correa-, Trasi, Niraj S.},

url = {https://www.sciencedirect.com/science/article/abs/pii/S0731708517320940?via%3Dihub},

doi = {https://doi.org/10.1016/j.jpba.2017.07.066},

year  = {2017},

date = {2017-08-24},

abstract = {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 ofthe 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, 15 wt.% drug loading) and ezetimibe (EZT, 30 wt.% 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 8 days of storage at 40 ?C/82% RH, whereas no evidence of crystallinity could be 

observed by XRPD until 26 days. Correspondingly, for FTM samples stored at 40 ?C/75% RH, crystals were 

detected after 11 days by SHG microscopy and after 53 days 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 (15 days) with SHG microscopy relative to with XRPD (60 days). 

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.},

keywords = {SONICC},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Second Harmonic Generation Guided Raman Spectroscopy for Sensitive Detection of Polymorph Transitions},

author = {Chowdhury, Azhad U., Ye, Dong Hye},

url = {https://pubs.acs.org/doi/10.1021/acs.analchem.7b00431},

doi = {https://doi.org/10.1021/acs.analchem.7b00431},

year  = {2017},

date = {2017-05-08},

abstract = {Second harmonic generation (SHG) was integrated with Raman spectroscopy for the 

analysis of pharmaceutical materials. Particulate formulations of clopidogrel bisulphate were 

prepared in two crystal forms (Form I and Form II). Image analysis approaches enable 

automated identification of particles by bright field imaging, followed by classification by SHG. 

Quantitative SHG microscopy enabled discrimination of crystal form on a per particle basis with 

99.95% confidence in a total measurement time of ~10 ms per particle. Complementary 

measurements by Raman and synchrotron XRD are in excellent agreement with the 

classifications made by SHG, with measurement times of ~1 minute and several seconds per 

particle, respectively. Coupling these capabilities with at-line monitoring may enable real-time 

feedback for reaction monitoring during pharmaceutical production to favor the more 

bioavailable but metastable Form I with limits of detection in the ppm regime.},

keywords = {SONICC},

pubstate = {published},

tppubtype = {article}

}