Webinar Summary

The fourth episode of the webinar series on electron diffraction explores how electron diffraction (ED) can be a transformative tool across the entire pharmaceutical development pipeline, from early discovery to final drug products.

Electron diffraction is an advanced technique used to analyze the structure of materials at the atomic level. Unlike conventional X-ray powder diffraction, ED can work with extremely small amounts of sample—nanograms or less—and provides detailed information grain by grain. This means it can analyze individual tiny crystals within a powder, even in highly impure or early-stage samples.

For pharmaceutical companies, this opens the door to much faster and more precise characterization of drug forms (polymorphs), especially in situations where time and material are limited.

Applications across the development pipeline

Early discovery and preformulation:

Electron diffraction excels at identifying crystalline forms as they appear during early solid formation. Even when only trace crystals are available, ED can identify whether the material is crystalline, determine its unit cell (the fundamental building block of a crystal structure), and even determine the connectivity of the atoms in the molecule.

Unlike other techniques that rely on comparing overall spectral patterns, ED yields unambiguous identification of distinct forms. This minimizes false positives when identifying polymorphs.

Automated high-throughput screening:

ED can be automated to rapidly test thousands of individual grains or liquid-deposited samples, making it well-suited for high-throughput environments. Liquid samples from microplates can be pipetted directly onto grids, evaporated, and analyzed. This is ideal for ligand screening or early-phase crystallization experiments.

Crystal structure prediction integration and early risk assessment:

With modern drug discovery relying on computational crystal structure prediction (CSP), ED bridges the gap by providing real experimental data on the real structures that form, even transiently. These can be used to calibrate CSP models, improving the accuracy of stability predictions and helping ensure the most stable form is chosen early in development.

Downstream utility

Formulation and drug product characterization:

As pharma moves toward nanoparticle delivery systems, ED becomes particularly powerful. It can image and analyze individual lipid nanoparticles, even determining if the active ingredient inside has crystallized. This granularity enables deep insights into how APIs behave inside formulations.

ED also helps study structural disorder, such as changes induced by milling, which can affect performance and manufacturability. It can detect very early phase separation in amorphous solid dispersions and explore atomic-level disorder using advanced techniques like total diffraction and pair distribution function (PDF) analysis.

Finished drug products and IP:

ED is an exceptional tool for reverse-engineering tablets or capsules. With just nanograms of powdered sample, it can identify multiple crystalline forms—e.g., different APIs or excipients—in a drug product. This makes it valuable for detecting counterfeits, validating composition, and checking for potential IP issues.

ED is also sensitive to hydrogen atoms, which means it can distinguish between salts and co-crystals—critical for regulatory classification and patent strategy.

Practical considerations

Electron diffraction requires very small and thin samples (30–300 nanometers thick) and operates under a vacuum. It uses electrons accelerated at very high voltages (e.g., 200 kV), giving it extremely high resolution. Despite concerns about so-called “dynamical diffraction” (where strong interaction between electrons and matter can complicate analysis), modern software tools and automation largely resolve these issues for routine pharmaceutical work.

Automation is a game-changer. Once samples are loaded, the system can align, analyze, and process data from thousands of grains with minimal manual effort. This makes ED viable even in industrial, non-academic settings.

Emerging and routine uses

Routine uses now include:

Identifying unknown polymorphs in commercial drug products.
Determining absolute structure (molecular “handedness”).
Resolving whether a compound is a salt or a cocrystal.
Protein crystal structure determination and ligand screening (surprisingly feasible with ED).
Characterizing amorphous and disordered systems using total diffraction analysis.

Emerging applications include detailed analysis of lipid nanoparticles and early-stage phase separation in amorphous solid dispersions, with the ultimate goal of understanding and predicting formulation behavior and risks at the nanoscale.

Electron diffraction has evolved into a robust, practical, and indispensable tool for pharmaceutical R&D. Its sensitivity, low sample requirements, and automation make it uniquely suited for today’s challenges—speed, miniaturization, complexity, and the growing importance of polymorph and particle-scale characterization. Whether you're formulating new drugs, investigating unknown substances, or reverse-engineering finished products, ED offers actionable insights no other technique can match.

Key questions answered in this webinar:

: Electron diffraction (ED) is a powerful technique that uses a beam of electrons to analyze the crystalline structure of materials. Unlike X-ray diffraction (XRD), which relies on X-rays, ED utilizes electrons that interact much more strongly with matter (10⁵ to 10⁶ times stronger than X-rays). This strong interaction allows ED to work with extremely small samples, typically in the nanogram to sub-microgram range, with thicknesses of 30 to 300 nanometers.

A key difference lies in their operational principles and the information they provide. While XRD typically provides a bulk average of a powder sample, ED can analyze individual powder grains. Furthermore, ED's short electron wavelength (e.g., 0.025 Å for 200 kV electrons) results in a very flat Ewald sphere, causing multiple Bragg peaks to scatter simultaneously (multibeam dynamical case). This contrasts with XRD, which typically intersects only one Bragg spot at a time. Despite this "dynamical effect" in ED, it turns out to be incredibly robust for determining unit cells and even molecular connectivity, as these are based on peak positions rather than exact intensities. ED is also uniquely sensitive to hydrogens, which can be crucial for distinguishing between salts and cocrystals in pharmaceutical research.
: Electron diffraction excels across the entire drug discovery and development pipeline, offering unique advantages, especially in polymorph screening. Its primary benefits include:

Minimal sample requirements: ED can perform polymorph screening with sub-microgram or even nanogram quantities of "as-made" powder, eliminating the need for time-consuming scale-up steps often required by other techniques.

Unit cell based characterization: Unlike spectral-based methods like X-ray powder diffraction or Raman, which compare data patterns, ED provides a unit cell-based characterization. Each unique unit cell (defined by its six parameters: a, b, c, alpha, beta, gamma) unequivocally identifies a distinct polymorph, making differentiation clear and unambiguous.

Early-stage screening: ED's ability to work with small, potentially impure, and kinetically crashed-out solids in early development allows for very early polymorph screening. This helps identify metastable forms that might otherwise be missed after purification, contributing to a more comprehensive understanding of the material's solid form landscape.

Calibration of Crystal Structure Prediction (CSP): By identifying multiple polymorphs at early stages, ED data can be used to calibrate quantum force fields used in CSP pipelines. This improves the accuracy of predicting the most stable crystalline form, which is crucial for drug product stability during manufacturing and storage.

Automation and high throughput: ED systems are highly amenable to automation, enabling high-throughput workflows. This allows for the rapid screening of thousands of candidates per week, making it ideal for discovery and early development phases. Grains are automatically aligned, checked for crystallinity, unit cells are determined, and further analysis (like molecular connectivity) can proceed if needed.
: Electron diffraction is particularly well-suited for characterizing materials at the nanoscale, a critical aspect in the growing field of nanopharmaceutics and nano delivery systems. Its capabilities include:

Imaging and diffraction of individual nanoparticles: ED can image individual nanoparticles and perform diffraction studies on them. This is exemplified by its application to lipid nanoparticles, such as Doxorubicin API encapsulated in pegylated liposomes, allowing for the characterization of individual delivery systems as small as 30-40 nanometers.

Evaluation of disorder: ED can be used to study atomic disorder introduced during particle size reduction processes (e.g., milling of API to sub-micron sizes). By isolating individual particles, researchers can characterize the structural changes and defects induced, which can impact dissolution rates and bioavailability.

Analysis of Amorphous Solid Dispersions (ASDs): A new application involves analyzing ASDs to detect early nucleation and phase separation of the API from the carrying polymer. ED can identify nanometer-sized nuclei forming within the polymers, providing insights into the stability and performance of these amorphous formulations.

Liquid-based automation and in situ studies: ED can integrate with liquid-based automation workflows, allowing for the deposition of picoliters of material from high-throughput screens onto grids. Furthermore, a wide range of in situ environmental stages (low/high temperatures, liquid/gas environments, cryo-stages, and nano-channel liquid cells) enable the study of crystallization and nucleation dynamics directly from mother liquor, observing the formation of protocrystals and early metastable polymorphs.
: Electron diffraction is adept at handling complex pharmaceutical samples, including final drug products and mixtures, providing detailed insights that might be difficult to obtain with other techniques:

Reverse engineering and contaminant identification: Tablets and solid dosage forms can be rendered into a powder, spread on a grid, and analyzed grain by grain using ED. This allows for the complete characterization of crystalline materials present, including different API polymorphs and excipients. This capability is invaluable for reverse engineering dosage forms, identifying contaminants (e.g., a "black spec" in a customer complaint sample), and supporting intellectual property protection.

Identification of elusive polymorphs: ED has successfully identified elusive crystalline forms of APIs within drug products that could not be characterized by other methods (e.g., an antihistamine drug product where the API polymorph was unknown). From just a few crystalline grains of the API, ED can provide sufficient data to determine the unit cell, crystal structure, molecular connectivity, and even the absolute handedness of the molecule.

Characterizing multicomponent crystals (salts vs. cocrystals): When crystals contain more than one molecular component (coformers), ED can unequivocally identify where the proton exists. This sensitivity to hydrogens allows for clear differentiation between salts (where the proton is transferred from the API) and cocrystals (where it remains attached to the API), which is crucial due to differing regulatory classifications by agencies.

Combined analysis with EDS: Newer ED systems often include an Energy Dispersive X-ray Spectroscopy (EDS) channel. This allows for the elemental makeup analysis of individual nanoparticles or grains, aiding in the identification of specific elements within the structure. This is highly useful for reverse engineering, identifying trace contaminants, or confirming the presence of key elements that might be difficult to distinguish through diffraction alone (e.g., iron vs. gallium in isostructural complexes).
: The typical workflow for electron diffraction in an industrial environment emphasizes automation and integrated processes to make it efficient and user-friendly. The process generally involves:

Sample preparation: Pharmaceutical powders are typically "dusted" onto a 3 mm TEM (Transmission Electron Microscopy) grid, which has an electron-transparent substrate (e.g., amorphous carbon, polymers, graphene, or silicon nitride). Different substrate types (flat carbon, lacy carbon, holy carbon) can be chosen based on the sample's habit or desired analysis (e.g., lacy carbon for varied grain orientation, holy carbon for total diffraction without substrate interference). For liquid-based samples, picoliters can be pipetted onto grids, with solvents evaporating rapidly to deposit solids.

Sample environment: Due to the high vacuum in the electron column, sensitive organic materials often require specialized environments. A cryo-stage is highly recommended for pharmaceutical materials to protect volatiles and prevent damage from the electron beam. Heating capabilities are also common. Gas environments and liquid cells are available for in situ studies, such as observing nucleation in mother liquor.

Automated grain identification and alignment: The system automatically steps through individual grains identified on the grid. Each grain is aligned into the electron beam.

Tiered data collection and analysis:

First pass (crystallinity check): The system first determines if the grain diffracts, indicating crystallinity. For high-throughput screening, simply knowing if it's crystalline may suffice.

Second pass (unit cell determination): If crystalline, more data is collected (multi-images) to automatically determine the unit cell parameters (a, b, c, alpha, beta, gamma). Unit cells are determined based on peak positions, which are unaffected by dynamical diffraction.

Third pass (molecular connectivity/structure solution): If needed, data from individual grains or combined data from multiple grains with the same unit cell can be used to derive the molecular connectivity and crystal structure.

Advanced analysis (absolute structure, hydrogen placement): For highly accurate placement of hydrogens or absolute structure determination, the data can be further processed through dynamic pipeline software (e.g., YANA, OLEX) to account for dynamical effects.

Multigrain analysis and clustering: The collected unit cells and associated data are fed into hierarchical clustering algorithms to group similar unit cells, indicating the same polymorph. This allows for the identification of different polymorphic forms present in the sample, even if unknown previously.

Post-measurement capabilities: Beyond diffraction, electron beam power can be increased for high-resolution imaging, tomography, and energy dispersive X-ray fluorescence (XRF/EDS) for elemental analysis, providing additional characterization of the material's composition.
: Dynamical diffraction refers to the phenomenon where electrons, due to their strong interaction with matter and short wavelength, can scatter multiple times within a perfect crystal as they pass through. Unlike X-ray diffraction, where single diffraction events are common, electron beams can interact with multiple Bragg peaks simultaneously, leading to a complex interference pattern. This means the intensity of diffracted beams is not simply proportional to the scattering power of atoms, but also influenced by these multiple scattering pathways.

While "dynamical effects" are often cited as a challenge in electron diffraction, they generally do not hinder routine single crystal structure determination, especially for small molecules and disordered materials. Here's why:

Robust pipeline for connectivity: The primary goal in much of pharmaceutical research is to determine molecular connectivity and unit cells. Unit cells are derived from peak positions, not intensities, making them unaffected by dynamical interactions. For connectivity, often distinguishing between strong and weak reflections is sufficient.

Prevalence of disordered crystals: Many samples in early pharma development are small, kinetically grown, or highly milled, leading to disordered crystals. Disordered crystals lack the perfection required to establish strong dynamical interference effects, thus exhibiting largely kinematic (single scattering) diffraction.

Narrow Darwin width: Strong dynamical diffraction only occurs when the crystal is precisely aligned "on zone axis" and within an extremely narrow angular range (around 0.002°), known as the Darwin width. Data collection methods that rock the sample through diffraction conditions ensure that most of the time, the diffraction is kinematic.

Dedicated dynamical correction pipelines: If absolute structure, precise hydrogen placement, or extremely accurate intensities are required, dedicated dynamic correction pipelines are available and integrated into ED software.

Utility for local structure: Paradoxically, dynamical effects can be useful. They act as a strong local probe, providing sensitive information for local structure evaluation (e.g., total diffraction and PDF analysis) and absolute structure determination, which can be critical for chiral molecules.

In essence, while understanding dynamical effects is important, they are rarely a barrier to obtaining actionable structural data in the context of pharmaceutical research with electron diffraction.
: Electron diffraction of pharmaceutical materials requires careful consideration of environmental conditions and sample preparation methods due to the high vacuum environment of the electron microscope and the sensitivity of many organic compounds.

Sample preparation:

TEM grids: Powders are typically "dusted" onto 3-millimeter Transmission Electron Microscopy (TEM) grids. These grids have an electron-transparent substrate, such as:

Amorphous carbon: Standard and widely used, typically 3-20 nanometers thick.

Lacy carbon: Features an undulating surface, which helps orient grains in varied directions. This is useful for improving data completeness when combining diffraction patterns from multiple grains, especially if the crystals have a preferred habit (e.g., plates that tend to lie flat).

Holy carbon: Contains holes in the film, allowing grains to span the holes. This is beneficial for total diffraction measurements, as it minimizes interference from the substrate material.

Polymers and graphene: Other options, with double-layer graphene films even allowing for small amounts of liquid environment to be sealed with crystals.

Liquid-based automation: For samples from high-throughput liquid screens, picoliters of material can be pipetted onto grids. The solvent typically evaporates quickly, depositing the solids onto the grid.

Environmental conditions:

Vacuum: The electron column operates under high vacuum, meaning the sample will automatically be in a vacuum unless specific measures are taken.

Cryo-stage: This is highly recommended for almost all pharmaceutical materials. A cryo-stage maintains the sample at very low temperatures (e.g., liquid nitrogen temperatures). This is crucial for:

Protecting volatiles: Prevents the evaporation of volatile components from the sample.

Electron beam protection: Mitigates damage to the material caused by the electron beam, which can be significant for organic compounds.

Temperature range studies: Many cryo-stages also offer heating capabilities, allowing researchers to explore a broad temperature range and observe its impact on crystal polymorphs.

Gas environments and liquid cells: These specialized sample holders enable in situ experiments that are otherwise impossible.

Nano-channel holders (liquid cells): Allow for the observation of nucleation and crystal growth directly from mother liquor. This opens up a vast energy space for polymorph screening, as it enables the study of early, metastable forms that emerge during the crystallization process. This information can then be used to refine quantum force fields in Crystal Structure Prediction (CSP).

These varied preparation and environmental control options allow ED to be highly versatile for different pharmaceutical research needs.
: Electron diffraction is continuously evolving, with new applications and future directions expanding its utility in pharmaceutical research:

Lipid nanoparticle delivery systems: With the significant growth in nanopharmaceutics, ED is being used to analyze individual nano delivery systems, such as lipid nanoparticles containing APIs. This involves imaging and diffraction studies on particles as small as 30-40 nanometers. The goal is to obtain structural fingerprints through techniques like total diffraction and PDF (Pairwise Distribution Function) analysis to understand their structure and ensure QC compliance.

Disordered systems: ED is increasingly applied to study disorder in APIs, particularly those induced by milling to reduce particle size. By analyzing the diffuse diffraction patterns from disordered crystals, researchers can gain insights into how the material's atomic structure is affected, which can influence dissolution rates, bioavailability, and overall drug product performance.

Amorphous solid dispersions (ASDs): A new area of focus is the analysis of ASDs to detect early indications of API phase separation and nucleation within polymer matrices. ED's ability to probe nanometer-sized nuclei allows for a better understanding of the stability and risk associated with amorphous formulations.

Total diffraction and pairwise distribution function (PDF) analysis: ED can collect total diffraction data from both crystalline and amorphous systems. This data can be converted into a PDF format, which provides a real-space representation of atomic arrangements. This is a powerful tool for characterizing the local structure, even in disordered or amorphous materials, and is being applied to understand the structure of ASDs.

High-throughput protein screening: While not new, its application in an industrial setting for high-throughput protein electron diffraction (MicroED) is gaining momentum. This allows for the routine solution of protein structures and screening for ligands binding to specific target sites, accelerating drug discovery efforts.

Integration with other techniques: The addition of post-measurement capabilities like high-resolution imaging, tomography, and Energy Dispersive X-ray Spectroscopy (EDXRF) to ED systems significantly enhances their analytical power. EDXRF, for instance, allows for elemental analysis of individual particles, aiding in reverse engineering, identifying contaminants, and verifying material composition at a very localized level.

Automation and speed enhancement: Ongoing efforts are focused on further increasing the speed of automated data collection. The current rate of 1-2 minutes per grain is being reduced, with a target of analyzing 1,000 grains per day, which would unlock even greater potential for high-throughput screening in discovery and early development.