Quality assessment of virus-like particle: A new transmission electron microscopy approach.

Transmission electron microscopy (TEM) is a gold standard analytical method for nanoparticle characterization and is playing a valuable role in virus-like particle (VLP) characterization extending to other biological entities such as viral vectors. A dedicated TEM facility is a challenge to both small and medium-sized enterprises (SMEs) and companies operating in low-and-middle income countries (LMICs) due to high start-up and running costs. A low-voltage TEM solution with assisted image acquisition and analysis such as the MiniTEM system, coupled with Vironova Imaging and Analysis Software (VIAS) could provide an affordable and practical alternative. The MiniTEM system has a small footprint and software that enables semi-automated data collection and image analysis workflows using built-in deep learning methods (convolutional neural networks) for automation in analysis, increasing speed of information processing and enabling scaling to larger datasets. In this perspective we outline the potential and challenges in the use of TEM as mainstream analytical tool in manufacturing settings. We highlight the rationale and preliminary findings from our proof-of-concept study aiming to develop a method to assess critical quality attributes (CQAs) of VLPs and facilitate adoption of TEM in manufacturing settings. In our study we explored all the steps, from sample preparation to data collection and analysis using synthetic VLPs as model systems. The applicability of the method in product development was verified at pilot-scale during the technology transfer of dengue VLPs development from a university setting to an LMIC- based vaccine manufacturing company, demonstrating the applicability of this analytical technique to VLP vaccine characterization.

Assessment of the percentage of full recombinant adeno-associated virus particles in a gene therapy drug using CryoTEM.

In spite of continuous development of gene therapy vectors with thousands of drug candidates in clinical drug trials there are only a small number approved on the market today stressing the need to have characterization methods to assist in the validation of the drug development process. The level of packaging of the vector capsids appears to play a critical role in immunogenicity, hence an objective quantitative method assessing the content of particles containing a genome is an essential quality measurement. As transmission electron microscopy (TEM) allows direct visualization of the particles present in a specimen, it naturally seems as the most intuitive method of choice for characterizing recombinant adeno-associated virus (rAAV) particle packaging. Negative stain TEM (nsTEM) is an established characterization method for analysing the packaging of viral vectors. It has however shown limitations in terms of reliability. To overcome this drawback, we propose an analytical method based on CryoTEM that unambiguously and robustly determines the percentage of filled particles in an rAAV sample. In addition, we show that at a fixed number of vector particles the portion of filled particles correlates well with the potency of the drug. The method has been validated according to the ICH Q2 (R1) guidelines and the components investigated during the validation are presented in this study. The reliability of nsTEM as a method for the assessment of filled particles is also investigated along with a discussion about the origin of the observed variability of this method.

A microfluidic device for TEM sample preparation.

Transmission electron microscopy (TEM) allows for visualizing and analyzing viral particles and has become a vital tool for the development of vaccines and biopharmaceuticals. However, appropriate TEM sample preparation is typically done manually which introduces operator-based dependencies and can lead to unreliable results. Here, we present a capillary-driven microfluidic single-use device that prepares a TEM grid with minimal and non-critical user interaction. The user only initiates the sample preparation process, waits for about one minute and then collects the TEM grid, ready for imaging. Using Adeno-associated virus (AAV) particles as the sample and NanoVan® as the stain, we demonstrate microfluidic consistency and show that the sample preparation quality is sufficient for automated image analysis. We further demonstrate the versatility of the microfluidic device by preparing two protein complexes for TEM investigations using two different stain types. The presented TEM sample preparation concept could alleviate the problems associated with human inconsistency in manual preparation protocols and allow for non-specialists to prepare TEM samples.

Multi-step control over self-assembled hydrogels of peptide-derived building blocks and a polymeric cross-linker.

We present a detailed study of self-assembled hydrogels of bundled and cross-linked networks consisting of positively charged amyloid-like nanofibers and a triblock copolymer with negatively charged end blocks as a cross-linker. In a first step small oligopeptides self-assemble into macrocycles which are held together by reversible disulfide bonds. Interactions between the peptides cause the macrocycles to assemble into nanofibers, which form a reversible hydrogel. The physical properties of the hydrogel are tuned using various methods such as control over the fibre length, addition of a cross-linking copolymer, and addition of salt. We establish a relationship between the bulk mechanical properties, the properties of the individual fibers and the hydrogel morphology using characterization techniques operating at different length scales such as rheology, atomic force microscopy (AFM) and cryo transmission electron microscopy (Cryo-TEM). This allows for a precise control of the elastic behaviour of these networks.

Instructable Nanoparticles Using Dynamic Combinatorial Chemistry.

The application of nanoparticles to the multivalent recognition of biomacromolecules or programmed self-assembly requires control over the relative placement of chemical groups on their surface. We have developed a method to direct the functionalization of surfaces of aldehyde-equipped gold nanoparticles using a DNA template. An error-correction mechanism is built into the functionalization process thanks to the thermodynamic control enabled by the hydrazone exchange reaction. This reversible reaction can be conveniently switched off by removing the catalyst, preserving the functionalization.

Template-Triggered Emergence of a Self-Replicator from a Dynamic Combinatorial Library.

Self-assembly of a specific member of a dynamic combinatorial library (DCL) may lead to self-replication of this molecule. However, if the concentration of the potential replicator in the DCL fails to exceed its critical aggregation concentration (CAC), then self-replication will not occur. We now show how addition of a template can raise the concentration of a library member-template complex beyond its CAC, leading to the onset of self-replication. Once in existence, the replicator aggregates promote further replication also in the absence of the template that induced the initial emergence of the replicator.

Exponential self-replication enabled through a fibre elongation/breakage mechanism.

Self-replicating molecules are likely to have played a central role in the origin of life. Most scenarios of Darwinian evolution at the molecular level require self-replicators capable of exponential growth, yet only very few exponential replicators have been reported to date and general design criteria for exponential replication are lacking. Here we show that a peptide-functionalized macrocyclic self-replicator exhibits exponential growth when subjected to mild agitation. The replicator self-assembles into elongated fibres of which the ends promote replication and fibre growth. Agitation results in breakage of the growing fibres, generating more fibre ends. Our data suggest a mechanism in which mechanical energy promotes the liberation of the replicator from the inactive self-assembled state, thereby overcoming self-inhibition that prevents the majority of self-replicating molecules developed to date from attaining exponential growth.

Controlling the Structure and Length of Self-Synthesizing Supramolecular Polymers through Nucleated Growth and Disassembly.

Directing self-assembly processes out-of-equilibrium to yield kinetically trapped materials with well-defined dimensions remains a considerable challenge. Kinetically controlled assembly of self-synthesizing peptide-functionalized macrocycles through a nucleation-growth mechanism is reported. Spontaneous fiber formation in this system is effectively shut down as most of the material is diverted into metastable non-assembling trimeric and tetrameric macrocycles. However, upon adding seeds to this mixture, well-defined fibers with controllable lengths and narrow polydispersities are obtained. This seeded growth strategy also allows access to supramolecular triblock copolymers. The resulting noncovalent assemblies can be further stabilized through covalent capture. Taken together, these results show that self-synthesizing materials, through their interplay between dynamic covalent bonds and noncovalent interactions, are uniquely suited for out-of-equilibrium self-assembly.

Localized template-driven functionalization of nanoparticles by dynamic combinatorial chemistry.

We have developed a method for the localized functionalization of gold nanoparticles using imine-based dynamic combinatorial chemistry. By using DNA templates, amines were grafted on the aldehyde-functionalized nanoparticles only if and where the nanoparticles interacted with the template molecules. Functionalization of the nanoparticles was controlled solely by the DNA template; only amines capable of interacting with DNA were bound to the surface. Interestingly, even though our libraries contained only a handful of simple amines, the DNA sequence influenced their attachment to the surface. Our method opens up new opportunities for the synthesis of multivalent, nanoparticle-based receptors for biomacromolecules.

An "ingredients" approach to functional self-synthesizing materials: a metal-ion-selective, multi-responsive, self-assembled hydrogel.

New methodology for making novel materials is highly desirable. Here, an "ingredients" approach to functional self-assembled hydrogels was developed. By designing a building block to contain the right ingredients, a multi-responsive, self-assembled hydrogel was obtained through a process of template-induced self-synthesis in a dynamic combinatorial library. The system can be switched between gel and solution by light, redox reactions, pH, temperature, mechanical energy and sequestration or addition of Mg(II) salt.

Transient substrate-induced catalyst formation in a dynamic molecular network.

In biology enzyme concentrations are continuously regulated, yet for synthetic catalytic systems such regulatory mechanisms are underdeveloped. We now report how a substrate of a chemical reaction induces the formation of its own catalyst from a dynamic molecular network. After complete conversion of the substrate, the network disassembles the catalyst. These results open up new opportunities for controlling catalysis in synthetic chemical systems.

Uncovering the selection criteria for the emergence of multi-building-block replicators from dynamic combinatorial libraries.

A family of self-replicating macrocycles was developed using dynamic combinatorial chemistry. Replication is driven by self-assembly of the replicators into fibrils and relies critically on mechanically induced fibril fragmentation. Analysis of separate dynamic combinatorial libraries made from one of six peptide-functionalized building blocks of different hydrophobicity revealed two selection criteria that govern the emergence of replicators from these systems. First, the replicators need to have a critical macrocycle size that endows them with sufficient multivalency to enable their self-assembly into fibrils. Second, efficient replication occurs only for library members that are of low abundance in the absence of a replication pathway. This work has led to spontaneous emergence of replicators with unrivalled structural complexity, being built from up to eight identical subunits and reaching a MW of up to 5.6 kDa. The insights obtained in this work provide valuable guidance that should facilitate future discovery of new complex self-replicating molecules. They may also assist in the development of new self-synthesizing materials, where self-assembly drives the synthesis of the very molecules that self-assemble. To illustrate the potential of this concept, the present system enables access to self-assembling materials made from self-synthesizing macrocycles with tunable ring size ranging from trimers to octamers.