Morphologically-Directed Raman Spectroscopy as an Analytical Method for Subvisible Particle Characterization in Therapeutic Protein Product Quality.

Subvisible particles (SVPs) are a critical quality attribute of injectable therapeutic proteins (TPs) that needs to be controlled due to potential risks associated with drug product quality. The current compendial methods routinely used to analyze SVPs for lot release provide information on particle size and count. However, chemical identification of individual particles is also important to address root-cause analysis. Herein, we introduce Morphologically-Directed Raman Spectroscopy (MDRS) for SVP characterization of TPs. The following particles were used for method development: (1) polystyrene microspheres, a traditional standard used in industry; (2) photolithographic (SU-8); and (3) ethylene tetrafluoroethylene (ETFE) particles, candidate reference materials developed by NIST. In our study, MDRS rendered high-resolution images for the ETFE particles (> 90%) ranging from 19 to 100 µm in size, covering most of SVP range, and generated comparable morphology data to flow imaging microscopy. Our method was applied to characterize particles formed in stressed TPs and was able to chemically identify individual particles using Raman spectroscopy. MDRS was able to compare morphology and transparency properties of proteinaceous particles with reference materials. The data suggests MDRS may complement the current TPs SVP analysis system and product quality characterization workflow throughout development and commercial lifecycle.

Testing Precision Limits of Neural Network-Based Quality Control Metrics in High-Throughput Digital Microscopy.

OBJECTIVE: Digital microscopy is used to monitor particulates such as protein aggregates within biopharmaceutical products. The images that result encode a wealth of information that is underutilized in pharmaceutical process monitoring. For example, images of particles in protein drug products typically are analyzed only to obtain particle counts and size distributions, even though the images also reflect particle characteristics such as shape and refractive index. Multiple groups have demonstrated that convolutional neural networks (CNNs) can extract information from images of protein aggregates allowing assignment of the likely stress at the "root-cause" of aggregation. A practical limitation of previous CNN-based approaches is that the potential aggregation-inducing stresses must be known a priori, disallowing identification of particles produced by unknown stresses. METHODS: We demonstrate an expanded CNN analysis of flow imaging microscopy (FIM) images incorporating judiciously chosen particle standards within a recently proposed "fingerprinting algorithm" (Biotechnol. & Bioeng. (2020) 117:3322) that allows detection of particles formed by unknown root-causes. We focus on ethylene tetrafluoroethylene (ETFE) microparticles as standard surrogates for protein aggregates. We quantify the sensitivity of the new algorithm to experimental parameters such as microscope focus and solution refractive index changes, and explore how FIM sample noise affects statistical testing procedures. RESULTS & CONCLUSIONS: Applied to real-world microscopy images of protein aggregates, the algorithm reproducibly detects complex, distinguishing "textural features" of particles that are not easily described by standard morphological measurements. This offers promise for quality control applications and for detecting shifts in protein aggregate populations due to stresses resulting from unknown process upsets.

Microscopic evaluation of pharmaceutical glass container-formulation interactions under stressed conditions.

Chemical incompatibility of the formulation with glass container can adversely impact the quality of parenteral products. The objective of this study is to investigate formulation-glass interactions at the inner surface of the glass containers that lead to the generation of particulates under stressed conditions (i.e., combinations of high pHs, temperatures and prolonged exposure selected to purposely cause failure of glass containers) using advanced microscopic techniques. The optical, electron microscopy and X-ray spectroscopy were used in tandem to investigate the nature of these interactions at the vial inner surface. These interactions were characterized by surface roughness and reaction zones on the inner surface of the vials and particulates in the formulation using two commercially available pharmaceutical glass containers (Vials 1 and 2). A nanoscale level examination of the inner surface of Vial 1 revealed layers flaking off from the inner surface of the vial resulting in typical particulate generation, while the reaction zone on the inner surface of Vial 2 exhibited a different layered structure. The results suggest that particulates observed in Vials 1 and 2 were generated through different failure modes.

Quality attributes and evaluation of pharmaceutical glass containers for parenterals.

Pharmaceutical containers for parenterals have been predominantly manufactured using glass as a packaging material of choice, especially Type-I glass, since it has been regarded as a chemically inert and an effective container closure system (CCS). Nevertheless, there have been reports and recalls related to glass quality issues, such as breakage, flakes, and particles observed in marketed products. The novelty of this research is based on the knowledge gathered from our previously conducted risk assessments and establishing a comprehensive testing platform focused on risk factors for glass container failure modes and applicability to other types of pharmaceutical containers. The evaluation of container quality attributes was performed for three model glass vials using a mechanical and chemical durability testing platform: freeze-thaw, lyophilization, compression, scratch tests; visual inspection, pH, particle size analyses, extractable, leachable and imaging studies that were conducted under normal (4 and 25 °C), and stress condition (60 °C), respectively. The performance between the glass containers tested under certain stress conditions (failure modes) were variable and differentiated. The systematic platform testing approach shows the importance of lab-based risk evaluation in assessing common failure modes of pharmaceutical containers, since the quality attributes for injectable products are complex and can impact final product quality.

Solid-Phase Nucleic Acid Sequence-Based Amplification and Length-Scale Effects during RNA Amplification.

Solid-phase oligonucleotide amplification is of interest because of possible applications to next-generation sequencing, multiplexed microarray-based detection, and cell-free synthetic biology. Its efficiency is, however, less than that of traditional liquid-phase amplification involving unconstrained primers and enzymes, and understanding how to optimize the solid-phase amplification process remains challenging. Here, we demonstrate the concept of solid-phase nucleic acid sequence-based amplification (SP-NASBA) and use it to study the effect of tethering density on amplification efficiency. SP-NASBA involves two enzymes, avian myeloblastosis virus reverse transcriptase (AMV-RT) and RNase H, to convert tethered forward and reverse primers into tethered double-stranded DNA (ds-DNA) bridges from which RNA- amplicons can be generated by a third enzyme, T7 RNA polymerase. We create microgels on silicon surfaces using electron-beam patterning of thin-film blends of hydroxyl-terminated and biotin-terminated poly(ethylene glycol) (PEG-OH, PEG-B). The tethering density is linearly related to the PEG-B concentration, and biotinylated primers and molecular beacon detection probes are tethered to streptavidin-activated microgels. While SP-NASBA is very efficient at low tethering densities, the efficiency decreases dramatically with increasing tethering density due to three effects: (a) a reduced hybridization efficiency of tethered molecular beacon detection probes; (b) a decrease in T7 RNA polymerase efficiency;

Molecular Crowding Effects on Microgel-Tethered Oligonucleotide Probes.

Microgel tethering is a nontraditional method with which to bind oligonucleotide hybridization probes to a solid surface. Microgel-tethering physically positions the probes away from the underlying hard substrate and maintains them in a highly waterlike environment. This paper addresses the question of whether molecular crowding affects the performance of microgel-tethered molecular beacon probes. The density of probe-tethering sites is controlled experimentally using thin-film blends of biotin-terminated [PEG-B] and hydroxyl-terminated [PEG-OH] poly(ethylene glycol) from which microgels are synthesized and patterned by electron beam lithography. Fluorescence measurements indicate that the number of streptavidins, linear DNA probes, hairpin probes, and molecular beacon probes bound to the microgels increases linearly with increasing PEG-B/PEG-OH ratio. For a given tethering-site concentration, more linear probes can bind than structured probes. Crowding effects emerge during the hybridization of microgel-tethered molecular beacons but not during the hybridization of linear probes, as the tethering density increases. Crowding during hybridization is associated with conformational constraints imposed by the close proximity of closed and hybridized structured probes. The signal-to-background ratio (SBR) of hybridized beacons is highest and roughly constant for low tethering densities and decreases at the highest tethering densities. Despite differences between microgel tethering and traditional oligonucleotide surface-immobilization approaches, these results show that crowding defines an optimum tethering density for molecular beacon probes that is less than the maximum possible, which is consistent with previous studies involving various linear and structured oligonucleotide probes.