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.

Evaluation of Abuse-Deterrent Characteristics of Tablets Prepared via Hot-Melt Extrusion.

In this study, the effect of formulation variables and process parameters on the abuse-deterrent (AD) characteristics of a matrix tablet manufactured using hot-melt extrusion (HME) was investigated. The formulation variables included polyethylene oxide (PEO) grades and its input level, while the HME process parameters varied were barrel temperature profile and die diameter. Depending on the diameter of the extrudate (2.5 mm or 5.0 mm), two different downstream processes were used to prepare the tablets: cryo-milling followed by compression for the 2.5 mm extrudate, and cutting followed by compression for the 5.0 mm extrudate. A D-optimal statistical design was used to evaluate the impact of formulation and process parameters on various responses, including tablet physical strength, particle size after manipulation, syringeability and injectability, solution viscosity, extractability in solvents, and dissolution rates. It was found that the post-HME extrusion processing method played a critical role in affecting the AD characteristics of abuse-deterrent formulations, likely through changing the tablet compactability and porosity. When the extrudates were cryo-milled-compressed, the tablets could be readily manipulated by milling, which led to high degree of extractability. Under high alcohol concentration, burst drug release was observed for the tablets compressed from cryo-milled extrudates. Additionally, heat exposure during HME process caused significant drop in PEO solution viscosity, likely due to thermal degradation.

Application of Optical Coherence Tomography Freeze-Drying Microscopy for Designing Lyophilization Process and Its Impact on Process Efficiency and Product Quality.

Optical coherence tomography freeze-drying microscopy (OCT-FDM) is a novel technique that allows the three-dimensional imaging of a drug product during the entire lyophilization process. OCT-FDM consists of a single-vial freeze dryer (SVFD) affixed with an optical coherence tomography (OCT) imaging system. Unlike the conventional techniques, such as modulated differential scanning calorimetry (mDSC) and light transmission freeze-drying microscopy, used for predicting the product collapse temperature (Tc), the OCT-FDM approach seeks to mimic the actual product and process conditions during the lyophilization process. However, there is limited understanding on the application of this emerging technique to the design of the lyophilization process. In this study, we investigated the suitability of OCT-FDM technique in designing a lyophilization process. Moreover, we compared the product quality attributes of the resulting lyophilized product manufactured using Tc, a critical process control parameter, as determined by OCT-FDM versus as estimated by mDSC. OCT-FDM analysis revealed the absence of collapse even for the low protein concentration (5 mg/ml) and low solid content formulation (1%w/v) studied. This was confirmed by lab scale lyophilization. In addition, lyophilization cycles designed using Tc values obtained from OCT-FDM were more efficient with higher sublimation rate and mass flux than the conventional cycles, since drying was conducted at higher shelf temperature. Finally, the quality attributes of the products lyophilized using Tc determined by OCT-FDM and mDSC were similar, and product shrinkage and cracks were observed in all the batches of freeze-dried products irrespective of the technique employed in predicting Tc.

Stability characterization and appearance of particulates in a lyophilized formulation of a model peptide hormone-human secretin.

Drug shortages and recalls are often caused due to particulate growth in parenteral products and can have serious clinical implications. Root cause analysis of such recalls and shortages may arise due to insufficient understanding of process, formulations issues and environmental effects than often reported filtration and inadequate personnel training. Therefore, the goal of this study was to use a model peptide hormone, secretin that is currently under drug shortage, and investigate the effect of excipients on the lyophilized secretin formulation and evaluate the effect of storage and excursion temperatures. Lyophilized formulation was assayed for secretin by reverse phase HPLC. Solid state characteristics of lyophilized formulation were determined by X-ray powder diffraction (XRPD), thermal and spectroscopic methods. Dynamic light scattering (DLS) was used to detect particulates in the formulation after reconstitution. To assess the environmental impact, the lyophilized samples were stored at -20°C, 4°C, 25°C and 25°C/60%RH and analyzed at time 0, 1, 4, and 8 weeks. HPLC analyses exhibited a decrease in secretin concentration by 8 week (20-27% fold decrease). Visual observation and DLS showed particulates and increased reconstitution time (e.g., at 25°C/60%RH, particle size of ∼390 nm at day 0 to >2 µm as early as week 1; reconstitution time of ∼20s at day 0 to ∼67s at week 8). XRPD, thermal and spectroscopic methods demonstrated polymorphic transitions of mannitol and increased crystallinity in the lyophilized formulations with time. These studies potentially address the effect of product excursions outside the proposed label storage conditions which is -20°C for secretin formulation and this is the first time it has been investigated. These observations indicate that both environmental factor and excipient may have an impact on the stability of secretin formulation and appearance of particles in the product.

Non-aqueous suspensions of antibodies are much less viscous than equally concentrated aqueous solutions.

PURPOSE: The aim of this study was to markedly lower the viscosities of highly concentrated protein, in particular antibody, formulations. An effective approach elaborated herein for γ-globulin and a monoclonal antibody is to replace aqueous solutions with equimolar suspensions in neat organic solvents. METHODS: Viscosities of aqueous solutions and non-aqueous suspensions of the model protein bovine γ-globulin and a murine monoclonal antibody were examined under a variety of experimental conditions. In addition, protein particle sizes were measured using dynamic light scattering and light microscopy. RESULTS: Concentrated suspensions of amorphous γ-globulin powders (up to 300 mg/mL, composed of multi-micron-sized particles) in absolute ethanol and a number of other organic solvents were found to have viscosities up to 38 times lower than the corresponding aqueous solutions. Monoclonal antibody follows the same general trend. Additionally, the higher the protein concentration and lower the temperature, the greater the viscosity benefit of a suspension over a solution. CONCLUSIONS: The viscosities of concentrated γ-globulin and monoclonal antibody suspensions in organic solvents are drastically reduced compared to the corresponding aqueous solutions; the magnitude of this reduction depends on the solvent, particularly its hydrogen-bonding properties.

Integrin-targeted stabilized nanoparticles for an efficient delivery of siRNAs in vitro and in vivo.

Utilizing small interfering RNAs (siRNAs) to silence disease-associated genes holds promise as a potential therapeutic strategy. However, the greatest challenge for RNAi remains the delivery of siRNA to target tissues or cells. Specifically lymphocytes are difficult to transduce by conventional methods but represent good targets for anti-inflammatory therapeutics. Integrins are an important class of cell adhesion receptors on leukocytes. Antibodies to integrins have been used to inhibit inflammatory reactions in patients. Here, we describe a strategy to deliver the siRNA cargo to leukocytes by stabilized nanoparticles surface-decorated with antibodies to integrin as targeting moieties. A detailed methodology for preparation of the integrin-targeted stabilized nanoparticles (I-tsNPs) and their delivery in vitro and in vivo is discussed.

Optimization and characterization of anionic lipoplexes for gene delivery.

Anionic lipoplexes, comprising divalent cations, DNA and anionic liposomes, were optimized for high transfection efficiency and low cytotoxicity. Different molar ratios of anionic to zwitterionic lipid, 1:9 to 1:1 (1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DOPG): 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), various cations (Ca2+, Mg2+ and Na+) and different anionic lipid/DNA ratios were investigated. The optimized formulation was composed of: anionic/zwitterionic lipid molar ratio 1:4 (DOPG:DOPE); 15-20 mM Ca2+; and 15-20 microg lipid for complexation with 0.8 microg plasmid DNA. Biophysical studies (particle size analysis, gel electrophoresis, transmission electron microscopy (TEM), and confocal microscopy) were conducted to characterize the different formulations. TEM revealed structural differences between the complexed and uncomplexed lipoplexes. Gel electrophoresis confirmed the formation of anionic lipoplexes with the amount of free DNA minimized for the optimized formulation. Confocal imaging showed cellular uptake of the anionic lipoplexes. Most significantly the anionic lipoplex formulation, optimized for the highest transfection efficiency (approximately 78% in the presence of serum) exhibited the highest cell viability (approximately 93%, (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide) MTT assay). This was compared to Lipofectamine 2000 which had a transfection efficiency and cell viability of approximately 68% and 35%, respectively. The anionic lipoplex formulation developed here shows promise as a non-viral vector with high transfection efficiency and low cytotoxicity.

Dual fluorescent labeling method to visualize plasmid DNA degradation.

The efficiency of nonviral vectors for gene delivery may be enhanced by understanding the key barriers that limit the translocation of the therapeutic DNA into the nucleus. One such barrier is the instability of DNA in the cytoplasm. In this work, we have developed a method to dual-label plasmid DNA to be utilized as a tool to elucidate DNA instability during its trafficking in the intracellular microenvironment. Plasmid DNA containing rhodamine and maleimide groups linked using peptide nucleic acid (PNA) linkers was utilized for conjugation. Covalent conjugation of the maleimide group with a second fluorophore, fluorescein, did not alter the electrophoretic mobility or the structural integrity of the DNA, as confirmed by gel electrophoresis. The intact DNA was visualized as a single color (yellow) due to the close proximity of the green and red fluorophores. DNA degradation was simulated using restriction endonucleases (BamH1 and PflMI) to cut the DNA at two or more sites resulting in color separation. Confocal time-lapse imaging was utilized to follow DNA stability upon incubation of Lipofectamine2000/dual-labeled DNA complexes in CHO-K1 cells. Yellow fluorescent voxels were seen in the cell cytoplasm indicating the presence of intact DNA. Red and green fluorescent voxels were also seen in a few cells, suggesting separation of the fluorophores and probable DNA degradation. The methodology developed in this report provides a new tracking tool for investigators to explore DNA degradation at the molecular level inside single cell.

Labeling and intracellular tracking of functionally active plasmid DNA with semiconductor quantum dots.

Semiconductor nanocrystal quantum dots (QDs) allow long-term imaging in the cellular environment with high photostability. QD biolabeling techniques have previously been developed for tagging proteins and peptides as well as oligonucleotides. In this contribution, QD-decorated plasmid DNA was utilized for the first time for long-term intracellular and intranuclear tracking studies. Conjugation of plasmid DNA with phospholipid-coated QDs was accomplished using a peptide nucleic acid (PNA)-N-succinimidyl-3-(2-pyridylthio) propionate linker. Gel electrophoresis and confocal and atomic force microscopy (AFM) were used to confirm the structure of QD-DNA conjugates. AFM imaging also revealed that multiple QDs were attached in a cluster at the PNA-reactive site of the plasmid DNA. These QD-DNA conjugates were capable of expressing the reporter protein, enhanced green fluorescent protein, following transfection in Chinese hamster ovary (CHO-K1) cells with an efficiency of ca. 62%, which was comparable to the control (unconjugated) plasmid DNA.