Imaging Flow Cytometry for Sizing and Counting of Subvisible Particles in Biotherapeutics.

Imaging flow cytometry (IFC), a technique originally designed for cellular imaging, is featured by the parallel acquisition in brightfield (BF), fluorescence (FL), and side scattering channels. Introduced to the field of subvisible particle analysis in biopharmaceuticals roughly ten years ago, it has the potential to yield additional information, e.g., on particle origin. Here, we present an extensive, systematic development of masks for IFC image analysis to optimize the accuracy of size determination of polystyrene beads and pharmaceutically relevant particles (protein, silicone oil) in BF and FL channels. Based on the developed masks, particle sizing and counting by IFC are compared to flow imaging microscopy (FIM). Mask verification based on fluorescent polystyrene particles revealed good agreement between sizes obtained from IFC and FIM. In the evaluation of counting accuracy, IFC reported lower concentrations for polystyrene particle standards than FIM. For the analysis of fluorescently stained silicone oil and protein particles however, IFC FL imaging reported higher particle concentrations in the low micrometer size range. Overall, we identified IFC as suitable tool to generate supportive data for particle characterization purposes or trouble shooting activities, but not as routine quantitative technique, e.g., for subvisible particle analysis during drug product development or quality control.

Forced degradation of cell-based medicinal products guided by flow imaging microscopy: Explorative studies with Jurkat cells.

Cell-based medicinal products (CBMPs) offer ground-breaking opportunities to treat diseases with limited or no therapeutic options. However, the intrinsic complexity of CBMPs results in great challenges with respect to analytical characterization and stability assessment. In our study, we submitted Jurkat cell suspensions to forced degradation studies mimicking conditions to which CBMPs might be exposed from procurement of cells to administration of the product. Flow imaging microscopy assisted by machine learning was applied for determination of cell viability and concentration, and quantification of debris particles. Additionally, orthogonal cell characterization techniques were used. Thawing of cells at 5 °C was detrimental to cell viability and resulted in high numbers of debris particles, in contrast to thawing at 37 °C or 20 °C which resulted in better stability. After freezing of cell suspensions at -18 °C in presence of dimethyl sulfoxide (DMSO), a DMSO concentration of 2.5% (v/v) showed low stabilizing properties, whereas 5% or 10% was protective. Horizontal shaking of cell suspensions did not affect cell viability, but led to a reduction in cell concentration. Fetal bovine serum (10% [v/v]) protected the cells during shaking. In conclusion, forced degradation studies with application of orthogonal analytical characterization methods allow for CBMP stability assessment and formulation screening.

Particulate impurities in cell-based medicinal products traced by flow imaging microscopy combined with deep learning for image analysis.

Cell-based medicinal products (CBMPs) are rapidly gaining importance in the treatment of life-threatening diseases. However, the analytical toolbox for characterization of CBMPs is limited. The aim of our study was to develop a method based on flow imaging microscopy (FIM) for the detection, quantification and characterization of subvisible particulate impurities in CBMPs. Image analysis was performed by using an image classification approach based on a convolutional neural network (CNN). Jurkat cells and Dynabeads were used in our study as a representation of cellular material and non-cellular particulate impurities, respectively. We demonstrate that FIM assisted with CNN is a powerful method for the detection and quantification of Dynabeads and cells with other process related impurities, such as cell agglomerates, cell-bead adducts and debris. By using CNN, we achieved a more than 50-fold lower misclassification rate compared with the use of output parameters from the FIM software. The limit of detection was ~15 000 beads/mL in the presence of ~500 000 cells/mL, making this approach suitable for the detection of these particulate impurities in CBMPs. In conclusion, CNN-assisted FIM is a powerful method for the detection and quantification of cells, Dynabeads and other subvisible process impurities potentially present in CBMPs.

Capillary electrophoresis-mass spectrometry using noncovalently coated capillaries for the analysis of biopharmaceuticals.

In this work, the usefulness of capillary electrophoresis-electrospray ionization time-of-flight-mass spectrometry for the analysis of biopharmaceuticals was studied. Noncovalently bound capillary coatings consisting of Polybrene-poly(vinyl sulfonic acid) or Polybrene-dextran sulfate-Polybrene were used to minimize protein and peptide adsorption, and achieve good separation efficiencies. The potential of the capillary electrophoresis-mass spectrometry (CE-MS) system to characterize degradation products was investigated by analyzing samples of the drugs, recombinant human growth hormone (rhGH) and oxytocin, which had been subjected to prolonged storage, heat exposure, and/or different pH values. Modifications could be assigned based on accurate masses as obtained with time-of-flight-mass spectrometry (TOF-MS) and migration times with respect to the parent compound. For heat-exposed rhGH, oxidations, sulfonate formation, and deamidations were observed. Oxytocin showed strong deamidation (up to 40%) upon heat exposure at low pH, whereas at medium and high pH, mainly dimer (>10%) and trisulfide formation (6-7%) occurred. Recombinant human interferon-ß-1a (rhIFN-ß) was used to evaluate the capability of the CE-MS method to assess glycan heterogeneity of pharmaceutical proteins. Analysis of this N-glycosylated protein revealed a cluster of resolved peaks which appeared to be caused by at least ten glycoforms differing merely in sialic acid and hexose N-acetylhexosamine composition. Based on the relative peak area (assuming an equimolar response per glycoform), a quantitative profile could be derived with the disialytated biantennary glycoform as most abundant (52%). Such a profile may be useful for in-process and quality control of rhIFN-ß batches. It is concluded that the separation power provided by combined capillary electrophoresis and TOF-MS allows discrimination of highly related protein species.

Optimization of a pharmaceutical freeze-dried product and its process using an experimental design approach and innovative process analyzers.

The aim of the present study was to examine the possibilities/advantages of using recently introduced in-line spectroscopic process analyzers (Raman, NIR and plasma emission spectroscopy), within well-designed experiments, for the optimization of a pharmaceutical formulation and its freeze-drying process. The formulation under investigation was a mannitol (crystalline bulking agent)-sucrose (lyo- and cryoprotector) excipient system. The effects of two formulation variables (mannitol/sucrose ratio and amount of NaCl) and three process variables (freezing rate, annealing temperature and secondary drying temperature) upon several critical process and product responses (onset and duration of ice crystallization, onset and duration of mannitol crystallization, duration of primary drying, residual moisture content and amount of mannitol hemi-hydrate in end product) were examined using a design of experiments (DOE) methodology. A 2-level fractional factorial design (2(5-1)=16 experiments+3 center points=19 experiments) was employed. All experiments were monitored in-line using Raman, NIR and plasma emission spectroscopy, which supply continuous process and product information during freeze-drying. Off-line X-ray powder diffraction analysis and Karl-Fisher titration were performed to determine the morphology and residual moisture content of the end product, respectively. In first instance, the results showed that - besides the previous described findings in De Beer et al., Anal. Chem. 81 (2009) 7639-7649 - Raman and NIR spectroscopy are able to monitor the product behavior throughout the complete annealing step during freeze-drying. The DOE approach allowed predicting the optimum combination of process and formulation parameters leading to the desired responses. Applying a mannitol/sucrose ratio of 4, without adding NaCl and processing the formulation without an annealing step, using a freezing rate of 0.9°C/min and a secondary drying temperature of 40°C resulted in efficient freeze-drying supplying end products with a residual moisture content below 2% and a mannitol hemi-hydrate content below 20%. Finally, using Monte Carlo simulations it became possible to determine how varying the factor settings around their optimum still leads to fulfilled response criteria, herewith having an idea about the probability to exceed the acceptable response limits. This multi-dimensional combination and interaction of input variables (factor ranges) leading to acceptable response criteria with an acceptable probability reflects the process design space.

Radiation therapy and mechanical dilation of endobronchial obstruction secondary to Wegener's granulomatosis.

A patient with Wegener's granulomatosis, which was otherwise well controlled with steroids and cyclophosphamide, had persistent total obstruction of an inflamed left mainstem bronchus. The inflammation resolved, and the lung expanded following radiation therapy. However, intermittent atelectasis and pneumonia occurred distal to a residual short stricture. Repeated dilation endoscopically with Plummer bougies has proven effective in maintaining good ventilation and preventing recurrence of the atelectasis and pneumonia in the left lung for 18 months.