Within this study, the performance and limitations of tunable resistive pulse sensing (TRPS) was evaluated to characterize submicron particles in unstressed and heat stressed monoclonal antibody ... Show moreWithin this study, the performance and limitations of tunable resistive pulse sensing (TRPS) was evaluated to characterize submicron particles in unstressed and heat stressed monoclonal antibody (mAb) solutions. These were compared with microfluidic resistive pulse sensing (MRPS), resonant mass measurement (RMM), and nanoparticle tracking analysis (NTA). For TRPS and MRPS measurements, an adjustment of ionic strength was required to achieve suitable measurement conditions. The addition of electrolytes is potentially critical for protein formulations and therefore the effect of salt concentration and pH on submicron particle levels was further investigated. Heat stress caused a sharp increase in particle levels between 250-900 nm, observable by all four techniques. Due to reduced colloidal stability, indicated by increased attractive forces and reduced aggregation onset temperatures in the presence of sodium chloride, protein aggregation was observed in heat stressed mAb only after the addition of sodium chloride. Achieving adequate ionic strength by replacing sodium chloride with other electrolytes similarly resulted in reduced colloidal stability and protein aggregation. It is recommended that protein samples prone for aggregation in the presence of high ionic strength should not be analyzed by RPS measurements after the addition of electrolytes. However, protein samples containing already required ionic strength can be analyzed by any of the four techniques. Show less
The measurement of polydisperse protein aggregates and particles in biotherapeutics remains a challenge, especially for particles with diameters of ≈ 1 µm and below (sub-micrometer). This paper... Show moreThe measurement of polydisperse protein aggregates and particles in biotherapeutics remains a challenge, especially for particles with diameters of ≈ 1 µm and below (sub-micrometer). This paper describes an interlaboratory comparison with the goal of assessing the measurement variability for the characterization of a sub-micrometer polydisperse particle dispersion composed of five sub-populations of poly(methyl methacrylate) (PMMA) and silica beads. The study included 20 participating laboratories from industry, academia, and government, and a variety of state-of-the-art particle-counting instruments. The received datasets were organized by instrument class to enable comparison of intralaboratory and interlaboratory performance. The main findings included high variability between datasets from different laboratories, with coefficients of variation from 13 % to 189 %. Intralaboratory variability was, on average, 37 % of the interlaboratory variability for an instrument class and particle sub-population. Drop-offs at either end of the size range and poor agreement on maximum counts of particle sub-populations were noted. The mean distributions from an instrument class, however, showed the size-coverage range for that class. The study shows that a poly-disperse sample can be used to assess performance capabilities of an instrument set-up (including hardware, software, and user settings) and provides guidance for the development of polydisperse reference materials. Show less
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... Show moreCell-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. Show less
Particles are omnipresent in biopharmaceutical products. In protein-based therapeutics such particles are generally associated with impurities, either derived from the drug product itself (e.g.... Show moreParticles are omnipresent in biopharmaceutical products. In protein-based therapeutics such particles are generally associated with impurities, either derived from the drug product itself (e.g. protein aggregates), or from extrinsic contaminations (e.g. cellulose fibers). These impurities can affect product stability, as well as cause adverse effects once introduced into the human body. Particulate impurities are present over a wide range of sizes (from nanometers to millimeters) making them difficult to characterize by using a single method.Novel drug products may also contain particles that act as the active pharmaceutical ingredient (e.g., living cells) or a drug delivery vehicle (e.g., lipid nanoparticles). Unwanted immunotoxicity and inconsistent in vivo functionality can result from particle instability and aggregate formation. Therefore, the efficacy and safety of these therapeutics is dependent on the particle composition, quantity and size distribution.Consequently, well-established methods are required to quantify and characterize particles in the submicron- and micron-size ranges. In this thesis, we developed new approaches which allow for comprehensive characterization of the particle populations present in biopharmaceutical products, both as impurities or as API. Furthermore, the performed work focused on comparing different particle characterization techniques to allow a better understanding of the limitations and strengths of each method applied. Show less
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... Show moreCell-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.(c) 2020 International Society for Cell & Gene Therapy. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) Show less
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... Show moreCell-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. Show less
Polysorbate 80 (PS80) is a commonly used surfactant in therapeutic protein formulations to mitigate adsorption and interface-induced protein aggregation. Several PS80 grades and qualities are... Show morePolysorbate 80 (PS80) is a commonly used surfactant in therapeutic protein formulations to mitigate adsorption and interface-induced protein aggregation. Several PS80 grades and qualities are available on the market for parenteral application. The role of PS80 grade on protein stability remains debatable, and the impact of (partially) degraded PS on protein aggregation is not yet well understood. In our study, a monoclonal antibody (IgG) was subjected to 3 different mechanical stress conditions in the presence of multicompendial (MC) and Chinese pharmacopeia (ChP) grade PS80. Furthermore, IgG formulations were spiked with (partly) hydrolyzed PS80 to investigate the effect of PS80 degradants on protein stability. PS80 functionality was assessed by measuring the extent of protein aggregation and particle formation induced during mechanical stress by using size-exclusion chromatography, dynamic light scattering, backgrounded membrane imaging, and flow imaging microscopy. No distinguishable differences in PS80 functionality between MC and ChP grade were observed in the 3 stress tests. However, with increasing degree of PS80 hydrolysis, higher counts of subvisible particles were measured after stress. Furthermore, higher levels of PS80 degradants at a constant PS80 concentration may destabilize the IgG. In conclusion, MC and ChP grade PS80 are equally protective, but PS80 degradants compromise IgG stability. Show less
Grabarek, A.D.; Weinbuch, D.; Jiskoot, W.; Hawe, A. 2018
The objective was to evaluate performance, strengths, and limitations of the microfluidic resistive pulse sensing (MRPS) technique for the characterization of particles in the size range from about... Show moreThe objective was to evaluate performance, strengths, and limitations of the microfluidic resistive pulse sensing (MRPS) technique for the characterization of particles in the size range from about 50 to 2000 nm. MRPS, resonant mass measurement (RMM), nanoparticle tracking analysis (NTA) and dynamic light scattering were compared for the analysis of nanometer-sized polystyrene (PS) beads, liposomes, bacteria, and protein aggregates. An electrical conductivity of at least 3 mS/cm (equivalent to 25 mM NaCl) was determined as a key requirement for reliable analysis with MRPS. Particle size distributions of PS beads determined by MRPS, NTA, and RMM correlated well. However, counting precision varied significantly among the techniques and was best for RMM followed by MRPS and NTA. As determined by measuring single and mixed PS bead populations, MRPS showed the highest peak resolution for sizing. RMM and MRPS were superior over dynamic light scattering and NTA for the characterization of stressed protein samples. Finally, MRPS proved to be the only analytical technique able to characterize both bacteria and liposomes. In conclusion, MRPS is an orthogonal technique alongside other established techniques for a comprehensive analysis of a samples particle size distribution and particle concentration. Show less
