Analyzing Subvisible Particles in Protein Drug Products: a Comparison of Dynamic Light Scattering (DLS) and Resonant Mass Measurement (RMM)

Aggregation is common in protein drug manufacture, and while the effects of protein particulates are under investigation, many techniques applicable for their characterization have been recently developed. Among the methods available to characterize and quantify protein aggregates, none is applicable over the full size range and different methods often give conflicting results. The studies presented here compare two such methods: dynamic light scattering (DLS) and resonant mass measurement (RMM). The performance of each method was first characterized using polystyrene particle size standards (20, 60, 100, 200, 400, and 1,000 nm) over a range of concentrations. Standard particles were measured both singly and in binary mixtures containing 20 nm particles at a fixed concentration (10¹⁴ particles/mL) and various concentrations of one of the other particle sizes (i.e., 60, 100, 200, 400, or 1,000 nm). DLS and RMM were then used to detect unknown aggregate content in stressed samples of IgG. Both instruments were shown to have a working range that depends on particle size and concentration. In binary mixtures and polydisperse solutions, DLS was able to resolve two species in a manner dependent on both concentration and particle size. RMM was able to resolve particles above 200 nm (150 nm for protein) at concentrations below 10⁹ particles/mL. In addition, dilution was evaluated as a technique to confirm and quantify the number of particles in solution.     

Submicron Protein Particle Characterization using Resistive Pulse Sensing and Conventional Light Scattering Based Approaches

Purpose

Characterizing submicron protein particles (approximately 0.1–1μm) is challenging due to a limited number of suitable instruments capable of monitoring a relatively large continuum of particle size and concentration. In this work, we report for the first time the characterization of submicron protein particles using the high size resolution technique of resistive pulse sensing (RPS).

Methods

Resistive pulse sensing, dynamic light scattering and size-exclusion chromatography with in-line multi-angle light scattering (SEC-MALS) are performed on protein and placebo formulations, polystyrene size standards, placebo formulations spiked with silicone oil, and protein formulations stressed via freeze-thaw cycling, thermal incubation, and acid treatment.

Results

A method is developed for monitoring submicron protein particles using RPS. The suitable particle concentration range for RPS is found to be approximately 4?×?10⁷-1?×?10¹¹ particles/mL using polystyrene size standards. Particle size distributions by RPS are consistent with hydrodynamic diameter distributions from batch DLS and to radius of gyration profiles from SEC-MALS. RPS particle size distributions provide an estimate of particle counts and better size resolution compared to light scattering.

Conclusion

RPS is applicable for characterizing submicron particles in protein formulations with a high degree of size polydispersity. Data on submicron particle distributions provide insights into particles formation under different stresses encountered during biologics drug development.

     

Near-infrared reflection spectroscopy (NIRS) as a successful tool for simultaneous identification and particle size determination of amoxicillin trihydrate

A successful application of NIR spectroscopy (NIRS) in combination with multivariate data analysis (MVA) for the simultaneous identification and particle size determination of amoxicillin trihydrate particles was developed. Particle size analysis was ascertained by NIRS in diffuse reflection mode on different particle size fractions of amoxicillin trihydrate with D90 particle diameters ranging from 6.9 to 21.7 μm. The present problem of fractionating the powder into good enough size fractions to achieve a stable calibration model was solved. By probing dried suspensions measurement parameters were optimized and further combined with the best suitable chemometric operations. Thereby the quality of established regression models could be improved considerably. A linear coherence between particle size and absorbance signal was found at specific wavenumbers. Satisfactory clustering by particle size was achieved by principal component analysis (PCA) whereas partial least squares regression (PLSR) and principal component regression (PCR) was compared for quantitatively calibrating the NIRS data. PLSR turned out to predict unknown test samples slightly better than PCR.     

Addressing new analytical challenges in protein formulation development

As the share of therapeutic proteins in the arsenal of modern medicine continue increasing, relatively little progress has been made in the development of analytical methods that would address specific needs encountered during the development of these new drugs. Consequently, the researchers resort to adaptation of existing instrumentation to meet the demands of rigorous bioprocess and formulation development. In this report, we present a number of such adaptations as well as new instruments that allow efficient and precise measurement of critical parameters throughout the development stage. The techniques include use of atomic force microscopy to visualize proteinacious sub-visible particles, use of extrinsic fluorescent dyes to visualize protein aggregates, particle tracking analysis, determination of the concentration of monoclonal antibodies by the analysis of second-derivative UV spectra, flow cytometry for the determination of subvisible particle counts, high-throughput fluorescence spectroscopy to study phase separation phenomena, an adaptation of a high-pressure liquid chromatography (HPLC) system for the measurement of solution viscosity and a variable-speed streamlined analytical ultracentrifugation method. An ex vivo model for understanding the factors that affect bioavailability after subcutaneous injections is also described. Most of these approaches allow not only a more precise insight into the nature of the formulated proteins, but also offer increased throughput while minimizing sample requirements.     

Identifying and measuring alcohol abuse among the elderly: serious problems with existing instrumentation

Existing instruments for identifying and measuring alcohol abuse are inappropriate for use with elderly populations because of differences between the younger populations on which these measures were standardized and the elderly. Five domains used in measuring alcohol abuse--level of consumption, alcohol-related social and legal problems, alcohol-related health problems, symptoms of drunkenness or dependence and self-recognition of the problem--and the extent to which these domains (as currently measured) apply to elderly populations are discussed. Some recommendations for developing instruments suitable for the elderly are made.     

Application of a Best Practice Approach Using Resonant Mass Measurement for Biotherapeutic Product Characterization

Characterizing and quantifying subvisible particles in protein drug products is critical to ensuring product quality. A variety of analytical methods are used to detect and make meaningful measurements of subvisible particles. Resonant mass measurement (RMM) is a novel technology that characterizes the subvisible particle content of samples on a particle-by-particle basis. The technology presents great promise in the study of therapeutic protein products. As an emerging tool in the biopharmaceutical field, the best practices and limitations of RMM for protein products have not been well established. One key challenge of particle analysis is producing robust and reliable data, with high precision and accuracy, for particle characterization. In this study, we develop a set of possible best practices for RMM using a model protein system. We test the effects of these practices on the repeatability and reproducibility of particle measurements. Additionally, we present the data collected under a rigorously controlled set of operating conditions at 3 collaborating sites as well as a summary of the resulting optimal practices. In employing these practices, we successfully obtained improved relative standard deviation values and achieved high reproducibility and repeatability in both sizing and concentration measurement results over a broad range of sample volumes.     

An Interlaboratory Comparison on the Characterization of a Sub-micrometer Polydisperse Particle Dispersion

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 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 polydisperse 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.     

Time-of-flight aerodynamic particle size analyzers: their use and limitations for the evaluation of medical aerosols.

Time-of-flight (TOF) aerosol analyzers are a class of instruments that measure the aerodynamic diameter of individual particles following a controlled acceleration in a well-defined flow field. Two instruments have been used to analyze the size of medical aerosols: Aerosizer particle size analyzer (TSI Particle Instruments/Amherst, Amherst, MA), Aerodynamic Particle Sizer (APS) aerosol spectrometer (TSI) Both instruments are capable of sizing several thousand particles a second, making it possible to obtain aerodynamic particle size distributions in a few seconds compared with up to 1 hour per measurement using compendial methods that are based on either the multistage liquid impinger or cascade impactor. This rapidity makes TOF analysis attractive for product development, as many different variables can potentially be investigated during a short period of time. The data thus obtained should be used with caution, however. Several issues, most notably the lack of a direct relationship with the mass of drug substance present and the vulnerability of the measurements to coincidence effects when sampling concentrated aerosols, may severely limit the value of data from many aerosol delivery systems, especially pressurized metered dose inhalers (pMDIs). A review of the literature illustrating the issues that are involved and providing guidance on the most appropriate uses of these analyzers is presented.     

Micro-Flow Imaging: Flow Microscopy Applied to Sub-visible Particulate Analysis in Protein Formulations

The need to monitor, measure, and control sub-visible proteinaceous particulates in biopharmaceutical formulations has been emphasized in recent publications and commentaries. Some of these particulates can be highly transparent, fragile, and unstable. In addition, for much of the size range of concern, no practical measurement method with adequate sensitivity and repeatability has been available. A complication in measuring protein particulates in many formulations is the simultaneous presence of other particle types such as silicone micro-droplets, air bubbles, and extrinsic contaminants. The need has therefore been identified for new analytical methods which can accurately measure and characterize sub-visible particulates in formulations. Micro-flow imaging has been shown to provide high sensitivity in detecting and imaging transparent protein particles and a unique capability to independently analyze such populations even when other particle types are present.Key words: light obscuration, micro-flow imaging, particle sizing, protein aggregation, protein formulation     

Utility of Three Flow Imaging Microscopy Instruments for Image Analysis in Evaluating four Types of Subvisible Particle in Biopharmaceuticals

Subvisible particles (SVPs) are a critical quality attribute of parenteral and ophthalmic products. United States Pharmacopeia recommends the characterizations of SVPs which are classified into intrinsic, extrinsic, and inherent particles. Flow imaging microscopy (FIM) is useful as an orthogonal method in both the quantification and classification of SVPs because FIM instruments provide particle images. In addition to the conventionally used FlowCam (Yokogawa Fluid Imaging Technologies) and Micro-Flow Imaging (Bio-Techne) instruments, the iSpect DIA-10 (Shimadzu) instrument has recently been released. The three instruments have similar detection principles but different optical settings and image processing, which may lead to different results of the quantification and classification of SVPs based on the information from particle images. The present study compares four types of SVP (protein aggregates, silicone oil droplets, and surrogates for solid free-fatty-acid particles, milled-lipid particles, and sprayed-lipid particles) to compare the results of size distributions and classification abilities obtained using morphological features and a deep-learning approach. Although the three FIM instruments were effective in classifying the four types of SVP through convolutional neural network analysis, there was no agreement on the size distribution for the same protein aggregate solution, suggesting that using the classifiers of the FIM instruments could result in different evaluations of SVPs in the field of biopharmaceuticals.     

Characterization of particles in protein solutions: reaching the limits of current technologies

Recent publications have emphasized the lack of characterization methods available for protein particles in a size range comprised between 0.1 and 10 μm and the potential risk of immunogenicity associated with such particles. In the present paper, we have investigated the performance of light obscuration, flow microscopy, and Coulter counter instruments for particle counting and sizing in protein formulations. We focused on particles 2-10 μm in diameter and studied the effect of silicon oil droplets originating from the barrel of pre-filled syringes, as well as the effect of high protein concentrations (up to 150 mg/ml) on the accuracy of particle characterization. Silicon oil was demonstrated to contribute significantly to the particle counts observed in pre-filled syringes. Inconsistent results were observed between different protein concentrations in the range 7.5-150 mg/ml for particles <10 μm studied by optical techniques (light obscuration and flow microscopy). However, the Coulter counter measurements were consistent across the same studied concentration range but required sufficient solution conductivity from the formulation buffer or excipients. Our results show that currently available technologies, while allowing comparisons between samples of a given protein at a fixed concentration, may be unable to measure particle numbers accurately in a variety of protein formulations, e.g., at high concentration in sugar-based formulations.     

A Platform for Preparing Homogeneous Proteinaceous Subvisible Particles With Distinct Morphologies

Regulatory authorities and scientific communities are increasingly attentive to the known and universal presence of small particulates in biological drug products. The underlying concern is that these particulates may cause unwanted formation of antidrug antibodies in patients. Pharmacological studies, however, have to date not succeeded in unambiguously identifying risk-prone particle properties. This lack of success may be partly due to a lack of available, well-defined, homogenous particle material. Protein particles arising from stress of protein drug products are by nature often highly heterogeneous in size, morphology, and structure of the constituent protein in the particles. Here, we present simple and pharmaceutically relevant stress conditions to produce 8 different highly homogenous micrometer-sized protein particles from human insulin, representing very different morphologies and conformation of the constituent protein molecules in the particles generated. Insulin's self-association patterns were varied by formulation approaches to create diverse starting materials. The resulting collection of homogenous particles underlines that the particle formation is not necessarily a random process but a consequence of formulation and specific stress condition. Owing to the inherent homogenicity of these populations, the particle materials can act as a standard platform for further studies on insulin subvisible particles in drug products.     

Particle sizing measurements in pharmaceutical applications: Comparison of in-process methods versus off-line methods

It has been previously described that when a sample’s particle size is determined using different sizing techniques, the results can differ considerably. The purpose of this study was to review several in-process techniques for particle size determination (Spatial Filtering Velocimetry, Focused Beam Reflectance Measurements, Photometric Stereo Imaging, and the Eyecon® technology) and compare them to well-known and widespread off-line reference methods (laser diffraction and sieve analysis). To start with, a theoretical explanation of the working mechanism behind each sizing technique is presented, and a comparison between them is established. Secondly, six batches of granules and pellets (i.e., spherical particles) having different sizes were measured using these techniques. The obtained size distributions and related D₁₀, D₅₀, and D₉₀ values were compared using the laser diffraction wet dispersion method as reference technique. As expected, each technique provided different size distributions with different D values. These dissimilarities were examined and explained considering the measurement principles behind each sizing technique. The particle property measured by each particle size analyzer (particle size or chord length) and how it is measured as well as the way in which size information is derived and calculated from this measured property and how results are presented (e.g., volume or mass distributions) are essential for the interpretation of the particle size data.     

洁净室中悬浮粒子的测量不确定度评估

目的：对洁净室悬浮粒子的结果进行测量不确定度评估。方法：通过建立数学模型,识别和评估悬浮粒子测量过程的不确定度分量。结果：量化了各分量的相对标准不确定度,并计算出合成标准不确定度。取k=2（置信概率95%）,最终得出测量结果的扩展不确定度并给出测量不确定度报告。结论：洁净室悬浮粒子的测量不确定度主要来源为仪器计量性能引入的不确定度及各采样点的布置和测量引入的不确定度。     

Development of a High-Volume Concentrated Ambient Particles System (CAPS) for Human and Animal Inhalation Toxicological Studies

A two-stage, high-volume, ambient particle concentrator was developed and characterized. This versatile system, depending on its operational parameters, can be used to fractionate and concentrate particles in three size ranges (PM 10-2.5, PM 10-1, PM 2.5-1) . The performance of this concentrated ambient particle system (CAPS), as well as its individual virtual impaction stages, was investigated as a function of several parameters, including minor-to-total flow ratios and acceleration nozzle Reynolds number. During these laboratory tests, performance parameters such as concentration enrichment factor (CF), particle losses, collection efficiency curves, cutpoint, and pressure drop were measured. The main objective of these investigations was to optimize the ability of the system to concentrate ambient PM 2.5-10 and PM 1-10 particles. PM 2.5-10 particles were concentrated by a factor of 70 to 150. The flow rate of the concentrated aerosol can range between 12.5 and 50 LPM (L/min). Other features of the system include relatively low-pressure drops in the major and minor flows, low particle losses, and a compact design. Performance evaluation of the system also confirmed that separation and concentration of the PM 2.5-10 particles occurred without any significant distortion of the size distribution, during the concentration process. Similar results were obtained for the PM 1-10 size range. For this size range, concentration enrichment was 70 times, and again, no particle size distribution distortion was observed. The overall performance of this versatile system makes it suitable for inhalation toxicological studies.     

Development of a high-volume concentrated ambient particles system (CAPS) for human and animal inhalation toxicological studies

A two-stage, high-volume, ambient particle concentrator was developed and characterized. This versatile system, depending on its operational parameters, can be used to fractionate and concentrate particles in three size ranges (PM(10-2.5), PM(10-1), PM(2.5-1)). The performance of this concentrated ambient particle system (CAPS), as well as its individual virtual impaction stages, was investigated as a function of several parameters, including minor-to-total flow ratios and acceleration nozzle Reynolds number. During these laboratory tests, performance parameters such as concentration enrichment factor (CF), particle losses, collection efficiency curves, cutpoint, and pressure drop were measured. The main objective of these investigations was to optimize the ability of the system to concentrate ambient PM(2.5-10) and PM(1-10) particles. PM(2.5-10) particles were concentrated by a factor of 70 to 150. The flow rate of the concentrated aerosol can range between 12.5 and 50 LPM (L/min). Other features of the system include relatively low-pressure drops in the major and minor flows, low particle losses, and a compact design. Performance evaluation of the system also confirmed that separation and concentration of the PM(2.5-10) particles occurred without any significant distortion of the size distribution, during the concentration process. Similar results were obtained for the PM(1-10) size range. For this size range, concentration enrichment was 70 times, and again, no particle size distribution distortion was observed. The overall performance of this versatile system makes it suitable for inhalation toxicological studies.     

Epidemiological evidence on health effects of ultrafine particles.

Evidence from epidemiologic studies linking ambient concentrations of particulate matter to morbidity and mortality influenced the guidelines for air quality standards worldwide. With the improvement of measurement techniques, clearer effects were observed with smaller particle sizes. Based on these effects and results from animal studies on the potential toxicity of ultrafine particles, recent epidemiologic studies focus on the health effects of particles which are less than 100nm in diameter. However, most of the studies are ongoing and only few results have been available so far. Six panel studies with patients suffering from chronic pulmonary diseases have been performed in Germany, Finland and the United Kingdom. Overall, a decrease of peak expiratory flow (PEF) and an increase of daily symptoms and medication use was found for elevated daily particle concentrations. Effects were seen with both fine and ultrafine particles. One large study on daily mortality from Germany showed comparable effects of fine and ultrafine particles in all size classes considered. However, fine particles showed more immediate effects while ultrafine particles showed more delayed effects on mortality. The limited number of epidemiological studies suggest that there are health effects of fine and ultrafine particles which might be independent of each other. If these effects are confirmed by ongoing research, monitoring and regulation of particulate air pollution may need to be revised.     

Nanoparticle analysis and characterization methodologies in environmental risk assessment of engineered nanoparticles

Environmental risk assessments of engineered nanoparticles require thorough characterization of nanoparticles and their aggregates. Furthermore, quantitative analytical methods are required to determine environmental concentrations and enable both effect and exposure assessments. Many methods still need optimization and development, especially for new types of nanoparticles in water, but extensive experience can be gained from the fields of environmental chemistry of natural nanomaterials and from fundamental colloid chemistry. This review briefly describes most methods that are being exploited in nanoecotoxicology for analysis and characterization of nanomaterials. Methodological aspects are discussed in relation to the fields of nanometrology, particle size analysis and analytical chemistry. Differences in both the type of size measures (length, radius, aspect ratio, etc.), and the type of average or distributions afforded by the specific measures are compared. The strengths of single particle methods, such as electron microscopy and atomic force microscopy, with respect to imaging, shape determinations and application to particle process studies are discussed, together with their limitations in terms of counting statistics and sample preparation. Methods based on the measurement of particle populations are discussed in terms of their quantitative analyses, but the necessity of knowing their limitations in size range and concentration range is also considered. The advantage of combining complementary methods is highlighted.     

Disassembly and reassembly improves morphology and thermal stability of human papillomavirus type 16 virus-like particles

Recombinant human papillomavirus (HPV) 16 L1 protein self-assembles into virus-like particles (VLPs) with diameters of 40 to 60 nm, which are key components in prophylactic HPV vaccines. Marked improvement in morphology and thermal stability on VLP disassembly and reassembly was demonstrated at production scale. Differential scanning calorimetry showed enhanced conformational stability as indicated by the unfolding temperatures and peak heights/areas. Cloud point studies indicated (1) a much lower propensity for post-reassembly VLPs to aggregate during a time course study and (2) much higher cloud point temperatures. In-solution atomic force microscopy showed more uniform size distribution and fully closed particles, with evidence of virion-like assembly revealed by the structural details from a single particle image. Similar approaches for the reassembly of other recombinant VLPs with intrinsic conformational switches would be expected to improve the particle properties and render nanoparticles more suitable for use as vaccines or therapeutics.From the Clinical EditorThe authors of this study demonstrated that recombinant human papillomavirus 16 L1 protein self-assembles into virus-like particles (VLPs) with marked improvement in morphology and thermal stability on VLP disassembly and reassembly at production scale. This is expected to render these nanoparticles more suitable for use as vaccines or therapeutics.     

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.