In-line Size Monitoring of a Twin Screw Granulation Process Using High-Speed Imaging

Purpose

This study aimed to evaluate the feasibility of implementing a 3D high-speed imaging camera for in-line monitoring of a continuous wet granulation process and its potential for real-time process control.

Methods

The Eyecon^TM camera was used for monitoring a twin screw granulated-placebo formulation composed of lactose (73.5 %), microcrystalline cellulose (20 %), hydroxypropylmethyl cellulose (5 %), and croscarmellose sodium (1.5 %). Water was used as the granulating liquid at liquid-to-solid (L/S) ratios from 0.15 to 0.35 at 0.05 increments. The lactose particle size was varied using three grades of lactose: pharmatose 200 M, impalpable, and SuperTab 30GR. The process was perturbed by changing the L/S ratio, while monitoring the process using the camera. A Shewhart control chart was constructed using control limits obtained under steady-state conditions. Statistical process control tools were used to assess the implications of choosing different quality characteristics on the detectability of process shifts.

Results

The camera showed sensitivity to variation in process parameters, which was evident in the calculated granule size and particle count. Larger particles had strong leverage on the converted volume distribution, which significantly affected the magnitude of variability in the statistics of particle diameters. It was shown that particle count and d ₁₀ were more sensitive to process shifts than d ₅₀ and d ₉₀.

Conclusions

The sensitivity of the high-speed camera, coupled with image analysis, to process perturbations and the variety of generated characteristics of particle attributes demonstrate the potential of this technique for continuous process monitoring and control.     

Critical Evaluation of Microfluidic Resistive Pulse Sensing for Quantification and Sizing of Nanometer- and Micrometer-Sized Particles in Biopharmaceutical Products

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 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.     

Sizing and phenotyping of cellular vesicles using Nanoparticle Tracking Analysis

Cellular microvesicles and nanovesicles (exosomes) are involved in many disease processes and have major potential as biomarkers. However, developments in this area are constrained by limitations in the technology available for their measurement. Here we report on the use of fluorescence nanoparticle tracking analysis (NTA) to rapidly size and phenotype cellular vesicles. In this system vesicles are visualized by light scattering using a light microscope. A video is taken, and the NTA software tracks the brownian motion of individual vesicles and calculates their size and total concentration. Using human placental vesicles and plasma, we have demonstrated that NTA can measure cellular vesicles as small as ≈ 50 nm and is far more sensitive than conventional flow cytometry (lower limit ≈ 300 nm). By combining NTA with fluorescence measurement we have demonstrated that vesicles can be labeled with specific antibody-conjugated quantum dots, allowing their phenotype to be determined. FROM THE CLINICAL EDITOR: The authors of this study utilized fluorescence nanoparticle tracking analysis (NTA) to rapidly size and phenotype cellular vesicles, demonstrating that NTA is far more sensitive than conventional flow cytometry.     

纳米颗粒跟踪分析和纳米流式细胞仪对脂肪间充质干细胞的细胞外囊泡检测结果评价

目的 探讨纳米颗粒跟踪分析（nanoparticle tracking analysis,NTA）和纳米流式细胞仪（nano-flow cytometry,nanoFCM）对对照品微球和脂肪间充质干细胞（adipose mesenchymal stem cells,AdMSCs）的细胞外囊泡（extracellular vesicles,EVs）的检测能力,为EVs的鉴定和质量控制提供依据。方法 首先使用对照品微球来验证NTA和nanoFCM的检测能力。随后,对于聚乙二醇6000沉淀-超速离心法制备的AdMSCs-EVs,通过Western blotting和电子透射显微镜进行一般特征鉴定后,使用NTA和nanoFCM来检测其粒径分布和PKH67染色后的荧光颗粒占比。结果 NTA和nanoFCM在颗粒浓度方面的检测能力相近,但是nanoFCM的精度更高,粒径区分度更好。用NTA检测EVs的检测结果显示NTA粒径分布较广,nanoFCM粒径分布较窄;NTA的PKH67的荧光检测阳性率显著低于nanoFCM。结论 NanoFCM拥有较高的精度,但也存在着一定应用局限性。NTA的粒径区分度相对较差,但是可检测的粒径范围更广。因此,NTA可以满足EVs的粒径分布检测,对荧光检测具有较高灵敏度的nanoFCM更适合分析EVs的表面标志物比例。     

Effects of Submicron Particles on Formation of Micron-Sized Particles During Long-Term Storage of an Interferon-Beta-1a Solution

We present evidence that homogeneous submicron particles can influence the growth rate of larger particles upon long-term storage in a temperature-dependent manner. Interferon-beta-1a was thermally stressed at 50°C for 6 h and characterized using nanoparticle tracking analysis (NTA), microflow digital imaging (MFI), and circular dichroism (CD) spectroscopy. This study showed selective formation of submicron particles exhibiting a perturbed protein conformation. These thermally induced submicron particles were spiked into an unstressed solution at three levels, and then monitored for micron-sized particle formation upon storage at 5°C and 25°C for 12 months. The resulting particle growth effects were temperature dependent. NTA and MFI results at 5°C showed little evidence that initial submicron particle levels impacted particle growth across the range ~0.03–25 μm. In contrast, MFI results at 25°C indicated that particle growth in the 1–10 μm size range correlated strongly with initial submicron particle levels, and particle counts in the 10–25 μm size range were highest after 12 months for the samples with highest initial submicron particle content. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:347–351, 2013     

Qualitative High-Throughput Analysis of Subvisible Particles in Biological Formulations Using Backgrounded Membrane Imaging

High-throughput analysis of low-volume samples for detection of subvisible particles (SVPs) in biologic formulations remains an unmet need in the pharmaceutical industry. Some commonly used methods, such as light obscuration and microflow imaging, for SVP analysis are not high throughput and require significant amounts of sample volume, which may impede the collection of SVP data when therapeutic protein amounts are limited, typically during early stages of formulation development. We evaluated backgrounded membrane imaging (BMI) as an orthogonal method for SVP analysis and identified critical experimental parameters. Protein concentration, sample viscosity, and membrane coverage area had to be adjusted for each sample, especially those with high protein concentrations. A comparative analysis of particle counts obtained from BMI, light obscuration, and microflow imaging for five protein samples revealed that particle counts obtained with BMI were significantly higher than those acquired with the other two techniques for all particle size categories. BMI could not accurately count particles in protein-containing samples, as the image analysis software could not accurately trace the boundaries of translucent particles. Based on our results, BMI could be used as an orthogonal method for particle characterization when sample material is limited, such as during the early stages of formulation development or screening.     

Quantification and Characterization of Subvisible Proteinaceous Particles in Opalescent mAb Formulations Using Micro-Flow Imaging

Micro-flow imaging (MFItm) has been shown to be more sensitive than light obscuration (LO) methods for measuring subvisible proteinaceous particles in protein formulations. Given the potential challenges in detecting particulates in opalescent mAb formulations, the accuracy of MFI to size and count particles in opalescent solutions was investigated and compared to LO and membrane microscopy methods. Proteinaceous monoclonal antibody (mAb) particles, generated either by chemical denaturation or agitation stress, polystyrene and glass particles were used as model systems for measurements in opalescent mAb solutions. The sizing and counting accuracies of MFI were unaffected by the opalescence of the medium. Using glass particles as a model system for proteinaceous particles, MFI was able to detect relatively low particle concentrations (~ 10/mL) in opalescent solutions. MFI showed excellent linearity (R2 = 0.9969) for quantifying proteinaceous particles in opalescent solutions over a wide range of particle concentrations (~ 20-160,000/mL). Analyses of MFI particle image intensities revealed significant differences in the transparency of proteinaceous particles as a function of their size and mode of generation. LO method significantly underestimated proteinaceous particles, particularly those in the 2–10 μm size range. The less opaque proteinaceous particles were relatively more underestimated by the LO method in opalescent solutions.     

Microscopic image analysis techniques for the morphological characterization of pharmaceutical particles: Influence of process variables

This study investigated the effects of various process variables on some of the shape factors most widely used in the morphological characterization of pharmaceutical particles (including circularity, aspect ratio, er, and the recently proposed Vr and Vp). After optimization of the illumination system and greyscale thresholds for discrimination of particle background, we evaluated the effects of process variables within the image capture and analysis system, including the image capture device (video or digital camera), image color information (24-bits-per-pixel RGB or 8-bits-per-pixel black and white), file type (JPG or TIFF), and JPG file compression ratio. A key aspect was evaluation of the effect of scaling factor (microm/pixel), dependent on real pixel size and optical magnification, on shape factor estimates. The results obtained indicate that accurate estimation of shape factors requires use of a scaling factor below a certain maximum; however, use of very low scaling factors will mean that the field of view is very small, so that it will contain very few particles, implying a time-consuming increase in the number of images that must be analyzed. Finally, we use statistical procedures to estimate the minimum number of particles that must be analyzed in order to provide accurate estimates of shape factors.     

Interference from Proteins and Surfactants on Particle Size Distributions Measured by Nanoparticle Tracking Analysis (NTA)

Purpose

Characterization of submicron protein particles continues to be challenging despite active developments in the field. NTA is a submicron particle enumeration technique, which optically tracks the light scattering signal from suspended particles undergoing Brownian motion. The submicron particle size range NTA can monitor in common protein formulations is not well established. We conducted a comprehensive investigation with several protein formulations along with corresponding placebos using NTA to determine submicron particle size distributions and shed light on potential non-particle origin of size distribution in the range of approximately 50–300 nm.

Methods

NTA and DLS are performed on polystyrene size standards as well as protein and placebo formulations.

Results

Protein formulations filtered through a 20 nm filter, with and without polysorbate-80, show NTA particle counts. As such, particle counts above 20 nm are not expected in these solutions. Several other systems including positive and negative controls were studied using NTA and DLS.

Conclusions

These apparent particles measured by NTA are not observed in DLS measurements and may not correspond to real particles. The intent of this article is to raise awareness about the need to interpret particle counts and size distribution from NTA with caution.

     

Compatibility, physical stability, and characterization of an IgG4 monoclonal antibody after dilution into different intravenous administration bags

The physical stability of an immunoglobulin G4 monoclonal antibody (mAb) upon dilution into intravenous (i.v.) bags containing 0.9% saline was examined. Soluble aggregates and subvisible particles were observed by size‐exclusion high‐performance liquid chromatography (SE‐HPLC) and light obscuration when formulated with suboptimal levels of polysorbate 20. The formation of soluble aggregates and particulates was further characterized by a combination of SE‐HPLC, nanoparticle tracking analysis (NTA), microflow‐digital imaging (MFI), and turbidity measurements. With sufficient PS20 levels, particle formation was minimized, although quantification of submicron sized particles by NTA was not possible because of the interference from PS20. Intravenous bags composed of polyvinyl chloride caused more protein particle formation than polyolefin bags. Differences between bag types were affected by removing headspace and by transferring the saline solution into glass vials. Characterization studies with Fourier transform infrared microscopy and extrinsic fluorescence spectroscopy demonstrated that isolated particles contained native‐like secondary structure with partially altered tertiary structure, compared with heat‐denatured and nonstressed controls. Transmission electron microscopy and MFI analysis showed particles had an amorphous morphology of varying sizes. Particles contained some non‐native disulfide bond crosslinks, potentially initiated by low levels of free thiol in the native mAb. The critical role of proper formulation design to stabilize proteins against physical instability during i.v. administration is discussed.     

Physical Characterization and Stabilization of a Lentiviral Vector Against Adsorption and Freeze-Thaw

A replication-deficient lentiviral vector encoding the tumor antigen gene NY-ESO-1 was characterized in terms of vector morphology, particle size range, concentration, and zeta potential using a variety of physical methods. Environmentally stressed vector samples were then evaluated in terms of viral vector particle size and concentration by nanoparticle tracking analysis (NTA). These NTA stability results correlated reasonably well with a quantitative polymerase chain reaction assay for quantitation of viral genome copy number (r² = 0.80). Approximately 40 pharmaceutical excipients were examined for their ability to stabilize the vector against exposure to an adsorptive container surface (glass) as well as freeze-thaw cycling using NTA as the screening method. Stabilizing additives that inhibited viral vector particle loss under these conditions included proline, lactose, and mannitol. Several candidate frozen liquid formulations that contained a combination of these lead excipients and various buffering agents were further evaluated for their ability to stabilize the viral vector. The additional benefit of lowering the Tris buffer concentration was observed. This study highlights the use of physical particle assays such as NTA for initial screening of stabilizing excipients to minimize vector loss due to container adsorption and freeze-thaw cycling to facilitate early formulation development of viral vector candidates in frozen liquid formulations.     

Application of computer image analysis for characterization of pellets

The morphological characteristics of pellets are critical parameters, because of their physico-chemical features depend on the size, shape and surface geometric of the particles. To ensure the spherical shape and required particle of pharmaceutical pellets size is a prerequisite. The detailed technology is basic requirement for the successful and cost efficient production of particles of acceptable quality. Since the determination of the particle size is influenced by the particle shape, therefore microscopic examination is always suggested, which together with image analysis is suitable for the assesment of the most typical parameters. The method of the microscopic image analysis is useful not only for particle size measurement, but also for particle shape and texture evaluation, with a high sensitivity. Using the microscopic method particle shape may be defined either qualitatively and quantitatively. Reviewing the related articles and results on the investigation of sugar pellets demonstrate that roundness characterization is strongly influenced by the applied statistical shape parameters.     

Quantitative Laser Diffraction for Quantification of Protein Aggregates: Comparison With Resonant Mass Measurement,Nanoparticle Tracking Analysis,Flow Imaging,and Light Obscuration

In the past, analysis of micron-sized (>1.0 μm) aggregates of therapeutic proteins has been limited to light obscuration (LO), and appropriate quantitative methods of evaluating protein aggregates need to be developed. Recently, novel methods with enhanced reliability and sensitivity, such as nanoparticle tracking analysis (NTA), resonant mass measurement (RMM), and flow imaging (FI), have emerged. We have found that quantitative laser diffraction (qLD) is also effective for quantitative evaluation of protein aggregates over a wide size range. However, the different detection principles of the methods potentially lead to inconsistencies in results. This study aimed to compare particle size distributions and concentrations of protein aggregates using the orthogonal methods. Protein aggregates were generated by stirring an immunoglobulin solution. Serial dilutions of the aggregates stock were analyzed by RMM, FI, and qLD to obtain the particle size distribution and concentration using each method. In addition, size distribution of a protein aggregates solution was compared by RMM, NTA, FI, LO, and qLD. Both particle size distribution and concentration were in good agreement between RMM and qLD (0.3-2 μm) and between FI and qLD (2-20 μm). Thus, we concluded that qLD enables covering of the overlapping particle size range between RMM and FI.     

The impact of material attributes and process parameters on the micronisation of lactose monohydrate

Dry powder inhalers (DPIs), which are important medicines for drug delivery to the lungs, require drug particles in the respirable size range of 1-6 μm for optimal lung deposition. Drugs administered by the oral route also derive benefit from particles in this size range owing to their large surface area to volume ratio, which provides potential for rapid dissolution. Micronisation used in the production of particles, however often leads to heterogeneous product containing mechanically activated surfaces with amorphous content. This study was therefore carried out to evaluate the effect of particle properties of three grades of lactose monohydrate, with sizes above and below the brittle-ductile transition (dcrit) and their interaction with process variables on the quality of micronised material. Following an experimental design, the impact of three factors (grinding pressure, injector pressure and feed rate) on the particulate attributes of micronised powders produced from the different size grades was assessed. Processing conditions were shown to be important determinants of powder properties only for the coarsest starting material. Ultrafine material was achieved by processing finer grade feed stock below dcrit. However the resultant product was more crystalline and transformed on heating to the anhydrous state with markedly reduced onset temperature with lower energy surfaces than powders produced from larger sized starting material. Thus the propensity for micronisation of lactose monohydrate can be altered through control of starting materials and optimal settings for process variables.     

Influence of cohesive properties of micronized drug powders on particle size analysis

Particle size analysis results with respect to micronized, mean particle size below 10 μm, furosemide, chloramphenicol palmitate and acetaminophen particles are dealt with in this paper. Special consideration was given to the effect of the agglomeration of particles on data generated by three size measurement techniques. The physicochemical basis for preparing sufficiently well dispersed and stable suspensions for analysis by employing mechanical methods of pretreatment are shown. Furthermore, methods to determine the state of dispersion and methods to assess the individual particle size before size analysis are described. An attempt was also made to establish the statistical confidence that can be assigned to a particular instrument and the confidence level that may be placed on comparative data obtained with the different particle size analysers. Results especially showed the impact of the agglomeration of very small furosemide particles, mean size 3 μm, on particle size analysis and the importance of controlling the cohesive properties of this drug. To overcome the problems associated with agglomeration more attention must be paid to the physical properties of the drug substance. Combining particle size analysis with bulk density, surface area and microscopical studies also helped to identify potential problems.     

Protein particles: What we know and what we do not know

All therapeutic protein products contain intrinsic particles formed by the aggregation of protein monomers. There is growing interest in understanding particles in biopharmaceutical products, fostered on one hand by significant advancements in particle analysis and on the other hand by concerns about potential impact of particles on product quality and safety. With currently available methods, particles in therapeutic proteins can be counted, sized, and characterized in a rudimentary way over a broad size range (from 10s of nanometers to 100s of micrometers). Here, we review the known attributes of common protein particles, and then discuss the gaps in our current knowledge. The capabilities, limitations, and opportunities for improvement of common particle counting and characterization methods are listed. We conclude that further analytical progress is needed to better classify and characterize the diversity of particles encountered in therapeutic proteins, which may vary in the degree of protein unfolding, the inclusion of nonprotein nucleation centers, and aggregate morphology. Very little is known about the potential correlation between specific particle attributes and increased immunogenicity. In this environment of uncertainty, a deeper understanding about specific particle attributes and potentially increased immunogenicity is greatly needed and will likely be an area of future intensive research. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 101:3568–3579, 2012     

Fluorescence Single Particle Tracking for the Characterization of Submicron Protein Aggregates in Biological Fluids and Complex Formulations

Purpose

To evaluate the potential of fluorescence single particle tracking (fSPT) for the characterization of submicron protein aggregates in human serum, plasma and formulations containing human serum albumin (HSA).

Methods

A monoclonal IgG was covalently labeled with a fluorescent dye and cross-linked with glutaraldehyde. IgG aggregates and fluorescent beads of 0.1 ??m (control) were diluted in buffer, serum and plasma, and their size distributions were analyzed by fSPT and nanoparticle tracking analysis (NTA). In a separate experiment, IgG and HSA, fluorescently labeled with different dyes, were mixed and subjected to heat stress. The stressed sample was analyzed by fSPT using a dual color mode and by NTA.

Results

The accuracy and precision of fSPT proved to be comparable to NTA. fSPT was able to successfully measure all the samples in buffer, serum and plasma. The average size of the cross-linked protein aggregates showed a slight increase in biological fluids. Moreover, fSPT analysis showed that a significant proportion of the aggregates formed by subjecting an IgG/HSA mixture to heat stress were composed of both proteins.

Conclusion

fSPT is a powerful technique for the characterization of submicron protein aggregates in biological fluids and complex formulations.     

Elucidating spray-dried dispersion dissolution mechanisms with focused beam reflectance measurement: contribution of polymer chemistry and particle properties to performance

Amorphous spray-dried dispersions (SDDs) are a key enabling technology for oral solid dosage formulations, used to improve dissolution behaviour and clinical exposure of poorly soluble active pharmaceutical ingredients (APIs). Appropriate assessment of amorphous dissolution mechanisms is an ongoing challenge. Here we outline the novel application using focused beam reflectance measurement (FBRM) to analyse particle populations orthogonal to USP 2 dissolution. The relative impact of polymer substitution and particle attributes on 25% BMS-708163/HPMC-AS SDD dissolution was assessed. Dissolution mechanisms for SDDs were categorized into erosion versus disintegration. Beyond an initial mixing period, FBRM particle counts diminish slowly and particles are detectable until the point where API dissolution is complete. There is correlation between FBRM particle count decay rate, representing loss of SDD particles in the dissolution media, and UV dissolution rate, measuring dissolved API. For the SDD formulation examined, the degree of succinoyl substitution for HPMC-AS, SDD particle size and surface area all had an impact on dissolution. These data indicate the SDD displayed an erosion mechanism and that FBRM is capturing a rate-limiting step. From this screening tool, the mechanistic understanding and measured impact of polymer chemistry and particle properties can inform a risk-assessment and control strategy for this compound.     

Ultrafine particle deposition in humans during rest and exercise

Ultrafine particles (diameter < 100 nm) may be important in the health effects of air pollution, in part because of their predicted high respiratory deposition. However, there are few measurements of ultrafine particle deposition during spontaneous breathing. The fractional deposition for the total respiratory tract of ultrafine carbon particles (count median diameter = 26 nm, geometric standard deviation = 1.6) was measured in 12 healthy subjects (6 female, 6 male) at rest (minute ventilation 9.0 +/- 1.3 L/min) using a mouthpiece exposure system. The mean +/- SD fractional deposition was 0.66 +/- 0.11 by particle number and 0.58 +/- 0.13 by particle mass concentration, similar to model predictions. The number deposition fraction increased as particle size decreased, reaching 0.80 +/- 0.09 for the smallest particles (midpoint count median diameter = 8.7 nm). No gender differences were observed. In an additional 7 subjects (2 female, 5 male) alternating rest with moderate exercise (minute ventilation 38.1 +/- 9.5 L/min), the deposition fraction during exercise increased to 0.83 +/- 0.04 and 0.76 +/- 0.06 by particle number and mass concentration, respectively, and reached 0.94 +/- 0.02 for the smallest particles. Experimental deposition data exceeded model predictions during exercise. The total number of deposited particles was more than 4.5-fold higher during exercise than at rest because of the combined increase in deposition fraction and minute ventilation. Fractional deposition of ultrafine particles during mouth breathing is high in healthy subjects, and increases further with exercise.