Contribution of Intravenous Administration Components to Subvisible and Submicron Particles Present in Administered Drug Product

Particulate matter present in drug products intended for parenteral administration to patients is typically monitored and controlled in the finished drug product to minimize potential risks to patients. In contrast to particulates found in drug products, the current study evaluated particulates representative of materials and operations typically used in the dose preparation and administration of drug products. A comprehensive assessment of intrinsic and extrinsic sources of subvisible and submicron particulates arising from materials associated with subcutaneous and intravenous dose preparation and administration was conducted. In particular, particles arising from disposable syringes, commercial sterile diluents, and intravenous supplies were quantitated using established methods for subvisible (light obscuration, flow imaging) and submicron particles (resistive pulse sensing). Each of these sources contributed varying amounts of particulates; therefore, owing to sources from materials required for administration, it is inadequate to assume that the total particulate load delivered to patients arises solely from the drug product. Careful consideration of the administration method and supplies used can improve the predictability of particulate levels present in dose preparations or administration volumes.     

The Coulter Principle for Analysis of Subvisible Particles in Protein Formulations

The Coulter principle can be used for analysis of subvisible particles in protein formulations. The approach has several advantages including: an orthogonal operating principle, high sensitivity, ability to detect very small particles, excellent reproducibility, and high-resolution size information. This minireview discusses some of the important considerations that must be taken into account when utilizing the Coulter principle for subvisible particle analysis in protein formulations.     

折光率对微流成像法检测蛋白制剂不溶性微粒的影响

目的：蛋白制剂中不溶性微粒的含量是衡量样品质量的重要指标之一,为了更为准确地检测不溶性微粒的含量和粒径,本研究探讨了溶液折光率对于微流成像系统检测不溶性微粒的影响。方法：本研究以牛血清白蛋白（BSA）为例,通过常见的外界刺激条件（冷冻-解冻）制备高浓度的蛋白质不溶性微粒,并将此微粒稀释至不同折光率的溶液（由PEG1000、海藻糖制备）中,利用微流成像系统检测不溶性微粒的含量。结果：当溶液的折光率接近蛋白质不溶性微粒折光率时,利用微流成像技术检测的不溶性微粒含量低于实际的微粒含量。此外,随着溶液折光率的增加,采用微流成像技术检测出的不溶性微粒的粒径也随之减小。结论：蛋白质溶液的折光率发生改变,会影响利用微流成像技术检测不溶性微粒的准确性。因此,利用微流成像技术检测蛋白制剂中不溶性微粒时,需要考虑到制剂处方的折光率对于检测不溶性微粒的影响,必要时可以采用稀释的方法降低折光率对蛋白质颗粒的屏蔽作用。     

Stress Factors in Protein Drug Product Manufacturing and Their Impact on Product Quality

Injectable protein-based medicinal products (drug products, or DPs) must be produced by using sterile manufacturing processes to ensure product safety. In DP manufacturing the protein drug substance, in a suitable final formulation, is combined with the desired primary packaging (e.g., syringe, cartridge, or vial) that guarantees product integrity and enables transportation, storage, handling and clinical administration. The protein DP is exposed to several stress conditions during each of the unit operations in DP manufacturing, some of which can be detrimental to product quality. For example, particles, aggregates and chemically-modified proteins can form during manufacturing, and excessive amounts of these undesired variants might cause an impact on potency or immunogenicity. Therefore, DP manufacturing process development should include identification of critical quality attributes (CQAs) and comprehensive risk assessment of potential protein modifications in process steps, and the relevant steps must be characterized and controlled. In this commentary article we focus on the major unit operations in protein DP manufacturing, and critically evaluate each process step for stress factors involved and their potential effects on DP CQAs. Moreover, we discuss the current industry trends for risk mitigation, process control including analytical monitoring, and recommendations for formulation and process development studies, including scaled-down runs.     

Deep Convolutional Neural Network Analysis of Flow Imaging Microscopy Data to Classify Subvisible Particles in Protein Formulations

Flow-imaging microscopy (FIM) is commonly used to characterize subvisible particles in therapeutic protein formulations. Although pharmaceutical companies often collect large repositories of FIM images of protein therapeutic products, current state-of-the-art methods for analyzing these images rely on low-dimensional lists of “morphological features” to characterize particles that ignore much of the information encoded in the existing image databases. Deep convolutional neural networks (sometimes referred to as “CNNs or ConvNets”) have demonstrated the ability to extract predictive information from raw macroscopic image data without requiring the selection or specification of “morphological features” in a variety of tasks. However, the inherent heterogeneity of protein therapeutics and optical phenomena associated with subvisible FIM particle measurements introduces new challenges regarding the application of ConvNets to FIM image analysis. We demonstrate a supervised learning technique leveraging ConvNets to extract information from raw images in order to predict the process conditions or stress states (freeze-thawing, mechanical shaking, etc.) that produced a variety of different protein particles. We demonstrate that our new classifier, in combination with a “data pooling” strategy, can nearly perfectly differentiate between protein formulations in a variety of scenarios of relevance to protein therapeutics quality control and process monitoring using as few as 20 particles imaged via FIM.     

预灌封注射器橡胶活塞表面硅油提取及向甲氨蝶呤注射液中迁移的研究

目的 研究预灌封注射器的橡胶活塞表面硅油的使用量,以及硅油在甲氨蝶呤注射液中的迁移量。方法 采用傅里叶变换红外光谱法测定预灌封注射器中溴化丁基橡胶活塞表面的硅油含量,并采用电感耦合等离子体质谱法测定甲氨蝶呤注射液中的硅油迁移量。结果 活塞表面的硅油含量为每个0.191 mg,0.5,0.2,0.15 mL规格甲氨蝶呤注射液的最大迁移量分别为2.65,1.26,1.04 μg。结论 傅里叶变换红外光谱法和电感耦合等离子体质谱法能分别快速测定橡胶活塞表面的硅油量和硅油在制剂中的迁移量。     

An Industry Perspective on the Monitoring of Subvisible Particles as a Quality Attribute for Protein Therapeutics

Concern around the lack of monitoring of proteinaceous subvisible particulates in the 0.1–10 mm range has been heightened (Carpenter et al., 2009, J Pharm Sci 98: 1202–1205), primarily due to uncertainty around the potential immunogenicity risk from these particles. This article, representing the opinions of a number of industry scientists, aims to further the discussion by developing a common understanding around the technical capabilities, limitations, as well as utility of monitoring this size range; reiterating that the link between aggregation and clinical immunogenicity has not been unequivocally established; and emphasizing that such particles are present in marketed products which remain safe and efficacious despite the lack of monitoring. Measurement of subvisible particulates in the < 10 μm size range has value as an aid in product development and characterization. Limitations in measurement technologies, variability from container/closure, concentration, viscosity, history, and inherent batch heterogeneity, make these measurements unsuitable as specification for release and stability or for comparability, at the present time. Such particles constitute microgram levels of protein with currently monitored sizes ≥ 10 μm representing the largest fraction. These levels are well below what is detected or reported for other product quality attributes. Subvisible particles remain a product quality attribute that is also qualified in clinical trials.     

Continuing HIV Risk in New York City Injection Drug Users: The Association of Syringe Source and Syringe Sharing

Sterile syringe access is an important means to reduce HIV risk, but many injection drug users (IDU) who obtain syringes from sterile sources continue to share syringes. We examined the factors associated with continuing syringe sharing in New York City. We recruited 500 active IDU in 2005 through respondent-driven sampling. In multiple logistic regression, not obtaining all syringes in the past year exclusively from sterile sources was associated with increased syringe sharing. Ensuring adequate syringe availability as well as engaging and retaining nonusers and inconsistent users in sterile syringe services may increase sterile syringe access and decrease syringe sharing.     

加药环境与加药器具对输液不溶性微粒污染的影响

考察加药环境与加药器具对输液不溶性微粒的影响。取一次性使用无菌加药器具,分别在净化洁净室和病房治疗室,模拟加药,按《中国药典》,以光阻法测定样品中的不溶性微粒,与未模拟加药的输液比较,在病房治疗室和净化洁净室制备的样品中,≥10μm的不溶性微粒均有显著增加(P<0.05),≥25μm的不溶性微粒均增加不明显(P>0.05);病房治疗室制备的样品与净化洁净室制备的样品比较,≥10μm的不溶性微粒增加显著(P<0.05),≥25μm的不溶性微粒增加不明显(P>0.05)。病房治疗室内加药环境及一次性加药器具可使输液中≥10μm的不溶性微粒明显增加。     

Optimization of Infrared Microscopy to Assess Secondary Structure of Insulin Molecules Within Individual Subvisible Particles in Aqueous Formulations

The analysis of subvisible particles is currently challenging but pivotal to the understanding and control of the quality of protein therapeutics. While a range of characterization methods is available for subvisible particles, information on the protein conformation in a particle—considered a possible parameter in eliciting unwanted immunogenicity of protein therapeutics—is especially challenging in the lower micrometer range using existing analytical technologies. Using 6 different protein particle populations, we show that transmission Fourier transform infrared (FTIR) microscopy can determine protein secondary structure in single particles down to 10 μm. The analytical setup presented here is able to immobilize protein particles and obtain transmission FTIR spectra on individual protein particles in their intact aqueous environment. Spectra of dried particles, on the other hand, were found to occasionally differ from spectra of particles in aqueous environment. In summary, using the analytical setup described in this study, transmission FTIR microscopy uniquely provides information on single protein particles in particle populations in their aqueous environment without interference from the background protein solution.     

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.     

Flow Microscopy Imaging Is Sensitive to Characteristics of Subvisible Particles in Peginesatide Formulations Associated With Severe Adverse Reactions

The presence of subvisible particles in formulations of therapeutic proteins is a risk factor for adverse immune responses. Although the immunogenic potential of particulate contaminants likely depends on particle structural characteristics (e.g., composition, size, and shape), exact structure-immunogenicity relationships are unknown. Images recorded by flow imaging microscopy reflect information about particle morphology, but flow microscopy is typically used to determine only particle size distributions, neglecting information on particle morphological features that may be immunologically relevant. We recently developed computational techniques that utilize the Kullback-Leibler divergence and multidimensional scaling to compare the morphological properties of particles in sets of flow microscopy images. In the current work, we combined these techniques with expectation maximization cluster analyses and used them to compare flow imaging microscopy data sets that had been collected by the U.S. Food and Drug Administration after severe adverse drug reactions (including 7 fatalities) were observed in patients who had been administered some lots of peginesatide formulations. Flow microscopy images of particle populations found in the peginesatide lots associated with severe adverse reactions in patients were readily distinguishable from images of particles in lots where severe adverse reactions did not occur.     

Characterization of Virus Particles and Submicron-Sized Particulate Impurities in Recombinant Adeno-Associated Virus Drug Product

Characterization of particulate impurities such as aggregates is necessary to develop safe and efficacious adeno-associated virus (AAV) drug products. Although aggregation of AAVs can reduce the bioavailability of the virus, only a limited number of studies focus on the analysis of aggregates. We explored three technologies for their capability to characterize AAV monomers and aggregates in the submicron (<1 µm) size range: (i) mass photometry (MP), (ii) asymmetric flow field flow fractionation coupled to a UV-detector (AF4-UV/Vis) and (iii) microfluidic resistive pulse sensing (MRPS). Although low counts for aggregates impeded a quantitative analysis, MP was affirmed as an accurate and rapid method for quantifying the genome content of empty/filled/double-filled capsids, consistent with sedimentation velocity analytical ultracentrifugation results. MRPS and AF4-UV/Vis enabled the detection and quantification of aggregate content. The developed AF4-UV/Vis method separated AAV monomers from smaller aggregates, thereby enabling a quantification of aggregates <200 nm. MRPS was experienced as a straightforward method to determine the particle concentration and size distribution between 250-2000 nm, provided that the samples do not block the microfluidic cartridge. Overall, within this study we explored the benefits and limitations of the complementary technologies for assessing aggregate content in AAV samples.     

Microfabricated Particles for Engineered Drug Therapies: Elucidation into the Mechanisms of Cellular Internalization of PRINT Particles

PURPOSE: To investigate the cellular internalization pathways of shape- and size-specific particles as a function of zeta potential in different cell types. METHODS: A top-down particle fabrication technique called PRINT was utilized to fabricate monodisperse 1 microm cylindrical particles. Cellular internalization of these PRINT particles was monitored using confocal microscopy, flow cytometry, and transmission electron microscopy. The endocytic pathway used by 1 microm cationic PRINT particles was evaluated using different inhibitory strategies. Cytotoxicity assays were used to determine the toxicity of both cationic and anionic PRINT particles in multiple cell types. RESULTS: Particle internalization was confirmed using confocal microscopy, flow cytometry and transmission electron microscopy. The mechanism of internalization of positively charged PRINT particles was found to be predominantly clathrin-mediated endocytosis and macropinocytosis with very few particles utilizing a caveolae-mediated endocytic pathway. The exposed charge on the surface of the particles had a significant effect on the rate of endocytosis in all cell types tested, except for the macrophage cells. No significant cytotoxicity was observed for all PRINT particles used in the present study. CONCLUSIONS: Cylindrical 1 microm PRINT particles were readily internalized into HeLa, NIH 3T3, OVCAR-3, MCF-7, and RAW 264.7 cells. Particles with a positive zeta potential exhibited an enhanced rate of endocytosis compared to negatively charged particles with identical sizes and shapes. It was found that PRINT particles with a positive zeta potential were endocytosed into HeLa cells using predominantely clathrin-mediated and macropinocytotic pathways.     

光阻法检测人血白蛋白、静注人免疫球蛋白中的不溶性微粒

目的对人血白蛋白、静注人免疫球蛋白中的不溶性微粒进行检查研究。方法按照《中国药典》2010年版三部中不溶性微粒检查法-光阻法进行检测。结果样品不经稀释,即可用于检测。液体制剂混匀后静置2min即可消除气泡,冻干制剂则需静置2~4h方可脱气。10μm粒子的回收率为98.5%,25μm粒子的回收率为100.1%。共检测人血白蛋白130批、静注人免疫球蛋白（pH 4）103批、冻干静注人免疫球蛋白（pH 4）44批,均符合规定。结论《中国药典》2010年版三部附录中的＂不溶性微粒检查法＂可用于人血白蛋白、静注人免疫球蛋白的检测,抽检的样品均能达到新版药典的要求。     

The Impact of N-nitrosamine Impurities on Clinical Drug Development

Over the past few years, an increasing number of commercially available drugs have been reported to contain N-nitrosamine impurities above acceptable intake limits. Consequent interruption or discontinuation of the manufacturing and distribution of several marketed drugs has culminated into shortages of marketed drugs, including the antidiabetic drug metformin and the potentially life-saving drug rifampin for the treatment of tuberculosis. Alarmingly, the clinical development of new investigational products has been complicated as well by the presence of N-nitrosamine impurities in batches of marketed drug. In particular, rifampin is a key clinical index drug employed in drug-drug interaction (DDI) studies, and as a result of nitrosamine impurities regulatory bodies no longer accept the administration of rifampin in DDI studies involving healthy subjects. Drug developers are now forced to look at alternative approaches for commonly employed perpetrators, which will be discussed in this review.     

Product-Specific Impact of Viscosity Modulating Formulation Excipients During Ultra-High Concentration Biotherapeutics Drug Product Development

Developing ultra-high concentration biotherapeutics drug products can be challenging due to increased viscosity, processing, and stability issues. Excipients used to alleviate these concerns are traditionally evaluated at lower protein concentrations. This study investigates whether classically known modulators of stability and viscosity at low (<50 mg/mL) to high (>50 – 150 mg/mL) protein concentrations are beneficial in ultra-high (>150 mg/mL) concentration protein formulations and drug products. This study evaluates the effect of arginine monohydrochloride, proline, and lysine monohydrochloride on viscosity and concentratability at different high and ultra-high protein concentrations using a monoclonal antibody, mAbN, formulation as a candidate protein system. The effect of excipients on the viscosity and concentratability (rate and extent) was different at high versus ultra-high protein concentrations. These results highlight that classical excipients in literature known to modulate protein interactions at low protein concentrations and reduce viscosity at high protein concentrations may need to be evaluated at target protein concentrations in a product-specific manner while developing ultra-high concentration biologics drug products.     

The Impact of Quality of Family Life on Drug Consumption

The nuclear family, particularly its role modeling during primary socialization, is found to exert an influence as strong as or stronger than the more stock-in-trade factors typically used for explaining alcohol and drug ingestion. With regard to the general North Queensland population quality of family life, past and current parental drinking are of almost equal importance.     

Extended Characterization and Impact of Visible Fatty Acid Particles - A Case Study With a mAb Product

In recent years, there has been increased scrutiny on the presence and formation of product-related particles in biopharmaceutical formulations. These types of particles, originating from the degradation of the active pharmaceutical ingredient or the excipients, can be challenging to identify and characterize due to their fragility. Additionally, the mechanisms of their formation as well as the impact of their presence on drug product safety can be complicated to elucidate. In this work, a case study is presented in which multiple batches of one formulated monoclonal antibody (mAb-A) were analyzed at different batch ages to better understand the formation of visible particles resulting from degradation of the surfactant polysorbate 20. The particle identity was determined by Raman spectroscopy as free fatty acid (FFA) and the particle composition over time was monitored by mass spectrometry. Further experimental work includes the counts and morphologies of subvisible particles by flow imaging microscopy. Finally, we evaluated the consequences of saline and human plasma exposure to the visible particles to better understand their fate upon dilution and/or administration which is routinely performed in the clinical setting. The experiments performed in this work can be used to support risk assessments of visible product-related particles.