Product quality, both in terms of biological activity and immunogenicity can, be highly influenced by the state of protein aggregation. Download this free white paper from protein simple to learn about. Interfacial shear rheology was used to detect the gelation time and strength of protein gels at the airwater interface. Microflow imaging analyzes protein aggregation for stability measurements following stress testing. Request pdf flow imaging microscopy for protein particle. Microflow imaging size, size distribution, shape and morphology combined in one instrument protein aggregation is a key challenge in the development of biologic formulations because it has crucial impact on product quality in terms of efficacy and immunogenicity. Get quantifiable morphological parameters to your micro flow imaging. Light exposure may lead to photolytic degradation of the protein through photooxidation of the sidechains of certain amino acids such as met, tyr, trp, his, cys, and phe 818283 and has been shown to induce protein aggregation. Feb 29, 2016 however, to the best of our knowledge, no studies have utilized micro flow imaging techniques for the assessment of protein aggregates when samples are formulated with an opaque vaccine adjuvant. Microflow imaging combines the direct imaging capabilities of digital microscopy with the precise control of microfluidics. Particleanalyzing technologies including microflow imaging mfi and dynamic light scattering dls are becoming more and more popular to characterize protein degradation, especially aggregation. Similar results have been reported using different protein based formulations in other pharmaceutical laboratories 5,6.
Protein particle analysis critical factors and new standards. Assays including ph, uvvis spectroscopy, size exclusion chromatography, intrinsic tryptophantyrosine fluorescence, and microflow imaging were performed to assess the impact on shortterm mab stability and aggregation. Particle characterization for a protein drug product. Protein aggregation, proteinaceous particles, nonproteinaceous particles, subvisible particles, mfi, mvas, lumetics link, discriminant analysis. Holographic characterization of protein aggregates nyu physics. Characterization and biological relevance of protein. Microflow imaging enables cutting edge biopharmaceuticals. Mfi provides you with the most accurate picture of protein aggregation in your sample. Microflow imaging has been shown to provide high sensitivity in.
Sep 01, 2009 microflow imaging mfi is an imaging technology that has recently been applied to detecting and measuring subvisible and visible particulate matter in protein containing solutions. Therapeutic proteins have greatly advanced treatment for a wide range of human medical conditions including cancer, infection, and autoimmune disease. Therefore, protein aggregation and subvisible particle content must be carefully monitored during inprocess and final product development. Di erentiation of protein aggregates from nonaggregates. Novel software analysis of protein aggregation and particle analysis using micro flow imaging mfi. Therefore, you get a complete image of what passes through the flow cell. Microflow imaging analyses reflect mechanisms of aggregate formation.
Evaluation of aggregate and siliconeoil counts in pre filled. Counting, sizing and visualizing aggregation in proteins. Flow microscopy applied to subvisible particulate analysis in protein formulations, authord. The aggregation behavior of two model proteins i bovine serum albumin bsa and ii. All formulated antibody drug products contain low levels of aggregates. Thus, a high peak indicates some elongated particles\3d histogram\.
Pdf classification of protein aggregates deepak sharma. Comparisons of two different analytical methodologies for. Measuring protein aggregation is a common way to get an indication of stability, and mfi is widely used in formulation studies and stability studies where aggregation measurements are needed. Flow microscopy applied to subvisible particulate analysis in protein formulations. These instruments typically cover a size range from about 1 to 400 m, while the upper particle concentration limit is typically higher than that of light obscuration systems. Quantitative analysis of monoclonal antibody formulations. Microflow imaging mfi has been used for formulation development for analyzing subvisible particles. European journal of pharmaceutics and biopharmaceutics. Protein aggregation and subvisible particle analysis is a breeze with microflow imaging mfi. Microflow imaging mfi has been shown to provide high sensitivity in detecting and imaging transparent particles and a unique capability to differentiate subpopulations of particles having different morphologies using image filtering 4,5. Flow imaging microscopy for protein particle analysisa. Particle characterization for a protein drug product stored. Using micro flow imaging mfi to measure protein aggregation white paper jan 08, 2018 subvisible particles are a critical quality attribute for pharmaceutical products, as protein aggregates can elicit an immunogenic response that affects the therapeutics efficacy.
Flow imaging microscopy for protein particle analysis a comparative evaluation of four. Protein subvisible particle and free radical formation of a. A novel approach to identify 5 types of simulated stresses that induce protein aggregation in prefilled syringetype biopharmaceuticals was developed. Similar acceleration of protein aggregation and particle formation was observed when unfiltered kgf. In this manuscript, we report an interesting phenomenon in which repeated dropping of freezedried monoclonal antibody x mabx formulation powder resulted in significant protein subvisible particles sbvps in the reconstituted liquid as determined by the sensitive particle analyzing technique microflow imaging mfi. Method development for subvisible protein particle micro. Micro flow imaging system mfi flow cell and digital photography are used to individually image, size, and count particles in liquid formulations.
There are multiple pathways to protein particle formation. Subvisible particle microflow imaging subvisible particle analysis in high concentration protein formulations, and silicone oil droplet classification using microflow imaging. Screening of monoclonal antibody protein charge variants during formulation development using simple western charge assays. Proteinsimple offers microflow imaging mfi, a powerful system to analyze protein aggregates and other subvisible. Proteinsimple offers microflow imaging mfi, a powerful system to analyze protein aggregates and other subvisible particles in your samples. Because its imagebased, you can add quantifiable morphological parameters. The clinical immunogenic risk of aggregates is uncertain, resulting in a high risk factor being assigned to the presence of protein aggregates in biologics. Automatic identification of the stress sources of protein. Inhibition of tungsteninduced protein aggregation by cetyl. Protein aggregates can be tough to measure but are important indicators of a pharmaceuticals stability.
This study utilized these two techniques to investigate the effectiveness of polysorbate ps80 in mitigating the particle formation of a. In a single run bot1 can handle up to 90 samples without manual intervention. Comparison of sample stability formulated in buffers ph 4. Dec 22, 2009 characterizing the state of aggregation in proteins is of paramount importance when trying to understand product stability and efficacy. Flow imaging microscopy fim is commonly used in both academia and industry to characterize subvisible particles those \le 25 \mu m in size. Guidelines for nano and micro sensor use information provided by malvern. However, more detailed guidelinesrecommendations on vaccine evaluation in terms of quality, safety and. The effects of excipients on protein aggregation during. Not only is mfi more sensitive than traditional particle detection techniques, it also lets you discriminate between translucent protein aggregates and other particulates. Microflow imaging analyses reflect mechanisms of aggregate.
The current study was performed to determine the contribution of several key variables to the variability of the subvisible particle counts e. Evaluation of aggregate and siliconeoil counts in pre. Proteostat protein aggregation standards enz51039 enzo. In a single run bot1 can handle up to 90 samples without manual interventi. Normal shipping and delivery methodologies are suspected of causing protein aggregation. An investigation of the stabilizing effects of surfactants. The presence of subvisible particles svps in parenteral formulations of biologics is a major challenge in the development of therapeutic protein formulations. Because its imagebased, you can add quantifiable morphological parameters to your analysis. Our microflow imaging technology mfi gives you more insight into the world of subvisible particlesway more than just size and count. Particles shed from syringe filters and their effects on. Flow imaging mfi is an imaging technology that has recently been applied to detecting and measuring subvisible and visible particulate matter in protein. Get quantifiable morphological parameters to your microflow imaging. Nta, micro flow imaging mfi, tunable resistive pulse sensing.
Guidelines on the quality, safety, and efficacy of. Microflow imaging imagebased particle analysis for biopharmaceuticals. Quantitation of protein particles in parenteral solutions. Di erentiation of protein aggregates from non aggregates. Protein aggregation is not only a challenge in the development of drug products, it is a significant barrier. Protein aggregation and particle analysis proteinsimple. Microflow imaging mfi 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. Comparing protein particle data sets using the kullbackleibler divergence nathaniel r. Micro flow imaging has been shown to provide high sensitivity in. Sep 12, 2016 microflow imaging mfi has been used for formulation development for analyzing subvisible particles.
Water nmr for protein aggregation characterization web. Comparisons of two different analytical methodologies for the. Microflow imaging and fluorescence spectroscopy tht fluorescence. Separation, characterization and discriminant analysis of. Using deep convolutional neural networks to circumvent. The proteostat protein aggregation standards are ideal for reliable and accurate quantification of protein aggregation in solution. Dynamic light scattering dls for monitoring protein aggregation. This study evaluates the utility of mdi particle analysis, specifically using the flowcam vs instrument, to assess a variety of subvisible. Ps80 functionality was assessed by measuring the extent of protein aggregation and particle formation induced during mechanical stress by using sizeexclusion chromatography, dynamic light scattering, backgrounded membrane imaging and flow imaging microscopy. Micro flow imaging mfi, were evaluated with respect to their. Comparison of fluorescence dyes in their applicability to study protein aggregation. Fi and lo, interlaboratories, and interinstruments micro flow imaging and flowcam. Protein subvisible particle and free radical formation of.
Characterisation of protein aggregation with the smoluchowski. Protein aggregation and the formation of subvisible particles can significantly impact the safety and efficacy of biopharmaceuticals. Characterization of protein particles in therapeutic. Novel software analysis of protein aggregation and particle analysis using microflow imaging mfi. In this manuscript, we report an interesting phenomenon in which repeated dropping of freezedried monoclonal antibody x mabx formulation powder resulted in significant protein subvisible particles sbvps in the reconstituted liquid as determined by the sensitive particle analyzing technique microflow imaging. Comparison of techniques reported for protein characterisation aggregation 39 table 1. Microflow imaging mfi is an imaging technology that has recently been applied to detecting and measuring subvisible and visible particulate matter in protein containing solutions. Sep 01, 2009 quantitation of protein particles in parenteral solutions using micro.
The tendency of proteins to aggregate into clusters. Nov 30, 2008 pdf aggregation or reversible selfassociation of protein therapeutics can arise through a number of different mechanisms. Coriolis operates several systems from different suppliers, such as microflow imaging devices from proteinsimple and flowcam systems from fluid imaging technology. Mfi microflow imaging ms mass spectrometry mtd maximum tolerated dose mw molecular weight nhps nonhuman primates. Even translucent protein aggregates that would be missed by lo are caught by mfi. This work investigates the effect of ultrasonic nebulization on protein aggregation as a function of impurity level, gasliquid surface to value ratios, protein concentration, solution viscosity, and nebulization time. Microflow imaging mfi, were evaluated with respect to their subvisible particle measurement and characterisation capabilities. Principal components analyses of texture metrics extracted from flow imaging microscopy images were used to define subgroups of particles. Using an autosampler or by manual injection, the sample is passed through the flow cell and pictures are captured after automatic illumination and precision focusing.
The unique analytical requirements required to successfully count and size subvisible particles present within protein formulations has been considered, and a protocol for method validation specific to the microflow imaging mfi analysis technique has been developed and successfully applied upon a model protein containing a stable suspension. The standards are used to determine low levels of aggregated protein in a sample by comparing the assay response of a test sample to that of the standard curve. Protein aggregation is not only a challenge in the development of drug products, it is a significant barrier to costeffective production of purified bulk drug substance. The system was flushed with 5 ml of purified water before each measurement. The lumetics linktm software platform scans network locations for new measurement data files, copies data directly to a centralized database, and provides a powerful user interface for rapid multimeasurement multitechnique data aggregation.
The effect of excipients on the extent of protein aggregation was determined by uvvisible spectroscopy and microflow imaging. Particle shedding from syringe filtersand the resulting acceleration of protein aggregation during agitationvaried greatly among the different syringe filters and individual units of. Comparing protein particle data sets using the kullbackleibler divergence. Archimedes, a novel technique for analyzing submicron particles, has been considered as an orthogonal method to currently existing techniques.
An investigation of the stabilizing effects of surfactants on. Subvisible particlemicroflow imaging microflow imaging. Control of protein particle formation during ultra. In addition, a relatively new technology micro flow digital imaging mdi has been introduced, which may be a viable addon to measure sbvp.
Monitoring protein aggregation in injectable vaccines. Microflow imagebased particle analysis for biopharmaceuticals. Using micro flow imaging mfi to measure protein aggregation. Collaborative study for analysis of subvisible particles using flow. Microflow imaging instruments analyze aggregation in up to 90 samples in one run. Inhibition of tungsteninduced protein aggregation by. Sharma and dave king and peter oma and clark merchant, journalthe aaps journal. Particle analysis was performed using microflow imaging on 156 a dpa 4100 brightwell technologies, inc. The evaluation of subvisible particles, including protein aggregates. Evaluation of microflow digital imaging particle analysis for.
See what light obscuration misses with microflow imaging. Pdf cell aggregation in thawed haematopoietic stem cell. Flow imaging microscopy for protein particle analysis a comparative evaluation of four different analytical instruments. Understanding the contribution of relevant factors to the analytical variability of the microflow imaging mfi technique is of prime importance because of the significance of the subvisible particulate data in biopharmaceutical product development.
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