In particular, microflow imaging mfi instruments became well established in the field of. The proteostat protein aggregation standards are ideal for reliable and accurate quantification of protein aggregation in solution. Normal shipping and delivery methodologies are suspected of causing protein aggregation. Automation of particle characterization using microflow. Protein aggregation and subvisible particles were analysed by micro flow imaging mfi.
In addition, a relatively new technology micro flow digital imaging mdi has been introduced, which may be a viable addon to measure sbvp. Dynamic imaging particle analysis is also biopharmaceutical research to characterize and analyze particles ranging from 300 nm to 5mm in size. Ron jones, tatiana perevozchikova, and katie weigandt. The aim of the present study was a comparative evaluation of four of the most relevant flow imaging microscopy. Classificationandcharacterizationoftherapeuticantibody. Protein aggregation is a field of increasing importance in the biopharmaceutical industry. Protein aggregation was evaluated by light obscuration for particle number and size distribution and by microflow imaging for morphology to determine the number and characteristics of particles present. There are a number of protein aggregation related diseases that can be investigated in a similar fashion as described here for parkinson disease. Sep 18, 20 a key focus of my work has been to evaluate and solve aggregation challenges of biopharmaceutical products, ranging from small peptides to large protein recombinant antibodies, including manual and automated analysis of subvisible particles by micro flow imaging.
Characterization and biological relevance of protein. Particles and fibrils may be formed through nucleation and growth mechanisms. Particles shed from syringe filters and their effects on. The measurements that can be made include particle size, particle shape morphology or shape analysis and grayscale or color, as well as distributions graphs of statistical population measurements. October 2006 page 2 of 11 summary the introduction of a growing number of targeted proteinbased drug formulations poses a significant challenge to. Application of mfi to the analysis of particles in parenteral fluids brightwell technologies inc. 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. Inhibition of tungsteninduced protein aggregation by. An investigation of the stabilizing effects of surfactants on biotherapeutics marianna fleischman surf program 20.
Di erentiation of protein aggregates from non aggregates. Protein aggregates and subvisible particles sbvp, inherently present in. Characterisation of protein aggregation with the smoluchowski coagulation approach for use in biopharmaceuticals. 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 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. For example, despite the proven safety and efficacy of biotechnological products, some publications have suggested.
Protein aggregation or impurities introduced during the manufacturing process of any biologic agent may stimulate antidrug antibody formation. An investigation of the stabilizing effects of surfactants on biotherapeutics marianna fleischman surf program 20 university of delaware advisors. The aggregation in these samples was quantified by size exclusion chromatography sec, dynamic light scattering dls, and. Subvisible particlemicroflow imaging microflow imaging. Flow imaging microscopy for protein particle analysisa. Linearity between fluorescence intensity and protein concentration should be verified by a standard curve if the fluorescence intensity at the plateau phase is used quantitatively. Characterization of protein particles in therapeutic. We speculate that particles shed from filters may affect the results of some protein aggregation studies such as those often conducted in the studies of amyloid formation and aggregation of therapeutic protein formulations. Flow imaging mfi is an imaging technology that has recently been applied to detecting and measuring subvisible and visible particulate matter in protein.
Using micro flow imaging mfi to measure protein aggregation. Imaging particle analysis is a technique for making particle measurements using digital imaging, one of the techniques defined by the broader term particle size analysis. However, in combination, the protein was destabilized enough to aggregate substantially. A key focus of my work has been to evaluate and solve aggregation challenges of biopharmaceutical products, ranging from small peptides to large protein recombinant antibodies, including manual and automated analysis of subvisible particles by microflow imaging. Because its imagebased, you can add quantifiable morphological parameters to your analysis. Samples were incubated with or without agitation, and in the presence or absence of the excipients heparin, sucrose, or polysorbate 80 ps80. Holographic characterization of protein aggregates department of. Micro flow imaging mfi proteinsimple 4100hm, 4100lm5100, 42005200 introduction the lumetics link tm 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. Micro flow imaging system mfi flow cell and digital photography are used to individually image, size, and count particles in liquid.
Many different analytical techniques are used today to study protein aggregates in the one to few hundreds m range like light obscuration lo, dynamic imaging particle analysis dipa techniques, microflow imaging mfi, and coulter counter cc. Few particles were measured in the unstressed sample compared to the stressed sample, suggesting that fluorescent labeling does not cause aggregation of monomeric protein. Our findings are consistent with earlier findings that surfactants affect protein aggregation and adsorption. After the protein solution and water for injection wfi were filled into both silicone oil and silicone oilfree systems, each syringe was shaken gently. Sharma,1,2 dave king,1 peter oma,1 and clark merchant1 received 21 september 2009.
Assays including ph, uvvis spectroscopy, size exclusion chromatography, intrinsic tryptophantyrosine fluorescence, and micro flow imaging were performed to assess the impact on shortterm mab stability and aggregation. Method development for subvisible protein particle micro. The effect of excipients on the extent of protein aggregation was determined by uvvisible spectroscopy and microflow imaging. 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. Microflow imaging as a quantitative tool to assess size and. Microflow imaging imagebased particle analysis for biopharmaceuticals. The clinical immunogenic risk of aggregates is uncertain, resulting in a high risk factor being assigned to the presence of protein aggregates in biologics. Application of microflow imaging mfi to the analysis of. The effects of excipients on protein aggregation during. Water nmr for protein aggregation characterization web. Lately, regulatory agencies have been focusing on subvisible particles because of the risk they pose to patient safety. Read unmasking translucent protein particles by improved micro. Thorough analysis of particles in therapeutic protein formulations is crucial due to regulatory requirements, the potential immunogenicity of protein aggregates and particles, and the need for quality. Counting, sizing and visualizing aggregation in proteins.
Evaluation of microflow digital imaging particle analysis for sub. Techniques to improve the characterization of protein. Quantitation of protein particles in parenteral solutions. Comparison of sample stability formulated in buffers ph 4. Flow imaging microscopy was introduced as a technique for protein particle analysis a few years ago and has strongly gained in importance ever since. Read microflow imaging and resonant mass measurement archimedes complementary methods to quantitatively differentiate protein particles and silicone oil droplets, journal of pharmaceutical sciences on deepdyve, the largest online rental service for scholarly research with thousands of academic publications available at your fingertips. Quantitation of protein particles in parenteral solutions using micro. Please use one of the following formats to cite this article in your essay, paper or report. Although the primary structures of biosimilars must be identical to those of. 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. Mfi systems detect, quantitate and differentiate particles that you may not see now.
May 27, 20 samples were incubated with or without agitation, and in the presence or absence of the excipients heparin, sucrose, or polysorbate 80 ps80. I need to measure translucent protein aggregates in my sample. Do you need to answer tough questions about subvisible particles. Screening of monoclonal antibody protein charge variants during formulation development using simple western charge assays. Two mab samples were incubated at 30c in different buffer systems of protein a chromatography for observing degradation due to aggregation. The methods developed during this research were utilized to characterize protein andor silicone oil particles, and to provide differentiation between protein and non protein particles. Microflow imaging instruments analyze aggregation in up to 90 samples in one run. Inhibition of tungsteninduced protein aggregation by cetyl. Quantitative analysis of monoclonal antibody formulations. There are a number of protein aggregation related diseases that can be investigated in a similar fashion as. 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. Microflow imaging video subvisible particle analysis. The methods developed during this research were utilized to characterize protein andor silicone oil particles, and to provide differentiation between protein and nonprotein particles. 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.
Proteostat protein aggregation standards enz51039 enzo. 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. Micro flow imaging mfi is a particle analysis technique that uses flow microscopy to quantify particles contained in a. Flow imaging algorithms, journal of pharmaceutical science on deepdyve, the largest online rental service for scholarly research with thousands of academic publications available at your fingertips. Microflow 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. Immunogenicity of protein aggregates of a monoclonal antibody. Shape characterization of subvisible particles using dynamic.
Unmasking translucent protein particles by improved microflow imaging algorithms article in journal of pharmaceutical sciences 1031 january 2014 with 287 reads how we measure reads. Microflow imaging and resonant mass measurement archimedes. Characterization of protein particles in therapeutic formulations using imaging flow cytometry. Dynamic light scattering dls for monitoring protein aggregation. Feb 10, 2020 please use one of the following formats to cite this article in your essay, paper or report. Jun 06, 2018 there are various different analytical techniques that can be used to investigate the presence of protein aggregates within the micrometer. Counting, sizing and visualizing aggregation in proteins using nanoparticle tracking analysis. See particles and protein aggregates for the first time with mfi. Di erentiation of protein aggregates from nonaggregates. An investigation of the stabilizing effects of surfactants on. Immunogenicity of protein aggregates of a monoclonal. Pdf evaluation of microflow digital imaging particle analysis for.
Mfi gives you more insight into the world of subvisible particlesway more than just size and count. The need to monitor, measure, and control subvisible proteinaceous particulates in. The tendency of proteins to aggregate into clusters. Mitja zidar ab, drago kuzman b and miha ravnik ac a faculty of mathematics and physics, university of ljubljana, jadranska 19, ljubljana, slovenia.
To evaluate the possibility that fluorescence labeling induced protein aggregation, unstressed and stressed protein samples at the same concentration were measured figs. Sep 17, 2019 protein aggregation is a process that can materialize at any stage in the lifetime of a therapeutic protein. Particles and fibrils may be formed through nucleation and. Indeed, protein aggregation can occur during all stages of the lifetime of a protein. Flow microscopy applied to subvisible particulate analysis in protein formulations deepak k.
The aggregation in these samples was quantified by size exclusion chromatography sec, dynamic light scattering dls, and micro flow imaging mfi. Proteostat was used to measure protein aggregation based on lack of covalent conjugation step, lack of a wash step, brighter signal compared to conventional extrinsic dyes e. 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. October 2006 page 2 of 11 summary the introduction of a growing number of targeted protein based drug formulations poses a significant challenge to. Please note that the general technical library page may contain additional product. Samples were diluted 2 10 in pbs stellar klh hmw and pierce imject hmw or water stellar klh subunit, biosyn vacmune subunit, and. Microflow imaging has been shown to provide high sensitivity in. Novel software analysis of protein aggregation and particle analysis using microflow imaging mfi. Aug 31, 20 flow imaging microscopy was introduced as a technique for protein particle analysis a few years ago and has strongly gained in importance ever since. No significant changes in particle counts were observed for polystyrene particle standards and lysozyme protein aggregates, however significant.
Characterisation of protein aggregation with the smoluchowski. Method development for subvisible protein particle. Jul 01, 20 read microflow imaging and resonant mass measurement archimedes complementary methods to quantitatively differentiate protein particles and silicone oil droplets, journal of pharmaceutical sciences on deepdyve, the largest online rental service for scholarly research with thousands of academic publications available at your fingertips. The characteristics of protein aggregates differed between the mab solution and shaken samples, which strongly indicates that silicone oil accelerates protein aggregation. Both aspects represent new challenges to the pharmaceutical development field 7, 8. The specific mechanism by which protein aggregation occurs is unknown. An investigation of the stabilizing effects of surfactants. All formulated antibody drug products contain low levels of aggregates. Manual selection of agglomerated particles within the monospheres. There are various different analytical techniques that can be used to investigate the presence of protein aggregates within the micrometer. Compared to lo, microflow imaging mfi was shown to be slightly more. Aggregation of proteins and active principle ingredients api in injectable biopharmaceutical products remains a major concern impacting the stability and usability of a product. Comparisons of two different analytical methodologies for.
Micro flow imaging mfi and resonant mass measurement rmm were tested for the differentiation of protein particles and silicone oil droplets which is highly relevant especially for pharmaceutical products in prefilled syringes. Unmasking translucent protein particles by improved micro. Protein aggregation is a process that can materialize at any stage in the lifetime of a therapeutic protein. Aug 28, 2019 two mab samples were incubated at 30c in different buffer systems of protein a chromatography for observing degradation due to aggregation. Micro flow imaging system mfi flow cell and digital photography are used to individually image, size, and count particles in liquid formulations. Dynamic light scattering dls for monitoring protein. The aim of this thesis was to identify and evaluate critical factors for protein particle analysis and to apply this knowledge for the development of novel standardized proteinlike particles. Microflow imaging combines the direct imaging capabilities of. 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 micro flow imaging mfi analysis technique has been developed and successfully applied upon a model protein containing a stable suspension. A microflow imaging system mfi5100, proteinsimple, equipped with a silanecoated flow cell 400 mm, 1. Analytical platform for monitoring aggregation of monoclonal.
Some examples of these methods include light obscuration lo, dynamic imaging particle analysis dipa, microflow imaging mfi and coulter counter cc. Monitoring protein aggregation in injectable vaccines. However, to the best of our knowledge, no studies have utilized micro flow imaging techniques for the assessment of protein aggregates when. Protein aggregates and particles are important quality attributes of. The effect of excipients on the extent of protein aggregation was determined by uvvisible spectroscopy and micro flow imaging.