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• W2017604966 abstract "Accurate counting and sizing of protein particles has been limited by discrepancies of counts obtained by different methods. To understand the bias and repeatability of techniques in common use in the biopharmaceutical community, the National Institute of Standards and Technology has conducted an interlaboratory comparison for sizing and counting subvisible particles from 1 to 25 μm. Twenty-three laboratories from industry, government, and academic institutions participated. The circulated samples consisted of a polydisperse suspension of abraded ethylene tetrafluoroethylene particles, which closely mimic the optical contrast and morphology of protein particles. For restricted data sets, agreement between data sets was reasonably good: relative standard deviations (RSDs) of approximately 25% for light obscuration counts with lower diameter limits from 1 to 5 μm, and approximately 30% for flow imaging with specified manufacturer and instrument setting. RSDs of the reported counts for unrestricted data sets were approximately 50% for both light obscuration and flow imaging. Differences between instrument manufacturers were not statistically significant for light obscuration but were significant for flow imaging. We also report a method for accounting for differences in the reported diameter for flow imaging and electrical sensing zone techniques; the method worked well for diameters greater than 15 μm. Accurate counting and sizing of protein particles has been limited by discrepancies of counts obtained by different methods. To understand the bias and repeatability of techniques in common use in the biopharmaceutical community, the National Institute of Standards and Technology has conducted an interlaboratory comparison for sizing and counting subvisible particles from 1 to 25 μm. Twenty-three laboratories from industry, government, and academic institutions participated. The circulated samples consisted of a polydisperse suspension of abraded ethylene tetrafluoroethylene particles, which closely mimic the optical contrast and morphology of protein particles. For restricted data sets, agreement between data sets was reasonably good: relative standard deviations (RSDs) of approximately 25% for light obscuration counts with lower diameter limits from 1 to 5 μm, and approximately 30% for flow imaging with specified manufacturer and instrument setting. RSDs of the reported counts for unrestricted data sets were approximately 50% for both light obscuration and flow imaging. Differences between instrument manufacturers were not statistically significant for light obscuration but were significant for flow imaging. We also report a method for accounting for differences in the reported diameter for flow imaging and electrical sensing zone techniques; the method worked well for diameters greater than 15 μm. INTRODUCTIONProtein particles, consisting of aggregated protein and possibly a nonprotein nucleating core, can form in biopharmaceutical drugs.1.Carpenter J.F. Randolph T.W. Jiskoot W. Crommelin D.J.A. Middaugh C.R. Winter G. Fan Y.-X. Kirshner S. Verthelyi D. Kozlowski S. Clouse K.A. Swann P.G. Rosenberg A. Cherney B. Overlooking subvisible particles in therapeutic protein products: Gaps that may compromise product quality.J Pharm Sci. 2009; 98: 1201-1205Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar, 2.Singh S.K. Afonina N. Awwad M. Bechtold-Peters K. Blue J.T. Chou D. Cromwell M. Krause H.-J. Mahler H.-C. Meyer B.K. Narhi L. Nesta D.P. Spitznagel T. An industry perspective on the monitoring of subvisible particles as a quality attribute for protein therapeutics.J Pharm Sci. 2010; 99: 3302-3321Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar Stresses that can lead to the formation of protein particles include changes in chemical environment, exposure to interfaces, agitation, elevation of temperature, or the introduction of nonprotein particles.3.Joubert M.K. Luo Q. Nashed-Samuel Y. Wypych J. Narhi L.O. Classification and characterization of therapeutic antibody aggregates.J Biol Chem. 2011; 286: 25118-25133Crossref PubMed Scopus (246) Google Scholar, 4.Bee J.S. Chiu D. Sawicki S. Stevenson J.L. Chatterjee K. Freund E. Carpenter J.F. Randolph T.W. Monoclonal antibody interactions with micro- and nanoparticles: Adsorption, aggregation, and accelerated stress studies.J Pharm Sci. 2009; 98: 3218-3238Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 5.Bee J.S. Randolph T.W. Carpenter J.F. Bishop S.M. Dimitrova M.N. Effects of surfaces and leachables on the stability of biopharmaceuticals.J Pharm Sci. 2011; 100: 4158-4170Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 6.Tyagi A.K. Randolph T.W. Dong A. Maloney K.M. Hitscherich Jr., C. Carpenter J.F. IgG particle formation during filling pump operation: A case study of heterogeneous nucleation on stainless steel nanoparticles.J Pharm Sci. 2009; 98: 94-104Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 7.Nayak A. Colandene J. Bradford V. Perkins M. Characterization of subvisible particle formation during the filling pump operation of a monoclonal antibody solution.J Pharm Sci. 2011; 100: 4198-4204Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar Counting and characterizing these particles is necessary to assure the quality of these drugs. Although the size of aggregated proteins may vary from 10s of nanometers to 100 s of micrometers, the most sensitive analytical techniques cover the approximate range from 1 to 100 μm.3.Joubert M.K. Luo Q. Nashed-Samuel Y. Wypych J. Narhi L.O. Classification and characterization of therapeutic antibody aggregates.J Biol Chem. 2011; 286: 25118-25133Crossref PubMed Scopus (246) Google Scholar, 8.Zolls S. Tantipolphan R. Wiggenhorn M. Winter G. Jiskoot W. Friess W. Hawe A. Particles in therapeutic protein formulations, part 1: Overview of analytical methods.J Pharm Sci. 2011; 101: 914-935Abstract Full Text Full Text PDF PubMed Scopus (166) Google ScholarIn contrast to possible nonprotein impurities (e.g., glass chips, stainless steel particles, and fibers) protein particles have low optical contrast (equivalent to a small refractive index difference from the matrix fluid) and are subject to dynamic changes in size and concentration as particles are formed or dissolve back into solution.3.Joubert M.K. Luo Q. Nashed-Samuel Y. Wypych J. Narhi L.O. Classification and characterization of therapeutic antibody aggregates.J Biol Chem. 2011; 286: 25118-25133Crossref PubMed Scopus (246) Google Scholar, 9.Ripple D.C. Dimitrova M.N. Protein particles: What we know and what we do not know.J Pharm Sci. 2012; 101: 3568-3579Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 10.Zolls S. Gregoritza M. Tantipolphan R. Wiggenhorn M. Winter G. Friess W. Hawe A. How subvisible particles become invisible— Relevance of the refractive index for protein particle analysis.J Pharm Sci. 2013; 102: 1434-1446Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar Industry has made great strides in adopting new technologies to count protein particles routinely down to sizes of approximately 2 μm, but particle counts obtained with different types of instruments often differ by as much as a factor of 10.11.Huang C.-T. Sharma D. Oma P. Krishnamurthy R. Quantitation of protein particles in parenteral solutions using micro-flow imaging.J Pharm Sci. 2009; 98: 3058-3071Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 12.Sharma D.K. King D. Oma P. Merchant C. Micro-flow imaging: Flow microscopy applied to sub-visible particulate analysis in protein formulations.AAPS J. 2010; 12: 455-464Crossref PubMed Scopus (124) Google Scholar Particle counting instruments are commonly calibrated using polystyrene latex (PSL) beads, which have high optical contrast and spherical shape; the ob-served count discrepancies indicate that instrument calibrations with PSL beads do not suffice to standardize instrument response to protein particles.Comparison of analytical measurements of particle size and count has been hampered by the instability ofthe protein particles themselves, which can aggregate further on shipping or revert back to smaller aggregates or monomer protein molecules. As an alternate path to producing a suitable reference material, the National Institute of Standards and Technology (NIST) is developing a reference material comprising irregular particles of a low-refractive index fluoropolymer, ethylene tetrafluoroethylene (ETFE). The morphology and optical contrast of this material closely resembles that of typical protein particles.As an initial step in implementing this reference material and to assess the level of agreement among different laboratories, NIST has conducted an interlaboratory comparison for sizing and counting subvisible particles from 1 to 25 μm, using a polydisperse polymer suspension that closely mimics actual protein particles. As listed in Table 1, a total of 23 laboratories participated, including 15 from biopharmaceutical manufacturers, one from biomedical device manufacturers, two from instrumentation manufacturers, three from government laboratories, and two from academic laboratories.Table 1List of Laboratories That Participated in the StudyAmgen, Inc., Formulation and Analytical Sciences, Thousand Oaks, CaliforniaBD Medical, Pharmaceutical Systems, Pharmaceutical technology/R&D, Pont de Claix, FranceBiogen Idec, QC Analytical Technology, Research Triangle Park, North CarolinaBristol Myers Squibb, Biologics Analytical Development and Testing, Pennington, New JerseyBoehringer Ingelheim Pharma GmbH and Company KG, Biopharmaceuticals, Biberach an der Riss, GermanyCoriolis Pharma, Martinsried, GermanyEli Lilly and Company, Biopharmaceutical Research and Development, Indianapolis, IndianaF. Hoffmann-La Roche Ltd, Pharma Technical Development Europe (Biologics), Basel, SwitzerlandFood and Drug Administration,aAlthough US FDA laboratory participated in the scientific study and/ or discussion, please note that FDA does not recommend, endorse, or recognize this standard development and further, the content of this communication represents the authors’ views and does not bind or obligate FDA. Laboratory of Plasma Derivatives, Center for Biologics Evaluation and Research, Bethesda, MarylandFluid Imaging Technologies, Yarmouth, MaineGenentech, Inc., Roche Group, Late Stage Pharmaceutical and Processing Development, South San Francisco, CaliforniaHach Company, Grants Pass, OregonGlaxoSmithKline R&D, Biopharm Product Sciences (BPS), King of Prussia, PennsylvaniaGlaxoSmithKline (formerly Human Genome Sciences), Gaithersburg, MarylandHealth Canada, Centre for Biologics Evaluation, Biologics and Genetic Therapies Directorate, Ottawa, CanadaJanssen R&D, Schaffhausen, SwitzerlandMedImmune, Formulation Sciences Department, Gaithersburg, MarylandNational Institute of Standards and Technology, Bioprocess Measurements Group, Gaithersburg, MarylandNovartis Pharma AG, Biologics Process R&D, Basel, SwitzerlandPfizer, Inc., Biotherapeutics Pharmaceutical Sciences, Chesterfield, MissouriSandoz Biopharmaceuticals, Pharmaceutical and Device Development, Drug Product Analytics, Sandoz GmbH, Langkampfen, AustriaUniversity of Kansas, Department of Pharmaceutical Chemistry, Macromolecule and Vaccine Stabilization Center, Lawrence, KansasUniversity of Leiden, Leiden/Amsterdam Center for Drug Research, Department of Drug Delivery Technology, Gorlaeus Laboratories, Leiden, The Netherlandsa Although US FDA laboratory participated in the scientific study and/ or discussion, please note that FDA does not recommend, endorse, or recognize this standard development and further, the content of this communication represents the authors’ views and does not bind or obligate FDA. Open table in a new tab This paper describes the design, production, and characterization of the particles (section Materials and Methods); gives an overview of the bias between different counting methods (section Results and Discussion); and then discusses results for the four methods8.Zolls S. Tantipolphan R. Wiggenhorn M. Winter G. Jiskoot W. Friess W. Hawe A. Particles in therapeutic protein formulations, part 1: Overview of analytical methods.J Pharm Sci. 2011; 101: 914-935Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 13.Narhi L.O. Jiang Y. Cao S. Benedek K. Shnek D. A critical review of analytical methods for subvisible and visible particles.Curr Pharm Biotechnol. 2009; 10: 373-381Crossref PubMed Scopus (114) Google Scholar used by participants: flow imaging,12.Sharma D.K. King D. Oma P. Merchant C. Micro-flow imaging: Flow microscopy applied to sub-visible particulate analysis in protein formulations.AAPS J. 2010; 12: 455-464Crossref PubMed Scopus (124) Google Scholar light obscuration, electrical sensing zone (ESZ),14.Demeule B. Messick S. Shire S.J. Liu J. Characterization of particles in protein solutions: Reaching the limits of current technologies.AAPS J. 2010; 12: 708-715Crossref PubMed Scopus (86) Google Scholar, 15.Barnard J.G. Rhyner M.N. Carpenter J.F. Critical evaluation and guidance for using the coulter method for counting subvisible particles in protein solutions.J Pharm Sci. 2012; 101: 140-153Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar and resonant mass measurement (RMM)16.Burg T.P. Godin M. Shen W. Carlson G. Foster J.S. Babcock K. Manalis S.R. Weighing of biomolecules, single cells, and single nanoparticles in fluid.Nature. 2007; 446: 1066-1069Crossref PubMed Scopus (980) Google Scholar (sections Overview and Identification of Outliers to Resonant Mass Measurement). Sections Particle Morphology and Resonant Mass Measurement also describe an initial attempt to adjust the reported diameter ofESZ instruments to be equivalent to the diameter reported by flow imaging instruments.The results give a snapshot of the level of agreement between different laboratories for the particle counting methods in common use today in the biopharmaceutical industry. As expected from published results on protein particles, particle counts differed significantly depending on the counting method. For each specific method, statistically significant deviations were observed primarily because of differences in instrument response. There were also several outliers (~10% of the reported data) likely related to insufficient resuspension of the ETFE particles and contamination of the ETFE particles by debris from vial-thread abrasion. Surprisingly, data obtained by light obscuration agreed well for small diameter particles [relative standard deviation (RSD) of <26% for lower diameter limits from 1 to 5 μm], but the level of agreement was significantly worse for large particles. For flow imaging, there were statistically significant differences between data sets acquired on different instrument models, resulting in a large variability of counts (RSD values of 33%-61% for all flow imaging data). For specified instrument settings and models, the variability was reduced, with RSD values of 13%-49% over the full size range of the comparison. ESZ instruments gave anomalously high counts for the lowest diameter limits.MATERIALS AND METHODSPreparation of the Particle SuspensionThe samples circulated for testing consisted of a polydisperse suspension of particles created from the polymer ETFE. ETFE is attractive because it has low refractive index17.French R.H. Rodríguez-Parada J.M. Yang M.K. Derryberry R.A. Lemon M.F. Brown M.J. Haeger C.R. Samuels S.L. Romano E.C. Richardson R.E. Optical properties of materials for concentrator photovoltaic systems. In Proc 34th IEEE Photovoltaic Specialists Conference (PVSC).Philadelphia, Pennsylvania, June. 2009; 7–12: 394-399Google Scholar (≈1.40, similar to that of protein films adsorbed on surfaces18.Voros J. The density and refractive index of adsorbing protein layers.Biophys J. 2004; 87: 553-561Abstract Full Text Full Text PDF PubMed Scopus (610) Google Scholar) and is chemically inert and mechanically strong.19.DuPont Tefzel® fluoropolymer resin. Properties handbook. Accessed November 19, 2014, at: http://www2.dupont.com/TeflonIndustrial/enUS/assets/downloads/h96518.pdf.Google ScholarThe particles were produced by abrading a solid polymer sample of ETFE against a diamond lapping disc. Although the process of producing the ETFE particles in no way corresponds to the aggregation mechanism of actual protein particles, the morphology of the ETFE particles is remarkably similar to protein particles. Thus, the ETFE particles can serve as a surrogate to actual protein particles, with similar morphology and optical contrast. Like actual protein particle suspensions, but unlike PSL standards, the ETFE suspensions are polydisperse, with particles ranging in approximate sizes from greater than 50 μm down to less than 0.5 μm.We produced polydisperse ETFE particles by first abrading ETFE against a diamond abrasive (45 μm nominal grit size, nickel bonded to a compliant backing) while submersed in an aqueous solution of 0.03 mol/L 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) and 0.1% mass concentration sodium dodecyl sulfate (SDS) buffered to pH 6 (we intended to use pH 7.5, but inadvertently used a pH 6 buffer for the particle fabrication). At approximately 1 h intervals, the particle suspension was withdrawn by pipette from the well holding the abrasive disc. To prevent clogging of analytical instruments, large particles were filtered out by passing the suspension through a nylon screen with nominal 53 μm square openings. The nylon screen did not shed an appreciable number of particles if it was securely mounted, not folded or manipulated during the filtering process, and thoroughly rinsed with particle-free water prior to use. As harvested, the particle count was too high for direct measurement in some instruments. The suspension was diluted to the desired particle count with additional HEPES/SDS solution buffered to a pH of 7.5. Prior to use, the HEPES/SDS solution was filtered through a 0.45-pm PVDF syringe filter (Millex-HV; EMD Millipore, Billerica,Massachusetts). (Certain commercial equipment, instruments, or materials are identified in this document. Such identification is not intended to imply recommendation or endorsement by the National Institute of Standards and Technology, nor is it intended to imply that the products identified are necessarily the best available for the purpose.) The large concentration of SDS proved necessary to promote dispersion of the highly hydrophobic ETFE particles: measurements on suspensions with an SDS concentration below 0.03% mass concentration had poor repeatability.The suspension was packaged in perfluorinated alkoxy (PFA) screw-top vials with flat interior bottoms. PFA has several desirable attributes. First, PFA, with its low refractive index (1.34), does not produce thread debris of high optical contrast on repeated opening and closing of the vials. Second, the PFA vials have minimal leachates. Initial studies with glass screw-top vials revealed unacceptable quantities of debris created by friction between the screw top and the vial. Studies with polypropylene vials revealed that strong agitation (necessary to resuspend the ETFE particles) would produce long-lasting, vesicle-like droplets, likely as a result of combining SDS with organic leachates. These observations motivated the switch to the more expensive PFA vials.Transfer ofhomogeneous aliquots ofETFE solution to a large number of vials is hampered by the fast settling of the ETFE particles relative to PSL beads. The ETFE particles were maintained in suspension in a 400 mL, open-top PFA jar by placing four stainless steel baffles around the periphery of the jar and then stirring at 200 revolutions per minute.20.Myers K.J. Reeder M.F. Fasano J.B. Optimize mixing by using the proper baffles.Chem Eng Prog. 2002; 98: 42-47Google Scholar From this stirred jar, the suspension could be transferred by pipet into the PFA vials. Once in the vials, the suspension was further diluted by addition of the 0.03 mol/L HEPES, pH 7.5, 0.1% mass concentration SDS solution.The threaded joint of the PFA vials can leak because of cold flow of the PFA over the course of several days. To prevent this problem, the vials were tightened after sitting overnight, and again after 7 days from the filling date. Rocking the lids back and forth while tightening also helped to promote a tight seal. Participants weighed the vials on receipt to ensure that the vials had not leaked during shipping; all vials were successfully shipped without leakage.The vials were stored and shipped to participants at ambient temperature.Characterization of the Particle SuspensionVariability of the Particle ConcentrationOn initial production, there are vial-to-vial variations in particle count because of irrepeatability of the dispensing and diluting process. After initial production, there are several potential mechanisms for further changes of the particle size distribution. As examples, agitation because of shipping or resuspension can potentially either break apart particles or cause abrasion of the PFA vials; sedimentation and natural convection can lead to entanglement of particles; and resealing the threaded closures can add abraded PFA particles.To quantify vial-to-vial variations and possible changes in particle size distribution during storage, five vials were measured by light obscuration over a 3-month period corresponding to the period of data collection by the participants. Changes in particle size distribution were estimated from the changes observed for a single vial shipped from Maryland to California and back again. Separate experiments were conducted to repeatedly reseal three vials (which can lead to creation of thread debris), and measure the particle size distribution periodically by both light obscuration and flow imaging, for a total of 30 sealing cycles. The vial stability tests were conducted with multiple vials, opened at staggered time points, to reduce the effects of vial resealing on the stability test.From the variations observed in these experiments, the standard uncertainty was determined for each effect, which is equivalent to the expected variations of any one vial from the mean of all vials at a confidence limit of 68% (i.e., equivalent to one standard deviation). The combined uncertainty uc (i.e., the uncertainty attributed to all terms) was calculated by taking the square root of the sum of the squares of all uncertainty components. (The results of this analysis are given in the Supplementary Information as Table S1.) The value of uc is an acceptable 8% for particle concentrations with a lower size limit of 5 μm or lower. For higher limits, uc climbs to 19% at a lower limit of 25 μm. The larger value of uc for a lower limit of 25 μm results at least in part from the low particle concentration in that size range, with N & 80 mL-1 (mL-1 is the unit symbol designating units of particles per milliliter). This low value increases the impact of small numbers of additional contaminants. Download .docx (.36 MB) Help with docx files Table S1Although the stability of the ETFE particle suspension was sufficient for the purposes of this comparison, some drift in results were noted by both NIST and one participant. Part of this observed drift is likely because of thread debris, as discussed in section Overview and Identification of Outliers. One possible additional source of drift is microbial growth: despite the ability of SDS to denature proteins, bacteria have been observed to degrade SDS at concentrations in excess of the 0.1% mass concentration used here.21.Chaturvedi V. Kumar A. Isolation of sodium dodecyl sulfate degrading strains from a detergent polluted pond situated in Varanasi city, India.J Cell Molec Bio. 2010; 8: 103-1110Google Scholar, 22.Louvado A. Coehlo F.J.R.C. Domingues P. Santos A.L. Gomes N.C.M. Almeida A. Cunha A. Isolation of surfactant-resistant pseudomonads from the estuarine surface microlayer.J Microbiol Biotechnol. 2012; 22: 283-291Crossref PubMed Scopus (17) Google Scholar Subsequent to the data acquisition phase of the comparison, a stability test was initiated on ETFE particle suspensions of similar particle size distribution, but with the addition of 0.02% mass concentration of sodium azide and with greater care in filtration of the buffer. The stability of this second lot over 6.5 months was a factor of 1.9 better than the stability of the comparison lot over 3 months (data not shown).Particle MorphologyWe examined the particle morphology using both optical and electron microscopy. Examples of both types of images are shown in Figure 1. Optical images of the ETFE particles were acquired in suspension, using a flow imaging system with 10 x magnification and a 100-μm thick flow cell.Optical imaging of the ETFE particles is limited by the resolution of optical microscopy; scanning electron microscopy (SEM) has the potential of providing images with much higher resolution. However, the particles must be deposited on a substrate in such a way that the particles do not agglomerate on drying and must have the high SDS and HEPES concentration washed off. For SEM imaging, the particles were first captured on anodized alumina filters (Anodisc brand; GE Healthcare Bio-Sciences, Pittsburgh, Pennsylvania) with 0.2 μm diameter pores. Prior to use, the filters were first washed with water and then with a mixture of photoresist remover and ethanol. After oven drying, the filters were sputter coated on both sides with 25 nm ofa Au/Pd alloy. The coated filter was placed on the glass-frit support of a vacuum flask, and 300 μL of water was pipetted nto the top of the filter to form a nearly hemispherical “dome” on the hydrophobic Au/Pd film, whereas a slight partial vacuum (≈1.3 kPa, as determined by a vacuum manometer) was applied to the flask, sufficient to draw liquid through the filter at approximately 0.6 mL/min. A 100-μL sample of the ETFE suspension was slowly pipetted into the center of the dome; as the dome returned to an approximate volume of 300 μL, 100 μL of water was slowly pipetted into the top of the dome. This process was repeated until the desired particle count was achieved. The particles were then rinsed by 10 successive additions of 100 μL of water. After the rinse step, the vacuum was maintained to draw the water fully through the filter. Following drying of the filter in place, the filter was removed and a 25-nm top coat ofAu/Pd film was sputter deposited. SEM images were obtained on a Zeiss scanning electron microscope at an electron energy of 5 keV, with both in-lens and conventional secondary electron detectors. Dispersive X-ray analysis conducted on the SEM confirmed that we were imaging fluorocarbon particles.Inspection of the microscopic images shows them to be irregular and somewhat fibrous. We wished to characterize the average fraction of solid ETFE within the overall envelope of the particle, in anticipation that this packing fraction would help us understand differences in the particle counts obtained by different methods. The technique of quantitative phase microscopy23.Paganin D. Nugent K.A. Noninterferometric phase imaging with partially coherent light.Phys Rev Lett. 1998; 80: 2586-2589Crossref Scopus (657) Google Scholar, 24.Bellair C.J. Curl C.L. Allman B.E. Harris P.J. Roberts A. Delbridge L.M.D. Nugent K.A. Quantitative phase amplitude microscopy IV: imaging thick specimens.J Microscopy. 2004; 214: 62-69Crossref PubMed Scopus (52) Google Scholar produces a phase map of the apparent optical phase difference N(x, y), which is proportional to the difference in optical thickness between the particle and the matrix liquid:φxy=2πΔnEzEλ0,(1) where ∆nE is the refractive index difference between solid ETFE and the matrix liquid, zE is the total thickness of ETFE in the vertical z direction, x and y are lateral dimensions in the image plane, and is the wavelength of light in vacuum. Integrating Eq. (1) over the whole area of the particle relates the integral of the optical phase difference over the whole particle area, Φ, to the total volume displaced by solid ETFE, V:Φ=2πΔnEVλ0.(2) From the literature, the refractive index of ETFE, at the wavelength of 527 nm used here, is 1.400.17.French R.H. Rodríguez-Parada J.M. Yang M.K. Derryberry R.A. Lemon M.F. Brown M.J. Haeger C.R. Samuels S.L. Romano E.C. Richardson R.E. Optical properties of materials for concentrator photovoltaic systems. In Proc 34th IEEE Photovoltaic Specialists Conference (PVSC).Philadelphia, Pennsylvania, June. 2009; 7–12: 394-399Google Scholar From measurements with a calibrated Abbe refractometer, the HEPES/SDS buffer has a refractive index of 1.337, giving ΔnE=0.063 in Eqs. (1) and (2).Published literature for objects with a thickness comparable to the width and length report that correcting phase values from the thin-object algorithm of Paganin and Nugent23.Paganin D. Nugent K.A. Noninterferometric phase imaging with partially coherent light.Phys Rev Lett. 1998; 80: 2586-2589Crossref Scopus (657) Google Scholar by a factor of either two to five (varying with object size and using white light illumination)24.Bellair C.J. Curl C.L. Allman B.E. Harris P.J. Roberts A. Delbridge L.M.D. Nugent K.A. Quantitative phase amplitude microscopy IV: imaging thick specimens.J Microscopy. 2004; 214: 62-69Crossref PubMed Scopus (52) Google Scholar or one (using highly coherent light).25.Frank J. Matrisch J. Horstmann J. Altmeyer S." @default.
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• W2017604966 title "An Interlaboratory Comparison of Sizing and Counting of Subvisible Particles Mimicking Protein Aggregates" @default.
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