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The degradation of polysorbate surfactants can limit the shelf life of biologic pharmaceutical products. Polysorbate is susceptible to degradation via either oxidation or hydrolysis pathways which releases free fatty acids (FFA) and other complex polymers. Degradants from Polysorbate 80 (PS80) can form particles and impact drug product quality. PS80 degradation products appear at low concentrations, and their refractive indexes are similar to that of the buffer, making them very challenging to detect. Furthermore, aggregates of FFA are similar in size and refractive index to protein aggregates adding complexity to characterizing these particles in protein solutions. Total Holographic Characterization (THC) is used in this work to characterize FFA particles of oleic acid and linoleic acid, the two most common degradation products of PS80. We demonstrate that the characteristic THC profile of the FFA oleic acid emulsion droplets can be used to monitor the degradation of PS80. THC can detect oleic acid at a concentration down to less than 100 ng/mL. Using the characteristic THC signal of oleic acid as a marker, the degradation of PS80 in protein solutions can be monitored quantitatively even in the presence of other contaminants of the same size, including silicone oil emulsion droplets and protein aggregates., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [Fook Chiong Cheong reports was provided by Spheryx, Inc. Fook Chiong Cheong reports a relationship with Spheryx Inc that includes employment and equity or stocks. Laura A. Philips reports a relationship with Spheryx Inc that includes employment and equity or stocks. Tiffany Markus reports a relationship with Spheryx Inc that includes employment. Juliana Lumer reports a relationship with Spheryx Inc that includes employment. Rivka Stasavage reports a relationship with Spheryx Inc that includes employment. David B. Ruffner reports a relationship with Spheryx Inc that includes employment. Fook Chiong Cheong has patent #20120135535 issued to New York University. Fook Chiong Cheong has patent #11747258 issued to New York University. David B. Ruffner has patent #11747258 issued to New York University. Laura A. Philips has patent #11747258 issued to New York University. no other relationship If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper]., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Total holographic characterization (THC) is presented here as an efficient, automated, label-free method of accurately identifying cell viability. THC is a single-particle characterization technology that determines the size and index of refraction of individual particles using the Lorenz-Mie theory of light scattering. Although assessment of cell viability is a challenge in many applications, including biologics manufacturing, traditional approaches often include unreliable labeling with dyes and/or time consuming methods of manually counting cells. In this work we measured the viability of Saccharomyces cerevisiae yeast in the presence of various concentrations of isopropanol as a function of time. All THC measurements were performed in the native environment of the sample with no dilution or addition of labels. Holographic measurements were made with an in-line holographic microscope using a 40[Formula: see text] objective lens with plane wave illumination. We compared our results with THC to manual counting of living and dead cells as distinguished with trypan blue dye. Our findings demonstrate that THC can effectively distinguish living and dead yeast cells by the index of refraction of individual cells., (© 2022. The Author(s).)

Quantifying the heterogeneous nature of protein aggregates is important to understanding the impact aggregates may have on the performance of antibody therapeutics. The spatially averaged density ρ p of aggregates, defined as the total mass, including water, divided by the volume, is a parameter that can be used to relate size distributions measured by orthogonal methods, to characterize protein particles, and perhaps to estimate the amount of aggregated protein in a sample. We report measurements by two methods on the distribution of density values for different aggregate sizes, where the aggregates were produced by stir-stressing fluorescently labeled monoclonal antibody (NISTmAb). A fluorescence microscope was used to image particles. Each particle was analyzed for brightfield equivalent circular diameter (ECD) and fluorescence intensity and the results converted to average density. Measurements were also obtained using video holography. The aggregates were highly porous with median density decreasing from 1.07 g/cm 3 to 1.02 g/cm 3 as the size increased from 0.9 μm to 6 μm by fluorescence, and similar results by video holography. The distribution in density for a given particle size was asymmetrical and broad. For example, particles with an ECD of 2.5 μm ranged in density from 1.005 g/cm 3 to 1.1 g/cm 3 ., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Inc.)

The measurement of polydisperse protein aggregates and particles in biotherapeutics remains a challenge, especially for particles with diameters of ≈ 1 µm and below (sub-micrometer). This paper describes an interlaboratory comparison with the goal of assessing the measurement variability for the characterization of a sub-micrometer polydisperse particle dispersion composed of five sub-populations of poly(methyl methacrylate) (PMMA) and silica beads. The study included 20 participating laboratories from industry, academia, and government, and a variety of state-of-the-art particle-counting instruments. The received datasets were organized by instrument class to enable comparison of intralaboratory and interlaboratory performance. The main findings included high variability between datasets from different laboratories, with coefficients of variation from 13 % to 189 %. Intralaboratory variability was, on average, 37 % of the interlaboratory variability for an instrument class and particle sub-population. Drop-offs at either end of the size range and poor agreement on maximum counts of particle sub-populations were noted. The mean distributions from an instrument class, however, showed the size-coverage range for that class. The study shows that a polydisperse sample can be used to assess performance capabilities of an instrument set-up (including hardware, software, and user settings) and provides guidance for the development of polydisperse reference materials., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: N. Do is an employee of Spectradyne LLC, which manufactures and sells microfluidic resistive pulse sensing-based instrumentation for particle quantification. D. Grier is a founder of Spheryx, Inc., the company that manufactures the xSight instrument that was used for part of the NYU study. J. Hadley was employed at Malvern Panalytical, which develops and sells the Archimedes and LM10, LM14, LM20, NS300, and NS500 instruments used in this study. C. Probst was employed by Luminex Corporation, the manufacturer of the Amnis® CellStream® and FlowSight® that were used in this study. During this study J. Tatarkiewicz was employed by MANTA Instruments, Inc. which since then was acquired by HORIBA Scientific, Inc. The measurements were done using ViewSizer 3000 instrument. Alpha Nano Tech LLC is a Contract Research Organization; it has actual contracts with Particle Metrix Inc., as well as with other instruments vendors, and provides services for the companies on request. In general, Alpha Nano Tech LLC has contracts with Particle Metrix and special terms on acquiring instruments. Alpha Nano Tech provides services on variety of instruments/techniques, including those which compete with each other. Spheryx Inc. sells the Holographic Characterization Instrument (xSight and xCell8) used in this publication. We also hold the patents associated to this technology. Yokogawa Fluid Imaging Technologies, Inc. develops and sells the FlowCam® Nano flow imaging microscope used in this study. All other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier Inc.)

We demonstrate the use of holographic video microscopy to detect individual subvisible particles dispersed in biopharmaceutical formulations and to differentiate them based on material characteristics measured from their holograms. The result of holographic analysis is a precise and accurate measurement of the concentrations and size distributions of multiple classes of subvisible contaminants dispersed in the same product simultaneously. We demonstrate this analytical technique through measurements on model systems consisting of human IgG aggregates in the presence of common contaminants such as silicone oil emulsion droplets and fatty acids. Holographic video microscopy also clearly identifies metal particles and air bubbles. Being able to differentiate and characterize the individual components of such heterogeneous dispersions provides a basis for tracking other factors that influence the stability of protein formulations including handling and degradation of surfactant and other excipients., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The in-line hologram of a micrometer-scale colloidal sphere can be analyzed with the Lorenz-Mie theory of light scattering to obtain precise measurements of the sphere's diameter and refractive index. The same technique also can be used to characterize porous and irregularly shaped colloidal particles provided that the extracted parameters are interpreted with effective-medium theory to represent the properties of an equivalent effective sphere. Here, we demonstrate that the effective-sphere model consistently accounts for changes in the refractive index of the medium as it fills the pores of porous particles and therefore yields quantitative information about such particles' structure and composition. In addition to the sample-averaged porosity, holographic perfusion porosimetry gauges the polydispersity of the porosity. We demonstrate these capabilities through measurements on mesoporous spheres, fractal protein aggregates and irregular nanoparticle agglomerates, all of which are noteworthy for their industrial significance.

Characterizing protein aggregates in the presence of silicone oil is a long standing challenge for the pharmaceutical industry. Silicone oil is often used as a lubricant in devices that deliver and store therapeutic protein products and has been linked to protein aggregation, which can compromise a drug's effectiveness or cause autoimmune responses in patients. Most traditional technologies cannot quantitatively distinguish protein aggregates and silicone oil in their native formulations for sizes less than 5 μm. We use holographic video microscopy to study protein aggregation to demonstrate its capability to quantitatively distinguish protein aggregates and silicone oil in the presence of varying amounts of the surfactants SDS and polysorbate 80 in the size range of 0.5-10 μm without the need for dilution or special sample preparation. We show that SDS denatures proteins and stabilizes silicone oil. We also show that polysorbate 80 may limit protein aggregate formation if it is added to an IgG solution before introducing silicone oil., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Micrometer sized particles can be accurately characterized using holographic video microscopy and Lorenz-Mie fitting. In this work, we explore some of the limitations in holographic microscopy and introduce methods for increasing the accuracy of this technique with the use of multiple wavelengths of laser illumination. Large high index particle holograms have near degenerate solutions that can confuse standard fitting algorithms. Using a model based on diffraction from a phase disk, we explain the source of these degeneracies. We introduce multiple color holography as an effective approach to distinguish between degenerate solutions and provide improved accuracy for the holographic analysis of sub-visible colloidal particles.

Determining the size distribution and composition of particles suspended in water can be challenging in heterogeneous multicomponent samples. Light scattering techniques can measure the distribution of particle sizes, but provide no basis for distinguishing different types of particles. Direct imaging techniques can categorize particles by shape, but offer few insights into their composition. Holographic characterization meets this need by directly measuring the size, refractive index, and three-dimensional position of individual particles in a suspension. The ability to measure an individual colloidal particle's refractive index is a unique capability of holographic characterization. Holographic characterization is fast enough, moreover, to build up population distribution data in real time, and to track time variations in the concentrations of different dispersed populations of particles. We demonstrate these capabilities using a model system consisting of polystyrene microbeads co-dispersed with bacteria in an oil-in-water emulsion. We also demonstrate how the holographic fingerprint of different contaminants can contribute to identifying their source., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

We demonstrate how holographic video microscopy can be used to detect, count, and characterize individual micrometer-scale protein aggregates as they flow down a microfluidic channel in their native buffer. Holographic characterization directly measures the radius and refractive index of subvisible protein aggregates and offers insights into their morphologies. The measurement proceeds fast enough to build up population averages for time-resolved studies and lends itself to tracking trends in protein aggregation arising from changing environmental factors. Information on individual particle's refractive indexes can be used to differentiate protein aggregates from such contaminants as silicone droplets. These capabilities are demonstrated through measurements on samples of bovine pancreas insulin aggregated through centrifugation and of bovine serum albumin aggregated by complexation with a polyelectrolyte. Differentiation is demonstrated with samples that have been spiked with separately characterized silicone spheres. Holographic characterization measurements are compared with results obtained with microflow imaging and dynamic light scattering., (Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Purpose: To compare higher-order aberrations (HOAs) after foldable phakic intraocular lens (pIOL) implantation and wavefront-guided photorefractive keratectomy (PRK) for the correction of myopia., Setting: Department of Ophthalmology, Medisch Centrum Haaglanden, Den Haag, The Netherlands., Design: Comparative case series., Methods: This retrospective comparative case series comprised eyes that had implantation of an Artiflex foldable pIOL or wavefront-guided PRK. Preoperative and postoperative refractive data, pupil diameter, endothelial cell density, and higher-order aberrations (HOAs) were measured and compared., Results: The median follow-up was 13 months in the pIOL group and 10 months in the PRK group. The mean spherical equivalent (SE) in the pIOL group was -5.50 diopters (D) ± 1.05 (SD) (range -3.25 to -7.13 D) preoperatively and -0.26 ± 0.28 D (range -0.88 to 0.38 D) postoperatively and the mean in the PRK group, -5.72 ± 0.88 D (range -4.25 to -7.50 D) and -0.03 ± 0.42 D (range -0.75 to 1.00 D), respectively. The postoperative SE was within ±0.50 D in 86.4% of eyes and 85.2% of eyes, respectively. Postoperatively, the significant changes in the pIOL group and the PRK group, respectively, were as follows: total HOAs, 0.03 μm and 0.26 μm (P = .001); trefoil-y, 0.09 μm and -0.04 μm (P = .004); spherical aberration, -0.05 μm and 0.21 μm (P = .002)., Conclusions: Foldable pIOL implantation increased total HOAs and spherical aberration less than wavefront-guided PRK to correct low to moderate myopia, suggesting foldable pIOL implantation maintains the cornea's prolate shape better., (Copyright © 2011 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.)

Purpose: To determine the effects of Artiflex phakic intraocular lens (PIOL) implantation on best spectacle-corrected high and low contrast visual acuity under photopic and mesopic conditions., Methods: Forty-two consecutive eyes had implantation of a foldable iris-supported PIOL to correct myopia. The mean preoperative spherical equivalent refraction was -7.26+/-1.91 diopters (D) (range: -4.90 to -13.60 D). The high (100%) and low (2.5%) contrast visual acuity with best spectacle correction was tested with translucent logarithmic visual acuity charts (Precision Vision) under photopic and mesopic conditions., Results: At mean 7.6+/-5.4 months after PIOL implantation, the mean high and low contrast visual acuity under photopic and mesopic conditions had significantly increased from preoperatively: photopic high contrast visual acuity from -0.07+/-0.11 logMAR to -0.14+/-0.08 logMAR (P<.001); photopic low contrast visual acuity from 0.48+/-0.14 logMAR to 0.36+/-0.11 logMAR (P=.011); mesopic high contrast visual acuity from -0.09+/-0.09 logMAR to -0.16+/-0.08 logMAR (P<.001); and mesopic low contrast visual acuity from 0.50+/-0.13 logMAR to 0.33+/-0.10 logMAR (P<.001). Mean postoperative spherical equivalent refraction was -0.21+/-0.28 D (range: +0.50 to -0.75 D). Of all eyes, 91.9% were within 0.50 D of attempted correction, and 67% gained 1 or more lines of best spectacle-corrected visual acuity (BSCVA). No eye lost 2 or more lines of BSCVA., Conclusions: Artiflex PIOL implantation for the correction of moderate to high myopia seems to be a safe and predictable procedure, which resulted in increased high and low contrast visual acuity under photopic and mesopic conditions., (Copyright 2010, SLACK Incorporated.)

Objective: To determine whether racial/ethnic differences in colon cancer screening are independent of socioeconomic and personal risk factors., Design: Baseline cross-section for a prospective cohort., Method: We recruited adults between 2000 and 2002 to undergo a questionnaire and venipuncture to study cancer risks., Results: Among 5595 adults over 50 years old, 40.3% reported sigmoidoscopy or colonoscopy within the prior five years; rates were 48.0% for Whites, 32.8% for Blacks, 27.9% for Hispanics, 30.3% for Asians, and 33.3% for others. Adjusting for age, gender, access to care (as income and insurance), and risk profile (as cancer in family, smoking, and obesity), Blacks and Hispanics were less likely to have been screened than Whites., Conclusions: Screening for colon cancer is low, especially among racial/ethnic minorities. Sociocultural factors merit closer attention.

The Parkin gene is an extremely large gene (1.5 Mb) within the highly unstable FRA6E common fragile site (CFS) region, which is frequently altered in ovarian, breast, and hepatocellular carcinomas. Because Parkin/FRA6E has genomic similarities to FHIT/FRA3B and WWOX/FRA16D, two other large tumor-suppressor genes that are within CFS regions, we were interested in characterizing Parkin gene alterations and their possible association with cancer. After analyzing 50 cancer-derived cell lines including 11 hepatocellular carcinoma (HCC) cell lines, we found that one HCC cell line, PLC/PRF/5, had a detectable homozygous deletion encompassing exon 3. Using quantitative duplex PCR and fluorescence in situ hybridization analysis to characterize the copy number changes of Parkin exons in HCC cell lines, we found that 4 of 11 HCC cell lines had heterozygous deletions of Parkin exons and one, Hep3B, had an exon duplication. Parkin protein expression was significantly decreased or absent in all 11 HCC cell lines. Furthermore, more than 50% of HCC primary tumors had decreased Parkin expression compared to that in normal liver tissue. Parkin gene-transfected PLC5 and Hep3B cells grew more slowly than vector-only transfectants and also showed increased sensitivity to apoptosis induced by cell-cycle inhibitors. Therefore, we suggest that Parkin may be involved in tumor suppression and that the loss of Parkin contributes to the development of hepatocarcinoma., (Copyright 2004 Wiley-Liss, Inc.)

High-resolution microwave and infrared molecular-beam spectra have been measured for 1,1,2,2-tetrafluoroethane (HFC134). For the higher energy, polar, C2 symmetry, gauche conformer, microwave spectra have been recorded for the normal and mono-13C isotopomers and analyzed to determine a C-C bond length of 1.512(4) Å, in good agreement with a recent ab initio value (MP2/6-31G**) of 1.515 Å [S. Papasavva, K. H. Illinger, and J. E. Kenny, J. Phys. Chem. 100, 10100-10110 (1996)]. A tunable microwave-sideband CO2 laser and electric-resonance optothermal spectrometer have been used to measure the infrared spectrum of the nu6, C-C stretch of the gauche conformer near 906 cm-1. Microwave-infrared double resonance and precise ground state combination differences provided by the microwave measurements guide the assignment of the spectrum. The observation of a c-type spectrum definitively establishes that the upper state vibration is of A symmetry in the C2 point group. The spectrum is fit to a Watson asymmetric-top Hamiltonian to a standard deviation of 0.24 MHz. A weak perturbation shifts the line positions for transitions near J = Kc = 20 by as much as 12 MHz. The identity of the perturber is unknown. Pulsed slit-jet diode-laser spectra have been recorded for the nu16 vibration of the anti conformer near 1127 cm-1. An a- and c-type hybrid band is observed, consistent with a Bu symmetry mode. Previous low-resolution studies have attributed the 1127-cm-1 mode to either a Bu or an Au symmetry vibration. A total of 522 nonblended transitions were assigned and fit to determine ground and excited state constants. The ground state constants of A = 5134.952(65) MHz, B = 3148.277(27) MHz, and C = 2067.106(43) MHz are the first experimental determinations of the rotational constants for this conformer. Here, type A standard uncertainties are given in the parentheses. Copyright 1998 Academic Press.

A case of a 40-year old marine with bilateral optic neuritis and a branch retinal artery occlusion after vaccination is presented. Blood investigations showed no abnormalities. Cerebrospinal fluid studies revealed a lymphocytic pleocytosis and IgG antibodies against hepatitis A and rabies. Computerized tomography and magnetic resonance imaging of the brain were negative. A diagnosis of vaccine-induced auto-immune demyelinative optic neuritis was made. The clinical picture improved after systemic corticosteroid treatment.