PNNL-13501 - Pacific Northwest National Laboratory

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Study Control Number: PN00079/1486 Real Time Biomarkers of Oxidative Stress Thomas J. Weber Oxygen free radicals have been implicated in the pathophysiology of a number of human diseases, including cancer, atherosclerosis, neurodegenerative disorders, and aging. Oxidative stress is also central to the adverse effects of ionizing radiation. In the present study, we have investigated mass changes detected in exhaled breath of mice following hyperoxia, and in cell culture following exposure to hydrogen peroxide with the goal of identifying a preliminary set of candidate biomarkers of oxidative stress that can be measured in real time. Project Description A breath analyzer device for the real time measurement of chemicals in exhaled breath using mass spectrometry is a tool that is ideally suited for noninvasive population and worker monitoring under a variety of conditions. This breath analyzer may also be useful for investigating the effect of experimental parameters such as geography and diet on defined mass targets in exhaled breath. In the present study, we have investigated mass changes detected in exhaled breath of mice following hyperoxia, and in cell culture following exposure to hydrogen peroxide with the goal of identifying preliminary biomarkers of oxidative stress that can be measured in real time. Since most, if not all, human disease is dependent on the generation of an oxidative stress, a noninvasive biomarker of oxygen free radical exposure may have wide application in human health monitoring. A number of candidate mass targets increased in response to oxidative stress were identified. Additional studies are required to identify these targets and further characterize their application as biomarkers. Introduction Oxygen is a necessary requirement of aerobic organisms, however, its more reactive metabolites, termed reactive oxygen species, are implicated in a number of diseases, including cancer, atherosclerosis, neurodegenerative disorders, and aging (Janssen et al. 1993). The adverse effects of ionizing radiation are also attributed to reactive oxygen species generation (Dahm-Daphi et al. 2000). Mammalian cells have developed an elaborate defense system, which includes enzymatic and nonenzymatic antioxidants, to protect themselves against the damaging effects of reactive oxygen species. However, these defense systems are not always adequate and can be overcome, leading to reactive oxygen species-dependent damage to cellular macromolecules. Current methods for analyzing reactive oxygen speciesinduced cellular damage are difficult and not easily extended to health monitoring on a broad scale. An accurate biomarker of oxidative stress that can be measured noninvasively in real time may eliminate this limitation. We have applied a noninvasive analytical tool (breath analyzer) to experimental model systems perturbed by an oxidative stress to determine if a preliminary set of candidate biomarkers could be identified. Volatile or exhaled masses from two experimental model systems were compared, namely, 1) cultured cells treated with hydrogen peroxide and 2) mice exposed to hyperoxia. Proper validation of these biomarkers may contribute to human health monitoring. Results and Accomplishments Mass Spectrometry Analysis of Cell Culture Head Space Following Treatment with Hydrogen Peroxide Cells in culture (LLC-PK1) were treated with hydrogen peroxide (0.27 mM) and volatile chemicals in the “headspace” monitored over time (1 to 75 minutes) by mass spectrometry. Mass differences between control and H2O2-treated cells were divided into two categories, termed persistent and transient changes. Specific masses categorized into these groups are summarized in Tables 1 through 3, and represent an increased appearance of the target mass, relative to control. As an additional control, we identified mass changes for volatile chemicals following treatment of cell culture media with hydrogen peroxide under identical conditions. In a second model system, mice were exposed to 95% oxygen for 24 hours (hyperoxia) and mass changes in exhaled breath were determined. Mass changes are summarized in Table 4. Human Health and Safety 299
Table 1. Transient mass changes associated with media following H 2O 2 treatment M/Z = 44, 59, 60, 63, 72, 87, 88, 89 Table 2. Transient mass changes associated with cells following H 2O 2 treatment M/Z = 24, 35, 37, 43, 44, 51, 56, 67, 75 Table 3. Persistent mass changes associated with cells following H 2O 2 treatment M/Z = 41, 52, 65, 88 Table 4. Mass changes in hyperoxic mice M/Z = 28, 31, 32, 33, 47, 36, 37, 39, 41, 42, 48, 49, 50, 52, 53, 54, 55, 57, 58, 59, 60, 61, 62, 64, 68, 69, 70, 72, 76, 77, 78, 80, 81, 82, 83, 84, 86, 88, 89, 92, 95, 96, 98, 99, 100, 101, 102, 103, 104, 105, 106, 108, 111, 112, 1113, 115, 116, 118, 119, 120, 122, 125, 127, 128, 129, 131, 133, 134, 135, 136, 137, 138, 140, 142, 145, 146, 151, 152, 154, 155, 156, 157, 158, 159, 160, 161, 1662, 163, 164, 165, 166, 180, 183, 200, 203, 205, 208, 209 Summary and Conclusions A number of mass targets were identified in our study. Clearly, a more complex data set was identified in our preliminary studies in a hyperoxic whole animal model, relative to cultured cells treated with hydrogen peroxide. Of particular interest, a candidate biomarker (mass/change ratio; = 88) was observed both in vitro and 300 FY 2000 <strong>Laboratory</strong> Directed Research and Development Annual Report in vivo in response to reactive oxygen species exposure, under conditions that are likely comparable to the steadystate response to reactive oxygen species. We have examined the literature for mass targets increased in pathologies associated with an oxidative stress to determine whether there is precedent for this M/Z = 88 target. A volatile M/Z = 88.2 was identified in burn patients with infection, a condition associated with the generation of an oxidative stress (Labows et al. 1980). The M/Z = 88.2 was identified as isopentanol. Therefore, future studies will be directed at determining whether the M/Z = 88 target identified in our studies is also isopentanol. The correlation between prooxidant states and an increased detection of M/Z = 88 raises the possibility that this mass may be a biomarker of oxidative stress that can be measured noninvasively in real time. References Dahm-Daphi J, C Sass, and W Alberti, W. 2000. “Comparison of biological effects of DNA damage induced by ionizing radiation and hydrogen peroxide in CHO cells.” Int. J. Radiat. Biol. 76, 67-75. Janssen YM, B Van Houten, PJ Borm, and BT Mossman. 1993. “Cell and tissue responses to oxidative damage.” Lab. Invest. 69, 261-274. Labows JN, KJ McGinley, GF Webster, and JJ Leyden. 1980. “Headspace analysis of volatile metabolites of Pseudomonas aeruginosa and related species by gas chromatography-mass spectrometry.” J Clin. Microbiol. 12(4):521-526.
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Laboratory Directed Research and De
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Laboratory Directed Research and De
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Computational Science and Engineeri
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Policy and Management Sciences Asse
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Introduction Department of Energy O
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5. After reviews by the Technical a
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• The Technical Council peer revi
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methods applicable to combustion, s
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Summary Each national laboratory mu
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Study Control Number: PN0004/1411 A
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Table 1. Positive and negative ions
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m/z Arbitrary Units Figure 1. Mass
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introduce low-volatility compounds
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arrangement, but the charge is reta
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two liquid chromatographic gradient
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Integrated Microfabricated Devices
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Matrix Assisted Laser Desorption/Io
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Molecular Beam Synthesis and Charac
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temperature distribution of the des
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expected to result in significant a
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Study Control Number: PN99069/1397
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Seuss DT and KA Prather. 1999. “M
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Analysis of Receptor-Modulated Ion
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laboratory (Lu and Xie 1997; Lu et
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Automated DNA Fingerprinting Microa
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Comparison of 4 Xanthomonas Isolate
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Approach Hematopoietic (bone marrow
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Anti-Human lgG Human lgG Protein G
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Characterization of Global Gene Reg
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PCR products for immobilization on
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identify novel proteins of importan
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Immunoprecipitaton of tyrosinephosp
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Coupled NMR and Confocal Microscopy
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In the optical microscopy image, th
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Development and Applications of a M
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Figure 3. Hepa-1 cells undergoing a
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cellular processing (such as post-t
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8. Initial demonstration of an off-
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expression systems for several year
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accumulation of protein products, i
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Fungal Conversion of Agricultural W
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Fungal Conversion of Waste Glucose/
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Summary and Conclusions We demonstr
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are shown in Figure 1(a) and 1(b),
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Anchorage-Independent Growth (% con
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selection of seedlings demonstratin
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NMR-Based Structural Genomics Micha
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Solution-State Structure and Fold-C
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Plant Root Exudates and Microbial G
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Summary and Conclusions • The gre
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Jones-Oliveira JB, JS Oliveira, HE
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• securely moves files to a targe
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Plenary invited lecture, “The Ele
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Figure 1. X3DGEN tetrahedral mesh o
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Figure 5a. OSO volume rendering of
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Development of Models of Cell-Signa
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SOS p 23 EGFR Professor Rodland at
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Oliveira JS, JB Jones-Oliveira, and
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Figure 2. Associating a dataset mar
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shown in Figure 1. Simulated result
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HostBuilder: A Tool for the Combina
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Invariant Discretization Methods fo
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Graphics, Inc., workstation. The co
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Mixed Hamiltonian Methods for Geoch
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guyanaite, and grimaldiite (see Fig
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in the Environmental Molecular Scie
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Simulation and Modeling of Electroc
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Summary and Conclusions We implemen
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Since the implementation of the gen
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asis of previous performance, perce
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Bioinformatics for High-Throughput
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Summary and Conclusions In previous
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User Cat - Supervisor (client) Anal
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The second goal was to research way
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Figure 1. A visualization technique
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and interactions. The flexibility o
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Figure 4. A filtered version of Fig
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Development of Modeling Tools for J
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Figure 2. Schematic of experimental
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Integrated Modeling and Simulation
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(a) (b) (c) (d) Figure 1. Results o
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Thurman DA. August 2000. Collaborat
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MARC Input initial m esh and result
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(Johnson 1999; Colligan 1999; Gould
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Modeling of High-Velocity Forming f
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Global divergence error 1 10 -14 0
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that users were unlikely to appreci
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Earth System Science
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the lateral extent of the plume so
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Biogeochemical Processes and Microb
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Analyses of populations of viable a
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The first task was to establish fiv
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purged for 2 minutes. The gas sampl
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developing code architecture to min
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Berkowitz, CM. June 2000. “Atmosp
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environmental monitoring, 2) portab
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corresponded to a current density o
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Enhancing Emissions Pre-Processing
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Environmental Fate and Effects of D
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nitrogen and unpredictable eutrophi
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Figure 1. Ordinary kriging of 2 m d
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precipitation of calcite occurred i
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Interrelationships Between Processe
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phenanthrene resistant fraction con
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injection of an immiscible gas phas
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still some key kinetic issues that
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Application of a Crop Model The res
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The Nature, Occurrence, and Origin
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Particle number concentration, 1/cm
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integrated voltage (V) 0.10 0.08 0.
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The first step was to implement thi
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Spontaneous Formation of Cu2O Nano-
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Actinide Chemistry at the Aqueous-M
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Figure 5. Close-up atomic force mic
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to 300°C and 400°C. Unfortunately
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Summary and Conclusions Transmutati
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Advanced Calibration/Registration T
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Figure 1a. Violet filter Figure 1b.
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Autonomous Ultrasonic Probe for Pro
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containing nitrous oxide, an optica
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Chemical Detection Using Cavity Enh
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Figure 3 is a color rendered simula
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appropriate study has been performe
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Exploitation of Conventional Chemic
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Figures 3 and 4 illustrate the impa
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enefits of using a modified spread-
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Single Channel Signal 0.0025 0.0020
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Therefore, in order to extract the
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A next-generation technology is nee
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Wind Dir. Figure 2. Negative ion co
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Volumetric Imaging of Materials by
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amplitude and peak frequency change
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Figure 3. 13 C NMR spectrum of corn
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Approach Two flow loops, one that o
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Modification of Ion Exchange Resins
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Permanganate Treatment for the Deco
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minimized by medium exchange. After
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Figure 1. Thermogravimetric analysi
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Validation and Scale-Up of Monosacc
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Concentration (g/100g solution) Con
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Analysis of High-Volume, Hyper-Dime
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ecent figure of merit values. Shoul
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Probabilistic Methods Development f
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a) Traditional PCA-based process co
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Acronyms and Abbreviations 2-D two-
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