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  1. Article ; Online: Features in Backgrounds of Microscopy Images Introduce Biases in Machine Learning Analyses.

    Greenblott, David N / Johann, Florian / Snell, Jared R / Gieseler, Henning / Calderon, Christopher P / Randolph, Theodore W

    Journal of pharmaceutical sciences

    2024  Volume 113, Issue 5, Page(s) 1177–1189

    Abstract: Subvisible particles may be encountered throughout the processing of therapeutic protein formulations. Flow imaging microscopy (FIM) and backgrounded membrane imaging (BMI) are techniques commonly used to record digital images of these particles, which ... ...

    Abstract Subvisible particles may be encountered throughout the processing of therapeutic protein formulations. Flow imaging microscopy (FIM) and backgrounded membrane imaging (BMI) are techniques commonly used to record digital images of these particles, which may be analyzed to provide particle size distributions, concentrations, and identities. Although both techniques record digital images of particles within a sample, FIM analyzes particles suspended in flowing liquids, whereas BMI records images of dry particles after collection by filtration onto a membrane. This study compared the performance of convolutional neural networks (CNNs) in classifying images of subvisible particles recorded by both imaging techniques. Initially, CNNs trained on BMI images appeared to provide higher classification accuracies than those trained on FIM images. However, attribution analyses showed that classification predictions from CNNs trained on BMI images relied on features contributed by the membrane background, whereas predictions from CNNs trained on FIM features were based largely on features of the particles. Segmenting images to minimize the contributions from image backgrounds reduced the apparent accuracy of CNNs trained on BMI images but caused minimal reduction in the accuracy of CNNs trained on FIM images. Thus, the seemingly superior classification accuracy of CNNs trained on BMI images compared to FIM images was an artifact caused by subtle features in the backgrounds of BMI images. Our findings emphasize the importance of examining machine learning algorithms for image analysis with attribution methods to ensure the robustness of trained models and to mitigate potential influence of artifacts within training data sets.
    MeSH term(s) Microscopy ; Machine Learning ; Neural Networks, Computer ; Algorithms ; Bias
    Language English
    Publishing date 2024-03-12
    Publishing country United States
    Document type Journal Article
    ZDB-ID 3151-3
    ISSN 1520-6017 ; 0022-3549
    ISSN (online) 1520-6017
    ISSN 0022-3549
    DOI 10.1016/j.xphs.2024.03.003
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  2. Article ; Online: Motion blur filtering: A statistical approach for extracting confinement forces and diffusivity from a single blurred trajectory.

    Calderon, Christopher P

    Physical review. E

    2016  Volume 93, Issue 5, Page(s) 53303

    Abstract: Single particle tracking (SPT) can aid in understanding a variety of complex spatiotemporal processes. However, quantifying diffusivity and confinement forces from individual live cell trajectories is complicated by inter- and intratrajectory kinetic ... ...

    Abstract Single particle tracking (SPT) can aid in understanding a variety of complex spatiotemporal processes. However, quantifying diffusivity and confinement forces from individual live cell trajectories is complicated by inter- and intratrajectory kinetic heterogeneity, thermal fluctuations, and (experimentally resolvable) statistical temporal dependence inherent to the underlying molecule's time correlated confined dynamics experienced in the cell. The problem is further complicated by experimental artifacts such as localization uncertainty and motion blur. The latter is caused by the tagged molecule emitting photons at different spatial positions during the exposure time of a single frame. The aforementioned experimental artifacts induce spurious time correlations in measured SPT time series that obscure the information of interest (e.g., confinement forces and diffusivity). We develop a maximum likelihood estimation (MLE) technique that decouples the above noise sources and systematically treats temporal correlation via time series methods. This ultimately permits a reliable algorithm for extracting diffusivity and effective forces in confined or unconfined environments. We illustrate how our approach avoids complications inherent to mean square displacement or autocorrelation techniques. Our algorithm modifies the established Kalman filter (which does not handle motion blur artifacts) to provide a likelihood based time series estimation procedure. The result extends A. J. Berglund's motion blur model [Phys. Rev. E 82, 011917 (2010)PLEEE81539-375510.1103/PhysRevE.82.011917] to handle confined dynamics. The approach can also systematically utilize (possibly time dependent) localization uncertainty estimates afforded by image analysis if available. This technique, which explicitly treats confinement and motion blur within a time domain MLE framework, uses an exact likelihood (time domain methods facilitate analyzing nonstationary signals). Our estimator is demonstrated to be consistent over a wide range of exposure times (5 to 100 ms), diffusion coefficients (1×10^{-3} to 1μm^{2}/s), and confinement widths (100 nm to 2μm). We demonstrate that neglecting motion blur or confinement can substantially bias estimation of kinetic parameters of interest to researchers. The technique also permits one to check statistical model assumptions against measured individual trajectories without "ground truth." The ability to reliably and consistently extract motion parameters in trajectories exhibiting confined and/or non-stationary dynamics, without exposure time artifacts corrupting estimates, is expected to aid in directly comparing trajectories obtained from different experiments or imaging modalities. A Python implementation is provided (open-source code will be maintained on GitHub; see also the Supplemental Material with this paper).
    Language English
    Publishing date 2016-05
    Publishing country United States
    Document type Journal Article
    ZDB-ID 2844562-4
    ISSN 2470-0053 ; 2470-0045
    ISSN (online) 2470-0053
    ISSN 2470-0045
    DOI 10.1103/PhysRevE.93.053303
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  3. Article ; Online: Supervised and unsupervised machine learning approaches for monitoring subvisible particles within an aluminum-salt adjuvanted vaccine formulation.

    Greenblott, David N / Wood, Caitlin V / Zhang, Jingtao / Viza, Nelia / Chintala, Ramesh / Calderon, Christopher P / Randolph, Theodore W

    Biotechnology and bioengineering

    2024  Volume 121, Issue 5, Page(s) 1626–1641

    Abstract: Suspensions of protein antigens adsorbed to aluminum-salt adjuvants are used in many vaccines and require mixing during vial filling operations to prevent sedimentation. However, the mixing of vaccine formulations may generate undesirable particles that ... ...

    Abstract Suspensions of protein antigens adsorbed to aluminum-salt adjuvants are used in many vaccines and require mixing during vial filling operations to prevent sedimentation. However, the mixing of vaccine formulations may generate undesirable particles that are difficult to detect against the background of suspended adjuvant particles. We simulated the mixing of a suspension containing a protein antigen adsorbed to an aluminum-salt adjuvant using a recirculating peristaltic pump and used flow imaging microscopy to record images of particles within the pumped suspensions. Supervised convolutional neural networks (CNNs) were used to analyze the images and create "fingerprints" of particle morphology distributions, allowing detection of new particles generated during pumping. These results were compared to those obtained from an unsupervised machine learning algorithm relying on variational autoencoders (VAEs) that were also used to detect new particles generated during pumping. Analyses of images conducted by applying both supervised CNNs and VAEs found that rates of generation of new particles were higher in aluminum-salt adjuvant suspensions containing protein antigen than placebo suspensions containing only adjuvant. Finally, front-face fluorescence measurements of the vaccine suspensions indicated changes in solvent exposure of tryptophan residues in the protein that occurred concomitantly with new particle generation during pumping.
    MeSH term(s) Aluminum ; Unsupervised Machine Learning ; Adjuvants, Immunologic/chemistry ; Vaccines/chemistry ; Antigens/chemistry
    Chemical Substances Aluminum (CPD4NFA903) ; Adjuvants, Immunologic ; Vaccines ; Antigens
    Language English
    Publishing date 2024-02-19
    Publishing country United States
    Document type Journal Article
    ZDB-ID 280318-5
    ISSN 1097-0290 ; 0006-3592
    ISSN (online) 1097-0290
    ISSN 0006-3592
    DOI 10.1002/bit.28671
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  4. Article ; Online: Machine learning approaches to root cause analysis, characterization, and monitoring of subvisible particles in monoclonal antibody formulations.

    Greenblott, David N / Zhang, Jingtao / Calderon, Christopher P / Randolph, Theodore W

    Biotechnology and bioengineering

    2022  Volume 119, Issue 12, Page(s) 3596–3611

    Abstract: Processing stresses on therapeutic proteins may cause formation of subvisible particles. Different stress mechanisms generate particle populations with characteristic morphological "fingerprints," and machine learning techniques like convolutional neural ...

    Abstract Processing stresses on therapeutic proteins may cause formation of subvisible particles. Different stress mechanisms generate particle populations with characteristic morphological "fingerprints," and machine learning techniques like convolutional neural networks (CNNs) allow classification of microscopy images of these particles according to known stresses at their root cause. Using CNNs to classify novel particle types not included during network training may lead to inaccurate classification, however, using CNNs to monitor the presence of particulate matter not explicitly used in training could serve as a useful process analytical technology. We used CNNs to classify and identify the root cause of particles generated by subjecting three monoclonal antibodies (mAbs) to various common manufacturing stresses. We probed the generality of particles generated by stressing different mAbs in different formulations and showed that CNN analyses were sensitive not only to the applied stress, but also the buffer conditions and the particular mAb that generated particle populations. Thus, models trained on images of particles created with one mAb and buffer system may not provide accurate root cause analysis when applied to particles generated by other mAb and buffer systems. A lever-rule analysis of CNN-derived fingerprints was used to characterize the composition of mixtures of particle types. Finally, we monitored the temporal evolution of CNN-derived fingerprints when novel populations of particles, which were not included during training, were generated by pumping mAb solutions through a peristaltic pump.
    MeSH term(s) Antibodies, Monoclonal ; Root Cause Analysis ; Drug Compounding ; Machine Learning ; Neural Networks, Computer
    Chemical Substances Antibodies, Monoclonal
    Language English
    Publishing date 2022-10-03
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 280318-5
    ISSN 1097-0290 ; 0006-3592
    ISSN (online) 1097-0290
    ISSN 0006-3592
    DOI 10.1002/bit.28239
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  5. Article ; Online: Correction: Calderon, C.P. Data-Driven Techniques for Detecting Dynamical State Changes in Noisily Measured 3D Single-Molecule Trajectories. Molecules 19, 18381-18398.

    Calderon, Christopher P

    Molecules (Basel, Switzerland)

    2015  Volume 20, Issue 2, Page(s) 2828–2830

    Abstract: The author wishes to make the following corrections to paper [1] (doi:10.3390/molecules 191118381, website: http://www.mdpi.com/1420-3049/19/11/18381): ...

    Abstract The author wishes to make the following corrections to paper [1] (doi:10.3390/molecules 191118381, website: http://www.mdpi.com/1420-3049/19/11/18381):
    MeSH term(s) Models, Theoretical
    Language English
    Publishing date 2015-02-09
    Publishing country Switzerland
    Document type Published Erratum
    ZDB-ID 1413402-0
    ISSN 1420-3049 ; 1431-5165 ; 1420-3049
    ISSN (online) 1420-3049
    ISSN 1431-5165 ; 1420-3049
    DOI 10.3390/molecules20022828
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  6. Article ; Online: Enhanced Diffusive Transport in Fluctuating Porous Media.

    Sarfati, Raphaël / Calderon, Christopher P / Schwartz, Daniel K

    ACS nano

    2021  Volume 15, Issue 4, Page(s) 7392–7398

    Abstract: Mass transport within porous structures is a ubiquitous process in biological, geological, and technological systems. Despite the importance of these phenomena, there is no comprehensive theory that describes the complex and diverse transport behavior ... ...

    Abstract Mass transport within porous structures is a ubiquitous process in biological, geological, and technological systems. Despite the importance of these phenomena, there is no comprehensive theory that describes the complex and diverse transport behavior within porous environments. While the porous matrix itself is generally considered a static and passive participant, many porous environments are in fact dynamic, with fluctuating walls, pores that open and close, and dynamically changing cross-links. While diffusion has been measured in fluctuating structures, notably in model biological systems, it is rarely possible to isolate the effect of fluctuations because of the absence of control experiments involving an identical static counterpart, and it is generally impossible to observe the dynamics of the structure. Here we present a direct comparison of the diffusion of nanoparticles of various sizes within a trackable, fluctuating porous matrix and a geometrically equivalent static matrix, in conditions spanning a range of regimes from
    Language English
    Publishing date 2021-04-01
    Publishing country United States
    Document type Journal Article ; Research Support, U.S. Gov't, Non-P.H.S.
    ISSN 1936-086X
    ISSN (online) 1936-086X
    DOI 10.1021/acsnano.1c00744
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  7. Article ; Online: Interfacial Adsorption Controls Particle Formation in Antibody Formulations Subjected to Extensional Flows and Hydrodynamic Shear.

    Thite, Nidhi G / Ghazvini, Saba / Wallace, Nicole / Feldman, Naomi / Calderon, Christopher P / Randolph, Theodore W

    Journal of pharmaceutical sciences

    2023  Volume 112, Issue 11, Page(s) 2766–2777

    Abstract: During their manufacturing and delivery to patients, therapeutic proteins are commonly exposed to various interfaces and to hydrodynamic shear forces. Although adsorption of proteins to solid-liquid interfaces is known to foster formation of protein ... ...

    Abstract During their manufacturing and delivery to patients, therapeutic proteins are commonly exposed to various interfaces and to hydrodynamic shear forces. Although adsorption of proteins to solid-liquid interfaces is known to foster formation of protein aggregates and particles, the impact of shear remains controversial, in part because of experimental challenges in separating the effects of shear from those caused by simultaneous exposure to interfaces. Extensional flows (occurring when solutions flow through sudden contractions) exert localized elongational forces that have been suspected to be damaging to proteins. In this work, we measured aggregation and particle formation in formulations of polyclonal and monoclonal antibodies subjected to extensional flow, high shear (10
    Language English
    Publishing date 2023-07-13
    Publishing country United States
    Document type Journal Article
    ZDB-ID 3151-3
    ISSN 1520-6017 ; 0022-3549
    ISSN (online) 1520-6017
    ISSN 0022-3549
    DOI 10.1016/j.xphs.2023.07.010
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  8. Article ; Online: Data-driven techniques for detecting dynamical state changes in noisily measured 3D single-molecule trajectories.

    Calderon, Christopher P

    Molecules (Basel, Switzerland)

    2014  Volume 19, Issue 11, Page(s) 18381–18398

    Abstract: Optical microscopes and nanoscale probes (AFM, optical tweezers, etc.) afford researchers tools capable of quantitatively exploring how molecules interact with one another in live cells. The analysis of in vivo single-molecule experimental data faces ... ...

    Abstract Optical microscopes and nanoscale probes (AFM, optical tweezers, etc.) afford researchers tools capable of quantitatively exploring how molecules interact with one another in live cells. The analysis of in vivo single-molecule experimental data faces numerous challenges due to the complex, crowded, and time changing environments associated with live cells. Fluctuations and spatially varying systematic forces experienced by molecules change over time; these changes are obscured by "measurement noise" introduced by the experimental probe monitoring the system. In this article, we demonstrate how the Hierarchical Dirichlet Process Switching Linear Dynamical System (HDP-SLDS) of Fox et al. [IEEE Transactions on Signal Processing 59] can be used to detect both subtle and abrupt state changes in time series containing "thermal" and "measurement" noise. The approach accounts for temporal dependencies induced by random and "systematic overdamped" forces. The technique does not require one to subjectively select the number of "hidden states" underlying a trajectory in an a priori fashion. The number of hidden states is simultaneously inferred along with change points and parameters characterizing molecular motion in a data-driven fashion. We use large scale simulations to study and compare the new approach to state-of-the-art Hidden Markov Modeling techniques. Simulations mimicking single particle tracking (SPT) experiments are the focus of this study.
    MeSH term(s) Models, Theoretical
    Language English
    Publishing date 2014-11-12
    Publishing country Switzerland
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 1413402-0
    ISSN 1420-3049 ; 1431-5165 ; 1420-3049
    ISSN (online) 1420-3049
    ISSN 1431-5165 ; 1420-3049
    DOI 10.3390/molecules191118381
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  9. Article ; Online: Statistical Inference of Transport Mechanisms and Long Time Scale Behavior from Time Series of Solute Trajectories in Nanostructured Membranes.

    Coscia, Benjamin J / Calderon, Christopher P / Shirts, Michael R

    The journal of physical chemistry. B

    2020  Volume 124, Issue 37, Page(s) 8110–8123

    Abstract: Appropriate time series modeling of complex diffusion in soft matter systems on the microsecond time scale can provide a path toward inferring transport mechanisms and predicting bulk properties characteristic of much longer time scales. In this work we ... ...

    Abstract Appropriate time series modeling of complex diffusion in soft matter systems on the microsecond time scale can provide a path toward inferring transport mechanisms and predicting bulk properties characteristic of much longer time scales. In this work we apply nonparametric Bayesian time series analysis, more specifically the sticky hierarchical Dirichlet process autoregressive hidden Markov model (HDP-AR-HMM) to solute center-of-mass trajectories generated from long molecular dynamics (MD) simulations in a cross-linked inverted hexagonal phase lyotropic liquid crystal (LLC) membrane in order to automatically detect a variety of solute dynamical modes. We can better understand the mechanisms controlling these dynamical modes by grouping the states identified by the HDP-AR-HMM into clusters based on multiple metrics aimed at distinguishing solute behavior based on their fluctuations, dwell times in each state, and positions within the inhomogeneous membrane structure. We analyze predominant clusters in order to relate their dynamical parameters to physical interactions between solutes and the membrane. Along with parameters of individual states, the HDP-AR-HMM simultaneously infers a transition matrix which allows us to stochastically propagate solute behavior from all of the independent trajectories onto arbitrary length time scales while still preserving the qualitative behavior characteristic of the MD trajectories. This affords a direct connection to important macroscopic observables used to characterize performance like solute flux and selectivity. This work provides a promising way to simultaneously identify transport mechanisms in nanoporous materials and project complex diffusive behavior on long time scales. Our enhanced understanding of the diverse range of solute behavior allows us to hypothesize design changes to LLC monomers aimed toward controlling the rates of solute passage, thus improving the selective performance of LLC membranes.
    Language English
    Publishing date 2020-09-02
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
    ISSN 1520-5207
    ISSN (online) 1520-5207
    DOI 10.1021/acs.jpcb.0c05010
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  10. Article ; Online: Machine learning and statistical analyses for extracting and characterizing "fingerprints" of antibody aggregation at container interfaces from flow microscopy images.

    Daniels, Austin L / Calderon, Christopher P / Randolph, Theodore W

    Biotechnology and bioengineering

    2020  Volume 117, Issue 11, Page(s) 3322–3335

    Abstract: Therapeutic proteins are exposed to numerous stresses during their manufacture, shipping, storage and administration to patients, causing them to aggregate and form particles through a variety of different mechanisms. These varied mechanisms generate ... ...

    Abstract Therapeutic proteins are exposed to numerous stresses during their manufacture, shipping, storage and administration to patients, causing them to aggregate and form particles through a variety of different mechanisms. These varied mechanisms generate particle populations with characteristic morphologies, creating "fingerprints" that are reflected in images recorded using flow imaging microscopy. Particle population fingerprints in test samples can be extracted and compared against those of particles produced under baseline conditions using an algorithm that combines machine learning tools such as convolutional neural networks with statistical tools such as nonparametric density estimation and Rosenblatt transform-based goodness-of-fit hypothesis testing. This analysis provides a quantitative method with user-specified type 1 error rates to determine whether the mechanisms that produce particles in test samples differ from particle formation mechanisms operative under baseline conditions. As a demonstration, this algorithm was used to compare particles within intravenous immunoglobulin formulations that were exposed to freeze-thawing and shaking stresses within a variety of different containers. This analysis revealed that seemingly subtle differences in containers (e.g., glass vials from different manufacturers) generated distinguishable particle populations after the stresses were applied. This algorithm can be used to assess the impact of process and formulation changes on aggregation-related product instabilities.
    MeSH term(s) Algorithms ; Antibodies/analysis ; Antibodies/chemistry ; Antibodies/metabolism ; Image Processing, Computer-Assisted/methods ; Immunoglobulins, Intravenous/analysis ; Immunoglobulins, Intravenous/chemistry ; Immunoglobulins, Intravenous/metabolism ; Machine Learning ; Microscopy/methods ; Protein Aggregates ; Protein Stability
    Chemical Substances Antibodies ; Immunoglobulins, Intravenous ; Protein Aggregates
    Language English
    Publishing date 2020-07-28
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ZDB-ID 280318-5
    ISSN 1097-0290 ; 0006-3592
    ISSN (online) 1097-0290
    ISSN 0006-3592
    DOI 10.1002/bit.27501
    Database MEDical Literature Analysis and Retrieval System OnLINE

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