Native SEC and Reversed-Phase LC-MS Reveal Impact of Fab Glycosylation of Anti-SARS-COV-2 Antibodies on Binding to the Receptor Binding Domain.

The binding affinity of monoclonal antibodies (mAbs) for their intended therapeutic targets is often affected by chemical and post-translational modifications in the antigen binding (Fab) domains. A new two-dimensional analytical approach is described here utilizing native size exclusion chromatography (SEC) to separate populations of antibodies and bound antibody-antigen complexes for subsequent characterization of these modifications by reversed-phase (RP) liquid chromatography-mass spectrometry (LC-MS) at the intact antibody level. Previously, we utilized peptide mapping to measure modifications impacting binding.

Lumbar multifidus muscle ultrasound imaging: Is handheld technology reliable?

Background: Advancement in ultrasound imaging technology has led to the development of handheld devices that are more accessible to physical therapists due to decreased cost, reduced size, and improved ease of use relative to current established units. Physical therapists use ultrasound imaging of the lumbar multifidus muscle (LMM) to assist in rehabilitation of patients with lumbar pathology.

Objectives: To identify the inter-device reliability of measuring the LMM thickness during a sustained contraction when comparing handheld (Butterfly iQ+) and established (SonoSite M-Turbo) ultrasound units.

The microbiome of an outpatient rehabilitation clinic and predictors of contamination: A pilot study.

Background: Understanding sources of microbial contamination in outpatient rehabilitation (REHAB) clinics is important to patients and healthcare providers.

Purpose: The purpose of this study was to characterize the microbiome of an outpatient REHAB clinic and examine relationships between clinic factors and contamination.

Methods: Forty commonly contacted surfaces in an outpatient REHAB clinic were observed for frequency of contact and swiped using environmental sample collection kits.

Non-targeted characterization of attributes affecting antibody-FcγRIIIa V158 (CD16a) binding via online affinity chromatography-mass spectrometry.

Antibodies facilitate targeted cell killing by engaging with immune cells such as natural killer cells through weak binding interactions with Fcγ receptors on the cell surface. Here, we evaluate the binding affinity of the receptor FcγRIIIa V158 (CD16a) for several therapeutic antibody classes, isoforms, and Fc-fusion proteins using an immobilized receptor affinity liquid chromatography (LC) approach coupled with online mass spectrometry (MS) detection. Aglycosylated FcγRIIIa was used in the affinity chromatography and compared with published affinities using glycosylated receptors.

Identification of critical chemical modifications by size exclusion chromatography of stressed antibody-target complexes with competitive binding.

Chemical modifications (attributes) in the binding regions of stressed therapeutic proteins may affect binding to target and efficacy of therapeutic proteins. The method presented here describes the criticality assessment of therapeutic antibody modifications by size-exclusion chromatography (SEC) of competitive binding between a stressed antibody and its target, human epidermal growth factor receptor-2 (HER2), followed by SEC fractionation and peptide mapping characterization of bound and unbound antibodies. When stressed antibody and its target were mixed at a stoichiometric molar ratio of 1:2, only antibody-receptor complex eluted from SEC, indicating that binding was not decreased to break the complex.

Identification of critical chemical modifications and paratope mapping by size exclusion chromatography of stressed antibody-target complexes.

Therapeutic proteins including antibodies and Fc-fusion proteins undergo a large number of chemical modifications during cell culture, purification, storage and in human circulation. They are also exposed to harsh conditions during stress studies, including elevated temperature, extremes of pH, forced oxidation, physiological pH, UV light to assess the possible degradation pathways and suitability of methods for detecting them. Some of these modifications are located on residues in binding regions, leading to loss of binding and potency and classified as critical quality attributes.

Characterization of therapeutic proteins by cation exchange chromatography-mass spectrometry and top-down analysis.

Recently, cation exchange chromatography (CEX) using aqueous volatile buffers was directly coupled with mass spectrometry (MS) and applied for intact analysis of therapeutic proteins and antibodies. In our study, chemical modifications responsible for charge variants were identified by CEX-UV-MS for a monoclonal antibody (mAb), a bispecific antibody, and an Fc-fusion protein. We also report post-CEX column addition of organic solvent and acid followed by mixing at elevated temperatures, which unfolded proteins, increased ion intensity (sensitivity) and facilitated top-down analysis.

Changes in Gait and Texting Ability During Progressively Difficult Gait Tasks.

To investigate the effects of a cell phone texting task on an individual's ability to perform three ambulation-based tasks, each with different and progressively more difficult demands. 36 participants (24 male/12 female, average age 23.4) performed: a Timed Up & Go (TUG), stair ambulation (STAIR), and tandem gait (TAN).

Impact of antibody subclass and disulfide isoform differences on the biological activity of CD200R and βklotho agonist antibodies.

Agonism of cell surface receptors by monoclonal antibodies is dependent not only on its ability to bind the target, but also to deliver a biological signal through receptors to the cell. Immunoglobulin G2 antibodies (IgG2s) are made up of a mixture of distinct isoforms (IgG2-A, -B and A/B), which differ by the disulfide connectivity at the hinge region. When evaluating panels of agonistic antibodies against CD200 receptor (CD200R) or βklotho receptor (βklotho), we noticed striking activity differences of IgG1 or IgG2 antibodies with the same variable domains.

Fabrication of petal-shaped masks for suppression of the on-axis Poisson spot in telescope systems.

The presence of a bright (Poisson) spot in the geometrical shadow of circular/spherical shapes has been known for the past two centuries. A broad class of telescopes that involve simultaneous transmit and receive require suppression of the reflected light from the secondary mirror on the detector. For instance, the on-axis design of optical telescope for the evolved Laser Interferometric Space Antenna (eLISA), a re-scoped version of the baseline LISA mission concept, requires suppression of reflected laser light from the secondary mirror on the detector.

Protected hinge in the immunoglobulin G2-A2 disulfide isoform.

Human IgG2 consists of disulfide-mediated structural isoforms, classified by the number of Fab arms disulfide-linked to the heavy chain hinge. In the IgG2-B isoform, both Fab arms are linked to the hinge region, and in IgG2-A, neither Fab arm are linked to the hinge. IgG2-A/B is a hybrid between these two forms, with only one Fab arm disulfide-linked to the hinge.

IgG2 disulfide isoform conversion kinetics.

Human IgG2 antibodies contain three types of disulfide isoforms, classified by the number of Fab arms having disulfide links to the heavy chain hinge region. In the IgG2-B form, both Fab arms have interchain disulfide bonds to the hinge region, and in IgG2-A, neither Fab arm are disulfide linked to the hinge. The IgG2-A/B is a hybrid between these two forms, with only one Fab arm disulfide linked to the hinge.

Passive 77 GHz millimeter-wave sensor based on optical upconversion.

A passive millimeter-wave (mmW) sensor operating at a frequency of 77 GHz is built and characterized. The sensor is a single pixel sensor that raster scans to create an image. Optical upconversion is used to convert the incident mmW signal into an optical signal for detection.

Field flow fractionation for assessing neonatal Fc receptor and Fcγ receptor binding to monoclonal antibodies in solution.

Analysis of the strength and stoichiometry of immunoglobulin G (IgG) binding to neonatal Fc receptor (FcRn) and Fcγ receptor (FcγR) is important for evaluating the pharmacokinetics and effector functions of therapeutic monoclonal antibody (mAb) products, respectively. The current standard for assessing FcγR and FcRn binding is composed of cell-based and surface plasmon resonance (SPR) assays. In this work, asymmetrical flow field flow fractionation (AF4) was evaluated to establish the true stoichiometry of IgG binding in solution.

Interchain disulfide bonding in human IgG2 antibodies probed by site-directed mutagenesis.

Human IgG2 exists as a mixture of disulfide-linked structural isoforms that can show different activities. To probe the contribution of specific cysteine residues to the formation of structural isoforms, we characterized a series of Cys-->Ser mutant IgG2 recombinant monoclonal antibodies, focused on the first C(H)1 cysteine and the first two hinge cysteines. These residues participate in the formation of structural isoforms that have been noted by nonreduced capillary sodium dodecyl sulfate polyacrylamide gel electrophoresis, reversed-phase high-performance liquid chromatography, and cation exchange chromatography.

Metformin inhibits breast cancer cell growth, colony formation and induces cell cycle arrest in vitro.

The anti-diabetic drug metformin reduces human cancer incidence and improves the survival of cancer patients, including those with breast cancer. We studied the activity of metformin against diverse molecular subtypes of breast cancer cell lines in vitro. Metformin showed biological activity against all estrogen receptor (ER) positive and negative, erbB2 normal and abnormal breast cancer cell lines tested.

Perceived quality benefits influenced by usefulness and documentation accuracy of information systems.

In the technology-rich environment of healthcare, hospital documentation systems need to be developed so that quality benefits are realized and are more evident. The effects of nurses' perceptions of ease of use, usefulness, and documentation accuracy on their perceptions of improved quality benefits from information systems were studied based on a proposed model. This model was developed from variables in both technology acceptance and healthcare literature.

Conformational implications of an inversed pH-dependent antibody aggregation.

Antibody formulation development relies on accelerated stability data at elevated temperatures to optimize formulation parameters. However, the pH- and temperature-dependence of aggregation is complicated for antibody formulations. In this study, a human monoclonal IgG2 antibody exhibited typical pH-dependent dimer formation under normal storage conditions (4 and/or 29 degrees C).

Human IgG2 antibody disulfide rearrangement in vivo.

Proteins destined to circulate in the blood are first folded and assembled in the endoplasmic reticulum of secretory cells. For antibodies, like many other serum proteins, the folding and assembly steps involve the formation of disulfide bonds. Such bonds have been thought to be static features of proteins, stabilizing domains, and linking polypeptide chains, although some cases of extracellular disulfide bond cleavage have been noted.

Fiber-to-waveguide coupler based on the parabolic reflector.

We present a fiber-to-waveguide coupling structure, the so-called vertical J coupler, based on the parabolic reflector. The device addresses the multiple objectives of high coupling efficiency, large bandwidth operation, polarization insensitivity, and compact footprint. The optical mode emanating from a fiber arranged normal to the plane of the substrate is incident underneath the parabolic reflector, turned through 90 degrees and focused into a dielectric waveguide.

Structural and functional characterization of disulfide isoforms of the human IgG2 subclass.

In the accompanying report ( Wypych, J., Li, M., Guo, A.

Human IgG2 antibodies display disulfide-mediated structural isoforms.

In this work, we present studies of the covalent structure of human IgG2 molecules. Detailed analysis showed that recombinant human IgG2 monoclonal antibody could be partially resolved into structurally distinct forms caused by multiple disulfide bond structures. In addition to the presently accepted structure for the human IgG2 subclass, we also found major structures that differ from those documented in the current literature.

Automated tryptic digestion procedure for HPLC/MS/MS peptide mapping of immunoglobulin gamma antibodies in pharmaceutics.

The rapid growth of antibody drugs and drug candidates in the biopharmaceutical industry has created a demand for automated proteolytic digestion to assist in pharmaceutical stability studies, identity assays and quality control of these therapeutic proteins. Here, we describe the development of a fully automated proteolytic digestion procedure for monoclonal antibodies in solution, which requires a high concentration of denaturants for unfolding. The antibody samples were placed in a 96-well plate or in 0.

Characterization of IgG1 immunoglobulins and peptide-Fc fusion proteins by limited proteolysis in conjunction with LC-MS.

A combinatory approach for the characterization of post-translational and chemical modifications in high molecular weight therapeutic proteins like antibodies and peptide-Fc fusion proteins (MW > or = 50 000 Da) is presented. In this approach, well-established techniques such as limited proteolysis, reversed-phase (RP) high-performance liquid chromatography (HPLC), and in-line mass spectrometry (MS) were combined for the characterization of a monoclonal IgG1 antibody and three different peptide-Fc fusion proteins. The one commonality of these molecules is the presence of a similarly accessible lysine residue either located in the flexible hinge region of the antibody or in the flexible linker of the peptide-Fc fusion proteins.

Isomerization of a single aspartyl residue of anti-epidermal growth factor receptor immunoglobulin gamma2 antibody highlights the role avidity plays in antibody activity.

A new isoform of the light chain of a fully human monoclonal immunoglobulin gamma2 (IgG2) antibody panitumumab against human epidermal growth factor receptor (EGFR) was generated by in vitro aging. The isoform was attributed to the isomerization of aspartate 92 located between phenylalanine 91 and histidine 93 residues in the antigen-binding region. The isomerization rate increased with increased temperature and decreased pH.