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. However, in this study, the large size of the modification (N-glycosylation) allowed assessing its impact from small amounts (∼20 ug) of intact antibody, without the need for peptide mapping. Here, we apply the native SEC-based competitive binding assay to quickly and qualitatively investigate the effects of Fab glycosylation of four antispike protein mAbs that were developed for use in the treatment of COVID-19 disease. Three of the mAbs were observed to have consensus N-glycosylation sites (N-X-T/S) in the Fab domains, a relatively rare occurrence in therapeutic mAbs. The goal of the study was to characterize the levels of Fab glycosylation present, as well as determine the impact of glycosylation on binding to the spike protein receptor binding domain (RBD) and the ability of the mAbs to inhibit RBD-ACE2 interaction at the intact antibody level, with minimal sample treatment and preparation. The three mAbs with Fab N-glycans were found to have glycosylation profiles ranging from full occupancy at each Fab (in one mAb) to partially glycosylated with mixed populations of two, one, or no glycan moieties. Competitive SEC analysis of mAb-RBD revealed that the glycosylated antibody populations outcompete their nonglycosylated counterparts for the available RBD molecules. This competitive SEC binding analysis was applied to investigate the three-body interaction of a glycosylated mAb blocking the interaction between endogenous binding partners RBD-ACE2, finding that both glycosylated and nonglycosylated mAb populations bound to RBD with high enough affinity to block RBD-ACE2 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. A secondary purpose was to determine the reliability of a student physical therapist using both devices. DESIGN: A reliability measurement study METHOD: A blinded examiner identified the LMM at the L4 vertebral level and measured the thickness of the contracted muscle utilizing both the handheld and established ultrasound devices. ICC values were calculated to determine the inter-device and intra-rater reliability. RESULTS: The study included 42 healthy participants, 30 females and 12 males, with a mean age of 38.5 years. The inter-device reliability during a sustained LMM contraction was excellent (ICC = 0.92, 95% CI: 0.87-0.94) and the intra-rater reliability was good for both the handheld (ICC = 0.85, 95% CI: 0.73-0.92) and established (ICC = 0.89, 95% CI: 0.82-0.93) ultrasound units. CONCLUSION: Results support the use of handheld ultrasound by physical therapists and students to measure the LMM thickness. Future studies could investigate the reliability of handheld ultrasound in a variety of musculoskeletal and pathological structures important to PT practice.

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. Surfaces were categorized based on frequency of contact and cleaning and surface type. Total bacterial and fungal load was assessed using primer sets specific for the 16S rRNA and ITS genes, respectively. Bacterial samples were sequenced using the Illumina system and analyzed using Illumina-utils, Minimum Entropy Decomposition, QIIME2 (for alpha and beta diversity), LEfSe and ANCOM-BC for taxonomic differential abundance and ADONIS to test for differences in beta diversity (p<0.05). RESULTS: Porous surfaces had more bacterial DNA compared to non-porous surfaces (median non-porous = 0.0016ng/µL, 95%CI = 0.0077-0.00024ng/µL, N = 15; porous = 0.0084 ng/µL, 95%CI = 0.0046-0.019 ng/µL, N = 18. p = 0.0066,DNA. Samples clustered by type of surface with non-porous surfaces further differentiated by those contacted by hand versus foot. ADONIS two-way ANOVA showed that the interaction of porosity and contact frequency (but neither alone) had a significant effect on 16S communities (F = 1.7234, R2 = 0.0609, p = 0.032). DISCUSSION: Porosity of surfaces and the way they are contacted may play an underestimated, but important role in microbial contamination. Additional research involving a broader range of clinics is required to confirm results. Results suggest that surface and contact-specific cleaning and hygiene measures may be needed for optimal sanitization in outpatient REHAB clinics.

Examining the effectiveness of psychological interventions for marginalised and disadvantaged women and individuals who have experienced gender-based violence: protocol for a scoping review.

INTRODUCTION: Exposure to gender-based violence (GBV) has devastating psychological outcomes for victims/survivors. Particularly in conditions where GBV intersects with multiple forms of oppression, the negative impacts of violence are more challenging to overcome and potential pathways for recovery become less accessible. However, evidence regarding the availability and effectiveness of mental health interventions for GBV survivors from marginalised and disadvantaged communities has yet to be systematically integrated and synthesised. The proposed scoping review will examine the relevant literature regarding the availability and effectiveness of psychological interventions for survivors of GBV from marginalised and disadvantaged backgrounds. This review will (i) document what psychological interventions have been available and empirically established for marginalised and disadvantaged women and individuals with experiences of GBV, (ii) provide a narrative examination of the treatment outcomes of identified interventions regarding their effectiveness and (iii) examine the degree to which GBV interventions in selected sources are designed and applied with a recognition of the social determinants of mental health. METHODS AND ANALYSIS: The search for the proposed scoping review will include five electronic databases: PsycINFO, Scopus, Web of Science, Ovid Medline, and CINAHL. The database search will be completed in June 2022. An additional search will be conducted before the completion of the study in December 2022. The search will target research studies published after 2010. The primary eligibility criterion for study selection is having a focus on psychological interventions for GBV survivors from marginalised and disadvantaged groups. Two reviewers will conduct screening and data extraction. The data will be evaluated to map the treatment outcomes of interventions and their effectiveness. Implications for clinical services will be discussed. ETHICS AND DISSEMINATION: No ethical consideration is foreseen for this scoping review. The dissemination will be done through a publication in a top-tier open access journal and conference presentations.

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. Affinity LC-MS differentiated the IgG1 antibodies primarily according to their Fc glycosylation patterns, with highly galactosylated species having greater affinity for the immobilized receptors and thus eluting later from the column (M5< G0F < G0 afucosylated ≅ G1F < G2F). Sialylated species bound weaker to their asialylated counterparts as reported previously. High mannose glycoforms bound weaker than G0F, contrary to previously published studies using glycosylated receptors. Also, increased receptor binding affinity associated with afucosylated antibodies was not observed with the aglycosylated FcγRIIIa. This apparent difference from previous findings highlighted the importance of the glycans on the receptors for mediating stronger binding interactions. Characterization of temperature-stressed samples by LC-MS peptide mapping revealed over 200 chemical and post-translational modifications, but only the Fc glycans, deamidation of EU N325, and an unknown modification to either proline or cysteine residues of the hinge region were found to have a statistically significant impact on binding.Abbreviations: Antibody-dependent cell-mediated cytotoxicity (ADCC), chimeric antigen receptor (CAR), Chinese hamster ovary (CHO), dithiothreitol (DTT), electrospray ionization (ESI), hydrogen-deuterium exchange (HDX), filter aided-sample preparation (FASP), Fcγ receptor (FcγR), fragment crystallizable (Fc), high-pressure liquid chromatography (HPLC), immunoglobulin G (IgG), liquid chromatography (LC), monoclonal antibody (mAb), mass spectrometry (MS), natural killer (NK), N-glycolylneuraminic acid (NGNA), N-acetylneuraminic acid (NANA), principal component analysis (PCA), surface plasmon resonance (SPR), trifluoroacetic acid (TFA), and extracted mass chromatogram (XMC).

From screen time to the digital level of analysis: a scoping review of measures for digital media use in children and adolescents.

OBJECTIVES: This scoping review aims to facilitate psychometric developments in the field of digital media usage and well-being in young people by (1) identifying core concepts in the area of "screen time" and digital media use in children, adolescents, and young adults, (2) synthesising existing research paradigms and measurement tools that quantify these dimensions, and (3) highlighting important areas of need to guide future measure development. DESIGN: A scoping review of 140 sources (126 database, 14 grey literature) published between 2014 and 2019 yielded 162 measurement tools across a range of domains, users, and cultures. Database sources from Ovid MEDLINE, PsycINFO and Scopus were extracted, in addition to grey literature obtained from knowledge experts and organisations relevant to digital media use in children. To be included, the source had to: (1) be an empirical investigation or present original research, (2) investigate a sample/target population that included children or young persons between the ages of 0 and 25 years of age, and (3) include at least one assessment method for measuring digital media use. Reviews, editorials, letters, comments and animal model studies were all excluded. MEASURES: Basic information, level of risk of bias, study setting, paradigm, data type, digital media type, device, usage characteristics, applications or websites, sample characteristics, recruitment methods, measurement tool information, reliability and validity. RESULTS: Significant variability in nomenclature surrounding problematic use and criteria for identifying clinical impairment was discovered. Moreover, there was a paucity of measures in key domains, including tools for young children, whole families, disadvantaged groups, and for certain patterns and types of usage. CONCLUSION: This knowledge synthesis exercise highlights the need for the widespread development and implementation of comprehensive, multi-method, multilevel, and multi-informant measurement suites.

Family Medicine With Refugee Newcomers During the COVID-19 Pandemic.

Certain members of society are disproportionately affected by the COVID-19 crisis and the added strain being placed on already overextended health care systems. In this article, we focus on refugee newcomers. We outline vulnerabilities refugee newcomers face in the context of COVID-19, including barriers to accessing health care services, disproportionate rates of mental health concerns, financial constraints, racism, and higher likelihoods of living in relatively higher density and multigenerational dwellings. In addition, we describe the response to COVID-19 by a community-based refugee primary health center in Ontario, Canada. This includes how the clinic has initially responded to the crisis as well as recommendations for providing services to refugee newcomers as the COVID-19 crisis evolves. Recommendations include the following actions: (1) consider social determinants of health in the new context of COVID-19; (2) provide services through a trauma-informed lens; (3) increase focus on continuity of health and mental health care; (4) mobilize International Medical Graduates for triaging patients based on COVID-19 symptoms; and (5) diversify communication efforts to educate refugees about COVID-19.

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. Currently, criticality of modifications is assessed by a laborious process of collecting antibody fractions from the soft chromatography techniques ion exchange and hydrophobic interaction chromatography and characterizing the fractions one-by-one for potency and chemical modifications. Here, we describe a method for large-scale, parallel identification of all critical chemical modifications in one experiment. In the first step, the antibody is stressed by one or several stress methods. It is then mixed with target protein and separated by size-exclusion chromatography (SEC) on bound antibody-target complex and unbound antibody. Peptide mapping of fractions and statistical analysis are performed to identify modifications on amino acid residues that affect binding. To identify the modifications leading to slight decreases in binding, competitive SEC of antibody and antigen mixtures was developed and described in a companion study by Shi et al, where target protein is provided at lower level, below the stoichiometry. The newly described method was successfully correlated to crystallography for assessing criticality of chemical modifications and paratope mapping. It is more sensitive to low-level modifications, better streamlined and platform ready.

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. When a smaller amount of the receptor was provided (1:1), the antibody species with modifications reducing binding eluted as unbound from SEC, while the antibody-receptor complex eluted as the bound fraction. Peptide mapping revealed ratios of modifications between unbound and bound fractions. Statistical analysis after triplicate measurements (n = 3) indicated that heavy chain (HC) D102 isomerization and light chain (LC) N30 deamidation were four-fold higher in unbound fraction with high statistical significance. Although HC N55 deamidation and M107 oxidation were also abundant, they were not statistically different between unbound and bound. Our findings agree with previously published potency measurements of collected CEX fractions and the crystal structure of antibody and HER2. Overall, competitive SEC of stressed antibody-receptor mixture followed by peptide mapping is a useful tool in revealing critical residues and modifications involved in the antibody-target binding, even if they elute as a complex from SEC when mixed at 1:2 stoichiometric ratio.

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. mAb stressed by hydrogen peroxide oxidation was used as a model system, which produced additional CEX peaks. The on-line CEX-UV-MS top-down analysis produced gas-phase fragments containing one or two methionine residues. Oxidation of some methionine residues contributed to earlier (acidic), some to later (basic) eluting peaks, while oxidation of other residues did not change CEX elution. The abundance of the oxidized and non-oxidized fragment ions also allowed estimation of the oxidation percentage of different methionine residues in stressed mAb. CEX-UV-MS measurement revealed a new intact antibody proteoform at 5% that eluted as a basic peak and included paired modifications: high-mannose glycosylation and remaining C-terminal lysine residue (M5/M5 + K). This finding was confirmed by peptide mapping and on-column disulfide reduction coupled with reversed-phase liquid chromatography - top-down MS analysis of the collected basic peak. Overall, our results demonstrate the utility of the on-line method in providing site-specific structural information of charge modifications without fraction collection and laborious peptide mapping.

From 'screen time' to the digital level of analysis: protocol for a scoping review of digital media use in children and adolescents.

INTRODUCTION: Research on the relationship between digital media exposure and child development is complex, inconsistent and fraught with debate. A highlighted area of inadequacy surrounds the methodological limitations of measuring digital media use for both researchers and clinicians, alike. This protocol aims to (1) identify core concepts in the area of screen time and digital media use in children and adolescents (2) map existing research paradigms and screening/measurement tools that serve to underpin and operationalise core concepts and (3) provide an initial step in integrating these findings into a consolidated screening toolkit. It is expected this enterprise will help advance research and clinical evaluation in fields concerned with digital media use, namely medicine, child development and the social sciences. METHODS AND ANALYSIS: The planned scoping review will search relevant electronic databases, including Ovid MEDLINE, PsycINFO and Scopus, in addition to grey literature. All empirical investigations and presentation of original research will be considered, and measurement/screening tools for digital media usage in children and adolescents will be identified and reported on. Two reviewers will pilot test the screening criteria, and data extraction forms prior to independently screening all relevant literature and extracting the data. A three-stage synthesis process will be used to map the existent measurement and screening tools for digital media usage in children and adolescents. ETHICS AND DISSEMINATION: There are no ethical considerations for this scoping review. Plans for dissemination include publication in a top-tier, open-access journal, public presentations and conference proceedings. Presentation of the full scoping review has been accepted to the American Academy of Child & Adolescent Psychiatry 66th Annual Meeting.

Impact of antibody subclass and disulfide isoform differences on the biological activity of CD200R and ßklotho 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 ßklotho receptor (ßklotho), we noticed striking activity differences of IgG1 or IgG2 antibodies with the same variable domains. For the CD200R antibody, the IgG2 antibody demonstrated higher activity than the IgG1 or IgG4 antibody. More significantly, for ßklotho, agonist antibodies with higher biological activity as either IgG2 or IgG1 were identified. In both cases, ion exchange chromatography was able to isolate the bioactivity to the IgG2-B isoform from the IgG2 parental mixture. The subclass-related increase in agonist activity was not correlated with antibody aggregation or binding affinity, but was driven by enhanced avidity for the CD200R antibody. These results add to the growing body of evidence that show that conformational differences in the antibody hinge region can have a dramatic impact on the antibody activity and must be considered when screening and engineering therapeutic antibody candidates. The results also demonstrate that the IgG1 (IgG2-A like) or the IgG2-B form may provide the most active form of agonist antibodies for different antibodies and targets.

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. In the past few years, the hypergaussian functions with petal-shaped realization have been shown to significantly suppress intensity along the optical axis. This work reports on fabrication of a series of petal-shaped masks using a variety of techniques such as 3D printing, photolithography, and wire Electro Discharge Machining. These masks are designed and fabricated to operate in the range of Fresnel numbers between 4 and 120. This paper discusses the challenges, successes, and failures of each fabrication technique and the optical performance of typical masks with suggestions for potential follow up work.

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. Within each of these isoform types are subtypes, with subtle disulfide-linkage differences. Here we explored the structural basis for the A1 and A2 isoform subtypes. Whereas A1 isoform converts into the A/B and B isoforms under mild redox conditions, A2 does not. Characterization of the disulfide connectivities of A2 isoform revealed a similar structure to A1 isoform, with parallel inter heavy chain disulfide linkages in the hinge region. However, the hinge disulfides in A2 isoform were resistant to reduction under conditions where A1 isoform hinge disulfides became reduced and they required thermal treatment (>55 °C) to obtain thiol-dependent disulfide reduction. Structural analysis of the hinge region indicated that the protected disulfides were restricted to cysteines 219 and 220 of the upper hinge. Disruption of the upper hinge through insertion mutagenesis eliminated A2 isoform behavior. (1)H NMR studies showed that the A1 isoform Fc glycan was more dynamic than that on A2 isoform and showed some other conformational differences. Results point to an IgG2-A2 upper hinge region that is more akin to the interior of a globular protein than the flexible hinge region expected on an IgG.

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. Changes in the relative levels of these forms over time are observed while IgG2 circulates in humans, suggesting IgG2-A→IgG2-A/B→IgG2-B conversion. Using a flow-through dialysis system, we studied the conversion kinetics of these forms in vitro under physiological conditions. For two IgG2κ antibodies, in vivo results closely matched the kinetics observed in vitro, indicating that the changes observed in vivo were solely conversions between isoforms, not differential clearance of specific forms. Moreover, the combined results validate the accuracy of the physiological model for the study of blood redox reactions. Further exploration of the conversion kinetics using material enriched in the IgG2-A forms revealed that the IgG2-A→IgG2-A/B rate was similar between IgG2κ and IgG2λ antibodies. In IgG2κ antibodies, conversion of IgG2-A/B→IgG2-B was slower than the IgG2-A→IgG2-A/B reaction. However, in IgG2λ antibodies, little IgG2-A/B→IgG2-B conversion was detected under physiological conditions. Thus, small differences in the C-terminus of the light chain sequences affect the disulfide conversion kinetics and impact the IgG2 disulfide isoforms produced in vivo.

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. Components were picked to be representative of a single element in a distributed aperture system. The performance of the system is analyzed, and the noise equivalent temperature difference is found to be 0.5 K (for a 1 s integration time) with a diffraction limited resolution of ~8 mrad. Representative images are shown that demonstrate the phenomenology associated with this spectrum.

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. AF4 and liquid chromatography-mass spectrometry (LC-MS) were applied to directly observe IgG/FcγR and IgG/FcRn complexes, which were not observed using nonequilibrium size exclusion chromatography (SEC) analysis. Human serum albumin (HSA), an abundant component of human blood and capable of binding FcRn, was studied in combination with FcRn and IgG. AF4 demonstrated that the majority of large complexes of IgG/FcRn/HSA were at an approximate 1:2:1 molar ratio. In addition, affinity measurements of the complex were performed in the sub-micromolar affinity range. A significant decrease in binding was detected for IgG molecules with increased oxidation in the Fc region. AF4 was useful in detecting weak binding between full-length IgG/Fc fragments and Fc receptors and the effect of chemical modifications on binding. AF4 is a useful technique in the assessment of mAb product quality attributes.

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. We show that single Cys-->Ser mutants can greatly reduce heterogeneous disulfide bonding in human IgG2 and maintain in vitro activity. The data demonstrate the feasibility of applying site-directed mutagenesis to reduce disulfide bond heterogeneity in human IgG2 while preserving the activity of this therapeutically important class of human antibodies.

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. It inhibited cellular proliferation, reduced colony formation and caused partial cell cycle arrest at the G(1) checkpoint. Metformin did not induce apoptosis (as measured by DNA fragmentation and PARP cleavage) in luminal A, B or erbB2 subtype breast cancer cell lines. At the molecular level, metformin treatment was associated with a reduction of cyclin D1 and E2F1 expression with no changes in p27(kip1) or p21(waf1). It inhibited mitogen activated protein kinase (MAPK) and Akt activity, as well as the mammalian target of rapamycin (mTOR) in both ER positive and negative, erbB2-overexpressing and erbB2-normal expressing breast cancer cells. In erbB2-overexpressing breast cancer cell lines, metformin reduced erbB2 expression at higher concentrations, and at lower concentrations within the therapeutic range, it inhibited erbB2 tyrosine kinase activity evidenced by a reduction of phosphorylated erbB2 (P-erbB2) at both auto- and Src- phosphorylation sites. These data suggest that metformin may have potential therapeutic utility against ER positive and negative, erbB2-overexpressing and erbB2-normal expressing breast cancer cells.

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). However, an inversed pH-dependence was discovered for high molecular weight aggregate formation at elevated temperatures (37 degrees C). The different stability profiles exhibited at the various storage conditions resulted in nonlinearity of the Arrhenius kinetics. Thermal unfolding at or below 37 degrees C was not evident by differential scanning calorimetry. Enriched populations of the structural isoforms of the IgG2 subclass were tested for their unique temperature and pH-dependence of aggregation. The Arrhenius kinetics of aggregation for each of the individual IgG2 isoforms was also nonlinear. However, the temperature-dependence of clipping suggested that clip-mediated aggregation was responsible for the increased higher order aggregates at low pH and elevated temperatures. Unique clip species resulting from the conformational differences between the IgG2 isoforms lead to increased aggregation. These results have implications on the mechanisms of antibody aggregation and on the validity of accelerated data to predict shelf-life accurately.