ABSTRACT
This study has employed mammalian transient expression systems to generate afucosylated antibodies and antibody Fc mutants for rapid candidate screening in discovery and early development. While chemical treatment with the fucose analogue 2-fluoro-peracetyl-fucose during transient expression only partially produced antibodies with afucosylated N-glycans, the genetic inactivation of the FUT8 gene in ExpiCHO-S™ by CRISPR/Cas9 enabled the transient production of fully afucosylated antibodies. Human IgG1 and murine IgG2a generated by the ExpiCHOfut8KO cell line possessed a 8-to-11-fold enhanced FcγRIIIa binding activity in comparison with those produced by ExpiCHO-S™. The Fc mutant S239D/S298A/I332E produced by ExpiCHO-S™ had an approximate 2-fold higher FcγRIIIa affinity than that of the afucosylated wildtype molecule, although it displayed significantly lower thermal-stability. When the Fc mutant was produced in the ExpiCHOfut8KO cell line, the resulting afucosylated Fc mutant antibody had an additional approximate 6-fold increase in FcγRIIIa binding affinity. This synergistic effect between afucosylation and the Fc mutations was further verified by a natural killer (NK) cell activation assay. Together, these results have not only established an efficient large-scale transient CHO system for rapid production of afucosylated antibodies, but also confirmed a cooperative impact between afucosylation and Fc mutations on FcγRIIIa binding and NK cell activation.
Subject(s)
Immunoglobulin G , Killer Cells, Natural , Humans , Animals , Mice , Immunoglobulin G/genetics , MammalsABSTRACT
INTRODUCTION: Physician wellness is a much broader concept than physician satisfaction, although physician satisfaction tends to be the predominant form of measurement. The purpose of this paper is to offer some key strategies for building a culture of physician wellness in the unique setting of a large, private group of orthopaedic surgeons. METHODS: Organizational culture was assessed formally through validated physician satisfaction surveys and data gathered through the Patient Advocacy Reporting System (PARS program, and informally through organic, organizational initiatives, and responses. In addition, the authors continuously reviewed literature related to physician engagement, satisfaction and wellness, and organizational interventions designed to promote the same. RESULTS: From 2014 to 2018, overall physician engagement improved and remained above the 90th percentile as compared with the vendor's database of over 95,000 physicians surveyed nationally. Physician resilience, a measure of the predictors of physician burnout, also improved from 2014 to 2018 and is now above the national physician average. During the same time period, significant improvement was noted as it relates to a physician's ability to disconnect and "recharge" outside of work (an important element of overall wellness). From 2016 to 2018, PARS interventions aimed at increasing professionalism and identifying at-risk physicians decreased by 23%. DISCUSSION: At OrthoCarolina, we believe that developing a nurturing environment with good interpersonal connections is foundational to our success. Physicians within the group also report that supportive avenues such as mentor programs and organized activities outside of the workplace amplified the sense of connection and community within the physician group. CONCLUSIONS: Organizational culture of a physician group is a key determinant of physician wellness. It is imperative to have the full support of the physicians within the organization and its leaders to promote and support a culture of wellness. Data-driven programs create awareness and foster curiosity, which in turn allows for the development of specific initiatives including informal, organic initiatives that build connection and community.
Subject(s)
Health Promotion , Occupational Health , Organizational Culture , Orthopedics , Burnout, Professional , Group Practice , Humans , North Carolina , PhysiciansABSTRACT
Healthy Siblings of Children with Autism Spectrum Disorders. A Mixed-methods Pilot Study Healthy siblings of chronically ill and/or disabled children may have an increased risk of developing behavioral problems; this is particularly given for siblings of children with Autism Spectrum Disorders (ASD). Quality of life and distress of five siblings (12-15 years) of children with ASD were examined using self- and parent-proxy-reports. Guideline-based interviews were conducted with the siblings and their parents and analyzed according to Mayring. Quality of life described by the children resembled the KINDLR´s standardized range of scores, but parents described a decreased quality of life. Both siblings and parents reported low to medium distress. Interviews revealed healthy children are asked by their parents to take responsibility for their siblings with ASD. Parents assumed their healthy children are less affected by the ASD of the sibling. The healthy siblings described being "annoyed" by ASD-associated behavior and partly feeling "treated unfairly". They wished their sibling no longer "suffered" from ASD or behaved "normally". The results of this pilot study, albeit with a small sample, show siblings do experience low to medium burden. This burden is often caused by the sibling relationship or the ASD-associated behavior. Suggestions for supporting the healthy siblings are given.
Subject(s)
Autism Spectrum Disorder , Siblings , Child , Family Health , Humans , Pilot Projects , Quality of Life , Sibling RelationsABSTRACT
A highly robust hydrophilic interaction liquid chromatography (HILIC) method that involves both fluorescence and mass spectrometric detection was developed for profiling and characterizing enzymatically released and 2-aminobenzamide (2-AB)-derivatized mAb N-glycans. Online HILIC/mass spectrometry (MS) with a quadrupole time-of-flight mass spectrometer provides accurate mass identifications of the separated, 2-AB-labeled N-glycans. The method features a high-resolution, low-shedding HILIC column with acetonitrile and water-based mobile phases containing trifluoroacetic acid (TFA) as a modifier. This column and solvent system ensures the combination of robust chromatographic performance and full compatibility and sensitivity with online MS in addition to the baseline separation of all typical mAb N-glycans. The use of TFA provided distinct advantages over conventional ammonium formate as a mobile phase additive, such as, optimal elution order for sialylated N-glycans, reproducible chromatographic profiles, and matching total ion current chromatograms, as well as minimal signal splitting, analyte adduction, and fragmentation during HILIC/MS, maximizing sensitivity for trace-level species. The robustness and selectivity of HILIC for N-glycan analyses allowed for method qualification. The method is suitable for bioprocess development activities, heightened characterization, and clinical drug substance release. Application of this HILIC/MS method to the detailed characterization of a marketed therapeutic mAb, Rituxan(®), is described.
