Development of in silico models to predict viscosity and mouse clearance using a comprehensive analytical data set collected on 83 scaffold-consistent monoclonal antibodies.

Biologic drug discovery pipelines are designed to deliver protein therapeutics that have exquisite functional potency and selectivity while also manifesting biophysical characteristics suitable for manufacturing, storage, and convenient administration to patients. The ability to use computational methods to predict biophysical properties from protein sequence, potentially in combination with high throughput assays, could decrease timelines and increase the success rates for therapeutic developability engineering by eliminating lengthy and expensive cycles of recombinant protein production and testing. To support development of high-quality predictive models for antibody developability, we designed a sequence-diverse panel of 83 effector functionless IgG1 antibodies displaying a range of biophysical properties, produced and formulated each protein under standard platform conditions, and collected a comprehensive package of analytical data, including in vitro assays and in vivo mouse pharmacokinetics. We used this robust training data set to build machine learning classifier models that can predict complex protein behavior from these data and features derived from predicted and/or experimental structures. Our models predict with 87% accuracy whether viscosity at 150 mg/mL is above or below a threshold of 15 centipoise (cP) and with 75% accuracy whether the area under the plasma drug concentration-time curve (AUC0-672 h) in normal mouse is above or below a threshold of 3.9 × 106 h x ng/mL.

Rational selection of building blocks for the assembly of bispecific antibodies.

Bispecific antibodies, engineered to recognize two targets simultaneously, demonstrate exceptional clinical potential for the therapeutic intervention of complex diseases. However, these molecules are often composed of multiple polypeptide chains of differing sequences. To meet industrial scale productivity, enforcing the correct quaternary assembly of these chains is critical. Here, we describe Chain Selectivity Assessment (CSA), a high-throughput method to rationally select parental monoclonal antibodies (mAbs) to make bispecific antibodies requiring correct heavy/light chain pairing. By deploying CSA, we have successfully identified mAbs that exhibit a native preference toward cognate chain pairing that enables the production of hetero-IgGs without additional engineering. Furthermore, CSA also identified rare light chains (LCs) that permit positive binding of the non-cognate arm in the common LC hetero-IgGs, also without engineering. This rational selection of parental mAbs with favorable developability characteristics is critical to the successful development of bispecific molecules with optimal manufacturability properties.

Two cases of upper-extremity swelling: Paget-Schroetter syndrome and non-Hodgkin lymphoma.

Two adolescents presented to our emergency department with isolated, acute onset upper-extremity swelling. In their initial emergency department evaluations, both patients were found to have a deep venous thrombosis. Despite their similar presentations, the etiologies of their deep venous thrombosis were very different. After further evaluation, one patient was diagnosed with Paget-Schroetter syndrome and the other with non-Hodgkin lymphoma. These cases illustrate the importance of maintaining a broad differential diagnosis and using a multidisciplinary approach to patients with the unusual and potentially life-threatening presentation of upper-extremity swelling.

High Mass Analysis with a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer: From Inorganic Salt Clusters to Antibody Conjugates and Beyond.

Analysis of proteins and complexes under native mass spectrometric (MS) and solution conditions was typically performed using time-of-flight (ToF) analyzers, due to their routine high m/z transmission and detection capabilities. However, over recent years, the ability of Orbitrap-based mass spectrometers to transmit and detect a range of high molecular weight species is well documented. Herein, we describe how a 15 Tesla Fourier transform ion cyclotron resonance mass spectrometer (15 T FT-ICR MS) is more than capable of analyzing a wide range of ions in the high m/z scale (>5000), in both positive and negative instrument polarities, ranging from the inorganic cesium iodide salt clusters; a humanized IgG1k monoclonal antibody (mAb; 148.2 kDa); an IgG1-mertansine drug conjugate (148.5 kDa, drug-to-antibody ratio; DAR 2.26); an IgG1-siRNA conjugate (159.1 kDa; ribonucleic acid to antibody ratio; RAR 1); the membrane protein aquaporin-Z (97.2 kDa) liberated from a C8E4 detergent micelle; the empty MSP1D1-nanodisc (142.5 kDa) and the tetradecameric chaperone protein complex GroEL (806.2 kDa; GroEL dimer at 1.6 MDa). We also investigate different regions of the FT-ICR MS that impact ion transmission and desolvation. Finally, we demonstrate how the transmission of these species and resultant spectra are highly consistent with those previously generated on both quadrupole-ToF (Q-ToF) and Orbitrap instrumentation. This report serves as an impactful example of how FT-ICR mass analyzers are competitive to Q-ToFs and Orbitraps for high mass detection at high m/z.

Discovery, characterization, and remediation of a C-terminal Fc-extension in proteins expressed in CHO cells.

Protein-based biotherapeutics are produced in engineered cells through complex processes and may contain a wide variety of variants and post-translational modifications that must be monitored or controlled to ensure product quality. Recently, a low level (~1-5%) impurity was observed in a number of proteins derived from stably transfected Chinese hamster ovary (CHO) cells using mass spectrometry. These molecules include antibodies and Fc fusion proteins where Fc is on the C-terminus of the construct. By liquid chromatography-mass spectrometry (LC-MS), the impurity was found to be ~1177 Da larger than the expected mass. After tryptic digestion and analysis by LC-MS/MS, the impurity was localized to the C-terminus of Fc in the form of an Fc sequence extension. Targeted higher-energy collision dissociation was performed using various normalized collision energies (NCE) on two charge states of the extended peptide, resulting in nearly complete fragment ion coverage. The amino acid sequence, SLSLSPEAEAASASELFQ, obtained by the de novo sequencing effort matches a portion of the vector sequence used in the transfection of the CHO cells, specifically in the promoter region of the selection cassette downstream of the protein coding sequence. The modification was the result of an unexpected splicing event, caused by the resemblance of the commonly used GGU codon of the C-terminal glycine to a consensus splicing donor. Three alternative codons for glycine were tested to alleviate the modification, and all were found to completely eliminate the undesirable C-terminal extension, thus improving product quality.

Improving Guideline-Based Care of Acute Asthma in a Pediatric Emergency Department.

BACKGROUND AND OBJECTIVE: Rapid repetitive administration of short-acting ß-agonists (SABA) is the most effective means of reducing acute airflow obstruction in asthma. Little evidence exists that assesses process measures (ie, timeliness) and outcomes for asthma. We used quality improvement (QI) methods to improve emergency department care in accordance with national guidelines including timely SABA administration and use of asthma severity scores. METHODS: The Model for Improvement was used and interventions were targeted at 4 key drivers: knowledge, engagement, decision support, and workflow enhancement. Time series analysis was performed and outcomes assessed on statistical process control charts. RESULTS: Asthma severity scoring increased from 0% to >95% in triage and to >75% for repeat scores. Time to first SABA (T1) improved by 32.8 minutes (47%). T1 for low severity patients improved by 17.6 minutes (28%). T1 for high severity patients improved by 3.1 minutes to 18.1 minutes (15%). Time to third SABA (T3) improved by 30 minutes (24%). T3 for low severity patients improved by 42.5 minutes (29%) and T3 for high severity patients improved by 21 minutes (23%). Emergency department length of stay for low severity patients discharged to home improved by 29.3 minutes (15%). The number of asthma-related visits between 48-hour return hospitalizations increased from 114 to 261. The admission rate decreased 6.0%. CONCLUSIONS: We implemented standardized asthma severity scoring with high rates of compliance, improved timely administration of ß-agonist treatments, demonstrated early improvements in Emergency department length of stay, and reduced admission rates without increasing unplanned return admissions.

Developing a Strategy Menu for Community-Level Obesity Prevention.

Childhood obesity is a complex problem influenced by policies, systems, and environments, and its prevention requires changes across a range of community settings. To address this, we developed an obesity prevention strategy menu and an ongoing study to pilot its use and provide technical support for its implementation. The strategy menu is comprised of a set of effective approaches communities can use to develop tailored, context-specific health interventions based on local community needs and capacity. It was developed by a multidisciplinary team of researchers and practitioners who reviewed evidence and organized it to incorporate effective policy, systems, and environmental changes for reducing and preventing childhood obesity. Eventually, it will be part of a web-based point of access that complements the foundational relationships built between communities, researchers, and practitioners. By developing a framework to engage communities in the selection and implementation of multisetting obesity prevention strategies, we aim to create and sustain momentum toward a long-term reduction in obesity in Wisconsin children.

Ligand-binding mass spectrometry to study biotransformation of fusion protein drugs and guide immunoassay development: strategic approach and application to peptibodies targeting the thrombopoietin receptor.

The knowledge of in vivo biotransformation (e.g., proteolysis) of protein therapeutic candidates reveals structural liabilities that impact stability. This information aids the development and confirmation of ligand-binding assays with the required specificity for bioactive moieties (including intact molecule and metabolites) for appropriate PK profiling. Furthermore, the information can be used for re-engineering of constructs to remove in vivo liabilities in order to design the most stable candidates. We have developed a strategic approach of ligand-binding mass spectrometry (LBMS) to study biotransformation of fusion proteins of peptides fused to human Fc ("peptibodies") using anti-human Fc immunoaffinity capture followed by tiered mass spectrometric interrogation. LBMS offers the combined power of selectivity of ligand capture with the specificity and detailed molecular-level information of mass spectrometry. In this paper, we demonstrate the preclinical application of LBMS to three peptibodies, AMG531 (romiplostim), AMG195(linear), and AMG195(loop), that target the thrombopoietin receptor. The data show that ligand capture offers excellent sample cleanup and concentration of intact peptibodies and metabolites for subsequent query by matrix-assisted laser desorption ionization time-of-flight mass spectrometry for identification of in vivo proteolytic points. Additional higher-resolution analysis by nanoscale liquid chromatography interfaced with electrospray ionization mass spectrometry is required for identification of heterogeneous metabolites. Five proteolytic points are accurately identified for AMG531 and two for AMG195(linear), while AMG195(loop) is the most stable construct in rats. We recommend the use of LBMS to assess biotransformation and in vivo stability during early preclinical phase development for all novel fusion proteins.

The impact of a brief expectation survey on parental satisfaction in the pediatric emergency department.

OBJECTIVES: To determine the effect of physician knowledge of parental expectations on satisfaction with emergency department (ED) care. METHODS: This was a prospective, controlled, interventional trial involving parents of children presenting to a children's hospital ED. Parents completed an expectation survey on arrival, which was either immediately placed back in the enrollment envelope (control) or shown to the physician caring for the child (intervention). The physician was instructed to initial the expectation survey to acknowledge receipt of the survey. Parents then completed a satisfaction survey at discharge. The primary outcomes were differences in satisfaction with physician review of the expectation survey, as measured by 1) parental ratings of overall care and 2) their willingness to recommend the ED to others. A third (baseline) group completed only a satisfaction survey at discharge. RESULTS: A total of 614 (66%) of the 930 enrolled parents completed the study. Intention-to-treat analysis did not show a significant increase in parental satisfaction ratings for either overall care or recommend the ED; however, only 42% of the intervention group surveys had documented physician review. When these initialed surveys were compared with the control group in a per-protocol analysis, there was a significant improvement in parental satisfaction. There were no differences between the control and baseline groups, indicating no effect of the expectation survey completion on satisfaction. CONCLUSIONS: Physician knowledge of written parental expectations may improve parental satisfaction during an ED visit. Further work is needed to overcome the barriers to physician review of the expectation survey to maximize parent satisfaction.