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.

VERITAS: Harnessing the power of nomenclature in biologic discovery.

We are entering an era in which therapeutic proteins are assembled using building block-like strategies, with no standardized schema to discuss these formats. Existing nomenclatures, like AbML, sacrifice human readability for precision. Therefore, considering even a dozen such formats, in combination with hundreds of possible targets, can create confusion and increase the complexity of drug discovery. To address this challenge, we introduce Verified Taxonomy for Antibodies (VERITAS). This classification and nomenclature scheme is extensible to multispecific therapeutic formats and beyond. VERITAS names are easy to understand while drawing direct connections to the structure of a given format, with or without specific target information, making these names useful to adopt in scientific discourse and as inputs to machine learning algorithms for drug development.

Successful management of hypothermic cardiopulmonary bypass in a malignant hyperthermia susceptible patient.

Malignant hyperthermia (MH) is a potentially lethal reaction in those that are genetically predisposed, frequently triggered by inhaled anesthetics. MH is often difficult to diagnose because it is accompanied by signs and symptoms that are shared with other disorders. The diagnosis is further obscured in cardiac surgical patients, as the signs of MH can be masked by the cardiopulmonary bypass circuit (CPB) and the use of induced hypothermia. In this case-report, we describe the successful anesthetic management of a 65-year-old MH-susceptible female, confirmed via caffeine halothane contracture test, with aortic regurgitation and ascending aortic dilatation who underwent a Bentall procedure. We have also identified certain key measures for the safe anesthetic management of these patients.

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.

Intermolecular interactions involving an acidic patch on immunoglobulin variable domain and the γ2 constant region mediate crystalline inclusion body formation in the endoplasmic reticulum.

Full-length immunoglobulins (Igs) are widely considered difficult to crystallize because of their large size, N-linked glycosylation, and flexible hinge region. However, numerous cases of intracellular Ig crystallization are reported in plasma cell dyscrasias. What makes some Ig clones more prone to crystallize during biosynthesis as well as the biochemical and cell biological requirements for this cryptic event are poorly understood. To investigate the underlying process of intracellular Ig crystallization we searched for model IgGs that can induce crystalline inclusions during recombinant overexpression. By testing various subunit combinations through mixing and matching of individual subunit chains derived from a panel of human IgG clones, we identified one secretion competent IgG2λ that induced needle-like crystalline inclusions in transfected HEK293 cells. Ig crystallization rarely occurred at steady-state cell growth conditions but was easily induced when ER-to-Golgi transport was pharmacologically blocked. Homology modeling revealed the presence of a prominent negatively-charged patch on the variable domain surface. The patch was composed of eight aspartic acids, of which five were in the heavy chain variable region and three were in the light chain. Crystallization occurred only when the two subunits were co-transfected and the intracellular crystals co-localized with ER resident proteins. Furthermore, subtype switching from IgG2 to IgG1 and stepwise neutralization of the acidic patch independently abrogated Ig crystallization events. The evidence supported that the formation of needle-like crystalline inclusions in the ER was underscored by multivalent intermolecular interactions between the acidic patch and undefined determinants present on the γ2 subunit constant region.

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.

Surgical site infection rates in dialysis patients undergoing endovascular procedures.

A surgical site infection (SSI) is an infection related to surgery that develops within 30 days after an operation or within 1 year of implant placement. Postoperative SSIs are the most common health-care-associated infections, occurring in up to 5% of surgical patients. Endovascular surgical procedures related to vascular access are common in the dialysis population and may cause SSIs. A large outpatient vascular access system developed and implemented a surveillance program to measure and monitor SSIs in their population. The health-care surveillance system extended to 76 ambulatory care centers across the United States and Puerto Rico. Based on a recorded 92,880 patient encounters, the surveillance system tabulated 12,541 valid patient survey responses documenting self-reported symptoms of infection within a 30-day postoperative period. The SSI rate was tabulated based on the presence of two or more specified indicators of infection: antibiotics, pus, dehiscence, pain, warmth, and swelling. Patients undergoing interventional procedures received surveys at discharge. Data were collected and analyzed using SPSS software. Survey analysis indicated a less than 3% superficial incisional SSI rate in hemodialysis patients undergoing endovascular procedures. The SSI rate for clean wound procedures is generally 2% or less. These data indicate that dialysis patients undergoing interventional procedures in vascular access centers may have a slightly greater risk of developing SSIs due to the presence of additional risk factors including obesity, diabetes, and age. This study was limited by a set of loose diagnostic criteria self-reported by patients, which may have overestimated the prevalence of infection. SSIs are a serious medical problem associated with increased morbidity and mortality and increased medical care costs. All providers should consider an active surveillance program following endovascular procedures given the comorbidities associated with the dialysis population.

Integrated experimental setup for angle resolved photoemission spectroscopy of transuranic materials.

We have developed the Angle Resolved Photoemission Spectroscopy (ARPES) system for transuranic materials. The ARPES transuranic system is an endstation upgrade to the Laser Plasma Light Source (LPLS) at Los Alamos National Laboratory. The LPLS is a tunable light source for photoemission with a photon energy range covering the vacuum ultraviolet (VUV) and soft x-ray regions (27-140 eV). The LPLS was designed and developed for transuranic materials. Transuranic photoemission is currently not permitted at the public synchrotrons worldwide in the VUV energy range due to sample encapsulation requirements. With the addition of the ARPES capability to the LPLS system there is an excellent opportunity to explore new details centered on the electronic structure of actinide and transuranic materials.

A review of postoperative feeding regimens in infantile hypertrophic pyloric stenosis.

Infantile hypertrophic pyloric stenosis is a condition well known to pediatric surgeons. Postoperative length of hospital stay is a financial concern and remains a potential target for reduction in hospital costs. Ultimately, these costs are directly affected by the ability to effectively advance postoperative enteral nutrition. This review will serve to: 1) identify clinically relevant postoperative feeding patterns following pyloromyotomy, 2) review the relevant literature to determine an optimal feeding pattern, and 3) identify possible preoperative predictors that may determine the success of postoperative feeding regiments.

Inhibition of WISE preserves renal allograft function.

Wnt-modulator in surface ectoderm (WISE) is a secreted modulator of Wnt signaling expressed in the adult kidney. Activation of Wnt signaling has been observed in renal transplants developing interstitial fibrosis and tubular atrophy; however, whether WISE contributes to chronic changes is not well understood. Here, we found moderate to high expression of WISE mRNA in a rat model of renal transplantation and in kidneys from normal rats. Treatment with a neutralizing antibody against WISE improved proteinuria and graft function, which correlated with higher levels of ß-catenin protein in kidney allografts. In addition, treatment with the anti-WISE antibody reduced infiltration of CD68(+) macrophages and CD8(+) T cells, attenuated glomerular and interstitial injury, and decreased biomarkers of renal injury. This treatment reduced expression of genes involved in immune responses and in fibrogenic pathways. In summary, WISE contributes to renal dysfunction by promoting tubular atrophy and interstitial fibrosis.