No abstract

We conducted a retrospective study to determine the yield of blood samples drawn for culture during the initial 72 h of antibiotic therapy given to 139 patients who were admitted to the hospital for community-acquired infections or fever. The yield of these blood cultures was predictable and rarely (in only 1 patient [0.72%]) isolated new pathogens.

No abstract

Liquid chromatography tandem mass spectrometry (LC-MS/MS) has been shown to be a viable tool for preclinical pharmacokinetic (PK) analysis of monoclonal antibody (mAb) therapeutics. This work describes free and total PK assays for the mAb PF-00547,659 in serum of ulcerative colitis patients in a First-In-Human study [Vermeire, S. et al. Gut2011, 60 (8), 1068-1075]. The assay to measure free PF-00547,659 used immuno-enrichment with a biotinylated anti-idiotypic antibody and streptavidin magnetic beads. The total assay used enrichment by protein G magnetic beads. Following elution of PF-00547,659 from the beads, addition of an extended sequence stable isotope labeled peptide and trypsin digestion, a proteotypic peptide derived from the CDR region of the light chain of PF-00547,659 was quantified by LC-MS/MS. The free assay had a calibration range from 7.03 ng/mL to 450 ng/mL. The assay was precise and accurate with interbatch imprecision <16.5%, and interbatch inaccuracy <13.7% at all concentrations investigated during assay qualification. Results from LC-MS/MS methodologies are compared with historical immunoassay data originally acquired during the course of the clinical study. PK parameter estimates were highly correlated between the two analytical approaches. This work provides precedence that immunoaffinity LC-MS/MS can effectively be used to measure the serum concentrations of mAb therapeutics in clinical studies.

BackgroundBiomarkers of inflammation and altered coagulation are of increasing interest as predictors of chronic disease and mortality in HIV patients, as well as the use of risk stratification scores such as the Framingham index and the Veterans Aging Cohort Study (VACS) score.MethodsDemographic and laboratory data for 252 HIV patients were assessed for their relationship with 5 biomarkers: hsCRP, D-dimer, Cystatin C, IL-6 and TNF-alpha. Analysis of variance was used to model the association between the number of elevated biomarkers patients had and their Framingham 10 year cardiovascular risk and VACS scores.Results87% of patients were male and 75.7% were virally suppressed (HIV RNA <48 copies/ml). The median and interquartile ranges for each biomarker were: hsCRP 1.65 ug/mL (0.73, 3.89), D-dimer 0.17 ug/mL (0.09, 0.31), Cystatin C 0.87 mg/L (0.78, 1.01), IL-6 2.13 pg/mL (1.3, 3.59), TNF-alpha 4.65 pg/mL (3.5, 5.97). 62.6% of patients had more than one biomarker >75th percentile, while 18.6% had three or more elevated biomarkers. Increased age, cigarette smoking, CD4 counts of <200 cells/mm3, Framingham scores and VACS scores were most strongly associated with elevations in biomarkers. When biomarkers were used to predict the Framingham and VACS scores, those with a higher number of elevated biomarkers had higher mean VACS scores, with a similar but less robust finding for Framingham scores.ConclusionsDespite viral suppression and immunological stability, biomarkers of inflammation and coagulation remain elevated in a significant number of patients with HIV and are associated with higher scores on risk stratification indices.

The administration of biological protein therapeutics can lead to an unwanted immune response resulting in the generation of anti-drug antibodies (ADA) with potentially harmful clinical consequences. Hence, to develop safe and efficacious biotherapeutics, the immunogenic potential needs to be examined during the development phase. Current assay technologies measuring ADAs are subject to interference by high circulating concentrations of the protein therapeutic, raising concerns about data reliability since protein therapeutic-free washout samples are not always available. Herein, we report the development and characterization of a magnetic bead based immunoprecipitation method followed by quantitative LC/MS to determine ADA in human and cynomolgus serum in the presence of high circulating concentrations of the protein therapeutic. Available ADA binding sites are saturated by the addition of excess therapeutic followed by magnetic bead based protein G isolation of IgG antibodies and their bound antigens before elution and digestion. Peptides of the target therapeutic proteins are then quantified by LC/MS using stable isotope labeled standards inferring the presence of total ADA. This approach complements established methodologies for the assessment of immunogenicity responses and currently supports clinical programs addressing the safety and tolerability of human growth hormone analogues.

While myriad molecular formats for bispecific antibodies have been examined to date, the simplest structures are often based on the scFv. Issues with stability and manufacturability in scFv-based bispecific molecules, however, have been a significant hindrance to their development, particularly for high-concentration, stable formulations that allow subcutaneous delivery. Our aim was to generate a tetravalent bispecific molecule targeting two inflammatory mediators for synergistic immune modulation. We focused on an scFv-Fc-scFv format, with a flexible (A4T)3 linker coupling an additional scFv to the C-terminus of an scFv-Fc. While one of the lead scFvs isolated directly from a naïve library was well-behaved and sufficiently potent, the parental anti-CXCL13 scFv 3B4 required optimization for affinity, stability, and cynomolgus ortholog cross-reactivity. To achieve this, we eschewed framework-based stabilizing mutations in favor of complementarity-determining region (CDR) mutagenesis and re-selection for simultaneous improvements in both affinity and thermal stability. Phage-displayed 3B4 CDR-mutant libraries were used in an aggressive “hammer-hug” selection strategy that incorporated thermal challenge, functional, and biophysical screening. This approach identified leads with improved stability and >18-fold, and 4,100-fold higher affinity for both human and cynomolgus CXCL13, respectively. Improvements were exclusively mediated through only 4 mutations in VL-CDR3. Lead scFvs were reformatted into scFv-Fc-scFvs and their biophysical properties ranked. Our final candidate could be formulated in a standard biopharmaceutical platform buffer at 100 mg/ml with <2% high molecular weight species present after 7 weeks at 4 °C and viscosity <15 cP. This workflow has facilitated the identification of a truly manufacturable scFv-based bispecific therapeutic suitable for subcutaneous administration.

African trypanosomiasis is a rare but well-documented cause of fever in United States travelers returning from areas where it is endemic. We report two recently diagnosed cases that involved tourists who went on safari in Tanzania. Review of these and 29 other published cases indicates that disease in returning United States travelers is nearly always of the East African form, a fulminant illness for which prompt diagnosis is necessary. In the United States, timely and appropriate therapy for this disease has resulted in favorable outcomes for most patients. Chemoprophylaxis for East African trypanosomiasis is not recommended, but travelers visiting areas of endemicity should practice appropriate preventive measures to prevent tsetse fly bites.