Profiling Active Enzymes for Polysorbate Degradation in Biotherapeutics by Activity-Based Protein Profiling.

Polysorbate is widely used to maintain stability of biotherapeutic proteins in pharmaceutical formulation development. Degradation of polysorbate can lead to particle formation in drug products, which is a major quality concern and potential patient risk factor. Enzymatic activity from residual host cell enzymes such as lipases and esterases plays a major role for polysorbate degradation. Their high activity, often at very low concentration, constitutes a major analytical challenge in the biopharmaceutical industry. In this study, we evaluated and optimized the activity-based protein profiling (ABPP) approach to identify active enzymes responsible for polysorbate degradation. Using an optimized chemical probe, we established the first global profile of active serine hydrolases in harvested cell culture fluid (HCCF) for monoclonal antibodies (mAbs) production from two Chinese hamster ovary (CHO) cell lines. A total of eight known lipases were identified by ABPP with enzyme activity information, while only five lipases were identified by a traditional abundance-based proteomics (TABP) approach. Interestingly, phospholipase B-like 2 (PLBL2), a well-known problematic HCP was not found to be active in process-intermediates from two different mAbs. In a proof-of-concept study with downstream samples, phospholipase A2 group VII (PLA2G7) was only identified by ABPP and confirmed to contribute to polysorbate-80 degradation for the first time. The established ABBP approach is approved to be able to identify low-abundance host cell enzymes and fills the gap between lipase abundance and activity, which enables more meaningful polysorbate degradation investigations for biotherapeutic development.

Evaluation of the toxicity of sodium dodecyl sulphate (SDS) in the MucilAir™ human airway model in vitro.

The aim of the study was to use multiple in vitro assays to assess the effects of a model irritant, sodium dodecyl sulphate (SDS) (≤10 mM (0.29 %, w/v)), on an in vitro model of the airway, MucilAir™. The use of MucilAir™ in recovery studies was also explored. A 24 h exposure increased IL-8 release at an SDS concentration ≥0.63 mM (0.018 %, w/v). Mucin secretion increased and transepithelial electrical resistance (TEER) decreased at SDS concentrations ≥1.25 mM (0.04 %, w/v). Cytotoxicity (lactate dehydrogenase (LDH) release into basolateral chamber) was observed at SDS concentrations of ≥2.5 mM (0.07 %, w/v). The sensitivity of the assays was IL-8 release > TEER = mucin secretion > LDH release. After 7 days, full or partial recovery was observed for intermediate concentrations of SDS using all assays but not at 5 and 10 mM SDS. Morphologically, erosion and cell loss were observed at these concentrations. Resazurin metabolism at 7 days tended to decrease in a dose-dependent manner at SDS concentrations above 2.5 mM (0.07 %, w/v). Together, these data support a No Observable Effect Level of 0.31 mM (0.009 % w/v) SDS and the use of MucilAir™ as a relevant model for airway toxicity studies.

Changes in Procoagulant Blood Biomarkers After Mechanical Thrombectomy.

OBJECTIVES: There is limited knowledge of the relationship between mechanical thrombectomy (MT) and endothelial inflammation in large-vessel occlusion (LVO) acute ischemic stroke (AIS). Intimal arterial damage releases tissue factor, a precipitant of the clotting cascade and thrombosis. We report changes in blood coagulation markers after MT treated with and without tissue plasminogen activator for AIS. MATERIALS AND METHODS: Cases of LVO-AIS treated with MT were included. Blood coagulation marker levels were measured within 10 h of stroke onset as a baseline and then 48 h later. Assayed biomarkers included: tissue factor procoagulant activity (TFPCA), factor VII (FVII), activated factor VII (FVIIa), factor VIII (FVIII), d-dimer, thrombin-antithrombin complex (TAT), plasminogen activator inhibitor-1 (PAI-1), and tissue factor pathway inhibitor (TFPI). Biomarker levels of MT with tissue plasminogen activator (TPA) or without (non-TPA) are reported. RESULTS: Biomarker levels from five patients with LVO-AIS treated with MT (three non-TPA, two TPA) were included. In non-TPA cases, TFPCA and PAI-1 increased while FVII, FVIIa, TAT, d-dimer, and TFPI decreased from baseline to 48 h. In TPA cases, TFPCA, FVIIa, d-dimer, TFPI, and PAI-1 decreased while FVIII increased from baseline to 48 h. CONCLUSIONS: TFPCA increased after MT in non-TPA but decreased in TPA treated patients. This finding suggests that MT is associated with elevated inflammation and procoagulation which may be reduced with TPA treatment. With further validation, the increase in TFPCA levels could help guide anticoagulant management of patients with MT without TPA.

Hepatopancreatoduodenectomy in North America: are the outcomes acceptable?

BACKGROUND: Hepatopancreatoduodenectomies (HPD) are historically associated with high morbidity and mortality. Currently, no data with hepatopancreatobiliary-specific complications have been available for HPD in North America. The aim of this retrospective analysis was to compare the outcomes of HPD to those of major hepatectomy (MH) and pancreatoduodenectomy (PD) in North America. METHODS: The 2014-16 American College of Surgeons-National Surgical Quality Improvement Program database was queried for MH, PD, and HPD. Partial hepatectomies, wedge liver biopsies, distal pancreatectomies, pancreatic enucleations and total pancreatectomies were excluded. Propensity score matching was utilized to match 23 HPDs to 92 MHs and 138 PDs by 28 demographic, comorbidity, laboratory, operative and pathologic variables. Outcomes were compared among these three groups. RESULTS: The overall morbidity and mortality for HPD were 87% and 26%, respectively, and were significantly higher (p < 0.01) compared to both MH (51%, 7.6%) and PD (52%, 1.4%). Post-hepatectomy liver failure (PHLF) was more common (p < 0.01) in HPD patients, but pancreatic fistula rates were similar. CONCLUSION: The morbidity and mortality after HPD are significantly higher than after MH or PD alone and may explain why HPD is performed so infrequently in North America. Centralization of HPD to a very few centers may be a strategy to improve outcomes.

Delayed Traumatic Intracerebral Hematoma: A Pathophysiological Classification and Literature Review.

Delayed traumatic intracerebral hematoma (DTICH) is a relatively common occurrence after a traumatic brain injury (TBI). Several case series have been performed to study DTICH, many of which offer different definitions of DTICH. Some definitions involve a delayed progression of an existing hemorrhage, and others involve a de novo intracerebral hematoma that was not evident on the initial trauma evaluation. We propose a classification system for DTICH that accounts for the subtleties in the clinical manifestation and pathophysiology of the different types of DTICH, with the ultimate goal of providing strategies to prevent and manage DTICH. Based on the senior author's clinical experience, we generated a classification system for DTICH, and each type of DTICH was illustrated with a case. We defined type 1A (case 1A), the classic presentation of DTICH as predominantly characterized in the literature, as an intracerebral hematoma unseen on initial computed tomography imaging that typically develops five days to one week following blunt or penetrating head trauma. We defined type 1B (case 1B) as a hematoma that forms after at least one week following trauma in areas of the brain initially hemorrhage-free. We defined type 2 (case 2) as a hematoma that develops rapidly following a surgical evacuation of a different hematoma. We defined type 3 (case 3) as a hematoma that develops after a traumatic head injury in areas of non-hemorrhagic contusion, usually frontal or temporal. A literature review was performed using select terms on PubMed to find articles related to DTICH, excluding articles describing DTICH from an underlying vascular injury. After performing the literature review and screening articles by title and/or abstract, a total of 79 articles were found to meet the inclusion and exclusion criteria. We recorded which type of DTICH from our classification system best correlated with the articles in our literature review. Taken together with results from the literature, the proposed classification system is based on the senior author's clinical experience. Overall, DTICH is a relatively common occurrence after head trauma, and our pathophysiologic classification has the potential to help outline future studies to recognize and prevent the development of DTICH.

Extensive Characterization of Polysorbate 80 Oxidative Degradation Under Stainless Steel Conditions.

Polysorbate-80 (PS-80) is a common surfactant used in biologics formulations. However, the tendency of oxidation to PS-80 when exposed to stainless steel surfaces brings various challenges during manufacturing processes, such as inconsistent shelf-life of PS-80 solutions, which can further impact the biologics and vaccines production. In this work, the root causes of PS-80 oxidation when in contact with stainless steel conditions were thoroughly investigated through the use of various complementary analytical techniques including U/HPLC-CAD, LC-MS, ICP-MS, peroxide assay, and EPR spectroscopy. The analytical tool kit used in this work successfully revealed a PS-80 degradation mechanism from the perspective of PS-80 content, PS-80 profile, iron content, peroxide production, and radical species. The combined datasets reveal that PS-80 oxidative degradation occurs in the presence of histidine and iron in addition to being combined with the hydroperoxides in PS-80 material. The oxidative pathway and potential degradants were identified by LC-MS. The PS-80 profile based on the U/HPLC-CAD assay provided an effective way to identify early-signs of PS-80 degradation. The results from a peroxide assay observed increased hydroperoxide along with PS-80 degradation. EPR spectra confirmed the presence of histidine-related radicals during PS-80 oxidation identifying how histidine is involved in the oxidation. All assays and findings introduced in this work will provide insight into how PS-80 oxidative degradation can be avoided, controlled, or detected. It will also provide valuable evaluations on techniques that can be used to identify PS-80 degradation related events that occur during the manufacturing process.

Incorporating new approach methodologies into regulatory nonclinical pharmaceutical safety assessment.

New approach methodologies (NAMs) based on human biology enable the assessment of adverse biological effects of pharmaceuticals and other chemicals. Currently, however, it is unclear how NAMs should be used during drug development to improve human safety evaluation. A series of 5 workshops with 13 international experts (regulators, preclinical scientists, and NAMs developers) was conducted to identify feasible NAMs and to discuss how to exploit them in specific safety assessment contexts. Participants generated four "maps" of how NAMs can be exploited in the safety assessment of the liver, respiratory, cardiovascular, and central nervous systems. Each map shows relevant endpoints measured and tools used (e.g., cells, assays, platforms), and highlights gaps where further development and validation of NAMs remains necessary. Each map addresses the fundamental scientific requirements for the safety assessment of that organ system, providing users with guidance on the selection of appropriate NAMs. In addition to generating the maps, participants offered suggestions for encouraging greater NAM adoption within drug development and their inclusion in regulatory guidelines. A specific recommendation was that pharmaceutical companies should be more transparent about how they use NAMs in-house. As well as giving guidance for the four organ systems, the maps provide a template that could be used for additional organ safety testing contexts. Moreover, their conversion to an interactive format would enable users to drill down to the detail necessary to answer specific scientific and regulatory questions.

Desiccating stress stimulates expression of matrix metalloproteinases by the corneal epithelium.

PURPOSE: To determine the effects of experimental dryness on production of matrix metalloproteinases (MMPs) and their physiological inhibitors (TIMPs) by the corneal epithelium. METHODS: Experimental dry eye (EDE) was created in two strains of mice: BALB/c and C57BL/6. Real-time PCR was performed with MMP and TIMP probes, and the results were analyzed by the comparative C(T) method, selecting the relative mRNA levels in untreated control samples as calibrator. Immunofluorescent staining with specific antibodies immunolocalized MMP proteins in situ. MMP enzymatic activity was evaluated in tears and corneal lysates. Corneal permeability to Oregon green dextran (OGD) and sodium fluorescein was measured. Corneal smoothness was evaluated by graded regularity of a ring reflected off the corneal surface. RESULTS: Desiccating stress significantly increased levels of MMP-1, -3, -9, and -10 transcripts in the corneal epithelium in C57BL/6 mice, but had no effect on levels of MMP transcripts in the corneal epithelium of BALB/c mice. There was no change in levels of TIMP transcripts except for TIMP-4 which significantly increased on day 10 in C57BL/6 mice. The MMP-1, -3, and -9 concentration in tears significantly increased compared with control levels after EDE for 4 and 6 days, respectively, in C57BL/6 and BALB/c. Changes in MMP protein expression detected by immunofluorescent staining were similar to changes in gene transcripts for most MMPs. EDE increased corneal permeability to OGD and fluorescein and corneal surface irregularity. CONCLUSIONS: Corneal dryness stimulates production of certain MMPs in a strain-dependent fashion and causes the disruption of the corneal barrier, thus increasing permeability and corneal irregularity.

Authorities and Mechanisms for Purchased Care at the Department of Veterans Affairs.

The Veterans Access, Choice, and Accountability Act of 2014 addressed the need for access to timely, high-quality health care for veterans. Section 201 of the legislation called for an independent assessment of various aspects of veterans' health care. The RAND Corporation was tasked with an assessment of the authorities and mechanisms by which the Department of Veterans Affairs (VA) pays for health care services from non-VA providers. Purchased care accounted for 10 percent, or around $5.6 billion, of VA's health care budget in fiscal year 2014, and the amount of care purchased from outside VA is growing rapidly. VA purchases non-VA care through an array of programs, each with different payment processes and eligibility requirements for veterans and outside providers. A review and analysis of statutes, regulations, legislation, and literature on VA purchased care, along with interviews with expert stakeholders, a survey of VA medical facilities, and an evaluation of local-level policy documents revealed that VA's purchased care system is complex and decentralized. Inconsistencies in procedures, unclear goals, and a lack of cohesive strategy for purchased care could have ramifications for veterans' access to care. Adding to the complexity of VA's purchased care system is a lack of systematic data collection on access to and quality of care provided through VA's purchased care programs. The analysis also explored concepts of "episodes of care" and their implications for purchased care by VA.

Hospital and emergency department factors associated with variations in missed diagnosis and costs for patients age 65 years and older with acute myocardial infarction who present to emergency departments.

OBJECTIVES: The objective was to measure the variation in missed diagnosis and costs of care for older acute myocardial infarction (AMI) patients presenting to emergency departments (EDs) and to identify the hospital and ED characteristics associated with this variation. METHODS: Using 2004-2005 Medicare inpatient and outpatient records, the authors identified a cohort of AMI patients age 65 years and older who presented to the ED for initial care. The primary outcome was missed diagnosis of AMI, i.e., AMI hospital admission within 7 days of an ED discharge for a condition suggestive of cardiac ischemia. Costs were defined as Medicare hospital payments for all services associated with and immediately resulting from the ED evaluation. The effect of ED and hospital characteristics on quality and costs were estimated using multilevel models with hospital random effects. RESULTS: There were 371,638 AMI patients age 65 and older included in the study, of whom 4,707 were discharged home from their initial ED visits and subsequently admitted to the hospital. The median unadjusted hospital-level missed diagnosis percentage was 0.52% (interquartile range [IQR] = 0 to 3.45%). ED characteristics protective of adverse outcomes include higher ED chest pain acuity (adjusted odds ratio [aOR] = 0.23, 99% confidence interval [CI] = 0.19 to 0.27) and American Board of Emergency Medicine (ABEM) certification (aOR = 0.60, 99% CI = 0.50 to 0.73). Protective hospital characteristics include larger hospital size (aOR = 0.46, 99% CI = 0.37 to 0.57) and academic status (aOR = 0.74, 99% CI = 0.58 to 0.94). All of these characteristics were associated with higher costs as well. CONCLUSIONS: The proportion of missed AMI diagnoses and cost of care for patients age 65 years and older presenting to EDs with AMI varies across hospitals. Hospitals with more board-certified emergency physicians (EPs) and higher average acuity are associated with significantly higher quality. All hospital characteristics associated with better ED outcomes are associated with higher costs.

As she lay dying: how I fought to stop medical errors from killing my mom.

A woman with cancer dies after receiving poor care for an infection. Her physician-son calls on the health system to involve patients and families in improving safety.