Kinetics and Characterization of Non-enzymatic Fragmentation of Monoclonal Antibody Therapeutics

Purpose

To understand non-enzymatic hydrolytic fragmentation of a monoclonal antibody therapeutic under temperature stressed conditions and investigating possible mechanism for the same.

Methods

The mAb therapeutic was incubated at 50°C in phosphate buffer at pH 6.5 and fragmentation was monitored at different ionic strengths under stressed conditions. The incubated mAb was sampled at regular time intervals by analytical Size Exclusion Chromatography (SEC).

Results

It was observed that 57% of the mAb product fragmented over 4 days into two fragment species – Fc-Fab and Fab with molecular weights of 97 KDa and 47 KDa, respectively, as measured by mass spectrometry (MS) and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The fragmentation rate was slow initially and then accelerated with time. No change in % aggregate level was observed in this duration, implying that degradation was primarily via fragmentation at high temperature. Kinetics of hydrolytic fragmentation was hypothesized and SEC data was fitted to estimate the kinetic rate constants. While degradation of the monomer into fragment species was non-Arrhenius with a negative activation energy, further degradation of Fab-Fc fragments into Fab or Fc fragments followed Arrhenius Law with an activation energy of 2.1 and 15.38 kcal/mol, respectively.

Conclusion

High temperature (50°C) causes mAb to cleave at the hinge region to form Fab-Fc and Fab/Fc, as confirmed by dynamic light scattering, SDS-PAGE, SEC, and MS. A kinetic model for hydrolytic fragmentation has been proposed. The results are expected to assist end users in formulation development as well as in monitoring stability of biotherapeutic products.

     

Results of the First Clinical Study in Humans That Combines Hyperbaric Oxygen Pretreatment with Autologous Peripheral Blood Stem Cell Transplantation

Patients undergoing high-dose chemotherapy and autologous hematopoietic cell transplantation (auto-HCT) are at risk for multiple morbidities, including mucosal inflammation and neutropenic fever, both related to neutropenia. Evidence from our preclinical work in an umbilical cord blood (UCB) transplantation murine model suggests that treatment with hyperbaric oxygen (HBO) before UCB infusion improves UCB CD34⁺ cell engraftment by reducing erythropoietin levels. A pilot clinical trial using HBO in patients undergoing UCB transplantation showed improvement in kinetics of blood count recovery. In this study, we evaluated HBO in combination with auto-HCT. Our primary aim was to determine the safety of HBO in this setting and secondarily to determine its efficacy in reducing time to neutrophil and platelet engraftment compared with matched historic controls. Patients with multiple myeloma, non-Hodgkin lymphoma, and Hodgkin disease eligible for auto-HCT were included. On day 0, patients received HBO treatment consisting of exposure to 2.5 atmosphere absolutes for a total of 90 minutes, in a monoplace hyperbaric chamber, breathing 100% oxygen. Six hours after the start of HBO, peripherally mobilized stem/progenitor cells were infused and patients were followed daily for toxicity and blood count recovery. All patients received daily granulocyte colony-stimulating factor starting on day +5 and until absolute neutrophil count (ANC) of ≥1500 or ANC of 500 for 3 consecutive days. A matched historic cohort of 225 patients who received auto-HCT between January 2008 and December 2012 was chosen for comparison and matched on sex, age, conditioning regimen, and disease type. We screened 26 patients for this study; 20 were treated and included in the primary analysis, and 19 completed the HBO therapy and were included in the secondary analysis. Although the median time to neutrophil count recovery was 11 days in both the HBO and control cohorts, the Kaplan-Meier estimates of the full distributions indicate that the time to neutrophil recovery was generally about 1 day sooner for HBO versus historical controls (log-rank P = .005; range, 9 to 13 for HBO patients and 7 to 18 for controls). The median time to platelet count recovery was 16 days (range, 14 to 21) for HBO versus 18 days (range, 11 to 86) for controls (log-rank P < .0001). In the secondary analysis comparing the HBO cohort who completed HBO therapy (n = 19) with our historical cohort, we evaluated neutropenic fever, growth factor use, mucositis, day +100 disease responses, and blood product use. HBO was associated with less growth factor use (median 6 days in HBO cohort versus median 8 days in controls, P < .0001). Packed RBC and platelet transfusion requirements were not statistically different between the 2 cohorts. Mucositis incidence was significantly lower in the HBO cohort (26.3% in HBO cohort versus 64.2% in controls, P = .002). HBO therapy appears to be well tolerated in the setting of high-dose therapy and auto-HCT. Prospective studies are needed to confirm potential benefits of HBO with respect to earlier blood count recovery, reduced mucositis, and growth factor use, and a cost-benefit analysis is warranted.© 2019 American Society for Blood and Marrow Transplantation.     

Microscopic Visualization of Metabotropic Glutamate Receptors on the Surface of Living Cells Using Bifunctional Magnetic Resonance Imaging Probes

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Treatment of non-union of humerus diaphyseal fractures: a prospective study comparing interlocking nail and locking compression plate

Background

The aim of this prospective comparative study was to compare outcomes and complications of humeral diaphyseal fracture non-unions managed with humerus interlocking nail (HIL) and locking compression plate (LCP).

Materials and methods

40 patients with non-union of humeral diaphyseal fractures were included in this study and were randomly allocated in two groups; group A had 20 cases treated with HIL and group B had 20 cases treated with LCP. Clinico-radiological assessments were done for each case up to 2-year follow-up period. Primary outcome measures (time to fracture union, union rate) and secondary outcome measures (functional outcome and complication such as infection, malunion, delayed union, implant failure, joint stiffness and iatrogenic radial nerve palsy) were compared between both the groups. Disabilities of the arm, shoulder and hand (DASH) scoring and Steward and Hundley’s scoring system were used to assess functional outcome of the fracture fixation.

Results

There was no significant difference (p = 0.12) in terms of mean fracture union time between group A (15.8 ± 4.2 weeks) and group B (17.2 ± 3.8 weeks). Group A had 95 % union rate and group B had 100 % union rate (p = 0.14). At the 2-year follow-up visit, there was no significant difference found between both the groups regarding range of motion of shoulder and elbow joint. There was no significant difference found in final functional outcomes between both the groups on comparing DASH score (p = 0.14) and Steward and Hundley’s score (p = 0.08). In terms of complications, there was insignificant difference found between both the groups.

Conclusions

This study concludes that both the implants can be used in non-union of humeral shaft fractures with good functional outcomes and acceptable rate of complications.     

Assessing Documentation of Critical Imaging Result Follow-up Recommendations in Emergency Department Discharge Instructions

To facilitate follow-up of critical test results across transitions in patient care settings, we implemented an electronic discharge module that enabled care providers to include follow-up recommendations in the discharge instructions. We assessed the impact of this module on documentation of follow-up recommendations for critical imaging findings in Emergency Department (ED) discharge instructions. We studied 240 patients with critical imaging findings discharged from the ED before (n?=?80) and after (n?=?160) implementation of the module. We manually reviewed hand-written forms and electronic discharge instructions to determine if follow-up recommendations were documented. Follow-up recommendations in ED discharge instructions increased from 60.0% (48/80) to 73.8% (118/160) post-module implementation (p?=?0.03), a relative increase of 23%. There was no significant change in the rate of documented critical imaging findings in the discharge instructions (77.5% [62/80] before the intervention and 76.9% [123/160] after the intervention; p?=?0.91). Implementation of a discharge module was associated with increased documentation of critical imaging finding follow-up recommendations in ED discharge instructions. However, one in four patients still did not receive adequate follow-up recommendations, suggesting further opportunities for performance improvement exist.     

Primary vs delayed primary closure in patients undergoing lower limb amputation following trauma: A randomised control study

Lower limb crush injury is a major source of mortality and morbidity in trauma patients. Complications, especially surgical site infections (SSIs) are a major source of financial burden to the institute and to the patient as it delays rehabilitation. As such, every possible attempt should be made to reduce any complications. We, thus, aimed to compare the outcomes in early vs delayed closure of lower extremity stumps in cases of lower limb crush injury requiring amputation, so as to achieve best possible outcome. A randomised controlled study was conducted in the Division of Trauma Surgery & Critical Care at Jai Prakash Narayan Apex Trauma Centre, All India Institute of Medical Sciences, New Delhi from 1 September 2018 to 30 June 2019 and included patients undergoing lower limb amputation below hip joint. Patients were randomised in two groups, in one group amputation stump was closed primarily, while in the second group delayed primary closure of stump was performed. We compared rate of SSI, length of hospital stay, and number of surgeries in both the groups. Fifty‐six patients with 63 amputation stumps were recruited in the study. Mean age of patients in the study was 34 years, of which about 95% patients were males. The most common mechanism of injury was road traffic injury in 66% of patients. Mean injury severity score was 12.28 and four patients had diabetes preoperatively. Total 63 extremities were randomised with 30 cases in group I and 33 cases in group II as per computer‐generated random number. Above knee amputations was commonest (57.14%) followed by below knee amputations (33.3%). Two patients died in the current study. In group I, In‐hospital infection was detected in 7 cases (23.3%) and in group II 9 cases (27.3%) had SSI during hospital admission (P > .05). Mean hospital stay in group I was 10.32 ± 7.68 days and in group II was 11 ± 8.17 days (P > .05). Road traffic injuries and train‐associated injuries are a major cause of lower limb crush injuries, leading to limb loss. Delayed primary closure of such wounds requires extra number of surgical interventions than primary closure. There is no difference in extra number of surgical interventions required in both the groups. Thus, primary closure can be safely performed in patients undergoing lower limb amputations following trauma, provided that a good lavage and wound debridement is performed.     

Fractional exhaled nitric oxide in children with acute exacerbation of asthma

Objective

To determine whether fractional exhaled nitric oxide (FENO) has a utility as a diagnostic or predictive maker in acute exacerbations of asthma in children.

Design

Analysis of data collected in a pediatric asthma cohort.

Setting

Pediatric Chest Clinic of a tertiary care hospital

Methods

A cohort of children with asthma was followed up every 3 months in addition to any acute exacerbation visits. Pulmonary function tests (PFT) and FENO were obtained at all visits. We compared the FENO values during acute exacerbations with those at baseline and those during the follow up.

Results

243 asthmatic children were enrolled from August 2009 to December 2011 [mean (SD) follow up — 434 (227) days]. FENO during acute exacerbations was not different from FENO during follow up; however, FENO was significantly higher than personal best FENO during follow up (P < 0.0001). FENO during acute exacerbation did not correlate with the severity of acute exacerbation (P=0.29). The receiver operating characteristics curve for FENO as a marker for acute exacerbation had an area under the curve of 0.59. Cut-off of 20 ppb had a poor sensitivity (44%) and specificity (68.7%) for acute exacerbation.

Conclusions

FENO levels during acute exacerbation increase from their personal best levels. However, no particular cut off could be identified that could help in either diagnosing acute exacerbation or predicting its severity.