Structure-function relationship study for sulfated protein therapeutics using hydrophobic interaction chromatography and mass spectrometry.

Protein tyrosine sulfation is a post-translational modification (PTM) that is rarely reported in recombinant therapeutic proteins. However, when sulfation does occur, the additional negative charge from the modification can influence intermolecular interactions and antigen-binding activity, making it a critical quality attribute that necessitates stringent control. In this study, we developed a unique hydrophobic interaction chromatography (HIC) method for the separation and quantification of a therapeutic bispecific antibody with varying degrees of sulfation. Despite the increased surface hydrophilicity of sulfated species, the HIC method provides enhanced retention. Baseline resolution was attained based on the degree of sulfation, independent of other PTMs such as C-terminal amidation and forced deamidation. Further structure-function relationship studies of enriched sulfated bispecific antibody species were conducted using mass spectrometry and fluorescence-linked immunosorbent assay (FLISA). These studies revealed that the tyrosine sulfation modification, which occurs in the complementarity-determining region (CDR), is a critical quality attribute and can adversely impact the antibody's binding to its cognate antigen. The evaluation of sulfation assay using HIC method confirmed it is an effective means for controlling this critical quality attribute.

Effect of Preoperative Weight Loss and Baseline Comorbidity on Short-Term Complications and Reoperations After Laparoscopic Roux-en-Y Gastric Bypass in 2,067 Patients.

PURPOSE: Decreasing popularity of Roux-en-Y gastric bypass (RYGB) in bariatric-metabolic surgery may be due to higher perceived peri-operative complications. There are few studies on whether preoperative weight loss can reduce complications or reoperations following RYGB. We investigated this using a standardised operative technique. MATERIALS AND METHODS: Retrospective single-centre study of RYGB from 2004 to 2019 using a prospective database. Preoperative behavioural management included intentional weight loss. Maximum preoperative weight, weight on the day of operation, and Obesity-Surgery Mortality Risk Score (OS-MRS) class were recorded. Short-term outcomes (post-operative stay, 30-day complication and reoperation rates) were analysed. RESULTS: In 2,067 RYGB patients (1,901 primary and 166 revisional), median preoperative total body weight loss (TWL) was 6.2% (IQR: 2.5-10.7%). The median age was 46 (interquartile range (IQR) 38-54) and 80.4% were female (n=1,661). For primary surgery, the median body mass index (BMI) was 47.6 kg/m2 (IQR: 43.1-53.3). Excluding the 100-procedure learning curve, the complication rate for primary cases was 4.4% and reoperation rate of 2.8% and one peri-operative mortality (0.06%). OS-MRS ≥2 (class B or C) predicted higher risk of complications (6.1%) compared to those with a score <2 (class A) (3.8%, p=0.021), but not reoperations. Five percent preoperative TWL did not decrease complications compared to <5% TWL. Patients with ≥10% TWL had greater baseline risk and had an increased risk of complications (6.6% vs 3.7%, p=0.017) and reoperations (4.5% vs 2.7%, p<0.001). CONCLUSIONS: RYGB performed using a standardised technique has low overall risk. The influence of preoperative weight loss on outcomes was inconsistent.

Laparoscopic-Assisted Endoscopic Retrograde Cholangiopancreatography (ERCP) for Bile Duct Stones After Roux-en-Y-Gastric Bypass: Single-Centre Experience.

PURPOSE: Gallstones are common in bariatric patients due to obesity and rapid weight loss. Bile duct stones after Roux-en-Y gastric bypass (RYGB) pose a technical challenge. We present our experience in management of bile duct stones following RYGB using laparoscopic-assisted endoscopic retrograde cholangiopancreatography (LA-ERCP). MATERIALS AND METHODS: Retrospective review of RYGB patients who had endoscopic intervention for bile duct stones between 2010 and 2019. We assessed demographic and clinical outcomes. RESULTS: There were 12 patients: 9 females, median age 64 years (range 34-73), median ASA score 3 (range 2-3), and median body mass index (BMI) 30 kg/m2 (range 24.4-46). Median time of presentation since RYGB was 5 years (range 6-96 months). Clinical presentations were biliary pain with deranged liver function tests (n = 8, 67%) and cholangitis (n = 4, 33%). Ten patients (83%) had cholecystectomy prior to presentation. LA-ERCP was performed in all 12 patients. It was successful in 10 patients (83%) of which 7 were performed as a primary intervention for bile duct stones and 3 were for residual stones following previous bile duct exploration. Two out of 12 LA-ERCPs (17%) were converted to open duct clearance. Median overall hospital stay was 2.5 days (range 1-10). One patient developed post-ERCP pancreatitis; one had chronic pain. There was no major complication or mortality. CONCLUSION: LA-ERCP is feasible for bile duct stones after RYGB and can clear the duct primarily or following previous surgical exploration. It also provides an opportunity to perform cholecystectomy and diagnostic laparoscopy.

Muon tomography for the analysis of in-container vitrified products.

Alternate treatment routes for radioactive waste are a key research area for much of the nuclear industry, with potentially significant savings available through volume reduction of waste. Achieving this requires a full and demonstrable understanding of waste product behaviour. For this purpose, the UK's National Nuclear Laboratory (NNL) has been collaborating with the University of Glasgow and Lynkeos Technology to develop passive techniques for analysis of waste containers over a number of years. In this instance, novel muon tomographic techniques have been applied to the analysis of thermally treated nuclear waste surrogates as part of a project to build and deploy a first of a kind muon imaging system for nuclear waste. The system has been deployed at NNL's Central Laboratory, Cumbria, UK, to analyse products from a series of thermal treatment technology trials, funded by the Nuclear Decommissioning Authority (NDA) through the Direct Research Portfolio (DRP). Analysis of the waste products using this technique has proven the value of muon analysis in the development of waste management technologies, proving an ability to understand the homogeneity of products and direct further destructive testing. Results from three different thermal treatment trials are presented, with three different surrogate intermediate level waste (ILW) forms in each.

Novel muon imaging techniques.

Owing to the high penetrating power of high-energy cosmic ray muons, muon imaging techniques can be used to image large bulky objects, especially objects with heavy shielding. Muon imaging systems work just like CT scanners in the medical imaging field-that is, they can reveal information inside of a target. There are two forms of muon imaging techniques: muon absorption imaging and muon multiple scattering imaging. The former is based on the flux attenuation of muons, and the latter is based on the multiple scattering of muons in matter. The muon absorption imaging technique is capable of imaging very large objects such as volcanoes and large buildings, and also smaller objects like spent fuel casks; the muon multiple scattering imaging technique is best suited to inspect smaller objects such as nuclear waste containers. Muon imaging techniques can be applied in a broad variety of fields, i.e. from measuring the magma thickness of volcanoes to searching for secret cavities in pyramids, and from monitoring the borders of countries checking for special nuclear materials to monitoring the spent fuel casks for nuclear safeguards applications. In this paper, the principles of muon imaging are reviewed. Image reconstruction algorithms such as Filtered Back Projection and Maximum Likelihood Expectation Maximization are discussed. The capability of muon imaging techniques is demonstrated through a Geant4 simulation study for imaging a nuclear spent fuel cask.This article is part of the Theo Murphy meeting issue 'Cosmic-ray muography'.

First-of-a-kind muography for nuclear waste characterization.

In the last decade, there has been a surge in the number of academic research groups and commercial companies exploiting naturally occurring cosmic-ray muons for imaging purposes in a range of industrial and geological applications. Since 2009, researchers at the University of Glasgow and the UK National Nuclear Laboratory (NNL) have pioneered this technique for the characterization of shielded nuclear waste containers with significant investment from the UK Nuclear Decommissioning Authority and Sellafield Ltd. Lynkeos Technology Ltd. was formed in 2016 to commercialize the Muon Imaging System (MIS) technology that resulted from this industry-funded academic research. The design, construction and performance of the Lynkeos MIS is presented along with first experimental and commercial results. The high-resolution images include the identification of small fragments of uranium within a surrogate 500-litre intermediate level waste container and metal inclusions within thermally treated GeoMelt® R&D Product Samples. The latter of these are from Lynkeos' first commercial contract with the UK National Nuclear Laboratory. The Lynkeos MIS will be deployed at the NNL Central Laboratory facility on the Sellafield site in Summer 2018 where it will embark upon a series of industry trials.This article is part of the Theo Murphy meeting issue 'Cosmic-ray muography'.

Influence of social deprivation on provision of bariatric surgery: 10-year comparative ecological study between two UK specialist centres.

OBJECTIVE: To investigate the effect of residential location and socioeconomic deprivation on the provision of bariatric surgery. DESIGN: Retrospective cross-sectional ecological study. SETTING: Patients resident local to one of two specialist bariatric units, in different regions of the UK, who received obesity surgery between 2003 and 2013. METHODS: Demographic data were collected from prospectively collected databases. Index of Multiple Deprivation (IMD 2010) was used as a measure of socioeconomic status. Obesity prevalences were obtained from Public Health England (2006). Patients were split into three IMD tertiles (high, median, low) and also tertiles of time. A generalised linear model was generated for each time period to investigate the effect of socioeconomic deprivation on the relationship between bariatric case count and prevalence of obesity. We used these to estimate surgical intervention provided in each population in each period at differing levels of deprivation. RESULTS: Data were included from 1163 bariatric cases (centre 1-414, centre 2-749). Incidence rate ratios (IRRs) were calculated to measure the associations between predictor and response variables. Associations were highly non-linear and changed over the 10-year study period. In general, the relationship between surgical case volume and obesity prevalence has weakened over time, with high volumes becoming less associated with prevalence of obesity. DISCUSSION: As bariatric services have matured, the associations between demand and supply factors have changed. Socioeconomic deprivation is not apparently a barrier to service provision more recently, but the positive relationships between obesity and surgical volume we would expect to find are absent. This suggests that interventions are not being taken up in the areas of need. We recommend a more detailed national analysis of the relationship between supply side and demand side factors in the provision of bariatric surgery.

Laparoscopic retrieval of an intra-abdominal air gun pellet.

A 19-year-old man presented to the emergency department following an air gun pellet injury to the abdomen. He was clinically stable and underwent laparoscopic retrieval of the pellet, which was found embedded in the small bowel mesentery. He recovered fully and was discharged after 2 days. We further discuss air gun-related injuries.

The By-Band study: gastric bypass or adjustable gastric band surgery to treat morbid obesity: study protocol for a multi-centre randomised controlled trial with an internal pilot phase.

BACKGROUND: The prevalence of severe and complex obesity is increasing worldwide and surgery may offer an effective and lasting treatment. Laparoscopic adjustable gastric band and Roux-en-Y gastric bypass surgery are the two main surgical procedures performed. DESIGN: This open parallel-group randomised controlled trial will compare the effectiveness, cost-effectiveness and acceptability of gastric band (Band) versus gastric bypass (Bypass) in adults with severe and complex obesity. It has an internal pilot phase (in two centres) with integrated qualitative research to establish effective and optimal methods for recruitment. Adults with a body mass index (BMI) of 40 kg/m2 or more, or a BMI of 35 kg/m2 or more and other co-morbidities will be recruited. At the end of the internal pilot the study will expand into more centres if the pre-set progression criteria of numbers and rates of eligible patients screened and randomised are met and if the expected rates of retention and adherence to treatment allocation are achieved. The trial will test the joint hypotheses that Bypass is non-inferior to Band with respect to more than 50% excess weight loss and that Bypass is superior to Band with respect to health related quality of life (HRQOL, EQ-5D) at three years. Secondary outcomes include other weight loss measures, waist circumference and remission/resolution of co-morbidities; generic and symptom-specific HRQOL; nutritional blood test results; resource use; eating behaviours and adverse events. A core outcome set for reporting the results of obesity surgery will be developed and a systematic review of the evidence for sleeve gastrectomy undertaken to inform the main study design. DISCUSSION: By-Band is the first pragmatic study to compare the two most commonly performed bariatric surgical procedures for severe and complex obesity. The design will enable and empower surgeons to learn to recruit and participate in a randomised study. Early evidence shows that timely recruitment is possible. TRIAL REGISTRATION: Current Controlled Trials ISRCTN00786323.

Living with a Gastric Band: A Qualitative Study.

UNLABELLED: Gastric banding is an established and effective form of weightloss surgery. Semi-structured interviews explored the experiences of gastric banding of twenty purposively recruited patients one year after surgery. Data was analysed using thematic analysis. RESULTS: Three themes emerged. They included 'Exercising choice' (restriction by the band was counterbalanced by new food-related choices.); 'Rediscovering life' (improved health, physical ability and energy enabled the patients to re-discover life.) and 'Goals achieved with no regrets' (patients had nearly achieved their self-set goals.) CONCLUSION: Beyond achieving weight loss and improved health, the participants had improved quality of life as defined by patients. Knowledge about this active process informs the care of these patients.

Effect of source of funding on weight loss up to 3 years after gastric banding.

BACKGROUND: Laparoscopic adjustable gastric banding (LAGB) is a popular choice for patients seeking weight loss surgery. Since behavioural change appears to play a role in weight loss outcomes we postulated that publicly funded patients might not do as well as self-payers. This series examines the effect of public funding versus self-pay on patients undergoing LAGB over 1, 2 and 3 years. METHODS: Consecutive non-randomised cohort series of patient undergoing LAGB over 5 years (September 2003 to December 2008) in a single unit. Age, sex, funding route, body mass index (BMI) and complications were recorded. Per cent excess weight loss (EWL) and the Reinhold criterion for success (proportion achieving 50 % EWL) were assessed. RESULTS: Ninety-nine patients were publicly funded, and 250 patients were self-payers. Initial BMI was significantly higher in publicly funded patients (46.6 vs. 42.3 kg/m(2), p < 0.001) with a higher proportion of males (22.2 vs. 6.0 %, p < 0.001). Mean % EWL was significantly less for publicly funded patients at 1 year (38.1 vs. 53.5 %, p < 0.001) and 2 years (49.6 vs. 64.1 %, p < 0.001), but not at 3 years (59.7 vs. 61.8 %, p = 0.784). Fewer publicly funded patients achieved 50 % EWL at 1 year (24.5 vs. 50.2 %, p < 0.001), but with no significant difference at 2 years (54.8 vs. 67.0 %, p = 0.140) or 3 years (55.2 vs. 66.0 %, p = 0.349). CONCLUSIONS: Self-pay patients initially achieved more % EWL and greater success in reaching 50 % EWL after LAGB, but this difference was not maintained. The results suggest that patient motivation, using self-pay as a surrogate marker, may affect early results, but the operation itself is the main determinant of weight loss at 3 years.

The Patient Journey to Gastric Band Surgery: A Qualitative Exploration.

AIMS: This study explored the views and experiences of obese people preparing to undergo laparoscopic gastric banding (LAGB) leading up to the time of surgery. BACKGROUND: Weight loss surgery (WLS) is the most successful intervention available for the treatment of morbid obesity, and LAGB is among the most commonly used procedures in bariatric surgery. So far, the patient experience of deciding to undergo LAGB has been explored rarely and predominantly retrospectively. DESIGN: Semi-structured interviews took place with 23 patients about to undergo LAGB between June 2011 and March 2012. Data were analyzed using thematic analysis. Demographic and quality of life data situated the sample within the LAGB patient population. RESULTS: Three overarching themes were described. Participants were "living with obesity," including the physical, social, and psychological challenges and consequences of being obese. These created in them a "desire to change," expressed in multiple unsuccessful attempts to lose weight, and a quest for information, finally focusing on WLS. Eventually, "expectations toward LAGB" were formed, mainly to hand back a measure of control that enabled them to achieve, as well as ultimately to maintain, weight loss. This active process resulted in the patients' decision to undergo LAGB. When combined, these themes outline a distinct patient journey toward gastric banding. CONCLUSION: Knowledge of the patient journey can inform both selection and care of patients awaiting gastric band surgery and is required by all health professionals working with this patient group.

The role of bile after Roux-en-Y gastric bypass in promoting weight loss and improving glycaemic control.

Gastric bypass leads to the remission of type 2 diabetes independently of weight loss. Our hypothesis is that changes in bile flow due to the altered anatomy may partly explain the metabolic outcomes of the operation. We prospectively studied 12 patients undergoing gastric bypass and six patients undergoing gastric banding over a 6-wk period. Plasma fibroblast growth factor (FGF)19, stimulated by bile acid absorption in the terminal ileum, and plasma bile acids were measured. In canine and rodent models, we investigated changes in the gut hormone response after altered bile flow. FGF19 and total plasma bile acids levels increased after gastric bypass compared with no change after gastric banding. In the canine model, both food and bile, on their own, stimulated satiety gut hormone responses. However, when combined, the response was doubled. In rats, drainage of endogenous bile into the terminal ileum was associated with an enhanced satiety gut hormone response, reduced food intake, and lower body weight. In conclusion, after gastric bypass, bile flow is altered, leading to increased plasma bile acids, FGF19, incretin. and satiety gut hormone concentrations. Elucidating the mechanism of action of gastric bypass surgery may lead to novel treatments for type 2 diabetes.

A platform for characterizing therapeutic monoclonal antibody breakdown products by 2D chromatography and top-down mass spectrometry.

With the growing commercialization of therapeutic monoclonal antibodies developed for the treatment of various diseases comes the need for increased analytical scrutiny of the impurity components contained within such drug products. Traditionally, relatively low performance and throughput analytical techniques were employed for elucidating the product-related breakdown components derived from the original molecule, including N-terminal Edman sequencing and matrix-assisted laser desorption time-of-flight (MALDI-TOF) mass spectrometry. Although N-terminal sequencing provides a definitive starting point of an unknown breakdown product, the resolution and mass accuracy of MALDI-TOF instruments are often insufficient for unambiguous sequence characterization. Described here is the implementation of existing advanced analytical technologies, including high-performance mass spectrometry (LTQ-Orbitrap XL-ETD) and a chip-based nanoelectrospray autosampling robot (TriVersa NanoMate), for the thorough identification and characterization of breakdown products derived from a force-degraded monoclonal antibody. Many anticipated breakdown products were identified, including Fab fragment (48,325 Da) and heavy chain polypeptide hydrolysis product (15,521 Da). Using high-resolution collisionally induced and electron transfer dissociation methods, additional identifications were made with specific localization of unpredicted modifications. As examples, a modified Fab fragment (N- and C-terminal cyclization, 47,902 Da) and a hydrolyzed free light chain impurity components (23,191 Da) were identified with a high degree of confidence (E value, <1e-5). This work describes the approach for top-down characterization of breakdown products and is readily applicable to additional monoclonal antibodies (mAb) characterization experiments, including charge isoform characterization and aggregate analysis, for a more thorough understanding of therapeutic mAb drug products.

Demonstrating ß-glucan and yeast peptide clearance in biopharmaceutical downstream processes.

The use of yeast- and plant-derived hydrolysates in cell culture production processes has sparked concerns over the potential immunogenicity risk posed by ß-glucans and yeast peptides contained in these raw materials. This article utilizes a combination of in-process testing from large-scale manufacturing and scale-down spiking studies to demonstrate the clearance of ß-glucans and yeast peptides through chromatographic steps in the downstream purification process for a monoclonal antibody. ß-Glucans were found to flow through most all three modes of chromatography (Protein A, cation and anion exchange) without binding to the resins or the product. Protein A affinity chromatography was found to provide the best clearance factor. The efficacy of the resin sanitization and storage procedures to prevent carryover from one run to the next was also demonstrated. Yeast peptides were found to be metabolized during the cell culture process and were undetectable after the Protein A purification step. The data presented here serve to allay concerns about the use of hydrolysates in cell culture production. The methodology presented here provides a template to demonstrate clearance of ß-glucans and yeast peptides through chromatographic steps in downstream processing.

Assessment of cosmetic outcome after laparoscopic cholecystectomy among women 4 years after laparoscopic cholecystectomy: is there a problem?

BACKGROUND: Advocates of single-port laparoscopic cholecystectomy (SPLC) claim that improved cosmetic outcome is one of its main benefits over conventional laparoscopic cholecystectomy (CLC). However, the published data quantifying the cosmetic outcome after CLC is sparse. This study aimed to determine the cosmetic outcome after CLC using a validated scar assessment tool. METHODS: The patient scar assessment questionnaire was sent to all women ages 20-50 years who had undergone CLC at the Norfolk and Norwich University Hospital (Norwich), the Homerton Hospital (London), and the Musgrove Park Hospital (Taunton) in 2005 (n = 380). In all cases, the operation had been performed using a four-port technique. The patients were asked to give scores related to the appearance and symptoms associated with the scars at the time the questionnaire was completed. RESULTS: Of the 380 patients, 195 responded to the questionnaire, giving a response rate of 51%. The median age of the responders was 39 years, and 63 (32%) of them had undergone previous surgery. The mean score for each section was low, indicating a favorable cosmetic outcome. This correlated with the global question answered with "excellent" for 4 of 5 categories and "good" for the remaining category. Nine patients highlighted dissatisfaction with the umbilical incision. CONCLUSIONS: Patients perceive the cosmetic results after CLC as excellent. Therefore, SPLC seems to have a limited role in terms of improving cosmesis for patients undergoing cholecystectomy. Anecdotal evidence from the questionnaire suggests that the umbilical port may be the site of problems for some patients. Further investigation is needed to determine whether this is significant, especially because it may be exaggerated after SPLC.

Reduced phenytoin levels in an epileptic patient following Roux-En-Y gastric bypass for obesity.

Despite widespread uptake of bariatric procedures for severe obesity, changes in pharmacodynamics after surgery are poorly understood. We report an epileptic patient who had a seizure following gastric bypass, although he had been asymptomatic for 30 years and without any change in his treatment. Phenytoin levels were undetectable despite a high dose. Drugs with a narrow therapeutic range such as phenytoin should be prescribed with caution after bariatric surgery.

Remission of type 2 diabetes after gastric bypass and banding: mechanisms and 2 year outcomes.

OBJECTIVE: To investigate the rate of type 2 diabetes remission after gastric bypass and banding and establish the mechanism leading to remission of type 2 diabetes after bariatric surgery. SUMMARY BACKGROUND DATA: Glycemic control in type 2 diabetic patients is improved after bariatric surgery. METHODS: In study 1, 34 obese type 2 diabetic patients undergoing either gastric bypass or gastric banding were followed up for 36 months. Remission of diabetes was defined as patients not requiring hypoglycemic medication, fasting glucose below 7 mmol/L, 2 hour glucose after oral glucose tolerance test below 11.1 mmol/L, and glycated haemoglobin (HbA1c) <6%. In study 2, 41 obese type 2 diabetic patients undergoing either bypass, banding, or very low calorie diet were followed up for 42 days. Insulin resistance (HOMA-IR), insulin production, and glucagon-like peptide 1 (GLP-1) responses after a standard meal were measured. RESULTS: In study 1, HbA1c as a marker of glycemic control improved by 2.9% after gastric bypass and 1.9% after gastric banding at latest follow-up (P < 0.001 for both groups). Despite similar weight loss, 72% (16/22) of bypass and 17% (2/12) of banding patients (P = 0.001) fulfilled the definition of remission at latest follow-up. In study 2, within days, only bypass patients had improved insulin resistance, insulin production, and GLP-1 responses (all P < 0.05). CONCLUSIONS: With gastric bypass, type 2 diabetes can be improved and even rapidly put into a state of remission irrespective of weight loss. Improved insulin resistance within the first week after surgery remains unexplained, but increased insulin production in the first week after surgery may be explained by the enhanced postprandial GLP-1 responses.

Acute giant gastric volvulus causing cardiac tamponade.

INTRODUCTION: Acute gastric volvulus is an uncommon condition which is rarely associated with cardiac impairment. DISCUSSION: We report a patient with an acute giant gastric volvulus causing cardiac tamponade. Prompt decompression was unsuccessful and the patient died prior to definitive treatment.