Increased Patient Age as a Risk Factor Following Free Flap Reconstruction after Breast Cancer: A Single Institutional Review of 2,598 Cases.

BACKGROUND: Autologous breast reconstruction (ABR) after mastectomy is increasing due to benefits over implant-based reconstruction. However, free flap reconstruction is not universally offered to patients of advanced age due to perceived increased perioperative risk. METHODS: Patients undergoing free flap breast reconstruction at our institution from 2005 to 2018 were included. Risk-adjusted logistic regression models were fit while controlling for demographic and comorbid characteristics to determine the association of age with the probability of venous thromboembolism (VTE), delayed healing, skin necrosis, surgical site infection (SSI), seroma, hematoma, hernia, and flap loss. Linear predictions from risk-adjusted logistic regression models were used to create spline curves and determine the risk of outcomes associated with age. RESULTS: A cohort of 2,598 patients underwent free flap breast reconstruction in the period examined. The median age was 51 with approximately 9% of patients being 65 or older. Increased age was associated with a greater risk of delayed healing, skin necrosis, and hematoma after surgery. There was no increased risk of medical complications such as VTE or complications such as flap loss, seroma, or SSI. CONCLUSION: A set age cutoff for patients undergoing free flap breast reconstruction does not appear warranted. There is no difference in major surgical complications such as flap loss with increasing age. However, older age does predispose patients to specific wound complications such as hematoma, skin necrosis, and delayed wound healing, which should guide preoperative counseling. Further, medical complications do not increase with advanced age. Overall, however, the safety of ABR in older patients appears uncompromised.

Extracorporeal Membrane Oxygenation-Associated Compartment Syndrome: Review of a National Database.

INTRODUCTION: Extracorporeal membrane oxygenation (ECMO)-associated compartment syndrome (CS) is a rare complication seen in critically ill patients. The epidemiology and management of ECMO-associated CS in the upper extremity (UE) and lower extremity (LE) are poorly defined in the literature. We sought to determine the epidemiology and characterize treatment and outcomes of UE-CS compared to LE-CS in the setting of ECMO therapy. METHODS: Adult patients undergoing ECMO therapy were identified in the Nationwide Readmission Database (2015-2019) and followed up for 6 months. Patients were stratified based on UE-CS versus LE-CS. Primary outcomes were fasciotomy and amputation. All-cause mortality and length of stay were also collected. Risk-adjusted modeling was performed to determine patient- and hospital-level factors associated with differences in the management UE-CS versus LE-CS while controlling for confounders. RESULTS: A total of 24,047 cases of ECMO during hospitalization were identified of which 598 were complicated by CS. Of this population, 507 cases were in the LE (84.8%), while 91 (15.5%) were in the UE. After multivariate analysis, UE-CS patients were less likely to undergo fasciotomy (50.5 vs. 70.9; P = 0.013) and were less likely to undergo amputation of the extremity (3.3 vs. 23.7; P = 0.001) although there was no difference in mortality (58.4 vs. 65.4; P = 0.330). CONCLUSIONS: ECMO patients with CS experience high mortality and morbidity. UE-CS has lower rates of fasciotomy and amputations, compared to LE-CS, with similar mortality. Further studies are needed to elucidate the reasons for these differences.

Factors associated with late surgical correction of craniosynostosis: A decade-long review of the United States nationwide readmission database.

Late-repair craniosynostosis (LRC), defined as craniosynostosis surgery beyond 1 year of age, is often associated with increased complexity and potential complications. Our study analyzed data from the 2010-2019 Nationwide Readmissions Database to investigate patient factors related to LRC. Of 10 830 craniosynostosis repair cases, 17% were LRC. These patients were predominantly from lower-income families and had more comorbidities, indicating that socioeconomic status could be a significant contributor. LRC patients were typically treated at teaching hospitals and privately owned investment institutions. Our risk-adjusted analysis revealed that LRC patients were more likely to belong to the lowest-income quartile, receive treatment at privately owned investment hospitals, and use self-payment methods. Despite these challenges, the hospital stay duration did not significantly differ between the two groups. Interestingly, LRC patients faced a higher predicted mean total cost compared with those who had surgery before turning 1. This difference in cost did not translate to a longer length of stay, further emphasizing the complexity of managing LRC. These findings highlight the urgent need for earlier intervention in craniosynostosis cases, particularly in lower-income communities. The medical community must strive to improve early diagnosis and treatment strategies in order to mitigate the socioeconomic and health disparities observed in LRC patients.

Obesity as a Risk Factor in Cosmetic Abdominal Body Contouring: A Systematic Review and Meta-Analysis.

BACKGROUND: The incidence of obesity is on the rise around the globe. Outside of the massive weight loss (MWL) patient population, knowledge of risk factors associated with abdominal body contouring (BC) is limited. This systematic review and meta-analysis assesses the impact of obesity has on cosmetic abdominal BC outcomes. METHODS: A systematic review conducted in accordance with PRISMA 2020 was done. PubMed, Embase, Scopus, and COCHRANE databases were reviewed under search syntax "obesity," "abdominoplasty," "panniculectomy," and "body contouring" for articles. Cosmetic was defined as abdominoplasty or panniculectomy outside the context of MWL. Obesity was defined as BMI ≥ 30 kg/m2. Studies reporting postoperative outcomes with less than 50% of their population involving MWL patients were included. Postoperative outcomes were assessed by pooled analysis and meta-analysis. RESULTS: Of 3088 initial studies, 16 met inclusion criteria, and nine were used for pooled and meta-analysis. Meta-analysis demonstrated that obesity was associated with more seromas (OR 1.45, 1.06-1.98, p = 0.02), hematomas (OR 2.21, 1.07-4.57, p = 0.03), and total surgical site occurrences (OR 1.99, 1.30-3.04, p = 0.0016). There was no significant difference in odds of any other complications. Analysis by obesity class showed no significant increase in odds in seromas or wound dehiscence. CONCLUSIONS: This review demonstrates obesity increased odds of postoperative complications following cosmetic abdominal BC. However, risk of complications does not continue to increase with higher obesity class. A BMI ≥ 30 kg/m2 should not be a strict contraindication to cosmetic abdominal BC. Instead, plastic surgeons should evaluate patients on a case-by-case basis. LEVEL OF EVIDENCE III: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

The Impact of Breast Cancer Type, Staging, and Treatment on Vascular Complications of Immediate Free-Flap Breast Reconstruction.

BACKGROUND: Patients with advanced cancer staging have a greater risk of developing venous thromboembolism than noncancer patients. The impact of breast cancer stage and treatment on outcomes after autologous free-flap breast reconstruction (ABR) is not well-established. The objective of this retrospective study is to determine the impact of breast cancer characteristics, such as cancer stage, hormone receptor status, and neoadjuvant treatments, on vascular complications of ABR. METHODS: A retrospective review was conducted examining patients who underwent ABR from 2009 to 2018. Breast cancer stage, cancer types, hormone receptor status, and treatments were collected in addition to demographic data. Intraoperative vascular concerns, postoperative vascular concerns, and flap loss were analyzed. Univariate analysis and fixed-effects models were used to associate breast cancer characteristics with outcomes. RESULTS: Neoadjuvant hormone therapy was associated with increased risk for intraoperative vascular concern (odds ratio, 1.059 [ P = 0.0441]). Neoadjuvant trastuzumab was associated with decreased risk of postoperative vascular concern (odds ratio, 0.941 [ P = 0.018]). Breast cancer stage, somatic genetic mutation, receptor types, neoadjuvant chemotherapy, and neoadjuvant radiation had no effect on any vascular complications of ABR. CONCLUSION: Autologous free-flap breast reconstruction is a reliable reconstructive option for patients with all stages and types of breast cancer. There is potentially increased risk of intraoperative microvascular compromise in patients who have neoadjuvant hormone therapy. Trastuzumab is potentially protective against postoperative microvascular compromise. Patients should feel confident that, despite higher stage cancer, they can pursue their desired reconstructive option without fear of vascular compromise.

Integration and Implementation of Patient-Reported Outcomes: A Prospective, Observational Clinical Quality Improvement Study.

BACKGROUND: Implementation of patient-reported outcomes (PROs) represents a critical barrier to their widespread use and poses challenges to workflow and patient satisfaction. The authors sought to implement PRO surveys into surgical practice and identify principles for successful and broader implementation. METHODS: Outpatient surgical encounters from 2016 through 2019 related to hernia, breast surgery, or postbariatric body contouring were assessed with the Abdominal Hernia-Q, BREAST-Q, or BODY-Q surveys, respectively. Outcomes were implementation rates per quarter and time to optimal implementation (≥80%). Successful implementation principles were identified during the first implemented PRO instrument and applied to subsequent ones. Logistic regression models were used to estimate increase in rate of implementation per quarter by instrument controlling for clinic volume. Risk-adjusted generalized linear models determined predicted mean differences in total clinic time and patient satisfaction. RESULTS: A total of 1206 encounters were identified. The overall survey implementation rate increased from 15% in the first quarter to 90% in the last quarter ( P < 0.01). Abdominal Hernia-Q optimal implementation was reached by 15 months. Principles for successful implementation of PROs were workflow optimization, appropriate patient selection, staff engagement, and electronic survey integration. Consistent application of these principles optimized time to optimal implementation for BREAST-Q [9 months; 18.1% increase in implementation per quarter (95% CI, 1.5 to 37.5); P < 0.01] and BODY-Q [3 months; 56.3% increase in implementation per quarter (95% CI, 26.8 to 92.6); P = 0.03]. Neither patient clinic time ( P = 0.16) nor patient satisfaction differed during the implementation of PROs process ( P = 0.98). CONCLUSIONS: Prospective implementation of PROs can be achieved in surgical practice without an adverse effect on patient satisfaction or workflow. The proposed principles of implementation may be used to optimize efficiency for implementation of PROs.

Impact of Panniculectomy and/or Abdominoplasty on Quality of Life: A Retrospective Cohort Analysis of Patient-Reported Outcomes.

BACKGROUND: Panniculectomy and abdominoplasty are uptrending procedures to address excess skin after weight loss which affects patient-reported quality of life. The authors aimed to identify factors associated with low preoperative quality of life, quantify the benefit of these procedures, and evaluate benefits across grades of obesity. METHODS: Patients seeking panniculectomy and abdominoplasty between 2018 and 2019 with a completed preoperative BODY-Q questionnaire were identified. Stratification by quality of life in tertiles for each BODY-Q domain allowed identification of characteristics associated with low quality of life using chi-square tests. Wilcoxon signed-rank tests were used to compare preoperative to postoperative change in quality of life. Differences in quality of life by obesity class (1-2 vs. 3) were ascertained using chi-square tests. RESULTS: A total of 183 patients completed preoperative quality-of-life questionnaires. Preoperative factors associated with low quality of life included age older than 40 years, Black race, public insurance, hypertension, and American Society of Anesthesiologists class (all p < 0.05). Of patients who completed a preoperative BODY-Q and underwent surgery, 46 (63 percent) completed both surveys. Quality of life improved postoperatively across all domains ( p < 0.01). The presence of a surgical site occurrence (e.g., infection, delayed healing, hematoma, seroma) did not impact postoperative quality of life in any domain ( p > 0.05). Obesity classification did not affect change in quality of life preoperatively to postoperatively ( p > 0.05). CONCLUSION: Quality of life is significantly lower at baseline in older, Black, publicly insured patients, and multimorbid patients, but improves dramatically after panniculectomy and abdominoplasty regardless of incidence of complications or degree of obesity. . CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.

The Impact of a Single Dual-Trained Surgeon in the Management of Mastectomy and Reconstruction.

BACKGROUND: Oncoplastic breast surgery is typically performed using a two-surgeon, two-team approach. The authors present their experience with patients undergoing mastectomy and immediate reconstruction performed by a single, dual-trained breast surgical oncologist and plastic and reconstructive microsurgeon. METHODS: Patients who underwent mastectomy and/or immediate reconstruction performed by the senior author between 2015 and 2019 were divided into single-surgeon or dual-surgeon cohorts, and matched by age, body mass index, reconstruction type, and cancer stage. RESULTS: The authors included 158 patients in their analysis (single-surgeon, n = 45; dual-surgeon, n = 113). Single-surgeon patients underwent surgery 13.2 days earlier than dual-surgeon patients (p < 0.01), and required significantly fewer preoperative (1.9 versus 3.4; p < 0.01) and postoperative visits (6.8 versus 10.7; p < 0.01). Operative duration was comparable (single-surgeon, 245 minutes; dual-surgeon, 245 minutes; p = 0.99). The authors found no significant difference in surgical-site infection, seroma, hematoma, abdominal donor-site healing, or flap and prosthesis loss between the groups. The authors did find that dual-surgeon patients had a significantly higher rate of mastectomy flap necrosis (20 percent versus 4 percent; p = 0.01), which held true on logistic regression when controlling for other variables. BREAST-Q data demonstrated that single-surgeon patients had significantly higher overall scores (p = 0.04), and were significantly more satisfied with their outcomes, surgeon, and the information provided (p = 0.03, p = 0.03, and p = 0.01, respectively). CONCLUSIONS: The single-surgeon approach has the potential to decrease patient burden by requiring fewer preoperative and postoperative visits without compromising surgical outcomes or oncologic safety. Further investigation is warranted into the financial implications and patient outcomes. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, III.

A Comparative Analysis of Fleur-de-Lis and Traditional Panniculectomy after Bariatric Surgery.

INTRODUCTION: The Fleur-de-Lis panniculectomy (FdL) adds a vertical component to correct complex contour deformities after massive weight loss by addressing supra-umbilical horizontal skin excess which is not addressed with infra-umbilical transverse panniculectomy (TP). We aim to perform a head-to-head comparison of clinical outcomes and patient reported outcomes (PROs) between FdL and TP. METHODS: A retrospective review of patients ≥18 with a history of bariatric surgery undergoing FdL or TP by a single plastic surgeon between 07/01/2015 and 05/31/2020 was conducted. Surgical site occurrences (SSOs) including surgical site infection (SSI), delayed healing, cellulitis, seroma, hematoma, surgical site occurrences requiring procedural intervention (SSOpi), and a composite of other postoperative outcomes were assessed. In addition, patient satisfaction was analyzed using the Body-Q questionnaire across 12 domains. RESULTS: The analysis included 56 patients; 26 and 30 patients who were treated with FdL and TP, respectively. Cohorts were statistically similar in terms of age, gender, BMI, and co-morbid conditions. The overall complication rate was 50% (FdL n = 14 (53.9%), TP n = 14 (46.7%); p = 0.592). On multivariate analysis, pannus weight was associated with the development of SSO (p = 0.04). FdL incision, however, was not an independent risk factor for adverse outcomes. Absolute improvement in PROs was similar in both cohorts across multiple domains. CONCLUSION: FdL showed a comparable safety and efficacy profile to TP when performed in post-bariatric surgery patients, with equivalent improvement in PROs across multiple domains. Preoperative weight loss should be encouraged in this population as pannus weight is an independent risk factor for complications. LEVEL OF EVIDENCE III: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Contemporary treatment of keloids: A 10-year institutional experience with medical management, surgical excision, and radiation therapy.

INTRODUCTION: We evaluate a single center's, decade-long experience utilizing 3 approaches to keloid treatment: corticosteroid medical management (MM), surgical excision (SE), and surgical excision + radiation therapy (SE + RT). STUDY DESIGN: Patients undergoing keloid treatment were identified (2008-2017). Outcomes were symptomatology/cosmesis for MM, and recurrence and complications for SE and SE + RT. Logistic regression was used to determine factors associated with recurrence and complications. RESULTS: 284 keloids (95 MM, 94 SE, 95 S E + RT) corresponded to patients with a median age of 39.1 (IQR: 26.1-53), 68.1% Black, and followed-up for 15.4 months (IQR: 5.6-30.7). For MM, 84.6% and 72.5% reported improvement in cosmesis and symptoms, respectively. SE and SE + RT recurrence were 37.2 and 37.9%, respectively. In adjusted analyses, higher radiation doses were associated with decreased recurrence whereas male gender (OR 3.3) and postoperative steroids (OR 9.5) were associated with increased recurrence (p < 0.01). There were more complications in the SE + RT group. CONCLUSIONS: MM resulted in at least some improvement. Recurrence rates after SE and SE + RT were similar. Female sex is protective, race does not affect outcomes.

Peptide Isolation via Spray Drying: Particle Formation, Process Design and Implementation for the Production of Spray Dried Glucagon.

PURPOSE: Spray drying plays an important role in the pharmaceutical industry for product development of sensitive bio-pharmaceutical formulations. Process design, implementation and optimisation require in-depth knowledge of process-product interactions. Here, an integrated approach for the rapid, early-stage spray drying process development of trehalose and glucagon on lab-scale is presented. METHODS: Single droplet drying experiments were used to investigate the particle formation process. Process implementation was supported using in-line process analytical technology within a data acquisition framework recording temperature, humidity, pressure and feed rate. During process implementation, off-line product characterisation provided additional information on key product properties related to residual moisture, solid state structure, particle size/morphology and peptide fibrillation/degradation. RESULTS: A psychrometric process model allowed the identification of feasible operating conditions for spray drying trehalose, achieving high yields of up to 84.67%, and significantly reduced levels of residual moisture and particle agglomeration compared to product obtained during non-optimal drying. The process was further translated to produce powders of glucagon and glucagon-trehalose formulations with yields of >83.24%. Extensive peptide aggregation or degradation was not observed. CONCLUSIONS: The presented data-driven process development concept can be applied to address future isolation problems on lab-scale and facilitate a systematic implementation of spray drying for the manufacturing of sensitive bio-pharmaceutical formulations.

Effect on metabolic bone disease markers in the neonatal intensive care unit with implementation of a practice guideline.

OBJECTIVES: To determine the effect of implementing a 2015 policy for the screening, prevention, and management of metabolic bone disease for very low birth weight (VLBW) infants in two Level IV NICUs. STUDY DESIGN: Retrospective cohort study of VLBW infants in the 2 years prior to (2013-2014) and after (2016-2017) policy implementation. RESULTS: We identified 316 VLBW infants in 2013-2014 and 292 in 2016-2017 who met study criteria. After policy implementation, vitamin D supplementation began earlier (20.1 ± 15.5 days vs 30.2 ± 20.1 days, p < 0.0005), the percentage of infants with alkaline phosphatase obtained increased (89.7% vs 76.3%, p < 0.0005), while the percentage of infants with alkaline phosphatase >800 IU/L (11.7 vs 4.5%, p = 0.0001) and phosphorous <4 mg/dL (14.2% vs 7.9%, p = 0.014) fell significantly. CONCLUSIONS: After policy implementation, vitamin D supplementation began significantly earlier and the rate of detecting abnormal biochemical markers of metabolic bone disease decreased significantly.

Examination of the effect of agitation on the potency of the Ebola Zaire vaccine rVSVΔG-ZEBOV-GP.

The Ebolavirus vaccine (rVSVΔG-ZEBOV-GP) is stored at -80 to -60 °C and should be kept frozen for transport. Due to significant logistical challenges, frozen transport is not feasible for some remote locations. To determine if local distribution at 2-8 °C is a potential option for these locations, a study was conducted to evaluate the impact of agitation on the thawed vaccine. After up to 7 days of constant agitation, no impact on vaccine potency was evident for the agitated vaccine versus the corresponding vaccine kept stationary at 2-8 °C. In conclusion, in-country transport of the Ebolavirus vaccine, rVSVΔG-ZEBOV-GP, at 2-8 °C appears to be a feasible option for those remote locations where significant logistical challenges prohibit transporting the vaccine at -80 to -60 °C.

The Role of a Resident Aesthetic Clinic in Addressing the Trainee Autonomy Gap.

BACKGROUND: Our institution supports a chief resident aesthetic clinic with the goal of fostering autonomy and preparedness for independent practice in a safe environment. OBJECTIVES: The aim of this study was to compare safety profiles and costs for common aesthetic procedures performed in our resident versus attending clinics. METHODS: A retrospective review was conducted of all subjects presenting for aesthetic face, breast, and/or abdominal contouring surgery at our institution from 2008 to 2017. Two cohorts were identified: subjects undergoing surgery through the chief resident versus attending clinics. Aesthetic procedures queried included: (1) blepharoplasty, rhinoplasty, or rhytidectomy; (2) augmentation mammaplasty, reduction mammaplasty, or mastopexy; (3) abdominoplasty; and (4) combination. Demographics, perioperative characteristics, costs, and postoperative complications were analyzed. RESULTS: In total, 262 and 238 subjects underwent aesthetic procedures in the resident and attending clinics, respectively. Subjects presenting to the residents were younger (P < 0.001), lower income (P < 0.001), and had fewer comorbidities (P < 0.001). Length of procedure differed between resident and attending cohorts at 181 and 152 minutes, respectively (P < 0.001), although hospital costs were not significantly increased. Total costs were higher in the attending cohort independent of aesthetic procedure (P < 0.001). Hospital readmissions (P < 0.05) and cosmetic revisions (P < 0.002) were more likely to occur in the attending physician cohort. Postoperative complications (P < 0.50) and reoperative rates (P < 0.39) were not significantly different. CONCLUSIONS: The resident aesthetic clinic provides a mechanism for increased autonomy and decision-making, while maintaining patient safety in commonly performed cosmetic procedures.

Cell confluency analysis on microcarriers by micro-flow imaging.

The productivity of cell culture-derived vaccines grown in anchorage-dependent animal cells is limited by bioreactor surface area. One way to increase the available surface area is by growing cells as monolayers on small spheres called microcarriers, which are approximately 100-250 µm in diameter. In order for microcarrier-based cell culture to be a success, it is important to understand the kinetics of cell growth on the microcarriers. Micro-flow imaging (MFI) is a simple and powerful technique that captures images and analyzes samples as they are drawn through a precision flow cell. In addition to providing size distribution and defect frequency data to compare microcarrier lots, MFI was used to generate hundreds of images to determine cell coverage and confluency on microcarriers. Same-day manual classification of these images provided upstream cell culture teams with actionable data that informed in-process decision making (e.g. time of infection). Additionally, an automated cell coverage algorithm was developed to increase the speed and throughput of the analyses.

Characterization of Propylene Glycol-Mitigated Freeze/Thaw Agglomeration of a Frozen Liquid nOMV Vaccine Formulation by Static Light Scattering and Micro-Flow Imaging.

UNLABELLED: The purpose of this work was to investigate the susceptibility of an aluminum adjuvant and an aluminum-adjuvanted native outer membrane vesicle (nOMV) vaccine formulation to freeze/thaw-induced agglomeration using static light scattering and micro-flow Imaging analysis; and to evaluate the use of propylene glycol as a vaccine formulation excipient by which freeze/thaw-induced agglomeration of a nOMV vaccine formulation could be mitigated. Our results indicate that including 7% v/v propylene glycol in an nOMV containing aluminum adjuvanted vaccine formulation, mitigates freeze/thaw-induced agglomeration. LAY ABSTRACT: We evaluated the effect of freeze-thawing on an aluminum adjuvant and an aluminum adjuvanted native outer membrane vesicle (nOMV) vaccine formulation. Specifically, we characterized the freeze/thaw-induced agglomeration through the use of static light scattering, micro-flow imaging, and cryo-electron microscopy analysis. Further, we evaluated the use of 0-9% v/v propylene glycol as an excipient which could be included in the formulation for the purpose of mitigating the agglomeration induced by freeze/thaw. The results indicate that using 7% v/v propylene glycol as a formulation excipient is effective at mitigating agglomeration of the nOMV vaccine formulation, otherwise induced by freeze-thawing.

Factor VII and protein C are phosphatidic acid-binding proteins.

Seven proteins in the human blood clotting cascade bind, via their GLA (γ-carboxyglutamate-rich) domains, to membranes containing exposed phosphatidylserine (PS), although with membrane binding affinities that vary by 3 orders of magnitude. Here we employed nanodiscs of defined phospholipid composition to quantify the phospholipid binding specificities of these seven clotting proteins. All bound preferentially to nanobilayers in which PS headgroups contained l-serine versus d-serine. Surprisingly, however, nanobilayers containing phosphatidic acid (PA) bound substantially more of two of these proteins, factor VIIa and activated protein C, than did equivalent bilayers containing PS. Consistent with this finding, liposomes containing PA supported higher proteolytic activity by factor VIIa and activated protein C toward their natural substrates (factors X and Va, respectively) than did PS-containing liposomes. Moreover, treating activated human platelets with phospholipase D enhanced the rates of factor X activation by factor VIIa in the presence of soluble tissue factor. We hypothesize that factor VII and protein C bind preferentially to the monoester phosphate of PA because of its accessibility and higher negative charge compared with the diester phosphates of most other phospholipids. We further found that phosphatidylinositol 4-phosphate, which contains a monoester phosphate attached to its myo-inositol headgroup, also supported enhanced enzymatic activity of factor VIIa and activated protein C. We conclude that factor VII and protein C bind preferentially to monoester phosphates, which may have implications for the function of these proteases in vivo.

Inhibition of tungsten-induced protein aggregation by cetyl trimethyl ammonium bromide.

TECHNICAL ABSTRACT: The purpose of this work was to investigate a potential mechanism for the inhibition of tungsten-mediated monoclonal antibody (mAb) biophysical modifications and sub-visible particle formation. A 1 mg/mL mAb formulated in 150 mM NaCl, 20 mM histidine, pH 6.0, was incubated with 1, 37, and 100 ppm of tungsten polyanions in the form of sodium tungstate both in the presence and absence of the anionic surfactant and chelating agent diethylene triamine pentaacetic acid (DTPA) or the cationic surfactant cetyl trimethyl ammonium bromide (CTAB) for 24 h at 25 °C. Assays including pH, UV-Vis spectroscopy, size exclusion chromatography, intrinsic tryptophan/tyrosine fluorescence, and micro-flow imaging were performed to assess the impact on short-term mAb stability and aggregation. We conclude that the use of micromolar concentrations of the formulation excipient and cationic surfactant CTAB equivalent to the anticipated tungsten concentration in solution effectively inhibits loss of protein concentration, fragmentation, changes in intrinsic fluorescence intensity, and the formation of sub-visible particles. LAY ABSTRACT: The purpose of this work was to investigate a potential mechanism for the inhibition of tungsten-mediated monoclonal antibody (mAb) biophysical modifications and sub-visible particle formation. A mAb formulation was incubated with tungsten polyanions in the presence and absence of the anionic surfactant and chelating agent diethylene triamine pentaacetic acid (DTPA) or the cationic surfactant cetyl trimethyl ammonium bromide (CTAB). Formulation was characterized by pH, UV-Vis spectroscopy, size exclusion chromatography, intrinsic tryptophan/tyrosine fluorescence, and micro-flow imaging. We conclude that the formulation excipient and cationic surfactant CTAB effectively inhibits biophysical modifications and sub-visible particle formation.

Influence of formulation pH and suspension state on freezing-induced agglomeration of aluminum adjuvants.

Freezing and thawing of vaccines containing aluminum adjuvants can lead to formation of aggregates and loss in vaccine potency. We sought to understand whether and to what extent the freeze-thaw damage to aluminum adjuvants would differ based on suspension state (flocculation and settlement) at the time of freezing. As flocculation and settlement characteristics of aluminum adjuvants are driven largely by the electrostatic charges on the adjuvant particles, which, in turn, are strongly influenced by the pH of the suspension, we conducted freeze-thaw studies on both Adjuphos and Alhydrogel™ samples at three pH levels (4, 6.5, and 7.2) in buffer solutions with 9% sucrose. Significantly less aggregation occurred in the buffered sucrose solutions at the pH furthest from the aluminum adjuvant point of zero charge during slow freezing at -20°C. The freezing-induced aggregation for the samples with 9% sucrose at each pH was minimal during fast freezing at -70°C and -115°C. Suspensions that were flocculated and settled to a greater extent experienced the most freeze-thaw aggregation, whereas suspensions that were frozen before significant flocculation and settlement occurred showed little or no aggregation. Because pH of formulation can affect flocculation and settling time, it indirectly affects the extent of freeze-thaw aggregation.

Molecular determinants of phospholipid synergy in blood clotting.

Many regulatory processes in biology involve reversible association of proteins with membranes. Clotting proteins bind to phosphatidylserine (PS) on cell surfaces, but a clear picture of this interaction has yet to emerge. We present a novel explanation for membrane binding by GLA domains of clotting proteins, supported by biochemical studies, solid-state NMR analyses, and molecular dynamics simulations. The model invokes a single "phospho-L-serine-specific" interaction and multiple "phosphate-specific" interactions. In the latter, the phosphates in phospholipids interact with tightly bound Ca(2+) in GLA domains. We show that phospholipids with any headgroup other than choline strongly synergize with PS to enhance factor X activation. We propose that phosphatidylcholine and sphingomyelin (the major external phospholipids of healthy cells) are anticoagulant primarily because their bulky choline headgroups sterically hinder access to their phosphates. Following cell damage or activation, exposed PS and phosphatidylethanolamine collaborate to bind GLA domains by providing phospho-L-serine-specific and phosphate-specific interactions, respectively.