Impact of fed-batch process intensification on the productivity and product quality of two CHO cell lines expressing unique novel molecular format proteins.

While monospecific antibodies have long been the foundational offering of protein therapeutics, recent advancements in antibody engineering have allowed for the development of far more complex antibody structures. Novel molecular format (NMF) proteins, such as bispecific antibodies (BsAbs), are structures capable of multispecific binding, allowing for expanded therapeutic functionality. As demand for NMF proteins continues to rise, biomanufacturers face the challenge of increasing bioreactor process productivity while simultaneously maintaining consistent product quality. This challenge is exacerbated when producing structurally complex proteins with asymmetric modalities, as seen in NMFs. In this study, the impact of a high inoculation density (HID) fed-batch process on the productivity and product quality attributes of two CHO cell lines expressing unique NMFs, a monospecific antibody with an Fc-fusion protein and a bispecific antibody, compared to low inoculation density (LID) platform fed-batch processes was evaluated. It was observed that an intensified platform fed-batch process increased product concentrations by 33 and 109% for the two uniquely structured complex proteins in a shorter culture duration while maintaining similar product quality attributes to traditional fed-batch processes.

Environmental Injustice Is Associated With Poorer Asthma Outcomes in School-Age Children With Asthma in Metropolitan Atlanta, Georgia.

BACKGROUND: Environmental justice mandates that no person suffers disproportionately from environmental exposures. The Environmental Justice Index (EJI) provides an estimate of the environmental burden for each census tract but has not yet been used in asthma populations. OBJECTIVE: We hypothesized that children from census tracts with high environmental injustice determined by the EJI would have a greater burden of asthma exacerbations, poorer asthma control, and poorer lung function over 12 months. METHODS: Children aged 6 to 18 years with asthma (N = 575) from metropolitan Atlanta, Georgia, completed a baseline research visit. Participant addresses were geocoded to obtain the EJI Social-Environmental Ranking for each participant's census tract, which was divided into tertiles. Medical records were reviewed for 12 months for asthma exacerbations. A subset of participants completed a second research visit involving spirometry and questionnaires. RESULTS: Census tracts with the greatest environmental injustice had more racial and ethnic minorities, lower socioeconomic status, more hazardous exposures (particularly to airborne pollutants), and greater proximity to railroads and heavily trafficked roadways. Children with asthma residing in high injustice census tracts had a longer duration of asthma, greater historical asthma-related health care utilization, poorer asthma symptom control and quality of life, and more impaired lung function. By 12 months, children from high injustice census tracts also had more asthma exacerbations with a shorter time to exacerbation and persistently more symptoms, poorer asthma control, and reduced lung function. CONCLUSIONS: Disparities in environmental justice are present in metropolitan Atlanta that may contribute to asthma outcomes in children. These findings require an additional study and action to improve health equity.

Altered Symptom Perception in Children With Asthma Is Associated With Poor Childhood Opportunity and Adverse Outcomes.

BACKGROUND: Effective asthma self-management requires that children recognize their asthma symptoms when they occur. However, some children have altered symptom perception, which impairs their ability to respond to their asthma symptoms in a timely manner. OBJECTIVE: To characterize the prevalence and features of altered symptom perception in children aged 5 to 18 years. We hypothesized that children with altered symptom perception would have more features of uncontrolled asthma, more health inequity, and poorer longitudinal asthma outcomes over 12 months. METHODS: Children (N = 371) completed an outpatient research visit for clinical characterization. Altered symptom perception was defined by discordance between child responses on the 6-item Asthma Control Questionnaire and medical provider-elicited symptoms. Electronic medical records were reviewed for 12 months for the occurrence of an asthma exacerbation treated with systemic corticosteroids and an asthma exacerbation prompting an emergency department visit. RESULTS: Approximately 15% of children had altered symptom perception and their asthma features were similar to those of children with uncontrolled asthma. Children with altered symptom perception were uniquely distinguished by non-White race and more severe prior exacerbations. These children also resided in ZIP codes with the poorest childhood opportunity (ie, poorest education, health and environmental features, and socioeconomic features). Outcomes of children with altered symptom perception were equally disparate with approximately 2-fold higher odds of a future exacerbation and approximately 3-fold higher odds of an emergency department visit for asthma. CONCLUSIONS: Altered symptom perception is present in a small but significant number of children with asthma and is related to poorer childhood opportunity and other health inequities that require additional intervention.

Metabolomics identifies disturbances in arginine, phenylalanine, and glycine metabolism as differentiating features of exacerbating atopic asthma in children.

Background: Asthma exacerbations are highly prevalent in children, but only a few studies have examined the biologic mechanisms underlying exacerbations in this population. Objective: High-resolution metabolomics analyses were performed to understand the differences in metabolites in children with exacerbating asthma who were hospitalized in a pediatric intensive care unit for status asthmaticus. We hypothesized that compared with a similar population of stable outpatients with asthma, children with exacerbating asthma would have differing metabolite abundance patterns with distinct clustering profiles. Methods: A total of 98 children aged 6 through 17 years with exacerbating asthma (n = 69) and stable asthma (n = 29) underwent clinical characterization procedures and submitted plasma samples for metabolomic analyses. High-confidence metabolites were retained and utilized for pathway enrichment analyses to identify the most relevant metabolic pathways that discriminated between groups. Results: In all, 118 and 131 high-confidence metabolites were identified in positive and negative ionization mode, respectively. A total of 103 unique metabolites differed significantly between children with exacerbating asthma and children with stable asthma. In all, 8 significantly enriched pathways that were largely associated with alterations in arginine, phenylalanine, and glycine metabolism were identified. However, other metabolites and pathways of interest were also identified. Conclusion: Metabolomic analyses identified multiple perturbed metabolites and pathways that discriminated children with exacerbating asthma who were hospitalized for status asthmaticus. These results highlight the complex biology of inflammation in children with exacerbating asthma and argue for additional studies of the metabolic determinants of asthma exacerbations in children because many of the identified metabolites of interest may be amenable to targeted interventions.

Automated Raman feed-back control of multiple supplemental feeds to enable an intensified high inoculation density fed-batch platform process.

Biologics manufacturing is increasingly moving toward intensified processes that require novel control strategies in order to achieve higher titers in shorter periods of time compared to traditional fed-batch cultures. In order to implement these strategies for intensified processes, continuous process monitoring is often required. To this end, inline Raman spectroscopy was used to develop partial least squares models to monitor changes in residual concentrations of glucose, phenylalanine and methionine during the culture of five different glutamine synthetase piggyBac® Chinese hamster ovary clones cultured using an intensified high inoculation density fed-batch platform process. Continuous monitoring of residual metabolite concentrations facilitated automated feed-rate adjustment of three supplemental feeds to maintain glucose, phenylalanine, and methionine at desired setpoints, while maintaining other nutrient concentrations at acceptable levels across all clones cultured on the high inoculation density platform process. Furthermore, all clones cultured on this process achieved high viable cell concentrations over the course of culture, indicating no detrimental impacts from the proposed feeding strategy. Finally, the automated control strategy sustained cultures inoculated at high cell densities to achieve product concentrations between 5 and 8.3 g/L over the course of 12 days of culture.

Social determinants of health influence preschool and caregiver experiences during symptoms and exacerbations of wheezing.

BACKGROUND: Social determinants of health have been inadequately studied in preschool children with wheezing and their caregivers but may influence the care received. OBJECTIVE: To evaluate the symptom and exacerbation experiences of wheezing preschool children and their caregivers, stratified by risk of social vulnerability, over 1 year of longitudinal follow-up. METHODS: A total of 79 caregivers and their preschool children with recurrent wheezing and at least 1 exacerbation in the previous year were stratified by a composite measure of social vulnerability into "low" (N = 19), "intermediate" (N = 27), and "high" (N = 33) risk groups. Outcome measures at the follow-up visits included child respiratory symptom scores, asthma control, caregiver-reported outcome measures of mental and social health, exacerbations, and health care utilization. The severity of exacerbations reflected by symptom scores and albuterol use and exacerbation-related caregiver quality of life were also assessed. RESULTS: Preschool children at high risk of social vulnerability had greater day-to-day symptom severity and more severe symptoms during acute exacerbations. High-risk caregivers were also distinguished by lower general life satisfaction at all visits and lower global and emotional quality of life during acute exacerbations which did not improve with exacerbation resolution. Rates of exacerbation or emergency department visits did not differ, but intermediate- and high-risk families were significantly less likely to seek unscheduled outpatient care. CONCLUSION: Social determinants of health influence wheezing outcomes in preschool children and their caregivers. These findings argue for routine assessment of social determinants of health during medical encounters and tailored interventions in high-risk families to promote health equity and improve respiratory outcomes.

Poorer Caregiver Mental and Social Health Is Associated With Worse Respiratory Outcomes in Preschool Children With Recurrent Wheezing.

BACKGROUND: Mental and social health in caregivers of preschool children has been inadequately studied, but it may influence respiratory symptom recognition and management. OBJECTIVE: To identify preschool caregivers at highest risk for poor mental and social health outcomes on the basis of patient-reported outcome measures. METHODS: Female caregivers 18 to 50 years old (N = 129) with a preschool child aged 12 to 59 months with recurrent wheezing and at least 1 exacerbation in the previous year completed 8 validated patient-reported outcome measures of mental and social health. k-means cluster analysis was performed using the T score for each instrument. Caregiver/child dyads were followed for 6 months. Primary outcomes included caregiver quality of life and wheezing episodes in their preschool children. RESULTS: Three clusters of caregivers were identified: low risk (n = 38), moderate risk (n = 56), and high risk (n = 35). The high-risk cluster had the lowest life satisfaction, meaning and purpose, and emotional support and the highest social isolation, depression, anger, perceived stress, and anxiety that persisted for more than 6 months. This cluster had the poorest quality of life and marked disparities in social determinants of health. Preschool children from caregivers in the high-risk cluster had more frequent respiratory symptoms and a higher occurrence of any wheezing episode, but a lower outpatient physician utilization for wheezing management. CONCLUSIONS: Caregiver mental and social health is associated with respiratory outcomes in preschool children. Routine assessment of mental and social health in caregivers is warranted to promote health equity and improve wheezing outcomes in preschool children.

Outcomes of children with life-threatening status asthmaticus requiring isoflurane therapy and extracorporeal life support.

BACKGROUND: Severe, refractory asthma is a life-threatening emergency that may be treated with isoflurane and extracorporeal life support. The objective of this study was to describe the clinical response to isoflurane and outcomes after discharge of children who received isoflurane and/or extracorporeal life-support for near-fatal asthma. METHODS: This was a retrospective descriptive study using electronic medical record data from two pediatric intensive care units within a single healthcare system in Atlanta, GA. RESULTS: Forty-five children received isoflurane, and 14 children received extracorporeal life support, 9 without a trial of isoflurane. Hypercarbia and acidosis improved within four hours of starting isoflurane. Four children died during the index admission for asthma. Twenty-seven percent had a change in Functional Status Score of three or more points from baseline to PICU discharge. Patients had median percent predicted FEV1 and FEV1/FVC ratios pre- and post-bronchodilator values below normal pediatric values. CONCLUSION: Children who received isoflurane and/or ECLS had a high frequency of previous PICU admission and intubation. Improvement in ventilation and acidosis occurred within the first four hours of starting isoflurane. Children who required isoflurane or ECLS may develop long-lasting deficits in their functional status. Children with near-fatal asthma are a high-risk group and require improved follow-up in the year following PICU discharge.

Continuous background correction of refractive index signal to improve monoclonal antibody concentration monitoring during UF/DF and SPTFF operations.

Inline refractive index (RI) has the potential for monitoring protein concentration during final bulk concentration. While useful for monitoring and controlling product concentration, RI is sensitive to the respective background buffer being used for processing. This raises concerns around variations in buffer preparations, and during diafiltration where the buffer background is a mixture of different buffers during exchange. This study evaluated whether the use of a RI probe in the permeate line could facilitate continuous background subtraction (dual RI) and improve concentration monitoring during ultrafiltration/diafiltration and single pass TFF concentration for IgG1 and IgG4 antibodies. The proposed dual RI strategy yielded reductions in % error compared to the use of a single refractive index estimate from the retentate line (6.18% vs 8.63% for IgG4 and 2.65% vs 8.85% for IgG1) during traditional ultrafiltration/diafiltration. The improvement in IgG estimates were best during diafiltration where the continuous background subtraction of the permeate RI-enabled continuous monitoring of antibody material without knowledge of what the background buffer was compared to the use of a single RI estimate (6.47% vs 10.79% for IgG4 and 3.29% vs 19.59% for IgG1). In contrast minimal improvement to accuracy was obtained when using SPTFF as a concentration step. The ability to monitor product concentration changes via the proposed dual RI approach removes the need for complex calibrations, minimal worry about changing buffer backgrounds during diafiltration, and could enable better process control during product concentration in the cGMP manufacture of biologics.

Geospatial Analysis of Social Determinants of Health Identifies Neighborhood Hot Spots Associated With Pediatric Intensive Care Use for Life-Threatening Asthma.

BACKGROUND: Social determinants of health are associated with asthma prevalence and healthcare use in children with asthma, but are multifactorial and complex. Whether social determinants similarly influence exacerbation severity is not clear. OBJECTIVE: Composite measures of social determinants of health and readmission outcomes were evaluated in a large regional cohort of 1,403 school-age children admitted to a pediatric intensive care unit (PICU) for asthma. METHODS: Residential addresses were geocoded and spatially joined to census tracts. Composite measures of social vulnerability and childhood opportunity, PICU readmission rates, and hospital length of stay were compared between neighborhood hot spots, where PICU admission rates per 1,000 children are at or above the 90th percentile, versus non-hot spots. RESULTS: A total of 228 children resided within a neighborhood hot spot (16%). Hot spots were associated with a higher (ie, poorer) composite Social Vulnerability Index ranking, reflecting differences in socioeconomic status, household composition and disability, and housing type and transportation. Hot spots also had a lower (ie, poorer) composite Childhood Opportunity Index percentile ranking, reflecting differences in the education, health and environment, and social and economic domains. Higher social vulnerability and lower childhood opportunity were associated with PICU readmission. Residing within a hot spot was further associated with a longer duration of hospital stay, individual inpatient bed days, and total census tract inpatient bed days. CONCLUSIONS: Social determinants of health identified by geospatial analyses are associated with more severe asthma exacerbation outcomes in children. Outpatient strategies that address both biological and social determinants of health are needed to care for and prevent PICU admissions optimally in children with asthma.

Simulation-based User-centered Design: An Approach to Device Development during COVID-19.

INTRODUCTION: Since the onset of COVID-19, intubations have become very high risk for clinical teams. Barrier devices during endotracheal intubation protect clinicians from the aerosols generated. Simulation-based user-centered design (UCD) was an iterative design process used to develop a pediatric intubation aerosol containment system (IACS). Simulation was anchored in human factor engineering and UCD to better understand clinicians' complex interaction with the IACS device, elicit user wants and needs, identify design inefficiencies, and unveil safety concerns. METHODS: This study was a prospective observational study of a simulation-based investigation used to design a pediatric IACS rapidly. Debriefing and Failure Mode and Effect Analysis identified latent conditions related to 5 device prototypes. Design iterations made were based on feedback provided to the engineering team after each simulation. RESULTS: Simulation identified 32 latent conditions, resulting in 5 iterations of the IACS prototype. The prototypes included an (1) intubation box; (2) IACS shield; (3) IACS frame with PVC pipes; (4) IACS plexiglass frame, and finally, (5) IACS frame without a plexiglass top. CONCLUSIONS: Integration of simulation with human factor ergonomics and UCD, in partnership with mechanical engineers, facilitated a novel context to design and redesign a pediatric IACS to meet user needs and address safety concerns.

Feedback control of two supplemental feeds during fed-batch culture on a platform process using inline Raman models for glucose and phenylalanine concentration.

The use of Raman models for glucose and phenylalanine concentrations to provide the signal for a control algorithm to continuously adjust the feed rate of two separate supplemental feeds during the fed-batch culture of a CHOK1SV GS-KO® cell line in a platform process was evaluated. Automated feed rate adjustment of the glucose feed using a Raman model for glucose concentration, maintained the glucose concentration within the desired target (average deviation ± 0.49 g/L). Automated feed rate adjustment of the nutrient feed using a Raman model for phenylalanine concentration, maintained phenylalanine concentrations within the target (average deviation ± 29.97 mg/L). The novel use of a Raman model for phenylalanine concentration, combined with a Raman model for glucose concentration, to maintain target glucose and phenylalanine concentrations through feed-rate adjustments, reduced the average cumulative glucose and nutrient feed additions (19% and 27% respectively) compared to manually adjusted cultures. Additionally, the proposed automation strategy led to lower osmolality during culture, maintained the nutrient environment more consistently, and achieved higher harvest product concentration (≈ 20% higher) compared to typical fed-batch process control for the cell line and platform process evaluated. Furthermore, the proposed feeding strategy yielded similar glycosylation and charge variant profiles compared to manually adjusted fed-batch process control. The ability to continuously adjust the feed rate addition of two separate feeds in this manner helps enable a shift away from the current daily offline sampling needed to control fed-batch mammalian cell culture during clinical and commercial manufacturing on platform processes.

Differential type I interferon response and primary airway neutrophil extracellular trap release in children with acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) is a heterogeneous condition characterized by the recruitment of large numbers of neutrophils into the lungs. Neutrophils isolated from the blood of adults with ARDS have elevated expression of interferon (IFN) stimulated genes (ISGs) associated with decreased capacity of neutrophils to kill Staphylococcus aureus and worse clinical outcomes. Neutrophil extracellular traps (NETs) are elevated in adults with ARDS. Whether pediatric ARDS (PARDS) is similarly associated with altered neutrophil expression of ISGs and neutrophil extracellular trap release is not known. Tracheal aspirate fluid and cells were collected within 72 h from seventy-seven intubated children. Primary airway neutrophils were analyzed for differential ISG expression by PCR, STAT1 phosphorylation and markers of degranulation and activation by flow cytometry. Airway fluid was analyzed for the release of NETs by myeloperoxidase-DNA complexes using an ELISA. Higher STAT1 phosphorylation, markers of neutrophil degranulation, activation and NET release were found in children with versus without PARDS. Higher NETs were detected in the airways of children with ventilator-free days less than 20 days. Increased airway cell IFN signaling, neutrophil activation, and NET production is associated with PARDS. Higher levels of airway NETs are associated with fewer ventilator-free days.

Development of generic raman models for a GS-KOTM CHO platform process.

The monitoring and control of bioprocesses is of the utmost importance in order to provide a consistent, safe, and high-quality product for consumers. Current monitoring and control schemes rely on infrequent and time consuming offline sampling methods, which inherently leads to some variability in the process which may impact the product quality profile. As part of Lonza's dedication to process analytical technology (PAT) initiatives this study evaluated the ability to generate generic calibration models, which are independent of the cell line, using Raman probes to monitor changes in glucose, lactate, glutamate, ammonium, viable cell concentration (VCC), total cell concentration (TCC) and product concentration. Calibration models were developed from cell culture using two different CHOK1SV GS-KOTM cell lines producing different monoclonal antibodies (mAbs). Developed predictive models, measured changes in glucose, lactate, ammonium, VCC, and TCC with average prediction errors of 0.44, 0.23, 0.03 g L-1 , 1.90 × 106 cells mL-1 , and 1.85 × 106 cells mL-1 , respectively over the course of cell culture with minimal cell line dependence. The development of these generic models allows the application of spectroscopic PAT techniques in clinical and commercial manufacturing environments, where processes are typically run once or twice in GMP manufacturing based on a common platform process. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:730-737, 2018.