Demonstration of Physicochemical and Functional Similarity of Biosimilar Pegfilgrastim-cbqv to Pegfilgrastim.

BACKGROUND: Pegfilgrastim-cbqv/CHS-1701 (UDENYCA®) (hereafter referred to as pegfilgrastim-cbqv) was approved in 2018 by the US Food and Drug Administration as a biosimilar for pegfilgrastim (Neulasta®) (hereafter referred to as pegfilgrastim). Both pegfilgrastim-cbqv and pegfilgrastim are conjugates of recombinant human granulocyte colony stimulating factor (r-metHuG-CSF) with a 20 kDa polyethylene glycol (PEG) indicated to decrease the incidence of infection, as manifested by febrile neutropenia, in patients receiving myelosuppressive anticancer drugs. The demonstration of analytical similarity for PEG-protein conjugates presents unique challenges since both the protein and PEG attributes must be characterized. OBJECTIVE: The current study demonstrates the analytical similarity of pegfilgrastim-cbqv and the reference product, pegfilgrastim. In addition to the physicochemical and functional characterization of the protein, the study assessed attributes specific to PEGylation including PEG size and polydispersity, site of attachment, linker composition, and PEGylation process-related variants. METHODS: The structural, functional, and stability attributes of pegfilgrastim-cbqv and pegfilgrastim were compared using state-of-the-art analytical methods. For the protein, the primary structure, disulfide structure, and secondary and tertiary structures were assessed using traditional protein characterization techniques such as mass spectrometry (MS), circular dichroism (CD), intrinsic fluorescence, and differential scanning calorimetry (DSC), as well as more advanced techniques such as two-dimensional (2D) nuclear magnetic resonance (NMR) and hydrogen deuterium exchange (HDX). For the PEG moiety, the site of attachment, occupancy, linker composition, size and polydispersity were compared using mass spectrometry (both intact and after endoprotease digestion), multiangle light scattering detection (MALS), and Edman degradation. Purity assessments included the assessment of both protein variants and PEGylation variants using chromatographic and electrophoretic analytical separation techniques. The functional similarity between pegfilgrastim-cbqv and pegfilgrastim was compared using both a cell-based bioassay and surface plasmon resonance (SPR). The degradation rates and stability profiles were compared under accelerated and stressed conditions. RESULTS: Biosimilarity was demonstrated by a thorough assessment of physiochemical and functional attributes, as well as comparative stability, of pegfilgrastim-cbqv relative to pegfilgrastim. These studies demonstrated identical primary structure and disulfide structure, highly similar secondary and tertiary structure, as well as functional similarity. The impurity profile of pegfilgrastim-cbqv was comparable to that of pegfilgrastim with only minor differences in PEGylation variants and a slight offset in the PEG molar mass. These differences were not clinically relevant. The degradation profiles were qualitatively and quantitatively similar under accelerated and stress conditions. CONCLUSION: The structural, functional, and stability data demonstrate that pegfilgrastim-cbqv is highly similar to the reference product, pegfilgrastim.

Demonstration of Physicochemical and Functional Similarity of Biosimilar Adalimumab-aqvh to Adalimumab.

BACKGROUND: Adalimumab-aqvh/CHS-1420 (YUSIMRYTM) (hereafter referred to as adalimumab-aqvh) was recently approved by the US Food and Drug Administration as a biosimilar for adalimumab. OBJECTIVE: The current study was conducted to investigate the analytical similarity of adalimumab-aqvh and the reference product, adalimumab. METHODS: The structural, functional, and stability attributes of adalimumab-aqvh and adalimumab were compared using state-of-the-art assays. The primary structure, disulfide structure, glycan profile, secondary and tertiary structures, molar mass, size variants, free thiol, charge variants, hydrophobic variants, post-translational modifications, subvisible particles, host cell proteins, and protein concentration were investigated. The functional similarity between adalimumab-aqvh and adalimumab was demonstrated by comparing fragment antigen-binding (Fab)-associated and fragment crystallizable (Fc)-associated biological activities. The stability of adalimumab-aqvh and of adalimumab was compared through forced degradation. RESULTS: The structural attributes of adalimumab-aqvh were identical to those of adalimumab or met the similarity criteria, with a few exceptions. Adalimumab-aqvh and adalimumab exhibited comparable stability profiles and functional activities. Any observed differences in the physiochemical attributes did not impact the conclusion of similarity because they did not influence any functional activities related to the adalimumab mechanism of action. CONCLUSION: The structural, functional, and stability data provide convincing evidence of biosimilarity between adalimumab-aqvh and the reference product, adalimumab.

Evaluation of an Opt-Out Protocol for Antibiotic De-Escalation in Patients With Suspected Sepsis: A Multicenter, Randomized, Controlled Trial.

BACKGROUND: Sepsis guidelines recommend daily review to de-escalate or stop antibiotics in appropriate patients. This randomized, controlled trial evaluated an opt-out protocol to decrease unnecessary antibiotics in patients with suspected sepsis. METHODS: We evaluated non-intensive care adults on broad-spectrum antibiotics despite negative blood cultures at 10 US hospitals from September 2018 through May 2020. A 23-item safety check excluded patients with ongoing signs of systemic infection, concerning or inadequate microbiologic data, or high-risk conditions. Eligible patients were randomized to the opt-out protocol vs usual care. Primary outcome was post-enrollment antibacterial days of therapy (DOT). Clinicians caring for intervention patients were contacted to encourage antibiotic discontinuation using opt-out language. If continued, clinicians discussed the rationale for continuing antibiotics and de-escalation plans. To evaluate those with zero post-enrollment DOT, hurdle models provided 2 measures: odds ratio of antibiotic continuation and ratio of mean DOT among those who continued antibiotics. RESULTS: Among 9606 patients screened, 767 (8%) were enrolled. Intervention patients had 32% lower odds of antibiotic continuation (79% vs 84%; odds ratio, 0.68; 95% confidence interval [CI], .47-.98). DOT among those who continued antibiotics were similar (ratio of means, 1.06; 95% CI, .88-1.26). Fewer intervention patients were exposed to extended-spectrum antibiotics (36% vs 44%). Common reasons for continuing antibiotics were treatment of localized infection (76%) and belief that stopping antibiotics was unsafe (31%). Thirty-day safety events were similar. CONCLUSIONS: An antibiotic opt-out protocol that targeted patients with suspected sepsis resulted in more antibiotic discontinuations, similar DOT when antibiotics were continued, and no evidence of harm. CLINICAL TRIALS REGISTRATION: NCT03517007.

Safety of endoscopist-directed nurse-administered balanced propofol sedation in patients with severe systemic disease (ASA class III).

BACKGROUND AND AIMS: The safety of endoscopist-directed nurse-administered propofol sedation (EDNAPS) has been demonstrated in low-risk patients (American Society of Anesthesiologists [ASA] class I and II). There are limited data regarding the safety of EDNAPS for endoscopic procedures in ASA class III patients. The purpose of this study was to determine the safety of EDNAPS for routine outpatient endoscopy in this population. METHODS: We retrospectively reviewed all outpatient EGDs and colonoscopies performed with EDNAPS at the University of Utah from January 2015 to November 2018. Exclusion criteria were inpatient procedures, combined procedures, ASA IV or higher, use of continuous or bilevel positive airway pressure at the start of the procedure, or procedures performed by a nongastroenterologist. Major adverse events were defined as intubation or death. Minor adverse events were defined as hypoxia, hypotension, bradycardia, or need for airway interventions. Patients were stratified by procedure type and ASA I/II status and were compared with patients with ASA III status and matched according to age, gender, and the involvement of a fellow in a 3 to 1 fashion. RESULTS: The final sample size was 18,910 colonoscopy procedures (17,205 patients) and 9178 EGD procedures (6827 patients). In both colonoscopy and EGD procedures, there were no major adverse events such as intubation, need for resuscitation, or death. The rates of any airway intervention, jaw thrust, oral nasal airway, or use of positive pressure ventilation were low in both procedure types and not different between ASA I/II and ASA III patients. CONCLUSION: EDNAPS is safe in both ASA I/II and ASA class III patients undergoing routine outpatient endoscopy.

Changes in alcohol use after the onset of spinal cord injury.

OBJECTIVE: Identify the prevalence of alcohol consumption and binge drinking at time of spinal cord injury (SCI) onset, compare these rates to data from the general population, and identify changes in alcohol use at an average of 17 months post-injury. DESIGN: Cross sectional, mailed self-report assessment. SETTING: A specialty hospital in the southeastern United States. PARTICIPANTS: Five hundred sixty-six inpatients completed the baseline measure. After eliminating those under age 18, there were 524 participants at baseline. 410 were approached for follow-up, with 201 of those responding. INTERVENTIONS: N/A. OUTCOME MEASURES: Self-reported assessments were completed during inpatient rehabilitation and at follow-up approximately 17 months later. The two primary outcomes were the number of days consuming 5 or more drinks (binge drinking) and the number of days consuming any alcoholic beverages within the 30 days prior to the assessment. Comparison data were taken from the Behavioral Risk Factor Surveillance System. RESULTS: At SCI onset, the prevalence of alcohol use, particularly binge drinking, was substantially higher than the general population (SCI = 44.9%; general population = 13%). Drinking rates decreased by 17 months post-injury. CONCLUSION: Alcohol use and binge drinking are elevated over the general population at the time of injury. Drinking patterns reflect a decrease following injury but remain slightly elevated, signifying a need for interventions to minimize long-term health consequences.

Preinjury cigarette smoking among those with traumatic spinal cord injury.

OBJECTIVE: Identify the prevalence of cigarette smoking at the time of spinal cord injury (SCI) onset, compare the rate of cigarette smoking to that of the general population in the same geographic area, and identify the relationship of cigarette smoking with demographic, injury, behavioral, and psychological characteristics. RESEARCH METHOD: Self-report assessments were completed during inpatient rehabilitation for new SCI. RESULTS: The prevalence of smokers at the time of their SCI onset was 37.9%, substantially higher than the rate for those in the general population from the same geographic region (22.8%). Those who were smokers at SCI onset, on average, were older, had less education, were more likely to have consumed alcohol in the month prior to SCI onset, and had higher personality scores indicative of Impulsive/Sensation Seeking and Neuroticism/Anxiety. CONCLUSIONS: At the time of SCI, cigarette smoking is elevated over the general population and is related to behavioral and psychological factors that may be important to consider with smoking cessation interventions.

Adoptive transfer of activated marrow-infiltrating lymphocytes induces measurable antitumor immunity in the bone marrow in multiple myeloma.

Successful adoptive T cell therapy (ACT) requires the ability to activate tumor-specific T cells with the ability to traffic to the tumor site and effectively kill their target as well as persist over time. We hypothesized that ACT using marrow-infiltrating lymphocytes (MILs) in multiple myeloma (MM) could impart greater antitumor immunity in that they were obtained from the tumor microenvironment. We describe the results from the first clinical trial using MILs in MM. Twenty-five patients with either newly diagnosed or relapsed disease had their MILs harvested, activated and expanded, and subsequently infused on the third day after myeloablative therapy. Cells were obtained and adequately expanded in all patients with anti-CD3/CD28 beads plus interleukin-2, and a median of 9.5 × 10(8) MILs were infused. Factors indicative of response to MIL ACT included (i) the presence of measurable myeloma-specific activity of the ex vivo expanded product, (ii) low endogenous bone marrow T cell interferon-γ production at baseline, (iii) a CD8(+) central memory phenotype at baseline, and (iv) the generation and persistence of myeloma-specific immunity in the bone marrow at 1 year after ACT. Achieving at least a 90% reduction in disease burden significantly increased the progression-free survival (25.1 months versus 11.8 months; P = 0.01). This study demonstrates the feasibility and efficacy of MILs as a form of ACT with applicability across many hematologic malignancies and possibly solid tumors infiltrating the bone marrow.

Stability of lyophilized sucrose formulations of an IgG1: subvisible particle formation.

Eight lyophilized formulations of a IgG1 monoclonal antibody (MAb) were prepared containing increasing levels of sucrose. In addition, three of the formulations had sorbitol added at a level of 5% w/w relative to sucrose. The samples were stored for up to 4 weeks at 40°C, which is well below the Tg. Upon reconstitution, the levels of subvisible particles were measured using microflow imaging (MFI). The formulation containing no sucrose contained exceedingly high levels of subvisible particles, accounting for as much as 25% of the weight of the protein. Addition of sucrose markedly decreased the number of subvisible particles, with the maximal sucrose:protein weight ratio being 2:1 (the highest level tested). Addition of sorbitol further decreased subvisible particle levels, even for formulations where the sucrose:protein ratio was relatively high. This suggests that even small amounts of a plasticizer like sorbitol can improve the storage stability of a lyophilized antibody formulation, probably by dampening ß-relaxations within the amorphous glass.

Structure, stability, and mobility of a lyophilized IgG1 monoclonal antibody as determined using second-derivative infrared spectroscopy.

There are many aspects of stabilization of lyophilized proteins. Of these various factors, retention of native structure, having sufficient amount of stabilizer to embed the protein within an amorphous matrix, and dampening ß-relaxations have been shown to be critical in optimizing protein stability during storage. In this study, an IgG1 was lyophilized with varying amounts of sucrose. In some formulations, a small amount of sorbitol was added as a plasticizer. The structure of the protein in dried state was monitored using infrared (IR) spectroscopy. The IR spectra indicated increasing retention of the native structure, which correlated with stability as indicated by size-exclusion chromatography as well as micro-flow imaging. Maximal stability was achieved with a 2:1 mass ratio of sucrose to protein, which is more than that would be expected based on earlier studies. Analysis of both high and low frequency bands associated with intramolecular ß-sheet structure provides additional information on the structure of antibodies in the solid state. Finally, there is a correlation between the bandwidth of the ß-sheet bands and the enthalpy of relaxation, suggesting that amide I bands can provide some indication of the degree of coupling to the sugar matrix, as well as structural heterogeneity of the protein.

Supply chain optimization for pediatric perioperative departments.

Economic challenges compel pediatric perioperative departments to reduce nonlabor supply costs while maintaining the quality of patient care. Optimization of the supply chain introduces a framework for decision making that drives fiscally responsible decisions. The cost-effective supply chain is driven by implementing a value analysis process for product selection, being mindful of product sourcing decisions to reduce supply expense, creating logistical efficiency that will eliminate redundant processes, and managing inventory to ensure product availability. The value analysis approach is an analytical methodology for product selection that involves product evaluation and recommendation based on consideration of clinical benefit, overall financial impact, and revenue implications.

Root Cause Analysis of Tungsten-Induced Protein Aggregation in Pre-filled Syringes.

Particles isolated from a pre-filled syringe containing a protein-based solution were identified as aggregated protein and tungsten. The origin of the tungsten was traced to the tungsten pins used in the supplier's syringe barrel forming process. A tungsten recovery study showed that the vacuum stopper placement process has a significant impact on the total amount of tungsten in solutions. The air gap formed in the syringe funnel area (rich in residual tungsten) becomes accessible to solutions when the vacuum is pulled. Leachable tungsten deposits that were not removed by the supplier's wash process are concentrated in this small area. Extraction procedures used to measure residual tungsten in empty syringes would under-report the tungsten quantity unless the funnel area is wetted during the extraction. Improved syringe barrel forming and washing processes at the supplier have lowered the residual tungsten content and significantly reduced the risk of protein aggregate formation. This experience demonstrates that packaging component manufacturing processes, which are outside the direct control of drug manufacturers, can have an impact on the drug product quality. Thus close technical communication with suppliers of product contact components plays an important role in making a successful biotherapeutic.

Tungsten-induced protein aggregation: solution behavior.

Tungsten has been associated with protein aggregation in prefilled syringes (PFSs). This study probed the relationship between PFSs, tungsten, visible particles, and protein aggregates. Experiments were carried out spiking solutions of two different model proteins with tungsten species obtained from the extraction of tungsten pins typically used in syringe manufacturing processes. These results were compared to those obtained with various soluble tungsten species from commercial sources. Although visible protein particles and aggregates were induced by tungsten from both sources, the extract from tungsten pins was more effective at inducing the formation of the soluble protein aggregates than the tungsten from other sources. Furthermore, our studies showed that the effect of tungsten on protein aggregation is dependent on the pH of the buffer used, the tungsten species, and the tungsten concentration present. The lower pH and increased tungsten concentration induced more protein aggregation. The protein molecules in the tungsten-induced aggregates had mostly nativelike structure, and aggregation was at least partly reversible. The aggregation was dependent on tungsten and protein concentration, and the ratio of these two and appears to arise through electrostatic interaction between protein and tungsten molecules. The level of tungsten required from the various sources was different, but in all cases it was at least an order of magnitude greater than the typical soluble tungsten levels measured in commercial PFS.

Domino liver transplantation in maple syrup urine disease.

Liver transplantation has been reported in a few cases of maple syrup urine disease (MSUD), but is controversial. Many patients with approved indications for liver transplantation die before grafts are available. A 25-yr-old man with MSUD underwent liver transplantation, and his liver was used as a domino graft for a 53-yr-old man with hepatocellular carcinoma who had low priority on the liver transplant waiting list and was unlikely to survive until routine organ procurement. Both transplants were performed as "piggy back" procedures, reconstructing the domino graft with caval segments from the cadaveric donor. Neither required veno-venous bypass. Whole body leucine oxidation was estimated by 13CO2 in breath after oral boluses of L-[1-13C]-leucine, before and after transplantation in both patients and a control subject. The surgical outcome was successful. The patient with MSUD had marked decreases in plasma branched-chain amino acids (BCAAs) and alloisoleucine (from 255 +/- 66 to 16 +/- 7 micromol/L), despite advancement of dietary protein from 6 to >40 gm/day. The domino recipient maintained near-normal levels of plasma amino acids with no detectable alloisoleucine on unrestricted diet. Leucine oxidation increased in the patient with MSUD (from 2.2 to 5.6% recovered in 4 hours) and decreased in the recipient (from 9.7 to 6.2%). Neither patient demonstrated any apparent symptoms of MSUD over more than 7 months. In conclusion, liver transplantation substantially corrects whole body BCAA metabolism in MSUD and greatly attenuates the disease. Livers from patients with MSUD may be considered as domino grafts for patients who might otherwise not survive until transplantation.

Detecting acute coronary syndrome in the emergency department with cardiac magnetic resonance imaging.

BACKGROUND: Managing chest pain in the emergency department remains a challenge with current diagnostic strategies. We hypothesized that cardiac MRI could accurately identify patients with possible or probable acute coronary syndrome. METHODS AND RESULTS: The diagnostic performance of MRI was evaluated in a prospective study of 161 consecutive patients. Enrollment required 30 minutes of chest pain compatible with myocardial ischemia but an ECG not diagnostic of acute myocardial infarction. MRI was performed at rest within 12 hours of presentation and included perfusion, left ventricular function, and gadolinium-enhanced myocardial infarction detection. MRI was interpreted qualitatively but also analyzed quantitatively. The sensitivity and specificity, respectively, for detecting acute coronary syndrome were 84% and 85% by MRI, 80% and 61% by an abnormal ECG, 16% and 95% for strict ECG criteria for ischemia (ST depression or T-wave inversion), 40% and 97% for peak troponin-I, and 48% and 85% for a TIMI risk score > or =3. The MRI was more sensitive than strict ECG criteria for ischemia (P<0.001), peak troponin-I (P<0.001), and the TIMI risk score (P=0.004), and MRI was more specific than an abnormal ECG (P<0.001). Multivariate logistic regression analysis showed MRI was the strongest predictor of acute coronary syndrome and added diagnostic value over clinical parameters (P<0.001). CONCLUSIONS: Resting cardiac MRI exhibited diagnostic operating characteristics suitable for triage of patients with chest pain in the emergency department. Performed urgently to evaluate chest pain, MRI accurately detected a high fraction of patients with acute coronary syndrome, including patients with enzyme-negative unstable angina.

A novel Web-based graduate medical education management system including ACGME compliance algorithms.

OBJECTIVE: Administration of graduate medical education programs has become more difficult as compliance with ACGME work guidelines has assumed increased importance. These guidelines have caused many changes in the resident work environment, including the emergence of complicated cross-cover arrangements. Many participating residents (each with his or her own individual scheduling requirements) usually generate these schedules. Accordingly, schedules are often not submitted in a timely fashion and they may not be in compliance with the ACGME guidelines for maximum on-call assignments and mandatory days off. Our objective was the establishment of a Web-based system that guides residents in creating on-call schedules that follow ACGME guidelines while still allowing maximum flexibility -- thus allowing each resident to maintain an internal locus of control. DESCRIPTION: A versatile and scalable system with password-protected user (resident) and administrator interfaces was created. An entire academic year is included, and past months and years are automatically archived. The residents log on within the first 15 days of the preceding month and choose their positions in a schedule template. They then make adjustments while receiving immediate summary feedback on compliance with ACGME guidelines. The schedule is electronically submitted to the educational administrator for final approval. If a cross-cover system is required, the program automatically generates an optimal schedule using both of the approved participating service schedules. The residents then have an additional five-day period to make adjustments in the cross-cover schedule while still receiving compliance feedback. The administrator again provides final approval electronically. The communication interface automatically pages or e-mails the residents when schedules are updated or approved. Since the information exists in a relational database, simple reporting tools are included to extract the information necessary to generate records for institutional GME management. DISCUSSION: Implementation of this program has been met with great enthusiasm from the institutional stakeholders. Specifically, residents have embraced the ability to directly control their schedules and have gained appreciation for the regulatory matrix in which they function. Institutional administrators have praised the improvement in compliance and the ease of documentation. We anticipate that the system will also meet with approval from reviewing regulatory bodies, as it generates and stores accurate information about the resident work environment. This program is robust and versatile enough to be modified for any GME training program in the country.