Quantitation and characterization of serotype 6A activation for pneumococcal conjugate vaccine by cryo-EM and SEC methods.

Pneumococcal conjugate vaccines (PCV) typically consist of capsular polysaccharides from different S. pneumoniae serotypes which are covalently attached to carrier protein. A well-established process to manufacture PCV is through activating polysaccharide by oxidation of vicinal diols to aldehydes, followed by protein conjugation via reductive amination. Polysaccharide activation is a crucial step that affects vaccine product critical attributes including conjugate size and structure. Therefore, it is highly desired to have robust analytical methods to well characterize this activation process. In this study, using pneumococcal serotype 6A as the model, we present two complimentary analytical methods for characterization of activated polysaccharide. First, a size exclusion chromatography (SEC) method was developed for quantitative measurement of polysaccharide activation levels. This SEC method demonstrated good assay characteristics on accuracy, precision and linearity. Second, a gold nanoparticle labeled cryo-electron microscopy (Cryo-EM) technique was developed to visualize activation site distribution along polysaccharide chain and provide information on activation heterogeneity. These two complimentary methods can be utilized to control polysaccharide activation process and ensure consistent delivery of conjugate vaccine products.

Characterization of High Molecular Weight Pneumococcal Conjugate by SEC-MALS and AF4-MALS.

Infections by Streptococcus pneumoniae can cause serious pneumococcal diseases and other medical complications among patients. Polysaccharide-based vaccines have been successfully developed as prophylactic agents against such deadly bacterial infections. In the 1980s, PNEUMOVAX® 23 were introduced as the first pneumococcal polysaccharide vaccines (PPSV). Later, pneumococcal polysaccharides were conjugated to a carrier protein to improve immune responses. Pneumococcal conjugate vaccines (PCV) such as PREVNAR® and VAXNEUVANCE™ have been developed. Of the more than 90 pneumococcal bacteria serotypes, serotype 1 (ST-1) and serotype 4 (ST-4) are the two main types that cause invasive pneumococcal diseases (IPD) that could lead to morbidity and mortality. Development of a novel multi-valent PCV against these serotypes requires extensive biophysical and biochemical characterizations of each monovalent conjugate (MVC) in the vaccine. To understand and characterize these high molecular weight (Mw) polysaccharide protein conjugates, we employed the multi-angle light scattering (MALS) technique coupled with size-exclusion chromatography (SEC) separation and asymmetrical flow field flow fractionation (AF4). MALS analysis of MVCs from the two orthogonal separation mechanisms helps shed light on the heterogeneity in conformation and aggregation states of each conjugate.

Multi-attribute characterization of pneumococcal conjugate vaccine by Size-exclusion chromatography coupled with UV-MALS-RI detections.

Streptococcus pneumoniae bacterial infection can cause serious diseases. Among more than 90 known streptococcus pneumoniae serotypes, more than 30 can cause invasive pneumococcal diseases that could lead to morbidity and mortality. Initially, a 23-valent polysaccharide vaccines (PPSV) PNEUMOVAX®23, was developed to generate an antigen-specific immune response and prevent diseases caused by these pneumoniae serotypes. Later, pneumococcal conjugate vaccines (PCV), such as PREVNAR® and VAXNEUVANCE™ have been developed to offer a more robust immune response in the pediatric population. In our effort to develop novel pneumococcal conjugate vaccines, each serotype of pneumococcal polysaccharide (Ps) is conjugated to a detoxified diphtheria toxin carrier protein CRM197 to form a monovalent conjugate (MVC). MVCs from multiple serotypes are formulated with vaccine adjuvant to form a multi-valent vaccine drug product. During the product development, critical attributes including conjugate molecular weight (Mw), protein and polysaccharide concentration, have been used to monitor process and product quality. To measure these attributes, a size-exclusion chromatography (SEC) method was developed with a series of in-line detectors including UV, multi-angle light scattering (MALS) and refractive index (RI). This SEC-UV-MALS-RI method is employed to characterize and monitor process intermediates and product during process development and for product release and stability testing. With this, we have expanded the multi-attribute SEC method to a 15-valent pneumococcal conjugate vaccine.

Structure of the cell-binding component of the Clostridium difficile binary toxin reveals a di-heptamer macromolecular assembly.

Targeting Clostridium difficile infection is challenging because treatment options are limited, and high recurrence rates are common. One reason for this is that hypervirulent C. difficile strains often have a binary toxin termed the C. difficile toxin, in addition to the enterotoxins TsdA and TsdB. The C. difficile toxin has an enzymatic component, termed CDTa, and a pore-forming or delivery subunit termed CDTb. CDTb was characterized here using a combination of single-particle cryoelectron microscopy, X-ray crystallography, NMR, and other biophysical methods. In the absence of CDTa, 2 di-heptamer structures for activated CDTb (1.0 MDa) were solved at atomic resolution, including a symmetric (SymCDTb; 3.14 Å) and an asymmetric form (AsymCDTb; 2.84 Å). Roles played by 2 receptor-binding domains of activated CDTb were of particular interest since the receptor-binding domain 1 lacks sequence homology to any other known toxin, and the receptor-binding domain 2 is completely absent in other well-studied heptameric toxins (i.e., anthrax). For AsymCDTb, a Ca2+ binding site was discovered in the first receptor-binding domain that is important for its stability, and the second receptor-binding domain was found to be critical for host cell toxicity and the di-heptamer fold for both forms of activated CDTb. Together, these studies represent a starting point for developing structure-based drug-design strategies to target the most severe strains of C. difficile.

Nationwide Analysis of 30-Day Readmissions After Esophagectomy: Causes, Costs, and Risk Factors.

BACKGROUND: Postsurgical readmissions are an increasingly scrutinized marker of health care quality. We sought to estimate the rate, risk factors, causes, and costs associated with readmissions after esophagectomy in a large, nationally representative cohort. METHODS: We studied patients from the Nationwide Readmissions Database undergoing esophagectomy from 2010 to 2014. Data were collected on the prevalence and indications for readmission within 30 days as well as the hospital-, procedure-, and patient-level risk factors as determined by multivariable logistic regression. RESULTS: Among 13,282 cases, the rate of 30-day readmission was 19.4%, with the most common indications for readmission being pulmonary (20.6%) and gastrointestinal complications (20%). Median cost of readmission was $9660 (interquartile range, $5392 to $20,447), and pulmonary complications accounted for the greatest total cost burden at 25.8% of all readmission-related costs. Independent risk factors for readmission on multivariable analysis included perioperative blood transfusion (adjusted odds ratio [AOR] 1.33; 95% confidence interval [CI], 1.08 to 1.65; P = .008), discharge to a nursing facility (AOR 1.83; 95% CI, 1.41 to 2.39; P < .001), high illness severity based on All Patients Refined Diagnosis-Related Groups scoring (AOR 1.49; 95% CI, 1.21 to 1.84; P < .001), chronic renal failure (AOR 1.61; 95% CI, 1.13 to 2.29; P = .009), and comorbid drug abuse (AOR 2.19; 95% CI, 1.08 to 4.41; P = .029). CONCLUSIONS: Nearly 1 in 5 patients undergoing esophagectomy are readmitted within 30 days of discharge, at a median cost of $9660 per readmission. Pulmonary complications account for the greatest number of readmissions and the greatest total cost burden. Targeting the causes of readmission, especially pulmonary causes, may help significantly reduce the total morbidity and health care costs associated with esophagectomy.

Multi-site N-Glycan mapping study 2: UHPLC.

In the first part of this publication, the results from an international study evaluating the precision (i.e., repeatability and reproducibility) of N-glycosylation analysis using capillary electrophoresis of APTS-labeled N-glycans were presented. The corresponding results from ultra-high performance liquid chromatography (UHPLC) with fluorescence detection are presented here from 12 participating sites. All participants used the same lot of samples, reagents, and columns to perform the assays. Elution time, peak area and peak area percent values were determined for all peaks ≥0.1% peak area, and statistical analysis was performed following ISO 5725-2 guideline principles. The results demonstrated adequate reproducibility, within any given site as well across all sites, indicating that standard UHPLC-based N-glycan analysis platforms are appropriate for general use.

Functional outcomes after inpatient rehabilitation for trauma-improved but unable to return home.

BACKGROUND: Twenty-five percent of trauma patients are discharged to postacute care, indicating a loss of physical function and need for rehabilitation. The purpose of this study was to quantify the functional improvements in trauma patients discharged from inpatient rehabilitation facility (IRF) and identify predictors of improvement. MATERIALS AND METHODS: A retrospective cohort study of trauma patients aged ≥ 18 years were admitted to an IRF after discharge from a level-1 trauma center. Data included demographics, injury characteristics, hospital, and IRF course. The functional independence measure (FIM) was used to measure change in physical and cognitive function. RESULTS: There were 245 patients with a mean age of 55.8 years and mean injury severity score (ISS) of 14.7. Fall was the leading mechanism of injury (45.7%). On IRF admission, 50.7% of patients required moderate or greater assistance. On discharge, the mean intraindividual change in FIM score was 29.9; 85.4% of the patients improved by ≥1 level of functioning. Before injury, 99.6% of patients were living at home, but only 56.0% were discharged home from the IRF, despite 81.8% requiring minimal assistance at most (23.5% to skilled nursing; 19.7% readmitted). Increasing age and lower ISS were associated with less FIM improvement, and increasing ISS was associated with increased FIM improvement. CONCLUSIONS: More than 80% of the trauma patients experienced meaningful functional improvements during IRF admission. However, only half were discharged home, and a quarter required further institutional care. Further research is needed to identify the additional impediments to return to preinjury functioning.

Development of a non-radiolabeled glucosyltransferase activity assay for C. difficile toxin A and B using ultra performance liquid chromatography.

Clostridium difficile infection (CDI) is the leading cause of gastroenteritis-associated death in the United States. The major virulent factors of C. difficile are toxin A (TcdA) and toxin B (TcdB). Toxicity is mediated by the glucosyltransferase domains on TcdA and TcdB wherein a glucose is transferred from UDP-glucose to Ras homolog family member A (RhoA) receptor. This modification results in disruption of critical cell signaling events. Vaccination against these toxins is considered the best way to combat the CDI. In order to produce non-toxic TcdA and TcdB antigens, their glucosyltransferase domains were genetically mutated to inactivate the toxin activity. We have developed a reverse phase ultra performance liquid chromatographic (RP-UPLC) method to measure this glucosyltransferase activity by separating RhoA and glucosylated RhoA. Glucosylated RhoA and RhoA have a retention time (RT) of 31.25 and 31.95min. We determine for the first time the glucosyltransferase kinetics (Km and kcat) of both full length TcdA and TcdB to RhoA and demonstrate that the genetically mutated TcdA and TcdB show no glucosyltransferase activity. Furthermore, two-dimensional electron microscopy (2D EM) data demonstrates that the overall global structures of mutated toxins do not change compared to native toxins.

Development of a Novel Vaccine Containing Binary Toxin for the Prevention of Clostridium difficile Disease with Enhanced Efficacy against NAP1 Strains.

Clostridium difficile infections (CDI) are a leading cause of nosocomial diarrhea in the developed world. The main virulence factors of the bacterium are the large clostridial toxins (LCTs), TcdA and TcdB, which are largely responsible for the symptoms of the disease. Recent outbreaks of CDI have been associated with the emergence of hypervirulent strains, such as NAP1/BI/027, many strains of which also produce a third toxin, binary toxin (CDTa and CDTb). These hypervirulent strains have been associated with increased morbidity and higher mortality. Here we present pre-clinical data describing a novel tetravalent vaccine composed of attenuated forms of TcdA, TcdB and binary toxin components CDTa and CDTb. We demonstrate, using the Syrian golden hamster model of CDI, that the inclusion of binary toxin components CDTa and CDTb significantly improves the efficacy of the vaccine against challenge with NAP1 strains in comparison to vaccines containing only TcdA and TcdB antigens, while providing comparable efficacy against challenge with the prototypic, non-epidemic strain VPI10463. This combination vaccine elicits high neutralizing antibody titers against TcdA, TcdB and binary toxin in both hamsters and rhesus macaques. Finally we present data that binary toxin alone can act as a virulence factor in animal models. Taken together, these data strongly support the inclusion of binary toxin in a vaccine against CDI to provide enhanced protection from epidemic strains of C. difficile.

Ion-Exchange Chromatography to Analyze Components of a Clostridium difficile Vaccine.

Ion-exchange (IEX) chromatography is one of many separation techniques that can be employed to analyze proteins. The separation mechanism is based on a reversible interaction between charged amino acids of a protein to the charged ligands attached to a column at a given pH. This interaction depends on both the pI and conformation of the protein being analyzed. The proteins are eluted by increasing the salt concentration or pH gradient. Here we describe the use of this technique to characterize the charge variant heterogeneities and to monitor stability of four protein antigen components of a Clostridium difficile vaccine. Furthermore, the IEX technique can be used to monitor reversion to toxicity for formaldehyde-treated Clostridium difficile toxins.

A Size-Exclusion Chromatography Method for Analysis of Clostridium difficile Vaccine Toxins.

High-performance size-exclusion chromatography (HPSEC or SEC) is a method that can be applied to measure size distribution of proteins, including aggregates, monomers, and fragments. In the biopharmaceutical industry the quantitation of aggregates contained in biotherapeutics and protein-based vaccines is critical given the potential impact on safety, immunogenicity, and efficacy. Hence, aggregation analysis of therapeutic proteins or protein-based vaccine products is almost always a requirement of regulatory agencies. SEC, also referred to as gel-filtration chromatography, separates molecules by size through a porous resin stationary phase. Under isocratic flow small molecules are retained on the column longer than large molecules. Here we describe the use of this SEC technique to characterize aggregation levels for four different protein antigens for a Clostridium difficile vaccine.

Applications of an Automated and Quantitative CE-Based Size and Charge Western Blot for Therapeutic Proteins and Vaccines.

Capillary Electrophoresis (CE) is a versatile and indispensable analytical tool that can be applied to characterize proteins. In recent years, labor-intensive SDS-PAGE and IEF slab gels have been replaced with CE-SDS (CGE) and CE-IEF methods, respectively, in the biopharmaceutical industry. These two CE-based methods are now an industry standard and are an expectation of the regulatory agencies for biologics characterization. Another important and traditional slab gel technique is the western blot, which detects proteins using immuno-specific reagents after SDS-PAGE separation. This technique is widely used across industrial and academic laboratories, but it is very laborious, manual, time-consuming, and only semi-quantitative. Here, we describe the applications of a relatively new CE-based western blot technology which is automated, fast, and quantitative. We have used this technology for both charge- and size-based CE westerns to analyze biotherapeutic and vaccine products. The size-based capillary western can be used for fast antibody screening, clone selection, product titer, identity, and degradation while the charge-based capillary western can be used to study product charge heterogeneity. Examples using this technology for monoclonal antibody (mAb), Enbrel, CRM197, and Clostridium difficile (C. difficile) vaccine proteins are presented here to demonstrate the utility of the capillary western techniques. Details of sample preparation and experimental conditions for each capillary western mode are described in this chapter.

Characterization of N-glycosylation profiles from mammalian and insect cell derived chikungunya VLP.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes severe arthralgia. The envelope of CHIKV is composed of 240 copies of two glycoproteins: E1 and E2. In this work, we have characterized the N-glycosylation patterns of CHIKV virus-like particles (VLPs), containing both E1 and E2 proteins, derived from mammalian and insect cells using hydrophilic interaction liquid chromatography (HILIC) with fluorescence (FL) and mass spectrometry (MS) detection. While HEK293 derived CHIKV VLPs contain oligomannose, hybrid and complex glycans, VLPs derived from SfBasic predominantly contain oligomannose glycans. This strong host dependence of N-glycosylation pattern resembles other alphaviruses such as SINV. The VLPs from HEK293 and SfBasic, with significantly different N-glycosylation profiles, are valuable reagents enabling future in-depth correlation studies between immunogenicity and glycosylation. In addition, the characterization tools presented here allow one to monitor glycosylation during vaccine process development and ensure process consistency.

Toxicity assessment of Clostridium difficile toxins in rodent models and protection of vaccination.

Clostridium difficile is the leading cause of hospital-acquired diarrhea, also known as C. difficile associated diarrhea. The two major toxins, toxin A and toxin B are produced by most C. difficile bacteria, but some strains, such as BI/NAP1/027 isolates, produce a third toxin called binary toxin. The precise biological role of binary toxin is not clear but it has been shown to be a cytotoxin for Vero cells. We evaluated the toxicity of these toxins in mice and hamsters and found that binary toxin causes death in both animals similar to toxins A and B. Furthermore, immunization of mice with mutant toxoids of all three toxins provided protection upon challenge with native toxins. These results support the concept that binary toxin contributes to the pathogenicity of C. difficile and provide a method for monitoring the toxicity of binary toxin components in vaccines.

Residual bovine serum albumin (BSA) quantitation in vaccines using automated Capillary Western technology.

Bovine serum albumin (BSA) is a major component of fetal bovine serum (FBS), which is commonly used as a culture medium during vaccine production. Because BSA can cause allergic reactions in humans the World Health Organization (WHO) has set a guidance of 50 ng or less residual BSA per vaccine dose. Vaccine manufacturers are expected to develop sensitive assays to detect residual BSA. Generally, sandwich enzyme-linked immunosorbent assays (ELISA) are used in the industry to detect these low levels of BSA. We report the development of a new improved method for residual BSA detection using the SimpleWestern technology to analyze residual BSA in an attenuated virus vaccine. The method is based on automated Capillary Western and has linearity of two logs, >80% spike recovery (accuracy), intermediate precision of CV <15%, and LOQ of 5.2 ng/ml. The final method was applied to analyze BSA in four lots of bulk vaccine products and was used to monitor BSA clearance during vaccine process purification.

Qualitative and quantitative evaluation of Simon™, a new CE-based automated Western blot system as applied to vaccine development.

Many CE-based technologies such as imaged capillary IEF, CE-SDS, CZE, and MEKC are well established for analyzing proteins, viruses, or other biomolecules such as polysaccharides. For example, imaged capillary isoelectric focusing (charge-based protein separation) and CE-SDS (size-based protein separation) are standard replacement methods in biopharmaceutical industries for tedious and labor intensive IEF and SDS-PAGE methods, respectively. Another important analytical tool for protein characterization is a Western blot, where after size-based separation in SDS-PAGE the proteins are transferred to a membrane and blotted with specific monoclonal or polyclonal antibodies. Western blotting analysis is applied in many areas such as biomarker research, therapeutic target identification, and vaccine development. Currently, the procedure is very manual, laborious, and time consuming. Here, we evaluate a new technology called Simple Western™ (or Simon™) for performing automated Western analysis. This new technology is based on CE-SDS where the separated proteins are attached to the wall of capillary by a proprietary photo activated chemical crosslink. Subsequent blotting is done automatically by incubating and washing the capillary with primary and secondary antibodies conjugated with horseradish peroxidase and detected with chemiluminescence. Typically, Western blots are not quantitative, hence we also evaluated the quantitative aspect of this new technology. We demonstrate that Simon™ can quantitate specific components in one of our vaccine candidates and it provides good reproducibility and intermediate precision with CV <10%.