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Background: Pneumococcal conjugate vaccines (PCVs) have significantly decreased pneumococcal disease worldwide; however, expanding serotype coverage may further reduce disease burden. A 20-valent PCV (PCV20) containing capsular polysaccharide conjugates of serotypes present in the 13-valent PCV (PCV13) and 7 new serotypes (8, 10A, 11A, 12F, 15B, 22F, and 33F) is currently in development. This phase 2 study evaluated safety, tolerability, and immunogenicity of PCV20 in adults without prior pneumococcal vaccination., Methods: In this randomized, active-controlled, double-blinded trial, 444 adults 60 through 64 years of age were randomized to receive either a single dose of PCV20 followed 1 month later by saline placebo or a single dose of PCV13 followed 1 month later by 23-valent polysaccharide vaccine. Local injection site reactions, select systemic symptoms, and adverse events (AEs) were recorded. Immunogenicity was assessed by measuring serotype-specific opsonophagocytic activity (OPA) titers before and approximately 1 month after each vaccination., Results: Local reaction and systemic event rates were similar after vaccination with PCV20 or PCV13; no serious vaccine-related AEs were reported. In the PCV20 group, functional immune responses as measured by OPA were robust for all 20 serotypes included in the vaccine, with geometric mean fold rises from baseline ranging from 6.0 to 113.4., Conclusions: PCV20 was well tolerated in adults 60 to 64 years of age, with a safety profile consistent with historical experience of PCVs in this age group. Substantial OPA responses were elicited against all serotypes. Results demonstrate the potential for PCV20 to expand pneumococcal disease protection., Clinical Trials Registration: NCT03313037., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America.)

Objectives: Assess Staphylococcus aureus (S. aureus) colonization in healthy Australian adults receiving an investigational S. aureus 3-antigen vaccine (SA3Ag)., Methods: In this phase 1, double-blind, sponsor-unblinded study, participants were randomized to receive a single dose (1 of 3 dose levels) of SA3Ag or placebo and a booster dose or placebo at 6 months. S. aureus isolates from nasal, perineal, and oropharyngeal swabs before and through 12 months post-vaccination were identified., Results: Baseline S. aureus colonization prevalence was 30.6% (any site), with nasal carriage (27.0%) more common than oropharyngeal/perineal (3.2% each). Following initial vaccination (low-dose: 102; mid-dose: 101; high-dose: 101; placebo: 102) and booster (low-dose: 45; mid-dose: 44; high-dose: 27; placebo: 181), placebo and SA3Ag groups showed similar S. aureus carriage through 12 months. Most colonized participants (74.0%) were colonized by single spa types. Placebo and SA3Ag groups had similar persistence of colonization, with 19.6-30.7% due to single spa types. Acquisition was observed in mid- and high-dose recipients (∼20%) and low-dose and placebo recipients (∼12%). Vaccination resulted in substantial increases in antibodies to all 3 antigens, irrespective of carriage status., Conclusions: Based on descriptive analyses of this small study, SA3Ag vaccination did not impact S. aureus acquisition or carriage. Carriage status did not impact antibody responses to SA3Ag., (Copyright © 2019. Published by Elsevier Ltd.)

Introduction: Streptococcus pneumoniae is a leading cause of bacteremia, bacterial pneumonia, and meningitis, and is associated with substantial morbidity and mortality, particularly in those under 2 years of age and those over 65 years of age. While significant progress against S. pneumoniae-related disease has been made as a result of the introduction of pneumococcal conjugate vaccines (PCV7, PCV10 and PCV13), there remains value in further expanding pneumococcal vaccine serotype coverage. Here we present the first report of a 20-valent pneumococcal conjugate vaccine (PCV20) containing capsular polysaccharide conjugates present in PCV13 as well as 7 new serotypes (8, 10A, 11A, 12F, 15B, 22F, and 33F) which are important contributors to pneumococcal disease., Methods: This Phase I first-in-human study was a randomized, controlled, observer-blinded study with a two-arm parallel design to assess the safety, tolerability, and immunogenicity of PCV20 in adults. A total of 66 healthy adults 18-49 years of age with no history of pneumococcal vaccination were enrolled and randomized to receive a single dose of PCV20 or a licensed tetanus, diphtheria, acellular pertussis combination vaccine (Tdap) control. Local injection site reactions, select systemic symptoms, laboratory studies, and adverse events were assessed. Opsonophagocytic activity (OPA) titers and IgG concentrations were measured in sera collected prior to, and approximately one month (28-35 days) after vaccination., Results: Vaccination with PCV20 elicited substantial IgG and functional bactericidal immune responses as demonstrated by increases in IgG geometric mean concentrations (GMCs) and OPA geometric mean titers (GMTs) to the 20 vaccine serotypes. The overall safety profile of PCV20 was similar to Tdap, and generally consistent with that observed after PCV13 administration., Conclusions: Vaccination with PCV20 was well tolerated and induced substantial functional (OPA) and IgG responses to all vaccine serotypes. There were no safety issues identified in this Phase 1 study, and the data supported further evaluation of PCV20., (Copyright © 2019. Published by Elsevier Ltd.)

Background: Staphylococcus aureus is a leading cause of healthcare-associated infections. No preventive vaccine is currently licensed. SA4Ag is an investigational 4-antigen S. aureus vaccine, composed of capsular polysaccharide conjugates of serotypes 5 and 8 (CP5 and CP8), recombinant surface protein clumping factor A (rmClfA), and recombinant manganese transporter protein C (rMntC). This Phase 1 study aimed to confirm the safety and immunogenicity of SA4Ag produced by the final manufacturing process before efficacy study initiation in a surgical population., Methods: Healthy adults (18-<65years) received one intramuscular SA4Ag injection. Serum functional antibodies were measured at baseline and Day 29 post-vaccination. An opsonophagocytic activity (OPA) assay measured the ability of vaccine-induced antibodies to CP5 and CP8 to kill S. aureus clinical isolates. For MntC and ClfA, antigen-specific immunogenicity was assessed via competitive Luminex® immunoassay (cLIA) and via fibrinogen-binding inhibition (FBI) assay for ClfA only. Reactogenicity and adverse event data were collected., Results: One hundred participants were vaccinated. SA4Ag was well tolerated, with a satisfactory safety profile. On Day 29, OPA geometric mean titers (GMTs) were 45,738 (CP5, 95% CI: 38,078-54,940) and 42,652 (CP8, 95% CI: 32,792-55,477), consistent with 69.2- and 28.9-fold rises in bacteria-killing antibodies, respectively; cLIA GMTs were 2064.4 (MntC, 95% CI: 1518.2-2807.0) and 3081.4 (ClfA, 95% CI: 2422.2-3920.0), consistent with 19.6- and 12.3-fold rises, respectively. Similar to cLIA results, ClfA FBI titers rose 11.0-fold (GMT: 672.2, 95% CI: 499.8-904.2). The vast majority of participants achieved the pre-defined biologically relevant thresholds: CP5: 100%; CP8: 97.9%, ClfA: 87.8%; and MntC 96.9%., Conclusions: SA4Ag was safe, well tolerated, and rapidly induced high levels of bacteria-killing antibodies in healthy adults. A Phase 2B efficacy trial in adults (18-85years) undergoing elective spinal fusion is ongoing to assess SA4Ag's ability to prevent postoperative invasive surgical site and bloodstream infections caused by S. aureus. Clinicaltrials.gov Identifier: NCT02364596., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

As an increasing number of recombinant therapeutic glycoproteins are manufactured and investigated, the importance of their attached glycans is becoming more widely reported and understood. Regulatory agencies expect detailed "extended characterization" of the glycoprotein as well as routine, well-controlled "release assays" with specifications to be employed for quality control of each manufactured lot. In this chapter we will briefly discuss relevant glycan issues in the area of therapeutic recombinant glycoprotein manufacture and describe in detail two assays that are employed in the development of, for example, recombinant Factor VIII for the treatment of hemophilia.

A long-acting factor VIII (FVIII) as a replacement therapy for hemophilia A would significantly improve treatment options for patients with hemophilia A. To develop a FVIII with an extended circulating half-life, but without a reduction in activity, we have engineered 23 FVIII variants with introduced surface-exposed cysteines to which a polyethylene glycol (PEG) polymer was specifically conjugated. Screening of variant expression level, PEGylation yield, and functional assay identified several conjugates retaining full in vitro coagulation activity and von Willebrand factor (VWF) binding.PEGylated FVIII variants exhibited improved pharmacokinetics in hemophilic mice and rabbits. In addition, pharmacokinetic studies in VWF knockout mice indicated that larger molecular weight PEG may substitute for VWF in protecting PEGylated FVIII from clearance in vivo. In bleeding models of hemophilic mice, PEGylated FVIII not only exhibited prolonged efficacy that is consistent with the improved pharmacokinetics but also showed efficacy in stopping acute bleeds comparable with that of unmodified rFVIII. In summary site-specifically PEGylated FVIII has the potential to be a long-acting prophylactic treatment while being fully efficacious for on-demand treatment for patients with hemophilia A.

Objective: Lurasidone is a novel psychotropic agent with high affinity for D(2) and 5-HT(2A) receptors, as well as for receptors implicated in the enhancement of cognition and mood and the reduction of negative symptoms (5-HT(7), 5-HT(1A), and alpha(2c)). The objective of the study was to evaluate the safety and efficacy of lurasidone in patients hospitalized for an acute exacerbation of DSM-IV-defined schizophrenia., Method: Patients were randomly assigned to 6 weeks of double-blind treatment with a fixed dose of lurasidone 80 mg (N = 90, 75.6% male, mean age = 39.7 years, mean baseline score on the Brief Psychiatric Rating Scale derived from the Positive and Negative Syndrome Scale [BPRSd] = 55.1) or placebo (N = 90, 77.8% male, mean age = 41.9 years, mean BPRSd score = 56.1). The primary efficacy measure was the BPRSd. The study was conducted from May to December 2004., Results: At day 42, last-observation-carried-forward endpoint, treatment with lurasidone was associated with significant improvement compared to placebo on the BPRSd (least squares mean +/- SE = -8.9 +/- 1.3 vs. -4.2 +/- 1.4; p = .012), as well as on all secondary efficacy measures, including the PANSS total score (-14.1 +/- 2.1 vs. -5.5 +/- 2.2; p = .004) and the PANSS positive (-4.3 +/- 0.7 vs. -1.7 +/- 0.7; p = .006), negative (-2.9 +/- 0.5 vs. -1.3 +/- 0.5; p = .025), and general psychopathology (-7.0 +/- 1.1 vs. -2.7 +/- 1.2; p = .0061) subscales. Significant improvement was seen as early as day 3, based on BPRSd, PANSS, and Clinical Global Impressions-Severity of Illness assessments. Treatment with lurasidone was generally well tolerated and was not associated with adverse changes in metabolic or electrocardiogram parameters. There were no clinically significant differences between lurasidone and placebo in objective measures of extrapyramidal symptoms., Conclusions: The results of this study suggest that the novel psychotropic agent lurasidone is a safe and effective treatment for patients with an acute exacerbation of schizophrenia., Trial Registration: (ClinicalTrials.gov) Identifier: NCT00088634., (Copyright 2009 Physicians Postgraduate Press, Inc.)

A PEGylated glucagon-like peptide-1 (GLP-1) agonist and glucagon antagonist hybrid peptide was engineered as a potential treatment for type 2 diabetes. To support preclinical development of this PEGylated dual-acting peptide for diabetes (DAPD), we developed a reproducible method for PEGylation, purification, and analysis. Optimal conditions for site-specific PEGylation with 22 and 43 kDa maleimide-polyethylene glycol (maleimide-PEG) polymers were identified by evaluating pH, reaction time, and reactant molar ratio parameters. A 3-step purification process was developed and successfully implemented to purify PEGylated DAPD and remove excess uncoupled PEG and free peptide. Five lots of 43 kDa PEGylated DAPD with starting peptide amounts of 100 mg were produced with overall yields of 53% to 71%. Analytical characterization by N-terminal sequencing, amino acid analysis, matrix-assisted laser desorption/ionization mass spectrometry, and GLP-1 receptor activation assay confirmed site-specific attachment of PEG at the engineered cysteine residue, expected molecular weight, correct amino acid sequence and composition, and consistent functional activity. Purity and safety analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), analytical ion-exchange chromatography, reversed-phase high-performance liquid chromatography, and limulus amebocyte lysate test showed that the final products contained <1% free peptide, <5% uncoupled PEG, and <0.2 endotoxin units per milligram of peptide. These results demonstrate that the PEGylation and purification process we developed was consistent and effective in producing PEGylated DAPD preclinical materials at the 100 mg (peptide weight basis) or 1.2 g (drug substance weight basis) scale.

Recombinant Factor VIII (rFVIII) is involved in the cascade of biochemical reactions leading to blood coagulation and is used for the treatment of haemophilia A. Plasma-derived FVIII (pdFVIII) has been reported to be post-translationally modified by sulfation of tyrosine residues at positions 346, 1664, 1680, 718, 719 and 723. This report describes the quantitation of tyrosine sulfate residues in BHK-derived, human rFVIII by amino acid composition analysis and the identification of their positions in the polypeptide sequence using a combination of liquid chromatography and electrospray ionization mass spectrometry in the analysis of proteolytic digests of the protein.

A capillary electrophoresis/electrospray ionization mass spectrometry (CE/ESI-MS) interface, based on an electric circuit across a microdialysis membrane surrounding a short capillary segment closely connected to the separation capillary terminus, is demonstrated to be sensitive, efficient, and rugged. A microspray type ionization emitter produces a stable electrospray at the low flow rates provided by CE and thus avoids both the need for a makeup liquid flow provided by liquid junction or sheath flow interfaces and the subsequent dilution and reduction in sensitivity. Reproducibility studies and comparisons with CE/UV and the CE/sheath flow interface with ESI-MS are presented. Additionally, postrun acidification via the microdialysis junction interface is demonstrated and shown to be capable of denaturing the holomyoglobin protein noncovalent complex while maintaining separation efficiency.

The interfacing of capillary electrophoresis (CE) with Fourier transform ion cyclotron resonance-mass spectrometry (FTICR-MS) and the factors which dictate obtainable performance (i.e., sensitivity, mass resolution, scan rate and duty cycle) are described. We demonstrate the current status of the technique with examples of capillary zone electrophoresis (CZE) and capillary isotachophoresis (CITP) with FTICR analyses of proteins and oligonucleotides, and describe current limitations on sensitivity and scan speed. The first on-line interfacing of capillary isoelectric focusing (CIEF) with FTICR is also demonstrated and shown to be effective for separating minor components of protein mixtures for on-line mass spectral analysis. Finally, the potential for greatly improved performance based upon recent advances in FTICR instrumentation and methods is briefly described.