Orthogonal Comparison of Analytical Methods by Theoretical Reconstruction from Bottom-up Assay Data.

In the never-ending endeavor to produce stable and efficacious protein therapeutics, biopharmaceutical companies often employ numerous analytical techniques to characterize and quantify a drug candidate's stability. Mass spectrometry, due to the information-rich data it produces, is commonly used in its numerous configurations to ascertain chemical and structural stability. At issue is the comparison of the various configurations utilized, that is, comparing bottom-up methods such as proteolytic digest followed by reversed phase LC-MS with intact LC-MS methods.

Surfactant Impact on Interfacial Protein Aggregation and Utilization of Surface Tension to Predict Surfactant Requirements for Biological Formulations.

Biological drug products are formulated with excipients to maintain stability over the shelf life of the product. Surfactants are added to the drug product to stabilize air-water interfaces known to induce protein aggregation. Early formulation development is focused on maintaining protein conformation and colloidal stability over the course of the drug product shelf life but rarely considers stability through dose preparation and administration.

Spectroscopic analysis of chlamydial major outer membrane protein in support of structure elucidation.

Chlamydial major outer membrane protein (MOMP) is the major protein constituent of the bacterial pathogen Chlamydia trachomatis. Chlamydia trachomatis Serovars D-K are the leading cause of genital tract infections which can lead to infertility or ectopic pregnancies. A vaccine against Chlamydia is highly desirable but currently not available.

Detection and Measurement of Methionine Oxidation in Proteins.

Methionine oxidation is a prevalent modification found in proteins both in biological settings and in the manufacturing of biotherapeutic molecules. In cells, the oxidation of specific methionine sites can modulate protein function or promote interactions that trigger signaling pathways. In biotherapeutic development, the formation of oxidative species could be detrimental to the efficacy or safety of the drug product.

Performance of the Verigene® enteric pathogens test, Biofire FilmArray™ gastrointestinal panel and Luminex xTAG® gastrointestinal pathogen panel for detection of common enteric pathogens.

Background: Multiplex syndromic panels have the capability of identifying causes of diarrheal illness. This study evaluated the performance characteristics of three multiplex molecular assays for the detection of common stool pathogens.

Methods: A total of 152 stool specimens were tested using three platforms: Verigene Enteric Pathogens Test (Verigene), Biofire FilmArray Gastrointestinal Panel (Biofire) and Luminex xTAG® Gastrointestinal Pathogen Panel (Luminex).

Protection of rhesus macaques against inhalational anthrax with a Bacillus anthracis capsule conjugate vaccine.

The efficacy of currently licensed anthrax vaccines is largely attributable to a single Bacillus anthracis immunogen, protective antigen. To broaden protection against possible strains resistant to protective antigen-based vaccines, we previously developed a vaccine in which the anthrax polyglutamic acid capsule was covalently conjugated to the outer membrane protein complex of Neisseria meningitidis serotype B and demonstrated that two doses of 2.5μg of this vaccine conferred partial protection of rhesus macaques against inhalational anthrax .

Immunogenicity in mice and non-human primates of the Group A Streptococcal J8 peptide vaccine candidate conjugated to CRM197.

Vaccine development for Group A streptococcal (GAS) infection has been extensively focused on the N-terminal hypervariable or the C-terminal conserved regions of the M protein, a major virulence factor of GAS. We evaluated the immunogenicity and functional activity of the conserved C-terminal peptide vaccine candidate, J8, conjugated to CRM197, in two mouse strains: C3H (H2(k)) and Balb/c (H2(d)), and in rhesus macaques. Mice were immunized with J8-CRM197 formulated with Amorphous Aluminum Hydroxyphosphate Sulfate Adjuvant (AAHSA), and non-human primates were immunized with J8-CRM197 formulated with AAHSA, ISCOMATRIX (TM) adjuvant, or AAHSA/ISCOMATRIX adjuvant.

Sporozoite neutralizing antibodies elicited in mice and rhesus macaques immunized with a Plasmodium falciparum repeat peptide conjugated to meningococcal outer membrane protein complex.

Antibodies that neutralize infectivity of malaria sporozoites target the central repeat region of the circumsporozoite (CS) protein, which in Plasmodium falciparum is comprised primarily of 30-40 tandem NANP tetramer repeats. We evaluated immunogenicity of an alum-adsorbed (NANP)(6) peptide conjugated to an outer membrane protein complex (OMPC) derived from Neisseria meningitidis, a carrier protein used in a licensed Haemophilus influenzae pediatric vaccine. Mice immunized with (NANP)(6)-OMPC adsorbed to Merck's alum adjuvant (MAA), with or without Iscomatrix® as co-adjuvant, developed high levels of anti-repeat peptide antibody that inhibited in vitro invasion of human hepatoma cells by transgenic P.

Efficacy of a capsule conjugate vaccine against inhalational anthrax in rabbits and monkeys.

Bacillus anthracis, the causative agent of anthrax, is recognized as one of the most serious bioterrorism threats. The current human vaccines are based on the protective antigen component of the anthrax toxins. Concern about possible vaccine resistant strains and reliance on a single antigen has prompted the search for additional immunogens.

High throughput monitoring of amyloid-β(42) assembly into soluble oligomers achieved by sensitive conformation state-dependent immunoassays.

Accumulation of small soluble assemblies of amyloid-β (Aβ)(42) in the brain is thought to play a key role in the pathogenesis of Alzheimer's disease. As a result, there has been much interest in finding small molecules that inhibit the formation of synaptotoxic Aβ(42) oligomers that necessitates sensitive methods for detecting the initial steps in the oligomerization of Aβ(42). Modeling suggests that oligomerized Aβ(42) adopts a conformation in which the C-terminus is embedded in the center, whereas the N-terminus is exposed at the periphery of the oligomer.

Small molecule mimetics of an HIV-1 gp41 fusion intermediate as vaccine leads.

We describe here a novel platform technology for the discovery of small molecule mimetics of conformational epitopes on protein antigens. As a model system, we selected mimetics of a conserved hydrophobic pocket within the N-heptad repeat region of the HIV-1 envelope protein, gp41. The human monoclonal antibody, D5, binds to this target and exhibits broadly neutralizing activity against HIV-1.

Design of an HA2-based Escherichia coli expressed influenza immunogen that protects mice from pathogenic challenge.

Influenza HA is the primary target of neutralizing antibodies during infection, and its sequence undergoes genetic drift and shift in response to immune pressure. The receptor binding HA1 subunit of HA shows much higher sequence variability relative to the metastable, fusion-active HA2 subunit, presumably because neutralizing antibodies are primarily targeted against the former in natural infection. We have designed an HA2-based immunogen using a protein minimization approach that incorporates designed mutations to destabilize the low pH conformation of HA2.

Vaccination with peptide mimetics of the gp41 prehairpin fusion intermediate yields neutralizing antisera against HIV-1 isolates.

Eliciting a broadly neutralizing polyclonal antibody response against HIV-1 remains a major challenge. One approach to vaccine development is prevention of HIV-1 entry into cells by blocking the fusion of viral and cell membranes. More specifically, our goal is to elicit neutralizing antibodies that target a transient viral entry intermediate (the prehairpin intermediate) formed by the HIV-1 gp41 protein.

Oligomers of beta-amyloid are sequestered into and seed new plaques in the brains of an AD mouse model.

Amyloid plaque deposition in the brain is a hallmark of Alzheimer's disease, but recent evidence indicates that the disease may be primarily caused by soluble amyloid-beta (1-42) (Abeta) oligomers or Abeta-derived diffusible ligands (ADDLs). ADDLs induce cognitive deficits in animal models and are thought to assemble in vitro by a mechanism apart from plaque formation. To investigate the in vivo relationship of ADDLs and plaques, biotin-labeled ADDLs (bADDLs) or amylin oligomers (bAMs) were injected into the hippocampus of hAPP overexpressing mice.

Staphylococcus aureus capsule type 8 antibodies provide inconsistent efficacy in murine models of staphylococcal infection.

Staphylococcus aureus is a clinically important capsule-forming bacterium. The capsule polysaccharide (CPs) occurs as different chemical structures depending on the serotype of the organism, but one form, capsular polysaccharide type 8 (CPs8) found in clinical isolates, is largely unstudied. The potential of CPs8 as a vaccine target was evaluated using two approaches.

Anti-ADDL antibodies differentially block oligomer binding to hippocampal neurons.

Abeta-derived diffusible ligands (ADDLs) are abundant in AD brain, bind to hippocampal neurons and induce deficits in rodent cognition. To further investigate ADDL binding to neurons and identify antibodies that block this association, a panel of anti-Abeta and anti-ADDL antibodies was characterized for their ability to immuno-detect neuronally bound ADDLs and attenuate the binding of ADDLs to neurons. The results showed that anti-Abeta and anti-ADDL antibodies were able to abate ADDLs binding to hippocampal neurons, but to different degrees.

Amino acid analysis of peptide loading ratios in conjugate vaccines: a comparison of direct electrochemical detection and 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate pre-column derivatization methods.

Amino acid analysis using direct electrochemical detection was compared with precolumn fluorescent derivatization using 6-aminoquinolyl- N-hydroxysuccinimidyl carbamate (AQC) for evaluation of the degree of covalent coupling of peptides to a carrier-protein complex derived from the bacteria Neisseria meningitidis. AQC derivatization was found to give superior sensitivity compared to electrochemical detection, with less interference from sample components such as carbohydrates or buffer salts. Hydrolysis time and temperature were optimized for maximal recoveries of the marker amino acid 6-aminohexanoic acid (epsilon-Ahx) and the unique amino acids S-dicarboxyethyl cysteine (SDCEC) and S-carboxymethyl homocysteine (SCHMC), which are generated upon the hydrolysis of the covalent linkage between the peptide and the carrier protein.

Solution state characterization of amyloid beta-derived diffusible ligands.

A growing body of evidence suggests that soluble oligomeric forms of the amyloid beta peptide known as amyloid-derived diffusible ligands (ADDLs) are the toxic species responsible for neurodegeneration associated with Alzheimer's disease. Accurate biophysical characterization of ADDL preparations is hampered by the peptide's strong tendency to self-associate and the effect of factors such as ionic strength, temperature, and pH on its behavior. In addition, amyloid peptides are known to interact with common laboratory excipients, specifically detergents, further complicating the results from standard analytical methods such as denaturing polyacrylamide gel electrophoresis.

Immunogenicity and protective efficacy of Bacillus anthracis poly-gamma-D-glutamic acid capsule covalently coupled to a protein carrier using a novel triazine-based conjugation strategy.

The capsular polypeptide of Bacillus anthracis is composed of a unique polyglutamic acid polymer in which D-glutamate monomers are joined by gamma-peptidyl bonds. The capsule is poorly immunogenic, and efforts at exploiting the polymer for vaccine development have focused on increasing its inherent immunogenicity through chemical coupling to immune-stimulating protein carriers. The usual strategy has employed carbodiimide-based condensing reagents for activation of free alpha-carboxyl groups, despite reports that this chemistry may lead to chain scission.

A recombinant 63-kDa form of Bacillus anthracis protective antigen produced in the yeast Saccharomyces cerevisiae provides protection in rabbit and primate inhalational challenge models of anthrax infection.

Infection by Bacillus anthracis is preventable by prophylactic vaccination with several naturally derived and recombinant vaccine preparations. Existing data suggests that protection is mediated by antibodies directed against the protective antigen (PA) component of the anthrax toxin complex. PA is an 83-kDa protein cleaved in vivo to yield a biologically active 63-kDa protein.

Isolation, structural characterization, and immunological evaluation of a high-molecular-weight exopolysaccharide from Staphylococcus aureus.

Colonization of implanted medical devices by coagulase-negative staphylococci such as Staphylococcus epidermidis is mediated by the bacterial polysaccharide intercellular adhesin (PIA), a polymer of beta-(1-->6)-linked glucosamine substituted with N-acetyl and O-succinyl constituents. The icaADBC locus containing the biosynthetic genes for production of PIA has been identified in both S. epidermidis and S.

Purification of virus-like particles of recombinant human papillomavirus type 11 major capsid protein L1 from Saccharomyces cerevisiae.

Recombinant major capsid protein, L1 (M(r) = 55,000), of human papillomavirus type 11 was expressed intracellularly at high levels in a galactose-inducible Saccharomyces cerevisiae expression system by an HPV6/11 hybrid gene. The capsid protein self-assembled into virus-like particles (VLPs) and accounted for 15% of the total soluble protein. A purification process was developed that consisted of two main steps: microfiltration and cation-exchange chromatography.

One-step fluorescent probe product-enhanced reverse transcriptase assay.

Recently developed PCR-based reverse transcriptase (RT) assays are useful in the detection of retroviruses since they are approximately a millionfold more sensitive than conventional RT assays. However, these assays are both labor- and time-intensive. The previously described product-enhanced reverse transcriptase (PERT) assay involves a two-step RT-PCR followed by detection and quantitation of PCR products by either Southern blot or enzyme-linked immunosorbent assay (ELISA).