Investigation of novel cyclic structure in glycoconjugate using a simple model system.

Bacterial capsular polysaccharide protein conjugates are a major class of vaccines for preventing severe bacterial infections. The conjugation of a polysaccharide to a carrier protein is critical for inducing adaptive immune response in healthy humans. Due to the high molecular mass and extensive structural heterogeneity of the glycoconjugate, the underlying sugar linkages and polypeptide site selectivity of the conjugation reaction are not well characterized and understood.

Practical approaches for overcoming challenges in heightened characterization of antibody-drug conjugates with new methodologies and ultrahigh-resolution mass spectrometry.

Antibody-drug conjugation strategies are continuously evolving as researchers work to improve the safety and efficacy of the molecules. However, as a part of process and product development, confirmation of the resulting innovative structures requires new, specialized mass spectrometry (MS) approaches and methods, as compared to those already established for antibody-drug conjugates (ADCs) and the heightened characterization practices used for monoclonal antibodies (mAbs), in order to accurately elucidate the resulting conjugate forms, which can sometimes have labile chemical bonds and more extreme chemical properties like hydrophobic patches. Here, we discuss practical approaches for characterization of ADCs using new methodologies and ultrahigh-resolution MS, and provide specific examples of these approaches.

Process development of a FGF21 protein-antibody conjugate.

A scalable, viable process was developed for the Fibroblast Growth Factor 21 (FGF21) protein-antibody conjugate, CVX-343, an extended half-life therapeutic for the treatment of metabolic disease. CVX-343 utilizes the CovX antibody scaffold technology platform that was specifically developed for peptide and protein half-life extension. CVX-343 is representative of a growing number of complex novel peptide- and protein-based bioconjugate molecules currently being explored as therapeutic candidates.

Expression of proto-oncogene cFMS protein in lung, breast, and ovarian cancers.

We performed immunohistochemistry for macrophage colony-stimulating factor 1 receptor (also known as c-fms proto-oncogene product) on tissue microarrays of human nontumor lung, pulmonary squamous cell carcinomas (SCC) and adenocarcinomas (ADC), and breast and ovarian carcinomas using a commercially available anti-cFMS antibody. The specificity of the antibody was validated by Western blot and mass spectrometry analysis. Staining of cFMS was restricted to stromal fibroblasts in pulmonary SCC and ADC specimens and was not identified in tumor epithelium or epithelium and stromal cells of nontumor lung.

Liquid chromatography/tandem mass spectrometry based targeted proteomics quantification of P-glycoprotein in various biological samples.

P-Glycoprotein (P-gp/ABCB1) is expressed in membrane barriers to exclude pharmacological substrates from cells, and therefore influences the ADME/Tox properties and efficacy of therapeutics. In the present study, a liquid chromatography/tandem mass spectrometry (LC/MS/MS)-mediated targeted proteomics was developed to quantitate P-gp protein. With the aid of in silico predictive tools, a unique 9-mer tryptic peptide of P-gp protein was synthesized (with the stable isotope labeled (SIL) peptide as internal standard) and applied for quantitative LC/MS/MS method development.

Formation of calcium deficient HAp/collagen composites by hydrolysis of alpha-TCP.

Bone-like composites containing calcium deficient hydroxyapatite (CDHAp) were formed by the hydrolysis of alpha-tricalcium phosphate (alpha-TCP) in the presence of type I collagen. CDHAp-collagen composites were synthesized using two techniques. In one technique alpha-TCP was mixed with non-milled (as-received) collagen prior to the addition of the aqueous solution.

Development and characterization of biodegradable nanocomposite injectables for orthopaedic applications based on polyphosphazenes.

Self-setting hydroxyapatite-biodegradable injectable composites are excellent candidates for applications in orthopaedics. We have previously demonstrated the feasibility of development of self-setting calcium-deficient nanocrystalline hydroxyapatite-polymer composites using different calcium phosphate precursors and biodegradable polyphosphazenes. This study aimed to evaluate these novel injectable composites as suitable materials for orthopaedic applications through evaluating their biomechanical properties, osteoblast cellular attachment and gene expression over time.

Biodegradable polyphosphazene-nanohydroxyapatite composite nanofibers: scaffolds for bone tissue engineering.

Bone is a natural composite comprised of hierarchically arranged collagen fibrils, hydroxyapatite and proteoglycans in the nanometer scale. This preliminary study reports the fabrication of biodegradable poly[bis(ethyl alanato)phosphazene]-nanohydroxyapatite (PNEA-nHAp) composite nanofiber matrices via electrospinning. Binary solvent compositions of THF and ethanol were used as a spinning solvent to attain better nanohydroxyapatite dispersibility in PNEA solution.

Mechanical properties and osteocompatibility of novel biodegradable alanine based polyphosphazenes: Side group effects.

The versatility of polymers for tissue regeneration lies in the feasibility to modulate the physical and biological properties by varying the side groups grafted to the polymers. Biodegradable polyphosphazenes are high-molecular-weight polymers with alternating nitrogen and phosphorus atoms in the backbone. This study is the first of its kind to systematically investigate the effect of side group structure on the compressive strength of novel biodegradable polyphosphazene based polymers as potential materials for tissue regeneration.

Effects of carbonate on hydroxyapatite formed from CaHPO(4) and Ca(4)(PO(4))(2)O.

Carbonated hydroxyapatites were formed via reactions in NaHCO(3)/NaH(2)PO(4) solutions from a mixture of particulate tetracalcium phosphate (TetCP) and anhydrous dicalcium phosphate (DCPA). Reactions were followed by determinations of pH and ion concentrations. The solids formed were analyzed by XRD and FTIR.

iTRAQ experimental design for plasma biomarker discovery.

There is considerable interest in using mass spectrometry for biomarker discovery in human blood plasma. We investigated aspects of experimental design for large studies that require analysis of multiple sample sets using iTRAQ reagents for sample multiplexing and quantitation. Immunodepleted plasma samples from healthy volunteers were compared to immunodepleted plasma from patients with osteoarthritis in eight separate iTRAQ experiments.

Formation of composites comprised of calcium deficient HAp and cross-linked gelatin.

Cross-linked gelatin/calcium deficient hydroxyapatite (CDHAp) composites were prepared at or near physiologic temperature. alpha-tricalcium phosphate (alpha-TCP) or a mixture of tetracalcium phosphate and dicalcium phosphate were used as CDHAp precursors. Glutaraldehyde was used to cross-link the gelatin fibers.

Novel low temperature setting nanocrystalline calcium phosphate cements for bone repair: osteoblast cellular response and gene expression studies.

Low temperature setting calcium phosphate cements (CPC) formed from reactive calcium phosphate precursors are receiving great attention in the fields of orthopaedics and tissue engineering. The purpose of this study was to evaluate the mechanical properties and osteocompatibility of a novel calcium deficient hydroxyapatite (CDSHA) with a Ca/P ratio of 1.6 developed in our laboratories and compare it to a previously developed calcium deficient hydroxyapatite (CDHA) with a Ca/P ratio of 1.

Effect of side group chemistry on the properties of biodegradable L-alanine cosubstituted polyphosphazenes.

Biodegradable polyphosphazenes have been investigated for a variety of applications, such as controlled drug delivery matrixes, tissue-engineering scaffolds, membranes, and bone-type composites. In this study we have evaluated the effect of side group chemistry on the properties of biodegradable phosphazene polymers that contain ethyl alanato side groups together with ethyl glycinato, p-methylphenoxy, or p-phenylphenoxy side groups. The polymers were synthesized by a macromolecular substitution route.

In vivo biodegradability and biocompatibility evaluation of novel alanine ester based polyphosphazenes in a rat model.

Amino acid ester substituted polyphosphazenes are attractive candidates for various biomedical applications because of their biocompatibility, controllable hydrolytic degradation rates, and nontoxic degradation products. In this study, the biocompatibility of three L-alanine ethyl ester functionalized polyphosphazenes was evaluated in a subcutaneous rat model. The polymers used in the study were poly[bis(ethylalanato)phosphazene] (PNEA), poly[(50% ethylalanato) (50% methylphenoxy) phosphazene] (PNEA(50)mPh(50)), and poly[(50% ethylalanato)(50% phenyl phenoxy) phosphazene] (PNEA(50)PhPh(50)).

Synthesis, characterization, and osteocompatibility evaluation of novel alanine-based polyphosphazenes.

This study deals with the synthesis and in vitro osteocompatibility evaluation of two novel alanine-containing biodegradable ester polyphosphazenes as candidates to form self-setting composites with hydroxyapatite (HAp) precursors. The two novel biodegradable polyphosphazenes synthesized were poly[(ethyl alanato)1.0(ethyl oxybenzoate)1.

Fabrication and optimization of methylphenoxy substituted polyphosphazene nanofibers for biomedical applications.

Electrospinning has developed as a unique and versatile process to fabricate ultrathin fibers in the form of nonwoven meshes or as oriented arrays from a variety of polymers. The very small dimension of these fibers can generate a high surface area, which makes them potential candidates for various biomedical and industrial applications. The objective of the present study was to develop nanofibers from polyphosphazenes, a class of inorganic-organic polymers known for high biocompatibility, high-temperature stability, and low-temperature flexibility.

Phase evolution during the formation of stoichiometric hydroxyapatite at 37.4 degrees C.

The mechanism of formation of stoichiometric hydroxyapatite in aqueous solution by the acid-base reaction of the precursor calcium phosphates tetracalcium phosphate [Ca4(PO4)2O, TetCP] and anhydrous dicalcium phosphate (CaHPO4, DCPA) was investigated. Phase evolution during the formation of hydroxyapatite was studied, and could be correlated with the kinetics of reaction. Initial dissolution of precursors is followed by hydroxyapatite nucleation and growth.

Formation of hydroxyapatite in water, Hank's solution, and serum at physiological temperature.

The influence of de-ionized water, Hank's saline solution, and bovine calf serum on formation of stoichiometric (Ca/P = 1.67) hydroxyapatite (SHAp) at physiological temperature was studied. SHAp formed in aqueous solution by acid-base reaction of particulate Ca(H(2)PO(4))(2).