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Yan C.,University of Delaware | MacKay M.E.,University of Delaware | Czymmek K.,University of Delaware | Nagarkar R.P.,University of Delaware | And 4 more authors.
Langmuir | Year: 2012

β-hairpin peptide-based hydrogels are a class of injectable solid hydrogels that can deliver encapsulated cells or molecular therapies to a target site via syringe or catheter injection as a carrier material. These physical hydrogels can shear-thin and consequently flow as a low-viscosity material under a sufficient shear stress but immediately recover back into a solid upon removal of the stress, allowing them to be injected as preformed gel solids. Hydrogel behavior during flow was studied in a cylindrical capillary geometry that mimicked the actual situation of injection through a syringe needle in order to quantify effects of shear-thin injection delivery on hydrogel flow behavior and encapsulated cell payloads. It was observed that all β-hairpin peptide hydrogels investigated displayed a promising flow profile for injectable cell delivery: a central wide plug flow region where gel material and cell payloads experienced little or no shear rate, and a narrow shear zone close to the capillary wall where gel and cells were subject to shear deformation. The width of the plug flow region was found to be weakly dependent on hydrogel rigidity and flow rate. Live-dead assays were performed on encapsulated MG63 cells 3 h after injection flow and revealed that shear-thin delivery through the capillary had little impact on cell viability and the spatial distribution of encapsulated cell payloads. These observations help us to fundamentally understand how the gels flow during injection through a thin catheter and how they immediately restore mechanically and morphologically relative to preflow, static gels. © 2012 American Chemical Society.

Zhang X.,Ohio State University | Zhang X.,KBI Biopharma | Yang S.-T.,Ohio State University
Journal of Biotechnology | Year: 2011

We have designed, built and tested a three-dimensional (3-D) cell culture system on modified microplates for high-throughput, real-time, proliferation and cytotoxicity assays. In this 3-D culture system, cells expressing the enhanced green fluorescent protein (EGFP) were cultured in nonwoven polyethylene terephthalate (PET) fibrous scaffolds. Compared to 2-D cultures in conventional microplates, 3-D cultures gave more than 10-fold higher fluorescence signals with significantly increased signal-to-noise ratio (SNR), thus extending the application of conventional fluorescence microplate readers for online monitoring of culture fluorescence. The 3-D system was successfully used to demonstrate the effects of fetal bovine serum, fibronectin coating of PET fibers, and cytotoxicity of dexamethasone on recombinant murine embryonic stem D3 cells. The dosage effects of 5-fluorouracil and gemcitabine on high-density colon cancer HT-29 cells were also tested. These studies demonstrated that the 3-D culture microplate system with EGFP expressing cells can be used as a high-throughput system in drug discovery and bioprocess development. © 2010 Elsevier B.V.

Shukla A.A.,KBI Biopharma | Gottschalk U.,Sartorius Stedim Biotech
Trends in Biotechnology | Year: 2013

The manufacture of protein biopharmaceuticals is conducted under current good manufacturing practice (cGMP) and involves multiple unit operations for upstream production and downstream purification. Until recently, production facilities relied on the use of relatively inflexible, hard-piped equipment including large stainless steel bioreactors and tanks to hold product intermediates and buffers. However, there is an increasing trend towards the adoption of single-use technologies across the manufacturing process. Technical advances have now made an end-to-end single-use manufacturing facility possible, but several aspects of single-use technology require further improvement and are continually evolving. This article provides a perspective on the current state-of-the-art in single-use technologies and highlights trends that will improve performance and increase the market penetration of disposable manufacturing in the future. © 2012 Elsevier Ltd.

Jianwu C.,University of Pittsburgh | Theoret J.R.,University of Pittsburgh | Archana S.,University of Pittsburgh | Smedley III J.G.,University of Pittsburgh | And 2 more authors.
Infection and Immunity | Year: 2012

Clostridium perfringens enterotoxin (CPE) causes the gastrointestinal symptoms of the second most common bacterial foodborne illness. Previous studies suggested that a region named TM1, which has amphipathic characteristics and spans from amino acids 81 to 106 of the native CPE protein, forms a Β-hairpin involved in Β-barrel pore formation. Tofurther explore the potential role of TM1 in pore formation, the single Cys naturally present in CPE at residue 186 was first altered to alanine by mutagenesis; the resultant rCPE variant, named C186A, was shown to retain cytotoxic properties. Cys-scanning mutagenesis was then performed in which individual Cys mutations were introduced into each TM1 residue of the C186A variant. When those Cys variants were characterized, three variants were identified that exhibit reduced cytotoxicity despite possessing binding and oligomerization abilities similar to those of the C186A variant from which they were derived. Pronase challenge experiments suggested that the reduced cytotoxicity of those two Cys variants, i.e., the F91Cand F95C variants, which model to the tip of the Β-hairpin, was attributable to a lessened ability of these variants toinsert into membranes after oligomerization. In contrast, another Cys variant, i.e., the G103C variant, with impaired cytotoxicity apparently inserted into membranes after oligomerization but could not form a pore with a fully functional channel. Collectively, these results support the TM1 region forming a Β-hairpin as an important step in CPE insertion and pore formation. Furthermore, this work identifies the first amino acid residues specifically involved inthose two steps in CPE action. © 2012, American Society for Microbiology.

Klapoetke S.,KBI Biopharma
Methods in Molecular Biology | Year: 2014

Three methods are introduced here to fully characterize glycosylation at protein, peptides, and glycan levels by LC-MS (ESI TOF MS). At protein level, glycosylation could be detected by comparing protein masses with and without glycan. At peptide level, glycosylation sites could be detected by removing glycan and site specific glycosylation could be examined with glycopeptides. At glycan level, glycan profiling and identification could be achieved by analyzing released glycans labeled with procainamide. All these methods are MS based and able to provide a whole picture of glycosylation of a glycoprotein. These methods are also simple to implement in industrial or academic laboratory with proper training and instruments. © 2014 Springer Science+Business Media, New York.

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