Charles L. Cooney

Enzymatic degradation of glycosaminoglycans.

Glycosaminoglycans (GAGs) play an intricate role in the extracellular matrix (ECM), not only as soluble components and polyelectrolytes, but also by specific interactions with growth factors and other transient components of the ECM. Modifications of GAG chains, such as isomerization, sulfation, and acetylation, generate the chemical specificity of GAGs. GAGs can be depolymerized enzymatically either by eliminative cleavage with lyases (EC 4.2.2.-) or by hydrolytic cleavage with hydrolases (EC 3.2.1.-). Often, these enzymes are specific for residues in the polysaccharide chain with certain modifications. As such, the enzymes can serve as tools for studying the physiological effect of residue modifications and as models at the molecular level of protein-GAG recognition. This review examines the structure of the substrates, the properties of enzymatic degradation, and the enzyme substrate-interactions at a molecular level. The primary structure of several GAGs is organized macroscopically by segregation into alternating blocks of specific sulfation patterns and microscopically by formation of oligosaccharide sequences with specific binding functions. Among GAGs, considerable dermatan sulfate, heparin and heparan sulfate show conformational flexibility in solution. They elicit sequence-specific interactions with enzymes that degrade them, as well as with other proteins, however, the effect of conformational flexibility on protein-GAG interactions is not clear. Recent findings have established empirical rules of substrate specificity and elucidated molecular mechanisms of enzyme-substrate interactions for enzymes that degrade GAGs. Here we propose that local formation of polysaccharide secondary structure is determined by the immediate sequence environment within the GAG polymer, and that this secondary structure, in turn, governs the binding and catalytic interactions between proteins and GAGs.

End-to-End Continuous Manufacturing of Pharmaceuticals: Integrated Synthesis, Purification, and Final Dosage Formation†

A series of tubes: The continuous manufacture of a finished drug product starting from chemical intermediates is reported. The continuous pilot-scale plant used a novel route that incorporated many advantages of continuous-flow processes to produce active pharmaceutical ingredients and the drug product in one integrated system.

Large-scale production of pharmaceutical-grade plasmid DNA for gene therapy: problems and bottlenecks

Gene therapy is a promising process for the prevention, treatment and cure of diseases such as cancer, acquired immunodeficiency syndrome (AIDS) and cystic fibrosis. One of the methods used to administer therapeutic genes is the direct injection of naked or lipid-coated plasmid DNA, but this requires considerable amounts of plasmid DNA. There are several problems and bottlenecks associated with the design and operation of large-scale processes for the production of pharmaceutical-grade plasmid DNA for gene therapy.

Preparative purification of supercoiled plasmid DNA using anion-exchange chromatography

Large scale manufacturing of gene vectors such as plasmid DNA is an important issue in gene therapy. Anion-exchange chromatography is fundamental in the downstream processing of plasmids both as a process and analytical technique. This work reports the use of Q-Sepharose columns (1, 10 and 40 ml) for the preparative purification of plasmid pUC18. NaCl gradient elution enabled the isolation of supercoiled plasmid from low-M(r) RNA, cDNA and plasmid variants. A compact covalently closed, supercoiled form of denatured plasmid carrying large stretches of single-stranded DNA was identified as one of the major contaminants. Anion-exchange HPLC on a Poros QE 20 column was used to quantify plasmid yield. Supercoiled plasmid was recovered in a single fraction with a 62 +/- 8% yield. Loadings higher than 40 micrograms/ml gel could be used but at the expense of a loss of resolution between open circular and supercoiled forms. Plasmid quality was evaluated by gel electrophoresis, restriction analysis, transformation experiments and protein assays.

Nondestructive and on-line monitoring of tablets using light-induced fluorescence technology.

A system using light-induced fluorescence (LIF) technology was developed for rapid and nondestructive analysis of active pharmaceutical ingredients on tablet surfaces. Nonhomogenous tablets with defined layer of active ingredients were made by 3-Dimensional Printing technology to determine penetration depths of the light source and the resultant fluorescence responses. The LIF method of analysis showed penetration to depths of up to 3 mm into tablets. A correlation between LIF signals from analysis of tablet surfaces and the total drug content of the respective tablets was established. This method of surface analysis was verified with UV spectrometric methods for the total drug content of each respective tablet. The results from a small sample population of tablets made from both homogeneous and nonhomogeneous powder mixtures established good correlation between LIF surface monitoring and total tablet content. The use of on-line monitoring of the individual tablet for surface content demonstrated consistent LIF profiles from simulated production rates up to 3000 tablets a minute. The instrument was also field tested successfully on a tablet analyzer.

White Paper on Continuous Bioprocessing May 20–21 2014 Continuous Manufacturing Symposium

There is a growing interest in realizing the benefits of continuous processing in biologics manufacturing, which is reflected by the significant number of industrial and academic researchers who are actively involved in the development of continuous bioprocessing systems. These efforts are further encouraged by guidance expressed in recent US FDA conference presentations. The advantages of continuous manufacturing include sustained operation with consistent product quality, reduced equipment size, high-volumetric productivity, streamlined process flow, low-process cycle times, and reduced capital and operating cost. This technology, however, poses challenges, which need to be addressed before routine implementation is considered. This paper, which is based on the available literature and input from a large number of reviewers, is intended to provide a consensus of the opportunities, technical needs, and strategic directions for continuous bioprocessing. The discussion is supported by several examples illustrating various architectures of continuous bioprocessing systems.

Effects of Oxygen on Recombinant Protein Expression

Efforts to increase cell growth and protein yields need to be complemented by the maintenance of the quality of the protein produced. Elevated oxygen pressure or rapid increases in oxygen content can cause oxidative stress within the cells, leading to oxidation of specific proteins and nucleotide sequences. In addition, transient or steady‐state anoxic conditions can cause limitations in amino acid production and plasmid stability. Major pathways and mechanisms of oxidative damage to proteins expressed in bacteria are reviewed. Damage to nucleic acids involved in gene expression also is considered. The methodologies for identifying oxidative damage to macromolecules are improving but are not yet adequate for on‐line feedback. This limits our ability to integrate information about these phenomena and the cellular responses into a quantitative model. Enough information is available, however, to consider changes in the time profile of dissolved oxygen as a cause for poor process performance.

Bioreactors: Design and Operation

The bioreactor provides a central link between the starting feedstock and the product. The reaction yield and selectivity are determined by the biocatalyst, but productivity is often determined by the process technology; as a consequence, biochemical reaction engineering becomes the interface for the biologist and engineer. Developments in bioreactor design, including whole cell immobilization, immobilized enzymes, continuous reaction, and process control, will increasingly reflect the need for cross-disciplinary interaction in the biochemical process industry.

Microbial utilization of methanol.

Publisher Summary This chapter focuses on the microbial utilization of methanol. Most attention has been devoted to the aerobic utilization of methanol, particularly by gram-negative bacteria. Significant progress has been made toward understanding the pathways of methanol. Considerably less is known about the anaerobic utilization of methanol. The chapter reveals that many university and industrial research teams have put forth a large effort toward exploring the potential of single-cell protein (SCP) as a novel protein source. In the course of this work, a wide variety of carbon-energy sources have been considered for use in protein production. Emphasis is given on the use of hydrocarbons, particularly normal alkanes including methane. The chapter states that the present and future cost structure, the solubility and ease of handling, and the purity are all factors that make methanol an attractive raw material for SCP production. As a low-cost carbon source, methanol has potential application in other fermentation processes than SCP production.

Fuel gas recovery from controlled landfilling of municipal wastes

Abstract Experiments were carried out in unstirred reactors for the digestion to fuel gas of shredded municipal solid waste and sewage sludge at high total solids concentration. Waste and sludge solids together comprised up to 48 percent by weight of the reactor contents. Finely divided calcium carbonate dispersed in the aqueous phase was employed as a pH buffer. Results of experiments showed that conversion to fuel gas of up to 0.128 m3/ kg (2.04 ft3 CH4 (STP)/lb) solid waste was obtained. In a separate experiment, alkaline pretreatment of the solid waste component preceding digestion further improved conversion to fuel gas. An engineering analysis was conducted for application of these results to a controlled landfill system. For an approximately 1.04 Gg/day (1150 U.S. tons/day) at 7 day/week municipal waste system, based on documented equipment costs and an accepted private utility financing method, the incremental capital cost to modify a landfill for fuel gas production was estimated to be