Author
Kenneth P. Karey
Bio: Kenneth P. Karey is an academic researcher from Genzyme. The author has contributed to research in topics: Acid sphingomyelinase & Niemann–Pick disease. The author has an hindex of 8, co-authored 9 publications receiving 708 citations.
Papers
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TL;DR: The first successful demonstration of the integration of a perfusion bioreactor and a four‐column periodic counter‐current chromatography system for the continuous capture of candidate protein therapeutics is reported, demonstrating the potential of integrated continuous bioprocessing as a universal platform for the manufacture of various kinds of therapeutic proteins.
Abstract: In the current environment of diverse product pipelines, rapidly fluctuating market demands and growing competition from biosimilars, biotechnology companies are increasingly driven to develop innovative solutions for highly flexible and cost-effective manufacturing. To address these challenging demands, integrated continuous processing, comprised of high-density perfusion cell culture and a directly coupled continuous capture step, can be used as a universal biomanufacturing platform. This study reports the first successful demonstration of the integration of a perfusion bioreactor and a four-column periodic counter-current chromatography (PCC) system for the continuous capture of candidate protein therapeutics. Two examples are presented: (1) a monoclonal antibody (model of a stable protein) and (2) a recombinant human enzyme (model of a highly complex, less stable protein). In both cases, high-density perfusion CHO cell cultures were operated at a quasi-steady state of 50-60 × 10(6) cells/mL for more than 60 days, achieving volumetric productivities much higher than current perfusion or fed-batch processes. The directly integrated and automated PCC system ran uninterrupted for 30 days without indications of time-based performance decline. The product quality observed for the continuous capture process was comparable to that for a batch-column operation. Furthermore, the integration of perfusion cell culture and PCC led to a dramatic decrease in the equipment footprint and elimination of several non-value-added unit operations, such as clarification and intermediate hold steps. These findings demonstrate the potential of integrated continuous bioprocessing as a universal platform for the manufacture of various kinds of therapeutic proteins.
365 citations
TL;DR: Results indicate that purified rhASM can be activated in vitro by loss of the free thiol on the C-terminal cysteine via chemical modification, dimerization, or deletion of this amino acid residue.
139 citations
TL;DR: This method provides an effective process for generating recombinant cell lines producing high levels of therapeutic proteins, with the benefits of rapid and accurate 96‐well plate clone screening and elimination of unstable clones at an earlier stage in the development process.
Abstract: Flow cytometry was partnered with a nonfluorescent reporter protein for rapid, early stage identification of clones producing high levels of a therapeutic protein. A cell surface protein, not normally expressed on CHO cells, is coexpressed, as a reporter, with the therapeutic protein and detected using a fluorescently labeled antibody. The genes encoding the reporter protein and the therapeutic protein are linked by an IRES, so that they are transcribed in the same mRNA but are translated independently. Since they each arise from a common mRNA, the reporter protein's expression level accurately predicts the relative expression level of the therapeutic protein for each clone. This method provides an effective process for generating recombinant cell lines producing high levels of therapeutic proteins, with the benefits of rapid and accurate 96-well plate clone screening and elimination of unstable clones at an earlier stage in the development process. Furthermore, because this method does not rely on the availability of an antibody specific for the therapeutic protein being expressed, it can be easily implemented into any cell line development process.
87 citations
TL;DR: The effectiveness of intracerebroventricular delivery of recombinant human ASM into ASMKO mice showed that ICV delivery of the enzyme led to widespread distribution of the hydrolase throughout the CNS and a significant reduction in lysosomal accumulation of sphingomyelin was observed throughout the brain and also within the spinal cord and viscera.
71 citations
TL;DR: The results suggest that the nonclinical safety assessment of novel therapeutics should include the use of specific animal models of disease whenever feasible, and the observed toxicity involves the rapid breakdown of large amounts of sphingomyelin into ceramide and/or other toxic downstream metabolites, which are known signaling molecules with cardiovascular and pro-inflammatory effects.
45 citations
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TL;DR: Chemical mechanisms for IR-induced modifications of biomolecules along with methods for their detection are described and the synergy of combined "omics" technologies such as genomics and epigenomics, proteomics, and metabolomics is highlighted.
Abstract: Significance: The detrimental effects of ionizing radiation (IR) involve a highly orchestrated series of events that are amplified by endogenous signaling and culminating in oxidative damage to DNA, lipids, proteins, and many metabolites. Despite the global impact of IR, the molecular mechanisms underlying tissue damage reveal that many biomolecules are chemoselectively modified by IR. Recent Advances: The development of high-throughput “omics” technologies for mapping DNA and protein modifications have revolutionized the study of IR effects on biological systems. Studies in cells, tissues, and biological fluids are used to identify molecular features or biomarkers of IR exposure and response and the molecular mechanisms that regulate their expression or synthesis. Critical Issues: In this review, chemical mechanisms are described for IR-induced modifications of biomolecules along with methods for their detection. Included with the detection methods are crucial experimental considerations and cav...
487 citations
TL;DR: It is shown that Cu2+ triggers hepatocyte apoptosis through activation of acid sphingomyelinase (Asm) and release of ceramide, which suggests a previously unidentified mechanism for liver cirrhosis and anemia in Wilson disease.
Abstract: Wilson disease is caused by accumulation of Cu(2+) in cells, which results in liver cirrhosis and, occasionally, anemia. Here, we show that Cu(2+) triggers hepatocyte apoptosis through activation of acid sphingomyelinase (Asm) and release of ceramide. Genetic deficiency or pharmacological inhibition of Asm prevented Cu(2+)-induced hepatocyte apoptosis and protected rats, genetically prone to develop Wilson disease, from acute hepatocyte death, liver failure and early death. Cu(2+) induced the secretion of activated Asm from leukocytes, leading to ceramide release in and phosphatidylserine exposure on erythrocytes, events also prevented by inhibition of Asm. Phosphatidylserine exposure resulted in immediate clearance of affected erythrocytes from the blood in mice. Accordingly, individuals with Wilson disease showed elevated plasma levels of Asm, and displayed a constitutive increase of ceramide- and phosphatidylserine-positive erythrocytes. Our data suggest a previously unidentified mechanism for liver cirrhosis and anemia in Wilson disease.
421 citations
TL;DR: The molecular mechanisms that regulate these enzymes in vivo and in vitro are focused on, especially the roles of oxidants (glutathione, peroxide, nitric oxide), proteins, and lipids (diacylglycerol, arachidonic acid, and ceramide).
Abstract: Ceramide, an emerging bioactive lipid and second messenger, is mainly generated by hydrolysis of sphingomyelin through the action of sphingomyelinases. At least two sphingomyelinases, neutral and a...
326 citations
TL;DR: Ceramide-enriched membrane platforms serve the clustering of receptors, the recruitment of intracellular signaling molecules and the exclusion of inhibitory signaling factors and, thus, facilitate signal transduction initiated by the specific stimulus.
313 citations
TL;DR: A large, multimeric complex is composed of the adapter protein, apoptotic protease-activating factor-1, and the cysteine protease, caspase-9, which stimulates formation of the "apoptosome" in stressed cells.
Abstract: Significance: The intrinsic apoptosis pathway is conserved from worms to humans and plays a critical role in the normal development and homeostatic control of adult tissues. As a result, numerous diseases from cancer to neurodegeneration are associated with either too little or too much apoptosis. Recent Advances: B cell lymphoma-2 (BCL-2) family members regulate cell death, primarily via their effects on mitochondria. In stressed cells, proapoptotic BCL-2 family members promote mitochondrial outer membrane permeabilization (MOMP) and cytochrome c (cyt c) release into the cytoplasm, where it stimulates formation of the “apoptosome.” This large, multimeric complex is composed of the adapter protein, apoptotic protease-activating factor-1, and the cysteine protease, caspase-9. Recent studies suggest that proteins involved in the processes leading up to (and including) formation of the apoptosome are subject to various forms of post-translational modification, including proteolysis, phosphorylation,...
292 citations