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Otol Neurotol ; 40(6): e592-e599, 2019 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-31135666


OBJECTIVE: To remove barriers and improve access for patients seeking cochlear implantation. STUDY DESIGN: Prospective quality improvement study at a large tertiary academic care center. METHODS: A Kaizen quality improvement model was applied over the course of a year. Four weeklong meetings were used to identify areas for improvement and remediation. Data were collected at baseline, 90-days, and 1-year postcompletion of the project. Outcome measures included lead times, defined as the wait time between first contact with the clinic and the first appointment, and the wait time between surgery and activation, and cycle times defined as the total test time needed to determine cochlear implant candidacy, and total time needed to complete initial activation. The total inventory kept as clinic stock was also calculated RESULTS:: Kaizen team members collected data for each outcome measure. After the Kaizen principles were applied, the following outcomes were observed: median lead times between first contact with the clinic to candidacy testing, candidacy testing to surgery, and surgery to activation of the implant remained stable from baseline to 1-year follow-up. Median cycle time for candidacy testing decreased from 7.3 hours at baseline to 3.0 hours at 1-year follow-up. Cycle times for initial activation of the device did not change over time. The total inventory of clinic stock was reduced by 31%. CONCLUSIONS: Though outcomes for lead and cycle times varied, implementation of Kaizen principles was found to be an effective method for completing this quality improvement project at a large cochlear implant program overall. LEVEL OF EVIDENCE: 3a.

Biotechnol Prog ; 33(5): 1393-1400, 2017 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-28722325


Most biopharmaceutical drugs, especially monoclonal antibodies (mAbs), bispecific antibodies (BsAbs) and Fc-fusion proteins, are expressed using Chinese Hamster Ovary (CHO) cell lines. CHO cells typically yield high product titers and high product quality. Unfortunately, CHO cell lines also generate high molecular weight (HMW) aggregates of the desired product during cell culture along with CHO host cell protein (HCP) and CHO DNA. These immunogenic species, co-purified during Protein A purification, must be removed in a multi-step purification process. Our colleagues have reported the use of a novel polymer-mediated flocculation step to simultaneously reduce HMW, HCP and DNA from stable CHO cell cultures prior to Protein A purification. The objective of this study was to evaluate this novel "smart polymer" (SmP) in a high throughput antibody discovery workflow using transiently transfected CHO cultures. SmP treatment of 19 different molecules from four distinct molecular categories (human mAbs, murine mAbs, BsAbs and Fabs) with 0.1% SmP and 25 mM stimulus resulted in minimal loss of monomeric protein. Treatment with SmP also demonstrated a variable, concentration-dependent removal of HMW aggregates after Protein A purification. SmP treatment also effectively reduced HCP levels at each step of mAb purification with final HCP levels being several fold lower than the untreated control. Interestingly, SmP treatment was able to significantly reduce high concentrations of artificially spiked levels of endotoxin in the cultures. In summary, adding a simple flocculation step to our existing transient CHO process reduced the downstream purification burden to remove impurities and improved final product quality. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1393-1400, 2017.

Anticuerpos Monoclonales/aislamiento & purificación , Floculación , Polímeros/química , Proteínas Recombinantes/normas , Animales , Células CHO , Cricetinae , Cricetulus , Endotoxinas/análisis , Endotoxinas/química , Endotoxinas/aislamiento & purificación , Humanos , Fragmentos Fab de Inmunoglobulinas/aislamiento & purificación , Proteínas/análisis , Proteínas/química , Proteínas/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificación
Biotechnol Prog ; 33(6): 1436-1448, 2017 11.
Artículo en Inglés | MEDLINE | ID: mdl-28547769


Generating purified protein for GLP toxicology studies (GLP-Tox) represents an important and often rate limiting step in the biopharmaceutical drug development process. Toxicity testing requires large amounts of therapeutic protein (>100 g), typically produced in a single 500-2,500 L bioreactor, using the final CHO clonally derived cell line (CDCL). One approach currently used to save time is to manufacture GLP-Tox material using pools of high-producing CHO CDCLs instead of waiting for the final CDCL. Recently, we reported CHO pools producing mAb titers >7 g/L using piggyBac-mediated gene integration (PB CHO pools). In this study, we wanted to leverage high titer PB CHO pools to produce GLP-Tox material. A detailed product quality attribute (PQA) assessment was conducted comparing PB CHO pools to pooled Top4 CDCLs. Four mAbs were evaluated. First, we found that PB CHO pools expressed all four mAbs at high titers (2.8-4.4 g/L in shake flasks). Second, all four PB CHO pools were aged to 55 generations (Gen). All four PB CHO Pools were found to be suitable over 55 Gen. Finally, we performed bioreactor scale-up. PB CHO pool titers (3.7-4.8 g/L) were similar or higher than the pooled Top 4 CDCLs in 5 L bioreactors (2.4-4.1 g/L). The PQAs of protein derived from PB CHO pools were very similar to pooled Top 4 CHO CDCLs according to multiple orthogonal techniques including peptide mapping analysis. Taken together, these results demonstrate the technical feasibility of using PB CHO pools to manufacture protein for GLP-Tox. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1436-1448, 2017.

Anticuerpos Monoclonales/genética , Reactores Biológicos , Células CHO/efectos de los fármacos , Proteínas Recombinantes/genética , Animales , Anticuerpos Monoclonales/farmacología , Células CHO/metabolismo , Cricetulus , Evaluación Preclínica de Medicamentos , Humanos , Proteínas Recombinantes/efectos adversos , Proteínas Recombinantes/aislamiento & purificación
Biotechnol Lett ; 37(12): 2379-86, 2015 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-26298077


OBJECTIVE: To develop a simple approach to increase titers of transient gene expression in CHO cells without relying on host cell line engineering as recent reports suggest that for PEI-mediated transfections, under optimized conditions, DNA delivery into cells and nuclei is not the limiting factor. RESULTS: N, N-Dimethyl acetamide (DMA) was utilized to enhance transcription. To target post-transcriptional events, we evaluated the co-expression of various genes involved in the unfolded protein response, namely XBP1S, ATF4, CHOP and HSPA5. XBP1S overexpression led to a 15-85 % increase in titer for multiple therapeutic proteins. Mechanistic studies confirmed that addition of 0.125 % DMA increased transgene mRNA levels as expected. However, overexpression of XBP1S had no effect on transgene mRNA levels, indicating that it influenced post-transcriptional events. Since DMA and XBP1S targeted different pathways, the combination of the two approaches led to an additive improvement in protein titer (150-250 % titer increase). CONCLUSION: Transcriptional and post-transcriptional pathways of transient gene expression can be targeted to increase titers without resorting to host cell line engineering in a simple, short, 7 day production process.

Expresión Génica , Proteínas Recombinantes/biosíntesis , Animales , Células CHO , Cricetulus , Procesamiento Postranscripcional del ARN/efectos de los fármacos , Proteínas Recombinantes/genética , Transcripción Genética/efectos de los fármacos
Psychopharmacology (Berl) ; 231(6): 1105-24, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-24429870


INTRODUCTION: Genetic causes, or predisposition, are increasingly accepted to be part of the ethiopathogenesis of many neuropsychiatric diseases. While genes can be studied in any type of cells, their physiological function in human brain cells is difficult to evaluate, particularly in living subjects. METHODS: As a first step towards the characterisation of human inducible pluripotent stem cell (iPSC)-derived neurons from autism spectrum disorder (ASD) patients, we used gene expression and functional studies to define the regional identity of the typical forebrain differentiation, demonstrate expression patterns of genes of interest in ASD and understand the properties of 'control' iPSC-derived neurons (iCell-Neurons™), with a focus on receptors and ion channels that play a central role in synaptic physio-pathology. RESULTS AND DISCUSSION: The gene expression profile of the iCell-Neurons™ closely resembled that observed in neonatal prefrontal cortex tissues. Functional studies, performed mainly using calcium flux assays, demonstrated the presence of ionotropic glutamate (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and N-methyl-D-aspartate) and gamma-aminobutyric acid type A receptors. Voltage-gated sodium and calcium channels were also identified using similar techniques. CONCLUSIONS: Overall, the results reported here suggest that iCell-Neurons™ are a good cellular model of a relatively immature forebrain human neuron population that can be used both as a control in comparison to patients cells, and as host cells in which mutations, insertions and deletions can be used in order to study the molecular mechanisms of ASD and other neurological disorders in an isogenic cellular background.

Células Madre Pluripotentes Inducidas/fisiología , Canales Iónicos/metabolismo , Neuronas/fisiología , Prosencéfalo/fisiología , Calcio/metabolismo , Canales de Calcio/metabolismo , Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Neuronas/efectos de los fármacos , Prosencéfalo/efectos de los fármacos , ARN Mensajero/metabolismo , Receptores de GABA/metabolismo , Receptores de GABA-A/metabolismo , Receptores Ionotrópicos de Glutamato/agonistas , Receptores Ionotrópicos de Glutamato/metabolismo , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Receptores de N-Metil-D-Aspartato/metabolismo , Factores de Tiempo , Canales de Sodio Activados por Voltaje/metabolismo
PLoS One ; 8(7): e71267, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23923059


Biofilm-related infections are a major contributor to human disease, and the capacity for surface attachment and biofilm formation are key attributes for the pathogenesis of microbes. Serratia marcescens type I fimbriae-dependent biofilms are coordinated by the adenylate cyclase, CyaA, and the cyclic 3',5'-adenosine monophosphate (cAMP)-cAMP receptor protein (CRP) complex. This study uses S. marcescens as a model system to test the role of cAMP-phosphodiesterase activity in controlling biofilm formation. Herein we describe the characterization of a putative S. marcescens cAMP-phosphodiesterase gene (SMA3506), designated as cpdS, and demonstrated to be a functional cAMP-phosphodiesterase both in vitro and in vivo. Deletion of cpdS resulted in defective biofilm formation and reduced type I fimbriae production, whereas multicopy expression of cpdS conferred a type I fimbriae-dependent hyper-biofilm. Together, these results support a model in which bacterial cAMP-phosphodiesterase activity modulates biofilm formation.

Bacterias/crecimiento & desarrollo , Bacterias/metabolismo , Biopelículas , Receptores de AMP Cíclico/metabolismo , Bacterias/genética , AMP Cíclico/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fimbrias Bacterianas/metabolismo , Fimbrias Bacterianas/ultraestructura , Regulación Bacteriana de la Expresión Génica , Orden Génico , Genoma Bacteriano , Hidrólisis , Datos de Secuencia Molecular , Mutación , Serratia marcescens/genética , Serratia marcescens/crecimiento & desarrollo , Serratia marcescens/metabolismo