Opportunities for collective advancement in the biopharmaceutical manufacturing community

Technology innovation, workforce development, and regulatory advancement have been a hallmark of the industry for the past several decades. As the industry continues to expand, mature and evolve, there are new opportunities for shared risk in technology innovation, for partnerships to facilitate training, and for new approaches to engage with health authorities. It can be argued that collaborative efforts in this space, done appropriately, can 1) reduce the burden and investment that would otherwise be required by individual organizations to advance their technology needs and address issues with speed, quality, and cost; 2) refocus academic faculty on the training of students who are better prepared for industry careers; and 3) help faculty better understand the current technology needs of existing industry. Collaborate public-private partnerships offer one way to bring stakeholders together as well as pay for relevant activities. This presentation is intended to catalyze a discussion regarding the opportunities and challenges associated with collaborative technology development and will include a discussion about the ways that stakeholders may collaborate to create opportunities for the biopharmaceutical industry, government, and academic partners

Investigating Difficult-to-Express mAb frameworks in transient and site-specific integration-based CHO expression systems

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Analysis of DNA DSB repair and production stability in CHO cells

Poster Number 13 ANALYSIS OF DNA DSB REPAIR AND PRODUCTION STABILITY IN CHO CELLS Xiaolin Zhang, Department of Chemical and Biomolecular Engineering, University of Delaware Delaware Biotechnology Institute, University of Delaware [email protected] Kelvin H. Lee, Department of Chemical and Biomolecular Engineering, University of Delaware Delaware Biotechnology Institute, University of Delaware Key Words: CHO cell, DSB repair, heterologous expression, production instability. Productivity of recombinant proteins in CHO cell lines often decreases over long-term cultivation. This production instability limits the use of CHO-based platforms and can negatively impact the capability of a manufacturing process to meet market demands. A method to prevent the production loss during long-term cultivation is highly desirable. Genome instability can reduce transgene copy number and is reported as a major cause for production instability. We hypothesize that the DNA double-strand break (DSB) repair system in CHO is deficient and associated with both genome and production instabilities. Our results indicated that CHO cells had a lower DSB repair rate compared to the bEnd.3 mouse endothelial cell line, which is consistent with our hypothesis. The ability to improve DSB repair in CHO may provide a strategy to prevent production instability. Therefore, we tested heterologous expression of eight DSB repair-related genes, and found that four genes could significantly improve DSB repair in CHO cells. To further assess the impact of improved DSB repair on protein production, each of the four heterologous genes was stably expressed in a secreted alkaline phosphatase (SEAP) producing cell line, and SEAP production in single clones was evaluated over three months in the absence of methotrexate (MTX). Our results showed that productivity correlated strongly with the SEAP copy number, and two heterologous genes could substantially improve the production retention during long-term cultivation

Host cell protein control via CHO genome engineering

Chinese hamster ovary (CHO) cells, a major mammalian platform in biomanufacturing, produce and secret recombinant proteins along with host cell proteins (HCPs). Because residual HCPs in the final drug product can adversely affect (1) patients by causing immune responses, (2) drug efficacy, and (3) product stability, the effective removal of HCPs is necessary. Unfortunately, many studies have reported that many HCPs can be difficult to remove through downstream purification processes because they share similar biophysical properties to biopharmaceuticals. In this study we employed a genome engineering approach using clustered regularly interspaced short palindromic repeats and associated protein 9 (CRISPR/Cas9) system-mediated knockout to address difficult-to-remove HCP problems. Three HCPs (Cathepsin D, Nidogen-1, and Prosaposin) that are known to be difficult to remove were selected, and respective knockout clones were isolated without using selective reagents or reporter genes. Clones for each HCP were characterized using various analysis methods. Taken together, we demonstrate the applicability of the CRISPR/Cas9 system to eliminate difficult-to-remove HCP expression in an industry-relevant setting

Mean field approaches to the totally asymmetric exclusion process with quenched disorder and large particles

The process of protein synthesis in biological systems resembles a one dimensional driven lattice gas in which the particles (ribosomes) have spatial extent, covering more than one lattice site. Realistic, nonuniform gene sequences lead to quenched disorder in the particle hopping rates. We study the totally asymmetric exclusion process with large particles and quenched disorder via several mean field approaches and compare the mean field results with Monte Carlo simulations. Mean field equations obtained from the literature are found to be reasonably effective in describing this system. A numerical technique is developed for computing the particle current rapidly. The mean field approach is extended to include two-point correlations between adjacent sites. The two-point results are found to match Monte Carlo simulations more closely

Genomic sequencing and analysis of a Chinese hamster ovary cell line using Illumina sequencing technology

<p>Abstract</p> <p>Background</p> <p>Chinese hamster ovary (CHO) cells are among the most widely used hosts for therapeutic protein production. Yet few genomic resources are available to aid in engineering high-producing cell lines.</p> <p>Results</p> <p>High-throughput Illumina sequencing was used to generate a 1x genomic coverage of an engineered CHO cell line expressing secreted alkaline phosphatase (SEAP). Reference-guided alignment and assembly produced 3.57 million contigs and CHO-specific sequence information for ~ 18,000 mouse and ~ 19,000 rat orthologous genes. The majority of these genes are involved in metabolic processes, cellular signaling, and transport and represent attractive targets for cell line engineering.</p> <p>Conclusions</p> <p>This demonstrates the applicability of next-generation sequencing technology and comparative genomic analysis in the development of CHO genomic resources.</p

PERIOD–TIMELESS Interval Timer May Require an Additional Feedback Loop

In this study we present a detailed, mechanism-based mathematical framework of Drosophila circadian rhythms. This framework facilitates a more systematic approach to understanding circadian rhythms using a comprehensive representation of the network underlying this phenomenon. The possible mechanisms underlying the cytoplasmic “interval timer” created by PERIOD–TIMELESS association are investigated, suggesting a novel positive feedback regulatory structure. Incorporation of this additional feedback into a full circadian model produced results that are consistent with previous experimental observations of wild-type protein profiles and numerous mutant phenotypes

Complement protein isoforms in CSF as possible biomarkers for neurodegenerative disease

Abstract. It has been suggested that the activation of the complement system is involved in the pathogenesis of several neurodegenerative diseases including Alzheimer&apos;s disease (AD), Parkinson&apos;s disease (PD), and multiple sclerosis (MS). Here, the CSF expression levels of complement proteins C3b, C4b, factor B, and factor H were compared between normal subjects and patients diagnosed with AD, PD, MS, and neurosyphilis. The CSF proteins were initially separated using two-dimensional gel electrophoresis, which allowed the comparison of some of the individual complement isoforms. Patients with AD, PD, and MS all showed more than one complement isoform with a significant change (p &lt; 0.05) in CSF expression level compared to normal subjects. PD patients were found to have the greatest number of significantly changed isoforms, all showing a decreased expression level in PD CSF. The complement isoforms examined were able to distinguish between some, but not all, of the diseases studied. The data suggest that when investigating a protein as a possible biomarker, it may be useful to compare individual protein isoform expression levels in addition to the more commonly measured total protein expression level

Synthetic alpha-synuclein fibrils cause mitochondrial impairment and selective dopamine neurodegeneration in part via iNOS-mediated nitric oxide production

Producción CientíficaIntracellular accumulation of α-synuclein (α-syn) are hallmarks of synucleinopathies, including Parkinson's disease (PD). Exogenous addition of preformed α-syn fibrils (PFFs) into primary hippocampal neurons induced α-syn aggregation and accumulation. Likewise, intrastriatal inoculation of PFFs into mice and non-human primates generates Lewy bodies and Lewy neurites associated with PD-like neurodegeneration. Herein, we investigate the putative effects of synthetic human PFFs on cultured rat ventral midbrain dopamine (DA) neurons. A time- and dose-dependent accumulation of α-syn was observed following PFFs exposure that also underwent phosphorylation at serine 129. PFFs treatment decreased the expression levels of synaptic proteins, caused alterations in axonal transport-related proteins, and increased H2AX Ser139 phosphorylation. Mitochondrial impairment (including modulation of mitochondrial dynamics-associated protein content), enhanced oxidative stress, and an inflammatory response were also detected in our experimental paradigm. In attempt to unravel a potential molecular mechanism of PFFs neurotoxicity, the expression of inducible nitric oxide synthase was blocked; a significant decline in protein nitration levels and protection against PFFs-induced DA neuron death were observed. Combined exposure to PFFs and rotenone resulted in an additive toxicity. Strikingly, many of the harmful effects found were more prominent in DA rather than non-DA neurons, suggestive of higher susceptibility to degenerate. These findings provide new insights into the role of α-syn in the pathogenesis of PD and could represent a novel and valuable model to study DA-related neurodegeneration