Microfiltration membranes are increasingly used to retain the cells inside bioreactors while continuous harvest of the desired biopharmaceutical occurs in perfusion processes. One method of ...microfiltration is Alternating Tangential Flow (ATF) filtration, which involves moving the cultivation broth tangentially back and forth across the membrane, which results in a backwash effect that reduces fouling. In this study, fouling was investigated with asymmetric polysulfone hollow fibers operated in ATF mode attached to a bioreactor producing a recombinant protein in Chinese Hamster Ovary (CHO) cells. Fouling was assessed through different approaches, including determination of critical flux using an improved flux-step method. Fouling was studied through measurements of Transmembrane Pressure (TMP), protein transmission, membrane pore size and staining of the membrane after operation to visualize the distribution of biological fouling inside the membrane. For critical flux determination, fluxes of up to 69 LMH were used without exceeding the critical flux. No sign of fouling was observed for the short-term (<3.5 h) critical flux experiment. However, during prolonged operation at 8.3 LMH the TMP jumped to 0.9–0.95 bar, indicating fouling. At this state, the protein transmission remains at the same high level (>88 %).
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•Mammalian cell culturing for production of recombinant protein.•Reverse asymmetric membrane hollow fiber filtration in perfusion process.•Critical flux determination in Alternating Tangential Flow Filtration.•Distribution of biological fouling in membrane by staining techniques.•Constantly high protein transmission even though significant fouling is present.
Host cell proteins (HCPs) are process‐related impurities that may copurify with biopharmaceutical drug products. Within this class of impurities there are some that are more problematic. These ...problematic HCPs can be considered high‐risk and can include those that are immunogenic, biologically active, or enzymatically active with the potential to degrade either product molecules or excipients used in formulation. Some have been shown to be difficult to remove by purification. Why should the biopharmaceutical industry worry about these high‐risk HCPs? What approach could be taken to understand the origin of its copurification and address these high‐risk HCPs? To answer these questions, the BioPhorum Development Group HCP Workstream initiated a collaboration among its 26‐company team with the goal of industry alignment around high‐risk HCPs. The information gathered through literature searches, company experiences, and surveys were used to compile a list of frequently seen problematic/high‐risk HCPs. These high‐risk HCPs were further classified based on their potential impact into different risk categories. A step‐by‐step recommendation is provided for establishing a comprehensive control strategy based on risk assessments for monitoring and/or eliminating the known impurity from the process that would be beneficial to the biopharmaceutical industry.
The authors through a 26‐company collaboration compiled a list of “high‐risk” host cell proteins (HCPs) and classified them based on their potential impact into four different risk categories. A step‐by‐step recommendation was provided for establishing a comprehensive control strategy based on risk assessments for monitoring and/or eliminating the high‐risk HCP from the process. Additionally, this article provides a link to a dynamic web database, which serves as a powerful resource to the biotherapeutic industry in dealing with high‐risk HCPs.
•MicroRNAs are powerful cellular engineering tools to improve CHO productivity.•Boosting mitochondrial metabolism has the potential to enhance recombinant protein production.•miR-31* increases cell ...specific productivity in IgG producer CHO cells.•Proteomic profiling is key to understanding cellular processes after miRNA transfection.
MicroRNAs are increasingly being used to enhance relevant pathways of interest during CHO cell line development and to optimise biopharmaceutical production processes. Previous studies have demonstrated that genetic manipulation of microRNAs has led to the development of highly productive phenotypes by increasing cell density through modifying the cell cycle, extending the culture lifespan by delaying apoptotic mechanisms, or improving the energetic flux by targeting mitochondrial metabolism. Re-programming mitochondrial metabolism has arisen as a potential area of interest due to the potential to decrease the Warburg effect and increase cell specific productivity with significant impact on the manufacture of recombinant therapeutic proteins. In this study, we have demonstrated a role for miR-31* to enhance specific productivity in CHO cells by boosting oxidative phosphorylation in the mitochondria. A detailed analysis of the mitochondrial metabolism revealed that miR-31* transfection increases basal oxygen consumption and spare respiratory capacity that leads to an increase in ATP production. Additionally, a proteomic analysis unveiled a number of potential targets involved in fatty acid metabolism and the TCA cycle, both implicated in mitochondrial metabolism. This data demonstrates a potential role for miR-31* to reprogramme the mitochondrial energetic metabolism and increase recombinant protein production in CHO cells.
Chinese hamster ovary (CHO) cells are widely used to manufacture biopharmaceuticals, most of all monoclonal antibodies (mAbs). Some CHO cell lines exhibit production instability, where the ...productivity of the cells decreases as a function of time in culture. To counter this, we designed a passaging strategy that, rather than maximizing the time spent in log-growth phase, mimics the first 7 days of a fed-batch production process. Cultures passaged using this method had lower net growth rates and were more oxidative throughout 6 weeks of passaging. Fed-batch cultures inoculated by cells passaged using this method had increased net growth rates, oxidative metabolism, and volumetric productivity compared to cells passaged using a conventional strategy. Cells from unstable cell lines passaged by this new method produced 80%-160% more mAbs per unit volume than cells passaged by a conventional method. This new method, named Super7, provides the ability to mitigate the impact of production instability in CHO-K1 cell lines without a need for further cell line creation, genetic engineering, or medium development.
The aim of this study was to synthesize a series of quinoline–pyrimidine hybrids and to evaluate their in vitro antimalarial activity as well as cytotoxicity. The hybrids were brought about in a ...two-step nucleophilic substitution process involving quinoline and pyrimidine moieties. They were screened alongside chloroquine (CQ), pyrimethamine (PM) and fixed combinations thereof against the D10 and Dd2 strains of Plasmodium falciparum. The cytotoxicity was determined against the mammalian Chinese Hamster Ovarian cell line. The compounds were all active against both strains. However, hybrid (21) featuring piperazine linker stood as the most active of all. It was found as potent as CQ and PM against the D10 strain, and possessed a moderately superior potency over CQ against the Dd2 strain (IC50: 0.157 vs 0.417μM, ∼threefold), and also displayed activity comparable to that of the equimolar fixed combination of CQ and PM against both strains.
Chinese hamster ovary (CHO) cells represent the most frequently applied host cell system for industrial manufacturing of recombinant protein therapeutics. CHO cells are capable of producing high ...quality biologics exhibiting human-like post-translational modifications in gram quantities. However, production processes for biopharmaceuticals using mammalian cells still suffer from cellular limitations such as limited growth, low productivity and stress resistance as well as higher expenses compared to bacterial or yeast based expression systems. Besides bioprocess, media and vector optimizations, advances in host cell engineering technologies comprising introduction, knock-out or post-transcriptional silencing of engineering genes have paved the way for remarkable achievements in CHO cell line development. Furthermore, thorough analysis of cellular pathways and mechanisms important for bioprocessing steadily unravels novel target molecules which might be addressed by functional genomic tools in order to establish superior production cell factories. This review provides a comprehensive summary of the most fundamental achievements in CHO cell engineering over the past three decades. Finally, the authors discuss the potential of novel and innovative methodologies that might contribute to further enhancement of existing CHO based production platforms for biopharmaceutical manufacturing in the future.
Heparin: Past, Present, and Future Oduah, Eziafa I; Linhardt, Robert J; Sharfstein, Susan T
Pharmaceuticals (Basel, Switzerland),
07/2016, Letnik:
9, Številka:
3
Journal Article
Recenzirano
Odprti dostop
Heparin, the most widely used anticoagulant drug in the world today, remains an animal-derived product with the attendant risks of adulteration and contamination. A contamination crisis in 2007-2008 ...increased the impetus to provide non-animal-derived sources of heparin, produced under cGMP conditions. In addition, recent studies suggest that heparin may have significant antineoplastic activity, separate and distinct from its anticoagulant activity, while other studies indicate a role for heparin in treating inflammation, infertility, and infectious disease. A variety of strategies have been proposed to produce a bioengineered heparin. In this review, we discuss several of these strategies including microbial production, mammalian cell production, and chemoenzymatic modification. We also propose strategies for creating "designer" heparins and heparan-sulfates with various biochemical and physiological properties.
Accurate and complete genome sequences are essential in biotechnology to facilitate genome‐based cell engineering efforts. The current genome assemblies for Cricetulus griseus, the Chinese hamster, ...are fragmented and replete with gap sequences and misassemblies, consistent with most short‐read‐based assemblies. Here, we completely resequenced C. griseus using single molecule real time sequencing and merged this with Illumina‐based assemblies. This generated a more contiguous and complete genome assembly than either technology alone, reducing the number of scaffolds by >28‐fold, with 90% of the sequence in the 122 longest scaffolds. Most genes are now found in single scaffolds, including up‐ and downstream regulatory elements, enabling improved study of noncoding regions. With >95% of the gap sequence filled, important Chinese hamster ovary cell mutations have been detected in draft assembly gaps. This new assembly will be an invaluable resource for continued basic and pharmaceutical research.
The authors completely resequenced the Chinese hamster genome using Single Molecule Real Time (SMRT) sequencing and merged the resulting SMRT‐based assembly with the publicly available draft Illumina‐based assemblies. The new assembly is more contiguous and complete than genome assemblies from either technology alone, reducing the number of scaffolds by >28‐fold, with 90% of the sequence in the 122 longest scaffolds. This new assembly will be an invaluable resource for continued basic and pharmaceutical CHO cell research.
Cell line development (CLD) represents a complex but highly critical process during the development of a biological drug. To shed light on this crucial workflow, a team of BioPhorum members (authors) ...has developed and executed surveys focused on the activities and effort involved in a typical CLD campaign. An average of 27 members from different companies that participate in the BioPhorum CLD working group answered surveys covering three distinguishable stages of a standard CLD process: (1) Pre-transfection, including vector design and construction; (2) Transfection, spanning the initial introduction of vector into cells and subsequent selection and analysis of the pools; and (3) Single Cell Cloning and Lead Clone Selection, comprising methods of isolating single cells and confirming clonal origin, subsequent expansion and screening processes, and methods for identifying and banking lead clones. The surveys were very extensive, including a total of 341 questions split between antibody and complex molecule CLD processes. In this survey review, the authors interpret and highlight responses for antibody development and, where relevant, contrast complex molecule development challenges to provide a comprehensive industry perspective on the typical time and effort required to develop a CHO production cell line.
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