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Understanding the impact of zinc on ...

Bhatia, Hemlata.

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Understanding the impact of zinc on CHO cell transcriptome.

紀錄類型:	書目-電子資源 : Monograph/item
正題名/作者:	Understanding the impact of zinc on CHO cell transcriptome./
作者:	Bhatia, Hemlata.
出版者:	Ann Arbor : ProQuest Dissertations & Theses, : 2018,
面頁冊數:	145 p.
附註:	Source: Dissertation Abstracts International, Volume: 79-10(E), Section: B.
Contained By:	Dissertation Abstracts International79-10B(E).
標題:	Biomedical engineering. -
電子資源:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=10836943
ISBN:	9780438006546

Understanding the impact of zinc on CHO cell transcriptome.
Bhatia, Hemlata.

Understanding the impact of zinc on CHO cell transcriptome. - Ann Arbor : ProQuest Dissertations & Theses, 2018 - 145 p.

Source: Dissertation Abstracts International, Volume: 79-10(E), Section: B.

Thesis (Ph.D.)--University of Massachusetts Lowell, 2018.

Recombinant protein therapeutics have revolutionized the area of modern medicine because of their use to cure variety of diseases from flu to cancer. Chinese hamster ovary (CHO) cells have been used in nearly 70% of the manufactured recombinant therapeutics with the current sales of over $30 billion worldwide annually. The protein yield has increased ten folds in last 2 decades due to multiple advances in cell line and media optimizations.

ISBN: 9780438006546Subjects--Topical Terms:

535387
Biomedical engineering.

Understanding the impact of zinc on CHO cell transcriptome.
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100 1 $a Bhatia, Hemlata. $3 3350730
245 1 0 $a Understanding the impact of zinc on CHO cell transcriptome.
260 1 $a Ann Arbor : $b ProQuest Dissertations & Theses, $c 2018
300 $a 145 p.
500 $a Source: Dissertation Abstracts International, Volume: 79-10(E), Section: B.
500 $a Adviser: Seongkyu Yoon.
502 $a Thesis (Ph.D.)--University of Massachusetts Lowell, 2018.
520 $a Recombinant protein therapeutics have revolutionized the area of modern medicine because of their use to cure variety of diseases from flu to cancer. Chinese hamster ovary (CHO) cells have been used in nearly 70% of the manufactured recombinant therapeutics with the current sales of over $30 billion worldwide annually. The protein yield has increased ten folds in last 2 decades due to multiple advances in cell line and media optimizations.
520 $a Advances in media include the replacement of animal protein sources from media. Traditional media contained serum as it is the source of amino acids, lipids, vitamins, trace metals and carbohydrates. However, the use of chemically defined media has become the current practice in industry. For the chemically defined media, different chemicals are added in optimized concentrations in order to enhance the efficiency of the process. A review of media optimization strategies is provided..
520 $a Careful consideration of media optimization strategy helps to balance the process optimization in a timely manner. The study presented in chapter 2 is based on an effort to optimize a class of media components, trace metals, with a goal of enhancing the productivity without compromising the product quality. Trace metals play an important role in serum free cell culture media as serum contains a basal amount of trace metals and it becomes necessary to supplement trace metals in chemically defined media. Trace metals act as cofactors for multiple reactions involved in important cellular functions such as cell growth, protein production and protein folding, may act as mRNA stabilizers, may act as an anti-oxidant etc. that are required to perform necessary cellular functions.
520 $a In this study, Zinc was found to have significant impact on CHO cell growth, metabolism, productivity and product quality. Cell culture media was supplemented with different concentrations of zinc (15microM - 100microM) in order to determine the best working condition for a specified cell line. The phenotypic changes observed in the cell culture are briefly mentioned:
520 $a (i) Increase in production of protein by 49.2% at 45microM zinc concentration (ii) Prevention of lactate consumption above 45microM zinc (iii) Increase in the stationary phase period of cell cycle for 2 days (iv) Decrease in peak VCD with increasing zinc concentration (v) Glycosylation profile changes (Increase in GO and decrease in GI and G2 species) of the protein with increasing concentration of zinc.
520 $a The lactate shift has been shown to increase the cellular efficiency. However, the zinc seems to interfere with the lactate metabolism. This dissertation uses the capacity of RNA-sequencing to measure the gene expression of CHO cells under the influence of zinc supplementation. Understanding the lactate mechanism change caused by zinc supplementation would help with identifying the cell engineering targets in order to make the lactate mechanism better.
520 $a The RNA-sequencing was performed in order to observe the changes caused at transcriptomic level by the supplementation of zinc. Genes from various pathways were observed to be affected: apoptosis, lipid synthesis, mevalonate pathway, gluconeogenesis, and anti-oxidant pathway. It was found that the glycolysis was upregulated, and gluconeogenesis and TCA were observed to be downregulated possibly causing the lactate to build up in zinc supplemented conditions. Specifically, gene for gluconeogenesis (pck2) was down regulated in zinc supplemented conditions. The zinc behavior interference with lactate metabolism is explained in Chapter 2. Also Zinc supplementation was also suggested to alter the pathways related with the production of acetyl CoA, so the data suggests the genes related Acss2, Act2, ACLY and Atoxl to be the potential targets in order to alter the lactate metabolism.
520 $a The attempts were made to understand the cause for the increased specific productivity. The genes for cell cycle, protein processing and product quality were studied. The 1.5-fold upregulation of gene for p21 suggests the possibility of longer stay of cells in GI phase, which in turn could explain the increased protein production. Also, the genes responsible for translation (Eif4a), protein processing in ER and Golgi (PGAP2, HSPA8, Pdia4 and DNajc3) are upregulated. Zinc has never been known before as a metal to change the product quality. As the zinc concentration increases, the GOF species are increased and all the other species (M5, G1F and G2F) are decreased. At transcriptomics level, the genes (Galnt2, Galnt7, Manla2, Man2a2, and Man2b2) were observed to be having low expression level. The transcriptomics study of zinc impact on protein production and quality is explained in chapter 3..
520 $a In Chapter 4, The Quality by design approach to maintain the product quality is explained. Quality by design is explained as the way to build quality into the product so that the quality after product production steps is minimized.
520 $a A unique "design space (DSp) exploration strategy," defined as a function of four key scenarios, was successfully integrated and validated to enhance the DSp building exercise, by increasing the accuracy of analyses and interpretation of processed data. The four key scenarios, defining the strategy, were based on cumulative analyses of individual models developed for the Critical Quality Attributes (23 Glycan Profiles) considered for the study. The analyses of the CQA estimates and model performances were interpreted as (1) Inside Specification/Significant Model (2) Inside Specification/Non-significant Model (3) Outside Specification/Significant Model (4) Outside Specification/Non-significant Model. DSp exploration strategy will aid the critical process of consistently and reproducibly achieving predefined quality of a product throughout its lifecycle. (Published: Int J Pharm. 2016 Oct 15; 512(1):242-252).
520 $a In Chapter 5, application of Process analytical technology (PAT) in cell culture and proof of concept for inline monitoring of cell culture media components (amino acids) is presented. Among various spectroscopic techniques used for real time cell culture data collection, Raman spectroscopy has been shown to have advantages over other techniques. Measurements of several process parameters such as glucose, lactate, glutamine, glutamate, ammonium, osmolality and VCD using Raman-based chemometric models have been reported in literature. The application of Raman spectroscopy, coupled with calibration models for amino acid measurement in cell cultures, has been assessed. The developed models cover four amino acids important for cell growth and production: tyrosine, tryptophan, phenylalanine and methionine. These Raman based models demonstrate the significant potential for the quantification of tyrosine, tryptophan and phenylalanine. (Published: Engineering in Life sciences 2017, DOI: 10.1002/elsc.201700084). .
590 $a School code: 0111.
650 4 $a Biomedical engineering. $3 535387
650 4 $a Systematic biology. $3 3173492
650 4 $a Cellular biology. $3 3172791
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710 2 $a University of Massachusetts Lowell. $3 1017839
773 0 $t Dissertation Abstracts International $g 79-10B(E).
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