Identification of novel modulators towards high cell density and high-producing Chinese hamster ovary suspension cell cultures as well as their application in biopharmaceutical protein production / Beat Thalmann.

Format:

Book

Author/Creator:

Thalmann, Beat, 1984- author.

Series:

Bielefelder Schriften zur molekularen Biotechnologie ; 15

Language:

English

German

Subjects (All):

Animal cell biotechnology.

Cell culture.

Ovaries--Physiology--Research.

Ovaries.

Hamsters as laboratory animals.

Physical Description:

1 online resource (viii, 207 pages) : illustrations.

Edition:

1st ed.

Place of Publication:

Berlin, Germany : Logos Verlag, [2015]

Language Note:

Text in English; series in German.

Summary:

Long description: Amongst the mammalian producer cell lines, the Chinese hamster ovary (CHO) cell lines are of predominant importance in biopharmaceutical production. Thus, novel factors increasing overall productivity are sought and bear the potential to reduce the unit costs of a production process. Furthermore, the current patent situation for several therapeutic proteins demands innovative tools to at least maintain or preferentially increase the cost-effectiveness of their production processes. In this thesis, hitherto unknown factors were revealed by next generation sequencing of chemically mutated and selected CHO-K1 suspension cell lines. Two factors were proven to improve CHO-based production processes: cgrSnord78 and cgrTtc36. The Cricetulus griseus Ttc36 increases the integral as well as the maximal viable cell density and abolishes the cell-cell aggregation whilst cgrSnord78 improves the specific as well as volumetric productivity without significant impact on cell growth. Based on the present results and discussion, foundations for future research on these functionally unrevealed factors are laid. Hence, this work represents the first step towards the application of the genuine biomolecules cgrTtc36 and cgrSnord78 in biopharmaceutical protein production.

Contents:

Intro

1 Aim of the work

2 Introduction

2.1 Short overview of Chinese hamster ovary cell lines

2.2 Unspecific mutations: A short survey of their use in cell line generation

2.3 Selection methods for cell line generation of distinct phenotypes

2.4 Identification of unknown regulatory cellular factors

2.4.1 Proteomics

2.4.2 Transcriptomics

2.5 Pathway engineering towards high cell density cultures

2.5.1 Cell cycle engineering

2.5.2 Modulation of cell death factors

2.5.3 Relevance of heat shock proteins, molecular chaperons and detoxifying enzymes

2.5.4 Hypothermic factors

2.5.5 Metabolic engineering towards high cell densities

2.5.6 Increasing the maximal cell density by pathway engineering - A summary

2.6 Towards high cell density growing CHO cell lines: A theoretical approach

3 Material and methods

3.1 Cultivation

3.2 Sampling

3.2.1 Cell culture supernatant recovery for analytical purpose

3.2.2 Cell pellet recovery for RNA and genomic DNA isolation

3.3 Manual cell counting

3.4 Cryopreservation and thawing

3.5 Next generation sequencing

3.6 Quantitative real-time PCR analysis

3.6.1 Total RNA isolation

3.6.2 cDNA generation

3.6.3 Quantitative real-time PCR analysis

3.7 Preparative Methods

3.7.1 Genomic DNA isolation

3.7.2 Preparative PCR

3.7.3 Custom gene synthesis

3.7.4 Restriction digest

3.7.5 DNA dephosphorylation

3.7.6 DNA phosphorylation

3.7.7 Isolation and Purification of DNA Fragments

3.7.8 Ligation

3.7.9 Transformation of competent E.coli DH5a cells

3.7.10 E.coli cultivation

3.7.11 Plasmid DNA preparation

3.7.12 Linearisation of vectors and sterile DNA precipitation

3.8 Vectors and cell line generation

3.8.1 Overview on basal vectors and their derivatives

3.8.2 Nucleofection of CHO-S cell lines.

3.8.3 Selection of positively transfected cells and further cultivation

3.8.4 Limited dilution and clonal cell line generation

3.9 Analytics

3.9.1 Fixation of cells and DAPI staining

3.9.2 Agarose Gel Electrophoresis

3.9.3 Plasmid DNA Sequencing

3.9.4 Determination of glucose concentration

3.9.5 hIgG ELISA

3.9.6 SDS-PAGE

3.10 Data compilation and computation

3.11 Material

3.12 Terms and acronyms

3.13 Cell line terminology

4 Results

4.1 Generation of a clonal CHO cell line with capability growing at HCD

4.1.1 Adaption of CHO-K1 cells to CM1035

4.1.2 Chemical mutagenesis and mutated cell regeneration

4.1.3 Selection of CHO-S-E400 towards growth at high osmolality and glutamine absence

4.1.4 Single cell cloning of CHO-S-E400HyOsm

4.1.5 Selection of CHO-S-E400HyOsm clones

4.1.6 Single cell cloning of 25-CHO-S

4.1.7 Transfection and productivity of CHO-S-E400/1.104 and 25-CHO-S/2.5C8

4.1.8 Accumulating cell culture CHO-S-E400/1.104 and 25-CHO-S/2.5C8

4.1.9 Optimised fedbatch cultures and sampling for factor identification

4.2 Factor identification and verification

4.2.1 Transcriptomics by HiSeq 2000

4.2.2 Factor verification using quantitative RT-PCR

4.3 Factor validation in non-producing and producer cell lines

4.3.1 Validation of cgrTPRp in non-producing 25-CHO-S/2.5C8 cells

4.3.2 Validation of LOC100759461/TPR repeat-containing protein C10orf93-like

4.3.3 Validation of cgrGas5 variants and cgrSnord78

4.3.4 Validation of cgrTtc36

5 Discussion

5.1 Increase of genomic variation by mutagenesis and the selection process

5.1.1 Mutagenesis and recovery of the 25-CHO-S cell line

5.1.2 Selection towards growth at high cell densities

5.2 Transcriptomic analysis and data verification

5.3 The tetratricopeptide repeat-containing proteins.

5.3.1 The effect upon the overexpression of cgrTPRp

5.3.2 The putative function of cgrTtc36

5.3.3 The putative localisation as well as function of cgrC10orf93-like/cgrTtc40-like

5.3.4 The inhibition of cell aggregation in cgrTtc36 and cgrC10orf93-like/cgrTtc40-like overexpressing cell lines

5.4 Arguments on cgrGas5 and its intronic sequence cgrSnord78

5.4.1 Insights into the function and structure of cgrGas5

5.4.2 Possible function and targets of cgrSnord78

5.5 Optimisation and industrial relevance of cgrSnord78 and cgrTtc36

6 Conclusion and perspectives

7 References

8 Abbreviations.

Notes:

"Dissertation for the completion of the doctoral degree doctor rerum naturalium at the technical faculty of the Bielefeld University, Germany."

Includes bibliographical references (pages 178-206).

Description based on print version record.

ISBN:

3-8325-8790-X

9783832587901