Cell Culture Bioprocess Engineering, Second Edition

Wei-Shou Hu

• Publisher: CRC Press
• Year: 2020
• eBook ISBN: 9780429531897
• Edition: 2
• Topic: Biowissenschaften
• Subtopic: Pharma-, Biotechnologie- & Gesundheitsindustrie

This book is the culmination of three decades of accumulated experience in teaching biotechnology professionals. It distills the fundamental principles and essential knowledge of cell culture processes from across many different disciplines and presents them in a series of easy-to-follow, comprehensive chapters. Practicality, including technological advances and best practices, is emphasized.

This second edition consists of major updates to all relevant topics contained within this work. The previous edition has been successfully used in training courses on cell culture bioprocessing over the past seven years. The format of the book is well-suited to fast-paced learning, such as is found in the intensive short course, since the key take-home messages are prominently highlighted in panels. The book is also well-suited to act as a reference guide for experienced industrial practitioners of mammalian cell cultivation for the production of biologics.

1

Overview of Cell Culture Processes

Contents

An Introduction to Cell Culture: A Historical Perspective

Early Enabling Innovations in Cell Culture

Differentiated Cell Lines

Differentiated Cells in Culture

Stem Cells

Guiding Cell Differentiation in Vitro

Cell Culture as Process Technology

Viral Vaccines

Protein Molecules as Therapeutics

Biosimilars and the Expanded Reach of Protein Therapeutics

Gene Therapy

Cell Therapy

Industrial Cell Lines

Other Production Systems

Insect Cell Culture

Yeasts

Transgenic Animals

Manufacturing

Upstream Process

Single-Use Systems and Continuous Process

Product Recovery

Product Quality in Cell Culture Processing

Quality of the Product

Glycosylation Profile

Protein Structural Variants

Process Control and Product Quality

From Discovery to Clinical Products

References

An Introduction to Cell Culture: A Historical Perspective

Early Enabling Innovations in Cell Culture

Cultured mammalian cells are the workhorse for the manufacturing of the class of pharmaceuticals known as biologics, including viral vaccines and many protein medicines. What may not be known to many is the art-like nature of growing cells at the dawn of cell culture, a skill that required a great deal of creativity, inquisitiveness, passion, and perseverance, in addition to all the good traits of a scientist. Around the turn of the twentieth century, explants of animal tissues began to be cultured on glass surfaces that were submerged in an animal’s tissue fluid. Cells grew from the tissue clumps and remained viable and observable for a few days.¹ From the outgrowth of tissue clumps, some cells could be isolated and eventually dissected out and expanded to new glass surfaces. The capability of passaging or expanding cell population was an important step towards genuine cell culture. Among the first cell lines established that could be continuously expanded in culture was the mouse L cell.² The early cell lines could be sustained in the lab only by continuous passaging; they could not be frozen, stored away, and later thawed to resume growth. In that era, the complex nutrient mixture could not be sterilized by heat. Rather, serum, ascetic fluids, or chicken embryo extract were carefully isolated from animals in order to maintain sterility for cell cultivation (Figure 1.1).³ Imagine the amount of work involved in maintaining cultured cells!

Figure 1.1. Milestones in cell culture technology.

The discovery of cryopreservation for freezing animal sperm and later other cells allowed cell growth to be paused and resumed in the lab.⁴ Another important advance was the use of trypsin for cell passaging, instead of relying on dissection to dissociate cells from the surface. The first human cell line, HeLa, derived from human cervical cancer, fully took advantage of this.^{3, 5}

Critical to cell culture advances was the arrival of membrane-filtration-based medium sterilization, first by ultrafiltration with a nitrocellulose membrane and later by microfiltration. While saline and media for microorganisms could be autoclaved for sterilization, the complex nutrients needed by animal cells are destroyed at high temperatures. Membrane sterilization accelerated the development of a chemical nutrient medium consisting of glucose, amino acids, vitamins, and balanced salts.⁶ This not only advanced our knowledge of the nutritional needs of cells, but also greatly simplified the logistics of growing cells, leading to the establishment of many important cell lines and eventually to the industrialization of cell culture.

The early cell lines that could be continuously passaged, including mouse L and HeLa, were derived from cancerous tissues (Panel 1.1). Morphologically, they looked abnormal and were distinct from primary cells first grown from normal tissues. Later continuous cell lines were isolated from various animal tissues, including baby hamster kidney (BHK) from the Syrian hamster,⁷ Vero from green monkey kidney,⁸ and Chinese hamster ovary (CHO) from Chinese hamster ovary.⁹ These cell lines carried mutations that allowed them to bypass their cells’ internal growth control mechanisms. These cells were not phenotypically normal. They often did not exhibit contact inhibition. With abundant nutrient supply, they grew into multiple layers of cells on a surface. Later, 3T3, a cell line which is adhesion dependent, exhibits contact inhibition, and does not undergo senescence, was established.¹⁰ But the karyotype (or chromosome composition) of 3T3 cells, as well as other continuous cell lines, was aneuploid, not diploid. Around the same time, phenotypically normal human fibroblastic cell strains, such as WI-38 and later MRC-5 and FS-4, were isolated.^{11, 12, 13} These cells were diploid and exhibited contact inhibition but were not continuous cell lines like 3T3. They senesced after repeated passaging in culture. For many decades, these cell lines and cell strains served important roles in biological science and medical research, and many were used in the industrial production of viral vaccines and other biologics.

Panel 1.1. Cell Substrate Example

Primary Cells

• Tissue explant, limited cell expansion

Cell Lines

• e.g., Mouse L, HeLa, BHK, CHO

Differentiated Cell Lines

• e.g., HepG2 (liver), PC12 (neuronal)

Stem Cells

• Multipotent, pluripotent
• Capable of directed differentiation in vitro

Different...

Table of contents

• Cover
• Half Title
• Title Page
• Copyright Page
• Table of Contents
• Preface
• 1 Overview of Cell Culture Processes
• 2 Cell Biology for Bioprocessing
• 3 Cell Physiology
• 4 Product Quality and Process
• 5 Stoichiometry and the Kinetics of Cell Cultivation
• 6 Cell Line Development
• 7 Medium Design for Cell Culture Processing
• 8 Cell Culture Bioreactors
• 9 Fed-Batch Culture Processes
• 10 Continuous Cell Culture Processes
• 11 Oxygen Transfer in Cell Culture Bioreactors
• 12 Scaling Up and Scaling Down for Cell Culture Bioreactors
• Appendix A: Cell Culture Process Data Analysis
• Appendix B
• Index